Interactome Analysis of Human Phospholipase D and Phosphatidic Acid-Associated Protein Network.

Mammalian phospholipase D (PLD) enzyme family consists of six members. Among them, PLD1/2/6 catalyzes phosphatidic acid (PA) production, while PLD3/4/5 has no catalytic activities. Deregulation of the PLD-PA lipid signaling has been associated with various human diseases including cancer. However, a comprehensive analysis of the regulators and effectors for this crucial lipid metabolic pathway has not been fully achieved. Using a proteomic approach, we defined the protein interaction network for the human PLD family of enzymes and PA and revealed diverse cellular signaling events involving them. Through it, we identified PJA2 as a novel E3 ubiquitin ligase for PLD1 involved in control of the PLD1-mediated mammalian target of rapamycin signaling. Additionally, we showed that PA interacted with and positively regulated sphingosine kinase 1. Taken together, our study not only generates a rich interactome resource for further characterizing the human PLD-PA lipid signaling but also connects this important metabolic pathway with numerous biological processes.

Effect of Intraoperative Arterial Hypotension on the Risk of Perioperative Stroke After Noncardiac Surgery: A Retrospective Multicenter Cohort Study.

BACKGROUND: Intraoperative cerebral blood flow is mainly determined by cerebral perfusion pressure and cerebral autoregulation of vasomotor tone. About 1% of patients undergoing noncardiac surgery develop ischemic stroke. We hypothesized that intraoperative hypotension within a range frequently observed in clinical practice is associated with an increased risk of perioperative ischemic stroke within 7 days after surgery. METHODS: Adult noncardiac surgical patients undergoing general anesthesia at Beth Israel Deaconess Medical Center and Massachusetts General Hospital between 2005 and 2017 were included in this retrospective cohort study. The primary exposure was intraoperative hypotension, defined as a decrease in mean arterial pressure (MAP) below 55 mm Hg, categorized into no intraoperative hypotension, short (<15 minutes, median [interquartile range {IQR}], 2 minutes [1-5 minutes]) and prolonged (≥15 minutes, median [IQR], 21 minutes [17-31 minutes]) durations. The primary outcome was a new diagnosis of early perioperative ischemic stroke within 7 days after surgery. In secondary analyses, we assessed the effect of a MAP decrease by >30% from baseline on perioperative stroke. Analyses were adjusted for the preoperative STRoke After Surgery (STRAS) prediction score, work relative value units, and duration of surgery. RESULTS: Among 358,391 included patients, a total of 1553 (0.4%) experienced an early perioperative ischemic stroke. About 42% and 3% of patients had a MAP of below 55 mm Hg for a short and a prolonged duration, and 49% and 29% had a MAP decrease by >30% from baseline for a short and a prolonged duration, respectively. In an adjusted analysis, neither a MAP <55 mm Hg (short duration: adjusted odds ratio [ORadj], 0.95; 95% confidence interval [CI], 0.85-1.07; P = .417 and prolonged duration: ORadj, 1.18; 95% CI, 0.91-1.55; P = .220) nor a MAP decrease >30% (short duration: ORadj, 0.97; 95% CI, 0.67-1.42; P = .883 and prolonged duration: ORadj, 1.30; 95% CI, 0.89-1.90; P = .176) was associated with early perioperative stroke. A high a priori stroke risk quantified based on preoperatively available risk factors (STRAS prediction score) was associated with longer intraoperative hypotension (adjusted incidence rate ratio, 1.04; 95% CI, 1.04-1.05; P < .001 per 5 points of the STRAS prediction score). CONCLUSIONS: This study found no evidence to conclude that intraoperative hypotension within the range studied was associated with early perioperative stroke within 7 days after surgery. These findings emphasize the importance of perioperative cerebral blood flow autoregulation to prevent ischemic stroke.

Testicular thermoregulation with respect to spermatogenesis and contraception.

There has been some evidence that a short heating exposure of the testicles can lead to temporary sterility. However, little attention has been paid to analyzing the temperature/time pattern throughout the testicle and how metabolism and blood perfusion affect the temperature profile. We use the Penne bioheat equation that accounts for the elements of conduction, perfusion, and metabolism along with the COMSOL Finite Element Method for the analysis of this thermoregulation problem. Typically, the testicles were modeled as two uniform spheres and exposed to an increase in surface temperature of 42 °C for 30 min. We found that heat produced by tissue metabolism had a minor effect on the temperature pattern. However, blood flow from the core (at 37 °C) could prevent the region where the testicles touch each other from reaching the target temperature of 42 °C. Further, we found that for animals, as rodents and monkeys that are used for contraception studies, the temperature distribution on heating testicles is much more uniform than for humans. Thus, extrapolation results from animal models to humans should be done with caution.

Genetic background alters dominance relationships between mat alleles in the ciliate Tetrahymena thermophila.

The pattern of inheritance and mechanism of sex determination can have important evolutionary consequences. We studied probabilistic sex determination in the ciliate Tetrahymena thermophila, which was previously shown to cause evolution of skewed sex ratios. We find that the genetic background alters the sex determination patterns of mat alleles in heterozygotes and that allelic interaction can differentially influence the expression probability of the 7 sexes. We quantify the dominance relationships between several mat alleles and find that A-type alleles, which specify sex I, are indeed recessive to B-type alleles, which are unable to specify that sex. Our results provide additional support for the presence of modifier loci and raise implications for the dynamics of sex ratios in populations of T. thermophila.

Provider-reported experiences, barriers, and perspectives on genetic testing as part of autism diagnosis.

Several professional organizations recommend conducting genetic testing as part of the autism diagnosis process, as it can provide additional information and benefits for autistic people and their families. However, there is disagreement among autism communities about whether genetic testing reflects autistic people's best interests. In practice, rates of clinical genetic testing for autism are much lower than diagnoses, creating a large gap between clinical guidelines and real clinical encounters. To investigate one potential source of this gap, we interviewed 14 healthcare providers about the autism diagnostic process and their actions related to autism genetic testing. We recruited a sample of primarily Ph.D. level-psychologists and analyzed our qualitative data using a five-step framework analysis method. Participants generally had positive or mixed views of genetic testing in autism. They described their current experiences of implementation of genetic testing, including that they did not often find it changed their clinical practice. Only some providers recommended it to everyone receiving an autism diagnosis. They also listed factors which discourage families from getting testing, including high costs, families feeling overwhelmed, other support needs taking priority, and ethical implications. Notably, providers highlighted a trend of referring patients to research genetic testing rather than clinical testing, which may provide a cheaper and easier alternative but is not likely to return results to participants. Finally, participants felt they needed more training in genetics and listed specific topics of uncertainty. Our research highlights a need to further educate clinicians in the uses and limitations of genetic testing for autism and suggests content areas of focus for genetics educators.

Culturing of SARS-CoV-2 from patient samples: Protocol for optimal virus recovery and assessment of infectious viral load.

Optimal sampling, preservation, and culturing of SARS-CoV-2 from COVID-19 patients are critical for successful recovery of virus isolates and to accurately estimate contagiousness of the patient. In this study, we investigated the influence of the type of sampling media, storage time, freezing conditions, sterile filtration, and combinations of these to determine the optimal pre-analytic conditions for virus recovery and estimation of infectious viral load in COVID-19 patients. Further, we investigated the viral shedding kinetics and mucosal antibody response in 38 COVID-19 hospitalized patients. We found Universal Transport Medium (Copan) to be the most optimal medium for preservation of SARS-CoV-2 infectivity. Our data showed that the probability of a positive viral culture was strongly correlated to Ct values, however some samples did not follow the general trend. We found a significant correlation between plaque forming units and levels of mucosal antibodies and found that high levels of mucosal antibodies correlated with reduced chance of isolating the virus. Our data reveals essential parameters to consider from specimen collection over storage to culturing technique for optimal chance of isolating SARS-CoV-2 and accurately estimating patient contagiousness.

A novel multitargeted self-assembling peptide-siRNA complex for simultaneous inhibition of SARS-CoV-2-host cell interaction and replication.

Effective therapeutics are necessary for managing severe COVID-19 disease despite the availability of vaccines. Small interfering RNA (siRNA) can silence viral genes and restrict SARS-CoV-2 replication. Cell-penetrating peptides is a robust method for siRNA delivery, enhancing siRNA stability and targeting specific receptors. We developed a peptide HE25 that blocks SARS-CoV-2 replication by various mechanisms, including the binding of multiple receptors involved in the virus's internalization, such as ACE2, integrins and NRP1. HE25 not only acts as a vehicle to deliver the SARS-CoV-2 RNA-dependent RNA polymerase siRNA into cells but also facilitates their internalization through endocytosis. Once inside endosomes, the siRNA is released into the cytoplasm through the Histidine-proton sponge effect and the selective cleavage of HE25 by cathepsin B. These mechanisms effectively inhibited the replication of the ancestral SARS-CoV-2 and the Omicron variant BA.5 in vitro. When HE25 was administered in vivo, either by intravenous injection or inhalation, it accumulated in lungs, veins and arteries, endothelium, or bronchial structure depending on the route. Furthermore, the siRNA/HE25 complex caused gene silencing in lung cells in vitro. The SARS-CoV-2 siRNA/HE25 complex is a promising therapeutic for COVID-19, and a similar strategy can be employed to combat future emerging viral diseases.

Examining the effect of parent participation in an adult weight management program on changes in children's weight.

Children of parents participating in weight management programs (WMPs) are more likely to adopt their parents' weight control practices. Little is known about the weight outcomes of children who have a parent participating in a WMP. This study aimed to assess this relationship. Children 2-17 years of age with a parent who participated in a WMP were included in the study. Multilevel linear mixed-effects regression models were used, stratified by child weight status at the time of parental WMP participation (healthy weight, overweight/obesity) to determine change in children's BMIz from before to after parents WMP participation, including covariates of parent BMI and parental feeding practices. Parents (N = 77) were mostly white (76%) and female (84%). Children (N = 114) had a mean age of 10.5 ± 4.6; 47% had overweight or obesity. Children with overweight or obesity prior to their parent's WMP had a decrease in BMIz (-0.68) after the WMP while children with a healthy weight had no significant change. Children with overweight or obesity had a decrease in BMIz from before to after parent's participation in a WMP. Further research is needed to understand changes in family eating practices that occur during and after parent WMP participation.

Comparison of behaviorally inhibited and typically developing children's play behaviors in the preschool classroom.

Introduction: Behavioral inhibition (BI) is a temperamental trait characterized by a bias to respond with patterns of fearful or anxious behavior when faced with unfamiliar situations, objects, or people. It has been suggested that children who are inhibited may experience early peer difficulties. However, researchers have yet to systematically compare BI versus typically developing children's observed asocial and social behavior in familiar, naturalistic settings. Method: We compared the in-school behaviors of 130 (M = 54 months, 52% female) highly inhibited preschoolers (identified using the parent-reported Behavioral Inhibition Questionnaire) to 145 (M = 53 months, 52% female) typically developing preschoolers. Both samples were observed on at least two different days for approximately 60 min. Observers used the Play Observation Scale to code children's behavior in 10-s blocks during free play. Teachers completed two measures of children's behavior in the classroom. Results: Regression models with robust standard errors controlling for child sex, age, and weekly hours in school revealed that preschoolers identified as BI engaged in significantly more observed reticent and solitary behavior, and less social play and teacher interaction than the typically developing sample. Children with BI also initiated social interaction with their peers and teachers less often than their counterparts who were not inhibited. Teachers reported that children identified as BI were more asocial and less prosocial than their non-BI counterparts. Discussion: Significantly, the findings indicated that inhibited children displayed more solitude in the context of familiar peers. Previous observational studies have indicated behavioral differences between BI and unfamiliar typical age-mates in novel laboratory settings. Children identified as BI did not receive fewer bids for social interaction than their typically developing peers, thereby suggesting that children who are inhibited have difficulty capitalizing on opportunities to engage in social interaction with familiar peers. These findings highlight the need for early intervention for children with BI to promote social engagement, given that the frequent expression of solitude in preschool has predicted such negative outcomes as peer rejection, negative self-regard, and anxiety during the elementary and middle school years.

Plasma Kallikrein Cleaved H-kininogen: An End-Point Marker for Contact Activation in vitro and ex vivo.

Objectives: The contact system consists of coagulation factor XII (FXII), prekallikrein, and H-kininogen (HK) and plays important roles in many diseases. Plasma kallikrein (PKa) cleaved HK (cHK) is a marker of contact activation. Presently, we developed a specific and precise enzyme-linked immunosorbent assay (ELISA) for determination of cHK in vitro and ex vivo. Methods: Cleaved HK specific mouse monoclonal antibodies (mAbs) were generated using a peptide corresponding to the PKa cleavage site on HK as immunogen. ELISA, surface plasmon resonance analysis, and immunoprecipitation established the specificity of the antibody, which subsequently was used in a sandwich ELISA. The analytical imprecision and the concentration of cHK in a reference population and in women receiving oral contraceptives (OC) were determined. cHK was assessed in vitro in plasma exposed to polytetrafluoroethylene, silicone, and glass tubes. Results: The selected mAb showed excellent specificity towards cHK. The intra-assay and inter-assay CV of the ELISA were 3.6 and 6.0%, respectively. The reference population (60 women, 60 men) displayed a median cHK plasma concentration of 1.38 µg/mL and a reference interval of 0.82 - 2.56 µg/mL. Women receiving OC had significantly higher concentrations, p < 0.001. cHK was significantly elevated in plasma exposed to polytetrafluoroethylene, p = 0.001, and glass, p < 0.0001. Conclusion: The ELISA showed excellent precision and specificity. cHK assessment ex vivo demonstrated ongoing contact activation in healthy individuals, augmented by OC. The cHK antibody and the ELISA could be promising tools in contact activation related diseases and in vitro investigations of the plasma compatibility of blood contacting biomaterials.

Depressed Kallikrein Generation in Women With Preeclampsia: A Matched Cross-Sectional Study.

Objective: The pathophysiology of preeclampsia is not fully understood. Disturbances in the contact system are associated with preeclampsia. Few studies have investigated the association between preeclampsia and alterations in the contact system in plasma. This study aims to elucidate whether this basic biological system is affected in preeclampsia using new methods focusing on the dynamic interactions and total capacity of the contact system in blood. Design: Cross-sectional study matching women with preeclampsia and controls without preeclampsia regarding age, pregestational body mass index, and gestational age at onset of the disease. Setting: Two Danish University hospitals. Sample: A cohort of 117 women with preeclampsia and 117 controls. Methods: The turnover and capacity of the contact system were determined with new methods. Paired t-test, Wilcoxon signed-pairs signed rank test, Mann-Whitney or Chi2-test were applied, as appropriate. Main Outcome Measurements: Kallikrein generation (peak kallikrein concentration and endogenous kallikrein potential), coagulation factor XII, prekallikrein, H-kininogen, cleaved H-kininogen, and complement C1 esterase inhibitor. Results: The endogenous kallikrein potential, peak kallikrein concentration, prekallikrein and cleaved H-kininogen were significantly lower in women with preeclampsia compared to the controls, p ≤ 0.005, whereas the concentration of coagulation factor XII, H-kininogen and complement C1 esterase inhibitor was not significantly different, p > 0.05. Conclusion: This study demonstrates significant reduction in kallikrein generating capacity, prekallikrein and cleaved H-kininogen indicating that the contact system is affected in preeclampsia suggesting a link to the pathophysiology of the disease.

Machine Learning to Delineate Surgeon and Clinical Factors That Anticipate Positive Surgical Margins After Robot-Assisted Radical Prostatectomy.

Purpose: Automated performance metrics (APMs), derived from instrument kinematic and systems events data during robotic surgery, are validated objective measures of surgeon performance. Our previous studies showed that APMs are strong outcome predictors of urinary continence after robot-assisted radical prostatectomy (RARP). We now use machine learning to investigate how surgeon performance (i.e., APMs) and clinical factors can predict positive surgical margins (PSMs) after RARP. Methods: We prospectively collected data of patients undergoing RARP at our institution from 2016 to 2019. Random Forest model predicted PSMs based on 15 clinical factors and 38 APMs from 11 standardized RARP steps. Out-of-bag Gini impurity index determined the top 10 variables of importance (VOI). APMs in the top 10 VOI were assessed for confounding effects by extracapsular extension (ECE) and pathologic T (pT) through Poisson regression with Generalized Estimating Equation. Results: 55/236 (23.3%) cases had PSMs. Of the 55 cases with PSMs, 9 (16.4%) were pT2 and 46 (83.6%), pT3. The full model, including clinical factors and APMs, achieved area under the curve (AUC) 0.74. When assessing clinical factors or APMs alone, the model achieved AUC 0.72 and 0.64, respectively. The strongest PSM predictors were ECE and pT stage, followed by APMs in specific steps. After adjusting for ECE and pT stage, most APMs remained as independent predictors of PSM. Conclusion: Using machine learning methods, we found that the strongest predictors of PSMs after RARP are nonmodifiable, disease-driven factors (ECE and pT). While APMs provide minimal additional insight into when PSMs may occur, they are nonetheless capable of independently predicting PSMs based on objective measures of surgeon performance.

Effects of Volatile Anesthetics on Postoperative Ischemic Stroke Incidence.

Background Preclinical studies suggest that volatile anesthetics decrease infarct volume and improve the outcome of ischemic stroke. This study aims to determine their effect during noncardiac surgery on postoperative ischemic stroke incidence. Methods and Results This was a retrospective cohort study of surgical patients undergoing general anesthesia at 2 tertiary care centers in Boston, MA, between October 2005 and September 2017. Exclusion criteria comprised brain death, age <18 years, cardiac surgery, and missing covariate data. The exposure was defined as median age-adjusted minimum alveolar concentration of all intraoperative measurements of desflurane, sevoflurane, and isoflurane. The primary outcome was postoperative ischemic stroke within 30 days. Among 314 932 patients, 1957 (0.6%) experienced the primary outcome. Higher doses of volatile anesthetics had a protective effect on postoperative ischemic stroke incidence (adjusted odds ratio per 1 minimum alveolar concentration increase 0.49, 95% CI, 0.40-0.59, P<0.001). In Cox proportional hazards regression, the effect was observed for 17 postoperative days (postoperative day 1: hazard ratio (HR), 0.56; 95% CI, 0.48-0.65; versus day 17: HR, 0.85; 95% CI, 0.74-0.99). Volatile anesthetics were also associated with lower stroke severity: Every 1-unit increase in minimum alveolar concentration was associated with a 0.006-unit decrease in the National Institutes of Health Stroke Scale (95% CI, -0.01 to -0.002, P=0.002). The effects were robust throughout various sensitivity analyses including adjustment for anesthesia providers as random effect. Conclusions Among patients undergoing noncardiac surgery, volatile anesthetics showed a dose-dependent protective effect on the incidence and severity of early postoperative ischemic stroke.

Replacement of microglia in the aged brain reverses cognitive, synaptic, and neuronal deficits in mice.

Microglia, the resident immune cell of the brain, can be eliminated via pharmacological inhibition of the colony-stimulating factor 1 receptor (CSF1R). Withdrawal of CSF1R inhibition then stimulates microglial repopulation, effectively replacing the microglial compartment. In the aged brain, microglia take on a "primed" phenotype and studies indicate that this coincides with age-related cognitive decline. Here, we investigated the effects of replacing the aged microglial compartment with new microglia using CSF1R inhibitor-induced microglial repopulation. With 28 days of repopulation, replacement of resident microglia in aged mice (24 months) improved spatial memory and restored physical microglial tissue characteristics (cell densities and morphologies) to those found in young adult animals (4 months). However, inflammation-related gene expression was not broadly altered with repopulation nor the response to immune challenges. Instead, microglial repopulation resulted in a reversal of age-related changes in neuronal gene expression, including expression of genes associated with actin cytoskeleton remodeling and synaptogenesis. Age-related changes in hippocampal neuronal complexity were reversed with both microglial elimination and repopulation, while microglial elimination increased both neurogenesis and dendritic spine densities. These changes were accompanied by a full rescue of age-induced deficits in long-term potentiation with microglial repopulation. Thus, several key aspects of the aged brain can be reversed by acute noninvasive replacement of microglia.

Frequency of trichomonads as coinfecting agents in Pneumocystis pneumonia.

OBJECTIVE: To investigate the incidence of the association of Trichomonas and Pneumocystis in the lung. STUDY DESIGN: Sixty-six bronchoalveolar lavage fluid (BALF) samples from immunocompromised patients with pneumocystosis were retrospectively examined microscopically. RESULTS: Trichomonads were found as coinfecting agents in 60% of BALF samples. The frequency and abundance of trichomonads was increased, up to 100%, in cases rich in Pneumocystis. CONCLUSION: The data suggest that pulmonary Trichomonas infection occurs frequently in the course of Pneumocystis pneumonia. The role of trichomonads in causing alveolar damage during Pneumocystis pneumonia is hypothetical.

Chromosome copy number variation and control in the ciliate Chilodonella uncinata.

Copy number variations are widespread in eukaryotes. The unusual genome architecture of ciliates, in particular, with its process of amitosis in macronuclear division, provides a valuable model in which to study copy number variation. The current model of amitosis envisions stochastic distribution of macronuclear chromosomes during asexual reproduction. This suggests that amitosis is likely to result in high levels of copy number variation in ciliates, as dividing daughter cells can have variable copy numbers of chromosomes if chromosomal distribution during amitosis is a stochastic process. We examined chromosomal distribution during amitosis in Chilodonella uncinata, a ciliate with gene-size macronuclear chromosomes. We quantified 4 chromosomes in evolving populations of C. uncinata and modeled the amitotic distribution process. We found that macronuclear chromosomes differ in copy number from one another but that copy number does not change as expected under a stochastic process. The chromosome carrying SSU increased in copy number, which is consistent with selection to increase abundance; however, two other studied chromosomes displayed much lower than expected among-line variance. Our models suggest that balancing selection is sufficient to explain the observed maintenance of chromosome copy during asexual reproduction.