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Singh, S.

Publications and source records attributed to Singh, S..

At least 325 records · Page 18Linked to original sources

Merging Bioactivity Predictions from Cell Morphology and Chemical Fingerprint Models by Leveraging Similarity to Training Data

The applicability domain of machine learning models trained on structural fingerprints for the prediction of biological endpoints is often limited by the lack of diversity of chemical space of the training data. In this work, we developed similarity-based merger models which combined the outputs of individual models trained on cell morphology (based on Cell Painting) and chemical structure (based on chemical fingerprints) and the structural and morphological similarities of the compounds in the test dataset to compounds in the training dataset. We applied these similarity-based merger models using logistic regression models on the predictions and similarities as features and predicted assay hit calls of 177 assays from ChEMBL, PubChem and the Broad Institute (where the required Cell Painting annotations were available). We found that the similarity-based merger models outperformed other models with an additional 20% assays (79 out of 177 assays) with an AUC>0.70 compared with 65 out of 177 assays using structural models and 50 out of 177 assays using Cell Painting models. Our results demonstrated that similarity-based merger models combining structure and cell morphology models can more accurately predict a wide range of biological assay outcomes and further expanded the applicability domain by better extrapolating to new structural and morphology spaces. O_FIG_DISPLAY_L [Figure 1] M_FIG_DISPLAY O_FLOATNOFigure:C_FLOATNO For TOC Only C_FIG_DISPLAY

bioinformatics↗

Learning representations for image-based profiling of perturbations

Measuring the phenotypic effect of treatments on cells through imaging assays is an efficient and powerful way of studying cell biology, and requires computational methods for transforming images into quantitative data that highlight phenotypic outcomes. Here, we present an optimized strategy for learning representations of treatment effects from high-throughput imaging data, which follows a causal framework for interpreting results and guiding performance improvements. We use weakly supervised learning (WSL) for modeling associations between images and treatments, and show that it encodes both confounding factors and phenotypic features in the learned representation. To facilitate their separation, we constructed a large training dataset with Cell Painting images from five different studies to maximize experimental diversity, following insights from our causal analysis. Training a WSL model with this dataset successfully improves downstream performance, and produces a reusable convolutional network for image-based profiling, which we call Cell Painting CNN-1. We conducted a comprehensive evaluation of our strategy on three publicly available Cell Painting datasets, discovering that representations obtained by the Cell Painting CNN-1 can improve performance in downstream analysis for biological matching up to 30% with respect to classical features, while also being more computationally efficient.

bioinformatics↗

Identification of a TNF-TNFR-like system in malaria vectors (Anopheles stephensi) likely to influence Plasmodium resistance

Identification of Plasmodium-resistance genes in malaria vectors remains an elusive goal despite the recent availability of high-quality genomes of several mosquito vectors. An. stephensi, with its three distinctly-identifiable forms at the egg stage, correlating with varying vector competence, offers an ideal species to discover functional mosquito genes implicated in Plasmodium resistance. Recently, the genomes of several strains of An. stephensi of the type-form, known to display high vectorial capacity, have been reported. Here, we report a chromosomal-level assembly of an intermediate-form of An. stephensi strain (IndInt), shown to have reduced vectorial capacity relative to a strain of type-form (IndCh). The contig level assembly with a L50 of 4 was scaffolded into chromosomes by using the genome of IndCh as the reference. The final assembly shows a heterozygous paracentric inversion, 3Li, involving 8 Mbp, which is syntenic to the extensively-studied 2La inversion implicated in Plasmodium resistance in An. gambiae involving 21 Mbp. Deep annotation of genes within the 3Li region in IndInt assembly using the state-of-the-art protein-fold prediction and other annotation tools reveals the presence of a TNF-like gene, which is the homolog of the eiger gene in Drosophila. Subsequent chromosome-wide searches revealed homologs of wengen (wgn) and grindelwald (grnd) genes in IndInt, which are known to be the receptors for eiger in Drosophila. We have identified all the genes in IndInt required for eiger-mediated signaling by analogy to TNF-TNFR system, suggesting the presence of a functionally active eiger signaling pathway present in IndInt. Comparative genomics of high-quality genome assemblies of the three type-forms with that of IndInt, reveals structurally disruptive mutations in eiger gene in all three strains of the type-form, alluding to compromised innate immunity in the type-form as the cause of high vectorial capacity in these strains. This is the first report of the presence of an intact evolutionarily-conserved TNF-TNFR signaling system in malaria vectors, with a potential role in Plasmodium resistance.

genomics↗

Characterization and Preclinical Treatment of Rotational Force-Induced Brain Injury

Millions of traumatic brain injuries (TBIs) occur annually. TBIs commonly result from falls, traffic accidents, and sports-related injuries, all of which involve rotational acceleration/deceleration of the brain. During these injuries, the brain endures a multitude of primary insults including compression of brain tissue, damaged vasculature, and diffuse axonal injury. All of these deleterious effects can contribute to secondary brain ischemia, cellular death, and neuroinflammation that progress for weeks to months after injury and impede neurological recovery. While the linear effects of head trauma have been extensively modeled, less is known about how rotational injuries mediate neuronal damage following injury. Here, we developed a new model of rotational head trauma in rodents and extensively characterized the pathological, behavioral, and electrophysiological effects of rotational TBI (rTBI). We identify aberrant cyclin dependent kinase 5 (Cdk5) activity as a principal mediator of rTBI and show pharmacological inhibition of Cdk5 reduces the cognitive and pathological consequences of injury. Finally, we utilize Cdk5-enriched phosphoproteomics to uncover potential downstream mediators of rTBI. These studies contribute meaningfully to our understanding of the mechanisms of rTBI and how they may be effectively treated.

neuroscience↗

Glucocorticoid signaling mediates CD8+ T cell memory differentiation

We provide evidence on the TRM potentiation by glucocorticoids in stimulated CD8+ T cells. Signaling via glucocorticoid receptor reciprocally regulated short-lived effector cells and memory precursor effector cells. Influenza A virus infected mice treated transiently with dexamethasone preferentially generated multipotent TRM cells that efficiently responded to the subsequent infection. Compromised memory following abrogation of glucocorticoid signaling further confirmed their role in memory potentiation. Transcriptomic and biochemical analysis of dexamethasone treated cells revealed a metabolic switch to oxidative phosphorylation via an engagement of AMPK signaling due to reduced glucose uptake. These cells exhibited an accumulation of phosphorylated STAT3 and STAT5 to drive memory differentiation. Therefore, glucocorticoids mediate tissue homing memory T cell differentiation. One-Sentence SummaryGlucocorticoid signaling in responding CD8+ T cells promotes memory differentiation

immunology↗

Reference compounds for characterizing cellular injury in high-content cellular morphology assays

Robust, generalizable approaches to identify compounds efficiently with undesirable mechanisms of action in complex cellular assays remain elusive. Such a process would be useful for hit triage during high-throughput screening and, ultimately, predictive toxicology during drug development. We generated cell painting and cellular health profiles for 218 prototypical cytotoxic and nuisance compounds in U-2 OS cells in a concentration-response format. A diversity of compounds causing cellular damage produced bioactive cell painting morphologies, including cytoskeletal poisons, genotoxins, nonspecific electrophiles, and redox-active compounds. Further, we show that lower quality lysine acetyltransferase inhibitors and nonspecific electrophiles can be distinguished from more selective counterparts. We propose that the purposeful inclusion of cytotoxic and nuisance reference compounds such as those profiled in this Resource will help with assay optimization and compound prioritization in complex cellular assays like cell painting.

pharmacology and toxicology↗

Effect of psilocybin on marble-burying in ICR mice: Role of 5-HT1A receptors and implications for the treatment of obsessive-compulsive disorder

BackgroundPreliminary clinical findings, supported by preclinical studies employing behavioral paradigms such as marble-burying, suggest that psilocybin may be effective in treating obsessive-compulsive disorder. AimsTo explore 1) the role of 5-HT2A and 5-HT1A receptors in the effect of psilocybin on marble-burying; 2) the effect of staggered versus bolus psilocybin administration and persistence of the effect; 3) the effect of the 5-HT1A partial agonist, buspirone, on marble-burying and the head-twitch response (HTR) induced by psilocybin, a rodent correlate of psychedelic effects. MethodsMale ICR mice were administered psilocybin 4.4 mg/kg, escitalopram 5 mg/kg, 8-OH-DPAT 2 mg/kg, M100907 2 mg/kg, buspirone 5 mg/kg, WAY100635 2 mg/kg or combinations, intraperitoneally, and were tested on the MBT. HTR was examined in a magnetometer-based assay. Results1) Psilocybin and escitalopram significantly reduced marble-burying. The effect of psilocybin was not attenuated by the 5-HT2A antagonist, M100907. The 5-HT1A agonist, 8-OH-DPAT reduced marble-burying as did the 5-HT1A partial agonist, buspirone. The effect of 8-OH-DPAT was additive to that of psilocybin but that of buspirone was not. The 5-HT1A antagonist, WAY100635, attenuated the effect of 8-OH-DPAT and buspirone but not the effect of psilocybin. 2) Psilocybin injections over 3.5 hours had no effect on marble-burying and the effect of bolus injection was not persistent. 3) Co-administration of buspirone with psilocybin blocked its effect on HTR ConclusionsNeither 5-HT2A nor 5-HT1A receptors are pivotally implicated in the effect of psilocybin on marble-burying. Co-administration with buspirone may block the psychedelic effects of psilocybin without impeding its anti-obsessional effects.

pharmacology and toxicology↗

Optimizing the Cell Painting assay for image-based profiling

In image-based profiling, software extracts thousands of morphological features of cells from multi-channel fluorescence microscopy images, yielding single-cell profiles that can be used for basic research and drug discovery. Powerful applications have been proven, including clustering chemical and genetic perturbations based on their similar morphological impact, identifying disease phenotypes by observing differences in profiles between healthy and diseased cells, and predicting assay outcomes using machine learning, among many others. Here we provide an updated protocol for the most popular assay for image-based profiling, Cell Painting. Introduced in 2013, it uses six stains imaged in five channels and labels eight diverse components of the cell: DNA, cytoplasmic RNA, nucleoli, actin, Golgi apparatus, plasma membrane, endoplasmic reticulum, and mitochondria. The original protocol was updated in 2016 based on several years experience running it at two sites, after optimizing it by visual stain quality. Here we describe the work of the Joint Undertaking for Morphological Profiling (JUMP) Cell Painting Consortium, aiming to improve upon the assay via quantitative optimization, based on the measured ability of the assay to detect morphological phenotypes and group similar perturbations together. We find that the assay gives very robust outputs despite a variety of changes to the protocol and that two vendors dyes work equivalently well. We present Cell Painting version 3, in which some steps are simplified and several stain concentrations can be reduced, saving costs. Cell culture and image acquisition take 1-2 weeks for a typically sized batch of 20 or fewer plates; feature extraction and data analysis take an additional 1-2 weeks. Key references using this protocolVirtual screening for small-molecule pathway regulators by image-profile matching (https://doi.org/10.1016/j.cels.2022.08.003) - recent work examining the ability to use collected Cell Painting profiles to screen for regulators of a number of diverse biological pathways. JUMP Cell Painting dataset: images and profiles from two billion cells perturbed by 140,000 chemical and genetic perturbations (DOI) - the description of the main JUMP master public data set, using this protocol in the production of >200 TB of image data and >200 TB of measured profiles. Key data used in this protocolCell Painting, a high-content image-based assay for morphological profiling using multiplexed fluorescent dyes (https://doi.org/10.1038/nprot.2016.105) - this paper provides the first step-by-step Cell Painting protocol ever released.

bioinformatics↗

Biological evaluation of novel side chain containing CQTrICh-analogs as antimalarials and their development as PfCDPK1 kinase inhibitors

To combat the emergence of drug resistance against the existing antimalarials, novel side chain containing 7-chloroquinoline-indole-chalcones tethered with a triazole (CQTrICh-analogs 7 (a-s) and 9) were designed and synthesized by reacting substituted 1-phenyl-3-(1-(prop-2-yn-1- yl)-1H-indol-3-yl) prop-2-en-1-one and 1-(prop-2-yn-1-yl)-1H-indole-3-carbaldehyde with 4- azido-7-chloroquinoline, respectively via a click reaction. The selected CQTrICh-analogs: 7l and 7r inhibited chloroquine-sensitive (3D7) and resistant (RKL-9) strains of Plasmodium falciparum, with IC50 values of 2.4 {micro}M & 1.8 {micro}M (7l), and 3.5 {micro}M & 2.7 {micro}M (7r), respectively, and showed insignificant hemolysis and cytotoxicity in mammalian cells. Intra-erythrocytic progression studies revealed that the active hybrids: 7l and 7r are effective against the mature stages of the parasite. Given the importance of Calcium-Dependent Protein Kinase 1 (PfCDPK1) in the parasite biology, notably during late schizogony and subsequent invasion of merozoites into host RBCs, we identified this protein as a possible molecular target of these active hybrids. In silico interaction analysis indicated that 7l and 7r stably interact with the catalytically active ATP-binding pocket of PfCDPK1, by the formation of energetically favorable H-bonds. Furthermore, in vitro Microscale Thermophoresis and kinase assays with recombinant PfCDPK1 demonstrated that the active hybrids interact with and inhibit the kinase activity, thus presumably responsible for the parasite growth inhibition. Interestingly, 7l and 7r showed no inhibitory effect on the human kinases, indicating that they are selective for the parasite kinase. Conceivably, we report the antiplasmodial potential of novel kinase targeting bio-conjugates, a step towards developing pan-kinase inhibitors, which is a prerequisite for cross-stage anti-malarial protection. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=115 SRC="FIGDIR/small/498981v1_ufig1.gif" ALT="Figure 1"> View larger version (22K): org.highwire.dtl.DTLVardef@113574borg.highwire.dtl.DTLVardef@c81a33org.highwire.dtl.DTLVardef@1301d22org.highwire.dtl.DTLVardef@1c8cac5_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

TLR4 signaling in presence of HIV-induced activation enhances programmed death ligand-1 expression on human plasmacytoid dendritic cells and modulates their function

Human Plasmacytoid dendritic cells (pDC) only comprise a minute fraction of human mononuclear leukocytes, but are important anti-viral responders that mediate both innate as well as adaptive immune responses. Persistent activation of pDC enhances HIV pathogenesis by promoting immune suppressive mechanisms such as T regulatory cells. It is therefore important to identify the sources of pDC activation in the context of HIV infection. HIV-associated disruption of gut mucosa associated lymphoid tissue introduces normal flora-lipopolysaccharide (LPS) into systemic circulation, which exacerbates HIV-induced immune activation. We report here that pDC are capable of mediating functional TLR4 signaling upon LPS stimulation, and that pDC of HIV-infected individuals have enhanced TLR4 expression compared to healthy individuals. How TLR4 signaling affects pDC function in HIV infection has not been examined before. Hence we examined the influence of TLR4 signaling in presence of HIVstimulation on pDC and found that it not only potentiated HIV-induced activation but also strongly up-regulated Programmed death ligand-1 (PD-L1) expression and Interleukin-6 synthesis. TLR4 signaling specifically up-regulated PD-L1 expression on activated pDC in presence of HIV stimulation. LPS and HIV co-stimulated pDC demonstrated enhanced migratory potential and repressed T cell proliferation. Together, these results suggest that in the setting of HIV infection enhancement of pDC immune suppressive mechanisms such as PD-L1 may be an outcome of HIV-associated immune activation potentiated by TLR4 signaling.

immunology↗

New Hydra Genomes Reveal Conserved Principles of Hydrozoan Transcriptional Regulation

The epithelial and interstitial stem cells of the freshwater polyp Hydra are the best characterized stem cell systems in any cnidarian, providing valuable insight into cell type evolution and the origin of stemness in animals. However, little is known about the transcriptional regulatory mechanisms that determine how these stem cells are maintained and how they give rise to their diverse differentiated progeny. To address such questions, a thorough understanding of transcriptional regulation in Hydra is needed. To this end, we generated extensive new resources for characterizing transcriptional regulation in Hydra, including new genome assemblies for Hydra oligactis and the AEP strain of Hydra vulgaris, an updated whole-animal single-cell RNA-seq atlas, and genome-wide maps of chromatin interactions, chromatin accessibility, sequence conservation, and histone modifications. These data revealed the existence of large chromatin interaction domains in the Hydra genome that likely influence transcriptional regulation in a manner distinct from topologically associating domains in bilaterians. We also uncovered the transcriptomic profiles of two previously molecularly uncharacterized cell types, isorhiza-containing nematocytes and somatic gonad ectoderm. We identified novel candidate regulators of cell-type-specific transcription, several of which have likely been conserved at least since the divergence of Hydra and the jellyfish Clytia hemisphaerica over 200 million years ago. The resources generated in this study, which collectively represent the most comprehensive characterization of transcriptional regulation in a cnidarian to date, are accessible through a newly created genome portal, available at research.nhgri.nih.gov/HydraAEP/.

developmental biology↗

More than Mycobacterium tuberculosis: specific site-of-disease microbial communities, functional capacities, and their distinct clinical profiles in tuberculous lymphadenitis

BackgroundLymphadenitis is the most common extrapulmonary tuberculosis (EPTB) manifestation and a major cause of death. The microbiome is important to human health but uninvestigated in EPTB. We profiled the site-of-disease lymph node microbiome in tuberculosis lymphadenitis (TBL). MethodsFine needle aspiration biopsies (FNABs) were collected from 159 pre-treatment presumptive TBL patients in Cape Town, South Africa. 16S Illumina MiSeq rRNA gene sequencing was done. ResultsWe analysed 89 definite TBLs (dTBLs) and 61 non-TBLs (nTBLs), which had similar -but different {beta}-diversities (p=0.001). Clustering identified five lymphotypes prior to TB status stratification: Mycobacterium-, Prevotella- and Streptococcus-dominant lymphotypes were more frequent in dTBLs whereas a Corynebacterium-dominantlymphotype and a fifth lymphotype (no dominant taxon) were more frequent in nTBLs. When restricted to dTBLs, clustering identified a Mycobacterium-dominant lymphotype with low -diversity and other non-Mycobacterium-dominated lymphotypes (termed Prevotella-Corynebacterium and Prevotella-Streptococcus). The Mycobacterium dTBL lymphotype was associated with HIV-positivity and clinical features characteristic of severe lymphadenitis (e.g., node size). dTBL microbial communities were enriched with potentially proinflammatory microbial short chain fatty acid metabolic pathways (propanoate, butanoate) vs. those in nTBLs. 11% (7/61) of nTBLs had Mycobacterium reads. ConclusionsTBL at the site-of-disease is not microbially homogenous and distinct microbial community clusters exist that are associated with different immunomodulatory potentials and clinical characteristics. Non-Mycobacterium-dominated dTBL lymphotypes, which contain taxa potentially targeted by TB treatment, represent less severe potentially earlier stage disease. These investigations lay foundations for studying the microbiomes role in lymphatic TB and the long-term clinical significance of lymphotypes requires prospective evaluation.

molecular biology↗

Water-Quality analysis and Fish diversity of Southern West Part of the West-Bengal

Marine fishes are one of the important factors to stabilize the local aquatic ecosystem and regulating socio-economy of local fishermen. In this study we emphasized upon finding out the interrelationship in the water quality, anthropogenic activity, and fish habitat through the 31km stretch of East Medinipur coast in West Bengal, India which is known for its tourist destinations. The study was conducted monthly on different trawler fish landing sites from Dec 2018 to Dec 2021. During this period, we took fish samples and identified them. We took water quality data such as SST, Concentration of Chl-a, Turbidity, and DO for further correlation between the water quality and species diversity. Total 154 numbers of commercially important marine fish species were documented. Among them, 12 species are from Elasmobranch and the rest is Actinopterygii. As per the IUCN database, 13% of the total fish species are under the red list category and 16% of the species showing decreasing population trend. After analyzing the water quality data, we found out that DO, SST, Turbidity, and Chlorophyll-A correlate with the species richness and it also differs during the seasons.

ecology↗

Small molecule mediated stabilization of PP2A modulates the Homologous Recombination pathway and potentiates DNA damage-induced cell death

High-Grade Serous Carcinoma (HGSC) is the most common and lethal ovarian cancer subtype. PARP-inhibitors (PARPi) have become the mainstay of HGSC targeted therapy, given that these tumors are driven by a high degree of genomic instability and Homologous Recombination (HR) defects. Nonetheless, only [~]30% of patients initially respond to treatment, ultimately relapsing with resistant disease. Thus, despite recent advances in drug development and increased understanding of genetic alterations driving HGSC progression, mortality has not declined, highlighting the need for novel therapies. Using a Small Molecule Activator of Protein Phosphatase 2A (PP2A) (SMAP-061), we investigated the mechanism by which PP2A stabilization induces apoptosis in Patient-Derived HGSC cells and Xenograft (PDX) models alone or in combination with PARPi. We uncovered that PP2A genes essential for transformation (B56,B56{gamma} and PR72) and basal phosphatase activity (PP2A-A and -C) are heterozygously lost in the majority of HGSC. Moreover, loss of these PP2A genes correlates with worse overall patient survival. We show that SMAP-061 stabilization of PP2A inhibits the HR output by targeting RAD51, leading to chronic accumulation of DNA damage and ultimately apoptosis. Furthermore, combination of SMAP-061 and PARPi leads to enhanced apoptosis in both HR-proficient and -deficient cells and in patient-derived xenograft models. Our studies identify PP2A as novel regulator of HR and introduces PP2A activators as a potential treatment for HGSC tumors. Our studies further emphasize the potential of PP2A modulators to overcome PARPi insensitivity, given that targeting RAD51 has presented benefits in overcoming PARPi-resistance driven by BRCA1/2 mutation reversions.

cancer biology↗

Integrating inflammatory biomarker analysis and artificial intelligence-enabled image-based profiling to identify drug targets for intestinal fibrosis

Intestinal fibrosis is a common complication of several enteropathies with inflammatory bowel disease being the major cause. The progression of intestinal fibrosis may lead to intestinal stenosis and obstruction. Even with an increased understanding of tissue fibrogenesis, there are no approved treatments for intestinal fibrosis. Historically, drug discovery for diseases like intestinal fibrosis has been impeded by a lack of screenable cellular phenotypes. Here we applied Cell Painting, a scalable image-based morphology assay, augmented with machine learning algorithms to identify small molecules that were able to morphologically reverse the activated fibrotic phenotype of intestinal myofibroblasts under pro-fibrotic TNF stimulus. In combination with measuring CXCL10, a common pro-inflammatory cytokine in intestinal fibrosis, we carried out a high-throughput small molecule chemogenomics screen of approximately 5000 compounds with known targets or mechanisms, which have achieved clinical stage or approval by the FDA. Through the use of two divergent analytical methods, we identified several compounds and target classes that are potentially able to treat intestinal fibrosis. The phenotypic screening platform described here represents significant improvements in identifying a wide range of drug targets over conventional methods by integrating morphological analyses and artificial intelligence using pathologically-relevant cells and disease-relevant stimuli.

pharmacology and toxicology↗

Characterization of prohibitins as novel target to block asexual and sexual stage growth of Plasmodium falciparum

Prohibitins (PHBs) are highly conserved pleiotropic proteins as they have been shown to mediate key cellular functions. Here, we characterize PHBs encoding putative genes of Plasmodium falciparum by exploiting different orthologous models. We demonstrated that PfPHB1 (PF3D7_0829200) and PfPHB2 (PF3D7_1014700) are expressed in asexual and sexual blood stages of the parasite. Immunostaining indicated these proteins as mitochondrial residents as they were found to be localized as punctate foci. We further validated PfPHBs as organellar proteins residing in the Plasmodium mitochondria, where they interact with each other. Functional characterization was done in Saccharomyces cerevisiae orthologous model by expressing PfPHB1 and PfPHB2 in cells harboring respective mutants. The PfPHBs functionally complemented the yeast PHB1 and PHB2 mutants, where the proteins were found to be involved in stabilizing the mitochondrial DNA, retaining mitochondrial integrity and rescuing yeast cell growth. Further, Rocaglamide (Roc-A), a known inhibitor of PHBs and anti-cancerous agent, was tested against PfPHBs and as an antimalarial. Roc-A treatment retarded the growth of PHB1, PHB2, and ethidium bromide petite yeast mutants. Moreover, Roc-A inhibited growth of yeast PHBs mutants that were functionally complemented with PfPHBs, validating P. falciparum PHBs as one of the molecular targets for Roc-A. Roc-A treatment led to growth inhibition of artemisinin-sensitive (3D7), artemisinin-resistant (R539T) and chloroquine-resistant (RKL-9) parasites in nanomolar ranges. The compound was able to retard gametocyte growth with significant morphological aberrations. Based on our findings, we propose the presence of functional mitochondrial PfPHB1 and PfPHB2 in P. falciparum and their druggability to block parasite growth.

molecular biology↗

Niclosamide loaded Eudragit EPO nanoparticles show enhanced Candida biofilm penetration, trigger biofilm detachment and protect from mucosal candidiasis

Candida albicans biofilms are a complex multilayer community of cells that are resistant to almost all classes of antifungal drugs. The bottommost layers of biofilms experience nutrient limitation where C. albicans cells are required to respire. We previously reported that a protein Ndu1 is essential for Candida mitochondrial respiration; loss of NDU1 causes inability of C. albicans to grow on alternative carbon sources and triggers early biofilm detachment. Here, we screened a repurposed library of FDA approved small molecule inhibitors, to identify those that prevent NDU1-associated functions. We identified an anti-helminthic drug, Niclosamide (NCL), which not only prevented growth on acetate, C. albicans hyphenation and early biofilm growth, but also completely disengaged fully grown biofilms of drug resistant C. albicans and C. auris from their growth surface. To overcome the sub-optimal solubility and permeability of NCL that is well-known to affect its in vivo efficacy, we developed NCL encapsulated Eudragit EPO (an FDA-approved polymer) nanoparticles (NCL-EPO-NPs) with high niclosamide loading, that also provided long-term stability. The developed NCL-EPO-NPs completely penetrated mature biofilms and attained anti-biofilm activity at low microgram concentrations. NCL-EPO-NPs induced ROS activity in C. albicans, and drastically reduced oxygen consumption rate in the fungus, similar to that seen in an NDU1 mutant. NCL-EPO-NPs also significantly abrogated mucocutaneous candidiasis by fluconazole resistant strains of C. albicans, in mice models of oropharyngeal and vulvovaginal candidiasis. To our knowledge, this is the first study that targets biofilm detachment as a target to get rid of drug-resistant Candida biofilms, and uses nanoparticles of an FDA approved non-toxic drug to improve biofilm penetrability and microbial killing.

microbiology↗

ABHD6 selectively controls Gq-dependent increases in 2-AG production

The most abundant endocannabinoid (eCB) in the brain, 2-arachidonoyl glycerol (2-AG), is hydrolyzed by /{beta}-hydrolase domain containing 6 (ABHD6); yet how ABHD6 controls stimuli-dependent increases in 2-AG production is unknown. To explore this question, we leveraged the recently developed 2-AG sensor, GRABeCB2.0, and found that stimulation of Neuro2a cells in culture with bradykinin (BK) acting at metabotropic B2K receptors and ATP acting at ionotropic P2X7 receptors led to differential increases in 2-AG levels. B2K triggered increases in 2-AG levels via diacylglycerol lipase (DAGL), and this mechanism was potentiated by increases in intracellular calcium and ABHD6 inhibition. By contrast, P2X7-triggered increases in 2-AG levels were dependent on DAGL and extracellular calcium but unaffected by ABHD6 inhibition. Thus, ABHD6 preferentially regulates metabotropic-dependent increases in 2-AG levels over ionotropic-dependent increases in 2-AG levels. Our study indicates that ABHD6 selectively controls stimuli-dependent increases in 2-AG production and emphasizes its specific role in eCB signaling.

cell biology↗