title: Differentiation of human adult-derived stem cells towards a neural lineage involves a de-differentiation event prior to re-differentiation to neural phenotypes

abstract:
Although it has been reported that mesenchymal stem cells isolated from adult tissues can be induced to overcome their mesenchymal fate and transdifferentiate into neural cells, the findings and their interpretation have been challenged. The main argument against this process is that the cells rapidly adopt neuron-like morphologies through retraction of the cytoplasm rather than active neurite extension.

In this study, we examined the sequence of biological events during neural differentiation of human periodontal ligament-derived stem cells (hPDLSCs), human bone marrow-derived stem cells (hBMSCs) and human dental pulp-derived stem cells (hDPSCs) by time-lapse microscopy.

We have demonstrated that hPDLSCs, hBMSCs and hDPSCs can directly differentiate into neuron-like cells without passing through a mitotic stage and that they shrink dramatically and change their morphology to that of neuron-like cells through active neurite extension. Furthermore, we observed micronuclei movement and transient cell nuclei lobulation concurrent to in vitro neurogenesis from hBMSCs and hDPSCs.

Our results demonstrate that the differentiation of hPDLSCs, hBMSCs and hDPSCs towards a neural lineage occurs through a de-differentiation step followed by re-differentiation to neural phenotypes, and therefore we definitively confirm that the rapid acquisition of the neural phenotype is via a differentiation trait.


title: Five hours total sleep deprivation does not affect CA1 dendritic length or spine density

abstract: Sleep is essential for long term memory function. However the neuroanatomical consequences of sleep loss are disputed. Sleep deprivation has been reported to cause both decreases and increases of dendritic spine density. Here we use Thy1-GFP expressing transgenic mice to investigate the effects of acute sleep deprivation on the dendritic architecture of hippocampal CA1 pyramidal neurons. We found that five hours of sleep deprivation had no effect on either dendritic length or dendritic spine density. Our work suggests that no major neuroanatomical changes result from one episode of sleep deprivation.

category: neuroscience


title: PML-Dependent Memory of Type I Interferon Treatment Results in a Restricted Form of HSV Latency

abstract: Herpes simplex virus (HSV) establishes latent infection in long-lived neurons. During initial infection, neurons are exposed to multiple inflammatory cytokines but the effects of immune signaling on the nature of HSV latency is unknown. We show that initial infection of primary murine neurons in the presence of type I interferon (IFN) results in a form of latency that is restricted for reactivation. We also found that the subnuclear condensates, promyelocytic leukemia-nuclear bodies (PML-NBs), are absent from primary sympathetic and sensory neurons but form with type I IFN treatment and persist even when IFN signaling resolves. HSV-1 genomes colocalized with PML-NBs throughout a latent infection of neurons only when type I IFN was present during initial infection. Depletion of PML prior to or following infection did not impact the establishment latency; however, it did rescue the ability of HSV to reactivate from IFN-treated neurons. This study demonstrates that viral genomes possess a memory of the IFN response during de novo infection, which results in differential subnuclear positioning and ultimately restricts the ability of genomes to reactivate.

category: immunology


title: Interleukin-33 regulates metabolic reprogramming of the retinal pigment epithelium in response to immune stressors.

abstract: It remains unresolved how retinal pigment epithelial (RPE) cell metabolism is regulated following immune activation to maintain retinal homeostasis and retinal function. We exposed RPE to several stress signals, particularly toll-like receptor stimulation, and uncovered an ability of RPE to adapt their metabolic preference on aerobic glycolysis or oxidative glucose metabolism in response to different immune stimuli. We have identified interleukin-33 (IL-33) as a key metabolic checkpoint that antagonises the Warburg effect to ensure the functional stability of the RPE. The identification of IL-33 as a key regulator of mitochondrial metabolism suggests roles for the cytokine that go beyond its extracellular "alarmin" activities. IL-33 exerts control over mitochondrial respiration in RPE by facilitating oxidative pyruvate catabolism. We have also revealed that in the absence of IL-33, mitochondrial function declines and resultant bioenergetic switching is aligned with altered mitochondrial morphology. Our data not only sheds new light in the molecular pathway of activation of mitochondrial respiration in RPE in response to immune stressors, but also uncovers a novel role of nuclear intrinsic IL-33 as a metabolic checkpoint regulator.


title: Evolution of butterfly-plant networks over time, as revealed by Bayesian inference of host repertoire

abstract: The study of herbivorous insects underpins much of the theory that concerns the evolution of species interactions. In particular, Pieridae butterflies and their host plants have served as a model system for studying evolutionary arms-races. To learn more about the coevolution of these two clades, we reconstructed ancestral ecological networks using stochastic mappings that were generated by a phylogenetic model of host-repertoire evolution. We then measured if, when, and how two ecologically important structural features of the ancestral networks (modularity and nestedness) evolved over time. Our study shows that as pierids gained new hosts and formed new modules, a subset of them retained or recolonized the ancestral host(s), preserving connectivity to the original modules. Together, host-range expansions and recolonizations promoted a phase transition in network structure. Our results demonstrate the power of combining network analysis with Bayesian inference of host-repertoire evolution to understand changes in complex species interactions over time.

category: ecology


title: Phylogenetic reconstruction of ancestral ecological networks through time for pierid butterflies and their host plants

category: evolutionary biology


title: The prevalence, predominance, and metabolic potentials of Candidatus Prochlorobium terpiosii in the coral-killing sponge, Terpios hoshinota

abstract:
Terpios hoshinota is a ferocious, space-competing sponge that kills a variety of stony corals by overgrowth. Outbreaks of this species have led to intense coral reef damage and declines in living corals on the square kilometer scale in many geographical locations. Our large-scale 16S rRNA gene survey across three oceans revealed that the core microbiome of T. hoshinota included operational taxonomic units (OTUs) related to Prochloron, Endozoicomonas, Pseudospirillum, SAR116, Magnetospira, and Ruegeria. A Prochloron- related OTU was the most dominant cyanobacterium in T. hoshinota in the western Pacific Ocean, South China Sea, and Indian Ocean. The complete metagenome-assembled genome of the Prochloron-related cyanobacterium and our pigment analysis revealed that this bacterium had phycobiliproteins and phycobilins and lacked chlorophyll b, inconsistent with the iconic definition of Prochloron. Furthermore, the phylogenetic analyses based on 16S rRNA genes and 120 single-copy genes demonstrated that the bacterium was phylogenetically distinct to Prochloron, strongly suggesting that it should be a sister taxon to Prochloron; we therefore proposed this symbiotic cyanobacterium as a novel species under a new genus: Candidatus Paraprochloron terpiosii. With the recovery of the complete genome, we characterized the metabolic potentials of the novel cyanobacterium in carbon and nitrogen cycling and proposed a model for the interaction between Ca. Pp. terpiosi LD05 and T. hoshinota. In addition, comparative genomics analysis revealed that Ca. Paraprochloron and Prochloron showed distinct features in transporter systems and DNA replication.

ImportanceThe finding that one species predominates cyanobacteria in T. hoshinota from different geographic locations indicates that this sponge and Ca. Pp. terpiosi LD05 share a tight relationship. This study builds the foundation for T. hoshinotas microbiome and paves a way for understanding the ecosystem, invasion mechanism, and causes of outbreak of this coral-killing sponge. Also, the first Prochloron-related complete genome enables us to study this bacterium with molecular approaches in the future and broadens our knowledge of the evolution of symbiotic cyanobacteria.


title: Prevalence, complete genome and metabolic potentials of a phylogenetically novel cyanobacterial symbiont in the coral-killing sponge, Terpios hoshinota


title: CRISPR interference identifies vulnerable cellular pathways with bactericidal phenotypes in Mycobacterium tuberculosis

abstract: Mycobacterium tuberculosis remains a leading cause of death for which new drugs are needed. The identification of drug targets has been advanced by high-throughput and targeted genetic deletion strategies. Each though has limitations including the inability to distinguish between levels of vulnerability, lethality and scalability as a molecular tool. Using mycobacterial CRISPR interference in combination with phenotypic screening we have overcome these individual issues to investigate essentiality, vulnerability and lethality for 96 target genes from a diverse array of cellular pathways, many of which are potential antibiotic targets. Essential genes involved in cell wall synthesis and central cellular functions were equally vulnerable and often had bactericidal consequences. Conversely, essential genes involved in metabolism, oxidative phosphorylation or amino acid synthesis were less vulnerable to inhibition and frequently bacteriostatic. In conclusion, this study provides novel insights into mycobacterial genetics and biology that will help to prioritise potential drug targets.

category: genomics


title: Ovine CD14- an immune response gene has a role against gastrointesinal nematode Haemonchus contortus -a novel report

abstract: CD14 (also known as monocyte differentiation antigen) is an important immune response gene known to be primarily responsible for innate immunity against bacterial pathogen and as pattern recognition receptor (PRR) binds with LPS (endotoxin), lipoproteins, lipotechoic acid of bacteria.So far very limited work has been conducted in parasitic immunology. In the current study, we reported the role of CD14 in parasitic immunology in livestock species (sheep) for the first time. Ovine CD14 is characterized as a horse-shoe shaped as a bent solenoid with a hydrophobic amino-terminal pocket for CD14 along with domains. High mutation frequency was observed, out of total 41 mutations identified, 23 mutations were observed to be thermodynamically unstable and 11 mutations were deleterious in nature, causing major functional alteration of important domains of CD14, an indicative for variations in individual susceptibility for sheeps against Haemonchus contortus infestations. In silico studies with molecular docking reveals a role of immune response against Haemonchus contortus in sheep, which is later confirmed with experimental evidence through differential mRNA expression analysis for sheep, which revealed better expression of CD14 in Haemonchus contortus infected sheep compared to that of healthy sheep. We confirmed the above findings with supportive evidences with haematological and biochemical analyses. Phylogenetic analysis was conducted to assess the evolutionary relationship with respect to human and observed that sheep may well be used as model organism due better genetic closeness compared to that of mouse.


title: Practical selection of representative sets of RNA-seq samples using a hierarchical approach

abstract: Despite numerous RNA-seq samples available at large databases, most RNA-seq analysis tools are evaluated on a limited number of RNA-seq samples. This drives a need for methods to select a representative subset from all available RNA-seq samples to facilitate comprehensive, unbiased evaluation of bioinformatics tools. In sequence-based approaches for representative set selection (e.g. a k-mer counting approach that selects a subset based on k-mer similarities between RNA-seq samples), because of the huge number of available RNA-seq samples and the large number of k-mers/sequences in each sample, computing the full similarity matrix between all samples using k-mers/sequences for the entire set of RNA-seq samples in a large database (e.g. the SRA) has memory and runtime challenges, making direct representative set selection infeasible with limited computing resources. Therefore, we developed a novel computational method called "hierarchical representative set selection" to handle this challenge. Hierarchical representative set selection is a divide-and-conquer-like algorithm that breaks the representative set selection into sub-selections and hierarchically selects representative samples through multiple levels. We demonstrate that hierarchical representative set selection can achieve performance close to that of direct representative set selection, while largely reducing the runtime and memory requirements of computing the full similarity matrix (up to 8.4X runtime reduction and 4.7X memory reduction for 10000 samples that could be practically run with direct subset selection). We show that hierarchical representative set selection substantially outperforms random sampling on the entire SRA set of RNA-seq samples, making it a practical solution to representative set selection on large databases such as the SRA.

category: bioinformatics


title: GRAFIMO: variant and haplotype aware motif scanning on pangenome graphs

abstract:
Transcription factors (TFs) are proteins that promote or reduce the expression of genes by binding short genomic DNA sequences known as transcription factor binding sites (TFBS). While several tools have been developed to scan for potential occurrences of TFBS in linear DNA sequences or reference genomes, no tool exists to find them in pangenome variation graphs (VGs). VGs are sequence-labelled graphs that can efficiently encode collections of genomes and their variants in a single, compact data structure. Because VGs can losslessly compress large pangenomes, TFBS scanning in VGs can efficiently capture how genomic variation affects the potential binding landscape of TFs in a population of individuals. Here we present GRAFIMO (GRAph-based Finding of Individual Motif Occurrences), a command-line tool for the scanning of known TF DNA motifs represented as Position Weight Matrices (PWMs) in VGs. GRAFIMO extends the standard PWM scanning procedure by considering variations and alternative haplotypes encoded in a VG. Using GRAFIMO on a VG based on individuals from the 1000 Genomes project we recover several potential binding sites that are enhanced, weakened or missed when scanning only the reference genome, and which could constitute individual-specific binding events. GRAFIMO is available as an open-source tool, under the MIT license, at https://github.com/pinellolab/GRAFIMO and https://github.com/InfOmics/GRAFIMO.

Author summaryTranscription factors (TFs) are key regulatory proteins and mutations occurring in their binding sites can alter the normal transcriptional landscape of a cell and lead to disease states. Pangenome variation graphs (VGs) efficiently encode genomes from a population of individuals and their genetic variations. GRAFIMO is an open-source tool that extends the traditional PWM scanning procedure to VGs. By scanning for potential TBFS in VGs, GRAFIMO can simultaneously search thousands of genomes while accounting for SNPs, indels, and structural variants. GRAFIMO reports motif occurrences, their statistical significance, frequency, and location within the reference or alternative haplotypes in a given VG. GRAFIMO makes it possible to study how genetic variation affects the binding landscape of known TFs within a population of individuals.


title: Machine learning for single cell genomics data analysis

abstract: Single-cell omics technologies produce large quantities of data describing the genomic, transcriptomic or epigenomic profiles of many individual cells in parallel. In order to infer biological knowledge and develop predictive models from these data, machine learning (ML)-based model are increasingly used due to their flexibility, scalability, and impressive success in other fields. In recent years, we have seen a surge of new ML-based method development for low-dimensional representations of single-cell omics data, batch normalization, cell type classification, trajectory inference, gene regulatory network inference or multimodal data integration. To help readers navigate this fast-moving literature, we survey in this review recent advances in ML approaches developed to analyze single-cell omics data, focusing mainly on peer-reviewed publications published in the last two years (2019-2020).


title: A Quantitative Data-Driven Analysis (QDA) Framework for Resting-state fMRI: a Study of the Impact of Adult Age

abstract:
PurposeThe objective of this study is to introduce a new quantitative data-driven analysis (QDA) framework for the analysis of resting-state fMRI (R-fMRI) and use it to investigate the effect of adult age on resting-state functional connectivity (RFC).

MethodsWhole-brain R-fMRI measurements were conducted on a 3T clinical MRI scanner in 227 healthy adult volunteers (N=227, aged 18-74 years old, male/female=99/128). With the proposed QDA framework we derived two types of voxel-wise RFC metrics: the connectivity strength index (CSI) and connectivity density index (CDI) utilizing the convolutions of the cross-correlation (CC) histogram with different kernels. Furthermore, we assessed the negative and positive portions of these metrics separately.

ResultsWith the QDA framework we found age-related declines of RFC metrics in the superior and middle frontal gyrus (MFG), posterior cingulate cortex (PCC), right insula and inferior parietal lobule (IPL) of the default mode network (DMN), which resembles previously reported results using other types of RFC data processing methods. Importantly, our new findings complement previously undocumented results in the following aspects: 1) the PCC and right insula are anti-correlated and tend to manifest simultaneously declines of both the negative and positive connectivity strength with subjects age; 2) separate assessment of the negative and positive RFC metrics provides enhanced sensitivity to the aging effect; 3) the sensorimotor network depicts enhanced negative connectivity strength with the adult age.

ConclusionThe proposed QDA framework can produce threshold-free, voxel-wise analysis of R-fMRI data the RFC metrics. The detected adult age effect is largely consistent with previously reported studies using different R-fMRI analysis approaches. Moreover, the separate assessment of the negative and positive contributions to the RFC metrics can enhance the RFC sensitivity and clarify some of the mixed results in the literature regarding to the DMN and sensorimotor network involvement in adult aging.

HighlightsO_LIA quantitative data-driven analysis (QDA) framework was proposed to analysis resting-state fMRI data.
C_LIO_LIThreshold-free resting-state functional connectivity (RFC) metrics were derived to assess brain changes with adult age.
C_LIO_LISeparate assessment of the positive and negative correlations improve sensitivity of the RFC metrics.
C_LIO_LIThe posterior cingulate and right insula cortices are anti-correlated and tend to manifest declines in both the negative and positive connectivity strength with adult age.
C_LIO_LINegative connectivity strength enhances with adult age in sensorimotor network.
C_LI


title: Interaction between decision-making and interoceptive representations of bodily arousal in frontal cortex

abstract:
Decision-making and representations of arousal are intimately linked. Behavioral investigations have classically shown that either too little or too much bodily arousal is detrimental to decision-making, indicating that there is an inverted  U relationship between bodily arousal and performance. How these processes interact at the level of single neurons as well as the neural circuits involved are unclear. Here we recorded neural activity from orbitofrontal cortex (OFC) and dorsal anterior cingulate cortex (dACC) of macaque monkeys while they made reward-guided decisions. Heart rate (HR) was also recorded and used as a proxy for bodily arousal. Recordings were made both before and after subjects received excitotoxic lesions of the bilateral amygdala. In intact monkeys, higher HR facilitated reaction times (RTs). Concurrently, a set of neurons in OFC and dACC selectively encoded trial-by-trial variations in HR independent of reward value. After amygdala lesions, HR increased and the relationship between HR and RTs was reversed. Concurrent with this change, there was an increase in the proportion of dACC neurons encoding HR. Applying a novel population-coding analysis, we show that after bilateral amygdala lesions the balance of encoding in dACC is skewed away from signaling either reward value or choice direction towards HR coding around the time that choices are made. Taken together, the present results provide insight into how bodily arousal and decision-making are signaled in frontal cortex.

Significance statementHow bodily arousal states influence decision-making has been a central question in psychology, but the neural mechanisms are unclear. We recorded heart rate, a measure of bodily arousal, while simultaneously monitoring neural activity in orbitofrontal cortex (OFC) and dorsal anterior cingulate cortex (dACC) of macaques making reward-guided decisions. In intact macaques higher HR was associated with shorter reaction times. Concurrently, the activity of a set of neurons in OFC and dACC selectively encoded HR. Following amygdala lesions, HR generally increased and now the relationship between HR and reaction times was reversed. At the neural level, the balance of encoding in dACC shifted towards signaling HR, suggesting a specific mechanism through which bodily arousal influences decision-making.


title: Neuron-derived extracellular vesicles extracted from plasma show altered size and miRNA cargo as a function of antidepressant drug response

abstract: Previous work has demonstrated that microRNAs (miRNAs) change as a function of antidepressant treatment (ADT) response. However, it is unclear how representative these peripherally detected miRNA changes are to those occurring in the brain. This study aimed to use peripherally extracted neuron-derived extracellular vesicles (NDEVs) to circumvent these limitations and investigate neuronal miRNA changes associated with antidepressant response. Samples were collected at two time points (baseline and after 8 weeks of follow-up) from depressed patients who responded (N=20) and did not respond (N=20) to escitalopram treatment, as well as controls (N=20). Total extracellular vesicles (EVs) were extracted from plasma, and then further enriched for NDEVs by immunoprecipitation with L1CAM. EV size was measured using tunable resistive pulse sensing, and NDEV miRNA cargo was extracted and sequenced. Subsequently, studies in cell lines and postmortem tissue were conducted. Characterization of NDEVs revealed they were smaller than other EVs isolated from plasma (p<0.0001), had brain-specific neuronal markers, and contained miRNAs enriched for brain functions (p<0.0001) Furthermore, NDEVs from depressed patients were smaller than controls (p<0.05), and NDEV size increased with ADT response (p<0.01). Finally, changes in NDEV cargo, specifically changes in miR-21-5p, miR-30d-5p and miR-486-5p together (p<0.01), were associated with ADT response. Targets of these three miRNAs were altered in brain tissue from depressed individuals (p<0.05). Together, this study indicates that changes in peripherally isolated NDEVs can act as both a clinically accessible and informative biomarker of ADT response specifically through size and cargo.


title: The molecular clockwork of the suprachiasmatic nucleus is sufficient to co-ordinate phasing and stabilisation of sleep-wake cycles and enhance memory deficits in a clockless mouse

abstract:
The timing and quality of sleep-wake cycles are regulated by interacting circadian and homeostatic mechanisms. Although the suprachiasmatic nucleus (SCN) is the principal circadian clock, local clocks are active across the brain and the respective sleep-regulatory roles of SCN and extra-SCN clocks are unclear. To determine the specific contribution(s) of the SCN, we used virally mediated genetic complementation, expressing Cryptochrome1 (Cry1) to restore circadian molecular competence to the SCN of globally clockless Cry1/Cry2-null mice. Under free-running conditions, the rest/activity behaviour of Cry1/Cry2-null controls which received EGFP (SCNCon) was arrhythmic, whereas Cry1-complemented mice (SCNCry1) had circadian behaviour comparable to that of Cry1,2-competent wild-types (WT). In SCNCon mice, sleep-wakefulness, assessed by electroencephalography/electromyography, also lacked circadian organisation. In SCNCry1 mice, however, it was comparable to WT, with consolidated vigilance states (wake, REM and NREM sleep) and rhythms in NREMS delta power and expression of REMS within total sleep. Wakefulness in SCNCon mice was more fragmented than in WT, with more wake-NREMS-wake transitions. This disruption was corrected in SCNCry1 mice. Following sleep deprivation, all mice showed an initial homeostatic increase in NREMS delta power. The SCNCon mice, however, had reduced, non-consolidated NREMS during the inactive phase of the recovery period. In contrast, the dynamics of homeostatic responses in the SCNCry1 mice were equivalent to WT. Finally, SCNCon mice exhibited poor sleep-dependent memory but this was corrected in SCNCry1mice. Therefore, the SCN clock is sufficient for circadian control of sleep-wake, facilitating initiation and maintenance of wake, promoting sleep consolidation, homeostatic dynamics, and sleep-dependent memory.

Significance statementThe circadian timing system regulates sleep-wake cycles. The hypothalamic suprachiasmatic nucleus (SCN) is the principal circadian clock, but local clocks are also active across the brain and the respective roles of SCN and local clocks in regulating sleep are unclear. To determine, explicitly, the contribution of the SCN, we used virally mediated genetic complementation to restore SCN molecular circadian functions in otherwise genetically clockless mice. This initiated circadian activity-rest cycles, accompanied by circadian sleep-wake cycles, circadian patterning to the intensity of NREM sleep and circadian control of REM sleep as a proportion of total sleep. Consolidation of sleep-wake established normal dynamics of sleep homeostasis and enhanced sleep-dependent memory. Thus, the SCN is the principal and sufficient circadian regulator of sleep-wake.


title: Psilocybin-induced changes in brain network integrity and segregation correlate with plasma psilocin level and psychedelic experience

abstract: The emerging novel therapeutic psilocybin produces psychedelic effects via engagement of cerebral serotonergic targets by psilocin (active metabolite). The serotonin 2A receptor critically mediates these effects by altering distributed neural processes that manifest as increased entropy, reduced functional connectivity (FC) within discrete brain networks (i.e., reduced integrity) and increased FC between networks (i.e., reduced segregation). Reduced integrity of the default mode network (DMN) is proposed to play a particularly prominent role in psychedelic phenomenology, including perceived ego-dissolution. Here, we investigate the effects of a psychoactive oral dose of psilocybin (0.2-0.3 mg/kg) on plasma psilocin level (PPL), subjective drug intensity (SDI) and their association in fifteen healthy individuals. We further evaluate associations between these measures and resting-state FC, measured with functional magnetic resonance imaging, acquired over the course of five hours after psilocybin administration. We show that PPL and SDI correlate negatively with measures of network integrity (including DMN) and segregation, both spatially constrained and unconstrained. We also find that the executive control network and dorsal attention network desegregate, increasing connectivity with other networks and throughout the brain as a function of PPL and SDI. These findings provide direct evidence that psilocin critically shapes the time course and magnitude of changes in the cerebral functional architecture and subjective experience following psilocybin administration. Our findings provide novel insight into the neurobiological mechanisms underlying profound perceptual experiences evoked by this emerging transnosological therapeutic and implicate the expression of network integrity and segregation in the psychedelic experience and consciousness.


title: Somatosensory innervation of healthy human oral tissues

abstract: The oral somatosensory system relays essential information about mechanical stimuli to enable oral functions such as feeding and speech. The neurochemical and anatomical diversity of sensory neurons across oral cavity sites have not been systematically compared. To address this gap, we analyzed healthy human tongue and hard palate innervation. Biopsies were collected from 12 volunteers and underwent multiplex fluorescent immunohistochemistry ([≥]2 specimens per marker/structure). Afferents were analyzed for markers of neurons ({beta}III tubulin), myelinated afferents (neurofilament heavy, NFH), and Merkel cells and taste cells (keratin 20, K20). Hard-palate innervation included Meissners corpuscles, glomerular endings, Merkel cell-neurite complexes, and free nerve endings. The organization of these somatosensory endings is reminiscent of fingertips, suggesting that the hard palate is equipped with a rich repertoire of sensory neurons for pressure sensing and spatial localization of mechanical inputs, which are essential for speech production and feeding. Likewise, the tongue is innervated by afferents that impart it with exquisite acuity and detection of moving stimuli that support flavor construction and speech. Filiform papillae contain end bulbs of Krause, as well as endings that have not been previously reported, including subepithelial neuronal densities, and NFH+ neurons innervating basal epithelium. Fungiform papillae had Meissners corpuscles and densities of NFH+ intraepithelial neurons surrounding taste buds. The differing compositions of sensory endings within filiform and fungiform papillae suggest that these structures have distinct roles in mechanosensation. Collectively, this study has identified previously undescribed afferent endings in human oral tissues and provides an anatomical framework for understanding oral mechanosensory functions.


title: Sublingual allergen immunotherapy with recombinant dog allergens prevents airway hyperresponsiveness in a model of asthma marked by vigorous TH2 and TH17 cell responses

abstract: Allergy to dogs affects around ten percent of the population in developed countries. Immune therapy of allergic patients with dog allergen extracts has shown limited therapeutic benefit. Herein, we established a mouse model of dog allergy and tested the efficacy of a recombinant protein containing Can f 1, f 2, f 4 and f 6 as a sublingual immune therapy (SLIT). Repeated inhalation of dog extracts induced infiltration of the airways by TH2 cells, eosinophils and goblet cells, reminiscent of the house dust mite (HDM) model of asthma. However, dog allergen extracts also induced robust TH17 cell responses, which was associated with a high neutrophilic infiltration of the airways and promoted airway hyperresponsiveness more potently than HDM allergens. scRNA-Seq analysis of T helper cells responding to dog allergens identified several unique clusters with TH17 cells being hallmarked by the expression of several receptors including IL-17RE. Analysis of T cell receptors also depicted a high frequency of clones that were shared between TH17, TH2 and suppressive Treg cells, indicative of the plasticity of T helper cells in this model. Importantly, prophylactic SLIT reduced airway hyperresponsiveness and type 2-mediated inflammation in this model supporting the use of recombinant allergens in immune therapy.


title: Akaike model selections of the vegetation structures and aerosphere factors in supporting lesser short nosed fruit bat (Cynopterus brachyotis Muller, 1838) populations in Asia mountainous paddy fields

abstract: As an aerial and arboreal fauna, the abundances and populations of fruit bat Cynopterus brachyotis were influenced by the vegetation structures and aerosphere condition variables of fruit bat ecosystems. While mountaineous paddy field is an unique habitat since the trees are scarce and has exposure to the aerosphere variables including air temperature and humidity. Here this paper aims to select the best vegetation structures and aerosphere factors that support the abundance of C. brachyotis in mountainous paddy field landscape in West Java. The model selection was using AIC methodology by testing 15 models including 5 single models and 10 combination models of explanatory variables. Based on the model, tree height and combinations of tree height and elevation produced the best prediction for the bat abundances, as described by low values of AIC and the highest values of R2 and adjusted R2. For the best models, the AIC values ranged from 16.674 to 17.603, from 0.3404 to 0.4144 (R2), and 0.2461 to 0.2192 for adjusted R2. Regarding conservation of C. brachyotis and learning from the model, the conservation approaches mainly in mountainous paddy fields are encouraged to protect and conserve high altitude landscapes and trees with height > 10 m. Whereas the AIC results show lack of aerosphere variable effects on C. brachyotis (AIC: 19.346-20.406, R2: 0.1124-0.001353, and adjusted R2: -0.01444 - -0.1413).


title: Brain transcriptional regulatory architecture and schizophrenia etiology converge between East Asian and European ancestral populations

abstract: Understanding the genetic architecture of gene expression and splicing in human brain is critical to unlocking the mechanisms of complex neuropsychiatric disorders like schizophrenia (SCZ). Large-scale brain transcriptomic studies are based primarily on populations of European (EUR) ancestry. The uniformity of mono-racial resources may limit important insights into the disease etiology. Here, we characterized brain transcriptional regulatory architecture of East Asians (EAS; n=151), identifying 3,278 expression quantitative trait loci (eQTL) and 4,726 spliceQTL (sQTL). Comparing these to PsychENCODE/BrainGVEX confirmed our hypothesis that the transcriptional regulatory architecture in EAS and EUR brains align. Furthermore, distinctive allelic frequency and linkage disequilibrium impede QTL translation and gene-expression prediction accuracy. Integration of eQTL/sQTL with genome-wide association studies reveals common and novel SCZ risk genes. Pathway-based analyses showing shared SCZ biology point to synaptic and GTPase dysfunction as a prospective pathogenesis. This study elucidates the transcriptional landscape of the EAS brain and emphasizes an essential convergence between EAS and EUR populations.

category: genetics


title: High similarity among ChEC-seq datasets

abstract: ChEC-seq is a method used to identify protein-DNA interactions across a genome. It involves fusing micrococcal nuclease (MNase) to a protein of interest. In principle, specific genome-wide interactions of the fusion protein with chromatin result in local DNA cleavages that can be mapped by DNA sequencing. ChEC-seq has been used to draw conclusions about broad gene-specificities of certain protein-DNA interactions. In particular, the transcriptional regulators SAGA, TFIID, and Mediator are reported to generally occupy the promoter/UAS of genes transcribed by RNA polymerase II in yeast. Here we compare published yeast ChEC-seq data performed with a variety of protein fusions across essentially all genes, and find high similarities with negative controls. We conclude that ChEC-seq patterning for SAGA, TFIID, and Mediator differ little from background at most promoter regions, and thus cannot be used to draw conclusions about broad gene specificity of these factors.


title: LevioSAM: Fast lift-over of alternate reference alignments

abstract:
MotivationAs more population genetics datasets and population-specific references become available, the task of translating ("lifting") read alignments from one reference coordinate system to another is becoming more common. Existing tools generally require a chain file, whereas VCF files are the more common way to represent variation. Existing tools also do not make effective use of threads, creating a post-alignment bottleneck.

ResultsLevioSAM is a tool for lifting SAM/BAM alignments from one reference to another using a VCF file containing population variants. LevioSAM uses succinct data structures and scales efficiently to many threads. When run downstream of a read aligner, levioSAM completes in less than 13% the time required by an aligner when both are run with 16 threads.

Availabilityhttps://github.com/alshai/levioSAM

Contacttmun1@jhu.edu, langmea@cs.jhu.edu


title: Insertions and deletions as phylogenetic signal in alignment-free sequence comparison

abstract: Most methods for phylogenetic tree reconstruction are based on sequence alignments; they infer phylogenies from substitutions that may have occurred at the aligned sequence positions. Gaps in alignments are usually not employed as phylogenetic signal. In this paper, we explore an alignment-free approach that uses insertions and deletions (indels) as an additional source of information for phylogeny inference. For a set of four or more input sequences, we generate so-called quartet blocks of four putative homologous segments each. For pairs of such quartet blocks involving the same four sequences, we compare the distances between the two blocks in these sequences, to obtain hints about indels that may have happened between the blocks since the respective four sequences have evolved from their last common ancestor. A prototype implementation is presented to infer phylogenetic trees from these data, using a quartet-tree approach or, alternatively, under the maximum-parsimony paradigm. This approach should not be regarded as an alternative to established methods, but rather as a complementary source of phylogenetic information. Interestingly, however, our software is able to produce phylogenetic trees from putative indels alone that are comparable to trees obtained with existing alignment-free methods.


title: Discovery and Functional Characterization of Pro-growth Enhancers in Human Cancer Cells

abstract: Precision medicine depends critically on developing treatment strategies that can selectively target cancer cells with minimal adverse effects. Identifying unique transcriptional regulators of oncogenic signaling, and targeting cancer-cell-specific enhancers that may be active only in specific tumor cell lineages, could provide the necessary high specificity, but a scarcity of functionally validated enhancers in cancer cells presents a significant hurdle to this strategy. We address this limitation by carrying out large-scale functional screens for pro-growth enhancers using highly multiplexed CRISPR-based perturbation and sequencing in multiple cancer cell lines. We used this strategy to identify 488 pro-growth enhancers in a colorectal cancer cell line and 22 functional enhancers for the MYC and MYB key oncogenes in an additional nine cancer cell lines. The majority of pro-growth enhancers are accessible and presumably active only in cancer cells but not in normal tissues, and are enriched for elements associated with poor prognosis in colorectal cancer. We further identify master transcriptional regulators and demonstrate that the cancer pro-growth enhancers are modulated by lineage-specific transcription factors acting downstream of growth signaling pathways. Our results uncover context-specific, potentially actionable pro-growth enhancers from cancer cells, yielding insight into altered oncogenic transcription and revealing potential therapeutic targets for cancer treatment.

category: cancer biology


title: Bone Marrow-Derived AXL Tyrosine Kinase Promotes Mitogenic Crosstalk and Cardiac Allograft Vasculopathy

abstract: Cardiac Allograft Vasculopathy (CAV) is a leading contributor to late transplant rejection. Although implicated, the mechanisms by which bone marrow-derived cells promote CAV remain unclear. Emerging evidence implicates the cell surface receptor tyrosine kinase AXL to be elevated in rejecting human allografts. AXL protein is found on multiple cell types, including bone marrow-derived myeloid cells. The causal role of AXL from this compartment and during transplant is largely unknown. This is important because AXL is a key regulator of myeloid inflammation. Utilizing experimental chimeras deficient in the bone marrow-derived Axl gene, we report that Axl antagonizes cardiac allograft survival and promotes CAV. Flow cytometric and histologic analyses of Axl-deficient transplant recipients revealed reductions in both allograft immune cell accumulation and vascular intimal thickness. Co-culture experiments designed to identify cell-intrinsic functions of Axl uncovered complementary cell-proliferative pathways by which Axl promotes CAV-associated inflammation. Specifically, Axl-deficient myeloid cells were less efficient at increasing the replication of both antigen-specific T cells and vascular smooth muscle cells (VSMCs), the latter a key hallmark of CAV. For the latter, we discovered that Axl-was required to amass the VSMC mitogen Platelet-Derived Growth Factor. Taken together, our studies reveal a new role for myeloid Axl in the progression of CAV and mitogenic crosstalk. Inhibition of AXL-protein, in combination with current standards of care, is a candidate strategy to prolong cardiac allograft survival.


title: A dynamically-structured matrix population model based on renewal processes for accumulative development under variable environmental conditions

abstract: Arthropod vectors are responsible for the transmission of pathogens in humans and other species. The transmission rate depends on the size and activity of the vector population, factors which are in turn strongly affected by environmental conditions. Therefore, in order to develop realistic representations of vector population dynamics, it is necessary to properly account for the impact of a changing environment on the duration of life processes. Here, we use a pseudo-stage-structured population to model the accumulative process of development as a renewal process with a variable rate that depends on the environment. We incorporate this into sPop, formerly an age-structured population dynamics model. This framework allows the modeller to represent realistic life stage durations by choosing from three alternative probability schemes: an Erlang distribution, a Pascal distribution, or a fixed duration, while enabling the model to respond appropriately to variations in stage duration characteristics. Using this approach, we demonstrate that introducing random variation into the environmental conditions, which results in fluctuating development rates, on average decreases the time required for stage completion. An exception to this is an already optimum development rate being perturbed by noise towards a less efficient course. The proposed framework is suitable for performing inverse modelling with data collected from highly variable environmental conditions, the results of which can be used to develop realistic climate-driven population dynamics models.


title: Catching SARS-CoV-2 by sequence hybridization: a comparative analysis

abstract: Controlling and monitoring the still ongoing SARS-CoV-2 pandemic regarding geographical distributions, evolution and emergence of new mutations of the SARS-CoV-2 virus is only possible due to continuous next-generation sequencing (NGS) and worldwide sequence data sharing. Efficient sequencing strategies enabling the retrieval of the maximum number of high quality, full-length genomes are hence indispensable. Here, we describe for the first time a combined approach of digital droplet PCR (ddPCR) and NGS to evaluate five commercially available sequence capture panels targeting SARS-CoV-2. In doing so, we were not only able to determine the most sensitive and specific capture panel, but to discriminate their mode of action and number of read pairs needed to recover a high quality full length genome. Thereby, we are providing essential information for all sequencing laboratories worldwide striving for maximizing the sequencing output and simultaneously minimizing time, costs and sequencing resources.


title: Tuberculostearic acid (TSA)-containing phosphatidylinositols as reliable marker to determine Mycobacterium tuberculosis bacterial burden

abstract:
It is estimated that approximately one-fourth of the world's population is infected with strains of the Mycobacterium tuberculosis complex (MTBC), the causative agents of tuberculosis (TB). In this study, we present rationally developed molecular markers for bacterial burden, which are derived from mycobacterial phospholipids. Using lipidomic approaches, we show that tuberculostearic acid (TSA)-containing phosphatidylinositols (PI) are present in all clinically relevant MTBC lineages investigated. For the major abundant lipid PI 16:0_19:0 (TSA), a detection limit equivalent to 102 colony forming units (CFU) was determined for bacterial cultures and approximately 103 for cell culture systems. We further developed a mass spectrometry based targeted lipid assay, which - in contrast to bacterial quantification on solid medium - can be performed within several hours including sample preparation. Translation of this indirect and culture-free detection approach allowed the determination of pathogen loads in infected murine macrophages, human neutrophils and murine lung tissue. We show that marker lipids inferred from the mycobacterial PIs are increased in peripheral blood mononuclear cells (PBMCs) of TB patients beyond the lipid metabolic background in comparison to healthy controls. In a small cohort of drug-susceptible TB patients elevated levels of these marker molecules were detected at therapy start and declined following successful anti-tuberculosis treatment. The concentration of TSA-containing PIs can be used as correlate for reliable and rapid quantification of Mycobacterium tuberculosis (Mtb) burden in experimental in vitro model systems and may also provide a clinically relevant tool for monitoring TB therapy.

One Sentence SummaryTuberculostearic acid containing phosphatidylinositols represent a novel, fast to measure, reliable correlate of Mycobacterium tuberculosis bacterial burden in experimental model systems, which makes a future clinical application conceivable.

category: biochemistry


title: SARS-CoV-2 ORF7b: is a bat virus protein homologue a major cause of COVID-19 symptoms?

abstract: ORF7b is an accessory protein of SARS-CoV-2, the virus behind the COVID-19 pandemic. Using cell-free synthesized ORF7b, we experimentally show that ORF7b assembles into stable multimers. The ORF7b sequence shows a transmembrane segment, which multimerizes through a leucine zipper. We hypothesize that ORF7b has the potential to interfere with important cellular processes that involve leucine-zipper formation, and present two particularly striking examples. First, leucine zippers are central in heart rhythm regulation through multimerization of phospholamban in cardiomyocytes. Second, epithelial cell-cell adhesion relies on E-cadherins, which dimerize using a transmembrane leucine zipper. Most common symptoms of SARS-CoV-2 infection, including heart arrythmias, odor loss, impaired oxygen uptake and intestinal problems, up to multiorgan failure, can be rationalized by a possible interference of ORF7b with the functions of these proteins. We ask whether this is pure coincidence, or whether our observations point to disruption by ORF7b of vital processes in COVID-19.


title: Collaboration between the Fab and Fc contribute to maximal protection against SARS-CoV-2 in nonhuman primates following NVX-CoV2373 subunit vaccine with Matrix-M™ vaccination

abstract:
Recently approved vaccines have already shown remarkable protection in limiting SARS-CoV-2 associated disease. However, immunologic mechanism(s) of protection, as well as how boosting alters immunity to wildtype and newly emerging strains, remain incompletely understood. Here we deeply profiled the humoral immune response in a cohort of non-human primates immunized with a stable recombinant full-length SARS-CoV-2 spike (S) glycoprotein (NVX-CoV2373) at two dose levels, administered as a single or two-dose regimen with a saponin-based adjuvant Matrix-M. While antigen dose had some effect on Fc-effector profiles, both antigen dose and boosting significantly altered overall titers, neutralization and Fc-effector profiles, driving unique vaccine-induced antibody fingerprints. Combined differences in antibody effector functions and neutralization were strongly associated with distinct levels of protection in the upper and lower respiratory tract, pointing to the presence of combined, but distinct, compartment-specific neutralization and Fc-mechanisms as key determinants of protective immunity against infection. Moreover, NVX-CoV2373 elicited antibodies functionally target emerging SARS-CoV-2 variants, collectively pointing to the critical collaborative role for Fab and Fc in driving maximal protection against SARS-CoV-2. Collectively, the data presented here suggest that a single dose may prevent disease, but that two doses may be essential to block further transmission of SARS-CoV-2 and emerging variants.

HighlightsO_LINVX-CoV2373 subunit vaccine elicits receptor blocking, virus neutralizing antibodies, and Fc-effector functional antibodies.
C_LIO_LIThe vaccine protects against respiratory tract infection and virus shedding in non-human primates (NHPs).
C_LIO_LIBoth neutralizing and Fc-effector functions contribute to protection, potentially through different mechanisms in the upper and lower respiratory tract.
C_LIO_LIBoth macaque and human vaccine-induced antibodies exhibit altered Fc-receptor binding to emerging mutants.
C_LI


title: LncRNA SNHG1 suppresses LPS-induced acute lung injury by regulating miR-421/TIMP3 axis

abstract: Extensive evidence has revealed the crucial roles of long non-coding RNAs (lncRNAs) in acute lung injury (ALI). This study aimed to explore the mechanism of lncRNA SNHG1 in lipopolysaccharides (LPS)-induced ALI. RT-qPCR was employed to test the levels of SNHG1, miR-421 and TIMP3 in A549 cells. Cell viability and apoptosis were assessed by CCK-8 assay and flow cytometry. ELISA assay was adopted to examine the levels of inflammatory-related cytokines, including IL-1{beta}, IL-6 and TNF-. The binding sequences of miR-421 and SNHG1 or TIMP3 were predicted using starBase software. Then dual-luciferase reporter and RIP assays were adopted to verify the interaction between miR-421 and SNHG1 or TIMP3. The protein level of TIMP3 was measured by western blotting. It was found that LPS stimulation downregulated SNHG1 level and SNHG1 addition decreased viability, and induced apoptosis as well as promoted inflammatory responses in LPS-treated A549 cells. SNHG1 could sponge miR-421 and SNHG1 protected A549 cells from LPS-induced injury via inhibiting miR-421. Moreover, TIMP3 was a target of miR-421. MiR-421 silence protected A549 cells against the LPS-triggered inhibition in viability, and promotion in apoptosis and inflammatory responses. SNHG1 could upregulate TIMP3 through acting as a ceRNA of miR-421 in A549 cells. Altogether, the present study elaborated that SNHG1 inhibited LPS-stimulated ALI by modulating the miR-421/TIMP3 axis.


title: Post-infection treatment with a protease inhibitor increases survival of mice with a fatal SARS-CoV-2 infection

abstract: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection continues to be a serious global public health threat. The 3C-like protease (3CLpro) is a virus protease encoded by SARS-CoV-2, which is essential for virus replication. We have previously reported a series of small molecule 3CLpro inhibitors effective for inhibiting replication of human coronaviruses including SARS-CoV-2 in cell culture and in animal models. Here we generated a series of deuterated variants of a 3CLpro inhibitor, GC376, and evaluated the antiviral effect against SARS-CoV-2. The deuterated GC376 displayed potent inhibitory activity against SARS-CoV-2 in the enzyme and the cell-based assays. The K18-hACE2 mice develop mild to lethal infection commensurate with SARS-CoV-2 challenge doses and was proposed as a model for efficacy testing of antiviral agents. We treated lethally infected mice with a deuterated derivative of GC376. Treatment of K18-hACE2 mice at 24 hr post infection with a derivative (compound 2) resulted in increased survival of mice compared to vehicle-treated mice. Lung virus titers were decreased, and histopathological changes were ameliorated in compound 2-treated mice compared to vehicle-treated mice. Structural investigation using high-resolution crystallography illuminated binding interactions of 3CLpro of SARS-CoV-2 and SARS-CoV with deuterated variants of GC376. Taken together, deuterated GC376 variants have excellent potential as antiviral agents against SARS-CoV-2.

category: microbiology


title: Cellular management of Zinc in group B Streptococcus supports bacterial resistance against metal intoxication and promotes disseminated infection

abstract:
Zinc (Zn) is an essential trace element for normal bacterial physiology but divergently, can intoxicate bacteria at high concentrations. Here, we define the molecular systems for Zn detoxification in Streptococcus agalactiae, also known as group B Streptococcus, and examine the effects of resistance to Zn stress on virulence. We compared the growth of wild-type bacteria and mutants deleted for the Zn exporter, czcD, and the response regulator, sczA, using Zn-stress conditions in vitro. Macrophage antibiotic protection assays and a mouse model of disseminated infection were used to assess virulence. Global bacterial transcriptional responses to Zn stress were defined by RNA-sequencing and qRTPCR. czcD and sczA enabled S. agalactiae to survive Zn stress, with the putative CzcD efflux system activated by SczA. Additional genes activated in response to Zn stress encompassed divalent cation transporters that contribute to regulation of Mn and Fe homeostasis. In vivo, the czcD-sczA Zn-management axis supported virulence in the blood, heart, liver and bladder. Additionally, several genes not previously linked to Zn stress in any bacterium, including most notably, arcA for arginine deamination also mediated resistance to Zn stress; representing a novel molecular mechanism of bacterial resistance to metal intoxication. Taken together, these findings show that S. agalactiae responds to Zn stress by sczA regulation of czcD, with additional novel mechanisms of resistance supported by arcA, encoding arginine deaminase. Cellular management of Zn stress in S. agalactiae supports virulence by facilitating bacterial survival in the host during systemic infection.

Importance StatementStreptococcus agalactiae, also known as group B streptococcus, is an opportunistic pathogen that causes various diseases in humans and animals. This bacterium has genetic systems that enable Zinc (Zn) detoxification in environments of metal stress, but these systems remain largely undefined. Using a combination of genomic, genetic and cellular assays we show that this pathogen controls Zn export through CzcD to manage Zn stress, and utilizes a system of arginine deamination never previously linked to metal stress responses in bacteria to survive metal intoxication. We show that these systems are crucial for survival of S. agalactiae in vitro during Zn stress and also enhance virulence during systemic infection in mice. These discoveries establish new molecular mechanisms of resistance to metal intoxication in bacteria; we suggest these mechanisms are likely to operate in other bacteria as a way to sustain microbial survival in conditions of metal stress, including in host environments.


title: High fiber, whole foods dietary intervention alters the human gut microbiome but not fecal short-chain fatty acids

abstract:
Dietary shifts can have a direct impact on the gut microbiome by preferentially selecting for microbes capable of utilizing the various dietary nutrients. Intake of dietary fiber has decreased precipitously in the last century, while consumption of processed foods has increased. Fiber, or microbiota-accessible carbohydrates (MACs), persist in the digestive tract and can be metabolized by specific bacteria encoding fiber degrading enzymes. Digestion of MACs results in the accumulation of short-chain fatty acids (SCFAs) and other metabolic byproducts that are critical to human health. Here, we implemented a two-week dietary fiber intervention aiming for 40-50 grams of fiber per day within the context of a course-based undergraduate research experience (CURE) (n = 20). By coupling shotgun metagenomic sequencing and targeted gas-chromatography mass spectrometry (GC/MS), we found that the dietary intervention significantly altered the composition of individual gut microbiomes, accounting for 8.3% of the longitudinal variability within subjects. Notably, microbial taxa that increased in relative abundance as a result of the diet change included known MAC degraders (i.e., Bifidobacterium and Lactobacillus). We further assessed the genetic diversity within Bifidobacterium, assayed by amplification of the groEL gene. Concomitant with microbial composition changes, we show an increase in the abundance of genes involved in inositol degradation. Despite these changes in gut microbiome composition, we did not detect a consistent shift in SCFA abundance. Collectively, our results demonstrate that on a short-term timescale of two weeks, increased fiber intake can induce compositional changes of the gut microbiome, including an increase in MAC degrading bacteria.

IMPORTANCEA profound decrease in the consumption of dietary fiber in many parts of the world in the last century may be associated with the increasing prevalence of Type II diabetes, colon cancer, and other health problems. A typical U.S. diet includes about [~]15 grams of fiber per day, far less fiber than daily recommended allowance. Changes in dietary fiber intake affect human health not only through the uptake of nutrients directly, but also indirectly through changes in the microbial community and their associated metabolism. Here we conducted a two-week diet intervention in healthy young adults to investigate the impact of fiber consumption on the gut microbiome. Participants increased their average fiber consumption by 25 grams/day on average for two weeks. The high fiber diet intervention altered the gut microbiome of the study participants, including increases in known fiber degrading microbes such as Bifidobacterium and Lactobacillus.


title: On the robustness of inference of association with the gut microbiota in stool, swab and mucosal tissue samples

abstract: The gut microbiota plays an important role in human health and disease. Stool, swab and mucosal tissue samples have been used in individual studies to survey the microbial community but the consequences of using these different sample types are not completely understood. We previously reported differences in microbial community composition with 16S rRNA amplicon sequencing between stool, swab and mucosal tissue samples. Here, we extended the previous study to a larger cohort and performed shotgun metagenome sequencing of 1,397 stool, swab and mucosal tissue samples from 240 participants. Consistent with previous results, taxonomic composition of stool and swab samples was distinct, but still more similar to each other than mucosal tissue samples, which had a substantially different community composition, characterized by a high relative abundance of the mucus metabolizers Bacteroides and Subdoligranulum, as well as bacteria with higher tolerance for oxidative stress such as Escherichia. As has been previously reported, functional profiles were more uniform across sample types than taxonomic profiles with differences between stool and swab samples smaller, but mucosal tissue samples remained distinct from the other two types. When the taxonomic and functional profiles of different sample types were used for inference in association with host phenotypes of age, sex, body mass index (BMI), antibiotics or non-steroidal anti-inflammatory drugs (NSAIDs) use, hypothesis testing using either stool or swab gave broadly similar results, but inference performed on mucosal tissue samples gave results that were generally less consistent with either stool or swab. Our study represents an important resource for the experimental design of studies aimed to understand microbiota perturbations specific to defined micro niches within the human intestinal tract.


title: Protein glycosylation is essential for SARS-CoV-2 infection

abstract: SARS-CoV-2 extensively N-glycosylates its spike proteins, which are necessary for host cell invasion and the target of both vaccines and immunotherapies. These sugars are predicted to help mediate spike binding to the host receptor by stabilizing its  open conformation and evading host immunity. Here, we investigated both the essentiality of the host N-glycosylation pathway and SARS-CoV-2 N-glycans for infection. Inhibition of host N-glycosylation using RNAi or FDA-approved drugs reduced virus infectivity, including that of several variants. Under these conditions, cells produced less virions and some completely lost their infectivity. Furthermore, partial deglycosylation of intact virions showed that surface-exposed N-glycans are critical for cell invasion. Altogether, spike N-glycosylation is a targetable pathway with clinical potential for treatment or prevention of COVID-19.


title: Protein N-glycosylation is essential for SARS-CoV-2 infection


title: Evolutionary implications of recombination differences across diverging populations of Anopheles

abstract: Recombination is one of the main evolutionary mechanisms responsible for changing the genomic architecture of populations; and in essence, it is the main mechanism by which novel combinations of alleles, haplotypes, are formed. A clear picture that has emerged across study systems is that recombination is highly variable, even among closely related species. However, it is only until very recently that we have started to understand how recombination variation between populations of the same species impact genetic diversity and divergence. Here, we used whole-genome sequence data to build fine-scale recombination maps for nine populations within two species of Anopheles, Anopheles gambiae and Anopheles coluzzii. The genome-wide recombination averages were on the same order of magnitude for all populations except one. Yet, we identified significant differences in fine-scale recombination rates among all population comparisons. We report that effective population sizes, and presence of a chromosomal inversion has major contribution to recombination rate variation along the genome and across populations. We identified over 400 highly variable recombination hotspots across all populations, where only 9.6% are shared between two or more populations. Additionally, our results are consistent with recombination hotspots contributing to both genetic diversity and absolute divergence (dxy) between populations and species of Anopheles. However, we also show that recombination has a small impact on population genetic differentiation as estimated with FST. The minimal impact that recombination has on genetic differentiation across populations represents the first empirical evidence against recent theoretical work suggesting that variation in recombination along the genome can mask or impair our ability to detect signatures of selection. Our findings add new understanding to how recombination rates vary within species, and how this major evolutionary mechanism can maintain and contribute to genetic variation and divergence within a prominent malaria vector.


title: Little evidence the standard genetic code is optimized for resource conservation

abstract: Selection for resource conservation can shape the coding sequences of organisms living in nutrient-limited environments. Recently, it was proposed that selection for resource conservation, specifically for nitrogen and carbon content, has also shaped the structure of the standard genetic code, such that the missense mutations it allows tend to cause small increases in the number of nitrogen and carbon atoms in amino acids. Moreover, it was proposed that this optimization is not confounded by known optimizations of the standard genetic code, such as for polar requirement or hydropathy. We challenge these claims. We show the proposed optimization for nitrogen conservation is highly sensitive to choice of null model and the proposed optimization for carbon conservation is confounded by the known conservative nature of the standard genetic code with respect to the molecular volume of amino acids. There is therefore little evidence the standard genetic code is optimized for resource conservation.


title: Expanded Asgard archaea shed new light on the origin of eukaryotes and support a 2-domain tree of life

abstract: The hypothesis that eukaryotes originated from within the domain Archaea has been strongly supported by recent phylogenomic analyses placing Heimdallarchaeota from the Asgard superphylum as the closest known archaeal sister-group to eukaryotes. At present, only seven phyla are described in the Asgard superphylum, which limits our understanding of the relationship between eukaryotes and archaea, as well as the evolution and ecological functions of Asgard archaea. Here, we describe five novel phylum-level Asgard archaeal lineages, tentatively named Tyr-, Sigyn-, Freyr-, Hoder- and Balderarchaeota. Comprehensive phylogenomic analyses supported a new Asgard lineage Tyrarchaeota was identified as a deeper branching lineage cluster with the eukaryotic nuclear host lineage than Heimdallarchaeota that were previously considered as the closest archaeal relatives of eukaryotes. Metabolic reconstruction of Tyrarchaeota suggests a mixotrophic lifestyle of this archaea, capable of peptides and amino acids utilization while having the potential using the Wood-Ljungdahl pathway for carbon fixation and acetogenesis. This study largely expands the Asgard superphylum, provides additional evidences to support the 2-domain life tree thus sheds new light on the evolution and geochemical functions of the Asgard archaea.


title: Drosophila STING protein has a role in lipid metabolism.

abstract: Stimulator of interferon genes (STING) plays an important role in innate immunity by controlling type I interferon response against invaded pathogens. In this work we describe a direct but previously unknown role of STING in lipid metabolism in Drosophila. Flies with STING deletion are sensitive to starvation and oxidative stress, have reduced lipid storage and downregulated expression of lipid metabolism genes. We found that Drosophila STING interacts with lipid synthesizing enzymes acetyl-CoA carboxylase (ACC) and fatty acid synthase (FAS). ACC and FAS also interact with each other, indicating that all three proteins may be components of a large multi-enzyme complex. The deletion of Drosophila STING leads to disturbed ACC localization and decreased FAS enzyme activity. Together, our results demonstrate a direct role of STING in lipid metabolism in Drosophila.


title: BGvar - a comprehensive resource for blood group immunogenetics

abstract:
BackgroundBlood groups form the basis of effective and safe blood transfusion. There are about 41 well recognized human blood group systems presently known. Blood groups are molecularly determined by the presence of specific antigens on the red blood cells and are genetically determined and inherited following Mendelian principles. The lack of a comprehensive, relevant, manually compiled and genome-ready dataset of red cell antigens limited the widespread application of genomic technologies to characterise and interpret the blood group complement of an individual from genomic datasets.

Materials and MethodsA range of public datasets were used to systematically annotate the variation compendium for its functionality and allele frequencies across global populations. Details on phenotype or relevant clinical importance were collated from reported literature evidence.

ResultsWe have compiled the Blood Group Associated Genomic Variant Resource (BGvar), a manually curated online resource comprising all known human blood group related allelic variants including a total of 1672 ISBT approved alleles and 1552 alleles predicted and curated from literature reports. This repository includes 1606 Single Nucleotide Variations (SNVs), 270 Insertions, Deletions (InDels) and Duplications and about 1310 combination mutations corresponding to 41 human blood group systems and 2 transcription factors. This compendium also encompasses gene fusion and rearrangement events occurring in human blood group genes.

ConclusionTo the best of our knowledge, BGvar is a comprehensive and a user friendly resource with most relevant collation of blood group alleles in humans. BGvar is accessible online at URL: http://clingen.igib.res.in/bgvar/


title: Human brain mitochondrial-nuclear cross-talk is cell-type specific and is perturbed by neurodegeneration

abstract: Mitochondrial dysfunction contributes to the pathogenesis of many neurodegenerative diseases as mitochondria are essential to neuronal function. The mitochondrial genome encodes a small number of core respiratory chain proteins, whereas the vast majority of mitochondrial proteins are encoded by the nuclear genome. Here we focus on establishing a profile of nuclear-mitochondrial transcriptional relationships in healthy human central nervous system tissue data, before examining perturbations of these processes in Alzheimer&#8217s disease using transcriptomic data originating from affected human brain tissue. Through cross-central nervous system analysis of mitochondrial-nuclear gene pair relationships, we find that the cell type composition underlies regional variation, and variation is driven at the subcellular level by heterogeneity of nuclear-mitochondrial coordination in post-synaptic regions. We show that nuclear genes causally implicated in sporadic Parkinson&#8217s disease and Alzheimer&#8217s disease show much stronger relationships with the mitochondrial genome than expected by chance, and that nuclear-mitochondrial relationships are significantly perturbed in Alzheimer&#8217s disease cases, particularly amongst genes involved in synaptic and lysosomal pathways. Finally, we present MitoNuclearCOEXPlorer, a web tool designed to allow users to interrogate and visualise key mitochondrial-nuclear relationships in multi-dimensional brain data. We conclude that mitochondrial-nuclear relationships differ significantly across regions of the healthy brain, which appears to be driven by the functional specialisation of different cell types. We also find that mitochondrial-nuclear co-expression in critical pathways is disrupted in Alzheimer&#8217s disease, potentially implicating the regulation of energy balance and removal of dysfunctional mitochondria in the etiology or progression of the disease and making the case for the relevance of bi-genomic co-ordination in the pathogenesis of neurodegenerative diseases.


title: Serological Profile Of Specific Antibodies Against Dominant Antigens Of SARS-CoV-2 In Chilean COVID-19 Patients.

abstract: Coronavirus disease 2019 (COVID-19) is caused by SARS-CoV-2 and has been a pandemic since March 2020. Currently, the virus has infected more than 50 million people worldwide and more than half a million in Chile. For many coronaviruses, Spike (S) and Nucleocapsid (N) proteins are described as major antigenic molecules, inducing seroconversion and production of neutralizing antibodies. In this work, we evaluated the presence in serum of IgM, IgA and IgG antibodies against N and S proteins of SARS-CoV-2 using western blot, and developed an ELISA test for the qualitative characterization of COVID-19 patients. Patients with an active infection or who have recovered from COVID-19 showed specific immunoblotting patterns for the recombinants S protein and its domains S1 and S2, as well as for the N protein of SARS-CoV-2. Anti-N antibodies were more frequently detected than anti-S or anti-S1-RBD antibodies. People who were never exposed to SARS-CoV-2 did not show reactivity. Finally, indirect ELISA assays using N and S1-RBD proteins, alone or in combination, were established with variable sensitivity and specificity depending on the antigen bound to the solid phase. Overall, Spike showed higher specificity than the nucleocapsid, and comparable sensitivity for both antigens. Both approaches confirmed the seroconversion after infection and allowed us to implement the analysis of antibodies in blood for research purposes in a local facility.


title: Furin cleavage of the SARS-CoV-2 spike is modulated by O-glycosylation

abstract: The SARS-CoV-2 coronavirus responsible for the global pandemic contains a unique furin cleavage site in the spike protein (S) that increases viral infectivity and syncytia formation. Here, we show that O-glycosylation near the furin cleavage site is mediated by specific members of the GALNT enzyme family and is dependent on the novel proline at position 681 (P681). We further demonstrate that O-glycosylation of S decreases furin cleavage. Finally, we show that GALNT family members capable of glycosylating S are expressed in human respiratory cells that are targets for SARS-CoV-2 infection. Our results suggest that O-glycosylation may influence viral infectivity/tropism by modulating furin cleavage of S and provide mechanistic insight into the potential role of P681 mutations in the recently identified, highly transmissible B.1.1.7 variant.


title: Caprin-1 binding to the critical stress granule protein G3BP1 is regulated by pH

abstract: G3BP is the central hub within the protein-RNA interaction network of stress-induced bio-molecular condensates known as stress granules (SG). The SG-associated proteins Caprin-1 and USP10 exhibit mutually exclusive binding to the structured NTF2-domain of G3BP1, thereby regulating G3BP1-mediated condensation, but with opposite effects: Caprin-1 promotes but USP10 inhibits SG formation. Herein, we present the crystal structure of G3BP1-NTF2 in complex with a Caprin-1 derived short linear motif (SLiM), which provides a molecular understanding for the mutually exclusive binding of USP10 and Caprin-1 to G3BP1. Caprin-1 but not USP10 contacts two G3BP1-NTF2 histidine residues, which was confirmed using biochemical, biophysical and cellular biological binding assays. G3BP1/Caprin-1 interactions disrupted via point mutations resulted in fewer and smaller SG condensates. In addition, biochemical binding assays demonstrated reduced binding of Caprin-1 to G3BP1 at lower pH values. Finally, ratiometric pH sensitive measurements of SGs revealed a substantial drop in pH compared to the adjacent cytosol, suggesting that reduced pH can fine-tune and regulate the G3BP1-mediated interaction network via a NTF2-mediated pH-sensitive SLiM-selection mechanism.


title: Focused X-ray Luminescence Computed Tomography using a Continuous Scanning Scheme

abstract: X-ray luminescence computed tomography (XLCT) imaging is a hybrid molecular imaging modality combining the merits of both conventional x-ray imaging (high spatial resolution) and optical imaging (high measurement sensitivity). The narrow x-ray beam based XLCT imaging has been shown to be promising. However due to the selective excitation scheme, the imaging speed is slow thus limiting its practical applications for in vivo imaging. In this work, we have introduced a continuous scanning scheme to acquire data for each angular projection in one motion, eliminating the previous stepping scheme and reducing the data acquisition time, which makes it feasible for multiple transverse scans for three-dimensional (3D) imaging. We have introduced a high accuracy vertical stage to our focused x-ray beam based XLCT imaging system to perform high-resolution and 3D XLCT imaging. We have also included a scintillator crystal coupled to a PMT to act as a single-pixel detector for boundary detection purposes to replace our previous flat panel x-ray detector. We have verified the feasibility of our proposed scanning scheme and imaging system by performing phantom experimental studies. A phantom was embedded with a set of cylindrical targets with 200 {micro}m edge-to-edge distance and was scanned in our imaging system with the proposed method. To test the feasibility for 3D scanning, we took measurements from 4 transverse slices with a vertical step size of 1 mm. The results of the experiments verified the feasibility of our proposed method to perform 3D XLCT imaging using a narrow x-ray beam in a reasonable time.

category: bioengineering


title: Inference of genome 3D architecture by modeling overdispersion of Hi-C data

abstract:
We address the challenge of inferring a consensus 3D model of genome architecture from Hi-C data. Existing approaches most often rely on a two step algorithm: first convert the contact counts into distances, then optimize an objective function akin to multidimensional scaling (MDS) to infer a 3D model. Other approaches use a maximum likelihood approach, modeling the contact counts between two loci as a Poisson random variable whose intensity is a decreasing function of the distance between them. However, a Poisson model of contact counts implies that the variance of the data is equal to the mean, a relationship that is often too restrictive to properly model count data.

We first confirm the presence of overdispersion in several real Hi-C data sets, and we show that the overdispersion arises even in simulated data sets. We then propose a new model, called Pastis-NB, where we replace the Poisson model of contact counts by a negative binomial one, which is parametrized by a mean and a separate dispersion parameter. The dispersion parameter allows the variance to be adjusted independently from the mean, thus better modeling overdispersed data. We compare the results of Pastis-NB to those of several previously published algorithms: three MDS-based methods (ShRec3D, ChromSDE, and Pastis-MDS) and a statistical methods based on a Poisson model of the data (Pastis-PM). We show that the negative binomial inference yields more accurate structures on simulated data, and more robust structures than other models across real Hi-C replicates and across different resolutions.

A Python implementation of Pastis-NB is available at https://github.com/hiclib/pastis under the BSD license

Supplementary information is available at https://nellev.github.io/pastisnb/


title: Multi-task deep learning for concurrent prediction of protein structural properties

abstract:
Protein structural properties are diverse and have the characteristics of spatial hierarchy, such as secondary structures, solvent accessibility and backbone angles. Protein tertiary structures are formed in close association with these features. Separate prediction of these structural properties has been improved with the increasing number of samples of protein structures and with advances in machine learning techniques, but concurrent prediction of these tightly related structural features is more useful to understand the overall protein structure and functions. We introduce a multi-task deep learning method for concurrent prediction of protein secondary structures, solvent accessibility and backbone angles ({phi}, {psi}). The new method has main two deep network modules: the first one is designed as a DenseNet architecture a using bidirectional simplified GRU (GRU2) network, and the second module is designed as an updated Google Inception network. The new method is named CRRNN2.

CRRNN2 is trained on 14,100 protein sequences and its prediction performance is evaluated by testing on public benchmark datasets: CB513, CASP10, CASP11, CASP12 and TS1199. Compared with state-of-the-art methods, CRRNN2 achieves similar, or better performance on the prediction of 3- and 8-state secondary structures, solvent accessibility and backbone angles ({phi}, {psi}). Online CRRN-N2 applications, datasets and standalone software are available at http://qianglab.scst.suda.edu.cn/crrnn2/.


title: Uncovering Differences in Hydration Free Energies for Model Compound Mimics of Charged Sidechains of Amino Acids

abstract: Free energies of hydration are of fundamental interest for modeling and understanding conformational and phase equilibria of macromolecular solutes in aqueous phases. Of particular relevance to systems such as intrinsically disordered proteins are the free energies of hydration and hydration structures of model compounds that mimic charged sidechains of Arg, Lys, Asp, and Glu. Here, we deploy a Thermodynamic Cycle based Proton Dissociation (TCPD) approach in conjunction with data from direct measurements to obtain estimates for the free energies of hydration for model compounds that mimic the sidechains of Arg+, Lys+, Asp-, and Glu-. Irrespective of the choice made for the hydration free energy of the proton, the TCPD approach reveals clear trends regarding the free energies of hydration for Arg+, Lys+, Asp-, and Glu-. These trends include asymmetries between the hydration free energies of acidic (Asp- and Glu-) and basic (Arg+ and Lys+) residues. Further, the TCPD analysis, which relies on a combination of experimental data, shows that the free energy of hydration of Arg+ is less favorable than that of Lys+. We sought a physical explanation for the TCPD derived trends free energies of hydration. To this end, we performed temperature dependent calculations of free energies of hydration and analyzed hydration structures from simulations that use the polarizable AMOEBA (Atomic Multipole Optimized Energetics for Biomolecular Applications) forcefield and water model. At 298 K, the AMOEBA model generates estimates of free energies of hydration that are consistent with TCPD values with a free energy of hydration for the proton of {approx} -259 kcal / mol. Analysis of temperature dependent simulations leads to a structural explanation for the observed differences in free energies of hydration of ionizable residues and reveals that the heat capacity of hydration is positive for Arg+ and Lys+ and negative for Asp- and Glu-.

category: biophysics


title: Uncovering Differences in Hydration Free Energies and Structures for Model Compound Mimics of Charged Sidechains of Amino Acids


title: Targeting of TP53-independent cell cycle checkpoints overcomes FOLFOX resistance in Metastatic Colorectal Cancer

abstract: Patients with colorectal cancer (CRC) frequently develop liver metastases during the course of their disease. A substantial proportion of them receive neoadjuvant FOLFOX (5-Fluorouracil, Oxaliplatin, Leucovorin) prior to surgery in an attempt to enable successful surgical removal of their metastases and to reduce the risk of recurrence. Yet, the majority of patients progress during treatment or recur following surgery, and molecular mechanisms that contribute to FOLFOX resistance remain poorly understood. Here, using a combination of phenotypic, transcriptomic and genomic analyses of both tumor samples derived from patients with metastatic CRC and matching patient-derived tumor organoids (PDTOs), we characterize a novel FOLFOX resistance mechanism and identify inhibitors that target this mechanism to resensitize metastatic organoids to FOLFOX. Resistant PDTOs, identified after in vitro exposure to FOLFOX, exhibited elevated expression of E2F pathway, S phase, G2/M and spindle assembly checkpoints (SAC) genes. Similar molecular features were detected in CRLM from patients with progressive disease while under neoadjuvant FOLFOX treatment, highlighting the relevance of this finding. FOLFOX resistant PDTOs displayed inactivating mutations of TP53 and exhibited transcriptional features of P53 pathway downregulation. We found that they accumulated in early S-phase and underwent significant DNA damage during FOLFOX exposure, thereafter arresting in G2/M while they repaired their DNA after FOLFOX withdrawal. In parallel, results of a large kinase inhibitor screen indicated that drugs targeting regulators of the DNA damage response, G2M checkpoint and SAC had cytotoxic effects on PDTOs generated from patients whose disease progressed during treatment with FOLFOX. Corroborating this finding, CHK1 and WEE1 inhibitors were found to synergize with FOLFOX and sensitize previously resistant PDTOs. Additionally, targeting the SAC master regulator MPS1 using empesertib after exposure to FOLFOX, when cells accumulate in G2M, was also very effective to kill FOLFOX-resistant PDTOs. Our results indicate that targeted and timely inhibition of specific cell cycle checkpoints shows great potential to improve response rates to FOLFOX in patients with metastatic CRC, for whom therapeutic alternatives remain extremely limited.


title: A theoretical approach to coupling the epithelial-mesenchymal transition (EMT) to extracellular matrix (ECM) stiffness via LOXL2

abstract: The epithelial-mesenchymal transition (EMT) plays a critical role in cancer progression, being responsible in many cases for the onset of the metastatic cascade and being integral in the ability of cells to resist drug treatment. Most studies of EMT focus on its induction via chemical signals such as TGF-{beta} or Notch ligands, but it has become increasingly clear that biomechanical features of the microenvironment such as ECM (extracellular matrix) stiffness can be equally important. Here, we introduce a coupled feedback loop connecting stiffness to the EMT transcription factor ZEB1, which acts via increasing the secretion of LOXL2 that leads to increased cross-linking of collagen fibers in the ECM. This increased cross-linking can effectively increase ECM stiffness and increase ZEB1 levels, thus setting a positive feedback loop between ZEB1 and ECM stiffness. To investigate the impact of this non-cell-autonomous effect, we introduce a computational approach capable of connecting LOXL2 concentration to increased stiffness and thereby to higher ZEB1 levels. Our results indicate that this positive feedback loop, once activated, can effectively lock the cells in a mesenchymal state.


title: Pharmacokinetics of Orally Administered GS-441524 in Dogs

abstract: Despite being FDA-approved for COVID-19, the clinical efficacy of remdesivir (Veklury(R)) remains contentious. We previously pointed out pharmacokinetic, pharmacodynamic and toxicology reasons for why its parent nucleoside GS-441524, is better suited for COVID-19 treatment. Here, we assess the oral bioavailability of GS-441524 in beagle dogs and show that plasma concentrations [~]24-fold higher than the EC50 against SARS-CoV-2 are easily and safely sustained. These data support translation of GS-441524 as an oral agent for COVID-19.

category: pharmacology and toxicology


title: Nutritional Redundancy in Human Diet

abstract: Studying human dietary intake may help us identify effective measures to treat or prevent many chronic diseases whose natural histories are influenced by nutritional factors. Through examining four comprehensive cohorts with dietary intake data collected on different time scales, we find that the food intake profile varies tremendously across individuals and over time, while the nutritional intake profile is highly stable across individuals and over time. We refer to this phenomenon as  nutritional redundancy. We find that this phenomenon cannot be simply explained by the fact that different foods contain similar nutrients. Instead, it is largely due to more sophisticated features (e.g., the highly nested structure) of the food-nutrient network --- a bipartite graph that connects foods to their nutrient constituents. The food-nutrient network also enables us to quantify the level of nutritional redundancy for each diet record of any individual, i.e., the personal nutritional redundancy. Interestingly, this personal nutritional redundancy measure does not strongly correlate with any classical healthy diet scores, but its performance of predicting healthy aging in a cohort of older women is as strong as those healthy diet scores. The concept of nutritional redundancy developed here offers us a new perspective on studying human diet and presumably could be used for more phenotype predictions.

category: systems biology


title: Standing Variations Modeling Captures Inter-Individual Heterogeneity in a Deterministic Model of Prostate Cancer Response to Combination Therapy

abstract: Sipuleucel-T (Provenge) is the first live cell vaccine approved for advanced, hormonally refractive prostate cancer. However, survival benefit is modest and the optimal combination or schedule of sipuleucel-T with androgen depletion remains unknown. We employ a nonlinear dynamical systems approach to modeling the response of hormonally refractive prostate cancer to sipuleucel-T. Our mechanistic model incorporates the immune response to the cancer elicited by vaccination, and the effect of androgen depletion therapy. Because only a fraction of patients benefit from sipuleucel-T treatment, inter-individual heterogeneity is clearly crucial. Therefore, we introduce our novel approach, Standing Variations Modeling, which exploits inestimability of model parameters to capture heterogeneity in a deterministic model. We use data from mouse xenograft experiments to infer distributions on parameters critical to tumor growth and to the resultant immune response. Sampling model parameters from these distributions allows us to represent heterogeneity, both at the level of the tumor cells and the individual (mouse) being treated. Our model simulations explain the limited success of sipuleucel-T observed in practice, and predict an optimal combination regime that maximizes predicted efficacy. This approach will generalize to a range of emerging cancer immunotherapies.


title: Modeling ex vivo tumor-infiltrating lymphocyte expansion from established solid malignancies

abstract: Adoptive transfer of tumor-infiltrating lymphocytes (TIL) elicits the regression of metastatic malignancies, yet a low proportion of patients achieve complete durable responses. The high incidence of relapse in these patients highlights the need to better understand mechanisms of tumor escape from T cell control. While melanoma has provided the foundation for developing TIL therapy, much less is known about TIL efficacy and relapse in other malignancies. We sought to investigate TIL characteristics in mouse tumors which have not been studied in this setting. Here, we expanded murine TIL ex vivo in IL-2 from fragments of multiple tumor models, including oral cavity cancer models of varying immunogenicity. Additionally, TIL was expanded from pmel-1 mice bearing B16 melanoma, yielding an enriched population of tumor-infiltrating TCR transgenic T cells. Murine TILs are similar to human TIL in that they express high levels of inhibitory receptors (PD-1, Tim-3, etc.) and can be expanded ex vivo in IL-2 extensively. Of clinical relevance, we draw parallels between murine and patient TIL, evaluating relationships between PD-1, Lag-3, and Tim-3 on TILs from a cohort of oral cavity cancer patients. This platform can be used by labs even in the absence of clinical specimens or clean cell facilities and will be important to more broadly understand TIL phenotypes across many different malignancies.


title: CRISPR-Cas9 gene editing and rapid detection of gene-edited mutants using high-resolution melting in the apple scab fungus, Venturia inaequalis

abstract:
BackgroundScab, or black spot, caused by the filamentous fungal pathogen Venturia inaequalis, is the most economically important disease of apple (Malus x domestica) worldwide. To develop durable control strategies against this disease, a better understanding of the genetic mechanisms underlying the growth, reproduction, virulence and pathogenicity of V. inaequalis is required. A major bottleneck for the genetic characterization of V. inaequalis is the inability to easily delete or disrupt genes of interest using homologous recombination. Indeed, no gene deletions or disruptions in V. inaequalis have yet been published. Recently, CRISPR-Cas9 has emerged as an efficient tool for gene editing in filamentous fungi. With this in mind, we set out to establish CRISPR-Cas9 as a gene editing tool in V. inaequalis.

ResultsWe showed that CRISPR-Cas9 can be used for gene inactivation in the apple scab fungus. As a proof of concept, we targeted the melanin biosynthesis pathway gene trihydroxynaphthalene reductase (THN), which has previously been shown to result in a light-brown colony phenotype when transcriptionally silenced using RNA interference. Using one of two CRISPR-Cas9 single guide RNAs (sgRNAs) targeted to the THN gene, delivered by a single autonomously replicating Golden Gate-compatible plasmid, we were able to identify six of 36 stable transformants with a light-brown phenotype, indicating an ~16.7% gene inactivation efficiency. Notably, of these six THN mutants, five had an independent mutation. As part of our pipeline, we also report a high-resolution melting (HRM) curve protocol for the rapid detection of CRISPR-Cas9 gene-edited mutants of V. inaequalis. This protocol identified a single base pair deletion mutation in a sample containing only 5% mutant genomic DNA, indicating high sensitivity for mutant screening.

ConclusionsIn establishing CRISPR-Cas9 as a tool for gene editing in V. inaequalis, we have provided a strong starting point for studies aiming to decipher the function of genes associated with the growth, reproduction, virulence and pathogenicity of this fungus. The associated HRM curve protocol will enable CRISPR-Cas9 transformants to be screened for gene inactivation in a high-throughput and low-cost manner, which will be particularly powerful in cases where the CRISPR-Cas9-mediated gene inactivation efficiency is low.


title: Mathematical model reveals that heterogeneity in the number of ion transporters regulates the fraction of mouse sperm capacitation

abstract:
Capacitation is a complex maturation process mammalian sperm must undergo in the female genital tract to be able to fertilize an egg. This process involves, amongst others, physiological changes in flagellar beating pattern, membrane potential, intracellular ion concentrations and protein phosphorylation. Typically, in a capacitation medium, only a fraction of sperm achieve this state. The cause for this heterogeneous response is still not well understood and remains an open question. Here, one of our principal results is to develop a discrete regulatory network, with mostly deterministic dynamics in conjunction with some stochastic elements, for the main biochemical and biophysical processes involved in the early events of capacitation. The model criterion for capacitation requires the convergence of specific levels of a select set of nodes. Besides reproducing several experimental results and providing some insight on the network interrelations, the main contribution of the model is the suggestion that the degree of variability in the total amount and individual number of ion transporters among spermatozoa regulates the fraction of capacitated spermatozoa. This conclusion is consistent with recently reported experimental results. Based on this mathematical analysis, experimental clues are proposed for the control of capacitation levels. Furthermore, cooperative and interference traits that become apparent in the modelling among some components also call for future theoretical and experimental studies.

Author summaryFertilization is one of the fundamental processes for the preservation of life. In mammals, sperm undergo a complex process during their passage through the female tract known as capacitation, which enables them for fertilization. At the present time, it is accepted from experimental observation, though not understood, that only a fraction of the sperm is capacitated. In this work, by means of a network mathematical model for regulatory sperm intracellular signaling processes involved in mice capacitation, we find that the variability in the distribution of the number of ion transporters intervenes in the regulation of the capacitation fraction. Experimental verification of this suggestion could open a line of research geared to the regulation of the degree of heterogeneity in the number of ion transporters as a fertility control. The model also uncovers, through in silico overactivation and loss of function of network nodes, synergetic traits which again call for experimental verification.

category: developmental biology