Lysophosphatidic Acid Receptor 4 Is Transiently Expressed during Cardiac Differentiation and Critical for Repair of the Damaged Heart.

Efficient differentiation of pluripotent stem cells (PSCs) into cardiac cells is essential for the development of new therapeutic modalities to repair damaged heart tissue. We identified a novel cell surface marker, the G protein-coupled receptor lysophosphatidic acid receptor 4 (LPAR4), specific to cardiac progenitor cells (CPCs) and determined its functional significance and therapeutic potential. During in vitro differentiation of mouse and human PSCs toward cardiac lineage, LPAR4 expression peaked after 3-7 days of differentiation in cardiac progenitors and then declined. In vivo, LPAR4 was specifically expressed in the early stage of embryonal heart development, and as development progressed, LPAR4 expression decreased and was non-specifically distributed. We identified the effective agonist octadecenyl phosphate and a p38 MAPK blocker as the downstream signal blocker. Sequential stimulation and inhibition of LPAR4 using these agents enhanced the in vitro efficiency of cardiac differentiation from mouse and human PSCs. Importantly, in vivo, this sequential stimulation and inhibition of LPAR4 reduced the infarct size and rescued heart dysfunction in mice. In conclusion, LPAR4 is a novel CPC marker transiently expressed only in heart during embryo development. Modulation of LPAR4-positive cells may be a promising strategy for repairing myocardium after myocardial infarction.

Viral Shedding 1 Year Following First-Episode Genital HSV-1 Infection.

Importance: Herpes simplex virus type 1 (HSV-1) is the leading cause of first-episode genital herpes in many countries. Objective: To inform counseling messages regarding genital HSV-1 transmission, oral and genital viral shedding patterns among persons with first-episode genital HSV-1 infection were assessed. The trajectory of the development of HSV-specific antibody and T-cell responses was also characterized. Design, Setting, and Participants: Prospective cohort followed up for up to 2 years, with 82 participants followed up between 2013 and 2018. Participants were recruited from sexual health and primary care clinics in Seattle, Washington. Persons with laboratory-documented first-episode genital HSV-1 infection, without HIV infection or current pregnancy, were referred for enrollment. Exposures: First-episode genital HSV-1 infection. Main Outcomes and Measures: Genital and oral HSV-1 shedding and lesion rates at 2 months, 11 months, and up to 2 years after initial genital HSV-1 infection. Participants self-collected oral and genital swabs for HSV polymerase chain reaction testing for 30 days at 2 and 11 months and up to 2 years after diagnosis of genital HSV-1. Blood samples were collected at serial time points to assess immune responses to HSV-1. Primary HSV-1 infection was defined as absent HSV antibody at baseline or evolving antibody profile using the University of Washington HSV Western Blot. HSV-specific T-cell responses were detected using interferon γ enzyme-linked immunospot. Results: Among the 82 participants, the median (range) age was 26 (16-64) years, 54 (65.9%) were women, and 42 (51.2%) had primary HSV-1 infection. At 2 months, HSV-1 was detected from the genital tract in 53 participants (64.6%) and in the mouth in 24 participants (29.3%). Genital HSV-1 shedding was detected on 275 of 2264 days (12.1%) at 2 months and declined significantly to 122 of 1719 days (7.1%) at 11 months (model-predicted rate, 6.2% [95% CI, 4.3%-8.9%] at 2 months vs 3.2% [95% CI, 1.8%-5.7%] at 11 months; relative risk, 0.52 [95% CI, 0.29-0.93]). Genital lesions were rare, reported on 65 of 2497 days (2.6%) at 2 months and 72 of 1872 days (3.8%) at 11 months. Oral HSV-1 shedding was detected on 88 of 2247 days (3.9%) at 2 months. Persons with primary HSV-1 infection had a higher risk of genital shedding compared with those with nonprimary infection (model-predicted rate, 7.9% [95% CI, 5.4%-11.7%] vs 2.9% [95% CI, 1.7%-5.0%]; relative risk, 2.75 [95% CI, 1.40-5.44]). Polyfunctional HSV-specific CD4+ and CD8+ T-cell responses were maintained during the follow-up period. Conclusions and Relevance: Genital HSV-1 shedding was frequent after first-episode genital HSV-1, particularly among those with primary infection, and declined rapidly during the first year after infection.

Fast Grid Search and Bootstrap-based Inference for Continuous Two-phase Polynomial Regression Models.

Two-phase polynomial regression models (Robison, 1964; Fuller, 1969; Gallant and Fuller, 1973; Zhan et al., 1996) are widely used in ecology, public health, and other applied fields to model nonlinear relationships. These models are characterized by the presence of threshold parameters, across which the mean functions are allowed to change. That the threshold is a parameter of the model to be estimated from the data is an essential feature of two-phase models. It distinguishes them, and more generally, multi-phase models, from the spline models and has profound implications for both computation and inference for the models. Estimation of two-phase polynomial regression models is a non-convex, non-smooth optimization problem. Grid search provides high quality solutions to the estimation problem, but is very slow when done by brute force. Building upon our previous work on piecewise linear two-phase regression models estimation, we develop fast grid search algorithms for two-phase polynomial regression models and demonstrate their performance. Furthermore, we develop bootstrap-based pointwise and simultaneous confidence bands for mean functions. Monte Carlo studies are conducted to demonstrate the computational and statistical properties of the proposed methods. Three real datasets are used to help illustrate the application of two-phase models, with special attention on model choice.

The Effect of Hormonal Contraception and Menstrual Cycle Timing on Genital Herpes Simplex Virus-2 Shedding and Lesions.

BACKGROUND: The effect of female sex hormones on herpes simplex virus (HSV)-2 shedding and lesion frequency is poorly understood. Previous studies suggest that hormonal contraception may increase the frequency of HSV-2 shedding. METHODS: We studied HSV-2 seropositive women who performed daily genital swabbing for HSV DNA and completed diaries for genital lesions and menses. We used Poisson mixed effects models to determine if HSV detection varied throughout the menstrual cycle, or in response to hormonal contraception. We used the Wilcoxon signed-rank test and rank-sum test to determine if lesion frequency differed by cycle phase or hormonal contraceptive use. RESULTS: In 189 women aged 19 to 46 years who collected swabs on 10,715 days and were not using hormonal contraception, HSV-2 DNA was detected on 20.9% of days in the follicular phase and 17.8% of days in the luteal phase (rate ratio, 1.19; 95% confidence interval, 1.03-1.37, P = 0.02). Genital lesions did not differ in the follicular versus luteal phase (12.8% vs. 10.7%, P = 0.07). In analyses of hormonal contraception, including 244 women, HSV-2 DNA was detected on 19.0% of days for women not using hormonal contraception and 18.3% of days for those using hormonal contraception (P = 0.50). Lesions were present on 11.1% of days for women not using hormonal contraception, and 8.7% of days for those using hormonal contraception (P = 0.66). CONCLUSIONS: In women with genital HSV-2 infection who are not using hormonal contraception, the follicular phase of the cycle may be associated with a higher frequency of HSV-2 shedding compared to the luteal phase. Lesion frequency is similar during the 2 menstrual phases. Hormonal contraception use was not observed to affect genital HSV-2 DNA detection or lesions.

Comparison of supramesenteric aortic cross-clamping with supraceliac aortic cross-clamping for aortic reconstruction.

OBJECTIVE: Supraceliac aortic cross-clamping (SCXC) is routinely used during open aortic reconstruction (OAR) of pararenal aortic disease when suprarenal control is not feasible. On occasion, however, aortic control may be obtained at the supramesenteric level by supramesenteric cross-clamping (SMXC) between the superior mesenteric artery and the celiac axis. The purpose of this study was to compare outcomes between patients who had SMXC vs SCXC during OAR for both aneurysmal and occlusive diseases. METHODS: A retrospective chart review identified 69 patients who underwent elective OAR requiring SMXC (n = 18) or SCXC (n = 51). All patients with thoracoabdominal aneurysms and those who had inframesenteric (suprarenal and infrarenal) aortic control were excluded. Propensity score-based matching was performed to adjust for confounding factors in a 1:1 ratio to compare outcomes. Late survival was estimated by Kaplan-Meier methods. RESULTS: Propensity score-based matching was performed at a 1:1 ratio; 18 SMXC cases were matched with 18 SCXC cases. The average age was 66.7 years, and men constituted 72%. Baseline characteristics were matched, except for the incidence of peripheral vascular occlusive disease (72.2% in the SMXC group vs 33.3% in the SCXC group; P = .04). A majority (80.6%) of patients underwent OAR for aneurysmal disease (72.2% in the SMXC group, 88.9% in the SCXC group). Intraoperatively, there were no differences in operative times (325 minutes for SMXC vs 298 minutes for SCXC; P = .48), but the SMXC group had a longer renal ischemia time (40 minutes vs 28 minutes; P = .03). There were no significant differences in intraoperative blood loss (2.4 L vs 1.6 L; P = .2) or blood product transfusion requirements (packed red blood cells, 2.2 units vs 1.6 units [P = .5]; Cell Saver, 1.3 L vs 0.7 L [P = .09]). Overall complication rates did not differ significantly (27.8% for SMXC vs 44.4% for SCXC; P = .24). Thirty-day mortality rates did not differ between the two groups (0% for SMXC vs 5.6% for SCXC; P = 1). CONCLUSIONS: In this study, there were no differences in early morbidity or mortality between SMXC and SCXC during aortic reconstruction. SMXC, however, can be performed safely and effectively in properly selected patients. A larger, multicenter prospective study would help elucidate the potential benefits.

SOX17-mediated LPAR4 expression plays a pivotal role in cardiac development and regeneration after myocardial infarction.

Lysophosphatidic acid receptor 4 (LPAR4) exhibits transient expression at the cardiac progenitor stage during pluripotent stem cell (PSC)-derived cardiac differentiation. Using RNA sequencing, promoter analyses, and a loss-of-function study in human PSCs, we discovered that SRY-box transcription factor 17 (SOX17) is an essential upstream factor of LPAR4 during cardiac differentiation. We conducted mouse embryo analyses to further verify our human PSC in vitro findings and confirmed the transient and sequential expression of SOX17 and LPAR4 during in vivo cardiac development. In an adult bone marrow transplantation model using LPAR4 promoter-driven GFP cells, we observed two LPAR4+ cell types in the heart following myocardial infarction (MI). Cardiac differentiation potential was shown in heart-resident LPAR4+ cells, which are SOX17+, but not bone marrow-derived infiltrated LPAR4+ cells. Furthermore, we tested various strategies to enhance cardiac repair through the regulation of downstream signals of LPAR4. During the early stages following MI, the downstream inhibition of LPAR4 by a p38 mitogen-activated protein kinase (p38 MAPK) blocker improved cardiac function and reduced fibrotic scarring compared to that observed following LPAR4 stimulation. These findings improve our understanding of heart development and suggest novel therapeutic strategies that enhance repair and regeneration after injury by modulating LPAR4 signaling.

Adhesion GPCR Latrophilin-2 Specifies Cardiac Lineage Commitment through CDK5, Src, and P38MAPK.

Identifying lineage-specific markers is pivotal for understanding developmental processes and developing cell therapies. Here, we investigated the functioning of a cardiomyogenic cell-surface marker, latrophilin-2 (LPHN2), an adhesion G-protein-coupled receptor, in cardiac differentiation. LPHN2 was selectively expressed in cardiac progenitor cells (CPCs) and cardiomyocytes (CMCs) during mouse and human pluripotent stem cell (PSC) differentiation; cell sorting with an anti-LPHN2 antibody promoted the isolation of populations highly enriched in CPCs and CMCs. Lphn2 knockdown or knockout PSCs did not express cardiac genes. We used the Phospho Explorer Antibody Array, which encompasses nearly all known signaling pathways, to assess molecular mechanisms underlying LPHN2-induced cardiac differentiation. LPHN2-dependent phosphorylation was the strongest for cyclin-dependent kinase 5 (CDK5) at Tyr15. We identified CDK5, Src, and P38MAPK as key downstream molecules of LPHN2 signaling. These findings provide a valuable strategy for isolating CPCs and CMCs from PSCs and insights into the still-unknown cardiac differentiation mechanisms.

Clusterin contributes to early stage of Alzheimer's disease pathogenesis.

While clusterin is reportedly involved in Alzheimer's disease (AD) pathogenesis, how clusterin interacts with amyloid-ß (Aß) to cause Aß neurotoxicity remains unclear in vivo. Using 5×FAD transgenic mice, which develop robust AD pathology and memory deficits when very young, we detected interactions between clusterin and Aß in the mouse brains. The two proteins were concurrently upregulated and bound or colocalized with each other in the same complexes or in amyloid plaques. Neuropathology and cognitive performance were assessed in the progeny of clusterin-null mice crossed with 5×FAD mice, yielding clu-/- ;5×FAD and clu+/+ ;5×FAD. We found far less of the various pools of Aß proteins, most strikingly soluble Aß oligomers and amyloid plaques in clu-/- ;5×FAD mice at 5 months of age. At that age, those mice also had higher levels of neuronal and synaptic proteins and better motor coordination, spatial learning and memory than age-matched clu+/+ ;5×FAD mice. However, at 10 months of age, these differences disappeared, with Aß and plaque deposition, neuronal and synaptic proteins and impairment of behavioral and cognitive performance similar in both groups. These findings demonstrate that clusterin is necessarily involved in early stages of AD pathogenesis by enhancing toxic Aß pools to cause Aß-directed neurodegeneration and behavioral and cognitive impairments, but not in late stage.

Characterization of the Ste20-like kinase Krs1 of Dictyostelium discoideum.

Ste20-like kinases constitute a ubiquitous and expanding group of serine/threonine kinases, homologous to Ste20 in Saccharomyces cerevisiae. The social amoeba Dictyostelium discoideum contains at least 17 members of this kinase family, 13 from the germinal center kinase (GCK) subgroup and 4 p21-activated kinases (PAK). Here, we describe the kinase Krs1 which is encoded by the gene krsA, and phylogenetic analysis groups it into subfamily GCK-II together with human MST2 and MST1 or Hippo from Drosophila melanogaster. Significant similarities are found especially in the catalytic domain and in a short regulatory region (SARAH) which is thought to be important for protein/protein interactions. Northern blot analysis showed a single krsA transcript throughout development with an upregulation at 12h after the onset of starvation. The protein levels as detected with anti-Krs1 polyclonal antibodies revealed a similar pattern. Gel filtration experiments suggested that AX2 wild-type cells harbored multimeric forms of Krs1. In vitro phosphorylation assays with recombinant protein showed that the kinase exhibits autophosphorylation and accepts myelin basic protein and D. discoideum severin as substrates. A series of C-terminal deletions of Krs1 indicated that the regulatory domain in the C-terminal half contains inhibitory elements, and highlighted the importance of two predicted alpha-helices following subdomain XI of the classical catalytic domain. GFP-Krs1-overexpressing wild-type cells showed an enrichment of the kinase in the cortex, and motility of these cells during aggregation was reduced. Krs1 knockout strains exhibited only subtle differences to wild-type cells which suggests a certain redundancy among Ste20-like kinases in D. discoideum.

Enhanced disinfection efficiency of mechanically mixed oxidants with free chlorine.

To the best of our knowledge, this study is the first investigation to be performed into the potential benefits of mechanically mixed disinfectants in controlling bacterial inactivation. The purpose of this study was to evaluate the disinfection efficiency of mechanically mixed oxidants with identical oxidant concentrations, which were made by adding small amounts of subsidiary oxidants, namely ozone (O3), chlorine dioxide (ClO2), hydrogen peroxide (H2O2) and chlorite (ClO2(-)), to free available chlorine (Cl2), using Bacillus subtilis spores as the indicator microorganisms. The mechanically mixed oxidants containing Cl2/O3, Cl2/ClO2 and Cl2/ClO2(-) showed enhanced efficiencies (of up to 52%) in comparison with Cl2 alone, whereas no significant difference was observed between the mixed oxidant, Cl2/H2O2, and Cl2 alone. This enhanced disinfection efficiency can be explained by the synergistic effect of the mixed oxidant itself and the effect of intermediates such as ClO2(-)/ClO2, which are generated from the reaction between an excess of Cl2 and a small amount of O3/ClO2(-). Overall, this study suggests that mechanically mixed oxidants incorporating excess chlorine can constitute a new and moderately efficient method of disinfection.