Early activation and redistribution of calpain activity in skeletal muscle during hindlimb unweighting and reweighting

The aims of this study were the following: (i) to determine whether activation of the Ca2+-activated protease, calpain, is an early event during hindlimb unweighting (HU) in skeletal muscle; and (ii) to assess whether calpain activity is greater during reweighting compared with HU alone. Rats were exposed to 12, 24, and 72 h, or 9 d of HU, followed by reweighting for 0, 12, or 24 h. Calpain activities were assayed for total, soluble, and particulate fractions. Total calpain activity was increased in the soleus at all HU time points, whereas activities were elevated in the gastrocnemius only after 9 d of HU. With reweighting, calpain activity remained elevated at all time points for both muscles. In general, reweighting the gastrocnemius increased its calpain activity more than during HU only, whereas reweighting the soleus did not produce additional increases in its calpain activity. The increases in calpain activity were associated with a proportional increase in activity of the particulate (membrane- and protein-associated) fraction. The results suggest that calpain activation is an early event during HU in the soleus, and that the increases in calpain activity in both muscles are associated with a redistribution of activity from cytosolic to particulate fractions.     

The effect of simulated censored data on estimates of heritability of longevity in the Thoroughbred racing industry

We examined the impact of censored data on estimates of heritability of longevity. Longevity, defined as the length of productive racing life of an individual, is influenced by many factors. A simulated data set, modelled on the Irish Thoroughbred industry, was used to estimate heritabilities of longevity. Several scenarios representing various levels of censoring of performance data were created. The heritability of longevity was estimated for each scenario and compared to the estimated heritability of 0.120 for the complete data set. It was found that the estimates of heritability (0.107, 0.106, 0.082) were biased downwards with (10, 20, and 25%, respectively) censoring of data from poor-performing animals. We found that for a complete reporting it is necessary to reduce bias in the estimation of heritability of longevity.     

The First Transmembrane Domain of Lipid Phosphatase SAC1 Promotes Golgi Localization

The lipid phosphatase Sac1 cycles between endoplasmic reticulum and cisternal Golgi compartments. In proliferating mammalian cells, a canonical dilysine motif at the C-terminus of Sac1 is required for coatomer complex-I (COP-I)-binding and continuous retrieval to the ER. Starvation triggers accumulation of Sac1 at the Golgi. The mechanism responsible for Golgi retention of Sac1 is unknown. Here we show that the first of the two transmembrane regions in human SAC1 (TM1) functions in Golgi localization. A minimal construct containing only TM1 and the adjacent flanking sequences is concentrated at the Golgi. Transplanting TM1 into transferrin receptor 2 (TfR2) induces Golgi accumulation of this normally plasma membrane and endosomal protein, indicating that TM1 is sufficient for Golgi localization. In addition, we determined that the N-terminal cytoplasmic domain of SAC1 also promotes Golgi localization, even when TM1 is mutated or absent. We conclude that the distribution of SAC1 within the Golgi is controlled via both passive membrane thickness-dependent partitioning of TM1 and a retention mechanism that requires the N-terminal cytoplasmic region.     

Mutations in B3GALNT2 Cause Congenital Muscular Dystrophy and Hypoglycosylation of α-Dystroglycan

Mutations in several known or putative glycosyltransferases cause glycosylation defects in α-dystroglycan (α-DG), an integral component of the dystrophin glycoprotein complex. The hypoglycosylation reduces the ability of α-DG to bind laminin and other extracellular matrix ligands and is responsible for the pathogenesis of an inherited subset of muscular dystrophies known as the dystroglycanopathies. By exome and Sanger sequencing we identified two individuals affected by a dystroglycanopathy with mutations in β-1,3-N-acetylgalactosaminyltransferase 2 (B3GALNT2). B3GALNT2 transfers N-acetyl galactosamine (GalNAc) in a β-1,3 linkage to N-acetyl glucosamine (GlcNAc). A subsequent study of a separate cohort of individuals identified recessive mutations in four additional cases that were all affected by dystroglycanopathy with structural brain involvement. We show that functional dystroglycan glycosylation was reduced in the fibroblasts and muscle (when available) of these individuals via flow cytometry, immunoblotting, and immunocytochemistry. B3GALNT2 localized to the endoplasmic reticulum, and this localization was perturbed by some of the missense mutations identified. Moreover, knockdown of b3galnt2 in zebrafish recapitulated the human congenital muscular dystrophy phenotype with reduced motility, brain abnormalities, and disordered muscle fibers with evidence of damage to both the myosepta and the sarcolemma. Functional dystroglycan glycosylation was also reduced in the b3galnt2 knockdown zebrafish embryos. Together these results demonstrate a role for B3GALNT2 in the glycosylation of α-DG and show that B3GALNT2 mutations can cause dystroglycanopathy with muscle and brain involvement.     

Suicide and suicide attempts in children and adolescents in the child welfare system

Background:

Few population studies have examined the psychiatric outcomes of children and adolescents in the child welfare system, and no studies have compared outcomes before and after entry into care. Our objective was to assess the relative rate (RR) of suicide, attempted suicide, admission to hospital and visits to physicians’ offices among children and adolescents in care compared with those not in care. We also examined these outcomes within the child welfare population before and after entry into care.

Methods:

We used population-level data to identify children and adolescents 5 to 17 years of age who were in care in Manitoba for the first time between Apr. 1, 1997, and Mar. 31, 2006, and a comparison cohort not in care. We compared the two cohorts to obtain RRs for the specified outcomes. We also determined RRs within the child welfare population relative to the same population two years before entry into care.

Results:

We identified 8279 children and adolescents in care for the first time and a comparison cohort of 353 050 children and adolescents not in care. Outcome rates were higher among those in care than in the comparison cohort for suicide (adjusted RR 3.54, 95% confidence interval [CI] 2.11–5.95), attempted suicide (adjusted RR 2.11, 95% CI 1.84–2.43) and all other outcomes. However, adjusted RRs for attempted suicide (RR 0.27, 95% CI 0.21–0.34), admissions to hospital and physician visits decreased after entry into care.

Interpretation:

Children and adolescents in care were at greater risk of suicide and attempting suicide than those who were not in care. Rates of suicide attempts and hospital admissions within this population were highest before entry into care and decreased thereafter.In Canada, about 76 000 children and adolescents are under the care of provincial child and family services.¹ In Manitoba, more than 7000 children and adolescents were in the care of child and family services in 2008. Many of them had experienced abuse and neglect, or death or conflict in their families, along with disability or emotional problems.^2–13 Concerns have been raised that the Canadian child welfare system does not provide adequate resources and supports to mitigate the effects of abuse and neglect.¹⁴ Although the health outcomes of this population are a frequent topic of concern in the media, population-based research describing these outcomes is limited.To our knowledge, only two studies of a population cohort of children and adolescents in care have been published to date, both describing the psychiatric morbidity and mortality of children and adolescents in care in Sweden.^4,6 These studies found greater rates of suicide, suicide attempts and psychiatric hospital admissions among children in care than in the general population. However, these studies had substantial limitations. Although they used the general population as a comparison group, they did not analyze for the presence of psychiatric morbidity in the period before entry into care. This omission limits the ability to draw conclusions about whether the poor outcomes of these children were associated with disruptions in their lives and families related to involvement in the child welfare system or whether they were a consequence of their life, health and psychological characteristics before they entered the care system.The first objective of the current study was to assess the relative rates (RRs) of suicide and attempted suicide and the number of hospital admissions and visits to physicians’ offices among children and adolescents with a history of being in the care of child and family services in Manitoba, relative to the general population of children and adolescents not in care. The second objective was to assess the RR of attempting suicide and the number of hospital admissions and physician visits in the child welfare population before and after entry into care.     

A novel mutation in the HSD17B10 gene of a 10-year-old boy with refractory epilepsy, choreoathetosis and learning disability

Hydroxysteroid (17beta) dehydrogenase 10 (HSD10) is a mitochondrial multifunctional enzyme encoded by the HSD17B10 gene. Missense mutations in this gene result in HSD10 deficiency, whereas a silent mutation results in mental retardation, X-linked, syndromic 10 (MRXS10). Here we report a novel missense mutation found in the HSD17B10 gene, namely c.194T>C transition (rs104886492), brought about by the loss of two forked methyl groups of valine 65 in the HSD10 active site. The affected boy, who possesses mutant HSD10 (p.V65A), has a neurological syndrome with metabolic derangements, choreoathetosis, refractory epilepsy and learning disability. He has no history of acute decompensation or metabolic acidosis whereas his urine organic acid profile, showing elevated levels of 2-methyl-3-hydroxybutyrate and tiglylglycine, is characteristic of HSD10 deficiency. His HSD10 activity was much lower than the normal control level, with normal β-ketothiolase activity. The c.194T>C mutation in HSD17B10 can be identified by the restriction fragment polymorphism analysis, thereby facilitating the screening of this novel mutation in individuals with intellectual disability of unknown etiology and their family members much easier. The patient's mother is an asymptomatic carrier, and has a mixed ancestry (Hawaiian, Japanese and Chinese). This demonstrates that HSD10 deficiency patients are not confined to a particular ethnicity although previously reported cases were either Spanish or German descendants.     

Microevolutionary analysis of Clostridium difficile genomes to investigate transmission

Background

The control of Clostridium difficile infection is a major international healthcare priority, hindered by a limited understanding of transmission epidemiology for these bacteria. However, transmission studies of bacterial pathogens are rapidly being transformed by the advent of next generation sequencing.

Results

Here we sequence whole C. difficile genomes from 486 cases arising over four years in Oxfordshire. We show that we can estimate the times back to common ancestors of bacterial lineages with sufficient resolution to distinguish whether direct transmission is plausible or not. Time depths were inferred using a within-host evolutionary rate that we estimated at 1.4 mutations per genome per year based on serially isolated genomes. The subset of plausible transmissions was found to be highly associated with pairs of patients sharing time and space in hospital. Conversely, the large majority of pairs of genomes matched by conventional typing and isolated from patients within a month of each other were too distantly related to be direct transmissions.

Conclusions

Our results confirm that nosocomial transmission between symptomatic C. difficile cases contributes far less to current rates of infection than has been widely assumed, which clarifies the importance of future research into other transmission routes, such as from asymptomatic carriers. With the costs of DNA sequencing rapidly falling and its use becoming more and more widespread, genomics will revolutionize our understanding of the transmission of bacterial pathogens.