Plasma heavy metal levels correlate with deregulated gene expression of detoxifying enzymes in osteoporotic patients.

Heavy metal levels appear to be associated with low bone mineral density (BMD) and the consequent osteoporosis risk, but the relationship with the disease has not been clearly defined. The altered expression pattern of numerous genes, including detoxifying genes, seems to play a pivotal role in this context, leading to increased susceptibility to several diseases, including osteoporosis. The purpose of this study is to analyse circulating heavy metals levels and the expression of detoxifying genes in osteoporotic patients (OPs, n = 31), compared with healthy subjects (CTRs, n = 32). Heavy metals concentration in plasma samples was determined by Inductively Coupled Plasma Mass Spectrometry (ICP-MS), and the subsequent expression analysis of NAD(P)H quinone dehydrogenase 1 (NQO1), Catalase (CAT), and Metallothionein 1E (MT1E) genes in Peripheral Blood Mononuclear Cells (PBMCs) was assessed by real-time polymerase chain reaction (qRT-PCR). Copper (Cu), mercury (Hg), molybdenum (Mo) and lead (Pb) were found to be significantly higher in the plasma of OPs compared to CTRs. Analysis of the expression levels of detoxifying genes showed a significant decrease in CAT and MT1E in OP group. In addition, Cu correlated positively with the expression levels of both CAT and MT1E in CTRs group and MT1E in OPs. This study shows an increased circulating concentration of certain metals combined with an altered expression pattern of detoxifying genes in OPs, highlighting a novel aspect to be investigated in order to better characterize the role of metals in the pathogenesis of osteoporosis.

A gait-based paradigm to investigate central body representation in cervical dystonia patients.

BACKGROUND: Cervical dystonia (CD) is a common adult-onset idiopathic form of dystonia characterized by an abnormal head posture caused by an excessive activity of the neck muscles. The position of the head is important to direct viewpoint in the rounding environment, and the body orientation, during gait, must be coherent with the subjective straight ahead (SSA). An alteration of the SSA, as in the case of CD patients, could affect gait when visual input is not available. The aim of this study was to probe the behavior of patients with CD during blindfolded walking, investigating the ability to walk straight ahead based only on somatosensory and vestibular information. METHODS: In this observational cross-sectional study, patients with CD and healthy control subjects (HC) were compared. All participants were evaluated through a gait analysis during blindfolded walking on a GAITRite carpet, relying on their own sense of straightness. RESULTS: Patients with CD showed lower values of path length (p < 0.001), a lower number of steps on the carpet (p < 0.001). A higher number of CD patients deviated during the task, walking out of the carpet, (p < 0.005) compared to HS. No relation was found between the dystonic side and the gait trajectory deviation. A significant correlation was found between pain symptom and gait performance. CONCLUSIONS: CD patients showed dysfunctions in controlling dynamic body location during walking without visual afferences, while the dystonic side does not seem to be related to the lateral deviation of the trajectory. Our results would assume that a general proprioceptive impairment could lead to an improper body position awareness in patients with CD.

A pilot study of lncRNAs expression profile in serum of progressive multiple sclerosis patients.

OBJECTIVE: Multiple Sclerosis (MS) is an inflammatory and neurodegenerative disease that affect both white and gray matter. The relapsing and the eventually progressive course of MS is heterogeneous; thus, a confident long-term prediction of individual prognosis is not possible yet. Recent studies have demonstrated the role of long non-coding RNA (lncRNAs) as potential biomarkers that could provide information to predict disease activity and progression. PATIENTS AND METHODS: By qRT-PCR, we analysed the lncRNAs expression in the serum of 16 secondary progressive MS (SP-MS), 12 primary progressive (PP-MS) patients and 8 healthy controls. RESULTS: We found that TUG1 was upregulated in SP-MS, while the comparison of PP-MS vs. controls showed a downregulation of non-protein coding RNA 188 (LRRC75A-AS1) and a significant upregulation of two lncRNAs: long intergenic non-protein coding RNA 293 (LINC00293) and RP11-29G8.3. Moreover, we performed an in-silico analysis using DIANA-LncBase v2 and HMDD v3.0 software, in order to predict the possible interaction of these four lncRNAs with miRNAs. We identified 21 miRNAs prediction targets possibly involved in MS. CONCLUSIONS: Our data indicate a regulatory function of these lncRNAs in autoimmune and inflammatory processes related to MS suggesting their potential role in progressive MS pathogenesis.

Complementemia in pregnancies with antiphospholipid syndrome.

Prognosis of pregnancies in women with antiphospholipid syndrome has dramatically improved over the past two decades using conventional treatment with low molecular weight heparin and low-dose aspirin. However, despite this regimen, 10-15% of antiphospholipid syndrome patients experience pregnancy losses. Several studies have been performed in order to identify risk factors predictive of complications. Thrombosis has been generally accepted as the key pathogenetic mechanism underlying pregnancy morbidity. However, the thrombogenic state alone is not able to explain all the different mechanisms leading to pregnancy failure. In fact, emerging evidence shows that complement pathway could play an important role in mediating clinical events in antiphospholipid syndrome. However, the exact mechanism through which complement mediates antiphospholipid syndrome complications remains unknown. Low complement levels (C3 and C4) are associated with poor pregnancy outcome in women with antiphospholipid syndrome in different studies. Hypocomplementemia could be indicated as an early predictor of adverse pregnancy outcome, available at the beginning of pregnancy for starting, if necessary, additional treatment to conventional therapy. However, future studies need to better understand the impact of low complement level on antiphospholipid syndrome pregnancy outcome.

Influence of Biliopancreatic Diversion on Pregnancy Outcomes in Comparison to Other Bariatric Surgery Procedures.

BACKGROUND: Pregnancy after bariatric surgery (BS) has an increased risk for small-for-gestational-age infants (SGA), shorter length of gestation, and probably perinatal mortality. The aim of this study was to investigate if biliopancreatic diversion could impair pregnancy outcomes in comparison to other bariatric surgery procedures. METHODS: We conducted a cohort retrospective single-center study in 65 women before and after BS. Thirty-one pregnancies occurred before BS, while 109 after BS, amongst which n = 51 after biliopancreatic diversion (BPD) and n = 58 after non-malabsorptive procedures. RESULTS: The pregnancy outcomes after BS in comparison with those before BS resulted less affected by diabetes, hypertensive disorders, macrosomia, and large-for-gestational-age (LGA), but more complicated by preterm births (14.5 versus 4.0%) and low birth weight (LBW) infants (28.9 versus 0%). Moreover, mean birth weight resulted lower after BS than before BS (p < 0.001). In pregnancies after BPD in comparison to those before BS, the LBW rate (42.5%) resulted a drastic increase (p < 0.001), and mean birth weight (p < 0.001) and mean birth weight centile (p < 0.001) were lower after BPD. When pregnancy outcomes after BPD were compared with those after non-malabsorptive procedures, the rate of congenital anomalies, preterm births, LBW, and SGA resulted an increase (p = 0.002, 0.008, 0.032, and < 0.001, respectively). CONCLUSIONS: BPD drastically reduced diabetes, hypertensive disorders, macrosomia, and LGA; however, it was associated with the poorest pregnancy outcomes in comparison to those observed after other BS procedures. On the basis of the present study, we recommend a cautious multidisciplinary selection of severely obese patients for BPD during the fertile age.

AFM nano-mechanical study of the beating profile of hiPSC-derived cardiomyocytes beating bodies WT and DM1.

Myotonic Dystrophy type 1 (DM1) is the most common form of muscular dystrophy in adults, characterized by a variety of multisystemic features and associated with cardiac anomalies. Among cardiac phenomena, conduction defects, ventricular arrhythmias, and dilated cardiomyopathy represent the main cause of sudden death in DM1 patients. Patient-specific induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) represent a powerful in vitro model for molecular, biochemical, and physiological studies of disease in the target cells. Here, we used an Atomic Force Microscope (AFM) to measure the beating profiles of a large number of cells, organized in CM clusters (Beating Bodies, BBs), obtained from wild type (WT) and DM1 patients. We monitored the evolution over time of the frequency and intensity of the beating. We determined the variations between different BBs and over various areas of a single BB, caused by morphological and biomechanical variations. We exploited the AFM tip to apply a controlled force over the BBs, to carefully assess the biomechanical reaction of the different cell clusters over time, both in terms of beating frequency and intensity. Our measurements demonstrated differences between the WT and DM1 clusters highlighting, for the DM1 samples, an instability which was not observed in WT cells. We measured differences in the cellular response to the applied mechanical stimulus in terms of beating synchronicity over time and cell tenacity, which are in good agreement with the cellular behavior in vivo. Overall, the combination of hiPSC-CMs with AFM characterization can become a new tool to study the collective movements of cell clusters in different conditions and can be extended to the characterization of the BB response to chemical and pharmacological stimuli.

Perception of dental students on risk factors of musculoskeletal disorders.

OBJECTIVE: To evaluate the validity and reliability of the Job Factors Questionnaire, and the influence of gender, academic level and pain/discomfort on dental students' perception regarding risk factors of musculoskeletal disorders. METHODS: One hundred forty-five dental students from Stony Brook School of Dental Medicine participated voluntarily in this study. The survey was organised in two sections: (i) Job Factors Questionnaire with 15 items that evaluated students' perceptions about job/study environmental risk factors and their potential contribution to musculoskeletal disorders. The tri-factorial structure (repetitiveness, work posture and external factors) was used; (ii) Part of the Nordic Questionnaire with questions referring to 9 body areas: Neck, shoulders, upper back, elbows, lower back, wrists/hands, hips, knees and ankles/feet. Students answered if they have had a job-related pain/discomfort in any of these body areas in the previous 12 months. The data were evaluated statistically by confirmatory factor analysis, convergent and discriminant validities, internal consistency and Z-test (α = .05). RESULTS: The tri-factorial structure was considered valid and reliable for the sample after excluding item 8 and inserting correlations between items 11 and 12, and between 14 and 15. The academic level presented a significant effect on the factors "Work Posture" (P = .02) and "External Factors" (P = .01). Most of the students reported pain in their neck (73.79%), lower back (62.06%) and shoulders (53.10%) in the previous 12 months. CONCLUSION: The Work Posture and External Factors were influenced only by academic level. The lower the students' academic level, the higher their perception for both factors of musculoskeletal disorders.

Association between false-positive TORCH and antiphospholipid antibodies in healthy pregnant women.

The present study investigated: (a) the presence of antiphospholipid antibodies and (b) the obstetric outcome in healthy pregnant women showing false-positive TORCH-Toxoplasmosis, Other: syphilis, varicella-zoster, Rubella, Cytomegalovirus (CMV), and Herpes infections-results. Data from 23 singleton healthy pregnancies with false-positive TORCH results were collected. Each woman was systematically screened for TORCH IgG and IgM during the pre-conception assessment and/or at the beginning of pregnancy. In the presence of IgM positivity, when indicated (CMV, toxoplasmosis, rubella, herpes simplex virus), IgG avidity was evaluated and, if possible, polymerase chain reaction was performed on an amniotic fluid sample in order to distinguish between primary infection or false positivity. The antiphospholipid antibodies tests were: lupus anticoagulant, anticardiolipin antibodies IgG, IgM, and anti-ß2glicoprotein I IgG, IgM. The antiphospholipid antibodies tests, if positive, were repeated after 12 weeks to confirm the results. In pregnant women with false-positive TORCH, the overall prevalence of positive antiphospholipid antibodies for one or more tests was 52.2%. To clarify the correlation of false-positive TORCH results with clinical practice, obstetric outcome was analyzed in terms of live births, week of delivery, neonatal birth weight, and neonatal birth weight percentile. A statistically significant lower neonatal birth weight and neonatal birth weight percentile were observed in women with false-positive TORCH associated with antiphospholipid antibodies positivity (Group A) in comparison with those in women with false-positive TORCH without antiphospholipid antibodies positivity (Group B). No statistically significant difference was found for the week of delivery between the two groups. It is hoped that future studies will verify the life-long persistence of antiphospholipid antibodies positivity by follow-up of these women and identify who will develop a classical antiphospholipid syndrome or other autoimmune disorders.

Myotonic dystrophy type 1: role of CCG, CTC and CGG interruptions within DMPK alleles in the pathogenesis and molecular diagnosis.

Myotonic dystrophy type 1 (DM1) is a multisystem neuromuscular disease caused by a CTG triplet expansion in the 3'-untranslated region (3'-UTR) of DMPK gene. This CTG array is usually uninterrupted in both healthy and DM1 patients, but recent studies identified pathological variant expansions containing unstable CCG, CTC and CGG interruptions with a prevalence of 3-5% of cases. In this review, we will describe the clinical, molecular and genetic issues related to the occurrence of variant expansions associated with DM1. Indeed, the identification of these complex DMPK alleles leads to practical consequences in DM1 genetic counseling and testing, because these exams can give false negative results. Moreover, DM1 patients carrying interrupted alleles can manifest either additional atypical neurological symptoms or, conversely, mild, late-onset forms. Therefore, the prognosis of the disease in these patients is difficult to determine because of the great uncertainty about the genotype-phenotype correlations. We will discuss the putative effects of the variant DM1 alleles on the pathogenic disease mechanisms, including mitotic and meiotic repeats instability and splicing alteration typical of DM1 tissues. Interruptions within the DMPK expanded alleles could also interfere with the chromatin structure, the transcriptional activity of the DM1 locus and the interaction with RNA CUG-binding proteins.

Validation of plasma microRNAs as biomarkers for myotonic dystrophy type 1.

Non-invasive and simple to measure biomarkers are still an unmet need for myotonic dystrophy type 1 (DM1). Indeed, muscle biopsies can be extremely informative, but their invasive nature limits their application. Extracellular microRNAs are emerging humoral biomarkers and preliminary studies identified a group of miRNAs that are deregulated in the plasma or serum of small groups of DM1 patients. Here we adopted very stringent selection and normalization criteria to validate or disprove these miRNAs in 103 DM1 patients and 111 matched controls. We confirmed that 8 miRNAs out of 12 were significantly deregulated in DM1 patients: miR-1, miR-27b, miR-133a, miR-133b, miR-206, miR-140-3p, miR-454 and miR-574. The levels of these miRNAs, alone or in combination, discriminated DM1 from controls significantly, and correlated with both skeletal muscle strength and creatine kinase values. Interestingly, miR-133b levels were significantly higher in DM1 female patients. Finally, the identified miRNAs were also deregulated in the plasma of a small group (n = 30) of DM2 patients. In conclusion, this study proposes that miRNAs might be useful as DM1 humoral biomarkers.

Is there any role for the hydroxychloroquine (HCQ) in refractory obstetrical antiphospholipid syndrome (APS) treatment?

The best therapy regimen for refractory obstetrical antiphospholipid syndrome remains to be determined. Additional treatments with steroids, plasma exchanges and immunoglobulins failed to show any beneficial effect. We present a case of a woman who had a better pregnancy outcome after the administration of hydroxychloroquine (HCQ) as additional treatment. Furthermore, we highlighted that HCQ was able to dramatically reduce the antiphospholipid antibodies levels.

SCN4A mutation as modifying factor of myotonic dystrophy type 2 phenotype.

In myotonic dystrophy type 2 (DM2), an association has been reported between early and severe myotonia and recessive chloride channel (CLCN1) mutations. No DM2 cases have been described with sodium channel gene (SCN4A) mutations. The aim is to describe a DM2 patient with severe and early onset myotonia and co-occurrence of a novel missense mutation in SNC4A. A 26-year-old patient complaining of hand cramps and difficulty relaxing her hands after activity was evaluated at our department. Neurophysiology and genetic analysis for DM1, DM2, CLCN1 and SCN4A mutations were performed. Genetic testing was positive for DM2 (2650 CCTG repeat) and for a variant c.215C>T (p.Pro72Leu) in the SCN4A gene. The variation affects the cytoplasmic N terminus domain of Nav1.4, where mutations have never been reported. The biophysical properties of the mutant Nav1.4 channels were evaluated by whole-cell voltage-clamp analysis of heterologously expressed mutant channel in tsA201 cells. Electrophysiological studies of the P72L variant showed a hyperpolarizing shift (-5 mV) of the voltage dependence of activation that may increase cell excitability. This case suggests that SCN4A mutations may enhance the myotonic phenotype of DM2 patients and should be screened for atypical cases with severe myotonia.

Cerebral sinovenous thrombosis in neonatal antiphospholipid syndrome: a new entity?

Neonatal antiphospholipid syndrome (neonatal APS) seems to be exceedingly rare, as the antiphospholipid antibodies (aPL) related thrombosis in the neonatal period. The pathogenesis of perinatal aPL related thrombosis may be explained both by the transplacental passage of the maternal antibodies and by the production of de novo antibodies by the neonate. However, few cases of neonatal APS are reported in the literature, especially regarding arterial thrombotic events. In particular, only two cases of neonatal aPL related isolated cerebral sinovenous thrombosis (CSVT) are described in the literature. Despite its frequency, CSVT results in significant mortality and morbidity, probably also due to the difficulty in early diagnosis and then in correct managing in the neonatal period. A diagnosis of neonatal APS should be considered in the evaluation of neonates with CSVT, as well as in any case of neonatal thrombosis, to correctly manage the affected neonates and counsel the mother for future pregnancies.

Glutathione administration reduces mitochondrial damage and shifts cell death from necrosis to apoptosis in ageing diabetic mice hearts during exercise.

BACKGROUND AND PURPOSE: The effect of antioxidants on ageing type 2 diabetic (T2D) hearts during exercise is unclear. We hypothesized that GSH therapy during exercise reduces mitochondrial oxidative stress (mOXS) and cell death in ageing db/db mice hearts. EXPERIMENTAL APPROACH: The effect of GSH on cardiac mOXS and cell death was evaluated both in vivo and in vitro. KEY RESULTS: During exercise, GSH treatment protected db/db hearts from exaggerated mOXS without reducing total cell death. Despite similar cell death, investigations on apoptosis-specific single-stranded DNA breaks and necrosis-specific damage provided the first in vivo evidence of a shift from necrosis to apoptosis, with reduced fibrosis following GSH administration in exercised db/db hearts. Further support for a GSH-regulated 'switch' in death phenotypes came from NIH-3T3 fibroblasts and H9c2 cardiomyocytes treated with H2 O2 , a reactive oxygen species (ROS). Similar to in vivo findings, augmenting GSH by overexpressing glutamyl cysteine ligase (GCLc) protected fibroblasts and cardiomyocytes from necrosis induced by H2 O2 , but elevated caspase-3 and apoptosis instead. Similar to in vivo findings, where GSH therapy in normoglycaemic mice suppressed endogenous antioxidants and augmented caspase-3 activity, GCLc overexpression during staurosporine-induced death, which was not characterized by ROS, increased GSH efflux and aggravated death in fibroblasts and cardiomyocytes, confirming that oxidative stress is required for GSH-mediated cytoprotection. CONCLUSIONS AND IMPLICATIONS: While GSH treatment is useful for reducing mOXS and attenuating necrosis and fibrosis in ageing T2D hearts during exercise, such antioxidant treatment could be counterproductive in the healthy heart during exercise.

Premature senescence in primary muscle cultures of myotonic dystrophy type 2 is not associated with p16 induction.

Myotonic dystrophy type 1 (DM1) and type 2 (DM2) are multisystemic disorders linked to two different genetic loci and characterized by several features including myotonia, muscle weakness and atrophy, cardiac dysfunctions, cataracts and insulin-resistance. In both forms, expanded nucleotide sequences cause the accumulation of mutant transcripts in the nucleus deregulating the activity of some RNAbinding proteins and providing an explanation for the multisystemic phenotype of DM patients. However this pathogenetic mechanism does not explain some histopathological features of DM skeletal muscle like muscle atrophy. It has been observed that DM muscle shares similarities with the ageing muscle, where the progressive muscle weakness and atrophy is accompanied by a lower regenerative capacity possibly due to the failure in satellite cells activation. The aim of our study is to investigate if DM2 satellite cell derived myoblasts exhibit a premature senescence as reported for DM1 and if alterations in their proliferation potential and differentiation capabilities might contribute to some of the histopathological features observed in DM2 muscles. Our results indicate that DM myoblasts have lower proliferative capability than control myoblasts and reach in vitro senescence earlier than controls. Differentely from DM1, the p16 pathway is not responsible for the premature growth arrest observed in DM2 myoblasts which stop dividing with telomeres shorter than controls. During in vitro senescence, a progressive decrease in fusion index is observable in both DM and control myotubes with no significant differences between groups. Moreover, myotubes obtained from senescent myoblasts appear to be smaller than those from young myoblasts. Taken together, our data indicate a possible role of DM2 premature myoblast senescence in skeletal muscle histopathological alterations i.e., dystrophic changes and type 2 fibre atrophy.

MBNL142 and MBNL143 gene isoforms, overexpressed in DM1-patient muscle, encode for nuclear proteins interacting with Src family kinases.

Myotonic dystrophy type-1 (DM1) is the most prevalent form of muscular dystrophy in adults. This disorder is an RNA-dominant disease, caused by expansion of a CTG repeat in the DMPK gene that leads to a misregulation in the alternative splicing of pre-mRNAs. The longer muscleblind-like-1 (MBNL1) transcripts containing exon 5 and the respective protein isoforms (MBNL142-43) were found to be overexpressed in DM1 muscle and localized exclusively in the nuclei. In vitro assays showed that MBNL142-43 bind the Src-homology 3 domain of Src family kinases (SFKs) via their proline-rich motifs, enhancing the SFK activity. Notably, this association was also confirmed in DM1 muscle and myotubes. The recovery, mediated by an siRNA target to Ex5-MBNL142-43, succeeded in reducing the nuclear localization of both Lyn and MBNL142-43 proteins and in decreasing the level of tyrosine phosphorylated proteins. Our results suggest an additional molecular mechanism in the DM1 pathogenesis, based on an altered phosphotyrosine signalling pathway.

[Genotoxicity risk assessment and oocytes: basis of genetic toxicology and application in reproductive science]. / Risque génotoxique et ovocytes : principes de toxicologie génétique et applications.

The aim of genotoxicity tests in germ cells is to assess the impact of exposure to environmental mutagens that may represent a risk for the fertility or for the offspring of exposed subject. The comet assay on mature mouse oocytes is a simple, reproductive and rapid test to study primary DNA damage in oocytes. This test is used to complete toxicology assays applied in first line to somatic cells, and could find many applications in reproductive toxicology to study impact of environmental factors on female germ cells. We describe a practical application of comet assay in reproductive biology to assess the genotoxicity of cryoprotectants used at high concentrations in oocyte vitrification protocols. This test allowed us to demonstrate that dimethylsulfoxide and ethylene glycol are non-genotoxic for the mouse oocytes and led us to hypothesize a genotoxic effect of 1,2-propanediol (PrOH) at high concentrations after having observed induction of significant DNA damage on CHO cell line and on mouse oocytes.

The relationship between TORCH complex false positivity and obstetric outcome in patients with antiphospholipid syndrome.

OBJECTIVE: The presence of TORCH IgM positivity is not a specific indicator of primary infection; the assessment of IgG avidity index has been shown to be useful in identifying or excluding primary infection in pregnant women with no pre-gestational TORCH serology. TORCH is an acronym for Toxoplasmosis, Others (HBV, syphilis, Varicella-Zoster virus, Epstein Barr virus, Coxsackie virus and Parvovirus), Rubella, Cytomegalovirus (CMV) and Herpes Simplex. PATIENTS AND METHODS: Data from 54 pregnancies in women with antiphospholipid syndrome (APS) were assessed in comparison with data from 222 healthy pregnant women as controls. Each woman in both groups was systematically screened for TORCH IgG and IgM during pre-conceptional evaluation and/or at the beginning of pregnancy. The assessment of IgG avidity was also evaluated in order to identify primary infection or false positivity. RESULTS: A significant increase of CMV IgM false positivity in APS in comparison with controls was detected. A worse pregnancy outcome was observed among APS patients having CMV IgM false positivity in comparison with APS patients without false positivity; in particular a statistically significant lower neonatal birth weight and a lower neonatal birth weight percentile were observed. CONCLUSION: Our data suggest that the presence of CMV IgM false positivity could represent a novel prognostic factor for poor pregnancy outcome in APS patients.