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Papers by alka saxena

Immunity, 2020

If citing, it is advised that you check and use the publisher's definitive version for pagination... more If citing, it is advised that you check and use the publisher's definitive version for pagination, volume/issue, and date of publication details. And where the final published version is provided on the Research Portal, if citing you are again advised to check the publisher's website for any subsequent corrections.

Classical dendritic cells (cDCs) are rare sentinel cells specialized in the regulation of adaptiv... more Classical dendritic cells (cDCs) are rare sentinel cells specialized in the regulation of adaptive immunity. Modeling cDC development is both crucial to study cDCs and harness their potential in immunotherapy. Here we addressed whether cDCs could differentiate in response to trophic cues delivered by mesenchymal components of the hematopoietic niche where they physiologically develop and maintain. We found that expression of the membrane bound form of human FLT3L and SCF together with CXCL12 in a bone marrow mesenchymal stromal cell line is sufficient to induce the contact-dependent specification of both type 1 and type 2 cDCs from CD34+ hematopoietic stem and progenitor cells (HSPCs). Engraftment of these engineered mesenchymal stromal cells (eMSCs) together with CD34+ HSPCs creates an in vivo synthetic niche in the dermis of immunodeficient mice. Cell-to-cell contact between HSPCs and stromal cells within these organoids drive the local specification of cDCs and CD123+AXL+CD327+ p...

1.BackgroundNeuropsychiatric disease has polygenic determinants but is often precipitated by envi... more 1.BackgroundNeuropsychiatric disease has polygenic determinants but is often precipitated by environmental pressures, including adverse perinatal events. However, the way in which genetic vulnerability and early-life adversity interact remains obscure. Preterm birth is associated with abnormal brain development and psychiatric disease. We hypothesised that the extreme environmental stress of premature extra-uterine life could contribute to neuroanatomic abnormality in genetically vulnerable individuals.MethodsWe combined Magnetic Resonance Imaging (MRI) and genome-wide single nucleotide polymorphism (SNP) data from 194 infants, born before 33 weeks of gestation, to test the prediction that: the characteristic deep grey matter abnormalities seen in preterm infants are associated with polygenic risk for psychiatric illness. Summary statistics from a meta-analysis of SNP data for five psychiatric disorders were used to compute individual polygenic risk scores (PRS). The variance explai...

Nature Communications, 2019

Frontal fibrosing alopecia (FFA) is a recently described inflammatory and scarring type of hair l... more Frontal fibrosing alopecia (FFA) is a recently described inflammatory and scarring type of hair loss affecting almost exclusively women. Despite a dramatic recent increase in incidence the aetiopathogenesis of FFA remains unknown. We undertake genome-wide association studies in females from a UK cohort, comprising 844 cases and 3,760 controls, a Spanish cohort of 172 cases and 385 controls, and perform statistical meta-analysis. We observe genome-wide significant association with FFA at four genomic loci: 2p22.2, 6p21.1, 8q24.22 and 15q2.1. Within the 6p21.1 locus, fine-mapping indicates that the association is driven by the HLA-B*07:02 allele. At 2p22.1, we implicate a putative causal missense variant in CYP1B1, encoding the homonymous xenobiotic- and hormone-processing enzyme. Transcriptomic analysis of affected scalp tissue highlights overrepresentation of transcripts encoding components of innate and adaptive immune response pathways. These findings provide insight into disease ...

Human Molecular Genetics, 2018

Facioscapulohumeral muscular dystrophy (FSHD) is a prevalent, incurable myopathy, linked to epige... more Facioscapulohumeral muscular dystrophy (FSHD) is a prevalent, incurable myopathy, linked to epigenetic derepression of D4Z4 repeats on chromosome 4q, leading to ectopic DUX4 expression. FSHD patient myoblasts have defective myogenic differentiation, forming smaller myotubes with reduced myosin content. However, molecular mechanisms driving such disrupted myogenesis in FSHD are poorly understood. We performed high-throughput morphological analysis describing FSHD and control myogenesis, revealing altered myogenic differentiation results in hypotrophic myotubes. Employing polynomial models and an empirical Bayes approach, we established eight critical time points during which human healthy and FSHD myogenesis differ. RNA-sequencing at these eight nodal time points in triplicate, provided temporal depth for a multivariate regression analysis, allowing assessment of interaction between progression of differentiation and FSHD disease status. Importantly, the unique size and structure of ...

Epigenetics & chromatin, 2015

The capacity for plasticity in the adult brain is limited by the anatomical traces laid down duri... more The capacity for plasticity in the adult brain is limited by the anatomical traces laid down during early postnatal life. Removing certain molecular brakes, such as histone deacetylases (HDACs), has proven to be effective in recapitulating juvenile plasticity in the mature visual cortex (V1). We investigated the chromatin structure and transcriptional control by genome-wide sequencing of DNase I hypersensitive sites (DHSS) and cap analysis of gene expression (CAGE) libraries after HDAC inhibition by valproic acid (VPA) in adult V1. We found that VPA reliably reactivates the critical period plasticity and induces a dramatic change of chromatin organization in V1 yielding significantly greater accessibility distant from promoters, including at enhancer regions. VPA also induces nucleosome eviction specifically from retrotransposon (in particular SINE) elements. The transiently accessible SINE elements overlap with transcription factor-binding sites of the Fox family. Mapping of transc...

BMC genomics, Jan 24, 2014

Mutations in three functionally diverse genes cause Rett Syndrome. Although the functions of Fork... more Mutations in three functionally diverse genes cause Rett Syndrome. Although the functions of Forkhead box G1 (FOXG1), Methyl CpG binding protein 2 (MECP2) and Cyclin-dependent kinase-like 5 (CDKL5) have been studied individually, not much is known about their relation to each other with respect to expression levels and regulatory regions. Here we analyzed data from hundreds of mouse and human samples included in the FANTOM5 project, to identify transcript initiation sites, expression levels, expression correlations and regulatory regions of the three genes RESULTS: Our investigations reveal the predominantly used transcription start sites (TSSs) for each gene including novel transcription start sites for FOXG1. We show that FOXG1 expression is poorly correlated with the expression of MECP2 and CDKL5. We identify promoter shapes for each TSS, the predicted location of enhancers for each gene and the common transcription factors likely to regulate the three genes. Our data imply Polyc...

Nature Genetics, 2014

The importance of microRNAs and long noncoding RNAs in the regulation of pluripotency has been do... more The importance of microRNAs and long noncoding RNAs in the regulation of pluripotency has been documented; however, the noncoding components of stem cell gene networks remain largely unknown. Here we investigate the role of noncoding RNAs in the pluripotent state, with particular emphasis on nuclear and retrotransposon-derived transcripts. We have performed deep profiling of the nuclear and cytoplasmic transcriptomes of human and mouse stem cells, identifying a class of previously undetected stem cell-specific transcripts. We show that long terminal repeat (LTR)-derived transcripts contribute extensively to the complexity of the stem cell nuclear transcriptome. Some LTR-derived transcripts are associated with enhancer regions and are likely to be involved in the maintenance of pluripotency.

Nature, 2012

Non-coding RNAs (ncRNAs) are involved in an increasing number of cellular events 1. Some ncRNAs a... more Non-coding RNAs (ncRNAs) are involved in an increasing number of cellular events 1. Some ncRNAs are processed by DICER and DROSHA ribonucleases to give rise to small doublestranded RNAs involved in RNA interference (RNAi) 2. The DNA-damage response (DDR) is a signaling pathway that originates from the DNA lesion and arrests cell proliferation 3. So far, DICER or DROSHA RNA products have not been reported to control DDR activation. Here we show that DICER and DROSHA, but not downstream elements of the RNAi pathway, are necessary to activate DDR upon oncogene-induced genotoxic stress and exogenous DNA damage, as studied also by DDR foci formation in mammalian cells and zebrafish and by checkpoint assays. DDR foci are sensitive to RNase A treatment, and DICER-and DROSHA-dependent RNA products are required to restore DDR foci in treated cells. Through RNA deep sequencing and studies of DDR activation at an inducible unique DNA double-strand break (DSB), we demonstrate that DDR foci formation requires site-specific DICER-and DROSHA-dependent small RNAs, named DDRNAs, which act in a MRE11-RAD50-NBS1 (MRN) complex-dependent manner. Chemically synthesized or in vitro-generated by DICER cleavage, DDRNAs are sufficient to restore DDR in RNase A-treated cells, also in the absence of other cellular RNAs. Our results describe an unanticipated direct role of a novel class of ncRNAs in the control of DDR activation at sites of DNA damage.

Nature, 2011

RNAi pathways have evolved as important modulators of gene expression that act in the cytoplasm b... more RNAi pathways have evolved as important modulators of gene expression that act in the cytoplasm by degrading RNA target molecules via the activity of short (21-30nt) RNAs 1-6 RNAi components have been reported to play a role in the nucleus as they are involved in epigenetic regulation and heterochromatin formation 7-10. However, although RNAi-mediated post-Users may view, print, copy, download and text and data-mine the content in such documents, for the purposes of academic research, subject always to the full Conditions of use:

ABSTRACTPreterm birth places newborn infants in an adverse environment that leads to brain injury... more ABSTRACTPreterm birth places newborn infants in an adverse environment that leads to brain injury linked to neuroinflammation. To characterise this pathology, we present a translational bioinformatics investigation, with integration of human and mouse molecular and neuroimaging datasets to provide a deeper understanding of the role of microglia in preterm white matter damage. We examined preterm neuroinflammation in a mouse model of encephalopathy of prematurity induced by IL1B exposure, carrying out a gene network analysis of the cell-specific transcriptomic response to injury, which we extended to analysis of protein-protein interactions, transcription factors, and human brain gene expression, including translation to preterm infants by means of imaging-genetics approaches in the brain. We identified the endogenous synthesis of DLG4 (PSD95) protein by microglia in mouse and human, modulated by inflammation and development. Systemic genetic variation in DLG4 was associated with str...

Circulation Research, 2015

Rationale: Platelets shed microRNAs (miRNAs). Plasma miRNAs change on platelet inhibition. It is ... more Rationale: Platelets shed microRNAs (miRNAs). Plasma miRNAs change on platelet inhibition. It is unclear whether plasma miRNA levels correlate with platelet function. Objective: To link small RNAs to platelet reactivity. Methods and Results: Next-generation sequencing of small RNAs in plasma revealed 2 peaks at 22 to 23 and 32 to 33 nucleotides corresponding to miRNAs and YRNAs, respectively. Among YRNAs, predominantly, fragments of RNY4 and RNY5 were detected. Plasma miRNAs and YRNAs were measured in 125 patients with a history of acute coronary syndrome who had undergone detailed assessment of platelet function 30 days after the acute event. Using quantitative real-time polymerase chain reactions, 92 miRNAs were assessed in patients with acute coronary syndrome on different antiplatelet therapies. Key platelet-related miRNAs and YRNAs were correlated with platelet function tests. MiR-223 ( r p =0.28; n=121; P =0.002), miR-126 ( r p =0.22; n=121; P =0.016), and other abundant plate...

BioEssays, 2011

Common themes are emerging in the molecular mechanisms of long non-coding RNA-mediated gene repre... more Common themes are emerging in the molecular mechanisms of long non-coding RNA-mediated gene repression. Long non-coding RNAs (lncRNAs) participate in targeted gene silencing through chromatin remodelling, nuclear reorganisation, formation of a silencing domain and precise control over the entry of genes into silent compartments. The similarities suggest that these are fundamental processes of transcription regulation governed by lncRNAs. These findings have paved the way for analogous investigations on other lncRNAs and chromatin remodelling enzymes. Here we discuss these common mechanisms and provide our view on other molecules that warrant similar investigations. We also present our concepts on the possible mechanisms that may facilitate the exit of genes from the silencing domains and their potential therapeutic applications. Finally, we point to future areas of research and put forward our recommendations for improvements in resources and applications of existing technologies towards targeted outcomes in this active area of research.

Nature communications, Sep 5, 2017

Preterm birth places infants in an adverse environment that leads to abnormal brain development a... more Preterm birth places infants in an adverse environment that leads to abnormal brain development and cerebral injury through a poorly understood mechanism known to involve neuroinflammation. In this study, we integrate human and mouse molecular and neuroimaging data to investigate the role of microglia in preterm white matter damage. Using a mouse model where encephalopathy of prematurity is induced by systemic interleukin-1β administration, we undertake gene network analysis of the microglial transcriptomic response to injury, extend this by analysis of protein-protein interactions, transcription factors and human brain gene expression, and translate findings to living infants using imaging genomics. We show that DLG4 (PSD95) protein is synthesised by microglia in immature mouse and human, developmentally regulated, and modulated by inflammation; DLG4 is a hub protein in the microglial inflammatory response; and genetic variation in DLG4 is associated with structural differences in ...

Liver Biopsy in Modern Medicine, 2011

Liver Biopsy in Modern Medicine 336 associated antigen, Glial Fibrillary Acidic Protein (GFAP), i... more Liver Biopsy in Modern Medicine 336 associated antigen, Glial Fibrillary Acidic Protein (GFAP), is induced on pro-fibrotic cells such as HSCs, and is a definitive marker of fibrogenic pathway activation in this latter cell type (29). Studies of fibrosis mechanisms in human liver are limited. One longitudinal study, after liver transplantation, reported that increased density of GFAP in liver biopsy specimens predicted subsequent advanced fibrosis or cirrhosis (9). Although cells harboring GFAP were only presumed to be activated HSCs, the study concluded that 30% of cells in cirrhotic livers may be activated HSCs. However, the possibility of a direct effect of HCV on GFAP expression in hepatocytes was not investigated. The present study therefore examined the effect of HCV on hepatic fibrosis marker expression, using two human hepatoma cell line model systems, capable of supporting either non-productive HCV replication (HCV replicon, (22)), or productive HCV infection (genotype 2a infectious clone JFH1; (44)). The study also examined liver biopsy samples from HCV infected patients for the simultaneous presence of GFAP and HCV replicative intermediate RNA. Finally, microarrays were used to analyze expression of multiple cellular genes linked with liver fibrosis, in human hepatoma cell lines plus or minus HCV. The effect of HCV on differential expression of 153 genes (1, 3, 17, 28, 37) either involved in, or associated with, with the process of liver fibrosis, is reported. 2. Methods 2.1 Human liver biopsy specimens Thirty-two liver biopsy specimens, obtained under informed consent and per IRB-approved protocol, were available for study. All 32 subjects had chronic, active (viremic) HCV genotype 1 infections. During procurement, the specimens were immediately preserved in OCT buffer and snap frozen at the bedside. Parallel sections of the liver biopsies were reviewed by a single pathologist who was blinded to HCV status and all other data. Liver fibrosis severity, staged as 0 (no fibrosis) through 4 (cirrhosis), was assigned according to the system described by Batts and Ludwig (5). For the present study, the liver specimens were de-identified for all information except HCV replication status and fibrosis severity. Fresh thin sections were obtained for the GFAP immunostaining experiments described below. Parallel sections of all 32 liver biopsy specimens were assayed for GFAP expression by immunocytochemistry. 29 of the specimens had been previously analyzed for both HCV genomic (G) and replicative intermediate (RI) RNAs by strand-specific in situ hybridization (ISH). Details of the ISH assay, and assay results for a larger sample of hepatitis C cases, were previously reported (31). Of 29 specimens with both GFAP and HCV replication data, HCV RNA was determined as either positive (G+RI+; 20 specimens), or negative (G-RI-; 9 specimens), and GFAP staining level (% of cells per biopsy staining positive for GFAP, or %GFAP) was then analyzed as a function of HCV infection/replication status, and fibrosis stage. 2.2 Hepatoma cell infection by JFH1 HCV Huh7.5.1 cells (48) were generously provided by Francis Chisari (Scripps Institute, La Jolla, CA). Infection of Huh7.5.1 cells with the HCV JFH1 genotype 2a clone was performed as previously described (43), including the preparation of the JFH1 viral stock, cell infection, and titration. Briefly, we inoculated naïve Huh7.5.1 cells with supernatant harvested from JFH1 RNA transfected cells. Naïve Huh7.5.1 cells were seeded 24 h before infection at a density of 1 x 10 6 per 10 cm dish. The cells were incubated with 2.5 ml of the JFH1 inoculum at an multiplicity of infection of 0.

International Journal for Parasitology, 2007

Transcription of protein-coding genes in Leishmania major and other trypanosomatids differs from ... more Transcription of protein-coding genes in Leishmania major and other trypanosomatids differs from that in most eukaryotes and bioinformatic analyses have failed to identify several components of the RNA polymerase (RNAP) complexes. To increase our knowledge about this basic cellular process, we used tandem affinity purification (TAP) to identify subunits of RNAP II and III. Mass spectrometric analysis of the complexes co-purified with TAP-tagged LmRPB2 (encoded by LmjF31.0160) identified seven RNAP II subunits: RPB1, RPB2, RPB3, RPB5, RPB7, RPB10 and RPB11. With the exception of RPB10 and RPB11, and the addition of RPB8, these were also identified using TAP-tagged constructs of one (encoded by LmjF34.0890) of the two LmRPB6 orthologues. The latter experiments also identified the RNAP III subunits RPC1 (C160), RPC2 (C128), RPC3 (C82), RPC4 (C53), RPC5 (C37), RPC6 (C34), RPC9 (C17), RPAC1 (AC40) and RPAC2 (AC19). Significantly, the complexes precipitated by TAP-tagged LmRPB6 did not contain any RNAP I-specific subunits, suggesting that, unlike in other eukaryotes, LmRPB6 is not shared by all three polymerases but is restricted to RNAP II and III, while the LmRPB6z (encoded by LmjF25.0140) isoform is limited to RNAP I. Similarly, we identified peptides from only one (encoded by LmjF18.0780) of the two RPB5 orthologues and one (LmjF13.1120) of the two RPB10 orthologues, suggesting that LmRPB5z (LmjF18.0790) and LmRPB10z (LmjF13.1120) are also restricted to RNAP I. In addition to these RNAP subunits, we also identified a number of other proteins that co-purified with the RNAP II and III complexes, including a potential transcription factor, several histones, an ATPase involved in chromosome segregation, an endonuclease, four helicases, RNA splicing factor PTSR-1, at least two RNA binding proteins and several proteins of unknown function.

Human Molecular Genetics, 2002

Poly(ADP-ribose) polymerase 2 (PARP-2) is a newly discovered member of the PARP family. We report... more Poly(ADP-ribose) polymerase 2 (PARP-2) is a newly discovered member of the PARP family. We report the association of PARP-2 with mammalian centromeres in a cell-cycle-dependent manner, accumulating at centromeres during prometaphase and metaphase, disassociating during anaphase, and disappearing from the centromeres by telophase. Analysis of a pseudodicentric chromosome and a human neocentromere indicates that PARP-2 binding occurs only at active centromeres in a sequence-independent manner. Centromere binding peaks at the outer centromere region, and is significantly enhanced upon treatment with microtubule-inhibiting drugs. Co-immunoprecipitation assay demonstrates interaction between PARP-2 and its functional homolog PARP-1, constitutive centromere proteins Cenpa and Cenpb, and spindle checkpoint protein Bub3, but not with a third constitutive centromere protein Cenpc. These results, together with our previous demonstration that PARP-1 displays an identical binding pattern with Cenpa, Cenpb and Bub3, but not Cenpc, and that all three proteins undergo significant poly(ADP-ribosyl)ation upon c-irradiation of cells, point to possible diverse roles of PARP-2 and PARP-1 in modulating the structure and checkpoint functions of the mammalian centromere, in particular during radiation-induced DNA damage.

Disease Markers, 2012

Mutations in the MECP2 gene are found in a large proportion of girls with Rett Syndrome. Despite ... more Mutations in the MECP2 gene are found in a large proportion of girls with Rett Syndrome. Despite extensive research, the principal role of MeCP2 protein remains elusive. Is MeCP2 a regulator of genes, acting in concert with co-activators and co-repressors, predominantly as an activator of target genes or is it a methyl CpG binding protein acting globally to change the chromatin state and to supress transcription from repeat elements? If MeCP2 has no specific targets in the genome, what causes the differential expression of specific genes in the Mecp2 knockout mouse brain? We discuss the discrepancies in current data and propose a hypothesis to reconcile some differences in the two viewpoints. Since transcripts from repeat elements contribute to piRNA biogenesis, we propose that piRNA levels may be higher in the absence of MeCP2 and that increased piRNA levels may contribute to the mis-regulation of some genes seen in the Mecp2 knockout mouse brain. We provide preliminary data showin...

Nucleic Acids Research, 2008

MECP2, a relatively small gene located in the human X chromosome, was initially described with th... more MECP2, a relatively small gene located in the human X chromosome, was initially described with three exons transcribing RNA from which the protein MeCP2 was translated. It is now known to have four exons from which two isoforms are translated; however, there is also evidence of additional functional genomic structures within MECP2, including exons potentially transcribing non-coding RNAs. Accompanying the recognition of a higher level of intricacy within MECP2 has been a recent surge of knowledge about the structure and function of human genes more generally, to the extent that the definition of a gene is being revisited. It is timely now to review the published and novel functional elements within MECP2, which is proving to have a complexity far greater than was previously thought.

Antimicrobial Agents and Chemotherapy, 2002

The opportunistic fungal pathogen Candida albicans is the major causative agent of oropharyngeal ... more The opportunistic fungal pathogen Candida albicans is the major causative agent of oropharyngeal candidiasis (OPC) in AIDS. The development of azoles, such as fluconazole, for the treatment of OPC has proven effective except in cases where C. albicans develops resistance to fluconazole during the course of treatment. In the present study, we used microarray technology to examine differences in gene expression from a fluconazole-susceptible and a fluconazole-resistant well-characterized, clinically obtained matched set of C. albicans isolates to identify genes which are differentially expressed in association with azole resistance. Among genes found to be differentially expressed were those involved in amino acid and carbohydrate metabolism; cell stress, cell wall maintenance; lipid, fatty acid, and sterol metabolism; and small molecule transport. In addition to CDR1 , which has previously been demonstrated to be associated with azole resistance, the drug resistance gene RTA3 , the e...