Biology subjects

Shah, D.

Publications and source records attributed to Shah, D..

At least 37 records · Page 2Linked to original sources

bioRxiv2024

R-Spondin Mimetic, SZN-043, Induced Proliferation and Wnt Activity, Two Features Deficient in Human Alcohol-Associated Liver Disease

Liver regeneration is impaired in patients suffering from alcohol-associated liver (ALD) diseases. Wnt ligands and their FZD receptors are dysregulated in diseased livers. R-spondin and their receptors are known to regulate Wnt activity via the stabilization of FZD receptors. Here, we investigated the components of the Wnt and R-Spondin-signaling pathways and their activity in patients with ALD. We found that while hepatocytes retained high levels of differentiation markers such as ASGR1 and ASGR2, the expression of two R-spondin co-receptors, LGR4 and LGR5, and of Wnt target genes, CYP1A2 and others, were strongly reduced. SZN-043, a hepatocyte-targeted R-Spondin mimetic, is a new investigational drug that stimulates the physiological Wnt repair pathway and proliferation of hepatocytes. Here, we show that SZN-043 induced hepatocyte proliferation in all models tested, including humanized mouse livers, a chronic-binge alcohol-induced liver injury, and a CCl4-induced fibrosis mouse model. Altogether, SZN-043 could be beneficial for the treatment of ALD.

pathology↗

bioRxiv2024

A scalable approach to absolute quantitation in metabolomics

Mass spectrometry-based metabolomics allows for the quantitation of metabolite levels in diverse biological samples. The traditional method of converting peak areas to absolute concentrations involves the use of matched heavy isotopologues. However, this approach is laborious and limited to a small number of metabolites. We addressed these limitations by developing PyxisTM, a machine learning-based technology which converts raw mass spectrometry data to absolute concentration measurements without the need for per-analyte standards. Here, we demonstrate Pyxis performance by quantifying metabolome concentration dynamics in murine blood plasma. Pyxis performed equivalently to traditional quantitation workflows used by research institutions, with a fraction of the time needed for analysis. We show that absolute quantitation by Pyxis can be expanded to include concentrations for additional metabolites, without the need to acquire new data. Furthermore, Pyxis allows for absolute quantitation as part of an untargeted metabolomics workflow. By removing the bottleneck of per-analyte standards, Pyxis allows for absolute quantitation in metabolomics that is scalable to large numbers of metabolites. The ability of Pyxis to make concentration-based measurements across the metabolome has the potential to deepen our understanding of diverse metabolic perturbations.

systems biology↗

bioRxiv2024

Chickpea NCR13 disulfide cross-linking variants exhibit profound differences in antifungal activity and modes of action

Small cysteine-rich antifungal peptides with multi-site modes of action (MoA) have potential for development as biofungicides. In particular, legumes of the inverted-repeat lacking clade express a large family of nodule-specific cysteine-rich (NCR) peptides that orchestrate differentiation of nitrogen-fixing bacteria into bacteroids. These NCRs can form 2 or 3 intramolecular disulfide bonds and a subset of these peptides with high cationicity exhibits antifungal activity. However, the importance of intramolecular disulfide pairing and MoA against fungal pathogens for most of these plant peptides remains to be elucidated. Our study focused on a highly cationic chickpea NCR13, which has a net charge of +9 and contains six cysteines capable of forming three disulfide bonds. NCR13 expression in Pichia pastoris resulted in formation of two peptide folding variants, NCR13_PFV1 and NCR13_PFV2, that differed in the pairing of two out of three disulfide bonds despite having an identical amino acid sequence. The NMR structure of each PFV revealed a unique three-dimensional fold with the PFV1 structure being more compact but less dynamic. Surprisingly, PFV1 and PFV2 differed profoundly in the potency of antifungal activity against several fungal plant pathogens and their multi-faceted MoA. PFV1 showed significantly faster fungal cell-permeabilizing and cell entry capabilities as well as greater stability once inside the fungal cells. Additionally, PFV1 was more effective in binding fungal ribosomal RNA and inhibiting protein translation in vitro. Furthermore, when sprayed on pepper and tomato plants, PFV1 was more effective in reducing disease symptoms caused by Botrytis cinerea, causal agent of gray mold disease in fruits, vegetables and flowers. In conclusion, our work highlights the significant impact of disulfide pairing on the antifungal activity and MoA of NCR13 and provides structural framework for design of novel, potent antifungal peptides for agricultural use. Author SummaryFungal pathogens cause significant pre-harvest and post-harvest losses of crop yield, making them a serious biological threat to global food security. Chemical fungicides are effective in controlling fungal diseases across various crops. However, rapid evolution of fungal pathogen resistance to single-site chemical fungicides in agriculture has created an urgent need for development of safe, sustainable, and cost-effective multi-site fungicides. Nodule-specific cysteine-rich (NCR) peptides expressed in the inverted-repeat lacking clade legume plants exhibit potent antifungal activity; however, their modes of action (MoA) are poorly understood. Particularly, the specific contribution of disulfide pairing to the potency and spectrum of antifungal activity against fungal plant pathogens and MoA of these peptides remains to be identified. Chickpea NCR13 expressed in P. pastoris generates two peptide variants that differ in their disulfide cross-linking pattern. These variants exhibit striking differences in their three-dimensional structures and potency of antifungal activity against multiple fungal pathogens and MoA. They also differ in their ability to confer resistance to gray mold in pepper and tomato plants. Our study highlights the major impact a specific pattern of disulfide pairing can have on the in vitro and in planta antifungal activity of an NCR peptide.

plant biology↗

bioRxiv2024

In-silico heart model phantom to validate cardiac strain imaging

The quantification of cardiac strains as structural indices of cardiac function has a growing prevalence in clinical diagnosis. However, the highly heterogeneous four-dimensional (4D) cardiac motion challenges accurate "regional" strain quantification and leads to sizable differences in the estimated strains depending on the imaging modality and post-processing algorithm, limiting the translational potential of strains as incremental biomarkers of cardiac dysfunction. There remains a crucial need for a feasible benchmark that successfully replicates complex 4D cardiac kinematics to determine the reliability of strain calculation algorithms. In this study, we propose an in-silico heart phantom derived from finite element (FE) simulations to validate the quantification of 4D regional strains. First, as a proof-of-concept exercise, we created synthetic magnetic resonance (MR) images for a hollow thick-walled cylinder under pure torsion with an exact solution and demonstrated that "ground-truth" values can be recovered for the twist angle, which is also a key kinematic index in the heart. Next, we used mouse-specific FE simulations of cardiac kinematics to synthesize dynamic MR images by sampling various sectional planes of the left ventricle (LV). Strains were calculated using our recently developed non-rigid image registration (NRIR) framework in both problems. Moreover, we studied the effects of image quality on distorting regional strain calculations by conducting in-silico experiments for various LV configurations. Our studies offer a rigorous and feasible tool to standardize regional strain calculations to improve their clinical impact as incremental biomarkers.

bioengineering↗

bioRxiv2023

Harnessing Bilayer Biomaterial Delivery of FTY720 as an Immunotherapy to Accelerate Oral Wound Healing

Orofacial clefts are the most common craniofacial congenital anomaly. Following cleft palate repair, up to 60% of surgeries have wound healing complications leading to oronasal fistula (ONF), a persistent connection between the roof of the mouth and the nasal cavity. The current gold standard methods for ONF repair use human allograft tissues; however, these procedures have risks of graft infection and/or rejection, requiring surgical revisions. Immunoregenerative therapies present a novel alternative approach to harness the bodys immune response and enhance the wound healing environment. We utilized a repurposed FDA-approved immunomodulatory drug, FTY720, to reduce the egress of lymphocytes and induce immune cell fate switching toward pro-regenerative phenotypes. Here, we engineered a bilayer biomaterial system using Tegaderm, a liquid-impermeable wound dressing, to secure and control the delivery of FTY720- nanofiber scaffolds (FTY720-NF). We optimized release kinetics of the bilayer FTY720-NF to sustain drug release for up to 7d with safe, efficacious transdermal absorption and tissue biodistribution. Through comprehensive immunophenotyping, our results illustrate a pseudotime pro-regenerative state transition in recruited hybrid immune cells to the wound site. Additional histological assessments established a significant difference in full thickness ONF closure in mice on Day 7 following treatment with bilayer FTY720-NF, compared to controls. These findings demonstrate the utility of immunomodulatory strategies for oral wound healing, better positing the field to develop more efficacious treatment options for pediatric patients. One Sentence SummaryLocal delivery of bilayer FTY720-nanofiber scaffolds in an ONF mouse model promotes complete wound closure through modulation of pro-regenerative immune and stromal cells.

bioengineering↗

bioRxiv2023

Sexually dimorphic mechanisms of VGLUT-mediated protection from dopaminergic neurodegeneration

Parkinsons disease (PD) targets some dopamine (DA) neurons more than others. Sex differences offer insights, with females more protected from DA neurodegeneration. The mammalian vesicular glutamate transporter VGLUT2 and Drosophila ortholog dVGLUT have been implicated as modulators of DA neuron resilience. However, the mechanisms by which VGLUT2/dVGLUT protects DA neurons remain unknown. We discovered DA neuron dVGLUT knockdown increased mitochondrial reactive oxygen species in a sexually dimorphic manner in response to depolarization or paraquat-induced stress, males being especially affected. DA neuron dVGLUT also reduced ATP biosynthetic burden during depolarization. RNA sequencing of VGLUT+ DA neurons in mice and flies identified candidate genes that we functionally screened to further dissect VGLUT-mediated DA neuron resilience across PD models. We discovered transcription factors modulating dVGLUT-dependent DA neuroprotection and identified dj-1{beta} as a regulator of sex-specific DA neuron dVGLUT expression. Overall, VGLUT protects DA neurons from PD-associated degeneration by maintaining mitochondrial health.

neuroscience↗

bioRxiv2023

Scaling laws of human transcriptional activity

Each human chromosome maintains its individuality during the cell cycle, and occupies a spatially limited volume, termed chromosome territory. Each linear chromosomal DNA is folded into multiple loops in the three dimensional space, and further organized into densely packed heterochromatin, less dense euchromatin and nucleosome-free regions that are accessible for transcription factor binding. As the average density of chromatin in the nucleus is very high, size exclusion potentially restricts access of large macromolecules such as RNA polymerase II and Mediator to DNA buried in chromosomal interiors. To examine this idea, we investigated whether increase in chromosome size leads to relative decrease in transcriptional activity of larger chromosomes. We found that the scaling of gene expression relative to chromosome size follows exactly the surface-area-to-volume ratio, suggesting that active genes are located at chromosomal surfaces. To directly test this hypothesis, we developed a scalable probe to assess chromatin accessibility to macromolecules of different sizes. We show that, at the chromosomal level, open chromatin landscapes of small and large molecules are strikingly similar. However, at a finer locus level, regions accessible to small transcription factors were primarily enriched around promoters, whereas regions accessible to large molecules were dispersed along gene bodies. Collectively, our results indicate that DNA accessibility is controlled at two different scales, and suggest that making chromatin accessible to large molecules is a critical step in the control of gene expression.

genetics↗

bioRxiv2023

Small cationic cysteine-rich defensin-derived antifungal peptide controls white mold in soybean

White mold disease caused by a necrotrophic ascomycete pathogen Sclerotinia sclerotiorum results in serious economic losses of soybean yield in the USA. Lack of effective genetic resistance to this disease in soybean germplasm and increasing pathogen resistance to fungicides makes white mold difficult to manage. Small cysteine-rich antifungal peptides with multi-faceted modes of action hold potential for development as sustainable spray-on bio-fungicides. We have previously reported that GMA4CG_V6 peptide, a 17-amino acid variant of the MtDef4 defensin-derived peptide GMA4CG containing the active {gamma}-core motif, exhibits potent antifungal activity against the gray mold fungal pathogen Botrytis cinerea in vitro and in planta. This peptide exhibited antifungal activity against an aggressive field isolate of S. sclerotiorum 555 in vitro. It markedly reduced white mold disease symptoms when applied to detached soybean leaves, pods, and stems. Spray-application on soybean plants provided robust control of the white mold disease. GMA4CG_V6 at sub-lethal concentrations reduced sclerotia production. It was also non-phytotoxic to soybean plants. Our results demonstrate that GMA4CG_V6 peptide has high potential for development as a bio-fungicide for white mold control in soybean.

plant biology↗

bioRxiv2023

The lateral septum plays a transforming role in acute stress-induced analgesia

AbstractStress is a potent modulator of pain. Specifically, acute stress due to physical restraint induces stress-induced analgesia (SIA). However, where and how acute stress and pain pathways interface in the brain are poorly understood. Here, we describe how the dorsal lateral septum (dLS), a forebrain limbic nucleus, facilitates SIA through its downstream targets in the lateral hypothalamic area (LHA) of mice. Taking advantage of transsynaptic viral-genetic, optogenetic, and chemogenetic techniques, we show that the dLS[->]LHA circuitry is sufficient to drive analgesia and is required for SIA. Further, our results reveal that the dLS[->]LHA pathway is opioid-dependent and modulates pain through the pro-nociceptive neurons in the rostral ventromedial medulla (RVM). Remarkably, we found that the inhibitory dLS neurons are recruited specifically when the mice struggle to escape under restraint and, in turn, inhibit excitatory LHA neurons. As a result, the RVM neurons downstream of LHA are disengaged, thus suppressing nociception. Together, we delineate a poly-synaptic pathway that can transform escape behavior in mice under restraint to acute stress into analgesia.

neuroscience↗

bioRxiv2022

IFNγ insufficiency during mouse intravaginal Chlamydia trachomatis infection exacerbates alternative activation in macrophages with compromised CD40 functions

Chlamydia trachomatis (C.tr), an obligate intracellular pathogen, causes asymptomatic genital infections in women and is also a leading cause of preventable blindness. Limited mouse model of chronic C.tr infection are available to study the host immune response. We have developed in vivo mice models of acute and chronic infections for C. trachomatis to explore the significance of macrophage-directed response in mediating immune activation/suppression. During chronic and repeated infections, IFN{gamma} secretion from T cells is abated while TGF{beta} and IL-10 secretion is enhanced. An increase in exhaustion (PD1, CTLA4) and anergic (Klrg3, Tim3) T cell markers is also observed during chronic infection. It was observed that alternatively-activated macrophages with low CD40 expression promote Th2 and Treg differentiation and lead to sustained C. trachomatis genital infection. Macrophages infected with C. trachomatis or treated with supernatant of infected epithelial cells drive them to alternately-activated phenotype. C. trachomatis infection prevents increase in CD40 expression. Low IFN{gamma}, as observed in chronic infection leads to incomplete clearance of bacteria and poor immune activation. C. trachomatis decapacitates IFN{gamma} responsiveness in macrophages via hampering IFN{gamma}RI and IFN{gamma}RII expression which can be correlated with poor expression of MHC-II, CD40, iNOS and NO release even following IFN{gamma} supplementation. Alternatively-activated macrophages during C. trachomatis infection express low CD40 rendering immunosuppressive, Th2 and Treg differentiation which could not be reverted even after IFN{gamma} supplementation. The alternative macrophages also harbour high bacterial load and are poor responders to IFN{gamma}, thus promoting immunosupression. Thus, C. trachomatis modulate the innate immune cells attenuating the anti-chlamydial functions of T cells in a manner that involves decreased CD40 expression on macrophages.

immunology↗

bioRxiv2022

Astrocyte calcium dysfunction causes early network hyperactivity in Alzheimer's Disease

Dysfunctions of network activity and functional connectivity (FC) represent early events in Alzheimers disease (AD), but the underlying mechanisms remain unclear. Astrocytes regulate neuronal activity in the healthy brain, but their involvement in early network hyperactivity in AD is unknown. We show increased FC in the human cingulate cortex, several years before amyloid deposition. We found the same early cingulate FC disruption and neuronal hyperactivity in AppNL-F mice. Crucially, these network disruptions are accompanied by decreased astrocyte calcium signaling. Recovery of astroglial calcium activity normalizes neuronal hyperactivity and FC, as well as seizure susceptibility and day/night behavioral hyperactivity. In conclusion, we show for the first time that astrocytes mediate initial features of AD and drive clinically relevant phenotypes.

neuroscience↗

bioRxiv2021

Redox-dependent Structure and Dynamics of Macrophage Migration Inhibitory Factor Reveal Sites of Latent Allostery

Macrophage migration inhibitory factor (MIF) is a multifunctional immunoregulatory protein that is a key player in the innate immune response. Given its overexpression at sites of inflammation in a wide range of diseases marked by increasingly oxidative cellular environment, a comprehensive structural understanding of how cellular redox conditions may impact the structure and function of MIF is necessary. We used solution NMR spectroscopy and mass spectrometry to investigate structural and dynamic signatures of MIF under varied solution redox conditions. Our results indicate that the MIF structure is modified and becomes increasingly dynamic in an oxidative environment, which may be a means to alter the MIF functional response in a redox-dependent manner. We identified latent allosteric sites within MIF that are redox-sensitive and mutational analysis reveals that loss of redox-responsive residues attenuates activation of the coreceptor CD74. Leveraging sites of redox-sensitivity therefore reveals an avenue to modulate MIF function in its "disease state" via structure-based drug design.

biophysics↗

bioRxiv2021

Heparan Sulfate-dependent RAGE oligomerization is indispensable for pathophysiological functions of RAGE

RAGE, a druggable inflammatory receptor, is known to function as an oligomer but the exact oligomerization mechanism remains poorly understood. Previously we have shown that heparan sulfate (HS) plays an active role in RAGE oligomerization. To understand the physiological significance of HS-induced RAGE oligomerization in vivo, we generated RAGE knock-in mice (RageAHA/AHA) by introducing point mutations to specifically disrupt HS-RAGE interaction. The RAGE mutant demonstrated normal ligand-binding but impaired capacity of HS-binding and oligomerization. Remarkably, RageAHA/AHA mice phenocopied Rage-/- mice in two different pathophysiological processes, namely bone remodeling and neutrophil-mediated liver injury, which demonstrates that HS-induced RAGE oligomerization is essential for RAGE signaling. Our findings suggest that it should be possible to block RAGE signaling by inhibiting HS-RAGE interaction. To test this, we generated a monoclonal antibody that targets the HS-binding site of RAGE. This antibody blocks RAGE signaling in vitro and in vivo, recapitulating the phenotype of RageAHA/AHA mice. By inhibiting HS-RAGE interaction genetically and pharmacologically, our work validated an alternative strategy to antagonize RAGE. Finally, we have performed RNA-seq analysis of neutrophils and lungs and found that while Rage-/- mice had a broad alteration of transcriptome in both tissues compared to wild-type mice, the changes of transcriptome in RageAHA/AHA mice were much more restricted. This unexpected finding suggests that by preserving the expression of RAGE protein (in a dominant-negative form), RageAHA/AHA mouse might represent a cleaner genetic model to study physiological roles of RAGE in vivo compared to Rage-/- mice.

cell biology↗

bioRxiv2021

Rigorous estimation of post-translational proteasomal splicing in the immunopeptidome

Recently, de novo peptide sequencing has made it possible to gain new insights into the human immunopeptidome without relying on peptide databases, while identifying peptides of unknown origin. Many recent studies have attributed post-translational proteasomal splicing as the origin of those peptides. Here, we describe a peptide source assignment workflow to rigorously assign the source of de novo sequenced peptides and find that the estimated extent of post-translational splicing in the immunopeptidome is much lower than previously reported. Our approach demonstrates that many peptides that were thought to be post-translationally spliced are likely linear peptides, and many peptides that were thought to be trans-spliced could be cis-spliced. We believe our approach furthers the understanding of post-translationally spliced peptides and thus improves the characterization of immunopeptidome which plays a critical role in the immune response to antigens in cancer, autoimmune disease, and infections.

bioinformatics↗

bioRxiv2021

The impact of viral mutations on recognition by SARS-CoV-2 specific T-cells

We identify amino acid variants within dominant SARS-CoV-2 T-cell epitopes by interrogating global sequence data. Several variants within nucleocapsid and ORF3a epitopes have arisen independently in multiple lineages and result in loss of recognition by epitope-specific T-cells assessed by IFN-{gamma} and cytotoxic killing assays. These data demonstrate the potential for T-cell evasion and highlight the need for ongoing surveillance for variants capable of escaping T-cell as well as humoral immunity.

immunology↗

bioRxiv2021

Medicago Sativa Defensin 1 (MsDef1), A Natural Tumor Targeted Sensitizer for Improving Chemotherapy: Translation from Anti-Fungal Agent to Potential Anti-Cancer Agent

IntroductionMsDef1, a 45-amino acid cysteine-rich peptide from the seed of Medicago sativa is an antifungal defensin small protein. It exhibits broad-spectrum antifungal activity against fungal pathogens of plants at low micromolar concentrations. The common vulnerability of fungal and cancer cells determines the utility of MsDef1 as a potential anti-tumor agent. ResultsThe solution dynamics of 15N-labeled MsDef1, 15N longitudinal relaxation (T1) and 15N-1H Nuclear Overhauser Effect (NOE) shows that GlcCer binds at two sites on the peptide molecule, i.e., Asp36-Cys39 and amino acids between 12-20 and 33-40. MsDef1 interacts with drug resistant breast cancer MCF-7R cells, permeates GlcCer-rich plasma membrane and releases apoptotic ceramide. This results in the activation of ceramide pathway involving interaction of the peptide with intracellular thioredoxin (Trx), another tumor specific biomarker. MsDef1 oxidizes Trx through four S-S bonds and in the process, gets reduced to thiols. Oxidation of Trx is correlated with the activation of Apoptosis Stimulating Kinase 1 (ASK1) which is known to sensitize cancer cells to chemotherapeutics including front-line drug Doxorubicin. A combination of MsDef1 and Doxorubicin exhibits 5-10-fold greater apoptosis in vitro in MDR triple negative breast cancer (TNBC) cells compared to either MsDef1 or Doxorubicin alone. ConclusionAn antifungal plant defensin MsDef1 is shown to be a cell permeating peptide (CPP) for MDR cancer cells targeted to two tumor specific targets activating two cell death pathways. That makes MsDef1, potentially a tumor targeted sensitizer neoadjuvant to cancer therapy.

cancer biology↗

bioRxiv2020

Immuno-Phenotyping of High-Grade Glioma Infiltrating Immune Cells Reveals Grade Specific Differences in Cells of Myeloid Origin

Gliomas are heavily infiltrated with immune cells of myeloid origin. Past studies have shown that high-grade gliomas have a higher proportion of alternatively activated and suppressive myeloid cells when compared to low-grade gliomas, which correlate with poor prognosis. However, the differences in immune cell phenotypes within high-grade gliomas (between grade III and IV) are relatively less explored, and a correlation of phenotypic characteristics between immune cells in the blood and high grade tumors has not been performed. Additionally, myeloid cells of granulocytic origin present in gliomas remain poorly characterized. Herein, we address these questions through phenotypic characterizations of monocytes and neutrophils present in blood and tumors of individuals with glioblastoma (GBM, grade IV) or grade III gliomas. Our data show that CD163 expressing M2 monocytes are present in greater proportions in GBM tissue when compared to grade III glioma tissue. In addition, we observe that neutrophils are highly heterogeneous among individuals with glioma, and a greater proportion of granulocytic myeloid-derived suppressor cells are present in grade III gliomas when compared to GBM. Finally, we show that the expression levels of CD86 and CD63 showed a high correlation between blood and tumor, and suggest that these may be used as possible markers for prognosis.

immunology↗

bioRxiv2020

Endoderm Nitric Oxide Signals to Regulate Nascent Development of Cardiac Progenitors in Chicken Embryos

Heart development in the chicken embryo is regulated by a concert of cardiogenic morphogens and signaling molecules, but the physiological signal molecule nitric oxide(NO) has not been studied in the context of heart formation. A dynamic investigation of endoderm NO formation demonstrates for the first time a correlation with the established development events of the cardiac heart fields and heart tube. Manipulation of endoderm NO signaling demonstrate a role of NO signaling in the differentiation and proliferation of cardiac progenitors for heart tube formation and cardiac heart field development. To investigate NO in the proliferation of myocardial cells in the heart tube embryos, a computer vision based artificial intelligence approach is followed to automate the long and tedious job of counting cells in a large image dataset. We document NO as an important signaling molecule in the regulation of nascent embryonic cardiogenesis whose effects on other early cardiogenic morphogens is unknown.

developmental biology↗