MRI Is a DNA Damage Response Adaptor during Classical Non-homologous End Joining.

The modulator of retrovirus infection (MRI or CYREN) is a 30-kDa protein with a conserved N-terminal Ku-binding motif (KBM) and a C-terminal XLF-like motif (XLM). We show that MRI is intrinsically disordered and interacts with many DNA damage response (DDR) proteins, including the kinases ataxia telangiectasia mutated (ATM) and DNA-PKcs and the classical non-homologous end joining (cNHEJ) factors Ku70, Ku80, XRCC4, XLF, PAXX, and XRCC4. MRI forms large multimeric complexes that depend on its N and C termini and localizes to DNA double-strand breaks (DSBs), where it promotes the retention of DDR factors. Mice deficient in MRI and XLF exhibit embryonic lethality at a stage similar to those deficient in the core cNHEJ factors XRCC4 or DNA ligase IV. Moreover, MRI is required for cNHEJ-mediated DSB repair in XLF-deficient lymphocytes. We propose that MRI is an adaptor that, through multivalent interactions, increases the avidity of DDR factors to DSB-associated chromatin to promote cNHEJ.

C-terminal processing of GlyGly-CTERM containing proteins by rhombosortase in Vibrio cholerae.

Vibrio cholerae and a subset of other Gram-negative bacteria, including Acinetobacter baumannii, express proteins with a C-terminal tripartite domain called GlyGly-CTERM, which consists of a motif rich in glycines and serines, followed by a hydrophobic region and positively charged residues. Here we show that VesB, a V. cholerae serine protease, requires the GlyGly-CTERM domain, the intramembrane rhomboid-like protease rhombosortase, and the type II secretion system (T2SS) for localization at the cell surface. VesB is cleaved by rhombosortase to expose the second glycine residue of the GlyGly-CTERM motif, which is then conjugated to a glycerophosphoethanolamine-containing moiety prior to engagement with the T2SS and outer membrane translocation. In support of this, VesB accumulates intracellularly in the absence of the T2SS, and surface-associated VesB activity is no longer detected when the rhombosortase gene is inactivated. In turn, when VesB is expressed without an intact GlyGly-CTERM domain, VesB is released to the extracellular milieu by the T2SS and does not accumulate on the cell surface. Collectively, our findings suggest that the posttranslational modification of the GlyGly-CTERM domain is essential for cell surface localization of VesB and other proteins expressed with this tripartite extension.

Acinetobacter baumannii Is Dependent on the Type II Secretion System and Its Substrate LipA for Lipid Utilization and In Vivo Fitness.

UNLABELLED: Gram-negative bacteria express a number of sophisticated secretion systems to transport virulence factors across the cell envelope, including the type II secretion (T2S) system. Genes for the T2S components GspC through GspN and PilD are conserved among isolates of Acinetobacter baumannii, an increasingly common nosocomial pathogen that is developing multidrug resistance at an alarming rate. In contrast to most species, however, the T2S genes are dispersed throughout the genome rather than linked into one or two operons. Despite this unique genetic organization, we show here that the A. baumannii T2S system is functional. Deletion of gspD or gspE in A. baumannii ATCC 17978 results in loss of secretion of LipA, a lipase that breaks down long-chain fatty acids. Due to a lack of extracellular lipase, the gspD mutant, the gspE mutant, and a lipA deletion strain are incapable of growth on long-chain fatty acids as a sole source of carbon, while their growth characteristics are indistinguishable from those of the wild-type strain in nutrient-rich broth. Genetic inactivation of the T2S system and its substrate, LipA, also has a negative impact on in vivo fitness in a neutropenic murine model for bacteremia. Both the gspD and lipA mutants are outcompeted by the wild-type strain as judged by their reduced numbers in spleen and liver following intravenous coinoculation. Collectively, our findings suggest that the T2S system plays a hitherto-unrecognized role in in vivo survival of A. baumannii by transporting a lipase that may contribute to fatty acid metabolism. IMPORTANCE: Infections by multidrug-resistant Acinetobacter baumannii are a growing health concern worldwide, underscoring the need for a better understanding of the molecular mechanisms by which this pathogen causes disease. In this study, we demonstrated that A. baumannii expresses a functional type II secretion (T2S) system that is responsible for secretion of LipA, an extracellular lipase required for utilization of exogenously added lipids. The T2S system and the secreted lipase support in vivo colonization and thus contribute to the pathogenic potential of A. baumannii.

Brain ACE2 overexpression reduces DOCA-salt hypertension independently of endoplasmic reticulum stress.

Endoplasmic reticulum (ER) stress was previously reported to contribute to neurogenic hypertension while neuronal angiotensin-converting enzyme type 2 (ACE2) overexpression blunts the disease. To assess which brain regions are important for ACE2 beneficial effects and the contribution of ER stress to neurogenic hypertension, we first used transgenic mice harboring a floxed neuronal hACE2 transgene (SL) and tested the impact of hACE2 knockdown in the subfornical organ (SFO) and paraventricular nucleus (PVN) on deoxycorticosterone acetate (DOCA)-salt hypertension. SL and nontransgenic (NT) mice underwent DOCA-salt or sham treatment while infected with an adenoassociated virus (AAV) encoding Cre recombinase (AAV-Cre) or a control virus (AAV-green fluorescent protein) to the SFO or PVN. DOCA-salt-induced hypertension was reduced in SL mice, with hACE2 overexpression in the brain. This reduction was only partially blunted by knockdown of hACE2 in the SFO or PVN, suggesting that both regions are involved but not essential for ACE2 regulation of blood pressure (BP). DOCA-salt treatment did not increase the protein levels of ER stress and autophagy markers in NT mice, despite a significant increase in BP. In addition, these markers were not affected by hACE2 overexpression in the brain, despite a significant reduction of hypertension in SL mice. To further assess the role of ER stress in neurogenic hypertension, NT mice were infused intracerebroventricularlly with tauroursodeoxycholic acid (TUDCA), an ER stress inhibitor, during DOCA-salt treatment. However, TUDCA infusion failed to blunt the development of hypertension in NT mice. Our data suggest that brain ER stress does not contribute to DOCA-salt hypertension and that ACE2 blunts neurogenic hypertension independently of ER stress.

Teaching Nursing Leadership: Comparison of Simulation versus Traditional Inpatient Clinical.

Nurse educators claim accountability to ensure their students are prepared to assume leadership responsibilities upon graduation. Although front-line nurse leaders and nurse executives feel new graduates are not adequately prepared to take on basic leadership roles, professional nursing organizations such as the American Nurses Association (ANA) and the Association of Colleges of Nursing (AACN) deem leadership skills are core competencies of new graduate nurses. This study includes comparison of a leadership-focused multi-patient simulation and the traditional leadership clinical experiences in a baccalaureate nursing leadership course. The results of this research show both environments contribute to student learning. There was no statistical difference in the overall score. Students perceived a statistically significant difference in communication with patients in the traditional inpatient environment. However, the students perceived a statistical significant difference in teaching-learning dyad toward simulation.

The Type II secretion system delivers matrix proteins for biofilm formation by Vibrio cholerae.

Gram-negative bacteria have evolved several highly dedicated pathways for extracellular protein secretion, including the type II secretion (T2S) system. Since substrates secreted via the T2S system include both virulence factors and degradative enzymes, this secretion system is considered a major survival mechanism for pathogenic and environmental species. Previous analyses revealed that the T2S system mediates the export of ≥ 20 proteins in Vibrio cholerae, a human pathogen that is indigenous to the marine environment. Here we demonstrate a new role in biofilm formation for the V. cholerae T2S system, since wild-type V. cholerae was found to secrete the biofilm matrix proteins RbmC, RbmA, and Bap1 into the culture supernatant, while an isogenic T2S mutant could not. In agreement with this finding, the level of biofilm formation in a static microtiter assay was diminished in T2S mutants. Moreover, inactivation of the T2S system in a rugose V. cholerae strain prevented the development of colony corrugation and pellicle formation at the air-liquid interface. In contrast, extracellular secretion of the exopolysaccharide VPS, an essential component of the biofilm matrix, remained unaffected in the T2S mutants. Our results indicate that the T2S system provides a mechanism for the delivery of extracellular matrix proteins known to be important for biofilm formation by V. cholerae. Because the T2S system contributes to the pathogenicity of V. cholerae by secreting proteins such as cholera toxin and biofilm matrix proteins, elucidation of the molecular mechanism of T2S has the potential to lead to the development of novel preventions and therapies.

Structural and functional studies on the interaction of GspC and GspD in the type II secretion system.

Type II secretion systems (T2SSs) are critical for secretion of many proteins from Gram-negative bacteria. In the T2SS, the outer membrane secretin GspD forms a multimeric pore for translocation of secreted proteins. GspD and the inner membrane protein GspC interact with each other via periplasmic domains. Three different crystal structures of the homology region domain of GspC (GspC(HR)) in complex with either two or three domains of the N-terminal region of GspD from enterotoxigenic Escherichia coli show that GspC(HR) adopts an all-ß topology. N-terminal ß-strands of GspC and the N0 domain of GspD are major components of the interface between these inner and outer membrane proteins from the T2SS. The biological relevance of the observed GspC-GspD interface is shown by analysis of variant proteins in two-hybrid studies and by the effect of mutations in homologous genes on extracellular secretion and subcellular distribution of GspC in Vibrio cholerae. Substitutions of interface residues of GspD have a dramatic effect on the focal distribution of GspC in V. cholerae. These studies indicate that the GspC(HR)-GspD(N0) interactions observed in the crystal structure are essential for T2SS function. Possible implications of our structures for the stoichiometry of the T2SS and exoprotein secretion are discussed.

Long helical filaments are not seen encircling cells in electron cryotomograms of rod-shaped bacteria.

How rod-shaped bacteria form and maintain their shape is an important question in bacterial cell biology. Results from fluorescent light microscopy have led many to believe that the actin homolog MreB and a number of other proteins form long helical filaments along the inner membrane of the cell. Here we show using electron cryotomography of six different rod-shaped bacterial species, at macromolecular resolution, that no long (> 80 nm) helical filaments exist near or along either surface of the inner membrane. We also use correlated cryo-fluorescent light microscopy (cryo-fLM) and electron cryo-tomography (ECT) to identify cytoplasmic bundles of MreB, showing that MreB filaments are detectable by ECT. In light of these results, the structure and function of MreB must be reconsidered: instead of acting as a large, rigid scaffold that localizes cell-wall synthetic machinery, moving MreB complexes may apply tension to growing peptidoglycan strands to ensure their orderly, linear insertion.

Docking and assembly of the type II secretion complex of Vibrio cholerae.

Secretion of cholera toxin and other virulence factors from Vibrio cholerae is mediated by the type II secretion (T2S) apparatus, a multiprotein complex composed of both inner and outer membrane proteins. To better understand the mechanism by which the T2S complex coordinates translocation of its substrates, we are examining the protein-protein interactions of its components, encoded by the extracellular protein secretion (eps) genes. In this study, we took a cell biological approach, observing the dynamics of fluorescently tagged EpsC and EpsM proteins in vivo. We report that the level and context of fluorescent protein fusion expression can have a bold effect on subcellular location and that chromosomal, intraoperon expression conditions are optimal for determining the intracellular locations of fusion proteins. Fluorescently tagged, chromosomally expressed EpsC and EpsM form discrete foci along the lengths of the cells, different from the polar localization for green fluorescent protein (GFP)-EpsM previously described, as the fusions are balanced with all their interacting partner proteins within the T2S complex. Additionally, we observed that fluorescent foci in both chromosomal GFP-EpsC- and GFP-EpsM-expressing strains disperse upon deletion of epsD, suggesting that EpsD is critical to the localization of EpsC and EpsM and perhaps their assembly into the T2S complex.

The three-dimensional structure of the cytoplasmic domains of EpsF from the type 2 secretion system of Vibrio cholerae.

The type 2 secretion system (T2SS), a multi-protein machinery that spans both the inner and the outer membranes of Gram-negative bacteria, is used for the secretion of several critically important proteins across the outer membrane. Here we report the crystal structure of the N-terminal cytoplasmic domain of EpsF, an inner membrane spanning T2SS protein from Vibrio cholerae. This domain consists of a bundle of six anti-parallel helices and adopts a fold that has not been described before. The long C-terminal helix alpha6 protrudes from the body of the domain and most likely continues as the first transmembrane helix of EpsF. Two N-terminal EpsF domains form a tight dimer with a conserved interface, suggesting that the observed dimer occurs in the T2SS of many bacteria. Two calcium binding sites are present in the dimer interface with ligands provided for each site by both subunits. Based on this new structure, sequence comparisons of EpsF homologs and localization studies of GFP fused with EpsF, we propose that the second cytoplasmic domain of EpsF adopts a similar fold as the first cytoplasmic domain and that full-length EpsF, and its T2SS homologs, have a three-transmembrane helix topology.

Synthesis of a 35-member stereoisomer library of bistramide A: evaluation of effects on actin state, cell cycle and tumor cell growth.

Synthesis and preliminary biological evaluation of a 35-member library of bistramide A stereoisomers are reported. All eight stereoisomers of the C1-C13 tetrahydropyran fragment of the molecule were prepared utilizing crotylsilane reagents 9 and 10 in our [4+2]-annulation methodology. In addition, the four isomers of the C14-C18 gamma-amino acid unit were accessed via a Lewis acid mediated crotylation reaction with use of both enantiomers of organosilane 11. The spiroketal subunit of bistramide A was modified at the C39-alcohol to give another point of stereochemical diversification. The fragments were coupled by using a standard peptide coupling protocol to provide 35 stereoisomers of the natural product. These stereochemical analogues were screened for their effects on cellular actin and cytotoxicity against cancer cell lines (UO-31 renal and SF-295 CNS). The results of these assays identified one analogue, 1.21, with enhanced potency relative to the natural product, bistramide A.

Enhancing Community Education Through Innovative Teaching Strategies in a Baccalaureate Nursing Program.

BACKGROUND: Nurse educators are relentlessly in search of innovative measures that enhance student learning and offer opportunities that prepare them for clinical practice. Collaborative opportunities between schools of nursing and community settings are beneficial for students and the community. METHOD: One strategy was developed by a southeastern baccalaureate nursing program through the assistance of the National Institutes of Health mini-grants program, related to their Safe Sleep campaign. Safe Sleep subject matter was integrated into the curriculum, where students learned content, developed teaching strategies, and taught community members components of Safe Sleep. RESULTS: The project provided faculty and baccalaureate nursing students with an opportunity to implement a community outreach education project addressing Safe Sleep. Students learned cultural assessment, teaching, and communication skills, and community members benefited from learned knowledge of health promotion programs. CONCLUSION: Future educational outreach projects are recommended to further examine program effectiveness and the value of teaching-learning strategies. [J Nurs Educ. 2018;57(4):240-244.].

Homeodomain Proteins Directly Regulate ATM Kinase Activity.

Ataxia-telangiectasia mutated (ATM) is a serine/threonine kinase that coordinates the response to DNA double-strand breaks and oxidative stress. NKX3.1, a prostate-specific transcription factor, was recently shown to directly stimulate ATM kinase activity through its highly conserved homeodomain. Here, we show that other members of the homeodomain family can also regulate ATM kinase activity. We found that six representative homeodomain proteins (NKX3.1, NKX2.2, TTF1, NKX2.5, HOXB7, and CDX2) physically and functionally interact with ATM and with the Mre11-Rad50-Nbs1 (MRN) complex that activates ATM in combination with DNA double-strand breaks. The binding between homeodomain proteins and ATM stimulates oxidation-induced ATM activation in vitro but inhibits ATM kinase activity in the presence of MRN and DNA and in human cells. These findings suggest that many tissue-specific homeodomain proteins may regulate ATM activity during development and differentiation and that this is a unique mechanism for the control of the DNA damage response.

Mapping critical interactive sites within the periplasmic domain of the Vibrio cholerae type II secretion protein EpsM.

The type II secretion (T2S) system is present in many gram-negative species, both pathogenic and nonpathogenic, where it supports the delivery of a variety of toxins, proteases, and lipases into the extracellular environment. In Vibrio cholerae, the T2S apparatus is composed of 12 Eps proteins that assemble into a multiprotein complex that spans the entire cell envelope. Two of these proteins, EpsM and EpsL, are key components of the secretion machinery present in the inner membrane. In addition to likely forming homodimers, EpsL and EpsM have been shown to form a stable complex in the inner membrane and to protect each other from proteolytic degradation. To identify and map the specific regions of EpsM involved in protein-protein interactions with both another molecule of EpsM and EpsL, we tested the interactions of deletion constructs of EpsM with full-length EpsM and EpsL by functional characterization and copurification as well as coimmunoprecipitation. Analysis of the truncated EpsM mutants revealed that the region of EpsM from amino acids 100 to 135 is necessary for EpsM to form homo-oligomers, while residues 84 to 99 appear to be critical for a stable interaction with EpsL.

Factors Associated With First-Time NCLEX-RN Success: A Descriptive Research Study.

BACKGROUND: The purpose of this study was to identify variables associated with scores achieved on the Health Education Systems, Inc. (HESI) exit examination and successful first-time NCLEX-RN® pass rates. METHOD: A retrospective descriptive study examined the administrative data of 211 baccalaureate nursing students. RESULTS: Students who completed the program in sequence and scored higher in certain HESI course examinations were more likely to have a better performance on the HESI exit examination. The higher the scores students achieved on the HESI exit examination, the more likely they were to pass the NCLEX-RN on their first attempt. CONCLUSION: These findings add to the growing body of literature seeking to identify variables associated with success in first-time NCLEX-RN success. Further research is needed to identify strategies that can be implemented to ensure timely progression, program completion, and licensure examination success. [J Nurs Educ. 2017;56(9):542-545.].

Targeting the Type II Secretion System: Development, Optimization, and Validation of a High-Throughput Screen for the Identification of Small Molecule Inhibitors.

Nosocomial pathogens that develop multidrug resistance present an increasing problem for healthcare facilities. Due to its rapid rise in antibiotic resistance, Acinetobacter baumannii is one of the most concerning gram-negative species. A. baumannii typically infects immune compromised individuals resulting in a variety of outcomes, including pneumonia and bacteremia. Using a murine model for bacteremia, we have previously shown that the type II secretion system (T2SS) contributes to in vivo fitness of A. baumannii. Here, we provide support for a role of the T2SS in protecting A. baumannii from human complement as deletion of the T2SS gene gspD resulted in a 100-fold reduction in surviving cells when incubated with human serum. This effect was abrogated in the absence of Factor B, a component of the alternative pathway of complement activation, indicating that the T2SS protects A. baumannii against the alternative complement pathway. Because inactivation of the T2SS results in loss of secretion of multiple enzymes, reduced in vivo fitness, and increased sensitivity to human complement, the T2SS may be a suitable target for therapeutic intervention. Accordingly, we developed and optimized a whole-cell high-throughput screening (HTS) assay based on secreted lipase activity to identify small molecule inhibitors of the T2SS. We tested the reproducibility of our assay using a 6,400-compound library. With small variation within controls and a dynamic range between positive and negative controls, the assay had a z-factor of 0.65, establishing its suitability for HTS. Our screen identified the lipase inhibitors Orlistat and Ebelactone B demonstrating the specificity of the assay. To eliminate inhibitors of lipase activity and lipase expression, two counter assays were developed and optimized. By implementing these assays, all seven tricyclic antidepressants present in the library were found to be inhibitors of the lipase, highlighting the potential of identifying alternative targets for approved pharmaceuticals. Although no T2SS inhibitor was identified among the compounds that reduced lipase activity by ≥30%, our small proof-of-concept pilot study indicates that the HTS regimen is simple, reproducible, and specific and that it can be used to screen larger libraries for the identification of T2SS inhibitors that may be developed into novel A. baumannii therapeutics.

Type II secretion: from structure to function.

Gram-negative bacteria use the type II secretion system to transport a large number of secreted proteins from the periplasmic space into the extracellular environment. Many of the secreted proteins are major virulence factors in plants and animals. The components of the type II secretion system are located in both the inner and outer membranes where they assemble into a multi-protein, cell-envelope spanning, complex. This review discusses recent progress, particularly newly published structures obtained by X-ray crystallography and electron microscopy that have increased our understanding of how the type II secretion apparatus functions and the role that individual proteins play in this complex system.

Outer Membrane Vesicle-Mediated Export of Processed PrtV Protease from Vibrio cholerae.

BACKGROUND: Outer membrane vesicles (OMVs) are known to release from almost all Gram-negative bacteria during normal growth. OMVs carry different biologically active toxins and enzymes into the surrounding environment. We suggest that OMVs may therefore be able to transport bacterial proteases into the target host cells. We present here an analysis of the Vibrio cholerae OMV-associated protease PrtV. METHODOLOGY/PRINCIPAL FINDINGS: In this study, we demonstrated that PrtV was secreted from the wild type V. cholerae strain C6706 via the type II secretion system in association with OMVs. By immunoblotting and electron microscopic analysis using immunogold labeling, the association of PrtV with OMVs was examined. We demonstrated that OMV-associated PrtV was biologically active by showing altered morphology and detachment of cells when the human ileocecum carcinoma (HCT8) cells were treated with OMVs from the wild type V. cholerae strain C6706 whereas cells treated with OMVs from the prtV isogenic mutant showed no morphological changes. Furthermore, OMV-associated PrtV protease showed a contribution to bacterial resistance towards the antimicrobial peptide LL-37. CONCLUSION/SIGNIFICANCE: Our findings suggest that OMVs released from V. cholerae can deliver a processed, biologically active form of PrtV that contributes to bacterial interactions with target host cells.

High throughput screening for cyanovirin-N mimetics binding to HIV-1 gp41.

The human immunodeficiency virus type-1 (HIV-1) envelope glycoprotein gp41 is an important mediator of viral entry into host cells. Previous studies showed that the virucidal protein cyanovirin-N (CV-N) bound to both gp120 and gp41, and that this binding was associated with its antiviral activity. We constructed an HTS assay based on the interaction of europium-labeled CV-N with recombinant glycosylated gp41 ectodomain to support identification of small-molecule mimetics of CV-N that might be developed as antiviral drug leads. Primary screening of over 107,000 natural product extracts in the assay yielded 347 confirmed hits. Secondary assays eliminated extracts that bound directly to labeled CV-N or for which the simple sugars mannose and N-acetylglucosamine blocked the interaction with gp41 (lectin activity). Extracts were further prioritized based on anti-HIV activity and other biological, biochemical, and chemical criteria. The distribution of source organism taxonomy of active extracts was analyzed, as was the cross-correlation of activity between the CV-N-gp41 binding competition assay and the previously reported CV-N-gp120 binding competition assay. A limited set of extracts was selected for bioassay-guided fractionation.