Biology subjects

Aggarwal, S.

Publications and source records attributed to Aggarwal, S..

At least 37 records · Page 2Linked to original sources

bioRxiv2023

A large-scale volumetric correlated light and electron microscopy study localizes Alzheimer's disease-related molecules in the hippocampus

Connectomics using volume-electron-microscopy enables mapping and analysis of neuronal networks, revealing insights into neural circuit function and dysfunction. In Alzheimers disease (AD), where amyloid-{beta} (A{beta}) and hyperphosphorylated-Tau (pTau) are implicated, connectomics offers an approach to unravel how these molecules contribute to circuit alterations by enabling the study of these molecules within the context of the complete local neuronal and glial milieu. We present a volumetric-correlated-light-and-electron microscopy (vCLEM) protocol using fluorescent nanobodies to localize A{beta} and pTau within a large-scale connectomics dataset from the hippocampus of the 3xTg AD mouse model. A key outcome of this work is a publicly accessible vCLEM dataset, featuring fluorescent labeling of A{beta} and pTau in the ultrastructural context with segmented neurons, glia, and synapses. This dataset provides a unique resource for exploring AD pathology in the context of connectomics and fosters collaborative opportunities in neurodegenerative disease research. As a proof-of-principle, we uncovered new localizations of A{beta} and pTau, including pTau-positive spine-like protrusions at the axon initial segment and changes in the number and size of synapses near A{beta} plaques. Our vCLEM approach facilitates the discovery of both molecular and structural alterations within large-scale EM data, advancing connectomics research in Alzheimers and other neurodegenerative diseases.

neuroscience↗

bioRxiv2023

EZH2 deletion does not impact acinar cell regeneration but restricts progression to pancreatic cancer in mice

Enhancer of Zeste Homologue 2 (EZH2) is part of the Polycomb Repressor Complex 2, which induces trimethylation of lysine 27 on histone 3 (H3K27me3) and promotes genes repression. EZH2 is overexpressed in many cancers including pancreatic ductal adenocarcinoma (PDAC). Previous studies in mice attributed both pro-oncogenic and tumor suppressive functions to EZH2. Deletion of the EZH2 enhances de novo KRAS-driven neoplasia following pancreatic injury by preventing acinar cell regeneration, while increased EZH2 expression in PDAC is correlated to poor prognosis, suggesting a context-dependant effect for EZH2 in PDAC progression. In this study, we examined EZH2 function in pre-and early neoplastic stages of PDAC. Using an inducible model to generate deletion of EZH2 only in adult acinar cells (EZH2{Delta}SET), we showed loss of EZH2 activity did not prevent acinar cell regeneration in the absence of oncogenic KRAS (KRASG12D), nor lead to increased PanIN formation in the presence of KRASG12D in adult mice. However, loss of EZH2 did reduce recruitment of inflammatory cells and, when combined with a PDAC model, promoted widespread PDAC progression. Loss of EZH2 function also correlated to remodeling of the tumor microenvironment, which favors cancer cell progression. This study suggests expression of EZH2 in adult acinar cells restricts PDAC initiation and progression by affecting both the tumour microenvironment and acinar cell differentiation.

cancer biology↗

bioRxiv2023

Sequential early-life viral infections modulate the microbiota and adaptive immune responses to systemic and mucosal vaccination

Increasing evidence points to the microbial exposome as a critical factor in maturing and shaping the host immune system, thereby influencing responses to immune challenges such as infections or vaccines. To investigate the effect of early-life viral exposures on immune development and vaccine responses, we inoculated mice with six distinct viral pathogens in sequence beginning in the neonatal period, and then evaluated their immune signatures before and after intramuscular or intranasal vaccination against SARS-CoV-2. Sequential viral infection drove profound changes in all aspects of the immune system, including increasing circulating leukocytes, altering innate and adaptive immune cell lineages in tissues, and markedly influencing serum cytokine and total antibody levels. Beyond these immune responses changes, these exposures also modulated the composition of the endogenous intestinal microbiota. Although sequentially-infected mice exhibited increased systemic immune activation and T cell responses after intramuscular and intranasal SARS-CoV-2 immunization, we observed decreased vaccine-induced antibody responses in these animals. These results suggest that early-life viral exposures are sufficient to diminish antibody responses to vaccination in mice, and highlight their potential importance of considering prior microbial exposures when investigating vaccine responses.

microbiology↗

bioRxiv2023

Profiling human hypothalamic neurons reveals a candidate combination drug therapy for weight loss

Obesity substantially increases the risk of type 2 diabetes, cardiovascular disease, and other diseases, making it a leading preventable cause of death in developed countries. It has a strong genetic basis, with obesity-associated genetic variants preferentially acting in the brain. This includes the hypothalamic pro-opiomelanocortin (POMC) neurons that inhibit food intake and are stimulated by drugs that agonise glucagon-like 1 peptide receptor (GLP1R) including Semaglutide (Ozempic/Wegovy). We therefore hypothesised that drugs which selectively activate human POMC neurons would suppress appetite and promote weight loss, and that focusing on drugs already approved for use would facilitate rapid clinical translation. We therefore generated POMC neurons from human pluripotent stem cells (hPSCs) and identified enriched genes that were genetically associated with obesity and targeted by approved drugs. We found that human POMC neurons are enriched in GLP1R, reliably activated by Semaglutide, and their responses are further increased by co-administration of Ceritinib, an FDA-approved drug potently and selectively inhibiting anaplastic lymphoma kinase (ALK). Ceritinib reduced food intake and body weight in obese but not lean mice, and upregulated the expression of GLP1R in the mouse hypothalamus and hPSC-derived human hypothalamic neurons. These studies reveal a new potential therapeutic strategy for reducing food intake and body weight, and demonstrate the utility of hPSC-derived hypothalamic neurons for drug discovery.

neuroscience↗

bioRxiv2023

Metformin may reduce dementia risk through neuroprotection not mitigation of diabetes

Dementia is a largely untreatable syndrome that is epidemiologically associated with metabolic diseases such as type 2 diabetes (T2D) and obesity. Drugs used to treat T2D such as metformin are inexpensive, safely given to millions of people, and have also been reported to slow neurodegeneration. We hypothesised that the neuroprotective benefits of metformin might extend to metabolically healthy individuals and tested this hypothesis in a mouse prion model that recapitulates key features of human neurodegenerative disease, including synaptic loss and motor impairment. These features and the time course of this model (24 weeks) allows the effects of metabolic risk factors and metformin to be tested and potentially generalised to other forms of neurodegenerative disease. Mice fed a high fat diet (HFD) developed high adiposity with impaired glucose and insulin homeostasis, similar to the effects of chronic obesity seen in humans whereas mice on matched control diet (CD) remain metabolically healthy. Chronic treatment with metformin in HFD-fed mice significantly increased survival and health span relative to vehicle-treated mice. Mice fed a HFD also had a modestly extended health span relative to mice fed CD, as measured by development of motor signs of prion disease. Metformin also significantly extended health span in metabolically healthy CD-fed mice. Using targeted mass spectrometry, we found that metformin reached deep brain structures at functional concentrations, driving a reduction in pPERK and changing the activity of microglia in vivo. Metformin was able to alter ER stress pathways at the same concentrations in healthy animals and using human iPSC-derived microglia and mouse organotypic slices we show that the action of metformin at these concentrations does not require a systemic mechanism, necessary for the treatment of diabetes, and is likely a result of direct secondary pharmacology in the brain. Together, these data broadly support the premise of repurposing metformin for neuroprotection, even in metabolically healthy individuals.

neuroscience↗

bioRxiv2023

Quality control and annotation of variant peptides identified through proteogenomics

Variant peptides resulting from translation of single nucleotide polymorphisms (SNPs) can lead to aberrant or altered protein functions and thus hold translational potential for disease diagnosis, therapeutics and personalized medicine. Variant peptides detected by proteogenomics are fraught with high number of false positives. Class-specific FDR along with ad-hoc post-search filters have been employed to tackle this issue, but there is no uniform and comprehensive approach to assess variant quality. These protocols are mostly manual or tedious, and not accessible across labs. We present a software tool, PgxSAVy, for the quality control of variant peptides. PgxSAVy provides a rigorous framework for quality control and annotations of variant peptides on the basis of (i) variant quality, (ii) isobaric masses, and (iii) disease annotation. PgxSAVy was able to segregate true and false variants with 98.43% accuracy on simulated data. We then used [~]2.8 million spectra (PXD004010 and PXD001468) and identified 12,705 variant PSMs, of which PgxSAVy evaluated 3028 (23.8%), 1409 (11.1%) and 8268 (65.1%) as confident, semi-confident and doubtful respectively. PgxSAVy also annotates the variants based on their pathogenicity and provides support for assisted manual validation. In these datasets, it identified previously found variants as well some novel variants not seen in original studies. The confident variants identified the importance of mutations in glycolysis and gluconeogenesis pathways in Alzheimers disease. The analysis of proteins carrying variants can provide fine granularity in discovering important pathways. PgxSAVy will advance personalized medicine by providing a comprehensive framework for quality control and prioritization of proteogenomics variants. AvailabilityPgxSAVy is freely available at https://github.com/anuragraj/PgxSAVy Key PointsO_LIVariant peptide in proteogenomics have high rates of false positives C_LIO_LIclass-specific FDR is not sufficiently effective, and tedious manual filtering is not scalable C_LIO_LIWe developed PgxSAVy for automated quality control and disease annotation of variant peptides from proteogenomics search results C_LIO_LIPgxSAVy was validated using simulation data and manually annotated variant PSMs C_LIO_LIIndependent application on large datasets on Alzheimers and HEK cell lines demonstrated that PgxSAVy discovered known and novel mutations with important biological roles. C_LI Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=161 HEIGHT=200 SRC="FIGDIR/small/542998v2_ufig1.gif" ALT="Figure 1"> View larger version (39K): org.highwire.dtl.DTLVardef@187a515org.highwire.dtl.DTLVardef@67471corg.highwire.dtl.DTLVardef@6d90e9org.highwire.dtl.DTLVardef@144c6c1_HPS_FORMAT_FIGEXP M_FIG C_FIG

bioinformatics↗

bioRxiv2023

Xiphoid nucleus of the midline thalamus controls cold-induced food seeking

Maintaining body temperature is calorically expensive for endothermic animals. Mammals eat more in the cold to compensate for energy expenditure, but the neural mechanism underlying this coupling is not well understood. Through behavioral and metabolic analyses, we found that mice dynamically switch between energy conservation and food-seeking states in the cold, the latter of which is primarily driven by energy expenditure rather than the sensation of cold. To identify the neural mechanisms underlying cold-induced food seeking, we use whole-brain cFos mapping and found that the xiphoid (Xi), a small nucleus in the midline thalamus, was selectively activated by prolonged cold associated with elevated energy expenditure but not with acute cold exposure. In vivo calcium imaging showed that Xi activity correlates with food-seeking episodes in cold conditions. Using activity-dependent viral strategies, we found that optogenetic and chemogenetic stimulation of cold-activated Xi neurons recapitulated cold-induced feeding, whereas their inhibition suppressed it. Mechanistically, Xi encodes a context-dependent valence switch promoting food-seeking behaviors in cold but not warm conditions. Furthermore, these behaviors are mediated by a Xi to nucleus accumbens projection. Our results establish Xi as a key region for controlling cold-induced feeding, an important mechanism for maintaining energy homeostasis in endothermic animals.

neuroscience↗

bioRxiv2023

Slope of the power spectral density flattens at low frequencies (<150 Hz) with healthy aging but also steepens at higher frequency (>200 Hz) in human electroencephalogram

Brain signals such as electroencephalogram (EEG) often show oscillations at various frequencies, which are represented as distinct "bumps" in the power spectral density (PSD) of these signals. In addition, the PSD also shows a distinct reduction in power with increasing frequency, which pertains to aperiodic activity and is often termed as the "1/f" component. While a change in periodic activity in brain signals with healthy aging and mental disorders has been reported, recent studies have shown a reduction in the slope of the aperiodic activity with these factors as well. However, these studies only analysed PSD slopes over a limited frequency range (<100 Hz). To test whether the PSD slope is affected over a wider frequency range with aging and mental disorder, we collected EEG data with high sampling rate (2500 Hz) from a large population of elderly subjects (>49 years) who were healthy (N=217) or had mild cognitive impairment (MCI; N=11) or Alzheimers Disease (AD; N=5), and analysed the PSD slope till 800 Hz. Consistent with previous studies, the 1/f slope up to ~150 Hz reduced with healthy aging. Surprisingly, we found the opposite at higher frequencies (>200 Hz): the slope increased with age. This result was observed in all electrodes, for both eyes open and eyes closed conditions, and for different reference schemes. Slopes were not significantly different in MCI/AD subjects compared to age and gender matched healthy controls. Overall, our results constrain the biophysical mechanisms that are reflected in the PSD slopes in healthy and pathological aging. Significance StatementAperiodic activity in the brain is characterized by measuring the slope of the power spectrum of brain signals. This slope has been shown to flatten with healthy aging, suggesting an increase in some sort of "neural noise". However, this flattening has been observed only over a limited frequency range (<150 Hz). We found that at higher frequencies (>200 Hz), the opposite happens: the slope steepens with age. This occurs at all electrodes, irrespective of state and referencing techniques. However, the slope is unchanged in subjects with early Alzheimers Disease (AD) and their controls. Our results shed new light on the properties of neural noise and the neurophysiological processes affecting AD and the aperiodic activity.

neuroscience↗

bioRxiv2022

Scalable early detection of grapevine virus infection with airborne imaging spectroscopy

Viral diseases, including Grapevine Leafroll-associated Virus Complex 3 (GLRaV-3), cause $3 billion in damages and losses to the United States wine and grape industry annually. GLRaV-3 has a well-studied, year-long latent period in which vines are infectious but do not yet display visible symptoms, making it an ideal model pathosystem to evaluate the scalability of symptomatic and asymptomatic imaging spectroscopy-based disease detection. Plant disease causes physiological and chemical changes to occur locally and systemically throughout a plant, which imaging spectroscopy can detect both directly and indirectly. Reliable and scalable disease detection during the latent period would greatly reduce management costs, as current detection methods are entirely ground-based, labor-intensive, and expensive. Here, we use data collected in September 2020 by the NASA Airborne Visible/Infrared Imaging Spectrometer Next Generation (AVIRIS-NG) to detect GLRaV-3 in Cabernet Sauvignon grapevines in Lodi, CA. During September 2020 and 2021, industry collaborators scouted 317 acres of Vitis vinifera winegrapes for visible disease symptoms, and collected a subset for confirmation molecular testing at a commercial facility. Grapevines identified as visibly diseased in 2021 were assumed to have been latently infected (asymptomatic) during the September 2020 AVIRIS-NG data collection. We combined random forest with synthetic minority oversampling technique (SMOTE) to train multiple spectral models able to distinguish between non-infected (NI) and GLRaV-3-infected grapevines. We observed clear spectral differences that allowed for differentiation between NI and GLRaV-3 infected vines both pre- and post-symptomatically at 1m through 5m resolution. Our two best performing models had 87% accuracy (0.73 Kappa) distinguishing between NI and asymptomatic (aSy), and 85% accuracy (0.71 Kappa) distinguishing between NI and (aSy + symptomatic [Sy]) respectively. We hypothesize these spectral differences are linked to changes in overall plant physiology induced by disease, as visible foliar symptoms were restricted to the lower canopy. HighlightsO_LIAirborne imaging spectroscopy allows for scalable early-detection models of grapevine leafroll-associated virus complex 3 (GLRaV-3). C_LIO_LIRandom Forest based models trained with scouting ground data and imaging spectroscopy are accurate up to 5 meter but perform best at 3 meter spatial resolution. C_LIO_LIGLRaV-3 detection via imaging spectroscopy will not replace existing field scouting strategies or molecular testing but supplement by allowing for more strategic resource deployment to improve the overall financial, environmental, and societal sustainability of winegrape production. C_LI

pathology↗

bioRxiv2022

Fluorescence lifetime enables high-resolution analysis of neuromodulator dynamics across time and animals

The dynamics of neuromodulators are essential for their functions. Optical sensors have transformed the study of neuromodulators because they capture neuromodulator dynamics with high spatial and temporal resolution. However, fluorescence intensity-based sensors are restricted to measure acute changes within one animal over a short period because intensity varies with sensor expression level and excitation light fluctuation. In contrast, fluorescence lifetime is impervious to sensor expression level or excitation light power, allowing comparison between individuals and across long periods. Here, we discover fluorescence lifetime response in multiple intensity-based neuromodulator sensors. Using the acetylcholine sensor GRABACh3.0 to investigate the power of lifetime measurement, we find that fluorescence lifetime correlates with animal behavior states accurately despite varying excitation power or changes in sensor expression level across weeks and animals. Thus, fluorescence lifetime of neuromodulator sensors enables comparison of neuromodulator dynamics at high resolution between animals and for chronic time scales.

neuroscience↗

bioRxiv2022

An ensemble method for designing phage-based therapy against bacterial infections

Phage therapy is a viable alternative to antibiotics for treating microbial infections, particularly managing drug-resistant strains of bacteria. One of the major challenges in designing phage based therapy is to identify the most appropriate phage to treat a bacterial infection. In this study, an attempt has been made to predict phage-host interaction with high accuracy to identify the best virus for treating a bacterial infection. All models have been developed on a training dataset containing 826 phage host-interactions, whereas models have been evaluated on a validation dataset comprising 1201 phage-host interactions. Firstly, alignment based models have been developed using similarity between phage-phage (BLASTPhage), host-host (BLASTHost) and phage-CRISPR (CRISPRPred) where we achieved accuracy between 42.4%-66.2% for BLASTPhage, 55%-78.4% for BLASTHost, and 43.7%-80.2% for CRISPRPred at five taxonomic levels. Secondly, alignment free models have been developed using machine learning techniques. Thirdly, hybrid models have been developed by integrating alignment-free models and similarity-score where we achieved maximum performance of (60.6%-93.5%). Finally, an ensemble model has been developed that combines hybrid and alignment based model. Our ensemble model achieved highest accuracy of 67.9%, 80.6%, 85.5%, 90%, 93.5% at Genus, Family, Order, Class and Phylum levels, which is better than existing methods. In order to serve the scientific community we have developed a webserver named PhageTB and standalone software package (https://webs.iiitd.edu.in/raghava/phagetb/). Key PointsO_LIPhage therapy provides an alternative to mange drug resistant strains of bacteria C_LIO_LIPrediction bacterial strains that can be treated by a given phage C_LIO_LIAlignment-based, alignment-free and ensemble models have been developed. C_LIO_LIPrediction of appropriate phage/virus that can lyse a given strain of bacteria. C_LIO_LIWebserver and standalone package provided to predict phage-host interactions. C_LI

bioinformatics↗

bioRxiv2022

Redox sensing by SENP1 augments insulin secretion early after high-fat feeding in mice

Pancreatic {beta}-cells respond to metabolic stress by upregulating insulin secretion, however the underlying mechanisms remain unclear. In {beta}-cells from overweight humans without diabetes, and mice fed a high-fat diet for 2 days, insulin exocytosis and secretion are enhanced without increased Ca2+ influx. {beta}-cell RNA-seq suggests altered metabolic pathways early following HFD, where we find increased basal oxygen consumption, proton leak, but a more reduced cytosolic redox state. Increased {beta}-cell exocytosis after 2-day HFD is dependent on this reduced intracellular redox and requires the sentrin-specific SUMO-protease-1 (SENP1). Mice with either pancreas- or {beta}-cell-specific SENP1 deletion fail to up-regulate exocytosis and become rapidly glucose intolerant after 2-day HFD. Mechanistically, redox-sensing by SENP1 requires a thiol group at C535 which together with Zn+-binding suppresses basal protease activity and unrestrained {beta}-cell exocytosis and increases SENP1 sensitivity to regulation by redox signals.

cell biology↗

bioRxiv2021

A reference induced pluripotent stem cell line for large-scale collaborative studies

Human induced pluripotent stem cell (iPSC) lines are a powerful tool for studying development and disease, but the considerable phenotypic variation between lines makes it challenging to replicate key findings and integrate data across research groups. To address this issue, we sub-cloned candidate iPSC lines and deeply characterised their genetic properties using whole genome sequencing, their genomic stability upon CRISPR/Cas9-based gene editing, and their phenotypic properties including differentiation to commonly-used cell types. These studies identified KOLF2.1J as an all-around well-performing iPSC line. We then shared KOLF2.1J with groups around the world who tested its performance in head-to-head comparisons with their own preferred iPSC lines across a diverse range of differentiation protocols and functional assays. On the strength of these findings, we have made KOLF2.1J and hundreds of its gene-edited derivative clones readily accessible to promote the standardization required for large-scale collaborative science in the stem cell field. SummaryThe authors of this collaborative study deeply characterized human induced pluripotent stem cell (iPSC) lines to rationally select a clonally-derived cell line that performs well across multiple modalities. KOLF2.1J was identified as a candidate reference cell line based on single-cell analysis of its gene expression in the pluripotent state, whole genome sequencing, genomic stability after highly efficient CRISPR-mediated gene editing, integrity of the p53 pathway, and the efficiency with which it differentiated into multiple target cell populations. Since it is deeply characterized and can be readily acquired, KOLF2.1J is an attractive reference cell line for groups working with iPSCs. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=200 SRC="FIGDIR/small/472643v6_ufig1.gif" ALT="Figure 1"> View larger version (84K): org.highwire.dtl.DTLVardef@cef106org.highwire.dtl.DTLVardef@31d5d8org.highwire.dtl.DTLVardef@1ce6d8corg.highwire.dtl.DTLVardef@17a19d2_HPS_FORMAT_FIGEXP M_FIG C_FIG

neuroscience↗

bioRxiv2021

Erythroid spectrin binding modulates peroxidase and catalase activity of heme proteins

Hemoglobin oxidation due to oxidative stress and disease conditions leads to generation of ROS (reactive oxygen species) and membrane attachment of hemoglobin in-vivo, where its redox activity leads to peroxidative damage of membrane lipids and proteins. Spectrin, the major component of the RBC membrane skeleton, is known to interact with hemoglobin and, here this interaction is shown to increase hemoglobin peroxidase activity in the presence of reducing substrate ABTS (2, 2-Azino-Bis-3-Ethylbenzothiazoline-6-Sulfonic Acid). It is also shown that in the absence of reducing substrate, spectrin forms covalently cross-linked aggregates with hemoglobin which display no peroxidase activity. This may have implications in the clearance of ROS and limiting peroxidative damage. Spectrin is found to modulate the peroxidase activity of different hemoglobin variants like A, E, and S, and of isolated globin chains from each of these variants. This may be of importance in disease states like sickle cell disease and HbE-{beta}-thalassemia, where increased oxidative damage and free globin subunits are present due to the defects inherent in the hemoglobin variants associated with these diseases. This hypothesis is corroborated by lipid peroxidation experiments. The modulatory role of spectrin is shown to extend to other heme proteins, namely catalase and cytochrome-c. Experiments with free heme and Raman spectroscopy of heme proteins in the presence of spectrin show that structural alterations occur in the heme moiety of the heme proteins on spectrin binding, which may be the structural basis of increased enzyme activity.

biochemistry↗

bioRxiv2021

Structural heterogeneity in biliverdin modulates spectral properties of Sandercyanin fluorescent protein

Sandercyanin, a blue homo-tetrameric lipocalin protein purified from Canadian walleye (Stizostedion vitreus), is the first far-red fluorescent protein reported in vertebrates(1-3). Sandercyanin binds non-covalently to biliverdin IX (BLA) and fluoresces at 675nm on excitation at 375nm and 635nm(1). Sandercyanin fluorescence can be harnessed for many in vivo applications when engineered into a stable monomeric form. Here, we report the spectral properties and crystal structures of engineered monomeric Sandercyanin-BLA complexes. Compared to wild-type protein, monomeric Sandercyanin ([~]18kDa) binds BLA with similar affinities and show a broad red-shifted absorbance spectra but possess reduced quantum efficiency. Crystal structures reveal D-ring pyrrole of BLA rotated around the C14-C15 bond, which is stabilized by neighboring aromatic residues and increased water-mediated polar contacts in the BLA-binding pocket. A tetrameric Sandercyanin variant (Tyr-142-Ala) co-displaying red- and far-red absorbing states, and reduced fluorescence shows similar conformational changes in BLA binding pocket. Our results suggest that D-ring flexibility of BLA and its rearrangement reduces the fluorescence quantum-yield of monomeric Sandercyanin. Structures of monomeric Sandercyanin could be utilized as prototypes to generate bright BLA-inducible fluorescent proteins. Further, our study postulates a mechanism for modulating photo-states in BLA-bound lipocalins, known only in phytochromes till date. Significance StatementSandercyanin is a tetrameric red fluorescent protein from a blue variant of walleye (Stizostedion vitreum) that binds to biliverdin IX (BLA). Its biophysical properties and structures have been published earlier(1). A bright and stable monomeric Sandercyanin could be utilized as a fusion protein for fluorescence-based applications. Here we report the first structures and spectral properties of fluorescent monomeric Sandercyanin-BLA complexes and describe the molecular basis of modulated spectral properties due to rotated D-ring pyrrole around C14-C15 bond and re-shuffling of BLA-binding pocket. BLA-bound monomeric Sandercyanin could be engineered into brighter variants for in-vivo applications. Our study also reveals an unfamiliar mechanism in BLA-binding lipocalins that regulates red- and far-red absorbance states.

bioengineering↗

bioRxiv2021

Immunological and cardio-vascular pathologies associated with SARS-CoV-2 infection in golden syrian hamster

Severe acute respiratory syndrome coronavirus (SARS-CoV)-2 infection in golden Syrian hamster (GSH) causes lung pathology and resembles human coronavirus disease (Covid-19). However, extra-pulmonary pathologies of SARS-CoV-2 infection that result in long Covid remains undefined in GSH. Here, using in silico modelling we show that hamster angiotensin-converting enzyme-2 (ACE-2) and neuropilin-1 (NRP-1) interaction with SARS-CoV-2 is similar to human. Intranasal SARS-CoV-2 infection in GSH resulted in early onset of lung pathologies marked by aggressive inflammatory response. Remarkably, late phase of SARS-CoV2 infection in GSH showed cardiovascular complications (CVC) characterized by ventricular hypertrophy, ventricular wall thickening, interstitial coronary fibrosis and altered lipidomics with elevated cholesterol, low-density lipoprotein and long chain fatty acid triglycerides. Moreover, serum metabolomics profile of infected GSH correlated with Covid19 patients. Together, we propose GSH as a suitable animal model to study immediate and long Covid19 pathologies that could be extended to therapeutics against Covid19 related CVC.

microbiology↗

bioRxiv2021

Secretome of senescent hepatoma cells modulate macrophage polarization and neutrophil extracellular traps formation

Presence of dysfunctional senescent hepatocyte is a hallmark feature of cirrhosis. We now report the presence of senescent hepatocytes (p21 and p53 positive) in vicinity of infiltrated immune cells in hepatocellular carcinoma. Hence, we checked if senescent cells can alter fate of macrophage polarization and neutrophil extracellular trap (NETs) formation. Using an in vitro assay, senescence was induced in hepatoma cells (HepG2 and Huh7 cells) by doxorubicin treatment and senescent cell showed secretory phenotype with strong expression of cytokines (IL1{beta}, IL6, IL8 and IL13) as evaluated by Flow cytometry. The senescent secretome from hepatoma cells induced macrophage differentiation predominantly with M2 markers (CD80, CD86) while that of non-senescent cell induced M1 phenotype (CD163, CD206) as analysed by flow cytometry. Human hepatocellular carcinoma harbouring senescent hepatocytes showed presence of M2 macrophages, while M1 macrophages were predominant in non-tumorous region. Additionally, the senescent secretome from Huh7 cells (p53mut) enhanced the NETs formation, while HepG2 (p53+/+) secretome had an inhibitory affect In conclusion, the "pro-inflammatory" senescent secretome drives non-inflammatory type M2 macrophage polarization and modulate neutrophil traps thereby modulating the microenvironment towards tumor promotion. Targeting senescent hepatocyte secretome appears a promising therapeutic target in liver cancer in future. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=126 SRC="FIGDIR/small/423905v1_ufig1.gif" ALT="Figure 1"> View larger version (27K): org.highwire.dtl.DTLVardef@1df4256org.highwire.dtl.DTLVardef@7c4f53org.highwire.dtl.DTLVardef@13bfe80org.highwire.dtl.DTLVardef@1c1c4b5_HPS_FORMAT_FIGEXP M_FIG Work Highlight (Diagrammatic Representation). C_FIG

cancer biology↗

bioRxiv2020

Site-specific Crosslinking Coupled with Mass Spectrometry as a Structural Tool in Studies of the Human α1 Glycine Receptor

Recent advances in mass spectrometry coupled with chemical crosslinking (CX-MS) can be applied for the structural interrogation of macromolecular complexes to identify statedependent distance constraints and provides a powerful complementary technique to other structural methods. In this study, we develop a CX-MS approach to identify the sites of crosslinking from a single targeted location within the human glycine receptor (GlyR) in a single apo/resting state. The GlyR belongs to the family of pentameric ligand-gated ion channel receptors that function in fast neuronal transmission. A single cysteine residue was re-introduced into Cys null GlyR construct at position 41 within the extracellular domain of an overexpressed human a1 homomeric GlyR. After purification and reconstitution into vesicles, a methanethiosulfonate benzophenone heterobifunctional crosslinker was attached via formation of a disulfide bond, and the resting receptor is subsequently photocrosslinked. Monomeric and oligomeric bands from SDS-PAGE gels were then trypsinized and analyzed by tandem MS in bottom-up studies. Dozens of intra- and inter-subunit sites of crosslinking were differentiated and identified from single gel bands (pmols of purified GlyR), showing the utility of this approach to identify a diverse array of distance constraints of GlyR in its resting state. These studies highlight the potential of CX-MS as an experimental approach to identify state-dependent crosslinks of full length integral membrane protein assemblies in a native-like lipid environment.Competing Interest StatementThe authors have declared no competing interest.View Full Text

biophysics↗