bluuebunny/crossref_metadata_2025 huggingface dataset:
A genome wide CRISPR/Cas9 screen identifies calreticulin as a selective repressor of ATF6α
Authors: Tung Joanne , Huang Lei , George Ginto , Harding Heather P. , Ron David , Ordóñez Adriana
Published: February 2024

Activating transcription factor 6 (ATF6) is one of three endoplasmic reticulum (ER) transmembrane stress sensors that mediate the unfolded protein response (UPR). Despite its crucial role in long-term ER stress adaptation, regulation of ATF6 alpha (α) signalling remains poorly understood, possibly because its activation involves ER-to-Golgi and nuclear trafficking. Here, we generated an ATF6α/IRE1 dual UPR reporter CHO-K1 cell line and performed an unbiased genome-wide CRISPR/Cas9 mutagenesis screen to systematically profile genetic factors that specifically contribute to ATF6α signalling in the presence and absence of ER stress. The screen identified both anticipated and new candidate genes that regulate ATF6α activation. Among these, calreticulin (CRT), a key ER luminal chaperone, selectively repressed ATF6α signalling: Cells lacking CRT constitutively activated a BiP::sfGFP ATF6α-dependent reporter, had higher BiP levels and an increased rate of trafficking and processing of ATF6α. Purified CRT interacted with the luminal domain of ATF6αin vitroand the two proteins co-immunoprecipitated from cell lysates. CRT depletion exposed a negative feedback loop implicating ATF6α in repressing IRE1 activity basally and overexpression of CRT reversed this repression. Our findings indicate that CRT, beyond its known role as a chaperone, also serves as an ER repressor of ATF6α to selectively regulate one arm of the UPR.
Critical Differential Expression Assessment for Individual Bulk RNA-Seq Projects
Authors: Warden Charles D. , Wu Xiwei
Published: February 2024

Finding the right balance of quality and quantity can be important, and it is essential that project quality does not drop below the level where important main conclusions are missed or misstated. We use knock-out and over-expression studies as a simplification to test recovery of a known causal gene in RNA-Seq cell line experiments. When single-end RNA-Seq reads are aligned with STAR and quantified with htseq-count, we found potential value in testing the use of the Generalized Linear Model (GLM) implementation of edgeR with robust dispersion estimation more frequently for either single-variate or multi-variate 2-group comparisons (with the possibility of defining criteria less stringent than |fold-change| > 1.5 and FDR < 0.05). When considering a limited number of patient sample comparisons with larger sample size, there might be some decreased variability between methods (except for DESeq1). However, at the same time, the ranking of the gene identified using immunohistochemistry (for ER/PR/HER2 in breast cancer samples from The Cancer Genome Atlas) showed as possible shift in performance compared to the cell line comparisons, potentially highlighting utility for standard statistical tests and/or limma-based analysis with larger sample sizes. If this continues to be true in additional studies and comparisons, then that could be consistent with the possibility that it may be important to allocate time for potential methods troubleshooting for genomics projects.

Analysis of public data presented in this study does not consider all experimental designs, and presentation of downstream analysis is limited. So, any estimate from this simplification would be an underestimation of the true need for some methods testing for every project. Additionally, this set of independent cell line experiments has a limitation in being able to determine the frequency of missing a highly important gene if the problem is rare (such as 10% or lower). For example, if there was an assumption that only one method can be tested for “initial” analysis, then it is not completely clear to the extent that using edgeR-robust might perform better than DESeq2 in the cell line experiments.

Importantly, we do not wish to cause undue concern, and we believe that it should often be possible to define a gene expression differential expression workflow that is suitable for some purposes for many samples. Nevertheless, at the same time, we provide a variety of measures that we believe emphasize the need to critically assess every individual project and maximize confidence in published results.
Extracellular disposal of nuclear waste by APP: a protective mechanism impaired in Alzheimer’s disease
Authors: Dougnon Godfried , Otsuka Takayoshi , Nakamura Yuka , Sakai Akiko , Yamanaka Tomoyuki , Matsui Noriko , Nakahara Asa , Ito Ai , Hatano Atsushi , Matsumoto Masaki , Igarashi Hironaka , Kakita Akiyoshi , Ueno Masaki , Matsui Hideaki
Published: February 2024

Although the amyloid beta (Aβ) hypothesis1has long been central to Alzheimer’s disease (AD) research, effective therapeutic strategies remain elusive2,3. Here we re-evaluate the functions of amyloid precursor protein (APP) and reveal its critical function in protecting against nuclear impairment-induced cell death and inflammation4,5. Overexpression of APP mitigated etoposide or lamin A knockdown-induced nuclear damage, while APP removal or mutations exacerbated these effects. Interestingly, neurons differentiated from induced pluripotent stem cells (iPSCs) exhibited similar patterns, and notably, familial AD-associated mutant APP failed to confer protection against nuclear impairment. We identify APP’s interaction with a cytoplasmic structure of nuclear origin, termed “nuclear waste”, and propose its role in extracellular waste disposal. Intriguingly, cells lacking APP showed impaired nuclear waste clearance, leading to abnormal cytoplasmic accumulation of the nuclear waste. Similarly, neuron-specific APP overexpression using adeno-associated virus (AAV) in mice reduced neuronal death and inflammation caused by nuclear damage. Conversely, shRNA-mediated APP exacerbated these effects, and mutant APP associated with familial AD lacked protective effects. Moreover, postmortem analysis of AD brains revealed accumulation of abnormal nuclear waste in the neurocytoplasm, irregular nuclear morphology, and reduced APP levels per neuron. Our data underscore APP’s crucial role in disposing of nuclear waste, maintaining cellular homeostasis, and suggest its dysregulation as a potential contributor to AD pathogenesis. Restoring APP waste clearance in AD could be a promising target for disease-modifying therapies.
Conformational basis of subtype-specific allosteric control of NMDA receptor gating
Authors: Bleier Julia , Ribeiro Furtado de Mendonca Philipe , Habrian Chris , Stanley Cherise , Vyklicky Vojtech , Isacoff Ehud Y.
Published: February 2024

N-methyl-D-aspartate receptors are ionotropic glutamate receptors that are integral to synaptic transmission and plasticity. Variable GluN2 subunits in diheterotetrameric receptors with identical GluN1 subunits set very different functional properties, which support their individual physiological roles in the nervous system. To understand the conformational basis of this diversity, we assessed the conformation of the common GluN1 subunit in receptors with different GluN2 subunits using single-molecule fluorescence resonance energy transfer (smFRET). We established smFRET sensors in the ligand binding domain and modulatory amino-terminal domain to study an apo-like state and partially liganded activation intermediates, which have been elusive to structural analysis. Our results demonstrate a strong, subtype- specific influence of apo and glutamate-bound GluN2 subunits on GluN1 rearrangements, suggesting a conformational basis for the highly divergent levels of receptor activity, desensitization and agonist potency. Chimeric analysis reveals structural determinants that contribute to the subtype differences. Our study provides a framework for understanding GluN2-dependent functional properties and could open new avenues for subtype-specific modulation.
Aligning with the predecessors and counterarguments: A systematic review of the anatomical correlates for the newly discovered meningeal layer in the existing literature
Authors: Kumar Ashutosh , Kumar Rajesh , Narayan Ravi K. , Nath Banshi , Datusalia Ashok K. , Rastogi Ashok K. , Jha Rakesh K. , Kumar Pankaj , Pareek Vikas , Prasoon Pranav , Faiq Muneeb A. , Agrawal Prabhat , Prasad Surya Nandan , Kumari Chiman , Asghar Adil
Published: February 2024

A recent study reported the existence of a subarachnoid lymphatic-like membrane (SLYM) –an intermediate leptomeningeal layer between the arachnoid and pia mater in mice and human brains – dividing the subarachnoid space (SAS) into two functional compartments. Despite being a macroscopic structure, how it missed detection in previous studies is surprising. We systematically reviewed the published reports in animals and humans to explore whether prior descriptions of this meningeal layer have existed. An electronic search was conducted in PubMed/Medline, EMBASE, Google Scholar, Science Direct, and Web of Science databases using combinations of MeSH terms and keywords with Boolean operators from inception until 31stDec 2023. We found at least eight studies that provided structural evidence of an intermediate leptomeningeal layer in the brain or spinal cord. However, unequivocal descriptions for this layer all along the central nervous system were scarce. Obscured names were used to describe it, i.e., the epipial layer, intermediate meningeal layer, intermediate lamella, and outer pial layer. Its microscopic/ultrastructural details closely resembled the SLYM. Further, we examined the counterarguments in current literature that are skeptical of this layer’s existence. Considering the significant physiological/clinical implications, exploring further structural and functional details of the new meningeal layer is a need of the hour.
Zebrafish reveal new roles for Fam83f in hatching and the DNA damage-mediated autophagic response
Authors: Jones Rebecca A. , Cooper Fay , Kelly Gavin , Barry David , Renshaw Matthew J. , Sapkota Gopal , Smith James C.
Published: February 2024

The FAM83 (Family with sequence similarity83) family is highly conserved in vertebrates, but little is known of the functions of these proteins beyond their association with oncogenesis. Of the family, FAM83F is of particular interest because it is the only membrane-targeted FAM83 protein. When over-expressed, FAM83F activates the canonical Wnt signalling pathway and binds to and stabilizes p53; it therefore interacts with two pathways often dysregulated in disease. Insights into gene function can often be gained by studying the roles they play during development, and here we report the generation offam83fknock-out (KO) zebrafish, which we have used to study the role of Fam83f in vivo. We show that endogenousfam83fis most strongly expressed in the hatching gland of developing zebrafish embryos, and thatfam83fKO embryos hatch earlier than their wild-type (WT) counterparts, despite developing at a comparable rate. We also demonstrate thatfam83fKO embryos are more sensitive to ionizing radiation than WT embryos—an unexpected finding, bearing in mind the previously-reported ability of FAM83F to stabilize p53. Transcriptomic analysis shows that loss offam83fleads to downregulation of phosphatidylinositol-3-phosphate (PI(3)P) binding proteins and impairment of cellular degradation pathways, particularly autophagy, a crucial component of the DNA damage response. Finally, we show that Fam83f protein is itself targeted to the lysosome when over-expressed in HEK293T cells, and that this localization is dependent upon a C’ terminal signal sequence. The zebrafish lines we have generated suggest that Fam83f plays an important role in autophagic/lysosomal processes, resulting in dysregulated hatching and increased sensitivity to genotoxic stress in vivo.
Induction andin silicostaging of human gastruloids with neural tube, segmented somites & advanced cell types
Authors: Hamazaki Nobuhiko , Yang Wei , Kubo Connor , Qiu Chengxiang , Martin Beth K. , Garge Riddhiman K. , Regalado Samuel G. , Nichols Eva , Lee Choli , Daza Riza M. , Srivatsan Sanjay , Shendure Jay
Published: February 2024

Embryonic organoids are emerging as powerful models for studying early mammalian development. For example, stem cell-derived ‘gastruloids’ form elongating structures containing all three germ layers1–4. However, although elongated, human gastruloids do not morphologically resemble post-implantation embryos. Here we show that a specific, discontinuous regimen of retinoic acid (RA) robustly induces human gastruloids with embryo-like morphological structures, including a neural tube and segmented somites. Single cell RNA-seq (sc-RNA-seq) further reveals that these human ‘RA-gastruloids’ contain more advanced cell types than conventional gastruloids, including neural crest cells, renal progenitor cells, skeletal muscle cells, and, rarely, neural progenitor cells. We apply a new approach to computationally stage human RA-gastruloids relative to somite-resolved mouse embryos, early human embryos and other gastruloid models, and find that the developmental stage of human RA-gastruloids is comparable to that of E9.5 mouse embryos, although some cell types show greater or lesser progression. We chemically perturb WNT and BMP signaling in human RA-gastruloids and find that these signaling pathways regulate somite patterning and neural tube length, respectively, while genetic perturbation of the transcription factors PAX3 and TBX6 markedly compromises the formation of neural crest and somites/renal cells, respectively. Human RA-gastruloids complement other embryonic organoids in serving as a simple, robust and screenable model for decoding early human embryogenesis.
Structure-based design of a soluble human cytomegalovirus glycoprotein B antigen stabilized in a prefusion-like conformation
Authors: Sponholtz Madeline R. , Byrne Patrick O. , Lee Alison G. , Ramamohan Ajit R. , Goldsmith Jory A. , McCool Ryan S. , Zhou Ling , Johnson Nicole V. , Hsieh Ching-Lin , Connors Megan , Karthigeyan Krithika , Campbell John D. , Permar Sallie R. , Maynard Jennifer A. , Yu Dong , Bottomley Matthew J. , McLellan Jason S.
Published: February 2024

Human cytomegalovirus (HCMV) glycoprotein B (gB) is a class III membrane fusion protein required for viral entry. HCMV vaccine candidates containing gB have demonstrated moderate clinical efficacy, but no HCMV vaccine has been FDA-approved. Here, we used structure-based design to identify and characterize amino acid substitutions that stabilize gB in its metastable prefusion conformation. One variant containing two engineered interprotomer disulfide bonds and two cavity-filling substitutions (gB-C7), displayed increased expression and thermostability. A 2.8 Å resolution cryo-electron microscopy structure shows that gB-C7 adopts a prefusion-like conformation, revealing additional structural elements at the membrane-distal apex. Unlike previous observations for several class I viral fusion proteins, mice immunized with postfusion or prefusion-stabilized forms of soluble gB protein displayed similar neutralizing antibody titers, here specifically against an HCMV laboratory strain on fibroblasts. Collectively, these results identify initial strategies to stabilize class III viral fusion proteins and provide tools to probe gB-directed antibody responses.

Teaser

A structure-based design campaign leads to stabilization of the class III viral fusion protein from HCMV in a prefusion-like conformation.
Microtubule forces drive nuclear damage inLMNAcardiomyopathy
Authors: Amiad Pavlov Daria , Heffler Julie , Suay-Corredera Carmen , Dehghany Mohammad , Shen Kaitlyn M. , Zuela-Sopilniak Noam , Randell Rani , Uchida Keita , Jain Rajan , Shenoy Vivek , Lammerding Jan , Prosser Benjamin
Published: February 2024

Nuclear homeostasis requires a balance of forces between the cytoskeleton and nucleus. Mutations in theLMNAgene, which encodes the nuclear envelope proteins lamin A/C, disrupt this balance by weakening the nuclear lamina. This results in nuclear damage in contractile tissues and ultimately muscle disease. Intriguingly, disrupting the LINC complex that connects the cytoskeleton to the nucleus has emerged as a promising strategy to ameliorateLMNA-associated cardiomyopathy. Yet how LINC complex disruption protects the cardiomyocyte nucleus remains unclear. To address this, we developed an assay to quantify the coupling of cardiomyocyte contraction to nuclear deformation and interrogated its dependence on the nuclear lamina and LINC complex. We found that, surprisingly, the LINC complex was mostly dispensable for transferring contractile strain to the nucleus, and that increased nuclear strain in lamin A/C-deficient cardiomyocytes was not rescued by LINC complex disruption. Instead, LINC complex disruption eliminated the cage of microtubules encircling the nucleus. Disrupting microtubules was sufficient to prevent nuclear damage and rescue cardiac function induced by lamin A/C deficiency. We computationally simulated the stress fields surrounding cardiomyocyte nuclei and show how microtubule forces generate local vulnerabilities that damage lamin A/C-deficient nuclei. Our work pinpoints localized, microtubule-dependent force transmission through the LINC complex as a pathological driver and therapeutic target forLMNA-cardiomyopathy.

Graphical abstract
Characterisation of RNA editing and gene therapy with a compact CRISPR-Cas13 in the retina
Authors: Kumar Satheesh , Hsiao Yi-Wen , Wong Vickie H Y , Aubin Deborah , Wang Jiang-Hui , Lisowski Leszek , Rakoczy Elizabeth P , Li Fan , Alarcon-Martinez Luis , Gonzalez-Cordero Anai , Bui Bang V , Liu Guei-Sheung
Published: February 2024

CRISPR-Cas13 nucleases are programmable RNA-targeting effectors that can silence gene expression in a reversible manner. Recent iterations of Cas13 nucleases are compact for adeno-associated virus (AAV) delivery to achieve strong and persistent expression in various organs in a safe manner. Here, we report significant transcriptomic signatures of Cas13bt3 expression in retinal cells and show all-in-one AAV gene therapy with Cas13bt3 can effectively silenceVEGFAmRNA in human retinal organoids and humanisedVEGFtransgenic mouse (trVEGF029, Kimba) models. Specifically, human embryonic stem cells (hESC)-derived retinal pigment epithelium cells show high expression of Cas13bt3 from virus delivery corresponding to a significant reduction ofVEGFAmRNA. We further show that intravitreal delivery of Cas13bt3 can transduce mouse retinal cells efficiently, reaching the photoreceptors for specific knockdown of humanVEGFAin the Kimba mouse. Our results reveal important considerations for assessing Cas13 activity and establish Cas13bt3 as a potential anti-VEGF agent that can achieve long-term control of VEGFA for the treatment of retinal neovascularization.
A Predictive Model: The Key to Success for Badminton Servers and Receivers
Authors: Liu Cheng
Published: February 2024

To explore the influence of technical actions on badminton competition outcomes, this study analyzes the frequency and impact of specific movements made by players, distinguishing between the server and receiver roles. Focusing on international competitions from 2019 to 2023, we collected data on 23 distinct technical actions (e.g., Net Front, Slice/Drop, Push) to construct a predictive model. The study distinguishes itself by employing a Random Forest algorithm to ascertain the significance of each technical action, utilizing forward stepwise selection and 5-fold cross-validation for feature refinement. SHAP value analysis further validated the pivotal roles of ‘Net Front’, ‘Slice/Drop’, and ‘Push’ across both sexes, linking higher frequencies of ‘Net Front’ with increased match-winning probabilities. Model validation on a test set demonstrated effective performance in both sexes, with the model based on male data exhibiting higher accuracy and predictive values, surpassing the performance of the female data model. This comprehensive examination, grounded in quantitative analysis, not only enhances our understanding of badminton gameplay dynamics but also offers valuable insights for coaching strategies and training methodologies.
Identification Drug Targets for Oxaliplatin-Induced Cardiotoxicity without Affecting Cancer Treatment through Inter Variability Cross-Correlation Analysis (IVCCA)
Authors: Du Junwei , Sudlow Leland C. , Biswas Hridoy , Mitchell Joshua D. , Mollah Shamim , Berezin Mikhail Y.
Published: February 2024

The successful treatment of side effects of chemotherapy faces two major limitations: the need to avoid interfering with pathways essential for the cancer-destroying effects of the chemotherapy drug, and the need to avoid helping tumor progression through cancer promoting cellular pathways. To address these questions and identify new pathways and targets that satisfy these limitations, we have developed the bioinformatics tool Inter Variability Cross-Correlation Analysis (IVCCA). This tool calculates the cross-correlation of differentially expressed genes, analyzes their clusters, and compares them across a vast number of known pathways to identify the most relevant target(s). To demonstrate the utility of IVCCA, we applied this platform to RNA-seq data obtained from the hearts of the animal models with oxaliplatin-induced CTX. RNA-seq of the heart tissue from oxaliplatin treated mice identified 1744 differentially expressed genes with False Discovery Rate (FDR) less than 0.05 and fold change above 1.5 across nine samples. We compared the results against traditional gene enrichment analysis methods, revealing that IVCCA identified additional pathways potentially involved in CTX beyond those detected by conventional approaches. The newly identified pathways such as energy metabolism and several others represent promising target for therapeutic intervention against CTX, while preserving the efficacy of the chemotherapy treatment and avoiding tumor proliferation. Targeting these pathways is expected to mitigate the damaging effects of chemotherapy on cardiac tissues and improve patient outcomes by reducing the incidence of heart failure and other cardiovascular complications, ultimately enabling patients to complete their full course of chemotherapy with improved quality of life and survival rates.
An Organism-Level Quantitative Flux Model of Energy Metabolism in Mice
Authors: Yuan Bo , Doxsey Will , Tok Özlem , Kwon Young Yon , Inouye Karen E. , Hotamışlıgil Gökhan S. , Hui Sheng
Published: February 2024

Mammalian tissues feed on nutrients in the blood circulation. At the organism-level, mammalian energy metabolism comprises of oxidation, storage, interconverting, and releasing of circulating nutrients. Though much is known about the individual processes and nutrients, a holistic and quantitative model describing these processes for all major circulating nutrients is lacking. Here, by integrating isotope tracer infusion, mass spectrometry, and isotope gas analyzer measurement, we developed a framework to systematically quantify fluxes through these metabolic processes for 10 major circulating energy nutrients in mice, resulting in an organism-level quantitative flux model of energy metabolism. This model revealed in wildtype mice that circulating nutrients have more dominant metabolic cycling fluxes than their oxidation fluxes, with distinct partition between cycling and oxidation flux for individual circulating nutrients. Applications of this framework in obese mouse models showed on a per animal basis extensive elevation of metabolic cycling fluxes in ob/ob mice, but not in diet-induced obese mice. Thus, our framework describes quantitatively the functioning of energy metabolism at the organism-level, valuable for revealing new features of energy metabolism in physiological and disease conditions.

Highlights

A flux model of energy metabolism integrating13C labeling of metabolites and CO2

Circulating nutrients have characteristic partition between oxidation and storage

Circulating nutrients’ total cycling flux outweighs their total oxidation flux

Cycling fluxes are extensively elevated in ob/ob but not in diet-induced obese mice

Graphical Abstract
Single molecule imaging of the central dogma reveals myosin-2A gene expression is regulated by contextual translational buffering
Authors: Wiggan O'Neil , Stasevich Timothy J
Published: February 2024

While protein homeostasis is a hallmark of gene regulation, unraveling the hidden regulatory mechanisms that maintain homeostasis is difficult using traditional methods. To confront this problem, we CRISPR engineered a human cell line with multiple tags in the endogenous MYH9 gene, which encodes the essential and ubiquitous myosin-2A cytoskeletal motor. Using these cells, we imaged MYH9 transcription, translation, and mature mRNA and protein in distinct colors, enabling a full dissection of the central dogma. Our data show that MYH9 transcription is upregulated in an SRF-dependent manner in response to cytoskeletal cues and that MYH9 translation can either buffer or match the transcriptional response depending on context. Upon knockdown of actin-depolymerizing proteins like cofilin, translation efficiency drops by a factor of two to buffer strong transcriptional upregulation, likely to help prevent excessive myosin activity. In contrast, following serum stimulation, translation matches the transcriptional response to readily reestablish steady state. Our results identify contextual translational buffering as an important regulatory mechanism driving stable MYH9 expression. They also demonstrate the power and broad applicability of our cell line, which can now be used to accurately quantify central dogma dynamics in response to diverse forms of cellular perturbations.
Whole-Plant Physiological Identification and Quantification of Disease Progression
Authors: Friedman Shani , Dalal Ahan , Batat Dor , Burdman Saul , Sela Yheonatan , Hipsch Matanel , Rosenwaser Shilo , Hadad Evgeniya Marcos , Covo Shay , Moshelion Menachem
Published: February 2024

Visual estimates of plant symptoms are traditionally used to quantify disease severity. Yet, the methodologies used to assess these phenotypes are often subjective and do not allow for tracking the disease’s progression from very early stages. Here, we hypothesized that quantitative analysis of whole-plant physiological vital functions can be used to objectively determine plant health, providing a more sensitive way to detect disease.

We studied the tomato wilt that is caused byFusarium oxysporumf. sp.lycopersici(Fol). Physiological performance of infected and non-infected tomato plants was compared using a whole-plant lysimeter functional-phenotyping system. Water-balance traits of the plants were measured continuously and simultaneously in a quantitative manner.

Infected plants exhibited early reductions in transpiration and biomass gain, which preceded visual disease symptoms. These changes in transpiration proved to be effective quantitative indicators for assessing both the plant’s susceptibility to infection and the virulence of the fungus. Physiological changes linked to fungal outgrowth and toxin release contributed to reduced hydraulic conductance during initial infection stages.

The functional-phenotyping method objectively captures early-stage disease progression, advancing plant disease research and management. This approach emphasizes the potential of quantitative whole-plant physiological analysis over traditional visual estimates for understanding and detecting plant diseases.
Functional dynamics and selectivity of two parallel corticocortical pathways from motor cortex to layer 5 circuits in somatosensory cortex
Authors: Kim Hye-Hyun , Bonekamp Kelly E. , Gillie Grant R. , Autio Dawn M. , Keller Tryton , Crandall Shane R.
Published: February 2024

In the rodent whisker system, active sensing and sensorimotor integration are mediated in part by the dynamic interactions between the motor cortex (M1) and somatosensory cortex (S1). However, understanding these dynamic interactions requires knowledge about the synapses and how specific neurons respond to their input. Here, we combined optogenetics, retrograde labeling, and electrophysiology to characterize the synaptic connections between M1 and layer 5 (L5) intratelencephalic (IT) and pyramidal tract (PT) neurons in S1 of mice (both sexes). We found that M1 synapses onto IT cells displayed modest short-term depression, whereas synapses onto PT neurons showed robust short-term facilitation. Despite M1 inputs to IT cells depressing, their slower kinetics resulted in summation and a response that increased during short trains. In contrast, summation was minimal in PT neurons due to the fast time course of their M1 responses. The functional consequences of this reduced summation, however, were outweighed by the strong facilitation at these M1 synapses, resulting in larger response amplitudes in PT neurons than IT cells during repetitive stimulation. To understand the impact of facilitating M1 inputs on PT output, we paired trains of inputs with single backpropagating action potentials, finding that repetitive M1 activation increased the probability of bursts in PT cells without impacting the time-dependence of this coupling. Thus, there are two parallel but dynamically distinct systems of M1 synaptic excitation in L5 of S1, each defined by the short-term dynamics of its synapses, the class of postsynaptic neurons, and how the neurons respond to those inputs.

SIGNIFICANCE STATEMENT

Normal sensorimotor integration depends in part on the dynamic interactions between the primary motor cortex and the somatosensory cortex, but the functional properties of the excitatory synapses interconnecting the motor cortex with the somatosensory cortex are poorly understood. Our results show that the short-term dynamics of excitatory motor cortex synapses and the nature of the postsynaptic response they generate onto layer 5 pyramidal neurons in the somatosensory cortex depend on the postsynaptic cell type and if their axons project to other cortical areas or subcortical regions. These two parallel but dynamically distinct channels of synaptic excitation constitute previously unknown synaptic circuits by which different temporal patterns of motor cortex activity can shape how signals propagate out of the somatosensory cortex.
Whole-Genome Sequencing of aJanthinobacterium sp. Isolated from the Patagonian Desert
Authors: Cavanaugh Nicole T , Kumar Girish , Reverdy Pearson Alicyn , Colbert Julia , Riquelme Carlos , Hudson André O , Chai Yunrong , Godoy-Carter Veronica
Published: February 2024

Janthinobacteriumis a genus of Gram-negative environmental bacteria that survive extreme conditions by forming biofilms and producing pigments.Janthinobacteriumsp. LS2A, an extremophile isolated from soil in the Chilean Patagonia, contains seven known biosynthetic gene clusters, including the purple pigment violacein, which may aid in its survival in harsh environments.
HiCrayon reveals distinct layers of multi-state 3D chromatin organization
Authors: Nolan Ben , Harris Hannah L. , Kalluchi Achyuth , Reznicek Timothy E. , Cummings Christopher T. , Rowley M. Jordan
Published: February 2024

The co-visualization of chromatin conformation with 1D ‘omics data is key to the multi-omics driven data analysis of 3D genome organization. Chromatin contact maps are often shown as 2D heatmaps and visually compared to 1D genomic data by simple juxtaposition. While common, this strategy is imprecise, placing the onus on the reader to align features with each other. To remedy this, we developed HiCrayon, an interactive tool that facilitates the integration of 3D chromatin organization maps and 1D datasets. This visualization method integrates data from genomic assays directly into the chromatin contact map by coloring interactions according to 1D signal. HiCrayon is implemented using R shiny and python to create a graphical user interface (GUI) application, available in both web or containerized format to promote accessibility. HiCrayon is implemented in R, and includes a graphical user interface (GUI), as well as a slimmed-down web-based version that lets users quickly produce publication-ready images. We demonstrate the utility of HiCrayon in visualizing the effectiveness of compartment calling and the relationship between ChIP-seq and various features of chromatin organization. We also demonstrate the improved visualization of other 3D genomic phenomena, such as differences between loops associated with CTCF/cohesin vs. those associated with H3K27ac. We then demonstrate HiCrayon’s visualization of organizational changes that occur during differentiation and use HiCrayon to detect compartment patterns that cannot be assigned to either A or B compartments, revealing a distinct 3rd chromatin compartment. Overall, we demonstrate the utility of co-visualizing 2D chromatin conformation with 1D genomic signals within the same matrix to reveal fundamental aspects of genome organization.

Local version:https://github.com/JRowleyLab/HiCrayon

Web version:https://jrowleylab.com/HiCrayon
Prevalence and Associated Risk Factors of Bovine Trypanosomosis in Diga District, Western Ethiopia
Authors: Gessese Abebe Tesfaye , Yadesa Gemechis
Published: February 2024

Trypanosomosis is a protozoal disease caused by different species of unicellular parasites found in the blood and other tissues of vertebrates including livestock, wildlife, and people. It is the most serious animal production problem in sub-Saharan Africa including Ethiopia. The present study was conducted to assess the prevalence of bovine trypanosomosis and prevailing species of Tyrpanosomes in Diga district and to identify host related risk factors for trypanosomosis prevalence in Western Ethiopia. Blood samples were collected from 384 randomly selected indigenous cattle (Bos indicus) and analyzed using parasitological and hematological techniques. Out of the 384 animals examined 22 (5.73%) were found trypanosomosis positive. Of which 11 (2.87%) were infected withT. vivax, 9 (2.34%) withT. congolenseand 2 (0.52%) with mixed infection ofT. vivaxandT. congolense. This difference in trypanosomes species prevalence was not statistically significant (P>0.05). The association of prevalence with the various risk factors including kebele, age, sex, body condition and coat color were measured. There was a significant variation in the prevalence of trypanosomosis (P<0.05) among cattles with different body conditions, poor body conditioned animals with the highest prevalence followed by medium and good body condition animals, respectively. There was no any statistically significant difference in other variables considered. The overall Packed Cell Volume (PCV) mean PCV value of 25.67 was recorded during the study period. The mean PCV value of infected animals was lower (20.18) than that of non-infected animals (26.0). The difference was statistically significant (P<0.05). The result of the present study indicated the prevailing occurrence of trypanosomosis in the study area necessitating integrated control measures.
Cytotoxicity and resistance evolution of a novel antifungal carbon nanoparticle
Authors: Poudel Sharma Sijan , Paudyal Suraj , Domena Justin , Zhou Yiqun , Cleven Elliot , Agatemor Christian , Van Dyken J. David , Leblanc Roger
Published: February 2024

Antifungal drug resistance is a major problem in healthcare and agriculture. Synthesizing new drugs is one of the major mitigating strategies for overcoming this problem. In this context, carbon-dots (CDs) are a newer category of nanoparticles that have wide applications, potentially including use as antibiotics. However, there is a lack of understanding of the effect of long-term use of CDs as antimicrobials, particularly the ability of microbes to evolve resistance to antibiotic CDs. In this study, we synthesized novel florescent the bottom-up method using two antifungal drugs fluconazole and nourseothricin sulphate (ClonNAT). We first extensively characterized the physical properties of the newly synthesized carbon dots, Flu-Clo CDs. We measured the cytotoxicity of Flu-Clo CDs on budding yeastSaccharomyces cerevisiaeand determined that it had comparable antifungal inhibition with extensively used drug fluconazole. Furthermore, we demonstrate that Flu-CLO CDs are not cytotoxic to human fibroblasts cell lines. Then, we quantified the ability of yeast to evolve resistance to Flu-Clo CDs. We evolved replicate laboratory yeast populations for 250 generations in the presence of Flu-Clo CDs or aqueous fluconazole. We found that yeast evolved resistance to Flu-Clo CDs and aqueous fluconazole at similar rates. Further, we found that resistance to Flu-Clo CDs conferred cross-resistance to aqueous fluconazole. Overall, the results demonstrate the efficacy of CDs as potential antifungal drugs. We can conclude that yeast populations can adapt quickly to novel antibiotics including CD based antibiotics, including CD-based antibiotics indicating the importance of proper use of antimicrobials in combating infections.
Cell-autonomous GP130 activation suppresses prostate cancer development via STAT3/ARF/p53-driven senescence and confers an immune-active tumor microenvironment
Authors: Sternberg Christina , Limberger Tanja , Raigel Martin , Trachtová Karolína , Schlederer Michaela , Lindner Desiree , Kodajova Petra , Yang Jiaye , Ziegler Roman , Neubauer Heidi A. , Dey Saptaswa , Redmer Torben , Stoiber Stefan , Hejret Václav , Tichy Boris , Tomberger Martina , Harbusch Nora S. , Tangermann Simone , Oberhuber Monika , Bystry Vojtech , Persson Jenny L. , Pospisilova Sarka , Wolf Peter , Sternberg Felix , Högler Sandra , Lagger Sabine , Rose-John Stefan , Kenner Lukas
Published: February 2024

Prostate cancer ranks as the second most frequently diagnosed cancer in men worldwide. Recent research highlights the crucial roles GP130-mediated signaling pathways play in the development and progression of various cancers, particularly through hyperactivated STAT3 signaling. Here, we find that genetic cell-autonomous activation of the GP130 receptor in prostate epithelial cells triggers active STAT3 signaling and significantly reduces tumor growthin vivo. Mechanistically, genetic activation of GP130 signaling mediates senescence via the STAT3/ARF/p53 axis and anti-tumor immunity via recruitment of cytotoxic T-cells, ultimately impeding tumor progression. In prostate cancer patients, highGP130mRNA expression levels correlate with better recurrence-free survival, increased senescence signals and a transition from an immune-cold to an immune-hot tumor. Our findings reveal a context-dependent role of GP130/STAT3 in carcinogenesis and a tumor-suppressive function in prostate cancer development. We challenge the prevailing concept of blocking GP130/STAT3 signaling as functional prostate cancer treatment and instead propose cell-autonomous GP130 activation as a novel therapeutic strategy.
Scalable nonparametric clustering with unified marker gene selection for single-cell RNA-seq data
Authors: Nwizu Chibuikem , Hughes Madeline , Ramseier Michelle L. , Navia Andrew W. , Shalek Alex K. , Fusi Nicolo , Raghavan Srivatsan , Winter Peter S. , Amini Ava P. , Crawford Lorin
Published: February 2024

Clustering is commonly used in single-cell RNA-sequencing (scRNA-seq) pipelines to characterize cellular heterogeneity. However, current methods face two main limitations. First, they require user-specified heuristics which add time and complexity to bioinformatic workflows; second, they rely on post-selective differential expression analyses to identify marker genes driving cluster differences, which has been shown to be subject to inflated false discovery rates. We address these challenges by introducing nonparametric clustering of single-cell populations (NCLUSION): an infinite mixture model that leverages Bayesian sparse priors to identify marker genes while simultaneously performing clustering on single-cell expression data. NCLUSION uses a scalable variational inference algorithm to perform these analyses on datasets with up to millions of cells. By analyzing publicly available scRNA-seq studies, we demonstrate that NCLUSION (i) matches the performance of other state-of-the-art clustering techniques with significantly reduced runtime and (ii) provides statistically robust and biologically relevant transcriptomic signatures for each of the clusters it identifies. Overall, NCLUSION represents a reliable hypothesis-generating tool for understanding patterns of expression variation present in single-cell populations.
Hemilineage-specific expression of the RHG genesGrimandReapersculpt neural network composition duringDrosophilaneurogenesis
Authors: Sproston Connor J , Rak Julia E , Kondo Shu , Williams Darren W
Published: February 2024

During development, nervous systems generate an incredible diversity of different cell types. One mechanism that appears critical for controlling whether a specific subtype is present or absent, and ensuring that neuronal numbers are appropriate for specific regions, is programmed cell death.

Here we show that the precisely patterned hemilineage-based cell death found in the ventral nerve cord ofDrosophila melanogasteris controlled by the transcription of the pro-apoptotic RHG genesreaperandgrim.

Using smFISH we show thatreaperandgrim, but nothid, are expressed in postembryonic lineages during neurogenesis. By generating novel T2A-GAL4 knock-in tools we have been able to mapreaperandgrimexpression on a lineage-by-lineage basis and show diverse expression patterns within different hemilineage populations. Analysis of null mutants for both genes reveal a ‘division of labour’ with distinct combinations being important in sculpting the nervous system.

Investigation of a specific sexually dimorphic lineage reveals temporal patterning of hemilineage-specific programmed cell death through Grim and Reaper function underpins the development of sex-specific circuitry in the VNC.

Our findings suggest that the precisely patterned expression of these pro-apoptotic genes is a critical fate determinant that generates segmental-specific circuity and instructs the appropriate assembly of sexually dimorphic neural circuits in the VNC.
Single-cell and Spatial Transcriptomics Identified Fatty Acid-binding Proteins Controlling Endothelial Glycolytic and Arterial Programming in Pulmonary Hypertension
Authors: Liu Bin , Yi Dan , Li Shuai , Ramirez Karina , Xia Xiaomei , Cao Yanhong , Zhao Hanqiu , Tripathi Ankit , Qiu Shenfeng , Kala Mrinalini , Rafikov Ruslan , Gu Haiwei , Perez Vinicio de jesus , Lemay Sarah-Eve , Glembotski Christopher C. , Knox Kenneth S , Bonnet Sebastien , Kalinichenko Vladimir V. , Zhao You-Yang , Fallon Michael B. , Boucherat Olivier , Dai Zhiyu
Published: February 2024

Pulmonary arterial hypertension (PAH) is a devastating disease characterized by obliterative vascular remodeling and persistent increase of vascular resistance, leading to right heart failure and premature death. Understanding the cellular and molecular mechanisms will help develop novel therapeutic approaches for PAH patients. Single-cell RNA sequencing (scRNAseq) analysis found that both FABP4 and FABP5 were highly induced in endothelial cells (ECs) ofEgln1Tie2Cre(CKO) mice, which was also observed in pulmonary arterial ECs (PAECs) from idiopathic PAH (IPAH) patients, and in whole lungs of pulmonary hypertension (PH) rats. Plasma levels of FABP4/5 were upregulated in IPAH patients and directly correlated with severity of hemodynamics and biochemical parameters using plasma proteome analysis. Genetic deletion of bothFabp4and 5 in CKO mice (Egln1Tie2Cre/Fabp4-5-/-, TKO) caused a reduction of right ventricular systolic pressure (RVSP) and RV hypertrophy, attenuated pulmonary vascular remodeling and prevented the right heart failure assessed by echocardiography, hemodynamic and histological analysis. Employing bulk RNA-seq and scRNA-seq, and spatial transcriptomic analysis, we showed thatFabp4/5deletion also inhibited EC glycolysis and distal arterial programming, reduced ROS and HIF-2α expression in PH lungs. Thus, PH causes aberrant expression of FABP4/5 in pulmonary ECs which leads to enhanced ECs glycolysis and distal arterial programming, contributing to the accumulation of arterial ECs and vascular remodeling and exacerbating the disease.
SLC-25A46 Regulates Mitochondrial Fusion through FZO-1/Mitofusin and is Essential for Maintaining Neuronal Morphology
Authors: Obinata Hiroyuki , Takahashi Hironori , Shimo Satoshi , Oda Toshiyuki , Sugimoto Asako , Niwa Shinsuke
Published: February 2024

Mitochondria are dynamic organelles shaped by sequential fission and fusion events. The mitochondrial protein SLC25A46 has been identified as a causative gene for mitochondrial neuropathies. However, the function of SLC25A46 in mitochondrial morphogenesis remains controversial, with several reports suggesting it acts as a mitochondrial fission factor, while others propose it as a fusion factor. In this study, employing forward genetics, we identifiedslc-25A46, aCaenorhabditis elegansorthologue of human SLC25A46, as an essential factor for mitochondrial fusion. Suppressor mutagenesis screening revealed loss-of-function mutations indrp-1, a mitochondrial fission factor, as suppressors ofslc-25A46. The phenotype ofslc-25A46mutants is similar to those offzo-1mutants, wherein the mitochondrial fusion factor Mitofusin is disrupted. Overexpressing FZO-1/Mitofusin mitigated mitochondrial defects inslc-25a46mutants, indicating SLC-25A46 promotes fusion through FZO-1/Mitofusin. Disease model worms carrying mutations associated with SLC25A46 exhibited mitochondrial fragmentation and accelerated neurodegeneration, suggestingslc-25A46maintains neuronal morphology through mitochondrial fusion regulation.
Oncogenic STIL-mediated loss of BRCA1 functionality causes DNA damage and centrosome amplification
Authors: Sanghi Srishti , Singh Priyanka
Published: February 2024

DNA damages increase centrosome number, a typical phenotype in many cancers. However, the molecular linkers between the two pathways remain elusive. STIL (SCL/TAL1 Interrupting locus) is a core centrosome-organizing protein overexpressed in different cancer types. This work shows that STIL forms a complex with the DNA damage response protein, BRCA1, and is required for regulating its protein stability. Subsequently, STIL depletion resulting in BRCA1 loss from the nucleus causes centrosome amplification and DNA damage. We also identified a heterozygous missense mutation (S76L) in STIL from the pan-cancer datasets, which does not affect total BRCA1 protein levels. However, it reduces BRCA1 nuclear localization with enhanced centrosome recruitment simultaneously at the S-phase. The enhanced BRCA1 at centrosome causes an increase in Aurora-A kinase levels at centrosomes, thus culminating in DNA damage and centrosome amplification. The expression of exogenous BRCA1 can push back this imbalance of the STIL-BRCA1 axis in oncogenic conditions. The amplified centrosomes in oncogenic STIL condition cluster in the M phase and cause pseudo-bipolar spindle organization. We could decluster them by the HSET(KIFC1)-specific inhibitor, CW069, suggesting a promising target for anti-cancer treatment.