Mariana Ferreira

SANDO: Two novel mutations in POLG1 gene

Sensory ataxia with neuropathy, dysarthria and ophthalmoparesis represent the clinical triad of SANDO, a specific mitochondrial phenotype first reported in 1997 in association with multiple mitochondrial DNA deletions and mutations in POLG1 or more rarely in the C10orf2 (twinkle-helicase) gene. We report a 44-year-old man with SANDO who harboured two novel mutations (P648R/R807C) in the POLG1 gene.

Progressive cavitating leukoencephalopathy associated with respiratory chain complex I deficiency and a novel mutation in NDUFS1

We present clinical, neuroimaging, and molecular data on the identification of a new homozygous c.1783A>G (p.Thr595Ala) mutation in NDUFS1 in two inbred siblings with isolated complex I deficiency associated to a progressive cavitating leukoencephalopathy, a clinical and neuroradiological entity originally related to unknown defects of the mitochondrial energy metabolism. In both sibs, the muscle biopsy showed severe reduction of complex I enzyme activity, which was not obvious in fibroblasts. We also observed complex I dysfunction in a Neurospora crassa model of the disease, obtained by insertional mutagenesis, and in patient fibroblasts grown in galactose. Altogether, these results indicate that the NDUFS1 mutation is responsible for the disease and complex I deficiency. Clinical presentation of complex I defect is heterogeneous and includes an ample array of clinical phenotypes. Expanding the number of allelic variants in NDUFS1, our findings also contribute to a better understanding on the function of complex I.

Solution and biological behaviour of enrofloxacin metalloantibiotics: a route to counteract bacterial resistance?

Solution behaviour of enrofloxacin complexes with copper(II), nickel(II), cobalt(II) and zinc(II) in the presence and absence of 1, 10-phenanthroline was studied in aqueous solution, by potentiometry. The results obtained show that under physiological conditions (micromolar concentration range and pH 7.4) only copper(II) forms stable complexes. Binary copper(II)/enrofloxacin and ternary copper(II)/enrofloxacin/phenanthroline complexes were synthesised and characterized by elemental analysis, UV-visible spectroscopy and FTIR. The antimicrobial activity of these complexes and of copper(II)/enrofloxacin and copper(II)/enrofloxacin/phenanthroline solutions, prepared by mixing of the individual components in the same stoichiometric proportion and concentration range used for the synthesised complexes, was tested against two different Escherichia coli strains. Although, at a glance, the results point to a possible use of both complexes as metalloantibiotics, a detailed analysis shows that, at biological concentrations, the copper(II) binary complex does not exist and the antimicrobial activity observed is a consequence of its dissociation into free enrofloxacin. Consequently, only the ternary complex seems worth pursuing as a possible antimicrobial agent candidate. Moreover, as the biological studies showed, both the synthesised complexes and the solutions prepared by mixing the components exhibited the same behaviour. Hence, a new, faster and accurate methodology to screen metalloantibiotics prior to synthesis of the complexes is proposed.

Salt intake and risk of gastric intestinal metaplasia: systematic review and meta-analysis.

The understanding of the association between salt intake and precancerous lesions may contribute to clarify the causal relation with gastric cancer. We systematically reviewed 17 articles addressing the association between dietary salt exposure and gastric intestinal metaplasia and conducted meta-analyses for quantitative synthesis (random effects model). Salt exposure was estimated assessing salted/salty food consumption, preference for salted/salty foods, use of table salt, or sodium urinary excretion. Heterogeneity was also large regarding food items evaluated, consumption categories, and data analysis. The combined odds ratio (OR) was 1.68 (95% confidence interval (CI) = 0.98–2.90; I2 = 55.4%) for the association between salted/salty meat and intestinal metaplasia (4 studies) and the OR was 1.53 (95% CI = 0.72–3.24; I2 = 76.8%) for salt preference. There was a positive, nonstatistically significant association between intestinal metaplasia and urinary sodium excretion. The heterogeneity of methodological options and results preclude quantitative synthesis or its proper interpretation, even if the available evidence may suggest a positive association between salt and intestinal metaplasia.

Relative frequency of known causes of multiple mtDNA deletions: Two novel POLG mutations

Diseases affecting mtDNA stability, termed nuclear-mitochondrial intergenomic communication disorders, are caused by a primary nuclear gene defect resulting in multiple mtDNA deletions. The aim of this study was to estimate the frequency of known etiologies and the spectrum of mutations in a cohort of 21 patients harboring multiple mtDNA deletions in skeletal muscle. We showed that 10 cases (48%) display mutations in POLG, including eight previously reported variants and two novel mutations (namely, p.Trp585X and p.Arg1081Gln). The novel mutations affect evolutionary conserved residues and were absent in a large set of control chromosomes. These findings expand the array of mutations associated with multiple rearranged mtDNA attributed to mutations in POLG. The relatively high diagnostic yield (about one in two cases) supports the notion that it is recommended to test POLG routinely in diagnostic laboratories whenever multiple mtDNA deletions are present, regardless of the age of onset of patients and their clinical phenotype.

Ciprofloxacin metalloantibiotic: an effective antibiotic with an influx route strongly dependent on lipid interaction?

AbstractFluoroquinolones are antibiotics that have a large spectrum of action against bacteria, especially Gram-negative. A strategy to enhance their pharmacological behavior, and try to counteract bacterial resistance, is their coordination to divalent metal ions and 1,10-phenanthroline. These stable complexes modify fluoroquinolones potency and specificity, possibly due to their alternative translocation through the bacterial membranes. In this work, we determined the interaction of ciprofloxacin and its copper(II) ternary complex with unilamellar liposomes of DMPC, POPE/POPG (0.75:0.25), POPE/POPG/cardiolipin (0.67:0.23:0.10), and E. coli total extract, using time-resolved and steady-state fluorescence spectroscopy. The association constants determined show that the interaction of both compounds depends on membrane lipids composition and is always higher for the metalloantibiotic, a trend already observed for other fluoroquinolone metalloantibiotics. Nevertheless, the interaction of ciprofloxacin metalloantibiotic is, normally, higher, which reflects the fluoroquinolone species that are present in solution at physiological pH. In overall, the results obtained suggest that ciprofloxacin and its metalloantibiotic have different translocation pathways, proposing that the diffusion of the metalloantibiotic is a hydrophobic mechanism and suggesting that this new metalloantibiotic may be a good choice to replace the pure ciprofloxacin and bypass, at least, one of the mechanisms of the bacterial resistance to fluoroquinolones.

Interactions of a non-fluorescent fluoroquinolone with biological membrane models: A multi-technique approach

Fluoroquinolones are antibiotics which act by penetrating into bacterial cells and inhibiting enzymes related to DNA replication, and metal complexes of these drugs have recently been investigated as one approach to counteracting bacterial resistance. In this work, we apply a multi-technique approach to studying the partition coefficient (Kp) for the non-fluorescent third-generation fluoroquinolone sparfloxacin or its copper-complex with lipid membrane models of Gram-negative bacteria. The techniques investigated are UV-vis absorption and (19)F NMR spectroscopies together with quenching of a fluorescent probe present in the lipids (using steady-state and time-resolved methods). (19)F NMR spectroscopy has previously been used to determine the Kp values of fluorinated drugs but in the case of sparfloxacin did not yield useful data. However, similar Kp values for sparfloxacin or its copper-complex were obtained for the absorption and fluorescence quenching methods confirming the usefulness of a multi-technique approach. The Kp values measured for sparfloxacin were significantly higher than those found for other fluoroquinolones. In addition, similar Kp values were found for sparfloxacin and copper-complex suggesting that in contrast to other fluoroquinolones hydrophobic diffusion occurs readily for both of these molecules.

Identification of novel L2HGDH gene mutations and update of the pathological spectrum

L-2-hydroxyglutaric aciduria (L-2-HGA, MIM 236792) is a neurometabolic disorder caused by the toxic accumulation of high concentration of L-2-hydroxyglutaric acid in plasma and cerebrospinal fluid. Distinct mutations on the L2HGDH gene have been associated with the clinical and biochemical phenotype. Here we present three novel mutations (Gln197X, Gly211Val and c.540+1 G>A), which increase the present deleterious collection of L2HGDH gene up to 35 mutations that we have compiled in this study. In addition, we used the haplotypic information based on polymorphic markers to demonstrate the common origin of Gly57Arg harboring chromosomes.

Evaluation of membrane fluidity of multidrug-resistant isolates of Escherichia coli and Staphylococcus aureus in presence and absence of antibiotics

In the face of the serious problem of antimicrobial resistance and the global dissemination of multidrug-resistant (MDR) bacteria, it is relevant to deeply study such bacteria both genetically and phenotypically. It is well known that bacteria have the ability to modify the biophysical properties of their cytoplasmic membranes, namely fluidity, in order to survive and thrive in hostile environments. The aim of this study was to assess and compare the membrane fluidity among multidrug-resistant (MDR) isolates of Escherichia coli and Staphylococcus aureus in absence and in presence of antibiotics (ceftazidime or ciprofloxacin). The membrane fluidity was monitored at 24-h intervals up to three days and at the sixth day, by measuring the anisotropy of 1,6-diphenyl-1,3,5-hexatriene (DPH) and the generalized polarization (GP) of Laurdan (6-dodecanoyl-2-dimethylaminonaphthalene). The anisotropy values as well as the Laurdan excitation GP (GPexc) values obtained from all three E. coli strains (two MDR isolates and one susceptible reference strain) were quite similar and indicative of a very alike membrane phospholipid composition, regardless harboring or not resistance to multiple antimicrobials. Nonetheless, in the case of S. aureus, the anisotropy values were more increased in methicillin-resistant S. aureus (MRSA) isolates in comparison to the reference strain, meaning they have a less fluid membrane. Equally, GPexc values were statistically different among the three S. aureus strains and showed that the two MRSA isolates had more rigid membranes than the susceptible strain. The exposition of MDR isolates of S. aureus to subinhibitory concentrations of ciprofloxacin did not affect neither the anisotropy values nor the GPexc values, therefore, not altering membrane fluidity. Membrane integrity, assessed by the Live/Dead staining, of all strains and conditions studied was maintained over the six days. Thus, these preliminary studies on membrane properties of MDR isolates demonstrate that i) MRSA seem to have a more rigid membrane that susceptible S. aureus and ii) the presence of subinhibitory concentrations of antibiotics does not significantly alter the membrane fluidity of S. aureus, regardless being MDR or susceptible, but slightly affect the membrane fluidity of E. coli.

Data on Laurdan spectroscopic analyses to compare membrane fluidity between susceptible and multidrug-resistant bacteria

The data presented are related to the research article entitled “Evaluation of membrane fluidity of multidrug-resistant isolates of Escherichia coli and Staphylococcus aureus in presence and absence of antibiotics” (Bessa et al., 2018) [1]. This data article provides a dataset that includes emission spectra of Laurdan-labeled bacteria, namely of three Escherichia coli and three Staphylococcus aureus strains. Laurdan (6-dodecanoyl-2-dimethylaminonaphthalene) is a popular fluorescence probe used to assess membrane fluidity. Laurdan detects changes in membrane phase properties through its sensitivity to the polarity of the environment in the bilayer. Polarity changes are reflected by shifts in the Laurdan fluorescence emission spectrum that can be quantified by calculating the excitation generalized polarization (GPexc).