Isolation and characterization of an anti‐leishmanial disintegrin from Cerastes cerastes venom

Investigating new antimicrobial and antiparasitic components from Viperidae venoms represents an alternative therapeutic strategy. In this study, we report the characterization of a disintegrin isolated from Cerastes cerastes venom, exhibiting antiparasitic activity on Leishmania infantum promastigotes. Indeed, isolated disintegrin, referred to Disintegrin_Cc, induced 84.75% of parasiticidal activity and deep morphological alterations on the parasites. SDS‐PAGE analysis indicated that this disintegrin was homogenous. This dimeric disintegrin of 14,193.97 Da contains an RGD domain and four intramolecular disulfide bridges. It presents a high percentage of identity with other related snake disintegrins. Predicted 3D structure indicated that this peptide shares partial homology with well‐known active antimicrobial peptides. Disintegrin_Cc inhibited 80% of arachidonic acid‐induced platelet aggregation. The obtained results suggest that the isolated molecule plays a dual role as a disintegrin and as an anti‐leishmanial compound. This component could be useful as a drug in the treatment of leishmaniasis.     

Contrasting effects of necrotrophic and biotrophic plant pathogens on the aphid Aphis fabae

Phytophagous insects have to contend with a wide variation in food quality brought about by a variety of factors intrinsic and extrinsic to the plant. One of the most important factors is infection by plant pathogenic fungi. Necrotrophic and biotrophic plant pathogenic fungi may have contrasting effects on insect herbivores due to their different infection mechanisms and induction of different resistance pathways, although this has been little studied and there has been no study of their combined effect. We studied the effect of the biotrophic rust fungus Uromyces viciae‐fabae (Pers.) Schroet (Basidiomycota: Uredinales: Pucciniaceae) and the necrotrophic fungus Botrytis cinerea Pers. (Ascomycota: Helotiales: Sclerotiniaceae) singly and together on the performance of the aphid Aphis fabae Scopoli (Hemiptera: Aphididae) on Vicia faba (L.) (Fabaceae). Alone, botrytis had an inhibitory effect on individual A. fabae development, survival, and fecundity, whereas rust infection consistently enhanced individual aphids' performance. These effects varied in linear relation to lesion or pustule density. However, whole‐plant infection by either pathogen resulted in a smaller aphid population of smaller aphids than on uninfected plants, indicating a lowering of aphid carrying capacity with infection. When both fungi were applied simultaneously to a leaf they generally cancelled the effect of each other out, resulting in most performance parameters being similar to the controls, although fecundity was reduced. However, sequential plant infection (pathogens applied 5 days apart) led to a 70% decrease in fecundity and 50% reduction in intrinsic rate of increase. The application of rust before botrytis had a greater inhibitory effect on aphids than applying botrytis before rust. Rust infection increased leaf total nitrogen concentration by 30%, whereas infection by botrytis with or without rust led to a 38% decrease. The aphids' responses to the two plant pathogens individually is consistent with the alteration in plant nutrient content by infection and also the induction of different plant defence pathways and the possible cross‐talk between them. This is the first demonstration of the complex effects of the dual infection of a plant by contrasting pathogens on insect herbivores.     

Airborne Ganoderma basidiospores in a country with desert environment

Aerobiological studies to identify Ganoderma basidiospores were conducted using Burkard Volumetric 7-Day Recording Sampler (Burkard Manufacturing Co. Ltd., England) at three separate cities in Saudi Arabia. At one site, Jizan, close to the coast of Red Sea, up to 17% of all basidiospores counted were identified as Ganoderma spp. while less than 1% Ganoderma spp. were identified at the two non-coastal sites. A clear seasonal pattern from late autumn to early summer (October-March) with a peak in December was recorded at Jizan and the maximum concentration of Ganoderma basidiospores reached 1.9×10³?m^?3 in December followed by 1.2×10³?m^?3 in January. The diurnal pattern of Ganoderma spore concentrations, when averaged over the year had late-evening maxima (a nocturnal pattern). However, other sites that showed low concentrations of Ganoderma basidiospores did not exhibit any peak or a high maximal level. The study demonstrates that even in a desert environment, airborne activities of Ganoderma basidiospores can be recorded. The impact of Ganoderma on asthmatic patients, particularly in such environments, needs to be investigated.     

Madurella Mycetomatis as an Agent of Brain Abscess: Case Report and Review of Literature

Fungal cerebral abscesses are rare and usually seen in immunocompromised individuals. We report a case and review published literature of Madurella mycetomatis as an agent of cerebral abscess. We found contiguous head and neck infections to be the principal cause of cerebral maduromycosis caused by M. mycetomatis. Early recognition of Madurella spp. as the causative agent is essential to avoid cerebral spread.     

Inhibitory effect of tumor necrosis factor α on gluconeogenesis in perfused rat liver

Tumor necrosis factor α (TNFα) is a cytokine involved in many metabolic responses in both normal and pathological states. Considering that the effects of TNFα on hepatic gluconeogenesis are inconclusive, we investigated the influence of this cytokine in gluconeogenesis from various glucose precursors. TNFα (10 μg/kg) was intravenously injected in rats; 6 h later, gluconeogenesis from alanine, lactate, glutamine, glycerol, and several related metabolic parameters were evaluated in situ perfused liver. TNFα reduced the hepatic glucose production (p < 0.001), increased the pyruvate production (p < 0.01), and had no effect on the lactate and urea production from alanine. TNFα also reduced the glucose production (p < 0.01), but had no effect on the pyruvate production from lactate. In addition, TNFα did not alter the hepatic glucose production from glutamine nor from glycerol. It can be concluded that the TNFα inhibited hepatic gluconeogenesis from alanine and lactate, which enter in gluconeogenic pathway before the pyruvate carboxylase step, but not from glutamine and glycerol, which enter in this pathway after the pyruvate carboxylase step, suggesting an important role of this metabolic step in the changes mediated by TNFα.     

Preferential HIV Infection of CCR6+ Th17 Cells Is Associated with Higher Levels of Virus Receptor Expression and Lack of CCR5 Ligands

Th17 cells are enriched in the gut mucosa and play a critical role in maintenance of the mucosal barrier and host defense against extracellular bacteria and fungal infections. During chronic human immunodeficiency virus (HIV) infection, Th17 cells were more depleted compared to Th1 cells, even when the patients had low or undetectable viremia. To investigate the differential effects of HIV infection on Th17 and Th1 cells, a culture system was used in which CCR6⁺ CD4⁺ T cells were sorted from healthy human peripheral blood and activated in the presence of interleukin 1β (IL-1β) and IL-23 to drive expansion of Th17 cells while maintaining Th1 cells. HIV infection of these cultures had minimal effects on Th1 cells but caused depletion of Th17 cells. Th17 loss correlated with greater levels of virus-infected cells and cell death. In identifying cellular factors contributing to higher susceptibility of Th17 cells to HIV, we compared Th17-enriched CCR6⁺ and Th17-depleted CCR6⁻ CD4 T cell cultures and noted that Th17-enriched CCR6⁺ cells expressed higher levels of α4β7 and bound HIV envelope in an α4β7-dependent manner. The cells also had greater expression of CD4 and CXCR4, but not CCR5, than CCR6⁻ cells. Moreover, unlike Th1 cells, Th17 cells produced little CCR5 ligand, and transfection with one of the CCR5 ligands, MIP-1β (CCL4), increased their resistance against HIV. These results indicate that features unique to Th17 cells, including higher expression of HIV receptors and lack of autocrine CCR5 ligands, are associated with enhanced permissiveness of these cells to HIV.     

Employing in vitro directed molecular evolution for the selection of α-amylase variant inhibitors with activity toward cotton boll weevil enzyme

Numerous species of insect pests attack cotton plants, out of which the cotton boll weevil (Anthonomus grandis) is the main insect in Brazil and must be controlled to avert large economic losses. Like other insect pests, A. grandis secretes a high level of α-amylases in the midgut lumen, which are required for digestion of carbohydrates. Thus, α-amylase inhibitors (α-AIs) represent a powerful tool to apply in the control of insect pests. Here, we applied DNA shuffling and phage display techniques and obtained a combinatorial library containing 10⁸α-AI variant forms. From this library, variants were selected exhibiting in vitro affinity for cotton boll weevil α-amylases. Twenty-six variant sequences were cloned into plant expression vectors and expressed in Arabidopsis thaliana. Transformed plant extracts were assayed in vitro to select specific and potent α-amylase inhibitors against boll weevil amylases. While the wild type inhibitors, used to create the shuffled library, did not inhibit the A. grandis α-amylases, three α-AI mutants, named α-AIC3, α-AIA11 and α-AIG4 revealed high inhibitory activities against A. grandis α-amylases in an in vitro assay. In summary, data reported here shown the potential biotechnology of new α-AI variant genes for cotton boll weevil control.     

Amplification of GC-rich DNA for High-Throughput Family-Based Genetic Studies

Researchers face a significant problem in PCR amplification of DNA fragments with high GC contents. Analysis of these regions is of importance since many regulatory regions of different genes and their first exons are GC-rich. There are a large number of protocols for amplification of GC-rich DNA, some of which perform well but are costly. Most of the economical protocols fail to perform consistently, especially on products with >80 % GC contents and a size of >300 bp. One of these protocols requires multiple additions of DNA polymerase during thermal cycling which therefore rules out its utility if a large number of samples have to be amplified. We have established a method for simultaneous amplification of specific PCR products from a large number of human DNA samples using general laboratory reagents. These amplicons have GC contents ranging from 65–85 % and sizes up to 870 bp. The protocol uses a PCR buffer containing co-solvents including 2-mercaptoethanol and bovine serum albumin for amplification of DNA. A specific thermal cycling profile is also used which incorporates a high annealing temperature in the first 7 cycles of the reactions. The PCR products are suitable for different molecular biology applications including sequencing.