Faculty Bibliography

The yeast Hsp26 protein, a conserved a-crystallin small heatshock chaperone, is assembled in to oligomeric complexes, microscopically visible as distinct cytoplasmic foci. We studied at single cell resolution the dynamics of Hsp26p foci assembly, the mode of their inheritance in to progeny cells and the physiological significance of Hsp26p function. We showed that Hsp26p foci are formed upon cells' entry in to stationary phase, but upon re-entry to proliferation they are asymmetrically retained in the mother cells and are absent from the newborn daughters. Despite the fact that Hsp26p assists re-solubilization of aggregation-prone proteins it does not extend chronological life span nor does it increase the tolerance of either mother or daughters against lethal stresses. Upon sequential HSP26 inductions, newly synthesized Hsp26p is readily incorporated in pre-existing foci, generating larger in size, but similar in appearance foci. At extreme heat-shock conditions, Hsp26p foci break apart into smaller granules dispersed in both mothers and growing buds, while recovery at normal temperature results in Hsp26p foci reassembly. These results suggested that such a complicated mechanism of dynamic Hsp26p assembly and disassembly, as well as asymmetric segregation may contribute to fine tuning regulation of protein aggregates' refolding, cell fitness and survival.

Bimodality in gene expression can generate phenotypic heterogeneity facilitating fitness and growth of isogenic cell populations in suboptimal environments. We investigated the mechanism by which, in conditions of limiting galactose, yeast cell populations activate GAL genes in a bimodal fashion with a cell fraction expressing GAL genes (ON), while the rest subpopulation is kept at the non-expressing (OFF) state. We show that a long non-coding RNA (GAL10-ncRNA) crossing the bidirectional GAL1-10 promoter, decreases the rate by which single cells commit transition to the ON state without affecting the rate of GAL transcription per se in ON cells. This is accomplished by repressing stochastic expression of the bifunctional Gal1p galactokinase, which besides its enzymatic activity acts as an essential inducer of the system under those conditions. We show that once single cells switch to the ON state, the GAL10-ncRNA effect is overridden by accumulating Gal1p levels sufficient to feedback positively on Gal4p, and not by the active transcription of GAL10 that occurs in opposite direction relative to that of GAL10-ncRNA. Conversely, GAL10-ncRNA does not influence transition of ON cells, where Gal4p is active, back to the OFF state. Our model suggests that the functional interplay between GAL10-ncRNA transcription, stochastic Gal1p expression and Gal1p positive feedback on Gal4p constitutes a novel molecular switch mechanism dictating the commitment of individual cells for either metabolic state.

Organisms integrate information of current environmental stressors and can adjust themselves against harmful events that might occur in the future. The molecular processes that lead to such "anticipatory" behaviors, although of great interest, are mostly unexplored and the minimal genetic requirements for reconfiguring key signaling networks in order either to create or to strengthen such vital "anticipatory" capabilities is largely unknown. We identified new "anticipatory" phenotypes in yeast cells by evolving yeast strains that strongly associate a present modest stress with a future deadly one. Whole genome sequencing and classic genetic analysis revealed that two dominant negative ras2 alleles (ras2-K23N and ras2-G17C) displayed a strong "anticipatory" ability being highly resistant to oxidative stress, extremely thermotolerant and long lived only following an initial mild heat shock. We suggest that such "anticipatory" phenotypes can be easily evolved by a single point mutation in a key signaling protein, the Ras2 small GTPase, and we propose a molecular model describing how specific ras2 alleles, and not null ras2 mutants, or mutations in other components of the Ras/cAMP pathway, can enhance the "predictive ability" of cells for future lethal stressors.

There is a substantial effort to increase the accuracy of conflicts of interest (COI) reporting, and reduce the influence of COI between physicians and industry, especially as it relates to clinical practice guidelines.We used the newly implemented Open Payments dataset to evaluate the accuracy of COI disclosures of authors of clinical practice guidelines that were either newly published or revised within 2014 and were included in the National Guideline Clearinghouse (NGC) website (maintained by the U.S. Department of Health and Human Services). Authors were considered as having inaccurate COI disclosure if they had not reported all companies from which they had received funds >$5000 in the 12 months preceding the guideline's publication.We identified 223 guidelines that were either newly published (109/223; 48.9%) or revised (114/223; 51.1%) within 2014 and were included in the NGC website. Among the 1329 guideline authors with available Open Payments data, 523 received >$5000 from at least 1 healthcare-associated entity. However, only 56 out of the 523 authors (10.7%) were found to have accurate COI disclosure. The percentage of authors with accurate COI disclosure in revised guidelines was significantly lower than in newly published guidelines (6.8% vs 14.3%; P < 0.01) and was also found to differ between specialties. Furthermore, authors were less likely to inaccurately disclose "research payments" (37/49, 75.5%) compared to "general payments" (488/559, 87.3%, P = 0.02) as well as "other/associated research funding" (430/506, 85.0%, P = 0.08). No statistically significant association was detected between funding amount and disclosure accuracy.The majority of guideline authors lacked significant COIs, but among authors that received significant funds from at least 1 healthcare-associated entity the frequency of accurate disclosure was low. These findings indicate that the current process of disclosing COIs may be suboptimal and a proactive approach should be adopted in order to minimize COI reporting discrepancies. Furthermore, every effort should be undertaken to ensure the completeness and accuracy of the data recorded in the Open Payments database.

Rapid diagnostic methods for fungal infections are long awaited and are expected to improve outcomes through early initiation of targeted antifungal therapy. T2Candida panel is a novel qualitative diagnostic platform that was recently approved by the US Food and Drug Administration (FDA) for diagnosis of candidemia with a mean time to species identification of less than 5 h. T2Candida panel is performed on the fully automated T2Dx instrument in whole blood K₂EDTA specimens and is able to detect 5 Candida spp., namely Candida albicans, Candida tropicalis, Candida parapsilosis, Candida krusei, and Candida glabrata. By combining magnetic resonance with molecular diagnostics, T2Candida panel amplifies DNA and detects the amplified product by amplicon-induced agglomeration of supermagnetic particles and T2 Magnetic Resonance (T2MR) measurement. Here we describe the materials and methods needed to diagnose candidemia with the T2Candida panel.

The elderly population is particularly vulnerable to Clostridium difficile infection (CDI), but the epidemiology of CDI in long-term care facilities (LTCFs) is unknown.We performed a retrospective cohort study and used US 2011 LTCF resident data from the Minimum Data Set 3.0 linked to Medicare claims. We extracted CDI cases based on International Classification of Diseases-9 coding, and compared residents with the diagnosis of CDI to those who did not have a CDI diagnosis during their LTCF stay. We estimated CDI prevalence rates and calculated 3-month mortality rates.The study population consisted of 2,190,613 admissions (median age 82 years; interquartile range 76-88; female to male ratio 2:1; >80% whites), 45,500 of whom had a CDI diagnosis. The nationwide CDI prevalence rate was 1.85 per 100 LTCF admissions (95% confidence interval [CI] 1.83-1.87). The CDI rate was lower in the South (1.54%; 95% CI 1.51-1.57) and higher in the Northeast (2.29%; 95% CI 2.25-2.33). Older age, white race, presence of a feeding tube, unhealed pressure ulcers, end-stage renal disease, cirrhosis, bowel incontinence, prior tracheostomy, chemotherapy, and chronic obstructive pulmonary disease were independently related to "high risk" for CDI. Residents with a CDI diagnosis were more likely to be admitted to an acute care hospital (40% vs 31%, P < 0.001) and less likely to be discharged to the community (46% vs 54%, P < 0.001) than those not reported with CDI during stay. Importantly, CDI was associated with higher mortality (24.7% vs 18.1%, P = 0.001).CDI is common among the elderly residents of LTCFs and is associated with significant increase in 3-month mortality. The prevalence is higher in the Northeast and risk stratification can be used in CDI prevention policies.

Eukaryotic promoters are tightly regulated and often securely repressed. However, recent reports indicated that transcripts originating from the strictly regulated GAL1-10 promoter can be detected by single-yeast cell imaging under repressive conditions. Such leaky, noisy transcription events were suppressed by a long non-coding RNA (GAL10-ncRNA) transcribed within the GAL1-10 locus. It was further suggested that GAL10-ncRNA repression of GAL1-10 promoter leakage tunes the bimodal expression pattern of the GAL network. Independent evidence has indicated that GAL10-ncRNA transcription establishes a repressive chromatin structure through the Set2 histone methyl-transferase and the Rpd3s histone deacetylase complex. In this report we set up a novel, simple genetic Cre recombinase assay in order to readily quantify transcriptional leakage from tightly repressed promoters. By applying this method we demonstrate that GAL10-ncRNA, Set2p and Rpd3p all suppress leaky GAL1-10 driven transcription. However, GAL10-ncRNA repression is not mediated by Set2p or Rpd3p. Moreover, as opposed to GAL10-ncRNA transcription, Set2 and Rpd3 do not influence the bimodal expression of GAL genes, despite their effect on GAL1-10 promoter leakage. We suggest that GAL10-ncRNA tunes the expression of GAL genes by additional mechanisms besides suppressing leaky transcription from the GAL1-10 promoter.

UNLABELLED:Background.  Intensive care unit (ICU) patients are at higher risk for Clostridium difficile infection (CDI). METHODS:We performed a systematic review and meta-analysis of published studies from 1983 to 2015 using the PubMed, EMBASE, and Google Scholar databases to study the prevalence and outcomes of CDI in this patient population. Among the 9146 articles retrieved from the studies, 22 articles, which included a total of 80 835 ICU patients, were included in our final analysis. Results.  The prevalence of CDI among ICU patients was 2% (95% confidence interval [CI], 1%-2%), and among diarrheic ICU patients the prevalence was 11% (95% CI, 6%-17%). Among CDI patients, 25% (95% CI, 5%-51%) were diagnosed with pseudomembranous colitis, and the estimated length of ICU stay before CDI acquisition was 10.74 days (95% CI, 5%-51%). The overall hospital mortality among ICU patients with CDI was 32% (95% CI, 26%-39%), compared with 24% (95% CI, 14%-36%) among those without CDI presenting a statistically significant difference in mortality risk (P = .030). It is worth noting that the length of ICU and hospital stay among CDI patients was significantly longer, compared with non-CDI patients (standardized mean of difference [SMD] = 0.49, 95% CI, .39%-.6%, P = .00 and SMD = 1.15, 95% CI, .44%-1.91%, P = .003, respectively). It is noteworthy that the morbidity score at ICU admission (Acute Physiology and Chronic Health Evaluation II [APACHE II]) was not statistically different between the 2 groups (P = .911), implying that the differences in outcomes can be attributed to CDI. Conclusions.  The ICU setting is associated with higher prevalence of CDI. In this setting, CDI is associated with increased hospital mortality and prolonged ICU and overall hospital stay. These findings highlight the need for additional prevention and treatment studies in this setting.

Clostridium difficile infection is the most common hospital-acquired infection. Besides infected patients, carriers have emerged as a key player in C. difficile epidemiology. In this study, we evaluated the impact of identifying and isolating carriers upon hospital admission on the incidence of CDI incidence and hospital-acquired C. difficile colonization, as a single policy and as part of bundle approaches. We simulated C. difficile transmission using a stochastic mathematical approach, considering the contribution of carriers based on published literature. In the baseline scenario, CDI incidence was 6.18/1,000 admissions (95% CI, 5.72-6.65), simulating reported estimates from U.S. hospital discharges. The acquisition rate of C. difficile carriage was 9.72/1,000 admissions (95% CI, 9.15-10.31). Screening and isolation of colonized patients on admission to the hospital decreased CDI incidence to 4.99/1,000 admissions (95% CI, 4.59-5.42; relative reduction (RR) = 19.1%) and led to 36.2% reduction in the rate of hospital-acquired colonization. Simulating an antimicrobial stewardship program reduced CDI rate to 2.35/1,000 admissions (95% CI, 2.07-2.65). In sensitivity analysis, CDI incidence was less than 2.32/1,000 admissions (RR = 62.4%) in 95% of 1,000 simulations. The combined bundle, focusing on reducing C. difficile transmission from colonized patients and the individual risk of these patients to develop CDI, decreased significantly the incidence of both CDI and hospital-acquired colonization. Implementation of this bundle to current practice is expected to have an important impact in containing CDI.

Clinical guidelines play a central role in day-to-day practice. We assessed the degree of incorporation of cost analyses to guidelines and identified modifiable characteristics that could affect the level of incorporation.We selected the 100 most cited guidelines listed on the National Guideline Clearinghouse (http://www.guideline.gov) and determined the number of guidelines that used cost analyses in their reasoning and the overall percentage of incorporation of relevant cost analyses available in PubMed. Differences between medical specialties were also studied. Then, we performed a case-control study using incorporated and not incorporated cost analyses after 1:1 matching by study subject and compared them by the Consolidated Health Economic Evaluation Reporting Standards (CHEERS) statement requirements and other criteria.We found that 57% of guidelines do not use any cost justification. Guidelines incorporate a weighted average of 6.0% (95% confidence interval [CI] 4.3-7.9) among 3396 available cost analyses, with cardiology and infectious diseases guidelines incorporating 10.8% (95% CI 5.3-18.1) and 9.9% (95% CI 3.9- 18.2), respectively, and hematology/oncology and urology guidelines incorporating 4.5% (95% CI 1.6-8.6) and 1.6% (95% CI 0.4-3.5), respectively. Based on the CHEERS requirements, the mean number of items reported by the 148 incorporated cost analyses was 18.6 (SD = 3.7), a small but significant difference over controls (17.8 items; P = 0.02). Included analyses were also more likely to directly relate cost reductions to healthcare outcomes (92.6% vs 81.1%, P = 0.004) and declare the funding source (72.3% vs 53.4%, P < 0.001), while similar number of cases and controls reported a noncommercial funding source (71% vs 72.7%; P = 0.8).Guidelines remain an underused mechanism for the cost-effective allocation of available resources and a minority of practice guidelines incorporates cost analyses utilizing only 6% of the available cost analyses. Fulfilling the CHEERS requirements, directly relating costs with healthcare outcomes and transparently declaring the funding source seem to be valued by guideline-writing committees.