Chain Transcripts Research Articles

Diversification of Antibodies: From V(D)J Recombination to Somatic Exon Shuffling.

Antibodies that gain specificity by a large insert encoding for an extra domain were described for the first time in 2016. In malaria-exposed individuals, an exon deriving from the leukocyte-associated immunoglobulin-like 1 (LAIR1) gene integrated via a copy-and-paste insertion into the immunoglobulin heavy chain encoding region. A few years later, a second example was identified, namely a dual exon integration from the leukocyte immunoglobulin-like receptor B1 (LILRB1) gene that is located in close proximity to LAIR1. A dedicated high-throughput characterization of chimeric immunoglobulin heavy chain transcripts unraveled, that insertions from distant genomic regions (including mitochondrial DNA) can contribute to human antibody diversity. This review describes the modalities of insert-containing antibodies. The role of known DNA mobility aspects, such as genomic translocation, gene conversion, and DNA fragility, is discussed in the context of insert-antibody generation. Finally, the review covers why insert antibodies were omitted from the past repertoire analyses and how insert antibodies can contribute to protective immunity or an autoreactive response.

Antigen-driven Convergent Evolution of Polysaccharide-specific "DH-less" B Cells in Glycoconjugate Immunized Mice.

Glycoconjugate vaccines elicit robust anti-polysaccharide Ab response by recruiting T-cell help. Multiple doses of glycoconjugate vaccine are required to induce long-lasting immunity. The characteristics of anti-polysaccharide Ab response have been reported previously. However, the effect of glycoconjugate booster immunization on anti-polysaccharide and anti-carrier protein Ab repertoire remains poorly understood. In this study, we used clinically relevant pneumococcal capsular polysaccharide type 14 (PCP14) conjugated with cross-reactive material 197 (CRM197) as a model glycoconjugate Ag (PCP14-CRM197). We performed a comprehensive sequence analysis of mouse mAbs generated against PCP14 and CRM197 following immunization with one or three doses of PCP14-CRM197. Analysis of the paired Ig H and L chain transcripts revealed that anti-PCP14 Ab repertoire is extremely restricted. The reoccurrence of five replacement mutations at identical positions in anti-polysaccharide mAbs generated from different mice provided evidence for Ag-driven selection in PCP14-specific B cells. Convergent evolution was observed wherein distinct V(D)J rearrangements resulted in identical or nearly identical CDR3 in anti-PCP14 mAbs. Abs that lacked DH encoded amino acids dominated the anti-PCP14 Ab response. In contrast, anti-CRM197 Ab response was quite diverse, with fewer mutations compared with the anti-PCP14 mAbs, suggesting that conjugation of the polysaccharide to a carrier protein interferes with the development of carrier protein-specific Ab responses. Our findings provide molecular insights into the maturation of Ab responses driven by booster doses of glycoconjugate. This has fundamental implications for the design of glycoconjugate vaccines, especially where the development of Ab response against the carrier protein is also crucial.

Ultralong CDR3 cattlebody use biased to switched immunoglobulin isotypes in bovine immune tissues

Abstract Alongside typical immunoglobulin (Ig), cattle express unique ultralong immunoglobulins (UL) that contain a CDR H3 comprised of a β-ribbon stalk and disulfide-linked knob that reaches over 70 amino acids in length. UL Ig exhibit potential "reach" to recessed pathogen epitopes and can provide broadly neutralizing antibodies to elusive antigens. This study explores the relative amounts of UL Ig in pre-switch isotypes (IgM and IgD) versus post-switch isotypes (IgG, IgA and IgE). Rearranged immunoglobulin heavy chain transcripts from primary and secondary lymphoid tissues of three steer were assessed with primers specific for the variable segments used in ultralong and canonical rearrangements as well as specific subisotype constant regions. Relative expression suggests biased selection for UL Ig in germinal centers and use in mucosal tissues of the ruminant gastrointestinal tract. Despite the explosion of studies exploring the immunogenetic diversification and binding possibilities of the UL Ig knob "picobody", our understanding of the use of the role of these antibodies in the Bovid immune system has been much slower. With a more complete understanding of tissue-specific expression patterns and isotype-specific immune responses using these exceptional paratopes, we'll be better poised to exploit them for improved bovine and human health.

Cannabidiol modulates hippocampal genes involved in mitochondrial function, ribosome biogenesis, synapse organization, and chromatin modifications.

Cannabidiol (CBD) is one of the main cannabinoids present in Cannabis sativa female flowers. Previous investigation has already provided insights into the CBD molecular mechanism; however, there is no transcriptome data for CBD effects on hippocampal subfields. Here, we investigate transcriptomic changes in dorsal and ventral CA1 of adult mice hippocampus after 100 mg/kg of CBD administration (i.p.) for one or seven consecutive days. C57BL/6JUnib mice were treated with either vehicle or CBD for 1 or 7 days. The collected brains were sectioned, and the hippocampal sub-regions were laser microdissected for RNA-Seq analysis. The transcriptome analysis following 7 days of CBD administration indicates the differential expression of 1559 genes in dCA1 and 2924 genes in vCA1. Furthermore, GO/KEGG analysis identified 88 significantly enriched biological process and 26 significantly enriched pathways for dCBD7, whereas vCBD7 revealed 128 enriched BPs and 24 pathways. This dataset indicates a widespread decrease of electron transport chain and ribosome biogenesis transcripts in CA1, while chromatin modifications and synapse organization transcripts were increased following CBD administration for 7 days.

Immunoglobulin light chain transcript detection by ultrasensitive RNA in situ hybridization for B-cell lymphoma diagnosis.

Evaluation of B-cell clonality can be challenging in the interpretation of lymphoid infiltrates on tissue sections. Clonality testing based on IG gene rearrangements analysis by PCR (IG-PCR) is the gold standard. Alternatively, B-cell clonality can be assessed by the recognition of immunoglobulin light chain (IgLC) restriction, by immunohistochemistry (IHC), chromogenic in situ hybridization (ISH) or flow cytometry (FC). IG-PCR requires molecular facilities, and FC requires cell suspensions, both not widely available in routine pathology units. This study evaluates the performance of B-cell clonality detection by IgLC-RNAscope® (RNAsc) in a group of 216 formalin-fixed, paraffin-embedded samples including 185 non-Hodgkin B-cell lymphomas, 11 Hodgkin lymphomas (HL) and 20 reactive samples. IgLC-RNAsc, performed in parallel with FC in 53 cases, demonstrated better performances (93% vs 83%), particularly in diffuse large B-cell lymphoma (98% vs 71%) and follicular lymphoma (93% vs 83%) diagnosis. IgLC-RNAsc was also superior to IHC and ISH especially in samples with limited tumor cell content, where IG-PCR was not informative. Performed for the first time on mediastinal lymphomas, IgLC-RNAsc identified monotypic IgLC transcripts in 69% of primary mediastinal large B-cell lymphoma (PMBCL) and 67% of mediastinal gray zone lymphomas (MGZL). IGK/L double-negative cells were detected in 1 PMBCL, 2 MGZL, and all classical HL, while monotypic IgLC expression appeared to be a hallmark in nodular lymphocyte-predominant HL. IgLC-RNAsc demonstrates to be a powerful tool in B-cell lymphoma diagnosis, above all in challenging cases with limited tumor cell content, ensuring in situ investigations on mechanisms of Ig regulation across lymphoma entities.

Genomic Features of Recombinant CHO Clones Arising from Transposon-based and Randomized Integration.

The use of transposase in cell line development (CLD) programs has experienced increased popularity over the past decade. However, few studies have described the mechanism of action and the genomic and phenotypic characteristics of clones derived from transposase. Additionally, how these traits impact long-term bioproduction is unknown. Here, we use chromosome painting, deep sequencing, and ddPCR to characterize the unique fingerprints associated with transposase-derived clones. Transposase reduces the cellular pool of transient vector as early as three days post transfection following transfection and expedites stable pool establishment by up to two weeks. Furthermore, recombinant DNA expression is significantly improved up to ~3 fold along with a greater balance of antibody heavy and light chain transcripts, resulting in higher titers in transposase generated pools. Transposase derived pools contained an often innumerable number of integration sites, representing a vast increase in integration site diversity over randomly generated pools, which were bottlenecked at 1-3 integration sites per pool. These transposase mediated integrations typically occurred in clean singlets, free of genomic scars such as deletions, inversions, and other modifications associated with legacy transfection methods which exhibited higher copy numbers per integration site. Relative declines in gene expression occur with copy number increase in the randomly generated, but not the transposase derived clones. Furthermore, transposase-derived clones were more likely to exhibit enhanced a long term stability profile, including product quality attributes such as mannose-5. This improved stability may result from circumventing mechanisms associated with the silencing of tandem repeats. Thus, transposase-mediated approaches can provide multifaceted molecular and phenotypic advantages in cell line development when compared to legacy random-integration methods.

Evidence of a Novel Mitochondrial Signature in Systemic Sclerosis Patients with Chronic Fatigue Syndrome.

Symptoms of myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS) are common in rheumatic diseases, but no studies report the frequency of these in early systemic sclerosis. There are no known biomarkers that can distinguish between patients with ME/CFS, although mitochondrial abnormalities are often demonstrated. We sought to assess the prevalence of ME/CFS in limited cutaneous SSc (lcSSc) patients early in their disease (<5 years from the onset of non-Raynaud's symptoms) and to determine if alterations in mitochondrial electron transport chain (ETC) transcripts and mitochondrial DNA (mtDNA) integrity could be used to distinguish between fatigued and non-fatigued patients. All SSc patients met ACR/EULAR classification criteria. ME/CFS-related symptoms were assessed through validated questionnaires, and the expression of ETC transcripts and mtDNA integrity were quantified via qPCR. SSc patients with ME/CFS could be distinguished from non-fatigued patients through ETC gene analysis; specifically, reduced expression of ND4 and CyB and increased expression of Cox7C. ND4 and CyB expression correlated with indicators of disease severity. Further prospective and functional studies are needed to determine if this altered signature can be further utilized to better identify ME/CFS in SSc patients, and whether ME/CFS in early SSc disease could predict more severe disease outcomes.

Identification cloning and functional analysis of novel natural antisense lncRNA CFL1-AS1 in cattle

ABSTRACT Long noncoding RNAs have been identified as important regulators of gene expression and animal development. The expression of natural antisense transcripts (NATs) transcribed in the opposite direction to protein-coding genes is usually positively correlated with the expression of homologous sense genes and is the key factor for expression. Here, we identified a conserved noncoding antisense transcript, CFL1-AS1, that plays an important role in muscle growth and development. CFL1-AS1 overexpression and knockout vectors were constructed and transfected into 293T and C2C12 cells. CFL1-AS1 positively regulated CFL1 gene expression, and the expression of CFL2 was also downregulated when CFL1-AS1 was knocked down. CFL1-AS1 promoted cell proliferation, inhibited apoptosis and participated in autophagy. This study expands the research on NATs in cattle and lays a foundation for the study of the biological function of bovine CFL1 and its natural antisense chain transcript CFL1-AS1 in bovine skeletal muscle development. The discovery of this NAT can provide a reference for subsequent genetic breeding and data on the characteristics and functional mechanisms of NATs.

Impaired Response to Polysaccharide Vaccine in Selective IgE Deficiency.

Immunoglobulin E deficiency (IgED) (defined as IgE < 2IU/mL) is enriched in patients with primary antibody deficiency (PAD). We hypothesized that selective IgED (sIgED) is a more sensitive predictor of the development of PAD than declining IgG, as IgE production typically requires two class switch recombination (CSR) events in contrast to IgG. Thus, the inability of patients with sIgED to mount an appropriate antibody response to a T-cell independent antigen or evidence of aberrant induction of ɛ germ line (ɛGL) or IgE heavy chain (IgEHC) transcripts in vitro would support the concept that sIgED is a biomarker for emerging PAD. We compared pre- and post-polysaccharide vaccination titers in healthy patients with sIgED without a history of recurrent infections or autoimmunity (n = 20) and in healthy controls (HCs) (n = 17). Subsequently, we assessed in vitro induction of εGL and IgEHC transcripts in patients with sIgED and HC (n = 6) in response to IL-4 + CD40L stimulation. Thirty percent of patients with sIgED did not have a robust vaccine response compared to 0% of HCs (p = 0.017). Individuals with sIgED with an abnormal vaccine response demonstrated persistent germline mRNA expression in their B-cells at day 5, with lower levels of IgEHC, compared to both HCs and sIgED participants with a normal vaccine response. Patients with sIgED are more likely to have abnormal antibody responses to a T cell-independent antigen and may have dysregulated CSR machinery. Following individuals with sIgED longitudinally may be beneficial in the early identification of PAD.

FLAIRR-Seq: A Method for Single-Molecule Resolution of Near Full-Length Antibody H Chain Repertoires.

Current Adaptive Immune Receptor Repertoire sequencing (AIRR-seq) using short-read sequencing strategies resolve expressed Ab transcripts with limited resolution of the C region. In this article, we present the near-full-length AIRR-seq (FLAIRR-seq) method that uses targeted amplification by 5' RACE, combined with single-molecule, real-time sequencing to generate highly accurate (99.99%) human Ab H chain transcripts. FLAIRR-seq was benchmarked by comparing H chain V (IGHV), D (IGHD), and J (IGHJ) gene usage, complementarity-determining region 3 length, and somatic hypermutation to matched datasets generated with standard 5' RACE AIRR-seq using short-read sequencing and full-length isoform sequencing. Together, these data demonstrate robust FLAIRR-seq performance using RNA samples derived from PBMCs, purified B cells, and whole blood, which recapitulated results generated by commonly used methods, while additionally resolving H chain gene features not documented in IMGT at the time of submission. FLAIRR-seq data provide, for the first time, to our knowledge, simultaneous single-molecule characterization of IGHV, IGHD, IGHJ, and IGHC region genes and alleles, allele-resolved subisotype definition, and high-resolution identification of class switch recombination within a clonal lineage. In conjunction with genomic sequencing and genotyping of IGHC genes, FLAIRR-seq of the IgM and IgG repertoires from 10 individuals resulted in the identification of 32 unique IGHC alleles, 28 (87%) of which were previously uncharacterized. Together, these data demonstrate the capabilities of FLAIRR-seq to characterize IGHV, IGHD, IGHJ, and IGHC gene diversity for the most comprehensive view of bulk-expressed Ab repertoires to date.

Cladribine treatment specifically affects peripheral blood memory B cell clones and clonal expansion in multiple sclerosis patients.

B cells are acknowledged as crucial players in the pathogenesis of multiple sclerosis (MS). Several disease modifying drugs including cladribine have been shown to exert differential effects on peripheral blood B cell subsets. However, little is known regarding functional changes within the peripheral B cell populations. In this study, we obtained a detailed picture of B cell repertoire changes under cladribine treatment on a combined immunoglobulin (Ig) transcriptome and proteome level. We performed next-generation sequencing of Ig heavy chain (IGH) transcripts and Ig mass spectrometry in cladribine-treated patients with relapsing-remitting multiple sclerosis (n = 8) at baseline and after 6 and 12 months of treatment in order to generate Ig transcriptome and Ig peptide libraries. Ig peptides were overlapped with the corresponding IGH transcriptome in order to analyze B cell clones on a combined transcriptome and proteome level. The analysis of peripheral blood B cell percentages pointed towards a significant decrease of memory B cells and an increase of naive B cells following cladribine therapy. While basic IGH repertoire parameters (e.g. variable heavy chain family usage and Ig subclasses) were only slightly affected by cladribine treatment, a significantly decreased number of clones and significantly lower diversity in the memory subset was noticeable at 6 months following treatment which was sustained at 12 months. When looking at B-cell clones comprising sequences from the different time-points, clones spanning between all three time-points were significantly more frequent than clones including sequences from two time-points. Furthermore, Ig proteome analyses showed that Ig transcriptome specific peptides could mostly be equally aligned to all three time-points pointing towards a proportion of B-cell clones that are maintained during treatment. Our findings suggest that peripheral B cell related treatment effects of cladribine tablets might be exerted through a reduction of possibly disease relevant clones in the memory B cell subset without disrupting the overall clonal composition of B cells. Our results -at least partially- might explain the relatively mild side effects regarding infections and the sustained immune response after vaccinations during treatment. However, exact disease driving B cell subsets and their effects remain unknown and should be addressed in future studies.

UNDETECTABLE IGE AS A SENTINEL BIOMARKER FOR HUMORAL IMMUNODEFICIENCY

<h3>Introduction</h3> The clinical significance of undetectable IgE (<2 IU/mL) in patients with otherwise normal immunoglobulins is unclear. We hypothesize that undetectable IgE reflects an impairment in isotype switching that could portend future development of a broader defect in humoral immunity. <h3>Methods</h3> We recruited 17 healthy adults with IgE <2 IU/mL. We compared their response to immunization with a Salmonella typhi polysaccharide vaccine relative to 16 randomly selected adults with IgE >2 IU/mL. We assessed pre-and post-vaccine IgG titers 4-6 weeks after vaccination, with a normal Salmonella typhi IgG index (STIGG index) defined as a >2-fold increase in titers. We isolated peripheral blood lymphocytes from 14 patients with undetectable IgE and induced IgE production <i>in vitro</i> via stimulation with CD40 ligand (CD40L) and IL-4. IgE production was assessed through semi-quantitative PCR of ε-germline and IgE heavy chain transcripts. <h3>Results</h3> 4/17 patients with undetectable IgE had a STIGG index ≤2, compared with 0/16 patients in the control group (p-value = 0.0582, Fisher's exact test). ε-germline and IgE heavy chain mRNA were detected in peripheral lymphocytes from all of the assessed patients with undetectable IgE. <h3>Conclusion</h3> An impaired polysaccharide vaccine response occurred more frequently in patients with undetectable IgE than in patients with an IgE >2 IU/mL, but the difference in this small pilot study was nonsignificant. A larger sample size is needed to clarify these findings. Peripheral blood lymphocytes isolated from patients with undetectable IgE are capable of producing IgE transcripts in response to CD40L/IL-4 stimulation.

Monoclonal Antibody Sequence Variants Disguised as Fragments: Identification, Characterization, and Their Removal by Purification Process Optimization

During early stage development of a therapeutic IgG1 monoclonal antibody, high levels of low molecular weight (LMW) peaks were observed by high performance size-exclusion chromatography and capillary electrophoresis. Further characterization of the LMW peak enriched HPSEC fractions using reversed phase liquid chromatography coupled to mass spectrometry showed these LMW species were 47 kDa and 50 kDa in size. However, the measured masses could not be matched to any fragments resulting from peptide bond hydrolysis. To identify these unknown LMW species, molecular characterization methods were employed, including high-throughput sequencing of RNA. Transcriptomic analysis revealed the LMW species were generated by mis-splicing events in the heavy chain transcript, which produced truncated heavy chain products that assembled with the light chain to mimic the appearance of fragments identified by routine purity assays. In an effort to improve product quality, an optimized purification process was developed. Characterization of the process intermediates confirmed removal of both LMW species by the optimized process. Our study demonstrates that deep-dive analytical characterization of biotherapeutics is critical to ensure product quality and inform process development. Transcriptomic analysis tools can help identify the cause of unknown species, and plays a key role in product and process characterization.

Mystifying Molecular Structure, Expression and Repertoire Diversity of IgM Heavy Chain Genes (Ighμ) in Clarias Catfish and Hybrids: Two Novel Transcripts in Vertebrates.

Two novel immunoglobulin heavy chain (Ighμ) transcripts encoding membrane-bound forms of IgM (mIgM) were discovered in bighead catfish, Clarias macrocephalus. The first transcript contains four constant and two transmembrane domains [Cμ1-Cμ2-Cμ3-Cμ4-TM1-TM2] that have never been reported in teleosts, and the second transcript is an unusual mIgM that has never been identified in any vertebrate [Cμ1-(Cδ2-Cδ3-Cδ4-Cδ5)-Cμ2-Cμ3-TM1-TM2]. Fluorescence in situ hybridization (FISH) in bighead catfish, North African catfish (C. gariepinus) and hybrid catfish revealed a single copy of Ighμ in individual parent catfish, while two gene copies were found in diploid hybrid catfish. Intensive sequence analysis demonstrated multiple distinct structural variabilities in the VH domain in Clarias, and hybrid catfish were defined and used to generate diversity with various mechanisms. Expression analysis of Ighμ in Aeromonas hydrophila infection of the head kidney, peripheral blood leukocytes and spleen revealed significantly higher levels in North African catfish and hybrid catfish than in bighead catfish.

Heat shock protein A4 ablation leads to skeletal muscle myopathy associated with dysregulated autophagy and induced apoptosis

BackgroundMolecular chaperones assist protein folding, facilitate degradation of misfolded polypeptides, and thereby maintain protein homeostasis. Impaired chaperone activity leads to defective protein quality control that is implicated in multiple skeletal muscle diseases. The heat shock protein A4 (HSPA4) acts as a co-chaperone for HSP70. Previously, we showed that Hspa4 deletion causes impaired protein homeostasis in the heart. However, its functional role in skeletal muscle has not been explored.MethodsWe performed a comparative phenotypic and biochemical analyses of Hspa4 knockout (KO) mice with wild-type (WT) littermates.ResultsHSPA4 is markedly upregulated in regenerating WT muscle in vivo, and in differentiated myoblasts in vitro. Hspa4-KO mice are marked by growth retardation and increased variability in body weight, accompanied by 35% mortality rates during the peri-weaning period. The surviving Hspa4-KO mice experienced progressive skeletal muscle myopathy, characterized by increased number of muscle fibers with centralized nuclei, heterogeneous myofiber size distribution, inflammatory cell infiltrates and upregulation of embryonic and perinatal myosin heavy chain transcripts. Hspa4-KO muscles demonstrated an accumulation of autophagosome-associated proteins including microtubule associated protein1 light chain 3-II (LC3-II) and p62/sequestosome accompanied by increased number of TUNEL-positive nuclei.ConclusionsOur findings underscore the indispensable role of HSPA4 in maintenance of muscle integrity through contribution in skeletal muscle autophagy and apoptosis, which might provide a novel therapeutic strategy for skeletal muscle morbidities.

Prevalence of Allelic Variants and Clonality of IGHV1-69 Expressing B-Cells in Patients with Different Severity of COVID 19 Disease

Introduction: Since the first months of the COVID-19 pandemic, efforts have been made to understand the importance of broadly neutralising natural antibodies in determining the response to SARS-CoV-2. Previous studies have shown that allelic variants of the IGHV1-69 gene play a dominant role in protective natural antibody responses to several other viral pathogens, including influenza virus, hepatitis C virus, human immunodeficiency virus and, most notably, the SARS-CoV-2-related viruses SARS-CoV and MERS-CoV. These allelic variants are commonly known as 51p1-related and differ from the other IGHV1-69 alleles (known as hv1263-related) in the presence of a Phe54 residue in the CDR2 region. Importantly, crystallographic studies have shown that the Phe54 residue is critical for the binding of IGHV1-69 antibodies to the SARS-CoV and MERS-CoV spike proteins. In this study, we evaluated the prevalence of 51p1 and hv1263 alleles and the clonality of 51p1- and hv1263-expressing B cells in a large cohort of healthy individuals and COVID-19 patients and correlated the findings with the severity of the disease.Мaterials and methods: A total of 419 samples were included in the study, of which 78 asymptomatic/mildly symptomatic individuals, 200 hospitalized patients with severe disease, 94 critically ill patients and 47 healthy donors. Peripheral blood was collected 8-20 days after the onset of symptoms and total cellular RNA was extracted from whole blood using an automated procedure. Аllelle-specific Ig-gene fingerprinting of IgM heavy chain transcripts was used to simultaneously analyse the clonality of the IgM+ B-cell population and the clonality of the 51p1- and hv1263-expressing B cell populations. The significance of the differences in the prevalence of clonal B-cell populations between healthy donors and patients and between patients with different severity of the disease was calculated with the Chi-Square test.Results: Analysis of the clonality of the IgM+ B-cell population showed a polyclonal pattern in most of the investigated healthy individuals (33/47, 70%) but in only 20% of all SARS-CoV-2 infected individuals (75/372, p<0.001). A significant difference was also observed between mildly affected and severely/critically ill patients [31/78 (39.7%) vs. 44/294 (15%), respectively) (p<0.001)], but not between severely and critically ill patients [28/200 (14,%) vs. 16/94 (17,1%), (p=n.s.)]. No 51p1 transcripts were detected in 74/372 (19.9%) of SARS-CoV-2 infected individuals and in 14/47 (29,8%) of the control group (p>0,01), while hv1263 transcripts were not detected in 155/289 (53,6%) and in 27/47 (68,6%) tasted patients and controls, respectively (p>0,05). We did not find a statistically significant difference in the prevalence of 51p1 and hv1263 alleles between patients with different disease severity. However, a significantly higher number of patients displayed clonal expansions of 51p1- or hv1263-expressing B cells (219/372(58.9%) and 118/244 (48,4%), respectively in comparison to healthy donors [5/47(10.6%) and 7/47(14.9%), respectively]. There was no statistically significant difference between mildly affected and severely/critically ill patients in the clonallity status of 51p1- 38/61 (62,3%) and 182/237 (76,7%) respectively or between hv1263- expressing B cells in the same two groups of patients [20/25 (80%) and 98/109 (89,9%), p>0.05].Conclusions: Our results show that SARS-CoV-2 infection stimulates clonal expansions of IGHV1-69 -expressing B-cells, but this is independent of the severity of the disease. In addition, no difference in the prevalence of IGHV1-69 alleles was observed between patients at different stages of the disease, indicating that natural neutralizing antibodies encoded by this gene are not an important determinant of COVID-19 severity and progression. DisclosuresNo relevant conflicts of interest to declare.

A Comprehensive Analysis of the Colonic Flora Diversity, Short Chain Fatty Acid Metabolism, Transcripts, and Biochemical Indexes in Heat-Stressed Pigs.

Heat stressed pigs show typical characteristics of inflammatory bowel disease (IBD). However, little is known about the pathogenesis of heat stress (HS)-induced IBD in pigs. In this study, we determined the effects of HS on colon morphology, intestinal microbiota diversity, transcriptome genes (transcripts), and short chain fatty acids (SCFAs) metabolism in pigs. In addition, the correlation among these parameters was analyzed by weighted gene co-expression network analysis. Results showed that the liver and kidney functions related to blood biochemical indexes were partially changed in pigs under HS. Furthermore, the levels of diamine oxidase and D-lactic acid were significantly increased, whereas the levels of secretory immunoglobulin A were decreased. The integrity of colonic tissue was damaged under HS, as bleeding, lymphatic infiltration, and villi injury were observed. The concentrations of SCFAs in the colon, such as acetic acid and butyric acid, were decreased significantly. In addition, the composition of colon microbiota, such as decrease in Lactobacillus johnsonii, Lactobacillus reuteri and increase in Clostridium sensu stricto 1 of day 7 and 14 while under HS. These changes were associated with changes in the concentration of SCFAs and biochemical indexes above mentioned. Differentially expressed genes were enriched in the nucleotide-binding oligomerization domain-like receptor signaling pathway, Th17 cell differentiation, and IBD pathway, which were also associated with the changes in SCFAs. Thus, the structure, diversity of intestinal microorganisms, and changes in the levels of SCFAs in colon of heat stressed pigs changed significantly, contributing to the activation of immune response and inflammatory signal pathways and causing abnormal physiological and biochemical indexes and intestinal mucosal damage. These results highlight the interconnections between intestinal microbiota, SCFAs, and immune response and their role in the pathogenesis of stress induced IBD therapy.

Detailed characterization of the transcriptome of single B cells in mantle cell lymphoma suggesting a potential use for SOX4

Mantle cell lymphoma (MCL) is a malignancy arising from naive B lymphocytes with common bone marrow (BM) involvement. Although t(11;14) is a primary event in MCL development, the highly diverse molecular etiology and causal genomic events are still being explored. We investigated the transcriptome of CD19+ BM cells from eight MCL patients at single-cell level. The transcriptomes revealed marked heterogeneity across patients, while general homogeneity and clonal continuity was observed within the patients with no clear evidence of subclonal involvement. All patients were SOX11+CCND1+CD20+. Despite monotypic surface immunoglobulin (Ig) κ or λ protein expression in MCL, 10.9% of the SOX11 + malignant cells expressed both light chain transcripts. The early lymphocyte transcription factor SOX4 was expressed in a fraction of SOX11 + cells in two patients and co-expressed with the precursor lymphoblastic marker, FAT1, in a blastoid case, suggesting a potential prognostic role. Additionally, SOX4 was found to identify non-malignant SOX11– pro-/pre-B cell populations. Altogether, the observed expression of markers such as SOX4, CD27, IgA and IgG in the SOX11+ MCL cells, may suggest that the malignant cells are not fixed in the differentiation state of naïve mature B cells, but instead the patients carry B lymphocytes of different differentiation stages.

Actinin BioID reveals sarcomere crosstalk with oxidative metabolism through interactions with IGF2BP2.

SUMMARYActinins are strain-sensing actin cross-linkers that are ubiquitously expressed and harbor mutations in human diseases. We utilize CRISPR, pluripotent stem cells, and BioID to study actinin interactomes in human cardiomyocytes. We identify 324 actinin proximity partners, including those that are dependent on sarcomere assembly. We confirm 19 known interactors and identify a network of RNA-binding proteins, including those with RNA localization functions. In vivo and biochemical interaction studies support that IGF2BP2 localizes electron transport chain transcripts to actinin neighborhoods through interactions between its K homology (KH) domain and actinin’s rod domain. We combine alanine scanning mutagenesis and metabolic assays to disrupt and functionally interrogate actinin-IGF2BP2 interactions, which reveal an essential role in metabolic responses to pathological sarcomere activation using a hypertrophic cardiomyopathy model. This study expands our functional knowledge of actinin, uncovers sarcomere interaction partners, and reveals sarcomere crosstalk with IGF2BP2 for metabolic adaptation relevant to human disease.

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