Search bioRxiv⌕ Search

Biology subjects

Hussain, N.

Publications and source records attributed to Hussain, N..

12 recordsLinked to original sources

Single cell functional immunogenomics of the fallopian tube reveals a precursor immune surveillance network for ovarian cancer prevention

The fallopian tube (FT) is increasingly recognized as the site of origin for high-grade serous ovarian cancer (HGSOC), yet its immune landscape and potential role in tumor surveillance remain poorly understood. Here, we employ single-cell RNA sequencing (scRNA-seq) and paired single-cell T-cell receptor sequencing (scTCR-seq) to profile tissue-resident memory-like T cells (TRMLs) from matched non- cancerous FT, metastatic omental tumors, and peripheral blood in HGSOC patients. Surprisingly, we identify a substantial clonal and functional overlap between TRMLs from non-cancerous FT and tumor- infiltrating TRMLs, with 18.4% of tumor TRML clonotypes being shared with FT TRMLs, significantly exceeding overlap with circulating T cells. Shared TCR clonotypes are preferentially enriched in exhausted CD8+ T cells (CD8-Tex) and proliferative exhausted CD8+ subsets, suggesting prior antigenic exposure. Notably, we identify a previously uncharacterized CD8+ SIK3-high subset enriched in tumors, with gene expression signatures implicating epigenetic plasticity and metabolic adaptation. Functionally, non-cancerous FT-derived TRMLs recognize autologous tumor antigens, as evidenced by robust IFN-{gamma} responses in tumor organoid co-culture assays and CD137 upregulation. Whole-genome and transcriptomic analyses reveal shared somatic mutations between the FT and tumor, supporting the FT as the tumors evolutionary origin. Predicted tumor neoantigens elicit TRML responses in the non-cancerous FT, and single-cell TCR profiling confirms that neoantigen-reactive T cells in FT share clonotypes with tumor-infiltrating TRMLs, further reinforcing their role in early immune surveillance. Importantly, FT-derived TRMLs exhibit lower exhaustion signatures than their tumor counterparts, suggesting their potential as a preferable source for adoptive T cell therapies. Our findings uncover a precursor immune surveillance network in the FT and provide a rationale for leveraging FT-resident T cells in cancer immunotherapy and prevention.

immunology↗

Expanded adaptive NKG2C+ NK cells exhibit potent ADCC and functional responses against HBV-infected hepatoma cell lines

BackgroundHepatitis B virus (HBV) infection remains a significant global health challenge, leading to chronic liver disease and hepatocellular carcinoma (HCC). Natural killer (NK) cells play an important role in the clearance of HBV-infected cells, but their efficacy is often compromised during chronic infection. Adaptive NK cells, characterised by NKG2C expression and enhanced functional responses, represent a promising therapeutic avenue for enhancing anti-HBV immunity and responses to HBV-driven cancers. MethodsWe applied an established protocol, involving K562-HLA-E expressing feeder cells and cytokines (IL-2), for the expansion of adaptive NK cells from cryopreserved T- and B cell depleted peripheral blood mononuclear cells (PBMCs) derived from donors with chronic HBV infection alone or with Human Immunodeficiency Virus (HIV) co-infection. We evaluated the adaptive profile of expanded NK cells, their antibody-dependent cellular cytotoxicity (ADCC) capacity and functional responses against hepatoma cell lines in the presence or absence of HBV infection. ResultsExpanded NK cells achieved >97% purity, with the NKG2C positive population exhibiting a mean 100-fold expansion. These cells demonstrated a predominantly adaptive phenotype with high surface expression of NKG2C and cytotoxic potential (Granzyme B). They maintained high levels of CD16 surface expression and upregulated CD2, essential for ADCC. Functionally, expanded adaptive NK cells showed enhanced ADCC capacity and functional responses to K562 targets, naive, HBV integrant-expressing, and de novo infected hepatoma cell lines. TGF-{beta} preconditioning induced tissue-resident features (CD103, CD49a) in expanded adaptive NK cells, while preserving their adaptive phenotype and functionality, enhancing their potential for liver targeted immunotherapy. Further, expanded adaptive NK cells demonstrated minimal reactivity against autologous activated T cells, suggesting limited off-target effects. ConclusionsOur study demonstrates the first successful expansion of adaptive NK cells with robust functional responses from donors with chronic viral infection. This approach creates opportunities for NK cell-based therapies alone or in combination with monoclonal antibodies contributing to HBV functional cure strategies and the treatment of HBV-driven cancers.

immunology↗

Molecular characterisation of Streptococcus pyogenes (StrepA) non-invasive isolates during the 2022-23 UK upsurge

At the end of 2022 into early 2023 the UK Health Security Agency reported unusually high levels of scarlet fever and invasive disease caused by Streptococcus pyogenes (StrepA or group A Streptococcus). During this time, we collected and genome sequenced 341 non-invasive throat and skin S. pyogenes isolates identified during routine clinical diagnostic testing in Sheffield, a large UK city. We compared the data with that obtained from a similar collection of 165 isolates from 2016-17. Numbers of throat-associated isolates collected peaked in early December 2022, reflecting the national scarlet fever upsurge, while skin infections peaked later in December. The most common emm-types in 2022-23 were emm1 (28.7%), emm12 (24.9%), and emm22 (7.7%) in throat; and emm1 (22%), emm12 (10%), emm76 (18%), and emm49 (7%) in skin. Whilst all emm1 isolates were the M1UK lineage, comparison with 2016-17 revealed diverse lineages in other emm-types, including emm12, and emergent lineages within other types including a new acapsular emm75 lineage, demonstrating that the upsurge was not completely driven by a single genotype. Analysis of the capsule locus predicted only 51% of throat isolates would produce capsule compared to 78% of skin isolates. 90% of throat isolates were also predicted to have high NADase and Streptolysin O (SLO) expression, based on the promoter sequence, compared to only 56% of skin isolates. Our study has highlighted the value in analysis of non-invasive isolates to characterise tissue tropisms, as well as changing strain diversity and emerging genomic features which may have implications for spillover into invasive disease and future S. pyogenes upsurges. Data summaryAll new genome sequence data is available on the NCBI short read archive under the bioproject PRJNA1062601 and individual accession numbers are listed in Supplementary Table 1 and Table 2. Impact statementThe human bacterial pathogen Streptococcus pyogenes, also known as group A Streptococcus or StrepA, caused a dramatic and sudden upsurge in scarlet fever in the UK at the end of 2022 into early 2023. We present molecular characterisation of this upsurge, through genome sequence analysis of throat, skin and other types of non-severe infection isolates collected by the microbiology diagnostic lab at the Northern General Hospital in Sheffield, England. We found that, whilst two strain types were the predominant cause of infections during the upsurge, other types had emerged or changed when compared to a similar collection from 2016-17. We also identified differences between throat-associated isolates and skin-associated isolates and highlighted important bacterial factors that might influence infection types. Isolates from non-severe throat/skin types of infections are rarely saved and therefore our knowledge of them is limited. However, here we demonstrate that study of such isolates may be key to understanding upsurges of more severe infections.

microbiology↗

Long noncoding RNA-mediated epigenetic regulation of auxin-related genes controlling shade avoidance syndrome in Arabidopsis thaliana

The long noncoding RNA (lncRNA) AUXIN-REGULATED PROMOTER LOOP (APOLO) recognizes a subset of target loci across the Arabidopsis thaliana genome by forming RNA-DNA hybrids (R-loop) and modulating local three-dimensional chromatin conformation. Here we show that APOLO is involved in regulating the shade avoidance syndrome (SAS) by dynamically modulating the expression of key factors. In response to far-red (FR) light, the expression of APOLO anticorrelates with its target BRANCHED1 (BRC1), a master regulator of shoot branching in Arabidopsis thaliana. APOLO deregulation results in BRC1 transcriptional repression and an increase in the number of branches. APOLO transcriptional accumulation fine-tunes the formation of a repressive chromatin loop encompassing the BRC1 promoter, which normally occurs only in leaves as well as in a late response to FR treatment in axillary buds. In addition, our data reveal that APOLO participates in leaf hyponasty, in agreement with its previously reported role in the control of auxin homeostasis through direct modulation of YUCCA2 (auxin synthesis), PID and WAG2 (auxin efflux). We found that direct application of APOLO RNA to leaves results in a rapid increase in auxin accumulation that is associated with changes in the response of the plants to FR light. Collectively, our data support the view that lncRNAs coordinate the shade avoidance syndrome in Arabidopsis thaliana and shed light on the potential of lncRNAs as bioactive exogenous molecules. Deploying exogenous RNAs that modulate plant-environment interactions are important new tools for sustainable agriculture.

plant biology↗

Attenuated humoral responses in HIV infection after SARS-CoV-2 vaccination are linked to global B cell defects and cellular immune profiles

People living with HIV (PLWH) on suppressive antiretroviral therapy (ART) can have residual immune dysfunction and often display poorer responses to vaccination. We assessed in a cohort of PLWH (n=110) and HIV negative controls (n=64) the humoral and spike-specific B-cell responses following 1, 2 or 3 SARS-CoV-2 vaccine doses. PLWH had significantly lower neutralizing antibody (nAb) titers than HIV-negative controls at all studied timepoints. Moreover, their neutralization breadth was reduced with fewer individuals developing a neutralizing response against the Omicron variant (BA.1) relative to controls. We also observed a delayed development of neutralization in PLWH that was underpinned by a reduced frequency of spike-specific memory B cells (MBCs) and pronounced B cell dysfunction. Improved neutralization breadth was seen after the third vaccine dose in PLWH but lower nAb responses persisted and were associated with global, but not spike-specific, MBC dysfunction. In contrast to the inferior antibody responses, SARS-CoV-2 vaccination induced robust T cell responses that cross-recognized variants in PLWH. Strikingly, a subset of PLWH with low or absent neutralization had detectable functional T cell responses. These individuals had reduced numbers of circulating T follicular helper cells and an enriched population of CXCR3+CD127+CD8+ T cells after two doses of SARS-CoV-2 vaccination, which may compensate for sub-optimal serological responses in the event of infection. Therefore, normalisation of B cell homeostasis could improve serological responses to vaccines in PLWH and evaluating T cell immunity could provide a more comprehensive immune status profile in these individuals and others with B cell imbalances.

immunology↗

Structural insights into pore dynamics of human Pannexin isoforms

Pannexins are single-membrane large-pore ion channels that release ATP upon activation. Three isoforms of pannexins, 1, 2, and 3, perform diverse cellular roles, including inflammation, differentiation, neuropathic pain, and ATP release. In this study, we report the cryoEM structure of pannexin 3 at 3.9 [A] and characterize the structural differences with pannexin isoforms 1 and 2. We observe the organization of the Pannexin 3 vestibule into two distinct chambers with a wider pore radius in comparison to both PANX1 and 2 isoforms. We further report the structure of pannexin1 congenital mutant R217H in the resolution range of 3.9 [A]. The congenital mutant R217H in transmembrane helix3 (TM3), R217H induce structural changes that leads to a partially closed pore and altered ATP interaction propensities. The channel conductance of the congenital mutant displays weakened voltage sensitivity. The results showcase a complete comparison of the three pannexin isoform structures that along with the structure of Pannexin 1 congenital mutant highlight distinct structural features of pannexin isoforms and the allosteric role of distant substitutions in dictating channel behavior in Pannexin 1.

biochemistry↗

An in vitro assessment of anti-SARS-CoV-2 activity of oral preparations of iodine complexes (RENESSANS)

Since the emergence of CoVID-19 pandemic in China in late 2019, scientists are striving hard to explore non-toxic, viable anti-SARS-CoV-2 compounds or medicines. We determined In Vitro anti-SARS-CoV-2 activity of oral formulations (syrup and capsule) of an Iodine-complex (Renessans). A monolayer of vero cells were exposed to SARS-CoV-2 in the presence and absence of different concentrations (equivalent to 50, 05 and 0.5 g/ml of I2) of Renessans. Anti-SARS-CoV-2 activity of each of the formulation was assessed in the form of cell survival, SARS-CoV-2-specific cytopathic effect (CPE) and genome quantization. With varying concentrations of syrup and capsule, a varying rate of inhibition of CPE, cells survival and virus replication was observed. Compared to 0.5 g/ml concentration of Renessans syrup, 5 and 50 g/ml showed comparable results where there was a 100% cell survival, no CPEs and a negligible viral replication ({Delta}CT= 0.11 and 0.13, respectively). This study indicates that Renessans, containing iodine, may have potential activity against SARS-CoV-2 which needs to be further investigated in human clinical trials.

microbiology↗

Exportin Crm1 is important for Swi6 nucleocytoplasmic shuttling and MBF transcription activation in Saccharomyces cerevisiae

The Swi6 protein acts as a transcription factor in budding yeast, functioning in two different heterodimeric complexes, SBF and MBF, that activate the expression of distinct but overlapping sets of genes. Swi6 undergoes regulated changes in nucleocytoplasmic localization throughout the cell cycle that correlate with changes in gene expression. While the process of Swi6 nuclear import is well understood, mechanisms underlying its nuclear export remain unclear. Here we investigate Swi6 nuclear export and its impact on Swi6 function. We show that the exportin Crm1, in addition to three other karyopherins previously shown to affect Swi6 localization, is important for Swi6 nuclear export and activity. A truncation of Swi6 that removes a putative Crm1 nuclear export signal results in the loss of changes in nucleocytoplasmic Swi6 localization that normally occur during progression through the cell cycle. Mutagenesis of the NES-like sequence or removal of Crm1 activity using leptomycin B results in a similar decrease in nuclear export as cells enter S-phase. Using two-hybrid analysis, we also show that Swi6 associates with Crm1 in vivo. Alteration of the Crm1 NES in Swi6 results in a decrease in MBF-mediated gene expression, but does not affect expression of an SBF reporter, suggesting that export of Swi6 by Crm1 regulates a subset of Swi6 transcription activation activity. Finally, alteration of the Crm1 NES in Swi6 results in cells that are larger than wild type, but not to the extent of those with a complete Swi6 deletion. Expressing a Swi6 NES mutant in combination with a deletion of Msn5, an exportin involved in Swi6 nuclear export and specifically affecting SBF activation, further increases the large cell phenotype, but still not to the extent observed in a Swi6 deletion mutant. These data suggest that Swi6 has at least two different exportins, Crm1 and Msn5, each of which interacts with a distinct nuclear export signal and influences expression of a different subset of Swi6-controlled genes. Summary StatementPrecise intracellular localization is important for the proper activity of proteins. Here we provide evidence that the Swi6 transcription factor important for cell cycle progression shuttles between the cell nucleus and cytoplasm, its nuclear export is important for its activity, and that it contains a nuclear export signal (NES) recognized by the Crm1 nuclear transport factor.

cell biology↗

CDK4 as a phytochemical based anticancer drug target

Success story of plant-based medicine had been overlooked during the advent of modern pharmaceutical industry. Despite the negligence of the multimillion-dollar drug industry, people entirely rely on medicinal plants in some part of the world. In this study, we have emphasized on going back to those traditional medicinal practices to figure out their underlying mechanism to move forward on phytochemical based drug development. We screened Medicinal Plant Database Bangladesh 1.0 (MPDB1.0) and on-going extension, MPDB2.0, of that database to find traditionally used medicinal plants and their active compounds. Here, Mangiferin, extracted from Mangifera indica, have been demonstrated to interact with cell cycle regulator Cyclin-dependent Kinase 4 (CDK4). CDK4 is differentially expressed during Glioblastoma multiforme (GBM), Brain Lower Grade Glioma (LGG), and Sarcoma (SARC). Expression of CDK4 is interlinked to the patients survival rate and its consistent expression throughout different stages have provided the advantage to use it as diagnostic tool and drug target. Altogether, this study demonstrated that simple mango tree extracted active compounds, mangiferin, can work as potential anticancer drug and leveraging the recent advancement of sequencing and gene expression data can accelerate the phytochemical based drug discovery process.

cancer biology↗

Learning to learn persistently modifies a neocortical-hippocampal excitatory-inhibitory subcircuit

Cognitive control, the judicious use of relevant information while ignoring distractions, is a feature of everyday cognitive experience, but its neurobiology is understudied. We investigated whether cognitive control training (CCT) changes hippocampal neural circuit function in mice, beyond the changes caused by place learning and memory formation. Mice learned and remembered a conditioned place avoidance during CCT that required ignoring irrelevant locations of shock. They were compared to controls that learned the same place avoidance under lower cognitive control demands. Weeks after CCT, mice learn new tasks in novel environments faster than controls; they learned to learn. We investigated entorhinal cortex-to-dentate gyrus neural circuit changes and report that CCT rapidly changes synaptic circuit function, resulting in an excitatory-inhibitory subcircuit change that persists for months. CCT increases inhibition that attenuates the dentate response to medial entorhinal cortical input, and through disinhibition, potentiates the response to strong inputs, pointing to overall signal-to-noise enhancement. These neurobiological findings support a neuroplasticity hypothesis that, beyond storing item/event associations, CCT persistently optimizes neural circuit information processing.

neuroscience↗

Importance of parental genome balance in the generation of novel yet heritable epigenetic and transcriptional states during doubled haploid breeding

BackgroundDoubling the genome contribution of haploid plants has accelerated breeding in most cultivated crop species. Although plant doubled haploids are isogenic in nature, they frequently display unpredictable phenotypes, thus limiting the potential of this technology. Therefore, being able to predict the factors implicated in this phenotypic variability could accelerate the generation of desirable genomic combinations and ultimately plant breeding.\n\nResultsWe use computational analysis to assess the transcriptional and epigenetic dynamics taking place during doubled haploids generation in the genome of Brassica oleracea. We observe that doubled haploid lines display unexpected levels of transcriptional and epigenetic variation, and that this variation is largely due to imbalanced contribution of parental genomes. We reveal that epigenetic modification of transposon-related sequences during DH breeding contributes to the generation of unpredictable yet heritable transcriptional states. Targeted epigenetic manipulation of these elements using dCas9-hsTET3 confirms their role in transcriptional regulation. We have uncovered a hitherto unknown role for parental genome balance in the transcriptional and epigenetic stability of doubled haploids.\n\nConclusionsThis is the first study that demonstrates the importance of parental genome balance in the transcriptional and epigenetic stability of doubled haploids, thus enabling predictive models to improve doubled haploid-assisted plant breeding.

plant biology↗

Dissection of protonation sites for antibacterial recognition and transport in QacA, a multi-drug efflux transporter.

QacA is a drug:H+ antiporter (DHA2) with 14 transmembrane helices, that renders antibacterial resistance to methicillin-resistant Staphylococcus aureus (MRSA) strains, with homologues in other pathogenic organisms. It is a highly promiscuous antiporter, capable of H+- driven efflux of a wide array of cationic antibacterial compounds and dyes. Our study, using a homology model of QacA, reveals a group of six protonatable residues in its vestibule. Systematic mutagenesis resulted in identification of D34 (TM1), and a cluster of acidic residues in TM13 including E407 and D411 and D323 in TM10, as being crucial for substrate recognition and transport of monovalent and divalent cationic antibacterial compounds. The transport and binding properties of QacA and its mutants were explored using whole cells, inside-out vesicles, substrate-induced H+ release and microscale thermophoresis. We identify two sites, D34 and D411 as vital players in substrate recognition while E407 facilitates substrate efflux as a protonation site. We also observe that E407 plays a moonlighting role as a substrate recognition site for dequalinium transport. These observations rationalize the promiscuity of QacA for diverse substrates. The study unravels the role of acidic residues in QacA with implications for substrate recognition, promiscuity and processive transport in multidrug efflux transporters, related to QacA.\n\nHighlightsO_LIA homology model of QacA with 14 TM helices was used to test the importance of acidic residues within the vestibule.\nC_LIO_LIMono and Divalent cationic substrate recognition requires two sites D34 (TM1) and D411 (TM13).\nC_LIO_LIE407 (TM13) is important for protonation driven efflux.\nC_LIO_LISubstrate recognition of a divalent substrate, dequalinium occurs at E407 instead of D411 providing glimpses into the promiscuity of substrate recognition in QacA.\nC_LI

biochemistry↗