Protease-Activated Receptor 2 Controls Vascular Smooth Muscle Cell Proliferation in Cyclic AMP-Dependent Protein Kinase/Mitogen-Activated Protein Kinase Kinase 1/2-Dependent Manner.

INTRODUCTION: Cardiovascular disorders are characterized by vascular smooth muscle (VSM) transition from a contractile to proliferative state. Protease-activated receptor 2 (PAR2) involvement in this phenotypic conversion remains unclear. We hypothesized that PAR2 controls VSM cell proliferation in phenotype-dependent manner and through specific protein kinases. METHODS: Rat clonal low (PLo; P3-P6) and high passage (PHi; P10-P15) VSM cells were established as respective models of quiescent and proliferative cells, based on reduced PKG-1 and VASP. Western blotting determined expression of cytoskeletal/contractile proteins, PAR2, and select protein kinases. DNA synthesis and cell proliferation were measured 24-72 h following PAR2 agonism (SLIGRL; 100 nM-10 µm) with/without PKA (PKI; 10 µm), MEK1/2 (PD98059; 10 µm), and PI3K (LY294002; 1 µm) blockade. RESULTS: PKG-1, VASP, SM22α, calponin, cofilin, and PAR2 were reduced in PHi versus PLo cells. Following PAR2 agonism, DNA synthesis and cell proliferation increased in PLo cells but decreased in PHi cells. Western analyses showed reduced PKA, MEK1/2, and PI3K in PHi versus PLo cells, and kinase blockade revealed PAR2 controls VSM cell proliferation through PKA/MEK1/2. DISCUSSION: Findings highlight PAR2 and PAR2-driven PKA/MEK1/2 in control of VSM cell growth and provide evidence for continued investigation of PAR2 in VSM pathology.

Matrix effects demystified: Strategies for resolving challenges in analytical separations of complex samples.

Matrix effects can significantly impede the accuracy, sensitivity, and reliability of separation techniques presenting a formidable challenge to the analytical process. It is crucial to address matrix effects to achieve accurate and precise measurements in complex matrices. The multifaceted nature of matrix effects which can be influenced by factors such as target analyte, sample preparation protocol, composition, and choice of instrument necessitates a pragmatic approach when analyzing complex matrices. This review aims to highlight common challenges associated with matrix effects throughout the entire analytical process with emphasis on gas chromatography-mass spectrometry, liquid chromatography-mass spectrometry, and sample preparation techniques. These techniques are susceptible to matrix effects that could lead to ion suppression/enhancement or impact the analyte signal at various stages of the analytical workflow. The assessment, quantification, and mitigation of matrix effects are necessary in developing any analytical method. Strategies can be implemented to reduce or eliminate the matrix effect by changing the type of ionization, improving extraction and clean-up methods, optimization of chromatography conditions, and corrective calibration methods. While development of an effective strategy to completely mitigate matrix effects remains elusive, an integrated approach that combines sample preparation, analytical extraction, and effective instrumental analysis remains the most promising avenue for identifying and resolving matrix effects.

Nutrient availability contributes to a graded refractory period for regeneration in Xenopus tropicalis.

Xenopus tadpoles are a unique model for regeneration in that they exhibit two distinct phases of age-specific regenerative competence. In Xenopus laevis, young tadpoles fully regenerate following major injuries such as tail transection, then transiently lose regenerative competence during the "refractory period" from stages 45-47. Regenerative competence is then regained in older tadpoles before being permanently lost during metamorphosis. Here we show that a similar refractory period exists in X. tropicalis. Notably, tadpoles lose regenerative competence gradually in X. tropicalis, with full regenerative competence lost at stage 47. We find that the refractory period coincides closely with depletion of maternal yolk stores and the onset of independent feeding, and so we hypothesized that it might be caused in part by nutrient stress. In support of this hypothesis, we find that cell proliferation declines throughout the tail as the refractory period approaches. When we block nutrient mobilization by inhibiting mTOR signaling, we find that tadpole growth and regeneration are reduced, while yolk stores persist. Finally, we are able to restore regenerative competence and cell proliferation during the refractory period by abundantly feeding tadpoles. Our study argues that nutrient stress contributes to lack of regenerative competence and introduces the X. tropicalis refractory period as a valuable new model for interrogating how metabolic constraints inform regeneration.

Demographic and technological factors influencing virtual seizure clinic visit satisfaction before and during the Covid-19 pandemic in rural Hawaii.

BACKGROUND: Telemedicine clinic visits traditionally originated from spoke clinic sites, but recent trends have favored home-based telemedicine, particularly in the time of Covid-19. Our study focused on identification of barriers and factors influencing perceptions of care with use of home-based telemedicine in patients with seizures living in rural Hawaii. We additionally compared characteristics of patients using telemedicine versus in-person clinic visits prior to the Covid-19 pandemic. METHODS: For the retrospective portion of our study, we queried charts of adult outpatients treated by the two full-time epileptologists at a Level 4 epilepsy center accredited by the National Association of Epilepsy Centers between November 2018 and December 2019. We included patients who live on the neighbor islands of Hawaii but not on Oahu, i.e., patients who would require air travel to see an epileptologist. There had been no set protocol at the epilepsy center for telemedicine referral; our practice had been to offer telemedicine visits to all neighbor island patients when felt to be appropriate. We collected demographic and clinic visit data. For the prospective portion we surveyed neighbor island patients or their caregivers, seen via home-based telemedicine between March 2020 and December 2020. We obtained verbal consent for study participation. Survey questions addressed satisfaction with clinical care, visit preferences, and potential barriers to care. RESULTS: In a 14-month period prior to the Covid-19 pandemic, 75 (61%) neighbor island patients were seen exclusively in-person in seizure clinic while 47 (39%) had at least one telemedicine visit. 39% of patients seen only in-person were female whereas 38% of patients seen by telemedicine were female. Patients seen in-person had an older median age (47.2 years) compared to those seen at least once by telemedicine (42.4 years). The no-show rate was 13% for in-person visits versus 4% for telemedicine visits. Among patients seen in person, 17% were Asian, 32% Native Hawaiian, and 47% White, whereas patients seen by telemedicine were 15% Asian, 23% Native Hawaiian, and 57% White. Patients who were seen in person lived in zip codes with median household income of $68,516 and patients who were seen by telemedicine lived in zip codes with median household income of $67,089. Patients who were seen in person lived in zip codes in which 78% of the population had access to broadband internet, whereas patients who were seen by telemedicine lived in zip codes in which 79% of the population had access to broadband internet. During the Covid-19 pandemic, we surveyed 47 consecutive patients seen by telemedicine, 45% female with median age of 33 years. Telemedicine connection was set up by the patient in 74% of cases, or by the patient's mother (15%), other family member (9%), or other caregiver (2 %). Median patient satisfaction score was 5 ("highly satisfied") on a 5-point Likert scale with mean score of 4.6. Telemedicine visit was done using a smartphone by 62% of patients, a computer by 36% of patients, and a tablet by 2% of patients. A home WiFi connection was used in 83% of patients. CONCLUSIONS: Home-based telemedicine visits provide a high-satisfaction method for seizure care delivery despite some obstacles. Demographic disparities may be an obstacle to telemedicine care and seem to relate to race and possibly age, rather than to sex/gender, household income, or access to broadband internet. Additionally, despite high satisfaction overall, more patients felt the physical exam was superior at in-person clinic visits and more patients expressed a preference for in-person visits. During the Covid-19 pandemic when there may be barriers to in-person clinic visits, home-based telemedicine is a feasible alternative.

Circulating miRNA profiles provide a biomarker for severity of stroke outcomes associated with age and sex in a rat model.

Small non-coding RNA [miRNA (microRNA)] found in the circulation have been used successfully as biomarkers and mechanistic targets for chronic and acute disease. The present study investigated the impact of age and sex on miRNA expression following ischaemic stroke in an animal model. Adult (6 month) and middle-aged (11-12 months) female and male rats were subject to MCAo (middle cerebral artery occlusion) using ET-1 (endothelin-1). Circulating miRNAs were analysed in blood samples at 2 and 5 days post-stroke, and brain miRNAs were analysed at 5 days post-stroke. Although stroke-associated infarction was observed in all groups, infarct volume and sensory-motor deficits were significantly reduced in adult females compared with middle-aged females, adult males or middle-aged males. At 2 days post-stroke, 21 circulating miRNAs were differentially regulated and PCA (principal component analysis) confirmed that most of the variance was due to age. At 5 days post-stroke, 78 circulating miRNAs exhibited significantly different regulation, and most of the variance was associated with sex. A small cohort (five) of miRNAs, miR-15a, miR-19b, miR-32 miR-136 and miR-199a-3p, were found to be highly expressed exclusively in adult females compared with middle-aged females, adult males and middle-aged males. Predicted gene targets for these five miRNAs analysed for KEGG (Kyoto Encyclopedia of Genes and Genomes) pathways revealed that the top ten KEGG pathways were related to growth factor signalling, cell structure and PI3K (phosphoinositide 3-kinase)/Akt and mTOR (mammalian target of rapamycin) signalling. Overall, the pattern of circulating miRNA expression suggests an early influence of age in stroke pathology, with a later emergence of sex as a factor for stroke severity.

Ex vivo drug testing of patient-derived lung organoids to predict treatment responses for personalized medicine.

Lung cancer is the leading cause of global cancer-related mortality resulting in âˆ¼ 1.8 million deaths annually. Systemic, molecular targeted, and immune therapies have provided significant improvements of survival outcomes for patients. However, drug resistance usually arises and there is an urgent need for novel therapy screening and personalized medicine. 3D patient-derived organoid (PDO) models have emerged as a more effective and efficient alternative for ex vivo drug screening than 2D cell culture and patient-derived xenograft (PDX) models. In this review, we performed an extensive search of lung cancer PDO-based ex vivo drug screening studies. Lung cancer PDOs were successfully established from fresh or bio-banked sections and/or biopsies, pleural effusions and PDX mouse models. PDOs were subject to ex vivo drug screening with chemotherapy, targeted therapy and/or immunotherapy. PDOs consistently recapitulated the genomic alterations and drug sensitivity of primary tumors. Although sample sizes of the previous studies were limited and some technical challenges remain, PDOs showed great promise in the screening of novel therapy drugs. With the technical advances of high throughput, tumor-on-chip, and combined microenvironment, the drug screening process using PDOs will enhance precision care of lung cancer patients.

The impact of race and ethnicity on diffuse large B-cell lymphoma outcomes within the veterans health administration (VHA).

We performed a retrospective chart review of 6266 randomly selected DLBCL patients treated in the VHA nationwide between 1/1/2011 and 12/31/2021. The 3178 patients who met inclusion criteria were predominantly male (97%) and white (75%). Median age of diagnosis for Black patients was 63 years vs 69 years for the entire cohort (p < 0.001). However, patients in each race/ethnicity subgroup presented with similar rates of stage I/II and III/IV disease, IPI score, cell of origin and HIT status. Outcomes analysis revealed similar treatment, response rates, median overall survival, and 1-, 3-, and 5-year survival across all subgroups. Hispanic patients had a 21% lower risk of death (HR = 0.79) than white patients, and Black patients had no significant difference in survival (HR = 0.98). This large retrospective study shows that when standard of care therapy is given within an equal access system, short-term treatment and survival outcomes are the same for all races.

Restriction endonuclease cleavage of phage DNA enables resuscitation from Cas13-induced bacterial dormancy.

Type VI CRISPR systems protect against phage infection using the RNA-guided nuclease Cas13 to recognize viral messenger RNA. Upon target recognition, Cas13 cleaves phage and host transcripts non-specifically, leading to cell dormancy that is incompatible with phage propagation. However, whether and how infected cells recover from dormancy is unclear. Here we show that type VI CRISPR and DNA-cleaving restriction-modification (RM) systems frequently co-occur and synergize to clear phage infections and resuscitate cells. In the natural type VI CRISPR host Listeria seeligeri, we show that RM cleaves the phage genome, thus removing the source of phage transcripts and enabling cells to recover from Cas13-induced cellular dormancy. We find that phage infections are neutralized more effectively when Cas13 and RM systems operate together. Our work reveals that type VI CRISPR immunity is cell-autonomous and non-abortive when paired with RM, and hints at other synergistic roles for the diverse host-directed immune systems in bacteria.

FoxO3 normalizes Smad3-induced arterial smooth muscle cell growth.

Transition of arterial smooth muscle (ASM) from a quiescent, contractile state to a growth-promoting state is a hallmark of cardiovascular disease (CVD), a leading cause of death and disability in the United States and worldwide. While many individual signals have been identified as important mechanisms in this phenotypic conversion, the combined impact of the transcription factors Smad3 and FoxO3 in ASM growth is not known. The purpose of this study was to determine that a coordinated, phosphorylation-specific relationship exists between Smad3 and FoxO3 in the control of ASM cell growth. Using a rat in vivo arterial injury model and rat primary ASM cell lysates and fractions, validated low and high serum in vitro models of respective quiescent and growth states, and adenoviral (Ad-) gene delivery for overexpression (OE) of individual and combined Smad3 and/or FoxO3, we hypothesized that FoxO3 can moderate Smad3-induced ASM cell growth. Key findings revealed unique cellular distribution of Smad3 and FoxO3 under growth conditions, with induction of both nuclear and cytosolic Smad3 yet primarily cytosolic FoxO3; Ad-Smad3 OE leading to cytosolic and nuclear expression of phosphorylated and total Smad3, with almost complete reversal of each with Ad-FoxO3 co-infection in quiescent and growth conditions; Ad-FoxO3 OE leading to enhanced cytosolic expression of phosphorylated and total FoxO3, both reduced with Ad-Smad3 co-infection in quiescent and growth conditions; Ad-FoxO3 inducing expression and activity of the ubiquitin ligase MuRF-1, which was reversed with concomitant Ad-Smad3 OE; and combined Smad3/FoxO3 OE reversing both the pro-growth impact of singular Smad3 and the cytostatic impact of singular FoxO3. A primary takeaway from these observations is the capacity of FoxO3 to reverse growth-promoting effects of Smad3 in ASM cells. Additional findings lend support for reciprocal antagonism of Smad3 on FoxO3-induced cytostasis, and these effects are dependent upon discrete phosphorylation states and cellular localization and involve MuRF-1 in the control of ASM cell growth. Lastly, results showing capacity of FoxO3 to normalize Smad3-induced ASM cell growth largely support our hypothesis, and overall findings provide evidence for utility of Smad3 and/or FoxO3 as potential therapeutic targets against abnormal ASM growth in the context of CVD.

Durvalumab Treatment Patterns for Patients with Unresectable Stage III Non-Small Cell Lung Cancer in the Veterans Health Administration (VHA): A Nationwide, Real-World Study.

BACKGROUND: Durvalumab is approved for the treatment of adults with unresectable stage III non-small cell lung cancer (NSCLC) post-chemoradiotherapy (CRT). This real-world study describes patient characteristics and durvalumab treatment patterns (number of doses and therapy duration; treatment initiation delays, interruptions, discontinuations, and associated reasons) among VHA-treated patients. METHODS: This was a retrospective cohort study of adults with unresectable stage III NSCLC receiving durvalumab at the VHA between 1 January 2017 and 30 June 2020. Patient characteristics and treatment patterns were presented descriptively. RESULTS: A total of 935 patients were included (median age: 69 years; 95% males; 21% Blacks; 46% current smokers; 16% ECOG performance scores ≥ 2; 50% squamous histology). Durvalumab initiation was delayed in 39% of patients (n = 367). Among the 200 patients with recorded reasons, delays were mainly due to physician preference (20%) and CRT toxicity (11%). Overall, patients received a median (interquartile range) of 16 (7-24) doses of durvalumab over 9.0 (2.9-11.8) months. Treatment interruptions were experienced by 19% of patients (n = 180), with toxicity (7.8%) and social reasons (2.6%) being the most cited reasons. Early discontinuation occurred in 59% of patients (n = 551), largely due to disease progression (24.2%) and toxicity (18.2%). CONCLUSIONS: These real-world analyses corroborate PACIFIC study results in terms of the main reasons for treatment discontinuation in a VHA population with worse prognostic factors, including older age, predominantly male sex, and poorer performance score. One of the main reasons for durvalumab initiation delays, treatment interruptions, or discontinuations was due to toxicities. Patients could benefit from improved strategies to prevent, identify, and manage CRT and durvalumab toxicities timely and effectively.

A Preliminary Assessment of the Qualities and Behaviors of Exemplary Practitioners: Perspectives From U.S.-Based Behavior Analysts.

Individuals with credentials (Board Certified Behavior Analyst-Doctoral and Board Certified Behavior Analyst) from the Behavior Analyst Certification Board throughout the United States were asked to identify the characteristics and corresponding behaviors of individuals they consider to be exemplary in the profession. From these responses, a list of 35 characteristics and attendant behaviors was compiled into the Exemplary Behavior Analyst Checklist. This checklist contains a number of characteristics that are traditionally representative of the field (e.g., analytical, applied, conceptually systematic, technological) and relate to technical and conceptual skills. Respondents also identified a number of characteristics associated with compassion and support of clients/individuals (e.g., client centered, culturally competent, empathetic, positive/encouraging). A "top 10" list of the qualities and behaviors of exemplary behavior analysts identified by participants is presented, and a discussion regarding the implications for the training of credentialed professionals is provided.

Clinicopathologic differences and mortality among Latinos and non-Latino whites with gastric cancer at a majority-minority cancer center in South Texas.

BACKGROUND: Latino patients have a higher incidence of gastric cancer compared to non-Latino white patients nationwide, with greater disparities in South Texas. However, the impact of Latino ethnicity on mortality in gastric cancer is controversial. We evaluated clinicopathological characteristics and survival outcomes in Latino vs. non-Latino white patients at our National Cancer Institute (NCI)-designated cancer center and its affiliated hospital. METHODS: We conducted a retrospective chart review of Latino and non-Latino white patients diagnosed with gastric cancer who were seen at Mays Cancer Center at the University of Texas Health in San Antonio, Texas, from 2000-2018. Median overall survival (mOS) was estimated from Kaplan-Meier curves and groups were compared with the log-rank test. RESULTS: A total of 193 patients met inclusion criteria and 65% (n=126) were Latino. Median age for all patients was 61 years. Female patients represented almost 50% of Latinos vs. 36% of non-Latino whites. There were no differences in Eastern Cooperative Oncology Group (ECOG) performance status, primary tumor location, stage, Helicobacter pylori status, HER2 status, or histologic subtype at diagnosis. Median overall survival was 14 months (95% CI: 13-36) for Latinos vs. 33 months (95% CI: 14 to n/a) for non-Latino whites (P=0.36). CONCLUSIONS: Compared to non-Latino white patients, Latino patients with gastric cancer at a majority-minority cancer center in South Texas did not have significant differences in baseline clinicopathologic features or survival outcomes. Further prospective studies are needed to evaluate epidemiologic, pathogenetic, and molecular differences in gastric cancer in order to identify variables associated with treatment efficacy and survival.

Chromatin accessibility dynamics and single cell RNA-Seq reveal new regulators of regeneration in neural progenitors.

Vertebrate appendage regeneration requires precisely coordinated remodeling of the transcriptional landscape to enable the growth and differentiation of new tissue, a process executed over multiple days and across dozens of cell types. The heterogeneity of tissues and temporally-sensitive fate decisions involved has made it difficult to articulate the gene regulatory programs enabling regeneration of individual cell types. To better understand how a regenerative program is fulfilled by neural progenitor cells (NPCs) of the spinal cord, we analyzed pax6-expressing NPCs isolated from regenerating Xenopus tropicalis tails. By intersecting chromatin accessibility data with single-cell transcriptomics, we find that NPCs place an early priority on neuronal differentiation. Late in regeneration, the priority returns to proliferation. Our analyses identify Pbx3 and Meis1 as critical regulators of tail regeneration and axon organization. Overall, we use transcriptional regulatory dynamics to present a new model for cell fate decisions and their regulators in NPCs during regeneration.