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During the pre-implantation period of pregnancy in eutherian mammals, changes to the uterine endometrium are required (both at the transcriptional and protein level) to facilitate the endometrium becoming receptive to an implanting embryo. We know that the developing conceptus (embryo and extraembryonic membranes) produces proteins during this developmental stage. We hypothesised that this common process in early pregnancy in eutheria may be facilitated by highly conserved conceptus-derived proteins such as macrophage capping protein CAPG. More specifically, we propose that CAPG may share functionality in modifying the transcriptome of the endometrial epithelial cells to facilitate receptivity to implantation in species with different implantation strategies, such as human and bovine. A recombinant bovine form of CAPG (91% sequence identity between bovine and human) was produced and bovine endometrial epithelial (bEECs) and stromal (bESCs) cells and human endometrial epithelial cells (hEECs) were cultured for 24 h with or without rbCAPG. RNA sequencing and quantitative real-time PCR analysis was used to assess the transcriptional response to rbCAPG (Control, vehicle, CAPG 10, 100, 1000 ng/ml: n=3 biological replicates per treatment per species). Treatment of bEECs with CAPG resulted in changes to 1052 transcripts (629 increased and 423 decreased) compared to vehicle controls, including those previously only identified as regulated by interferon-tau, the pregnancy recognition signal in cattle. Treatment of hEECs with bovine CAPG increased expression of transcripts previously known to interact with CAPG in different systems (CAPZB, CAPZA2, ADD1 and ADK) compared with vehicle controls (P<0.05). In conclusion, we have demonstrated that CAPG, a highly conserved protein in eutherian mammals elicits a transcriptional response in the endometrial epithelium in two species with different implantation strategies that may facilitate uterine receptivity.
Daily resetting of the circadian clock to the 24h natural photoperiod might induce marginal costs that would accumulate over time and forward affect fitness. It was proposed as the circadian resonance theory by Pittendrigh in 1972. For the first time, we aimed to evaluate these physiological and cognitive costs that would partially explain the mechanisms of the circadian resonance hypothesis. We evaluated the potential costs of imposing a 26h photoperiodic regimen compared to the classical 24h entrainment measuring several physiological and cognitive parameters (body temperature, energetic expenditure, oxidative stress, cognitive performances). We found significant higher resting body temperature and energy expenditure and lower cognitive performances when the photoperiodic cycle length was 26h. Together these results suggest that a great deviation of external cycles from 24h leads to daily greater synchronization costs, and lower cognitive capacities. To our knowledge, this study is the first to highlight potential mechanisms of circadian resonance theory.
Pigs are an important translational research model for biomedical imaging studies, and especially for modeling diseases of the liver. Dynamic contrast enhanced (DCE)-MRI is experimentally used to measure liver function in humans, but has never been characterized in pig liver. Here we performed DCE-MRI of pig liver following the delivery of two FDA approved hepato-specific MRI contrast agents, Gd-EOB-DTPA (Eovist) and Gd-BOPTA (Multihance), and the non-hepatospecific agent Magnevist, and optimized the anesthesia and animal handling protocol to acquire robust data. A single pig underwent 5 scanning sessions over six weeks, each time injected at clinical dosing either with Eovist (twice), Multihance (twice) or Magnevist (once). DCE-MRI was performed at 1.5T for 60 minutes. DCE-MRI showed rapid hepatic MRI signal enhancement following IV injection of Eovist or Multihance. Efflux of contrast agent from liver exhibited kinetics similar to that in humans, except for one hyperthermic animal where efflux was very fast. As expected, Magnevist was non-enhancing in the liver. The hepatic signal enhancement from Eovist matched that seen in humans and primates, while the hepatic signal enhancement from Multihance was different, similar to rodents and dogs, likely the result of differential hepatic organic anion transport polypeptides. This first experience with these agents in pigs provides valuable information on contrast agent dynamics in normal pig liver. Given the disparity in contrast agent uptake kinetics with humans for Multihance, Eovist should be used in porcine models for biomedical imaging. Proper animal health maintenance, especially temperature, seems essential for accurate and reproducible results.
Backgrounds & AimsAs the bile acid (BA) pool composition is of major impact on liver pathophysiology, we studied its regulation by the BA receptor TGR5, promoting hepatoprotection against BA overload. MethodsWT, total and hepato-specific TGR5-KO, and TGR5-overexpressing mice were used in: partial and 90% extended hepatectomies (EH) upon normal, ursodeoxycholic acid (UDCA)- or cholestyramine (CT)-enriched diet, bile duct ligation (BDL), cholic acid (1%)-enriched diet, and TGR5 agonist (RO) treatments. We thereby studied TGR5 impact on: BA pool composition, liver injury, regeneration and survival. Particular focus was made on gut microbiota (GM) and gallbladder (GB) function analysis. BA pool composition was analyzed in patients undergoing major hepatectomy. ResultsThe TGR5-KO hyperhydrophobic BA pool was not related to BA synthesis alteration, nor to the TGR5-KO GM dysbiosis, as supported by hepatocyte-specific KO mice and cohousing experiments. The TGR5-dependent control of GB dilatation was crucial for BA pool composition, as determined by experiments including RO treatment +/- cholecystectomy. The poor TGR5-KO post-EH survival rate, related with exacerbated peribiliary necrosis and BA overload, was improved by shifting the BA pool towards a more hydrophilic composition (CT and UDCA treatments). After either BDL or CA-enriched diet +/- cholecystectomy, we found that GB dilatation had strong TGR5-dependent hepatoprotective properties. In patients, a more hydrophobic BA pool was correlated with an unfavorable outcome after hepatectomy. ConclusionBA pool composition is crucial for hepatoprotection in mice and humans. We point TGR5 as a key regulator of BA profile and thereby as a potential hepatoprotective target under BA overload conditions. Lay summaryThrough multiple in vivo experimental approaches in mice, together with a patients study, this work brings some new light on the relationships between biliary homeostasis, gallbladder function and liver protection. We showed that the bile acid pool composition is crucial for optimal liver repair, not only in mice but also in human patients undergoing major hepatectomy.
ObjectiveCirculating endothelial progenitor cells (EPCs) were shown to be affected in cardiovascular and metabolic diseases. As interventional strategies, hypoxia and exercise are both known to increase the number and enhance the function of EPCs, potentially by extending their lifespan induced by a reduced senescence. Therefore, this pilot study investigated the effect of exercise under normobaric hypoxia on the senescence of EPCs by in vitro cultivation with autologous human serum (AHS). MethodsFour healthy trained young males (23 {+/-} 2 years) performed an incremental cycling step test until exhaustion in a normobaric hypoxic-chamber with an average altitude of 4,000 m (O2 12.3%). Blood serum was taken at pre, 10 min post and 4 h post, which was later used for in vitro cultivation of EPCs. Senescence was investigated by {beta}-galactosidase staining. ResultsThe participants spent 30-40 min in normobaric hypoxia. The EPC senescence rate was reduced 10 min (0.72 {+/-} 0.57%) and 4 hours (0.67 {+/-} 0.52%) after exercise compared to pre (1.89 {+/-} 0.37%). ConclusionThis pilot study indicates that intense exercise under normobaric hypoxia may enhance EPC function by slowing down their senescence.
White fat stores excess energy, while brown and beige fat dissipate energy as heat1. These thermogenic adipose tissues markedly improve glucose and lipid homeostasis in mouse models, though the extent to which brown adipose tissue (BAT) influences metabolic and cardiovascular disease in humans is unclear2, 3, 4. Here, we categorized 139,224 18F-FDG PET/CT scans from 53,475 patients by presence or absence of BAT and used propensity score matching to assemble a study cohort. Individuals with BAT showed lower prevalences of cardiometabolic diseases. Additionally, BAT independently correlated with lower odds of type II diabetes, coronary artery disease and congestive heart failure. These findings were supported by improved glucose, triglyceride and high-density lipoprotein values. The effects of BAT were more pronounced in overweight and obesity, indicating that BAT can offset the deleterious effects of obesity. Strikingly, we also found lower rates of hypertension among patients with BAT. Studies in a mouse model with genetic ablation of beige fat demonstrated elevated blood pressure due to increased sensitivity to angiotensin II in peripheral resistance arteries. In addition to highlighting a role for BAT in promoting overall cardiometabolic health, this study reveals a new link between thermogenic adipose tissue and blood pressure regulation.
The mitochondrial GTP (mtGTP)-dependent phosphoenolpyruvate (PEP) cycle is an anaplerotic-cataplerotic mitochondrial shuttle utilizing mitochondrial PEPCK (PCK2) and pyruvate kinase (PK). PEP cycling stimulates insulin secretion via OxPhos-independent lowering of ADP by PK. We assess in vivo whether islet PCK2 is necessary for glucose sensing and if speeding the PEP cycle via pharmacological PK activators amplifies insulin secretion. Pck2-/- mice had severely impaired insulin secretion during islet perifusion, oral glucose tolerance tests and hyperglycemic clamps. Acute and chronic pharmacologic PK activator therapy improved islet insulin secretion from normal, high-fat diet (HFD) fed, or Zucker diabetic fatty (ZDF) rats, and glucolipotoxic or diabetic humans. A similar improvement in insulin secretion was observed in regular chow and HFD rats in vivo. Insulin secretion and cytosolic Ca2+ during PK activation were dependent on PCK2. These data provide a preclinical rationale for strategies, such as PK activation, that target the PEP cycle to improve glucose homeostasis. HighlightsO_LILoss of mitochondrial phosphoenolpyruvate (PEP) impairs insulin release in vivo. C_LIO_LIPyruvate kinase (PK) activators stimulate beta-cells in preclinical diabetes models. C_LIO_LIPEP cycling in vivo depends on PK and mitochondrial PEPCK (PCK2) for insulin release. C_LIO_LIAcute and 3-week oral PK activator amplifies insulin release during hyperglycemia. C_LI eTOC BlurbAbudukadier et al. show that small molecule pyruvate kinase activation in vivo and in vitro increases insulin secretion in rodent and human models of diabetes. The phosphoenolpyruvate (PEP) cycling mechanism and its amplification are dependent on mitochondrial PEPCK (PCK2).
Diastolic dysfunction is a prominent feature of cardiac aging in both mice and humans. We show here that 8-week treatment of old mice with the mitochondrial targeted peptide SS-31 (elamipretide) can substantially reverse this deficit. SS-31 normalized the increase in proton leak and reduced mitochondrial ROS in cardiomyocytes from old mice, accompanied by reduced protein oxidation and a shift towards a more reduced protein thiol redox state in old hearts. Improved diastolic function was concordant with increased phosphorylation of cMyBP-C Ser282 but was independent of titin isoform shift. Late-life viral expression of mitochondrial-targeted catalase (mCAT) produced similar functional benefits in old mice and SS-31 did not improve cardiac function of old mCAT mice, implicating normalizing mitochondrial oxidative stress as an overlapping mechanism. These results demonstrate that pre-existing cardiac aging phenotypes can be reversed by targeting mitochondrial dysfunction and implicate mitochondrial energetics and redox signaling as therapeutic targets for cardiac aging.
The sport performance relies on hard work, intensity, duration of workout and undoubtedly on genetic background. These components fold on multicausal character of sport achievements. One of the best known genes associated with sport performance is angiotensin converting enzyme gene (ACE). In this study, we showed ACE distribution in women horse riders and the correlation between physiological and genetic background, that should be taken into account when deciding to train sports discipline. Data from 40 healthy female adults were collected. Participants trained horse-riding for at least 3 years and participated in sport competition at least at national level and control group consist of out of training persons. We analysed BMI, %FAT, cardiorespiratory efficiency (VO2max), Complex Reaction Time, body balance, force and Lower Limbs Explosive Force between both calculated groups. Physiological data was calculated by non-parametric Mann-Whitney test with Dunnetts post hoc test. The DNA amplification from saliva was performed by PCR method to analyze the ACE genotype distribution. In our study, for the first time we showed that the women training dressage have higher force and complex reaction time than the control group. It was correlated with possession of the D allele in genotype that is associated with muscle strength, efficiency and muscle power. There is no research relating to ACE polymorphism among horse riders. This paper showed angiotensin converting enzyme gene distribution polymorphism in female Polish professional horse riders. This article confirms correlation between genotype, sports results and physiological parameters.
The mechanistic target of rapamycin (mTOR) complex 1 (mTORC1) regulates cell size and growth in response to nutrients, however, the mechanisms by which nutrient levels are sensed by mTORC1 in human skeletal muscle are yet to be fully elucidated. The Class III PI3Kinase Vps34 has recently been proposed as a sensor essential for mTORC1 activation following nutrient stimulation. We therefore investigated the effects of increasing nutrient availability through protein-carbohydrate (PRO-CHO) feeding on Vps34 kinase activity and cellular localization in human skeletal muscle. Eight young, healthy males (age - 21 {+/-} 0.5yrs, mean {+/-} SEM) ingested a PRO-CHO beverage containing 20/44/1g PRO/CHO/FAT respectively, with skeletal muscle biopsies obtained at baseline and 1h and 3h post-feeding. PRO-CHO feeding did not alter Vps34 kinase activity, but did stimulate Vps34 translocation toward the cell periphery (PRE (mean{+/-}SEM) - 0.273{+/-}0.021, 1h - 0.347{+/-}0.022, Pearsons Coefficient (r)) where it co-localized with mTOR (PRE - 0.312{+/-}0.018, 1h - 0.348{+/-}0.024, Pearsons Coefficient (r))). These alterations occurred in parallel to an increase in S6K1 kinase activity - 941{+/-}164% of PRE at 1h post-feeding). Subsequent in vitro experiments in C2C12 and human primary myotubes displayed no effect of the Vps34-specific inhibitor SAR405 on mTORC1 signalling responses to elevated nutrient availability. Therefore, in summary, PRO-CHO ingestion does not increase Vps34 activity in human skeletal muscle, whilst pharmacological inhibition of Vps34 does not prevent nutrient stimulation of mTORC1 in vitro. However, PRO-CHO ingestion promotes Vps34 translocation to the cell periphery, enabling Vps34 to associate with mTOR. Therefore, our data suggests that interaction between Vps34 and mTOR, rather than changes in Vps34 activity per se may be involved in PRO-CHO activation of mTORC1 in human skeletal muscle.
Proopiomelanocortin (POMC) neurons are major regulators of energy balance and glucose homeostasis. In addition to being regulated by hormones and nutrients, POMC neurons are controlled by glutamatergic input originating from multiple brain regions. However, the factors involved in the formation of glutamatergic inputs and how they contribute to bodily functions remain largely unknown. Here, we show that during the development of glutamatergic inputs, POMC neurons exhibit enriched expression of the Efnb1 (EphrinB1) and Efnb2 (EphrinB2) genes, which are known to control excitatory synapse formation. In vitro silencing and in vivo loss of Efnb1 or Efnb2 in POMC neurons decreases the amount of glutamatergic inputs into these neurons. We found that mice lacking Efnb1 in POMC neurons display impaired glucose tolerance due to blunted vagus nerve activity and decreased insulin secretion. However, mice lacking Efnb2 in POMC neurons showed no deregulation of insulin secretion and only mild alterations in feeding behavior and gluconeogenesis. Collectively, our data demonstrate the role of ephrins in controlling excitatory input amount into POMC neurons and show an isotype-specific role of ephrins on the regulation of glucose homeostasis and feeding.
Both Achilles tendon stiffness and walking patterns influence the energy cost of walking, but their relative contributions remain unclear. These independent contributions can only be investigated using simulations. We created models for 16 young (24{+/-}2 years) and 15 older (75{+/-}4 years) subjects, with individualized (using optimal parameter estimations) and generic triceps surae muscle-tendon parameters. We varied Achilles tendon stiffness and calculated the energy cost of walking. Both in young and older adults, Achilles tendon stiffness independently contributed to the energy cost of walking. However, overall, a 25% increase in Achilles tendon stiffness increased the triceps surae and whole-body energy cost of walking with approximately 7% and 1.5%, respectively. Therefore, the influence of Achilles tendon stiffness is rather limited. Walking patterns also independently contributed to the energy cost of walking because the plantarflexor (including, but not limited to the triceps surae) energy cost of walking was lower in older than in young adults. Hence, training interventions should probably rather target specific walking patterns than Achilles tendon stiffness to decrease the energy cost of walking. However, based on the results of previous experimental studies, we expected that the calculated hip extensor and whole-body energy cost of walking would be higher in older than in young adults. This was not confirmed in our results. Future research might therefore assess the contribution of the walking pattern to the energy cost of walking by individualizing maximal isometric muscle force and by using three-dimensional models of muscle contraction. Summary statementAchilles tendon stiffness and walking patterns independently contribute to the energy cost in simulations of walking in young and older adults. The influence of Achilles tendon stiffness is rather small.
Saliva plays important roles in the mastication, swallowing and digestion of food, speech and lubrication of oral mucosa, antimicrobial and anti-inflammatory activity and control of body temperature in grooming animals. The salivary protein BPIFA2 (BPI fold containing family A member 2; former names: Parotid Secretory Protein, PSP, SPLUNC2, C20orf70) is related to lipid-binding and LPS-binding proteins expressed in mucosa. Indeed, BPIFA2 binds LPS but the physiological role of BPIFA2 remains to be determined. To address this question, Bpifa2 knockout (Bpifa2tm1(KOMP)Vlcg) (KO) mice were phenotyped with a special emphasis on saliva and salivary glands. Saliva collected from KO mice was less able to spread on a hydrophobic surface than wild-type saliva and the surface tension of KO saliva was close to that of water. These data suggest that BPIFA2 is a salivary surfactant that is mainly responsible for the low surface tension of mouse saliva. The reduced surfactant activity of KO saliva did not affect consumption of dry food or grooming, but saliva from KO mice contained less LPS than wild-type saliva. Indeed, mice lacking BPIFA2 responded to ingested LPS with an increased stool frequency, suggesting that BPIFA2 plays a role in the solubilization and activity of ingested LPS. Consistent with these findings, BPIFA2-depleted mice also showed increased insulin secretion and metabolomic changes that were consistent with a mild endotoxemia. These results support the distal physiological function of a salivary protein and reinforce the connection between oral biology and systemic disease.
Browning of white fat reduces obesity in many preclinical models. Vitamin A metabolites (retinoids) have been linked to thermogenic programming of adipose tissue (AT), however the physiologic importance of systemic retinoid metabolism for AT browning is unknown. Here we show that cold stimulation in mice and humans increases circulating retinol and its plasma transporter, retinol binding protein (RBP). Cold exposure shifts retinol abundance from liver towards subcutaneous white AT which correlates with enhanced thermogenic gene transcription. Cold-mediated retinoid flux is abrogated in Rbp deficient (Rbp-/-) mice and AT browning is dramatically impaired, which renders Rbp-/- mice cold intolerant. Rbp deficiency attenuates cold-induced lipid clearance due to decreased oxidative capacity. In humans, cold-mediated retinol increase is associated with enhanced lipid utilization. Retinol stimulation in primary human adipocytes promotes thermogenic gene expression and mitochondrial respiration. In conclusion, coordinated retinol delivery is essential for cold-induced thermogenic programming of white fat.
Our previous study demonstrated that the loss of growth hormone releasing hormone (GHRH) results in increased lifespan and improved metabolic homeostasis in the mouse model generated by classical embryonic stem cell based gene-targeting method. In this study, we targeted the GHRH gene using the CRISPR/Cas9 technology to avoid passenger alleles/mutations and performed in-depth physiological and metabolic characterization. In agreement with our previous observation, male and female GHRH-/- mice have significantly reduced body weight and enhanced insulin sensitivity when compared to wild type littermates. Dual-energy X-ray absorptiometry showed that there were significant decreases in lean mass, bone mineral content and density, and a dramatic increase in fat mass of GHRH-/- mice when compared to wild type littermates. Indirect calorimetry measurements including oxygen consumption, carbon dioxide production and energy expenditure were dramatically reduced in GHRH-/- mice compared to wild type mice. Respiratory exchange ratio was significantly lower in GHRH-/- mice during the light cycle, but not during the dark cycle, indicating a circadian related metabolic shift towards fat utilization in the growth hormone deficient mice. The novel CRISPR/Cas9 GHRH-/- mice are exhibiting the consistent and unique physiological and metabolic characteristics, which might mediate the longevity effects of growth hormone deficiency in mice.
In the Bruce effect, pregnant mice remember the odor of the fathering male, and miscarry in response to the odor of a male stranger. Humans experience a high rate of unexplained spontaneous miscarriage. Could it be that a portion of these miscarriages reflect a Bruce-like effect? Given ethical constraints on a direct test, we instead probed for circumstantial evidence in women with repeated pregnancy loss (RPL). Consistent with a Bruce-like effect, women with RPL remembered the body-odor of their spouse, but controls could not. Also consistent with a Bruce-like effect, body-odor from a stranger man caused increased activity in the hypothalamus of women experiencing RPL, yet decreased activity in the hypothalamus of women controls. Finally, RPL was associated with reduced olfactory-bulb volume. Although not causal, these observations link RPL with an altered behavioral and brain response to mens body-odor, implicating the olfactory system in this poorly understood or managed condition.
Sepsis is a systemic inflammatory response to infection and a leading cause of death. Mucosal-associated invariant T (MAIT) cells are innate-like T cells enriched in mucosal tissues that recognize bacterial ligands. We investigated MAIT cells during clinical and experimental sepsis, and their contribution to host responses. In experimental sepsis, MAIT-deficient mice had significantly increased mortality and bacterial load, and reduced tissue-specific cytokine responses. MAIT cells of WT mice expressed lower levels of IFN-{gamma} and IL-17a during sepsis compared to sham surgery, changes not seen in non-MAIT T cells. MAIT cells of patients presenting with sepsis were significantly reduced in frequency, more activated, and had decreased IFN-{gamma} production when stimulated, compared to healthy donors and paired 90-day post-sepsis samples. Our data suggest that MAIT cells are highly activated and become dysfunctional during clinical sepsis, and contribute to tissue-specific cytokine responses that are protective against mortality during experimental sepsis.
The actin cytoskeletal regulator Wiskott Aldrich syndrome protein (WASp) has been implicated in maintenance of the autophagy-inflammasome axis in innate murine immune cells. Here, we show that WASp deficiency is associated with impaired rapamycin-induced autophagosome formation and trafficking to lysosomes in primary human monocyte-derived macrophages (MDMs). WASp reconstitution in vitro and in WAS patients following clinical gene therapy restores autophagic flux and is dependent on the actin-related protein complex ARP2/3. Induction of mitochondrial damage with CCCP, as a model of selective autophagy, also reveals a novel ARP2/3-dependent role for WASp in formation of sequestrating actin cages and maintenance of mitochondrial network integrity. Furthermore, mitochondrial respiration is suppressed in WAS patient MDMs and unable to achieve normal maximal activity when stressed, indicating profound intrinsic metabolic dysfunction. Taken together, we provide evidence of new and important roles of human WASp in autophagic processes and immunometabolic regulation, which may mechanistically contribute to the complex WAS immunophenotype.
Behavioural flexibility allows ectotherms to exploit the environment to govern their metabolic physiology, including in response to environmental stress. Hydrogen sulfide (H2S) is a widespread environmental toxin that can lethally inhibit metabolism. However, H2S can also alter behaviour and physiology, including a hypothesised induction of hibernation-like states characterised by downward shifts of the innate thermal setpoint (anapyrexia). Support for this hypothesis has proved controversial because it is difficult to isolate active and passive components of thermoregulation, especially in animals with high resting metabolic heat production. Here, we directly test this hypothesis by leveraging the natural behavioural thermoregulatory drive of fish to move between environments of different temperatures in accordance with their current physiological state and thermal preference. We observed a decrease in adult zebrafish (Danio rerio) preferred body temperature with exposure to 0.02% H2S, which we interpret as a shift in thermal setpoint. Individuals exhibited consistent differences in shuttling behaviour and preferred temperatures, which were reduced by a constant temperature magnitude during H2S exposure. Seeking lower temperatures alleviated H2S-induced metabolic stress, as measured by reduced rates of aquatic surface respiration rate. Our findings highlight the interactions between individual variation and sublethal impacts of environmental toxins on behaviour.
Ischemic injury to the heart causes a loss of mitochondria function due to an increase in oxidative stress. MG53, also known as TRIM72, is highly expressed in striated muscle and is essential to repair damage to plasma membrane. We have shown that mg53-/- mice are more susceptible to ischemia-reperfusion injury, whereas treatment with exogenous recombinant human MG53 (rhMG53) reduces both infarct damage and restores cardiac function. This study assesses whether MG53 protects and repairs mitochondria injury after oxidative stress associated with myocardial infarction. We hypothesize that in addition to known cell membrane repair function, MG53 acts as a myokine to protect cardiomyocytes by maintaining mitochondrial function. A combination of in vivo and in vitro ischemia/reperfusion models were used to assess MG53s effect on mitochondria using biochemical assays and confocal microscopic imaging. Treatment with rhMG53 allowed cells to maintain a healthy mitochondrial membrane potential, reduced release of mitochondrial reactive oxygen species, and mitigated mitophagy. Mitochondrial localization of rhMG53 is mediated by exposure of and interaction with cardiolipin on the mitochondrial membrane. Our data demonstrates that rhMG53 protein preserves mitochondria integrity in cardiomyocytes during ischemia reperfusion-induced oxidative stress.
BackgroundAcute coronary syndrome (ACS) is a growing global health problem, and precision medicine techniques hold promise for the development of diagnostic indicators of ACS. In this pilot, we sought to assess the utility of an integrated analysis of metabolomic and microRNA data in peripheral blood to distinguish patients with abnormal cardiac stress testing from matched controls. MethodsWe used prospectively collected samples from emergency department (ED) patients placed in an ED-based observation unit who underwent stress testing for ACS. We isolated microRNA and quantified metabolites from plasma collected before and after stress testing in patients with myocardial ischemia on stress testing versus those with normal stress tests. The combined metabolomic and microRNA data were analyzed jointly for case (ischemia) and 1:1 matched control patients in a supervised, dimension-reducing discriminant analysis. Two integrative models were implemented: a baseline model utilizing data collected prior to stress-testing (T0) and a stress-delta model, which included the difference between post-stress test (T1) and pre-stress test (T0). ResultsSeven case patients with myocardial ischemia on ED cardiac stress testing (6 females, 85% Caucasian, mean Thrombolysis In Myocardial Infarction Score=3, 4 patients ultimately received percutaneous coronary intervention) were 1:1 age and sex-matched to controls. Several metabolites and microRNAs were differentially expressed between cases and controls. Integrative analysis of the baseline levels of metabolites and microRNA expression showed modest performance for distinguishing cases from controls with an overall error rate of 0.143. The stress-delta model showed worse performance for distinguishing cases from controls, with an overall error rate of 0.500. ConclusionsGiven our small sample size, results are hypothesis-generating. However, this pilot study shows a potential method for a precision medicine approach to cardiac stress testing in patients undergoing workup for ACS.
Placenta-mediated pregnancy complications are a major challenge in the management of maternal-fetal health. Maternal thrombophilia is a suspected risk factor but the role of thrombotic processes in these complications and the potential for antithrombotic treatment have remained unclear. Endothelial Protein C Receptor (EPCR) is an anticoagulant protein highly expressed in the placenta. EPCR autoantibodies and specific gene variants of EPCR are associated with poor pregnancy outcomes. In mice, fetal EPCR deficiency results in placental failure and in utero death. Adult EPCR-deficient mice generated by maintaining placental expression exhibit plasma markers of thrombophilia without overt thrombosis. We demonstrate that inactivation of clotting factor VIII or Protease Activated Receptor 4 (Par4), Par3 or integrin IIb in the mother allows placental development and intrauterine survival of murine embryos lacking EPCR. Rescued EPCR-deficient embryos exhibit thrombosis in placental venous sinuses at late gestation and a high rate of neonatal lethality. In contrast to fetal EPCR deficiency, maternal deficiency of EPCR results in frequent stillbirths and maternal death accompanied by pathological findings that resemble placental abruption and consumptive coagulopathy. Inactivation of Par4, but not clotting factor VIII, prevents maternal death and restores normal pregnancy outcomes. These observations establish a cause-effect relationship between maternal thrombophilia and placental abruption. They demonstrate that sites of uteroplacental thrombosis and the potential response to antithrombotic intervention may differ with gestational age and maternal versus fetal origin of thrombophilia. Our findings highlight the potential for therapeutic inhibition of thrombin-mediated platelet activation in a subset of pregnancy complications. KEY POINTSO_LIMurine model establishes a cause-effect relationship between maternal thrombophilia, retroplacental hemorrhage and severe pregnancy complications. C_LIO_LIThrombin-mediated activation of maternal platelets is a key event in thrombophilia-associated pregnancy complications and a potential target of therapeutic intervention. C_LIO_LIMaternal venous channels in uteroplacental circulation are additional sites of thrombotic pathology associated with adverse neonatal outcomes. C_LI
Reducing insulin-like growth factor (IGF) signaling is one of the best conserved and characterized mechanisms to extend longevity. Pregnancy associated plasma protein A (PAPP-A) is a secreted metalloprotease that increases IGF availability by cleaving IGF binding proteins. PAPP-A inhibition reduces local IGF signaling, limits the progression of multiple age-related diseases, and extends lifespan, but the mechanisms behind these pleiotropic effects remains unknown. Here, we developed and utilized a PAPP-A neutralizing antibody to discover that adulthood inhibition of this protease reduced collagen and extracellular matrix (ECM) gene expression in multiple tissues in mice. Using bone marrow to explore this effect, we identified mesenchymal stromal cells (MSCs) as the source of PAPP-A and primary responders to PAPP-A inhibition. Short-term treatment with anti-PAPP-A reduced IGF signaling in MSCs, altered MSC expression of collagen/ECM, and decreased MSC number. This affected MSC-dependent functions, decreasing myelopoiesis and osteogenesis. Our data demonstrate that PAPP-A inhibition reduces the activity and number of IGF-dependent mesenchymal progenitor cells and their differentiated progeny, and that this reduction leads to functional changes at the tissue level. MSC-like cells are present in virtually all tissues, and aberrant collagen and ECM production from mesenchymal cells drives aspects of aging and age-related diseases, thus this may be a mechanism by which PAPP-A deficiency enhances lifespan and healthspan. SummaryInhibition of PAPP-A, a regulator of IGF signaling, decreases multi-tissue collagen and extracellular matrix gene expression and modulates mesenchymal stromal cell activity in murine bone marrow.
Mediator of Cell Motility 1 (MEMO1) is an ubiquitously expressed modulator of cellular responses to growth factors including FGF23 signaling, and Memo1-deficient mice share some phenotypic traits with Fgf23- or Klotho-deficient mouse models. Here, we tested whether Memo1 gene expression is regulated by calciotropic hormones or by changing the dietary mineral load. MLO-Y4 osteocyte-like cells were cultured and treated with 1,25(OH)2-vitamin D3. Wildtype C57BL/6N mice underwent treatments with 1,25(OH)2-vitamin D3, parathyroid hormone (PTH), 17{beta}-estradiol or vehicle. Other cohorts of C57BL/6N mice were fed diets varying in calcium or phosphate content. Expression of Memo1 and control genes was assessed by qPCR. 1,25(OH)2-vitamin D3 caused an acute decrease in Memo1 transcript levels in vitro, but not in vivo. None of the hormones tested had an influence on Memo1 transcripts, whereas the assessed control genes reacted the expected way. Dietary interventions with calcium and phosphate did not affect Memo1 transcripts but altered the chosen control genes expression. We observed that Memo1 was not regulated by calciotropic hormones or change in mineral load, suggesting major differences between the regulation and physiological roles of Klotho, Fgf23 and Memo1.
Nicotine consumption in both human and animal studies has been strongly associated with changes in feeding-related behaviors and metabolism. The current dogma is that chronic nicotine decreases food intake and increases metabolism, leading to lower body weight. However, the effect of acute nicotine intake on feeding is unclear. The present study employed microstructural and macrostructural behavioral analyses to elucidate changes in feeding behavior in animals that intravenously self-administered nicotine. At the microstructural level (seconds to minutes), nicotine increased feeding and drinking behavior during the first 5 minutes after nicotine self-administration. This effect was also observed in animals that passively received nicotine, but the effect was not observed in animals that self-administered saline or passively received saline. At the macrostructural level (hours to days), nicotine decreased body weight gain, decreased feeding, and was associated with increases in feeding and body weight gain during abstinence. These results suggest that nicotine first produces anti-anorectic effects before producing long-term anorexigenic effects. These results challenge the notion that nicotine is an anorexigenic drug and paradoxically suggest that the anorexigenic effects of nicotine may be a long-term consequence of acute anti-anorectic effects of nicotine.
The current study determined the area-per-player during small- or large-sided games with or without goalkeeper that replicates the relative (m{middle dot}min-1) total distance, high-intensity running distance, sprint distance and metabolic power covered during official matches. Time-motion analysis was performed on twenty-five elite soccer-players during 26 home-matches. A total of 2565 individual samples for SSGs using different pitch sizes and different number of players were collected and classified as SSGs with (SSG-G) or without goalkeeper (SSG-P). A between-position comparison was also performed. The area-per-player needed to replicate the official match demands was largely greater in SSG-G vs SSG-P for total distance [187{+/-}53 vs 115{+/-}35 m2, effect size (ES): 1.60 95%CI 0.94/2.21], high-intensity running distance [262{+/-}72 vs 166{+/-}39 m2, ES: 1.66(0.99/2.27)] and metabolic power [177{+/-}42 vs 94{+/-}40, ES: 1.99(1.31/2.67)], but similar for sprint distance [(316{+/-}75 vs 295{+/-}99 m2, ES: 0.24(-0.32/0.79)] with direction of larger area-per-player for sprint distance > high-intensity running > total distance {cong} metabolic power for both SSG-G and SSG-P. In SSG-G, forwards required greater area-per-player than central-defenders [ES: 2.96(1.07/4.35)], wide-midfielders [ES: 2.45(0.64/3.78)] and wide-defenders [ES: 3.45(1.13/4.99)]. Central-midfielders required greater area-per-player than central-defenders [ES: 1.69(0.20/2.90)] and wide-midfielders [ES: 1.35(-0.13/2.57)]. In SSG-P, central defenders need smaller area-per-player (ES: -6.01/-0.92) to overall replicate the match demands compared to all other positions. The current results highlight that soccer players need a specific area-per-player during the small-side games with or without goalkeeper to replicate the overall match demands, especially to perform high-intensity running or sprint distance. Additionally, central defenders, central midfielders and forwards need to be trained with tailored area-per-player or specific rules/additional exercises.
PurposeTo determine whether there was a change in the fundus of the eye in diabetic patients without retinopathy after insulin therapy. MethodsThe diabetic patients without retinopathy were included in this study. A swept-source optical coherence tomography/angiography (SS-OCT/A) was used to obtain the measurements of macular retinal nerve fibre layer (mRNFL) thickness, ganglion cell-inner plexiform layer (GC-IPL) thickness, retinal thickness (RT), macular choroidal thickness (MCT), peripapillary retinal nerve fibre layer (pRNFL) thickness, peripapillary choroidal thickness (PCT), and perfused vascular density (PVD). Univariable and multivariable regression analyses were performed to explore the influence of insulin use on measurements of OCT/A. ResultsA total of 1140 patients used insulin (using group), and 126 patients did not use insulin (without group). The average MCT of the using group was 171.3{+/-}67.8 m, which was thinner than that in the without group (190.2{+/-}74.7 m) (P=0.012). The average PVD of the using group (48.0{+/-}2.1 m) was less than that in the without group (48.7{+/-}2.1 m) (P<0.001). After adjusting for age, gender, axial length, duration, HbA1c, systolic blood pressure, diastolic blood pressure, cholesterol, serum creatinine, insulin use was significantly associated with thinner MCT (beta=-16.12m; 95%CI:-29.42, -2.81m; P= 0.018) and lower PVD (beta=-0.79; 95%CI: -1.22, -0.36; P<0.001). ConclusionThe use of insulin by diabetic patients without retinopathy might decrease the MCT and PVD compared to patients who did not use insulin, which helps to better understanding the role of insulin use on higher risk for diabetic retinopathy.
Endometriosis affects [~]176 million women worldwide, yet on average, women experience pain [~]10 years from symptom onset before being properly diagnosed. Standard treatments (drugs or surgery) often fail to provide long-term pain relief. Elevated levels of reactive aldehydes such as 4-hydroxynonenal (4-HNE) have been implicated in the peritoneal fluid of women with endometriosis and upon accumulation, reactive aldehydes can form protein-adducts and/or generate pain. A key enzyme in detoxifying reactive aldehydes to less reactive forms, is the mitochondrial enzyme aldehyde dehydrogenase-2 (ALDH2). Here, we tested the hypothesis that aberrant reactive aldehyde detoxification by ALDH2, underlies endometriosis and its associated pain. We determined, in the eutopic and ectopic endometrium of women with severe (stage IV) peritoneal endometriosis, that ALDH2 enzyme activity was decreased, which was associated with decreased ALDH2 expression and increased 4-HNE adduct formation compared to the eutopic endometrium of controls in the proliferative phase. Using a rodent model of endometriosis and an ALDH2*2 knock-in mouse with decreased ALDH2 activity, we determined that increasing ALDH2 activity with the enzyme activator Alda-1 could prevent endometriosis lesion development as well as alleviate pain-associated behaviors in proestrus. Overall, our findings suggest that targeting the ALDH2 enzyme in endometriosis may lead to better treatment strategies and in the proliferative phase, that increased 4-HNE adduct formation within the endometrium may serve as a less invasive diagnostic biomarker to reduce years of suffering in women. One Sentence SummaryALDH2 activity influences endometriosis and its associated pain.
A phenotypic screening of 12 industrial yeast strains and the well-studied laboratory strain CEN.PK113-7D at cultivation temperatures between 12 {degrees}C and 40 {degrees}C revealed significant differences in maximum growth rates and temperature tolerance. Two Saccharomyces cerevisiae strains, one performing best at sub-, and the other at supra-optimal temperatures, plus the laboratory strain, were selected for further physiological characterization in well-controlled bioreactors. The strains were grown in anaerobic chemostats, at a fixed specific growth rate of 0.03 h-1 and sequential batch cultures at 12, 30, and 39 {degrees}C. We observed significant differences in biomass and ethanol yields on glucose, biomass protein and storage carbohydrate contents, and biomass yields on ATP between strains and cultivation temperatures. Increased temperature tolerance coincided with higher energetic efficiency of cell growth, indicating that temperature intolerance is a result of energy wasting processes, such as increased turnover of cellular components (e.g. proteins) due to temperature induced damage.
Employing self-labelling protein tags for the attachment of fluorescent dyes has become a routine and powerful technique in optical microscopy to visualize and track fused proteins. However, membrane permeability of the dyes and the associated background signals can interfere with the analysis of extracellular labeling sites. Here we describe a novel approach to improve extracellular labeling by functionalizing the SNAP-tag substrate benzyl guanine ("BG") with a charged sulfonate ("SBG"). This chemical manipulation improves solubility, reduces non-specific staining and renders the bioconjugation handle impermeable while leaving its cargo untouched. We report SBG-conjugated fluorophores across the visible spectrum, which cleanly label SNAP-fused proteins in the plasma membrane of living cells. We demonstrate the utility of SBG-conjugated fluorophores to interrogate class A, B and C G protein-coupled receptors (GPCRs) using a range of imaging approaches including nanoscopic super-resolution imaging, analysis of GPCR trafficking from intra- and extracellular pools, in vivo labelling in mouse brain and analysis of receptor stoichiometry using single molecule pull down.
Obesity is associated with alterations in hepatic lipid metabolism. We previously identified the prorenin receptor (PRR) as a potential contributor to liver steatosis. Therefore, we aimed to determine the relative contribution of PRR and its soluble form, sPRR, to lipid homeostasis. PRR-floxed male mice were treated with an adeno-associated virus with thyroxine-binding globulin promoter driven Cre to delete specifically PRR in hepatocytes (Liver PRR KO mice). Hepatic PRR deletion did not change the body weight but increased liver weights. Liver PRR KO mice exhibited higher plasma cholesterol levels and lower hepatic LDLR protein than control mice. Surprisingly, hepatic PRR deletion elevated hepatic cholesterol, and up-regulated hepatic SREBP2 and HMG CoA-R genes. In addition, hepatic PRR deletion increased plasma sPRR levels. In vitro studies in Hep-G2 cells demonstrated that sPRR treatment up-regulated SREBP2 suggesting that elevated plasma sPRR could contribute to hepatic cholesterol biosynthesis. Interestingly, PPAR{gamma}, PRR and total sPRR were elevated in the adipose tissue of Liver PRR KO mice suggesting that elevated plasma sPRR originated from the adipose tissue. In 3T3-L1 cells, sPRR treatment up-regulated PPAR{gamma} indicating that sPRR stimulates master regulator of adipocyte differentiation. Overall, this work support a new role for sPRR in lipid metabolism and adipose tissue - liver crosstalk.
The development of obesity-related metabolic syndrome (MetS) involves a complex interaction of genetic and environmental factors. One environmental factor found to be significantly associated with MetS is early life stress (ELS). We have previously reported on our mouse model of ELS, induced by neonatal maternal separation (NMS), that displays altered regulation of the hypothalamic-pituitary-adrenal (HPA) axis and increased sensitivity in the urogenital organs, which was attenuated by voluntary wheel running. Here, we are using our NMS model to determine if ELS-induced changes in the HPA axis also influence weight gain and MetS. Naive (non-stressed) and NMS male mice were given free access to a running wheel and a low-fat control diet at 4-weeks of age. At 16-weeks of age, half of the mice were transitioned to a high fat/sucrose (HFS) diet to investigate if NMS influences the effectiveness of voluntary exercise to prevent diet-induced obesity and MetS. Overall, we observed a greater impact of voluntary exercise on prevention of HFS diet-induced outcomes in naive mice, compared to NMS mice. Although body weight and fat mass were still significantly higher, exercise attenuated fasting insulin levels and mRNA levels of inflammatory markers in epididymal adipose tissue in HFS diet-fed naive mice. Only moderate changes were observed in exercised NMS mice on a HFS diet, although this could partially be explained by reduced running distance within this group. Interestingly, sedentary NMS mice on a control diet displayed impaired glucose homeostasis and moderately increased pro-inflammatory mRNA levels in epididymal adipose, suggesting that early life stress alone impairs metabolic function and negatively impacts the therapeutic effect of voluntary exercise.
PurposeTo characterize the effects of netarsudil on the aqueous humor outflow tract distal to the trabecular meshwork (TM). We hypothesized that netarsudil increases outflow facility in eyes with and without circumferential ab interno trabeculectomy (AIT) that removes the TM. Methods64 porcine anterior segment cultures were randomly assigned to groups with (n=32) and without circumferential AIT (n=32). Cultures were exposed to 0.1, 1, and 10 M netarsudil (N= 8 eyes per concentration). For each concentration, IOP and vessel diameters were compared to their respective pretreatment baselines. Outflow tract vessel diameters were assessed by spectral-domain optical coherence tomography (SDOCT) and rendered in 4D (XYZ time-series). ResultsNetarsudil at 1 M reduced IOP in both eyes with TM (-0.60{+/-}0.24 mmHg, p = 0.01) and in eyes without TM (-1.79{+/-}0.42 mmHg, p<0.01). At this concentration, vessels of the distal outflow tract dilated by 72%. However, at 0.1 M netarsudil elevated IOP in eyes with TM (1.59{+/-}0.36 mmHg, p<0.001) as well as in eyes without TM (0.23{+/-}0.32 mmHg, p<0.001). Vessels of the distal outflow tract constricted by 31%. Similarly, netarsudil at a concentration of 10 M elevated IOP both in eyes with TM (1.91{+/-}0.193, p<0.001) and in eyes without TM (3.65{+/-}0.86 mmHg, p<0.001). At this concentration, outflow tract vessels constricted by 27%. ConclusionIn the porcine anterior segment culture, the dose-dependent IOP changes caused by netarsudil matched the diameter changes of distal outflow tract vessels. Hyper- and hypotensive properties of netarsudil persisted after TM removal.
Cardiac lipotoxicity is associated with structural remodeling and functional changes that are features of obesity-related cardiomyopathy. Both high fat diet and the ob/ob mutation lead to increased fatty acid (FA) uptake, elevated triacylglycerol (TAG) content, hypertrophy, and systolic and diastolic dysfunction in murine hearts. Cardiomyocyte-specific long-chain acyl-CoA synthetase 1 (ACSL1) deficiency (Acsl1H-/-) results in a 90% reduction in FA activation, suggesting that Acsl1 ablation might alleviate obesity-associated myocardial dysfunction. Double knockout ob-Acsl1H-/- and ob-Acsl1flox/flox control mice were treated with tamoxifen at 20 weeks of age; heart function, TAG content, and relevant gene expression were assessed immediately before and 2 and 5 weeks after treatment. Heart weights initially increased in lean and obese Acsl1H-/- mice, but normalized in ob-Acsl1H-/- mice by 5 weeks. Ventricular TAG content was decreased by 51% and 61% in ob-Acsl1H-/- mice 2 and 5 weeks after Acsl1 knockout induction, respectively. Moreover, ACSL1 knockout resulted in increased survival of ob/ob mice, suggesting that lack of ACSL1 protected obese hearts subjected to stress. Our results indicate that partial knockdown of cardiac ACSL1 is sufficient to reverse cardiac TAG accumulation and to ameliorate heart dysfunction even in the context of established obesity-related cardiomyopathy.
PurposeRisk for glaucoma is driven by the microanatomy and function of the anterior segment. We performed a computation-intense, high-resolution, full-thickness ribbon-scanning confocal microscopy (RSCM) of the outflow tract of two human eyes. We hypothesized this would reveal important species differences when compared to existing data of porcine eyes, an animal that does not spontaneously develop glaucoma. MethodsAfter perfusing two human octogenarian eyes with lectin-fluorophore conjugate and optical clearance with benzyl alcohol benzyl benzoate (BABB), anterior segments were scanned by RSCM and reconstructed in 3D for whole-specimen rendering. Morphometric analyses of the outflow tract were performed for the trabecular meshwork (TM), limbal, and perilimbal outflow structures and compared to existing porcine data. ResultsRSCM provided high-resolution data for IMARIS-based surface reconstruction of outflow tract structures in 3D. Different from porcine eyes with an abundance of highly interconnected, narrow, and short collector channels (CCs), human eyes demonstrated fewer CCs which had a 1.5x greater cross-sectional area (CSA) and 2.6x greater length. Proximal CC openings at the level of Schlemms canal (SC) had a 1.3x larger CSA than distal openings into the scleral vascular plexus (SVP). CCs were 10.2x smaller in volume than the receiving SVP vessels. Axenfeld loops, projections of the long ciliary nerve, were also visualized. ConclusionIn this high-resolution, volumetric RSCM analysis, human eyes had far fewer outflow tract vessels than porcine eyes. Human CCs spanned several clock-hours and were larger than in porcine eyes. These species differences may point to factors downstream of the TM that increase our vulnerability to glaucoma. Grant informationNational Eye Institute K08EY022737 (NAL); Initiative to Cure Glaucoma of the Eye and Ear Foundation of Pittsburgh (NAL); Wiegand Fellowship of the Eye and Ear Foundation of Pittsburgh (YD); P30-EY08098 (NAL); Department grant by Research to Prevent Blindness (NAL); an unrestricted fellowship grant from the Xiangya Hospital of Central South University (SC).
Large conductance calcium-activated potassium (BK) channels play a critical role in electrical resonance, a mechanism of frequency selectivity in chicken hair cells. We determine that BK currents are dependent on inward flow of Ca2+, and intracellular buffering of Ca2+. Entry of Ca2+ is further amplified locally by Ca2+ induced Ca2+ release (CICR) in close proximity to plasma membrane BK channels. Ca2+ imaging reveals peripheral clusters of high concentrations of Ca2+ that are suprathreshold to that needed to activate BK channels. PKA activation increases BK currents likely by recruiting more BK channels due to spatial spread of high Ca2+ concentrations in turn from increasing CICR. STORM imaging confirms the presence of nanodomains with ryanodine and IP3 receptors in close proximity to the Slo subunit of BK channels. Together, these data require a rethinking of how electrical resonance is brought about and suggest effects of CICR in synaptic release. Both genders were included in this study.
MAP kinase-activated protein kinase-2 (MK2) is protein serine/threonine kinase activated by p38/{beta}. Herein we examined the cardiac phenotype of pan MK2-null (MK2-/-) mice. Survival curves for male MK2+/+ and MK2-/- mice did not differ (Mantel-Cox test, P = 0.580). At 12-weeks of age, MK2-/- mice exhibited normal systolic function along with signs of possible early diastolic dysfunction; however, ageing was not associated with an abnormal reduction in diastolic function. Both R-R interval and P-R segment durations were prolonged in MK2-deficient mice. However, heart rates normalized when isolated hearts were perfused ex vivo in working mode. Ca2+ transients evoked by field stimulation or caffeine were similar in ventricular myocytes from MK2+/+ and MK2-/- mice. MK2-/- mice had lower body temperature and an age-dependent reduction in body weight. mRNA levels of key metabolic genes, including Ppargc1a, Acadm, Lipe, and Ucp3 were increased in hearts from MK2-/- mice. For equivalent respiration rates, mitochondria from MK2-/- hearts showed a significant decrease in Ca2+-sensitivity to mitochondrial permeability transition pore (mPTP) opening. Finally, the pressure overload-induced increase in heart weight/tibia length and decrease in systolic function were attenuated in MK2-/- mice two weeks, but not eight weeks, after constriction of the transverse aorta. Collectively, these results implicate MK2 in (i) autonomic regulation of heart rate, (ii) cardiac mitochondrial function, and (iii) the early stages of myocardial remodeling in response to chronic pressure overload. Key points summaryThe cardiac characterization of pan MK2-null mice showed: O_LIaltered autonomic regulation of heart rate C_LIO_LIincreased expression of key metabolic genes C_LIO_LIdecreased Ca2+-sensitivity for MPTP opening C_LIO_LIdelayed hypertrophic remodeling in response to increased afterload C_LI
The aim of this study was to provide support for the performance, localization and expression of the epithelial calcium transporter channels, calbindin-D28k (Calb) and TRPV6, and of the morphology of the digestive and reproductive system of laying quails under heat stress, and with methionine supplementation. Therefore, the present study characterized the positivity (immunohistochemistry) and expression (real-time PCR) of calcium channels (Calb and TRPV6) in the kidneys, intestine and uterus of 504 laying quails that were submitted to different methionine supplementation (100, 110 and 120%) and temperatures (20, 24, 28 and 32{degrees}C). The animals under thermal stress had lower villus height, villus:crypt ratio, and goblet cell index in the duodenum and jejunum, fewer secondary and tertiary uterine folds, smaller hepatic steatosis, and increased number of distal convoluted renal tubules (CT) positive to Calb (protein), and increased positivity in proximal CTs. The deleterious effects of heat stress were minimized with methionine supplementation for the following variables: duodenal crypts, number of goblet cells of the jejunum, number of uterine folds, decreased Calb positivity in intestines and kidney, increased positivity of Calb in the uterus and increased TRPV6 gene expression in the kidney. Calcium transporters were altered due to less need for calcium absorption and reabsorption due to more calcium available with the supplementation, increasing egg production and quality. Methionine supplementation further increased intestinal villus absorption area and height, increased steatosis, decreased Calb positivity in the intestine and kidney, increased uterine positivity and Calb expression, and increased TRPV6 expression in the uterus under thermoneutrality. This is the first study that describes the gene and protein expression of calcium transporters in the intestine, kidney and uterus of laying quails, and concludes that the use of methionine supplementation is justifiable in order to partially reverse the deleterious effects of thermal stress on the production.
The metabolic intermediates of nitrogen source have been proved to have multiple functions on the metabolism of mehthanotrophs. In this study, accumulation and assimilation mechanism of the nitrate metabolic intermediate ammonium in the fast growing Methylomonas sp. ZR1 was analyzed. Although, nitrate salt was the best nitrogen source supporting the growth of ZR1, its metabolic intermediate ammonium would accumulate and inhibit ZR1. Kinetic studies indicated that accumulation of NH4+ was deduced from the imbalance of nitrogen and carbon metabolism. Compensation of carbon skeleton -keto-glutaramate could effectively relieve the inhibition of NH4+ to ZR1, which further approved the assumption. qPCR analysis indicated a third ammonium assimilation pathway Glycine synthesis system may function in ZR1 under high ammonium tension. In the presence of ammonium, ZR1 might employ two strategies to relieve the ammonium stress, one was assimilating the excess ammonium, and another one was cutting off the nitrogen reduction reactions. Investigation of the nitrogen metabolism and its influence to the carbon metabolism is meaningful to systematically understand and control the C1 feedstock bioconversion process in methanotrophs. ImportanceThe nitrogen metabolism in methanotrophs has long been concerned. However, there are lots of research problems yet to be solved. In this study, the accumulation and assimilation mechanism of the nitrogen metabolic intermediate ammonium in the fast growing Methylomonas sp. ZR1 was analyzed. Owing to the imbalance metabolism of carbon and nitrogen source, ammonium would accumulate to high concentrations to inhibit cell growth. Compensation of carbon skeleton was an effective strategy to relieve the inhibition of NH4+. A third ammonium assimilation pathway related genes were proved actively expressing in ZR1 when it confronted with high ammonium tension. When confronted with ammonium tension, ZR1 might employ different strategies to relieve the ammonium stress according to the edible carbon source. Revealing the endogenous ammonium accumulation mechanism and its metabolic adjustment effect on the central metabolism of methanotrophs, was meaningful to reveal the complex coordination metabolic mechanism of nitrogen and carbon in methanotrophs.
BackgroundBiological methylation requires S-adenosylmethionine (SAM) and participates in a range of processes from modulation of gene expression via histone modifications to neurotransmitter synthesis. An important factor in all methylation reactions is the concentration ratio of SAM to methylation byproduct S-adenosylhomocysteine (SAH). SAH hydrolase, also known as adenosylhomocysteinase, depletes SAH and thereby facilitates metabolite recycling and maintains the methylation permissive SAM/SAH ratio. While the importance of SAH hydrolase in sustaining methylation is obvious on the cellular level, the function of this metabolic process on the organismal scale is not clear. ResultsWe used planarian Dugesia japonica to investigate the role SAH hydrolase in physiological homeostasis on the body-wide scale. Remarkably, pharmacological inhibition of the SAH hydrolase results in regression of anterior tissues and is accompanied by extensive apoptosis throughout the planarian body. Moreover, exposure to the SAHH inhibitor AdOx leads to changes in brain morphology and spatial shift in the expression of Wnt-modulator Notum. Strikingly, planarians are able to overcome these destructive patterning defects through regeneration of the anterior tissues and adaptation to the used inhibitor. Transcriptome analysis indicates that resistance to the SAHH inhibitor is at least partly mediated by changes in folate cycle and lipid metabolism. ConclusionsSAH hydrolase plays a critical role in planarian homeostasis and anterior patterning potentially through modulation of Wnt signaling. Moreover, planarian adaptation to the SAHH inhibitor via metabolic reprogramming suggests potential targets for addressing methylation-related human conditions.
We previously reported that cell-free heme (CFH) is increased in the plasma of patients with acute and chronic lung injury and causes pulmonary edema in animal model of acute respiratory distress syndrome (ARDS) post inhalation of halogen gas. However, the mechanisms by which CFH causes pulmonary edema are unclear. Herein we report for the first time the presence of CFH and chlorinated lipids (formed by the interaction of halogen gas, Cl2, with plasmalogens) in the plasma of patients and mice exposed to Cl2 gas. Ex vivo incubation of red blood cells (RBC) with halogenated lipids caused oxidative damage to RBC cytoskeletal protein spectrin, resulting in hemolysis and release of CFH. A single intramuscular injection of the heme-scavenging protein hemopexin (4 {micro}g/kg body weight) in mice, one hour post halogen exposure, reversed RBC fragility and decreased CFH levels to those of air controls. Patch clamp and short circuit current measurements revealed that CFH inhibited the activity of amiloride-sensitive (ENaC) and cation sodium (Na+) channels in mouse alveolar cells and trans-epithelial Na+ transport across human airway cells with EC50 of 125 nM and 500 nM, respectively. Molecular modeling identified 22 putative heme-docking sites on ENaC (energy of binding range: 86-1563 kJ/mol) with at least 2 sites within its narrow transmembrane pore, potentially capable of blocking Na+ transport across the channel. In conclusion, results suggested that CFH mediated inhibition of ENaC activity may be responsible for pulmonary edema post inhalation injury.
AO_SCPLOWBSTRACTC_SCPLOWMetabolic rate is often measured as a phenotype in evolutionary genetics studies because it impacts organismal fitness, is repeatable and heritable, and is responsive to numerous environmental variables. Despite a wide body of literature about metabolic rates, key questions remain unanswered: 1) why do individuals from the same population exhibit up to three fold differences in metabolic rate, 2) how does metabolic rate change during an individuals lifetime, and 3) what metabolic rate is advantageous in a specific environment? Current low throughput approaches to measure metabolic rate make it difficult to answer these and other relevant ecological and evolutionary questions that require a much larger sample size. Here we describe a scalable high-throughput intermittent flow respirometer (HIFR) design and use it to measure the metabolic rates of 20 aquatic animals simultaneously while reducing equipment costs and time by more than 50%.
Skeletal muscle function is inferred from the spatial arrangement of myofiber architecture and the molecular and metabolic features of myofibers. Features of myofiber types can be distinguished by the expression of myosin heavy chain (MyHC) isoforms, indicating contraction properties. In most studies, a local sampling, typically obtained from the median part of the muscle, is used to represent the whole muscle. It remains largely unknown to what extent this local sampling represents the entire muscle. Here we studied myofiber architecture over the entire wild type mouse tibialis anterior muscle, using a high-throughput procedure combining automatic imaging and image processing analyses. We reconstructed myofiber architecture from consecutive cross-sections stained for laminin and MyHC isoforms. The data showed a marked variation in myofiber geometric features, as well as MyHC expression and the distribution of neuromuscular junctions, and suggest that muscle regions with distinct properties can be defined along the entire muscle. We show that in these muscle regions myofiber geometric properties align with biological function and propose that future studies on muscle alterations in pathological or physiological conditions should consider the entire muscle.
Extreme heat directly limits an organisms survival and reproduction, but scientists cannot agree on what causes organisms to lose function or die during heating. According to the theory of oxygen- and capacity-limitation of thermal tolerance, heat stress occurs when a warming organisms demand for oxygen exceeds its supply, triggering a widespread drop in ATP concentration. This model predicts that an organisms heat tolerance should decrease under hypoxia, yet most terrestrial organisms tolerate the same amount of warming across a wide range of oxygen concentrations. This point is especially true for adult insects, who deliver oxygen through highly efficient respiratory systems. However, oxygen limitation at high temperatures may be more common during immature life stages, which have less developed respiratory systems. To test this hypothesis, we measured the effects of heat and hypoxia on the survival of locusts (Schistocerca cancellata) throughout development. We demonstrate that the heat tolerance of locusts depends on oxygen supply during the first instar but not during later instars. This finding provides further support for the idea that oxygen limitation of thermal tolerance depends on respiratory performance, especially during immature life stages.
ContextCold-induced activation of thermogenesis modulates energy metabolism, but the role of humoral mediators is not completely understood. ObjectiveTo investigate the role of parathyroid and thyroid hormones in acute and adaptive response to cold in humans. DesignCross-sectional study examining acute response to ice-water swimming and to experimental non-shivering thermogenesis (NST) induction in individuals acclimatized and non-acclimatized to cold. Seasonal variation in energy metabolism of ice-water swimmers and associations between circulating PTH and molecular components of thermogenic program in brown adipose tissue (BAT) of neck-surgery patients were evaluated. SettingClinical Research Center. Patients, ParticipantsIce-water swimmers (winter swim n=15, NST-induction n=6), non-acclimatized volunteers (NST-induction, n=11, elective neck surgery n = 36). Main Outcomes and ResultsIn ice-water swimmers, PTH and TSH increased in response to 15min winter swim, while activation of NST failed to regulate PTH and lowered TSH. In non-acclimatized men, NST-induction decreased PTH and TSH. Positive correlation between systemic levels of PTH and whole-body metabolic preference for lipids as well as BAT 18F-FDG uptake was found across the two populations. Moreover, NST-cooling protocol-induced changes in metabolic preference for lipids correlated positively with changes in PTH. Finally, variability in circulating PTH correlated positively with UCP1/UCP1, PPARGC1A and DIO2 in BAT from neck surgery patients. ConclusionsRegulation of PTH and thyroid hormones during cold exposure in humans depends on the cold acclimatization level and/or cold stimulus intensity. Role of PTH in NST is substantiated by its positive relationships with whole-body metabolic preference for lipids, BAT volume and UCP1 content.
The intrinsic lymphatic contractile activity is necessary for proper lymph transport. Mesenteric lymphatic vessels from high-fructose diet-induced metabolic syndrome (MetSyn) rats exhibited impairments in its intrinsic phasic contractile activity; however, the molecular mechanisms responsible for the weaker lymphatic pumping activity in MetSyn conditions are unknown. Several metabolic disease models have shown that dysregulation of sarcoplasmic reticulum Ca2+ ATPase (SERCA) pump is one of the key determinants of the phenotypes seen in various muscle tissues. Hence, we hypothesized that a decrease in SERCA pump expression and/or activity in lymphatic muscle influences the diminished lymphatic vessel contractions in MetSyn animals. Results demonstrated that SERCA inhibitor, thapsigargin, significantly reduced lymphatic phasic contractile frequency and amplitude in control vessels, whereas, the reduced MetSyn lymphatic contractile activity was not further diminished by thapsigargin. While SERCA2a expression was significantly decreased in MetSyn lymphatic vessels, myosin light chain 20, MLC20 phosphorylation was increased in these vessels. Additionally, insulin resistant lymphatic muscle cells exhibited elevated intracellular calcium and decreased SERCA2a expression and activity. The SERCA activator, CDN 1163 increased phasic contractile frequency in the vessels from MetSyn, thereby, partially restored lymph flow. Thus, our data provide the first evidence that SERCA2a modulates the lymphatic pumping activity by regulating phasic contractile amplitude and frequency, but not the lymphatic tone. Diminished lymphatic contractile activity in the vessels from the MetSyn animal is associated with the decreased SERCA2a expression and impaired SERCA2 activity in lymphatic muscle.
This physio-ecological study investigated the physiology and distribution of Lamellodysidea sp. in Moorea, French Polynesia. Specifically, its distribution was described across three reef types--fringing, mid-barrier, and barrier reefs--as well as across sites between Cooks Bay and Opunohu Bay. Additionally, filtration experiments were conducted to test how temperature impacts filtration efficiency. This is informative when predicting how future ocean temperatures are going to affect sponges success and distribution. Sponge abundance was found to decrease from the fringing reef out to the barrier reef, with the highest number of sponges in the fringing reef, less in the mid-barrier reef, and none found in the barrier reef. Sponges were also unevenly distributed across sites, with sponge abundance clearly increasing as you move away from Cooks Bay. Together, these data show that sponges demonstrate habitat preference that is related to their physiological tolerances. Filtration data showed that over a 3-hour period sponges increase their physiological output when introduced to environments 3-6 degrees Celsius above normal. This suggests that as ocean temperatures warm, sponges are likely to increase their filtration efficiency, thus increasing their ecological role as filter feeders, or struggle to survive at this new level of physiological function.
ZIKV infection is associated with pregnancy loss, fetal microcephaly and other malformations. While Aedes sp. of mosquito are the primary vector for ZIKV, sexual transmission of ZIKV is a significant route of infection. ZIKV has been documented in human, mouse and non-human primate (NHP) semen. It is critical to establish NHP models of vertical transfer of ZIKV that recapitulate human ZIKV pathogenesis. We hypothesized that vaginal deposition of ZIKV infected baboon semen would lead to maternal infection and vertical transfer in the olive baboon (Papio anubis). Timed pregnant baboons (n=6) were inoculated via vaginal deposition of baboon semen containing 106 ffu ZIKV (n=3, French Polynesian isolate:H/PF/2013, n=3 Puerto Rican isolate:PRVABC59) at mid-gestation (86-95 days gestation [dG]; term 183dG) on day (d) 0 (all dams), and then at 7 day intervals through three weeks. Maternal blood, saliva and cervico-vaginal washes were obtained at select days post-inoculation. Animals were euthanized at 28 days post initial inoculation (dpi; n=5) or 39 dpi (n=1) and maternal/fetal tissues collected. vRNA was quantified by qPCR. Viremia was achieved in 3/3 FP ZIKV infected dams and 2/3 PR ZIKV. ZIKV RNA was detected in cvw (5/6 dams;). ZIKV RNA was detected in lymph nodes, but not ovary, uterus, cervix or vagina in the FP ZIKV dams but was detected in uterus, vagina and lymph nodes. Placenta, amniotic fluid and all fetal tissues were ZIKV RNA negative in the FP infected dams whereas 2/3 PR infected dam placentas were ZIKV RNA positive. We conclude that ZIKV infected semen is a means of ZIKV transmission during pregnancy in primates. The PR isolate appeared more capable of wide spread dissemination to tissues, including placenta compared to the FP strain. IMPORTANCEDue to its established link to pregnancy loss, microcephaly and other major congenital anomalies, Zika virus (ZIKV) remains a worldwide health threat. Although mosquitoes are the primary means of ZIVK transmission, sexual transmission in human populations is well documented and provides a means for widespread dissemination of the virus. Differences in viremia, tissue distribution, immune responses and pregnancy outcome from sexually transmitted ZIKV compared to the subcutaneous route of infection are needed to better clinically manage ZIKV in pregnancy. Through our previous work, we have developed the olive baboon as a non-human primate model of ZIKV infection that is permissible to ZIKV infection via the subcutaneous route of inoculation and transfer of ZIKV to the fetus in pregnancy. The current study evaluated the course of ZIKV infection after vaginal inoculation of ZIKV in pregnant baboons at mid-gestation using baboon semen as the carrier and comparing two isolates of ZIKV, the French Polynesian isolate first associated with microcephaly and the Puerto Rican isolate, associated with an increased risk of microcephaly observed in the Americas.
Mesial temporal lobe epilepsy (MTLE) is the most common form of focal epilepsy in adults and is typically associated with hippocampal sclerosis and drug-resistant seizures. As an alternative to curative epilepsy surgery, brain stimulation evolves as a promising approach for seizure-interference. However, particularly in MTLE with severe hippocampal sclerosis, current stimulation protocols are often not effective. Here, we show that optogenetic low-frequency stimulation (oLFS) of entorhinal afferents exhibits unprecedented anti-ictogenic effects in chronically epileptic mice. Photostimulation at 1 Hz resulted in an almost complete suppression of focal seizures, independent of the degree of hippocampal sclerosis. Furthermore, by performing oLFS for 30 min before a pro-convulsive stimulus, seizure generalization was successfully prevented. Finally, acute slice experiments revealed a decreased excitability upon oLFS, which may partially explain the observed anti-epileptic effects. Taken together, our results suggest that oLFS of entorhinal afferents constitutes a promising approach for seizure control in MTLE.
BackgroundProtease-activated receptor 2 (PAR-2) is activated by proteases involved in allergy and triggers airway epithelial secretion and inflammation. PAR-2 is normally expressed basolaterally in differentiated nasal ciliated cells. ObjectiveWe tested if epithelial remodeling during diseases characterized by loss of cilia and squamous metaplasia may alter PAR-2 polarization. MethodsEndogenous PAR-2 responses were measured by live cell calcium and cilia imaging, measurement of fluid secretion, and quantification of cytokines. We utilized airway squamous cell lines, primary differentiated air-liquid interface cultures, and tissue explants. Cells were exposed to disease-related modifiers that alter epithelial morphology, including IL-13, cigarette smoke condensate, and retinoic acid deficiency. We used concentrations and exposure times that altered epithelial morphology without causing breakdown of the epithelial barrier, likely reflecting early disease states. ResultsPAR-2 signaling in airway squamous cells activated calcium and inflammatory responses. Squamous cells cultured at air liquid interface (ALI) responded to PAR-2 agonists applied both apically and basolaterally. Primary well-differentiated nasal epithelial ALI cultures responded only to basolateral PAR-2 stimulation. Primary cultures exposed to IL-13, cigarette smoke condensate, or reduced retinoic acid responded to both apical and basolateral PAR-2 stimulation. Nasal polyp tissue, but not control middle turbinate, exhibited apical calcium responses to PAR-2 stimulation. However, isolated ciliated cells from both polyp and turbinate maintained basolateral PAR-2 polarization. ConclusionsSquamous metaplasia and/or loss of cilia enhances apical PAR-2 responses. Altered PAR-2 polarization in dedifferentiated or remodeled epithelia may contribute to increased sensitivity to inhaled protease allergens in inflammatory airway diseases.
There are many situations in physiological and pharmacological analyses where multivariate data is collected. Frequently these are analysed with t-tests and multiple (Bonferroni) comparisons or ANOVA with post-hoc test. Increasingly, even with more powerful computers many variables and it seems that feature reduction would be a useful approach. The most commonly used method is principle component analyses, but in this report we compare this to a technique developed for genetic analyses, discriminant analysis of principle component (DAPC) analyses. A simple to use and well-maintained library exists for DAPC analyses, Adegenet2, and using this we find that DAPC detects differences between synthetic physiological datasets with significantly greater accuracy than traditional PCA.
Type 2 diabetes (T2D) is a metabolic disorder characterized by hyperglycemia, hyperinsulinemia and insulin resistance (IR). During the early phase of T2D, insulin synthesis and secretion by pancreatic {beta} cells is enhanced, which can lead to proinsulin (ProIns) misfolding that aggravates endoplasmic reticulum (ER) homeostasis in {beta} cells. Moreover, increased insulin in the circulation may contribute to fatty liver disease. Medical interventions aimed at alleviating ER stress in {beta} cells while maintaining optimal insulin secretion are therefore an attractive therapeutic strategy for T2D. Previously, we demonstrated that germline Chop gene deletion preserved {beta} cells in high fat diet (HFD) fed mice and in leptin receptor-deficient db/db mice. In the current study, we further investigated whether targeting Chop/Ddit3 specifically in murine {beta} cells confers therapeutic benefits. First, we show that Chop deletion in {beta} cells alleviates {beta} cell ER stress and delays glucose-stimulated insulin secretion (GSIS) in HFD fed mice. Second, importantly, {beta} cell-specific Chop deletion prevented liver steatosis and hepatomegaly in aged HFD fed mice without affecting basal glucose homeostasis. Third, we provide the first mechanistic evidence that ER remodeling secondary to Chop deletion modulates glucose-induced islet Ca2+ oscillations. Finally, using state-of-the-art GLP1-conjugated Chop AntiSense Oligonucleotides (GLP1-Chop ASO), we demonstrated that the Chop deletion induced GSIS change is a long term complex event in {beta} cells. In summary, our results demonstrate that Chop depletion in {beta} cells is a new therapeutic strategy to alleviate dysregulated insulin secretion and the consequently fatty liver disease in T2D.
Insects use rapid acclimation to enhance their tolerance of abiotic stresses within minutes to hours. These responses are critical adaptations for insects and other small ectotherms to tolerate drastic changes in temperature, hydration, or other factors that can fluctuate precipitously with ambient conditions or as a result of behavior. Rapid cold-hardening, where insects use brief exposure to modest chilling as a cue to enhance their cold tolerance, is the most thoroughly-studied of these responses and relatively little is known about rapid acclimation that is either triggered by or enhances tolerance of other abiotic stresses. Here, we used larvae of the Antarctic midge, Belgica antarctica, a polar extremophile that routinely experiences numerous stresses in nature, to investigate how 2 h exposure to modest environmental stresses affect stress tolerance in insects. Brief pretreatment by various stresses, including hyperosmotic challenge, hypoosmotic challenge, acidity, basicity, and UV irradiation enhanced stress tolerance in B. antarctica larvae relative to untreated controls. These results indicate that numerous environmental cues can trigger rapid acclimation in insects and that these responses can enhance tolerance of multiple stresses.
A growing number of long non-coding RNAs (lncRNAs) have emerged as vital metabolic regulators in research animals suggesting that lncRNAs could also play an important role in human metabolism. However, most human lncRNAs are non-conserved, vastly limiting our ability to identify human lncRNA metabolic regulators (hLMRs). As the sequence-function relation of lncRNAs has yet to be established, the identification of lncRNA metabolic regulators in animals often relies on their regulations by experimental metabolic conditions. But it is very challenging to apply this strategy to human lncRNAs because well-controlled human data are much limited in scope and often confounded by genetic heterogeneity. In this study, we establish an efficient pipeline to identify putative hLMRs that are metabolically sensitive, disease-relevant, and population applicable. We first progressively processed human transcriptome data to select human liver lncRNAs that exhibit highly dynamic expression in the general population, show differential expression in a metabolic disease population, and response to dietary intervention in a small disease cohort. We then experimentally demonstrated the responsiveness of selected hepatic lncRNAs to defined metabolic milieus in a liver-specific humanized mouse model. Furthermore, by extracting a concise list of protein-coding genes that are persistently correlated with lncRNAs in general and metabolic disease populations, we predicted the specific function for each hLMR. Using gain- and loss-of-function approaches in humanized mice as well as ectopic expression in conventional mice, we were able to validate the regulatory role of one non-conserved hLMR in cholesterol metabolism. Mechanistically, this hLMR binds to an RNA-binding protein, PTBP1, to modulate the transcription of cholesterol synthesis genes. In summary, our study provides a pipeline to overcome the variabilities intrinsic to human data to enable the efficient identification and functional definition of hLMRs. The combination of this bioinformatic framework and humanized murine model will enable broader systematic investigation of the physiological role of disease-relevant human lncRNAs in metabolic homeostasis.
BackgroundFemales have higher inflammatory pain representation. However, sex differences in central pain sensitization and the regulation of nociceptive response to peripheral inflammation remain unclear. The central pain sensitization is mediated by inhibitory neurotransmission and glial cell activity dysregulation where spinal glycine and GLP-1 receptors have described play a critical role. ObjectivesThe aim of this study was to compare the mechanical withdrawal nociceptive threshold with spinal glycine receptor subunits and GLP-1 expression in adult male and female rats after inflammatory hypersensitivity. MethodsSex differences in inflammatory nociception were evaluated before and after intraplantar hindpaw Zymosan A injection in Sprague-Dawley rats. Mechanical paw withdrawal thresholds were tested using von Frey filaments. Western blot was used to measure GlyRs subunits protein levels in the spinal cord. GLP-1 was determined using the Magnetic Luminex Assay. ResultsA reduced nociceptive threshold was observed in males and females rats after 4 hours of inflammatory Zymosan A injection. However, this reduction was significantly major in females. Western blot analysis demonstrated significantly increased 1, 2, 3 and {beta} GlyR subunit levels in male rats. Female rats only increased 3 and {beta} GlyR subunits after Zymosan A injection. GLP-1 was reduced in female spinal tissues after an inflammatory injury. ConclusionsOur study indicates that sex differences in nociceptive threshold after inflammatory Zymosan A rat pain sensitization is related to the sex differences in glycine receptor subunits and GLP-1 expression at the spinal cord.
Speech fluency can be impaired in stressful situations. The aim of the present study was to investigate whether a verbal fluency task without any further stress induction by itself induces responses of the hypothalamic-pituitary adrenal (HPA) axis and of the sympathetic nervous system (SNS). The sample consisted of n = 85 participants (68.2% female; 33.3 {+/-} 15.2 years, BMI = 23.7 {+/-} 4.3 kg/m2) who performed two consecutive verbal fluency tasks for two minutes each. The categories were either stress or disease and animals or foods which were presented in a randomized order. Three saliva samples were collected, prior to the task (t0), immediately after (t1), and ten minutes after (t2). Salivary -amylase and cortisol were assessed. Furthermore, blood pressure, heart rate, and subjective ratings of actual stress perception, level of effort, and tiredness were measured. The verbal fluency task induced an HPA axis response with a maximum cortisol level at t2 (p < .001, {eta}p2 = .19) which was independent of task performance. Furthermore, subjective stress and effort as well of tiredness increased immediately after the task (all p < .001; all d [≥] 1.0). Tiredness immediately after the task was negatively correlated with task performance (p = .002). No -amylase, blood pressure or heart rate responses were found. We conclude that a verbal fluency task acts like an acute stressor that induces a cortisol response without the need of further (e.g., social-evaluative) stress components. Therefore, it is a little time-consuming alternative to other stress tasks that can be used in field studies with little effort.
Despite high metabolic activity, the retina and optic nerve head lack traditional lymphatic drainage. We here identified a novel ocular glymphatic clearance route for fluid and wastes via the proximal optic nerve. Amyloid-{beta} (A{beta}) was cleared from the vitreous via a pathway driven by the ocular-cranial pressure difference. After traversing the lamina barrier, intra-axonal A{beta} was cleared via the perivenous space and subsequently drained to lymphatic vessels. Light-induced pupil constriction enhanced, while atropine or raising intracranial pressure blocked efflux. In two distinct murine models of glaucoma, A{beta} leaked from the eye via defects in the lamina barrier instead of directional axonal efflux. The discovery of a novel pathway for removal of fluid and metabolites from the intraocular space prompts a reevaluation of the core principles governing eye physiology and provides a framework for new therapeutic approaches to treat common eye diseases, including glaucoma. One Sentence SummaryGlymphatic pathway clears ocular amyloid-{beta} via optic nerve and fails in glaucoma.
To forage in fast, turbulent flow environments where prey are abundant, predatory fishes must deal with the high associated costs of locomotion. Prevailing theory suggests that many species exploit hydrodynamic refuges to minimize the cost of locomotion while foraging. Here we challenge this theory based on direct oxygen consumption measurements of drift-feeding trout (Oncorhynchus mykiss) foraging in the freestream and from behind a flow refuge at velocities up to 100 cm s-1. We demonstrate that refuging is not energetically beneficial when foraging in fast flows due to a high attack cost and low prey capture success associated with leaving a station-holding refuge to intercept prey. By integrating optimum foraging theory with empirical data from respirometry and video imaging, we develop a mathematical model to predict when drift-feeding fishes should exploit or avoid refuges based on prey density, size and flow velocity. Our foraging and refuging model provides new mechanistic insights into the locomotor costs, habitat use, and prey selection of fishes foraging in current-swept habitats.
Type 2 diabetes and obesity are associated with back pain in juveniles and adults and are implicated in intervertebral disc (IVD) degeneration. Hypercaloric Western diets are associated with both obesity and type 2 diabetes. The objective of this study was to determine if obesity and type 2 diabetes result in spinal pathology in a sex-specific manner using in vivo diabetic and dietary mouse models. Leptin is an appetite-regulating hormone, and its deficiency leads to polyphagia, resulting in obesity and diabetes. Leptin is also associated with IVD degeneration, and increased expression of its receptor was identified in degenerated IVDs. We used young, leptin receptor deficient (Db/Db) mice to mimic the effect of diet and diabetes on adolescents. Db/Db and Control mice were fed either Western or Control diets, and were sacrificed at 3 months of age. Db/Db mice were obese, while only female mice developed diabetes. Female Db/Db mice displayed altered IVD morphology, with increased intradiscal notochordal band area, suggesting delayed IVD cell proliferation and differentiation, rather than IVD degeneration. Motion segments from Db/Db mice exhibited increased failure risk with decreased torsional failure strength. Db/Db mice also had inferior bone quality, which was most prominent in females. We conclude that obesity and diabetes due to impaired leptin signaling contribute to pathological changes in vertebrae, as well as an immature IVD phenotype, particularly of females, suggesting a sex-dependent role of leptin in the spine.
Aberrant lipid metabolism promotes the development of skeletal muscle insulin resistance, but the exact identity of lipid-mediated mechanisms relevant to human obesity remains unclear. A comprehensive lipidomic analyses of primary myocytes from lean insulin-sensitive (LN) and obese insulin-resistant (OB) individuals revealed several species of lysophospholipids (lyso-PL) that were differentially-abundant. These changes coincided with greater expression of lysophosphatidylcholine acyltransferase 3 (LPCAT3), an enzyme involved in phospholipid transacylation (Lands cycle). Strikingly, mice with skeletal muscle-specific knockout of LPCAT3 (LPCAT3-MKO) exhibited greater muscle lyso-PC/PC, concomitant with greater insulin sensitivity in vivo and insulin-stimulated skeletal muscle glucose uptake ex vivo. Absence of LPCAT3 reduced phospholipid packing of the cellular membranes and increased plasma membrane lipid clustering, suggesting that LPCAT3 affects insulin receptor phosphorylation by modulating plasma membrane lipid organization. In conclusion, obesity accelerates the skeletal muscle Lands cycle, whose consequence might induce the disruption of plasma membrane organization that suppresses muscle insulin action.
Human reproductive success depends on a properly decidualized uterine endometrium that allows implantation and the formation of the placenta. At the core of the decidualization process are endometrial stromal fibroblasts (ESF) that differentiate to decidual stromal cells (DSC). As variations in oxygen levels are functionally relevant in endometrium both upon menstruation and during placentation, we assessed the transcriptomic responses to hypoxia in ESF and DSC. In both cell types hypoxia upregulated genes in classical hypoxia pathways such as glycolysis and the epithelial mesenchymal transition. In DSC hypoxia restored an ESF like transcriptional state for a subset of transcription factors that are known targets of the progesterone receptor, suggesting that hypoxia partially interferes with progesterone signaling. In both cell types hypoxia modified transcription of several inflammatory transcription factors that are known regulators of decidualization, including decreased transcription of STATs and increased transcription of CEBPs. We observed that hypoxia upregulated genes had a significant overlap with genes previously detected to be upregulated in endometriotic stromal cells. Promoter analysis of the genes in this overlap suggested the hypoxia upregulated Jun/Fos and CEBP transcription factors as potential drivers of endometriosis-associated transcription. Using immunohistochemistry we observed increased expression of JUND and CEBPD in endometriosis lesions compared to healthy endometria. Overall the findings suggest that hypoxic stress establishes distinct transcriptional states in ESF and DSC, and that hypoxia influences the expression of genes that contribute to the core gene regulation of endometriotic stromal cells.
The identification of genes and interventions that slow or reverse aging is hampered by the lack of non-invasive metrics that can predict life expectancy of pre-clinical models. Frailty Indices (FIs) in mice are composite measures of health that are cost-effective and non-invasive, but whether they can accurately predict health and lifespan is not known. Here, mouse FIs were scored longitudinally until death and machine learning was employed to develop two clocks. A random forest regression was trained on FI components for chronological age to generate the FRIGHT (Frailty Inferred Geriatric Health Timeline) clock, a strong predictor of chronological age. A second model was trained on remaining lifespan to generate the AFRAID (Analysis of Frailty and Death) clock, which accurately predicts life expectancy and the efficacy of a lifespan-extending intervention up to a year in advance. Adoption of these clocks should accelerate the identification of novel longevity genes and aging interventions.
The expiratory neurons of the Botzinger complex (BotC) provide inhibitory inputs to the respiratory network, which, during eupnea, are critically important for respiratory phase transition and duration control. Herein, we investigated how the BotC neurons interact with the expiratory oscillator located in the parafacial respiratory group (pFRG) and control the abdominal activity during active expiration. Using the decerebrated, arterially perfused in situ rat preparations, we recorded the neuronal activity and performed pharmacological manipulations of the BotC and pFRG during hypercapnia or after the exposure to short-term sustained hypoxia - conditions that generate active expiration. The experimental data were integrated in a mathematical model to gain new insights in the inhibitory connectome within the respiratory central pattern generator. Our results reveal a complex inhibitory circuitry within the BotC that provides inhibitory inputs to the pFRG thus restraining abdominal activity under resting conditions and contributing to abdominal expiratory pattern formation during active expiration.
When exposed to prolonged anoxia insects enter a reversible coma during which neural and muscular systems temporarily shut down. Nervous system shut down is a result of spreading depolarization throughout neurons and glial cells. Upon return to normoxia, recovery occurs following the restoration of ion gradients. However, there is a delay in the functional recovery of synaptic transmission following membrane repolarization. In mammals, the build-up of extracellular adenosine following spreading depolarization contributes to this delay. Adenosine accumulation is a marker of metabolic stress and it has many downstream effects through the activation of adenosine receptors. Here we demonstrate that adenosine lengthens the time to functional recovery following anoxic coma in locusts. Caffeine, used as an adenosine receptor antagonist, decreased the time to recovery in intact animals and lengthened the time to recovery in semi-intact animals. Our results show that the rate of recovery in insect systems is affected by the presence of adenosine.
Vitamin D-binding protein (DBP) or GC-globulin carries vitamin D metabolites from the circulation to target tissues. DBP expression is highly-localized to the liver and pancreatic -cells. While DBP serum levels, gene polymorphisms and autoantigens have all been associated with diabetes risk, the underlying mechanisms remain unknown. Here, we show that DBP regulates -cell morphology, -cell function and glucagon secretion. Deletion of DBP led to smaller and hyperplastic -cells, altered Na+ channel conductance, impaired -cell activation by low glucose, and reduced rates of glucagon secretion. Mechanistically, this involved reversible changes in islet microfilament abundance and density, as well as changes in glucagon granule distribution. Defects were also seen in {beta}-cell and {delta}-cell function. Immunostaining of human pancreata revealed generalized loss of DBP expression as a feature of late-onset and longstanding, but not early-onset type 1 diabetes. Thus, DBP is a critical regulator of -cell phenotype, with implications for diabetes pathogenesis. HIGHLIGHTSO_LIDBP expression is highly-localized to mouse and human -cells C_LIO_LILoss of DBP increases -cell number, but decreases -cell size C_LIO_LI-cells in DBP knockout islets are dysfunctional and secrete less glucagon C_LIO_LIDBP expression is decreased in -cells of donors with late-onset or longstanding type 1 diabetes C_LI
Adult body size is determined by the quality and quantity of nutrients available to animals. In insects, nutrition affects adult size primarily during the nymphal or larval stages. However, measures of adult size like body weight are likely to also change with adult nutrition. In this study, we sought to the roles of nutrition throughout the life cycle on adult body weight and the size of two appendages, the wing and the femur, in the fruit fly Drosophila melanogaster. We manipulated nutrition in two ways: by varying the protein to carbohydrate content of the diet, called macronutrient restriction, and by changing the caloric density of the diet, termed caloric restriction. We employed a fully factorial design to manipulate both the larval and adult diets for both diet types. We found that manipulating the larval diet had greater impacts on all measures of adult size. Further, macronutrient restriction was more detrimental to adult size than caloric restriction. For adult body weight, a rich adult diet mitigated the negative effects of poor larval nutrition for both types of diets. In contrast, small wing and femur size caused by poor larval diet could not be increased with the adult diet. Taken together, these results suggest that appendage size is fixed by the larval diet, while those related to body composition remain sensitive to adult diet. Further, our studies provide a foundation for understanding how the nutritional environment of juveniles affects how adults respond to diet.
Alpha adrenergic stimulation is known to produce vasoconstriction. We have earlier shown that, in spiral strips of small arteries Phenylephrine (PE) caused vasorelaxation under high nitric oxide (NO) environment. However on further experimentation it was realized that the PE-induced vasorelaxant response occurred only with longitudinal strips of small arteries even under normal NO environment while circular strips showed contraction with PE even under high NO environment. Such PE-induced vasorelaxation of longitudinal strips was blocked by Phentolamine, an alpha-adrenergic receptor blocker. On delineation of specific receptor subtype, PE-induced relaxation was found to be mediated through alpha 1D receptor. However, this phenomenon is specific to small artery, as longitudinal smooth muscle of aorta showed only contractile response to adrenergic stimulation. There is no prior report of longitudinal smooth muscle in small artery up to our knowledge. The results of this study and histological examination of vessel sections suggest the presence of longitudinal smooth muscle in small artery and their relaxant response to alpha adrenergic stimulation is a novel phenomenon.
Human food waste in cities presents urban wildlife with predictable, easily accessible high-calorie food sources, but this can be both beneficial and harmful for individual health. We analyzed body condition and serum chemistry (electrolyte levels, markers of kidney and liver function, protein, glucose, and cholesterol) in an urban and rural population of eastern grey squirrels (Sciurus carolinensis) to assess whether proximity to the human food waste that is associated with urban habitats had ill effects on health. We found no differences in body condition between habitats and no evidence of malnutrition at either site. However, urban squirrels had higher blood glucose, lower potassium, phosphorus, chloride, and albumin:globulin ratios. These results align with previous findings of increased dietary sugar in cities, and suggest that urban populations of grey squirrels are under greater environmental stress than rural populations.
AO_SCPLOWBSTRACTC_SCPLOWAgeing is the dominant risk factor for cardiovascular diseases. A great body of experimental data has been gathered on cellular remodelling in the Ageing myocardium from animals. Very few experimental data are available on age-related changes in the human cardiomyocyte. We have used our combined electromechanical model of the human cardiomyocyte and the population modelling approach to investigate the variability in the response of cardiomyocytes to age-related changes in the model parameters. To generate the model population, we varied nine model parameters and excluded model samples with biomarkers falling outside of the physiological ranges. We evaluated the response to age-related changes in four electrophysiological model parameters reported in the literature: reduction in the density of the K+ transient outward current, maximal velocity of SERCA, and an increase in the density of NaCa exchange current and CaL-type current. The sensitivity of the action potential biomarkers to individual parameter variations was assessed. Each parameter modulation caused an increase in APD, while the sensitivity of the model to changes in GCaL and Vmax_up was much higher than to those in the effects of Gto and KNaCa. Then 60 age-related sets of the four parameters were randomly generated and each set was applied to every model in the control population. We calculated the frequency of model samples with repolarisation anomalies (RA) and the shortening of the electro-mechanical window in the ageing model populations as an arrhythmogenic ageing score. The linear dependence of the score on the deviation of the parameters showed a high determination coefficient with the most significant impact due to the age-related change in the CaL current. The population-based approach allowed us to classify models with low and high risk of age-related RA and to predict risks based on the control biomarkers.
In this study we assessed the effects of increased cortisol levels during early embryonic development (0-6 hours post-fertilisation (hpf)), thereby mimicking maternal stress, on metabolism in zebrafish (Danio rerio) larvae. In two series of experiments fertilized eggs were exposed to a cortisol-containing, a dexamethasone-containing (to stimulate the glucocorticoid receptor (GR) specifically) or a control medium for 6 hours post-fertilisation (0-6 hpf). In the first series we measured oxygen consumption as a proxy for metabolism, in the second series gene-expression of genes related to gluconeogenesis and glucose transport. Previously we have found that at 5 days post-fertilisation (dpf) baseline cortisol levels are increased following cortisol pre-treatment but not following dexamethasone pre-treatment, suggesting a higher hypothalamus-pituitary-interrenal cells (HPI-axis) activity. Hence, we hypothesized that oxygen consumption and gene-expression were stronger in cortisol-treated than in dexamethasone-treated and control-treated subjects at 5 dpf. Indeed, we observed increased oxygen consumption in cortisol-treated subjects compared to dexamethasone-treated or control-treated subjects. However, gene-expression levels were not different between treatments, which may have been due to a developmental delay in this second series. We also reasoned that both cortisol-treated and dexamethasone-treated subjects would show a higher metabolism at 1 dpf than control-treated subjects as the HPI-axis is not functional as yet and more general processes are being stimulated by cortisol through GR stimulation. Indeed, we observed increased oxygen consumption and increased expression of genes related to gluconeogenesis and glucose transport in cortisol-treated and dexamethasone-treated subjects than control-treated subjects. These data show that early-life exposure to cortisol, mimicking thereby maternal stress, increased metabolism at different life stages, i.e. 1 and 5 dpf, involving the GR.
The aim of this study was to determine which physiological variables could predict performance during a CrossFit competition. Fifteen male CrossFit athletes (35 {+/-} 9 years) participated and performed a series of tests (incremental load test for full squat and bench press, jump tests, incremental running test, and Wingate test) that were used as potential predictors of CrossFit performance. Thereafter, they performed the five Workouts of the Day (WODs) corresponding to the CrossFit Games Open 2019, and the relationship between each variable and CrossFit performance was analyzed. Overall Crossfit performance (i.e., final ranking considering all WODs) was significantly related to jump ability, mean and peak power output during the Wingate test, relative maximum strength for the full squat and the bench press, and maximum oxygen uptake and maximum speed during an incremental running test (all p<0.05, r=0.58-0.75), although the relationship of most markers varied depending on the analyzed WOD. Multiple linear regression analysis showed that the combination of maximum oxygen uptake, squat jump ability, and reactive strength index accounted for 81% of the variance in overall CrossFit performance (p=0.0003). CrossFit performance seems dependent on a variety of power-, strength-, and aerobic-related markers, which reflects the complexity of this sport. Improvements in aerobic capacity may help people and athletes in CrossFit performance and well-being. Also, focus on lower body power could be the key to obtain better performance markers.
Toxic ions begin to accumulate in tissues of salt-stressed plants after the initial osmotic shock. In glycophytes, the ability to mobilize or sequester excess ions define tolerance mechanisms. Mobilization and sequestration of excess Na+ involves three transport mechanisms facilitated by the plasma membrane H+/Na+ antiporter (SOS1), vacuolar H+/Na+ antiporter (NHX1), and Na+/K+ transporter in vascular tissues (HKT1). While the cultivated Gossypium hirsutum (upland cotton) is significantly more tolerant to salinity relative to other crops, the critical factors contributing to the observed variation for tolerance potential across the germplasm has not been fully scrutinized. In this study, the spatio-temporal patterns of Na+ accumulation at different severities of salt stress were investigated across a minimal comparative panel representing the spectrum of genetic diversity across the improved cotton germplasm. The goal was to define the importance of integrative or network effects relative to the direct effects of Na+ homeostasis mechanisms mediated by GhHKT1, GhSOS1, and GhNHX1. Multi-dimensional physio-morphometric attributes were investigated in univariate and multivariate statistical contexts, as well as the relationship between variables using structural equation modeling. Results showed that mobilized or sequestered Na+ may contribute to the baseline salinity tolerance, but the observed variance in overall tolerance potential across a meaningful subset of the germplasm were more significantly associated to antioxidant capacity, maintenance of stomatal conductance, chlorophyll content, and divalent cations, and other physiological interactions occurring through complex networks. One-Sentence SummaryVariation in salinity tolerance potential across the tetraploid cultivated Gossypium germplasm is better explained by complex physiological networks rather than just cellular Na+ homeostasis.
Relatively little is known about regulated glucagon secretion by human islet cells compared to insulin secretion from {beta} cells, despite conclusive evidence of dysfunction in both cell types in diabetes mellitus. Distinct insulin sequences in humans and mice permit in vivo studies of {beta} cell regulation after human islet transplantation in immunocompromised mice, whereas identical glucagon sequences prevent analogous in vivo measures of glucagon output from human cells. We used CRISPR/Cas9 genome editing to remove glucagon-encoding codons 2-29 in immunocompromised (NSG) mice, preserving production of other proglucagon-derived hormones, like Glucagon-like-peptide 1. These NSG-Glucagon knockout (NSG-GKO) mice had phenotypes associated with glucagon signaling deficits, including hypoglycemia, hyperaminoacidemia, hypoinsulinemia, and islet cell hyperplasia. NSG-GKO host metabolic and islet phenotypes reverted after human islet transplantation, and human islets retained regulated glucagon and insulin secretion. NSG-GKO mice provide an unprecedented resource to investigate unique, species-specific human cell regulation in vivo.
Fibrosis can affect any organ resulting in the loss of tissue architecture and function with often life-threatening consequences. Pathologically, fibrosis is characterised by expansion of connective tissue due to excessive deposition of extracellular matrix proteins (ECM), including the fibrillar forms of collagen. A significant hurdle for discovering cures for fibrosis is the lack of suitable models and techniques to quantify mature collagen deposition in tissues. Here we have extensively characterized an ex-vivo cultured human lung derived, precision-cut lung slices model (hPCLS) using live fluorescence light microscopy as well as mass spectrometry-based techniques to obtain a proteomic and metabolomic fingerprint. Using an integrated approach of multiple readouts such as quantitative label-free Second Harmonic Generation (SHG) imaging to measure fibrillar collagen in the extracellular matrix and ELISA-based methods to measure soluble ECM biomarkers, we investigated TGFbeta1-mediated pro-fibrotic signalling in hPCLS. We demonstrate that hPCLS are viable and metabolically active with mesenchymal, epithelial, endothelial, and immune cells surviving for at least two weeks in ex vivo culture. Analysis of hPCLS-conditioned supernatants showed strong induction of ECM synthesis proteins P1NP and fibronectin upon TGFb stimulation. Importantly, this effect translated into an increased deposition of fibrillar collagen in ECM of cultured hPCLS as measured by a novel quantitative SHG-based imaging method only following addition of a metalloproteinase inhibitor (GM6001). Together the data show that an integrated approach of measuring soluble pro-fibrotic markers and quantitative SHG-based analysis of fibrillar collagen is a valuable tool for studying pro-fibrotic signalling and testing anti-fibrotic agents.
Boron (B) is indispensable for plant growth and has been reported in the mitigation of aluminum (Al) toxicity in different plants. This study unraveled the efficacy of B in reducing the toxicity of Al to trifoliate orange seedlings in a hydroponic experiment. In the current study, B supply had a positive effect on root length and plant growth-related parameters, and attenuated Al-induced inhibition of plasma membrane H+-ATPase activity. The results of XPS and SEM-EDS revealed that B reduces the Al accumulation in root cell wall (CW), especially acts on pectin fractions (alkali-soluble pectin), accompanied by suppressing the pectin synthesis, inhibiting pectin methylesterase (PME) activity and PME expression. Furthermore, B application inhibits NRAT1 expression while increases ALS1 expression, which are responsible for restraining Al transport from external cells to the cytoplasm and accelerating Al divert to vacuoles, and the results can be further demonstrated by TEM-EDS analysis. Taken together, our results indicated that B mainly promotes the efflux of H+ by regulating the plasma membrane H+-ATPase activity, futhur reduce the demethylation of pectin to weaken Al binding ability to carboxyl. More importantly, B alleviated some of the toxic effects of Al by decreasing the deposition of Al in cytoplasm and compartmentalizes Al into vacuoles. One-sentence summaryBoron can reduce the binding amount of carboxyl group to Al in pectin, decreasing the deposition of Al in cytoplasm and compartmentalizes Al into vacuoles, thereby reduce the toxicity of Al to plants..
Brassinosteroid (BR) is a pivotal phytohormone involved in regulating root development. Boron (B) is an essential micronutrient for plant growth and development, and root growth of plants is rapidly inhibited under B deficiency condition, but the mechanisms are still elusive. Here, we demonstrate that BR plays crucial roles in these processes. We identify BR-related processes underlying B deficiency at the physiological, genetic, molecular/cell biological and transcriptome levels, and provide strong evidences that B deficiency can affect BR signalling, thereby altering root growth. RNA-sequencing analysis reveals a high co-regulation between BR-regulated genes and B deficiency-responsive genes. We found that low B negatively regulates BR signalling to control BR signalling-dependent root elongation, bes1-D exhibits insensitivity to low B stress, and bri1-301 mutants fails to respond to B depletion. Exogenous eBL application can rescue the inhibition of root growth under B deficiency condition, and application of BR biosynthesis inhibitor BRZ aggravates root growth inhibition of wild-type under B deficiency condition. B deficiency reduces the nuclear signal of BES1. We further found that B deficiency reduces the accumulation of brassinolide (BL) by reducing BR6ox1 and BR6ox2 mRNA level to down-regulate BR signalling and modulate root elongation. Altogether, our results uncover a role of BR signalling in root elongation under B deficiency. One-sentence summaryB deficiency reduces the accumulation of brassinolide by reducing BR6ox1 and BR6ox2 mRNA level to down-regulate BR signalling and modulate root elongation.
ObjectivesTo determine the influences of age and gender on the taste functions of healthy Taiwanese. MethodsWe evaluated taste functions of healthy Taiwanese using the whole mouth suprathreshold taste test, along with the taste quad test. In the whole-mouth test, we applied in a counterbalanced order sweet, sour, salty, and bitter solutions, each at 5 different suprathreshold concentrations to subjects, who were instructed to sip and swish in mouth twice. Each subject had to indicate the taste quality, and to rate the intensity and unpleasantness/pleasantness of each taste of the solutions. In the quad test, the 4 quadrants of the tongue surface were tested by dripping one concentration of sweet, sour, salty, or bitter solutions for 6 times. Subjects then indicated the taste quality, and rated the intensity of the solution. ResultsSubjects were divided into groups based on their gender and age: 20-39 years, 40-59 years, or [≥] 60 years. We found that in the whole mouth taste test, the total correct identification score dropped with age. But identifying sweet and salty qualities was not affected by age. No differences were found between male and female, except women scored better than men for sweet quality in the age group of 40-59 years. The total correct identification score of the taste quad test also decreased with an increased age of the taste quad test, without gender differences. ConclusionBoth age and gender affected the taste functions in healthy Taiwanese to some extent, and differences were dependent on age, tongue region, and taste quality.
Temperature is a major factor that impacts tick populations by limiting geographic range of different species. Little is known about the thermal characteristics of these pests outside of a few studies on survival related to thermal tolerance. In this study, thermal tolerance limits, thermal preference, impact of temperature on metabolic rate, and temperature-activity dynamics were examined in larvae for six species of ixodid ticks. Tolerance of low temperatures ranged from -15 to -24{degrees}C with Dermacentor andersoni surviving at the lowest temperatures. High temperature survival ranged from 41 to 47 {degrees}C, with Rhipicephalus sanguineus having the highest upper lethal limit. Ixodes scapularis showed the lowest survival at both low and high temperatures. Thermal preference temperatures were tested from 0-41{degrees}C. D. variabilis exhibited a significant distribution of individuals in the lower temperatures, while the majority of other species gathered around 20-30{degrees}C. Activity was measured from 10-60{degrees}C, and the highest activity was observed in most species was near 30{degrees}C. Metabolic rate was the highest for most species around 40{degrees}C. Both activity and metabolic rate dropped dramatically at temperatures below 10{degrees}C and above 50{degrees}C. In summary, tick species vary greatly in their thermal characteristics, and our results will be critical to predict distribution of these ectoparasites with changing climates.

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