title: Microbial Energy Metabolism Fuels a CSF2-dependent Intestinal Macrophage Niche within Tertiary Lymphoid Organs

abstract: Maintaining intestinal macrophage (MP) heterogeneity is critical to ensure tissue homeostasis and host defense. The gut microbiota and host factors are thought to synergistically shape colonic MP development, although there remains a fundamental gap in our understanding of the details of such collaboration. Here, we report tertiary lymphoid organs (TLOs), enriched in group 3 innate lymphoid cells (ILC3s), as a microbiota-operated intestinal niche for the development of monocyte-derived MPs. ILC3-derived colony stimulating factor 2 (CSF2) serves as a developmental and functional determinant for MPs and required microbe-derived extracellular adenosine 5-triphosphate (ATP) as a trigger. Microbial communities rich in extracellular ATP promoted MP turnover via ILC3 activity in an NLRP3-dependent fashion. Single cell RNA-sequencing of MPs revealed unique TLO-associated, CSF2-dependent MP populations critical for anti-microbial defense against enteric infection. Collectively, these findings describe a fundamental framework that constitutes an intestinal MP niche fueled by microbial energy metabolism.


title: Genomic diversity across Candida auris clinical isolates shapes rapid development of antifungal resistance in vitro and in vivo

abstract:
Antifungal drug resistance and tolerance poses a serious threat to global public health. In the human fungal pathogen, Candida auris, resistance to triazole, polyene, and echinocandin antifungals is rising, resulting in multidrug resistant isolates. Here, we use genome analysis and in vitro evolution of seventeen new clinical isolates of C. auris from clades I and IV to determine how quickly resistance mutations arise, the stability of resistance in the absence of drug, and the impact of genetic background on evolutionary trajectories. We evolved each isolate in the absence of drug as well as in low and high concentrations of fluconazole. In just three passages, we observed genomic and phenotypic changes including karyotype alterations, aneuploidy, acquisition of point mutations, and increases in MIC values within the populations. Fluconazole resistance was stable in the absence of drug, indicating little to no fitness cost associated with resistance. Importantly, two isolates substantially increased fluconazole resistance to [≥]256{micro}g/ml fluconazole. Multiple evolutionary pathways and mechanisms to increase fluconazole resistance occurred simultaneously within the same population. Strikingly, the sub-telomeric regions of C. auris were highly dynamic as deletion of multiple genes near the sub-telomeres occurred during the three passages in several populations. Finally, we discovered a mutator phenotype in a clinical isolate of C. auris. This isolate had elevated mutation rates compared to other isolates and acquired substantial resistance during evolution in vitro and in vivo supporting that the genetic background of clinical isolates can have a significant effect on evolutionary potential.

ImportanceDrug resistant Candida auris infections are recognized by the CDC as an urgent threat. Here, we obtained and characterized a set of clinical isolates of C. auris including multiple isolates from the same patient. To understand how drug resistance arises, we evolved these isolates and found that resistance to fluconazole, the most commonly prescribed antifungal, can occur rapidly and that there are multiple pathways to resistance. During our experiment, resistance was gained, but it was not lost, even in the absence of drug. We also found that some C. auris isolates have higher mutation rates than others and are primed to acquire antifungal resistance mutations. Furthermore, we found that multidrug resistance can evolve within a single patient. Overall, our results highlight the high stability and high rates of acquisition of antifungal resistance of C. auris that allow evolution of pan-resistant, transmissible isolates in the clinic.

category: microbiology


title: Genetic glyco-profiling and rewiring of insulated flagellin glycosylation pathways

abstract: Glycosylation of surface structures diversifies cells chemically and physically. Sialic acids commonly serve as glycosyl donors, particularly pseudaminic (Pse) or legionaminic acid (Leg) that prominently decorate eubacterial and archaeal surface layers or appendages. We investigated a new class of FlmG protein glycosyltransferases that modify flagellin, the structural subunit of the flagellar filament. Functional insulation of orthologous Pse and Leg biosynthesis pathways accounted for the flagellin glycosylation specificity and motility conferred by the cognate FlmG in the -proteobacteria Caulobacter crescentus and Brevundimonas subvibrioides, respectively. Exploiting these functions, we conducted genetic glyco-profiling to classify Pse or Leg biosynthesis pathways and we used heterologous reconstitution experiments to unearth a signature determinant of Leg biosynthesis in eubacteria and archaea. These findings and our chimeric FlmG analyses reveal two modular determinants that govern flagellin glycosyltransferase specificity: a glycosyltransferase domain that accepts either Leg or Pse and that uses specialized flagellin-binding domain to identify the substrate.


title: Distribution and genomic variation of thermophilic cyanobacteria in diverse microbial mats at the upper temperature limits of photosynthesis

abstract:
Thermophilic cyanobacteria have been extensively studied in Yellowstone National Park (YNP) hot springs, particularly from decades of work on the thick laminated mats of Octopus and Mushroom Springs. However, focused studies of cyanobacteria outside of these two hot springs have been lacking, especially regarding how physical and chemical parameters along with community morphology influence the genomic makeup of these organisms. Here, we used a metagenomic approach to examine cyanobacteria existing at the upper temperature limits of photosynthesis. We examined 15 alkaline hot spring samples across six geographic areas of YNP, all with varying physical and chemical parameters, and community morphology. We recovered 22 metagenome-assembled genomes (MAGs) belonging to thermophilic cyanobacteria, notably an uncultured Synechococcus-like taxon recovered from the upper temperature limit of photosynthesis, 73{degrees}C, in addition to thermophilic Gloeomargarita. Furthermore, we found that three distinct groups of Synechococcus-like MAGs recovered from different temperature ranges vary in their genomic makeup. MAGs from the uncultured very high temperature (up to 73{degrees}C) Synechococcus-like taxon lack key nitrogen metabolism genes and have genes implicated in cellular stress responses that diverge from other Synechococcus-like MAGs. Across all parameters measured, temperature was the primary determinant of taxonomic makeup of recovered cyanobacterial MAGs. However, Fe, community morphology, and biogeography played an additional role in the distribution and abundance of upper temperature limit-adapted Synechococcus-like MAGs.These findings expand our understanding of cyanobacterial diversity in YNP and provide a basis for interrogation of understudied thermophilic cyanobacteria.

ImportanceOxygenic photosynthesis arose early in microbial evolution - approx. 2.5-3.5 billion years ago - and entirely reshaped the biological makeup of Earth. However, despite the span of time in which photosynthesis has been refined, it is strictly limited to temperatures below 73{degrees}C, a barrier that many other biological processes have been able to overcome. Furthermore, photosynthesis at temperatures above 56{degrees}C is limited to circumneutral and alkaline pH. Hot springs in Yellowstone National Park (YNP), which have a large diversity in temperatures, pH and geochemistry provide a natural laboratory to study thermophilic microbial mats, and the cyanobacteria within. While cyanobacteria in YNP microbial mats have been studied for decades, a vast majority of work has focused on two springs within the same geyser basin, both containing similar community morphologies. Thus, the drivers of cyanobacterial adaptations to the upper limits of photosynthesis across a variety of environmental parameters have been understudied. Our findings provide new insights into the influence of these parameters on both taxonomic diversity and genomic content of cyanobacteria across a range of hot spring samples.


title: Infection dynamics of co-transmitted reproductive symbionts are mediated by sex, tissue, and development

abstract:
One of the most prevalent intracellular infections on earth is with Wolbachia: a bacterium in the Rickettsiales that infects a range of insects, crustaceans, chelicerates, and nematodes. Wolbachia is maternally transmitted to offspring and has profound effects on the reproduction and physiology of its hosts, which can result in reproductive isolation, altered vectorial capacity, mitochondrial sweeps, and even host speciation. Some populations stably harbor multiple Wolbachia strains, which can further contribute to reproductive isolation and altered host physiology. However, almost nothing is known about the requirements for multiple intracellular microbes to be stably maintained across generations while they likely compete for space and resources. Here we use a coinfection of two Wolbachia strains ("wHa" and "wNo") in Drosophila simulans to define the infection and transmission dynamics of an evolutionarily stable double infection. We find that a combination of sex, tissue, and host development contribute to the infection dynamics of the two microbes and that these infections exhibit a degree of niche partitioning across host tissues. wHa is present at a significantly higher titer than wNo in most tissues and developmental stages, but wNo is uniquely dominant in ovaries. Unexpectedly, the ratio of wHa to wNo in embryos does not reflect those observed in the ovaries, indicative of strain-specific transmission dynamics. Understanding how Wolbachia strains interact to establish and maintain stable infections has important implications for the development and effective implementation of Wolbachia-based vector biocontrol strategies, as well as more broadly defining how cooperation and conflict shape intracellular communities.

IMPORTANCEWolbachia are maternally transmitted intracellular bacteria that manipulate the reproduction and physiology of arthropods, resulting in drastic effects on the fitness, evolution, and even speciation of their hosts. Some hosts naturally harbor multiple strains of Wolbachia that are stably transmitted across generations, but almost nothing is known about the factors that limit or promote these co-infections which can have profound effects on the hosts biology and evolution, and are under consideration as an insect-management tool. Here we define the infection dynamics of a known stably transmitted double infection in Drosophila simulans with an eye towards understanding the patterns of infection that might facilitate compatibility between the two microbes. We find that a combination of sex, tissue, and development all contribute how the coinfection establishes.


title: Epigenetic modulators link mitochondrial redox homeostasis to cardiac function

abstract:
Excessive production of reactive oxygen species (ROS) is characteristic of numerous diseases, but most studies in this area have not considered the impact of endogenous antioxidative defenses. Here, utilizing multi-omics, we demonstrate that in cardiomyocytes mitochondrial isocitrate dehydrogenase (IDH2) constitutes a major antioxidant defense. In both male and female mice and humans the paradoxical reduction in expression of IDH2 associated with heart failure is compensated for by an increase in the enzymes activity. We describe extensive mutual regulation of the antioxidant activities of IDH2 and NRF2 by a network involving 2-oxoglutarate and L2-hydroxyglutarate and mediated in part through unconventional hydroxymethylation of cytosine residues present in introns. Conditional targeting of ROS in a murine model of heart failure improves cardiac function. Together, these insights may explain why previous attempts to treat heart failure with antioxidants have been unsuccessful and open new approaches to personalizing and, thereby, improving such treatment.

Graphical abstract

O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=200 SRC="FIGDIR/small/485908v1_ufig1.gif" ALT="Figure 1">
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C_LIO_LIAn antioxidative loop involving IDH2 coordinates other antioxidative defenses, such as NRF2.
C_LIO_LIThis loop produces epigenetic modifications that link oxidative stress to mitochondrial function.
C_LIO_LIThe conclusion that enhancing antioxidative capacity improves cardiac function only when the endogenous capacity is insufficient opens new approaches to individualized treatment of patients with heart failure.
C_LI

category: molecular biology


title: Korean endemic species no more: on the occurrence of Pelophylax chosenicus in China

abstract: Understanding species distribution is the first requirement to work on their behaviour, conservation, and phylogeography. Over the last decades, the number of species described on the Korean peninsula has significantly increased, but areas around the boundaries of the Korean peninsula still have to be surveyed for the presence of these species, especially where the environment is similar and connected. Here, we conducted surveys in the continuous landscapes of the Republic of Korea, the Democratic Peoples Republic of Korea and the Peoples Republic of China to determine the range of the Gold-spotted pond frog, Pelophylax chosenicus. The surveys were conducted between 2016 and 2020 through visual and call encounters. We also used molecular tools to confirm the species identity of the northernmost population, in China, sequencing the mitochondrial DNA 16S gene fragment. We then determined the importance of landscape types for the species, and especially rice paddies, and used landscape models to define suitable habitats across the region. We found the species to be widespread in low elevation wetlands along the coast of the Yellow Sea, with two isolated populations on the south-east of the Korean Peninsula, and the northernmost population in the vicinity of Dandong in PR China. As this species is listed as threatened in the Republic of Korea, knowing its exact distribution will be important for conservation practices, and this first record for PR China provides a baseline for further surveys.

category: ecology


title: Polytypy and systematics: diversification of Papilio swallowtail butterflies in the biogeographically complex Indo-Australian Region

abstract: A long-standing problem in evolutionary biology and systematics is defining patterns of diversification and speciation, which is compounded by allopatric distributions of polytypic taxa in biogeographically fragmented landscapes. In this paper we revisit this enduring systematic challenge using Mormon swallowtail butterflies (Papilio subgenus Menelaides)--an evolutionary and genetic model system. Menelaides is speciose and intensively sampled, with nearly 260 years of systematic study complicated by polytypy resulting from discontinuous morphological variation. This variation is structured by the mainland-island matrix of the geologically complex Indo-Australian Region, where drawing species boundaries has been difficult. We sampled variation across the biogeographic range of Menelaides, covering 97% of currently recognized species and nearly half of all subspecies. We generated a well-supported mito-nuclear phylogeny, on which we delineated species based on two species delimitation methods (GMYC and mPTP) and strongly supported reciprocal monophyly. These analyses showed that the true species diversity in this group may be up to 25% greater than traditional taxonomy suggests, and prompts extensive taxonomic restructuring. Biogeographic analyses showed that Menelaides have diversified largely in allopatry in Indo-Australian subregions by repeated dispersals across key biogeographic barriers. These results provide critical insights into the diversification process in this morphologically diverse and taxonomically complicated model group. These results will also be informative in future studies on systematics, biogeography, speciation and morphological diversification in the Indo-Australian Region--arguably the most complex geological land/seascape in the world.

category: evolutionary biology


title: Spatiotemporal variations in retrovirus-host interactions among Darwin's finches

abstract: Endogenous retroviruses (ERVs) are inherited remnants of past retroviruses that colonized host germline over millions of years, providing a sampling of retroviral diversity across time. The utility of ERVs as markers of host genome evolution is augmented by their ability to reveal historic retrovirus-host interactions, given that ERVs are examples of active retroviruses at the time of germline invasion. Here, we utilize the strength of Darwins finches, a system synonymous with evolutionary studies, for investigating ERV history, revealing recent retrovirus-host interactions in natural populations. By mapping ERV variation across all species of Darwins finches and comparing with outgroup species, we highlight geographical and historical patterns of retrovirus-host occurrence, utilizing the system as a natural model for evaluating the extent and timing of retroviral activity in hosts undergoing adaptive radiation and colonization of new environments. We found shared ERVs among all samples indicating retrovirus-host associations pre-dating host speciation, as well as considerable ERV variation across populations of the entire Darwins finches radiation. Unexpected ERV variation in finch species on different islands suggest historical changes in gene flow and selection. Non-random distribution of ERVs along and between chromosomes, as well as across finch species, suggests association between ERV accumulation and the rapid speciation of Darwins finches.


title: Intestinal epithelial Axin1 deficiency protects against colitis via altered gut microbiota

abstract:
Background and AimsIntestinal homeostasis is maintained by specialized host cells and the gut microbiota. Wnt/{beta}-catenin signaling is essential for gastrointestinal development and homeostasis, and its dysregulation has been implicated in inflammation and colorectal cancer. Axin1 negatively regulates activated Wnt/{beta}-catenin signaling, but little is known regarding its role in regulating host-microbial interactions in health and disease. Here, we aim to demonstrate that intestinal Axin1 determines gut homeostasis and host response to inflammation.

MethodsThe expression of Axin1 was analyzed in human IBD datasets. To explore the effects and mechanism of intestinal Axin1 in regulating intestinal homeostasis and colitis, we generated mouse models with Axin1 conditional knockout in intestinal epithelial (Axin1{Delta}IEC) and Paneth cells (Axin1{Delta}PC) to compare with control (Axin1LoxP) mice.

ResultsWe found increased Axin1 expression in the colonic epithelium of human IBD. Axin1{Delta}IEC mice exhibited altered goblet cell spatial distribution, Paneth cell morphology, reduced lysozyme expression, and enriched Akkermansia muciniphila. Absence of intestinal epithelial and Paneth cell Axin1 decreased susceptibility to DSS-induced colitis in vivo. Axin1{Delta}IEC and Axin1{Delta}PC became more susceptible to DSS-colitis after cohousing with control mice, suggesting the non-colitogenic effect is driven by the gut microbiota.

ConclusionWe found loss of intestinal Axin1 protects against colitis, which is likely driven through Paneth cell Axin1 and the microbiota. Our study demonstrates a novel role of Axin1 in mediating intestinal homeostasis and the microbiota. Further mechanistic studies using specific Axin1 mutations elucidating how Axin1 modulates microbiome and host inflammatory response, will provide new therapeutic strategies for human IBD.

What you Need to Know1. Background and ContextWnt/beta-catenin is a fundamental molecular pathway that affects intestinal proliferation and differentiation. Axin1 negatively regulates activated Wnt/{beta}-catenin signaling, but little is known regarding its role in the microbiome. Dysfunction of Wnt/beta-catenin was reported in human inflammatory bowel disease (IBD) and Axin1 serum level was elevated in patients with UC.

2. New FindingsWe found increased Axin1 expression at both the mRNA and protein level in human IBD. Specifically, we identified increased Axin1 expression positive correlated with pro-inflammatory cytokines IL-6 and TNF- in CD. Our study, for the first time, identifies links between the gut microbiota and intestinal Axin1 in intestinal inflammation through utilization of innovative deletion mouse models in intestinal epithelium and Paneth cells. Loss of intestinal Axin1 plays a novel role in intestinal inflammation by altering the Paneth cells and microbiome (e.g., enriched Akkermansia mucinlphila). Our study has provided insights into the molecular mechanism that might contribute to IBD, especially the novel role of Paneth cell Axin1 in colitis.

3. LimitationsThere are no human or mice studies assessing the role of intestinal epithelial and Paneth cell Axin1 in inflammation and the microbiome.

4. ImpactFurther explorations of the gut microbiota and Axin1 interaction as we report will provide novel mechanistic strategies for therapeutic approaches for human IBD by targeting intestinal Axin1 and Axin1-associated microbiome.

category: pathology


title: Cyb5r3-based mechanism and reversal of secondary failure to sulfonylurea

abstract: Sulfonylureas (SU) are effective and affordable anti-diabetic drugs. But chronic use leads to secondary failure, limiting their utilization. The mechanism of secondary failure is unknown. Here we identify Cyb5r3 downregulation as a mechanism of SU failure and successfully reverse it. Chronic exposure to SU impairs Cyb5r3 levels and reduces islet glucose utilization with a metabolomics signature characterized by low acetyl-CoA and amino acid levels. Cyb5r3 engages in a glucose-dependent interaction that stabilizes glucokinase (Gck) to maintain glucose utilization. Accordingly, activating Gck mutations in patients with hyperinsulinemia reduce Cyb5r3 binding, whereas inactivating MODY mutations increase it, providing evidence for a role of Cyb5r3 in determining flux through Gck. The Cyb5r3 activator tetrahydroindenoindole (THII) rescues secondary failure to SU in an animal model of chronic SU treatment and restores insulin secretion from ex vivo islets. We conclude that Cyb5r3 loss-of-function is a key factor in the secondary failure to SU and a potential target for its prevention, which may lead to a rehabilitation of SU use in diabetes.

category: physiology


title: Thiazides induce glucose intolerance through inhibition of mitochondrial carbonic anhydrase 5b in β-cells

abstract:
Thiazides are associated with glucose intolerance and new onset diabetes mellitus, but the molecular mechanisms remain elusive. The aim of this study was to decipher the molecular basis of thiazide-induced glucose intolerance. In mice, hydrochlorothiazide induced a pathological glucose tolerance, characterized by reduced first phase insulin secretion but normal insulin sensitivity. In vitro, thiazides inhibited glucose-and sulfonylurea-stimulated insulin secretion in islets and the murine {beta}-cell line Min6 at pharmacologically relevant concentrations. Inhibition of insulin secretion by thiazides was CO2/HCO3--dependent, not additive to unselective carbonic anhydrase (CA) inhibition with acetazolamide and independent of extracellular potassium. In contrast, insulin secretion was unaltered in islets of mice lacking the known molecular thiazide targets NCC (SLC12A3) or NDCBE (SLC4A8). CA expression profiling with subsequent knock-down of individual CA isoforms suggested mitochondrial CA5b as molecular target. In support of these findings, thiazides significantly attenuated Krebs cycle anaplerosis through reduction of mitochondrial oxalacetate synthesis. CA5b KO mice were resistant to thiazide-induced glucose intolerance, and insulin secretion of islets isolated from CA5b KO mice was unaffected by thiazides.

In summary, our study reveals attenuated insulin secretion due to inhibition of the mitochondrial CA5b isoform in {beta}-cells as molecular mechanism of thiazide-induced glucose intolerance.


title: Unleashing the Potential of Noncanonical Amino Acid Biosynthesis for Creation of Cells with Site-Specific Tyrosine Sulfation

abstract:
Incorporation of noncanonical amino acids (ncAAs) into proteins holds great promise for modulating the structure and function of those proteins and for influencing evolutionary dynamics in organisms. Despite significant progress in improving the efficiency of translational machinery needed for incorporating ncAAs, exogenous feeding of high concentrations of chemically-synthesized ncAAs, especially in the case of polar ncAAs, is required to ensure adequate intracellular ncAA levels. Here, we report the creation of autonomous cells, both prokaryotic and eukaryotic, with the ability to biosynthesize and genetically encode sulfotyrosine (sTyr), an important protein post-translational modification with low membrane permeability. We discovered the first enzyme catalyzing tyrosine sulfation, sulfotransferase 1C1 from Nipponia nippon (NnSULT1C1), using a sequence similarity network (SSN). The unique specificity of NnSULT1C1 for tyrosine has been systematically explored using both bioinformatics and computational methods. This NnSULT1C1 was introduced into both bacterial and mammalian cells so as to yield organisms capable of biosynthesizing high levels of intracellular sTyr. These engineered cells produced site-specifically sulfated proteins at a higher yield than cells fed exogenously even with the highest level of sTyr reported in literature. We have used these autonomous cells to prepare highly potent thrombin inhibitors with site-specific sulfation. By enhancing ncAA incorporation efficiency, this added ability of cells to biosynthesize ncAAs and genetically incorporate them into proteins greatly extends the utility of genetic code expansion methods.

TOC

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category: synthetic biology


title: In silico Investigation of Molecular Networks Linking Gastrointestinal Diseases, Malnutrition, and Sarcopenia

abstract: Malnutrition is a common primary or secondary complication in gastrointestinal diseases. The patients nutritional status also influences muscle mass and function, which can be impaired up to the degree of sarcopenia. The molecular interactions in diseases leading to sarcopenia are complex and multifaceted, affecting muscle physiology, the intestine (nutrition), and the liver at different levels. Although extensive knowledge of individual molecular factors is available, their regulatory interplay is not yet fully understood. A comprehensive overall picture of pathological mechanisms and resulting phenotypes is lacking. In silico approaches that convert existing knowledge into computationally readable formats can help to unravel such complex systems. We compiled available experimental evidence for molecular interactions involved in the development of sarcopenia into a knowledge base, referred to as the Sarcopenia Map. By including specific diseases, namely liver cirrhosis, and intestinal dysfunction, and considering their effects on nutritional status and blood secretome, we investigated their contribution to the development of sarcopenia. The Sarcopenia Map is publicly available as an open-source, interactive online resource, providing tools that allow users to explore the information on the map and perform in silico perturbation experiments.

category: systems biology


title: SCAMPR: Single-Cell Automated Multiplex Pipeline for RNA Quantification and Spatial Mapping

abstract:
Spatial gene expression, achieved classically through in situ hybridization, is a fundamental tool for topographic phenotyping of cell types in the nervous system. Newly developed techniques allow for the visualization of multiple mRNAs at single-cell resolution, greatly expanding the ability to link gene expression to tissue topography. Yet, methods for efficient and accurate quantification and analysis of high dimensional in situ hybridization are limited. To this end, the Single-Cell Automated Multiplex Pipeline for RNA (SCAMPR) was developed, facilitating rapid and accurate segmentation of neuronal cell bodies using a dual immunohistochemistry-RNAscope protocol and quantification of low and high abundance mRNA signals using open-source image processing and automated segmentation tools. Proof of principle using SCAMPR focused on spatial mapping of gene expression by peripheral (vagal nodose) and central (visual cortex) neurons. The analytical effectiveness of SCAMPR is demonstrated by identifying the impact of early life stress on differential gene expression by vagal neuron subtypes.

MotivationQuantitative analysis of spatial mRNA expression in neurons can lack accuracy and be both computationally and time intensive. Existing methods that rely on nuclear labeling (DAPI) to distinguish adjoining cells lack the precision to detect mRNA expression in the cytoplasm. In addition, quantification methods that rely on puncta counts can generate large, variable datasets that potentially undercount highly expressed mRNAs. To overcome these methodological barriers, we developed the SCAMPR pipeline that allows for fast, accurate segmentation of neuronal cell body boundaries, topographic gene expression mapping, and high dimensional quantification and analysis of mRNA expression in tissue sections.

category: neuroscience


title: Fast-synaptic axo-axonal transmission from striatal cholinergic interneurons onto dopamine fibers.

abstract: Transmission from striatal cholinergic interneurons (CINs) controls dopamine release through nicotinic acetylcholine receptors (nAChRs) on dopaminergic axons. Anatomical studies suggest that cholinergic terminals signal predominantly through non-synaptic volume transmission. However, the influence of cholinergic transmission on electrical signaling in axons remains unclear. We examined axo-axonal transmission from CINs onto dopaminergic axons using perforated-patch recordings which revealed rapid spontaneous EPSPs with properties characteristic of fast synapses. Pharmacology showed that axonal EPSPs (axEPSPs) were mediated primarily by high-affinity 6-containing receptors. Remarkably, axEPSPs triggered spontaneous action potentials locally in dopaminergic axons, suggesting these axons perform integration to convert synaptic input into spiking, a function associated with somatodendritic compartments. We investigated cross-species validity of cholinergic axo-axonal transmission by recording dopaminergic axons in macaque putamen and found similar axEPSPs. Thus, we reveal that fast synaptic-like neurotransmission underlies cholinergic signaling onto dopaminergic axons, providing insight into how nicotinic receptors shape electrical signaling directly in axon terminals.


title: Nr2f1-dependent shaping of mitochondrial architecture in newborn mouse hippocampal neurons unravels novel insights into the neurodevelopmental disorder BBSOAS

abstract: The nuclear receptor Nr2f1 plays a key role during cortical development by acting as a strong transcriptional regulator in embryonic and postnatal neural cells. In humans, its mutations cause the Bosch-Boonstra-Schaaf optic atrophy-intellectual syndrome (BBSOAS), a rare neurodevelopmental disorder characterized by multiple clinical features including optic nerve atrophy, intellectual disability, and autistic traits. In this study by genome-wide and in silico analyses we found that key factors for mitochondrial function and dynamics represent potential genomic targets under direct Nr2f1 transcriptional control in neurons. Although mitochondrial dysfunction is increasingly implicated in neurodevelopmental disorders, whether and how Nr2f1 can regulate mitochondria function in neural cells is still completely unknown. To address these questions, we have combined mouse genetics, neuroanatomical and imaging approaches. To genetically manipulate Nr2f1 function in newborn neurons we focused on the adult mouse hippocampal dentate gyrus, a site of persistent neurogenesis where Nr2f1 is highly expressed. We showed that conditional Nr2f1 loss-of-function within the hippocampal neurogenic niche lead to a reduced mitochondrial mass associated to mitochondrial fragmentation in newborn neurons, likely reflecting alteration in mitochondrial dynamics. Accordingly, we showed that the fusion factor Mfn2, which is as a putative Nr2f1 target, is downregulated following both Nr2f1 adult depletion and embryonic haploinsufficiency in mice. Overall, our study provides the first evidence of a crucial role of Nr2f1 in shaping mitochondria in neurons and opens a promising avenue for the identification of new mechanisms implicated in BBSOAS pathogenesis involving mitochondrial dysfunction.


title: Nr2f1 controls mitochondrial dynamics in mouse adult-born hippocampal neurons


title: mTORC2 coordinates the leading and trailing edge cytoskeletal programs during neutrophil migration

abstract: By acting both upstream and downstream of biochemical organizers of the cytoskeleton, physical forces function as central integrators of cell shape and movement. Here we use a combination of genetic, pharmacological, and optogenetic perturbations to probe the role of the conserved mechanoresponsive mTORC2 program in neutrophil polarity and motility. We find that the tension-based inhibition of leading edge signals (Rac, F-actin) that underlies protrusion competition is gated by the kinase-independent role of the complex, whereas the mTORC2 kinase arm is essential for regulation of Rho activity and Myosin II-based contraction at the trailing edge. Cells required mTORC2 for spatial and temporal coordination between the front and back polarity programs and persistent migration under confinement. mTORC2 is in a mechanosensory cascade, but membrane stretch did not suffice to stimulate mTORC2 unless the co-input PIP3 was also present. Our work suggests that different signalling arms of mTORC2 regulate spatially and molecularly divergent cytoskeletal programs allowing efficient coordination of neutrophil shape and movement.

category: cell biology


title: Degeneracy in the neurological model of auditory speech repetition

abstract: In the neurological model of language, repeating heard speech involves four left hemisphere regions: primary auditory cortex for processing sounds; Wernickes area for processing auditory images of speech; Brocas area for processing motor images of speech; and primary motor cortex for overt speech articulation. Previous functional-MRI (fMRI) studies confirm that auditory repetition activates these regions. Here, we used dynamic causal modelling (DCM) to test how the four regions interact with each other during single word and pseudoword auditory repetition. Contrary to expectation, we found that, for both word and pseudoword repetition, the effective connectivity between Wernickes and Brocas areas was predominantly bidirectional and inhibitory; activity in the motor cortex could be driven by either Wernickes area or Brocas area; and the latter effect varied both within and between individuals. Such variability speaks to degenerate functional architectures that support auditory repetition and may explain resilience to functional loss after brain damage.


title: Inter-brain coupling reflects disciplinary differences in real-world classroom learning

abstract: Classroom learning is one of the most critical scenarios in educational practices worldwide. Classroom learning was always organized into different disciplines (such as Math or language courses) in educational practice. However, we know little about how disciplinary differences are manifested in students neural dynamics supporting successful classroom learning. In order to address this question, we investigated disciplinary differences during the successful classroom learning from the framework of hard and soft disciplines by recording thirty-four high-school students EEGs simultaneously during their regular Chinese (a soft discipline) and Math (a hard discipline) courses across a whole semester. Ones theta-band inter-brain coupling to all other classmates during both the Chinese and Math courses was found to be positively correlated with their learning outcomes in Math. In contrast, ones alpha-band inter-brain coupling to their excellent peers was positively correlated with their learning outcomes in Chinese. These results demonstrated the value of inter-brain coupling as neural correlates of successful classroom learning and highlighted excellent peers as a candidate to represent successful learning for soft disciplines. Our findings provide real-world neuroscience evidence for understanding disciplinary differences and suggest possibly different neurocognitive mechanisms to support successful learning in different disciplines.


title: Indirect decoding of behavior from brain signals via reconstruction of experimental conditions

abstract: The data acquired during a functional magnetic resonance imaging (fMRI) experiment usually comprise experimental conditions, brain signals and behavioral responses. This reflects the underlying causal flow where the experimental conditions evoke brain responses that in turn result in behavior. In multivariate analyses, a common approach is to focus on the second step of that chain and to decode behavioral responses from brain signals. However, a different approach would be to first reconstruct the experimental conditions from brain signals and in turn use these to predict behavior. While this indirect approach would go against the causal chain of events, it might work better under certain circumstances, especially when the experimental conditions evoke much stronger measurable brain signals than the overt motor behavior. Here we tested this question directly by assessing the various mappings between conditions, brain signals and behavior in an open dataset. We found that the path of first decoding experimental conditions works surprisingly well, even though in our example data set, it is still outperformed by directly decoding the behavior from brain responses.


title: Gata2-regulated Gfi1b expression controls endothelial programming during endothelial-to-hematopoietic transition

abstract:
The first hematopoietic stem cells (HSCs) are formed through endothelial-to-hematopoietic transition (EHT) events during embryonic development. The transcription factor GATA2 is a crucial regulator of EHT and HSC function throughout life. Because GATA2 haploinsufficiency patients have inborn mutations, prenatal defects are likely to have an influence on disease development. In mice, Gata2 haploinsufficiency (Gata2+/-) reduces the number and the functionality of embryonic hematopoietic stem and progenitor cells (HSPCs) generated through EHT. However, the embryonic HSPC pool is heterogeneous and the mechanisms underlying this defect in Gata2+/- embryos are unclear. Here, we investigated whether Gata2 haploinsufficiency selectively affects a cellular subset undergoing EHT. We show that Gata2+/- HSPCs initiate but cannot fully activate hematopoietic programming during EHT. In addition, due to reduced activity of the endothelial repressor Gfi1b, Gata2+/- HSPCs cannot repress the endothelial identity to complete maturation. Finally, we show that hematopoietic-specific induction of gfi1b can restore HSC production in gata2b-null (gata2b-/-) zebrafish embryos. This study illustrates pivotal roles of Gata2 on the regulation of transcriptional network governing HSPC identity throughout EHT.

HighlightsO_LIMaturation of embryonic Gata2+/- HSPCs is disturbed due to aberrant endothelial gene expression and incomplete activation of hematopoietic transcriptional programming.
C_LIO_LIGata2 activates Gfi1b to repress endothelial identity of embryonic HSPCs during maturation.
C_LIO_LIHematopoietic-specific induction of gfi1b restores the number of embryonic HSCs in gata2b-/- zebrafish.
C_LI

category: developmental biology


title: Modeling phytoplankton-zooplankton interactions: opportunities for species richness and challenges for modern coexistence theory

abstract: Many potential mechanisms can sustain biodiversity, but we know little about their relative importance. To compare multiple mechanisms, we modeled a two-trophic planktonic food-web based on mechanistic species interactions and empirically measured species traits. We simulated thousands of communities under realistic and altered trait distributions to assess the relative importance of three potential drivers of species richness: resource competition, predator-prey interactions, and trait trade-offs. Next, we computed niche and fitness differences of competing zooplankton to obtain a deeper understanding of how these mechanisms limit species richness. We found that predator-prey interactions were the most important driver of species richness and that fitness differences were a better predictor of species richness than niche differences. However, for many communities we could not apply modern coexistence theory to compute niche and fitness differences due to complications arising from trophic interactions. We therefore need to expand modern coexistence theory to investigate multi-trophic communities.


title: Environmental response in gene expression and DNA methylation reveals factors influencing the adaptive potential of Arabidopsis lyrata

abstract: Understanding what factors influence plastic and genetic variation is valuable for predicting how organisms respond to changes in the selective environment. Here, using gene expression and DNA methylation as molecular phenotypes, we study environment-induced variation among Arabidopsis lyrata plants grown at lowland and alpine field sites. Our results show that gene expression is highly plastic, as many more genes are differentially expressed between the field sites than between populations. The environmentally responsive genes evolve under strong selective constraint - the strength of purifying selection on the coding sequence is high, while the rate of adaptive evolution is low. We find, however, that positive selection on cis-regulatory variants has likely contributed to genetically variable environment-responses, but such variants segregate only between distantly related populations. In contrast to gene expression, DNA methylation at genic regions is largely insensitive to the environment, and plastic methylation changes are not associated with differential gene expression. Besides genes, we detect environmental effects at transposable elements (TEs): TEs at the high-altitude field site have higher expression and methylation levels, suggestive of a broad-scale TE activation. Compared to the lowland population, plants native to the alpine environment harbor and excess of recent TE insertions, and we observe that specific TE families are enriched within environmentally responsive genes. In sum, although our results suggest that strong evolutionary constraint at environmentally responsive genes may limit the species adaptive potential, plastic responses at TEs could rapidly create novel heritable variation that is primed towards environmental adaptation.


title: The maintenance of self-incompatibility: overdominance of inbreeding depression and S-linked genetic load

abstract:
Inbreeding depression plays a fundamental role in evolution. To help detect and characterize viability loci that underlie inbreeding depression, plants from self-incompatible populations of Leavenworthia alabamica were force self-pollinated to produce families of progeny that were genotyped at hundreds of single nucleotide polymorphism (SNP) loci.

We used Bayesian analysis of segregation data for each SNP to explore support for different dominance and selection coefficients at linked viability loci.

There was strong support for overdominance (or pseudo-overdominance) at many viability loci. There was some support for recessivity and underdominance. One recessive viability locus mapped to the genomic region of the novel self-incompatibility locus in Leavenworthia alabamica.

The results are consistent with earlier findings showing that inbreeding depression is recalcitrant to purging in Leavenworthia alabamica. The results also help account for the maintenance of self-incompatibility in this species and are consistent with expectations from evolutionary genetic theory that recessive, deleterious alleles linked to loci under balancing selection are sheltered from selection.


title: Study of aging using circuit tests and parametric analysis

abstract:
Mouse development and aging were investigated in 11 tests. The test data were parametrically analysed, normalized, and integrated using principal component analysis (PCA). Changes in the indexing items displayed two trends: power and accuracy in the trials. Power decreased monotonically with age, whereas accuracy peaked at 6 months. Features, such as fear or anxiety, could not have been measured. The noise level and sensitivity of each test were determined. In an open field test that involved a video recording of movement, the noise level was the lowest and the sensitivity was high. This test contributed the most to the PCA axes, and reproduced the two main directions.

Significance StatementThe mathematical aspect may have been neglected in behavioural tests. Here, the circuit test data were objectively analysed by focusing on the data distributions during the aging of mice. The test results revealed two clear directions for the data: power, which was gradually reduced, and accuracy, which peaked at sixth months old. Compared with the open field test evaluated using video recordings, the other tests had higher noise levels and weaker signals. Other tests can be improved by using movie recordings and identifying appropriate items.

category: animal behavior and cognition


title: Designing Cost-Effective, Open-Source, Multi-Head Bioprinters via Conversion of Hobby-Grade 3D Printers

abstract: Over the past decade, additive manufacturing has resulted in significant advances towards fabricating anatomic-size, patient-specific scaffolds for tissue models and regenerative medicine. This can be attributed to the development of advanced bioinks capable of precise deposition of cells and biomaterials. The combination of additive manufacturing with advanced bioinks is enabling researchers to fabricate intricate tissue scaffolds that recreate the complex spatial distributions of cells and bioactive cues found in the human body. However, the expansion of this promising technique has been hampered by the high cost of commercially available bioprinters and proprietary software. In contrast, conventional 3D printing has become increasingly popular with home hobbyists and caused an explosion of both low-cost thermoplastic 3D printers and open source software to control the printer. In this work, we bring these benefits into the field of bioprinting by converting widely available and cost-effective 3D printers into fully functional, open source, and customizable multi-head bioprinters. We demonstrate the practicality of this approach by designing bioprinters customized with multiple extruders, automatic bed leveling, and temperature controls for approximately $400. These bioprinters were then used for in vitro and ex vivo bioprinting to demonstrate their utility for tissue engineering.

category: bioengineering


title: Extended longevity geometrically-inverted proximal tubule organoids for protein uptake studies

abstract: While some in vitro platforms have been adapted to study proteinuric kidney disease, organoids have been challenging to study the disease. This is because apical access is historically difficult, and this is the surface on which megalin (LRP2), an endocytic receptor responsible for tubular reabsorption of filtered plasma proteins, resides. Based on our previous geometrically-inverted organoids, this study established high-throughput basal-in and apical-out proximal tubule organoids to study proteinuric kidney disease in a more physiologically consistent manner. Organoids successfully formed around a minimal Matrigel scaffold, and were maintained in culture for 90+ days, the longest reported hanging drop culture to date. The proximal tubule organoids exhibited good polarization, showed upregulation of maturity markers, such as aquaporin-1 and megalin, and experienced less epithelial-to-mesenchymal (EMT) transition compared to 2D cells. To assess protein uptake, fluorescent albumin was placed in the surrounding media, facing the apical surface, and organoids demonstrated functional protein uptake even at 90 days. To mimic proteinuric conditions, organoids were exposed to human serum albumin and released kidney injury molecule-1 (KIM-1), a common biomarker for kidney injury, in both dose- and time-dependent manners. While this study focuses on applications for modeling proteinuric kidney disease conditions, these organoids are envisioned to have broad utility where apical proximal tubule cell access is required.


title: Gfastats: conversion, evaluation and manipulation of genome sequences using assembly graphs

abstract:
MotivationWith the current pace at which reference genomes are being produced, the availability of tools that can reliably and efficiently generate genome assembly summary statistics has become critical. Additionally, with the emergence of new algorithms and data types, tools that can improve the quality of existing assemblies through automated and manual curation are required.

ResultsWe sought to address both these needs by developing gfastats, as part of the Vertebrate Genomes Project (VGP) effort to generate high-quality reference genomes at scale. Gfastats is a standalone tool to compute assembly summary statistics and manipulate assembly sequences in fasta, fastq, or gfa [.gz] format. Gfastats stores assembly sequences internally in a gfa-like format. This feature allows gfastats to seamlessly convert fast* to and from gfa [.gz] files. Gfastats can also build an assembly graph that can in turn be used to manipulate the underlying sequences following instructions provided by the user, while simultaneously generating key metrics for the new sequences.

Availability and implementationGfastats is implemented in C++. Precompiled releases (Linux, MacOS, Windows) and commented source code for gfastats are available under MIT license at https://github.com/vgl-hub/gfastats. Examples of how to run gfastats are provided in the Github. Gfastats is also available in Bioconda, in Galaxy (https://assembly.usegalaxy.eu) and as a MultiQC module [1] (https://github.com/ewels/MultiQC). An automated test workflow is available to ensure consistency of software updates.

Supplementary informationSupplementary data are available at Bioinformatics online.

category: bioinformatics


title: bpRNA-align: Improved RNA Secondary Structure Global Alignment for Comparing and Clustering RNA Structures

abstract: Ribonucleic acid (RNA) is a polymeric molecule that is fundamental to biological processes, with structure being more highly conserved than primary sequence and often key to its function. Advances in RNA structure characterization have resulted in an increase in the number of accurate secondary structures. The task of uncovering common RNA structural motifs with a collective function through structural comparison remains challenging and could be used to discover new RNA families. In this work, we present a novel secondary structure alignment and clustering method, bpRNA-align. bpRNA-align is a customized global structural alignment method, utilizing an inverted (gap extend costs more than gap open) context-specific affine gap penalty and a structural, feature-specific substitution matrix to provide similarity scores. We evaluate our similarity scores in comparison to other methods, using affinity propagation clustering, applied to a benchmarking data set of known structure types.


title: CellSium - Versatile Cell Simulator for Microcolony Ground Truth Generation

abstract:
SummaryTo address the need for ground truth data for deep learning based segmentation algorithms in microfluidic live-cell imaging, we present CellSium, a cell simulator primarily aimed at synthesizing realistic image sequences of bacterial microcolonies suitable for training neural networks.

Availability and ImplementationCellSium is free and open source software under the BSD license, implemented in Python, available at https://github.com/modsim/cellsium (DOI: 10.5281/zenodo.6193033), along with documentation, usage examples and Docker images.

Contactk.noeh@fz-juelich.de


title: diemr: Genome polarisation for detecting barriers to geneflow in R

abstract:
MotivationWe extend classic allele counting measures of geneflow to the case of genomescale data. Using a deterministic data labelling, small numbers of diagnostic markers can be replaced by large numbers of markers, each with a diagnostic index. Individuals hybrid indices can then be calculated genome wide conditioned on marker diagnosticity; within diploid, haplodiploid and/or haploid genome compartments; or indeed over any subset of markers, allowing standard cline width/barrier strength comparisons.

ResultsDiagnostic index expectation maximisation (diem) estimates bistate marker labelling polarities with respect to the sides of a barrier, also returning polarity support and a likelihoodbased diagnostic index. Polarised markers allow detection of geneflow at the scale of whole genomes.

Availability and implementationThe diem method is implemented in Mathematica and R. The Mathematica code is available at github through https://github.com/StuartJEBaird, and the R package diemr is available at CRAN through https://CRAN.R-project.org/package=diemr


title: StrainXpress: strain aware metagenome assembly from short reads

abstract: Next-generation sequencing based metagenomics has enabled to identify microorganisms in characteristic habitats without the need for lengthy cultivation. Importantly, clinically relevant phenomena such as resistance to medication, virulence or interactions with the environment can vary already within species. Therefore, a major current challenge is to reconstruct individual genomes from the sequencing reads at the level of strains, and not just the level of species. However, strains of one species can differ only by minor amounts of variants, which makes it difficult to distinguish them. Despite considerable recent progress, related approaches have remained fragmentary so far. Here, we present StrainXpress, as a comprehensive solution to the problem of strain aware metagenome assembly from next-generation sequencing reads. In experiments, StrainXpress reconstructs strain-specific genomes from metagenomes that involve up to more than 1000 strains, and proves to successfully deal with poorly covered strains. The amount of reconstructed strain-specific sequence exceeds that of the current state-of-the-art approaches by on average 26.75% across all data sets (first quartile: 18.51%, median: 26.60%, third quartile: 35.05%).


title: L-GIREMI uncovers RNA editing sites in long-read RNA-seq

abstract: Using third-generation sequencers, long-read RNA-seq is increasingly applied in transcriptomic studies given its major advantage in characterizing full-length transcripts. A number of methods have been developed to analyze this new type of data for transcript isoforms and their abundance. Another application, which is significantly under-explored, is to identify and analyze single nucleotide variants (SNVs) in the RNA. Identification of SNVs, such as genetic mutations or RNA editing sites, is fundamental to many biomedical questions. In long-read RNA-seq, SNV analysis presents significant challenges, due to the well-known relatively high error rates of the third-generation sequencers. Here, we present the first study to detect and analyze RNA editing sites in long-read RNA-seq. Our new method, L-GIREMI, effectively handles sequencing errors and biases in the reads, and uses a model-based approach to score RNA editing sites. Applied to PacBio long-read RNA-seq data, L-GIREMI affords a high accuracy in RNA editing identification. In addition, the unique advantage of long reads allowed us to uncover novel insights about RNA editing occurrences in single molecules and double-stranded RNA (dsRNA) structures. L-GIREMI provides a valuable means to study RNA nucleotide variants in long-read RNA-seq.


title: Proton-transporting heliorhodopsins from marine giant viruses

abstract: Rhodopsins convert light into signals and energy in animals and microbes. Heliorhodopsins (HeRs), a recently discovered new rhodopsin family, are widely present in archaea, bacteria, unicellular eukaryotes, and giant viruses, but their function remains unknown. Here we report that a viral HeR from Emiliania huxleyi virus 202 (V2HeR3) is a light-gated proton channel. V2HeR3 absorbs blue-green lights, and the active intermediate contains the deprotonated retinal Schiff base. Site-directed mutagenesis study revealed that E191 in TM6 constitutes the gate together with the retinal Schiff base. E205 and E215 form a proton accepting group of the Schiff base, whose mutations converted the protein into an outward proton pump. Three environmental viral HeRs from the same group, as well as a more distantly related HeR exhibited similar proton-transport activity, indicating that HeR functions might be diverse similarly to type-1 microbial rhodopsins. Some strains of E. huxleyi contain one HeR that is related to the viral HeRs, while its viruses EhV-201 and EhV-202 contain two and three HeRs, respectively. Except for V2HeR3 from EhV-202, none of these proteins exhibit ion-transport activity. Thus, when expressed in the E. huxleyi cell membranes, only V2HeR3 has the potential to depolarize the host cells by light, possibly to overcome the host defense mechanisms or to prevent superinfection. The neuronal activity generated by V2HeR3 suggests that it can potentially be used as an optogenetic tools, like type-1 microbial rhodopsins.

category: biophysics


title: TCGADEPMAP -- Mapping Translational Dependencies and Synthetic Lethalities within The Cancer Genome Atlas

abstract:
The Cancer Genome Atlas (TCGA) has yielded unprecedented genetic and molecular characterization of the cancer genome, yet the functional consequences and patient-relevance of many putative cancer drivers remain undefined. TCGADEPMAP is the first hybrid map of TCGA patient dependencies that was built from 6,581 expression-based predictive models of gene essentiality across 791 cancer cell models within the Cancer Dependency Map (DEPMAP). TCGADEPMAP captured well-known cancer lineage and sub-lineage dependencies (e.g., ESR1, ERBB2, etc.), genetic drivers (e.g., KRAS, BRAF, etc.), and synthetic lethalities (e.g., STAG1/2, SMARCA2/4, etc.), demonstrating the robustness of TCGADEPMAP as a translational dependency map. TCGADEPMAP also unveiled novel synthetic lethalities that were experimentally confirmed using multiplexed CRISPR screening across multiple cancer cell lines. Other map "extensions" were built to capture unique aspects of patient-relevant tumor dependencies, including translating gene essentiality to drug response in patient-derived xenograft (PDX) models (i.e., PDXEDEPMAP) and predicting gene tolerability within normal tissues (GTEXDEPMAP). Collectively, this study demonstrates how translational dependency maps can be used to leverage the rapidly expanding catalog of patient genomic datasets to identify and prioritize novel therapeutic targets with the best therapeutic indices.

Statement of SignificanceThe translational dependency maps described here provide novel insights into the essentiality and tolerability of target genes in human patients. Starting with 20 million data points from the Cancer Dependency Map (DEPMAP), the Cancer Genome Atlas (TCGA), the Patient-Derived Xenograft Encyclopedia (PDXE), and the Genotype-Tissue Expression (GTEx) projects, machine learning models for gene dependency and tolerability were built to predict: O_LI6,581 predictive models of gene essentiality in TCGADEPMAP and PDXEDEPMAP, with corresponding prediction of gene tolerability in GTEXDEPMAP
C_LIO_LI1,966 predictive models of selective gene essentiality were robustly cross-validated and accurately predict pan-cancer lineages (AUC>0.99), breast cancer sub-lineages (AUC>0.88), synthetic lethalities (SMARCA2/4, STAG1/2, EP300/CREBBP etc.), and oncogenic drivers (e.g., KRAS, BRAF, ERBB2, etc.)
C_LIO_LI881 synthetic lethality candidates were tested using a unique multiplexed CRISPR screen by AsCas12a across multiple cancer models, revealing multiple translatable and potentially druggable synthetic lethalities (STAG1/2, EP300/CREBBP, PAPSS1/2, etc.)
C_LI

category: cancer biology


title: Multi-omic analysis of the cardiac cellulome defines a vascular contribution to cardiac diastolic dysfunction in obese female mice

abstract: Coronary microvascular dysfunction (CMD) is associated with cardiac dysfunction and predictive of cardiac mortality in obesity, especially in females. Emerging evidence suggests development of heart failure with preserved ejection fraction in females with CMD and that mineralocorticoid receptor (MR) antagonism may be more efficacious in obese female, versus male, HFpEF patients. Accordingly, we examined the hypothesis that smooth muscle cell (SMC)-specific MR deletion prevents obesity-associated coronary and cardiac diastolic dysfunction in females. Obesity was induced in female mice via western diet (WD) feeding alongside littermates fed standard diet. Initial studies revealed that global MR blockade with spironolactone prevented impaired coronary vasodilation and diastolic dysfunction in obese females. Importantly, specific deletion of SMC-MR similarly prevented obesity-associated coronary and cardiac dysfunction. Cardiac gene expression profiling suggested reduced cardiac inflammation in WD-fed mice with SMC-MR deletion independent of blood pressure, aortic stiffening, and cardiac hypertrophy. Further mechanistic studies utilizing single-cell RNA sequencing of non-cardiomyocyte cell populations revealed novel impacts of SMC-MR deletion on the cardiac cellulome in obese mice. Specifically, WD feeding induced inflammatory gene signatures in multiple non-myocyte populations (B/T cells, macrophages, and endothelium), independent of cardiac fibrosis, that was prevented by SMC-MR deletion. Further, SMC-MR deletion induced a basal reduction in cardiac mast cells and prevented WD-induced cardiac pro-inflammatory chemokine expression and leukocyte recruitment. These data reveal a central role for SMC-MR signaling in obesity-associated coronary and cardiac dysfunction thus supporting the emerging paradigm of a vascular origin of cardiac dysfunction in obesity.


title: Spatiotemporal patterns of sleep spindle activity in human anterior thalamus and cortex

abstract: Sleep spindles (8 - 16 Hz) are transient electrophysiological events during non-rapid eye movement sleep. While sleep spindles are routinely observed in the cortex using scalp electroencephalography (EEG), recordings of their thalamic counterparts have not been widely studied in humans. Based on a few existing studies, it has been hypothesized that spindles occur as largely local phenomena. To see whether they also facilitate thalamocortical communication, we investigated intra-thalamic and thalamocortical spindle co-occurrence. We obtained scalp EEG and thalamic recordings from 7 patients that received bilateral deep brain stimulation (DBS) electrodes to the anterior thalamus for the treatment of drug resistant focal epilepsy. Spindles were categorized into subtypes based on their main frequency (i.e., slow (10{+/-}2 Hz) or fast (14{+/-}2 Hz)) and their level of thalamic involvement (spanning one channel, or spreading uni- or bilaterally within the thalamus). For the first time, we contrasted observed spindle patterns with permuted data to estimate random spindle co-occurrence. We found that multichannel spindle patterns were systematically coordinated at the thalamic and thalamocortical level. Importantly, distinct topographical patterns of thalamocortical spindle overlap were associated with slow and fast subtypes of spindles. These observations provide further evidence for coordinated spindle activity in thalamocortical networks.


title: Two modes of mid-frontal theta suggest a role in conflict and error processing

abstract: Mid-frontal theta is well-known to be involved in conflict and error-processing. In paradigms known to evoke response conflict, the temporal sequence of events leading up to conflict or error resolution and thetas involvement in such scenarios is not well understood. Using single-trial analyses of electrophysiological data from participants performing the Flanker (N = 28) and Simon paradigm (N = 18), we probed the relationship between transient theta activity and the underlying metrics of response conflict. We specifically investigated "partial errors", in which a small burst of muscle activity in the incorrect response effector occurred, quickly followed by a correction. We found evidence for two modes of theta activity, one of which was strongly related to the partial errors. The temporal distribution of this mode of theta was different in the partial error trials compared to the correct trials with no partial error. Furthermore, we saw that this mode of theta occurred later in trials in which a full error was committed. Our findings point to a possible role of theta in error-correction. This adds to the wider theoretical understanding of mid-frontal theta and the possibility of multiple functional roles of theta in different contexts.


title: Stable working memory and perceptual representations in macaque lateral prefrontal cortex during naturalistic vision in virtual environments

abstract:
Primates use perceptual and mnemonic visuospatial representations to perform everyday functions. Neurons in the lateral prefrontal cortex (LPFC) have been shown to encode both of these representations during tasks where eye movements are strictly controlled and visual stimuli are reduced in complexity. This raises the question of whether perceptual and mnemonic representations encoded by LPFC neurons remain robust during naturalistic vision -- in the presence of a rich visual scenery and during eye movements. Here we investigate this issue by training macaque monkeys to perform working memory and perception tasks in a visually complex virtual environment that requires navigation using a joystick and allows for free visual exploration of the scene. We recorded the activity of 3950 neurons in the LPFC (areas 8a and 9/46) of two rhesus macaques using multi-electrode arrays, and measured eye movements using video tracking. We found that navigation trajectories to target locations and eye movement behavior differed between the perception and working memory tasks suggesting that animals employed different behavioral strategies. Single neurons were tuned to target location during cue encoding and working memory delay and neural ensemble activity was predictive of the animals behavior. Neural decoding of target location was stable throughout the working memory delay epoch. However, neural representations of similar target locations differed between the working memory and perception tasks. These findings indicate that during naturalistic vision, LPFC neurons maintain robust and distinct neural codes for mnemonic and perceptual visuospatial representations.

Significance StatementWe show that LPFC neurons encode working memory and perceptual representations during a naturalistic task set in a virtual environment. We show that despite eye movement and complex visual input, neurons maintain robust working memory representations of space which are distinct from neuronal representations for perception. We further provide novel insight on the use of virtual environments to construct behavioral tasks for electrophysiological experiments.


title: Local 5-HT signal bi-directionally regulates the coincidence time window of associative learning

abstract: Temporal coincidence between the conditioned stimulus (CS) and unconditioned stimulus (US) is essential for associative learning across species. Despite its ubiquitous presence, the mechanism that may regulate this time window duration remains unclear yet. Using olfactory associative learning in Drosophila as a model, we find that suppressing or promoting serotonin (5-HT) signal could respectively shorten or prolong the coincidence time window of odor-shock associative learning and synaptic plasticity in mushroom body (MB) Kenyon cells (KCs). Capitalizing on GPCR-activation based (GRAB) sensors for 5-HT and acetylcholine (ACh), we characterized the in vivo 5-HT dynamics in MB lobes during odor and shock stimulations and further dissected this microcircuit. Interestingly, local KC-released ACh activates nicotinic receptors on the dorsal paired medial (DPM) neuron, and in turn the DPM neuron releases 5-HT to inhibit the ACh signal via the 5-HT1a receptor. Finally, we demonstrated that the DPM-mediated serotonergic feedback circuit is sufficient and necessary to regulate the coincidence time window. This work provides a model for studying the temporal contingency of environmental events and their causal relationship.


title: HALO: A software tool for real-time head alignment in the MR scanner

abstract: Magnetic resonance imaging (MRI) studies in human subjects often require multiple scanning sessions/visits. Changes in a subjects head position across sessions result in different alignment between brain tissues and the magnetic field which leads to changes in magnetic susceptibility. These changes can have considerable impacts on acquired signals. Therefore, we developed the Head Alignment Optimization (HALO) tool. HALO provides real-time visual feedback of a subjects current head position relative to the position in a previous session. We verified that HALO enabled subjects to actively align their head positions to the desired position of their initial sessions. Our pilot sample of healthy subjects were able to improve their head alignment significantly using HALO and achieved good alignment with their first session meeting stringent criteria similar to that used for within-run head motion (less than 2mm translation or 2 degrees rotation in any direction from the desired position). Utilization of HALO in longitudinal studies will reduce the noise across sessions related to changes in magnetic susceptibility. HALO has been made publicly available.


title: Temporal dynamics of cognitive flexibility in adolescents with anorexia nervosa: a high density EEG study

abstract:
Cognitive rigidity is a core symptom of anorexia nervosa (AN) and is associated with treatment resistance. Nevertheless, studies on the neural basis of cognitive flexibility (CF) in adolescent AN are rare.

The aim of this study was to investigate temporal brain dynamics of CF in adolescents with AN and their tendency to experience anxiety. To address this aim, participants performed a Dimensional Change Card Sorting task during high-density EEG recording. Transient state and stable traits of anxiety were measured with the State-Trait Anxiety Inventory. Data were collected on 22 girls with AN, and 23 healthy controls (HC) (aged 12-20 years old). Evoked potentials were investigated using microstates analysis. Effects of trait and state anxiety on microstates parameters were investigated performing correlations across groups and in the AN group only.

Adolescents with AN did not differ from HC with respect to behavioral switching cost, despite a greater overall accuracy. Adolescents with AN showed altered visual orienting processes (N100 micro-state), and earlier attentional processing to task switching (P300b microstate) when compared to HC. Reduced activation in a network involving the pre-central cortex, the cerebellum, and the posterior cingulate was identified in adolescents with AN. Trait and state anxiety were correlated with atypical AN microstates across groups.

The present findings suggest CF manifests with inefficient behavioral and neural strategies in adolescents with AN, at early stages in the illness. Additionally, this study provides evidence that transient and stable tendencies to experience anxiety could represent a vulnerability to developing CF impairments in adolescent AN.


title: Structural and functional MRI data differentially predict chronological age and behavioral memory performance

abstract: Human cognitive abilities decline with increasing chronological age, with decreased explicit memory performance being most strongly affected. However, some older adults show "successful aging", that is, relatively preserved cognitive ability in old age. One explanation for this could be higher brain structural integrity in these individuals. Alternatively, the brain might recruit existing resources more efficiently or employ compensatory cognitive strategies. Here, we approached this question by testing multiple candidate variables from structural and functional neuroimaging for their ability to predict chronological age and memory performance, respectively. Prediction was performed using support vector machine (SVM) classification and regression across and within two samples of young (N = 106) and older (N = 153) adults. The candidate variables were (i) behavioral response frequencies in an episodic memory test, (ii) recently described fMRI scores reflecting preservation of functional memory networks, (iii) whole-brain fMRI contrasts for novelty processing and subsequent memory, (iv) resting-state fMRI maps quantifying voxel-wise signal fluctuation and (v) gray matter volume estimated from structural MR images. While age group could be reliably decoded from all variables, chronological age within young and older subjects was best predicted from gray matter volume. In contrast, memory performance was best predicted from task-based fMRI contrasts and particularly single-value fMRI scores, whereas gray matter volume has no predictive power with respect to memory performance in healthy adults. Our results suggest that superior memory performance in healthy older adults is better explained by efficient recruitment of memory networks rather than by preserved brain structure.


title: Suboptimal foraging decisions and involvement of the ventral tegmental area in human opioid addiction

abstract:
Addiction is marked by a tendency to exploit sources of reward despite diminishing returns. This behavior is aptly captured by animal patch-foraging models that have recently been extended to humans. Dopamine and norepinephrine centrally mediate addictive behavior and activity in both catecholaminergic systems is proposed to reflect the computations necessary for optimal foraging. However, the specific neural bases of excessive foraging and their role in human addiction are largely unknown. To address this gap, we studied the behavior of people with and without opioid use disorder (OUD) on a patch-foraging task in which they made serial decisions to "harvest" a depleting resource ("patch") for reward or incur a varying cost to "travel" to a replenished patch. In a subset of participants, we used high-resolution neuromelanin-sensitive MRI to image neuromelanin concentration, a proxy for long-term catecholaminergic function, in distinct dopaminergic nuclei (ventral tegmental area, substantia nigra subregions) and the noradrenergic locus coeruleus. While all participants were sensitive to the long-run reward rates of different patch-foraging environments, OUD participants stayed in reward patches longer than optimal--markedly overharvesting a source of reward despite its declining value--and this correlated with more chronic drug use. Overharvesting was selectively associated with lower neuromelanin signal in the ventral tegmental area but not other dopaminergic nuclei, nor the locus coeruleus. Our findings suggest that foraging decisions relevant to addiction involve a ventral-tegmental-area circuit that may signal reward rates in dynamic environments and implicate this circuit in maladaptive reward pursuit in human addiction to opioids.

Significance statementPatch-foraging provides a potentially important translational framework for understanding addictive behavior by revealing how maladaptive reward pursuit emerges in more ecologically valid decision contexts. Here, we show that the tendency to exploit sources of reward despite diminishing returns is associated with chronic drug use in people with opioid use disorder, a particularly devastating form of addiction. We further use neuromelanin-sensitive MRI, a neuroimaging measure of the long-term function of dopamine neurons, to reveal that variation in ventral tegmental area neuromelanin signal selectively underlies individual differences in this overharvesting bias. These findings establish a role for specific dopaminergic circuits in patch-foraging decisions and advance understanding of the neurobiology of human addiction to opioids that has so far eluded the field.


title: A novel maxillofacial technique to deliver drugs to the visual pathway, the optic nerve and the retina using the glymphatic system.

abstract:
The blood neural barriers are formidable barriers that prevent delivery of drugs into the eye and the brain. Currently, large biologics are used for treating many diseases of the eye and the brain. But the blood retinal barrier and the blood brain barrier prevent entry of these useful drugs into the eye and the brain respectively. Hence, there is a need for a drug delivery technique to bypass these natural barriers.

Bevacizumab (Avastin) is a full length humanized monoclonal antibody with a molecular weight of 149kda. It binds to circulating vascular endothelial growth factor A and prevents interaction with vascular endothelial growth receptors. This results in blocking endothelial response and tumor vascularization. Bevacizumab is used to prevent choroidal neovascularization in age related macular degeneration and to prevent retinal neovascularization in diabetic retinopathy. It is also used for treating certain types of brain cancer. But it crosses neither the blood retinal barrier nor the blood brain barrier.

This in vivo mouse study assessed the delivery of a commercial formulation of bevacizumab into the retina of mice using different ocular techniques like topical drops, subconjunctival injection, intravitreal injection and a novel maxillofacial technique. The objective was to assess whether the novel maxillofacial drug delivery technique could deliver drugs into the retina and to compare the efficacy of the maxillofacial technique with the mentioned ocular techniques. The distribution of bevacizumab was further compared with retinoschisin which is a small protein with a molecular weight of 24kda.

Intravitreal injection and maxillofacial technique were effective in delivering drugs into the retina. In addition, the maxillofacial technique could target the brain including regions involved in the visual pathway and the optic nerve. The glymphatic pathway could also be targeted for drug delivery. Drug was also detected in the contralateral optic nerve and retina.

Based on our study findings, we propose a new concept to explain the presence of the drug in the contralateral eye. We propose that after maxillofacial drug delivery, early distribution of the drug can occur in the CSF at the optic chiasma from the brain via the glymphatic system. In the case of the intravitreal injection, the drug from the experimental eye may be cleared through the glymphatic pathway of the ipsilateral optic nerve into the CSF surrounding the nerve. The crossover of the ipsilateral optic nerve fibers at the optic chiasma can result in further distribution of the drug in the CSF at the optic chiasma. From the region of the optic chiasma, the drug can distribute into the CSF surrounding the contralateral optic nerve. The drug can be then distributed into the contralateral optic nerve through the glymphatic pathway and be delivered into the contralateral eye. The drug can be further cleared into the aqueous of the contralateral eye through the anterior clearance pathway.


title: Peripheral blood T-cell deficiency and hyperinflammatory monocyte responses associate with MAC lung disease

abstract:
RationaleAlthough nontuberculous mycobacterial (NTM) disease is a growing problem, available treatments are suboptimal and diagnostic tools are inadequate. Immunological mechanisms of susceptibility to NTM disease are poorly understood.

ObjectiveTo understand NTM pathogenesis, we evaluated innate and antigen-specific adaptive immune responses to Mycobacterium avium complex (MAC) in individuals with MAC lung disease (MACDZ).

MethodsWe synthesized 15mer MAC-, NTM-, or MAC/Mtb-specific peptides and stimulated peripheral blood mononuclear cells (PBMC) with pools of these peptides. We measured T-cell responses by cytokine production, expression of surface markers, and analysis of global gene expression in 27 MACDZ individuals and 32 healthy controls. We also analyzed global gene expression in Mav-infected and uninfected peripheral blood monocytes from 17 MACDZ and 17 healthy controls.

Measurements and Main ResultsWe were unable to detect T-cell responses against the peptide libraries or Mav lysate that has increased reactivity in MACDZ subjects compared to controls. T-cell responses to non-mycobacteria derived antigens were preserved.

MACDZ individuals had a lower frequency of Th1 and Th1* T-cell populations. By contrast, global gene expression analysis demonstrated upregulation of proinflammatory pathways in uninfected and Mav-infected monocytes derived from MACDZ subjects compared to controls.

ConclusionsPeripheral blood T-cell responses to Mycobacterial antigens and the frequency of Th1 and Th1* cell populations are diminished in individuals with MAC disease. In contrast, MACDZ subjects had hyperinflammatory monocyte responses. Together, these data suggest a novel immunologic defect which underlies MAC pathogenesis and includes concurrent innate and adaptive dysregulation.

category: immunology


title: Longitudinal single cell transcriptional and epigenetic mapping of effector, memory, and exhausted CD8 T cells reveals shared biological circuits across distinct cell fates

abstract: Naive CD8 T cells can differentiate into effector (TEFF), memory (TMEM), or exhausted (TEX) CD8 T cells. These developmental pathways are associated with distinct transcriptional and epigenetic changes that endow cells with different functional capacities and therefore therapeutic potential. The molecular circuitry underlying these developmental trajectories and the extent of heterogeneity within TEFF, TMEM, TEX populations remain poorly understood. Here, we used the lymphocytic choriomeningitis virus model of acutely-resolved and chronic infection and addressed these gaps by applying longitudinal scRNA-seq and scATAC-seq analysis. These analyses uncovered new subsets, including a subpopulation of TEX expressing NK cell-associated genes, as well as multiple distinct TCF1+ stem/progenitor-like subsets in acute and chronic infection. These data also revealed insights into the reshaping of TEX subsets following PD1 pathway blockade and identified a key role for the cell stress regulator, Btg1, in TEX differentiation. Finally, these results highlighted how the same biological circuits such as cytotoxicity or stem/progenitor pathways can be used by CD8 T cells with highly divergent underlying chromatin landscapes. Thus, this transcriptional and chromatin accessibility landscape map elucidates developmental biology and underlying mechanisms governing TEFF, TMEM, and TEX differentiation.


title: Spatial exploration of immune cell heterogeneity in skin functional scar-less wound regeneration model at single-cell resolution

abstract: Fibrotic scar devoid of skin appendages bring great challenge to tissue engineering, particularly in severe large area wound. Intriguingly, HF (hair follicle) can regenerate specifically in the large wound center of adult mice, termed wound-induced hair follicle neogenesis (WIHN). Single-cell RNA sequencing (scRNA-seq) comparison of wound in different size showed increased activation of adaptive immune systems along with predominance of HF precursor cells in large wound. Integration the gene expression profiles of spatial transcriptomic (ST) and scRNA-seq highlighted the preferential aggregation of T cells, pro- inflammatory macrophage (PIM) and monocyte in the margin and center of large wound co-localizing with HF precursor cells. Local pro- regenerative immune response among them provided potential therapeutic target for the further HF regeneration. Aligned electrospinning scaffolds were previously shown to accelerate small wound healing with the immunomodulatory effect of T cells and macrophages in small wound. Here we observed that wound recapitulated normal skin architecture with the implantation of biodegradable biosynthetic scaffold despite wound size. The scaffolds did not trigger a severe foreign body response (FBR) but showed the immunomodulatory effect on adaptive immune system. Enlarged distribution of the "pro-regenerative" immune niche overlapping with HF precursor cells may account for the well-proportioned de novo HF in scaffold-implanted group. Collectively, our study illustrated that manipulating the adaptive immune system may stimulate de novo HF regeneration via local cell-cell interactions. And the easy-fabricating electrospinning membrane with appropriate biocompatibility also offered available treatment options in severe skin defect regeneration.


title: AIRRscape: an interactive tool for exploring B-cell receptor repertoires and antibody responses

abstract:
The sequencing of antibody repertoires of B-cells at increasing coverage and depth has led to the identification of vast numbers of immunoglobulin heavy and light chains. However, the size and complexity of these Adaptive Immune Receptor Repertoire sequencing (AIRR-seq) datasets makes it difficult to perform exploratory analyses. To aid in data exploration, we have developed AIRRscape, an R Shiny-based interactive web browser application that enables B-cell receptor (BCR) and antibody feature discovery through comparisons among multiple repertoires. Using AIRR-seq data as input, AIRRscape starts by aggregating and sorting repertoires into interactive and explorable bins of germline V-gene, germline J-gene, and CDR3 length, providing a high-level view of the entire repertoire. Interesting subsets of repertoires can be quickly identified and selected, and then network topologies of CDR3 motifs can be generated for further exploration. Here we demonstrate AIRRscape using patient BCR repertoires and sequences of published monoclonal antibodies to investigate patterns of humoral immunity to three viral pathogens: SARS-CoV-2, HIV-1, and DENV (dengue virus). AIRRscape reveals convergent antibody sequences among datasets for all three pathogens, although HIV-1 antibody datasets display limited convergence and idiosyncratic responses. We have made AIRRscape available on GitHub to encourage its open development and use by immuno-informaticians, virologists, immunologists, vaccine developers, and other scientists that are interested in exploring and comparing multiple immune receptor repertoires.

Author SummaryTechnological advances in next generation sequencing have allowed for broad experimental sampling of immune repertoires, providing insight into how our immune system responds to infection, vaccination, autoimmunity, and cancer. The scale of these "big data", however, make it difficult to bioinformatically extract the key sequence features that are shared across multiple repertoires. With AIRRscape, we enable large-scale immune repertoire visualization and analysis that requires no knowledge of the command line or advanced programming. By providing the community with an open-source, interactive, and user-friendly interface, we reduce the barriers to exploring immune repertoires at scale. We demonstrate the use of AIRRscape to characterize features of immune responses to viral infection that are shared across multiple repertoire datasets.


title: PIP4K2B is a mechanosensor and induces heterochromatin-driven nuclear softening through UHRF1.

abstract: Phosphatidylinositol-5-phosphate (PtdIns5P)-4-kinases (PIP4Ks) are stress-regulated phosphoinositide kinases able to phosphorylate PtdIns5P to PtdIns(4, 5)P2. In cancer patients their expression is typically associated with bad prognosis. Among the three PIP4K isoforms expressed in mammalian cells, PIP4K2B is the one with more prominent nuclear localization. Here, we unveil the role for PIP4K2B as mechanosensor. PIP4K2B protein level, indeed, strongly decreases in cells growing on soft substrates. Its direct silencing or pharmacological inhibition, mimicking cell response to soft, triggers a concomitant reduction of the epigenetic regulator UHRF1 and induces changes in nuclear polarity, nuclear envelope tension and chromatin compaction. This substantial rewiring of the nucleus mechanical state drives YAP cytoplasmic retention and impairment of its activity as transcriptional regulator, finally leading to defects in cell spreading and motility. Since YAP signalling is essential for initiation and growth of human malignancies, our data suggest that potential therapeutic approaches targeting PIP4K2B could be beneficial in the control of the altered mechanical properties of cancer cells.


title: The role of hepatitis B virus surface protein in inducing Sertoli cell ferroptosis

abstract:
Hepatitis B virus infection could result in male infertility by inhibiting sperm function and viability. Sertoli cell death contributes to spermatogenesis impairment, which is associated with sperm defects and dysfunction. Ferroptosis-mediated cell death of Sertoli cells was found to contribute to spermatogenesis disorder and poor sperm quality. However, the effects of hepatitis B virus infection on ferroptosis of Sertoli cells remain to be elucidated. Human Sertoli cells were cultured in vitro with 25, 50, and 100 mg/mL of hepatitis B virus surface protein for 48 hours. Cell viability was measured with CCK-8. Levels of glutathione, malondialdehyde, iron, and m6A in human Sertoli cells were determined. Lipid peroxidation was assessed using C11-BODIPY. Luminescence analysis was performed to detect the binding of METTL3 and 3{cents}-UTR of TRIM37 containing the m6A motifs. Immunoprecipitation was applied to determine the relationship between TRIM37 and GPX4. qPCR and immunoblotting were performed to measure mRNA and protein levels. Hepatitis B virus surface protein exposure significantly increased TRIM37 expression, malondialdehyde level, and ferroptosis, and decreased cell viability and glutathione level of human Sertoli cells. TRIM37 silencing inhibits the effect of HBs exposure-regulated cell viability and ferroptosis in human Sertoli cells. TRIM37 inhibits GPX4 expression through ubiquitination. GPX4 overexpression inhibits the effect of TRIM37 on cell viability and ferroptosis in human Sertoli cells.

Administration of ferroptosis inhibitor recovers the cell viability decreased by TRIM37. Mechanism study showed HBs increases the level of TRIM37 3-UTR m6A by promoting the expression of METTL3, and the binding of m6A reader IGF2BP2 and TRIM37 3-UTR promotes the stability of TRIM37 mRNA.HBs inhibit Sertoli cell viability by promoting ferroptosis of Sertoli cells through TRIM37-mediated ubiquitination of GPX4. The findings highlight the importance of TRIM37/GPX4 signaling in the ferroptosis of Sertoli cells.


title: DiSiR: a software framework to identify ligand-receptor interactions at subunit level from single-cell RNA-sequencing data

abstract: Most of cell-cell interactions and crosstalks are mediated by ligand-receptor interactions. The advent of single-cell RNA-sequencing (scRNA-seq) techniques has enabled characterizing tissue heterogeneity at single-cell level. Over the past recent years, several methods have been developed to study ligand-receptor interactions at cell type level using scRNA-seq data. However, there is still no easy way to query the activity of a specific user-defined signaling pathway in a targeted way or map the interactions of the same subunit with different ligands as part of different receptor complexes. Here, we present DiSiR, a fast and easy-to-use permutation-based software framework to investigate how individual cells are interacting with each other by analyzing signaling pathways of multi-subunit ligand-activated receptors from scRNA-seq data, not only for available curated databases of ligand-receptor interactions, but also for interactions that are not listed in these databases. We show that, when utilized to infer melanoma disease map on a gold-standard dataset, DiSiR outperforms other well-known permutation-based methods, e.g., CellPhoneDB and ICELLNET. To demonstrate DiSiRs utility in exploring data and generating biologically relevant hypotheses, we apply it to COVID lung and rheumatoid arthritis (RA) synovium scRNA-seq data and highlight potential differences between inflammatory pathways at cell type level for control vs. disease samples.


title: Germline/soma distinction in Drosophila embryos requires regulators of zygotic genome activation

abstract: In Drosophila melanogaster embryos, somatic versus germline identity is the first cell fate decision. Zygotic genome activation (ZGA) orchestrates regionalized gene expression, imparting specific identity on somatic cells. ZGA begins with a minor wave that commences at nuclear cycle (NC) 8 under the guidance of chromatin accessibility factors (Zelda, CLAMP, GAF), followed by the major wave during NC14. By contrast, primordial germ cell (PGC) specification requires maternally deposited and posteriorly anchored germline determinants. This is accomplished by a centrosome coordinated release and sequestration of germ plasm during the precocious cellularization of PGCs in NC10. Here we report a novel requirement for Zelda and CLAMP during the establishment of the germline/soma distinction. When their activity is compromised, PGC determinants are not properly sequestered, and specification is disrupted. Conversely, the spreading of PGC determinants from the posterior pole adversely influences transcription in the neighboring somatic nuclei. These reciprocal aberrations can be correlated with defects in centrosome duplication/separation that are known to induce inappropriate transmission of the germ plasm. Interestingly, consistent with the ability of BMP signaling to influence specification of embryonic PGCs, reduction in the transcript levels of a BMP family ligand, decapentaplegic (dpp), is exacerbated at the posterior pole.


title: Scalable Generation of Pseudo-Unipolar Sensory Neurons from Human Pluripotent Stem Cells

abstract: Development of new non-addictive analgesics requires advanced strategies to differentiate human pluripotent stem cells (hPSCs) into relevant cell types amenable for translational research. Here, we developed a highly efficient and reproducible method that differentiates hPSCs into peptidergic and non-peptidergic nociceptors. By modulating specific cell signaling pathways, hPSCs were first converted into SOX10+ neural crest cells, followed by differentiation into sensory neurons with an in vivo-like pseudo-unipolar morphology. Detailed characterization confirmed that the hPSC-derived nociceptors displayed molecular and cellular features comparable to native dorsal root ganglion (DRG) neurons, and expressed high-threshold primary sensory neuron markers, transcription factors, neuropeptides, and over 150 ion channels and receptors, including critical pain-relevant drug targets (e.g., TRPV1, TAC1, CALCA, NAV1.7, NAV1.8). Moreover, after confirming robust functional activities and differential response to noxious stimuli and specific drugs, a robotic cell culture system was employed to produce large quantities of human sensory neurons, which can be used to develop nociceptor-selective analgesics.