title: Next Generation Opto-Jasplakinolides Enable Local Remodeling of Actin Networks

abstract:
The natural product jasplakinolide is a widely used tool compound to stabilize F-actin and influence actin dynamics. We have previously introduced photoswitchable jasplakinolides (optojasps) that are activated with violet light and deactivated with blue light. Based on insights from cryo-electron microscopy and structure-activity relationship (SAR) studies, we now developed a new generation of functionally superior optojasps that are better suited for biological investigations. These compounds are procured through chemical total synthesis and feature rationally designed red-shifted azobenzene photoswitches. Our new optojasps can be activated with longer wavelengths in the visible range (e.g. 440-477 nm) and rapidly return to their inactive state through thermal relaxation. This has enabled the reversible control of F-actin dynamics, as shown through live-cell imaging and cell migration, as well as cell proliferation assays. Brief sub-cellular activation with blue-green light resulted in highly localized F-actin clusters that gradually dissolved in the dark. Our light-responsive tools can be useful in diverse fields to study actin dynamics with outstanding spatiotemporal precision.

Graphical Abstract

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category: cell biology


title: Mechanical competition promotes selective listening to receptor inputs to resolve directional dilemmas in neutrophil migration

abstract: As neutrophils navigate complex environments to reach sites of infection, they may encounter obstacles that force them to split their front into multiple leading edges, raising the question of how the cell selects which front to maintain and which front(s) to abandon. Here we challenge chemotaxing HL60 neutrophil-like cells with symmetric bifurcating microfluidic channels, enabling us to probe the cell-intrinsic properties of their decision-making process. Using supervised statistical learning, we demonstrate that cells commit to one leading edge late in the decision- making process, rather than amplifying early pre-existing asymmetries. Furthermore, we use optogenetic tools to show that receptor inputs only bias the decision similarly late, once mechanical stretching begins to weaken each front. Finally, optogenetic attempts to reverse cell decisions reveal that, once an edge begins retracting, it commits to this fate, with the kinase ROCK limiting its sensitivity to inputs until the retraction is complete. Collectively our results suggest a "selective listening" model in which both actively protruding cell fronts and actively retracting cell rears have strong commitment to their current migratory program.


title: Aberrant cortical development is driven by impaired cell cycle and translational control in a DDX3X syndrome model

abstract: Mutations in the RNA helicase, DDX3X, are a leading cause of Intellectual Disability and present as DDX3X syndrome, a neurodevelopmental disorder associated with cortical malformations and autism. Yet the cellular and molecular mechanisms by which DDX3X controls cortical development are largely unknown. Here, using a mouse model of Ddx3x loss-of-function we demonstrate that DDX3X directs translational and cell cycle control of neural progenitors, which underlies precise corticogenesis. First, we show brain development is highly sensitive to Ddx3x dosage; Complete Ddx3x loss from neural progenitors causes microcephaly in females, whereas hemizygous males and heterozygous females show reduced neurogenesis without marked microcephaly. In addition, Ddx3x loss is sexually dimorphic, as its paralog, Ddx3y, compensates for Ddx3x in the developing male neocortex. Using live imaging of progenitors, we show that DDX3X promotes neuronal generation by regulating both cell cycle duration and neurogenic divisions. Finally, we use ribosome profiling in vivo to discover the repertoire of translated transcripts in neural progenitors, including those which are DDX3X-dependent and essential for neurogenesis. Our study reveals invaluable new insights into the etiology of DDX3X syndrome, implicating dysregulated progenitor cell cycle dynamics and translation as pathogenic mechanisms.

category: developmental biology


title: Pneumococcal within-host diversity during colonisation, transmission and treatment

abstract: Characterising the genetic diversity of pathogens within the host promises to greatly improve surveillance and reconstruction of transmission chains. For bacteria, it also informs our understanding of inter-strain competition, and how this shapes the distribution of resistant and sensitive bacteria. Here we study the genetic diversity of Streptococcus pneumoniae within individual infants and their mothers by deep sequencing whole pneumococcal populations from longitudinal nasopharyngeal samples. We demonstrate deep sequencing has unsurpassed sensitivity for detecting multiple colonisation, doubling the rate at which highly invasive serotype 1 bacteria were detected in carriage compared to gold-standard methods. The greater resolution identified an elevated rate of transmission from mothers to their children in the first year of the childs life. Comprehensive treatment data demonstrated infants were at an elevated risk of both the acquisition, and persistent colonisation, of a multidrug resistant bacterium following antimicrobial treatment. Some alleles were enriched after antimicrobial treatment, suggesting they aided persistence, but generally purifying selection dominated within-host evolution. Rates of co-colonisation imply that in the absence of treatment, susceptible lineages outcompeted resistant lineages within the host. These results demonstrate the many benefits of deep sequencing for the genomic surveillance of bacterial pathogens.

category: genomics


title: From bacteria to plants: a repurposing strategy in the pursuit for novel herbicides

abstract: Herbicide resistance represents one of the biggest threats to our natural environment and agricultural sector. Thus, new herbicides are urgently needed to tackle the rise in herbicideresistant weeds. Here, we employed a novel strategy to repurpose a  failed antibiotic into a new and target-specific herbicidal compound. Specifically, we identified an inhibitor of bacterial dihydrodipicolinate reductase (DHDPR), an enzyme involved in lysine biosynthesis in plants and bacteria, that exhibited no antibacterial activity but severely attenuated germination of the plant Arabidopsis thaliana. We confirmed that the inhibitor targets plant DHDPR orthologues in vitro, and exhibits no toxic effects against human cell lines. A series of analogues were then synthesised with improved efficacy in germination assays and against soil-grown A. thaliana plants. We also showed that our lead compound is the first lysine biosynthesis inhibitor with herbicidal activity against a weed species, providing proof-of-concept that DHDPR inhibition may represent a much-needed new herbicide mode of action. Furthermore, this study exemplifies the untapped potential of repurposing  failed antibiotic scaffolds to fast-track the development of herbicide candidates targeting the respective plant enzymes to combat the global rise in herbicide-resistant weeds.

category: biochemistry


title: PBRM1 is a vulnerability in ARID1A deficient multicellular tumor spheroids

abstract: ARID1A, a subunit of SWI/SNF, has been shown to play a major role in recruitment of the chromatin remodeler to enhancers for transcriptional regulation. Mutations inARID1A have been found in various cancers, many of which form solid tumors. Recent studies have revealed vulnerabilities in cells lacking ARID1A, specifically ARID1B, an ortholog and mutually exclusive subunit, in 2D cell culture. However, identification of vulnerabilities within SWI/SNF for loss of ARID1A in a multicellular tumor spheroid, that mimic in vivo condition within tumors, has not been explored. Here we show in the absence of ARID1A in a MTS model, ARID1B continues to be a vulnerability but we have identified PBRM1 as a new vulnerability within SWI/SNF. Levels of ARID1B and PBRM1 are elevated on loss of ARID1A. Further, reduction of ARID1B and PBRM1 protein levels, decreases cell survival and reduces induction of several hypoxia regulated genes in ARID1A deficient MTSs. Our studies have identified PBRM1 as a new vulnerability in ARID1a deficient cancers and which provides a new target for therapeutic strategies.


title: Allosteric interactions prime androgen receptor dimerization and activation

abstract: The androgen receptor (AR) is a steroid receptor and master transcription factor that governs gene expression programs required for luminal development of prostate epithelium, formation of muscle tissue and maintenance of the male phenotype. AR misregulation is a hallmark of multiple malignancies, including prostate cancer, where AR hyperactivation and expansion of its transcriptome occur in part through AR gene amplification and interaction with oncoprotein cofactors. Despite its biological importance, how ARs individual domains and its protein cofactors cooperate to bind DNA have remained elusive. Using a combination of reconstitution biochemistry and single particle cryo-electron microscopy (EM), we have isolated three conformational states of AR bound to DNA. We observe that AR forms a non-obligate dimer, with the buried dimer interface utilized by related ancestral nuclear receptors repurposed to facilitate cooperative DNA binding. We identify surfaces bridging ARs domains responsible for allosteric communication, that are compromised in partial androgen insensitivity syndrome (PAIS), and are reinforced by ARs oncoprotein cofactor, ERG, and DNA binding site motifs. Finally, we present evidence that this plastic dimer interface for transcriptional activation may have been adopted by AR at the expense of DNA binding. Our work highlights how fine-tuning of ARs cooperative interactions translate to consequences in development and disease.


title: Soluble extracts from amnion and chorion membranes improve hMSC osteogenic response in a mineralized collagen scaffold

abstract: Craniomaxillofacial (CMF) bone injuries present a major surgical challenge and cannot heal naturally due to their large size and complex topography. Approximately 26% of injured Iraq war veterans sustained CMF injuries in the form of blast wounds, and 0.1% of births involve CMF defects like cleft palate. We previously developed a class of mineralized collagen scaffolds designed to mimic native extracellular matrix (ECM) features of bone. These scaffolds induce in vitro human mesenchymal stem cell (hMSC) osteogenic differentiation and in vivo bone formation without the need for exogenous osteogenic supplements. Here, we seek to enhance cellular bioactivity and osteogenic activity via inclusion of placental-derived products in the scaffold architecture. The amnion and chorion membranes are distinct components of the placenta that individually have displayed anti-inflammatory, immunogenic, and osteogenic properties. They represent a potentially powerful compositional modification to the mineralized collagen scaffolds to improve bioactivity. Here we examine introduction of the placental-derived amnion and chorion membranes or soluble extracts derived from these membranes into the collagen scaffolds, comparing the potential for these modifications to improve hMSC osteogenic activity. We report structural analysis of the scaffolds via mechanical compression testing, imaging via scanning electron microscopy (SEM), and assessments of various metrics for osteogenesis including gene expression (Nanostring), protein elution (ELISA), alkaline phosphatase (ALP) activity, inductively coupled plasma mass spectrometry (ICP) for mineralization, and cell viability (AlamarBlue). Notably, a post fabrication step to incorporate soluble extracts from the amnion membrane induces the highest levels of metabolic activity and performs similarly to the conventional mineralized collagen scaffolds in regard to mineral deposition and elution of the osteoclast inhibitor osteoprotegerin (OPG). Together, these findings suggest that mineralized collagen scaffolds modified using elements derived from amnion and chorion membranes, particularly their soluble extracts, represent a promising environment conducive to craniomaxillofacial bone repair.

category: bioengineering


title: Structural insights into the peptide selectivity and activation of human neuromedin U receptors

abstract: Neuromedin U receptors (NMURs), including NMUR1 and NMUR2, are a group of Gq/11-coupled G protein-coupled receptors (GPCRs) related to pleiotropic physiological functions. Upon stimulation by two endogenous neuropeptides, neuromedin U and S (NMU and NMS) with similar binding affinities, NMUR1 and NMUR2 primarily display distinct peripheral tissue and central nervous system (CNS) functions, respectively, due to their distinct tissue distributions. These NMU receptors have triggered extensive attention as drug targets for obesity and immune inflammation. Specifically, selective agonists for NMUR1 in peripheral tissue show promising long-term anti-obesity effects with fewer CNS-related side effects. However, the mechanisms of peptide binding specificity and receptor activation remain elusive due to the lack of NMU receptor structures, which hamper drug design targeting NMU receptors. Here, we report four cryo-electron microscopy structures of Gq chimera-coupled NMUR1 and NMUR2 bound with NMU and NMS. These structures present the conserved overall peptide-binding mode and reveal the mechanism of peptide selectivity for specific NMURs, as well as the common activation mechanism of the NMUR subfamily. Together, these findings provide insights into the molecular basis of the peptide recognition selectivity and offer a new opportunity for designing selective drugs targeting NMURs.

category: biophysics


title: Cadmium-induced endocytosis of the broad spectrum root metal transporter of Arabidopsis

abstract: Iron is an essential micronutrient for plant growth and development. Under low iron conditions, Arabidopsis plants take up soil iron using the root iron transporter IRT1. In addition to iron, IRT1 also transports others divalent metals including cadmium that consequently accumulates into plant tissues and enters the food chain. IRT1 expression was shown to be regulated at the transcriptional and post-translational levels by its essential metal substrates to maximize iron uptake while limiting the accumulation of zinc, manganese or cobalt. Here, we characterized the regulation of IRT1 by cadmium and uncovered a cadmium-mediated downregulation of IRT1 protein by endocytosis. A short term exposure to cadmium indeed decreased the celle surface levels of IRT1 through endocytosis and degradation. This is mediated through the direct binding of cadmium to histidine residues within the regulatory loop of IRT1. Moreover, we demonstrated that cadmium-induced IRT1 degradation uses ubiquitin-mediated endocytosis driven by the IDF1 E3 ligase. Altogether, this work sheds light on the mechanisms of cadmium-mediated downregulation of IRT1 and offers a unique opportunity to boost plant cadmium uptake in phytoremediation/phytoextraction strategies.

category: plant biology


title: Dynamic changes in uterine NK cell subset frequency and function over the menstrual cycle and pregnancy

abstract: Uterine Natural Killer cells (uNK) play an important role in promoting successful pregnancy by regulating trophoblast invasion and spiral artery remodelling in the first trimester. Recently, single cell RNA sequencing (scRNAseq) on first trimester decidua showed that uNKs can be divided into three subsets, which may have different roles in pregnancy. Here we present an integration of previously published scRNAseq datasets, together with novel flow cytometry data to interrogate the frequency, phenotype and function of uNK1-3 in seven stages of the reproductive cycle (menstrual, proliferative, secretory phases; first, second, third trimester; and postpartum). We found that uNK1 and -2 peak in the first trimester, but by the third trimester the majority of uNK are uNK3. All three subsets are most able to degranulate and produce cytokines during the secretory phase of the menstrual cycle and express KIR2D molecules, which allow them to interact with HLA-C expressed by placental extravillous trophoblast cells, at the highest frequency during the first trimester. Taken together, our findings suggest that uNK are particularly active and able to interact with placental cells at the time of implantation, and that uNK1 and 2 may be particularly involved in these processes. Our findings are the first to establish how uNK frequency and function changes dynamically across the healthy reproductive cycle. This serves as a platform from which the relationship between uNK function and impaired implantation and placentation can be investigated. This will have important implications for the study of subfertility, recurrent miscarriage, pre-eclampsia and pre-term labour.

category: immunology


title: Gut microbiome disturbances of altricial Blue and Great tit nestlings are countered by continuous microbial inoculations from parental microbiomes

abstract: Gut microbial communities are complex and heterogeneous and play critical roles for animal hosts. Early-life disruptions to microbiome establishment can negatively impact host fitness and development. However, the consequences of such early-life disruptions are unknown in wild birds. To help fill this gap, after validating the disruptive influence of antibiotic and probiotic treatments on the gut microbiome in adult Great tits (Parus major) (efficacy experiment), we investigated the effect of continuous early-life gut microbiome disruptions on the establishment and development of gut communities in wild Great and Blue tit (Cyanistes caeruleus) nestlings (field experiment). Despite negative impacts of treatments on microbial alpha and beta diversities in the efficacy experiment, treatment did not affect the composition of nestling microbiomes in the field experiment. Independent of treatment, nestling gut microbiomes of both species grouped by brood, sharing high numbers of bacterial taxa with both the nest environment and their mother. The distance between nests increased inter-brood microbiome dissimilarity, but only in Great tits, indicating species-specific influence of environment on microbiomes. The strong maternal effect, driven by continuous recolonization from the nest environment and vertical transfer of microbes during feeding thus appear to provide resilience towards early-life disruptions in nestling gut microbiomes.

category: microbiology


title: Epstein-Barr virus latency programs dynamically sensitize B-cells to ferroptosis

abstract:
Epstein-Barr virus (EBV) causes 200,000 cancers annually. Upon B-cell infection, EBV induces lipid metabolism to support B-cell proliferation. Yet, little is known about how latent EBV infection, or human B-cell stimulation more generally, alter sensitivity to ferroptosis, a non-apoptotic form of programmed cell death driven by iron-dependent lipid peroxidation and membrane damage. To gain insights, we analyzed lipid reactive oxygen species (ROS) levels and ferroptosis vulnerability in primary human CD19+ B-cells infected by EBV or stimulated by key B-cell receptors. Prior to the first mitosis, EBV-infected cells were exquisitely sensitive to blockade of glutathione biosynthesis, a phenomenon not observed with B-cell receptor stimulation. Subsequently, EBV-mediated Burkitt-like hyper-proliferation generated elevated levels of lipid ROS, which necessitated SLC7A11-mediated cystine import and glutathione peroxidase 4 (GPX4) activity to prevent ferroptosis. By comparison, B-cells were sensitized to ferroptosis induction by combinatorial CD40-ligand and interleukin-4 stimulation or anti-B-cell receptor and Toll-like receptor 9 stimulation upon GPX4 inhibition, but not with SLC7A11 blockade. EBV transforming B-cells became progressively resistant to ferroptosis induction upon switching to the latency III program and lymphoblastoid physiology. Similarly, latency I Burkitt cells were particularly vulnerable to blockade of SLC7A11 or GPX4 or cystine withdrawal, while latency III Burkitt and lymphoblastoid cells were comparatively resistant. The selenocysteine biosynthesis kinase PSTK was newly implicated as a cellular target for ferroptosis induction including in Burkitt cells, likely due to roles in GPX4 biosynthesis. These results highlight ferroptosis as an intriguing therapeutic target for the prevention or treatment of particular EBV-driven B-cell malignancies.

SignificanceEBV contributes to B-cell Burkitt and post-transplant lymphoproliferative disease (PTLD). EBV transforming programs activate lipid metabolism to convert B-cells into immortalized lymphoblastoid cell lines (LCL), a PTLD model. We found that stages of EBV transformation generate lipid reactive oxygen species (ROS) byproducts to varying degrees, and that a Burkitt-like phase of B-cell outgrowth is dependent on lipid ROS detoxification by glutathione peroxidase 4 and its cofactor glutathione. Perturbation of this redox defense in early stages of B-cell transformation or in Burkitt cells triggered ferroptosis, a programmed cell death pathway. LCLs were less dependent on this defense, a distinction tied to EBV latency programs. This highlights ferroptosis induction as a novel therapeutic approach for prevention or treatment of EBV+ lymphomas.


title: Checks and balances of the RTG pathway under arginine deprivation and canavanine exposure in Saccharomyces cerevisiae

abstract: Decades of research in Saccharomyces cerevisiae underlie the current dogma of mitochondrial retrograde (RTG) signaling: Rtg2-dependent translocation of the heterodimer Rtg1/Rtg3 from the cytoplasm to the nucleus induces the transcription of RTG-target genes under glutamate starvation or loss of respiration. We previously found that RTG mutants show severe growth inhibition from arginine deprivation and are highly sensitive to canavanine when grown on glucose. Here, we show that on solid media, RTG mutants are also sensitive to thialysine, a toxic lysine analog, although lysine deprivation causes a milder growth defect. Growth on an alternative carbon source restores RTG mutants ability to grow without arginine or lysine and improves their tolerance of toxic analogs; deletion of MIG1 affords a similar rescue on glucose and improves canavanine tolerance, except for in rtg2{Delta}. It is well known that the target of rapamycin (TOR) signaling pathway inhibits the RTG pathway. Batch growth experiments with or without TOR inhibition reveal phenotypic and regulatory differences between RTG mutants. rtg1{Delta} can sustain simultaneous canavanine exposure and TOR inhibition via rapamycin, but rtg2{Delta} and rtg3{Delta} cannot. Surprisingly, our data show that under fermentative lifestyle and arginine deprivation, both RTG signaling and TOR activity are required. This expands the universe of TOR and RTG signaling, suggesting bilateral communication rather than unidirectional RTG regulation by TOR. To the best of our knowledge, this work shows for the first time that Rtg3 activity can be separate from its role as a heterodimer with Rtg1. This work also strongly suggests a specific role for Rtg2 in canavanine tolerance.


title: Staphylococcus aureus overcomes anaerobe-derived short-chain fatty acid stress via FadX and the CodY regulon

abstract:
Chronic rhinosinusitis (CRS) is characterized by immune dysfunction, mucus hypersecretion, and persistent infection of the paranasal sinuses. While Staphylococcus aureus is a primary CRS pathogen, recent sequence-based surveys have found increased relative abundances of anaerobic bacteria, suggesting that S. aureus may experience altered metabolic landscapes in CRS relative to healthy airways. To test this possibility, we characterized the growth kinetics and transcriptome of S. aureus in supernatants of the abundant CRS anaerobe Fusobacterium nucleatum. While growth was initially delayed, S. aureus ultimately grew to similar levels as in the control medium. The transcriptome was significantly affected by F. nucleatum metabolites, with the agr quorum sensing system notably repressed. Conversely, expression of fadX, encoding a putative propionate coA-transferase, was significantly increased, leading to our hypothesis that short chain fatty acids (SCFAs) produced by F. nucleatum could mediate S. aureus growth behavior and gene expression. Supplementation with propionate and butyrate, but not acetate, recapitulated delayed growth phenotypes observed in F. nucleatum supernatants. A fadX mutant was found to be more sensitive than wild type to propionate, suggesting a role for FadX in the S. aureus SCFA stress response. Interestingly, spontaneous resistance to butyrate, but not propionate, was frequently observed. Whole genome sequencing and targeted mutagenesis identified codY mutants as resistant to butyrate inhibition. Together, these data show that S. aureus physiology is dependent on its co-colonizing microbiota and metabolites they exchange, and indicate that propionate and butyrate may act on different targets in S. aureus to suppress its growth.

ImportanceS. aureus is an important CRS pathogen, yet is found in the upper airways of 30-50% of people without complications. The presence of strict and facultative anaerobic bacteria in CRS sinuses has recently spurred research into bacterial interactions and how they influence S. aureus physiology and pathogenesis. We show here that propionate and butyrate produced by one such CRS anaerobe, F. nucleatum, alter the growth and gene expression of S. aureus. We show that fadX is important for S. aureus to resist propionate stress, and that the CodY regulon mediates growth in inhibitory concentrations of butyrate. This work highlights the possible complexity of S. aureus-anaerobe interactions, and implicates membrane stress as a possible mechanism influencing S. aureus behavior in CRS sinuses.


title: Alternate response of Bacillus anthracis atxA and acpA to serum, HCO3- and CO2

abstract: Bacillus anthracis overcome host immune response by producing capsule and secreting toxins. Production of these virulence factors in response to host environment was shown to be regulated by atxA, the major virulence regulator, that was shown to be activated by HCO3- and CO2. While toxin production is regulated directly by atxA, capsule production is mediated by either one of two regulators; acpA and acpB. In addition, it was demonstrated that acpA gene have at least two promotors were one of them was linked to atxA. We used a genetic approach to study capsule and toxin production under different conditions. Unlike previous works that used NBY-HCO3- medium in a CO2 enriched atmosphere, we used a sDMEM based medium in which toxins and capsule production can be induced in B. anthracis in ambient or CO2 enriched atmosphere. Using this system, we could differentiate between induction by 10% NRS, 10% CO2 or 0.75% HCO3-. In response to high CO2, capsule production is induced by acpA in an atxA independent manner, while little or no toxins (protective antigen PA) is produced. atxA is activated in response to serum in CO2 independent manner and induce toxins production and capsule production in an acpA or acpB dependent manner. HCO3- activates atxA as well, however in non-physiological concentrations. Our findings might be relevant to the first stages of inhalational infection were in the dendritic cell the germinating spore must be protected by the capsule without effecting cell migration to the draining lymph-node by toxin secretion.


title: TFIIB-related factor 1 is a nucleolar protein that promotes RNA polymerase I-directed transcription and tumour cell growth

abstract: RNA polymerase I (Pol I) products play pivotal roles in ribosomal assembly, protein synthesis and cell growth. Dysregulation of Pol I product synthesis closely correlates with tumourigenesis. However, the factors and pathways governing Pol I-dependent transcription remain to be identified. Here, we report that TFIIB-related factor 1 (BRF1), a subunit of TFIIIB required for Pol III-mediated transcription, is a nucleolar protein and modulates the rRNA synthesis directed by Pol I. We show that BRF1 can be co-localized with nucleolar protein markers in several human cell types. BRF1 expression positively correlates with Pol I product levels and tumour cell growth in vitro and in vivo. Mechanistically, BRF1 binds to the Pol I transcription machinery factors and is recruited to the rDNA promoter with them. Alteration of BRF1 expression affected the recruitment of Pol I transcription machinery at the rDNA promoter, the rDNA promoter activity and expression of TBP and TAF1A, indicating that BRF1 modulates Pol I-direct transcription by controlling the expression of selective factor 1 subunits. Collectively, we demonstrate that BRF1 acts as a positive factor to regulate Pol I-directed transcription, suggesting that BRF1 can concurrently modulate Pol I and Pol III transcription and acts as a key coordinator between them.

category: molecular biology


title: Tyrosine phosphorylation of mitofusin 2 regulates endoplasmic reticulum-mitochondria tethering

abstract: Contact sites between the mitochondria and endoplasmic reticulum (ER) are irregulates the exchange of lipids, Ca2+, and reactive oxygen species (ROS) across the two organelles. Mitofusin 2 (Mfn2) has been shown as one of the major components tethering these two organelles. Several post-translational modifications (PTMs) of Mfn2 have been identified to modulate canonical (i.e., mitochondrial fusion) and non- canonical functions, such as mitophagy and activation of ER stress signaling, however there is little information whether any PTMs can regulate mitochondrial and ER tethering. Basal tyrosine phosphorylation of Mfn2 was detected by mass spectroscopy, but it is unknown whether Mfn2 is a substrate of mitochondria-localized tyrosine kinases. Here we show that the mitochondria-localized Src family tyrosine kinases including c-Src can phosphorylate Mfn2, which decreases distance between the mitochondria and ER, and increases Ca2+ transfer from the ER to mitochondria, followed by changes in ROS generation and mitochondrial bioenergetics. Our findings suggest that tyrosine phosphorylation of Mfn2 may uniquely work to fine-tune ER- mitochondrial Ca2+ transport under physiological conditions, without activating mitophagy or ER stress signaling.


title: Cigarette smoke induces pulmonary arterial dysfunction through an imbalance in the guanylyl cyclase redox status.

abstract:
Chronic obstructive pulmonary disease (COPD), whose main risk factor is cigarette smoking (CS), is one of the most common diseases globally. Many COPD patients also develop pulmonary hypertension (PH), a severe complication that leads to premature death. Evidence suggests reactive oxygen species (ROS) involvement in COPD and PH, especially regarding pulmonary artery smooth muscle cells (PASMC) dysfunction. However, the effects of CS on the pulmonary vasculature are not completely understood. Herein we provide evidence on the effects of CS extract (CSE) exposure on PASMC regarding ROS production, antioxidant response and its consequences on vascular tone dysregulation. Our results indicate that CSE exposure promotes mitochondrial fission, mitochondrial membrane depolarization and increased mitochondrial superoxide levels. However, the increase in superoxide did not parallel a counterbalancing antioxidant response in human pulmonary artery (PA) cells. Interestingly, the mitochondrial superoxide chelator mitoTEMPO reduced mitochondrial fission and membrane potential depolarization caused by CSE. As we have previously shown, CSE reduces PA vasoconstriction and vasodilation. In this respect, mitoTEMPO prevented the impaired nitric oxide-mediated vasodilation, while vasoconstriction remained reduced. Finally, we observed a CSE-driven downregulation of the Cyb5R3 enzyme, which prevents soluble guanylyl cyclase oxidation in PASMC. This might explain the CSE-mediated decrease in PA vasodilation. These results provide evidence that there might be a connection between mitochondrial ROS and altered vasodilation responses in PH secondary to COPD, and strongly support the potential of antioxidant strategies specifically targeting mitochondria as a new therapy for these diseases.

Graphical abstract O_FIG_DISPLAY_L [Figure 1] M_FIG_DISPLAY C_FIG_DISPLAY


title: A phospholipase assay screen identifies synergistic inhibitors of the P. aeruginosa toxin ExoU

abstract: The opportunistic pathogen Pseudomonas aeruginosa is a leading cause of disability and mortality worldwide and the World Health Organisation has listed it with the highest priority for the need of new therapies. P. aeruginosa strains that express ExoU are implicated in the worst clinical outcomes. ExoU is phospholipase that is secreted by P. aeruginosa directly into the cytoplasm of target host cells, where its catalytic activity, directed towards plasma membranes, causes rapid cell lysis. Inhibition of ExoU may be a novel strategy to combat acutely cytotoxic ExoU expressing P. aeruginosa infections. Using an in vitro phospholipase assay, we performed a high throughput screen to identify compounds that might be repurposed as therapeutic ExoU inhibitors. We discovered a panel of compounds that appeared to inhibit ExoU through distinct mechanisms. Compound C prevented ExoU membrane localisation in HEK293T cells and caused colocalization with lysosomes, whereas compound D prevented PIP2 dependent oligomerisation of ExoU in vitro suggestive of synergistic action. Indeed, the concentrations required by compounds C and D to inhibit in vitro ExoU catalytic activity, when used in combination, was in the nanomolar region. In corneal scratch and infection assays, these compounds reduced ExoU mediated cytotoxicity, as assessed by fluorescence microscopy and lactate dehydrogenase release assays.


title: Gradients of orientation, composition and hydration of proteins for efficient light collection by the cornea of the horseshoe crab

abstract:
The lateral eyes of the horseshoe crab, Limulus polyphemus, are the largest compound eyes within recent Arthropoda. While this visual system has been extensively described before, the precise mechanism allowing vision has remained controversial. Correlating quantitative refractive index (RI) mapping and detailed structural analysis, we demonstrate how gradients of RI in the cornea result from the hierarchical organization of chitin-protein fibers, heterogeneity in protein composition and bromine doping, as well as spatial variation in water content. Combining the realistic cornea structure and measured RI gradients with full-wave optical modelling and ray-tracing approaches, we show that the light collection mechanism depends on both refraction-based graded index (GRIN) optics and total internal reflection. The optical properties of the cornea are governed by different mechanisms at different hierarchical levels, demonstrating the remarkable versatility of arthropod cuticle.

One-sentence summaryStructural hierarchy and protein hydration determine the optical performance of the cornea of L. polyphemus.


title: Structural and functional determinants inferred from deep mutational scans

abstract:
Mutations that affect protein binding to a cognate partner primarily occur either at buried residues or at exposed residues directly involved in partner binding. Distinguishing between these two categories based solely on mutational phenotypes is challenging. The bacterial toxin CcdB kills cells by binding to DNA Gyrase. Cell death is prevented by binding to its cognate antitoxin CcdA, at an extended interface that partially overlaps with the GyrA binding site. Using the CcdAB toxin-antitoxin (TA) system as a model, a comprehensive site-saturation mutagenesis library of CcdB was generated in its native operonic context. The mutational sensitivity of each mutant was estimated by evaluating the relative abundance of each mutant in two strains, one resistant and the other sensitive to the toxic activity of the CcdB toxin, through deep sequencing. The ability to bind CcdA was inferred through a RelE reporter gene assay, since the CcdAB complex binds to its own promoter, repressing transcription. By analysing mutant phenotypes in the CcdB sensitive, CcdB resistant and RelE reporter strains, it was possible to assign residues to buried, CcdA interacting or GyrA interacting sites. A few mutants were individually constructed, expressed, and biophysically characterised to validate molecular mechanisms responsible for the observed phenotypes. Residues inferred to be important for antitoxin binding, are also likely to be important for rejuvenating CcdB from the CcdB-Gyrase complex. Therefore, even in the absence of structural information, when coupled to appropriate genetic screens, such high-throughput strategies can be deployed for predicting structural and functional determinants of proteins.

Broader Impact StatementPartial loss-of-function mutations predominantly occur either at buried-site or exposed, active-site residues. We report a facile method to identify multiple binding sites for different interacting partners for a protein, and distinguish them from buried site and exposed non active-site residues, solely from mutational data.


title: Decoding mechanism of action and susceptibility to drug candidates from integrated transcriptome and chromatin state

abstract: Omics-based technologies are driving major advances in precision medicine but efforts are still required to consolidate their use in drug discovery. In this work, we exemplify the use of multi-omics to support the development of 3-chloropiperidines (3-CePs), a new class of candidate anticancer agents. Combined analyses of transcriptome and chromatin accessibility elucidated the mechanisms underlying sensitivity to test agents. Further, we implemented a new versatile strategy for the integration of RNA-seq and ATAC-seq data, able to accelerate and extend the standalone analyses of distinct omic layers. This platform guided the construction of a perturbation-informed basal signature able to predict cancer cell lines sensitivity and to further direct compound development against specific tumor types. Overall, this approach offered a scalable pipeline to support the early phases of drug discovery, understanding of mechanism and potentially inform the positioning of therapeutics in the clinic.

category: systems biology


title: Comparison of two sampling, quenching and extraction methods for quantitative yeasts metabolomics

abstract: Knowing intracellular metabolite concentrations is important for fundamental metabolism research as well as for biotechnology. The first steps in a quantitative metabolomics workflow, i.e., sampling, quenching, and extraction, are key to obtaining unbiased quantitative data. A qualified quenching and extraction method not only needs to rapidly terminate the in vivo biochemical reaction activities to preserve the endogenous metabolite levels but also has to fully extract all metabolites from cells. Recently, two different filtration-based sampling, quenching, and extraction methods have been proposed and used for quantitative yeast metabolomics. One method integrates the quenching and extraction into one step using a methanol-acetonitrile-water solution after a filtration step, while the other --more conventional-- method quenches with cold methanol and extracts with boiling ethanol. In this study, we tested whether these two methods are equally well suited for quantitative metabolome analyses with yeast. Using isotope dilution mass spectrometry (IDMS) with GC-MS and LC-MS as analytical methods, in combination with precise quantification of cell volumes, we determined absolute concentrations of 63 intracellular metabolites covering amino acids, organic acids, phosphorylated sugars, and nucleotides in two S. cerevisiae strains with different physiology. By analyzing the data from samples generated with the two methods, we found that while both methods yielded essentially identical concentrations for amino and organic acids, the cold-solvent extraction yielded significantly lower concentrations for particularly phosphorylated sugars and nucleotides, presumably because of lower quenching or extraction efficiency of this method. Our results show that methanol-quenching combined with boiling-ethanol-extraction is the more accurate approach when aiming to quantify a broad range of different metabolites from yeast cells. The significant discrepancy observed between both common metabolite extraction methods demonstrates the importance of method optimization for quantitative studies in particular when working with microbes with rigid cell walls such as those found in yeast.


title: HIV-1 Vpu restricts Fc-mediated effector functions in vivo

abstract: Non-neutralizing antibodies (nnAbs) can eliminate HIV-1-infected cells via antibody-dependent cellular cytotoxicity (ADCC) and were identified as a correlate of protection in the RV144 vaccine trial. Fc-mediated effector functions of nnAbs were recently shown to alter the course of HIV-1 infection in vivo using a vpu-defective virus. Since Vpu is known to downregulate cell surface CD4, which triggers conformational changes in the viral envelope glycoprotein (Env), we ask whether the lack of Vpu expression was linked to the observed nnAbs activity. We found that restoring Vpu expression greatly reduces nnAb recognition of infected cells, rendering them resistant to ADCC responses. Moreover, administration of a nnAb in humanized mice reduces viral loads only in animals infected with a vpu-defective but not with a wildtype virus. Finally, nnAb Fc-effector functions are observed only on cells expressing Env in the "open" conformation. This work highlights the importance of Vpu-mediated evasion of humoral responses.


title: Influenza A virus agnostic receptor tropism revealed using a novel biological system with terminal sialic acid-knockout cells

abstract:
Avian or human influenza A viruses bind preferentially to avian- or human-type sialic acid receptors, respectively, indicating that receptor tropism is an important factor for determining the viral host range. However, there are currently no reliable methods for analyzing receptor tropism biologically under physiological conditions. Here, we established a novel system using MDCK cells with avian- or human-type sialic acid receptors and with both sialic acid receptors knocked out (KO). When we examined the replication of human and avian influenza viruses in these KO cells, we observed unique viral receptor tropism that could not be detected using a conventional solid-phase sialylglycan binding assay, which directly assesses physical binding between the virus and sialic acids. Furthermore, we serially passaged an engineered avian-derived H4N5 influenza virus, whose PB2 gene was deleted, in avian-type receptor-KO cells stably expressing PB2 to select a mutant with enhanced replication in KO cells; however, its binding to human-type sialylglycan was undetectable using the solid-phase binding assay. These data indicate that a panel of sialic acid receptor-KO cells could be a useful tool for determining the biological receptor tropism of influenza A viruses. Moreover, the PB2-KO virus experimental system could help to safely and efficiently identify the mutations required for avian influenza viruses to adapt to human cells that could trigger a new influenza pandemic.

Author summaryInfluenza A virus initiates infection via hemagglutinin by binding to avian- or human-type receptors. The acquisition of mutations that allow avian virus hemagglutinins (HAs) to recognize human-type receptors is mandatory for the transmission of avian influenza viruses to humans, which could lead to a pandemic. Therefore, it is important to detect such mutation(s) in animal influenza viruses for pandemic surveillance and risk assessment. In this study, we established a novel system using a set of genetically engineered MDCK cells with knocked out sialic acid receptors to biologically evaluate the receptor tropism for influenza A viruses. Using this system, we observed unique receptor tropism in several virus strains that was undetectable using conventional solid-phase binding assays that measure physical binding between the virus and artificially synthesized sialylglycans. This study makes a significant contribution to the literature because our findings suggest the pitfall of conventional receptor binding assay and the existence of a sialic acid-independent pathway for viral infection. In addition, our system could be safely used to identify mutations that could acquire human-type receptor tropism. Thus, this system could contribute not only toward basic analyses, such as elucidating the mechanism of influenza virus host range determination, but also the surveillance of viruses of animal origin that could be capable of infecting via human-type receptors, triggering a new influenza pandemic.


title: Comprehensive analysis of PNA-based antisense antibiotics targeting various essential genes in uropathogenic Escherichia coli

abstract: Antisense peptide nucleic acids (PNAs) that target mRNAs of essential bacterial genes exhibit specific bactericidal effects in several microbial species, but our mechanistic understanding of PNA activity and their target gene spectrum is limited. Here, we present a systematic analysis of PNAs targeting eleven essential genes with varying expression levels in uropathogenic Escherichia coli (UPEC). We demonstrate that UPEC is susceptible to killing by peptide-conjugated PNAs, especially when targeting the widely-used essential gene acpP. Our evaluation yields three additional promising target mRNAs for effective growth inhibition, i.e., dnaB, ftsZ, and rpsH. The analysis also shows that transcript abundance does not predict target vulnerability and that PNA-mediated growth inhibition is not universally associated with target mRNA depletion. Global transcriptomic analyses further reveal PNA sequence-dependent but also -independent responses, including the induction of envelope stress response pathways. Importantly, we show that the growth inhibitory capacity of 9mer PNAs is generally as effective as their 10mer counterparts. Overall, our systematic comparison of a range of PNAs targeting mRNAs of different essential genes in UPEC suggests important features for PNA design, reveals a general bacterial response to PNA conjugates and establishes the feasibility of using PNA antibacterials to combat UPEC.


title: Antisense non-coding transcription represses the PHO5 model gene via remodelling of promoter chromatin structure

abstract: Pervasive transcription of eukaryotic genomes generates non-coding transcripts with regulatory potential. We examined the effects of non-coding antisense transcription on the regulation of expression of the yeast PHO5 gene, a paradigmatic case for gene regulation through promoter chromatin remodeling. By enhancing or impairing the level of overlapping antisense transcription through specific mutant backgrounds and the use of the CRISPRi system, we demonstrated a negative role for antisense transcription at the PHO5 gene locus. Furthermore, enhanced elongation of PHO5 antisense leads to a more repressive chromatin configuration at the PHO5 gene promoter, which is remodeled more slowly upon gene induction. The negative effect of antisense transcription on PHO5 gene transcription is mitigated by inactivation of the histone deacetylase Rpd3, showing that PHO5 antisense RNA acts via histone deacetylation. This regulatory pathway leads to Rpd3-dependent decreased recruitment of the RSC chromatin remodeling complex to the PHO5 gene promoter upon induction of antisense transcription. Overall, we extend the model of PHO5 gene regulation by demonstrating a gene silencing function of antisense transcription through a chromatin-based mechanism.


title: Quantitative genetic study suggests sex-specific genetic architecture for fetal testosterone in a wild mammal

abstract: Testosterone plays a critical role in mediating fitness-related traits in many species. Although it is highly responsive to environmental and social conditions, evidence from several species show a heritable component to its individual variation. Despite the known effects that in utero testosterone exposure have on adult fitness, the heritable component of individual testosterone variation in fetuses is mostly unexplored. Furthermore, testosterone has sex-differential effects on fetal development, i.e., a specific level may be beneficial for male fetuses but detrimental for females. The above mentioned may lead to a different genetic structure underlying the heritability of testosterone between the sexes. Here, we used a wild animal model, the feral nutria, quantified testosterone using hair-testing and estimated its heritability between parent and offspring from the same and opposite sex. We found that in utero accumulated hair testosterone levels were heritable between parents and offspring of the same sex. However, there was a low additive genetic covariance between the sexes, and a relatively low cross-sex genetic correlation, suggesting a potential for sex-dependent trait evolution, expressed early on, in utero.


title: Insights into the ribosome function from the structures of non-arrested ribosome nascent chain complexes.

abstract: During protein synthesis, the growing polypeptide chain threads through the nascent peptide exit tunnel that spans the body of the large ribosomal subunit while simultaneously acting as a modulator of ribosomal activity by itself or by sensing various small molecules, such as metabolites or antibiotics appearing in the tunnel. While arrested ribosome nascent chain complexes (RNCCs) have been extensively studied structurally, little attention has been given to the RNCCs that represent the functionally active state of the ribosome. This is in part due to the lack of a simple and reliable procedure for the large-scale preparation of peptidyl-tRNAs. Here we report a new chemoenzymatic approach based on native chemical ligation reaction for the facile synthesis of stably linked peptidyl-tRNAs that were used to determine several structures of RNCCs in the functional pre-attack state of the peptidyl transferase center (PTC) at the highest resolution available to date. These structures reveal a previously unknown role of the ribosome in stabilization of the growing polypeptide within the PTC and suggest an extended entropic trap model that mechanistically rationalizes how ribosome acts with comparable efficiencies upon a multitude of possible growing peptides having various sequences. Our structures also provide new insights into the mechanism of PTC functioning and explain what makes ribosome a versatile catalyst.


title: Activation of Nedd4L Ubiquitin Ligase by FCHO2-generated Membrane Curvature

abstract: The C2-WW-HECT domain ubiquitin ligase Nedd4L regulates sorting in endocytosis by mediating ubiquitination of cargo molecules, such as the epithelial sodium channel (ENaC)1, 2. Defects in ENaC ubiquitination cause Liddle syndrome, a hereditary hypertension. Nedd4L is catalytically autoinhibited by an intramolecular interaction between the C2 and HECT domains3, but the activation mechanism is poorly understood. Here, we show that Nedd4L is activated by membranes sculpted by FCHO2, a Bin-Amphiphysin-Rsv (BAR) domain protein that regulates endocytosis. We found that FCHO2 was required for Nedd4L-mediated ubiquitination and endocytosis of ENaC. Nedd4L co-localized with FCHO2 at clathrin-coated pits where it likely became activated. Nedd4L was specifically recruited to and activated by the FCHO2 BAR domain exogenously expressed in cells. Furthermore, we reconstituted in vitro FCHO2-induced recruitment and activation of Nedd4L. Both the recruitment and activation were mediated by membrane curvature rather than protein-protein interactions. The Nedd4L C2 domain recognized a specific degree of membrane curvature that was generated by the FCHO2 BAR domain. Consequently, this curvature activated Nedd4L by relieving autoinhibition. Thus, we show for the first time a specific functionality (i.e., recruitment and activation of an enzyme regulating cargo sorting) of membrane curvature by a BAR domain protein.


title: Photoreceptors generate neuronal diversity in their target field through a Hedgehog morphogen gradient in Drosophila

abstract: Defining the origin of neuronal diversity is a major challenge in developmental neurobiology. The Drosophila visual system is an excellent paradigm to study how cellular diversity is generated during development. Photoreceptors from the eye disc grow their axons into the optic lobe and secrete Hedgehog (Hh) to induce the lamina, such that for every unit eye there is a corresponding lamina unit made up of post-mitotic precursors stacked into columns. Each differentiated column contains five lamina neuron types (L1-L5), making it the simplest neuropil in the optic lobe, yet how this diversity is generated was unknown. Here, we found that Hh pathway activity is graded along the distal-proximal axis of lamina columns and further determined that this gradient in pathway activity arises from a gradient of Hh ligand. We manipulated Hh pathway activity both cell-autonomously in lamina precursors and by inactivating the Hh ligand, and showed that different thresholds of activity specify unique cell identities, with more proximal cell types specified in response to progressively lower Hh levels. Thus, our data establish that Hh acts as a morphogen to pattern the lamina. Although, this is the first such report during Drosophila nervous system development, our work uncovers a remarkable similarity with the vertebrate neural tube, which is patterned by Sonic Hedgehog. Altogether, we show that differentiating neurons can regulate the neuronal diversity of their distant target fields through morphogen gradients.


title: Landscape diversity and local temperature, but not climate, affect arthropod predation among habitat types

abstract: Arthropod predators are important for ecosystem functioning by providing top-down regulation of insect herbivores. As predator communities and activity are influenced by biotic and abiotic factors on different spatial scales, the strength of top-down regulation ( arthropod predation) is also likely to vary. Understanding the combined effects of potential drivers on arthropod predation is urgently needed with regard to anthropogenic climate and land-use change. In a large-scale study, we recorded arthropod predation rates using artificial caterpillars on 113 plots of open herbaceous vegetation embedded in contrasting habitat types (forest, grassland, arable field, settlement) along climate and land-use gradients in Bavaria, Germany. As potential drivers we included habitat characteristics (habitat type, plant species richness, local mean temperature and mean relative humidity during artificial caterpillar exposure), landscape diversity (0.5-3.0-km, six scales), climate (multi-annual mean temperature,  MAT) and interactive effects of habitat type with other drivers. We observed no substantial differences in arthropod predation rates between the studied habitat types, related to plant species richness and across the Bavarian-wide climatic gradient, but predation was limited when local mean temperatures were low and tended to decrease towards higher relative humidity. Arthropod predation rates increased towards more diverse landscapes at a 2-km scale. Interactive effects of habitat type with local weather conditions, plant species richness, landscape diversity and MAT were not observed. We conclude that landscape diversity favours high arthropod predation rates in open herbaceous vegetation independent of the dominant habitat in the vicinity. This finding may be harnessed to improve top-down control of herbivores, e.g. agricultural pests, but further research is needed for more specific recommendations on landscape management. The absence of MAT effects suggests that high predation rates may occur independent of moderate increases of MAT in the near future.

category: ecology


title: Construction of a High-Density Genetic Map Based on Large-Scale Marker Development in  Coix lacryma-jobi  L .  Using Specifific-Locus Amplifified Fragment Sequencing (SLAF-seq)

abstract: Coix lacryma-jobi L. is one of the most important economical and medicine corn. In this study, a high genetic linkage map has been constructed for Coix lacryma-jobi L. from a cross F2 community of "Qianyi NO.2" x "Wenyi NO.2" and their parents through high-throughput sequencing and specfic-locus amplified fragment (SLAF) library construction. After pre-processing, 325.49 Gb of raw data containing 1,628 M reads were obtained. A total of 22,944 high-quality SLAFs were detected, among which 3,952 SLAFs and 3,646 of the polymorphic markers could meet the requirements for construction of a genetic linkage map. The integrated map contains 3,605 high quality SLAFs, which are grouped to 10 genetic linkage groups. The total length of the linkage map is 1,620.39 cM with an average distance of 0.45 cM and 360.5 markers in average of per linkage group. This work will provide an important molecular biology basis for investigating their characteristics, gene cloning, molecular marker-assisted breeding, functional genomics and so on for Jobs tears.

category: genetics


title: Closely related type II-C Cas9 orthologs recognize diverse PAMs

abstract: Autosomal dominant diseases can be treated by allele-specific disruption of mutant alleles. If the missense mutation generates a novel protospacer-adjacent motif (PAM), CRISPR-Cas9 can distinguish mutant alleles from wild-type alleles by a PAM-specific approach. Therefore, it is crucial to develop a CRISPR toolbox capable of recognizing multiple PAMs. Here, by using a GFP-activation assay, we tested the activities of 29 type II-C orthologs closely related to Nme1Cas9, 25 of which are active in human cells. These orthologs recognize diverse PAMs with variable length and nucleotide preference, including purine-rich, pyrimidine-rich, and mixed purine and pyrimidine PAMs. We characterized in depth the activity and specificity of Nsp2Cas9. We also generated a chimeric Cas9 nuclease that recognizes a simple N4C PAM, representing the most relaxed PAM preference for compact Cas9s to date. These Cas9 nucleases significantly enhance our ability to perform allele-specific genome editing.


title: Monomeric α-Synuclein activates the Plasma Membrane Calcium Pump

abstract: Alpha-synuclein (aSN) is a membrane-associated and intrinsically disordered protein, well-known for pathological aggregation in neurodegeneration. The physiological function of aSN however is disputed. Pull-down experiments have pointed to plasma membrane Ca2+-ATPase (PMCA) as a potential interaction partner. From proximity ligation assays we find that aSN and PMCA colocalize at neuronal synapses, and that calcium expulsion is activated by aSN and PMCA. From PMCA activity studies we show that soluble, monomeric aSN activates PMCA at par with CaM, yet independent of the autoinhibitory domain of PMCA, but highly dependent on acidic phospholipids and membrane-anchoring of aSN. On PMCA, the key site is mapped to the acidic lipid-binding site, located within a disordered PMCA-specific loop region connecting the cytosolic A domain and transmembrane segment 3. Our studies point towards a novel physiological role of monomeric aSN as a stimulator of calcium clearance in neurons through activation of PMCA.


title: Structural basis for the activation and ligand recognition of the human oxytocin receptor

abstract: The small cyclic neuropeptide hormone oxytocin (OT) and its cognate receptor play a central role in the regulation of social behaviour and sexual reproduction. Here we report the single-particle cryo-electron microscopy structure of the active oxytocin receptor (OTR) in complex with its cognate ligand oxytocin. Our structure provides high-resolution insights into the OT binding mode, the OTR activation mechanism as well as the subtype specificity within the oxytocin/vasopressin receptor family.


title: A Facile Method to Produce N-Terminally Truncated 	α-Synuclein

abstract: -Synuclein is a key protein of the nervous system, which regulates the release and recycling of neurotransmitters in the synapses. It is also involved in several neurodegenerative conditions, including Parkinsons disease and Multiple System Atrophy, where it forms toxic aggregates. The N-terminus of -synuclein is of particular interest as it has been linked to both the physiological and pathological functions of the protein and undergoes post-translational modification. One such modification, N-terminal truncation, affects the aggregation propensity of the protein in vitro and is also found in aggregates from patients brains. To date, our understanding of the role of this modification has been limited by the many challenges of introducing biologically relevant N-terminal truncations with no overhanging starting methionine. Here, we present a method to produce N-terminally truncated variants of -synuclein that do not carry extra terminal residues. We show that our method can generate highly pure protein to facilitate the study of this modification and its role in physiology and disease. Thanks to this method, we have determined that the first six residues of -synuclein play an important role in the formation of the amyloids.


title: AFM Microfluidic Cantilevers as Weight Sensors for Single Cell Mass Measurements

abstract: Reliably measuring small mass changes at the single-cell level is challenging. In this manuscript, we report the use of microfluidic cantilevers in liquid with sub-nanogram scale weight sensing capability for the measurement of cellular mass changes of living single cells. With this instrumentation, we were able to perform fast mass measurements within 3 minutes. We show results of mass measurements of polystyrene and metal beads of various sizes (smallest weight measured at 280 {+/-} 95 pg) and live single-cell mass measurements in a physiologically relevant environment. We also performed finite element analysis to simulate and optimize the structural design and materials of cantilevers. Our simulation results indicate that using polymer materials, such as SU8 and polyimide, could improve the minimal detectable mass by 3-fold compared to conventional silicon cantilevers. The simulations also suggest that smaller dimensions of length, width, and thickness would improve the mass detection capability of microfluidic cantilevers.


title: Brassinosteroids modulate autophagy through phosphorylation of RAPTOR1B by the GSK3-like kinase BIN2 in Arabidopsis

abstract: Macroautophagy/autophagy is a conserved recycling process that maintains cellular homeostasis during environmental stress. Autophagy is negatively regulated by TARGET OF RAPAMYCIN (TOR), a nutrient-regulated protein kinase that in plants is activated by several phytohormones, leading to increased growth. However, the detailed molecular mechanisms by which TOR integrates autophagy and hormone-signaling are poorly understood. Here, we show that TOR modulates brassinosteroid (BR)-regulated plant growth and stress-response pathways. Active TOR was required for full BR-induced growth in Arabidopsis thaliana. Autophagy was constitutively up-regulated upon blocking BR biosynthesis or signaling, and down-regulated by increasing the activity of the BR pathway. BRASSINOSTEROID-INSENSITIVE 2 (BIN2) kinase, a GSK3-like kinase functioning as a negative regulator in BR signaling, directly phosphorylated Regulatory-Associated Protein of TOR 1B (RAPTOR1B), a substrate-recruiting subunit in the TOR complex, at a conserved serine residue within a typical BIN2 phosphorylation motif. Mutation of RAPTOR1B serine 916 to alanine, to block phosphorylation by BIN2, repressed autophagy and increased phosphorylation of the TOR substrate autophagy-related protein 13a (ATG13a). By contrast, this mutation had only a limited effect on growth. We present a model in which RAPTOR1B is phosphorylated and inhibited by BIN2 when BRs are absent, activating the autophagy pathway. When BRs signal and inhibit BIN2, RAPTOR1B is thus less inhibited by BIN2 phosphorylation. This leads to increased TOR activity and ATG13a phosphorylation, and decreased autophagy activity. Our studies define a new mechanism by which coordination between BR and TOR signaling pathways helps to maintain the balance between plant growth and stress responses.


title: Cobalamin riboswitches are broadly sensitive to corrinoid cofactors to enable an efficient gene regulatory strategy

abstract: In bacteria, many essential metabolic processes are controlled by riboswitches, gene regulatory RNAs that directly bind and detect metabolites. Highly specific effector binding enables riboswitches to respond to a single biologically relevant metabolite. Cobalamin riboswitches are a potential exception because over a dozen chemically similar but functionally distinct cobalamin variants (corrinoid cofactors) exist in nature. Here, we measured cobalamin riboswitch activity in vivo using a Bacillus subtilis fluorescent reporter system and found that among 38 tested riboswitches, a subset responded to corrinoids promiscuously, while others were semi-selective. Analyses of chimeric riboswitches and structural models indicate that, unlike other riboswitch classes, cobalamin riboswitches indirectly differentiate among corrinoids by sensing differences in their structural conformation. This regulatory strategy aligns riboswitch-corrinoid specificity with cellular corrinoid requirements in a B. subtilis model. Thus, bacteria can employ broadly sensitive riboswitches to cope with the chemical diversity of essential metabolites.


title: Proteasomal downregulation of the pro-apoptotic MST2 pathway contributes to BRAF inhibitor resistance in melanoma

abstract: The RAS-RAF-MEK-ERK pathway is hyperactivated in almost all malignant melanomas, and mutations in BRAF or NRAS account for most of these cases. BRAF inhibitors are highly efficient for treating patients with BRAFV600E mutations, but tumours frequently acquire resistance within a few months. Multiple resistance mechanisms have been identified, due to mutations or network adaptations that revive ERK signalling. We have previously shown that RAF proteins inhibit the MST2 proapoptotic pathway in a kinase independent fashion. Here, we have investigated the role of the MST2 pathway in mediating resistance to BRAF inhibitors. We show that the BRAFV600E mutant protein, but not the wildtype BRAF protein, strongly binds to MST2 and inhibits MST2 pro-apoptotic signalling. Downregulation of MST2 reduces BRAF inhibitor induced apoptosis. In BRAF inhibitor resistant cell lines MST2 pathway proteins are downregulated by ubiquitination and subsequent proteasomal degradation rendering cells refractory to MST2 pathway induced apoptosis. Restoration of apoptosis can be achieved by increasing MST2 pathway protein expression using proteasome inhibitors. In summary, we show that the MST2 pathway plays a role in the acquisition of BRAF inhibitor resistance in melanoma.


title: Molecular Signatures and Cellular Diversity During Mouse Habenula Development

abstract: The habenula plays a key role in various motivated and pathological behaviors and is composed of molecularly distinct neuron subtypes. Despite progress in identifying mature habenula neuron subtypes, how these subtypes develop and organize into functional brain circuits remains largely unknown. Here we performed single-cell transcriptional profiling of mouse habenular neurons at critical developmental stages instructed by detailed three-dimensional anatomical data. Our data reveal cellular and molecular trajectories during embryonic and postnatal development leading to different habenular subtypes. Further, based on this analysis our work establishes the distinctive functional properties and projection target of a previously uncharacterized subtype of Cartpt+ habenula neurons. Finally, we show how comparison of single-cell transcriptional profiles and GWAS data links specific developing habenular subtypes to psychiatric disease. Together, our study begins to dissect the mechanisms underlying habenula neuron subtype-specific development and creates a framework for further interrogation of habenular development in normal and disease states.


title: Quantitative Proteomic Profiling of Murine Embryonic Heart Development Reveals a Role for the Mevalonate Pathway in Cardiomyocyte Proliferation

abstract: Defining the molecular mechanisms that govern heart development is essential for identifying the etiology of congenital heart disease. Here, quantitative proteomics was used to measure temporal changes in the cardiac proteome at eight critical stages of murine embryonic heart development. Global temporal profiles of the over 7,300 identified proteins uncovered signature cardiac protein interaction networks that linked protein dynamics with molecular pathways. Using this integrated dataset, we identified and established a functional role for the mevalonate pathway in the regulation of embryonic cardiomyocyte proliferation and cell signaling. Overall, our proteomic datasets are an invaluable resource for studying molecular events that regulate embryonic heart development and contribute to congenital heart disease.


title: The Impact of Protection Measures and Treatment onPneumonia Infection Model

abstract: Pneumonia has been a major airborne transmitted disease and continues to pose a major public health burden in both developed and developing countries of the world. In this study, we constructed and analyzed a nonlinear deterministic compartmental mathematical model for assessing the community-level impacts of vaccination, other protection measures like practicing good hygiene, avoiding close contacts with sick people and limiting exposure to cigarette smoke, etc. and treatment on the transmission dynamics of pneumonia disease in a population of varying size. Our model exhibits two kinds of equilibrium points: pneumonia disease-free equilibrium point, and pneumonia endemic equilibrium point(s). Using center manifold criteria, we have verified that the pneumonia model exhibits backward bifurcations whenever its effective reproduction number [R]P < 1 and in the same region, the model shows the existence of more than one endemic equilibrium point where some of which are stable and others are unstable. Thus, for pneumonia infection, the necessity of the pneumonia effective reproduction number [R]P < 1, although essential, it might not be enough to completely eradicate the pneumonia infection from the considered community. Our examination of sensitivity analysis shows that the pneumonia infection transmission rate denoted by {beta} plays a crucial role to change the qualitative dynamics of pneumonia infection. By taking standard data from published literature, our numerical computations show that the numerical value of pneumonia infection model effective reproduction number is [R]P = 8.31 at {beta} = 4.21 it implies that the disease spreads throughout the community. Finally, our numerical simulations show that protection, vaccination, and treatment against pneumonia disease have the effect of decreasing pneumonia expansion.


title: The Zpr-3 antibody recognizes the 320-354 region of Rho and labels both rods and green cones in zebrafish

abstract: Retinal markers with high quality and specificity are important for the observation of pathologic changes of retinal cells during retinal development, degeneration, and regeneration. Zpr-3 is a mouse monoclonal antibody with uncharacterized antigen usually used to label rod photoreceptors. In this study, we provided evidence to demonstrate that the antigen gene of zpr-3 is rho, which encodes the rod opsin, and the exact epitope of zpr-3 is the 320-354 region of Rho protein. More importantly, our immunofluorescence assays indicated that zpr-3 labels both the outer segments of rods and green-cones on zebrafish retinal sections, probably due to the cross-reaction with the green-cone opsin. Our work is valuable for the scientific community to interpret the experimental data involving the zpr-3 antibody.


title: Consistent measurement of LAG-3 expression across multiple staining platforms with the 17B4 antibody clone

abstract:
ContextAn immunohistochemistry (IHC) assay developed to detect lymphocyte-activation gene 3 (LAG-3), a novel immune checkpoint inhibitor target, has demonstrated high analytical precision and interlaboratory reproducibility using a Leica staining platform, but has not been investigated on other IHC staining platforms.

ObjectiveTo evaluate the performance of LAG-3 IHC assays using the 17B4 antibody clone across widely used IHC staining platforms: Agilent/Dako Autostainer Link 48 (ASL-48) and VENTANA BenchMark ULTRA (VBU) compared with Leica BOND-RX (BOND-RX).

DesignEighty formalin-fixed paraffin-embedded melanoma tissue blocks were cut into consecutive sections and evaluated using staining platform-specific IHC assays with the 17B4 antibody clone. Duplicate testing was performed on the BOND-RX platform to assess intraplatform agreement. LAG-3 expression using a numerical score was evaluated by a pathologist and with a digital scoring algorithm. LAG-3 positivity was determined from manual scores using a [≥] 1% cutoff.

ResultsLAG-3 IHC staining patterns and intensities were visually similar across all 3 staining platforms. Pearson correlation was [≥] 0.88 for interplatform and BOND-RX intraplatform concordance when LAG-3 expression was evaluated with a numerical score determined by a pathologist. Correlation increased with a numerical score determined with a digital scoring algorithm (Pearson correlation [≥] 0.93 for all comparisons). Overall percentage agreement was [≥] 77.5% for interplatform and BOND-RX intraplatform comparisons when a [≥] 1% cutoff was used to determine LAG-3 positivity.

ConclusionsData from this study demonstrate that LAG-3 expression can be robustly and reproducibly assessed across 3 major commercial IHC staining platforms using the 17B4 antibody clone.

category: pathology


title: Interpreting how machine learning models make predictions in biological studies

abstract: Explainable artificial intelligence aims to interpret how the machine learning models make decisions, and many model explainers have been developed in the computer vision field. However, the understandings of the applicability of these model explainers to biological data are still lacking. In this study, we comprehensively evaluated multiple explainers by interpreting pretrained models of predicting tissue types from transcriptomic data, and by identifying top contributing genes from each sample with the greatest impacts on model prediction. To improve the reproducibility and interpretability of results generated by model explainers, we proposed a series of optimization strategies for each explainer on two different model architectures of Multilayer Perceptron (MLP) and Convolutional Neural Network (CNN). We observed three groups of explainer and model architecture combinations with high reproducibility. Group II, which contains three model explainers on aggregated MLP models, identified top contributing genes in different tissues that exhibited tissue-specific manifestation and were potential cancer biomarkers. In summary, our work provides novel insights and guidance for exploring biological mechanisms using explainable machine learning models.

category: bioinformatics


title: Assessment and Optimization of the Interpretability of Machine Learning Models Applied to Transcriptomic Data


title: Tensor decomposition- and principal component analysis-based unsupervised feature extraction to select more reasonable differentially expressed genes: Optimization of standard deviation versus state-of-art methods

abstract:
BackgroundTensor decomposition- and principal component analysis-based unsupervised feature extraction were proposed almost 5 and 10 years ago, respectively; although these methods have been successfully applied to a wide range of genome analyses, including drug repositioning, biomarker identification, and disease-causing genes identification, some fundamental problems have been identified: the number of genes identified was too small to assume that there were no false negatives, and the histogram of P-values derived was not fully coincident with the null hypothesis that principal component and singular value vectors follow the Gaussian distribution.

ResultsOptimizing the standard deviation such that the histogram of P-values is as much as possible coincident with the null hypothesis results in an increase in the number and biological reliability of the selected genes.

ConclusionsTensor decomposition- and principal component analysis-based unsupervised feature extraction are perhaps better than state-of-art methods in regard to predicting differentially expressed genes because they achieve the desired property that the less expressed differentially expressed genes should be less likely selected or even associated with the same amount of logarithmic fold change, although they assume neither negative binomial distribution nor dispersion relation, which is usually assumed in state-of-art methods.


title: The Effect of Genome Graph Expressiveness on the Discrepancy Between Genome Graph Distance and String Set Distance

abstract:
MotivationIntra-sample heterogeneity describes the phenomenon where a genomic sample contains a diverse set of genomic sequences. In practice, the true string sets in a sample are often unknown due to limitations in sequencing technology. In order to compare heterogeneous samples, genome graphs can be used to represent such sets of strings. However, a genome graph is generally able to represent a string set universe that contains multiple sets of strings in addition to the true string set. This difference between genome graphs and string sets is not well characterized. As a result, a distance metric between genome graphs may not match the distance between true string sets.

ResultsWe extend a genome graph distance metric, Graph Traversal Edit Distance (GTED) proposed by Ebrahimpour Boroojeny et al., to FGTED to model the distance between heterogeneous string sets and show that GTED and FGTED always underestimate the Earth Movers Edit Distance (EMED) between string sets. We introduce the notion of string set universe diameter of a genome graph. Using the diameter, we are able to upper-bound the deviation of FGTED from EMED and to improve FGTED so that it reduces the average error in empirically estimating the similarity between true string sets. On simulated TCR sequences and Hepatitis B virus genomes, we show that the diameter-corrected FGTED reduces the average deviation of the estimated distance from the true string set distances by more than 250%.

AvailabilityData and source code for reproducing the experiments are available at: https://github.com/Kingsford-Group/gtedemedtest/

Contactcarlk@cs.cmu.edu


title: Noncontact and high-precision sensing system for piano keys identified fingerprints of virtuosity

abstract: Dexterous tool use is typically characterized by fast and precise motions performed by multiple fingers. One representative task is piano playing, which involves fast performance of a sequence of complex motions with high spatiotemporal precision. However, for several decades, a lack of contactless sensing technologies that are capable of precision measurement of piano key motions has been a bottleneck for unveiling how such an outstanding skill is cultivated. Here, we developed a novel sensing system that can record the vertical position of all piano keys with a time resolution of 1 ms and a spatial resolution of 0.01 mm in a noncontact manner. Using this system, we recoded the piano key motions while 49 pianists played a complex sequence of tones that required both individuated and coordinated finger movements to be performed as fast and accurately as possible. Penalized regression using various feature variables of the key motions identified distinct characteristics of the key-depressing and key-releasing motions in relation to the speed and accuracy of the performance. For the maximum rate of the keystrokes, individual differences across the pianists were associated with the peak key descending velocity, the key depression duration, and key-lift timing. For the timing error of the keystrokes, the interindividual differences were associated with the peak ascending velocity of the key and the interstrike variability of both the peak key descending velocity and the key depression duration. These results highlight the importance of dexterous control of the vertical motions of the keys for fast and accurate piano performance.

category: neuroscience


title: Reactive astrocytes transduce blood-brain barrier dysfunction through a TNFα-STAT3 signaling axis and secretion of alpha 1-antichymotrypsin

abstract: Astrocytes are critical components of the neurovascular unit that support blood-brain barrier (BBB) function in brain microvascular endothelial cells (BMECs). Transformation of astrocytes to a reactive state in response to injury and disease can be protective or harmful to BBB function, but the underlying mechanisms for these effects remain mostly unclear. Using a human induced pluripotent stem cell (iPSC)-derived coculture model of BMEC-like cells and astrocytes, we found that tumor necrosis factor alpha (TNF) transitions astrocytes to an inflammatory reactive state through activated STAT3 signaling, whereby the resultant astrocytes disrupt passive BBB function and induce vascular cell adhesion molecule 1 (VCAM-1) expression in the BMEC-like cells. These associations between inflammatory reactive astrocytes, STAT3 activation, and vascular VCAM-1 expression were corroborated in human postmortem tissue. Bioinformatic analyses coupled with CRISPR interference techniques in the iPSC model revealed that inflammatory reactive astrocytes transduce BBB disruption in part through SERPINA3, which encodes alpha 1-antichymotrypsin (1ACT), a secreted serine protease inhibitor associated with aging, neuroinflammation, and Alzheimers disease. In murine ex vivo cortical explant cultures, shRNA-mediated silencing of Serpina3n in astrocytes reduced vascular VCAM-1 expression after TNF challenge. Further, direct treatment with recombinant Serpina3n in both ex vivo explant cultures and the brain in vivo (via intracerebroventricular injection into wild-type mice) was sufficient to induce vascular VCAM-1 expression and reduce tight junction integrity. Overall, our results define the TNF-STAT3 signaling axis as a driver of an inflammatory reactive astrocyte subtype responsible for BBB dysfunction. Our results also identify 1ACT as an explicit mediator of BBB damage and suggest that inhibition of 1ACT expression or activity could represent a therapeutic avenue for reversing BBB deficits in aging and neurodegenerative disease.


title: The effects of caloric restriction on adipose tissue and metabolic health are sex- and age-dependent

abstract:
Caloric restriction (CR) is a nutritional intervention that reduces the risk of age-related diseases in numerous species, including humans. CRs metabolic effects, including decreased fat mass and improved insulin sensitivity, play an important role in its broader health benefits. However, the extent and basis of sex differences in CRs health benefits are unknown. We found that 30% CR in young (3-month-old) male mice decreased fat mass and improved glucose tolerance and insulin sensitivity, whereas these effects were blunted or absent in young female mice. Resistance to fat and weight loss in females was not explained by decreased energy expenditure compared to males. Positron emission tomography-computed tomography (PET/CT) with 18F-fluorodeoxyglucose (18F-FDG) showed that peripheral glucose uptake was comparable between sexes. Instead, CR caused a pronounced suppression of hepatic gluconeogenesis and altered liver ceramide content in males but not in females. In aged mice (18-months old), when females are anoestrus, CR decreased fat mass and improved glucose homeostasis to a similar extent in both sexes. CR-induced fat loss in humans was also sex- and age-dependent: younger females (<45 years) resisted fat loss compared to younger males while in older subjects (>45 years) this sex difference was absent. Collectively, these studies identify age-dependent sex differences in the metabolic effects of CR and highlight adipose tissue, the liver and oestrogen as key determinants of CRs metabolic benefits. This has important implications for understanding the interplay between diet and health and for maximising the benefits of CR in humans.

HIGHLIGHTSO_LICaloric restriction (CR) decreases fat mass and improves glucose homeostasis in young male mice, but young females resist these effects.
C_LIO_LIThese effects are associated with improved hepatic insulin sensitivity and suppressed gluconeogenesis, without marked differences in peripheral glucose uptake.
C_LIO_LICRs effects on fat loss and glucose homeostasis are comparable in aged male and female mice, implicating oestrogen as the driver of the sexually dimorphic effects in young mice.
C_LIO_LIIn humans, females resist CR-induced fat loss in an age-dependent manner, further supporting the role of oestrogen in the sexually dimorphic effects of CR.
C_LI

category: physiology


title: Voxel-wise Intermodal Coupling Analysis of Two or More Modalities using Local Covariance Decomposition

abstract: When individual subjects are imaged with multiple modalities, biological information is present not only within each modality, but also between modalities - that is, in how modalities covary at the voxel level. Previous studies have shown that local covariance structures between modalities, or intermodal coupling (IMCo), can be summarized for two modalities, and that two-modality IMCo reveals otherwise undiscovered patterns in neurodevelopment and certain diseases. However, previous IMCo methods are based on the slopes of local weighted linear regression lines, which are inherently asymmetric and limited to the two-modality setting. Here, we present a generalization of IMCo estimation which uses local covariance decompositions to define a symmetric, voxel-wise coupling coefficient that is valid for two or more modalities. We use this method to study coupling between cerebral blood flow, amplitude of low frequency fluctuations, and local connectivity in 803 subjects ages 8 through 22. We demonstrate that coupling is spatially heterogeneous, varies with respect to age and sex in neurodevelopment, and reveals patterns that are not present in individual modalities. As availability of multi-modal data continues to increase, principal-component-based IMCo (pIMCo) offers a powerful approach for summarizing relationships between multiple aspects of brain structure and function. An R package for estimating pIMCo is available at: https://github.com/hufengling/pIMCo.


title: Identifying the midline thalamus in humans in vivo

abstract:
The midline thalamus is essential to flexible cognition, memory, and stress regulation and its dysfunction is associated with several neurological and mental health disorders including Alzheimers disease, schizophrenia, and depression. Despite the pervasive role of the midline thalamus in cognition and disease, almost nothing is known about it in humans due to the lack of a rigorous methodology for finding this brain region using noninvasive imaging technologies. Here, we introduce a new method for identifying the midline thalamus in vivo using probabilistic tractography and k-means clustering with diffusion weighted imaging data. This method clusters thalamic voxels based on data-driven cortical and subcortical connectivity profiles, and then segments midline thalamic nuclei based on connectivity profiles established in rodent and macaque tracer studies. Results from two different diffusion weighted imaging sets, including adult data (22-35yrs) from the Human Connectome Project (n=127) and adolescent data (9-14yrs) collected at Florida International University (n=34) showed that this approach reliably classifies midline thalamic clusters. As expected, these clusters were most evident along the dorsal/ventral extent of the third ventricle and primarily connected to the agranular medial prefrontal cortex (e.g., anterior cingulate cortex), nucleus accumbens, and medial temporal lobe regions. The midline thalamic clusters were then bisected based on a human brain atlas (Ding et al., 2016) into a dorsal midline thalamic cluster (paraventricular and paratenial nuclei) and a ventral midline thalamic cluster (rhomboid and reuniens nuclei). This anatomical connectivity-based identification procedure for the midline thalamus offers new opportunities to study this region in vivo in healthy populations and in those with psychiatric and neurological disorders.

Significance StatementThe midline thalamus is essential for flexible cognition, decision making, and stress regulation. Abnormal development, dysfunction, and neurodegeneration of the midline thalamus is thought to be a critical pathology in several psychiatric and neurological disorders. Yet, little is known about the role of the human midline thalamus. Here we solve the problem of localization in vivo by using probabilistic tractography and data driven clustering (k-means), a method capable of capitalizing on known connectivity in non-human animal tracer studies. Localizing the midline thalamus based on its connectivity patterns provides a useful tool for future functional and structural human MRI studies to investigate clinical implications of the midline thalamus in diseases such as Alzheimers and schizophrenia.


title: Competition contributes to both warm and cool range edges

abstract: Competition plays an important role in shaping species spatial distributions. However, it remains unclear where and how competition regulates species range limits. In a field experiment with plants originating from low and high elevations and conducted across an elevation gradient in the Swiss Alps, we find that both lowland and highland species can better persist in the presence of competition within than beyond their elevation ranges, suggesting that competition helps set both lower and upper elevation range limits of these species. Furthermore, the reduced ability of pairs of lowland or highland species to coexist beyond their range edges is mainly driven by diminishing niche differences; changes in both niche differences and relative fitness differences drive weakening competitive dominance of lowland over highland species with increasing elevation. These results highlight the need to account for competitive interactions and investigate underlying coexistence mechanisms to understand current and future species distributions.


title: Quantifying variability in population abundances

abstract: The variability in population abundances is of central concern for the quantification of evolutionary patterns and an indispensable tool for ecological analysis. A new metric proportional variability(PV), which corresponds to the expected percentage difference between abundances, is introduced here. Although it can be readily calculated from the differences between all combinations of abundances, a more efficient method is expounded that can also accommodate zero values, if desired. The presented analysis compares PV with standard measures of population variability. The findings reveal that this truly proportional measure has appealing properties and advantages over standard measures. Furthermore, it can provide a common ground for evaluating the variability of populations undergoing very different dynamics.


title: Close-kin mark-recapture methods to estimate demographic parameters of mosquitoes

abstract:
Close-kin mark-recapture (CKMR) methods have recently been used to infer demographic parameters such as census population size and survival for fish of interest to fisheries and conservation. These methods have advantages over traditional mark-recapture methods as the mark is genetic, removing the need for physical marking and recapturing that may interfere with parameter estimation. For mosquitoes, the spatial distribution of close-kin pairs has been used to estimate mean dispersal distance, of relevance to vector-borne disease transmission and novel biocontrol strategies. Here, we extend CKMR methods to the life history of mosquitoes and comparable insects. We derive kinship probabilities for mother-offspring, father-offspring, full-sibling and half-sibling pairs, where an individual in each pair may be a larva, pupa or adult. A pseudo-likelihood approach is used to combine the marginal probabilities of all kinship pairs. To test the effectiveness of this approach at estimating mosquito demographic parameters, we develop an individual-based model of mosquito life history incorporating egg, larva, pupa and adult life stages. The simulation labels each individual with a unique identification number, enabling close-kin relationships to be inferred for sampled individuals. Using the dengue vector Aedes aegypti as a case study, we find the CKMR approach provides unbiased estimates of adult census population size, adult and larval mortality rates, and larval life stage duration for logistically feasible sampling schemes. Considering a simulated population of 3,000 adult mosquitoes, estimation of adult parameters is accurate when a total of 1,000 adult females are sampled biweekly-to-fortnightly over a three month period. Estimation of larval parameters is accurate when adult sampling is supplemented with a total of 4,000 larvae sampled biweekly over the same period. As the cost of genome sequencing declines, these methods hold great promise for characterizing the demography of mosquitoes and comparable insects of epidemiological and agricultural significance.

Author summaryClose-kin mark-recapture (CKMR) methods are a genetic analogue of traditional mark-recapture methods in which the frequency of marked individuals in a sample is used to infer demographic parameters such as census population size and mean dispersal distance. In CKMR, the mark is a close-kin relationship between individuals (parents and offspring, siblings, etc.). While CKMR methods have mostly been applied to aquatic species to date, opportunities exist to apply them to insects and other terrestrial species. Here, we explore the application of CKMR to mosquitoes, with Aedes aegypti, a primary vector of dengue, chikungunya and yellow fever, as a case study. By analyzing simulated Ae. aegypti populations, we find the CKMR approach provides unbiased estimates of adult census population size, adult and larval mortality rates, and larval life stage duration. Optimal sampling schemes are consistent with Ae. aegypti ecology and field studies, requiring only minor adjustments to current mosquito surveillance programs. This study represents the first theoretical exploration of the application of CKMR to an insect species, and demonstrates its potential for characterizing the demography of insects of epidemiological and agricultural importance.


title: Joint effect of changing selection and demography on the site frequency spectrum

abstract: The site frequency spectrum (SFS) is an important statistic that summarizes the molecular variation in a population, and used to estimate population-genetic parameters and detect natural selection. While the equilibrium SFS in a constant environment is quite well studied, recent research has focused on nonequilibrium SFS to elucidate the role of demography when selection is constant in time and of fluctuating selection in a population of constant size. However, the joint effect of time-dependent selection and population size has not been investigated so far. Here, we study the SFS in a randomly mating, diploid population in which both the population size and selection coefficient vary periodically with time using a diffusion theory approach, and derive simple analytical expressions for the time-averaged SFS in slowly and rapidly changing environments. We show that for strong selection and in slowly changing environments, the time-averaged SFS differs significantly from the equilibrium SFS when the population experiences both positive and negative cycles of the selection coefficient. The deviation depends on the time spent by the population in the deleterious part of the selection cycle and the phase difference between the selection coefficient and population size. In particular, we find that the time-averaged SFS in slowly to moderately fast varying, on-average neutral environment has the same qualitative shape as the equilibrium SFS for positively selected mutant but differs quantitatively from it which can be captured by an effective population size.

category: evolutionary biology