Expression and purification of a small heat shock protein from the plant pathogen Xylella fastidiosa

The small heat shock proteins (smHSPs) belong to a family of proteins that function as molecular chaperones by preventing protein aggregation and are also known to contain a conserved region termed alpha-crystallin domain. Here, we report the expression, purification, and partial characterization of a novel smHSP (HSP17.9) from the phytopathogen Xylella fastidiosa, causal agent of the citrus variegated chlorosis (CVC). The gene was cloned into a pET32-Xa/LIC vector to over-express the protein coupled with fusion tags in Escherichia coli BL21(DE3). The expressed HSP17.9 was purified by immobilized metal affinity chromatography (IMAC) and had its identity determined by mass spectrometry (MALDI-TOF). The correct folding of the purified recombinant protein was verified by circular dichroism spectroscopy. Finally, the HSP17.9 protein also proved to efficiently prevent induced aggregation of insulin, strongly indicating a chaperone-like activity.     

Free radical scavenging and antiproliferative properties of Biginelli adducts

A series of Biginelli adducts bearing different substituents at C-4 position were synthesized by using p-sulfonic acid calix[4]arene as a catalyst. The in vitro potential to scavenge reactive nitrogen/oxygen species (RNS and ROS) and the ability to inhibit cancer cells growth were then investigated. Four adducts were found to be potent scavengers of 2,2-diphenyl-1-picrylhydrazyl (RNS) and/or superoxide anion (ROS) radicals. The antiproliferative activity against cancer cells was disclosed for the first time for 16 monastrol analogs. The capacity of all compounds to inhibit cancer cells growth was dependent on the histological origin of cells, except for BA24, which was highly active against all cell lines. BA20 and BA33 were as potent as the reference drug doxorubicin against adriamycin-resistant ovarian and prostate cancer cells, respectively. These results highlight some monastrol analogs as lead compounds for the design of new free radical scavengers and anticancer agents.     

Imported submicroscopic malaria in Madrid

ABSTRACT: BACKGROUND: Submicroscopic malaria (SMM) can be defined as low-density infections of Plasmodium that are unlikely to be detected by conventional microscopy. Such submicroscopic infections only occasionally cause acute disease, but they are capable of infecting mosquitoes and contributing to transmission. This entity is frequent in endemic countries; however, little is known about imported SMM. The goals of this study were two-fold: a) to know the frequency of imported SMM, and b) to describe epidemiological, laboratorial and clinical features of imported SMM. METHODS: A retrospective study based on review of medical records was performed. The study population consisted of patients older than 15 years attended at the Tropical Medicine Unit of Hospital Carlos III, between January 1, 2002 and December 31, 2007. Routinely detection techniques for Plasmodium included Field staining and microscopic examination through thick and thin blood smear. A semi-nested multiplex malaria PCR was used to diagnose or to confirm cases with low parasitaemia. RESULTS: SMM was diagnosed in 104 cases, representing 35.5% of all malaria cases. Mean age (IC95%) was 40.38 years (37.41-43.34), and sex distribution was similar. Most cases were in immigrants, but some cases were found in travellers. Equatorial Guinea was the main country where infection was acquired (81.7%). Symptoms were present only in 28.8% of all SMM cases, mainly asthenia (73.3% of symptomatic patients), fever (60%) and arthromialgias (53.3%). The associated laboratory abnormalities were anaemia (27.9%), leukopaenia (15.4%) and thrombopaenia (15.4%). Co-morbidity was described in 75 cases (72.1%). CONCLUSIONS: Results from this study suggest that imported SMM should be considered in some patients attended at Tropical Medicine Units. Although it is usually asymptomatic, it may be responsible of fever, or laboratory abnormalities in patients coming from endemic areas. The possibility of transmission in SMM has been previously described in endemic zones, and presence of vector in Europe has also been reported. Implementation of molecular tests in all asymptomatic individuals coming from endemic area is not economically feasible. So reemergence of malaria (Plasmodium vivax) in Europe may be speculated.     

Chemical Composition of Magonia pubescens Essential Oils and Gamma-Radiation Effects on Its Constituents and Cytotoxic Activity in Leukemia and Breast Cancer Model

Magonia pubescens A. St.-Hil. is a Brazilian species often used in ethnopharmacology for wound and pain healing and seborrhea treatment. For the first time, essential oils (EOs) obtained from M. pubescens inflorescences were studied. The plant materials (Montes Claros, Brazil, 2018) were submitted to different gamma-radiation doses and their chemical compositions were analyzed by GC/MS and GC-FID. The cytotoxic activity of the EOs was evaluated against K562 and MDA-MB-231 cancer cell lines. A total of 30 components were identified, being 24 compounds detected for the first time in M. pubescens. The main obtained components were hotrienol (35.9 %), cis-linalool oxide (17.0 %) and trans-linalool oxide (10.2 %). The chemical composition of the EO was slightly affected by the applied radiation doses. Irradiated and non-irradiated EOs showed cytotoxic activity against both cell lines and the non-irradiated EO sample was the most active against the K562 cell lines (IC₅₀=22.10±1.98).     

Functional Electrospun Poly (Lactic Acid) Scaffolds for Biomedical Applications: Experimental Conditions,Degradation and Biocompatibility Study

The electrospinning technique is a method used to produce nano and microfibers using the influence of electrostatic forces. Porous three dimensional networks of continuous and interconnected fibers as scaffolds were obtained from a poly (lactic acid) solution. The concentration of the polymeric solution, 12.5% m/w, as well as the conditions of voltage (V=11kV) and tip-metallic collector distance (H=13cm) were established to develop these scaffolds through the electrospinning process. The characteristics of the scaffolds, such as fiber diameter, sintering and the biomimetics of the characteristics of a native extra cellular matrix were verified by Scanning Electron Microscopy (SEM). The orientation induced in the material as a consequence of the electrospinning forces was studied by Differential Scanning Calorimetry (DSC) and X-Ray Diffraction (XRD).The same techniques were used to study the hydrolytic degradation of samples in a ringer solution (pH=7-7.4 at 37oC) for 12 weeks and showed evidences of superficial degradation on the microfibers. The suitability of these scaffolds for tissue engineering was studied through the primary cell culture of chondrocytes, by observing adhesion and cellular proliferation developed during 14 days of assay.     

Changes in water status and osmolyte contents in leaves and roots of olive plants (Olea europaea L.) subjected to water deficit

Two-year-old olive trees (Olea europaea L., cv. Coratina) were subjected to a 15-day period of water deficit, followed by 12 days of rewatering. Water deficit caused decreases in predawn leaf water potential (Ψ_w), relative water content and osmotic potential at full turgor (Ψ _π100) of leaves and roots, which were normally restored upon the subsequent rewatering. Extracts of leaves and roots of well-watered olive plants revealed that the most predominant sugars are mannitol and glucose, which account for more than 80% of non-structural carbohydrates and polyols. A marked increase in mannitol content occurred in tissues of water-stressed plants. During water deficit, the levels of glucose, sucrose and stachyose decreased in thin roots (with a diameter <1 mm), whereas medium roots (diameter of 1–5 mm) exhibited no differences. Inorganic cations largely contribute to Ψ _π100 and remained stable during the period of water deficit, except for the level of Ca²⁺, which increased of 25% in water-stressed plants. The amount of malate increased in both leaves and roots during the dry period, whereas citrate and oxalate decreased. Thin roots seem to be more sensitive to water deficit and its consequent effects, while medium roots present more reactivity and a higher osmotic adjustment. The results support the hypothesis that the observed decreases in Ψ_w and active osmotic adjustment in leaves and roots of water-stressed olive plants may be physiological responses to tolerate water deficit.     

Employing phage display to study the mode of action of Bacillus thuringiensis Cry toxins

Phage display is an in vitro method for selecting polypeptides with desired properties from a large collection of variants. The insecticidal Cry toxins produced by Bacillus thuringiensis are highly specific to different insects. Various proteins such as cadherin, aminopeptidase-N (APN) and alkaline phosphatase (ALP) have been characterized as potential Cry-receptors. We used phage display to characterize the Cry toxin-receptor interaction(s). By employing phage-libraries that display single-chain antibodies (scFv) from humans or from immunized rabbits with Cry1Ab toxin or random 12-residues peptides, we have identified the epitopes that mediate binding of lepidopteran Cry1Ab toxin with cadherin and APN receptors from Manduca sexta and the interaction of dipteran Cry11Aa toxin with the ALP receptor from Aedes aegypti. Finally we displayed in phages the Cry1Ac toxin and discuss the potential for selecting Cry variants with improved toxicity or different specificity.