A combined computational and experimental approach to human osteocalcin and GPCR Family C Group 6 Member A

INTRODUCTION. The use of mouse genetics recently highlighted coordination by endocrine regulation between bone, energy metabolism, and endogenous sex hormones. Osteocalcin, a bone matrix protein that regulates hydroxyapatite size and shape through its vitamin‑K-dependent γ‑carboxylated form, unraveled endocrinological functions in its circulating forms, that are not only simple markers of bone formation. Undercarboxylated osteocalcin administration regulates gene and protein expression in adipocytes, in pancreatic β cells, and in Leydig cells by a G protein-coupled receptor, GPRC6A receptor, with a potential gender selectivity. Total serum osteocalcin concentrations in humans are inversely associated with measures of glucose metabolism and cardiovascular risk factors; however, human data are inconclusive with regard to the role of uncarboxylated osteocalcin because most studies do not account for the influence of vitamin K or differentiate between the different γ‑carboxylated forms of osteocalcin. Intriguingly GPRC6A receptor mediates also the non-genomic effects of androgens and, although the information about SHBG-receptor structure is not conclusive, evidence seems to suggest that a G protein-coupled receptor is involved. AIM of the STUDY. Starting from clinical observations, we investigated the protein-protein interaction computational predictors of osteocalcin and SHBG with GPRC6A receptor, and we validated experimentally in vitro in a two-step approach. MATHERIAL and METHODS. Clinical and biochemical characteristics were studied in a cohort of 91 obese patients and healthy controls in a cross-sectional study. 3-D protein structure alignment analysis and protein docking analysis were resolved with a four (i.e. TM-align, FATCAT, TriangleMatch, TopMatch) and three (i.e. GRAMM-X, ZDOCK, PatchDock) different algorithms approach, respectively. ClickMD-min script was used as a molecular dynamic platform for computational analysis of the conformational structure of osteocalcin in presence or absence of Ca2+, developed with MOE software. Steered molecular dynamics simulations related to the carboxylation state were performed, and the data compared with the spectroscopic techniques results. Gene and protein expression analysis and cell-surface receptor binding assays (i.e. immunofluorescence and flow cytometry analysis) on HEK-293T cells experimentally validated the former results in vitro. RESULTS. Our study shows for the first time the existence of the competition for a specific binding site between osteocalcin and SHBG on human cells expressing GPRC6A receptor. The results are supported by a computational prediction analysis, describing amino acid residues of SHBG from Gly145 to Leu161 as a highly predicted interface. Our experiments describe the structure of human osteocalcin in its carboxylated and undercarboxylated forms for the first time. The influence of the binding of calcium on osteocalcin structure seems to be stronger than the presence of γ-carboxylated glutamic acid residues. Our clinical data show an imbalance between the γ‑carboxylated forms of osteocalcin in a cohort of hypogonadic obese an overweight patients, with decreased levels of SHBG and altered levels of the other sex hormones. CONCLUSIONS. The current two-step approach offers a target approach of investigation directly in humans, that has the potential to identify novel pathophysiological pathways as well as novel therapeutic possibilities. The computational basis of the possible binding of SHBG and osteocalcin has been experimentally validated and can directly lead to the synthesis of a peptide, whose physiological and therapeutic implications represent a feasible perspective for future research in an area of paramount importance, such as the cross-talk between bone, energy metabolism, and endogenous sex hormones

Human Physiology During Exposure to the Cave Environment: A Systematic Review With Implications for Aerospace Medicine

Background: Successful long-duration missions outside low-Earth orbit will depend on technical and physiological challenges under abnormal environmental conditions. Caves, characterized by absence of light, confinement, three-dimensional human movement and long-duration isolation, are identifiably one of the earliest examples of scientific enquiry into space analogs. However, little is known about the holistic human physiological response during cave exploration or prolonged habitation.Objectives: The aim of our review was to conduct a systematic bibliographic research review of the effects of short and prolonged exposure to a cave environment on human physiology, with a view to extend the results to implications for human planetary exploration missions.Methods: A systematic search was conducted following the structured PRISMA (Preferred Reporting Items for Systematic Reviews and Meta-Analyses) guidelines for electronic databases.Results: The search retrieved 1,519 studies. There were 50 articles selected for further consideration, of which 31 met our inclusion criteria. Short-term cave exposure studies have investigated visual dysfunction, cardiovascular, endocrine-metabolic, immunologic-hematological and muscular responses in humans. Augmentations of heart rate, muscular damage, initial anticipatory stress reaction and inflammatory responses were reported during caving activity. Prolonged exposure studies mainly investigated whether biological rhythms persist or desist in the absence of standard environmental conditions. Changes were evident in estimated vs. actual rest-activity cycle periods and external desynchronization, body temperature, performance reaction time and heart rate cycles. All studies have shown a marked methodological heterogeneity and lack reproduction under controlled conditions.Conclusions: This review facilitates a further comparison of the proposed physiological impact of a subterranean space analog environment, with existing knowledge in related disciplines pertaining to human operative preparation under challenging environmental conditions. This comprehensive overview should stimulate more reproducible research on this topic and offer the opportunity to advance study design and focus future human research in the cave environment on noteworthy, reproducible projects

Monitoring body temperature during moderate intensity exercise and inactive recovery in the cold: a pilot study

Exposure to cold ambient conditions during outdoor recreation can lead to significant heat loss. It is unknown how fast body temperature decreases or how fast a person could become hypothermic in cold temperatures. We present a series of pilot tests involving moderate intensity exercise and inactive recovery in the cold to monitor how body temperature changes with exposure to -10°C. The primary aim of this pilot study was to test the feasibility of the proposed protocol with the intention to design a main study. The primary questions were: (i) to what degree does body temperature increase or decrease with this protocol, (ii) whether epitympanic temperature is a suitable measure of core temperature using a recently developed, non-invasive device and (iii) if participants are able to tolerate the cold during inactive recovery. This pilot series included seven participants. After an acclimatization phase (15 minutes), participants exercised at 60% peak heart rate (20 minutes) followed by a seated, inactive recovery phase (15 minutes) in the cold. The mean ambient conditions were -10.0±0.4°C and 66.1±8.6% relative humidity and no wind. The primary findings based on the feasibility criteria were that body temperature increased while exercising at an intensity of 60% HRpeak and decreased during inactive recovery by -0.3±0.1°C (epitympanic temperature). Secondly, the agreement between epitympanic and esophageal temperature (mean difference 0.2°C, 95% confidence interval -0.5 to 0.0, p=0.095) was better than in previous studies. Finally, all participants were able to tolerate the cold and complete the study despite thermal discomfort and shivering in the recovery phase. This protocol was successful in showing small changes in body temperature during exercise and recovery in the cold, though some modifications to the current protocol are recommended to elicit a larger effect size

Efficacy of warming systems in mountain rescue : an experimental manikin study

Mountain accident casualties are often exposed to cold and windy weather. This may induce post-traumatic hypothermia which increases mortality. The aim of this study was to assess the ability of warming systems to compensate for the victim’s estimated heat loss in a simulated mountain rescue operation. We used thermal manikins and developed a thermodynamic model of a virtual patient. Manikins were placed on a mountain rescue stretcher and exposed to wind chill indices of 0 °C and - 20 °C in a climatic chamber. We calculated the heat balance for two simulated clinical scenarios with both a shivering and non-shivering victim and measured the heat gain from gel, electrical, and chemical warming systems for 3.5 h. The heat balance in the simulated shivering patient was positive. In the non-shivering patient, we found a negative heat balance for both simulated weather conditions (- 429.53 kJ at 0 °C and -1469.78 kJ at - 20 °C). Each warming system delivered about 300 kJ. The efficacy of the gel and electrical systems was higher within the first hour than later (p < 0.001). We conclude that none of the tested warming systems is able to compensate for heat loss in a simulated model of a non-shivering patient whose physiological heat production is impaired during a prolonged mountain evacuation. Additional thermal insulation seems to be required in these settings

Surgical masks and filtering facepiece class 2 respirators (FFP2) have no major physiological effects at rest and during moderate exercise at 3000 m altitude. A randomised controlled trial

Background: During the COVID-19 pandemic, the use of face masks has been recommended or enforced in several situations, however their effects on physiological parameters and cognitive performance at high altitude are unknown. Methods: Eight healthy participants (four females) rested and exercised (cycling, 1 W/kg) while wearing no mask, a surgical mask, or a filtering facepiece class 2 respirator (FFP2), both in normoxia and hypobaric hypoxia corresponding to an altitude of 3000 m. Arterialised oxygen saturation (SaO2), partial pressure of oxygen (PaO2) and carbon dioxide (PaCO2), heart and respiratory rate, pulse oximetry (SpO2), cerebral oxygenation, visual analogue scales for dyspnoea and mask's discomfort were systematically investigated. Resting cognitive performance and exercising tympanic temperature were also assessed. Results: Mask use had a significant effect on PaCO2 (overall +1.2 ± 1.7 mmHg). There was no effect of mask use on all other investigated parameters except for dyspnoea and discomfort, which were highest with FFP2. Both masks were associated with a similar non-significant decrease in SaO2 during exercise in normoxia (-0.5% ± 0.4%) and, especially, in hypobaric hypoxia (-1.8% ± 1.5%), with similar trends for PaO2 and SpO2. Conclusions: Although mask use was associated with higher rates of dyspnoea, it had no clinically relevant impact on gas exchange at 3000 m at rest and during moderate exercise, and no detectable effect on resting cognitive performance. Wearing a surgical mask or an FFP2 can be considered safe for healthy people living, working, or spending their leisure time in mountains, high-altitude cities, or other hypobaric environments (e.g. aircrafts) up to an altitude of 3000 m

Avalanche survival depends on the time of day of the accident: A retrospective observational study.

INTRODUCTION We aimed to investigate the relationship between the time of the day and the probability of survival of completely buried avalanche victims. We explored the frequency of avalanche burials occurring after sunset, and described victims' characteristics, duration of burial and rescue circumstances compared to daytime avalanches. METHODS In this retrospective, observational study, we analysed avalanche data from the registry of the Swiss Institute for Snow and Avalanche Research, from 1998 to 2020. RESULTS A total of 3,892 avalanche victims were included in the analysis, with 72 of the accidents (1.85%) occurring in the nighttime. Nearly 50% of the victims involved in nighttime avalanche accidents were completely buried, compared to about 25% of victims in daytime avalanches. Completely buried victims were rescued by a companion less often at night than in the daytime (15% vs. 51%, p<.001). The search and rescue of completely buried avalanche victims took longer during the nighttime compared to the daytime (median 89 min vs 20 min, p=.002). The probability of survival decreased as the day progressed; it was highest at around midday (63.0%), but decreased at sunset (40.4%) and was the lowest at midnight (28.7%). CONCLUSIONS Avalanche accidents at night are a rare event, and probability of survival after complete burial is lower during the nighttime compared to the daytime. The most relevant reason for this is the longer duration of burial, which is explained in part by the lower rate of companion rescue and the lower rate of victim localisation with an avalanche transceiver

Effects of hypothermia, hypoxia, and hypercapnia on brain oxygenation and hemodynamic parameters during simulated avalanche burial: a porcine study.

Avalanche patients who are completely buried but still able to breathe are exposed to hypothermia, hypoxia, and hypercapnia (triple H syndrome). In a porcine model, there was no clinically relevant reduction in cerebral oxygenation during hypothermia and initial reduction of fraction of inspiratory oxygen ([Formula: see text]), as observed during hypercapnia. Hypercapnia may be the main cause of cardiovascular instability, which seems to be the major trigger for a decrease in cerebral oxygenation in triple H syndrome despite severe hypothermia

Isolated high altitude psychosis, delirium at high altitude, and high altitude cerebral edema: are these diagnoses valid?

Psychosis is a psychopathological syndrome that can be triggered or caused by exposure to high altitude (HA). Psychosis can occur alone as isolated HA psychosis or can be associated with other mental and often also somatic symptoms as a feature of delirium. Psychosis can also occur as a symptom of high altitude cerebral edema (HACE), a life-threatening condition. It is unclear how psychotic symptoms at HA should be classified into existing diagnostic categories of the most widely used classification systems of mental disorders, including the Diagnostic and Statistical Manual of Mental Disorders (DSM-V) and the International Statistical Classification of Diseases and Related Health Problems (ICD-11). We provide a diagnostic framework for classifying symptoms using the existing diagnostic categories: psychotic condition due to a general medical condition, brief psychotic disorder, delirium, and HACE. We also discuss the potential classification of isolated HA psychosis into those categories. A valid and reproducible classification of symptoms is essential for communication among professionals, ensuring that patients receive optimal treatment, planning further trips to HA for individuals who have experienced psychosis at HA, and advancing research in the field