title: Conventional polymers may unintentionally refill in vivo with unassociated drugs

abstract: Polymers used as drug delivery devices are ultimately limited by how much drug they can hold; with the device failing if the drug is depleted before the disease is cured. Our lab discovered a means to use thermodynamic driving forces to refill certain classes of polymer after implantation, for additional drug delivery windows. These same, refillable polymers can be used as additives, to provide refilling capacity to classical, non-refillable polymers such as poly(methyl methacrylate) (PMMA). In this paper, we investigated the refilling capacity of another conventional polymer: poly(lactic-co-glycolic acid) or PLGA. We explored both unmodified PLGA implants as well as implants supplemented with polymerized cyclodextrin (pCD) in microparticle form, previously shown to add refillability to poly(methyl methacrylate) (PMMA) implants which were otherwise not refillable. Assessments of in situ forming PLGA implants with and without pCD additives were made, including drug loading capacity in a liquid medium, drug refilling through a tissue-mimicking gel medium, and refilling in ex vivo and in vivo conditions. Implant cross-sections were imaged via fluorescence microscopy. Drug release from refilled implants, polymer swelling, degradation, phase inversion characteristics were assessed, and drug/monomer computational simulation studies were performed. While generally, the incorporation of cyclodextrin into implants led to significant increases in the amount of refilled drug; unexpectedly, PLGA implants with no incorporated pCD also showed refilling capability. Moreover, in two out of three in vivo conditions in rats, PLGA alone showed the potential to refill with comparable, if not greater, amounts of drug than PLGA with pCD incorporated. This contrasts predictions, since PLGA has no specifically designed affinity structure, like pCD does. We theorize that the mechanism for PLGAs refilling depends on nano-patterning of hydrophilic and hydrophobic molecular domains, giving rise to its affinity-like behavior. The fact that PLGA implants can be refilled with unassociated drugs, gives rise to concerns about the fate of all implants made of poly alpha-hydroxy esters, and likely other polymers as well, and will likely lead to new directions of study such as of unintended polymer / drug interactions.


title: Mooney Face Image Processing in Deep Convolutional Neural Networks Compared to Humans

abstract: Deep Convolutional Neural Networks (CNNs) are criticised for their reliance on local shape features and texture rather than global shape. We test whether CNNs are able to process global shape information in the absence of local shape cues and texture by testing their performance on Mooney stimuli, which are face images thresholded to binary values. More specifically, we assess whether CNNs classify these abstract stimuli as face-like, and whether they exhibit the face inversion effect (FIE), where upright stimuli are classified positively at a higher rate compared to inverted. We tested two standard networks, one (CaffeNet) trained for general object recognition and another trained specifically for facial recognition (DeepFace). We found that both networks perform perceptual completion and exhibit the FIE, which is present over all levels of specificity. By matching the false positive rate of CNNs to humans, we found that both networks performed closer to the human average (85.73% for upright, 57.25% for inverted) for both conditions (61.31% and 62.70% for upright, 48.61% and 42.26% for inverted, for CaffeNet and DeepFace respectively). Rank order correlation between CNNs and humans across individual stimuli shows a significant correlation in upright and inverted conditions, indicating a relationship in image difficulty between observers and the model. We conclude that in spite of the texture and local shape bias of CNNs, which makes their performance distinct from humans, they are still able to process object images holistically.

category: bioinformatics


title: REVERSE: A user-friendly web server for analyzing next-generation sequencing data from in vitro selection/evolution experiments

abstract: Next-generation sequencing (NGS) enables the identification of functional nucleic acid sequences from in vitro selection/evolution experiments and illuminates the evolutionary process at single nucleotide resolution. However, analyzing the vast output from NGS can be daunting, especially with limited programming skills. No single platform exists that performs all the steps necessary to generate publishable results starting with raw sequence data. We developed REVERSE (Rapid EValuation of Experimental RNA Selection/Evolution) (https://www.reverseserver.org/), a web server that incorporates an integrated computational pipeline through a graphical user interface, which performs both pre-processing and sequence level analyses within minutes. FASTQ files from multiple rounds are quality filtered, dereplicated, and trimmed before being analyzed by two pipelines. The first pipeline counts, sorts, and tracks enrichment of unique sequences and tracks the enrichment of sequence motifs. It also identifies mutational intermediates present in the sequence data that connect two input sequences. The second pipeline sorts similar sequences into clusters and tracks enrichment of peak sequences. It also performs nucleotide conservation analysis on the cluster of choice. Both pipelines generate downloadable high-resolution figures. Collectively, REVERSE is a one stop-solution for the rapid analysis of NGS data obtained from in vitro selection/evolution experiments that obviates the need for computational expertise.


title: Targeting Reductive Metabolic Shifts by T315I Mutation in BCR-ABL Myeloid Leukemia for Therapy

abstract: T315I mutation of Bcr-Abl in chronic myeloid leukemia (CML) leads to therapeutic resistance. It is known that Bcr-Abl transformation causes ROS-induced DNA damages and replication stress, which can be exploited for anti-nucleotide therapy. We developed a small compound, JMF4073, which inhibited pyrimidylate kinases and selectively eliminated Bcr-Abl-transformed, but not untransformed myeloid cells, due to dTTP exhaustion and ROS-induced replication stress. However, T315I-Bcr-Abl-transformed cells were less vulnerable to JMF4073 because of higher dTTP pool and low replication stress. Unlike WT-Bcr-Abl-transformed cells, T315I-Bcr-Abl cells lacked Sirt1- regulated OXPHOS with increased glutamine flux to reductive carboxylation in TCA cycle and glutathione synthesis. Blocking mitochondrial pyruvate carrier (MPC) by UK-5099 reduced NADH and glutathione levels with replication stress induction, thereby converting T315I-Bcr-Abl cells sensitive to JMF4073 with dTTP and dCTP depletion. The combination of JMF4073 with UK-5099 showed in vivo eradication of T315I-Bcr-Abl-CML. These data reveal that T315I mutation causes reductive metabolic shifts in Bcr-Abl-CML, and demonstrate the therapeutic option by co-targeting MPC and pyrimidylate kinases.

category: cancer biology


title: Identifying targetable metabolic dependencies across colorectal cancer progression

abstract: Colorectal cancer (CRC) is a multi-stage process initiated through the formation of a benign adenoma, progressing to an invasive carcinoma and finally metastatic spread. Tumour cells must adapt their metabolism to support the energetic and biosynthetic demands associated with disease progression. As such, targeting cancer cell metabolism is a promising therapeutic avenue in CRC. However, to identify tractable nodes of metabolic vulnerability specific to CRC stage, we must understand how metabolism changes during CRC development. Here, we use a unique model system - comprising human early adenoma to late adenocarcinoma. We show that adenoma cells transition to elevated glycolysis at the early stages of tumour progression but maintain oxidative metabolism. Progressed adenocarcinoma cells rely more on glutamine-derived carbon to fuel the TCA cycle, whereas glycolysis and TCA cycle activity remain tightly coupled in early adenoma cells. Adenocarcinoma cells are more flexible with respect to fuel source, enabling them to proliferate in nutrient-poor environments. Despite this plasticity, we identify asparagine (ASN) synthesis as a node of metabolic vulnerability in late-stage adenocarcinoma cells. We show that loss of asparagine synthetase (ASNS) blocks their proliferation, whereas early adenoma cells are largely resistant to ASN deprivation. Mechanistically, we show that late-stage adenocarcinoma cells are dependent on ASNS to support mTORC1 signalling and maximal glycolytic and oxidative capacity. Resistance to ASNS loss in early adenoma cells is likely due to a feedback loop, absent in late-stage cells, allowing them to sense and regulate ASN levels and supplement ASN by autophagy. Together, our study defines metabolic changes during CRC development and highlights ASN synthesis as a targetable metabolic vulnerability in later stage disease.


title: SCREENING OF RELEVANT METABOLISM-DISRUPTING CHEMICALS ON PANCREATIC β-CELLS: EVALUATION OF MURINE AND HUMAN IN VITRO MODELS

abstract: Endocrine-disrupting chemicals (EDCs) are chemical substances that can interfere with the normal function of the endocrine system. EDCs are ubiquitous and can be found in a variety of consumer products such as food packaging materials, personal care and household products, plastic additives, and flame retardants. Over the last decade, the impact of EDCs on human health has been widely acknowledged as they have been associated with different endocrine diseases. Among them, a subset called metabolism-disrupting chemicals (MDCs) are able to promote metabolic changes that can lead to the development of metabolic disorders such as diabetes, obesity, hepatic steatosis, and metabolic syndrome, among others. Despite this, today, there are still no definitive and standardized in vitro tools to support the metabolic risk assessment of existing and emerging MDCs for regulatory purposes. Here, we evaluated two different pancreatic cell-based in vitro systems, the murine pancreatic {beta}-cell line MIN6 as well as the human pancreatic {beta}-cell line EndoC- {beta}H1. Both were challenged with a range of relevant concentrations of seven well-known EDCs (bisphenol-A (BPA), bisphenol-S (BPS), bisphenol-F (BPF), perfluorooctanesulfonic acid (PFOS), di(2-ethylhexyl) phthalate (DEHP), cadmium chloride (CdCl2) and dichlorodiphenyldichloroethylene (DDE)). The screening revealed that most of the tested chemicals have detectable deleterious effects on glucose-stimulated insulin release, insulin content, electrical activity, gene expression, and/or viability. Our data provide new molecular information on the direct effects of the selected chemicals on key aspects of pancreatic {beta}-cell function such as the stimulus-secretion coupling and ion channel activity. In addition, we found that, in general, the sensitivity and responses were comparable to those from other in vivo studies reported in the literature. Overall, our results suggest that both systems can serve as effective tools for rapid screening of potential MDC effects on pancreatic {beta}-cell physiology as well for deciphering and better understanding the molecular mechanisms that underlie their action.

category: pharmacology and toxicology


title: Development of in vitro test methods in a model of human pancreatic β-cells to identify metabolism disrupting chemicals with diabetogenic activity

abstract: There is a need to develop identification tests for Metabolism Disrupting Chemicals (MDCs) with diabetogenic activity. Here we used the human EndoC-{beta}H1 {beta}-cell line, the rat {beta}-cell line INS-1E and dispersed mouse islet cells to assess the effects of endocrine disruptors on cell viability and glucose-stimulated insulin secretion (GSIS). We tested six chemicals at concentrations within human exposure (from 0.1 pM to 1 M). Bisphenol-A (BPA) and tributyltin (TBT) were used as controls while four other chemicals, namely perfluorooctanoic acid (PFOA), triphenylphosphate (TPP), triclosan (TCS) and dichlorodiphenyldichloroethylene (DDE), were used as "unknowns". Regarding cell viability, BPA and TBT increased cell death as previously observed. Their mode of action involved the activation of estrogen receptors and PPAR{gamma}, respectively. ROS production was a consistent key event in BPA- and TBT-treated cells. None of the other MDCs tested modified viability or ROS production. Concerning GSIS, TBT increased insulin secretion while BPA produced no effects. PFOA decreased GSIS, suggesting that this chemical could be a "new" diabetogenic agent. Our results indicate that the EndoC-{beta}H1 cell line is a suitable human {beta}-cell model for testing diabetogenic MDCs. Optimization of the test methods proposed here could be incorporated into tier protocols for the identification of MDCs.


title: Association of seminal polyaromatic hydrocarbons exposome with idiopathic male factor infertility: A proteomic insight into sperm function

abstract: Oxidative stress (OS) is implicated in 80% of idiopathic male infertility (IMI) patients where exposure to redox active environmental toxicants such as polyaromatic hydrocarbons (PAHs) may play. In the present study the seminal exposome of various PAH was analyzed in two separate cohorts including 43 fertile donors and 60 IMI patients by HPLC and receiver operator characteristic curve was applied to find out the cut-off limits. Furthermore, spermatozoa from both the groups were subjected to label free liquid chromatography mass spectroscopy (LC-MS/MS) followed by bioinformatics analysis to elucidate the molecular mechanism(s) involved and the key proteins from the affected pathways were validated by western blot along with oxidative modification of proteins. Of the 16 standard PAH 13 were detected in the semen. Receiver Operator Character (ROC) Curve analysis (AUCROC) revealed the PAHs having most significant effect on fertility are of the following order Anthracene<benzo(a)pyrene<benzo[b]fluoranthene<Fluoranthene<benzo(a)anthracene<indol (123CD)pyrene<pyrene<naphthalene<dibenzo(AH)anthracene<fluorene<2bromonaphthalene <chrysene<benzo(GH1)perylene. Benzo[a] pyrene is invariably present in all infertile patients while naphthalene is present in both fertile and IMI group. Of the total 773 detected proteins (Control: 631 and PAH: 717); 71 were differentially expressed (13 underexpressed, 58 overexpressed) in IMI patients resulting in impaired mitochondrial dysfunction and oxidative phosphorylation, DNA damage, Aryl hydrocarbon receptor (AHR) signalling, xenobiotic metabolism and induction of NRF-2 mediated oxidative stress response (increased in 4-hydroxynonenal and nitrosylated protein adduct formation, and declined antioxidant defence). The increased GSH/GSSG ratio in patients may be an adaptive response to metabolize the xenobiotics via conjugation as evidenced by overexpression of AHR and Heat shock protein 90 beta (HSP90{beta}) in patients. Seminal PAH concentrations, oxidative protein modification along with protein markers (e.g. AHR and HSP90{beta}) may help in better prediction and management of IMI. Contribution of environment borne PAH in semen should not undermined in infertility evaluation.


title: Modulation of the DA1 pathway in maize shows that translatability of information from Arabidopsis to crops is complex

abstract: Modern agriculture is struggling to meet the increasing food, silage and raw material demands due to the rapid growth of population and climate change. In Arabidopsis, DA1 and DAR1 are proteases that negatively regulate cell proliferation and control organ size. DA1 and DAR1 are activated by ubiquitination catalyzed by the E3 ligase BIG BROTHER (BB). Here, we characterized the DA1, DAR 1 and BB gene families in maize and analyzed whether perturbation of these genes regulates organ size similar to what was observed in Arabidopsis. We generated da1_dar1a_dar1b triple CRISPR maize mutants and bb1_bb2 double mutants. Detailed phenotypic analysis showed that the size of leaf, stem, cob, and seed was not consistently enlarged in these mutants. Also overexpression of a dominant-negative DA1R333K allele, resembling the da1-1 allele of Arabidopsis which has larger leaves and seeds, did not alter the maize phenotype. The mild negative effects on plant height of the DA1R333K_bb1_bb2 mutant indicate that the genes in the DA1 pathway may control organ size in maize, albeit less obvious than in Arabidopsis.

category: plant biology


title: A wheat resistosome defines common principles of immune receptor channels

abstract: Plant intracellular nucleotide-binding leucine-rich repeat (NLRs) receptors detect pathogen effectors to trigger immune responses. Indirect recognition of a pathogen effector by the dicotyledonous Arabidopsis thaliana coiled-coil (CC) domain containing NLR (CNL) ZAR1 induces the formation of a large hetero-oligomeric protein complex, termed the ZAR1 resistosome, which functions as a calcium channel required for ZAR1-mediated immunity (1-3). Whether the resistosome and channel activities are conserved among plant CNLs remains unknown. We report here a cryogenic electron microscopy (cryo-EM) structure of the wheat CNL Sr35 in complex with the effector AvrSr35 of the wheat stem rust pathogen at 3.0 [A] resolution. Direct effector binding to the leucine-rich repeats (LRRs) of Sr35 results in the formation of a pentameric Sr35-AvrSr35 complex, which we designate the Sr35 resistosome. Wheat Sr35 and Arabidopsis ZAR1 resistosomes bear striking structural similarity, including a previously unnoticed arginine cluster in the LRR domain that co-occurs and forms intramolecular interactions with the  EDVID motif in the CC domain. Electrophysiological measurements show that the Sr35 resistosome exhibits non-selective cation channel activity. These structural insights allowed us to generate novel variants of closely related wheat and barley orphan NLRs that recognize AvrSr35. Our data support the evolutionary conservation of CNL resistosomes in plants and demonstrate proof of principle for structure-based engineering of NLRs for crop improvement.


title: BNIP3 upregulation characterizes cancer cell subpopulation with increased fitness and proliferation

abstract: BNIP3 is a BH3 only protein with both pro-apoptotic and pro-survival roles depending on the cellular context. It remains unclear how BNIP3 RNA level dictates cell fate decisions of cancer cells. Here we undertook a quantitative analysis of BNIP3 expression and functions in single cell datasets of various epithelial malignancies. Our results demonstrated that BNIP3 upregulation characterizes cancer cell subpopulations with increased fitness and proliferation. We further validated the upregulation of BNIP3 in liver cancer organoids compared with 2D culture. Taken together, the combination of in silico perturbations using public single cell datasets and experimental cancer modeling using organoids ushered in a new approach to address cancer heterogeneity.

category: systems biology


title: Gene expression noise accelerates the evolution of a biological oscillator

abstract:
Gene expression is a biochemical process, where stochastic binding and unbinding events naturally generate fluctuations and cell-to-cell variability in gene dynamics. These fluctuations typically have destructive consequences for proper biological dynamics and function (e.g., loss of timing and synchrony in biological oscillators). Here, we show that gene expression noise counter-intuitively accelerates the evolution of a biological oscillator and, thus, can impart a benefit to living organisms. We used computer simulations to evolve two mechanistic models of a biological oscillator at different levels of gene expression noise. We first show that gene expression noise induces oscillatory-like dynamics in regions of parameter space that cannot oscillate in the absence of noise. We then demonstrate that these noise-induced oscillations generate a fitness landscape whose gradient robustly and quickly guides evolution by mutation towards robust and self-sustaining oscillation. These results suggest that noise can help dynamical systems evolve or learn new behavior by revealing cryptic dynamic phenotypes outside the bifurcation point.

Graphical Abstract

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title: Density-dependent attributes of schooling in small pelagic fishes

abstract: Many small pelagic fishes obligately form schools; some of these schools attain a remarkable size. Although the school is a fundamental and important ecological unit and is the site of biological interactions such as competition and predation, information on schooling processes in the field remains scarce. Here, we examined the quantitative relationships between population density and school size, the number of schools, and other school characteristics (i.e., packing density, volume, and cross-sectional area) in three species of small pelagic fishes: chub mackerel Scomber japonicus, Japanese sardine Sardinops melanostictus, and Japanese anchovy Engraulis japonicus. We found that school size increased almost linearly with population density, whereas the number of schools and other characteristics increased non-linearly with population density, whereby the rate of increase slowed radically as population density increased. These results indicate that, at low population densities, an increase in density causes an increase in both school size and the number of schools, whereas at higher population densities, an increase in density triggers the formation of larger schools rather than more schools. Furthermore, we found that the shapes of the relationships of all school characteristics with population density differed among species. Our results indicate that the schooling behaviour of small pelagic fishes is density-dependent, and responses to changes in density are species-specific. Our results provide insight into of how biological interactions such as intra- and inter-specific competition and predator-prey interactions mediate the density-dependent processes that underlie the population dynamics and community structure of small pelagic fishes in marine ecosystems.

category: zoology


title: Rethinking the Methods and Algorithms for Inner Speech Decoding - and Making them Reproducible

abstract: This study focuses on the automatic decoding of inner speech using noninvasive methods, such as electroencephalography (EEG)). While inner speech has been a research topic in philosophy and psychology for half a century, recent attempts have been made to decode nonvoiced spoken words by using various brain-computer interfaces. The main shortcomings of existing work are reproducibility and the availability of data and code. In this work, we investigate various methods (using Convolutional Neural Network (CNN), Gated Recurrent Unit (GRU), Long Short-Term Memory Networks (LSTM)) for the detection task of 5 vowels and 6 words on a publicly available EEG dataset. The main contributions of this work are (1) subject dependent vs. subject-independent approaches, (2) the effect of different preprocessing steps (Independent Component Analysis (ICA), down-sampling and filtering), and (3) word classification (where we achieve state-of-the-art performance on a publicly available dataset). Overall we achieve a performance accuracy of 35.20% and 29.21% when classifying 5 vowels and 6 words respectively in a publicly available dataset, using our tuned iSpeech-CNN architecture. All of our code and processed data are publicly available to ensure reproducibility. As such, this work contributes to a deeper understanding and reproducibility of experiments in the area of inner speech detection.

category: neuroscience


title: Galanin Analogs Prevent Seizure-Induced Respiratory Arrest

abstract:
ObjectiveSudden Unexpected Death in Epilepsy (SUDEP) accounts for 20% of mortality in those with recurrent seizures. While risk factors, monitoring systems, and standard practices are in place, the pathophysiology of SUDEP is still not well understood. Better knowledge of SUDEP and its potential mechanisms of action is crucial to reducing risk in this patient population and developing potential treatment options. Clinical studies and animal models of SUDEP suggest that diminished post-ictal respiratory control may be the dominant mechanism contributing to mortality. Recently, it was demonstrated that the depletion of the neuropeptide galanin in the amygdala occurs in human SUDEP. The amygdala plays a key role in the central integration of respiratory signaling; the depletion of galanin may represent a critical change that predisposes individuals to SUDEP.

MethodsTo evaluate the potential benefit of enhancing galaninergic signaling as a means to protect against SUDEP, we studied seizure-induced respiratory arrest (S-IRA) following central (intracerebroventricular, intra-amygdala) and systemic (intraperitoneal, subcutaneous) administration of galanin agonists. Seizure naive and seizure experienced (fully kindled) mice were tested.

ResultsCentral and systemically-administered galanin analogs protect against S-IRA in naive C57Bl/6J mice. Differential efficacy between receptor subtype-selective analogs varied based on the route of administration. Sub-chronic systemic administration at doses that reduced 6 Hz seizures also protected against S-IRA. Acute treatment benefits also extended to fully kindled mice subjected to tonic extension.

SignificanceThese data demonstrate that galanin agonists may be protective against post-ictal respiratory collapse.

KEY POINTSO_LICentral and systemic galanin agonists prevent seizure-induced respiratory arrest.
C_LIO_LIEfficacy was observed in three separate mouse strains under various experimental conditions.
C_LIO_LISub-chronic administration demonstrated galanin agonist protection against respiratory arrest.
C_LIO_LIAcute systemic administration also conferred protection against respiratory arrest following tonic extension.
C_LIO_LIGalanin analogs may represent a novel potential therapy in SUDEP-susceptible individuals.
C_LI


title: Fast & Scrupulous: Gesture-Based Alarms Improve Performances Under Several Mental Workloads. An ERSP Study.

abstract: In high-risk environments, fast and accurate responses to warning systems are essential to efficiently handle emergency situations and prevent potential catastrophic events. The aim of the present experiment was twofold: 1) investigating whether hand action videos, referred as to gesture alarms, are more efficient to trigger fast and accurate responses than text alarm messages (referred as written alarms), especially when the mental workload is high; and 2) investigating the brain activity in response to both types of alarms as a function of the level of mental workload. Regardless the level of mental workload, participants were found to be both faster and more accurate when responding to the gesture alarms than to the written alarms. Brain electrophysiological results suggest that this greater efficiency might be due to the fact that (compared to written alarms) gesture alarms facilitate the action execution reflected by the mu and beta power decrease observed around the response time window. Taken together, these results demonstrate that gesture alarm systems may help improving operators performances in emergency situations.


title: Neuronal temperature perception induces specific defenses that enable C. elegans to cope with the enhanced reactivity of hydrogen peroxide at high temperature

abstract: Hydrogen peroxide is the most common reactive chemical that organisms face on the microbial battlefield. The rate with which hydrogen peroxide damages biomolecules required for life increases with temperature, yet little is known about how organisms cope with this temperature-dependent threat. Here, we show that Caenorhabditis elegans nematodes use temperature information perceived by sensory neurons to cope with the temperature-dependent threat of hydrogen peroxide produced by the pathogenic bacterium Enterococcus faecium. These nematodes preemptively induce the expression of specific hydrogen peroxide defenses in response to perception of high temperature by a pair of sensory neurons. These neurons communicate temperature information to target tissues expressing those defenses via an insulin/IGF1 hormone. This strategy, which we call "enhancer sensing," is the first example of a multicellular organism inducing their defenses to a chemical when they sense an inherent enhancer of the reactivity of that chemical.


title: A Single-Cell Molecular Atlas of White Adipose Tissue Shows Differences in Myeloid and Lymphoid Cell Polarization in Type 2 Diabetes and HIV Infection

abstract: Subcutaneous white adipose tissue (scWAT) is a critical regulator of systemic metabolic homeostasis. HIV infection is accompanied by alterations in scWAT immune cells, which may contribute to the increased risk for cardiometabolic diseases in persons with HIV (PWH) and serve as a model to investigate cellular relationships. We generated the largest molecular atlas of scWAT in humans to date from 91 individuals using paired single-cell transcriptomic and proteogenomics to determine the scWAT immune profile in non-diabetic, pre-diabetic, and diabetic PWH, and HIV-negative diabetic persons. We identified a substantial increase in lipid- associated macrophages (LAMs) with glucose intolerance, and, in PWH only, CD4+ and CD8+ T effector memory cells were correlated with LAM proportion. Finally, we resolved adipogenic and fibrogenic cells at high resolution and found a strong association between LAMs and myofibroblasts. These results provide a high-resolution, multi-platform characterization of changes in scWAT composition that occur with metabolic disease and HIV.

category: immunology


title: Wolbachia wAlbB inhibit dengue and Zika infection in the mosquito Aedes aegypti with an Australian background

abstract:
Biological control of mosquito vectors using the insect-specific bacteria Wolbachia is an emerging strategy for the management of human arboviral diseases. We recently described the development of a strain of Ae. aegypti infected with the Wolbachia strain wAlbB (referred to as the wAlbB2-F4 strain) through simple backcrossing of wild type Australian mosquitoes with a wAlbB infected Ae. aegypti strain from the USA. Field releases of male wAlbB2-F4 mosquitoes resulted in the successful suppression of a wild population of mosquitoes in the trial sites by exploiting the strains Wolbachia-induced cytoplasmic incompatibility. We now demonstrate that the strain is resistant to infection by dengue and Zika viruses and is genetically similar to endemic Queensland populations. There was a fourfold reduction in the proportion of wAlbB2-F4 mosquitoes that became infected following a blood meal containing dengue 2 virus (16.7%) compared to wild type mosquitoes (69.2%) and a 6-7 fold reduction in the proportion of wAlbB2-F4 mosquitoes producing virus in saliva following a blood meal containing an epidemic strain of Zika virus (8.7% in comparison to 58.3% in wild type mosquitoes). Restriction-site Associated DNA (RAD) sequencing revealed that wAlbB2-F4 mosquitoes have > 98% Australian ancestry, confirming the successful introduction of the wAlbB2 infection into the Australian genomic background through backcrossing. Genotypic and phenotypic analyses showed the wAlbB2-F4 strain retains the insecticide susceptibility phenotype and genotype of the Australian mosquitoes. We demonstrate that the Wolbachia wAlbB2-F4, in addition to being suitable for suppression programs, can be effective in population replacement programs given its high inhibition of virus infection in mosquitoes. The ease at which a target mosquito population can be transfected with wAlbB2, while retaining genotypes and phenotypes of the target population, shows the robustness of this strain as a biocontrol agent against the Ae. aegypti mosquito itself as well as the pathogens it transmits.

IMPORTANCEEpidemics of arthopod-borne virus (arbovirus) diseases affect millions of people and are becoming more frequent and widespread. A successful strategy to control these diseases is by infecting mosquito populations with benign, insect-specific Wolbachia bacteria that render mosquitoes refractory to infection with pathogenic arboviruses. Here we show that a strain of the major mosquito vector Ae. aegypti that was infected with Wolbachia following a simple back-cross mating procedure is refractory to infection with dengue and Zika viruses. Importantly, the genetic background of the strain is equivalent to the target population, which is important for persistence of the strain and regulatory approval.

category: microbiology


title: The Venturia inaequalis effector repertoire is expressed in waves, and is dominated by expanded families with predicted structural similarity to avirulence proteins from other fungi

abstract:
BackgroundScab, caused by the biotrophic fungus Venturia inaequalis, is the most economically important disease of apples worldwide. During infection, V. inaequalis occupies the subcuticular environment, where it secretes virulence factors, termed effectors, to promote host colonization. However, in the presence of corresponding host resistance proteins, these effectors are recognized as avirulence determinants to activate plant defences. To develop durable control strategies against scab, a better understanding of the molecular mechanisms underpinning subcuticular growth by V. inaequalis is required. Likewise, more information is needed on the role that effectors play in activating, suppressing or circumventing resistance protein-mediated defences.

ResultsWe generated the first comprehensive RNA-seq transcriptome of V. inaequalis during colonization of apple. Analysis of this transcriptome revealed five in planta gene expression clusters or waves corresponding to three specific infection stages: early, mid and mid-late infection. Early infection was characterized by genes encoding plant cell wall-degrading enzymes (PCWDEs) and proteins associated with oxidative stress responses. Mid infection was characterized by genes encoding transporter proteins. Finally, mid-late infection was characterized by genes encoding PCWDEs and effector candidates (ECs), with most ECs belonging to expanded protein families. To gain insights into function, AlphaFold2 was used to predict the tertiary structures of proteinaceous ECs. Strikingly, many ECs were predicted to have structural similarity to avirulence proteins from other plant-pathogenic fungi, including members of the MAX, LARS, ToxA and FOLD structural effector families. In addition, several other ECs, including an EC family with amino acid similarity to the AvrLm6 effector from Leptosphaeria maculans, were predicted to adopt a KP6/ferredoxin-like fold. Thus, proteins with a KP6/ferredoxin-like fold may represent yet another structural family of effectors shared among plant-pathogenic fungi.

ConclusionsOur study reveals the transcriptomic profile underpinning subcuticular growth by V. inaequalis, and reinforces the idea that fungal effectors share a limited number of structural folds. Importantly, our study also provides an enriched list of V. inaequalis ECs that can be investigated for roles in virulence and avirulence, and raises the possibility that apple resistance proteins can be engineered to recognize EC family folds or host components targeted by multiple EC family members.


title: SUMO4 regulates DNA double-strand break repair independently of conjugation.

abstract: The small ubiquitin-like modifier (SUMO) family is composed of five members, SUMO1, the highly similar SUMO2/SUMO3, SUMO4 and the tissue-specific SUMO5 (SUMO1P1). Sequence variation in SUMO4 is thought to prevent its maturation, resulting in an un-conjugatable SUMO isoform, and consequently, its functions are poorly understood. Here we show for the first time that SUMO4 promotes DNA double-strand break signalling in a manner distinct from SUMO1 or SUMO2/3. We show that SUMO4 function depends on interaction with partner proteins through SUMO interacting motifs and, on its inability, to be conjugated. We show that SUMO4 promotes the activity of the SUMO protease SENP1. In the absence of SUMO4, reduced SENP1 catalytic activity results in hyperSUMOylation that unbalances the recruitment of several DSB repair factors, including RAP80. These data reveal that SUMO4 acts as a buffer for the SUMOylation system.

category: molecular biology


title: Structural architecture and brain network efficiency links polygenic scores to intelligence

abstract: Intelligence is highly heritable. Genome-wide association studies (GWAS) have shown that thousands of alleles contribute to variation in intelligence with small effect sizes. Polygenic scores (PGS), which combine these effects into one genetic summary measure, are increasingly used to investigate polygenic effects in independent samples. Whereas PGS explain a considerable amount of variance in intelligence, it is largely unknown how brain structure and function mediate this relationship. Here we show that individuals with higher PGS for educational attainment and intelligence had higher scores on cognitive tests, larger surface area, and more efficient fiber connectivity derived by graph theory. Fiber network efficiency as well as surface of brain areas partly located in parieto-frontal regions were found to mediate the relationship between PGS and cognitive performance. These findings are a crucial step forward in decoding the neurogenetic underpinnings of intelligence, as they identify specific regional networks that link polygenic predisposition to intelligence.

category: genetics


title: Occurrence and quantities of DNA modifications across the tree of life

abstract: Enzymatic DNA modifications like methylcytosine (5mdC), methyladenine (N6mdA), or hydroxymethylcytosine (5hmdC) are key for chromatin function, gene expression regulation, and antiviral defense, but they remain understudied in non-model organisms. We established a mass spectrometric method for the sensitive and accurate quantification of enzymatic DNA modifications, and analyzed 85 bacterial genomes, 19 plant samples, 41 tissues from 12 animal species, 6 yeast species, and two archaeal species. We report no or only very low concentrations of DNA modifications in yeast and insects, but find DNA modifications universal to both bacteria and higher eukaryotes. Specifically for prokaryotes, our dataset indicates that evolutionary relationships and host-pathogen interactions, but not the ecological niche in general, select for a similar degree of DNA modification. In higher eukaryotes, largest concentration differences between tissues are detected for 5hmdC. Our dataset further reveals unique biological cases that warrant attention in the study of DNA modifications. For instance, while our data shows that most species contain just one dominating DNA modification, we detect all dominianting DNA modifications (5mdC, N6mdA, and 5hmdC) to exist in parallel in Raphanus sativus. Other plant species, like onion, sunflower, or the grass big bluestem, can have more than 35% of cytosines methylated. Finally, 5hmdC, so far mostly studied in the vertebrate central nervous system, was identified to reach a concentration of up to 8% of all cytosines in the Oman garra brain, and was also detected in several plants, like Lepidium sativum. The present study underscores the exploitation of biological diversity for studying DNA modifications.

category: genomics


title: DCATS: differential composition analysis for complex single-cell experimental designs

abstract: Differential composition analysis - the identification of cell types that have statistically significantly change in abundance between multiple experimental conditions - is one of the most common tasks in single cell omic data analysis. However, it remains challenging to perform differential composition analysis in the presence of complex experimental designs and uncertainty in cell type assignment. Here, we introduce a statistical model and an open source R package, DCATS, for differential composition analysis based on a beta-binomial regression framework that addresses these challenges. Our empirical evaluation shows that DCATS consistently maintain high sensitively and specificity compared to state-of-the-art methods.


title: Strain design optimization using reinforcement learning

abstract:
Engineered microbial cells present a sustainable alternative to fossil-based synthesis of chemicals and fuels. Cellular synthesis routes are readily assembled and introduced into microbial strains using state-of-the-art synthetic biology tools. However, the optimization of the strains required to reach industrially feasible production levels is far less efficient. It typically relies on trial-and-error leading into high uncertainty in total duration and cost. New techniques that can cope with the complexity and limited mechanistic knowledge of the cellular regulation are called for guiding the strain optimization.

In this paper, we put forward a multi-agent reinforcement learning (MARL) approach that learns from experiments to tune the metabolic enzyme levels so that the production is improved. Our method is model-free and does not assume prior knowledge of the microbes metabolic network or its regulation. The multi-agent approach is well-suited to make use of parallel experiments such as multi-well plates commonly used for screening microbial strains.

We demonstrate the methods capabilities using the genome-scale kinetic model of Escherichia coli, k-ecoli457, as a surrogate for an in vivo cell behaviour in cultivation experiments. We investigate the methods performance relevant for practical applicability in strain engineering i.e. the speed of convergence towards the optimum response, noise tolerance, and the statistical stability of the solutions found. We further evaluate the proposed MARL approach in improving L-tryptophan production by yeast Saccharomyces cerevisiae, using publicly available experimental data on the performance of a combinatorial strain library.

Overall, our results show that multi-agent reinforcement learning is a promising approach for guiding the strain optimization beyond mechanistic knowledge, with the goal of faster and more reliably obtaining industrially attractive production levels.

Author summaryEngineered microbial cells offer a sustainable alternative solution to chemical production from fossil resources. However, to make the chemical production using microbial cells economically feasible, they need to be substantially optimized. Due to the biological complexity, this optimization to reach sufficiently high production is typically a costly trial and error process.

This paper presents an Artificial Intelligence (AI) approach to guide this task. Our tool learns a model from previous experiments and uses the model to suggest improvements to the engineering design, until a satisfactory production performance is reached. This paper evaluates the behaviour of the proposed AI method from several angles, including the amount of experiments needed, the tolerance to noise as well as the stability of the proposed designs.


title: scMAGS: Marker gene selection from scRNA-seq data for spatial transcriptomics studies

abstract: Single-Cell RNA sequencing (scRNA-seq) has provided unprecedented opportunities for exploring gene expression and thus uncovering regulatory relationships between genes at the single cell level. However, scRNA-seq relies on isolating cells from tissues. Thus, the spatial context of the regulatory processes is lost. A recent technological innovation, spatial transcriptomics, allows to measure gene expression while preserving spatial information. A first step in the spatial transcriptomic analysis is to identify the cell type which requires a careful selection of cell-specific marker genes. For this purpose, currently scRNA-seq data is used to select limited number of marker genes from among all genes that distinguish cell types from each other. This study proposes scMAGS (single-cell MArker Gene Selection), a new approach for marker gene selection from scRNA-seq data for spatial transcriptomics studies. scMAGS uses a filtering step in which the candidate genes are extracted prior to the marker gene selection step. For the selection of marker genes, cluster validity indices, Silhouette index or Calinski-Harabasz index (for large datasets) are utilized. Experimental results showed that, in comparison to the existing methods, scMAGS is scalable, fast and accurate. Even for the large datasets with millions of cells, scMAGS could find the required number of marker genes in a reasonable amount of time with less memory requirements.


title: An Orobanche cernua x Orobanche cumana segregating population provides insight into the regulation of germination specificity in a parasitic plant

abstract: Germination of seeds of Orobanche species requires specific chemicals exuded by host roots. A family of "divergent" KARRIKIN INSENSITIVE2 (KAI2d) genes encode proteins that recognize strigolactone (SL) class germination simulants. We explored specificity of germination stimulant detection by analyzing interspecific segregants of a cross between Orobanche cernua and O. cumana, closely related species that differ in stimulant response. O. cernua parasitizes tomato and germinates in response to the SL orobanchol, while O. cumana parasitizes sunflower and responds to dehydrocostus lactone (DCL). KAI2d genes were catalogued in parents and in segregants that showed stimulant specificity. KAI2d genes were also functionally assayed in the Arabidopsis kai2 mutant background. We identified five full-length KAI2d genes in O. cernua and eight in O. cumana. The O. cernua KAI2d2, as well as its ortholog in O. cumana, are associated with SL perception. A cluster of O. cumana KAI2d genes was genetically linked to DCL perception, although no specific receptor gene was identified by heterologous complementation. These findings support the KAI2d-mediated perception of SLs, but fall short of explaining how O. cumana perceives DCL. The ability of some O. cumana KAI2d genes to detect SLs points to the involvement of additional factors in regulating stimulant specificity.


title: Information maximization explains state-dependent synaptic plasticity and memory reorganization during non-rapid eye movement sleep

abstract: Slow waves during the non-rapid eye movement (NREM) sleep reflect the alternating up and down states of cortical neurons; global and local slow waves promote memory consolidation and forgetting, respectively. Furthermore, distinct spike-timing-dependent plasticity (STDP) operates in these up and down states. The contribution of different plasticity rules to neural information coding and memory reorganization remains unknown. Here, we show that optimal synaptic plasticity for information maximization in a cortical neuron model provides a unified explanation for these phenomena. The model indicates that the optimal synaptic plasticity is biased towards depression as the baseline firing rate increases. This property explains the distinct STDP observed in the up and down states. Furthermore, it explains how global and local slow waves predominantly potentiate and depress synapses, respectively, if the background firing rate of excitatory neurons declines with the spatial scale of waves as the model predicts. The model provides a unifying account of the role of NREM sleep, bridging neural information coding, synaptic plasticity, and memory reorganization.


title: Oscillatory dynamics underlying emotion-cognition integration: differential role of theta and alpha oscillations

abstract: A fundamental aspect of human mental life is the seamless ability for integration of emotion and cognition. Despite progress regarding the spatial architecture of Emotion-Cognition Integration (ECI), the time course of ECI processes remains unclear. To examine the temporal organization of brain oscillations underpinning ECI, we simultaneously manipulated emotional valence of stimuli and cognitive task demand while recording electrophysiological responses of 61 participants. They were asked to complete tasks with low (body-part judgement) and high (laterality judgement) cognitive demand while viewing other people photographs that varied on dimensions of laterality (left or right), body-part (hand or foot), and emotional valence (pain or no pain). We found increased reaction times and error rates in pain versus no pain during laterality judgement relative to body-part judgement, suggesting reciprocal inhibition between emotion and cognition. EEG results showed that 1) emotion processing (valence) occurred first in the theta band from 144 to 372 ms; 2) cognitive processing (laterality) took place in the theta band from 332 to 608 ms; 3) emotional and cognitive processes were integrated in the alpha band from 268 ms and lasted to 800 ms. These findings reveal oscillatory dynamics of the processing and integration of emotion and cognition, providing further insights into the underlying neurophysiology. This may ultimately contribute to our understanding of ECI processing in psychopathology.


title: Multi-finger Receptive Field Properties in Primary Somatosensory Cortex: A Revised Account of the Spatio-Temporal Integration Functions of Area 3b

abstract: AO_SCPLOWBSTRACTC_SCPLOWThe leading view in the somatosensory system indicates that area 3b serves as a cortical relay site that encodes cutaneous (tactile) features limited to individual digits. Our recent work argues against this model by showing that cells in area 3b integrate information from cutaneous and proprioceptive modalities. Here, we further test this model, by studying the multi-finger neural integration properties of area 3b. In contrast to the prevailing view, we found that most cells in area 3b have a receptive field (RF) that extends to multiple digits. Responses to tactile stimulation emerged earlier in cells with a multi-digit (MD) vs. single-digit (SD) RF. We also found that the RF size of MD cells (the number of responsive digits) increased across time, and the orientation preference across digits was highly correlated. Taken together, these data provide strong evidence that area 3b plays a larger role in generating neural representations of tactile objects, as opposed to just being a  feature detector relay site.


title: Single cell Transcriptome and T cell Repertoire Mapping of the Mechanistic Signatures and T cell Trajectories Contributing to Vascular and Dermal Manifestations of Behcet Disease

abstract: Behcets disease (BD) is a form of vasculitis characterized by complex multi-organ manifestations that may frequently recur and induce major tissue damage. Although genetic association studies have identified a number of risk factors, the etiology of BD and its tissue manifestations remains unknown, and the landscape of immune responses in BD is opaque, particularly in terms of inflammatory recurrence. In this study, we mapped the transcriptomes of the immune cell compartment in BD at single-cell resolution, sampling both circulation and affected skin in order to chart the immune interplay driving pathogenesis. Through comprehensive expression and communication analysis of the twenty major cell types identified, we observe striking mechanistic differences in immune response between BD skin lesions and peripheral circulation involving TNF signaling and T cell migration. Through integrated TCR sequencing, we further discover a pattern of clonal sharing between circulating and skin CD8+T cell populations along a trajectory defined by the acquisition of tissue-residential properties. In addition, we also identify a population of expanded CD4+ Tregs with the propensity to produce IL-32. Instead of suppressing effector T cell proliferation and function, IL-32 triggers increased expression of CD97, and may thus encourage prolonged local T cell activity in the skin. Collectively, our data serve to advance understandings of contributions of varying immune cell types to BD pathogenesis in the vasculature and skin, as well as the lifecycle patterns of T cells clones in this context. These data may also assist in further investigations of the mechanisms contributing to Treg dysfunction in systemic autoimmunity, while generating a conceptual model of T cell function contributing to BD recurrence.


title: Human CD4+/CD8α+ regulatory T cells induced by Faecalibacterium prausnitzii protect against intestinal inflammation

abstract: Faecalibacterium prausnitzii (F. prausnitzii), a dominant bacterium of the human microbiota, is decreased in patients with inflammatory bowel diseases (IBD) and exhibits anti-inflammatory effects. In human, colonic lamina propria contains IL-10-positive, Foxp3-negative regulatory T cells (Treg) characterized by a double expression of CD4 and CD8 (DP8) and a specificity for F. prausnitzii. This Treg subset is decreased in IBD. The in vivo effect of DP8 cells has not been evaluated yet. Here, using a humanized model of NOD.Prkcscid IL2r{gamma}-/- (NSG) immunodeficient mouse strain that expresses human leucocyte antigen D-related 4 (HLA-DR4) but not murine class II (NSG-Ab{degrees} DR4), we demonstrated a protective effect of DP8 Tregs combined with F. prausnitzii administration in a colitis model. In a cohort of patients with IBD, we showed an independent association between the frequency of circulating DP8 cells and disease activity. Finally, we pointed out a positive correlation between F. prausnitzii-specific DP8 Tregs and the amount of F. prausnitzii in fecal microbiota in healthy individuals and patients with ileal Crohns disease.


title: Commensal fungi Candida albicans modulates dietary high-fat induced alterations in metabolism, immunity, and gut microbiota

abstract: Obesity and its associated diseases represent a growing health concern. Despite a strong correlation between gut microbiota and obesity, a limited study is available to suggest the direct involvement of fungus in host physiology. Candida albicans survives as a commensal fungus in the gastrointestinal tract, and its excessive growth causing infections in immune-suppressed individuals is widely accepted. However, any mutualistic relationship that may exist between C. albicans and the host remains outstanding. Here we show that the dietary C. albicans do not cause any noticeable infections, and our metagenomics analyses suggest that it colonizes in the gut and modulates microbiome dynamics which in turn negates the high-fat diet induced uncontrolled body weight gain, metabolic hormonal imbalances, and inflammatory response. Interestingly, adding C. albicans to a non-obesogenic diet stimulates the appetite regulated hormones and helped the mice gain healthy body weight. In concert, our results suggest a mutualism between C. albicans and the host, therefore, contrary to the notion, C. albicans is not always an adversary rather a bonafide admirable companion of the host. Finally, we discuss its potential translational implication as a probiotic especially in obese people or people dependent on high fat calorie intakes to manage obesity and associated complications.


title: Comparative analysis of genome-wide protein-DNA interactions across domains of life reveals unique binding patterns for hypersaline archaeal histones

abstract:
DNA-binding proteins with roles in chromatin architecture and transcriptional regulation are present in all three domains of life. Histones package DNA and regulate gene expression in eukaryotes, and find their evolutionary origin in the domain of life Archaea. Previously characterised archaeal histones have a somewhat conserved functional role in nucleosome formation and DNA packaging. However, previous research has indicated that the histone-like proteins of high salt-adapted archaea, or halophiles, appear to function differently. The sole histone protein encoded by the model halophilic species Halobacterium salinarum is non-essential, is involved in direct and indirect transcriptional regulation, and does not appear to package DNA. Here we use protein-DNA binding assays, computational analysis, and quantitative phenotyping to compare DNA binding patterns across halophilic histone proteins, bacterial and archaeal TFs, NAPs, and eukaryotic histones. Like TFs, halophilic histones bind the genome too sparsely to compact the genome. However, unlike TFs, binding occurs in both coding and intergenic regions. Unlike histones, halophilic histone occupancy is not depleted at the start sites of genes, and halophilic genomes lack the dinucleotide periodicity known to facilitate histone binding. We detect unique sequence preferences for histone binding in halophiles. Together these data suggest that the non-essentiality and genome-wide binding features of halophilic histone-like proteins are conserved across halophiles; they bind DNA in ways resembling both TFs and chromatin proteins, but do not appear to play a role in forming chromatin.

IMPORTANCEMost cells in eukaryotic species - from yeast to humans- possess histone proteins that pack and unpack DNA in response to environmental cues. These essential proteins regulate the genes necessary for important cellular processes, including development and stress protection. The domain of life Archaea represent the evolutionary progenitors of eukaryotes. The universal conservation of the primary sequences of histone proteins across archaeal lineages suggests that eukaryotic histones originated in the Archaea. However, archaeal histones lack N-terminal tails and, in some species, package DNA in a continuous helix with no linker DNA between nucleosomes. We recently discovered that histones in hypersaline adapted archaeal species do not package DNA, and can act like transcription factors (TFs) to regulate stress response gene expression. Here we compare hypersaline histone function to a variety of DNA binding proteins across the tree of life, revealing a mosaic of functions for hypersaline-adapted histones.


title: Enhancing the Fitness of Embryoid Bodies and Organoids by Chemical Cytoprotection

abstract: Embryoid bodies (EBs) and self-organizing organoids derived from human pluripotent stem cells (hPSCs) recapitulate tissue development in a dish and hold great promise for disease modeling and drug development. However, current protocols are hampered by cellular stress and apoptosis during cell aggregation, resulting in variability and impaired cell differentiation. Here, we demonstrate that EBs and various organoid models (e.g., brain, gut, and kidney) can be optimized by using the CEPT small molecule cocktail, a polypharmacology approach that ensures cytoprotection and cell survival. Application of CEPT (chroman 1, emricasan, polyamines, trans-ISRIB) for just 24 hours during cell aggregation has long-lasting consequences affecting morphogenesis, gene expression, and cellular differentiation. Various qualification methods confirmed that CEPT treatment consistently improved EB and organoid fitness as compared to the widely used ROCK inhibitor Y-27632. Collectively, we discovered that stress-free cell aggregation and superior cell survival in the presence of CEPT are critical quality control determinants that establish a robust foundation for bioengineering complex tissue and organ models.

category: developmental biology


title: An uncommon garden experiment: microenvironment has stronger influence on phenotypic variation than epigenetic memory in the clonal Lombardy poplar

abstract: Environmental changes can trigger phenotypic variation in plants through epigenetic mechanisms, but strong genetic influences on epigenetic variation and phenotypes make it difficult to isolate and study these effects. We investigated phenotypic plasticity using the Lombardy poplar (Populus nigra cv.  Italica Duroi), a globaly distributed clonal tree. We surveyed 14 functional traits related to tree growth, ecophysiological and phenological processes in poplar ramets collected along a wide geographical range in Europe and planted under common garden conditions. We investigated whether phenotypic variation was related to geography and historical bioclimatic data of the ramets sites of origin using linear mixed effect models. We found significant differences in among ramets from different geographic origins in tree height, number of stems per ramet and duration of bud flush. However, microenvironmental variation in the common garden, captured via block effects, had an even bigger impact on phenotypic variation than the environmental conditions at the sites of origin. Our results show that phenotypic variation in the ramets might be associated to the climate origin from different climates, suggesting possible epigenetic memory. However, such legacy effects might be quickly outweighed by new environmental conditions.

category: ecology


title: Classifying the activity states of small vertebrates using automated VHF telemetry

abstract:
O_LIThe most basic behavioural states of animals can be described as active or passive. However, while high-resolution observations of activity patterns can provide insights into the ecology of animal species, few methods are able to measure the activity of individuals of small taxa in their natural environment. We present a novel approach in which the automated VHF radio-tracking of small vertebrates fitted with lightweight transmitters (< 0.2 g) is used to distinguish between active and passive behavioural states.
C_LIO_LIA dataset containing > 3 million VHF signals was used to train and test a random forest model in the assignment of either active or passive behaviour to individuals from two forest-dwelling bat species (Myotis bechsteinii (n = 50) and Nyctalus leisleri (n = 20)). The applicability of the model to other taxonomic groups was demonstrated by recording and classifying the behaviour of a tagged bird and by simulating the effect of different types of vertebrate activity with the help of humans carrying transmitters. The random forest model successfully classified the activity states of bats as well as those of birds and humans, although the latter were not included in model training (F-score 0.96-0.98).
C_LIO_LIThe utility of the model in tackling ecologically relevant questions was demonstrated in a study of the differences in the daily activity patterns of the two bat species. The analysis showed a pronounced bimodal activity distribution of N. leisleri over the course of the night while the night-time activity of M. bechsteinii was relatively constant. These results show that significant differences in the timing of species activity according to ecological preferences or seasonality can be distinguished using our method.
C_LIO_LIOur approach enables the assignment of VHF signal patterns to fundamental behavioural states with high precision and is applicable to different terrestrial and flying vertebrates. To encourage the broader use of our radio-tracking method, we provide the trained random forest models together with an R-package that includes all necessary data-processing functionalities. In combination with state-of-the-art open-source automated radio-tracking, this toolset can be used by the scientific community to investigate the activity patterns of small vertebrates with high temporal resolution, even in dense vegetation.
C_LI

category: animal behavior and cognition


title: Population genetic analysis of Ophidiomyces ophidiicola, the causative agent of snake fungal disease, indicates recent introductions to the USA

abstract: Snake fungal disease (SFD; ophidiomycosis), caused by the pathogen Ophidiomyces ophidiicola (Oo), has been documented in wild snakes in North America and Eurasia, and is considered an emerging disease in the eastern USA. However, a lack of historical disease data has made it challenging to determine whether Oo is a recent arrival to the USA or whether SFD emergence is due to other factors. Here, we examined the genomes of 82 Oo strains to determine the pathogens history in the eastern USA. Oo strains from the USA formed a clade (Clade II) distinct from European strains (Clade I), and molecular dating indicated that these clades diverged too recently ([~]2,000 years ago) for transcontinental dispersal of Oo to have occurred via natural snake movements across Beringia. A lack of nonrecombinant intermediates between clonal lineages in Clade II indicates that Oo has actually been introduced multiple times to North America from an unsampled source population, and molecular dating indicates that several of these introductions occurred within the last few hundred years. Molecular dating also indicated that the most common Clade II clonal lineages have expanded recently in the USA, with time of most recent common ancestor mean estimates ranging from 1985-2007 CE. The presence of Clade II in captive snakes worldwide demonstrates a potential mechanism of introduction and highlights that additional incursions are likely unless action is taken to reduce the risk of pathogen translocation and spillover into wild snake populations.


title: A multiple comorbidities mouse model to assess atherosclerosis progression following lung infection in ApoE deficient mice

abstract:
BackgroundInflammation is a risk factor for atherosclerosis progression. Hospitalisation for pneumonia is associated with increased risk of cardiovascular disease. Herein, we describe a multiple comorbidities murine model to study the impact of bacterial pneumonia on atherosclerosis.

MethodsFirstly, a minimal infectious dose of Streptococcus pneumoniae (TIGR4 strain) to produce clinical pneumonia with a low mortality rate (20%) was established. C57Bl/6 ApoE-/- mice were fed a high-fat diet prior to administering intranasally 105 colony forming units of TIGR4 or phosphate buffered saline (PBS). At days 2, 7 and 28 post inoculation (PI), the lungs of mice were imaged by MRI and PET. Mice were euthanised and investigated for changes in systemic inflammation and changes in lung morphology using ELISA, Luminex assay and real-time PCR.

ResultsTIGR4 inoculated mice presented with varying degreess of lung infiltrate, pleural effusion and consolidation on MRI at all timepoints up to 28 days PI. Moreover, PET scans identified significantly higher FDG uptake in the lungs of TIGR4 inoculated mice up to 28 days PI. Majority (90%) TIGR4-inoculated mice developed pneumococcal-specific IgG antibody response at 28 days PI. Consistent with these observations, TIGR4 inoculated mice displayed significantly increased inflammatory gene expression (IL-1{beta} & IL6) in the lungs and significantly increased levels of circulating inflammatory protein (CCL3) at 7- and 28-days PI respectively.

ConclusionsOur mouse model presents a discovery tool to understand the link between acute infections, including pneumonia, and increased cardiovascular disease risk in humans with inflammation as the mechanistic catalyst.

Graphical Abstract

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title: Pin1-promoted SUMOylation of RNF168 restrains its chromatin accumulation.

abstract: The E3 ubiquitin ligase RNF168 is a rate-limiting component of DNA double-strand break signalling that acts to amplify histone ubiquitylation. The confining of RNF168 chromatin spreading around DNA break sites has been related to constraining its expression levels and to the removal of ubiquitin-conjugates to which it binds. Here we identify a new mechanism that suppresses RNF168 amplification at chromatin. We show that depletion of Peptidyl-prolyl cis-trans isomerase NIMA-interacting 1 (Pin1) or mutation of the Pin1-binding site of RNF168 increases DNA-damage site accumulation of RNF168 to supraphysiological levels without impacting RNF168 expression levels. Pin1 promotes SUMOylation of RNF168 by SUMO2/3, and a SUMO conjugation site on RNF168 is required to restrict RNF168 accumulations. We find Pin1-SUMO mediated-regulation of RNF168 is associated with cellular radioresistance. These data demonstrate that the regulation of chromatin ubiquitylation is actively suppressed by modification of RNF168 to counteract excessive RNF168-ubiquitin spreading.


title: Cdk1-mediated threonine phosphorylation of Sam68 modulates its RNA binding, alternative splicing activity, and cellular functions

abstract: Sam68 is a member of the STAR family of proteins that directly link signal transduction with post-transcriptional gene regulation. Sam68 controls the alternative splicing of many oncogenic proteins and its role is modulated by post-translational modifications, including serine/threonine phosphorylation, that differ at various stages of the cell cycle. However, the molecular basis and mechanisms of these modulations remain largely unknown. Here, we combined mass spectrometry, NMR spectroscopy, and cell biology techniques to provide a comprehensive post-translational modification (PTM) mapping of Sam68 at different stages of the cell cycle in HEK293 and HCT116 cells. We established that Sam68 is specifically phosphorylated at T33 and T317 by Cdk1, and demonstrated that these phosphorylation events reduce the binding of Sam68 to RNA, control its cellular localization, and reduce its alternative splicing activity, leading to a reduction in the induction of apoptosis and an increase in the proliferation of HCT116 cells.


title: Gene editing for accelerated breeding in wheat

abstract: Diseases adversely affect grain yield of crop plants. Leaf rust is a major disease of wheat. As race-specific resistance breaks down, introduction of newer sources of resistance often from older accessions is necessary. Linkage drag from the donor accessions adversely impacts grain yield. As a result, CIMMYT breeding effort has shifted to using durable resistance, also referred to as race non-specific host resistance, which allows slow rusting but maintains grain yield. One of the key genes for durable resistance is Lr67 and the resistant form of this gene is absent in CIMMYT elite lines. Hence, we have initiated efforts to convert the susceptible copy of Lr67 into its resistant form directly in these elite lines using gene editing. This would eliminate backcrossing and thus save time as well as eliminate linkage drag that would accompany the resistant copy of the gene if it were to be introgressed from an older accession. As first steps, we have isolated and sequenced the genomic copies from each of the A, B, and D genome of the Lr67 gene from three elite lines and an experimental line. Identification of more than 50 single nucleotide polymorphisms (SNPs) in the open reading frames (ORF) among these lines would be useful in designing the guide RNA molecules with precision. Further, we have streamlined genetic transformation of these elite lines, a prerequisite step for gene editing.


title: Agrobacterium mediated transformation and deciphering SNPs in TaLr67 gene homeologs for gene editing in wheat.


title: Cryo-EM structures of human zinc transporter ZnT7 reveal the mechanism of Zn2+</var> uptake into the Golgi apparatus

abstract: Zinc ions (Zn2+) are vital to most cells, with the intracellular concentrations of Zn2+ being tightly regulated by multiple zinc transporters located at the plasma and organelle membranes. We herein present the 2.8-2.9 [A]-resolution cryo-EM structures of a Golgi-localized human Zn2+/H+ antiporter ZnT7 (hZnT7) in its outward- and inward-facing forms. Cryo-EM analyses showed that hZnT7 exists as a homodimer via tight interactions in both the cytosolic and transmembrane (TM) regions of two protomers, each of which contains a single Zn2+-binding site in its TM domain. hZnT7 undergoes a TM-helix rearrangement to create a negatively charged cytosolic cavity for Zn2+ entry in the inward-facing form and a widened luminal cavity for Zn2+ release in the outward-facing form. An exceptionally long cytosolic histidine-rich loop characteristic of hZnT7 can bind at least two Zn2+ ions, likely facilitating Zn2+ recruitment from the cytosol. Unique mechanisms of hZnT7-mediated Zn2+ uptake into the Golgi are proposed.


title: N1-methylpseudouridine-incorporated mRNA enhances exogenous protein expression and suppresses immunogenicity in primary human fibroblast-like synoviocytes

abstract: Studies conducted using murine arthritis models have indicated that the use of in vitro-transcribed messenger RNA (IVT mRNA) is an effective therapeutic approach for joint diseases. However, the use of IVT mRNA in human synovial cells has not been widely studied. Recently, the outbreak of the novel coronavirus disease has accelerated the development of innovative mRNA vaccines such as those containing a modified nucleic acid, N1-methylpseudouridine-5'-triphosphate (m1{psi}). IVT mRNA is an attractive tool for biological experiments and drug discovery. To verify the protein expression of IVT mRNA in vitro, primary cultured human fibroblast-like synoviocytes (FLS) were transfected with enhanced green fluorescent protein (EGFP) mRNA with or without m1{psi} incorporation. EGFP was detected using western blotting and fluorescence microscopy. A multiplex assay was performed to comprehensively understand IVT mRNA-induced immunogenicity. FLS transfected EGFP mRNA containing m1{psi} generated higher levels of EGFP than unmodified EGFP mRNA or control RNAs. The multiplex assay of the FLS culture supernatant revealed that concentrations of IL-6, TNF-, and CXCL10 were upregulated by unmodified EGFP mRNA, whereas they were suppressed by EGFP mRNA with m1{psi}. Overall, m1{psi} incorporation enhanced protein expression and decreased cytokine expressions in primary cultured FLS. The findings may contribute to arthritis research.


title: Stem cell homeostasis regulated by hierarchy and neutral competition

abstract: Tissue stem cells maintain themselves through self-renewal while constantly supplying differentiating cells. As a mechanism of stem cell homeostasis, two distinct models were proposed: the classical model states that there are hierarchy among stem cells and master stem cells provide stem cells by asymmetric divisions, whereas the recent model states that stem cells are equipotent and neutrally competing each other. However, its mechanism is still controversial in several tissues and species. Here, we developed a mathematical model linking these two models. Our theoretical analysis showed that the model with the hierarchy and neutral competition, called the hierarchical neutral competition (hNC) model, exhibited bursts in clonal expansion, unlike existing models. Furthermore, the scaling law in clone size distribution, thought to be a unique characteristic of the recent model, was satisfied in the hNC model. Based on these findings, we proposed a criterion for distinguishing the three models by experiments.


title: Full-length transcriptomic analysis in murine and human heart reveals diversity of PGC-1α promoters and isoforms regulated distinctly in myocardial ischemia and obesity

abstract:
BackgroundPeroxisome proliferator-activated receptor gamma coactivator-1 alpha (PGC-1) acts as a transcriptional coactivator and regulates mitochondrial function. Various isoforms are generated by alternative splicing and differentially regulated promoters. In the heart, total PGC-1 deficiency knockout leads to dilatative cardiomyopathy, but knowledge on the complexity of cardiac isoform expression of PGC-1 remains sparse. Thus, this study aims to generate a reliable dataset on cardiac isoform expression pattern by long-read mRNA sequencing, followed by investigation of differential regulation of PGC-1 isoforms under metabolic and ischemic stress, using high-fat-high-sucrose-diet-induced obesity and a murine model of myocardial infarction.

Methods and ResultsMurine (C57Bl/6J) or human heart tissue (obtained during LVAD-surgery), was used for long-read mRNA sequencing, resulting in full-length transcriptomes including 58,000 mRNA isoforms with 99% sequence accuracy. Automatic bioinformatic analysis as well as manual similarity search against exonic sequences lead to identification of putative coding PGC-1 isoforms, validated by PCR and Sanger-Sequencing. Thereby, 12 novel transcripts generated by hitherto unknown splicing events were detected. In addition, we postulate a novel promoter with homologous and strongly-conserved sequence in human heart. High-fat-diet as well as ischemia/reperfusion (I/R) injury transiently reduced cardiac expression of PGC-1-isoforms, with the most pronounced effect in the infarcted area. Recovery of PGC-1-isoform expression was even more decelerated when I/R was performed in diet-induced obese mice.

ConclusionsWe deciphered for the first time a complete full-length-transcriptome of the murine and human heart, identifying novel putative PGC-1 coding transcripts including a novel promoter. These transcripts are differentially regulated in I/R and obesity suggesting transcriptional regulation and alternative splicing that may modulate PGC-1 function in the injured and metabolically challenged heart.


title: XY sex determination in a cnidarian

abstract:
Sex determination occurs across animal species, but most of our knowledge about the mechanisms of sex determination comes from only a handful of bilaterian taxa. This limits our ability to infer the evolutionary history of sex determination within animals. In this study, we generated a linkage map of the genome of the colonial cnidarian Hydractinia symbiolongicarpus and used this map to determine that this species has an XX/XY sex determination system. We delineate the pseudoautosomal and non-recombining regions of the Y chromosome and show that the latter encodes a number of genes with male gonad-specific expression. These findings establish Hydractinia as a tractable non-bilaterian model system for the study of sex determination.

Significance StatementWhat determines whether an animal is male or female? The answer depends on the species. Some rely on signals from their environment, while others take cues from their genome. Most of what we understand about sex determination comes from traditional model organisms (such as mice, flies, and worms) or groups of well-studied vertebrates and insects. Studying sex determination in other animals, especially those from phyla that diverged early in animal evolution, would allow us to better understand how sex has evolved across the animal kingdom and could reveal pathways present in the eumetazoan ancestor >600 million years ago. In this study, we show that the cnidarian Hydractinia symbiolongicarpus has XY sex determination, establishing this species as a model system for sex determination in an understudied group of animals.


title: Epigenetic and genetic population structure is coupled in a marine invertebrate

abstract:
Delineating the relative influence of genotype and the environment on DNA methylation is critical for characterizing the spectrum of organism fitness as driven by adaptation and phenotypic plasticity. In this study, we integrated genomic and DNA methylation data for two distinct Olympia oyster (Ostrea lurida) populations while controlling for within-generation environmental influences. In addition to providing the first characterization of genome-wide DNA methylation patterns in the oyster genus Ostrea, we identified 3,963 differentially methylated loci between populations. Our results show a clear coupling between genetic and epigenetic patterns of variation, with 27% of variation in inter-individual methylation differences explained by genotype. Underlying this association are both direct genetic changes in CpGs (CpG-SNPs) and genetic variation with indirect influence on methylation (mQTLs). The association between genetic and epigenetic patterns breaks down when comparing measures of population divergence at specific genomic regions, which has implications for the methods used to study epigenetic and genetic coupling in marine invertebrates.

Significance statementWe know that genotype and epigenetic patterns are primarily responsible for phenotype, yet there is a lack of understanding to what degree the two are linked. Here we characterized the mechanisms and the degree by which genetic variation and DNA methylation variation are coupled in a marine invertebrate, with almost a third of the methylation variation attributable to genotype. This study provides a framework for future studies in environmental epigenetics to take genetic variation into account when teasing apart the drivers of phenotypic variation. By identifying methylation variation that cannot be attributed to genotype or environmental changes during development, our results also highlight the need for future research to characterize molecular mechanisms adjacent to genetic adaptation for producing long-term shifts in phenotype.


title: Filter paper disks as a matrix for manipulation of recombinant proteins

abstract: Filter paper provides an excellent matrix for retention of proteins containing a cellulose binding domain. To use this capability for manipulating recombinant fusion proteins, binding and elution parameters were explored and procedures developed for small scale purification, modification and assay. Proteins were tagged with the cellulose binding domain from the C thermocellum CipB gene via a cleavable linker. Filter paper disks of 6mm diameter were able to bind up to 80 g protein although there was a substantial dependence on molecular size. Different means of introducing fusion proteins to the disks allow either binding within 20 minutes from microliter volumes or slower binding from milliliter volumes. Elution with protease in small volumes yielded greater than 10 g amounts with concentrations in the 1-2 mg/ml range. To demonstrate their utility, disks were used for small scale protein purification, covalent modification of protein, immunoprecipitation, and in a binding assay. These versatile methods allow parallel processing of multiple samples and may find many uses when only small amounts of protein are needed.

category: biochemistry


title: IsarPipeline: Combining MMseqs2 and PSI-BLAST to Quickly Generate Extensive Protein Sequence Alignment Profiles

abstract: Many of the machine learning (ML) models used in the field of bioinformatics and computational biology to predict either function or structure of proteins rely on the evolutionary information as summarized in multiple-sequence alignments (MSAs) or the resulting position-specific scoring matrices (PSSMs), as generated by PSI-BLAST. The current procedure used in protein structure and function prediction is computationally exhaustive and time-consuming. The main issue relies on the PSI-BLAST software being forced to load the current database of sequences (about 220 GB) in batches and search for similar sequence alignments to a query sequence. This leads to an average runtime of about 40-60 min for a medium-sized (450 Amino Acids) query protein. This average runtime is strictly dependent on the hardware used to run the software. The issue is becoming more problematic since the bio-sequence data pools are increasing in size exponentially over time, hence raising PSI-BLAST runtime as well. A prominent solution claims to speed up the current process by 100 folds. The MMseqs2 method, given enough memory, will load the whole database in memory and apply certain heuristics to retrieve the relevant set of aligned sequences. However, this solution cannot be used directly to generate the final output in the desired PSI-BLAST alignment and PSSM profile data format. In this research project, we analyzed the runtime performance of each tool separately. Furthermore, we built a pipeline that combines both MMseqs2 and PSI-BLAST to obtain a robust, optimized and very fast hybrid alignment tool, faster than PSI-BLAST by two orders of magnitude. It is implemented in C++ and is freely available under the MIT license at https://github.com/issararab/IsarPipeline. The output of our pipeline was evaluated on two previously built predictive models.


title: Recovering false negatives in CRISPR fitness screens with JLOE

abstract: It is widely accepted that pooled library CRISPR knockout screens offer greater sensitivity and specificity than prior technologies in detecting genes whose disruption leads to fitness defects, a critical step in identifying candidate cancer targets. However, the assumption that CRISPR screens are saturating has been largely untested. Through integrated analysis of screen data in cancer cell lines generated by the Cancer Dependency Map, we show that a typical CRISPR screen has a [~]20% false negative rate, in addition to library-specific false negatives. Replicability falls sharply as gene expression decreases, while cancer subtype-specific genes within a tissue show distinct profiles compared to false negatives. Cumulative analyses across tissues improves our understanding of core essential genes and suggest only a small number of lineage-specific essential genes, enriched for transcription factors that define pathways of tissue differentiation. To recover false negatives, we introduce a method, Joint Log Odds of Essentiality (JLOE), which builds on our prior work with BAGEL to selectively rescue the false negatives without an increased false discovery rate.


title: Context-dependent triggering of STING-interferon signaling by CD11b agonists supports anti-tumor immunity in mouse models and human cancer patients

abstract:
Chronic activation of inflammatory pathways and suppressed interferon signaling are hallmarks of myeloid cells in immunosuppressive tumors that drive poor responsiveness to conventional and immune therapies. Previous studies have identified agonistic activation of the CD11b integrin as a potential strategy to enhance anti-tumor immunity. However, the mechanisms by which CD11b-agonism reprogram tumor immunity are poorly understood, and this may impair patient selection and identification of effective treatment combinations. Herein we used a combination of in vitro systems, animal models, and samples from first in human clinical trials of the CD11b-agonist GB1275 to identify the mechanism of action of this approach and identify combinations for further testing. We found that CD11b agonism altered tumor-associated macrophage (TAM) phenotypes by simultaneously repressing NF{kappa}B/IL1-signaling and activating interferon (IFN) gene expression. Repression of NF{kappa}B/IL-1 signaling was due to rapid degradation of p65 protein by the proteosome and was not context dependent. In contrast, CD11b agonism triggered mitochondrial dysfunction to stimulate STING-induced, STAT1-mediated interferon signaling. The magnitude of CD11b agonist induction of STING/IFN signaling was dependent on the tumor microenvironment and was significantly amplified by cytotoxic therapies. Using tissues from phase I clinical trials, we demonstrated that GB1275 treatment activated STING and STAT1 signaling in TAMs in human tumors. Together, these mechanisms allowed macrophages to augment anti-tumor T cell immunity. These studies identified potential mechanism-based therapeutic strategies for CD11b agonist use and identified potential patient populations more likely to benefit from them.

Statement of significanceCD11b agonists are a novel approach to reprogram myeloid cells in solid tumors. We show that GB1275, a CD11b-agonist, amplified STING/IFN signaling in TAMs to support anti-tumor immunity and this signaling is amplified further by cytotoxic therapy. These studies support new treatment strategies for advanced solid tumors with myeloid immunosuppression.


title: Proteomic analysis reveals microvesicles containing NAMPT as mediators of radiation resistance in glioma

abstract:
Glioma is a malignant brain tumor that is highly resistant to radiation and chemotherapy, where patients survive on average only 15 months after diagnosis. Furthering the understanding of mechanisms leading to radiation resistance of glioma is paramount to identify novel therapeutic targets. Previous studies have shown that glioma stem cells (GSCs) play an important role in promoting radiation resistance and disease recurrence. Herein we analyze the proteomic alterations occurring in patient-derived GSCs upon radiation treatment in order to identify molecular drivers of resistance. We show that proteome changes upon radiation accurately predict the resistance status of the cells, whereas resistance to radiation does not correlate with glioma transcriptional subtypes. We further show that the radio-resistant GSC-267 cell line sheds microvesicles (MVs) enriched in the metabolic enzyme nicotinamide phosphoribosyltransferase (NAMPT). These MVs can be transferred to recipient fibroblasts and radio-sensitive GSCs, enhancing their intracellular total NAD+ and NADH level, and their ability to proliferate when cultured in low serum, treated with a radio-mimetic drug or irradiated. The NAMPT enzymatic inhibitor FK-866 blocked the ability of MVs from GSC-267 cells to mediate these effects. Similarly, GSC-267 cells where NAMPT was knocked-down using shRNA, which produced MVs depleted of this enzyme, were unable to promote cell proliferation. Collectively, our findings demonstrates that proteome-level regulation can accurately predict the radio-resistance status of GSCs, and identifies NAMPT transfer via MVs as a mechanism for spreading radiation resistance within the glioma tumor microenvironment.

SignificanceThe highly aggressive and deadly brain cancer glioma is commonly resistant to standard chemo- and radio-therapy. We used systems biology approaches to study patient-derived glioma stem cells (GSCs), which are known to be responsible for therapeutic resistance, and cell-to-cell communication mediated by extracellular vesicles (EVs), which plays an important role in tumor progression. Analysis of the proteome of GSCs and of the EVs they release led us to determine that the EV-mediated transfer of the metabolic enzyme nicotinamide phosphorybosyltransferase (NAMPT) from radio-resistant to less aggressive cells confers resistance to radiation. Our findings identify a mechanism of therapy resistance in glioma, and suggest that NAMPT inhibition could enhance the efficacy of radiation for the treatment of glioma.


title: Identifying competition phenotypes in synthetic biochemical circuits

abstract: Synthetic gene circuits require cellular resources, which are often limited. This leads to competition for resources by different genes, which alter a synthetic genetic circuits behavior. However, the manner in which competition impacts behavior depends on the identity of the "bottleneck" resource which might be difficult to discern from input-output data. In this paper, we aim at classifying the mathematical structures of resource competition in biochemical circuits. We find that some competition structures can be distinguished by their response to different competitors or resource levels. Specifically, we show that some response curves are always linear, convex, or concave. Furthermore, high levels of certain resources protect the behavior from low competition, while others do not. We also show that competition phenotypes respond differently to various interventions. Such differences can be used to eliminate candidate competition mechanisms when constructing models based on given data. On the other hand, we show that different networks can display mathematically equivalent competition phenotypes.

category: synthetic biology


title: Phosphoprotein Phosphatase Activity Positively Regulates Oligomeric Pyrin to Trigger Inflammasome Assembly in Response to Bacterial Effectors and Toxins that Inactivate RhoA in Macrophages

abstract: Pyrin is a pattern-recognition receptor in phagocytes that triggers capase-1 inflammasome assembly in response to bacterial toxins and effectors that inactivate RhoA. Pyrin contains coiled coil and B-box domains that promote oligomerization. Pyrin is negatively regulated by phosphorylation of two linker domain residues, S205 and S241 (murine) or S208 and S242 (human), via the kinase PKN, which is in turn activated by RhoA. Inactivation of RhoA by the TcdB toxin of Clostridioides difficile, the YopE and YopT effectors of Yersinia, or the TecA effector of Burkholderia cenocepacia triggers pyrin inflammasome assembly in macrophages. Inactivation of RhoA removes the negative regulation but a phosphoprotein phosphatase (PPP) is needed to positively regulate pyrin by dephosphorylation. No PPP that dephosphorylates pyrin has been identified, the oligomeric state of murine pyrin has not been determined, and the phosphorylation status of pyrin oligomers is unknown. We used primary murine macrophages and human THP-1 cells combined with the use of inhibitors, native PAGE, immunoblotting with phospho-specific pyrin antibodies and siRNA knockdowns to determine if a PPP positively regulates oligomeric pyrin. Results obtained with inhibitor calyculin A indicate that PPP activity is required to dephosphorylate S205 or S242 in murine or human pyrin, respectively, in response to inactivation of RhoA by TcdB, YopE/T or TecA. Findings from native PAGE show that murine pyrin exists as dimers and higher order oligomers that are phosphorylated on S205 prior to RhoA inactivation. Calyculin A treatment causes reduced mobility of murine pyrin on native PAGE and hyperphosphorylation of S242 in human pyrin, suggesting a PPP constitutively counterbalances PKN to keep the second linker site hypophosphorylated. Data from low dose okadaic acid and siRNA knockdown experiments implicates PP2A in dephosphorylation of S205 and positive regulation of pyrin in response to RhoA inactivation.


title: Bacteriophages Playing Nice: Lysogenic bacteriophage replication stable in the human gut microbiota

abstract:
1.1.The human gut is a dense microbial community, of which bacteria and bacteriophages are the majority. Bacteriophages, viruses of bacteria, exist stably, without major fluctuations in the gut of healthy individuals. This stability appears to be due to an absence of  kill-the-winner dynamics, and the existence of  piggy-back-the-winner dynamics, where lysogenic replication rather than lytic replication occurs. Revisiting the deep-viral sequencing data of a healthy individual studied over 2.4 years, we were able to improve our understanding of how these dynamics occur in healthy individuals. We assembled prophages from bacterial metagenomic data and show that these prophages were continually switching from lysogenic to lytic replication. Prophages were the source of a stable extracellular phage population continually present in low abundance, in comparison to the lytic-phage population, where taxonomic diversity diverged over 2.4 years. The switch to lytic replication, or prophage induction, appears to occur mostly through spontaneous prophage induction. The observed phage dynamics of regular spontaneous induction are ecologically important as they allow prophages to maintain their ability to replicate, avoiding degradation and their loss from the gut microbiota.

1.2. Significance StatementIt has been eight years since Minot and colleagues published their landmark longitudinal study of phages in the gut. In the years following, the bioinformatic field improved in great strides, including the methods of bacterial-genome assembly, phage-identification, and prophage detection. We leveraged the unprecedented deep sequencing of phages in this dataset by adding bacterial assembly and prophage detection analyzes. We show clearly for the first time that  piggy-back-the-winner dynamics are maintained in the gut through spontaneous prophage induction, and not widespread triggered prophage induction. These dynamics play an important ecological role by creating a stable subpopulation of phages, which could help explain how phages are maintained over the 2.4 years timeframe that this individual was studied.


title: An optimized method for high-quality RNA extraction from distinctive intrinsic laryngeal muscles in the rat model.

abstract: Challenges related to high-quality RNA extraction from post-mortem tissue have limited RNA-sequencing (RNA-seq) application in certain skeletal muscle groups, including the intrinsic laryngeal muscles (ILMs). The present study identified critical factors contributing to substandard RNA extraction from the ILMs and established a suitable method that permitted high-throughput analysis. Here, standard techniques for tissue processing were adapted, and an effective means to control confounding effects during specimen preparation was determined. The experimental procedure consistently provided sufficient intact total RNA (N = 68) and RIN ranging between 7.0 and 8.6, which was unprecedented using standard RNA purification protocols. This study confirmed the reproducibility of the workflow through repeated trials at different postnatal time points and across the distinctive ILMs. High-throughput diagnostics from 90 RNA samples indicated no sequencing alignment scores below 70%, validating the extraction strategy. Significant differences between the standard and experimental conditions suggest circumvented challenges and broad applicability to other skeletal muscles. This investigation remains ongoing given the prospect of therapeutic insights to voice, swallowing, and airway disorders. The present methodology supports pioneering global transcriptome investigations in the larynx previously unfounded in literature.