Aquaporins contribute to diarrhoea caused by attaching and effacing bacterial pathogens

Attaching and effacing (A/E) pathogens such as enterohemorrhagic Escherichia coli (EHEC) and enteropathogenic E. coli (EPEC) cause serious global health problems. These bacteria colonize the gastrointestinal system, attach to intestinal epithelial cells, efface (collapse) infected cell microvilli and cause overt diarrhoea that may ultimately result in death of the host. Although pathogenically induced diarrhoea is a significant global health issue, the molecular mechanisms that underlie this disease remain largely unknown. A natural murine infection model, employing the A/E pathogen Citrobacter rodentium, has been helpful in studying the diseases in vivo. C. rodentium colonize the colon at high levels, attach to colonocytes, efface microvilli and cause hyperplasia and inflammation in infected mice. As the disease progresses, the mice develop a diarrhoea-like phenotype. Aquaporin (AQP) water channels have been proposed to play a role in the normal dehydration of faecal contents. Here we examine whether C. rodentium infection may alter AQP localization in colonocytes. We demonstrate that during infection, AQP2 and AQP3 are mislocalized from their normal location along cell membranes to the cell cytoplasm. The change in localization of these proteins correlates with the diarrhoea-like phenotype present in infected mice. Mice that recover from the infection at 28-35 days post inoculum regain their normal membrane AQP localization. The altered localization of AQPs is partially dependent on the bacterial type III effector proteins EspF and EspG. We conclude that altered AQP localization may be a contributing factor to diarrhoea during bacterial infection.     

The ATM Signaling Cascade Promotes Recombination-Dependent Pachytene Arrest in Mouse Spermatocytes

Most mutations that compromise meiotic recombination or synapsis in mouse spermatocytes result in arrest and apoptosis at the pachytene stage of the first meiotic prophase. Two main mechanisms are thought to trigger arrest: one independent of the double-strand breaks (DSBs) that initiate meiotic recombination, and another activated by persistent recombination intermediates. Mechanisms underlying the recombination-dependent arrest response are not well understood, so we sought to identify factors involved by examining mutants deficient for TRIP13, a conserved AAA+ ATPase required for the completion of meiotic DSB repair. We find that spermatocytes with a hypomorphic Trip13 mutation (Trip13^mod/mod) arrest with features characteristic of early pachynema in wild type, namely, fully synapsed chromosomes without incorporation of the histone variant H1t into chromatin. These cells then undergo apoptosis, possibly in response to the arrest or in response to a defect in sex body formation. However, TRIP13-deficient cells that additionally lack the DSB-responsive kinase ATM progress further, reaching an H1t-positive stage (i.e., similar to mid/late pachynema in wild type) despite the presence of unrepaired DSBs. TRIP13-deficient spermatocytes also progress to an H1t-positive stage if ATM activity is attenuated by hypomorphic mutations in Mre11 or Nbs1 or by elimination of the ATM-effector kinase CHK2. These mutant backgrounds nonetheless experience an apoptotic block to further spermatogenic progression, most likely caused by failure to form a sex body. DSB numbers are elevated in Mre11 and Nbs1 hypomorphs but not Chk2 mutants, thus delineating genetic requirements for the ATM-dependent negative feedback loop that regulates DSB numbers. The findings demonstrate for the first time that ATM-dependent signaling enforces the normal pachytene response to persistent recombination intermediates. Our work supports the conclusion that recombination defects trigger spermatocyte arrest via pathways than are genetically distinct from sex body failure-promoted apoptosis and confirm that the latter can function even when recombination-dependent arrest is inoperative. Implications of these findings for understanding the complex relationships between spermatocyte arrest and apoptosis are discussed.     

Pat1 protects centromere-specific histone H3 variant Cse4 from Psh1-mediated ubiquitination

Evolutionarily conserved histone H3 variant Cse4 and its homologues are essential components of specialized centromere (CEN)-specific nucleosomes and serve as an epigenetic mark for CEN identity and propagation. Cse4 is a critical determinant for the structure and function of the kinetochore and is required to ensure faithful chromosome segregation. The kinetochore protein Pat1 regulates the levels and spatial distribution of Cse4 at centromeres. Deletion of PAT1 results in altered structure of CEN chromatin and chromosome segregation errors. In this study, we show that Pat1 protects CEN-associated Cse4 from ubiquitination in order to maintain proper structure and function of the kinetochore in budding yeast. PAT1-deletion strains exhibit increased ubiquitination of Cse4 and faster turnover of Cse4 at kinetochores. Psh1, a Cse4-specific E3-ubiquitin ligase, interacts with Pat1 in vivo and contributes to the increased ubiquitination of Cse4 in pat1∆ strains. Consistent with a role of Psh1 in ubiquitination of Cse4, transient induction of PSH1 in a wild-type strain resulted in phenotypes similar to a pat1∆ strain, including a reduction in CEN-associated Cse4, increased Cse4 ubiquitination, defects in spatial distribution of Cse4 at kinetochores, and altered structure of CEN chromatin. Pat1 interacts with Scm3 and is required for its maintenance at kinetochores. In conclusion, our studies provide novel insights into mechanisms by which Pat1 affects the structure of CEN chromatin and protects Cse4 from Psh1-mediated ubiquitination for faithful chromosome segregation.     

A complex endeavour: an ethnographic study of the implementation of the Sepsis Six clinical care bundle

Background

Implementation of the ‘Sepsis Six’ clinical care bundle within an hour of recognition of sepsis is recommended as an approach to reduce mortality in patients with sepsis, but achieving reliable delivery of the bundle has proved challenging. There remains little understanding of the barriers to reliable implementation of bundle components. We examined frontline clinical practice in implementing the Sepsis Six.

Methods

We conducted an ethnographic study in six hospitals participating in the Scottish Patient Safety Programme Sepsis collaborative. We conducted around 300 h of non-participant observation in emergency departments, acute medical receiving units and medical and surgical wards. We interviewed a purposive sample of 43 members of hospital staff. Data were analysed using a constant comparative approach.

Results

Implementation strategies to promote reliable use of the Sepsis Six primarily focused on education, engaging and motivating staff, and providing prompts for behaviour, along with efforts to ensure that equipment required was readily available. Although these strategies were successful in raising staff awareness of sepsis and engagement with implementation, our study identified that completing the bundle within an hour was not straightforward. Our emergent theory suggested that rather than being an apparently simple sequence of six steps, the Sepsis Six actually involved a complex trajectory comprising multiple interdependent tasks that required prioritisation and scheduling, and which was prone to problems of coordination and operational failures. Interventions that involved allocating specific roles and responsibilities for completing the Sepsis Six in ways that reduced the need for coordination and task switching, and the use of process mapping to identify system failures along the trajectory, could help mitigate against some of these problems.

Conclusions

Implementation efforts that focus on individual behaviour change to improve uptake of the Sepsis Six should be supplemented by an understanding of the bundle as a complex trajectory of work in which improving reliability requires attention to coordination of workflow, as well as addressing the mundane problems of interruptions and operational failures that obstruct task completion.

     

Structure, Function, and Evolution of the Crimean-Congo Hemorrhagic Fever Virus Nucleocapsid Protein

Crimean-Congo hemorrhagic fever virus (CCHFV) is an emerging tick-borne virus of the Bunyaviridae family that is responsible for a fatal human disease for which preventative or therapeutic measures do not exist. We solved the crystal structure of the CCHFV strain Baghdad-12 nucleocapsid protein (N), a potential therapeutic target, at a resolution of 2.1 Å. N comprises a large globular domain composed of both N- and C-terminal sequences, likely involved in RNA binding, and a protruding arm domain with a conserved DEVD caspase-3 cleavage site at its apex. Alignment of our structure with that of the recently reported N protein from strain YL04057 shows a close correspondence of all folds but significant transposition of the arm through a rotation of 180 degrees and a translation of 40 Å. These observations suggest a structural flexibility that may provide the basis for switching between alternative N protein conformations during important functions such as RNA binding and oligomerization. Our structure reveals surfaces likely involved in RNA binding and oligomerization, and functionally critical residues within these domains were identified using a minigenome system able to recapitulate CCHFV-specific RNA synthesis in cells. Caspase-3 cleaves the polypeptide chain at the exposed DEVD motif; however, the cleaved N protein remains an intact unit, likely due to the intimate association of N- and C-terminal fragments in the globular domain. Structural alignment with existing N proteins reveals that the closest CCHFV relative is not another bunyavirus but the arenavirus Lassa virus instead, suggesting that current segmented negative-strand RNA virus taxonomy may need revision.     

Phospholipase D2 (PLD2) shortens the time required for myeloid leukemic cell differentiation: mechanism of action

Cell differentiation is compromised in acute leukemias. We report that mammalian target of rapamycin (mTOR) and S6 kinase (S6K) are highly expressed in the undifferentiated promyelomonocytic leukemic HL-60 cell line, whereas PLD2 expression is minimal. The expression ratio of PLD2 to mTOR (or to S6K) is gradually inverted upon in vitro induction of differentiation toward the neutrophilic phenotype. We present three ways that profoundly affect the kinetics of differentiation as follows: (i) simultaneous overexpression of mTOR (or S6K), (ii) silencing of mTOR via dsRNA-mediated interference or inhibition with rapamycin, and (iii) PLD2 overexpression. The last two methods shortened the time required for differentiation. By determining how PLD2 participates in cell differentiation, we found that PLD2 interacts with and activates the oncogene Fes/Fps, a protein-tyrosine kinase known to be involved in myeloid cell development. Fes activity is elevated with PLD2 overexpression, phosphatidic acid or phosphatidylinositol bisphosphate. Co-immunoprecipitation indicates a close PLD2-Fes physical interaction that is negated by a Fes-R483K mutant that incapacitates its Src homology 2 domain. All these suggest for the first time the following mechanism: mTOR/S6K down-regulation→PLD2 overexpression→PLD2/Fes association→phosphatidic acid-led activation of Fes kinase→granulocytic differentiation. Differentiation shortening could have a clinical impact on reducing the time of return to normalcy of the white cell counts after chemotherapy in patients with acute promyelocytic leukemia.