Increased Virtual Visits to Physicians During the COVID-19 Pandemic and Estimated Impact on Physician Compensation: The Case of Lung and Colorectal Cancers, Chronic Obstructive Pulmonary Diseases, and Heart Failure in Alberta, Canada.

Introduction: The COVID-19 pandemic started in Alberta in March 2020 and significantly increased telehealth service use and provision reducing the risk of virus transmission. We examined the change in the number and proportion of virtual visits by physician specialty and condition (chronic obstructive pulmonary diseases [COPD], heart failure [HF], colorectal and lung cancers), as well as associated changes in physician compensation. Methods: A population-based design was used to analyze all processed physician claims comparing the number and proportion of virtual visits and associated physician billings relative to in-person between pre- (2019/2020) and intra-pandemic (2020/2021). Physician compensations were the claim amounts paid by the health insurance. Results: Pre-pandemic (intra-), there were 8,981 (8,897) lung cancer, 9,245 (9,029) colorectal, 37,558 (36,292) HF, and 68,270 (52,308) COPD patients. Each patient had totally 2.3-4.7 (of which 0.4-0.6% were virtual) general practitioner (GP) visits and 0.9-2.3 (0.2-0.7% were virtual) specialist visits per year pre-pandemic. The average number and proportion of per-patient virtual visits to GPs and specialists grew significantly pre- to intra-pandemic by 2,138-4,567%, and 2,201-7,104%, respectively. Given the lower fees of virtual compared with in-person visits, the reduction in physician compensation associated with the increased use of virtual care was estimated at $3.85 million, with $2.44 million attributed to specialist and $1.41 million to GP. Discussion: Utilization of telehealth increased significantly, while the physician billings per patient and physician compensation declined early in the pandemic in Alberta for the four chronic diseases considered. This study forms the basis for future study in understanding the impact of virtual care, now part of the fabric of health care delivery, on quality of care and patient safety, overall health service utilization (such as diagnostic imaging and other investigations), as well as economic impacts to patients, health care systems, and society.

Hormonal effects on glucose and ketone metabolism in a perfused liver of an elasmobranch, the North Pacific spiny dogfish, Squalus suckleyi.

Hormonal influence on hepatic function is a critical aspect of whole-body energy balance in vertebrates. Catecholamines and corticosteroids both influence hepatic energy balance via metabolite mobilization through glycogenolysis and gluconeogenesis. Elasmobranchs have a metabolic organization that appears to prioritize the mobilization of hepatic lipid as ketone bodies (e.g. 3-hydroxybutyrate [3-HB]), which adds complexity in determining the hormonal impact on hepatic energy balance in this taxon. Here, a liver perfusion was used to investigate catecholamine (epinephrine [E]) and corticosteroid (corticosterone [B] and 11-deoxycorticosterone [DOC]) effects on the regulation of hepatic glucose and 3-HB balance in the North Pacific Spiny dogfish, Squalus suckleyi. Further, hepatic enzyme activity involved in ketogenesis (3-hydroxybutyrate dehydrogenase), glycogenolysis (glycogen phosphorylase), and gluconeogenesis (phosphoenolpyruvate carboxykinase) were assessed in perfused liver tissue following hormonal application to discern effects on hepatic energy flux. mRNA transcript abundance key transporters of glucose (glut1 and glut4) and ketones (mct1 and mct2) and glucocorticoid function (gr, pepck, fkbp5, and 11ßhsd2) were also measured to investigate putative cellular components involved in hepatic responses. There were no changes in the arterial-venous difference of either metabolite in all hormone perfusions. However, perfusion with DOC increased gr transcript abundance and decreased flow rate of perfusions, suggesting a regulatory role for this corticosteroid. Phosphoenolpyruvate carboxykinase activity increased following all hormone treatments, which may suggest gluconeogenic function; E also increased 3-hydroxybutyrate dehydrogenase activity, suggesting a function in ketogenesis, and decreased pepck and fkbp5 transcript abundance, potentially showing some metabolic regulation. Overall, we demonstrate hormonal control of hepatic energy balance using liver perfusions at various levels of biological organization in an elasmobranch.

The chondrichthyan glucagon-like peptide 3 regulates hepatic ketone metabolism in the Pacific spiny dogfish Squalus suckleyi.

Chondrichthyans have a novel proglucagon-derived peptide, glucagon-like peptide (GLP)-3, in addition to GLP-1 and GLP-2 that occur in other vertebrates. Given that the GLPs are important regulators of metabolic homeostasis across vertebrates, we sought to investigate whether GLP-3 displays functional actions on metabolism within a representative chondrichthyan, the Pacific spiny dogfish Squalus suckleyi. There were no observed effects of GLP-3 perfusion (10 nM for 15 min) on the rate of glucose or oleic acid acquisition at the level of the spiral valve nor were there any measured effects on intermediary metabolism within this tissue. Despite no effects on apparent glucose transport or glycolysis in the liver, a significant alteration to ketone metabolism occurred. Firstly, ketone flux through the perfused liver switched from a net endogenous production to consumption following hormone application. Accompanying this change, significant increases in mRNA transcript abundance of putative ketone transporters and in the activity of ß-hydroxybutyrate dehydrogenase (a key enzyme regulating ketone flux in the liver) were observed. Overall, while these results show effects on hepatic metabolism, the physiological actions of GLP are distinct between this chondrichthyan and those of GLP-1 on teleost fishes. Whether this is the result of the particular metabolic dependency on ketone bodies in chondrichthyans or a differential function of a novel GLP remains to be fully elucidated.

Understanding olfactory and behavioural responses to dietary cues in age-1 lake sturgeon Acipenser fulvescens.

Detection of environmental cues is essential for all vertebrates and is typically established by the olfactory epithelium and olfactory sensory neurons (OSNs). In fishes, microvillous and ciliated OSNs are the principal types, typically detecting amino acids and bile salts, respectively. Activation of OSN receptors by specific ligands initiate downstream signal processing often leading to behavioural responses. In this study we used electrophysiological and behavioural techniques to evaluate olfactory detection and behaviour in juvenile lake sturgeon Acipenser fulvescens in response to hatchery- and natural dietary cues. We hypothesized that electro-olfactogram (EOG) and behavioural responses would be dependent on diet type. We predicted that inhibition of the phospholipase C/inositol 1,4,5-triphosphate (PLC/IP3) secondary transduction pathway would reduce EOG responses to dietary cues and, inhibition of the adenylyl cyclase/adenosine 3,5-cyclic monophosphate (cAMP) pathway, would have no effect. Furthermore, we predicted a strong EOG response would be manifested in a change in behaviour. We observed that both the PLC/IP3 and cAMP pathways were significantly involved in the detection of dietary cues. However, EOG responses did not manifest to behavioural responses, although the foraging activity to the hatchery cue was significantly greater compared to the control. Our results support the notion that lake sturgeon raised in a hatchery and fed a commercial pelleted diet may become accustomed to it prior to release into the wild. Further, this study suggests that, in conservation aquaculture settings, lake sturgeon should be exposed to natural dietary cues prior to release as one strategy to promote food recognition.

Performance assessment of surgical tracking systems based on statistical process control and longitudinal QA.

A system for performance assessment and quality assurance (QA) of surgical trackers is reported based on principles of geometric accuracy and statistical process control (SPC) for routine longitudinal testing. A simple QA test phantom was designed, where the number and distribution of registration fiducials was determined drawing from analytical models for target registration error (TRE). A tracker testbed was configured with open-source software for measurement of a TRE-based accuracy metric ε and Jitter (J). Six trackers were tested: 2 electromagnetic (EM - Aurora); and 4 infrared (IR - 1 Spectra, 1 Vega, and 2 Vicra) - all NDI (Waterloo, ON). Phase I SPC analysis of Shewhart mean (x¯) and standard deviation (s) determined system control limits. Phase II involved weekly QA of each system for up to 32 weeks and identified Pass, Note, Alert, and Failure action rules. The process permitted QA in <1 min. Phase I control limits were established for all trackers: EM trackers exhibited higher upper control limits than IR trackers in ε (EM: x¯Îµ âˆ¼2.8-3.3 mm, IR: x¯Îµ âˆ¼1.6-2.0 mm) and Jitter (EM: x¯jitter âˆ¼0.30-0.33 mm, IR: x¯jitter âˆ¼0.08-0.10 mm), and older trackers showed evidence of degradation - e.g. higher Jitter for the older Vicra (p-value < .05). Phase II longitudinal tests yielded 676 outcomes in which a total of 4 Failures were noted - 3 resolved by intervention (metal interference for EM trackers) - and 1 owing to restrictive control limits for a new system (Vega). Weekly tests also yielded 40 Notes and 16 Alerts - each spontaneously resolved in subsequent monitoring.

Comparison of metabolic rate between two genetically distinct populations of lake sturgeon.

Environmental temperatures differ across latitudes in the temperate zone, with relatively lower summer and fall temperatures in the north leading to a shorter growing season prior to winter. As an adaptive response, during early life stages, fish in northern latitudes may grow faster than their conspecifics in southern latitudes, which potentially manifests as different allometric relationships between body mass and metabolic rate. In the present study, we examined if population or year class had an effect on the variation of metabolic rate and metabolic scaling of age-0 lake sturgeon (Acipenser fulvescens) by examining these traits in both a northern (Nelson River) and a southern (Winnipeg River) population. We compiled 6 years of data that used intermittent flow respirometry to measure metabolic rate within the first year of life for developing sturgeon that were raised in the same environment at 16°C. We then used a Bayesian modeling approach to examine the impacts of population and year class on metabolic rate and mass-scaling of metabolic rate. Despite previous reports of genetic differences between populations, our results showed that there were no significant differences in standard metabolic rate, routine metabolic rate, maximum metabolic rate, and metabolic scaling between the two geographically separated populations at a temperature of 16°C. Our analysis implied that the lack of metabolic differences between populations could be due to family effects/parental contribution, or the rearing temperature used in the study. The present research provided insights for conservation and reintroduction strategies for these populations of lake sturgeon, which are endangered or threatened across most of their natural range.

Physiological responses to acute warming at the agitation temperature in a temperate shark.

Thermal tolerance and associated mechanisms are often tested via the critical thermal maximum (CTmax). The agitation temperature is a recently described thermal limit in fishes that has received little mechanistic evaluation. The present study used a temperate elasmobranch fish to test the hypothesis that this thermal tolerance trait is partially set by the onset of declining cardiorespiratory performance and the cellular stress response. Pacific spiny dogfish (Squalus suckleyi) were screened for cardiorespiratory and whole-organism thermal limits to test for associations between thermal performance and tolerance. Then, biochemical markers of secondary stress, aerobic and anaerobic enzyme activities, and molecular markers of cellular stress were determined for various tissues at the agitation temperature and secondary stress markers were determined at CTmax. In dogfish, the agitation temperature was characterised by increased turning activity within experimental chambers and was equal to the temperature at which dogfish exhibited maximum heart rate. Citrate synthase activity increased at the agitation temperature in white muscle relative to unmanipulated dogfish. Furthermore, lactate dehydrogenase activity and accumulated lactate in the plasma and muscle were not affected by acute warming. Cellular stress was apparent in hypothalamus, gill filament and ventricle, denoted by elevated transcript abundance of the stress response gene hsp70 but not the oxygen homeostasis gene hif1α. Conversely, CTmax was characterised by metabolic acidosis driven by anaerobic lactate production, signifying an increased reliance on anaerobic metabolism between the agitation temperature and CTmax. Together, these data provide partial support for our hypothesis, in that cellular stress, but not declining thermal performance, occurred at the agitation temperature.

An adapted liver perfusion in a shark species, Squalus suckleyi: investigation of energy mobilization.

The liver is an essential energy storage organ in vertebrates. In teleosts and elasmobranchs, previous studies examining hepatic energy balance have used isolated hepatocytes. Although these studies have been informative, the high-fat content in the elasmobranch liver limits isolation of hepatocytes and therefore the utility of this method to understand hepatic metabolic processes. In the present study, we developed an in situ liver perfusion in the North Pacific spiny dogfish Squalus suckleyi. Perfusions were conducted by cannulating the hepatic portal vein (inflowing cannulation) and the sinus venosus through the heart (outflowing cannulation). Changes in major elasmobranch metabolites (glucose and 3-hydroxybutarate [3-HB]) were determined by the arterial (inflow)-venous (outflow) difference in metabolite concentration. Liver preparations were considered viable due to consistent oxygen consumption over 3 h and the maintenance of predictable vasoconstriction following administration of homologous 10-7 M angiotensin II (ANG II). Removal and reintroduction from the perfusate of metabolites showed endogenous 3-HB production in the isolated perfused livers but did not affect glucose balance. However, the arterial-venous difference of both metabolites did not change following perfusion with heterologous insulin and homologous glucagon, which may be due to the glucose intolerant nature of elasmobranchs. Ultimately, we show the viability of this perfusion for the investigation of hepatic energy mobilization in sharks.NEW & NOTEWORTHY We describe a viable liver perfusion in a shark species for the first time as determined by oxygen consumption and hormone-mediated changes in hemodynamics (angiotensin II, ANG II). In addition, removal of major energy metabolites confirms hepatic ketone [3-hydroxybutyrate (3-HB)] production by an elasmobranch liver. Perfusion with heterologous insulin and homologous glucagon did not cause changes in glucose balance, however, possibly demonstrating differences in glucose metabolism in this taxon as compared with more derived vertebrates.

Tissue-specific transcriptomes reveal potential mechanisms of microbiome heterogeneity in an ancient fish.

The lake sturgeon (Acipenser fulvescens) is an ancient, octoploid fish faced with conservation challenges across its range in North America, but a lack of genomic resources has hindered molecular research in the species. To support such research, we created a transcriptomic database from 13 tissues: brain, esophagus, gill, head kidney, heart, white muscle, liver, glandular stomach, muscular stomach, anterior intestine, pyloric cecum, spiral valve and rectum. The transcriptomes for each tissue were sequenced and assembled individually from a mean of 98.3 million (±38.9 million SD) reads each. In addition, an overall transcriptome was assembled and annotated with all data used for each tissue-specific transcriptome. All assembled transcriptomes and their annotations were made publicly available as a scientific resource. The non-gut transcriptomes provide important resources for many research avenues. However, we focused our analysis on messenger ribonucleic acid (mRNA) observations in the gut because the gut represents a compartmentalized organ system with compartmentalized functions, and seven of the sequenced tissues were from each of these portions. These gut-specific analyses were used to probe evidence of microbiome regulation by studying heterogeneity in microbial genes and genera identified from mRNA annotations. Gene set enrichment analyses were used to reveal the presence of photoperiod and circadian-related transcripts in the pyloric cecum, which may support periodicity in lake sturgeon digestion. Similar analyses were used to identify different types of innate immune regulation across the gut, while analyses of unique transcripts annotated to microbes revealed heterogeneous genera and genes among different gut tissues. The present results provide a scientific resource and information about the mechanisms of compartmentalized function across gut tissues in a phylogenetically ancient vertebrate. Database URL: https://figshare.com/projects/Lake_Sturgeon_Transcriptomes/133143.

In situ method for the determination of nutrient acquisition and its hormonal regulation in the spiral valve of two chondrichthyan fishes.

Chondrichthyans play an important role in nutrient cycling of many marine ecosystems, yet little is known about their nutritional physiology particularly relating to nutrient acquisition in the spiral valve intestine. This unique organ poses challenges for examining nutrient transport physiology using traditional reductionist methods owing to its scroll-like morphology. Thus, we established a method for the characterization of nutrient uptake rates in two representative chondrichthyans, the Pacific spiny dogfish (Squalus suckleyi) and the Pacific spotted ratfish (Hydrolagus colliei). We validated a dual-cannulation method wherein perfusate was circulated through the vasculature of the spiral valve via the anterior and posterior intestinal arteries, and [14C]glucose or [3H]oleic acid was accumulated from the static spiral valve lumen into the anterior and posterior intestinal veins. Radiotracer accumulated at a stable rate in the venous effluent in comparison with measures of mucosal disappearance. Interestingly, similar anterior and posterior glucose uptake was observed in dogfish, yet significantly more oleic acid was accumulated in the posterior veins of ratfish. Further validation of the preparation in dogfish demonstrated sodium dependence of glucose transport as well as an effect of bovine insulin administration to the arterial circulation. Each of these manipulations resulted in significant differences in glucose handling between the anterior and posterior veins, suggestive of heretofore unknown heterogenous functions along the intestine. This preparation demonstrates a new and reliable method for the measurement of nutrient acquisition and regulation thereof in a unique digestive organ. Furthermore, it presents avenues for investigation of differential functional along the spiral valve.NEW & NOTEWORTHY We describe a novel dual cannulation method for investigating radiolabeled nutrient uptake from a unique organ, the spiral valve. Furthermore, we identify functional differences in nutrient transport along the length of the spiral valve which consists of a homogenous gross morphology. Finally, this method reveals a useful way in which to manipulate the arterial supply to better understand postprandial physiology as it varies with metabolites and endocrine factors.

Antiviral and Antibacterial Cold Spray Coating Application on Rubber Substrate, Disruption in Disease Transmission Chain.

The objective of this study was to prepare a copper-coated rubber surface using cold spray technology with improved virucidal and antimicrobial properties to fight against highly transmissible viruses and bacteria. A successful cold spray coating was produced using irregular-shaped pure Cu powder on an escalator handrail rubber. The powder particles and the deposited coatings (single and double pass) were characterized in terms of particle morphology and size distribution, coating surface and coat/substrate cross-section properties. The bonding between powder and rubber surfaces was purely mechanical interlocking. The Cu powder penetration depth within the rubber surface increases with a number of depositions pass. The virucidal properties of the coated surface were tested utilizing surrogates for SARS-CoV-2: HCoV-229E, a seasonal human coronavirus, and baculovirus, a high-titer enveloped insect cell virus. A double-pass coated surface showed significant baculovirus inactivation relative to a bare rubber control surface after 2-h (approximately 1.7-log) and 4-h (approximately 6.2-log), while a 4-h exposure reduced HCoV-229E titer to below the limit of detection. A similar microbial test was performed using E. coli, showing a 4-log microbial reduction after 2-h exposure relative to the bare rubber. These promising results open a new application for cold spray in the health sector. Supplementary Information: The online version contains supplementary material available at 10.1007/s11666-023-01553-x.

Molecular and pharmacological analysis of the melanocortin-2 receptor and its accessory proteins Mrap1 and Mrap2 in a Squalomorph shark, the Pacific spiny dogfish.

The hypothalamus-pituitary-adrenal/interrenal (HPA/I) axis is a conserved vertebrate neuroendocrine mechanism regulating the stress response. The penultimate step of the HPA/I axis is the exclusive activation of the melanocortin-2 receptor (Mc2r) by adrenocorticotropic hormone (ACTH), requiring an accessory protein, Mrap1 or Mrap2. Limited data for only three cartilaginous fishes support the hypothesis that Mc2r/Mrap1 function in bony vertebrates is a derived trait. Further, Mc2r/Mrap1 functional properties appear to contrast among cartilaginous fishes (i.e., the holocephalans and elasmobranchs). This study sought to determine whether functional properties of Mc2r/Mrap1 are conserved across elasmobranchs and in contrast to holocephalans. The deduced amino acid sequences of Pacific spiny dogfish (Squalus suckleyi; pd) pdMc2r, pdMrap1, and pdMrap2 were obtained from a de novo transcriptome of the interrenal gland and validated against the S. suckleyi genome. pdMc2r showed high primary sequence similarity with elasmobranch and holocephalan Mc2r except at extracellular domains 1 and 2, and transmembrane domain 5. pdMraps showed similarly high sequence similarity with holocephalan and other elasmobranch Mraps, with all cartilaginous fish Mrap1 orthologs lacking an activation motif. cAMP reporter gene assays demonstrated that pdMc2r requires an Mrap for activation, and can be activated by stingray (sr) ACTH(1-24), srACTH(1-13)NH2 (i.e., α-MSH), and γ-melanocyte-stimulating hormone at physiological concentrations. However, pdMc2r was three orders of magnitude more sensitive to srACTH(1-24) than srACTH(1-13)NH2. Further, pdMc2r was two orders of magnitude more sensitive to srACTH(1-24) when expressed with pdMrap1 than with pdMrap2. These data suggest that functional properties of pdMc2r/pdMrap1 reflect other elasmobranchs and contrast what is seen in holocephalans.

Using 15N to determine the metabolic fate of dietary nitrogen in North Pacific spiny dogfish (Squalus acanthias suckleyi).

Marine elasmobranchs are ureosmotic, retaining large concentrations of urea to balance their internal osmotic pressure with that of the external marine environment. The synthesis of urea requires the intake of exogenous nitrogen to maintain whole-body nitrogen balance and satisfy obligatory osmoregulatory and somatic processes. We hypothesized that dietary nitrogen may be directed toward the synthesis of specific nitrogenous molecules in post-fed animals; specifically, we predicted the preferential accumulation and retention of labelled nitrogen would be directed towards the synthesis of urea necessary for osmoregulatory purposes. North Pacific spiny dogfish (Squalus acanthias suckleyi) were fed a single meal of 7 mmol l-1 15NH4Cl in a 2% ration by body mass of herring slurry via gavage. Dietary labelled nitrogen was tracked from ingestion to tissue incorporation and the subsequent synthesis of nitrogenous compounds (urea, glutamine, bulk amino acids, protein) in the intestinal spiral valve, plasma, liver and muscle. Within 20 h post-feeding, we found labelled nitrogen was incorporated into all tissues examined. The highest δ15N values were seen in the anterior region of the spiral valve at 20 h post-feeding, suggesting this region was particularly important in assimilating the dietary labelled nitrogen. In all tissues examined, enrichment of the nitrogenous compounds was sustained throughout the 168 h experimental period, highlighting the ability of these animals to retain and use dietary nitrogen for both osmoregulatory and somatic processes.

Investigating nitrogen movement in North Pacific spiny dogfish (Squalus acanthias suckleyi), with focus on UT, Rhp2, and Rhbg mRNA abundance.

For ureosmotic marine elasmobranchs, the acquisition and retention of nitrogen is critical for the synthesis of urea. To better understand whole-body nitrogen homeostasis, we investigated mechanisms of nitrogen trafficking in North Pacific spiny dogfish (Squalus acanthias suckleyi). We hypothesized that the presence of nitrogen within the spiral valve lumen would affect both the transport of nitrogen and the mRNA abundance of a urea transporter (UT) and two ammonia transport proteins (Rhp2, Rhbg) within the intestinal epithelium. The in vitro preincubation of intestinal tissues in NH4Cl, intended to simulate dietary nitrogen availability, showed that increased ammonia concentrations did not significantly stimulate the net uptake of total urea or total methylamine. We also examined the mRNA abundance of UT, Rhp2, and Rhbg in the gills, kidney, liver, and spiral valve of fasted, fed, excess urea fed, and antibiotic-treated dogfish. After fasting, hepatic UT mRNA abundance was significantly lower, and Rhp2 mRNA in the gills was significantly higher than the other treatments. Feeding significantly increased Rhp2 mRNA levels in the kidney and mid spiral valve region. Both excess urea and antibiotics significantly reduced Rhbg mRNA levels along all three spiral valve regions. The antibiotic treatment also significantly diminished UT mRNA abundance levels in the anterior and mid spiral valve, and Rhbg mRNA levels in the kidney. In our study, no single treatment had significantly greater influence on the overall transcript abundance of the three transport proteins compared to another treatment, demonstrating the dynamic nature of nitrogen balance in these ancient fish.

Elevated temperatures reduce population-specific transcriptional plasticity in developing lake sturgeon (Acipenser fulvescens).

Rising mean and variance in temperatures elevates threats to endangered freshwater species such as lake sturgeon, Acipenser fulvescens. Previous research demonstrated that higher temperatures during development result in physiological consequences for lake sturgeon populations throughout Manitoba, Canada, with alteration of metabolic rate, thermal tolerance, transcriptional responses, growth and mortality. We acclimated lake sturgeon (30-60 days post fertilization, a period of high mortality) from northern and southern populations (56°02'46.5″N, 96°54'18.6″W and 50°17'52″N, 95°32'51″W, respectively, separated by approximately 650 km) within Manitoba to current (summer highs of 20-23°C) and future projected (+2-3°C) environmental temperatures of 16, 20 and 24°C for 30 days, and we measured gill transcriptional responses using RNAseq. Transcripts revealed SNPs consistent with genetically distinct populations and transcriptional responses altered by acclimation temperature. There were a higher number of differentially expressed transcripts observed in the southern, compared to the northern, population as temperatures increased, indicating enhanced transcriptional plasticity. Both lake sturgeon populations responded to elevated acclimation temperatures by downregulating the transcription of genes involved in protein synthesis and energy production. Furthermore, there were population-specific thresholds for the downregulation of processes promoting transcriptional plasticity as well as mitochondrial function as the northern population showed decreases at 20°C, while this capacity was not diminished until 24°C in the southern population. These transcriptional responses highlight the molecular impacts of increasing temperatures for divergent lake sturgeon populations during vulnerable developmental periods and the critical influence of transcriptome plasticity on acclimation capacity.

Elevated temperatures dampen the innate immune capacity of developing lake sturgeon (Acipenser fulvescens).

Chronic exposure to high temperatures may leave freshwater fishes vulnerable to opportunistic pathogens, particularly during early life stages. Lake sturgeon, Acipenser fulvescens, populations within the northern expanse of their range in Manitoba, Canada, may be susceptible to high temperature stress and pathogenic infection. We acclimated developing lake sturgeon for 22 days to two ecologically relevant, summer temperatures (16 and 20°C). Individuals from both acclimation treatments were then exposed to 0, 30 and 60 µg ml-1 bacterial lipopolysaccharides (endotoxins), as an immune stimulus, for 48 h and sampled 4 and 48 h during trial exposures and following a 7 day recovery period. We then measured whole-body transcriptional (mRNA) responses involved in the innate immune, stress and fatty acid responses following acute exposure to the bacterial endotoxins. Data revealed that overall levels of mRNA transcript abundance were higher in 20°C-reared sturgeon under control conditions. However, following exposure to a bacterial stimulus, lake sturgeon acclimated to 16°C produced a more robust and persistent transcriptional response with higher mRNA transcript abundance across innate immune, stress and fatty acid responses than their 20°C-acclimated counterparts. Additional whole-animal performance metrics (critical thermal maximum, metabolic rate, cortisol concentration and whole-body and mucosal lysozyme activity) demonstrated acclimation-specific responses, indicating compromised metabolic, stress and enzymatic capacity following the initiation of immune-related responses. Our study showed that acclimation to 20°C during early development impaired the immune capacity of developing lake sturgeon as well as the activation of molecular pathways involved in the immune, stress and fatty acid responses. The present study highlights the effects of ecologically relevant, chronic thermal stress on seasonal pathogen susceptibility in this endangered species.

Nitrogen transporters along the intestinal spiral valve of cloudy catshark (Scyliorhinus torazame): Rhp2, Rhbg, UT.

As part of their osmoregulatory strategy, marine elasmobranchs retain large quantities of urea to balance the osmotic pressure of the marine environment. The main source of nitrogen used to synthesize urea comes from the digestion and absorption of food across the gastrointestinal tract. In this study we investigated possible mechanisms of nitrogen movement across the spiral valve of the cloudy catshark (Scyliorhinus torazame) through the molecular identification of two Rhesus glycoprotein ammonia transporters (Rhp2 and Rhbg) and a urea transporter (UT). We used immunohistochemistry to determine the cellular localizations of Rhp2 and UT. Within the spiral valve, Rhp2 was expressed along the apical brush border membrane, and UT was expressed along the basolateral membrane and the blood vessels. The mRNA abundance of Rhp2 was significantly higher in all regions of the spiral valve of fasted catsharks compared to fed catsharks. The mRNA abundance of UT was significantly higher in the anterior spiral valve of fasted catsharks compared to fed. The mRNA transcript of four ornithine urea cycle (OUC) enzymes were detected along the length of the spiral valve and in the renal tissue, indicating the synthesis of urea via the OUC occurs in these tissues. The presence of Rhp2, Rhbg, and UT along the length of the spiral valve highlights the importance of ammonia and urea movement across the intestinal tissues, and increases our understanding of the mechanisms involved in maintaining whole-body nitrogen homeostasis in the cloudy catshark.

Effects of N-acetylcysteine on Growth, Viability, and Ultrastructure of In Vitro Cultured Bovine Secondary Follicles.

This study aimed to investigate the effects of different concentrations of N-acetylcysteine (NAC) on the growth, antrum formation, viability, and ultrastructure of bovine secondary follicles cultured in vitro for 18 days. To this end, the follicles were cultured in TCM-199+ medium alone or supplemented with 1.0, 5.0, or 25.0 mM NAC. Follicular growth, antrum formation, viability (calcein-AM and ethidium homodimer-1) and ultrastructure were evaluated at the end of culture period. The results showed that 1.0 mM NAC increased the percentage of growing follicles and the fluorescence intensity for calcein-AM when compared to other treatments (p < 0.05). On the other hand, follicles cultured with 25.0 mM NAC had higher fluorescence intensity for ethidium homodimer-1, which is a sign of degeneration. Ultrastructural analysis showed that oocytes from follicles cultured in control medium alone or with 1 mM NAC had intact zonae pellucidae in close association with oolemmae, but the ooplasm showed mitochondria with a reduced number of cristae. On the other hand, oocytes from follicles cultured with 5 or 25 mM NAC had extremely vacuolated cytoplasm and no recognizable organelles. In conclusion, 1 mM NAC increases cytoplasmic calcein staining and the growth rate in bovine secondary follicles cultured in vitro, but the presence of 5 or 25 mM NAC causes damage in cellular membranes and organelles, as well as reducing the percentages of growing follicles.

Spatiotemporal variation of ringed seal blubber cortisol levels in the Canadian Arctic.

Climate change in the Arctic has widespread and complex effects on the health of animals and their populations. We used radioimmunoassay to measure blubber cortisol in ringed seals (Pusa hispida) sampled in Ulukhaktok, Inuvialuit Settlement Region, Northwest Territories, Canada (spring, 2002, 2004-2005, 2007-2012) and Arviat, Nunavut, Canada (autumn, 2003-2012) to examine chronic stress relative to biology (age, sex, length), body condition (blubber depth), and diet (δ13C, δ15N, and isotopic niche size). Ulukhaktok ringed seals had higher cortisol concentrations overall (0.46 ± 0.04 ng/g) than Arviat ringed seals (0.36 ± 0.03 ng/g), and these higher concentrations were associated with higher muscle δ15N and lower blubber thickness. In contrast, blubber cortisol concentrations for Arviat ringed seals decreased with blubber depth and increased with age, though testing of age effects individually suggests that age-related patterns are weak. Annual mean cortisol concentration increased from 2003 to 2012 in Arviat ringed seals, but low sample sizes precluded analysis of annual patterns for Ulukhaktok ringed seals. The trend of increased cortisol over time in Arviat ringed seals suggests that they might be experiencing greater chronic stress over time, which could have implications for numerous population health metrics including reproduction and pup recruitment.

Effects of Aloe vera extract on growth, viability, ultrastructure and expression of mRNA for antioxidant enzymes in bovine secondary follicles cultured in vitro.

This study aimed to investigate the effects of Aloe vera extract on follicular growth, viability, ultrastructure, and mRNA levels for superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase 1 (GPX1) and peroxiredoxin 6 (PRDX6) in bovine secondary follicles cultured in vitro. To this end, secondary follicles were mechanically isolated from the ovarian cortex and cultured at 38.5 °C, with 5% CO2 in air, for 18 days in TCM-199+ alone or supplemented with 2.5%, 5.0%, 10.0% and 20.0% Aloe vera extract. Follicular growth, morphology and antrum formation were evaluated every 6 days, while ultrastructure was evaluated at the end of culture. Analysis of viability was performed by calcein-AM and ethidium homodimer-1, while mRNA levels for SOD, CAT, GPX1 and PRDX6 were evaluated by real-time PCR at the end of culture. The results show that follicles cultured with 2.5% Aloe vera had increased the rate of antrum formation, while 2.5% and 5.0% Aloe vera improved follicular viability rate. Follicles cultured with 2.5% and 10.0% Aloe vera increased the levels of mRNA for SOD and GPX1 respectively, but the levels of CAT were reduced in follicles cultured with 2.5%, 5.0%, 10.0% and 20.0%. Additionally, follicles cultured with 2.5% of Aloe vera had their ultrastructure well preserved, while those cultured with 5.0%, 10.0% and 20.0% exhibited increased oocyte vacuolization and damaged organelles. In conclusion, 2.5% Aloe vera increases antrum formation, viability and expression of mRNA for SOD in cultured secondary follicles, but higher concentrations of Aloe vera have negative effects on follicular ultrastructure.