Novel Determinants That Influence Azole Susceptibility in Candida glabrata and Candida albicans

Despite the scientific and medical communities’ best efforts, the incidence of fungal infections in susceptible populations continues to rise. The most common cause of these opportunistic fungal infections is Candida. In fact, Candida is the fourth most common pathogen associated with nosocomial blood stream infections. Reported mortality rates for patients with candidemia vary, but have not decreased in the past fifteen years and are reported to be as high as 50%. Candida glabrata, second only to Candida albicans among Candida infections, expresses high rates of resistance to treatment with arguably the best class of currently available antifungals - the azoles. Other available antifungals have associated toxicities, are not available as oral dosage forms or are cost prohibitive. As multidrug resistant C. glabrata have been reported, the need to find ways to overcome resistance to azoles is more pressing than ever. The work described here highlights our efforts to develop a better understanding of azole resistance in Candida, with a focus on C. glabrata, which can then be utilized to inform better strategies for decreasing or preventing resistance. In C. glabrata clinical azole resistance is mediated almost exclusively by activating mutations in the zinc cluster transcription factor Pdr1, which controls the genes encoding the multidrug resistance transporters Cdr1, Pdh1, and Snq2. However, the specific relative contribution of these transporters to resistance is not known. In order to determine this, the SAT1 flipper method was used to delete CDR1, PDH1, and SNQ2 in a strain of C. glabrata engineered to carry a clinically relevant activating mutation in PDR1. Susceptibility testing was performed according to the CLSI guidelines with minor modifications and confirmed with Etest strips. Of the single transporter deletion strains, only CDR1 deletion resulted in decreased azole MIC. Deletion of PDH1 in combination with CDR1 resulted in a moderate decrease in MIC from that observed with deletion of CDR1 alone. SNQ2 deletion only decreased the MIC in the triple deletion strain in the absence of both CDR1 and PDH1. Deletion of all three transporters in combination decreased the MIC to the level observed in the PDR1 deletion strains for some, but not all of the azoles tested, which indicates additional Pdr1 targets likely play a minor role in this process. These results demonstrate that Cdr1 is the most important Pdr1-mediated multidrug resistance transporter for azole resistance in C. glabrata, suggesting that targeting this transporter alone might be sufficient to overcome this clinical problem. Upc2 and Ecm22 in S. cerevisiae and Upc2 in C. albicans are the transcriptional regulators of ERG11, the gene encoding the target of azoles in the ergosterol biosynthesis pathway. Recently two homologs for these transcription factors, UPC2A and UPC2B, were identified in C. glabrata. One of these, UPC2A, was shown to influence azole susceptibility. We hypothesized that due to the global role for Upc2 in sterol biosynthesis in S. cerevisiae and C. albicans, disruption of UPC2A would enhance the activity of fluconazole in both azole-susceptible-dose dependent (SDD) and -resistant C. glabrata clinical isolates. To test this hypothesis, we constructed mutants disrupted for UPC2A and UPC2B alone and in combination in a matched pair of clinical azole-SDD and - resistant isolates. Disruption of UPC2A in both the SDD and resistant isolates resulted in increased susceptibility to sterol biosynthesis inhibitors, including a reduction in fluconazole minimum inhibitory concentration and minimum fungicidal concentration, enhanced azole activity by time-kill analysis, a decrease in ergosterol content, and downregulation of baseline and inducible expression of several sterol biosynthesis genes. Our results indicate that Upc2A is a key regulator of ergosterol biosynthesis and is essential for resistance to sterol biosynthesis inhibitors in C. glabrata. As such, the UPC2A pathway may represent a potential co-therapeutic target for enhancing azole activity against this organism. The importance of Pdr1 in azole resistance in C. glabrata is well established. Our understanding of how Pdr1 is being regulated, however, is predominantly informed by regulation of similar systems in other organisms. In order to identify genes that interact with the Pdr1 transcriptional pathway, and influence the susceptibility of C. glabrata to fluconazole, we screened a collection of deletion mutants for those exhibiting increased resistance to fluconazole. Deletion of the gene coding for a protein homologous to the S. cerevisiae J protein Jjj1 resulted in decreased fluconazole susceptibility. We used the SAT1 flipper method to generate independent deletion mutants for JJJ1 in a SDD clinical isolate. Expression of both CDR1 and PDR1 was increased in the absence of JJJ1. In the absence of CDR1 or PDR1, deletion of JJJ1 had only a modest effect on fluconazole susceptibility. Transcriptional profiling using RNA-Seq revealed up-regulation of genes of the Pdr1 regulon in the absence of JJJ1. Jjj1 appears to be a negative regulator of fluconazole resistance in C. glabrata and acts primarily through up-regulation of the ABC transporter gene CDR1 via activation of the Pdr1 transcriptional pathway. Unlike C. glabrata which has essentially one mechanism of resistance, in C. albicans clinical azole resistance can be attributed to multiple mechanisms, often in combination. The RTA3 gene, coding for a member of the Rta1p-like lipid-translocating exporter family, is coordinately upregulated with the ABC transporter genes CDR1 and CDR2 in azole-resistant clinical isolates of C. albicans that carry activating mutations in the transcription factor Tac1p. We show here that deleting RTA3 in an azole-resistant clinical isolate carrying a Tac1p activating mutation lowered fluconazole resistance by two-fold, while overexpressing RTA3 in an azole-susceptible clinical isolate resulted in enhanced fluconazole tolerance associated with trailing growth in a liquid microtiter plate assay. We also demonstrate that an Rta3p-GFP fusion protein localizes predominantly to the plasma membrane, consistent with a putative function for Rta3p as a lipid translocase

Optimal Eventfulness of Narratives

OpenAccess Series in Informatics (OASICS)This study examines whether there is an optimal degree of eventfulness of short narratives. We ask whether there is a specific degree of eventfulness (unexpectedness) that makes them “stick” better than other stories so that they are maintained more faithfully in serial reproduction (telephone games). The result is: probably not. The finding is that there is an impressive correlation of eventfulness rankings of original stories and resulting retellings in serial reproduction, despite the change of many other story elements and almost regardless of low or high eventfulness. Put more simply, people remember and retell “eventfulness” accurately, even when the actual events and circumstances of a story are changed

Perceptions of the Delivery of Group and Individual Writing Telehealth Interventions for Students 7 to 12 Years of Age using Two Virtual Platforms: A Pilot Study

Background: Existing evidence supports occupational therapy’s (OT) role in improving handwriting skills; however, evidence is limited regarding the delivery of virtual intervention. Method: This pilot study reviewed the use of a virtual interprofessional writing program for five children 7 to 12 years of age that addressed both handwriting (OT) and spelling (speech-language pathology) skills. The program included eight weekly sessions using Microsoft Teams (first four sessions) and Cisco WebEx (last four sessions). A parent survey consisting of 14 questions was conducted to gauge overall satisfaction with the program as well as learn about platform preferences. In addition, the occupational therapists provided insights from using the features of the two platforms. Results: Overall, the families were satisfied with the virtual program, the primary strengths being the engagement of their child, the materials used in the program, and skill development. The occupational therapists identified various features of the virtual platforms that impacted their use for individual and group interventions. Conclusion: Information from this pilot study can be used to help clinicians when preparing for the virtual delivery of OT services. It also provided feedback from parents that is consistent with previous literature about strengths and weaknesses of virtual services

Dental Fluoride Varnish Application During Medical Visits Among Children Who Are Privately Insured

This cross-sectional study examines fluoride varnish application rates during well-child medical visits and identify characteristics associated with fluoride varnish receipt

What makes an operational Farm Soil Carbon Code? Insights from a global comparison of existing soil carbon codes using a structured analytical framework

Soils have the potential to sequester and store significant amounts of carbon, contributing towards climate change mitigation. Soil carbon markets are now emerging to pay farmers for changes in land use or management that absorb carbon from the atmosphere, governed by codes that ensure additionality, permanence and non-leakage whilst protecting against unintentional reversals. This paper represents the first global comparative analysis of agricultural soil carbon codes, providing new insights into the wide range of approaches currently used to govern these emerging markets internationally. To do this, the paper first develops an analytical framework for the systematic comparison of codes, which could be applied to the analysis of codes in other land uses and habitats. This framework was then used to identify commonalities and differences in methods, projects, administration and commercialisation and associated code documents for 12 publicly available codes from the UK, France, Australia, USA and international bodies. Codes used a range of mechanisms to manage: additionality (including legal, adoption, financial viability and investment tests); uncertainty and risks around soil carbon sequestration (including minimum permanence periods, carbon buffers, contractual arrangements and/or insurance policies); leakage (including restriction of eligible practices and monitoring to subtract leakage from verified sequestration); baselines (including multi-year and variable buffers based on empirical data or models); measurement, reporting and verification methods (stipulating time intervals, methods, data sources and assessments of uncertainty); auditing; resale of carbon units; stakeholder engagement; and approaches to ensure market integrity (such as buyer checks). The paper concludes by discussing existing MRV methods and codes that could be adapted for use in the UK and evaluates the need for an over-arching standard for soil carbon codes in the UK, to which existing codes and other schemes already generating soil carbon credits could be assessed and benchmarked

Fact vs. Affect in the Telephone Game: All Levels of Surprise Are Retold With High Accuracy, Even Independently of Facts

When people retell stories, what guides their retelling? Most previous research on story retelling and story comprehension has focused on information accuracy as the key measure of stability in transmission. This paper suggests that there is a second, affective, dimension that provides stability for retellings, namely the audience affect of surprise. In a large-sample study with multiple iterations of retellings, we found evidence that people are quite accurate in preserving all degrees of surprisingness in serial reproduction – even when the event that produced the surprisingness in the original story is dropped or changed. Thus, we propose that the preservation of affect is an implicit goal of retelling: merely do retellers not recall highly surprising events better, but rather they register all levels of surprisingness precisely and aim to surprise their implied audience to same degree. This study used 2,389 participants.Significance Statement: Story retelling is a process whereby cultural information is transmitted horizontally across social networks and vertically down generations. For the most part, retelling research has focused on the relevance and stability of factual information, “who did what, where, when, and why”; comparatively little is known about the transmission of affective information. We suggest that affect can serve as a second axis of stability for retelling, partially independent from factual information. In serial reproduction tasks modeled after the telephone game, we find that surprisingness of stories is well preserved across retellings – even when the facts and events of the story are not. The findings are significant for the communication of information, and thereby also the stability and transformation of culture in general

Guidelines of the American Society of Mammalogists for the use of wild mammals in research

Guidelines for use of wild mammal species are updated from the American Society of Mammalogists (ASM) 2007 publication. These revised guidelines cover current professional techniques and regulations involving mammals used in research and teaching. They incorporate additional resources, summaries of procedures, and reporting requirements not contained in earlier publications. Included are details on marking, housing, trapping, and collecting mammals. It is recommended that institutional animal care and use committees (IACUCs), regulatory agencies, and investigators use these guidelines as a resource for protocols involving wild mammals. These guidelines were prepared and approved by the ASM, working with experienced professional veterinarians and IACUCs, whose collective expertise provides a broad and comprehensive understanding of the biology of nondomesticated mammals in their natural environments. The most current version of these guidelines and any subsequent modifications are available at the ASM Animal Care and Use Committee page of the ASM Web site (http://mammalsociety.org/committees/index.asp).American Society of Mammalogist

Comparative effects of RRR-alpha- and RRR-gamma-tocopherol on proliferation and apoptosis in human colon cancer cell lines

BACKGROUND: Mediterranean societies, with diets rich in vitamin E isoforms, have a lower risk for colon cancer than those of northern Europe and the Americas. Vitamin E rich diets may neutralize free radicals generated by fecal bacteria in the gut and prevent DNA damage, but signal transduction activities can occur independent of the antioxidant function. The term vitamin E represents eight structurally related compounds, each differing in their potency and mechanisms of chemoprevention. The RRR-γ-tocopherol isoform is found primarily in the US diet, while RRR-α-tocopherol is highest in the plasma. METHODS: The effectiveness of RRR-α- and RRR-γ-tocopherol at inhibiting cell growth and inducing apoptosis in colon cancer cell lines with varying molecular characteristics (SW480, HCT-15, HCT-116 and HT-29) and primary colon cells (CCD-112CoN, nontransformed normal phenotype) was studied. Colon cells were treated with and without RRR-α- or RRR-γ-tocopherol using varying tocopherol concentrations and time intervals. Cell proliferation and apoptosis were measured using the trypan blue assay, annexin V staining, DNA laddering and caspase activation. RESULTS: Treatment with RRR-γ-tocopherol resulted in significant cell death for all cancer cell lines tested, while RRR-α-tocopherol did not. Further, RRR-γ-tocopherol treatment showed no cytotoxicity to normal colon cells CCD-112CoN at the highest concentration and time point tested. RRR-γ-tocopherol treatment resulted in cleavage of PARP, caspase 3, 7, and 8, but not caspase 9. Differences in the percentage cell death and apoptosis were observed in different cell lines suggesting that molecular differences in these cell lines may influence the ability of RRR-γ-tocopherol to induce cell death. CONCLUSION: This is the first study to demonstrate that multiple colon cancer cell lines containing varying genetic alterations will under go growth reduction and apoptosis in the presence of RRR-γ-tocopherol without damage to normal colon cells. The amount growth reduction was dependent upon the molecular signatures of the cell lines. Since RRR-γ-tocopherol is effective at inhibition of cell proliferation at both physiological and pharmacological concentrations dietary RRR-γ-tocopherol may be chemopreventive, while pharmacological concentrations of RRR-γ-tocopherol may aid chemotherapy without toxic effects to normal cells demonstrated by most chemotherapeutic agents

Effect of Deutetrabenazine on Chorea Among Patients With Huntington Disease A Randomized Clinical Trial

Importance Deutetrabenazine is a novel molecule containing deuterium, which attenuates CYP2D6 metabolism and increases active metabolite half-lives and may therefore lead to stable systemic exposure while preserving key pharmacological activity. Objective To evaluate efficacy and safety of deutetrabenazine treatment to control chorea associated with Huntington disease. Design, Setting, and Participants Ninety ambulatory adults diagnosed with manifest Huntington disease and a baseline total maximal chorea score of 8 or higher (range, 0-28; lower score indicates less chorea) were enrolled from August 2013 to August 2014 and randomized to receive deutetrabenazine (n = 45) or placebo (n = 45) in a double-blind fashion at 34 Huntington Study Group sites. Interventions Deutetrabenazine or placebo was titrated to optimal dose level over 8 weeks and maintained for 4 weeks, followed by a 1-week washout. Main Outcomes and Measures Primary end point was the total maximal chorea score change from baseline (the average of values from the screening and day-0 visits) to maintenance therapy (the average of values from the week 9 and 12 visits) obtained by in-person visits. This study was designed to detect a 2.7-unit treatment difference in scores. The secondary end points, assessed hierarchically, were the proportion of patients who achieved treatment success on the Patient Global Impression of Change (PGIC) and on the Clinical Global Impression of Change (CGIC), the change in 36-Item Short Form– physical functioning subscale score (SF-36), and the change in the Berg Balance Test. Results Ninety patients with Huntington disease (mean age, 53.7 years; 40 women [44.4%]) were enrolled. In the deutetrabenazine group, the mean total maximal chorea scores improved from 12.1 (95% CI, 11.2-12.9) to 7.7 (95% CI, 6.5-8.9), whereas in the placebo group, scores improved from 13.2 (95% CI, 12.2-14.3) to 11.3 (95% CI, 10.0-12.5); the mean between-group difference was –2.5 units (95% CI, –3.7 to –1.3) (P < .001). Treatment success, as measured by the PGIC, occurred in 23 patients (51%) in the deutetrabenazine group vs 9 (20%) in the placebo group (P = .002). As measured by the CGIC, treatment success occurred in 19 patients (42%) in the deutetrabenazine group vs 6 (13%) in the placebo group (P = .002). In the deutetrabenazine group, the mean SF-36 physical functioning subscale scores decreased from 47.5 (95% CI, 44.3-50.8) to 47.4 (44.3-50.5), whereas in the placebo group, scores decreased from 43.2 (95% CI, 40.2-46.3) to 39.9 (95% CI, 36.2-43.6), for a treatment benefit of 4.3 (95% CI, 0.4 to 8.3) (P = .03). There was no difference between groups (mean difference of 1.0 unit; 95% CI, –0.3 to 2.3; P = .14), for improvement in the Berg Balance Test, which improved by 2.2 units (95% CI, 1.3-3.1) in the deutetrabenazine group and by 1.3 units (95% CI, 0.4-2.2) in the placebo group. Adverse event rates were similar for deutetrabenazine and placebo, including depression, anxiety, and akathisia. Conclusions and Relevance Among patients with chorea associated with Huntington disease, the use of deutetrabenazine compared with placebo resulted in improved motor signs at 12 weeks. Further research is needed to assess the clinical importance of the effect size and to determine longer-term efficacy and safety