Biology subjects

Sharma, J.

Publications and source records attributed to Sharma, J..

33 records · Page 2Linked to original sources

bioRxiv2023

Most azole antifungal resistance mutations in the drug target provide cross-resistance and carry no intrinsic fitness cost

Azole antifungals are among the most frequently used drugs to treat fungal infections. Amino acid substitutions in and around the binding site of the azole target Erg11 (Cyp51) are a common resistance mechanism in pathogenic yeasts such as Candida albicans. How many and which mutations confer resistance, and at what cost, is however largely unknown. Here, we measure the impact of nearly 4,000 amino acid variants of the Erg11 ligand binding pocket on the susceptibility to six medical azoles. We find that a large fraction of amino acid substitutions lead to resistance (33%), most resistance mutations confer cross-resistance to two or more azoles (88%) and most importantly, only a handful of resistance mutations show a significant fitness cost in the absence of drug (9%). Our results reveal that resistance to azoles can arise through a large set of mutations and this will likely lead to azole pan-resistance, with very little evolutionary compromise. Such a resource will help inform treatment choices in clinical settings and guide the development of new drugs.

microbiology↗

bioRxiv2023

TFEB degradation is regulated by an IKK/β-TrCP2 phosphorylation-ubiquitination cascade

Transcription factor EB (TFEB) is a master regulator of lysosomal biogenesis and autophagy that plays a key role in the regulation of cellular clearance pathways. TFEB is regulated via a complex array of post-translational modifications (PTMs), but the exact molecular mechanism that regulates TFEB stability has remained elusive. Here, we show that TFEB levels are critically regulated by a defined phosphorylation-ubiquitination cascade. A human kinome screen identifies IKK (inhibitor of {kappa}B kinase) as a TFEB modifier, and a combination of phosphorylation assays, mass spectrometry analyses, and site-specific mutagenesis unveils a previously unrecognized TFEB phospho-degron (423SPFPSLS429) as the target of IKK. We show that IKK-mediated phosphorylation of TFEB triggers ubiquitination of adjacent lysine residues (K430 and K431) by the E3 ligase {beta}-TrCP2 ({beta}-Transducin repeat-containing protein 2), thereby tagging TFEB for degradation. Modified TFEB constructs that abolish these PTMs show much increased stability and expression levels but remain equally sensitive to autophagy- or stress- related stimuli while maintaining the capability to promote the expression of TFEB target genes and the clearance of Alzheimers associated tau in a cellular model of disease. Our results therefore uncover an IKK/{beta}-TrCP2 phosphorylation-ubiquitination cascade as a major mechanism that governs TFEB stability independently of other TFEB regulators.

cell biology↗

bioRxiv2023

SpatialPrompt: spatially aware scalable and accurate tool for spot deconvolution and clustering in spatial transcriptomics

Spatial transcriptomics has advanced our understanding of tissue biology by enabling sequencing while preserving spatial coordinates. In sequencing-based spatial technologies, each measured spot typically consists of multiple cells. Deconvolution algorithms are required to decipher the cell-type distribution at each spot. Existing spot deconvolution algorithms for spatial transcriptomics often neglect spatial coordinates and lack scalability as datasets get larger. We introduce SpatialPrompt, a spatially aware and scalable method for spot deconvolution as well as domain identification for spatial transcriptomics. Our method integrates gene expression, spatial location, and single-cell RNA sequencing (scRNA-seq) reference data to infer cell-type proportions of spatial spots accurately. At the core, SpatialPrompt uses non-negative ridge regression and an iterative approach inspired by graph neural network (GNN) to capture the local microenvironment information in the spatial data. Quantitative assessments on the human prefrontal cortex dataset demonstrated the superior performance of our tool for spot deconvolution and domain identification. Additionally, SpatialPrompt accurately decipher the spatial niches of the mouse cortex and the hippocampus regions that are generated from different protocols. Furthermore, consistent spot deconvolution prediction from multiple references on the mouse kidney spatial dataset showed the impressive robustness of the tool. In response to this, SpatialPromptDB database is developed to provide compatible scRNA-seq references with cell-type annotations for seamless integration. In terms of scalability, SpatialPrompt is the only method performing spot deconvolution and clustering in less than 2 minutes for large spatial datasets with 50,000 spots. SpatialPrompt tool along with the SpatialPromptDB database are publicly available as open source software for large-scale spatial transcriptomics analysis.

bioinformatics↗

bioRxiv2023

Bowel dysmotility and enteric neuron degeneration in lysosomal storage disease mice is prevented by gene therapy

Background and aimsChildren with neurodegenerative disease often have debilitating gastrointestinal (GI) symptoms that may be due at least in part to underappreciated involvement of neurons in the enteric nervous system (ENS), the master regulator of bowel function. MethodsWe investigated bowel motility in mouse models of CLN1 and CLN2 disease, neurodegenerative lysosomal storage disorders caused by deficiencies in palmitoyl protein thioesterase-1 (PPT1) and tripeptidyl peptidase-1 (TPP1), respectively. We then explored the integrity of ENS anatomy in immunostained bowel wholemount preparations from these mice. Lastly, we administered adeno-associated viral gene therapy to neonatal mice and determined if this would prevent these newly identified bowel phenotypes. ResultsMouse models of CLN1 and CLN2 disease both displayed slow bowel transit in vivo that worsened with age. Although the ENS appeared to develop normally, there was a progressive and profound loss of myenteric plexus neurons accompanied by changes in enteric glia in adult mice. Neonatal administration of adeno-associated virus-mediated gene therapy prevented bowel transit defects and the loss of many ENS neurons. ConclusionsWe show that two neurodegenerative lysosomal storage diseases cause profound and progressive damage to the mouse enteric nervous system and impair bowel motility. We also provide proof-of-principle evidence that gene therapy can prevent enteric nervous system disease. This study may have general therapeutic implications for many inherited neurodegenerative disorders. What you need to knowO_ST_ABSBackground and ContextC_ST_ABSMany pediatric central nervous system disorders also have debilitating gastrointestinal symptoms. For most of these diseases, it is not known if the enteric nervous system (ENS) is also affected and to what degree ENS defects contribute to GI symptoms. To date, no attempts have been made to directly treat or prevent enteric nervous system disease via gene therapy. New FindingsThe enteric nervous system is severely affected in mouse models of CLN1 and CLN2 disease, profoundly neurodegenerative lysosomal storage disorders. Bowel transit defects and most of the enteric nervous system pathology can be prevented by neonatal administration of gene therapy. LimitationsInformation about enteric nervous system disease in human children is still lacking, and methods will need to be developed to treat the human bowel. ImpactThese findings identify an underappreciated effect of neurodegenerative disease upon the bowel and demonstrate that enteric nervous system degeneration can be prevented in mice. This provides a new perspective on these childhood disorders that may be applicable to many other conditions that affect the bowel. Lay SummaryIn childrens diseases where the brain degenerates, nerve cells in the bowel also die causing gastrointestinal problems, but this can be prevented by gene therapy.

neuroscience↗

bioRxiv2023

The soluble guanylyl cyclase pathway is inhibited to evade androgen deprivation-induced senescence and enable progression to castration resistance

Castration-resistant prostate cancer (CRPC) is fatal and therapeutically under-served. We describe a novel CRPC-restraining role for the vasodilatory soluble guanylyl cyclase (sGC) pathway. We discovered that sGC subunits are dysregulated during CRPC progression and its catalytic product, cyclic GMP (cGMP), is lowered in CRPC patients. Abrogating sGC heterodimer formation in castration-sensitive prostate cancer (CSPC) cells inhibited androgen deprivation (AD)-induced senescence, and promoted castration-resistant tumor growth. We found sGC is oxidatively inactivated in CRPC. Paradoxically, AD restored sGC activity in CRPC cells through redox-protective responses evoked to protect against AD-induced oxidative stress. sGC stimulation via its FDA-approved agonist, riociguat, inhibited castration-resistant growth, and the anti-tumor response correlated with elevated cGMP, indicating on-target sGC activity. Consistent with known sGC function, riociguat improved tumor oxygenation, decreasing the PC stem cell marker, CD44, and enhancing radiation-induced tumor suppression. Our studies thus provide the first evidence for therapeutically targeting sGC via riociguat to treat CRPC. Statement of significanceProstate cancer is the second highest cancer-related cause of death for American men. Once patients progress to castration-resistant prostate cancer, the incurable and fatal stage, there are few viable treatment options available. Here we identify and characterize a new and clinically actionable target, the soluble guanylyl cyclase complex, in castration-resistant prostate cancer. Notably we find that repurposing the FDA-approved and safely tolerated sGC agonist, riociguat, decreases castration-resistant tumor growth and re-sensitizes these tumors to radiation therapy. Thus our study provides both new biology regarding the origins of castration resistance as well as a new and viable treatment option.

cancer biology↗

bioRxiv2023

Salmonella Typhimurium effector SseI regulates host peroxisomal dynamics to acquire lysosomal cholesterol for better intracellular growth

Intracellular Salmonella resides and multiplies in cholesterol-rich specialized compartment called Salmonella-containing vacuoles (SCVs) and avoids fusion with acidic lysosomes. Given, lysosomes are primary organelle that redistributes LDL derived cholesterol to other organelles; we questioned how lysosomal cholesterol can be transported to SCV. We demonstrate here that peroxisomes are recruited to SCVs in human primary macrophages, epithelial cells and functions as pro-bacterial organelles. Further, this interaction is assisted by SseI, a Salmonella effector protein containing mammalian peroxisome targeting sequence. SseI localizes to peroxisome, interacts and activates a host Ras GTPase, ARF-1 on the peroxisome membrane. Activation of ARF-1 leads to recruitment of phosphatidylinsolitol-5- phosphate-4 kinase to generate phosphatidylinsolitol-4-5-bisphosphate on peroxisomes. Accordingly, the{Delta} sseI strain showed reduced virulence in cell lines and during mice infection. Taken together, our work identified a fascinating mechanism by which a pathogen targets host organelles via its secretory effectors and exploits host metabolic intermediates for its intracellular proliferation.

cell biology↗

bioRxiv2022

A marmoset brain cell census reveals persistent influence of developmental origin on neurons

The mammalian brain is composed of many brain structures, each with its own ontogenetic and developmental history. Transcriptionally-based cell type taxonomies reveal cell type composition and similarity relationships within and across brain structures. We sampled over 2.4 million brain cells across 18 locations in the common marmoset, a New World monkey primed for genetic engineering, and used single-nucleus RNA sequencing to examine global gene expression patterns of cell types within and across brain structures. Our results indicate that there is generally a high degree of transcriptional similarity between GABAergic and glutamatergic neurons found in the same brain structure, and there are generally few shared molecular features between neurons that utilize the same neurotransmitter but reside in different brain structures. We also show that in many cases the transcriptional identities of cells are intrinsically retained from their birthplaces, even when they migrate beyond their cephalic compartments. Thus, the adult transcriptomic identity of most neuronal types appears to be shaped much more by their developmental identity than by their primary neurotransmitter signaling repertoire. Using quantitative mapping of single molecule FISH (smFISH) for markers for GABAergic interneurons, we found that the similar types (e.g. PVALB+ interneurons) have distinct biodistributions in the striatum and neocortex. Interneuron types follow medio-lateral gradients in striatum but form complex distributions across the neocortex that are not described by simple gradients. Lateral prefrontal areas in marmoset are distinguished by high relative proportions of VIP+ neurons. We further used cell-type-specific enhancer driven AAV-GFP to visualize the morphology of molecularly-resolved interneuron classes in neocortex and striatum, including the previously discovered novel primate-specific TAC3+ striatal interneurons. Our comprehensive analyses highlight how lineage and functional class contribute to the transcriptional identity and biodistribution of primate brain cell types. One-Sentence SummaryAdult primate neurons are imprinted by their region of origin, more so than by their functional identity.

neuroscience↗

bioRxiv2022

The effect of subconjunctival TNF-α inhibitors on retinal ganglion cell apoptosis and optic nerve degeneration after corneal surgery or trauma--of possible prophylactic value against secondary glaucoma.

AbstractO_ST_ABSBackground and PurposeC_ST_ABSLate secondary glaucoma is an often-severe complication after acute events like anterior segment surgery, trauma, infection, etc. TNF- is a major mediator that is rapidly upregulated, diffusing also to the retina and causes apoptosis of the ganglion cells and degeneration of their optic nerve axons (mediating steps to glaucomatous damage). Anti-TNF- antibodies are in animals very effective in protecting the retinal cells and the optic nerve--and might therefore be useful prophylactically against secondary glaucoma in future such patients.Here we evaluate 1) toxicity and 2) efficacy of two TNF- inhibitors (adalimumab and infliximab), in rabbits by subconjunctival administration. MethodsFor drug toxicity, animals with normal, unburned corneas were injected with adalimumab (0.4, 4, or 40 mg), or infliximab (1, 10, or 100 mg). For drug efficacy, other animals were subjected to alkali burn before such injection, or steroids (for control). The rabbits were evaluated clinically with slit lamp and photography, electroretinography, optical coherence tomography, and intraocular pressure manometry. A sub-set of eyes were stained ex vivo after 3 days for retinal cell apoptosis (TUNEL). In other experiments the optic nerves were evaluated by paraphenylenediamine staining after 50 or 90 days. Loss of retinal cells and optic nerve degeneration were quantified. ResultsSubconjunctival administration of 0.4 mg or 4.0 mg adalimumab were well tolerated, whereas 40.0 mg was toxic to the retina. 1, 10, or 100 mg infliximab were also well tolerated.Analysis of the optic nerve axons after 50 days confirmed the safety of 4.0 mg adalimumab and of 100 mg infliximab. For efficacy, 4.0 mg adalimumab subconjunctivally in 0.08 mL provided practically full protection against retinal cell apoptosis 3 days following alkali burn, and infliximab 100 mg only slightly less. At 90 days following burn injury, control optic nerves showed about 50% axon loss as compared to 8% in the adalimumab treatment group. ConclusionsSubconjunctival injection of 4.0 mg adalimumab in rabbits shows no eye toxicity and provides excellent neuroprotection, both short (3 days) and long-term (90 days). Ourtotal accumulated data from several of our studies, combined with the present paper, suggest that corneal injuries, including surgery, might benefit from routine administration of anti-TNF- biologics to reduce inflammation and future secondary glaucoma.

pathology↗

bioRxiv2022

Development and applications of a CRISPR activation system for facile genetic overexpression in Candida albicans

For the fungal pathogen Candida albicans, genetic overexpression readily occurs via a diversity of genomic alterations, such as aneuploidy and gain-of-function mutations, with important consequences for host adaptation, virulence, and evolution of antifungal drug resistance. Given the important role of overexpression on C. albicans biology, it is critical to develop and harness tools that enable the analysis of genes expressed at high levels in the fungal cell. Here, we describe the development, optimization, and application of a novel, single-plasmid-based CRISPR activation (CRISPRa) platform for targeted genetic overexpression in C. albicans, which employs a guide RNA to target an activator complex to the promoter region of a gene of interest, thus driving transcriptional expression of that gene. Using this system, we demonstrate the ability of CRISPRa to drive high levels of gene expression in C. albicans, and we assess optimal guide RNA targeting for robust and constitutive overexpression. We further demonstrate the specificity of the system via RNA sequencing. We highlight the application of CRISPRa to overexpress genes involved in pathogenesis and drug resistance and contribute towards the identification of novel phenotypes. Together, this tool will facilitate a broad range of applications for the study of C. albicans genetic overexpression.

microbiology↗

bioRxiv2022

Aromatase Inhibitor Induced Musculoskeletal Inflammation is Observed Independent of Oophorectomy in a Novel Mouse Model

BackgroundAromatase Inhibitors (AIs) block physiological estrogen production in peripheral tissues and are used clinically to reduce disease recurrences and improve overall survival rates in hormone receptor-positive breast cancer patients. However, half of patients taking these drugs develop aromatase inhibitor induced arthralgia (AIIA), which is characterized by severe pain and inflammation in various joints and the surrounding musculoskeletal tissue. While the pathophysiology is not currently understood, it has been proposed to be associated with systemic estrogen deficiency resulting from AI treatment. Since AIIA leads to suspension of therapy in 20-30% of patients, reducing AIIA incidence may provide sustained AI treatment and enhance long-term survival. ObjectiveIn order to establish a better understanding of disease pathology and to create a platform that can be used to explore future interventional strategies, our objective in this study was to design a novel animal model of AIIA. MethodsFemale BALB/C-Tg(NF{kappa}B-RE-luc)-Xen mice, which have a firefly luciferase cDNA reporter transgene under the regulation of NF{kappa}B binding sites, were oophorectomized and treated with AI (letrozole) by daily subcutaneous injections for 5 weeks. Control groups included oophorectomized mice receiving vehicle injections and non-oophorectomized mice treated with AI. Knee joints and surrounding muscle tissue were imaged on the BioSpec 94/30 micro-MRI. The primary weight-bearing joint (hind limb) was examined histopathologically and NF{kappa}B activity was measured by bioluminescent imaging. Serum was collected for cytokine analysis. Additionally, healthy human PBMCs were treated with letrozole, estrogen, or both, and RNA sequencing was performed at 36 hrs. ResultsBioluminescent imaging showed significantly enhanced NF{kappa}B activation with AI treatment in the hind limbs compared to controls receiving vehicle treatment. Moreover, analysis of knee joints and legs by MRI showed enhanced signal detection in the joint space and surrounding tissue following daily AI injections. Surprisingly, the enhanced MRI detection and NF{kappa}B activation was observed with AI treatment independent of the oophorectomy procedure. This indicates that the induction of musculoskeletal-directed inflammation by AI is not mediated by changes in physiological estrogen levels, which is contrary to proposed mechanisms of disease pathogenesis. Similarly, histopathological analysis showed tenosynovitis and musculoskeletal infiltrates in all mice receiving AI with or without oophorectomy. IHC analysis of the infiltrates demonstrated a predominantly macrophage-mediated inflammatory response with scattered CD4+ T cells. Additionally, serum cytokine levels of IL-2, IL-4, IL-6, and CXCL1 were significantly elevated in mice with AI treatment. RNA sequencing of human PBMCs after in vitro AI stimulation did not demonstrate an AI-specific gene expression pattern associated with immune system activation directly, suggesting that the pathogenesis of AIIA may be mediated through cells in other tissues in vivo. ConclusionsCollectively, these data establish a novel mouse model of AIIA and identify an estrogen-independent stimulation of disease pathology via AI-mediated induction. This suggests that the pathogenesis of AIIA may not be mediated by estrogen deficiency, as previously hypothesized, and indicates that AI-induced inflammation may not be regulated directly through a pathogenic mechanism initially derived from circulating mononuclear cells. Future studies aim to characterize this inflammatory mechanism in vivo with a focus on other cells, including macrophages, synovial cells and chondrocytes, to provide insight into putative therapeutic strategies directed at mitigating disease pathology.

immunology↗

bioRxiv2021

Hemodynamic molecular imaging of tumor-associated enzyme activity in the living brain.

Molecular imaging could have great utility for detecting, classifying, and guiding treatment of brain disorders, but existing probes offer limited capability for assessing relevant physiological parameters. Here we describe a potent approach for noninvasive mapping of cancer-associated enzyme activity using a molecular sensor that acts on the vasculature, providing a diagnostic readout via local changes in hemodynamic image contrast. The sensor is targeted at the fibroblast activation protein (FAP), an extracellular dipeptidase and clinically relevant biomarker of brain tumor biology. Optimal FAP sensor variants were identified by screening a series of prototypes for responsiveness in a cell-based bioassay. The best variant was then applied for quantitative neuroimaging of FAP activity in rats, where it reveals nanomolar-scale FAP expression by xeno-grafted tumor cells. The activated probe also induces robust hemodynamic contrast in nonhuman primate brain. This work thus demonstrates a translatable strategy for ultrasensitive functional imaging of molecular targets in neuromedicine.

bioengineering↗

bioRxiv2020

Decoding the role of CBLB for innate immune responses regulating systemic dissemination during Non-Tuberculous Mycobacteria infection

Non-Tuberculous Mycobacteria (NTM) are ubiquitous in nature, present in soil and water, and cause primary leading to disseminated infections in immunocompromised individuals. NTM infections are surging in recent years due to an increase in an immune-suppressed population, medical interventions, and patients with underlying lung diseases. Host regulators of innate immune responses, frontiers for controlling infections and dissemination, are poorly defined during NTM infections. Here, we describe the role of CBLB, an E3-ubiquitin ligase, for innate immune responses and disease progression in a mouse model of NTM infection under compromised T-cell immunity. We found that CBLB thwarted NTM growth and dissemination in a time- and infection route- dependent manner. Mechanistically, we uncovered defects in many innate immune cells in the absence of Cblb, including poor responses of NK cells, inflammatory monocytes, and conventional dendritic cells. Strikingly, Cblb-deficient macrophages were competent to control NTM growth in vitro. Histopathology suggested the lack of early formation of granulomatous inflammation in the absence of CBLB. Collectively, CBLB is essential to mount productive innate immune responses and help prevent the dissemination during an NTM infection under T-cell deficiency.

immunology↗

bioRxiv2019

Close range vocal interaction in the common marmoset (Callithrix Jacchus)

Vocal communication in animals often involves taking turns vocalizing. In humans, turn taking is a fundamental rule in conversation. Among non-human primates, the common marmoset is known to engage in antiphonal calling using phee calls and trill calls. Calls of the trill type are the most common, yet difficult to study, because they are not very loud and uttered in conditions when animals are in close proximity to one another. Here we recorded trill calls in captive pair-housed marmosets using wearable microphones, while the animals were together with their partner or separated, but within trill call range. Trills were exchanged mainly with the partner and not with other animals in the room. Animals placed outside the home cage increased their trill call rate and uttered more trills in response more to their partner. The fundamental frequency, F0, of trills increased when animals were placed outside the cage. Our results indicate that trill calls can be monitored using wearable audio equipment. Relatively minor changes in social context affect trill call interactions and spectral properties of trill calls, indicating that marmosets can communicate subtle information to their partner vocally.

animal behavior and cognition↗

bioRxiv2019

Viral manipulation of functionally distinct neurons from mice to humans

Recent success in identifying gene regulatory elements in the context of recombinant adeno-associated virus vectors have enabled cell type-restricted gene expression. However, within the cerebral cortex these tools are presently limited to broad classes of neurons. To overcome this limitation, we developed a strategy that led to the identification of multiple novel enhancers to target functionally distinct neuronal subtypes. By investigating the regulatory landscape of the disease gene Scn1a, we identified enhancers that target the breadth of its expression, including two that are selective for parvalbumin and vasoactive intestinal peptide cortical interneurons. Demonstrating the functional utility of these elements, we found that the PV-specific enhancer allowed for the selective targeting and manipulation of these neurons across species, from mice to humans. Finally, we demonstrate that our selection method is generalizable to other genes and characterize four additional PV-specific enhancers with exquisite specificity for distinct regions of the brain. Altogether, these viral tools can be used for cell-type specific circuit manipulation and hold considerable promise for use in therapeutic interventions.

neuroscience↗

bioRxiv2019

A CLN6-CLN8 complex recruits lysosomal enzymes at the ER for Golgi transfer

Lysosomal enzymes are synthesized in the endoplasmic reticulum (ER) and transferred to the Golgi complex by interaction with the Batten disease protein CLN8. Here we investigated the relationship of this pathway with CLN6, an ER-associated protein of unknown function that is defective in a different Batten disease subtype. Experiments focused on protein interaction and trafficking identified CLN6 as an obligate component of a CLN6-CLN8 complex (herein referred to as EGRESS: ER-to-Golgi relaying of enzymes of the lysosomal system) which recruits lysosomal enzymes at the ER to promote their Golgi transfer. Simultaneous deficiency of CLN6 and CLN8 did not aggravate mouse pathology compared to the single deficiencies, indicating that the EGRESS complex works as a functional unit. Mutagenesis experiments showed that the second luminal loop of CLN6 is required for the interaction of CLN6 with the enzymes but dispensable for interaction with CLN8, and in vitro and in vivo studies showed that CLN6 deficiency results in inefficient ER export of lysosomal enzymes and diminished levels of the enzymes at the lysosome. These results identify CLN6 and the EGRESS complex as key players in lysosome biogenesis and shed light on the molecular etiology of Batten disease caused by defects in CLN6.

cell biology↗