Conformational Differences between Open and Closed States of the Eukaryotic Translation Initiation Complex.

Translation initiation in eukaryotes begins with the formation of a pre-initiation complex (PIC) containing the 40S ribosomal subunit, eIF1, eIF1A, eIF3, ternary complex (eIF2-GTP-Met-tRNAi), and eIF5. The PIC, in an open conformation, attaches to the 5' end of the mRNA and scans to locate the start codon, whereupon it closes to arrest scanning. We present single particle cryo-electron microscopy (cryo-EM) reconstructions of 48S PICs from yeast in these open and closed states, at 6.0 Å and 4.9 Å, respectively. These reconstructions show eIF2ß as well as a configuration of eIF3 that appears to encircle the 40S, occupying part of the subunit interface. Comparison of the complexes reveals a large conformational change in the 40S head from an open mRNA latch conformation to a closed one that constricts the mRNA entry channel and narrows the P site to enclose tRNAi, thus elucidating key events in start codon recognition.

eIF2α interactions with mRNA control accurate start codon selection by the translation preinitiation complex.

In translation initiation, AUG recognition triggers rearrangement of the 48S preinitiation complex (PIC) from an open conformation to a closed state with more tightly-bound Met-tRNAi. Cryo-EM structures have revealed interactions unique to the closed complex between arginines R55/R57 of eIF2α with mRNA, including the -3 nucleotide of the 'Kozak' context. We found that R55/R57 substitutions reduced recognition of a UUG start codon at HIS4 in Sui- cells (Ssu- phenotype); and in vitro, R55G-R57E accelerated dissociation of the eIF2·GTP·Met-tRNAi ternary complex (TC) from reconstituted PICs with a UUG start codon, indicating destabilization of the closed complex. R55/R57 substitutions also decreased usage of poor-context AUGs in SUI1 and GCN4 mRNAs in vivo. In contrast, eIF2α-R53 interacts with the rRNA backbone only in the open complex, and the R53E substitution enhanced initiation at a UUG codon (Sui- phenotype) and poor-context AUGs, while reducing the rate of TC loading (Gcd- phenotype) in vivo. Consistently, R53E slowed TC binding to the PIC while decreasing TC dissociation at UUG codons in vitro, indicating destabilization of the open complex. Thus, distinct interactions of eIF2α with rRNA or mRNA stabilize first the open, and then closed, conformation of the PIC to influence the accuracy of initiation in vivo.

Foam-Based Drug Delivery: A Newer Approach for Pharmaceutical Dosage Form.

Foam is a dispersal of gas in solid foam or liquid foam. Solid foams are generally formed through quick curing of liquid foams. Because of their positive characteristics, simplicity of application, and improved patient acceptability/compliance, foams are an appealing and promising delivery strategy for medical, cosmetic, and pharmaceutical, applications. Recent breakthroughs in topical foams for cosmetic and dermal applications are described here, as well as categorization based on foam formulation and recent assessment techniques of critical physical properties of the topical foam. Despite the expanding amount of knowledge on topical foams for cutaneous applications, the majority of research has focused on the stability and structure of foam in contact with solid nonporous surfaces. It is still difficult to figure out how such foams destabilize when they come into touch with porous surfaces like skin or skin-like membranes. The foam-covered wide surface area and easily applied to the affected area. This type of delivery system also eliminates the chance of secondary infection that is associated with ointment and creams, which need to be applied to the affected area with help of fingers or an applicator.

A network of eIF2ß interactions with eIF1 and Met-tRNAi promotes accurate start codon selection by the translation preinitiation complex.

In translation initiation, a 43S preinitiation complex (PIC) containing eIF1 and a ternary complex (TC) of GTP-bound eIF2 and Met-RNAi scans the mRNA for the start codon. AUG recognition triggers eIF1 release and rearrangement from an open PIC conformation to a closed state with more tightly-bound Met-tRNAi (PIN state). Cryo-EM models reveal eIF2ß contacts with eIF1 and Met-tRNAi exclusive to the open complex that should destabilize the closed state. eIF2ß or eIF1 substitutions disrupting these contacts increase initiation at UUG codons, and compound substitutions also derepress translation of GCN4, indicating slower TC recruitment. The latter substitutions slow TC loading while stabilizing TC binding at UUG codons in reconstituted PICs, indicating a destabilized open complex and shift to the closed/PIN state. An eIF1 substitution that should strengthen the eIF2ß:eIF1 interface has the opposite genetic and biochemical phenotypes. eIF2ß is also predicted to restrict Met-tRNAi movement into the closed/PIN state, and substitutions that should diminish this clash increase UUG initiation in vivo and stabilize Met-tRNAi binding at UUG codons in vitro with little effect on TC loading. Thus, eIF2ß anchors eIF1 and TC to the open complex, enhancing PIC assembly and scanning, while impeding rearrangement to the closed conformation at non-AUG codons.

eIF1 Loop 2 interactions with Met-tRNAi control the accuracy of start codon selection by the scanning preinitiation complex.

The eukaryotic 43S preinitiation complex (PIC), bearing initiator methionyl transfer RNA (Met-tRNAi) in a ternary complex (TC) with eukaryotic initiation factor 2 (eIF2)-GTP, scans the mRNA leader for an AUG codon in favorable context. AUG recognition evokes rearrangement from an open PIC conformation with TC in a "POUT" state to a closed conformation with TC more tightly bound in a "PIN" state. eIF1 binds to the 40S subunit and exerts a dual role of enhancing TC binding to the open PIC conformation while antagonizing the PIN state, necessitating eIF1 dissociation for start codon selection. Structures of reconstituted PICs reveal juxtaposition of eIF1 Loop 2 with the Met-tRNAi D loop in the PIN state and predict a distortion of Loop 2 from its conformation in the open complex to avoid a clash with Met-tRNAi We show that Ala substitutions in Loop 2 increase initiation at both near-cognate UUG codons and AUG codons in poor context. Consistently, the D71A-M74A double substitution stabilizes TC binding to 48S PICs reconstituted with mRNA harboring a UUG start codon, without affecting eIF1 affinity for 40S subunits. Relatively stronger effects were conferred by arginine substitutions; and no Loop 2 substitutions perturbed the rate of TC loading on scanning 40S subunits in vivo. Thus, Loop 2-D loop interactions specifically impede Met-tRNAi accommodation in the PIN state without influencing the POUT mode of TC binding; and Arg substitutions convert the Loop 2-tRNAi clash to an electrostatic attraction that stabilizes PIN and enhances selection of poor start codons in vivo.

Rps3/uS3 promotes mRNA binding at the 40S ribosome entry channel and stabilizes preinitiation complexes at start codons.

The eukaryotic 43S preinitiation complex (PIC) bearing Met-tRNAiMet in a ternary complex (TC) with eukaryotic initiation factor (eIF)2-GTP scans the mRNA leader for an AUG codon in favorable "Kozak" context. AUG recognition provokes rearrangement from an open PIC conformation with TC bound in a state not fully engaged with the P site ("POUT") to a closed, arrested conformation with TC tightly bound in the "PIN" state. Yeast ribosomal protein Rps3/uS3 resides in the mRNA entry channel of the 40S subunit and contacts mRNA via conserved residues whose functional importance was unknown. We show that substitutions of these residues reduce bulk translation initiation and diminish initiation at near-cognate UUG start codons in yeast mutants in which UUG selection is abnormally high. Two such substitutions-R116D and R117D-also increase discrimination against an AUG codon in suboptimal Kozak context. Consistently, the Arg116 and Arg117 substitutions destabilize TC binding to 48S PICs reconstituted in vitro with mRNA harboring a UUG start codon, indicating destabilization of the closed PIN state with a UUG-anticodon mismatch. Using model mRNAs lacking contacts with either the mRNA entry or exit channels of the 40S subunit, we demonstrate that Arg116/Arg117 are crucial for stabilizing PIC-mRNA contacts at the entry channel, augmenting the function of eIF3 at both entry and exit channels. The corresponding residues in bacterial uS3 promote the helicase activity of the elongating ribosome, suggesting that uS3 contacts with mRNA enhance multiple phases of translation across different domains of life.

Comparison and evaluation of single-use LMA supreme versus the reusable proseal LMA in paralyzed patients undergoing surgery with controlled ventilation.

BACKGROUND AND AIMS: The objective of this prospective randomized blinded study was to assess the safety and efficacy of the laryngeal mask airway (LMA) Supreme as compared with the LMA Proseal. MATERIAL AND METHODS: A total of 60 patients were randomised into two groups to either receive a Proseal LMA (PLMA) or Supreme LMA (SLMA) for airway management. The primary outcome was to measure oropharyngeal leak pressure (OLP) in both groups. The secondary outcomes were the measurement of insertion time, insertion success rate, fibreoptic grading, intracuff pressure, ease of ventilation, and airway pressure on standard ventilatory settings and postoperative complications. RESULTS: Intracuff pressure increase after 60 minutes of induction was significantly higher in the PLMA group (PLMA 97.43 ± 11.03 cm of H2O and SLMA 75.17 ± 8.95 cm of H2O). OLP was recorded after device insertion, after 30 min and after 60 min in each group and was found to be 28.71 ± 2.97, 30.93 ± 2.87, and 31.93 ± 2.72 cm of H2O in PLMA and 24.84 ± 2.08, 26.73 ± 2.26, and 27.95 ± 2.55 cm of H2O in SLMA group, respectively. The mean OLP with the SLMA was significantly (p=<.001) lower than PLMA. All the other parameters were comparable in both groups. CONCLUSION: PLMA is better than SLMA as airway device to ventilate at higher airway pressure in paralyzed adult patients. On the basis of our study, we recommend Proseal over Supreme LMA.

Two dimensional allotropes of arsenene with a wide range of high and anisotropic carrier mobility.

Considering the rapid development of experimental techniques for fabricating 2D materials in recent years, various monolayers are expected to be experimentally realized in the near future. Motivated by the recent research activities focused on the honeycomb arsenene monolayers, the stability and carrier mobility of non-honeycomb and porous allotropic arsenene are determined using first principles calculations. In addition to five honeycomb structures of arsenene, a total of eight other structures are considered in this study. An extensive analysis comprising energetics, phonon spectra and mechanical properties confirms that these structures are energetically and dynamically stable. All these structures are semiconductors with a broad range of band gaps varying from ∼1 eV to ∼2.5 eV. Significantly, these monolayer allotropes possess anisotropic carrier mobilities as high as several hundred cm2 V-1 s-1 which is comparable with well-known 2D materials such as black phosphorene and monolayer MoS2. Combining such broad band gaps and superior carrier mobilities, these monolayer allotropes can be promising candidates for the superior performance of the next generation nanoscale devices. We further explore these monolayer allotropes for photocatalytic water splitting and find that arsenene monolayers have potential for usage in visible light driven photocatalytic water splitting.

Substrate specificity and mapping of residues critical for transport in the high-affinity glutathione transporter Hgt1p.

The high-affinity glutathione transporter Hgt1p of Saccharomyces cerevisiae belongs to a relatively new and structurally uncharacterized oligopeptide transporter (OPT) family. To understand the structural features required for interaction with Hgt1p, a quantitative investigation of substrate specificity of Hgt1p was carried out. Hgt1p showed a higher affinity for reduced glutathione (GSH), whereas it transported oxidized glutathione (GSSG) and other glutathione conjugates with lower affinity. To identify the residues of Hgt1p critical for substrate binding and translocation, all amino acid residues of the 13 predicted transmembrane domains (TMDs) have been subjected to mutagenesis. Functional evaluation of these 269 mutants by growth and biochemical assay followed by kinetic analysis of the severely defective mutants including previous mutagenic studies on this transporter have led to the identification of N124 (TMD1), V185 (TMD3), Q222, G225 and Y226 (TMD4), P292 (TMD5), Y374 (TMD6), L429 (TMD7) and F523 and Q526 (TMD9) as critical for substrate binding with at least 3-fold increase in Km upon mutagenesis to alanine. In addition residues Y226 and Y374 appeared to be important for differential substrate specificity. An ab initio model of Hgt1p was built and refined using these mutagenic data that yielded a helical arrangement that includes TMD3, TMD4, TMD5, TMD6, TMD7, TMD9 and TMD13 as pore-lining helices with the functionally important residues in a channel-facing orientation. Taken together the results of this study provides the first mechanistic insights into glutathione transport by a eukaryotic high-affinity glutathione transporter.

Ophthalmic diseases and disorders in free-ranging rhesus macaque (Macaca mulatta) of Shivalik hill area of Himachal Pradesh, Northern India.

BACKGROUND: The studies on ophthalmic diseases and disorders in a population of rhesus macaques, in particular with free-ranging macaques, are limited mainly by the difficulty in capturing animals and obtaining samples. METHODS: From October 2011 to 2013, prevalence of various ophthalmic affections was recorded and analyzed on the basis of sex, age-group, and disease condition both in urban and peri-urban free-ranging rhesus macaques (Macaca mulatta) from various locations in Shivalik hill areas of Himachal Pradesh in northern India, as a part of clinical health examination. RESULTS AND CONCLUSION: Ophthalmic diseases were more prevalent in macaques captured from urban settings than those from peri-urban, and difference between two groups was statistically significant (P < 0.01). Further classification of ophthalmic diseases revealed that traumatic injuries, corneal opacity, and cataract were significantly higher in urban than those in peri-urban macaque (P < 0.01). Similarly, the ophthalmic disorders among different age-groups were also found statistically significant (P < 0.01). The effect of sex on ophthalmic disorders was not found significant. Overviewing these results, disturbances in environment, close proximity to humans, and increasing age may lead to ophthalmic occurrences in these free-range macaques.

Glutathione transporters.

BACKGROUND: Glutathione (GSH) is synthesized in the cytoplasm but there is a requirement for glutathione not only in the cytoplasm, but in the other organelles and the extracellular milieu. GSH is also imported into the cytoplasm. The transports of glutathione across these different membranes in different systems have been biochemically demonstrated. However the molecular identity of the transporters has been established only in a few cases. SCOPE OF REVIEW: An attempt has been made to present the current state of knowledge of glutathione transporters from different organisms as well as different organelles. These include the most well characterized transporters, the yeast high-affinity, high-specificity glutathione transporters involved in import into the cytoplasm, and the mammalian MRP proteins involved in low affinity glutathione efflux from the cytoplasm. Other glutathione transporters that have been described either with direct or indirect evidences are also discussed. MAJOR CONCLUSIONS: The molecular identity of a few glutathione transporters has been unambiguously established but there is a need to identify the transporters of other systems and organelles. There is a lack of direct evidence establishing transport by suggested transporters in many cases. Studies with the high affinity transporters have led to important structure-function insights. GENERAL SIGNIFICANCE: An understanding of glutathione transporters is critical to our understanding of redox homeostasis in living cells. By presenting our current state of understanding and the gaps in our knowledge the review hopes to stimulate research in these fields. This article is part of a Special Issue entitled Cellular functions of glutathione.

Structure and genetic diversity of natural populations of Morus alba in the trans-Himalayan Ladakh region.

Sequence-related amplified polymorphism markers were used to assess the genetic structure in three natural populations of Morus alba from trans-Himalaya. Multilocation sampling was conducted across 14 collection sites. The overall genetic diversity estimates were high: percentage polymorphic loci 89.66%, Nei's gene diversity 0.2286, and Shannon's information index 0.2175. At a regional level, partitioning of variability assessed using analysis of molecular variance (AMOVA), revealed 80% variation within and 20% among collection sites. Pattern appeared in STRUCTURE, BARRIER, and AMOVA, clearly demonstrating gene flow between the Indus and Suru populations and a geographic barrier between the Indus-Suru and Nubra populations, which effectively hinders gene flow. The results showed significant genetic differentiation, population structure, high to restricted gene flow, and high genetic diversity. The assumption that samples collected from the three valleys represent three different populations does not hold true. The fragmentation present in trans-Himalaya was more natural and less anthropogenic.

QbD-driven development of phospholipid-embedded lipidic nanocarriers of raloxifene: extensive in vitro and in vivo evaluation studies.

Raloxifene (RLX) is popularly indicated in treatment of osteoporosis and prevention of breast cancer. Owing to its poor aqueous solubility, high pre-systemic metabolism, intestinal glucuronidation, and P-glycoprotein (P-gp) efflux, however, it demonstrates low (< 2%) and inconsistent oral bioavailability. The current work, Quality by Design (QbD)-driven development of phospholipid-embedded nanostructured lipidic carriers (NLCs) of RLX, accordingly, was undertaken to potentiate its lymphatic uptake, augment oral bioavailability, and possibly reduce drug dosage. Factor screening and failure mode effect analysis (FMEA) studies were performed to delineate high-risk factors using solid lipid (glyceryl monostearate), liquid lipid (vitamin E), and surfactant (Tween 80). Response surface optimization studies were performed employing the Box-Behnken design. Mathematical and graphical methods were adopted to embark upon the selection of optimized NLCs with various critical quality attributes (CQAs) of mean particle size as 186 nm, zeta potential of - 23.6 mV, entrapment efficiency of 80.09%, and cumulative drug release at 12 h of 83.87%. The DSC and FTIR studies, conducted on optimized NLCs, indicated successful entrapment of drug into the lipid matrix. In vitro drug release studies demonstrated Fickian diffusion mechanism. In vivo pharmacokinetic studies in rats construed significant improvement in AUC0-72 h (4.48-folds) and in Cmax (5.11-folds), unequivocally indicating markedly superior (p < 0.001) oral bioavailability of RLX-NLCs vis-à-vis marketed tablet formulation. Subsequently, level "A" in vitro/in vivo correlation (IVIVC) was also successfully attempted between the percentages of in vitro drug dissolved and of in vivo drug absorbed at the matching time points. In vitro cytotoxicity and cellular uptake studies also corroborated higher efficacy and successful localization of coumarin-6-loaded NLCs into MG-63 cells through microfluidic channels.

Tofacitinib in dermatology: a potential opportunity for topical applicability through novel drug-delivery systems.

Tofacitinib is a first-generation JAK inhibitor approved by the US FDA for treating rheumatoid arthritis. It exhibits a broad-spectrum inhibitory effect with abilities to block JAK-STAT signalling. The primary objective of this review is to obtain knowledge about cutting-edge methods for effectively treating a variety of skin problems by including tofacitinib into formulations that are based on nanocarriers. The review also highlights clinical trials and offers an update on published clinical patents. Nanocarriers provide superior performance compared to conventional treatments in terms of efficacy, stability, drug bioavailability, target selectivity and sustained drug release. Current review has the potential to make significant contributions to the ongoing discussion involving dermatological treatments and the prospective impact of nanotechnology on transforming healthcare within this field.

Mutations in the N-terminal region of the Schizosaccharomyces pombe glutathione transporter pgt1⁺ allows functional expression in Saccharomyces cerevisiae.

Pgt1p encodes a glutathione transporter in Schizosaccharomyces pombe, orthologous to the Saccharomyces cerevisiae glutathione transporter, Hgt1p. Despite high similarity to Hgt1p, Pgt1p failed to display functionality during heterologous expression in S. cerevisiae. In the present study we employed a genetic strategy to investigate the reason behind the non-functionality of pgt1⁺ in S. cerevisiae. Functional mutants were isolated after in vitro mutagenesis. Several mutants were obtained and four mutants analysed. Among these, three yielded different point mutations in the N-terminal region (301-350 bp) of the transporter before the first transmembrane domain, while one mutant contained a deletion of 42 nucleotides within the same region. The mutant pgt1⁺ proteins not only expressed and localized correctly, but displayed high-affinity glutathione transport capabilities in S. cerevisae. Comparison of wild-type pgt1⁺ with the functional mutants revealed that a loss in protein expression was responsible for lack of functionality of wild-type pgt1⁺ in S. cerevisiae. The mRNA levels in wild-type and mutants were comparable, suggesting that the block was in translation. The formation of a strong stem-loop structure appeared to be responsible for inefficient translation in pgt1⁺ and disruption of these structures in the mutants was probably permitting translation. This was confirmed by making silent mutations in this region of wild-type pgt1⁺, which led to their functionality in S. cerevisiae. This genetic strategy to relieve functional blocks in expression should greatly facilitate the study of these and other transporters from more intractable genetic organisms in a heterologous expression system.

Intrathecal clonidine as an adjuvant to hyperbaric bupivacaine in patients undergoing inguinal herniorrhaphy: A randomized double-blinded study.

BACKGROUND: Clonidine is added to intrathecal local anesthetics to improve intraoperative analgesia and to increase the duration of sensory and motor block. Aim of this study was to evaluate and compare the effects of addition of two different doses of clonidine (15 and 30 mcg) to 11 mg hyperbaric bupivacaine in patients undergoing inguinal herniorrhaphy surgery under spinal anesthesia. MATERIALS AND METHODS: Seventy-five patients enrolled in the study were randomly divided into three groups of 25 each. Group I patients received 11 mg hyperbaric bupivacaine, whereas groups II and III received 15 mcg and 30 mcg clonidine, respectively, as an adjuvant to 11 mg hyperbaric bupivacaine. The volume of solution was kept constant to 2.4 ml by adding saline wherever needed. RESULTS: Highest level of sensory block, time to achieve this level, and highest Bromage scale recorded were comparable among the groups. The mean time to two-segment regression, regression of sensory block to L3 dermatome, and mean duration of motor block were the greatest in group III followed by group II and group I. There was significant fall in mean arterial pressure (MAP) in groups II and III as compared to group I (P = 0.04). Episodes of hypotension were more in group III than in group II. CONCLUSION: 30 mcg clonidine was associated with more incidence and duration of hypotension than 15 µg of clonidine. 15 mcg clonidine added to 11 mg hyperbaric bupivacaine provides better sensory and motor blockade for inguinal herniorrhaphy.

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.

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.

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.

Glutathione utilization by Candida albicans requires a functional glutathione degradation (DUG) pathway and OPT7, an unusual member of the oligopeptide transporter family.

Candida albicans lacks the ability to survive within its mammalian host in the absence of endogenous glutathione biosynthesis. To examine the ability of this yeast to utilize exogenous glutathione, we exploited the organic sulfur auxotrophy of C. albicans met15Δ strains. We observed that glutathione is utilized efficiently by the alternative pathway of glutathione degradation (DUG pathway). The major oligopeptide transporters OPT1-OPT5 of C. albicans that were most similar to the known yeast glutathione transporters were not found to contribute to glutathione transport to any significant extent. A genomic library approach to identify the glutathione transporter of C. albicans yielded OPT7 as the primary glutathione transporter. Biochemical studies on OPT7 using radiolabeled GSH uptake revealed a K(m) of 205 µm, indicating that it was a high affinity glutathione transporter. OPT7 is unusual in several aspects. It is the most remote member to known yeast glutathione transporters, lacks the two highly conserved cysteines in the family that are known to be crucial in trafficking, and also has the ability to take up tripeptides. The transporter was regulated by sulfur sources in the medium. OPT7 orthologues were prevalent among many pathogenic yeasts and fungi and formed a distinct cluster quite remote from the Saccharomyces cerevisiae HGT1 glutathione transporter cluster. In vivo experiments using a systemic model of candidiasis failed to detect expression of OPT7 in vivo, and strains disrupted either in the degradation (dug3Δ) or transport (opt7Δ) of glutathione failed to show a defect in virulence.