Polarization of the endoplasmic reticulum by ER-septin tethering.

Polarization of the plasma membrane (PM) into domains is an important mechanism to compartmentalize cellular activities and to establish cell polarity. Polarization requires formation of diffusion barriers that prevent mixing of proteins between domains. Recent studies have uncovered that the endoplasmic reticulum (ER) of budding yeast and neurons is polarized by diffusion barriers, which in neurons controls glutamate signaling in dendritic spines. The molecular identity of these barriers is currently unknown. Here, we show that a direct interaction between the ER protein Scs2 and the septin Shs1 creates the ER diffusion barrier in yeast. Barrier formation requires Epo1, a novel ER-associated subunit of the polarisome that interacts with Scs2 and Shs1. ER-septin tethering polarizes the ER into separate mother and bud domains, one function of which is to position the spindle in the mother until M phase by confining the spindle capture protein Num1 to the mother ER.

Histone H3K4 demethylation is negatively regulated by histone H3 acetylation in Saccharomyces cerevisiae.

Histone H3 lysine 4 trimethylation (H3K4me3) is a hallmark of transcription initiation, but how H3K4me3 is demethylated during gene repression is poorly understood. Jhd2, a JmjC domain protein, was recently identified as the major H3K4me3 histone demethylase (HDM) in Saccharomyces cerevisiae. Although JHD2 is required for removal of methylation upon gene repression, deletion of JHD2 does not result in increased levels of H3K4me3 in bulk histones, indicating that this HDM is unable to demethylate histones during steady-state conditions. In this study, we showed that this was due to the negative regulation of Jhd2 activity by histone H3 lysine 14 acetylation (H3K14ac), which colocalizes with H3K4me3 across the yeast genome. We demonstrated that loss of the histone H3-specific acetyltransferases (HATs) resulted in genome-wide depletion of H3K4me3, and this was not due to a transcription defect. Moreover, H3K4me3 levels were reestablished in HAT mutants following loss of JHD2, which suggested that H3-specific HATs and Jhd2 serve opposing functions in regulating H3K4me3 levels. We revealed the molecular basis for this suppression by demonstrating that H3K14ac negatively regulated Jhd2 demethylase activity on an acetylated peptide in vitro. These results revealed the existence of a general mechanism for removal of H3K4me3 following gene repression.

Association of pre-hospital time intervals and clinical outcomes in ST-elevation myocardial infarction patients.

Study Objectives: Timely coronary reperfusion is critical for favorable outcomes after ST-elevation myocardial infarction (STEMI). A substantial proportion of the total ischemic time is patient related, occurring before first medical contact (FMC). We aimed to expand the limited current understanding of the associations between prehospital intervals and clinical outcomes. Methods: We conducted a retrospective analysis of consecutive STEMI patients who underwent primary percutaneous coronary intervention (pPCI) (January 2009-March 2016) and assessed the associations between prehospital intervals and the incidence of new heart failure, cardiogenic shock, and hospital length of stay (LOS), adjusting for important clinical variables. Results: A total of 773 patients (77% men, median age 65 years) met eligibility criteria. The median pre-911 activation interval was 29 minutes (interquartile range: 11, 89); the median 911 call to FMC interval was 12 minutes (interquartile range: 9, 15). In multivariable analysis, there was a V-shaped relationship between the pre-911 activation interval and outcomes: a lower likelihood of new heart failure (odds ratio [OR] 0.51; 95% confidence interval [CI]: 0.30, 0.87), cardiogenic shock (OR 0.40; 95% CI: 0.21, 0.75) and prolonged LOS (OR 0.24; 95% CI: 0.14, 0.42) for midrange intervals (11-88 minutes) when compared to the early (< 11-minute) interval. There was no statistically significant relationship between total pre-FMC time and FMC to device activation time. Conclusions: Among ambulance-transported STEMI patients receiving pPCI, the shortest and longest pre-911 activation time intervals were associated with poorer outcomes. However, variation in post-FMC interval alone was not associated with outcomes, suggesting that interventions to reduce pre-FMC intervals must be prioritized.

Transcription Promotes the Interaction of the FAcilitates Chromatin Transactions (FACT) Complex with Nucleosomes in Saccharomyces cerevisiae.

The FACT (FAcilitates Chromatin Transactions) complex is a conserved complex that maintains chromatin structure on transcriptionally active genes. Consistent with this, FACT is enriched on highly expressed genes, but how it is targeted to these regions is unknown. In vitro, FACT binds destabilized nucleosomes, supporting the hypothesis that FACT is targeted to transcribed chromatin through recognition of RNA polymerase (RNAP)-disrupted nucleosomes. In this study, we used high-resolution analysis of FACT occupancy in Saccharomyces cerevisiae to test this hypothesis. We demonstrate that FACT interacts with nucleosomes in vivo and that its interaction with chromatin is dependent on transcription by any of the three RNAPs. Deep sequencing of micrococcal nuclease-resistant fragments shows that FACT-bound nucleosomes exhibit differing nuclease sensitivity compared to bulk chromatin, consistent with a modified nucleosome structure being the preferred ligand for this complex. Interestingly, a subset of FACT-bound nucleosomes may be "overlapping dinucleosomes," in which one histone octamer invades the ∼147-bp territory normally occupied by the adjacent nucleosome. While the differing nuclease sensitivity of FACT-bound nucleosomes could also be explained by the demonstrated ability of FACT to alter nucleosome structure, transcription inhibition restores nuclease resistance, suggesting that it is not due to FACT interaction alone. Collectively, these results are consistent with a model in which FACT is targeted to transcribed genes through preferential interaction with RNAP-disrupted nucleosomes.

Composition of human breast milk in acute kidney injury.

BACKGROUND: Breastfeeding is a widely encouraged practice due to its benefits for mother and the infant. Little is known about the impact of disease states, such as kidney dysfunction and childbirth complications, on the composition of breast milk. METHODS: We describe a case of a 35-year-old woman who suffered a postpartum hemorrhage, was administered a contrast dye prior to computer tomography, and developed an acute kidney injury. Using nuclear magnetic resonance spectrometry, we measured composition of milk in acute kidney injury. The amount of dye secreted into milk was determined using a spectroscopic assay. RESULTS: Here we show that acute kidney injury results in changes in milk composition, but it does not significantly affect major macronutrients. We also determine that iodinated computer tomography contrast dye does not accumulate in milk in appreciable amounts. CONCLUSION: Acute kidney injury has impact on breast milk. Intravenous administration of computer tomography contrast dye does not result in significantly elevated levels in milk.

Pilot-testing an adverse drug event reporting form prior to its implementation in an electronic health record.

BACKGROUND: Adverse drug events (ADEs), harmful unintended consequences of medication use, are a leading cause of hospital admissions, yet are rarely documented in a structured format between care providers. We describe pilot-testing structured ADE documentation fields prior to integration into an electronic medical record (EMR). METHODS: We completed a qualitative study at two Canadian hospitals. Using data derived from a systematic review of the literature, we developed screen mock-ups for an ADE reporting platform, iteratively revised in participatory workshops with diverse end-user groups. We designed a paper-based form reflecting the data elements contained in the mock-ups. We distributed them to a convenience sample of clinical pharmacists, and completed ethnographic workplace observations while the forms were used. We reviewed completed forms, collected feedback from pharmacists using semi-structured interviews, and coded the data in NVivo for themes related to the ADE form. RESULTS: We completed 25 h of clinical observations, and 24 ADEs were documented. Pharmacists perceived the form as simple and clear, with sufficient detail to capture ADEs. They identified fields for omission, and others requiring more detail. Pharmacists encountered barriers to documenting ADEs including uncertainty about what constituted a reportable ADE, inability to complete patient follow-up, the need for inter-professional communication to rule out alternative diagnoses, and concern about creating a permanent record. CONCLUSION: Paper-based pilot-testing allowed planning for important modifications in an ADE documentation form prior to implementation in an EMR. While paper-based piloting is rarely reported prior to EMR implementations, it can inform design and enhance functionality. Piloting with other groups of care providers and in different healthcare settings will likely lead to further revisions prior to broader implementations.

Designing an Adverse Drug Event Reporting System to Prevent Unintentional Reexposures to Harmful Drugs: Study Protocol for a Multiple Methods Design.

BACKGROUND: Adverse drug events (ADEs) are unintended and harmful events related to medication use. Up to 30% of serious ADEs recur within six months because culprit drugs are unintentionally represcribed and redispensed. Improving the electronic communication of ADE information between care providers, and across care settings, has the potential to reduce recurrent ADEs. OBJECTIVE: We aim to describe the methods used to design Action ADE, a novel electronic ADE reporting system that can be leveraged to prevent unintentional reexposures to harmful drugs in British Columbia, Canada. METHODS: To develop the new system, our team will use action research and participatory design, approaches that employ social scientific research methods and practitioner participation to generate insights into work settings and problem resolution. We will develop a systematic search strategy to review existing ADE reporting systems identified in academic and grey literature, and analyze the content of these systems to identify core data fields used to communicate ADE information. We will observe care providers in the emergency departments and on the wards of two urban tertiary hospitals and one urban community hospital, in one rural ambulatory care center, and in three community pharmacies in British Columbia, Canada. We will also conduct participatory workshops with providers to understand their needs and priorities related to communicating ADEs and preventing erroneous represcribing or redispensing of culprit medications. These methods will inform the iterative development of a preliminary paper-based reporting form, which we will then pilot test with providers in a real-world setting. RESULTS: This is an ongoing project with results being published as analyses are completed. The systematic review has been completed; field observations, focus groups, and pilot testing of a preliminary paper-based design are ongoing. Results will inform the development of software that will enable clinically useful user-friendly documentation and communication of ADEs. CONCLUSIONS: We take this approach with the recognition that information technology-based solutions in health care often fall short of expectations as a result of designers' failure to account for organizational and work practice considerations, and the needs of end-users. We describe how integrating qualitative methods into an iterative participatory design process (planned in partnership with end-users) will allow us to address specific clinical needs, conceptualize linkages between systems, integrate the reporting system into clinicians' workflow, and design the system to optimize its uptake into practice.

Critical determinants for chromatin binding by Saccharomyces cerevisiae Yng1 exist outside of the plant homeodomain finger.

The temporal and spatial regulation of histone post-translational modifications is essential for proper chromatin structure and function. The Saccharomyces cerevisiae NuA3 histone acetyltransferase complex modifies the amino-terminal tail of histone H3, but how NuA3 is targeted to specific regions of the genome is not fully understood. Yng1, a subunit of NuA3 and a member of the Inhibitor of Growth (ING) protein family, is required for the interaction of NuA3 with chromatin. This protein contains a C-terminal plant homeodomain (PHD) finger that specifically interacts with lysine 4-trimethylated histone H3 (H3K4me3) in vitro. This initially suggested that NuA3 is targeted to regions bearing the H3K4me3 mark; however, deletion of the Yng1 PHD finger does not disrupt the interaction of NuA3 with chromatin or result in a phenotype consistent with loss of NuA3 function in vivo. In this study, we uncovered the molecular basis for the discrepancies in these data. We present both genetic and biochemical evidence that full-length Yng1 has two independent histone-binding motifs: an amino-terminal motif that binds unmodified H3 tails and a carboxyl-terminal PHD finger that specifically recognizes H3K4me3. Although these motifs can bind histones independently, together they increase the apparent association of Yng1 for the H3 tail.