Chronic inhibition of endoplasmic reticulum calcium-release channels and calcium-ATPase lengthens the period of hepatic clock gene Per1

Abstract Background The role played by calcium as a regulator of circadian rhythms is not well understood. The effect of the pharmacological inhibition of the ryanodine receptor (RyR), inositol 1,4,5-trisphosphate receptor (IP3R), and endoplasmic-reticulum Ca2+-ATPase (SERCA), as well as the intracellular Ca2+-chelator BAPTA-AM was explored on the 24-h rhythmicity of the liver-clock protein PER1 in an experimental model of circadian synchronization by light and restricted-feeding schedules. Methods Liver explants from Period1-luciferase (Per1-luc) transgenic rats with either free food access or with a restricted meal schedule were treated for several days with drugs to inhibit the activity of IP3Rs (2-APB), RyRs (ryanodine), or SERCA (thapsigargin) as well as to suppress intracellular calcium fluctuations (BAPTA-AM). The period of Per1-luc expression was measured during and after drug administration. Results Liver explants from rats fed ad libitum showed a lengthened period in response to all the drugs tested. The pharmacological treatments of the explants from meal-entrained rats induced the same pattern, with the exception of the ryanodine treatment which, unexpectedly, did not modify the Per1-luc period. All effects associated with drug application were reversed after washout, indicating that none of the pharmacological treatments was toxic to the liver cultures. Conclusions Our data suggest that Ca2+ mobilized from internal deposits modulates the molecular circadian clock in the liver of rats entrained by light and by restricted meal access.</p

Changes in the 24 h Rhythmicity of Liver PPARs and Peroxisomal Markers When Feeding Is Restricted to Two Daytime Hours

Restricted feeding (RF) during daytime is associated with anticipatory activity before feeding, marked hyperphagia after mealtime, adjustments in hepatic metabolism, and the expression of a food-entrained oscillator (FEO). 24 h rhythmicity of liver PPARα, β, and γ, peroxisomal markers (PMP70, AOX, and catalase), and free fatty acids (FFAs) during RF was evaluated. The effect of fasting-refeeding was also studied. Results showed (1) higher levels of FFA before feeding, (2) a shift of PPARα and PPARγ before and of PPARβ peaks after feeding, (3) an increase in peroxisomal markers, (4) a shift of PMP70 and AOX peaks before feeding, and of maximal catalase activity in the dark period, (5) changes in the fasting-refeeding response, and (6) high correlations (>0.9) of serum corticosterone with PPARα and PPARγ and of PMP70 with PPARβ. In conclusion, 24 h rhythmicity of FFA, liver PPARs, and peroxisomal markers are biochemical adaptations associated with daytime RF and FEO expression

Diurnal and nutritional adjustments of intracellular Ca2+ release channels and Ca2+ ATPases associated with restricted feeding schedules in the rat liver

Background: Intracellular calcium is a biochemical messenger that regulates part of the metabolic adaptations in the daily fed-fast cycle. The aim of this study was to characterize the 24-h variations of the liver ryanodine and IP3 receptors (RyR and IP3R) as well as of the endoplasmic-reticulum and plasma-membrane Ca2+-ATPases (SERCA and PMCA) in daytime restricted feeding protocol. Methods: A biochemical and immunohistochemical approach was implemented in this study: specific ligand-binding for RyR and IP3R, enzymatic activity (SERCA and PMCA), and protein levels and zonational hepatic-distribution were determined by immunoblot and immunohistochemistry respectively under conditions of fasting, feeding, and temporal food-restriction. Results: Binding assays and immunoblots for IP3R1 and 2 showed a peak at the light/dark transition in the'ad-libitum' (AL) group, whereas in the restricted-feeding (RF) group the peak shifted towards the food-access time. In the case of RyR binding experiments, both AL and RF groups showed a modest elevation during the dark period, with the RF rats exhibiting increased binding in response to feeding. The AL group showed 24-h rhythmicity in SERCA level; in contrast, RF group showed a pronounced amplitude elevation and a peak phase-shift during the light-period in SERCA level and activity. The activity of PMCA was constant along day in both groups; PMCA1 levels showed a 24-h rhythmicity in the RF rats (with a peak in the light period), meanwhile PMCA4 protein levels showed rhythmicity in both groups. The fasted condition promoted an increase in IP3R binding and protein level; re-feeding increased the amount of RyR; neither the activity nor expression of SERCA and PMCA protein was affected by fasting–re-feeding conditions. Histochemical experiments showed that the distribution of the Ca2+-handling proteins, between periportal and pericentral zones of the liver, varied with the time of day and the feeding protocol. Conclusions: Our findings show that RF influences mainly the phase and amplitude of hepatic IP3R and SERCA rhythms as well as discrete zonational distribution for RyR, IP3Rs, SERCA, and PMCA within the liver acinus, suggesting that intracellular calcium dynamics could be part of the rheostatic adaptation of the liver due to diurnal meal entrainment/food entrained oscillator expression

Endoparasitic infections in captive wild mammals under human care in San Luis Potosí, Mexico

Objective: Determine parasite prevalence, richness and load in wild mammals of Tangamanga I and II Parks in San Luis Potosí, SLP. Evaluate if infections are related to diet and/or climatic seasons. Design/methodology/approach: Analysis of fresh feces collected during three consecutive days in two sampling periods. Passive flotation techniques with sodium nitrate (qualitative) and McMaster (quantitative) were performed. Results: General prevalence observed was 36% (n=242). The parasite richness is formed by protozoa: Eimeria sp, Isospora sp and Cystoisospora sp; by the cestode Moniezia expansa and nematodes: Toxocara sp, Toxascaris sp, Ancylostoma sp/Uncinaria sp, Strongyloides sp, Trichuris suis and eggs from the Strongyles group. The parasite load observed in the case of protozoa ranged from 0 – 8,505 Oocysts per Gram of Feces, and from 0 – 1,400 Eggs per Gram of Feces in the case of helminths. Statistical analyzes showed that the prevalence of parasites does not depend on the climatic season, and only in Tangamanga II Park is the prevalence dependent on the diet (herbivores). Study limitations/implications: The preservation method used limits the coproparasitoscopic techniques that can be used (coproculture or coccidia sporulation). Findings/conclusions: The observed endoparasitic infections can be a potential risk to the health of animals, especially those of great genetic value (i.e., endangered species). In addition, the potentially zoonotic parasites observed represent a threat to the health of their keepers.Objective: To determine the prevalence, richness and parasitic load in wild mammals of Tangamanga I and II Parks in San Luis Potosí, SLP. To assess whether infections are related to the type of feeding and weather seasons. Design/methodology/approach: Analysis of fresh feces collected for three consecutive days at two sampling stations. Passive flotation techniques with sodium nitrate (qualitative) and McMaster (quantitative) were used. Results: The overall prevalence observed was 36% (n=242). The parasitic wealth is formed by protozoa: Eimeria sp., Isospora sp. and Cystoisospora sp.; by the cestode Moniezia expansa, nematodes: Toxocara sp., Toxascaris sp., Ancylostoma sp./Uncinaria sp., Strongyloides sp., Trichuris suis and Strongylid eggs. The parasite load in the case of protozoa was in the range of 0 – 8505 oocysts per gram of faeces, and 0 – 1400 eggs per gram of faeces in the case of helminths. Statistical analyses showed that the prevalence of parasites does not depend on the climatic season, and only in Tangamanga II Park is the prevalence dependent on the type of feeding (herbivores). Study limitations/implications: The conservation method used limits the stool test techniques that can be employed (stool culture or sporulation). Findings/conclusions: Endoparasitic infections can be a potential risk to the health of animals. In particular to those of great genetic value such as species threatened with extinction. In addition, the potentially zoonotic parasites observed pose a threat to the health of caregiver

The evolution of the ventilatory ratio is a prognostic factor in mechanically ventilated COVID-19 ARDS patients

Background: Mortality due to COVID-19 is high, especially in patients requiring mechanical ventilation. The purpose of the study is to investigate associations between mortality and variables measured during the first three days of mechanical ventilation in patients with COVID-19 intubated at ICU admission. Methods: Multicenter, observational, cohort study includes consecutive patients with COVID-19 admitted to 44 Spanish ICUs between February 25 and July 31, 2020, who required intubation at ICU admission and mechanical ventilation for more than three days. We collected demographic and clinical data prior to admission; information about clinical evolution at days 1 and 3 of mechanical ventilation; and outcomes. Results: Of the 2,095 patients with COVID-19 admitted to the ICU, 1,118 (53.3%) were intubated at day 1 and remained under mechanical ventilation at day three. From days 1 to 3, PaO2/FiO2 increased from 115.6 [80.0-171.2] to 180.0 [135.4-227.9] mmHg and the ventilatory ratio from 1.73 [1.33-2.25] to 1.96 [1.61-2.40]. In-hospital mortality was 38.7%. A higher increase between ICU admission and day 3 in the ventilatory ratio (OR 1.04 [CI 1.01-1.07], p = 0.030) and creatinine levels (OR 1.05 [CI 1.01-1.09], p = 0.005) and a lower increase in platelet counts (OR 0.96 [CI 0.93-1.00], p = 0.037) were independently associated with a higher risk of death. No association between mortality and the PaO2/FiO2 variation was observed (OR 0.99 [CI 0.95 to 1.02], p = 0.47). Conclusions: Higher ventilatory ratio and its increase at day 3 is associated with mortality in patients with COVID-19 receiving mechanical ventilation at ICU admission. No association was found in the PaO2/FiO2 variation

Treatment with tocilizumab or corticosteroids for COVID-19 patients with hyperinflammatory state: a multicentre cohort study (SAM-COVID-19)

Objectives: The objective of this study was to estimate the association between tocilizumab or corticosteroids and the risk of intubation or death in patients with coronavirus disease 19 (COVID-19) with a hyperinflammatory state according to clinical and laboratory parameters. Methods: A cohort study was performed in 60 Spanish hospitals including 778 patients with COVID-19 and clinical and laboratory data indicative of a hyperinflammatory state. Treatment was mainly with tocilizumab, an intermediate-high dose of corticosteroids (IHDC), a pulse dose of corticosteroids (PDC), combination therapy, or no treatment. Primary outcome was intubation or death; follow-up was 21 days. Propensity score-adjusted estimations using Cox regression (logistic regression if needed) were calculated. Propensity scores were used as confounders, matching variables and for the inverse probability of treatment weights (IPTWs). Results: In all, 88, 117, 78 and 151 patients treated with tocilizumab, IHDC, PDC, and combination therapy, respectively, were compared with 344 untreated patients. The primary endpoint occurred in 10 (11.4%), 27 (23.1%), 12 (15.4%), 40 (25.6%) and 69 (21.1%), respectively. The IPTW-based hazard ratios (odds ratio for combination therapy) for the primary endpoint were 0.32 (95%CI 0.22-0.47; p < 0.001) for tocilizumab, 0.82 (0.71-1.30; p 0.82) for IHDC, 0.61 (0.43-0.86; p 0.006) for PDC, and 1.17 (0.86-1.58; p 0.30) for combination therapy. Other applications of the propensity score provided similar results, but were not significant for PDC. Tocilizumab was also associated with lower hazard of death alone in IPTW analysis (0.07; 0.02-0.17; p < 0.001). Conclusions: Tocilizumab might be useful in COVID-19 patients with a hyperinflammatory state and should be prioritized for randomized trials in this situatio