Intraspecific diversity of the Cassava Green Mite Mononychellus progresivus (Acari: Tetranychidae) using comparisons of mitochondrial and nuclear ribosomal DNA sequences and cross-breeding

Intraspecific diversity in Cassava Green Mite Mononychellus progresivus Doreste was examined using individuals collected in Benin and the Congo and in Columbia and Brazil. Comparisons were based on mitochondrial and ribosomal DNA sequences and the results of several cross-breeding experiments. Sequence variation was examined in a total of 1139 base pairs (bp) constituting the ITS2 ribosomal DNA (805 bp) and a fragment of the Cytochrome Oxidase I (COI) gene (334 bp). Sequence divergence is low, ranging from 0% to 2.1% for COI and from 0% to 0.4% for ITS2. Inter-strain comparisons have shown that the two African populations appear to be identical. They were similar to the Colombian population while the Brazilian population was clearly different. The data support the hypothesis of a single introduction of the species in the two African populations. Crossing experiments have shown partial hybrid sterility, suggesting a genetic incompatibility consistent with differences detected by sequence data. The results show the usefulness of molecular markers as a tool for determining taxonomic status and dispersion paths in spider mites.     

Brain cholecystokinin-8 immunoreactivity in rats with experimental liver cirrhosis

Cholecystokinin-8 like immunoreactivity (CCK-8 IR) was measured in different brain regions of rats with experimental liver cirrhosis. A statistically significant reduction of CCK-8 content was observed in the hypothalamus of cirrhotic rats. No significant modification of brain CCK fractionation pattern was observed in treated animals as compared to controls. The decrease of CCK-8 IR parallels the recently reported hypothalamic depletion of beta endorphin in cirrhotic rats confirming that central neuropeptides are affected by chronic liver failure.     

Prevalence of Ventricular Arrhythmia and Its Associated Factors in Nondialyzed Chronic Kidney Disease Patients

Background and Objectives

Sudden cardiac death is the most common cause of mortality in chronic kidney disease patients, and it occurs mostly due to ventricular arrhythmias. In this study, we aimed at investigating the prevalence of ventricular arrhythmia and the factors associated with its occurrence in nondialyzed chronic kidney disease patients.

Design, Setting, Participants and Measurements

This cross-sectional study evaluated 111 chronic kidney disease patients (estimated glomerular filtration rate 34.7±16.1 mL/min/1.73 m², 57±11.4 years, 60% male, 24% diabetics). Ventricular arrhythmia was assessed by 24-hour electrocardiogram. Left ventricular hypertrophy (echocardiogram), 24-hour ambulatory blood pressure monitoring, and coronary artery calcification (multi-slice computed tomography) and laboratory parameters were also evaluated.

Results

Ventricular arrhythmia was found in 35% of the patients. Non-controlled hypertension was observed in 21%, absence of systolic decency in 29%, left ventricular hypertrophy in 27%, systolic dysfunction in 10%, and coronary artery calcification in 49%. Patients with ventricular arrhythmia were older (p<0.001), predominantly men (p = 0.009), had higher estimated glomerular filtration rate (p = 0.03) and hemoglobin (p = 0.005), and lower intact parathyroid hormone (p = 0.024) and triglycerides (p = 0.011) when compared to patients without ventricular arrhythmia. In addition, a higher left ventricular mass index (p = 0.002) and coronary calcium score (p = 0.002), and a lower ejection fraction (p = 0.001) were observed among patients with ventricular arrhythmia. In the multiple logistic regression analysis, aging, increased hemoglobin levels and reduced ejection fraction were independently related to the presence of ventricular arrhythmia.

Conclusions

Ventricular arrhythmia is prevalent in nondialyzed chronic kidney disease patients. Age, hemoglobin levels and ejection fraction were the factors associated with ventricular arrhythmia in these patients.     

Resuscitation rate in different enterococcal species in the viable but non-culturable state

AIMS: The viable but non-culturable (VBNC) state is a survival strategy adopted by bacteria when exposed to environmental stress. When in this state bacteria are no longer culturable on conventional growth media, but cells display metabolic activity and maintain pathogenicity factors/genes and, in some cases, resuscitation from the VBNC state has been shown. This state has been described for both human pathogens and faecal pollution indicators. In this study, we present evidence for entry of different enterococcal species into the VBNC state in an oligotrophic microcosm. METHODS AND RESULTS: The duration of the viability of the cells in the VBNC state was measured either by detecting the presence of pbp5 mRNA or by quantifying their resuscitation capability. Enterococci showed different behaviours. Enterococcus faecalis and Enterococcus hirae entered into the VBNC state within 2 weeks and remained in that state for 3 months. In the experiments described the resuscitation rate was 1:10 000 cells as soon as the cells entered the VBNC state and decreased gradually to undetectable levels over the following 3 months. Enterococcus faecium, however, remained culturable up to 4 weeks. After this time period, when the population was totally unculturable, the cells were far less resuscitable than other enterococci and only over a narrow time interval (2 weeks). CONCLUSIONS: These results suggest that Ent. faecalis and Ent. hirae enter the VBNC state but that Ent. faecium, in an oligotrophic laboratory environment, tends to die instead of entering the VBNC state. SIGNIFICANCE AND IMPACT OF THE STUDY: These experiments may mimic what happens when enterococci are released by humans and animals in natural environments.     

Spontaneous lipid vesicle fusion with electropermeabilized cells

Fusion is obtained between electropermeabilized mammalian cells and intact large unilamellar lipid vesicles. This is monitored by a fluorescence assay. Prepulse contact is obtained by Ca2+ when negatively charged lipids are present in the liposomes. The mixing of the liposome content in the cell cytoplasm is observed under conditions preserving cell viability. Electric conditions are such that free liposomes are not affected by the external field. Therefore destabilization of only one of the two membranes of the partners is sufficient for fusion. The comparison between the efficiency of dye delivery for different liposome preparations (multilamellar vesicles, large unilamellar vesicles, small unilamellar vesicles) is indicative that more metastable liposomes are more fusable with electropulsated cells. This observation is discussed within the framework of the recent hypothesis that occurrence of a contact induced electrostatic destabilization of the plasma membrane is a key step in the exocytosis process.     

Involvement of rpoS in the survival of Escherichia coli in the viable but non-culturable state

When exposed to stress-provoking environmental conditions such as those of ground waters, many medically important bacteria have been shown to be capable of activating a survival strategy known as the viable but non-culturable (VBNC) state. In this state bacteria are no longer culturable on conventional growth media, but the cells maintain their viability and pathogenicity genes/factors and can start dividing again, in a part of the cell population, upon restoration of favourable environmental conditions. Little is known about the genetic mechanisms underlying the VBNC state. In this study we show evidence of involvement of the rpoS gene in persistence of Escherichia coli in the VBNC state. The kinetics of entry into the non-culturable state and duration of cell viability were measured in two E. coli mutants carrying an inactivated rpoS gene and compared with those of the parents. For these experiments, laboratory microcosms consisting of an artificial oligotrophic medium incubated at 4 degrees C were used. The E. coli parental strains reached the non-culturable state in 33 days when the plate counts were evaluated on Luria-Bertani agar containing sodium pyruvate, whereas cells of the rpoS mutants lost their culturability in only 21 days. Upon reaching unculturability the parents yielded respiring cells and cells with intact membranes for at least the next three weeks and resuscitation was allowed during this time. In contrast, the RpoS- mutant cells demonstrated intact membranes for only two weeks and a very restricted (<7 days) resuscitation capability. Guanosine 3',5'-bispyrophosphate (ppGpp) acts as a positive regulator during the production and functioning of RpoS. A mutant deficient in ppGpp production behaved like the rpoS mutants, while overproducers of ppGpp displayed a vitality at least comparable to that of RpoS+ strains. These results suggest that the E. coli parental strains enter the VBNC state which lasts for, at least, three weeks, after which apparently all the cells die. The rpoS mutants do not activate this survival strategy and early die. This implies involvement of the rpoS gene in E. coli persistence in the VBNC state.     

Racemization and degradation of thioridazine and thioridazine 2-sulfone in human plasma and aqueous solutions

We present two methods for the enantioselective analysis of thioridazine (THD) and thioridazine 2-sulfone (THD 2-SO(2)) in human plasma based on liquid-liquid extraction with diethyl ether and chiral resolution of the enantiomers on Chiralpak AD and Chiralcel OD-H columns, respectively. After validation, the methods were used to study the degradation and racemization of both drug and metabolite. Our results showed that both enantiomers of THD and THD 2-SO(2) were stable at varying temperatures, pH, and ionic strengths; however, solubility problems for THD and THD 2-SO(2) enantiomers were observed, mainly at pH 8.5. The influence of light on the stability of the THD and THD 2-SO(2) enantiomers was also studied. Degradation of the THD enantiomers was observed under UV light (254 and 366 nm) while THD 2-SO(2) enantiomers were stable at these wavelengths and also when exposed to visible light.     

Simultaneous determination of albendazole sulfoxide enantiomers and albendazole sulfone in plasma

A high-performance liquid chromatographic method has been developed for the simultaneous determination of albendazole sulfoxide (ABZSO) enantiomers and albendazole sulfone (ABZSO₂) in human plasma. The resolution of ABZSO enantiomers and ABZSO₂ was obtained on a Chiralpak^® AD column using hexane–isopropanol–ethanol (81:14.25:4.75, v/v/v) as the mobile phase. The drugs were detected by fluorescence (λ_exc=280 nm, λ_em=320 nm). The drugs were extracted from 500 μl plasma with ethyl acetate, and after solvent evaporation, the residues were dissolved in the mobile phase and chromatographed. The method was precise and accurate for the three compounds, as judged by the coefficients of variation and relative errors observed. Linear standard curves were obtained in the concentration range of 5–2500 ng/ml for ABZSO enantiomers and 1–500 ng/ml for ABZSO₂. A typical plasma concentration–time profile is presented for one patient under treatment for neurocysticercosis.     

Inferring the degree of incipient speciation in secondary contact zones of closely related lineages of Palearctic green toads (Bufo viridis subgroup)

Reproductive isolation between lineages is expected to accumulate with divergence time, but the time taken to speciate may strongly vary between different groups of organisms. In anuran amphibians, laboratory crosses can still produce viable hybrid offspring >20 My after separation, but the speed of speciation in closely related anuran lineages under natural conditions is poorly studied. Palearctic green toads (Bufo viridis subgroup) offer an excellent system to address this question, comprising several lineages that arose at different times and form secondary contact zones. Using mitochondrial and nuclear markers, we previously demonstrated that in Sicily, B. siculus and B. balearicus developed advanced reproductive isolation after Plio-Pleistocene divergence (2.6 My, 3.3–1.9), with limited historic mtDNA introgression, scarce nuclear admixture, but low, if any, current gene flow. Here, we study genetic interactions between younger lineages of early Pleistocene divergence (1.9 My, 2.5–1.3) in northeastern Italy (B. balearicus, B. viridis). We find significantly more, asymmetric nuclear and wider, differential mtDNA introgression. The population structure seems to be molded by geographic distance and barriers (rivers), much more than by intrinsic genomic incompatibilities. These differences of hybridization between zones may be partly explained by differences in the duration of previous isolation. Scattered research on other anurans suggests that wide hybrid zones with strong introgression may develop when secondary contacts occur <2 My after divergence, whereas narrower zones with restricted gene flow form when divergence exceeds 3 My. Our study strengthens support for this rule of thumb by comparing lineages with different divergence times within the same radiation.     

A novel design for an instrumented stairway

Kinetics during stair ambulation is currently studied via either the use of sensing elements embedded in the steps of the stairway or simple rigid blocks of different height positioned on top of existing force platforms, typically embedded in a walkway for gait analysis. Neither of these approaches is truly satisfactory for gait analysis laboratories. The first one is expensive and requires setting up a dedicated space. The second approach is limited by the number of platforms utilized in the laboratory for evaluating level walking. This communication proposes a novel design, referred to as "interlaced stairway", that allows one to measure ground reaction force and position of the center of pressure (CoP) for four foot contacts during stair ambulation using only two force platforms embedded in a walkway. Accuracy and precision of the CoP estimates and natural frequency of the stairway structure were derived from experimental data. Test results indicate that the interlaced stairway structure does not appreciably reduce the quality of the measures gathered by the existing force platforms. Specifically, the estimated CoP coordinates show good agreement with the horizontal coordinates of the geometric center of the calibration object utilized to assess accuracy and precision of the CoP estimates (max difference < 6 mm). The natural frequency of the stairway structure is lower than the one for the unloaded force platform but higher than the frequency components of interest in stair ambulation analysis.