Biology subjects

Ramamoorthy, S.

Publications and source records attributed to Ramamoorthy, S..

11 recordsLinked to original sources

bioRxiv2026

Benchmarking Molecular Methods of Protein Engineering and Surveying Protein Search Space to Bolster Artificial Intelligence-Based Designs

Proteins are complex macromolecules that drive functionality in cells across all life on Earth. Protein engineering and AI-based designs facilitate exploration of the vast search space. Herein, we explore and compare mutagenic methodologies in combination with fast and efficient phenotypic evaluation of a pink fluorophore (Magenta) to map protein search space and inform AI-based designer algorithms. We binned mutated variants as neutral, deleterious, or catastrophic based on the observed phenotype and fitness measurement (relative fluorescent units, RFUs). Importantly, we found that for a given fluorophore, about 80% of random mutations were deleterious for the artificially selected phenotype resulting in decreased fitness, 20% were neutral mutations, and 0.8% enhanced the artificially selected phenotype. Using error prone PCR (epPCR), we were able to identify four candidates in the first round and one candidate in the second round of epPCR for enhanced fluorescence and in toto enhanced RFUs by 35% after two rounds of epPCR. We compared these rates of true positive enhanced RFU generated sequences against a state of the art evolutionary algorithm approach and found that biological computation beats it in its current state. We conclude with suggestions for key improvements to enhance algorithm performance in the future. Taken together, we demonstrated that iterative rounds of variant prediction improved our algorithm, and our study can serve as a reference point for future AI driven modeling studies in red fluorescent protein engineering.

bioinformatics↗

bioRxiv2026

Hippocampal/medial temporal sclerosis is associated with clinical severity, regional TDP-43, and hippocampal atrophy in Alzheimer disease

Alzheimer disease neuropathologic change (ADNC) does not fully explain clinical severity, suggesting important contributions from coexisting pathologies. We examined major co-pathologies in a multicenter autopsy cohort, focusing on hippocampal/medial temporal sclerosis (HS/MTL sclerosis), clinical severity, regional TDP43 topography, clinical status near death, and gross hippocampal atrophy. We analyzed longitudinal clinical and neuropathology data from the National Alzheimers Coordinating Center autopsy cohort. Form specific definitions harmonized AD pathology, major co-pathologies, HS/MTL sclerosis, and regional TDP43 findings across neuropathology versions 8 to10. HS/MTL sclerosis was present in 13.0% of assessed AD cases and was associated with greater CDR Dementia Staging Instrument sum-of-boxes severity in non AD (odds ratio [OR], 4.93; 95% confidence interval [CI], 3.66 to 6.63) and AD strata (OR, 2.56; 95% CI, 2.15 to 3.04). In version 10, HS/MTL sclerosis was associated with higher regional TDP43 topography (OR, 6.54; 95% CI, 3.88 to11.02) and greater gross hippocampal atrophy (OR, 6.43; 95% CI, 4.97 to 8.32). Among AD cases assessed within 2 years of death, HS negative participants had greater odds of non-dementia status than HS positive participants (OR, 5.81; 95% CI, 2.80 to 12.06). HS/MTL sclerosis marked greater clinical severity in both AD and non-AD strata and co-occurred with regional TDP43 involvement and hippocampal atrophy. Its absence identified a subset with intermediate/high AD pathology but no dementia near death. Clinical severity therefore cannot be inferred from ADNC alone.

neuroscience↗

bioRxiv2026

Kappa opioid receptors regulate cocaine effects on nucleus accumbens dopamine through phosphorylation of dopamine transporter at the threonine 53 site

Chronic cocaine exposure leads to tolerance of the dopamine transporter (DAT) to cocaine and heightened kappa opioid receptor (KOR)-induced inhibition of dopamine (DA) release in the nucleus accumbens (NAc). KORs on presynaptic DA terminals inhibit DA release and modulate DAT function, but whether KOR signaling causes DAT tolerance is unknown. We demonstrate that rats self-administering cocaine escalate their intake, and the KOR antagonist norbinaltorphimine (nBNI) blunts this escalation. We employed fast-scan cyclic voltammetry (FSCV) in acute brain slices containing the NAc core from these rats and found that the cocaine exposure paradigm resulted in tolerance of the DAT to cocaine, an effect absent in animals pre-treated with nBNI. In drug-naive mice, either superfusion of the KOR agonist U50,488 or a single in vivo injection of U69,593 inhibited DA release and reduced cocaine-induced inhibition of DA reuptake, reproducing the tolerance phenotype without any drug history. We replicated this finding in the NAc of rats, and with the uptake inhibitor nomifensine in rhesus macaques, indicating conservation across rodents and non-human primates. As KOR activation results in phosphorylation of the threonine-53 site on the DAT, we tested whether this is required for the KOR-mediated DAT tolerance to cocaine. We show that a mouse line with a phosphorylation-insensitive alanine-53 mutation of the DAT has enhanced DA release and uptake. Remarkably, KOR agonism no longer mediated DAT tolerance to cocaine in these mice, demonstrating the dependence of this effect on the threonine-53 site and highlighting a KOR-driven mechanism underlying cocaine tolerance at the DAT.

neuroscience↗

bioRxiv2026

Spatio-temporal coordination of virulence, metabolism, and stress responses shapes infection dynamics of Xanthomonas perforans

Plants provide distinct ecological niches for diverse microbial communities, with each member adopting strategies tailored to the specific ecological niche it inhabits. Two foliar niches, the leaf surface (epiphytic environment) and the apoplast, impose distinct physiological constraints on microbial fitness, particularly for hemibiotrophic pathogens. In this study, we investigated how these environments shape the transcriptional responses of Xanthomonas perforans (Xp), a tomato pathogen, and how its virulence factors, metabolic pathways, and regulatory networks are spatially and temporally coordinated during disease progression. Transcriptome profiling of a pathogen recovered from the leaf surface and apoplast revealed pronounced niche-specific and colonization stage-specific gene expression patterns. Early epiphytic colonization was characterized by activation of chemosensing, and motility pathways that facilitate pathogen relocation and acquisition of limiting nutrients such as iron and phosphate. This stage also featured induction of DNA and protein repair systems, quorum sensing pathways, phenylalanine degradation and tyrosine conversion to counter phenylpropanoid defenses, genes involved in mitigating osmotic and oxidative stress, active DNA exchange machinery, and type VI secretion system-mediated microbial competition. Upon entry into the apoplast, Xp shifted toward active metabolism and replication, accompanied by investment in type II and III secreted virulence factor expression. Genes involved in evasion of plant immunity and overcoming of host-mediated nutrient sequestration were also upregulated, including those involved in quinone detoxification, phosphate and sulfur uptake, and fatty acid, xanthan, and LPS biosynthesis. During late apoplastic colonization, the pathogen transitioned again towards strong stress response activation, followed by renewed expression of flagellar motility and chemotaxis genes, suggesting preparation for dissemination. Notably, genes associated with oxidative and nutrient stress were enriched across both niches, although specific components differed. Type IV pili, conjugation genes, and plasmid-borne type III effectors were induced early in both niches, suggesting their niche-independent role in initial establishment. Together, these findings reveal a coordinated spatio-temporal regulatory strategy during the transition from the leaf surface to the apoplast. Author SummaryXanthomonas perforans is a foliar bacterial pathogen that infects tomato plants and leads to severe yield losses. To establish a successful infection, the pathogen must overcome a series of environmental and host-imposed challenges. This study characterizes the traits activated at distinct stages of infection, during both early and late pathogenesis, and across different niches, including the leaf surface and its interior (apoplastic) space. On the leaf surface Xanthomonas mainly focuses on movement, communication, and survival against stress and starvation with the major functions related to motility, nutrient uptake, and DNA transfer during early stages. Once inside the leaf, the bacteria switches tactics to focus primarily on reproduction, defense against the plant immune response, production of factors that weaken the plants defenses and gaining access to nutrients the plant normally restricts. Understanding the different stages of infection may inform how the crosstalk among host and pathogen unfolds during pathogenesis allowing us to understand the host environment. These findings can help us discover pathogen weaknesses that could be targeted for disease management.

microbiology↗

bioRxiv2025

Identifying Key Hub Genes that Attribute Varying Host Responses: A Longitudinal RNA-seq Analysis of SARS-CoV-2 Delta and Omicron Infections.

Coronavirus disease 2019 (COVID-19), caused by SARS-CoV-2, has severely impacted global health, with different Variants of Concern (VOCs) resulting in varied clinical outcomes. Among these, the Delta and Omicron variants have drawn significant attention due to their distinct characteristics--Delta is associated with higher virulence and Omicron has greater transmissibility but reduced severity. This study compares host immune responses to the Delta and Omicron variants using RNA-seq data from infected primary human airway epithelial cells. Both variants triggered a robust antiviral innate immune response by 2 days post-infection (dpi). However, Omicron was found to elicit a more rapid immune response, showing pathway enrichment at 1 dpi. In contrast, Delta displayed no immune-related pathway activation within the first 24 hours, suggesting it may evade early immune detection, promoting increased viral replication. By 3 dpi, Delta induced a more aggressive immune response, particularly in pathways related to cell death and pro-inflammatory signaling, such as "programmed cell death" and "regulation of cell death." Weighted gene co-expression network analysis revealed distinct immune-related genes: Delta infections were characterized by hub genes like MYD88 and IL1R1 involved in pro-inflammatory responses, HLA-A & B, NLRC5 and PSMB9 involved in antigen presentation and TNSF10 and IFR1 involved in pro-apoptotic processes. Conversely, Omicron infections were marked by hub genes such as CXCL1 and CXCL8 involved in immune cells recruitment, MET and LYN involved in reducing hyperinflammatory responses and maintaining immune balance, and IFI44, EIF2AK2 and IFIT5 responsible for the sensing of viral RNA among others.

bioinformatics↗

bioRxiv2024

Dopamine transporter threonine-53 phosphorylation dictates kappa opioid receptor mediated locomotor suppression and conditioned place aversion via transporter upregulation

Dynorphin (DYN)/kappa opioid receptor (KOR) activation contributes to aversion, dysphoria, sedation, depression, and enhanced psychostimulant-rewarding effects, which have been attributed to the inhibition of dopamine (DA) release. DYN fibers synapse onto DA terminals which express both KOR and dopamine transporter (DAT). DAT activity is critical in the regulation of DA dynamics and dopaminergic neurotransmission. Previously, we demonstrated that KOR agonists upregulate DAT activity via ERK1/2 signaling involving phospho-Thr53 DAT (pT53-DAT). However, whether pT53-DAT is involved in KOR-mediated DAT regulation in-vivo and whether such phenomenon contributes to the behavioral effects of KOR agonism are unknown. Here, we investigated the role of endogenous pT53-DAT in KOR-mediated DAT regulation and the effect of KOR agonists on locomotor suppression and aversive behaviors using DAT-Ala53 knock-in mice expressing DAT carrying non-phosphorylatable Ala at position 53 replacing Thr. Acute systemic administration of KOR agonist, U69593 resulted in KOR antagonist-sensitive increases in DAT activity in parallel to increases in pT53-DAT, and DAT Vmax and surface expression in the ventral and dorsal striatum (containing the nucleus accumbens and caudate putamen respectively) of WT, but not DAT-Ala53 mice. KOR agonists produced conditioned place aversion (CPA) and locomotor suppression in WT but not DAT-Ala53 mice. However, both WT and DAT-Ala53 mice exhibited similar lithium chloride-induced CPA and morphine-induced conditioned place preference (CPP). These findings provide the first evidence that locomotor suppression and aversive responses to KOR agonists manifest due to the modulation of DAT activity via DAT-T53 phosphorylation establishing a causal relationship of pT53-DAT in KOR-mediated DAT regulation and KOR agonist-induced adverse effects.

animal behavior and cognition↗

bioRxiv2024

Enhancing cellular production of fucoxanthin through machine learning assisted predictive approach in Isochrysis galbana

The marine microalgae, Isochrysis galbana is a prolific producer of fucoxanthin which is a xanthophyll carotenoid with substantial global market value boasting extensive applications in the food, nutraceutical, pharmaceutical, and cosmetic industries. Although supplementation of different phytohormones to medium enhances fucoxanthin production, the quantification of pigment by conventional means is time-consuming and labor-intensive. This study addressed the multiple methodological limitations of HPLC-based fucoxanthin quantification and emphasized the need to develop a Machine Learning (ML) model as optimization and precise prediction remain a challenging task. Hence, an integrated experimental approach coupled with ML models was employed to predict fucoxanthin yield by supplementation of various phytohormones. The accuracy of fucoxanthin prediction excluding and including hormone descriptors was compared and evaluated using the ML models namely Random Forest (RF), Linear Regression (LR), Artificial Neural Network (ANN), and Support Vector Machine (SVM). RF model provided the most accurate prediction excluding hormone descriptors with coefficient of determination (R2 = 0.809) and root-mean-square error (RMSE = 0.776) followed by the ANN model with (R2 = 0.722) and (RMSE = 0.937). The inclusion of hormone descriptors for training and pre-processing of data further improved the fucoxanthin prediction accuracy of the RF model to (R2 = 0.839) and (RMSE = 0.712) and ANN model to (R2 = 0.738) and (RMSE = 0.909). These results indicated that the combination of low-cost, Ultraviolet (UV) spectrometric-based fucoxanthin quantification coupled with ML algorithms can be efficiently used for reliable prediction and enhanced fucoxanthin production, therefore highlighting a promising approach and furnishing invaluable insights towards the commercialization of microalgal fucoxanthin production.

systems biology↗

bioRxiv2023

Transcriptome profiling of type VI secretion system core gene tssM mutant of Xanthomonas perforans highlights regulators controlling diverse functions ranging from virulence to metabolism

Type VI secretion system (T6SS) is a versatile, contact dependent contractile nano-weapon in Gram-negative bacteria that fires proteinaceous effector molecules directly into prokaryotic and eukaryotic cells aiding in manipulation of the host and killing of competitors in complex niches. In plant pathogenic xanthomonads, T6SS has been demonstrated to play these diverse roles in individual pathosystems. However, the regulatory circuit involved in mediating biological functions carried out by T6SS are still elusive in Xanthomonas sp. To bridge this knowledge gap, we conducted an in vitro transcriptome screen using plant apoplast mimicking minimal medium, XVM2 medium, to decipher the effect of tssM deletion, a core gene belonging to T6SS-cluster i3*, on the regulation of gene expression in Xanthomonas perforans strain AL65. Transcriptomic data revealed that a total of 277 and 525 genes were upregulated, while 307 and 392 genes were downregulated in the mutant strain post 8 and 16 hours of growth in XVM2 medium. The transcript abundance of several genes associated with flagellum and pilus biogenesis as well as type III secretion system were downregulated in the mutant strain. Deletion of tssM of cluster-i3* resulted in upregulation of several T6SS genes belonging to cluster-i3*** and genes involved in biofilm and cell wall biogenesis. Similarly, transcription regulators like rpoN, Pho regulon, rpoE and csrA were identified to be upregulated in the mutant strain. Our results suggest that T6SS modulates the expression of global regulators like csrA, rpoN and pho regulons triggering a signaling cascade and co-ordinates the expression of suite of virulence factors, stress response genes and metabolic genes. ImportanceType VI secretion system (T6SS) has received attention due to its significance in mediating inter-organismal competition through contact-dependent release of effector molecules into prokaryotic and eukaryotic cells. Reverse-genetic studies targeting core genes of T6SS have indicated the role of T6SS in virulence in a variety of plant pathogenic bacteria, including Xanthomonas studied here. However, it is not clear whether such effect on virulence is merely because of a shift in the microbiome-mediated protection or if T6SS is involved in a complex regulatory network governing virulence in plant pathogens involving type III secretion system or c-di-GMP signaling pathways. In this study, we conducted in vitro transcriptome profiling in minimal medium to decipher the signaling pathways regulated by tssM-i3* in Xanthomonas perforans strain AL65. We show that TssM-i3* regulates the expression of a suite of genes associated with virulence and metabolism either directly or indirectly by altering the transcription of several regulators. These findings further expand our knowledge on the intricate molecular circuits regulated by T6SS in phytopathogenic bacteria.

microbiology↗

bioRxiv2023

Kappa Opioid Receptor Antagonism Rescues Genetic Perturbation of Dopamine Homeostasis: Molecular, Physiological and Behavioral Consequences

Aberrant dopamine (DA) signaling is implicated in schizophrenia, bipolar disorder (BPD), autism spectrum disorder (ASD), substance use disorder, and attention-deficit/hyperactivity disorder (ADHD). Treatment of these disorders remains inadequate, as exemplified by the therapeutic use of d-amphetamine and methylphenidate for the treatment of ADHD, agents with high abuse liability. In search for an improved and non-addictive therapeutic approach for the treatment of DA-linked disorders, we utilized a preclinical mouse model expressing the human DA transporter (DAT) coding variant DAT Val559, previously identified in individuals with ADHD, ASD, or BPD. DAT Val559, like several other disease-associated variants of DAT, exhibits anomalous DA efflux (ADE) that can be blocked by d-amphetamine and methylphenidate. Kappa opioid receptors (KORs) are expressed by DA neurons and modulate DA release and clearance, suggesting that targeting KORs might also provide an alternative approach to normalizing DA-signaling disrupted by perturbed DAT function. Here we demonstrate that KOR stimulation leads to enhanced surface trafficking and phosphorylation of Thr53 in wildtype DAT, effects achieved constitutively by the Val559 mutant. Moreover, these effects can be rescued by KOR antagonism of DAT Val559 in ex vivo preparations. Importantly, KOR antagonism also corrected in vivo DA release as well as sex-dependent behavioral abnormalities observed in DAT Val559 mice. Given their low abuse liability, our studies with a construct valid model of human DA associated disorders reinforce considerations of KOR antagonism as a pharmacological strategy to treat DA associated brain disorders.

neuroscience↗

bioRxiv2021

Neurotoxic astrocytic glypican-4 drives APOE4-dependent tau pathology

The aggregation of tau proteins into insoluble filaments and the spread of these filaments across brain regions are the major drivers of neurodegeneration in tauopathies, including in Alzheimers disease (AD). Apolipoprotein E4 (APOE4), a crucial genetic risk factor for late-onset AD, has been shown to exacerbate tau pathology in mouse models. However, the exact mechanisms through which APOE4 induces tau pathology remains unknown. Here, we report that the astrocyte-secreted protein glypican-4 (GPC-4), which we identify as a novel binding partner of APOE4, drives tau pathology. We discovered that GPC-4 preferentially interacts with APOE4 in comparison to APOE2, considered to be a protective allele to AD, and post-mortem APOE4-carrying AD brains highly express GPC-4 in neurotoxic astrocytes. The astrocyte-secreted GPC-4 induced both tau accumulation and propagation in vitro. CRISPR/dCas9 mediated activation of GPC-4 in a tauopathy mouse model robustly induced tau pathology. Using in vitro Tau FRET-biosensor cells, human iPSCs-derived astrocytes and an in vivo mouse model, we found that APOE4-induced tau pathology was greatly diminished in the absence of GPC-4. We further show that APOE4-mediated surface trafficking of APOE receptor low-density lipoprotein receptor-related protein 1 (LRP1) through GPC-4 can be a gateway to tau spreading. Together, our data comprehensively demonstrate that APOE4-induced tau pathologies are directly mediated by GPC-4.

neuroscience↗

bioRxiv2019

A mutation in Hnrnph1 that decreases methamphetamine-induced reinforcement, reward, and dopamine release and increases synaptosomal hnRNP H and mitochondrial proteins

Individual variation in the addiction liability of amphetamines has a heritable genetic component. We previously identified Hnrnph1 (heterogeneous nuclear ribonucleoprotein H1) as a quantitative trait gene underlying decreased methamphetamine-induced locomotor activity in mice. Here, mice (both male and female) with a heterozygous mutation in the first coding exon of Hnrnph1 (H1+/-) showed reduced methamphetamine reinforcement and intake and dose-dependent changes in methamphetamine reward as measured via conditioned place preference. Furthermore, H1+/- mice showed a robust decrease in methamphetamine-induced dopamine release in the nucleus accumbens with no change in baseline extracellular dopamine, striatal whole tissue dopamine, dopamine transporter protein, or dopamine uptake. Immunohistochemical and immunoblot staining of midbrain dopaminergic neurons and their forebrain projections for tyrosine hydroxylase did not reveal any major changes in staining intensity, cell number, or in the number of forebrain puncta. Surprisingly, there was a two-fold increase in hnRNP H protein in the striatal synaptosome of H1+/- mice with no change in whole tissue levels. To gain insight into the molecular mechanisms linking increased synaptic hnRNP H with decreased methamphetamine-induced dopamine release and behaviors, synaptosomal proteomic analysis identified an increased baseline abundance of several mitochondrial complex I and V proteins that rapidly decreased at 30 min post-methamphetamine administration in H1+/- mice. In contrast, the much lower level of basal synaptosomal mitochondrial proteins in wild-type mice showed a rapid increase in response to methamphetamine. We conclude that H1+/- decreases methamphetamine-induced dopamine release, reward, and reinforcement and induces dynamic changes in basal and methamphetamine-induced synaptic mitochondrial function.\n\nSIGNIFICANCE STATEMENTMethamphetamine dependence is a significant public health concern with no FDA-approved treatment. We discovered a role for the RNA binding protein hnRNP H in methamphetamine reward and reinforcement. Hnrnph1 mutation also blunted methamphetamine-induced dopamine release in the nucleus accumbens - a key neurochemical event contributing to methamphetamine addiction liability. Finally, Hnrnph1 mutants showed a marked increase in basal level of synaptosomal hnRNP H and mitochondrial proteins that decreased in response to methamphetamine whereas wild-type mice showed a methamphetamine-induced increase in synaptosomal mitochondrial proteins. Thus, we identified a potential role for hnRNP H in basal and dynamic mitochondrial function that informs methamphetamine-induced cellular adaptations associated with reduced addiction liability.

neuroscience↗