Human Gene Research Articles

Disparate and shared transcriptomic signatures associated with cortical atrophy in genetic behavioral variant frontotemporal degeneration

BackgroundCortical atrophy is a common manifestation in behavioral variant frontotemporal degeneration (bvFTD), exhibiting spatial heterogeneity across various genetic subgroups, which may be driven by distinct biological mechanisms.MethodsWe employed an integrative imaging transcriptomics approach to identify both disparate and shared transcriptomic signatures associated with cortical thickness in bvFTD with C9orf72 repeat expansions or pathogenic variants in GRN or MAPT. Functional enrichment analyses were conducted on each gene list significantly associated with cortical thickness. Additionally, we mapped neurotransmitter receptor/transporter density maps to the cortical thickness maps, to uncover different correlation patterns for each genetic form. Furthermore, we examined whether the identified genes were enriched for pathology-related genes by using previously identified genes linked to TDP-43 positive neurons and genes associated with tau pathology.ResultsFor each genetic form of bvFTD, we identified cortical thickness signatures and gene sets associated with them. The cortical thickness associated genes for GRN-bvFTD were significantly involved in neurotransmitter system and circadian entrainment. The different patterns of spatial correlations between synaptic density and cortical thinning, further confirmed the critical role of neurotransmission and synaptic signaling in shaping brain structure, especially in the GRN-bvFTD group. Furthermore, we observed significant overlap between genes linked to TDP-43 pathology and the gene sets associated with cortical thickness in C9orf72-bvFTD and GRN-bvFTD but not the MAPT-bvFTD group providing specificity for our associations. C9orf72-bvFTD and GRN-bvFTD also shared genes displaying consistent directionality, with those exhibiting either positive or negative correlations with cortical thickness in C9orf72-bvFTD showing the same direction (positive or negative) in GRN-bvFTD. MAPT-bvFTD displayed more pronounced differences in transcriptomic signatures compared to the other two genetic forms. The genes that exhibited significantly positive or negative correlations with cortical thickness in MAPT-bvFTD showed opposing directionality in C9orf72-bvFTD and GRN-bvFTD.ConclusionsOverall, this integrative transcriptomic approach identified several new shared and disparate genes associated with regional vulnerability with increased biological interpretation including overlap with synaptic density maps and pathologically-specific gene expression. These findings illuminated the intricate molecular underpinnings contributing to the heterogeneous nature of disease distribution in bvFTD with distinct genetic backgrounds.

Sampling techniques and genomic analysis of biological material from artworks.

The genomic analysis of biological material from artworks can be used to guide curation, preservation, and restoration. Additionally, human DNA recovered from artworks may provide other insights. However, the recovery of biological samples from artworks is dependent on the sampling technique used and the media from which the biological materials are recovered. The ideal sampling method should be noninvasive, yet robust. We studied five artworks on paper and compared three sampling methods, each with increasing degrees of invasiveness. Minimally invasive swabbing techniques collect samples from the surface, whereas more aggressive techniques such as wet vacuuming were expected to yield more biological material from within the support media and more likely to produce authentic DNA from the artwork. We report a comparison of collection techniques to generate microbial DNA sequence data, the conserved human gene RNase P, and Y-STRs from artworks on paper. We observed that wet vacuuming resulted in higher DNA recovery than double swabbing and core punches. Diverse microbial populations existed on the corners and centers of the five artworks studied, but the distribution of the total biomass was relatively even across the surfaces of the works sampled. Studies of peripheral regions, where sampling is less likely to cause alterations to the artwork, could thus yield useful results in microbiome and human DNA studies. These results provide a framework for sampling artworks on paper to obtain biological material. The methods described may provide microbiome identification to facilitate restoration and preservation, and might also contribute to the determination of provenance.

Whole genome sequencing in early onset advanced heart failure

The genetic contributions to early onset heart failure (HF) are incompletely understood. Genetic testing in advanced HF patients undergoing heart transplantation (HTx) may yield clinical benefits, but data is limited. We performed deep-coverage whole genome sequencing (WGS) in 102 Swedish HTx recipients. Gene lists were compiled through a systematic literature review. Variants were prioritized for pathogenicity and classified manually. We also compared polygenic HF risk scores to a population-based cohort. We found a pathogenic (LP/P) variant in 34 individuals (34%). Testing yield was highest in hypertrophic (63% LP/P carriers), dilated (40%) and arrhythmogenic right ventricular (33%) cardiomyopathy and lower in ischemic cardiomyopathy (10%). A family history was more common in LP/P variant carriers than in non-carriers but was present in less than half of carriers (44% vs 13%, P < 0.001), whereas age was similar. Polygenic risk scores were similar in HTx recipients and the population cohort. In conclusion, we observed a high prevalence of pathogenic cardiomyopathy gene variants in individuals with early-onset advanced HF, which could not accurately be ruled out by family history and age. In contrast, we did not observe higher polygenic risk scores in early onset advanced HF cases than in the general population.

Modification of the telomerase gene with human regulatory sequences resets mouse telomeres to human length

Telomeres shorten with each cell division, serving as biomarkers of aging, with human tissues exhibiting short telomeres and restricted telomerase expression. In contrast, mice have longer telomeres and widespread telomerase activity, limiting their relevance as models for human telomere biology. To address this, we engineer a mouse strain with a humanized mTert gene (hmTert), replacing specific non-coding sequences with human counterparts. The hmTert gene, which is repressed in adult tissues except the gonads and thymus, closely mimics human TERT regulation. This modification rescues telomere dysfunction in mTert-knockout mice. Successive intercrosses of Terth/- mice stabilized telomere length below 10 kb, while Terth/h mice achieve a human-like average length of 10–12 kb, compared to 50 kb in wildtype mice. Despite shortened telomeres, Terth/h mice maintain normal body weight and cell homeostasis. These mice, with humanized telomere regulation, represent a valuable model to study human aging and cancer.

Construction of a TAT-Cas9-EGFP Site-Specific Integration Eukaryotic Cell Line Using Efficient PEG10 Modification

The CRISPR/Cas9 system enables precise and efficient modification of eukaryotic genomes. Among its various applications, homology-directed repair (HDR) mediated knock-in (KI) is crucial for creating human disease models, gene therapy, and agricultural genetic enhancements. Despite its potential, HDR-mediated knock-in efficiency remains relatively low. This study investigated the impact of 5′ end PEG10 modification on site-specific integration of the target gene. The HEK293 cell line is considered a highly attractive expression system for the production of recombinant proteins, with the construction of site-specific integration cell lines at the AAVS1 locus enabling stable protein expression. This study investigated the impact of the 5′ end PEG10 modification on the site-specific integration of the target gene at the AAVS1 locus in the 293T cell line. Utilizing this 5′ end PEG10 modification resulted in a 1.9-fold increase in knock-in efficiency for a 1.8 kb target fragment, improving efficiency from 26% to 49%. An optimized system was utilized to successfully establish a high-expression, site-specific integration 293T cell line for TAT-Cas9-EGFP, providing a reliable resource of seed cells for subsequent protein production.

Sudden cardiac death, arrhythmogenic cardiomyopathy and intercalated disc pathology due to reduced filamin C protein levels: a matter of life and death.

Mutations in the human FLNC gene encoding filamin C (FLNc) cause a broad spectrum of sporadic and familial cardiomyopathies and myopathies. We report on the genetic, clinical, morphological and biochemical findings in a German family harboring an FLNC variant that leads to severe cardiac disease comprising sudden cardiac death and arrhythmogenic cardiomyopathy. Genetic analysis identified a novel heterozygous FLNC variant in exon 16 (NM_001458.4:c.2495_2498delAGTA, het; p.K832TfsX45) in i) the index patient suffering from dilated cardiomyopathy necessitating heart transplantation, ii) a son, who died from sudden cardiac death, iii) a second son, who survived an episode of sudden cardiac arrest and iv) a third son affected by isolated skeletal muscle myopathy. FLNc protein levels were markedly reduced in cardiac tissue obtained from the index patient, implying that the p.K832TfsX45 FLNc variant most probably caused nonsense-mediated decay of the corresponding mRNA. Morphological analysis of the diseased cardiac tissue revealed extensive fibrotic remodeling, and marked degenerative changes of the contractile apparatus of cardiomyocytes and severe structural alterations of intercalated discs. Connexin-43 signal intensity at intercalated discs was diminished and FLNc labelling of myofibrils was attenuated or even absent. Proteome analyses demonstrated complex alterations of extracellular matrix and intercalated disc proteins. Our findings demonstrate that this novel, truncating FLNC mutation likely leads to haploinsufficiency, thereby causing a deleterious sequence of degenerative changes of cardiac tissue with extensive fibrotic remodeling and intercalated disc pathology as the structural basis for FLNC-related cardiomyopathy with life-threatening cardiac arrhythmias.

Toward a comprehensive profiling of alternative splicing proteoform structures, interactions and functions.

The mRNA splicing machinery has been estimated to generate 100,000 known protein-coding transcripts for 20,000 human genes (Ensembl, Sept. 2024). However, this set is expanding with the massive and rapidly growing data coming from high-throughput technologies, particularly single-cell and long-read sequencing. Yet, the implications of splicing complexity at the protein level remain largely uncharted. In this review, we describe the current advances toward systematically assessing the contribution of alternative splicing to proteome function diversification. We discuss the potential and challenges of using artificial intelligence-based techniques in identifying alternative splicing proteoforms and characterising their structures, interactions, and functions.

Comparative Study of Lactogenic Effect and Milk Mutritional Density of Oral Galactagogues in Female Rabbit.

Hypogalactia and agalactia in lactating mothers are the major causes of child malnutrition, mortality, morbidity, and overall ill health. The development of such treatments requires a well-designed preclinical study with suitable laboratory animals, which needs to be improved. Thus, a suitably designed study with a laboratory animal to analyse galactagogue activity, along with an assessment of the quality and quantity of milk, is required. This study aimed to evaluate the potential of rabbits as an animal model for studying lactogenic activity. The structural homology of prolactin, prolactin, and prolactin in humans, rabbits, and rats was studied using BLAST and PyMol to assess similarity in the lactogenic system. Daily and cumulative milk production and pre-treatment (control) and post-treatment (three drugs) in rabbits were recorded and evaluated by analysing protein, fat, lactose, solid non-fat, and ash values. All parameters were recorded on the 0th day and at the end of weeks 1, 2, and 3. Mammary gland histopathology was performed to evaluate the effects on mammary glands. Homology studies revealed that the sequences of the human and rabbit prolactin genes, receptors, and hormones had a high similarity index. Treatment with Domperidone, Metoclopramide, and Shatavari significantly enhanced milk production by enhancing prolactin secretion; only Shatavari increased milk nutrition. Enlargement of the tubuloalveolar ducts of the mammary glands was observed. Our findings suggest that rabbits are robust, reproducible, ethically superior, and preclinically relevant animals for assessing lactogenic activity.

NRG-GI007: Secondary endpoints of safety assessment and clinical complete response (cCR) of chemoradiation with endoscopically injected oncolytic virus OBP-301 in medically inoperable esophageal cancer.

435 Background: Definitive chemoradiation (CRT) is a standard-of-care for patients (Pts) with medically inoperable esophageal cancer (EC). The NRG/RTOG 0436 study of cisplatin/paclitaxel and RT (+/- cetuximab) as definitive therapy showed that 58% of control arm Pts have locally persistent disease. OBP-301 is a conditionally-restricted, replication-competent adenovirus derived from human adenovirus type 5 that adds a human Telomerase Reverse Transcriptase gene promoter, replicating only in tumor cells to cause lysis.It may cause immunogenic cell death, enhance RT increasing radiosensitization by blocking DNA repair and improve local control. Methods: In NRG-GI007, a phase 1 study (NCT04391049), OBP-301 was added to weekly carboplatin/paclitaxel and RT (50.4 Gy/28 fxs) for medically inoperable EC Pts with pathologically proven adenocarcinoma or squamous cell carcinoma (SCC) of the esophagus or Siewert Type I/II gastroesophageal junction. Pts receive 1-2mL of intratumoral OBP-301 1×10 12 virus particles/ml via endoscopy 3 days prior to and then at days 12 and 26 of CRT. The primary endpoint was protocol-defined dose-limiting toxicity, already reported. Secondary endpoints reported here are treatment-related (definitely or probably related to any protocol treatment) AEs - all and grade 4+ non-hematologic, and cCR in the primary tumor, defined as negative EGD/biopsy 6-8 weeks post-CRT. Per the protocol design, no statistical testing is done for these endpoints. Results: Initially, 6 evaluable (eligible and started protocol treatment) Pts were enrolled from June 2020 to April 2023. As initial dose level was deemed safe, an additional 9 Pts were enrolled from August 2023 to July 2024, totaling 15 evaluable Pts for secondary endpoints. Median age was 74 years, 9 Pts (60%) with ECOG PS 1. 11 Pts (73%) had adenocarcinoma and 4 had SCC; 11 Pts had N+ disease. 14 Pts (93%) received all planned OBP-301 injections and received 50.4 Gy RT. The most common treatment-related grade 3/4 AEs were decreased neutrophil (6 Pts; 40%) and lymphocyte counts (5 Pts; 33%), expected with CRT. No grade ≥3 OBP-301-related non-hematologic AEs were reported. 2 Pts died prior to restaging neitherdefinitely or probably related to OBP-301: 1 Pt developed grade 5 respiratory failure 6 weeks after CRT (suspected RT pneumonitis), while a 2 nd Pt had an unrecognized tumor invasion of the bronchus at baseline, which worsened during week 3 of CRT, resulting in a grade 5 tracheo-esophageal fistula. 2 Pts have not yet reached time for restaging. All 11 Pts who have been restaged had a cCR, for a preliminary cCR rate of 100% (95% CI: 74.1%, 100%). Conclusions: OBP-301 + CRT is safe and the preliminary cCR rate compares favorably to the historical control from NRG/RTOG 0436. Updated safety and cCR data will be presented. A randomized study is being planned. Clinical trial information: NCT04391049 .

Model organisms for investigating the functional involvement of NRF2 in non-communicable diseases.

Non-communicable chronic diseases (NCDs) are most commonly characterized by age-related loss of homeostasis and/or by cumulative exposures to environmental factors, which lead to low-grade sustained generation of reactive oxygen species (ROS), chronic inflammation and metabolic imbalance. Nuclear factor erythroid 2-like 2 (NRF2) is a basic leucine-zipper transcription factor that regulates the cellular redox homeostasis. NRF2 controls the expression of more than 250 human genes that share in their regulatory regions a cis-acting enhancer termed the antioxidant response element (ARE). The products of these genes participate in numerous functions including biotransformation and redox homeostasis, lipid and iron metabolism, inflammation, proteostasis, as well as mitochondrial dynamics and energetics. Thus, it is possible that a single pharmacological NRF2 modulator might mitigate the effect of the main hallmarks of NCDs, including oxidative, proteostatic, inflammatory and/or metabolic stress. Research on model organisms has provided tremendous knowledge of the molecular mechanisms by which NRF2 affects NCDs pathogenesis. This review is a comprehensive summary of the most commonly used model organisms of NCDs in which NRF2 has been genetically or pharmacologically modulated, paving the way for drug development to combat NCDs. We discuss the validity and use of these models and identify future challenges.

Viral influencers: deciphering the role of endogenous retroviral LTR12 repeats in cellular gene expression.

The human genome is like a museum of ancient retroviral infections. It contains a large number of endogenous retroviruses (ERVs) that bear witness to past integration events. About 5,000 of them are so-called long terminal repeat 12 (LTR12) elements. Compared with 20,000 human genes, this is a remarkable number. Although LTR12 elements can act as promoters or enhancers of cellular genes, the function of most of these retroviral elements has remained unclear. In our mini-review, we show that different LTR12 elements share many similarities, including common transcription factor binding sites. Furthermore, we summarize novel insights into the epigenetic mechanisms governing their silencing and activation. Specific examples of genes and pathways that are regulated by LTR12 loci are used to illustrate the regulatory network built by these repetitive elements. A particular focus is on their role in the regulation of antiviral immune responses, tumor cell proliferation, and senescence. Finally, we describe how a targeted activation of this fascinating ERV family could be used for diagnostic or therapeutic purposes.

Pathogenicity assessment of seven RYR1 variants in patients with confirmed susceptibility to malignant hyperthermia in the Netherlands.

Malignant hyperthermia (MH) susceptibility is associated with variants in RYR1, the gene encoding the skeletal muscle ryanodine receptor-1 (RyR1), in 70-75% of patients. Functional characterisation demonstrating an increased sensitivity to RyR1 agonists is necessary among other criteria for inclusion in the European Malignant Hyperthermia Group list of MH susceptibility diagnostic variants. Seven variants in the RYR1 gene, p.Glu342Lys, p.Leu2288Ser, p.Phe2340Leu, p.Arg2676Trp, p.Val3324Ala, p.Phe4076Leu, and p.Trp5020Cys, identified in MH-susceptible individuals were introduced into the cDNA for the human RYR1 gene. These variants were tested in cultured human embryonic kidney HEK293 cells for their effect on calcium release in response to the RyR1 agonist 4-chloro-m-cresol. Calcium release of each variant was compared with wild-type and benign and pathogenic controls. Each variant was subjected to curation using the European Malignant Hyperthermia Group scoring matrix and ClinGen RYR1 Variant Curation Expert Panel guidelines. Six of seven RYR1 variants (p.Glu342Lys, p.Leu2288Ser, p.Phe2340Leu, p.Arg2676Trp, p.Val3324Ala, p.Phe4076Leu) showed hypersensitivity to 4-chloro-m-cresol compared with wild-type. The p.Trp5020Cys variant did not release calcium in response to 4-chloro-m-cresol. All variants had minor allele frequencies <0.1%. Rare exome variant ensemble learner scores of p.Glu342Lys, p.Leu2288Ser, p.Phe4076Leu, and p.Trp5020Cys were >0.85, supporting pathogenicity. The variants p.Glu342Lys, p.Leu2288Ser p.Phe2340Leu, and p.Arg2676Trp are pathogenic or likely pathogenic for MH and can be used for presymptomatic testing for MH susceptibility. As current knowledge on the p.Val3324Ala, p.Phe4076Leu, and p.Trp5020Cys variants remains insufficient, they are still classified as variants of uncertain significance.

Complete primer set for amplification and expression of full-length recombinant human monoclonal antibodies from single human B cells.

Human monoclonal antibodies (mAbs) are an important segment in precision therapeutics. Various methodologies are available for generating them. Recombinant human mAbs expression from sorted single B cells is preferred for its rapid expression using mammalian vectors while maintaining in vivo immunoglobulin (Ig) pairing. The success rate of generating recombinant mAbs from single sorted human B cells directly relies on Ig heavy (IgH) and light (IgL) gene coverage of the PCR primers. Existing primer sets fail to cover all functional human Ig gene rearrangements, exhibit high degeneracy leading to non-specific amplifications and mutations arising from primer mismatch/degeneracy, and require high amplification cycles. Some existing primer sets have high coverage but are not designed for expression as recombinant mAbs. Here, we have designed a primer set to amplify all functional V(D)J transcripts in human B cell repertoire using a nested RT-PCR approach. The resultant amplicons can be cloned into mammalian vectors for expression of recombinant mAb. Non-specific amplifications were minimized using isotype-specific primers for cDNA synthesis and limiting primer degeneracy. We validated the designed primers on single sorted B cells, bulk sorted B cells and peripheral blood mononuclear cells. We were successfully able to amplify paired heavy and light chain transcripts in 38.46 % (80/208) from naive, memory and B1 B cell subsets sorted as single B cells. Paired Ig transcripts from five single B cells were cloned into expression vectors and purified from mammalian cells as recombinant mAbs. Thus, our new primer set offers significant advantages over existing primers as it allows amplification of all functional V(D)J rearrangements, facilitating rapid generation of antigen-specific recombinant antibodies from diverse human B cell repertoires following vaccinations and infections previously inaccessible due to primer limitations.

Identification of gene expression change associated with isoflurane anesthesia and neurocognitive disorders using bioinformatics methods.

Isoflurane, a commonly used anesthetic, has been linked to neurocognitive dysfunction (NCD). However, the precise relationship between isoflurane anesthesia and NCD remains unclear. Datasets related to isoflurane anesthesia were obtained from a public database. The hub genes of isoflurane anesthesia were selected using logistic regression with the least absolute shrinkage and selection operator (LASSO). Human neuroblastoma cell line SH-SY5Y was induced to differentiate into terminal neuron-like cells. The signaling pathways involved in isoflurane anesthesia and NCD were subjected to analysis, resulting in the identification of 35 signaling pathways, including the PI3K/AKT pathway, the AGE-RAGE signaling pathway and the apelin signaling pathway, which were found to be associated with both NCD and isoflurane anesthesia. A total of nine cross-expressed genes (Nfe2l2, Fgf2, Edn1, Spp1, Hmox1, Picalm, Gnb5, Eif2s1 and Gls) were identified between isoflurane anesthesia and NCD. The LASSO logistic regression model was employed to identify three hub genes (Fgf2, Gnb5, Spp1). The expression of these three hub genes was validated using other expression datasets. In human neuron-like cells, isoflurane also significantly affected the expression of three corresponding human homologous genes. The expression trends of these three genes in human cells were consistent with the expression in rat brains after isoflurane treatment, providing further evidence for the rationality of the three hub genes. The present study revealed key candidate genes and related functional signaling pathways through bioinformatics analysis and cell experiments, which may serve as the basis for isoflurane-related NCD.

Is DEXI a Multiple Sclerosis Susceptibility Gene?

The genetic landscape of multiple sclerosis (MS) has been extensively mapped, yielding significant insights into the molecular mechanisms of the disorder. Early studies highlighted key genes associated with the immune system, particularly T cells, as critical for MS susceptibility. Subsequent large-scale genome-wide association studies (GWASs) identified over 200 genetic variants linked to MS, revealing a complex interplay between MS risk and genes involved in various processes within adaptive and innate immune cells, as well as brain-resident microglia. Recently, a groundbreaking GWAS pinpointed the first gene variant associated with MS disease progression, distinguishing the mechanisms driving disease onset from those influencing progression. The C-type lectin domain family 16, member A (CLEC16A) gene within the 16p13 region has consistently been shown to be associated with increased risk of developing both MS and other autoimmune disorders. Notably, several autoimmune-associated genetic variants in CLEC16A introns act as expression quantitative trait loci for the dexamethasone-induced protein (DEXI gene, adding DEXI to the growing list of MS susceptibility genes. This review explores the molecular and functional characterization of DEXI with a particular focus on recent advances in understanding its role in autoimmunity, specifically in the context of multiple sclerosis. We underscore the importance of continued molecular investigation of susceptibility loci for MS identified in genetic studies, with the goal of translating this knowledge into clinical applications.

Development of a Genetic Test Indicating Increased AVP/V1b Signalling in Patients with Acute Depression.

A subgroup of patients with acute depression show an impaired regulation of the hypothalamic-pituitary-adrenocortical axis, which can be sensitively diagnosed with the combined dexamethasone (dex)/corticotropin releasing hormone (CRH)-test. This neuropathological alteration is assumed to be a result of hyperactive AVP/V1b signalling. Given the complicated procedure of the dex/CRH-test, this study aimed to develop a genetic variants-based alternative approach to predict the outcome of the dex/CRH-test in acute depression.Using data of a representative cohort of 352 patients with severe depression participating in the dex/CRH-test, a genome-wide interaction analysis was performed starting with an anchor single nucleotide polymorphism located in the upstream transcriptional region of the human V1b-receptor gene to predict the adrenocorticotropic hormone (ACTH) response to this test. A probabilistic neural-network-algorithm was used to develop the optimal prediction model.Overall prediction accuracy for correctly identifying high ACTH responders in the dex/CRH-test was 93.5% (sensitivity 90%; specificity 95%). Analysis of pituitary RNAseq expression data confirmed that the identified genetic interactions of the gene test translate into an interactive network of corresponding transcripts in the pituitary gland, which is the biologically relevant target tissue, with the aggregated strength of the transcript interactions significantly stronger than expected from chance.The findings suggest the suitability of the presented gene test as a proxy for hyperactive AVP/V1b signalling during an acute depressive episode, highlighting its potential as companion test for identifying patients with acute depression whose pathology can be optimally treated by specific drugs targeting the AVP/V1b-signaling cascade.

P-1825. Investigating the Impact of Immunomodulatory Drugs on Severe COVID-19 Through Transcriptomic Analysis

Abstract Background The Coronavirus disease (COVID-19) has highlighted the role of hyperinflammatory states and cytokine storms in severe SARS-CoV-2 infections, leading to extensive research into immunomodulatory drugs previously used for autoimmune diseases. Despite their promising effects, the mechanisms underlying their efficacy remain unclear. This study aims to elucidate these mechanisms through RNA-sequence data analysis.Table 1.Clinical characteristics of 30 COVID-19 patient cohort Methods Bulk RNA sequencing data from PBMC samples of COVID-19 patients was used to identify two sets of differentially expressed genes (DEGs). The first set was made by comparing COVID-19 patient data to healthy individuals using public datasets, while the second set involved DEGs from samples treated with immunomodulatory drugs versus untreated samples. Comparative analysis focused on genes with opposite expression changes, particularly those showing contrasting fold changes between COVID-19 and drug-treated samples.Figure 1.DEG array Results All COVID-19 patients met the criteria for severe or critical illness, requiring at least a high-flow nasal cannula. The median age was 66.5 years. Clinically, 46.7% underwent mechanical ventilation and the mortality rate stood at 46.7%. By arraying the table with all the samples combined from COVID-19 patient samples and immunomodulatory drug samples, DEGs that show opposite fold change signs were selected as having potential significance in COVID-19 pathways and being affected by immunomodulatory drugs. Specifically, 18 genes were upregulated in response to COVID-19 yet downregulated in immunomodulatory drug use, including key inflammatory and immune response genes such as FAP, RGS16, and IDO1. Conversely, 39 genes showed downregulation in response to COVID-19 but were upregulated in immunomodulatory drug use, featuring genes like DUSP1, TOB1, and RGMA.Table 2.List of genes found in DEG array Conclusion Through transcriptomic analysis and differential expression screening between COVID-19 and medication-treated samples, 57 were found to exhibit reversed fold changes in expression. Further investigation of these genes is expected. This research aims to uncover the shared biological mechanisms between autoimmune diseases and COVID-19, which could lead to effective drug repurposing strategies for treating COVID-19. Disclosures All Authors: No reported disclosures

Transgenic rabbits with genes of human granulocyte colony-stimulating factor and green fluorescent protein

Human granulocyte–colony stimulating factor (GCSF) is one of the pharmacological proteins that can be isolated from the milk of transgenic (TG) animals. The plasmid containing the human GCSF gene under the control of regulatory elements of the bovine β-lactoglobulin gene and the reporter green fluorescent protein (EGFP) gene under the cytomegalovirus (cmv) promoter were obtained. The use of the selected promoters ensures tissue-specific expression of the target protein in the mammary gland of the TG producing animal and a high level of early expression of the reporter protein in eukaryotic cells, which makes it possible to detect TG embryos at the cultivation stage and perform their preimplantation selection. Testing of the gene construct effectiveness was carried out on TG rabbits obtained by microinjection into the male pronucleus of zygotes. It was concluded that GFP is toxic to embryos in the early stages of development due to overexpression of the EGFP gene under a strong cmv promoter. The TG female rabbit (F0) was obtained, in which the level of human GKSF in milk and blood serum was assessed by the ELISA method. Of the 22 baby rabbits obtained from her in four kindling, two were transgenic. Offspring (F1) was obtained from the TG male F0, 56 % of which were males, of which 88 % were TG and did not differ from ordinary rabbits in terms of health. Among females, TG was 10 %, and they died within two weeks after birth.

Amelioration of a von Willebrand disease type 2B phenotype invivo upon treatment with allele-selective siRNAs.

Treatment options for the bleeding disorder von Willebrand disease type 2B (VWD2B) are insufficient and fail to address the negative effects of circulating mutant von Willebrand factor (VWF). The dominant-negative nature of VWD2B makes functionally defective VWF an interesting therapeutic target. Previous invitro studies have demonstrated the feasibility of allele-selective silencing of mutant VWF using small interfering RNAs (siRNAs) targeting common single nucleotide polymorphisms (SNPs) in the human VWF gene, an approach that can be applied irrespective of the disease-causing VWF mutation. This study aims to extend this concept to a heterozygous VWD2B mouse model (c.3946G>A; p.Val1316Met) here using mouse strain-specific genetic differences as proxy for human SNPs. Homozygous VWD2B C57BL/6J (2B-B6) mice were crossed with homozygous wild-type 129S1/SvImJ (129S) mice to create heterozygous 2B-B6.129S F1 offspring. These 2B-B6.129S mice were intravenously injected with endothelial-specific lipid nanoparticles loaded with the allele-selective siVwf.B6 or control and 96 hours later, lung Vwf messenger RNA, plasma VWF levels, and phenotypic characteristics were evaluated. Treatment with siVwf.B6 reduced total VWF levels by 50%, with an expected selective reduction in mutant VWF protein. This coincided with normalization of multimeric structure, improved VWF collagen binding/VWF antigen ratio, and normalized bleeding times in two-thirds of heterozygous 2B-B6.129S mice. Being a novel approach in the field of hemostasis, we proved, for VWD, in mice, the concept of selectively inhibiting a mutant dominant-negative allele with siRNAs targeting a single nucleotide variation rather than the disease-causing mutation. For dominant-negative VWD, this offers potential for a customized therapeutic strategy.

Marigold: a machine learning-based web app for zebrafish pose tracking

BackgroundHigh-throughput behavioral analysis is important for drug discovery, toxicological studies, and the modeling of neurological disorders such as autism and epilepsy. Zebrafish embryos and larvae are ideal for such applications because they are spawned in large clutches, develop rapidly, feature a relatively simple nervous system, and have orthologs to many human disease genes. However, existing software for video-based behavioral analysis can be incompatible with recordings that contain dynamic backgrounds or foreign objects, lack support for multiwell formats, require expensive hardware, and/or demand considerable programming expertise. Here, we introduce Marigold, a free and open source web app for high-throughput behavioral analysis of embryonic and larval zebrafish.ResultsMarigold features an intuitive graphical user interface, tracks up to 10 user-defined keypoints, supports both single- and multiwell formats, and exports a range of kinematic parameters in addition to publication-quality data visualizations. By leveraging a highly efficient, custom-designed neural network architecture, Marigold achieves reasonable training and inference speeds even on modestly powered computers lacking a discrete graphics processing unit. Notably, as a web app, Marigold does not require any installation and runs within popular web browsers on ChromeOS, Linux, macOS, and Windows. To demonstrate Marigold’s utility, we used two sets of biological experiments. First, we examined novel aspects of the touch-evoked escape response in techno trousers (tnt) mutant embryos, which contain a previously described loss-of-function mutation in the gene encoding Eaat2b, a glial glutamate transporter. We identified differences and interactions between touch location (head vs. tail) and genotype. Second, we investigated the effects of feeding on larval visuomotor behavior at 5 and 7 days post-fertilization (dpf). We found differences in the number and vigor of swimming bouts between fed and unfed fish at both time points, as well as interactions between developmental stage and feeding regimen.ConclusionsIn both biological experiments presented here, the use of Marigold facilitated novel behavioral findings. Marigold’s ease of use, robust pose tracking, amenability to diverse experimental paradigms, and flexibility regarding hardware requirements make it a powerful tool for analyzing zebrafish behavior, especially in low-resource settings such as course-based undergraduate research experiences. Marigold is available at: https://downeslab.github.io/marigold/.