Biology subjects

Singh, T.

Publications and source records attributed to Singh, T..

At least 37 records · Page 2Linked to original sources

bioRxiv2025

The Notch1 intracellular domain orchestrates mechanotransduction of fluid shear stress

Hemodynamic shear stress regulates endothelial phenotype through activation of Notch1 signaling, yet the mechanistic basis for this activation is unclear. Here, we establish a fluid shear stress-dependent mechanism of Notch1 activation that is distinct from canonical ligand trans-endocytosis. Application of laminar flow triggers the rapid spatial polarization of full-length Notch1 heterodimers into downstream membrane microdomains. Unlike canonical transactivation, this response occurs independently of ligand redistribution, and Notch1 receptors are cis-endocytosed into the receptor-bearing cell within polarized microdomains prior to proteolytic activation. We discover that the Notch1 intracellular domain (ICD) critically orchestrates receptor polarization and proteolytic activation in response to flow but is dispensable for canonical ligand transactivation. Shear stress increases ICD interaction with annexin A2 and caveolar proteins which control Notch1 endocytosis and proteolytic compartmentalization. These findings define a flow-specific Notch1 mechanotransduction pathway linking receptor polarization and endocytosis with proteolytic activation and establish new mechanisms regulating Notch receptor activation.

cell biology↗

bioRxiv2025

Sensory Appendage Protein triggers alarm to pyrethroid in Indian malarial vector Anopheles culicifacies

Sensory appendage proteins (SAPs) serve as molecular arms of odorant-binding protein family members and mediate chemical communication from the external to the internal environment of the insect body. We recently reported that SAP members might have an important role in blood-feeding and/or insecticide resistance-associated behavioral physiologies, but the mechanism remains unclear. Here we show how SAP contributes (i) to the host attraction during navigation for blood-feeding, and (ii) triggers an alert via integrated actions of eyes and peripheral sensory organs to circumvent the toxic effect of insecticide in the susceptible strain of An. culicifacies. Among the six identified SAP members, AcSAP1 and AcSAP2 are abundantly expressed in the olfactory and legs, and their expression is significantly modulated at night in naive adult female mosquitoes. We noticed that AcSAP1 silencing significantly impairs the host-attraction properties of the mosquitoes. Its strong binding affinity to the synthetic pyrethroid prompted us to evaluate the AcSAP1 response to insecticide exposure. Confocal microscopy and molecular profiling data reveal a heightened modulation in the expression of AcSAP1 in eyes, and other peripheral sensory organs including the olfactory (****p<0.00001), legs (**p < 0.004), wings (***p < 0.0004). These findings collectively suggest a crucial role of AcSAP1 in facilitating mosquito navigation, and/or insecticide avoidance. Unraveling the basis of SAP interactions with peripheral sensory systems could pave the way for the development of novel vector control tools.

animal behavior and cognition↗

bioRxiv2025

Smooth pursuit eye movements contribute to long-latency reflex modulation in the lower extremity

Somatosensory mediated reactions play a fundamental role in adapting to environmental changes, particularly through long-latency responses (LLRs)--rapid corrective muscle responses (50-100ms) following limb perturbations that account for limb biomechanics and task goals. We investigated how smooth pursuit eye movements (SPEM), which are slow eye movements used to track moving objects, influence LLRs of the upper and lower limb during mechanical interactions with moving objects. In the first experiment, participants stood and stabilized their arm against a colliding virtual object. This occurred while subjects either visually pursued the moving object or fixated a central location. The robot occasionally applied a mechanical perturbation to the arm either 200ms or 60ms before the anticipated collision. As in previous studies, LLRs were observed in leg muscles to a perturbation of the upper limb. Moreover, leg LLRs were modulated by gaze, being larger during pursuit than fixation but only during the late perturbations. This timing-specific modulation aligns with previous reports of policy transitions in feedback control roughly 60ms before impact. Upper limb LLRs were not significantly impacted by gaze. This lack of modulation could reflect the context of upright stance, so we conducted a second experiment which was the same in all ways except that the subjects remained seated. Again, the upper limb LLRs were not impacted by gaze. The selective impact of gaze modulation on stance control highlights the sophisticated nature of coordinating eye movements, arm control, and whole-body postural responses. NEW and NOTEWORTHYSmooth pursuit eye movements are used to track moving objects. We show for the first time that smooth pursuit eye movements contribute to modulation of long-latency reflexes in the lower limb during virtual object interactions in upright stance. These findings suggest a neurophysiological link between predictive control of eye movements and feedback control of upright stance.

neuroscience↗

bioRxiv2024

In Silico Toxicity Assessment of Organophosphates: A DFT and Molecular Docking Study on Their Interaction with Human Serum Albumin (HSA)

This study presents an in silico analysis of the toxicity of Organophosphates (OPs) through their interaction with Human Serum Albumin (HSA) protein using density functional theory (DFT) and molecular docking approaches. Organophosphates, known for their widespread use as pesticides, have raised significant concerns due to their potential toxicological effects. To investigate the molecular mechanisms underlying OP toxicity, we conducted DFT calculations to determine the electronic properties and reactivity of selective OP compounds. Molecular docking simulations were performed to explore the binding affinity, interaction sites, and conformational changes of HSA upon exposure to OPs. The DFT analysis revealed key electronic descriptors, such as HOMO-LUMO gap and electrostatic potential, that indicate high reactivity of OPs with biological molecules. Docking results showed strong binding affinities between OPs and HSA, particularly at sites involved in metabolite and drug transport, suggesting potential interference with the proteins native function. The interaction of OPs with HSA was further supported by molecular dynamics simulations, which confirmed the stability of the OP-HSA complex over time. These findings provide critical insights into the molecular basis of organophosphate toxicity, emphasizing the importance of their interaction with HSA. The combined DFT and molecular docking approach offers a valuable framework for predicting the toxicological behavior of OPs and lays the foundation for further in vitro and in vivo studies. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=158 SRC="FIGDIR/small/628551v3_ufig1.gif" ALT="Figure 1"> View larger version (46K): org.highwire.dtl.DTLVardef@881814org.highwire.dtl.DTLVardef@e4cb5forg.highwire.dtl.DTLVardef@e3cb74org.highwire.dtl.DTLVardef@1426bb4_HPS_FORMAT_FIGEXP M_FIG C_FIG

bioinformatics↗

bioRxiv2024

Pharmacological inhibition of 11βhydroxysteroid dehydrogenase type 1 after myocardial infarction targets extracellular matrix processing and preserves cardiac function in a translational mini-pig model

Background and PurposePlasma glucocorticoids (GCs) increase acutely after myocardial infarction (MI), thereafter tissue levels are amplified selectively within cells expressing 11{beta}Hydroxysteroid Dehydrogenase type 1 (11{beta}HSD1) that regenerates active GCs from circulating metabolites. GCs initially protect cardiomyocytes and prevent excessive inflammation after MI but can also suppress subsequent wound repair leading to functional decline. The present study aimed to investigate the potential of pharmacological 11{beta}HSD1 inhibition after MI to prevent deterioration of cardiac function and its impact on wound repair. Experimental ApproachAdult female Gottingen mini-pigs underwent percutaneous balloon MI/reperfusion and were randomised to receive either oral 11{beta}HSD1 inhibitor AZD8329 (n=11), or vehicle (n=9), from 2 until 27 days later, with concurrent administration of clinically relevant therapeutic intervention (anti-platelet, statin and ACE inhibitor). Key ResultsAZD8329 treatment increased plasma accumulation of cortisone substrate consistent with successful 11{beta}HSD1 inhibition. Gadolinium-enhanced MRI showed equivalent infarct size in both groups prior to commencing treatment. 28 days after MI cardiac function and LV area were preserved in the AZD8329 treated group relative to vehicle. There was no impact of 11{beta}HSD1 inhibitor on neovascularisation or infarct area. Mass spectrometry imaging revealed AZD8329 binding to the healing infarct and altered regulation of extracellular matrix (ECM) processing was highlighted by birefringence microscopy and proteomic analysis. Conclusions and ImplicationsPharmacological inhibition of 11{beta}HSD1 after MI prevents deterioration of cardiac function and detrimental remodelling. 11{beta}HSD1 inhibitors have safely reached phase 2 clinical trials in diabetes and dementia and could be repurposed as an addition to standard care after MI to prevent the development of heart failure. Bullet Point Summary What is already known?O_LIGCs are released from the adrenal gland after MI, but also regenerated within the heart from circulating precursors by the enzyme 11{beta}HSD1 in cardiomyocytes, fibroblasts and macrophages. C_LIO_LIGenetic suppression of Hsd11b1 expression in the mouse promotes neovascularisation, prevents infarct expansion during infarct repair after MI and the development of heart failure. C_LI What does this study add?O_LIOral pharmacological inhibition of 11{beta}HSD1 after MI/reperfusion in a translational mini-pig model of MI receiving concurrent clinically relevant therapy prevents cardiac functional deterioration and adverse ventricular remodelling over the following 4 weeks. C_LIO_LIMass spectrometry imaging reveals target engagement of the 11{beta}HSD1i in the repairing infarct. C_LIO_LIThe mechanism is independent of neovascularisation but does involve modification of extracellular matrix remodelling during repair and scar formation. C_LI What is the clinical significance?O_LITissue 11{beta}HSD1 expression is increased in aging when the risk of MI is higher. C_LIO_LIPharmacological inhibitors of 11{beta}HSD1 have safely reached phase 2 clinical trials for dementia and metabolic disease and could be repurposed for use post-MI to prevent the development of heart failure. C_LI

pharmacology and toxicology↗

bioRxiv2024

HOXB6 and HOXB8 control immune-cancer cell interactions in pancreatic cancer.

Pancreatic ductal adenocarcinoma (PDAC) is a lethal cancer lacking effective drugs and therefore new treatment targets are needed. Transcriptomic analysis comparing human embryonic and PDAC tissue identified a large overlap of expression profiles suggesting a re-initiation of developmental programs in pancreatic cancer. Specifically, we identified the transcription factors HOXB6 and HOXB8 as potential key regulators in PDAC. Loss of HOXB6 and HOXB8 in pancreatic cancer cells inhibited cell proliferation, induced apoptosis and senescence and enhanced gemcitabine sensitivity. Moreover, reduced HOXB6 and HOXB8 expression in pancreatic and lung adenocarcinoma cell lines affected transcription of immune response pathways which resulted in an increased sensitivity of cancer cells to anti-tumorigenic activities of macrophages suggesting that the HOXB6 and HOXB8 immune regulatory pattern is conserved in different cancer types. Additionally, naive M0 macrophages exposed to HOXB8 deficient PDAC cells were unable to differentiate into tumor associated macrophages, suggesting that HOXB8 promotes the transition of initial anti-tumor macrophage to a tumor-promoting macrophage phenotype in pancreatic cancer. Our findings indicate that HOXB6 and HOXB8 play important roles in regulating cell proliferation, immune response and treatment resistance to promote pancreatic cancer tumorigenesis and could be useful therapeutic targets.

cancer biology↗

bioRxiv2024

Modelling genetic risk of β-cell dysfunction in human induced pluripotent stem cells from patients carrying the MTNR1B risk variant.

Disruptions in circadian rhythm, partly controlled by the hormone melatonin, increase the risk of type 2 diabetes (T2D). Accordingly, a variant of the gene encoding the melatonin receptor 1B (MTNR1B) is robustly associated with increased risk of T2D. This single nucleotide polymorphism (SNP; rs10830963; G-allele) is an expression quantitative trait locus (eQTL) in human pancreatic islets, conferring increased expression of MTNR1B, which is thought to perturb pancreatic {beta}-cell function. To understand this pathogenic mechanism in detail, we utilized human induced pluripotent stem cells (hiPSC), derived from individuals with T2D carrying the MTNR1B G-allele. Patient-derived fibroblasts were reprogrammed to hiPSC and single-base genome editing by CRISPR/Cas9 was employed to create isogenic lines of either the C/C or G/G genotypes (non-risk and risk, respectively). In addition, the human embryonic stem cell (hESC) line (HUES4) was subjected to genome editing to create isogenic lines of either the C/C or G/G genotypes. hiPSC and hESC were differentiated into {beta}-cells, using a 50-day 2D protocol. Single-base genome editing generated cells with the desired genotype at a success rate of >90%. Expression of stage-specific markers confirmed differentiation of both hiPSC and hESC into {beta}-cells. MTNR1B mRNA levels were consistently low in differentiated {beta}-cells, precluding quantitative analysis of gene expression. However, Western blot analysis showed higher levels of MTNR1B in differentiated {beta}-cells carrying the risk allele, consistent with rs10830963 (G-allele) being an eQTL in {beta}-cells. Insulin secretion in response to glucose and IBMX was similar between the genotypes, whereas addition of melatonin reduced secretion in G-allele carriers. We conclude that the stem cell-derived {beta}-cells are not sufficiently mature to allow determination of eQTL status at the mRNA level. However, we did observe increased MTNR1B protein and increased sensitivity of {beta}-cells from risk allele carriers (G-allele) to melatonin with regard to insulin secretion, thus supporting a functional role for the rs10830963 SNP in {beta}-cell dysfunction.

cell biology↗

bioRxiv2024

Impairments in proprioceptively-referenced limb and eye movements in chronic stroke

BackgroundUpper limb proprioceptive impairments are common after stroke and can affect daily function. Recent work has shown that stroke survivors have difficulty using visual information to improve proprioception. It is unclear how eye movements are impacted to guide action of the arm after stroke. Here, we aimed to understand how upper limb proprioceptive impairments impact eye movements in individuals with stroke. MethodsControl (N=20) and stroke participants (N=20) performed a proprioceptive matching task with upper limb and eye movements. A KINARM exoskeleton with eye tracking was used to assess limb and eye kinematics. The upper limb was passively moved by the robot and participants matched the location with either an arm or eye movement. Accuracy was measured as the difference between passive robot movement location and the active limb matching (Hand-End Point Error) or the active eye movement matching (Eye-End Point Error). ResultsWe found that individuals with stroke had significantly larger Hand and Eye-End Point Errors compared to controls. Further, we found that stroke participants had proprioceptive errors of the hand and eye were highly correlated [r=0.67, p=0.001], a relationship not observed for controls. ConclusionsEye movement accuracy declined as a function of proprioceptive impairment of the more-affected limb, which was used as a proprioceptive reference. The inability to use proprioceptive information of the arm to coordinate eye movements suggests that disordered proprioception impacts integration of sensory information across different modalities. These results have important implications for how vision is used to actively guide limb movement during rehabilitation.

neuroscience↗

bioRxiv2024

The genetic architecture and evolutionary consequences of the human pelvic form

Human pelvic shape has undergone significant evolutionary change since the divergence from the chimpanzee lineage. This transformation, involving the reduction of the pelvic canal size to support bipedal locomotion, is thought to give rise to the obstetrical dilemma, a hypothesis highlighting the mismatch between the large brain size of infants and the narrowed birth canal in females. Empirical evidence for this classic hypothesis has been equivocal, largely due to a lack of sample size and appropriate types of data. To elucidate the genetic underpinnings of pelvic morphology, we applied a deep learning model to 31,115 dual-energy X-ray absorptiometry (DXA) from the UK Biobank, extracting a set of seven pelvic proportion (PP) phenotypes, including measures of the birth canal. All PPs were found to be highly heritable ([~]25-40%) and a genome-wide association study of these traits identified 179 independent loci. Unlike other skeletal proportions including long bone lengths, the subpubic angle associated with the birth canal exhibits a genetic correlation between sexes significantly less than 1, in line with sex-specific reproductive function. PPs were also left-right asymmetric but not heritable and instead associated with handedness. We conducted phenotypic and genetic association analyses to link PPs to 3 facets of the dilemma: locomotion, pelvic floor function and childbirth. Larger birth canal phenotypes were associated with reduced walking pace, decreased risk of back pain, and increased risk of hip osteoarthritis - phenotypes linked to locomotor efficiency. We also observed that a narrower birth canal width was associated with a reduced risk of pelvic floor disorders. When examining childbirth-related outcomes, narrower birth canal phenotypes were associated with increased risk of emergency cesarean sections and obstructed labor due to insufficient dilation, but not obstructed labor due to positioning of the fetus. Finally, we examined whether the dilemma might have been alleviated through evolution. We found no association between any PPs and gestational duration, contrary to the initial prediction by Washburn in 1960. However, we found that the birth weight of the child, a proxy for skull and brain size, was genetically correlated with birth canal width but not with other PPs. Collectively, our study offers fresh insight on a 60-year-old debate in human evolutionary studies. Our results support the idea that the obstetrical dilemma has played a central role in the co-evolution of the human brain and pelvis, while also highlighting the potential role of associated factors such as pelvic floor health.

genetics↗

bioRxiv2024

Predictive posture stabilization before contact with moving objects: equivalence of smooth pursuit tracking and peripheral vision

Postural stabilization is essential to effectively interact with our environment. Humans preemptively adjust their posture to counteract impending disturbances, such as those encountered during interactions with moving objects, a phenomenon known as anticipatory postural adjustments (APAs). APAs are thought to be influenced by predictive models that incorporate object motion via retinal motion and extra-retinal signals. Building on our previous work that examined APAs in relation to the perceived momentum of moving objects, here we explored the impact of object motion within different visual field sectors on the human capacity to anticipate motion and prepare APAs for contact between virtual moving objects and the limb. Participants interacted with objects moving towards them under different gaze conditions. In one condition, participants fixated on either a central point (central fixation) or left-right of the moving object (peripheral fixation), while in another, they followed the moving object with smooth pursuit eye movements (SPEM). We found that APAs had the smallest magnitude in the central fixation condition and that no notable differences in APAs were apparent between the SPEM and peripheral fixation conditions. This suggests that the visual system can accurately perceive motion of objects in peripheral vision for posture stabilization. Using Bayesian Model Averaging, we also evaluated the contribution of different gaze variables, such as eye velocity and gain (ratio of eye and object velocity) and showed that both eye velocity and gain signals were significant predictors of APAs. Taken together, our study underscores the roles of oculomotor signals in modulation of APAs. New and NoteworthyWe show that the human visuomotor system can detect motion in peripheral vision and make anticipatory adjustments to posture before contact with moving objects, just as effectively as when the eye movement system tracks those objects through smooth pursuit eye movements. These findings pave the way for research into how age-induced changes in spatial vision, eye movements, and motion perception could affect the control of limb movements and postural stability during motion-mediated interactions with objects.

neuroscience↗

bioRxiv2024

Short-term performance impairment in adaptive reaching following exercise induced neuromuscular fatigue in lower-limb postural muscles

Neuromuscular fatigue (NMF) induces temporary reductions in muscle force production capacity, affecting various aspects of motor function. While studies have extensively explored NMFs impact on muscle activation patterns and postural stability, its influence on motor adaptation processes remains less understood. This paper investigates the effects of localized NMF on motor adaptation during upright stance, focusing on reaching tasks. Utilizing a force field perturbation paradigm, participants performed reaching movements while standing upright before and after inducing NMF in the ankle dorsiflexor muscles. Results revealed that despite maintained postural stability, participants in the NMF group exhibited larger movement errors during reaching tasks, suggesting impaired motor adaptation. This was evident in both initial and terminal phases of adaptation, indicating a disruption in learning processes rather than a decreased adaptation rate. Analysis of electromyography activation patterns highlighted distinct strategies between groups, with the NMF group showing altered activation of both fatigued and non-fatigued muscles. Additionally, differences in co-activation patterns suggested compensatory mechanisms to prioritize postural stability despite NMF-induced disruptions. These findings underscore the complex interplay between NMF, motor adaptation, and postural control, suggesting a potential role for central nervous system mechanisms in mediating adaptation processes. Understanding these mechanisms has implications for sports performance, rehabilitation, and motor skill acquisition, where NMF may impact the learning and retention of motor tasks. Further research is warranted to elucidate the transient or long-term effects of NMF on motor adaptation and its implications for motor rehabilitation interventions. New & NoteworthyWe assessed motor adaptation during force field reaching following exercise-induced neuromuscular fatigue (NMF) on postural muscles. NMF impaired adaptation in performance. Similarly, diverging activation strategies were observed in the muscles. No effects were seen on measures of postural control. These results suggest the remodulation of motor commands to the muscles in presence of NMF, which may be relevant in settings where participants could be exposed to NMF while learning, such as sports and rehabilitation.

physiology↗

bioRxiv2024

Regulation of Diseases-Associated Microglia in the Optic Nerve by Lipoxin B4 and Ocular Hypertension

BackgroundThe resident astrocyte-retinal ganglion cell (RGC) lipoxin circuit is impaired during retinal stress, which includes ocular hypertension-induced neuropathy. Lipoxin B4 produced by homeostatic astrocytes directly acts on RGCs to increase survival and function in ocular hypertension-induced neuropathy. RGC death in the retina and axonal degeneration in the optic nerve are driven by the complex interactions between microglia and macroglia. Whether LXB4 neuroprotective actions include regulation of other cell types in the retina and/or optic nerve is an important knowledge gap. MethodsCellular targets and signaling of LXB4 in the retina were defined by single-cell RNA sequencing. Retinal neurodegeneration was induced by injecting silicone oil into the anterior chamber of the mouse eyes, which induced sustained and stable ocular hypertension. Morphological characterization of microglia populations in the retina and optic nerve was established by MorphOMICs and pseudotime trajectory analyses. The pathways and mechanisms of action of LXB4 in the optic nerve were investigated using bulk RNA sequencing. Transcriptomics data was validated by qPCR and immunohistochemistry. Differences between experimental groups were assessed by Students t-test and one-way ANOVA. ResultsSingle-cell transcriptomics identified microglia as a primary target for LXB4 in the healthy retina. LXB4 downregulated genes that drive microglia environmental sensing and reactivity responses. Analysis of microglial function revealed that ocular hypertension induced distinct, temporally defined, and dynamic phenotypes in the retina and, unexpectedly, in the distal myelinated optic nerve. Microglial expression of CD74, a marker of disease-associated microglia in the brain, was only induced in a unique population of optic nerve microglia, but not in the retina. Genetic deletion of lipoxin formation correlated with the presence of a CD74 optic nerve microglia population in normotensive eyes, while LXB4 treatment during ocular hypertension shifted optic nerve microglia toward a homeostatic morphology and non-reactive state and downregulated the expression of CD74. Furthermore, we identified a correlation between CD74 and phospho-phosphoinositide 3-kinases (p-PI3K) expression levels in the optic nerve, which was reduced by LXB4 treatment. ConclusionWe identified early and dynamic changes in the microglia functional phenotype, reactivity, and induction of a unique CD74 microglia population in the distal optic nerve as key features of ocular hypertension-induced neurodegeneration. Our findings establish microglia regulation as a novel LXB4 target in the retina and optic nerve. LXB4 maintenance of a homeostatic optic nerve microglia phenotype and inhibition of a disease-associated phenotype are potential neuroprotective mechanisms for the resident LXB4 pathway.

molecular biology↗

bioRxiv2024

Enhancement of Mediodorsal Thalamus Rescues Aberrant Belief Dynamics in a Novel Mouse Model for Schizophrenia

Optimizing behavioral strategy requires belief updating based on new evidence, a process that engages higher cognition. In schizophrenia, aberrant belief dynamics may lead to psychosis, but the mechanisms underlying this process are unknown, in part, due to lack of appropriate animal models and behavior readouts. Here, we address this challenge by taking two synergistic approaches. First, we generate a mouse model bearing patient-derived point mutation in Grin2a (Grin2aY700X+/-), a gene that confers high-risk for schizophrenia and recently identified by large-scale exome sequencing. Second, we develop a computationally trackable foraging task, in which mice form and update belief-driven strategies in a dynamic environment. We found that Grin2aY700X+/- mice perform less optimally than their wild-type (WT) littermates, showing unstable behavioral states and a slower belief update rate. Using functional ultrasound imaging, we identified the mediodorsal (MD) thalamus as hypofunctional in Grin2aY700X+/- mice, and in vivo task recordings showed that MD neurons encoded dynamic values and behavioral states in WT mice. Optogenetic inhibition of MD neurons in WT mice phenocopied Grin2aY700X+/- mice, and enhancing MD activity rescued task deficits in Grin2aY700X+/- mice. Together, our study identifies the MD thalamus as a key node for schizophrenia-relevant cognitive dysfunction, and a potential target for future therapeutics.

neuroscience↗

bioRxiv2023

PMTPred: Machine Learning Based Prediction of Protein Methyltransferases using the Composition of k-spaced Amino Acid Pairs

Protein methyltransferases (PMTs) are a group of enzymes that help to catalyze the transfer of a methyl group to its substrates. These enzymes play an important role in epigenetic regulation and are able to methylate various substrates with DNA, RNA, protein, and smallmolecule secondary metabolites. Dysregulation of methyltransferases is involved in different types of human cancers. However, in light of the well-recognized significance of PMTs, it becomes crucial to have reliable and fast methods for identifying these proteins. In the present work, we propose a machine-learning-based method for the identification of PMTs. Various sequence-based features were calculated and prediction models were develped using different machine-learning methods. A ten-fold cross-validation technique was used for model training. The SVM-based CKSAAP model gave the best prediction and achieved the highest accuracy of 87.94% with balance sensitivity (88.8%) and specificity (87.11%) with MCC of 0.759 and AUROC of 0.945. Also, SVM performed better than the compared deep learning algorithms for the prediction of PMTs. Finally, the best model was implemented in standalone software of PMTPred to facilitate the prediction of PMTs. The PMTPred achieved 86.50% prediction accuracy with 82.33% sensitivity, 90.67% specificity and ROC value 0.939 on the blind dataset. The standalone software of PMTPred is freely available for download at https://github.com/ArvindYadav7/PMTPred for research and academic use.

bioinformatics↗

bioRxiv2023

Anatomy of a mega-radiation: Biogeography and niche evolution in Astragalus

Astragalus (Fabaceae), with more than 3,000 species, represents a successful radiation of morphologically highly similar species found across the Northern Hemisphere. It has attracted attention from systematists and biogeographers, who have asked what factors might be behind the extraordinary diversity of this important arid-adapted clade and what sets it apart from close relatives with far less species richness. Here, for the first time using extensive taxonomic sampling in a phylogenetic analysis, we ask whether (1) Astragalus is uniquely characterized by bursts of radiation or is instead similar to related taxa. Then we test whether the species diversity of Astragalus is attributable specifically to its predilection for (2) cold and arid habitats or (3) particular soils. Finally, we test (4) whether Astragalus originated in central Asia as proposed and (5) whether niche evolutionary shifts were associated with the colonization of other continents. Our results point to the importance of heterogeneity in the diversification of Astragalus, with upshifts associated with the earliest divergences but attributable to no abiotic factor or biogeographic regionalization tested here. The only potential correlate with diversification we identified was chromosome number. We find strong evidence for a central Asian origin and direct dispersals from this region responsible for much of the present-day distribution, highlighting the importance of central Asia as a biogeographic gateway. In contrast to diversification shifts, biogeographic shifts have a strong association with the abiotic environment. Our most important result was a fundamental divide in soil types and diurnal temperature variation between the Eastern and Western Hemisphere species; this divergence does not reflect differences in available habitat among these biogeographic domains but may reflect unique local gains of edaphic and abiotic stress adaptations. While large clades are logistically difficult to tackle, our investigation shows the importance of phylogenetic and evolutionary studies of "mega-radiations." Our findings reject any simple key innovation behind the dominance and richness of Astragalus and underline the often nuanced, multifactorial processes leading to species-rich clades.

plant biology↗

bioRxiv2023

Discovery of a Novel 3site State as the Multi-Substrate Bound State of P450cam

Archetypal metalloenzyme Cytochrome P450cam (CYP101A1) catalyzes regioselective hydroxylation of its native substrate camphor in heme active site. However, the proposal of potential existence of additional substrate binding modes distal from the active site in P450cam and their concomitant roles in regulating recognition at active site have remained a matter of recurring discourse. Herein we report the discovery of a novel 3site state in P450cam, where three substrate molecules were observed to simultaneously bind to P450cam at three distinct sites including the heme active site. These three binding modes, hereby referred as catalytic, waiting and allosteric binding modes in 3site state, are allosterically inter-linked and function in mutually synergistic fashion. The 3site state possesses regio-selective conformations of substrate essential for catalysis and establishes substrate-ingress and product exit process to and from the active site via two distinct channels. The ensemble of three-state binding modes are found to be self-consistent with NMR pseudo-contact shift data obtained from TROSY-HSQC measurements and DEER based predictions. Binding of redox partner Putidaredoxin with 3site model retains closed conformation of 3site state, siding with NMR based hypothesis that the catalysis would take place in closed insulation of P450cam even in presence of its redox partner. Statement of SignificanceUbiquitous superfamily of mono-oxygenases cytochrome P450s are involved in broad range of metabolic process in all domains of life and are also important drug targets. Apart from the well known and established binding mode in heme active site, the substrate bindings at additional distal sites have been postulated in multitude of P450s. Using the archetypal bacterial cytochrome P450 i.e., P450cam, a novel 3site state of cytochrome P450 is elucidated in this work. The novel 3site state has two additional binding modes namely waiting and allosteric (also postulated previously), apart from known binding mode catalytic in the active site. The known functions of P450cam are found to be most optimally explained by this 3site state, instead of single substrate bound catalytic state. This state can be of critical importance for CYP superfamily at large and potentially be useful in understanding the non-Michaelis behaviour, observed in many P450s.

biochemistry↗

bioRxiv2023

Smooth pursuit eye movements contribute to anticipatory force control during mechanical stopping of moving objects

When stopping a closing door or catching an object, humans process the motion of inertial objects and apply reactive limb force over short period to interact with them. One way in which the visual system processes motion is through extraretinal signals associated with smooth pursuit eye movements (SPEM). We conducted three experiments to investigate how SPEM contribute to anticipatory and reactive hand force modulation when interacting with a virtual object moving in the horizontal plane. We hypothesized that SPEM signals are critical for timing motor responses, anticipatory control of hand force, and task performance. Participants held a robotic manipulandum and attempted to stop an approaching simulated object by applying a force impulse (area under force-time curve) that matched the objects virtual momentum upon contact. We manipulated the objects momentum by varying either its virtual mass or its speed under free gaze or constrained gaze conditions. We examined gaze variables, timing of hand motor responses, anticipatory force control, and overall task performance. Our results show that when SPEM were constrained, anticipatory modulation of hand force prior to contact decreased. However, constraining SPEM did not seem to affect the timing of the motor response or the task performance. Together, these results suggest that SPEM may be important for anticipatory control of hand force prior to contact and may also play a critical role in anticipatory stabilization of limb posture when humans interact with moving objects. New and NoteworthyWe show for the first time that smooth pursuit eye movements (SPEM) play a role in modulation of anticipatory control of hand force to stabilize posture against contact forces. SPEM are critical for tracking moving objects, facilitate processing motion of moving objects, and are impacted during aging and in many neurological disorders, such as Alzheimers disease and Multiple Sclerosis. These results provide a novel basis to probe how changes in SPEM could contribute to deficient limb motor control in older adults and patients with neurological disorders.

neuroscience↗

bioRxiv2023

Notch1 cortical signaling regulates epithelial architecture and cell-cell adhesion

Notch receptors control tissue morphogenic processes that involve coordinated changes in cell architecture and gene expression, but how a single receptor can produce these diverse biological outputs is unclear. Here we employ a 3D organotypic model of a ductal epithelium to reveal tissue morphogenic defects result from loss of Notch1, but not Notch1 transcriptional signaling. Instead, defects in duct morphogenesis are driven by dysregulated epithelial cell architecture and mitogenic signaling which result from loss of a transcription-independent Notch1 cortical signaling mechanism that ultimately functions to stabilize adherens junctions and cortical actin. We identify that Notch1 localization and cortical signaling are tied to apical-basal cell restructuring and discover a Notch1-FAM83H interaction underlies stabilization of adherens junctions and cortical actin. Together, these results offer new insights into Notch1 signaling and regulation, and advance a paradigm in which transcriptional and cell adhesive programs might be coordinated by a single receptor.

cell biology↗