Mitochondrial NADP(H) deficiency due to a mutation in NADK2 causes dienoyl-CoA reductase deficiency with hyperlysinemia.

Dienoyl-CoA reductase (DECR) deficiency with hyperlysinemia is a rare disorder affecting the metabolism of polyunsaturated fatty acids and lysine. The molecular basis of this condition is currently unknown. We describe a new case with failure to thrive, developmental delay, lactic acidosis and severe encephalopathy suggestive of a mitochondrial disorder. Exome sequencing revealed a causal mutation in NADK2. NADK2 encodes the mitochondrial NAD kinase, which is crucial for NADP biosynthesis evidenced by decreased mitochondrial NADP(H) levels in patient fibroblasts. DECR and also the first step in lysine degradation are performed by NADP-dependent oxidoreductases explaining their in vivo deficiency. DECR activity was also deficient in lysates of patient fibroblasts and could only be rescued by transfecting patient cells with functional NADK2. Thus NADPH is not only crucial as a cosubstrate, but can also act as a molecular chaperone that activates and stabilizes enzymes. In addition to polyunsaturated fatty acid oxidation and lysine degradation, NADPH also plays a role in various other mitochondrial processes. We found decreased oxygen consumption and increased extracellular acidification in patient fibroblasts, which may explain why the disease course is consistent with clinical criteria for a mitochondrial disorder. We conclude that DECR deficiency with hyperlysinemia is caused by mitochondrial NADP(H) deficiency due to a mutation in NADK2.

Next-generation sequencing of microRNAs uncovers expression signatures in polarized macrophages.

microRNAs (miRNAs) are small noncoding RNAs that regulate gene expression at a posttranscriptional level and play a crucial role in the development of cells of the immune system. Macrophages are essential for generating inflammatory reactions upon tissue damage and encountering of invading pathogens, yet modulation of their immune responses is critical for maintaining tissue homeostasis. Macrophages can present different phenotypes, depending on the cytokine environment they encounter in the affected tissues. In this study, we have identified expression signatures of miRNAs that are differentially regulated during maturation of monocytes and polarization of macrophages by cytokines. We present a comprehensive characterization of miRNA expression in human monocytes and M1, M2a, and M2c polarized macrophages, using next-generation sequencing. Furthermore, we show that miRNA expression signatures are closely related to the various immune functions of polarized macrophages and therefore are involved in shaping the diverse phenotypes of these cells. The miRNAs identified here serve as markers for identification of inflammatory macrophages involved in the development of immune responses. Our findings contribute to understanding the role of miRNAs in determining the macrophage function in healthy and diseased tissues.

Recessive MYL2 mutations cause infantile type I muscle fibre disease and cardiomyopathy.

A cardioskeletal myopathy with onset and death in infancy, morphological features of muscle type I hypotrophy with myofibrillar disorganization and dilated cardiomyopathy was previously reported in three Dutch families. Here we report the genetic cause of this disorder. Multipoint parametric linkage analysis of six Dutch patients identified a homozygous region of 2.1 Mb on chromosome 12, which was shared between all Dutch patients, with a log of odds score of 10.82. Sequence analysis of the entire linkage region resulted in the identification of a homozygous mutation in the last acceptor splice site of the myosin regulatory light chain 2 gene (MYL2) as the genetic cause. MYL2 encodes a myosin regulatory light chain (MLC-2V). The myosin regulatory light chains bind, together with the essential light chains, to the flexible neck region of the myosin heavy chain in the hexameric myosin complex and have a structural and regulatory role in muscle contraction. The MYL2 mutation results in use of a cryptic splice site upstream of the last exon causing a frameshift and replacement of the last 32 codons by 20 different codons. Whole exome sequencing of an Italian patient with similar clinical features showed compound heterozygosity for two other mutations affecting the same exon of MYL2, also resulting in mutant proteins with altered C-terminal tails. As a consequence of these mutations, the second EF-hand domain is disrupted. EF-hands, assumed to function as calcium sensors, can undergo a conformational change upon binding of calcium that is critical for interactions with downstream targets. Immunohistochemical staining of skeletal muscle tissue of the Dutch patients showed a diffuse and weak expression of the mutant protein without clear fibre specificity, while normal protein was absent. Heterozygous missense mutations in MYL2 are known to cause dominant hypertrophic cardiomyopathy; however, none of the parents showed signs of cardiomyopathy. In conclusion, the mutations in the last exon of MYL2 are responsible for a novel autosomal recessive lethal myosinopathy due to defects changing the C-terminal tail of the ventricular form of the myosin regulatory light chain. We propose 'light chain myopathy' as a name for this MYL2-associated myopathy.

CRB1 is required for recycling by RAB11A+ vesicles in human retinal organoids.

CRB1 gene mutations can cause early- or late-onset retinitis pigmentosa, Leber congenital amaurosis, or maculopathy. Recapitulating human CRB1 phenotypes in animal models has proven challenging, necessitating the development of alternatives. We generated human induced pluripotent stem cell (iPSC)-derived retinal organoids of patients with retinitis pigmentosa caused by biallelic CRB1 mutations and evaluated them against autologous gene-corrected hiPSCs and hiPSCs from healthy individuals. Patient organoids show decreased levels of CRB1 and NOTCH1 expression at the retinal outer limiting membrane. Proximity ligation assays show that human CRB1 and NOTCH1 can interact via their extracellular domains. CRB1 patient organoids feature increased levels of WDFY1+ vesicles, fewer RAB11A+ recycling endosomes, decreased VPS35 retromer complex components, and more degradative endolysosomal compartments relative to isogenic control organoids. Taken together, our data demonstrate that patient-derived retinal organoids enable modeling of retinal degeneration and highlight the importance of CRB1 in early endosome maturation receptor recycling in the retina.

p27kip1-838C>A single nucleotide polymorphism is associated with restenosis risk after coronary stenting and modulates p27kip1 promoter activity.

BACKGROUND: The cyclin-dependent kinase inhibitor p27(kip1) is a key regulator of smooth muscle cell and leukocyte proliferation in vascular disease, including in-stent restenosis. We therefore hypothesized that common genetic variations or single nucleotide polymorphisms in p27(kip1) may serve as a useful tool in risk stratification for in-stent restenosis. METHODS AND RESULTS: Three single nucleotide polymorphisms concerning the p27(kip1) gene (-838C>A, rs36228499; -79C>T, rs34330; +326G>T, rs2066827) were determined in a cohort of 715 patients undergoing coronary angioplasty and stent placement. We discovered that the p27(kip1)-838C>A single nucleotide polymorphism is associated with clinical in-stent restenosis; the -838AA genotype decreases the risk of target vessel revascularization (hazard ratio, 0.28; 95% confidence interval, 0.10 to 0.77). This finding was replicated in another cohort study of 2309 patients (hazard ratio, 0.61; 95% confidence interval, 0.40 to 0.93). No association was detected between this end point and the p27(kip1)-79C>T and +326G>T single nucleotide polymorphisms. We subsequently studied the functional importance of the -838C>A single nucleotide polymorphism and detected a 20-fold increased basal p27(kip1) transcriptional activity of the -838A allele containing promoter. CONCLUSIONS: Patients with the p27(kip1)-838AA genotype have a decreased risk of in-stent restenosis corresponding with enhanced promoter activity of the -838A allele of this cell-cycle inhibitor, which may explain decreased smooth muscle cell proliferation.

Quantification of Movement Characteristics in Women's English Premier Elite Domestic Rugby Union.

This study aims were to determine the positional physical requirements of English domestic women's rugby union match-play. Global positioning system data (Catapult Minimax S4) were collected at 10 Hz of 129 competitive player games from the Tyrrells Premier15 league. Players were classified according to broad (Forwards, Backs) and specific positions (front-, second-, back-row, scrum-half, inside-, and outside-backs). Total distances, maximum speed, and player loads were calculated. Mean total distance was 4982 m and was similar between the Forwards and Backs, with second-row players covering the most (5297 m) and outside-backs the least (4701 m). Inside- and outside-backs covered a significantly greater distance at high speed running (134 m; 178 m) and sprinting (74 m; 92 m) speeds, respectively, whereas the second- and back-row covered greater distances jogging (1966 m; 1976 m) and the front-row spent the greatest overall distance walking (2613 m). Outside-backs reached greater maximum speed than all other positions (24.9 km.h-1). The mean player load was highest in the back-row (562 AU) and second-row (555 AU) and these were higher than the outside-backs (476 AU). These findings indicate that the demands placed on female rugby players are position specific and differ from male players. Additionally, the data are the first obtained from the 10 Hz GPS and from within English domestic women's rugby, thus adding to the overall limited data available on women's rugby union.

Effect of the PreBind Engagement Process on Scrum Timing and Stability in the 2013-16 Six Nations.

This study examined whether changes in scrum engagement laws from the "crouch-touch-set" in 2013 to the "PreBind" engagement from 2014 onward have led to changes in scrum characteristics, specifically timing, in international rugby union. Duration and outcomes were identified for all scrums occurring in the 2013-16 Six Nations (N = 60 games) using video analysis. Scrum duration increased after the introduction of the PreBind engagement from 59 s in 2013 to 69 s in 2016 (P = .024, effect size = 0.93). A significant increase in mean contact duration per scrum occurred when prebinding was adopted (P < .05), moving from 7.5 s under the crouch-touch-set process to 8.5, 10.0, and 10.8 s with PreBind in 2014, 2015, and 2016 (effect size = 0.71, 2.05, and 3.0, respectively). The number of scrum resets and collapsed scrums, along with early engagement and pulling down infringements, was lower under the PreBind process. Overall, the PreBind engagement resulted in longer scrums with significant increases observed in overall and contact durations, with improved stability-related characteristics. The longer contact time is a consequence of increased stability with a shift from high-energy impact to a sustained push phase with a lower force that is a benefit to player welfare.

Differentiating technical skill and motor abilities in selected and non-selected 3-5 year old team-sports players.

This study examined the difference in 22 3-5year old boys selected to an advanced or non-advanced group on an English community-based professional club training program. Time to complete 15m linear sprint and 15m zig-zag agility tests, with and without a ball, were used to assess the children's technical skill and motor ability. Age and body mass of both groups were the same, whereas height was greater and BMI was lower in the selected group (p<0.01). Linear sprint times without and with the ball were 3.98±0.35 and 4.44±0.36s, respectively for the selected and corresponding times were 4.64±1.04 and 11.2±5.37s for the non-selected (p<0.01, ES 0.8, 1.8). Similar results were found when a change of movement was included, both with and without the ball. A model of selection indicated that performance in an agility test with the ball and height had the greatest discriminatory power and explained 95.5% of between group variance. Selected players performed significantly better in tests when ball control was required. These findings suggest that technical proficiency and physical differences may influence team selection in three to five year old children.

Effect of Hyperglycemia on Gene Expression during Early Organogenesis in Mice.

BACKGROUND: Cardiovascular and neural malformations are common sequels of diabetic pregnancies, but the underlying molecular mechanisms remain unknown. We hypothesized that maternal hyperglycemia would affect the embryos most shortly after the glucose-sensitive time window at embryonic day (ED) 7.5 in mice. METHODS: Mice were made diabetic with streptozotocin, treated with slow-release insulin implants and mated. Pregnancy aggravated hyperglycemia. Gene expression profiles were determined in ED8.5 and ED9.5 embryos from diabetic and control mice using Serial Analysis of Gene Expression and deep sequencing. RESULTS: Maternal hyperglycemia induced differential regulation of 1,024 and 2,148 unique functional genes on ED8.5 and ED9.5, respectively, mostly in downward direction. Pathway analysis showed that ED8.5 embryos suffered mainly from impaired cell proliferation, and ED9.5 embryos from impaired cytoskeletal remodeling and oxidative phosphorylation (all P ≤ E-5). A query of the Mouse Genome Database showed that 20-25% of the differentially expressed genes were caused by cardiovascular and/or neural malformations, if deficient. Despite high glucose levels in embryos with maternal hyperglycemia and a ~150-fold higher rate of ATP production from glycolysis than from oxidative phosphorylation on ED9.5, ATP production from both glycolysis and oxidative phosphorylation was reduced to ~70% of controls, implying a shortage of energy production in hyperglycemic embryos. CONCLUSION: Maternal hyperglycemia suppressed cell proliferation during gastrulation and cytoskeletal remodeling during early organogenesis. 20-25% of the genes that were differentially regulated by hyperglycemia were associated with relevant congenital malformations. Unexpectedly, maternal hyperglycemia also endangered the energy supply of the embryo by suppressing its glycolytic capacity.

Next-generation sequencing of microRNAs in primary human polarized macrophages.

Macrophages are important for mounting inflammatory responses to tissue damage or infection by invading pathogens, and therefore modulation of their cellular functions is essential for the success of the immune system as well as for maintaining tissue homeostasis. Small non-coding RNAs are important regulatory elements of gene expression and microRNAs are the most widely known to be fundamental for the proper development of cells of the immune system. Macrophages can exhibit different phenotypes, depending on the cytokine environment they encounter in the affected tissues. We have analyzed the microRNA expression profiles during maturation of human primary monocytes into macrophages and polarization by pro- or anti-inflammatory cytokines. Here we describe the analysis of next-generation sequencing data deposited in EMBL-EBI ArrayExpress under accession number E-MTAB-1969 and associated with the study published by Cobos Jiménez and collaborators in Physiological Genomics in 2014 (1). The data presented here contributes to our understanding of microRNA expression profiles in human monocytes and macrophages and will also serve as a resource for novel microRNAs and other small RNA species expressed in these cells.