Bioprospecting of Saprobe Fungi from the Semi‐Arid North‐East of Brazil for the Control of Anthracnose on Sorghum

Anthracnose, caused by the hemiotrophic fungus Colletotrichum sublineolum, is one of the most important diseases affecting sorghum production worldwide. The main goal of this study was to select saprobe fungi from the semi‐arid north‐east of Brazil that could increase sorghum resistance to anthracnose and investigate this increased resistance at both physiological and biochemical levels. Plants were sprayed with Curvularia inaequalis, Gonytrichum macroladum, Memnoniella levispora, Pithomyces chartarum, Periconia hispidula, Phaeoisaria clematidia, Dictyochaeta heteroderae, Sarcopodium circinatum, Periconia byssoides, Moorella speciosa, Stachybotrys chartarum, Pseudobotrytis terrestres, Memnoniella echinata, Stachybotrys globosa and Gonytrichum clamydosporium 24 h before inoculation with C. sublineolum. Plants sprayed with water served as the control treatment. The area under the anthracnose progress curve was significantly reduced in comparison with the control treatment only for plants sprayed with C. inaequalis. Therefore, C. inaequalis was selected for physiological and biochemical evaluations. The peroxidases, chitinases and β‐1,3‐glucanases activities were significantly higher for plants sprayed with C. inaequalis and inoculated with C. sublineolum than for plants not sprayed with C. inaequalis and inoculated with C. sublineolum. There was no apparent decrease in the values of net carbon assimilation rate, stomatal conductance to water vapour or transpiration rate for plants sprayed with C. inaequalis and infected by C. sublineolum in comparison with plants not sprayed with C. inaequalis and infected by C. sublineolum. In conclusion, sorghum resistance against C. sublineolum infection was greatly potentiated by C. inaequalis without any apparent change in the photosynthetic capacity of the infected plants.     

Identification of NF-κB and PLCL2 as new susceptibility genes and highlights on a potential role of IRF8 through interferon signature modulation in systemic sclerosis

IntroductionSystemic sclerosis (SSc) and primary biliary cirrhosis (PBC) are rare polygenic autoimmune diseases (AIDs) characterized by fibroblast dysfunction. Furthermore, both diseases share some genetic bases with other AIDs, as evidenced by autoimmune gene pleiotropism. The present study was undertaken to investigate whether single-nucleotide polymorphisms (SNPs) identified by a large genome-wide association study (GWAS) in PBC might contribute to SSc susceptibility.MethodsSixteen PBC susceptibility SNPs were genotyped in a total of 1,616 patients with SSc and 3,621 healthy controls from two European populations (France and Italy).ResultsWe observed an association between PLCL2 rs1372072 (odds ratio (OR) = 1.22, 95% confidence interval (CI) 1.12 to 1.33, P_adj = 7.22 × 10⁻⁵), nuclear factor-kappa-B (NF-κB) rs7665090 (OR = 1.15, 95% CI 1.06 to 1.25, P_adj = 0.01), and IRF8 rs11117432 (OR = 0.75, 95% CI 0.67 to 0.86, P_adj = 2.49 × 10⁻⁴) with SSc susceptibility. Furthermore, phenotype stratification showed an association between rs1372072 and rs11117432 with the limited cutaneous subgroup (lcSSc) (P_adj = 4.45 × 10⁻⁴ and P_adj = 0.001), whereas rs7665090 was associated with the diffuse cutaneous subtype (dcSSc) (P_adj = 0.003). Genotype-mRNA expression correlation analysis revealed that the IRF8 protective allele was associated with increased interferon-gamma (IFN-γ) expression (P = 0.03) in patients with SSc but decreased type I IFN (IFIT1) expression in patients and controls (P = 0.02). In addition, we found an epistatic interaction between NF-κB and IRF8 (OR = 0.56, 95% CI 0.00 to 0.74, P = 4 × 10⁻⁴) which in turn revealed that the IRF8 protective effect is dependent on the presence of the NF-κB susceptibility allele.ConclusionsAn analysis of pleiotropic genes identified two new susceptibility genes for SSc (NF-κB and PLCL2) and confirmed the IRF8 locus. Furthermore, the IRF8 variant influenced the IFN signature, and we found an interaction between IRF8 and NF-κB gene variants that might play a role in SSc susceptibility.

Electronic supplementary material

The online version of this article (doi:10.1186/s13075-015-0572-y) contains supplementary material, which is available to authorized users.     

BALB/c mice infected with antimony treatment refractory isolate of Leishmania braziliensis present severe lesions due to IL-4 production

Background

Leishmania braziliensis is the main causative agent of cutaneous leishmaniasis in Brazil. Protection against infection is related to development of Th1 responses, but the mechanisms that mediate susceptibility are still poorly understood. Murine models have been the most important tools in understanding the immunopathogenesis of L. major infection and have shown that Th2 responses favor parasite survival. In contrast, L. braziliensis–infected mice develop strong Th1 responses and easily resolve the infection, thus making the study of factors affecting susceptibility to this parasite difficult.

Methodology/Principal Findings

Here, we describe an experimental model for the evaluation of the mechanisms mediating susceptibility to L. braziliensis infection. BALB/c mice were inoculated with stationary phase promastigotes of L. braziliensis, isolates LTCP393(R) and LTCP15171(S), which are resistant and susceptible to antimony and nitric oxide (NO), respectively. Mice inoculated with LTCP393(R) presented larger lesions that healed more slowly and contained higher parasite loads than lesions caused by LTCP15171(S). Inflammatory infiltrates in the lesions and production of IFN-γ, TNF-α, IL-10 and TGF-β were similar in mice inoculated with either isolate, indicating that these factors did not contribute to the different disease manifestations observed. In contrast, IL-4 production was strongly increased in LTCP393(R)-inoculated animals and also arginase I (Arg I) expression. Moreover, anti-IL-4 monoclonal antibody (mAb) treatment resulted in decreased lesion thickness and parasite burden in animals inoculated with LTCP393(R), but not in those inoculated with LTCP15171(S).

Conclusion/Significance

We conclude that the ability of L. braziliensis isolates to induce Th2 responses affects the susceptibility to infection with these isolates and contributes to the increased virulence and severity of disease associated with them. Since these data reflect what happens in human infection, this model could be useful to study the pathogenesis of the L. braziliensis infection, as well as to design new strategies of therapeutic intervention.     

The Vesicular Acetylcholine Transporter Is Required for Neuromuscular Development and Function

The vesicular acetylcholine (ACh) transporter (VAChT) mediates ACh storage by synaptic vesicles. However, the VAChT-independent release of ACh is believed to be important during development. Here we generated VAChT knockout mice and tested the physiological relevance of the VAChT-independent release of ACh. Homozygous VAChT knockout mice died shortly after birth, indicating that VAChT-mediated storage of ACh is essential for life. Indeed, synaptosomes obtained from brains of homozygous knockouts were incapable of releasing ACh in response to depolarization. Surprisingly, electrophysiological recordings at the skeletal-neuromuscular junction show that VAChT knockout mice present spontaneous miniature end-plate potentials with reduced amplitude and frequency, which are likely the result of a passive transport of ACh into synaptic vesicles. Interestingly, VAChT knockouts exhibit substantial increases in amounts of choline acetyltransferase, high-affinity choline transporter, and ACh. However, the development of the neuromuscular junction in these mice is severely affected. Mutant VAChT mice show increases in motoneuron and nerve terminal numbers. End plates are large, nerves exhibit abnormal sprouting, and muscle is necrotic. The abnormalities are similar to those of mice that cannot synthesize ACh due to a lack of choline acetyltransferase. Our results indicate that VAChT is essential to the normal development of motor neurons and the release of ACh.Cholinergic neurotransmission has key functions in life, as it regulates several central and peripheral nervous system outputs. Acetylcholine (ACh) is synthesized in the cytoplasm by the enzyme choline acetyltransferase (ChAT) (16). Choline supplied by the high-affinity choline transporter (CHT1) is required to maintain ACh synthesis (52). A lack of ChAT (4, 35) or the high-affinity choline transporter (21) in genetically modified mice is incompatible with life. ACh plays an important role in wiring the neuromuscular junction (NMJ) during development (38, 43). Embryonic synthesis of ACh is fundamental for the development of proper nerve-muscle patterning at the mammalian NMJ, as ChAT-null mice present aberrant nicotinic ACh receptor (nAChR) localization and increased motoneuron (MN) survival, axonal sprouting, and branching (4, 35).The vesicular ACh transporter (VAChT) exchanges cytoplasmic ACh for two vesicular protons (37, 41). Previously reported electrophysiological studies showed that quantal size is decreased by vesamicol, an inhibitor of VAChT, but only in nerve terminals that have been electrically stimulated (19, 59, 60, 63). VAChT overexpression in developing Xenopus MNs increases both the size and frequency of miniature-end-plate currents (54). In Caenorhabditis elegans, mutations in VAChT affect behavior (65). Moreover, a decrease in VAChT expression has functional consequences for mammals, as mutant mice with a 70% reduction in the expression levels of this transporter (VAChT knockdown [KD^HOM] mice) are myasthenic and have cognitive deficits (47). Hence, vesicular transport activity is rate limiting for neurotransmission “in vivo” (18, 47).Exocytosis of synaptic vesicle contents is the predominant mechanism for the regulated secretion of neurotransmitters (55). However, alternative mechanisms of secretion have been proposed (20, 56, 61). Quantal ACh release, comparable to that seen in developing nerve terminals, has been detected in myocytes and fibroblasts in culture, which presumably do not express VAChT (14, 24). More recently, it was found that the correct targeting of Drosophila photoreceptor axons is disrupted in flies with null mutations in ChAT (64). Remarkably, the inactivation of VAChT did not produce the same result (64). The result suggests that the release of ACh during development is not dependent on VAChT, perhaps because it is nonvesicular or because vesicular storage can occur without VAChT.To test if the VAChT-independent secretion of ACh has any physiological role in the mammalian nervous system, we generated a mouse line in which the VAChT gene is deleted. These mice lack the stimulated release of ACh from synaptosomes, die after birth, and show several alterations in neuromuscular wiring consistent with a severe decrease in the cholinergic input to muscles during development. These experiments indicate that VAChT has an important role in maintaining activity-dependent ACh release that supports life and the correct patterning of innervation at the NMJ.     

Complications after breast reconstruction with alloplastic material in breast cancer patients submitted or not to post mastectomy radiotherapy

Background and purposeBreast reconstruction following mastectomy is a relevant element of breast cancer treatment. The purpose of this study was to evaluate the influence of radiotherapy (RT) on local complications in patients with breast cancer that had undergone breast reconstruction with alloplastic material.Materials and methodsRetrospective study of breast cancer patients submitted to mastectomy and breast reconstruction from 2009 to 2013. Clinical and treatment variables were correlated with early and late complications.Results251 patients were included; mean age was 49.7 (25 to 78) years. Reconstruction was immediate in 94% of the patients, with 88% performed with a temporary tissue expander. Postoperative radiotherapy (RT) was delivered to 167 patients (66.5%). Early complications were present in 26.3% of the patients. Irradiated patients presented 5.4% incidence of late complications versus 2.4% for non-irradiated patients (p = 0.327). Diabetes (OR = 3.41 95% CI: 1.23–9.45, p = 0.018) and high body mass index (BMI) (OR = 2.65; 95% CI: 1.60–4.37, p < 0.0001) were the main risk factors. The overall incidence of late complications was 4.4%, with predominance of severe capsular contracture (8/11). Arterial hypertension (OR = 4.78; 95% CI: 1.97–11.63, p = 0.001), BMI (OR = 0.170; 95% CI: 0.048–0.607, p = 0.006) and implant placement (OR = 3.55; 95% CI: 1.26–9.99, p = 0.016) were related to late complications.ConclusionsThe overall rate of complications was low in this population. Radiotherapy delivery translated into a higher but not statistically significant risk of late complications when compared with the non-irradiated patients. Already well-known clinical risk factors for complications after breast reconstruction were identified.     

The effects of intraspinal L-NOARG or SIN-1 on the control by descending pathways of incisional pain in rats

The modulation by spinal nitric oxide (NO) of descending pathways travelling through the dorsal lateral funiculus (DLF) is a mechanism proposed for the antinociceptive effects of drugs that changes the NO metabolism. In this study we confirm that a surgical incision in the mid-plantar hind paw of rats reduces the threshold to mechanical stimulation with von Frey filaments. The incisional pain was further increased in rats with ipsilateral DLF lesion. Intrathecal L-NOARG (50-300 microg), or SIN-1 (0.1-5.0 microg) reduced, while SIN-1 (10 and 20 microg) intensified the incisional pain in rats with sham or effective lesion of the DLF. Stimulation of the dorsal raphe (DRN) or anterior pretectal (APtN) nuclei with stepwise increased electrical currents (7, 14, 21, 28 and 35 microA r.m.s.) produced a current-related reduction of the incisional pain. These nuclei activate pain inhibitory pathways that descend to the spinal cord mainly through the DLF. Intrathecal SIN-1 (5 microg) reduced, SIN-1 (20 microg) decreased and L-NOARG (150 microg) did not change the EC50 for the DRN or APtN stimulation-induced reduction of incisional pain. We conclude that the antinociceptive effects of L-NOARG or low doses of SIN-1 are independent on the activity of descending pain control pathways travelling via the DLF, but the antinociceptive effect of stimulating electrically the DRN or APtN can be summated to the effect of low dose of SIN-1 or overcome by the high dose of SIN-1.     

Cystyl aminopeptidase activity in the plasma, viscera and brain of the snake Bothrops jararaca

The relationship between plasma osmolality and cystyl aminopeptidase was characterized in the snake Bothrops jararaca and comparisons were made with the emerging picture of this relationship in rats. The profile of cystyl aminopeptidase activity under basal conditions was determined in the soluble and membrane-bound forms in visceral organs and in the central nervous system in comparison with that of alanyl aminopeptidase. The regional localization of cystyl and alanyl aminopeptidase activities was studied in the central nervous system. The basal level of plasma cystyl aminopeptidase, four- to six-fold higher than in rats, suggests its importance to help regulate circulating levels of neurohypophysial peptides in B. jararaca snake. The osmotic sensitivity of this plasma enzyme, undetectable in male, but about three-fold higher in female snakes than in rats, reveals a sexual dimorphism. In marked contrast to those observed in rats, low levels of soluble and particulate forms in the kidney indicate that cystyl aminopeptidase plays a minor metabolizing role at this anatomical location in B. jararaca. Despite of the regional-specific divergence between the levels of rat and snake enzymes, the bilaterally symmetric pattern of the diencephalic distribution of alanyl aminopeptidase reflects functional homologies between these two distantly related species.     

Aqueous extraction of recombinant human proinsulin from transgenic maize endosperm

Different plant species have been used as systems to produce recombinant proteins. Maize is a crop considered to have a large potential to produce high levels of recombinant proteins and is the host for the recombinant proteins from plants currently available on the market. In the development of a plant system to produce a recombinant proteins it is important to consider the costs related to downstream processing. Also, the steps necessary to achieve the protein purity required will be highly influenced by the quality of the extract obtained. In this study, we analyzed aqueous extracts from the endosperm of transgenic maize expressing recombinant human proinsulin (rhProinsulin). A study of the effects of the variables pH and ionic strength on the extraction efficiency was carried out using experimental design and response surface methodology. Besides the concentration of the recombinant protein, the characteristics of the extracts were evaluated in terms of concentration of native components (proteins, carbohydrates, and phenolic compounds) and extract filterability. The highest rhProinsulin concentration (97.33 ng/mL) was found with a 200 mM NaCl pH 10.0 extraction solution. Under this experimental condition the concentrations of total soluble proteins, carbohydrates, and phenolics were 2.01 mg/mL, 2.21 mg/mL, and 0.11 mmol/L, respectively.     

Transfection of mammalian cells with connexins and measurement of voltage sensitivity of their gap junctions

Vertebrate gap junction channels are formed by a family of more than 20 connexin proteins. These gap junction proteins are expressed with overlapping cellular and tissue specificity, and coding region mutations can cause human hereditary diseases. Here we present a summary of what has been learned from voltage clamp studies performed on cell pairs either endogenously expressing gap junctions or in which connexins are exogenously expressed. General protocols presented here are currently used to transfect mammalian cells with connexins and to study the biophysical properties of the heterologously expressed connexin channels. Transient transfection is accomplished overnight with maximal expression occurring at about 36 h; stable transfectants normally can be generated within three or four weeks through colony selection. Electrophysiological protocols are presented for analysis of voltage dependence and single-channel conductance of gap junction channels as well as for studies of chemical gating of these channels.     

Increased corticosterone levels in mice subjected to the rat exposure test

In recent years, there has been a notable interest in studying prey-predator relationships to develop rodent-based models for the neurobehavioral aspects of stress and emotion. However, despite the growing use of transgenic mice and results showing important differences in the behavioral responses of rats and mice, little research has been conducted regarding the responses of mice to predators. The rat exposure test (RET), a recently developed and behaviorally validated prey-predator (mouse-rat)-based model, has proven to be a useful tool in evaluating the defensive responses of mice facing rats. To further validate the RET, we investigated the endocrine and behavioral responses of mice exposed to this apparatus. We first constructed a plasma corticosterone secretion curve in mice exposed to a rat or to an empty cage (control). Rat-exposed mice showed a pronounced rise in corticosterone levels that peaked 15 min from the beginning of the predator exposure. The corticosterone levels and behavioral responses of mice exposed to a rat or to a toy in the RET apparatus were then measured. We observed high plasma corticosterone levels along with clear avoidance behaviors represented by decreases in tunnel and surface area exploration and increases in risk assessment behaviors and freezing. This strongly suggests that the test elicits a repertoire of behavioral responses compatible with an aversion state and indicates that it is a promising model for the evaluation of prey-predator interactions. However, more physiological, neurochemical, and pharmacological studies are needed to further validate the test.