PubReading: Recent Episodes
Mando Mourad
Researchers would love if they can expand time to fit in that publication, article, or review of their topic of interest; if that were true, however, there is no end to bottomless scrolling and missing the important details. PubReading would read out the abstract, results, and discussions to allow hassle-free information and a chance to create connections with like-minded individuals.
A workshop held last June by the National Institutes of Health (NIH) Director’s Office, Nature Publishing Group, and Science focused on the role that journals play in supporting scientific research that is reproducible, robust, and transparent. The “Principles and Guidelines for Reporting Preclinical Research” that emerged from the workshop have since been endorsed by nearly 80 societies, journals, and associations.VOL.290,NO.50,pp.29692–29694 - 2015
The gender imbalance in science, technology, engineering, and math (STEM) fields has remained constant for decades and increases the farther up the STEM career pipeline one looks. Why does the underrepresentation of women endure? This study investigated the role of parenthood as a mechanism of gender-differentiated attrition from STEM employment. Using a nationally representative 8-year longitudinal sample of US STEM professionals, we examined the career trajectories of new parents after the birth or adoption of their first child. We found substantial attrition of new mothers: 43% of women leave full-time STEM employment after their first child. New mothers are more likely than new fathers to leave STEM, to switch to part-time work, and to exit the labor force. These gender differences hold irrespective of variation by discipline, race, and other demographic factors. However, parenthood is not just a “mother’s problem”; 23% of new fathers also leave STEM after their first child. Suggesting the difficulty of combining STEM work with caregiving responsibilities generally, new parents are more likely to leave full-time STEM jobs than otherwise similar childless peers and even new parents who remain employed full time are more likely than their childless peers to exit STEM for work elsewhere. These results have implications for policymakers and STEM workforce scholars; whereas parenthood is an important mechanism of women’s attrition, both women and men leave at surprisingly high rates after having children. Given that most people become parents during their working lives, STEM fields must do more to retain professionals with children.doi/10.1073/pnas.1810862116 - 2019
Carbon nanomaterials (CNMs) are an incredibly versatile class of materials that can be used as scaffolds to construct anticancer nanocarrier systems. The ease of chemical functionalisation, biocompatibility, and intrinsic therapeutic capabilities of many of these nanoparticles can be leveraged to design effective anticancer systems. This article is the first comprehensive review of CNM-based nanocarrier systems that incorporate approved chemotherapy drugs, and many different types of CNMs and chemotherapy agents are discussed. Almost 200 examples of these nanocarrier systems have been analysed and compiled into a database. The entries are organised by anticancer drug type, and the composition, drug loading/release metrics, and experimental results from these systems have been compiled. Our analysis reveals graphene, and particularly graphene oxide (GO), as the most frequently employed CNM, with carbon nanotubes and carbon dots following in popularity. Moreover, the database encompasses various chemotherapeutic agents, with antimicrotubule agents being the most common payload due to their compatibility with CNM surfaces. The benefits of the identified systems are discussed, and the factors affecting their efficacy are detailed.doi.org/10.3390/pharmaceutics15051545 - 2023
Human tissues are invaluable resources for pharmaceutical research. They provide information about disease pathophysiology - and equally importantly, healthy function; confirmation (or refutation) of potential drug targets; validation (or otherwise) of other models employed; and functional models for assessing drugs’ effects, whether beneficial or undesirable, in the most appropriate environment that can be replicated outside the human body. While human tissues have long been prized by pathologists in furthering our under- standing of disease processes, there is a growing appreciation of their value at the late pre-clinical stage of drug discovery. Human tissues’ potential to contribute to earlier phases of the process, before significant resources have been expended, is also now gaining recognition. Mounting concern over high rates of clinical stage drug failures mandates exploration of avenues for improving efficiency. Human tissue-based assays could play a key role in improving the translation process, as well as in moving towards stratified or personalised medicines. This editorial highlights some of the potential benefits of introducing human biosamples at each stage of the research process as a drug moves from concept to clinic. Some of the challenges with respect to obtaining tissues, minimising variability and gaining acceptance are also discussed.https://doi.org/10.1517/17460441.2012.689282 - 2012
Albert Einstein once eloquently stated “We still do not know one thousandth of one percent of what nature has revealed to us.” Mother Nature has proven time and again to be the best engineer, architect, scientist, and doctor. This fact has not been lost on mankind, and since time immemorial, we have looked to nature for answers to human conditions and diseases. Indeed, our first evidence dates back to preliterary history as long as 50,000 years ago, in archaeological discoveries from a Middle Eastern grave site at Shanidar, Iraq, of a Neanderthal man which contained plant specimens, of which are still used in local traditional medicine. Since then, the use and application of herbal medicine has been recorded in every society from Traditional Chinese, Ayurvedic, Yunani, and other cultures in the developing world.DOI: 10.1097/FJC.0000000000001278 - 2022
Triple negative breast cancer (TNBC) remains a therapeutic challenge due to the lack of targetable genetic alterations and the frequent development of resistance to the standard cisplatin-based chemotherapies. Here, we have taken a systems biology approach to investigate kinase signal transduction networks that are involved in TNBC resistance to cisplatin. Treating a panel of cisplatin- sensitive and cisplatin-resistant TNBC cell lines with a panel of kinase inhibitors allowed us to reconstruct two kinase signalling networks that characterise sensitive and resistant cells. The analysis of these networks suggested that the activation of the PI3K/AKT signalling pathway is critical for cisplatin resistance. Experimental validation of the computational model predictions confirmed that TNBC cell lines with activated PI3K/AKT signalling are sensitive to combinations of cisplatin and PI3K/AKT pathway inhibitors. Thus, our results reveal a new therapeutic approach that is based on identifying targeted therapies that synergise with conventional chemotherapies.doi.org/10.3390/jpm12081277 - 2022
Meta-research, or the science of science, is a powerful technique that scientists can use to improve science, however most scientists are unaware that meta-research exists and courses are rare. This initiative demonstrates the feasibility of a participant-guided “learn by doing” approach, in which a multidisciplinary, global team of early career researchers learned meta-research skills by working together to design, conduct and publish a meta-research study.https://doi.org/10.1371/journal.pbio.3001073 - 2021
Mechanically interlocked molecules such as rotaxanes and catenanes contain free-moving components that cannot dissociate and have enabled the investigation and control of various translational and rotational molecular motions. The architecture of pseudo-rotaxanes and of some kinetically labile rotaxanes is comparable to that of rotaxanes but their components are reversibly associated and not irreversibly interlocked. In other words, pseudo-rotaxanes may fall apart. This Account focuses on a peculiar family of rotaxane-like architectures termed foldaxanes. Foldaxanes consist of a helically folded oligomer wound around a rod-like dumbbell-shaped guest. Winding of the helix around the rod thus entails an unwinding−rewinding process that creates a kinetic barrier. It follows that foldaxanes, albeit reversibly assembled, have significant lifetimes and may not fall apart while defined molecular motions are triggered. Foldaxanes based on helically folded aromatic oligoamide hosts and oligo(alkyl carbamate) guests can be designed rationally through the inclusion of complementary binding motifs on the rod and at the inner rim of the helix so that helix length and rod length match. Single helical foldaxanes (bimolecular species) and double helical foldaxanes (trimolecular species) have thus been produced as well as poly[n]foldaxanes, in which several helices bind to long rods with multiple binding stations. When the binding stations differ and are organized in a certain sequence, a complementary sequence of different stacked helices, each matching with their binding station, can be assembled, thus reproducing in an artificial system a sort of translation process.https://doi.org/10.1021/acs.accounts.2c00050 - 2022
[Ru(phen)2 (dppz)]2+ has been studied since the 1990s due to its ‘light-switch’ properties. It can be used as a luminescent DNA probe, with emission switched on through DNA binding. The luminescence observed is dependent on the solvent accessibility of the pyrazine nitrogen atoms, and therefore is sensitive to changes in both binding site of the cation and chromophore orientation. The compound is also chiral, and there are distinct differences between the enantiomers in terms of the emission behaviour when bound to a variety of DNA sequences. Whilst a number of binary DNA-complex X-ray crystal structures are available, most include the lambda enantiomer and there is very little structural information about binding of the delta enantiomer. Here, we present the first X-ray crystal structure of a delta enantiomer bound to well-matched DNA, in the absence of the other, lamda enantiomer. We show how the binding site observed here can be related to a more general pattern of motifs in the crystallographic literature and propose that the delta enantiomer can bind with five different binding modes, offering a new hypothesis for the interpretation of solution data.doi: 10.1093/nar/gkw753 - 2016
The movement towards open science is a consequence of seemingly pervasive failures to replicate previous research. This transition comes with great benefits but also significant challenges that are likely to affect those who carry out the research, usually early career researchers (ECRs). Here, we describe key benefits, including reputational gains, increased chances of publication, and a broader increase in the reliability of research. The increased chances of publication are supported by exploratory analyses indicating null findings are substantially more likely to be published via open registered reports in comparison to more conventional methods. These benefits are balanced by challenges that we have encountered and that involve increased costs in terms of flexibility, time, and issues with the current incentive structure, all of which seem to affect ECRs acutely. Although there are major obstacles to the early adoption of open science, overall open science practices should benefit both the ECR and improve the quality of research. We review 3 benefits and 3 challenges and provide suggestions from the perspective of ECRs for moving towards open science practices, which we believe scientists and institutions at all levels would do well to consider.https:// doi.org/10.1371/journal.pbio.3000246 - 2019
We describe an isothermal, single-reaction method for assembling multiple overlapping DNA molecules by the concerted action of a 5' exonuclease, a DNA polymerase and a DNA ligase. First we recessed DNA fragments, yielding single-stranded DNA overhangs that specifically annealed, and then covalently joined them. This assembly method can be used to seamlessly construct synthetic and natural genes, genetic pathways and entire genomes, and could be a useful molecular engineering tool.DOI:10.1038/NMETH.1318 - 2009
Formation of natural intramolecular triple-helical structures of DNA is still an intriguing research topic in view of the possible involvement of these structures in biological processes. The biochemical and biophysical properties of DNA triplex structures have been extensively studied, and experimental data show that H-DNA is likely to form in vivo and may regulate the expression of various genes. However, direct and unambiguous evidence of the possible biological roles of these structures is yet elusive. This review focuses on the basic facts that are in favor of, or against, the hypothesis of the presence and function of natural DNA triple-helical structures in vivo, and outlines the different methods and probes that have been used to support these facts.DOI 10.1007/s00018-003-3046-3 - 2003
In STEM, and particularly in science, many early career researchers find themselves isolated and lacking guidance. There is an enormous need to connect early career scientists with experienced professionals outside their immediate work environment. A new initiative aims to create a supportive community to foster communication between scientists through all stages of their career.https://doi.org/10.1016/j.molmed.2019.08.007 - 2019
Pneumocystis, a major opportunistic pathogen in patients with a broad range of immunodeficiencies, contains abundant surface proteins encoded by a multicopy gene family, termed the major surface glycoprotein (Msg) gene superfamily. This superfamily has been identified in all Pneumocystis species characterized to date, highlighting its important role in Pneumocystis biology. In this report, through a comprehensive and in-depth characterization of 459 msg genes from 7 Pneumocys- tis species, we demonstrate, for the first time, the phylogeny and evolution of conserved domains in Msg proteins and provide a detailed description of the classification, unique characteristics, and phylogenetic relatedness of five Msg families. We further describe, for the first time, the relative expression levels of individual msg families in two rodent Pneumocystis species, the substantial variability of the msg repertoires in P. carinii from laboratory and wild rats, and the distinct features of the expression site for the classic msg genes in Pneumocystis from 8 mammalian host species. Our analysis suggests multiple functions for this superfamily rather than just conferring antigenic variation to allow immune evasion as previously believed. This study provides a rich source of information that lays the foundation for the continued experimental exploration of the functions of the Msg superfamily in Pneumocystis biology.https://doi.org/10 .1128/mBio.02878-19 - 2020
Myeloid derived suppressors cells (MDSC) play major roles in regulating immune homeostasis and immune responses in many conditions, including cancer. MDSC interact with cancer cells within the tumor microenvironment (TME) with direct and indirect mechanisms: production of soluble factors and cytokines, expression of surface inhibitory molecules, metabolic rewiring and exosome release. The two-way relationship between MDSC and tumor cells results in immune evasion and cancer outgrowth. In multiple myeloma (MM), MDSC play a major role in creating protumoral TME conditions. In this minireview, we will discuss the interplay between MDSC and MM TME and the possible strategies to target MDSC.doi: 10.3389/fimmu.2022.1102471 - 2023
Early career researchers face uncertainties with respect to their job prospects due to dwindling job markets, decreased availability of funding and undefined career paths. As basic researchers and clinicians tend to have different approaches to scientific problems, there are many advantages from successful collaborations between them. Here, we discuss how collaborations between basic and clinical scientists should be promoted early in their careers. To achieve this, researchers, both basic and clinical, must be proactive during their training and early stages of their careers. Mentors can further augment collaborative links in many ways. We suggest that universities and institutions might reassess their involvement in promoting collaborations between basic and clinical researchers. We hope that this paper will serve as a reminder of the importance of such collaborations, and provide the opportunity for all members of the scientific community to reflect on and ame- liorate their own contributions.DOI: 10.1111/jth.13447 - 2016
DNA has emerged as an attractive medium for archival data storage due to its durability and high information density. Scalable parallel random access to information is a desirable property of any storage system. For DNA-based storage systems, however, this still needs to be robustly established. Here we report on a thermoconfined polymerase chain reaction, which enables multiplexed, repeated random access to compartmentalized DNA files. The strategy is based on localizing biotin-functionalized oligonucleotides inside thermoresponsive, semipermeable microcapsules. At low temperatures, microcapsules are permeable to enzymes, primers and amplified products, whereas at high temperatures, membrane collapse prevents molecular crosstalk during amplification. Our data show that the platform outperforms non-compartmentalized DNA storage compared with repeated random access and reduces amplification bias tenfold during multiplex polymerase chain reaction. Using fluorescent sorting, we also demonstrate sample pooling and data retrieval by microcapsule barcoding. Therefore, the thermoresponsive microcapsule technology offers a scalable, sequence-agnostic approach for repeated random access to archival DNA files.https://doi.org/10.1038/s41565-023-01377-4 - 2023
Improving the reproducibility of neuroscience research is of great concern, especially to early-career researchers (ECRs). Here I outline the potential costs for ECRs in adopting practices to improve reproducibility. I highlight the ways in which ECRs can achieve their career goals while doing better science and the need for established researchers to support them in these efforts.https://doi.org/10.1016/j.neuron.2018.11.030 - 2019
High dropout rates, delay, and dissatisfaction among PhD students are common problems in doctoral education. Research shows that many different factors are associated with doctoral success, but these factors have not often been studied simultaneously. Moreover, characteristics of the PhD project are mostly neglected. In this study, we investigate which supervision, psychosocial, and project characteristics are related to satisfaction, progress, and quit intentions in a sample of 839 PhD candidates at a university in the Netherlands. Results of regression analyses show that experienced workload was negatively related to satisfaction and progress and positively to quit intentions. The quality of the supervisor-PhD candidate relationship, the PhD candidate’s sense of belonging, the amount of freedom in the project, and working on a project closely related to the supervisor’s research were positively related to satisfaction and negatively to quit intentions. The high workload of PhD candidates should be a major point of attention for universities who wish to increase their rates of PhD completion and PhD candidates’ satisfaction. In addition, the ‘match’ between PhD candidate and supervisor is crucial, both personally – a good relationship – and academically, i.e. that the PhD candidate works on a topic closely related to the supervisor’s research.https://doi.org/10.1080/0158037X.2019.1652158 -2021
Artificial deep neural networks (DNNs) initially inspired by the brain enable computers to solve cognitive tasks at which humans excel. In the absence of explanations for such cognitive phenomena, in turn cognitive scientists have started using DNNs as models to investigate biological cognition and its neural basis, creating heated debate. Here, we reflect on the case from the perspective of philosophy of science. After putting DNNs as scientific models into context, we discuss how DNNs can fruitfully contribute to cognitive science. We claim that beyond their power to provide predictions and explanations of cognitive phenomena, DNNs have the potential to contribute to an often overlooked but ubiquitous and fundamental use of scientific models: exploration.https://doi.org/10.1016/j.tics.2019.01.009 - 2019
In 2006, to address the global inequitable access to influenza vaccines in the event of an influenza pandemic, WHO, with support of donors and partners, embarked on an ambitious project, the Technology Transfer Initiative (TTI), to facilitate influenza vaccine production capacity-building in low- and middle-income countries (LMICs). This commentary briefly summarizes the high-level lessons learned, key challenges encountered, and critical components needed for success.https://doi.org/10.1016/j.vaccine.2022.06.057 - 2022
The methods section of a research paper provides the information by which a study’s validity is judged. Therefore, it requires a clear and precise description of how an experiment was done, and the rationale for why specific experimental procedures were chosen. The methods section should describe what was done to answer the research question, describe how it was done, justify the experimental design, and explain how the results were analyzed. Scientific writing is direct and orderly. Therefore, the methods section structure should: describe the materials used in the study, explain how the materials were prepared for the study, describe the research protocol, explain how measurements were made and what calculations were performed, and state which statistical tests were done to analyze the data. Once all elements of the methods section are written, subsequent drafts should focus on how to present those elements as clearly and logically as possibly. The description of preparations, measurements, and the protocol should be organized chronologically. For clarity, when a large amount of detail must be presented, information should be presented in sub-sections according to topic. Material in each section should be organized by topic from most to least important.49 (10) 1229-1232; - 2004
The May 2012 Sackler Colloquium on “The Science of Science Communication” brought together scientists with research to communicate and scientists whose research could facilitate that communication. The latter include decision scientists who can identify the scientific results that an audience needs to know, from among all of the scientific results that it would be nice to know; behavioral scientists who can design ways to convey those results and then evaluate the success of those attempts; and social scientists who can create the channels needed for trustworthy communications. This overview offers an introduction to these communication sciences and their roles in science-based communication programs.
Randomisation is the process of assigning clinical trial participants to treatment groups. Randomisation gives each participant a known (usually equal) chance of being assigned to any of the groups. Successful randomisation requires that group assignment cannot be predicted in advance.
DOI: 10.5694/j.1326-5377.2002.tb04955.x - 2002
The Covid-19 pandemic disrupted many clinical trials that were potentially bringing new therapeutics to market—an additional untallied cost of the pandemic in lives and quality of life owing to delays in releasing potentially beneficial therapeutics to patients in need. A separate side effect of the pandemic has been swift adoption of virtual interactions between physicians and patients to provide continuity of care while maintaining social distancing. This comes at a time of rapid advancement of technology permitting those interactions, such as enhanced internet connectivity, electronic health records, real-time video conferencing, smartphone health applications, and remotely connectable health monitoring devices that are becoming both more accurate, practical, and affordable. Interest in decentralized clinical trials (DCTs) that use “virtual elements” like these has grown in parallel with acceptance of “virtual medicine,” accelerating shifts in clinical trial design that many feel are long overdue.
https://doi.org/10.1016/j.jacbts.2021.01.011 - 2021
In this era of a pandemic, why do we need the placebo controls for regulatory agency or Food and Drug Administration (FDA)-approved clinical trials aimed at COVID-19 patients? The answer is always that we have to establish a ruler with a baseline onto which efficacy measurements can be statistically judged.
https://doi.org/10.1007/s00005-021-00612-x - 2021
Advocates bring unique and important viewpoints to the cancer research process, ensuring that scientific and medical advances are patient-centered and relevant. In this article, we discuss the benefits of engaging advocates in cancer research and underscore ways in which both the scientific and patient communities can facilitate this mutually beneficial collaboration. We discuss how to establish and nurture successful scientist-advocate relationships throughout the research process. We review opportunities that are available to advocates who want to obtain training in the evaluation of cancer research. We also suggest practical solutions that can strengthen communication between scientists and advocates, such as introducing scientist-advocate interactions at the trainee level. Finally, we highlight the essential role social media can play in disseminating patient-supported cancer research findings to the patient community and in raising awareness of the importance of promoting cancer research. Our perspective offers a model that Georgetown Breast Cancer Advocates have found effective and which could be one option for those interested in developing productive, successful, and sustainable collaborations between advocates and scientists in cancer research.
DOI: 10.1158/0008-5472.CAN-18-1600 - 2018
The rapid and exponential growth of genome editing has posed many challenges for bioethics. This article briefly explains the nature of the technique and the particularly rapid development of Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR) technology. The international and, specifically, European-level systems for assessing the ethical issues consequent on these developments are outlined and discussed. The challenges posed by cases in China are summarized to raise concerns about how a more shared, universally consistent appraisal of bioethical issues can be promoted.
https://doi.org/10.11613/BM.2019.020202 - 2019
Our current economic and political structures have an increasingly devastating impact on the Earth's climate and ecosystems: we are facing a biospheric emergency, with catastrophic consequences for both humans and the natural world on which we depend. Life scientists - including biologists, medical scientists, psychologists and public health experts - have had a crucial role in documenting the impacts of this emergency, but they have failed to drive governments to take action in order to prevent the situation from getting worse. Here we, as members of the movement Scientist Rebellion, call on life scientists to re-embrace advocacy and activism - which were once hallmarks of academia - in order to highlight the urgency and necessity of systemic change across our societies. We particularly emphasise the need for scientists to engage in nonviolent civil resistance, a form of public engagement which has proven to be highly effective in social struggles throughout history.
doi: 10.7554/eLife.83292 - 2022
Peer review is a cornerstone of scientific publication, and consequently, predatory journals are feared to be a threat to the credibility of science as they perform no or low-quality peer review. The question of why researchers decide to publish in a questionable journal remains relatively unexplored. This paper provides an overview of the existing literature on why researchers decide to publish papers in questionable journals, specifically whether or not they search for a low-barrier way to getting published while being aware that the chosen journal probably does not adhere to acceptable academic standards. The choice of a publication outlet can be seen as a submission tree that consists of various incentives, and explaining why authors publish in deceptive journals may thus consist of a combination of awareness and motivational factors. Awareness and motivation of diligent authors is very different from that of unethical authors. Unethical authors may use a lack of awareness to excuse their actions, but they may actively search for a low-barrier way to getting published. As there are different types of authors who publish in deceptive journals, we need different approaches to solve the problem.
doi: 10.1002/leap.1214 - 2018
A literature review article provides a comprehensive overview of literature related to a theme/theory/method and synthesizes prior studies to strengthen the foundation of knowledge. In the growing International Business (IB) research field, systematic literature reviews have great value, yet there are not many reviews published describing how researchers can design and develop classic review articles. In explaining the purpose, methodology, and structure of a systematic review, we provide guidelines for developing most insightful and useful review articles. By outlining steps and thumb rules to keep in mind, we present an overview of different types of review articles and explain how future researchers could potentially find them useful. In addition, we introduce nine articles finally selected for this special issue of systematic literature review-Looking back to look forward International Business research in the days to come.
https://doi.org/10.1016/j.ibusrev.2020.101717 - 2020
Doctorate graduates are expected to contribute original knowledge and possess advanced skills essential for addressing complex problems. Embedding doctorateness in doctorate programmes could help ensure that the productivity of doctoral research is explicitly demonstrated. Doctorateness represents independent scholarship, the transition from knowledge consumption to knowledge creation, original scholarly contribution, and research integrity. A research proposal is the backbone of a doctoral journey, as it acts as an academic development plan for supervisors and students. Hence, understanding the role of supervisors in enabling doctorateness in preparing a research proposal is essential for improving student satisfaction and competencies in their academic development. Here, we develop a framework for facilitating doctorateness in preparing a research proposal and provide recommen- dations on supervisor functions.
https://doi.org/10.1080/14703297.2022.2124186 - 2022
Health researchers are often challenged on how to integrate a sex and gender lens into their work. Reviews completed across a range of health research studies show there are several commonly overlooked opportunities to do better in this regard. Nine ways to improve the integration of a sex and gender lens in health research proposals have been identified.
https://doi.org/10.1186/s41073-020-00102-2 - 2020
This is the first question one should consider before submitting a paper to an international journal. The answer is simple: If researchers or practitioners from another country can learn something from your paper that can influence a practice or a research they are involved in, then your paper is relevant for an international audience. There are many elements that can influence in this cross-border transferability. One could think that having a big “n”, or performing complex statistical calculations, or using complicated study designs makes the paper more attractive to colleagues from other countries. These elements can help, but they are not sufficient. On the other hand, one could think that a study performed in a small hospital in a given country will never be of interest for these foreign colleagues. That is not necessarily correct. Let’s burst some myths.
https://doi.org/10.18549/PharmPract.2020.2.1924 - 2020
This paper, on writing research questions, is the first in a series that aims to support novice researchers within clinical education, particularly those undertaking their first qualitative study. Put simply, a research question is a question that a research project sets out to answer. Most research questions will lead to a project that aims to generate new insights, but the target audience and the methodology will vary widely. The term ‘evaluation question’ is used less commonly, but the same principles apply. The key difference is that evaluation questions are typically more focused on the immediate context: for example, the effectiveness of an educational intervention in a particular setting. Whether your ambition is for research or evaluation, we hope that you will find this paper helpful for designing your own educational projects.
DOI: doi: 10.1111/tct.12776 - 2018
A graphical abstract (GA) represents a piece of artwork that is intended to summarize the main findings of an article for readers at a single glance. Many publishers currently encourage authors to supplement their articles with GAs, in the hope that such a convenient visual summary will facilitate readers with a clearer outline of papers that are of interest and will result in improved overall visibility of the respective publication. To test this assumption, we statistically compared publications with or without GA published in Moleculesbetween March 2014 and March 2015 with regard to several output parameters reflecting visibility. Contrary to our expectations, manuscripts published without GA performed significantly better in terms of PDF downloads, abstract views, and total citations than manuscripts with GA. To the best of our knowledge, this is the first empirical study on the effectiveness of GA for attracting attention to scientific publications.
doi:10.3390/molecules21091247 - 2016
Publishing articles in international scientific journals is the primary method for the communication of validated research findings and ideas. Journal articles are commonly used as a major input for evaluations of researchers and institutions. Few articles have been published previously about the different aspects needed for writing high-quality articles. In this manuscript, we provide an updated and brief guide for the multiple dimensions needed for writing manuscripts in the health and biological sciences, from current, international and interdisciplinary perspectives and from our expertise as authors, peer reviewers and editors. We provide key suggestions for writing major sections of the manuscript (e.g. title, abstract, introduction, methods, results and discussion), for submitting the manuscript and bring an overview of the peer review process and of the post-publication impact of the articles.
https://doi.org/10.1186/s12967-020-02596-2 - 2022
The impostor “syndrome” refers to the notion that some individuals feel as if they ended up in esteemed roles and positions not because of their competencies, but because of some oversight or stroke of luck. Such individuals therefore feel like frauds or “impostors.” Despite the fact that impostor feelings are often linked to marginalized groups in society, to date, research predominantly approaches this phenomenon as an issue of the individual: pointing toward individuals for the roots and solutions of the “syndrome.” Drawing from a rich body of social and organizational psychology research, in this perspectives piece, we propose a shift in how scholars conceptualize and empirically examine this phenomenon. Instead of framing the insecurities of individuals belonging to marginalized groups solely as a problem that arises within these individuals, we argue that it is critical for future research to consider the important role of the environment in eliciting their impostor feelings as well. By doing so, we can address the contextual roots of individuals’ impostor feelings, and offer more structural and effective solutions.
doi: 10.3389/fpsyg.2020.575024 - 2020
Knowledge representation and reasoning (KR&R) has been successfully imple- mented in many fields to enable computers to solve complex problems with AI methods. However, its application to biomedicine has been lagging in part due to the daunting complexity of molecular and cellular pathways that govern human physiology and pathology. In this article, we describe concrete uses of Scalable PrecisiOn Medicine Knowledge Engine (SPOKE), an open knowledge network that connects curated information from thirty-seven specialized and human- curated databases into a single property graph, with 3 million nodes and 15 mil- lion edges to date. Applications discussed in this article include drug discovery, COVID-19 research and chronic disease diagnosis, and management. - DOI: 10.1002/aaai.12037 - 2021
Although DNA and RNA sequencing has a history spanning five decades, large-scale massively parallel sequencing, or next-generation sequencing (NGS), has only been commercially available for about 10 years. Nonetheless, the meteoric increase in sequencing throughput with NGS has dramatically changed our understanding of our genome and our- selves. Sequencing the first human genome as a haploid reference took nearly 10 years but now a full diploid human genome sequence can be accomplished in just a few days. NGS has also reduced the cost of generating sequence data and a plethora of sequence-based methods for probing a genome have emerged using NGS as the readout and have been applied to many species. NGS methods have also entered the medical realm and will see increasing use in diagnosis and treatment. NGS has largely been driven by short-read generation (150 bp) but new platforms have emerged and are now capable of generating long multikilobase reads. These latter platforms enable reference-independent genome assemblies and long-range haplotype generation. Rapid DNA and RNA sequencing is now mainstream and will continue to have an increasing impact on biology and medicine. - oi: 10.1101/cshperspect.a036798 - 2019
DNA methylation plays a fundamental role in the control of gene expression and genome integrity. Although there are multiple tools that enable its detection from Nanopore sequencing, their accuracy remains largely unknown. Here, we present a systematic benchmarking of tools for the detection of CpG methylation from Nanopore sequencing using individual reads, control mixtures of methylated and unmethylated reads, and bisulfite sequencing. We found that tools have a tradeoff between false positives and false negatives and present a high dispersion with respect to the expected methylation frequency values. We described various strategies to improve the accuracy of these tools, including a consensus approach, METEORE (https://github.com/comprna/METEORE), based on the combination of the predictions from two or more tools that shows improved accuracy over individual tools. Snakemake pipelines are also provided for reproducibility and to enable the systematic application of our analyses to other datasets. - doi.org/10.1038/s41467-021-23778-6 - 2021
Human immunodeficiency virus (HIV-1) remains a major health threat. Viral capsid uncoating and nuclear import of the viral genome are critical for productive infection. The size of the HIV-1 capsid is generally believed to exceed the diameter of the nuclear pore complex (NPC), indicating that capsid uncoating has to occur prior to nuclear import. Here, we combined correlative light and electron microscopy with subtomogram averaging to capture the structural status of reverse transcription-competent HIV-1 complexes in infected T cells. We demonstrated that the diameter of the NPC in cellulo is sufficient for the import of apparently intact, cone-shaped capsids. Subsequent to nuclear import, we detected disrupted and empty capsid fragments, indicating that uncoating of the replication complex occurs by breaking the capsid open, and not by disassembly into individual subunits. Our data directly visualize a key step in HIV-1 replication and enhance our mechanistic understanding of the viral life cycle. - doi.org/10.1016/j.cell.2021.01.025 - 2021
The definition of epigenetics refers that molecular modifications on DNA that can regulate gene activity are independent of DNA sequence and mitotically stable. Notably, epigenetics studies have grown exponentially in the past few years. Recent progresses that lead to exciting discoveries and groundbreaking nature of this area demand thorough methodologies and advanced technologies to move epigenetics to the forefront of molecular biology. The most recognized epigenetic regulations are DNA methylation, histone modifications, and non-coding RNAs (ncRNAs). This review will discuss the modern techniques that are available to detect locus-specific and genome-wide changes for all epigenetic codes. Furthermore, updated analysis of technologies, newly developed methods, recent breakthroughs and bioinformatics pipelines in epigenetic analysis will be presented. These methods, as well as many others presented in this specific issue, provide comprehensive guidelines in the area of epigenetics that facilitate further developments in this promising and rapidly developing field. - doi.org/10.1016/j.ymeth.2020.06.022 - 2020
Chimeric antigen receptor (CAR) T‐cell therapy has dramatically shifted the landscape of treatment for lymphoid malignancies, especially diffuse large B‐cell lymphoma (DLBCL) and acute lymphoblastic leukemia (ALL). However, there continue to be significant limitations of this therapy, such as incomplete or nonsustained responses and severe toxicities in a subset of patients. Furthermore, expanding the role of CAR T‐cell therapy to new disease types is an important next step. In this review, we will highlight landmark trials for anti‐CD19 CAR T cells and first‐in‐human trials of novel CARs, as well as discuss promising innovative CAR designs that are still undergoing preclinical development. Lastly, we will discuss toxicity and mechanisms of CAR T‐cell resistance and failure, as well as potential future treatment approaches to these common issues. - DOI: 10.1002/hon.2591 - 2019
As RNA-binding proteins (RBPs) play essential roles in cellular physiology by interacting with target RNA molecules, binding site identification by UV crosslinking and immunoprecipitation (CLIP) of ribonucleoprotein complexes is critical to understanding RBP function. However, current CLIP protocols are technically demanding and yield low-complexity libraries with high experimental failure rates. We have developed an enhanced CLIP (eCLIP) protocol that decreases requisite amplification by ∼1,000-fold, decreasing discarded PCR duplicate reads by ∼60% while maintaining single-nucleotide binding resolution. By simplifying the generation of paired IgG and size-matched input controls, eCLIP improves specificity in the discovery of authentic binding sites. We generated 102 eCLIP experiments for 73 diverse RBPs in HepG2 and K562 cells (available at https://www.encodeproject.org), demonstrating that eCLIP enables large-scale and robust profiling, with amplification and sample requirements similar to those of ChIP-seq. eCLIP enables integrative analysis of diverse RBPs to reveal factor-specific profiles, common artifacts for CLIP and RNA-centric perspectives on RBP activity. - DOI: 10.1038/nmeth.3810 - 2016
The development of real-life applicable ion sensors, in particular those capable of repeat use and long-term monitoring, remains a formidable challenge. Herein, we demonstrate, in a proof-of-concept, the real-time voltammet- ric sensing of anions under continuous flow in a 3D-printed microfluidic system. Electro-active anion receptive halogen bonding (XB) and hydrogen bonding (HB) ferrocene-isophtha- lamide-(iodo)triazole films were employed as exemplary sensory interfaces. Upon exposure to anions, the cathodic perturbations of the ferrocene redox-transducer are monitored by repeat square-wave voltammetry (SWV) cycling and peak fitting of the voltammograms by a custom-written MATLAB script. This enables the facile and automated data processing of thousands of SW scans and is associated with an over one order-of-magnitude improvement in limits of detection. In addition, this improved analysis enables tuning of the measurement parameters such that high temporal resolution can be achieved. More generally, this new flow methodology is extendable to a variety of other analytes, including cations, and presents an important step towards translation of voltammetric ion sensors from laboratory to real-world applications. - doi.org/10.1002/chem.202103249 - 2021
Protein structure prediction can be used to determine the three-dimensional shape of a protein from its amino acid sequence1. This problem is of fundamental importance as the structure of a protein largely determines its function2; however, protein structures can be difficult to determine experimentally. Considerable progress has recently been made by leveraging genetic information. It is possible to infer which amino acid residues are in contact by analysing covariation in homologous sequences, which aids in the prediction of protein structures3. Here we show that we can train a neural network to make accurate predictions of the distances between pairs of residues, which convey more information about the structure than contact predictions. Using this information, we construct a potential of mean force4 that can accurately describe the shape of a protein. We find that the resulting potential can be optimized by a simple gradient descent algorithm to generate structures without complex sampling procedures. The resulting system, named AlphaFold, achieves high accuracy, even for sequences with fewer homologous sequences. In the recent Critical Assessment of Protein Structure Prediction5 (CASP13)—a blind assessment of the state of the field—AlphaFold created high-accuracy structures (with template modelling (TM) scores6 of 0.7 or higher) for 24 out of 43 free modelling domains, whereas the next best method, which used sampling and contact information, achieved such accuracy for only 14 out of 43 domains. AlphaFold represents a considerable advance in protein-structure prediction. We expect this increased accuracy to enable insights into the function and malfunction of proteins, especially in cases for which no structures for homologous proteins have been experimentally determined. - doi.org/10.1038/s41586-019-1923-7 - 2020
Structural biology is the study of the molecular architecture of proteins and nucleic acids, which are the basis for all life forms. Knowledge of these structures alone is not enough to understand their functions, but it has become clear that a detailed mechanistic picture of function is not possible without structural information. Studying structure can reveal how molecules have evolved, and this type of insight would otherwise be lost by looking at only the molecule’s sequence. - .doi.org/10.1016/j.bpj.2016.02.001 - 2016
Growth hormone–secreting (GH-secreting) pituitary tumors are driven by oncogenes that induce cAMP signaling. In this issue of the JCI, Ben-Shlomo et al. performed a whole-exome study of pituitary adenomas. GH-secreting tumors had a high frequency of whole chromosome or chromosome arm copy number alterations and were associated with an increase in the tumor protein p53 and the cyclin-dependent kinase inhibitor p21WAF1/CIP1, which are findings consistent with induction of a response to DNA damage. Further, treatment of mouse pituitary cells with cAMP pathway agonists in vitro and in vivo elicited biomarkers of DNA replication stress or double-strand breaks. The findings of Ben Shlomo et al. indicate that oncoproteins that drive constitutively high cAMP signaling pathway output in susceptible cell types can elicit DNA replication stress and may promote genomic instability. - doi.org/10.1172/JCI142237. - 2020
Proteins are essential to life, and understanding their structure can facilitate a mechanistic understanding of their function. Through an enormous experimental effort, the structures of around 100,000 unique proteins have been determined5, but this represents a small fraction of the billions of known protein sequences. Structural coverage is bottlenecked by the months to years of painstaking effort required to determine a single protein structure. Accurate computational approaches are needed to address this gap and to enable large-scale structural bioinformatics. Predicting the three-dimensional structure that a protein will adopt based solely on its amino acid sequence—the structure prediction component of the ‘protein folding problem’—has been an important open research problem for more than 50 years9. Despite recent progress existing methods fall far short of atomic accuracy, especially when no homologous structure is available. Here we provide the first computational method that can regularly predict protein structures with atomic accuracy even in cases in which no similar structure is known. We validated an entirely redesigned version of our neural network-based model, AlphaFold, in the challenging 14th Critical Assessment of protein Structure Prediction (CASP14), demonstrating accuracy competitive with experimental structures in a majority of cases and greatly outperforming other methods. Underpinning the latest version of AlphaFold is a novel machine learning approach that incorporates physical and biological knowledge about protein structure, leveraging multi-sequence alignments, into the design of the deep learning algorithm. - doi.org/10.1038/s41586-021-03819-2 - 2021
Liquid-electron microscopy (EM), the room-temperature correlate to cryo-EM, is a rapidly growing field providing high-resolution insights of macromolecules in solution. Here, we describe how liquid-EM experiments can incorporate automated tools to propel the field to new heights. We demonstrate fresh workflows for specimen preparation, data collection, and computing processes to assess biological structures in liquid. Adeno-associated virus (AAV) and the SARS-CoV-2 nucleocapsid (N) were used as model systems to highlight the technical advances. These complexes were selected based on their major differences in size and natural symmetry. AAV is a highly symmetric, icosahedral assembly with a particle diameter of ∼25 nm. At the other end of the spectrum, N protein is an asymmetric monomer or dimer with dimensions of approximately 5–7 nm, depending upon its oligomerization state. Equally important, both AAV and N protein are popular subjects in biomedical research due to their high value in vaccine development and therapeutic efforts against COVID-19. Overall, we demonstrate how automated practices in liquid-EM can be used to decode molecules of interest for human health and disease. - doi:10.1017/S1431927621013921 - 2022
Cellular reprogramming can manipulate the identity of cells to generate the desired cell types1–3. The use of cell intrinsic components, including oocyte cytoplasm and transcription factors, can enforce somatic cell reprogramming to pluripotent stem cells4–7. By contrast, chemical stimulation by exposure to small molecules offers an alternative approach that can manipulate cell fate in a simple and highly controllable manner8–10. However, human somatic cells are refractory to chemical stimulation owing to their stable epigenome2,11,12 and reduced plasticity13,14; it is therefore challenging to induce human pluripotent stem cells by chemical reprogramming. Here we demonstrate, by creating an intermediate plastic state, the chemical reprogramming of human somatic cells to human chemically induced pluripotent stem cells that exhibit key features of embryonic stem cells. The whole chemical reprogramming trajectory analysis delineated the induction of the intermediate plastic state at the early stage, during which chemical-induced dedifferentiation occurred, and this process was similar to the dedifferentiation process that occurs in axolotl limb regeneration. Moreover, we identified the JNK pathway as a major barrier to chemical reprogramming, the inhibition of which was indispensable for inducing cell plasticity and a regeneration-like program by suppressing pro-inflammatory pathways. Our chemical approach provides a platform for the generation and application of human pluripotent stem cells in biomedicine. This study lays foundations for developing regenerative therapeutic strategies that use well-defined chemicals to change cell fates in humans. - doi.org/10.1038/s41586-022-04593-5 - 2022
The hallmarks of cancer conceptualization is a heuristic tool for distilling the vast complexity of cancer phenotypes and genotypes into a provisional set of underlying principles. As knowledge of cancer mechanisms has progressed, other facets of the disease have emerged as potential refinements. Herein, the prospect is raised that phenotypic plasticity and disrupted differentiation is a discrete hallmark capability, and that nonmutational epigenetic reprogram- ming and polymorphic microbiomes both constitute distinctive enabling characteristics that facilitate the acquisition of hallmark capabilities. Additionally, senescent cells, of varying origins, may be added to the roster of functionally important cell types in the tumor microenvironment. - doi: 10.1158/2159-8290.CD-21-1059 - 2022
The development of metastatic disease is a complex interplay of genetic and epigenetic factors from the host and cancer cells acting in a patient-specific manner. Inhibiting key driver traits of metastasis should yield survival benefit at any stage of the disease, and we look forward to the next generation of personalized medicines for cancer therapy that target cancer cell motility for increased therapeutic efficacy. - doi.org/10.1080/14737140.2020.1718496 - 2020
Brain imaging, specifically magnetic resonance imaging (MRI), plays a key role in the clinical and research aspects of neuro-oncology. Novel neuroimaging techniques enable the transformation of a brain MRI into a so-called average brain. This allows projects using already acquired brain MRIs to perform group analyses and draw conclusions. Once the data are in this average brain, several types of analyses can be performed. For example, determining the most vulnerable locations for certain tumor types or perhaps even the underlying circuitry and gene expression that might cause predisposition to tumor growth. This information may further our understanding of tumor behavior, leading to better patient counseling, surgery timing, and treatment monitoring. - doi.org/10.3390/ cancers14030464 - 2022
Mutations in Dystrophin, one of the largest proteins in the mammalian body, are causative for a severe form of muscle disease, Duchenne Muscular Dystrophy (DMD), affecting not only skeletal muscle, but also the heart. In particular, exons 45–52 constitute a hotspot for DMD mutations. A variety of molecular therapies have been developed, comprising vectors encoding micro-and minidystrophins as well as utrophin, a protein with partially overlapping functions. With the advent of the CRISPR-Cas9-nuclease, genome editing offers a novel option of correction of the disease-causing mutations. Full restoration of the healthy gene by homology directed repair is a rare event. However, non-homologous end-joining (NHEJ) may restore the reading frame by causing exon excision. This approach has first been demonstrated in mice and then translated to large animals (dogs, pigs). This review discusses the potential opportunities and limitations of genome editing in DMD, including the generation of appropriate animal models as well as new developments in genome editing tools. - doi.org/10.1038/s41434-021-00222-4 - 2021
RNA-containing structures, including ribonucleotide insertions, DNA:RNA hybrids and R-loops, have recently emerged as critical players in the maintenance of genome integrity. Strikingly, different enzymatic activities classically involved in genome maintenance contribute to their generation, their processing into genotoxic or repair intermediates, or their removal. Here we review how this substrate promiscuity can account for the detrimental and beneficial impacts of RNA insertions during genome metabolism. We summarize how in vivo and in vitro experiments support the contribution of DNA polymerases and homologous recombination proteins in the formation of RNA-containing structures, and we discuss the role of DNA repair enzymes in their removal. The diversity of pathways that are thus affected by RNA insertions likely reflects the ancestral function of RNA molecules in genome maintenance and transmission.
Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR)-based technology has revolutionized the field of biomedicine with broad applications in genome editing, therapeutics and diagnostics. While a majority of applications involve the RNA-guided site-specific DNA or RNA cleavage by CRISPR enzymes, recent successes in nucleic acid detection rely on their collateral and non-specific cleavage activated by viral DNA or RNA. Ranging in enzyme composition, the mechanism for distinguishing self- from foreign-nucleic acids, the usage of second messengers, and enzymology, the CRISPR enzymes provide a diverse set of diagnosis tools in further innovations. Structural biology plays an important role in elucidating the mechanisms of these CRISPR enzymes. Here we summarize and compare structures of three types of CRISPR enzymes used in nucleic acid detection captured in their respective functional forms and illustrate the current understanding of their activation mechanism. - doi:10.1016/j.jsb.2022.107838 - 2022
Despite the abundance of available software tools, optimal particle selection is still a vital issue in single-particle cryoelectron microscopy (cryo-EM). Regardless of the method used, most pickers struggle when ice thickness varies on a micrograph. IceBreaker allows users to estimate the relative ice gradient and flatten it by equalizing the local contrast. It allows the differentiation of particles from the background and improves overall particle picking performance. Furthermore, we introduce an additional parameter corresponding to local ice thickness for each particle. Particles with a defined ice thickness can be grouped and filtered based on this parameter during processing. These functionalities are especially valuable for on-the-fly processing to automatically pick as many particles as possible from each micrograph and to select optimal regions for data collection. Finally, estimated ice gradient distributions can be stored separately and used to inspect the quality of prepared samples. - doi.org/10.1016/j.str.2022.01.005 - 2022
Over the past few decades, neuroimaging has become a ubiquitous tool in basic research and clinical studies of the human brain. However, no reference standards currently exist to quantify individual differences in neuroimaging metrics over time, in contrast to growth charts for anthropometric traits such as height and weight1. Here we assemble an interactive open resource to benchmark brain morphology derived from any current or future sample of MRI data (http://www.brainchart.io/). With the goal of basing these reference charts on the largest and most inclusive dataset available, acknowledging limitations due to known biases of MRI studies relative to the diversity of the global population, we aggregated 123,984 MRI scans, across more than 100 primary studies, from 101,457 human participants between 115 days post-conception to 100 years of age. MRI metrics were quantified by centile scores, relative to non-linear trajectories2 of brain structural changes, and rates of change, over the lifespan. Brain charts identified previously unreported neurodevelopmental milestones, showed high stability of individuals across longitudinal assessments, and demonstrated robustness to technical and methodological differences between primary studies. Centile scores showed increased heritability compared with non-centiled MRI phenotypes, and provided a standardized measure of atypical brain structure that revealed patterns of neuroanatomical variation across neurological and psychiatric disorders. In summary, brain charts are an essential step towards robust quantification of individual variation benchmarked to normative trajectories in multiple, commonly used neuroimaging phenotypes. - doi.org/10.1038/s41586-022-04554-y - 2022
Hyperglycemia is associated with poor clinical outcomes in critically ill patients. Initial clinical trials of intensive insulin therapy targeting blood glucose levels of 80 to 110 mg/dL showed improved outcomes, but subsequent trials found no benefits and even increased harm with this approach. Emerging literature has evaluated other glycemic indices including time-in-target blood glucose range, glycemic variability, and stress hyperglycemia ratio. These indices, while well described in observational studies, have not been addressed in the initial trials. Additionally, the patient’s preexisting diabetes status and preadmission diabetic control may modulate the outcomes of stringent glycemic control, with worse outcomes of hyperglycemia being observed in patients without diabetes and in those with well-controlled diabetes. Most medical societies recommend less stringent glucose control in the range of 140 to 180 mg/dL for critically ill patients. - doi:10.3949/ccjm.89a.20171 - 2022
Since its initial release in 2000, the human reference genome has covered only the euchromatic fraction of the genome, leaving important heterochromatic regions unfinished. Addressing the remaining 8% of the genome, the Telomere-to-Telomere (T2T) Consortium presents a complete 3.055 billion–base pair sequence of a human genome, T2T-CHM13, that includes gapless assemblies for all chromosomes except Y, corrects errors in the prior references, and introduces nearly 200 million base pairs of sequence containing 1956 gene predictions, 99 of which are predicted to be protein coding. The completed regions include all centromeric satellite arrays, recent segmental duplications, and the short arms of all five acrocentric chromosomes, unlocking these complex regions of the genome to variational and functional studies.- DOI: 10.1126/science.abj6987 - 2022
This article documents a keynote seminar presented at the IUCr Congress in Prague, 2021. The cryo-EM method microcrystal electron diffraction is described and put in the context of macromolecular electron crystallography from its origins in 2D crystals of membrane proteins to today’s application to 3D crystals a millionth the size of that needed for X-ray crystallography. Milestones in method development and applications are described with an outlook to the future. - doi.org/10.1107/S2052252521013063 - 2022
Pancreatic cancer is the third leading cause of cancer mortality in the United States, recently surpassing breast cancer. A key component of pancreatic cancer’s lethality is its acquired immune privilege, which is driven by an immunosuppressive microenvironment, poor T-cell infiltration, and a low mutational burden. Although immunotherapies such as checkpoint blockade or engineered T cells have yet to demonstrate efficacy, a growing body of evidence suggests that orthogonal combinations of these and other strategies could unlock immunotherapy in pancreatic cancer. In this review, we will discuss promising immunotherapies currently under investigation in pancreatic cancer and provide a roadmap for the development of prevention vaccines for this and other cancers. - doi:10.1016/j.trecan.2018.04.001 - 2018
Radiotherapy is a cornerstone of both curative and palliative cancer care. However, radiotherapy is severely limited by radiation-induced toxicities. If these toxicities could be reduced, a greater dose of radiation could be given therefore facilitating a better tumor response. Initial pre-clinical studies have shown that irradiation at dose rates far exceeding those currently used in clinical contexts reduce radiation-induced toxicities whilst maintaining an equivalent tumor response. This is known as the FLASH effect. To date, a single patient has been subjected to FLASH radiotherapy for the treatment of subcutaneous T-cell lymphoma resulting in complete response and minimal toxicities. The mechanism responsible for reduced tissue toxicity following FLASH radiotherapy is yet to be elucidated, but the most prominent hypothesis so far proposed is that acute oxygen depletion occurs within the irradiated tissue. This review examines the tissue response to FLASH radiotherapy, critically evaluates the evidence supporting hypotheses surrounding the biological basis of the FLASH effect, and considers the potential for FLASH radiotherapy to be translated into clinical contexts. - doi: 10.3389/fonc.2019.01563 - 2020
Protein structures can provide invaluable information, both for reasoning about biological processes and for enabling interventions such as structure-based drug development or targeted mutagenesis. After decades of effort, 17% of the total residues in human protein sequences are covered by an experimentally determined structure1. Here we markedly expand the structural coverage of the proteome by applying the state-of-the-art machine learning method, AlphaFold2, at a scale that covers almost the entire human proteome (98.5% of human proteins). The resulting dataset covers 58% of residues with a confident prediction, of which a subset (36% of all residues) have very high confidence. We introduce several metrics developed by building on the AlphaFold model and use them to interpret the dataset, identifying strong multi-domain predictions as well as regions that are likely to be disordered. Finally, we provide some case studies to illustrate how high-quality predictions could be used to generate biological hypotheses. We are making our predictions freely available to the community and anticipate that routine large-scale and high-accuracy structure prediction will become an important tool that will allow new questions to be addressed from a structural perspective.
Lithium is the most effective mood stabilizer for the treatment of bipolar disorder, but it is toxic at only twice the therapeutic dosage and has many undesirable side effects. It is likely that a small molecule could be found with lithium-like efficacy but without toxicity through target-based drug discovery; however, therapeutic target of lithium remains equivocal. Inositol monophosphatase is a possible target but no bioavailable inhibitors exist. Here we report that the antioxidant ebselen inhibits inositol monophosphatase and induces lithium-like effects on mouse behaviour, which are reversed with inositol, consistent with a mechanism involving inhibition of inositol recycling. Ebselen is part of the National Institutes of Health Clinical Collection, a chemical library of bioavailable drugs considered clinically safe but without proven use. Therefore, ebselen represents a lithium mimetic with the potential both to validate inositol monophosphatase inhibition as a treatment for bipolar disorder and to serve as a treatment itself. - DOI: 10.1038/ncomms2320 - 2013
The 3D architecture of tissues bearing tumors impacts on the mechanical microenvironment of cancer, the accessibility of stromal cells, and the routes of invasion. A myriad of intrinsic and extrinsic forces exerted by the cancer cells, the host tissue, and the molecular and cellular microenvironment modulate the morphology of the tumor and its malignant potential through mechanical, biochemical, genetic, and epigenetic cues. Recent studies have investigated how tissue architecture influences cancer biology from tumor initiation and progression to distant metastatic seeding and response to therapy. With a focus on carcinoma, the most common type of cancer, this review discusses the latest discoveries on how tumor architecture is built and how tissue morphology affects the biology and progression of cancer cells.-doi:10.1016/j.trecan.2018.04.001 - 2018
Choline is an essential nutrient for humans. It is a precursor of membrane phospholipids (e.g., phosphatidylcholine (PC)), the neurotransmitter acetylcholine, and via betaine, the methyl group donor S-adenosylmethionine. High choline intake during gestation and early postnatal development in rat and mouse models improves cognitive function in adulthood, prevents age-related memory decline, and protects the brain from the neuropathological changes associated with Alzheimer’s disease (AD), and neurological damage associated with epilepsy, fetal alcohol syndrome, and inherited conditions such as Down and Rett syndromes. These effects of choline are correlated with modifications in histone and DNA methylation in brain, and with alterations in the expression of genes that encode proteins important for learning and memory processing, suggesting a possible epigenomic mechanism of action. Dietary choline intake in the adult may also influence cognitive function via an effect on PC containing eicosapentaenoic and docosahexaenoic acids; polyunsaturated species of PC whose levels are reduced in brains from AD patients, and is associated with higher memory performance, and resistance to cognitive decline. - doi:10.3390/nu9080815 - 2017
The X inactive-specific transcript (Xist) gene is the master regulator of X chromosome inactivation in mammals. Xist produces a long noncoding (lnc)RNA that accumulates over the entire length of the chromosome from which it is transcribed, recruiting factors to modify under-lying chromatin and silence X-linked genes in cis. Recent years have seen significant progress in identifying important functional elements in Xist RNA, their associated RNA-binding proteins (RBPs), and the downstream pathways for chromatin modification and gene silencing. In this review, we summarize progress in understanding both how these pathways function in Xist-mediated silencing and the complex interplay between them. - doi/10.1101/gad.337196 - 2022
Nucleic acids bear the genetic information and participate in its expression and evolution during replication, repair, recombination, transcription, and translation. These phenomena are mostly based on recognition of nucleic acids by proteins. The major factor enabling the specific recognition is structure. Circular dichroism (CD) spectroscopy is very useful to study secondary structures of nucleic acids, in general, and DNA, in particular. CD sensitively reflects isomerizations among distinct conformational states. The isomerizations may operate as molecular switches regulating various physiological or pathological processes. Here, we review CD spectra of nucleic acids, beginning with early studies on natural DNA molecules through analyses of synthetic polynucleotides to study of selected genomic fragments. - DOI: 10.1002/chir.22064 - 2012
Engineered and native enzymes are poised to solve challenges in medicine, bioremediation, and biotechnology. One important goal is the possibility of upcycling polymers using enzymes. However, enzymes are often inactive in industrial, nonbiological conditions. It is particularly difficult to protect water-soluble enzymes at elevated temperatures by methods that preserve their functionality. Through atomistic and coarse-grained molecular dynamics simulations that capture protein conformational change, we show that an enzyme, PETase (polyethylene terephthalate [PET]), can be stabilized at elevated temperatures by complexation with random copolymers into nanoscale aggregates that do not precipitate into macroscopic phases. We demonstrated the efficiency of the method by simulating complexes of random copolymers and the enzyme PETase, which depolymerizes PET, a highly used polymer. These polymers are more industrially viable than peptides and can target specific domains on an enzyme. We design the mean composition of the random copolymers to control the polymer–enzyme surface contacts and the polymer conformation. When positioned on or near the active site, these polymer contacts can further stabilize the conformation of the active site at elevated temperatures. We explore the experimental implications of this active site stabilization method and show that PETase-random copolymer complexes have enhanced activity on both small molecule substrates and solid PET films. These results provide guidelines for engineering enzyme–polymer complexes with enhanced enzyme functionality in nonbiological environments. - doi.org/10.1073 - 2022
Psoriasis and psoriatic arthritis, together known as psoriatic disease, is highly prevalent chronic relapsing inflammatory disease affecting skin, joints or both and is associated with several comorbidities such as cardiovascular, metabolic, psychiatric, renal disease etc. The etiopathogenesis of psoriasis is complex and mainly driven by aberrant immune response owing to the genetic susceptibility and various environmental factors such as trauma, infections and drugs. Recent advances in understanding molecular and cellular pathways have identified tumor necrosis factor-α (TNF-α), interleukin-17 (IL-17), IL-23, IL-22 as major contributors in psoriasis pathogenesis. Advances in the knowledge of pathophysiology, the interaction of autoinflammation and clinical phenotypes have led to the development of highly effective targeted therapeutic agents which include TNF-α, IL-17, IL-23, IL-1 α/β or IL-36 inhibitors or receptor blockers, small molecule drugs like phosphodiesterase-4 inhibitors (apremilast), Janus kinase (JAK) inhibitors, retinoic acid receptor-related orphan receptor γt (RORγt) inhibitors. These novel drugs have promised the potential of improved disease control. In recent years, the transition from biologics to biosimilars especially with TNF-α inhibitors had significant impact on decreasing health care cost and increasing therapeutic options to the patients. However, selection of right treatment for an individual patient still remains challenging. Moreover, interplay between different epigenetic mechanisms such as the DNA methylation, chromatin modifications and noncoding RNA regulation has recently been started to be deciphered. Enzymes inhibitors involved in epigenetic pathways such as DNA methyltransferases and histone deacetylases demonstrated to restore normal epigenetic patterns in clinical settings and have provided the potential as novel therapeutic targets for psoriasis. In this review, we will discuss novel biologic agents and newer therapeutic approaches in treatment of psoriatic disease.
There is concern that the COVID-19 pandemic and its aftermath will result in excess suicides by increasing known risk factors such as self-harm, but evidence on how pandemic-related risk factors contribute to changes in these outcomes is lacking.
In total, 26.1% and 7.9% of respondents reported self-harm thoughts and behaviours respectively at least once over the study period. The number of adverse experiences was more strongly related to outcomes than the number of worries. The largest specific adversity contributing to increases in both outcomes was having experienced physical or psychological abuse. Financial worries increased the likelihood of both outcomes in most age groups, and having had COVID-19 increased the likelihood of both outcomes in young (18–29 years) and middle-aged (45–59 years) adults. - 10.1192/bjp.2021.130 - 2022
The role of adipose tissue (AT) inflammation in obesity and its multiple related- complications is a rapidly expanding area of scientific interest. Within the last 30 years, the role of the adipocyte as an endocrine and immunologic cell has been progressively established. Like the macrophage, the adipocyte is capable of linking the innate and adaptive immune system through the secretion of adipokines and cytokines; exosome release of lipids, hormones, and microRNAs; and contact interaction with other immune cells. Key innate immune cells in AT include adipocytes, macrophages, neutrophils, and innate lymphoid cells type 2 (ILC2s). The role of the innate immune system in promoting adipose tissue inflammation in obesity will be highlighted in this review. T cells and B cells also play important roles in contributing to AT inflammation and are discussed in this series in the chapter on adaptive immunity. - doi: 10.3389/fimmu.2021.650768 - 2021
The three probes of the structure of matter (X-rays, neutrons and electrons) in biology have complementary properties and strengths. The balance between these three probes within their strengths and weaknesses is perceived to change, even dramatically so at times. For the study of combined states of order and disorder, NMR crystallography is also applicable. Of course, to understand biological systems the required perspectives are surely physiologically relevant temperatures and relevant chemical conditions, as well as a minimal perturbation owing to the needs of the probe itself. These remain very tough challenges because, for example, cryoEM by its very nature will never be performed at room temperature, crystallization often requires nonphysiological chemical conditions, and X-rays and electrons cause beam damage. However, integrated structural biology techniques and functional assays provide a package towards physiological relevance of any given study. Reporting of protein crystal structures, and their associated database entries, could usefully indicate how close to the biological situation they are, as discussed in detail in this feature article. - doi.org/10.1107/S2059798320000546 - 2020
Metformin, the most prescribed antidiabetic medicine, has shown other benefits such as anti-ageing and anticancer effects1–4. For clinical doses of metformin, AMP-activated protein kinase (AMPK) has a major role in its mechanism of action4,5; however, the direct molecular target of metformin remains unknown. Here we show that clinically relevant concentrations of metformin inhibit the lysosomal proton pump v-ATPase, which is a central node for AMPK activation following glucose starvation6. We synthesize a photoactive metformin probe and identify PEN2, a subunit of γ-secretase7, as a binding partner of metformin with a dissociation constant at micromolar levels. Metformin-bound PEN2 forms a complex with ATP6AP1, a subunit of the v-ATPase8, which leads to the inhibition of v-ATPase and the activation of AMPK without effects on cellular AMP levels. Knockout of PEN2 or re-introduction of a PEN2 mutant that does not bind ATP6AP1 blunts AMPK activation. In vivo, liver-specific knockout of Pen2 abolishes metformin-mediated reduction of hepatic fat content, whereas intestine-specific knockout of Pen2 impairs its glucose-lowering effects. Furthermore, knockdown of pen-2 in Caenorhabditis elegans abrogates metformin- induced extension of lifespan. Together, these findings reveal that metformin binds PEN2 and initiates a signalling route that intersects, through ATP6AP1, the lysosomal glucose-sensing pathway for AMPK activation. This ensures that metformin exerts its therapeutic benefits in patients without substantial adverse effects. - https://doi.org/10.1038/s41586-022-04431-8 - 2022
Traumatic brain injury (TBI) affects a growing portion of the population and continues to take national spotlight with advances in imaging technology and understanding of long-term effects. However, there is large variance in TBI treatment protocols due to injury variability and lack of both mechanistic understanding and strong treatment recommendations. Recent practice suggests three disparate treatment approaches, all which aim at promoting neuroprotection after TBI, show promise: immediate hypothermia, hyper- baric oxygen, and progesterone supplementation. The research is controversial at times, yet there are abundant opportunities to develop the technology behind hypothermia and hyperbaric oxygen treatments which would surely aid in aligning the current data. Additionally, while progesterone has already been packaged in nanoparticle form it may benefit from continued formulation and administration research. The treatments and the avenues for improvement are reviewed in the present paper. - 10.4137/BECB.S10968 - 2013
Fibrosis affects millions of people with cardiac disease. We developed a therapeutic approach to generate transient antifibrotic chimeric antigen receptor (CAR) T cells in vivo by delivering modified messenger RNA (mRNA) in T cell–targeted lipid nanoparticles (LNPs). The efficacy of these in vivo–reprogrammed CAR T cells was evaluated by injecting CD5-targeted LNPs into a mouse model of heart failure. Efficient delivery of modified mRNA encoding the CAR to T lymphocytes was observed, which produced transient, effective CAR T cells in vivo. Antifibrotic CAR T cells exhibited trogocytosis and retained the target antigen as they accumulated in the spleen. Treatment with modified mRNA-targeted LNPs reduced fibrosis and restored cardiac function after injury. In vivo generation of CAR T cells may hold promise as a therapeutic platform to treat various diseases. - DOI: 10.1126/science.abm0594 - 2022
Evidence-based medicine (EBM) is the combination of the best available research evidence with clinical experience and patient needs. The concept of EBM as a part of clinical decision making has become increasingly popular over the last decade. In the hierarchy of studies meta-analysis and systematic reviews occupy the highest levels. A systematic review of a clinical question can be performed by following a relatively standard form. These techniques as described here can be performed without formal training. Systematic reviews conducted in this fashion can be used as a higher form of current concepts or as review articles and replace the traditional expert opinion narrative review. - DOI: 10.1097/BLO.0b013e31802c9098
Physical exercise is generally beneficial to all aspects of human and animal health, slowing cognitive ageing and neurodegeneration1. The cognitive benefits of physical exercise are tied to an increased plasticity and reduced inflammation within the hippocampus2–4, yet little is known about the factors and mechanisms that mediate these effects. Here we show that ‘runner plasma’, collected from voluntarily running mice and infused into sedentary mice, reduces baseline neuroinflammatory gene expression and experimentally induced brain inflammation. Plasma proteomic analysis revealed a concerted increase in complement cascade inhibitors including clusterin (CLU). Intravenously injected CLU binds to brain endothelial cells and reduces neuroinflammatory gene expression in a mouse model of acute brain inflammation and a mouse model of Alzheimer’s disease. Patients with cognitive impairment who participated in structured exercise for 6 months had higher plasma levels of CLU. These findings demonstrate the existence of anti-inflammatory exercise factors that are transferrable, target the cerebrovasculature and benefit the brain, and are present in humans who engage in exercise. - https://doi.org/10.1038/s41586-021-04183-x - 2021
Cellular homeostasis requires the sensing of and adaptation to intracellular oxygen (O2) and reactive oxygen species (ROS). The Arg/N-degron pathway targets proteins that bear destabilizing N-terminal residues for degradation by the proteasome or via autophagy. Under normoxic conditions, the N-terminal Cys (Nt-Cys) residues of specific substrates can be oxidized by dioxygenases such as plant cysteine oxidases and cysteamine (2-aminoethane- thiol) dioxygenases and arginylated by ATE1 R-transferases to generate Arg-CysO2(H) (R-CO2). Proteins bearing the R-CO2 N-degron are targeted via Lys48 (K48)–linked ubiquitylation by UBR1/UBR2 N-recognins for proteasomal degradation. During acute hypoxia, such proteins are partially stabilized, owing to decreased Nt-Cys oxidation. Here, we show that if hypoxia is prolonged, the Nt-Cys of regulatory proteins can be chemically oxidized by ROS to generate Arg-CysO3(H) (R-CO3), a lysosomal N-degron. The resulting R-CO3 is bound by KCMF1, a N-recognin that induces K63-linked ubiquitylation, followed by K27-linked ubiquitylation by the noncanonical N-recognin UBR4. Autophagic targeting of Cys/N-degron substrates is mediated by the autophagic N-recognin p62/SQTSM- 1/Sequestosome-1 through recognition of K27/K63-linked ubiquitin (Ub) chains. This Cys/N-degron–dependent reprogramming in the proteolytic flux is important for cellular homeostasis under both chronic hypoxia and oxidative stress. A small-compound ligand of p62 is cytoprotective under oxidative stress through its ability to accelerate proteolytic flux of K27/K63-ubiquitylated Cys/N-degron substrates. Our results suggest that the Nt-Cys of conditional Cys/ N-degron substrates acts as an acceptor of O2 to maintain both O2 and ROS homeostasis and modulates half-lives of substrates through either the proteasome or lysosome by reprogramming of their Ub codes. - https://doi.org/10.1073/pnas.2107993118 - 2021
Rapid advances in nanopore technologies for sequencing single long DNA and RNA molecules have led to substantial improvements in accuracy, read length and throughput. These breakthroughs have required extensive development of experimental and bioinformatics methods to fully exploit nanopore long reads for investigations of genomes, transcriptomes, epigenomes and epitranscriptomes. Nanopore sequencing is being applied in genome assembly, full-length transcript detection and base modification detection and in more specialized areas, such as rapid clinical diagnoses and outbreak surveillance. Many opportunities remain for improving data quality and analytical approaches through the development of new nanopores, base-calling methods and experimental protocols tailored to particular applications. - https://doi.org/10.1038/s41587-021-01108-x - 2021
The new European X-ray Free-Electron Laser is the first X-ray free-electron laser capable of delivering X-ray pulses with a megahertz inter-pulse spacing, more than four orders of magnitude higher than previously possible. However, to date, it has been unclear whether it would indeed be possible to measure high-quality diffraction data at megahertz pulse repetition rates. Here, we show that high-quality structures can indeed be obtained using currently available operating conditions at the European XFEL. We present two complete data sets, one from the well-known model system lysozyme and the other from a so far unknown complex of a β-lactamase from K. pneumoniae involved in antibiotic resistance. This result opens up megahertz serial femtosecond crystallography (SFX) as a tool for reliable structure determination, substrate screening and the efficient measurement of the evolution and dynamics of molecular structures using megahertz repetition rate pulses available at this new class of X-ray laser source. - DOI: 10.1038/s41467-018-06156-7 - 2018
Intestinal failure (IF), following extensive anatomical or functional loss of small intestine (SI), has debilitating long-term consequences on children1. The priority of patient care is to increase the length of functional intestine, particularly the jejunum, to promote nutritional independence2. Here we construct autologous jejunal mucosal grafts using pediatric patient biomaterials and show that patient-derived organoids (PDO) can be expanded efficiently in vitro. In parallel, we generate decellularized human intestinal matrix with intact nanotopography, that forms biological scaffolds. Proteomic and Raman spectroscopy analyses reveal highly analogous biochemical profiles of human SI and colon scaffolds, indicating that they can be used interchangeably as platforms for intestinal engineering. Indeed, seeding of jejunal organoids onto either type of scaffold reliably reconstructs grafts that exhibit several aspects of physiological jejunal function, and that survive to form luminal structures after transplantation into the kidney capsule or subcutaneous pockets for up to 2 weeks. Our findings provide proof-of-concept data for engineering patient-specific jejunal grafts for children with IF, ultimately aiding in the restoration of nutritional autonomy. - doi:10.1038/s41591-020-1024-z. - 2020
Despite their significant potential, catalytic asymmetric reactions of olefins with formaldehyde are rare and metal-free approaches have not been previously disclosed. Here we describe an enantioselective intermolecular Prins reaction of styrenes and paraformaldehyde to form 1,3-dioxanes, using confined imino-imidodiphosphate (iIDP) Brønsted acid catalysts. Isotope labeling experiments and computations suggest a concerted, highly asynchronous addition of an acid-activated formaldehyde oligomer to the olefin. The enantioenriched 1,3-dioxanes can be transformed into the corresponding optically active 1,3-diols, which are valuable synthetic building blocks. - https://doi.org/10.1021/jacs.1c10245 - 2021
Background: The pathogenesis of experimental diabetic cardiomyopathy may involve the activator protein 1 (AP-1) member, JunD. Using non-diabetic heart transplant (HTX) in recipients with diabetes, we examined the effects of the diabetic milieu (hyperglycemia and insulin resistance) on cardiac JunD expression over 12 months. Because sodium/glucose cotransporter-2 inhibitors (SGLT2i) significantly reverse high glucose-induced AP-1 binding in the proximal tubular cell, we investigated JunD expression in a subgroup of type 2 diabetic recipients receiving SGLT2i treatment.
Methods: We evaluated 77 first HTX recipients (40 and 37 patients with and without diabetes, respectively). Among the recipients with diabetes, 17 (45.9%) were receiving SGLT2i treatment. HTX recipients underwent standard clinical evaluation (metabolic status, echocardiography, coronary computed tomography angiography, and endomyocardial biopsy). In the biopsy samples, we evaluated JunD, insulin receptor substrates 1 and 2 (IRS1 and IRS2), peroxisome proliferator-activated receptor-γ (PPAR-γ), and ceramide levels using real-time polymerase chain reaction and immunofluorescence. The biopsy evaluations in this study were performed at 1–4 weeks (basal), 5–12 weeks (intermediate), and up to 48 weeks (final, end of 12-month follow-up) after HTX.
Results: There was a significant early and progressive increase in the cardiac expression of JunD/PPAR-γ and ceramide levels, along with a significant decrease in IRS1 and IRS2 in recipients with diabetes but not in those without diabetes. These molecular changes were blunted in patients with diabetes receiving SGLT2i treatment. Conclusion: Early pathogenesis in human diabetic cardiomyopathy is associated with JunD/PPAR-γ overexpression and lipid accumulation following HTX in recipients with diabetes. Remarkably, this phenomenon was reduced by concomitant therapy with SGLT2i, which acted directly on diabetic hearts. - doi.org/10.1016/j.metabol.2021.154936 - 2021
Accurate prediction of disease risk based on the genetic make-up of an individual is essential for effective prevention and personalized treatment. Nevertheless, to date, individual genetic variants from genome-wide association studies have achieved only moderate prediction of disease risk. The aggregation of genetic variants under a polygenic model shows promising improvements in prediction accuracies. Increasingly, electronic health records (EHRs) are being linked to patient genetic data in biobanks, which provides new opportunities for developing and applying polygenic risk scores in the clinic, to systematically examine and evaluate patient susceptibilities to disease. However, the heterogeneous nature of EHR data brings forth many practical challenges along every step of designing and implementing risk prediction strategies. In this Review, we present the unique considerations for using genotype and phenotype data from biobank-linked EHRs for polygenic risk prediction. - doi.org/10.1038/s41576-020-0224-1 - 2020
With the development of biotherapy, biomacromolecular drugs have gained tremendous attention recently, especially in drug development field due to the sophisticated functions in vivo. Over the past few years, a motley variety of drug delivery strategies have been developed for bio- macromolecular drugs to overcome the difficulties in the druggability, e.g., the instability and easily restricted by physiologic barriers. The application of novel delivery systems to deliver bio- macromolecular drugs can usually prolong the half-life, increase the bioavailability, or improve patient compliance, which greatly improves the efficacy and potentiality for clinical use of biomacromolecular drugs. In this review, recent studies regarding the drug delivery strategies for macromolecular drugs in cancer therapy are summarized, mainly drawing on the development over the last five years. - doi.org/10.1016/j.apsb.2020.01.009 - 2019
The morphological characteristics of tendons have been thoroughly evaluated via microscopy. Optical microscopy and electron microscopy are the most commonly used techniques for tendon tissue observation. According to the principles of both microscopy types, preparation and evaluation methods vary. Simple optical microscopy is commonly used in the observation of cells and extracellular matrix, and many stains, including hematoxylineosin, Van Gieson, Prussian blue, Alcian blue, and toluidine blue, are used for evaluating cells, collagen fiber arrangement, and non-collagenous proteins. Histological scoring systems have been used in many studies for semi-quantification. Scanning electron microscopy (SEM) and transmission electron microscopy (TEM) are the most commonly used electron microscopy types, and special consideration is needed for the fixation and embedding protocols. Glutaraldehyde followed by osmium is most commonly used in the chemical fixation of tendon tissue, followed by epoxy resin embedment. Longitudinal sections captured in SEM images show the arrangement of collagen fibrils and the cells and lipid drops among them, while cross-sections captured in TEM images show the diameter and distribution of collagen fibrils. SEM and TEM are used together for comprehensive evaluations. This mini review is focused on the preparation methodology and related evaluation indexes for the morphological evaluation of tendons. - DOI: 10.1111/os.12637 - 2020
Flavonoids are polyphenolic compounds subdivided into 6 groups: isoflavonoids, flavanones, flavanols, flavonols, flavones and anthocyanidins found in a variety of plants. Fruits, vegetables, plant-derived beverages such as green tea, wine and cocoa-based products are the main dietary sources of flavonoids. Flavonoids have been shown to possess a wide variety of anticancer effects: they modulate reactive oxygen species (ROS)-scavenging enzyme activities, participate in arresting the cell cycle, induce apoptosis, autophagy, and suppress cancer cell proliferation and invasiveness. Flavonoids have dual action regarding ROS homeostasis—they act as antioxidants under normal conditions and are potent pro-oxidants in cancer cells triggering the apoptotic pathways and downregulating pro-inflammatory signaling pathways. This article reviews the biochemical properties and bioavailability of flavonoids, their anticancer activity and its mechanisms of action. - doi:10.3390/nu12020457 - 2020
Ineffective antibody-mediated responses are a key characteristic of chronic viral infection. However, our understanding of the intrinsic mechanisms that drive this dysregulation are unclear. Here, we identify that targeting the epigenetic modifier BMI-1 in mice improves humoral responses to chronic lymphocytic choriomeningitis virus. BMI-1 was upregulated by germinal center B cells in chronic viral infection, correlating with changes to the accessible chromatin landscape, compared to acute infection. B cell-intrinsic deletion of Bmi1 accelerated viral clearance, reduced splenomegaly and restored splenic architecture. Deletion of Bmi1 restored c-Myc expression in B cells, concomitant with improved quality of antibody and coupled with reduced antibody-secreting cell numbers. Specifically, BMI-1-deficiency induced antibody with increased neutralizing capacity and enhanced antibody-dependent effector function. Using a small molecule inhibitor to murine BMI-1, we could deplete antibody-secreting cells and prohibit detrimental immune complex formation in vivo. This study defines BMI-1 as a crucial immune modifier that controls antibody-mediated responses in chronic infection. - doi.org/10.1038/s41590-021-01077-y- 2021
Long-chain n-3 polyunsaturated fatty acids (n-3 PUFA) have been shown to modulate the immune response and have therapeutic effects in inflammatory disorders. PUFA are also peroxisome proliferators-activator receptor-gamma (PPARγ) ligands; a family of ligand-activated transcription factors, which when activated antagonise the pro-inflammatory capability of nuclear factor κB (NF-κB). PPARγ plays a role in dendritic cell (DC) maturation and n-3 PUFA have been shown to affect DC maturation by decreasing activation of NF-κB. While n-3 PUFA can function as PPAR ligands, it is not known whether the NF-κB-mediated immunomodulatory properties of n-3 PUFA are PPARγ-dependent. In this study we examined whether the immunomodulatory effects of n-3 PUFA on DC activation were mediated through activation of PPARγ. Treatment of murine bone marrow derived DCs with docosahexaenoic acid (DHA; 25 μM) and eicosapentaenoic acid (EPA; 25 μM) attenuated LPS-induced DC maturation. This was characterised by suppression of IL-12 production and expression of CD40, CD80, CD86 and MHC II and enhanced production of IL-10 and expression of IL-10R. This was coincident with enhanced PPARγ expression, suppressed NF-κB activity and increased the physical interaction and cellular colocalization between NF-κB with PPARγ. To understand the functional implication of the physical association of PPARγ with NF-κB, we determined whether the specific PPARγ inhibitor, GW9662 could abolish the anti-inflammatory effect of n-3 PUFA Inhibiting PPARγ did not impede the NF-κB-mediated anti-inflammatory cytokine profile induced by EPA and DHA alone. Thus n-3 PUFA activate PPARγ and interact with NF-κB in DC. However, the anti-inflammatory effects of EPA and DHA on DCs are independent of PPARγ. - 10.1016/j.jnutbio.2010.06.009 - 2011
BACKGROUND: Studies in non-human mammals suggest that environmental factors can influence spermatozoal DNA methylation, and some research suggests that spermatozoal DNA methylation is also implicated in conditions such as subfertility and imprinting disorders in the offspring. Together with an increased availability of cost-effective methods of interrogating DNA methylation, this premise has led to an increasing number of studies investigating the DNA methylation landscape of human spermatozoa. However, how the human spermatozoal DNA methylome is influenced by environmental factors is still unclear, as is the role of human spermatozoal DNA methylation in subfertility and in influencing offspring health.
OBJECTIVE AND RATIONALE: The aim of this systematic review was to critically appraise the quality of the current body of literature on DNA methylation in human spermatozoa, summarize current knowledge and generate recommendations for future research.
SEARCH METHODS: A comprehensive literature search of the PubMed, Web of Science and Cochrane Library databases was conducted using the search terms ‘semen’ OR ‘sperm’ AND ‘DNA methylation’. Publications from 1 January 2003 to 2 March 2020 that studied human sperm and were written in English were included. Studies that used sperm DNA methylation to develop methodologies or forensically identify semen were excluded, as were reviews, commentaries, meta-analyses or editorial texts. The Grading of Recommendations, Assessment, Development and Evaluations (GRADE) criteria were used to objectively evaluate quality of evidence in each included publication.
OUTCOMES: The search identified 446 records, of which 135 were included in the systematic review. These 135 studies were divided into three groups according to area of research; 56 studies investigated the influence of spermatozoal DNA methylation on male fertility and abnormal semen parameters, 20 studies investigated spermatozoal DNA methylation in pregnancy outcomes including offspring health and 59 studies assessed the influence of environmental factors on spermatozoal DNA methylation. Findings from studies that scored as ‘high’ and ‘moderate’ quality of evidence according to GRADE criteria were summarized. We found that male subfertility and abnormal semen parameters, in particular oligozoospermia, appear to be associated with abnormal spermatozoal DNA methylation of imprinted regions. However, no specific DNA methylation signature of either subfertility or abnormal semen parameters has been convincingly replicated in genome-scale, unbiased analyses. Furthermore, although findings require independent replication, current evidence suggests that the spermatozoal DNA methylome is influenced by cigarette smoking, advanced age and environmental pollutants. Importantly however, from a clinical point of view, there is no convincing evidence that changes in spermatozoal DNA methylation influence pregnancy outcomes or offspring health.
WIDER IMPLICATIONS: Although it appears that the human sperm DNA methylome can be influenced by certain environmental and physiological traits, no findings have been robustly replicated between studies. We have generated a set of recommendations that would enhance the reliability and robustness of findings of future analyses of the human sperm methylome. Such studies will likely require multicentre collaborations to reach appropriate sample sizes, and should incorporate phenotype data in more complex statistical models.
doi:10.1093/humupd/dmaa025 - 2020
The 2021 Nucleic Acids Research database Issue contains 189 papers spanning a wide range of biological fields and investigation. It includes 89 papers reporting on new databases and 90 covering recent changes to resources previously published in the Issue. A further ten are updates on databases most recently published elsewhere. Seven new databases focus on COVID-19 and SARS-CoV- 2 and many others offer resources for studying the virus. Major returning nucleic acid databases include NONCODE, Rfam and RNAcentral. Protein family and domain databases include COG, Pfam, SMART and Panther. Protein structures are covered by RCSB PDB and dispersed proteins by PED and MobiDB. In metabolism and signalling, STRING, KEGG and WikiPathways are featured, along with returning KLIFS and new DKK and KinaseMD, all focused on kinases. IMG/M and IMG/VR update in the microbial and viral genome resources section, while human and model organism genomics resources include Flybase, Ensembl and UCSC Genome Browser. Cancer studies are covered by updates from canSAR and PINA, as well as newcomers CNCdatabase and Oncovar for cancer drivers. Plant comparative genomics is catered for by updates from Gramene and GreenPhylDB. The entire Database Issue is freely available online on the Nucleic Acids Research website (https://academic.oup.com/nar). The NAR online Molecular Biology Database Collection has been substantially updated, revisiting nearly 1000 entries, adding 90 new resources and eliminating 86 obsolete databases, bringing the current total to 1641 databases. It is available at https://www. oxfordjournals.org/nar/database/c/. - doi: 10.1093/nar/gkaa1216 - 2021
MicroRNAs (miRNAs) are a class of small non-coding RNA molecules involved in the regulation of gene expression. They are involved in the fine-tuning of fundamental biological processes such as proliferation, differentiation, survival and apoptosis in many cell types. Emerging evidence suggests that miRNAs regulate critical pathways involved in stem cell function. Several miRNAs have been suggested to target transcripts that directly or indirectly coordinate the cell cycle progression of stem cells. Moreover, previous studies have shown that altered expression levels of miRNAs can contribute to pathological conditions, such as cancer, due to the loss of cell cycle regulation. However, the precise mechanism underlying miRNA- mediated regulation of cell cycle in stem cells is still incompletely understood. In this review, we discuss current knowledge of miRNAs regulatory role in cell cycle progression of stem cells. We describe how specific miRNAs may control cell cycle associated molecules and checkpoints in embryonic, somatic and cancer stem cells. We further outline how these miRNAs could be regulated to influence cell cycle progression in stem cells as a potential clinical application. - doi.org/10.1007/s12015-018-9808-y - 2018
The majority of therapies that target individual proteins rely on specific activity- modulating interactions with the target protein—for example, enzyme inhibition or ligand blocking. However, several major classes of therapeutically relevant proteins have unknown or inaccessible activity profiles and so cannot be targeted by such strategies. Protein-degradation platforms such as proteolysis-targeting chimaeras (PROTACs)1,2 and others (for example, dTAGs3, Trim-Away4, chaperone-mediated autophagy targeting5 and SNIPERs6) have been developed for proteins that are typically difficult to target; however, these methods involve the manipulation of intracellular protein degradation machinery and are therefore fundamentally limited to proteins that contain cytosolic domains to which ligands can bind and recruit the requisite cellular components. Extracellular and membrane-associated proteins—the products of 40% of all protein-encoding genes7—are key agents in cancer, ageing-related diseases and autoimmune disorders8, and so a general strategy to selectively degrade these proteins has the potential to improve human health. Here we establish the targeted degradation of extracellular and membrane-associated proteins using conjugates that bind both a cell-surface lysosome-shuttling receptor and the extracellular domain of a target protein. These initial lysosome-targeting chimaeras, which we term LYTACs, consist of a small molecule or antibody fused to chemically synthesized glycopeptide ligands that are agonists of the cation-independent mannose-6-phosphate receptor (CI-M6PR). We use LYTACs to develop a CRISPR interference screen that reveals the biochemical pathway for CI-M6PR-mediated cargo internalization in cell lines, and uncover the exocyst complex as a previously unidentified—but essential—component of this pathway. We demonstrate the scope of this platform through the degradation of therapeutically relevant proteins, including apolipoprotein E4, epidermal growth factor receptor, CD71 and programmed death-ligand 1. Our results establish a modular strategy for directing secreted and membrane proteins for lysosomal degradation, with broad implications for biochemical research and for therapeutics. - doi.org/10.1038/s41586-020-2545-9 - 2020
The simultaneous measurement of multiple modalities represents an exciting frontier for single-cell genomics and necessitates computational methods that can define cellular states based on multimodal data. Here, we introduce ‘‘weighted-nearest neighbor’’ analysis, an unsupervised framework to learn the relative utility of each data type in each cell, enabling an integrative analysis of multiple modalities. We apply our procedure to a CITE-seq dataset of 211,000 human peripheral blood mononuclear cells (PBMCs) with panels extending to 228 antibodies to construct a multimodal reference atlas of the circulating immune system. Multimodal analysis substantially improves our ability to resolve cell states, allowing us to identify and validate previously unreported lymphoid subpopulations. Moreover, we demonstrate how to leverage this reference to rapidly map new datasets and to interpret immune responses to vaccination and coronavirus disease 2019 (COVID-19). Our approach represents a broadly applicable strategy to analyze single-cell multimodal data- sets and to look beyond the transcriptome toward a unified and multimodal definition of cellular identity. - doi.org/10.1016/j.cell.2021.04.048 - 2021
DNA nanotechnology has generated a wealth of structures uniquely suited for nanoscale patterning; however, scalability, affordability, and recyclability are important preconditions for the industrial production and widespread use of DNA-based materials. In this perspective, we propose that ‘‘printing’’ programmed particles from transient DNA templates provides a practical pathway toward overcoming these hurdles. Just as a printing press transfers ink to paper in a cyclical process, DNA can translocate materials from one substrate to another while preserving spatial information. DNA printing decorates a particle with arbitrarily designed patterns that break its symmetry, enabling its autonomous assembly into complex structures. Printing thus organizes anisotropic particles with high throughput: a single DNA scaffold engenders template recyclability and hybrid material scale-up. By applying DNA-based printing methods to increasingly diverse materials, we will export DNA nanotechnology to other research areas and generate multivalent, asymmetric structures for scalable, nanoscale control of structure and function. - doi.org/10.1016/j.chempr.2020.06.012 - 2020
One of the hallmarks of Alzheimer’s disease (AD) are deposits of amyloid-beta (Aβ) protein in amyloid plaques in the brain. The Aβ peptide exists in several forms, including full-length Aβ1-42 and Aβ1-40 – and the N-truncated species, pyroglutamate Aβ3-42 and Aβ4-42, which appear to play a major role in neurodegeneration. We previously identified a murine antibody (TAP01), which binds specifically to soluble, non-plaque N-truncated Aβ species. By solving crystal structures for TAP01 family antibodies bound to pyroglutamate Aβ3-14, we identified a novel pseudo β-hairpin structure in the N-terminal region of Aβ and show that this underpins its unique binding properties. We engineered a stabilised cyclic form of Aβ1-14 (N-Truncated Amyloid Peptide AntibodieS; the ‘TAPAS’ vaccine) and showed that this adopts the same 3-dimensional conformation as the native sequence when bound to TAP01. Active immunisation of two mouse models of AD with the TAPAS vaccine led to a striking reduction in amyloid- plaque formation, a rescue of brain glucose metabolism, a stabilisation in neuron loss, and a rescue of memory deficiencies. Treating both models with the humanised version of the TAP01 antibody had similar positive effects. Here we report the discovery of a unique conformational epitope in the N-terminal region of Aβ, which offers new routes for active and passive immunisation against AD. - doi.org/10.1038/s41380-021-01385-7. -2021
Helicases are in the spotlight of DNA metabolism and are critical for DNA repair in all domains of life. At their biochemical core, they bind and hydrolyze ATP, converting this energy to translocate unidirectionally, with different strand polarities and substrate binding specificities, along one strand of a nucleic acid. In doing so, DNA and RNA helicases separate duplex strands or remove nucleoprotein complexes, affecting DNA repair and the architecture of replication forks. In this review, we focus on recent advances on the roles and regulations of DNA helicases in homologous recombination repair, a critical pathway for mending damaged chromosomes and for ensuring genome integrity. - doi.org/10.1016/j.gde.2021.06.009 - 2021
Many viruses evolve rapidly. This is due, in part, to their high mutation rates. Mutation rate estimates for over 25 viruses are currently available. Here, we review the population genetics of virus mutation rates. We specifically cover the topics of mutation rate estimation, the forces that drive the evolution of mutation rates, and how the optimal mutation rate can be context-dependent. - doi.org/10.1128/JVI .01031-17. - 2018
Compact and versatile CRISPR-Cas systems will enable genome engineering applications through high-efficiency delivery in a wide variety of contexts. Here, we create an efficient miniature Cas system (CasMINI) engineered from the type V-F Cas12f (Cas14) system by guide RNA and protein engineering, which is less than half the size of currently used CRISPR systems (Cas9 or Cas12a). We demonstrate that CasMINI can drive high levels of gene activation (up to thousands-fold increases), while the natural Cas12f system fails to function in mammalian cells. We show that the CasMINI system has comparable activities to Cas12a for gene activation, is highly specific, and allows robust base editing and gene editing. We expect that CasMINI can be broadly useful for cell engineering and gene therapy applications ex vivo and in vivo. - doi.org/10.1016/j.molcel.2021.08.008 - 2021
Obesity and in particular the excessive visceral fat distribution is accompanied by several alterations at hormonal, inflammatory and endothelial level. These alterations induce a stimulation of several other mechanisms that contribute to the hypertensive state and on the other side to increase the cardiovascular morbidity. In these chapter we will examine the main mechanisms of obesity and obesity-related hypertension and in particular the role of sympathetic nervous system, the alterations of the renal function and at the microvascular level. We will also depict the role of insulin resistance as factor stimulating and potentiating the other mechanisms. The second part will be focalized on the major target organ damage linked with obesity and obesity-related hypertension. We will finally describe the management and treatment of obesity and the antihypertensive drug therapies more effective in hypertensive obeses. - doi.org/10.1016/j.phrs.2017.05.013 - 2017
Hypertension is the strongest or one of the strongest risk factors for almost all different cardiovascular diseases acquired during life, including coronary disease, left ventricular hypertrophy and valvular heart diseases, cardiac arrhythmias including atrial fibrillation, cerebral stroke and renal failure. The continuous relationship between blood pressure and cardiovascular and renal events makes the distinction between high normal blood pressure and hypertension based on arbitrary cut-off values for blood pressures. Overall the prevalence of hypertension in different European countries appears to be around 30–45% of the general population, with a steep increase with ageing. The prevention of cardiovascular disease and treatment recommendations should be related to quantification of total cardiovascular risk which could be estimated from several different models. However the impact of age on risk is so strong that young adults (particularly women) are unlikely to reach high-risk levels even when they have more than one major risk factor and a clear increase in relative risk. Therefore age-adjusted models, models assessing relative risks compared to others of same age and models including thorough assessments of target organ damage and ambulatory 24 h blood pressure are needed together with national models because of the large variations between countries. - doi.org/10.1016/j.phrs.2017.11.003 - 2017
Liquid-liquid phase separation (LLPS) in cells is known as a complex physicochemical process causing the formation of membrane-less organelles (MLOs). Cells have well-defined different membrane-surrounded organelles like mitochondria, endoplasmic reticulum, lysosomes, peroxisomes, etc., however, on demand they can create MLOs as stress granules, nucleoli and P bodies to cover vital functions and regulatory activities. However, the mechanism of intracellular molecule assembly into functional compartments within a living cell remains till now not fully understood. in vitro and in vivo investigations unveiled that MLOs emerge after preceding liquid-liquid, liquid gel, liquid-semi-crystalline, or liquid-crystalline phase separations. Liquid-liquid and liquid-gel MLOs form the majority of cellular phase separation events, while the occurrence of micro-sized crystals in cells was only rarely observed, however can be considered as a result of a preceding protein phase separation event. In vivo, also known and termed as in cellulo crystals, are reported since 1853. In some cases, they have been linked to vital cellular functions, such as storage and detoxification. How- ever, the occurrence of in cellulo crystals is also associated to diseases like cataract, hemoglobin C diseases, etc. Therefore, better knowledge about the involved molecular processes will support drug discovery investigations to cure diseases related to in cellulo crystallization. We summarize physical and chemical determinants known today required for phase separation initiation and formation and in cellulo crystal growth. In recent years it has been demonstrated that LLPS plays a crucial role in cell compartmentalization and formation of MLOs. Here we discuss potential mechanisms and potential crowding agents involved in protein phase separation and in cellulo crystallization. - DOI: 10.1111/tra.12711 - 2019
Diabetes is a complex metabolic disorder affecting the glucose status of the human body. Chronic hyperglycaemia related to diabetes is associated with end organ failure. The clinical relationship between diabetes and atherosclerotic cardiovascular disease is well established. This makes therapeutic approaches that simultaneously target diabetes and atherosclerotic disease an attractive area for research. The majority of people with diabetes fall into two broad pathogenetic categories, type 1 or type 2 diabetes. The role of obesity, adipose tissue, gut microbiota and pancreatic beta cell function in diabetes are under intensive scrutiny with several clinical trials to have been completed while more are in development. The emerging role of inflammation in both type 1 and type 2 diabetes (T1D and T1D) pathophysiology and associated metabolic disorders, has generated increasing interest in targeting inflammation to improve prevention and control of the disease. After an extensive review of the possible mechanisms that drive the metabolic pattern in T1D and T2D and the inflammatory pathways that are involved, it becomes ever clearer that future research should focus on a model of combined suppression for various inflammatory response pathways. - doi.org/10.15420/ecr.2018.33.1 - 2019
Glioblastomas are intrinsic brain tumors thought to originate from neuroglial stem or progenitor cells. More than 90% of glioblastomas are isocitrate dehydrogenase (IDH)-wildtype tumors. Incidence increases with age, males are more often affected. Beyond rare instances of genetic predisposition and irradiation exposure, there are no known glioblastoma risk factors. Surgery as safely feasible followed by involved-field radiotherapy plus concomitant and maintenance temozolomide chemotherapy define the standard of care since 2005. Except for prolonged progression-free, but not overall survival afforded by the vascular endothelial growth factor antibody, bevacizumab, no pharmacological intervention has been demonstrated to alter the course of disease. Specifically, targeting cellular pathways frequently altered in glioblastoma, such as the phosphoinositide 3- kinase (PI3K)/protein kinase B (AKT)/mammalian target of rapamycin (mTOR), the p53 and the retinoblastoma (RB) pathways, or epidermal growth factor receptor (EGFR) gene amplification or mutation, have failed to improve outcome, likely because of redundant compensatory mechanisms, insufficient target coverage related in part to the blood brain barrier, or poor tolerability and safety. Yet, uncommon glioblastoma subsets may exhibit specific vulnerabilities amenable to targeted interventions, including, but not limited to: high tumor mutational burden, BRAF mutation, neurotrophic tryrosine receptor kinase (NTRK) or fibroblast growth factor receptor (FGFR) gene fusions, and MET gene amplification or fusions. There is increasing interest in targeting not only the tumor cells, but also the microenvironment, including blood vessels, the monocyte/macrophage/microglia compartment, or T cells. Improved clinical trial designs using pharmacodynamic endpoints in enriched patient populations will be required to develop better treatments for glioblastoma. - doi.org/10.1016/j.ctrv.2019.101896 - 2019
Anyone who has ever attempted to crystallise a protein or other biological macromolecule has encountered at least one, if not all of the following scenarios: No crystals at all, tiny low quality that do not diffract at all. In this paper, we review a number of simple ways to overcome such problems, which have worked well in our hands and in other laboratories. It brings together information that has been dispersed in various publications and lectures over the years and includes further information that has not been previously published. - doi:10.3390/cryst9020106 - 2019
The importance of cancer-cell-autonomous functions of the tumour suppressor p53 (encoded by TP53) has been established in many studies, but it is now clear that the p53 status of the cancer cell also has a profound impact on the immune response. Loss or mutation of p53 in cancers can affect the recruitment and activity of myeloid and T cells, allowing immune evasion and promoting cancer progression. p53 can also function in immune cells, resulting in various outcomes that can impede or support tumour development. Understanding the role of p53 in tumour and immune cells will help in the development of therapeutic approaches that can harness the differential p53 status of cancers compared with most normal tissue. - doi:10.1242/jcs.237453 - 2020
Ki-67 serves as a prominent cancer marker. We describe how expression of the MKI67 gene coding for Ki-67 is controlled during the cell cycle. MKI67 mRNA and Ki-67 protein are maximally expressed in G2 phase and mitosis. Expression is dependent on two CHR elements and one CDE site in the MKI67 promoter. DREAM transcriptional repressor complexes bind to both CHR sites and downregulate the expression in G0/G1 cells. Upregulation of MKI67 transcription coincides with binding of B-MYB-MuvB and FOXM1-MuvB complexes from S phase into G2/M. Importantly, binding of B-MYB to the two CHR elements correlates with loss of CHR-dependent MKI67 promoter activation in B-MYB-knockdown experiments. In knockout cell models, we find that DREAM/MuvB-dependent transcriptional control cooperates with the RB Retinoblastoma tumor suppressor. Furthermore, the p53 tumor suppressor indirectly downregulates transcription of the MKI67 gene. This repression by p53 requires p21/CDKN1A. These results are consistent with a model in which DREAM, B-MYB-MuvB, and FOXM1-MuvB together with RB cooperate in cell cycle-dependent transcription and in transcriptional repression following p53 activation. In conclusion, we present mechanisms how MKI67 gene expression followed by Ki-67 protein synthesis is controlled during the cell cycle and upon induction of DNA damage, as well as upon p53 activation. - doi.org/10.1038/s41418-021-00823-x - 2021
The use of photodynamic therapy (PDT) against cancer has received increasing attention over recent years. However, the application of the currently approved photosensitizers (PSs) is limited by their poor aqueous solubility, aggregation, photobleaching and slow clearance from the body. To overcome these limitations, there is a need for the development of new classes of PSs with ruthenium(II) polypyridine complexes currently gaining momentum. However, these compounds generally lack significant absorption in the biological spectral window, limiting their application to treat deep-seated or large tumors. To overcome this drawback, ruthenium(II) polypyridine complexes designed in silico with (E,E′)-4,4′-bisstyryl- 2,2′-bipyridine ligands show impressive 1- and 2-Photon absorption up to a magnitude higher than the ones published so far. While nontoxic in the dark, these compounds are phototoxic in various 2D monolayer cells, 3D multicellular tumor spheroids and are able to eradicate a multiresistant tumor inside a mouse model upon clinically relevant 1-Photon and 2-Photon excitation. - doi.org/10.1038/s41467-020-16993-0 - 2020
The polyubiquitin gene ubiquitin C (UBC) is considered a stress protective gene and is upregulated under various stressful conditions, which is probably a consequence of an increased demand for ubiquitin in order to remove toxic misfolded proteins. We previously identified heat shock elements (HSEs) within the UBC promoter, which are responsible for heat shock factor (HSF)1-driven induction of the UBC gene and are activated by proteotoxic stress. Here, we determined the molecular players driving the UBC gene transcriptional response to arsenite treatment, mainly addressing the role of the nuclear factor-erythroid 2-related factor 2 (Nrf2)-mediated antioxidant pathway. Exposure of HeLa cells to arsenite caused a time-dependent increase of UBC mRNA, while cell viability and proteasome activity were not affected. Nuclear accumulation of HSF1 and Nrf2 transcription factors were detected upon both arsenite and MG132 treatment, while HSF2 nuclear levels increased in MG132-treated cells. Notably, siRNA-mediated knockdown of Nrf2 did not reduce UBC transcription under either basal or stressful conditions, but significantly impaired the constitutive and inducible expression of well-known antioxidant response element-dependent genes. A chromatin immunoprecipitation assay consistently failed to detect Nrf2 binding to the UBC promoter sequence. By contrast, depletion of HSF1, but not HSF2, significantly compromised stress-induced UBC expression. Critically, HSF1-mediated UBC trans-activation upon arsenite exposure relies on transcription factor binding to previously mapped distal HSEs, as demonstrated to occur under proteasome inhibition. These data highlight HSF1 as the pivotal transcription factor that translates different stress signals into UBC gene transcriptional induction. - doi:10.1002/2211-5463.12484 - 2018
Renal artery aneurysms are rare in the general population, although the true incidence and natural history remain elusive. Controversy over criteria for repair persists across decades. Indications for repair presently include aneurysm size >2 cm, female gender within childbearing age, symptoms like pain and hematuria, medically refractory hypertension including that associated with functionally important renal artery stenosis, thromboembolism, dissection, and rupture. Conventional surgical reconstruction options are variable and continue to offer technically sound and durable results. Endovascular therapies with novel devices also offer technical success with few major adverse events, and are increasingly employed as indications for intervention broaden. This review summarizes the accumulated evidence on true renal artery aneurysms with a particular focus on contemporary treatment criteria, natural history, options for repair and outcomes following such. -doi.org/10.1016/j.jvs.2015.05.034 - 2015
The ability to predictably control the coassembly of multiple nanoscale building blocks, especially those with disparate chemical and physical properties such as biomolecules and inorganic nanoparticles, has far-reaching implications in catalysis, sensing, and photonics, but a generalizable strategy for engineering specific contacts between these particles is an outstanding challenge. This is especially true in the case of proteins, where the types of possible interparticle interactions are numerous, diverse, and complex. Herein, we explore the concept of trading protein–protein interactions for DNA–DNA interactions to direct the assembly of two nucleic-acid–functionalized proteins with distinct surface chemstries into six unique lattices composed of catalytically active proteins, or of a combination of proteins and DNA-modified gold nanoparticles. The programmable nature of DNA–DNA interactions used in this strategy allows us to control the lattice symmetries and unit cell constants, as well as the compositions and habit, of the resulting crystals. This study provides a potentially generalizable strategy for constructing a unique class of materials that take advantage of the diverse morphologies, surface chemistries, and functionalities of proteins for assembling functional crystalline materials. - doi/10.1073/pnas.1503533112 - 2015
The use of microneedles has facilitated the painless localized delivery of drugs across the skin. However, their efficacy has been limited by slow diffusion of molecules and often requires external triggers. Herein, an autonomous and degradable, active microneedle delivery platform is introduced, employing magnesium microparticles loaded within the microneedle patch, as the built-in engine for deeper and faster intradermal payload delivery. The magnesium particles react with the interstitial fluid, leading to an explosive-like rapid production of H2 bubbles, providing the necessary force to breach dermal barriers and enhance payload delivery. The release kinetics of active microneedles is evaluated in vitro by measuring the amount of IgG antibody (as a model drug) that passed through phantom tissue and a pigskin barrier. In vivo experiments using a B16F10 mouse melanoma model demonstrate that the active delivery of anti-CTLA-4 (a checkpoint inhibitor drug) results in greatly enhanced immune response and significantly longer survival. Moreover, spatially resolved zones of active and passive microneedles allow a combinatorial rapid burst response along with slow, sustained release, respectively. Such versatile and effective autonomous dynamic microneedle delivery technology offers considerable promise for a wide range of therapeutic applications, toward a greatly enhanced outcome, convenience, and cost. - DOI: 10.1002/adma.201905740 - 2020
Human genome function is underpinned by the primary storage of genetic information in canonical B-form DNA, with a second layer of DNA structure providing regulatory control. I-motif structures are thought to form in cytosine-rich regions of the genome and to have regulatory functions; however, in vivo evidence for the existence of such structures has so far remained elusive. Here we report the generation and characterization of an antibody fragment (iMab) that recognizes i-motif structures with high selectivity and affinity, enabling the detection of i-motifs in the nuclei of human cells. We demonstrate that the in vivo formation of such structures is cell-cycle and pH dependent. Furthermore, we provide evidence that i-motif structures are formed in regulatory regions of the human genome, including promoters and telomeric regions. Our results support the notion that i-motif structures provide key regulatory roles in the genome. - doi.org/10.1038/s41557-018-0046-3 - 2018
Metallodrugs provide important first-line treatment against various forms of human cancer. To overcome chemotherapeutic resistance and widen treatment possibilities, new agents with improved or alternative modes of action are highly sought after. Here, we present a click chemistry strategy for developing DNA damaging metallodrugs. The approach in- volves the development of a series of polyamine ligands where three primary, secondary or tertiary alkyne-amines were selected and ‘clicked’ using the copper-catalysed azide-alkyne cycloaddition reac- tion to a 1,3,5-azide mesitylene core to produce a family of compounds we call the ‘Tri-Click’ (TC) se- ries. From the isolated library, one dominant ligand (TC1) emerged as a high-affinity copper(II) binding agent with potent DNA recognition and damaging properties. Using a range of in vitro biophysical and molecular techniques––including free radical scavengers, spin trapping antioxidants and base excision repair (BER) enzymes–the oxidative DNA damaging mechanism of copper-bound TC1 was elucidated. This activity was then compared to intracellular results obtained from peripheral blood mononuclear cells exposed to Cu(II)–TC1 where use of BER enzymes and fluorescently modified dNTPs enabled the characterisation and quantification of genomic DNA lesions produced by the complex. The approach can serve as a new avenue for the design of DNA damaging agents with unique activity profiles. - doi.org/10.1093/nar/gkab817 - 2021
We present the results of our technical validation process in establishing the analysis of circulating tumor DNA (ctDNA) as a diagnostic tool. Like most cells in our body, tumor cells shed DNA in the blood flow. Analysis of ctDNA mutational content can provide invaluable information on the genetic makeup of a tumor, and assist oncologists in deciding on therapy, or in following residual disease. However, low absolute amounts of circulating DNA and low tumor fraction constitute formidable analytical challenges. A key step is to avoid contamination with genomic DNA from cell lysis. Several brands of specialized blood collection tubes are available to prevent leukocyte lysis. We show that they are not equally efficient, depending on storage temperature and time before plasma preparation. We report our analysis of preanalytical factors pertaining to ctDNA analysis (tubes, transportation time, temperature) and our conclusions in terms of instructions to prescribing physicians. We also stress the importance of proper DNA quality control and compare several methods, including a differential amplicon length PCR technique which allows determination of multiple QC parameters from minimal amounts of DNA. Altogether, these data provide useful practical information to diagnostic laboratories wishing to implement the assay of ctDNA in clinical practice. - doi.org/10.1016/j.ab.2017.11.004 - 2018
Apoptosis, the cell’s natural mechanism for death, is a promising target for anticancer therapy. Both the intrinsic and extrinsic pathways use caspases to carry out apoptosis through the cleavage of hundreds of proteins. In cancer, the apoptotic pathway is typically inhibited through a wide variety of means including overexpression of antiapoptotic proteins and under-expression of proapoptotic proteins. Many of these changes cause intrinsic resistance to the most common anticancer therapy, chemotherapy. Promising new anticancer therapies are plant-derived compounds that exhibit anticancer activity through activating the apoptotic pathway. doi:10.3390/ijms19020448 - 2018
Tumor necrosis factor (TNF) is one of the few cytokines successfully targeted by therapies against inflammatory diseases. However, blocking this well studied and pleiotropic ligand can cause dramatic side-effects. Here, we reason that a systems-level proteomic analysis of TNF signaling could dissect its diverse functions and offer a base for developing more targeted therapies. Therefore, we combine phosphoproteomics time course experiments with sub-cellular localization and kinase inhibitor analysis to identify functional modules of protein phosphorylation. The majority of regulated phosphorylation events can be assigned to an upstream kinase by inhibiting master kinases. Spatial proteomics reveals phosphorylation- dependent translocations of hundreds of proteins upon TNF stimulation. Phosphoproteome analysis of TNF-induced apoptosis and necroptosis uncovers a key role for transcriptional cyclin-dependent kinase activity to promote cytokine production and prevent excessive cell death downstream of the TNF signaling receptor. This resource of TNF-induced pathways and sites can be explored at http://tnfviewer.biochem.mpg.de/. - doi.org/10.1038/s41467-021-26289-6 - 2021
Particulate methane monooxygenase (pMMO) is an integral membrane metalloenzyme that catalyses the conversion of methane to methanol. Knowledge of how pMMO performs this extremely challenging chemistry may have an impact on the use of methane as an alternative energy source by facilitating the development of new synthetic catalysts. We have determined the structure of pMMO from the methanotroph Methylococcus capsulatus (Bath) to a resolution of 2.8A ̊. The enzyme is a trimer with an a3b3g3 polypeptide arrangement. Two metal centres, modelled as mononuclear copper and dinuclear copper, are located in soluble regions of each pmoB subunit, which resembles cytochrome c oxidase subunit II. A third metal centre, occupied by zinc in the crystal, is located within the membrane. The structure provides new insight into the molecular details of biological methane oxidation. - doi.org/10.1038/nature03311 - 2005
Identification of HLA class I ligands from the tumor surface (ligandome or immunopeptidome) is essential for designing T-cell mediated cancer therapeutic approaches. However, the sensitivity of the process for isolating MHC-I restricted tumor-specific peptides has been the major limiting factor for reliable tumor antigen characterization, making clear the need for technical improvement. Here, we describe our work from the fabrication and development of a microfluidic-based chip (PeptiCHIP) and its use to identify and characterize tumor-specific ligands on clinically relevant human samples. Specifically, we assessed the potential of immobilizing a pan-HLA antibody on solid surfaces via well-characterized streptavidin−biotin chemistry, overcoming the limitations of the cross-linking chemistry used to prepare the affinity matrix with the desired antibodies in the immunopeptidomics workflow. Furthermore, to address the restrictions related to the handling and the limited availability of tumor samples, we further developed the concept toward the implementation of a microfluidic through-flow system. Thus, the biotinylated pan-HLA antibody was immobilized on streptavidin-functionalized surfaces, and immune-affinity purification (IP) was carried out on customized microfluidic pillar arrays made of thiol−ene polymer. Compared to the standard methods reported in the field, our methodology reduces the amount of antibody and the time required for peptide isolation. In this work, we carefully examined the specificity and robustness of our customized technology for immunopeptidomics workflows. We tested this platform by immunopurifying HLA-I complexes from 1 × 106 cells both in a widely studied B-cell line and in patients-derived ex vivo cell cultures, instead of 5 × 108 cells as required in the current technology. After the final elution in mild acid, HLA-I-presented peptides were identified by tandem mass spectrometry and further investigated by in vitro methods. These results highlight the potential to exploit microfluidics-based strategies in immunopeptidomics platforms and in personalized immunopeptidome analysis from cells isolated from individual tumor biopsies to design tailored cancer therapeutic vaccines. Moreover, the possibility to integrate multiple identical units on a single chip further improves the throughput and multiplexing of these assays with a view to clinical needs. - doi.org/10.1021/acsnano.1c04371 - 2021
Automated data acquisition is used widely for single-particle reconstruction of three-dimensional (3D) volumes of biological complexes preserved in vitreous ice and imaged in a transmission electron microscope. Automation has become integral to this method because of the very large number of particle images required in order to overcome the typically low signal-to-noise ratio of these images. For optimal efficiency, automated data acquisition software packages typically employ some beam-image shift targeting as this method is both fast and accurate ( ± 0.1 μm). In contrast, using only stage movement, relocation to a targeted area under low-dose conditions can only be achieved in combination with multiple iterations or long relaxation times, both reducing efficiency. Nevertheless it is well known that applying beam- image shift induces beam-tilt and with it a potential structure phase error with a phase error π/4 the highest acceptable value. This theory has been used as an argument against beam-image shift for high resolution data collection. Nevertheless, in practice many small beam-image shift datasets have resulted in 3D reconstructions beyond the π/4 phase error limit. To address this apparent contradiction, we performed cryo-EM single-particle reconstructions on a T20S proteasome sample using applied beam-image shifts corresponding to beam tilts from 0 to 10 mrad. To evaluate the results we compared the FSC values, and examined the water density peaks in the 3D map. We conclude that the phase error does not limit the validity of the 3D reconstruction from single-particle averaging beyond the π/4 resolution limit. - doi.org/10.1016/j.jsb.2018.07.015 - 2018
Before a deleterious DNA lesion can be replaced with its undamaged counterpart, the lesion must first be removed from the genome. This process of removing and replacing DNA lesions is accomplished by the careful coordination of several protein factors during DNA repair. One such factor is the multifunctional enzyme human apurinic/apyrimidinic endonuclease 1 (APE1), known best for its DNA backbone cleavage activity at AP sites during base excision repair (BER). APE1 preforms AP site incision with surgical precision and skill, by sculpting the DNA to place the cleavage site in an optimal position for nucleophilic attack within its compact protein active site. APE1, however, has demonstrated broad surgical expertise, and applies its DNA cleavage activity to a wide variety of DNA and RNA substrates. Here, we discuss what is known and unknown about APE1 cleavage mechanisms, focusing on structural and mechanistic considerations. Importantly, disruptions in the biological functions associated with APE1 are linked to numerous human maladies, including cancer and neurodegenerative diseases. The continued elucidation of APE1 mechanisms is required for rational drug design towards novel and strategic ways to target its associated repair pathways. - doi.org/10.1016/j.dnarep.2018.08.012 - 2018
Recombinant adeno-associated viruses (AAVs) are among the most commonly used vehicles for in vivo gene delivery. However, their tropism is limited, and additionally their efficacy can be negatively affected by prevalence of neutralizing antibodies in sera. Methodologies to systematically engineer AAV capsid properties would thus be of great relevance. In this regard, we develop here multi-functional AAVs by engineering precision tethering of oligonucleotides onto the AAV surface, and thereby enabling a spectrum of nucleic-acid programmable functionalities. Towards this, we engineered genetically encoded incorporation of unnatural amino acids (UAA) bearing bio-orthogonal chemical handles onto capsid proteins. Via these we enabled site-specific coupling of oligonucleotides onto the AAV capsid surface using facile click chemistry. The resulting oligo-AAVs could be sequence specifically labeled, and also patterned in 2D using DNA array substrates. Additionally, we utilized these oligo conjugations to engineer viral shielding by lipid-based cloaks that efficaciously protected the AAV particles from neutralizing serum. We confirmed these ‘cloaked AAVs’ retained full functionality via their ability to transduce a range of cell types, and also enable robust delivery of CRISPR-Cas9 effectors. Taken together, we anticipate this programmable oligo-AAV system will have broad utility in synthetic biology and AAV engineering applications. - DOI:10.1038/s41598-018-21742-x - 2018
Replication protein A (RPA), a major eukaryotic ssDNA- binding protein, is essential for all metabolic processes that involve ssDNA, including DNA replication, repair, and damage signaling. To perform its functions, RPA binds ssDNA tightly. In contrast, it was presumed that RPA binds RNA weakly. However, recent data suggest that RPA may play a role in RNA metabolism. RPA stimulates RNA-templated DNA repair in vitro and associates in vivo with R-loops, the three-stranded structures consisting of an RNA-DNA hybrid and the displaced ssDNA strand. R-loops are common in the genomes of pro- and eukaryotes, including humans, and may play an important role in transcription-coupled homologous recombination and DNA replication restart. However, the mechanism of R-loop formation remains unknown. Here, we investigated the RNA-binding properties of human RPA and its possible role in R-loop formation. Using gel-retardation and RNA/ DNA competition assays, we found that RPA binds RNA with an unexpectedly high affinity (KD ’ 100 pM). Furthermore, RPA, by forming a complex with RNA, can promote R-loop formation with homologous dsDNA. In reconstitution experiments, we showed that human DNA polymerases can utilize RPA-generated R-loops for initiation of DNA synthesis, mimicking the process of replication restart in vivo. These results demonstrate that RPA binds RNA with high affinity, supporting the role of this protein in RNA metabolism and suggesting a mechanism of genome maintenance that depends on RPA- mediated DNA replication restart. - DOI 10.1074/jbc.RA120.013812 - 2020
A systematic study of several new types of hybrids of Cu-chelated clamped phenanthroline artificial metallonuclease (AMN) with triplex-forming oligonucleotides (TFO) for sequence-specific cleavage of double-stranded DNA (dsDNA) is reported. The synthesis of these AMN–TFO hybrids is based on application of the alkyne–azide cycloaddition click reaction as the key step. The AMN was attached through different linkers at either the 5’- or 3’-ends or in the middle of the TFO stretch. The diverse hybrids efficiently formed triplexes with the target purine-rich sequence and their copper complexes were studied for their ability to cleave dsDNA in the presence of ascorbate as a reductant. In all cases, the influence of the nature and length of the AMN–TFO, time, conditions and amounts of ascorbate were studied, and optimum conjugates and a procedure that gave reasonably efficient (up to 34%) cleavage of the target sequence, while rendering an off-target dsDNA intact, were found. The footprint of cleavage on PAGE was identified only in one case, with low conversion; this means that cleavage does not proceed with single nucleotide precision. On the other hand, these AMN–TFO hybrids are useful for the selective degradation of target dsDNA sequences. Future improvements to this design may provide higher resolution and selectivity. - doi.org/10.1002/cbic.201900670. - 2019
High-quality crystals are essential to ensure high-resolution structural information. Protein crystals are controlled by many factors, such as pH, temperature, and the ion concentration of crystalline solutions. We previously reported the development of a device dedicated to protein crystallization. In the current study, we have further modified and improved our device. Exposure to external magnetic field leads to alignment of the crystal toward a preferred direction depending on the magnetization energy. Each material has different magnetic susceptibilities depending on the individual direction of their unit crystal cells. One of the strategies to acquire a large crystal entails controlling the nucleation rate. Furthermore, exposure of a crystal to a magnetic field may lead to new morphologies by affecting the crystal volume, shape, and quality. - doi:10.3390/cryst10090821 - 2020
TLR8 is among the highest-expressed pattern-recognition receptors in the human myeloid compartment, yet its mode of action is poorly understood. TLR8 engages two distinct ligand binding sites to sense RNA degradation products, although it remains unclear how these ligands are formed in cellulo in the context of complex RNA molecule sensing. Here, we identified the lysosomal endoribonuclease RNase T2 as a non-redundant upstream component of TLR8- dependent RNA recognition. RNase T2 activity is required for rendering complex single-stranded, exogenous RNA molecules detectable for TLR8. This is due to RNase T2’s preferential cleavage of single-stranded RNA molecules between purine and uridine residues, which critically contributes to the supply of catabolic uridine and the generation of purine-2',3'-cyclophosphate-terminated oligoribonucleotides. Thus-generated molecules constitute agonistic ligands for the first and second binding pocket of TLR8. Together, these results establish the identity and origin of the RNA-derived molecular pattern sensed by TLR8. - doi.org/10.1016/j.cell.2019.11.001 - 2019
Human immunodeficiency virus (HIV-1) remains a major health threat. Viral capsid uncoating and nuclear import of the viral genome are critical for productive infection. The size of the HIV-1 capsid is generally believed to exceed the diameter of the nuclear pore complex (NPC), indicating that capsid uncoating has to occur prior to nuclear import. Here, we combined correlative light and electron microscopy with subtomogram averaging to capture the structural status of reverse transcription-competent HIV-1 complexes in infected T cells. We demonstrated that the diameter of the NPC in cellulo is sufficient for the import of apparently intact, cone-shaped capsids. Subsequent to nuclear import, we detected disrupted and empty capsid fragments, indicating that uncoating of the replication complex occurs by breaking the capsid open, and not by disassembly into individual subunits. Our data directly visualize a key step in HIV-1 replication and enhance our mechanistic understanding of the viral life cycle. - doi.org/10.1016/j.cell.2021.01.025 - 2021
DNA-templated synthesis takes advantage of the programmability of DNA-DNA interactions to accelerate chemical reactions under diluted conditions upon sequence-specific hybridization. While this strategy has proven advantageous for a variety of applications, including sensing and drug discovery, it has been so far limited to the use of nucleic acids as templating elements. Here, we report the rational design of DNA templated synthesis controlled by specific IgG antibodies. Our approach is based on the co-localization of reactants induced by the bivalent binding of a specific IgG antibody to two antigen-conjugated DNA templating strands that triggers a chemical reaction that would be otherwise too slow under diluted conditions. This strategy is versatile, orthogonal and adaptable to different IgG antibodies and can be employed to achieve the targeted synthesis of clinically-relevant molecules in the presence of specific IgG biomarker antibodies. - doi.org/10.1038/s41467-020-20024-3 - 2020
Single-stranded guanine-rich DNA sequences can fold into four-stranded DNA structures called G-quadruplexes (G4s) that arise from the self-stacking of two or more guanine quartets. There has been considerable recent progress in the detection and mapping of G4 structures in the human genome and in biologically relevant contexts. These advancements, many of which align with predictions made previously in computational studies, provide important new insights into the functions of G4 structures in, for example, the regulation of transcription and genome stability, and uncover their potential relevance for cancer therapy. - doi:10.1038/nrm.2017.3 - 2017
In recent years, long noncoding RNAs (lncRNAs) have emerged as multifaceted regulators of gene expression, controlling key developmental and disease pathogenesis processes. However, due to the paucity of lncRNA loss-of-function mouse models, key questions regarding the involvement of lncRNAs in organism homeostasis and (patho)-physiology remain difficult to address experimentally in vivo. The clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 platform provides a powerful genome-editing tool and has been successfully applied across model organisms to facilitate targeted genetic mutations, including Caenorhabditis elegans, Drosophila melanogaster, Danio rerio and Mus musculus. However, just a few lncRNA-deficient mouse lines have been created using CRISPR/Cas9-mediated genome engineering, presumably due to the need for lncRNA-specific gene targeting strategies considering the absence of open-reading frames in these loci. Here, we describe a step-wise procedure for the generation and validation of lncRNA loss-of-function mouse models using CRISPR/Cas9-mediated genome engineering. In a proof-of-principle approach, we generated mice deficient for the liver-enriched lncRNA Gm15441, which we found downregulated during development of metabolic disease and induced during the feeding/fasting transition. Further, we discuss guidelines for the selection of lncRNA targets and provide protocols for in vitro single guide RNA (sgRNA) validation, assessment of in vivo gene-targeting efficiency and knockout confirmation. The procedure from target selection to validation of lncRNA knockout mouse lines can be completed in 18–20 weeks, of which
Circular dichroism (CD) spectroscopy is a widely-used method in biochemistry, structural biology and pharmaceutical chemistry. More than 24000 papers published in the past decade have included CD characterisations of proteins; many of those studies have also included other complementary chemical, biophysical, and computational chemistry methods. This tutorial review describes the background to the technique of CD spectroscopy and good practice methods for high quality data collection. It specifically focuses on both established and new methods and tools available for experimental design and interpretation, data processing, visualisation, analysis, validation, archiving, and accession, including tools developed to enhance the complementarity of this method with other structural and chemical biology studies. - DOI: 10.1039/d0cs00558d - 2021
Monocytes are part of the mononuclear phagocytic system. Monocytes play a central role during inflammatory conditions and a better understanding of their dynamics might open therapeutic opportunities. In the present study, we focused on the characterization and impact of monocytes on brown adipose tissue (BAT) functions during tissue remodeling. Single-cell RNA sequencing analysis of BAT immune cells uncovered a large diversity in monocyte and macrophage populations. Fate-mapping experiments demonstrated that the BAT macrophage pool requires constant replenishment from monocytes. Using a genetic model of BAT expansion, we found that brown fat monocyte numbers were selectively increased in this scenario. This observation was confirmed using a CCR2-binding radiotracer and positron emission tomography. Importantly, in line with their tissue recruitment, blood monocyte counts were decreased while bone marrow hematopoiesis was not affected. Monocyte depletion prevented brown adipose tissue expansion and altered its architecture. Podoplanin engagement is strictly required for BAT expansion. Together, these data redefine the diversity of immune cells in the BAT and emphasize the role of monocyte recruitment for tissue remodeling. - doi.org/10.1038/s41467-021-25616-1 - 2021
The ribosome is an ancient molecular fossil that provides a telescope to the origins of life. Made from RNA and protein, the ribosome translates mRNA to coded protein in all living systems. Universality, economy, centrality and antiquity are ingrained in translation. The translation machinery dominates the set of genes that are shared as orthologues across the tree of life. The lineage of the translation system defines the universal tree of life. The function of a ribosome is to build ribosomes; to accomplish this task, ribosomes make ribosomal proteins, polymerases, enzymes, and signaling proteins. Every coded protein ever produced by life on Earth has passed through the exit tunnel, which is the birth canal of biology. During the root phase of the tree of life, before the last common ancestor of life (LUCA), exit tunnel evolution is dominant and unremitting. Protein folding coevolved with evolution of the exit tunnel. The ribosome shows that protein folding initiated with intrinsic disorder, supported through a short, primitive exit tunnel. Folding progressed to thermodynamically stable β- structures and then to kinetically trapped α-structures. The latter were enabled by a long, mature exit tunnel that partially offset the general thermodynamic tendency of all polypeptides to form β-sheets. RNA chaperoned the evolution of protein folding from the very beginning. The universal common core of the ribosome, with a mass of nearly 2 million Daltons, was finalized by LUCA. The ribosome entered stasis after LUCA and remained in that state for billions of years. Bacterial ribosomes never left stasis. Archaeal ribosomes have remained near stasis, except for the superphylum Asgard, which has accreted rRNA post LUCA. Eukaryotic ribosomes in some lineages appear to be logarithmically accreting rRNA over the last billion years. Ribosomal expansion in Asgard and Eukarya has been incremental and iterative, without substantial remodeling of pre-existing basal structures. The ribosome preserves information on its history. - dx.doi.org/10.1021/acs.chemrev.9b00742 - 2020
Epigenetic programming of cells requires methylation of deoxycytidines (dC) to 5-methyl-dC (mdC) followed by oxidation to 5-hydroxymethyl-dC (hmdC), 5-formyl-dC (fdC), and 5-carboxy-dC (cadC). Subsequent transformation of fdC and cadC back to dC by various pathways establishes a chemical intra-genetic control circle. One of the discussed pathways involves the Tdg-independent deformylation of fdC directly to dC. Here we report the synthesis of a fluorinated fdC feeding probe (F-fdC) to study direct deformylation to F-dC. The synthesis was performed along a novel pathway that circumvents any F-dC as a reaction intermediate to avoid contamination interference. Feeding of F-fdC and observation of F-dC formation in vivo allowed us to gain insights into the Tdg-independent removal process. While deformylation was shown to occur in stem cells, we here provide data that prove deformylation also in different somatic cell types. We also investigated active demethylation in a non-dividing neurogenin-inducible system of iPS cells that differentiate into bipolar neurons. - doi.org/10.1002/anie.202107089. - 2021
Advanced spatiotemporal control of CRISPR-Cas9 activity is demonstrated through the use of chemically modified photoactivatable guide RNA. - doi.org/10.1021/acscentsci.0c00350 - 2020
P. Shing Ho and his colleagues at Oregon State and Berkeley publish in this issue of PNAS an interesting study (1) of helical structure in the DNA hexamer GGCGCC, finding that various states that appear to be logical intermediates between A-DNA and B-DNA can be induced by methylation or bromination of cytosine or by crystal packing. Their results bear on three issues that have been argued over in the past: (i) the differences between A-DNA and B- DNA and transitions between them, (ii) the intrinsic sequence-dependent malleability of a DNA duplex, and (iii) the effects of local helix packing on DNA fine-structure. - doi-10.1073-pnas.141238898 - 2001
In 1595, French anatomist Andre du Laurens first described the structure of a rope-like tail of fibers at the caudal end of the spinal cord. This bundle of numerous axons was termed the cauda equina, from the Latin translation meaning “horse’s tail,” and it contains nerves which innervate both sensory and motor targets within lumbar, sacral, and coccygeal spinal cord levels. Epidemiologic assessments regard lesions to the cauda equina as uncommon, with a prevalence of 1 to 3 per 100,000 subjects. Typically caused by a herniated intervertebral disc at the L5-S1 levels, such lesions affect females as often as males and manifest as a number of urogenital and neuromuscular symptoms in the namesake “cauda equina syndrome." - StatPearls Publishing -2021
According to guidelines, it is safe for low-risk patients with myocardial infarction (MI) to be discharged within 72 h of hospitalization. However, results coming from registries show that the hospital stay is often much longer in a real-life situation. Data on the length of the hospital stay (LOS) of MI patients in Polish centers are lacking. We enrolled 212 consecutive patients with acute MI. Low-risk patients were defined according to PAMI II criteria: age 45%, no persistent ventricular arrhythmia, and no multi-vessel disease (MVD). The median of the hospitalization length was eight days (Q1: 6; Q3: 9). In low-risk patients (25%), the median of LOS was six days (Q1: 5; Q3: 7) (p < 0.001). In a logistic regression analysis patients age, LVEF, ST-segment-elevation MI and the presence of MVD were independent predictors of longer hospitals stay (≥8 days). During follow up, there were no significant differences in the rates of clinical events between patients with shorter (
Many bacterial and archaeal organisms use CRISPR-Cas (clustered regularly interspaced short palindromic repeats-CRISPR associated) systems to defend themselves from mobile genetic elements. These CRISPR-Cas systems are classified into six types based on their composition and mechanism. CRISPR-Cas enzymes are widely used for genome editing and offer immense therapeutic opportunity to treat genetic diseases. To realize their full potential, it is important to control the timing, duration, efficiency and specificity of CRISPR-Cas enzyme activities. In this review we discuss the mechanisms of natural CRISPR-Cas regulatory biomolecules that enhance or inhibit CRISPR-Cas immunity by altering enzyme function. We also discuss the potential applications of these CRISPR regulators and highlight unanswered questions about their evolution and purpose in nature. - doi:10.1038/s41589-020-00700-7 - 2021
Antisense oligonucleotides can regulate gene expression in living cells. As such, they regulate cell function and division, and can modulate cellular responses to internal and external stresses and stimuli. Although encouraging results from preclinical and clinical studies have been obtained and significant progress has been made in developing these agents as drugs, they are not yet recognized as effective therapeutics. Several major hurdles remain to be overcome, including problems with efficacy, off-target effects, delivery and side effects. The lessons learned from antisense drug development can help in the development of other oligonucleotide-based therapeutics such as CpG oligonucleotides, RNAi and miRNA. -doi:10.1016/j.drudis.2008.03.014 - 2008
The temporal dynamics of cancer evolution remain elusive, because it is impractical to longitudinally observe cancers unperturbed by treatment. Consequently, our knowledge of how cancers grow largely derives from inferences made from a single point in time – the endpoint in the cancer’s evolution, when it is removed from the body and studied in the laboratory. Fortuitously however, the cancer genome, by virtue of ongoing mutations that uniquely mark clonal lineages within the tumour, provides a rich, yet surreptitious, record of cancer development. In this review, we describe how a cancer’s genome can be analysed to reveal the temporal history of mutation and selection, and discuss why both selective and neutral evolution feature prominently in carcinogenesis. We argue that selection in cancer can only be properly studied once we have some understanding of what the absence of selection looks like. We review the data describing punctuated evolution in cancer, and reason that punctuated phenotype evolution is consistent with both gradual and punctuated genome evolution. We conclude that, to map and predict evolutionary trajectories during carcinogenesis, it is critical to better understand the relationship between genotype change and phenotype change.- DOI: 10.1002/path.4821 - 2016
The successful introduction of antibody-based protein therapeutics into the arsenal of treatments for patients has within a few decades fostered intense innovation in the production and engineering of antibodies. Reviewed here are the methods currently used to produce antibodies along with how our knowledge of the structural and functional characterization of immunoglobulins has resulted in the engineering of antibodies to produce protein therapeutics with unique properties, both biological and biophysical, that are leading to novel therapeutic approaches. Antibody engineering includes the introduction of the antibody combining site (variable regions) into a host of architectures including bi and multi-specific formats that further impact the therapeutic properties leading to further advantages and successes in patient treatment. - http://dx.doi.org/10.1016/j.sbi.2016.07.012 - 2016
Crohn’s disease is an autoimmune disease that predominantly affects the gastrointestinal tract. Crohn’s disease is diagnosed at a young age and runs a chronic course with acute flare-ups. When patients with Crohn’s disease present with flare-ups at the emergency department, they are usually managed in a way similar to patients with acute abdomen; there is no consensus about the most appropriate imaging work-up for patients with flare-ups of Crohn’s disease. Thus, we decided to review the literature about the imaging tests indicated (whether related to their diagnostic performance or to lower exposure to ionizing radiation) for acute flare-ups in patients with Crohn’s disease. - DOI: 10.1016/j.rx.2018.12.003 - 2019
Herpes simplex virus type I (HSV-1) is the causative agent of several pathologies ranging in severity from the common cold sore to life-threatening encephalitic infection. During productive lytic infection, over 80 viral proteins are expressed in a highly regulated manner, resulting in the replication of viral genomes and assembly of progeny virions. The virion of all herpesviruses consists of an external membrane envelope, a proteinaceous layer called the tegument, and an icosahedral capsid containing the double-stranded linear DNA genome. The capsid shell of HSV-1 is built from four structural proteins: a major capsid protein, VP5, which forms the capsomers (hexons and pentons), the triplex consisting of VP19C and VP23 found between the capsomers, and VP26 which binds to VP5 on hexons but not pentons. In addition, the dodecameric pUL6 portal complex occupies one of the 12 capsid vertices, and the capsid vertex specific component (CVSC), a heterotrimer complex of pUL17, pUL25 and pUL36 binds specifically to the triplexes adjacent to each penton. The capsid is assembled in the nucleus where the viral genome is packaged into newly assembled closed capsid shells. Cleavage and packaging of replicated, concatemeric viral DNA requires the seven viral proteins encoded by the UL6, UL15, UL17, UL25, UL28, UL32, and UL33 genes. Considerable advances have been made in understanding the structure of the herpesvirus capsid and the function of several of the DNA packaging proteins by applying biochemical, genetic, and structural techniques. This review is a summary of recent advances with respect to the structure of the HSV-1 virion capsid and what is known about the function of the seven packaging proteins and their interactions with each other and with the capsid shell. - doi:10.1007/978-3-319-53168-7_6. - 2017
Platinum (Pt)-based anticancer drugs such as cisplatin have been used to treat various cancers. However, they have some limitations including poor selectivity and toxicity towards normal cells and increasing chemoresistance. Therefore, there is a need for novel metallo-anticancers, which has not been met for decades. Since the initial introduction of ruthenium (Ru) polypyridyl complex, a number of attempts at structural evolution have been conducted to improve efficacy. Among them, half-sandwich Ru-arene complexes have been the most prominent as an anticancer platform. Such complexes have clearly shown superior anticancer profiles such as increased selectivity toward cancer cells and ameliorating toxicity against normal cells compared to existing Pt-based anticancers. Currently, several Ru complexes are under human clinical trials. For improvement in selectivity and toxicity associated with chemotherapy, Ru complexes as photodynamic therapy (PDT), and photoactivated chemotherapy (PACT), which can selectively activate prodrug moieties in a specific region, have also been investigated. With all these studies on these interesting entities, new metallo- anticancer drugs to at least partially replace existing Pt-based anticancers are anticipated. This review covers a brief description of Ru-based anticancer complexes and perspectives. https://doi.org/10.2147/DDDT.S275007 - 2020
Background: Early reports indicate that AKI is common during COVID-19 infection. Different mortality rates of AKI due to SARS-CoV-2 have been reported, based on the degree of organic dysfunction and varying from public to private hospitals. However, there is a lack of data about AKI among critically ill patients with COVID-19.
Methods: We conducted a multicenter cohort study of 424 critically ill adults with severe acute respiratory syndrome (SARS) and AKI, both associated with SARS-CoV-2, admitted to six public ICUs in Brazil. We used multivariable logistic regression to identify risk factors for AKI severity and in-hospital mortality.
Results: The average age was 66.42±13.79years, 90.3% were on mechanical ventilation (MV), 76.6% were at KDIGO stage 3, and 79% underwent hemodialysis. The overall mortality was 90.1%. We found a higher frequency of dialysis (82.7% versus 45.2%), MV (95% versus 47.6%), vasopressors (81.2% versus 35.7%) (p
With the discovery in the late 1980s that the DNA-repair gene RAD6encodes a ubiquitin-conjugating enzyme, it became clear that protein modification by ubiquitin conjugation has a much broader significance than had previously been assumed. Now, two decades later, ubiquitin and its cousin SUMO are implicated in a range of human diseases, including breast cancer and Fanconi anaemia, giving fresh momentum to studies focused on the relationships between ubiquitin, SUMO and DNA-repair pathways. - NATURE - 2009 -. doi:10.1038/nature07963
Despite several critical steps forward in the management of chronic heart failure (CHF), the area of acute heart failure (AHF) has remained relatively stagnant. As stated in the updated ESC HF guidelines, clinicians responsible for managing patients with AHF must frequently make treatment decisions without adequate evidence, usually on the basis of expert opinion consensus.2 Specifically, the treatment of acute HF remains largely opinion-based with little good evidence to guide therapy. Acute heart failure is a syndrome in which emergency physicians, cardiologists, intensivists, nurses, and other healthcare providers have to cooperate to provide ‘rapid’ benefit to the patients. We hereby would like to underscore the wider experience grown in different settings of the area of intensive care on acute heart failure, actually larger and more composite than that got in specialized Care Units. The distillate of such different experiences is discussed and integrated in the present document. Hence, the authors of this consensus paper believe a common working definition of AHF covering all dimensions and modes of presentations has to be made, with the understanding that most AHF presentations are either acute decompensations of chronic underlying HF or the abrupt onset of dyspnoea associated with significantly elevated blood pressure. Secondly, recent data show that, much like acute coronary syndrome, AHF might have a ‘time to therapy’ concept. Accordingly, ‘pre-hospital’ management is considered a critical component of care. Thirdly, most patients with AHF have normal or high blood pressure at presentation and are admitted with symptoms and/or signs of congestion. This is in contradiction to the presentation where low cardiac output leads to symptomatic hypotension and signs/symptoms of hypoperfusion, a circumstance that is relatively rare, present in coronary care unit/ intensive care unit (CCU/ICU) but associated with a particularly poor outcome. Hence, it is important to note that appropriate therapy requires appropriate identification of the specific AHF pheno-type.3 The aim of the current paper is not to replace guidelines, but, to provide contemporary perspective for early hospital management within the context of the most recent data and to provide guidance, based on expert opinions, to practicing physicians and other healthcare professionals (Figure 1). We believe that the experience accrued in the different settings from the emergency department through to the ICU/CCU is collectivel valuable in determining how best to manage the patients with AHF. Herein, a shortened version mainly including group recommendations is provided. Full version of the consensus paper is provided as Supplementary material online. - Alexandre Mebazaa - http://onlinelibrary.wiley.com/doi/10.1002/ejhf.289/full - 2015
Since determination of the myoglobin structure in 1957, X-ray crystallography, as the anchoring tool of structural biology, has played an instrumental role in deciphering the secrets of life. Knowledge gained through X-ray crystallography has fundamentally advanced our views on cellular processes and greatly facilitated development of modern medicine. In this brief narrative, I describe my personal understanding of the evolution of structural biology through X-ray crystallography—using as examples mechanistic understanding of protein kinases and integral membrane proteins—and comment on the impact of technological development and outlook of X-ray crystallography - dx.doi.org/10.1016/j.cell.2014.10.051 - 2014
Modified nucleic acids, also called xeno nucleic acids (XNAs), offer a variety of advantages for biotechnological applications and address some of the limitations of first-generation nucleic acid therapeutics. Indeed, several therapeutics based on modified nucleic acids have recently been approved and many more are under clinical evaluation. XNAs can provide increased biostability and furthermore are now increasingly amenable to in vitro evolution, accelerating lead discovery. Here, we review the most recent discoveries in this dynamic field with a focus on progress in the enzymatic replication and functional exploration of XNAs. K. Duffy, S. Arangundy-Franklin and P. Holliger -doi.org/10.1186/s12915-020-00803-6 - 2020
Genome editing employs targeted nucleases as powerful tools to precisely alter the genome of target cells and regulate functional genes. Various strategies have been risen so far as the molecular scissors-mediated genome editing that includes zinc finger nuclease, transcription activator-like effector nucleases, and clustered regularly interspaced short palindromic repeats—CRISPR-related protein 9. These tools allow researchers to understand the basics of manipulating the genome, create animal models to study human diseases, understand host–pathogen interactions and design disease targets. Targeted genome modification utilizing RNA-guided nucleases are of recent curiosity, as it is a fast and effective strategy that enables the researchers to manipulate the gene of interest, carry out functional studies, understand the molecular basis of the disease and design targeted therapies. CRISPR-Cas9, a bacterial defense system employed against viruses, consists of a single-strand RNA-guided Cas9 nuclease connected to the corresponding complementary target sequence. This powerful and versatile tool has gained tremendous attention among the researchers, owing to its ability to correct genetic disorders. To help illus- trate the potential of this gene editor in unexplored corners of oncology, we describe the history of CRISPR-Cas9, its rapid progression in cancer research as well as future perspectives. - P Daisy, K Shreyas, T. S. Anitha -doi.org/10.1007/s12033-020-00289-1 - 2021
Telomeres are DNA-protein complexes that cap and protect the ends of linear chromosomes. In almost all species, telomeric DNA has a G/C strand bias, and the short tandem repeats of the G-rich strand have the capacity to form into secondary structures in vitro, such as four-stranded G-quadruplexes. This has long prompted speculation that G-quadruplexes play a positive role in telomere biology, resulting in selection for G-rich tandem telomere repeats during evolution. There is some evidence that G-quadruplexes at telomeres may play a protective capping role, at least in yeast, and that they may positively affect telomere maintenance by either the enzyme telomerase or by recombination-based mechanisms. On the other hand, G-quadruplex formation in telomeric DNA, as elsewhere in the genome, can form an impediment to DNA replication and a source of genome instability. This review summarizes recent evidence for the in vivo existence of G-quadruplexes at telomeres, with a focus on human telomeres, and highlights some of the many unanswered questions regarding the location, form, and functions of these structures. - Tracy Bryan - doi:10.3390/molecules25163686 - 2020
DNA self-assembly has proved to be a useful bottom- up strategy for the construction of user-defined nanoscale objects, lattices and devices. The design of these structures has largely relied on exploiting simple base-pairing rules and the formation of double-helical domains as secondary structural elements. However, other helical forms involving specific non-canonical base-base interactions have introduced a novel paradigm into the process of engineering with DNA. The most notable of these is a three-stranded complex generated by the binding of a third strand within the duplex major groove, generating a triple-helical (‘triplex’) structure. The sequence, structural and assembly requirements that differentiate triplexes from their duplex counterparts has allowed the design of nanostructures for both dynamic and/or structural purposes, as well as a means to target non-nucleic acid components to precise locations within a nanostructure scaffold. Here, we review the properties of triplexes that have proved useful in the engineering of DNA nanostructures, with an emphasis on applications that hitherto have not been possible by duplex formation alone. Arun Chandrasekaran, David Rusling - doi: 10.1093/nar/gkx1230 - 2018
There are three major families of DNA helices: A-DNA, B-DNA and Z-DNA. The helical
structure of DNA is variable and depends on the sequence as well as the environment. David W Ussery
We show that groove recognition of nucleic acid triple helices can be achieved with aminosugars. Among these aminosugars, neomycin is the most effective aminoglycoside (groove binder) for stabilizing a DNA triple helix. It stabilizes both the T·A·T triplex and mixed-base DNA triplexes better than known DNA minor groove binders (which usually destabilize the triplex) and polyamines. Neomycin selectively stabilizes the triplex (T·A·T and mixed base) without any effect on the DNA duplex. The selectivity of neomycin likely originates from its potential and shape complementarity to the triplex Watson–Hoogsteen groove, making it the first molecule that selectively recognizes a triplex groove over a duplex groove. The groove recognition of aminoglycosides is not limited to DNA triplexes, but also extends to RNA and hybrid triple helical structures. Intercalator–neomycin conjugates are shown to simultaneously probe the base stacking and groove surface in the DNA triplex. Calorimetric and spectrosocopic studies allow the quantification of the effect of surface area of the intercalating moiety on binding to the triplex. These studies outline a novel approach to the recognition of DNA triplexes that incorporates the use of non-competing binding sites. These principles of dual recognition should be applicable to the design of ligands that can bind any given nucleic acid target with nanomolar affinities and with high selectivity. Dev P. Arya
Molecular Structure of Nucleic Acids - A structure for Deoxyribose Nucleic Acid - We wish to suggest astructure for the salt of deoxyribose nucleic acid (D.N.A.). This structure has novel features which are of considerable biological interest. J. D. Watson, F. H. C Crick.