Biology subjects

Das, S.

Publications and source records attributed to Das, S..

At least 181 records · Page 10Linked to original sources

bioRxiv2024

VASCilia (Vision Analysis StereoCilia): A Napari Plugin for Deep Learning-Based 3D Analysis of Cochlear Hair Cell Stereocilia Bundles

Cochlear hair cells are essential for hearing, and their stereocilia bundles are critical for mechanotransduction. However, analyzing the 3D morphology of these bundles can be challenging due to their complex organization and the presence of other cellular structures in the tissue. To address this, we developed VASCilia (Vision Analysis StereoCilia), a Napari plugin suite that automates the analysis of 3D confocal microscopy datasets of phalloidin-stained cochlear hair cell bundles. VASCilia includes five deep learning-based models that streamline the analysis process, including: (1) Z-Focus Tracker (ZFT) for selecting relevant slices in a 3D image stack; (2) PCPAlignNet (Planar Cell Polarity Alignment Network) for automated orientation of image stacks; (3) a segmentation model for identifying and delineating stereocilia bundles; (4) a tonotopic Position Prediction tool; and (5) a classification tool for identifying hair cell subtypes. In addition, VASCilia provides automated computational tools and measurement capabilities. Using VASCilia, we demonstrate its utility on challenging datasets, including neonatal wildtype and Eps8 KO 5-day old mice), revealing that total actin content does not necessarily scale with bundle height. We further showcase its power by quantifying complex bundle disorganization in Cdh23-/- cochleae via texture analysis, which revealed systematically more heterogeneous and less regular bundles than in controls. These novel biological findings demonstrate the power of VASCilia in facilitating detailed quantitative analysis of stereocilia. VASCilia also provides a user-friendly interface that allows researchers to easily navigate and use the tool, with the added capability to reload all their analyses for review or sharing purposes. We believe that VASCilia will be a valuable resource for researchers studying cochlear hair cell development and function, addressing a longstanding need in the hair cell research community for specialized deep learning-based tools capable of high-throughput image quantitation. We have released our code along with a manually annotated dataset that includes approximately 55 3D stacks featuring instance segmentation (https://github.com/ucsdmanorlab/Napari-VASCilia). This dataset comprises a total of 502 inner and 1,703 outer hair cell bundles annotated in 3D. As the first open-source dataset of its kind, we aim to establish a foundational resource for constructing a comprehensive atlas of cochlea hair cell images. Ultimately, this initiative will support the development of foundational models adaptable to various species, markers, and imaging scales to accelerate advances within the hearing research community.

bioinformatics↗

bioRxiv2024

Molecular profiling of frontal and occipital subcortical white matter hyperintensities in Alzheimer's disease

White matter hyperintensities (WMHs) are commonly detected on T2-weighted magnetic resonance imaging (MRI) scans, occurring in both typical aging and Alzheimers disease. Despite their frequent appearance and their association with cognitive decline, the molecular factors contributing to WMHs remain unclear. In this study, we investigated the transcriptomic profiles of two commonly affected brain regions with coincident AD pathology--frontal subcortical white matter (frontal-WM) and occipital subcortical white matter (occipital-WM)--and compared with age-matched healthy controls. Through RNA-sequencing in frontal- and occipital-WM bulk tissues, we identified an upregulation of genes associated with brain vasculature function in AD white matter. To further elucidate vasculature-specific transcriptomic features, we performed RNA-seq analysis on blood vessels isolated from these white matter regions, which revealed an upregulation of genes related to protein folding pathways. Finally, comparing gene expression profiles between AD individuals with high-versus low-WMH burden showed an increased expression of pathways associated with immune function. Taken together, our study characterizes the diverse molecular profiles of white matter changes in AD compared to normal aging and provides new mechanistic insights processes underlying AD-related WMHs.

neuroscience↗

bioRxiv2024

Ribosome-Associated Vesicles promote activity-dependent local translation

Local protein synthesis in axons and dendrites underpins synaptic plasticity. However, the composition of the protein synthesis machinery in distal neuronal processes and the mechanisms for its deployment to local translation sites remain unclear. Here, we employed a multi-scale imaging approach combining cryo-electron tomography, volume electron microscopy, and live-cell imaging to identify endoplasmic reticulum-derived Ribosome-Associated Vesicles (RAVs) as a dynamic platform for moving ribosomes to distal processes and promoting activity-dependent local translation. We demonstrate that neuronal stimulation triggers compartment-specific RAV responses: dendrites accumulate stationary RAVs at sites of enhanced translation, while axons accelerate RAV transport. Real-time imaging of translation at single mRNA resolution reveals that RAVs boost local translation output compared to RAV-independent mechanisms. These findings establish RAVs as specialized platforms that integrate activity-dependent signals with local protein synthesis, providing a mechanistic framework for understanding how neurons achieve precise spatiotemporal control of protein synthesis.

neuroscience↗

bioRxiv2024

TYK2 as a novel therapeutic target in Alzheimer Disease

Neuroinflammation is a pathological feature of many neurodegenerative diseases, including Alzheimers disease (AD)1,2 and amyotrophic lateral sclerosis (ALS)3, raising the possibility of common therapeutic targets. We previously established that cytoplasmic double-stranded RNA (cdsRNA) is spatially coincident with cytoplasmic pTDP-43 inclusions in neurons of patients with C9ORF72-mediated ALS4. CdsRNA triggers a type-I interferon (IFN-I)-based innate immune response in human neural cells, resulting in their death 4. Here, we report that cdsRNA is also spatially coincident with pTDP-43 cytoplasmic inclusions in brain cells of patients with AD pathology and that type-I interferon response genes are significantly upregulated in brain regions affected by AD. We updated our machine-learning pipeline DRIAD-SP (Drug Repurposing In Alzheimers Disease with Systems Pharmacology) to incorporate cryptic exon (CE) detection as a proxy of pTDP-43 inclusions and demonstrated that the FDA-approved JAK inhibitors baricitinib and ruxolitinib that block interferon signaling show a protective signal only in cortical brain regions expressing multiple CEs. Furthermore, the JAK family member TYK2 was a top hit in a CRISPR screen of cdsRNA-mediated death in differentiated human neural cells. The selective TYK2 inhibitor deucravacitinib, an FDA-approved drug for psoriasis, rescued toxicity elicited by cdsRNA. Finally, we identified CCL2, CXCL10, and IL-6 as candidate predictive biomarkers for cdsRNA-related neurodegenerative diseases. Together, we find parallel neuroinflammatory mechanisms between TDP-43 associated-AD and ALS and nominate TYK2 as a possible disease-modifying target of these incurable neurodegenerative diseases.

neuroscience↗

bioRxiv2024

Janus Mazebots and Cellbots Navigating Obstacles in Dense Mammalian Environment

The field of microrobotics has immensely grown in the last few decades, exhibiting several challenges as new features such as shapes, sizes, and actuation mechanisms are explored. Two of the biggest challenges faced in microrobotics are the development of a control system suited for precise microrobotic manipulation, and the ability to navigate microrobots in densely populated environments. In this paper, we fabricate the Mazebot microrobots using silica spheres and ferromagnetic coating, and we use them to create cellbots with genetically modified Chinese Hamster Ovary (CHO) cells. Subsequently, we navigate both the Mazebots and the cellbots through a dense environment populated by CHO cells. The Mazebots navigation is done with a control system that allows the Mazebots to swim on their own, or guide a specific cell from a given origin to a target location while avoiding cell obstacles. The control system operates in open and closed-loop modes, where the first one allows the microrobot to reorient the cell using self-induced fluid vortices, and the second one closely follows a predefined trajectory along the origin and destination. On the other hand, the cellbots navigation is done in closed-loop operation. This enables cell manipulation for potential applications in cell and tissue engineering when in a confined space. Biocompatibility of the Mazebots is confirmed through the exposure of CHO cells to the robots for 24 hours. Experimental results demonstrate the functionality of our algorithm and its potential for biomedical applications, showcasing our system as a powerful and efficient solution for precise cellular manipulation.

bioengineering↗

bioRxiv2024

Microarray Integrated Spatial Transcriptomics (MIST) for Affordable, Robust, and Comprehensive Digital Pathology

10X Visium, a popular Spatial transcriptomics (ST) method, faces limited adoption due to its high cost and restricted sample usage per slide. To address these issues, we propose Microarray Integrated Spatial Transcriptomics (MIST), combining conventional tissue microarray (TMA) with Visium, using laser-cutting and 3D printing to enhance slide throughput. Our design facilitates independent replication and customization in individual labs to suit specific experimental needs. We provide a step-by-step guide from designing TMAs to the library preparation step. We demonstrate MISTs cost-effectiveness and technical benefits over Visium and GeoMx Nanostring. We also introduce AnnotateMap, a novel computational tool for efficient analysis of multiple ROIs processed through MIST.

bioinformatics↗

bioRxiv2024

In Silico Identification of Potential Inhibitors of Mycobacterium tuberculosis DNA Gyrase from Phytoconstituents of Indian Medicinal Plants

DNA gyrase, the sole type-II topoisomerase in Mycobacterium tuberculosis, plays a significant role in the bacterias survival by catalyzing the DNA topology change and is considered a crucial drug target. The emergence of drug resistance in Mycobacterium tuberculosis specifically against the second line of drugs; fluoroquinolones, targeting DNA gyrase, demands new potential compounds that would efficiently interact with it. To find a common suitable inhibitor for the apo and mutated forms of DNA gyrase, in this study, the compounds from an Indian medicinal plant database were screened for selecting potential drug-like molecules and two compounds IM5 and IM7 were selected after successful virtual screening and hit optimization with ADMET predictions, and chosen for MD simulation. The MMPBSA analysis of binding free energy validates the docking results of the two molecules. The principal component analysis also supported the stability of the complexes and thus, these two molecules have turned out to be promising candidates against Mycobacterium tuberculosis. Overall, this work sheds light on the potential of DNA gyrase inhibitors as therapeutic agents for Mycobacterium tuberculosis treatment, and IM5 and IM7 showed promise as future research compounds.

bioinformatics↗

bioRxiv2024

Molecular Signatures for Microbe-Associated Colorectal Cancers

BackgroundGenetic factors and microbial imbalances play crucial roles in colorectal cancers (CRCs), yet the impact of infections on cancer initiation remains poorly understood. While bioinformatic approaches offer valuable insights, the rising incidence of CRCs creates a pressing need to precisely identify early CRC events. We constructed a network model to identify continuum states during CRC initiation spanning normal colonic tissue to pre-cancer lesions (adenomatous polyps) and examined the influence of microbes and host genetics. MethodsA Boolean network was built using a publicly available transcriptomic dataset from healthy and adenoma affected patients to identify an invariant Microbe-Associated Colorectal Cancer Signature (MACS). We focused on Fusobacterium nucleatum (Fn), a CRC-associated microbe, as a model bacterium. MACS-associated genes and proteins were validated by RT-qPCR, RNA seq, ELISA, IF and IHCs in tissues and colon-derived organoids from genetically predisposed mice (CPC-APCMin+/-) and patients (FAP, Lynch Syndrome, PJS, and JPS). ResultsThe MACS that is upregulated in adenomas consists of four core genes/proteins: CLDN2/Claudin-2 (leakiness), LGR5/leucine-rich repeat-containing receptor (stemness), CEMIP/cell migration-inducing and hyaluronan-binding protein (epithelial-mesenchymal transition) and IL8/Interleukin-8 (inflammation). MACS was induced upon Fn infection, but not in response to infection with other enteric bacteria or probiotics. MACS induction upon Fn infection was higher in CPC-APCMin+/- organoids compared to WT controls. The degree of MACS expression in the patient-derived organoids (PDOs) generally corresponded with the known lifetime risk of CRCs. ConclusionsComputational prediction followed by validation in the organoid-based disease model identified the early events in CRC initiation. MACS reveals that the CRC-associated microbes induce a greater risk in the genetically predisposed hosts, suggesting its potential use for risk prediction and targeted cancer prevention.

microbiology↗

bioRxiv2024

Novel phylogenomic inference and 'Out of Asia' biogeography of cobras, coral snakes, and their allies

Estimation of evolutionary relationships among lineages that rapidly diversified can be challenging, and, in such instances, inaccurate or unresolved phylogenetic estimates can lead to erroneous conclusions regarding historical geographical ranges of lineages. One example underscoring this issue has been the historical challenge posed by untangling the biogeographic origin of elapoid snakes, which includes numerous dangerously venomous species as well as species not known to be dangerous to humans. The worldwide distribution of this lineage makes it an ideal group for testing hypotheses related to historical faunal exchanges among the many continents and other landmasses occupied by contemporary elapoid species. We developed a novel suite of genomic resources, included worldwide sampling, and inferred a robust estimate of evolutionary relationships, which we leveraged to quantitatively estimate geographical range evolution through the deep-time history of this remarkable radiation. Our phylogenetic and biogeographical estimates of historical ranges definitively reject a lingering former Out of Africa hypothesis and support an Out of Asia scenario involving multiple faunal exchanges between Asia, Africa, Australasia, the Americas, and Europe.

evolutionary biology↗

bioRxiv2024

Overexpression of α-SNAPRhg1 can improve rhg1-a mediated soybean resistance to soybean cyst nematode

(i)The rhg1-a and rhg1-b haplotypes of the soybean Rhg1 locus are economically effective tools for the control of soybean cyst nematode (SCN; Heterodera glycines), but ongoing SCN evolution requires improved sources of resistance. Both Rhg1 haplotypes carry multiple tandem repeat copies of a four-gene block encoding four disparate proteins; resistance efficacy scales with copy number. The haplotypes encode different variants of an unusual -SNAP protein whose abundance increases in the nematode-reprogrammed plant cells that form the syncytium (nematode feeding site), which subsequently collapses. Simultaneous presence of the two -SNAPRhg1 protein types, or substantially elevated expression of either type, was hypothesized to improve SCN resistance but has not been achievable by conventional plant breeding methods. We accomplished both, by transgenic additions to an rhg1-a Rhg4 soybean line. Existing resistance to HG type 0 and HG type 2.5.7 SCN was strengthened, but resistance was not improved against an HG type 1.3.6.7 SCN population that was already highly virulent on the parent line. No significant yield penalties from constitutive overexpression of transgenic -SNAP genes were observed in greenhouse or initial field studies. Cisgenic combination or elevated expression of the different -SNAP genes may extend the efficacy and/or durability of Rhg1-mediated resistance. We also observed differential protein abundances for some -SNAPRhg1 isoforms after inoculation with different HG type populations of SCN. These approaches provide opportunities to understand the different mechanisms of the two Rhg1 haplotypes and to possibly fine-tune them for better management of SCN.

plant biology↗

bioRxiv2024

Unequal genetic redundancies among MYC bHLH transcription factors underlie seedling photomorphogenesis in Arabidopsis

Light is one of the most critical ecological cues controlling plant growth and development. Plants have evolved complex mechanisms to cope with fluctuating light signals. In Arabidopsis, bHLH transcription factors MYC2, MYC3, and MYC4 have been shown to play a vital role in protecting plants against herbivory and necrotrophic pathogens. While the role of MYC2 in light-mediated seedling development has been studied in some detail, the role of MYC3 and MYC4 still needs to be discovered. Here, we show that MYC4 negatively regulates seedling photomorphogenesis, while the MYC3 function seems redundant. However, the genetic analysis reveals that MYC3/MYC4 together act as positive regulators of seedling photomorphogenic growth as the myc3myc4 double mutants showed exaggerated hypocotyl growth compared to myc4 single mutants and Col-0. Intriguingly, the loss of MYC2 function in the myc3myc4 double mutant background (myc2myc3myc4) resulted in further enhancement in the hypocotyl growth than myc3myc4 double mutants in WL, BL and FRL, suggesting that MYC2/3/4 together play an essential and positive role in meditating optimal seedling photomorphogenesis. Besides, MYC3/MYC4 genetically and physically interact with HY5 to partially inhibit its function in controlling hypocotyl and photo-pigment accumulation. Moreover, our results suggest that COP1 physically interacts and degrades MYC3 and MYC4 through the 26S proteasomal pathway and controls their response to dark and light for fine-tuning HY5 function and seedling photomorphogenesis.

plant biology↗

bioRxiv2024

Proteome profiling identifies a link between the mitochondrial pathways and host-microbial sensor ELMO1 following Salmonella infection.

The host EnguLfment and cell MOtility protein 1 (ELMO1) is a cytosolic microbial sensor that facilitates bacterial sensing, internalization, clearance, and inflammatory responses. We have shown previously that ELMO1 binds bacterial effector proteins, including pathogenic effectors from Salmonella and controls host innate immune signaling. To understand the ELMO1-regulated host pathways, we have performed liquid chromatography Multinotch MS3-Tandem Mass Tag (TMT) multiplexed proteomics to determine the global quantification of proteins regulated by ELMO1 in macrophages during Salmonella infection. Comparative proteome analysis of control and ELMO1-depleted murine J774 macrophages after Salmonella infection quantified more than 7000 proteins with a notable enrichment in mitochondrial-related proteins. Gene ontology enrichment analysis revealed 19 upregulated and 11 downregulated proteins exclusive to ELMO1-depleted cells during infection, belonging to mitochondrial functions, metabolism, vesicle transport, and the immune system. By assessing the cellular energetics via Seahorse analysis, we found that Salmonella infection alters mitochondrial metabolism, shifting it from oxidative phosphorylation to glycolysis. Importantly, these metabolic changes are significantly influenced by the depletion of ELMO1. Furthermore, ELMO1 depletion resulted in a decreased ATP rate index following Salmonella infection, indicating its importance in counteracting the effects of Salmonella on immunometabolism. Among the proteins involved in mitochondrial pathways, mitochondrial fission protein DRP1 was significantly upregulated in ELMO1-depleted cells and in ELMO1-KO mice intestine following Salmonella infection. Pharmacological Inhibition of DRP1 revealed the link of the ELMO1-DRP1 pathway in regulating the pro-inflammatory cytokine TNF- following infection. The role of ELMO1 has been further characterized by a proteome profile of ELMO1-depleted macrophage infected with SifA mutant and showed the involvement of ELMO1-SifA on mitochondrial function, metabolism and host immune/defense responses. Collectively, these findings unveil a novel role for ELMO1 in modulating mitochondrial functions, potentially pivotal in modulating inflammatory responses. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=149 SRC="FIGDIR/small/592405v1_ufig1.gif" ALT="Figure 1"> View larger version (43K): org.highwire.dtl.DTLVardef@72862org.highwire.dtl.DTLVardef@1ec4a5borg.highwire.dtl.DTLVardef@5b805org.highwire.dtl.DTLVardef@17466d7_HPS_FORMAT_FIGEXP M_FIG C_FIG Significance StatementHost microbial sensing is critical in infection and inflammation. Among these sensors, ELMO1 has emerged as a key regulator, finely tuning innate immune signaling and discriminating between pathogenic and non-pathogenic bacteria through interactions with microbial effectors like SifA of Salmonella. In this study, we employed Multinotch MS3-Tandem Mass Tag (TMT) multiplexed proteomics to determine the proteome alterations mediated by ELMO1 in macrophages following WT and SifA mutant Salmonella infection. Our findings highlight a substantial enrichment of host proteins associated with metabolic pathways and mitochondrial functions. Notably, we validated the mitochondrial fission protein DRP1 that is upregulated in ELMO1-depleted macrophages and in ELMO1 knockout mice intestine after infection. Furthermore, we demonstrated that Salmonella-induced changes in cellular energetics are influenced by the presence of ELMO1. This work shed light on a possible novel link between mitochondrial dynamics and microbial sensing in modulating immune responses.

microbiology↗

bioRxiv2024

Caspar determines primordial germ cell identity in Drosophila melanogaster

Repurposing of pleiotropic factors during execution of diverse cellular processes has emerged as a regulatory paradigm. Embryonic development in metazoans is controlled by maternal factors deposited in the egg during oogenesis. Here, we explore maternal role(s) of Caspar (Casp), the Drosophila orthologue of human Fas-associated factor-1 (FAF1) originally implicated in host-defense as a negative regulator of NF-{kappa}B signaling. Maternal loss of either Casp or its protein partner, Transitional endoplasmic reticulum 94 (TER94) leads to partial embryonic lethality correlated with aberrant centrosome behavior, cytoskeletal abnormalities, and defective gastrulation. Although ubiquitously distributed, both proteins are enriched in the primordial germ cells (PGCs), and in keeping with the centrosome problems, mutant embryos display a significant reduction in the PGC count. Moreover, the total number of pole buds is directly proportional to the level of Casp. Consistently, its loss and gain results in respective reduction and increase in the Oskar protein levels, the master determinant of PGC fate. To elucidate this regulatory loop, we analyzed several known components of mid-blastula transition and identify the translational repressor Smaug, a zygotic regulator of germ cell specification, as a potential critical target. We present a detailed structure-function analysis of Casp aimed at understanding its novel involvement during PGC development.

genetics↗

bioRxiv2024

Insulin signaling accelerates the anterograde movement of Rab4 vesicles in axons through Klp98a/KIF16B recruitment via Vps34-PI3Kinase

Rab4 GTPase organizes endosomal sorting essential for maintaining the balance between recycling and degradative pathways. Rab4 localizes to many cargos whose transport in neurons is critical for regulating neurotransmission and neuronal health. Furthermore, elevated Rab4 levels in the CNS are associated with synaptic atrophy and neurodegeneration in Drosophila and humans, respectively. However, how the transport of Rab4-associated vesicles is regulated in neurons remains unknown. Using in vivo time-lapse imaging of Drosophila larvae, we show that activation of insulin signaling via Dilp2 and dInR increases the anterograde velocity, run length, and flux of Rab4 vesicles in the axons. Molecularly, we show that activation of neuronal insulin signaling further activates Vps34, elevates the levels of PI(3)P on Rab4-associated vesicles, recruits Klp98A (a PI(3)P-binding kinesin-3 motor) and activates their anterograde transport. Together, these observations delineate the role of insulin signaling in regulating axonal transport and synaptic homeostasis. HighlightsO_LIDilp2-mediated insulin signaling activates anterograde transport of Rab4 vesicles C_LIO_LIVps34 regulates anterograde velocity, run length and flux of Rab4 vesicles C_LIO_LILocal PI(3)P signaling on Rab4 vesicles regulates their motility in the axons C_LIO_LIPI(3)P production upon acute insulin stimulation recruits Klp98A on Rab4 vesicles C_LI

cell biology↗

bioRxiv2024

PIP4K2C inhibition reverses autophagic flux impairment induced by SARS-CoV-2

In search for broad-spectrum antivirals, we discovered a small molecule inhibitor, RMC-113, that potently suppresses the replication of multiple RNA viruses including SARS-CoV-2 in human lung organoids. We demonstrated selective dual inhibition of the lipid kinases PIP4K2C and PIKfyve by RMC-113 and target engagement by its clickable analog. Advanced lipidomics revealed alteration of SARS-CoV-2-induced phosphoinositide signature by RMC-113 and linked its antiviral effect with functional PIP4K2C and PIKfyve inhibition. We discovered PIP4K2Cs roles in SARS-CoV-2 entry, RNA replication, and assembly/egress, validating it as a druggable antiviral target. Integrating proteomics, single-cell transcriptomics, and functional assays revealed that PIP4K2C binds SARS-CoV-2 nonstructural protein 6 and regulates virus-induced impairment of autophagic flux. Reversing this autophagic flux impairment is a mechanism of antiviral action of RMC-113. These findings reveal virus-induced autophagy regulation via PIP4K2C, an understudied kinase, and propose dual inhibition of PIP4K2C and PIKfyve as a candidate strategy to combat emerging viruses.

microbiology↗

bioRxiv2024

Ether lipids influence cancer cell fate by modulating iron uptake

Cancer cell fate has been widely ascribed to mutational changes within proteincoding genes associated with tumor suppressors and oncogenes. In contrast, the mechanisms through which the biophysical properties of membrane lipids influence cancer cell survival, dedifferentiation and metastasis have received little scrutiny. Here, we report that cancer cells endowed with high metastatic ability and cancer stem celllike traits employ ether lipids to maintain low membrane tension and high membrane fluidity. Using genetic approaches and lipid reconstitution assays, we show that these ether lipid-regulated biophysical properties permit non-clathrin-mediated iron endocytosis via CD44, resulting in significant increases in intracellular redox-active iron and enhanced ferroptosis susceptibility. Using a combination of in vitro threedimensional microvascular network systems and in vivo animal models, we show that loss of ether lipids from plasma membranes also strongly attenuates extravasation, metastatic burden and cancer stemness. These findings illuminate a mechanism whereby ether lipids in carcinoma cells serve as key regulators of malignant progression while conferring a unique vulnerability that can be exploited for therapeutic intervention.

cancer biology↗

bioRxiv2024

Post-GWAS functional analyses of CNTNAP5 suggests its role in glaucomatous neurodegeneration

Primary angle closure glaucoma (PACG) affects more than 20 million people worldwide, with an increased prevalence in south-east Asia. In a prior haplotype-based GWAS, we identified a novel CNTNAP5 genic region, significantly associated with PACG. In the current study, we have extended our perception of CNTNAP5 involvement in glaucomatous neurodegeneration in a zebrafish model, through investigating phenotypic consequences pertinent to retinal degeneration upon knockdown of cntnap5 by translation-blocking morpholinos. While cntnap5 knockdown was successfully validated using an antibody, immunofluorescence followed by western blot analyses in cntnap5-morphant (MO) zebrafish revealed increased expression of acetylated tubulin indicative of perturbed cytoarchitecture of retinal layers. Moreover, significant loss of Nissl substance is observed in the neuro-retinal layers of cntnap5-MO zebrafish eye, indicating neurodegeneration. Additionally, in spontaneous movement behavioural analysis, cntnap5-MO zebrafish have a significantly lower average distance traversed in light phase compared to mismatch-controls, whereas no significant difference was observed in the dark phase, corroborating with vision loss in the cntnap5-MO zebrafish. This study provides the first direct functional evidence of a putative role of CNTNAP5 in visual neurodegeneration.

genetics↗

bioRxiv2024

Discovery of free glycated amines and glycated urea as potential markers of diabetic nephropathy

The role of protein glycation in the pathogenesis of diabetes is well established. Akin to proteins, free amino acids and other small-molecule amines are also susceptible to glycation in hyperglycemic conditions and may have a role in the pathogenesis of the disease. However, the information on glycation of free amino acids and other small- molecule amines is relatively obscure. In the quest to discover small molecule glycated amines in the plasma, we have synthesized glycated amino acids, glycated creatine, and glycated urea, and by using a high-resolution accurate mass spectrometer, a mass spectral library was developed comprising the precursor and predominant fragment masses of glycated amines. Using this information, we report the discovery of glycation of free lysine, arginine, and leucine/isoleucine from the plasma of diabetic patients. This has great physiological significance as glycation of these amino acids may create their deficiency and affect vital physiological processes such as protein synthesis, cell signaling and insulin secretion. Also, these glycated amino acids could serve as potential markers of diabetes and its complications. On the other hand, accumulation of creatinine and urea in the plasma act as biomarkers of diabetic nephropathy diagnosis. For the first time, we report the detection of glycated urea in diabetes, which is confirmed by matching the precursor and fragment masses with the in vitro synthesized glycated urea by using 12C6 and 13C6-glucose. Further, we quantified glycated urea detected in two forms, monoglycated urea (MGU) and diglycated urea (DGU), by a targeted mass spectrometric approach in the plasma of healthy, diabetes, and diabetic nephropathy subjects. Both MGU and DGU showed a positive correlation with clinical parameters such as blood glucose and HbA1c. Given that urea gets converted to glycated urea in hyperglycemic conditions, it is crucial to quantify MGU and DGU along with the urea for the diagnosis of diabetic nephropathy.

biochemistry↗