bluuebunny/crossref_metadata_2025 huggingface dataset:
Quantifying community responses to environmental variation from replicate time series
Authors: Phillips Joseph S. , Nell Lucas A. , Botsch Jamieson C.
Published: January 2021

Time-series data for ecological communities are increasingly available from long-term studies designed to track species responses to environmental change. However, classical multivariate methods for analyzing community composition have limited applicability for time series, as they do not account for temporal autocorrelation in community-member abundances. Furthermore, traditional approaches often obscure the connections between responses at the community level and those for individual taxa, limiting their capacity to infer mechanisms of community change. We show how linear mixed models that account for group-specific temporal autocorrelation and observation error can be used to infer both taxon- and community-level responses to environmental predictors from replicated time-series data. Variation in taxon-specific responses to predictors is modeled using random effects, which can be used to characterize variation in community composition. Moreover, the degree of autocorrelation is estimated separately for each taxon, since this is likely to vary due to differences in their underlying population dynamics. We illustrate the utility of the approach by analyzing the response of a predatory arthropod community to spatiotemporal variation in allochthonous resources in a subarctic landscape. Our results show how mixed models with temporal autocorrelation provide a unified approach to characterizing taxon- and community-level responses to environmental variation through time.
Decoding shared versus divergent transcriptomic signatures across cortico-amygdala circuitry in PTSD and depressive disorders
Authors: Jaffe Andrew E. , Tao Ran , Tran Matthew N. , Page Stephanie C. , Maynard Kristen R. , Pattie Elizabeth A. , Nguyen Claudia V. , Deep-Soboslay Amy , Bharadwaj Rahul , Young Keith A. , Friedman Matthew J. , Williamson Douglas E. ,   Traumatic Stress Brain Research Group, Shin Joo Heon , Hyde Thomas M. , Martinowich Keri , Kleinman Joel E.
Published: January 2021

Post-traumatic stress disorder (PTSD) is a debilitating neuropsychiatric disease with a projected lifetime risk of 8.7%. PTSD is highly comorbid with depressive disorders including major depressive disorder (MDD) and bipolar disorder (BD). It is hypothesized that the overlap in symptoms stems from partially shared underlying neurobiological mechanisms. To better understand shared and unique transcriptional patterns of PTSD and MDD we performed RNA-sequencing in the postmortem brain of two prefrontal cortex (PFC) regions and two amygdala (AMY) regions, from neurotypical donors (N=109) as well as donors with diagnoses of PTSD (N=107) or MDD (N=109) across 1285 RNA-seq samples. We identified a small number of differentially expressed genes (DEGs) specific to PTSD, mostly in the cortex compared to amygdala. PTSD-specific DEGs were preferentially enriched in cortistatin-expressing cells, a subpopulation of somatostatin interneurons. These PTSD DEGs also showed strong enrichment for gene sets associated with immune-related pathways and microglia, largely driven by decreased expression of these genes in PTSD donors. While we identified a greater number of DEGs for MDD, there were only a few that were specific to MDD as they showed high overlap with PTSD DEGs. Finally, we used weighted gene co-expression network analysis (WGCNA) as an orthogonal approach to confirm the observed cellular and molecular associations. These findings highlight the sub-population of cortistatin-expressing interneurons as having potential functional significance in PTSD and provide supporting evidence for dysregulated neuroinflammation and immune signaling in MDD and PTSD pathophysiology.
Arsenic hexoxide has differential effects on cell proliferation and genome-wide gene expression in human primary mammary epithelial and MCF7 cells
Authors: Kim Donguk , Park Na Yeon , Kang Keunsoo , Calderwood Stuart K. , Cho Dong-Hyung , Bae Ill Ju , Bunch Heeyoun
Published: January 2021

Arsenic is reportedly a biphasic inorganic compound for its toxicity and anticancer effects in humans [1, 2]. Recent studies have shown that certain arsenic compounds including arsenic hexoxide (AS4O6; hereafter, AS6) induce programmed cell death and cell cycle arrest in human cancer cells and murine cancer models [3, 4]. However, the mechanisms by which AS6 suppresses cancer cells are incompletely understood. In this study, we report the mechanisms of AS6 through transcriptome analyses. In particular, the cytotoxicity and global gene expression regulation by AS6 were compared in human normal and cancer breast epithelial cells. Using RNA-sequencing and bioinformatics analyses, differentially expressed genes in significantly affected biological pathways in these cell types were validated by real-time quantitative polymerase chain reaction and immunoblotting assays. Our data show markedly differential effects of AS6 on cytotoxicity and gene expression in human mammary epithelial normal cells (HUMEC) and Michigan Cancer Foundation 7 (MCF7), a human mammary epithelial cancer cell line. AS6 selectively arrests cell growth and induces cell death in MCF7 cells without affecting the growth of HUMEC in a dose-dependent manner. AS6 alters the transcription of a large number of genes in MCF7 cells, but much fewer genes in HUMEC. Importantly, we found that the cell proliferation, cell cycle, and DNA repair pathways are significantly suppressed whereas cellular stress response and apoptotic pathways increase in AS6-treated MCF7 cells. Together, we provide the first evidence of differential effects of AS6 on normal and cancerous breast epithelial cells, suggesting that AS6 at moderate concentrations induces cell cycle arrest and apoptosis through modulating genome-wide gene expression, leading to compromised DNA repair and increased genome instability selectively in human breast cancer cells.
Genes encoding teleost orthologs of human haplo-insufficient and monoallelic genes remain in duplicate more frequently than the whole genome
Authors: Picolo Floriane , Grandchamp Anna , Piégu Benoît , Veitia Reiner A. , Monget Philippe
Published: January 2021

Gene dosage is important is an important issue both in cell and evolutionary biology. Most genes are present in two copies in eukaryotic cells. The first outstanding exception is monoallelic gene expression (MA) that concerns genes localized on the X chromosome or in regions undergoing parental imprinting in eutherians, and many other genes scattered throughout the genome. The second exception concerns haploinsufficiency (HI), responsible for the fact that a single functional copy of a gene in a diploid organism is insufficient to ensure a normal biological function. One of the most important mechanisms ensuring functional innovation during evolution is Whole genome duplication (WGD). In addition to the two WGDs that have occurred in vertebrate genomes, the teleost genomes underwent an additional WGD, after their divergence from tetrapod. In the present work, we have studied on 57 teleost species whether the orthologs of human MA or HI genes remain more frequently in duplicates or returned more frequently in singleton than the rest of the genome. Our results show that the teleost orthologs of HI human genes remained more frequently in duplicate than the rest of the genome in all the teleost species studied. No signal was observed for the orthologs of genes localized on the human X chromosome or subjected to parental imprinting. Surprisingly, the teleost orthologs of the other human MA genes remained in duplicate more frequently than the rest of the genome for most teleost species. These results suggest that the teleost orthologs of MA and HI human genes also undergo selective pressures either related to absolute protein amounts and/or of dosage balance issues. However, these constraints seem to be different for MA genes in teleost in comparison with human genomes.
Comparative analysis of whole genome sequences ofLeptospiraspp. from RefSeq database provide interspecific divergence and repertoire of virulence factors
Authors: Abdullah Mohd , Kadivella Mohammad , Sharma Rolee , Baig Mirza. S. , Faisal Syed M. , Azam Sarwar
Published: January 2021

Leptospirosis is an emerging zoonotic and neglected disease across the world causing huge loss of life and economy. The disease is caused byLeptospiraof which 605 sequenced genomes representing 72 species are available in RefSeq database. A comparative genomics approach based on Average Amino acid Identity (AAI), Average Nucleotide Identity (ANI), and Insilco DNA-DNA hybridization provide insight that taxonomic and evolutionary position of few genomes needs to be changed and reclassified. Clustering on the basis of AAI of core and pan-genome contradict clustering pattern on basis of ANI into 4 clusters. Amino acid identity based hierarchical clustering clearly established 3 clusters ofLeptospiracorrelating with level of virulence. Whole genome tree supported three cluster classifications and groupedLeptospirainto three clades termed as pathogenic, intermediate and saprophytic.Leptospiragenus consist of diverse species and exist in heterogeneous environment, it contains relatively large and closed core genome of 1038 genes. Analysis provided pan genome remains open with 20822 genes. COG analysis revealed that mobilome related genes were found mainly in pan-genome of pathogenic clade. Clade specific genes mined in the study can be used as marker for determining clade and associating level of virulence of any newLeptospiraspecies. Many knownLeptospiravirulent genes were absent in set of 78 virulent factors mined using Virulence Factor database. A deep search approach provided a repertoire of 496 virulent genes in pan-genome. Further validation of virulent genes will help in accurately targeting pathogenicLeptospiraand controlling leptospirosis.

Graphical Abstract
Trans Fats Consumption and Body Mass Index in Cross Sectional Analysis
Authors: Golomb Beatrice A. , Pavlovsky Arthur , Koslik Hayley J.
Published: January 2021

Background

Trans fats remain on the market in parts of the world. Emerging evidence suggests that factors beyond calorie balance may affect fat deposition and body mass index (BMI). Trans fats are prooxidant, proinflammatory, and have shown adverse metabolic effects and increased fat deposition in animals.

Objective

To assess the relation of dietary trans fatty acid consumption (dTFA) to BMI in humans.

Design

Cross-sectional analysis of baseline data from a broadly-sampling study.

Setting

Community-dwelling adults from Southern California.

Participants

1018 adult men and women aged 20-85 without known diabetes, CVD, or cancer, with screening LDL 115-190mg/dL. Women of procreative potential and children were excluded.

Measurements

Height, weight, and waist circumference were measured, and BMI calculated (kg/m2). The Fred Hutchinson Food Frequency Questionnaire provided data on dTFA (grams/day) and calories consumed. Additional covariates included activity, chocolate consumption frequency, and mood. Regression assessed the relation of BMI (outcome) to dTFA, unadjusted and in models adjusting for age and sex, and adding calories and activity, chocolate consumption frequency, and mood.

Results

Higher dTFA was associated with higher BMI and waist circumference. The coefficient was strengthened, not attenuated, with adjustment for calories and activity, and other assessed covariates. In the fully adjusted model, each gram/day of dTFAs was associated with 0.44 higher BMI (SE=0.12;95%CI=0.21,0.67); P<0.001 and 1cm greater waist circumference (SE=0.35;95%CI=0.37,1.7); P=0.003.

Conclusion

Findings relating greater dTFA to higher BMI in humans comport with experimental data in animals, extend adverse metabolic associations of dTFA, and buttress evidence that foods’ composition, as well as caloric number, bears on BMI. Findings are cross-sectional but strength and consistency of association, biological gradient, and biological plausibility add “weight” to the prospect of a causal connection.

Strengths and limitations of this study

Findings are cross-sectional and rely on dietary recall.

Observational studies carry risks of bias and confounding; but randomized trials are problematic where products with potential for harm are under evaluation.

Although some nations have implemented plans to remove or reduce dTFAs from the food supply, dTFA remain on the market in other nations, rendering findings of continued interest.

The association of increased dTFA with increased BMI and waist circumference fits with other documented adverse metabolic associations of dTFA and is buttressed by experimental documentation that dTFA (without excess calories) increases visceral fat in animals, supporting prospects for causality in the observed association.
Increased infections, but not viral burden, with a new SARS-CoV-2 variant
Authors: Walker A. Sarah , Vihta Karina-Doris , Gethings Owen , Pritchard Emma , Jones Joel , House Thomas , Bell Iain , Bell John I , Newton John N , Farrar Jeremy , Diamond Ian , Studley Ruth , Rourke Emma , Hay Jodie , Hopkins Susan , Crook Derrick , Peto Tim , Matthews Philippa C. , Eyre David W. , Stoesser Nicole , Pouwels Koen B. ,   COVID-19 Infection Survey team
Published: January 2021

Background

A new variant of SARS-CoV-2, B.1.1.7/VOC202012/01, was identified in the UK in December-2020. Direct estimates of its potential to enhance transmission are limited.

Methods

Nose and throat swabs from 28-September-2020 to 2-January-2021 in the UK’s nationally representative surveillance study were tested by RT-PCR for three genes (N, S and ORF1ab). Those positive only on ORF1ab+N, S-gene target failures (SGTF), are compatible with B.1.1.7/VOC202012/01. We investigated cycle threshold (Ct) values (a proxy for viral load), percentage of positives, population positivity and growth rates in SGTF vs non-SGTF positives.

Results

15,166(0.98%) of 1,553,687 swabs were PCR-positive, 8,545(56%) with three genes detected and 3,531(23%) SGTF. SGTF comprised an increasing, and triple-gene positives a decreasing, percentage of infections from late-November in most UK regions/countries, e.g. from 15% to 38% to 81% over 1.5 months in London. SGTF Ct values correspondingly declined substantially to similar levels to triple-gene positives. Population-level SGTF positivity remained low (<0.25%) in all regions/countries until late-November, when marked increases with and without self-reported symptoms occurred in southern England (to 1.5-3%), despite stable rates of non-SGTF cases. SGTF positivity rates increased on average 6% more rapidly than rates of non-SGTF positives (95% CI 4-9%) supporting addition rather than replacement with B.1.1.7/VOC202012/01. Excess growth rates for SGTF vs non-SGTF positives were similar in those up to high school age (5% (1-8%)) and older individuals (6% (4-9%)).

Conclusions

Direct population-representative estimates show that the B.1.1.7/VOC202012/01 SARS-CoV-2 variant leads to higher infection rates, but does not seem particularly adapted to any age group.
Antimicrobial resistance determinants are associated with Staphylococcus aureus bacteraemia and adaptation to the hospital environment: a bacterial genome-wide association study
Authors: Young Bernadette C , Wu Chieh-Hsi , Charlesworth Jane , Earle Sarah , Price James R , Gordon N Claire , Cole Kevin , Dunn Laura , Liu Elian , Oakley Sarah , Godwin Heather , Fung Rowena , Miller Ruth , Knox Kyle , Votintseva Antonia , Quan T Phuong , Tilley Robert , Scarborough Matthew , Crook Derrick W , Peto Timothy E , Walker A Sarah , Llewelyn Martin J , Wilson Daniel J
Published: January 2021

Background

Staphylococcus aureus is a major bacterial pathogen in humans, and a dominant cause of severe bloodstream infections. Globally, antimicrobial resistance (AMR) in S. aureus remains challenging. While human risk factors for infection have been defined, contradictory evidence exists for the role of bacterial genomic variation in S. aureus disease.

Methods

To investigate the contribution of bacterial lineage and genomic variation to the development of bloodstream infection, we undertook a genome-wide association study comparing bacteria from 1017 individuals with bacteraemia to 984 adults with asymptomatic S. aureus nasal carriage. Within 984 carriage isolates, we also compared healthcare-associated (HA) carriage with community-associated (CA) carriage.

Results

All major global lineages were represented in both bacteraemia and carriage, with no evidence for different attack rates. However, kmers tagging trimethoprim resistance-conferring mutation F99Y in dfrB were significantly associated with bacteraemia-vs-carriage (p=10−8.9-10−9.3). Pooling variation within genes, bacteraemia-vs-carriage was associated with the presence of mecA (HMP=10−5.3) as well as the presence of SCCmec (HMP=10−4.4).

Among S. aureus carriers, no lineages were associated with HA-vs-CA carriage. However, we found a novel signal of HA-vs-CA carriage in the foldase protein prsA, where kmers representing conserved sequence allele were associated with CA carriage (p=10−7.1-10−19.4), while in gyrA, a ciprofloxacin resistance-conferring mutation, L84S, was associated with HA carriage (p=10−7.2).

Conclusions

In an extensive study of S. aureus bacteraemia and nasal carriage in the UK, we found strong evidence that all S. aureus lineages are equally capable of causing bloodstream infection, and of being carried in the healthcare environment.

Genomic variation in the foldase protein prsA is a novel genomic marker of healthcare origin in S. aureus but was not associated with bacteraemia. AMR determinants were associated with both bacteraemia and hospital-associated carriage, suggesting that AMR increases the propensity not only to survive in hospital environments, but also to cause invasive disease.
A phenomics approach forin vitroantiviral drug discovery
Authors: Rietdijk Jonne , Tampere Marianna , Pettke Aleksandra , Georgieva Polina , Lapins Maris , Berglund Ulrika Warpman , Spjuth Ola , Puumalainen Marjo-Riitta , Carreras-Puigvert Jordi
Published: January 2021

Background

The current COVID-19 pandemic has highlighted the need for new and fast methods to identify novel or repurposed therapeutic drugs. Here we present a method for untargeted phenotypic drug screening of virus-infected cells, combining Cell Painting with antibody-based detection of viral infection in a single assay. We designed an image analysis pipeline for segmentation and classification of virus-infected and non-infected cells, followed by extraction of morphological properties.

Results

We show that the methodology can successfully capture virus-induced phenotypic signatures of MRC-5 human lung fibroblasts infected with Human coronavirus 229E (CoV-229E). Moreover, we demonstrate that our method can be used in phenotypic drug screening using a panel of nine host- and virus-targeting antivirals. Treatment with effective antiviral compounds reversed the morphological profile of the host cells towards a non-infected state.

Conclusions

The method can be used in drug discovery for morphological profiling of novel antiviral compounds on both infected and non-infected cells.
Modulation of the IKSchannel by PIP2requires two binding sites per monomer
Authors: Kongmeneck Audrey Deyawe , Kasimova Marina A. , Tarek Mounir
Published: January 2021

The phosphatidyl-inositol-4,5-bisphosphate (PIP2) lipid has been shown to be crucial for the coupling between the voltage sensor and the pore of the potassium voltage-gated KV7 channel family, especially the KV7.1 channel. The latter, expressed in the myocardium membrane is complexed with its auxiliary subunits, KCNE1 to generate the so-called IKScurrent. We present here molecular models of transmembrane domain of this complex in its three known states, namely the Resting/Closed (RC), the Intermediate/Closed (IC), and the Activated/Open (AO), robustness of which is assessed by agreement with a range of biophysical data. Molecular Dynamics (MD) simulations of these models embedded in a lipid bilayer including phosphatidyl-inositol-4,5-bisphosphate (PIP2) lipids show that in presence of KCNE1, two PIP2lipids are necessary to stabilize each state. The simulations also show that KCNE1 interacts with both PIP2binding sites, forming a tourniquet around the pore and preventing its opening. The present investigation provides therefore key molecular elements that govern the role of PIP2in KCNE1 modulation of IKSchannels, possibly a common mechanism by which auxiliary KCNE subunits might modulate a variety of other ion channels.
Natural variations in the P-type ATPase heavy metal transporter ZmCd1 controlling cadmium accumulation in maize grains
Authors: Tang Bin , Luo Meijie , Zhang Yunxia , Guo Huanle , Li Jingna , Song Wei , Zhang Ruyang , Feng Zhen , Kong Mengsi , Li Han , Cao Zhongyang , Lu Xiaoduo , Li Delin , Zhang Jianhua , Wang Ronghuan , Wang Yuandong , Chen Zhihui , Zhao Yanxin , Zhao Jiuran
Published: January 2021

Cadmium (Cd) accumulation in maize grains is detrimental to human health. Developing maize varieties with low-Cd contents via marker-assisted selection is important for ensuring the production of maize grains safe for consumption. However, the key gene controlling maize grain Cd accumulation has not been cloned. In this study, we identified two major loci for maize grain Cd accumulation (qCd1andqCd2) on chromosome 2 during a genome-wide association study (GWAS). TheqCd1locus was analyzed by bulked segregant RNA-seq and fine mapping with a biparental segregating population of Jing724 (low-Cd line) and Mo17 (high-Cd line). TheZmCd1candidate gene in theqCd1locus encodes a vacuolar membrane-localized heavy metal P-type ATPase transporter, ZmHMA3, which is orthologous to the tonoplast Cd transporter OsHMA3. Genomic DNA sequence and transcript analyses suggested that a transposon in intron 1 ofZmCd1is responsible for the abnormal amino acid sequence in Mo17. An EMS mutant analysis and an allelism test confirmedZmCd1influences maize grain Cd accumulation. The natural variations inZmCd1were used to develop four PCR-based molecular markers, which revealed fiveZmCd1haplotypes in the GWAS population. The molecular markers were also used to predict the grain Cd contents in commonly cultivated maize germplasms in China. The predicted Cd contents for 36 inbred lines and 13 hybrids were consistent with the measured Cd contents. Furthermore, several low-Cd elite inbred lines and hybrids were identified, including Jing2416, MC01, Jingnonke728, and Jingke968. Therefore, the molecular markers developed in this study are applicable for molecular breeding and developing maize varieties with low grain Cd contents.
Dysregulated functional and metabolic response in multiple sclerosis patient macrophages correlate with a more inflammatory state, reminiscent of trained immunity
Authors: Fransson J. , Bachelin C. , Deknuydt F. , Ichou F. , Guillot-Noël L. , Ponnaiah M. , Gloaguen A. , Maillart E. , Stankoff B. , Tenenhaus A. , Mochel F. , Fontaine B. , Louapre C. , Zujovic V.
Published: January 2021

In multiple sclerosis (MS), immune cells invade the central nervous system and destroy myelin. Macrophages contribute to demyelination and myelin repair, and their role in each process depends on their ability to acquire specific phenotypes in response to external signals. Here, we assess whether defects in MS patient macrophage responses may lead to increased inflammation or lack of neuro-regenerative effects.

To test this hypothesis, CD14+CD16-monocytes from MS patients and healthy controls were activatedin vitroto obtain homeostatic-like, pro-inflammatory and pro-regenerative macrophages. Myelin phagocytic capacity and surface molecule expression of CD14, CD16 and HLA-DR were evaluated with flow cytometry. In parallel, macrophages were assessed through RNA sequencing and metabolomics.

We observed that MS patient monocytesex vivorecapitulate their preferential activation toward a CD16+phenotype, a subset of pro-inflammatory cells present in MS lesions. Even in the absence of pro-inflammatory stimuli, MS patient macrophages exhibit a pro-inflammatory transcriptomic profile with higher levels of cytokine/chemokine suggesting increased recruitment capacities. Interestingly, MS patient macrophages exhibit a specific metabolic signature with a mitochondrial energy metabolism blockage resulting in a shift from oxidative phosphorylation to glycolysis. Furthermore, we observe a failure to up-regulate apoptosis effector genes in the pro inflammatory state suggesting a longer-lived pro-inflammatory macrophage population.

Our results highlight an intrinsic defect of MS patient macrophages that provide evidence of innate immune cell memory in MS.
Muscle Activation Profile During Perturbed Walking is Modulated According to Body State
Authors: Rosenblum Uri , Melzer Itshak , Zeilig Gabi , Plotnik Meir
Published: January 2021

During an unexpected loss of balance, avoiding a fall requires people to readjust their footing rapidly and effectively. We investigated the activation patterns of the ankle and knee muscles, and muscle fiber type recruitment resulting from unannounced, medio-lateral (i.e., right/left) horizontal-surface walking perturbations in twenty healthy adults (27.00±2.79 years, 10 females). Surface electromyography (sEMG) total spectral power for specific frequency bands (40-60Hz, 60-150Hz, 150-250Hz, 250-400Hz and 400-1000Hz), from tibialis anterior (TA) and vastus lateralis (VL) muscles were analyzed. Compared to non-perturbed walking, we found a significant increase in the total spectral power of lower-extremity muscles during the first 3 seconds after perturbation. When two feet were on the ground in time of perturbation we found a different muscle fiber type recruitment pattern between VL and TA muscles. This was not significant for perturbations implemented when one foot was on the ground. Our findings suggest that muscle operating frequency is modulated in real time to fit body state and somatosensory input in the context of functional goal requirements such as a rapid change of footing in response to unexpected loss of balance in single and double-support phases of gait.

New & Noteworthy

To study muscle spectral profiles in response to loss of balance, we investigated the dynamics of muscle spectral power changes, across different frequency bands after unannounced mechanical perturbations during walking. We showed increased activation of high-frequency motor units of the lower-limb muscles, subside 3 seconds after perturbation. Differences in power increase of specific frequency bands suggest that muscle activation is modulated in real time to fit body state in the context of functional goal requirements.
A theoretical approach for quantifying the impact of changes in effective population size and expression level on the rate of coding sequence evolution
Authors: Latrille T. , Lartillot N.
Published: January 2021

Molecular sequences are shaped by selection, where the strength of selection relative to drift is determined by effective population size (Ne). Populations with highNeare expected to undergo stronger purifying selection, and consequently to show a lower substitution rate for selected mutations relative to the substitution rate for neutral mutations (ω). However, computational models based on biophysics of protein stability have suggested thatωcan also be independent ofNe, a result proven under general conditions. Together, the response ofωto changes inNedepends on the specific mapping from sequence to fitness. Importantly, an increase in protein expression level has been found empirically to result in decrease ofω, an observation predicted by theoretical models assuming selection for protein stability. Here, we derive a theoretical approximation for the response ofωto changes inNeand expression level, under an explicit genotype-phenotype-fitness map. The method is generally valid for additive traits and log-concave fitness functions. We applied these results to protein undergoing selection for their conformational stability and corroborate out findings with simulations under more complex models. We predict a weak response ofωto changes in eitherNeor expression level, which are interchangeable. Based on empirical data, we propose that fitness based on the conformational stability may not be a sufficient mechanism to explain the empirically observed variation inωacross species. Other aspects of protein biophysics might be explored, such as protein-protein interactions, which can lead to a stronger response ofωto changes inNe.
“An intrinsically disordered intracellular domain of PIEZO2 is required for force-from-filament activation of the channel”
Authors: Verkest Clement , Schaefer Irina , Jegelka Juri M. , Nees Timo A. , Na Wang , Taberner Francisco J. , Lechner Stefan G.
Published: January 2021

A central question in mechanobiology is how mechanical forces acting in or on a cell are transmitted to mechanically-gated PIEZO channels that convert these forces into biochemical signals. Here we show that PIEZO2 is sensitive to force-transmission via the membrane (force-from-lipids) as well as force transmission via the cytoskeleton (force-from-filament) and demonstrate that the latter requires the intracellular linker between the transmembrane helices nine and ten (IDR5). Moreover, we show that rendering PIEZO2 insensitive to force-from-filament by deleting IDR5 abolishes PIEZO2-mediated inhibition of neurite outgrowth, which relies on the detection of cellgenerated traction forces, while it only partially affects its sensitivity to cell indentation and does not at all alter its sensitivity to membrane stretch. Hence, we propose that PIEZO2 is a polymodal mechanosensor that detects different types of mechanical stimuli via different force transmission pathways, which highlights the importance of utilizing multiple complementary assays when investigating PIEZO channel function.
Layer 6A pyramidal cells subtypes form synaptic microcircuits with distinct functional and structural properties
Authors: Yang Danqing , Qi Guanxiao , Feldmeyer Dirk
Published: January 2021

Neocortical layer 6 plays a crucial role in sensorimotor coordination and integration through functionally segregated circuits linking intracortical and subcortical areas. However, because of the high neuronal heterogeneity and sparse intralaminar connectivity data on the cell-type specific synaptic microcircuits in layer 6 remain few and far between. To address this issue, whole-cell recordings combined with morphological reconstructions have been used to identify morpho-electric types of layer 6A pyramidal cells (PCs) in rat barrel cortex. Cortico-thalamic (CT), cortico-cortical (CC) and cortico-claustral (CCla) pyramidal cells have been distinguished based on to their distinct dendritic and axonal morphologies as well as their different electrophysiological properties. Here we demonstrate that these three types of layer 6A pyramidal cells innervate neighboring excitatory neurons with distinct synaptic properties: CT PCs establish weak facilitating synapses to other L6A PCs; CC PCs form synapses of moderate efficacy; while synapses made by putative CCla PCs display the highest release probability and a marked short-term depression. Furthermore, for excitatory-inhibitory synaptic connections in layer 6 we were able to show that both the presynaptic PC type and the postsynaptic interneuron type govern the dynamic properties of the of the respective synaptic connections. We have identified a functional division of local layer 6A excitatory microcircuits which may be responsible of the differential temporal engagement of layer 6 feed-forward and feedback networks. Our results provides a basis for further investigations on the long-range cortico-cortical, cortico-thalamic and cortico-claustral pathways.
Genetic engineering forin vivooptical interrogation of neuronal responses to cell type-specific silencing
Authors: Terzi Firat , Knabbe Johannes , Cambridge Sidney B.
Published: January 2021

Genetic engineering of quintuple transgenic brain tissue was used to establish a low background, Cre-dependent version of the inducible Tet-On system for fast, cell type-specific transgene expressionin vivo. Co-expression of a constitutive, Cre-dependent fluorescent marker selectively allowed single cell analysesbeforeandafterinducible, tet-dependent transgene expression. Here, we used this method for acute, high-resolution manipulation of neuronal activity in the living brain. Single induction of the potassium channel Kir2.1 produced cell type-specific silencing within hours that lasted for at least three days. Longitudinalin vivoimaging of spontaneous calcium transients and neuronal morphology demonstrated that prolonged silencing did not alter spine densities or synaptic input strength. Furthermore, selective induction of Kir2.1 in parvalbumin interneurons increased the activity of surrounding neurons in a distance-dependent manner. This high-resolution, inducible interference and interval imaging of individual cells (high I5, ‘HighFive’) method thus allows visualizing temporally precise, genetic perturbations of defined cells.
An integrative approach unveils a distal encounter site for rPTPε and phospho-Src complex formation
Authors: EswarKumar Nadendla , Yang Cheng-Han , Tewary Sunilkumar , Yeh Yi-Qi , Yang Hsiao-Ching , Ho Meng-Chiao
Published: January 2021

Protein tyrosine phosphatase: phospho-protein complex structure determination, which requires to understand how specificity is achieved at the protein level remains a significant challenge for protein crystallography and cryoEM due to the transient nature of binding interactions. Using rPTPεD1 and phospho-SrcKD as a model system, we established an integrative workflow involving protein crystallography, SAXS and pTyr-tailored MD simulations to reveal the complex formed between rPTPεD1 and phospho-SrcKD, revealing transient protein–protein interactions distal to the active site. To support our finding, we determined the associate rate between rPTPεD1 and phospho-SrcKD and showed that a single mutation on rPTPεD1 disrupts this transient interaction, resulting in the reduction of association rate and activity. Our simulations suggest that rPTPεD1 employs a binding mechanism involving conformational change prior to the engagement of cSrcKD. This integrative approach is applicable to other PTP: phospho-protein complex determination and is a general approach for elucidating transient protein surface interactions.
An intracellular pathway controlled by the N-terminus of the pump subunit inhibits the bacterial KdpFABC ion pump in high K+conditions
Authors: Khandelia Himanshu , Stokes David , Pedersen Bjørn Panyella , Dubey Vikas
Published: January 2021

The heterotetrameric bacterial KdpFABC transmembrane protein complex is an ion channel-pump hybrid that consumes ATP to import K+against its transmembrane chemical potential gradient in low external K+environments. The KdpB ion-pump subunit of KdpFABC is a P-type ATPase, and catalyses ATP hydrolysis. Under high external K+conditions, K+can diffuse into the cells through passive ion channels. KdpFABC must therefore be inhibited in high K+conditions to conserve cellular ATP. Inhibition is thought to occur via unusual phosphorylation of residue Ser162 of the TGES motif of the cytoplasmic A domain. It is proposed that phosphorylation most likely traps KdpB in an inactive E1-P like conformation, but the molecular mechanism of phosphorylation-mediated inhibition and the allosteric links between phosphorylation on the A domain and the inactivation of the pump remain unknown. Here, we employ molecular dynamics (MD) simulations of the dephosphorylated and phosphorylated versions of KdpFABC to demonstrate that phosphorylated KdpB is trapped in a conformation where the ion-binding site is hydrated by an intracellular pathway between transmembrane helices M1 and M2 which opens in response to the rearrangement of cytoplasmic domains resulting from phosphorylation. Cytoplasmic access of water to the ion-binding site is accompanied by a remarkable loss of secondary structure of the KdpB N-terminus and disruption of a key salt bridge between Glu87 in the A domain and Arg212 in the P domain. Our results provide the molecular basis of a unique mechanism of regulation amongst P-type ATPases, and suggest that the N-terminus has a significant role to play in the conformational cycle and regulation of KdpFABC
Ultra-conserved sequences in the genomes of highly diverseAnophelesmosquitoes, with implications for malaria vector control
Authors: O’Loughlin Samantha M. , Forster Annie J. , Fuchs Silke , Dottorini Tania , Nolan Tony , Crisanti Andrea , Burt Austin
Published: January 2021

DNA sequences that are exactly conserved over long evolutionary time scales have been observed in a variety of taxa. Such sequences are likely under strong functional constraint and they have been useful in the field of comparative genomics for identifying genome regions with regulatory function. A potential new application for these ultra-conserved elements has emerged in the development of gene drives to control mosquito populations. Many gene drives work by recognising and inserting at a specific target sequence in the genome, often imposing a reproductive load as a consequence. They can therefore select for target sequence variants that provide resistance to the drive. Focusing on highly conserved, highly constrained sequences lowers the probability that variant, gene drive-resistant alleles can be tolerated.

Here we search for conserved sequences of 18bp and over in an alignment of 21Anophelesgenomes, spanning an evolutionary timescale of 100 million years, and characterise the resulting sequences according to their location and function. Over 8000 ultra-conserved elements were found across the alignment, with a maximum length of 164 bp. Length-corrected gene ontology analysis revealed that genes containingAnophelesultra-conserved elements were over-represented in categories with structural or nucleotide binding functions. Known insect transcription factor binding sites were found in 48% of intergenicAnophelesultra-conserved elements. When we looked at the genome sequences of 1142 wild-caught mosquitoes we found that 15% of theAnophelesultra-conserved elements contained no polymorphisms. Our list ofAnophelesultra-conserved elements should provide a valuable starting point for the selection and testing of new targets for gene-drive modification in the mosquitoes that transmit malaria.
Targeting conserved viral virulence determinants by single domain antibodies to block SARS-CoV2 infectivity
Authors: Singh Sudhakar , Dahiya Surbhi , Singh Yuviana J. , Beeton Komal , Jain Ayush , Sarkar Roman , Dubey Abhishek , Tehseen Syed Azeez , Sehrawat Sharvan
Published: January 2021

We selected SARS-CoV2 specific single domain antibodies (sdAbs) from a previously constructed phage display library using synthetic immunogenic peptides of the virus spike (S) protein as bait. The sdAbs targeting the cleavage site (CS) and the receptor binding domain (RBD) in S protein efficiently neutralised the infectivity of a pseudovirus expressing SARS-CoV2 S protein. Anti-CS sdAb blocked the virus infectivity by inhibiting proteolytic processing of SARS-CoV2 S protein. Both the sdAbs retained characteristic structure within the pH range of 2 to 12 and remained stable upto 65°C. Furthermore, structural disruptions induced by a high temperature in both the sdAbs were largely reversed upon their gradual cooling and the resulting products neutralised the reporter virus. Our results therefore suggest that targeting CS in addition to the RBD of S protein by sdAbs could serve as a viable option to reduce SARS-CoV2 infectivity and that proteolytic processing of the viral S protein is critical for infection.
Dynlt3, a novel fundamental regulator of melanosome movement, distribution, maturation and transfer
Authors: Aktary Zackie , Conde-Perez Alejandro , Rambow Florian , Marco Mathilde Di , Amblard François , Hurbain Ilse , Raposo Graça , Delevoye Cédric , Coscoy Sylvie , Larue Lionel
Published: January 2021

Skin pigmentation is dependent on cellular processes including melanosome biogenesis, transport, maturation and transfer to keratinocytes. However, how the cells finely control these processes in space and time to ensure proper pigmentation remains unclear. Here, we show that a component of the cytoplasmic dynein complex, Dynlt3, is required for efficient melanosome transport, maturation and transfer. In melanocytes with decreased levels of Dynlt3, pigmented melanosomes undergo a more directional (convective) motion leading to their peripheral location in the cell, and are not fully matured. Stage IV melanosomes are more acidic, but still heavily pigmented, resulting in a less efficient melanosome transfer. Finally, the level of Dynlt3 is dependent on β-catenin activity, revealing a novel function of the Wnt/β-catenin signalling pathway during melanocyte and skin pigmentation, by coupling the transport, position and maturation of melanosomes required for their transfer.
Gating current noise produced by Brownian models of a voltage sensor
Authors: Catacuzzeno Luigi , Franciolini Fabio , Bezanilla Francisco , Eisenberg Robert S.
Published: January 2021

The activation of voltage-dependent ion channels is associated with the movement gating charges, that give rise to gating currents. Although gating currents originating from a single channel are too small to be detected, analysis of the fluctuations of macroscopic gating currents originating from a population of channels can make a good guess of their magnitude. The analysis of experimental gating current fluctuations, when interpreted in terms of a Markov model of channel activation, are in accordance with the presence of a main step along the activation pathway carrying 2.3-2.4 e0of charge. To give a physical interpretation to these results and to relate them to the known atomic structure of the voltage sensor domain, we employed a Brownian model of voltage-dependent gating that we recently developed using structural information and applying the laws of electrodynamics. The model was capable to reproduce gating currents and gating current fluctuations essentially similar to those experimentally observed. The detailed study of this model output, also performed by making several simplifications aimed at understanding the basic dependencies of the gating current fluctuations, suggests that in real ion channels the voltage sensor does not move in a fully Markovian regimen due to the relatively low (<5 kT) energy barriers separating successive intermediate states. As a consequence, the simultaneous jump of multiple gating charges through the gating pore becomes frequent, and this occurrence is at the origin of the relatively high single-step charge detected by assuming Markovian behavior.
Chemical synthesis and purification of a non-hydrolyzable CTP analog CTPγS
Authors: Rejzek M , Le Tung B. K.
Published: January 2021

Slowly- or non-hydrolyzable analogs of ATP and GTP, for example adenosine 5’-(γ-thio)triphosphate (ATPγS) and guanosine 5’-(γ-thio)triphosphate (GTPγS), have been widely employed to probe the roles of ATP and GTP in biosystems, and these compounds are readily available from commercial sources. In contrast, cytosine 5’-(γ-thio)triphosphate (CTPγS) is not widely available commercially. The recent discovery of ParB as the founding member of a new class of CTPase enzyme (Osorio-Valerianoet al., 2019; Sohet al., 2019) and the possibility of multiple other undiscovered CTPases necessitate the use of the CTPγS to elucidate the roles of CTP hydrolysis in these systems. Here, we adapt a procedure for the synthesis of modified phosphoanhydrides (Hoferet al., 2015) to chemically synthesize and purify a milligram-scale of CTPγS.
The epigenetic eraser LSD1 lies at the apex of a reversible erythroid to myeloid cell fate decision
Authors: Yu Lei , Myers Greggory , Ku Chia-Jui , Schneider Emily , Wang Yu , Singh Sharon A. , Jearawiriyapaisarn Natee , White Andrew , Moriguchi Takashi , Khoriaty Rami , Yamamoto Masayuki , Rosenfeld M. Geoffrey , Pedron Julien , Bushweller John H. , Lim Kim-Chew , Engel James Douglas
Published: January 2021

H3K4Me demethylase KDM1a/LSD1 is a therapeutic target for multiple diseases, including the β-globinopathies (sickle cell disease and β-thalassemia) since its inactivation has been shown to lead to robust induction of the fetal globin genes. Here we examined the consequences of conditional inactivation ofLsd1in adult red blood cells using a newGata1creERT2BAC transgene. Loss ofLsd1activity in mice blocked erythroid differentiation and expanded GMP-like cells, converting hematopoietic differentiation potential from an erythroid to a myeloid fate. The analogous phenotype was also observed in human HSPC, coincident with induction of myeloid transcription factors (e.g. PU.1 andCEBPα). Finally, blocking the activity of myeloid transcription factors PU.1 or RUNX1 at the same time as LSD1 reverted myeloid lineage conversion to an erythroid phenotype. The data show that LSD1 promotes erythropoiesis by repressing myeloid cell fate, and that inhibition of myeloid differentiation reverses the lineage switch caused by LSD1 inactivation.
BRN2 is a non-canonical melanoma tumor-suppressor
Authors: Hamm Michael , Sohier Pierre , Petit Valérie , Raymond Jérémy H , Delmas Véronique , Le Coz Madeleine , Gesbert Franck , Kenny Colin , Aktary Zackie , Pouteaux Marie , Rambow Florian , Sarasin Alain , Bellacosa Alfonso , Sanchez-del-Campo Luis , Mosteo Laura , Lauss Martin , Meijer Dies , Steingrimsson Eirikur , Jönsson Göran B , Cornell Robert A , Davidson Irwin , Goding Colin R , Larue Lionel
Published: January 2021

While the major drivers of melanoma initiation, including activation of NRAS/BRAF and loss ofPTENorCDKN2A, have been identified, the role of key transcription factors that impose altered transcriptional states in response to deregulated signaling is not well understood. The POU domain transcription factor BRN2 is a key regulator of melanoma invasion, yet its role in melanoma initiation remains unknown. Here, we show thatBRN2haplo-insufficiency is sufficient to promote melanoma initiation and metastasis, acting as a non-canonical tumor suppressor. Mechanistically, BRN2 directly modulatesPTENexpression, and PI3K signaling, to drive tumor initiation and progression. Collectively our results reveal that somatic deletion of oneBRN2allele elicits melanoma initiation and progression.

SIGNIFICANCE

Here, we report frequent mono-allelic loss of the transcription factor BRN2 in human cutaneous melanoma metastases. We developed a mouse model for Brn2-deficient melanoma based on the most common alterations (BrafV600EandPtenloss) in human melanoma and established the role of Brn2 as a functional regulator of tumor initiation, tumor growth, and the formation of metastasesin vivo. Mechanistically, BRN2 loss increases PI3K-signaling through PTEN repression, either via MITF induction or not. Overall, we describe a novel tumor suppressor of high prevalence in human melanoma that regulates several steps ofin vivomelanomagenesis through two previously unknown molecular mechanisms.