title: Pseudomonas aeruginosa infection reveals a Caspase-1-dependent neutrophil pyroptosis pathway that restrains damaging Histone release

abstract:
Multiple neutrophil death programs contribute to host defense against infections. Although expressing all necessary components, neutrophils specifically fail to undergo pyroptosis, a lytic form of cell death triggered by the activation of the pro-inflammatory complex inflammasome. In the light of the arm race, we hypothesized that intrinsic neutrophil pyroptosis resistance might be bypassed in response to specific microbial species. We show that Pseudomonas aeruginosa (P. aeruginosa) stimulates Caspase-1-dependent pyroptosis in human and murine neutrophils. Mechanistically, activated NLRC4 inflammasome supports Caspase-1-driven Gasdermin-D (GSDMD) activation, IL-1{beta} cytokine release and neutrophil pyroptosis. Furthermore, GSDMD activates Peptidyl Arginine Deaminase-4 which drives an "incomplete NETosis" where neutrophil DNA fills the cell cytosol but fails crossing plasma membrane. Finally, we show that neutrophil Caspase-1 account for IL-1{beta} production and contributes to various P. aeruginosa strains spread in mice. Overall, we demonstrate that neutrophils are fully competent for Caspase-1-dependent pyroptosis, which drives an unsuspected "incomplete NETosis".

SummaryNeutrophils play an essential roles against infections. Although multiple neutrophil death programs contribute to host defense against infections, they fail to undergo pyroptosis, a pro-inflammatory form of cell death. Upon Infections, pyroptosis can be induced in macrophages or epithelial cells upon activation of pro-inflammatory complexes, inflammasomes that trigger Caspase-1-driven Gasdermin dependent plasma membrane lysis. In the light of host-microbe interactions, we hypothesized that yet to find microbial species might hold the capacity to overcome neutrophil resistance to inflammasome-driven pyroptosis. Among several bacterial species, we describe that the bacterium Pseudomonas aeruginosa specifically engages the NLRC4 inflammasome, which promotes Caspase-1-dependent Gasdermin-D activation and subsequent neutrophil pyroptosis. Furthermore, inflammasome-driven pyroptosis leads to DNA decondensation and expansion into the host cell cytosol but not to the so called Neutrophil Extracellular Trap (NET) release as DNA fails breaching the plasma membrane. Finally, in vivo P. aeruginosa infections highlight that Caspase-1-driven neutrophil pyroptosis is functional and is detrimental upon P. aeruginosa infection. Altogether, our results unexpectedly underline neutrophil competence for Caspase-1-dependent pyroptosis, a process that contributes to host susceptibility to P. aeruginosa infection.


title: Caspase-1-driven neutrophil pyroptosis promotes an incomplete NETosis upon Pseudomonas aeruginosa infection

category: immunology


title: Repurposing of the enhancer-promoter communication underlies the compensation of Mesp2 by Mesp1

abstract: Organisms are inherently equipped with buffering systems against genetic perturbations. Upregulation of homologous genes responding to gene loss, termed genetic compensation, is one such buffering mechanism. Recently, a well-conserved compensatory mechanism was proposed: transcriptional adaptation of homologs under the nonsense-mediated mRNA decay pathways. However, this model cannot explain the onset of all compensatory events. We report a novel genetic compensation mechanism operating over the Mesp gene locus. Mesp1 and Mesp2 are homologs located adjacently in the genome. Mesp2 loss is partially rescued by Mesp1 upregulation in the presomitic mesoderm (PSM). Using a cultured PSM induction system, we reproduced the compensatory response in vitro and found that the Mesp2-enhancer is required to promote Mesp1. We revealed that the Mesp2-enhancer directly interacts with the Mesp1 promoter, thereby upregulating Mesp1 expression upon the loss of Mesp2. Of note, this interaction is established by genomic arrangement upon PSM development independently of Mesp2 disruption. We propose that the repurposing of this established enhancer-promoter communication is the mechanism underlying this compensatory response for the upregulation of the adjacent homolog.

category: molecular biology


title: FGF/ERK autocrine signaling is enhanced by NANOG in a subpopulation of pluripotent stem cells to execute autoregulation and induce heterogeneity

abstract: The self-renewal and differentiation potential of Embryonic stem cells (ESCs) is maintained by the regulated expression of core pluripotency factors. The expression level of core pluripotency factor Nanog is tightly regulated by a negative feedback autorepression loop. However, it remains unclear how the ESCs perceive the NANOG levels and execute autorepression. Here, we show that a dose-dependent induction of Fgfbp1 and Fgfr2 by NANOG activates an autocrine mediated ERK signaling in high-Nanog cells to trigger autorepression. pERK recruits NONO to Nanog locus to repress transcription by preventing POL2 loading. The Nanog autorepression process establishes a self-perpetuating NANOG-pERK reciprocal regulatory circuit. We further demonstrate that the reciprocal regulatory circuit induces the pERK heterogeneity and ERK signaling dynamics in pluripotent stem cells.

category: cell biology


title: A NANOG-pERK reciprocal regulatory circuit mediates Nanog autoregulation and ERK signaling dynamics.


title: TLR4 regulation in human fetal membranes as an explicative mechanism of a pathological preterm case.

abstract: The integrity of human fetal membranes is crucial for harmonious fetal development throughout pregnancy. Their premature rupture is often the consequence of a physiological phenomenon previously exacerbated. Beyond all biological processes implied, inflammation is of primary importance and is qualified as "sterile" at the end of pregnancy. Complementary methylomic and transcriptomic strategies on amnion and choriodecidua explants taken from the altered (cervix zone) and intact fetal membranes at term and before labor were used in this study. By cross-analyzing genome-wide studies strengthened by in vitro experiments, we deciphered how the expression of Toll-like receptor 4 (TLR4), a well-known actor of pathological fetal membrane rupture, is controlled. Indeed, it is differentially regulated in the altered zone and between both layers by a dual mechanism: 1) the methylation of TLR4 and miRNA promoters and 2) targeting by miRNA (let-7a-2 and miR-125b-1) acting on the 3-UTR of TLR4. Consequently, this study demonstrates that a fine regulation of TLR4 is required for sterile inflammation establishment at the end of pregnancy and that it may be dysregulated in the pathological premature rupture of membranes.


title: Population structure and environmental niches of Rimicaris shrimps from the Mid Atlantic Ridge

abstract: Among the endemic and specialized fauna from hydrothermal vents, Rimicaris shrimp surely constitutes one of the most important and emblematic components of these ecosystems. In the Mid Atlantic Ridge, two species affiliated to this genus co-occur: Rimicaris exoculata and Rimicaris chacei that differ by their morphology, their trophic regime as well as by their abundance. The first forms large and dense aggregations on active vent chimney walls in close proximity to vent fluid emissions, whereas the second is recognized as much less conspicuous, living mostly in scattered groups or solitary further away from the fluids. However, the recent revision of Rimicaris juvenile stages from the Mid Atlantic Ridge shows that R. chacei abundance would be higher than expected at these early life stages. Here, we describe and compare the population structures of R. exoculata and R. chacei at the Snake Pit and TAG vent fields. We show distinct population demographics between the two co-occurring shrimps with a large post settlement collapse in R. chacei population suggesting a large juvenile mortality for this species. We also observe important spatial segregation patterns between the two species and their different life stages. Additionally, our results highlight distinct niches for the earliest juvenile stages of both R. exoculata and R. chacei, compared to all the other life stages. Finally, we discuss the potential factors - predation and competitive interactions among others - that could explain the differences we observe in the population structure of these two species.

category: ecology


title: Gut microbiota of ring-tailed lemurs (Lemur catta) vary across natural and captive populations and correlate with environmental microbiota

abstract:
BackgroundInter-population variation in host-associated microbiota reflects differences in the hosts environments, but this characterization is typically based on studies comparing few populations. The diversity of natural habitats and captivity conditions occupied by any given host species has not been captured in these comparisons. Moreover, intraspecific variation in gut microbiota, generally attributed to diet, may also stem from differential acquisition of environmental microbes - an understudied mechanism by which host microbiomes are directly shaped by environmental microbes. To more comprehensively characterize gut microbiota in an ecologically flexible host, the ring-tailed lemur (Lemur catta; n = 209), while also investigating the role of environmental acquisition, we used 16S rRNA sequencing of lemur gut and soil microbiota sampled from up to 13 settings, eight in the wilderness of Madagascar and five in captivity in Madagascar or the U.S. Based on matched fecal and soil samples, we used source-sink ecological theory to examine covariation between the two types of consortia.

ResultsThe diversity of lemur gut microbes varied markedly within and between settings. Microbial diversity was not consistently greater in wild than in captive lemurs, indicating that this metric is not necessarily an indicator of host habitat or condition. Variation in microbial composition was inconsistent with either a single, representative gut community for wild conspecifics or a universal  signal of captivity that homogenizes the gut consortia of captive animals. Despite the similar, commercial diets of captive lemurs on both continents, lemurs within Madagascar were compositionally most similar, suggesting that non-dietary factors govern some of the variability. In particular, soil microbial communities were most distinct between the two continents, and there was significant and context-specific covariation between lemur gut and soil microbiota.

ConclusionsAs one of the broadest, single-species investigations of primate microbiota, our study highlights that gut consortia are sensitive to multiple scales of environmental differences. This finding begs a reevaluation of the simple  captive vs. wild dichotomy. Beyond the important implications for animal care, health, and conservation, our finding that environmental acquisition may mediate aspects of host-associated consortia further expands the framework for how host-associated and environmental microbes interact across different microbial landscapes.


title: Fishing for biodiversity

abstract: Fisheries are damaging, and seemingly incompatible with the conservation of marine ecosystems. Yet fish are an important source of food, and support the lives of many people in coastal communities. This paper considers an idea that a moderate intensity of fishing, appropriately scaled across species, could help in maintaining biodiversity, rather than reducing it. The scaling comes from an intuition that rates of fishing mortality of species should be kept in line with production rates of the species, a notion known as balanced harvesting. This places species conservation and exploitation on an equal footing in a single equation, showing quantitatively the relative levels of fishing mortality that species of different abundance can support. Using a dynamic model of a marine ecosystem, we give numerical evidence showing for the first time that fishing, if scaled in this way, can protect rarer species, while allowing some exploitation of species with greater production. This works because fishing mortality rates, when scaled by production, are density-dependent. Such fishing, operating adaptively to follow species production rates over time, contains a feedback that would help to protect species from overfishing in the presence of uncertainty about how marine ecosystems work.


title: Fishing for biodiversity by balanced harvesting


title: Single-cell reconstitution reveals persistence of clonal heterogeneity in the murine hematopoietic system

abstract: The persistence of patterns of monoallelic expression is a controversial matter. We report a genome-wide in vivo transcriptomics approach based on allelic expression imbalance to evaluate whether the transcriptional allelic patterns of single murine hematopoietic stem cells (HSC) are still present in the respective differentiated clonal B-cell populations. For 14 genes, we show conclusive evidence for a remarkable persistence in HSC-derived B clonal cells of allele-specific autosomal transcriptional states already present in HSCs. In a striking contrast to the frequency of genes with clonal allelic expression differences in clones expanded without differentiation (up to 10%), we find that clones that have undergone multiple differentiation steps in vivo are more similar to each other. These data suggest that most of the random allele-specific stable transcriptional states on autosomal chromosomes are established de novo during cell lineage differentiation. Given that allele-specific transcriptional states are more stable in cells not undergoing extensive differentiation than in the clones we assessed after full lineage differentiation in vivo, we introduce the "Punctuated Disequilibria" model: random allelic expression biases are stable if the cells are not undergoing differentiation, but may change during differentiation between developmental stages and reach a new stable equilibrium that will only be challenged if the cell engages in further differentiation. Thus, the transcriptional allelic states may not be a stable feature of the differentiating clone, but phenotypic diversity between clones of a population at any given stage of the cell lineage is still ensured.

category: genetics


title: Contrasting a reference cranberry genome to a crop wild relative provides insights into adaptation, domestication, and breeding

abstract: Cranberry (Vaccinium macrocarpon) is a member of the Heath family (Ericaceae) and is a temperate low-growing woody perennial native to North America that is both economically important and has significant health benefits. While some native varieties are still grown today, breeding programs over the past 50 years have made significant contributions to improving disease resistance, fruit quality and yield. An initial genome sequence of an inbred line of the wild selection  Ben Lear, which is parent to multiple breeding programs, provided insight into the gene repertoire as well as a platform for molecular breeding. Recent breeding efforts have focused on leveraging the circumboreal V. oxycoccos, which forms interspecific hybrids with V. macrocarpon, offering to bring in novel fruit chemistry and other desirable traits. Here we present an updated, chromosome-resolved V. macrocarpon reference genome, and compare it to a high-quality draft genome of V. oxycoccos. Leveraging the chromosome resolved cranberry reference genome, we confirmed that the Ericaceae has undergone two whole genome duplications that are shared with blueberry and rhododendron. Leveraging resequencing data for  Ben Lear inbred lines, as well as several wild and elite selections, we identified common regions that are targets of improvement. These same syntenic regions in V. oxycoccos, were identified and represent environmental response and plant architecture genes. These data provide insight into early genomic selection in the domestication of a native North American berry crop.

category: genomics


title: LIM domain-wide comprehensive mutagenesis reveals the role of leucine in CSRP3 protein stability

abstract: Cardiomyopathies are a severe and chronic cardiovascular burden worldwide, affecting a large cohort in the general population. Cysteine and glycine-rich protein 3 (CSRP3) is one of key proteins implicated in dominant dilated cardiomyopathy (DCM) and hypertrophic cardiomyopathy (HCM). In this study, we device a rapid in-silico screening protocol that creates a mutational landscape map for all possible allowed and disallowed substitutions in the protein of interest. This map provides the structural and functional insights on the stability of LIM domains of CSRP3. Further, the sequence analysis delineates the eukaryotic CSRP3 protein orthologs which complements the mutational map. Next, we also evaluated the effect of HCM/DCM mutations on these domains. One of highly destabilising mutations - L44P (also disease causing) and a neutral mutation - L44M were further subjected to molecular dynamics (MD) simulations. The results establish that L44P substitution affects the LIM domain structure. The present study provides a useful perspective to our understanding of the role of mutations in the CSRP3 LIM domains and their evolution. Experimentally verifying every reported mutation can become challenging both in time and resources used. This study provides a novel screening method for quick identification of key mutation sites for specific protein structures that can reduce the burden on experimental research.

category: bioinformatics


title: Predicting and interpreting large scale mutagenesis data using analyses of protein stability and conservation

abstract: Understanding and predicting the functional consequences of single amino acid is central in many areas of protein science. Here we collected and analysed experimental measurements of effects of >150,000 variants in 29 proteins. We used biophysical calculations to predict changes in stability for each variant, and assessed them in light of sequence conservation. We find that the sequence analyses give more accurate prediction of variant effects than predictions of stability, and that about half of the variants that show loss of function do so due to stability effects. We construct a machine learning model to predict variant effects from protein structure and sequence alignments, and show how the two sources of information are able to support one another. Together our results show how one can leverage large-scale experimental assessments of variant effects to gain deeper and general insights into the mechanisms that cause loss of function.

category: biophysics


title: GTL1 is required for a robust root hair growth response to avoid nutrient overloading

abstract:
O_LIRoot hair growth is tuned in response to the environment surrounding plants. While most of previous studies focused on the enhancement of root hair growth during nutrient starvation, few studies investigated the root hair response in the presence of excess nutrients.
C_LIO_LIWe report that the post-embryonic growth of wild-type Arabidopsis plants is strongly suppressed with increasing nutrient availability, particularly in the case of root hair growth. We further used gene expression profiling to analyze how excess nutrient availability affects root hair growth, and found that RHD6 subfamily genes, which are positive regulators of root hair growth, are down-regulated in this condition.
C_LIO_LIOn the other hand, defects in GTL1 and DF1, which are negative regulators of root hair growth, cause frail and swollen root hairs to form when excess nutrients are supplied. Additionally, we observed that the RHD6 subfamily genes are mis-expressed in gtl1-1 df1-1. Furthermore, overexpression of RSL4, an RHD6 subfamily gene, induces swollen root hairs in the face of a nutrient overload, while mutation of RSL4 in gtl1-1 df1-1 restore root hair swelling phenotype.
C_LIO_LIIn conclusion, our data suggest that GTL1 and DF1 prevent unnecessary root hair formation by repressing RSL4 under excess nutrient conditions.
C_LI

category: plant biology


title: Herbivore feeding behavior validates optimal defense theory for specialized metabolites within plants

abstract: Numerous plants protect themselves from attackers using specialized metabolites. The biosynthesis of these deterrent, often toxic metabolites is costly, as their synthesis diverts energy and resources on account of growth and development. How plants diversify investments into growth and defense is explained by the optimal defense theory. The central prediction of the optimal defense theory is that plants maximize growth and defense by concentrating specialized metabolites in tissues that are decisive for fitness. To date, supporting physiological evidence merely relies on the correlation between plant metabolite distribution and animal feeding preference. Here, we use glucosinolates as a model to examine the effect of changes in chemical defense distribution on actual feeding behavior. Taking advantage of the uniform glucosinolate distribution in transporter mutants, we show that high glucosinolate accumulation in tissues important to fitness protects them by guiding larvae of a generalist herbivore to feed on other tissues. Moreover, we show that mature leaves of Arabidopsis thaliana supply young leaves with glucosinolates to optimize defense against herbivores. Our study provides physiological evidence for the central hypothesis of the optimal defense theory and sheds light on the importance of integrating glucosinolate biosynthesis and transport for optimizing plant defense.


title: Stress decreases serotonin tone in the nucleus accumbens to promote aversion and potentiate cocaine preference via decreased stimulation of 5-HT1B receptors

abstract: Stress-induced release of dynorphins (Dyn) activates kappa opioid receptors (KOR) in monoaminergic neurons to produce dysphoria and potentiate drug reward; however, the circuit mechanisms responsible for this effect are not known. We found that conditional deletion of KOR from Slc6a4 (SERT)-expressing neurons blocked stress-induced potentiation of cocaine conditioned place preference (CPP). Within the dorsal raphe nucleus (DRN), two overlapping populations of KOR-expressing neurons: Slc17a8 (VGluT3) and SERT, were distinguished functionally and anatomically. Optogenetic inhibition of these SERT+ neurons potentiated subsequent cocaine CPP, whereas optical inhibition of the VGluT3+ neurons blocked subsequent cocaine CPP. SERT+/VGluT3- expressing neurons were concentrated in the lateral aspect of the DRN. SERT projections from the DRN were observed in the medial nucleus accumbens (mNAc), but VGluT3 projections were not. Optical inhibition of SERT+ neurons produced place aversion, whereas optical stimulation of SERT+ terminals in the mNAc attenuated stress-induced increases in forced swim immobility and subsequent cocaine CPP. KOR neurons projecting to mNAc were confined to the lateral aspect of the DRN, and the principal source of dynorphinergic (Pdyn) afferents in the mNAc was from local neurons. Excision of Pdyn from the mNAc blocked stress-potentiation of cocaine CPP. Prior studies suggested that stress-induced dynorphin release within the mNAc activates KOR to potentiate cocaine preference by a reduction in 5-HT tone. Consistent with this hypothesis, a transient pharmacological blockade of mNAc 5-HT1B receptors potentiated subsequent cocaine CPP. 5-HT1B is known to be expressed on 5-HT terminals in NAc, and 5-HT1B transcript was also detected in Pdyn+, Adora2a+ and ChAT+ (markers for direct pathway, indirect pathway, and cholinergic interneurons, respectively). Following stress exposure, 5-HT1B transcript was selectively elevated in Pdyn+ cells of the mNAc. These findings suggest that Dyn/KOR regulates serotonin activation of 5HT1B receptors within the mNAc and dynamically controls stress response, affect, and drug reward.

category: neuroscience


title: Modified defence peptides from horseshoe crab target and kill bacteria inside host cells

abstract: Bacteria that occupy an intracellular niche can evade extracellular host immune responses and antimicrobial molecules. In addition to classic intracellular pathogens, other bacteria including uropathogenic Escherichia coli (UPEC) can adopt both extracellular and intracellular lifestyles. UPEC intracellular survival and replication complicates treatment, as many therapeutic molecules do not effectively reach all components of the infection cycle. In this study, we explored cell penetrating antimicrobial peptides from distinct structural classes as alternative molecules for targeting bacteria. We identified two {beta}-hairpin peptides from the horseshoe crab, tachyplesin I and polyphemusin I, with broad antimicrobial activity toward a panel of pathogenic and non-pathogenic bacteria in planktonic form. Peptide analogues [I11A]tachyplesin I and [I11S]tachyplesin I maintained activity toward bacteria, but were less toxic to mammalian cells than native tachyplesin I. This important increase in therapeutic window allowed treatment with higher concentrations of [I11A]tachyplesin I and [I11S]tachyplesin I, to significantly reduce intramacrophage survival of UPEC in an in vitro infection model. Mechanistic studies using bacterial cells, model membranes and cell membrane extracts, suggest that tachyplesin I and polyphemusin I peptides kill UPEC by selectively binding and disrupting bacterial cell membranes. Moreover, treatment of UPEC with sublethal peptide concentrations increased zinc toxicity and enhanced innate macrophage antimicrobial pathways. In summary, our combined data show that cell penetrating peptides are attractive alternatives to traditional small molecule antibiotics for treating UPEC infection, and that optimization of native peptide sequences can deliver effective antimicrobials for targeting bacteria in extracellular and intracellular environments.

category: microbiology


title: Population structure and reproduction of the alvinocaridid shrimp Rimicaris exoculata on the Mid-Atlantic Ridge: variations between habitats and vent fields

abstract: Rimicaris exoculata is a dominant species of deep Mid-Atlantic Ridge (MAR) vent fields and inhabits areas close to vent emissions at depths below 2000 m. Its high abundance and strong genetic connectivity along the MAR point at a remarkable ability to produce dispersing larval stages. However, the reproduction of this species long remained enigmatic because brooding females were rarely observed. Here, we describe the population structure and reproduction of R. exoculata at the Snake Pit and TAG vent fields (3600m depth) for the months of January-February. We observed major differences in population structure between habitats within a single vent field: females widely dominate the large swarms near active venting while inactive peripheries are inhabited by large males. Low temperature diffusion zones are mainly colonized by small juveniles of R. chacei instead of R. exoculata. Size structure of populations from dense active areas is polymodal at both fields, suggesting discontinuous recruitment. Male and female sizes did not vary across habitats and vent fields, with sexually mature female being slightly larger than males. In contrast to previous studies, hundreds of ovigerous females were observed at both vent fields, suggesting seasonal reproduction. Proportion of ovigerous females among sexually mature females were similar between vent fields (36.7 %). However, reproductive output was lower at TAG, where ovigerous females had smaller size-specific fecundity and egg size, and more aborted broods. Broods were colonized by the dirivultid copepod Stygiopontius pectinatus at both vent fields, apparently without deleterious effect on egg development. In the light of the observed variability in R. exoculata population structure, we propose a hypothetical scenario depicting its mating system and brooding behavior, and discuss more generally intraspecific interactions during its benthic life stages.


title: CTCF and transcription influence chromatin structure re-configuration after mitosis

abstract: During mitosis, transcription is globally attenuated and chromatin architecture is dramatically reconfigured. Here we exploited the M- to G1-phase progression to interrogate the contributions of the architectural factor CTCF and the process of transcription to re-sculpting the genome in newborn nuclei. Depletion of CTCF specifically during the M- to G1-phase transition altered the re-establishment of local short-range compartmentalization after mitosis. Chromatin domain boundary reformation was impaired upon CTCF loss, but a subset ([~]27%) of boundaries, characterized by transitions in chromatin states, was established normally. Without CTCF, structural loops failed to form, leading to illegitimate contacts between cis-regulatory elements (CREs). Transient CRE contacts that are normally resolved after telophase persisted deeply into G1-phase in CTCF depleted cells. CTCF loss-associated gains in transcription were often linked to increased, normally illegitimate enhancer-promoter contacts. In contrast, at genes whose expression declined upon CTCF loss, CTCF seems to function as a conventional transcription activator, independent of its architectural role. CTCF-anchored structural loops facilitated formation CRE loops nested within them, especially those involving weak CREs. Transcription inhibition did not elicit global architectural changes and left transcription start site-associated boundaries intact. However, ongoing transcription contributed considerably to the formation of gene domains, regions of enriched contacts spanning the length of gene bodies. Notably, gene domains formed rapidly in ana/telophase prior to the completion of the first round of transcription, suggesting that epigenetic features in gene bodies contribute to genome reconfiguration prior to transcription. The focus on the de novo formation of nuclear architecture during G1 entry yielded novel insights into how CTCF and transcription contribute to the dynamic re-configuration of chromatin architecture during the mitosis to G1 phase progression.


title: Patient-derived tumor spheroid cultures as a promising tool to assist personalized therapeutic decisions in breast cancer

abstract:
Breast cancer is the most common cause of cancer related death in women. Treatment of breast cancer has many limitations including a lack of accurate biomarkers to predict success of chemotherapy and intrinsic resistance of a significant group of patients to the gold standard of therapy. Therefore, new tools are needed to provide doctors with guidance in choosing the most effective treatment plan for a particular patient and thus to increase the survival rate for breast cancer patients.

Here, we present a successful method to grow in vitro spheroids from primary breast cancer tissue. Samples were received in accordance with relevant ethical guidelines and regulations. After tissue dissociation, in vitro spheroids were generated in a scaffold-free 96-well plate format. Spheroid composition was investigated by immunohistochemistry (IHC) of epithelial (Pan Cytokeratin (panCK)), stromal (Vimentin) and breast cancer-specific markers (ER, PR, HER2, GATA-3). Growth and cell viability of the spheroids were assessed upon treatment with multiple anti-cancer compounds. Students t-test and two-way ANOVA test were used to determine statistical significance.

We were able to successfully grow spheroids from 27 out of 31 samples from surgical resections of breast cancer tissue from previously untreated patients. Recapitulation of the histopathology of the tissue of origin was confirmed. Furthermore, a drug panel of standard first- and second-line chemotherapy drugs used to treat breast cancer was applied to assess the viability of the patient-derived spheroids and revealed variation between samples in the response of the spheroids to different drug treatments.

We investigated the feasibility and the utility of an in vitro patient-derived spheroid model for breast cancer therapy, and we conclude that spheroids serve as a highly effective platform to explore cancer therapeutics and personalized treatment efficacy. These results have significant implications for the application of this model in clinical personalized medicine.

category: cancer biology


title: Mutation in Abl kinase with altered drug binding kinetics indicates a novel mechanism of imatinib resistance

abstract:
Protein kinase inhibitors are potent anti-cancer therapeutics (1). For example, the Bcr-Abl kinase inhibitor imatinib decreases mortality for Chronic Myeloid Leukemia (CML) by 80% (2, 3), but 22-41% of patients acquire resistance to imatinib (4). About 70% of relapsed patients harbor mutations in the Bcr-Abl kinase domain (5), in which more than a hundred different mutations have been identified (6-8). Some mutations are located near the imatinib binding site and cause resistance through altered interactions with the drug. However, many resistance mutations are located far from the drug binding site (9) and it remains unclear how these mutations confer resistance. Additionally, earlier studies on small sets of patient-derived imatinib resistance mutations indicated that some of these mutant proteins were in fact sensitive to imatinib in cellular and biochemical studies (10). Here, we surveyed the resistance of 94 patient-derived Abl kinase domain mutations annotated as disease-relevant or resistance-causing using an engagement assay in live cells. We found that only two-thirds of mutations weaken imatinib affinity by more than two-fold compared to Abl wild type. Surprisingly, one-third of mutations in Abl kinase domain still remain sensitive to imatinib and bind with similar or higher affinity than wild type. Intriguingly, we identified a clinical Abl mutation that binds imatinib with wild type-like affinity but dissociates from imatinib three times faster. Given the relevance of residence time for drug efficacy (11-14), mutations that alter binding kinetics could cause resistance in the non-equilibrium environment of the body where drug export and clearance play critical roles.

SignificanceWe performed the first in cell screen of imatinib binding against a library of Abl kinase mutants derived from patients with imatinib-resistant CML. The majority of mutations readily bind imatinib, posing the question of how these mutations cause resistance in patients. We identified a kinetic mutant that binds imatinib with wild type affinity but dissociates considerably faster from the mutant kinase. Using NMR and molecular dynamics, we found that this mutation increases the conformational dynamics of the mutant protein, linking conformational dynamics of the protein to drug dissociation. The results underline the importance of drug dissociation kinetics for drug efficacy and propose a novel kinetic resistance mechanism that may be targetable by altering drug treatment schedules.

category: biochemistry


title: Pan-cancer landscape of AID-related mutations, composite mutations and its potential role in the ICI response

abstract:
Activation-induced cytidine deaminase, AICDA or AID, is a driver of somatic hypermutation and class-switch recombination in immunoglobulins. In addition, this deaminase belonging to the APOBEC family, may have off-target effects genome-wide, but its effects at pan-cancer level are not well elucidated. Here, we used different pan-cancer datasets, totaling more than 50,000 samples analyzed by whole-genome, whole-exome or targeted sequencing. AID synergizes initial hotspot mutations by a second composite mutation. Analysis of 2.5 million cells, normal and oncogenic, revealed AICDA expression activation after oncogenic transformation and cell cycle regulation loss. AID mutational load was found to be independently associated with favorable outcome in immune-checkpoint inhibitors (ICI) treated patients across cancers after analyzing 2,000 samples. Finally, we found that AID related neoepitopes, resulting from mutations at more frequent hotspots if compared to other mutational signatures, enhance CXCL13/CCR5 expression, immunogenicity and T-cell exhaustion, which may increase ICI sensitivity.

In BriefA combined bulk and single cell multi-omic analysis of over 50,000 patients and 2.5 million cells across 80 tumor types reveals oncogenic acquired AICDA expression inducing composite mutations and clonal immunogenic neoepitopes that are associated with favorable outcome in patients treated by immune-checkpoint inhibitors.

Highlights* Pan-cancer analysis of AID mutations using > 50,000 samples, 2,000 ICI treated cases and 2.5 million cells with genome, exome and transcriptome data
* Oncogenic transient AICDA expression induces mutations mainly during transcription of its off-target genes in virtually all cancers
* AID is implicated in composite mutations on weakly functional alleles and immunogenic clonal neoepitopes at hotspots with greater positive selection
* AID mutational load predicts response and is associated with favorable outcome in ICI treated patients


title: TRIOBP promotes bidirectional radial stiffness gradients within the organ of Corti

abstract: Hearing depends on complex mechanical properties of the inner ear sensory epithelium. Yet, the individual contributions of different cell types to the stiffness spectrum of the sensory epithelium have not been thoroughly investigated. Using sub-100 nanometer spatial resolution PeakForce Tapping Atomic Force Microscopy (PFT-AFM), we mapped the Youngs modulus (stiffness) of the apical surface of different cells of freshly-dissected cochlear epithelium from wild-type mice and mice lacking the F-actin bundling protein TRIOBP-5 or TRIOBP-4 and TRIOBP-5. Variants of the genes encoding human and mouse TRIOBP are associated with deafness. We show that TRIOBP deficiency affects formation of supporting cell apical phalangeal microfilaments and bundled cortical F-actin of hair cell cuticular plates, softening the apical surface of the sensory epithelium. Unexpectedly, high-resolution PFT-AFM-mapping also revealed previously unrecognized reticular lamina radial stiffness gradients of opposite orientations in wild-type supporting and hair cells. Deafness-associated TRIOBP deficiencies significantly modified these bidirectional radial stiffness gradients.


title: Relaxin/insulin-like family peptide receptor 4 (Rxfp4) expressing hypothalamic neurons modulate food intake and preference in mice

abstract: Relaxin/insulin-like-family peptide receptor-4 (RXFP4), the cognate receptor for insulin-like peptide 5 (INSL5), has been implicated in feeding behaviour as Rxfp4 knockout mice display shorter meal durations and reduced high fat diet (HFD) intake. Here, we generated transgenic Rxfp4-Cre mice to explore Rxfp4 expression and physiology. Using this model, we identified Rxfp4 expression in the central nervous system, including in the ventromedial hypothalamus (VMH). Intra-VMH infusion of INSL5 increased HFD and highly palatable liquid meal intake (HPM) of ad libitum fed wildtype mice. Single-cell RNA-sequencing of VMH Rxfp4-expressing cells (RXFP4VMH) defined a cluster of Rxfp4-labelled neurons expressing Esr1, Tac1 and Oxtr, alongside known appetite-modulating neuropeptide receptors (Mc4r, Cckar and Nmur2). Viral tracing demonstrated RXFP4VMH neural projections to the bed nucleus of the stria terminalis, paraventricular hypothalamus, paraventricular thalamus and central nucleus of the amygdala. Utilising designer receptors exclusively activated by designer drugs (DREADDs), we found that whole body chemogenetic inhibition (Di) of Rxfp4-expressing cells, mimicking native INSL5-RXFP4 signalling, increased intake of HFD and HPM, whilst activation (Dq), either at whole body level or specifically within the VMH, reduced HFD and HPM intake and altered food preference. Ablating VMH Rxfp4-expressing cells recapitulated the lower HFD intake phenotype of Rxfp4 knockout mice, resulting in reduced body weight. These findings identify a discrete Rxfp4-expressing neuronal population as a key regulator of food intake and preference and reveal hypothalamic RXFP4 signalling as a target for feeding behaviour manipulation.

category: physiology


title: Genome-wide identification, expression and bioinformatic analyses of GRAS transcription factor genes in rice

abstract: Our group has previously identified the activation tagging of a GRAS transcription factor (TF)gene in the gain-of-function mutant population of rice (indica rice variety BPT 5204) screened for water use efficiency (Moin et al, 2016a). This family of GRAS transcription factors has been well known for their diverse roles in gibberellin signaling, light responses, root development, gametogenesis etc. Recent studies indicated their role in biotic and abiotic responses as well. Although this family of TFs received significant attention, not many genes were identified specifically for their roles in mediating stress tolerance in rice. Only OsGRAS23 (here named as OsGRAS22) was reported to code for a TF that induces drought tolerance in rice. In the present study, we have analyzed the expression patterns of rice GRAS TF genes under abiotic (NaCl and ABA treatments) and biotic (leaf samples infected with pathogens, Xanthomonas oryzae pv. oryzae that causes bacterial leaf blight and Rhizoctonia solani that causes sheath blight) stress conditions. In addition, their expression patterns were also analyzed in thirteen different developmental stages. We studied their spatio-temporal regulation and correlated them with in-silico studies. Fully annotated genomic sequences available in rice database have enabled us to study the protein properties, ligand interactions, domain analysis and presence of cis-regulatory elements in a bioinformatics analysis. Most of the genes were induced immediately after the onset of stress particularly in the roots of ABA treated plants. OsGRAS39 was found to be very highly expressive gene under sheath blight infection and both abiotic stress treatments while OsGRAS8, OsSHR1 and OsSLR1 were also responsive. Our earlier functional characterization (Moin et al., 2016a) followed by the genome wide characterization of the GRAS gene family members in the present study clearly show that they are highly appropriate candidate genes for manipulating stress tolerance in rice and other crop plants.


title: Developmental Effects on Relative Use of PEPCK and NADP-ME Pathways of C4 Photosynthesis in Maize

abstract:
C4 photosynthesis is an adaptive photosynthetic pathway which concentrates CO2 around Rubisco in specialized bundle sheath cells to reduce photorespiration. Historically, the pathway has been characterized into three different subtypes based on the decarboxylase involved, although recent work has provided evidence that some plants can use multiple decarboxylases, with maize in particular using both the NADP-malic enzyme (NADP-ME) pathway and phosphoenolpyruvate carboxykinase (PEPCK) pathway. Parallel C4 pathways could be advantageous in balancing energy and reducing equivalents between bundle sheath and mesophyll cells, in decreasing the size of the metabolite gradients between cells and may better accommodate changing environmental conditions or source to sink demands on growth. The enzyme activity of C4 decarboxylases can fluctuate with different stages of leaf development, but it remains unclear if the pathway flexibility is an innate aspect of leaf development or an adaptation to the leaf microenvironment that is regulated by the plant. In this study, variation in the two C4 pathways in maize were characterized at nine plant ages throughout the life cycle. Two positions in the canopy were examined for variation in physiology, gene expression, metabolite concentration, and enzyme activity, with particular interest in asparagine as a potential regulator of C4 decarboxylase activity. Variation in C4 and C3 metabolism was observed for both leaf age and canopy position, reflecting the ability of C4 pathways to adapt to changing microenvironments.

One Sentence SummaryThe proportion of the two C4 pathways in maize plants is dependent on canopy position and not the age of the leaf.


title: Embedded control of cell growth using tunable genetic systems

abstract: We present an embedded feedback control strategy to control the density of a bacterial population, allowing cells to self-regulate their growth rate so as to reach a desired density at steady state. We consider a static culture condition, where cells are provided with a limited amount of space and nutrients. The control strategy is built using a tunable expression system (TES), which controls the production of a growth inhibitor protein, complemented with a quorum sensing mechanism for the sensing of the population density. We show on a simplified population-level model that the TES endows the control system with additional flexibility by allowing the set-point to be changed online. Finally, we validate the effectiveness of the proposed control strategy by means of realistic in silico experiments conducted in BSim, an agent-based simulator explicitly designed to simulate bacterial populations, and we test the robustness of our design to disturbances and parameters variations due, for instance, to cell-to-cell variability.

category: synthetic biology


title: LncRNAs of Saccharomyces cerevisiae dodge the cell cycle arrest imposed by the ethanol stress

abstract: Ethanol impairs many subsystems of Saccharomyces cerevisiae, including the cell cycle. Cyclins and damage checkpoints drive the cell cycle. Two ethanol-responsive lncRNAs in yeast interact with cell cycle proteins, and here we investigated the role of these RNAs on the ethanol-stressed cell cycle. Our network dynamic modeling showed that the higher and lower ethanol tolerant strains undergo a cell cycle arrest during the ethanol stress. However, lower tolerant phenotype arrest in a later phase leading to its faster population rebound after the stress relief. Two lncRNAs can skip the arrests mentioned. The in silico overexpression of lnc9136 of SEY6210 (a lower tolerant strain), and CRISPR-Cas9 partial deletions of this lncRNA, evidenced that the one induces a regular cell cycle even under ethanol stress; this lncRNA binds to Gin4 and Hsl1, driving the Swe1p, Clb1/2, and cell cycle. Moreover, the lnc10883 of BY4742 (a higher tolerant strain) interacts to the Mec1p and represses Bub1p, circumventing the DNA and spindle damage checkpoints keeping a normal cell cycle even under DNA damage. Overall, we present the first evidence of the direct roles of lncRNAs on cell cycle proteins, the dynamics of this system in different ethanol tolerant phenotypes, and a new cell cycle model.

category: systems biology


title: Sparse connectivity for MAP inference in linear models using sister mitral cells

abstract:
Sensory processing is hard because the variables of interest are encoded in spike trains in a relatively complex way. A major goal in studies of sensory processing is to understand how the brain extracts those variables. Here we revisit a common encoding model in which variables are encoded linearly. Although there are typically more variables than neurons, this problem is still solvable because only a small number of variables appear at any one time (sparse prior). However, previous solutions require all-to-all connectivity, inconsistent with the sparse connectivity seen in the brain. Here we propose an algorithm that provably reaches the MAP (maximum a posteriori) inference solution, but does so using sparse connectivity. Our algorithm is inspired by the circuit of the mouse olfactory bulb, but our approach is general enough to apply to other modalities. In addition, it should be possible to extend it to nonlinear encoding models.

SummarySensory systems must infer latent variables from noisy and ambiguous input. MAP inference - choosing the most likely latent variable given the sensory input - is one of the simplest methods for doing that, but its neural implementation often requires all-to-all connectivity between the neurons involved. In common sensory contexts this can require a single neuron to connect to hundreds of thousands of others, which is biologically implausible. In this work we take inspiration from the  sister mitral cells of the olfactory system - groups of neurons associated with the same input channel - to derive a method for performing MAP inference using sparse connectivity. We do so by assigning sister cells to random subsets of the latent variables and using additional cells to ensure that sisters correctly share information. We then derive the circuitry and dynamics required for the sister cells to compute the original MAP inference solution. Our work yields a biologically plausible circuit that provably solves the MAP inference problem and provides experimentally testable predictions. While inspired by the olfactory system, our method is quite general, and is likely to apply to other sensory modalities.


title: Residual errors in visuomotor adaptation persist despite extended motor preparation periods

abstract:
A consistent finding in sensorimotor adaptation is a persistent undershoot of full compensation, such that performance asymptotes with residual errors greater than seen at baseline. This behaviour has been attributed to limiting factors within the implicit adaptation system, which reaches a sub-optimal equilibrium between trial-by-trial learning and forgetting. However, recent research has suggested that allowing longer motor planning periods prior to movement eliminates these residual errors. The additional planning time allows required cognitive processes to be completed before movement onset, thus increasing accuracy. Here we looked to extend these findings by investigating the relationship between increased motor preparation time and the size of imposed visuomotor rotation (30{degrees}, 45{degrees} or 60{degrees}), with regards to the final asymptotic level of adaptation. We found that restricting preparation time to 0.35 seconds impaired adaptation for moderate and larger rotations, resulting in larger residual errors compared to groups with additional preparation time. However, we found that even extended preparation time failed to eliminate persistent errors, regardless of magnitude of cursor rotation. Thus, the asymptote of adaptation was significantly less than the degree of imposed rotation, for all experimental groups. Additionally, there was a positive relationship between asymptotic error and implicit retention. These data suggest that a prolonged motor preparation period is insufficient to reliably achieve complete adaptation and therefore our results provide support for the proposal that limitations within the implicit learning system contributes to asymptotic adaptation levels.

New & NoteworthyResidual errors in sensorimotor adaptation are commonly attributed to an equilibrium between trial-by-trial learning and forgetting. Recent research suggested that allowing sufficient time for mental rotation eliminates these errors. In a number of experimental conditions, we show that while restricted motor preparation time does limit adaptation - consistent with mental rotation - extending preparation time fails to eliminate the residual errors in motor adaptation.


title: Dynamic causal communication channels between neocortical areas

abstract: Dynamic pathways of information flow between distributed brain regions underlie the diversity of behaviour. However, it remains unclear how neuronal activity in one area causally influences ongoing population activity in another, and how such interactions change over time. Here we introduce a causal approach to quantify cortical interactions by pairing simultaneous electrophysiological recordings with neural perturbations. We found that the influence visual cortical areas had on each other was surprisingly variable over time. Both feedforward and feedback pathways reliably affected different subpopulations of target neurons at different moments during processing of a visual stimulus, resulting in dynamically rotating communication dimensions between the two cortical areas. The influence of feedback on primary visual cortex (V1) became even more dynamic when visual stimuli were associated with a reward, impacting different subsets of V1 neurons within tens of milliseconds. This, in turn, controlled the geometry of V1 population activity in a behaviourally relevant manner. Thus, distributed neural populations interact through dynamically reorganizing and context-dependent communication channels to evaluate sensory information.


title: Circadian modulation of human sleep spindle frequency

abstract: Homeostatic and circadian processes play a pivotal role in determining sleep structure, timing and quality. In sharp contrast with the wide accessibility of the EEG index of sleep homeostasis, an electrophysological measure of the circadian modulation of sleep is still non-available. Evidence suggests that sleep spindle frequencies decelerate during biological night. In order to test the feasibility of measuring this marker in common polysomnographic protocols, the Budapest-Munich database of sleep records (N = 251 healthy subjects, 122 females, age range: 4-69 years), as well as an afternoon nap sleep record database (N = 112 healthy subjects, 30 females, age range: 18-30 years) were analysed by the Individual Adjustment Method of sleep spindle analysis. Slow and fast sleep spindle frequencies were characterized by U-shaped overnight dynamics, with highest values in the first and the fourth-to-fifth sleep cycle and the lowest values in the middle of the sleeping period (cycles 2-3). Age-related attenuation of sleep spindle deceleration was evident. Estimated phases of the nadirs in sleep spindle frequencies were advanced in children as compared to other age groups. Additionally, nap sleep spindles were faster than night sleep spindles (0.57 and 0.39 Hz difference for slow and fast types, respectively). The fine frequency resolution analysis of sleep spindles is a feasible method of measuring the assumed circadian modulation of sleep. Moreover, age-related attenuation of circadian sleep modulation might be measurable by assessing the overnight dynamics in sleep spindle frequency. Phase of the minimal sleep spindle frequency is a putative biomarker of chronotype.


title: Sleep spindle frequency: overnight dynamics, afternoon nap effects, and possible circadian modulation


title: Cerebello-cerebral Functional Connectivity Networks in Major Depressive Disorder: A CAN-BIND-1 Study Report

abstract:
ObjectiveNeuroimaging studies have demonstrated aberrant structure and function of the "cognitive-affective cerebellum" in Major Depressive Disorder (MDD), although the specific role of the cerebello-cerebral circuitry in this population remains largely uninvestigated. The objective of this study was to delineate the role of cerebellar functional networks in depression.

MethodsA total of 308 unmedicated participants completed resting-state functional magnetic resonance imaging scans, of which 247 (148 MDD; 99 Healthy Controls, HC) were suitable for this study. Seed-based resting-state functional connectivity (RsFc) analysis was performed using three cerebellar regions of interest (ROIs): ROI1 corresponded to default mode network (DMN) / inattentive processing; ROI2 corresponded to attentional networks including frontoparietal, dorsal attention, and ventral attention; ROI3 corresponded to motor processing. These ROIs were delineated based on prior functional gradient analyses of the cerebellum. A general linear model was used to perform within-group and between-group comparisons.

ResultsIn comparison to HC, participants with MDD displayed increased RsFc within the cerebello-cerebral DMN (ROI1) and significantly elevated RsFc between the cerebellar ROI1 and bilateral angular gyrus at a voxel threshold (p < 0.001, two-tailed) and at a cluster level (p < 0.05, FDR-corrected). Group differences were non-significant for ROI2 and ROI3.

ConclusionsThese results contribute to the development of a systems neuroscience approach to the diagnosis and treatment of MDD. Specifically, our findings confirm previously reported associations between MDD, DMN, and cerebellum, and highlight the promising role of these functional and anatomical locations for the development of novel imaging-based biomarkers and targets for neuromodulation therapies.


title: A Reevaluation of the Effect of Dietary Restriction on Different Recombinant Inbred (RI) Lines of Male and Female Mice

abstract:
Dietary restriction (DR) was reported to either have no effect or reduced the lifespan of the majority of the 41-recombinant inbred (RI)-lines studied (Liao et al., 2010). In an appropriately power longevity study (n > 30 mice/group), we measured the lifespan of the four RI-lines (115-RI, 97-RI, 98-RI, and 107-RI) that were reported to have the greatest decrease in lifespan when fed 40% DR. DR increased the median lifespan of female and male 115-RI mice and female 97-RI and 107-RI mice. DR had little effect (less than 4%) on the median lifespan of female and male 98-RI mice and male 97-RI mice and reduced the lifespan of male 107-RI mice over 20%. While our study was unable to replicate the effect of DR on the lifespan of the RI-mice (except male 107-RI mice) reported by Liao et al. (2010), we found that the genotype of a mouse had a major impact on the effect of DR on lifespan, with the effect of DR ranging from a 50% increase to a 22% decrease. No correlation was observed between the changes in either body composition or glucose tolerance induced by DR and the changes observed in lifespan of the four RI-lines of male and female mice. These four RI-lines of mice give the research community a unique resource where investigators for the first time can study the anti-aging mechanism of DR by comparing mice in which DR increases lifespan to mice where DR has either no effect or reduces lifespan.

Graphical Abstract

O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=112 SRC="FIGDIR/small/449984v1_ufig1.gif" ALT="Figure 1">
View larger version (11K):
org.highwire.dtl.DTLVardef@1a11d22org.highwire.dtl.DTLVardef@7fd855org.highwire.dtl.DTLVardef@153b5bborg.highwire.dtl.DTLVardef@c013f3_HPS_FORMAT_FIGEXP  M_FIG C_FIG Brief AbstractIn this study we reevaluate the effect of the genotype and sex of the recombinant inbred strains of mice on their response to dietary restriction. Out of the eight lines studied, half of the groups showed an increase in lifespan when the diet was restricted by 40% and the other half either did not respond or showed a decrease in lifespan. The recombinant inbred lines used in this study are potentially important resource for aging research to identify pathways and understand the mechanism involved in the anti-aging action of DR.


title: The effect of Stachys inflata on the performance of broiler chickens and Newcastle Disease Antibody Titer

abstract: This study shows how performance and NDHI titers of broiler chickens are affected by the plant Stachys inflatay. One hundred and eighty, one day old Ross male broiler chickens were randomly divided into 5 groups of 36 (consisting of 3 replicates) and placed in 12 separate pens. Groups 1, 2, 3, 4 and 5 received 0, 0.15, 0.3, 0.45 and 0.6% S.inflata in their feed respectively from 8 to 42 days of age. Performance indexes were recorded on a weekly basis. At 42 days of age, serum and mid gut samples were taken for NDHI test and histopathological studies. The results were analyzed by the One Way ANOVA, Tukey test. The body weight of the second group was significantly (P [≤] 0.05) higher than the control from the third week to the end. All treatment groups had HI titers higher than the control but the differences were not significant (P > 0.05). In the third group (0.3% S.inflata), relative liver and heart weight were significantly lower than control. No microscopic lesions were observed in mid gut in all experimental groups. It seems that S. inflata reduces harmful intestinal flora which causes better use of feed.

category: developmental biology


title: Detecting quantitative trait loci and exploring chromosomal pairing in autopolyploids using polyqtlR

abstract:
MotivationThe investigation of quantitative trait loci (QTL) is an essential component in our understanding of how organisms vary phenotypically. However, many important crop species are polyploid (carrying more than two copies of each chromosome), requiring specialised tools for such analyses. Moreover, deciphering meiotic processes at higher ploidy levels is not straightforward, but is necessary to understand the reproductive dynamics of these species, or uncover potential barriers to their genetic improvement.

ResultsHere we present polyqtlR, a novel software tool to facilitate such analyses in (auto)polyploid crops. It performs QTL interval mapping in F1 populations of outcrossing polyploids of any ploidy level using identity-by-descent (IBD) probabilities. The allelic composition of discovered QTL can be explored, enabling favourable alleles to be identified and tracked in the population. Visualisation tools within the package facilitate this process, and options to include genetic co-factors and experimental factors are included. Detailed information on polyploid meiosis including prediction of multivalent pairing structures, detection of preferential chromosomal pairing and location of double reduction events can be performed.

Availability and implementationpolyqtlR is freely available from the Comprehensive R Archive Network (CRAN) at http://cran.r-project.org/package=polyqtlR.

Contactpeter.bourke@wur.nl or chris.maliepaard@wur.nl

Supplementary informationSupplementary data are available


title: Analysis at single-cell resolution identifies a stable mammalian tRNA-mRNA interface and increased translation efficiency in neurons

abstract: The correlation between codon and anticodon pools influences the efficiency of translation, but whether differences exist in these pools across individual cells is unknown. We determined that codon usage and amino acid demand are highly stable across different cell types using single-cell RNA-sequencing atlases of adult mouse and fetal human. After demonstrating the robustness of ATAC-sequencing for analysis of tRNA gene usage, we quantified anticodon usage and amino acid supply in adult mouse and fetal human single-cell ATAC-seq atlases. We found that tRNA gene usage is overall coordinated across cell types, except in neurons which clustered separately from other cell types. Integration of these datasets revealed a strong and statistically significant correlation between amino acid supply and demand across almost all cell types. Neurons have an enhanced translation efficiency over other cell types, driven by an increased supply of tRNAAla (AGC) anticodons. This results in faster decoding of the Ala-GCC codon, as determined by cell-type specific ribosome profiling, and a reduction of tRNAAla (AGC) anticodon pools may be implicated in neurological pathologies. This study, the first such examination of codon usage, anticodon usage, and translation efficiency at single-cell resolution, identifies conserved features of translation elongation across mammalian cellular diversity and evolution.


title: An immune-related E2F gene expression signature provides prognostic information in patients with hepatocellular

abstract: Hepatocellular carcinoma (HCC) is one of the most common primary hepatic malignancies. E2F transcription factors play an important role in the tumorigenesis and progression of HCC, mainly through the RB/E2F pathway. Prognostic models for HCC based on gene signatures have been developed rapidly in recent years; however, their discriminating ability at the single cell level remains elusive, which could reflect the underlying mechanisms driving the sample bifurcation. In this study, we constructed and validated a predictive model based on E2F expression, successfully stratifying HCC patients into two groups with different survival risks. Then we used a single-cell dataset to test the discriminating ability of the predictive model on infiltrating T cells, demonstrating remarkable cellular heterogeneity as well as altered cell fates. We identified distinct cell subpopulations with diverse molecular characteristics. We also found that the distribution of cell subpopulations varied considerably across onset stages among patients, providing fundamental basis for patient-oriented precision evaluation. Moreover, single-sample gene set enrichment analysis revealed that subsets of CD8+ T cells with significantly different cell adhesion level could be associated with different patterns of tumor cell dissemination. Therefore, our findings linked the conventional prognostic gene signature to the immune microenvironment and cellular heterogeneity at the single-cell level, thereby providing deeper insights into the understanding of HCC tumorigenesis.


title: Risk modelling of single cell populations revealed the heterogeneity of immune infiltration in hepatocellular carcinoma


title: ARID1A loss derepresses human endogenous retrovirus-H to modulate BRD4-dependent transcription

abstract: The transposable elements (TEs) through evolutionary exaptation have become an integral part of human genome, offering ample regulatory sequences and shaping chromatin 3D architecture. While the functional impacts of TE-derived sequences on early embryogenesis are recognized, their role in malignancy has only started to emerge. Here we show that many TEs, especially the pluripotency-related endogenous retrovirus H (HERVH), are abnormally activated in colorectal cancer (CRC) samples. The transcriptional upregulation of HERVH is associated with mutations of several tumor suppressors including ARID1A. Knockout of ARID1A in CRC cells leads to increased accessibility at HERVH loci and enhanced transcription, which is dependent on ARID1B. Suppression of HERVH in CRC cells and patient-derived organoids impairs tumor growth. Mechanistically, HERVH transcripts colocalize with nuclear BRD4 foci, modulate their dynamics, and co-regulate many target genes. Altogether, we uncover a critical role for ARID1A in restraining HERVH, which can promote tumorigenesis by stimulating BRD4-dependent transcription when ARID1A is mutated.


title: DeepMP: a deep learning tool to detect DNA base modifications on Nanopore sequencing data

abstract:
MotivationDNA Methylation plays a key role in a variety of biological processes. Recently, Nanopore long-read sequencing has enabled direct detection of these modifications. As a consequence, a range of computational methods have been developed to exploit Nanopore data for methylation detection. However, current approaches rely on a human-defined threshold to detect the methylation status of a genomic position and are not optimized to detect sites methylated at low frequency. Furthermore, most methods employ either the Nanopore signals or the basecalling errors as the model input and do not take advantage of their combination.

ResultsHere we present DeepMP, a convolutional neural network (CNN)-based model that takes information from Nanopore signals and basecalling errors to detect whether a given motif in a read is methylated or not. Besides, DeepMP introduces a threshold-free position modification calling model sensitive to sites methylated at low frequency across cells. We comprehensively benchmarked DeepMP against state-of-the-art methods on E. coli, human and pUC19 datasets. DeepMP outperforms current approaches at read-based and position-based methylation detection across sites methylated at different frequencies in the three datasets.

AvailabilityDeepMP is implemented and freely available under MIT license at github.com/pepebonet/DeepMP

Contactjose.bonet@irbbarcelona.org -- mandiche@kth.se


title: DNA METHYLTRANSFERASE 3 (MET3) is regulated by Polycomb Group complex during Arabidopsis endosperm development

abstract:
Complex epigenetic changes occur during plant reproduction. These regulations ensure the proper transmission of epigenetic information as well as allowing for zygotic totipotency. In Arabidopsis, the main DNA methyltransferase is called MET1 and is responsible for methylating cytosine in the CG context. The Arabidopsis genome encodes for three additional reproduction-specific homologs of MET1, namely MET2a, MET2b and MET3. In this paper, we show that the DNA methyltransferase MET3 is expressed in the seed endosperm and its expression is further restricted to the chalazal endosperm. MET3 is biallelically expressed in the endosperm but displays a paternal expression bias. We found that MET3 expression is regulated by the Polycomb complex proteins FIE and MSI1. Seed development is not impaired in met3 mutant, and we could not observe significant transcriptional changes in met3 mutant. Interestingly, we found that MET3 regulates gene expression in a Polycomb mutant background suggesting a further complexification of the interplay between H3K27me3 and DNA methylation in the seed endosperm.

Key messageThe DNA METHYLTRANSFERASE MET3 is controlled by Polycomb group complex during endosperm development.


title: Rad52 mediates class-switch DNA recombination to IgD

abstract: While the biology of IgD begins to be better understood, the mechanism of expression of this phylogenetically old and highly conserved Ig remains unknown. In B cells, IgD is expressed together with IgM as transmembrane receptor for antigen through alternative splicing of long primary VHDJH-C-s-m-C{delta}-s-m RNAs, which also underpin secreted (s)IgD. IgD is also expressed through class switch DNA recombination (CSR), as initiated by AID-mediated double-strand DNA breaks (DSBs) in S and {sigma}{delta}, and resolution of such DSBs by a still unknown mechanism. This synapses S with {sigma}{delta} region DSB resected ends leading to insertion of extensive S-S junction microhomologies, unlike Ku70/Ku86-dependent NHEJ which resolves DSB blunt ends in CSR to IgG, IgA and IgE with little or no microhomologies. Our previous demonstration of a novel role of Rad52 in a Ku70/Ku86-independent "short-range" microhomology-mediated synapsis of intra-S region DSBs led us to hypothesize that this homologous recombination DNA annealing factor is also involved in short-range microhomology-mediated alternative endjoining (A-EJ) recombination of S with {sigma}{delta}. We found that induction of IgD CSR by selected stimuli downregulated Zfp318 (the suppressor of C-s-m transcription termination), promoted Rad52 phosphorylation and Rad52 recruitment to S and {sigma}{delta}, leading to S-{sigma}{delta} recombination with extensive microhomologies, VHDJH-C{delta}s transcription and sustained IgD secretion. Rad52 ablation in mouse Rad52-/- B cells aborted IgD CSR in vitro and in vivo and dampened the specific IgD antibody response to OVA. Further, Rad52 knockdown in human B cells virtually abrogated IgD CSR. Finally, Rad52 phosphorylation was associated with high levels of IgD CSR and anti-nuclear IgD autoantibodies in lupus-prone mice and lupus patients. Thus, Rad52 effects CSR to IgD through microhomology-mediated A-EJ and in concert with Zfp318 modulation. This is a previously unrecognized, critical and dedicated role of Rad52 in mammalian DNA repair that provides a mechanistic underpinning to CSR A-EJ.

category: immunology


title: A bacterial extracellular vesicle-based intranasal vaccine against SARS-CoV-2

abstract: Several vaccines have been introduced to combat the coronavirus infectious disease-2019 (COVID-19) pandemic, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Current SARS-CoV-2 vaccines include mRNA-containing lipid nanoparticles or adenoviral vectors that encode the SARS-CoV-2 Spike (S) protein of SARS-CoV-2, inactivated virus, or protein subunits. Despite growing success in worldwide vaccination efforts, additional capabilities may be needed in the future to address issues such as stability and storage requirements, need for vaccine boosters, desirability of different routes of administration, and emergence of SARS-CoV-2 variants such as the Delta variant. Here, we present a novel, well-characterized SARS-CoV-2 vaccine candidate based on extracellular vesicles (EVs) of Salmonella typhimurium that are decorated with the mammalian cell culture-derived Spike receptor-binding domain (RBD). RBD-conjugated outer membrane vesicles (RBD-OMVs) were used to immunize the golden Syrian hamster (Mesocricetus auratus) model of COVID-19. Intranasal immunization resulted in high titers of blood anti-RBD IgG as well as detectable mucosal responses. Neutralizing antibody activity against wild-type and Delta variants was evident in all vaccinated subjects. Upon challenge with live virus, hamsters immunized with RBD-OMV, but not animals immunized with unconjugated OMVs or a vehicle control, avoided body mass loss, had lower virus titers in bronchoalveolar lavage fluid, and experienced less severe lung pathology. Our results emphasize the value and versatility of OMV-based vaccine approaches.


title: A bacterial extracellular vesicle-based intranasal vaccine against SARS-CoV-2 protects against disease and elicits neutralizing antibodies to wild-type virus and Delta variant


title: Myeloid cell interferon responses correlate with clearance of SARS-CoV-2

abstract: The emergence of mutant SARS-CoV-2 strains associated with an increased risk of COVID-19-related death necessitates better understanding of the early viral dynamics, host responses and immunopathology. While studies have reported immune profiling using single cell RNA sequencing in terminal human COVID-19 patients, performing longitudinal immune cell dynamics in humans is challenging. Macaques are a suitable model of SARS-CoV-2 infection. We performed longitudinal single-cell RNA sequencing of bronchoalveolar lavage (BAL) cell suspensions from adult rhesus macaques infected with SARS-CoV-2 (n=6) to delineate the early dynamics of immune cells changes. The bronchoalveolar compartment exhibited dynamic changes in transcriptional landscape 3 days post-SARS-CoV-2-infection (3dpi) (peak viremia), relative to 14-17dpi (recovery phase) and pre-infection (baseline). We observed the accumulation of distinct populations of both macrophages and T-lymphocytes expressing strong interferon-driven inflammatory gene signature at 3dpi. Type I IFN response was highly induced in the plasmacytoid dendritic cells. The presence of a distinct HLADR+CD68+CD163+SIGLEC1+ macrophage population exhibiting higher angiotensin converting enzyme 2 (ACE2) expression was also observed. These macrophages were significantly recruited to the lungs of macaques at 3dpi and harbored SARS-CoV-2, while expressing a strong interferon-driven innate anti-viral gene signature. The accumulation of these responses correlated with decline in viremia and recovery. The recruitment of a myeloid cell-mediated Type I IFN response is associated with the rapid clearance of SARS-CoV-2 infection in macaques.


title: Single-cell imaging reveals that Staphylococcus aureus is highly competitive against Pseudomonas aeruginosa on surfaces

abstract: Pseudomonas aeruginosa and Staphylococcus aureus frequently occur together in polymicrobial infections, and their interactions can complicate disease progression as well as treatment options. While interactions between P. aeruginosa and S. aureus have been extensively described using planktonic batch cultures, little is known about whether and how individual cells interact with each other on solid substrates. This is important, because in infections, both species frequently colonize surfaces to form microcolony aggregates and biofilms. Here, we performed single-cell time-lapse fluorescence microscopy combined with automated image analysis to describe interactions between P. aeruginosa PAO1 with three different S. aureus strains (Cowan I, 6850, JE2) during microcolony growth on agarose surfaces. While P. aeruginosa is usually considered the dominant species, we found that the competitive balance tips in favor of S. aureus on surfaces. We observed that all S. aureus strains accelerated the onset of microcolony growth in competition with P. aeruginosa and significantly compromised P. aeruginosa growth prior to physical contact. These results suggest that S. aureus deploys mechanisms of both resource competition and interference competition via diffusible compounds. JE2 was the most competitive S. aureus strain, simply usurping P. aeruginosa microcolonies when coming into direct contact, while 6850 was the weakest competitor itself suppressed by P. aeruginosa. Moreover, P. aeruginosa reacted to the assault of S. aureus by showing increased directional growth and expedited expression of quorum sensing regulators controlling the synthesis of competitive traits. Altogether, our results reveal that quantitative single-cell live imaging has the potential to uncover microbial behaviors that cannot be predicted from batch culture studies, and thereby contribute to our understanding of interactions between pathogens that co-colonize host-associated surfaces during polymicrobial infections.


title: A SARS-CoV-2 mini-genome assay based on negative-sense RNA to study replication inhibitors and emerging mutations

abstract:
Severe Acute Respiratory Coronavirus 2 (SARS-CoV-2) is a positive-sense single-stranded RNA virus and the causative agent of the Coronavirus disease 2019 (COVID-19) pandemic. Efforts to identify inhibitors of SARS-CoV-2 replication enzymes and better understand the mechanisms underlying viral RNA synthesis have largely relied on biosafety level 3 (BSL3) laboratories, limiting throughput and accessibility. Recently, replicon systems have been proposed that involve ~30 kb RNA-based replicons or large plasmids that express the viral structural and non-structural proteins (nsp) in addition to a positive-sense reporter RNA. Unfortunately, these assays are not user-friendly due to plasmid instability or a poor signal to background ratio. We here present a simple mini-genome assay consisting of a ~2.5 kb-long negative-sense, nanoluciferase-encoding sub-genomic reporter RNA that is expressed from a plasmid, and amplified and transcribed by the SARS-CoV-2 RNA polymerase core proteins nsp7, nsp8 and nsp12. We show that expression of nsp7, 8 and 12 is sufficient to obtain robust positive- and negative-sense RNA synthesis in cell culture, that addition of other nsps modulates expression levels, and that replication of the reporter RNA can be inhibited by active site mutations in nsp12 or the SARS-CoV-2 replication inhibitor remdesivir. The mini-genome assay provides a signal that is 170-fold above background on average, providing excellent sensitivity for high-throughput screens, while the use of small plasmids facilitates site-directed mutagenesis for fundamental analyses of SARS-CoV-2 RNA synthesis.

Importance statementThe impact of the COVID-19 pandemic has made it essential to better understand the basic biology of SARS-CoV-2, and to search for compounds that can block the activity of key SARS-CoV-2 replication enzymes. However, studies with live SARS-CoV-2 require biosafety level 3 facilities, while existing replicon systems depend on long positive-sense subgenomes that are often difficult to manipulate or produce a high background signal, limiting drug-screens and a rapid analysis of emerging SARS-CoV-2 mutations during the COVID-19 pandemic. To make it easier to study emerging SARS-CoV-2 mutants and screen for inhibitors, we developed a simple mini-replicon that produces a minimal background signal, that can be used in any tissue culture lab, and that only requires four small plasmids to work.


title: Effect of prophylactic use of intra-nasal oil formulations in the hamster model of Covid-19

abstract: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV2) infection initiates with viral entry in upper respiratory tract leading to coronavirus disease 2019 (Covid-19). Severe Covid-19 is characterized by pulmonary pathologies associated with respiratory failure. Thus, therapeutics aimed at inhibiting entry of the virus or its internalization in the upper respiratory tract, are of interest. Herein, we report the prophylactic application of two intra-nasal formulations provided by the National Medicinal Plant Board (NMPB), Anu oil and Til tailya in SARS-CoV2 infection hamster model. Prophylactic nasal instillation of these oil formulations exhibited reduced viral load in lungs, and resulted in reduced body weight loss and pneumonitis. In line with reduced viral load, histopathlogical analysis revealed a reduction in lung pathology in Anu oil group as compared to the control infected group. However, Til tailya group did not show a significant reduction in lung pathology. Furthermore, molecular analysis using mRNA expression profiling indicated reduced expression of pro-inflammatory cytokines genes, including Th1 and Th17 cytokines for both the intra-nasal formulations as a result of decreased viral load. Together, the prophylactic intra-nasal application of Annu oil seems to be useful in limiting both the viral load and disease severity disease in SARS-CoV2 infection in hamster model.


title: Global analysis of putative phospholipases in the malaria parasite  Plasmodium falciparum  reveals critical factors for parasite proliferation

abstract: For its replication within red blood cells, the malaria parasite is highly dependent on correctly regulated lipid metabolism. Enzymes involved in lipid metabolic processes are therefore potential drug targets. We here provide a functional analysis of the 20 putative phospholipases that are expressed by asexual blood stages of Plasmodium falciparum. We reveal a high level of redundancy among members of this group, but using conditional mislocalization and gene disruption techniques we show that the phosphoinositide-specific phospholipase C (PF3D7_1013500) has a previously unrecognized essential role in intracellular parasite maturation. In addition, we demonstrate that the patatin-like phospholipase PF3D7_1358000 localizes to the mitochondrion. Parasites lacking this enzyme display a severe growth phenotype and defects in mitochondrial morphogenesis and function leading to hypersensitivity towards proguanil and inhibitors of the mitochondrial electron transport chain including atovaquone. This demonstrates that regulated mitochondrial lipid homeostasis is necessary for mitochondrial function and coordinated division during parasite multiplication.


title: Serum Neutralizing Activity of mRNA-1273 against SARS-CoV-2 Variants

abstract: The emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants has led to growing concerns over increased transmissibility and the ability of some variants to partially escape immunity. Sera from participants immunized on a prime-boost schedule with the mRNA-1273 COVID-19 vaccine were tested for neutralizing activity against several SARS-CoV-2 variants, including variants of concern (VOCs) and variants of interest (VOIs), compared to neutralization of the wild-type SARS-CoV-2 virus (designated as D614G). Results showed minimal effects on neutralization titers against the B.1.1.7 (Alpha) variant (1.2-fold reduction compared with D614G); other VOCs such as B.1.351 (Beta, including B.1.351-v1, B.1.351-v2, and B.1.351-v3), B.1.617.2 (Delta), and P.1 (Gamma) showed decreased neutralization titers ranging from 2.1-fold to 8.4-fold reductions compared with D614G, although all remained susceptible to mRNA-1273-elicited serum neutralization.


title: Solar simulated ultraviolet radiation inactivates HCoV-NL63 and SARS-CoV-2 coronaviruses at environmentally relevant doses

abstract: The germicidal properties of short wavelength ultraviolet C (UVC) light are well established and used to inactivate many viruses and other microbes. However, much less is known about germicidal effects of terrestrial solar UV light, confined exclusively to wavelengths in the UVA and UVB regions. Here, we have explored the sensitivity of the human coronaviruses HCoV-NL63 and SARS-CoV-2 to solar-simulated full spectrum ultraviolet light (sUV) delivered at environmentally relevant doses. First, HCoV-NL63 coronavirus inactivation by sUV-exposure was confirmed employing (i) viral plaque assays, (ii) RT-qPCR detection of viral genome replication, and (iii) infection-induced stress response gene expression array analysis. Next, a detailed dose-response relationship of SARS-CoV-2 coronavirus inactivation by sUV was elucidated, suggesting a half maximal suppression of viral infectivity at low sUV doses. Likewise, extended sUV exposure of SARS-CoV-2 blocked cellular infection as revealed by plaque assay and stress response gene expression array analysis. Moreover, comparative (HCoV-NL63 versus SARS-CoV-2) single gene expression analysis by RT-qPCR confirmed that sUV exposure blocks coronavirus-induced redox, inflammatory, and proteotoxic stress responses. Based on our findings, we estimate that solar ground level full spectrum UV light impairs coronavirus infectivity at environmentally relevant doses. Given the urgency and global scale of the unfolding SARS-CoV-2 pandemic, these prototype data suggest feasibility of solar UV-induced viral inactivation, an observation deserving further molecular exploration in more relevant exposure models.


title: N4-hydroxycytidine and inhibitors of dihydroorotate dehydrogenase synergistically suppress SARS-CoV-2 replication

abstract:
Effective therapeutics to inhibit the replication of SARS-CoV-2 in infected individuals are still under development. The nucleoside analogue N4-hydroxycytidine (NHC), also known as EIDD-1931, interferes with SARS-CoV-2 replication in cell culture. It is the active metabolite of the prodrug Molnupiravir (MK-4482), which is currently being evaluated for the treatment of COVID-19 in advanced clinical studies. Meanwhile, inhibitors of dihydroorotate dehydrogenase (DHODH), by reducing the cellular synthesis of pyrimidines, counteract virus replication and are also being clinically evaluated for COVID-19 therapy. Here we show that the combination of NHC and DHODH inhibitors such as teriflunomide, IMU-838/vidofludimus, and BAY2402234, strongly synergizes to inhibit SARS-CoV-2 replication. While single drug treatment only mildly impaired virus replication, combination treatments reduced virus yields by at least two orders of magnitude. We determined this by RT-PCR, TCID50, immunoblot and immunofluorescence assays in Vero E6 and Calu-3 cells infected with wildtype and the Alpha and Beta variants of SARS-CoV-2. We propose that the lack of available pyrimidine nucleotides upon DHODH inhibition increases the incorporation of NHC in nascent viral RNA, thus precluding the correct synthesis of the viral genome in subsequent rounds of replication, thereby inhibiting the production of replication competent virus particles. This concept was further supported by the rescue of replicating virus after addition of pyrimidine nucleosides to the media. Based on our results, we suggest combining these drug candidates, which are currently both tested in clinical studies, to counteract the replication of SARS-CoV-2, the progression of COVID-19, and the transmission of the disease within the population.

SIGNIFICANCEO_LIThe strong synergy displayed by DHODH inhibitors and the active compound of Molnupiravir might enable lower concentrations of each drug to antagonize virus replication, with less toxicity.
C_LIO_LIBoth Molnupiravir and DHODH inhibitors are currently being tested in advanced clinical trials or are FDA-approved for different purposes, raising the perspective of rapidly testing their combinatory efficacy in clinical studies.
C_LIO_LIMolnupiravir is currently a promising candidate for treating early stages of COVID-19, under phase II/III clinical evaluation. However, like Remdesivir, it appears only moderately useful in treating severe COVID-19. Since the combination inhibits virus replication far more strongly, and since DHODH inhibitors may also suppress excessive immune responses, the combined clinical application bears the potential of alleviating the disease burden even at later stages.
C_LI

category: molecular biology


title: Anoikis resistance in mammary epithelial cells is mediated by semaphorin-7a.

abstract: Semaphorin-7a (SEMA7A), best known as a neuroimmune molecule, plays a diverse role in many cellular processes and pathologies. Here, we show that SEMA7A promotes anoikis resistance in cultured mammary epithelial cells through integrins and activation of pro-survival kinase AKT, which led us to investigate a role for SEMA7A during postpartum mammary gland involution--a normal developmental process where cells die by anoikis. Our results reveal that SEMA7A is expressed on live mammary epithelial cells during involution, that SEMA7A expression is primarily observed in 6-integrin expressing cells, and that luminal progenitor cells, specifically, are decreased in mammary glands of SEMA7A-/- mice during involution. We further identify a SEMA7A-6/{beta}1-integrin dependent mechanism of mammosphere formation and chemoresistance in mammary epithelial cells and suggest that this mechanism is relevant for recurrence in breast cancer patients.

category: cell biology


title: Convergent adaptation and ecological speciation result from unique genomic mechanisms in sympatric extremophile fishes

abstract:
Divergent selection along ecological gradients can lead to speciation, and replicated speciation events occur when populations of multiple lineages undergo divergence following colonization of similar environments. In such instances, it remains unclear to what extent reproductive isolation evolves via convergent mechanisms at the genomic level due to biases associated with shared ancestry and variation in selection associated with geographic replicates typically studied in this context. We used a unique system in which three species of poeciliid fishes occur in sympatry in a toxic, hydrogen sulfide (H2S)-rich spring and an adjacent nonsulfidic stream to examine shared patterns of adaptive divergence across multiple levels of biological organization. Despite small spatial scales, we found strong genetic differentiation between populations in sulfidic and nonsulfidic habitats mediated by strong selection against migrants between habitat types. High levels of reproductive isolation were accompanied by convergent patterns of adaptation in morphological and physiological traits, as well as genome-wide patterns of gene expression across all three species. Furthermore, the mitochondrial genomes of each species exhibit shared signatures of selection on key genes involved in H2S toxicity. However, contrary to predictions of speciation theory, analyses of divergence across the nuclear genomes neither revealed evidence for clear genomic islands of speciation nor substantial congruence of outlier regions across population pairs. Instead, heterogenous regions of divergence spread across the genome suggest that selection for polygenic physiological adaptations likely facilitated the rapid evolution of high levels of reproductive isolation. Overall, we demonstrate that substantial convergence across multiple levels of biological organization can be mediated by non-convergent modifications at the genomic level. By disentangling variation in natural selection from lineage-specific evolution in this system of highly divergent, yet sympatric lineages, our results emphasize the outsized role of the genomic substrate upon which selection acts in driving convergent evolution at the phenotypic level.

Significance StatementDivergent lineages that coexist in sympatry and are exposed to the same sources of natural selection provide a unique opportunity to study convergent evolution across levels of organization because confounding factors associated with geographic replications are eliminated. Using three sympatric lineages of livebearing fishes inhabiting toxic and adjacent nontoxic habitats, we show that the convergent evolution of phenotypic adaptation and reproductive isolation can evolve in the absence of substantial convergence at the genomic level. Our results suggest that the nature of selection is less important in producing genomic convergence than the initial genomic substrates that are available for selection to act on.

category: evolutionary biology


title: Reactivities of the Front Pocket N-Cap Cysteines in Human Kinases

abstract: Discovery of targeted covalent inhibitors directed at nucleophilic cysteines is attracting enormous interest. The front pocket (FP) N-cap cysteine has been the most popular site of covalent modification in kinases. Curiously, a long-standing hypothesis associates the N-cap position with cysteine hyper-reactivity; however, traditional computational methods suggest that the FP N-cap cysteines in all human kinases are predominantly unreactive at physiological pH. Here we applied a newly developed GPU-accelerated continuous constant pH molecular dynamics (CpHMD) tool to test the N-cap hypothesis and elucidate the cysteine reactivities. Simulations showed that the N-cap cysteines in BTK/BMX/TEC/ITK/TXK, JAK3, and MKK7 sample the reactive thiolate form to varying degrees at physiological pH; however, those in BLK and EGFR/ERBB2/ERBB4 which contain an Asp at the N-cap+3 position adopt the unreactive thiol form. The latter argues in favor of the base-assisted thiol-Michael addition mechanisms as suggested by the quantum mechanical calculations and experimental structure-function studies of EGFR inhibitors. Analysis revealed that the reactive N-cap cysteines are stabilized by hydrogen bond as well as electrostatic interactions, and in their absence a N-cap cysteine is unreactive due to desolvation. To test a corollary of the N-cap hypothesis, we also examined the reactivities of the FP N-cap+2 cysteines in JNK1/JNK2/JNK3 and CASK. Additionally, our simulations predicted the reactive cysteine and lysine locations in all 15 kinases. Our findings offer a systematic understanding of cysteine reactivities in kinases and demonstrate the predictive power and physical insights CpHMD can provide to guide the rational design of targeted covalent inhibitors.


title: Radiolabeling of PSMA-617 with 89Zr: A Novel Use of DMSO for Radiochemical Yield Enhancement and Preliminary Small-Animal PET Results

abstract:
IntroductionProstate-specific membrane antigen (PSMA)-targeted ligands, including PSMA-617, have been developed for theranostics of prostate cancer. 68Ga-PSMA-617 is the de facto standard of PSMA Positron Emission Tomography (PET) for imaging in prostate cancer patients prior to radioligand therapy (RLT) with 177Lu-PSMA-617. The dose-limiting toxicity for PSMA-RLT is damage to the kidney. PET scans using 68Ga-PSMA-617 have to be performed within a few hours of injection due to its short half-life (68 min). However, the presence of radioactivity in urine at the PET imaging timepoint hampers the dose optimization of 177Lu (half-life 6.6 d)-labeled PSMA-617. Thus, the long-lived positron emitter 89Zr (half-life 3.3 d) is suited for optimizing the doses of 177Lu-PSMA-617 because PET scans can be performed after excretion of radioactive urine. Although 89Zr has great potential for PET imaging, its inadequate incorporation into 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA), limits its applications. Here, we report the radiolabeling of PSMA-617 with 89Zr and preliminary PET imaging studies using 89Zr-PSMA-617.

MethodsDMSO and HEPES buffer were used to label PSMA-617 with 89Zr. The dissociation constant (Kd) of 89Zr-PSMA-617 was determined using a cell-binding assay. Delayed-PET scans using 89Zr-PSMA-617 were performed at 24 h (N = 5).

Results89Zr-PSMA-617 was prepared with a radiochemical yield of 70 {+/-} 9%. The Kd value was 6.8 nM. In PET imaging, standardized uptake value (SUV) was highest in LNCaP tumors (SUVmax = 0.98 {+/-} 0.32), whereas it was low in kidney (SUVmax = 0.18 {+/-} 0.7).

ConclusionThe preparation of 89Zr-PSMA-617 was achieved by using the DMSO and HEPES buffer. 89Zr-PSMA-617 visualize the PSMA positive LNCaP tumors without accumulation in bladder.

Advances in knowledge and implications for patient careThe use of 89Zr-PSMA-617 to predict the radiation doses in normal tissues lead to safe and effective RLT with 177Lu-PSMA-617.


title: A novel small molecule that induces cytotoxicity in lung cancer cells inhibits disulfide reductases GSR and TXNRD1

abstract: High-throughput phenotype-based screening of large libraries of novel compounds without known targets can identify small molecules that elicit a desired cellular response, but additional approaches are required to find and characterize their targets and mechanisms of action. Here we show that a compound termed lung cancer screen 3 (LCS3), previously selected for its ability to impair the growth of human lung adenocarcinoma (LUAD) cell lines, but not normal lung cells, induces oxidative stress and activates the NRF2 signaling pathway by generating reactive oxygen species (ROS) in sensitive LUAD cell lines. To identify the target that mediates this effect, we applied thermal proteome profiling (TPP) and uncovered the disulfide reductases GSR and TXNRD1 as LCS3 targets. Through enzymatic assays using purified protein, we confirmed that LCS3 inhibits disulfide reductase activity through a reversible, uncompetitive mechanism. Further, we demonstrate that LCS3-sensitive LUAD cells are correspondingly sensitive to the synergistic inhibition of glutathione and thioredoxin pathways. Lastly, a genome-wide CRISPR knockout screen identified the loss of NQO1 as a mechanism of LCS3 resistance. This work highlights the ability of TPP to uncover targets of small molecules identified by high-throughput screens and demonstrates the potential utility of inhibiting disulfide reductases as a therapeutic strategy for LUAD.