Biology subjects

Bansal, R.

Publications and source records attributed to Bansal, R..

18 recordsLinked to original sources

bioRxiv2026

Pleiotropic antimalarial activities and immunomodulation exhibited by Himalayan Buransh (Rhododendron arboreum) in human and rodent malaria models

Emerging drug resistance against the malaria parasite is worrisome and necessitates the development of novel antimalarials. Himalayan Buransh (Rhododendron arboreum) is a well-known medicinal plant found in the northern states of India. In this study, we observed the pleiotropic antimalarial activities and immunomodulation exhibited by the aqueous extract of Buransh flower (AEBF). AEBF demonstrated significant IC50 values (16-29 {micro}g/ml) against the asexual stages of various P. falciparum strains (3D7, Dd2-chloroquine-resistant and C580Y-artemisinin resistant). The oral administration of AEBF (200 mg/kg) in mice, suppressed [~]80% P. berghei parasitemia, improved mean survival time (MST-23.5 days) and prevented splenomegaly. Notably, the combination of AEBF and artesunate not only cleared primary infection, but also conferred sustained immunity. This immunomodulatory effect, driven by protective IFN-{gamma} resulted in reduced parasitemia during a homologous challenge without the need for further treatment. It is important to highlight the malaria transmission blocking activity of AEBF, resulting in reduced sexual stage male gametocyte exflagellation. Furthermore, the virtual drug screening of selected bioactive constituents from Buransh flower demonstrated potent binding against multiple P. falciparum proteins, suggested a pleiotropic mode of action. Altogether, our results corroborated the first ever evidence of the multistage antimalarial potential of Buransh flower, supported by in vitro cell studies, in vivo rodent malaria model and in silico docking analyses. Based on our studys findings and the traditional use of Buransh juice as a medicinal beverage in Uttarakhand, India, we propose exploring it as an adjunct therapy for drug-resistant malaria, subject to further clinical validation.

pathology↗

bioRxiv2026

Gut microbiota-derived indole-3 acetic acid mitigates key hallmarks of metabolic dysfunction-associated steatotic liver disease (MASLD) in an NLRP3-dependent manner in in vitro and diet-induced mouse models

BackgroundMetabolic-dysfunction associated steatotic liver disease(MASLD) is a global epidemic affecting >30 % global population with no approved therapies till date. Recent reports suggest that activation of NLRP3 inflammasome may be involved in the pathogenesis of MASLD. We, therefore, aimed to identify small molecule inhibitor(s) of NLRP3 inflammasome as a potential therapeutic strategy to manage MASLD and validate their efficacy using in vitro and in vivo models of MASLD. MethodologyWe screened an in-house library of natural products using an in vitro phenotypic assay and identified a gut microbiota derived metabolite of dietary Tryptophan; indole-3 acetic acid (I3A) for its ability to inhibit the levels of IL-1{beta} and IL-18, which are elevated as a result of activation of NLRP3 inflammasome in differentiated THP1 cells. Subsequently, we carried out several in vitro and in vivo studies to confirm the mechanism of action of I3A and its ability to mitigate the key hallmarks of MASLD ResultsOur in vitro data suggest that I3A is an inhibitor of NLRP3 inflammasome. I3A was also found to improve the blood glucose level, plasma lipid profile, hepatic fat, and liver function in high-fat-high-fructose diet induced model of MASLD using C57BL/6 mice. ConclusionOur results show that I3A, which has been previously reported to be a gut microbiota-derived metabolite of dietary tryptophan, mitigates the key hallmarks of MASLD in an NLRP3 dependent manner. A dedicated structure-activity-relationship (SAR) study around the I3A chemotype may be carried out in future to identify novel NLRP3 inhibitors with desirable pharmacological profile.

pharmacology and toxicology↗

bioRxiv2025

Selective targeting of Plasmodium falciparum hexose transporter by phytochemical Ginsenoside Rg1 disrupts glucose metabolism and block development of parasite

The emergence of resistance to first-line antimalarial therapies highlights the critical need for next-generation drugs that target distinct molecular pathways and employ novel mechanisms of action. Notably, the intra-erythrocytic parasite development is highly dependent on a sustained glucose supply as their fundamental energy source. Therefore, exploiting a "selective starvation" strategy, by targeting the parasites reliance on glucose metabolism, particularly through the Plasmodium falciparum hexose transporter (PfHT1), which is critical for parasite survival can serve as a promising therapeutic approach to combat multidrug-resistant Plasmodium parasites. Through molecular docking and structure-based drug design approach, we identified a natural compound, Ginsenoside Rg1 (G-Rg1) from drug bank database library, as a potential PfHT1 inhibitor. The PfHT1 specificity of G-Rg1 was validated using yeast complementation model. Subsequently, to investigate the role of PfHT1 in drug resistant Pf parasites we investigated the stage-specific expression of PfHT1 in both artemisinin (ART)-sensitive and resistant Pf parasites and reported its elevated expression in resistant parasites, predicting its role in their survival. Notably, in vitro growth inhibition studies demonstrated that G-Rg1 effectively suppressed the growth of both ART-sensitive and resistant Pf parasites. Additionally, G-Rg1 potentiated the efficacy of dihydroartemisinin in combination and ring survival assays, indicating its potential to circumvent resistance mechanisms. G-Rg1 administration alone and in combination with ART, in P. berghei ANKA-infected mice reduced parasite multiplication and increased mean survival time. Our findings support G-Rg1 as a promising candidate for drug development against malaria, highlighting the potential of targeting PfHT1 to combat drug-resistant malaria.

pharmacology and toxicology↗

bioRxiv2025

Targeting Leishmania donovani Sphingosine Kinase 1 using PF-543 enhances immune response and limits parasite load

BackgroundSphingosine-1-phosphate (S1P) is a bioactive lipid mediator regulating apoptosis, proliferation, and immune responses. While S1Ps presence in Leishmania donovani phagolysosomes has been reported, the role of sphingosine kinases, especially SphK1, in parasite survival and host immune modulation remains underexplored. This study investigates the molecular and functional role of L. donovani SphK1 (LdSphK1) and evaluates the antileishmanial potential of PF-543, a specific SphK1 inhibitor. MethodsLdSphK1 and human SphK1 (rhSphK1) were cloned, expressed in E. coli, purified, and analyzed by SDS-PAGE. Enzymatic activity and inhibition by PF543 were assessed using NBD-S1P-based fluorometric assays. Protein-ligand interactions were analyzed using Microscale Thermophoresis (MST). Leishmania promastigotes overexpressing LdSphK1 were studied via confocal microscopy, and their viability and infectivity were assessed in vitro. THP-1 macrophages infected with L. donovani were treated with PF543 alone or with Amphotericin B and analyzed by MTT assay, RT-PCR, Giemsa staining, ELISA and immunoblotting. In vivo efficacy was tested in L. donovani-infected Swiss mice. ResultsrLdSphK1 ([~]102 kDa) and rhSphK1 ([~]50 kDa) were enzymatically active and significantly inhibited by PF-543. MST confirmed high-affinity binding of PF-543 (KD [~]29 microMolar). In L. donovani SphK1 overexpressor (LdSphKa) promastigotes, PF543 inhibited SphK1 activity and reduced parasite infectivity, more than in wildtype L. donovani promastigotes. Notably, PF543 treatment reduced parasite infectivity in vitro, lowered amastigote load by [~]40%, and promoted a pro-inflammatory cytokine shift ({uparrow}IL-12, {uparrow}TNF-, {downarrow}IL-10). Inhibition of ceramide synthesis and S1P supplementation revealed that S1P rescues ceramide-induced parasite death, implicating SphK1 in parasite survival. PF543 and Amphotericin B demonstrated synergistic anti-parasitic effects both in vitro and in vivo, with >90% reduction in parasite burden in mice. ConclusionPF543 is a potent inhibitor of SphK1, impairing parasite survival and modulating host immune responses. When combined with Amphotericin B, it offers a synergistic therapeutic strategy against visceral leishmaniasis, warranting further clinical exploration. Author SummaryLeishmaniasis, a neglected tropical disease, has limited available treatments and is becoming more resistant to medications. In this study, we explored the therapeutic potential of PF-543, a potent sphingosine kinase 1 (SphK1) inhibitor (demonstrated anticancer effects in various preclinical models) against Leishmania donovani. We successfully cloned and purified both Leishmania and human SphK1 proteins and confirmed PF-543 binding through biochemical and biophysical assays. Overexpression of LdSphK1 in parasites enhanced their survival and infectivity. In vitro, PF-543 treatment of infected macrophages decreased amastigote burden, shifted cytokine profiles towards a pro-inflammatory state, and enhanced host cell apoptosis. Notably, PF-543 acted synergistically with Amphotericin B, the current clinical drug, both in vitro and in vivo in Swiss mice, drastically lowering parasite burden. This study highlights the possibility of combination therapy and finds PF-543 to be a promising irresistible host-targeted antileishmanial drug. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=151 SRC="FIGDIR/small/652326v2_ufig1.gif" ALT="Figure 1"> View larger version (36K): org.highwire.dtl.DTLVardef@1926575org.highwire.dtl.DTLVardef@4305f4org.highwire.dtl.DTLVardef@3c10c7org.highwire.dtl.DTLVardef@171801e_HPS_FORMAT_FIGEXP M_FIG C_FIG

pathology↗

bioRxiv2024

Evaluation of Immunopharmacological Efficacy of Live Leishmania donovani Overexpressing Ld_ζ1domain for Protection Against Experimental Human Visceral Leishmaniasis

ObjectiveTo evaluate the efficacy and immunogenicity of the zeta domain over-expressing Leishmania donovani (Ld_{zeta}1domain) as a vaccination candidate against visceral leishmaniasis (VL). MethodsIn this study, Leishmania overexpressor Ld_{zeta}1domain (OE) were transformed by electroporation using a GFP-tagged Ld_{zeta}1domain recombinant plasmid. The resulting overexpressing cells were analysed in vitro to assess their growth dynamics and infectivity. We also investigated the immune-protective potential of these overexpressor in a mouse model challenged with Leishmania donovani. The immune response, including Th1 and Th2 pathways, was thoroughly characterized using RT-PCR and ELISA assays. In addition, the study conducted a thorough evaluation of the mouses spleen and liver parasites, as well as quantitative evaluation of tissue pathological changes. ResultsLd_{zeta}1 domain (OE) parasites exhibited significantly lower viability and replication rates than WT parasites, and in vivo studies showed that mice immunized with the Ld_{zeta}1(OE) domain had lower parasite numbers than mice infected with LdWT. Spleen and liver showed significant histological changes suggestive of protection. Parasite burden in the spleen and liver of vaccinated mice were significantly reduced. The immune response showed increased IFN-{gamma} levels and lower IL-10 production, resulting in a greater IFN-{gamma}/IL-10 ratio, indicating parasite elimination. The vaccination also caused a significant IgG humoral response and increased nitric oxide production in immunized mice. ConclusionOur findings demonstrated that overexpressing the zeta toxin resulted in controlled parasite attenuation, lowering pathogenicity while retaining immunogenic features. Our work established the zeta over-expressors protective efficacy, immunogenicity, and proliferation in response to a Leishmania challenge in vitro and in vivo. This preliminary prototype study suggested that Ld_{zeta}1domain (OE) parasites may be suitable for developing an attenuated vaccine against leishmaniasis. Graphical representation O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=88 SRC="FIGDIR/small/630021v1_ufig1.gif" ALT="Figure 1"> View larger version (18K): org.highwire.dtl.DTLVardef@32dfc0org.highwire.dtl.DTLVardef@1ffe89dorg.highwire.dtl.DTLVardef@d9feorg.highwire.dtl.DTLVardef@1e7bae7_HPS_FORMAT_FIGEXP M_FIG C_FIG A schematic representation on the protective effectiveness, immunogenicity, and proliferation of the zeta over-expressor in response to Leishmania challenge in vitro and in vivo model.

pathology↗

bioRxiv2024

Chemical degradation identified Human p38-MAPK as a host target to combat parasitic infections

The interplay between host and parasite determines parasite burden and disease outcome. Parasite exploits host signaling pathways like p38-MAPK for its survival and pathogenesis. NR-7h, a proteolysis-targeting chimera (PROTAC) targeting human p38-MAPK was used to assess p38-MAPKs role in Leishmania donovani and Plasmodium falciparum infection in their respective hosts. NR-7h degraded host p38-MAPK in a time- and dose-dependent manner. Degradation of host p38-MAPK by NR-7h reduced parasite load in host cells dose-dependently, implicating the role of p38-MAPK in parasite survival. The modulation of cytokine profiling and oxidative burst upon NR-7h mediated degradation of host p38-MAPK was further correlated with parasite death. The synergistic effect of host p38-MAPK degradation by NR-7h with Amphotericin B enhances the efficacy of parasite-directed therapy. This study underscores the importance of host p38-MAPK for L. donovani and P. falciparum progression and highlights NR-7hs potential in antiparasitic therapy by targeting this pathway.

microbiology↗

bioRxiv2024

The Mag-Click-Capture-Release Technology for Selective Capture and Release of Hepatocyte-Derived Extracellular Vesicles as Biomarkers for Liver Disease

Chronic liver diseases, such as liver cirrhosis and hepatocellular carcinoma (HCC), present major global health challenges, often diagnosed late. Circulating extracellular vesicles (EVs), which carry disease-specific biomolecular cargo, is emerging as an early diagnostic and prognostic biomarker for several diseases including cancer. However, current EV purification methods including ultracentrifugation and size exclusion chromatography present several limitations. Here, we present the Mag-Click-Capture-Release Technology for selective capture and release of EVs that combines magnetic beads, trans-cyclooctene (TCO) and tetrazine (Tz) click chemistry, immuno(antibody)-based capture and disulfide-driven release of EVs. Importantly, the Mag-Click-Capture-Release Technology is customizable, whereby using specific antibodies conjugated to TCO antibodies, different EV subtypes can be selectively captured and released for further analysis. With our Mag-Click-Capture-Release Technology, we successfully isolated hepatocyte-derived EVs from human serum with good recovery, high specificity and purity when compared with standard ultracentrifugation. Validation in serum samples obtained from cirrhosis and HCC patients with alcohol-associated liver disease evidenced an increasing trend in hepatocyte-EV levels correlating with disease severity, suggesting potential for early diagnosis and prognosis. In conclusion, we present here the Mag-Click-Capture-Release Technology, a customizable and efficient approach for selective isolation of organ-, cell-specific, and disease-relevant EVs from biological samples that can be subsequently released for downstream molecular EV analysis and EV-related functional assays.

bioengineering↗

bioRxiv2023

The rules of sex and strain: deciphering the chemical language of inbred and wild mouse conspecific scents

In most mammals, conspecific chemosensory communication relies on semiochemical release within complex bodily secretions and subsequent stimulus detection by the vomeronasal organ (VNO). Urine, a rich source of ethologically relevant chemosignals, conveys detailed information about sex, social hierarchy, health and reproductive state, which becomes accessible to a conspecific via vomeronasal sampling. So far, however, numerous aspects of social chemosignaling along the vomeronasal pathway remain unclear. Moreover, since virtually all research on vomeronasal physiology is based on secretions derived from inbred laboratory mice, it remains uncertain whether such stimuli provide a true representation of potentially more relevant cues found in the wild. Here, we combine a robust low-noise VNO activity assay with comparative molecular profiling of sex- and strain-specific mouse urine samples from two inbred laboratory strains as well as from wild mice. With comprehensive molecular portraits of these secretions, VNO activity analysis now enables us to (i) assess whether and, if so, how much sex- / strain-selective "raw" chemical information in urine is accessible via vomeronasal sampling; (ii) identify which chemicals exhibit sufficient discriminatory power to signal an animals sex, strain, or both; (iii) determine the extent to which wild mouse secretions are unique; and (iv) analyze whether vomeronasal response profiles differ between strains. We report both sex- and, in particular, strain-selective VNO representations of chemical information. Within the urinary secretome, both volatile compounds and proteins exhibit sufficient discriminative power to provide sex- and strain-specific molecular fingerprints. While total protein amount is substantially enriched in male urine, females secrete a larger variety at overall comparatively low concentrations. Surprisingly, the molecular spectrum of wild mouse urine does not dramatically exceed that of inbred strains. Finally, vomeronasal response profiles differ between C57BL/6 and BALB/c animals, with particularly disparate representations of female semiochemicals.

neuroscience↗

bioRxiv2023

Dual functioning by the PhoR sensor is a key determinant to Mycobacterium tuberculosis virulence

PhoP-PhoR empowers M. tuberculosis to adapt to diverse environmental conditions, and remains essential for virulence. Although PhoP and PhoR have been structurally characterized, the signal(s) that this TCS responds to remains unknown. In this study, we show that PhoR is a sensor of acidic pH/high salt conditions, which activate PhoP via phosphorylation. Transcriptomic studies uncover that acidic pH-inducible expression of PhoP regulon is significantly inhibited in a PhoR-deleted M. tuberculosis. Using genome-wide screening we further identify a non-canonical mechanism of PhoP phosphorylation by the sensor kinase PrrB. To investigate how phosphorylation of PhoP is regulated, we discovered that PhoR functions as a phosphatase. Our results identify the motif/residues responsible for contrasting kinase/phosphatase dual functioning of PhoP, and collectively determine the homeostatic regulation of intra-mycobacterial P~PhoP which controls the final output of PhoP regulon. Together, these data uncover that PhoR plays a central role in mycobacterial adaptation to low pH conditions within the host macrophage phagosome. Consistent with these results a PhoR-deleted M. tuberculosis remains significantly attenuated in macrophages and animal models.

microbiology↗

bioRxiv2022

Physiological Condition Dependent Changes in Ciliary GPCR Localization in the Brain

Primary cilia are small immotile cellular appendages which mediate diverse types of singling and are found on most mammalian cell types including throughout the central nervous system. Cilia are known to localize certain G protein-coupled receptors (GPCRs) and are critical for mediating the signaling of these receptors. Several of these neuronal GPCRs have recognized roles in feeding behavior and energy homeostasis. Heterologous cell line and model systems like C. elegans and Chlamydomonas have implicated both dynamic GPCR cilia localization and cilia length and shape changes as key for signaling. However, it is unclear if mammalian ciliary GPCRs utilize similar mechanisms in vivo and under what physiological conditions these processes may occur. Here, we use the ciliary GPCRs, melanin concentrating hormone receptor 1 (MCHR1) and neuropeptide-Y receptor 2 (NPY2R) as model ciliary receptors to determine if dynamic localization to cilia occurs. We tested physiological conditions in which these GPCRs have been implicated such as feeding behavior, obesity, and circadian rhythm. Cilia were imaged using confocal microscopy and analyzed with a computer assisted approach allowing for unbiased and high throughput analysis of cilia. We analyzed GPCR positive cilia, cilia frequency as well as cilia length and receptor occupancy. Interestingly we observed changes in ciliary length, receptor occupancy, and cilia frequency under different conditions, but no consistent theme across GPCRs or brain nuclei was observed. A better understanding of the subcellular localization dynamics of ciliary GPCRs could reveal unrecognized molecular mechanisms regulating behaviors like feeding. Significance StatementOften, primary cilia localize specific G protein-coupled receptors (GPCRs) for subcellular signaling. Cell lines and model systems have indicated that cilia deploy dynamic GPCR localization and change their shape or length to modulate signaling. We used mice to assess neuronal cilia GPCRs under physiological conditions associated with both the receptors known functions and ciliopathy clinical features like obesity. We show that certain cilia with specific GPCRs appear to dynamically alter their length while others appear relatively stable under these conditions. These results implicate multiple themes across cilia GPCR mediated signaling and indicate that not all cilia modulate GPCR signaling using the same mechanisms. These data will be important for potential pharmacological approaches to target cilia GPCR-mediated signaling.

neuroscience↗

bioRxiv2022

Single cell transcriptomic profiling of human brain organoids reveals developmental timing- and cell-type-specific vulnerabilities induced by NRXN1 CNVs in schizophrenia

De novo mutations and copy number deletions in NRXN1 (2p16.3) pose a significant risk for schizophrenia (SCZ). It is unclear how NRXN1 deletions impact cortical development in a cell type-specific manner and disease background modulates these phenotypes. Here, we leveraged human pluripotent stem cell-derived forebrain organoid models carrying NRXN1 heterozygous deletions in isogenic and SCZ patient genetic backgrounds and conducted single-cell transcriptomic analysis over the course of brain organoid development from 3 weeks to 3.5 months. Intriguingly, while both deletions similarly impacted molecular pathways associated with ubiquitin-proteasome system, alternative splicing, and synaptic signaling in maturing glutamatergic and GABAergic neurons, SCZ-NRXN1 deletions specifically perturbed developmental trajectories of early neural progenitors and accumulated disease-specific transcriptomic signatures. Using calcium imaging, we found that both deletions led to long-lasting changes in spontaneous and synchronous neuronal networks, implicating synaptic dysfunction. Our study reveals developmental-timing- and cell-type-dependent actions of NRXN1 deletions in unique genetic contexts.

neuroscience↗

bioRxiv2022

A type IV Autotaxin inhibitor ameliorates acute liver injury and non-alcoholic steatohepatitis in mice

An important but rather underexplored pathway implicated in liver disease is the lysophosphatidic acid (LPA) signaling axis. LPA acts through G-protein coupled receptors inducing downstream signaling pathways related to cell proliferation, differentiation, and migration, and is predominantly produced by the extracellular phosphodiesterase, Autotaxin (ATX). ATX has gained significant attention lately with an impressive number of ATX inhibitors (type I-IV) reported. Here, we aim to evaluate the therapeutic potential of a (yet unexplored) type IV ATX inhibitor, Cpd17, in liver injury. In this study, we first confirmed the involvement of the ATX/LPA signaling axis in human and murine diseased livers. Thereafter, we evaluated the effects of Cpd17, in comparison with the classic type I ATX inhibitor PF8380, in vitro. While both inhibitors attenuated induced cell injury phenotypes as assessed using various assays and specific readout parameters in hepatocytes, macrophages, and hepatic stellate cells (HSCs), Cpd17 appeared more effective. This prompted us to characterize the mechanism of action of both inhibitors in situ and in vitro in macrophages and HSCs, demonstrating that Cpd17 was more potent in inhibiting relevant signaling pathways, namely RhoA-mediated cytoskeletal remodeling, and phosphorylation of MAPK/ERK and AKT/PKB. Finally, we investigated the therapeutic potential of Cpd17 in two liver disease mouse models, CCl4-induced acute liver injury and diet-induced non-alcoholic steatohepatitis. We demonstrate that Cpd17 has an excellent potential for reducing liver injury in both disease models in vivo. We conclude that ATX inhibition, by type IV inhibitor in particular, has an excellent potential for clinical application in liver diseases.

molecular biology↗

bioRxiv2021

An N-terminal Fusion Allele to Study Melanin Concentrating Hormone Receptor 1

Cilia on neurons play critical roles in both the development and function of the central nervous system (CNS). While it remains challenging to elucidate the precise roles for neuronal cilia, it is clear that a subset of G-protein-coupled receptors (GPCRs) preferentially localize to the cilia membrane. Further, ciliary GPCR signaling has been implicated in regulating a variety of behaviors. Melanin concentrating hormone receptor 1 (MCHR1), is a GPCR expressed centrally in rodents known to be enriched in cilia. Here we have used MCHR1 as a model ciliary GPCR to develop a strategy to fluorescently tag receptors expressed from the endogenous locus in vivo. Using CRISPR/Cas9, we inserted the coding sequence of the fluorescent protein mCherry into the N-terminus of Mchr1. Analysis of the fusion protein (mCherryMCHR1) revealed its localization to neuronal cilia in the CNS, across multiple developmental time points and in various regions of the adult brain. Our approach simultaneously produced fortuitous in/dels altering the Mchr1 start codon resulting in a new MCHR1 knockout line. Functional studies using electrophysiology show a significant alteration of synaptic strength in MCHR1 knockout mice. A reduction in strength is also detected in mice homozygous for the mCherry insertion, suggesting that while the strategy is useful for monitoring the receptor, activity could be altered. However, both lines should aid in studies of MCHR1 function and contribute to our understanding of MCHR1 signaling in the brain. Additionally, this approach could be expanded to aid in the study of other ciliary GPCRs.

neuroscience↗

bioRxiv2021

Artificial Intelligence Approaches to Assessing Primary Cilia

Cilia are microtubule based cellular appendages that function as signaling centers for a diversity of signaling pathways in many mammalian cell types. Cilia length is highly conserved, tightly regulated, and varies between different cell types and tissues and has been implicated in directly impacting their signaling capacity. For example, cilia have been shown to alter their lengths in response to activation of ciliary G protein-coupled receptors. However, accurately and reproducibly measuring the lengths of numerous cilia is a time-consuming and labor-intensive procedure. Current approaches are also error and bias prone. Artificial intelligence (Ai) programs can be utilized to overcome many of these challenges due to capabilities that permit assimilation, manipulation, and optimization of extensive data sets. Here, we demonstrate that an Ai module can be trained to recognize cilia in images from both in vivo and in vitro samples. After using the trained Ai to identify cilia, we are able to design and rapidly utilize applications that analyze hundreds of cilia in a single sample for length, fluorescence intensity and colocalization. This unbiased approach increased our confidence and rigor when comparing samples from different primary neuronal preps in vitro as well as across different brain regions within an animal and between animals. Moreover, this technique can be used to reliably analyze cilia dynamics from any cell type and tissue in a high-throughput manner across multiple samples and treatment groups. Ultimately, Ai-based approaches will likely become standard as most fields move toward less biased and more reproducible approaches for image acquisition and analysis. SUMMARYThe use of Artificial Intelligence (Ai) to analyze images is emerging as a powerful, less biased, and rapid approach compared with commonly used methods. Here we trained Ai to recognize a cellular organelle, primary cilia, and analyze properties such as length and staining intensity in a rigorous and reproducible manner.

cell biology↗

bioRxiv2021

Spinophilin limits GluN2B-containing NMDAR activity and sequelae associated with excessive hippocampal NMDAR function.

N-methyl-D-Aspartate receptors (NMDARs) are calcium-permeable ion channels that are ubiquitously expressed within the glutamatergic postsynaptic density. Phosphorylation of NMDAR subunits defines receptor activity and surface localization. Modulation of NMDAR phosphorylation by kinases and phosphatases regulates calcium entering the cell and subsequent activation of calcium-dependent processes. Spinophilin is the major synaptic protein phosphatase 1 (PP1) targeting protein that controls phosphorylation of myriad substrates via targeting or inhibition of PP1. Spinophilin limits NMDAR function in a PP1-dependent manner and we have previously shown that spinophilin sequesters PP1 away from the GluN2B subunit of the NMDAR, which results in increased phosphorylation of Ser-1284. However, how spinophilin modifies NMDAR function is unclear. Herein, we detail that while Ser-1284 phosphorylation increases calcium influx via GluN2B-containing NMDARs, overexpression of spinophilin decreases GluN2B-containing NMDAR activity by decreasing its surface expression. In hippocampal neurons isolated from spinophilin knockout animals there is an increase in cleaved caspase-3 levels compared to wildtype mice; however, this effect is not exclusively due to NMDAR activation; suggesting multiple putative mechanisms by which spinophilin may modulate caspase cleavage. Behaviorally, our data suggest that spinophilin knockout mice have deficits in spatial cognitive flexibility, a behavior associated GluN2B function within the hippocampus. Taken together, our data demonstrate a unique mechanism by which spinophilin modulates GluN2B containing NMDAR phosphorylation, channel function, and trafficking and that loss of spinophilin promotes pathological sequelae associated with GluN2B dysfunction. HIGHLIGHTSO_LISpinophilin bidirectionally regulates GluN2B-containing NMDAR function. C_LIO_LILoss of spinophilin in primary hippocampal neurons increases a pro-apoptotic marker. C_LIO_LILoss of spinophilin in vivo decreases measures of spatial cognitive flexibility. C_LI Graphical AbstractSpinophilin increases the phosphorylation of Ser-1284 on GluN2B, thereby enhancing calcium influx through the GluN2B containing NMDARs. In contrast, spinophilin limits GluN2B-containing surface expression putatively due to modulation of GluN2B interactions with endocytotic proteins. Since the second effect of spinophilin occurs independent of the first, we observe an overall decrease in calcium influx through GluN2B containing NMDARs when spinophilin is present. This low, basal calcium influx is less likely to be promote calcium-dependent activation of caspase and downstream apoptotic pathways and permits flexible search strategies and behaviors. In the absence of spinophilin, the spinophilin-driven internalization of the receptors is decreased, more receptors are expressed on the surface and calcium influx into the cell is increased. This high levels of intracellular calcium triggers apoptotic pathways leading to cell death. This impact may be more dramatic in cells with high expression of GluN2B-containing NMDA receptors. This loss of spinophilin reduces cognitive flexibility in hippocampal dependent tasks. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=116 SRC="FIGDIR/small/424812v1_ufig1.gif" ALT="Figure 1"> View larger version (37K): org.highwire.dtl.DTLVardef@1548b5org.highwire.dtl.DTLVardef@158a0aaorg.highwire.dtl.DTLVardef@19c185dorg.highwire.dtl.DTLVardef@103848b_HPS_FORMAT_FIGEXP M_FIG C_FIG

neuroscience↗

bioRxiv2019

A Comparison of Drug Delivery into Skin Using Topical Solutions, Needle Injections and Jet Injections

Drug diffusion within the skin with a needle-free micro-jet injection (NFI) device was compared with two well-established delivery methods: topical application and solid needle injection. A permanent make-up (PMU) machine, normally used for dermal pigmentation, was utilized as a solid needle injection method. For NFIs a continuous wave (CW) laser diode was used to create a bubble inside a microfluidic device containing a light absorbing solution. Each method delivered two different solutions into ex-vivo porcine skin. The first solution consisted of a red dye (direct red 81) and rhodamine B in water. The second solution was direct red 81 and rhodamine B in water and glycerol. For PMU experiments, the skin samples were kept stationary and the diffusion depth, width and surface area were measured. The NFI has a higher vertical dispersion velocity of 3 x 105 m/s compared to topical (0.1 m/s) and needle injection (53 m/s). The limitations and advantages of each method are discussed, and we conclude that the micro-jet injector represents a fast and minimally invasive injection method, while the solid needle injector causes notably tissue damage. In contrast, the topical method had the slowest diffusion rate but causes no visible damage to the skin.

bioengineering↗

bioRxiv2018

Neuregulin 1 type III reduces severity in a mouse model of Congenital Hypomyelinating Neuropathy

Myelin sheath thickness is precisely regulated and essential for rapid propagation of action potentials along myelinated axons. In the peripheral nervous system, extrinsic signals from the axonal protein neuregulin 1 type III regulate Schwann cell fate and myelination. Here we ask if modulating neuregulin 1 type III levels in neurons would restore myelination in a model of congenital hypomyelinating neuropathy (CHN). Using a mouse model of CHN, we rescued the myelination defects by early overexpression of neuregulin 1 type III. Surprisingly, the rescue was independent from the upregulation of Egr2 or essential myelin genes. Rather, we observed the activation of MAPK/ERK and other myelin genes such as peripheral myelin protein 2 (Pmp2) and oligodendrocyte myelin glycoprotein (Omg). We also confirmed that the permanent activation of MAPK/ERK in Schwann cells has detrimental effects on myelination. Our findings demonstrate that the modulation of axon-to-glial neuregulin 1 type III signaling has beneficial effects and restores myelination defects during development in a model of CHN.\n\n\n\nO_TBL View this table:\norg.highwire.dtl.DTLVardef@103281aorg.highwire.dtl.DTLVardef@3dc68eorg.highwire.dtl.DTLVardef@1b6c5feorg.highwire.dtl.DTLVardef@80e419org.highwire.dtl.DTLVardef@1ff2591_HPS_FORMAT_FIGEXP M_TBL C_TBL

neuroscience↗

bioRxiv2018

Targeting integrin alpha5 receptor in pancreatic stellate cells to diminish tumor-promoting effects in pancreatic cancer

Pancreatic stellate cells (PSCs) are the main precursors of cancer-associated fibroblasts (CAFs) in pancreatic ductal adenocarcinoma (PDAC), known to induce cancer aggressiveness. Integrin alpha5 (ITGA5), a fibronectin receptor, was found to be overexpressed by CAFs in stroma and linked to poor overall survival (log-rank p=0.022, n=137) of patients with PDAC. In vitro, knockdown of ITGA5 in human PSCs (hPSCs) inhibited their adhesion, migration, and proliferation and also inhibited TGF-{beta}-mediated differentiation. In vivo, co-injection of PANC-1 tumor cells and hPSCs (sh-ITGA5) developed tumors with reduced fibrosis and slower growth rate compared to those composed of PANC-1 and hPSC (sh-Ctrl). Furthermore, we developed a ITGA5-antagonizing peptidomimetic (AV3) which inhibited TGF{beta}-mediated hPSC differentiation by blocking ITGA5/FAK pathway. In vivo, treatment with AV3 intraperitoneally attenuated tumor fibrosis and thereby enhanced the efficacy of gemcitabine in patient-derived xenografts in mice. Altogether, this study reports the therapeutic importance of ITGA5 in PDAC and provides novel therapeutic peptidomimetic to enhance the effect of chemotherapy.

cancer biology↗