Paradoxical Activation of a Type VI Secretion System Phospholipase Effector by Its Cognate Immunity Protein.

Type VI secretion systems (T6SSs) deliver cytotoxic effector proteins into target bacteria and eukaryotic host cells. Antibacterial effectors are invariably encoded with cognate immunity proteins that protect the producing cell from self-intoxication. Here, we identify transposon insertions that disrupt the tli immunity gene of Enterobacter cloacae and induce autopermeabilization through unopposed activity of the Tle phospholipase effector. This hyperpermeability phenotype is T6SS dependent, indicating that the mutants are intoxicated by Tle delivered from neighboring sibling cells rather than by internally produced phospholipase. Unexpectedly, an in-frame deletion of tli does not induce hyperpermeability because Δtli null mutants fail to deploy active Tle. Instead, the most striking phenotypes are associated with disruption of the tli lipoprotein signal sequence, which prevents immunity protein localization to the periplasm. Immunoblotting reveals that most hyperpermeable mutants still produce Tli, presumably from alternative translation initiation codons downstream of the signal sequence. These observations suggest that cytosolic Tli is required for the activation and/or export of Tle. We show that Tle growth inhibition activity remains Tli dependent when phospholipase delivery into target bacteria is ensured through fusion to the VgrG ß-spike protein. Together, these findings indicate that Tli has distinct functions, depending on its subcellular localization. Periplasmic Tli acts as a canonical immunity factor to neutralize incoming effector proteins, while a cytosolic pool of Tli is required to activate the phospholipase domain of Tle prior to T6SS-dependent export. IMPORTANCE Gram-negative bacteria use type VI secretion systems deliver toxic effector proteins directly into neighboring competitors. Secreting cells also produce specific immunity proteins that neutralize effector activities to prevent autointoxication. Here, we show the Tli immunity protein of Enterobacter cloacae has two distinct functions, depending on its subcellular localization. Periplasmic Tli acts as a canonical immunity factor to block Tle lipase effector activity, while cytoplasmic Tli is required to activate the lipase prior to export. These results indicate Tle interacts transiently with its cognate immunity protein to promote effector protein folding and/or packaging into the secretion apparatus.

Paradoxical activation of a type VI secretion system (T6SS) phospholipase effector by its cognate immunity protein.

Type VI secretion systems (T6SS) deliver cytotoxic effector proteins into target bacteria and eukaryotic host cells. Antibacterial effectors are invariably encoded with cognate immunity proteins that protect the producing cell from self-intoxication. Here, we identify transposon insertions that disrupt the tli immunity gene of Enterobacter cloacae and induce auto-permeabilization through unopposed activity of the Tle phospholipase effector. This hyper-permeability phenotype is T6SS-dependent, indicating that the mutants are intoxicated by Tle delivered from neighboring sibling cells rather than by internally produced phospholipase. Unexpectedly, an in-frame deletion of tli does not induce hyper-permeability because Δ tli null mutants fail to deploy active Tle. Instead, the most striking phenotypes are associated with disruption of the tli lipoprotein signal sequence, which prevents immunity protein localization to the periplasm. Immunoblotting reveals that most hyper-permeable mutants still produce Tli, presumably from alternative translation initiation codons downstream of the signal sequence. These observations suggest that cytosolic Tli is required for the activation and/or export of Tle. We show that Tle growth inhibition activity remains Tli-dependent when phospholipase delivery into target bacteria is ensured through fusion to the VgrG ß-spike protein. Together, these findings indicate that Tli has distinct functions depending on its subcellular localization. Periplasmic Tli acts as a canonical immunity factor to neutralize incoming effector proteins, while a cytosolic pool of Tli is required to activate the phospholipase domain of Tle prior to T6SS-dependent export.

The ß-encapsulation cage of rearrangement hotspot (Rhs) effectors is required for type VI secretion.

Bacteria deploy rearrangement hotspot (Rhs) proteins as toxic effectors against both prokaryotic and eukaryotic target cells. Rhs proteins are characterized by YD-peptide repeats, which fold into a large ß-cage structure that encapsulates the C-terminal toxin domain. Here, we show that Rhs effectors are essential for type VI secretion system (T6SS) activity in Enterobacter cloacae (ECL). ECL rhs- mutants do not kill Escherichia coli target bacteria and are defective for T6SS-dependent export of hemolysin-coregulated protein (Hcp). The RhsA and RhsB effectors of ECL both contain Pro-Ala-Ala-Arg (PAAR) repeat domains, which bind the ß-spike of trimeric valine-glycine repeat protein G (VgrG) and are important for T6SS activity in other bacteria. Truncated RhsA that retains the PAAR domain is capable of forming higher-order, thermostable complexes with VgrG, yet these assemblies fail to restore secretion activity to ∆rhsA ∆rhsB mutants. Full T6SS-1 activity requires Rhs that contains N-terminal transmembrane helices, the PAAR domain, and an intact ß-cage. Although ∆rhsA ∆rhsB mutants do not kill target bacteria, time-lapse microscopy reveals that they assemble and fire T6SS contractile sheaths at ∼6% of the frequency of rhs+ cells. Therefore, Rhs proteins are not strictly required for T6SS assembly, although they greatly increase secretion efficiency. We propose that PAAR and the ß-cage provide distinct structures that promote secretion. PAAR is clearly sufficient to stabilize trimeric VgrG, but efficient assembly of T6SS-1 also depends on an intact ß-cage. Together, these domains enforce a quality control checkpoint to ensure that VgrG is loaded with toxic cargo before assembling the secretion apparatus.

Discovery and Development of Gaseous Nitric Oxide Under Increased Atmospheric Pressure as an Antimicrobial: In Vitro and In Vivo Testing of Nitric Oxide Against Multidrug-Resistant Organisms.

Gaseous nitric oxide under increased atmospheric pressure (gNOp) has shown ability to kill multidrug-resistant bacteria in an in vitro model and in a live mammalian (porcine) model. Factors impacting the kill rate of the multidrug-resistant bacteria include atmospheric pressures, concentration of gaseous NO, flow rate, and duration of application. Using successful in vitro parameters, gNOp showed multilog reduction of bacteria in a live mammalian (porcine) model. The in vitro testing system, using the EpiDerm-FT skin model (stem cell grown skin), was used to develop an infected wound model for Acinetobacter baumannii, Pseudomonas aeruginosa, Staphylococcus aureus, and methicillin-resistant S aureus.

The next generation core competencies for emergency management.

The Next Generation Core Competencies (NGCC) guide the professional development of future emergency managers. Once familiar roles are evolving as the world grows more interdependent; at the same time, disaster risk factors are intensified by the changing interactions between the social, built, and physical environments. The updated edition of emergency management core competencies is particularly important for refining the trajectory of the emergency management discipline and developing capacities requisite to reducing disaster risk and building resilient communities in the midst of a turbulent, complex, and uncertain future. The NGCC project was a multiphase study conducted by a FEMA-sponsored focus group. Oriented toward future needs, the competencies have been built on the current emergency management competencies, a review of related competencies and global risk trends, a multiphase Delphi study, and wider emergency management community listening sessions. Behavioral anchors and key actions for measurement accompany the new core competencies. The overarching goal of the work is to establish the next generation emergency management core competencies, which are likely to underpin the emergency management workforce of 2030 and beyond. The 13 core competencies fall into three nested categories that are interrelated, but have attributes that build the individual, the practitioner, or relationships.

Reactions: Antibacterial and bioactive dental restorative materials: Do they really work?

PURPOSE: The study and development of antibacterial materials for use in dental applications is growing with the development of novel materials and procedures. Examination of the effects of such antibacterial materials on oral pathogens as well as on stability and longevity of dental restorations is of paramount importance to the field. RESULTS: This review addressed the range of topics covered by the manuscripts presented at the Seoul symposium on antibacterial dental materials. CLINICAL SIGNIFICANCE: Based on the presented works, it seems that the emerging antibacterial and bioactive mate-rials can potentially benefit restorative dentistry; however, like many other subjects in clinical dentistry, good quality evidence on their effectiveness under clinical situations is yet to be accumulated.

Assessing the Impact of a Human Rights-Based Approach across a Spectrum of Change for Women's, Children's, and Adolescents' Health.

Global momentum around women's, children's, and adolescents' health, coupled with the ambitious and equalizing agenda of the Sustainable Development Goals (SDGs), has exposed a tension between the need for comprehensive, multi-actor, rights-based approaches that seek to "close the gaps" and a growing economic and political imperative to demonstrate efficiency, effectiveness, and returns on specific investments. To address this challenge, this paper proposes a framework to measure "results" in a way that offers a more nuanced understanding of the impact of human rights-based approaches and their complexity, as well as their contextual, multi-sectoral, and evolving nature. We argue that the impact of human rights-based approaches is best measured across a spectrum of change-at the individual, programmatic, structural, and societal levels. Such an analysis would allow for more accurate assessments of the cumulative effect of these changes. The paper also underscores the long-overdue need to better define the parameters of a human rights-based approach to health. This is an important part of the research agenda on human rights and health in the context of the SDGs and the Global Strategy for Women's, Children's and Adolescents' Health, and amid calls for better measurement and greater accountability for resources, results, and rights at all levels. While this paper focuses on women's, children's, and adolescents' health, the proposed framework can apply as readily to other areas of health and provides a new frame of reference for assessing the impact of human rights-based approaches.

Chief executive officers in US hospitals: an examination of workforce demographics and educational issues.

A study was conducted to determine if workforce demographics of chief executive officers within hospitals in the United States were changing. It sought to analyze the retirement patterns and the current gender mix of chief executive officers in hospital settings within the United States. It also sought to capture the perspectives of those in top-level executive positions in regard to the educational requirements of future health care executives.

Determining return on investment for training using simple regression: a hypothetical case study for the health care industry.

Wisely allocating financial resources is essential to the success of every health care organization. Therefore, health care managers must be able to determine if dedicating the necessary funds for employee training results in an adequate return on investment. This case study examines how training programs can be evaluated in terms of business results and describes one method, simple regression analysis, that health care managers may use to help determine if the training was financially beneficial to the organization.

Employee satisfaction and employee retention: catalysts to patient satisfaction.

Over the last few years, most health care facilities have become intensely aware of the need to increase patient satisfaction. However, with today's more consumer-driven market, this can be a daunting task for even the most experienced health care manager. Recent studies indicate that focusing on employee satisfaction and subsequent employee retention may be strong catalysts to patient satisfaction. This study offers a review of how employee satisfaction and retention correlate with patient satisfaction and also examines the current ways health care organizations are focusing on employee satisfaction and retention.

Measuring ultrashort optical pulses in the presence of noise: an empirical study of the performance of spectral phase interferometry for direct electric field reconstruction.

We have measured the performance of a real spectral phase interferometry for direct electric field reconstruction (SPIDER) apparatus operating under suboptimal conditions. We analyzed the errors in SPIDER's measurements of the temporal phases and intensities of 50-fs ultrashort laser pulses as a function of the additive noise in the detected signal. It was found that SPIDER performs exceptionally well, particularly in the case of additive noise. Specifically, a signal with 10% noise yields a pulse that has a mere 2% error in its intensity profile and a phase that differs from the nominal value by 0.2 rad. Furthermore, we quantified SPIDER's performance with limited detector resolution and as a function of signal averaging.

Is continence enough?

The ideal of virtue demands not only right choice and right behavior but also right desire in the emotions. Homosexual desire, then, even if it does not result in overt homosexual behavior, is contrary to virtue, and the completion of virtue demands right desire. If a homosexual has no plan to marry, then right desire implies only the removal of homosexual desire and not the revival of heterosexual desire at which reorientation therapy aims. On the other hands, if a homosexual is married or plans to marry, then right desire includes heterosexual desire for one's spouse. If homosexual desire is viewed as an emotional disorder, and not merely as a moral defect, then it may require reorientation therapy to bring about heterosexual desire. The pursuit of virtue, then, may recommend reorientation therapy, but even this recommendation is fairly limited, since reorientation therapy may be burdensome and may not be that effective. In conclusion, married homosexuals or homosexuals planning to marry may have a limited moral obligation to pursue reorientation therapy.

Thin-section CT findings in flock worker's lung, a work-related interstitial lung disease.

PURPOSE: To characterize the thin-section computed tomographic (CT) features of flock worker's lung (FWL) and to determine whether these features may be used to distinguish workers with FWL from flock workers who do not fulfill diagnostic criteria for FWL. MATERIALS AND METHODS: Thin-section CT images obtained in 43 flock workers (including 11 with FWL) were reviewed independently by radiologists blinded to occupational and clinical details. CT features recorded included ground-glass opacities, consolidation, micronodules, reticular abnormality, and septal thickening. Thirty-five of the CT scans (including nine obtained in patients with FWL) were also studied by using quantitative image analysis. The Student t test was used to compare mean lung attenuation between the workers with FWL and those without it. RESULTS: Every patient with FWL and 19 (59%) of the 32 exposed flock workers who did not meet criteria for the disease had an abnormal thin-section CT scan. The most common findings in FWL were ground-glass opacities and micronodules. Quantitative analysis showed a mean lung attenuation of -736.4 HU in patients with FWL, compared with -775.0 HU in workers without the disease (P <.05). CONCLUSION: While ground-glass opacities, micronodules, or both were found in all cases of FWL, these abnormalities were also present in a substantial proportion of symptomatic flock workers who did not satisfy current criteria for FWL. Although nonspecific, these findings should suggest the diagnosis of FWL in exposed individuals.