Biology subjects

Ahmad, S.

Publications and source records attributed to Ahmad, S..

At least 55 records · Page 3Linked to original sources

bioRxiv2024

UltimateSynth: MRI Physics for Pan-Contrast AI

Magnetic resonance imaging (MRI) is commonly used in healthcare for its ability to generate diverse tissue contrasts without ionizing radiation. However, this flexibility complicates downstream analysis, as computational tools are often tailored to specific types of MRI and lack generalizability across the full spectrum of scans used in healthcare. Here, we introduce a versatile framework for the development and validation of AI models that can robustly process and analyze the full spectrum of scans achievable with MRI, enabling model deployment across scanner models, scan sequences, and age groups. Core to our framework is UltimateSynth, a technology that combines tissue physiology and MR physics in synthesizing realistic images across a comprehensive range of meaningful contrasts. This pan-contrast capability bolsters the AI development life cycle through efficient data labeling, generalizable model training, and thorough performance benchmarking. We showcase the effectiveness of UltimateSynth by training an off-the-shelf U-Net to generalize anatomical segmentation across any MR contrast. The U-Net yields highly robust tissue volume estimates, with variability under 4% across 150,000 unique-contrast images, 3.8% across 2,000+ low-field 0.3T scans, and 3.5% across 8,000+ images spanning the human lifespan from ages 0 to 100.

bioinformatics↗

bioRxiv2024

PACT prevents aberrant activation of PKR by endogenous dsRNA without sequestration

The innate immune sensor PKR for double-stranded RNA (dsRNA) is critical for antiviral defense, but its aberrant activation by cellular dsRNA is linked to various diseases. The dsRNA-binding protein PACT plays a critical yet controversial role in the PKR pathway. We demonstrate that PACT is a direct and specific suppressor of PKR against endogenous dsRNA ligands like inverted-repeat Alu RNAs, which robustly activate PKR in the absence of PACT. PACT-mediated inhibition does not involve competition for dsRNA binding. Instead, PACT impairs PKRs ability to scan along dsRNA--a process necessary for PKR molecules to encounter and autophoshorylate each other for activation. By scanning along dsRNA and directly interacting with PKR, PACT restricts PKRs movement on dsRNA, reducing the likelihood of PKR molecular collisions and subsequent autophosphorylation, effectively inhibiting PKR without sequestering dsRNA. Consequently, PKR inhibition is more robust with longer and less abundant dsRNA, and minimal with abundant or short dsRNA. Thus, PACT functions to adjust the PKR activation threshold for long endogenous dsRNA without altering its inherent activity, revealing new mechanisms for establishing self-tolerance.

biochemistry↗

bioRxiv2024

The Growing Little Brain: Cerebellar Functional Development from Cradle to School

Despite the cerebellums crucial role in brain functions, its early development, particularly in relation to the cerebrum, remains poorly understood. Here, we examine cerebellocortical connectivity using over 1,000 high-quality resting-state functional MRI scans of children from birth to 60 months. By mapping cerebellar topography with fine temporal detail for the first time, we show the hierarchical organization of cerebellocortical functional connectivity from infancy. We observe dynamic shifts in cerebellar network gradients, which become more focal with age while generally maintaining stable anchor points similar to adults, highlighting the cerebellums evolving yet stable role in functional integration during early development. Our findings provide the first evidence of cerebellar connections to higher-order networks at birth, which generally strengthen with age, emphasizing the cerebellums early role in cognitive processing beyond sensory and motor functions. Our study provides insights into early cerebellocortical interactions, reveals functional asymmetry and sex-specific patterns in cerebellar development, and lays the groundwork for future research on cerebellum-related disorders in children.

neuroscience↗

bioRxiv2024

Leveraging clinical sleep data across multiple pediatric cohorts for insights into neurodevelopment: the Retrospective Analysis of Sleep in Pediatric (RASP) cohorts study

Sleep disturbances are prominent across neurodevelopmental disorders (NDDs) and may reflect specific abnormalities in brain development and function. Overnight polysomnography (PSG) allows for detailed investigation of sleep architecture, offering a unique window into neurocircuit function. A better understanding of sleep in NDDs compared to typically developing children could therefore define mechanisms underlying abnormal development in NDDs and provide avenues for the development of therapeutic interventions to improve sleep quality and developmental outcomes. Here, we introduce and characterize a collection of 1527 pediatric overnight PSGs across five different sites. We first developed an automated stager trained on independent pediatric sleep data, which yielded better performance compared to a stager trained on adults. Using consistent staging across cohorts, we derived a panel of EEG micro-architectural features. This unbiased approach replicated broad trajectories previously described in typically developing sleep architecture. Further, we found sleep architecture disruptions in children with Downs Syndrome (DS) that were consistent across independent cohorts. Finally, we built and evaluated a model to predict age from sleep EEG metrics, which recapitulated our previous findings of younger predicted brain age in children with DS. Altogether, by creating a resource pooled from existing clinical data we expanded the available datasets and computational resources to study sleep in pediatric populations, specifically towards a better understanding of sleep in NDDs. This Retrospective Analysis of Sleep in Pediatric (RASP) cohorts dataset, including staging annotation derived from our automated stager, will be deposited at https://sleepdata.org. Statement of significanceWe introduce the RASP cohorts, a collection of 1527 clinical pediatric overnight polysomnographies that includes typically developing and neurodevelopmental disorder cases. As a first step towards addressing the analytic bottleneck inherent in manual sleep staging, we developed and validated a pediatric-specific sleep stager. Leveraging the retrospective RASP cohorts dataset, we redemonstrated known developmental trajectories in sleep architecture. To summarize changes in brain function reflected in sleep, we developed a model to predict brain age from sleep measures. We recapitulate younger predicted age in RASP Downs Syndrome cases. This work not only enhances our understanding of sleep disturbances in NDDs, but also provides a valuable resource for future research and underscores the utility of existing clinical polysomnography studies.

neuroscience↗

bioRxiv2024

Predicting the Impact of Dialyzer Choice and Binder Dialysate Flow Rate on Bilirubin Removal

Liver failure is the 12th leading cause of death worldwide. Protein bound toxins such as bilirubin are responsible for many complications of the disease. Binder dialysis systems use albumin dialysate and detoxifying sorbent columns to remove these toxins. Systems like the Molecular Adsorbent Recirculating System (MARS) and BioLogic-DT have existed since the 1990s, but survival benefit in randomized controlled trials have not been consistent. Thus, a new generation of binder dialysis systems, including Open Albumin Dialysis (OPAL) and the Advanced Multi-Organ Replacement System (AMOR) are being developed. Optimal conditions for binder dialysis have not been established. We developed and validated a computational model of bound solute dialysis using established thermodynamic theories. Our objective is to improve AMOR therapy. We confirmed our models validity by predicting the impact of changing between two benchtop dialysis setups using different polysulfone dialyzers (F3 and F6HPS). We then applied it to predict the impact of varying dialysate flow rate on toxin removal. We found that bilirubin removal is independent of dialysate flow rate within the clinically relevant range (20 mL/min - 800 mL/min), matching our models predictions. At very low dialysate flow rates (2 mL/min), bilirubin removal declines, deviating from the thermodynamic model. This model may be useful to achieving optimal clinical outcomes by setting optimal dialyzer and flow rate conditions. Further improvement is possible by accounting for toxin adsorption onto the dialyzer membrane.

bioengineering↗

bioRxiv2024

Next-generation snow leopard population assessment tool: multiplex-PCR SNP panel for individual identification from feces

Snow leopards, Panthera uncia, are under threat from numerous pressures and are the focus of a great deal of conservation efforts. However, their elusive nature makes it difficult to estimate population sizes. Current methods used to monitor local population sizes include visually identifying individuals from camera trap photos and genetically identifying individuals from fecal samples using microsatellite loci. Here, we present a new method for identifying snow leopard individuals from fecal samples using a multiplex PCR single nucleotide polymorphism (SNP) panel method. The SNP panel we present consists of 144 SNPs and utilizes next-generation sequencing technology, making it cheaper and easier than current microsatellite methods. We validate our SNP panel with paired tissue and fecal samples from zoo individuals, showing a minimum of 96.7% accuracy in allele calls per run. We then generate SNP data from 235 field-collected fecal samples from across Pakistan to show that the panel can reliably identify individuals from low-quality fecal samples of unknown age and is robust to contamination. We also show that our SNP panel has the capability to identify first-order relatives and provides insights into the geographic origin of samples. This SNP panel will empower the snow leopard research community in their efforts to assess local and global snow leopard population sizes. More broadly, we present a method for developing a SNP panel that utilizes open source software for SNP selection and primer design, Illumina sequencing technology, and a streamlined lab and bioinformatics protocol which can be used to create similar SNP panels for any species of interest for which adequate genomic reference data is available.

genetics↗

bioRxiv2024

The urinary microbiome in association with diabetes and diabetic kidney disease: A systematic review

BackgroundThe urinary microbiome, or urobiome, is a novel area of research that has been gaining attention recently, as urine was thought to be sterile for years. There is limited information about the composition of the urobiome in health and disease. The urobiome may be affected by several factors and diseases such as diabetes, a disease that often leads to kidney damage. Thus, we need to understand the role of the urobiome to assess and monitor kidney disease related to diabetes over time. MethodsWe conducted a systematic review to summarize knowledge about the urobiome in association with diabetes mellitus and diabetic kidney disease. The search was conducted in several electronic databases until November 2024. ResultsEighteen studies were selected including cross-sectional case-control studies, cross-sectional surveys and one prospective longitudinal study. In total, the urobiome of 1,571 people was sequenced, of which 662 people had diabetes, and of these 36 had confirmed diabetic kidney disease; 609 were healthy individuals, 179 had prediabetes or were at risk of type 2 diabetes mellitus and 121 did not have diabetes but had other comorbidities. Eight studies analysed data from females, one was focused on male data, and the other nine had mixed female-male data. Most of the studies had a small sample size, used voided midstream urine, and used 16S rRNA sequencing. ConclusionThis systematic review summarizes trends seen throughout published data available to have a first baseline knowledge of the urinary microbiome, and its microbiota, in association with diabetes including the decreased richness and -diversity in urinary microbiota in individuals with diabetes compared to healthy controls and the decreased -diversity with the evolution of kidney disease independently of the cause.

microbiology↗

bioRxiv2024

Soil bacteria may be the natural reservoirs of drug resistance genes

Soil bacteria are the main source of antibiotics because they produce them naturally to get territorial advantage. We collected deep soil samples and characterized cultivable microbes. We used morphological and cultural characters, biochemical reactions, 16s RNA PCR and DNA sequencing to identify the isolates. The isolates included Pseudomonas spp (12), Shigella spp (2), E. coli (1), Klebsiella/Citrobacter spp (1), Micrococcus spp (2), unidentified Bacillus spp.(18), Bacillus paramycoides (3), Paenibacillus lautus (2), Bacillus pacificus (2), and Lysinibacillus pakistanensis (1). Out of these 44 isolates, 33 (75%) were multi-drug resistant. Clinically relevant and clinically irrelevant bacteria had similar drug resistance patterns (88.9% and 88.0%, 88.9% and 88.0%, 100 and 96.0%, 61.1% and 92%, 83.3% and 96%, 33.3% and 36%, 55.6% and 68.0%, and 83.3% and 60%) towards Ampicillin, Amoxicillin, Oxacillin, Azithromycin, Streptomycin, Gentamicin, Ceftriaxone and Sulfamethoxazole respectively. The observation that bacteria which cannot colonize humans/animals and therefore cannot enter the horizontal drug resistance gene transfer cycle in clinical settings also have a large and similar arsenal of drug resistance genes, may indicate that they are actually the natural reservoirs. Because they are more dynamic in their ability to survive in different conditions, they provide better fitness for maintenance of these reservoirs. Impact StatementWe think this study has raised interesting questions and further probing at a larger scale and a greater depth will provide new insight that may be very helpful for understanding the phenomenon of spread of drug resistance among bacteria which is one of the paramount challenges for humanity.

microbiology↗

bioRxiv2024

Functional Hierarchy of the Human Neocortex from Cradle to Grave

Recent evidence indicates that the organization of the human neocortex is underpinned by smooth spatial gradients of functional connectivity (FC). These gradients provide crucial in-sight into the relationship between the brains topographic organization and the texture of human cognition. However, no studies to date have charted how intrinsic FC gradient archi-tecture develops across the entire human lifespan. In this work, we model developmental trajectories of the three primary gradients of FC using a large, high-quality, and temporally-dense functional MRI dataset spanning from birth to 100 years of age. The gradient axes, denoted as sensorimotor-association (SA), visual-somatosensory (VS), and modulation-representation (MR), encode crucial hierarchical organizing principles of the brain in development and aging. By tracking their development throughout the human lifespan, we provide the first ever comprehensive low-dimensional normative reference of global FC hierarchical architecture. We observe significant age-related changes in global network features, with global markers of hierarchical organization increasing from birth to early adulthood and decreasing there-after. During infancy and early childhood, FC organization is shaped by primary sensory processing, dense short-range connectivity, and immature association and control hierarchies. Functional differentiation of transmodal systems supported by long-range coupling drives a convergence toward adult-like FC organization during late childhood, while adolescence and early adulthood are marked by the expansion and refinement of SA and MR hierarchies. While gradient topographies remain stable during late adulthood and aging, we observe decreases in global gradient measures of FC differentiation and complexity from 30 to 100 years. Examining cortical microstructure gradients alongside our functional gradients, we observed that structure-function gradient coupling undergoes differential lifespan trajectories across multiple gradient axes.

neuroscience↗

bioRxiv2024

An expanded metabolic pathway for androgen production by host-associated bacteria

A growing body of literature implicates host-associated microbiota in the modulation of circulating androgen levels in the host, which could have far-reaching implications for androgen-mediated diseases. However, the microbial genetic pathways involved in androgen production remain unknown. Here, we report the first host-associated microbial gene (desF) encoding an enzyme that catalyzes conversion of androstenedione to epitestosterone (epiT) in the gut bacterium, Clostridium scindens. Despite current dogma that epiT is a nuclear androgen-receptor (AR) antagonist, we demonstrate that epiT is a potent androgen, as assessed by its ability to promote prostate cancer cell growth and expression of prostate specific antigen (PSA). We then quantified the desF gene in fecal samples collected from individuals with advanced prostate cancer (rising blood PSA) undergoing androgen deprivation therapy combined with abiraterone acetate and prednisone (AA/P). Strikingly, fecal desF levels were elevated in a subset of individuals progressing on AA/P versus samples taken during AA/P response (stable). Importantly, we observed that AA does not inhibit the bacterial desmolase enzyme that is analogous to the human drug target of AA. We then determined that bacterial isolates from urine or prostatectomy tissue are capable of androgen production. From these isolates we detected 17{beta}-hydroxysteroid dehydrogenase (17{beta}-HSDH) activity, which has not been previously reported in urinary tract bacteria, and discovered the desG gene in urinary isolates encoding 17{beta}-HSDH that catalyzed conversion of androstenedione to testosterone. Applying advanced artificial intelligence and molecular dynamics, we predict the structures and ligand binding to DesF and DesG. Using a novel bioengineered microencapsulation technique, we demonstrate that urinary androgen-producing bacterial strains can also promote prostate cancer cell growth through steroid metabolism. Taken together, our results are a significant advance for steroid microbiology in humans and suggest that these microbial biotransformations should be further studied in the context of androgen-mediated physiological processes and diseases.

microbiology↗

bioRxiv2024

A resource to enable chemical biology and drug discovery of WDR Proteins

Protein class-focused drug discovery has a long and successful history in pharmaceutical research, yet most members of druggable protein families remain unliganded, often for practical reasons. Here we combined experiment and computation to enable discovery of ligands for WD40 repeat (WDR) proteins, one of the largest human protein families. This resource includes expression clones, purification protocols, and a comprehensive assessment of the druggability for hundreds of WDR proteins. We solved 21 high resolution crystal structures, and have made available a suite of biophysical, biochemical, and cellular assays to facilitate the discovery and characterization of small molecule ligands. To this end, we use the resource in a hit-finding pilot involving DNA-encoded library (DEL) selection followed by machine learning (ML). This led to the discovery of first-in-class, drug-like ligands for 9 of 20 targets. This result demonstrates the broad ligandability of WDRs. This extensive resource of reagents and knowledge will enable further discovery of chemical tools and potential therapeutics for this important class of proteins.

biochemistry↗

bioRxiv2024

Heterologous expression and characterization of mutant cellulase from indigenous strain of Aspergillus niger

The current study highlights the effect of mutagenesis on endoglucanase B activity of indigenous strain of Aspergillus niger and its heterologous expression studies in the pET28a+ vector. The physical and chemical mutagens were employed to incorporate mutations in A. niger. For determination of mutations, mRNA was isolated followed by cDNA synthesis and cellulase gene was amplified, purified and sequenced both from native and mutant A. niger. On comparison of gene sequences, it was observed that 5 nucleotide base pairs have been replaced in the mutant cellulase. The mutant recombinant enzyme showed 4.5 times higher activity (428.5 {micro}mol/mL/min) as compared to activity of native enzyme (94 {micro}mol/mL/min). The mutant gene was further investigated using Phyre2 and I-Tesser tools which exhibited 71% structural homology with Endoglucanase B of Thermoascus aurantiacus. The root mean square deviation (RMSD), root mean square fluctuation (RMSF), solvent accessible surface area (SASA), radius of gyration (Rg) and hydrogen bonds analysis were carried at 35 {degrees}C and 50 {degrees}C to explore the integrity of structure of recombinant mutant endoglucanase B which corresponded to its optimal temperature. Hydrogen bonds analysis showed more stability of recombinant mutant endoglucanase B as compared to native enzyme. Both native and mutant endoglucanase B genes were expressed in pET 28a+ and purified with nickel affinity chromatography. Theoretical masses determined through ExPaSy Protparam were found 38.7 and 38.5 kDa for native and mutant enzymes, respectively. The optimal pH and temperature values for the mutant were 5.0 and 50 {degrees}C while for native these were found 4.0 and 35 {degrees}C, respectively. On reacting with carboxy methyl cellulose (CMC) as substrate, the mutant enzyme exhibited less Km (0.452 mg/mL) and more Vmax (50.25 {micro}mol/ml/min) as compared to native having 0.534 mg/mL as Km and 38.76 {micro}mol/ml/min as Vmax. Among metal ions, Mg2+ showed maximum inducing effect (200 %) on cellulase activity at 50 mM concentration followed by Ca2+ (140%) at 100 mM concentration. Hence, expression of a recombinant mutant cellulase from A. niger significantly enhanced its cellulytic potential which could be employed for further industrial applications at pilot scale.

biochemistry↗

bioRxiv2023

Video Diffusion models for the apoptosis forcasting

Reliable and early prediction of cell death (apoptosis) is critically important in various areas of biology, particularly in characterizing the effectiveness of cell-based infusion products utilized for cancer immunotherapy. While deep Convolutional Neural Networks (CNNs) are often used in state-of-the-art approaches for apoptosis classification, they typically focus solely on individual cells and ignore cell-cell interaction. To address this limitation, we propose a novel generative approach based on a video diffusion model, which predicts future cellular behaviors for early detection of apoptosis events, even before molecular markers like Annexin-V or visual indications like membrane blebbing become apparent. Our approach accounts for the interactions of multiple target cells and their spatial and temporal relationships at each time frame. We condition our generative model on two starting frames and utilize an auto-regressive framework to predict the subsequent five frames. Our model achieves a 0.88 F1 score on the cell death event classification and 2.11 mean absolute death-time error, significantly outperforming state-of-the-art methods.

cell biology↗

bioRxiv2023

MicroRNA-541-3p alters lipoproteins to reduce atherosclerosis by degrading Znf101 and Casz1 transcription factors

High apoB-containing low-density lipoproteins (LDL) and low apoA1-containing high-density lipoproteins (HDL) are associated with atherosclerosis. In search of a molecular regulator that could simultaneously and reciprocally control both LDL and HDL levels, we screened a microRNA (miR) library using human hepatoma Huh-7 cells. We identified miR-541-3p that both decreases apoB and increases apoA1 expression by inducing mRNA degradation of two different transcription factors, Znf101 and Casz1. Znf101 enhances apoB expression while Casz1 represses apoA1 expression. The hepatic knockdown of orthologous Zfp961 and Casz1 genes in mice altered plasma lipoproteins and reduced atherosclerosis without causing hepatic lipid accumulation, most likely by lowering hepatic triglyceride production, increasing HDL cholesterol efflux capacity, and reducing lipogenesis. Notably, human genetic variants in the MIR541, ZNF101, and CASZ1 loci are significantly associated with plasma lipids and lipoprotein levels. This study identifies miR-541-3p and Znf101/Casz1 as potential therapeutic agent and targets, respectively, to reduce plasma lipoproteins and atherosclerosis without causing liver steatosis. O_FIG O_LINKSMALLFIG WIDTH=153 HEIGHT=200 SRC="FIGDIR/small/565110v1_ufig1.gif" ALT="Figure 1"> View larger version (15K): org.highwire.dtl.DTLVardef@741dd1org.highwire.dtl.DTLVardef@151ad9aorg.highwire.dtl.DTLVardef@15c96a9org.highwire.dtl.DTLVardef@1a73b2d_HPS_FORMAT_FIGEXP M_FIG Graphical Abstract. A schematic diagram depicting the role of miR-541-3p in the control of plasma lipoproteins and atherosclerosis.Our data show that miR-541-3p downregulates ZNF101 and CASZ1 by enhancing post-transcriptional degradation of mRNAs after interacting with their 3'-UTRs. Furthermore, our data indicate that ZNF101 is an enhancer of APOB, and CASZ1 is a repressor of APOA1. Hepatic knockdown (KD) of Zfp961, an orthologue of ZNF101, reduces plasma apoB-containing lipoproteins, whereas KD of Casz1 increases high density lipoproteins in mice. Furthermore, we show that hepatic KDs of these transcription factors reduces atherosclerosis in mice induced by the expression of mutant PCSK9. C_FIG

physiology↗

bioRxiv2023

Exosomes promote axon outgrowth and a polarized neuronal morphology by engaging the Wnt-Planar Cell Polarity pathway

In neurons, the acquisition of a polarized morphology is achieved upon the outgrowth of a single axon from one of several neurites. Exosomes or small extracellular vesicles (sEVs) from diverse sources are known to promote the neurite outgrowth and thus may have therapeutic potential. However, the effect of fibroblast-derived exosomes on axon elongation in neurons of the central nervous system under growth permissive conditions remains unclear. Here, we show that fibroblast-derived sEVs promote axon outgrowth and a polarized neuronal morphology in mouse primary embryonic cortical neurons. Mechanistically, we demonstrate that the sEV-induced increase in axon outgrowth requires endogenous Wnts and core PCP components including Prickle, Vangl, Frizzled and Dishevelled. We demonstrate that sEVs are internalized by neurons, colocalize with Wnt7b and induce relocalization of Vangl2 to the distal axon during axon outgrowth. In contrast, sEVs derived from neurons or astrocytes do not promote axon outgrowth, while sEVs from activated astrocytes inhibit elongation. Thus, our data reveals that fibroblast-derived sEVs promote axon elongation through the Wnt-PCP pathway in a manner that is dependent on endogenous Wnts.

neuroscience↗

bioRxiv2023

Tumor-targeted delivery of Tetanus toxoid by Listeria improves immunotherapy against ovarian cancer in mice

Ovarian cancer is known for its poor neoantigen expression and strong immunosuppression. Here, we utilized an attenuated non-pathogenic bacterium Listeria monocytogenes to deliver a highly immunogenic Tetanus Toxoid protein (Listeria-TT), as a neoantigen surrogate, into tumor cells through infection in a metastatic mouse ovarian cancer model (Id8p53-/-Luc). Gemcitabine (GEM) was added to reduce immune suppression. Listeria-TT+GEM treatments resulted in tumors expressing TT and reactivation of pre-existing CD4 and CD8 memory T cells to TT (generated early in life). These T cells were then attracted to the TT-expressing tumors now producing perforin and granzyme B. This correlated with a strong reduction in tumor burden, and significant improvement of the survival time compared to all control groups. Checkpoint inhibitors have little effect on ovarian cancer partly because of low neoantigen expression. Here we demonstrated that Listeria-TT+GEM+anti-PD1 was significantly more effective (efficacy and survival) than anti-PD1 or Listeria-TT+GEM alone. Of clinical interest, high doses of anti-PD1 (PD1H) (when added to Listeria-TT+GEM) were less effective than the low doses (PD1L). IHC and ELISPOT demonstrated that high doses of anti-PD1 inhibited T cell function in the TME. Using RNAseq, Differentially Expressed Genes (DEG) analysis and Genes Set Enrichment Analysis (GSEA) showed that gene expression levels and biological pathways were predominantly upregulated in the PD1H compared to the PD1L group, in correlation with low immune infiltration in tumors, more immune suppression, and more aggressive ovarian cancer. In summary, this study suggests that our approach may benefit ovarian cancer patients. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=101 SRC="FIGDIR/small/561944v3_ufig1.gif" ALT="Figure 1"> View larger version (24K): org.highwire.dtl.DTLVardef@197942eorg.highwire.dtl.DTLVardef@8180d4org.highwire.dtl.DTLVardef@3113e6org.highwire.dtl.DTLVardef@116851_HPS_FORMAT_FIGEXP M_FIG Graphical Abstract Human concept: Childhood vaccinations with the highly immunogenic tetanus toxoid (TT) generate TT-specific memory T cells, which circulate in the blood for life. After appearance of ovarian cancer (late in life), the patients will receive one high dose with Listeria-TT to deliver TT into tumor cells, followed by multiple low doses of Listeria-TT over a period of 2 weeks to restimulate the pre-existing memory T cells to TT. MDSC are involved in the delivery of Listeria-TT to the TME. Reactivated memory T cells will in turn destroy the tumor cells expressing TT. Multiple low doses of GEM will be added after TT has been delivered at the tumor site, which reduce immune suppression by eliminating MDSC and TAM (not shown here). Since individuals have seen TT earlier in life (during childhood vaccinations) and since TT is highly immunogenic (attracting T cells to the TME) but not expressed in normal cells, TT functions here as a vaccine recall antigen and as a neoantigen surrogate, respectively. C_FIG

cancer biology↗

bioRxiv2023

Discovery of a first-in-class small molecule ligand for WDR91 using DNA-encoded chemical library selection followed by machine learning

WD40 repeat-containing protein 91 regulates endosomal phosphatidylinositol 3-phosphate levels at the critical stage of endosome maturation and plays vital roles in endosome fusion, recycling, and transport by mediating protein-protein interactions. Due to its various roles in endocytic pathways, WDR91 has recently been identified as a potential host factor responsible for viral infection. We employed DNA-Encoded Chemical Library (DEL) selection against the WDR domain of WDR91, followed by machine learning to generate a model that was then used to predict ligands from the synthetically accessible Enamine REAL database. Screening of predicted compounds enabled us to identify the hit compound 1, which binds selectively to WDR91 with a KD of 6 {+/-} 2 M by surface plasmon resonance. The co-crystal structure confirmed the binding of 1 to the WDR91 side pocket, in proximity to cysteine 487. Machine learning-assisted structure activity relationship-by-catalog validated the chemotype of 1 and led to the discovery of covalent analogs 18 and 19. Intact mass LC-MS and differential scanning fluorimetry confirmed the formation of a covalent adduct, and thermal stabilization, respectively. The discovery of 1, 18, 19, accompanying SAR, and co-crystal structures will provide valuable insights for designing more potent and selective compounds against WDR91, thus accelerating the development of novel chemical tools to evaluate the therapeutic potential of WDR91 in disease.

molecular biology↗

bioRxiv2023

Zebrafish models of Mucopolysaccharidosis types IIIA, B, & C show hyperactivity and changes in oligodendrocyte state

Sanfilippo syndrome, also known as mucopolysaccharidosis type III (MPS III), is a group of rare inherited lysosomal storage disorders which causes childhood dementia. Subtypes of MPS III are caused by deficiencies in one of four enzymes required for degradation of the glycosaminoglycan heparan sulfate (HS). An inability to degrade HS leads to progressive neurodegeneration and death, often in the second or third decades of life in the classical course of the disease. Knowledge of MPS III pathogenesis is incomplete, and no effective therapies are yet approved for human use. We generated hypomorphic mutations in the endogenous zebrafish genes orthologous to those associated with MPS IIIA, MPS IIIB and MPS IIIC. Our models display the primary MPS III disease signature of significant brain accumulation of HS, and behavioural analyses revealed hyperactivity phenotypes in MPS IIIA and MPS IIIB zebrafish. Brain transcriptome analysis revealed changes related to lysosomal, glycosaminoglycan and immune system biology in all three models but also distinct differences in brain transcriptome state between models, which may be mutation dependent. The transcriptome analysis also indicated marked disturbance of oligodendrocyte-related genes in the brains of MPS III zebrafish, supporting that effects on this cell type are a consistent characteristic of MPS III. Overall, our zebrafish models recapture key characteristics of the human disease and phenotypes seen in mouse models. Our models will allow exploitation of the zebrafishs extreme fecundity and accessible anatomy to dissect the pathological mechanisms in MPS IIIA, IIIB, and IIIC.

genetics↗