Re-Evaluation of the Action Potential Upstroke Velocity as a Measure of the Na+ Current in Cardiac Myocytes at Physiological Conditions

Background: The SCN5A encoded sodium current (INa) generates the action potential (AP) upstroke and is a major determinant of AP characteristics and AP propagation in cardiac myocytes. Unfortunately, in cardiac myocytes, investigation of kinetic properties of INa with near-physiological ion concentrations and temperature is technically challenging due to the large amplitude and rapidly activating nature of INa, which may seriously hamper the quality of voltage control over the membrane. We hypothesized that the alternating voltage clamp-current clamp (VC/CC) technique might provide an alternative to traditional voltage clamp (VC) technique for the determination of INa properties under physiological conditions. Principal Findings: We studied INa under close-to-physiological conditions by VC technique in SCN5A cDNA-transfected HEK cells or by alternating VC/CC technique in both SCN5A cDNA-transfected HEK cells and rabbit left ventricular myocytes. In these experiments, peak INa during a depolarizing VC step or maximal upstroke velocity, dV/dtmax, during VC/CC served as an indicator of available INa. In HEK cells, biophysical properties of INa, including current density, voltage dependent (in)activation, development of inactivation, and recovery from inactivation, were highly similar in VC and VC/CC experiments. As an application of the VC/CC technique we studied INa in left ventricular myocytes isolated from control or failing rabbit hearts

Fragile X syndrome: Diagnostic and carrier testing

The following are the recommendations of the American College of Medical Genetics (ACMG) Professional Practice and Guidelines Committee, convened to assist health care professionals in making decisions regarding genetic diagnosis and testing. The purpose of this document is to provide a brief overview of fragile X syndrome (FXS), and to make recommendations that can serve as general guidelines to aid clinicians in making referrals for diagnostic and carrier testing for this condition. Fragile X syndrome is the most common cause of inherited mental retardation and is caused by a mutation in the X-linked FMR1 gene. DNA studies are used for testing individuals with symptoms of FXS and individuals at risk for carrying the mutation. Genotypes are determined by examining the size of the trinucleotide repeat segment and the methylation status of the FMR1 gene. These guidelines supersede the 1994 ACMG statement of the same name

Tourette disorder spectrum maps to chromosome 14q31.1 in an Italian kindred

Tourette syndrome (TS) is a frequent neuropsychiatric disorder of unknown etiology. A number of chromosomal regions have been nominated as TS loci in linkage studies, but confirmation has met with limited success and causative mutations have not yet been definitely identified. Furthermore, TS, chronic tics, and obsessive–compulsive disorder (OCD) occur at increased frequencies among TS relatives, supporting the view that these phenotypes represent parts of the same genetically determined spectrum. We ascertained a four-generation Italian kindred segregating TS, chronic multiple motor tics (CMT), and OCD, and we performed a ten-centimorgan (cM) genome-wide linkage scan in order to map the underlying genetic defect. Suggestive linkage to chromosome 14q31.1 (multipoint LOD = 2.4) was detected by affected-only analysis under an autosomal dominant model and a narrower phenotype definition (only the subjects with TS and CMT were considered as affected). The linkage peak increased and it approached genome-wide significance (LOD = 3.29) when a broader phenotype definition was adopted (subjects with TS, CMT, and OCD considered as affected). Haplotype analysis defined a ∼2.3 cM critical region, shared by all the relatives with TS, CMT, or OCD. In conclusion, we provide strong evidence for linkage of TS spectrum to chromosome 14q31.1. Suggestive linkage to an overlapping region of chromosome 14q was reported in a recent scan of TS sibling pairs. This region might therefore contain an important gene for TS, and it should be prioritized for further study

Prognostic implications of the Quebec Task Force classification of back-related leg pain: An analysis of longitudinal routine clinical data

Background: Low back pain (LBP) patients with related leg pain have a more severe profile than those with local LBP and a worse prognosis. Pain location above or below the knee and the presence of neurological signs differentiate patients with different profiles, but knowledge about the prognostic value of these subgroups is sparse. The objectives of this study were (1) to investigate whether subgroups consisting of patients with Local LBP only, LBP + leg pain above the knee, LBP + leg pain below the knee, and LBP + leg pain and neurological signs had different prognoses, and (2) to determine if this was explained by measured baseline factors. Methods. Routine clinical data were collected during the first visit to an outpatient department and follow-ups were performed after 3 and 12 months. Patients were divided into the four subgroups and associations between subgroups and the outcomes of activity limitation, global perceived effect (GPE) after 3 months, and sick leave after 3 months were tested by means of generalised estimating equations. Models were univariate (I), adjusted for duration (II), and adjusted for all baseline differences (III). Results: A total of 1,752 patients were included, with a 76% 3-month and 70% 12-month follow-up. Subgroups were associated with activity limitation in all models (p < 0.001). Local LBP had the least and LBP + neurological signs the most severe limitations at all time-points, although patients with neurological signs improved the most. Associations with GPE after 3 months were only significant in Model I. Subgroups were associated with sick leave after 3 months in model I and II, with sick leave being most frequent in the subgroup with neurological signs. No significant differences were found in any pairwise comparisons of patients with leg pain above or below the knee. Conclusions: Subgrouping LBP patients, based on pain location and neurological signs, was associated with activity limitation and sick leave, but not with GPE. The presence of neurological signs and pain in the leg both have prognostic implications but whether that leg pain without neurological signs is above or below the knee does not

Factors affecting milk cortisol in mid lactating dairy cows

Background: Whether the measurement of cortisol in dairy cows can be used as a biomarker of adverse environmental or pathophysiological conditions is still under of scientific debate. In these situations, several systems mainly the hypothalamic-pituitary-adrenal axis, the autonomic nervous system, and the immune system are recruited to reestablish homeostasis. A first aim of the present study was to compare milk and blood cortisol concentrations and to consider its variability in milk in relation to farm, milk yield and days in milk. A second study investigates the effects of breed, class of somatic cell count (SCC) and farm on milk cortisol levels in a larger number of cows and farms, with the aim to validate the results obtained in the pilot study. Methods: For study 1, 135 cows were sampled from 2 Italian Simmental and 2 Italian Holstein commercial farms, whilst in the second study, 542 cows were sampled from 6 commercial farms of Italian Simmental and 499 cows from 4 commercial farms of Italian Holstein. Results: In study 1, the values of cortisol content in milk were significantly higher in Holstein than Simmental cows. Significant differences between farms were observed for milk and plasma cortisol concentrations. Cortisol content in milk was not correlated to plasma content in study 1 and the mean milk to plasma cortisol ratio was about 1:30. In study 2, for Holstein cows, significantly higher values of milk cortisol in comparison to Simmental cows was reported. A significant effect of class of SCC was observed, cows belonging to class 3 (SCC higher than 400.000/ml) showed the highest mean values of milk cortisol. The farm effect was significant also in the study 2, confirming the results obtained in the first study. Conclusions: Milk can be considered a preferential site of sampling in dairy cows to point out short term stimulation of the hypothalamic-pituitary-adrenal axis. Further studies are needed to investigate the physiological basis of the relationship between milk cortisol content and bree

MicroRNA-277 Modulates the Neurodegeneration Caused by Fragile X Premutation rCGG Repeats

Fragile X-associated tremor/ataxia syndrome (FXTAS), a late-onset neurodegenerative disorder, has been recognized in older male fragile X premutation carriers and is uncoupled from fragile X syndrome. Using a Drosophila model of FXTAS, we previously showed that transcribed premutation repeats alone are sufficient to cause neurodegeneration. MiRNAs are sequence-specific regulators of post-transcriptional gene expression. To determine the role of miRNAs in rCGG repeat-mediated neurodegeneration, we profiled miRNA expression and identified selective miRNAs, including miR-277, that are altered specifically in Drosophila brains expressing rCGG repeats. We tested their genetic interactions with rCGG repeats and found that miR-277 can modulate rCGG repeat-mediated neurodegeneration. Furthermore, we identified Drep-2 and Vimar as functional targets of miR-277 that could modulate rCGG repeat-mediated neurodegeneration. Finally, we found that hnRNP A2/B1, an rCGG repeat-binding protein, can directly regulate the expression of miR-277. These results suggest that sequestration of specific rCGG repeat-binding proteins could lead to aberrant expression of selective miRNAs, which may modulate the pathogenesis of FXTAS by post-transcriptionally regulating the expression of specific mRNAs involved in FXTAS

Reliability of Eye Tracking and Pupillometry Measures in Individuals with Fragile X Syndrome

Recent insight into the underlying molecular and cellular mechanisms of fragile X syndrome (FXS) has led to the proposal and development of new pharmaceutical treatment strategies, and the initiation of clinical trials aimed at correcting core symptoms of the developmental disorder. Consequently, there is an urgent and critical need for outcome measures that are valid for quantifying specific symptoms of FXS and that are consistent across time. We used eye tracking to evaluate test–retest reliability of gaze and pupillometry measures in individuals with FXS and we demonstrate that these measures are viable options for assessing treatment-specific outcomes related to a core behavioral feature of the disorder

A Novel RNA Transcript with Antiapoptotic Function Is Silenced in Fragile X Syndrome

Several genome-wide transcriptomics efforts have shown that a large percentage of the mammalian genome is transcribed into RNAs, however, only a small percentage (1–2%) of these RNAs is translated into proteins. Currently there is an intense interest in characterizing the function of the different classes of noncoding RNAs and their relevance to human disease. Using genomic approaches we discovered FMR4, a primate-specific noncoding RNA transcript (2.4 kb) that resides upstream and likely shares a bidirectional promoter with FMR1. FMR4 is a product of RNA polymerase II and has a similar half-life to FMR1. The CGG expansion in the 5′ UTR of FMR1 appears to affect transcription in both directions as we found FMR4, similar to FMR1, to be silenced in fragile X patients and up-regulated in premutation carriers. Knockdown of FMR4 by several siRNAs did not affect FMR1 expression, nor vice versa, suggesting that FMR4 is not a direct regulatory transcript for FMR1. However, FMR4 markedly affected human cell proliferation in vitro; siRNAs knockdown of FMR4 resulted in alterations in the cell cycle and increased apoptosis, while the overexpression of FMR4 caused an increase in cell proliferation. Collectively, our results demonstrate an antiapoptotic function of FMR4 and provide evidence that a well-studied genomic locus can show unexpected functional complexity. It cannot be excluded that altered FMR4 expression might contribute to aspects of the clinical presentation of fragile X syndrome and/or related disorders

The neural basis of auditory temporal discrimination in girls with fragile X syndrome

Fragile X syndrome (FXS) is a common genetic disorder in which temporal processing may be impaired. To our knowledge however, no studies have examined the neural basis of temporal discrimination in individuals with FXS using functional magnetic resonance imaging (fMRI). Ten girls with fragile X syndrome and ten developmental age-matched typically developing controls performed an auditory temporal discrimination task in a 3T scanner. Girls with FXS showed significantly greater brain activation in a left-lateralized network, comprising left medial frontal gyrus, left superior and middle temporal gyrus, left cerebellum, and left brainstem (pons), when compared to a developmental age-matched typically developing group of subjects who had similar in-scanner task performance. There were no regions that showed significantly greater brain activation in the control group compared to individuals with FXS. These data indicate that networks of brain regions involved in auditory temporal processing may be dysfunctional in FXS. In particular, it is possible that girls with FXS employ left hemispheric resources to overcompensate for relative right hemispheric dysfunction