HERV1-env Induces Unfolded Protein Response Activation in Autoimmune Liver Disease: A Potential Mechanism for Regulatory T Cell Dysfunction.

Regulatory T cells (Tregs) are not terminally differentiated but can acquire effector properties. Here we report an increased expression of human endogenous retrovirus 1 (HERV1-env) proteins in Tregs of patients with de novo autoimmune hepatitis and autoimmune hepatitis, which induces endoplasmic reticulum (ER) stress. HERV1-env-triggered ER stress activates all three branches (IRE1, ATF6, and PERK) of the unfolded protein response (UPR). Our coimmunoprecipitation studies show an interaction between HERV1-env proteins and the ATF6 branch of the UPR. The activated form of ATF6α activates the expression of RORC and STAT3 by binding to promoter sequences and induces IL-17A production. Silencing of HERV1-env results in recovery of Treg suppressive function. These findings identify ER stress and UPR activation as key factors driving Treg plasticity (species: human).

Carbon substrates promotes stress resistance and drug tolerance in clinical isolates of Candida tropicalis.

Candida tropicalis is a human pathogen and one of the most prevalent non-Candida albicans Candida (NCAC) species causing invasive infections. Azole antifungal resistance in C. tropicalis is also gradually increasing with the increasing incidence of infections. The pathogenic success of C. tropicalis depends on its effective response in the host microenvironment. To become a successful pathogen, cellular metabolism, and physiological status determine the ability of the pathogen to counter diverse stresses inside the host. However, to date, limited knowledge is available on the impact of carbon substrate metabolism on stress adaptation and azole resistance in C. tropicalis. In this study, we determined the impact of glucose, fructose, and sucrose as the sole carbon source on the fluconazole resistance and osmotic (NaCl), oxidative (H2O2) stress adaptation in C. tropicalis clinical isolates. We confirmed that the abundance of carbon substrates influences or increases drug resistance and osmotic and oxidative stress tolerance in C. tropicalis. Additionally, both azole-resistant and susceptible isolates showed similar stress adaptation phenotypes, confirming the equal efficiency of becoming successful pathogens irrespective of drug susceptibility profile. To the best of our knowledge, our study is the first on C. tropicalis to demonstrate the direct relation between carbon substrate metabolism and stress tolerance or drug resistance.

BAFF antagonism via the BAFF receptor 3 binding site attenuates BAFF 60-mer-induced classical NF-κB signaling and metabolic reprogramming of B cells.

Human recombinant B cell activating factor (BAFF) is secreted as 3-mers, which can associate to form 60-mers in culture supernatants. However, the presence of BAFF multimers in humans is still debated and it is incompletely understood how BAFF multimers activate the B cells. Here, we demonstrate that BAFF can exist as 60-mers or higher order multimers in human plasma. In vitro, BAFF 60-mer strongly induced the transcriptome of B cells which was partly attenuated by antagonism using a soluble fragment of BAFF receptor 3. Furthermore, compared to BAFF 3-mer, BAFF 60-mer strongly induced a transient classical and prolonged alternate NF-κB signaling, glucose oxidation by both aerobic glycolysis and oxidative phosphorylation, and succinate utilization by mitochondria. BAFF antagonism selectively attenuated classical NF-κB signaling and glucose oxidation. Altogether, our results suggest critical roles of BAFF 60-mer and its BAFF receptor 3 binding site in hyperactivation of B cells.

SARS-CoV-2 Infection in Pediatric Solid Organ Transplant Recipients: A Single Center Observation.

OBJECTIVES: This is a descriptive study to characterize rates of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection in pediatric solid organ transplant (SOT) recipients during the early days of the pandemic. We hypothesized that asymptomatic infection may represent a large proportion of SARS-CoV-2 infection in pediatric SOT recipients. METHODS: We queried Organ Transplant Tracking Record (OTTR) for all pediatric SOT recipients followed at our center and reviewed medical records to identify patients tested for SARS-CoV-2 between March 15, 2020 and June 30, 2021. Patients were tested by polymerase chain reaction (PCR): prior to planned procedures or because of symptoms; OR: tested by measurement of IgG to spike protein with their routine labs q 2-monthly. A positive PCR was called acute infection. A positive IgG with negative PCR was called convalescence. For immunologic studies, blood was obtained when the PCR or IgG was positive. Statistical comparisons were made between (1) acute infection versus convalescence; (2) acute infection versus SOT recipients without infection (called healthy controls); (3) liver transplant (LT) versus small bowel (SB)/multivisceral transplant (MVT); (4) positive versus negative test result. RESULTS: Of 257 LT recipients, 99 were tested: 6 were PCR positive, 13 were antibody positive. Of 150 SB/MVT recipients, 55 were tested: 4 were PCR positive, 6 were antibody positive. Of 8 simultaneous liver, kidney transplant recipients, 3 were tested: 1 was PCR positive. Symptoms when present were mostly mild. Patients with a positive test result were younger (6.3 vs 10.0 years; P = 0.017). We observed a rapid decline in viral load within 96 hours without a change in immunosuppression. Antibody lasted >8 months beyond the time it was monitored. Acute infection was associated with increased CD4 and CD8 T EM cell frequency ( P = 0.04, P = 0.03, respectively), decreased interferon (IFN)-γ production from T-cells (2.8% vs 11.3%; P = 0.006), and decreased CD8 TEMRA frequency (4.56% vs 11.70%; P = 0.006). CONCLUSIONS: Early in the pandemic, COVID-19 disease was mostly mild in pediatric SOT recipients with no rejection, patient death, or graft loss observed.

Matrix-assisted laser desorption/ionisation-time of flight mass spectrometry: Protocol standardisation, comparison and database expansion for faster and reliable identification of dermatophytes.

BACKGROUND: Accurate and early identification of dermatophytes enables prompt antifungal therapy. However, phenotypic and molecular identification methods are time-consuming. MALDI-TOF MS-based identification is rapid, but an optimum protocol is not available. OBJECTIVES: To develop and validate an optimum protein extraction protocol for the efficient and accurate identification of dermatophytes by MALDI-TOF MS. MATERIALS/METHODS: Trichophyton mentagrophytes complex (n = 4), T. rubrum (n = 4) and Microsporum gypseum (n = 4) were used for the optimisation of protein extraction protocols. Thirteen different methods were evaluated. A total of 125 DNA sequence confirmed clinical isolates of dermatophytes were used to create and expand the existing database. The accuracy of the created database was checked by visual inspection of MALDI spectra, MSP dendrogram and composite correlation index matrix analysis. The protocol was validated further using 234 isolates. RESULT: Among 13 protein extraction methods, six correctly identified dermatophytes but with a low log score (≤1.0). The modified extraction protocol developed provided an elevated log score of 1.6. Significant log score difference was observed between the modified protocol and other existing protocols (T. mentagrophytes complex: 1.6 vs. 0.2-1.0, p < .001; T. rubrum: 1.6 vs. 0.4-1.0, p < .001; M. gypseum:1.6 vs. 0.2-1.0, p < .001). Expansion of the database enabled the identification of all 234 isolates (73.5% with log score ≥2.0 and 26.4% with log scores range: 1.75-1.99). The results were comparable to DNA sequence-based identification. CONCLUSION: MALDI-TOF MS with an updated database and efficient protein extraction protocol developed in this study can identify dermatophytes accurately and also reduce the time for identifying them.

Regulation of physiological aspects in plants by hydrogen sulfide and nitric oxide under challenging environment.

Plants are exposed to a plethora of abiotic stresses such as drought, salinity, heavy metal and temperature stresses at different stages of their life cycle, from germination to seedling till the reproductive phase. As protective mechanisms, plants release signaling molecules that initiate a cascade of stress-signaling events, leading either to programmed cell death or plant acclimation. Hydrogen sulfide (H2 S) and nitric oxide (NO) are considered as new 'gasotransmitter' molecules that play key roles in regulating gene expression, posttranslational modification (PTM), as well as cross-talk with other hormones. Although the exact role of NO in plants remains unclear and is species dependent, various studies have suggested a positive correlation between NO accumulation and environmental stress in plants. These molecules are also involved in a large array of stress responses and act synergistically or antagonistically as signaling components, depending on their respective concentration. This study provides a comprehensive update on the signaling interplay between H2 S and NO in the regulation of various physiological processes under multiple abiotic stresses, modes of action and effects of exogenous application of these two molecules under drought, salt, heat and heavy metal stresses. However, the complete picture of the signaling cascades mediated by H2 S and NO is still elusive. Recent researches indicate that during certain plant processes, such as stomatal closure, H2 S could act upstream of NO signaling or downstream of NO in response to abiotic stresses by improving antioxidant activity in most plant species. In addition, PTMs of antioxidative pathways by these two molecules are also discussed.

Anthropogenic forcing exacerbating the urban heat islands in India.

Urban heat island (UHI) phenomena is among the major consequences of the alteration of earth's surface due to human activities. The relatively warmer temperatures in urban areas compared to suburban areas (i.e. UHI) has potential health hazards, such as mortality due to high temperatures and heat waves. In addition, UHI situation demands more energy (e.g. fans and air-conditioners) that would trigger greenhouse gas emissions. Studies on UHI intensity help to assess its impact on urban population, city planning, and urban health planning. This is particularly important for a country like India, where 32% people (~7% of total world population), live in urban areas. We conducted a detailed study on surface UHI intensity (SUHII), which is the difference between urban and surrounding rural land surface temperatures, across all seasons in 44 major cities of India, which shows that mean daytime SUHII is positive (up to 2 °C) for most cities, as analysed from satellite temperature measurements for the period 2000-2017, in contrast to previous studies. However, although statistically insignificant, most cities show a positive trend in SUHII for monsoon and post-monsoon periods, but negative for winter and summer seasons. The increasing night-time SUHII in all seasons for most cities suggest increasing trend in temperature in cities due to the impact of the rapid urbanisation, and thus, suggesting the influence of anthropogenic forcing on SUHII. This is also supported by the analysis of aerosols, night lights, precipitation and vegetation in the study regions. Therefore, this study shall aid planning and management of urban areas by giving insights about the effects of nature and intensity of development, land cover and land use mix and the structure of cities on SUHII.

Stable isotope labelling: an approach for MALDI-TOF MS-based rapid detection of fluconazole resistance in Candida tropicalis.

BACKGROUND: With the rise of fluconazole resistance in Candida tropicalis in Asian countries, the rapid detection of resistance is required to optimize therapy. OBJECTIVES: We evaluated a stable isotope labelling by/with amino acids in cell culture (SILAC) approach for rapid detection of fluconazole resistance in C. tropicalis by MALDI-TOF MS. METHODS: Twenty-six fluconazole-resistant and 19 -susceptible C. tropicalis isolates were used. Isolates were grown in media containing normal lysine (NL), isotopically labelled ('heavy') lysine (HL) and fluconazole (FLC) with labelled lysine (HL + FLC). MALDI-TOF MS was performed, acquired spectra were visually compared and composite correlation index (CCI) values were calculated. The results were analysed by CCI matrix, virtual gel and principal component analysis (PCA). RESULTS: The MICs of fluconazole in 26 resistant and 19 susceptible isolates were 16-256 mg/L and 0.5-1 mg/L, respectively. The m/z values of 15 peaks specific for media containing NL and their corresponding peaks specific for media with HL were detected to differentiate resistant and susceptible isolates. The CCI cut-off values for susceptible and resistant isolates were significant (P < 0.05). The CCI matrix, virtual gel and PCA dendrogram confirmed the results. The sensitivity, specificity, positive predictive value, negative predictive value and accuracy of this method for detection of fluconazole resistance were 92.31%, 100%, 100%, 90.48% and 95.56%, respectively. CONCLUSIONS: SILAC, a promising approach for rapid resistance detection in C. tropicalis using MALDI-TOF MS, may be used in the routine laboratory in the near future.

Ecosystem service value assessment of a natural reserve region for strengthening protection and conservation.

Ecosystem Services (ESs) refer to the direct and indirect contributions of ecosystems to human well-being and subsistence. Ecosystem valuation is an approach to assign monetary values to an ecosystem and its key ecosystem goods and services, generally referred to as Ecosystem Service Value (ESV). We have measured spatiotemporal ESV of 17 key ESs of Sundarbans Biosphere Reserve (SBR) in India using temporal remote sensing (RS) data (for years 1973, 1988, 2003, 2013, and 2018). These mangrove ecosystems are crucial for providing valuable supporting, regulatory, provisioning, and cultural ecosystem services. We have adopted supervised machine learning algorithms for classifying the region into different ecosystem units. Among the used machine learning models, Support Vector Machine (SVM) and Random Forest (RF) algorithms performed the most accurate and produced the best classification estimates with maximum kappa and an overall accuracy value. The maximum ESV (derived from both adjusted and non-adjusted units, million US$ year-1) is produced by mangrove forest, followed by the coastal estuary, cropland, inland wetland, mixed vegetation, and finally urban land. Out of all the ESs, the waste treatment (WT) service is the dominant ecosystem service of SBR. Additionally, the mangrove ecosystem was found to be the most sensitive to land use and land cover changes. The synergy and trade-offs between the ESs are closely associated with the spatial extent. Therefore, accurate estimates of ES valuation and mapping can be a robust tool for assessing the effects of poor decision making and overexploitation of natural resources on ESs.

Molecular Typing and Antifungal Susceptibility of Candida viswanathii, India.

We report invasive candidiasis caused by Candida viswanathii over 2 time periods during 2013-2015 in a tertiary care hospital in Chandigarh, India. Molecular typing revealed multiple clusters of the isolates. We detected high MICs for fluconazole in the second time period.

Rapid detection of fluconazole resistance in Candida tropicalis by MALDI-TOF MS.

With the changing epidemiology and emergence of antifungal resistance among Candida species, rapid antifungal susceptibility testing (AFST) is crucial for optimization of antifungal therapy. This study was conducted to standardize a matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI -TOF MS) based AFST method (ms-AFST) for susceptibility of Candida tropicalis isolates. Clinical isolates of C. tropicalis were confirmed for fluconazole resistance by the CLSI (M27-A3) method. The incubation period and drug concentration were optimized to determine the minimal profile change concentration (MPCC) by MALDI-TOF MS. The data were analyzed first by direct visual observation of the spectra followed by composite correlation index (CCI) matrix analysis, virtual gel analysis, and cluster analysis for confirmation. Finally, the correlation between minimum inhibitory concentrations (MICs) and MPCCs was evaluated. A total of 15 fluconazole resistant (MICs ranging from 16 to 128 µg/ml) and 19 fluconazole susceptible C. tropicalis isolates (MIC ≤1 µg/ml) were included in this study. All C. tropicalis isolates had significant spectral changes after 4h incubation with fluconazole. Of 34 isolates, MPCCs and MICs were equivalent for 16 isolates, and the MPCC was one dilution lower than the respective MIC in the remaining 18 isolates. This finding was further supported by visual analysis, CCI matrix analysis, virtual gel and principal component analysis dendrogram analysis. The correlation between MPCC and MIC was significant (P < .05). Therefore, a MALDI-TOF MS based AFST assay may be used as a rapid screening technique for fluconazole resistance in C. tropicalis.

Estimating global ecosystem service values and its response to land surface dynamics during 1995-2015.

Changes in land use due to the industrial revolution, increasing population, ever-increasing desire for economic growth is a global concern. The aforementioned changes can have a significant impact on global and regional ecosystem services which are indispensable for human well-being and their subsistence. This study identifies several approaches (Costanza et al., de Groot et al., and Xie et al.) to estimate the value of global terrestrial ecosystem services. High resolution (300 m) land use products provided by European Space Agency-Climate Change Initiative (ESA-CCI) were used to quantify the global ecosystem service values (ESV) for 1995, 2000, 2005, 2010 and 2015 respectively. The coefficient of elasticity (CE) and coefficient of sensitivity (CS) was calculated to compute the response of ESV's corresponding to land use land cover (LULC) change. The results estimated the mean global ESV's (Trillion US$ year-1) to be 58.97 in 1995 and 57.76 in 2015, indicating a net loss of ESV (1.21 Trillion US$ year-1) during the analysis period (1995-2015) due to depletion of forest cover and wetland/water surface. The overall ESV (Trillion US$ year-1) increased in cropland (4.8 in 1995 to 4.9 in 2015) and urban coverage (0.3 in 1995 to 0.59 in 2015) whereas, it reduced substantially in forests (17.59 in 1995 to 17.42 in 2015), grasslands (9.1 in 1995 to 8.9 in 2015), wetland (22.19 in 1995 to 21.11 in 2015) and water bodies (5.29 in 1995 to 5.27). The forestland, wetland, and water bodies are the highest sensitive eco-regions defined by all valuation methods. The current research provides a way to quantify the overall economic loss or gain due to changes in the past, present, and future land use. This will bridge the gap between economic evaluations of current assets concerning the changes in land use. It will also help planners to provide an in-depth thought to the changes in the overall economic value of a particular land use in future (keeping biodiversity in mind) while validating long-term policies concerning to ecological conservation of a country.

Stress response in medically important Mucorales.

Mucorales are saprobes, ubiquitously distributed and able to infect a heterogeneous population of human hosts. The fungi require robust stress responses to survive in human host. We tested the growth of Mucorales in the presence of different abiotic stress. Eight pathogenic species of Mucorales, including Rhizopus arrhizus, Rhizopus microsporus, Rhizomucor pusillus, Apophysomyces elegans, Licthemia corymbifera, Cunninghamella bertholletiae, Syncephalastrum racemosum and Mucor racemosus, were exposed to different stress inducers: osmotic (sodium chloride and d-sorbitol), oxidative (hydrogen peroxide and menadione), pH, cell wall and metal ions (Cu, Zn, Fe and Mg). Wide variation in stress responses was noted: R. arrhizus showed maximum resistance to both osmotic and oxidative stresses, whereas R. pusillus and M. indicus were relatively sensitive. Rhizopus arrhizus and R. microsporus showed maximum resistance to alkaline pH, whereas C. bertholletiae, L. corymbifera, M. racemosus and A. elegans were resistant to acidic pH. Maximum tolerance was noted in R. microsporus to Cu, R. microsporus and R. arrhizus to Fe and C. bertholletiae to Zn. In contrast, L. corymbifera, A. elegans and M. indicus were sensitive to Cu, Zn and Fe respectively. In conclusion, R. arrhizus showed high stress tolerance in comparison to other species of Mucorales, and this could be the possible reason for high pathogenic potential of this fungi.

Fungal Keratitis in North India: Spectrum of Agents, Risk Factors and Treatment.

To assess the prevalence of fungal keratitis, we conducted a retrospective study over 7 years (2005 through 2011) at a tertiary care center in North India. Effort has been made to analyze the disease burden, spectrum of agents and treatment history. The findings were compared with an earlier study at the same center for any change in the epidemiology of the disease. Microbiology records were screened at the Postgraduate Institute of Medical Education and Research, Chandigarh, India, to identify fungal keratitis cases, and available clinical records of those cases were analyzed. Of 2459 clinically suspected fungal keratitis cases, 765 (31 %) cases were direct microscopy confirmed. Of these microscopy-confirmed cases, fungi were isolated in 393 (51.4 %), with Aspergillus spp. ranked top (n = 187, 47.6 %), followed by melanized fungi (n = 86, 21.9 %) and Fusarium spp. (n = 64, 16 %). A male predominance of 78.7 % was noted with a peak in the incidence of fungal keratitis during post-monsoon season (September to November). A delay in diagnosis was significantly associated (p < 0.001) with keratitis cases due to melanized fungi. In comparison with an earlier study, higher isolation of melanized fungi was noted with a widening of the spectrum of agents identified. Thus, fungal keratitis due to Aspergillus spp. remains a serious ocular illness among the active male population in North India with relative rise of keratitis due to melanized fungi. The spectrum of agents causing fungal keratitis has broadened with many rare fungi that are implicated.

Tracking the messengers: Emerging advances in mRNA-based plant communication.

Messenger RNAs (mRNAs) are the templates for protein translation but can also act as non-cell-autonomous signaling molecules. Plants input endogenous and exogenous cues to mobile mRNAs and output them to local or systemic target cells and organs to support specific plant responses. Mobile mRNAs form ribonucleoprotein (RNP) complexes with proteins during transport. Components of these RNP complexes could interact with plasmodesmata (PDs), a major mediator of mRNA transport, to ensure mRNA mobility and transport selectivity. Based on advances in the last two to three years, this review summarizes mRNA transport mechanisms in local and systemic signaling from the perspective of RNP complex formation and PD transport. We also discuss the physiological roles of endogenous mRNA transport and the recently revealed roles of non-cell-autonomous mRNAs in inter-organism communication.

A fungal metabolic regulator underlies infectious synergism during Candida albicans - Staphylococcus aureus intra-abdominal co-infection.

Candida albicans and Staphylococcus aureus are two commonly associated pathogens that cause nosocomial infections with high morbidity and mortality. Our prior and current work using a murine model of polymicrobial intra-abdominal infection (IAI) uncovered synergistic lethality that was driven by Candida -induced upregulation of functional S. aureus ⍺-toxin leading to polymicrobial sepsis and organ damage. In order to determine the candidal effector(s) mediating enhanced virulence, an unbiased screen of C. albicans transcription factor mutants was undertaken and revealed that zcf13 Δ/Δ failed to drive augmented ⍺-toxin or lethal synergism during co-infection. Using a combination of transcriptional and phenotypic profiling approaches, ZCF13 was shown to regulate genes involved in pentose metabolism, including RBK1 and HGT7 that contribute to fungal ribose catabolism and uptake, respectively. Subsequent experiments revealed that ribose inhibited the staphylococcal agr quorum sensing system and concomitantly repressed toxicity. Unlike wild-type C. albicans , zcf13 Δ/Δ was unable to effectively utilize ribose during co-culture or co-infection leading to exogenous ribose accumulation and agr repression. Forced expression of RBK1 and HGT7 in the zcf13 Δ/Δ mutant fully restored pathogenicity during co-infection. Collectively, our results detail the interwoven complexities of cross-kingdom interactions and highlight how intermicrobial metabolism impacts polymicrobial disease pathogenesis with devastating consequences for the host.

Cross-talk between abscisic acid-dependent and abscisic acid-independent pathways during abiotic stress.

Salinity, drought and low temperature are the common forms of abiotic stress encountered by land plants. To cope with these adverse environmental factors, plants execute several physiological and metabolic responses. Both osmotic stress (elicited by water deficit or high salt) and cold stress increase the endogenous level of the phytohormone abscisic acid (ABA). ABA-dependent stomatal closure to reduce water loss is associated with small signaling molecules like nitric oxide, reactive oxygen species and cytosolic free calcium, and mediated by rapidly altering ion fluxes in guard cells. ABA also triggers the expression of osmotic stress-responsive (OR) genes, which usually contain single/multiple copies of cis-acting sequence called abscisic acid-responsive element (ABRE) in their upstream regions, mostly recognized by the basic leucine zipper-transcription factors (TFs), namely, ABA-responsive element-binding protein/ABA-binding factor. Another conserved sequence called the dehydration-responsive element (DRE)/C-repeat, responding to cold or osmotic stress, but not to ABA, occurs in some OR promoters, to which the DRE-binding protein/C-repeat-binding factor binds. In contrast, there are genes or TFs containing both DRE/CRT and ABRE, which can integrate input stimuli from salinity, drought, cold and ABA signaling pathways, thereby enabling cross-tolerance to multiple stresses. A strong candidate that mediates such cross-talk is calcium, which serves as a common second messenger for abiotic stress conditions and ABA. The present review highlights the involvement of both ABA-dependent and ABA-independent signaling components and their interaction or convergence in activating the stress genes. We restrict our discussion to salinity, drought and cold stress.

Candida-bacterial cross-kingdom interactions.

While the fungus Candida albicans is a common colonizer of healthy humans, it is also responsible for mucosal infections and severe invasive disease. Understanding the mechanisms that allow C. albicans to exist as both a benign commensal and as an invasive pathogen have been the focus of numerous studies, and recent findings indicate an important role for cross-kingdom interactions on C. albicans biology. This review highlights how C. albicans-bacteria interactions influence healthy polymicrobial community structure, host immune responses, microbial pathogenesis, and how dysbiosis may lead to C. albicans infection. Finally, we discuss how cross-kingdom interactions represent an opportunity to identify new antivirulence compounds that target fungal infections.

Stable isotope labeling as a promising tool for rapid drug susceptibility testing in Neisseria gonorrhoeae.

The world is heading towards an era of intractable and impending untreatable N. gonorrhoeae, thereby underlining the significance of rapid and accurate prediction of drug resistance as an indispensable need of the hour. In the present study, we optimized and evaluated a stable isotope labeling-based approach using the MALDI-TOF MS (Matrix-Assisted Laser Desorption/Ionization-Time of Flight Mass Spectrometry) for rapid and reliable detection of ciprofloxacin and azithromycin resistance in N. gonorrhoeae. All the isolates were cultured under three varied condition setups viz. medium supplemented with normal lysine, heavy lysine (isotope), and heavy lysine along with the antibiotics (ciprofloxacin/azithromycin), respectively. After incubation, spectra were acquired using the MALDI-TOF MS which were further screened for unique patterns (media-specific spectra) to differentiate drug-susceptible and resistant isolates. The results of the stable isotope labeling assay were comparable to the results of phenotypic methods used for susceptibility testing.

Mechanisms of azole antifungal resistance in clinical isolates of Candida tropicalis.

This study was designed to understand the molecular mechanisms of azole resistance in Candida tropicalis using genetic and bioinformatics approaches. Thirty-two azole-resistant and 10 azole-susceptible (S) clinical isolates of C. tropicalis were subjected to mutation analysis of the azole target genes including ERG11. Inducible expression analysis of 17 other genes potentially associated with azole resistance was also evaluated. Homology modeling and molecular docking analysis were performed to study the effect of amino acid alterations in mediating azole resistance. Of the 32 resistant isolates, 12 (37.5%) showed A395T and C461T mutations in the ERG11 gene. The mean overexpression of CDR1, CDR3, TAC1, ERG1, ERG2, ERG3, ERG11, UPC2, and MKC1 in resistant isolates without mutation (R-WTM) was significantly higher (p<0.05) than those with mutation (R-WM) and the sensitive isolates (3.2-11 vs. 0.2-2.5 and 0.3-2.2 folds, respectively). Although the R-WTM and R-WM had higher (p<0.05) CDR2 and MRR1 expression compared to S isolates, noticeable variation was not seen among the other genes. Protein homology modelling and molecular docking revealed that the mutations in the ERG11 gene were responsible for structural alteration and low binding efficiency between ERG11p and ligands. Isolates with ERG11 mutations also presented A220C in ERG1 and together T503C, G751A mutations in UPC2. Nonsynonymous mutations in the ERG11 gene and coordinated overexpression of various genes including different transporters, ergosterol biosynthesis pathway, transcription factors, and stress-responsive genes are associated with azole resistance in clinical isolates of C. tropicalis.