Trends in time-loss injuries during the 2011-2016 South African Rugby Youth Weeks.

Youth rugby is a popular sport in South Africa (SA) with a high injury incidence. The annual SA Rugby Youth Week tournaments attract the top age-group players in the country providing a sample of players for reliable injury surveillance. The aim of the study was to analyze the changes in time-loss injury rates at the SA Rugby Youth Week tournaments between 2011 and 2016, differences between age-groups, and to investigate associated injury risk factors. All confirmed time-loss injuries at the 4 age-group tournaments (under-13, under-16, and 2 under-18) from 2011 to 2016 were recorded. Injury incidence densities (IID) for years, tournaments, and injury risk factors were calculated and Poisson regression analyses were performed to determine differences. Time-loss injuries (n = 494) were reported over 24 240 exposure hours, with an overall IID of 20.4 (18.6-22.2) injuries per 1000 player hours. The year 2013 had a significantly lower IID compared to 2011. Injury risk decreased with increasing age; under-13 and under-16 had significantly higher IID compared to under-18 Craven Week. Tackling was the phase of play at highest risk, with an IID of 7.4 (6.3-8.5) injuries per 1000 player hours. Central/peripheral nervous system (CNS/PNS) and, therefore, the head/neck were the most commonly occurring injuries/location injured. In conclusion, within the SA Rugby tournament structure, the older players had a decreased rate of injury. The tackle event was still the phase of play with the highest injury incidence regardless of age. This increase in incidence is largely due to an increase in CNS/PNS injuries.

Non-sanctioning of illegal tackles in South African youth community rugby.

OBJECTIVES: The tackle event in rugby union ('rugby') contributes to the majority of players' injuries. Referees can reduce this risk by sanctioning dangerous tackles. A study in elite adult rugby suggests that referees only sanction a minority of illegal tackles. The aim of this study was to assess if this finding was similar in youth community rugby. DESIGN: Observational study. METHODS: Using EncodePro, 99 South African Rugby Union U18 Youth Week tournament matches were coded between 2011 and 2015. All tackles were coded by a researcher and an international referee to ensure that laws were interpreted correctly. The inter- and intra-rater reliabilities were 0.97-1.00. A regression analysis compared the non-sanctioned rates over time. RESULTS: In total, 12 216 tackles were coded, of which less than 1% (n=113) were 'illegal'. The majority of the 113 illegal tackles were front-on (75%), high tackles (72%) and occurred in the 2nd/4th quarters (29% each). Of the illegal tackles, only 59% were sanctioned. The proportions of illegal tackles and sanctioning of these illegal tackles to all tackles improved by 0.2% per year from 2011-2015 (p<0.05). CONCLUSIONS: In these youth community rugby players, 59% of illegal tackles were not sanctioned appropriately. This was better than a previous study in elite adult rugby, where only 7% of illegal tackles were penalised. Moreover, the rates of illegal tackles and non-sanctioned illegal tackles both improved over time. However, it is critical that referees consistently enforce all laws to enhance injury prevention efforts. Further studies should investigate the reasons for non-sanctioning.

Cyclin A1 directly interacts with B-myb and cyclin A1/cdk2 phosphorylate B-myb at functionally important serine and threonine residues: tissue-specific regulation of B-myb function.

Cyclin A1 is tissue-specifically expressed during spermatogenesis, but it is also highly expressed in acute myeloid leukemia (AML). Its pathogenetic role in AML and in the cell cycle of leukemic blasts is unknown. B-myb is essential for G1/S transition and has been shown to be phosphorylated by the cyclin A2/cdk2 complex. Here it is demonstrated that cyclin A1 interacts with the C-terminal portion of B-myb as shown by glutathione S-transferase (GST) precipitation. This interaction is confined to cyclin A1 because binding could not be detected between cyclin A2 and B-myb. Also, cdk2 was not pulled down by GST-B-myb from U937 lysates. In addition, co-immunoprecipitation of cyclin A1 and B-myb in leukemic cells evidenced protein interaction in vivo. Baculovirus-expressed cyclin A1/cdk2 complexes were able to phosphorylate human as well as murine B-myb in vitro. Tryptic phosphopeptide mapping revealed that cyclin A1/cdk2 complexes phosphorylated the C-terminal part of B-myb at several sites including threonine 447, 490, and 497 and serine 581. These phosphorylation sites have been demonstrated to be important for the enhancement of B-myb transcriptional activity. Further studies showed that cyclin A1 cooperated with B-myb to transactivate myb binding site containing promoters including the promoter of the human cyclin A1 gene. Taken together, the data suggest that cyclin A1 is a tissue-specific regulator of B-myb function and activates B-myb in leukemic blasts. (Blood. 2001;97:2091-2097)

Methylation of the cyclin A1 promoter correlates with gene silencing in somatic cell lines, while tissue-specific expression of cyclin A1 is methylation independent.

Gene expression in mammalian organisms is regulated at multiple levels, including DNA accessibility for transcription factors and chromatin structure. Methylation of CpG dinucleotides is thought to be involved in imprinting and in the pathogenesis of cancer. However, the relevance of methylation for directing tissue-specific gene expression is highly controversial. The cyclin A1 gene is expressed in very few tissues, with high levels restricted to spermatogenesis and leukemic blasts. Here, we show that methylation of the CpG island of the human cyclin A1 promoter was correlated with nonexpression in cell lines, and the methyl-CpG binding protein MeCP2 suppressed transcription from the methylated cyclin A1 promoter. Repression could be relieved by trichostatin A. Silencing of a cyclin A1 promoter-enhanced green fluorescent protein (EGFP) transgene in stable transfected MG63 osteosarcoma cells was also closely associated with de novo promoter methylation. Cyclin A1 could be strongly induced in nonexpressing cell lines by trichostatin A but not by 5-aza-cytidine. The cyclin A1 promoter-EGFP construct directed tissue-specific expression in male germ cells of transgenic mice. Expression in the testes of these mice was independent of promoter methylation, and even strong promoter methylation did not suppress promoter activity. MeCP2 expression was notably absent in EGFP-expressing cells. Transcription from the transgenic cyclin A1 promoter was repressed in most organs outside the testis, even when the promoter was not methylated. These data show the association of methylation with silencing of the cyclin A1 gene in cancer cell lines. However, appropriate tissue-specific repression of the cyclin A1 promoter occurs independently of CpG methylation.

Distinct phenotypes associated with increasing dosage of the PLP gene: implications for CMT1A due to PMP22 gene duplication.

Increased dosage of the proteolipid protein (Plp) gene causes CNS disease (Pelizaeus-Merzbacher disease [PMD]), which has many similarities to disorders of the PNS associated with duplication of the peripheral myelin protein-22 (PMP22) gene locus. Transgenic mice carrying extra copies of the wild-type Plp gene provide a valid model of PMD. Variations in gene dosage can cause a wide range of phenotypes from severe, lethal dysmyelination through late-onset demyelination. A predilection for different fiber diameters may occur within the various phenotypes with dysmyelination being more obvious in large fibers and late-onset degeneration predominantly affecting small fibers. Although the frequency of apoptotic oligodendrocytes is increased with high gene dosage, the number of mature oligodendrocytes appears adequate. Oligodendrocytes in the dysmyelinated CNS express a range of genes typical of mature cells, yet are unable to assemble sufficient myelin. Oligodendrocytes contain abnormal vacuoles and stain intensely for PLP and other proteins such as MAG. The findings suggest that with high gene dosage much of the PLP, and possibly other proteins, is missorted and degraded in the lysosomal system.

Formation of compact myelin is required for maturation of the axonal cytoskeleton.

Although traditional roles ascribed to myelinating glial cells are structural and supportive, the importance of compact myelin for proper functioning of the nervous system can be inferred from mutations in myelin proteins and neuropathologies associated with loss of myelin. Myelinating Schwann cells are known to affect local properties of peripheral axons (de Waegh et al., 1992), but little is known about effects of oligodendrocytes on CNS axons. The shiverer mutant mouse has a deletion in the myelin basic protein gene that eliminates compact myelin in the CNS. In shiverer mice, both local axonal features like phosphorylation of cytoskeletal proteins and neuronal perikaryon functions like cytoskeletal gene expression are altered. This leads to changes in the organization and composition of the axonal cytoskeleton in shiverer unmyelinated axons relative to age-matched wild-type myelinated fibers, although connectivity and patterns of neuronal activity are comparable. Remarkably, transgenic shiverer mice with thin myelin sheaths display an intermediate phenotype indicating that CNS neurons are sensitive to myelin sheath thickness. These results indicate that formation of a normal compact myelin sheath is required for normal maturation of the neuronal cytoskeleton in large CNS neurons.

Distinct Phenotypes Associated with Increasing Dosage of the PLP Gene: Implications for CMT1A Due to PMP22 Gene Duplication.

Increased dosage of the proteolipid protein (Plp) gene causes CNS disease (Pelizaeus-Merzbacher disease [PMD]), which has many similarities to disorders of the PNS associated with duplication of the peripheral myelin protein-22 (PMP22) gene locus. Transgenic mice carrying extra copies of the wild-type Plp gene provide a valid model of PMD. Variations in gene dosage can cause a wide range of phenotypes from severe, lethal dysmyelination through late-onset demyelination. A predilection for different fiber diameters may occur within the various phenotypes with dysmyelination being more obvious in large fibers and late-onset degeneration predominantly affecting small fibers. Although the frequency of apoptotic oligodendrocytes is increased with high gene dosage, the number of mature oligodendrocytes appears adequate. Oligodendrocytes in the dysmyelinated CNS express a range of genes typical of mature cells, yet are unable to assemble sufficient myelin. Oligodendrocytes contain abnormal vacuoles and stain intensely for PLP and other proteins such as MAG. The findings suggest that with high gene dosage much of the PLP, and possibly other proteins, is missorted and degraded in the lysosomal system.

Pituitary-directed leukemia inhibitory factor transgene causes Cushing's syndrome: neuro-immune-endocrine modulation of pituitary development.

Leukemia inhibitory factor (LIF) regulates the mature hypothalamic-pituitary-adrenal axis in vivo. In vitro, LIF determines corticotroph cell proliferation and induces POMC transcription. To explore LIF action on pituitary development, transgenic mice expressing LIF driven by the pituitary glycoprotein hormone alpha-subunit (alphaGSU) promoter were generated. Transgenic mice exhibited dwarfism with low IGF-I (29 +/- 9 ng/ml vs. wild type (WT) 137 +/- 16 ng/ml; P < 0.001), hypogonadism with low FSH (0.04 +/- 0.023 ng/ml vs. WT 0.63 +/- 0.18 ng/ml; P < 0.001), and Cushingoid features of thin skin and truncal obesity with elevated cortisol levels (86 +/- 22 ng/ml vs. WT 50 +/- 14 ng/ml; P = 0.002). Their pituitary glands showed corticotroph hyperplasia, striking somatotroph and gonadotroph hypoplasia, and multiple Rathke-like cysts lined by ciliated cells. LIF, overexpressed in Rathke's pouch at embryonal day 10, diverts the differentiation stream of hormone-secreting cells toward the corticotroph lineage and ciliated nasopharyngeal-like epithelium. Thus, inappropriate expression of LIF, a neuro-immune interfacing cytokine, plays a key role in the terminal differentiation events of pituitary development and mature pituitary function.

MR microscopy of transgenic mice that spontaneously acquire experimental allergic encephalomyelitis.

Pathology of fixed spinal cords from transgenic mice with a myelin basic protein (MBP) specific T cell receptor was investigated. These mice spontaneously acquire the demyelinating disease experimental allergic encephalomyelitis (EAE). Several complementary imaging modalities, all on the same tissues, were used to visualize lesions; these included high-field (11.7-T) microscopic diffusion tensor imaging (DTI), T2*-weighted imaging, and optical microscopy on histological sections. Lesions were predominantly in white matter around meninges and vasculature and appeared hyperintense in anatomical images. DTIs showed reduced diffusion anisotropy in the same hyperintense regions, consistent with inflammation and edema. Histology in the same tissues exhibited the characteristic pathology of EAE. Two techniques for visualizing the effective diffusion tensor fields are presented, which display direction, organization, and integrity of neuronal fibers. It is shown that DTI offers intriguing possibilities for visualizing axonal organization and lesions within white matter.

Characterization of myelin basic protein charge microheterogeneity in developing mouse brain and in the transgenic shiverer mutant.

Myelin basic protein (MBP) is a highly heterogeneous family of membrane proteins consisting of several isoforms resulting from alternative splicing and charge isomers arising from posttranslational modifications. Although well characterized in the bovine and human species, those in the mouse are not. With the availability of a number of transgenic and knockout mice, the need to understand the chemical nature of the MBPs has become very important. To isolate and characterize the MBP species in murine brain, two methods were adapted for use with the small amounts of MBP available from mice. The first was a scaled-down version of the preparative CM-52 chromatographic system commonly used to isolate MBP charge isomers; the second was an alkaline-urea slab gel technique that required five times less material than the conventional tube gel system and, from these gels, western blots were readily obtained. Murine MBP was resolved into two populations of charge isomers: the 18.5- and 14-kDa isoforms. Isolation and characterization of these charge isomers or components permitted us to assign possible posttranslational modifications to some of them. Component 1 (C-1), the most cationic isomer, had a molecular weight of 14,140.38 +/- 0.79. C-2 consisted of two 14-kDa species, 14,136.37 +/- 0.74 and 14,204.45 +/- 0.70. Two variants, 14,215.57 +/- 0.94 and 18,413.57 +/- 0.76, constituted C-3. C-4, C-5, and C-8 (the least cationic isomer) each consisted of both 14- and 18.5-kDa isoforms. During myelinogenesis, the 18.5-kDa isoform appeared first (day 4); the 14-kDa isoform appeared at day 16 and subsequently became the dominant isoform. The transgenic shiverer mutant synthesized mainly the 18.5-kDa isoform, but none of the 14-kDa isoform, similar to the 4-day-old mouse. We concluded that the transgenic shiverer was able to initiate myelinogenesis with the 18.5-kDa isoform, but was unable to complete myelinogenesis because of the absence of the 14-kDa isoform.

Pituitary-directed leukemia inhibitory factor transgene forms Rathke's cleft cysts and impairs adult pituitary function. A model for human pituitary Rathke's cysts.

Leukemia inhibitory factor (LIF) and LIF receptors are expressed in adenohypophyseal cells and LIF regulates pituitary hormone transcription and cell replication in vitro. Therefore, transgenic mice expressing pituitary-directed LIF driven by the rat growth hormone (GH) promoter were generated to evaluate the impact of LIF on pituitary development. Three founders were established with diminished linear growth and body weight (57-65% of wild type [WT]), and intense anterior pituitary LIF immunoreactivity. Cystic cavities observed in pituitary anterior lobes were lined by cuboidal, ciliated epithelial cells, focally immunopositive for cytokeratin and S-100 protein and immunonegative for adenohypophyseal hormones. Transgenic pituitaries showed decreased GH (40%) and prolactin (PRL) (26%) cells, and decreased GH and PRL mRNAs by in situ hybridization. ACTH cells increased 2.2-fold, whereas gonadotrophs and thyrotrophs were unchanged. Serum GH was undetectable (< 0.78 ng/ml), PRL levels were one third of WT (P < 0.05), IGF-I levels were 30% of WT (P < 0. 001), and T4 was normal. 10 human pituitary Rathke's cysts studied all showed conclusive LIF immunoreactivity in cyst-lining cells. Thus, intrapituitary murine LIF overexpression causes cystic invaginations from the anterior wall of Rathke's cleft, suggesting failed differentiation of Rathke's epithelium to hormone-secreting cells. Arrested murine pituitary maturation with formation of pituitary Rathke's cleft cysts, GH deficiency, and short stature provide a model to study human Rathke's cyst pathogenesis.

Dominant-negative action of the jimpy mutation in mice complemented with an autosomal transgene for myelin proteolipid protein.

Mutations in genes encoding membrane proteins have been associated with cell death of unknown cause from invertebrate development to human degenerative diseases. A point mutation in the gene for myelin proteolipid protein (PLP) underlies oligodendrocyte death and dysmyelination in jimpy mice, an accurate model for Pelizaeus-Merzbacher disease. To distinguish the loss of PLP function from other effects of the misfolded protein, we took advantage of the X chromosomal linkage of the gene and have complemented jimpy with a wild-type PLP transgene. In this artificial heterozygous situation, the jimpy mutation emerged as genetically dominant. At the cellular level oligodendrocytes showed little increase in survival although endogenous PLP gene and autosomal transgene were truly coexpressed. In surviving oligodendrocytes, wild-type PLP was functional and immunodetectable in myelin. Moreover, compacted myelin sheaths regained their normal periodicity. This strongly suggests that, despite the presence of functional wild-type PLP, misfolded jimpy PLP is by itself the primary cause of abnormal oligodendrocyte death.

Premature arrest of myelin formation in transgenic mice with increased proteolipid protein gene dosage.

Proteolipid protein (PLP) is an integral membrane protein of CNS myelin. Mutations of the X chromosome-linked PLP gene cause glial cell death and myelin deficiency in jimpy mice and other neurological mutants. As part of an attempt to rescue these mutants by transgenic complementation, we generated normal mouse lines expressing autosomal copies of the entire wild-type PLP gene. Surprisingly, increase of the PLP gene dosage in nonmutant mice with only 2-fold transcriptional overexpression results in a novel phenotype characterized by severe hypomyelination and astrocytosis, seizures, and premature death. This demonstrates that precise control of the PLP gene is a critical determinant of terminal oligodendrocyte differentiation. Dysmyelination of PLP transgenic mice provides experimental evidence that Pelizaeus-Merzbacher disease, previously associated with a partial duplication of the human X chromosome, can be caused by doubling of the PLP gene dosage.

Identification of an embryonic isoform of myelin basic protein that is expressed widely in the mouse embryo.

We have identified a myelin basic protein (MBP) isoform in mouse embryos that includes an exon upstream of the usual transcription initiation site. This isoform, embryonic-neonatal MBP (E-MBP), is expressed at the protein level in the embryonic nervous system at a time when other MBP isoforms are not detected. In addition to the central and peripheral nervous systems of the embryo and neonate, the thymus, spleen, and testes also express E-MBP at the protein level. The expression of E-MBP in cell types distinct from the nervous system strongly suggests that this MBP isoform has a role apart from the formation of myelin.

A myelin proteolipid protein-LacZ fusion protein is developmentally regulated and targeted to the myelin membrane in transgenic mice.

Transgenic mice were generated with a fusion gene carrying a portion of the murine myelin proteolipid protein (PLP) gene, including the first intron, fused to the E. coli LacZ gene. Three transgenic lines were derived and all lines expressed the transgene in central nervous system white matter as measured by a histochemical assay for the detection of beta-galactosidase activity. PLP-LacZ transgene expression was regulated in both a spatial and temporal manner, consistent with endogenous PLP expression. Moreover, the transgene was expressed specifically in oligodendrocytes from primary mixed glial cultures prepared from transgenic mouse brains and appeared to be developmentally regulated in vitro as well. Transgene expression occurred in embryos, presumably in pre- or nonmyelinating cells, rather extensively throughout the peripheral nervous system and within very discrete regions of the central nervous system. Surprisingly, beta-galactosidase activity was localized predominantly in the myelin in these transgenic animals, suggesting that the NH2-terminal 13 amino acids of PLP, which were present in the PLP-LacZ gene product, were sufficient to target the protein to the myelin membrane. Thus, the first half of the PLP gene contains sequences sufficient to direct both spatial and temporal gene regulation and to encode amino acids important in targeting the protein to the myelin membrane.

Morphometric analysis of normal, mutant, and transgenic CNS: correlation of myelin basic protein expression to myelinogenesis.

The neurological mutant mice shiverer (shi) and myelin deficient (shimld) lack a functional gene for the myelin basic proteins (MBP), have virtually no myelin in their CNS, shiver, seize, and die early. Mutant mice homozygous for an MBP transgene have MBP mRNA and MBP in net amounts approximately 25% of normal, have compact myelin, do not shiver or seize, and live normal life spans. We bred mice with various combinations of the normal, transgenic, shi, and shimld genes to produce mice that expressed MBP mRNA at levels of 0, 5, 12.5, 17.5, 50, 62.5, and 100% of normal. The CNS of these mice were analyzed for MBP content, tissue localization of MBP, degree of myelination, axon size, and myelin thickness. MBP protein content correlated with predicted MBP gene expression. Immunocytochemical staining localized MBP to white matter in normal and transgenic shi mice with an intensity of staining comparable to the degree of MBP gene expression. An increase in the percentage of myelinated axons and the thickness of myelin correlated with increased gene expression up to 50% of normal. The percentage of myelinated axons and myelin thickness remained constant at expression levels greater than 50%. The presence of axons loosely wrapped with oligodendrocytic membrane in mice expressing lower amounts of MBP mRNA and protein suggested that the oligodendroglia produced sufficient MBP to elicit axon wrapping but not enough to form compact myelin. Mean axon circumference of myelinated axons was greater than axon circumference of unmyelinated axons at each level of gene expression, further evidence that oligodendroglial cells preferentially myelinate axons of larger caliber.(ABSTRACT TRUNCATED AT 250 WORDS)

The shiverer mouse mutation shi/shi: rescue and preimplantation detection.

The shiverer mouse mutation has been used as a model in this series of experiments. Germ-line therapy of this disorder has been demonstrated by producing mice transgenic for the wild-type gene for myelin basic protein (MBP). The mutation has also been diagnosed in preimplantation mouse blastocysts.

Rapid preimplantation detection of mutant (shiverer) and normal alleles of the mouse myelin basic protein gene allowing selective implantation and birth of live young.

As a model for the detection of human genetic disease in preimplantation embryos, we describe a method in which trophectoderm biopsy samples from viable mouse blastocysts are simultaneously analyzed for the presence of a normal or mutant allele of the myelin basic protein gene by the polymerase chain reaction. The biopsied embryos are kept in culture during analysis of biopsied material and later reintroduced to a foster mother. Prenatal diagnosis can be completed in less than 7 hr. The identity of either amplification product was proved conclusively by direct sequence analysis of amplified products. Ninety-six percent of recovered blastocysts survived biopsy, as judged by re-formation of a blastocyst cavity in culture. Fifty-nine percent of the biopsied embryos established pregnancy by day 6.5, compared to 88% of unmanipulated controls. This approach can be applied to preimplantation diagnosis of human genetic diseases by using extraembryonic cells from blastocysts obtained after in vitro fertilization or uterine lavage. It will make possible the elimination of a mutant allele from a family in a single generation.

The dysmyelinating mouse mutations shiverer (shi) and myelin deficient (shimld).

Shiverer (shi/shi) is an autosomal recessive mouse mutation that produces a shivering phenotype in affected mice. A shivering gait can be seen from a few weeks after birth until their early death, which occurs between 50 and 100 days. The central nervous system of the mutant mouse is hypomyelinated but the peripheral nervous system appears normal. The myelin of the CNS, wherever present, is not well compacted and lacks the major dense line. Myelin basic protein (MBP), which is associated with the major dense line, is absent, and this is due to a deletion of the major part of the gene encoding MBP. Transgenic shiverer mice that have integrated and express the wild-type mouse MBP transgene no longer shiver and have normal life spans. Conversely, normal mice that have integrated an antisense MBP transgene, shiver. Myelin deficient shimld/shimld is allelic to shiverer (shi/shi) but the mutant mouse is less severely affected. Although MBP is present in the CNS, it is low in quantity and is not developmentally regulated. The gene encoding MBP has been both duplicated and inverted. Transgenic shimld/shimld mice with the wild-type MBP transgene have normal phenotypes.