Biology subjects

Iyer, S. S.

Publications and source records attributed to Iyer, S. S..

At least 19 recordsLinked to original sources

bioRxiv2026

Zyxin and LPP differ in their sensing of strained actin filaments at tricellular junctions

The actomyosin cytoskeleton plays important roles in cell-cell adhesion by generating and responding to forces. Actin-binding proteins support actomyosin networks by reinforcing actin filaments, promoting actin remodeling, and transmitting forces to transmembrane adhesion proteins. One family of actin-binding proteins, LIM domain-containing proteins, is recruited to strained actin filaments. Here, we investigate two members of the LIM domain family, Zyxin and Lipoma-Preferred Partner (LPP), using the embryonic epithelium of Xenopus laevis, where actin-associated cell-cell junctions connect cells to promote tissue integrity and barrier function. Specifically, we compare Zyxin and LPP's response to increased tension at tricellular junctions (TCJs), sites of heightened mechanical strain within the tissue. Upon increased tension, Zyxin and LPP mechanoaccumulate to different extents relative to F-actin, suggesting distinct mechanisms. Our results demonstrate that Zyxin's and LPP's LIM domain-containing regions (LCRs) are sufficient for mechanoaccumulation as well as responsible for the difference in mechanoaccumulation, while their N-termini play a regulatory role in their mechanosensitive responses. Docking simulations show that a hydrophobic interaction between the LCR and the "crack site" that forms in F-actin filaments under high force may underlie the LCR's sensing of strained F-actin. Additionally, the docking simulations reveal that previously-identified conserved residues are present at the binding interface. Mutations of conserved residues in Zyxin or LPP reduce the extent of mechanoaccumulation at TCJs, supporting the docking prediction. Together, this study advances our understanding of Zyxin and LPP's sensing of strained actin at TCJs under mechanical challenge.

cell biology↗

bioRxiv2025

Taxane-Induced Conformational Changes in the Microtubule Lattice Activate GEF-H1-Dependent RhoA Signaling

Taxanes are widely used chemotherapeutic agents that perturb cell division. They also exert effects during interphase, but the underlying mechanisms are poorly understood. Here, we show that taxanes activate RhoA signaling and induce actin remodeling by displacing the RhoA activator GEF-H1 from microtubules. This taxane-induced release of GEF-H1 occurs rapidly, is independent of tubulin post-translational modifications, and can be recapitulated using purified proteins. In vitro reconstitution assays combined with analyses of microtubule structure revealed that microtubule binding by GEF-H1 is inhibited by microtubule-stabilizing agents that expand the microtubule lattice, such as taxanes and GMPCPP, but not by others, including GTP{gamma}S and discodermolide, which stabilize a compacted microtubule lattice. Our findings demonstrate that alterations in microtubule lattice conformation can activate key signaling pathways, offering new insights into the mode of action of taxanes and the possible origins of their side effects.

cell biology↗

bioRxiv2025

On the Mechanism of Ezrin Activation

Ezrin is a peripheral membrane protein that contributes to the organization and stability of cellular membrane structures by reversibly linking the plasma membrane to actin filaments. The formation of this membrane-actin linkage has been experimentally shown to require ezrin N-terminal (FERM) domain binding to PI(4,5)P2 phospholipid-enriched membrane sites and the phosphorylation of the ezrin C-terminal domain (CTD) at residue T567. Collectively, membrane association and T567 phosphorylation are believed to promote separation of the FERM and CTD domains; however, the underlying molecular mechanism remains less clear. In this study, we investigate the mechanistic steps of ezrin activation and the thermodynamic free energy landscape of FERM-CTD dissociation using enhanced sampling molecular dynamics (MD). We find that upon ezrin attachment to a lipid membrane, PI(4,5)P2 molecules outcompete other phospholipids at the surface of the FERM F1 and F3 subdomains. This interaction triggers a major conformational rearrangement within the FERM domain that destabilizes the FERM F2-CTD interface and initiates dissociation between the FERM and CTD. By employing well-tempered metadynamics (WTMetaD) with a contact-map collective variable, we determine that the principal barrier to FERM-CTD dissociation comes from F3-CTD interactions and that this dissociation can happen spontaneously with a moderate free energy barrier. We also show that the FERM-CTD reassociation after ezrin T567 phosphorylation is impeded due to reduced dissociation energy barrier. The free energy profile of dissociation between FERM and the CTD-replacing EBP50 protein is similar to that of the FERM-CTD system with nonphosphorylated T567, which agrees well with an in vivo experimental observation that EBP50 competes with the CTD for F2-F3 binding after CTD is dissociated. Together, our results help establish a revised view on the ezrin activation mechanism where FERM binding to PI(4,5)P2 enables spontaneous dissociation of the nonphosphorylated CTD. SIGNIFICANCEEzrin and related ERM proteins control how cells link their plasma membrane to the actin cytoskeleton, a process fundamental to cell shape, signaling and motility. Despite decades of study, the molecular basis of ezrin activation - how it transitions from a self-inhibited to an active membrane-bound state - has remained unresolved. Using atomistic and enhanced sampling molecular dynamics together with biochemical validation, we show that binding of the FERM domain to PI(4,5)P2-enriched membranes alone is sufficient to trigger spontaneous dissociation of the nonphosphorylated C-terminal domain. Phosphorylation of T567 subsequently stabilizes the open conformation and prevents domain reassociation, enabling actin engagement and binding of FERM partners such as EBP50. Collectively, these findings advance a more integrated view of ezrin activation, highlighting how membrane interactions, conformational flexibility and phosphorylation act in concert to regulate membrane-cytoskeleton coupling.

biophysics↗

bioRxiv2025

Reaching the full potential of cryo-EM reconstructions with molecular dynamics simulations at 310 K: Actin filaments as an example

Cryo-electron microscopy (cryo-EM) structures of multi-protein complexes such as actin filaments help explain the mechanisms of assembly and interactions with partner proteins. Yet, rapid cooling during freezing may not preserve the conformations at physiological temperature. All-atom molecular dynamics simulations starting with cryo-EM reconstructions can provide additional insights. For example, at 310 K the states of ADP-actin filaments consistent with higher entropy favor partly twisted subunits and smaller rotations along short-pitch helix than the cryo-EM reconstructions, while cryogenic temperatures favor flattened conformations. In the active site, the positions of Q137 and the catalytic water 1 and activating water 2 optimal for in line attack on the {gamma}-phosphate of ATP are very rare at 310 K, explaining in part the slow rate of ATP hydrolysis in filaments. This favorable arrangement of the waters is not observed in simulations of actin monomers. At 310 K subunits in ADP-Pi-actin filaments have their backdoor gates open 60% of the time for phosphate release, a conformation not observed by cryo-EM. Rare fluctuations open binding sites for cofilin and phalloidin. The twisted conformations of pointed end subunits and interactions of the D-loop of the penultimate subunit explain the slow association of new subunits. The terminal subunit at the barbed end is tethered to its neighbor along the long-pitch helix but transiently dissociates from its lateral neighbor. These effects of subfreezing temperatures on actin filaments are surely not an isolated example, so MD simulations of structures of other frozen proteins will be informative. Significance statementFunctionally important properties of proteins are tightly linked to their conformations at physiological temperatures. While cryo-EM reconstructions and crystal structure of frozen proteins provide a molecular resolution of protein structures, they might differ from the conformations at physiological temperature. Using the actin filament as a case study, we find cryo-EM reconstructed structures correspond to low entropy conformations that differ from the ensemble of structures in molecular dynamics simulations at 310 K. The fluctuations of subunit dihedral angles, short-pitch rotations and some side chains explain functionally important properties of actin filaments, including the slow rates of ATP hydrolysis and phosphate release as well as the slow binding of the protein cofilin and cyclic peptide phalloidin.

biophysics↗

bioRxiv2024

Spherical Echo-Planar Time-resolved Imaging (sEPTI) for rapid 3D quantitative T2* and Susceptibility imaging

PurposeTo develop a 3D spherical EPTI (sEPTI) acquisition and a comprehensive reconstruction pipeline for rapid high-quality whole-brain submillimeter T2* and QSM quantification. MethodsFor the sEPTI acquisition, spherical k-space coverage is utilized with variable echo-spacing and maximum kx ramp-sampling to improve efficiency and incoherency when compared to existing EPTI approaches. For reconstruction, an iterative rank-shrinking B0 estimation and odd-even high-order phase correction algorithms were incorporated into the reconstruction to better mitigate artifacts from field imperfections. A physics-informed unrolled network was utilized to boost the SNR, where 1-mm and 0.75-mm isotropic whole-brain imaging were performed in 45 and 90 seconds, respectively. These protocols were validated through simulations, phantom, and in vivo experiments. Ten healthy subjects were recruited to provide sufficient data for the unrolled network. The entire pipeline was validated on additional 5 healthy subjects where different EPTI sampling approaches were compared. Two additional pediatric patients with epilepsy were recruited to demonstrate the generalizability of the unrolled reconstruction. ResultssEPTI achieved 1.4 x faster imaging with improved image quality and quantitative map precision compared to existing EPTI approaches. The B0 update and the phase correction provide improved reconstruction performance with lower artifacts. The unrolled network boosted the SNR, achieving high-quality T2* and QSM quantification with single average data. High-quality reconstruction was also obtained in the pediatric patient using this network. ConclusionsEPTI achieved whole-brain distortion-free multi-echo imaging and T2* and QSM quantification at 0.75 mm in 90 seconds which has the potential to be useful for wide clinical applications.

bioengineering↗

bioRxiv2023

CCR7+ CD4 T Cell Immunosurveillance Disrupted in Chronic SIV-Induced Neuroinflammation in Rhesus Brain.

CD4 T cells survey and maintain immune homeostasis in the brain, yet their differentiation states and functional capabilities remain unclear. Our approach, combining single-cell transcriptomic analysis, ATAC-seq, spatial transcriptomics, and flow cytometry, revealed a distinct subset of CCR7+ CD4 T cells resembling lymph node central memory (TCM) cells. We observed chromatin accessibility at the CCR7, CD28, and BCL-6 loci, defining molecular features of TCM. Brain CCR7+ CD4 T cells exhibited recall proliferation and interleukin-2 production ex vivo, showcasing their functional competence. We identified the skull bone marrow as a local niche for these cells alongside other CNS border tissues. Sequestering TCM cells in lymph nodes using FTY720 led to reduced CCR7+ CD4 T cell frequencies in the cerebrospinal fluid, accompanied by increased monocyte levels and soluble markers indicating immune activation. In macaques chronically infected with SIVCL57 and experiencing viral rebound due to cessation of antiretroviral therapy, a decrease in brain CCR7+ CD4 T cells was observed, along with increased microglial activation and initiation of neurodegenerative pathways. Our findings highlight a role for CCR7+ CD4 T cells in CNS immune surveillance and their decline during chronic SIV-induced neuroinflammation highlights their responsiveness to neuroinflammatory processes. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=87 SRC="FIGDIR/small/555037v1_ufig1.gif" ALT="Figure 1"> View larger version (20K): org.highwire.dtl.DTLVardef@191f9caorg.highwire.dtl.DTLVardef@a67d2dorg.highwire.dtl.DTLVardef@645443org.highwire.dtl.DTLVardef@d81fd6_HPS_FORMAT_FIGEXP M_FIG C_FIG In BriefUtilizing single-cell and spatial transcriptomics on adult rhesus brain, we uncover a unique CCR7+ CD4 T cell subset resembling central memory T cells (TCM) within brain and border tissues, including skull bone marrow. Our findings show decreased frequencies of this subset during SIV- induced chronic neuroinflammation, emphasizing responsiveness of CCR7+ CD4 T cells to CNS disruptions. HighlightsO_LICCR7+ CD4 T cells survey border and parenchymal CNS compartments during homeostasis; reduced presence of CCR7+ CD4 T cells in cerebrospinal fluid leads to immune activation, implying a role in neuroimmune homeostasis. C_LIO_LICNS CCR7+ CD4 T cells exhibit phenotypic and functional features of central memory T cells (TCM) including production of interleukin 2 and the capacity for rapid recall proliferation. Furthermore, CCR7+ CD4 T cells reside in the skull bone marrow. C_LIO_LICCR7+ CD4 T cells are markedly decreased within the brain parenchyma during chronic viral neuroinflammation. C_LI

neuroscience↗

bioRxiv2023

CD4 T cell Responses in the CNS during SIV infection

Virologic suppression with antiretroviral therapy (ART) has significantly improved health outcomes for people living with HIV, yet challenges related to chronic inflammation in the central nervous system (CNS) - known as Neuro-HIV-persist. As primary targets for HIV-1 with the ability to survey and populate the CNS and interact with myeloid cells to co-ordinate neuroinflammation, CD4 T cells are pivotal in Neuro-HIV. Despite their importance, our understanding of CD4 T cell distribution in virus-targeted CNS tissues, their response to infection, and potential recovery following initiation of ART remain limited. To address these gaps, we studied ten SIVmac251-infected rhesus macaques using an ART regimen simulating suboptimal adherence. We evaluated four macaques during the acute phase pre-ART and six during the chronic phase. Our data revealed that HIV target CCR5+ CD4 T cells inhabit both the brain parenchyma and adjacent CNS tissues, encompassing choroid plexus stroma, dura mater, and the skull bone marrow. Aligning with the known susceptibility of CCR5+ CD4 T cells to viral infection and their presence within the CNS, high levels of viral RNA were detected in the brain parenchyma and its border tissues during acute SIV infection. Single-cell RNA sequencing of CD45+ cells from the brain revealed colocalization of viral transcripts within CD4 clusters and significant activation of antiviral molecules and specific effector programs within T cells, indicating CNS CD4 T cell engagement during infection. Despite viral suppression with ART, acute infection led to significant depletion of CNS CD4 T cells, persisting into the chronic phase. These findings underscore the functional involvement of CD4 T cells within the CNS during SIV infection, enhancing our understanding of their role in establishing CNS viral presence. Our results offer insights for potential T cell-focused interventions while also underscoring the challenges in eradicating HIV from the CNS, even with effective ART. AUTHOR SUMMARYAntiretroviral therapy (ART) has improved health outcomes of people living with HIV. However, there are still challenges, especially in the central nervous system (CNS), where ongoing inflammation can lead to neurological disorders. Our study focused on understanding the role of CD4 T cells in the brain during HIV infection and treatment. We used a model with SIV-infected rhesus monkeys to study the AIDS virus in the brain and surrounding tissues. We discovered that a subset of CD4 T cells, which are vulnerable to HIV, are present throughout the CNS. During the early stages of infection, we noticed high levels of the virus in both the brain and nearby tissues. By examining these CD4 T cells at a single-cell level, we found that they actively respond to the virus by activating specific antiviral effector functions to fight it. Although ART controlled virus replication in the brain, CD4 T cells were continuously depleted, indicative of immune perturbations. Overall, our study helps us understand the role of CD4 T cells within the CNS during both acute and chronic HIV infection. This knowledge could help us develop new ways to target the virus in the CNS and devise treatments for complications related to Neuro-HIV.

neuroscience↗

bioRxiv2023

Tailoring Tfh Profiles Enhances Antibody Persistence to a Clade C HIV-1 Vaccine in Rhesus Macaques

CD4 T follicular helper cells (Tfh) are essential for establishing serological memory and have distinct helper attributes that impact both the quantity and quality of the antibody response. Insights into Tfh subsets that promote antibody persistence and functional capacity can critically inform vaccine design. Based on the Tfh profiles evoked by the live attenuated measles virus vaccine, renowned for its ability to establish durable humoral immunity, we investigated the potential of a Tfh1/17 recall response during the boost phase to enhance persistence of HIV-1 Envelope (Env) antibodies in rhesus macaques. Using a DNA-prime encoding gp160 antigen and Tfh polarizing cytokines (interferon protein-10 (IP-10) and interleukin-6 (IL-6)), followed by a gp140 protein boost formulated in a cationic liposome-based adjuvant (CAF01), we successfully generated germinal center (GC) Tfh1/17 cells. In contrast, a similar DNA-prime (including IP-10) followed by gp140 formulated with monophosphoryl lipid A (MPLA)+QS-21 adjuvant predominantly induced GC Tfh1 cells. While the generation of GC Tfh1/17 cells with CAF01 and GC Tfh1 cells with MPLA+QS-21 induced comparable peak Env antibodies, the latter group demonstrated significantly greater antibody concentrations at week 8 after final immunization which persisted up to 30 weeks (gp140 IgG ng/ml-MPLA; 5500; CAF01, 2155; p <0.05). Notably, interferon {gamma}+ Env-specific Tfh responses were consistently higher with gp140 in MPLA+QS-21 and positively correlated with Env antibody persistence. These findings suggest that vaccine platforms maximizing GC Tfh1 induction promote persistent Env antibodies, important for protective immunity against HIV.

immunology↗

bioRxiv2023

Deep Learning Initialized Compressed Sensing (Deli-CS) in Volumetric Spatio-Temporal Subspace Reconstruction

IntroductionSpatio-temporal MRI methods enable whole-brain multi-parametric mapping at ultra-fast acquisition times through efficient k-space encoding, but can have very long reconstruction times, which limit their integration into clinical practice. Deep learning (DL) is a promising approach to accelerate reconstruction, but can be computationally intensive to train and deploy due to the large dimensionality of spatio-temporal MRI. DL methods also need large training data sets and can produce results that dont match the acquired data if data consistency is not enforced. The aim of this project is to reduce reconstruction time using DL whilst simultaneously limiting the risk of deep learning induced hallucinations, all with modest hardware requirements. MethodsDeep Learning Initialized Compressed Sensing (Deli-CS) is proposed to reduce the reconstruction time of iterative reconstructions by "kick-starting" the iterative reconstruction with a DL generated starting point. The proposed framework is applied to volumetric multi-axis spiral projection MRF that achieves whole-brain T1 and T2 mapping at 1-mm isotropic resolution for a 2-minute acquisition. First, the traditional reconstruction is optimized from over two hours to less than 40 minutes while using more than 90% less RAM and only 4.7 GB GPU memory, by using a memory-efficient GPU implementation. The Deli-CS framework is then implemented and evaluated against the above reconstruction. ResultsDeli-CS achieves comparable reconstruction quality with 50% fewer iterations bringing the full reconstruction time to 20 minutes. ConclusionDeli-CS reduces the reconstruction time of subspace reconstruction of volumetric spatio-temporal acquisitions by providing a warm start to the iterative reconstruction algorithm.

neuroscience↗

bioRxiv2023

Western diet dampens T regulatory cell function to fuel hepatic inflammation in nonalcoholic fatty liver disease

Background and aimsThe immunosuppressive T regulatory cells (Tregs) regulate immune responses and maintain immune homeostasis, yet their functions in nonalcoholic fatty liver disease (NAFLD) pathogenesis remains controversial. MethodsMice were fed a normal diet (ND) or a western diet (WD) for 16 weeks to induce NAFLD. Diphtheria toxin injection to deplete Tregs in Foxp3DTR mice or Treg induction therapy in WT mice to augment Treg numbers was initiated at twelve and eight weeks, respectively. Liver tissues from mice and NASH human subjects were analyzed by histology, confocal imaging, and qRT-PCR. ResultsWD triggered accumulation of adaptive immune cells, including Tregs and effector T cells, within the liver parenchyma. This pattern was also observed in NASH patients, where an increase in intrahepatic Tregs was noted. In the absence of adaptive immune cells in Rag1 KO mice, WD promoted accumulation of intrahepatic neutrophils and macrophages and exacerbated hepatic inflammation and fibrosis. Similarly, targeted Treg depletion exacerbated WD-induced hepatic inflammation and fibrosis. In Treg-depleted mice, hepatic injury was associated with increased accumulation of neutrophils, macrophages, and activated T cells within the liver. Conversely, induction of Tregs using recombinant IL2/IL2 mAb cocktail reduced hepatic steatosis, inflammation, and fibrosis in WD-fed mice. Analysis of intrahepatic Tregs from WD-fed mice revealed a phenotypic signature of impaired Treg function in NAFLD. Ex vivo functional studies showed that glucose and palmitate, but not fructose, impaired the immunosuppressive ability of Treg cells. ConclusionsOur findings indicate that the liver microenvironment in NAFLD impairs ability of Tregs to suppress effector immune cell activation, thus perpetuating chronic inflammation and driving NAFLD progression. These data suggest that targeted approaches aimed at restoring Treg function may represent a potential therapeutic strategy for treating NAFLD. Lay summaryIn this study, we elucidate the mechanisms contributing to the perpetuation of chronic hepatic inflammation in nonalcoholic fatty liver disease (NAFLD). We show that dietary sugar and fatty acids promote chronic hepatic inflammation in NAFLD by impairing immunosuppressive function of regulatory T cells. Finally, our preclinical data suggest that targeted approaches aimed at restoring T regulatory cell function have the potential to treat NAFLD.

pathology↗

bioRxiv2023

Selective targeting of α4β7/MAdCAM-1 axis suppresses fibrosis progression in chronic liver disease

Integrin 4{beta}7+ T cells perpetuate tissue injury in chronic inflammatory diseases, yet their role in hepatic fibrosis progression remains poorly understood. Here we report increased accumulation of 4{beta}7+ T cells in the liver of people with cirrhosis relative to disease controls. Similarly, hepatic fibrosis in the established mouse model of CCl4-induced liver fibrosis was associated with enrichment of intrahepatic 4{beta}7+ CD4 and CD8 T cells. Monoclonal antibody (mAb)-mediated blockade of 4{beta}7 or its ligand mucosal addressin cell adhesion molecule (MAdCAM)-1 attenuated hepatic inflammation and prevented fibrosis progression in CCl4 treated mice. Improvement in liver fibrosis was associated with a significant decrease in the infiltration of 4{beta}7+ CD4 and CD8 T cells suggesting that 4{beta}7/MAdCAM-1 axis regulates both CD4 and CD8 T cell recruitment to the fibrotic liver, and 4{beta}7+ T cells promote hepatic fibrosis progression. Analysis of hepatic 4{beta}7+ and 4{beta}7-CD4 T cells revealed that 4{beta}7+ CD4 T cells enriched for markers of activation and proliferation demonstrating an effector phenotype. Notably, blockade of 4{beta}7 or MAdCAM-1 did not affect the recruitment of Foxp3+ regulatory T cells, demonstrating the specificity of 4{beta}7/MAdCAM-1 axis in regulating effector T cell recruitment to the liver. The findings suggest that 4{beta}7+ T cells play a critical role in promoting hepatic fibrosis progression, and mAb-mediated blockade of 4{beta}7 or MAdCAM-1 represents a promising therapeutic strategy for slowing hepatic fibrosis progression in chronic liver diseases.

immunology↗

bioRxiv2022

Inducible Bronchus-Associated Lymphoid Tissue in SARS-CoV-2 Infected Rhesus Macaques

Pulmonary immunity against SARS-CoV-2 infection has not been well studied. This study investigated the distribution of immune cells int the lungs of 8 rhesus macaques experimentally infected with SARS-CoV-2, and euthanized 11-14 days later. Using immunohistochemistry, inducible bronchus-associated lymphoid tissue was found in all animals. The inducible bronchus-associated lymphoid tissues were composed of B cells, T cells, and follicular dendritic cells with evidence of lymphocyte priming and differentiation. This suggests local immunity plays an important role in the SARS-CoV-2 infection. Further study of local immunity in the lung would benefit our understanding of SARS-CoV-2 pathogenesis and could lead to new interventions to control the SARS-CoV-2 infection and disease.

pathology↗

bioRxiv2022

Distinct upper airway epithelium interferon-stimulated and profibrotic gene expression between adult and infant rhesus macaques infected with SARS-CoV-2

The global spread of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) and its associated coronavirus disease (COVID-19) has led to a pandemic of unprecedented scale. An intriguing feature of the infection is the minimal disease in most children, a demographic at higher risk for respiratory viral diseases. To elucidate age-dependent effects of SARS-CoV-2 pathogenesis, we inoculated two rhesus macaque monkey dam-infant pairs with SARS-CoV-2 and conducted virological and transcriptomic analysis of the respiratory tract and evaluated systemic cytokine and antibody responses. Viral RNA levels in all sampled mucosal secretions were comparable across dam-infant pairs in the respiratory tract. Despite comparable viral loads, adult macaques showed higher IL-6 in serum while CXCL10 was induced in all animals. Both groups mounted neutralizing antibody (nAb) responses, with infants showing a more rapid induction at day 7. Transcriptome analysis of tracheal tissue isolated at day 14 post-infection revealed significant upregulation of multiple interferon-stimulated genes in infants compared to adults. In contrast, a profibrotic transcriptomic signature with genes associated with cilia structure and function, extracellular matrix (ECM) composition and metabolism, coagulation, angiogenesis, and hypoxia was induced in adults compared to infants. Our observations suggest age-dependent differential airway responses to SARS-CoV-2 infection that could explain the distinction in pathogenesis between infants and adults.

immunology↗

bioRxiv2021

Enthalpic and Entropic Contributions to Interleaflet Coupling Drive Domain Registration and Anti-registration in Biological Membrane

Biological membrane is a complex self-assembly of lipids, sterols and proteins organized as a fluid bilayer of two closely stacked lipid leaflets. Differential molecular interactions among its diverse constituents give rise to heterogeneities in the membrane lateral organization. Under certain conditions, heterogeneities in the two leaflets can be spatially synchronised and exist as registered domains across the bilayer. Several contrasting theories behind mechanisms that induce registration of nanoscale domains have been suggested[1-3]. Following a recent study[4] showing the effect of position of lipid tail unsaturation on domain registration behavior, we decided to develop an analytical theory to elucidate the driving forces that create and maintain domain registry across leaflets. Towards this, we formulated a Hamiltonian for a stacked lattice system where site variables capture the lipid molecular properties such as the position of unsaturation and various other interactions that could drive phase separation and interleaflet coupling. We solve the Hamiltonian using Monte Carlo simulations and create a complete phase diagram that reports the presence or absence of registered domains as a function of various Hamiltonian parameters. We find that the interleaflet coupling should be described as a competing enthalpic contribution due to interaction of lipid tail termini, primarily due to saturated-saturated interactions, and an interleaflet entropic contribution from overlap of unsaturated tail termini. A higher position of unsaturation is seen to provide weaker interleaflet coupling. Thermodynamically stable nanodomains could also be observed for certain points in the parameter space in our bilayer model, which were further verified by carrying out extended Monte Carlo simulations. These persistent non-coalescing registered nanodomains close to the lower end of the accepted nanodomain size range also point towards a possible "nanoscale" emulsion description of lateral heterogeneities in biological membrane leaflets.

biophysics↗

bioRxiv2021

Early post-infection treatment of SARS-CoV-2 infected macaques with human convalescent plasma with high neutralizing activity reduces lung inflammation

Early in the SARS-CoV-2 pandemic, there was a high level of optimism based on observational studies and small controlled trials that treating hospitalized patients with convalescent plasma from COVID-19 survivors (CCP) would be an important immunotherapy. However, as more data from controlled trials became available, the results became disappointing, with at best moderate evidence of efficacy when CCP with high titers of neutralizing antibodies was used early in infection. To better understand the potential therapeutic efficacy of CCP, and to further validate SARS-CoV-2 infection of macaques as a reliable animal model for testing such strategies, we inoculated 12 adult rhesus macaques with SARS-CoV-2 by intratracheal and intranasal routes. One day later, 8 animals were infused with pooled human CCP with a high titer of neutralizing antibodies (RVPN NT50 value of 3,003), while 4 control animals received normal human plasma. Animals were monitored for 7 days. Animals treated with CCP had detectable levels of antiviral antibodies after infusion. In comparison to the control animals, they had similar levels of virus replication in the upper and lower respiratory tract, but had significantly reduced interstitial pneumonia, as measured by comprehensive lung histology. By highlighting strengths and weaknesses, data of this study can help to further optimize nonhuman primate models to provide proof-of-concept of intervention strategies, and guide the future use of convalescent plasma against SARS-CoV-2 and potentially other newly emerging respiratory viruses. Author summaryThe results of treating SARS-CoV-2 infected hospitalized patients with COVID-19 convalescent plasma (CCP), collected from survivors of natural infection, have been disappointing. The available data from various studies indicate at best moderate clinical benefits only when CCP with high titer of neutralizing antibodies was infused early in infection. The macaque model of SARS-CoV-2 infection can be useful to gain further insights in the value of CCP therapy. In this study, animals were infected with SARS-CoV-2 and the next day, were infused with pooled human convalescent plasma, selected to have a very high titer of neutralizing antibodies. While administration of CCP did not result in a detectable reduction in virus replication in the respiratory tract, it significantly reduced lung inflammation. These data, combined with the results of monoclonal antibody studies, emphasize the need to use products with high titers of neutralizing antibodies, and guide the future development of CCP-based therapies.

immunology↗

bioRxiv2021

Hydrophobic residues advance the onset of simple coacervation in intrinsically disordered proteins at low densities: Insights from field theoretical simulations studies

Intrinsically disordered proteins (IDPs) have engendered a definitive change in the way we think about the classical "sequence-structure-function" dogma. Their conformational pliability and rich molecular recognition features endow them with the ability to bind to diverse partners and predispose them to an elaborate functional armory. And of late, with studies on IDP-based liquid-liquid phase separation (LLPS) leading to formation of functional subcellular coacervates - best described as "membrane-less organelles (MLOs)", IDPs are also bringing about paradigmatic changes in the way we think about biomolecular assemblies and subcellular organization. Though it is well recognized that the phase behavior of a given IDP is tightly coupled to its amino-acid sequences, there are only a few theories to model polyampholyte coacervation for IDPs. Recently, Joan-Emma Shea and co-workers used field theoretical simulations (FTS) to elucidate the complete phase diagram for LLPS of IDPs by considering different permutations of 50-residues chain representing 25 Lysine and 25 Glutamic acid [1]. Our work is an extension of that FTS framework where we develop and solve an augmented Hamiltonian that also accounts for hydrophobic interactions in the chain. We show that incorporation of hydrophobic interactions result in an advanced onset of coacervation at low densities. The patterning of hydrophobic, positive and negative residues plays important role in determining relative differences in the onset of phase separation. Though still very coarse-grained, once additional chemical specificities are incorporated, these high throughput analytical theory methods can be used as a starting point for designing sequences that drive LLPS.

biophysics↗

bioRxiv2019

Interpretation of Phase Boundary Fluctuation Spectra in Biological Membranes with Nanoscale Organization

In this work, we use Support Vector Machine algorithm to detect simple and complex interfaces in atomistic and coarse-grained molecular simulation trajectories of phase separating lipid bilayer systems. We show that the power spectral density of the interfacial height fluctuations and in turn the line tension of the lipid bilayer systems depend on the order parameter used to identify the intrinsic interface. To highlight the effect of artificial smoothing of the interface on the fluctuation spectra and the ensuing line tension calculations, we perform a convolution of the boundaries identified at molecular resolution with a 2D Gaussian function of variance{varepsilon} 2 equal to the resolution limit, (1/2{pi}{varepsilon}2)exp(-|r|2/2{varepsilon}2). The convolution function is given by h{otimes}g, where h is the instantaneous height fluctuation and g is the Gaussian function. This is similar to the effect of point spread functions in experiments. We find that the region of fluctuation spectra that scales according to capillary wave theory formalism depends on the complexity of the interfacial geometry, which may not always be detected at experimental resolutions. We propose that the different q-regimes in the fluctuation spectra can be used to characterize mode dependent inter-facial tensions to understand the interfaces beyond the linear line tension calculations. This could also be useful in interpretation of fluctuating boundaries in out-of-equilibrium in-vivo membrane systems that carry information about the nature of non-thermal (active) fluctuations in these systems.

biophysics↗

bioRxiv2019

Aging-related inflammation driven by cellular senescence enhances NAD consumption via activation of CD38+ macrophages

Decline in tissue NAD levels during aging is linked to aging and its associated diseases. However, the mechanism for aging-associated NAD decline remains unclear. Here we report that pro-inflammatory M1-like macrophages, but not naive or M2 macrophages, accumulate in metabolic tissues including visceral white adipose tissue and the liver during aging. Remarkably, these M1-like macrophages highly express the NAD consuming enzyme CD38 and have enhanced CD38-dependent NADase activity. We also find that senescent cells progressively accumulate in visceral white adipose tissue during aging and that inflammatory cytokines found in the supernatant from senescent cells (Senescence associated secretory proteins, SASP) induce macrophages to proliferate and to express CD38. These results highlight a new causal link between visceral tissue senescence and tissue NAD decline during aging and represent a novel therapeutic opportunity targeting maintenance of NAD levels during aging.

immunology↗