OpenSAFELY: The impact of COVID-19 on azathioprine, leflunomide and methotrexate monitoring, and factors associated with change in monitoring rate.

AIMS: The COVID-19 pandemic created unprecedented pressure on healthcare services. This study investigates whether disease-modifying antirheumatic drug (DMARD) safety monitoring was affected during the COVID-19 pandemic. METHODS: A population-based cohort study was conducted using the OpenSAFELY platform to access electronic health record data from 24.2 million patients registered at general practices using TPP's SystmOne software. Patients were included for further analysis if prescribed azathioprine, leflunomide or methotrexate between November 2019 and July 2022. Outcomes were assessed as monthly trends and variation between various sociodemographic and clinical groups for adherence with standard safety monitoring recommendations. RESULTS: An acute increase in the rate of missed monitoring occurred across the study population (+12.4 percentage points) when lockdown measures were implemented in March 2020. This increase was more pronounced for some patient groups (70-79 year-olds: +13.7 percentage points; females: +12.8 percentage points), regions (North West: +17.0 percentage points), medications (leflunomide: +20.7 percentage points) and monitoring tests (blood pressure: +24.5 percentage points). Missed monitoring rates decreased substantially for all groups by July 2022. Consistent differences were observed in overall missed monitoring rates between several groups throughout the study. CONCLUSION: DMARD monitoring rates temporarily deteriorated during the COVID-19 pandemic. Deterioration coincided with the onset of lockdown measures, with monitoring rates recovering rapidly as lockdown measures were eased. Differences observed in monitoring rates between medications, tests, regions and patient groups highlight opportunities to tackle potential inequalities in the provision or uptake of monitoring services. Further research should evaluate the causes of the differences identified between groups.

Specific dynamic action: the energy cost of digestion or growth?

The physiological processes underlying the post-prandial rise in metabolic rate, most commonly known as the 'specific dynamic action' (SDA), remain debated and controversial. This Commentary examines the SDA response from two opposing hypotheses: (i) the classic interpretation, where the SDA represents the energy cost of digestion, versus (ii) the alternative view that much of the SDA represents the energy cost of growth. The traditional viewpoint implies that individuals with a reduced SDA should grow faster given the same caloric intake, but experimental evidence for this effect remains scarce and inconclusive. Alternatively, we suggest that the SDA reflects an organism's efficacy in allocating the ingested food to growth, emphasising the role of post-absorptive processes, particularly protein synthesis. Although both viewpoints recognise the trade-offs in energy allocation and the dynamic nature of energy distribution among physiological processes, we argue that equating the SDA with 'the energy cost of digestion' oversimplifies the complexities of energy use in relation to the SDA and growth. In many instances, a reduced SDA may reflect diminished nutrient absorption (e.g. due to lower digestive efficiency) rather than increased 'free' energy available for somatic growth. Considering these perspectives, we summarise evidence both for and against the opposing hypotheses with a focus on ectothermic vertebrates. We conclude by presenting a number of future directions for experiments that may clarify what the SDA is, and what it is not.

Influence of a single locking throw on the in vitro holding security of five friction throws.

OBJECTIVE: To compare the in vitro security of four different friction throws (square, surgeon's, Miller's, and strangle), with and without a locking throw, and a slip knot, on a vascular ligation model. STUDY DESIGN: Experimental study. SAMPLE POPULATION: Nine groups with nine samples per group. METHODS: Four throws (square, surgeon's, Miller's, and strangle), with and without a locking throw, and a square throw locked in slip knot fashion, were tested to measure leakage pressure. RESULTS: The square and surgeon's throws leaked at a lower pressure than the Miller's and strangle throws (p < .0001). The leakage pressure was below physiologic arterial pressure for the square and the surgeon's throws. After the addition of a locking throw, the five knots leaked at a similar pressure (p = .5233) above physiologic arterial pressure. CONCLUSION: Following the addition of a locking throw, all the constructs tested in this study leaked at a similar pressure. The leakage pressure for all knots exceeded physiologic arterial pressures. CLINICAL SIGNIFICANCE: Any throw tested may be appropriate for secure vascular ligation if the initial throw is applied correctly.

The biotic ligand model as a promising tool to predict Cu toxicity in amazon blackwaters.

The Rio Negro basin of Amazonia (Brazil) is a hotspot of fish biodiversity that is under threat from copper (Cu) pollution. The very ion-poor blackwaters have a high dissolved organic carbon (DOC) concentration. We investigated the Cu sensitivity of nine Amazonian fish species in their natural blackwaters (Rio Negro). The acute lethal concentration of Cu (96 h LC50) was determined at different dilutions of Rio Negro water (RNW) in ion-poor well water (IPW), ranging from 0 to 100%. The IPW was similar to RNW in pH and ionic composition but deficient in DOC, allowing this parameter to vary 20-fold from 0.4 to 8.3 mg/L in tests. The Biotic Ligand Model (BLM; Windward version 3.41.2.45) was used to model Cu speciation and toxicity over the range of tested water compositions, and to estimate lethal Cu accumulations on the gills (LA50). The modeling predicted a high relative abundance of Cu complexes with DOC in test waters. As these complexes became more abundant with increasing RNW content, a concomitant decrease in free Cu2+ was observed. In agreement with this modeling, acute Cu toxicity decreased (i.e. 96 h LC50 values increase) with increasing RNW content. The three most sensitive species (Hemigrammus rhodostomus, Carnegiella strigatta and Hyphessobrycon socolofi) were Characiformes, whereas Corydoras schwartzi (Siluriformes) and Apistogramma agassizii (Cichliformes) were the most tolerant. These sensitivity differences were reflected in the BLM-predicted lethal gill copper accumulation (LA50), which were generally lower in Characiformes than in Cichliformes. Using these newly estimated LA50 values in the BLM allowed for accurate prediction of acute Cu toxicity in the nine Amazonian fish. Our data emphasize that the BLM approach is a promising tool for assessing Cu risk to Amazonian fish species in blackwater conditions characterized by very low concentrations of major ions but high concentrations of DOC.

Evaluation of stable chest pain following emergency department presentation: Impact of first-line cardiac computed tomography diagnostic strategy in an Australian setting.

OBJECTIVE: International guidelines provide increasing support for computed tomography coronary angiography (CTCA) in investigating chest pain. A pathway utilising CTCA first-line for outpatient stable chest pain evaluation was implemented in an Australian ED. METHODS: In pre-post design, the impact of the pathway was prospectively assessed over 6 months (August 2021 to January 2022) and compared with a 6-month pre-implementation group (February 2021 to July 2021). CTCA was recommended first-line in suspected stable cardiac chest pain, followed by chest pain clinic review. Predefined criteria were provided recommending functional testing in select patients. The impact of CTCA versus functional testing was evaluated. Data were obtained from digital medical records. RESULTS: Three hundred and fifteen patients were included, 143 pre-implementation and 172 post-implementation. Characteristics were similar except age (pre-implementation: 58.9 ± 12.0 vs post-implementation: 62.8 ± 12.3 years, P = 0.004). Pathway-guided management resulted in higher first-line CTCA (73.3% vs 46.2%, P < 0.001), lower functional testing (30.2% vs 56.6%, P < 0.001) and lower proportion undergoing two non-invasive tests (4.7% vs 10.5%, P = 0.047), without increasing investigation costs or invasive coronary angiography (ICA) (pre-implementation: 13.3% vs post-implementation: 9.3%, P = 0.263). In patients undergoing CTCA, 40.7% had normal coronaries and 36.2% minimal/mild disease, with no difference in disease burden post-implementation. More medication changes occurred following CTCA compared with functional testing (aspirin: P = 0.005, statin: P < 0.001). In patients undergoing ICA, revascularisation to ICA ratio was higher following CTCA compared with functional testing (91.7% vs 18.2%, P < 0.001). No 30-day myocardial infarction or death occurred. CONCLUSIONS: The pathway increased CTCA utilisation and reduced downstream investigations. CTCA was associated with medication changes and improved ICA efficiency.

The first direct measurements of ventilatory flow and oxygen utilization after exhaustive exercise and voluntary feeding in a teleost fish, Oncorhynchus mykiss.

A new "less invasive" device incorporating an ultrasonic flow probe and a divided chamber, but no stitching of membranes to the fish, was employed to make the first direct measurements of ventilatory flow rate (V̇w) and % O2 utilization (%U) in juvenile rainbow trout (37 g, 8ºC) after exhaustive exercise (10-min chasing) and voluntary feeding (2.72% body mass ration). Under resting conditions, the allometrically scaled V̇w (300 ml kg-1 min-1 for a 37-g trout = 147 ml kg-1 min-1 for a 236-g trout exhibiting the same mass-specific O2 consumption rate, MO2) and the convection requirement for O2 (CR = 4.13 L mmol-1) were considerably lower, and the %U (67%) was considerably higher than in previous studies using surgically attached masks or the Fick principle. After exhaustive exercise, V̇w and MO2 approximately doubled whereas frequency (fr) and %U barely changed, so increased ventilatory stroke volume (Vsv) was the most important contributor to increased MO2. CR declined slightly. Values gradually returned to control conditions after 2-3 h. After voluntary feeding, short-term increases in V̇w, Vsv and MO2 were comparable to those after exercise, and fr again did not change. However, %U increased so CR declined even more. The initial peaks in V̇w, Vsv and MO2, similar to those after exercise, were likely influenced by the excitement and exercise component of voluntary feeding. However, in contrast to post-exercise fish, post-prandial fish exhibited second peaks in these same parameters at 1-3 h after feeding, and %U increased further, surpassing 85%, reflecting the true "specific dynamic action" response. We conclude that respiration in trout is much more efficient than previously believed.

The physiological consequences of a very large natural meal in a voracious marine fish, the staghorn sculpin (Leptocottus armatus).

Little information exists on physiological consequences when wild fish eat natural food. Staghorn sculpins at 10-13°C voluntarily consumed 15.8% of their body mass in anchovies. Gastric clearance was slow with >60% of the meal retained in the stomach at 48 h, and was not complete until 84 h. At 14-24 h post-feeding, pH was depressed by 3 units and Cl- concentration was elevated 2-fold in gastric chyme, reflecting HCl secretion, while in all sections of the intestine, pH declined by 1 pH unit but Cl- concentration remained unchanged. PCO2 and total ammonia concentration were greatly elevated throughout the tract, whereas PNH3 and HCO3- concentration were depressed. Intestinal HCO3- secretion rates, measured in gut sacs in vitro, were also lower in fed fish. Whole-animal O2 consumption rate was elevated approximately 2-fold for 72 h post-feeding, reflecting 'specific dynamic action', whereas ammonia and urea-N excretion rates were elevated about 5-fold. Arterial blood exhibited a modest 'alkaline tide' for about 48 h, but there was negligible excretion of metabolic base to the external seawater. PaCO2 and PaO2 remained unchanged. Plasma total amino acid concentration and total lipid concentration were elevated about 1.5-fold for at least 48 h, whereas small increases in plasma total ammonia concentration, PNH3 and urea-N concentration were quickly attenuated. Plasma glucose concentration remained unchanged. We conclude that despite the very large meal, slow processing with high efficiency minimizes internal physiological disturbances. This differs greatly from the picture provided by previous studies on aquacultured species using synthetic diets and/or force-feeding. Questions remain about the role of the gastro-intestinal microbiome in nitrogen and acid-base metabolism.

Osmo-respiratory compromise in the mosshead sculpin (Clinocottus globiceps): effects of temperature, hypoxia, and re-oxygenation on rates of diffusive water flux and oxygen uptake.

In nature, mosshead sculpins (Clinocottus globiceps) are challenged by fluctuations in temperature and oxygen levels in their environment. However, it is unclear how mosshead sculpins modulate the permeability of their branchial epithelia to water and O2 in response to temperature or hypoxia stress. Acute decrease in temperature from 13 to 6 oC reduced diffusive water flux rate by 22% and MO2 by 51%, whereas acute increase in temperature from 13 to 25 oC increased diffusive water flux rate by 217% and MO2 by 140%, yielding overall Q10 values of 2.08 and 2.47 respectively. Acute reductions in oxygen tension from >95% to 20% or 10% air saturation did not impact diffusive water flux rates, however, MO2 was reduced significantly by 36% and 65% respectively. During 1-h or 3-h recovery periods diffusive water flux rates were depressed while MO2 exhibited overshoots beyond the normoxic control level. Many responses differed from those seen in our parallel earlier study on the tidepool sculpin, a cottid with similar hypoxia tolerance but much smaller gill area that occupies a similar environment. Overall, our data suggest that during temperature stress, diffusive water flux rates and MO2 follow the traditional osmo-respiratory compromise pattern, but during hypoxia and re-oxygenation stress, diffusive water flux rates are decoupled from MO2.

Role of the kidneys in acid-base regulation and ammonia excretion in freshwater and seawater fish: implications for nephrocalcinosis.

Maintaining normal pH levels in the body fluids is essential for homeostasis and represents one of the most tightly regulated physiological processes among vertebrates. Fish are generally ammoniotelic and inhabit diverse aquatic environments that present many respiratory, acidifying, alkalinizing, ionic and osmotic stressors to which they are able to adapt. They have evolved flexible strategies for the regulation of acid-base equivalents (H+, NH4 +, OH- and HCO3 -), ammonia and phosphate to cope with these stressors. The gills are the main regulatory organ, while the kidneys play an important, often overlooked accessory role in acid-base regulation. Here we outline the kidneys role in regulation of acid-base equivalents and two of the key 'urinary buffers', ammonia and phosphate, by integrating known aspects of renal physiology with recent advances in the molecular and cellular physiology of membrane transport systems in the teleost kidneys. The renal transporters (NHE3, NBC1, AE1, SLC26A6) and enzymes (V-type H+ATPase, CAc, CA IV, ammoniagenic enzymes) involved in H+ secretion, bicarbonate reabsorption, and the net excretion of acidic and basic equivalents, ammonia, and inorganic phosphate are addressed. The role of sodium-phosphate cotransporter (Slc34a2b) and rhesus (Rh) glycoproteins (ammonia channels) in conjunction with apical V-type H+ ATPase and NHE3 exchangers in these processes are also explored. Nephrocalcinosis is an inflammation-like disorder due to the precipitation of calcareous material in the kidneys, and is listed as one of the most prevalent pathologies in land-based production of salmonids in recirculating aquaculture systems. The causative links underlying the pathogenesis and etiology of nephrocalcinosis in teleosts is speculative at best, but acid-base perturbation is probably a central pathophysiological cause. Relevant risk factors associated with nephrocalcinosis are hypercapnia and hyperoxia in the culture water. These raise internal CO2 levels in the fish, triggering complex branchial and renal acid-base compensations which may promote formation of kidney stones. However, increased salt loads through the rearing water and the feed may increase the prevalence of nephrocalcinosis. An increased understanding of the kidneys role in acid-base and ion regulation and how this relates to renal diseases such as nephrocalcinosis will have applied relevance for the biologist and aquaculturist alike.

Comparison of coronary artery calcium score and cardiovascular risk-stratification by European Society of Cardiology Guidelines and Steno Type 1 Risk Engine in statin-naïve adults with type 1 diabetes.

BACKGROUND: Coronary artery calcium (CAC) is a marker of atherosclerotic cardiovascular disease (CVD). However, for patients with type 1 diabetes (T1D), its relationship with T1D-specific cardiovascular (CV) risk-stratification tools is unknown. AIMS: Assess prevalence of CAC and evaluate relationship between CAC and T1D-specific CV risk-stratification methods in T1D. METHODS: Cross-sectional study of adults with T1D age 20-60 years, statin-naïve and no history of CVD. Data was obtained from electronic medical records and by interview. Presence of CAC was assessed using non-contrast cardiac computed tomography and quantified by Agatston Units (AU). CV risk-stratification was assessed using the 2019 European Society of Cardiology (ESC) Guidelines and the Steno T1 Risk Engine (ST1RE). RESULTS: 85 patients were included with mean age 35.4 ± 10.3 years, HbA1c 8.3 ± 1.5 % and T1D duration 17.0 ± 10.1 years. 67 patients (78.9 %) had a CAC score of 0 AU, 17 (20.0 %) >0-100 AU, and one (1.2 %) >100 AU. Duration of T1D (p = 0.009), body mass index (p = 0.029), neuropathy (p = 0.016) and low-density lipoprotein cholesterol levels (p = 0.016) were independently associated with a positive CAC score on multivariate analysis. Positive predictive value for a positive CAC score was 85.7 % for the ST1RE high risk category and 31.3 % for the 2019 ESC Guidelines very high risk category. CONCLUSIONS: One-fifth of this T1D cohort had a positive CAC score. The ST1RE was superior in identifying positive CAC compared to the 2019 ESC Guidelines. Further studies are required to elucidate the role of CAC in personalising CV risk-stratification and statin initiation in T1D.

Emersion and recovery alter oxygen consumption, ammonia and urea excretion, and oxidative stress parameters, but not diffusive water exchange or transepithelial potential in the green crab (Carcinus maenas).

The green crab (Carcinus maenas) is an inshore species affected by intertidal zonation patterns, facing periods of emersion during low tide and submersion during high tide. During these periods of air and subsequent water exposure, these species can face physiological challenges. We examined changes in O2 consumption rate (MO2), and ammonia and urea excretion rates over sequential 14 h periods in seawater (32 ppt, control), in air and during recovery in seawater after air exposure (13°C throughout). At the end of each exposure, the anterior (5th) and posterior (8th) gills and the hepatopancreas were removed for measurements of oxidative stress parameters (TBARs and catalase in the gills and hepatopancreas, and protein carbonyls in the gills). MO2 remained unchanged during air exposure, but increased greatly (3.4-fold above control levels) during the recovery period. Ammonia and urea net fluxes were reduced by 98% during air exposure, but rebounded during recovery to >2-fold the control rates. Exchangeable water pools, rate constants of diffusive water exchange, unidirectional diffusive water flux rates (using tritiated water) and transepithelial potential were also measured during control and recovery treatments, but exhibited no significant changes. Damage to proteins was not observed in either gill. However, lipid damage occurred in the anterior (respiratory) gill after the air exposure but not in the posterior (ionoregulatory) gill or hepatopancreas. Catalase activity also decreased significantly in recovery relative to levels during air exposure in both the anterior gill and hepatopancreas, but not in the posterior gill. The crabs did not modify water metabolism or permeability. We conclude that MO2 was maintained but not enhanced during air exposure, while ammonia and urea-N excretion were impaired. As a result, all of these parameters increase greatly during re-immersion recovery, and oxidative stress also occurs. Clearly, emersion is not without physiological costs.

Do extreme postprandial levels of oxygen, carbon dioxide, and ammonia in the digestive tract equilibrate with the bloodstream in the freshwater rainbow trout (Oncorhynchus mykiss)?

The gastrointestinal tract (GIT) lumen of teleosts harbors extreme conditions, especially after feeding: high PCO2 (20-115 Torr), total ammonia (415-3710 µM), PNH3 (79-1760 µTorr in the intestine), and virtual anoxia (PO2 < 1 Torr). These levels could be dangerous if they were to equilibrate with the bloodstream. Thus, we investigated the potential equilibration of O2, CO2, and ammonia across the GIT epithelia in freshwater rainbow trout by monitoring postprandial arterial and venous blood gases in vivo and in situ. In vivo blood was sampled from the indwelling catheters in the dorsal aorta (DA) and subintestinal vein (SIV) draining the posterior intestine in the fasting state and at 4 to 48 h following catheter-feeding. To investigate possible ammonia absorption in the anterior part of the GIT, blood was sampled from the DA, SIV and hepatic portal vein (HPV) from anaesthetized fish in situ following voluntary feeding. We found minimal equilibration of all three gases between the GIT lumen and the SIV blood, with the latter maintaining pre-feeding levels (PO2 = 25-49 Torr, PCO2 = 6-8 Torr, and total ammonia = 117-134 µM and PNH3 = 13-30 µTorr at 48 h post-feeding). In contrast to the SIV, we found that the HPV total ammonia more than doubled 24 h after feeding (128 to 297 µM), indicative of absorption in the anterior GIT. Overall, the GIT epithelia of trout, although specialized for absorption, prevent dangerous levels of PO2, PCO2 and ammonia from equilibrating with the blood circulation.

Exercise and emersion in air and recovery in seawater in the green crab (Carcinus maenas): Effects on nitrogenous wastes and branchial chamber fluid chemistry.

At low tide, the green crab, which is capable of breathing air, may leave the water and walk on the foreshore, carrying branchial chamber fluid (BCF). N-waste metabolism was examined in crabs at rest in seawater (32 ppt, 13°C), and during 18-h recovery in seawater after 1 h of exhaustive exercise (0.25 BL s-1 ) on a treadmill in air (20°C-23°C), or 1 h of quiet emersion in air. Measurements were made in parallel to O2 consumption (MO2 ), acid-base, cardio-respiratory, and ion data reported previously. At rest, the ammonia-N excretion rate (MAmm = 44 µmol-N kg-1 h-1 ) and ammonia quotient (AQ; MAmm /MO2 = 0.088) were low for a carnivore. Immediately after exercise and return to seawater, MAmm increased by 65-fold above control rates. After emersion alone and return to seawater, MAmm increased by 17-fold. These ammonia-N bursts were greater, but transient relative to longer-lasting elevations in MO2 , resulting in temporal disturbances of AQ. Intermittent excretion of urea-N and urate-N at rest and during recovery indicated the metabolic importance of these N-wastes. Hemolymph glutamate, glutamine, and PNH3 did not change. Hemolymph ammonia-N, urea-N, and urate-N concentrations increased after exercise and more moderately after emersion, with urate-N exhibiting the largest absolute increments, and urea-N the longest-lasting elevations. All three N-wastes were present in the BCF, with ammonia-N and PNH3 far above hemolymph levels even at rest. BCF volume declined by 34% postemersion and 77% postexercise, with little change in osmolality but large increases in ammonia-N concentrations. Neither rapid flushing of stored BCF nor clearance of hemolymph ammonia-N could explain the surges in MAmm after return to seawater.

Ammonia transport is independent of PNH3 gradients across the gastrointestinal epithelia of the rainbow trout: A role for the stomach.

Although the gastrointestinal tract (GIT) is an important site for nitrogen metabolism in teleosts, the mechanisms of ammonia absorption and transport remain to be elucidated. Both protein catabolism in the lumen and the metabolism of the GIT tissues produce ammonia which, in part, enters the portal blood through the anterior region of the GIT. The present study examined the possible roles of different GIT sections of rainbow trout (Oncorhynchus mykiss) in transporting ammonia in its unionized gas form-NH3 -by changing the PNH3 gradient across GIT epithelia using in vitro gut sac preparations. We also surveyed messenger RNA expression patterns of three of the identified Rh proteins (Rhbg, Rhcg1, and  Rhcg2) as potential NH3 transporters and NKCC as a potential ammonium ion (NH4 + ) transporter along the GIT of rainbow trout. We found that ammonia absorption is not dependent on the PNH3 gradient despite expression of Rhbg and Rhcg2 in the intestinal tissues, and Rhcg2 in the stomach. We detected no expression of Rhbg in the stomach and no expression of Rhcg1 in any GIT tissues. There was also a lack of correlation between ammonia transport and [NH4 + ] gradient despite NKCC expression in all GIT tissues. Regardless of PNH3 gradients, the stomach showed the greatest absorption and net tissue consumption of ammonia. Overall, our findings suggest nitrogen metabolism zonation of GIT, with stomach serving as an important site for the absorption, handling and transport of ammonia that is independent of the PNH3 gradient.

Lessons learnt from the global iodinated contrast media shortage in head and neck imaging.

A recent shortage in the global supply of iodinated contrast media (ICM) has required health service providers to review their contrast administration policies and implement strategies to conserve inventory. This article will review the current best practices in head and neck imaging for a variety of common presentations and provide examples where alternative imaging can be considered due to the recent ICM shortage. Ultrasound and MRI techniques can feature heavily in many diagnostic processes in head and neck pathology, and a variety of common presentations can be appropriately investigated through clinical evaluation or naso-endoscopy. In many instances, for the routine assessment of non-acute adult and paediatric head and neck presentations, the use of contrast-enhanced CT can be safely minimised to conserve ICM if required.

How the green crab Carcinus maenas copes physiologically with a range of salinities.

To evaluate the physiological ability to adjust to environmental variations of salinity, Carcinus maenas were maintained in 10, 20, 32 (control), 40, and 50 ppt (13.8 ± 0.6 °C) for 7 days. Closed respirometry systems were used to evaluate oxygen consumption ([Formula: see text]), ammonia excretion (Jamm), urea-N excretion (Jurea-N) and diffusive water fluxes (with 3H2O). Ions, osmolality, metabolites, and acid-base status were determined in the hemolymph and seawater, and transepithelial potential (TEP) was measured. At the lowest salinity, there were marked increases in [Formula: see text] and Jamm, greater reliance on N-containing fuels to support aerobic metabolism, and a state of internal metabolic alkalosis (increased [HCO3-]) despite lower seawater pH. At higher salinities, an activation of anaerobic metabolism and a state of metabolic acidosis (decreased [HCO3-] and increased [lactate]), in combination with respiratory compensation (decreased PCO2), were detected. TEP became more negative with decreasing salinity. Osmoregulation and osmoconformation occurred at low and high salinities, respectively, with complex patterns in individual ions; hemolymph [Mg2+] was particularly well regulated at levels well below the external seawater at all salinities. Diffusive water flux rates increased at higher salinities. Our results show that C. maenas exhibits wide plasticity of physiological responses when acclimated to different salinities and tolerates substantial disturbances of physiological parameters, illustrating that this species is well adapted to invade and survive in diverse habitats.

SETD4 cells contribute to brain development and maintain adult stem cell reservoir for neurogenesis.

Cellular quiescence facilitates maintenance of neural stem cells (NSCs) and their subsequent regenerative functions in response to brain injury and aging. However, the specification and maintenance of NSCs in quiescence from embryo to adulthood remain largely unclear. Here, using Set domain-containing protein 4 (SETD4), an epigenetic determinant of cellular quiescence, we mark a small but long-lived NSC population in deep quiescence in the subventricular zone of adult murine brain. Genetic lineage tracing shows that SETD4+ cells appear before neuroectoderm formation and contribute to brain development. In the adult, conditional knockout of Setd4 resulted in quiescence exit of NSCs, generating newborn neurons in the olfactory bulb and contributing to damage repair. However, long period deletion of SETD4 lead to exhaustion of NSC reservoir or SETD4 overexpression caused quiescence entry of NSCs, leading to suppressed neurogenesis. This study reveals the existence of long-lived deep quiescent NSCs and their neurogenetic capacities beyond activation.

Investigating the mechanisms of dissolved organic matter protection against copper toxicity in fish of Amazon's black waters.

We investigated how natural dissolved organic matter (DOM) of the Rio Negro (Amazon) affects acute copper (Cu) toxicity to local fish: the cardinal tetra (Paracheirodon axelrodi) and the dwarf cichlid (Apistogramma agassizii). It is established that Cu2+ complexation with DOM decreases Cu bioavailability (and thus toxicity) to aquatic organisms, as conceptualized by the Biotic Ligand Model (BLM). However, we also know that Rio Negro's DOM can interact with fish gills and have a beneficial effect on Na+ homeostasis, the main target of acute Cu toxicity in freshwater animals. We aimed to tease apart these potential protective effects of DOM against Cu-induced Na+ imbalances in fish. In the laboratory, we acclimated fish to Rio Negro water (10 mg L-1 DOC) and to a low-DOM water (1.4 mg L-1 DOC) with similar ion composition and pH (5.9). We measured 3-h Cu uptake in gills and unidirectional and net Na+ physiological fluxes across a range of Cu concentrations in both waters. Various DOM pre-acclimation times (0, 1 and 5 days) were evaluated in experiments with P. axelrodi. Copper exposure led to similar levels of net Na+ loss in the two fish, but with distinct effects on Na+ influx and efflux rates reflecting their different ionoregulation strategies. Rio Negro DOM protected against Cu uptake and toxicity in the two fish species. Both Cu uptake in fish gills and Na+ regulation disturbances were relatively well predicted by the modelled aqueous free Cu2+ ion concentration. These findings suggest that protection by DOM occurs mainly from Cu complexation under the tested conditions. The prevalence of this geochemical-type protection over a physiological-type protection agrees with the BLM conceptual framework, supporting the use of the BLM to assess the risk of Cu in these Amazonian waters.

Osmorespiratory compromise in an elasmobranch: oxygen consumption, ventilation and nitrogen metabolism during recovery from exhaustive exercise in dogfish sharks (Squalus suckleyi).

The functional trade-off between respiratory gas exchange versus osmolyte and water balance that occurs at the thin, highly vascularized gills of fishes has been termed the osmorespiratory compromise. Increases in gas exchange capacity for meeting elevated oxygen demands can end up favoring the passive movement of osmolytes and water, potentially causing a disturbance in osmotic balance. This phenomenon has been studied only sparsely in marine elasmobranchs. Our goal was to evaluate the effects of exhaustive exercise (as a modulator of oxygen demand) on oxygen consumption (MO2), branchial losses of nitrogenous products (ammonia and urea-N), diffusive water exchange rates, and gill ventilation (frequency and amplitude), in the Pacific spiny dogfish (Squalus suckleyi). To that end, MO2, osmolyte fluxes, diffusive water exchange rate, and ventilation dynamics were first measured under resting control conditions, then sharks were exercised until exhaustion (20 min), and the same parameters were monitored for the subsequent 4 h of recovery. While MO2 nearly doubled immediately after exercise and remained elevated for 2 h, ventilation dynamics did not change, suggesting that fish were increasing oxygen extraction efficiency at the gills. Diffusive water flux rates (measured over 0-2 h of recovery) were not affected. Ammonia losses were elevated by 7.6-fold immediately after exercise and remained elevated for 3 h into recovery, while urea-N losses were elevated only 1.75-fold and returned to control levels after 1 h. These results are consistent with previous investigations using different challenges (hypoxia, high temperature) and point to a tighter regulation of urea-N conservation mechanisms at the gills, likely due to the use of urea as a prized osmolyte in elasmobranchs. Environmental hyperoxia offered no relief from the osmorespiratory compromise, as there were no effects on any of the parameters measured during recovery from exhaustive exercise.

The roles of calcium and salinity in protecting against physiological symptoms of waterborne zinc toxicity in the euryhaline killifish (Fundulus heteroclitus).

In fresh water, environmental Ca ameliorates Zn toxicity because Ca2+ and Zn2+ compete for uptake at the gills. Zn toxicity is also lower in sea water, but it is unclear whether this is due to increased Ca2+ concentration, and/or to the other ions present at higher salinity. Using the euryhaline killifish, we evaluated the relative roles of Ca2+ (as CaNO3) versus the other ions contributing to salinity in protecting against physiological symptoms of Zn2+ toxicity. Killifish were exposed to a sublethal level of Zn (500 µg/L, as ZnSO4) for 96 h in either fresh water (0 % salinity) at low (1 mmol/L) and High Ca (10 mmol/L) or 35 ppt sea water (100 % salinity) at low (1 mmol/L) and High Ca (10 mmol/L). At 0 % salinity, High Ca partly or completely protected against the following effects of Zn seen at Low Ca: elevated plasma Zn, hypocalcaemia, inhibited unidirectional Ca2+ influx, inhibited branchial Na+/K+ATPase and Ca2+ATPase activities, and oxidative stress in gills, liver, intestine, and muscle. At 100 % salinity, in the presence of 1 mmol/L (Low Ca), Zn caused no disturbances in most of these same parameters, showing that the "non-Ca" components of sea water alone provided complete protection. However, for a few endpoints (inhibited intestinal Ca2+ATPase activity, oxidative stress in gill and liver), High Ca (10 mmol/L) was needed to provide full protection against Zn in 100 % salinity. There was no instance where the combination of 100 % salinity and High Ca failed to provide complete protection against Zn-induced disturbances in sea water.