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The study compared the effect of the novel "Gear" exercise programmes on inflammatory markers and cardiorespiratory fitness measurements. The participants mean age was 38 ± 5.74 years, with a mean body mass of 83 ± 16.93kg. Participants were randomly assigned to either the "Gear" exercise programme repeated at different times during the day (GEP-DT): cycled for 90 seconds, repeated three times per day, three days per week; the "Gear" exercise programme repeated at one point in time (GEP-OT): cycled for 90 seconds followed by four minutes and 30 seconds rest, repeated three times at one point in time, three days per week; moderate-intensity continuous exercise group (MICE): cycled for 30 minutes at 55-69% of HR

Background: Knee Osteoarthritis (KOA) is a prevalent, chronic disorder with excessive functional, social and economic burdens. The goal of treatment is to alleviate the symptoms and slow the progression. Documenting the effects of exercise and LLLT as co-modalities in the management of KOA allows practitioners to implement this management tool as part of KOA rehabilitation, resulting in the earlier discharge from a supervised rehabilitation setting. Objective: The purpose of this study was to determine the effect of low-level laser therapy (LLLT) in the treatment of knee osteoarthritis (KOA). A randomised controlled trial (RCT) was conducted on 111 participants (aged between 40-75 years) diagnosed with KOA. Participants were randomised into an exercise (n=39), LLLT (n=40), or a combined exercise-LLLT (n=32) group. Methods: The Western Ontario and McMaster Universities Osteoarthritis Index (WOMAC) scale was used to assess pain and functionality. Knee range of motion was assessed using a goniometer, and the one-minute timed sit–to-stand test measured physical functionality at four time points: (T1) baseline, (T2) post 12-session intervention, (T3) one-month post intervention and (T4) three-month’s post intervention. Knee circumference was measured using a measuring tape. Results: WOMAC pain and functionality scale and knee circumference scores decreased in all three groups (P

The thermal conductivity of nanofluids has been studied experimentally using the transient hot-wire method, and it is shown that a significant increase can be obtained. Existing methods for the prediction and correlation of the thermal conductivity are discussed. It is shown that a lot of work still needs to be done in this area.

We report a case of mild osteogenesis imperfecta in a 56-year-old male undergoing aortic valve replacement surgery. The primary defect in this patient was the substitution of arginine for glycine 85 in one of the two chains of alpha 1(I) procollagen. The thermal stability of the type I collagen synthesized by the patient's cultured skin fibroblasts was examined by enzymatic digestion. Digestion of the mutant type I collagen with trypsin and chymotrypsin at increasing temperatures sequentially generated three discrete collagenous fragments, approximately 90, 170, and 230 amino acids shorter than normal type I collagen. This incremental thermal denaturation is indicative of cooperative melting blocks within the type I collagen. This is the first demonstration of such cooperative blocks of melting in intact, essentially normal post-translationally modified type I collagen. This direct evidence for cooperative melting domains of uncut type I collagen suggests that discrete blocks of amino acids function as core sites stabilizing the collagen helix. The location of mutations of the alpha chains of type I collagen relative to these discrete blocks of amino acids may influence the severity of the disease phenotype.

Skin fibroblasts from a proband with lethal osteogenesis imperfecta synthesized a type I procollagen containing a cysteine residue in the alpha 1(I) helical domain. Assay of thermal stability of the triple helix by proteinase digestion demonstrated a decreased temperature for thermal unfolding of the protein. Of special importance was the observation that assays of thermal stability by proteinase digestion revealed two bands present in a 2:1 ratio of about 140 and 70 kDa; the 140 kDa band was reducible to a 70 kDa band. Further analysis of the fragments demonstrated that the cysteine mutation produced a local unfolding of the triple helix around residue 700 and apparently exposed the arginine residue at position 704 in both the alpha 1(I) and alpha 2(I) chains. Analysis of cDNAs and genomic DNAs demonstrated a single-base mutation that changed the GGT codon for glycine-691 of the alpha 1(I) chain to a TGT codon for cysteine. The mutation was not found in DNA from either of the proband's parents. Since the proteinase assay of helical stability generated a fragment of 700 residues that retained disulphide-bonded cysteine residues at alpha 1-691, the results provide one of the first indications that glycine substitutions in type I procollagen can alter the conformation of the triple helix at a site that is C-terminal to the site of the substitution.

Two substitutions for glycine in the triple-helical domain were found in type I procollagen synthesized by skin fibroblasts from two probands with lethal osteogenesis imperfecta. One was a substitution of valine for glycine alpha 1-637, and the other was a substitution of arginine for glycine alpha 2-694. The effects of the mutations on the zipper-like folding of the collagen triple helix were similar, since there was post-translational overmodification of the collagenase A fragments (amino acids 1-775) but not of more COOH-terminal fragments of the protein. The mutations differed markedly, however, on their effects on thermal unfolding of the triple helix. The collagenase A fragment from the collagen containing the arginine alpha 2-694 substitution was cleaved at about amino acid 700 when incubated with trypsin at 30-35 degrees C. Therefore, there was micro-unfolding of the triple helix at a site close to the glycine substitution. Surprisingly, however, the collagenase A fragment with the valine alpha 1-637 substitution was also cleaved at about amino acid 700 under the same conditions. The results, therefore, demonstrated that although most glycine substitutions delay folding of the triple helix in regions that are NH2-terminal to the site of the substitution, the effects on unfolding can be transmitted to regions that are COOH-terminal to the site of the glycine substitution.

Type VI collagen cDNAs of human and avian origin were recently obtained and characterized by screening cDNA libraries in k phage. Based on the published sequences of these cDNAs, we constructed partially degenerate oligonucleotide primers that we used in polymerase chain reactions for the generation of α2(VI) collagen clones of murine origin. As template, we used cDNA derived from murine total RNA. We amplified, cloned and sequenced a 1043-bp fragment that contains the coding sequence for the entire triple-helical domain except for the first two amino acids that are Gly-Pro in both man and chicken. Comparison of the nucleotide and derived amino acid sequences revealed 84.6% and 92.5% identity between mouse and man at the DNA and protein levels, respectively. Comparison with chicken sequences showed 72% and 79.1% identity. The third base usage showed a distinct preference for A in the glycine codons for the three species; whereas, U is preferred in all human fibrillar collagen genes previously defined. The preference for third base codon in Y position prolines is U for the α2(VI) collagen as it is for the human fibrillar collagen genes. A single cysteine at position 89, two Arg-Gly-Asp sequences, and one triple helix-interruption are conserved in mouse, man and chicken. Comparison of hydropathy plots showed great similarity between those of murine and human α2(VI) collagen chains and to a lesser extent between murine and chick. Northern blot hybridization of murine poly A+ RNA with a nick-translated radiolabeled ±2(VI) collagen probe detected one major transcript of 3.7 kb. Two less abundant transcripts of 2.7 and 2.1 kb were also detected. The presence of multiple transcripts suggests the existence of either multiple polyadenylation signals or alternative splicing events or both.

The development of selective inhibitors for individual PLA2 enzymes is necessary in order to target PLA2-specific signaling pathways, but it is challenging due to the observed promiscuity of known PLA2 inhibitors. In the current work, we present the development and application of a variety of synthetic routes to produce pentafluoro, tetrafluoro, and trifluoro derivatives of activated carbonyl groups in order to screen for selective inhibitors and characterize the chemical properties that can lead to selective inhibition. Our results demonstrate that the pentafluoroethyl ketone functionality favors selective inhibition of the GVIA iPLA2, a very important enzyme for which specific, potent, reversible inhibitors are needed. We find that 1,1,1,2,2-pentafluoro-7-phenyl-heptan-3-one (FKGK11) is a selective inhibitor of GVIA iPLA2 (XI(50) = 0.0073). Furthermore, we conclude that the introduction of an additional fluorine atom at the α′ position of a trifluoromethyl ketone constitutes an important strategy for the development of new potent GVIA iPLA2 inhibitors. © 2008 American Chemical Society.

A variety of lipophilic 2-oxoamides containing sulfonamide analogs of γ-amino acids as well as acyl sulfonamides of γ-aminobutyric acid were synthesized. Their ability to inhibit intracellular GIVA cPLA2 and GVIA iPLA2 as well as secreted GV sPLA2 was evaluated. The sulfonamide group seems a bioisosteric group suitable to replace the carboxyl group in 2-oxoamide inhibitors of GVIA cPLA2. Copyright © 2008 European Peptide Society and John Wiley & Sons, Ltd.

Background. Injuries occurring at the popular schoolboy rugby festivals in South Africa have not previously been evaluated. A rugby festivalis a unique event with multiple matches occurring over a 5-day period and a potentially increased risk of injury compared with adult games.Objectives. To analyse the prevalence and type of injuries over 2 years of a Johannesburg High School rugby festival, to compare the injuriesbetween the 2 years and to compare the injuries between the 3 days of the festivals.Methods. The study design was a retrospective, descriptive and observational study. The study population were participating rugby playersat the two rugby festivals in 2010 and 2011 who came to the medical tent provided. A standardised medical form was used to capture data.Results. A total of 626 players participated with 100 injury data sets analysed over the 2 years. The injury rate per player was 17% in year 1and 15% in year 2. There was no statistical difference (p=0.65) in the injury numbers between the 2 years. The injury profiles between therespective days and between the 2 years were not statistically different. Most injuries were to the head/face (30%), with the majority beingconcussion related (6%). Tackles were the most common mechanism of injury. Overall 24% of injuries were deemed severe enough to stopthe players from continuing play. Few injuries required referral for investigations or specialist physician care (19% and 2%, respectively)and most were managed with simple first aid at the primary care level.Conclusion. The number, nature and mechanisms of rugby injuries at this rugby festival were similar to numerous local and internationalstudies of schoolboy rugby players. Adequate standardised record keeping is recommended to increase knowledge and monitor trends.

All of the type I collagen in connective tissue is the product of one structural gene for the pro alpha 1(I) chain and another for the pro alpha 2(I) chain of type I procollagen. An intriguing question therefore is how the expression of the two genes differs in mineralizing and non-mineralizing tissues. One approach that our laboratory has pursued to answer this and related questions is to develop a new system whereby one can examine the self-assembly of collagen fibrils de novo by controlled enzymic cleavage of procollagen to collagen under physiological conditions. The system has made it possible for the first time to define thermodynamic parameters for the self-assembly process. We are now using the system to define the normal kinetics for fibril formation. The results should make it possible to study the effects of other components of extracellular matrix on fibril assembly, including the effects of bone-specific components that initiate mineralization. A second approach has been to define mutations in type I procollagen genes that cause increased brittleness of bone. Over a dozen mutations in type I procollagen genes have been found in probands with osteogenesis imperfecta. One of the surprises has been that at least 25% of the probands with lethal variants of osteogenesis imperfecta have mutations in type I procollagen genes. Another surprise has been the observation that a number of the mutations are tissue specific in terms of their phenotypic manifestations even though the same abnormal pro alpha chains are being synthesized in a variety of tissues.

Background: In contrast to the recommendations of the Joint Committee on Infant Hearing, neonatal hearing screening programs are still not universally available, and many countries implement elective screening in high-risk newborns. Objective: To assess the failure rates of neonates in hearing screening and the relative importance of risk factors for hearing impairment, both in neonatal intensive care units and in well-baby nursery neonates. The impact on cost-effectiveness is also evaluated. Subjects: In the current study, 25,288 newborns were assessed; 23,574 were full-term newborns in the well-baby nursery and 1,714 neonates were in neonatal intensive care units. Methods: All neonates had a general examination (including assessment for congenital anomalies and related history) and were assessed using transient evoked otoacoustic emissions. All newborns were older than 36 weeks at examination and thus had reliable transient evoked otoacoustic emissions. Results: From the 23,574 full-term neonates in the well-baby nursery, 23,123 (98.1%) passed the test and 451 failed (1.9%). Fifty-three of the 23,574 neonates (0.2%) had a risk factor for hearing impairment; 44 (83%) passed the test and 9 failed (17%). Family history of congenital hearing loss and congenital anomalies were the most frequent risk factors for hearing loss. From the 1,714 neonates in neonatal intensive care units, 1,590 (93%) passed the test and 124 failed (7%). Two hundred thirty-two of the 1,714 neonates (14%) had a risk factor for hearing impairment; 205 (88%) passed the test and 27 failed (12%). In neonatal intensive care unit neonates, toxic levels of ototoxic drugs, mechanical ventilation for more than 24 hours, prematurity, and low birth weight were the most frequent risk factors for hearing loss. Congenital anomalies/syndromes were the most important risk factors for failing screening in both the neonatal intensive care unit and the well-baby nursery, as they showed the highest risk of failing hearing screening. The second most important factor in neonatal intensive care unit newborns was low birth weight, and the third was prematurity in relation to the possibility of failing hearing screening. Conclusion: The present study found 575 neonates failing hearing screening of 25,288 tested newborns (2.3%). The fact that 78% of newborns who failed hearing screening were in the well-baby nurseries further supports the necessity of universal hearing screening instead of selective screening in neonatal intensive care units, even with the obvious impact on cost-effectiveness. Even if limited funding lead to selective screening in neonatal intensive care units, this should not be applied to high-risk newborns but to all neonatal intensive care unit neonates. Continuous assessment of risk factors and the related possibility of failing hearing screening are of paramount importance in designing hearing screening programs and refining the respective criteria.

Background: In contrast to the recommendations of the Joint Committee on Infant Hearing, neonatal hearing screening programs are still not universally available, and many countries implement elective screening in high-risk newborns. Objective: To assess the failure rates of neonates in hearing screening and the relative importance of risk factors for hearing impairment, both in neonatal intensive care units and in well-baby nursery neonates. The impact on cost-effectiveness is also evaluated. Subjects: In the current study, 25,288 newborns were assessed; 23,574 were full-term newborns in the well-baby nursery and 1,714 neonates were in neonatal intensive care units. Methods: All neonates had a general examination (including assessment for congenital anomalies and related history) and were assessed using transient evoked otoacoustic emissions. All newborns were older than 36 weeks at examination and thus had reliable transient evoked otoacoustic emissions. Results: From the 23,574 full-term neonates in the well-baby nursery, 23,123 (98.1%) passed the test and 451 failed (1.9%). Fifty-three of the 23,574 neonates (0.2%) had a risk factor for hearing impairment; 44 (83%) passed the test and 9 failed (17%). Family history of congenital hearing loss and congenital anomalies were the most frequent risk factors for hearing loss. From the 1,714 neonates in neonatal intensive care units, 1,590 (93%) passed the test and 124 failed (7%). Two hundred thirty-two of the 1,714 neonates (14%) had a risk factor for hearing impairment; 205 (88%) passed the test and 27 failed (12%). In neonatal intensive care unit neonates, toxic levels of ototoxic drags, mechanical ventilation for more than 24 hours, prematurity, and low birth weight were the most frequent risk factors for hearing loss. Congenital anomalies/syndromes were the most important risk factors for failing screening in both the neonatal intensive care unit and the well-baby nursery, as they showed the highest risk of failing hearing screening. The second most important factor in neonatal intensive care unit newborns was low birth weight, and the third was prematurity in relation to the possibility of failing hearing screening. Conclusion: The present study found 575 neonates failing hearing screening of 25,288 tested newborns (2.3%). The fact that 78% of newborns who failed hearing screening were in the well-baby nurseries further supports the necessity of universal hearing screening instead of selective screening in neonatal intensive care units, even with the obvious impact on cost-effectiveness. Even if limited funding lead to selective screening in neonatal intensive care units, this should not be applied to high-risk newborns but to all neonatal intensive care unit neonates. Continuous assessment of risk factors and the related possibility of failing hearing screening are of paramount importance in designing hearing screening programs and refining the respective criteria. © 2005, Otology & Neurotology, Inc.

The expansion of the trinucleotide repeat (CGG)n in successive generations through maternal meiosis is the cause of fragile X syndrome. Analysis of CA repeat polymorphisms flanking the FMR-1 gene provides evidence of a limited number of "founder" chromosomes and predisposing high-risk haplotypes related to the mutation. To investigate the origin of mutations in the fragile X syndrome in the Hellenic populations of Greece and Cyprus, we studied the alleles and haplotypes at DXS548 and FRAXAC2 loci of 16 independent fragile X and 70 normal control chromosomes. In addition, we studied 191 unrelated normal X chromosomes for the distribution and frequencies of CGG alleles. At DXS548, 6 alleles were found, 2 (194 and 196) of which were represented on fragile X chromosomes. At FRAXAC2, 6 alleles were found, 4 of which were present on fragile X chromosomes. Sixteen haplotypes were identified, but only 5 were present on fragile X chromosomes. The highest number of CGG repeats (or = 33) were associated with haplotypes 194-147, 194-151, 194-153, and 204-155. The data provide evidence for founder chromosomes and high-risk haplotypes in the Hellenic population.

The expansion of the trinucleotide repeat (CGG){sub n} in successive generations through maternal meiosis is the cause of fragile X syndrome. Analysis of CA repeat polymorphisms flanking the FMR-1 gene provides evidence of a limited number of {open_quotes}founder{close_quotes} chromosomes and predisposing high-risk haplotypes related to the mutation. To investigate the origin of mutations in the fragile X syndrome in the Hellenic populations of Greece and Cyprus, we studied the alleles and haplotypes at DXS548 and FRAXAC2 loci of 16 independent fragile X and 70 normal control chromosomes. In addition, we studied 191 unrelated normal X chromosomes for the distribution and frequencies of CGG alleles. At DXS548, 6 alleles were found, 2 (194 and 196) of which were represented on fragile X chromosomes. At FRAXAC2, 6 alleles were found, 4 of which were present on fragile X chromosomes. Sixteen haplotypes were identified, but only 5 were present on fragile X chromosomes. The highest number of CGG repeats ({ge} 33) were associated with haplotypes 194-147, 194-151, 194-153, and 204-155. The data provide evidence for founder chromosomes and high-risk haplotypes in the Hellenic population. 20 refs., 3 figs., 2 tabs.

The nucleotide sequences of the 3'-half of the human gene for the pro alpha(I) chain of type I procollagen (COL1A1) is presented. The results provide the nucleotide sequences for 26 introns not previously analyzed. The sequences that are presented, together with those previously published, make it possible to design primers for the polymerase chain reaction for amplifying and sequencing the gene. The availability of such primers will greatly facilitate the current search for mutations that can cause common and rare diseases of connective tissue.

Recent data from several laboratories have demonstrated that most forms of osteogenesis imperfecta (OI) are caused by mutations in one of the two structural genes for type I procollagen. Few, if any, are in the many other genes expressed in bone. This surprising conclusion has several implications for other genetic diseases that involve connective tissues as well as several more common diseases.

Type I procollagen was examined in cultured skin fibroblasts from a patient with a lethal variant of osteogenesis imperfecta. About half of the pro-α chains were post-translationally overmodified and had a decreased thermal stability. The vertebrate collagenase A fragment had a normal thermal stability, but the B fragment had a decreased thermal stability. Therefore, there was a change in primary structure in amino acids 776–1014 of either the α1(I) or α2(I) chain. Three of five cDNA clones for the α2(I) chain contained a single-base substitution of an A for a G that converted the codon for glycine at amino acid position 907 to aspartate. Complete nucleotide sequencing of bases coding for amino acids 776 to 1014 of the α2(I) chain was carried out in one cDNA clone that contained the mutation in the glycine codon and in one that did not. Also, nucleotide sequencing was performed of bases coding for amino acids 776–1014 of the α1(I) chain in seven independent cDNA clones. No other mutations were found. Therefore, the single base substitution that converts glycine 907 in the α2(I) chain to aspartate is solely responsible for the decreased thermal stability of the type I procollagen synthesized by the proband's fibroblasts. Also, glycine 907 of the α2(I) chain is an important component of a cooperative block that determines the melting temperature of the whole molecule.

Recent data from several laboratories have established that most variants of osteogenesis imperfecta (OI) are caused by mutations in the 2 structural genes for type I procollagen. There are 2 general reasons for the large number of mutations in type I procollagen in OI. One reason is that most of the structure of the procollagen monomer is essential for normal biological function of the protein. The second reason is that most of the mutations cause synthesis of structurally altered pro alpha chains of type I procollagen. The deleterious effects of the structurally altered pro alpha chains are then amplified by at least 3 mechanisms. One mechanism is a phenomenon referred to as "procollagen suicide" whereby altered pro alpha chains cause degradation of normal pro alpha chains synthesized by the same cell. Another mechanism involves the fact that many of the structurally altered pro alpha chains prevent normal processing of the N-propeptides of procollagen and persistence of the N-propeptide interferes with normal fibril assembly. A third mechanism is a recently discovered phenomenon in which a substitution of a bulkier amino acid for glycine can cause a kink in the triple helix of the molecule. The kinked collagen, in turn, causes formation of abnormally branched fibrils. Because the deleterious effects of abnormal pro alpha chains are amplified by these 3 mechanisms, most of the mutations are dominant and many are dominant lethal. The conclusion that most variants of OI are caused by mutations in the structural genes for type I procollagen has broad implications for other diseases that affect connective tissue, diseases such as chondrodystrophies, osteoarthritis, and osteoporosis.

Recent reports have demonstrated that a series of probands with severe osteogenesis imperfecta had single base mutations in one of the two structural genes for type I procollagen that substituted amino acids with bulkier side chains for glycine residues and decreased the melting temperature of the triple helix. Here we demonstrate that the type I procollagen synthesized by cultured fibroblasts from a proband with a severe form of osteogenesis imperfecta consisted of normal molecules and molecules over-modified by post-translational reactions. The thermal stability of the intact type I collagen was normal as assayed by protease digestion under conditions in which a decrease in thermal stability was previously observed with eight other substitutions for glycine in the alpha 1(I) chain. In contrast, the thermal stability of the one-quarter length B fragment generated by digestion with vertebrate collagenase was decreased by 2-3 degrees C under the same conditions. Nucleotide sequencing of cDNAs and genomic DNA established that the proband had a substitution of A for G in one allele of the pro alpha 1(I) gene that converted the codon for alpha 1-glycine 844 to a codon for serine. The results also established that the alpha 1-serine 844 was the only mutation that could account for the decrease in thermal stability of the collagenase B fragment. There are at least two possible explanations for the failure of the alpha 1-serine 844 substitution to decrease the thermal stability of the collagen molecule whereas eight similar mutations decreased the melting temperature. One possibility is that the effects of glycine substitutions are position specific because not all glycine residues make equivalent contributions to cooperative blocks of the triple helix that unfold in the predenaturation range of temperatures. A second possible explanation is that substitutions of glycine by serine have much less effect on the stability of protein than the substitutions by arginine, cysteine, and aspartate previously studied.

A fraction of the pro alpha 1(I) and pro alpha 2(I) chains in type I procollagen synthesized by the fibroblasts from a proband with a lethal variant of osteogenesis imperfecta were overmodified by posttranslational reactions. After digestion with pepsin, some of the alpha 1(I) chains were recovered as disulfide-linked dimers. Mapping of cyanogen bromide peptides indicated that the disulfide link was contained in alpha 1-CB6, the cyanogen bromide fragment containing amino acid residues 823-1014 of the alpha 1(I) chain. Nucleotide sequencing of cDNA clones demonstrated a substitution of T for G that converted glycine 904 of the alpha 1(I) chain to cysteine. A large fraction of the type I procollagen synthesized by the proband's fibroblasts had a thermostability that was 3-4 degrees C lower than the normal type I procollagen as assayed by brief proteinase digestion. In addition, the type I procollagen synthesized by the proband's fibroblasts was secreted with an abnormal kinetic pattern in that there was a lag period of about 30 min in pulse-chase experiments. The mutation of glycine to cysteine was not found in type I procollagen synthesized by fibroblasts from the proband's parents. Therefore, the mutation was a sporadic one. However, the mother's fibroblasts synthesized a type I procollagen in which part of the pro alpha chains were overmodified and had a lower thermostability. Therefore, the proband may have inherited a mutated allele for type I procollagen from her mother that contributed to the lethal phenotype. The mother was asymptomatic. She was somewhat short and had slightly blue sclerae but no definitive signs of a connective tissue abnormality. The observations on the mother indicated, therefore, that a mutation that causes synthesis of a type I procollagen with a lowered thermal stability does not necessarily produce a heritable disorder of connective tissue.