Tit-for-tat: type VI secretion system counterattack during bacterial cell-cell interactions.

The bacterial type VI secretion system (T6SS) is a dynamic organelle that bacteria use to target prey cells for inhibition via translocation of effector proteins. Time-lapse fluorescence microscopy has documented striking dynamics of opposed T6SS organelles in adjacent sister cells of Pseudomonas aeruginosa. Such cell-cell interactions have been termed "T6SS dueling" and likely reflect a biological process that is driven by T6SS antibacterial attack. Here, we show that T6SS dueling behavior strongly influences the ability of P. aeruginosa to prey upon heterologous bacterial species. We show that, in the case of P. aeruginosa, T6SS-dependent killing of either Vibrio cholerae or Acinetobacter baylyi is greatly stimulated by T6SS activity occurring in those prey species. Our data suggest that, in P. aeruginosa, T6SS organelle assembly and lethal counterattack are regulated by a signal that corresponds to the point of attack of the T6SS apparatus elaborated by a second aggressive T6SS(+) bacterial cell. PAPERFLICK:

For pet's sake: Discovering a naturally occurring zebrafish virus.

Zebrafish are often used to model host-pathogen interactions, but few models of natural virus infection have been established. A new study in PLOS Biology shows that metatranscriptomics and cohousing experiments can uncover a natural pathogenic virus of zebrafish for laboratory study.

Use of zebrafish to identify host responses specific to type VI secretion system mediated interbacterial antagonism.

Interbacterial competition is known to shape the microbial communities found in the host, however the interplay between this competition and host defense are less clear. Here, we use the zebrafish hindbrain ventricle (HBV) as an in vivo platform to investigate host responses to defined bacterial communities with distinct forms of interbacterial competition. We found that antibacterial activity of the type VI secretion system (T6SS) from both Vibrio cholerae and Acinetobacter baylyi can induce host inflammation and sensitize the host to infection independent of any individual effector. Chemical suppression of inflammation could resolve T6SS-dependent differences in host survival, but the mechanism by which this occurred differed between the two bacterial species. By contrast, colicin-mediated antagonism elicited by an avirulent strain of Shigella sonnei induced a negligible host response despite being a more potent bacterial killer, resulting in no impact on A. baylyi or V. cholerae virulence. Altogether, these results provide insight into how different modes of interbacterial competition in vivo affect the host in distinct ways.

Extended DNA-binding interfaces beyond the canonical SAP domain contribute to the function of replication stress regulator SDE2 at DNA replication forks.

Elevated DNA replication stress causes instability of the DNA replication fork and increased DNA mutations, which underlies tumorigenesis. The DNA replication stress regulator silencing-defective 2 (SDE2) is known to bind to TIMELESS (TIM), a protein of the fork protection complex, and enhances its stability, thereby supporting replisome activity at DNA replication forks. However, the DNA-binding activity of SDE2 is not well defined. Here, we structurally and functionally characterize a new conserved DNA-binding motif related to the SAP (SAF-A/B, Acinus, PIAS) domain in human SDE2 and establish its preference for ssDNA. Our NMR solution structure of the SDE2SAP domain reveals a helix-extended loop-helix core with the helices aligned parallel to each other, consistent with known canonical SAP folds. Notably, we have shown that the DNA interaction of this SAP domain extends beyond the core SAP domain and is augmented by two lysine residues in the C-terminal tail, which is uniquely positioned adjacent to the SAP motif and conserved in the pre-mRNA splicing factor SF3A3. Furthermore, we found that mutation in the SAP domain and extended C terminus not only disrupts ssDNA binding but also impairs TIM localization at replication forks, thus inhibiting efficient fork progression. Taken together, our results establish SDE2SAP as an essential element for SDE2 to exert its role in preserving replication fork integrity via fork protection complex regulation and highlight the structural diversity of the DNA-protein interactions achieved by a specialized DNA-binding motif.

Exopolysaccharide protects Vibrio cholerae from exogenous attacks by the type 6 secretion system.

The type 6 secretion system (T6SS) is a nanomachine used by many Gram-negative bacteria, including Vibrio cholerae, to deliver toxic effector proteins into adjacent eukaryotic and bacterial cells. Because the activity of the T6SS is dependent on direct contact between cells, its activity is limited to bacteria growing on solid surfaces or in biofilms. V. cholerae can produce an exopolysaccharide (EPS) matrix that plays a role in adhesion and biofilm formation. In this work, we investigated the effect of EPS production on T6SS activity between cells. We found that EPS produced by V. cholerae cells functions as a unidirectional protective armor that blocks exogenous T6SS attacks without interfering with its own T6SS functionality. This EPS armor is effective against both same-species and heterologous attackers. Mutations modulating the level of EPS biosynthesis gene expression result in corresponding modulation in V. cholerae resistance to exogenous T6SS attack. These results provide insight into the potential role of extracellular biopolymers, including polysaccharides, capsules, and S-layers in protecting bacterial cells from attacks involving cell-associated macromolecular protein machines that cannot readily diffuse through these mechanical defenses.

3D imaging for hindfoot alignment assessment: a comparative study between non-weight-bearing MRI and weight-bearing CT.

PURPOSE: The authors compared measurements of hindfoot alignment on MR imaging with weight-bearing CT (WB-CT) to establish the degree of correlation. Forty-seven feet in 44 patients had weight-bearing CT and MRI studies performed on the same day. MATERIALS AND METHODS: Hindfoot alignment on MRI was assessed by two radiologists who calculated tibiocalcaneal angle (TCA) and calcaneofibular ligament angle (CFLA). On WB-CT, foot ankle offset (FAO), calcaneal offset (CO) and hindfoot angle (HA) were assessed by a senior Foot and Ankle Surgeon using dedicated software. Pearson correlation coefficient was used to evaluate the correlation between these measurements. RESULTS: The study group comprised 27 males and 17 females with a mean age of 45 years (range 13-79 years). A statistically significant positive correlation was identified between TCA on MRI and all measurements of hindfoot alignment on WB-CT (p = 0.001-0.005). The CFLA on MRI only had significant correlation with CO on WB-CT (p = 0.03). A significant negative correlation was observed between both MRI parameters (p < 0.001). CONCLUSION: A highly significant correlation between tibiocalcaneal angle on non-weight-bearing ankle MR imaging and hindfoot alignment measurements on weight-bearing CT was identified.

The awareness of hindfoot malalignment on non-weight-bearing ankle MRI.

OBJECTIVE: Hindfoot malalignment is a relatively common clinical finding and several studies have suggested that hindfoot valgus can be identified on non-weight-bearing ankle MRI. The aim of this study was to determine the awareness of hindfoot malalignment on ankle MRI amongst consultant musculoskeletal radiologists. MATERIALS AND METHODS: All MRI studies referred by Foot and Ankle Unit Consultants reported by one of 14 consultant musculoskeletal radiologists between March 2016 and August 2019 were retrieved from the Hospital Radiology Information System. These were reviewed independently by a radiology fellow and a consultant radiologist. Tibiocalcaneal angle (TCA) was measured, and extra-articular talocalcaneal (EA-TCI) and calcaneofibular impingement (EA-CFI) were recorded. Radiology reports were then analysed for mention of hindfoot malalignment and the presence of EA-TCI and EA-CFI. RESULTS: The study group comprised 129 patients, 46 males and 83 females with a mean age of 46.8 years (range 8-84 years). Based on review, hindfoot valgus was present in 78-80 cases (60.5-62%), EA-TCI in 30-36 cases (23.2-27.9%) and EA-CFI in 18-21 cases (14-16.3%). By comparison, MRI reports mentioned hindfoot valgus in 18 cases (2 incorrectly), EA-TCI in 8 cases (1 incorrectly) and EA-CFI in 10 cases (1 incorrectly). CONCLUSION: Hindfoot valgus, EA-TCI and EA-CFI were present relatively commonly on review of ankle MRI studies in patients referred from a specialist Foot and Ankle Unit but were commonly under-reported highlighting a relative lack of awareness of hindfoot malalignment on ankle MRI amongst musculoskeletal radiologists, which could impact negatively on patient management.

Therapeutic effects of a small molecule agonist of the relaxin receptor ML290 in liver fibrosis.

Fibrosis is an underlying cause of cirrhosis and hepatic failure resulting in end stage liver disease with limited pharmacological options. The beneficial effects of relaxin peptide treatment were demonstrated in clinically relevant animal models of liver fibrosis. However, the use of relaxin is problematic because of a short half-life. The aim of this study was to test the therapeutic effects of recently identified small molecule agonists of the human relaxin receptor, relaxin family peptide receptor 1 (RXFP1). The lead compound of this series, ML290, was selected based on its effects on the expression of fibrosis-related genes in primary human stellate cells. RNA sequencing analysis of TGF-ß1-activated LX-2 cells showed that ML290 treatment primarily affected extracellular matrix remodeling and cytokine signaling, with expression profiles indicating an antifibrotic effect of ML290. ML290 treatment in human liver organoids with LPS-induced fibrotic phenotype resulted in a significant reduction of type I collagen. The pharmacokinetics of ML290 in mice demonstrated its high stability in vivo, as evidenced by the sustained concentrations of compound in the liver. In mice expressing human RXFP1 gene treated with carbon tetrachloride, ML290 significantly reduced collagen content, α-smooth muscle actin expression, and cell proliferation around portal ducts. In conclusion, ML290 demonstrated antifibrotic effects in liver fibrosis.-Kaftanovskaya, E. M., Ng, H. H., Soula, M., Rivas, B., Myhr, C., Ho, B. A., Cervantes, B. A., Shupe, T. D., Devarasetty, M., Hu, X., Xu, X., Patnaik, S., Wilson, K. J., Barnaeva, E., Ferrer, M., Southall, N. T., Marugan, J. J., Bishop, C. E., Agoulnik, I. U., Agoulnik, A. I. Therapeutic effects of a small molecule agonist of the relaxin receptor ML290 in liver fibrosis.

Second generation of temporary implantable nitinol device (iTind) in men with LUTS: 2 year results of the MT-02-study.

OBJECTIVES: Assessing medium-term functional results of a novel minimally-invasive treatment for lower urinary tract symptoms due to BPO with the second generation of the temporary implantable nitinol device (iTind; Medi-Tate Ltd®, Israel): 2-year follow-up of a single-arm, prospective, international multicenter study. Further, we aimed to identify preoperative baseline parameters predicting response to iTind treatment. METHODS: Following local ethical committee approval in every participating centre, 81 men with symptomatic BPO (IPSS ≥ 10, peak urinary flow < 12 ml/s, and prostate volume < 75 ml) were enrolled in this study. Patients with PVR > 250 ml, obstructive median lobe, previous prostatic surgery, confounding bladder or sphincter dysfunction based on medical history, active urinary infection and unable to interrupt antithrombotic or antiplatelet treatment were exclusion criteria. A wash-out period of 1 month for alpha-blockers and 6 months for 5-ARIs was mandatory to avoid confounders. The procedure was performed as previously described: implantation under light sedation and removal 5-7 days later with topical sedation. Patients were assessed for perioperative results including OR-time, pain (VAS) and complications (Clavien-Dindo-Grading System); and for functional results (PVR, Qmax, IPSS) and quality of life (QoL) including sexual and ejaculatory function using two yes/no questions. Follow-up assessments were done at 1, 3, and 6 months, and 1 and 2 years. RESULTS: Of the 81 patients initially enrolled in this study, follow-up included 67 men at 1 year and 51 men at 2 years. For the 51 men included in the present analysis, the median age was 65 years, median prostate volume 37 ml (range 16-65 ml). Baseline values for IPSS and QoL were 20.51 ± 4.58, 3.96 ± 0.87. Qmax and PVR were 7.62 ± 2.25 ml/s and 65.84 ± 38.46, respectively. No intraoperative complications were observed and the average pain level recorded on the visual analogue scale (VAS) was 3.2 ± 1.6. A significant reduction in symptoms and improvement in urinary flow was observed (p < 0.0001) at all assessment points: IPSS-score and QoL improved to 8.51 ± 5.51 and 1.76 ± 1.32, respectively; and Qmax increased to 16.00 ± 7.43 ml/s. None of the patients who were previously sexually active reported a deterioration in sexual or ejaculatory functions according to two yes/no questions over the follow-up period. Excluding the patients lost at follow-up, five patients underwent surgery between 12 and 24 months. Upon investigation, it was discovered that four of the five patients requiring surgery had median lobes and were protocol deviators. A failure analysis was carried out for all 81 patients in order to identify baseline parameters that could predict treatment failure. 58.33% of patients in the failure group (7 out of 12) had median lobes which was found to be statistically significant (p < 0.0001). None of the other preoperative variables (age, prostate volume, IPSS scores, Qmax, PVR, and PSA) were found to predict response to iTind treatment. CONCLUSION: iTind treatment for BPO-related LUTS showed marked and durable reduction in symptoms and improvement of functional parameters and quality of life at 24 months of follow-up. It was found that median lobe may predict failure of iTind treatment. According to the yes/no questions, ejaculatory and sexual functions do not seem to be effected following treatment, however, this finding must be supported with further studies using the accepted tools.

Vibrio cholerae type 6 secretion system effector trafficking in target bacterial cells.

The type 6 secretion system (T6SS) is used by many Gram-negative bacterial species to deliver toxic effector proteins into nearby bacteria prey cells to kill or inhibit their growth. VgrG proteins are core conserved secretion substrates of the T6SS and one subset of T6SS effectors consists of VgrG proteins with C-terminal extension domains carrying various enzymatic activities. In Vibrio cholerae, VgrG3 has a hydrolase extension domain and degrades peptidoglycan in the periplasm of target bacteria. In this study, we replaced this domain with a nuclease domain from Salmonella enterica subsp. arizonae This modified V. cholerae strain was able to kill its parent using its T6SS. This result also demonstrated that V. cholerae T6SS is capable of delivering effectors that could attack substrates found either in the periplasm or cytosol of target bacteria. Additionally, we found that effectors VgrG3 and TseL, despite lacking a classical Sec or TAT secretion signal, were able to reach the periplasm when expressed in the bacterial cytosol. This effector trafficking likely represents an evolutionary strategy for T6SS effectors to reach their intended substrates regardless of which subcellular compartment in the target cell they happen to be delivered to by the T6SS apparatus.

Implementation of an interdisciplinary tracheostomy care protocol to decrease rates of tracheostomy-related pressure ulcers and injuries.

OBJECTIVES: Tracheostomy-related pressure injuries (TRPI) have been demonstrated to occur in approximately 10% of tracheostomy patients. In this study, we present TRPI outcomes after implementation of a standardized tracheostomy care protocol. METHODS: A tracheostomy care protocol was developed by an interdisciplinary quality improvement program and implemented on July 1, 2016. The protocol was designed to minimize factors that contribute to the development of TRPI. Rates of TRPI over the subsequent 20 months were compared to the year before implementation. RESULTS: 9 out of 85 patients (10.6%) developed TRPI in the pre-protocol cohort compared to 0 of 137 (0%) in the post-protocol cohort, which was a statistically significant decrease by Fisher's exact test with a p-value of 0.0001. Pearson's correlation coefficient demonstrated a negative correlation between age and post-operative day of diagnosis (r = -0.641, p = 0.063), indicating that older patients develop TRPI more quickly. CONCLUSIONS: Interdisciplinary peri-operative tracheostomy care protocols can be effective in decreasing rates of TRPI.

From Wall to Wall: How the Type 6 Secretion System Knows To Stop Growing.

There is an inseverable link between the size of a cell and the size of its subcellular components. The type 6 secretion system (T6SS) is no exception. In this issue, Stietz et al. report their analysis of the T6SS under conditions in which the cell size was altered to an extreme degree (M. S. Stietz, X. Liang, M. Wong, S. Hersch, and T. G. Dong, J Bacteriol 202:e00425-19, 2020, https://doi.org/10.1128/JB.00425-19). That study and others investigating the regulation of T6SS filament polymerization have provided insight into how the T6SS apparatus matches its size to the cell that contains it.

Second-generation of temporary implantable nitinol device for the relief of lower urinary tract symptoms due to benign prostatic hyperplasia: results of a prospective, multicentre study at 1 year of follow-up.

OBJECTIVES: To report the clinical experience with a second-generation of temporary implantable nitinol device (iTIND; Medi-Tate Ltd, Or-Akiva, Israel) for the treatment of lower urinary tract symptoms (LUTS) due to benign prostatic hyperplasia (BPH) after 1 year of follow-up. PATIENTS AND METHODS: In all, 81 patients with LUTS, International Prostate Symptom Score (IPSS) ≥10, maximum urinary flow rate (Qmax ) ≤12 mL/s, and prostate volume <75 mL, were enrolled in this prospective Research Ethics Committee-approved multicentre study. The main exclusion criteria were: haemostatic disorders, post-void residual urine volume (PVR) >250 mL, obstructive median lobe, and previous prostate surgery. The iTIND was implanted within the bladder neck and the prostatic urethra under light sedation, using a rigid cystoscope. The device was removed 5-7 days later in an outpatient setting. Demographics, perioperative results, complications (according to the Clavien-Dindo system), functional results and quality of life (QoL) were evaluated. Follow-up assessments were conducted at 1, 3, 6 and 12 months postoperatively. RESULTS: The mean (sd) patient age was 65 (8.9) years, prostate volume was 40.5 (12.25) mL, Qmax was 7.3 (2.6) mL/s, IPSS was 22.5 (5.6), and the median (interquartile range) IPSS QoL score was 4 (2-5). All the implantations were successful, with no intraoperative complications recorded; all patients were discharged on the same day of surgery. The devices were retrieved at a mean (SD) of 5.9 (1.1) days after implantation, typically under topical anaesthesia. No Clavien-Dindo Grade >II complications were recorded. The mean (SD) Qmax at the 1 month follow-up visit was 11.2 (5.7) mL/s and continued to improve thereafter, reaching 14.7 (8.1) mL/s at the 12-month follow-up visit (+100%). The mean (SD) IPSS urinary symptom scores were 11.7 (8.0) after 1 month and further improved to 8.8 (6.4) at the 12-month follow-up (-60%). In parallel, the mean (SD) IPSS QoL score drop reached 1.6 (1.3) by the end of the study. During the 12-month period, two patients (2.4%) required medical therapy for BPH, two patients (2.4%) required transurethral resection of the prostate, whilst 10 patients were lost to follow-up (12.3%). As compared to baseline, none of the 61 sexually active patients who completed the 12-month follow-up period reported sexual or ejaculatory dysfunction. CONCLUSION: iTIND implantation is feasible, safe and effective in providing relief of BPH-related symptoms, at least until 12 months postoperatively. Sexual and ejaculatory functions are fully preserved. Further studies with a longer follow-up period are needed to assess the durability of these results and to clearly define the indications for iTIND implantation.

PAAR-repeat proteins sharpen and diversify the type VI secretion system spike.

The bacterial type VI secretion system (T6SS) is a large multicomponent, dynamic macromolecular machine that has an important role in the ecology of many Gram-negative bacteria. T6SS is responsible for translocation of a wide range of toxic effector molecules, allowing predatory cells to kill both prokaryotic as well as eukaryotic prey cells. The T6SS organelle is functionally analogous to contractile tails of bacteriophages and is thought to attack cells by initially penetrating them with a trimeric protein complex called the VgrG spike. Neither the exact protein composition of the T6SS organelle nor the mechanisms of effector selection and delivery are known. Here we report that proteins from the PAAR (proline-alanine-alanine-arginine) repeat superfamily form a sharp conical extension on the VgrG spike, which is further involved in attaching effector domains to the spike. The crystal structures of two PAAR-repeat proteins bound to VgrG-like partners show that these proteins sharpen the tip of the T6SS spike complex. We demonstrate that PAAR proteins are essential for T6SS-mediated secretion and target cell killing by Vibrio cholerae and Acinetobacter baylyi. Our results indicate a new model of the T6SS organelle in which the VgrG-PAAR spike complex is decorated with multiple effectors that are delivered simultaneously into target cells in a single contraction-driven translocation event.

Multi-material three dimensional printed models for simulation of bronchoscopy.

BACKGROUND: Bronchoscopy involves exploration of a three-dimensional (3D) bronchial tree environment using just two-dimensional (2D) images, visual cues and haptic feedback. Sound knowledge and understanding of tracheobronchial anatomy as well as ample training experience is mandatory for technical mastery. Although simulated modalities facilitate safe training for inexperienced operators, current commercial training models are expensive or deficient in anatomical accuracy, clinical fidelity and patient representation. The advent of Three-dimensional (3D) printing technology may resolve the current limitations with commercial simulators. The purpose of this report is to develop and test the novel multi-material three-dimensional (3D) printed airway models for bronchoscopy simulation. METHODS: Using material jetting 3D printing and polymer amalgamation, human airway models were created from anonymized human thoracic computed tomography images from three patients: one normal, a second with a tumour obstructing the right main bronchus and third with a goitre causing external tracheal compression. We validated their efficacy as airway trainers by expert bronchoscopists. Recruited study participants performed bronchoscopy on the 3D printed airway models and then completed a standardized evaluation questionnaire. RESULTS: The models are flexible, life size, anatomically accurate and patient specific. Five expert respiratory physicians participated in validation of the airway models. All the participants agreed that the models were suitable for training bronchoscopic anatomy and access. Participants suggested further refinement of colour and texture of the internal surface of the airways. Most respondents felt that the models are suitable simulators for tracheal pathology, have a learning value and recommend it to others for use in training. CONCLUSION: Using material jetting 3D printing to create patient-specific anatomical models is a promising modality of simulation training. Our results support further evaluation of the printed airway model as a bronchoscopic trainer, and suggest that pathological airways may be simulated using this technique.

Risk Factors for Early ACL Reconstruction Failure in Pediatric and Adolescent Patients: A Review of 561 Cases.

BACKGROUND: Anterior cruciate ligament (ACL) reconstruction failure is relatively common in young high-risk athletes. The purpose of this study was to examine a single center's 10-year experience with ACL reconstructions in pediatric and adolescent patients to better define short-term failure rates and risk factors for revision ACL surgery. METHODS: This institutional review board-approved retrospective study included all patients who underwent a primary ACL reconstruction between 2002 and 2013. Chart and radiographic review was performed to assess patient demographic, injury, and surgical data including growth plate status, concomitant ligament/meniscus/cartilage injury, surgical procedures, femoral drilling technique, graft source and type, femoral and tibial fixation devices, and graft size. Graft failures had to be confirmed both with clinical examination and magnetic resonance imaging or the patient had to undergo a revision ACL reconstruction. Potential factors associated with failure were evaluated using either parametric or nonparametric analysis as appropriate. RESULTS: A total of 561 ACL reconstructions were performed that met our inclusion criteria. The average patient age was 15.4 years (range, 5 to 19 y) and 53% of the patients were male. In all, 54 failures were identified for a 9.6% failure rate. Soft tissue grafts were twice as likely to fail compared with patellar tendon grafts (13% vs. 6%; P<0.001). Multivariate analysis revealed that graft choice (soft tissue vs. patellar tendon) was the primary variable predictive of failure (P<0.05), with interactions/mediating effects contributed by maturity (growth plate status) and ACL technique (P<0.05). The average time to failure was 13.6 months and hamstring grafts and anatomic femoral tunnels were both found to fail earlier (P<0.05). During the study period, approximately 8% of patients sustained a contralateral ACL injury. CONCLUSIONS: ACL failure rates in adolescent and pediatric patients vary based on patient age, graft selection, and surgical technique. Bone patellar tendon bone autografts had the lowest failure rate in this high-risk population. LEVEL OF EVIDENCE: Level IV-retrospective case series.

Adoption of innovative and evidence-based practices for children and adolescents in state-supported mental health clinics: a qualitative study.

BACKGROUND: This study examined how mental health clinic administrators decided whether or not to adopt evidence-based and other innovative practices by exploring their views of implementation barriers and facilitators and operation of these views in assessment of implementation costs and benefits. METHODS: Semi-structured interviews were conducted with 75 agency chief executive officers and program directors of 34 New York State-licensed mental health clinics serving children and adolescents. RESULTS: Three interconnected themes relating to barriers and facilitators were identified, namely costs and benefits associated with adoption, capacity for adoption, and acceptability of new practices. The highest percentage of participants (86.7%) mentioned costs as a barrier, followed by limited capacity (55.9%) and lack of acceptability (52.9%). The highest percentage (82.3%) of participants identified available capacity as a facilitator, followed by acceptability (41.2%) and benefits or limited costs (24.0%). Assessment of costs and benefits exhibited several principles of behavioural economics, including loss aversion, temporal discounting use of heuristics, sensitivity to monetary incentives, decision fatigue, framing, and environmental influences. CONCLUSIONS: The results point to opportunities for using agency leader models to develop strategies to facilitate implementation of evidence-based and innovative practices for children and adolescents.

Structural Insights into the Activation of Human Relaxin Family Peptide Receptor 1 by Small-Molecule Agonists.

The GPCR relaxin family peptide receptor 1 (RXFP1) mediates the action of relaxin peptide hormone, including its tissue remodeling and antifibrotic effects. The peptide has a short half-life in plasma, limiting its therapeutic utility. However, small-molecule agonists of human RXFP1 can overcome this limitation and may provide a useful therapeutic approach, especially for chronic diseases such as heart failure and fibrosis. The first small-molecule agonists of RXFP1 were recently identified from a high-throughput screening, using a homogeneous cell-based cAMP assay. Optimization of the hit compounds resulted in a series of highly potent and RXFP1 selective agonists with low cytotoxicity, and excellent in vitro ADME and pharmacokinetic properties. Here, we undertook extensive site-directed mutagenesis studies in combination with computational modeling analysis to probe the molecular basis of the small-molecule binding to RXFP1. The results showed that the agonists bind to an allosteric site of RXFP1 in a manner that closely interacts with the seventh transmembrane domain (TM7) and the third extracellular loop (ECL3). Several residues were determined to play an important role in the agonist binding and receptor activation, including a hydrophobic region at TM7 consisting of W664, F668, and L670. The G659/T660 motif within ECL3 is crucial to the observed species selectivity of the agonists for RXFP1. The receptor binding and activation effects by the small molecule ML290 were compared with the cognate ligand, relaxin, providing valuable insights on the structural basis and molecular mechanism of receptor activation and selectivity for RXFP1.

Identification of T6SS-dependent effector and immunity proteins by Tn-seq in Vibrio cholerae.

Type VI protein secretion system (T6SS) is important for bacterial competition through contact-dependent killing of competitors. T6SS delivers effectors to neighboring cells and corresponding antagonistic proteins confer immunity against effectors that are delivered by sister cells. Although T6SS has been found in more than 100 gram-negative bacteria including many important human pathogens, few T6SS-dependent effector and immunity proteins have been experimentally determined. Here we report a high-throughput approach using transposon mutagenesis and deep sequencing (Tn-seq) to identify T6SS immunity proteins in Vibrio cholerae. Saturating transposon mutagenesis was performed in wild type and a T6SS null mutant. Genes encoding immunity proteins were predicted to be essential in the wild type but dispensable in the T6SS mutant. By comparing the relative abundance of each transposon mutant in the mutant library using deep sequencing, we identified three immunity proteins that render protection against killing by T6SS predatory cells. We also identified their three cognate T6SS-secreted effectors and show these are important for not only antibacterial and antieukaryotic activities but also assembly of T6SS apparatus. The lipase and muramidase T6SS effectors identified in this study underscore the diversity of T6SS-secreted substrates and the distinctly different mechanisms that target these for secretion by the dynamic T6SS organelle.

Psychometric properties of Functional Assessment of Cancer Therapy-Prostate (FACT-P) in Chinese patients with prostate cancer.

PURPOSE: The aim of the study was to assess the validity, reliability and sensitivity of the FACT-P (version 4) in Chinese males with prostate cancer. METHODS: Construct validity was assessed using Spearman's correlation test against the 12-item Short Form Health Survey (SF-12v2). Internal consistency and test-retest reliability were assessed using Cronbach's α coefficient and intra-class correlation coefficient, respectively. Sensitivity was determined by performing known-group comparisons by independent t test. RESULTS: FACT-P subscale scores had a moderate correlation with the corresponding SF-12v2 domain score that conceptually measures the similar construct providing evidence for adequate construct validity. Internal consistency was acceptable (α: 0.687-0.900) for all subscales aside from the Prostate Cancer Subscale (α: 0.505) and Trial Outcome Index (α: 0.562). FACT-P subscale and total scores showed good test-retest reliability (range 0.753-0.913). All total scales and most of the subscales were sensitive in detecting differences between patients with different levels of functional impairment but not different cancer stages or levels of prostate-specific antigen. CONCLUSIONS: The measure is a valid and reliable measure to assess the health-related quality of life of Chinese males with prostate cancer. The FACT-P is sensitive to detect difference between patients with varying functional status.