Biology subjects

Ali, A.

Publications and source records attributed to Ali, A..

At least 91 records · Page 5Linked to original sources

bioRxiv2023

Unraveling the Global Proteome and Phosphoproteome of Prostate Cancer Patient-Derived Xenografts

Resistance to androgen deprivation therapies leads to metastatic castration-resistant prostate cancer (mCRPC) of adenocarcinoma (AdCa) origin that can transform to emergent aggressive variant prostate cancer (AVPC) which has neuroendocrine (NE)-like features. To this end, we used LuCaP patient-derived xenograft (PDX) tumors, clinically relevant models that reflects and retains key features of the tumor from advanced prostate cancer patients. Here we performed proteome and phosphoproteome characterization of 48 LuCaP PDX tumors and identified over 94,000 peptides and 9,700 phosphopeptides corresponding to 7,738 proteins. When we compared 15 NE versus 33 AdCa PDX samples, we identified 309 unique proteins and 476 unique phosphopeptides that were significantly altered and corresponded to proteins that are known to distinguish these two phenotypes. Assessment of protein and RNA concordance from these tumors revealed increased dissonance in transcriptionally regulated proteins in NE and metabolite interconversion enzymes in AdCa.

cancer biology↗

bioRxiv2023

Surface crosslinking of virus-like particles increases resistance to proteases, low pH and mechanical stress for mucosal applications

AbstractVirus-like particles (VLPs) are emerging as nano-scaffolds in a variety of biomedical applications including the delivery of vaccine antigens to mucosal surfaces. These soft, colloidal, and proteinaceous structures (capsids) are nevertheless susceptible to mucosal environmental factors which limit their usefulness. We addressed this issue by crosslinking multiple capsid surface reactive residues using polyethylene glycol tethers. Surface crosslinking enhanced the colloidal stability and mechanical strength of VLPs against low pH, proteases, and mechanical agitation, while it did not interfere with function as vaccine. Chemical crosslinking thus offers a viable means to enhance the resilience of VLPs in mucosal applications.

biophysics↗

bioRxiv2023

Indigenous probiotics Lactobacillus reuteri and Enterococcus faecium exhibit positive growth performance and disease prevention against extended-spectrum cephalosporin and fluoroquinolones resistant Salmonella enterica in broiler chicks.

ABSTRACTThe rapid increase in antibiotic resistance poses a global threat to public health, necessitating the development of effective antimicrobial alternatives. This study compared an indigenous probiotic mix containing Lactobacillus reuteri and two strains of Enterococcus faecium to a commercial probiotic blend ProtexinR on the growth performance, mortality rate, histomorphology, serum immunoglobulins, and intestinal microflora of broiler chickens challenged with two multi drug resistant Salmonella serovars, Typhimurium and Enteritidis. Two hundred and forty day-old broiler chicks were randomly assigned to six treatment groups for 4 weeks: the treatment groups were; birds continuously supplemented with only indigenous probiotic strains (108 CFU/mL) (IPRO-); birds challenged with Salmonella serovars 106 (CFU/mL) (PC+); birds continuously supplemented with indigenous probiotic strains and challenged with Salmonella serovars (IPRO+); birds supplemented with ProtexinR and challenged with Salmonella serovars (CM+); birds supplemented with only ProtexinR (CM-); and birds with no Salmonella challenge or probiotics (negative control; PC-). The results revealed that IPRO- diets significantly improved feed conversion ratio (FCR) and increased body weight (BW) (P [≤] 0.05). No effect of probiotic treatments was observed on IPRO- and CM- on relative organ weights as compared to the negative control (PC-). The Salmonella- challenged group PC+ had the highest (20%) mortality rate and lowest BW. The IPRO- had significantly lower FCR (1.55) compared to PC- (1.86) and PC+ (1.95). The broilers in the IPRO- group showed significantly increased serum concentrations of IgA and IgG relative to both control groups (P [≤] 0.05). Morphological analysis of the ileum revealed significant increases (P [≤] 0.05) in the villus height and villus height/crypt depth in birds fed IPRO- compared with the PC+. Cecal Lactobacillus and Enterococcus counts were the highest (P [≤] 0.05) and Salmonella counts were the lowest (P [≤] 0.05) in the IPRO- group compared to the Salmonella infected group PC+. These results indicated that indigenous probiotic strains Lactobacillus reuteri and Enterococcus faecium can be an effective and low-cost alternative compared to commercial probiotics in the Pakistan poultry industry.

microbiology↗

bioRxiv2023

An exosome-based gene delivery platform for cell-specific CRISPR/Cas9 genome editing

Exosomes are naturally occurring vesicles that have the potential to be manipulated to become promising drug delivery vehicles for on-demand in vitro and in vivo gene editing. Here, we developed the modular safeEXO platform, a prototype exosome delivery vehicle that is mostly devoid of endogenous RNA and can efficaciously deliver RNA and ribonucleoprotein (RNP) complexes to their intended intracellular targets manifested by downstream biologic activity. We also successfully engineered producer cells to produce safeEXO vehicles that contain endogenous Cas9 (safeEXO-CAS) to effectively deliver efficient ribonucleoprotein (RNP)-mediated CRISPR genome editing machinery to organs or diseased cells in vitro and in vivo. We confirmed that safeEXO-CAS exosomes could co-deliver ssDNA, sgRNA and siRNA, and efficaciously mediate gene insertion in a dose-dependent manner. We demonstrated the potential to target safeEXO-CAS exosomes by engineering exosomes to express a tissue-specific moiety, integrin alpha-6 (safeEXO-CAS-ITGA6), which increased their uptake to lung epithelial cells in vitro and in vivo. We tested the ability of safeEXO-CAS-ITGA6 loaded with EMX1 sgRNAs to induce lung-targeted editing in mice, which demonstrated significant gene editing in the lungs with no signs of morbidity or detectable changes in immune cell populations. Our results demonstrate that our modular safeEXO platform represents a targetable, safe and efficacious vehicle to deliver nucleic acid-based therapeutics that successfully reach their intracellular targets. Furthermore, safeEXO producer cells can be genetically manipulated to produce safeEXO vehicles containing CRISPR machinery for more efficient RNP-mediated genome editing. This platform has the potential to improve current therapies and increase the landscape of treatment for various human diseases using RNAi and CRISPR approaches.

molecular biology↗

bioRxiv2023

The direction of local adaptation on pathogen exploitation rates reverses with environmental context

Host heterogeneity and spatial population structure each influence parasite evolution but may also interact because space structures contacts between host types. Here, we experimentally evolve granulosis virus in microcosms of its Plodia interpunctella (Indian meal moth) host that differ in both spatial structure and host genetic diversity. We control spatial structure by manipulating the viscosity of the food that the larvae live within and host genetic diversity by adding larvae from either a single or two inbred lines to opposite ends of the microcosm. We preserve spatial structure across passages and assay virus from different positions within the microcosm on both host genotypes. We find that the lower contact rates between host genotypes resulting from spatial structure can lead to the evolution of locally specialized virus, even when the host population is genetically diverse overall. We also find that spatial structure changes how viruses specialize: viruses evolved in well-mixed environments had lower exploitation of familiar hosts, while those in spatially structured environments exhibited higher exploitation of familiar hosts. These results demonstrate that spatial structure and host heterogeneity interact to shape pathogen specialization and that the evolutionary consequences of host diversity depends on the population structure.

evolutionary biology↗

bioRxiv2023

Loss of Grem1-articular cartilage progenitor cells causes osteoarthritis.

Osteoarthritis (OA), which carries an enormous disease burden across the world, is characterised by irreversible degeneration of articular cartilage (AC), and subsequently bone. The cellular cause of OA is unknown. Here, using lineage tracing in mice, we show that the BMP-antagonist Gremlin 1 (Grem1) marks a novel chondrogenic progenitor (CP) cell population in the articular surface that generates joint cartilage and subchondral bone during development and adulthood. Notably, this CP population is depleted in injury-induced OA, and with age. OA is also induced by toxin-mediated ablation of Grem1 CP cells in young mice. Transcriptomic analysis and functional modelling in mice revealed articular surface Grem1-lineage cells are dependent on Foxo1; ablation of Foxo1 in Grem1-lineage cells led to early OA. This analysis identified FGFR3 signalling as a therapeutic target, and injection of its activator, FGF18, caused proliferation of Grem1-lineage CP cells, increased cartilage thickness, and reduced OA pathology. We propose that OA arises from the loss of CP cells at the articular surface secondary to an imbalance in progenitor cell homeostasis and present a new progenitor population as a locus for OA therapy.

cell biology↗

bioRxiv2023

Screening in serum-derived medium reveals differential response to compounds targeting metabolism

INTRODUCTION INTRODUCTION RESULTS DISCUSSION KEY RESOURCES TABLE CONTACT FOR REAGENT AND... EXPERIMENTAL MODEL AND SUBJECT... METHOD DETAILS QUANTIFICATION AND STATISTICAL... Data resources DATA AND SOFTWARE AVAILABILITY AUTHOR CONTRIBUTIONS DECLARATION OF INTERESTS REFERENCES Studies of cancer cells in standard culture conditions have long been used as a tractable tool for drug discovery. Cell culture provides unparalleled experimental flexibility, scalability, and low cost to identify and understand the response to cancer therapeutics; however, drug responses in culture are not always predictive of drug response in animal models or in patients1 ...

cancer biology↗

bioRxiv2023

Comprehensive codon usage analyses of the mutations in the Parkin protein leading to Parkinsonism

Neurodegenerative diseases typically occur due to the abnormal behavior of the proteins. One of the most prevalent neurodegenerative diseases is the Parkinsons disease. Parkin protein encoded by PARK2 gene is the central player in the central nervous system. Mutations in Parkin lead to the onset of Parkinsonism. In this work, we made an attempt to decipher the codon usage patterns of the amino acid residues in the different domains of Parkin protein. We observed that mutations at the first two codon positions have statistically significant disease associations. This is the first such report that analyzes the correlation between the disease severity of Parkinsonism and its association with the pattern of codon usages.

bioinformatics↗

bioRxiv2023

The maternal microbiome regulates infant respiratory disease susceptibility via intestinal Flt3L expression and plasmacytoid dendritic cell hematopoiesis

Severe lower respiratory infection (sLRI) are a major cause of infant morbidity and mortality, and predispose to later chronic respiratory diseases such as asthma. Poor maternal diet during pregnancy is a risk factor for sLRI in the offspring. Here we demonstrate in mice that a maternal low-fibre diet (LFD) disrupts plasmacytoid and conventional dendritic cell (DC) hematopoiesis in the offspring, predisposing to sLRI and subsequent asthma. The LFD alters the composition of the maternal milk microbiome and assembling infant gut microbiome, ablating the induction of a developmental wave of the non-redundant DC growth factor Flt3L by neonatal intestinal epithelial cells. Therapy with a propionate-producing bacteria isolated from the milk of high-fibre diet-fed mothers, or supplementation with propionate, confers protection against sLRI by restoring gut Flt3L expression and pDC hematopoiesis. Our findings identify a microbiome-dependent Flt3L axis in the gut that regulates pDC hematopoiesis in early life and confers disease resistance. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=51 SRC="FIGDIR/small/522516v1_ufig1.gif" ALT="Figure 1"> View larger version (15K): org.highwire.dtl.DTLVardef@1a39990org.highwire.dtl.DTLVardef@1b6fe67org.highwire.dtl.DTLVardef@f8440dorg.highwire.dtl.DTLVardef@13bc67e_HPS_FORMAT_FIGEXP M_FIG C_FIG

immunology↗

bioRxiv2023

Highly multiplexed ddPCR-amplicon sequencing reveals strong Plasmodium falciparum population structure and isolated populations amenable to local elimination efforts in Zanzibar

Over the past 15 years, Zanzibar has made great strides towards malaria elimination; yet progress has stalled. Parasite genetic data of Plasmodium falciparum may inform strategies for malaria elimination by helping to identify contributory factors to parasite persistence. Here we elucidate fine-scale parasite population structure and infer relatedness and connectivity of infections using an identity-by-descent (IBD) approach. We sequenced 518 P. falciparum samples from 5 districts covering both main islands using a novel, highly multiplexed droplet digital PCR (ddPCR)-based amplicon deep sequencing method targeting 35 microhaplotypes and drug-resistance loci. Despite high genetic diversity, we observe strong fine-scale spatial and temporal structure of local parasite populations, including isolated populations on Pemba Island and genetically admixed populations on Unguja Island, providing evidence of ongoing local transmission. We observe a high proportion of highly related parasites in individuals living closer together, including between clinical index cases and the mostly asymptomatic cases surrounding them, consistent with isolation-by-distance. We identify a substantial fraction (2.9%) of related parasite pairs between Zanzibar, and mainland Tanzania and Kenya, consistent with recent importation. We identify haplotypes known to confer resistance to known antimalarials in all districts, including multidrug-resistant parasites, but most parasites remain sensitive to current first-line treatments. Our study provides a high-resolution view of parasite genetic structure across the Zanzibar archipelago and reveals actionable patterns, including isolated parasite populations, which may be prioritized for malaria elimination.

genomics↗

bioRxiv2022

Adaptive preservation of orphan ribosomal proteins in chaperone-stirred condensates

Ribosome biogenesis is among the most resource-intensive cellular processes, with ribosomal proteins accounting for up to half of all newly synthesized proteins in eukaryotic cells. During stress, cells shut down ribosome biogenesis in part by halting rRNA synthesis, potentially leading to massive accumulation of aggregation-prone "orphan" ribosomal proteins (oRPs). Here we show that during heat shock in yeast and human cells, oRPs accumulate as reversible condensates at the nucleolar periphery recognized by the Hsp70 co-chaperone Sis1/DnaJB6. oRP condensates are liquid-like in cell-free lysate but solidify upon depletion of Sis1 or inhibition of Hsp70. When cells recover from heat shock, oRP condensates disperse in a Sis1-dependent manner, and their ribosomal protein constituents are incorporated into functional ribosomes in the cytosol, enabling cells to efficiently resume growth. One sentence summaryDuring stress, molecular chaperones preserve "orphan" ribosomal proteins (RPs) - RPs that are not bound to rRNA - in liquid-like condensates, maintaining the RPs in a usable form and enabling cells to efficiently resume growth upon recovery from stress.

cell biology↗

bioRxiv2022

Single-cell analysis of myeloid cells in HPV+ tonsillar cancer

The incidence of Human Papillomavirus positive (HPV+) tonsillar cancer has been sharply rising during the last two decades. Myeloid cells represent an appropriate therapeutic target due to their ability to orchestrate antigen-specific immunity within the tonsil, the availability of viral antigens, and the proximity of the tumor and the underlying lymphoid tissue. However, the interrelationship of steady-state and inflammatory myeloid cell subsets, and their impact on patient survival remains unexplored. Here, we used single-cell RNA-sequencing to map the myeloid compartment in HPV+ tonsillar cancer. Our analysis unveiled the existence of four dendritic cell lineages, two macrophage polarization processes, and their sequential maturation profiles. We observed an expansion of the myeloid compartment in HPV+ tonsillar cancer, accompanied by interferon-induced cellular responses both in DCs and monocyte-macrophages. Within the DC lineages, we describe a balance shift in the frequency of progenitor and mature cDC favoring the cDC1 lineage in detriment of cDC2s, in HPV+ lesions. Furthermore, we observed that all DC lineages apart from DC5s matured into a common activated DC profile. In turn, the monocyte-macrophage lineage was subjected to early monocyte polarization events, which gave raise to inflammatory-activated, and chemokine-producing macrophages. We validated the existence of most of the single-cell RNA-seq clusters using 26-plex flow cytometry, and described a positive impact of cDC1, activated DCs and macrophages in patient survival using signature scoring. The current study contributes towards the understanding of myeloid ontogeny and dynamics in human papilloma driven tonsillar cancer, and details myeloid biomarkers that can be used to predict therapy effects and assess patient prognosis.

immunology↗

bioRxiv2022

The influence of genetic structure on phenotypic diversity in the Australian mango (Mangifera indica) gene pool

Genomic selection is a promising breeding technique for tree crops to accelerate the development of new cultivars. However, factors such as genetic structure can create spurious associations between genotype and phenotype due to the shared history between populations with different trait values. Genetic structure can therefore reduce the accuracy of the genotype to phenotype map, a fundamental requirement of genomic selection models. Here, we employed 272 single nucleotide polymorphisms from 208 Mangifera indica cultivars to explore whether the genetic structure of the Australian mango gene pool explained variation in tree size, fruit blush colour and intensity. Our results show that genetic structure is weak, but cultivars imported from Southeast Asia (mainly those from Thailand) were genetically differentiated across multiple population genetic analyses. We find that genetic structure was strongly associated with phenotypic diversity in M. indica, suggesting that the history of these cultivars could drive spurious associations between loci and key mango phenotypes in the Australian mango gene pool. Incorporating such genetic structure in associations between genotype and phenotype has the potential to improve the accuracy of genomic selection, which can assist the development of new cultivars.

evolutionary biology↗

bioRxiv2022

Continuous Fish Muscle Cell Line with Capacity for Myogenic and Adipogenic-like Phenotypes

Cell-cultivated fish offers the potential for a more ethical, sustainable, and safe seafood system. However, fish cell culture is relatively understudied in comparison to mammalian cells. Here, we established and characterized a continuous Atlantic mackerel (Scomber scombrus) skeletal muscle cell line ("Mack" cells). The cells were isolated from muscle biopsies of fresh-caught fish, with separate isolations performed from two distinct fish. Mack1 cells (cells from the first isolation) were cultured for over a year and subcultured over 130 times. The cells proliferated at initial doubling times of 63.9 hr ({+/-} 19.1 SD). After a spontaneous immortalization crisis from passages 37-43, the cells proliferated at doubling times of 24.3 hr ({+/-} 4.91 SD). A muscle phenotype was confirmed through characterization of muscle stemness and differentiation via paired-box protein 7 (PAX7) and myosin heavy chain (MHC) immunostaining, respectively. An adipocyte-like phenotype was also demonstrated for the cells through lipid accumulation, confirmed via Oil Red O (ORO) staining and quantification of neutral lipids. New qPCR primers (HPRT, PAX3B, MYOD1, MYOG, TNNT3A, and PPARG) were tailored to the mackerel genome and used to characterize mackerel cell genotypes. This work provides the first spontaneously immortalized fish muscle cell line for research, ideally serving as a reference for subsequent investigation.

bioengineering↗

bioRxiv2022

Induction of Sis1 promotes fitness but not feedback in the heat shock response

Previously, we combined modeling and experiments to demonstrate that the heat shock response (HSR) functions as a negative feedback loop in which undefined chaperone clients activate the HSR by sequestering Hsp70, and subsequent induction of Hsp70 deactivates the response (Zheng et al., 2016; Krakowiak et al., 2018). Here, we formally define newly synthesized proteins (NSPs) as a major class of HSR activators and determine the role of Sis1, a co-chaperone of Hsp70, in HSR regulation. We develop and experimentally validate a new mathematical model that incorporates NSPs and Sis1. Unexpectedly, genetic decoupling and pulse-labeling experiments reveal that Sis1 induction promotes fitness during prolonged stress rather than providing negative feedback to the HSR. These results support an overall model in which NSPs signal the HSR by sequestering Sis1 and Hsp70, while induction of Hsp70 - but not Sis1 - attenuates the response.

cell biology↗

bioRxiv2022

Development of a monoclonal antibody to a vibriophage as a proxy for Vibrio cholerae detection

Cholera is an acute watery diarrheal disease that causes high rates of morbidity and mortality without treatment. Early detection of the etiologic agent of toxigenic Vibrio cholerae is important to mobilize treatment and mitigate outbreaks. Monoclonal antibody (mAb) based rapid diagnostic tests (RDTs) enable early detection in settings without laboratory capacity. However, the odds of an RDT testing positive are reduced by nearly 90% when the common virulent bacteriophage ICP1 is present. We hypothesize that adding a mAb for the common, and specific, virulent bacteriophage ICP1 as a proxy for V. cholerae to an RDT will increase diagnostic sensitivity when virulent ICP1 phage are present. In this study, we used an in-silico approach to identify immunogenic ICP1 protein targets that were conserved across disparate time periods and locations. Specificity of targets to cholera patients with known ICP1 was determined, and specific targets were used to produce mAbs in a murine model. Candidate mAbs to the head protein demonstrated specificity to ICP1 by ELISA and an ICP1 phage neutralization assay. The limit of detection of the final mAb candidate for ICP1 phage particles spiked into cholera stool matrix was 8 x 105 plaque forming units by Western blot analysis. This mAb will be incorporated into a RDT prototype for evaluation in a future diagnostic study to test the guiding hypothesis behind this study.

immunology↗

bioRxiv2021

Deciphering the Molecular Mechanism of HCV Protease Inhibitor Fluorination as a General Approach to Avoid Drug Resistance

Third generation Hepatitis C virus (HCV) NS3/4A protease inhibitors (PIs), glecaprevir and voxilaprevir, are highly effective across genotypes and against many resistant variants. Unlike earlier PIs, these compounds have fluorine substitutions on the P2-P4 macrocycle and P1 moieties. Fluorination has long been used in medicinal chemistry as a strategy to improve physicochemical properties and potency. However, the molecular basis by which fluorination improves potency and resistance profile of HCV NS3/4A PIs is not well understood. To systematically analyze the contribution of fluorine substitutions to inhibitor potency and resistance profile, we used a multi-disciplinary approach involving inhibitor design and synthesis, enzyme inhibition assays, co-crystallography, and structural analysis. A panel of inhibitors in matched pairs were designed with and without P4 cap fluorination, tested against WT protease and the D168A resistant variant, and a total of 22 high-resolution co-crystal structures were determined. While fluorination did not significantly improve potency against the WT protease, PIs with fluorinated P4 caps retained much better potency against the D168A protease variant. Detailed analysis of the co-crystal structures revealed that PIs with fluorinated P4 caps can sample alternate binding conformations that enable adapting to structural changes induced by the D168A substitution. Our results elucidate molecular mechanisms of fluorine-specific inhibitor interactions that can be leveraged in avoiding drug resistance.

biochemistry↗

bioRxiv2021

Evasion of Toll-like Receptor Recognition by Escherichia coli is mediated via Population Level Regulation of Flagellin Production

Uropathogenic Escherichia coli (UPEC) is a major cause of urinary tract infections. Analysis of the innate immune response in immortalised urothelial cells suggests that the bacterial flagellar subunit, flagellin, is key in inducing host defences. A panel of 40 clinical uro-associated Escherichia coli isolates recovered from either asymptomatic bacteruria (ASB), cystitis or pyelonephritis patients, were characterised for motility and their ability to induce an innate response in urothelial cells stably transfected with a NF{kappa}B luciferase reporter. Twenty-four isolates (60%) were identified as motile with strains recovered from cystitis patients exhibiting a bipolar motility distribution pattern (P < 0.005) and associated with a 2-5 fold increase in NF{kappa}B signalling. Although two isolates were associated with swarm sizes of >7 cm and NF{kappa}B activities of >30 fold (P = 0.029), data overall suggested bacterial motility and the NF{kappa}B signalling response were not directly correlated. To explore whether the signalling response reflected antigenic variation flagellin was purified from 11 different isolates and the urothelial cell challenges repeated. Purified flagellin filaments generated comparable (30.4{+/-}1.8 to 46.1{+/-}2.5 fold, P = NS) NF{kappa}B signalling responses, irrespective of either the source of the isolate or H-serotype. These data argued against any variability between isolates being related to flagellin itself. To determine the roles, if any, of flagellar abundance in inducing these responses flagellar hook numbers of a range of cystitis and ABU isolates were quantified using a plasmid encoded flagellar hook gene flgEA240C. Foci data suggested isolates were averaging between 1 and 2 flagella per cell, while only 10 to 60% each isolates population exhibited foci. These data suggested selective pressures exist in the urinary tract that allow uro-associated E. coli strains to maintain motility exploiting population heterogeneity to prevent host TLR5 recognition.

microbiology↗