Biology subjects

Alam, F.

Publications and source records attributed to Alam, F..

10 recordsLinked to original sources

bioRxiv2026

XIAP-associated factor 1 protects against viral-induced neuropathogenesis.

XIAP-associated factor 1 (XAF1) is a proapoptotic protein known to be involved in tumor suppression and regression whose gene expression has been reported to be dysregulated in a wide variety of tumor malignancies by different molecular mechanisms. Using a sterile alpha and TIR motif containing 1 (SARM1) knockout mouse model, we previously showed that XAF1 could be a candidate gene for protection against neurotropic virus infection. Here, using a CRISPR-knockout XAF1 mouse model, we show that XAF1 knockout mice are more susceptible to disease after VSV infection, a well-known neurotropic virus in mice. Interestingly, VSV-increased sensitivity in XAF1 knockout mice was not accompanied by differences in viral replication in the central nervous system (CNS). Nevertheless, infection of XAF1 knockout mice resulted in an increased pro-inflammatory response and immune cell infiltration into the CNS compared to that in wild-type mice. Similarly, XAF1 knockout mice showed slight increase to disease after infection with a different neurotropic virus, West Nile Virus (WNV). However, no differences in viral disease due to the absence of XAF1 were found upon infection with a respiratory virus such as influenza A virus (IAV). In vitro, XAF1-deficient cells showed a significant increase in interferon-stimulated genes (ISGs) expression upon stimulation with IFN and with different PAMPs, such as Poly(I:C), HT-DNA and LPS. Consistently, ectopic overexpression of XAF1 decreased IFN-signaling in a dose-dependent manner. Altogether, the data presented here suggest that the host factor XAF1 has a protective role in viral-induced neuropathogenesis due to excessive IFN responses. Author summaryWe previously identified XIAP-associated factor 1 (XAF1) as a candidate cell factor involved in viral phenotypes attributed to SARM1 deficiency. Even though the role of this factor has been studied in the cancer field as a proapoptotic tumor suppressor, its relevance in the context of viral infections has remained unclear. Here, we show that XAF1-deficient mice show increased susceptibility upon neurotropic virus infection and augmented levels of proinflammatory cytokines. We observe higher immune cell infiltration into the brain and disease exacerbation upon infection in mice lacking XAF1. This increased pathology is restricted to the brain, since no morbidity was observed upon infection with a respiratory virus, such as influenza virus. Gene expression analysis unveiled an unbalanced immune response in XAF1-deficient mice resulting in an elevated proinflammatory response and diminished capacity to restore homeostasis. Our data demonstrates the protective capacity of XAF1 and provides new insights into the host response against virus infections.

microbiology↗

bioRxiv2025

An optimised method for generation of murine CAR-T cells by CRISPR/Cas9.

Development of the next generation of chimeric antigen receptor (CAR) T-cells requires assessment in systems that better recapitulate the suppressive tumour microenvironment of solid tumours. CRISPR-Cas9 knock-in of promoter-less homology directed repair templates (HDRT) into the T-cell receptor locus has been shown to result in physiological expression of CARs with improved tumour control. We initially compared the use of dsDNA and adenovirus associated virus (AAV) HDRTs in mouse T cells. We have subsequently developed an optimised method for AAV transduction resulting in high editing efficiencies with minimal toxicity. In contrast with our experience of retroviral transduction of mouse T cells, our CRISPR/Cas9 AAV transduction method results in sustained CAR expression and T cell expansion in vitro as well as in vivo persistence. This approach allows for pre-clinical assessment of individual and libraries of CAR constructs in relevant immune-competent mouse models.

molecular biology↗

bioRxiv2025

In search of nonlipogenic ABCA1 inducers (NLAI): precision coregulator TR-FRET identifies diverse signatures for LXR ligands

APOE4, the major genetic risk factor for Alzheimers disease (AD), and ABCA1, required for lipidation of APOE are gene products of the liver X receptor (LXR) receptor. LXR agonists have been validated in animal models as therapeutics for AD, atherosclerosis, and many other diseases. Clinical progress has been thwarted by unwanted hepatic lipogenesis. Structurally diverse LXR ligands were profiled in coregulator TR-FRET (CRT) assays analyzing ligand-induced coactivator recruitment, coactivator selectivity, corepressor dissociation, and LXR isoform selectivity. A multiplex CRT assay was developed to measure synchronous ligand-induced displacement of corepressor by coactivator. Potency for coactivator recruitment to LXR{beta} correlated with induction of ABCA1 in human astrocytoma cells. Correlation with lipogenic activation of sterol response element (SRE) in hepatocarcinoma cells, was more complex. CRT response was diverse revealing ligands with theoretical full agonist, partial agonist, antagonist, and inverse agonist, and other signatures within the same chemical series, suggesting the scope for precision CRT to guide nonlipogenic LXR agonist design.

pharmacology and toxicology↗

bioRxiv2025

Self-assembly is important for the target membrane recruitment of a nuclear dynamin-related protein

Dynamin superfamily proteins are large GTPases that perform their cellular functions by self-assembling on their target membranes. Dynamin-related protein 6 (Drp6) associates with the nuclear membrane and performs nuclear remodeling. However, the mechanism of its recruitment to the target membrane is not known. Here, we discover that self-assembly of Drp6 is essential for its nuclear membrane recruitment. We identified four residues, 411-GKFR-414 to be essential for its self-assembly. We also demonstrated that the mutant Drp6 (Drp6GKFR-AAAA) failed to recruit to the nuclear membrane. This loss of nuclear membrane recruitment is not due to the lack of membrane binding capability, since the mutated protein was able to bind membrane prepared in vitro. Together, our results suggest that in addition to membrane binding, self-assembly of a nuclear dynamin-related protein is also important for the target membrane recruitment.

cell biology↗

bioRxiv2024

The prion-like protein Doppel: A soluble biomarker steering ovarian cancers peritoneal to circulatory dissemination

Detecting ovarian cancer (OC) early using existing biomarkers, e.g., cancer antigen 125 (CA125), is challenging due to its ubiquitous expression in many tissues. Doppel, a prion-like protein, expresses in male reproductive organ but absent in female reproductive systems and healthy tissues, but plays an important role in neoangiogenesis. Here, we have shown two platforms, soluble Doppel in sera/ascites and Doppel expressed circulating tumor cells (Dpl+CTC) in the whole blood, to detect subsets of epithelial OC (EOC). Increased level of Doppel in the sera of OC patients, in three different cohorts, confirm Doppel as OC specific biomarker. Serum Doppel level distinguishes EOC subtypes and early stages HGSOCs from non-cancerous conditions with high sensitivity and specificity. Stratifying the EOCs based on Doppel level, we categorized them into Doppel-high (Dplhi) and Doppel-low (Dpllow) groups. Using ascites-derived organoids and single cell sequencing of whole ascites of Dplhi and Dpllow patients, we identify that Doppel induces epithelial-mesenchymal transition (EMT) and creates an immunosuppressive microenvironment, respectively. Doppel levels in the sera/ascites correlate with the changes of Dpl+CTC number in whole blood, highlighting the association of Doppel-induced EMT with CTC dissemination in circulation. Thus, Doppel-based detection of EOC subtypes could be a promising platform as clinical biomarker and link Doppel-axis with OC dissemination.

cancer biology↗

bioRxiv2022

Pseudomonas aeruginosa increases the susceptibility of Candida albicans to amphotericin B in dual species biofilms

Biofilms are the leading cause of nosocomial infections, and are hard to eradicate due to their inherent antimicrobial resistance. Candida albicans is the leading cause of nosocomial fungal infections, and is frequently co-isolated with the bacterium Pseudomonas aeruginosa from biofilms in the Cystic Fibrosis lung and severe burn wounds. The presence of C. albicans in multi-species biofilms is associated with enhanced antibacterial resistance, which is largely mediated through fungal extracellular carbohydrates sequestering the antibiotics. However, significantly less is known regarding the impact of polymicrobial biofilms on antifungal resistance. Here we show that, in dual species biofilms, P. aeruginosa enhances the sensitivity of C. albicans to amphotericin B, an effect that was biofilm specific. Transcriptional analysis combined with gene ontology enrichment analysis identified several C. albicans processes associated with oxidative stress to be differentially regulated in dual species biofilms, suggesting that P. aeruginosa exerts oxidative stress on C. albicans, likely through the actions of secreted phenazines. However, the mitochondrial superoxide dismutase SOD2 was significantly downregulated in the presence of P. aeruginosa. Monospecies biofilms of the sod2{Delta} mutant were more susceptible to amphotericin B, and the susceptibility of these biofilms was further enhanced by the addition of exogenous phenazines. Therefore, we propose that in dual species biofilms, P. aeruginosa simultaneously induces mitochondrial oxidative stress, whilst downregulating key detoxification enzymes, which prevent C. albicans mounting an appropriate oxidative stress response to amphotericin B, leading to fungal cell death. This work highlights the importance of understanding the impact of polymicrobial interactions on antimicrobial susceptibility. ImportanceBiofilms are aggregates of cells encased in an extracellular matrix, and are responsible for the majority of infections in hospitals. The Gram-negative bacterium Pseudomonas aeruginosa, and the fungal pathogen Candida albicans are frequently co-isolated from biofilms in the Cystic Fibrosis lung, and in burn wounds. Here we show that in these biofilms, P. aeruginosa secreted phenazines induce mitochondrial reactive oxygen species stress, which enhances the sensitivity of C. albicans to the antifungal amphotericin B. This work highlights the importance of understanding the impact of polymicrobial interactions on antimicrobial susceptibility.

microbiology↗

bioRxiv2022

Impact of SARS-CoV-2 ORF6 and its variant polymorphisms on host responses and viral pathogenesis.

We and others have previously shown that the SARS-CoV-2 accessory protein ORF6 is a powerful antagonist of the interferon (IFN) signaling pathway by directly interacting with Nup98-Rae1 at the nuclear pore complex (NPC) and disrupting bidirectional nucleo-cytoplasmic trafficking. In this study, we further assessed the role of ORF6 during infection using recombinant SARS-CoV-2 viruses carrying either a deletion or a well characterized M58R loss-of-function mutation in ORF6. We show that ORF6 plays a key role in the antagonism of IFN signaling and in viral pathogenesis by interfering with karyopherin(importin)-mediated nuclear import during SARS-CoV-2 infection both in vitro, and in the Syrian golden hamster model in vivo. In addition, we found that ORF6-Nup98 interaction also contributes to inhibition of cellular mRNA export during SARS-CoV-2 infection. As a result, ORF6 expression significantly remodels the host cell proteome upon infection. Importantly, we also unravel a previously unrecognized function of ORF6 in the modulation of viral protein expression, which is independent of its function at the nuclear pore. Lastly, we characterized the ORF6 D61L mutation that recently emerged in Omicron BA.2 and BA.4 and demonstrated that it is able to disrupt ORF6 protein functions at the NPC and to impair SARS-CoV-2 innate immune evasion strategies. Importantly, the now more abundant Omicron BA.5 lacks this loss-of-function polymorphism in ORF6. Altogether, our findings not only further highlight the key role of ORF6 in the antagonism of the antiviral innate immune response, but also emphasize the importance of studying the role of non-spike mutations to better understand the mechanisms governing differential pathogenicity and immune evasion strategies of SARS-CoV-2 and its evolving variants. ONE SENTENCE SUMMARYSARS-CoV-2 ORF6 subverts bidirectional nucleo-cytoplasmic trafficking to inhibit host gene expression and contribute to viral pathogenesis.

microbiology↗

bioRxiv2021

Nicotinamide riboside alleviates Parkinson's disease symptoms but downregulates dopamine metabolism upon lactacystin-induced proteostasis failure

Activation of mitochondrial metabolism and proteostasis with the NAD+ precursor nicotinamide riboside (NR) has emerged as a potential therapeutic approach for neurodegenerative disorders including Parkinsons disease (PD). However, despite recently started clinical trials, studies on NR in animal models of PD are scarce. In this study, we investigated the effect of NR in multiple models of PD. In transgenic C. elegans overexpressing -synuclein, a protein of which aggregation is believed to promote PD, NR rescued PD-like phenotypes including mitochondrial dysfunction and motility defects, decreased oxidative stress, and age-related dopamine (DA) neuron loss. We found that NR eased symptoms of disease by activating the mitochondrial unfolded protein response (UPRmt) via the transcription factor atfs-1. Similarly, in a proteasome inhibitor, lactacystin, -induced mouse model of PD, NR rescued mitochondrial dysfunction and behavioural deficits caused by lactacystin lesion. However, long-term NR supplementation, in conjunction with proteasome inhibition, resulted in decreased DA levels in both the lesioned and unlesioned sides of the substantia nigra with concomitant downregulation of key genes in DA metabolism. Our results suggest specific endpoints that should be monitored in ongoing NR clinical trials.

neuroscience↗

bioRxiv2020

Minimal information for Chemosensitivity assays (MICHA): A next-generation pipeline to enable the FAIRification of drug screening experiments

Chemosensitivity assays are commonly used for preclinical drug discovery and clinical trial optimization. However, data from independent assays are often discordant, largely attributed to uncharacterized variation in the experimental materials and protocols. We report here the launching of MICHA (Minimal Information for Chemosensitivity Assays), accessed via https://micha-protocol.org. Distinguished from existing efforts that are often lacking support from data integration tools, MICHA can automatically extract publicly available information to facilitate the assay annotation including: 1) compounds, 2) samples, 3) reagents, and 4) data processing methods. For example, MICHA provides an integrative web server and database to obtain compound annotation including chemical structures, targets, and disease indications. In addition, the annotation of cell line samples, assay protocols and literature references can be greatly eased by retrieving manually curated catalogues. Once the annotation is complete, MICHA can export a report that conforms to the FAIR principle (Findable, Accessible, Interoperable and Reusable) of drug screening studies. To consolidate the utility of MICHA, we provide FAIRified protocols from five major cancer drug screening studies, as well as six recently conducted COVID-19 studies. With the MICHA webserver and database, we envisage a wider adoption of a community-driven effort to improve the open access of drug sensitivity assays.

bioinformatics↗

bioRxiv2017

A genetic screen suggests an alternative mechanism for inhibition of SecA by azide

Sodium azide prevents bacterial growth by inhibiting the activity of SecA, which is required for translocation of proteins across the cytoplasmic membrane. Azide inhibits ATP turnover in vitro, but its mechanism of action in vivo is unclear. To investigate how azide inhibits SecA in cells, we used transposon directed insertion-site sequencing (TraDIS) to screen a library of transposon insertion mutants for mutations that affect the susceptibility of E. coli to azide. Insertions disrupting components of the Sec machinery generally increased susceptibility to azide, but insertions truncating the C-terminal tail (CTT) of SecA decreased susceptibility of E. coli to azide. Treatment of cells with azide caused increased aggregation of the CTT, suggesting that azide disrupts its structure. Analysis of the metal-ion content of the CTT indicated that SecA binds to iron and the azide disrupts the interaction of the CTT with iron. Azide also disrupted binding of SecA to membrane phospholipids, as did alanine substitutions in the metal-coordinating amino acids. Furthermore, treating purified phospholipid-bound SecA with azide in the absence of added nucleotide disrupted binding of SecA to phospholipids. Our results suggest that azide does not inhibit SecA by inhibiting the rate of ATP turnover in vivo. Rather, azide inhibits SecA by causing it to \"backtrack\" from the ADP-bound to the ATP-bound conformation, which disrupts the interaction of SecA with the cytoplasmic membrane.\n\nSignificance statementSecA is a bacterial ATPase that is required for the translocation of a subset of secreted proteins across the cytoplasmic membrane. Sodium azide is a well-known inhibitor of SecA, but its mechanism of action in vivo is poorly understood. To investigate this mechanism, we examined the effect of azide on the growth of a library of [~]1 million transposon insertion mutations. Our results suggest that azide causes SecA to backtrack in its ATPase cycle, which disrupts binding of SecA to the membrane and to its metal cofactor, which is iron. Our results provide insight into the molecular mechanism by which SecA drives protein translocation and how this essential biological process can be disrupted.

microbiology↗