Biology subjects

Amin, A.

Publications and source records attributed to Amin, A..

At least 19 recordsLinked to original sources

bioRxiv2026

Single-cell RNA-seq reveals conserved and divergent cellular states across wound types and species

Chronic wounds, such as diabetic foot ulcers, fail to progress through the normal healing process and impose a significant burden on healthcare systems. While previous single-cell studies have characterized specific wound conditions, a unified understanding of the shared and distinct cellular landscapes across diverse wound microenvironments has been lacking. Therefore, we integrated over 500,541 cells from patients and mice across multiple wound conditions, including acute wound, diabetic foot ulcer, and venous ulcer as well as their healing outcome. Fibroblast-focused analysis identified a bifurcation in differentiation trajectories and identified STAT3 as a potential regulator of a reparative program in chronic wound. Furthermore, we discovered immune dysfunctions in non-healed chronic wounds, contrasting the quiescent memory-like T cells and TIMP1+ macrophages in healed chronic wounds with the exhausted T cells and foamy SPP1+ macrophage enriched in non-healed chronic wounds. Finally, we translated these results into a clinically applicable three-gene signature (CHI3L1, TIMP1, and SPP1) that accurately predicts chronic wound healing. To support wound biology community, we developed WoundSCAtlas, an interactive web resource for exploring diverse wound pathologies. In conclusion, this work provides a comprehensive and cross-species landscape of chronic wound healing, identifying conversed wound outcome-associated molecular programs, predictive biomarker, and interactive data resource.

bioinformatics↗

bioRxiv2026

The CNS-penetrant MAP4K inhibitor famlasertib restrains medulloblastoma dissemination without developmental toxicity

Cerebellum tissue invasion and dissemination are major drivers of recurrence and metastatic spread in medulloblastoma (MB), yet no invasion-inhibitory therapy is currently available. The serine/threonine kinase MAP4K4 is highly expressed in MB and promotes invasive behavior downstream of growth factor signaling, while its physiological postnatal downregulation suggests that disrupted developmental control may contribute to tumor pathogenesis. Here, we investigate pharmacological targeting of MAP4K4 using famlasertib, a CNS-penetrant, neuroprotective MAP4K inhibitor, as a strategy to suppress invasion in MB. Using 3D invasion assays and quantitative live-cell microscopy, we demonstrate that famlasertib markedly reduces invasive behavior and single-cell motility of MB cells. Organotypic cerebellum tissue and orthotopically implanted zebrafish larval models further confirm anti-invasive efficacy without detectable developmental toxicity at effective concentrations. Phospho-proteomics and functional analyses revealed that MAP4K inhibition alters kinase signaling linked to cytoskeletal remodeling, leading to suppressed F-actin dynamics, increased cell clustering, and enhanced cortical accumulation of tight junction protein 1 (TJP1). Collectively, our findings confirm MAP4Ks as therapeutically targetable regulators of MB invasion and establish famlasertib as a candidate migrastatic agent that restrains tumor cell dissemination while preserving developmental integrity.

cancer biology↗

bioRxiv2026

A low-cost rpoB-based multiplex MAMA PCR for differentiation of the Klebsiella pneumoniae species complex

The Klebsiella pneumoniae species complex (KpSC) is a clinically important group of closely related pathogens associated with invasive infections. The complex comprises seven closely related members, which are often reported as K. pneumoniae, particularly in resource-limited settings. Accurate differentiation of KpSC members remains challenging because routine laboratory methods lack sufficient resolution, and approaches like mass spectrometry and whole genome sequencing (WGS) are not widely available. Consequently, the epidemiology and clinical significance of non-K. pneumoniae members of the KpSC remain underrecognized. We developed a conventional multiplex mismatch amplification mutation assay (MAMA) PCR targeting species- and subspecies-specific single-nucleotide polymorphisms in the housekeeping gene rpoB, with six primer sets for differentiation of common KpSC members. The assay was validated against 49 genomically characterized clinical isolates, after which 179 wastewater-derived isolates provisionally identified as Klebsiella spp. by standard microbiological methods were tested. Of these, 174 were assigned to specific KpSC members by the assay, while 5 produced inconclusive amplification patterns. A subset of 16 environmental isolates was selected for WGS, including four of the five inconclusive isolates. All environmental isolates with interpretable MAMA PCR patterns were concordant with WGS. The four inconclusive environmental isolates were identified as Enterobacter spp. Overall, comparison of MAMA PCR with WGS showed 100% sensitivity and 100% specificity for all tested targets, and the total cost was approximately US$1. This rpoB-based multiplex MAMA PCR provides a simple, accurate, and low-cost approach for differentiation of KpSC members in routine laboratories and may support improved identification and surveillance in resource-limited settings. ImportanceThe Klebsiella pneumoniae species complex (KpSC) has seven members but is often reported as a single organism in routine laboratories, masking clinically and epidemiologically important diversity. As a result, the contribution of non-K. pneumoniae KpSC members to human and environmental microbiology remains poorly defined, especially in low-resource settings. We developed a conventional multiplex mismatch amplification mutation assay (MAMA) PCR based on discriminatory rpoB single nucleotide polymorphisms for differentiation of common KpSC members using standard PCR and agarose gel electrophoresis. The assay demonstrated 100% sensitivity and 100% specificity against whole-genome sequencing and excluded non-Klebsiella environmental isolates initially identified as Klebsiella pneumoniae using standard microbiological procedures. With an estimated per-test cost of about US$1, this method offers an affordable and scalable option for laboratories seeking more accurate KpSC identification and improved surveillance.

microbiology↗

bioRxiv2025

Genomic Flexibility Through Extrachromosomal Amplifications: A Leishmania Survival Strategy

Leishmania parasites modulate gene copy number through extrachromosomal DNA (ecDNA) amplification, enabling adaptation to environmental stress. Under drug pressure, both linear and circular ecDNA amplifications (amplicons) carrying resistance genes emerge. However, how these ecDNA structures form, diversify, and coexist remains poorly understood. Here, using experimental evolution and Oxford Nanopore long-read sequencing, we show that a single clonal population of drug resistant Leishmania produces a variety of linear and circular amplicons. As antimonial pressure increases, linear amplicons transition into circular forms, with high-stress conditions favoring circular amplicons carrying at least two copies of the resistance gene. Using the Nanopore long reads, we map recombination events driving linear and circular amplicon formation. Our model suggests that gene duplication in the amplicons originates from inter-chromatid homologous recombination, leading to an intermediate intra-chromosomal duplication, followed by a second homologous recombination event. Additionally, different Leishmania species exhibited distinct biases toward linear or circular amplification under identical drug conditions, suggesting species-specific adaptive strategies. Together, these findings define recombination-driven ecDNA dynamics as a central axis of genomic plasticity in Leishmania and underscore the potential for targeting ecDNA in therapeutic and diagnostic strategies against Leishmania and related pathogens.

microbiology↗

bioRxiv2025

The Adipomyokine Follistatin-like-1 Restores Cardiovascular Function in a Swine Model of Diabetic Myocardial Infarction

Obesity, diabetes, and metabolic syndrome increase the incidence and complicate the management of myocardial infarction (MI). Current treatments do not adequately blunt the progression to chronic ischemic heart disease and heart failure, making these diseases the largest cause of mortality worldwide. Insulin resistance and metabolic syndrome were induced in obese Ossabaw swine prior to ischemia/reperfusion injury-induced MI. Subcutaneous administration of the adipomyokine Follistatin-like-1 (recombinant non-glycosylated human FSTL1, ngFSTL1) for two weeks, beginning one month following myocardial infarction, decreased infarct necrosis, increased blood flow, and increased cardiomyocyte proliferation within the infarct region, leading to improved systolic and diastolic function. ngFSTL-1 also enhanced coronary and peripheral vascular function by increasing BKCa channel-dependent vasodilatory capacity. We conclude that subcutaneous ngFSTL1 ameliorated clinically relevant parameters of cardiac and vascular dysfunction in a preclinical swine model of diabetic MI, and suggest FSTL1 treatment may be broadly efficacious in humans. ARTICLE HIGHLIGHTSO_LISubcutaneous administration of recombinant human non-glycosylated FSTL1 (ngFSTL1) for 2 weeks, 1 month after infarction/reperfusion injury, improved cardiac function in a preclinical obese swine model of diabetic MI. C_LIO_LIngFSTL1 treatment induced cardiomyocyte proliferation and improved coronary perfusion, resulting in reduced necrosis of the infarct region. C_LIO_LIngFSTL1 treatment increased blood flow through peripheral arterioles, including cerebral and skeletal muscle, in addition to cardiac arterioles. C_LIO_LISystemic delivery of the non-glycosylated form of the adipomyokine FSTL1 might be an effective treatment for diabetic MI. C_LI

physiology↗

bioRxiv2025

Time-Resolved Transcriptomics Reveal Spliceosomal Disruption and Senescence Pathways in Crocin-Treated Hepatocellular Carcinoma Cells

Saffron-derived crocin exhibits anti-cancer properties, but the pathways underlying its effects remain incompletely characterized. Here, we utilized a high-dose perturbation strategy (1-2 mM crocin) to probe maximal pathway engagement in HepG2 hepatocellular carcinoma cells via time-series transcriptomics. We treated cells for 2, 6, 12, and 24 h and analyzed transcriptomic and splicing profiles at each timepoint. We identified 7400-12,100 differentially expressed genes (DEGs) per condition, with the higher dose (CR2) producing more total DEGs but the lower dose (CR1) demonstrating differential pathway prioritization. The spliceosome pathway ranked first among downregulated pathways for CR1 across multiple timepoints (false discovery rate, FDR p = 10-21 to 10-36) but only fourth for CR2, suggesting dose-dependent differences in pathway prioritization. Differential splicing analysis revealed functional spliceosome disruption, with 2000-2600 significant exon skipping events per condition and aberrant splicing of spliceosome components including HNRNPH1 (change in percent spliced in, dPSI = -0.78 to -0.89). Additionally, 66 genes implicated in non-alcoholic fatty liver disease were downregulated at 24 h (FDR p = 8x10-8). Crocin exposure consistently downregulated spliceosomal machinery genes while upregulating senescence and autophagy pathways. These findings identify spliceosome components and RNA processing machinery as crocin-sensitive pathways.

cancer biology↗

bioRxiv2025

Telomere-to-telomere assembly detects genomic diversity in Canadian strains of Borrelia burgdorferi

Borrelia burgdorferi, the bacteria causing Lyme disease, has a complex genome comprising a linear chromosome and a combination of linear and circular plasmids. The atypical hairpin structure at the telomere of linear replicons and the highly paralogous plasmids make the genome assembly challenging. We developed a genome assembly pipeline using Oxford Nanopore Technologies (ONT) long read and Illumina short read to overcome these challenges. Using ONT reads enabled us to completely assemble the hairpin telomeres of the linear replicons along with a novel lp28 subtype plasmid and the complete circular plasmids of nine B. burgdorferi strains from five geographical regions in Canada. Although these strains are highly similar across the conserved genomic regions, variability was observed predominantly at the right telomeric ends. Comparative analyses revealed that all nine strains carry a [~]2-10 kb right telomeric end identical to the linear plasmid lp28-1, which leads to variability in the telomere length and the gene content. Additionally, we observed diversity at the hairpin telomeric sequences of the linear chromosomes. Further analysis showed that the nine strains belong to seven ospC types and have diverse plasmid profile, highlighting the genomic diversity among the strains from the same geographical locations. Overall, these findings suggest that even the B. burgdorferi strains from close geographical locations can carry substantial genomic variation, especially at the telomeres and with respect to their plasmid content, emphasizing it to be a possible mechanism of rapid evolution within these Canadian strains.

genomics↗

bioRxiv2025

Chronobot: Deep learning guided time-resolved cryo-EM captures molecular choreography of RecA in homology search

The function of proteins and other biological macromolecules is regulated by conformational dynamics1. Many functional changes take place on millisecond timescales which cannot be experimentally captured by manual sample preparation for cryo-EM2. Here we introduce Chronobot, a robust, data-driven platform enabling reproducible, time-resolved cryo-EM sample preparation to visualize these transient intermediates. We quantify ice thickness, an important precondition and thus a reliable predictor of 3D reconstruction quality, using two complementary methods: a bespoke deep learning model analysing high-speed camera videos of the grid just before vitrification, and detailed ice thickness quantifications of outputs from common TEM screening workflows like the EPU software. Combining these methods enables rapid optimisation, resulting in an 11-fold improvement in cryo-EM sample quality compared to our previously reported workflow3. To demonstrate the Chronobot in capturing transient reaction intermediates visualised through cryo-EM and single particle analysis we focused on RecA homology search. The RecA family of recombinases perform the essential task of rapidly scanning for homologous dsDNA sequences to initiate homologous recombination. The dynamics of these RecA-dsDNA interactions occur on millisecond timescales, limiting structural insights4. We capture time-resolved homology search intermediates at 250 milliseconds. These structures reveal the involvement of the secondary DNA binding site in initial capture of dsDNA before homology sampling occurs. We also observe three-strand homology sampling intermediates, where the homologous strand is not fully displaced, and homology is not stably bound. Our results suggest a model of how RecA-family recombinases function in early homologous recombination, by coordinating the incoming DNA between RecAs various DNA binding sites depending on the stage of homology search and the presence of suUicient homology. We anticipate the Chronobot method to be broadly applicable to processes which cannot be captured by manual sample preparation methods. In addition, by leveraging AI inference, our rapid user feedback mechanisms allow for per-sample optimisation of grid conditions, increasing the likelihood of success and reducing the sample requirements of each time-resolved experiment.

biochemistry↗

bioRxiv2025

Single-cell characterization of the human C2 dorsal root ganglion recovered from C1-2 arthrodesis surgery: implications for neck pain

Neurons in the dorsal root ganglion (DRG) receive and transmit sensory information from the tissues they innervate and from the external environment. Upper cervical (C1-C2) DRGs are functionally unique as they receive input from the neck, head, and occipital cranial dura, the latter two of which are also innervated by the trigeminal ganglion (TG). The C2 DRG also plays an important role in neck pain, a common and disabling disorder that is poorly understood. Advanced transcriptomic approaches have significantly improved our ability to characterize RNA expression patterns at single-cell resolution in the DRG and TG, but no previous studies have characterized the C2 DRG. Our aim was to use single-nucleus and spatial transcriptomic approaches to create a molecular map of C2 DRGs from patients undergoing arthrodesis surgery with ganglionectomy. Patients with acute (<3 months) or chronic ([≥]3 months) neck pain were enrolled and completed patient-reported outcomes and quantitative sensory testing prior to surgery. C2 DRGs were characterized with bulk, single nucleus, and spatial RNA sequencing technologies from 22 patients. Through a comparative analysis to published datasets of the lumbar DRG and TG, neuronal clusters identified in both TG and DRG were identified in the C2 DRG. Therefore, our study definitively characterizes the molecular composition of human C2 neurons and establishes their similarity with unique characteristics of subsets of TG neurons. We identified differentially expressed genes in endothelial, fibroblast and myelinating Schwann cells associated with chronic pain, including FGFBP2, C8orf34 and EFNA1 which have been identified in previous genome and transcriptome wide association studies (GWAS/TWAS). Our work establishes an atlas of the human C2 DRG and identifies altered gene expression patterns associated with chronic neck pain. This work establishes a foundation for the exploration of painful disorders in humans affecting the cervical spine.

neuroscience↗

bioRxiv2025

Envirotyping Facilitates Understanding of Genotype x Environment Interactions and Highlights the Potential of Stay-green Traits in Wheat

Better understanding genotype by environment interaction (GxE) can help breeding for better adapted varieties. Envirotyping for environmental water status was applied to assist interpretation of GxE interactions for wheat yield in multi-environment trials conducted in drought-prone Australian environments. Genotypes from a multi-reference parent nested association mapping (MR-NAM) population were tested in 10 trials across the Australian wheatbelt. Genotype yield and phenology were measured in all trials, while traits associated with the stay-green phenotype were assessed for a subset of 5 trials. Envirotyping was conducted by characterizing water stress experienced by genotypes at each trial using crop modelling. Envirotyping facilitated the understanding of GxE interactions by explaining 75, 67, and 66% of the genotypic variance for yield in severe water-limited (ET3), mild terminal water-stress (ET2), and water-sufficient (ET1) environments, respectively. Yield and stay-green were negatively correlated with flowering time in most trials. However, when focusing on genotypes flowering at similar times within a trial, no significant correlation was found between yield and flowering. Importantly stay-green traits remained significantly correlated with yield. Stay-green traits such as delayed onset of senescence and slower senescence rate benefited yield by 0.2 to 1.1 t ha-1 across environments, highlighting the breeding potential for stay-green traits in both water-sufficient and water-limited environments. Hence, sustaining green leaf area during grain filling helped to enhance yield. Envirotyping to better understand GxE interactions for yield, coupled with screening for traits exhibiting superior adaptive mechanisms, are powerful assets in assisting plant breeders to select more effectively drought adapted genotypes. HighlightsGenotype x environment interaction for yield could be reduced with envirotyping. Envirotyping enabled a 75% gain in genotypic variance in severe drought conditions. Envirotyping clarified breeding potential of stay-green traits across environments. Stay-green has potential to enhance wheat yield across diverse environments.

physiology↗

bioRxiv2025

A critical role for the ER Membrane Complex (EMC) in lipid droplet homeostasis

The ER Membrane complex (EMC) facilitates insertion of multiple classes of integral membrane protein into the lipid bilayer of the endoplasmic reticulum (ER). Recent years have seen significant progress in understanding the structural aspects of EMC function, but the client protein/s enforcing its evolutionary conservation throughout eukaryotic kingdoms remain to be determined. Given reported role/s for the EMC in lipid homeostasis, we deleted the essential EMC3 subunit in the liver and adipose tissues of mice and examined the impact of EMC loss in a range of metabolic models. Intriguingly, absence of EMC3 in either the liver or adipose tissues results in defective storage of triglycerides and perturbations in lipid droplet (LD) homeostasis. EMC-deficient mouse adipose tissues were defective in their ability to mediate fat storage in response to a high fat diet and were unable to support non-shivering thermogenesis. Proteomic analyses of EMC3-deficient tissues found reduced levels of FITM2, a key regulator of LD biogenesis and ER homeostasis, identifying it as a novel EMC client. Strikingly, deletion of the EMC3 homolog dPob in the Drosophila fat body, which shares overlapping functions with mammalian liver/adipose tissues, revealed similar defects in LD homeostasis. Together, our results indicate that the EMC plays a key evolutionarily conserved role in biogenesis of machinery maintaining triglyceride storage in metazoans.

biochemistry↗

bioRxiv2024

Optimization of citric acid production from sugarcane molasses using Aspergillus niger by submerged Fermentation

The potentiality of citric acid on economy is high because of its multi-purpose uses, particularly in the food and pharmaceutical industries. Bangladesh spent more than million US dollars to import citric acid mostly from India and China. Its consumption is increasing 3.5-4%, annually, indicating the need for better manufacturing alternatives. Globally, citric acid is primarily produced through microbial fermentation with Aspergillus niger. To support the massive scale of production of citrate, the manufacturing process must be eco-friendly which should be inexpensive and available raw materials for maintaining high yielding in a cost-effective manner. In Bangladesh prospective, the current study has undertaken to optimize citric acid production using one of the most abundant raw materials sugarcane molasses. Moreover, the aim of this study was to determine the optimum conditions to produce citric acid from sugarcane molasses using Aspergillus niger (F-81) by submerged fermentation. The amount of citric acid production was determined by Marier-Boulet colorimetric method. The optimization data suggested that 10% substrate (from processed cane molasses), 4% inoculum size of A. niger, and initial pH 6.0 allowed to produce around 25.8 g/L citric acid. Further study is warranted to assess the feasibility of citrate production in an industrial level as well as improvement of microbial strains is needed to further enhance citric acid production.

microbiology↗

bioRxiv2023

Comparative Analysis, Diversification and Functional Validation of Plant Nucleotide-Binding Site Domain Genes

Nucleotide-binding site (NBS) domain genes are one of a superfamily of resistance genes involved in plant responses to pathogens. The current study identified presumably identified 12,820 NBS-containing genes across 34 species covering from mosses to monocots and dicots. These identified genes classified into 168 classes with several novel domain architectures patterns encompassing significant diversity among plant species. Several classical (NBS, NBS-LRR, TIR-NBS, TIR-NBS-LRR etc.) and species-specific structural patterns (TIR-NBS-TIR-Cupin_1-Cupin_1, TIR-NBS-Prenyltransf, Sugar_tr-NBS etc.) were discovered. We observed 603 orthogroups (OGs) with some core (most common orthogroups; OG0, OG1, OG2 etc.) and unique (highly specific to species; OG80, OG82 etc) OGs with tandem duplications. The expression profiling presented the putative upregulation of OG2, OG6, and OG15 in different tissues under various biotic and abiotic stresses in susceptible and tolerant plants to CLCuD. The genetic variation between susceptible (Coker 312) and tolerant (Mac7) G. hirsutum accessions identified several unique variants in NBS genes of Mac7 (6,583 varaints) and Coker312 (5,173 variants). The protein-ligand and proteins-protein interaction showed a strong interaction of some putative NBS proteins with ADP/ATP and different core proteins of cotton leaf curl disease virus. The silencing of GaNBS (OG2) in resistant cotton through virus-induced gene silencing (VIGS) demonstrated its putative role in virus tittering. The presented study will be further helpful to understand the plant adaptation mechanism.

plant biology↗

bioRxiv2023

Microevolution and phylogenomic characterization with perspectives in the 2022-2023 outbreak of child Respiratory Syncytial Virus type A

A communal respiratory syncytial virus (RSV) causes mild to severe illness, predominantly in older adults, or people with certain chronic medical conditions, and in particular, in young children. Symptoms may include runny nose, cough, fever, and difficulty breathing. In most cases, the infection is mild and resolves on its own, but in some cases, it can lead to more serious illness such as bronchiolitis or pneumonia. The RSV genome codes for ten proteins, NS1, NS2, N, P, M, SH, G, F, M2 and L. We aimed to identify the RSV geographical distribution and transmission pattern using site parsimonious frequencies, and investigate hotspot regions across the complete RSV genomes. These results indicated that RSV strains circulating in South and North America are not mixed to the European samples, however, genomes reported from Australia are the direct decedents of European samples. Samples reported from the United Kingdom were found diverse. Further, this report provides a comprehensive mutational analysis of all the individual RSV genes and in particular the 32 hotspot substituting regions circulating across the globe in RSV type A samples. This is the first comprehensive analysis of RSV type A that features mutational frequencies across the whole genome providing more clues for epidemiological control and drug development.

evolutionary biology↗

bioRxiv2022

Semaphorin 4B is an ADAM17-cleaved inhibitor of adipocyte thermogenesis

ObjectiveThe metalloprotease ADAM17 (also called TACE) plays fundamental roles in homeostasis by shedding key signaling molecules from the cell surface. Although its importance for the immune system and epithelial tissues is well-documented, little is known about the role of ADAM17 in metabolic homeostasis. The purpose of this study was to determine the impact of ADAM17 expression, specifically in adipose tissues, on metabolic homeostasis. MethodsWe used histopathology, molecular, proteomic, transcriptomic, in vivo integrative physiological and ex vivo biochemical approaches to determine the impact of adipose tissue-specific deletion of ADAM17 upon adipocyte and whole organism metabolic physiology. ResultsADAM17adipoq-cre{Delta}/{Delta} mice exhibited a hypermetabolic phenotype characterized by elevated energy consumption and increased levels of adipocyte thermogenic gene expression. On a high fat diet, these mice were more thermogenic, while exhibiting elevated expression levels of genes associated with lipid oxidation and lipolysis. This hypermetabolic phenotype protected mutant mice from obesogenic challenge, limiting weight gain, hepatosteatosis and insulin resistance. Activation of beta-adrenoceptors by the neurotransmitter norepinephrine, a key regulator of adipocyte physiology, triggered the shedding of ADAM17 substrates, and regulated ADAM17 expression at the mRNA and protein levels, hence identifying a functional connection between thermogenic licensing and the regulation of ADAM17. Proteomic studies identified Semaphorin 4B (SEMA4B), as a novel ADAM17-shed adipokine, whose expression is regulated by physiological thermogenic cues that acts to dampen thermogenic responses in adipocytes. Transcriptomic data showed that cleaved SEMA4B acts in an autocrine manner in brown adipocytes to dampen the expression of genes involved in thermogenesis, adipogenesis, lipid uptake, storage and catabolism. ConclusionOur findings identify a novel ADAM17-dependent axis, regulated by beta-adrenoceptors and mediated by the ADAM17-cleaved form of SEMA4B, that may act to limit uncontrolled energy depletion during thermogenesis.

physiology↗

bioRxiv2022

The ADAM17 sheddase complex regulator iTAP modulates inflammation, epithelial repair, and tumor growth

The metalloprotease ADAM17 catalyzes the shedding of key signalling molecules from the cell surface, including the inflammatory cytokine TNF (tumour necrosis factor) and activating ligands of the EGFR (epidermal growth factor receptor). ADAM17 exists within an assemblage called the "sheddase complex" containing a rhomboid pseudoprotease (iRhom1 or iRhom2). iRhoms control multiple aspects of ADAM17 biology, including its vesicular trafficking, maturation from its precursor pro-form, activation on the cell surface and specificity for subsets of proteolytic targets. Previous studies from our laboratory and others identified the FERM domain-containing protein Frmd8/iTAP as an iRhom-binding protein. iTAP is required to maintain the cell surface stability of the sheddase complex, thereby preventing the precocious shunting of ADAM17 and iRhom2 to lysosomes and their consequent degradation. As pathophysiological role(s) of iTAP have not been addressed, here we sought to characterize the impact of loss of iTAP on ADAM17-associated phenotypes in mice. Our data show that iTAP KO mice exhibit defects in ADAM17 activity in inflammatory and intestinal epithelial barrier repair functions, but do not exhibit the collateral effects associated with global loss of ADAM17. Furthermore, we show that iTAP promotes cancer cell growth in a cell-autonomous manner, and by modulating the tumor microenvironment. Our work suggests that pharmacological intervention at the level of iTAP may be beneficial to target ADAM17 activity in specific compartments during chronic inflammatory diseases or cancer, avoiding the deleterious impact on vital functions associated with the widespread inhibition of ADAM17 in normal tissues.

molecular biology↗

bioRxiv2021

Modelling liver cancer microenvironment: novel 3D culture system as a potential anti-cancer drug screening tool

The tumor microenvironment and its contribution to tumorigenesis has been a focal highlight in recent years. A two-way communication between the tumor and the surrounding microenvironment sustains and contributes to the growth and metastasis of tumors. Progression and metastasis of hepatocellular carcinoma have been reported to be exceedingly influenced by diverse microenvironmental cues. In this study, we present a 3D-culture model of liver cancer to better mimic in vivo tumor settings. By creating novel 3D co-culture model that combines free-floating and scaffold based 3D-culture techniques of liver cancer cells and fibroblasts, we aimed to establish a simple albeit reproducible ex vivo cancer microenvironment model that captures tumor-stroma interactions. The model presented herein exhibited unique gene expression and protein expression profiles when compared to 2D and 3D mono-cultures of liver cancer cells. Our results showed that in vivo like conditions cannot be mimicked by simply growing cancer cells as spheroids, but by co-culturing them with 3D fibroblast with which they were able to cross-talk. This was evident by the upregulation of several pathways involved in HCC, and the increase in secreted factors by co-cultured cancer cells, many of which are also involved in tumor-stroma interactions. Compared to the conventional 2D culture, the proposed model exhibits an increase in the expression of genes associated with development, progression, and poor prognosis of HCC. Our results correlated with an aggressive outcome that better mirrors in vivo HCC, and therefore, a more reliable platform for molecular understanding of HCC and possibly better anti-cancer drug screening.

cancer biology↗

bioRxiv2021

Tonic inhibition of the chloride/proton antiporter ClC-7 by PI(3,5)P2 is crucial for lysosomal pH maintenance

The acidic luminal pH of lysosomes, maintained within a narrow range, is essential for proper degrative function of the organelle and is generated by the action of a V-type H+ ATPase, but other pathways for ion movement are required to dissipate the voltage generated by this process. ClC-7, a Cl-/H+ antiporter responsible for lysosomal Cl- permeability, is a candidate to contribute to the acidification process as part of this "counterion pathway". The signaling lipid PI(3,5)P2 modulates lysosomal dynamics, including by regulating lysosomal ion channels, raising the possibility that it could contribute to lysosomal pH regulation. Here we demonstrate that depleting PI(3,5)P2 by inhibiting the PIKfyve kinase causes lysosomal hyperacidification, primarily via an effect on ClC-7. We further show that PI(3,5)P2 directly inhibits ClC-7 transport and that this inhibition is eliminated in a disease-causing gain-of-function ClC-7 mutation. Together these observations suggest an intimate role for ClC-7 in lysosomal pH regulation.

biophysics↗