Biology subjects

Maity, S.

Publications and source records attributed to Maity, S..

At least 37 records · Page 2Linked to original sources

bioRxiv2025

Antibody-Free Immunopeptide Nano-Conjugates for Brain-Targeted Drug Delivery in Glioblastoma Multiforme

Glioblastoma Multiforme (GBM) represents a significant clinical challenge amongst central nervous system (CNS) tumors, with a dismal mean survival rate of less than 8 months, a statistic that has remained largely unchanged for decades (National Brain Society, 2022). The specialized intricate anatomical features of the brain, notably the blood-brain barrier (BBB), pose significant challenges to effective therapeutic interventions, limiting the potential reach of modern advancements in immunotherapy to impact these types of tumors. This study introduces an innovative, actively targeted immunotherapeutic nanoconjugate (P12/AP-2/NCs) designed to serve as an immunotherapeutic agent capable of traversing the BBB via LRP-1 receptor-mediated transcytosis. P12/AP-2/NCs exert its immune-modulating effects by inhibiting the PD-1/PD-L1 axis through a small-size PD-L1/ PD-L2 antagonist peptide Aurigene NP-12 (P12). P12/AP-2/NCs are synthesized from completely biodegradable, functionalized high molecular weight {beta}-poly(L-malic acid) (PMLA) polymer, conjugated with P12 and Angiopep-2 (AP2) to yield P12/AP-2/NCs. Evaluating nanoconjugates for BBB permeability and 3-D tumor model efficacy using an in vitro BBB-Transwell spheroid based model demonstrating successful crossing of the BBB and internalization in brain 3D tumor environments. In addition, the nanoconjugate mediated T cells cytotoxicity on 3D tumor region death in a U87 GBM 3-D spheroid model. AP2/P12/NCs is selectively inhibited in PD1/PDL1 interaction on T cells and tumor site, increasing inflammatory cytokine secretion and T cell proliferation. In an in-vivo murine brain environment, rhodamine fluorophore-labeled AP2/P12/NCs displayed significantly increased accumulation in the brain during 2-6 h time intervals post-injection with a prolonged bioavailability over unconjugated peptides. AP2/P12/NCs demonstrated a safety profile at both low and high doses based on major organ histopathology evaluations. Our findings introduce a novel, programmable nanoconjugate platform capable of penetrating the BBB for directed delivery of small peptides and significant immune environment modulation without utilizing antibodies, offering promise for treating challenging brain diseases like glioblastoma multiforme and beyond.

immunology↗

bioRxiv2025

Inhibition of methylthioadenosine phosphorylase provides protection from experimental acute kidney injury

Acute kidney injury (AKI) increases mortality risk and predisposes individuals to chronic kidney disease. Metabolic pathways play a crucial role in AKI pathophysiology. Here, we investigate the potential of methylthioadenosine phosphorylase (MTAP) inhibition as a novel renoprotective strategy in AKI. Using AKI mouse models, we demonstrate that a small molecule MTAP inhibitor significantly reduces kidney injury markers and improves renal histology. RNA sequencing reveals that MTAP inhibition modulates pathways associated with inflammation, oxidative phosphorylation, and cell survival. Additionally, analysis of human single-cell RNA sequencing data links MTAP expression to kidney injury marker in AKI. This study provides evidence of MTAP inhibition as a potential therapeutic strategy for AKI, highlighting metabolic dysregulation as a target for future clinical interventions.

pathology↗

bioRxiv2024

Direct observation of secondary nucleation in huntingtin amyloid formation by High-Speed Atomic Force Microscopy

Amyloid fibril formation is a hallmark of various neurodegenerative diseases such as Huntingtons (HD), Alzheimers and Parkinsons disease. The protein aggregation process involves slow nucleation events followed by rapid growth and elongation of formed fibrils. Understanding the pathways of amyloid formation is key to development of novel therapeutic agents that can interfere with the pathogenic protein misfolding events. Recent studies of aggregation by polypeptides from Alzheimers and Huntingtons disease have identified the importance of a poorly understood secondary nucleation process that may even be the dominant source of protein aggregate formation. Here we focus on the polyglutamine-expansion disorder HD and employ mechanistic and structural studies to study different aspects of secondary nucleation in the aggregation of huntingtin Exon 1 (HttEx1). Notably, we apply high-speed atomic force microscopy (HS-AFM) to directly observe the process on the single-particle level and in real time. Our observations show unique features of the amyloid formation dynamics in real time, including secondary nucleation, elongation and the formation of large bundles of fibrils as a result of nucleated branching. We examine the role of HttEx1 flanking segments during the aggregation process, revealing that the N-terminal HttNT segment exhibits a clear primary nucleation-aggregation-enhancing ability, however, that it does not seem to induce or affect the secondary nucleation process. The obtained results illuminate the complex aggregation process of HttEx1 and have implications for attempts to inhibit or modulate it for therapeutic purposes.

molecular biology↗

bioRxiv2024

Adipocyte progenitors are primary contributors to the disrupted epithelial niche that is sustained following abrupt mammary gland involution

A short duration of breastfeeding is a risk factor for the development of high-mortality, postpartum, triple-negative breast cancer. The intrinsic properties of cancer-initiating epithelial cells that persist following breastfeeding cessation and mammary gland remodeling are poorly understood. Previously, we showed that Platelet-Derived Growth Factor Receptor alpha (PDGFR)-expressing stromal mammary adipocyte progenitors (MAPs) differentiate into epithelial luminal progenitors in the adult gland. In the current study, we demonstrate that MAP-derived luminal progenitors retain a mesenchymal transcriptomic signature. In an abrupt involution model that mimics a short breastfeeding duration, MAP-derived luminal progenitors persist and dominate luminal epithelia, undergoing transcriptomic alterations that signify a distinct ferrometabolic state linked to cancer. Concurrently, MAPs adopt an alternative interferon-mediated profibrotic and invasive stromal fate. Our work uncovers MAPs to be the primary cellular origin of a pathological stromal and epithelial microenvironment following abrupt involution, presenting a potential therapeutic target in postpartum breast cancer.

cancer biology↗

bioRxiv2024

An alternative mechanism for activation of innate immune signaling by MDA5

Long double-stranded (ds) RNA in the cytosol acts as a potent inflammatory molecule recognized by the receptor MDA5, triggering the innate immune response. Mutations affecting MDA5 ATPase activity lead to severe pathological conditions. MDA5 nucleoprotein filament assembly-disassembly dynamic is proposed to regulate dsRNA recognition, though the exact mechanism remains unclear. Here, we employed magnetic tweezers to monitor the assembly and manipulate MDA5 filaments at the single dsRNA level. Following a slow nucleation event, MDA5 assembles cooperatively and directionally into (partial) filaments and utilizes ATP hydrolysis to compact dsRNA via single-stranded (ss) RNA loop extrusion, even against a significant opposing force. This compacted state is further stabilized by oligomerization of MDA5s caspase recruitment domain (CARD) and requires high force to be disrupted. ssRNA gaps impaired compaction, suggesting a new mechanism for dsRNA recognition. We propose that MDA5 mediated dsRNA compaction captures viral dsRNA, preventing further usage for viral replication.

biophysics↗

bioRxiv2024

Microphysiological system modeling pericyte-induced temozolomide resistance in glioblastoma

Glioblastoma (GBM) is a malignancy with poor survival and high rates of chemoresistance. Temozolomide (TMZ), the standard-of-care chemotherapy for GBM patients, but GBM cells can be resistant to TMZ, resulting in limited clinical efficacy. Elucidating the complex mechanisms of TMZ chemoresistance in GBM requires novel in vitro models replicating the complex tumor microenvironment (TME). We present an multicellular 3D GBM model recapitulating the biomechanical characteristics of brain tissues and pericyte-mediated TMZ resistance. The composite hydrogel used to encapsulate GBM spheroids (U87, LN229, and PDM140), pericytes, or GBM spheroids with pericytes, mimics the rheological properties of brain tissues (G[~]800Pa and G"[~]100Pa). When untreated, the GBM models remain viable and proliferative for 14 days. PDM140 spheroids were most sensitive to TMZ (IC50=73M), followed by LN229 (IC50=278M) and U87 (IC50=446M). With pericytes, the viability of TMZ-treated GBM spheroids significantly increases by 22.7% for PDM140, 32.5% for LN229, and 22.1% for U87, confirming pericyte-induced GBM chemoresistance responses. The upregulation (380-fold) of C-C motif chemokine ligand 5 (CCL5) in pericytes upon TMZ treatment could explain the chemoresistance responses. This innovative brain-mimicking 3D GBM model represents a novel in vitro platform for testing the efficacy of TMZ and novel drugs targeting CCL5-mediated chemoresistance pathways in GBM.

bioengineering↗

bioRxiv2024

Androgen receptor plays critical role in regulating cervical cancer cell migration

Cervical cancer (CC) is the second most common cancer among women in India and the fourth worldwide. While major genes and pathways have been studied, further research is needed to identify candidates for targeted therapy in metastatic disease. This study used a network biology approach to identify key genes in disease progression. Stage-specific cervical cancer protein-protein interaction networks (PPIN) were constructed by overlaying stage-specific, patient-derived transcriptomics data onto a human protein-protein interaction network (HPPIN). Graph-theory-based network analysis identified important interacting proteins (IIPs) with maximum connectivity, high centrality scores, and significant global and local network perturbation scores. Among the identified IIPs, the Androgen receptor (AR) emerged as one of the crucial yet understudied regulator in cervical cancer. Patient samples and in vitro experiments showed significant downregulation in cervical cancer. Ligand-dependent overexpression of AR reduced cancer cell migration while failed to induce apoptosis in CC cell lines. Downregulation of mesenchymal markers and restoration of epithelial markers suggested ARs potential in reversing invasive properties of cervical cancer cells. AR overexpression upregulated its downstream target PTEN and restored GSK3{beta} activity by interfering with AKT phosphorylation, probably leading to degradation of mesenchymal markers. Further studies showed AR reduced cell motility by hindering focal adhesion formation and Actin filament assembly. An increased G-Actin ratio suggested AR disrupted cytoskeletal dynamics through the RhoA/ROCK1/LIMK1/CFL1 pathway, impeding cervical cancer cell spread.

cancer biology↗

bioRxiv2024

The impact of ERUPR on mitochondrial integrity mediated by PDK4

ER and mitochondrial stress are often interconnected and considered as major contributors to aging as well as neurodegeneration. Co-ordinated induction of ERUPR and mitoUPR has been observed in diabetes and pulmonary disorders. However, in the context of aging and neurodegeneration, regulation of this intra-organellar crosstalk has remained relatively elusive. Here, we demonstrate that pyruvate dehydrogenase kinase 4 (PDK4), a mitochondrial protein accumulates at the ER-mitochondrial contact sites (MAMs) during ER stress. Classically, PDK4 is known to phosphorylate PDHA1 (pyruvate dehydrogenase E1 subunit alpha 1), and plays a significant role in regulating the oxidative phosphorylation driven ATP production. In this study, we propose a non-canonical function of PDK4; we show that it acts as a connecting link between ERUPR and mitoUPR with significance in aging and neurodegeneration. Transcriptomics analyses show increased PDK4 levels upon drug induced ER stress. We detect elevated PDK4 levels in brain lysates of Alzheimers disease (AD) patients, as well as in in vivo and ex vivo AD models. Additionally, exogenous expression of PDK4 was found to refine ER-mitochondrial communication, significantly altering mitochondrial morphology and function. Further, we also observe defective autophagic clearance of mitochondria under such conditions. It is prudent to suggest that elevated PDK4 levels could be one of the key factors connecting ERUPR with mitoUPR, a phenotypic contributor in aging and at least some neurodegenerative diseases.

molecular biology↗

bioRxiv2024

Quinolinic acid links kidney injury to brain toxicity

Kidney dysfunction often leads to neurological impairment, yet the complex kidney-brain relationship remains elusive. We employed spatial and bulk metabolomics to investigate a mouse model of rapid kidney failure induced by mouse double minute 2 (Mdm2) conditional deletion in the kidney tubules to interrogate kidney and brain metabolism. Pathway enrichment analysis of focused plasma metabolomics panel pinpointed tryptophan metabolism as the most altered pathway with kidney failure. Spatial metabolomics showed toxic tryptophan metabolites in the kidneys and brains, revealing a novel connection between advanced kidney disease and accelerated kynurenine degradation. In particular, the excitotoxic metabolite quinolinic acid was localized in ependymal cells adjacent to the ventricle in the setting of kidney failure. These findings were associated with brain inflammation and cell death. A separate mouse model of acute kidney injury also had an increase in circulating toxic tryptophan metabolites along with altered brain inflammation. Patients with advanced CKD similarly demonstrated elevated plasma kynurenine metabolites and quinolinic acid was uniquely correlated with fatigue and reduced quality of life in humans. Overall, our study identifies the kynurenine pathway as a bridge between kidney decline, systemic inflammation, and brain toxicity, offering potential avenues for diagnosis and treatment of neurological issues in kidney disease.

molecular biology↗

bioRxiv2024

The large GTPase Guanylate-Binding Protein-1 (GBP-1) promotes mitochondrial fission in glioblastoma.

Glioblastomas (aka Glioblastoma multiforme (GBMs)) are the most deadly of the adult brain tumors. Even with aggressive treatment, the prognosis is extremely poor. The large GTPase Guanylate-Binding Protein-1 (GBP-1) contributes to the poor prognosis of GBM by promoting migration and invasion. GBP-1 is substantially localized to the cytosolic side of the outer membrane of mitochondria in GBM cells. Because mitochondrial dynamics, particularly mitochondrial fission, can drive cell migration and invasion, the potential interactions between GBP-1 and mitochondrial dynamin-related protein 1 (Drp1) were explored. Drp1 is the major driver of mitochondrial fission. While GBP-1 and Drp1 both had punctate distributions within the cytoplasm and localized to regions of the cytoplasmic side of the plasma membrane of GBM cells, the proteins were only molecularly co-localized at the mitochondria. Subcellular fractionation showed that the presence of elevated GBP-1 promoted the movement of Drp1 from the cytosol to the mitochondria. Migration of U251 cells treated with the Drp1 inhibitor, Mdivi-1, was less inhibited in the cells with elevated GBP-1. Elevated GBP-1 in GBM cells resulted in shorter and wider mitochondria, most likely from mitochondrial fission. Mitochondrial fission can drive a number of important cellular processes, including cell migration, invasion, and metastasis. Simple SummaryGlioblastomas are the most common and most aggressive adult brain tumors arising from astrocytes. Up to 60% of glioblastomas are promoted by either amplification and/or mutation of the Epidermal Growth Factor Receptor (EGFR). The large GTPase Guanylate-Binding Protein-1 (GBP-1) is one of the most robustly induced proteins following EGFR signaling in glioblastomas. How GBP-1 promotes tumor progression in glioblastomas is still under investigation. This study shows that GBP-1 promotes tumor progression by associating with Drp1 at glioblastoma mitochondria and enhancing their fission. Specifically, increased mitochondrial fission can promote tumor cell migration, invasion, and metastasis.

cancer biology↗

bioRxiv2024

Improved synthesis and application of an alkyne-functionalized isoprenoid analogue to study the prenylomes of motor neurons, astrocytes and their stem cell progenitors

Protein prenylation is one example of a broad class of post-translational modifications where proteins are covalently linked to various hydrophobic moieties. To globally identify and monitor levels of all prenylated proteins in a cell simultaneously, our laboratory and others have developed chemical proteomic approaches that rely on the metabolic incorporation of isoprenoid analogues bearing bio-orthogonal functionality followed by enrichment and subsequent quantitative proteomic analysis. Here, several improvements in the synthesis of the alkyne-containing isoprenoid analogue C15AlkOPP are reported to improve synthetic efficiency. Next, metabolic labeling with C15AlkOPP was optimized to obtain useful levels of metabolic incorporation of the probe in several types of primary cells. Those conditions were then used to study the prenylomes of motor neurons (ES-MNs), astrocytes (ES-As), and their embryonic stem cell progenitors (ESCs), which allowed for the identification of 54 prenylated proteins from ESCs, 50 from ES-MNs and 84 from ES-As, representing all types of prenylation. Bioinformatic analysis revealed specific enriched pathways, including nervous system development, chemokine signaling, Rho GTPase signaling, and adhesion. Hierarchical clustering showed that most enriched pathways in all three cell types are related to GTPase activity and vesicular transport. In contrast, STRING analysis showed significant interactions in two populations that appear to be cell type dependent. The data provided herein demonstrates that robust incorporation of C15AlkOPP can be obtained in ES-MNs and related primary cells purified via magnetic-activated cell sorting allowing the identification and quantification of numerous prenylated proteins. These results suggest that metabolic labeling with C15AlkOPP should be an effective approach for investigating the role of prenylated proteins in primary cells in both normal cells and disease pathologies, including ALS. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=96 SRC="FIGDIR/small/583211v1_ufig1.gif" ALT="Figure 1"> View larger version (28K): org.highwire.dtl.DTLVardef@193cfe1org.highwire.dtl.DTLVardef@134edccorg.highwire.dtl.DTLVardef@1e81deforg.highwire.dtl.DTLVardef@56406_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2024

Local Delivery of Soluble Fractalkine (CX3CL1) Peptide Restore Ribbon Synapses After Noise-Induced Cochlear Synaptopathy

Efficacy of chemokine fractalkine isoforms was evaluated for restoration of loss of inner hair cell ribbon synapses and hearing after noise-induced cochlear synaptopathy (NICS). Previously, we have demonstrated a critical role for fractalkine signaling axis (CX3CL1-CX3CR1) in synaptic repair where in the presence of fractalkine receptor (CX3CR1) expressed by cochlear macrophages, the damaged synapses are spontaneously repaired. Here, we examined whether overexpression of fractalkine ligand (CX3CL1 or FKN) in the form of a peptide is effective in restoring the lost synapses and hearing after NICS. Remarkably, single transtympanic (TT) injection of soluble isoform of FKN (sFKN) peptide at 1 day after synaptopathic noise trauma showed significant recovery of ABR thresholds, ABR peak I amplitudes and ribbon synapses in both FKN-wildtype and knockout mice when compared to mice injected with full length membrane-bound FKN peptide (mFKN). Mechanistically, sFKN peptide treatment increased macrophage numbers in the cochlea and in the absence of those macrophages, sFKN failed to restore loss of synapses and hearing after NICS. Furthermore, sFKN treatment attenuated cochlear inflammation after noise overexposure without altering the expression of CX3CR1. Finally, sFKN peptide was detectable inside the cochlea localized to the sensory epithelium for 24 hours after TT injection. These data provide a robust proof-of-principle that local delivery of an immune factor, sFKN is effective in restoring lost ribbon synapses and hearing after NICS in a macrophage-dependent manner and highlights the potential of sFKN as an immunotherapy for cochlear synaptopathy due to noise or aging. SummaryTranstympanic delivery of soluble FKN peptide is effective in restoring lost inner hair cell ribbon synapses and hearing after noise-induced cochlear synaptopathy in a macrophage-dependent manner.

neuroscience↗

bioRxiv2023

Obesity modifies cell fate plasticity of Pdgfrα-expressing mammary adipocyte progenitors to promote an aberrant mammary microenvironment

Excess fat gain culminating in obesity is a mounting global pandemic associated with a myriad of diseases including a higher risk of developing multiple cancers such as breast cancer. The underlying biological basis for obesity-linked breast cancer is attributed to alterations in adipose tissue-derived endocrine, metabolic and inflammatory factors. However, the precise cell types responsible for generating a cancer-susceptible mammary gland in obesity are not well understood. Using diet-induced obesity in conjunction with genetic reporter mouse models, we reveal elevated mammary adipocyte progenitors (MAPs) expressing Platelet-Derived Growth Factor Receptor alpha (PDGFR) in the mammary tissue microenvironment during obesity. Single-cell RNA sequencing analysis demonstrate intensified obesity-driven trajectories of MAPs to immune and epithelial progenitor cells. Further, lineage tracing indicates an invasive cellular state of MAPs that confers greater access of MAP descendants into the mammary epithelium concomitant with their transition into immune and epithelial cell fates. MAPs in obesity exhibit heightened activation of cancer-associated and inflammatory pathways where Egfr is identified as a common target upregulated in MAPs. Mechanistic studies on purified MAPs with a major obesity diet component and EGFR stimulation corroborate dynamic EGFR-mediated MAP responses. Our findings uncover MAPs as key contributors to forging an aberrant mammary gland in obesity, providing insight into the potential utility of targeting this cell lineage for eradicating cancer risk related to augmented adiposity.

cell biology↗

bioRxiv2023

Optimizing Phosphopeptide Structures That Target 14-3-3ϵ in Cutaneous Squamous Cell Carcinoma

14-3-3{varepsilon} is involved in various types of malignancies by increasing cell proliferation, promoting cell invasion or inhibiting apoptosis. In cutaneous squamous cell carcinoma (cSCC), 14-3-3{varepsilon} is over expressed and mislocalized from the nucleus to the cytoplasm where it interacts with the cell division cycle 25 A (CDC25A) and suppresses apoptosis. Hence inhibition of the 14-3-3{varepsilon} - CDC25A interaction is an attractive target for promoting apoptosis in cSCC. In this work, we optimized the structure of our previously designed inhibitor of 14-3-3{varepsilon} - CDC25A interaction, pT, a phosphopeptide fragment corresponding to one of the two binding regions of CDC25A to 14-3-3{varepsilon}. Starting from pT, we developed peptide analogs that bind 14-3-3{varepsilon} with nanomolar affinities. Peptide analogs were designed by shortening the pT peptide, and introducing modifications at position 510 of the pT(502-510) analog. Both molecular dynamics (MD) simulations and biophysical methods were used to determine peptides binding to 14-3-3{varepsilon}. Shortening the pT peptide from 14 to 9 amino acid residues resulted in a peptide (pT(502-510)) that binds 14-3-3{varepsilon} with a KD value of 45.2 nM. Gly to Phe substitution in position 510 of pT(502-510) led to further improvement in affinity (KD: 22.0 nM) of the peptide for 14-3-3{varepsilon}. Our results suggest that the designed peptide analogs are potential candidates for inhibiting 14-3-3{varepsilon} -CDC25A interactions in cSCC cells; thus, inducing their apoptosis.

biochemistry↗

bioRxiv2023

A new antibiotic from an uncultured bacterium binds to an immutable target

Antimicrobial resistance is a leading mortality factor worldwide. Here we report the discovery of clovibactin, a new antibiotic, isolated from uncultured soil bacteria. Clovibactin efficiently kills drug-resistant bacterial pathogens without detectable resistance. Using biochemical assays, solid-state NMR, and atomic force microscopy, we dissect its mode of action. Clovibactin blocks cell wall synthesis by targeting pyrophosphate of multiple essential peptidoglycan precursors (C55PP, Lipid II, LipidWTA). Clovibactin uses an unusual hydrophobic interface to tightly wrap around pyrophosphate, but bypasses the variable structural elements of precursors, accounting for the lack of resistance. Selective and efficient target binding is achieved by the irreversible sequestration of precursors into supramolecular fibrils that only form on bacterial membranes that contain lipid-anchored pyrophosphate groups. Uncultured bacteria offer a rich reservoir of antibiotics with new mechanisms of action that could replenish the antimicrobial discovery pipeline.

microbiology↗

bioRxiv2023

Lateral membrane organization as target of an antimicrobial peptidomimetic compound

Antimicrobial resistance is one of the leading concerns in medical care. Here we resolve the functional mechanism of the antimicrobial action of the cationic tripeptide AMC-109 by combining high speed-atomic force microscopy, molecular dynamics, fluorescence assays, and lipidomic analysis. We show that AMC-109 activity on the negatively charged plasma membrane of Staphylococcus aureus consists of two crucial steps. First, AMC-109 self-assembles into stable aggregates with specificity for negatively charged membranes. Second, by incorporation into the S. aureus membrane the lateral membrane organization is affected, dissolving membrane nanodomains. Domain dissolution affects membrane functions such as protein sorting and cell wall synthesis, and is suggested to cause a loss of resistance of methicillin-resistant S. aureus (MRSA) to methicillin. As the AMC-109 mode of action is similar to the activity of the disinfectant benzalkonium chloride (BAK), a broad applicability, but with low cytotoxicity to human cells, is expected.

biophysics↗

bioRxiv2022

Complex changes in serum protein levels upon recovery from SARS-CoV2 infection

The COVID-19 pandemic, triggered by severe acute respiratory syndrome coronavirus 2, has affected millions of people worldwide. Much research has been dedicated to our understanding of COVID-19 disease heterogeneity and severity, but less is known about recovery associated changes. To address this gap in knowledge, we quantified the proteome from serum samples from 29 COVID-19 convalescents and 29 age-, race-, and sex-matched healthy controls. Samples were acquired within the first months of the pandemic. Many proteins from pathways known to change during acute COVID-19 illness, such as from the complement cascade, coagulation system, inflammation and adaptive immune system, had returned to levels seen in healthy controls. In comparison, we identified 22 and 15 proteins with significantly elevated and lowered levels, respectively, amongst COVID-19 convalescents compared to healthy controls. Some of the changes were similar to those observed for the acute phase of the disease, i.e. elevated levels of proteins from hemolysis, the adaptive immune systems, and inflammation. In contrast, some alterations opposed those in the acute phase, e.g. elevated levels of CETP and APOA1 which function in lipid/cholesterol metabolism, and decreased levels of proteins from the complement cascade (e.g. C1R, C1S, and VWF), the coagulation system (e.g. THBS1 and VWF), and the regulation of the actin cytoskeleton (e.g. PFN1 and CFL1) amongst COVID-19 convalescents. We speculate that some of these shifts might originate from a transient decrease in platelet counts upon recovery from the disease. Finally, we observed race-specific changes, e.g. with respect to immunoglobulins and proteins related to cholesterol metabolism.

immunology↗

bioRxiv2022

Triggered functional dynamics of AsLOV2 by time-resolved electron paramagnetic resonance at high magnetic fields

We present time-resolved Gd-Gd electron paramagnetic resonance (TiGGER) at 240 GHz for tracking inter-residue distances during a proteins mechanical cycle in the solution state. TiGGER makes use of Gd-sTPATCN as spin labels, whose favorable qualities include a spin-7/2 EPR-active center, short linker, narrow intrinsic linewidth, and virtually no anisotropy at high fields (8.6 T) when compared to nitroxide spin labels. Using TiGGER, we determined that upon light activation, the C-terminus and N-terminus of AsLOV2 separate in less than 1 s and relax back to equilibrium with a time constant of approximately 60 s. TiGGER revealed that the light-activated long-range mechanical motion is slowed in the Q513A variant of AsLOV2 and is correlated to the similarly slowed relaxation of the optically excited chromophore as described in recent literature. TiGGER has the potential to valuably complement existing methods for the study of triggered functional dynamics in proteins.

biophysics↗