Biology subjects

Ram, A.

Publications and source records attributed to Ram, A..

12 recordsLinked to original sources

bioRxiv2025

Psilocybin has no immediate or persistent analgesic effect in acute and chronic mouse pain models

The psychedelic psilocybin may have lasting therapeutic effects for patients with chronic pain syndromes. Some clinical and preclinical data suggest these putative benefits derive from direct analgesic effects. However, this possibility has not been comprehensively tested in preclinical models. Here, we show that psilocybin is not analgesic over a range of doses across multiple pain assays and models of acute and chronic inflammatory, neuropathic, or musculoskeletal pain in mice.

neuroscience↗

bioRxiv2024

Influenza A defective viral genomes and non-infectious particles are in-creased by host PI3K inhibition via anti-cancer drug alpelisib

RNA viruses produce abundant defective viral genomes during replication, setting the stage for interactions between viral genomes that alter the course of pathogenesis. Harnessing these interactions to develop antivirals has become a recent goal of intense research focus. Despite decades of research, the mechanisms that regulate the production and interactions of Influenza A defective viral genomes are still unclear. The role of the host is essentially unexplored; specifically, it remains unknown whether host metabolism can influence the formation of defective viral genomes and the particles that house them. To address this question, we manipulated host cell anabolic signaling activity and monitored the production of defective viral genomes and particles by A/H1N1 and A/H3N2 strains, using a combination of single-cell immunofluorescence quantification, third-generation long-read sequencing, and the cluster-forming assay, a method we developed to titer defective and fully-infectious particles simultaneously. Here we show that alpelisib (Piqray), a highly selective inhibitor of mammalian Class 1a phosphoinositide-3 kinase (PI3K) receptors, significantly changed the proportion of defective particles and viral genomes (specifically deletion-containing viral genomes) in a strain-specific manner, under conditions that minimize multiple cycles of replication. Alpelisib pre-treatment of cells led to an increase in defective particles in the A/H3N2 strain, while the A/H1N1 strain showed a decrease in total viral particles. In the same infections, we found that defective viral genomes of polymerase and antigenic segments increased in the A/H1N1 strain, while the total particles decreased suggesting defective interference. We also found that the average deletion size in polymerase complex viral genomes increased in both the A/H3N2 and A/H1N1 strains. The A/H1N1 strain, additionally showed a dose-dependent increase in total number of defective viral genomes. In sum, we provide evidence that host cell metabolism can increase the production of defective viral genomes and particles at an early stage of infection, shifting the makeup of the infection and potential interactions among virions. Given that Influenza A defective viral genomes can inhibit pathogenesis, our study presents a new line of investigation into metabolic states associated with less severe flu infection and the potential induction of these states with metabolic drugs.

microbiology↗

bioRxiv2024

Deciphering the History of ERK Activity from Fixed-Cell Immunofluorescence Measurements

The Ras/ERK pathway drives cell proliferation and other oncogenic behaviors, and quantifying its activity in situ is of high interest in cancer diagnosis and therapy. Pathway activation is often assayed by measuring phosphorylated ERK. However, this form of measurement overlooks dynamic aspects of signaling that can only be observed over time. In this study, we combine a live, single-cell ERK biosensor approach with multiplexed immunofluorescence staining of downstream target proteins to ask how well immunostaining captures the dynamic history of ERK activity. Combining linear regression, machine learning, and differential equation models, we develop an interpretive framework for immunostains, in which Fra-1 and pRb levels imply long term activation of ERK signaling, while Egr-1 and c-Myc indicate recent activation. We show that this framework can distinguish different classes of ERK dynamics within a heterogeneous population, providing a tool for annotating ERK dynamics within fixed tissues.

molecular biology↗

bioRxiv2023

Finding needles in haystacks: identification of novel conserved PETase enzymes in Streptomyces

The rising use of plastic results in an appalling amount of waste which scatters into the environment affecting environmental, animal, and human health. One of these plastics is PET which is mainly used for bottles and textiles. In this research, we investigate the PET degrading ability of the IsPETase homolog ScLipA from Streptomyces coelicolor. Of 96 different Streptomyces strains screened, 18 % were able to degrade the model substrate BHET. Three different variants of lipase A, named ScLipA, S2LipA and S92LipA were identified and analyzed in detail. The lipA gene was deleted from S. coelicolor M145 using CRISPR/Cas9, resulting in reduced BHET degradation. LipA overexpression in the knock-out background significantly enhanced BHET degradation. All three enzymes were expressed in E. coli BL21 for protein purification and biochemical analysis, showing that enzymatic activity most likely resides in a dimeric form of the enzyme. The optimum pH and temperature were determined to be pH 7 and 25 {degrees}C for all three variants. Using these conditions, the activity on BHET and amorphous PET film was investigated. S2LipA efficiently degraded BHET and caused roughening and small indents on the surface of PET films, consistent with PET-degrading activity. The frequent occurrence of the S2LipA variant in Streptomyces suggests an environmental advantage towards the degradation of more hydrophobic substrates such as these polluting plastics in the environment.

microbiology↗

bioRxiv2023

Sequence-context dependency shown by eIF5G31R mutant for UUG start codon selection in Saccharomyces cerevisiae

In higher eukaryotes, the efficiency of start codon selection depends upon its sequence context A/GxxAUGG, especially at the -3 and +4 position. However, S. cerevisiae prefers AAA/UAUGU sequence context surrounding the AUG start codon. Furthermore, it was demonstrated that the first AUG codon on the mRNA serves as a signal for ribosomal recognition of the initiation site selection. In yeast, the G31R mutation in eIF5 shows Suppressor of initiation codon (Sui-) phenotype enabling 40S ribosome to initiate at UUG start codon. To understand whether the UUG codon recognition by the eIF5G31R mutant is sensitive to the sequence context, we made different HIS4-LacZ reporter constructs with UUG as start codon and changed the sequence context at -3 and -2 positions. The HIS4UUG-LacZ transcripts that carry purine (A/G) at the -3-position showed better {beta}-Galactosidase activity than pyrimidine (U/C). Furthermore, on changing the -1 position for the AAA context, we discovered purines are more preferred at the -1 position for the UUG start codon selection.

molecular biology↗

bioRxiv2022

Oral Delivery of Kidney Targeting Nanotherapeutics for Polycystic Kidney Disease

Autosomal dominant polycystic kidney disease (ADPKD) is the most common inherited renal disorder. Although a variety of candidate drugs have been found to modulate cystogenesis in animal studies, results from clinical trials have often been unfavorable due to low renal bioavailability and drug-induced side effects. To mitigate this, nanoparticles can be designed to deliver drugs directly to the target organ to increase effective dose while limiting off-target side effects. Unfortunately, there are no kidney-targeted nanomedicines clinically available, and most of the existing FDA-approved nanoparticles require intravenous administration which is not suitable for ADPKD that require lifelong therapy. To address this, we developed an oral drug delivery system using chitosan nanoparticles (CS-NP) that were loaded with peptide amphiphile micelles carrying metformin (met), an ADPKD drug candidate (CS-KM-met). We previously showed that CS-NP can shield met in the gastrointestinal tract; thus, we hypothesized that CS-NP could also enhance bioavailability of kidney-targeting micelles (KMs) upon oral administration. Specifically, we measured the loading capacity of KM-met in CS-NP, evaluated the stability of CS-KM-met under acidic conditions that mimic the gastric environment, and measured in vitro therapeutic effects. Upon oral administration in C57BL/6J mice, CS-KM-met showed significantly greater bioavailability and accumulation in the kidneys as compared to KM-met without CS-NP or free met for up to 24 hours. As such, CS-KM-met showed enhanced therapeutic efficacy in vivo upon oral administration in PKD mice (Pkd1fl/fl; Pax8-rtTA; Tet-O-Cre) compared to KM-met only. Herein, we demonstrate the potential of an oral delivery nanoformulation for the treatment of chronic kidney diseases such as ADPKD for the first time.

bioengineering↗

bioRxiv2022

A small molecule ligand for the novel pain target GPR171 produces minimal reward in mice

ProSAAS is one of the most abundant proteins in the brain and is processed into several smaller peptides. One of which, BigLEN, is an endogenous ligand for the G protein-coupled receptor, GPR171. Recent work in rodent models has shown that a small-molecule ligand for GPR171, MS15203, increases morphine antinociception and is effective in lessening chronic pain. While these studies provide evidence for GPR171 as a possible pain target, its abuse liability has not yet been assessed which we evaluate in the current study. We first mapped the distribution of GPR171 and ProSAAS throughout the reward circuit of the brain using immunohistochemistry and showed that GPR171 and ProSAAS are localized in the hippocampus, basolateral amygdala, nucleus accumbens, prefrontal cortex. In the major dopaminergic structure, the ventral tegmental area (VTA), GPR171 appeared to be primarily localized in dopamine neurons while ProSAAS is outside of dopamine neurons. Next, MS15203 was administered to mice with or without morphine, and VTA slices were stained for the immediate early gene c-Fos as a marker of neuronal activation. Quantification of c-Fos-positive cells revealed no statistical difference between MS15203 and saline, suggesting that MS15203 does not increase VTA activation and dopamine release. Similarly, the results of a conditioned place preference experiment showed that treatment with MS15203 or MS15203 + Morphine produced no place preference compared to Saline indicating a lack of reward-related behavior. Taken together this data provides evidence that the novel pain therapeutic, MS15203, has minimal reward liability. Therefore, GPR171 deserves further exploration as a pain target. Significance StatementMS15203, a drug that activates the receptor GPR171, was previously shown to increase morphine analgesia. The authors use in vivo and histological techniques to show that it fails to activate the rodent reward circuitry, providing support for the continued exploration of MS15203 as a novel pain drug, and GPR171 a novel pain target.

neuroscience↗

bioRxiv2022

LaeA-dependent production of small molecules of Aspergillus niger that compete with specific antibodies that bind to human immune receptors

Microorganisms secrete a variety of compounds into their environment such as proteins, carbohydrates, and secondary metabolites. These molecules play diverse roles in the interaction of microbes with their abiotic and biotic environment. Little is known about secreted fungal molecules mediating immune evasion. Here we screened culture media of three Aspergilli to assess whether these fungi secrete molecules that can compete with specific antibodies that bind to human immune receptors. Culture media of Aspergillus fumigatus Af293, Aspergillus tubingensis CBS 133792 and the non-acidifying mutant strain Aspergillus niger D15#26 contained components that showed competition for binding to a total of 13 receptors, of which PSGL-1, CXCR1, and CXCR2, were shared between the three species. Filtration experiments showed that most, if not all, interacting components were [≤] 3 kDa. Production of the components that competed with antibodies to bind to CD88 and CXCR2 was shown to be regulated by LaeA. The component(s) that competed for binding to CXCR1 was not only produced in the non-acidifying strain Aspergillus niger D15#26 but also in the non-acidifying oahA deletion strain of Aspergillus niger. Together, these data show that Aspergillus species might produce small molecules that interact with human immune receptors.

microbiology↗

bioRxiv2021

GPR171 Agonist Reduces Chronic Neuropathic and Inflammatory Pain in Male, but not in Female Mice

Chronic pain is a growing public health crisis that requires exigent and efficacious therapeutics. GPR171 is a promising therapeutic target that is widely expressed through the brain, including within the descending pain modulatory regions. Here, we explore the therapeutic potential of the GPR171 agonist, MS15203, in its ability to alleviate chronic pain in male and female mice using a once-daily systemic dose (10mg/kg, i.p.) of MS15203 over the course of 5 days. We found that in our models of Complete Freunds Adjuvant (CFA)-induced inflammatory pain and chemotherapy-induced peripheral neuropathy (CIPN), MS15203 did not reduce thermal hypersensitivity and allodynia, respectively, in female mice. On the other hand, MS15203 treatment decreased the duration of thermal hyper-sensitivity in CFA-treated male mice following 3 days of once-daily administration. MS15203 treatment also produced an improvement in allodynia in male mice, but not female mice, in neuropathic pain after 5 days of treatment. Gene expression of GPR171 and that of its endogenous ligand BigLEN, encoded by the gene PCSK1N, were unaltered within the periaqueductal gray in both male and female mice following inflammatory and neuropathic pain. However, following neuropathic pain in male mice, the protein levels of GPR171 were decreased in the periaqueductal gray. Treatment with MS15203 then rescued the protein levels of GPR171 in the periaqueductal gray of these mice. Taken together, our results identify GPR171 as a GPCR that displays sexual dimorphism in alleviation of chronic pain. Further, our results suggest that GPR171 and MS15203 have demonstrable therapeutic potential in the treatment of chronic pain.

pharmacology and toxicology↗

bioRxiv2020

Host infection by the grass-symbiotic fungus Epichloë festucaerequires catalytically active H3K9 and H3K36 methyl transferases

Recent studies have identified key genes in Epichloe festucae that control the symbiotic interaction of this filamentous fungus with its grass host. Here we report on the identification of specific fungal genes that determine its ability to infect and colonize the host. Deletion of setB, which encodes a homolog of the H3K36 histone methyltransferase Set2/KMT3, specifically reduced histone H3K36 trimethylation and led to severe defects in colony growth and hyphal development. The E. festucae {Delta}clrD mutant, which lacks the gene encoding the homolog of the H3K9 methyltransferase KMT1, displays similar developmental defects. Both mutants are completely defective in their ability to infect the host grass, and mutational studies of key residues in the catalytic SET domains from these proteins show that these phenotypes are dependent on the methyltransferase activities of SetB and ClrD. A comparison of the differences in the host transcriptome between seedlings inoculated with wild-type versus mutants suggests that the inability of these mutants to infect the host was not due to an aberrant host defense response. Co-inoculation of either {Delta}setB or {Delta}clrD with the wild-type strain enables these mutants to colonize the host. However, successful colonization by the mutants resulted in death or stunting of the host plant. Transcriptome analysis at the early infection stage identified four fungal candidate genes, three of which encode small-secreted proteins, that are differentially regulated in these mutants compared to wild-type. Deletion of crbA, which encodes a putative carbohydrate binding protein, resulted in significantly reduced host infection rates by E. festucae. Author SummaryThe filamentous fungus Epichloe festucae is an endophyte that forms highly regulated symbiotic interactions with the perennial ryegrass. Proper maintenance of such interactions is known to involve several signalling pathways, but much less is understood about the infection capability of this fungus in the host. In this study, we uncovered two epigenetic marks and their respective histone methyltransferases that are required for E. festucae to infect perennial ryegrass. Null mutants of the histone H3 lysine 9 and lysine 36 methyltransferases are completely defective in colonizing the host intercellular space, and these defects are dependent on the methyltransferase activities of these enzymes. Importantly, we observed no evidence for increased host defense response to these mutants that can account for their non-infection. Rather, these infection defects can be rescued by the wild-type strain in co-inoculation experiments, suggesting that failure of the mutants to infect is due to altered expression of genes encoding infection factors that are under the control of the above epigenetic marks that can be supplied by the wild-type strain. Among genes differentially expressed in the mutants at the early infection stage is a putative small-secreted protein with a carbohydrate binding function, which deletion in E. festucae severely reduced infection efficiency.

genetics↗

bioRxiv2019

Reprogramming of the apoplast metabolome of Lolium perenne upon infection with the mutualistic symbiont Epichloë festucae

Epichloe festucae is an endophytic fungus that forms a mutualistic symbiotic association with Lolium perenne. Here we analysed how the metabolome of the ryegrass apoplast changed upon infection of this host with sexual and asexual isolates of E. festucae. A metabolite fingerprinting approach was used to analyse the metabolite composition of apoplastic wash fluid from non-infected and infected L. perenne. Metabolites enriched or depleted in one or both of these treatments were identified using a set of interactive tools. A genetic approach in combination with tandem mass spectrometry was used to identify a novel product of a secondary metabolite gene cluster. Metabolites likely to be present in the apoplast were identified using the MarVis Pathway in combination with the BioCyc and KEGG databases, and an in-house Epichloe metabolite database. We were able to identify the known endophyte-specific metabolites, peramine and epichloecyclins, as well as a large number of unknown markers. To determine whether these methods can be applied to the identification of novel Epichloe-derived metabolites, we deleted a gene encoding a NRPS (lgsA) that is highly expressed in planta. Comparative mass spectrometric analysis of apoplastic wash fluid from wild-type- versus mutant- infected plants identified a novel Leu/Ile glycoside metabolite present in the former.

microbiology↗

bioRxiv2018

Repeat elements organize 3D genome structure and mediate transcription in the filamentous fungus Epichloë festucae

Structural features of genomes, including the three-dimensional arrangement of DNA in the nucleus, are increasingly seen as key contributors to the regulation of gene expression. However, studies on how genome structure and nuclear organization influence transcription have so far been limited to a handful of model species. This narrow focus limits our ability to draw general conclusions about the ways in which three-dimensional structures are encoded, and to integrate information from three-dimensional data to address a broader gamut of biological questions. Here, we generate a complete and gapless genome sequence for the filamentous fungus, Epichloe festucae. Coupling it with RNAseq and HiC data, we investigate how the structure of the genome contributes to the suite of transcriptional changes that an Epichloe species needs to maintain symbiotic relationships with its grass host. Our results reveal a unique \"patchwork\" genome, in which repeat-rich blocks of DNA with discrete boundaries are interspersed by gene-rich sequences. In contrast to other species, the three-dimensional structure of the genome is anchored by these repeat blocks, which act to isolate transcription in neighbouring gene-rich regions. Genes that are differentially expressed in planta are enriched near the boundaries of these repeat-rich blocks, suggesting that their three-dimensional orientation partly encodes and regulates the symbiotic relationship formed by this organism.

genomics↗