Regulatory features aid interpretation of 3'UTR variants.

Our ability to determine the clinical impact of variants in 3' untranslated regions (UTRs) of genes remains poor. We provide a thorough analysis of 3' UTR variants from several datasets. Variants in putative regulatory elements, including RNA-binding protein motifs, eCLIP peaks, and microRNA sites, are up to 16 times more likely than variants not in these elements to have gene expression and phenotype associations. Variants in regulatory motifs result in allele-specific protein binding in cell lines and allele-specific gene expression differences in population studies. In addition, variants in shared regions of alternatively polyadenylated isoforms and those proximal to polyA sites are more likely to affect gene expression and phenotype. Finally, pathogenic 3' UTR variants in ClinVar are up to 20 times more likely than benign variants to fall in a regulatory site. We incorporated these findings into RegVar, a software tool that interprets regulatory elements and annotations for any 3' UTR variant and predicts whether the variant is likely to affect gene expression or phenotype. This tool will help prioritize variants for experimental studies and identify pathogenic variants in individuals.

Aficamten for Symptomatic Obstructive Hypertrophic Cardiomyopathy.

BACKGROUND: One of the major determinants of exercise intolerance and limiting symptoms among patients with obstructive hypertrophic cardiomyopathy (HCM) is an elevated intracardiac pressure resulting from left ventricular outflow tract obstruction. Aficamten is an oral selective cardiac myosin inhibitor that reduces left ventricular outflow tract gradients by mitigating cardiac hypercontractility. METHODS: In this phase 3, double-blind trial, we randomly assigned adults with symptomatic obstructive HCM to receive aficamten (starting dose, 5 mg; maximum dose, 20 mg) or placebo for 24 weeks, with dose adjustment based on echocardiography results. The primary end point was the change from baseline to week 24 in the peak oxygen uptake as assessed by cardiopulmonary exercise testing. The 10 prespecified secondary end points (tested hierarchically) were change in the Kansas City Cardiomyopathy Questionnaire clinical summary score (KCCQ-CSS), improvement in the New York Heart Association (NYHA) functional class, change in the pressure gradient after the Valsalva maneuver, occurrence of a gradient of less than 30 mm Hg after the Valsalva maneuver, and duration of eligibility for septal reduction therapy (all assessed at week 24); change in the KCCQ-CSS, improvement in the NYHA functional class, change in the pressure gradient after the Valsalva maneuver, and occurrence of a gradient of less than 30 mm Hg after the Valsalva maneuver (all assessed at week 12); and change in the total workload as assessed by cardiopulmonary exercise testing at week 24. RESULTS: A total of 282 patients underwent randomization: 142 to the aficamten group and 140 to the placebo group. The mean age was 59.1 years, 59.2% were men, the baseline mean resting left ventricular outflow tract gradient was 55.1 mm Hg, and the baseline mean left ventricular ejection fraction was 74.8%. At 24 weeks, the mean change in the peak oxygen uptake was 1.8 ml per kilogram per minute (95% confidence interval [CI], 1.2 to 2.3) in the aficamten group and 0.0 ml per kilogram per minute (95% CI, -0.5 to 0.5) in the placebo group (least-squares mean between-group difference, 1.7 ml per kilogram per minute; 95% CI, 1.0 to 2.4; P<0.001). The results for all 10 secondary end points were significantly improved with aficamten as compared with placebo. The incidence of adverse events appeared to be similar in the two groups. CONCLUSIONS: Among patients with symptomatic obstructive HCM, treatment with aficamten resulted in a significantly greater improvement in peak oxygen uptake than placebo. (Funded by Cytokinetics; SEQUOIA-HCM ClinicalTrials.gov number, NCT05186818.).

Species-resolved, single-cell respiration rates reveal dominance of sulfate reduction in a deep continental subsurface ecosystem.

Rates of microbial processes are fundamental to understanding the significance of microbial impacts on environmental chemical cycling. However, it is often difficult to quantify rates or to link processes to specific taxa or individual cells, especially in environments where there are few cultured representatives with known physiology. Here, we describe the use of the redox-enzyme-sensitive molecular probe RedoxSensor™ Green to measure rates of anaerobic electron transfer physiology (i.e., sulfate reduction and methanogenesis) in individual cells and link those measurements to genomic sequencing of the same single cells. We used this method to investigate microbial activity in hot, anoxic, low-biomass (~103 cells mL-1) groundwater of the Death Valley Regional Flow System, California. Combining this method with electron donor amendment experiments and metatranscriptomics confirmed that the abundant spore formers including Candidatus Desulforudis audaxviator were actively reducing sulfate in this environment, most likely with acetate and hydrogen as electron donors. Using this approach, we measured environmental sulfate reduction rates at 0.14 to 26.9 fmol cell-1 h-1. Scaled to volume, this equates to a bulk environmental rate of ~103 pmol sulfate L-1 d-1, similar to potential rates determined with radiotracer methods. Despite methane in the system, there was no evidence for active microbial methanogenesis at the time of sampling. Overall, this method is a powerful tool for estimating species-resolved, single-cell rates of anaerobic metabolism in low-biomass environments while simultaneously linking genomes to phenomes at the single-cell level. We reveal active elemental cycling conducted by several species, with a large portion attributable to Ca. Desulforudis audaxviator.

A randomized trial of behavioral interventions yielding sustained reductions in distracted driving.

Distracted driving is responsible for nearly 1 million crashes each year in the United States alone, and a major source of driver distraction is handheld phone use. We conducted a randomized, controlled trial to compare the effectiveness of interventions designed to create sustained reductions in handheld use while driving (NCT04587609). Participants were 1,653 consenting Progressive® Snapshot® usage-based auto insurance customers ages 18 to 77 who averaged at least 2 min/h of handheld use while driving in the month prior to study invitation. They were randomly assigned to one of five arms for a 10-wk intervention period. Arm 1 (control) got education about the risks of handheld phone use, as did the other arms. Arm 2 got a free phone mount to facilitate hands-free use. Arm 3 got the mount plus a commitment exercise and tips for hands-free use. Arm 4 got the mount, commitment, and tips plus weekly goal gamification and social competition. Arm 5 was the same as Arm 4, plus offered behaviorally designed financial incentives. Postintervention, participants were monitored until the end of their insurance rating period, 25 to 65 d more. Outcome differences were measured using fractional logistic regression. Arm 4 participants, who received gamification and competition, reduced their handheld use by 20.5% relative to control (P < 0.001); Arm 5 participants, who additionally received financial incentives, reduced their use by 27.6% (P < 0.001). Both groups sustained these reductions through the end of their insurance rating period.

Rationally seeded computational protein design of ɑ-helical barrels.

Computational protein design is advancing rapidly. Here we describe efficient routes starting from validated parallel and antiparallel peptide assemblies to design two families of α-helical barrel proteins with central channels that bind small molecules. Computational designs are seeded by the sequences and structures of defined de novo oligomeric barrel-forming peptides, and adjacent helices are connected by loop building. For targets with antiparallel helices, short loops are sufficient. However, targets with parallel helices require longer connectors; namely, an outer layer of helix-turn-helix-turn-helix motifs that are packed onto the barrels. Throughout these computational pipelines, residues that define open states of the barrels are maintained. This minimizes sequence sampling, accelerating the design process. For each of six targets, just two to six synthetic genes are made for expression in Escherichia coli. On average, 70% of these genes express to give soluble monomeric proteins that are fully characterized, including high-resolution structures for most targets that match the design models with high accuracy.

Molecular basis of product recognition during PIP5K-mediated production of PI(4,5)P2 with positive feedback.

The ability for cells to localize and activate peripheral membrane binding proteins is critical for signal transduction. Ubiquitously important in these signaling processes are phosphatidylinositol phosphate (PIP) lipids, which are dynamically phosphorylated by PIP lipid kinases on intracellular membranes. Functioning primarily at the plasma membrane, phosphatidylinositol-4-phosphate 5-kinases (PIP5K) catalyzes the phosphorylation of PI(4)P to generate most of the PI(4,5)P2 lipids found in eukaryotic plasma membrane. Recently, we determined that PIP5K displays a positive feedback loop based on membrane-mediated dimerization and cooperative binding to its product, PI(4,5)P2. Here, we examine how two motifs contribute to PI(4,5)P2 recognition to control membrane association and catalysis of PIP5K. Using a combination of single molecule TIRF microscopy and kinetic analysis of PI(4)P lipid phosphorylation, we map the sequence of steps that allow PIP5K to cooperatively engage PI(4,5)P2. We find that the specificity loop regulates the rate of PIP5K membrane association and helps orient the kinase to more effectively bind PI(4,5)P2 lipids. After correctly orienting on the membrane, PIP5K transitions to binding PI(4,5)P2 lipids near the active site through a motif previously referred to as the substrate or PIP binding motif (PIPBM). The PIPBM has broad specificity for anionic lipids and serves a role in regulating membrane association in vitro and in vivo. Overall, our data supports a two-step membrane binding model where the specificity loop and PIPBM act in concert to help PIP5K orient and productively engage anionic lipids to drive the positive feedback during PI(4,5)P2 production.

Effect of Sacubitril/Valsartan on Cognitive Function in Patients With Heart Failure With Preserved Ejection Fraction: A Prespecified Analysis of PARAGON-HF.

BACKGROUND: A hypothetical concern has been raised that sacubitril/valsartan might cause cognitive impairment because neprilysin is one of several enzymes degrading amyloid-ß peptides in the brain, some of which are neurotoxic and linked to Alzheimer-type dementia. To address this, we examined the effect of sacubitril/valsartan compared with valsartan on cognitive function in patients with heart failure with preserved ejection fraction in a prespecified substudy of PARAGON-HF (Prospective Comparison of Angiotensin Receptor Neprilysin Inhibitor With Angiotensin Receptor Blocker Global Outcomes in Heart Failure With Preserved Ejection Fraction). METHODS: In PARAGON-HF, serial assessment of cognitive function was conducted in a subset of patients with the Mini-Mental State Examination (MMSE; score range, 0-30, with lower scores reflecting worse cognitive function). The prespecified primary analysis of this substudy was the change from baseline in MMSE score at 96 weeks. Other post hoc analyses included cognitive decline (fall in MMSE score of ≥3 points), cognitive impairment (MMSE score <24), or the occurrence of dementia-related adverse events. RESULTS: Among 2895 patients included in the MMSE substudy with baseline MMSE score measured, 1453 patients were assigned to sacubitril/valsartan and 1442 to valsartan. Their mean age was 73 years, and the median follow-up was 32 months. The mean±SD MMSE score at randomization was 27.4±3.0 in the sacubitril/valsartan group, with 10% having an MMSE score <24; the corresponding numbers were nearly identical in the valsartan group. The mean change from baseline to 96 weeks in the sacubitril/valsartan group was -0.05 (SE, 0.07); the corresponding change in the valsartan group was -0.04 (0.07). The mean between-treatment difference at week 96 was -0.01 (95% CI, -0.20 to 0.19; P=0.95). Analyses of a ≥3-point decline in MMSE, decrease to a score <24, dementia-related adverse events, and combinations of these showed no difference between sacubitril/valsartan and valsartan. No difference was found in the subgroup of patients tested for apolipoprotein E ε4 allele genotype. CONCLUSIONS: Patients with heart failure with preserved ejection fraction in PARAGON-HF had relatively low baseline MMSE scores. Cognitive change, measured by MMSE, did not differ between treatment with sacubitril/valsartan and treatment with valsartan in patients with heart failure with preserved ejection fraction. REGISTRATION: URL: https://www.clinicaltrials.gov; Unique identifier: NCT01920711.

Sodium-Glucose Cotransporter-2 Inhibitors and Major Adverse Cardiovascular Outcomes: A SMART-C Collaborative Meta-Analysis.

BACKGROUND: Sodium-glucose cotransporter-2 inhibitors (SGLT2i) consistently improve heart failure and kidney-related outcomes; however, effects on major adverse cardiovascular events (MACE) across different patient populations are less clear. METHODS: This was a collaborative trial-level meta-analysis from the SGLT2i Meta-analysis Cardio-Renal Trialists Consortium, which includes all phase 3, placebo-controlled, outcomes trials of SGLT2i across 3 patient populations (patients with diabetes at high risk for atherosclerotic cardiovascular disease, heart failure [HF], or chronic kidney disease). The outcomes of interest were MACE (composite of cardiovascular death, myocardial infarction , or stroke), individual components of MACE (inclusive of fatal and nonfatal events), all-cause mortality, and death subtypes. Effect estimates for SGLT2i versus placebo were meta-analyzed across trials and examined across key subgroups (established atherosclerotic cardiovascular disease, previous myocardial infarction, diabetes, previous HF, albuminuria, chronic kidney disease stages, and risk groups). RESULTS: A total of 78 607 patients across 11 trials were included: 42 568 (54.2%), 20 725 (26.4%), and 15 314 (19.5%) were included from trials of patients with diabetes at high risk for atherosclerotic cardiovascular disease, HF, or chronic kidney disease, respectively. SGLT2i reduced the rate of MACE by 9% (hazard ration [HR], 0.91 [95% CI, 0.87-0.96], P<0.0001) with a consistent effect across all 3 patient populations (I2=0%) and across all key subgroups. This effect was primarily driven by a reduction in cardiovascular death (HR, 0.86 [95% CI, 0.81-0.92], P<0.0001), with no significant effect for myocardial infarction in the overall population (HR, 0.95 [95% CI, 0.87-1.04], P=0.29), and no effect on stroke (HR, 0.99 [95% CI, 0.91-1.07], P=0.77). The benefit for cardiovascular death was driven primarily by reductions in HF death and sudden cardiac death (HR, 0.68 [95% CI, 0.46-1.02] and HR, 0.86 [95% CI, 0.78-0.95], respectively) and was generally consistent across subgroups, with the possible exception of being more apparent in those with albuminuria (Pinteraction=0.02). CONCLUSIONS: SGLT2i reduce the risk of MACE across a broad range of patients irrespective of atherosclerotic cardiovascular disease, diabetes, kidney function, or other major clinical characteristics at baseline. This effect is driven primarily by a reduction of cardiovascular death, particularly HF death and sudden cardiac death, without a significant effect on myocardial infarction in the overall population, and no effect on stroke. These data may help inform selection for SGLT2i therapies across the spectrum of cardiovascular-kidney-metabolic disease.

Estimates of congenital cytomegalovirus-attributable infant mortality in high-income countries: A review.

As many as 5%-10% of infants with symptomatic congenital cytomegalovirus (cCMV) disease, or 0.4%-0.8% of all liveborn infants with cCMV infection, die in early infancy in high-income countries. However, estimates are uncertain due to several potential biases that can result from data limitations and study designs. First, infants with cCMV infections who die prior to diagnosis, which usually occurs at 1-4 weeks after birth, may be excluded from both the count of deaths and the denominator of cCMV births, resulting in left truncation and immortal time biases. These 'biases' are features of the data and do not reflect bias on the part of researchers, but understanding the potential existence of threats to validity can help with interpretation of findings. Left truncation of infant deaths occurring prior to diagnosis of cCMV can result in understatement of the burden of infant deaths due to cCMV. Conversely, overestimation of infant deaths associated with symptomatic cCMV may occur in clinical case series owing to greater representation of relatively severely affected infants owing to ascertainment and referral biases. In this review, we summarise the characteristics of 26 studies that reported estimates of cCMV-associated infant deaths, including potential biases or limitations to which those estimates may have been subject. We discuss study designs whose implementation might generate improved estimates of infant deaths attributable to cCMV. More complete estimates of the overall public health impact of cCMV could inform current and future screening, prevention, and vaccine research.

Autologous bone marrow mononuclear cells to treat severe traumatic brain injury in children.

Autologous bone marrow mononuclear cells (BMMNCs) infused after severe traumatic brain injury have shown promise for treating the injury. We evaluated their impact in children, particularly their hypothesized ability to preserve the blood-brain barrier and diminish neuroinflammation, leading to structural CNS preservation with improved outcomes. We performed a randomized, double-blind, placebo-sham-controlled Bayesian dose-escalation clinical trial at two children's hospitals in Houston, TX and Phoenix, AZ, USA (NCT01851083). Patients 5-17 years of age with severe traumatic brain injury (Glasgow Coma Scale score ≤ 8) were randomized to BMMNC or placebo (3:2). Bone marrow harvest, cell isolation and infusion were completed by 48 h post-injury. A Bayesian continuous reassessment method was used with cohorts of size 3 in the BMMNC group to choose the safest between two doses. Primary end points were quantitative brain volumes using MRI and microstructural integrity of the corpus callosum (diffusivity and oedema measurements) at 6 months and 12 months. Long-term functional outcomes and ventilator days, intracranial pressure monitoring days, intensive care unit days and therapeutic intensity measures were compared between groups. Forty-seven patients were randomized, with 37 completing 1-year follow-up (23 BMMNC, 14 placebo). BMMNC treatment was associated with an almost 3-day (23%) reduction in ventilator days, 1-day (16%) reduction in intracranial pressure monitoring days and 3-day (14%) reduction in intensive care unit (ICU) days. White matter volume at 1 year in the BMMNC group was significantly preserved compared to placebo [decrease of 19 891 versus 40 491, respectively; mean difference of -20 600, 95% confidence interval (CI): -35 868 to -5332; P = 0.01], and the number of corpus callosum streamlines was reduced more in placebo than BMMNC, supporting evidence of preserved corpus callosum connectivity in the treated groups (-431 streamlines placebo versus -37 streamlines BMMNC; mean difference of -394, 95% CI: -803 to 15; P = 0.055), but this did not reach statistical significance due to high variability. We conclude that autologous BMMNC infusion in children within 48 h after severe traumatic brain injury is safe and feasible. Our data show that BMMNC infusion led to: (i) shorter intensive care duration and decreased ICU intensity; (ii) white matter structural preservation; and (iii) enhanced corpus callosum connectivity and improved microstructural metrics.

Gene Therapy Versus Common Care for Eligible Individuals With Sickle Cell Disease in the United States : A Cost-Effectiveness Analysis.

BACKGROUND: Sickle cell disease (SCD) and its complications contribute to high rates of morbidity and early mortality and high cost in the United States and African heritage community. OBJECTIVE: To evaluate the cost-effectiveness of gene therapy for SCD and its value-based prices (VBPs). DESIGN: Comparative modeling analysis across 2 independently developed simulation models (University of Washington Model for Economic Analysis of Sickle Cell Cure [UW-MEASURE] and Fred Hutchinson Institute Sickle Cell Disease Outcomes Research and Economics Model [FH-HISCORE]) using the same databases. DATA SOURCES: Centers for Medicare & Medicaid Services claims data, 2008 to 2016; published literature. TARGET POPULATION: Persons eligible for gene therapy. TIME HORIZON: Lifetime. PERSPECTIVE: U.S. health care sector and societal. INTERVENTION: Gene therapy versus common care. OUTCOME MEASURES: Incremental cost-effectiveness ratios (ICERs), equity-informed VBPs, and price acceptability curves. RESULTS OF BASE-CASE ANALYSIS: At an assumed $2 million price for gene therapy, UW-MEASURE and FH-HISCORE estimated ICERs of $193 000 per QALY and $427 000 per QALY, respectively, under the health care sector perspective. Corresponding estimates from the societal perspective were $126 000 per QALY and $281 000 per QALY. The difference in results between models stemmed primarily from considering a slightly different target population and incorporating the quality-of-life (QOL) effects of splenic sequestration, priapism, and acute chest syndrome in the UW model. From a societal perspective, acceptable (>90% confidence) VBPs ranged from $1 million to $2.5 million depending on the use of alternative effective metrics or equity-informed threshold values. RESULTS OF SENSITIVITY ANALYSIS: Results were sensitive to the costs of myeloablative conditioning before gene therapy, effect on caregiver QOL, and effect of gene therapy on long-term survival. LIMITATION: The short-term effects of gene therapy on vaso-occlusive events were extrapolated from 1 study. CONCLUSION: Gene therapy for SCD below a $2 million price tag is likely to be cost-effective when applying a societal perspective at an equity-informed threshold for cost-effectiveness analysis. PRIMARY FUNDING SOURCE: National Heart, Lung, and Blood Institute.

Clinical Outcomes With Electronic Nudges to Increase Influenza Vaccination : A Prespecified Analysis of a Nationwide, Pragmatic, Registry-Based, Randomized Implementation Trial.

BACKGROUND: In the NUDGE-FLU (Nationwide Utilization of Danish Government Electronic letter system for increasing inFLUenza vaccine uptake) trial, electronic letters incorporating cardiovascular (CV) gain-framing and repeated messaging increased influenza vaccination by approximately 1 percentage point. OBJECTIVE: To evaluate the effects of the successful nudging interventions on downstream clinical outcomes. DESIGN: Prespecified exploratory analysis of a nationwide randomized implementation trial. (ClinicalTrials.gov: NCT05542004). SETTING: The 2022 to 2023 influenza season. PARTICIPANTS: 964 870 Danish citizens aged 65 years or older. INTERVENTION: Usual care or 9 different electronically delivered behavioral nudging letters. MEASUREMENTS: Cardiovascular, respiratory, and other clinical end points during follow-up from intervention delivery (16 September 2022) through 31 May 2023. RESULTS: The analysis set included 691 820 participants. Hospitalization for pneumonia or influenza occurred in 3354 of 346 327 (1.0%) participants in the usual care group, 396 of 38 586 (1.0%) in the CV gain-framing group (hazard ratio [HR], 1.06 [95% CI, 0.95 to 1.18]; versus usual care), and 403 of 38 231 (1.1%) in the repeated letter group (HR, 1.09 [CI, 0.98 to 1.21]; versus usual care). In the usual care group, 44 682 (12.9%) participants were hospitalized for any cause, compared with 5002 (13.0%) in the CV gain-framing group (HR, 1.00 [CI, 0.97 to 1.03]; versus usual care) and 4965 (13.0%) in the repeated letter group (HR, 1.01 [CI, 0.98 to 1.04]; versus usual care). A total of 6341 (1.8%) participants died in the usual care group, compared with 721 (1.9%) in the CV gain-framing group (HR, 1.02 [CI, 0.94 to 1.10]; versus usual care) and 646 (1.7%) in the repeated letter group (HR, 0.92 [CI, 0.85 to 1.00]; versus usual care). LIMITATION: Prespecified but exploratory analysis, potential misclassification of events in routinely collected registry data, and results may not be generalizable to other health systems or countries with other racial compositions and/or cultural or societal norms. CONCLUSION: In a prespecified exploratory analysis, modest increases in influenza vaccination rates seen with electronic nudges did not translate into observable improvements in clinical outcomes. Seasonal influenza vaccination should remain strongly recommended. PRIMARY FUNDING SOURCE: Sanofi.

Seroreversion to Chlamydia trachomatis Pgp3 Antigen Among Children in a Hyperendemic Region of Amhara, Ethiopia.

Monitoring trachoma transmission with antibody data requires characterization of decay in IgG to Chlamydia trachomatis antigens. In a 3-year longitudinal cohort in a high-transmission setting, we estimated a median IgG half-life of 3 years and a seroreversion rate of 2.5 per 100 person-years (95% confidence interval, 1.6-3.5). Clinical Trials Registration NCT02754583.

Pacemaking in the lymphatic system.

Lymphatic collecting vessels exhibit spontaneous phasic contractions that are critical for lymph propulsion and tissue fluid homeostasis. This rhythmic activity is driven by action potentials conducted across the lymphatic muscle cell (LMC) layer to produce entrained contractions. The contraction frequency of a lymphatic collecting vessel displays exquisite mechanosensitivity, with a dynamic range from <1 to >20 contractions per minute. A myogenic pacemaker mechanism intrinsic to the LMCs was initially postulated to account for pressure-dependent chronotropy. Further interrogation into the cellular constituents of the lymphatic vessel wall identified non-muscle cell populations that shared some characteristics with interstitial cells of Cajal, which have pacemaker functions in the gastrointestinal and lower urinary tracts, thus raising the possibility of a non-muscle cell pacemaker. However, recent genetic knockout studies in mice support LMCs and a myogenic origin of the pacemaker activity. LMCs exhibit stochastic, but pressure-sensitive, sarcoplasmic reticulum calcium release (puffs and waves) from IP3R1 receptors, which couple to the calcium-activated chloride channel Anoctamin 1, causing depolarisation. The resulting electrical activity integrates across the highly coupled lymphatic muscle electrical syncytia through connexin 45 to modulate diastolic depolarisation. However, multiple other cation channels may also contribute to the ionic pacemaking cycle. Upon reaching threshold, a voltage-gated calcium channel-dependent action potential fires, resulting in a nearly synchronous calcium global calcium flash within the LMC layer to drive an entrained contraction. This review summarizes the key ion channels potentially responsible for the pressure-dependent chronotropy of lymphatic collecting vessels and various mechanisms of IP3R1 regulation that could contribute to frequency tuning.

Loss of anoctamin 1 reveals a subtle role for BK channels in lymphatic muscle action potentials.

Ca2+ signalling plays a crucial role in determining lymphatic muscle cell excitability and contractility through its interaction with the Ca2+-activated Cl- channel anoctamin 1 (ANO1). In contrast, the large-conductance (BK) Ca2+-activated K+ channel (KCa) and other KCa channels have prominent vasodilatory actions by hyperpolarizing vascular smooth muscle cells. Here, we assessed the expression and contribution of the KCa family to mouse and rat lymphatic collecting vessel contractile function. The BK channel was the only KCa channel consistently expressed in fluorescence-activated cell sorting-purified mouse lymphatic muscle cell lymphatic muscle cells. We used a pharmacological inhibitor of BK channels, iberiotoxin, and small-conductance Ca2+-activated K+ channels, apamin, to inhibit KCa channels acutely in ex vivo isobaric myography experiments and intracellular membrane potential recordings. In basal conditions, BK channel inhibition had little to no effect on either mouse inguinal-axillary lymphatic vessel (MIALV) or rat mesenteric lymphatic vessel contractions or action potentials (APs). We also tested BK channel inhibition under loss of ANO1 either by genetic ablation (Myh11CreERT2-Ano1 fl/fl, Ano1ismKO) or by pharmacological inhibition with Ani9. In both Ano1ismKO MIALVs and Ani9-pretreated MIALVs, inhibition of BK channels increased contraction amplitude, increased peak AP and broadened the peak of the AP spike. In rat mesenteric lymphatic vessels, BK channel inhibition also abolished the characteristic post-spike notch, which was exaggerated with ANO1 inhibition, and significantly increased the peak potential and broadened the AP spike. We conclude that BK channels are present and functional on mouse and rat lymphatic muscle cells but are otherwise masked by the dominance of ANO1. KEY POINTS: Mouse and rat lymphatic muscle cells express functional BK channels. BK channels make little contribution to either rat or mouse lymphatic collecting vessel contractile function in basal conditions across a physiological pressure range. ANO1 limits the peak membrane potential achieved in the action potential and sets a plateau potential limiting the voltage-dependent activation of BK. BK channels are activated when ANO1 is absent or blocked and slightly impair contractile strength by reducing the peak membrane potential achieved in the action potential spike and accelerating the post-spike repolarization.

Formation of Chromophores from cis-Pinonaldehyde Aged in Highly Acidic Conditions.

Sulfuric acid in the atmosphere can participate in acid-catalyzed and acid-driven reactions, including those within secondary organic aerosols (SOA). Previous studies have observed enhanced absorption at visible wavelengths and significant changes in the chemical composition when SOA was exposed to sulfuric acid. However, the specific chromophores responsible for these changes could not be identified. The goals of this study are to identify the chromophores and determine the mechanism of browning in highly acidified α-pinene SOA by following the behavior of specific common α-pinene oxidation products, namely, cis-pinonic acid and cis-pinonaldehyde, when they are exposed to highly acidic conditions. The products of these reactions were analyzed with ultra-performance liquid chromatography coupled with photodiode array spectrophotometry and high-resolution mass spectrometry, UV-vis spectrophotometry, and nuclear magnetic resonance spectroscopy. cis-Pinonic acid (2) was found to form homoterpenyl methyl ketone (4), which does not absorb visible radiation, while cis-pinonaldehyde (3) formed weakly absorbing 1-(4-(propan-2-ylidene)cyclopent-1-en-1-yl)ethan-1-one (5) and 1-(4-isopropylcyclopenta-1,3-dien-1-yl)ethan-1-one (6) via an acid-catalyzed aldol condensation. This chemistry could be relevant for environments characterized by high sulfuric acid concentrations, for example, during the transport of organic compounds from the lower to the upper atmosphere by fast updrafts.

Spatial memory encoding is associated with the anterior and posterior hippocampus: An fMRI activation likelihood estimation meta-analysis.

It has been hypothesized that differential processing occurs along the longitudinal (anterior-posterior) axis of the hippocampus. One hypothesis is that spatial memory (during both encoding and retrieval) is associated with the posterior hippocampus. An alternative hypothesis is that memory encoding (either spatial or nonspatial) is associated with the anterior hippocampus and memory retrieval is associated with the posterior hippocampus. Of importance, during spatial memory encoding, the spatial-posterior hypothesis predicts posterior hippocampal involvement, whereas the encoding-retrieval hypothesis predicts anterior hippocampal involvement. To distinguish between these hypotheses, we conducted a coordinate-based fMRI activation likelihood estimation (ALE) meta-analysis of 26 studies (with a total of 435 participants) that reported hippocampal activity during spatial memory encoding and/or spatial memory retrieval. Both spatial memory encoding and spatial memory retrieval produced extensive activity along the longitudinal axis of the hippocampus as well as the entorhinal cortex, the perirhinal cortex, and the parahippocampal cortex. Critically, the contrast of spatial memory encoding and spatial memory retrieval produced activations in both the anterior hippocampus and the posterior hippocampus. That spatial memory encoding produced activity in both the anterior and posterior hippocampus can be taken to reject strict forms of the spatial-posterior hypothesis, which stipulates that all forms of spatial memory produce activity in the posterior hippocampus, and the encoding-retrieval hypothesis, which stipulates that all forms of encoding versus retrieval produce activity in only the anterior hippocampus. Our results indicate that spatial memory encoding can involve the anterior hippocampus and the posterior hippocampus.

A MicroRNA Approach to Evaluating Elevated Prostate Cancer Risk in Cancer-Free Men.

Objective criteria are required for prostate cancer (PCa) risk assessment, treatment decisions, evaluation of therapy, and initial indications of recurrence. Circulating microRNAs were utilized as biomarkers to distinguish PCa patients from cancer-free subjects or those encountering benign prostate hyperplasia. A panel of 60 microRNAs was developed with established roles in PCa initiation, progression, metastasis, and recurrence. Utilizing the FirePlex® platform for microRNA analysis, we demonstrated the efficacy and reproducibility of a rapid, high-throughput, serum-based assay for PCa biomarkers that circumvents the requirement for extraction and fractionation of patient specimens supporting feasibility for expanded clinical research and diagnostic applications.

Outcomes from 871,441 Consecutive Surgical Procedures without Overlap or with Maximally Permissible Non-Concurrent Overlap.

OBJECTIVE: To isolate the impact of subsumed surgery (a shorter procedure completed entirely during overlapping non-critical portions of a longer antecedent procedure) on patient outcomes. SUMMARY BACKGROUND DATA: The American College of Surgeons recently recommended the elimination of "concurrent surgery" with overlap during a procedure's critical portions. Guidelines for non-concurrent overlap have been established, but the safety of subsumed surgery remains to be examined. METHODS: All consecutive procedures from 2013 to 2021 within a multihospital academic medical center were included (n=871,441). Simple logistic regression was performed to compare postoperative events between patients undergoing non-overlap surgery (n=533,032) and completely subsumed surgery (n=11,319). Thereafter, coarsened exact matching was used to match patients with non-overlap and subsumed surgery 1:1 on CPT code, 18 demographic features, baseline health characteristics, and procedural variables (n=7,146). Exact-matched cases were subsequently limited to pairs performed by the same surgeon (n=5,028). Primary outcomes included 30-day readmission, ED visits, and reoperations. RESULTS: Univariate analysis suggested that subsumed surgery had a higher 30-day risk of readmission (OR 1.55, P<0.0001), ED evaluation (OR 1.19, P<0.0001), and reoperation (OR 1.98, P<0.0001). When comparison was limited to the exact same procedure and patients were matched on demographics and health characteristics, there were no outcome differences between patients with subsumed surgery and non-overlapping surgery, even when limiting analyses to the same surgeon. CONCLUSIONS: Similar surgeries for similar patients result in similar outcomes whether there is completely subsumed or no overlap. Individual surgeons performing a specific procedure have no outcome differences with subsumed and non-overlapping cases.

Integrating Cost into Shared Decision-Making for Heart Failure with Reduced Ejection Fraction (POCKET-COST-HF): A Trial Providing Out-of-Pocket Costs for Heart Failure Medications during Clinical Encounters.

BACKGROUND: Evidence-based medical therapy for heart failure with reduced ejection fraction (HFrEF) often entails substantial out-of-pocket costs that can vary appreciably between patients. This has raised concerns regarding financial toxicity, equity, and adherence to medical therapy. In spite of these concerns, cost discussions in the HFrEF population appear to be rare, partly because out-of-pocket costs are generally unavailable during clinical encounters. In this trial, out-of-pocket cost information is given to patients and clinicians during outpatient encounters with the aim to assess the impact of providing this information on medication discussions and decisions. HYPOTHESIS: Cost-informed decision-making will be facilitated by providing access to patient-specific out-of-pocket cost estimates at the time of clinical encounter. DESIGN: Integrating Cost into Shared Decision-Making for Heart Failure with Reduced Ejection Fraction (POCKET-COST-HF) is a multicenter trial based at Emory Healthcare and University of Colorado Health. Adapting an existing patient activation tool from the EPIC-HF trial, patients and clinicians are presented a checklist with medications approved for treatment of HFrEF with or without patient-specific out-of-pocket costs (obtained from a financial navigation firm). Clinical encounters are audio-recorded, and patients are surveyed about their experience. The trial utilizes a stepped-wedge cluster randomized design, allowing for each site to enroll control and intervention group patients while minimizing contamination of the control arm. DISCUSSION: This trial will elucidate the potential impact of robust cost disclosure efforts and key information regarding patient and clinician perspectives related to cost and cost communication. It also will reveal important challenges associated with providing out-of-pocket costs for medications during clinical encounters. Acquiring medication costs for this trial requires an involved process and outsourcing of work. In addition, costs may change throughout the year, raising questions regarding what specific information is most valuable. These data will represent an important step towards understanding the role of integrating cost discussions into heart failure care. GOV IDENTIFIER: NCT04793880.