Biology subjects

Shah, M.

Publications and source records attributed to Shah, M..

At least 37 records · Page 2Linked to original sources

bioRxiv2024

SMYD5 is a ribosomal methyltransferase which trimethylates RPL40 lysine 22 through recognition of a KXY motif

The eukaryotic ribosome is highly modified by protein methylation, yet many of the responsible methyltransferases remain unknown. Here we have identified SMYD5 as a ribosomal protein methyltransferase that catalyses trimethylation of RPL40/eL40 at lysine 22. Through a systematic mass spectrometry-based approach, we show that the human ribosome has 12 primary sites of protein methylation, including at RPL40 K22. Through in vitro methylation of synthetic RPL40 using fractionated lysate, we then identify SMYD5 as a candidate RPL40 K22 methyltransferase. We show that recombinant SMYD5 has robust activity towards RPL40 K22 in vitro, and that active site mutations ablate this activity. Knockouts of SMYD5 in K562 cells show a complete loss of RPL40 K22 methylation and decrease polysome levels. By systematic analysis of its recognition motif, we show that SMYD5 requires a tyrosine in the +2 position, and thereby is incapable of methylating its previously reported histone substrates.

molecular biology↗

bioRxiv2024

Detecting microbial engraftment after FMT: sorting signals from noise using placebo sequencing and culture-enriched metagenomics

Fecal microbiota transplantation (FMT) has shown efficacy for the treatment of ulcerative colitis but with variable response between patients and trials. The mechanisms underlying FMTs therapeutic effects remains poorly understood but is generally assumed to involve engraftment of donor microbiota into the recipients microbiome. Previous studies have reported microbial engraftment following FMT in various disease contexts but with inconsistent results between studies. Here we investigate engraftment in UC patients receiving FMT from a single donor applying amplicon-based profiling, shotgun metagenomics and culture-enriched metagenomics. Placebo samples were included to estimate engraftment noise, and a significant level of false-positive engraftment was observed which confounds the prediction of true engraftment. We show that analyzing engraftment across multiple patients from a single donor enhances the accuracy of detection. We identified a unique set of genes engrafted in responders to FMT which supports strain displacement as the primary mechanism of engraftment in our cohort.

microbiology↗

bioRxiv2024

Morpho-physiological and transcriptomic responses of field pennycress to waterlogging

Field pennycress (Thlaspi arvense) is a new biofuel winter annual crop with extreme cold hardiness and a short life cycle, enabling off-season integration into corn and soybean rotations across the Midwest. Pennycress fields are susceptible to winter snow melt and spring rainfall, leading to waterlogged soils. The objective of this research was to determine if waterlogging during the reproductive stage had a significant effect on gene expression, morphology, physiology, recovery, and yield of two pennycress lines (SP32-10 and MN106). In a controlled environment, total pod number, shoot/root dry weight, and total seed count/weight were significantly reduced in SP32-10 in response to waterlogging, whereas primary branch number, shoot dry weight, and single seed weight were significantly reduced in MN106. This indicated waterlogging had a greater negative impact on seed yield in SP32-10 than MN106. The number of differentially expressed genes (DEGs) between waterlogged and control roots were doubled in MN106 (3,424) compared to SP32-10 (1,767). Functional enrichment analysis of upregulated DEGs revealed Gene Ontology (GO) terms associated with hypoxia and decreased oxygen, with genes in these categories involved in alcoholic fermentation and glycolysis. Interestingly, MN106 waterlogged roots exhibited significant stronger upregulation of these genes than SP32-10. Additionally, downregulated DEGs revealed GO terms associated with cell wall biogenesis and secondary metabolite biogenesis, indicating suppressed growth and energy conservation. Together, these results reveal the reconfiguration of cellular and metabolic processes in response to the severe energy crisis invoked by waterlogging in pennycress.

plant biology↗

bioRxiv2024

MENTOR: Multiplex Embedding of Networks for Team-Based Omics Research

While the proliferation of data-driven omics technologies has continued to accelerate, methods of identifying relationships among large-scale changes from omics experiments have stagnated. It is therefore imperative to develop methods that can identify key mechanisms among one or more omics experiments in order to advance biological discovery. To solve this problem, here we describe the network-based algorithm MENTOR - Multiplex Embedding of Networks for Team-Based Omics Research. We demonstrate MENTORs utility as a supervised learning approach to successfully partition a gene set containing multiple ontological functions into their respective functions. Subsequently, we used MENTOR as an unsupervised learning approach to identify important biological functions pertaining to the host genetic architectures in Populus trichocarpa associated with microbial abundance of multiple taxa. Moreover, as open source software designed with scientific teams in mind, we demonstrate the ability to use the output of MENTOR to facilitate distributed interpretation of omics experiments.

bioinformatics↗

bioRxiv2024

Loss of TDP-43 induces synaptic dysfunction that is rescued by UNC13A splice-switching ASOs

TDP-43 loss of function induces multiple splicing changes, including a cryptic exon in the amyotrophic lateral sclerosis and fronto-temporal lobar degeneration risk gene UNC13A, leading to nonsense-mediated decay of UNC13A transcripts and loss of protein. UNC13A is an active zone protein with an integral role in coordinating pre-synaptic function. Here, we show TDP-43 depletion induces a severe reduction in synaptic transmission, leading to an asynchronous pattern of network activity. We demonstrate that these deficits are largely driven by a single cryptic exon in UNC13A. Antisense oligonucleotides targeting the UNC13A cryptic exon robustly rescue UNC13A protein levels and restore normal synaptic function, providing a potential new therapeutic approach for ALS and other TDP-43-related disorders.

neuroscience↗

bioRxiv2024

Three Modes of Viral Adaption by the Heart

Viruses elicit long-term adaptive responses in the tissues they infect. Understanding viral adaptions in humans is difficult in organs such as the heart, where primary infected material is not routinely collected. In search of asymptomatic infections with accompanying host adaptions, we mined for cardio-pathogenic viruses in the unaligned reads of nearly one thousand human hearts profiled by RNA sequencing. Among virus-positive cases ([~]20%), we identified three robust adaptions in the host transcriptome related to inflammatory NF{kappa}B signaling and post-transcriptional regulation by the p38-MK2 pathway. The adaptions are not determined by the infecting virus, and they recur in infections of human or animal hearts and cultured cardiomyocytes. Adaptions switch states when NF{kappa}B or p38-MK2 are perturbed in cells engineered for chronic infection by the cardio-pathogenic virus, coxsackievirus B3. Stratifying viral responses into reversible adaptions adds a targetable systems-level simplification for infections of the heart and perhaps other organs.

systems biology↗

bioRxiv2024

Prophages block cell surface receptors to ensure survival of their viral progeny

In microbial communities, viruses compete for host cells to infect, and thus evolved diverse ways to inhibit their competitors. One mechanism is Superinfection exclusion (Sie), whereby a virus that has established an infection prevents a secondary infection. We identified a Pseudomonas prophage Sie protein that alters pilus dynamics through the pilus assembly chaperone, PilZ. This protein, known as Zip for PilZ interacting protein, does not abrogate pilus activity, but fine tunes it, providing strong phage resistance without a fitness cost. This tuning is modulated through quorum sensing, which coordinates Zip production in concert with bacterial cell density to ensure maximal protection when bacterial populations are at the highest risk of phage infection. Most notably, Zip activity prevents internalization and destruction of phage progeny. We refer to this as the "anti-Kronos effect" after the Greek god who devoured his own children and show that it is a conserved feature of diverse prophage-encoded Sie systems.

microbiology↗

bioRxiv2023

Functional Screening in human HSPCs identifies optimized protein-based enhancers of Homology Directed Repair

Homology Directed Repair (HDR) enables precise genome editing and holds great promise in the gene therapy field. However, the implementation of HDR-based therapies is hindered by limited efficiency in comparison to methods that exploit alternative DNA repair routes, such as Non-Homologous End Joining (NHEJ). In this study, we demonstrate the development of a functional, pooled screening platform utilizing an HDR-based readout to identify protein-based reagents that improve HDR outcomes in human hematopoietic stem and progenitor cells (HSPCs), a clinically relevant cell type for gene therapy. We leveraged this screening platform to explore sequence diversity at the binding interface of the NHEJ inhibitor i53 and its target, 53BP1, and we identified optimized i53 variants that enable new intermolecular bonds and robustly increase HDR. These variants specifically reduce insertion-deletion outcomes and also synergize with a DNAPK inhibitor to increase HDR rates. When applied at manufacturing scale, the incorporation of improved variants results in a significant increase in cells with at least one repaired allele and improved HDR in long-term HSPCs subpopulations, while not increasing off-target editing or gross chromosomal rearrangements. We anticipate the pooled screening platform will enable discovery of future gene editing reagents that improve HDR outcomes, such as the i53 variants reported here.

molecular biology↗

bioRxiv2023

Computational characterization of the interactions between novel Boronic Acid derivatives with urokinase-type plasminogen activator (uPA) and their binding energy

1.Urokinase type plasminogen activator is expected to play a significant role in metastasis therefore various inhibitors are being prepared for this target protein. However, the binding site with residues that are involved in binding and inhibition is unidentified. Hence, comprehensive computational techniques are applied for finding the binding pocket, important amino acid residues and for the characterization of the binding energy of the best ligand among seven novel boronic acid derivative inhibitors within the binding pocket. Among seven test compounds, C14H21BN2O2S showed best results in structure based molecular docking through Molecular Operating Environment (MOE) and GOLD suit with -3.2481 kcal/mol binding affinity and 46.4523 GOLD Score. C14H21BN2O2S also showed high binding affinity within the binding pocket in DFT (Density Functional Theory) studies. DFT was carried out using hybrid functional B3LYP in combination with basis set LANL2DZ level of density functional theory on the extracted geometry of bound ligand C14H21BN2O2S to the binding pocket of uPA with a -2 charge on amino-acid residue ASP189. Computational analysis values on Geometric Optimization (opt), Single Point Energy (SPE) and Self-Consistent Reaction Field (SCRF) were 53.9, -66.3 and -49.0 respectively. Hence it is concluded that C14H21BN2O2S shows better binding with uPA binding pocket when there is a negative two charge on it ASP189 amino acid residue in the binding pocket. These seven ligands were also used for generating pharmacophore model through random selection with genetic algorithm by MOE having sensitivity of 79% towards the test set, specificity of 78% towards test set and 51% calculated Matthews coefficient correlation. 2. Author SummaryBoronic-acid based proteasome inhibitor like Bortezomib and Ixazomib are Food and Drug Administration (FDA) approved drugs, which are being used for fighting cancer. They can be considered as a template for understanding the pharmacokinetics and role of Boronic-acid ligands in the process. Boron-based warheads with stabilised functionality along with reduced toxicity are beneficent therapeutically. We have utilized computational quantum mechanical techniques in predicting binding free energies for ligands and proteins in a solvent environment. Instead of providing precise estimations, these techniques are more suitable for prediction purposes. The main challenge is developing inhibitors for uPA sub-sites that have high selectivity, potency, and improved pharmacokinetic properties. We have used Molecular docking and ligand-based techniques to analyze the binding interactions between seven ligands and uPA. Among these ligands, C14H21BN2O2S is identified as the most appropriate inhibitor based on scores and its interactions with specific receptor amino acid residues. Computational quantum mechanical studies are conducted using electron density and hybrid functional B3LYP to determine the binding energy. A pharmacophore model is designed to identify crucial descriptors and search for compounds that can effectively inhibit uPA. The models accuracy is assessed through QSAR analysis, which reveals favorable hydrogen bond donor and acceptor groups as well as aromatic hydrophobic rings in proximity to the ligands. The designed model demonstrates good sensitivity, specificity, and calculated Matthews coefficient correlation.

bioinformatics↗

bioRxiv2023

The Interplay between Mutagenesis and Extrachromosomal DNA Shapes Urothelial Cancer Evolution

Advanced urothelial cancer is a frequently lethal disease characterized by marked genetic heterogeneity. In this study, we investigate the evolution of the genomic signatures caused by endogenous and external mutagenic stimuli and their interplay with complex structural variants. We superimposed mutational signatures and phylogenetic analyses of matched serial tumors from patients with urothelial cancer to define the evolutionary patterns of these processes. We show that APOBEC3-induced mutations are clonal and early, whereas mutational bursts comprising hundreds of late subclonal mutations are induced by chemotherapy. Using a novel genome graph computational paradigm, we observed frequent circular high copy-number amplicons characteristic of extrachromosomal DNA (ecDNA) involving double-minutes, breakage-fusion-bridge, and tyfonas events. We characterized the distinct temporal patterns of APOBEC3 mutations and chemotherapy-induced mutations within ecDNA, gaining new insights into the timing of these events relative to ecDNA biogenesis. Finally, we discovered that most CCND1 amplifications in urothelial cancer arise within circular ecDNA amplicons. These CCND1 ecDNA amplification events persisted and increased in complexity incorporating additional DNA segments potentially contributing selective fitness advantage to the evolution of treatment resistance. Our findings define fundamental mechanisms driving urothelial cancer evolution and have therapeutic implications for treating this disease.

genomics↗

bioRxiv2023

Multiple behavioral mechanisms shape social behavioral development in a highly social cichlid fish

Early-life social experiences shape adult phenotype, yet the underlying behavioural mechanisms remain poorly understood. We manipulated early-life social experience in the highly social African cichlid fish Astatotilapia burtoni to investigate the effects on behaviour and neuroendocrine stress axis function. Juveniles experienced different numbers of early-life social partners in stable pairs (1 partner), stable groups (6 fish; 5 partners), and socialized pairs (a novel fish was exchanged every 5 days; 5 partners). Treatments differed in group size (groups vs. pairs) and stability (stable vs. socialized). We then measured behaviour in multiple contexts and collected water-borne cortisol. We found effects of treatment on behaviour across all assays: open field exploration, social cue investigation, dominant behaviour, and subordinate behaviour. Cortisol did not differ across treatments. Principal components (PC) analysis revealed robust co- variation of behaviour across contexts, including with cortisol, to form behavioural syndromes sensitive to early-life social experience. PC1 (25.1%) differed by numbers of social partners: juveniles with more social partners were more active during the social cue investigation, spent less time in the territory, and were more interactive as dominants. Differences in PC5 (8.5%) were based on stability: socialized pairs were more dominant, spent less time in and around the territory, were more socially investigative, and had lower cortisol than stable groups or pairs. Behaviour observations in the home tanks provided further insights into the behavioural mechanisms underlying these effects. These results contribute to our understanding of how early- life social experiences are accrued and exert strong, lasting effects on adult phenotype.

animal behavior and cognition↗

bioRxiv2023

Coordinated stimulation of axon regenerative and neurodegenerative transcriptional programs by Atf4 following optic nerve injury

Stress signaling is important for determining the fates of neurons following axonal insults. Previously we showed that the stress-responsive kinase PERK contributes to injury-induced neurodegeneration (Larhammar et al., 2017). Here we show that PERK acts primarily through Activating Transcription Factor-4 (ATF4) to stimulate not only pro-apoptotic but also pro-regenerative responses following optic nerve damage. Using conditional knockout mice, we find an extensive PERK/ATF4-dependent transcriptional response that includes canonical ATF4 target genes and modest contributions by C/EBP Homologous Protein (CHOP). Overlap with c-Jun-dependent transcription suggests interplay with a parallel stress pathway that orchestrates regenerative and apoptotic responses. Accordingly, neuronal knockout of ATF4 recapitulates the neuroprotection afforded by PERK deficiency, and PERK or ATF4 knockout impairs optic axon regeneration enabled by disrupting the tumor suppressor PTEN. These findings reveal an integral role for PERK/ATF4 in coordinating neurodegenerative and regenerative responses to CNS axon injury.

neuroscience↗

bioRxiv2023

Prevalence of Slam-dependent hemophores in Gram-negative bacteria

Iron acquisition systems are crucial for pathogen growth and survival in iron-limiting host environments. To overcome nutritional immunity, bacterial pathogens evolved to use diverse mechanisms to acquire iron. Here, we examined a heme acquisition system driven by hemophores called HphAs from several Gram-negative bacteria. Structural determination of HphAs revealed a N-terminal clamp-like domain that binds heme and a C-terminal eight-stranded {beta}-barrel domain that shares the same architecture as the Slam-dependent Neisserial surface lipoproteins. The structure of these HphAs is strikingly similar to a novel hemophore discovered by Latham et al. (2019), named hemophilin1. The genetic organization of HphAs consist of genes encoding a Slam homolog and a TonB-dependent receptor (TBDR). We investigated the Slam-HphA system in the native organism or the reconstituted system in E. coli cells and found that the efficient secretion of HphA is dependent on Slam. The TBDR also played an important role for heme uptake and conferred specificity for its cognate HphA. Furthermore, bioinformatic analysis of HphA homologs revealed that HphAs are conserved in the alpha, beta, and gammaproteobacteria Together, these results show that HphA presents a new class of hemophores in Gram-negative bacteria and further expands the role of Slams in transporting soluble proteins supporting it role as a type 11 secretion system.

microbiology↗

bioRxiv2023

VAP spatially stabilizes dendritic mitochondria to locally fuel synaptic plasticity

Synapses are pivotal sites of memory formation and undergo plasticity in response to external inputs. Consequently, synapses are hotspots of energy consumption and are susceptible to dysfunction when their energy supplies are perturbed. Mitochondria are stabilized near synapses via cytoskeletal tethering and serve as local energy supplies to fuel synaptic plasticity. However, the mechanisms that tether and stabilize neuronal mitochondria for long durations and determine the spatial dendritic segment supported during synaptic plasticity are unknown. We identified a list of novel mitochondrial-cytoskeletal interactors in neurons using APEX-based proximity labeling. We narrowed down the protein candidates that exclusively tether mitochondria to actin near postsynaptic spines using high-resolution Airyscan confocal imaging. We find that VAP, the vesicle-associated membrane protein-associated protein implicated in Amyotrophic Lateral Sclerosis and interacts with the endoplasmic reticulum, stabilizes mitochondria via actin near the spines. To test if the VAP-dependent stable mitochondrial compartments can locally support synaptic plasticity, we investigated individual spines stimulated by two-photon glutamate uncaging for spine plasticity induction and their adjacent spines. We find that, along with actin, VAP functions as a spatial stabilizer of mitochondrial compartments to sustain synaptic plasticity for up to ~60 min and as a spatial ruler that determines the ~30 m length of the dendritic segment supporting synaptic plasticity.

neuroscience↗

bioRxiv2022

Childhood cancer mutagenesis caused by a domesticated DNA transposase

Genomic rearrangements are a hallmark of most solid tumors, including medulloblastoma, one of the most common brain tumors in children. Childhood cancers involve dysregulated cell development, but their mutational causes remain largely unknown. One of the most common forms of medulloblastoma is caused by ectopic activation of Sonic Hedgehog (SHH) signaling in cerebellar granule cell progenitors, associated with genetic deletions, amplifications, and other oncogenic chromosomal rearrangements. Here, we show that PiggyBac Transposable Element Derived 5 (Pgbd5) promotes tumor development in multiple developmentally-accurate mouse models of SHH medulloblastoma. Most mice with Pgbd5 deficiency do not develop tumors, while Pgbd5-deficient mice maintain largely normal cerebellar development. Mouse medulloblastomas expressing Pgbd5 exhibit significantly increased numbers of somatic structural DNA rearrangements, with PGBD5-specific transposon sequences at their breakpoints. Similar sequence breakpoints recurrently affect somatic DNA rearrangements of known tumor suppressors and oncogenes in medulloblastomas in 329 children. Therefore, this study identifies PGBD5 as a primary medulloblastoma mutator and provides a genetic mechanism responsible for the generation of somatic oncogenic DNA rearrangements in childhood cancer. One-Sentence SummaryInduction of somatic oncogenic mutations by the DNA transposase PGBD5 in cerebellar progenitor cells promotes medulloblastoma development.

cancer biology↗

bioRxiv2022

Machine learning guided signal enrichment for ultrasensitive plasma tumor burden monitoring

In solid tumor oncology, circulating tumor DNA (ctDNA) is poised to transform care through accurate assessment of minimal residual disease (MRD) and therapeutic response monitoring. To overcome the sparsity of ctDNA fragments in low tumor fraction (TF) settings and increase MRD sensitivity, we previously leveraged genome-wide mutational integration through plasma whole genome sequencing (WGS). We now introduce MRD-EDGE, a composite machine learning-guided WGS ctDNA single nucleotide variant (SNV) and copy number variant (CNV) detection platform designed to increase signal enrichment. MRD-EDGE uses deep learning and a ctDNA-specific feature space to increase SNV signal to noise enrichment in WGS by 300X compared to our previous noise suppression platform MRDetect. MRD-EDGE also reduces the degree of aneuploidy needed for ultrasensitive CNV detection through WGS from 1Gb to 200Mb, thereby expanding its applicability to a wider range of solid tumors. We harness the improved performance to track changes in tumor burden in response to neoadjuvant immunotherapy in non-small cell lung cancer and demonstrate ctDNA shedding in precancerous colorectal adenomas. Finally, the radical signal to noise enrichment in MRD-EDGE enables de novo mutation calling in melanoma without matched tumor, yielding clinically informative TF monitoring for patients on immune checkpoint inhibition.

genomics↗

bioRxiv2022

NETISCE: A Network-Based Tool for Cell Fate Reprogramming

The search for effective therapeutic targets in fields like regenerative medicine and cancer research has generated interest in cell fate reprogramming. This cellular reprogramming paradigm can drive cells to a desired target state from any initial state. However, methods for identifying reprogramming targets remain limited for biological systems that lack large sets of experimental data or a dynamical characterization. We present NETISCE, a novel computational tool for identifying cell fate reprogramming targets in static networks. In combination with machine learning algorithms, NETISCE estimates the attractor landscape and predicts reprogramming targets using Signal Flow Analysis and Feedback Vertex Set Control, respectively. Through validations in studies of cell fate reprogramming from developmental, stem cell, and cancer biology, we show that NETISCE can predict previously identified cell fate reprogramming targets and identify potentially novel combinations of targets. NETISCE extends cell fate reprogramming studies to larger-scale biological networks without the need for full model parameterization and can be implemented by experimental and computational biologists to identify parts of a biological system relevant to the desired reprogramming task.

systems biology↗

bioRxiv2021

Omicron: A heavily mutated SARS-CoV-2 variant exhibits stronger binding to ACE2 and potently escape approved COVID-19 therapeutic antibodies

The new SARS-CoV-2 variant of concern "Omicron" was recently (Nov. 24th. 2021) spotted in South Africa and already spread around the world due to its enhanced transmissibility. The variant became conspicuous as it harbors more than thirty mutations in the spike protein with 15 mutations in the RBD region alone, potentially dampening the potency of therapeutic antibodies and enhancing the ACE2 binding. More worrying, Omicron infections have been reported in individuals who have received vaccines jabs in South Africa and Hong Kong. Here, we investigated the binding strength of Omicron with ACE2 and seven monoclonal antibodies that are either approved by FDA for COVID-19 therapy or undergoing phase III clinical trials. Computational mutagenesis and binding free energies could confirm that Omicron Spike binds ACE2 stronger than prototype SARS-CoV-2. Notably, three substitutions, i.e., T478K, Q493K, and Q498R, significantly contribute to the binding energies and doubled electrostatic potential of the RBDOmic-ACE2 complex. Instead of E484K substitution that helped neutralization escape of Beta, Gamma, and Mu variants, Omicron harbors E484A substitution. Together, T478K, Q493K, Q498R, and E484A substitutions contribute to a significant drop in the electrostatic potential energies between RBDOmic-mAbs, particularly in Etesevimab, Bamlanivimab, and CT-p59. CDR diversification could help regain the neutralization strength of these antibodies; however, we could not conduct this analysis to this end. Conclusively, our findings suggest that Omicron binds ACE2 with greater affinity, enhancing its infectivity and transmissibility. Mutations in the Spike are prudently devised by the virus that enhances the receptor binding and weakens the mAbs binding to escape the immune response.

genomics↗