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Metrics play a vital part in the valuation and funding of research for scientists worldwide. We review the challenges that metrics pose in providing a fair and equitable system for research funding. We highlight the attempts with declarations, including the San Francisco Declaration on Research Assessment (SF‐DORA), to improve the research environment and specific impacts that metric choice can have on the evaluation and progression of Early Career Lecturers (ECLs). While there is much evidence that metrics will never be entirely satisfactory, we conclude there are opportunities that would benefit ECLs and reason for optimism for researchers. [ABSTRACT FROM AUTHOR]

The FEBS Journal publishes primary papers as well as reviews in the molecular life sciences relating to the molecules and mechanisms underpinning biological processes. Editor‐in‐Chief Seamus Martin shares some thoughts on the nature of conducting research, some highlights of the past year at the journal, and what is in store for 2025. [ABSTRACT FROM AUTHOR]

This editorial highlights and celebrates the winner of the 2024 Richard Perham prize. This was selected from shortlisted original articles that were published in The FEBS Journal in 2023 and received prize nominations from the Editorial Board. The winning paper, by Matteo Brindisi, Luca Frattaruolo, Federica Sotgia, Michael P Lisanti, Anna Rita Cappello and colleagues, shows how high cholesterol levels promote breast cancer aggressiveness. [ABSTRACT FROM AUTHOR]

Human metallothioneins (MTs) are involved in binding the essential elements, Cu(I) and Zn(II), and the toxic element, Cd(II), in metal‐thiolate clusters using 20 reduced cysteines. The brain‐specific MT3 binds a mixture of Cu(I) and Zn(II) in vivo. Its metallation properties are critically important because of potential connections between Cu, Zn and neurodegenerative diseases. We report that the use of isotopically pure 63Cu(I) and 68Zn(II) greatly enhances the element resolution in the ESI‐mass spectral data revealing species with differing Cu:Zn ratios but the same total number of metals. Room temperature phosphorescence and circular dichroism spectral data measured in parallel with ESI‐mass spectral data identified the presence of specific Cu(I)‐thiolate clusters in the presence of Zn(II). A series of Cu(I)‐thiolate clusters form following Cu(I) addition to apo MT3: the two main clusters that form are a Cu6 cluster in the β domain followed by a Cu4 cluster in the α domain. 63Cu(I) addition to 68Zn7‐MT3 results in multiple species, including clustered Cu5Zn5‐MT3 and Cu9Zn3‐MT3. We assign the domain location of the metals for Cu5Zn5‐MT3 as a Cu5Zn1‐β cluster and a Zn4‐α cluster and for Cu9Zn3‐MT3 as a Cu6‐β cluster and a Cu3Zn3‐α cluster. While many reports of the average MT3 metal content exist, determining the exact Cu,Zn stoichiometry has proven very difficult even with native ESI‐MS. The work in this paper solves the ambiguity introduced by the overlap of the naturally abundant Cu(I) and Zn(II) isotopes. Contrary to other reports, there is no indication of a major fraction of Cu4‐β‐Znn‐α‐MT3 forming. [ABSTRACT FROM AUTHOR]

John F. Cryan is Chair of the Department of Anatomy and Neuroscience, Principal Investigator in the APC Microbiome Ireland Institute and Vice President for Research and Innovation at University College of Cork, Ireland. He obtained his bachelor's and doctoral degrees (in Biochemistry and Pharmacology, respectively) from the University of Galway and undertook postdoctoral research experience in the USA, at the University of Pennsylvania and The Scripps Research Institute. He was also a visiting fellow at the Department of Psychiatry, University of Melbourne, for a couple of years post‐PhD. In 2002, John took up a position in industry as a laboratory head at the Novartis Institutes for BioMedical Research in Basel, Switzerland. After a 4‐year stint at Novartis, he returned to academia in late 2005 as a Lecturer and then Senior Lecturer in Pharmacology at University College Cork (UCC). The Cryan lab now focuses on determining the impact of the gut microbiota on the human brain and behaviour, with a particular interest in the brain–gut–microbiome axis in the context of depression. John has published more than 600 peer‐reviewed papers and has received many honours and accolades in recognition of his contributions to neuropharmacology and microbiome research, including the Datta lecture award from FEBS in 2022. In this interview, he outlines how he became interested in the role of the microbiome in brain development and disease, provides advice to budding scientists and highlights the broader public health implications of his research. [ABSTRACT FROM AUTHOR]

B Cover Illustration b Dissected trachea from hypoxia-treated wild-type (blue) and LUBEL-mutant (white) I Drosophila i larvae. M1-ubiquitination is induced in response to hypoxia in wild-type flies. Read the paper by Annika Meinander and co-authors in this issue (pages 5180-5197). [Extracted from the article]

One of the hottest topics in biomedical research is to decipher the functional implications of the Gasdermin (GSDM) protein family in human pathologies. These proteins are the key effectors of a lytic and pro‐inflammatory cell death type termed pyroptosis (also known as "Gasdermin‐mediated programmed cell death"). However, ever‐growing evidence showed that GSDMs can play multiple and complex roles in a context‐dependent manner. In this sense, Gasdermin‐B (GSDMB; the only GSDM gene absent in mice and rats) has been implicated in antibacterial defense, numerous inflammatory pathologies (e.g., asthma, ulcerative colitis), and cancer, but both cell death‐dependent and ‐independent functions have been reported in these diseases, fueling the debate on whether GSDMB has genuine pyroptotic capacity. Recently, a series of seminal papers cast light on the GSDMB multitasking capacity by showing that different GSDMB transcriptional isoforms have distinct biological activities. Nonetheless, there are still obscure areas to be clarified on the precise functional involvement of GSDMB translated variants in physiological and pathological conditions. In this viewpoint, we critically discuss the most recent and exciting data on this topic and propose a series of relevant challenges that need to be overcome before GSDMB‐driven biomedical applications (as a biomarker of disease risk/progression/outcome or as specific therapeutic target) become a reality in clinical settings. [ABSTRACT FROM AUTHOR]

Single‐cell RNA sequencing (scRNA‐seq) is a technique that has proven to be a powerful tool for a wide range of fields and research studies. However, scRNA‐seq data analysis has been dominated by scientists highly trained in bioinformatics or those with extensive computational experience and understanding. Recently, this trend has begun to shift as more user‐friendly web‐based scRNA‐seq analysis tools have been developed that require little computational experience to use. However, barriers persist for nonbioinformaticians in using this technique. Complex, unfamiliar language and scarce comprehensive literature guidance to provide a framework for understanding scRNA‐seq analysis outputs are among the obstacles. This work introduces many popular web‐based tools for scRNA‐seq and provides a general overview of their user interfaces and features. Then, a comprehensive start‐to‐finish introductory scRNA‐seq analysis pipeline is described in detail, which aims to enable researchers to carry out scRNA‐seq analysis, regardless of computational experience. Companion video tutorials can be found at "EasyScRNAseqTutorials" on YouTube (https://www.youtube.com/@scrnaseqtutorials). However, as scRNA‐seq continues to penetrate new fields and expand in importance, there remains a need for more literature to help overcome barriers to its use by explaining further the highly complex and advanced analyses that are introduced within this paper. [ABSTRACT FROM AUTHOR]

The document is the issue information for Volume 291 Number 9 of the FEBS Journal, published in May 2024. It includes the editorial board members, guidelines for authors, and the scope of the journal. The journal focuses on publishing high-quality papers in the molecular life sciences and welcomes interdisciplinary research. The issue also includes state-of-the-art reviews, mini-reviews, commentaries, and original articles on various topics in biochemistry, molecular biology, and other related fields. [Extracted from the article]

Protein coding mutations in leucine‐rich repeat kinase 2 (LRRK2) cause familial Parkinson's disease (PD), and noncoding variations around the gene increase the risk of developing sporadic PD. It is generally accepted that pathogenic LRRK2 mutations increase LRRK2 kinase activity, resulting in a toxic hyperactive protein that is inferred to lead to the PD phenotype. LRRK2 has long been linked to different membrane trafficking events, but the specific role of LRRK2 in these events has been difficult to resolve. Recently, several papers have reported the activation and translocation of LRRK2 to cellular organelles under specific conditions, which suggests that LRRK2 may influence intracellular membrane trafficking. Here, we review what is known about the role of LRRK2 at various organelle compartments. [ABSTRACT FROM AUTHOR]

Andrew Wyllie was a key figure in the discovery of the programmed cell death process termed apoptosis. He pioneered the study of apoptosis when this was a little‐known phenomenon and discovered the first biochemical marker of the process, internucleosomal DNA degradation. Andrew recently passed away, aged 78, but leaves a legacy of scientific achievement that opened up a new field of research. [ABSTRACT FROM AUTHOR]

Auxiliary sections that support the reader's understanding of the review, such as keywords and glossaries, are also useful to include, and author contributions and any conflicts of interest should be clearly outlined, usually in dedicated sections of the review. This shouldn't preclude early career researchers from writing review articles, with the mentorship of experienced colleagues - gaining experience in writing review articles early on in your career is a great way to hone your analytical and writing skills. Literature reviews are valuable resources for the scientific community. The advice given here is mostly relevant for the writing of a traditional literature-based review rather than other forms of review such as a systematic review or meta-analysis, which have their own criteria and guidelines [2,3]. [Extracted from the article]

The FEBS Journal publishes research on diverse topics in the molecular life sciences relating to the molecules and mechanisms underpinning biological processes. Editor‐in‐Chief Seamus Martin discusses the rewards of pursuing a career in basic research, some highlights of the past year at the journal, and what's in store for 2024. [ABSTRACT FROM AUTHOR]

The FEBS Journal publishes high impact research on diverse topics in the life sciences, with a focus on molecular mechanisms underpinning biological processes. Here, Editor‐in‐Chief Seamus Martin discusses highlights of the journal from the past year and gives a glimpse into what's in store for 2023. [ABSTRACT FROM AUTHOR]

Lori Passmore is a Group Leader at the MRC Laboratory of Molecular Biology (MRC‐LMB). She studied Biochemistry at the University of British Columbia in Vancouver (Canada), before moving to the UK in 1999 for a PhD at the Institute of Cancer Research. After completing her PhD, Lori moved to Cambridge, where she became a Post‐Doctoral Fellow at the MRC‐LMB. In 2009, Lori started her own group at the MRC‐LMB and was subsequently awarded an ERC Starting Grant (2011), an ERC Consolidator Grant (2017) and a Wellcome Discovery Award (2023). She was also elected into the EMBO Young Investigator Programme (2015) and EMBO Membership (2018). Lori's research focusses on the determination of the structures of protein complexes that regulate gene expression, using primarily cryo‐electron microscopy and in vitro assays. Her work has contributed significantly to our understanding of the underlying molecular mechanisms of cellular processes, giving insights into human physiology and disease. In this interview, Lori provides an overview of her research and discusses current challenges in the field, recalls the key events and collaborations that have helped shape her successful research career and offers advice to early career scientists. [ABSTRACT FROM AUTHOR]

Dame Carol Robinson is a professor of chemistry and Director of the Kavli Institute for Nanoscience Discovery at the University of Oxford. Carol's career in science began at the age of 16 as a lab technician at Pfizer (based in Kent), during which time she studied part‐time and took evening classes to obtain a degree in chemistry. This was followed by a master's degree at the University of Swansea and a PhD at the University of Cambridge. Carol's postdoctoral training was undertaken in Peter Bennett's lab at the Department of Pathology and Microbiology, University of Bristol. Subsequently, she took a career break of 8 years to spend time with her family but made a strong comeback, taking up a position at the University of Oxford, where she began to explore protein folding. It was here that she first demonstrated, using the GroEL chaperonin−substrate complex as a prototype, that protein secondary structure can be analysed in the gas phase. Carol later made history as the first female professor of chemistry at the University of Cambridge (2001) and again as the first female professor of chemistry at the University of Oxford (2009). In her research, she has also continuously pushed boundaries, pioneering the application of mass spectrometry for the elucidation of the 3D architecture of macromolecular complexes, including membrane‐bound assemblies. She has received many awards and honours in recognition of her significant contributions to the field of gas‐phase structural biology, including the Royal Society Fellowship, the Davy Medal, the Rosalind Franklin Award and the FEBS/EMBO Women in Science Award. In this interview, she discusses some of her career highlights and ongoing research aspirations and draws on her unique experiences to offer advice to early career scientists. [ABSTRACT FROM AUTHOR]

RETRACTION: H. Li, R. Yan, W. Chen, X. Ding, J. Liu, G. Chen, Q. Zhao, Y. Tang, S. Lv, S. Liu, and Y. Yu, "Long Non Coding RNA SLC26A4‐AS1 Exerts Antiangiogenic Effects in Human Glioma by Upregulating NPTX1 Via NFKB1 Transcriptional Factor," The FEBS Journal 288, no. 1 (2021): 212–228, https://doi.org/10.1111/febs.15325. The above article, published online on July 15, 2020, in Wiley Online Library (wileyonlinelibrary.com), has been retracted by agreement between the journal Editor‐in‐Chief; the Federation of European Biochemical Societies; and John Wiley & Sons Ltd. The retraction has been agreed upon following an investigation into concerns raised by a third party, which revealed inappropriate image duplication between this article (Fig. 1E) and another article previously published elsewhere by a different group of authors in a different scientific context. The authors were unable to provide a satisfactory explanation, and the partial raw data they supplied could not explain the identified issues. Consequently, the editors have lost confidence in the presented data and decided to retract the paper. [ABSTRACT FROM AUTHOR]

In this special interview series, we profile members of The FEBS Journal editorial board to highlight their research focus, perspectives on the journal and future directions in their field. Rudi Beyaert is Full Professor in Molecular Biology at the Department of Biomedical Molecular Biology, Faculty of Sciences, of the University of Ghent (Belgium). He also serves as Vice‐Science Director of the Center for Inflammation Research of the VIB in Ghent, where he is heading the Unit of Molecular Signal Transduction in Inflammation. He has served as an Editorial Board Member of The FEBS Journal since 2016. [ABSTRACT FROM AUTHOR]

In this special interview series, we profile members of The FEBS Journal editorial board to highlight their research focus, perspectives on the journal and future directions in their field. Manuela Baccarini is Professor of Cell Signaling at the University of Vienna, Coordinator of the International PhD Program 'Signaling Mechanisms in Cellular Homeostasis' and Director of the Vienna BioCenter PhD Program, a graduate school of the University and Medical University of Vienna in collaboration with the Institute of Molecular Pathology and the Austrian Academy of Sciences, Institute for Medical Biotechnology and Gregor Mendel Institute, as well as EMBO member and corresponding member of the Austrian Academy of Sciences. She has served as an editorial board member of The FEBS Journal since 2016. [ABSTRACT FROM AUTHOR]

Corrigendum to: "hhLIM Is a novel F-Actin binding protein involved in Actin cytoskeleton remodelling" by bin Zheng, Jin-Kun Wen and Mei Han. Extracts from C2C12 cells were precleared with GST-Sepharose beads and then incubated with GST-hhLIM Sepharose beads or its derivative proteins. Western blot of GST pull-down assay fractions using an actin antibody showing similar amounts of actin in samples with GST Sepharose versus GST-tagged hhLIM. [Extracted from the article]

Acetogenic bacteria such as the thermophilic anaerobic model organism Moorella thermoacetica reduce CO2 with H2 as a reductant via the Wood‐Ljungdahl pathway (WLP). The enzymes of the WLP of M. thermoacetica require NADH, NADPH, and reduced ferredoxin as reductants. Whereas an electron‐bifurcating ferredoxin‐ and NAD+‐reducing hydrogenase HydABC had been described, the enzyme that reduces NADP+ remained to be identified. A likely candidate is the HydABCDEF hydrogenase from M. thermoacetica. Genes encoding for the HydABCDEF hydrogenase are expressed during growth on glucose and dimethyl sulfoxide (DMSO), an alternative electron acceptor in M. thermoacetica, whereas expression of the genes hydABC encoding for the electron‐bifurcating hydrogenase is downregulated. Therefore, we have purified the hydrogenase from cells grown on glucose and DMSO to apparent homogeneity. The enzyme had six subunits encoded by hydABCDEF and contained 58 mol of iron and 1 mol of FMN. The enzyme reduced methyl viologen with H2 as reductant and of the physiological acceptors tested, only NADP+ was reduced. Electron bifurcation with pyridine nucleotides and ferredoxin was not observed. H2‐dependent NADP+ reduction was optimal at pH 8 and 60 °C; the specific activity was 8.5 U·mg−1 and the Km for NADP+ was 0.086 mm. Cell suspensions catalyzed H2‐dependent DMSO reduction, which is in line with the hypothesis that the NADP+‐reducing hydrogenase HydABCDEF is involved in electron transfer from H2 to DMSO. [ABSTRACT FROM AUTHOR]

Hydroxymethylbilane synthase (HMBS), involved in haem biosynthesis, catalyses the head‐to‐tail coupling of four porphobilinogens (PBGs) via a dipyrromethane (DPM) cofactor. DPM is composed of two PBGs, and a hexapyrrole is built before the tetrapyrrolic 1‐hydroxymethylbilane product is released. During this elongation, stable enzyme (E) intermediates are formed from the holoenzyme, with additional PBG substrates (S): ES, ES2, ES3 and ES4. Native PAGE and mass spectrometry of the acute intermittent porphyria (AIP)‐associated HMBS variant p.Arg167Gln demonstrated an increased amount of ES3. Kinetic parameters indicated catalytic dysfunction, however, the product release was not entirely prevented. Isolation and crystal structure analysis of the ES3 intermediate (PDB: 8PND) showed that a pentapyrrole was fully retained within the active site, revealing that polypyrrole elongation proceeds within the active site via a third interaction site, intermediate pyrrole site 3 (IPS3). The AIP‐associated HMBS variant p.Arg195Cys, located on the opposite side to p.Arg167Gln in the active site, accumulated the ES4 intermediate in the presence of excess PBG, implying that product hydrolysis was obstructed. Arg167 is thus involved in all elongation steps and is a determinant for the rate of enzyme catalysis, whereas Arg195 is important for releasing the product. Moreover, by substituting residues in the vicinity of IPS3, our results indicate that a fully retained hexapyrrole could be hydrolysed in a novel site in proximity of the IPS3. [ABSTRACT FROM AUTHOR]

CC and CXC chemokines are distinct chemokine subfamilies. CC chemokines usually do not bind CXC‐chemokine receptors and vice versa. CCR5 and CXCR4 receptors are activated by CCL5 and CXCL12 chemokines, respectively, and are also used as HIV‐1 coreceptors. CCL5 contains one conserved binding site for a sulfated tyrosine residue, whereas CXCL12 is unique in having two additional sites for sulfated/nonsulfated tyrosine residues. In this study, N‐terminal (Nt) CXCR4 peptides were found to bind CCL5 with somewhat higher affinities in comparison to those of short Nt‐CCR5(8–20) peptides with the same number of sulfated tyrosine residues. Similarly, a long Nt‐CCR5(1–27)(sY3,sY10,sY14) peptide cross reacts with CXCL12 and with lower KD in comparison to its binding to CCL5. Intermolecular nuclear overhauser effect (NOE) measurements were used to decipher the mechanism of the chemokine/Nt‐receptor peptide binding. The Nt‐CXCR4 peptides interact with the conserved CCL5 tyrosine sulfate‐binding site by an allovalency mechanism like that observed for CCL5 binding of Nt‐CCR5 peptides. Nt‐CCR5 peptides bind CXCL12 in multiple modes analogous to their binding to HIV‐1 gp120 and interact with all three tyrosine/sulfated tyrosine‐binding pockets of CXCL12. We suggest that the chemokine‐receptors Nt‐segments bind promiscuously to cognate and non‐cognate chemokines and in a mechanism that is dependent on the number of binding pockets for tyrosine residues found on the chemokine. In conclusion, common features shared among the chemokine‐receptors' Nt‐segments such as multiple tyrosine residues that are potentially sulfated, and a large number of negatively charged residues are the reason of the cross binding observed in this study. [ABSTRACT FROM AUTHOR]

Xanthine oxidoreductase (XOR) catalyzes the oxidation of purines (hypoxanthine and xanthine) to uric acid. XOR is widely used in various therapeutic and biotechnological applications. In this study, we characterized the biophysical and mechanistic properties of a novel bacterial XOR from Sulfobacillus acidophilus TPY (SaXOR). Our results showed that SaXOR is a heterotrimer consisting of three subunits, namely XoA, XoB, and XoC, which denote the molybdenum cofactor (Moco), 2Fe‐2S, and FAD‐binding domains, respectively. XoC was found to be stable when co‐expressed with XoB, forming an XoBC complex. Furthermore, we prepared a fusion of XoB and XoC via a flexible linker (fusXoBC) and evaluated its function in comparison to that of XoBC. Spectroscopic analysis revealed that XoB harbors two 2Fe‐2S clusters, whereas XoC bears a single‐bound FAD cofactor. Electron transfer from reduced forms of XoC, XoBC, and fusXoBC to molecular oxygen (O2) during oxidative half‐reaction yielded no flavin semiquinones, implying ultrafast single‐electron transfer from 2Fe‐2Sred to FAD. In the presence of XoA, XoBC and fusXoBC exhibited comparable XoA affinity and exploited a shared overall mechanism. Nonetheless, the linkage may accelerate the two‐step, single‐electron transfer cascade from 2Fe‐2Sred to FAD while augmenting protein stability. Collectively, our findings provide novel insights into SaXOR properties and oxidation mechanisms divergent from prior mammalian and bacterial XOR paradigms. [ABSTRACT FROM AUTHOR]

StarD7 is a member of the START protein family required for phosphatidylcholine delivery to the mitochondria, thus key to maintain mitochondrial structure. Its deficiency has been associated with an impairment of cellular processes, such as proliferation and migration, and it has also been reported that it is needed in myogenic differentiation. Here, we show that StarD7 deficiency in C2C12 muscle cells results in the accumulation of abnormal mitochondria, a reduced number of mitochondria per cell area and increased glycolysis. In addition, StarD7‐deficient cells undergo an increase in mitochondria‐ER contact sites, reduced connexin 43 expression, and disturbances in lipid handling, evidenced by lipid droplet accumulation and decreased levels in phosphatidylserine synthase 1 and 2 expression. Interestingly, StarD7‐deficient cells showed alterations in mitophagy markers. We observed accumulation of LC3B‐II and BNIP3 proteins in mitochondria‐enriched fractions and accumulation of autophagolysosomal and lysosomal vesicles in StarD7‐deficient cells. Furthermore, live‐cell imaging experiments of StarD7 knockdown cells expressing mitochondria‐targeted mKeima indicated an enhanced mitochondria delivery into lysosomes. Importantly, StarD7 reconstitution in StarD7‐deficient cells restores LC3B‐II expression in mitochondria‐enriched fractions at similar levels to those observed in control cells. Collectively, these findings suggest that StarD7‐deficient C2C12 myoblasts are associated with altered cristae structure, disturbances in neutral lipid accumulation, glucose metabolism, and increased mitophagy flux. The alterations mentioned above allow for the maintenance of mitochondrial function. [ABSTRACT FROM AUTHOR]

During periods of fasting, the body undergoes a metabolic shift from carbohydrate utilization to the use of fats and ketones as an energy source, as well as the inhibition of de novo lipogenesis and the initiation of gluconeogenesis in the liver. The transcription factor sterol regulatory element‐binding protein‐1 (SREBP‐1), which plays a critical role in the regulation of lipogenesis, is suppressed during fasting, resulting in the suppression of hepatic lipogenesis. We previously demonstrated that the interaction of fasting‐induced Kruppel‐like factor 15 (KLF15) with liver X receptor serves as the essential mechanism for the nutritional regulation of SREBP‐1 expression. However, the underlying mechanisms of KLF15 induction during fasting remain unclear. In this study, we show that the glucocorticoid receptor (GR) regulates the hepatic expression of KLF15 and, subsequently, lipogenesis through the KLF15‐SREBP‐1 pathway during fasting. KLF15 is necessary for the suppression of SREBP‐1 by GR, as demonstrated through experiments using KLF15 knockout mice. Additionally, we show that GR is involved in the fasting response, with heightened binding to the KLF15 enhancer. It has been widely known that the hypothalamic–pituitary–adrenal (HPA) axis regulates the secretion of glucocorticoids and plays a significant role in the metabolic response to undernutrition. These findings demonstrate the importance of the HPA‐axis‐regulated GR‐KLF15 pathway in the regulation of lipid metabolism in the liver during fasting. [ABSTRACT FROM AUTHOR]

Mammals have potent innate immune systems that work together to fight against a variety of distinct viruses. In addition to interferon (IFN) response, which has been intensively studied, antiviral RNA interference (RNAi) is gradually being studied. However, previous studies indicated low Dicer activity on double‐stranded RNA (dsRNA) substrates in vitro and that IFN response masks or inhibits antiviral RNAi in mammals. Therefore, whether or not the RNAi is functional for antiviral response in mammalian somatic cells is still an ongoing area of research. In this review, we will present the current advances in antiviral RNAi in mammals and focus on three fundamental questions critical to the intense debate about whether RNAi can function as an innate antiviral immunity in mammals. [ABSTRACT FROM AUTHOR]

The SARS‐CoV‐2 main protease (Mpro) holds significant importance as a biological target in combating coronaviruses due to its importance in virus replication. Considering the emergence of novel SARS‐CoV‐2 variants and the mutations observed in the Mpro sequence, we hypothesized that these mutations may have a potential impact on the protease's specificity. To test this, we expressed Mpro corresponding to the original strain and variants Beta1, Beta2, and Omicron and analyzed their activity on protein‐based and peptide substrates. Although we observed differential activity on the protein‐based substrate, there was very little difference when analyzed on the peptide substrate. We conclude that mutations on the Mpro sequence, despite having a minor effect on a peptide substrate cleavage, did not change the catalytic site environment enough to build resistance to inhibition. Therefore, we propose that inhibitors initially designed for the Mpro of the original strain will be effective in all the variants. Thus, Mpro is likely to continue to be a target of therapeutic interest as mutations in its sequence are rare and, as we show here, have a minor effect on the protease's recognition of peptide‐based molecules. [ABSTRACT FROM AUTHOR]

Invasion of brain endothelium protein A (IbeA) is a virulence factor specific to pathogenic Escherichia coli. Originally identified in the K1 strain causing neonatal meningitis, it was more recently found in avian pathogenic Escherichia coli (APEC) and adherent invasive Escherichia coli (AIEC). In these bacteria, IbeA facilitates host cell invasion and intracellular survival, in particular, under harsh conditions like oxidative stress. Furthermore, IbeA from AIEC contributes to intramacrophage survival and replication, thus enhancing the inflammatory response within the intestine. Therefore, this factor is a promising drug target for anti‐AIEC strategies in the context of Crohn's disease. Despite such an important role, the biological function of IbeA remains largely unknown. In particular, its exact nature and cellular localization, i.e., membrane‐bound invasin versus cytosolic factor, are still of debate. Here, we developed an efficient protocol for recombinant expression of IbeA under native conditions and demonstrated that IbeA from AIEC is a soluble, homodimeric flavoprotein. Using mass spectrometry and tryptophan fluorescence measurements, we further showed that IbeA preferentially binds flavin adenine dinucleotide (FAD), with an affinity in the one‐hundred nanomolar range and optimal binding under reducing conditions. 3D‐modeling with AlphaFold revealed that IbeA shares strong structural homology with FAD‐dependent oxidoreductases. Finally, we used ligand docking, mutational analyses, and molecular dynamics simulations to identify the FAD binding pocket within IbeA and characterize possible conformational changes occurring upon ligand binding. Overall, we suggest that the role of IbeA in the survival of AIEC within host cells, notably macrophages, is linked to modulation of redox processes. [ABSTRACT FROM AUTHOR]

Protein aggregation is a biological phenomenon caused by the accumulation of misfolded proteins. Amyloid beta (Aβ) peptides are derived from the cleavage of a larger membrane protein molecule and accumulate to form plaques extracellularly. According to the amyloid hypothesis, accumulation of Aβ aggregates in the brain is primarily responsible for the pathogenesis of Alzheimer's disease (AD). Therefore, the disassembly of Aβ aggregates may provide opportunities for alleviating or treating AD. Here, we show that the novel protein targeting machinery from chloroplast, chloroplast signal recognition particle 43 (cpSRP43), is an ATP‐independent membrane protein chaperone that can both prevent and reverse Aβ aggregation effectively. Using of thioflavin T dye, we obtained the aggregation kinetics of Aβ aggregation and determined that the chaperone prevents Aβ aggregation in a concentration‐dependent manner. Size exclusion chromatography and sedimentation assays showed that 10‐fold excess of cpSRP43 can keep Aβ in the soluble monomeric form. Electron microscopy showed that the fibril structure was disrupted in the presence of this chaperone. Importantly, cpSRP43 utilizes the binding energy to actively remodel the preformed Aβ aggregates without assistance by a co‐chaperone and ATP, emphasizing its unique function among protein chaperones. Moreover, when sodium chloride concentration is higher than 25 mm, the Aβ aggregation rate increases drastically to form tightly associated aggregates and generate more oligomers. Our results demonstrate that the presence of cpSRP43 and low NaCl levels inhibit or retard Aβ peptide aggregation, potentially opening new avenues to strategically develop an effective treatment for AD. [ABSTRACT FROM AUTHOR]

In the present study, cryo‐electron tomography was used to investigate the localization of 2‐oxoacid dehydrogenase complexes (OADCs) in cardiac mitochondria and mitochondrial inner membrane samples. Two classes of ordered OADC inner cores with different symmetries were distinguished and their quaternary structures modeled. One class corresponds to pyruvate dehydrogenase complexes and the other to dehydrogenase complexes of α‐ketoglutarate and branched‐chain α‐ketoacids. OADCs were shown to be localized in close proximity to membrane‐embedded respirasomes, as observed both in densely packed lamellar cristae of cardiac mitochondria and in ruptured mitochondrial samples where the dense packing is absent. This suggests the specificity of the OADC–respirasome interaction, which allows localized NADH/NAD+ exchange between OADCs and complex I of the respiratory chain. The importance of this local coupling is based on OADCs being the link between respiration, glycolysis and amino acid metabolism. The coupling of these basic metabolic processes can vary in different tissues and conditions and may be involved in the development of various pathologies. The present study shows that this important and previously missing parameter of mitochondrial complex coupling can be successfully assessed using cryo‐electron tomography. [ABSTRACT FROM AUTHOR]

Errors made by DNA polymerases contribute to both natural variation and, in extreme cases, genome instability and its associated diseases. Recently, the importance of polymerase misincorporation in disease has been highlighted by the identification of cancer‐associated polymerase variants with mutations in the exonuclease domain. A subgroup of these variants have a hypermutation phenotype in tumours, and when modelled in yeast, they show mutation rates in excess of that seen with polymerase with simple loss of proofreading activity. We have developed a bypass assay to rapidly determine the tendency of a polymerase to misincorporate in vitro. We have used the assay to compare misincorporation by wild‐type, exonuclease‐defective and two hypermutating human DNA polymerase ε variants, P286R and V411L. The assay clearly distinguished between the misincorporation rates of wild‐type, exonuclease dead and P286R polymerases. However, the V411L polymerase showed misincorporation rate comparable to the exonuclease dead enzyme rather than P286R, suggesting that there may be some differences in the way that these variants cause hypermutation. Using this assay, misincorporation opposite a templated C nucleotide was consistently higher than for other nucleotides, and this caused predominantly C‐to‐T transitions. This is consistent with the observation that C‐to‐T transitions are commonly seen in DNA polymerase ε mutant tumours. [ABSTRACT FROM AUTHOR]

Lysosomal degradation of the endoplasmic reticulum (ER) and its components through the autophagy pathway has emerged as a major regulator of ER proteostasis. Commonly referred to as ER‐phagy and ER‐to‐lysosome‐associated degradation (ERLAD), how the ER is targeted to the lysosome has been recently clarified by a growing number of studies. Here, we summarize the discoveries of the molecular components required for lysosomal degradation of the ER and their proposed mechanisms of action. Additionally, we discuss how cells employ these machineries to create the different routes of ER‐lysosome‐associated degradation. Further, we review the role of ER‐phagy in viral infection pathways, as well as the implication of ER‐phagy in human disease. In sum, we provide a comprehensive overview of the current field of ER‐phagy. [ABSTRACT FROM AUTHOR]

YES‐associated protein (YAP) is a part of the Hippo pathway, with pivotal roles in several developmental processes and dual functionality as both a tumor suppressor and an oncogene. In the present study, we identified YAP activity as a microtubular scaffold protein that maintains the stability of the mitotic spindle and midbody by physically interacting with α‐tubulin during mitotic progression. The interaction of YAP and α‐tubulin was evident in co‐immunoprecipitation assays, as well as observing their co‐localization in the microtubular structure of the mitotic spindle and midbody in immunostainings. With YAP depletion, levels of ECT2, MKLP‐1, and Aurora B are reduced, which is consistent with YAP functioning in midbody formation during cytokinesis. The concomitant decrease in α‐tubulin and increase in acetyl‐α‐tubulin during YAP depletion occurred at the post‐transcriptional level. This suggests that YAP maintains the stability of the mitotic spindle and midbody, which ensures appropriate chromosome segregation during mitotic division. The increase in acetyl‐α‐tubulin during YAP depletion may provide a lesion‐halting mechanism in maintaining the microtubule structure. The depletion of YAP also results in multinuclearity and aneuploidy, which supports its role in stabilizing the mitotic spindle and midbody. [ABSTRACT FROM AUTHOR]

Pyridoxine 4‐dehydrogenase (PdxI), a NADPH‐dependent pyridoxal reductase, is one of the key players in the Escherichia coli pyridoxal 5′‐phosphate (PLP) salvage pathway. This enzyme, which catalyses the reduction of pyridoxal into pyridoxine, causes pyridoxal to be converted into PLP via the formation of pyridoxine and pyridoxine phosphate. The structural and functional properties of PdxI were hitherto unknown, preventing a rational explanation of how and why this longer, detoured pathway occurs, given that, in E. coli, two pyridoxal kinases (PdxK and PdxY) exist that could convert pyridoxal directly into PLP. Here, we report a detailed characterisation of E. coli PdxI that explains this behaviour. The enzyme efficiently catalyses the reversible transformation of pyridoxal into pyridoxine, although the reduction direction is thermodynamically strongly favoured, following a compulsory‐order ternary‐complex mechanism. In vitro, the enzyme is also able to catalyse PLP reduction and use NADH as an electron donor, although with lower efficiency. As with all members of the aldo‐keto reductase (AKR) superfamily, the enzyme has a TIM barrel fold; however, it shows some specific features, the most important of which is the presence of an Arg residue that replaces the catalytic tetrad His residue that is present in all AKRs and appears to be involved in substrate specificity. The above results, in conjunction with kinetic and static measurements of vitamins B6 in cell extracts of E. coli wild‐type and knockout strains, shed light on the role of PdxI and both kinases in determining the pathway followed by pyridoxal in its conversion to PLP, which has a precise regulatory function. [ABSTRACT FROM AUTHOR]

The basic structures of membrane lipids that compose biomembranes differ among species; i.e., in mammals, the primary structure of long‐chain base (LCB), the common backbone of ceramides and complex sphingolipids, is sphingosine, whereas, in yeast Saccharomyces cerevisiae, it is phytosphingosine, and S. cerevisiae does not have sphingosine. In addition, the sterol, which is coordinately involved in various functions with complex sphingolipids, is cholesterol in mammals, while in yeast it is ergosterol. Previously, it was found that yeast cells are viable when the structure of LCBs is replaced by sphingosine by supplying an exogenous LCB to cells lacking LCB biosynthesis. Here, we characterized yeast cells having sphingosine instead of phytosphingosine (sphingosine cells). Sphingosine cells exhibited a strong growth defect when biosynthesis of ceramides or complex sphingolipids was inhibited, indicating that, in the sphingosine cells, exogenously added sphingosine is required to be further metabolized. The sphingosine cells exhibited hypersensitivity to various environmental stresses and had abnormal plasma membrane and cell wall properties. Furthermore, we also established a method for simultaneous replacement of both LCB and sterol structures with those of mammals (sphingosine/cholesterol cells). The multiple stress hypersensitivity and abnormal plasma membrane and cell wall properties observed in sphingosine cells were also observed in sphingosine/cholesterol cells, suggesting that simultaneous replacement of both LCB and sterol structures with those of mammals cannot prevent these abnormal phenotypes. This is the first study to our knowledge showing that S. cerevisiae can grow even if LCB and sterol structures are simultaneously replaced with mammalian types. [ABSTRACT FROM AUTHOR]

Magnesium (Mg2+) is the most abundant divalent cation in cells and plays key roles in almost all biological processes. CBS‐pair domain divalent metal cation transport mediators (CNNMs) are a newly characterized class of Mg2+ transporters present throughout biology. Originally discovered in bacteria, there are four CNNM proteins in humans, which are involved in divalent cation transport, genetic diseases, and cancer. Eukaryotic CNNMs are composed of four domains: an extracellular domain, a transmembrane domain, a cystathionine‐β‐synthase (CBS)‐pair domain, and a cyclic nucleotide‐binding homology domain. The transmembrane and CBS‐pair core are the defining features of CNNM proteins with over 20 000 protein sequences known from over 8000 species. Here, we review the structural and functional studies of eukaryotic and prokaryotic CNNMs that underlie our understanding of their regulation and mechanism of ion transport. Recent structures of prokaryotic CNNMs confirm the transmembrane domain mediates ion transport with the CBS‐pair domain likely playing a regulatory role through binding divalent cations. Studies of mammalian CNNMs have identified new binding partners. These advances are driving progress in understanding this deeply conserved and widespread family of ion transporters. [ABSTRACT FROM AUTHOR]

The ability to view structures of proteins at atomic resolution, facilitated by the rise of macromolecular crystallography, has had a tremendous impact in many areas of sciences, including molecular pharmacology, drug discovery and biotechnology. However, the teaching of macromolecular crystallography in universities across the globe has been less than optimal. This could be attributed to the interdisciplinary nature of this subject, making it appear esoteric and incomprehensible, at least at first glance, for students who have exclusive training in only one specific discipline. For the instructor, this problem is compounded further by the plethora of complex concepts and specialized terminologies that the science of macromolecular crystallography has accumulated over the course of its evolution. Moreover, the advent of robotics and several sophisticated software algorithms have reduced the incentive to understand the beautiful conceptual bedrock on which this subject is based. As a way of addressing some of the challenges delineated above, this Words of Advice article attempts to formulate the broad framework within which the teaching and learning of macromolecular crystallography should be approached. It advocates the acknowledgement that this is an interdisciplinary field, with substantial contributions from chemical, physical, biological and mathematical sciences, requiring the evolution of teaching approaches that acknowledge this reality. Moreover, it suggests the use of visual tools, use of computational resources and history to make the subject more relatable to students. [ABSTRACT FROM AUTHOR]

Loss of myocardial mass in a neonatal rat cardiomyocyte culture is studied to determine whether there is a distinguishable cellular response based on the origin of mechano‐signals. The approach herein compares the sarcomeric assembly and disassembly processes in heart cells by imposing mechano‐signals at the interface with the extracellular matrix (extrinsic) and at the level of the myofilaments (intrinsic). Experiments compared the effects of imposed internal (inside/out) and external (outside/in) loading and unloading on modifications in neonatal rat cardiomyocytes. Unloading of the cellular substrate by myosin inhibition (1 μm mavacamten), or cessation of cyclic strain (1 Hz, 10% strain) after preconditioning, led to significant disassembly of sarcomeric α‐actinin by 6 h. In myosin inhibition, this was accompanied by redistribution of intracellular poly‐ubiquitin K48 to the cellular periphery relative to the poly‐ubiquitin K48 reservoir at the I‐band. Moreover, loading and unloading of the cellular substrate led to a three‐fold increase in post‐translational modifications (PTMs) when compared to the myosin‐specific activation or inhibition. Specifically, phosphorylation increased with loading while ubiquitination increased with unloading, which may involve extracellular signal‐regulated kinase 1/2 and focal adhesion kinase activation. The identified PTMs, including ubiquitination, acetylation, and phosphorylation, are proposed to modify internal domains in α‐actinin to increase its propensity to bind F‐actin. These results demonstrate a link between mechanical feedback and sarcomere protein homeostasis via PTMs of α‐actinin that exemplify how cardiomyocytes exhibit differential responses to the origin of force. The implications of sarcomere regulation governed by PTMs of α‐actinin are discussed with respect to cardiac atrophy and heart failure. [ABSTRACT FROM AUTHOR]

Arsenic is a carcinogen that can cause skin, lung, and bladder cancer. While DNA double‐strand breaks (DSBs) have been implicated in arsenic‐induced carcinogenesis, the exact mechanism remains unclear. In this study, we performed genetic analysis to examine the impact of arsenic trioxide (As2O3) on four different DSB repair pathways using the human pre‐B cell line Nalm‐6. Random integration analysis showed that As2O3 does not negatively affect non‐homologous end joining or polymerase theta‐mediated end joining. In contrast, chromosomal DSB repair analysis revealed that As2O3 decreases the efficiency of homologous recombination (HR) and, less prominently, single‐strand annealing. Consistent with this finding, As2O3 decreased gene‐targeting efficiency, owing to a significant reduction in the frequency of HR‐mediated targeted integration. To further verify the inhibitory effect of arsenic on HR, we examined cellular sensitivity to olaparib and camptothecin, which induce one‐ended DSBs requiring HR for precise repair. Intriguingly, we found that As2O3 significantly enhances sensitivity to those anticancer agents in HR‐proficient cells. Our results suggest that arsenic‐induced genomic instability is attributed to HR suppression, providing valuable insights into arsenic‐associated carcinogenesis and therapeutic options. [ABSTRACT FROM AUTHOR]

The prostate is an androgen‐responsive organ, but the complex cellular and molecular interactions that mediate these responses remain incompletely defined. Here, I synthesise the existing literature to derive a simple conceptual framework describing the androgen‐dependent regulation of prostate epithelial dynamics. In this framework, epithelial androgen receptor (AR) cell‐autonomously controls luminal cell height, whereas stromal AR regulates the synthesis of growth factors that promote luminal cell survival and proliferation. With the additional aid of a reanalysis of single‐cell RNA‐seq data, I also propose that insulin‐like growth factor 1 (IGF1) functions as a key androgen‐dependent growth factor coordinating stromal‐to‐epithelial paracrine communication. A novel mathematical model based on this framework was able to quantitatively fit experimental data describing prostate regression and regeneration. Model analysis demonstrates how the luminal cell population can maintain a stable equilibrium size via competition for and degradation of stroma‐derived IGF1 and how this population size can be controlled by androgen levels, without a requirement for distinct luminal cell subsets. Moreover, model simulations were able to qualitatively recapitulate experimental observations in inflammatory and cancerous states, thereby providing insights into potential disease mechanisms. This simple model could therefore serve as a foundation for more comprehensive modelling of both the healthy and diseased prostate. [ABSTRACT FROM AUTHOR]

Transthyretin (TTR) is a carrier protein for thyroid hormone thyroxine (T4) in plasma, placental cytosol, and cerebrospinal fluid. While the potential toxicity of small molecules that compete with T4 for binding to TTR should be carefully studied, these small molecules can also serve as anti‐ATTR amyloidosis drugs by stabilizing the TTR structure. Here, we demonstrated that rafoxanide, an EU‐approved anthelmintic drug for domesticated animals, binds to the T4‐binding site of TTR. An intrinsic fluorescence quenching assay showed that rafoxanide also binds to the thyroid hormone‐related proteins, including serum albumin and thyroid hormone receptor β. Rafoxanide strongly inhibited TTR amyloidogenesis in fibrillization assay, but the binding of rafoxanide to TTR was interfered with in human plasma, probably due to interactions with thyroid hormone‐related proteins. Protein crystallography provided clues for the optimization of binding affinity and selectivity. Our findings emphasize the importance of considering rafoxanide as both a possible thyroid‐disrupting chemical and a lead compound for the development of new ATTR amyloidosis inhibitors. [ABSTRACT FROM AUTHOR]

Transcription factor RUNX1 plays important roles in hematopoiesis and leukemogenesis. RUNX1 function is tightly controlled through posttranslational modifications, including ubiquitination and acetylation. However, its regulation via ubiquitination, especially proteasome‐independent ubiquitination, is poorly understood. We previously identified DTX2 as a RUNX1‐interacting E3 ligase using a cell‐free AlphaScreen assay. In this study, we examined whether DTX2 is involved in the regulation of RUNX1 using in vitro and ex vivo analyses. DTX2 bound to RUNX1 and other RUNX family members RUNX2 and RUNX3 through their C‐terminal region. DTX2‐induced RUNX1 ubiquitination did not result in RUNX1 protein degradation. Instead, we found that the acetylation of RUNX1, which is known to enhance the transcriptional activity of RUNX1, was inhibited in the presence of DTX2. Concomitantly, DTX2 reduced the RUNX1‐induced activation of an MCSFR luciferase reporter. We also found that DTX2 induced RUNX1 cytoplasmic mislocalization. Moreover, DTX2 overexpression showed a substantial growth‐inhibitory effect in RUNX1‐dependent leukemia cell lines. Thus, our findings indicate a novel aspect of the ubiquitination and acetylation of RUNX1 that is modulated by DTX2 in a proteosome‐independent manner. [ABSTRACT FROM AUTHOR]

The filamentous fungus Aspergillus niger is well known for its high protein secretion capacity and a preferred host for homologous and heterologous protein production. To improve the protein production capacity of A. niger even further, a set of dedicated protein production strains was made containing up to 10 glucoamylase landing sites (GLSs) at predetermined sites in the genome. These GLSs replace genes encoding enzymes abundantly present or encoding unwanted functions. Each GLS contains the promotor and terminator region of the glucoamylase gene (glaA), one of the highest expressed genes in A. niger. Integrating multiple gene copies, often realized by random integration, is known to boost protein production yields. In our approach the GLSs allow for rapid targeted gene replacement using CRISPR/Cas9‐mediated genome editing. By introducing the same or different unique DNA sequences (dubbed KORE sequences) in each GLS and designing Cas9‐compatible single guide RNAs, one is able to select at which GLS integration of a target gene occurs. In this way a set of identical strains with different copy numbers of the gene of interest can be easily and rapidly made to compare protein production levels. As an illustration of its potential, we successfully used the expression platform to generate multicopy A. niger strains producing the Penicillium expansum PatE::6xHis protein catalysing the final step in patulin biosynthesis. The A. niger strain expressing 10 copies of the patE::6xHis expression cassette produced about 70 μg·mL−1 PatE protein in the culture medium with a purity just under 90%. [ABSTRACT FROM AUTHOR]

Patulin synthase (PatE) from Penicillium expansum is a flavin‐dependent enzyme that catalyses the last step in the biosynthesis of the mycotoxin patulin. This secondary metabolite is often present in fruit and fruit‐derived products, causing postharvest losses. The patE gene was expressed in Aspergillus niger allowing purification and characterization of PatE. This confirmed that PatE is active not only on the proposed patulin precursor ascladiol but also on several aromatic alcohols including 5‐hydroxymethylfurfural. By elucidating its crystal structure, details on its catalytic mechanism were revealed. Several aspects of the active site architecture are reminiscent of that of fungal aryl‐alcohol oxidases. Yet, PatE is most efficient with ascladiol as substrate confirming its dedicated role in biosynthesis of patulin. [ABSTRACT FROM AUTHOR]

Neonatal brachial plexus injury (NBPI), a leading cause of pediatric upper limb paralysis, results in disabling and incurable muscle contractures that are driven by impaired longitudinal growth of denervated muscles. A rare form of NBPI, which maintains both afferent and sympathetic muscle innervation despite motor denervation, protects against contractures. We have previously ruled out a role for NRG/ErbB signaling, the predominant pathway governing antegrade afferent neuromuscular transmission, in modulating the formation of contractures. Our current study therefore investigated the contributions of sympathetic innervation of skeletal muscle in modulating NBPI‐induced contractures. Through chemical sympathectomy and pharmacologic modification with a β2‐adrenergic agonist, we discovered that sympathetic innervation alone is neither required nor sufficient to modulate contracture formation in neonatal mice. Despite this, sympathetic innervation plays an intriguing sex‐specific role in mediating neonatal muscle growth, as the cross‐sectional area (CSA) and volume of normally innervated male muscles were diminished by ablation of sympathetic neurons and increased by β‐adrenergic stimulation. Intriguingly, the robust alterations in CSA occurred with minimal changes to normal longitudinal muscle growth as determined by sarcomere length. Instead, β‐adrenergic stimulation exacerbated sarcomere overstretch in denervated male muscles, indicating potentially discrete regulation of muscle width and length. Future investigations into the mechanistic underpinnings of these distinct aspects of muscle growth are thus essential for improving clinical outcomes in patients affected by muscle disorders in which both length and width are affected. [ABSTRACT FROM AUTHOR]

The P450 monooxygenase CYP109A2 from Bacillus megaterium DSM319 was previously found to convert vitamin D3 (VD3) to 25‐hydroxyvitamin D3. Here, we show that this enzyme is also able to convert testosterone in a highly regio‐ and stereoselective manner to 16β‐hydroxytestosterone. To reveal the structural determinants governing the regio‐ and stereoselective steroid hydroxylation reactions catalyzed by CYP109A2, two crystal structures of CYP109A2 were solved in similar closed conformations, one revealing a bound testosterone in the active site pocket, albeit at a nonproductive site away from the heme‐iron. To examine whether the closed crystal structures nevertheless correspond to a reactive conformation of CYP109A2, docking and molecular dynamics (MD) simulations were performed with testosterone and vitamin D3 (VD3) present in the active site. These MD simulations were analyzed for catalytically productive conformations, the relative occurrences of which were in agreement with the experimentally determined stereoselectivities if the predicted stability of each carbon‐hydrogen bond was taken into account. Overall, the first‐time determination and analysis of the catalytically relevant 3D conformation of CYP109A2 will allow for future small molecule ligand screening in silico, as well as enabling site‐directed mutagenesis toward improved enzymatic properties of this enzyme. [ABSTRACT FROM AUTHOR]

The MYC proto‐oncogene and BRD4, a BET family protein, are two cardinal proteins that have a broad influence in cell biology and disease. Both proteins are expressed ubiquitously in mammalian cells and play central roles in controlling growth, development, stress responses and metabolic function. As chromatin and transcriptional regulators, they play a critical role in regulating the expression of a burgeoning array of genes, maintaining chromatin architecture and genome stability. Consequently, impairment of their function or regulation leads to many diseases, with cancer being the most predominant. Interestingly, accumulating evidence indicates that regulation of the expression and functions of MYC are tightly intertwined with BRD4 at both transcriptional and post‐transcriptional levels. Here, we review the mechanisms by which MYC and BRD4 are regulated, their functions in governing various molecular mechanisms and the consequences of their dysregulation that lead to disease. We present a perspective of how the regulatory mechanisms for the two proteins could be entwined at multiple points in a BRD4‐MYC nexus that leads to the modulation of their functions and disease upon dysregulation. [ABSTRACT FROM AUTHOR]

Assembly of cell division protein FtsZ into the Z‐ring at the division site is a key step in bacterial cell division. The Min proteins can restrict the Z‐ring to the middle of the cell. MinC is the main protein that obstructs Z‐ring formation by inhibiting FtsZ assembly. Its N‐terminal domain (MinCN) regulates the localization of the Z‐ring by inhibiting FtsZ polymerization, while its C‐terminal domain (MinCC) binds to MinD as well as to FtsZ. Previous studies have shown that MinC and MinD form copolymers in vitro. This copolymer may greatly enhance the binding of MinC to FtsZ, and/or prevent FtsZ filaments from diffusing to the ends of the cell. Here, we investigated the assembly properties of MinCC–MinD of Pseudomonas aeruginosa. We found that MinCC is sufficient to form the copolymers. Although MinCC–MinD assembles into larger bundles, most likely because MinCC is spatially more readily bound to MinD, its copolymerization has similar dynamic properties: the concentration of MinD dominates their copolymerization. The critical concentration of MinD is around 3 μm and when MinD concentration is high enough, a low concentration MinCC could still be copolymerized. We also found that MinCC–MinD can still rapidly bind to FtsZ protofilaments, providing direct evidence that MinCC also interacts directly with FtsZ. However, although the presence of minCC can slightly improve the division defect of minC‐knockout strains and shorten the cell length from an average of 12.2 ± 6.7 to 6.6 ± 3.6 μm, it is still insufficient for the normal growth and division of bacteria. [ABSTRACT FROM AUTHOR]