The impact of sleep, physical activity and sedentary behaviour on symptoms of depression and anxiety before and during the COVID-19 pandemic in a sample of South African participants.

During lockdowns associated with the COVID-19 pandemic, individuals have experienced poor sleep quality and sleep regularity, changes in lifestyle behaviours, and heightened depression and anxiety. However, the inter-relationship and relative strength of those behaviours on mental health outcomes is still unknown. We collected data between 12 May and 15 June 2020 from 1048 South African adults (age: 32.76 ± 14.43 years; n = 767 female; n = 473 students) using an online questionnaire. Using structural equation modelling, we investigated how insomnia symptoms, sleep regularity, exercise intensity/frequency and sitting/screen-use (sedentary screen-use) interacted to predict depressive and anxiety-related symptoms before and during lockdown. We also controlled for the effects of sex and student status. Irrespective of lockdown, (a) more severe symptoms of insomnia and greater sedentary screen-use predicted greater symptoms of depression and anxiety and (b) the effects of sedentary screen-use on mental health outcomes were mediated by insomnia. The effects of physical activity on mental health outcomes, however, were only significant during lockdown. Low physical activity predicted greater insomnia symptom severity, which in turn predicted increased depressive and anxiety-related symptoms. Overall, relationships between the study variables and mental health outcomes were amplified during lockdown. The findings highlight the importance of maintaining physical activity and reducing sedentary screen-use to promote better sleep and mental health.

Prognostic indicators of mortality of mechanically ventilated patients with acute leukemia in a comprehensive cancer center.

BACKGROUND: The prognosis for adult acute leukemia patients that require intensive care unit (ICU) admission and invasive mechanical ventilation is poor. We aimed to identify prognostic indicators of 30-day hospital mortality in adult patients who had acute leukemia and respiratory failure, who had received invasive mechanical ventilation in the ICU but who had not received blood and marrow transplantation, were not admitted due to cardiopulmonary arrest or myocardial infarction and, had not recently undergone surgery. METHODS: In this case-control study, we retrospectively reviewed the medical records of relevant patients >16 year old who had been admitted to the ICU at our institution over a 4-year period. The main outcome measure was 30-day hospital mortality. Univariate and multivariate analyses were conducted to determine significant predictors of death. RESULTS: For the 167 patients meeting our eligibility criteria, the median age was 61 years. The majority was admitted due to respiratory insufficiency/failure (69%). The 30-day hospital mortality rate was 62%. Independent predictors of 30-day hospital mortality were advanced disease status (odds ratio [OR]=3.34; 95% confidence interval [CI], 1.65-6.77) and increased organ failure at the time of intubation (OR=1.17; 95% CI, 1.03-1.33) per point increase in the SOFA score. Patients who had received endotracheal intubation within the first 24 h of ICU admission were less likely than others to die (OR=0.46, 95% CI, 0.23-0.91) within the next 30 days after admission to the hospital. CONCLUSION: Advanced disease status and elevated SOFA scores at intubation are strong predictors of 30-day mortality in patients with acute leukemia and respiratory failure. The protective effect of early endotracheal intubation warrants further investigation.

Induction of bone formation by transforming growth factor-beta2 in the non-human primate Papio ursinus and its modulation by skeletal muscle responding stem cells.

OBJECTIVES: Four adult non-human primates Papio ursinus were used to study induction of bone formation by recombinant human transforming growth factor-beta(2) (hTGF-beta(2)) together with muscle-derived stem cells. MATERIALS AND METHODS: The hTGF-beta(2) was implanted in rectus abdominis muscles and in calvarial defects with and without addition of morcellized fragments of striated muscle, harvested from the rectus abdominis or temporalis muscles. Expression of osteogenic markers including osteogenic protein-1, bone morphogenetic protein-3 and type IV collagen mRNAs from generated specimens was examined by Northern blot analysis. RESULTS: Heterotopic intramuscular implantation of 5 and 25 microg hTGF-beta(2) combined with 100 mg of insoluble collagenous bone matrix yielded large corticalized mineralized ossicles by day 30 with remodelling and induction of haematopoietic marrow by day 90. Addition of morcellized rectus abdominis muscle to calvarial implants enhanced induction of bone formation significantly by day 90. CONCLUSIONS: In Papio ursinus, in marked contrast to rodents and lagomorphs, hTGF-beta(2) induced large corticalized and vascularized ossicles by day 30 after implantation into the rectus abdominis muscle. This striated muscle contains responding stem cells that enhance the bone induction cascade of hTGF-beta(2). Induction of bone formation by hTGF-beta(2) in the non-human primate Papio ursinus may occur as a result of expression of bone morphogenetic proteins on heterotopic implantation of hTGF-beta(2); the bone induction cascade initiated by mammalian TGF-beta proteins in Papio ursinus needs to be re-evaluated for novel molecular therapeutics for induction of bone formation in clinical contexts.

Picking out parallels: plant circadian clocks in context.

Molecular models have been described for the circadian clocks of representatives of several different taxa. Much of the work on the plant circadian system has been carried out using the thale cress, Arabidopsis thaliana, as a model. We discuss the roles of genes implicated in the plant circadian system, with special emphasis on Arabidopsis. Plants have an endogenous clock that regulates many aspects of circadian and photoperiodic behaviour. Despite the discovery of components that resemble those involved in the clocks of animals or fungi, no coherent model of the plant clock has yet been proposed. In this review, we aim to provide an overview of studies of the Arabidopsis circadian system. We shall compare these with results from different taxa and discuss them in the context of what is known about clocks in other organisms.

The molecular genetics of circadian rhythms in Arabidopsis.

While a number of physiological and biochemical processes in plants have been found to be regulated in a circadian manner, the mechanism underlying the circadian oscillator remains to be elucidated. Advances in the identification and characterization of components of the plant circadian system have been made largely through the use of genetics in Arabidopsis thaliana. Results so far indicate that the generation of rhythmicity by the Arabidopsis clock relies on molecular mechanisms that are similar to those described for other organisms, but that a totally different set of molecular components has been recruited to perform these functions.

Biosynthesis of heparin/heparan sulfate: kinetic studies of the glucuronyl C5-epimerase with N-sulfated derivatives of the Escherichia coli K5 capsular polysaccharide as substrates.

The D-glucuronyl C5-epimerase involved in the biosynthesis of heparin and heparan sulfate was investigated with focus on its substrate specificity, its kinetic properties, and a comparison of epimerase preparations from the Furth mastocytoma and bovine liver, which synthesize heparin and heparan sulfate, respectively. New substrates for the epimerase were prepared from the capsular polysaccharide of Escherichia coli K5, which had been labeled at C5 of its D-glucuronic and N-acetyl-D-glucosamine moieties by growing the bacteria in the presence of D-[5-(3)H]glucose. Following complete or partial ( approximately 50%) N-deacetylation of the polysaccharide by hydrazinolysis, the free amino groups were sulfated by treatment with trimethylamine.SO(3)complex, which yielded products that were recognized as substrates by the epimerase and released tritium from C5 of the D-glucuronyl residues upon incubation with the enzyme. Comparison of the kinetic properties of the two substrates showed that the fully N-sulfated derivative was the best substrate in terms of its K(m)value, which was significantly lower than that of its partially N-acetylated counterpart. The V(max)values for the E.coli polysaccharide derivatives were essentially the same but were both lower than that of the O-desulfated [(3)H]heparin used in our previous studies. Surprisingly, the apparent K(m)values for all three substrates increased with increasing enzyme concentration. The reason for this phenomenon is not entirely clear at present. Partially purified C5-epimerase preparations from the Furth mastocytoma and bovine liver, respectively, behaved similarly in terms of their reactivity towards the various substrates, but the variation in apparent K(m)values with enzyme concentration precluded a detailed comparison of their kinetic properties.

Dermatan is a better substrate for 4-O-sulfation than chondroitin: implications in the generation of 4-O-sulfated, L-iduronate-rich galactosaminoglycans.

The biosynthesis of dermatan sulfate is a complex process that involves, inter alia, formation of L-iduronic acid residues by C5-epimerization of D-glucuronic acid residues already incorporated into the growing polymer. It has been shown previously that this reaction is promoted by the presence of the sulfate donor 3'-phosphoadenosine-5'-phosphosulfate. In the present investigation, the role of sulfation in the biosynthesis of L-iduronic acid-rich galactosaminoglycans was examined more closely by a study of the substrate specificities and kinetic properties of the sulfotransferases involved in dermatan sulfate biosynthesis. Comparison of the acceptor reactivities of oligosaccharides from chondroitin and dermatan, in an in vitro system containing microsomes from cultured human skin fibroblasts and 3'-phosphoadenosine-5'-phosphosulfate, showed that Km values for the dermatan fragments were substantially lower than those for their chondroitin counterparts. Calculation of Vmax values likewise showed that dermatan was the better substrate. Whereas dermatan incorporated [35S]sulfate exclusively at the C4 position of N-acetylgalactosamine residues, approximately equal amounts of radioactivity were found at the C4 and C6 positions in the labelled chondroitin. Under standard assay conditions, the 4-O-sulfation of dermatan proceeded about six times faster than the 4-O-sulfation of chondroitin. On the basis of these results, we propose that L-iduronic acids, formed in the course of the biosynthesis of dermatan sulfates, enhance sulfation of their adjacent N-acetylgalactosamine residues, and will thereby be locked in the L-ido configuration.

Actions of sulfhydryl reagents on single ryanodine receptor Ca(2+)-release channels from sheep myocardium.

Effects of the reactive disulfides, 2,2'- and 4,4'-dithiodipyridine, on single cardiac ryanodine receptor (RyR) ion channels incorporated into lipid bilayers are reported. RyRs are activated within minutes of addition of the reactive disulfides (10(-7) to 10(-3) M) with an irreversible loss of channel activity after the activation at concentrations > or = 10(-4) M. This activation, followed by loss of activity, is seen over a wide range of cytoplasmic (cis) Ca2+ concentration between 10(-9) and 2 x 10(-2) M and occurs more rapidly with higher reactive disulfide concentrations or when RyRs are initially active at 10(-5) or 10(-3) M Ca2+. The reactive disulfides increase the channel open probability by introducing long components into the open time distributions, increasing the mean channel open time by up to 50-fold. Closed time distributions are not altered by the sulfhydryl reagents. The effects of the reactive disulfides are prevented by the reducing agents dithiothreitol and glutathione (1-10 mM). The results suggest that cysteine residues on the RyR complex can regulate the ion channel gating mechanisms.

Alkylglycosides as artificial primers for glycogen biosynthesis.

Glycogenin is a 37 kDa self-glycosylating protein which has been demonstrated to be the initiating enzyme and primer for glycogen biosynthesis in liver, skeletal muscle and other tissues. We have recently shown that glycogenin will use alkylglucosides and alkylmaltosides as artificial acceptors in glycosyl transfer from UDP-glucose and UDP-xylose in vitro and have suggested that such substrates might be used to promote the synthesis of glycogen in vitro and in vivo. We now report that alkylglycosides can also serve as acceptors for transfer of glucose by glycogen synthase, yielding alkylmaltooligosaccharide products which may potentially be elongated to glycogen. alpha-Glucosides were better substrates than the corresponding beta-glucosides, and alkylmaltosides were preferred over alkylglucosides. The hydrophobicity of the substrates markedly affected their acceptor activity, less hydrophobic substrates being more active. This is in contrast to the behavior of glycogenin, which acted preferentially upon the more hydrophobic substrates tested. Aromatic glycosides were also substrates for glycogen synthase, e.g., naphthyl-alpha-D- and beta-D-glucoside. The substrates were active in vitro both with partially purified rabbit muscle glycogen synthase and in incubations with crude muscle and liver homogenates from rat. In vivo experiments with mice further proved that intraperitoneal administration of alkylglucosides and alkylmaltosides increased the uptake of 14C-glucose in liver. The elevated uptake was due to an increase in both hydrophobic products, isolated by adsorption to Sep-Pak C18 columns, and more hydrophilic material that co-fractionated with glycogen upon treatment of the tissue with alkali and precipitation with ethanol. These results demonstrate the ability of alkylglycosides to serve as artificial primers for glycogen biosynthesis in vivo.

Analysis of the Morgan-Elson chromogens by high-performance liquid chromatography.

The Morgan-Elson method for quantitative N-acetylhexosamine analysis is a two-step procedure comprising alkali treatment of the sugar and subsequent condensation of the resulting chromogens with p-dimethylaminobenzaldehyde (Ehrlich's reagent) to yield a colored product. In the present investigation, the products formed in the first step of the procedure were analyzed by high-performance liquid chromatography (HPLC) on a reversed-phase (C18) column, which was eluted with a water-methanol gradient; the absorbance of the effluent was monitored at 229 nm. The profile generated from alkali-treated N-acetylglucosamine exhibited two major peaks, in a ratio of approximately 2.5:1, which accounted for 94% of the total peak area. A third peak, accounting for 3% of the peak area, was eluted in an intermediate position, and several smaller peaks were also observed. The three predominant components, isolated by preparative HPLC, all gave a purple color on addition of Ehrlich's reagent, indicating that they were Morgan-Elson chromogens. The HPLC profile of alkali-treated N-acetylmannosamine was identical to that of the products generated from N-actylglucosamine, as was expected because of the elimination of the asymmetry at C-2 during formation of the chromogens. N-Acetylgalactosamine yielded two major peaks, which were eluted in the same positions as the two major products formed from N-acetylglucosamine, but the intermediate peak seen in the N-acetylglucosamine pattern was absent. The HPLC procedure allowed detection of as little as approximately 25 ng of N-acetylglucosamine and may therefore be of value as an alternative to the complete Morgan-Elson procedure when only small amounts of sample are available for quantitative analysis.

N-acetylglucosamine kinase and N-acetylglucosamine 6-phosphate deacetylase in normal human erythrocytes and Plasmodium falciparum.

The major pathways of glucose metabolism in the malaria parasite, Plasmodium falciparum, have now been elucidated, and the structures and properties of parasite-specific enzymes are presently being investigated. Little is known, however, about the enzymes catalysing monosaccharide interconversions in the parasite. In the present investigation we have examined the pathway of N-acetylglucosamine catabolism which, in higher organisms, involves the following reaction sequence: N-acetylglucosamine -->N-acetylglucosamine 6-phosphate-->glucosamine 6-phosphate-->fructose 6-phosphate. Assay of the specific kinase (E.C. 2.7.1.59) catalysing the phosphorylation of the sugar showed that the enzyme is present in Plasmodium extracts as well as in normal human erythrocytes; specific activities of 7.2 and 5.3 nmol/h/mg protein were measured for the parasite and erythrocyte extracts, respectively, N-Acetylglucosamine 6-phosphate deacetylase (E.C. 3.5.1.25), catalysing the second reaction, was also detected in both normal and Plasmodium-infected erythrocytes. At 75% parasitaemia, the deacetylase activity was close to 3 times higher than that of normal control cells. The erythrocyte deacetylase was purified approximately 16,000-fold by chromatography on DE52 cellulose, chromatofocusing, and size exclusion chromatography. Attempts to purify the parasite enzyme by the same procedures were unsuccessful due to loss of activity. A partially purified erythrocyte deacetylase preparation (eluted from DE52 cellulose) had a pH optimum of 7.5, a pI of 6.0, as indicated by chromatofocusing, and a K(m) of 29 microM. In conjunction with previous investigations, the present study indicated that all three enzymes required for N-acetylglucosamine utilization are present in Plasmodium parasites as well as in normal erythrocytes.

Global analysis of a macromolecular interaction measured on BIAcore.

We demonstrate that the interaction between myoglobin and an immobilized anti-myoglobin antibody measured on BIAcore 2000 can be described by a simple bimolecular reaction mechanism. We improved the quality of the sensor data by correcting for refractive index changes and nonspecific binding using a blank sensor surface. Applying nonlinear least squares analysis, we simultaneously fitted the association and dissociation phase data generated for a range of myoglobin concentrations injected across the antibody surface. The ability to globally fit these data to a simple binding model indicates that effects related to the sensor surface, like mass transport and the dextran matrix, did not complicate the observed binding responses. These results illustrate the potential of BIAcore to monitor macromolecular interactions in real-time and the utility of global analysis to resolve the reaction kinetics.

Glucosamine 6-phosphate deaminase in Plasmodium falciparum.

The pathways of glucose utilization for energy production in the malaria parasite, Plasmodium falciparum, have been studied extensively. Little is known, however, about the reactions by which glucose is converted into complex carbohydrates in the parasite, and knowledge of the catabolism of these substances is likewise scanty. The present investigation was undertaken to determine whether the parasites possess a key enzyme of glucosamine catabolism, i.e. glucosamine 6-phosphate deaminase (EC 5.3.1.40), which catalyses the conversion of the sugar phosphate to fructose 6-phosphate and ammonia. Lysates of Plasmodium-infected erythrocytes had substantially higher deaminase activity than control samples from normal erythrocytes, and an even higher specific activity was observed in extracts of isolated parasites, amounting to 20-40 times that of uninfected cells. Anion exchange chromatography indicated that the parasite deaminase eluted in a retarded position when compared to the elution profile of the erythrocyte enzyme. The charge difference suggested by these findings was established more directly by chromatofocusing, which indicated pI values of 6.85 and 8.55 for the parasite and erythrocyte deaminases, respectively. Other differences were also observed, notably a greater thermolability on the part of the parasite enzyme. These results indicated that the parasites synthesize a specific deaminase that is distinct from the normal erythrocyte enzyme. Studies on synchronized parasite cultures further indicated that the parasite deaminase is developmentally regulated, because a dramatic increase in activity levels occurred during the later stages of parasite development.

Cytoplasmic Ca2+ inhibits the ryanodine receptor from cardiac muscle.

Ca(2+)-dependent inhibition of native and isolated ryanodine receptor (RyR) calcium release channels from sheep heart and rabbit skeletal muscle was investigated using the lipid bilayer technique. We found that cytoplasmic Ca2+ inhibited cardiac RyRs with an average Km = 15 mM, skeletal RyRs with Km = 0.7 mM and with Hill coefficients of 2 in both isoforms. This is consistent with measurements of Ca2+ release from the sarcoplasmic reticulum (SR) in skinned fibers and with [3H]-ryanodine binding to SR vesicles, but is contrary to previous bilayer studies which were unable to demonstrate Ca(2+)-inhibition in cardiac RyRs (Chu, Fill, Stefani & Entman (1993) J. Membrane Biol. 135, 49-59). Ryanodine prevented Ca2+ from inhibiting either cardiac or skeletal RyRs. Ca(2+)-inhibition in cardiac RyRs appeared to be the most fragile characteristic of channel function, being irreversibly disrupted by 500 mM Cs+, but not by 500 mM K+, in the cis bath or by solublization with the detergent CHAPS. These treatments had no effect on channel regulation by AMP-PNP, caffeine, ryanodine, ruthenium red, or Ca(2+)-activation. Ca(2+)-inhibition in skeletal RyRs was retained in the presence of 500 mM Cs+. Our results provide an explanation for previous findings in which cardiac RyRs in bilayers with 250 mM Cs+ in the solutions fail to demonstrate Ca(2+)-inhibition, while Ca(2+)-inhibition of Ca2+ release is observed in vesicle studies where K+ is the major cation. A comparison of open and closed probability distributions from individual RyRs suggested that the same gating mechanism mediates Ca(2+)-inhibition in skeletal RyRs and cardiac RyRs, with different Ca2+ affinities for inhibition. We conclude that differences in the Ca(2+)-inhibition in cardiac and skeletal channels depends on their Ca2+ binding properties.

Glucosamine 6-phosphate deaminase in normal human erythrocytes.

In the course of an investigation of hexosamine catabolism in the human malaria parasite, Plasmodium falciparum, it became apparent that a basic understanding of the relevant enzymatic reactions in the host erythrocyte is lacking. To acquire the necessary basic knowledge, we have determined the activities of several enzymes involved in hexosamine metabolism in normal human red blood cells. In the present communication we report the results of studies of glucosamine 6-phosphate deaminase (GlcN6-P) using a newly developed sensitive radiometric assay. The mean specific activity in extracts of fresh erythrocytes assayed within 4h of collection was 14.7 nmol/h/mg protein, whereas preparations from older erythrocytes that had been stored at 4 degrees C for up to 4 weeks had a mean specific activity of 6.2 nmol/h/mg. Characterization of the deaminase by chromatofocusing gave a pI of 8.55. The enzyme was optimally active at pH 9.0 and had a Km of 41 microM. The metal chelators EDTA and EGTA were non-inhibitory; however, inhibition was observed in the presence of metal ions, especially Cu2+, Ni2+ and Zn2+. In addition, the deaminase was also inhibited by several sugar phosphates including the reaction product, fructose 6-phosphate.

Inhibition of glycogenin-catalyzed glucosyl and xylosyl transfer by cytidine 5'-diphosphate and related compounds.

The self-glucosylation of beef kidney glycogenin was inhibited by the following pyrimidine nucleotides and nucleotide sugars, listed in order of decreasing effectiveness: CDP-glucose, CDP, UDP-xylose, UDP-N-acetylglucosamine, UDP-galactose, UDP, CTP, CDP-choline, UDP-glucuronic acid, beta-S-UDP-glucose, and CMP. In contrast, the purine nucleotide sugars, ADP-glucose and GDP-glucose, were essentially ineffective, as was the pyrimidine nucleoside, cytidine. UDP-Xylose may be utilized by glycogenin as an alternative sugar donor instead of UDP-glucose (Rodén, L., Ananth, S., Campbell, P., Manzella, S., and Meezan, E. (1994) J. Biol. Chem. 269, 11509-11513) and therefore presumably inhibited the glucosyl transfer reaction by being a competitive substrate. Like glucosyl transfer, xylosyl incorporation into glycogenin was also inhibited effectively by CDP. On the other hand, UDP-xylose:proteoglycan core protein xylosyltransferase (EC 2.4.2.26) was not affected by CDP, nor was it inhibited by UDP-glucose. Addition of CDP or UDP-glucose to reaction mixtures containing both enzymes therefore made it possible to assay xylosyltransferase EC 2.4.2.26 reliably without the extensive product characterization that is otherwise necessary. The CDP effect on glycogenin further allowed the development of an improved procedure for the purification of this enzyme, in which specific elution of an affinity matrix (UDP-glucuronic acid-agarose) was carried out with CDP as the eluant.

The effect of Escherichia coli J5 and modified live Salmonella dublin vaccines in artificially reared neonatal calves.

One thousand neonatal calves, allocated in a factorial design into four groups, were vaccinated subcutaneously with two doses each of either killed Escherichia coli (0111:B4) J5 bacterin or a UC Davis modified live, genetically altered (aro-) Salmonella dublin vaccine, or both, or with a placebo. In this prospective double-blind study to determine the immunogenicity and protective effects of both vaccines on bovine neonates in field conditions, calves were observed daily until 2 months of age, and serum samples from selected study calves were obtained at five different time points. No clinical adverse vaccine reactions were observed. Overall mortality was 7.5% (75 of 1000), E. coli and S. dublin infection being the most commonly associated aetiological agents of deaths. Both J5 (p < 0.01) and Salmonella (p = 0.05) vaccines were significantly effective in reducing the mortality rate but without an additive effect. The role of passive transfer was important in calf survival. The E. coli J5 and (aro-) S. dublin vaccination schedule employed significantly (p < 0.001) elevated J5 and Salmonella-specific serum ELISA antibody titres, respectively, by the sixth week of age.