title: Changes in chromatin accessibility are not concordant with transcriptional changes for single-factor perturbations

abstract: A major goal in the field of transcriptional regulation is the mapping of changes in the binding of transcription factors to the resultant changes in gene expression. Recently, methods for measuring chromatin accessibility have enabled us to measure changes in accessibility across the genome, which are thought to correspond to transcription factor binding events. In concert with RNA-sequencing, these data in principle enable such mappings; however, few studies have looked at their concordance over short duration treatments with specific perturbations. Here, we used tandem, bulk ATAC-seq and RNA-seq measurements from MCF-7 breast carcinoma cells to systematically evaluate the concordance between changes in accessibility and changes in expression in response to retinoic acid and TGF-{beta}. We found two classes of genes whose expression showed a significant change: those that showed some change in accessibility of nearby chromatin, and those that showed virtually no change despite strong changes in expression. The peaks associated with genes in the former group had a lower baseline accessibility prior to exposure to signal. Focusing the analysis specifically on peaks with motifs for transcription factors associated with retinoic acid and TGF-{beta} signaling did not reduce the lack of correspondence. Analysis of paired chromatin accessibility and gene expression data from distinct paths along the hematopoietic differentiation trajectory showed a much stronger correspondence, suggesting that the multifactorial biological processes associated with differentiation may lead to changes in chromatin accessibility that reflect rather than drive altered transcriptional status. Together, these results show many gene expression changes can happen independent of changes in accessibility of local chromatin in the context of a single-factor perturbation and suggest that some changes to accessibility changes may occur after changes to expression, rather than before.


title: Dissociable Roles of FEF Neurons in Initiating Saccades

abstract: The frontal eye field (FEF) plays a key role in initiating saccades. To explain how single neurons in this region may enable the initiation of saccades, Jeffery Schall proposed the "variable rate model" (Hanes and Schall, 1996) which assumes that the discharge of a neuron must reach a fixed activation threshold for a saccade to be evoked. However, the validity of this model has been questioned as results from later work testing saccades in different behavioral contexts seemed to require the assumption of variable thresholds. Moreover, even when sticking to the original behavioral context, not all types of frontal eye field neurons seemed to behave according to a variable rate model. In an attempt to reconsider the viability of the variable rate model avoiding limitations of previous research, we studied single neurons recorded from the FEF of two rhesus monkeys while they performed a memory-guided saccade task. We evaluated the degree to which each type of FEF neurons complied with the variable rate model by quantifying how precisely their discharge predicted an imminent saccade based on their immediate presaccadic activity. In addition, we asked whether there might be a hierarchical relation between visuomotor and motor neurons with only the latter responsible for the release of saccades and therefore satisfying the assumption of the variable rate model. We show that decoders trained on single motor neurons presaccadic activity performed better than decoders trained on visuomotor neurons in predicting an imminent saccade, in line with the assumption of a top position in a putative processing hierarchy. While this position might have suggested significant resilience to perturbation of the visual input to the FEF, we found quite the opposite that motor neurons but not visuomotor neurons were susceptible to perturbation. As a matter of fact, they lost the ability to predict the initiation of a saccade based on their immediate pressacadic discharge rate. Our results support the notion that motor neurons and visuomotor neurons have dissociable functions in information processing for saccades with motor neurons more closely related to saccade initiation, in line with the tenets of the variable rate model. Yet, they also clearly indicate that even these supposedly top layer neurons exhibit an unexpected degree of dependence on afferent input questions the viability of the assumption of a simple processing hierarchy.

category: neuroscience


title: Sex and age differences in cognitive bias and neural activation in response to cognitive bias

abstract:
Cognitive symptoms of depression, including negative cognitive bias, are more severe in women than in men. Current treatments to reduce negative cognitive bias are not effective and sex differences in the neural activity underlying cognitive bias may play a role. Here we examined sex and age differences in cognitive bias and functional connectivity in a novel paradigm. Male and female rats underwent an 18-day cognitive bias procedure, in which they learned to discriminate between two contexts (shock paired context A, no-shock paired context B), during either adolescence (postnatal day (PD 40)), young adulthood (PD 100), or middle-age (PD 210). Cognitive bias was measured as freezing behaviour in response to an ambiguous context (context C), with freezing levels akin to the shock paired context coded as negative bias. All animals learned to discriminate between the two contexts, regardless of sex or age. However, adults (young adults, middle-aged) displayed a greater negative cognitive bias compared to adolescents, and middle-aged males had a greater negative cognitive bias than middle-aged females. Females had greater neural activation of the nucleus accumbens, amygdala, and hippocampal regions to the ambiguous context compared to males, and young rats (adolescent, young adults) had greater neural activation in these regions compared to middle-aged rats. Functional connectivity between regions involved in cognitive bias differed by age and sex, and only adult males had negative correlations between the frontal regions and hippocampal regions. These findings highlight the importance of examining age and sex when investigating the underpinnings of negative cognitive bias and lay the groundwork for determining what age- and sex-specific regions to target in future cognitive bias studies.

HighlightsO_LIMiddle-aged males had a greater negative cognitive bias than middle-aged females
C_LIO_LIAdult rats displayed a greater negative cognitive bias compared to adolescents
C_LIO_LIGreater neural activity in females than males in limbic and reward regions
C_LIO_LIGreater role of the frontal cortex activation in the cognitive bias of adults
C_LIO_LIFunctional connectivity in response to cognitive bias differed by age and sex
C_LI


title: Pan-cancer association of DNA repair deficiencies with whole-genome mutational patterns

abstract:
BackgroundCancer genomes are shaped by DNA damage and repair. DNA Repair deficiencies in cancers may result in characteristic mutational patterns, as exemplified by deficiency of BRCA1/2 and efficacy prediction for PARP-inhibitors. We systematically evaluated the ability to identify and predict mono- or biallelic deficiencies of 736 DDR genes from associations with genome-wide mutational patterns, including structural variants, across 6,065 whole-genome sequenced cancers.

ResultsWe assembled 535 cancer-specific patient cohorts with shared deficiency of a DNA damage response gene. We trained and evaluated a predictive model for each cohort, and shortlisted 24 gene deficiencies that could be predicted with high accuracy from mutational patterns. These included expected gene deficiency models for BRCA1/2, MSH3/6, TP53, and CDK12. CDK12 is associated with tandem-duplications, and we demonstrate that this association can predict gene deficiency with high accuracy (AUROC=0.97) in prostate cancers.

Our novel associations include deficiencies of ATRX, IDH1, HERC2, CDKN2A, PTEN, and SMARCA4. We found that loss of ATRX or IDH1, which are often co-mutated, were associated with the same mutational phenotype in central nervous system cancers; HERC2 loss was associated with an increased number of short deletions in skin cancers and significantly co- occurring with TP53 loss; and PTEN loss was associated with decreased levels of structural rearrangements in cancers of the uterus and the central nervous system.

ConclusionOur systematic, generic approach yielded a catalogue of predictive models, which may provide targets for research and development of treatment, and potentially help guide therapy.

category: genomics


title: Beyond G1/S regulation: how cell size homeostasis is tightly controlled throughout the cell cycle?

abstract: Proliferating cells require control mechanisms to counteract stochastic noise and stabilize the cell mass distribution in a population. It is widely believed that size-dependent timing of the G1/S transition is the predominant control process in mammalian cells. However, a model based only on such a checkpoint cannot explain why cell lines with deficient G1/S control are still able to maintain a stable cell mass distribution or how cell mass is maintained throughout the subsequent phases of the cell cycle. To answer such questions, we used a recently developed form of Quantitative Phase Microscopy (ceQPM), which provides much-improved accuracy of individual cell mass measurement, to investigate the factors contributing to cell mass homeostasis. We find that cell mass homeostasis is robustly maintained, despite disruptions of the normal G1/S transition or cell growth rate. The coefficient of variation in mass, nevertheless declines well after the G1/S transition and throughout the cell cycle in both transformed and non-transformed cells. Furthermore, the cell growth rate responds to cell mass in different ways in different cell types. Growth rate modulation is conveyed by mTORC1-dependent and mTORC1-independent processes, which are independently regulated. Slightly reduced mass accumulation, below exponential growth can effectively reduce cell mass variation in a population. Both size-dependent cell cycle regulation and size-dependent growth rate modulation contribute to cell mass homeostasis. Together they form a compensatory network that strictly controls the coefficient of variation of cell mass in a population. These findings opens new avenues for the discovery of underlying molecular mechanisms. Furthermore, understanding feedback directly on cell growth may provide insights into fundamental principles of cell size control in normal proliferating and terminally differentiated cells, as well as cells in pathological circumstances.

category: cell biology


title: Chromatin sequesters pioneer transcription factor Sox2 from exerting force on DNA

abstract: Formation of biomolecular condensates constitutes an emerging mechanism for transcriptional regulation. Recent studies suggest that the co-condensation between transcription factors (TFs) and DNA can generate mechanical forces driving genome rearrangements. However, the reported forces generated by such protein-DNA co-condensation are typically below one piconewton (pN), questioning its physiological significance. Moreover, the force-generating capacity of these condensates in the chromatin context remains unknown. Using single-molecule biophysical techniques, we show that Sox2, a nucleosome-binding pioneer TF, forms co-condensates with DNA, thereby exerting considerable mechanical tension on DNA strands both in cis and trans. Sox2 can generate forces up to 7 pN--similar in magnitude to other cellular forces. Sox2:DNA condensates are highly stable, withstanding disruptive forces high enough to melt DNA. We find that the disordered domains of Sox2 are required for maximum force generation but not condensate formation per se. Finally, we show that nucleosomes dramatically attenuate the mechanical stress exerted by Sox2 via sequestering it from coalescing on bare DNA. Our findings reveal that TF-mediated DNA condensation can exert significant mechanical stress which can nonetheless be alleviated by the chromatin organization, suggesting a new function of eukaryotic chromatin in protecting the genome from potentially deleterious nuclear forces.

category: biophysics


title: Effects of clozapine-N-oxide and compound 21 on sleep in laboratory mice

abstract: Designer Receptors Exclusively Activated by Designer Drugs (DREADDs) are chemogenetic tools for remote control of targeted cell populations using chemical actuators that bind to modified receptors. Despite the popularity of DREADDs in neuroscience and sleep research, potential effects of the DREADD actuator clozapine-N-oxide (CNO) on sleep have never been systematically tested. Here we show that intraperitoneal injections of commonly used CNO doses (1, 5, and 10 mg/kg) alter sleep in wild-type male laboratory mice. Using electroencephalography (EEG) and electromyography (EMG) to analyse sleep, we found a dose-dependent suppression of rapid eye-movement (REM) sleep, changes in EEG spectral power during non-REM (NREM) sleep, and altered sleep architecture in a pattern previously reported for clozapine. Effects of CNO on sleep could arise from back-metabolism to clozapine or binding to endogenous neurotransmitter receptors. Interestingly, we found that the novel DREADD actuator, compound 21 (C21, 3 mg/kg), similarly modulates sleep despite a lack of back-metabolism to clozapine. Our results demonstrate that both CNO and C21 can modulate sleep of mice not expressing DREADD receptors. This implies that back-metabolism to clozapine is not the sole mechanism underlying side effects of chemogenetic actuators. Therefore, any chemogenetic experiment should include a DREADD-free control group injected with the same CNO, C21 or newly developed actuator. We suggest that electrophysiological sleep assessment could serve as a sensitive tool to test the biological inertness of novel chemogenetic actuators.


title: Transcriptomic analysis of aging mouse sciatic nerve reveals early pathways leading to sarcopenia

abstract:
BackgroundSarcopenia, the age-associated decline in skeletal muscle mass and strength, has long been considered a disease of muscle only, but accumulating evidence suggests that sarcopenia could originate from the neural components controlling muscles. To identify early molecular changes in the efferent nerves that may drive sarcopenia initiation, we performed a longitudinal transcriptomic analysis of the sciatic nerve in aging mice.

MethodsSciatic nerve and gastrocnemius muscle were obtained from young adult, middleaged, old, and sarcopenic (5,18, 21 and 24 months old, respectively) C57BL/6J female mice (n=6 per age group). Sciatic nerve RNA was extracted and subjected to RNA sequencing (RNA-seq), with real-time quantitative reverse transcription PCR (qRT-PCR) validation of differentially expressed genes (DEGs). Functional enrichment analysis of clusters of genes associated with patterns of gene expression across age groups was performed. Sarcopenia was confirmed with qRT-PCR of previously established markers of sarcopenia onset in gastrocnemius muscle.

ResultsWe detected 33 significant DEGs in sciatic nerve of 18-month-old mice compared to 5-month-old mice (absolute value of fold change > 2; false discovery rate [FDR] < 0.05) which we validated with qRT-PCR of the three top up- and down-regulated genes. Up-regulated genes were associated with circadian rhythm and the AMPK signaling pathway, while down-regulated genes were associated with biosynthesis and metabolic pathways and circadian rhythm. Strikingly, we detected a significant increase in Myog expression (log2 fold change = 18.93, FDR q-value = 1.54x10-12) in sciatic nerve of 18-month-old mice, before up-regulation in muscle was observed. We identified seven clusters of genes with similar expression patterns across groups. Functional enrichment analysis of these clusters revealed biological processes that may be implicated in sarcopenia initiation including extracellular matrix organization and circadian regulation of gene expression.

ConclusionsGene expression changes in mouse peripheral nerve can be detected prior to overt clinical onset of sarcopenia. These early molecular changes we report shed a new light on biological processes that may be implicated in sarcopenia initiation and pathogenesis. Future studies will validate which of the key changes we reported have disease modifying and/or biomarker potential.


title: Patterns of variation in canal and root number in human post-canine teeth

abstract: Descriptive morphology of tooth roots traditionally focuses on number of canals and roots. However, how or if canal and root number are related is poorly understood. While it is often assumed that canal number is concomitant with root number and morphology, in practice canal number and morphology do not always covary with external root features. To investigate the relationship between canal and root number, fully developed, adult post-canine teeth were examined and quantified from medical computerized tomography scans from a global sample of 945 modern humans. We tested the hypotheses that root and canal number do not follow a 1:1 ratio, that canal to root ratios differ between teeth, and that canal to root ratios differ across populations. Results indicate that not only is root number dependent on canal number, but that this relationship become more variable as canal number increases, varies both between individual teeth and by population, and changes as populations increase in distance from Sub-Saharan Africa. These results show that the ratio of canal number to root number is an important indicator of variation in dental phenotypes.

category: developmental biology


title: Windborne migration amplifies insect-mediated pollination services

abstract: Worldwide, hoverflies (Syrphidae: Diptera) provide crucial ecosystem services (ES) such as pollination and biological pest control. Although many hoverfly species exhibit migratory behavior, the spatiotemporal facets of these movement dynamics and their ES implications are poorly understood. In this study, we use long-term (16 yr) trapping records, trajectory analysis and intrinsic (i.e., isotope, genetic, pollen) markers to describe migration patterns of the hoverfly Episyrphus balteatus in China. Our work reveals long-range, windborne migration with spring migrants originating in northern China and exhibiting return migration during autumn. Given the substantial night-time dispersal of E. balteatus, this species possibly adopts a  dual migration strategy. The extensive genetic mixing and high genetic diversity of E. balteatus populations underscore its adaptive capacity to environmental disturbances e.g., climate change. Pollen markers and molecular gut-analysis further illuminate how E. balteatus visits min. 1,012 flowering plant species (39 orders) over space and time. By thus delineating E. balteatus trans-regional movements and pollination networks, we advance our understanding of its migration ecology and facilitate the design of targeted strategies to conserve and enhance its ecosystem services.

category: ecology


title: Discovery of a new but established population of the guppy in Germany

abstract: Feral populations of tropical fish species in temperate climates like Central Europe are a rare but repeatedly observed phenomenon. Due to the influence of industrial or geothermal heated water, released tropical fish may be able to survive harsh winter conditions. Here we characterize a newly discovered thermally polluted river, with an established population of the guppy (Poecilia reticulata) co-occurring with native species. Through a mark-recapture approach, we estimated the population size of the guppies close to the warm water inflow to be around 2000 individuals during summer and we further provide descriptive demographics of this population which allow us to assume it is well established in that river. Further, we found some of the sampled specimen being parasitized by Camallanus roundworms, thus showing the guppies host potential for this genus of internal parasites. The popularity and widespread distribution of guppies as ornamental fish often leads to their intentional or unintentional release into the wild where they are often pioneer species in anthropogenically heavily modified habitats. Guppies threaten native species through niche competition and transmission of diseases. Accordingly, early awareness and knowledge on the status of non-native populations is crucial for effective management strategies.


title: COVID-19 infection enhances susceptibility to oxidative-stress induced parkinsonism

abstract:
BackgroundViral induction of neurological syndromes has been a concern since parkinsonian-like features were observed in patients diagnosed with encephalitis lethargica subsequent to the 1918 influenza pandemic. Given the similarities in the systemic responses following SARS-CoV-2 infection with those observed after pandemic influenza, there is a question if a similar syndrome of post-encephalic parkinsonism could follow COVID-19 infection.

ObjectivesTo determine if prior infection with SARS-CoV-2 increased sensitivity to a mitochondrial toxin known to induce parkinsonism.

MethodshACE2 mice were infected with SARS-CoV-2 to induce mild to moderate disease. After 31 days recovery, mice were administered a non-lesion inducing dose of the parkinsonian toxin MPTP. Subsequent neuroinflammation and SNpc dopaminergic neuron loss was determined and compared to SARS-CoV-2 or MPTP alone.

ResultshACE2 mice infected with SARS-CoV-2 or MPTP showed no SNpc DA neuron loss following MPTP. In mice infected and recovered from SARS-CoV-2 infection, MPTP induced a 23% or 19% greater loss of SNpc dopaminergic neurons than SARS-CoV-2 or MPTP, respectively (p{square}<{square}0.05).

Examination of microglial activation showed a significant increase in the number of activated microglia in the SARS-CoV-2 + MPTP group compared to SARS-CoV-2 or MPTP alone.

ConclusionsOur observations have important implications for long-term public health, given the number of people that have survived SARS-CoV-2 infection as well as for future public policy regarding infection mitigation. However, it will be critical to determine if other agents known to increase risk of PD also have synergistic effects with SARS-CoV-2 and if are abrogated by vaccination.

FundingThis work was supported by grant from the State of North Carolina (PS, JE, DOR, RJS) and R21 NS122280 (RJS).


title: Development and optimisation of a high-throughput screening assay for in vitro anti-SARS-CoV-2 activity: evaluation of 5676 phase 1 passed structures

abstract:
Although vaccines are currently used to control the coronavirus disease 2019 (COVID-19) pandemic, treatment options are urgently needed for those who cannot be vaccinated and for future outbreaks involving new severe acute respiratory syndrome coronavirus virus 2 (SARS-CoV-2) strains or coronaviruses not covered by current vaccines. Thus far, few existing antivirals are known to be effective against SARS-CoV-2 and clinically successful against COVID-19.

As part of an immediate response to the COVID-19 pandemic, a high-throughput, high content imaging-based SARS-CoV-2 infection assay was developed in VeroE6-eGFP cells and was used to screen a library of 5676 compounds that passed phase 1 clinical trials. Eight candidates (nelfinavir, RG-12915, itraconazole, chloroquine, hydroxychloroquine, sematilide, remdesivir, and doxorubicin) with in vitro anti-SARS-CoV-2 activity in VeroE6-eGFP and/or Caco-2 cell lines were identified. However, apart from remdesivir, toxicity and pharmacokinetic data did not support further clinical development of these compounds for COVID-19 treatment.

category: immunology


title: Genomic rearrangements of mobile genetic elements associated with carbapenem resistance of Acinetobacter baumannii

abstract: With the excessive genome plasticity, Acinetobacter baumannii has the capability to acquire and disseminate antimicrobial resistance genes that are often associated with mobile genetic elements (MGE). Analyzing the genetic environment of resistance genes often provides valuable information on the origin, emergence, evolution and spread of resistance. Thus, we characterized the genomic features of some clinical isolates of carbapenem-resistant A. baumannii to understand the role of diverse MGE and their genetic context that are responsible for the dissemination of carbapenem resistance genes. For this, a total of 17 clinical isolates of A. baumannii obtained from multiple hospitals in India between the years 2018 and 2019 were analysed. Antimicrobial resistance determinants, genetic context of resistance genes and molecular epidemiology were studied using whole genome sequencing. A high prevalence of blaOXA-23 was observed followed by the presence of dual carbapenemase, blaOXA-23 and blaNDM. Three novel Oxford sequence types were identified. Majority of the isolates belonged to dominant clone, IC2 followed by less prevalent clones such as IC7 and IC8. Complex diverse AbaR4 like and AbGRI-like islands belonging to IC2 lineage were identified. To the best of our knowledge, this is the first study that provides a comprehensive profiling of resistance islands along with the MGE, acquired antimicrobial resistance genes and the distribution of clonal lineages of carbapenem resistant A. baumannii from India.

category: microbiology


title: Rapid aggregation of Staphylococcus aureus in synovial fluid is influenced by synovial fluid concentration, viscosity, and fluid dynamics-with evidence of polymer bridging

abstract:
Early bacterial survival in the post-surgical joint is still a mystery. Recently, synovial fluid-induced aggregation was proposed as a potential mechanism of bacterial protection upon entry into the joint. As synovial fluid is secreted back into the joint cavity following surgery, rapid fluctuations in synovial fluid concentration, composition, and viscosity occur. These changes, along with fluid movement from post-operative joint motion, will modify the environment and potentially affect the kinetics of aggregate formation. Through this work, we sought to evaluate the influence of exposure time, synovial fluid concentration, viscosity, and fluid dynamics on aggregation. Furthermore, we aimed to elucidate the primary mechanism of aggregate formation by assessing the interaction of bacterial adhesins with synovial fluid polymer, fibrinogen. Following incubation in each simulated post-operative joint condition, the aggregates were imaged using confocal microscopy. Our analysis revealed the formation of two distinct aggregate phenotypes dependent on whether the incubation was conducted under static or dynamic conditions. Using a surface adhesin mutant, we have narrowed down the genetic determinants for synovial fluid aggregate formation and identified essential host polymers required. We report here that synovial fluid-induced aggregation is influenced by various changes specific to the post-surgical joint environment. While we now have evidence that select synovial fluid polymers facilitate bridging aggregation through essential bacterial adhesins, we suspect that their utility is limited by the increasing viscosity under static conditions. Furthermore, dynamic fluid movement recovers the ability of the bacteria with present surface proteins to aggregate under high viscosity conditions, yielding large, globular aggregates.

ImportanceInfection is a major complication of knee and hip joint replacement surgery which is used to treat arthritis or joint damage. We have shown that Staphylococcus aureus, a common bacterial pathogen, aggregates upon contact with synovial fluid. Within seconds, the bacterial cells will interact with synovial fluid polymers in the joint fluid through their cell wall adhesins. The rapid formation of these aggregates likely aids in early bacterial survival in the joint-potentially contributing to the likelihood of developing an infection. By strengthening our basic understanding of the mechanics of synovial fluid aggregate formation under clinically relevant conditions, we hope to expand the knowledge of how to prevent or disrupt aggregation and reduce and more successfully treat these joint infections.


title: Synergistic Activity of Repurposed Peptide Drug Glatiramer Acetate with Tobramycin Against Cystic Fibrosis Pseudomonas aeruginosa

abstract: Pseudomonas aeruginosa is the most common pathogen infecting the lungs of people with cystic fibrosis (CF), causing both acute and chronic infections. Intrinsic and acquired antibiotic resistance, coupled with the physical barriers resulting from desiccated CF sputum, allow P. aeruginosa to colonise and persist in spite of antibiotic treatment. As well as the specific difficulties in eradicating P. aeruginosa from CF lungs, P. aeruginosa is also subject to the wider, global issue of antimicrobial resistance. Glatiramer acetate (GA) is a peptide drug, used in the treatment of multiple sclerosis (MS), which has been shown to have moderate anti-pseudomonal activity. Other antimicrobial peptides (AMPs) have been shown to be antibiotic resistance breakers; potentiating the activities of antibiotics when given in combination restoring and/or enhancing antibiotic efficacy. Growth, viability, minimum inhibitory concentration (MIC)-determination and synergy analysis showed that GA improved the efficacy of TOB against reference strains of P. aeruginosa, reducing TOB MICs and synergising with the aminoglycoside. This was also the case for clinical strains from people with CF. GA significantly reduced the concentration of TOB required to inhibit 50% (MIC50) of viable cells (from 1.69 [95%CI 0.26-8.97] to 0.62 [95%CI 0.15-3.94] mg/L, p=0.002) and inhibit 90% (MIC90) (from 7.00 [95%CI 1.18-26.50] to 2.20 [95%CI 0.99-15.03] mg/L, p=0.001) compared with TOB-only. Investigating mechanisms of GA activity showed that GA resulted in significant disruption of outer membranes, depolarisation of cytoplasmic membranes and permeabilisation of P. aeruginosa and was the only agent tested (including cationic AMPs) to significantly affect all three.


title: Two broadly conserved families of polyprenyl-phosphate transporters

abstract:
Peptidoglycan and virtually all surface glycopolymers in bacteria are built in the cytoplasm on the lipid carrier undecaprenyl-phosphate (UndP). These UndP-linked precursors are transported across the membrane and polymerized or directly transferred to surface polymers, lipids, or proteins. UndP is then flipped to regenerate the pool of cytoplasmic-facing UndP. The identity of the flippase that catalyzes transport has eluded identification for decades. Here, using the antibiotic amphomycin that targets UndP, we discovered two broadly conserved families that catalyze UndP recycling. One (UptA) is a member of the DedA superfamily; the other (PopT) contains the domain DUF368. We show that family members from gram-positive and gram-negative bacteria catalyze UndP transport in Bacillus subtilis. Inhibitors of these flippases could potentiate the current arsenal of cell envelope-targeting antibiotics.

One Sentence SummaryWe define two transporter families that recycle the universal lipid carrier in surface glycopolymer biogenesis.


title: Comprehensive comparison between azacytidine and decitabine treatment in an acute myeloid leukemia cell line

abstract: Azacytidine (AzaC) and decitabine (AzadC) are cytosine analogs that covalently trap DNA methyltransferases, which place the important epigenetic mark 5-methyl-2-deoxycytidine by methylating 2-deoxycytidine (dC) at the C5 position. AzaC and AzadC are used in the clinic as antimetabolites to treat myelodysplastic syndrome and acute myeloid leukemia and are explored against other types of cancer. Although their principal mechanism of action is known, the downstream effects of AzaC and AzadC treatment are not well understood and the cellular prerequisites that determine sensitivity towards AzaC and AzadC remain elusive. Here, we investigated the effects and phenotype of AzaC and AzadC exposure on the acute myeloid leukemia cell line MOLM-13. We found that while AzaC and AzadC share many effects on the cellular level, including decreased global DNA methylation, increased formation of DNA double strand breaks, transcriptional downregulation of important oncogenes and similar changes on the proteome level, AzaC failed in contrast to AzadC to induce apoptosis in MOLM-13. The only cellular marker that correlated with this clear phenotypical outcome was the level of hydroxy-methyl-dC, an additional epigenetic mark that is placed by TET enzymes and repressed in cancer cells. Whereas AzadC increased hmdC substantially in MOLM-13, AzaC treatment did not result in any increase at all. This suggests that hmdC levels in cancer cells should be monitored as a response towards AzaC and AzadC and considered as a biomarker to judge whether AzaC or AzadC treatment leads to cell death in leukemic cells.

category: molecular biology


title: Splicing inactivation generates hybrid mRNA-snoRNA transcripts targeted by cytoplasmic RNA decay

abstract: Many small nucleolar RNAs are processed from introns of host genes, but the importance of splicing for proper biogenesis and the fate of the snoRNAs is not well understood. Here we show that inactivation of splicing factors or mutation of splicing signals leads to the accumulation of incorrectly processed hybrid mRNA-snoRNA transcripts (hmsnoRNA). HmsnoRNAs are processed to the mature 3'-end of the snoRNA by the nuclear exosome and bound by snoRNP proteins, but they are targeted by the major cytoplasmic mRNA decay pathway due to their mRNA-like 5'-extensions. These results show that completion of splicing is required for full and accurate processing of intron-encoded snoRNAs and that splicing defects lead to degradation of hybrid mRNA-snoRNA species by cytoplasmic decay, uncovering a novel aspect of the importance of splicing for the biogenesis of intron encoded snoRNAs.


title: Real-time luminescence enables continuous drug-response analysis in adherent and suspension cell lines

abstract: The drug-induced proliferation (DIP) rate is a metric of in vitro drug response that avoids inherent biases in commonly used metrics such as 72h viability. However, DIP rate measurements rely on direct cell counting over time, a laborious task that is subject to numerous challenges, including the need to fluorescently label cells and automatically segment nuclei. Moreover, it is incredibly difficult to directly count cells and accurately measure DIP rates for cell populations in suspension. As an alternative, we use real-time luminescence measurements derived from the cellular activity of NAD(P)H oxidoreductase to efficiently estimate drug response in both adherent and suspension cell populations to a panel of known anticancer agents. For the adherent cell lines, we collect both luminescence reads and direct cell counts over time simultaneously to assess their congruency. Our results demonstrate that the proposed approach significantly speeds up data collection, avoids the need for cellular labels and image segmentation, and opens the door to significant advances in high-throughput screening of anticancer drugs.


title: Global chromatin mobility induced by a DSB is dictated by chromosomal conformation and defines the outcome of Homologous Recombination.

abstract: Repair of DNA double-strand breaks (DSBs) is crucial for genome integrity. A conserved response to DSBs is an increase in chromatin mobility that can be local, at the site of the DSB, or global, at undamaged regions of the genome. Here we address the function of global chromatin mobility during homologous recombination (HR) of a single, targeted, controlled DSB. We set up a system that tracks HR in vivo over time and show that two types of DSB-induced global chromatin mobility are involved in HR, depending on the position of the DSB. In spatial proximal regions such as the pericentromeric region, a DSB therein induces global mobility that depends solely on H2A(X) phosphorylation and accelerates repair kinetics, but is not essential. In contrast, the global mobility induced by a DSB away from the centromere, becomes essential for HR repair and is triggered by homology search through a mechanism that depends on H2A(X) phosphorylation, checkpoint progression and Rad51. Our data demonstrate that global mobility is governed by chromosomal conformation and differentially coordinates repair by HR.


title: A Mouse Model with Complete Penetrance for Atrioventricular Septal Defect/Complete AV Canal

abstract: Complete Atrioventricular Septal Defect/complete AV canal (AVSD/ cAVC) occurs via defective formation of the Dorsal Mesenchymal Protrusion (DMP) which grows from the interior dorsal wall of the fetal heart. The DMP is derived from cells of the second heart field (SHF). AVSD occurs in about 1/10,000 births in the general population, but is 2000 times more prevalent in individuals with Down syndrome (DS). The low penetrance of AVSD in the general population remains a fundamental challenge to investigation of the genetic and developmental basis for this condition. Analysis of the etiology of this condition in mouse models is limited by the fact that the developmental events producing the DMP begin around embryonic day 9 (E9) but AVSD cannot be diagnosed until E14, and no model has been described previously in which AVSD occurs in 100% of embryos. We describe a trisomic mouse DS model with conditional mutations in the SHH pathway that produces AVSD with 100% penetrance. This new mouse model can serve as an important tool to understand the mechanisms underlying AVSD pathogenesis.


title: Exploring the lncRNA localization landscape within the retinal pigment epithelium under normal and stress conditions

abstract: Long noncoding RNAs (lncRNAs) are emerging as a class of genes whose importance has yet to be fully realized. It is becoming clear that the primary function of lncRNAs is to regulate gene expression, and they do so through a variety of mechanisms that are critically tied to their subcellular localization. Although most lncRNAs are poorly understood, mapping lncRNA subcellular localization can provide a foundation for understanding these mechanisms. Here, we present an initial step toward uncovering the localization landscape of lncRNAs in the human retinal pigmented epithelium (RPE) using high throughput RNA-Sequencing (RNA-Seq). To do this, we differentiated human induced pluripotent stem cells (iPSCs) into RPE, isolated RNA from nuclear and cytoplasmic fractions, and performed RNA-Seq on both. Furthermore, we investigated lncRNA localization changes that occur in response to oxidative stress. We discovered that, under normal conditions, most lncRNAs (76%) are seen in both the nucleus and the cytoplasm to a similar degree, but of the transcripts that are highly enriched in one compartment, more are nuclear (18.6%) than cytoplasmic (5.6%). Interestingly, under oxidative stress conditions, we observed an increase in lncRNA localization in both nuclear (23.5%) and cytoplasmic (9.7%) fractions. In addition, we found that nuclear localization was partially attributable to the presence of previously described nuclear retention motifs, while adenosine to inosine (A-to-I) RNA editing appeared to play a very minimal role. Our findings regarding lncRNA localization in the RPE provide two avenues for future research: 1) how lncRNAs function in the RPE, and 2) how one environmental factor, in isolation, may play a role in retinal disease pathogenesis.

category: genetics


title: SKN-1 is a metabolic surveillance factor that monitors amino acid catabolism to control stress resistance

abstract: The deleterious potential to generate oxidative stress and damage is a fundamental challenge to metabolism. The oxidative stress response transcription factor, SKN-1/NRF2, can sense and respond to changes in metabolic state, although the mechanism and physiological consequences of this remain unknown. To explore this connection, we performed a genetic screen in C. elegans targeting amino acid catabolism and identified multiple metabolic pathways as regulators of SKN-1 activity. We found that genetic perturbation of the conserved amidohydrolase T12A2.1/amdh-1 activates a unique subset of SKN-1 regulated detoxification genes. Interestingly, this transcriptional program is independent of canonical P38-MAPK signaling components but requires the GATA transcription factor ELT-3, nuclear hormone receptor NHR-49, and mediator complex subunit MDT-15. This activation of SKN-1 is dependent on upstream histidine catabolism genes HALY-1 and Y51H4A.7/UROC-1 and may occur through accumulation of a catabolite, 4-imidazolone-5-propanoate (IP). Triggering SKN-1 activation results in a physiological trade off of increased oxidative stress resistance but decreased survival to heat stress. Together, our data suggest that SKN-1 is a key surveillance factor which senses and responds to metabolic perturbations to influence physiology and stress resistance.


title: Millennium-Old Pathogenic Mendelian Mutation Discovery for MultipleOsteochondromas from a Gaelic Medieval Graveyard.

abstract: Only a limited number of genetic diseases are diagnosable in archaeological individuals and none have had causal mutations identified in genome-wide screens. Two individuals from the Gaelic Irish Medieval burial ground of Ballyhanna, Co. Donegal, showed evidence of bone tumors consistent with the autosomal dominant condition multiple osteochondromas. Genome sequencing of the earlier individual uncovered a missense mutation in the second exon of EXT1, a specific lesion that has been identified in several modern patients. The later individual lacked this but displayed a novel frameshift mutation leading to a premature stop codon and loss of function in the same gene. These molecular confirmations of a paleopathological diagnosis within a single rural ancient context are surprisingly disjunct, given the observation of clusters of this disease in modern isolated populations and a de novo mutation rate of only 10%.


title: Telomeres are shorter in wild Saccharomyces cerevisiae isolates than in domesticated ones

abstract: Telomeres are ribonucleoproteins that cap chromosome-ends and their DNA length is controlled by counteracting elongation and shortening processes. The budding yeast Saccharomyces cerevisiae has been a leading model to study telomere DNA length control and dynamics. Its telomeric DNA is maintained at a length that slightly varies between laboratory strains, but little is known about its variation at the species level. The recent publication of the genomes of over 1000 S. cerevisiae strains enabled us to explore telomere DNA length variation at an unprecedented scale. Here, we developed a bioinformatic pipeline (YeaISTY) to estimate telomere DNA length from whole-genome-sequences and applied it to the sequenced 1011 S. cerevisiae collection. Our results revealed broad natural telomere DNA length variation among the isolates. Notably, telomere DNA length is shorter in those derived from wild rather than domesticated environments. Wild isolates are enriched in loss-of-function mutations in genes known to regulate telomere DNA length and the return of domesticated yeasts to a wild habitat coincides with shorter telomeres. Moreover, telomere DNA length variation is associated with mitochondrial metabolism, and this association is driven by wild strains. Overall, these findings suggest that budding yeasts telomere DNA length regulation might be shaped by ecological life-styles.


title: Enhancing high-temperature degradation of polyethylene terephthalate through a synergistic division of enzyme labour between a solid-degrading thermostable cutinase and a reaction intermediate-degrading thermostable carboxylesterase

abstract: Cutinases degrade PET (polyethylene terephthalate) into various degradation intermediates (DIs) such as OET (oligoethylene terephthalate), BHET (bis-hydroxyethyl terephthalate), and MHET (mono-hydroxyethyl terephthalate), and eventually into TPA (terephthalic acid), which is the terminal product of degradation. Unlike PET, which is insoluble, TPA and the DIs are sparingly soluble in water. This causes both DIs and TPA to be partitioned into aqueous solution, where DIs accumulate without undergoing significant further degradation, despite being better substrates of cutinase than solid PET. This frustrates the creation of a circular economy involving PET and TPA (since TPA must be separated from DIs before re-condensation into PET). We argue that the non-degradation of DIs owes to cutinase becoming progressively depleted from solution, through binding to solid PET. This creates a conundrum, in that degradation of PET is anticipated to be inversely correlated with degradation of DIs (at least while solid PET remains available to deplete cutinase from solution), causing any improvement of the cutinases PET-binding efficiency to only further ensure non-degradation of released DIs. Here, we propose the deployment of a second DI-degrading enzyme; one that remains in solution, and acts as an  assistant to the  master PET-invading cutinase acting upon PETs surface. We demonstrate that one such dual-enzyme system, consisting of a thermostable Thermus thermophilus carboxylesterase (TTCE), characterized here for the first time, and the already-used thermostable leaf-branch compost cutinase (LCC), allows complete degradation of all products of PET hydrolysis into TPA in solution, at 60 {degrees}C, even in the presence of residual solid PET.

category: biochemistry


title: Describing Chemical Kinetics in the Time Dimension with Mean Reaction Time

abstract: The chemical kinetics is such a fundamental topic in chemistry. By analyzing how the experimental conditions and parameters influence the reaction rate, chemists have deciphered the molecular details on how the chemical reactions occur. The quantitative analysis of the reaction rate requires the formulation of the rate equation, which describes the dependence of the reaction rate on the concentrations of the reactants and also the rate constants of the elementary steps. Though the methods to derive the rate equation from the kinetic model have been known for a century, it is still mathematically challenging to derive the rate equation for complex reactions with multiple steps, which requires a solution for simultaneous differential equations. Therefore, chemists frequently resort to the steady-state approximation or the numerical simulation. One way to avoid the mathematical difficulty is to describe the chemical kinetics in the time dimension. Describing the mean reaction time, the average of the time required for the completion of the chemical reaction, using the elementary rate constants of the kinetic model is much simpler mathematically than deriving the rate equation for the kinetic model. Here, we describe the basic rules for the derivation of the formula of the mean reaction time, derive the generalized equation for the mean reaction time, and analyze the components of the formula to determine how the individual steps in the complex reaction contribute to the mean reaction time. Being the ensemble-averaged value, the mean reaction time does not provide the information on the actual distribution of the reaction time of individual chemical entity. However, the formula of the mean reaction time reveals invaluable insights on how the energy levels of the ground state and the transition states affect the kinetics of the complex reaction and offers a way to identify the most time-consuming process of the complex reaction in a straightforward manner. We also apply the mean reaction time to enzyme kinetics and demonstrate that one can derive the expressions of the kinetic parameters (kcat/KM and kcat) in a surprisingly simple way even without resorting to the steady-state application.


title: A mono- and intralink filter (mi-filter) to improve false-discovery rates in cross-linking mass spectrometry data

abstract: Cross-Linking Mass Spectrometry (XL-MS) has become an indispensable tool for the emerging field of systems structural biology over the recent years. However, the confidence in individual protein-protein interactions (PPIs) depends on the correct assessment of individual inter protein cross-links. This can be challenging, in particularly in samples where relatively few PPIs are detected, as is often the case in complex samples containing low abundant proteins or in in-vivo settings. In this manuscript we are describing a novel mono- and intralink filter (mi-filter) that is applicable to any kind of crosslinking data and workflow. It stipulates that only proteins for which at least one monolink or intra-protein crosslink has been identified within a given dataset are considered for an inter-protein cross-link and therefore participate in a PPI. We show that this simple and intuitive filter has a dramatic effect on different types of crosslinking-data ranging from single protein complexes, over medium-complexity affinity enrichments to proteome-wide cell lysates and significantly lowers the number of false-positive identifications resulting in improved false-discovery rates for inter-protein links in all these types of XL-MS data.


title: Proteostasis in ice: The role of heat shock proteins and ubiquitin in the freeze tolerance of the intertidal mussel, Mytilus trossulus

abstract: The bay mussel, Mytilus trossulus, is one of the few animals that can survive internal ice formation. Freeze tolerant intertidal animals, like M. trossulus, may freeze and thaw many times during the winter, depending on air and ocean temperatures. Freezing can cause protein denaturation, leading to an induction of the heat shock response with expression of proteins like HSP70, and an increase in ubiquitin conjugated proteins. There has been little work on the mechanisms of freeze tolerance in intertidal species, limiting our understanding of this survival strategy. Additionally, this limited research has focused solely on the effects of single freezing events, but the act of repeatedly crossing the freezing threshold may present novel physiological or biochemical stressors that have yet to be discovered. We predicted that repeated freeze exposures would increase mortality, upregulate HSP70 expression, and increase ubiquitin conjugates in mussels, relative to single, prolonged freeze exposures. Mytilus trossulus from Vancouver, Canada were repeatedly frozen for a combination of 1 x 8 hours, 4 x 2 hours, or 2 x 4 hours. We then compared mortality, HSP70 expression, and ubiquitin quantity across experimental groups. We found a single 8-hour freeze caused significantly more mortality than repeated freeze-thaw cycles. We also found that HSP70 and ubiquitin expression was upregulated exclusively after freeze-thaw cycles, suggesting that freeze-thaw cycles offer a period of damage repair between freezes. This indicates that freeze-thaw cycles, which happen naturally in the intertidal, are crucial for M. trossulus survival in sub-zero temperatures.


title: In silico structure-based analysis of the predicted protein-protein interaction of Syntaxin-18, a putative receptor of  Peregrinus maidis  Ashmead (Hemiptera: Delphacidae) with Maize mosaic virus glycoprotein

abstract: The corn planthopper, Peregrinus maidis, is a widely distributed insect pest which serves as a vector of two phytopathogenic viruses, Maize mosaic virus (MMV) and Maize stripe virus (MStV). It transmits the viruses in a persistent and propagative manner. MMV is an alphanucleorhabdovirus with a negative-sense, single-stranded (ss) RNA unsegmented genome. One identified insect vector protein that may serve as receptor to MMV is Syntaxin-18 (PmStx18) which belongs to the SNAREs (soluble N-ethylmaleimide-sensitive factor attachment protein receptors). SNAREs play major roles in the final stage of docking and subsequent fusion of diverse vesicle-mediated transport events. In this work, in silico analysis of the interaction of MMV glycoprotein (MMV G) and PmStx18 was performed. Various freely available protein-protein docking web servers were used to predict the 3D complex of MMV G and PmStx18. Analysis and protein-protein interaction (PPI) count showed that the complex predicted by the ZDOCK server has the highest number of interaction and highest affinity, as suggested by the calculated solvation free energy gain upon formation of the interface ({Delta}iG = -31 kcal/mol). Molecular dynamics simulation of the complex revealed important interactions at the interface over the course of 50 ns. This is the first in silico analysis performed for the interaction on a putative receptor of P. maidis and MMV G. The results of the protein-protein interaction prediction provide novel information for studying the role of STX18 in the transport, docking and fusion events involved in virus particle transport in the insect vector cells and its release.


title: 2'-O	-methylation of the second transcribed nucleotide within mRNA 5' cap impacts protein production level in a cell specific manner and contributes to RNA immune evasion

abstract:
In higher eukaryotes, m7G-adjacent nucleotides undergo extensive modifications. Ribose of the first or first and second transcribed nucleotides can be subjected to 2-O-methylation to form cap1 or cap2, respectively. Additionally, when the first transcribed nucleotide is adenosine, it can not only undergo 2-O-methylation but can also be methylated at position N6 forming N6,2-O-dimethyladenosine (m6Am). Recent studies have shed some light on the functions of cap1, showing that cap1 in mammalian cells plays a crucial role in distinguishing between  self and  non-self RNA during viral infection. Here, we attempted to understand the impact of other cap methylations on RNA-related processes. Therefore, we synthesized tetranucleotide cap analogs and used them for efficient co-transcriptional RNA capping during in vitro transcription. Using this tool, we found that 2-O-methylation of the second transcribed nucleotide within the mRNA 5 cap influences protein production levels in a cell-specific manner. The presence of this modification can strongly hamper protein biosynthesis or do not influence protein production levels. Interestingly, 2-O-methylation of the second transcribed nucleotide as well as the presence of N6,2-O-dimethyladenosine as the first transcribed nucleotide serve as determinants that define transcripts as  self and contribute to transcript escape from the host innate immune response. Additionally, cap methylation status does not influence transcript affinity towards translation initiation factor 4E or in vitro susceptibility to decapping by DCP2; however what we observe is resistance of RNA capped with cap2 to DXO-mediated decapping and degradation.

Significance StatementMethylation of mRNA cap structure regulates protein biosynthesis in a cell-dependent manner. Among the three known m7G cap modifications, the 2-O-methylation is dominant. 2-O-methylation of the first transcribed nucleotide can boost protein production, whereas the same modification of the second transcribed nucleotide can strongly decrease translation. Interestingly, we show that in the JAWS II cell line, 2-O-methylation of mRNA cap had a prominent impact on the composition of the protein interactome associated with the RNA bearing mentioned modifications. Further analysis revealed that 2-O-methylation of the second transcribed nucleotide and N6-methylation of adenosine as the first transcribed nucleotide serve as determinants defining transcripts as  self and contribute to transcript escape from the host innate immune response.


title: Structural features of mTORC2 that control substrate-specific activities.

abstract: mTORC2 is a multi-subunit kinase complex that is central to multiple essential signaling pathways. Two core subunits, Rictor and mSin1 distinguish it from its relative, mTORC1 and support context-dependent phosphorylation of its substrates. mTORC2 structures have been determined previously, however, important questions remain, particularly regarding structural determinants of substrate specificity and context dependent activities. We used cryo-EM to obtain high resolution structures of the human mTORC2 apo-complex, as well as structures in the presence of substrates, Akt and SGK1. Specific predictions suggested by substrate-induced structural changes were tested in functional assays. First, side chain interactions between Rictor and mTOR that prevent recruitment of mTORC1 substrates and confer resistance to the mTORC1 inhibitor rapamycin were visualized for the first time in the apo-state, demonstrating the steric occlusion that prevents mTORC2 interaction with mTORC1 substrates and rapamycin. Also in the apo-state, mSin1 was seen to form extensive contacts with Rictor, including a pair of short -helices nestled between two Rictor helical repeat clusters, followed by an extended strand, which makes multiple weak contacts with Rictor helical cluster 1. In co-complex structures, SGK1, but not Akt, markedly altered the conformation of the mSin1 N-terminal extended strand, disrupting multiple weak interactions while inducing a large rotation of mSin1/Arg-83, which comes to interact with a negative patch within Rictor. Mutation of Arg-83 to Ala selectively disrupted mTORC2 dependent phosphorylation of SGK1 but not of Akt, supporting context-dependent substrate selection. These findings provide new structural and functional insights into mTORC2 specificity and context-dependent activities.


title: Activating ligands of Uncoupling protein 1 identified by rapid membrane protein thermostability shift analysis

abstract: Uncoupling protein 1 (UCP1) catalyzes mitochondrial proton leak in brown adipose tissue for heat production, and may combat metabolic disease if activated in humans. During the adrenergic stimulation of brown adipocytes, free fatty acids generated from lipolysis activate UCP1 via an unclear interaction. Here, we have utilized membrane protein thermostability shift analysis to characterize the interaction of activating molecules with purified UCP1. We reveal that activators influence the protein through a specific destabilizing interaction, behaving as transport substrates that shift UCP1 to a less stable conformation of a transport cycle. Through the detection of specific stability shifts in screens, we identify novel activators, including the drug ibuprofen, where ligand analysis indicates a relatively wide structural specificity for interacting molecules. Ibuprofen induces UCP1 activity in liposomes and isolated brown fat mitochondria, but not in cultured brown adipocytes. Though the drug does induce activity in UCP1-expressing HEK293 cells, demonstrating that the targeting of UCP1 in cells by approved drugs is in principle achievable as a therapeutic avenue, but requires variants with more effective delivery in brown adipocytes.


title: Unraveling the Enzymatic Mechanism of the SARS-CoV-2 RNA-Dependent-RNA-Polymerase. An Unusual Active Site Leading to High Replication Rates.

abstract: Viral infection relies on the hijacking of cellular machineries to enforce the reproduction of the infecting virus and its subsequent diffusion. In this context the replication of the viral genome is a key step performed by specific enzymes, i.e. polymerases. The replication of SARS-CoV-2, the causative agent of the COVID-19 pandemics, is based on the duplication of its RNA genome, an action performed by the viral RNA-dependent-RNA polymerase. In this contribution, for the first time and by using two-dimensional enhanced sampling quantum mechanics/ molecular mechanics, we have determined the chemical mechanisms leading to the inclusion of a nucleotide in the nascent viral RNA strand. We prove the high efficiency of the polymerase, which lowers the activation free energy to less than 10 kcal/mol. Furthermore, the SARS-CoV-2 polymerase active site is slightly different from those found usually found in other similar enzymes, and particularly it lacks the possibility to enforce a proton shuttle via a nearby histidine. Our simulations show that this absence is partially compensate by lysine, whose proton assist the reaction opening up an alternative, but highly efficient, reactive channel. Our results present the first mechanistic resolution of SARS-CoV-2 genome replication and shed light on unusual enzymatic reactivity paving the way for future rational design of antivirals targeting emerging RNA viruses.


title: Meis1 supports leukemogenesis through stimulation of ribosomal biogenesis and Myc

abstract: The homeobox transcription factors HoxA9 and Meis1 are causally involved in the etiology of acute myeloid leukemia. While HoxA9 immortalizes cells, cooperation with Meis1 is necessary to induce malignancy. Here, we apply degron techniques to elucidate the leukemogenic contribution of Meis1. ChIP-seq demonstrated that Meis1 localized mainly to H3K27ac and H3K4me1 modified enhancers pre-bound by HoxA9. HoxA9 was epistatic to Meis1 as degradation of HoxA9 caused an immediate release of Meis1 from chromatin. Nascent-RNA sequencing revealed the Meis1 gene expression pattern to be dominated by Myc, ribosome biogenesis and rRNA synthesis. While Myc accounted for cell-cycle stimulation, it could not substitute the leukemogenic effects of Meis1. Enhanced ribosomal biogenesis was accompanied by elevated resistance against RNA polymerase I and translation blocking inhibitors without affecting steady-state protein synthesis. HoxA9 and Meis1 protein stability was controlled by casein kinase 2 (CK2). CK2 inhibition caused rapid degradation of HoxA9 and Meis1 suggesting a potentially exploitable regulatory pathway.

category: cancer biology


title: Platinum-Induced Mitochondrial OXPHOS Contributes to Cancer Stem Cell Enrichment in Ovarian Cancer

abstract:
BackgroundPlatinum based agents - cisplatin and carboplatin in combination with taxanes are used for the treatment of ovarian cancer (OC) patients. However, the majority of OC patients develop recurrent, platinum resistant disease that is uniformly fatal. Acute platinum treatment enriches for chemoresistant aldehyde dehydrogenase (ALDH) + ovarian cancer stem cells (OCSCs), which contribute to tumor recurrence and disease relapse. Acquired platinum resistance includes metabolic reprograming and switching to oxidative phosphorylation (OXPHOS). Chemosensitive cells rely on glycolysis while chemoresistant cells have the ability to switch between glycolysis and OXPHOS, depending on which pathway drives a selective advantage for growth and chemoresistance. High expression of genes involved in OXPHOS and high production of mitochondrial ROS are characteristics of OCSCs, suggesting that OCSCs favor OXPHOS over glycolysis. Based on connections between OCSCs, chemoresistance and OXPHOS, we hypothesize that platinum treatment induces changes in metabolism that contribute to platinum-induced enrichment of OCSCs.

MethodsThe effect of cisplatin on mitochondrial activity was assessed by JC1 staining and expression of OXPHOS genes by quantitative RTPCR. Cisplatin-induced changes in Sirtuin 1 (SIRT1) levels and activity were assessed by Western blot. Small molecule inhibitors of mitochondrial complex I and SIRT1 were used to determine if their enzymatic activity contributes to the platinum-induced enrichment of OCSCs. The percentage of ALDH+ OCSCs in OC cells and tumor tissue from xenograft models across different treatment conditions was analyzed using ALDEFLUOR assay and flow cytometry.

ResultsWe demonstrate that acute platinum treatment increases mitochondrial activity. Combined treatment of platinum agents and OXPHOS inhibitors blocks the platinum-induced enrichment of ALDH+ OCSCs in vitro and in vivo. Furthermore, platinum treatment increases SIRT1 levels and subsequent deacetylase activity, which likely contributes to the increase in platinum-induced mitochondrial activity.

ConclusionsThese findings on metabolic pathways altered by platinum-based chemotherapy have uncovered key targets that can be exploited therapeutically to block the platinum-induced enrichment of OCSCs, ultimately improving the survival of OC patients.


title: Lymphatic-Preserving Treatment Sequencing with Immune Checkpoint Inhibition Unleashes cDC1-Dependent Antitumor Immunity in HNSCC

abstract:
Immune checkpoint inhibition (ICI) with anti-CTLA-4 and anti-PD-1 has revolutionized oncology; however, response rates remain limited in most cancer types, highlighting the need for more effective immune oncology (IO) treatment strategies. Paradoxically, head and neck squamous cell carcinoma (HNSCC), which bears a mutational burden and immune infiltrate commensurate with cancers that respond robustly to ICI, has demonstrated no response to anti- CTLA-4 in any setting or to anti-PD-1 for locally-advanced disease. Scrutiny of the landmark clinical trials defining current IO treatments in HNSCC reveals that recruited patients necessarily received regional ablative therapies per standard of care, prompting us to hypothesize that standard therapies, which by design ablate locoregional lymphatics, may compromise host immunity and the tumor response to ICI. To address this, we employed tobacco-signature HNSCC murine models in which we mapped tumor-draining lymphatics and developed models for regional lymphablation with surgery or radiation. Remarkably, we found that lymphablation eliminates the tumor ICI response, significantly worsening overall survival and repolarizing the tumor- and peripheral-immune compartments. Mechanistically, within tumor-draining lymphatics, we observed an upregulation of cDC1 cells and IFN-I signaling, showed that both are necessary for the ICI response and lost with lymphablation. Ultimately, we defined rational IO sequences that mobilize peripheral immunity, achieve optimal tumor responses, confer durable immunity and control regional lymphatic metastasis. In sum, we provide a mechanistic understanding of how standard regional, lymphablative therapies impact the response to ICI, which affords insights that can be applied to define rational, lymphatic-preserving IO treatment sequences for cancer.

One Sentence SummaryDespite the promise of immune checkpoint inhibition, therapeutic responses remain limited, raising the possibility that standard of care treatments delivered in concert may compromise the tumor response; here, we provide a mechanistic understanding of how standard oncologic therapies targeting regional lymphatics impact the tumor response to immune-oncology therapy in order to define rational treatment sequences that mobilize systemic antitumor immunity, achieve optimal tumor responses, confer durable antitumor immunity, and control regional metastatic disease.

Graphical Abstract

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title: Therapeutic vulnerability to ATR inhibition in concurrent NF1 and ATRX-deficient high-grade solid tumors

abstract: Subsets of Neurofibromatosis Type 1 (NF1)-associated solid tumors have been shown to display high frequencies of ATRX mutations and presence of alternative lengthening of telomeres (ALT). In addition, a potential biologic vulnerability of ALT-positive cancer cells is ATR inhibition, a regulator of homologous recombination. We studied the phenotype of combined NF1 and ATRX deficiency in malignant solid tumors. For these experiments, cell lines derived from NF1-deficient sporadic glioblastomas (U251, SF188), a NF1-associated ATRX mutant glioblastoma cell line (JHH-NF1-GBM1), a NF1-derived sarcoma cell line (JHH-CRC65), and two NF1-deficient MPNST cell lines (ST88-14, NF90.8) were utilized. Cancer cells were treated with ATR inhibitors (AZD6738 and VE-822), in vitro, with or without a MEK inhibitor (AZD6244, selumetinib) or temozolomide. In contrast to the glioma cell line SF188, combined ATRX knockout (KO) and TERC KO led to ALT-like properties and sensitized U251 glioma cells to ATR inhibition (AZD6738 and VE-822) in vitro and in vivo. In addition, ATR inhibitors sensitized U251 cells to temozolomide, but not MEK inhibition (AZD6244), irrespective of ATRX level manipulation; whereas, the JHH-NF1-GBM1 cell line (ATRX loss/ALT-positive) demonstrated sensitivity to ATR inhibition (AZD6738), but not temozolomide. Similar effects were noted using the MPNST cell line NF90.8 after combined ATRX knockdown and TERC KO; however, not in the MPNST cell line ST88-14. Taken together, our study supports the feasibility of targeting the ATR pathway in subsets of NF1-deficient and associated tumors. Tumors with pre-existing ALT, or that subsequently develop ALT after ATRX downregulation, are particularly vulnerable to this therapeutic approach.


title: Epigenetic cell memory: The gene's inner chromatin modification circuit

abstract: Epigenetic cell memory allows distinct gene expression patterns to persist in different cell types despite a common genotype. Although different patterns can be maintained by the concerted action of transcription factors (TFs), it was proposed that long-term persistence hinges on chromatin state. Here, we study how the dynamics of chromatin state affect memory, and focus on a biologically motivated circuit motif, among histones and DNA modifications, that mediates the action of TFs on gene expression. Memory arises from time-scale separation among three circuits constituent processes: basal erasure, auto and cross-catalysis, and recruited erasure of modifications. When the two latter processes are sufficiently faster than the former, the circuit exhibits bistability and hysteresis, allowing active and repressed gene states to coexist and persist after TF stimulus removal. The duration of memory is stochastic with a mean value that increases as time-scale separation increases, but more so for the repressed state. This asymmetry stems from the cross-catalysis between repressive histone modifications and DNA methylation and is enhanced by the relatively slower decay rate of the latter. Nevertheless, TF-mediated positive autoregulation can rebalance this asymmetry and even confers robustness of active states to repressive stimuli. More generally, by wiring positively autoregulated chromatin modification circuits under time scale separation, long-term distinct gene expression patterns arise, which are also robust to failure in the regulatory links.

category: synthetic biology


title: Ecological insights into soil health according to the life-history traits and environment-wide associations of bacteria in agricultural soils

abstract: Soil health assessment may be enhanced by monitoring changes in bacterial populations that are indicators of various biological, physical, and chemical properties of soil. However, the lack of ecological information for many abundant bacteria in agricultural soils limits our understanding of indicator responses and, thus, their utility for guiding management. We identified bacterial indicators of twelve conventional measures of soil health, and tillage intensity, from a 16S rRNA gene-based survey of farmland across North America. We then analyzed trends according to bacterial life-history frameworks and an environment-wide association survey (EWAS) to gain ecological insights. Life-history traits were assessed using genomic traits inferred from taxonomic classifications and included: genome size, rrn copy number, and coding density. An EWAS was conducted using 89 studies of agricultural land management. Most bacterial indicators were positively correlated with biological measures and negatively correlated with physical and chemical measures of soil health, revealing broad differences in the way management shapes bacterial associations with soil health. High soil health ratings corresponded with life-history traits associated with metabolic dependency (smaller genome and lower coding density), while lower health scores corresponded with traits selected for by environmental instability and disturbance (larger genome and multiple rrn). Trade-offs in community-weighted genome size explained most variation in overall health score. EWAS confirmed the importance of disturbance-adapted bacterial indicators, underscoring the impacts of tillage on soil bacterial communities. These findings provide insights into the ecological relationships between bacterial indicators and soil health and illustrate new approaches for interpreting patterns in microbiome data.


title: SdiA, a Quorum Sensing Transcriptional Regulator, Enhanced the Drug Resistance of Cronobacter sakazakiiand Suppressed its Motility, Adhesion and Biofilm Formation

abstract:
Cronobacter sakazakii is a common foodborne pathogen, and the mortality rate of its infection is as high as 40-80%. Quorum sensing is a regulation system of bacterial density-dependent multigene expression and is an important regulatory mechanism involved in adhesion, biofilm formation and virulence. C. sakazakii contains a QS signal molecular receiver, which is the LuxR receptor homolog SdiA, but its regulatory mechanism in C. sakazakii QS has not been defined. Here, we further determined the effect of SdiA on the QS system of C. sakazakii. The SdiA gene in C. sakazakii was knocked out by gene editing technology, and the biological characteristics of the {Delta}sdiA gene deletion strain of C. sakazakii were studied, followed by transcriptome analysis to elucidate its effects. The results suggested that SdiA enhanced the drug resistance of C. sakazakii but diminished its motility, adhesion and biofilm formation ability and had no effect on its growth. Transcriptome analysis showed that the deletion of the SdiA gene upregulated the expression levels of D-galactose operon genes (including dgoR, dgoK, dgoA, dgoD and dgoT) and flagella-related genes (FliA and FliC) in C. sakazakii and downregulated the expression levels of related genes in the type VI secretion system (VasK gene was downregulated by 1.53-fold) and ABC transport system (downregulated by 1.5-fold), indicating that SdiA was related to the physiological metabolism of C. sakazakii. The results of this study may be useful for clarifying the pathogenic mechanism of C. sakazakii and provide a theoretical basis for controlling bacterial infection.

IMPORTANCECronobacter sakazakii, as an emerging opportunistic foodborne pathogen, was associated with sepsis, meningitis and necrotizing enterocolitis in neonates and infants, with a mortality rate of 40-80%. Quorum sensing plays an important regulatory role in the pathogenicity of C. sakazakii. Nevertheless, the regulatory mechanism of QS in C. sakazakii remains unknown. Here, we studied the QS transcriptional regulator SdiA of C. sakazakii. We revealed the regulatory mechanisms of SdiA in C. sakazakii cell adhesion, motility, biofilm formation and drug resistance. It was helpful to further explore the function of the SdiA gene, revealing the pathogenic mechanism of C. sakazakii. It will also provide a new target for therapeutic interventions targeting the pathogenicity of C. sakazakii and developing quorum-sensing inhibitors.


title: Perilipin 5 interacts with Fatp4 at membrane contact sites to promote lipid droplet-to-mitochondria fatty acid transport

abstract: Cells adjust their metabolism by remodeling membrane contact sites that channel metabolites to different fates. Lipid droplet (LD)-mitochondria contacts change in response to fasting, cold exposure, and exercise. However, their function and mechanism of formation have remained controversial. We focused on perilipin 5 (Plin5), an LD protein that tethers mitochondria, to probe the function and regulation of LD-mitochondria contacts. We demonstrate that efficient LD-to-mitochondria fatty acid (FA) trafficking and {beta}-oxidation during starvation of myoblasts requires both phosphorylation of Plin5 and an intact Plin5 mitochondrial tethering domain. We further identified the acyl-CoA synthetase, Fatp4 (ACSVL4) as a novel mitochondrial interactor of Plin5. The C-terminal domains of Plin5 and Fatp4 constitute a minimal protein interaction capable of inducing organelle contacts. Our work suggests that starvation leads to phosphorylation of Plin5, lipolysis, and subsequent channeling of FAs from LDs to Fatp4 on mitochondria for conversion to fatty-acyl-CoAs and subsequent oxidation.


title: Fetal origin of sex-bias brain aging

abstract: DNA methylation plays crucial roles during fetal development as well as aging. Whether the aging of the brain is programmed at the fetal stage remains untested. To test this hypothesis, mouse epigenetic clock (epiclock) was profiled in fetal (gestation day 15), postnatal (day 5), and aging (week 70) brain of male and female C57BL/6J inbred mice. Data analysis showed that on week 70 the female brain was epigenetically younger than the male brain. Predictive modeling by neural network identified specific methylations in the brain at the developing stages that were predictive of epigenetic state of the brain during aging. Transcriptomic analysis showed coordinated changes in expression of epiclock genes in the fetal brain relative to placenta. Whole-genome bisulfite sequencing identified sites that were methylated both in the placenta and fetal brain in a sex-specific manner. Epiclock genes and genes associated with specific signaling pathways, primarily the gonadotropin-releasing hormone receptor (GnRHR) pathway, were associated with these sex-bias methylations in the placenta as well as fetal brain. Transcriptional crosstalk among the epiclock and GnRHR pathway genes was evident in the placenta that was maintained in the brain during development as well as aging. Collectively, these findings suggest that sex differences in the aging of brain are of fetal origin and epigenetically linked to the placenta.


title: Epithelia are multiscale active liquid crystals

abstract: Biological processes such as embryogenesis, wound healing and cancer progression, crucially rely on the ability of epithelial cells to coordinate their mechanical activity over length scales order of magnitudes larger than the typical cellular size. While regulated by signalling pathways, such as YAP (yes-associated protein), MAPK (mitogen-activated protein kinase) and Wnt, this behavior is believed to additionally hinge on a minimal toolkit of physical mechanisms, of which liquid crystal order is the most promising candidat. Yet, experimental and theoretical studies have given so far inconsistent results in this respect: whereas nematic order is often invoked in the interpretation of experimental data, computational models have instead suggested that hexatic order could in fact emerge in the biologically relevant region of parameter space. In this article we resolve this dilemma. Using a combination of in vitro experiments on Madin-Darby canine kidney cells (MDCK), numerical simulations and analytical work, we demonstrate that both nematic and hexatic order is in fact present in epithelial layers, with the former being dominant at large length scales and the latter at small length scales. In MDCK GII cells on uncoated glass, these different types of liquid crystal order crossover at 34 {micro}m, corresponding approximatively to clusters of 21 cells. Our work sheds light on the emergent organization of living matter, provides a new set of tools for analyzing the structure of epithelia and paves the way toward a comprehensive and predictive mesoscopic theory of tissues.


title: Identification of a suitable endogenous control miRNA in bone aging and senescence

abstract:
ObjectiveMicroRNAs (miRNAs) are promising tools as biomarkers and therapeutic agents in various chronic diseases such as osteoporosis, cancers, type I and II diabetes, and cardiovascular diseases. Considering the rising interest in the regulatory role of miRNAs in bone metabolism, aging, and cellular senescence, accurate normalization of qPCR-based miRNA expression data using an optimal endogenous control becomes crucial.

MethodsWe used a systematic approach to select candidate endogenous control miRNAs that exhibit high stability with aging from our miRNA sequence data and literature search. Validation of miRNA expression was performed using qPCR and their comprehensive stability was assessed using the RefFinder tool which is based on four statistical algorithms: GeNorm, NormFinder, BestKeeper, and comparative delta CT. The selected endogenous control was then validated for its stability in mice and human bone tissues, and in bone marrow stromal cells (BMSCs) following induction of senescence and senolytic treatment. Finally, the utility of selected endogenous control versus U6 was tested by using each as a normalizer to measure the expression of miR-34a, a miRNA known to increase with age and senescence.

ResultsOur results show that Let-7f did not change across the groups with aging, senescence or senolytic treatment, and was the most stable miRNA, whereas U6 was the least stable. Moreover, using Let-7f as a normalizer resulted in significantly increased expression of miR-34a with aging and senescence and decreased expression following senolytic treatment. However, the expression pattern for miR-34a reversed for each of these conditions when U6 was used as a normalizer.

ConclusionsWe show that optimal endogenous control miRNAs, such as Let-7f, are essential for accurate normalization of miRNA expression data to increase the reliability of results and prevent misinterpretation. Moreover, we present a systematic strategy that is transferrable and can easily be used to identify endogenous control miRNAs in other biological systems and conditions.


title: High-Resolution Ultrasound and Speckle Tracking: a non-invasive approach to assess in vivo gastrointestinal motility during development

abstract: Gastrointestinal motor activity has been extensively studied in adults, conversely only few studies have investigated fetal motor skills. When the gastrointestinal tract starts to contract during the embryonic period and how this function evolves during development are not known. Here, we adapted a non-invasive high-resolution echography technique combined with speckle tracking analysis to examine the gastrointestinal tract motor activity dynamics during chick embryo development. We provided the first recordings of fetal gastrointestinal motility in living embryos. We found that although gastrointestinal contractions appear very early during development, they become synchronized only at the end of the fetal period. To validate this approach, we used various pharmacological inhibitors and BAPX1 gene overexpression in vivo. We found that the enteric nervous system determinates the onset of the synchronized contractions in the stomach. Moreover, alteration of smooth muscle fiber organization led to an impairment of this functional activity. Altogether, our findings show that non-invasive high-resolution echography and speckle tracking analysis allow visualizing and quantifying gastrointestinal motility during development and highlight the progressive acquisition of functional and coordinated gastrointestinal motility before birth.


title: Identification of enhancer-like elements defines regulatory networks active in planarian adult stem cells

abstract: Planarians have become an established model system to study regeneration and stem cells, but the regulatory elements in the genome remain almost entirely undescribed. Here, by integrating epigenetic and expression data we use multiple sources of evidence to identify enhancer elements active in the adult stem cell populations that drive regeneration. We have used ChIP-seq data to identify regions with histone modifications consistent with enhancer identity and activity, and ATAC-seq data to identify accessible chromatin. Overlapping these signals allowed for the identification of a set of high confidence candidate enhancers predicted to be active in planarian adult stem cells. These enhancers are enriched for conserved transcription factor (TF) binding sites for TFs and TF families expressed in planarian adult stem cells. Foot-printing analyses provided further evidence that these potential TF binding sites are occupied in adult stem cells. We integrated these analyses to build testable hypotheses for the regulatory function of transcription factors in stem cells, both with respect to how pluripotency might be regulated, and to how lineage differentiation programs are controlled. Our work identifies active enhancers regulating adult stem cells and regenerative mechanisms.


title: Friction-driven membrane scission by the human ESCRT-III proteins CHMP1B and IST1

abstract: The endosomal sorting complexes required for transport (ESCRT) system is an ancient and ubiquitous membrane scission machinery that catalyzes the budding and scission of membranes. ESCRT-mediated scission events, exemplified by those involved in the budding of HIV-1, are usually directed away from the cytosol ( reverse-topology), but they can also be directed towards the cytosol ( normal-topology). Of the ESCRT complexes 0-III, ESCRT-III is most directly implicated in membrane severing. Various subunits of ESCRT-III recruit the AAA+ ATPase VPS4, which is essential for ESCRT disassembly and reverse topology membrane scission. The ESCRT-III subunits CHMP1B and IST1 can coat and constrict positively curved membrane tubes, suggesting that these subunits could catalyze normal topology membrane severing, perhaps in conjunction with a AAA+ ATPase. CHMP1B and IST1 bind and recruit the microtubule-severing AAA+ ATPase spastin, a close relative of VPS4, suggesting that spastin could have a VPS4-like role in normal topology membrane scission. In order to determine whether CHMP1B and IST1 are capable of membrane severing on their own or in concert with VPS4 or spastin, we sought to reconstitute the process in vitro using membrane nanotubes pulled from giant unilamellar vesicles (GUVs) using an optical trap. CHMP1B and IST1 copolymerize on membrane nanotubes, forming stable scaffolds that constrict the tubes, but do not, on their own, lead to scission. However, CHMP1B-IST1-scaffolded tubes were severed when an additional extensional force was applied, consistent with a friction-driven scission mechanism. Spastin colocalized with CHMP1B enriched sites but did not disassemble the CHMP1B-IST1 coat from the membrane. VPS4 resolubilized CHMP1B and IST1 but did not lead to scission. These data show that the CHMP1B and IST1 tubular coat contributes to membrane scission. Constriction alone is insufficient for scission. However, the dynamical extension of the coated tube does lead to scission. Finally, we find that in the normal topology setting analyzed here, scission is independent of VPS4 and spastin. These observations show that the CHMP1B-IST1 ESCRT-III combination is capable of severing membranes by a friction-driven mechanism.


title: Macromolecular crowding and supersaturation protect hemodialysis patients from the onset of dialysis-related amyloidosis

abstract: Dialysis-related amyloidosis (DRA), a serious complication among long-term hemodialysis patients, is caused by amyloid fibrils of {beta}2-microglobulin ({beta}2m). Although high serum {beta}2m levels and a long dialysis vintage are the primary and secondary risk factors for the onset of DRA, respectively, patients with these do not always develop DRA, indicating that there are additional risk factors. To clarify these unknown factors, we investigated the effects of human sera on {beta}2m amyloid fibril formation. Although sera markedly inhibited amyloid fibril formation, the inhibitory effects were weaker for sera collected from dialysis patients than for control sera. When sera collected before and after maintenance dialysis treatments were compared, the latter inhibited amyloid fibril formation more than the former. These results indicate that, although the inhibitory effects of sera were deteriorated in long-term dialysis patients, they were ameliorated by maintenance dialysis treatments in the short term. Maintenance dialysis decreases the body weight by 5% and consequently increases the concentrations of serum components. Among these components, we found that serum albumin prevented amyloid fibril formation based on macromolecular crowding effects, and that the decreased serum albumin concentration in dialysis patients is a tertiary risk factor for the onset of DRA. The model was constructed assuming accumulative effects of three risk factors and may be useful for predicting the onset of DRA. Furthermore, the model suggested the importance of monitoring temporary and accumulated risks to prevent the development of amyloidoses in general, which occurs based on supersaturation-limited amyloid fibril formation in a crowded milieu.


title: Force sensing on cells and tissues by atomic force microscopy

abstract:
Biosensors are aimed to detect tiny physical and chemical stimuli in biological systems. Physical forces are ubiquitous, being implied in all cellular processes, including cell adhesion, migration, and differentiation. Given the strong interplay between cells and their microenvironment, the extracellular matrix (ECM), the structural and mechanical properties of the ECM play an important role in the transmission of external stimuli to single cells within the tissue. Vice versa, also cells themselves use self-generated forces to probe the biophysical properties of the ECM. ECM mechanics influences cell fate, regulates tissue development and show peculiar features in health and disease conditions of living organisms. Force sensing in biological systems is therefore crucial to dissect and understand complex biological processes, such as mechanotransduction.

Atomic Force Microscopy (AFM), which can both sense and apply forces at the nanoscale, with sub-nanoNewton sensitivity, represents an enabling technology and a crucial experimental tool in biophysics and mechanobiology.

In this work, we report on the application of AFM to study of biomechanical fingerprints of different components of biological systems, such as the ECM, the whole cell, and cellular components, like the nucleus and the glycocalyx. We show that physical observables like the (spatially resolved) Youngs modulus of elasticity of ECMs or cells, and the effective thickness and stiffness of the glycocalyx, can be quantitatively characterised by AFM. Their modification can be correlated to changes of the microenvironment, physio-pathological conditions, or gene regulation.


title: A fractal polymer with loops recapitulates key features of chromosome organization

abstract: Chromosomes are exceedingly long polymers compacted in a cell nucleus. While it has long been suggested that mammalian chromosomes during interphase are folded into loops, experimental detection of such loops has remained a daunting task. The most comprehensive experimental information about chromosome spatial organization is provided by Hi-C experiments that measure the frequency of contacts between all chromosomal positions. The lack of a tractable physical model of a polymer folded into loops limits our ability to interpret experimental data. It thus remains unknown how to obtain accurate and quantitative information about the nature of chromosomal looping from Hi-C. Here, we introduce a model of a polymer with random loops, solve it analytically and extend it by simulations for real chains. Remarkably, our model faithfully reproduces complex shapes of experimental contact probability curves universal among mammalian cells. Furthermore, our model allows one to estimate sizes of randomly positioned chromosomal loops from experimental data. We also show that excluded volume in real chains can induce osmotic and topological repulsion between loops, stiffening interphase chromosomes. Thus, our new framework allows interpretation of experimental data and suggests that interphase chromosomes are crumpled polymers further folded into a sequence of randomly positioned loops.


title: Fractal polymer with loops recapitulates key features of chromosome organization


title: Apolipoprotein E4 has extensive conformational heterogeneity in lipid free and bound forms

abstract: The {varepsilon}4-allele variant of Apolipoprotein E (ApoE4) is the strongest genetic risk factor for Alzheimers disease, though it only differs from its neutral counterpart ApoE3 by a single amino acid substitution. While ApoE4 influences the formation of plaques and neurofibrillary tangles, the structural determinants of pathogenicity remain undetermined due to limited structural information. We apply a combination of single-molecule spectroscopy and molecular dynamics simulations to construct an atomically-detailed model of monomeric ApoE4 and probe the effect of lipid association. Our data reveal that ApoE4 is far more disordered than previously thought and retains significant conformational heterogeneity after binding lipids. In particular, the behavior of the hinge region and C-terminal domain of ApoE4 differs substantially from that proposed in previous models and provides a crucial foundation for understanding how ApoE4 differs from non-pathogenic and protective variants of the protein.


title: Perivascular macrophages collaborate to facilitate chemotherapy resistance in cancer

abstract: A subset of tumor associated macrophages (TAMs) identified by their expression of the lymphatic vessel endothelial hyaluronan receptor-1 (Lyve-1) reside proximal to blood vasculature and contribute to disease progression. Using a spontaneous murine model of mammary adenocarcinoma (MMTV-PyMT), we show that Lyve-1+ TAMs, which co-express heme oxygenase-1, form coordinated multi-cellular  nest structures in the perivascular niche. We show that TAM nest formation is dependent on IL-6 and a communication axis involving CCR5 and its cognate ligands CCL3/4. We demonstrate that Lyve-1+ TAM nests are associated with CD8+ T-cell exclusion from the tumor and the resistance to immune-stimulating chemotherapeutics. This study highlights an unappreciated collaboration between TAMs and uncovers a spatially driven therapeutic resistance mechanism of these cells in cancer which can be therapeutically targeted.


title: p53 mutation in normal esophagus promotes multiple stages of carcinogenesis but is constrained by clonal competition

abstract: Aging normal human epithelia, such as the esophagus, accumulate a substantial burden of TP53 mutant clones. These are the origin of most esophageal squamous carcinomas, in which biallelic TP53 disruption is almost frequent. However, the cellular mechanisms by which p53 mutants colonize the esophagus and participate in the subsequent stages of transformation are unclear. Here we show that inducing the p53R245W mutant in single esophageal progenitor cells in transgenic mice confers a proliferative advantage that drives clonal expansion but does not disrupt normal epithelial structure or function. Loss of the remaining p53 allele in mutant cells does not increase their competitive fitness, creating a bottleneck to the development of chromosomally unstable p53R245W/null epithelium. In carcinogenesis, p53 mutation does not initiate tumor formation, but tumors developing from areas with p53 mutation and LOH are larger and show extensive chromosomal instability compared to lesions arising in wild type epithelium. We conclude that p53 has distinct functions at different stages of carcinogenesis and that LOH within p53 mutant clones in normal epithelium is a critical step in malignant transformation.


title: PRMT5 regulates ATF4 transcript splicing and oxidative stress response

abstract: Protein methyltransferase 5 (PRMT5) symmetrically dimethylates arginine residues leading to regulation of transcription and splicing programs. Although PRMT5 has emerged as an attractive oncology target, the molecular determinants of PRMT5 dependency in cancer remain incompletely understood. Our transcriptomic analysis identified PRMT5 regulation of the activating transcription factor 4 (ATF4) pathway in acute myelogenous leukemia (AML). PRMT5 inhibition resulted in the expression of unstable, intron-retaining ATF4 mRNA that is detained in the nucleus. Concurrently, the decrease in the spliced cytoplasmic transcript of ATF4 led to lower levels of ATF4 protein and downregulation of ATF4 target genes. Upon loss of functional PRMT5, cells with low ATF4 displayed increased oxidative stress, growth arrest, and cellular senescence. Interestingly, leukemia cells with EVI1 oncogene overexpression demonstrated dependence on PRMT5 function. EVI1 and ATF4 regulated gene signatures were inversely correlated. We show that EVI1-high AML cells have reduced ATF4 levels, elevated baseline reactive oxygen species and increased sensitivity to PRMT5 inhibition. Thus, EVI1-high cells demonstrate dependence on PRMT5 function and regulation of oxidative stress response. Overall, our findings identify the PRMT5-ATF4 axis to be safeguarding the cellular redox balance that is especially important in high oxidative stress states, such as those that occur with EVI1 overexpression.


title: Augmented efficacy of uttroside B over sorafenib in a murine model of human hepatocellular carcinoma

abstract:
BackgroundWe previously reported the potency of S. nigrum-derived uttroside B (Utt-B). Recently Utt-B is flagged as an  orphan drug against hepatocellular carcinoma (HCC) by the US FDA. The current study aims to validate the enhanced in vivo efficacy of Utt-B over sorafenib, the first-line treatment option against HCC.

MethodsHuman liver cancer cell line, HepG2 was employed as an HCC model and the comparison between Utt-B vs sorafenib therapeutic efficacies against HCC in vivo were evaluated in NOD.CB17-Prkdcscid/J mice that bear HepG2-induced HCC xenografts.

ResultsOur data indicate that Utt-B is a more potent anti-HCC drug than sorafenib, in vivo. Apart from the superior therapeutic benefit over sorafenib, Utt-B is pharmacologically safer in vivo, and owing to this virtue, the drug-induced side effects are largely alleviated in the context of HCC chemotherapy.

ConclusionsOur data demonstrate the superior therapeutic index of Utt-B over sorafenib against HCC. Clinical studies in HCC patients utilizing Utt-B, which now holds the US FDA approval as an  orphan drug, is an essential step to promote this drug from bench to bedside.