Agrobacterium-mediated transformation of Guignardia citricarpa: An efficient tool to gene transfer and random mutagenesis

Guignardia citricarpa is the causal agent of Citrus Black Spot (CBS), an important disease in Citriculture. Due to the expressive value of this activity worldwide, especially in Brazil, understanding more about the functioning of this fungus is of utmost relevance, making possible the elucidation of its infection mechanisms, and providing tools to control CBS. This work describes for the first time an efficient and successful methodology for genetic transformation of G. citricarpa mycelia, which generated transformants expressing the gene encoding for the gfp (green fluorescent protein) and also their interaction with citrus plant. Mycelia of G. citricarpa were transformed via Agrobacterium tumefaciens, which carried the plasmid pFAT-gfp, contains the genes for hygromycin resistance (hph) as well as gfp. The optimization of the agrotransformation protocol was performed testing different conditions (type of membrane; inductor agent concentration [acetosyringone – AS] and cocultivation time). Results demonstrated that the best condition occurred with the utilization of cellulose's ester membrane; 200 μM of AS and 96 h as cocultivation time. High mitotic stability (82 %) was displayed by transformants using Polymerase Chain Reaction (PCR) technique to confirm the hph gene insertion. In addition, the presence of gfp was observed inside mycelia by epifluorescence optical microscopy. This technique easy visualization of the behaviour of the pathogen interacting with the plant for the first time, allowing future studies on the pathogenesis of this fungus. The establishment of a transformation method for G. citricarpa opens a range of possibilities and facilitates the study of insertional mutagenesis and genetic knockouts, in order to identify the most important genes involved in the pathogenesis mechanisms and plant–pathogen interaction.     

Abundance of Lutzomyia longipalpis (Diptera: Psychodidae: Phlebotominae) and urban transmission of visceral leishmaniasis in Campo Grande, state of Mato Grosso do Sul, Brazil

The outspread and urbanization of visceral leishmaniasis (VL) in Campo Grande, state of Mato Grosso do Sul, lead us to undertake the present study over diversity and abundance of sand flies in the urban area to compare with previous search carried out during 1999/2000, before the identification of the disease in the human population.The captures were carried out with automatic light traps, weekly, from February 2004 to February 2005 on three sites including a forested area (Zé Pereira), two peridomicilies (shelters of domestic animals and cultivation areas), and intradomicilie. In the present study 110 collections were obtained during 13 months for 1320 h of collections, resulting in 5004 specimens, 3649 males and 1355 females belonging to the 20 following species: Brumptomyia avellari, Brumptomyia sp., Bichromomyia flaviscutellata, Evandromyia lenti, E. termitophila, E. cortelezzii, E. borrouli, Lutzomyia sp., L. longipalpis, Micropygomyia quinquefer, N. antunesi, N. whitmani, Pintomyia christenseni, Pi. damascenoi, Psathyromyia aragaoi, Ps. campograndensis, Ps. hermanlenti, Ps. shannoni, Pychodopygus claustrei, and Sciopemyia sordellii. L. longipalpis was the most abundant species in the anthropic environment with 92.22% of the captures. This shows an increase of sixty times in the density of L. longipalpis compared to the last sand fly evaluation in 1999/2000. The high density of L. longipalpis in Campo Grande is the main factor of risk in transmission of the disease to human in the urban area. The capture of N. antunesi, typical specie from Amazonian region, in Mato Grosso do Sul is reported for the first time.     

Epidemiology of Invasive Fungal Infections in Patients with Acquired Immunodeficiency Syndrome at a Reference Hospital for Infectious Diseases in Brazil

Invasive fungal infections (IFIs) represent one of the main causes of morbimortality in immunocompromised patients. Pneumocystosis, cryptococcosis and histoplasmosis are the most frequently occurring IFIs in patients with acquired immunodeficiency syndrome (AIDS). Fungi, such as Candida spp. and Aspergillus spp., may cause severe diseases during the course of an HIV infection. Following the introduction of highly active anti-retroviral therapy, there has been a marked reduction of opportunistic fungal infections, which today is 20–25 % of the number of infections observed in the mid-1990s. This study is an observational and retrospective study aimed at the characterising IFI incidence and describing the epidemiology, clinical diagnostic and therapeutic features and denouement in HIV/AIDS patients. In HIV/AIDS patients, the IFI incidence is 54.3/1,000 hospitalisation/year, with a lethality of 37.7 %. Cryptococcosis represents the main opportunistic IFI in the population, followed by histoplasmosis. Nosocomial pathogenic yeast infections are caused principally by Candida spp., with a higher candidemia incidence at our institution compared to other Brazilian centres.     

Changes in calcium-dependent membrane permeability properties in mitochondria of livers from arthritic rats

The involvement of the mitochondrial permeability transition pore (PTP) in the responses of mitochondria from adjuvant-induced arthritic rats to Ca(2+) addition was investigated. The respiratory activity, the Ca(2+)-induced osmotic swelling and the electrophoretic (45)Ca(2+) uptake were evaluated in the absence and in the presence of cyclosporin A (CsA), a well-known inhibitor of the mitochondrial PTP. The Ca(2+)-induced mitochondrial permeability transition (MPT) process occurred in mitochondria from arthritic rats even in the presence of a low Ca(2+) concentration. Whereas in the normal condition, the Ca(2+)-induced uncoupling of oxidative phosphorylation and osmotic swelling was observed in the presence of 10 or 20 microM Ca(2+) concentration, in the arthritic condition, these events occurred at 1.0 microM concentration. In addition, mitochondria from arthritic rats presented an impaired ability to accumulate (45)Ca(2+). All these effects were completely prevented by the administration of CsA. The results of the present study suggest that the higher sensitivity of mitochondria from arthritic rats to Ca(2+)-induced MPT may be an important factor in the pathogenesis of the arthritis disease.     

Warming and water deficit impact leaf photosynthesis and decrease forage quality and digestibility of a C4 tropical grass

Global warming is predicted to cause more intense extreme events such as heat waves, flooding and severe droughts, producing significant effects on agriculture. In tropics, climate change will severely impact livestock production affecting water availability, forage quality and food for cattle. We investigated the isolated and combined effects of soil water deficit (wS) and + 2°C increase in canopy temperature (eT) on leaf gas exchange, chlorophyll fluorescence, carbohydrate content, forage quality and in vitro dry matter digestibility (IVDMD) of a field‐grown C4 tropical forage grass Panicum maximum Jacq. using a temperature‐free air‐controlled enhancement (T‐FACE) system. The wS and eT treatments showed no effects on photosystem II photochemistry. However, wS under ambient temperature decreased net photosynthesis rate (A), stomatal conductance (g_s) and maximum rate of carboxylation of Rubisco (V_cmax), leading to a reduced starch content in leaves. A 16% reduction in leaf dry mass (LDM) and reduction in forage quality by increasing fibers, reducing crude protein (CP) and decreasing the IVDMD was also observed by effect of wS. Warming under adequate soil moisture (eT) significantly increased LDM by 25% but reduced the forage quality, increasing the lignin content and reducing starch, CP and digestibility. The combined wSeT treatment reduced A, g_s, V_cmax and the forage quality. When compared to control, the lignin content in leaves increased by 43, 28 and 17% in wS, eT and wSeT, respectively, causing a significant reduction in IVDMD. We concluded that despite physiological mechanisms to acclimate to warming, both warming and water deficit will impair the quality and digestibility of C4 tropical pastures.     

Impacto de la actividad física sobre el control metabólico y el desarrollo de complicaciones crónicas en pacientes con diabetes mellitus tipo 1

Together with a balanced diet, regular physical activity is one of the pillars of diabetes mellitus (DM) management. Physical activity theoretically provides the same advantages in people with DM as in the general population and also has some beneficial effects in controlling metabolic factors, such as improving blood glucose levels and insulin sensitivity. In this article, we analyze the main clinical studies published to date that evaluate the impact of physical activity on metabolic control or the development of chronic complications in patients with type 1 diabetes mellitus. In conclusion, most of the evaluated studies show that regular physical activity favorably affects metabolic control in DM (or at least does not have adverse effects). However, there is insufficient information about the impact of physical activity on the development and progression of chronic complications.     

Multiple mechanisms involved in the large-spectrum therapeutic potential of cannabidiol in psychiatric disorders

Cannabidiol (CBD) is a major phytocannabinoid present in the Cannabis sativa plant. It lacks the psychotomimetic and other psychotropic effects that the main plant compound Δ⁹-tetrahydrocannabinol (THC) being able, on the contrary, to antagonize these effects. This property, together with its safety profile, was an initial stimulus for the investigation of CBD pharmacological properties. It is now clear that CBD has therapeutic potential over a wide range of non-psychiatric and psychiatric disorders such as anxiety, depression and psychosis. Although the pharmacological effects of CBD in different biological systems have been extensively investigated by in vitro studies, the mechanisms responsible for its therapeutic potential are still not clear. Here, we review recent in vivo studies indicating that these mechanisms are not unitary but rather depend on the behavioural response being measured. Acute anxiolytic and antidepressant-like effects seem to rely mainly on facilitation of 5-HT1A-mediated neurotransmission in key brain areas related to defensive responses, including the dorsal periaqueductal grey, bed nucleus of the stria terminalis and medial prefrontal cortex. Other effects, such as anti-compulsive, increased extinction and impaired reconsolidation of aversive memories, and facilitation of adult hippocampal neurogenesis could depend on potentiation of anandamide-mediated neurotransmission. Finally, activation of TRPV1 channels may help us to explain the antipsychotic effect and the bell-shaped dose-response curves commonly observed with CBD. Considering its safety profile and wide range of therapeutic potential, however, further studies are needed to investigate the involvement of other possible mechanisms (e.g. inhibition of adenosine uptake, inverse agonism at CB2 receptor, CB1 receptor antagonism, GPR55 antagonism, PPARγ receptors agonism, intracellular (Ca²⁺) increase, etc.), on CBD behavioural effects.     

Purification, characterization, and cloning of a serine proteinase inhibitor from the ectoparasite Haematobia irritans irritans (Diptera: Muscidae)

The fly Haematobia irritans irritans is one of the most important ectoparasites in cattle production, due to its ability to suck large amounts of blood. This report describes the purification and characterization of a serine proteinase inhibitor (HiTI) present in H. i. irritans head and thorax extracts. The HiTI purified by affinity chromatography on trypsin-Sepharose has a molecular mass of 7029Da by MALDI-TOF mass spectrometry. HiTI inhibited bovine trypsin, human neutrophil elastase, and a trypsin-like enzyme purified from H. i. irritans abdomen with dissociation constants of 0.57, 1.30, and 0.20nM, respectively. The HiTI partial amino acid sequence allowed its classification into the BPTI-Kunitz-type family. An HiTI cDNA fragment was cloned in the pGEMT vector using RT-PCR. The translated amino acid sequence of HiTI cDNA confirmed a unique Kunitz-type-domain protein. Our results suggest that HiTI could control some endogenous enzyme, e.g., the H. i. irritans trypsin-like protein.     

Evaluation of the catalytic specificity,biochemical properties,and milk clotting abilities of an aspartic peptidase from <Emphasis Type="Italic">Rhizomucor miehei</Emphasis>

In this study, we detail the specificity of an aspartic peptidase from Rhizomucor miehei and evaluate the effects of this peptidase on clotting milk using the peptide sequence of k-casein (Abz-LSFMAIQ-EDDnp) and milk powder. Molecular mass of the peptidase was estimated at 37 kDa, and optimum activity was achieved at pH 5.5 and 55 °C. The peptidase was stable at pH values ranging from 3 to 5 and temperatures of up 45 °C for 60 min. Dramatic reductions in proteolytic activity were observed with exposure to sodium dodecyl sulfate, and aluminum and copper (II) chloride. Peptidase was inhibited by pepstatin A, and mass spectrometry analysis identified four peptide fragments (TWSISYGDGSSASGILAK, ASNGGGGEYIFGGYDSTK, GSLTTVPIDNSR, and GWWGITVDRA), similar to rhizopuspepsin. The analysis of catalytic specificity showed that the coagulant activity of the peptidase was higher than the proteolytic activity and that there was a preference for aromatic, basic, and nonpolar amino acids, particularly methionine, with specific cleavage of the peptide bond between phenylalanine and methionine. Thus, this peptidase may function as an important alternative enzyme in milk clotting during the preparation of cheese.