Biology subjects

Thomas, R.

Publications and source records attributed to Thomas, R..

At least 55 records · Page 3Linked to original sources

bioRxiv2024

Longitudinal cellular and humoral immune responses following Covid-19 BNT162b2-mRNA-based booster vaccination of craft and manual workers in Qatar.

IntroductionIn March 2020, the rapid spread of SARS-CoV-2 prompted global vaccination campaigns to mitigate COVID-19 disease severity and mortality. The 2-dose BNT162b2- mRNA vaccine effectively reduced infection and mortality rates, however, waning vaccine effectiveness necessitated the introduction of a third vaccine dose or booster. To assess the magnitude and longevity of booster-induced immunity, we conducted a longitudinal study of SARS-CoV-2 specific cellular and humoral immune responses among Qatars vulnerable craft and manual worker community. We also investigated the impact of prior naturally acquired immunity on booster vaccination efficacy. MethodsSeventy healthy participants were enrolled in the study, of whom half had prior SARS-CoV-2 infection. Blood samples were collected before and after booster vaccination to evaluate immune responses through SARS- CoV-2 specific ELISpots, IgG ELISA, neutralization assays, and flow cytometric immunophenotyping ResultsT cell analysis revealed increased Th1 cellular responses, marked by enhanced IFN-{gamma} release, in recently infected participants, which was further enhanced by booster vaccination for up to 6-months. Furthermore, booster vaccination stimulated cytotoxic T cell responses in infection-naive participants, characterized by granzyme B production. Both natural SARS-CoV-2 infection and booster vaccination induced robust and durable SARS-CoV-2 specific humoral immune responses, with high neutralizing antibody levels. Prior natural infection was also linked to an increased number of class- switched B cells prior to booster vaccination. ConclusionThese findings underscore the importance of booster vaccination in enhancing anti-viral immunity across both infection-naive and previously infected individuals, enhancing distinct arms of the anti-viral immune response and prolonging naturally acquired immunity.

immunology↗

bioRxiv2024

Immunomodulatory effects of tumor Lactate Dehydrogenase C (LDHC) in breast cancer

BackgroundImmunotherapy has significantly improved outcomes for cancer patients; however, its clinical benefits vary among patients and its effectiveness across breast cancer subtypes remains uncertain. To enhance its efficacy, it is important to gain more insight into tumor-intrinsic immunomodulatory factors that could be used as therapeutic targets. We previously identified Lactate Dehydrogenase C (LDHC) to be a promising anti-cancer target due to its role in regulating cancer cell genomic integrity. In this study, we investigated the effects of tumor LDHC expression on immune responses. MethodsTIMER AND TIDE deconvolution methods were used to investigate the relationship between tumor LDHC expression, immune cell infiltration and T cell dysfunction. Multiplex cytokine assays and flow cytometry analyses of breast cancer cell monocultures, and direct and indirect cancer cell-immune cell co-culture models were performed to assess the effect of LDHC knockdown on the secretion of inflammatory mediators and the expression of immune checkpoint molecules. T cell activity was determined by IFN-{gamma} ELISPot assays and 7-AAD viability flow cytometry of cancer cells in direct co-culture. ResultsTIMER and TIDE analyses revealed that tumor LDHC expression is associated with T cell dysfunction in breast cancer and worse post-immunotherapy survival in melanoma. Depletion of LDHC in three breast cancer cell lines (MDA-MB-468, BT-549, HCC-1954) enhanced T cell activation and cytolytic function (4-hour direct co-culture). Analysis of cancer cell monocultures revealed an increase in secreted pro-inflammatory cytokines (IFN-{gamma}, GM- CSF, MCP-1, CXCL1), a decrease in immunosuppressive factors (IL-6, Gal-9) and a reduction in tumor cell surface PD-L1 expression following LDHC knockdown. Using 72-hour direct co- cultures with LDHC-silenced cancer cells, we observed a decrease in tumor-promoting cytokines (IL-1{beta}, IL-4 and IL-6) and an increase in the tumor-inhibiting cytokine CXCL1. Furthermore, LDHC knockdown reduced the number of CD8+ T cells expressing PD-1 and CTLA-4, as well as the cell surface expression of CTLA-4, TIGIT, TIM3, and VISTA. ConclusionsOur findings suggest that targeting LDHC may improve anti-tumor immune responses by modulating the secretion of pro- and anti-tumorigenic cytokines and impairing immune checkpoint signaling. Further studies are needed to elucidate the molecular mechanisms by which LDHC modulates these responses in breast cancer.

cancer biology↗

bioRxiv2024

Single-cell analyses reveal that monocyte gene expression profiles influence HIV-1 reservoir size in acutely treated cohorts

Elimination of latent HIV-1 is a major goal of AIDS research but the host factors determining the size of these reservoirs are poorly understood. Here, we investigated whether differences in host gene expression modulate the size of the HIV-1 reservoir during suppressive ART. Peripheral blood mononuclear cells (PBMC) from fourteen individuals initiating ART during acute infection who demonstrated effective viral suppression but varying magnitude of total HIV-1 DNA were characterized by single-cell RNA sequencing (scRNA-seq). Differentially expressed genes and enriched pathways demonstrated increased monocyte activity in participants with undetectable HIV-1 reservoirs. IL1B expression in CD14+ monocytes showed the greatest fold difference. The inverse association of IL1B with reservoir size was validated in an independent cohort comprised of 38 participants with different genetic backgrounds and HIV-1 subtype infections, and further confirmed with intact proviral DNA assay (IPDA(R)) measurements of intact HIV-1 proviruses in a subset of the samples. Modeling interactions with cell population frequencies showed that monocyte IL1B expression associated inversely with reservoir size in the context of higher frequencies of central memory CD4+ T cells, implicating an indirect effect of IL1B via the cell type well established to be a reservoir for persistent HIV-1. Signatures consisting of co-expressed genes including IL1B were highly enriched in the "TNF signaling via NF-{kappa}B" geneset. Functional analyses in cell culture revealed that IL1B activates NF-{kappa}B, thereby promoting productive HIV-1 infection while simultaneously suppressing viral spread, suggesting a natural latency reversing activity to deplete the reservoir in ART treated individuals. Altogether, unbiased high throughput scRNA-seq analyses revealed that monocyte IL1B variation could decrease HIV-1 proviral reservoirs in individuals initiating ART during acute infection.

immunology↗

bioRxiv2024

Role of the SAF-A SAP domain in X inactivation, transcription, splicing, and cell proliferation

SAF-A is conserved throughout vertebrates and has emerged as an important factor regulating a multitude of nuclear functions, including lncRNA localization, gene expression, and splicing. SAF-A has several functional domains, including an N-terminal SAP domain that binds directly to DNA. Phosphorylation of SAP domain serines S14 and S26 are important for SAF-A localization and function during mitosis, however whether these serines are involved in interphase functions of SAF-A is not known. In this study we tested for the role of the SAP domain, and SAP domain serines S14 and S26 in X chromosome inactivation, protein dynamics, gene expression, splicing, and cell proliferation. Here we show that the SAP domain serines S14 and S26 are required to maintain XIST RNA localization and polycomb-dependent histone modifications on the inactive X chromosome in female cells. In addition, we present evidence that an Xi localization signal resides in the SAP domain. We found that that the SAP domain is not required to maintain gene expression and plays only a minor role in mRNA splicing. In contrast, the SAF-A SAP domain, in particular serines S14 and S26, are required for normal protein dynamics, and to maintain normal cell proliferation. We propose a model whereby dynamic phosphorylation of SAF-A serines S14 and S26 mediates rapid turnover of SAF-A interactions with DNA during interphase.

cell biology↗

bioRxiv2024

Exploring the impacts of human breast milk functional lipidome on infant health and growth outcomes in early life using lipid bioinformatics

Human breast milk lipidome is complex, and how changes in the functional lipid metabolism converge systematically to alter infants health outcomes is poorly understood. We used human breast milk and infant-mother dyads as a test system to demonstrate how the application of improved lipid bioinformatics can be effective in discerning systematic changes in functional lipid metabolism providing novel discoveries of how lactational programming in early life can influence infant health and growth outcomes. The study consisted of 40 mother-infant dyads where breast milk, maternal diet, infant anthropometrics [fat mass index (FMI), length z score, BMI z score, fat-free mass index (FFMI)], and infant atopic disease outcome (ear infection, cold, wheezing, diarrhea, and eczema) were collected at one and four months postpartum. Integrated Lipid Bioinformatics analyses were conducted using XLSTAT, Metaboanalyst 5.0. R software, Lipid Search, Xcalibur, and Cytoscape software. The results showed breast milk lipidome ordinated into distinct clusters based on maternal BMI status, and differences in developmental and atopic disease outcomes following redundancy analysis. Specifically, lipids from obese mothers clustered with FMI and eczema, while lipids from non-obese mothers clustered with FFM and wheezing. Receiver operating analysis was effective in identifying potential lipid biomarkers that were significantly associated with infant FMI, FFMI, and eczema during early life. Sphingolipid and glycerophospholipid pathways were significantly associated with the altered breast milk lipidome impacting infant development and atopic disease outcome during the first year of life. The findings following the advanced lipid bioinformatics suggest that the breastmilk functional lipid metabolism appears to play a key role in lipid-mediated lactational programming influencing development and atopic disease outcome, and present opportunities for potential dietary intervention in early life.

bioinformatics↗

bioRxiv2024

Return of intracranial beta oscillations and traveling waves with recovery from traumatic brain injury

Traumatic brain injury (TBI) remains a pervasive clinical problem associated with significant morbidity and mortality. However, TBI remains clinically and biophysically ill-defined, and prognosis remains difficult even with the standardization of clinical guidelines and advent of multimodality monitoring. Here we leverage a unique data set from TBI patients implanted with either intracranial strip electrodes during craniotomy or quad-lumen intracranial bolts with depth electrodes as part of routine clinical practice. By extracting spectral profiles of this data, we found that the presence of narrow-band oscillatory activity in the beta band (12-30 Hz) closely corresponds with the neurological exam as quantified with the standard Glasgow Coma Scale (GCS). Further, beta oscillations were distributed over the cortical surface as traveling waves, and the evolution of these waves corresponded to recovery from coma, consistent with the putative role of waves in perception and cognitive activity. We consequently propose that beta oscillations and traveling waves are potential biomarkers of recovery from TBI. In a broader sense, our findings suggest that emergence from coma results from recovery of thalamo-cortical interactions that coordinate cortical beta rhythms.

neuroscience↗

bioRxiv2024

Parkinson's LRRK2-G2019S risk gene mutation drives sex-specific behavioral and cellular adaptations to chronic variable stress

Anxiety is a psychiatric non-motor symptom of Parkinsons that can appear in the prodromal period, prior to significant loss of brainstem dopamine neurons and motor symptoms. Parkinsons-related anxiety affects females more than males, despite the greater prevalence of Parkinsons in males. How stress, anxiety and Parkinsons are related and the basis for a sex-specific impact of stress in Parkinsons are not clear. We addressed this using young adult male and female mice carrying a G2019S knockin mutation of leucine-rich repeat kinase 2 (Lrrk2G2019S) and Lrrk2WT control mice. In humans, LRRK2G2019S significantly elevates the risk of late-onset Parkinsons. To assess within-sex differences between Lrrk2G2019S and control mice in stress-induced anxiety-like behaviors in young adulthood, we used a within-subject design whereby Lrrk2G2019S and Lrrk2WT control mice underwent tests of anxiety-like behaviors before (baseline) and following a 28 day (d) variable stress paradigm. There were no differences in behavioral measures between genotypes in males or females at baseline, indicating that the mutation alone does not produce anxiety-like responses. Following chronic stress, male Lrrk2G2019S mice were affected similarly to male wildtypes except for novelty-suppressed feeding, where stress had no impact on Lrrk2G2019S mice while significantly increasing latency to feed in Lrrk2WT control mice. Female Lrrk2G2019S mice were impacted by chronic stress similarly to wildtype females across all behavioral measures. Subsequent post-stress analyses compared cFos immunolabeling-based cellular activity patterns across several stress-relevant brain regions. The density of cFos-activated neurons across brain regions in both male and female Lrrk2G2019S mice was generally lower compared to stressed Lrrk2WT mice, except for the nucleus accumbens of male Lrrk2G2019S mice, where cFos-labeled cell density was significantly higher than all other groups. Together, these data suggest that the Lrrk2G2019S mutation differentially impacts anxiety-like behavioral responses to chronic stress in males and females that may reflect sex-specific adaptations observed in circuit activation patterns in stress-related brain regions.

neuroscience↗

bioRxiv2024

Purine and pyrimidine synthesis differently affect the strength of the inoculum effect for aminoglycoside and B-lactam antibiotics.

The inoculum effect has been observed for nearly all antibiotics and bacterial species. However, explanations accounting for its occurrence and strength are lacking. We previously found that growth productivity, which captures the relationship between [ATP] and growth, can account for the strength of the inoculum effect for bactericidal antibiotics. However, the molecular pathway(s) underlying this relationship, and therefore determining the inoculum effect, remain undiscovered. We show that nucleotide synthesis can determine the relationship between [ATP] and growth, and thus the strength of inoculum effect in an antibiotic class-dependent manner. Specifically, and separate from activity through the tricarboxylic acid cycle, we find that transcriptional activity of genes involved in purine and pyrimidine synthesis can predict the strength of the inoculum effect for {beta}-lactam and aminoglycosides antibiotics, respectively. Our work highlights the antibiotic class-specific effect of purine and pyrimidine synthesis on the severity of the inoculum effect and paves the way for intervention strategies to reduce the inoculum effect in the clinic.

microbiology↗

bioRxiv2024

A TBX5-dependent compartment boundary patterns the cardiac interventricular septum

Failure of septation of the interventricular septum (IVS) is the most common congenital heart defect (CHD), but mechanisms for patterning the IVS are largely unknown. Here, we show that a Tbx5+/Mef2cAHF+ progenitor lineage forms a compartment boundary bisecting the IVS. This coordinated population originates at a first- and second heart field interface. Ablation of Tbx5+/Mef2cAHF+ progenitors cause IVS disorganization, right ventricular hypoplasia and mixing of IVS lineages. Reduced dosage of the CHD transcription factor TBX5 disrupts boundary position and integrity, resulting in ventricular septation defects (VSDs) and patterning defects, including misexpression of Slit2 and Ntn1, which encode guidance cues. Reducing NTN1 dosage partly rescues cardiac defects in Tbx5 mutant embryos. Loss of Slit2 or Ntn1 causes VSDs and perturbed septal lineage distributions. Thus, we identify Tbx5 as a candidate selector gene, directing progenitors and regulating essential cues, to pattern a compartment boundary for proper cardiac septation, revealing mechanisms for cardiac birth defects.

developmental biology↗

bioRxiv2023

A genome-wide CRISPR/Cas9 screen identifies genes that regulate the cellular uptake of α-synuclein fibrils by modulating heparan sulfate proteoglycans

Synucleinopathies are characterized by the accumulation and propagation of -synuclein (-syn) aggregates throughout the brain, leading to neuronal dysfunction and death. Understanding how these aggregates propagate from cell to cell in a prion-like fashion thus holds great therapeutic promises. Here, we focused on understanding the cellular processes involved in the entry and accumulation of pathological -syn aggregates. We used an unbiased FACS-based genome-wide CRISPR/Cas9 knockout (KO) screening to identify genes that regulate the accumulation of -syn preformed fibrils (PFFs) in cells. We identified key genes and pathways specifically implicated in -syn PFFs intracellular accumulation, including heparan sulfate proteoglycans (HSPG) biosynthesis and Golgi trafficking. We show that all confirmed hits affect heparan sulfate (HS), a post-translational modification known to act as a receptor for proteinaceous aggregates including of -syn and tau. Intriguingly, KO of SLC39A9 and C3orf58 genes, encoding respectively a Golgi-localized exporter of Zn2+, and the Golgi-localized putative kinase DIPK2A, specifically impaired the uptake of -syn PFFs uptake but not of tau oligomers, by preventing the binding of PFFs to the cell surface. Mass spectrometry-based analysis of HS chains indicated major defects in HS maturation in SLC39A9 and C3orf58 KO cells, explaining the cell surface binding deficit. Our findings now clearly establish these two genes as HSPG-modulating factors. Interestingly, C3orf58 KO human iPSC-derived microglia exhibited a strong reduction in their ability to internalize -syn PFFs. Altogether, our data establish HSPGs as major receptors for -syn PFFs binding on the cell surface and identifies new players in -syn PFFs cell surface binding and uptake.

cell biology↗

bioRxiv2023

Transmission of highly virulent CXCR4 tropic HIV-1 through the mucosal route in an individual with a wild-type CCR5 genotype

Nearly all transmitted/founder (T/F) HIV-1 are CCR5 (R5)-tropic. While previous evidence suggested that CXCR4 (X4)-tropic HIV-1 are transmissible, detection was not at the earliest stages of acute infection. Here, we identified an X4-tropic T/F HIV-1 in a participant in acute infection cohort. Coreceptor assays demonstrated that this T/F virus is strictly CXCR4 tropic. The participant experienced significantly faster CD4 depletion compared with R5 virus infected participants in the same cohort. Naive and central memory CD4 subsets declined faster than effector and transitional memory subsets. All CD4 subsets, including naive, were productively infected. Increased CD4+ T cell activation was observed over time. This X4-tropic T/F virus is resistant to broadly neutralizing antibodies (bNAbs) targeting V1/V2 and V3 regions. These findings demonstrate that X4-tropic HIV-1 is transmissible through the mucosal route in people with the wild-type CCR5 genotype and have implications for understanding the transmissibility and immunopathogenesis of X4-tropic HIV-1.

microbiology↗

bioRxiv2023

New insights into the systematics of the Afrotropical Amblyomma marmoreum complex (Acari, Ixodidae) and a novel Rickettsia africae strain using morphological and metagenomic approaches

The Amblyomma marmoreum complex includes some Afrotropical species, such as Amblyomma sparsum, a three-host tick that parasitizes reptiles, birds, and mammals, and is a recognized vector of Ehrlichia ruminatum. However, the lack of morphological, genetic and ecological data on A. sparsum has caused considerable confusion in its identification among A. marmoreum complex members. In this study, we used microscopy and metagenomic approaches to analyze A. sparsum ticks collected from a puff adder snake (Bitis arietans) in southwest Senegal (an endemic rickettsioses area) in order to supplement previous morphological descriptions, provide novel genomic data for the A. marmoreum complex, and search for some associated spotted fever agent. Based on stereoscope and scanning electron microscopy (SEM) morphological evaluations, we provided high-quality images and new insights about punctation and enameling in the male of A. sparsum to facilitate identification for future studies. Additionally, the metagenomic approach allowed us assembly the complete mitochondrial genome of A. sparsum, as well as the nearly entire chromosome and complete plasmid sequences of a novel Rickettsia africae strain. Phylogenomic analyses showed a close relationship between A. sparsum and A. nuttalli for the first time and confirmed the position of A. sparsum within the A. marmoreum complex. Our results provide new insights into the systematic of A. sparsum and A. marmoreum complex, as well as the genetic diversity of R. africae in Afrotropical region. Future studies should consider the possibility that A. sparsum may be a competent vector for R. africae.

zoology↗

bioRxiv2023

TOM20 Gates PINK1 Activity And Mediates Its Tethering Of The TOM And TIM23 Translocases Upon Mitochondrial Stress

Mutations in PTEN-induced putative kinase 1 (PINK1) cause autosomal recessive early onset Parkinson disease (PD). PINK1 is a Ser/Thr kinase that regulates mitochondrial quality control by triggering mitophagy mediated by the ubiquitin ligase Parkin. Upon mitochondrial damage, PINK1 accumulates on the outer mitochondrial membrane (OMM) forming a high molecular weight complex with the translocase of the outer membrane (TOM). PINK1 then phosphorylates ubiquitin, which enables recruitment and activation of Parkin followed by autophagic clearance of the damaged mitochondrion. Thus, Parkin-dependent mitophagy hinges on the stable accumulation of PINK1 on the TOM complex. Yet, the mechanism linking mitochondrial stressors to PINK1 accumulation and whether the translocases of the inner membrane (TIMs) are also involved, remain unclear. Herein, we demonstrate that mitochondrial stress induces the formation of a PINK1-TOM-TIM23 supercomplex in human cultured cell lines, dopamine neurons, and midbrain organoids. Moreover, we show that PINK1 is required to stably tether the TOM to TIM23 complexes in response to stress, such that the supercomplex fails to accumulate in cells lacking PINK1. This tethering is dependent on an interaction between the PINK1 NT-CTE module and the cytosolic domain of the Tom20 subunit of the TOM complex, the disruption of which, by either designer or PD-associated PINK1 mutations, inhibits downstream mitophagy. Together, the findings provide key insight into how PINK1 interfaces with the mitochondrial import machinery, with important implications for the mechanisms of mitochondrial quality control and PD pathogenesis.

biochemistry↗

bioRxiv2023

Post-acute immunological and behavioral sequelae in mice after Omicron infection

Progress in understanding long COVID and developing effective therapeutics is hampered in part by the lack of suitable animal models. Here we used ACE2-transgenic mice recovered from Omicron (BA.1) infection to test for pulmonary and behavioral post-acute sequelae. Through in-depth phenotyping by CyTOF, we demonstrate that naive mice experiencing a first Omicron infection exhibit profound immune perturbations in the lung after resolving acute infection. This is not observed if mice were first vaccinated with spike-encoding mRNA. The protective effects of vaccination against post-acute sequelae were associated with a highly polyfunctional SARS-CoV-2-specific T cell response that was recalled upon BA.1 breakthrough infection but not seen with BA.1 infection alone. Without vaccination, the chemokine receptor CXCR4 was uniquely upregulated on multiple pulmonary immune subsets in the BA.1 convalescent mice, a process previously connected to severe COVID-19. Taking advantage of recent developments in machine learning and computer vision, we demonstrate that BA.1 convalescent mice exhibited spontaneous behavioral changes, emotional alterations, and cognitive-related deficits in context habituation. Collectively, our data identify immunological and behavioral post-acute sequelae after Omicron infection and uncover a protective effect of vaccination against post-acute pulmonary immune perturbations.

microbiology↗

bioRxiv2023

Isoform-specific translational control is evolutionarily conserved in primates

Alternative splicing (AS) alters messenger RNA (mRNA) coding capacity, localization, stability, and translation. Here we use comparative transcriptomics to identify cis-acting elements coupling AS to translational control (AS-TC). We sequenced total cytosolic and polyribosome-associated mRNA from human, chimpanzee, and orangutan induced pluripotent stem cells (iPSCs), revealing thousands of transcripts with splicing differences between subcellular fractions. We found both conserved and species-specific polyribosome association patterns for orthologous splicing events. Intriguingly, alternative exons with similar polyribosome profiles between species have stronger sequence conservation than exons with lineage-specific ribosome association. These data suggest that sequence variation underlies differences in the polyribosome association. Accordingly, single nucleotide substitutions in luciferase reporters designed to model exons with divergent polyribosome profiles are sufficient to regulate translational efficiency. We used position specific weight matrixes to interpret exons with species-specific polyribosome association profiles, finding that polymorphic sites frequently alter recognition motifs for trans-acting RNA binding proteins. Together, our results show that AS can regulate translation by remodeling the cis-regulatory landscape of mRNA isoforms.

molecular biology↗

bioRxiv2023

Modulation of SLFN11 induces changes in DNA Damage response

BackgroundLack of Schlafen family member 11 (SLFN11) expression has been recently identified as a dominant genomic determinant of response to DNA damaging agents in numerous cancer types. Thus, strategies aimed at increasing SLFN11 could be used to restore chemosensitivity of refractory cancers. As oncogenic downregulation is often driven by methylation of the promotor region, we explore the demethylation effect of 5-aza-2-deoxycytidine (decitabine), on the SLFN11 gene methylation. Since SLFN11 has been reported as an interferon inducible gene, and interferon is secreted during an active anti-tumor immune response, we investigated the in vitro effect of IFN-{gamma} on SLFN11 expression in breast cancer cell lines. A second broader approach to show cross talk between immune cells and SLFN11 expression is indirect co-culture of breast cancer cells with activated PBMCs and evaluate if this can drive SLFN11 upregulation. Finally, as a definitive and specific way to modulate SLFN11 expression we implemented SLFN11 dCas9 (dead CRISPR associated protein 9) systems to specifically increase or decrease SLFN11 expression. ResultsWe first confirmed a correlation previously reported between methylation of SLFN11 promoter and its expression across multiple cell lines. We showed in-vitro that decitabine and IFN-{gamma} could increase moderately the expression of SLFN11 in both BT- 549 and T47D cell lines, but not in strongly methylated cell lines such as MDA-MB-231. Though, in-vitro, the co-culture of the same cell lines with CD8-CD25 activated PBMC failed to increase SLFN11 expression. On the one hand, the use of a CRISPR-dCas9 UNISAM system could increase SLFN11 expression significantly (up to 5-fold), stably and specifically in BT-549 and T47D cancer cell lines. Though, this system also failed to force a strong expression of SLFN11 in cell lines with robust SLFN11 promoter methylation such as MDA-MB-231. On the other hand, the use of CRISPR-dCas9 KRAB could significantly reduce the expression of SLFN11 in BT-549 and T47D. We then used the modified cell lines to confirm the alteration in chemo sensitivity of those cells to treatment with DNA Damaging Agents (DDAs) such as Cisplatin and Epirubicin or DNA Damage Response (DDRs) drugs like Olaparib. RNAseq was used to elucidate the mechanisms of action affected by the alteration in SLFN11 expression. ConclusionTo our knowledge this is the first report of the stable non-lethal increase of SLFN11 expression in a cancer cell line. Our results show that induction of SLFN11 expression can enhance DDA and DDR sensitivity in breast cancer cells and dCas9 systems may represent a novel approach to increase SLFN11 and achieve higher sensitivity to chemotherapeutic agents, improving outcome or decreasing required drug concentrations. SLFN11-targeting therapies might be explored pre-clinically to develop personalized approaches.

cancer biology↗

bioRxiv2023

Incidences of Helicobacter infection in pigs and tracing occupational hazard in pig farmers

Helicobacter species (H. sp.) is a gram-negative spiral-shaped motile bacteria that causes gastritis in pigs and also colonizes the human stomach. The current study seeks to assess the prevalence of various H. sp. in the gastric mucosa of slaughtered and dead pigs, as well as the prevalence of Helicobacter infection among pig farmers. A total of 403 stomach samples from various pig slaughter points, 74 necropsy samples from various pig farms and 97 stool samples from pig farmers were collected from Assam, India. Among 477 pig stomach samples tested, 214 samples with gastritis (20.09%) showed Gram negative, spiral-shaped organisms in brush cytology from the mucosal surface, and the rest of the 263 stomach samples without any gastric lesion showed only 3.04% Gram negative, spiral-shaped organisms. In ultrastructure investigation, Scanning Electron Microscopy (SEM) of the four urease positive stomach samples revealed a tightly coiled Helicobacter bacterium (spiral-shaped) found in the mucous lining of the stomach. In histopathological examination of pars esophagia, cardiac and fundic mucosa showed chronic gastritis associated with hemorrhagic necrosis, leucocytic infiltration with neutrophils and macrophages, and lymphoid aggregates (lymphoid follicles) etc. PCR confirmed 16S rRNA genes of Helicobacter suis (H. suis) where a total of 42 (19.63%) out of 214 pig stomach samples and 2 (2.08%) out of 96 stool samples of pig farmers were found positive for H. suis. of these 96 stool samples of pig farmers 3 (3.12%) were confirmed positive for Helicobacter pylori (H. pylori) Phosphoglucosamine mutase gene in PCR. Phylogenic analysis of the 16S rRNA gene of H. suis showed distinct clusters with other H. sp. In conclusion, this study provides evidence for the prevalence of Helicobacter both in pig gastric mucosa and human stool. The findings highlight the need for improved sanitation and hygiene practices among pig farmers to minimize the risk of Helicobacter infection in humans.

microbiology↗

bioRxiv2023

Long COVID manifests with T cell dysregulation, inflammation, and an uncoordinated adaptive immune response to SARS-CoV-2

Long COVID (LC), a type of post-acute sequelae of SARS-CoV-2 infection (PASC), occurs after at least 10% of SARS-CoV-2 infections, yet its etiology remains poorly understood. Here, we used multiple "omics" assays (CyTOF, RNAseq/scRNAseq, Olink) and serology to deeply characterize both global and SARS-CoV-2-specific immunity from blood of individuals with clear LC and non-LC clinical trajectories, 8 months following infection and prior to receipt of any SARS-CoV-2 vaccine. Our analysis focused on deep phenotyping of T cells, which play important roles in immunity against SARS-CoV-2 yet may also contribute to COVID-19 pathogenesis. Our findings demonstrate that individuals with LC exhibit systemic inflammation and immune dysregulation. This is evidenced by global differences in T cell subset distribution in ways that imply ongoing immune responses, as well as by sex-specific perturbations in cytolytic subsets. Individuals with LC harbored increased frequencies of CD4+ T cells poised to migrate to inflamed tissues, and exhausted SARS-CoV-2-specific CD8+ T cells. They also harbored significantly higher levels of SARS-CoV-2 antibodies, and in contrast to non-LC individuals, exhibited a mis-coordination between their SARS-CoV-2-specific T and B cell responses. RNAseq/scRNAseq and Olink analyses similarly revealed immune dysregulatory mechanisms, along with non-immune associated perturbations, in individuals with LC. Collectively, our data suggest that proper crosstalk between the humoral and cellular arms of adaptive immunity has broken down in LC, and that this, perhaps in the context of persistent virus, leads to the immune dysregulation, inflammation, and clinical symptoms associated with this debilitating condition.

immunology↗