Biology subjects

Singh, P.

Publications and source records attributed to Singh, P..

At least 109 records · Page 6Linked to original sources

bioRxiv2024

Bridging fungal resistance and plant growth through constitutive overexpression of Thchit42 gene in Pelargonium graveolens

Pelargonium graveolens essential oil possesses significant attributes, known for perfumery and aromatherapy. However, optimal yield and propagation are predominantly hindered by biotic stress. All biotechnological approaches have yet to prove effective in addressing fungal resistance. The current study developed transgenic geranium bridging molecular mechanism of fungal resistance and plant growth by introducing cassette 35S::Thchit42. Furthermore, 120 independently putative transformed explants were regenerated on kanamycin fortified medium. Primarily transgenic lines were demonstrated peak pathogenicity and antifungal activity against formidable Colletotrichum gloeosporioides and Fusarium oxysporum. Additionally, phenotypic analysis revealed [~]2fold increase in leaf size and [~]2.1fold enhanced oil content. To elucidate the molecular mechanisms for genotypic cause, De novo transcriptional profiles were analyzed to indicate that the auxin-regulated longifolia gene is accountable for augmentation in leaf size, and ZF RICESLEEPER attributes growth upregulation. Collectively, data provides valuable insights into unravelling the mechanism of Thchit42-mediated crosstalk between morphological and chemical alteration in transgenic plants. This knowledge might create novel opportunities to cultivate fungal-resistant geranium throughout all seasons to fulfil demand.

plant biology↗

bioRxiv2024

Cryo-EM confirms a common fibril fold in the heart of four patients with ATTRwt amyloidosis

ATTR amyloidosis results from the conversion of transthyretin into amyloid fibrils that deposit in tissues causing organ failure and death. This conversion is facilitated by mutations in ATTRv amyloidosis, or aging in ATTRwt amyloidosis. ATTRv amyloidosis exhibits extreme phenotypic variability, whereas ATTRwt amyloidosis presentation is consistent and predictable. Previously, we found an unprecedented structural variability in cardiac amyloid fibrils from polyneuropathic ATTRv-I84S patients. In contrast, cardiac fibrils from five genotypically-different patients with cardiomyopathy or mixed phenotypes are structurally homogeneous. To understand fibril structures impact on phenotype, it is necessary to study the fibrils from multiple patients sharing genotype and phenotype. Here we show the cryo-electron microscopy structures of fibrils extracted from four cardiomyopathic ATTRwt amyloidosis patients. Our study confirms that they share identical conformations with minimal structural variability, consistent with their homogenous clinical presentation. Our study contributes to the understanding of ATTR amyloidosis biopathology and calls for further studies. One-Sentence Summary: Wild-type cardiac ATTR fibrils are structurally homogeneous.

biophysics↗

bioRxiv2024

Oncogenic STIL-mediated loss of BRCA1 functionality causes DNA damage and centrosome amplification

DNA damages increase centrosome number, a typical phenotype in many cancers. However, the molecular linkers between the two pathways remain elusive. STIL (SCL/TAL1 Interrupting locus) is a core centrosome-organizing protein overexpressed in different cancer types. This work shows that STIL forms a complex with the DNA damage response protein, BRCA1, and is required for regulating its protein stability. Subsequently, STIL depletion resulting in BRCA1 loss from the nucleus causes centrosome amplification and DNA damage. We also identified a heterozygous missense mutation (S76L) in STIL from the pan-cancer datasets, which does not affect total BRCA1 protein levels. However, it reduces BRCA1 nuclear localization with enhanced centrosome recruitment simultaneously at the S-phase. The enhanced BRCA1 at centrosome causes an increase in Aurora-A kinase levels at centrosomes, thus culminating in DNA damage and centrosome amplification. The expression of exogenous BRCA1 can push back this imbalance of the STIL-BRCA1 axis in oncogenic conditions. The amplified centrosomes in oncogenic STIL condition cluster in the M phase and cause pseudo-bipolar spindle organization. We could decluster them by the HSET(KIFC1)-specific inhibitor, CW069, suggesting a promising target for anti-cancer treatment.

cell biology↗

bioRxiv2024

Developmental tinkering of gene regulation facilitated super rapid adaptive radiation

Developmental shifts in gene regulation underlying key innovations remain largely uncharacterized over short evolutionary timescales. Here, we investigate the gene regulatory landscape of trophic innovations in the fastest vertebrate adaptive radiation: cichlid fishes from Lake Victoria. By analyzing the whole-transcriptomes of the oral and pharyngeal jaws from two life stages in five species adapted to divergent trophic niches, we find that both gene and isoform expression are developmentally dynamic. Expression signatures are most similar across jaws at the early stage and then diverge into species-specific developmental programs in adults. However, even at the early stage, expression in the oral jaws of species adapted to herbivory is distinct from those of carnivores, a pattern not observed for the pharyngeal jaws. We further show that differentially expressed and spliced genes between herbivorous and carnivorous species regulate different genes and pathways, particularly in adults. Interestingly, we find that splicing-mediated exonization of a craniofacial development gene, kaznb, may have contributed to the evolution of herbivory in Lake Victoria cichlids. Overall, our results contribute to our understanding of how ontogenetic shifts in gene regulation can facilitate rapid adaptive evolution.

evolutionary biology↗

bioRxiv2024

Multiomics analysis reveals B. MO1 as a distinct Babesia species and provides insights into its evolution and virulence.

Babesiosis, caused by protozoan parasites of the genus Babesia, is an emerging tick-borne disease of significance for both human and animal health. Babesia parasites infect erythrocytes of vertebrate hosts where they develop and multiply rapidly to cause the pathological symptoms associated with the disease. The identification of various Babesia species underscores the ongoing risk of new zoonotic pathogens capable of infecting humans, a concern amplified by anthropogenic activities and environmental shifts impacting the distribution and transmission dynamics of parasites, their vectors, and reservoir hosts. One such species, Babesia MO1, previously implicated in severe cases of human babesiosis in the midwestern United States, was initially considered closely related to B. divergens, the predominant agent of human babesiosis in Europe. Yet, uncertainties persist regarding whether these pathogens represent distinct variants of the same species or are entirely separate species. We show that although both B. MO1 and B. divergens share similar genome sizes, comprising three nuclear chromosomes, one linear mitochondrial chromosome, and one circular apicoplast chromosome, major differences exist in terms of genomic sequence divergence, gene functions, transcription profiles, replication rates and susceptibility to antiparasitic drugs. Furthermore, both pathogens have evolved distinct classes of multigene families, crucial for their pathogenicity and adaptation to specific mammalian hosts. Leveraging genomic information for B. MO1, B. divergens, and other members of the Babesiidae family within Apicomplexa provides valuable insights into the evolution, diversity, and virulence of these parasites. This knowledge serves as a critical tool in preemptively addressing the emergence and rapid transmission of more virulent strains.

microbiology↗

bioRxiv2024

Dihydrothiazolo ring-fused 2-pyridone antimicrobial compounds treat Streptococcus pyogenes skin and soft tissue infection

We have developed GmPcides from a peptidomimetic dihydrothiazolo ring-fused 2-pyridone scaffold that have antimicrobial activities against a broad-spectrum of Gram-positive pathogens. Here we examine the treatment efficacy of GmPcides using skin and soft tissue infection (SSTI) and biofilm formation models by Streptococcus pyogenes. Screening our compound library for minimal inhibitory (MIC) and minimal bactericidal (MBC) concentrations identified GmPcide PS757 as highly active against S. pyogenes. Treatment of S. pyogenes biofilm with PS757 revealed robust efficacy against all phases of biofilm formation by preventing initial biofilm development, ceasing biofilm maturation and eradicating mature biofilm. In a murine model of S. pyogenes SSTI, subcutaneous delivery of PS757 resulted in reduced levels of tissue damage, decreased bacterial burdens and accelerated rates of wound-healing, which were associated with down-regulation of key virulence factors, including M protein and the SpeB cysteine protease. These data demonstrate that GmPcides show considerable promise for treating S. pyogenes infections.

microbiology↗

bioRxiv2023

Vitamin B5 Metabolism is Essential for Vacuolar and Mitochondrial Integrity and Xenobiotic Detoxification in Fungi

Fungal infections, a leading cause of mortality among eukaryotic pathogens, pose a growing global health threat due to the rise of drug-resistant strains. New therapeutic strategies are urgently needed to combat this challenge. The PCA pathway for biosynthesis of Co-enzyme A (CoA) and Acetyl-CoA (AcCoA) from vitamin B5 (pantothenic acid) has been validated as an excellent target for the development of new antimicrobials against fungi and protozoa. The pathway regulates key cellular processes including metabolism of fatty acids, amino acids, sterols, and heme. In this study, we provide genetic evidence that disruption of the PCA pathway results in a significant alteration in the susceptibility of fungi to a wide range of xenobiotics, including clinically approved antifungal drugs through alteration of vacuolar integrity and drug detoxification. The drug potentiation-mediated by genetic regulation of genes in the PCA pathway could be recapitulated using the pantazine analog PZ-2891 as well as the celecoxib derivative, AR-12 through inhibition of fungal AcCoA synthase activity. Collectively, the data validate the PCA pathway as a suitable target for enhancing the efficacy and safety of current antifungal therapies.

microbiology↗

bioRxiv2023

DIGEST: An online tool for designing of multiple reaction monitoring assays

Targeted proteomics using multiple reaction monitoring (MRM) assays enables fast and sensitive detection of a preselected set of target peptides. This technique utilizes the specificity of precursors to product transitions for quantitative analysis of multiple proteins in a single sample. The success of an MRM experiment depends on the selection of transitions however, given the existing resources, accurately predicting signal intensity of peptides and their fragmentation patterns ab initio is challenging task. We present an alternative for rapid design of MRM transitions for proteomics research: DIGEST. Our method predicts the b and y ions with +1 and +2 charge produced in a collision cell of a mass spectrometer from peptides of multiple proteotypically digested proteins. Additionally, by using the existing knowledge of the fundamental rules for designing transitions, the tool provides optimal MRM transitions, negating the need to undertake prior "discovery" MS studies. We demonstrate that our algorithm is directed toward the selection of MRM precursor and product-ions pairs, and can avoid the pitfalls of interference due to cross-contamination of samples by selecting ion combinations that uniquely map to target peptides. Comparison with SRMAtlas showed that DIGEST successfully predicted the peptide and production pairs in the majority of cases. We believe that DIGEST will facilitate rapid design of MRM assays with increased specificity, reducing the overall time required to design an MRM assay for routine mass-spectrometry. DIGEST is available as a web-based tool at https://digest.raylab.iiitd.edu.in/

bioinformatics↗

bioRxiv2023

PLK4 homodimerization is required for CEP152 centrosome localization and spindle organization

Polo-Like Kinase 4 (PLK4) is a unique serine/threonine kinase family member that homodimerizes using its cryptic polo-box (CPB) region. Homodimerization of PLK4 causes transphosphorylation, which activates its ubiquitin-mediated degradation. Interestingly, CPB is also involved in interaction with the upstream centrosome recruiters, CEP152 and CEP192 in human cells. However, the effect of PLK4 homodimerization on the CEP192-CEP152 network remains unexplored. In this work, we identified a frequently occurring cancerous PLK4 variant (E774*), which truncated the protein at the 774 position. The truncated PLK4 is unable to homodimerize and interact with CEP152 and CEP192. During the S-phase progression, we show that CEP152 recruits PLK4 to centrosomes. The homodimerization of PLK4, in turn, is needed for maintaining CEP152 centrosome levels. CEP152 levels correlate to pericentrin at S-phase centrosomes, which generate focused spindles by the M-phase. The homodimerization mutant exhibits reduced levels of CEP152 and pericentrin at S-phase centrosomes, which causes unfocused spindles at the M-phase and reduces cell viability. This work shows the requirement of PLK4 homodimerization for proper centrosome and spindle organization, which is disrupted in cancer.

cell biology↗

bioRxiv2023

Pervasive translational control of photosynthesis genes during photomorphogenesis is acquired by C4 genes

C4 photosynthesis allows increased efficiency and has evolved in more than sixty-six plant lineages. Underpinning this repeated appearance of the C4 pathway is a major transcriptional reprogramming of photosynthesis genes. Here we investigated whether evolution has also significantly modified translational control by defining the translational dynamics of C3 rice and C4 sorghum during photomorphogenesis. In the dark rice photosynthesis transcripts are low abundance but highly translated. After exposure to light translational efficiency declines. The same phenomena occur in sorghum but in addition C4 cycle genes show this response. We propose a model in which translational control of photosynthesis genes permits a rapid response to light and that this translational regulation is gained by C4 genes during the evolution of the C4 pathway.

molecular biology↗

bioRxiv2023

Optimization of RNAi efficiency in PVD neuron of C. elegans

PVD neuron of C. elegans has become an attractive model for the study of dendrite development and regeneration due to the elaborate and stereotype dendrite morphology in this neuron. The molecular basis for dendrite maintenance and regeneration is poorly understood. RNA interference (RNAi) by feeding E. coli expressing dsRNA has been the basis of several genome wide screens performed using C. elegans. However, the feeding method often fails when it comes to nervous system. Using an optimal induction condition for the dsRNA expression in E coli, we fed the worm strains with HT115 bacteria expressing dsRNA against genes like mec-3, hpo-30, and tiam-1, whose loss of function are known to show dendrite morphology defects in PVD neuron. We found that RNAi of these genes in the strains such as nre-1(-) lin-15b(-), lin-15b(-) and sid-1(-); lin-15b(-); Punc-119::sid-1[+] resulted in significant reduction of dendrite branching. However, the phenotypes were significantly modest compared to the respective loss of function mutants. To obtain stronger phenotype for PVD specific genes, we have made a strain, which strongly expresses sid-1 under mec-3 promoter specific for PVD. When Pmec-3::sid-1 is expressed in either nre-1(-);lin-15b(-) or lin-15b(-) background, the higher order branching phenotype after RNAi of mec-3, hpo-30, and tiam-1 was significantly enhanced as compared to nre-1(-);lin-15b(-) and lin-15b(-) background alone. Next we tested the nre-1(-) lin-15b(-), Pmec-3-sid-1[+] strain for the knockdown of genes playing role in dendrite regeneration process. We found that when aff-1 and ced-10 genes were knocked down in the nre-1(-) lin-15b(-), Pmec-3-sid-1[+] background, the dendrite regeneration was significantly reduced and the extent of reduction was comparable to that of the mutants of aff-1 and ced-10. Essentially, our strain expressing sid-1 in PVD neuron optimizes the condition for RNAi for high throughput screening for PVD development, maintenance and regeneration.

genetics↗

bioRxiv2023

Gene co-expression patterns in Atlantic salmon adipose tissue provide a molecular link between seasonal changes, energy balance and age at maturity

Sexual maturation in many fishes requires a major physiological change that involves a rapid transition between energy storage and usage. In Atlantic salmon, this transition for the initiation of maturation is tightly controlled by seasonality and requires a high-energy status. Lipid metabolism is at the heart of this transition since lipids are the main energy storing molecules. The balance between lipogenesis (lipid accumulation) and lipolysis (lipid use) determines energy status transitions. A genomic region containing a transcription co-factor of the Hippo pathway, vgll3, is the main determinant of maturation timing in Atlantic salmon. Interestingly, vgll3 acts as an inhibitor of adipogenesis in mice and its genotypes are potentially associated with seasonal heterochrony in lipid storage and usage in juvenile Atlantic salmon. Here, we explored changes in expression of more than 300 genes directly involved in the processes of adipogenesis, lipogenesis and lipolysis, as well as the Hippo pathway in the adipose tissue of immature and mature Atlantic salmon with distinct vgll3 genotypes. We found molecular evidence consistent with a scenario in which immature males exhibiting vgll3 genotype specific seasonal direction of change in their lipid profiles. We also identified components of the Hippo signaling pathway as potential major drivers of vgll3 genotype specific differences in adipose tissue gene expression. This study demonstrates the importance of adipose gene expression patterns for directly linking environmental changes with energy balance and age at maturity through genetic factors bridging lipid metabolism, seasonality and sexual maturation.

physiology↗

bioRxiv2023

Structural and functional vascular dysfunction within brain metastases is linked to pembrolizumab inefficacy

Structurally and functionally aberrant vasculature is a hallmark of tumor angiogenesis and treatment resistance. Given the synergistic link between aberrant tumor vasculature and immunosuppression, we analyzed perfusion MRI for 44 patients with brain metastases (BM) undergoing treatment with pembrolizumab. To date, vascular-immune communication, or the relationship between immune checkpoint inhibitor (ICI) efficacy and vascular architecture, has not been well-characterized in human imaging studies. We found that ICI-responsive BM possessed a structurally balanced vascular makeup, which was linked to improved vascular efficiency and an immune-stimulatory microenvironment. In contrast, ICI-resistant BM were characterized by a lack of immune cell infiltration and a highly aberrant vasculature dominated by large-caliber vessels. Peri-tumor region analysis revealed early functional changes predictive of ICI resistance before radiographic evidence on conventional MRI. This study was one of the largest functional imaging studies for BM and establishes a foundation for functional studies that illuminate the mechanisms linking patterns of vascular architecture with immunosuppression, as targeting these aspects of cancer biology may serve as the basis for future combination treatments.

cancer biology↗

bioRxiv2023

Histone Lactylation Drives CD8 T Cell Metabolism and Function

The activation and functional differentiation of CD8 T cells are linked to metabolic pathways that result in the production of lactate. Lactylation is a lactate-derived histone post-translational modification (hPTM); however, the relevance of histone lactylation in the context of CD8 T cell activation and function is not known. Here, we show the enrichment of H3K18-lactylation (H3K18la) and H3K9-lactylation (H3K9la) in human and murine CD8 T cells which act as transcription initiators of key genes regulating CD8 T cell phenotype and function. Further, we note distinct impacts of H3K18la and H3K9la on CD8 T cell subsets linked to their specific metabolic profiles. Importantly, we demonstrate that modulation of H3K18la and H3K9la by targeting metabolic and epigenetic pathways regulates CD8 T cell effector function including anti-tumor immunity in preclinical models. Overall, our study uncovers the unique contributions of H3K18la and H3K9la in modulating CD8 T cell phenotype and function intricately associated with metabolic state.

immunology↗

bioRxiv2023

Integrative phenotypic-transcriptomic analysis of soybean plants subjected to multifactorial stress combination

Global warming, climate change, and industrial pollution are altering our environment subjecting crops to an increasing number and complexity of abiotic stress conditions, concurrently or sequentially. Recent studies revealed that a combination of 3 or more stresses simultaneously impacting a plant (termed multifactorial stress combination; MFSC) can cause a drastic decline in plant growth and survival, even if the level of each stress involved in the MFSC has a negligible effect on plants. However, the impacts of MFSC on crops are largely unknown. We subjected soybean plants to a MFSC of up to five different stresses (water deficit, salinity, low phosphate, acidity, and cadmium), in an increasing level of complexity, and conducted integrative transcriptomic-phenotypic analysis of reproductive and vegetative tissues. We reveal that MFSC has a negative cumulative effect on soybean yield, that each set of MFSC condition elicits a unique transcriptomic response (that is different between flowers and leaves), and that selected genes expressed in leaves or flowers are linked to the effects of MFSC on different vegetative, physiological, and/or reproductive parameters. We further reveal that the transcriptomic response of soybean and Arabidopsis to MFSC shares common features associated with reactive oxygen and iron/copper signaling/metabolism. Our study provides unique phenotypic and transcriptomic datasets for dissecting the mechanistic effects of MFSC on the vegetative, physiological, and reproductive processes of a crop plant.

plant biology↗

bioRxiv2023

Functional recovery associated with dendrite regeneration in PVD neuron of C. elegans

PVD neuron of C. elegans is a highly polarized cell with well-defined axonal, and dendritic compartments. PVD neuron operates in multiple sensory modalities controlling both nociceptive touch sensation and body posture. Although both axon and dendrites of this neuron show regeneration response following laser-assisted injury, it is rather unclear how the behavior associated with this neuron is affected by the loss of these structures. It is also unclear whether neurite regrowth would lead to functional restoration in these neurons. Upon axotomy, using a femtosecond laser, we saw that harsh touch response was specifically affected leaving the body posture unperturbed. Subsequently, recovery in the touch response is highly correlated to the axon regrowth, which was dependent on DLK-1 MAP Kinase. Dendrotomy of both major and minor primary dendrites affected the wavelength and amplitude of sinusoidal movement without any apparent effect on harsh touch response. We further correlated the recovery in posture behavior to the type of dendrite regeneration events. We found that dendrite regeneration with the fusion and reconnection between the proximal and distal branches of the injured dendrite corresponded to improved recovery of posture. Our data revealed that the axons and dendrites differentially regulate the functions of PVD neurons. It also revealed that dendrite and axon regeneration are both functionally and molecularly distinct.

neuroscience↗

bioRxiv2023

Highly scalable and standardized organ-on-chip platform with TEER for biological barrier modeling

The development of new therapies is hampered by the lack of predictive, and patient-relevant in vitro models. Organ-on-chip (OOC) technologies can potentially recreate physiological features and hold great promise for tissue and disease modeling. However, the non-standardized design of these chips and perfusion control systems has been a barrier to quantitative high-throughput screening (HTS). Here we present a scalable OOC microfluidic platform for applied kinetic in vitro assays (AKITA) that is applicable for high, medium, and low throughput. Its standard 96-well plate and 384-well plate layouts ensure compatibility with existing laboratory workflows and high-throughput data collection and analysis tools. The AKITA plate is optimized for the modeling of vascularized biological barriers, primarily the blood-brain barrier, skin, and lung, with precise flow control on a custom rocker. The integration of trans-epithelial electrical resistance (TEER) sensors allows rapid and repeated monitoring of barrier integrity over long time periods. Together with automated confocal imaging and compound permeability testing analyses, we demonstrate the flexibility of the AKITA platform for establishing human-relevant models for preclinical drug and precision medicines efficacy, toxicity, and permeability under physiological conditions.

bioengineering↗

bioRxiv2023

Vasodilators activate TMEM16A channels in endothelial cells to reduce blood pressure

Endothelial cells (ECs) regulate vascular contractility to control regional organ blood flow and systemic blood pressure. Several cation channels are expressed in ECs which regulate arterial contractility. In contrast, the molecular identity and physiological functions of anion channels in ECs is unclear. Here, we generated tamoxifen-inducible, EC-specific TMEM16A knockout (TMEM16A ecKO) mice to investigate the functional significance of this chloride (Cl-) channel in the resistance vasculature. Our data demonstrate that TMEM16A channels generate calcium-activated Cl- currents in ECs of control (TMEM16Afl/fl) mice that are absent in ECs of TMEM16A ecKO mice. Acetylcholine (ACh), a muscarinic receptor agonist, and GSK101, a TRPV4 agonist, activate TMEM16A currents in ECs. Single molecule localization microscopy data indicate that surface TMEM16A and TRPV4 clusters locate in very close nanoscale proximity, with [~]18% exhibiting overlap in ECs. ACh stimulates TMEM16A currents by activating Ca2+ influx through surface TRPV4 channels without altering the size or density of TMEM16A or TRPV4 surface clusters, their spatial proximity or colocalization. ACh-induced activation of TMEM16A channels in ECs produces hyperpolarization in pressurized arteries. ACh, GSK101 and intraluminal ATP, another vasodilator, all dilate pressurized arteries through TMEM16A channel activation in ECs. Furthermore, EC-specific knockout of TMEM16A channels elevates systemic blood pressure in conscious mice. In summary, these data indicate that vasodilators stimulate TRPV4 channels, leading to Ca2+-dependent activation of nearby TMEM16A channels in ECs to produce arterial hyperpolarization, vasodilation and a reduction in blood pressure. We identify TMEM16A as an anion channel present in ECs that regulates arterial contractility and blood pressure. One sentence summaryVasodilators stimulate TRPV4 channels, leading to calcium-dependent activation of nearby TMEM16A channels in ECs to produce arterial hyperpolarization, vasodilation and a reduction in blood pressure.

physiology↗