title: Mg2+-dependent conformational equilibria in CorA: an integrated view on transport regulation

abstract: The CorA family of proteins regulates the homeostasis of divalent metal ions in many bacteria, archaea, and eukaryotic mitochondria, making it an important target in the investigation of the mechanisms of transport and its functional regulation. Although numerous structures of open and closed channels are now available for the CorA family, the mechanism of the transport regulation remains elusive. Here, we investigated the conformational distribution and associated dynamic behaviour of the pentameric Mg2+ channel CorA at room temperature using small-angle neutron scattering (SANS) in combination with molecular dynamics (MD) simulations and solid-state nuclear magnetic resonance spectroscopy (NMR). We find that neither the Mg2+-bound closed structure nor the Mg2+-free open forms are sufficient to explain the average conformation of CorA. Our data support the presence of conformational equilibria between multiple states, and we further find a variation in the behaviour of the backbone dynamics with and without Mg2+. We propose that CorA must be in a dynamic equilibrium between different non-conducting states, both symmetric and asymmetric, regardless of bound Mg2+ but that conducting states become more populated in Mg2+-free conditions. These properties are regulated by backbone dynamics and are key to understanding the functional regulation of CorA.


title: Subtype specific responses in hKv7.4 and hKv7.5 channels to polyunsaturated fatty acids

abstract: The KV7.4 and KV7.5 subtypes of voltage-gated potassium channels are expressed in several tissues where they play a role in physiological processes such as sound amplification in the cochlea and adjusting vascular smooth muscle tone. Therefore, the mechanisms that regulate KV7.4 and KV7.5 channel function are of interest. Here, we study the effect of polyunsaturated fatty acids (PUFAs) on human KV7.4 and KV7.5 channels expressed in Xenopus oocytes. We report that KV7.5 is activated by PUFAs, which shift the V50 of the conductance versus voltage (G(V)) curve towards more negative voltages. This response depends on the charge of the head group as an uncharged PUFA analogue has no effect and a positively charged PUFA analogue induces positive V50 shifts. In contrast, we find that the KV7.4 channel is inhibited by PUFAs, which shift V50 towards more positive voltages. No effect on V50 of KV7.4 is observed by an uncharged or a positively charged PUFA analogue. Oocytes co-expressing KV7.4 and KV7.5 display an intermediate response to PUFAs. Altogether, the KV7.5 channels response to PUFAs is like that previously observed in KV7.1-7.3 channels, whereas the KV7.4 channel response is opposite, revealing subtype specific responses to PUFAs.

category: biophysics


title: Epigenetic MRI: Noninvasive Imaging of DNA Methylation in the Brain

abstract: Both neuronal and genetic mechanisms regulate brain function. While there are excellent methods to study neuronal activity in vivo, there are no nondestructive methods to measure global gene expression in living brains. Here we present a method, epigenetic magnetic resonance imaging (eMRI), that overcomes this limitation via direct imaging of DNA methylation, a major gene expression regulator. eMRI exploits the methionine metabolic pathways for DNA methylation to label genomic DNA through 13C-enriched diets. A novel 13C magnetic resonance spectroscopic imaging method then maps the spatial distribution of labeled DNA. We validated eMRI using pigs, whose brains have stronger similarity to humans in volume and anatomy than rodents, and confirmed efficient 13C labeling of brain DNA. We also discovered strong regional differences in global DNA methylation. Just as MRI measurements of regional neuronal activity have had a transformational effect on neuroscience, we expect that the eMRI signal as a surrogate for regional gene expression will enable many new investigations into the roles of gene expression in human brain function, behavior, and disease.

category: bioengineering


title: Evidence of long-term purging of mutation load in killer whale genomes

abstract: Genomes of high latitude killer whales harbour signatures of post-glacial founding and expansion. Here, we investigate whether reduced efficacy of selection increased mutation load in founder populations, or whether recessive deleterious mutations exposed to selection in homozygous genotypes were purged. Comparing the accumulation of synonymous and non-synonymous mutations across pairs of globally sampled genomes reveals that the most significant outliers are high latitude North Atlantic genomes, which have accumulated significantly fewer non-synonymous mutations than all other populations. Comparisons with the genome of a 7.5-Kyr-old North Atlantic killer whale, inferred to be closely related to the population directly ancestral to present-day Icelandic and Norwegian populations, calibrates the timing of the action of selection on non-synonymous mutations predominantly to the mid-late Holocene. Non-synonymous mutations purged in modern Norwegian killer whale genomes are found as globally shared standing variation in heterozygote genotypes more often than expected, suggesting overdominance. Taken together, our findings are consistent with purging of recessive non-synonymous mutations exposed to selection in founder-associated homozygous genotypes.

category: evolutionary biology


title: Disruption of Brachypodium Lichenase Alters Metabolism of Mixed-linkage Glucan and Starch

abstract: Mixed-linkage glucan (MLG), which is widely distributed in grasses, is a polysaccharide highly abundant in cell walls of grass endosperm and young vegetative tissues. Lichenases are enzymes that hydrolyze MLG first identified in MLG-rich lichens. In this study, we identify a gene encoding a lichenase we name Brachypodium distachyon LICHENASE 1 (BdLCH1), which is highly expressed in the endosperm of germinating seeds and coleoptiles and at lower amounts in mature shoots. RNA in situ hybridization showed that BdLCH1 is primarily expressed in chlorenchyma cells of mature leaves and internodes. Disruption of BdLCH1 resulted in an eight-fold increase in MLG content in senesced leaves. Consistent with the in situ hybridization data, immunolocalization results showed that MLG was not removed in chlorenchyma cells of lch1 mutants as it was in wild type and implicate the BdLCH1 enzyme in removing MLG in chlorenchyma cells in mature vegetative tissues. We also show that MLG accumulation in lch1 mutants was resistant to dark induced degradation, and eight-week-old lch1 plants showed a faster rate of starch breakdown than wild type in darkness. Our results suggest a role for BdLCH1 in modifying the cell wall to support highly metabolically active cells.

category: plant biology


title: Integrative molecular roadmap for direct conversion of fibroblasts into myocytes and myogenic progenitor cells

abstract: Transient MyoD overexpression in concert with small molecules treatment reprograms mouse fibroblasts into induced myogenic progenitor cells (iMPCs). However, the molecular landscape and mechanisms orchestrating this cellular conversion remain unknown. Here, we undertook an integrative multi-omics approach to delineate the process of iMPC reprogramming in comparison to myogenic transdifferentiation mediated solely by MyoD. Utilizing transcriptomics, proteomics and genome-wide chromatin accessibility assays, we unravel distinct molecular trajectories which govern the two processes. Notably, iMPC reprogramming is characterized by gradual upregulation of stem and progenitor cell markers, unique signaling pathways, chromatin remodelers and cell cycle regulators which manifest via rewiring of the chromatin in core myogenic promoters. Furthermore, we determine that only iMPC reprogramming is mediated by Notch pathway activation, which is indispensable for iMPC formation and self-renewal. Collectively, this study charts divergent molecular blueprints for myogenic transdifferentiation or reprogramming and underpins the heightened capacity of iMPCs in capturing myogenesis ex vivo.

category: cell biology


title: Genomic characterization of a pathogenic isolate of Saccharomyces cerevisiae reveals an extensive and dynamic landscape of structural variation

abstract: The budding yeast Saccharomyces cerevisiae has been extensively characterized for many decades and is a critical resource for the study of numerous facets of eukaryotic biology. Recently, the analysis of whole genome sequencing data from over 1000 natural isolates of S. cerevisiae has provided critical insights into the evolutionary landscape of this species by revealing a population structure comprised of numerous genomically diverse lineages. These survey-level analyses have been largely devoid of structural genomic information, mainly because short read sequencing is not suitable for detailed characterization of genomic architecture. Consequently, we still lack a complete perspective of the genomic variation the exists within the species. Single molecule long read sequencing technologies, such as Oxford Nanopore and PacBio, provide sequencing-based approaches with which to rigorously define the structure of a genome, and have empowered yeast geneticists to explore this poorly described realm of eukaryotic genomics. Here, we present the comprehensive genomic structural analysis of a pathogenic isolate of S. cerevisiae, YJM311. We used long read sequence analysis to construct a haplotype-phased, telomere-to-telomere length assembly of the YJM311 diploid genome and characterized the structural variations (SVs) therein. We discovered that the genome of YJM311 contains significant intragenomic structural variation, some of which imparts notable consequences to the genomic stability and developmental biology of the strain. Collectively, we outline a new methodology for creating accurate haplotype-phased genome assemblies and highlight how such genomic analyses can define the structural architectures of S. cerevisiae isolates. It is our hope that through continued structural characterization of S. cerevisiae genomes, such as we have reported here for YJM311, we will comprehensively advance our understanding of eukaryotic genome structure-function relationships, structural diversity, and evolution.

category: genomics


title: Integration of whole genome sequencing and transcriptomics reveals a complex picture of insecticide resistance in the major malaria vector Anopheles coluzzii

abstract: Insecticide resistance is a major threat to gains in malaria control, which have been stalling and potentially reversing since 2015. Studies into the causal mechanisms of insecticide resistance are painting an increasingly complicated picture, underlining the need to design and implement targeted studies on this phenotype. In this study, we compare three populations of the major malaria vector An. coluzzii: a susceptible and two resistant colonies with the same genetic background. The original colonised resistant population rapidly lost resistance over a 6-month period, a subset of this population was reselected with pyrethroids a third population of this colony that did not lose resistance was also available. The original resistant, susceptible and re-selected colonies were subject to RNAseq and whole genome sequencing, which identified a number of changes across the transcriptome and genome linked with resistance. Firstly, an increase in the expression of genes within the oxidative phosphorylation pathway were seen in both resistant populations compared to the susceptible control; this translated phenotypically through an increased respiratory rate, indicating that elevated metabolism is linked directly with resistance. Genome sequencing highlighted several blocks clearly associated with resistance, including the 2Rb inversion. Finally, changes in the microbiome profile were seen, indicating that the microbial composition may play a role in the resistance phenotype. Taken together, this study reveals a highly complicated phenotype in which multiple transcriptomic, genomic and microbiome changes combine to result in insecticide resistance.


title: Genome-wide in vivo screen of circulating tumor cells identifies SLIT2 as a regulator of metastasis

abstract: Circulating tumor cells (CTCs) break free from primary tumors and travel through the bloodstream and lymphatic system to seed metastatic tumors, which are the major cause of death from cancer. The identification of the major genetic factors that enhance production and persistence of CTCs in the bloodstream at a whole genome level would enable more comprehensive molecular mechanisms of metastasis to be elucidated and the identification of novel therapeutic targets, but this remains a challenging task due to the heterogeneity and extreme rarity of CTCs. Here, we describe the first in vivo genome-wide CRISPR KO screen using CTCs directly isolated from a mouse xenograft. This screen elucidated SLIT2 - a gene encoding a secreted protein acting as a cellular migration cue - as the most significantly represented gene knockout in the CTC population. SLIT2 knockout cells are highly metastatic with hypermigratory and mesenchymal phenotype. Reduced expression of SLIT2 is observed in human tumors, indicating its role as a negative modulator of tumor progression and metastasis.

category: cancer biology


title: Lytic Replication and Reactivation from B cells Is Not Required for Maintaining Gammaherpesvirus Latency in vivo

abstract:
Gammaherpesviruses (GHVs) are lymphotropic tumor viruses with a biphasic infectious cycle. Lytic replication at the primary site of infection is necessary for GHVs to spread throughout the host and establish latency in distal sites. Dissemination is mediated by infected B cells that traffic hematogenously from draining lymph nodes to peripheral lymphoid organs, such as the spleen. B cells serve as the major reservoir for viral latency, and it is hypothesized that periodic reactivation from latently infected B cells contributes to maintaining long-term chronic infection. While fundamentally important to an understanding of GHV biology, aspects of B cell infection in latency establishment and maintenance are incompletely defined, especially roles for lytic replication and reactivation in this cell type. To address this knowledge gap and overcome limitations of replication-defective viruses, we generated a recombinant murine gammaherpesvirus 68 (MHV68) in which ORF50, the gene that encodes the essential immediate-early replication and transcription activator protein (RTA), was flanked by loxP sites to enable conditional ablation of lytic replication by ORF50 deletion in cells that express Cre recombinase. Following infection of mice that encode Cre in B cells with this virus, splenomegaly and viral reactivation from splenocytes were significantly reduced, however the number of latently infected splenocytes was equivalent to WT MHV68. Despite ORF50 deletion, MHV68 latency was maintained over time in spleens of mice at levels approximating WT, reactivation-competent MHV68. Stimulation of polyclonal B cell activation and proliferation by treating mice with lipopolysaccharide (LPS), which promotes MHV68 reactivation ex vivo, yielded equivalent increases in the number of latently infected cells for both ORF50-deleted and WT MHV68, even when mice were simultaneously treated with the antiviral drug cidofovir. Together, these data demonstrate that lytic replication in B cells is not required for MHV68 latency establishment and maintenance and further indicate that B cell proliferation, and not reactivation per se, is a major mechanism for maintaining latent viral genomes in the host.

IMPORTANCEGammaherpesviruses establish lifelong chronic infections in cells of the immune system and place infected hosts at risk for developing lymphomas and other diseases. It is hypothesized that gammaherpesviruses must initiate acute infection in these cells to establish and maintain long-term infection, but this has not been directly tested. We report here the use of a viral genetic system that allows for cell-type-specific deletion of a viral gene that is essential for replication and reactivation. We employ this system in an in vivo model to reveal that viral replication is not required to initiate or maintain infection within immune cells.

category: microbiology


title: Monitoring the Antimicrobial Resistance Dynamics of Salmonella enterica in Healthy Dairy Cattle Populations at the Individual Farm Level Using Whole-Genome Sequencing

abstract: Livestock represent a possible reservoir for facilitating the transmission of the zoonotic foodborne pathogen Salmonella enterica to humans; there is also concern that strains can acquire resistance to antimicrobials in the farm environment. Here, we use whole-genome sequencing (WGS) to characterize Salmonella strains (n = 128) isolated from healthy dairy cattle and their associated environments on 13 New York State farms to assess the diversity and microevolution of this important pathogen at the level of the individual herd. Additionally, the accuracy and concordance of multiple in silico tools are assessed, including: (i) two in silico serotyping tools, (ii) combinations of five antimicrobial resistance (AMR) determinant detection tools and one to five AMR determinant databases, and (iii) one antimicrobial minimum inhibitory concentration (MIC) prediction tool. For the isolates sequenced here, in silico serotyping methods outperformed traditional serotyping and resolved all un-typable and/or ambiguous serotype assignments. Serotypes assigned in silico showed greater congruency with the Salmonella whole-genome phylogeny than traditional serotype assignments, and in silico methods showed high concordance (99% agreement). In silico AMR determinant detection methods additionally showed a high degree of concordance, regardless of the pipeline or database used ([≥]98% agreement between susceptible/resistant assignments for all pipeline/database combinations). For AMR detection methods that relied exclusively on nucleotide BLAST, accuracy could be maximized by using a range of minimum nucleotide identity and coverage thresholds, with thresholds of 75% nucleotide identity and 50-60% coverage adequate for most pipeline/database combinations. In silico characterization of the microevolution and AMR dynamics of each of six serotype groups (S. Anatum, Cerro, Kentucky, Meleagridis, Newport, Typhimurium/Typhimurium variant Copenhagen) revealed that some lineages were strongly associated with individual farms, while others were distributed across multiple farms. Numerous AMR determinant acquisition and loss events were identified, including the recent acquisition of cephalosporin resistance-conferring blaCMY- and blaCTX-M-type beta-lactamases. The results presented here provide high-resolution insight into the temporal dynamics of AMR Salmonella at the scale of the individual farm and highlight both the strengths and limitations of WGS in tracking zoonotic pathogens and their associated AMR determinants at the livestock-human interface.


title: OsSWEET11b, a sixth leaf blight susceptibility gene involved in sugar transport-dependent male fertility

abstract: SWEETs play important roles in intercellular sugar transport. Induction of SWEET sugar transporters by transcription activator-like effectors (TALe) of Xanthomonas ssp. is a key factor for bacterial leaf blight (BLB) infection of rice, cassava and cotton. Here, we identified the so far unknown OsSWEET11b with roles in male fertility and BLB susceptibility in rice. While single ossweet11a or b mutants were fertile, double mutants were sterile. Since clade III SWEETs can transport gibberellin (GA), a key hormone for rice spikelet fertility, sterility and BLB susceptibility might be explained by GA transport deficiencies. However, in contrast to the Arabidopsis homologs, OsSWEET11b did not mediate detectable GA transport. Fertility and susceptibility must therefore depend on SWEET11b-mediated sucrose transport. Ectopic induction of OsSWEET11b by designer TALe enables TALe-free Xanthomonas oryzae pv. oryzae (Xoo) to cause disease, identifying OsSWEET11b as a BLB susceptibility gene and demonstrating that the induction of host sucrose uniporter activity is key to virulence of Xoo. Notably, only three of now six clade III SWEETs are targeted by known Xoo strains from Asia and Africa. The identification of OsSWEET11b has relevance in the context of fertility and for protecting rice against emerging Xoo strains that evolve TALes to exploit OsSWEET11b.


title: Spacer2PAM: A computational framework for identification of functional PAM sequences for endogenous CRISPR systems

abstract: RNA-guided nucleases from clustered regularly interspaced short palindromic repeats (CRISPR) systems expand opportunities for precise, targeted genome modification. Endogenous CRISPR systems in many bacteria and archaea are particularly attractive to circumvent expression, functionality, and unintended activity hurdles posed by heterologous CRISPR effectors. However, each CRISPR system recognizes a unique set of PAM sequences, which requires extensive screening of randomized DNA libraries. This challenge makes it difficult to develop endogenous CRISPR systems, especially in organisms that are slow-growing or have transformation idiosyncrasies. To address this limitation, we present Spacer2PAM, an easy-to-use, easy-to-interpret R package built to identify potential PAM sequences for any CRISPR system given its corresponding CRISPR array as input. Spacer2PAM can be used in "Quick" mode to generate a single PAM prediction that is likely to be functional or in "Comprehensive" mode to inform targeted, unpooled PAM libraries small enough to screen in difficult to transform organisms. We demonstrate Spacer2PAM by predicting PAM sequences for industrially relevant organisms and experimentally identifying seven PAM sequences that mediate interference from the Spacer2PAM-predicted PAM library for the type I-B CRISPR system from Clostridium autoethanogenum. We anticipate that Spacer2PAM will facilitate the use of endogenous CRISPR systems for industrial biotechnology and synthetic biology.

category: synthetic biology


title: Evolving a generalist biosensor for bicyclic monoterpenes

abstract: Prokaryotic transcription factors can be repurposed as analytical and synthetic tools for precise chemical measurement and regulation. Monoterpenes encompass a broad chemical family that are commercially valuable as flavors, cosmetics, and fragrances, but have proven difficult to measure, especially in cells. Herein, we develop genetically-encoded, generalist monoterpene biosensors by using directed evolution to expand the effector specificity of the camphor-responsive TetR-family regulator CamR from Pseudomonas putida. Using a novel negative selection coupled with a high-throughput positive screen (Seamless Enrichment of Ligand-Inducible Sensors, SELIS), we evolve CamR biosensors that can recognize four distinct monoterpenes: borneol, fenchol, eucalyptol, and camphene. Different evolutionary trajectories surprisingly yielded common mutations, emphasizing the utility of CamR as a platform for creating generalist biosensors. Systematic promoter optimization driving the reporter increased the systems signal-to-noise ratio to 150-fold. These sensors can serve as a starting point for the high-throughput screening and dynamic regulation of bicyclic monoterpene production strains.


title: The properties of human disease mutations at protein interfaces

abstract: The assembly of proteins into complexes and interactions with other biomolecules are often vital for their biological function. While it is known that mutations at protein interfaces have a high potential to be damaging and cause human genetic disease, there has been relatively little consideration for how this varies between different types of interfaces. Here we investigate the properties of human pathogenic and putatively benign missense variants at homomeric (isologous and heterologous), heteromeric, DNA, RNA and other ligand interfaces, and at different regions with respect to those interfaces. We find that different types of interfaces vary greatly in their propensity to be associated with pathogenic mutations, with homomeric heterologous and DNA interfaces being particularly enriched in disease. We also find that residues that do not directly participate in an interface, but are close in 3D space, also show a significant disease enrichment. Finally, we show that mutations at different types of interfaces tend to have distinct property changes when undergoing amino acid substitutions associated with disease, and that this is linked to substantial variability in their identification by computational variant effect predictors.

category: bioinformatics


title: AlphaFold2 transmembrane protein structure prediction shines

abstract: Transmembrane (TM) proteins are major drug targets, indicated by the high percentage of prescription drugs acting on them. For a rational drug design and an understanding of mutational effects on protein function, structural data at atomic resolution are required. However, hydrophobic TM proteins often resist experimental structure determination and in spite of the increasing number of cryo-EM structures, the available TM folds are still limited in the Protein Data Bank. Recently, the DeepMinds AlphaFold2 machine learning method greatly expanded the structural coverage of sequences, with high accuracy. Since the employed algorithm did not take specific properties of TM proteins into account, the validity of the generated TM structures should be assessed. Therefore, we investigated the quality of structures at genome scales, at the level of ABC protein superfamily folds, and also in specific individual cases. We tested template-free structure prediction also with a new TM fold, dimer modeling, and stability in molecular dynamics simulations. Our results strongly suggest that AlphaFold2 performs astoundingly well in the case of TM proteins and that its neural network is not overfitted. We conclude that a careful application of its structural models will advance TM protein associated studies at an unexpected level.


title: StainedGlass: Interactive visualization of massive tandem repeat structures with identity heatmaps

abstract:
SummaryVisualization and analysis of genomic repeats is typically accomplished through the use of dot plots; however, the emergence of telomere-to-telomere assemblies with multi-megabase repeats requires new visualization strategies. Here, we introduce StainedGlass which can generate publication quality figures and interactive visualizations that depict the identity and orientation of multi-megabase repeat structures at a genome-wide scale. The tool can rapidly reveal higher-order structures and improve the inference of evolutionary history for some of the most complex regions of genomes.

Availability and implementationStainedGlass is implemented using Snakemake and is available open source under the MIT license at https://mrvollger.github.io/StainedGlass/.

Contactmvollger@uw.edu


title: brca2-mutant zebrafish exhibit context- and tissue-dependent alterations in cell phenotypes and response to injury

abstract: Cancer cells frequently co-opt molecular programs that are normally activated in specific contexts, such as embryonic development and the response to injury. Determining the impact of cancer-associated mutations on cellular phenotypes within these discrete contexts can provide new insight into how such mutations lead to dysregulated cell behaviors and subsequent cancer onset. Here we assess the impact of heritable BRCA2 mutation on embryonic development and the injury response using a zebrafish model (Danio rerio). Unlike most mouse models for BRCA2 mutation, brca2-mutant zebrafish are fully viable and thus provide a unique tool for assessing both embryonic and adult phenotypes. We find that maternally provided brca2 is critical for normal oocyte development and embryonic survival in zebrafish, suggesting that embryonic lethality associated with BRCA2 mutation is likely to reflect defects in both meiotic and embryonic developmental programs. On the other hand, we find that adult brca2-mutant zebrafish exhibit aberrant proliferation of several cell types under basal conditions and in response to injury in tissues at high risk for cancer development. These divergent effects exemplify the often-paradoxical outcomes that occur in embryos (embryonic lethality) versus adult animals (cancer predisposition) with mutations in cancer susceptibility genes such as BRCA2. The altered cell behaviors identified in brca2-mutant embryonic and adult tissues, particularly in adult tissues at high risk for cancer, indicate that the effects of BRCA2 mutation on cellular phenotypes are both context- and tissue-dependent.


title: Different neural codes serve long and short-term memory functions in primate Hippocampus and Lateral Prefrontal Cortex during virtual navigation

abstract: The hippocampus (HPC) and the lateral prefrontal cortex (LPFC) are two cortical areas of the primate brain deemed essential to cognition. Here we hypothesize that the codes mediating neuronal communication in HPC and LPFC microcircuits have distinctively evolved to serve plasticity and memory function at different spatiotemporal scales. We used a virtual reality task in which animals navigated through a maze using a joystick and selected one of two targets in the arms of the maze according to a learned context-color rule. We found that neurons and neuronal populations in both regions encode similar information about the task. Moreover, we demonstrate that many HPC neurons concentrate spikes into bursts, whereas most layer II/III LPFC neurons sparsely distribute spikes over time. As the animals learned the task HPC neurons, but not LPFC neurons, increased their burst rate as a function of performance. When integrating spike rates over short intervals, HPC neuronal ensembles reached maximum decoded information with fewer neurons than LPFC ensembles. Our results show that during associative learning HPC principal cells concentrate spikes in bursts enabling temporal summation and fast synaptic plasticity in small populations of neurons and ultimately facilitating rapid encoding of associative memories. On the other hand, layers II/III LPFC pyramidal cells fire spikes more sparsely distributed in time and over a larger number of neurons. The latter would facilitate broadcasting of signals loaded in short term memory across neuronal populations without necessarily triggering fast synaptic plasticity.

category: neuroscience


title: Distinct neural codes in primate Hippocampus and Lateral Prefrontal Cortex during associative learning in virtual environments


title: A small molecule drug screening identifies colistin sulfate as an enhancer of Natural Killer cell cytotoxicity.

abstract: Because of their crucial role in tumor immunity, NK cells have quickly become a prime target for immunotherapies, with adoptive transfer of NK cells and the use of NK cell engagers quickly moving to clinical stage. On the other hand, only few studies have focused on small molecule drugs capable of unleashing NK cell against cancer. In this context, repurposing small molecule is an attractive strategy to identify new immunotherapies from already approved drugs. Here, we screened 1,200 FDA-approved drugs from the Prestwick Chemical Library, to identify compounds that increase NK cell cytotoxic potential. Using a high-throughput luciferase-release cytotoxicity assay, we found that the antibiotic colistin sulfate increased cytotoxicity of human NK cells towards cancer cells. The effect of colistin was short lived and was not observed when NK cells were pretreated with the drug, showing how NK cell activity was potentiated only when the compound was present at the time of recognition of cancer cells. Further studies are needed to uncover the mechanism of action and the pre-clinical efficacy of colistin sulfate in mouse cancer models.

category: immunology


title: Identification and targeting of a pan-genotypic influenza A virus RNA structure that mediates packaging and disease.

abstract:
Currently approved anti-influenza drugs target viral proteins, are subtype limited, and are challenged by rising antiviral resistance. To overcome these limitations, we sought to identify a conserved essential RNA secondary structure within the genomic RNA predicted to have greater constraints on mutation in response to therapeutics targeting this structure. Here, we identified and genetically validated an RNA stemloop structure we termed PSL2, which serves as a packaging signal for genome segment PB2 and is highly conserved across influenza A virus (IAV) isolates. RNA structural modeling rationalized known packaging-defective mutations and allowed for predictive mutagenesis tests. Disrupting and compensating mutations of PSL2s structure give striking attenuation and restoration, respectively, of in vitro virus packaging and mortality in mice. Antisense Locked Nucleic Acid oligonucleotides (LNAs) designed against PSL2 dramatically inhibit IAV in vitro against viruses of different strains and subtypes, possess a high barrier to the development of antiviral resistance, and are equally effective against oseltamivir carboxylate-resistant virus. A single dose of LNA administered 3 days after, or 14 days before, a lethal IAV inoculum provides 100% survival. Moreover, such treatment led to the development of strong immunity to rechallenge with a ten-fold lethal inoculum. Together, these results have exciting implications for the development of a versatile novel class of antiviral therapeutics capable of prophylaxis, post-exposure treatment, and "just-in-time" universal vaccination against all IAV strains, including drug-resistant pandemics.

One Sentence SummaryTargeting a newly identified conserved RNA structure in the packaging signal region of influenza segment PB2 abrogates virus production in vitro and dramatically attenuates disease in vivo.


title: Ecological dynamics of the gut microbiome in response to dietary fiber

abstract: Dietary fibers are generally thought to benefit intestinal health. Their impacts on the composition and metabolic function of the gut microbiome, however, vary greatly across individuals. Previous research showed that each individuals response to fibers depends on their baseline gut microbiome, but the ecology driving microbiota remodeling during fiber intake remained unclear. Here, we studied the long-term dynamics of gut microbiome and short-chain fatty acids (SCFAs) in isogenic mice with distinct microbiota baselines fed with the fermentable fiber inulin compared to the non-fermentable fiber cellulose. We found that inulin produced generally rapid response followed by gradual stabilization to new equilibria, and those dynamics were baseline-dependent. We parameterized an ecology model from the timeseries data, which revealed a group of bacteria whose growth significantly increases in response to inulin. and whose baseline abundance and interspecies competition explains the baseline-dependence of microbiome density and community composition dynamics. Fecal levels of of SCFAs, such as propionate, is associated with the abundance of inulin responders, yet inter-individual variation of gut microbiome impedes the prediction of SCFAs by machine learning models. Finally, we showed that our methods and major findings are generalizable to dietary resistant starch. This study emphasizes the importance of ecological modeling to understand microbiome responses to dietary changes and the need for personalized interventions.

category: ecology


title: Camera trap based niche partitioning model of medium size ungulates and its implications on tigers prey availability in SE Asian rainforest

abstract: The presence and survival of the Sumatran tiger were determined by the presence, density, and activity of its prey. Several medium size mammal belongs to the ungulate were known as the potential prey for the tiger. While, the information on the temporal availability of this prey in the rainforest is still limited. In here this study aimed to aims to model the niche partitioning of several mammalian species that was potential prey for tiger. The studied species were including barking deer (Muntiacus muntjak), bearded pig (Sus barbatus), and wild boar (Sus scrofa). The method was using camera trap with data analyses including the calculation of Kernel density, diel activity, and niche partitioning determined using overlap index. . Based on the results, barking deer and wild boar showed only singular activity peak while bearded pig has several activity peaks. Only wild boar showed a strict diurnal activity pattern between 06:00 h and 12:00 h. Barking deer showed a crepuscular behavior with several activities observed at 18:00 h. While bearded pig showed a nocturnal behavior and showed at least two peaks of activity, one between 09:00 to 13:00 and another between 18:00 to 24:00 h. Barking deer and bearded pig uses almost similar niches since those species have the highest overlap indices value equals 0.504(95%CI:0.193-0.824). The lowest overlap indices value was observed for barking deer and wild boar with overlap indice values of 0.032(95%CI:0.0-0.162). Considering the diurnal activity pattern of the tiger that is mostly active at day, then the available preys were either barking deer or bearded pigs. While since barking deer and bearded pigs were using the same niche, then there will be potential competition.


title: Chromosomal rearrangements preserve adaptive divergence in ecological speciation

abstract: Despite increasing empirical evidence that chromosomal rearrangements may play an important role in adaptive divergence and speciation, the degree to which divergent genomic regions are associated with chromosomal rearrangements remains unclear. In this study, we provide the first whole-genome analyses of ecological speciation and chromosomal evolution in a Daphnia species complex, using chromosome-scale assemblies and natural-population sequencing of the recently diverged species pair, Daphnia pulicaria and Daphnia pulex, which occupy distinct yet overlapping habitats in North America, and the outgroup species Daphnia obtusa. Our results describe a mixed mode of geographic divergence (isolation with secondary contact) resulting in a heterogeneous landscape of genomic divergence. Large contiguous "continents of divergence" encompass over one third of the genome (36%) and contain nearly all the fixed differences (94%) between the species, while the background genome has been homogenized. Chromosomal rearrangements between species, including inversions and intrachromosomal translocations, are associated with the continents of divergence and capture multiple adaptive alleles in genes and pathways thought to contribute to the species phenotypic differences.


title: Physiological and genetic drivers underpinning canopy development are associated with durum wheat yield in rainfed environments

abstract: New durum wheat (Triticum turgidum L. ssp. Durum) cultivars with improved adaptation to variable rainfall environments are required to sustain productivity in the face of climate change. Physiological traits related to canopy development underpin the production of biomass and yield, as they interact with solar radiation and affect the timing of water use throughout the growing season. This study explored the temporal canopy dynamics of durum wheat using a nested-association mapping population evaluated for longitudinal normalized difference vegetation index (NDVI) measurements. Association mapping was performed to identify quantitative trait loci (QTL) for time-point NDVI and spline-smoothed NDVI trajectory traits. Yield effects associated with QTL for canopy development were investigated using data from four rainfed field trials. Four QTL associated with slower canopy closure, improved yield in specific environments, and notably, were not associated with a yield penalty in any environment. This was likely due to optimised timing of water-use and pleiotropic effects on yield component traits, including spike number and spike length. Overall, this study suggests that slower canopy closure is beneficial for durum wheat production in rainfed environments. Selection for traits or loci associated with canopy development may improve yield stability of durum wheat in water limited environments.

category: genetics


title: Liver enhancer signature and regulatory network of non-alcoholic steatohepatitis resistance in humans with obesity

abstract: The molecular underpinnings of non-alcoholic steatohepatitis (NASH) development in patients are poorly understood. Active enhancer landscapes are known to determine cell states and behaviors. Super-enhancers, in particular, have helped reveal key disease drivers in several cancer types; however, they remain unexplored in human NASH. To define the enhancer signature of NASH-prone (NP) and NASH-resistant (NR) phenotypes in humans with obesity, we performed chromatin run-on sequencing (ChRO-seq) analysis on liver biopsies of individuals with obesity who were stratified into either NP or NR. We first demonstrated that NP and NR groups exhibit distinct active enhancer signatures. The subsequent identification of NP- and NR-specific super-enhancers revealed the specific genes that are likely the most critical for each of the phenotypes, including HES1 for NP and GATM for NR. Integrative analysis with results from genome-wide association studies of NAFLD and related traits identified disease/trait-loci specific to NP or NR enhancers. Further analysis of the ChRO-seq data pointed to critical roles for serine/glycine metabolism in NASH resistance, which was corroborate by profiling of circulating amino acids in the same patients. Overall, the distinct enhancer signatures of human NP and NR phenotypes revealed key genes, pathways, and transcription factor networks that promote NASH development.


title: Super-enhancer signature reveals key mechanisms associated with resistance to non-alcoholicsteatohepatitis in humans with obesity


title: Engineering large-scale perfused tissues via synthetic 3D soft microfluidics

abstract: The vascularization of engineered tissues and organoids has remained a major unresolved challenge in regenerative medicine. While multiple approaches have been developed to vascularize in vitro tissues, it has thus far not been possible to generate sufficiently dense networks of small-scale vessels to perfuse large de novo tissues. Here, we achieve the perfusion of multi-mm3 tissue constructs by generating networks of synthetic capillary-scale 3D vessels. Our 3D soft microfluidic strategy is uniquely enabled by a 3D-printable 2-photon-polymerizable hydrogel formulation, which allows for precise microvessel printing at scales below the diffusion limit of living tissues. We demonstrate that these large-scale engineered tissues are viable, proliferative and exhibit complex morphogenesis during long-term in-vitro culture, while avoiding hypoxia and necrosis. We show by scRNAseq and immunohistochemistry that neural differentiation is significantly accelerated in perfused neural constructs. Additionally, we illustrate the versatility of this platform by demonstrating long-term perfusion of developing liver tissue. This fully synthetic vascularization platform opens the door to the generation of human tissue models at unprecedented scale and complexity.


title: The origin of universal cell shape variability in a confluent epithelial monolayer

abstract: Cell shape is fundamental in biology. The average cell shape can influence crucial biological functions, such as cell fate and division orientation. But cell-to-cell shape variability is often regarded as noise. In contrast, recent works reveal that shape variability in diverse epithelial monolayers follows a nearly universal distribution. However, the origin and implications of this universality are unclear. Here, assuming contractility and adhesion are crucial for cell shape, characterized via aspect ratio (AR), we develop a mean-field analytical theory for shape variability. We find that a single parameter, , containing all the system-specific details, describes the probability distribution function (PDF) of AR; this leads to a universal relation between the standard deviation and the average of AR. The PDF for the scaled AR is not strictly but almost universal. The functional form is not related to jamming, contrary to common beliefs, but a consequence of a mathematical property. In addition, we obtain the scaled area distribution, described by the parameter {micro}. We show that  and {micro} together can distinguish the effects of changing physical conditions, such as maturation, on different system properties. The theory is verified in simulations of two distinct models of epithelial monolayers and agrees well with existing experiments. We demonstrate that in a confluent monolayer, average shape determines both the shape variability and dynamics. Our results imply the cell shape variability is inevitable, where a single parameter describes both statics and dynamics and provides a framework to analyze and compare diverse epithelial systems.


title: NeuralDock: Rapid and conformation-agnostic docking of small molecules

abstract: Virtual screening is a cost- and time-effective alternative to traditional high-throughput screening in the drug discovery process. Both virtual screening approaches, structure-based molecular docking and ligand-based cheminformatics, suffer from computational cost, low accuracy, and/or reliance on prior knowledge of a ligand that binds to a given target. Here, we propose a neural network framework, NeuralDock which accelerates the process of high-quality computational docking by a factor of 106, and does not require prior knowledge of a ligand that binds to a given target. By approximating both protein-small molecule conformational sampling and energy-based scoring, NeuralDock accurately predicts the binding energy and affinity of a protein-small molecule pair, based on protein pocket 3D structure and small molecule topology. We use NeuralDock and 25 GPUs to dock 937 million molecules from the ZINC database against superoxide dismutase-1 in 21 hours, which we validate with physical docking using MedusaDock. Due to its speed and accuracy, NeuralDock may be useful in brute-force virtual screening of massive chemical libraries and training of generative drug models.


title: Bioequivalence Assessment of High-Capacity Polymeric Micelle Nanoformulation of Paclitaxel and Abraxane(R) in Rodent and Non-Human Primate Models Using a Stable Isotope Tracer Assay

abstract:
The in vivo fate of nanoformulated drugs is governed by the physicochemical properties of the drug and the functionality of nanocarriers. Nanoformulations such as polymeric micelles, which physically encapsulate poorly soluble drugs, release their payload into the bloodstream during systemic circulation. This results in three distinct fractions of the drug-nanomedicine: encapsulated, protein-bound, and free drug. Having a thorough understanding of the pharmacokinetic (PK) profiles of each fraction is essential to elucidate mechanisms of nanomedicine-driven changes in drug exposure and PK/PD relationships pharmacodynamic activity. Here, we present a comprehensive preclinical assessment of the poly(2-oxazoline)-based polymeric micelle of paclitaxel (PTX) (POXOL hl-PM), including bioequivalence comparison to the clinically approved paclitaxel nanomedicine, Abraxane(R). Physicochemical characterization and toxicity analysis of POXOL hl-PM was conducted using standardized protocols by the Nanotechnology Characterization Laboratory (NCL). The bioequivalence of POXOL hl-PM to Abraxane(R) was evaluated in rats and rhesus macaques using the NCLs established stable isotope tracer ultrafiltration assay (SITUA) to delineate the plasma PK of each PTX fraction. The SITUA study revealed that POXOL hl-PM and Abraxane(R) had comparable PK profiles not only for total PTX but also for the distinct drug fractions, suggesting bioequivalence in given animal models. The comprehensive preclinical evaluation of POXOL hl-PM in this study showcases a series of widely-applicable standardized studies by NCL for assessing nanoformulations prior to clinical investigation.

GRAPHICAL ABSTRACT

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category: pharmacology and toxicology


title: Why be thrifty? Sex-specific heterothermic patterns in wintering captive Microcebus murinus do not translate into differences in energy balance.

abstract: The physiological mechanisms of the responses toward stressors are the core of ecophysiology studies to understand the limits of an organisms flexibility and better predict the impact of environmental degradation on natural populations. However, little information is available when we question inter-individual variability of these physiological responses, even though they can be particularly important. Some observations of intersexual differences in heterothermy raised the question of a difference in energy management between sexes. Here we assess male and female differences in a mouse lemur model (Microcebus murinus), a highly seasonal Malagasy primate, studying their physiological flexibility toward caloric restriction, and examining the impact on their reproductive success. These animals are adapted for naturally changing food availability and climate conditions, and can express deep torpor, allowing them to spare their energy expenses over the dry and cold season. We monitored body mass and body temperature on 12 males and 12 females over winter, applying a chronic 40% caloric restriction to 6 individuals of each group. Our results showed variability of Tb modulations throughout winter and in response to caloric treatment depending on the sex, as females entered deep torpor regardless of food restriction, while only CR males had a similar response. The use of deep torpor, however, did not translate into better body condition either in females, or in response to CR, and did not clearly affect reproductive success. The favorable captive context potentially buffered the depth of torpor and minimized the positive effects of using torpor on energy savings. However, our results may emphasize the existence of other benefits of heterothermic responses than fat reserves.

category: physiology


title: Mechano-arrhythmogenicity is enhanced during late repolarisation in ischemia and driven by a TRPA1-, calcium-, and reactive oxygen species-dependent mechanism

abstract:
BackgroundCardiac dyskinesis in regional ischemia results in arrhythmias through mechanically-induced changes in electrophysiology ( mechano-arrhythmogenicity) that involve ischemic alterations in voltage-calcium (Ca2+) dynamics, creating a vulnerable period (VP) in late repolarisation.

ObjectiveTo determine cellular mechanisms of mechano-arrhythmogenicity in ischemia and define the importance of the VP.

Methods and ResultsVoltage-Ca2+ dynamics were simultaneously monitored in rabbit ventricular myocytes by dual-fluorescence imaging to assess the VP in control and simulated ischemia (SI). The VP was longer in SI than in control (146{+/-}7 vs 54{+/-}8ms; p<0.0001) and was reduced by blocking KATP channels with glibenclamide (109{+/-}6ms; p<0.0001). Cells were rapidly stretched (10-18% increase in sarcomere length over 110-170ms) with carbon fibres during diastole or the VP. Mechano-arrhythmogenicity, associated with stretch and release in the VP, was greater in SI than control (7 vs 1% of stretches induced arrhythmias; p<0.005) but was similar in diastole. Arrhythmias during the VP were more complex than in diastole (100 vs 69% had sustained activity; p<0.05). In the VP, incidence was reduced with glibenclamide (2%; p<0.05), by chelating intracellular Ca2+ (BAPTA; 2%; p<0.05), blocking mechano-sensitive TRPA1 (HC-030031; 1%; p<0.005), or by scavenging (NAC; 1%; p<0.005) or blocking reactive oxygen species (ROS) production (DPI; 2%; p<0.05). Ratiometric Ca2+ imaging revealed that SI increased diastolic Ca2{+/-} (+9{+/-}1%, p<0.0001), which was not prevented by HC-030031 or NAC.

ConclusionIn ischemia, mechano-arrhythmogenicity is enhanced specifically during the VP and is mediated by ROS, TRPA1, and Ca2+.


title: Age-related changes in Tau and Autophagy in human brain in the absence of neurodegeneration

abstract: Tau becomes abnormally hyper-phosphorylated and aggregated in tauopathies like Alzheimers disease (AD). As age is the greatest risk factor for developing AD, it is important to understand how tau protein itself, and the pathways implicated in its turnover, change during aging. We investigated age-related changes in total and phosphorylated tau in brain samples from two cohorts of cognitively normal individuals spanning 19-74 years, without overt neurodegeneration. One cohort utilised resected tissue and the other used post-mortem tissue. Total soluble tau levels declined with age in both cohorts. Phosphorylated tau was undetectable in the post-mortem tissue but was clearly evident in the resected tissue and did not undergo significant age-related change. To ascertain if the decline in soluble tau was correlated with age-related changes in autophagy, three markers of autophagy were tested but only two appeared to increase with age and the third was unchanged. This implies that in individuals who do not develop neurodegeneration, there is an age-related reduction in soluble tau which could potentially be due to age-related changes in autophagy. Thus, to explore how an age-related increase in autophagy might influence tau-mediated dysfunctions in vivo, autophagy was enhanced in a Drosophila model and all age-related tau phenotypes were significantly ameliorated. These data shed light on age-related physiological changes in proteins implicated in AD and highlights the need to study pathways that may be responsible for these changes. It also demonstrates the therapeutic potential of interventions that upregulate turnover of aggregate-prone proteins during aging.


title: Habitat fragmentation induces rapid divergence of migratory and isolated sticklebacks

abstract:
The adaptive capacity of many organisms is seriously challenged by human-imposed environmental change, which currently happens at unprecedented rates and magnitudes. For migratory fish, habitat fragmentation is a major challenge that can compromise their survival and reproduction. Therefore, it is important to study if fish populations can adapt to such modifications of their habitat. Here, we study whether originally anadromous three-spined stickleback populations (Gasterosteus aculeatus;  migrants) changed in behavior and morphology in response to human-induced isolation. We made use of a natural field-experiment, where the construction of pumping stations and sluices in the 1970s unintendedly created replicates of land-locked stickleback populations ( resident) in the Netherlands. For two years, we systematically tested populations of residents and migrants for differences in morphology and behavioral traits (activity, aggressiveness, exploration, boldness and shoaling) in lab-based assays. We detected differences between migrant and resident populations in virtually all phenotypic traits studied: compared to the ancestral migrants, residents were smaller in size, had fewer and smaller plates and were significantly more active, aggressive, exploratory and bolder and shoaled less. Despite large ecological differences between 2018 and 2019, results were largely consistent across the two years. Our study shows that human-induced environmental change has led to the rapid and consistent morphological and behavioral divergence of stickleback populations in about 50 generations. Such changes may be adaptive but this remains to be tested.

Lay summaryThe adaptive capacity of many organisms is seriously challenged by human-imposed environmental changes. For example, migratory fish encounter man-made barriers that impede their movements and force them to adopt a resident lifecycle. Here we study whether and how populations of three-spined sticklebacks diverged in response to human-induced isolation. We show that about 50 generations of isolation were sufficient to induce substantial morphological and behavioral differentiation between land-locked populations ( residents) and their migratory ancestors ( migrants).


title: Decoding cellular deformation from pseudo-simultaneously observed Rho GTPase activities

abstract: The inability to simultaneously observe all of the important Rho GTPases (Cdc42, Rac1, and RhoA) has prevented us from obtaining evidence of their coordinated regulation during cell deformation. Here, we propose Motion-Triggered Average (MTA), an algorithm that converts individually observed GTPases into pseudo-simultaneous observations. Using the time series obtained by MTA and mathematical model, we succeeded for the first time in decoding the cell edge velocity from the three GTPase activities to provide clear numerical evidence for coordinated cell edge regulation by the three GTPases. We found that the characteristics of the obtained activities were consistent with those of previous studies, and that GTPase activities and their derivatives were involved in edge regulation. Our approach provides an effective strategy for using single-molecule observations to elucidate problems hampered by the lack of simultaneous observations.

category: systems biology


title: Beyond dimension reduction: Stable electric fields emerge from and allow representational drift

abstract: It is known that the exact neurons maintaining a given memory (the neural ensemble) change from trial to trial. This raises the question of how the brain achieves stability in the face of this representational drift. Here, we demonstrate that this stability emerges at the level of the electric fields that arise from neural activity. We show that electric fields carry information about working memory content. The electric fields, in turn, can act as "guard rails" that funnel higher dimensional variable neural activity along stable lower dimensional routes. We obtained the latent space associated with each memory. We then confirmed the stability of the electric field by mapping the latent space to different cortical patches (that comprise a neural ensemble) and reconstructing information flow between patches. Stable electric fields can allow latent states to be transferred between brain areas, in accord with modern engram theory.


title: Mechanistic basis for the emergence of EPS1 as a catalyst in plant salicylic acid biosynthesis

abstract:
Unique to plants in the Brassicaceae family, the production of the plant defense hormone salicylic acid (SA) from isochorismate is accelerated by an evolutionarily young isochorismoyl-glutamate pyruvoyl-glutamate lyase, EPS1, which belongs to the BAHD acyltransferase protein family. Here, we report the crystal structures of apo and substrate-analog-bound EPS1 from Arabidopsis thaliana. Assisted by microsecond molecular dynamics simulations, we uncover a unique pericyclic rearrangement lyase mechanism facilitated by the active site of EPS1. We reconstitute the isochorismate-derived pathway of SA biosynthesis in Saccharomyces cerevisiae, which serves as an in vivo platform that helps identify active-site residues critical for EPS1 activity. This study describes the birth of a new catalyst in plant phytohormone biosynthesis by reconfiguring the ancestral active site of a progenitor enzyme to catalyze alternative reaction.

One sentence summaryBy reconfiguring the active site of a progenitor acyltransferase-fold, EPS1 acquired the unique, evolutionarily new lyase activity that accelerates phytohormone salicylic acid production in Brassicaceae plants.

category: biochemistry


title: Rational Discovery of New Folds for Human Solute Carrier Proteins

abstract: Solute carrier (SLC) superfamily is the largest group responsible for transmembrane transport of substances in human cells. It includes more than 400 members which are organized into 65 families according to their physiological function and sequence similarity. Different families of solute transporters can adopt the same or different folds that determine the working mechanism and reflect the evolutionary relationship between SLC members. Analysis of structural data in literatures shows that there are 13 different folds in the solute carrier superfamily covering 40 families and 343 members. To further study their working mechanism, we systematically explored the SLC superfamily to look for more folds. Based on our results, at least three new folds were found for SLC superfamily, and one was experimentally verified in the SLC44 family. Our work has laid a foundation and provided important insights for the systematic and comprehensive study on the structure and function of solute carriers.

category: biophysics


title: Rational Exploration of Fold Atlas for Human Solute Carrier Proteins


title: MEK1/2 inhibition transiently alters the tumor immune microenvironment to enhance immunotherapy efficacy against head and neck cancer

abstract:
Although the mitogen-activated protein kinases (MAPK) pathway is hyperactive in head and neck cancer (HNC), inhibition of MEK1/2 in HNC patients has not shown clinically meaningful activity. Using pre-clinical HNC models, we demonstrated that treatment with the MEK1/2 blocker trametinib delays HNC initiation and progression by reducing tumor cell proliferation and enhancing the anti-tumor immunity of CD8+ T cells. Further activation of CD8+ T cells by supplementation with anti-programmed death-1 (PD-1) antibody eliminated tumors and induced an immune memory in the cured mice. Mechanistically, an early response to trametinib treatment sensitized tumors to PD-1-supplementation by attenuating the expression of tumor-derived colony-stimulating factor-1 (CSF-1), which reduced the abundance of two CSF-1R+CD11c+ myeloid-derived suppressor cell (MDSC) populations in the tumor microenvironment (TME). In contrast, prolonged treatment with trametinib abolished the anti-tumor activity of PD-1, because tumor cells undergoing the epithelial to mesenchymal transition (EMT) in response to trametinib restored CSF-1 expression and re-created an immune-suppressive TME. These findings provide the rationale for testing the trametinib/PD-1 combination in HNC and highlight the importance of sensitizing tumors to immunotherapies by using targeted therapies to interfere with the host-tumor interaction.

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title: Chemistry, Taxonomy and Ecology of the potentially chimpanzee-dispersed Vepris teva sp.nov. (Rutaceae) of coastal thicket in the Congo Republic

abstract: Continuing a survey of the chemistry of species of the largely continental African genus Vepris, we investigate a species previously referred to as Vepris sp. 1 of Congo. From the leaves of Vepris sp. 1 we report six compounds. The compounds were three furoquinoline alkaloids, kokusaginine (1), maculine (2), and flindersiamine (3), two acridone alkaloids, arborinine (4)and 1-hydroxy-3-methoxy-10-methylacridone (5), and the triterpenoid, {beta}-amyrin (6). Compounds 1-4 are commonly isolated from other Vepris species, compound 5 has been reported before once, from Malagasy Vepris pilosa, while this is the first report of {beta}-amyrin from Vepris. This combination of compounds has never before been reported from any species of Vepris. We test the hypothesis that Vepris sp.1 is new to science and formally describe it as Vepris teva, unique in the genus in that the trifoliolate leaves are subsessile, with the median petiolule far exceeding the petiole in length. Similar fleshy-leathery 4-locular syncarpous fruits are otherwise only known in the genus in Vepris glaberrima (formerly the monotypic genus Oriciopsis Engl.), a potential sister species, but requiring further investigation to confirm this phylogenetic position. We briefly characterise the unusual and poorly documented Atlantic coast equatorial ecosystem, where Vepris teva is restricted to evergreen thicket on white sand, unusual in a genus usually confine to evergreen forest. This endemic-rich ecosystem with a unique amphibian as well as plants, extends along the coastline from the mouth of the Congo River to southern Rio Muni, a distance of about 1000 km, traversing five countries. We map and illustrate Vepris teva and assess its extinction risk as Endangered (EN B1ab(iii)+B2ab(iii)) using the IUCN standard. Only three locations are known, and threats include port and oil refinery construction and associated activities, with only one protected location, the Jane Goodall Institutes Tchimpounga Reserve. Initial evidence indicates that the seeds of Vepris teva are dispersed by chimpanzees, previously unreported in the genus


title: A deep mutational scanning platform to characterize the fitness landscape of anti-CRISPR proteins

abstract: Deep mutational scanning is a powerful method to explore the mutational fitness landscape of proteins. Its adaptation to anti-CRISPR proteins, which are natural CRISPR-Cas inhibitors and key players in the co-evolution of microbes and phages, would facilitate their in-depth characterization and optimization. Here, we developed a robust anti-CRISPR deep mutational scanning pipeline in Escherichia coli combining synthetic gene circuits based on CRISPR interference with flow cytometry-coupled sequencing and mathematical modeling. Using this pipeline, we created and characterized comprehensive single point mutation libraries for AcrIIA4 and AcrIIA5, two potent inhibitors of Streptococcus pyogenes Cas9. The resulting mutational fitness landscapes revealed that both Acrs possess a considerable mutational tolerance as well as an intrinsic redundancy with respect to Cas9 inhibitory features, suggesting evolutionary pressure towards high plasticity and robustness. Finally, to demonstrate that our pipeline can inform the optimization and fine-tuning of Acrs for genome editing applications, we cross-validated a subset of AcrIIA4 mutants via gene editing assays in mammalian cells and in vitro affinity measurements. Together, our work establishes deep mutational scanning as powerful method for anti-CRISPR protein characterization and optimization.


title: Eliminating Aedes aegypti from its southern margin in Australia: insights from genomic data and simulation modeling

abstract:
A rare example of a successful long-term elimination of the mosquito Aedes aegypti is in Brisbane, Queensland, where the legislatively-enforced removal of rainwater tanks drove its disappearance by the mid-1950s. However, a decade-long drought led to the mass installation of rainwater tanks throughout the region, re-introducing critical breeding sites for the mosquitos persistence in this subtropical region. With Ae. aegypti re-invading towns just 150 km north of Brisbane, we examined the potential for their sustained elimination. Through genomic analyses, we estimated historical expansion and current isolation between neighboring populations as close as 15 kilometers. The estimated recent migration rate, entomological and meteorological data were used to calibrate the simulations of elimination campaigns in the two southernmost populations. Our simulations indicate that Ae. aegypti could be eliminated with moderate release numbers of incompatible Wolbachia-infected (IIT) males (sorted with an error rate [≤]10-6) if non-compliant rainwater tanks are removed first. With this combined campaign, highly effective suppression (>99%) was predicted in both towns, and complete elimination was predicted in 35% of simulations in one town. Without tank removal, however, IIT led to a moderate suppression (61-93%) even with a 40:1 ratio of released IIT males to local males. Moreover, with a ratio of >20:1, Wolbachia establishment was predicted when the sorting error was >10-7. Our conservative estimates of intervention outcomes inform the planning of Ae. aegypti elimination in the region, and offer insight into the effective combinations of conventional and novel control tools, particularly for vulnerable mosquito populations at range margins.

SignificanceAfter decades of range stagnation in Australia, the Aedes aegypti mosquito is expanding southward, approaching the most-densely-populated areas of Queensland. Using population genomics and simulation modeling of elimination campaigns, we show that Australias southernmost populations of this disease vector are genetically isolated and could be eliminated with moderate releases of incompatible Wolbachia-infected males if major larval breeding sites (non-compliant rainwater tanks) are removed first. The risk of Wolbachia establishment for this approach is low, and so is the risk of quick mosquito re-invasion. Our conservative estimates of intervention outcomes inform the planning of Ae. aegypti elimination in the region, and offer new insight into the benefits of combining conventional and novel control tools, particularly for mosquito populations at range margins.


title: BIFURCATING NEURONS IN THE ANTERIOR THALAMIC NUCLEI

abstract: The anterior thalamic nuclei (ATN) form a nodal point within a distributed memory network. The conventional view of ATN function describes segregated efferents to different terminal regions. By contrast, we found that bifurcating neurons are common within the anteromedial nucleus (AM) of the ATN. A substantial proportion of AM neurons ([~]36% within a region of interest) showed collateral projections when one of two retrograde neurotracers, Cholera Toxin Subunit B (CTB) conjugated to either Alexa Fluor(R) 488 or 594, was placed in the medial prefrontal cortex (mPFC) or dorsal subiculum (dSub). A marked degree of collateralization ([~]20% AM neurons) was also found when neurotracers were placed in mPFC and caudal retrosplenial cortex (cRSC); about 10% showed collaterals when the cRSC was paired with either dSub or vHF; the fewest (6%) was found for mPFC paired with the ventral hippocampal formation (vHF). A generally similar range of percentages of bifurcating neurons was found in the adjacent nucleus reuniens (Re). Evidence that AM neurons project simultaneously to many distantly-located structures provides a new perspective on ATN function. These neurons would facilitate direct coordination among key neural structures to support memory and may explain the strong association between the ATN and diencephalic amnesia.


title: Bifurcating Neurons In The Anterior Thalamic Nuclei


title: Bantam regulates the adult sleep circuit in Drosophila

abstract: Sleep is a highly conserved feature of animal life characterized by dramatic changes in behavior, neural physiology and gene expression. The gene regulatory factors responsible for these sleep-dependent changes remain largely unknown. microRNAs are post-transcriptional modulators of gene expression which have been implicated in sleep regulation. Our previous screen identified 25 sleep-regulating microRNAs in Drosophila melanogaster, including the developmental regulator bantam (ban). Here we show that ban promotes early nighttime sleep through a population of glutamatergic neurons- the {gamma}5{beta}'2a/{beta}'2mp/{beta}'2mp_bilateral Mushroom Body Output Neurons (MBONs). We found that knockdown of ban in these neurons led to a reduction in early night sleep. The {gamma}5{beta}'2a/{beta}'2mp/{beta}'2mp_bilateral MBONs were previously shown to be wake-promoting, suggesting that ban acts to inhibit these neurons. GCaMP calcium imaging revealed that bantam inhibits the neural activity of the {gamma}5{beta}'2a/{beta}'2mp/{beta}'2mp_bilateral MBONs during the night but not the day. Blocking synaptic transmission in the {gamma}5{beta}'2a/{beta}'2mp/{beta}'2mp_bilateral MBONs rescued the effect of ban knockdown on sleep. Together these results suggest that ban promotes night sleep via the inhibition of the {gamma}5{beta}'2a/{beta}'2mp/{beta}'2mp_bilateral MBONs. RNAseq further revealed that bantam negatively regulates the wake-promoting mRNAs Kelch and CCHamide-2 receptor in the {gamma}5{beta}'2a/{beta}'2mp/{beta}'2mp_bilateral MBONs. These experiments establish bantam as an active regulator of sleep and neural activity within the fly brain.


title: Pro-IL-18 secreted by keratinocytes detects the group A streptococcal protease SpeB

abstract: Group A Streptococcus (GAS, Streptococcus pyogenes) is a professional human pathogen that commonly infects the skin. Keratinocytes are one of the first cells to contact GAS, and by inducing inflammation, they can initiate the earliest immune responses to pathogen invasion. Here, we characterized the proinflammatory cytokine repertoire produced by primary human keratinocytes and surrogate cell lines commonly used in vitro. Infection induces several cytokines and chemokines, but keratinocytes constitutively secrete IL-18 in a form that is inert (pro-IL-18) and lacks proinflammatory activity. Canonically, IL-18 activation and secretion are coupled through a single proteolytic event that is regulated intracellularly by the inflammasome protease caspase-1 in myeloid cells. The pool of extracellular pro-IL-18 generated by keratinocytes is poised to sense extracellular proteases and is directly processed into a mature active form by SpeB, a secreted GAS protease that is a critical virulent factor during skin infection. This mechanism contributes to the proinflammatory response against GAS, resulting in T cell activation and the secretion of IFN-{gamma} that restrict GAS growth. Other major bacterial pathogens and microbiota of this skin did not have significant IL-18-maturing ability. Taken together, these results suggest keratinocyte-secreted IL-18 is a sentinel that sounds an early alarm that is highly sensitive to GAS, yet tolerant to non-invasive members of the microbiota.


title: Teasing apart the host-related, nutrient-related and temperature-related effects shaping the phenology and microbiome of the tropical seagrass Halophila stipulacea

abstract:
BackgroundHalophila stipulacea seagrass meadows are an ecologically important and threatened component of the ecosystem in the Gulf of Aqaba. Recent studies have demonstrated correlated geographic patterns for leaf endophytic community composition and leaf morphology, also coinciding with different levels of water turbidity and nutrient concentrations. Based on these observations, workers have suggested an environmental microbial fingerprint, which may reflect various environmental stress factors seagrasses have experienced, and may add a holobiont level of plasticity to seagrasses, assisting their acclimation to changing environments and through range expansion. However, it is difficult to tease apart environmental effects from host-diversity dependent effects, which have covaried in field studies, although this is required in order to establish that differences in microbial community compositions among sites are driven by environmental conditions rather than by features governed by the host.

ResultsIn this study we carried out a mesocosm experiment, in which we studied the effects of warming and nutrient stress on the composition of epiphytic bacterial communities and on some phenological traits. We studied H. stipulacea collected from two different meadows in the Gulf of Aqaba, representing differences in the host and the environment alike. We found that the source site from which seagrasses were collected was the major factor governing seagrass phenology, although heat increased shoot mortality and nutrient loading delayed new shoot emergence. Bacterial diversity, however, mostly depended on the environmental conditions. The most prominent pattern was the increase in Rhodobacteraceae under nutrient stress without heat stress, along with an increase in Microtrichaceae. Together, the two taxa have the potential to maintain nitrate reduction followed by an anammox process, which can together buffer the increase in nutrient concentrations across the leaf surface.

ConclusionsOur results thus corroborate the existence of environmental microbial fingerprints, which are independent from the host diversity, and support the notion of a holobiont level plasticity, both important to understand and monitor H. stipulacea ecology under the changing climate.


title: Genotype-specific Features Reduce the Susceptibility of South American Yellow Fever Virus Strains to Vaccine-Induced Antibodies

abstract: The resurgence of yellow fever in South America has prompted mitigation through vaccination against the etiologic agent, yellow fever virus (YFV). Current vaccines are based on a virulent African isolate, and their capacity to induce neutralizing antibodies against the vaccine strain is widely used as a surrogate for protection. However, the sensitivity of genetically distinct South American strains to vaccine-induced antibodies is unknown. Here, we show that antiviral potency of the polyclonal antibody response in both U.S. and Brazilian vaccinees is attenuated against an emergent Brazilian strain. This reduction was attributable to genetic changes at two sites in the central domain II of the glycoprotein E, including the acquisition of an N-linked glycosylation site, which are unique to and shared among most South American YFV strains. Our findings call for a reevaluation of current approaches to YFV immunological surveillance in South America and suggest approaches for designing updated vaccines.


title: Effects of face masks and ventilation on the risk of SARS-CoV-2 respiratory transmission in public toilets: a quantitative microbial risk assessment

abstract:
Public toilets could increase the risk of COVID-19 infection via airborne transmission; however, related research is limited. We aimed to estimate SARS-CoV-2 infection risk through respiratory transmission using a quantitative microbial risk assessment framework by retrieving SARS-CoV-2 concentrations from the swab tests of 251 Thai patients. Three virus-generating scenarios were investigated: an infector breathing, breathing with a cough, and breathing with a sneeze. Infection risk (97.5th percentile) was as high as 10-3 with breathing and increased to 10-1 with a cough or sneeze, thus all higher than the risk benchmark of 5 x 10-5 per event. No significant gender differences for toilet users (receptors) were noted. The highest risk scenario of breathing and a sneeze was further evaluated for risk mitigation measures. Risk mitigation to lower than the benchmark succeeded only when the infector and receptor simultaneously wore an N95 respirator or surgical mask and when the receptor wore an N95 respirator and the infector wore a denim fabric mask. Ventilation up to 20 air changes per hour (ACH), beyond the 12-ACH suggested by the WHO, did not mitigate risk. Virus concentration, volume of expelled droplets, and receptor dwell time were identified as the main contributors to transmission risk.

Highlights- The use of public toilets poses a risk of SARS-CoV-2 respiratory transmission
- Highest risks generated in the order of sneezing, coughing, and breathing
- No gender differences in risk by counteracting dwell times and inhalation rates
- Ventilation did not reduce risk even at 20 ACH, beyond the WHO-recommended value
- N95 and surgical masks offer the most effective risk mitigation to toilet users

Graphical abstract

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title: Active anaerobic methane oxidation and sulfur disproportionation in the deep terrestrial subsurface

abstract:
Microbial life is widespread in the terrestrial subsurface and present down to several kilometers depth, but the energy sources that fuel metabolism in deep oligotrophic and anoxic environments remain unclear. In the deep crystalline bedrock of the Fennoscandian Shield at Olkiluoto, Finland, opposing gradients of abiotic methane and ancient seawater-derived sulfate create a terrestrial sulfate-methane transition zone (SMTZ). We used chemical and isotopic data coupled to genome-resolved metaproteogenomics to demonstrate active life and, for the first time, provide direct evidence of active anaerobic oxidation of methane (AOM) in a deep terrestrial bedrock. Proteins from Methanoperedens (formerly ANME-2d) are readily identifiable despite the low abundance ([≤]1%) of this genus and confirm the occurrence of AOM. This finding is supported by 13C-depleted dissolved inorganic carbon. Proteins from Desulfocapsaceae and Desulfurivibrionaceae, in addition to 34S-enriched sulfate, suggest that these organisms use inorganic sulfur compounds as both electron donor and acceptor. Zerovalent sulfur in the groundwater may derive from abiotic rock interactions, or from a non-obligate syntrophy with Methanoperedens, potentially linking methane and sulfur cycles in Olkiluoto groundwater. Finally, putative episymbionts from the candidate phyla radiation (CPR) and DPANN archaea represented a significant diversity in the groundwater (26/84 genomes) with roles in sulfur and carbon cycling. Our results highlight AOM and sulfur disproportionation as active metabolisms and show that methane and sulfur fuel microbial activity in the deep terrestrial subsurface.

Significance StatementThe deep terrestrial subsurface remains an environment in which there is limited understanding of the extant microbial metabolisms, despite its reported large contribution to the overall biomass on Earth. It is much less well studied than deep marine sediments. We show that microorganisms in the subsurface are active, and that methane and sulfur provide fuel in the oligotrophic and anoxic subsurface. We also uncover taxonomically and metabolically diverse ultra-small organisms that interact with larger host cells through surface attachment (episymbiosis). Methane and sulfur are commonly reported in terrestrial crystalline bedrock environments worldwide and the latter cover a significant proportion of the Earths surface. Thus, methane- and sulfur-dependent microbial metabolisms have the potential to be widespread in the terrestrial deep biosphere.


title: Enzyme fatigue limits the detoxification of aflatoxin by Rhodococcus species

abstract: Bacterial detoxification of mycotoxins has the potential to offer a low-cost solution to ensure that feed and food commodities contaminated by fungal growth become safe to consume. Among bacteria, Rhodococcus species are of particular interest because they can be metabolically versatile, non-pathogenic, and environment-friendly. However, the native response of Rhodococcus environmental isolates appears inadequate for current detoxification needs. By analyzing the detoxification of aflatoxin by two Rhodococcus species: R. pyridinivorans and R. erythropolis, we examine important features of the dynamics that could guide future optimization of bacterial detoxification. Our results for Rhodococcus species suggest that detoxification happens through a regulated process of secreting extracellular enzymes. We show that enzyme fatigue in the presence of the toxin determines the lifetime of the enzyme and limits the overall detoxification performance of these species. Additionally, we show that the regulation of enzyme production can be both species- and environment-dependent. Overall, our quantitative approach reveals that enzyme fatigue is a major determinant of overall detoxification and needs to be accounted for in assessing the performance of detoxification by live cells or cell-free filtrates.


title: Genome sequencing and molecular characterisation of XDR Acinetobacter baumannii reveal complexities in resistance: Novel combination of Sulbactam-Durlobactam holds promise for therapeutic intervention

abstract: Acinetobacter baumannii is an emerging nosocomial strain expressing extensive drug resistance (XDR). Whole-genome sequencing and molecular characterisation analysis revealed the presence of carbapenemase in 92.86% of studied Indian isolates having blaOXA-51, blaOXA-23, blaOXA-58, and blaNDM genes, with a few evidences of dual carbapenemase genes. As per the MLST scheme, IC2Oxf/CC2Pas was the predominant clone, with 57.14% isolates belonging to this lineage. The presence of {beta}-lactamases has rendered sulbactam (SUL) resistance (MIC: 16-256{micro}g/ml) in all the studied isolates. The efficacy of novel durlobactam (DUR) in inhibiting {beta}-lactamases and PBP2 was assessed through in-silico inter-molecular interaction analysis. Several non-synonymous single nucleotide polymorphisms (nsSNPs) were identified in PBP2 (G264S, I108V, S259T) and PBP3 (A515V, T526S) sequences. Minimal variations were recorded in the protein-backbone dynamics in active-site motifs of wild-type (WT) and mutants (MT), which correlated with the negligible binding energy fluctuations for PBP3-SUL (-5.85{+/-}0.04Kcal/mol) and PBP2-DUR (-5.16{+/-}0.66Kcal/mol) complexes. Furthermore, stronger binding affinities and low inhibition constants were noted in DUR complexed with OXA23 (-7.36Kcal/mol; 4.01{micro}M), OXA58 (-6.44Kcal/mol; 19.07{micro}M) and NDM (-6.82Kcal/mol; 10.01{micro}M) when compared with conventional drugs avibactam and aztreonam. Stable interaction profiles of DUR, can possibly restore SUL activity against both PBP3WT and PBP3MTs. The study establishes the efficacy of novel SUL-DUR combination as a successful treatment strategy to combat emerging XDR strains.


title: Multiple knockout mutants reveal a high redundancy of phytotoxic compounds that determine necrotrophic pathogenesis of Botrytis cinerea

abstract: Botrytis cinerea is a major pathogen of more than 1400 plant species. During infection, the kills host cells during infection and spreads through necrotic tissue, which is believed to be supported by induction of programmed plant cell death. To comprehensively evaluate the contributions of most of the currently known plant cell death inducing proteins (CDIPs) and metabolites for necrotrophic infection, an optimized CRISPR/Cas protocol was established which allowed serial marker-free mutagenesis to generate Botrytis mutants lacking up to 12 different CDIPs. Infection analysis revealed a decrease in virulence with increasing numbers of knockouts, and differences in the effects of knockouts on different host plants. The on planta secretomes obtained from these mutants revealed substantial remaining necrotic activity after infiltration into leaves. Our study has addressed for the first time the functional redundancy of virulence factors of a fungal pathogen, and demonstrates that B. cinerea releases a highly redundant cocktail of proteins and metabolites to achieve necrotrophic infection of a wide variety of host plants.

category: molecular biology


title: A standard knockout procedure alters expression of adjacent loci at the translational level

abstract: The S. cerevisiae gene deletion collection is widely used for functional gene annotation and genetic interaction analyses. However, the standard G418-resistance cassette used to produce knockout mutants delivers strong regulatory elements into the target genetic loci. To date, its side effects on the expression of neighboring genes have never been systematically assessed. Here, using ribosome profiling data, RT-qPCR, and reporter expression, we investigated perturbations induced by the KanMX module. Our analysis revealed significant alterations in the transcription efficiency of neighboring genes and, more importantly, severe impairment of their mRNA translation, leading to changes in protein abundance. In the "head-to-head" orientation of the neighbor and the deleted gene, knockout often led to a shift of the transcription start site of the neighboring gene, introducing new uAUG codon(s) into the expanded 5 untranslated region (5 UTR). In the "tail-to-tail" arrangement, knockout led to activation of alternative polyadenylation signals in the neighboring gene, thus altering its 3 UTR. These events may explain the so-called neighboring gene effect (NGE), i.e. false genetic interactions of the deleted genes. We estimate that in as much as [~]1/5 of knockout strains the expression of neighboring genes may be substantially (>2-fold) deregulated at the level of translation.


title: Bayesian inference of multi-point macromolecular architecture mixtures at nanometre resolution

abstract: AO_SCPLOWBSTRACTC_SCPLOWGaussian spot fitting methods have significantly extended the spatial range where fluorescent microscopy can be used, with recent techniques approaching nanometre (nm) resolutions. However, small inter-fluorophore distances are systematically over-estimated for typical molecular scales ([lsim] 50nm). This bias can be corrected computationally, but current algorithms are limited to correcting distances between pairs of fluorophores. Here we present a flexible Bayesian computational approach that infers the distances and angles between multiple markers and has several advantages over these previous methods. Specifically it improves confidence intervals for small lengths, estimates measurement errors of each fluorescent marker individually and infers the correlations between polygon lengths. The latter is essential for determining the full multi-fluorophore 3D architecture. We further developed the algorithm to infer the mixture composition of a heterogeneous population of multiple polygon states. We use our algorithm to analyse the 3D architecture of the human kinetochore, a macro-molecular complex that is essential for high fidelity cell division. We examine the conformational change induced by microtubule attachment using triple fluorophore marked data and demonstrate for the first time that in metaphase kinetochore conformation is heterogeneous.


title: Multiplexed fluorescence imaging of GPCR downstream signaling dynamics at the single-cell level.

abstract: G-protein-coupled receptors (GPCRs) play an important role in sensing various extracellular stimuli, such as neurotransmitters, hormones, and tastants, and transducing the input information into the cell. While the human genome encodes more than 800 GPCR genes, only four G-proteins (Gs, Gi/o, Gq/11, and G12/13) are known to couple with GPCRs. It remains unclear how such divergent GPCR information is translated into the downstream G-protein signaling dynamics. To answer this question, we report a live-cell fluorescence imaging system for monitoring GPCR downstream signaling dynamics at the single-cell level. Genetically encoded biosensors for cAMP, Ca2+, RhoA, and ERK were selected as markers for GPCR downstream signaling, and were stably expressed in HeLa cells. GPCR was further transiently overexpressed in the cells. As a proof-of-concept, we visualized GPCR signaling dynamics of 5 dopamine receptors and 12 serotonin receptors, and found heterogeneity between GPCRs and between cells. Even when the same G proteins were known to be coupled, the patterns of dynamics in GPCR downstream signaling, including the signal strength and duration, were substantially distinct among GPCRs. These results suggest the importance of dynamical encoding in GPCR signaling.


title: Quantitative live-cell fluorescence imaging of GPCR downstream signaling dynamics at the single-cell level.


title: Polycomb Repressive Complex 2 shields naïve human pluripotent cells from trophectoderm differentiation

abstract: The first lineage choice made in human embryo development separates trophectoderm from the inner cell mass, which proceeds to form the pluripotent epiblast and primitive endoderm. Trophectoderm on the other hand gives rise to the placenta. Naive pluripotent stem cells are derived from the pluripotent epiblast of the blastocyst and offer possibilities to explore how lineage integrity is maintained. Here, we discover that Polycomb repressive complex 2 (PRC2) restricts an intrinsic capacity of naive pluripotent stem cells to give rise to trophectoderm. Through quantitative epigenome profiling, we find that broad histone H3 lysine 27 trimethylation (H3K27me3) hypermethylation is a common feature of naive pluripotency across species. We define a previously unappreciated, naive-specific set of bivalent promoters, featuring PRC2-mediated H3K27me3 concomitant with H3K4me3. Naive bivalency maintains key trophectoderm transcription factors in a transcriptionally poised state that is resolved to an active state upon depletion of H3K27me3 via inhibition of the enzymatic subunits of PRC2, EZH1/2. Conversely, primed human embryonic stem cells cannot be driven towards trophectoderm development via PRC2 inhibition. While naive and primed hESCs share the majority of bivalent promoters, PRC2 contributes to the repression of largely non-overlapping subsets of these promoters in each state, hence H3K27me3-mediated repression provides a highly adaptive mechanism to restrict lineage potential during early human development.

category: developmental biology


title: Acquisition of cell identity in the brown alga Ectocarpus: which of time, cell shape or position matters most?

abstract: During development, cells undergo simultaneous changes of different types that together depict cell "identity". In the multicellular brown alga Ectocarpus sp., while ageing, cells change shape and relative position within the filament. Understanding how these factors act and interact to specify cell identity requires markers of cell identity and the ability to genetically separate age, shape and position. Here we used laser capture microdissection (LCM) to isolate specific cell types from young sporophytes of Ectocarpus, and performed differential RNA-seq analysis. Transcriptome profiles of cell types in the wild-type strain provided signatures of the five cell types that can be identified by shape and position. In two mutants, where the relationship between cell shape, position and age are altered, transcriptome signatures revealed that little differential expression could be identified when only shape was perturbed. More generally, although the two mutants are characterised by opposite morphological phenotypes, their transcriptomes were remarkably similar. We concluded that despite the robustness of cell differentiation during WT development, neither the shape nor the position of the cell could serve as a faithful gauge for tracking differentiation.


title: Diversification of small RNA pathways underlies germline RNAi incompetence in wild C. elegans strains

abstract: The discovery that experimental delivery of dsRNA can induce gene silencing at target genes revolutionized genetics research, by both uncovering essential biological processes and creating new tools for developmental geneticists. However, the efficacy of exogenous RNAi varies dramatically within the C. elegans population, raising questions about our understanding of RNAi in the lab relative to its activity and significance in nature. Here, we investigate why some wild-type strains fail to mount a robust RNAi response to germline targets. We observe diversity in mechanism: in some strains, the response is stochastic, either on or off among individuals, while in others the response is consistent but delayed. Increased activity of the Argonaute PPW-1, which is required for germline RNAi in the laboratory strain N2, rescues the response in some strains, but dampens it further in others. Among wild-type strains, genes known to mediate RNAi exhibited very high expression variation relative to other genes in the genome as well as allelic divergence and strain-specific instances of pseudogenization at the sequence level. Our results demonstrate functional diversification in the small RNA pathways in C. elegans, and suggest that RNAi processes are evolving rapidly and dynamically in nature.

category: evolutionary biology


title: Limited evidence of a genetic basis for sex determination in the common creek chub, Semotilus atromaculatus

abstract: 1Sexual reproduction is almost universal in the animal kingdom; therefore, each species must have a mechanism for designating sex as male or female. Fish especially have a wide range of sex determining systems. While incompatible systems are thought to increase reproductive isolation, interspecific hybridization is common among groups such as cyprinid minnows, thus, studies such as this can provide insight into hybridization and evolutionary diversification of this clade. In the present study, we aimed to identify a genetic basis for sex determination in the common creek chub (Semotilus atromaculatus) using genotyping-by-sequencing (GBS) data. No sex-associated markers were found by RADSex or a GWAS using GEMMA, however, our Weir and Cockerham locus-specific FST analysis and discriminant analysis of principal components revealed some genetic differentiation between the sexes at several loci. While no explicit sex determination mechanism has been yet discovered in creek chub, these loci are potential candidates for future studies. This study also highlights technical challenges involved in studying sex determination in species with extremely variable mechanisms.