American Journal of Physiology-Regulatory, Integrative and Comparative Physiology

In marine teleosts, gill ionocytes play central roles in the excretion of excess monovalent ions (Na+, Cl-, and K+). Basolateral K+ channels are considered essential for sustaining ion secretion by maintaining K+ homeostasis and membrane potential; however, the molecular identity of basolateral K+ channels in teleost gill ionocytes remains incompletely understood. Kcnj15 is a K+ channel expressed in multiple osmoregulatory tissues of the Japanese pufferfish (Takifugu rubripes), and its localization changes depending on tissue type and osmotic conditions; however, its localization in the gills has remained unresolved. Here, we determined the cellular localization of Kcnj15 in the gills and examined its expression in response to changes in environmental salinity. In seawater-acclimated fish, Kcnj15 was localized to the basolateral membrane of Nka- and Nkcc1-positive ionocytes. Following acclimation to hypoosmotic brackish water (1 ppt), transcript levels of cftr, nkcc1, and kcnj1a decreased, whereas kcnj15 expression remained unchanged. Under this condition, Kcnj15 was localized to the basolateral membrane of Nka-positive ionocytes. These results demonstrate that Kcnj15 is localized to the basolateral membrane of gill ionocytes and suggest that it may function as a basolateral K+ leak channel supporting ionocyte homeostasis under both seawater and low-salinity conditions.

BackgroundHydrogen sulfide (H2S) is an important endothelial-derived vasodilator, but the signaling mechanism remains incompletely understood. We previously demonstrated that H2S-mediated vasodilation requires transient receptor potential vanilloid type 4 (TRPV4) channels. Because H2S has been reported to enhance heme oxygenase (HO) activity and HO-derived carbon monoxide (CO) regulates endothelial signaling, we hypothesized that H2S-mediated vasodilation requires HO-2-derived CO. MethodsPressure myography was performed in isolated rat mesenteric arteries to determine the contribution of HO, TRPV4, eBK, and SK/IK channels to H2S-mediated vasodilation. HO-2 sulfhydration was assessed using a maleimide assay, and spatial association among HO-2 and TRPV4 was examined using proximity ligation assays in human aortic endothelial cells. ResultsH2S Selicited concentration-dependent vasodilation that was abolished by HO inhibition. Repletion of CO restored H2S-mediated vasodilation in the presence of HO inhibition. CO-mediated vasodilation was abolished by TRPV4 and SK/IK inhibition but was unaffected by eBK inhibition. H2S increased HO-2 sulfhydration and enhanced HO activity. In endothelial cells, HO-2 and TRPV4 exhibited close spatial association. ConclusionsThese findings support a model in which H2S stimulates HO-2-derived CO production, leading to TRPV4-dependent endothelial signaling, SK/IK activation, and vasodilation. Together, the data support the existence of an endothelial HO-2/TRPV4/SK/IK signaling domain that contributes to H2S-mediated vascular reactivity.

Arginine vasopressin (AVP) increases excitability of myometrial smooth muscle cells (MSMCs) through Gq-coupled AVP receptors. Although excitability requires membrane depolarization, the mechanisms linking AVP receptor activation to membrane depolarization and Ca{superscript 2} signaling are incompletely understood. Here, we show that AVPR1 is the predominant AVP receptor in primary MSMCs. In Xenopus oocytes, AVP signals through AVPR1 to inhibit SLO2.1-mediated potassium currents, reducing current amplitude to approximately 60% of control currents. Consistent with suppression of a hyperpolarizing conductance, AVP depolarized a myometrial cell line (hTERT-HM) and increased intracellular Ca{superscript 2} signaling. Analysis of Ca{superscript 2} dynamics revealed that the initial Ca{superscript 2} peak was largely preserved under conditions limiting extracellular Ca{superscript 2} entry, consistent with intracellular store release. Conversely, the oscillatory phase depended on extracellular Ca{superscript 2} influx and was reduced by SLO2.1 knockdown. Together, these findings support a model in which AVP preferentially signals through AVPR1A to inhibit SLO2.1, depolarize myometrial cells, enhance VDCC-dependent Ca{superscript 2} entry, and promote excitability, enhancing conditions for uterine contraction.

The urinary bladder functions to store and release urine, yet how the sensation of bladder fullness is conveyed and perceived to the central nervous system is not understood. During bladder filling, the detrusor smooth muscle (DSM) generates phasic contractions, resulting in pressure fluctuations within the bladder. These transient pressure events drive bursts of afferent nerve activity, yet the underlying mechanism leading to rhythmic contractions remains unclear. Here, we examined the role of Gq protein-coupled receptor (GqPCR) activity on DSM excitability and contractility. Using ex vivo pressurized urinary bladder preparations and sharp microelectrode experiments on bladder strips from mice, we evaluated whole bladder transient pressure events, whole bladder DSM Ca2+ activity, and membrane potential in bladder strips. We found that global inhibition of urinary bladder GqPCR activity with YM-254890 abates phasic contractility and transient pressure events through a reduction in DSM Ca2+ activity and propagation of Ca2+ waves. Further, we found inhibition of GqPCR significantly hyperpolarizes DSM, reducing action potentials and decreasing excitability, and activation of protein kinase C restores membrane potential to baseline levels. These findings highlight that GqPCR activity mediates DSM excitability and contractility in such a way as to result in phasic detrusor contractions and transient pressure events.

Cerebrospinal fluid (CSF) provides a specialized extracellular environment for the central nervous system, which is predominantly produced by the choroid plexus, a highly vascularized epithelial structure whose ion transport processes are fundamental to CSF secretion, composition, and homeostasis. The mechanisms of Na+ entry into choroid plexus epithelial cells (CPECs) from the interstitial side remain disputed. The slc4a10 gene product encoding the Na+-dependent Cl-/HCO3- exchanger, Ncbe, was suggested as a key transport mechanism based on its impact on the cell's Na+-dependent regulation of intracellular pH and its basolateral membrane expression. The current study was undertaken to directly assess the contribution of Ncbe to the Na+ uptake into CPECs. Intracellular Na+ was recorded by fluorometry using the Na+ probe Sodium Binding Fluorescent Indicator in clusters of CPECs with access to both the luminal and basolateral membranes. Removal of extracellular Na+ reduced the apparent ex vivo intracellular [Na+] to ~5 mM from a baseline of ~43 mM in the absence of CO2/HCO3- and ~54 mM in the presence of CO2/HCO3-. Flame photometry estimated the intracellular [Na+] ex vivo to ~28 mM. The CO2/HCO3--dependent rate of [Na+] recovery amounted to ~53% of the total recovery rate upon re-addition of Na+. Experiments with access to only the luminal membrane show a [Na+] recovery of a similar rate as observed in the absence of CO2/HCO3- in the clusters. The CO2/HCO3--independent [Na+] recovery was inhibited to ~50% by the NKCC1 inhibitor bumetanide and to ~30% by the TRPv4 inhibitor RN1734. NHE contributed to a minor extent to the CO2/HCO3--independent transport. The HCO3- transport inhibitor DIDS, however, inhibited the total [Na+] recovery rate to ~50%, indicating a role for Ncbe rather than NBCn1 in the cellular [Na+] recovery. Indeed, docking of DIDS into Ncbe and NBCn1 indicated that both proteins can accommodate the binding of DIDS. However, the orientation of the DIDS poses in Ncbe suggests a binding mode more similar to that found in the Anion Exchangers (SLC4A1-3), which seems to accommodate the covalent-type docking more than NBCn1. The Ncbe inhibition by DIDS was supported by the rate of [Na+] recovery that was significantly higher in CPECs from Ncbe-wt than Ncbe-ko mice in the presence of CO2/HCO3-. As both NKCC1 and TRPv4 are localized to the luminal membrane, the findings collectively suggest that Ncbe is the most prominent mechanism for Na+ entry into CPECs expressed at the basolateral side. We suggest Ncbe as the rate-limiting mechanism in the vectorial Na+ transport driving CSF secretion.

Introduction: With global warming, heatwaves are increasing in severity, affecting species worldwide. Recent studies show associations between heat exposure in pregnancy and adverse birth outcomes such as preterm birth, low birthweight and stillbirth across multiple species. This suggests potentially conserved mechanisms such as maternal inflammation, dehydration and changes in placental physiology in response to heat exposure. Moreover, the gestational timing of antenatal heat likely impacts the type and severity of outcome, yet this is not fully understood. Methods: We conducted a scoping review of studies on heat exposure and pregnancy outcomes in humans and other animals, to identify common mechanisms and temporal dynamics of effects. Temperature-, trimester- and species- specific patterns were reviewed across 197 studies and ten species (human, cow, sheep, pig, goat, rat, mouse, guinea pig, lizard and rabbit). Results: Qualitative trends indicate that high temperatures are associated with adverse birth outcomes. In particular, third trimester exposure increases risk of preterm birth and low birthweight. Many studies emphasise placental involvement in adverse outcomes and point to potential aberrations in placental physiology or cortisol signaling pathways. Conclusions: Overall, literature presents that heat exposure increases risk of adverse birth outcomes across studies, and taxa. Understanding the relationship between heat exposure and adverse birth outcomes, as well as the mechanisms and timing, is vital for informing public health policies and creating mitigating strategies for expecting mothers.

Nitrogenous waste excretion is essential for all developmental stages of fish. Embryonic fish excrete urea, transitioning to cutaneous and later branchial ammonia excretion. In zebrafish, ammonia excretion involves rhesus glycoproteins Rhbg and Rhcgb in keratinocytes and ionocytes, but the developmental moment they appear in the gill remains unclear. Potential redundancy between Rhbg and Rhcgb in ammonia excretion is also not fully investigated, nor is the difference in response to low pH. We hypothesized that rhesus glycoproteins are partially redundant, and that they differ in their response to low pH as ammonia excretion enables ionocytes to exchange Na+ and H+ (Rh-NHE-metabolon). We predicted that a loss of rhbg or rhcgb induces compensatory responses. We characterized the transition from urea to branchial ammonia excretion from 0 to 8 days-post fertilization (dpf) and the response to pH 5.0 on the expression and localization of rhesus glycoproteins in control zebrafish and rhbg or rhcgb-crispants. Effects of high external ammonia (HEA, 500 M NH4Cl) and 10 mM HEPES-buffering were further characterized in rhcgb-crispants. Rhag and Rhbg appeared in the gill at 5 dpf, while Rhcgb appeared at 6 dpf. A loss of rhbg or rhcgb did not impact baseline N-excretion, illustrating that zebrafish can maintain ammonia excretion without the full complement of rhesus glycoproteins. We observed no compensatory increase in rhesus glycoproteins, but expression of the transporter hippocampus-abundant transcript 1b increased. HEA-exposed rhcgb-crispants switched to urea as primary nitrogen waste. Together, these findings underline the plasticity of the larval in dealing with nitrogenous waste.

Aldosterone is a key regulator of systemic sodium, potassium, and fluid homeostasis. Chronic elevation of aldosterone, as observed in primary aldosteronism, chronic heart and kidney diseases, promotes cardiac hypertrophy, inflammation, fibrosis, and heart failure, which are further enhanced in aging. Although these pathological effects are primarily attributed to the mineralocorticoid receptor (MR), emerging evidence suggests roles for nonclassical G protein-coupled receptor (GPCR) signaling. We identified lysophosphatidic acid receptor 2 (LPAR2) as a novel GPCR candidate that mediates aldosterone signaling in cardiomyocytes. Using AC16 human cardiomyocyte cells and reporter gene assays, we demonstrate that aldosterone activates a Gi/o-dependent GPCR pathway. Pharmacological inhibition of LPAR2 internalization confirms its specific reaction to aldosterone. Functional studies revealed that aldosterone-induced cardiomyocyte injury requires both MR and LPAR2, where specific antagonists to both receptors reduced the ability of aldosterone to induce collagen expression and cell death markers. These findings uncover a previously unrecognized non-classical mechanism of aldosterone action in the heart and highlight LPAR2 as a promising therapeutic target for aldosterone-driven cardiovascular disease.

Axonal action potential propagation critically depends on tightly regulated intra and extracellular electrolyte concentrations and acid base homeostasis. Experimental studies have demonstrated that changes in extracellular sodium, potassium, and calcium concentrations alter axonal excitability and conduction properties. However, it remains unknown whether small variations in electrolyte concentrations within the physiological range are associated with measurable changes in nerve conduction velocity in humans. In this study, data from neurologically healthy children who underwent both nerve conduction studies and blood gas analysis were analyzed to investigate the relationship between electrolyte concentrations and median motor nerve conduction velocity. Nerve conduction velocity was significantly negatively correlated with ionized calcium concentration and positively correlated with sodium concentration, whereas no significant correlation was observed for potassium. In an a priori defined multiple linear regression model, ionized calcium, sodium, and bicarbonate remained significantly associated with nerve conduction velocity after correction for multiple testing. These findings indicate that even small variations in electrolyte concentrations within the physiological range are associated with measurable differences in peripheral nerve conduction velocity and suggest an additional contribution of acid base status to nerve conduction.

The formation of inert gas bubbles during decompression can lead to decompression sickness (DCS), a major operational risk for divers, compressed-gas workers, astronauts, and high-altitude aviators. In diving, DCS risk is typically inferred from post-dive ultrasound detection of venous gas emboli (VGE), precluding modification of decompression schedules based on real-time physiological feedback. Two-dimensional ultrasound imaging could provide additional insight into decompression-related physiological changes; however, its use in hyperbaric environments has been largely precluded by fire risk associated with elevated oxygen partial pressures (ppO2) in enclosed spaces. Here, we developed a workflow for operating a programmable ultrasound system under hyperbaric conditions and acquiring ultrasound data from the subclavian vein and calf muscle during decompression. A total of 42 dives were conducted by 26 individuals using a previously characterized dive profile to 132 feet seawater (FSW) for 20 min with 9 min of decompression. Three exposure conditions were evaluated: non-exercising, exercising, and a brief pause at 20 FSW during compression. Twelve dives included programmable ultrasound imaging during decompression. Post-dive VGE responses were consistent with prior reports while demonstrating substantial inter-individual variability and sensitivity to modest profile modifications. VGE were detected in the subclavian vein during decompression in two participants and subsequently confirmed by post-dive echocardiography. Calf muscle ultrasound brightness typically increased from pre-dive to decompression measurements, before decreasing below baseline in the 120 min post dive measurement period. These findings demonstrate the feasibility of programmable ultrasound imaging during human decompression and establish a practical framework for ultrasound operation under hyperbaric conditions. This approach may support future physiological studies and development of automated decompression monitoring technologies. New and NoteworthyThis study demonstrates the first use of a programmable ultrasound system to acquire and quantitatively analyze ultrasound data during human decompression. The approach enabled direct visualization of venous gas emboli during decompression and revealed calf muscle ultrasound signal changes, providing a new tool for investigating physiological responses during decompression that are not accessible through conventional post-dive monitoring.

Objective: Circadian rhythms are frequently disrupted in patients in the intensive care unit (ICU), potentially worsening clinical outcomes. Continuous enteral nutrition throughout the day and night is common in the ICU, but eliminates feeding-fasting cycles that serve as important timing cues for the circadian system. The objective of this study was to determine the effect of providing enteral nutrition in a cyclic daytime pattern, compared with continuous administration, on circadian rhythmicity in critically ill patients in the ICU. Design: Single-center randomized controlled trial Setting: Mixed medical-surgical tertiary intensive care unit in the Netherlands Patients: Adult ICU patients ([≥]18 yr) receiving enteral nutrition. Intervention: Patients were randomized to receive either continuous, or cyclic daytime enteral feeding (08:00-20:00), initiated from the start of nutritional support. Measurements and Main Results: Sixty-two ICU patients were enrolled, of whom 51 were included in the per-protocol analysis. While the amplitude of the 24-hour rhythm in core body temperature did not differ significantly between the cyclic daytime and continuous feeding groups (0.17 [interquartile range: 0.09-0.24] vs. 0.20 [0.13-0.30], p=0.182), the 24-hour rhythm in heart rate was enhanced in patients receiving cyclic daytime feeding, as reflected by significantly higher amplitudes and more synchronized peak times. No significant differences in 24-hour rhythmicity were observed between groups for the other vital signs or melatonin. Conclusions: Our findings suggest that cyclic daytime feeding may strengthen circadian rhythms in critically ill patients. Further studies are warranted to evaluate its impact on clinical outcomes.

Globally, hypercholesterolemia affects over 20% of the population; and while many studies have examined its impact on cardiovascular health, little is known about its effects on the lymphatic system. In mice, hypercholesterolemia has been linked to multiple aspects of lymphatic dysfunction; and a recent study demonstrated that cholesterol depletion by cyclodextrins promoted lymphatic vessel regeneration and restored lymphatic drainage in mouse models of lymphedema. Collecting lymphatic vessels rely on the spontaneous and highly entrained contractions of lymphatic muscle cells (LMCs) and competent unidirectional on-way valves to propel lymph forward. Critical to lymphatic pacemaking and contractility is the proper functioning of ion channels, which are known to be modulated by the cholesterol content in the plasma membrane. Therefore, we sought to understand the role cholesterol plays in regulating lymphatic contractility. The effects of cholesterol depletion by the cyclodextrins M{beta}CD and HP{beta}CD were assessed in cannulated and pressurized inguinal-axillary collecting lymphatic vessels (CLVs) from C57BL6/J (WT) mice. Noteworthy, studies have shown that HP{beta}CD is safe for human use, and in fact, it is commonly used as a drug excipient. Acute treatment with both cyclodextrins significantly increased the pumping capacity of CLVs, as demonstrated by the increased contraction amplitudes by [~]50{+/-}12% and calculated fluid volume displacement by each contraction by [~]35{+/-}11%. Calcium imaging demonstrated that HP{beta}CD increased the amplitude and duration of the large Cav1.2-mediated calcium events (termed calcium flashes. In contrast, cholesterol supplementation by incubation with BODIPY-cholesterol, which presumably incorporates cholesterol into the cell membrane, significantly impaired the contractile activity of CLVs compared to controls by decreasing contraction amplitude (control: 42{+/-}2 {micro}m versus BODIPY-cholesterol: 20{+/-}7{micro}m) and calculated fluid volume displacement (control: 9.2{+/-}3.9nL versus BODIPY cholesterol: 3.3{+/-}1.2nL) which were significantly restored with subsequent cholesterol depletion using HP{beta}CD (amplitude: 36{+/-}11{micro}m, volume displacement: 5.5{+/-}2.4nL). Similarly, treatment with HP{beta}CD significantly improved the contractile capacity of dysfunctional CLVs isolated from hypercholesterolemic ApoEKO mice. In conclusion, changes to cell membrane cholesterol content acutely and significantly altered CLV contractility with depletion improving contractility associated with recruitment of voltage-gated Cav1.2 channels in lymphatic muscle cells (LMCs). Future studies from our lab will determine whether pharmacological depletion of membrane cholesterol can be therapeutic strategy to improve and/or restore lymphatic contractile function in secondary lymphedema, including obesity/hypercholesterolemia-induced and cancer-related lymphedemas.

BackgroundHypertension, the persistent elevation of blood pressure (BP), is characterized by chronic low-grade inflammation and systemic cytokine release. Circulating cytokines contribute to the development of hypertension and end-organ damage. However, the specific immune profile associated with the progression of hypertension remains unclear. We hypothesize that a plasma cytokine signature reflects early BP changes in older adults. MethodsSeventy participants aged 57-81 years were categorized as normotensive (n = 17), elevated BP (n = 10), or hypertensive (n = 43) based on 24-hour ambulatory BP monitoring and antihypertensive treatment status. Plasma IL-1{beta}, IL-6, IL-10, IL-17A, IL-21, IL-22, IL-23, and TNF- were quantified using immunoassays. Partial Pearson correlations adjusted for demographic and biochemical covariates were used to assess associations between cytokines, BP, and cytokine-cytokine networks. ResultsIn untreated hypertensive individuals, plasma IL-23 was positively correlated with 24-hour diastolic BP. Antihypertensive treatment was associated with reduced IL-17A concentrations, which are negatively associated with 24-hour systolic BP. In the elevated BP group, IL-21 concentrations were higher than in normotensive individuals. To further characterize the cytokine signature, cytokine-cytokine correlations were examined. IL-23 and IL-17A were positively correlated with most interleukins, whereas TNF- showed few associations. IL-1{beta} exhibited strong correlations with both IL-23 and IL-17A, particularly in untreated participants. ConclusionIL-23 and IL-17A are associated with BP status and are broadly interconnected with other inflammatory cytokines, highlighting the potential importance of the IL-23/IL-17A axis in the hypertension of development. Early alterations in IL-21 in elevated BP may reflect immune changes that precede the onset of hypertension.

Background: Prone cardiopulmonary resuscitation (CPR) may be necessary when turning a prone patient supine would delay chest compressions. Although prone compressions can generate arterial pressures comparable with or greater than supine CPR, the pathway of pressure transmission is uncertain. We examined synchronized intrathoracic, intra-abdominal, and central arterial pressures in both supine and prone positions. Methods: Two thawed fresh-frozen adult cadavers underwent three, 2-minute mechanical CPR trials per position in a counterbalanced crossover sequence. Solid-state catheters recorded pleural, peritoneal, and central arterial pressures simultaneously. Trial-level outcomes included peak pressure, mean pressure, pressure-time area, and the mean peritoneal-to-pleural pressure gradient. Exploratory fixed-effects models included position, cadaver, and their interaction. Results: Prone CPR increased peak intrathoracic pressure by 7.04 mmHg, peak intra-abdominal pressure by 21.69 mmHg, and peak arterial pressure by 15.40 mmHg. Mean intra-abdominal and arterial pressures increased by 16.22 and 9.90 mmHg, respectively. The mean peritoneal-to-pleural gradient reversed direction from -8.46 mmHg supine to 4.85 mmHg prone. Intrathoracic pressure-time area increased 3.4-fold, from 1.62 to 5.46 mmHg{middle dot}s, and arterial pressure-time area increased 2.2-fold, from 2.96 to 6.42 mmHg{middle dot}s. Conclusions: Compared to supine, prone mechanical CPR generated higher arterial pressures and reversed the pressure relationship across the thoracoabdominal boundary in both cadavers. Higher abdominal pressure coincided with a larger intrathoracic pressure-time area, a pattern compatible with reduced caudal pressure dissipation.

Background: Loss- and gain-of-function variants in SCN5A, encoding the cardiac sodium channel Nav1.5, are major causes of inherited cardiac channelopathies. Their pathophysiology has almost exclusively been attributed to altered cardiomyocyte excitability and cardiac conduction. However, growing evidence indicates that autonomic dysfunction contributes to these disorders, raising the possibility that Nav1.5 dysfunction may also affect the intrinsic cardiac nervous system. Objectives: We investigated whether Nav1.5 is functionally expressed in murine intrinsic cardiac neurons and evaluated its contribution to neuronal excitability. Methods: Scn5a expression was investigated by digital PCR and immunocytochemistry in isolated murine intracardiac neurons. Whole-cell patch-clamp recordings were used to characterize sodium currents and action potential properties in wild-type and Scn5a+/- mice. Results: Scn5a transcripts and Nav1.5 protein were detected in intrinsic cardiac neurons. Voltage-clamp recordings identified a tetrodotoxin-resistant sodium current accounting for approximately 8% of the total sodium current and displaying pharmacological and biophysical properties consistent with Nav1.5. Activation and inactivation analyses indicated that Nav1.5 is the predominant sodium conductance available at most polarized membrane potentials. Accordingly, Scn5a haploinsufficiency selectively altered action potential threshold, maximal upstroke velocity, overshoot and duration in neurons resting below -60 mV, whereas more depolarized neurons were essentially unaffected. No structural or neurochemical remodeling of intrinsic cardiac ganglia was detected. Conclusion: Nav1.5 is a functional determinant of intrinsic cardiac neuronal excitability. These findings identify the intrinsic cardiac nervous system as a new cellular target of SCN5A dysfunction and suggest that inherited cardiac sodium channelopathies could involve alterations in both myocardial and intrinsic neuronal electrophysiology.

Tear fluid is amenable to non-invasive and repeated collection, making it a practical specimen for evaluating exercise-related physiological responses. However, the immediate proteome-wide alterations in tear fluid following acute exercise have not been characterised. In this study, we performed quantitative proteomic profiling of paired tear samples from healthy female participants before and immediately after a single exercise session using data-independent acquisition liquid chromatography-tandem mass spectrometry (DIA-LC-MS/MS). Among the 3,173 identified proteins, 744 were significantly altered post-exercise, of which 484 were up-regulated and 260 down-regulated. Functional enrichment analysis revealed that up-regulated proteins were predominantly associated with translation and ribosome biogenesis, whereas down-regulated proteins were involved in glycan metabolism, lysosomal processing, and extracellular matrix organisation. Collectively, these findings indicate that acute exercise elicits a rapid and coordinated reconfiguration of the tear proteome. This investigation provides a molecular basis for understanding exercise-mediated modulation of tear composition and ocular surface homeostasis.

Environmental temperature strongly influences muscle contractile mechanics and locomotor performance in ectotherms, yet animals routinely develop across a range of temperatures while maintaining effective movement. We tested the hypothesis that developmental temperature induces compensatory changes in the intrinsic mechanical properties of the muscles that power the fast-start escape response in larval zebrafish (Danio rerio). Larvae were reared at 25{degrees}C, 28{degrees}C, or 32{degrees}C, and contractile properties of intact tail myotomal muscles were measured across experimental temperatures. Acute changes in experimental temperature strongly affected twitch kinetics, particularly relaxation rate (Q10 = 2.1), resulting in substantial changes in twitch duration. In contrast, rearing temperature produced adaptive changes that opposed these acute thermal effects. At a common experimental temperature, muscles from cold-reared larvae exhibited faster intrinsic relaxation and greater force production during shortening at a physiologically relevant velocity, whereas warm-reared larvae showed slower relaxation and reduced shortening force. As a result, twitch kinetics were largely normalized when measurements were made at each group's rearing temperature, reducing the apparent thermal sensitivity of relaxation rate (Q10 = 1.1). To identify molecular correlates of these functional adaptations, we performed label-free quantitative LCMS proteomic analysis. Cold rearing increased the abundance of Sarco/Endoplasmic Reticulum Calcium-ATPase (SERCA) proteins, driven primarily by elevated atp2a1 expression, while warm rearing reduced the abundance of the major parvalbumin isoforms pvalb1 and pvalb2. These changes implicate remodeling of intracellular calcium handling as a mechanism underlying thermal compensation of muscle function. Together, our results demonstrate that developmental temperature modifies the intrinsic mechanical properties of larval zebrafish muscle in ways that counteract the direct effects of environmental temperature, thereby preserving the timing and power-generating capacity required for fast-start escape performance.

ObjectivesBycatch-related decompression after forced submersion can result in severe gas embolic disease in sea turtles. This work used qualitative and quantitative ultrasound analyses, including gas grading, brightness analysis, and texture feature extraction, to investigate organ-specific gas burden and temporal evolution in the hearts, kidneys, and livers of bycaught sea turtles. Materials and MethodsUltrasound imaging of the hearts, kidneys, and livers of bycaught turtles was performed as part of veterinary evaluation either onboard fishing vessels immediately after surfacing (boat group, n=47) or after longer periods at shore-based facilities (shore group, n=30). Gas burden in each ultrasound scan was graded on an ordinal scale from 0 (no gas) to 5 (gas completely shadowing organ anatomy). Temporal differences in gas burden were compared between the shore and boat groups. Quantitative brightness and texture features were extracted from all organs, including contrast, correlation, homogeneity, and energy from liver and kidney data. A multivariate logistic regression model with leave-one-out cross-validation was conducted, with shore versus boat as a binary outcome (surrogate of post-surfacing decompression state) and ultrasound texture metrics as independent variables. ResultsMedian grades from the first ultrasound scan were significantly higher in the boat group than in the shore group for the liver, kidney, and heart (3, 3, and 3 vs 1, 1, and 0, respectively). This pattern coincided with a difference in the mean duration until the first scan which was conducted being 54 min for the onboard studies vs 330 minutes in the shore group. Mean pixel brightness within cardiac and liver regions of interest increased with rising bubble grade before decreasing at the highest grades, consistent with acoustic shadowing at severe gas burden. Texture features demonstrated significant organ-specific changes with increasing gas burden, and the regression models achieved areas under the receiver operating characteristic curve of 0.92 for liver texture features and 0.83 for kidney texture features. ConclusionsThese findings demonstrate organ-specific differences in gas evolution over time. Quantitative ultrasound features were associated with gas burden and post-surfacing interval in bycaught sea turtles. These findings support the feasibility of quantitative ultrasound biomarkers for assessment of decompression-related gas burden.

Background: The placenta has a unique transcriptomic profile, including microRNAs that are secreted into maternal circulation throughout pregnancy. MicroRNAs are small, non-coding RNA that post-transcriptionally regulate gene expression. Spontaneous preterm birth (sPTB) is associated with substantial differences in both placental pathophysiology and placental gene expression compared to term birth. We aimed to generate microRNA signatures of sPTB and map them to target genes using a microRNA-mRNA network. Methods: This study was conducted within the Conditions Affecting Neurocognitive Development and Learning in Early childhood (CANDLE) study. Placental samples were collected at delivery, and RNA was isolated for mRNA and microRNA sequencing. To investigate sPTB, this study excluded placental samples of participants with iatrogenic indications for PTB or induced labor. We examined differences in microRNA expression in participants who delivered before 37 weeks (N=35) compared to term participants (N=404) in a series of covariate-adjusted linear regression models. We used paired placental microRNA and mRNA expression data from this cohort to validate associations between computationally predicted microRNA-mRNA pairs and establish a microRNA-mRNA network. Results: Expression of 7 microRNAs were increased in sPTB (FDR<0.05) and were inversely correlated with sPTB-associated genes involved in immune signaling. Expression of 12 microRNAs were decreased in sPTB, including 4 members of the maternally expressed chromosome 14 microRNA cluster (miR-376a-3p, miR-376c-3p, miR-377-3p, and miR-381-3p). These microRNAs were predicted to negatively regulate oxidative phosphorylation genes that were increased in sPTB. The associations between miR-376c-3p and miR-377-3p and oxidative phosphorylation were confirmed in microRNA knockdown experiments. Conclusions: This study highlights potential biological mechanisms by which placental microRNA dysfunction might contribute to sPTB and highlights putative sPTB biomarkers that may be detectable in maternal circulation.

Background: Venous thromboembolism (VTE), which encompasses deep vein thrombosis (DVT) and pulmonary embolism (PE), is a frequent disease associated with thrombus formation and vein wall remodeling. Hence, fibrosis might result from endothelial-to-mesenchymal transition (EndMT), characterized by the loss of endothelial markers and the acquisition of mesenchymal markers. In chronic thromboembolic pulmonary hypertension, transforming growth factor (TGF{beta}), the most potent inducer of EndMT, impairs thrombus resolution. However, the molecular mechanisms implicated in TGF[beta] signaling in the context of VTE are unknown. We hypothesized that epigenetic processes regulate the TGF{beta} signaling pathway in endothelial cells promoting EndMT and vascular fibrosis. Aims: To determine if the histone deacetylase 6 (HDAC6) regulates the TGF{beta} signaling pathway in endothelial cells promoting EndMT and delays venous thrombosis. Methods: To study the role of HDAC6 in EndMT, endothelial cells were treated with a pharmacological inhibitor (TCS20b) and incubated with TGF{beta} and thrombin for 2, 3, and 5 days. Real time PCR and Western blot were performed to analyze endothelial and mesenchymal marker expression and TGF{beta} signaling. An experimental model of VTE was used to study the role of HDAC6 on thrombus size overtime. Animals were treated or not with a specific HDAC6 inhibitor (tubastatin A) for 7 to 21 days. Analysis of RNAseq data sets publicly available were used to confirm our main results. Within group and treatment differences were analyzed using two-way ANOVA and Tukeys multiple comparisons. Results: Expression of the mesenchymal markers, calponin and transgelin, was increased by TGF{beta} and thrombin. Interestingly these changes were inhibited in presence of TCS20b. TGF{beta} mediated these effects through ERK1/2 and HDAC6 activation. Inhibition of HDAC6 in vivo reduced thrombus size 7 days after surgery compared to controls. This was associated with reduced expression of the EndMT marker transgelin in endothelial cells compared to the control animals. We found that FN1-EDA expression was associated with EndMT and regulated by HDAC6 in vitro. This marker was also associated with thrombosis in the RNAseq data set that we analyzed and potentially in patients with recurrent DVT. Conclusion: We found that HDAC6 regulates EndMT in venous thrombosis and impairs thrombus resolution. HDAC6 also regulates expression FN1-EDA that appears to be a strong marker associated with DVT and DVT recurrence. Thus, HDAC6 might represent an attractive therapeutic target for patients with a high risk of recurrent VTE.