Preanalytical considerations for clinical assays of circulating human miRNA-451a, miRNA-423-5p and miRNA-199a-3p for diagnostic purposes.

Circulating miRNA has recently emerged as important biomolecules with potential clinical values as diagnostic markers for several diseases. However, to be used as such, it is critical to accurately quantify miRNAs in the clinic. Yet, preanalytical factors that can affect an error-free quantification of these miRNAs have not been explored. This study aimed at investigating several of these preanalytical factors that may affect the accurate quantification of miRNA-451a, miRNA-423-5p and miRNA-199a-3p in human blood samples. We initially evaluated levels of these three miRNAs in red blood cells (RBCs), white blood cells (WBCs), platelets, and plasma by droplet digital PCR (ddPCR). Next, we monitored miRNA levels in whole blood or platelet rich plasma (PRP) stored at different temperatures for different time periods by ddPCR. We also investigated the effects of hemolysis on miRNA concentrations in platelet-free plasma (PFP). Our results demonstrate that more than 97% of miRNA-451a and miRNA-423-5p in the blood are localized in RBCs, with only trace amounts present in WBCs, platelets, and plasma. Highest amount of the miRNA-199a-3p is present in platelets. Hemolysis had a significant impact on both miRNA-451a and miRNA-423-5p concentrations in plasma, however miRNA-199a levels remain unaffected. Importantly, PRP stored at room temperature (RT) or 4°C showed a statistically significant decrease in miRNA-451a levels, while the other two miRNAs were increased, at days 1, 2, 3 and 7. PFP at RT caused statistically significant steady decline in miRNA-451a and miRNA-423-5p, observed at 12, 24, 36, 48 and 72 hours. Levels of the miRNA-199a-3p in PFP was stable during first 72 hours at RT. PFP stored at -20°C for 7 days showed declining stability of miRNA-451a over time. However, at -80°C miRNA-451a levels were stable up to 7 days. Together, our data indicate that hemolysis and blood storage at RT, 4°C and -20°C may have significant negative effects on the accuracy of circulating miRNA-451a and miRNA-423-5p quantification.

Assessing methods for detecting Alexandrium catenella (Dinophyceae) and paralytic shellfish toxins in Southeast Alaska.

Blooms of Alexandrium catenella threaten to disrupt subsistence, recreational, and commercial shellfish harvest in Alaska, as the paralytic shellfish toxins (PSTs) produced pose a serious public health risk and can lead to costly shutdowns for shellfish farmers. Current methods of PST detection in the region range from monitoring programs utilizing net tows to detect A. catenella to direct shellfish tissue testing via mouse bioassay (MBA) for commercial aquaculture harvest, as well as various optional testing methods for subsistence and recreational harvesters. The efficacy and feasibility of these methods vary, and they have not been directly compared in Southeast Alaska. In this study, we sought to assess and compare A. catenella and PST early detection methods to determine which can provide the most effective and accurate warning of A. catenella blooms or PST events. We found microscope counts to be variable and prone to missing lower numbers of A. catenella, which may be indicative of bloom formation. However, quantitative polymerase chain reaction (qPCR) significantly correlated with microscope counts and was able to effectively detect even low numbers of A. catenella on all sampling days. Paralytic shellfish toxin concentrations measured by enzyme-linked immunosorbent assay and MBA significantly correlated with each other, qPCR, and some microscope counts. These results show that qPCR is an effective tool for both monitoring A. catenella and serving as a proxy for PSTs. Further work is needed to refine qPCR protocols in this system to provide bloom warnings on an actionable timescale for the aquaculture industry and other shellfish harvesters. Integr Environ Assess Manag 2024;00:1-14. © 2024 The Authors. Integrated Environmental Assessment and Management published by Wiley Periodicals LLC on behalf of Society of Environmental Toxicology & Chemistry (SETAC). This article has been contributed to by U.S. Government employees and their work is in the public domain in the USA.

Mesenchymal stromal cells with chimaeric antigen receptors for enhanced immunosuppression.

Allogeneic mesenchymal stromal cells (MSCs) are a safe treatment option for many disorders of the immune system. However, clinical trials using MSCs have shown inconsistent therapeutic efficacy, mostly owing to MSCs providing insufficient immunosuppression in target tissues. Here we show that antigen-specific immunosuppression can be enhanced by genetically modifying MSCs with chimaeric antigen receptors (CARs), as we show for E-cadherin-targeted CAR-MSCs for the treatment of graft-versus-host disease in mice. CAR-MSCs led to superior T-cell suppression and localization to E-cadherin+ colonic cells, ameliorating the animals' symptoms and survival rates. On antigen-specific stimulation, CAR-MSCs upregulated the expression of immunosuppressive genes and receptors for T-cell inhibition as well as the production of immunosuppressive cytokines while maintaining their stem cell phenotype and safety profile in the animal models. CAR-MSCs may represent a widely applicable therapeutic technology for enhancing immunosuppression.

An estrogen receptor α-derived peptide improves glucose homeostasis during obesity.

Estrogen receptor α (ERα) plays a crucial role in regulating glucose and energy homeostasis during type 2 diabetes mellitus (T2DM). However, the underlying mechanisms remain incompletely understood. Here we find a ligand-independent effect of ERα on the regulation of glucose homeostasis. Deficiency of ERα in the liver impairs glucose homeostasis in male, female, and ovariectomized (OVX) female mice. Mechanistic studies reveal that ERα promotes hepatic insulin sensitivity by suppressing ubiquitination-induced IRS1 degradation. The ERα 1-280 domain mediates the ligand-independent effect of ERα on insulin sensitivity. Furthermore, we identify a peptide based on ERα 1-280 domain and find that ERα-derived peptide increases IRS1 stability and enhances insulin sensitivity. Importantly, administration of ERα-derived peptide into obese mice significantly improves glucose homeostasis and serum lipid profiles. These findings pave the way for the therapeutic intervention of T2DM by targeting the ligand-independent effect of ERα and indicate that ERα-derived peptide is a potential insulin sensitizer for the treatment of T2DM.

A qualitative study of low-load resistance training with blood flow restriction in people with advanced multiple sclerosis.

BACKGROUND: Blood flow restriction (BFR) combined with low-load resistance training could minimize exercise barriers and offer strength and mobility improvements for people with advanced Multiple Sclerosis (MS); but patient experience has not been evaluated. PURPOSE: The purpose of this study was to assess the satisfaction, acceptability, and impact of combining low-load resistance training with BFR for individuals with advanced MS (Expanded Disability Status Scale: EDSS 6.0-7.0). METHODS: We used an interpretive phenomenological research design and post-intervention interviews to explore participant experiences of a novel low-load resistance training program with BFR. RESULTS: Interview participants (n = 14) were 55.4 ± 6.2 years old and were diagnosed with MS for 19.1 ± 10.7 years. Four themes were identified (satisfaction, acceptability, impact, program refinement) with 14 subthemes. Satisfaction was mixed based on overall expectations, yet all participants recommended the intervention. Acceptability was evidenced by all participants identifying comfortable/easy aspects, and modifiable elements that could improve comfort. Impact was evidenced with translation to everyday life activities, strength/self-efficacy/psychological improvements, effectiveness, and fatigue reduction. CONCLUSIONS: Participants found BFR combined with low-load resistance training to be satisfactory (mixed initial expectations), acceptable (comfortable/easy), and impactful (translating to life improvements). Areas for program refinement were identified that should be targeted in future iterations.

ZnPP-laden nanoparticles improve glucose homeostasis and chronic inflammation during obesity.

BACKGROUND AND PURPOSE: Chronic inflammation plays a pivotal role in the development of Type 2 diabetes mellitus (T2DM). Previous studies have shown that haem oxygenase-1 (HO-1) plays a proinflammatory role during metabolic stress, suggesting that HO-1 inhibition could be an effective strategy to treat T2DM. However, the application of HO-1 inhibitors is restricted due to solubility-limited bioavailability. In this study, we encapsulated the HO-1 inhibitor, zinc protoporphyrin IX (ZnPP), within nanoparticles and investigated their role in regulating glucose homeostasis and chronic inflammation during obesity. EXPERIMENTAL APPROACH: We delivered DMSO-dissolved ZnPP (DMSO-ZnPP) and ZnPP-laden nanoparticles (Nano-ZnPP) to diet-induced obese male mice for 6 weeks. Glucose and insulin tolerance tests were carried out, liver and adipose tissue gene expression profiles analysed, and systemic inflammation analysed using flow cytometry. KEY RESULTS: Nanoparticles significantly increased the delivery efficiency of ZnPP in both cells and mice. In mice with diet-induced obesity, inhibition of HO-1 by Nano-ZnPP significantly decreased adiposity, increased insulin sensitivity, and improved glucose tolerance. Moreover, Nano-ZnPP treatment attenuated both local and systemic inflammatory levels during obesity. Mechanistically, Nano-ZnPP significantly attenuated glucagon, TNF, and fatty acid synthesis signalling pathways in the liver. In white adipose tissue, the oxidative phosphorylation signalling pathway was enhanced and the inflammation signalling pathway diminished by Nano-ZnPP. Our results show that Nano-ZnPP has better effects on the improvement of glucose homeostasis and attenuation of chronic inflammation, than those of DMSO-dissolved ZnPP. CONCLUSIONS AND IMPLICATIONS: These findings indicate that ZnPP-laden nanoparticles are potential therapeutic agents for treating T2DM.

Dissymmetrical Chiral Peropyrenes: Synthesis via Iridium-Catalyzed C-H Activation/Alkyne Benzannulation and Study of Their Properties.

Dissymmetrical chiral peropyrenes with electron-rich and electron-deficient aryl substituents in the bay regions were synthesized via iridium-catalyzed C-H activation and alkyne benzannulation. The electronic properties were studied using cyclic and differential pulse voltammetry. The enantiomers were separated and exhibited high glum and gabs values in circularly polarized luminescence (CPL) and circular dichroism (CD), respectively. Variable-temperature NMR experiments were conducted on symmetrical and dissymmetrical chiral peropyrenes to compare the barrier to rotation of the aryl groups in the bay region.

Demographic patterns of walleye (Sander vitreus) reproductive success in a Wisconsin population.

Harvest in walleye Sander vitreus fisheries is size-selective and could influence phenotypic traits of spawners; however, contributions of individual spawners to recruitment are unknown. We used parentage analyses using single nucleotide polymorphisms to test whether parental traits were related to the probability of offspring survival in Escanaba Lake, Wisconsin. From 2017 to 2020, 1339 adults and 1138 juveniles were genotyped and 66% of the offspring were assigned to at least one parent. Logistic regression indicated the probability of reproductive success (survival of age-0 to first fall) was positively (but weakly) related to total length and growth rate in females, but not age. No traits analyzed were related to reproductive success for males. Our analysis identified the model with the predictors' growth rate and year for females and the models with year and age and year for males as the most likely models to explain variation in reproductive success. Our findings indicate that interannual variation (i.e., environmental conditions) likely plays a key role in determining the probability of reproductive success in this population and provide limited support that female age, length, and growth rate influence recruitment.

Program Directors' Perspectives on Residency Applications in the Post‒United States Medical Licensing Examination Step 1 Era: A Case for Standardized Letters of Recommendation?

INTRODUCTION: The United States Medical Licensing Examination (USMLE) Step 1 test evolved into a key metric utilized by program directors (PDs) in assessing candidates for residency. The transition to a USMLE Step 1 binary pass/fail scoring system has resulted in a loss of an important objective assessment. With national movements toward pass/fail systems for clerkship grading and trends toward abandonment of class ranking, assessing residency applications has become increasingly challenging. METHODS: The Society of Academic Urologists convened a task force to, in part, assess the perspectives of urology PDs regarding the importance of various aspects of a residency application for predicting clinical performance. An anonymous survey was disseminated to all urology PDs in the US. Perspectives on 11 potential application predictors of clinical performance and demographics were recorded. Descriptive statistics characterized PD responses. Friedman test and pairwise Wilcoxon tests were used to evaluate the relative ranks assigned to application elements by PDs. RESULTS: There was a 60.5% response rate (89/147). Letters of recommendation (LORs) were ranked as the most important predictor, with a mean rank of 2.39, median of 2 (IQR 1-3). Clerkship grades and USMLE Step 1 were comparable and ranked second. Medical school reputation ranked the lowest. There was significant subjective heterogeneity among categories; however, this was less so for LORs, which predominated as the most important factor among application elements (P < .001). CONCLUSIONS: To our knowledge, this is the largest sample size assessing PD perspectives on application factors that predict clinical performance. The second (clerkship grades) and third (USLME Step 1) most important factors moving toward binary pass/fail systems create an opportunity for actionable change to improve assessment objectivity. Our data demonstrate LORs to be the most important factor of residency applications, making a compelling argument for moving toward a standardized LOR to maximize this tool, mitigate bias, and improve interreviewer reliability.

Genomic evidence for domestication selection in three hatchery populations of Chinook salmon, Oncorhynchus tshawytscha.

Fish hatcheries are widely used to enhance fisheries and supplement declining wild populations. However, substantial evidence suggests that hatchery fish are subject to differential selection pressures compared to their wild counterparts. Domestication selection, or adaptation to the hatchery environment, poses a risk to wild populations if traits specific to success in the hatchery environment have a genetic component and there is subsequent introgression between hatchery and wild fish. Few studies have investigated domestication selection in hatcheries on a genomic level, and even fewer have done so in parallel across multiple hatchery-wild population pairs. In this study, we used low-coverage whole-genome sequencing to investigate signals of domestication selection in three separate hatchery populations of Chinook salmon, Oncorhynchus tshawytscha, after approximately seven generations of divergence from their corresponding wild progenitor populations. We sequenced 192 individuals from populations across Southeast Alaska and estimated genotype likelihoods at over six million loci. We discovered a total of 14 outlier peaks displaying high genetic differentiation (F ST) between hatchery-wild pairs, although no peaks were shared across the three comparisons. Peaks were small (53 kb on average) and often displayed elevated absolute genetic divergence (D xy) and linkage disequilibrium, suggesting some level of domestication selection has occurred. Our study provides evidence that domestication selection can lead to genetic differences between hatchery and wild populations in only a few generations. Additionally, our data suggest that population-specific adaptation to hatchery environments likely occurs through different genetic pathways, even for populations with similar standing genetic variation. These results highlight the need to collect paired genotype-phenotype data to understand how domestication may be affecting fitness and to identify potential management practices that may mitigate genetic risks despite multiple pathways of domestication.

Effect of Medicaid expansion on inflammatory bowel disease and healthcare utilization.

BACKGROUND: Kentucky was among the first to adopt Medicaid expansion, resulting in reducing uninsured rates from 14.3% to 6.4%. We hypothesize that Medicaid expansion resulted in increased elective healthcare utilization and reductions in emergency treatments by patients suffering Inflammatory Bowel Disease (IBD). METHODS: The Hospital Inpatient Discharge and Outpatient Services Database (HIDOSD) identified all encounters related to IBD from 2009 to 2020 in Kentucky. Several demographic variables were compared in pre- and post-Medicaid expansion adoption. RESULTS: Our study analyzed 3386 pre-expansion and 24,255 post-expansion encounters for IBD patients. Results showed that hospitalization rates dropped (47.7%-8.4%), outpatient visits increased (52.3%-91.6%) and Emergency visits decreased (36.7%-11.4%). Admission following a clinical referral similarly increased with a corresponding drop in emergency room admissions. Hospital costs and lengths of stay also dropped following Medicaid expansion. CONCLUSION: In the IBD population, Medicaid expansion improved access to preventative care, reduced hospital costs by decreasing emergency care, and increased elective care pathways.

Complete Intraureteral Stents Decrease Urinary Symptoms Compared with Conventional Ureteral Stents: A Systematic Review and Meta-Analysis.

Introduction: Up to 80% of stent patients report urinary discomfort, negatively impacting their daily activities and quality of life. Conventional Double-J ureteral stents (DJSs) can cause adverse kidney and bladder-related symptoms. Complete intraureteral stents (CISs) may reduce bothersome bladder symptoms by reducing foreign material in the bladder. We sought to aggregate and analyze ureteral stent symptom questionnaire (USSQ) data from the available randomized controlled trials comparing CISs with conventional ureteral stents. Methods: In February 2023, a Preferred Reporting Items for Systematic Reviews and Meta-Analyses-compliant systematic review was performed to identify studies that evaluated the use of CISs and reported outcomes using the USSQ score. Two authors (D.E.H.-G. and G.S.) independently extracted and analyzed data using Review Manager 5.41. Heterogeneity was assessed using Higgins I2%, with values >50% considered heterogeneous and analyzed with a random effects model; otherwise, a fixed effects model was used. The results are presented as the mean difference (MD) with 95% confidence intervals (CIs). We set our significance level at p = 0.05. Results: Six randomized controlled clinical trials compared CISs with DJSs, but only five trials reported the USSQ score. Among these patients, 235 had CISs, whereas the remaining 259 had DJSs or loop-tail stents and served as controls for 494 patients. Urinary symptoms scores were lower in the CIS group (MD -5.19, 95% CI: [-5.89 to -4.50], p < 0.0001). Pain scores were also lower in the CIS group (MD -1.90 [-2.63 to -1.16] p < 0.00001). General health and work performance domains were similar between the groups. A 2.5% stent failure or migration rate requiring endoscopic intervention was reported in the CIS group compared with 0.3% in the DJS group (odds ratio 4.01 [0.96-16.76] p = 0.06). Conclusions: CISs significantly decrease urinary symptoms and pain associated with conventional indwelling ureteral stents. However, further trials are needed to determine the optimal patient selection for this type of stent.

A Single Dermatome Clinical Prediction Rule for Independent Walking 1 Year After Spinal Cord Injury.

OBJECTIVE: To derive and validate a simple, accurate CPR to predict future independent walking ability after SCI at the bedside that does not rely on motor scores and is predictive for those initially classified in the middle of the SCI severity spectrum. DESIGN: Retrospective cohort study. Binary variables were derived, indicating degrees of sensation to evaluate predictive value of pinprick and light touch variables across dermatomes. The optimal single sensory modality and dermatome was used to derive our CPR, which was validated on an independent dataset. SETTING: Analysis of SCI Model Systems dataset. PARTICIPANTS: Individuals with traumatic SCI. The data of 3679 participants (N=3679) were included with 623 participants comprising the derivation dataset and 3056 comprising the validation dataset. INTERVENTIONS: Not applicable. MAIN OUTCOME MEASURES: Self-reported ability to walk both indoors and outdoors. RESULTS: Pinprick testing at S1 over lateral heels, within 31 days of SCI, accurately identified future independent walkers 1 year after SCI. Normal pinprick in both lateral heels provided good prognosis, any pinprick sensation in either lateral heel provided fair prognosis, and no sensation provided poor prognosis. This CPR performed satisfactorily in the middle SCI severity subgroup. CONCLUSIONS: In this large multi-site study, we derived and validated a simple, accurate CPR using only pinprick sensory testing at lateral heels that predicts future independent walking after SCI.

Comparisons of Pediatric and Adult SARS-CoV-2-Specific Antibodies up to 6 Months after Infection, Vaccination, or Hybrid Immunity.

BACKGROUND: Characterization of longitudinal SARS-CoV-2-specific antibody responses in children following infection and vaccination is needed to inform SARS-CoV-2 vaccine policy decisions for children, which may differ from adults. METHODS: We enrolled individuals at the time of SARS-CoV-2 infection or vaccination for longitudinal serological testing and compared SARS-CoV-2-spike-specific IgG and neutralization activity in children and adults stratified by infection and vaccination status using enzyme-linked immunosorbent and virus neutralization assays. RESULTS: Between June 2020 and December 2022, we collected sera from 669 participants aged 40 days to 55 years, including 330 unvaccinated individuals with laboratory-confirmed SARS-CoV-2 infection, 180 vaccinated SARS-CoV-2-naïve individuals, and 159 vaccinated previously infected individuals. Half (n = 330, 49.3%) were children. SARS-CoV-2-specific IgG and neutralization activity in children < 12 years old in response to infection persisted at higher levels than those of adults through at least 6 months (spike-specific IgG levels, 2.05 [95% CI: 1.4-3.1] times higher than adults; neutralizing activity, median 88.8 vs 75.2%, respectively, p = .04). In addition, all pediatric participants had significantly higher IgG levels compared with adults at 6 months following infection or vaccination, regardless of prior infection status. Vaccine-induced SARS-CoV-2-specific IgG responses in previously infected individuals persisted at higher levels than those from infection alone at 6 months (median AUC, children 5-11 years old, 9115 vs 368; adolescents 3613 vs 475; adults 1956 vs 263, all p < .001). CONCLUSIONS: These data demonstrate the robust and persistent immunologic response of SARS-CoV-2 vaccination in children and emphasize the benefit of vaccination after SARS-CoV-2 infection.

Electrical signalling and plant response to herbivory: A short review.

For a long time, electrical signaling was neglected at the expense of signaling studies in plants being concentrated with chemical and hydraulic signals. Studies conducted in recent years have revealed that plants are capable of emitting, processing, and transmitting bioelectrical signals to regulate a wide variety of physiological functions. Many important biological and physiological phenomena are accompanied by these cellular electrical manifestations, which supports the hypothesis about the importance of bioelectricity as a fundamental 'model' for response the stresses environmental and for activities regeneration of these organisms. Electrical signals have also been characterized and discriminated against in genetically modified plants under stress mediated by sucking insects and/or by the application of systemic insecticides. Such results can guide future studies that aim to elucidate the factors involved in the processes of resistance to stress and plant defense, thus aiding in the development of successful strategies in integrated pest management. Therefore, this mini review includes the results of studies aimed at electrical signaling in response to biotic stress. We also demonstrated how the generation and propagation of electrical signals takes place and included a description of how these electrical potentials are measured.

Hybridization led to a rewired pluripotency network in the allotetraploid Xenopus laevis.

After fertilization, maternally contributed factors to the egg initiate the transition to pluripotency to give rise to embryonic stem cells, in large part by activating de novo transcription from the embryonic genome. Diverse mechanisms coordinate this transition across animals, suggesting that pervasive regulatory remodeling has shaped the earliest stages of development. Here, we show that maternal homologs of mammalian pluripotency reprogramming factors OCT4 and SOX2 divergently activate the two subgenomes of Xenopus laevis, an allotetraploid that arose from hybridization of two diploid species ~18 million years ago. Although most genes have been retained as two homeologous copies, we find that a majority of them undergo asymmetric activation in the early embryo. Chromatin accessibility profiling and CUT&RUN for modified histones and transcription factor binding reveal extensive differences in predicted enhancer architecture between the subgenomes, which likely arose through genomic disruptions as a consequence of allotetraploidy. However, comparison with diploid X. tropicalis and zebrafish shows broad conservation of embryonic gene expression levels when divergent homeolog contributions are combined, implying strong selection to maintain dosage in the core vertebrate pluripotency transcriptional program, amid genomic instability following hybridization.