bluuebunny/crossref_metadata_2025 huggingface dataset:
Contributions of adaptation and purifying selection to SARS-CoV-2 evolution
Authors: Neher Richard A
Published: December 2022

Continued evolution and adaptation of SARS-CoV-2 has led to more transmissible and immune-evasive variants with profound impacts on the course of the pandemic. Here I analyze the evolution of the virus over 2.5 years since its emergence and estimate the rates of evolution for synonymous and non-synonymous changes separately for evolution within clades—well-defined monophyletic groups with gradual evolution—and for the pandemic overall. The rate of synonymous mutation is found to be around 6 changes per year. Synonymous rates within variants vary little from variant to variant and are compatible with the overall rate of 7 changes per year (or $7.5 \times 10^{-4}$ per year and codon). In contrast, the rate at which variants accumulate amino acid changes (non-synonymous mutations) was initially around 12-16 changes per year, but in 2021 and 2022 it dropped to 6-9 changes per year. The overall rate of non-synonymous evolution, that is across variants, is estimated to be about 26 amino acid changes per year (or $2.7 \times 10^{-3}$ per year and codon). This strong acceleration of the overall rate compared to within clade evolution indicates that the evolutionary process that gave rise to the different variants is qualitatively different from that in typical transmission chains and likely dominated by adaptive evolution. I further quantify the spectrum of mutations and purifying selection in different SARS-CoV-2 proteins and show that the massive global sampling of SARS-CoV-2 is sufficient to estimate site-specific fitness costs across the entire genome. Many accessory proteins evolve under limited evolutionary constraints with little short-term purifying selection. About half of the mutations in other proteins are strongly deleterious.
Emergent dynamics of underlying regulatory network links EMT and androgen receptor-dependent resistance in prostate cancer
Authors: Jindal Rashi , Nanda Abheepsa , Pillai Maalavika , Ware Kathryn E , Singh Divyoj , Sehgal Manas , Armstrong Andrew J. , Somarelli Jason A , Jolly Mohit Kumar
Published: December 2022

Advanced prostate cancer patients initially respond to hormone therapy, be it in the form of androgen deprivation therapy or second-generation hormone therapies, such as abiraterone acetate or enzalutamide. However, most men with prostate cancer eventually develop hormone therapy resistance. This resistance emerges in several ways, such as through genetic mutations, epigenetic mechanisms, or through non-genetic pathways, such as lineage plasticity along epithelial-mesenchymal or neuroendocrine-like axes. These mechanisms of hormone therapy resistance often co-exist within a single patient’s tumor and can overlap within a single cell. There exists a growing need to better understand how phenotypic heterogeneity and plasticity results from emergent dynamics of the regulatory networks governing androgen independence. Here, we investigated the dynamics of a regulatory network connecting the drivers of androgen receptor (AR) splice variant-mediated androgen independence and those of epithelial-mesenchymal transition. Model simulations for this network revealed four possible phenotypes: epithelial-sensitive (ES), epithelial-resistant (ER), mesenchymal-resistant (MR) and mesenchymal-sensitive (MS), with the latter phenotype occurring rarely. We observed that well-coordinated “teams” of regulators working antagonistically within the network enable these phenotypes. These model predictions are supported by multiple transcriptomic datasets both at single-cell and bulk levels, includingin vitroEMT induction models and clinical samples. Further, our simulations reveal spontaneous stochastic switching between the ES and MR states. Addition of the immune checkpoint molecule, PD-L1, to the network was able to capture the interactions between AR, PD-L1, and the mesenchymal marker SNAIL, which was also confirmed through quantitative experiments. This systems-level understanding of the driver of androgen independence and EMT could aid in understanding non-genetic transitions and progression of such cancers and help in identifying novel therapeutic strategies or targets.
Plastid double-strand RNA transgenes trigger small RNA-based gene silencing of nuclear-encoded genes
Authors: Bélanger Sébastien , Kramer Marianne C. , Payne Hayden A. , Slotkin R. Keith , Meyers Blake C. , Staub Jeffrey M.
Published: December 2022

Plastid transformation technology has been widely used to express traits of potential commercial importance, though the technology has been limited to traits that function while sequestered in the organelle. Prior research indicates that plastid contents can escape from the organelle, suggesting a possible mechanism for engineering plastid transgenes to function in other cellular locations. To test this hypothesis, we created tobacco plastid transformants that express a fragment of the nuclear-encodedPhytoene desaturase(PDS) gene capable of catalyzing post-transcriptional gene silencing if RNA escape to the cytoplasm occurs. We found multiple lines of direct evidence that plastid-encodedPDStransgenes affect nuclearPDSgene silencing: knockdown of the nuclear-encodedPDSmRNA and/or its apparent translational inhibition, biogenesis of 21-nucleotide (nt) phased small interfering RNAs (phasiRNAs), and pigment deficient plants. Furthermore, plastid-expressed double-stranded RNA (dsRNA) with no cognate nuclear-encoded pairing partner also produced abundant 21-nt phasiRNAs in the cytoplasm, demonstrating that a nuclear-encoded template is not required for siRNA biogenesis. Our results indicate that RNA escape from plastids to the cytoplasm occurs broadly, with functional consequences that include entry into the gene silencing pathway. Furthermore, we uncover a method to produce plastid-encoded traits with functions outside of the organelle and open new fields of study in plastid development, compartmentalization and small RNA biogenesis.
Novel Peptide Inhibitor of Human Tumor Necrosis Factor-α has Antiarthritic Activity
Authors: Sahu Debasis , Gupta Charu , Yennamalli Ragothaman , Sharma Shikha , Roy Saugata , Hasan Sadaf , Gupta Pawan , Sharma Vishnu Kumar , Kashyap Sujit , Kumar Santosh , Dwivedi Ved Prakash , Panda Amulya Kumar , Das Hasi Rani , Liu Chuan-Ju
Published: December 2022

The inhibition of tumor necrosis factor-α (TNFα) trimer formation renders it inactive for binding to its receptors thus mitigating the vicious cycle of inflammation. We designed a peptide (PIYLGGVFQ) that simulates a sequence strand of human TNFα monomer using a series ofin silicomethods, such as active site finding (Acsite), protein-protein interaction (PPI), docking studies (GOLD and Modeller) followed by molecular dynamics (MD) simulation studies. The MD studies confirmed the intermolecular interaction of the peptide with the TNFα. Fluorescence-activated cell sorting (FACS) and fluorescence microscopy revealed that the peptide effectively inhibited the binding of TNF to the cell surface receptors. The cell culture assays showed that the peptide significantly inhibited the TNFα-mediated cell death. In addition, the nuclear translocation of the nuclear factor kappa B (NFκB) was significantly suppressed in the peptide-treated A549 cells as observed in immunofluorescence and gel mobility-shift assays. Furthermore, peptide protected against joint damage in collagen-induced arthritis (CIA) mouse model as revealed in the microfocal-CT scans. In conclusion, this TNFα antagonist would be useful for the prevention and repair of inflammatory bone destruction and subsequent loss in the mouse model of CIA as well as human rheumatoid arthritis (RA) patients. This calls upon further clinical investigation to utilize its potential effect as an anti-arthritic drug.
Predicting the evolution of lung squamous cell carcinoma in situ using deep learning
Authors: Vigdorovits Alon , Olteanu Gheorghe-Emilian , Tica Ovidiu , Boros Monica , Pop Ovidiu
Published: December 2022

Lung squamous cell carcinomain situ(SCIS) is the pre-invasive precursor lesion of lung squamous cell carcinoma (SCC). Only half of these lesions progress to invasive cancer, while a third undergo spontaneous regression. The ability to predict the evolution of SCIS lesions can significantly impact the management of lung cancer patients.

Here, we present the use of the deep learning (DL) approach in order to predict the progression of SCIS. The dataset consisted of 112 H&E stained whole slide images (WSI) that were obtained from the Image Data Resource public repository. The data set corresponded to tumors of patients who underwent biopsies of SCIS lesions and were subsequently followed up by bronchoscopy and CT to monitor for progression to SCC. We show that a deep convolutional neural network (DCNN) can predict if a SCIS lesion will progress to SCC. The model achieved a per-tile AUC of 0.78 (SD = 0.01) on the test set, an F1 score of 0.84 (SD = 0.05), and a sensitivity of 0.94 (SD = 0.01). Class activation maps were created in order to explore how the DCNN made decisions.

To our knowledge, this study is the first to demonstrate that DL has the ability to predict the evolution of SCIS from H&E WSI. DL has the potential to be used as a low-cost method that could provide prognostic information for patients with preinvasive lesions.
Variable organization of symbiont-containing tissue across planthoppers hosting different heritable endosymbionts
Authors: Michalik Anna , Franco Diego C. , Deng Junchen , Szklarzewicz Teresa , Kobiałka Michał , Stroiński Adam , Łukasik Piotr
Published: December 2022

Sap-feeding hemipteran insects live in associations with diverse heritable endosymbiotic bacteria and fungi that provide essential nutrients deficient in their diets. These symbionts typically reside in bacteriomes, dedicated organs made up of specialized cells termed bacteriocytes. The bacteriome organization varies between insect clades that are ancestrally associated with different microbes. As these symbioses evolve and additional microorganisms complement or replace the ancient associates, the organization of the symbiont-containing tissue becomes even more variable.

Planthoppers (Hemiptera: Fulgoromorpha) are ancestrally associated with bacterial symbiontsSulciaandVidania, but in many of the planthopper lineages, these symbionts are now accompanied or have been replaced by other heritable bacteria (e.g.,Sodalis, Arsenophonus, Purcelliella) or fungi. We know the identity of many of these microbes, but the symbiont distribution within the host tissues and the bacteriome organization have not been systematically studied using modern microscopy techniques.

Here, we combine light, fluorescence, and transmission electron microscopy with phylogenomic data to compare symbiont tissue distributions and the bacteriome organization across planthoppers representing 15 families. We identify and describe seven primary types of symbiont localization and seven types of the organization of the bacteriome. We show thatSulciaandVidania, when present, occupy distinct bacteriomes distributed within the body cavity. The more recently acquired gammaproteobacterial and fungal symbionts generally occupy separate groups of cells organized into distinct bacteriomes or mycetomes, distinct from those withSulciaandVidania. They can also be localized in the cytoplasm of fat body cells. Alphaproteobacterial symbionts colonize a wider range of host body habitats:Asaia-like symbionts often colonize the host gut lumen, whereasWolbachiaandRickettsiaare usually scattered across tissues and cell types, including bacteriocytes containing other symbionts, bacteriome sheath, fat body cells, gut epithelium, as well as hemolymph. However, there are exceptions, including Gammaproteobacteria that share bacteriome withVidania, or Alphaproteobacteria that colonizeSulciacells.

We discuss how planthopper symbiont localization correlates with their acquisition and replacement patterns and the symbionts’ likely functions. We also discuss the evolutionary consequences, constraints, and significance of these findings.
Targeting Damaged Collagen for Intra-Articular Delivery of Therapeutics using Collagen Hybridizing Peptides
Authors: Luke E. N. , Na Bhuket P. Ratnatilaka , Yu S. M. , Weiss J. A.
Published: December 2022

The objective of this study was to investigate the potential of collagen hybridizing peptides (CHPs), which bind to denatured collagen, to extend the retention time of near-infrared fluorophores (NIRF) following intra-articular (IA) injection in rat knee joints. CHPs were synthesized with a NIRF conjugated to N-terminus. Male Sprague-Dawley rats were assigned to one of four experimental groups: healthy, CHP; osteoarthritis (OA), CHP; healthy, scrambled-sequence CHP (sCHP), which has no collagen binding affinity; or OA, sCHP. Animals in the OA groups received an IA injection of monosodium iodoacetate to induce OA. All animals then received the corresponding CHP injection. Animals were imaged repeatedly over two weeks using anin vivofluorescence imaging system. Joint components were isolated and imaged to determine CHP binding distribution. Safranin-O and Fast Green histological staining was performed to confirm the development of OA. CHPs were found to be retained within the joint following IA injection in both healthy and OA animals for the full study period. In contrast, sCHPs were cleared within 24-48 hours. CHP signal was significantly greater (p<0.05) in OA joints when compared to healthy joints. At the two-week end point, multiple joint components retained CHPs, including cartilage, meniscus, and synovium. CHPs extended the retention time of NIRFs following IA injection in healthy and OA knee joints by binding to multiple collagenous tissues in the joint. These results support the pursuit of further research on CHP based therapeutics for IA treatment of OA.
Genome-wide association reveals novel insights into the molecular mechanisms regulating stem volume inPinus taeda
Authors: Aono Alexandre Hild , Bajay Stephanie Karenina , Francisco Felipe Roberto , de Souza Anete Pereira
Published: December 2022

Pinus taeda(loblolly pine [LP]) is a long-lived tree species and one of the most economically significant forest species. Among growth traits, volume is the most widely considered trait in tree improvement programs. However, deciphering the genetic variants responsible for growth trait variations in conifers, such as LP, is particularly challenging due to the vast size and intricate complexity ofPinusgenomes. We present a comprehensive genetic analysis of LP, focusing on markers associated with stem volume variation, to elucidate the molecular mechanisms governing high-performance phenotypes. We used a population of 1,692 individuals phenotyped for stem volume and genotyped these individuals using sequence capture probes. To conduct genome-wide associations, we utilized both genome-wide association study (GWAS) analysis and machine learning (ML) approaches. The markers identified in association with volume were found to be linked with the genes assembled from three distinct transcriptomes. These genes were subsequently used to construct gene coexpression networks, and through topological evaluations, we identified key genes with potential regulatory roles within stem volume configurations. Using a set of 31,589 SNPs, we defined 7 GWAS-associated SNPs and 128 ML-associated markers, all of which were correlated with multiple genes involved in diverse biological functions. Gene coexpression analysis revealed a group of 270 genes potentially associated with the regulation of genetic material. Key genes directly implicated in the regulation of growth and response to stress were identified, and inferences about their impact on pine development were subsequently elucidated. Our study not only offers insights into SNPs associated with stem volume but also elucidates a subset of genes characterized by unique regulatory features. These findings significantly advance our understanding of the genetic factors influencing growth traits, reveal candidate genes for future functional studies, and contribute to a broader comprehension of the genetic architecture underlying volume traits in LP.
Leptospermum extract (QV0) suppresses pleural mesothelioma tumour growthin vitroandin vivoby mitochondrial dysfunction associated apoptosis
Authors: Shi Huaikai , Zhang Le , Yu Ta-Kun , Zheng Ling , Ke Helen , Johnson Ben , Rath Emma , Lee Kenneth , Klebe Sonja , Kao Steven , Qin Karl Lijun , Pham Hong Ngoc Thuy , Vuong Quan , Cheng Yuen Yee
Published: December 2022

Pleural mesothelioma (PM) is a highly aggressive, fast-growing asbestos-induced cancer with limited effective treatments. There has been an interest in using naturally occurring anticancer agents derived from plant materials for the treatment of PM. However, it is unclear if aqueous extract from theLeptospermum polygalifolium(QV0) has activity against PM. Here we investigated the anti-cancer property of QV0in vitroandin vivo.

Animals treated with Defender®(QV0 dietary supply) exhibited a reduced tumour size over 30 days, which was associated with an average extended of seven days mouse life. There was no liver toxicity, nor increased blood glucose post-treatment in animals treated with Defender®. Moreover, QV0 suppressed the growth of 13 cancer cell lines in a dose-dependent manner, effective at concentrations as low as 0.02% w/v. This response was found to be associated with inhibited cell migration, proliferation, and colony formation, but without evident cell cycle alteration. We observed mitochondrial dysfunction post QV0 treatment, as evidenced by significantly decreased basal and maximal oxygen consumption rates. Significantly enhanced tumour apoptosis was observed in the Defender®-treated animals, correlating with mitochondrial dysfunction. To the best of our knowledge, this study constitutes the first demonstration of an improved host survival (without adverse effects) response in a QV0-treated PM mouse model, associated with an evident inhibition of PM cell growth and mitochondrial dysfunction-related enhancement of tumour apoptosis.

Importance

A major problem with cancer chemotherapy or immunotherapy is the severe adverse effects associated with normal tissue damage. PM is known to be treatment resistant and has poor a prognosis, therefore new therapeutic treatment options are urgently needed. In the present study, we explored the potential utility of aLeptospermumextract (QV0) as a treatment option for mesothelioma. We demonstrated for the first time that QV0 exhibits an anti-tumour response in mesothelioma, without any associated adverse effects observed in the PM mouse model. These findings provide a rationale for early-stage clinical trials. We anticipate that prospective translational research will lead to the clinical implementation of a novel QV0-based treatment strategy that will ultimately benefit PM patients.
Ribosomal s6 kinase (RSK) is a mediator of aquaporin 2 S256 phosphorylation and membrane accumulation after EGFR inhibition with erlotinib
Authors: Babicz Richard Samuel Edward , Cheung Pui W. , Baylor Noah , Bouley Richard , Brown Dennis
Published: December 2022

Vasopressin (VP) activates PKA, resulting in phosphorylation and membrane accumulation of aquaporin-2 (AQP2). Epidermal growth factor receptor (EGFR) inhibition with erlotinib also induces AQP2 membrane trafficking with a phosphorylation pattern similar to VP, but without increasing PKA activity. Here, we identify the ribosomal s6 kinase (RSK) as the final mediator phosphorylating AQP2 in this novel, erlotinib-induced pathway. We found that RSK was expressed in medullary principal cells in rat kidneys. RSK inhibition with BI-D1870 or siRNA blocked erlotinib-induced AQP2 S256 phosphorylation and membrane trafficking. CRISPR-generated RSK knockout cells failed to show increased S256 phosphorylation in response to erlotinib. Like PKA, RSK was able to phosphorylate AQP2 S256in vitro. Stimulation of PDK1, a known activator of RSK, caused AQP2 S256 phosphorylation and membrane accumulation similar to erlotinib and VP. We conclude that RSK is the terminal kinase phosphorylating AQP2 at S256 upon EGFR inhibition by erlotinib.
General stain deconvolution of histopathology images with physics-guided deep learning
Authors: Chen Jianan , Liu Lydia Y. , Han Wenchao , Wang Dan , Cheung Alison M. , Tsui Hubert , Martel Anne L.
Published: December 2022

Advances have been made in the use of deep learning to extract quantitative and predictive information from digital pathology slides, yet many barriers remain before clinical translation and deployment. In particular, models need to be generalizable despite the wide variations in image characteristics due to inter-scanner variability and differences in slide preparation protocols. This has led to an interest in stain deconvolution methods that could correct for the variability in image appearances. However, most existing stain deconvolution methods were developed and validated on specific datasets and perform poorly on unseen data. We developed Physics-Guided Deep Image Prior network for Stain deconvolution (PGDIPS), a method that combines a novel optical physics model and a self-supervised deep neural network to perform deconvolution for various classes and any number of stains, without the need of training data. PGDIPS outperformed state-of-the-art approaches for the deconvolution of conventional stain combinations, enabled analysis of previously unsupported special stains, and provided superior interpretability by explicitly encoding representations for stain properties and the light transmittance/absorbance process. PGDIPS is publicly available as an end-to-end off-the-shelf tool that does not require data curation, domain knowledge or high computation power.
Competition between chemoattractants causes unexpected complexity and can explain negative chemotaxis
Authors: Dowdell Adam , Paschke Peggy , Thomason Peter , Tweedy Luke , Insall Robert H.
Published: December 2022

Negative chemotaxis, where eukaryotic cells migrate away from repellents, is important throughout biology, for example in nervous system patterning and resolution of inflammation. However, the mechanisms by which molecules repel migrating cells are unknown. Here, we use a combination of modelling and experiments with Dictyostelium cells to show that competition between different ligands that bind to the same receptor leads to effective chemorepulsion. 8-CPT-cAMP, widely described as a simple chemorepellent, is inactive on its own, and only repels cells if it interacts with the attractant cAMP. If cells degrade either competing ligand, the pattern of migration becomes more complex; cells may be repelled in one part of a gradient but attracted elsewhere, leading to populations moving in different directions in the same assay, or converging in an arbitrary place. More counterintuitively still, two chemicals can each attract cells on their own, but repel cells when combined together. We have thus identified a new mechanism that drives reverse chemotaxis, verified by mathematical models and experiments with real cells, and important anywhere several ligands compete for the same receptors.
A Host-Harbored Metabolic Susceptibility of Coronavirus Enables Broad-Spectrum Targeting
Authors: Fang Huan , Wang Yonglun , Liu Lu , Cheng Kunlun , Li Pei , Tan Ya , Hao Xingjie , Mei Miao , Xu Xinxuan , Yao Yuanhang , Zan Fuwen , Wu Linzhi , Zhu Yuangang , Xu Bolin , Huang Dong , Wang Chaolong , Tan Xu , Qian Zhaohui , Chen Xiao-Wei
Published: December 2022

Host-based antivirals could offer broad-spectrum therapeutics and prophylactics against the constantly-mutating viruses including the currently-ravaging coronavirus, yet must target cellular vulnerabilities of viruses without grossly endangering the host. Here we show that the master lipid regulator SREBP1 couples the phospholipid scramblase TMEM41B to constitute a host “metabolism-to-manufacture” cascade that maximizes membrane supplies to support coronaviral genome replication, harboring biosynthetic enzymes including Lipin1 as druggable viral-specific-essential (VSE) host genes. Moreover, pharmacological inhibition of Lipin1, by a moonlight function of the widely-prescribed beta-blocker Propranolol, metabolically uncouples the SREBP1-TMEM41B cascade and consequently exhibits broad-spectrum antiviral effects against coronaviruses, Zika virus, and Dengue virus. The data implicate a metabolism-based antiviral strategy that is well tolerated by the host, and a potential broad-spectrum medication against current and future coronavirus diseases.
Whole-Genome Sequencing and Variant Discovery ofCitrus reticulata‘Kinnow’ from Pakistan
Authors: Jabeen Sadia , Saif Rashid , Haq Rukhama , Hayat Akbar , Naz Shagufta
Published: December 2022

Citrus is a source of many nutritional and medicinal advantages, which is cultivated worldwide with major citrus groups of sweet oranges, mandarins, grapefruits, kumquats, lemons and limes. Pakistan produces all of its major citrus groups with mandarin (Citrus reticulata) being the prominent group that includes local commercial cultivars such as Feutral’s Early, Dancy, Honey and Kinnow. The present study was designed to understand the genetic architecture of this unique variety ofCitrus reticulata -’Kinnow’. The whole-genome resequencing and variant calling was performed to map the genomic variability that might be responsible for its particular characteristics like taste, seededness, juice content, thickness of peel and its shelf-life. A total of 139,436,350 raw sequence reads using Illumina platform were generated with 20.9 Gb data in Fastq format having 98% effectiveness and 0.2% base call error rate. Overall, a total of 3,503,033 SNPs, 176,949 MNPs, 323,287 INS and 333,083 DEL were identified using GATK4 variant calling pipeline againstCitrus clementinaas a reference genome. Further, g:Profiler bioinformatics tool was applied for annotating the newly found variants, harbor genes/transcripts and their involved pathways. A total of 73,864 transcripts harbors 4,336,352 variants, most of the observed variants were predicted in non-coding regions and 1,009 transcripts were found well annotated by different databases. Out of total aforementioned transcripts, 588 involved in biological processes, 234 in molecular functions and 167 transcripts involved in cellular components inCitrus reticulata. In a nutshell, 18,153 high-impact variants and 216 genic-variants found in the current study which may be used for marker assisted breeding programs of ‘Kinnow’ to identify this particular variety among others and to propagate its valued traits to improve the contemporary citrus varieties as well.

Graphical Abstract
The impact of sex and physical activity on the local immune response to muscle pain
Authors: Lesnak Joseph B. , Hayashi Kazuhiro , Plumb Ashley N. , Janowski Adam J. , Chimenti Michael S. , Sluka Kathleen A.
Published: December 2022

Induction of muscle pain triggers a local immune response to produce pain and this mechanism may be sex and activity level dependent. The purpose of this study was to measure the immune system response in the muscle following induction of pain in sedentary and physically active mice. Muscle pain was produced via an activity-induced pain model using acidic saline combined with fatiguing muscle contractions. Prior to induction of muscle pain, mice (C57/BL6) were sedentary or physically active (24hr access to running wheel) for 8 weeks. The ipsilateral gastrocnemius was harvested 24hr after induction of muscle pain for RNA sequencing or flow cytometry. RNA sequencing revealed activation of several immune pathways in both sexes after induction of muscle pain, and these pathways were attenuated in physically active females. Uniquely in females, the antigen processing and presentation pathway with MHC II signaling was activated after induction of muscle pain; activation of this pathway was blocked by physical activity. Blockade of MHC II attenuated development of muscle hyperalgesia exclusively in females. Induction of muscle pain increased the number of macrophages and T-cells in the muscle in both sexes, measured by flow cytometry. In both sexes, the phenotype of macrophages shifted toward a pro-inflammatory state after induction of muscle pain in sedentary mice (M1+M1/2) but toward an anti-inflammatory state in physically active mice (M2+M0). Thus, induction of muscle pain activates the immune system with sex-specific differences in the transcriptome while physical activity attenuates immune response in females and alters macrophage phenotype in both sexes.
AutoGater: A Weakly Supervised Neural Network Model to Gate Cells in Flow Cytometric Analyses
Authors: Eslami Mohammed , Moseley Robert C. , Eramian Hamed , Bryce Daniel , Haase Steven B.
Published: December 2022

Flow cytometry is a useful and efficient method for the rapid characterization of a cell population based on the optical and fluorescence properties of individual cells. Ideally, the cell population would consist of only healthy viable cells as dead cells can confound the analysis. Thus, separating out healthy cells from dying and dead cells, and any potential debris, is an important first step in analysis of flow cytometry data. While gating of debris can be conducted using measured optical properties, identifying dead and dying cells often requires utilizing fluorescent stains (e.g. Sytox, a nucleic acid stain that stains cells with compromised cell membranes) to identify cells that should be excluded from downstream analyses. These stains prolong the experimental preparation process and use a flow cytometer’s fluorescence channels that could otherwise be used to measure additional fluorescent markers within the cells (e.g. reporter proteins). Here we outline a stain-free method for identifying viable cells for downstream processing by gating cells that are dying or dead. AutoGater is a weakly supervised deep learning model that can separate healthy populations from unhealthy and dead populations using only light-scatter channels. In addition, AutoGater harmonizes different measurements of dead cells such as Sytox and CFUs.
An Integrated Approach to the Characterization of Immune Repertoires Using AIMS: An Automated Immune Molecule Separator
Authors: Boughter Christopher T. , Meier-Schellersheim Martin
Published: December 2022

The adaptive immune system employs an array of receptors designed to respond with high specificity to pathogens or molecular aberrations faced by the host organism. Binding of these receptors to molecular fragments - collectively referred to as antigens - initiates immune responses. These antigenic targets are recognized in their native state on the surfaces of pathogens by antibodies, whereas T cell receptors (TCR) recognize processed antigens as short peptides, presented on major histocompatibility complex (MHC) molecules. Recent research has led to a wealth of immune repertoire data that are key to interrogating the nature of these molecular interactions. However, existing tools for the analysis of these large datasets typically focus on molecular sets of a single type, forcing researchers to separately analyze strongly coupled sequences of interacting molecules. Here, we introduce a software package for the integrated analysis of immune repertoire data, capable of identifying distinct biophysical differences in isolated TCR, MHC, peptide, antibody, and antigen sequence data. This integrated analytical approach allows for direct comparisons across immune repertoire subsets and provides a starting point for the identification of key interaction hotspots in complementary receptor-antigen pairs. The software (AIMS - Automated Immune Molecule Separator) is freely available as an open access package in GUI or command-line form.
Micronutrient Optimization Using Design of Experiments Approach in Tissue Engineered Articular Cartilage for Production of Type II Collagen
Authors: Cruz Maria A. , Gonzalez Yamilet , Vélez Toro Javier A. , Karimzadeh Makan , Rubbo Anthony , Morris Lauren , Medam Ramapaada , Splawn Taylor , Archer Marilyn , Fernandes Russell J. , Dennis James E. , Kean Thomas J.
Published: December 2022

Tissue Engineering of cartilage has been hampered by the inability of engineered tissue to express native levels of type II collagenin vitro. Inadequate levels of type II collagen are, in part, due to a failure to recapitulate the physiological environment in culture. In this study, we engineered primary rabbit chondrocytes to express a secreted reporter,GaussiaLuciferase, driven by the type II collagen promoter, and applied a Design of Experiments approach to assess chondrogenic differentiation in micronutrient-supplemented medium. Using a Response Surface Model, 240 combinations of micronutrients absent in standard chondrogenic differentiation medium, were screened and assessed for type II collagen expression. Five conditions predicted to produce the greatest Luciferase expression were selected for further study. Validation of these conditions in 3D aggregates identified an optimal condition for type II collagen expression. Engineered cartilage grown in this condition, showed a 170% increase in type II collagen expression (Day 22 Luminescence) and in Young’s tensile modulus compared to engineered cartilage in basal media alone. Collagen cross-linking analysis confirmed formation of type II-type : II collagen and type II-type : IX collagen cross-linked heteropolymeric fibrils, characteristic of mature native cartilage. Combining a Design of Experiments approach and secreted reporter cells in 3D aggregate culture enabled a high-throughput platform that can be used to identify more optimal physiological culture parameters for chondrogenesis.
Expression of mRNA encoding two gain-of-functioncyfip2variants associated with DEE65 results in spontaneous seizures inXenopus laevistadpoles
Authors: Panthi Sandesh , Szyszka Paul , Beck Caroline W.
Published: December 2022

Developmental and epileptic encephalopathies (DEE) are a genetically diverse group of disorders with similar early clinical presentations. DEE65 is caused byde novo, non-synonymous, gain-of-function mutations in CYFIP2. It presents in early infancy as hypotonia, epileptic spasms and global developmental delay. While modelling loss-of-function mutations can be done using knockdown or knockout techniques to reduce the amount of functional protein, modelling gain-of-function mutations requires different approaches. Here, we show that transient ectopic expression of the Arg87Cys pathogenic variant ofcyfip2mRNA inXenopus laevistadpoles resulted in increased seizure-related behaviours such as rapid darting and swimming in circles. In contrast, expression of a second pathological variant, Tyr108Cys, did not alter tadpole behaviour. Expression of either pathogenic variant resulted in spontaneous epileptic activity in the brain. For both variants, neuronal hyperactivity was reduced by treating the tadpole with 5 mM of the anti-seizure drug valproate (VPA). mRNA overexpression of gain-of-function variants inX. laevistadpoles may be useful both for understanding the aetiology of DEE and for pre-clinical drug testing.
MAPK/ERK Activation in Macrophages PromotesLeishmaniaInternalization and Pathogenesis
Authors: Barrie Umaru , Floyd Katherine , Datta Arani , Wetzel Dawn M.
Published: December 2022

The obligate intracellular protozoan parasiteLeishmaniabinds several host cell receptors to trigger its uptake by phagocytic cells, ultimately resulting in visceral or cutaneous leishmaniasis. AfterLeishmaniaengages receptors on macrophages and other phagocytes, a series of signaling pathways in the host cell are activated during its internalization, which are critical for establishment and persistence ofLeishmaniainfection. Thus, preventingLeishmaniainternalization by phagocytes could be a novel therapeutic strategy for leishmaniasis. However, the host cellular machinery that mediates promastigote and amastigote uptake is not well understood. Here, using small molecule inhibitors of Mitogen-activated protein kinases/Extracellular signal regulated kinases (MAPK/ERK), we demonstrate that ERK1/2 mediatesLeishmania amazonensisuptake and (to a lesser extent) phagocytosis of beads by macrophages. We find that inhibition of MEK1/2 or ERK1/2 leads to inefficient amastigote uptake by macrophages. Moreover, using inhibitors and primary macrophages lacking spleen tyrosine kinase (SYK) or Abl family kinases, we show that SYK and Abl family kinases mediate Raf, MEK, and ERK1/2 activity and are necessary for efficient uptake. Finally, we demonstrate that trametinib, a MEK1/2 inhibitor used clinically to treat certain cancers, significantly reduces disease severity and parasite burden inLeishmania-infected mice, even if it is started significantly after lesions develop. Our results show that maximalLeishmaniainfection requires MAPK/ERK and highlight the potential for MAPK/ERK-mediated signaling pathways to be novel therapeutic targets for leishmaniasis.

Lay summary

Leishmaniais a single-celled parasite that causes skin ulcers or a disseminated disease in humans. Our goal is to identify new drugs to treatLeishmaniainfection.Leishmaniamust live inside human immune cells to cause disease. IfLeishmaniais not able to enter human immune cells, it dies. Our studies demonstrate howLeishmaniainfection permits a set of proteins called MAP kinases to pass signals from one protein to the next within mammalian immune cells. The resulting signals allowLeishmaniato enter into these immune cells and survive within its host. Importantly, trametinib, a drug that prevents these signals from MAP kinases, decreases the development of skin ulcers when it is given to mice that are infected withLeishmania. Our findings suggest that leishmaniasis could be treated with drugs that act on kinases found in humans rather than the parasites themselves.

One sentence summary

An Abl2-SYK-Raf-MEK-ERK pathway facilitatesLeishmaniauptake by phagocytic cells and promotes disease severity inLeishmania-infected mice.
A mechanical wave travels along a genetic guide to drive the formation of an epithelial furrow
Authors: Popkova Anna , Pagnotta Sophie , Rauzi Matteo
Published: December 2022

Epithelial furrowing is a morphogenetic process that is pivotal during embryo gastrulation, neurulation and the shaping of the animal body. A furrow often results from a fold that propagates along a line. How fold formation and propagation are initiated, driven and controlled is still poorly understood. To shed new light on this fundamental morphogenetic process, we study the formation of the cephalic furrow: a fold that runs along the dorsal-ventral axis of the embryo during earlyDrosophilagastrulation and the developmental role of which is still unknown. Here, we provide evidence of its function and show that the cephalic furrow is initiated by two groups of cells located on the left and right lateral sides of the embryo. These cellular clusters work as a pacemaker triggering a bi-directional morphogenetic wave powered by actomyosin contractions and sustained byde novomedial apex-to-apex cell adhesion. The Cartesian position of the pacemakers is under the cross-control of the embryo anterior-posterior and dorsal-ventral gene patterning systems. Thus, furrow initiation and propagation are driven by a mechanical trigger wave that travels under the control of a multidimensional genetic guide.
SARM1 detection in oligodendrocytes but not Schwann cells thoughsarm1/Sarm1deletion does not perturb CNS nor PNS myelination in zebrafish and mice
Authors: Fazal Shaline V. , Mutschler Clara , Chen Civia Z. , Turmaine Mark , Chen Chiung-Ya , Hsueh Yi-Ping , Loreto Andrea , Casillas-Bajo Angeles , Cabedo Hugo , Franklin Robin J.M. , Barker Roger A. , Monk Kelly R. , Steventon Benjamin J. , Coleman Michael P. , Gomez-Sanchez Jose A. , Arthur-Farraj Peter
Published: December 2022

SARM1 is a central regulator of programmed axon death and is required to initiate axon self-destruction after traumatic and toxic insults to the nervous system. Abnormal activation of this axon degeneration pathway is increasingly recognized as a contributor to human neurological disease and SARM1 knockdown or inhibition has become an attractive therapeutic strategy to preserve axon loss in a variety of disorders of the peripheral and central nervous system. Despite this, it remains unknown whetherSarm1/SARM1 is present in myelinating glia and whether it plays a role in myelination in the PNS or CNS. It is important to answer these questions to understand whether future therapies inhibiting SARM1 function may have unintended deleterious impacts on myelination. Here we show thatSarm1mRNA is present in oligodendrocytes in zebrafish but only detectable at low levels in Schwann cells in both zebrafish and mice. We find SARM1 protein is readily detectable in murine oligodendrocytesin vitro and in vivoand activation of endogenous SARM1 in oligodendrocytes induces cell death. In contrast, SARM1 protein is not detectable in Schwann cells and satellite glia in the adult murine nervous system. Cultured Schwann cells contain negligible functional SARM1 and are insensitive to specific SARM1 activators. Using zebrafish and mouseSarm1mutants, we show that SARM1 is not required for initiation of myelination nor myelin sheath maintenance by oligodendrocytes and Schwann cells. Thus, strategies to inhibit SARM1 function in the nervous system to treat neurological disease are unlikely to perturb myelination in humans.

Main Points

SARM1 protein is detectable in oligodendrocytes but not in Schwann cells

Oligodendrocytes but not Schwann cells die in response to endogenous SARM1 activation

CNS nor PNS myelination, in zebrafish and mice, is hindered by loss ofsarm1/Sarm1
FiNuTyper: an automated deep learning-based platform for simultaneous fiber and nucleus type analysis in human skeletal muscle
Authors: Lundquist August , Lázár Enikő , Han Nan Sophia , Emanuelsson Eric B , Reitzner Stefan M , Chapman Mark A , Alkass Kanar , Druid Henrik , Petri Susanne , Sundberg Carl Johan , Bergmann Olaf
Published: December 2022

While manual quantification is still considered the gold standard for skeletal muscle histological analysis, it is time-consuming and prone to investigator bias. We assembled an automated image analysis pipeline, FiNuTyper (Fiber and Nucleus Typer), from recently developed deep learning-based image segmentation methods, optimized for unbiased evaluation of fresh and postmortem human skeletal muscle. We validated and utilized SERCA1 and SERCA2 as type-specific myonucleus and myofiber markers. Parameters including myonuclei per fiber, myonuclear domain, central myonuclei per fiber, and grouped myofiber ratio were determined in a fiber type-specific manner, revealing a large degree of gender- and muscle-related heterogeneity. Our platform was also tested on pathological muscle tissue (ALS) and adapted for the detection of other resident cell types (leukocytes, satellite cells, capillary endothelium). In summary, we present an automated image analysis tool for the simultaneous quantification of myofiber and myonuclear types, to characterize the composition of healthy and diseased human skeletal muscle.

Highlights

A deep learning-based automated platform for skeletal muscle microscopic analysis

High-fidelity identification and characterization of myonuclei and myofibers

Validation of SERCA1 and SERCA2 as markers for myofiber and myonuclear subtypes

Characterization of healthy and pathological human skeletal muscle tissue features

Adaptations provided for studies on other resident cell types like satellite cells

eTOC Blurb

An automated platform for unbiased analysis of skeletal muscle immunohistochemical images, focusing on type-specific myofiber-myonucleus relationships, facilitating high-throughput studies of healthy and diseased tissues.
Different hemispheric lateralization for periodicity and formant structure of vowels in the auditory cortex and its changes between childhood and adulthood
Authors: Orekhova E.V. , Fadeev K.A. , Goiaeva D.E. , Obukhova T.S. , Ovsiannikova T.M. , Prokofyev A.O. , Stroganova T.A.
Published: December 2022

The spectral formant structure and periodicity pitch are the major features that determine the identity of vowels and the characteristics of the speaker. However, very little is known about how the processing of these features in the auditory cortex changes during development. To address this question, we independently manipulated the periodicity and formant structure of vowels while measuring auditory cortex responses using MEG in children aged 7-12 years and adults. We analyzed the sustained negative shift of source current associated with these vowel properties, which was present in the auditory cortex in both age groups despite differences in the transient components of the auditory response. In adults, the sustained activation associated with formant structure was lateralized to the left hemisphere early in the auditory processing stream requiring neither attention nor semantic mapping. This lateralization was not yet established in children, in whom the right hemisphere contribution to formant processing was strong and decreased during or after puberty. In contrast to the formant structure, periodicity was associated with a greater response in the right hemisphere in both children and adults. These findings suggest that left-lateralization for the automatic processing of vowel formant structure emerges relatively late in ontogenesis and pose a serious challenge to current theories of hemispheric specialization for speech processing.
Enhancer grammar of liver cell types and hepatocyte zonation states
Authors: González-Blas Carmen Bravo , Matetovici Irina , Hillen Hanne , Taskiran Ibrahim Ihsan , Vandepoel Roel , Christiaens Valerie , Sansores-García Leticia , Verboven Elisabeth , Hulselmans Gert , Poovathingal Suresh , Demeulemeester Jonas , Psatha Nikoleta , Mauduit David , Halder Georg , Aerts Stein
Published: December 2022

Cell type identity is encoded by gene regulatory networks (GRN), in which transcription factors (TFs) bind to enhancers to regulate target gene expression. In the mammalian liver, lineage TFs have been characterized for the main cell types, including hepatocytes. Hepatocytes cover a relatively broad cellular state space, as they differ significantly in their metabolic state, and function, depending on their position with respect to the central or portal vein in a liver lobule. It is unclear whether this spatially defined cellular state space, called zonation, is also governed by a well-defined gene regulatory code. To address this challenge, we have mapped enhancer-GRNs across liver cell types at high resolution, using a combination of single cell multiomics, spatial omics, GRN inference, and deep learning. We found that cell state changes in transcription and chromatin accessibility in hepatocytes, liver sinusoidal endothelial cells and hepatic stellate cells depend on zonation. Enhancer-GRN mapping suggests that zonation states in hepatocytes are driven by the repressors Tcf7l1 and Tbx3, that modulate the core hepatocyte GRN, controlled by Hnf4a, Cebpa, Hnf1a, Onecut1 and Foxa1, among others. To investigate how these TFs cooperate with cell type TFs, we performed anin vivomassively parallel reporter assay on 12,000 hepatocyte enhancers and used these data to train a hierarchical deep learning model (called DeepLiver) that exploits both enhancer accessibility and activity. DeepLiver confirms Cebpa, Onecut, Foxa1, Hnf1a and Hnf4a as drivers of enhancer specificity in hepatocytes; Tcf7l1/2 and Tbx3 as regulators of the zonation state; and Hnf4a, Hnf1a, AP-1 and Ets as activators. Finally, taking advantage ofin silicomutagenesis predictions from DeepLiver and enhancer assays, we confirmed that the destruction of Tcf7l1/2 or Tbx3 motifs in zonated enhancers abrogates their zonation bias. Our study provides a multi-modal understanding of the regulatory code underlying hepatocyte identity and their zonation state, that can be exploited to engineer enhancers with specific activity levels and zonation patterns.