Skeletal muscle overexpression of sAnk1.5 in transgenic mice does not predispose to type 2 diabetes.

Genome-wide association studies (GWAS) and cis-expression quantitative trait locus (cis-eQTL) analyses indicated an association of the rs508419 single nucleotide polymorphism (SNP) with type 2 diabetes (T2D). rs508419 is localized in the muscle-specific internal promoter (P2) of the ANK1 gene, which drives the expression of the sAnk1.5 isoform. Functional studies showed that the rs508419 C/C variant results in increased transcriptional activity of the P2 promoter, leading to higher levels of sAnk1.5 mRNA and protein in skeletal muscle biopsies of individuals carrying the C/C genotype. To investigate whether sAnk1.5 overexpression in skeletal muscle might predispose to T2D development, we generated transgenic mice (TgsAnk1.5/+) in which the sAnk1.5 coding sequence was selectively overexpressed in skeletal muscle tissue. TgsAnk1.5/+ mice expressed up to 50% as much sAnk1.5 protein as wild-type (WT) muscles, mirroring the difference reported between individuals with the C/C or T/T genotype at rs508419. However, fasting glucose levels, glucose tolerance, insulin levels and insulin response in TgsAnk1.5/+ mice did not differ from those of age-matched WT mice monitored over a 12-month period. Even when fed a high-fat diet, TgsAnk1.5/+ mice only presented increased caloric intake, but glucose disposal, insulin tolerance and weight gain were comparable to those of WT mice fed a similar diet. Altogether, these data indicate that sAnk1.5 overexpression in skeletal muscle does not predispose mice to T2D susceptibility.

Exercise-induced alterations and loss of sarcomeric M-line organization in the diaphragm muscle of obscurin knockout mice.

We recently reported that skeletal muscle fibers of obscurin knockout (KO) mice present altered distribution of ankyrin B (ankB), disorganization of the subsarcolemmal microtubules, and reduced localization of dystrophin at costameres. In addition, these mice have impaired running endurance and increased exercise-induced sarcolemmal damage compared with wild-type animals. Here, we report results from a combined approach of physiological, morphological, and structural studies in which we further characterize the skeletal muscles of obscurin KO mice. A detailed examination of exercise performance, using different running protocols, revealed that the reduced endurance of obscurin KO animals on the treadmill depends on exercise intensity and age. Indeed, a mild running protocol did not evidence significant differences between control and obscurin KO mice, whereas comparison of running abilities of 2-, 6-, and 11-mo-old mice exercised at exhaustion revealed a progressive age-dependent reduction of the exercise tolerance in KO mice. Histological analysis indicated that heavy exercise induced leukocyte infiltration, fibrotic connective tissue deposition, and hypercontractures in the diaphragm of KO mice. On the same line, electron microscopy revealed that, in the diaphragm of exercised obscurin KO mice, but not in the hindlimb muscles, both M-line and H-zone of sarcomeres appeared wavy and less defined. Altogether, these results suggest that obscurin is required for the maintenance of morphological and ultrastructural integrity of skeletal muscle fibers against damage induced by intense mechanical stress and point to the diaphragm as the skeletal muscle most severely affected in obscurin-deficient mice.

A quantitative assessment of circulating progenitor cells in competitive athletes and in sedentary subjects.

AIM: Hematopoietic stem cells (HSCs) and endothelial progenitor cells (EPCs) are known to play a role in the vascular responses and adaptations to exercise. We performed a quantitative assessment of HSCs and EPCs in adolescents in order to investigate whether resting levels of circulating HSCs and EPCs are comparable between elite athletes and sedentary healthy subjects. METHODS: HSCs and EPCs levels were measured in adolescent competitive football players and in age- and sex-matched sedentary controls. A laboratory testing was also performed to determine the white blood cells count and the lipid profile. All athletes were evaluated at the same stage of their training program, after 6 months of training. Controls were not engaged in any kind of routine training program. RESULTS: Twenty male competitive athletes (18.4 ± 0.5 years) and 9 sedentary controls (18.7 ± 0.4 years) participated in the study. As expected, HDL cholesterol was higher in athletes as compared with controls (P<0.05). No significant differences in the other laboratory parameters were observed among groups. Circulating levels of HSCs were significantly lower in athletes in comparison with sedentary controls (P<0.05). Conversely, EPCs and KDR+ cell subpopulations did not substantially differ between athletes and controls. CONCLUSION: Adolescent athletes exhibit lower levels of circulating HSCs but not of EPCs compared to sedentary controls. The process of tissue repair associated with intensive training can contribute to this difference, acting as a stimulus for mobilization and homing of HSCs in the site of injuries.

Deletion of small ankyrin 1 (sAnk1) isoforms results in structural and functional alterations in aging skeletal muscle fibers.

Muscle-specific ankyrins 1 (sAnk1) are a group of small ankyrin 1 isoforms, of which sAnk1.5 is the most abundant. sAnk1 are localized in the sarcoplasmic reticulum (SR) membrane from where they interact with obscurin, a myofibrillar protein. This interaction appears to contribute to stabilize the SR close to the myofibrils. Here we report the structural and functional characterization of skeletal muscles from sAnk1 knockout mice (KO). Deletion of sAnk1 did not change the expression and localization of SR proteins in 4- to 6-mo-old sAnk1 KO mice. Structurally, the main modification observed in skeletal muscles of adult sAnk1 KO mice (4-6 mo of age) was the reduction of SR volume at the sarcomere A band level. With increasing age (at 12-15 mo of age) extensor digitorum longus (EDL) skeletal muscles of sAnk1 KO mice develop prematurely large tubular aggregates, whereas diaphragm undergoes significant structural damage. Parallel functional studies revealed specific changes in the contractile performance of muscles from sAnk1 KO mice and a reduced exercise tolerance in an endurance test on treadmill compared with control mice. Moreover, reduced Qγ charge and L-type Ca(2+) current, which are indexes of affected excitation-contraction coupling, were observed in diaphragm fibers from 12- to 15-mo-old mice, but not in other skeletal muscles from sAnk1 KO mice. Altogether, these findings show that the ablation of sAnk1, by altering the organization of the SR, renders skeletal muscles susceptible to undergo structural and functional alterations more evident with age, and point to an important contribution of sAnk1 to the maintenance of the longitudinal SR architecture.

3.0 Tesla MRI in the early evaluation of inferior alveolar nerve neurological complications after mandibular third molar extraction: a prospective study.

OBJECTIVES: To evaluate the use of 3.0 T MRI in the prognosis of inferior alveolar nerve (IAN) sensory disorders after mandibular third molar extraction, in the early post-operative period. METHODS: 343 IANs were examined before and 3 days after surgery. Two radiologists evaluated the course of the nerve and the relative signal intensity (RSI). Cohen's kappa coefficient (κ) and intraclass correlation coefficient (ICC) were used to evaluate the interobserver (k = 0.891) and intra-observer variability (ICC = 0.927; 0.914, respectively). The IANs were divided into four groups on the basis of neurosensory disorders recovery time. ANOVA was used to evaluate the differences among the RSIs of the four groups, and multiple comparisons were performed with Tukey's range test. RESULTS: No differences in the course of IANs were found before and after surgery. In 280 IANs, no iatrogenic paraesthesia was found (Group A). 63 IANs showed a neurosensory impairment. 38 IANs showed recovery of post-operative paraesthesia at 3-month follow-up (Group B). 16 IANs showed a full recovery of iatrogenic paraesthesia at 6-month follow-up (Group C). Seven IANs displayed a full recovery at 12-month follow-up and two IANs showed persistence of neurosensory disorders at 18-month follow-up (Group D). The one-way ANOVA results indicated statistically significant difference among all groups (p < 0.05), except between Groups C and D (p = 0.504). CONCLUSIONS: The early evaluation of RSI values represents a valid tool to determine the prognosis of IAN sensory disorders after mandibular third molar extraction.

Densitometric kneecap changes after unilateral knee arthroplasty.

PURPOSE: To assess bone density of kneecaps in subjects with femoro-tibial prosthesis before and after surgery by means of DEXA examination. SUBJECTS AND METHODS: We examined 34 patients with unilateral femoro-tibial prosthesis, 20 healthy subjects of the same age and non-carriers of knee replacement and 14 healthy young adult subjects. All the data sets were analysed by two radiologists (AS and AM). The coincidence of the results between the two specialists was evaluated by means of Cohen's Kappa index and the results were considered statistically significative if p value is < of 0.05. RESULTS: The values of patellar BMD in the group of 34 patients, were: a minimum of 0.386 g/cm(2) (K = 0.879, p = 0.0012), a maximum 1.707 g/cm(2) (K = 0.886, p = 0.0016). The comparison between the left and right knee showed the following data: minimum difference 0.034 g/cm2 (K = 0.901, p = 0.0015), maximum difference of 0.622 g/cm(2) (K = 0.908, p = 0.0017), the average was found to be of 0.277 g/cm(2) (K = 0.894, p = 0.0018). But this difference tends to decrease 6 months after surgery. In the group of healthy young adults, we obtained the following values: a minimum of 0.782 g/cm(2) (K = 0.907, p = 0.0025), maximum 1.503 g/cm(2) (K = 0.932, p = 0.0012). Between both knees, the difference was minimal 0.006 g/cm(2) (K = 0.951, p = 0.0035) and maximum 0.096 g/cm(2) (K = 0.926, p = 0.0007) with an average difference of 0.058 g/cm(2) (K = 0.954, p = 0.0026). In the group of healthy subjects of the same age and non-carriers of knee replacement the values were average higher. A maximum value of 1.134 g/cm(2) (K = 0.894, p = 0.0028) and a minimum value of 0.944 g/cm(2) (K = 0.892, p = 0.0023) were found; between both knees a minimum difference of 0.010 g/cm(2) (K = 0.918, p = 0.0047) and a maximum of 0.090 g/cm(2) (K = 0.937, p = 0.0017) were found, with an average difference of 0.052 g/cm(2) (K = 0.956, p = 0.0024). CONCLUSIONS: DEXA examination of the patellar is recommended as a supplementary study to the clinical and radiological standard exams because it is able to provide additional information to determine when to intervene surgically, on the basis of patellar bone density values.

The KSR2-calcineurin complex regulates STIM1-ORAI1 dynamics and store-operated calcium entry (SOCE).

Store-operated calcium entry (SOCE) is the predominant Ca(2+) entry mechanism in nonexcitable cells and controls a variety of physiological and pathological processes. Although significant progress has been made in identifying the components required for SOCE, the molecular mechanisms underlying it are elusive. The present study provides evidence for a direct involvement of kinase suppressor of Ras 2 (KSR2) in SOCE. Using lymphocytes and fibroblasts from ksr2(-/-) mice and shKSR2-depleted cells, we find that KSR2 is critical for the elevation of cytosolic Ca(2+) concentration. Specifically, our results show that although it is dispensable for Ca(2+)-store depletion, KSR2 is required for optimal calcium entry. We observe that KSR2 deficiency affects stromal interaction molecule 1 (STIM1)/ORAI1 puncta formation, which is correlated with cytoskeleton disorganization. Of interest, we find that KSR2-associated calcineurin is crucial for SOCE. Blocking calcineurin activity impairs STIM1/ORAI1 puncta-like formation and cytoskeleton organization. In addition, we observe that calcineurin activity and its role in SOCE are both KSR2 dependent.

Aarskog-Scott syndrome: clinical update and report of nine novel mutations of the FGD1 gene.

Mutations in the FGD1 gene have been shown to cause Aarskog-Scott syndrome (AAS), or facio-digito-genital dysplasia (OMIM#305400), an X-linked disorder characterized by distinctive genital and skeletal developmental abnormalities with a broad spectrum of clinical phenotypes. To date, 20 distinct mutations have been reported, but little phenotypic data are available on patients with molecularly confirmed AAS. In the present study, we report on our experience of screening for mutations in the FGD1 gene in a cohort of 60 European patients with a clinically suspected diagnosis of AAS. We identified nine novel mutations in 11 patients (detection rate of 18.33%), including three missense mutations (p.R402Q; p.S558W; p.K748E), four truncating mutations (p.Y530X; p.R656X; c.806delC; c.1620delC), one in-frame deletion (c.2020_2022delGAG) and the first reported splice site mutation (c.1935+3A>C). A recurrent mutation (p.R656X) was detected in three independent families. We did not find any evidence for phenotype-genotype correlations between type and position of mutations and clinical features. In addition to the well-established phenotypic features of AAS, other clinical features are also reported and discussed.

Novel PTEN mutations in neurodevelopmental disorders and macrocephaly.

Somatic mutations of the phosphatase and tensin (PTEN) gene have been frequently detected in many types of human cancer. However, germline mutations can determine multiple hamartoma syndromes and, as more recently ascertained, syndromes clinically characterized by autism associated with macrocephaly. To determine whether germline mutations of PTEN may lead to different phenotypes, we screened all the nine exons of the PTEN gene in 40 patients with neurodevelopmental disorders, with or without features of autism spectrum disorder, associated with macrocephaly. Three novel de novo missense mutations were found (p.H118P, p.Y176C, p.N276S) in two severely mentally retarded patients with autism and in a subject with neurodevelopmental disorders without autistic features. Our results provide evidence that PTEN germline mutations may sustain a more wide phenotypical spectrum than previously suggested.

Endothelial progenitor cells and colony-forming units in rheumatoid arthritis: association with clinical characteristics.

OBJECTIVE: To compare levels of a range of endothelial progenitor cells (EPCs) and endothelial colony-forming units (CFUs) in control participants and RA patients, in addition to verifying whether levels of EPCs or CFUs are associated with clinical characteristics in RA patients. METHODS: Peripheral blood mononuclear cells (PBMCs) from 36 RA patients and 30 control participants were analysed by flow cytometry for EPCs defined by the expression of CD34/CD133, CD34/CD117, CD34/CD31, CD34/KDR and CD34/CD133/KDR. Endothelial cell colonies derived from culture of PBMCs were also assessed by CFU assay. RESULTS: No differences in levels of EPCs were observed in RA patients compared with controls. However, levels of EPCs were negatively associated with prognostic markers of poor disease status, but not cardiovascular (CV)-related risk factors. Furthermore, the majority of EPCs examined were negatively correlated with levels of RF. In contrast, CFU number was significantly reduced in RA patients compared with controls and was negatively associated with CV risk factors only. CONCLUSION: These findings indicate that more informative than comparing changes in absolute levels of EPCs, the examination of their relationship with clinical characteristics of RA patients can reveal significant associations, which may provide important clinical insights.

Generalised reduction of putative endothelial progenitors and CXCR4-positive peripheral blood cells in type 2 diabetes.

AIMS/HYPOTHESIS: In patients with type 2 diabetes, reduced levels of circulating endothelial progenitor cells have been reported and these have been correlated with disease severity. In this study, we examined a panel of markers widely used to identify progenitor and/or stem cells, and determined their association with disease severity in diabetic patients. Since expression of chemokine (C-X-C motif) receptor 4 (CXCR4) has been associated with mobilisation and recruitment of progenitor cells, CXCR4 expression was also analysed. METHODS: Peripheral blood mononuclear cells (PBMCs) from 98 patients with type 2 diabetes and 39 control individuals were analysed by flow cytometry for surface marker expression. RESULTS: Cells expressing different combinations of progenitor and/or stem cell markers were severely reduced in PBMCs of diabetic patients compared with those of control participants. Moreover, a number of these putative progenitor cell populations were negatively associated with disease severity. Reduced expression of CXCR4 and CD34/CXCR4-positive cells was also observed in diabetic patients. PBMCs expressing CXCR4 positively correlated with levels of progenitor cells in control participants but not in diabetic patients. Levels of putative progenitor and CXCR4-positive cells were further decreased in patients with diabetic complications, including cardiovascular and microvascular diseases. CONCLUSIONS/INTERPRETATION: A generalised decrease in a range of progenitor cell populations was observed in type 2 diabetic patients. This reduction was also negatively associated with disease severity.

Generation and expansion of multipotent mesenchymal progenitor cells from cultured human pancreatic islets.

Cellular models and culture conditions for in vitro expansion of insulin-producing cells represent a key element to develop cell therapy for diabetes. Initial evidence that human beta-cells could be expanded after undergoing a reversible epithelial-mesenchymal transition has been recently negated by genetic lineage tracing studies in mice. Here, we report that culturing human pancreatic islets in the presence of serum resulted in the emergence of a population of nestin-positive cells. These proliferating cells were mainly C-peptide negative, although in the first week in culture, proliferating cells, insulin promoter factor-1 (Ipf-1) positive, were observed. Later passages of islet-derived cells were Ipf-1 negative and displayed a mesenchymal phenotype. These human pancreatic islet-derived mesenchymal (hPIDM) cells were expanded up to 10(14) cells and were able to differentiate toward adipocytes, osteocytes and chondrocytes, similarly to mesenchymal stem/precursor cells. Interestingly, however, under serum-free conditions, hPIDM cells lost the mesenchymal phenotype, formed islet-like clusters (ILCs) and were able to produce and secrete insulin. These data suggest that, although these cells are likely to result from preexisting mesenchymal cells rather than beta-cells, hPIDM cells represent a valuable model for further developments toward future replacement therapy in diabetes.

Unusually severe expression of craniofacial features in Aarskog-Scott syndrome due to a novel truncating mutation of the FGD1 gene.

Aarskog-Scott syndrome (AAS) is a rare, clinically and genetically heterogeneous condition characterized by facial dysmorphic features, short stature, brachydactyly, and genital anomalies. The X-linked form is caused by mutations of the FGD1 gene. Although clinical manifestations and diagnostic criteria are well established, diagnosis is not simple, as the spectrum of phenotypical features may be extremely variable. Here, we report on the clinical and genetic characterization of a family in which molecular analyses revealed the inheritance of a novel truncating mutation of the FDG1 gene (c.945insC) in two affected brothers, with one of them displaying unusually severe craniofacial abnormalities. This previously unreported combination of anomalies might be due to the occurrence of two distinct disorders (AAS and hemifacial microsomia) or may represent an extension of the AAS phenotypic spectrum. Our findings highlight the phenotypic heterogeneity of AAS, supporting the opinion that the FGD1 mutations result in a broad spectrum of severity and, in some cases, may express a clinical appearance very different than typically described.

[Voluminous ectopic polycystic ovaries in Mayer-Rokitansky-Küster-Hauser syndrome]. / Voluminose ovaie policistiche ectopiche nella sindrome di Mayer-Rokitansky-Küster-Hauser.

We report the case of a 20-year-old woman with primary amenorrhea, normally developed sexual characteristics, infantile external genitals and absence of vaginal orifice. Blood chemistry studies showed elevated serum levels of gonadotropin, estrogens, testosterone, 17OH-progesterone, prolactin and TSH and low concentrations of FT(3) and FT(4). Cytogenetic karyotyping and in situ fluorescence hybridization revealed a 46,XX karyotype and the presence of Barr chromatin body. Laparoscopy disclosed absence of a vagina, apparently normal, enlarged tubes, multifolicular ectopic gonads and symmetric bilateral uterine buds. Diagnostic work-up included skeletal radiography, renal ultrasonography, intravenous pyelography, pelvic echography, pelvic phlebography and magnetic resonance imaging studies to demonstrate possible associations between the genitourinary and skeletal anomalies. Based on the clinical and laboratory findings, a diagnosis of Mayer-Rokitansky-Küster-Hauser syndrome was established, associated with the presence of enlarged ectopic polycystic ovaries in the abdominal cavity.

Recent advances in the diagnosis of malignant hyperthermia susceptibility: how confident can we be of genetic testing?

Malignant hyperthermia (MH) is a condition that manifests in susceptible individuals only on exposure to certain anaesthetic agents. Although genetically heterogeneous, mutations in the RYR1 gene (19q13.1) are associated with the majority of reported MH cases. Guidelines for the genetic diagnosis for MH susceptibility have recently been introduced by the European MH Group (EMHG). These are designed to supplement the muscle biopsy testing procedure, the in vitro contracture test (IVCT), which has been the only means of patient screening for the last 30 years and which remains the method for definitive diagnosis in suspected probands. Discordance observed in some families between IVCT phenotype and susceptibility locus genotype could limit the confidence in genetic diagnosis. We have therefore assessed the prevalence of 15 RYR1 mutations currently used in the genetic diagnosis of MH in a sample of over 500 unrelated European MH susceptible individuals and have recorded the frequency of RYR1 genotype/IVCT phenotype discordance. RYR1 mutations were detected in up to approximately 30% of families investigated. Phenotype/genotype discordance in a single individual was observed in 10 out of 196 mutation-positive families. In five families a mutation-positive/IVCT-negative individual was observed, and in the other five families a mutation-negative/IVCT-positive individual was observed. These data represent the most comprehensive assessment of RYR1 mutation prevalence and genotype/phenotype correlation analysis and highlight the possible limitations of MH screening methods. The implications for genetic diagnosis are discussed.