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.

Second Harmonic Generation Microscopy as a Novel Intraoperative Assessment of Rat Median Nerve Injury.

PURPOSE: Nerves that are functionally injured but appear macroscopically intact pose the biggest clinical dilemma. Second Harmonic Generation (SHG) Microscopy may provide a real-time assessment of nerve damage, with the ultimate goal of allowing surgeons to accurately quantify the degree of nerve damage present. The aim of this study was to demonstrate the utility of SHG microscopy to detect nerve damage in vivo in an animal model. METHODS: Ten Sprague-Dawley rats were anesthetized and prepared for surgery. After surgical exposure and using a custom-made stretch applicator, the right median nerves were stretched by 20%, corresponding to a high strain injury, and held for 5 minutes. The left median nerve served as a sham control (SC), only being placed in the applicator for 5 minutes with no stretch. A nerve stimulator was used to assess the amount of stimulation required to induce a flicker and contraction of the paw. Nerves were then imaged using a multiphoton laser scanning microscope. RESULTS: Immediately after injury (day 0), SHG images of SC median nerves exhibited parallel collagen fibers with linear, organized alignment. In comparison with SC nerves, high strain nerves demonstrated artifacts indicative of nerve damage consisting of wavy, undulating fibers with crossing fibers and tears, as well as a decrease in the linear organization, which correlated with an increase in the mean stimulation required to induce a flicker and contraction of the paw. CONCLUSIONS: Second Harmonic Generation microscopy may provide the ability to detect an acute neural stretch injury in the rat median nerve. Epineurial collagen disorganization correlated with the stimulation required for nerve function. CLINICAL RELEVANCE: In the future, SHG may provide the ability to visualize nerve damage intraoperatively, allowing for better clinical decision-making. However, this is currently a research tool and requires further validation before translating to the clinical setting.

MicroRNA-21-Dependent Macrophage-to-Fibroblast Signaling Determines the Cardiac Response to Pressure Overload.

BACKGROUND: Cardiac macrophages (cMPs) are increasingly recognized as important regulators of myocardial homeostasis and disease, yet the role of noncoding RNA in these cells is largely unknown. Small RNA sequencing of the entire miRNomes of the major cardiac cell fractions revealed microRNA-21 (miR-21) as the single highest expressed microRNA in cMPs, both in health and disease (25% and 43% of all microRNA reads, respectively). MiR-21 has been previously reported as a key microRNA driving tissue fibrosis. Here, we aimed to determine the function of macrophage miR-21 on myocardial homeostasis and disease-associated remodeling. METHODS: Macrophage-specific ablation of miR-21 in mice driven by Cx3cr1-Cre was used to determine the function of miR-21 in this cell type. As a disease model, mice were subjected to pressure overload for 6 and 28 days. Cardiac function was assessed in vivo by echocardiography, followed by histological analyses and single-cell sequencing. Cocultures of macrophages and cardiac fibroblasts were used to study macrophage-to-fibroblast signaling. RESULTS: Mice with macrophage-specific genetic deletion of miR-21 were protected from interstitial fibrosis and cardiac dysfunction when subjected to pressure overload of the left ventricle. Single-cell sequencing of pressure-overloaded hearts from these mice revealed that miR-21 in macrophages is essential for their polarization toward a M1-like phenotype. Systematic quantification of intercellular communication mediated by ligand-receptor interactions across all cell types revealed that miR-21 primarily determined macrophage-fibroblast communication, promoting the transition from quiescent fibroblasts to myofibroblasts. Polarization of isolated macrophages in vitro toward a proinflammatory (M1-like) phenotype activated myofibroblast transdifferentiation of cardiac fibroblasts in a paracrine manner and was dependent on miR-21 in cMPs. CONCLUSIONS: Our data indicate a critical role of cMPs in pressure overload-induced cardiac fibrosis and dysfunction and reveal macrophage miR-21 as a key molecule for the profibrotic role of cMPs.

Outcomes of Arthroscopic Elbow Contracture Release: Improvement for Severe Prosupination and Flexion Contracture.

PURPOSE: The purpose of this study was to investigate outcomes following arthroscopic elbow contracture release to describe the use of arthroscopy for improvement in extension/flexion and pronation/supination arcs of motion at a single institution for degenerative and posttraumatic etiologies. METHODS: Consecutive arthroscopic elbow arthrolysis performed between 2003 and 2015 were retrospectively reviewed. Basic patient demographics, indications for surgery, preoperative and postoperative elbow range of motion, postoperative patient outcome score, and all complications were recorded and analyzed. RESULTS: Fifty-two patients were included with an average follow-up of 5.1 years (range 1.4 to 9.4). Severe contractures made up 50% of cases, followed by 23% moderate, and 27% mild. Average extension/flexion for the post-traumatic group (n = 30) increased by 63° ± 31 and by 29° ± 24 for the degenerative group (n = 22). Average gain in pronosupination was 38° ± 62 in the post-traumatic group and 13°±23 in the degenerative group. Postoperative DASH scores were 17.5 ± 18.4 for post-traumatic cases and 12.8 ± 19.3 for degenerative cases. CONCLUSION: Arthroscopic elbow contracture release is an effective intervention for degenerative and post-traumatic elbow contracture for both flexion/extension and pronosupination contracture. Furthermore, a two-stage release should be considered when both flexion and pronosupinaton contractures are present. LEVEL OF EVIDENCE: IV, case series, treatment study.

Cra and cAMP Receptor Protein Have Opposing Roles in the Regulation of fruB in Vibrio cholerae.

The Gram-negative bacterium Vibrio cholerae adapts to changes in the environment by selectively producing the necessary machinery to take up and metabolize available carbohydrates. The import of fructose by the fructose-specific phosphoenolpyruvate (PEP) phosphotransferase system (PTS) is of particular interest because of its putative connection to cholera pathogenesis and persistence. Here, we describe the expression and regulation of fruB, which encodes an EIIA-FPr fusion protein as part of the fructose-specific PTS in V. cholerae Using a series of transcriptional reporter fusions and additional biochemical and genetic assays, we identified Cra (catabolite repressor/activator) and cAMP receptor protein (CRP) as regulators of fruB expression and determined that this regulation is dependent upon the presence or absence of PTS sugars. Cra functions as a repressor, downregulating fruB expression in the absence of fructose when components of PTSFru are not needed. CRP functions as an activator of fruB expression. We also report that Cra and CRP can affect fruB expression independently; however, CRP can modulate cra expression in the presence of fructose and glucose. Evidence from this work provides the foundation for continued investigations into PTSFru and its relationship to the V. cholerae life cycle.IMPORTANCEVibrio cholerae is the causative agent of cholera disease. While current treatments of care are accessible, we still lack an understanding of the molecular mechanisms that allow V. cholerae to survive in both aquatic reservoirs and the human small intestine, where pathogenesis occurs. Central to V. cholerae's survival is its ability to use available carbon sources. Here, we investigate the regulation of fruB, which encodes a protein central to the import and metabolism of fructose. We show that fruB expression is controlled by the transcriptional regulators Cra and CRP. This work contributes toward a clearer understanding of how carbon source availability impacts the physiology and, potentially, the persistence of the pathogen.

Impact of CT scanner location on door to imaging time for emergency department stroke evaluation.

BACKGROUND AND OBJECTIVES: Stroke is a potentially serious condition commonly diagnosed in the ED. Time to diagnosis can be crucial to maximizing outcome in a majority of ischemic stroke cases amenable to thrombolytic therapy. METHODS: An analysis of 148 consecutive adults transported by EMS to an urban emergency department with a diagnosis of cerebro-vascular accident during a 12 month period was performed to determine the impact of CT scanner location on door-to-head CT [DTCT] scan time. The CT scanner was relocated from an upper floor of the hospital to within the ER department midway through the study period. RESULTS: The rate of DTCT scan time ≤20 min increased significantly from 47% [pre-relocation] to 74% [post-relocation]; and the rate of DTCT ultra-rapid scan time ≤10 min more than doubled. CONCLUSIONS: Hospitals providing ED care for stroke patients can expedite management by ensuring CT scanner location is in closest possible proximity to the ED.

Identification of a target cell permissive factor required for contact-dependent growth inhibition (CDI).

Bacterial contact-dependent growth inhibition (CDI) is mediated by the CdiB/CdiA family of two-partner secretion proteins. CdiA effector proteins are exported onto the surface of CDI(+) inhibitor cells, where they interact with susceptible bacteria and deliver effectors/toxins derived from their C-terminal regions (CdiA-CT). CDI(+) cells also produce an immunity protein that binds the CdiA-CT and blocks its activity to prevent autoinhibition. Here, we show that the CdiA-CT from uropathogenic Escherichia coli strain 536 (UPEC536) is a latent tRNase that requires activation by the biosynthetic enzyme CysK (O-acetylserine sulfhydrylase A). UPEC536 CdiA-CT exhibits no nuclease activity in vitro, but cleaves within transfer RNA (tRNA) anti-codon loops when purified CysK is added. CysK and CdiA-CT form a stable complex, and their binding interaction appears to mimic that of the CysK/CysE cysteine synthase complex. CdiA-CT activation is also required for growth inhibition. Synthesis of CdiA-CT in E. coli cysK(+) cells arrests cell growth, whereas the growth of ΔcysK mutants is unaffected by the toxin. Moreover, E. coli ΔcysK cells are completely resistant to inhibitor cells expressing UPEC536 CdiA, indicating that CysK is required to activate the tRNase during CDI. Thus, CysK acts as a permissive factor for CDI, providing a potential mechanism to modulate growth inhibition in target cells.

Unraveling the essential role of CysK in CDI toxin activation.

Contact-dependent growth inhibition (CDI) is a widespread mechanism of bacterial competition. CDI(+) bacteria deliver the toxic C-terminal region of contact-dependent inhibition A proteins (CdiA-CT) into neighboring target bacteria and produce CDI immunity proteins (CdiI) to protect against self-inhibition. The CdiA-CT(EC536) deployed by uropathogenic Escherichia coli 536 (EC536) is a bacterial toxin 28 (Ntox28) domain that only exhibits ribonuclease activity when bound to the cysteine biosynthetic enzyme O-acetylserine sulfhydrylase A (CysK). Here, we present crystal structures of the CysK/CdiA-CT(EC536) binary complex and the neutralized ternary complex of CysK/CdiA-CT/CdiI(EC536) CdiA-CT(EC536) inserts its C-terminal Gly-Tyr-Gly-Ile peptide tail into the active-site cleft of CysK to anchor the interaction. Remarkably, E. coli serine O-acetyltransferase uses a similar Gly-Asp-Gly-Ile motif to form the "cysteine synthase" complex with CysK. The cysteine synthase complex is found throughout bacteria, protozoa, and plants, indicating that CdiA-CT(EC536) exploits a highly conserved protein-protein interaction to promote its toxicity. CysK significantly increases CdiA-CT(EC536) thermostability and is required for toxin interaction with tRNA substrates. These observations suggest that CysK stabilizes the toxin fold, thereby organizing the nuclease active site for substrate recognition and catalysis. By contrast, Ntox28 domains from Gram-positive bacteria lack C-terminal Gly-Tyr-Gly-Ile motifs, suggesting that they do not interact with CysK. We show that the Ntox28 domain from Ruminococcus lactaris is significantly more thermostable than CdiA-CT(EC536), and its intrinsic tRNA-binding properties support CysK-independent nuclease activity. The striking differences between related Ntox28 domains suggest that CDI toxins may be under evolutionary pressure to maintain low global stability.

Comparative strength of elbow splint designs: a new splint design as a stronger alternative to posterior splints.

BACKGROUND: Musculoskeletal injuries of the upper extremity are frequently treated with temporary external immobilization. Traditionally, long arm posterior splints have been used to limit flexion/extension of the elbow. However, long arm posterior splints have been observed to fail clinically, necessitating a stronger alternative. In this study, we assessed the biomechanical strength of the long arm posterior splint compared with a new spiral splint design. METHODS: One male and one female participant were placed 10 times in long arm posterior splints and 10 times in spiral splints. Both splint types were subjected to a downward mechanical load of 39.2 N (4 kg) and assessed for a change in both flexion/extension and pronation/supination. RESULTS: There was no significant difference in starting position or starting flexion/extension between the 2 splint designs. Posterior splints allowed significantly greater initial pronation/supination compared with spiral splints. Both splint groups had significant increases in flexion/extension and pronation/supination compared with their starting ranges of motion. There was no significant difference in the change in pronation/supination between the 2 splint groups. Finally, posterior splints allowed a significantly greater change in flexion/extension compared with spiral splints. CONCLUSION: Spiral splints offered less initial pronation/supination than long arm posterior splints. Furthermore, spiral splints are able to resist flexion/extension of the elbow after application of a downward mechanical load better than posterior splints, thus suggesting spiral splints are mechanically superior to long arm posterior splints.

CdiA Effectors from Uropathogenic Escherichia coli Use Heterotrimeric Osmoporins as Receptors to Recognize Target Bacteria.

Many Gram-negative bacterial pathogens express contact-dependent growth inhibition (CDI) systems that promote cell-cell interaction. CDI+ bacteria express surface CdiA effector proteins, which transfer their C-terminal toxin domains into susceptible target cells upon binding to specific receptors. CDI+ cells also produce immunity proteins that neutralize the toxin domains delivered from neighboring siblings. Here, we show that CdiAEC536 from uropathogenic Escherichia coli 536 (EC536) uses OmpC and OmpF as receptors to recognize target bacteria. E. coli mutants lacking either ompF or ompC are resistant to CDIEC536-mediated growth inhibition, and both porins are required for target-cell adhesion to inhibitors that express CdiAEC536. Experiments with single-chain OmpF fusions indicate that the CdiAEC536 receptor is heterotrimeric OmpC-OmpF. Because the OmpC and OmpF porins are under selective pressure from bacteriophages and host immune systems, their surface-exposed loops vary between E. coli isolates. OmpC polymorphism has a significant impact on CDIEC536 mediated competition, with many E. coli isolates expressing alleles that are not recognized by CdiAEC536. Analyses of recombinant OmpC chimeras suggest that extracellular loops L4 and L5 are important recognition epitopes for CdiAEC536. Loops L4 and L5 also account for much of the sequence variability between E. coli OmpC proteins, raising the possibility that CDI contributes to the selective pressure driving OmpC diversification. We find that the most efficient CdiAEC536 receptors are encoded by isolates that carry the same cdi gene cluster as E. coli 536. Thus, it appears that CdiA effectors often bind preferentially to "self" receptors, thereby promoting interactions between sibling cells. As a consequence, these effector proteins cannot recognize nor suppress the growth of many potential competitors. These findings suggest that self-recognition and kin selection are important functions of CDI.

Varus posteromedial rotatory instability: a biomechanical analysis of posterior bundle of the medial ulnar collateral ligament reconstruction.

BACKGROUND: Recently, there has been growing interest in the involvement of the posterior bundle of the medial ulnar collateral ligament (pMUCL) in varus posteromedial rotatory instability (PMRI). Varus PMRI has been observed clinically, but the degree of involvement of the pMUCL remains unclear. This study assessed the degree to which the pMUCL is involved in stabilizing the elbow and the feasibility of a pMUCL reconstruction to restore stability. METHODS: Movements simulating PMRI were performed in 8 cadaveric elbows. Joint gapping values were obtained by 3-dimensional motion capture for the proximal and distal aspects of the ulnohumeral joint. Specimens were assessed at "intact," "cut coronoid + pMUCL," "reconstruction," and "cut anterior aspect MUCL + reconstruction" conditions with mechanical testing at 30°, 60°, and 90° of elbow flexion. RESULTS: Proximal joint gapping significantly increased from intact to cut coronoid + pMUCL at 60° and 90°, and distal joint gapping significantly increased at 90°. In the reconstruction condition, joint gapping across the proximal joint at 60° and 90° significantly recovered, as did distal joint gapping at 90°. In the cut anterior aspect MUCL + reconstruction condition, no significant increase occurred in proximal or distal joint gapping. CONCLUSIONS: Transection of the pMUCL with a coronoid fracture leads to increased joint gapping, suggesting the presence of PMRI. PMRI can still occur with an intact lateral ligamentous complex. A pMUCL tendon graft reconstruction confers some elbow stability in this injury mechanism.

Risk factors for and timing of adverse events after total shoulder arthroplasty.

BACKGROUND: Total shoulder arthroplasty (TSA) is a likely target for future bundled payment initiatives, necessitating accurate preoperative risk stratification. The purpose of this study was to identify risk factors for unplanned readmission and severe adverse events, to risk stratify TSA patients based on these risk factors, and to assess timing of complications after TSA. METHODS: Data were collected from patients undergoing TSA from 2009 to 2014 in the American College of Surgeons National Surgical Quality Improvement Program. Bivariate and multivariate analyses of risk factors for severe adverse events or readmission were assessed. Patients were risk stratified, and timing of severe adverse events and cause of readmission were evaluated. RESULTS: The analysis included 5801 TSA patients; 146 (2.5%) suffered severe adverse events, and 158 (2.7%) had a 30-day unplanned readmission. The most common severe adverse events were reoperation (40%), thrombolic event (deep venous thrombosis or pulmonary embolism; 14%), cardiac event (10%), and death (8.2%). Pneumonia (8.9%) and thrombolic event (7.6%) were the most common medically related causes, whereas dislocation (7.6%) and postoperative infection or wound complication (5.1%) were the most common surgical causes for readmission. Multivariate analysis identified inflammatory arthritis (P = .026), male gender (P = .019), age (P < .001), functional status (P = .024), and American Society of Anesthesiologists class 3/4 (P = .01) as independent predictors for unplanned 30-day readmission and all but inflammatory arthritis for severe adverse events (P ≤ .05 for all). Patients with ≥3 risk factors had an 11.56 (P = .002) and 3.43 (P = .013) times increased odds of unplanned readmission and severe adverse events occurring within 2 weeks after surgery, respectively, compared with patients with 0 risk factors. CONCLUSIONS: Patients at high risk of TSA complications and readmission should be identified preoperatively to improve outcomes and to lower costs. Bundled payment initiatives must account for both patient- and procedure-related risk factors.

Moonlighting O-acetylserine sulfhydrylase: New functions for an old protein.

O-acetylserine sulfhydrylase A (CysK) is the pyridoxal 5'-phosphate-dependent enzyme that catalyzes the final reaction of cysteine biosynthesis in bacteria. CysK was initially identified in a complex with serine acetyltransferase (CysE), which catalyzes the penultimate reaction in the synthetic pathway. This "cysteine synthase" complex is stabilized by insertion of the CysE C-terminus into the active-site of CysK. Remarkably, the CysK/CysE binding interaction is conserved in most bacterial and plant systems. For the past 40years, CysK was thought to function exclusively in cysteine biosynthesis, but recent studies have revealed a repertoire of additional "moonlighting" activities for this enzyme. CysK and its paralogs influence transcription in both Gram-positive bacteria and the nematode Caenorhabditis elegans. CysK also activates an antibacterial nuclease toxin produced by uropathogenic Escherichia coli. Intriguingly, each moonlighting activity requires a binding partner that invariably mimics the C-terminus of CysE to interact with the CysK active site. This article is part of a Special Issue entitled: Cofactor-dependent proteins: evolution, chemical diversity and bio-applications.

Day of Surgery and Surgical Start Time Affect Hospital Length of Stay After Total Hip Arthroplasty.

BACKGROUND: The United States spends $12 billion each year on ∼332,000 total hip arthroplasty (THA) procedures with the postoperative period accounting for ∼40% of costs. The purpose of this study was to evaluate the effect of surgical scheduling (day of week and start time) on clinical outcomes, hospital length of stay (LOS), and rate of nonhome discharge in THA patients. METHODS: Analysis of perioperative variables was performed for patients who underwent THA at an urban tertiary care teaching hospital from 2009 to 2014. RESULTS: A total of 580 THA patients were included for analysis. LOS was higher for the Thursday/Friday cohort compared to Monday/Tuesday (3.7 vs 3.4 days; P = .03). Patients who had a surgical start time after 2 PM had longer LOS compared to patients operated on before 2 PM (3.9 vs 3.5 days; P = .03). After controlling for patient comorbidities and THA surgical approach (direct anterior vs posterior), Thursday/Friday THAs were associated with a 3.27 times risk of extended LOS (>75th percentile LOS) compared to Monday/Tuesday THAs (P < .001). Additionally, case start before 2 PM was protective and associated with a 0.46 times odds of extended LOS (P = .01). LOS reduction opportunity for changing surgical start time to before 2 PM was 0.9 days for high-risk patients (American Society of Anesthesiology class 3/4 and/or liver disease) and 0.2 days for low-risk patients (American Society of Anesthesiology class 1/2). CONCLUSION: Patients who underwent THA Thursday/Friday or had start times after 2 PM had significantly extended hospital LOS. Preoperative risk modification along with adjustments to surgical scheduling and/or perioperative staffing may reduce LOS and thus hospital expenditures for THA procedures.

The F pilus mediates a novel pathway of CDI toxin import.

Contact-dependent growth inhibition (CDI) is a widespread form of inter-bacterial competition that requires direct cell-to-cell contact. CDI(+) inhibitor cells express CdiA effector proteins on their surface. CdiA binds to specific receptors on susceptible target bacteria and delivers a toxin derived from its C-terminal region (CdiA-CT). Here, we show that purified CdiA-CT(536) toxin from uropathogenic Escherichia coli 536 translocates into bacteria, thereby by-passing the requirement for cell-to-cell contact during toxin delivery. Genetic analyses demonstrate that the N-terminal domain of CdiA-CT(536) is necessary and sufficient for toxin import. The CdiA receptor plays no role in this import pathway; nor do the Tol and Ton systems, which are exploited to internalize colicin toxins. Instead, CdiA-CT(536) import requires conjugative F pili. We provide evidence that the N-terminal domain of CdiA-CT(536) interacts with F pilin, and that pilus retraction is critical for toxin import. This pathway is reminiscent of the strategy used by small RNA leviviruses to infect F(+) cells. We propose that CdiA-CT(536) mimics the pilin-binding maturation proteins of leviviruses, allowing the toxin to bind F pili and become internalized during pilus retraction.

The proton-motive force is required for translocation of CDI toxins across the inner membrane of target bacteria.

Contact-dependent growth inhibition (CDI) is a mode of bacterial competition orchestrated by the CdiB/CdiA family of two-partner secretion proteins. The CdiA effector extends from the surface of CDI(+) inhibitor cells, binds to receptors on neighbouring bacteria and delivers a toxin domain derived from its C-terminal region (CdiA-CT). Here, we show that CdiA-CT toxin translocation requires the proton-motive force (pmf) within target bacteria. The pmf is also critical for the translocation of colicin toxins, which exploit the energized Ton and Tol systems to cross the outer membrane. However, CdiA-CT translocation is clearly distinct from known colicin-import pathways because ΔtolA ΔtonB target cells are fully sensitive to CDI. Moreover, we provide evidence that CdiA-CT toxins can be transferred into the periplasm of de-energized target bacteria, indicating that transport across the outer membrane is independent of the pmf. Remarkably, CDI toxins transferred under de-energized conditions remain competent to enter the target-cell cytoplasm once the pmf is restored. Collectively, these results indicate that outer- and inner-membrane translocation steps can be uncoupled, and that the pmf is required for CDI toxin transport from the periplasm to the target-cell cytoplasm.

Genetically distinct pathways guide effector export through the type VI secretion system.

Bacterial secretion systems often employ molecular chaperones to recognize and facilitate export of their substrates. Recent work demonstrated that a secreted component of the type VI secretion system (T6SS), haemolysin co-regulated protein (Hcp), binds directly to effectors, enhancing their stability in the bacterial cytoplasm. Herein, we describe a quantitative cellular proteomics screen for T6S substrates that exploits this chaperone-like quality of Hcp. Application of this approach to the Hcp secretion island I-encoded T6SS (H1-T6SS) of Pseudomonas aeruginosa led to the identification of a novel effector protein, termed Tse4 (type VI secretion exported 4), subsequently shown to act as a potent intra-specific H1-T6SS-delivered antibacterial toxin. Interestingly, our screen failed to identify two predicted H1-T6SS effectors, Tse5 and Tse6, which differ from Hcp-stabilized substrates by the presence of toxin-associated PAAR-repeat motifs and genetic linkage to members of the valine-glycine repeat protein G (vgrG) genes. Genetic studies further distinguished these two groups of effectors: Hcp-stabilized effectors were found to display redundancy in interbacterial competition with respect to the requirement for the two H1-T6SS-exported VgrG proteins, whereas Tse5 and Tse6 delivery strictly required a cognate VgrG. Together, we propose that interaction with either VgrG or Hcp defines distinct pathways for T6S effector export.

Blurring personal health and public priorities: an analysis of celebrity health narratives in the public sphere.

This article explores the functions of personal celebrity health narratives in the public sphere. This study examines data about 157 celebrities, including athletes, actors, musicians, and politicians, who have shared private information regarding a personal health situation (or that of a loved one) with others in the public domain. Part of a larger project on celebrity health narratives, this article highlights three key functions that celebrity health narratives perform--education, inspiration, and activism--and discusses the implications for celebrities and for public conversations about health-related issues.

Facial Soft Tissue Lesions in Children.

Facial soft tissue lesions in children are often classified based on their structure or cellular origin and can be benign or malignant. This review focuses on common facial soft tissue lesions in children, their clinical morphology, natural history, and medical and surgical management, with an emphasis on those considerations unique to soft tissue lesions present at this anatomic site.