Exploring the Anti-Hypoxaemia Effect of Hydromethylthionine: A Prospective Study of Phase 3 Clinical Trial Participants.

Methylthioninium chloride (MTC) is a standard treatment for methaemoglobinaemia. A preparation of reduced MTC has been reported to increase blood oxygen saturation (SpO2) and lower respiratory rates in patients with severe COVID-19. We have developed a stable form of reduced methylthionine (hydromethylthionine-mesylate, HMTM) having a benign safety profile in two Phase 3 trials in Alzheimer's disease. The aim of this prospective study was to determine the effects of oral HMTM on SpO2 and methaemoglobin (metHb) levels in a cohort of patients with mild hypoxaemia not due to COVID-19. Eighteen participants randomised to a single dose of 4, 75, 100 or 125 mg doses of HMTM had SpO2 levels below 94% at baseline. Patients were routinely monitored by pulse oximetry after 4 h, and after 2 and 6 weeks of twice daily dosing. Significant ~3% increases in SpO2 occurred within 4 h and were sustained over 2 and 6 weeks with no dose differences. There were small dose-dependent increases (0.060-0.162%) in metHb levels over 2 to 6 weeks. Minimum-energy computational chemistry revealed that HMT can bind within 2.10 Å of heme iron by donating a pair of electrons from the central nitrogen of HMT to d orbitals of heme iron, but with lower affinity than oxygen. In conclusion, HMTM can increase SpO2 without reducing metHb by acting as a strong displaceable field ligand for heme iron. We hypothesise that this facilitates a transition from the low oxygen affinity T-state of heme to the higher affinity R-state. HMTM has potential as an adjunctive treatment for hypoxaemia.

Neurochemical, histological, and behavioral profiling of the acute, sub-acute, and chronic MPTP mouse model of Parkinson's disease.

Parkinson's disease (PD) is a heterogeneous multi-systemic disorder unique to humans characterized by motor and non-motor symptoms. Preclinical experimental models of PD present limitations and inconsistent neurochemical, histological, and behavioral readouts. The 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) mouse model of PD is the most common in vivo screening platform for novel drug therapies; nonetheless, behavioral endpoints yielded amongst laboratories are often discordant and inconclusive. In this study, we characterized neurochemically, histologically, and behaviorally three different MPTP mouse models of PD to identify translational traits reminiscent of PD symptomatology. MPTP was intraperitoneally (i.p.) administered in three different regimens: (i) acute-four injections of 20 mg/kg of MPTP every 2 h; (ii) sub-acute-one daily injection of 30 mg/kg of MPTP for 5 consecutive days; and (iii) chronic-one daily injection of 4 mg/kg of MPTP for 28 consecutive days. A series of behavioral tests were conducted to assess motor and non-motor behavioral changes including anxiety, endurance, gait, motor deficits, cognitive impairment, circadian rhythm and food consumption. Impairments in balance and gait were confirmed in the chronic and acute models, respectively, with the latter showing significant correlation with lesion size. The sub-acute model, by contrast, presented with generalized hyperactivity. Both, motor and non-motor changes were identified in the acute and sub-acute regime where habituation to a novel environment was significantly reduced. Moreover, we report increased water and food intake across all three models. Overall, the acute model displayed the most severe lesion size, while across the three models striatal dopamine content (DA) did not correlate with the behavioral performance. The present study demonstrates that detection of behavioral changes following MPTP exposure is challenging and does not correlate with the dopaminergic lesion extent.

Apolipoprotein E loss of function: Influence on murine brain markers of physiology and pathology.

The canonical role of Apolipoprotein E (ApoE) is related to lipid and cholesterol metabolism, however, additional functions of this protein have not been fully described. Given the association of ApoE with diseases such as Alzheimer's Disease (AD), it is clear that further characterisation of its roles, especially within the brain, is needed. Therefore, using protein and gene expression analyses of neonatal and 6-month old brain tissues from an ApoE knockout mouse model, we examined ApoE's contribution to several CNS pathways, with an emphasis on those linked to AD. Early neonatal changes associated with ApoE-/- were observed, with decreased soluble phosphorylated tau (p-tau, -40 %), increased synaptophysin (+36 %) and microglial Iba1 protein levels (+25 %) vs controls. Progression of the phenotype was evident upon analysis of 6-month-old tissue, where decreased p-tau was also confirmed in the insoluble fraction, alongside reduced synaptic and increased amyloid precursor protein (APP) protein levels. An age comparison further underlined deviations from WT animals and thus the impact of ApoE loss on neuronal maturation. Taken together, our data implicate ApoE modulation of multiple CNS roles. Loss of function is associated with alterations from birth, and include synaptic deficits, neuroinflammation, and changes to key AD pathologies, amyloid-ß and tau.

The BACE1 inhibitor LY2886721 improves diabetic phenotypes of BACE1 knock-in mice.

AIM: The ß-site amyloid precursor protein (APP) cleaving enzyme 1 (BACE1) has been identified as the central initiator of amyloid ß (Aß) generation in the brain, the key hallmark of Alzheimer's disease (AD). However, recent studies provided evidence that BACE1 also plays a crucial role in metabolic regulation, and we have shown that neuronal human BACE1 knock-in mice (PLB4) display type 2 diabetes mellitus (T2DM)-like symptoms alongside AD-like impairments. Hence, we here investigated if targeted BACE1 inhibition using LY2886721, an active site BACE1 inhibitor, would improve glucose homeostasis, insulin sensitivity and motor performance in PLB4 mice. MATERIALS AND METHODS: LY2886721 was administered as a dietary supplement (0.02% wt/wt) for six consecutive weeks. Physiological, metabolic and motor assessments were performed during the last two weeks of treatment, followed by molecular tissue analyses post-mortem. RESULTS: LY2886721 treatment improved glucose homeostasis and hepatic gluconeogenesis in diabetic PLB4 mice, as determined by improvements in basal glucose and glucose/pyruvate tolerance tests. Furthermore, LY2886721 improved hepatic insulin sensitivity, as indicated by enhanced basal hyperphosphorylation of insulin receptors. In PLB4 brains, we detected altered basal conditions of APP expression and processing, with beneficial effects on APP processing achieved by LY2886721 treatment. No improvements in motor coordination were found. CONCLUSIONS: Our data provide support for a role of BACE1 as a regulator of systemic glucose homeostasis and suggest BACE1 inhibitors for the treatment of T2DM-associated pathologies, especially in cases where diabetes is comorbid to AD.

NLRP3 inflammasome inhibition with MCC950 improves insulin sensitivity and inflammation in a mouse model of frontotemporal dementia.

The NOD-like receptor pyrin domain-containing protein 3 (NLRP3) inflammasome has been implicated as a crucial component in both neurodegeneration and diabetes. However, the role of metabolic signalling pathways and the NLRP3 inflammasome in frontotemporal dementia remain largely elusive. We therefore investigated the effects of an NLRP3 inhibitor (MCC950) in a murine tau knock-in (PLB2TAU) model vs. wild-type (PLBWT) control mice. In male PLB2TAU mice (4 months at start of study), MCC950 treatment (20 mg/kg, for 12 weeks) improved insulin sensitivity and reduced circulating plasma insulin levels. Further molecular analysis suggested normalisation in insulin signalling pathways in both liver and muscle tissue. Treatment also resulted in improvements in inflammation and ER stress signalling, both peripherally and centrally, alongside a partial normalisation of phospho-tau levels. Overall, we provide evidence that MCC950 improved metabolic, inflammatory and frontotemporal dementia (FTD) relevant phenotypes in multiple tissues. NLRP3 inhibition may therefore offer a therapeutic approach to ameliorate FTD pathology.

Knock-in of Mutated hTAU Causes Insulin Resistance, Inflammation and Proteostasis Disturbance in a Mouse Model of Frontotemporal Dementia.

Diabetes and obesity have been implicated as risk factors for dementia. However, metabolic mechanisms and associated signalling pathways have not been investigated in detail in frontotemporal dementia. We therefore here characterised physiological, behavioural and molecular phenotypes of 3- and 8-month-old male tau knock-in (PLB2TAU) vs wild-type (PLBWT) mice. Homecage analysis suggested intact habituation but a dramatic reduction in exploratory activity in PLB2TAU mice. Deficits in motor strength were also observed. At 3 months, PLB2TAU mice displayed normal glucose handling but developed hyperglycaemia at 8 months, suggesting a progressive diabetic phenotype. Brain, liver and muscle tissue analyses confirmed tissue-specific deregulation of metabolic and homeostatic pathways. In brain, increased levels of phosphorylated tau and inflammation were detected alongside reduced ER regulatory markers, overall suggesting a downregulation in essential cellular defence pathways. We suggest that subtle neuronal expression of mutated human tau is sufficient to disturb systems metabolism and protein handling. Whether respective dysfunctions in tauopathy patients are also a consequence of tau pathology remains to be confirmed, but could offer new avenues for therapeutic interventions.

Effects of Liraglutide and Fenretinide treatments on the diabetic phenotype of neuronal human BACE1 knock-in mice.

We recently reported that brain-specific human ß-secretase 1 (BACE1) knock-in (PLB4), a mouse model of sporadic Alzheimer's disease (AD), also develops a severe diabetic phenotype characterised by impaired glucose homeostasis, decreased insulin sensitivity and a fatty liver phenotype. Hence, we here aimed to assess if targeted anti-diabetic therapies (Liraglutide and Fenretinide) would attenuate the diabetic and behavioural phenotype of these mice. PLB4 mice and wild-type (WT) controls were administered Liraglutide or Fenretinide for ten consecutive weeks alongside vehicle-treated mice. Physiological (body weight and mass composition, glucose tolerance, serum hormone concentration), behavioural (locomotor activity) and molecular assessments were performed in mice pre- and post-treatment. Liraglutide and Fenretinide treatments inhibited adiposity gain and decreased circulating serum triglyceride (with Liraglutide) and leptin (with Fenretinide) levels in PLB4 mice. We also found that PLB4 mice exhibited increased levels of serum dipeptidyl peptidase 4 (DPP4), together with up-regulated hepatic expression of Dpp4, retinol binding protein 4 (Rbp4) and sterol regulatory element-binding 1c (Srebp1c), which was normalised by both treatments. Interestingly, Liraglutide treatment slowed down habituation to a novel environment and increased secondary night activity peak in WT mice, suggesting an impact on circadian activity regulation. However, neither treatment improved glucose homeostasis in PLB4 mice, implying that impaired glucose metabolism in this genotype may not be associated with glucagon like peptide 1 (GLP-1) and/or RBP4-mediated pathways. In summary, this study provides new insights into molecular mechanisms underlying neuronal BACE1-mediated metabolic regulation and implicates BACE1 as a putative regulator of systemic DPP4 levels.

Distinctive temporal profiles of detergent-soluble and -insoluble tau and Aß species in human Alzheimer's disease.

Alzheimer's disease (AD) pathology relevant proteins tau and beta-amyloid (Aß) exist as an array of post-translationally modified and conformationally altered species with varying abundance, solubility and toxicity. Insoluble neurofibrillary tau tangles and Aß plaques are end-stage AD hallmarks, yet may carry less disease significance compared to soluble species. At present, it is unclear how soluble and insoluble tau and Aß relate to each other as well as to disease progression. Here, detergent soluble and insoluble fractions generated from post-mortem human temporal lobe samples (Brodmann area 21) were probed for tau and Aß markers in immuno-dot assays. Measures were quantified according to diagnosis (AD cf. Non-AD), neuropathological severity, and correlated with disease progression (Braak stages). All markers were elevated within AD cases cf. non-AD controls (p < 0.05) independent of solubility. However, when considered according to neuropathological severity, phospho-tau (detected via CP13 and AT8 antibodies) was elevated early within the soluble fraction (p < 0.05 intermediate cf. low severity) and emerged only later within the insoluble fraction (p < 0.05 high cf. low severity). In contrast, PHF1 phospho-tau, TOC1 reactive tau oligomers and amyloid markers rose within the two fractions simultaneously. Independent of solubility, cognitive correlations were observed for tau makers and for fibrillary amyloid (OC), however only soluble total Aß was significantly correlated with intellectual impairment. Following the exclusion of end-stage cases, only soluble total Aß remained correlated with cognition. The data indicate differential rates of protein aggregation during AD progression and confirm the disease relevance of early emerging soluble Aß species.

Co-production of 11α-hydroxyprogesterone and ethanol using recombinant yeast expressing fungal steroid hydroxylases.

BACKGROUND: Bioethanol production from sustainable sources of biomass that limit effect on food production are needed and in a biorefinery approach co-products are desirable, obtained from both the plant material and from the microbial biomass. Fungal biotransformation of steroids was among the first industrial biotransformations allowing corticosteroid production. In this work, the potential of yeast to produce intermediates needed in corticosteroid production is demonstrated at laboratory scale following bioethanol production from perennial ryegrass juice. RESULTS: Genes encoding the 11α-steroid hydroxylase enzymes from Aspergillus ochraceus (11α-SHAoch) and Rhizopus oryzae (CYP509C12) transformed into Saccharomyces cerevisiae for heterologous constitutive expression in p425TEF. Both recombinant yeasts (AH22:p11α-SHAoch and AH22:p509C12) exhibited efficient progesterone bioconversion (on glucose minimal medial containing 300 µM progesterone) producing either 11α-hydroxyprogesterone as the sole metabolite (AH22:p11α-SHAoch) or a 7:1 mixture of 11α-hydroxyprogesterone and 6ß-hydroxyprogesterone (AH22:p509C12). Ethanol yields for AH22:p11α-SHAoch and AH22:p509C12 were comparable resulting in ≥75% conversion of glucose to alcohol. Co-production of bioethanol together with efficient production of the 11-OH intermediate for corticosteroid manufacture was then demonstrated using perennial ryegrass juice. Integration of the 11α-SHAoch gene into the yeast genome (AH22:11α-SHAoch+K) resulted in a 36% reduction in yield of 11α-hydroxyprogesterone to 174 µmol/L using 300 µM progesterone. However, increasing progesterone concentration to 955 µM and optimizing growth conditions increased 11α-hydroxyprogesterone production to 592 µmol/L product formed. CONCLUSIONS: The progesterone 11α-steroid hydroxylases from A. ochraceus and R. oryzae, both monooxygenase enzymes of the cytochrome P450 superfamily, have been functionally expressed in S. cerevisiae. It appears that these activities in fungi are not associated with a conserved family of cytochromes P450. The activity of the A. ochraceous enzyme was important as the specificity of the biotransformation yielded just the 11-OH product needed for corticosteroid production. The data presented demonstrate how recombinant yeast could find application in rural biorefinery processes where co-production of value-added products (11α-hydroxyprogesterone and ethanol) from novel feedstocks is an emergent and attractive possibility.

Deep Vein Thrombosis and Pulmonary Embolism in a Mountain Guide: Awareness, Diagnostic Challenges, and Management Considerations at Altitude.

High intensity exercise is associated with several potentially thrombogenic risk factors, including dehydration and hemoconcentration, vascular trauma, musculoskeletal injuries, inflammation, long-distance travel, and contraceptive usage. These are well documented in case reports of venous thrombosis in track and field athletes. For mountaineers and those working at high altitude, additional risks exist. However, despite there being a high degree of vigilance for "classic" conditions encountered at altitude (eg, acute mountain sickness, high altitude pulmonary edema, and high altitude cerebral edema), mainstream awareness regarding thrombotic conditions and their complications in mountain athletes is relatively low. This is significant because thromboembolic events (including deep vein thrombosis, pulmonary embolism, and cerebral vascular thrombosis) are not uncommon at altitude. We describe a case of deep vein thrombosis and pulmonary embolism in a male mountain guide and discuss the diagnostic issues encountered by his medical practitioners. Potential risk factors affecting blood circulation (eg, seated car travel and compression of popliteal vein) and blood hypercoagulability (eg, hypoxia, environmental and psychological stressors [avalanche risk, extreme cold]) relevant to the subject of this report and mountain athletes in general are identified. Considerations for mitigating and managing thrombosis in addition to personalized care planning at altitude are discussed. The prevalence of thrombosis in mountain athletes is uncharted, but lowlanders increasingly go to high altitude to trek, ski, or climb. Blood clots can and do occur in physically active people, and thrombosis prevention and recognition will demand heightened awareness among participants, healthcare practitioners, and the altitude sport/leisure industry at large.

Venous Thromboembolism in Physically Active People: Considerations for Risk Assessment, Mainstream Awareness and Future Research.

The global healthcare burden of venous thromboembolism (VTE) and associated comorbidities (e.g., obesity, heart disease and cancer) is significant. Physical activity-especially cardiovascular exercise-is popularly acclaimed for gold-standard prevention. Paradoxically, intensive training can expose athletes to several potentially thrombogenic risk factors (e.g., heat stress, dehydration, blood vessel injury and inflammation). However, awareness regarding the risk of VTE in physically active people is generally lacking. Given that the overall incidence of asymptomatic and/or occult blood clots that resolve spontaneously is uncharted, and because symptoms and sequelae are not always 'textbook', triage evaluation and diagnosis of VTE at large can be challenging. Front-line clinical evaluations, including the major Wells scoring criteria, are (versus the total number of possible factors and diagnoses) comparably reductionist, and the point at which a minor risk might be considered significant in one person-but not in another-is subjective. Considering the popular associations between VTE and inactivity, athletes might be at greater risk of a missed diagnosis quite simply because their cardiovascular conditioning presents as the polar opposite to standard assessment criteria. Undoubtedly, risk factors for VTE associated with exercise are not unique to cardiovascular training or athletes, but the extent to which they might increase the chances of blood clot precipitation in certain participants warrants attention. A multi-agency approach, including research to inform mainstream understanding and awareness about risk factors for VTE in patient groups across age, comorbidity and activity spectra, is required. In this article, the potential for pre-participatory thrombophilia screening, haemostatic monitoring and personalized prophylactic guidelines is discussed.

Use of 70% alcohol for the routine removal of microbial hard surface bioburden in life science cleanrooms.

Alcohol-based disinfectants are used for the removal of microbial hard surface bioburden in Life science Cleanrooms. Evidence for using formulations containing 70% alcohol has been lost over time but probably originates from historical observations of the activity of 60-70% alcohol. Tradition is no longer adequate to inform contemporary cleaning practice. We evaluated the efficacy of ethanol, isopropanol and trade-specific denatured alcohol 7 against vegetative Pseudomonas aeruginosa, Staphylococcus aureus, Escherichia coli and Enterococcus hirae using standardized European Suspension and Hard Surface tests. All three alcohols were effective at lower concentrations than the 70% standard. This constitutes the first evaluation of disinfectant formulations containing ≤70% alcohol using standard methodology. The utility of trade-specific denatured alcohol #7 and evidence-based cleanroom practice warrant further validation.

Co-production of bioethanol and probiotic yeast biomass from agricultural feedstock: application of the rural biorefinery concept.

Microbial biotechnology and biotransformations promise to diversify the scope of the biorefinery approach for the production of high-value products and biofuels from industrial, rural and municipal waste feedstocks. In addition to bio-based chemicals and metabolites, microbial biomass itself constitutes an obvious but overlooked by-product of existing biofermentation systems which warrants fuller attention. The probiotic yeast Saccharomyces boulardii is used to treat gastrointestinal disorders and marketed as a human health supplement. Despite its relatedness to S. cerevisiae that is employed widely in biotechnology, food and biofuel industries, the alternative applications of S. boulardii are not well studied. Using a biorefinery approach, we compared the bioethanol and biomass yields attainable from agriculturally-sourced grass juice using probiotic S. boulardii (strain MYA-769) and a commercial S. cerevisiae brewing strain (Turbo yeast). Maximum product yields for MYA-769 (39.18 [±2.42] mg ethanol mL(-1) and 4.96 [±0.15] g dry weight L(-1)) compared closely to those of Turbo (37.43 [±1.99] mg mL(-1) and 4.78 [±0.10] g L(-1), respectively). Co-production, marketing and/or on-site utilisation of probiotic yeast biomass as a direct-fed microbial to improve livestock health represents a novel and viable prospect for rural biorefineries. Given emergent evidence to suggest that dietary yeast supplementations might also mitigate ruminant enteric methane emissions, the administration of probiotic yeast biomass could also offer an economically feasible way of reducing atmospheric CH4.

Co-production of ethanol and squalene using a Saccharomyces cerevisiae ERG1 (squalene epoxidase) mutant and agro-industrial feedstock.

BACKGROUND: Genetically customised Saccharomyces cerevisiae that can produce ethanol and additional bio-based chemicals from sustainable agro-industrial feedstocks (for example, residual plant biomass) are of major interest to the biofuel industry. We investigated the microbial biorefinery concept of ethanol and squalene co-production using S. cerevisiae (strain YUG37-ERG1) wherein ERG1 (squalene epoxidase) transcription is under the control of a doxycycline-repressible tet0 7 -CYC1 promoter. The production of ethanol and squalene by YUG37-ERG1 grown using agriculturally sourced grass juice supplemented with doxycycline was assessed. RESULTS: Use of the tet0 7 -CYC1 promoter permitted regulation of ERG1 expression and squalene accumulation in YUG37-ERG1, allowing us to circumvent the lethal growth phenotype seen when ERG1 is disrupted completely. In experiments using grass juice feedstock supplemented with 0 to 50 µg doxycycline mL(-1), YUG37-ERG1 fermented ethanol (22.5 [±0.5] mg mL(-1)) and accumulated the highest squalene content (7.89 ± 0.25 mg g(-1) dry biomass) and yield (18.0 ± 4.18 mg squalene L(-1)) with supplements of 5.0 and 0.025 µg doxycycline mL(-1), respectively. Grass juice was found to be rich in water-soluble carbohydrates (61.1 [±3.6] mg sugars mL(-1)) and provided excellent feedstock for growth and fermentation studies using YUG37-ERG1. CONCLUSION: Residual plant biomass components from crop production and rotation systems represent possible substrates for microbial fermentation of biofuels and bio-based compounds. This study is the first to utilise S. cerevisiae for the co-production of ethanol and squalene from grass juice. Our findings underscore the value of the biorefinery approach and demonstrate the potential to integrate microbial bioprocess engineering with existing agriculture.

Clotrimazole as a potent agent for treating the oomycete fish pathogen Saprolegnia parasitica through inhibition of sterol 14α-demethylase (CYP51).

A candidate CYP51 gene encoding sterol 14α-demethylase from the fish oomycete pathogen Saprolegnia parasitica (SpCYP51) was identified based on conserved CYP51 residues among CYPs in the genome. It was heterologously expressed in Escherichia coli, purified, and characterized. Lanosterol, eburicol, and obtusifoliol bound to purified SpCYP51 with similar binding affinities (Ks, 3 to 5 µM). Eight pharmaceutical and six agricultural azole antifungal agents bound tightly to SpCYP51, with posaconazole displaying the highest apparent affinity (Kd, ≤3 nM) and prothioconazole-desthio the lowest (Kd, ∼51 nM). The efficaciousness of azole antifungals as SpCYP51 inhibitors was confirmed by 50% inhibitory concentrations (IC50s) of 0.17 to 2.27 µM using CYP51 reconstitution assays. However, most azole antifungal agents were less effective at inhibiting S. parasitica, Saprolegnia diclina, and Saprolegnia ferax growth. Epoxiconazole, fluconazole, itraconazole, and posaconazole failed to inhibit Saprolegnia growth (MIC100, >256 µg ml(-1)). The remaining azoles inhibited Saprolegnia growth only at elevated concentrations (MIC100 [the lowest antifungal concentration at which growth remained completely inhibited after 72 h at 20°C], 16 to 64 µg ml(-1)) with the exception of clotrimazole, which was as potent as malachite green (MIC100, ∼1 µg ml(-1)). Sterol profiles of azole-treated Saprolegnia species confirmed that endogenous CYP51 enzymes were being inhibited with the accumulation of lanosterol in the sterol fraction. The effectiveness of clotrimazole against SpCYP51 activity (IC50, ∼1 µM) and the concentration inhibiting the growth of Saprolegnia species in vitro (MIC100, ∼1 to 2 µg ml(-1)) suggest that clotrimazole could be used against Saprolegnia infections, including as a preventative measure by pretreatment of fish eggs, and for freshwater-farmed fish as well as in leisure activities.

Mitigation of human-pathogenic fungi that exhibit resistance to medical agents: can clinical antifungal stewardship help?

Reducing indiscriminate antimicrobial usage to combat the expansion of multidrug-resistant human-pathogenic bacteria is fundamental to clinical antibiotic stewardship. In contrast to bacteria, fungal resistance traits are not understood to be propagated via mobile genetic elements, and it has been proposed that a global explosion of resistance to medical antifungals is therefore unlikely. Clinical antifungal stewardship has focused instead on reducing the drug toxicity and high costs associated with medical agents. Mitigating the problem of human-pathogenic fungi that exhibit resistance to antimicrobials is an emergent issue. This article addresses the extent to which clinical antifungal stewardship could influence the scale and epidemiology of resistance to medical antifungals both now and in the future. The importance of uncharted selection pressure exerted by agents outside the clinical setting (agricultural pesticides, industrial xenobiotics, biocides, pharmaceutical waste and others) on environmentally ubiquitous spore-forming molds that are lesserstudied but increasingly responsible for drug-refractory infections is considered.

Basic skills workshop for physician assistant educators: effects of participation on perceived mastery of teaching skills and job satisfaction.

PURPOSE: The study examined participation in the Basic Skills Faculty Development Workshops (BSW) offered by the Physician Assistant Education Association (PAEA). The aim was to determine the effects of participation on perceived mastery of teaching skills and job satisfaction. METHODS: The 1,290 faculty and program director members of PAEA were invited to complete an electronic survey regarding their past participation in a BSW, levels of satisfaction with various aspects of their work, and their perception of their level of mastery of various teaching skills. Additionally, those who had participated in these workshops completed a section on colleague relationships that were developed or strengthened through workshop participation. RESULTS: Approximately half (n = 248) of the 493 respondents had participated in a BSW. Mean scores for satisfaction with salary, rank, position, and overall satisfaction did not differ significantly according to BSW participation. Perceived mastery of various teaching skills was significantly higher for nonattendees of BSW. However, controlling for "years in physician assistant education" nullified that association. Attendees reported a mean of 1.02 (SD = 1.47) new mentoring relationships and 2.45 (SD = 2.97) new peer relationships. Satisfaction with current position was significantly positively correlated with the number of colleague relationships. The number of new and strengthened mentor relationships correlated significantly with perceived mastery of advising students. CONCLUSIONS: Basic Skills Workshop attendees experience acceleration in their perceived mastery of teaching skills, closing the proficiency gap between them and their more-experienced colleagues who did not attend a Basic Skills Workshop. Also, participation is associated with an increased number of colleague relationships, which has a positive effect on satisfaction.

Two clinical isolates of Candida glabrata exhibiting reduced sensitivity to amphotericin B both harbor mutations in ERG2.

Two novel isolates of Candida glabrata exhibiting reduced sensitivity to amphotericin B (MIC, 8 µg ml(-1)) were found to be ERG2 mutants, wherein Δ(8)-sterol intermediates comprised >90% of the total cellular sterol fraction. Both harbored an alteration at Thr(121) in ERG2; the corresponding residue (Thr(119)) in Saccharomyces cerevisiae is essential for sterol Δ8-Δ7 isomerization. This constitutes the first report of C. glabrata harboring mutations in ERG2 and exhibiting reduced sensitivity to amphotericin B.