Increased retention of functional fusions to toxic genes in new two-hybrid libraries of the E. coli strain MG1655 and B. subtilis strain 168 genomes,prepared without passaging through E. coli

Background  

Cloning of genes in expression libraries, such as the yeast two-hybrid system (Y2H), is based on the assumption that the loss of target genes is minimal, or at worst, managable. However, the expression of genes or gene fragments that are capable of interacting with E. coli or yeast gene products in these systems has been shown to be growth inhibitory, and therefore these clones are underrepresented (or completely lost) in the amplified library.     

Carvacrol Codrugs: A New Approach in the Antimicrobial Plan

Objective

The increasing prevalence of antibiotic-resistant bacterial infections led to identify alternative strategies for a novel therapeutic approach. In this study, we synthesized ten carvacrol codrugs – obtained linking the carvacrol hydroxyl group to the carboxyl moiety of sulphur-containing amino acids via an ester bond – to develop novel compounds with improved antimicrobial and antibiofilm activities and reduced toxicity respect to carvacrol alone.

Method

All carvacrol codrugs were screened against a representative panel of Gram positive (S. aureus and S. epidermidis), Gram negative (E. coli and P. aeruginosa) bacterial strains and C. albicans, using broth microdilution assays.

Findings

Results showed that carvacrol codrug 4 possesses the most notable enhancement in the anti-bacterial activity displaying MIC and MBC values equal to 2.5 mg/mL for all bacterial strains, except for P. aeruginosa ATCC 9027 (MIC and MBC values equal to 5 mg/mL and 10 mg/mL, respectively). All carvacrol codrugs 1-10 revealed good antifungal activity against C. albicans ATCC 10231. The cytotoxicity assay showed that the novel carvacrol codrugs did not produce human blood hemolysis at their MIC values except for codrugs 8 and 9. In particular, deepened experiments performed on carvacrol codrug 4 showed an interesting antimicrobial effect on the mature biofilm produced by E. coli ATCC 8739, respect to the carvacrol alone. The antimicrobial effects of carvacrol codrug 4 were also analyzed by TEM evidencing morphological modifications in S. aureus, E. coli, and C. albicans.

Conclusion

The current study presents an insight into the use of codrug strategy for developing carvacrol derivatives with antibacterial and antibiofilm potentials, and reduced cytotoxicity.     

Understanding the Impact of Hazardous and Harmful Use of Alcohol and/or Other Drugs on ARV Adherence and Disease Progression

The objective of this study was to understand the impact of hazardous and harmful use of alcohol and/or other drugs on ARV adherence and disease progression among HIV patients. A cross-sectional study design was used. A total of 1503 patients attending HIV clinics in Cape Town, South Africa were screened for problematic substance use. A sub-sample of 607 patients (303 patients who screened positive for problematic substance use and 304 who did not) participated in this study. Hazardous or harmful alcohol use and problematic drug use predicted missing and stopping ARVs which, in turn, was associated with a decrease in CD4 counts and more rapid HIV-disease progression and poorer health outcomes in people living with HIV/AIDS (PLWHA). The findings of this study underscore the need for an integrated approach to managing substance-use disorders in PLWHA.     

Mitochondrial impairment observed in fibroblasts from South African Parkinson’s disease patients with parkin mutations

Parkinson’s disease (PD), defined as a neurodegenerative disorder, is characterized by the loss of dopaminergic neurons in the substantia nigra in the midbrain. Loss-of-function mutations in the parkin gene are a major cause of autosomal recessive, early-onset PD. Parkin has been implicated in the maintenance of healthy mitochondria, although previous studies show conflicting findings regarding mitochondrial abnormalities in fibroblasts from patients harboring parkin-null mutations. The aim of the present study was to determine whether South African PD patients with parkin mutations exhibit evidence for mitochondrial dysfunction. Fibroblasts were cultured from skin biopsies obtained from three patients with homozygous parkin-null mutations, two heterozygous mutation carriers and two wild-type controls. Muscle biopsies were obtained from two of the patients. The muscle fibers showed subtle abnormalities such as slightly swollen mitochondria in focal areas of the fibers and some folding of the sarcolemma. Although no differences in the degree of mitochondrial network branching were found in the fibroblasts, ultrastructural abnormalities were observed including the presence of electron-dense vacuoles. Moreover, decreased ATP levels which are consistent with mitochondrial dysfunction were observed in the patients’ fibroblasts compared to controls. Remarkably, these defects did not manifest in one patient, which may be due to possible compensatory mechanisms. These results suggest that parkin-null patients exhibit features of mitochondrial dysfunction. Involvement of mitochondria as a key role player in PD pathogenesis will have important implications for the design of new and more effective therapies.     

Sex pheromone and period gene characterization of Lutzomyia longipalpis sensu lato (Lutz & Neiva) (Diptera: Psychodidae) from Posadas, Argentina

Lutzomyia longipalpis s.l. is the primary vector of Leishmania (L.) infantum in the New World. In this study, male Lutzomyia longipalpis specimens from Posadas, Argentina were characterized for two polymorphic markers: the male sex pheromone and the period (per) gene. The male sex pheromone was identified as (S)-9-methylgermacrene-B, the same compound produced by Lu. longipalpis from Paraguay and many populations from Brazil. The analysis of per gene sequences revealed that the population from Argentina is significantly differentiated from previously studied Brazilian populations. Marker studies could contribute to the understanding of the distribution and spread of urban American visceral leishmaniasis, thus aiding in the design of regional surveillance and control strategies.     

Model-assisted analysis of tomato fruit growth in relation to carbon and water fluxes

This work proposed a model of tomato growth adapted from the Fishman and Génard model developed to predict carbon and water accumulation in peach fruit. The main adaptations relied on the literature on tomato and mainly concerned: (i) the decrease in cell wall extensibility coefficient during fruit development; (ii) the increase in the membrane reflection coefficient to solute from 0 to 1, which accounted for the switch from symplasmic to apoplasmic phloem unloading, and (iii) the negative influence of the initial fruit weight on the maximum rate of active carbon uptake based on the assumption of higher competition for carbon among cells in large fruits containing more cells. A sensitivity analysis was performed and the model was calibrated and evaluated with satisfaction on 17 experimental datasets obtained under contrasting environmental (temperature, air vapour pressure deficit) and plant (plant fruit load and fruit position) conditions. Then the model was used to analyse the variations in the main fluxes involved in tomato fruit growth and accumulation of carbon in response to virtual carbon and water stresses. The conclusions are that this model, integrating simple biophysical laws, was able to simulate the complex fruit behaviour in response to external or internal factors and thus it may be a powerful tool for managing fruit growth and quality.     

Final height in patients with idiopathic short stature and high growth hormone responses to stimulation tests

Children with idiopathic short stature (ISS) may have normal or increased growth hormone (GH) responses to provocation tests and achieve a final height (FH) below -2.0 standard deviation score (SDS) if untreated. FH of subjects with high stimulated GH levels has not been studied in detail. AIM: It was the aim of this study to analyse FH in ISS patients with high GH peak responses to the provocation test. PATIENTS AND METHODS: We studied 16 patients (9 pre-pubertal) with ISS and a GH peak >or=40 mU/l to insulin-induced hypoglycaemia. The patients were recalled at age 19.7 +/- 2.5 years for measurement of FH when blood samples were obtained for serum insulin-like growth factor (IGF)-I, IGF binding protein 3, acid-labile subunit and GH binding protein measurements. GH bioactivity was determined using the Nb2 bioassay. RESULTS: FH was -3.1 +/- 1.0 SDS, being significantly lower than target height (TH). At FH, IGF-I levels were within -1.5 and +1.5 SDS for age and sex in 10 patients and higher than +1.5 SDS in 6 patients. IGF binding protein 3, acid-labile subunit, GH binding protein levels and GH bioactivity values were normal. SUMMARY: These data suggest that patients with ISS and high GH levels during a GH stimulation test may have a more compromised FH. The association of severe ISS with a peak GH >40 mU/l might suggest a degree of insensitivity for the GH-IGF-I axis.     

Coxiella burnetii infection in C57BL/6 mice aged 1 or 14 months

The objective of this study was to investigate the effects of age on infection with Coxiella burnetii, the agent of Q fever. Bacterial burden and granuloma number were increased in the spleens of 14-month-old as compared with 1-month-old mice. This increase was not the result of an anti-inflammatory macrophage response, because inflammatory and anti-inflammatory cytokines were induced in macrophages from young mice but were repressed in mature mice. In addition, macrophage microbicidal competence was similar in mature and young mice. These results suggest the importance of individual host factors in the pathophysiology of an infectious disease such as Q fever.