In silico genome wide identification of long non-coding RNAs differentially expressed during Candida auris host pathogenesis.

Candida auris is an invasive fungal pathogen of high concern due to acquired drug tolerance against antifungals used in clinics. The prolonged persistence on biotic and abiotic surfaces can result in onset of hospital outbreaks causing serious health threat. An in depth understanding of pathology of C. auris is highly desirable for development of efficient therapeutics. Non-coding RNAs play crucial role in fungal pathology. However, the information about ncRNAs is scanty to be utilized. Herein our aim is to identify long noncoding RNAs with potent role in pathobiology of C. auris. Thereby, we analyzed the transcriptomics data of C. auris infection in blood for identification of potential lncRNAs with regulatory role in determining invasion, survival or drug tolerance under infection conditions. Interestingly, we found 275 lncRNAs, out of which 253 matched with lncRNAs reported in Candidamine, corroborating for our accurate data analysis pipeline. Nevertheless, we obtained 23 novel lncRNAs not reported earlier. Three lncRNAs were found to be under expressed throughout the course of infection, in the transcriptomics data. 16 of potent lncRNAs were found to be coexpressed with coding genes, emphasizing for their functional role. Noteworthy, these ncRNAs are expressed from intergenic regions of the genes associated with transporters, metabolism, cell wall biogenesis. This study recommends for possible association between lncRNA expression and C. auris pathogenesis.

A Novel Robust Method Mimicking Human Substratum To Dissect the Heterogeneity of Candida auris Biofilm Formation.

Candida auris is a pathogen of urgent threat level as marked by the CDC. The formation of biofilms is an essential property of this fungus to establish infection and escape drug treatment. However, our understanding of pathogenesis through biofilm is hampered by heterogeneity in C. auris biofilms observed in different studies. It is imperative to replicate in vivo conditions for studying C. auris biofilm formation in vitro. Different methods are standardized, but the surface used to form biofilms lacks consistency as well as the architecture of a typical biofilm. Here, we report an in vitro technique to grow C. auris biofilms on gelatin-coated coverslips. Interestingly, C. auris cells grown on gelatin-coated coverslips either on modified synthetic sweat media or RPMI 1640 resulted in similar multilayer biofilm formation with extracellular polymeric substances (EPS). This method is also consistent with the biofilm formation of other Candida species, such as Candida glabrata and Candida albicans. Biofilms of C. glabrata developed through this method show pseudohyphae and EPS. This method can be used to understand the molecular basis of biofilm formation, associated pathogenesis, and drug tolerance. The technique is cost-effective and would thus serve in rightful screening and repurposing drug libraries for designing new therapeutics against the less-studied high-alarm pathogen C. auris. IMPORTANCE Heterogeneity is seen when multidrug-resistant C. auris biofilm is cultured using different reported methods. Biofilm formed on the gelatin surface mimics the condition of a host environment that has multilayers and EPS. This method has feasibility for drug screening and analyzing biofilms through three-dimensional (3D) reconstruction. This in vitro biofilm formation technique is also exploited to study the formation of biofilm of other Candida species. The biofilms of C. glabrata and C. albicans can also be correctly mimicked using gelatin in the biofilm-forming environment. Thus, the novel in vitro method for biofilm formation reported here can be widely used to understand the mechanism of biofilm formation, related virulence properties, and drug tolerance of C. auris and other Candida species. This simple and low-cost technique is highly suitable for screening novel inhibitors and repurposed libraries and to design new therapeutics against Candida species.

Liver organoids as a primary human model to study HBV-mediated Hepatocellular carcinoma. A review.

Hepatitis B Virus (HBV) is the prevailing cause of chronic liver disease, which progresses to Hepatocellular carcinoma (HCC) in 75% of cases. It represents a serious health concern being the fourth leading cause of cancer-related mortality worldwide. Treatments available to date fail to provide a complete cure with high chances of recurrence and related side effects. The lack of reliable, reproducible, and scalable in vitro modeling systems that could recapitulate the viral life cycle and represent virus-host interactions has hindered the development of effective treatments so far. The present review provides insights into the current in-vivo and in-vitro models used for studying HBV and their major limitations. We highlight the use of three-dimensional liver organoids as a novel and suitable platform for modeling HBV infection and HBV-mediated HCC. HBV organoids can be expanded, genetically altered, patient-derived, tested for drug discovery, and biobanked. This review also provides the general guidelines for culturing HBV organoids and highlights their several prospects for HBV drug discovery and screening.

Candida auris biofilm: a review on model to mechanism conservation.

INTRODUCTION: Candida auris is included in the fungal infection category 'critical' by WHO because of associated high drug tolerance and spread at an alarming rate which if remains untouched may result in serious outbreaks. Since its discovery in 2009, several assiduous efforts by mycologists across the world have deciphered its biology including growth physiology, drug tolerance, biofilm formation, etc. The differential response of various strains from different clades poses a hurdle in drawing a final conclusion. AREAS COVERED: This review provides brief insights into the understanding of C. auris biofilm. It includes information on various models developed to understand the biofilms and conservation of different signaling pathways. Significant development has been made in the recent past with the generation of relevant in vivo and ex vivo models. The role of signaling pathways in the development of biofilm is largely unknown. EXPERT OPINION: The selection of an appropriate model system is a must for the accuracy and reproducibility of results. The conservation of major signaling pathways in C. auris with respect to C. albicans and S. cerevisiae highlights that initial inputs acquired from orthologs will be valuable in getting insights into the mechanism of biofilm formation and associated pathogenesis.

Rapamycin and Torin2 inhibit Candida auris TOR: Insights through growth profiling, docking, and MD simulations.

The fungus Candida auris is a pathogen of utmost concern due to its rapid emergence across the globe, acquired antifungal drug tolerance, thermotolerance, and ability to survive in hospital settings and preserved foods. Recent incidences of comorbidity of corona patients with its infection in hospital settings highlighted the importance of understanding the pathobiology and drug tolerance of this fungus on priority. The Target of rapamycin (TOR) is a central regulator of growth across eukaryotes with an illustrated role in fungal pathology. The role of the TOR signalling pathway in the growth of C. auris is yet to be described. In-silico, analysis revealed the presence of highly conserved Tor kinase, components of TORC, and key downstream components in C. auris. Rapamycin and Torin2, the specific inhibitors of Tor reduce the growth of C. auris. An inhibition of Tor leads to cell cycle arrest at the G1 phase with a defect in cytokinesis. Interestingly, with an insignificant difference in growth at 30 and 37 °C, a sharp decline in growth is seen with Torin2 at 37 °C. The heterogeneous response emphasizes the importance of physiology-based differential cellular response at different temperatures. In addition, the inhibition of Tor suppresses the biofilm formation. In silico studies through docking and simulations showed rapamycin and torin2 as specific inhibitors of C. auris Tor kinase (CauTor kinase) and hence can be exploited for a thorough understanding of the TOR signalling pathway in pathobiology and drug tolerance of C. auris. HIGHLIGHTSConservation of TOR signalling pathway in Candida aurisRapamycin and torin2 are specific inhibitors of Cau TorUnderstanding of the role of TOR signalling pathway through the use of inhibitors rapamycin and torin2.Heterogenous response of C. auris to torin2 at different physiological conditions.Communicated by Ramaswamy H. Sarma.

Long non-coding RNA in Non-alcoholic fatty liver disease.

Nonalcoholic fatty liver disease (NAFLD) is a chronic disease of worldwide impact. The disease process begins with steatosis, i.e., fat accumulation in the liver, and proceeds to nonalcoholic steatohepatitis (NASH). Liver biopsy is the gold standard for NASH diagnosis, but the procedure is invasive, expensive, error prone and poses considerable risk. Unfortunately, there are currently no precise FDA-approved therapies for NAFLD, the only options being lifestyle change and symptomatic treatment. Recently, much research has focused on the identification of molecular mechanisms that could be translated into novel diagnostics and therapeutics. With the advent of high throughput genomics and transcriptomics, noncoding RNAs, including long non-coding RNAs (lncRNAs) have been identified as key players of NAFLD pathogenesis and have accordingly attracted much attention as potential diagnostics and therapeutics. In this chapter, we reviewed various lncRNAs and their functions at different stages of NAFLD. We also highlighted how these unique molecules can be developed as stage-specific non-invasive diagnostic biomarkers for NAFLD.

Immunonano-Lipocarrier-Mediated Liver Sinusoidal Endothelial Cell-Specific RUNX1 Inhibition Impedes Immune Cell Infiltration and Hepatic Inflammation in Murine Model of NASH.

BACKGROUND: Runt-related transcription factor (RUNX1) regulates inflammation in non-alcoholic steatohepatitis (NASH). METHODS: We performed in vivo targeted silencing of the RUNX1 gene in liver sinusoidal endothelial cells (LSECs) by using vegfr3 antibody tagged immunonano-lipocarriers encapsulated RUNX1 siRNA (RUNX1 siRNA) in murine models of methionine choline deficient (MCD) diet-induced NASH. MCD mice given nanolipocarriers-encapsulated negative siRNA were vehicle, and mice with standard diet were controls. RESULTS: Liver RUNX1 expression was increased in the LSECs of MCD mice in comparison to controls. RUNX1 protein expression was decreased by 40% in CD31-positive LSECs of RUNX1 siRNA mice in comparison to vehicle, resulting in the downregulation of adhesion molecules, ICAM1 expression, and VCAM1 expression in LSECs. There was a marked decrease in infiltrated T cells and myeloid cells along with reduced inflammatory cytokines in the liver of RUNX1 siRNA mice as compared to that observed in the vehicle. CONCLUSIONS: In vivo LSEC-specific silencing of RUNX1 using immunonano-lipocarriers encapsulated siRNA effectively reduces its expression of adhesion molecules, infiltrate on of immune cells in liver, and inflammation in NASH.

Evolutionary conservation of long non-coding RNAs in non-alcoholic fatty liver disease.

Non-alcoholic fatty liver disease (NAFLD) encompasses a spectrum of conditions ranging from hepatic steatosis to steatohepatitis (NASH) to fibrosis in the absence of alcohol consumption. Its pathogenesis involves both genetic and environmental factors with a multitude of underlying molecular mechanisms and mediators at each stage. Recent transcriptomic-based studies have led to the identification and association of long non-coding RNAs (lncRNAs) with disease pathology in NAFLD patients and in vivo rodent models. However, the knowledge of function of most of the lncRNAs in NAFLD pathology remains obscure. In the current review, we give a comprehensive catalogue of well reported lncRNAs in NAFLD and classify them using sequence and synteny-based evolutionary conservation across rodents, nonhuman primate and human species. The conserved lncRNAs across all the three species may be dissected in larger clinical studies of NAFLD and can be explored as biomarkers and therapeutic targets. In addition, we also review and analyse single nucleotide polymorphisms (SNPs) in these lncRNAs. It adds another facet to the regulatory role of NAFLD-associated lncRNAs and underscores the significance of a novel genetic landscape of non-coding genome in determining the genetic susceptibility of NAFLD.

A regulatory circuit between lncRNA and TOR directs amino acid uptake in yeast.

Long non coding RNAs (lncRNAs) have emerged as crucial players of several central cellular processes across eukaryotes. Target of Rapamycin (TOR) is a central regulator of myriad of fundamental cellular processes including amino acid transport under diverse environmental conditions. Here we investigated the role of lncRNA in TOR regulated amino acid uptake in S. cerevisiae. Transcription of lncRNA regulates local gene expression in eukaryotes. In silico analysis of many genome wide studies in S. cerevisiae revealed that transcriptome includes conditional expression of numerous lncRNAs in proximity to amino acid transporters (AATs). Considering regulatory role of these lncRNAs, we selected highly conserved TOR regulated locus of a pair of AATs present in tandem BAP2 and TAT1. We observed that the expression of antisense lncRNA XUT_2F-154 (TBRT) and AATs BAP2 and TAT1 depends on activities of TOR signaling pathway. The expression of TBRT is induced, while that of BAP2 TAT1 is repressed upon TOR inhibition by Torin2. Notably, upon TOR inhibition loss of TBRT contributed to enhanced activities of Bap2 and Tat1 leading to improved growth. Interestingly, nucleosome scanning assay reveal that TOR signaling pathway governs chromatin remodeling at BAP2 biphasic promoter to control the antagonism of TBRT and BAP2 expression. Further TBRT also reprograms local chromatin landscapes to decrease the transcription of TAT1. The current work demonstrates a functional correlation between lncRNA production and TOR governed amino acid uptake in yeast. Thus this work brings forth a novel avenue for identification of potential regulators for therapeutic interventions against TOR mediated diseases.

MYOD and HAND transcription factors have conserved recognition sites in mTOR promoter: insights from in silico analysis.

mTOR regulates multiple cellular processes that are critical for proper maintenance of cell growth and development. However, mechanisms and factors responsible for transcriptional regulation of mTOR are partially known. To identify different transcription factor binding sites in promoter region of mTOR, we performed in silico phylogenetic foot printing analysis of diverse set of human orthologs. Phylogenetic tree for the orthologs was generated to establish the evolutionary relationships among them. Conserved binding sites among the species were predicted by tool MEME. The predicted conserved sites were further analyzed for binding of transcription factors by MatInspector program. Predicted TFs were then integrated with known physical interactions and coexpression data to decipher the important transcriptional regulators of mTOR signaling. Our study suggests that motifs AGGCGGG (+ 15 to + 21) and GGCGGC (+ 60 to + 65) are highly conserved across the species and are recognition sequence for HAND and MYOD transcription factors, respectively. Also these two transcription factors show direct physical interaction in protein-protein interaction map, indicating their regulatory role on expression of mTOR for control of myogenesis. Our study provides novel clues on differential regulation of mTOR under diverse environmental conditions.

Invitro Evaluation of Torin2 and 2, 6-Dihydroxyacetophenone in Colorectal Cancer Therapy.

Colorectal cancer (CRC) is one of the most prevalent cancers diagnosed worldwide. Despite recent advances, resistance to cytotoxic and targeted therapy remains one of the greatest challenges in long-term management of colorectal cancer therapy. Recently established role of mTOR signaling in proliferation of CRC has incited for evaluation of mTOR kinase specific inhibitors in CRC therapy. Second generation mTOR kinase inhibitors including Torin2 has demonstrated efficient anticancer properties against variety of cancers and are in various stages of drug development. The time and financial constraints concomitant from discovery to development of efficient chemical inhibitors has redirected attention towards investigation of wide spread naturally occurring largely inexpensive compounds for their therapeutic potential. One such naturally occurring compound acetophenone derivative polyphenolic compound 2, 6-Dihydroxyacetophenone (DHAP) inhibits cell growth in different conditions. We investigated anticancer properties of both Torin2 and DHAP against colorectal cancer in HCT8 cell lines. Both Torin2 and DHAP inhibited growth of CRC cells at different concentrations by restricting multiple cellular functions e.g., cell cycle progression, cell migration and induced apoptosis. Treatment of HCT8 cells with natural compound DHAP resulted in reduced expression of mTOR pathway specific genes p70S6K1 and AKT1. In silico docking studies showed affinity of DHAP to mTOR kinase like Torin2. Taken together, our result vouches for role of Torin2 in CRC therapy and recommends DHAP an mTOR inhibitor, as a potential lead in the development of new therapeutic regimes against colorectal cancer.

Novel insights into TOR signalling in Saccharomyces cerevisiae through Torin2.

Target of rapamycin (TOR) regulates cellular homeostasis by coordinating cellular growth pathways in response to different environmental signals. Rapamycin, an allosteric TOR complex 1 (TORC1) inhibitor, has proven to be invaluable for elucidating various aspects of the TOR signalling pathway; however, its applications are limited due to its inability to completely suppress TORC2. In the present study, we examined the effects of a newly discovered potent TOR inhibitor, Torin2, which inhibits both TORC1 and TORC2, on Saccharomyces cerevisiae growth. Genome-scale expression profiling of Torin2 treated yeast cells showed an expression profile similar to that of other TOR inhibitors such as rapamycin and caffeine. Distinct inhibition of cell growth by Torin2 treatment is indicated by the fact that a smaller number of transcripts are altered, compared to the changes after rapamycin and caffeine treatments. Our results revealed that Torin2 leads to increased expression of the calcineurin pathway genes favouring a synergistic therapeutic response of Torin2 in combination with calcineurin inhibitors. Further, Torin2 causes defective bud site selection during asymmetric cell division, indicating a role of TOR signalling in regulation of the budding pattern. Torin2 treated yeast cells exhibit increased expression of metalloreductases which affects iron homeostasis leading to iron toxicity. Notably, the enhanced expression of TOR1 and TOR2 rescue the Torin2 augmented iron toxicity of yeast cell. This study has revealed novel conduits and our results suggest that using Torin2 will enable the dissection of TORC2 mediated functions of the TOR signalling pathway.

HER2+ breast cancer therapy: by CPP-ZFN mediated targeting of mTOR?

A significant fraction of HER2+ patients develop resistance to available therapies such as trastuzumab. The acquired resistance is primarily due to hyper activation of HER2 downstream PI3K/Akt/mTOR signalling pathway. Hence, identification of inhibitors of components of this pathway, particularly mTOR, is an area of intense investigation. Interestingly, mTOR specific inhibitors (rapamycin/rapalogs) have been tested and shown to potentiate the effect of HER2 inhibitors. However, the use of mTOR inhibitors will also be associated with the limitations inherently linked with extensive use of anticancer drugs e.g., toxicity and acquired drug resistance. Hereby, we hypothesize development of an alternative novel molecular therapeutic intervention based on cell penetrating peptide (CPP), a highly efficient carrier, conjugated to zinc finger nuclease (ZFN), a precise molecular scissor. The use of HER2 specific CPP conjugated to mTOR specific ZFN, will make the mTOR locus non-functional and inhibit the PI3K/Akt/mTOR pathway, essential for growth and proliferation of cancerous cells. With the availability of HER21 cancerous cell specific CPP and proved applications of ZFN in targeted genome engineering of over 11 species, the prospects of success of CPP-ZFN anti-cancer therapy are very high.

Critical role of RPI1 in the stress tolerance of yeast during ethanolic fermentation.

Stress tolerance of yeast Saccharomyces cerevisiae during ethanolic fermentation is poorly understood due to the lack of genetic screens and conventional plate assays for studying this phenotype. We screened a genomic expression library of yeast to identify gene(s) that, upon overexpression, would prolong the survival of yeast cells during fermentation, with the view to understand the stress response better and to use the identified gene(s) in strain improvement. The yeast RPI1 (Ras-cAMP pathway inhibitor 1) gene was identified in such a screen performed at 38 degrees C; introducing an additional copy of RPI1 with its native promoter helped the cells to retain their viability by over 50-fold better than the wild type (WT) parent strain, after 36 h of fermentation at 38 degrees C. Disruption of RPI1 resulted in a drastic reduction in viability during fermentation, but not during normal growth, further confirming the role of this gene in fermentation stress tolerance. This gene seems to improve viability by fortifying the yeast cell wall, because RPI1 overexpression strain is highly resistant to cell lytic enzyme zymolyase, compared with the WT strain. As the RPI1 overexpression strain substantially retains cell viability at the end of fermentation, the cells can be reused in the subsequent round of fermentation, which is likely to facilitate economical production of ethanol.

Human protein phosphatase PP6 regulatory subunits provide Sit4-dependent and rapamycin-sensitive sap function in Saccharomyces cerevisiae.

In the budding yeast Saccharomyces cerevisiae the protein phosphatase Sit4 and four associated proteins (Sap4, Sap155, Sap185, and Sap190) mediate G(1) to S cell cycle progression and a number of signaling events controlled by the target of rapamycin TOR signaling cascade. Sit4 and the Sap proteins are ubiquitously conserved and their human orthologs, PP6 and three PP6R proteins, share significant sequence identity with their yeast counterparts. However, relatively little is known about the functions of the PP6 and PP6R proteins in mammalian cells. Here we demonstrate that the human PP6R proteins physically interact with Sit4 when expressed in yeast cells. Remarkably, expression of PP6R2 and PP6R3 but not expression of PP6R1 rescues the growth defect and rapamycin hypersensitivity of yeast cells lacking all four Saps, and these effects require Sit4. Moreover, PP6R2 and PP6R3 enhance cyclin G(1) gene expression and DNA synthesis, and partially abrogate the G(1) cell cycle delay and the budding defect of the yeast quadruple sap mutant strain. In contrast, the human PP6R proteins only modestly support nitrogen catabolite gene expression and are unable to restore normal levels of eIF2alpha phosphorylation in the quadruple sap mutant strain. These results illustrate that the human PP6-associated proteins are capable of providing distinct rapamycin-sensitive and Sit4-dependent Sap functions in the heterologous context of the yeast cell. We hypothesize that the human Saps may play analogous roles in mTORC1-PP6 signaling events in metazoans.

Nutritional control via Tor signaling in Saccharomyces cerevisiae.

The yeast Saccharomyces cerevisiae senses and responds to nutrients by adapting its growth rate and undergoing morphogenic transitions to ensure survival. The Tor pathway is a major integrator of nutrient-derived signals that in coordination with other signaling pathways orchestrates cell growth. Recent advances have identified novel Tor kinase substrates and established the protein trafficking membranous network and the nucleus as platforms for Tor signaling. These and other recent findings delineate distinct signaling branches emanating from membrane-associated Tor complexes to control cell growth.

Nuclear translocation of Gln3 in response to nutrient signals requires Golgi-to-endosome trafficking in Saccharomyces cerevisiae.

The yeast Saccharomyces cerevisiae has developed specialized mechanisms that enable growth on suboptimal nitrogen sources. Exposure of yeast cells to poor nitrogen sources or treatment with the Tor kinase inhibitor rapamycin elicits activation of Gln3 and transcription of nitrogen catabolite-repressed (NCR) genes whose products function in scavenging and metabolizing nitrogen. Here, we show that mutations in class C and D Vps components, which mediate Golgi-to-endosome vesicle transport, impair nuclear translocation of Gln3, NCR gene activation, and growth in poor nitrogen sources. In nutrient-replete conditions, a significant fraction of Gln3 is peripherally associated with light membranes and partially colocalizes with Vps10-containing foci. These results reveal a role for Golgi-to-endosome vesicular trafficking in TORC1-controlled nuclear translocation of Gln3 and support a model in which Tor-mediated signaling in response to nutrient cues occurs in these compartments. These findings have important implications for nutrient sensing and growth control via mTor pathways in metazoans.

Rapamycin bypasses vesicle-mediated signaling events to activate Gln3 in Saccharomyces cerevisiae.

Growth of Saccharomyces cerevisiae in poor nitrogen sources or exposure to the Tor inhibitor rapamycin results in expression of the nitrogen catabolite repressed (NCR) genes whose products are involved in scavenging and metabolizing nitrogen. The NCR genes are regulated by the GATA-like transactivators Gln3 and Gat1, which are thought to be under control of the rapamycin-sensitive Tor complex 1 (TORC1). We have recently shown that Gln3 nuclear translocation in response to nitrogen source quality but not in response to rapamycin requires Golgi to endosome trafficking. These and previous findings that several TORC1 components localize to low density endomembranes are discussed in a model that underscores a prominent role for the vesicular trafficking system in facilitating molecular interactions in response to nitrogen source. In addition, these findings have important implications for Tor signaling and rapamycin mechanism of action, both in yeast and in metazoans.

Efficient Tor signaling requires a functional class C Vps protein complex in Saccharomyces cerevisiae.

The Tor kinases regulate responses to nutrients and control cell growth. Unlike most organisms that only contain one Tor protein, Saccharomyces cerevisiae expresses two, Tor1 and Tor2, which are thought to share all of the rapamycin-sensitive functions attributable to Tor signaling. Here we conducted a genetic screen that defined the global TOR1 synthetic fitness or lethal interaction gene network. This screen identified mutations in distinctive functional categories that impaired vacuolar function, including components of the EGO/Gse and PAS complexes that reduce fitness. In addition, tor1 is lethal in combination with mutations in class C Vps complex components. We find that Tor1 does not regulate the known function of the class C Vps complex in protein sorting. Instead class C vps mutants fail to recover from rapamycin-induced growth arrest or to survive nitrogen starvation and have low levels of amino acids. Remarkably, addition of glutamate or glutamine restores viability to a tor1 pep3 mutant strain. We conclude that Tor1 is more effective than Tor2 at providing rapamycin-sensitive Tor signaling under conditions of amino acid limitation, and that an intact class C Vps complex is required to mediate intracellular amino acid homeostasis for efficient Tor signaling.