Biology subjects

S, A.

Publications and source records attributed to S, A..

At least 19 recordsLinked to original sources

bioRxiv2026

CDK1 facilitates RAD51-mediated DNA repair to protect dictyate stage arrested oocytes from genotoxic stress

Oocytes arrested at the dictyate stage of meiosis I must maintain genomic integrity for prolonged periods to preserve female fertility. During this extended arrest, DNA lesions arising from endogenous and exogenous sources threaten oocyte survival, yet the molecular mechanisms coordinating DNA repair in dormant oocytes remain poorly understood. Here, we identify cyclin-dependent kinase 1 (CDK1) as a critical regulator of the oocyte DNA damage response and homologous recombination (HR) repair under genotoxic stress. Using cisplatin-induced DNA damage models in fetal goat ovaries and neonatal mouse ovaries, we investigated repair mechanisms operating within the ovarian reserve. Label-free proteomic profiling revealed significant enrichment of DNA damage response pathways following cisplatin exposure, with CDK1 emerging as one of the most prominently upregulated kinases. Pharmacological inhibition of CDK1 had little effect on follicle survival under physiological conditions but aggravated oocyte and follicle loss following DNA damage, indicating a stress-dependent role for CDK1 in preserving ovarian follicle pool integrity. Mechanistically, DNA damage activated a Chk2-dependent signaling pathway that promoted p63 phosphorylation and altered the WEE1-CDK1 regulatory axis, resulting in reduced inhibitory CDK1 phosphorylation (Thr14/Tyr15) and increased activating phosphorylation (Thr161). Activated CDK1 was associated with enhanced RAD51 phosphorylation and accumulation at DNA damage foci, supporting homologous recombination (HR)-mediated repair in dictyate-arrested oocytes. In contrast, CDK1 inhibition reduced phospho-RAD51 levels, impaired RAD51 localization, increased persistent {gamma}H2AX accumulation, and elevated oocyte apoptosis. Notably, suppression of CDK1 was accompanied by increased expression of the non-homologous end joining (NHEJ) marker Ku80 and the nucleotide excision repair (NER) factor XPA, suggesting increased engagement of alternative DNA repair pathways. Furthermore, inhibition of Chk2 abolished the DNA damage-associated CDK1 activation signature and restored WEE1 expression, supporting a model in which CDK1 functions downstream of Chk2 signaling during the oocyte DNA damage response. Collectively, our findings identify a previously unrecognized Chk2-CDK1-RAD51 signaling axis that coordinates homologous recombination repair in dormant oocytes and safeguards ovarian follicular pool integrity under genotoxic stress. These findings provide new mechanistic insight into how dictyate-arrested oocytes maintain genome stability during prolonged meiotic arrest.

Cell Biology↗

bioRxiv2026

A Multiscale Computational Framework Linking Cortical Microtubule Dynamics to Plant Tissue Morphogenesis

Plant morphogenesis emerges through the coordinated regulation of cell growth and mechanical interactions across multiple spatial scales. A central role in this process is played by cortical microtubule (MT) arrays, which guide cellulose deposition and thereby regulate anisotropic cell expansion. Here, we develop a coupled multiscale computational framework integrating a dynamic vertex model of tissue mechanics with a stochastic model of cortical MT dynamics. Within this framework, MT organization regulates anisotropic cell-wall stiffness, while evolving cell geometry feeds back to influence MT alignment through bidirectional mechanochemical coupling. Simulations show that distinct regimes of MT self-organization generate qualitatively different tissue growth behaviors, ranging from isotropic expansion to strongly anisotropic elongation. Together, our results demonstrate that stochastic a complex coupling of MT self-organization with cell geometry and tissue mechanics is sufficient to generate emergent tissue-scale growth anisotropy, establishing a minimal multiscale framework linking cytoskeletal dynamics to plant tissue morphogenesis.

Systems Biology↗

bioRxiv2026

Herbivory-induced alterations in cytosolic proteins of pigeon pea (Cajanus cajan) leaves

Insect herbivory triggers cytosolic proteome reprogramming by activating defense pathways and modulating key metabolic processes. We found that simulated herbivory in pigeon pea (Cajanus cajan) induced reactive oxygen species (ROS) production and molecular alterations within 12 hours (h) of post treatment. We compared the leaf proteome profiles of two cultivated genotypes, ICPL 332 (moderately resistant) and ICPL 87 (susceptible), using two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) coupled with mass spectrometry (MS). More than 220 protein spots were detected in ICPL 332 and over 200 in ICPL 87. Comparative analysis revealed 75 differentially accumulated proteins (DAPs), of which 40 were consistently reproducible across biological replicates. These included 11 unique to ICPL 87, 9 unique to ICPL 332, and 10 common to both genotypes. Among the shared DAPs, ICPL 332 showed five upregulated and five downregulated, whereas ICPL 87 exhibited only two upregulated and eight downregulated. Functional categorization grouped DAPs into primary metabolism, stress response, and growth and development. Proteins related to primary metabolism were largely downregulated in both genotypes, while stress-associated proteins exhibited substantial downregulation in ICPL 87 compared to ICPL 332. Overall, the results demonstrate proteomic adjustments underlying defense responses in pigeon pea genotypes.

plant biology↗

bioRxiv2026

HIPK2-and IKKβ-dependent phosphorylation stabilizes TAp63α during the oocyte DNA damage response

TAp63, a member of the p53 family, serves as a central quality control factor in oocytes, safeguarding genomic integrity during the prolonged dictyate arrest of meiosis. In healthy oocytes, TAp63 is maintained in an inactive dimeric state; upon DNA damage, it undergoes phosphorylation-dependent tetramerization, enabling transcriptional activation of pathways that determine oocyte fate. While the upstream activation cascade of TAp63 has been well characterized, the mechanisms that regulate its stability during the DNA damage response remain incompletely understood. Here, we identify the kinases HIPK2 and IKK{beta} as key regulators of TAp63 stability. We show that TAp63 interacts with both kinases and is phosphorylated at distinct residues, T452 by HIPK2 and S4/S12 by IKK{beta} in vitro and in vivo, in addition to previously described CHK2 and CK1-mediated phosphorylation. Functionally, these phosphorylation events do not primarily contribute to activation, but instead stabilize TAp63 by limiting MDM4-dependent ubiquitination and subsequent proteasomal degradation. Mechanistically, our data support a model in which CHK2 and CK1 initiate TAp63 phosphorylation, while HIPK2 and IKK{beta} act in a complementary manner to maintain protein stability during genotoxic stress. Disruption of HIPK2 or IKK{beta} activity reduces TAp63 stability, whereas their inhibition in vivo attenuates oocyte loss following DNA damage, resulting in increased preservation of the follicle pool. Importantly, these effects are observed across multiple systems, including mouse models and ex vivo goat ovary cultures, supporting an evolutionarily conserved role for this regulatory axis. Together, our findings uncover a previously unidentified layer of TAp63 regulation, in which phosphorylation not only contributes to its activation but also enhances protein stability, thereby fine-tuning oocyte responses to DNA damage. Our results further indicate that HIPK2 and IKK{beta}-mediated phosphorylation modulates oocyte survival under genotoxic stress, highlighting this pathway as a potential target for strategies aimed at limiting oocyte loss.

cell biology↗

bioRxiv2025

Lipid Droplet Remodeling Safeguards Redox Balance through the DGAT1-PANK2-NRF2 Axis in Mammalian Oocytes and Drives Age-Associated Decline

How lipid droplets (LDs) buffer metabolic stress and redox imbalance in aging oocytes remains poorly understood. Here, we identify de novo LD remodeling as a metabolic capacitor that couples lipid storage to mitochondrial fitness and oxidative resilience in mammalian oocytes. Live imaging revealed pronounced LD dynamics, with LD number peaking at metaphase I and declining by metaphase II, while LD area shifted inversely. Despite stable triacylglyceride and free fatty acid pools, {beta}-oxidation increased sharply, indicating elevated lipid turnover during meiotic progression. Spatial mapping and fatty-acid tracing demonstrated that newly synthesized lipids are actively incorporated into LDs, which arise primarily from the endoplasmic reticulum and engage with lysosomes and mitochondria. Acute inhibition of DGAT1, the rate-limiting enzyme of LD biogenesis, disrupted meiotic maturation and triggered oxidative stress, mitochondrial aggregation, and ultrastructural damage. Proteomic profiling revealed robust PANK2 upregulation and suppression of NRF2-linked antioxidant pathways. Mechanistic analyses showed that {beta}-oxidation blockade, PANK2 inhibition, antioxidant supplementation, or NRF2 activation each partially rescued DGAT1-dependent defects, and genetic validation in NRF2-null oocytes confirmed pathway dependence. Notably, aged oocytes exhibited reduced de novo LD biogenesis and impaired DGAT1-ER organization despite increased LD accumulation, resulting in smaller, metabolically inert droplets and a mismatch between lipid formation and utilization. Inhibiting PANK2 alleviated oxidative stress in aged oocytes, further implicating the DGAT1-PANK2-NRF2 axis in redox control and oocyte quality. Together, these findings establish LD biogenesis as a core metabolic capacitor safeguarding mitochondrial and organelle integrity during meiosis and reveal dysfunction of the DGAT1-PANK2-NRF2 axis as a mechanistic driver of reproductive aging.

cell biology↗

bioRxiv2025

RNA-Binding moonlighting function of metabolic enzymes reveals deep evolutionary roots in Cyanobacteria

RNA-binding proteins (RBPs) have emerged as key regulators of diverse physiological and metabolic processes in cells. Notably, many metabolic enzymes exhibit moonlighting RNA-binding functions, and a substantial fraction localize to chloroplasts, the primary hub of photosynthesis and cellular metabolic homeostasis. Since chloroplasts originated from free-living cyanobacteria, understanding the RBP repertoire in these ancient phototrophs holds particular evolutionary and functional significance. A comprehensive characterization of the cyanobacterial RBPome is still lacking. Here, we employed Synechococcus elongatus PCC 7942, a model cyanobacterium, to define its RBPome using an RNA-interactome capture approach. We identified 136 RBPs, of which nearly 30% are associated with metabolic pathways, a proportion notably higher than that observed in bacteria, algae, plants, flies, worms, or animals. Strikingly, several enzymes from core metabolic pathways, including glycolysis/gluconeogenesis, the TCA cycle, and the pentose phosphate pathway, that are known RNA binders in humans are also conserved as RBPs in cyanobacteria. We identified a wide array of proteins from the photosynthetic apparatus exhibiting RNA-binding activity, many of which are conserved across the green lineage. In silico structural alignments of RNA-binding metabolic enzymes with their NAD(P)-binding pockets, a potential site for RNA-binding, suggests a broad conservation of RNA-binding capacity of core metabolic enzymes across species. Recent discoveries have revealed that RNA-binding can modulate enzymatic activity. In this context, our findings suggest that RNA-mediated control of core cellular metabolic processes may be widespread in cyanobacteria and riboregulation might be an evolutionarily ancient mechanism, potentially tracing its origins back to cyanobacteria.

plant biology↗

bioRxiv2025

Automated Eight-Stage Classification of Drosophila melanogaster Using Transfer-Learning CNNs with Mobile Live-Inference Deployment

Drosophila melanogaster is a foundational model organism whose rapid development and genetic tractability underpin research in genetics, development, and disease. Manual staging of its embryonic, larval, and pupal phases is slow and error-prone. An automated eight-class classifier is introduced to distinguish egg, first-, second-, and third-instar larvae, as well as white, brown, eye, and black pupae, from stereo-microscope images. By fine-tuning ImageNet-pretrained CNNs (ResNet-50, InceptionV3, ConvNeXtSmall) on a balanced dataset ([~]300 images per class), the best-performing model (ResNet-50) achieves 85% accuracy (F1 = 0.85) on a held-out validation set, significantly outperformed alternatives. Primary misclassifications align with subtle morphological transitions between adjacent stages. To facilitate broad adoption, the ResNet-50 model has been deployed in a lightweight Streamlit app offering live-camera inference ({approx}8 FPS on mobile). All code, pretrained weights, and data are publicly available, enabling scalable, high-throughput Drosophila staging for diverse experimental workflows.

bioinformatics↗

bioRxiv2025

Super-resolution Fluorescence Microscopy Reveals Densely Packed EboV Membrane Glycoprotein Clusters in the Cell Plasma-membrane During Ebola Infection

The glycoprotein (GP) of Ebola virus plays a central role in the viral entry into host cells. The detailed study of Ebola glycoprotein in the cellular system is unknown, mainly due to the resolution limit ([~]{lambda} /2) of optical fluorescence microscopes. To overcome this, we propose to use single-molecule localization microscopy (SMLM), which enables the study of the role of glycoproteins at both the single-molecule and ensemble levels. Accordingly, a photoactivable recombinant probe Dendra2 - EboV Glyc is developed by linking Ebola glycoprotein with Dendra2. The transient expression of photoactivable recombinant protein in NIH3T3 cells indicated the dominant presence of glycoprotein in the plasma membrane with regions of densely packed glycoproteins. Super-resolution single-molecule imaging gives direct proof of glycoprotein clusters in the plasma membrane. The corresponding single-molecule analysis using DBSCAN clustering algorithm reveals a dense packing of glycoprotein with an estimated average density of 5345 mol/{micro}m2 compared to the density in other regions of the membrane. The number of glycoproteins is estimated to be as high as 642 molecules per cluster and clusters spread area of {approx} 0.1219 {micro}m2 after 48 hrs post-transfection. Noting that glycoprotein is involved in cellular attachment, endosomal entry, and membrane fusion, the formation of glycoprotein clusters in the membrane is significant in the progression of Ebola pathogenesis. A drug capable of dispersing the glycoproteins clusters could be of therapeutic potential to lessen the intensity of infection and to slow down the disease progression. Statement of SignificanceThe fact that powerful fluorescence microscopes (such as SMLM) are available is a boon to disease biology, specifically to study the function of viral proteins at the cellular level. Ebola, one of the nocturnal viral diseases, is a threat and calls for study at the single-molecule level to understand the dynamics (kinetics and ensemble) of glycoprotein during viral infection. In this respect, the super-resolution microscopy techniques may help reveal the biological mechanism behind the infection process at the single-molecule level. A photoactivable fluorescent probe (Dendra2 - EboV Glyc) containing the gene-of-interest (EboVGP) is developed, and transfection studies are carried out, showing densely packed glycoprotein clusters in the cell plasma membrane with specific density, number, and size. We believe that dispersing these glycoprotein protein clusters could be of therapeutic potential.

biophysics↗

bioRxiv2025

pH induced motility pattern change in a marine dinoflagellate

Flagella-driven motility is a conserved feature across eukaryotic lineages, from unicellular plankton to mammals. In marine dinoflagellates, such as Lingulodinium polyedra, motility underlies diel vertical migration (DVM), a key adaptive strategy that enables access to spatio-temporally segregated resources in the water column. To investigate how pH influences motility, we used L. polyedra and two other dinoflagellates as a model and used a multi-particle tracking algorithm to monitor and quantitatively analyze cellular motility. Under normal pH conditions, L. polyedra displays linear, random motility with variable speeds. Upon CO{superscript 2}-induced acidification, we observed a dose-dependent decrease in motility speed accompanied by a striking behavioral shift: within minutes of pH reduction, over 90% of the cells transitioned to spiral motility. This effect was both reversible and reproducible, including when pH was modified chemically rather than via CO{superscript 2}. The rapid onset of these changes suggests a non-genomic mode of regulation, an area that remains largely unexplored in phytoplankton. We hypothesize that external pH modulates flagellar dynamics in dinoflagellates. Our findings offer new insights into the link between environmental pH and flagellar motility and provide a platform for investigating non-genomic responses to ocean acidification in marine phytoplankton.

ecology↗

bioRxiv2025

Deep Learning-Driven Gender Classification of Drosophila melanogaster: Advancing Real-Time Analysis with Mobile Integration

Researchers extensively use Drosophila melanogaster as a model organism in genetic research and developmental studies to better understand human diseases such as cancer and diabetes. For such investigations, it is necessary to identify and classify male and female flies. This study uses deep learning techniques for gender classification of Drosophila melanogaster via models YOLOv8, Detectron2, ResNet-50, InceptionV3, and MobileNet. Benchmarking revealed that YOLOv8 achieved a maximum accuracy of 98.0%. The classification and object detection models are integrated into a mobile application, facilitating real-time gender classification. This study demonstrates the possibility of integrating deep learning models with mobile technology while ensuring an efficient way for gender prediction, leading to better reproducibility and generating new opportunities in biological research.

scientific communication and education↗

bioRxiv2025

Chiral Single Molecule Localization Microscopy (chiralSMLM)

Chiral fingerprints are unique to a molecule and convey information related to binding sites, conformation, and its immediate chemical environment. Deciphering this information is expected to aid a better understanding of the biological processes (protein trafficking, kinetics, and aggregation) in a cellular environment at the scale of a single molecule. Here, we present a new optical technique called chiralSM LM, to selectively detect chiral enantiomers (both left-handed and right-handed) of single molecules and generate a super-resolved map to understand the role of chirality associated with the biological processes in a cell. Accordingly, calibration, characterization, and optimization of the system are carried out using a polarized light source (linear polarized, left and right circularly polarized) and by recording known structures (Actin filaments) in a cell. The system is employed to carry out cell transfection studies on two different disease models (Hemagglutinin protein for Influenza type-A and NS3 protein for Dengue type-2) to understand the role of molecular chirality on disease specific biological processes leading to clustering. Single-molecule cluster analysis revealed that left-handed Dendra2-HA and Dendra2-NS3 molecules have a larger footprint, suggesting the role of chiral molecules in promoting cluster formation. In addition, the presence of left-handed molecules at the cluster-periphery is perplexing. This is interesting, as it demonstrates the active role of single molecule handedness (left or right) during protein clustering in a transfected cell. The new classification of single molecules purely based on their chiral nature is expected to advance single-molecule imaging and provide new insights in disease biology. Summary & SignificanceThe chirality of single protein molecules is crucial to its functioning. This provides a new perspective on understanding biological functions, based solely on the chiral nature of proteins, and deciphers its role in a cellular processes. We developed a chiralSM LM microscopy system to access the role of chirality on the clustering behavior of viral proteins (HA for Influenza type-A and NS3 in Dengue type-2) in a cell. Hence, the technique offers a new approach to quantify chirality aided molecular interactions in disease biology.

biophysics↗

bioRxiv2024

Romiumeter: An Open-Source Inertial Measurement Unit-Based Goniometer for Range of Motion Measurements

Range of motion (ROM) serves as a crucial metric for assessing movement impairments. Traditionally, clinicians use goniometers to measure the ROM, but this method relies on the clinicians skill, in particular for difficult joints such as the shoulder and neck joints. Recent studies have explored the use of wearable inertial measurement units (IMUs) as an alternative. IMUs exhibit excellent agreement with goniometers, but the lack of affordable, accessible, and clinically validated tools remains an issue. This paper introduces the Romiumeter, a single IMU-based device designed to measure the ROM of the neck and shoulder movements. To validate its accuracy, the Romiumeter was tested on 34 asymptomatic individuals for shoulder and neck movements, using an optical motion capture system as the ground truth. The device demonstrated good accuracy, with a maximum absolute error of less than 5{degrees} with moderate to good reliable measurements(inter-rater reliability: 0.69 - 0.87 and intra-rater reliability: 0.76 - 0.87). Additionally, the Romiumeter underwent validation for different algorithms, including the complementary and Madgwick filters. Interestingly, no significant differences were found between the algorithms. Overall, the Romiumeter provides reliable measurements for assessing shoulder and neck ROM in asymptomatic individuals.

bioengineering↗

bioRxiv2023

Event-based Single Molecule Localization Microscopy (eventSMLM) for High Spatio-Temporal Super-resolution Imaging

Photon emission by single molecules is a random event with a well-defined distribution. This calls for event-based detection in single-molecule localization microscopy. The detector has the advantage of providing a temporal change in photons and emission characteristics within a single blinking period (typically, [~] 30 ms) of a single molecule. This information can be used to better localize single molecules within a user-defined collection time (shorter than average blinking time) of the event detector. The events collected over every short interval of time / collection time ([~] 3 ms) give rise to several independent temporal photon distributions (tPSFs) of a single molecule. The experiment showed that single molecules intermittently emit photons. So, capturing events over a shorter period / collection time than the entire blinking period gives rise to several realizations of the temporal PSFs (tPSFs) of a single molecule. Specifically, this translates to a sparse collection of active pixels per frame on the detector chip (image plane). Ideally, multiple realizations of single-molecule tPSF give several position estimates of the single-molecules, leading to multiple tPSF centroids. Fitting these centroid points by a circle provides an approximate position (circle center) and geometric localization precision (determined by the FWHM of the Gaussian) of a single molecule. Since the single-molecule estimate (position and localization precision) is directly driven by the data (photon detection events on the detector pixels) and the recorded tPSF, the estimated value is purely experimental rather than theoretical (Thomsons formula). Moreover, the temporal nature of the event camera and tPSF substantially reduces noise and background in a low-noise environment. The method is tested on three different test samples (1) Scattered Cy3 dye molecules on a coverslip, (2) Mitochondrial network in a cell, and (3) Dendra2HA transfected live NIH3T3 cells (Influenza-A model). A super-resolution map is constructed and analyzed based on the detection of events (temporal change in the number of photons). Experimental results on transfected NIH3T3 cells show a localization precision of [~] 10 nm, which is [~] 6 fold better than standard SMLM. Moreover, imaging HA clustering in a cellular environment reveals a spatio-temporal PArticle Resolution (PAR) (2.3lp x{tau} ) of 14.11 par where 1 par = 10-11 meter.second. However, brighter probes (such as Cy3) are capable of [~] 3.16 par. Cluster analysis of HA molecules shows > 81% colocalization with standard SMLM, indicating the consistency of the proposed eventSMLM technique. The single-molecule imaging on live cells reveals temporal dynamics (migration, association, and dissociation) of HA clusters for the first time over 60 minutes. With the availability of event-based detection and high temporal resolution, we envision the emergence of a new kind of microscopy that is capable of high spatio-temporal particle resolution in the sub-10 par regime.

biophysics↗

bioRxiv2023

The Anti-inflammatory Drug Leflunomide Inhibits NS2B3 Cluster Formation During Dengue Viral Infection as Revealed by Single Molecule Imaging

A prerequisite for Dengue viral infection is the clustering of NS2B3 viral protein in the infected cell. This calls for drugs capable of reversing the biological processes leading to the declustering of NS2B3 viral complex. In this work, we report a new drug (leflunomide) that shows reversal of NS2B3 clustering, post 24 hours of cell transfection with a recombinant probe (Dendra2-NS2B3) containing the viral complex of interest (NS2B3). To study, we constructed a photoactivable recombinant plasmid for visualizing the activity of the target protein-of-interest (Dendra2-NS2B3). This enabled a better understanding of the underlying biological processes involved in Dengue and the role of NS2B3. The study was performed in a cellular system by transfecting the cell (NIH3T3 -mouse fibroblast cell line), followed by drug treatment studies. A range of physiologically relevant concentrations (250 nM - 10 M) of the FDA-approved drug (leflunomide) was used. The single molecule super-resolution microscopy (scanSM LM) study showed declustering of NS2B3 clusters for concentrations > 250 nM and near complete disappearance of clusters at concentrations > 5 M . Moreover, the associated critical biophysical parameters suggest a substantial decrease in clustered molecules (from 53.2 {+/-} 1.77% for control to 14.89 {+/-} 4.80% at 250 nM, and further reduction to 10.55 {+/-} 2.91% at 500 nM). Moreover, the number of clusters reduced from 46 {+/-} 15 to 13 {+/-} 4, and the number of molecules per cluster decreased from 133 {+/-} 29 to 62 {+/-} 3, with a depletion in large clusters (from 24 to 12). The parameters collectively indicate the clustering nature of NS2B3 viral protein during the infection process at a cellular level and the effect of leflunomide in declustering. The results supported by statistical analysis suggest strong declustering promoted by leflunomide, which holds the promise to contain/treat dengue viral infection. Statement of SignificanceThe fact that there is no approved antiviral approach for Dengue makes it life-threatening and calls for ways to tackle viral infection. Hence, understanding Dengue biology at a single molecule level plays a vital role. In the present super-resolution study, we noted the formation of key viral protein (NS2B3) clusters post 24 hours of transfection in a cellular system. We identified a repurposed FDA-approved drug (Leflunomide) that inhibits the clustering process and promotes declustering at higher drug concentrations. This may become the basis of future studies, which may have therapeutic potential against Dengue.

biophysics↗

bioRxiv2022

Correlation Single Molecule Localization Microscopy (corrSMLM) Detects Fortunate Molecules for Better Localization Precision and High Signal-to-Noise Ratio

Single-molecule localization microscopy can decipher fine details that are otherwise not possible using diffraction-limited microscopy. Often the reconstructed super-resolved image contains unwanted noise, random background and is prone to false detections. This cause spurious data that necessitates several trials, multiple experimentations, and repeated preparation of specimens. Moreover, this is not suitable for experiments that require time-lapse imaging and real-time microscopy. To overcome these limitations, we propose a technique (corrSMLM) that can recognize and detect fortunate molecules (molecules with long fluorescence cycles) from the recorded data. The technique uses correlation between two or more consecutive frames to extract fortunate molecules that blink for longer than the standard blinking time. Accordingly, strongly-correlated spots (single molecule signatures) are compared in consecutive frames, followed by data integration (mean centroid position and the total number of photons) and estimation of critical parameters (position and localization precision). The technique addresses two major problems that plague SMLM : (1) random noise due to false detection that contributes to strong background, and (2) poor localization precision offered by standard SMLM techniques. On the brighter side, corrSMLM allows only fortunate molecules contribute to the super-resolved image, thereby suppressing the background and improving localization precision by a factor of 2-4 times as compared to standard SMLM. To substantiate, corrSMLM is used for imaging fixed cell samples (Dendra2-Actin and Dendra2-Tubulin transfected NIH3T3 cells). Results show multi-fold reduction in noise and localization precision with a marked improvement in overall resolution and SBR. We anticipate corrSMLM to improve overall image quality and offer a better understanding of single molecule dynamics in cell biology.

biophysics↗

bioRxiv2022

Single-Molecule Super-Resolution Microscopy Reveals Formation of NS2B3 Protein Clusters on Mitochondrial Network Leading to its Fragmentation during the Onset of Dengue (Denv-2) Viral Infection

Understanding viral proteins at single-molecule level and visualizing their dynamics in a cellular environment is challenging. This calls for sophisticated microscopy techniques (such as SMLM) and image-based study that can reveal details with the precision of a single-molecule. Specifically, NS2B3 is recognized as a critical protein complex responsible for proteolytic activity and processing of viral polyprotein during dengue type 2 (Denv - 2) infection. Using single-molecule-based super-resolution imaging, we study the dynamics of NS2B3 protein, its kinetics and its interaction with cell organelles. Two distinct photoactivable recombinant plasmids (mEos-NS2B3 and PAGFP - NS2B3) and a fluorescent recombinant plasmid (eGFP-NS2B3) were constructed to exemplify the role of NS2B3 protein complex. The study was conducted on NIH3T3 cells and optimized transfection protocol was developed. Studies confirmed the formation of NS2B3 clusters on the mitochondrial network. Statistical analysis of super-resolution data (images) helped determine cluster dynamics and facilitated the estimation of critical biophysical parameters (such as, cluster density, number of molecules / cluster and its spread). Results indicate an average NS2B3 cluster area / spread of [~] 0.050 {micro}m2 with a density of 3500mol./ {micro}m2, and an average of [~] 120 NS2B3 molecules per cluster. In addition, regional analysis suggests a direct positive correlation between NS2B3 cluster formation (single-molecule localization microscopy study) and fragmentation of mitochondrial network (confocal microscopy study). To further exemplify, we have carried out time-lapse imaging to visualize formation of NS2B3 clusters and its dynamics. The corresponding cluster parameters (#clusters, #mol/cluster and cluster area) suggests an increase in average #mol/cluster and cluster area. For the first time, single-molecule-based super-resolution imaging study helped reveal the dynamics of NS2B3 clusters in a cellular system. Understanding the underlying dynamics of NS2B3 clustering at the single-molecule level may help to decipher potential drug targets and the ways to disrupt the NS2B3 clusters. The image-based study has immediate implications in the broad field of single molecule imaging, fluorescence microscopy and disease biology. Statement of SignificanceThe arrival of single-molecule super-resolution imaging techniques and image-based study have advanced the field of cell biology, and our understanding of sub-cellular processes with single-molecule precision. Here, we report the first ever application of super-resolution imaging to visualize NS2B3 clusters in a cellular system that directly links to the fragmentation of mitochondrial network. To facilitate the study, two new photoactivable probes (mEos - NS2B3 and P AGF P- NS2B3) with key protein complex, NS2B3 of dengue virus were developed. The study involves cell transfection studies followed by confocal and single-molecule imaging. The correlative biophysical study (comprising of confocal and single-molecule imaging) and estimated cluster parameters corroborates our findings.

pathology↗

bioRxiv2022

Unveiling the anti-glioma potential of a marine derivative, Fucoidan: its synergistic cytotoxicity with Temozolomide-an in vitro and in silico experimental study

IntroductionGlioma, coined as a "butterfly" tumor associated with a dismal prognosis. Marine algal compounds with the richest sources of bioactive components, act as significant anti-tumor therapeutics. However, there is a paucity of studies conducted on Fucoidan to enhance the anti-glioma efficacy of Temozolomide. Therefore, the present study aimed to evaluate the synergistic anti-proliferative, anti-inflammatory and pro-apoptotic effects of Fucoidan with Temozolomide in in vitro and in silico experimental setup. MethodologyThe anti-proliferative effects of Temozolomide and Fucoidan was evaluated on C6 glioma cells by MTT and migration assay. Modulation of inflammatory markers and apoptosis induction was affirmed at the morphological and transcriptional level, by dual staining and gene expression. Molecular docking (MD) and molecular dynamics simulation (MDS) studies were performed against the targets to rationalize the inhibitory effect. ResultsThe dual-drug combination significantly reduced the cell viability and migration of glioma cells in a synergistic dose-dependent manner. At the molecular level, the dual-drug combination significantly down-regulated inflammatory genes with a concomitant upregulation of pro-apoptotic marker. In consensus with our in vitro findings, molecular docking and simulation studies revealed that the anti-tumor ligands: Temozolomide, Fucoidan with 5-(3-Methy1-trizeno)-imidazole-4-carboxamide (MTIC), and 4-amino-5-imidazole-carboxamide (AIC) had the potency to bind to the inflammatory proteins at their active sites, mediated by H-bonds and other non-covalent interactions. Discussion and ConclusionThe dual-drug combinatorial treatment synergistically inhibited the proliferation, migration of glioma cells and promoted apoptosis; conversely with the down-regulation of inflammatory genes. However, pre-clinical experimental evidence is warranted for the possible translation of this combination.

cancer biology↗

bioRxiv2022

Simultaneous Multisheet Multichannel Imaging Cytometry (SMMIC) System Powered by Adaptive Vertically-Aligned Multi-Sheet Array (VAMSA) PSF

Volumetric interrogating of a large population of live specimens at high throughput is a challenging task that necessitates new technology. We propose vertical-aligned multi-sheet array (VAMSA) illumination PSF that enables interrogation of specimens flowing simultaneously through multiple microfluidic channels. The very geometry of PSF enables high quality cross-sectional imaging, and facilitates volumetric interrogation of specimens flowing through commercial microfluidic chip (consists of multiple flow-channels), which is a step towards large population screening. The SMMIC technique employs a unique combination of transmission grating, beam-expander and high NA objective system in a specific optical configuration to generate diffraction-limited illumination PSF (VAMSA-PSF). However, the detection is accomplished by a large field-of-view widefield 4f-system that consists of low NA objective lens, high performance fluorescence filters, and tube lens. Studies show high quality sectional images (resolution [~] 2.5m, and SBR [~] 4.8dB) of HeLa cancerous cells at high flow throughput (flow-rate of, 2500 nl/min). A cell count of > 1k and volume reconstruction efficiency of [~] 121 cells/min is noteworthy. In addition, SMMIC system demonstrate organelle-level resolution with a SBR comparable to that of confocal especially at low flow-rates. It is hoped that the proposed system may accelerates drug-treatment studies for a large population of live specimens to advance the evolving field of translational medicine and health-care.

biophysics↗