Biology subjects

Rai, J.

Publications and source records attributed to Rai, J..

14 recordsLinked to original sources

bioRxiv2025

An integrated workflow for structural virology with a 100 keV electron microscope

Cryo-EM has revolutionized structural biology, especially for flexible and heterogeneous samples, although access to high end microscopes that enable these studies remains a bottleneck. While 300 keV microscopes have been the go-to for high-resolution structural determination, they are expensive and restricted to institutional and national facilities needing specialized expertise, with access falling far short of the demand. Here, we present the user-managed operation of a cheaper 100 keV electron microscope within a structural biology laboratory enabling close integration with protein production, biochemical and biophysical studies. We provide details and considerations for the installation of the microscope, its day-to-day maintenance, and operations. Using virus surface glycoproteins as case studies, we illustrate the workflow from grid screening, data collection, and data processing, and provide examples of data quality. This user-administered setup provides a training platform for researchers at all levels, with beginners in cryo-EM achieving proficiency to independently operate the microscope within a month of regular use and training. We have demonstrated routine high-quality low-resolution reconstructions using a Ceta CMOS camera and high-resolution reconstructions enabling building of atomic models using a Falcon C direct detector. While there are several examples of facilities that manage cryo-EM and individual laboratories leveraging cryo-EM, we provide here the first demonstration of a modern group independently doing both successfully, something that has been talked about frequently but rarely seen.

biochemistry↗

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↗

bioRxiv2023

Mouse model of multiple sclerosis induced by disrupting vesicular transport in oligodendrocytes

Multiple Sclerosis is an autoimmune demyelination disorder with unknown etiology. Despite the myelin damage, the roles of myelinating oligodendrocytes in driving disease progression remain unknown. We hypothesize that disrupting vesicular transport in oligodendrocytes during adolescence will disrupt myelin integrity and causes neuroinflammation. By creating a mouse model of SNAP-23 conditional knockout in mature oligodendrocytes, we showed that impairment in vesicular trafficking in oligodendrocytes causes demyelination. Neuroinflammation with infiltration of peripheral immune T cells into the central nervous system was observed accompanied by demyelination. Mechanistically, SNAP-23 removal in oligodendrocytes caused abnormal axon-myelin structures and impaired myelin protein trafficking, both can contribute to autoimmune activation and demyelination. With our novel animal model, we propose that oligodendrocyte injury is an endogenous early event in triggering Multiple Sclerosis. One-Sentence SummaryImpaired vesicular transport in oligodendrocytes in adults caused demyelination and inflammation driving Multiple Sclerosis

neuroscience↗

bioRxiv2022

Conformational Changes Regulate Metal Coordination in the Catalytic Sites of Cas9

The control of CRISPR-Cas9 activities plays an essential role in its safe adoption for clinical and research applications. Previous studies have identified conformational dynamics of Cas9 as a key regulatory element for its enzymatic activity. The molecular basis for how conformations influence the catalytic processes at both nuclease domains, however, remains elusive. Here we present well-resolved cryo-EM structures of the active Acidothermus cellulolyticus Cas9 (AceCas9) complexes representing pre-cleavage (2.9 [A]), two reaction intermediate (2.2 [A] and 2.4 [A]) and two post-cleavage (2.6 [A] and 2.7 [A]) states that reveal active site rearrangement during DNA binding and phosphodiester bond breakage by Cas9. Strikingly, large domain rearrangements in Cas9 triggered by the cognate DNA result in subtle changes in active sites that facilitate coordination of metal ions required for catalysis. The two reaction intermediate structures further reveal small oscillations in domain conformations, which alternates the reactive states of the two catalytic centers, thereby coupling cleavage of the two DNA strands. Consistent with the roles of conformations in organizing the active sites, adjustments of metal coordination ligands lead to altered metal specificity.

biochemistry↗

bioRxiv2022

Human-sloth bear conflict in Nepal - case study from an unprotected Churia forest

Human-sloth bear conflict, generally in the form of human attacks, is common throughout most areas where sloth bears co-occur with humans. The situation is more prevalent in multi-use forest landscapes outside protected areas. Although sloth bears are a conflict-prone species in Nepal, there is a clear lack of systematic studies that can inform human safety and conflict mitigation. We used data from questionnaire-based interviews with conflict-affected people and witnesses to provide detailed information about human-sloth bear conflict (1990- 2021) in the Trijuga forest, an important sloth bear stronghold outside protected areas in Nepal. The data were analyzed using descriptive statistics, chi-square tests, and regression analysis. For the time period, 66 conflict incidents involving 69 human individuals were recorded, with an annual average of 2.06 (SD = 1.48) incidents and 1.75 (SD = 1.34) attacks. Conflicts primarily impacted working-age group (25-55 years old) men, whose primary occupation was farming and who frequented the forest regularly. They typically occurred between 0900 and 1500, inside forests, and in habitats associated with poor land cover visibility. Poor visibility was also a significant positive determinant of bear attacks on humans. Fifty-six conflict incidents resulted in attacks that injured 59 people, with a fatality rate of 8.47%. Victims of bear attacks frequently had serious injuries, especially to the head and neck areas of the body. Serious injuries were more likely to occur to lone individuals than to people who were in groups of two or more. We suggest identification of conflict-risk habitats through a participatory mapping approach and education programs for the local people for effective human-sloth bear conflict management in Nepals unprotected forests.

ecology↗

bioRxiv2022

Neuronal SNAP-23 scales hippocampal synaptic plasticity and memory

Soluble NSF Attachment protein REceptor (SNARE)-mediated membrane fusion plays a crucial role not only in presynaptic vesicle exocytosis but also in postsynaptic receptor delivery. The latter is considered particularly important for long-term synaptic plasticity and learning and memory, yet underlying mechanisms including the identity of the key SNARE proteins remain elusive. Here, we investigate the role of neuronal Synaptosomal-Associated Protein-23 (SNAP-23) by analyzing pyramidal-neuron specific SNAP-23 conditional knockout (cKO) mice. SNAP-23 immunostaining in postsynaptic spines was effectively decreased in the SNAP-23 cKO hippocampus. Electrophysiological analysis of SNAP-23 deficient neurons using acute hippocampal slices showed normal basal neurotransmission in CA3-CA1 synapses with unchanged AMPA and NMDA currents. Nevertheless, we found theta-burst stimulation induced long-term potentiation (LTP) was vastly diminished in SNAP-23 cKO. Moreover, unlike syntaxin-4 cKO mice in which both basal neurotransmission and LTP decrease manifested changes in a broad set of behavioral tasks, deficits of SNAP-23 cKO is more limited to spatial memory. Our data reveal that neuronal SNAP-23 is selectively crucial for synaptic plasticity and spatial memory without affecting basal glutamate receptor function.

neuroscience↗

bioRxiv2022

Molecular Mechanism of Active Cas7-11 in Processing CRISPR RNA and Interfering Target RNA

Cas7-11 is a Type III-E CRISPR Cas effector that confers programmable RNA cleavage and has potential applications in RNA interference. Cas7-11 encodes a single polypeptide containing four Cas7- and one Cas11-like segments that obscures the distinction between the multi-subunit Class 1 and the single-subunit Class-2 CRISPR-Cas systems. We report a cryo-EM structure of the active Cas7-11 from Desulfonema ishimotonii (DiCas7-11) that reveals the molecular basis for RNA processing and interference activities. DiCas7-11 arranges its Cas7- and Cas11-like domains in an extended form that resembles the backbone made up by four Cas7 and one Cas11 subunits in the multi-subunit enzymes. Unlike the multi-subunit enzymes, however, the backbone of DiCas7-11 contains evolutionarily different Cas7 and Cas11 domains, giving rise to their unique functionality. The first Cas7-like domain nearly engulfs the last 15 direct repeat nucleotides and is responsible for processing and recognition of the CRISPR RNA. Whereas both the second and the third Cas7-like domains mediate target RNA cleavage, they differ in metal requirement for catalysis. The long variable insertion to the fourth Cas7-like domain has little impact to RNA processing or targeting, suggesting the possibility for engineering a compact and programmable RNA interference tool. One Sentence SummaryStructures of Cas7-11 reveal the molecular basis for processing CRISPR RNA and for cleaving target RNA.

biophysics↗

bioRxiv2022

Mmp14-controlled extracellular matrix homeostasis is required for circadian rhythm

The circadian clock in tendon regulates the daily rhythmic synthesis of collagen-I and the appearance and disappearance of small-diameter collagen fibrils in the extracellular matrix. How the fibrils are assembled and removed is not fully understood. Here, we first showed that the collagenase, membrane type I-matrix metalloproteinase (MT1-MMP, encoded by Mmp14), is regulated by the circadian clock in postnatal mouse tendon. Next, we generated tamoxifen-induced Col1a2-Cre-ERT2::Mmp14 KO mice (Mmp14 conditional knockout (CKO)). The CKO mice developed hind limb dorsiflexion and thickened tendons, which accumulated narrow-diameter collagen fibrils causing ultrastructural disorganization. Mass spectrometry of control tendons identified 1195 proteins of which 212 showed time-dependent abundance. In Mmp14 CKO mice 19 proteins had reversed temporal abundance and 176 proteins lost time dependency. Among these, the collagen crosslinking enzymes lysyl oxidase-like 1 (LOXL1) and lysyl hydroxylase 1 (LH1; encoded by Plod2) were elevated and had lost time-dependent regulation. High-pressure chromatography confirmed elevated levels of hydroxylysine aldehyde (pyridinoline) crosslinking of collagen in CKO tendons. As a result, collagen-I was refractory to extraction. We also showed that CRISPR-Cas9 deletion of Mmp14 from cultured fibroblasts resulted in loss of circadian clock rhythmicity of period 2 (PER2), and recombinant MT1-MMP was highly effective at cleaving soluble collagen-I but less effective at cleaving collagen pre-assembled into fibrils. In conclusion, our study shows that circadian clock-regulated Mmp14 controls the rhythmic synthesis of small diameter collagen fibrils, regulates collagen crosslinking, and its absence disrupts the circadian clock and matrisome in tendon fibroblasts.

cell biology↗

bioRxiv2021

Pseudouridine-free Ribosome Exhibits Distinct Inter-subunit Movements

Pseudouridine, the most abundant form of RNA modification, is known to play important roles in ribosome function. Mutations in human DKC1, the pseudouridine synthase responsible for catalyzing the ribosome RNA modification, cause translation deficiencies and are associated with a complex cancer predisposition. The structural basis for how pseudouridine impacts ribosome function remains uncharacterized. Here we report electron cryomicroscopy structures of a fully modified and a pseudouridine-free ribosome from Saccharomyces cerevisiae. In the modified ribosome, the rearranged N1 atom of pseudouridine is observed to stabilize key functional motifs by establishing predominately water-mediated close contacts with the phosphate backbone. The pseudouridine-free ribosome, however, is devoid of such interactions and displays conformations reflective of abnormal inter-subunit movements. The erroneous motions of the pseudouridine-free ribosome may explain its observed deficiencies in translation.

biochemistry↗

bioRxiv2021

Age-Related Type I Collagen Modifications Reveal Tissue-Defining Differences Between Ligament and Tendon

Tendons and ligaments tend to be pooled into a single category as dense elastic bands of collagenous connective tissue. They do have many similar properties, for example both tissues are flexible cords of fibrous tissue that join bone to either muscle or bone. Tendons and ligaments are both prone to degenerate and rupture with only limited capacity to heal, however; healing outcomes for tendons are often faster than ligament injuries. Type I collagen constitutes about 80% of the dry weight of tendons and ligaments and is principally responsible for the core strength of each tissue. Collagen synthesis is a complex process with multiple steps and numerous post-translational modifications including proline and lysine hydroxylation, hydroxylysine glycosylation and covalent cross-linking. The chemistry, placement and quantity of intramolecular and intermolecular cross-links are believed to be key contributors to the tissue-specific variations in material strength and biological properties of collagens. As tendons and ligaments grow and develop, the collagen cross-links are known to chemically mature, strengthen and change in profile. Accordingly, changes in cross-linking and other post-translational modifications are likely associated with tissue development and degeneration. Using mass spectrometry, we have compared tendon and ligaments from fetal and adult bovine knee joints to investigate changes in collagen post-translational properties. Although hydroxylation levels at the type I collagen helical cross-linking lysine residues were similar in all adult tissues, ligaments had significantly higher levels of glycosylation at these sites compared to tendon. Differences in lysine hydroxylation were also found between the tissues at the telopeptide cross-linking sites. Total collagen cross-linking analysis, including mature trivalent cross-links and immature divalent cross-links, revealed unique cross-linking profiles between tendon and ligament tissues. Tendons were found to have a significantly higher frequency of smaller diameter collagen fibrils compared with ligament, which we propose is a consequence of the unique cross-linking profile of each tissue. Understanding the specific molecular characteristics that define and distinguish these specialized tissues will be important for designing improved orthopedic treatment approaches.

biochemistry↗

bioRxiv2021

MITOCHONDRIAL GENOME REVEALS CONTRASTING PATTERN OF ADAPTIVE SELECTION IN TURTLES AND TORTOISES

Testudinoidea represents an evolutionarily unique taxon comprising both turtles and tortoises. The contrasting habitats that turtles and tortoises inhabit are associated with unique physio-ecological challenges hence enable distinct adaptive evolutionary strategies. To comparatively understand the pattern and strength of Darwinian selection and physicochemical evolution in turtle and tortoise mitogenomes, we employed adaptive divergence and selection analyses. We evaluated changes in structural and biochemical properties, and codon models on the mitochondrial protein-coding genes (PCGs) among three turtles and a tortoise lineage. We used mitochondrial PCGs that constitute the crucial oxidative phosphorylation (OXPHOS) respiratory system, a critical metabolic regulator which assumes key significance in energy regulation of ectotherms. We detected strong evidence of positive selection along the turtle lineages: Geoemydidae, Emydidae, and Platysternidae, but relatively weak signals in tortoises. The Platysternidae turtles revealed the highest gene and site-wise positive selection. In turtles, positively selected sites were prevalent in NAD2 and NAD4 genes in OXPHOS Complex I, and COB gene of Complex III, indicating convergent adaptive evolution. Besides, NAD3 was the only subunit that showed adaptive selection in both turtles and tortoises, expressing its relevance for all Testudinoidea. Structural and functional analysis revealed many sites and physiochemical changes in important conserved as well as biomedically significant regions, suggesting the influence of adaptive pressure on mitogenome functions. Hence, our study furnished novel evidence of contrasting evolutionary selective pressure acting on closely related groups such as turtles and tortoises with unique habitat preferences and associated eco-physiological challenges.

evolutionary biology↗

bioRxiv2021

Structure and Function of an in vivo Assembled Type III-A CRISPR-Cas Complex Reveal Critical Roles of Dynamics in Activity Control

The small RNA-mediated immunity in bacteria depends on foreign RNA-activated and self RNA-inhibited enzymatic activities. The multi-subunit Type III-A CRISPR-Cas effector complex (Csm) exemplifies this principle, but its molecular basis for regulation remains unexplained. Recognition of the foreign RNA, or cognate target RNA (CTR), triggers its single-stranded deoxyribonuclease (DNase) and cyclic oligoadenylate (cOA) synthesis activities. The same activities remain dormant in the presence of the self-RNA, or noncognate target RNA (NTR) that differs from CTR only in its 3-protospacer flanking sequence. Here we captured four structures of in vivo assembled Lactococcus lactis Csm (LlCsm) by electron cryomicroscopy representing both the active and the inactive states. Surprisingly, in absence of bound RNA, LlCsm largely forms a minimal assembly lacking the Csm2 subunit with a stably bound catalytic subunit Csm1. Comparison of the minimal LlCsm structure and activities, both in vitro and in vivo, with those of fully assembled LlCsm reveals a molecular mechanism responsible for the viral RNA-activated and self RNA-inhibited activity of Csm1 through protein dynamics. Graphic Art Summary O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=91 SRC="FIGDIR/small/428455v1_ufig1.gif" ALT="Figure 1"> View larger version (30K): org.highwire.dtl.DTLVardef@1e9c428org.highwire.dtl.DTLVardef@4d3397org.highwire.dtl.DTLVardef@76184dorg.highwire.dtl.DTLVardef@1359dd5_HPS_FORMAT_FIGEXP M_FIG C_FIG

biophysics↗

bioRxiv2020

Physioxia stimulates extra-cellular matrix deposition and increases mechanical properties of human chondrocyte-derived tissue-engineered cartilage

Cartilage tissue has been recalcitrant to tissue engineering approaches. In this study, human chondrocytes were formed into self-assembled cartilage sheets, cultured in physiologic (5%) and atmospheric (20%) oxygen conditions and underwent biochemical, histological and biomechanical analysis at one- and two-months. The results indicated that sheets formed at physiological oxygen tension were thicker, contained greater amounts of glycosaminoglycans (GAGs) and type II collagen, and had greater compressive and tensile properties than those cultured in atmospheric oxygen. In all cases, cartilage sheets stained throughout for extracellular matrix components. Type II-IX-XI collagen heteropolymer formed in the neo-cartilage and fibrils were stabilized by trivalent pyridinoline cross-links. Collagen cross-links were not significantly affected by oxygen tension but increased with time in culture. Physiological oxygen tension and longer culture periods both served to increase extracellular matrix components. The foremost correlation was found between compressive stiffness and the GAG to collagen ratio. SummaryTissue-engineered cartilage formed from human articular chondrocytes produces thicker, stiffer, more extracellular-matrix rich cartilage tissue when grown under physiological (5%) vs. atmospheric oxygen (20%) tension.

cell biology↗

bioRxiv2019

Subunit joining exposes nascent pre-40S rRNA for processing and quality control

During their maturation, nascent 40S subunits enter a translation-like quality control cycle, where they are joined by mature 60S subunits to form 80S-like ribosomes. While these assembly intermediates are essential for maturation and quality control, how they form, and how their structure promotes quality control remains unknown. To address these questions, we determined the structure of an 80S-like ribosome assembly intermediate to an overall resolution of 3.4 [A]. The structure, validated by biochemical data, resolves a large body of previously paradoxical data and illustrates how assembly and translation factors cooperate to promote the formation of an interface that lacks many mature subunit contacts but is stabilized by the universally conserved Dim1. We also show how Tsr1 enables this interface by blocking the canonical binding of eIF5B to 40S subunits, while maintaining its binding to 60S. The structure also shows how this interface leads to unfolding of the platform, which allows for temporal regulation of the ATPase Fap7, thus linking 40S maturation to quality-control during ribosome assembly.

biochemistry↗