Stress during dormancy: effect on recovery rates and life-history traits of anhydrobiotic animals

To test the influence of storage conditions on recovery and life-history traits of dormant animals we subjected cohorts of a bdelloid rotifer (Macrotrachela quadricornifera) and a nematode (Panagrolaimus rigidus) to three different conditions during a 26-d anhydrobiosis period. These conditions were (1) standard conditions in our laboratory (AC), (2) a flight to the U.S.A. and storage there (KSC), and (3) a flight to the U.S.A. followed by a flight on a Shuttle-to-Mir mission (Shuttle). Another cohort (HC) was maintained under hydrated conditions and served as reference for life-history traits of the desiccated samples. The recovery after dormancy and the residual life-history traits of the different cohorts were compared. Results indicate that (a) during dormancy the animals were indifferent to the test conditions, as evidenced from both recovery capacity and life history performances, (b) the anhydrobiotic period affected the experimental species differently. At recovery both animals resumed their life cycle, but while the rotifer appeared indifferent to the time spent in desiccation, the nematode modified its life history according to the duration of anhydrobiosis. Third, (c) induction of anhydrobiosis can act as a switching mechanism for animals capable of desiccation. Anhydrobiosis probably represents a suitable method to preserve organisms during stressful conditions allowing them to return to a normal life when convenient.     

Demographic and Health Related Data of Users of a Mobile Application to Support Drug Adherence is Associated with Usage Duration and Intensity

Purpose

A wealth of mobile applications are designed to support users in their drug intake. When developing software for patients, it is important to understand the differences between individuals who have, who will or who might never adopt mobile interventions. This study analyzes demographic and health-related factors associated with real-life “longer usage” and the “usage-intensity per day” of the mobile application “Medication Plan”.

Methods

Between 2010-2012, the mobile application “Medication Plan” could be downloaded free of charge from the Apple-App-Store. It was aimed at supporting the regular and correct intake of medication. Demographic and health-related data were collected via an online questionnaire. This study analyzed captured data.

Results

App-related activities of 1799 users (1708 complete data sets) were recorded. 69% (1183/1708) applied “Medication Plan” for more than a day. 74% were male (872/1183), the median age 45 years. Variance analysis showed a significant effect of the users´ age with respect to duration of usage (p = 0.025). While the mean duration of use was only 23.3 days for users younger than 21 years, for older users, there was a substantial increase over all age cohorts up to users of 60 years and above (103.9 days). Sex and educational status had no effect. “Daily usage intensity” was directly associated with an increasing number of prescribed medications and increased from an average of 1.87 uses per day and 1 drug per day to on average 3.71 uses per day for users stating to be taking more than 7 different drugs a day (p<0.001). Demographic predictors (sex, age and educational attainment) did not affect usage intensity.

Conclusion

Users aged 60+ as well as those with complicated therapeutic drug regimens relied on the service we provided for more than three months on average. Mobile applications may be a promising approach to support the treatment of patients with chronic conditions.     

Effect of age at first mating on primary sex-ratio of the two-spotted spider mite

This paper determines the influence of initial age at mating on the primary sex-ratio of the two-spotted spider miteTetranychus urticae Koch. The specific question asked is whether females that mate at ages beyond ecdysis, or are inseminated at ecdysis by virgin males of set ages, have daughter productions different from control females mated at ecdysis by randomly aged males.Age of initial female mating did not effect survival. Females mated at ecdysis produced more daughters than those mated at age 5 or 10 days. However, daughter production was equal for all female treatments in the post-mating period. All treatments stopped producing daughters at approximately the same age, regardless of when mated. Son production was lowest for females mated at ecdysis. However, son production by females mated when 5 or 10 days old was lower than that of females mated at ecdysis.Male age at mating did not effect female survival. Females exposed to virgin males either 1 or 7 days old produced more daughters than those exposed to males either 11 or 20 days of age. However, a substantial proportion of females in these latter cohorts were not mated. If only mated females are considered, daughter production is the same between all cohorts.Delayed age at first mating influenced sex-ratio only via the females, specifically by changes in daughter production (as opposed to changes in survivorship). Daughters that would have been produced between ecdysis and mating were never recovered by females that mated at ages beyond ecdysis. Thus, these daughters were lost from a sex-ratio viewpoint. Initial age of male mating did not change sex-ratio.     

Membrane fatty acid composition of tissues is related to body mass of mammals

Phospholipids were extracted from tissues (heart, skeletal muscle, kidney cortex, liver and brain) of mammals representing a 9,000-fold range in body mass (mouse, rat, rabbit, sheep and cattle) and their fatty acid composition was determined. In heart, skeletal muscle and kidney cortex, there were significant allometric decreases in the Unsaturation Index (UI; average number of double bonds per 100 fatty acid molecules) with increasing body mass. There were significant inverse allometric relationships between body mass and the proportion of docosahexaenoic acid (2263) in heart and skeletal muscle. In heart, skeletal muscle and kidney cortex, larger mammals also had shorter fatty acid chains in their phospholipids and a higher proportion of monounsaturates. In liver, smaller mammals had a higher UI than larger mammals (except the rabbit, which had the lowest UI and very low proportions of 3 fatty acids). The brain of all mammals maintained a high UI with similar levels of polyunsaturated fatty acids, especially 2263. Our results suggest that in heart, skeletal muscle and kidney cortex the activity of the elongases and desaturases are reduced in large mammals compared to small mammals. The allometric trends in membrane composition may be involved in modifying membrane permeability. It is proposed that the elevated degree of polyunsaturation in the membranes of several tissues from small mammals is related to their higher metabolic activity.This work was supported by an Australian Commonwealth Postgraduate Research Scholarship from the University of Wollongong to P. Couture and by a grant from the Australian Research Council to A.J. Hulbert. We wish to thank Voytek Mantaj for technical assistance.     

Further examination of the production-line hypothesis in mouse foetal oocytes

Chromosome pairing has been examined in foetal oocytes of mice heterozygous either for an X-linked inversion, In(X)1H, or an autosomal inversion, In(2)2H. The patterns of chromosome pairing have been screened systematically in foetuses of different gestational ages in a search for a production-line effect particularly affecting the inversion-bearing bivalents. The proportion of pachytene oocytes with a loop fell with increasing gestational age for both inversions. The decrease was linear for In(X)1H but best described by a quadratic function for In(2)2H. Examination of late zygotene cells and a comparison of loop frequency in early, mid and late pachytene oocytes suggested this age-related decrease to be principally due to synaptic adjustment and not to a production-line effect. However, two particular observations were somewhat at variance with this conclusion. Firstly, in In(X)1H heterozygotes, the presence of an inversion loop and the occurrence of partial pairing of long/long-medium bivalents at pachytene were independent of each other only on day 19. Secondly, although the proportion of oocytes with a loop fell overall, there was a rise at 19 days in In(2)2H heterozygotes. Thus in both inversions there is some evidence of a change in pairing behaviour affecting the inversion-bearing bivalents at the latest gestational age, as would be expected under the production-line hypothesis.     

Multifaceted intervention to improve diagnosis and treatment of osteoporosis in patients with recent wrist fracture: a randomized controlled trial

Background

Older patients who experience a fragility fracture are at high risk of future fractures but are rarely tested or treated for osteoporosis. We developed a multifaceted intervention directed at older patients with wrist fractures (in the form of telephone-based education) and their physicians (in the form of guidelines endorsed by opinion leaders, supported by reminders) to improve the quality of osteoporosis care.

Methods

In a randomized controlled trial with blinded ascertainment of outcomes, we compared our intervention with usual care (provision of printed educational materials to patients). Eligible patients were those older than 50 years of age who had experienced a wrist fracture and were seen in emergency departments and fracture clinics; we excluded those who were already being treated for osteoporosis. The primary outcome was bisphosphonate treatment within 6 months after the fracture. Secondary outcomes included bone mineral density testing, “appropriate care” (consisting of bone mineral density testing with treatment if bone mass was low) and quality of life.

Results

We screened 795 patients for eligibility and randomly assigned 272 to the intervention (137 patients) or control (135 patients) group. The median age was 60 years; 210 (77%) of the subjects were women, and 130 (48%) reported a previous fracture as an adult. Six months after the fracture, 30 (22%) of the intervention patients, as compared with 10 (7%) of the control patients, were receiving bisphosphonate therapy for osteoporosis (adjusted relative risk [RR] 2.6, 95% confidence interval [CI] 1.3–5.1, p = 0.008). Intervention patients were more likely than control patients to undergo bone mineral density testing (71/137 [52%] v. 24/135 [18%]; adjusted RR 2.8, 95% CI 1.9–4.2, p < 0.001) and to receive appropriate care (52/137 [38%] v. 15/135 [11%]; adjusted RR 3.1, 95% CI 1.8–5.3, p < 0.001). There were no differences between the groups in other outcomes. One patient died, and 4 others experienced recurrent fracture.

Interpretation

A multifaceted intervention directed at high-risk patients and their physicians substantially increased rates of testing and treatment for osteoporosis. Nevertheless, more than half of the patients in the intervention group were not receiving appropriate care 6 months after their fracture, which suggests that additional strategies should be explored. (ClinicalTrials.gov trial register no. NCT00152321.)Osteoporosis is a common, chronic and costly condition affecting at least 25% of women and 12% of men over 50 years of age.^1–3 Without better prevention strategies, the incidence of and costs related to osteoporotic fractures are expected to increase by 50% over the next 2 decades.³ Case-finding and secondary prevention (e.g., by identifying patients who have experienced a fragility fracture, ensuring that their bone mineral density is tested and offering efficacious osteoporosis treatments to those with low bone mass) constitute the most cost-effective strategy for reducing future fractures.^4–6An obvious target group for case-finding consists of older patients who experience a wrist fracture. Wrist fracture is the most common symptomatic fracture related to osteoporosis; its occurrence is a powerful forecaster of future fractures, and these fractures typically occur 10–20 years before the more devastating osteoporosis-related fractures of the spine or the hip.⁷ Unfortunately, although most older patients with wrist fractures have low bone mass and are eligible for treatment,^4,7 less than about 10% to 20% are tested or treated for osteoporosis in the 6 to 12 months after a wrist fracture.^4–9We previously reported a nonrandomized study of an intervention that incorporated patient education, physician reminders and treatment guidelines endorsed by opinion leaders, to improve osteoporosis treatment in patients with wrist fractures; in that study, which involved 102 patients, the rate of treatment was 40% in the intervention group but only 10% in the group receiving usual care.⁷ Several concerns were raised about the internal and external validity of that small study, so we conducted a randomized controlled trial of the intervention, which is reported here.     

Comparison of the egg flotation and egg candling techniques for estimating incubation day of Canada Goose nests

ABSTRACT Both egg flotation and egg candling have been used to estimate incubation day (often termed nest age) in nesting birds, but little is known about the relative accuracy of these two techniques. We used both egg flotation and egg candling to estimate incubation day for Canada Geese (Branta canadensis interior) nesting near Cape Churchill, Manitoba, from 2000 to 2007. We modeled variation in the difference between estimates of incubation day using each technique as a function of true incubation day, as well as, variation in error rates with each technique as a function of the true incubation day. We also evaluated the effect of error in the estimated incubation day on estimates of daily survival rate (DSR) and nest success using simulations. The mean difference between concurrent estimates of incubation day based on egg flotation minus egg candling at the same nest was 0.85 ± 0.06 (SE) days. The positive difference in favor of egg flotation and the magnitude of the difference in estimates of incubation day did not vary as a function of true incubation day. Overall, both egg flotation and egg candling overestimated incubation day early in incubation and underestimated incubation day later in incubation. The average difference between true hatch date and estimated hatch date did not differ from zero ( days) for egg flotation, but egg candling overestimated true hatch date by about 1 d (true – estimated; days). Our simulations suggested that error associated with estimating the incubation day of nests and subsequently exposure days using either egg candling or egg flotation would have minimal effects on estimates of DSR and nest success. Although egg flotation was slightly less biased, both methods provided comparable and accurate estimates of incubation day and subsequent estimates of hatch date and nest success throughout the entire incubation period.     

Gas exchange, heat production and oxidation of fat in chicken embryos from a fast or slow growing line

The experiment comprised 48 chicken (Gallus gallus) embryos from a modern, fast growing line, Ross 308 (RO) and 48 from a slow growing line, Labresse (LA). The O(2) consumption and CO(2) production were measured in an open-air-circuit respiration unit, and heat production (HE) from embryos was calculated at an age of 10, 13, 16 and 19 days. Gas exchange was below 10 ml/h for RO and LA by an age of 10-13 days, increasing steeply to a "peak" on day 16 and then slowing down between 16 and 19 days. The pattern of curves for gas exchange was identical for RO and LA, but on a lower level for LA. HE followed the pattern of gas exchange, with a mean around 50 J/h on day 10, increasing to 528 (RO) and 402 (LA) J/h on day 19. The main source of HE was oxidized fat. In addition to respiration experiments chemical analyses were carried out on 60 eggs from RO and 60 from LA. Prior to chemical analyses the eggs were incubated for 7, 13 and 19 days. Since fat oxidation was the main energy fuel the content of fat in the eggs decreased by 2.0 (RO) and 1.6 g (LA), while protein content was fairly constant in each line. It is remarkable that the differences in heat production between chickens from fast and slow growing lines were already manifested during their embryonic development.     

Effects of growth period,plant age and changes in solution aluminium concentrations on aluminium toxicity in wheat

The effects of growth period (time between transplanting and harvesting), plant age at which aluminium (Al) was added to solution, changes in Al concentration, and solution culture techniques (monitoring and adjusting solution Al concentrations thrice weekly or weekly replacement of the solutions) were investigated using a low ionic strength (2.7×10^–3 M) solution culture technique. The wheat (Triticum aestivum L.) cultivars Waalt (Al-tolerant) and Warigal (Al-sensitive), or the near isogenic lines bred from these cultivars (RR for the Al-tolerant line and SS for the Al-sensitive line) were grown. In all experiments and treatments, Al additions were required to maintain the nominal concentration. The decline in solution Al concentrations was partially attributed to formation of an Al-hydroxy-phosphate precipitate with an Al:P molar ratio of 2.8 to 4.0. Increasing the growth period from 14 to 28 days increased Al sensitivity in Warigal but not in Waalt. When plants were exposed to Al for the same time, increasing the age of the plants that Al was added to solution decreased sensitivity to Al. Differential Al tolerance between the two lines was evident when solutions were monitored thrice weekly or replaced weekly. However, the Al concentration required to reduce relative yield by a given amount when the solutions were replaced weekly was about twice that when the solutions were monitored. With a constant growth period of 28 days, increasing solution Al concentrations for 3 or more days resulted in decreased yields at harvest. The exact effect depended on the cultivar, plant part (tops or roots), when solution Al concentrations were increased and the duration of the increase. For example, increasing Al concentrations from 5 M to 20 M for 10 days reduced yield in the RR line by approximately 50% in the tops and 30% in the roots beyond the effect of 5 M but had no effect in the SS line due to yields already being low at 5 M. Adding 10 M Al to solution for 6 days at the beginning of the experiment reduced yield by 25% in the RR line and 50% in the SS line. In contrast, adding 10 M Al for 6 days in the middle of the growth cycle had no effect on the RR line but reduced yield by approximately 25% in the SS line. These results show that growth period, the age of the plants at which Al is added and the technique used (monitored or weekly replacement) all need to be considered when comparing results from different experiments. These results also show that the Al concentrations in solution need to be regularly monitored in long term experiments.     

The nematode Neoaplectana carpocapsae and the beet armyworm Spodoptera exigua: Infectivity of prepupae and pupae in soil and of adults during emergence from soil

The nematode, Neoaplectana carpocapsae, infected >90% of the prepupae of Spodoptera exigua in soil even at concentrations as low as five nematodes/cm² of soil surface. Pupae were less susceptible to nematode infection in soil than prepupae, with mortality ranging from 10 to 24% and 10 to 83% for pupae exposed 3–5 days and 6–8 days to the nematode, respectively. Longer exposure (6–8 days) of the pupae to the nematode resulted in higher mortality with a positive relationship with increasing concentrations. Adults of S. exigua were susceptible to nematode infections as they emerged from the soil. The higher nematode concentrations (25 and 50 nematodes/cm²) resulted in higher adult mortality. The majority of nematode-induced mortality occurred within 24 hr after emergence. The susceptibility of emerging S. exigua adults to N. carpocapsae offers a new dimension for insect control.     

Effects of 60 Hz electric and magnetic fields on the development of the rat cerebellum

The effects of extremely low frequency (ELF) electromagnetic (EM) fields on the maturation of the rat cerebellum were studied. Newborn rats were exposed to 60 Hz electric and magnetic fields under three different combinations in a specially constructed apparatus. The pups were irradiated for 7–8 h daily, with a 30-min interruption for nursing. Pups were kept with their mothers for the remainder of the time. After approximately 1, 2, or 3 weeks of exposure, the pups were killed. Control pups were sham exposed. The somatic growth of the irradiated rats did not show any significant difference from shamexposed controls. At 1 kV/m and 10 gauss exposure, there was a small but statistically significant decrease in cerebellar mass. In rats exposed at 1 kV/m and 10 gauss, DNA and RNA levels were significantly higher than those in shara-exposed controls at 6 and 13 days of age, but at 20 days, these two biochemical constituents were similar in both groups of rats. The ELF-EM treatment had no effect on protein and cerebroside concentrations. In terms of age effects. DNA and RNA exhibited increases from 6 to 13 days of age, and declined from 13 to 20 days. Protein and cerebroside levels exhibited increases during the 6–20 day periods. In rats exposed at 100 kV/m and 1 gauss, the DNA levels were initially less than those of sham-exposed controls at 8 days of age, reached approximately the same levels at 14 days, and then were higher than those of controls at 22 days. There was. therefore, a significant ELF-EM effect as well as a significant interaction between age and ELF-EM exposure. In terms of age effects, DNA levels for both control and exposed animals increased from 8 to 14 days. From 14 to 22 days, DNA levels of exposed rats continued to increase while those of the controls decreased. This age effect was significant. RNA levels in both groups of animals showed increases from 8 to 14 days of age, but the increase was less for the irradiated animals than for the controls. From days 14 to 22. RNA levels for both groups showed a reduction, but the decrease was greater in the irradiated than in control rats. ELF-EM treatment significantly reduced protein levels at 8 days of age. but at 14 to 22 days, protein levels of exposed rats were higher than those of controls. The cerebroside levels were not affected by exposure treatments but increased with the age of the animals. Exposure to 100 kV/m and 10 gauss did not exert any effect on the concentrations of DNA, RNA, protein, and cerebroside at all three time points examined. Both DNA and RNA exhibited increases with age from 6 to 13 days, and leveled off from 13 to 20 days. Protein and cerebroside levels also showed corresponding increases with the age of the animals. Morphological observations revealed no detectable changes in the irradiated animals in any experimental group. Thus, only biochemical studies indicate that exposure at certain ELF-EM field combinations induces alterations in cerebellar maturation. These changes were clearly detectable in the early postnatal period but gradually diminished with time. ©1993 Wiley-Liss, Inc.     

Bisecting Galactose as a Feature of N-Glycans of Wild-type and Mutant Caenorhabditis elegans

The N-glycosylation of the model nematode Caenorhabditis elegans has proven to be highly variable and rather complex; it is an example to contradict the existing impression that “simple” organisms possess also a rather simple glycomic capacity. In previous studies in a number of laboratories, N-glycans with up to four fucose residues have been detected. However, although the linkage of three fucose residues to the N,N′-diacetylchitobiosyl core has been proven by structural and enzymatic analyses, the nature of the fourth fucose has remained uncertain. By constructing a triple mutant with deletions in the three genes responsible for core fucosylation (fut-1, fut-6 and fut-8), we have produced a nematode strain lacking products of these enzymes, but still retaining maximally one fucose residue on its N-glycans. Using mass spectrometry and HPLC in conjunction with chemical and enzymatic treatments as well as NMR, we examined a set of α-mannosidase-resistant N-glycans. Within this glycomic subpool, we can reveal that the core β-mannose can be trisubstituted and so carries not only the ubiquitous α1,3- and α1,6-mannose residues, but also a “bisecting” β-galactose, which is substoichiometrically modified with fucose or methylfucose. In addition, the α1,3-mannose can also be α-galactosylated. Our data, showing the presence of novel N-glycan modifications, will enable more targeted studies to understand the biological functions and interactions of nematode glycans.Nematodes represent, along with arthropods, one of the largest groups of animals to exist on the planet; 25.000 species are described, but the existence of up to one million has been estimated (1, 2). They have various ecological niches and include free-living “worms” in the soil, fungivorous, entomopathogenic, and necromenic species as well as parasites of plants and mammals, which share the basic conserved body plan (more-or-less a digestive tube surrounded with muscle, whether larger or smaller). There are five major clades (Rhabditina, Enoplia, Spirurina, Tylenchina, and Dorylaimia) (2), yet the glycosylation of only a few nematode species has been studied with an inevitable focus on the model nematode Caenorhabditis elegans and parasitic species (3). Thereby, the use of C. elegans mutants has been highly valuable in dissecting aspects of nematode N-glycan biosynthesis and revealing the in vivo substrates for certain glycosyltransferases (4).As many nematodes are parasites, their interactions with the immune systems of their hosts have attracted attention; particularly, there are relationships between autoimmunity, allergy, vaccination, and helminth infections. The “old friends” hypothesis seeks to understand the evolutionary factors that have shaped the immune system and to explain correlations between lifestyles in the developed world and modern “epidemics,” which are due to immunological misbalance (5–7). Promising data have suggested that “worm therapy” may bring advantages to some patients with Crohn''s disease or allergies (8, 9); however, such approaches are controversial. Nevertheless, crude extracts even of Caenorhabditis elegans were shown to induce a glycan-dependent Th2 response (10), whereas the excretory-secretory products of some nematodes also have immunomodulatory activity (11). Furthermore, the native glycoproteins of some nematodes have proven effective in vaccination trials, whereas recombinant forms are not, which is suggestive that post-translational modifications may have a role in an efficacious immune response (12).As at least some of the molecules relevant to nematode immunomodulation or vaccination are glycoproteins, a proper understanding of nematode glycosylation is of biomedical and veterinary relevance. Over the years, it has become apparent that the core chitobiosyl region of nematode N-glycans is subject to a range of modifications, with up to three core fucose residues being present (α1,3- and α1,6-linked on the reducing-terminal “proximal” GlcNAc and α1,3-linked on the second “distal” GlcNAc). However, up to four fucose residues have been detected on C. elegans N-glycans and the exact nature of the linkage of the fourth fucose has remained obscure despite work in our own and other laboratories (3, 13–15). Combined with the latest knowledge regarding the specificity of C. elegans core fucosyltransferases (13, 16, 17) as well as our recent data regarding the exact structures of N-glycans from the C. elegans double hexosaminidase mutant and other nematodes (18–20), we concluded that some models for the tri- and tetrafucosylated N-glycans were incorrect. By preparing a triple mutant unable to core fucosylate its N-glycans, we generated a C. elegans strain containing maximally one fucose residue on the N-linked oligosaccharides. Thereby a pool of unusual mannosidase-resistant N-glycans was identified and, using mass spectrometry (MS) and NMR, we reveal their modification with bisecting galactose frequently capped with fucose or methylfucose.     

Differentiation of muscle and the determination of ultimate tissue size

Summary The factors that determine ultimate muscle size have been studied using a model that involves two strains of mice, which had been especially bred for largeness (QL) and for smallness (QS). The difference in muscle size was not found to be due to a difference in fibre size, but due to a difference in fibre number. The muscles from the QL mice contained about 30% more fibres.The reason for this increased fibre number was investigated. During early development, the fusion of mononucleated presumptive myoblasts to form multinucleated myotubes took place at the same time in both QL and OS mice, as indicated by the appearance and increase in activity of ATP: creatine phosphotransferase. At this stage fibre (and cell) number and size could be determined by measuring nuclear number and protein/DNA ratio respectively. No difference in fibre (and cell) size could be found in mice at 5 days before birth, newly born, or at 20 days of age. At these ages it was found that the muscles from the QL mice contained a greater number of nuclei (muscle cells). The amount of RNA/nucleus was used as an index of protein synthetic rate, and no difference could be found between the large mice and the small mice.It was concluded that, in the case of the QL mice, the increased fibre number was not brought about by: (i) a delay in time of fusion of presumptive myoblasts; (ii) a smaller number of myoblasts fusing to form myotubes; or (iii) extensive fibre formation after fusion. Differences in fibre number, and hence muscle size, must therefore, presumably be caused by initial differences in the rate of proliferation of myoblasts before fusion.     

Long-Term Administration of Valacyclovir Reduces the Number of Epstein-Barr Virus (EBV)-Infected B Cells but Not the Number of EBV DNA Copies per B Cell in Healthy Volunteers

Epstein-Barr virus (EBV) establishes a latent infection in B cells in the blood, and the latent EBV load in healthy individuals is generally stable over time, maintaining a “set point.” It is unknown if the EBV load changes after long-term antiviral therapy in healthy individuals. We treated volunteers with either valacyclovir (valaciclovir) or no antiviral therapy for 1 year and measured the amount of EBV DNA in B cells every 3 months with a novel, highly sensitive assay. The number of EBV-infected B cells decreased in subjects receiving valacyclovir (half-life of 11 months; P = 0.02) but not in controls (half-life of 31 years; P = 0.86). The difference in the slopes of the lines for the number of EBV-infected B cells over time for the valacyclovir group versus the control group approached significance (P = 0.054). In contrast, the number of EBV DNA copies per B cell remained unchanged in both groups (P = 0.62 and P = 0.92 for the control and valacyclovir groups, respectively). Valacyclovir reduces the frequency of EBV-infected B cells when administered over a long period and, in theory, might allow eradication of EBV from the body if reinfection does not occur.Primary infection with Epstein-Barr virus (EBV) is frequently asymptomatic in infants and children, but infection of adolescents and young adults can result in infectious mononucleosis. EBV is associated with several malignancies, including Burkitt''s lymphoma, nasopharyngeal carcinoma, Hodgkin''s disease, and lymphoproliferative disease, in immunocompromised and immunocompetent persons (6, 20).In healthy EBV-seropositive persons, about 1 to 10 in 10⁵ peripheral B cells are infected with EBV (14). The virus establishes latency in memory B cells. The level of the latent EBV load in healthy individuals remains stable over time, maintaining a “set point” for each individual (19). It is uncertain how this “set point” is maintained, but the latent EBV load is thought to reflect a balance between removal of EBV-infected cells due to the half-life of memory B cells and reinfection of new memory B cells during virus reactivation. The EBV genome replicates when B cells latently infected with EBV divide using the host DNA polymerase, which is not sensitive to the action of acyclovir. However, when the virus reactivates in latently infected B cells, EBV replicates using the viral DNA polymerase, which is inhibited by the phosphorylated form of acyclovir. Therefore, blocking production of new virus with acyclovir should decrease the latent EBV load at a rate equivalent to the half-life of memory B cells. Patients with zoster who were treated with oral acyclovir for 28 days showed no reduction in the EBV load in the blood, despite complete inhibition of EBV shedding in the saliva (23). These results suggested that antiviral therapy for longer than 28 days is necessary to detect a reduction in the EBV load in the blood.In this study, we administered either valacyclovir (valaciclovir) (which is absorbed more effectively than acyclovir and metabolized to acyclovir) or no antiviral to healthy volunteers for 12 months and measured the level of EBV DNA in the blood every 3 months.     

Effect of hypoxia on amnion rhythmic contractions and heart rate in the embryonic European pond turtle <Emphasis Type="Italic">Emys orbicularis</Emphasis> (Reptilia: Emydidae)

The effect of acute hypoxia (10% O₂ for 30 min) on the rate of amnion rhythmic contractions and heart rate (HR) was studied in two age groups of European pond turtle (Emys orbicularis) embryos, on days 19–27 and 37–43 of incubation (30–40 and 60–70% of the period until hatching). Under the control conditions, the two age groups of embryos did not differ from each other in either parameter. Hypoxia did not affect significantly the amnion contraction frequency but decreased the HR. The time course of the HR during hypoxia depended on the embryo age. The mean HR in the first group of embryos was 8% decreased by minutes 10–14 of hypoxia and did not change afterwards; in the second group, it was 18% decreased by minutes 3–7 and then partly or completely restored before the end of hypoxic exposure. It has been assumed that the capacity of European pond turtle embryos for restoring the HR when exposed to acute hypoxia during the second half of embryogenesis is related to the development of neurohumoral control mechanisms.     

High-dose versus standard-dose vitamin D supplementation in older adults with COVID-19 (COVIT-TRIAL): A multicenter,open-label,randomized controlled superiority trial

BackgroundVitamin D supplementation has been proposed as a treatment for Coronavirus Disease 2019 (COVID-19) based on experimental data and data from small and uncontrolled observational studies. The COvid19 and VITamin d TRIAL (COVIT-TRIAL) study was conducted to test whether a single oral high dose of cholecalciferol (vitamin D3) administered within 72 hours after the diagnosis of COVID-19 improves, compared to standard-dose cholecalciferol, the 14-day overall survival among at-risk older adults infected with Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2).Methods and findingsThis multicenter, randomized, controlled, open-label, superiority trial involved collaboration of 9 medical centers in France. Patients admitted to the hospital units or living in nursing homes adjacent to the investigator centers were eligible if they were ≥65 years, had SARS-CoV-2 infection of less than 3 days, and at least 1 COVID-19 worsening risk factor (among age ≥75 years, SpO2 ≤94%, or PaO₂/FiO₂ ≤300 mm Hg). Main noninclusion criteria were organ failure requiring ICU, SpO2 ≤92% despite 5 L/min oxygen, life expectancy <3 months, vitamin D supplementation >800 IU/day during the preceding month, and contraindications to vitamin D supplements. Eligible and consenting patients were randomly allocated to either a single oral high-dose (400,000 IU) or standard-dose (50,000 IU) cholecalciferol administered under medical supervision within 72 hours after the diagnosis of COVID-19. Participants and local study staff were not masked to the allocated treatment, but the Steering Committee and the Data and Safety Monitoring Board were masked to the randomization group and outcome data during the trial. The primary outcome was 14-day overall mortality. Between April 15 and December 17, 2020, of 1,207 patients who were assessed for eligibility in the COVIT-TRIAL study, 254 met eligibility criteria and formed the intention-to-treat population. The median age was 88 (IQR, 82 to 92) years, and 148 patients (58%) were women. Overall, 8 (6%) of 127 patients allocated to high-dose cholecalciferol, and 14 (11%) of 127 patients allocated to standard-dose cholecalciferol died within 14 days (adjusted hazard ratio = 0.39 [95% confidence interval [CI], 0.16 to 0.99], P = 0.049, after controlling for randomization strata [i.e., age, oxygen requirement, hospitalization, use of antibiotics, anti-infective drugs, and/or corticosteroids] and baseline imbalances in important prognostic factors [i.e., sex, ongoing cancers, profuse diarrhea, and delirium at baseline]). The number needed to treat for one person to benefit (NNTB) was 21 [NNTB 9 to ∞ to number needed to treat for one person to harm (NNTH) 46]. Apparent benefits were also found on 14-day mortality due to COVID-19 (7 (6%) deaths in high-dose group and 14 (11%) deaths in standard-dose group; adjusted hazard ratio = 0.33 [95% CI, 0.12 to 0.86], P = 0.02). The protective effect of the single oral high-dose administration was not sustained at 28 days (19 (15%) deaths in high-dose group and 21 (17%) deaths in standard-dose group; adjusted hazard ratio = 0.70 [95% CI, 0.36 to 1.36], P = 0.29). High-dose cholecalciferol did not result in more frequent adverse effects compared to the standard dose. The open-label design and limited study power are the main limitations of the study.ConclusionsIn this randomized controlled trial (RCT), we observed that the early administration of high-dose versus standard-dose vitamin D3 to at-risk older patients with COVID-19 improved overall mortality at day 14. The effect was no longer observed after 28 days.Trial registrationClinicalTrials.gov {"type":"clinical-trial","attrs":{"text":"NCT04344041","term_id":"NCT04344041"}}NCT04344041.

In a randomized trial, Cedric Annweiler and colleagues evaluate whether a single high dose of vitamin D3 improves survival among older adults in France with SARS-CoV-2 infection.     

Remdesivir for the treatment of patients in hospital with COVID-19 in Canada: a randomized controlled trial

Background:The role of remdesivir in the treatment of patients in hospital with COVID-19 remains ill defined in a global context. The World Health Organization Solidarity randomized controlled trial (RCT) evaluated remdesivir in patients across many countries, with Canada enrolling patients using an expanded data collection format in the Canadian Treatments for COVID-19 (CATCO) trial. We report on the Canadian findings, with additional demographics, characteristics and clinical outcomes, to explore the potential for differential effects across different health care systems.Methods:We performed an open-label, pragmatic RCT in Canadian hospitals, in conjunction with the Solidarity trial. We randomized patients to 10 days of remdesivir (200 mg intravenously [IV] on day 0, followed by 100 mg IV daily), plus standard care, or standard care alone. The primary outcome was in-hospital mortality. Secondary outcomes included changes in clinical severity, oxygen- and ventilator-free days (at 28 d), incidence of new oxygen or mechanical ventilation use, duration of hospital stay, and adverse event rates. We performed a priori subgroup analyses according to duration of symptoms before enrolment, age, sex and severity of symptoms on presentation.Results:Across 52 Canadian hospitals, we randomized 1282 patients between Aug. 14, 2020, and Apr. 1, 2021, to remdesivir (n = 634) or standard of care (n = 648). Of these, 15 withdrew consent or were still in hospital, for a total sample of 1267 patients. Among patients assigned to receive remdesivir, in-hospital mortality was 18.7%, compared with 22.6% in the standard-of-care arm (relative risk [RR] 0.83 (95% confidence interval [CI] 0.67 to 1.03), and 60-day mortality was 24.8% and 28.2%, respectively (95% CI 0.72 to 1.07). For patients not mechanically ventilated at baseline, the need for mechanical ventilation was 8.0% in those assigned remdesivir, and 15.0% in those receiving standard of care (RR 0.53, 95% CI 0.38 to 0.75). Mean oxygen-free and ventilator-free days at day 28 were 15.9 (± standard deviation [SD] 10.5) and 21.4 (± SD 11.3) in those receiving remdesivir and 14.2 (± SD 11) and 19.5 (± SD 12.3) in those receiving standard of care (p = 0.006 and 0.007, respectively). There was no difference in safety events of new dialysis, change in creatinine, or new hepatic dysfunction between the 2 groups.Interpretation:Remdesivir, when compared with standard of care, has a modest but significant effect on outcomes important to patients and health systems, such as the need for mechanical ventilation. Trial registration: ClinicalTrials.gov, no. {"type":"clinical-trial","attrs":{"text":"NCT04330690","term_id":"NCT04330690"}}NCT04330690.

The role of remdesivir in treating patients in hospital with COVID-19 remains ill defined.¹ Remdesivir, a repurposed antiviral medication, has full or emergency approval from a number of regulators — including Health Canada — for the treatment of COVID-19, based on clinical trial data documenting a benefit on improving time to recovery.² An interim report of the larger World Health Organization (WHO) Solidarity trial showed no difference regarding mortality or need for mechanical ventilation, with a number of smaller trials being inconclusive on these important outcomes.^3–6 Recommendations of clinical guidelines are mixed, with some recommending remdesivir as standard of care, and others weakly recommending against.^7,8 Its impact on other clinical outcomes, including resource utilization and post–hospital stay outcomes, has not been fully defined, and there remains a possibility of an important treatment effect, particularly in certain groups of patients.⁹Solidarity is a global pragmatic clinical trial examining the effects of various therapeutics in patients with COVID-19.^3,10 Canadian Treatments for COVID-19 (CATCO) is a substudy of Solidarity, funded by the Canadian Institutes of Health Research (CIHR), in which added data elements are collected to better understand the effects of specific agents. We aimed to estimate the effect of treatment with remdesivir compared with standard care, for patients in hospital with COVID-19 in Canada; global data, which include Canadian patients randomized before Jan. 29, 2021, will also be available in a separate publication.     

A selective somatostatin type-2 receptor agonist inhibits neointimal thickening and enhances endothelial cell growth and morphology following aortic balloon injury in the rabbit

Somatostatin analogs have been shown to inhibit vascular smooth muscle cell (VSMC) proliferation and attenuate neointimal thickening following experimental balloon catheter injury. In this study, the effects of a selective agonist for the somatostatin receptor subtype 2, PRL-2486, on neointimal thickening and endothelial cell regrowth 2 weeks following balloon catheterization of male New Zealand White rabbits were determined. Rabbits treated 2 days prior to and 2 weeks after catheter injury with 10 g/kg/day PRL-2486 (PRL-tx) had decreased I/M ratios (intimal area/medial area × 100; p < 0.05) but had no effect at lower (5 g/kg/day) or higher (20 g/kg/day) doses. PRL-tx had significantly decreased VSMC proliferation compared to untreated animals. PRL-tx increased endothelial regrowth by over 2-fold (p < 0.002) and improved endothelial cell morphology. Endothelial-dependent relaxation responses to acetylcholine were attenuated by catheter injury, and were not improved with PRL-tx. These data suggest that the PRL-2486-mediated inhibition of neointimal thickening exhibits a bell-shaped dose-response curve. This inhibition may be due in part to decreased VSMC proliferation, which may be a function of enhanced endothelial regrowth, but not the return of endothelium-dependent vascular function.     

Vitamin B6-deficient diet plus 4-deoxypyridoxine (4-DPD) reduces the inflammatory response induced by T. spiralis in diaphragm,masseter and heart muscle tissue of mice

Animals fed diets deficient in vitamin B6 develop microcytic anemia, alterations of growth, and other pathologies. 4-deoxypirydoxine is a potent antagonist of vitamin B6 coenzyme which depresses IL-1, TNF and IL-6 and has anti-inflammatory properties. The aim of this study was to show the anti-infl ammatory effects of 4-DPD on chronic inflammation caused by the nematode parasite T. spiralis, specifically on the recruitment and the activation of inflammatory cells. Two groups of mice, 6 weeks of age, were used: one was maintained on a vitamin B6-deficient synthetic pellet diet for 15 days before injection of the nematode, and administered an intraperitoneal injection (i.p.) of 4-DPD (250 g/mouse) for 15 days (the first, 5 days before infection), and the second group was maintained on a normal diet for the total duration of the experiment. These two groups were then injected with 150 larvae (L1-T. spiralis) per os.Chronic inflammation was caused by infection of treated or untreated mice with T. spiralis parasite. After 14 days post-infection all mice developed a chronic inflammatory response. Mice fed with a B6-deficient diet showed a significant decrease in the number of cysts found in the diaphragm when compared to mice treated with normal diet. In addition, in all mice treated with vitamin B6-deficient diet plus 4-DPD the average body weight was significantly lower, compared to the mice on normal diet in all weeks examined. Moreover, in sections of the diaphragm, masseter and miocardium muscles, the infiltration of inflammatory cells, such as macrophages, lymphocytes, and eosinophils were more intense in untreated mice compared to those fed a vitamin B6-deficient diet.These results show that BALB/c mice infected with T. spiralis and fed a vitamin B6-deficient diet plus the vitamin B6 antagonist, 4-DPD, prolong the time of invasion of the larvae in the muscle cells, influence the recruitment of inflammatory cells and the intensity of the inflammatory reaction compared to infected untreated mice (control)     

5′-Nucleotidase and adenosine deaminase in developing fetal guinea pig brain and the effect of maternal hypoxia

The activity of key enzymes of adenosine metabolism was studied in the developing fetal guinea pig brain. The activities of 5-nucleotidase and adenosine deaminase were determined in the brains of fetal guinea pigs at 30, 35, 40, 45, 50, 55, and 60 days of gestation. The level of 5-nucleotidase activity was extremely low at 30 and 35 days of gestation but increased rapidly during the 40 to 60 day period. The enzyme activity increased in the presence of Mg²⁺ with the Mg²⁺-dependent activation increasing with the age of gestation. This Mg²⁺-dependent activity was primarily associated with the membrane fraction. Prenatal hypoxia significantly increased the fetal brain M²⁺-independent 5-nucleotidase activity at 45 days of gestational age and beyond. Prior to this age, no effect was evident. Furthermore, following hypoxia, the Mg²⁺-dependent activation of 5-nucleotidase activity was lost. The activity of adenosine deaminase was present at 30 days of gestation and, unlike 5-nucleotidase, it remained at the same level until 60 days. The results indicate that the term fetal guinea pig brain has the enzymatic mechanisms of adenosine metabolism and thus the potential for adenosine-mediated regulation of cerebrovasculature during hypoxia.