Miracle-dz/UniFAR-FTUs huggingface dataset:
Orc6 at replication fork enables efficient mismatch repair

In eukaryotes, the Origin Recognition Complex (ORC) is required for the initiation of DNA replication. The smallest subunit of ORC, Orc6, is essential for pre-replication complex (pre-RC) assembly and cell viability in yeast and for cytokinesis in metazoans. However, unlike other ORC components, the role of human Orc6 in replication remains to be resolved. Here, we identify an unexpected role for hOrc6, which is to promote S-phase progression post pre-RC assembly and DNA damage response. Orc6 localizes at the replication fork and is an accessory factor of the mismatch repair (MMR) complex. In response to oxidative damage during S-phase, often repaired by MMR, Orc6 facilitates MMR complex assembly and activity, without which the checkpoint signaling is abrogated. Mechanistically, Orc6 directly binds to MutSα and enhances the chromatin-association of MutLα, thus enabling efficient mismatch repair. Based on this, we conclude that hOrc6 plays a fundamental role in genome surveillance during S-phase. Highlights Human Orc6 is dispensable for G1 licensing, but required for S-phase progression Human Orc6 at the replication fork is an accessory factor for MMR complex Depletion of hOrc6 sensitizes cells to DNA damage and impairs ATR activation Human Orc6 regulates MMR complex assembly and activity
Hyperinflammation evokes different antiviral TMPRSS2 and ADAM17 expression responses in human gut xenograft versus host mouse gut although overall genomic responses are similar

The global spread of the newly emerged severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has led to the pandemic outbreak of coronavirus disease 2019 (COVID-19), an inflammatory disease that is primarily affecting the respiratory system. However, gastrointestinal symptoms in COVID-19 patients suggests that the gut may present another viral target organ. Disease development and severity is dependent on viral interaction with two cell surface human proteins, ACE2 and TMPRSS2, and on antiviral response which may lead to systemic hyperinflammatory syndrome and multiorgan dysfunction. Understanding the host response to SARS-CoV-2 infection and the pathology of the disease will be greatly enhanced by the development of appropriate animal models. Laboratory mice have been the mainstay of therapeutic and vaccine development, however, the virus does not grow in wild type mice and only induced mild disease in transgenic animals expressing human ACE2. As there are known differences between immune response in laboratory mice and humans we evaluated the response of human gut developed as xenografts and host mouse gut following systemic LPS injections as a hyperinflammation model system. The orthologous gene expression levels in the mouse and human gut were highly correlated (Spearman’s rank correlation coefficient: 0.28–0.76) and gene set enrichment analysis of significantly upregulated human and mouse genes revealed that a number of inflammatory and immune response pathways are commonly regulated in the two species. However, species differences were also observed, most importantly, in the inflamed human gut but not in the mouse gut, there was clear upregulation of mRNAs coding for TMPRSS2, ADAM17 and for RIG-I-like receptors, which are involved in the recognition of viruses and in antiviral innate immune response. Moreover, using species-specific immunofluorescence microscopy, we demonstrated the expression and localization of human ACE2 and TMPRSS2 proteins, which are essential elements of the molecular machinery that enables SARS-CoV-2 to infect and replicate in human gut cells. Our findings demonstrate that the intestinal immune response to inflammation in humans and mice are generally very similar. However, certain human-specific diseases, such as COVID-19, can only be successfully studied in an experimental model of human tissue, such as the gut xenograft.
Embeddings of genomic region sets capture rich biological associations in lower dimensions

Motivation Genomic region sets summarize functional genomics data and define locations of interest in the genome such as regulatory regions or transcription factor binding sites. The number of publicly available region sets has increased dramatically, leading to challenges in data analysis. Results We propose a new method to represent genomic region sets as vectors, or embeddings, using an adapted word2vec approach. We compared our approach to two simpler methods based on interval unions or term frequency-inverse document frequency and evaluated the methods in three ways: First, by classifying the cell line, antibody, or tissue type of the region set; second, by assessing whether similarity among embeddings can reflect simulated random perturbations of genomic regions; and third, by testing robustness of the proposed representations to different signal thresholds for calling peaks. Our word2vec-based region set embeddings reduce dimensionality from more than a hundred thousand to 100 without significant loss in classification performance. The vector representation could identify cell line, antibody, and tissue type with over 90% accuracy. We also found that the vectors could quantitatively summarize simulated random perturbations to region sets and are more robust to subsampling the data derived from different peak calling thresholds. Our evaluations demonstrate that the vectors retain useful biological information in relatively lower-dimensional spaces. We propose that vector representation of region sets is a promising approach for efficient analysis of genomic region data. Availability https://github.com/databio/regionset-embedding
White matter microstructure characterisation in temporal lobe epilepsy

Temporal lobe epilepsy (TLE) is the most common form of focal epilepsy. Parameters of microstructural abnormalities derived from diffusion tensor imaging(DTI) have been reported to be helpful in differentiating between Left and Right TLE (L-TLE and R-TLE) but few of them compared L-TLE and R-TLE with a voxelwise approach. In this study, a whole brain tract based spatial statistical analysis was performed on DTI, diffusion kurtosis and NODDI derived parameters of 88 subjects to identify specific white matter patterns of alteration in patient affected by L-TLE and R-TLE with respect to healthy controls. Our findings demonstrated the presence of specific patterns of white matter alterations, with L-TLE more widely affected both in cerebral and cerebellar regions. This result supports the need to consider patients separately, according to the side of their pathology.
Acorde: unraveling functionally-interpretable networks of isoform co-usage from single cell data

Alternative splicing (AS) is a highly-regulated post-transcriptional mechanism known to modulate isoform expression within genes and contribute to cell-type identity. However, the extent to which alternative isoforms establish co-expression networks that may relevant in cellular function has not been explored yet. Here, we present acorde, a pipeline that successfully leverages bulk long reads and single-cell data to confidently detect alternative isoform co-expression relationships. To achieve this, we developed and validated percentile correlations, a novel approach that overcomes data sparsity and yields accurate co-expression estimates from single-cell data. Next, acorde uses correlations to cluster co-expressed isoforms into a network, unraveling cell type-specific alternative isoform usage patterns. By selecting same-gene isoforms between these clusters, we subsequently detect and characterize genes with co-differential isoform usage (coDIU) across neural cell types. Finally, we predict functional elements from long read-defined isoforms and provide insight into biological processes, motifs and domains potentially controlled by the coordination of post-transcriptional regulation.
Can charge-reversal be considered as a strategy for attaining thermal stability in proteins?

Do charge reversal mutations (CRM) naturally occur in mesophilic-thermophilic/hyperthermophilic (M-T/HT) orthologous proteins? Do they contribute to thermal stability by altering charge-charge interactions? A careful investigation on 1550 M-T/HT orthologous protein pairs with remarkable structural and topological similarity extracts the role of buried and partially exposed CRMs in enhancing thermal stability. Our findings could assist in engineering thermo-stable variants of proteins. SIGNIFICANCE Protein engineering is one of the hot topics for decades specifically for its applications in different fields like de-novo protein design, directed evolution, making highly stable variants for food and drug industry etc. Proteins from organisms living in extreme environments are therefore a matter of common interest for scientists from different disciplines. Over three decades of study has already found several sequence and structural adaptations related to thermal stability, while charge reversal study remains ignored to a large extent. Influenced by nature’s strategy, our study provides a systemic understanding of how proper designing of few partially exposed and buried CRMs significantly contributes to thermal stability by altering the short distance electrostatic interactions.
C. elegans PEZO-1 is a mechanosensitive ion channel involved in food sensation

PIEZO channels are force sensors essential for physiological processes, including baroreception and proprioception. The Caenorhabditis elegans genome encodes an ortholog gene of the Piezo family, pezo-1, which is expressed in several tissues, including the pharynx. This myogenic pump is an essential component of the C. elegans alimentary canal, whose contraction and relaxation are modulated by mechanical stimulation elicited by food content. Whether pezo-1 encodes a mechanosensitive ion channel and contributes to pharyngeal function remain unknown. Here, we leverage genome editing, genetics, microfluidics, and electropharyngeogram recording to establish that pezo-1 is expressed in the pharynx, including in a proprioceptive-like neuron, and regulates pharyngeal function. Knockout (KO) and gain-of-function (GOF) mutants reveal that pezo-1 is involved in fine-tuning pharyngeal pumping frequency, sensing osmolarity, and food mechanical properties. Using pressure-clamp experiments in primary C. elegans embryo cultures, we determine that pezo-1 KO cells do not display mechanosensitive currents, whereas cells expressing wild-type or GOF PEZO-1 exhibit mechanosensitivity. Moreover, infecting the Spodoptera frugiperda cell line with a baculovirus containing the G-isoform of pezo-1 (among the longest isoforms) demonstrates that pezo-1 encodes a mechanosensitive channel. Our findings reveal that pezo-1 is a mechanosensitive ion channel that regulates food sensation in worms.
Genetic determinants of chromatin reveal prostate cancer risk mediated by context-dependent gene regulation

Methods that link genetic variation to steady-state gene expression levels, such as expression quantitative trait loci (eQTLs), are widely used to functionally annotate trait-associated variants, but they are limited in identifying context-dependent effects on transcription. To address this challenge, we developed the cistrome-wide association study (CWAS), a framework for nominating variants that impact traits through their effects on chromatin state. CWAS associates the genetic determinants of cistromes (e.g., the genome-wide profiles of transcription factor binding sites or histone modifications) with traits using summary statistics from genome-wide association studies (GWAS). We performed CWASs of prostate cancer and androgen-related traits, using a reference panel of 307 prostate cistromes from 165 individuals. CWAS nominated susceptibility regulatory elements or androgen receptor (AR) binding sites at 52 out of 98 known prostate cancer GWAS loci and implicated an additional 17 novel loci. We functionally validated a subset of our results using CRISPRi and in vitro reporter assays. At 28 of the 52 risk loci, CWAS identified regulatory mechanisms that are not observable via eQTLs, implicating genes with complex or context-specific regulation that are overlooked by current approaches that relying on steady-state transcript measurements. CWAS genes include transcription factors that govern prostate development such as NKX3-1, HOXB13, GATA2, and KLF5. Moreover, CWAS boosts discovery power in modestly sized GWAS, identifying novel genetic associations mediated through AR binding for androgen-related phenotypes, including resistance to prostate cancer therapy. CWAS is a powerful and biologically interpretable paradigm for studying variants that influence traits by affecting context-dependent transcriptional regulation.
Emergent population activity in metric-free and metric networks of neurons with stochastic spontaneous spikes and dynamic synapses

We show that networks of excitatory neurons with stochastic spontaneous spiking activity and short-term synaptic plasticity can exhibit spontaneous repetitive synchronization in so-called population spikes. The major reason for this is that synaptic plasticity nonlinearly modulates the interaction between neurons. For large-scale two-dimensional networks, where the connection probability decreases exponentially with increasing distance between the neurons resulting in a small-world network connectome, a population spike occurs in the form of circular traveling waves diverging from seemingly non-stationary nucleation sites. The latter is in drastic contrast to the case of networks with a fixed fraction of steady pacemaker neurons, where the set of a few spontaneously formed nucleation sites is stationary. Despite the spatial non-stationarity of their nucleation, population spikes may occur surprisingly regularly. From a theoretical viewpoint, these findings show that the regime of nearly-periodic population spikes, which mimics respiratory rhythm, can occur strictly without stochastic resonance. In addition, the observed spatiotemporal effects serve as an example of transient chimera patterns.
Rapid generation of conditional knockout mice using the CRISPR-Cas9 system and electroporation for neuroscience research

The Cre/LoxP-based conditional knockout technology is a powerful tool for gene function analysis that allows region- and time-specific gene manipulation. However, inserting a pair of LoxP cassettes to generate conditional knockout can be technically challenging and thus time- and resource-consuming. This study proposes an efficient, low-cost method to generate floxed mice using in vitro fertilization and the CRISPR-Cas9 system over two consecutive generations. This method allowed us to produce floxed mice targeting exons 5 and 6 of CaMK1 in a short period of 125 days, using only 16 mice. In addition, we directly edited the genome of fertilized eggs of mice with our target genetic background, C57BL/6 N, to eliminate additional backcrossing steps. We confirmed that the genome of the generated floxed mice was responsive to the Cre protein. This low-cost, time-saving method for generating conditional knockout will facilitate comprehensive, tissue-specific genome analyses.
Inhibition-Dominated Rich-Club Shapes Dynamics in Cortical Microcircuits

Functional networks of cortical neurons contain highly interconnected hubs, forming a rich-club structure. However, the cell type composition within this distinct subnetwork and how it influences large-scale network dynamics is unclear. Using spontaneous activity recorded from hundreds of cortical neurons in orbitofrontal cortex of awake behaving mice and from organotypic cultures, we show that the rich-club is disproportionately composed of inhibitory neurons, and that inhibitory neurons within the rich-club are significantly more synchronous than other neurons. At the population level, neurons in the rich-club exert higher than expected Granger causal influence on overall population activity at a broad range of frequencies compared to other neurons. Finally, neuronal avalanche duration is significantly correlated with the fraction of rich neurons that participate in the avalanche. Together, these results suggest an unexpected role of a highly connected, inhibition-rich subnetwork in driving and sustaining activity in local cortical networks. SIGNIFICANCE STATEMENT It is widely believed that the relative abundance of excitatory and inhibitory neurons in cortical circuits is roughly 4:1. This relative abundance has been widely used to construct numerous cortical network models. Here we show that contrary to this notion, a sub-network of highly connected hub neurons (rich-club) consists of a higher abundance of inhibitory neurons compared to that found in the entire network or the non-rich subnetwork. Inhibitory hub neurons contribute to higher synchrony within the rich club compared to the rest of the network. Strikingly, higher activation of the inhibition-dominated rich club strongly correlates with longer avalanches in cortical circuits. Our findings reveal how network topology combined with cell-type specificity orchestrates population wide activity in cortical microcircuits.
In vitro Anticancer Effects of JI017 on Two Prostate Cancer Cell Lines Involve Endoplasmic Reticulum Stress Mediated by Elevated Levels of Reactive Oxygen Species

Prostate cancer is the second most commonly diagnosed cancer, and prostate cancer is the second most common cause of cancer death in United States men after lung cancer. Many therapies are used to treat prostate cancer, and chemotherapy is one of the most relevant treatments. However, chemotherapy has many side effects, and repeated administration of chemotherapeutic agents leads to acquired resistance. Thus, new drugs with few side effects are needed. We investigated the molecular mechanism of action of JI017 in human prostate cancer cells. We identified an endoplasmic reticulum (ER) stress pathway that depended on the reactive oxygen species (ROS) pathway and played a crucial role in JI017-induced apoptosis. We measured cell viability by the MTS assay to determine the effect of JI017. Analysis of apoptosis, mitochondrial dysfunction, and cell cycle features was performed by flow cytometry. We used western blot and RT-PCR to measure the levels of the proteins of the unfolded protein response (UPR) pathway and apoptosis markers. Immunoprecipitation assay and transfection were used to determine the expression levels of proteins interacting with the pathways influenced by JI017 in prostate cancer cells. The anticancer effects induced by JI017 were evaluated. JI017 induced cell death that regulated apoptotic molecules and caused cell cycle arrest that inhibited the proliferation of cancer cells. Moreover, JI017 generated ROS. Accumulation of ROS caused ER stress through the PERK–eIF2α–CHOP and IRE1α-CHOP pathways. Furthermore, persistent activation of the UPR pathway induced by JI017 treatment triggered mitochondrial dysfunction, including dissipation of mitochondrial membrane potential, which activated intrinsic apoptotic pathway in human prostate cancer cells. The data indicated that N-acetyl-L-cysteine diminished apoptosis. We demonstrated that JI017 induced ER stress and cell death. Anticancer properties of JI017 in prostate cancer cells and in a human prostate cancer model involved ROS-mediated ER stress. Thus, JI017 treatment provides a new strategy for chemotherapy of prostate cancer.
Comparison of a Point-of-Care Analyzer With a Chemiluminescent Immunoassay for Serum Progesterone Measurement in Breeding Management of the Bitch

Accurate serum progesterone measurements for timing bitches during breeding management is critical for reproductive practice, especially as artificial insemination has become routine to facilitate breeding of animals that are geographically or temporally separated. To measure serum progesterone, chemiluminescent immunoassay (CLIA) has replaced radioimmunoassay as the current standard in the bitch due to its high correlation and increased practicality. In January 2019, a colorimetric point-of-care (POC) immunoassay for quantitative in-clinic canine serum progesterone measurements in <30 min was released. This study provides an independent comparison of the POC (Catalyst One, IDEXX) to the current industry standard, CLIA (Immulite-2000, Siemens). To assess inter-assay imprecision of POC and agreement of the POC and CLIA results, 100 canine serum samples were analyzed on three analyzers (POC-1, POC-2, and CLIA), of which, 74 (POC-1) and 75 (POC-2) results were within POCs' reportable range of 0.2–20 ng/mL and included in the study. To assess intra-assay imprecision, pooled canine serum samples at low (L1), intermediate (L2), and high (L3) progesterone concentrations were analyzed ten times each on POC-1 and CLIA. Relative to CLIA, POC values showed good correlation (POC-1, r2 = 0.9366; POC-2, r2 = 0.9438, P < 0.0001) and significant positive proportional bias at values >2 ng/mL. The POC inter-assay coefficients of variation (CVs) were 13.2% (0.2–2.9 ng/mL, 0.6–9.2 nmol/L, L1), 10.0% (3.0–9.9 ng/mL, 9.5–31.5 nmol/L, L2), 7.1% (10.0–20.0 ng/mL, 31.8–63.6 nmol/L, L3), and 11.2% (all samples). The intra-assay CVs for POC (L1, 15.3%; L2, 7.0%; L3, 4.7%) were higher than those for CLIA (L1, 5.89%; L2, 4.89%; L3, 3.44%). Based on the more rapid increase in serial serum progesterone concentrations in ovulating bitches and the greater imprecision of the POC, the clinical interpretations of serum progesterone measurements as they relate to canine breeding management should be made with caution.
Autism Spectrum Disorder Risk Factor Met Regulates the Organization of Inhibitory Synapses

A common hypothesis explains autism spectrum disorder (ASD) as a neurodevelopmental disorder linked to excitatory/inhibitory (E/I) imbalance in neuronal network connectivity. Mutation of genes including Met and downstream signaling components, e.g., PTEN, Tsc2 and, Rheb are involved in the control of synapse formation and stabilization and were all considered as risk genes for ASD. While the impact of Met on glutamatergic synapses was widely appreciated, its contribution to the stability of inhibitory, GABAergic synapses is poorly understood. The stabilization of GABAergic synapses depends on clustering of the postsynaptic scaffolding protein gephyrin. Here, we show in vivo and in vitro that Met is necessary and sufficient for the stabilization of GABAergic synapses via induction of gephyrin clustering. Likewise, we provide evidence for Met-dependent gephyrin clustering via activation of mTOR. Our results support the notion that deficient GABAergic signaling represents a pathomechanism for ASD.