Influence of Mitochondrial Genetics on the Mitochondrial Toxicity of Linezolid in Blood Cells and Skin Nerve Fibers.

The antibiotic linezolid is a ribosomal inhibitor with excellent efficacy. Although the administration period has been reduced to 28 days, side effects, usually of hematologic or neuropathic origin, are still reported due to secondary inhibition of mitochondrial protein synthesis. Susceptibility to linezolid toxicity remains unknown. Therefore, the objective of this study was to gain an understanding of clinical heterogeneity in response to identical linezolid exposures through exhaustive examination of the molecular basis of tissue-dependent mitotoxicity, consequent cell dysfunction, and the association of mitochondrial genetics with adverse effects of linezolid administered for the recommended period. Peripheral blood mononuclear cells (PBMC) and skin nerve fibers from 19 and 6 patients, respectively, were evaluated before and after a 28-day linezolid treatment in order to assess toxic effects on mitochondria and cells. Mitochondrial DNA haplotypes and single nucleotide polymorphisms (SNPs) in ribosomal sequences where linezolid binds to mitochondrial ribosomes were also analyzed to investigate their genetic contributions. We found that linezolid reduced mitochondrial protein levels, complex IV activity, and mitochondrial mass in PBMC and was associated with a trend toward an increase in the rate of apoptosis. In skin tissue, mitochondrial mass increased within nerve fibers, accompanied by subclinical axonal swelling. Mitochondrial haplogroup U, mutations in 12S rRNA, and the m.2706A→G, m.3197T→C, and m.3010G→A polymorphisms in 16S rRNA showed a trend toward an association with increased mitochondrial and clinical adverse effects. We conclude that even when linezolid is administered for a shorter time than formerly, adverse effects are reported by 63% of patients. Linezolid exerts tissue-dependent mitotoxicity that is responsible for downstream cellular consequences (blood cell death and nerve fiber swelling), leading to adverse hematologic and peripheral nervous side effects. Multicentric studies should confirm genetic susceptibility in larger cohorts.

Differences in Neisseria gonorrhoeae population structure and antimicrobial resistance pattern between men who have sex with men and heterosexuals.

This study compared the antimicrobial susceptibility and genotypes of strains of Neisseria gonorrhoeae isolated from men who have sex with men (MSM) and from heterosexuals. One hundred and eleven strains were characterized from 107 patients, comprising 57 strains from 54 heterosexuals and 54 strains from 53 MSM. Antimicrobial resistance rates were higher in strains from heterosexual patients, with resistance to cefixime (P = 0·0159) and ciprofloxacin (P = 0·002) being significantly higher. Typing by N. gonorrhoeae multi-antigen sequence typing (NG-MAST) showed that the most prevalent sequence types (ST) and genogroups (G) respectively were ST2400, ST2992, and ST5793, and G1407, G2992, and G2400. A statistically significant association was observed for MSM and genogroups G2400 (P = 0·0005) and G2992 (P = 0·0488), and G1407 with heterosexuals (P = 0·0002). We conclude that in our region distinct populations of gonococci are circulating among subjects with different sexual practices, with their corresponding transmission patterns. Furthermore, the high prevalence of genotype G2400 in MSM, has not to our knowledge been previously described.

Neisseria gonorrhoeae antimicrobial susceptibility in Barcelona: penA, ponA, mtrR, and porB mutations and NG-MAST sequence types associated with decreased susceptibility to cephalosporins.

The aims of this study were to determine the antimicrobial susceptibility of Neisseria gonorrhoeae (NG) in our area, to analyze the molecular mechanisms involved in cephalosporins resistance, and to undertake molecular typing of our NG strains. Antimicrobial susceptibility was determined using the Etest. The genes penA, mtrR, penB, and ponA were studied. Molecular typing was performed by N. gonorrhoeae multiantigen sequence typing. Of 329 strains analyzed in 2013, none showed high-level cephalosporin resistance, but 8.2 % had resistance to cefixime [minimum inhibitory concentration (MIC) > 0.125 µg/mL] and 0.6 % to ceftriaxone (MIC > 0.125 µg/mL). Azithromycin resistance was documented in 4.3 % and ciprofloxacin resistance in 49.2 %. Among 48 strains with an MIC ≥ 0.125 µg/mL to cefixime, 58.3 % showed the penA mosaic pattern XXXIV, 98 % a Leu → Pro substitution at position 421 of the ponA gene, 100 % amino acid changes at positions 101 and 102 of the PorB1b porin, and 87.5 % of strains an adenine deletion in the promoter region of the MtrC-D-E efflux pump. A significant difference between strains with and without decreased cephalosporin susceptibility (MIC ≥ 0.125 µg/mL) was observed for these four genes. Of the 48 strains with an MIC ≥ 0.125 µg/mL to cefixime, 43.8 % belonged to the genogroup G1407 and 27.1 % belonged to the genogroup G2400. A significant association of G1407 with decreased susceptibility (MIC ≥ 0.125 µg/mL) and G2992 with susceptibility was found, and also between G1407 and mosaic pattern XXXIV and between G2400 and A501T substitution in penA. The NG resistance rate in our area is higher than the median of Europe. We have detected the emergence of G2400, which may be a source of antimicrobial resistance.

Gas-Phase Production of Hydroxylated Silicon Oxide Cluster Cations: Structure, Infrared Spectroscopy, and Astronomical Relevance.

The interaction of free cationic silicon oxide clusters, Si x O y + (x = 2-5, y ≥ x), with dilute water vapor, was investigated in a flow tube reactor. Product mass distributions indicate cluster size-dependent dissociative water adsorption. To probe the structure and vibrational spectra of the resulting Si x O y H2 + (x = 2-4) clusters, we employed infrared multiple photon dissociation spectroscopy and density functional theory calculations. The planar rhombic cluster core of the disilicon oxides (x = 2) appears to be retained upon dissociative adsorption of one H2O unit, whereas a significant structural transformation of the tri- and tetra-silicon oxides (x = 3 and 4) is induced, resulting in an increased coordination of the Si atoms and more 3D cluster structures. In an astronomical context, we discuss the potential relevance of Si x O y H z + clusters as seeds for dust nucleation and catalysts for carbon-based chemistry in diffuse or translucent interstellar clouds, where all the necessary conditions for producing these species are found. In the produced clusters, the frequency of the isolated silanol Si-OH stretching vibrational mode is considerably blue-shifted compared to that in hydroxylated bulk silica and small inorganic compounds. This mode has a characteristic frequency range between 1200 cm-1 (8.3 µm) and 1090 cm-1 (9.2 µm) and is associated with the anomalously small Si-OH bond lengths in these ionised species. In infrared observations such high frequency Si-O stretching modes are usually associated with a pure bulk silica component of silicate cosmic dust. The presence of Si x O y H2 + clusters in low silica astrophysical environments could thus potentially be detected via their signature Si-O band using the James Webb space telescope.

Effect of an ambulatory diagnostic and treatment programme in patients with sleep apnoea.

The aim of this study to evaluate the efficacy of a home-based programme on clinical response, continuous positive airway pressure (CPAP) compliance and cost in a population of high pre-test probability of suffering obstructive sleep apnoea syndrome (OSAS). Patients were randomised into the following three groups. Group A: home respiratory polygraphy (RP) and home follow-up; group B: hospital polysomnography and hospital follow-up; and group C: home RP and hospital follow-up. Evaluation during 6 months included Epworth Sleepiness Scale (ESS), Functional Outcomes Sleep Questionnaire (FOSQ), and daily activity and symptom questionnaires. Compliance was assessed by memory cards (group A) and using an hourly counter (groups B and C). 66 patients were included (22 per branch), 83% were males, aged mean±sd 52±10 yrs, body mass index 34±7kg·m(-2), apnoea/hypopnoea index 43±20 h(-1), CPAP pressure 8±2 cmH(2)O, with no between-group differences. Clinical response showed an ESS of mean±sd 15±3 to 6±4, a FOSQ of 16±3 to 18±2, symptoms of 43±7 to 25±7, and activity of 37±11 to 25±8. At the end of the study, compliance was: group A 73%, group B 68% and group C 57%. The cost per patient was: group A €590±43, group B €894±11 and group C €644±93 (p<0.001). In conclusion, patients with a high initial probability of having OSAS can be diagnosed and treated in a home setting, with a high level of CPAP compliance and lower cost than using either a hospital-based approach or home RP/hospital follow-up.

Lymphogranuloma venereum: a hidden emerging problem, Barcelona, 2011.

From the beginning of 2007 until the end of 2011, 146 cases of lymphogranuloma venereum (LGV) were notified to the Barcelona Public Health Agency. Some 49% of them were diagnosed and reported in 2011, mainly in men who have sex with men. Almost half of them, 32 cases, were reported between July and September. This cluster represents the largest since 2004. This article presents the ongoing outbreak of LGV in Barcelona.

Low aerobic capacity in McArdle disease: A role for mitochondrial network impairment?

BACKGROUND: McArdle disease is caused by myophosphorylase deficiency and results in complete inability for muscle glycogen breakdown. A hallmark of this condition is muscle oxidation impairment (e.g., low peak oxygen uptake (VO2peak)), a phenomenon traditionally attributed to reduced glycolytic flux and Krebs cycle anaplerosis. Here we hypothesized an additional role for muscle mitochondrial network alterations associated with massive intracellular glycogen accumulation. METHODS: We analyzed in depth mitochondrial characteristics-content, biogenesis, ultrastructure-and network integrity in skeletal-muscle from McArdle/control mice and two patients. We also determined VO2peak in patients (both sexes, N = 145) and healthy controls (N = 133). RESULTS: Besides corroborating very poor VO2peak values in patients and impairment in muscle glycolytic flux, we found that, in McArdle muscle: (a) damaged fibers are likely those with a higher mitochondrial and glycogen content, which show major disruption of the three main cytoskeleton components-actin microfilaments, microtubules and intermediate filaments-thereby contributing to mitochondrial network disruption in skeletal muscle fibers; (b) there was an altered subcellular localization of mitochondrial fission/fusion proteins and of the sarcoplasmic reticulum protein calsequestrin-with subsequent alteration in mitochondrial dynamics/function; impairment in mitochondrial content/biogenesis; and (c) several OXPHOS-related complex proteins/activities were also affected. CONCLUSIONS: In McArdle disease, severe muscle oxidative capacity impairment could also be explained by a disruption of the mitochondrial network, at least in those fibers with a higher capacity for glycogen accumulation. Our findings might pave the way for future research addressing the potential involvement of mitochondrial network alterations in the pathophysiology of other glycogenoses.

Hematopoietic gene therapy restores thymidine phosphorylase activity in a cell culture and a murine model of MNGIE.

Mitochondrial neurogastrointestinal encephalomyopathy (MNGIE) is an autosomal recessive disorder caused by mutations in the TYMP gene, which encodes thymidine phosphorylase (TP). TP dysfunction results in systemic thymidine (dThd) and deoxyuridine (dUrd) overload, which selectively impair mitochondrial DNA replication. Allogeneic hematopoietic transplantation has been used to treat MNGIE patients; however, this approach has serious adverse effects, including the toxicity of myeloablative conditioning, graft rejection and graft-versus-host disease. With the aim of testing the feasibility of gene therapy for MNGIE, we transduced TP-deficient B-lymphoblastoid cells from two MNGIE patients, with lentiviral vectors carrying a functional copy of the human TYMP DNA coding sequence. This restored TP activity in the cells, which reduced the excretion of dThd and dUrd and their concentrations when added in excess. Additionally, lentiviral-mediated hematopoietic gene therapy was used in partially myeloablated double Tymp/Upp1 knockout mice. In spite of the relatively low levels of molecular chimerism achieved, high levels of TP activity were observed in the peripheral blood of the transplanted mice, with a concomitant reduction of nucleoside concentrations. Our results suggest that hematopoietic gene therapy could be an alternative treatment for this devastating disorder in the future.

Group B streptococci causing neonatal infections in barcelona are a stable clonal population: 18-year surveillance.

We analyzed 212 group B streptococci (GBS) from newborns with invasive infections in the area of Barcelona, Spain, between 1992 and 2009, with the aim of documenting changes in the prevalences of serotypes, antimicrobial resistance, and genetic lineages and evaluating their associations with either early-onset disease (EOD) or late-onset disease (LOD). Serotypes III (n = 118) and Ia (n = 47) together accounted for nearly 78% of the isolates. All isolates carried an alpha or alpha-like protein gene, and specific associations between genes and serotypes, such as serotype Ib and bca, serotype II and bca, serotype III and rib, and serotype V and alp3, reflected the presence of particular genetic lineages. Macrolide resistance (14.2%) was significantly associated with serotype V. Pulsed-field gel electrophoresis (PFGE) clustering was an excellent predictor of serotype and antibiotic resistance. The combination of PFGE and multilocus sequence typing revealed a large number of genetically distinct lineages. Still, specific lineages were dominant in our collection, particularly the serotype III/ST17/rib lineage, which had enhanced potential to cause LOD. Serotype Ia was concentrated in a single PFGE cluster composed of two genetic lineages: ST23/eps and ST24/bca. The ST24/bca sublineage of serotype Ia, which is found infrequently elsewhere, may be emerging as an important cause of neonatal invasive infections in the Mediterranean region. In spite of the introduction of prophylaxis, resulting in a pronounced decline in the frequency of EOD, the study revealed a remarkably stable clonal structure of GBS causing neonatal infections in Barcelona over a period of 18 years.

Professional burnout and work satisfaction in Spanish allergists: analysis of working conditions in the specialty.

BACKGROUND: Burnout is a worrying problem in the medical profession and has proven to be highly prevalent in all the care settings and specialty areas in which it has been studied.We applied 2 widely used questionnaires to analyze the working conditions of Spanish allergists in terms of quality of professional life and degree of burnout perceived. METHODS: Participants completed 2 questionnaires: the 22-item Maslach scale, a structured questionnaire covering different aspects of the feelings and attitudes of professionals toward their work and patients; and the Spanish Quality of Professional Life Questionnaire (CPV-35), a 35-item questionnaire evaluating job satisfaction and perceived quality of life at work. RESULTS: We received 404 questionnaires from throughout Spain. The main sources of motivation were better pay (94.4%), more available resources/technology (85.1%), access to research activities (81%), and promotion in one's professional career (80.1%). Analysis of the scores from the Maslach scale revealed that two-thirds of the allergists interviewed experienced medium and high levels of negative burnout (67.9% and 66.2%, respectively, for emotional exhaustion and depersonalization). This situation was in part compensated for by the fact that a slightly lower proportion of the group (59.2%) obtained very high scores on personal accomplishment in their work. Analysis of the scores from the CPV-35 questionnaire revealed 3 complementary aspects of job satisfaction: perceptions of the workload borne (5.8), management support available to cope with daily patient workload (5.6), and levels of intrinsic motivation for work (7.7), which was the highest value. The score for the item summarizing self-perceived overall quality of working life was acceptable (6.4). CONCLUSION: Promoting intrinsic motivation of Spanish allergists using the motivating factors identified in this study could protect against professional burnout.

Proof-of-concept trial on the efficacy of sodium tungstate in human obesity.

AIM: Considering the poor long-term success of current dietary and pharmacological interventions, we aimed to evaluate the potential effect of sodium tungstate in the treatment of grade I and II obesity (ClinicalTrials.gov identifier: NCT00555074). METHODS: Prospective, randomized, placebo-controlled, double-blind, proof-of-concept study was carried out. Following a 2-week lead-in period, 30 obese (body mass index, BMI 30.0-39.9 kg/m(2)), non-diabetic subjects were randomized to receive either sodium tungstate (100 mg bid) or placebo for 6 weeks. The primary study endpoint was the absolute change in body weight relative to the time of randomization. RESULTS: Treatment with sodium tungstate [-0.135 ± 0.268 kg (95% CI -0.686 to +0.416 kg)] was not associated with a significant weight loss compared to placebo [-0.063 ± 0.277 kg (95% CI -0.632 to +0.507 kg)] (p = 0.854). Likewise, treatment with sodium tungstate was not associated with significant changes in fat mass (DEXA), resting energy expenditure (indirect calorimetry) or caloric consumption (3-day food records). CONCLUSION: Our data do not support sodium tungstate as a pharmacological agent in the treatment of human obesity.

NARP syndrome in a patient harbouring an insertion in the MT-ATP6 gene that results in a truncated protein.

BACKGROUND: Neurogenic muscle weakness, ataxia and retinitis pigmentosa (NARP) syndrome have been associated to m.8993T>G/C mutations in the subunit 6 of the ATP synthase (p.MT-ATP6). METHODS: We have performed a mutational screening of the mitochondrial DNA gene encoding for this protein in 62 patients with the disease, that do not carry any of the common mutations described to date. RESULTS: We report clinical and molecular data in one patient who harbours a de novo insertion in the MT-ATP6 gene that results in a truncated subunit. The mutation was heteroplasmic (85%) in muscle DNA and the BN-PAGE analysis showed a clear decrease in the amount of ATP synthase. CONCLUSION: Molecular analysis of NARP patients cannot be limited to the search of the m.8993T>G/C and either the ATP6 or the whole mtDNA should be sequenced.

Novel mutations in patients with McArdle disease by analysis of skeletal muscle mRNA.

OBJECTIVE: To identify pathogenic mutant alleles of the PYGM gene in "genetic manifesting heterozygous" patients with McArdle disease-that is, those in whom we could only find a sole mutant allele by genomic DNA analysis. METHODS: We studied four unrelated patients. PCR-RFLP, gene sequencing, and muscle cDNA analysis were performed to search for mutations in the PYGM gene. The effects of the mutations were evaluated by in silico analysis, and gene expression was assessed by real-time polymerase chain reaction (PCR). RESULTS: Patient 1 was a compound heterozygous for the p.G205S missense mutation and for a novel "in frame" mutation, p.Q176_M177insVQ, resulting from a retention of six nucleotides from the 3'-end sequence of intron 4. Patient 2 was heterozygous for the common nonsense mutation p.R50X, and for a 1094 bp, c.1969+214_2177+369del mutation, spanning from intron 16 to intron 17 sequences. Furthermore, mRNA expression level was dramatically reduced consistent with nonsense mediated decay. Patient 3 was heterozygous for the p.R50X substitution, and patient 4 was heterozygous for the relatively common private Spanish mutation p.W798R. These two patients harboured a heterozygous exonic synonymous variant, p.K215K. Quantification of gene transcripts in patient 3 revealed a drastic decrease in the relative expression of the gene, which strongly supports the possibility of nonsense mediated decay. CONCLUSIONS: Our results indicate that skeletal muscle cDNA studies in "genetic manifesting heterozygous" patients with McArdle disease are prone to identify their second mutant allele.

McArdle disease: another systemic low-inflammation disorder?

McArdle disease is caused by inherited deficit of human muscle glycogen phosphorylase with subsequent blockade in muscle glycogenolysis. Patients usually experience severe exercise intolerance and 'chronic' skeletal muscle damage. We determined circulating levels of 27 cytokines in a group of 31 adult McArdle patients (15 male 16 female; mean (+/-S.E.M.) age: 39+/-3 years) and 29 healthy sedentary controls (14 male, 15 female) before and after an acute exercise bout involving no muscle damage (cycling). Patients had an ongoing state of muscle breakdown even when following a sedentary lifestyle (serum creatine kinase activity at baseline of 2590+/-461 Ul(-1) vs. 97+/-5 Ul(-1) in controls). Under resting conditions, neutrophil count (+20%) and circulating levels of several cytokines were significantly higher (P

Mitochondrial DNA oxidation and manganese superoxide dismutase activity in peripheral blood mononuclear cells from type 2 diabetic patients.

AIM: To investigate the balance between parameters of oxidative stress and antioxidant defences in the mitochondria of peripheral blood mononuclear cells (PBMCs) of type 2 diabetic patients with late complications. METHODS: Ten type 2 diabetic patients with late diabetic complications and 10 age-matched healthy volunteers (controls) were prospectively recruited. Mitochondrial DNA (mtDNA) oxidative damage and mtDNA content were measured as indices of oxidative stress. Manganese superoxide dismutase (MnSOD) activity has been used as an index of mitochondrial antioxidant defence. Mitochondrial respiratory-chain function (cytochrome C oxidase activity) was also assessed. RESULTS: Mitochondrial DNA (mtDNA) oxidation was significantly higher in the PBMCs of diabetic patients than in control subjects (P<0.0001) and, although mtDNA content was lower in the diabetic group, this was not statistically significant. MnSOD activity was significantly increased in PBMCs of type 2 diabetic patients compared with healthy controls (1366+/-187 versus 686+/-167 U/g of protein; P=0.01), and was related to mtDNA oxidative damage. No differences in mitochondrial respiratory-chain function were found between diabetic patients and controls. CONCLUSION: PMBCs from type 2 diabetic patients with late diabetic complications exhibit high mtDNA oxidative damage. The degree of mtDNA oxidation was associated with an increase in MnSOD as an adaptive response to oxidative stress. The consequences of mtDNA oxidative damage on PBMC function and the progression of diabetic complications remain to be elucidated.

[Horseshoes kidney isthmus carcinoma. A case report]. / Carcinoma renal de istmo en riñn en herradura. A propósito de un caso.

Horseshoe kidney is the most frequent fusion abnormality of the kidney. The incidence of renal carcinoma in patients with horseshoe kidney is similar to those with normal anatomy. Its special anatomical features must be borne in mind for both surgical approach and conservative surgery. We present a horseshoe kidney isthmus carcinoma case report in which we performed conservative surgery of both renal units.

Sexually transmitted Chlamydia trachomatis, Neisseria gonorrhoeae, and HIV-1 infections in two at-risk populations in Barcelona: female street prostitutes and STI clinic attendees.

OBJECTIVE: To determine the prevalence of Chlamydia trachomatis (CT), Neisseria gonorrhoeae (NG), and HIV-1 infections in female street prostitutes and STI clinic attendees in Barcelona. DESIGN: This was a prospective study carried out in two four-month periods over two years. Urine specimens were tested for CT and NG using a PCR pooling algorithm. Among street prostitutes HIV-1 testing in urine was also carried out. RESULTS: The prevalences of CT, NG, and HIV-1 in female street prostitutes (n=301) were 4.7%, 3.7%, and 1.0%, respectively. Women from Eastern Europe had the highest prevalence of CT (p=0.01). Prevalences of CT, NG, and HIV-1 among all clinic attendees (n=536) were 4.3%, 4.5%, and 4.4%, respectively. Prevalence of HIV-1 infection among homosexual men was higher compared with heterosexual men and women (p<0.001). CONCLUSIONS: Overall CT prevalence is currently lower than in other European countries, although it could increase as a result of immigration. Rates of HIV-1 and of NG are higher among homosexual than among heterosexual men.

McArdle disease: molecular genetic update.

McArdle disease or Glycogenosis type V is an autosomal recessive metabolic disorder caused by a deficiency of the muscle isoform of glycogen phosphorylase (myophosphorylase, PYGM), the specific skeletal muscle enzyme that initiates glycogen breakdown. Since the first clinical description by Brian McArdle in 1951, several patients have been identified worldwide and significant advances have been made in the study of molecular genetics of the disease. Molecular heterogeneity has been demonstrated by the identification to date of more than 65 mutations in the PYGM gene.

Can patients with McArdle's disease run?

Patients with McArdle's disease commonly adopt a sedentary lifestyle. This sedentary behaviour, however, usually worsens the limited exercise capacity of these patients. Although eccentric muscle work can be associated with rhabdomyolysis, supervised eccentric training with gradually increasing loads has important advantages compared with conventional concentric work, particularly for patients with a poor cardiorespiratory system. We report the beneficial effects (particularly, increased VO(2peak) (from 14.6 to 30.8 ml/kg/min) and increased gross muscle efficiency (from 13.8% to 17.2%)) induced by a supervised aerobic training programme of 7 months duration including 3-4 running sessions (< or =60 min/session) per week in a 38-year-old patient. These preliminary data suggest the potential therapeutic value of this type of exercise in these patients.