Molecularly Imprinted Polymer-Based Electrochemical Sensor for the Detection of Azoxystrobin in Aqueous Media.

This work presents an electrochemical sensor detecting a fungicide-azoxystrobin (AZO) in aqueous environments. This AZO sensor utilizes a thin-film metal electrode (TFME) combined with an AZO-selective molecularly imprinted polymer (AZO-MIP). The AZO-MIP was directly generated on TFME through electrochemical polymerization from the solution containing two functional monomers: aniline (Ani) and m-phenylenediamine (mPD), and the template: AZO, which was afterwards removed to form AZO-selective cavities in the polymer matrix. The AZO-MIP preparation was characterized by electrochemical and ellipsometry measurements. Optimization of the synthesis parameters, including the charge density applied during electrodeposition, the monomer-to-template ratio, was performed to enhance the sensor's performance. The results demonstrated that the AZO sensor achieved a low limit of detection (LOD) of 3.6 nM and a limit of quantification (LOQ) of 11.8 nM in tap water, indicating its sensitivity in a complex aqueous environment. The sensor also exhibited satisfactory selectivity for AZO in both ultrapure and tap-water samples and achieved a good recovery (94-119%) for the target analyte. This study highlights the potential of MIP-based electrochemical sensors for the rapid and accurate detection of fungicide contaminants in water, contributing to the advancement of analytical tools for water-quality monitoring and risk assessment.

Altered skin microbiome, inflammation, and JAK/STAT signaling in Southeast Asian ichthyosis patients.

BACKGROUND: Congenital ichthyosis (CI) is a collective group of rare hereditary skin disorders. Patients present with epidermal scaling, fissuring, chronic inflammation, and increased susceptibility to infections. Recently, there is increased interest in the skin microbiome; therefore, we hypothesized that CI patients likely exhibit an abnormal profile of epidermal microbes because of their various underlying skin barrier defects. Among recruited individuals of Southeast Asian ethnicity, we performed skin meta-genomics (i.e., whole-exome sequencing to capture the entire multi-kingdom profile, including fungi, protists, archaea, bacteria, and viruses), comparing 36 CI patients (representing seven subtypes) with that of 15 CI age-and gender-matched controls who had no family history of CI. RESULTS: This case-control study revealed 20 novel and 31 recurrent pathogenic variants. Microbiome meta-analysis showed distinct microbial populations, decreases in commensal microbiota, and higher colonization by pathogenic species associated with CI; these were correlated with increased production of inflammatory cytokines and Th17- and JAK/STAT-signaling pathways in peripheral blood mononuclear cells. In the wounds of CI patients, we identified specific changes in microbiota and alterations in inflammatory pathways, which are likely responsible for impaired wound healing. CONCLUSIONS: Together, this research enhances our understanding of the microbiological, immunological, and molecular properties of CI and should provide critical information for improving therapeutic management of CI patients.

Calcineurin is required for Candida glabrata Pdr1 transcriptional activation.

Fluconazole is one of the most commonly used antifungals today. A result of this has been the inevitable selection of fluconazole-resistant organisms. This is an especially acute problem in the pathogenic yeast Candida glabrata. Elevated minimal inhibitory concentrations for fluconazole in C. glabrata are frequently associated with substitution mutations within the Zn2Cys6 zinc cluster-containing transcription factor-encoding gene PDR1. These mutant Pdr1 regulators drive constitutively high expression of target genes like CDR1 that encodes an ATP-binding cassette transporter thought to act as a drug efflux pump. Exposure of C. glabrata to fluconazole induced expression of both Pdr1 and CDR1, although little is known of the molecular basis underlying the upstream signals that trigger Pdr1 activation. Here, we show that the protein phosphatase calcineurin is required for fluconazole-dependent induction of Pdr1 transcriptional regulation. Calcineurin catalytic activity is required for normal Pdr1 regulation, and a hyperactive form of this phosphatase can decrease susceptibility to the echinocandin caspofungin but does not show a similar change for fluconazole susceptibility. Loss of calcineurin from strains expressing two different gain-of-function forms of Pdr1 also caused a decrease in CDR1 expression and increased fluconazole susceptibility, demonstrating that even these hyperactive Pdr1 regulatory mutants cannot bypass the requirement for calcineurin. Our data implicate calcineurin activity as a link tying azole and echinocandin susceptibility together via the control of transcription factor activity.IMPORTANCEDrug-resistant microorganisms are a problem in the treatment of all infectious diseases; this is an especially acute problem with fungi due to the existence of only three major classes of antifungal drugs, including the azole drug fluconazole. In the pathogenic yeast Candida glabrata, mutant forms of a transcription factor called Pdr1 are commonly associated with decreased fluconazole susceptibility and poor clinical outcomes. Here, we identify a protein phosphatase called calcineurin that is required for fluconazole-dependent induction of Pdr1 transcriptional activation and associated drug susceptibility. Gain-of-function mutant forms of Pdr1 still required the presence of calcineurin to confer normally decreased fluconazole susceptibility. Previous studies showed that calcineurin controls susceptibility to the echinocandin class of antifungal drugs, and our data demonstrate that this protein phosphatase is also required for normal azole drug susceptibility. Calcineurin plays a central role in susceptibility to two of the three major classes of antifungal drugs in C. glabrata.

Rapid, efficient auxin-inducible protein degradation in Candida pathogens.

A variety of inducible protein degradation (IPD) systems have been developed as powerful tools for protein functional characterization. IPD systems provide a convenient mechanism for rapid inactivation of almost any target protein of interest. Auxin-inducible degradation (AID) is one of the most common IPD systems and has been established in diverse eukaryotic research model organisms. Thus far, IPD tools have not been developed for use in pathogenic fungal species. Here, we demonstrate that the original AID and the second generation, AID2, systems work efficiently and rapidly in the human pathogenic yeasts, Candida albicans and Candida glabrata. We developed a collection of plasmids that support AID system use in laboratory strains of these pathogens. These systems can induce >95% degradation of target proteins within minutes. In the case of AID2, maximal degradation was achieved at low nanomolar concentrations of the synthetic auxin analog 5-adamantyl-indole-3-acetic acid. Auxin-induced target degradation successfully phenocopied gene deletions in both species. The system should be readily adaptable to other fungal species and to clinical pathogen strains. Our results define the AID system as a powerful and convenient functional genomics tool for protein characterization in fungal pathogens. IMPORTANCE Life-threatening fungal infections are an escalating human health problem, complicated by limited treatment options and the evolution of drug resistant pathogen strains. Identification of new targets for therapeutics to combat invasive fungal infections, including those caused by Candida species, is an urgent need. In this report, we establish and validate an inducible protein degradation methodology in Candida albicans and Candida glabrata that provides a new tool for protein functional characterization in these, and likely other, fungal pathogen species. We expect this tool will ultimately be useful for the identification and characterization of promising drug targets and factors involved in virulence and drug resistance.

A Case of Severe Metabolic Acidosis in the Setting of a Strict Ketogenic Diet.

Patients with metabolic acidosis often present with obscure, multifactorial etiologies, making efficient diagnosis and treatment key to preventing poor clinical outcomes. This case report describes a patient with severe metabolic acidosis in which the underlying cause was not immediately apparent. After a thorough work-up and history taking, the patient's strict ketogenic diet was identified as the most likely source of his illness. Over multiple days, the patient improved as he resumed a normal diet and was treated for refeeding syndrome. This case highlights the importance of taking a comprehensive social and diet history when assessing a patient with metabolic acidosis. It also highlights the need for physicians to understand and be ready to counsel on the possible effects of fad diets, such as the ketogenic diet.

Rapid, efficient auxin-inducible protein degradation in Candida pathogens.

A variety of inducible protein degradation (IPD) systems have been developed as powerful tools for protein functional characterization. IPD systems provide a convenient mechanism for rapid inactivation of almost any target protein of interest. Auxin-inducible degradation (AID) is one of the most common IPD systems and has been established in diverse eukaryotic research model organisms. Thus far, IPD tools have not been developed for use in pathogenic fungal species. Here, we demonstrate that the original AID and the second generation AID2 systems work efficiently and rapidly in the human pathogenic yeasts Candida albicans and Candida glabrata . We developed a collection of plasmids that support AID system use in laboratory strains of these pathogens. These systems can induce >95% degradation of target proteins within minutes. In the case of AID2, maximal degradation was achieved at low nanomolar concentrations of the synthetic auxin analog 5-adamantyl-indole-3-acetic acid (5-Ad-IAA). Auxin-induced target degradation successfully phenocopied gene deletions in both species. The system should be readily adaptable to other fungal species and to clinical pathogen strains. Our results define the AID system as a powerful and convenient functional genomics tool for protein characterization in fungal pathogens.

Caring for the whole person: transgender-competent HIV pre-exposure prophylaxis as part of integrated primary healthcare services in Vietnam.

INTRODUCTION: Although HIV prevalence among transgender women who have sex with men in Vietnam is high (16-18%), uptake of pre-exposure prophylaxis (PrEP) is low compared to other populations. When PrEP was initiated in 2017, gender-affirming healthcare was largely unavailable. Lack of access to competent, stigma-free healthcare is a well-documented barrier to transgender women's uptake of PrEP and primary healthcare (PHC). We aimed to demonstrate the utility of a PrEP quality improvement intervention in pinpointing and addressing barriers to PrEP use among transgender women in Vietnam. METHODS: We applied a real-world participatory continuous quality improvement (CQI) and Plan-Do-Study-Act (PDSA) methodology to ascertain barriers to PrEP uptake among transgender women and determine priority actions for quality improvement. A CQI team representing transgender women leaders, key population (KP)-clinic staff, public-sector HIV managers and project staff applied PDSA to test solutions to identified barriers that addressed the primary quality improvement outcome of the monthly change in PrEP uptake among transgender women and secondary outcomes, including month-3 PrEP continuation, the impact of offering PHC on PrEP uptake and unmet PrEP need. We utilized routine programmatic data and a descriptive cross-sectional study enrolling 124 transgender women to measure these outcomes from October 2018 to September 2021. RESULTS: Five key barriers to PrEP uptake among transgender women were identified and corresponding solutions were put in place: (1) offering gender-affirming care training to KP-clinics and community-based organizations; (2) integrating gender-affirming services into 10 KP-clinics; (3) offering PHC through five one-stop shop (OSS) clinics; (4) implementing a campaign addressing concerns related to hormone use and PrEP interactions; and (5) developing national HIV and transgender healthcare guidelines. New PrEP enrolment and month-3 PrEP continuation increased significantly among transgender women. Of 235 transgender women who initially sought healthcare other than PrEP at OSS clinics, 26.4% subsequently enrolled in PrEP. About one-third of transgender women reported unmet PrEP need, while two-thirds indicated an interest in long-acting cabotegravir. CONCLUSIONS: Offering gender-competent, integrated PHC can increase PrEP enrolment and continuation, and can be an entry-point for PrEP among those seeking care within PHC clinics. More work is needed to expand access to transgender women-led and -competent healthcare in Vietnam.

Nonidentical function of Upc2A binding sites in the Candida glabrata CDR1 promoter.

Increased expression of the Candida glabrata CDR1 gene, encoding an ATP-binding cassette membrane transporter, is routinely observed in fluconazole-resistant isolates of this pathogenic yeast. CDR1 transcription has been well-documented to be due to activity of the Zn2Cys6 zinc cluster-containing transcription factor Pdr1. Gain-of-function mutations in the gene encoding this factor are the most commonly observed cause of fluconazole hyper-resistance in clinical isolates. We have recently found that the sterol-responsive transcription factor Upc2A also acts to control CDR1 transcription, providing a direct link between ergosterol biosynthesis and expression of Pdr1 target genes. While this earlier work implicated Upc2A as an activator of CDR1 transcription, our further analyses revealed the presence of a second Upc2A binding site that negatively regulated CDR1 expression. This Upc2A binding site designated a sterol-responsive element (SRE) was found to have significant lower affinity for Upc2A DNA-binding than the previously described SRE. This new SRE was designated SRE2 while the original, positively acting site was named SRE1. A mutant version of SRE2 prevented in vitro DNA-binding by recombinant Upc2A and, when introduced into the CDR1 promoter, caused decreased fluconazole susceptibility and increased CDR1 expression. This negative effect caused by loss of SRE2 was shown to be Pdr1 independent, consistent with the presence of at least one additional activator of CDR1 transcription. The ability of Upc2A to exert either positive or negative effects on gene expression resembles behavior of mammalian nuclear receptor proteins and reveals an unexpectedly complex nature for SRE effects on gene regulation.

Advances in Detection of Antibiotic Pollutants in Aqueous Media Using Molecular Imprinting Technique-A Review.

Antibiotics constitute one of the emerging categories of persistent organic pollutants, characterised by their expansion of resistant pathogens. Antibiotic pollutants create a major public health challenge, with already identifiable detrimental effects on human and animal health. A fundamental aspect of controlling and preventing the spread of pollutants is the continuous screening and monitoring of environmental samples. Molecular imprinting is a state-of-the-art technique for designing robust biomimetic receptors called molecularly imprinted polymers (MIPs), which mimic natural biomolecules in target-selective recognition. When integrated with an appropriate sensor transducer, MIP demonstrates a potential for the needed environmental monitoring, thus justifying the observed rise in interest in this field of research. This review examines scientific interventions within the last decade on the determination of antibiotic water pollutants using MIP receptors interfaced with label-free sensing platforms, with an expanded focus on optical, piezoelectric, and electrochemical systems. Following these, the review evaluates the analytical performance of outstanding MIP-based sensors for environmentally significant antibiotics, while highlighting the importance of computational chemistry in functional monomer selection and the strategies for signal amplification and performance improvement. Lastly, the review points out the future trends in antibiotic MIP research, as it transits from a proof of concept to the much demanded commercially available entity.

Azole-Resistant Alleles of ERG11 in Candida glabrata Trigger Activation of the Pdr1 and Upc2A Transcription Factors.

Azoles, the most commonly used antifungal drugs, specifically inhibit the fungal lanosterol α-14 demethylase enzyme, which is referred to as Erg11. Inhibition of Erg11 ultimately leads to a reduction in ergosterol production, an essential fungal membrane sterol. Many Candida species, such as Candida albicans, develop mutations in this enzyme which reduces the azole binding affinity and results in increased resistance. Candida glabrata is also a pathogenic yeast that has low intrinsic susceptibility to azole drugs and easily develops elevated resistance. In C. glabrata, these azole resistant mutations typically cause hyperactivity of the Pdr1 transcription factor and rarely lie within the ERG11 gene. Here, we generated C. glabrata ERG11 mutations that were analogous to azole resistance alleles from C. albicans ERG11. Three different Erg11 forms (Y141H, S410F, and the corresponding double mutant (DM)) conferred azole resistance in C. glabrata with the DM Erg11 form causing the strongest phenotype. The DM Erg11 also induced cross-resistance to amphotericin B and caspofungin. Resistance caused by the DM allele of ERG11 imposed a fitness cost that was not observed with hyperactive PDR1 alleles. Crucially, the presence of the DM ERG11 allele was sufficient to activate the Pdr1 transcription factor in the absence of azole drugs. Our data indicate that azole resistance linked to changes in ERG11 activity can involve cellular effects beyond an alteration in this key azole target enzyme. Understanding the physiology linking ergosterol biosynthesis with Pdr1-mediated regulation of azole resistance is crucial for ensuring the continued efficacy of azole drugs against C. glabrata.

Loss-of-Function ROX1 Mutations Suppress the Fluconazole Susceptibility of upc2AΔ Mutation in Candida glabrata, Implicating Additional Positive Regulators of Ergosterol Biosynthesis.

Two of the major classes of antifungal drugs in clinical use target ergosterol biosynthesis. Despite its importance, our understanding of the transcriptional regulation of ergosterol biosynthesis genes in pathogenic fungi is essentially limited to the role of hypoxia and sterol-stress-induced transcription factors such as Upc2 and Upc2A as well as homologs of sterol response element binding (SREB) factors. To identify additional regulators of ergosterol biosynthesis in Candida glabrata, an important human fungal pathogen with reduced susceptibility to ergosterol biosynthesis inhibitors relative to other Candida spp., we used a serial passaging strategy to isolate suppressors of the fluconazole hypersusceptibility of a upc2AΔ deletion mutant. This led to the identification of loss-of-function mutations in two genes: ROX1, the homolog of a hypoxia gene transcriptional suppressor in Saccharomyces cerevisiae, and CST6, a transcription factor that is involved in the regulation of carbon dioxide response in C. glabrata. Here, we describe a detailed analysis of the genetic interaction of ROX1 and UPC2A. In the presence of fluconazole, loss of Rox1 function restores ERG11 expression to the upc2AΔ mutant and inhibits the expression of ERG3 and ERG6, leading to increased levels of ergosterol and decreased levels of the toxic sterol 14α methyl-ergosta-8,24(28)-dien-3ß, 6α-diol, relative to the upc2AΔ mutant. Our observations establish that Rox1 is a negative regulator of ERG gene biosynthesis and indicate that a least one additional positive transcriptional regulator of ERG gene biosynthesis must be present in C. glabrata. IMPORTANCE Candida glabrata is one of the most important human fungal pathogens and has reduced susceptibility to azole-class inhibitors of ergosterol biosynthesis. Although ergosterol is the target of two of the three classes of antifungal drugs, relatively little is known about the regulation of this critical cellular pathway. Sterols are both essential components of the eukaryotic plasma membrane and potential toxins; therefore, sterol homeostasis is critical for cell function. Here, we identified two new negative regulators in C. glabrata of ergosterol (ERG) biosynthesis gene expression. Our results also indicate that in addition to Upc2A, the only known activator of ERG genes, additional positive regulators of this pathway must exist.

The Candida glabrata Upc2A transcription factor is a global regulator of antifungal drug resistance pathways.

The most commonly used antifungal drugs are the azole compounds, which interfere with biosynthesis of the fungal-specific sterol: ergosterol. The pathogenic yeast Candida glabrata commonly acquires resistance to azole drugs like fluconazole via mutations in a gene encoding a transcription factor called PDR1. These PDR1 mutations lead to overproduction of drug transporter proteins like the ATP-binding cassette transporter Cdr1. In other Candida species, mutant forms of a transcription factor called Upc2 are associated with azole resistance, owing to the important role of this protein in control of expression of genes encoding enzymes involved in the ergosterol biosynthetic pathway. Recently, the C. glabrata Upc2A factor was demonstrated to be required for normal azole resistance, even in the presence of a hyperactive mutant form of PDR1. Using genome-scale approaches, we define the network of genes bound and regulated by Upc2A. By analogy to a previously described hyperactive UPC2 mutation found in Saccharomyces cerevisiae, we generated a similar form of Upc2A in C. glabrata called G898D Upc2A. Analysis of Upc2A genomic binding sites demonstrated that wild-type Upc2A binding to target genes was strongly induced by fluconazole while G898D Upc2A bound similarly, irrespective of drug treatment. Transcriptomic analyses revealed that, in addition to the well-described ERG genes, a large group of genes encoding components of the translational apparatus along with membrane proteins were responsive to Upc2A. These Upc2A-regulated membrane protein-encoding genes are often targets of the Pdr1 transcription factor, demonstrating the high degree of overlap between these two regulatory networks. Finally, we provide evidence that Upc2A impacts the Pdr1-Cdr1 system and also modulates resistance to caspofungin. These studies provide a new perspective of Upc2A as a master regulator of lipid and membrane protein biosynthesis.

Prepped for PrEP? Acceptability, continuation and adherence among men who have sex with men and transgender women enrolled as part of Vietnam's first pre-exposure prophylaxis program.

Background HIV prevalence among men who have sex with men (MSM) and transgender women (TGW) in Vietnam is high, whereas coverage of effective HIV prevention services has been inadequate. Studies have measured MSM and TGW demand for pre-exposure prophylaxis (PrEP) services, which led to the design of the first ever PrEP program in Vietnam, Prepped for PrEP (P4P). METHODS: In March 2017, PrEP services were offered in Ho Chi Minh City as part of the P4P demonstration project, enabling same-day enrolment in three key population (KP)-led clinics and four public clinics. P4P aimed to assess acceptability and feasibility of PrEP services through calculating the rate of PrEP enrolment over time, and quarterly measures of continuation and adherence over an 18-month period. RESULTS: A total of 1069 MSM and 62 TGW were enrolled in P4P. Average monthly PrEP enrolment among MSM increased five-fold from the first 3 months (March-June 2017) to the last 3 months of active enrolment (March-June 2018), whereas for TGW, no increased trend in PrEP enrolment per quarter was seen. Self-reported PrEP adherence was >90% at all time points among MSM, but varied from 11.1% to 88.9% among TGW. PrEP continuation was calculated at months 3, 6, 9, 12, 15 and 18. For MSM, it was 88.7% at month 3, 68.8% at month 12 and 46.6% at month 18, whereas for TGW, it was 87.1%, 54.8% and 52.8%, respectively. Multivariable regression identified that MSM with lower-than-average income (adjusted odds ratio (aOR) 2.38 (95% confidence interval (CI): 1.59-3.54), P = 0.000), aged >30 years (aOR 2.03 (95% CI: 1.30-3.40), P = 0.007) and with an increasing number of sex partners (aOR: 1.06 (1.01-1.11), P = 0.011) had greater odds of remaining on PrEP. For TGW, being aged >30 years was associated with continuing on PrEP (aOR 5.62 (95% CI: 1.05-29.9), P = 0.043). CONCLUSIONS: We found PrEP to be highly acceptable among MSM and moderately acceptable among TGW. Continuation rates were relatively high for the first roll-out of PrEP; however, those aged ≤30 years were much more likely to discontinue services. Scaling-up PrEP through differentiated and community-led and engaged PrEP service delivery will be key to effectively increase access and uptake over the next 5 years.

Demedicalisation of HIV interventions to end HIV in the Asia-Pacific.

Despite the challenges to the HIV response in the Asia-Pacific, a demedicalisation of HIV intervention has been demonstrated to be an important strategy to maximise the uptake of HIV prevention tools among key populations in this region. Demedicalisation of HIV interventions translates medical discourse and shifts the paradigm from a disease-focused to a people-centred approach. It also recognises real-life experiences of key populations in the HIV response by empowering them to voice their needs and be at the forefront of the epidemic control. We further categorise a demedicalisation approach into three frameworks: (1) the demystification of clinical or medical concerns; (2) the destigmatisation of people living with HIV; and (3) the decentralisation of healthcare services. This article reviewed the demedicalisation framework by looking at the HIV intervention examples from countries in the Asia-Pacific, which included: (1) a study on drug-drug interaction between pre-exposure prophylaxis and feminising hormone treatment for transgender women; (2) the roles of key population-led health services; and (3) certification of key population lay providers.

Evidence that Ergosterol Biosynthesis Modulates Activity of the Pdr1 Transcription Factor in Candida glabrata.

A crucial limitation in antifungal chemotherapy is the limited number of antifungal drugs currently available. Azole drugs represent the most commonly used chemotherapeutic, and loss of efficacy of these drugs is a major risk factor in successful treatment of a variety of fungal diseases. Candida glabrata is a pathogenic yeast that is increasingly found associated with bloodstream infections, a finding likely contributed to by its proclivity to develop azole drug resistance. C. glabrata often acquires azole resistance via gain-of-function (GOF) mutations in the transcription factor Pdr1. These GOF forms of Pdr1 drive elevated expression of target genes, including the ATP-binding cassette transporter-encoding CDR1 locus. GOF alleles of PDR1 have been extensively studied, but little is known of how Pdr1 is normally regulated. Here we test the idea that reduction of ergosterol biosynthesis (as occurs in the presence of azole drugs) might trigger activation of Pdr1 function. Using two different means of genetically inhibiting ergosterol biosynthesis, we demonstrated that Pdr1 activity and target gene expression are elevated in the absence of azole drug. Blocks at different points in the ergosterol pathway lead to Pdr1 activation as well as to induction of other genes in this pathway. Delivery of the signal from the ergosterol pathway to Pdr1 involves the transcription factor Upc2A, an ERG gene regulator. We show that Upc2A binds directly to the PDR1 and CDR1 promoters. Our studies argue for a physiological link between ergosterol biosynthesis and Pdr1-dependent gene regulation that is not restricted to efflux of azole drugs.IMPORTANCE A likely contributor to the increased incidence of non-albicans candidemias involving Candida glabrata is the ease with which this yeast acquires azole resistance, in large part due to induction of the ATP-binding cassette transporter-encoding gene CDR1 Azole drugs lead to induction of Pdr1 transactivation, with a central model being that this factor binds these drugs directly. Here we provide evidence that Pdr1 is activated without azole drugs by the use of genetic means to inhibit expression of azole drug target-encoding gene ERG11 These acute reductions in Erg11 levels lead to elevated Pdr1 activity even though no drug is present. A key transcriptional regulator of the ERG pathway, Upc2A, is shown to directly bind to the PDR1 and CDR1 promoters. We interpret these data as support for the view that Pdr1 function is responsive to ergosterol biosynthesis and suggest that this connection reveals the normal physiological circuitry in which Pdr1 participates.

Interaction of carbohydrate binding module 20 with starch substrates.

CBM20s are starch-binding domains found in many amylolytic enzymes, including glucoamylase, alpha-amylase, beta-amylases, and a new family of starch-active polysaccharide monooxygenases (AA13 PMOs). Previous studies of CBM20-substrate interaction only concerned relatively small or soluble amylose molecules, while amylolytic enzymes often work on extended chains of insoluble starch molecules. In this study, we utilized molecular simulation techniques to gain further insights into the interaction of CBM20 with substrates of various sizes via its two separate binding sites, termed as BdS1 and BdS2. Results show that substrate binding at BdS1 involving two conserved tryptophan residues is about 2-4 kcal mol-1 stronger than that at BdS2. CBM20 exhibits about two-fold higher affinity for helical substrates than for the amylose random coils. The affinity for amylose individual double helices does not depend on the helices' length. At least three parallel double helices are required for optimal binding. The binding affinity for a substrate containing 3 or more double helices is ∼-15 kcal mol-1, which is 2-3 kcal mol-1 larger than that for individual double helices. 100 ns molecular dynamics simulations were carried out for the binding of CBM20 to an extended substrate containing 3 layers of 9 60-unit double helices (A3L). A stable conformation of CBM20-A3L was found at BdS1. However, when CBM20 binds A3L viaBdS2, it moves across the surface of the substrate and does not form a stable complex. MD simulations show that small amylose helices are quickly disrupted upon binding to CBM20. Our results provide some important molecular insights into the interactions of CBM20 with starch substrates, which will serve as the basis for further studies of CBM20-containing enzymes, including AA13 PMOs.

From conventional to disruptive: upturning the HIV testing status quo among men who have sex with men in Vietnam.

INTRODUCTION: HIV prevalence among men who have sex with men (MSM) in Vietnam is increasing, while annual HIV testing uptake has remained consistently low, posing a significant challenge to reaching the 90-90-90 goals. Barriers to MSM seeking HIV testing include concerns regarding confidentiality and lack of convenient testing options. Two new HIV testing strategies-HIV lay provider and HIV self-testing (HIVST)-were piloted alongside intensive social media outreach to increase access to and uptake of HIV testing among MSM not actively engaged in services. METHODS: We measured the proportion of first-time MSM HIV testers opting for HIV lay or self-testing, and factors that were associated with first-time testing, as part of a larger HIV lay and self-testing study among key populations in Vietnam. We also assessed MSM satisfaction with HIV lay or self-testing, and testing location and provider preferences. Finally, we calculated linkage to care cascade among MSM that were diagnosed and enrolled in anti-retroviral therapy (ART) services. RESULTS: Among MSM that sought HIV lay and self-testing, 57.9% (n = 320) and 51.3% (n = 412) were first-time testers respectively. In the final adjusted models, the odds of being a first-time tester and opting for HIV lay testing were higher among MSM who were young, had lower levels of income and had never exchanged sex for money; for HIVST, the odds of being a first-time HIV tester were higher among MSM that had attained lower levels of education. HIV lay and self-testing resulted in higher detection of new HIV cases (6.8%) compared to conventional HIV testing among key populations (estimated at 1.6% in 2016), while MSM linked to testing through social media interventions presented with even higher HIV-positivity (11%). Combined, 1655 HIV cases were diagnosed and more than 90% were registered for ART services. CONCLUSIONS: Our findings suggest that MSM-delivered HIV testing and self-testing, promoted through online or face-to-face interactions, offer important additions to MSM HIV testing services in Vietnam, and could significantly contribute to epidemic control by increasing HIV testing among harder-to-reach and higher-risk MSM, effectively enrolling them in ART, and reducing onward transmission.

Construction and Use of a Recyclable Marker To Examine the Role of Major Facilitator Superfamily Protein Members in Candida glabrata Drug Resistance Phenotypes.

Candida glabrata is the second most common species causing candidiasis. C. glabrata can also readily acquire resistance to azole drugs, complicating its treatment. Here we add to the collection of disruption markers to aid in genetic analysis of this yeast. This new construct is marked with a nourseothricin resistance cassette that produces an estrogen-activated form of Cre recombinase in a methionine-regulated manner. This allows eviction and reuse of this cassette in a facile manner. Using this new disruption marker, we have constructed a series of strains lacking different members of the major facilitator superfamily (MFS) of membrane transporter proteins. The presence of 15 MFS proteins that may contribute to drug resistance in C. glabrata placed a premium on development of a marker that could easily be reused to construct multiple gene-disrupted strains. Employing this recyclable marker, we found that loss of the MFS transporter-encoding gene FLR1 caused a dramatic increase in diamide resistance (as seen before), and deletion of two other MFS-encoding genes did not influence this phenotype. Interestingly, loss of FLR1 led to an increase in levels of oxidized glutathione, suggesting a possible molecular explanation for this enhanced oxidant sensitivity. We also found that while overproduction of the transcription factor Yap1 could suppress the fluconazole sensitivity caused by loss of the important ATP-binding cassette transporter protein Cdr1, this required the presence of FLR1. IMPORTANCE Export of drugs is a problem for chemotherapy of infectious organisms. A class of membrane proteins called the major facilitator superfamily contains a large number of proteins that often elevate drug resistance when overproduced but do not impact this phenotype when the gene is removed. We wondered if this absence of a phenotype for a disruption allele might be due to the redundancy of this group of membrane proteins. We describe the production of an easy-to-use recyclable marker cassette that will allow construction of strains lacking multiple members of the MFS family of transporter proteins.

Lay provider HIV testing: A promising strategy to reach the undiagnosed key populations in Vietnam.

BACKGROUND: In Vietnam, reaching the remaining one-third of undiagnosed people living with HIV and facilitating their antiretroviral therapy (ART) enrollment requires breakthrough approaches. We piloted lay provider HIV testing as an innovative approach to reach at-risk populations that never or infrequently HIV test at facility-based services. METHODS: We conducted a cross-sectional survey and analysis of routine program data in two urban provinces (Hanoi and Ho Chi Minh City) and two rural mountainous provinces (Nghe An and Dien Bien) from October 2015 through September 2017. Acceptability of lay provider testing was defined as the proportion of first-time HIV testers utilizing the service, and effectiveness was measured by HIV positivity and ART initiation rates. Univariate and multivariate analyses were used to determine lay provider testing preference and factors associated with that preference. RESULTS: Among 1,230 individuals recruited for face-to-face interviews, 74% belonged to key populations: people who inject drugs accounted for 31.4%; men who have sex with men, 60.4%; and female sex workers, 8.2%. Most clients (67%) reported being first-time HIV testers, and the majority (85.8%) preferred lay provider testing to facility-based testing. Multivariate analysis found that clients in urban areas (adjusted odds ratio [aOR] = 2.50; 95% confidence interval [CI]: 1.30-4.90) and those who had a university or higher education (aOR = 1.83; 95% CI: 1.05-3.20) were more likely to prefer lay provider testing. Lay provider testing yielded a higher HIV positivity rate (4.1%), particularly among first-time testers (6.8%), compared to facility-based testing (nationally estimated at 1.6% in 2016) and had a high ART initiation rate (91%). CONCLUSIONS: Our findings suggest that lay provider HIV testing is an effective approach to reach previously unreached at-risk populations, and, therefore, a critical addition to accelerating Vietnam's attainment of the Joint United Nations Programme on HIV/AIDS 90-90-90 goals.