A novel mutation in the glycogen synthase 2 gene in a child with glycogen storage disease type 0

Background: Glycogen storage disease type 0 is an autosomal recessive disease presenting in infancy or early childhood and characterized by ketotic hypoglycemia after prolonged fasting and postprandial hyperglycemia and hyperlactatemia. Sixteen different mutations have been identified to date in the gene which encodes hepatic glycogen synthase, resulting in reduction of glycogen storage in the liver. Case Presentation: Biochemical evaluation as well as direct sequencing of exons and exon-intron boundary regions of the GYS2 gene were performed in a patient presenting fasting hypoglycemia and postprandial hyperglycemia and her parents. The patient was found to be compound heterozygous for one previously reported nonsense mutation (c. 736 C>T; R243X) and a novel frameshift mutation (966_967delGA/insC) which introduces a stop codon 21 aminoacids downstream from the site of the mutation that presumably leads to loss of 51% of the COOH-terminal part of the protein. The glycemia and lactatemia of the parents after an oral glucose tolerance test were evaluated to investigate a possible impact of the carrier status on the metabolic profile. The mother, who presented a positive family history of type 2 diabetes, was classified as glucose intolerant and the father, who did not exhibit metabolic changes after the glucose overload, had an antecedent history of hypoglycemia after moderate alcohol ingestion. Conclusion: The current results expand the spectrum of known mutations in GYS2 and suggest that haploinsufficiency could explain metabolic abnormalities in heterozygous carriers in presence of predisposing conditions.

The Effects of a 785-nm AlGaInP Laser on the Regeneration of Rat Anterior Tibialis Muscle After Surgically-Induced Injury

Objective: This study aims to investigate the effects of low-level laser therapy (LLLT) on muscle regeneration. For this purpose, the anterior tibialis muscle of 48 male Wistar rats received AlGaInP laser treatment (785 nm) after surgically-induced injury. Background Data: Few studies have been conducted on the effects of LLLT on muscle regeneration at different irradiation doses. Materials and Methods: The animals were randomized into four groups: uninjured rats (UN); uninjured and laser-irradiated rats (ULI); injured rats (IN); and injured and laser-irradiated rats (ILI). The direct contact laser treatment was started 24 h after surgery. An AlGaInP diode laser emitting 75 mW of continuous power at 785 nm was used for irradiation. The laser probe was placed at three treatment points to deliver 0.9 J per point, for a total dose of 2.7 J per treatment session. The animals were euthanized after treatment sessions 1, 2, and 4. Mounted sections were stained with hematoxylin and eosin and used for quantitative morphological analysis, in which the number of leukocytes and fibroblasts were counted over an area of 4480 mu m(2). The data were statistically analyzed by analysis of variance (ANOVA) and the Bonferroni t-test. Results: Quantitative data showed that the number of both polymorphonuclear and mononuclear leukocytes in the inflammatory infiltrate at the injury site was smaller in the ILI(1), ILI(2), and ILI(4) subgroups compared with their respective control subgroups (IN(1), IN(2), and IN(4)) for sessions 1, 2, and 4, respectively (p < 0.05). On the other hand, the number of fibroblasts increased after the fourth treatment session (p < 0.05). With regard to the regeneration of muscle fibers following injury, only after the fourth treatment session was it possible to find muscle precursor cells such as myoblasts and some myotubes in the ILI(4) subgroup. Conclusion: During the acute inflammatory phase, the AlGaInP laser treatment was found to have anti-inflammatory effects, reducing the number of leukocytes at the injury site and accelerating the regeneration of connective tissue.

Evaluation of muscle strength and motor abilities in children with type II and III spinal muscle atrophy treated with valproic acid

Background: Spinal muscular atrophy (SMA) is an autosomal recessive disorder that affects the motoneurons of the spinal anterior horn, resulting in hypotonia and muscle weakness. The disease is caused by deletion or mutation in the telomeric copy of SMN gene (SMN1) and clinical severity is in part determined by the copy number of the centromeric copy of the SMN gene (SMN2). The SMN2 mRNA lacks exon 7, resulting in a production of lower amounts of the full-length SMN protein. Knowledge of the molecular mechanism of diseases has led to the discovery of drugs capable of increasing SMN protein level through activation of SMN2 gene. One of these drugs is the valproic acid (VPA), a histone deacetylase inhibitor. Methods: Twenty-two patients with type II and III SMA, aged between 2 and 18 years, were treated with VPA and were evaluated five times during a one-year period using the Manual Muscle Test (Medical Research Council scale-MRC), the Hammersmith Functional Motor Scale (HFMS), and the Barthel Index. Results: After 12 months of therapy, the patients did not gain muscle strength. The group of children with SMA type II presented a significant gain in HFMS scores during the treatment. This improvement was not observed in the group of type III patients. The analysis of the HFMS scores during the treatment period in the groups of patients younger and older than 6 years of age did not show any significant result. There was an improvement of the daily activities at the end of the VPA treatment period. Conclusion: Treatment of SMA patients with VPA may be a potential alternative to alleviate the progression of the disease.

Sensitization Prevalence, Antibody Cross-Reactivity and Immunogenic Peptide Profile of Api g 2, the Non-Specific Lipid Transfer Protein 1 of Celery

Background: Celery (Apium graveolens) represents a relevant allergen source that can elicit severe reactions in the adult population. To investigate the sensitization prevalence and cross-reactivity of Api g 2 from celery stalks in a Mediterranean population and in a mouse model. Methodology: 786 non-randomized subjects from Italy were screened for IgE reactivity to rApi g 2, rArt v 3 (mugwort pollen LTP) and nPru p 3 (peach LTP) using an allergen microarray. Clinical data of 32 selected patients with reactivity to LTP under investigation were evaluated. Specific IgE titers and cross-inhibitions were performed in ELISA and allergen microarray. Balb/c mice were immunized with purified LTPs; IgG titers were determined in ELISA and mediator release was examined using RBL-2H3 cells. Simulated endolysosomal digestion was performed using microsomes obtained from human DCs. Results: IgE testing showed a sensitization prevalence of 25.6% to Api g 2, 18.6% to Art v 3, and 28.6% to Pru p 3 and frequent co-sensitization and correlating IgE-reactivity was observed. 10/32 patients suffering from LTP-related allergy reported symptoms upon consumption of celery stalks which mainly presented as OAS. Considerable IgE cross-reactivity was observed between Api g 2, Art v 3, and Pru p 3 with varying inhibition degrees of individual patients' sera. Simulating LTP mono-sensitization in a mouse model showed development of more congruent antibody specificities between Api g 2 and Art v 3. Notably, biologically relevant murine IgE cross-reactivity was restricted to the latter and diverse from Pru p 3 epitopes. Endolysosomal processing of LTP showed generation of similar clusters, which presumably represent T-cell peptides. Conclusions: Api g 2 represents a relevant celery stalk allergen in the LTP-sensitized population. The molecule displays common B cell epitopes and endolysosomal peptides that encompass T cell epitopes with pollen and plant-food derived LTP.

Effects of high-dose creatine supplementation on kidney and liver responses in sedentary and exercised rats

This study evaluated the effects of high-dose of short-term creatine supplementation (5g.kg(-1). day(-1) to 1 week) and long-term creatine supplementation (1g.kg(-1). day(-1) to 4-8 weeks) on kidney and liver structure and function of sedentary and exercised Wistar rats ( Exercise sessions consisted of swimming at 80% of maximal work load supported during 5 days per week with daily sessions of 60 minutes throughout the duration of the supplementation). Seventy-two animals ( 245 +/- 5g) were divided into four groups (n = 18): control diet Sedentary ( SED), Creatine diet Sedentary (CRE), control diet Exercised (EXE), and Creatine diet Exercised (EXECRE). Histological and blood biochemical studies were performed after one, four, and eight weeks of creatine supplementation and exercise ( n = 6). No differences were found when comparing SED, EXE and EXECRE groups for kidney and liver structure and function at one, four and eight weeks. However, the CRE group showed higher levels of creatinine (1.1 +/- 0.2 vs. 0.4 +/- 0.1 mg.dl(-1); p < 0.05), and urea ( 37 +/- 3 vs. 19 +/- 1 mg. dl(-1); p < 0.05) when compared with all others groups at four and eight weeks. At eight weeks, the CRE group presented increased levels of ALT (41 +/- 7 vs. 23 +/- 7 U.L(-1); p < 0.05), AST (89 +/- 6 vs. 62 +/- 5 U. L(-1); p < 0.05), GGT (8.0 +/- 0.9 vs. 3.9 +/- 1.0 U. L(-1); p < 0.05), and AP (125 +/- 10 vs. 69 +/- 9 U. L(-1); p < 0.05) also when compared with all others groups. Moreover, the CRE group demonstrated some structural alterations indicating renal and hepatic damage at four and eight weeks, respectively. These results suggest that long-term creatine supplementation (up to 4-8 weeks) may adversely affect kidney and liver structure and function of sedentary but not of exercised rats.

FBXO25-associated nuclear domains: A novel subnuclear structure

Skp1, Cul1, Rbx1, and the FBXO25 protein form a functional ubiquitin ligase complex. Here, we investigate the cellular distribution of FBXO25 and its colocalization with some nuclear proteins by using immunochemical and biochemical approaches. FBXO25 was monitored with affinity-purified antibodies raised against the recombinant fragment spanning residues 2-62 of the FBXO25 sequence. FBXO25 protein was expressed in all mouse tissues tested except striated muscle, as indicated by immunoblot analysis. Confocal analysis revealed that the endogenous FBXO25 was partially concentrated in a novel dot-like nuclear domain that is distinct from clastosomes and other well-characterized structures. These nuclear compartments contain a high concentration of ubiquitin conjugates and at least two other components of the ubiquitin-proteasome system: 20S proteasome and Skp1. We propose to name these compartments FBXO25-associated nuclear domains. Interestingly, inhibition of transcription by actinomycin D or heat-shock treatment drastically affected the nuclear organization of FBXO25-containing structures, indicating that they are dynamic compartments influenced by the transcriptional activity of the cell. Also, we present evidences that an FBXO25-dependent ubiquitin ligase activity prevents aggregation of recombinant polyglutamine-containing huntingtin protein in the nucleus of human embryonic kidney 293 cells, suggesting that this protein can be a target for the nuclear FBXO25 mediated ubiquitination.

Global gene expression profile in myelodysplastic syndromes using SAGE

The molecular pathogenesis of myelodysplastic syndromes (MDS) is poorly understood. In order to expand our knowledge of genetic defects in MDS, we determined the overall profile of genes expressed in bone marrow from patients with refractory anemia with excess blasts ( RAEB) by serial analysis of gene expression ( SAGE). The present report describes a partial transcriptome of RAEB bone marrow derived from 56,694 sequenced tags that provides information about expressed gene products. This is the first attempt to determine an overall profile of gene expression specifically in RAEB at diagnosis using SAGE, which should be useful in the understanding of the physiopathology of MDS and in identifying the genes involved.

Influence of Laser Photobiomodulation on Collagen IV During Skeletal Muscle Tissue Remodeling After Injury in Rats

Objective: The aim of the present study was to determine the effect of GaAlAs low-level laser therapy (LLLT) on collagen IV remodeling of the tibialis anterior (TA) muscle in rats after cryolesion. Background: Considerable interest exists in skeletal muscle regeneration in situations such as repair after exercise-induced muscle injury, after muscle transplantation, in muscular dystrophy, exercise-induced muscle injury, and the recovery of strength after atrophy due to disuse. A number of studies have demonstrated the potential of LLLT in facilitating the muscle-healing process; however, no consensus is found in the literature regarding the best laser-irradiation parameters. Methods: Adult male Wistar rats (n = 45) were used and randomly divided into three groups: control (n = 5); nontreated cryolesioned group (n = 20), and LLLT-cryolesioned group (n = 20). The cryolesioned groups were analyzed at 1, 7, 14, and 21 days after the injury procedure. Laser irradiation was performed 3 times per week on the injured region by using the GaAlAs laser (660 nm; beam spot of 0.04 cm(2), output power of 20 mW, power density of 500 mW/cm(2), and energy density of 5 J/cm(2), for 10 sec). The muscles were removed, frozen, cryosectioned, and then stained with hematoxylin-eosin for the visualization of general morphology or used for immunohistochemical analysis of collagen IV. Results: It was demonstrated that LLLT promotes an increase in collagen IV immunolabeling in skeletal muscle in the first 7 days after acute trauma caused by cryoinjury, but does not modify the duration of the tissue-repair process. Even with LLLT, the injured muscle tissue needs similar to 21 days to achieve the same state of organization as that in the noninjured muscle. Conclusion: The collagen IV content is modulated in regenerating skeletal muscle under LLLT, which might be associated with better tissue outcome, although the histologic analysis did not detect tissue improvement in the LLLT group.

New Source of Muscle-Derived Stem Cells with Potential for Alveolar Bone Reconstruction in Cleft Lip and/or Palate Patients

Cleft lip and palate (CLP), one of the most frequent congenital malformations, affects the alveolar bone in the great majority of the cases, and the reconstruction of this defect still represents a challenge in the rehabilitation of these patients. One of the current most promising strategy to achieve this goal is the use of bone marrow stem cells (BMSC); however, isolation of BMSC or iliac bone, which is still the mostly used graft in the surgical repair of these patients, confers site morbidity to the donor. Therefore, in order to identify a new alternative source of stem cells with osteogenic potential without conferring morbidity to the donor, we have used orbicular oris muscle (OOM) fragments, which are regularly discarded during surgery repair (cheiloplasty) of CLP patients. We obtained cells from OOM fragments of four unrelated CLP patients (CLPMDSC) using previously described preplating technique. These cells, through flow cytometry analysis, were mainly positively marked for five mesenchymal stem cell antigens (CD29, CD90, CD105, SH3, and SH4), while negative for hematopoietic cell markers, CD14, CD34, CD45, and CD117, and for endothelial cell marker, CD31. After induction under appropriate cell culture conditions, these cells were capable to undergo chondrogenic, adipogenic, osteogenic, and skeletal muscle cell differentiation, as evidenced by immunohistochemistry. We also demonstrated that these cells together with a collagen membrane lead to bone tissue reconstruction in a critical-size cranial defects previously induced in non-immunocompromised rats. The presence of human DNA in the new bone was confirmed by PCR with human-specific primers and immunohistochemistry with human nuclei antibodies. In conclusion, we showed that cells from OOM have phenotypic and behavior characteristics similar to other adult stem cells, both in vitro and in vivo. Our findings suggest that these cells represent a promising source of stem cells for alveolar bone grafting treatment, particularly in young CLP patients.

Soft tissue profile in white Brazilian adults with normal occlusions and well-balanced faces

Objective: To analyze anteroposterior soft tissue facial parameters for a sample of white Brazilian adults and to compare these measurements with the values proposed for white North American adults. Materials and Methods: Facial profile photographs were taken of 59 white Brazilians (30 men and 29 women) with normal occlusions and balanced faces with ages ranging from 18 to 30 years. The independent Student's t-test (P < .05) was used to compare the soft tissue parameters of the Brazilians with those of the North Americans. Results: White Brazilian women presented a less protruded face compared with white American women except for the glabella region. White Brazilian women showed a smaller nasal projection, less protruded upper and lower lips, a more obtuse nasolabial angle, and a smaller projection of the B' point and chin than white American women. Conversely, the two male groups demonstrated less evident soft tissue profile differences, with the exception of the nose projection, which was smaller in white Brazilian men than in white American men. Conclusions: A universal standard of facial esthetic is not applicable to diverse white populations. Differences regarding the soft tissue profile features were found between white Brazilians and white Americans. These differences should be considered in the orthodontic/orthognathic surgery diagnosis and treatment plan for white Brazilians together with the patient's individual opinion and perception of beauty.

Low-Level Laser Intensity Application in Masseter Muscle for Treatment Purposes

Objective: This study evaluated with histochemical analysis how the number of laser applications can affect the masseter muscle. Background: In dentistry today, the laser is used in patients with temporomandibular disorders (TMDs), mainly for radiating pain in the masticatory muscles, whose origins may be associated with malocclusion, although the laser effects are not well understood on the cellular level. Materials and Methods: Thirty mice (HRS/J lineage) were randomly distributed into groups according to the number of laser applications (three, six, and 10). For each group of laser applications (experimental, n = 5), it was considered the control group (n = 5), which was not irradiated. All animals inhaled halothane (2-bromo-2-chloro-1, 1, 1-trifluoroethane, minimum 99%, Sigma Aldrich, India) before each laser irradiation performed on the left masseter muscle region, on alternate days with 20 J/cm(2), 40mW, for 20 sec. The muscle samples were collected for histochemical analysis with succinate dehydrogenase (SDH) enzyme 72 h after the last application. Results: (a) A decrease in area of light fibers type (35.91% +/- 6.9%; 32.08% +/- 6.3%, and 27.88% +/- 6.3%), according to the increase of laser applications (p < 0.05); (b) significant increase (p < 0.05) in the area of intermediate fibers, with an increase of laser application (11.08% +/- 3.9%; 16.52% +/- 5.7%, and 15.96% +/- 3.9%), although the increase with 10 applications was small; (c) area increase of dark fibers in the group with three laser applications (0.16% +/- 0.3%) (p < 0.05), and in groups with six and 10 laser applications, respectively (9.68% +/- 6.0% and 9.60% +/- 4.0%). Conclusions: The SDH enzyme activity revealed that the number of laser applications increases the metabolic pattern of the muscle fibers. A minimal difference in metabolic activity between six and 10 applications of a laser suggests that further analyses should be done to confirm that six applications are enough to produce the same clinical effects, thereby contributing data to professionals from different fields in regard to the cost-benefit ratio of this therapy.

The viscosity parameter's dependence on the profile of the disk scale height

It is shown that, for accretion disks, the height scale is a constant whenever hydrostatic equilibrium and the subsonic turbulence regime hold in the disk. In order to have a variable height scale, processes are needed that contribute an extra term to the continuity equation. This contribution makes the viscosity parameter much greater in the outer region and much smaller in the inner region. Under these circumstances, turbulence is the presumable source of viscosity in the disk.

Human fallopian tube: a new source of multipotent adult mesenchymal stem cells discarded in surgical procedures

Background: The possibility of using stem cells for regenerative medicine has opened a new field of investigation. The search for sources to obtain multipotent stem cells from discarded tissues or through non-invasive procedures is of great interest. It has been shown that mesenchymal stem cells (MSCs) obtained from umbilical cords, dental pulp and adipose tissue, which are all biological discards, are able to differentiate into muscle, fat, bone and cartilage cell lineages. The aim of this study was to isolate, expand, characterize and assess the differentiation potential of MSCs from human fallopian tubes (hFTs). Methods: Lineages of hFTs were expanded, had their karyotype analyzed, were characterized by flow cytometry and underwent in vitro adipogenic, chondrogenic, osteogenic, and myogenic differentiation. Results: Here we show for the first time that hFTs, which are discarded after some gynecological procedures, are a rich additional source of MSCs, which we designated as human tube MSCs (htMSCs). Conclusion: Human tube MSCs can be easily isolated, expanded in vitro, present a mesenchymal profile and are able to differentiate into muscle, fat, cartilage and bone in vitro.

Effect of 655-nm Low-Level Laser Therapy on Exercise-Induced Skeletal Muscle Fatigue in Humans

Objective: To investigate if development of skeletal muscle fatigue during repeated voluntary biceps contractions could be attenuated by low-level laser therapy (LLLT). Background Data: Previous animal studies have indicated that LLLT can reduce oxidative stress and delay the onset of skeletal muscle fatigue. Materials and Methods: Twelve male professional volleyball players were entered into a randomized double-blind placebo-controlled trial, for two sessions (on day 1 and day 8) at a 1-wk interval, with both groups performing as many voluntary biceps contractions as possible, with a load of 75% of the maximal voluntary contraction force (MVC). At the second session on day 8, the groups were either given LLLT (655 nm) of 5 J at an energy density of 500 J/cm(2) administered at each of four points along the middle of the biceps muscle belly, or placebo LLLT in the same manner immediately before the exercise session. The number of muscle contractions with 75% of MVC was counted by a blinded observer and blood lactate concentration was measured. Results: Compared to the first session (on day 1), the mean number of repetitions increased significantly by 8.5 repetitions (+/- 1.9) in the active LLLT group at the second session (on day 8), while in the placebo LLLT group the increase was only 2.7 repetitions (+/- 2.9) (p = 0.0001). At the second session, blood lactate levels increased from a pre-exercise mean of 2.4 mmol/L (+/- 0.5 mmol/L), to 3.6 mmol/L (+/- 0.5 mmol/L) in the placebo group, and to 3.8 mmol/L (+/- 0.4 mmol/L) in the active LLLT group after exercise, but this difference between groups was not statistically significant. Conclusion: We conclude that LLLT appears to delay the onset of muscle fatigue and exhaustion by a local mechanism in spite of increased blood lactate levels.

beta-Hydroxy-beta-methylbutyrate (HM beta) supplementation stimulates skeletal muscle hypertrophy in rats via the mTOR pathway

beta-Hydroxy-beta-methylbutyrate (HM beta) supplementation is used to treat cancer, sepsis and exercise-induced muscle damage. However, its effects on animal and human health and the consequences of this treatment in other tissues (e. g., fat and liver) have not been examined. The purpose of this study was to evaluate the effects of HM beta supplementation on skeletal muscle hypertrophy and the expression of proteins involved in insulin signalling. Rats were treated with HM beta (320 mg/kg body weight) or saline for one month. The skeletal muscle hypertrophy and insulin signalling were evaluated by western blotting, and hormonal concentrations were evaluated using ELISAs. HM beta supplementation induced muscle hypertrophy in the extensor digitorum longus (EDL) and soleus muscles and increased serum insulin levels, the expression of the mammalian target of rapamycin (mTOR) and phosphorylation of p70S6K in the EDL muscle. Expression of the insulin receptor was increased only in liver. Thus, our results suggest that HM beta supplementation can be used to increase muscle mass without adverse health effects.