Shoulder structures and strength in competitive preadolescent swimmers: A longitudinal ultrasonographic study.

BACKGROUND: Repetitive shoulder movements during competitive training may cause changes in the strength of periarticular shoulder structures in preadolescent swimmers. OBJECTIVE: To prospectively determine the effects of training on shoulder periarticular structures and muscle strength in preadolescent swimmers. DESIGN: Prospective cohort study. SETTING: Community-based natatorium. PARTICIPANTS: Twenty-four preadolescent swimmers aged 10-12 years. INTERVENTIONS: Not applicable. MAIN OUTCOME MEASURES: Measurements were repeated in three periods as preseason, midseason, and postseason. Ultrasonographic measurements (supraspinatus tendon thickness, humeral head cartilage thickness, deltoid muscle thickness, and acromiohumeral distance) were performed using a portable device and a linear probe. Shoulder (flexion, extension, abduction, internal and external rotation) and back (serratus anterior, lower, and middle trapezius) isometric muscle strength were measured with a handheld dynamometer. RESULTS: Supraspinatus tendon thickness and acromiohumeral distance were similar in all periods (all p > .05); however, deltoid muscle and humeral head cartilage thicknesses increased throughout the season (p = .002, p = .008, respectively). Likewise, whereas shoulder muscle strength increased (all p < .05), back muscle strength was similar in all periods (all p > .05). CONCLUSIONS: In preadolescent swimmers, acromiohumeral distance and supraspinatus tendon thickness seem to not change; but humeral head cartilage and deltoid muscle thicknesses as well as shoulder muscle strength increase throughout the season.

Countermovement push-up test to assess the upper extremity force-time characteristics in swimmers during a macrocycle.

Although it is known that swimming training can improve upper extremity performance, the force-time characteristics of the upper extremity during different training periods are not well understood. The objective of this study was to measure changes in the force-time characteristics of the upper extremity of young swimmers during different training periods within a season. Seventeen young swimmers, comprising 5 males (age: 15.4 ± 0.54 years); 12 females (16.4 ± 2.6 years) participated in this study. They were tested at four experimental test time points: baseline (E1), post-general preparation (E2), post-specific preparation (E3), and taper season (E4). The countermovement push-up test was performed using a force plate to measure force time parameters. Differences in force, time, velocity and impulse parameters were evaluated between the different periods. The study found that vertical take off velocity significantly increased across the assessed periods (F = 11.79; p = .001; η2 = .424), with significant increases from E1 to E2 (p < .001) and from E3 to E4 (p = .016). Flight Time also significantly increased across the assessed periods (F = 11.79; p = .001; η2 = .424), with significant increases from E1 to E2 (p < .001), from E1 to E4(p = .001), and from E3 to E4 (p = .005). The Force Impulse significantly increased throughout the assessed periods (F = 5.84; p = .012; η2 = .267), with significant increases from E1 to E2, (p = .006), from E1 to E3 (p = .016), and from E1 to E4 (p = .003). As this study shows, periods of increased training intensity can affect athletic progression, even though training aims to improve strength, speed, and performance. While some practical aspects such as strength, flight time, and impulse parameters may change during a macrocycle, the countermovement push-up test can provide trainers with an alternative and convenient way to monitor anaerobic force, speed, and performance, as well as measure explosive force-time performance in the upper body.

Apitherapy products enhance the recovery of CCL4-induced hepatic damages in rats.

BACKGROUND/AIM: Our objective was to identify the antioxidant properties of honeybee products from Turkey, chestnut honey, pollen, propolis, and royal jelly, and their hepatoprotective activity against CCl4-induced hepatic damage in rats. MATERIALS AND METHODS: Animals were fed with honeybee products for 7 days following CCl4 injection. Development of liver damage and oxidative stress were monitored by measuring the activities of the enzymes alanine transaminase, aspartate transaminase, malondialdehyde, superoxide dismutase, and catalase. Antioxidant capacities of the bee products were identified using FRAP and DPPH assays, as well as by measuring total phenolic and flavonoid contents. RESULTS: The antioxidant activities of the honeybee products were highest in propolis, followed, in order, by pollen, honey, and royal jelly. Despite their different levels of antioxidant capacity, their roles in the prevention of liver damage induced by CCl4 were very similar, which can be explained through their bioavailability to the treated animals. CONCLUSIONS: Our results suggest that honey, propolis, pollen, and royal jelly significantly enhanced the healing of CCl4-induced liver damage, partially due to their antioxidant properties and bioavailability.

Nicotine Exposure During Pregnancy Results in Persistent Midline Epithelial Seam With Improper Palatal Fusion.

INTRODUCTION: Nonsyndromic cleft palate is a common birth defect (1:700) with a complex etiology involving both genetic and environmental risk factors. Nicotine, a major teratogen present in tobacco products, was shown to cause alterations and delays in the developing fetus. METHODS: To demonstrate the postpartum effects of nicotine on palatal development, we delivered three different doses of nicotine (1.5, 3.0, and 4.5mg/kg/d) and sterile saline (control) into pregnant BALB/c mice throughout their entire pregnancy using subcutaneous micro-osmotic pump. Dams were allowed to deliver (~day 21 of pregnancy) and neonatal assessments (weight, length, nicotine levels) were conducted, and palatal tissues were harvested for morphological and molecular analyses, as well as transcriptional profiling using microarrays. RESULTS: Consistent administration of nicotine caused developmental retardation, still birth, low birth weight, and significant palatal size and shape abnormality and persistent midline epithelial seam in the pups. Through microarray analysis, we detected that 6232 genes were up-regulated and 6310 genes were down-regulated in nicotine-treated groups compared to the control. Moreover, 46% of the cleft palate-causing genes were found to be affected by nicotine exposure. Alterations of a subset of differentially expressed genes were illustrated with hierarchal clustering and a series of formal pathway analyses were performed using the bioinformatics tools. CONCLUSIONS: We concluded that nicotine exposure during pregnancy interferes with normal growth and development of the fetus, as well results in persistent midline epithelial seam with type B and C patterns of palatal fusion. IMPLICATIONS: Although there are several studies analyzing the genetic and environmental causes of palatal deformities, this study primarily shows the morphological and large-scale genomic outcomes of gestational nicotine exposure in neonatal mice palate.The previous version was incorrect. New authors Ali Nawshad, Hasan Otu, Janki Sharma, and Elizabeth Sheldon have been included in this version; the funding and acknowledgement sections have been updated accordingly; the article title, some text, and one supplementary data file have been edited; and the corresponding author has been changed. The original corresponding author regrets these earlier errors.

Progresses towards safe and efficient gene therapy vectors.

The emergence of genetic engineering at the beginning of the 1970's opened the era of biomedical technologies, which aims to improve human health using genetic manipulation techniques in a clinical context. Gene therapy represents an innovating and appealing strategy for treatment of human diseases, which utilizes vehicles or vectors for delivering therapeutic genes into the patients' body. However, a few past unsuccessful events that negatively marked the beginning of gene therapy resulted in the need for further studies regarding the design and biology of gene therapy vectors, so that this innovating treatment approach can successfully move from bench to bedside. In this paper, we review the major gene delivery vectors and recent improvements made in their design meant to overcome the issues that commonly arise with the use of gene therapy vectors. At the end of the manuscript, we summarized the main advantages and disadvantages of common gene therapy vectors and we discuss possible future directions for potential therapeutic vectors.

TGFß3 regulates periderm removal through ΔNp63 in the developing palate.

The periderm is a flat layer of epithelium created during embryonic development. During palatogenesis, the periderm forms a protective layer against premature adhesion of the oral epithelia, including the palate. However, the periderm must be removed in order for the medial edge epithelia (MEE) to properly adhere and form a palatal seam. Improper periderm removal results in a cleft palate. Although the timing of transforming growth factor ß3 (TGFß3) expression in the MEE coincides with periderm degeneration, its role in periderm desquamation is not known. Interestingly, murine models of knockout (-/-) TGFß3, interferon regulatory factor 6 (IRF6) (-/-), and truncated p63 (ΔNp63) (-/-) are born with palatal clefts because of failure of the palatal shelves to adhere, suggesting that these genes regulate palatal epithelial differentiation. However, despite having similar phenotypes in null mouse models, no studies have analyzed the possible association between the TGFß3 signaling cascade and the IRF6/ΔNp63 genes during palate development. Recent studies indicate that regulation of ΔNp63, which depends on IRF6, facilitates epithelial differentiation. We performed biochemical analysis, gene activity and protein expression assays with palatal sections of TGFß3 (-/-), ΔNp63 (-/-), and wild-type (WT) embryos, and primary MEE cells from WT palates to analyze the association between TGFß3 and IRF6/ΔNp63. Our results suggest that periderm degeneration depends on functional TGFß3 signaling to repress ΔNp63, thereby coordinating periderm desquamation. Cleft palate occurs in TGFß3 (-/-) because of inadequate periderm removal that impedes palatal seam formation, while cleft palate occurs in ΔNp63 (-/-) palates because of premature fusion.

Hepatoprotective potential of chestnut bee pollen on carbon tetrachloride-induced hepatic damages in rats.

Bee pollen has been used as an apitherapy agent for several centuries to treat burns, wounds, gastrointestinal disorders, and various other diseases. The aim of our study was to investigate the hepatoprotective effects of chestnut bee pollen against carbon tetrachloride (CCI4)-induced liver damage. Total phenolic content, flavonoid, ferric reducing/antioxidant power, and DPPH radical activity measurements were used as antioxidant capacity determinants of the pollen. The study was conducted in rats as seven groups. Two different concentrations of chestnut bee pollens (200 and 400 mg/kg/day) were given orally and one group was administered with silibinin (50 mg/kg/day, i.p.) for seven days to the rats following the CCI4 treatment. The protective effect of the bee pollen was monitored by aspartate transaminase (AST) and alanine transaminase (AST) activities, histopathological imaging, and antioxidant parameters from the blood and liver samples of the rats. The results were compared with the silibinin-treated and untreated groups. We detected that CCI4 treatment induced liver damage and both the bee pollen and silibinin-treated groups reversed the damage; however, silibinin caused significant weight loss and mortality due, severe diarrhea in the rats. The chestnut pollen had showed 28.87 mg GAE/g DW of total phenolic substance, 8.07 mg QUE/g DW of total flavonoid, 92.71 mg Cyn-3-glu/kg DW of total anthocyanins, and 9 mg ß -carotene/100 g DW of total carotenoid and substantial amount of antioxidant power according to FRAP and DPPH activity. The results demonstrated that the chestnut bee pollen protects the hepatocytes from the oxidative stress and promotes the healing of the liver damage induced by CCI4 toxicity. Our findings suggest that chestnut bee pollen can be used as a safe alternative to the silibinin in the treatment of liver injuries.

Systematic analysis of palatal transcriptome to identify cleft palate genes within TGFß3-knockout mice alleles: RNA-Seq analysis of TGFß3 Mice.

BACKGROUND: In humans, cleft palate (CP) accounts for one of the largest number of birth defects with a complex genetic and environmental etiology. TGFß3 has been established as an important regulator of palatal fusion in mice and it has been shown that TGFß3-null mice exhibit CP without any other major deformities. However, the genes that regulate cellular decisions and molecular mechanisms maintained by the TGFß3 pathway throughout palatogenesis are predominantly unexplored. Our objective in this study was to analyze global transcriptome changes within the palate during different gestational ages within TGFß3 knockout mice to identify TGFß3-associated genes previously unknown to be associated with the development of cleft palate. We used deep sequencing technology, RNA-Seq, to analyze the transcriptome of TGFß3 knockout mice at crucial stages of palatogenesis, including palatal growth (E14.5), adhesion (E15.5), and fusion (E16.5). RESULTS: The overall transcriptome analysis of TGFß3 wildtype mice (C57BL/6) reveals that almost 6000 genes were upregulated during the transition from E14.5 to E15.5 and more than 2000 were downregulated from E15.5 to E16.5. Using bioinformatics tools and databases, we identified the most comprehensive list of CP genes (n = 322) in which mutations cause CP either in humans or mice, and analyzed their expression patterns. The expression motifs of CP genes between TGFß3+/- and TGFß3-/- were not significantly different from each other, and the expression of the majority of CP genes remained unchanged from E14.5 to E16.5. Using these patterns, we identified 8 unique genes within TGFß3-/- mice (Chrng, Foxc2, H19, Kcnj13, Lhx8, Meox2, Shh, and Six3), which may function as the primary contributors to the development of cleft palate in TGFß3-/- mice. When the significantly altered CP genes were overlaid with TGFß signaling, all of these genes followed the Smad-dependent pathway. CONCLUSIONS: Our study represents the first analysis of the palatal transcriptome of the mouse, as well as TGFß3 knockout mice, using deep sequencing methods. In this study, we characterized the critical regulation of palatal transcripts that may play key regulatory roles through crucial stages of palatal development. We identified potential causative CP genes in a TGFß3 knockout model, which may lead to a better understanding of the genetic mechanisms of palatogenesis and provide novel potential targets for gene therapy approaches to treat cleft palate.

Transforming growth factor-ß activates c-Myc to promote palatal growth.

During palatogenesis, the palatal mesenchyme undergoes increased cell proliferation resulting in palatal growth, elevation and fusion of the two palatal shelves. Interestingly, the palatal mesenchyme expresses all three transforming growth factor (TGF) ß isoforms (1, 2, and 3) throughout these steps of palatogenesis. However, the role of TGFß in promoting proliferation of palatal mesenchymal cells has never been explored. The purpose of this study was to identify the effect of TGFß on human embryonic palatal mesenchymal (HEPM) cell proliferation. Our results showed that all isoforms of TGFß, especially TGFß3, increased HEPM cell proliferation by up-regulating the expression of cyclins and cyclin-dependent kinases as well as c-Myc oncogene. TGFß activated both Smad-dependent and Smad-independent pathways to induce c-Myc gene expression. Furthermore, TBE1 is the only functional Smad binding element (SBE) in the c-Myc promoter and Smad4, activated by TGFß, binds to the TBE1 to induce c-Myc gene activity. We conclude that HEPM proliferation is manifested by the induction of c-Myc in response to TGFß signaling, which is essential for complete palatal confluency. Our data highlights the potential role of TGFß as a therapeutic molecule to correct cleft palate by promoting growth.

Implications of TGFß on Transcriptome and Cellular Biofunctions of Palatal Mesenchyme.

Development of the palate comprises sequential stages of growth, elevation, and fusion of the palatal shelves. The mesenchymal component of palates plays a major role in early phases of palatogenesis, such as growth and elevation. Failure in these steps may result in cleft palate, the second most common birth defect in the world. These early stages of palatogenesis require precise and chronological orchestration of key physiological processes, such as growth, proliferation, differentiation, migration, and apoptosis. There is compelling evidence for the vital role of TGFß-mediated regulation of palate development. We hypothesized that the isoforms of TGFß regulate different cellular biofunctions of the palatal mesenchyme to various extents. Human embryonic palatal mesenchyme (HEPM) cells were treated with TGFß1, ß2, and ß3 for microarray-based gene expression studies in order to identify the roles of TGFß in the transcriptome of the palatal mesenchyme. Following normalization and modeling of 28,869 human genes, 566 transcripts were detected as differentially expressed in TGFß-treated HEPM cells. Out of these altered transcripts, 234 of them were clustered in cellular biofunctions, including growth and proliferation, development, morphology, movement, cell cycle, and apoptosis. Biological interpretation and network analysis of the genes active in cellular biofunctions were performed using IPA. Among the differentially expressed genes, 11 of them are known to be crucial for palatogenesis (EDN1, INHBA, LHX8, PDGFC, PIGA, RUNX1, SNAI1, SMAD3, TGFß1, TGFß2, and TGFßR1). These genes were used for a merged interaction network with cellular behaviors. Overall, we have determined that more than 2% of human transcripts were differentially expressed in response to TGFß treatment in HEPM cells. Our results suggest that both TGFß1 and TGFß2 orchestrate major cellular biofunctions within the palatal mesenchyme in vitro by regulating expression of 234 genes.

Expression levels of the PiT-2 receptor explain, in part, the gestational age-dependent alterations in transduction efficiency after in utero retroviral-mediated gene transfer.

BACKGROUND: A fundamental obstacle to using retroviral-mediated gene transfer (GT) to treat human diseases is the relatively low transduction levels that have been achieved in clinically relevant human cells. We previously showed that performing GT in utero overcomes this obstacle and results in significant levels of transduction within multiple fetal organs, with different tissues exhibiting optimal transduction at different developmental stages. We undertook the present study aiming to elucidate the mechanism for this age-dependent transduction, testing the two factors that we hypothesized could be responsible: (i) the proliferative status of the tissue at the time of GT and (ii) the expression level of the amphotropic PiT-2 receptor. METHODS: Immunofluorescence was performed on tissues from sheep of varying developmental stages to assess the proliferative status of the predominant cells within each organ as a function of age. After developing an enzyme-linked immunosorbent assay (ELISA) and a quantitative reverse transcription chain reaction (qRT-PCR) assay, we then quantified PiT-2 expression at the protein and mRNA levels, respectively. RESULTS: The results obtained indicate that the proliferative status of organs at the time of fetal GT is not the major determinant governing transduction efficiency. By contrast, our ELISA and qRT-PCR analyses demonstrated that PiT-2 mRNA and protein levels vary with gestational age, correlating with the observed differences in transduction efficiency. CONCLUSIONS: The findings of the present study explain the age-related differences that we previously observed in transduction efficiency after in utero GT. They also suggest it may be possible to achieve relatively selective GT to specific tissues by performing in utero GT when levels of PiT-2 are maximal in the desired target organ.

Factors determining the risk of inadvertent retroviral transduction of male germ cells after in utero gene transfer in sheep.

The possibility of permanent genetic changes to the germline is central to the bioethics of in utero gene therapy (IUGT) because of the concern of inadvertent potentially deleterious alterations to the gene pool. Despite presumed protection of the male germline due to early germ cell (GC) compartmentalization, we reported that GCs within the developing ovine testes are transduced at low levels after retrovirus-mediated IUGT, thus underscoring the need for a thorough understanding of GC development in clinically predictive models to determine the optimal time to perform IUGT and avoid germline modification. In the present studies, we used the fetal sheep model to analyze GCs for phenotype, location, proliferation, and incidence of transduction after IUGT at various fetal ages to learn when during development the nascent germline is likely to be at greatest risk of retrovirus-mediated alteration. Our studies show that although GCs were transduced at all injection ages, the levels of transduction varied by nearly 700-fold as a function of the age at transfer. After remaining largely quiescent as they migrated to/settled within nascent sex cords, GCs began active cycling before cord closure was complete, suggesting this is likely the point at which they would be most susceptible to retroviral transduction.Furthermore, we observed that compartmentalization of GCs continued into early postnatal life, suggesting the male germline may be vulnerable to low-level inadvertent retroviral vector modification throughout fetal life, but that this risk can be minimized by performing IUGT later in gestation.

Male germ-line cells are at risk following direct-injection retroviral-mediated gene transfer in utero.

The recent observation of vector sequences in the semen of men undergoing clinical gene therapy for hemophilia has highlighted the need to evaluate the risk of inadvertent germ-line transduction in a clinically relevant animal model. In the present study, we used three different approaches to investigate whether the germ line is at risk of inadvertent alteration following in utero retroviral gene transfer in the clinically relevant, random-bred sheep model. First, we conducted breeding studies. All organs from the 10 resultant offspring were devoid of proviral DNA, suggesting that the germ line had not been altered. As a second approach, we performed PCR on gradient-enriched, forensically purified sperm cells from in utero-transduced rams. The purified sperm cells from 6 of 19 of these rams were PCR positive for provirus, providing compelling evidence that the germ line had been transduced. As a third approach, we performed immunohistochemistry on sections of the testis from in utero-transduced sheep. Numerous somatic cells and very low levels of germ cells within the male reproductive tissues were transduced. In conclusion, our analysis on over 3 x 10(9) sperm cells suggests that the direct-injection approach employed in these studies may result in the inadvertent transduction of very low numbers of male germ cells.

Gestational age of recipient determines pattern and level of transgene expression following in utero retroviral gene transfer.

The direct vector injection approach was used in the fetal sheep model of in utero gene therapy to determine the effects of the recipient gestational age on the efficacy and pattern of liver, lung, and brain transduction and transgene expression. The livers contained foci of transgene-expressing hepatocytes and demonstrated an inverse correlation between recipient age and hepatocyte transduction/transgene expression, with higher levels of gene transfer/expression early in gestation and lower levels late in gestation. Conversely, the percentage of transgene-expressing cells within the lungs of these same animals increased with gestational age, with the majority of transduction occurring in epithelium and fibroblasts. In contrast to the lung and liver, transgene-expressing cells within the brain were extremely limited at all gestational ages tested. Our results demonstrate that numerous nonhematopoietic cells within the liver and lung are transduced following direct injection of murine retroviral vectors into fetal sheep and suggest that the developmental stage of each organ at the time of injection may determine its susceptibility to in utero gene transfer and subsequent levels of transgene expression. Our results suggest that with further vector optimization this approach may be useful for treating diseases that involve the lung and liver early in development.