Construction of a Kluyveromyces lactis ku80 − Host Strain for Recombinant Protein Production: Extracellular Secretion of Pectin Lyase and a Streptavidin–Pectin Lyase Chimera

In several organisms used for recombinant protein production, integration of the expression cassette into the genome depends on site-specific recombination. In general, the yeast Kluyveromyces lactis shows low gene-targeting efficiency. In this work, two K. lactis ku80 ^? strains defective in the non-homologous end-joining pathway (NHEJ) were constructed using a split-marker strategy and tested as hosts for heterologous gene expression. The NHEJ pathway mediates random integration of exogenous DNA into the genome, and its function depends on the KU80 gene. KU80-defective mutants were constructed using a split-marker strategy. The vectors pKLAC1/Plg1 and pKLAC1/cStpPlg1 were used to evaluate the recovered mutants as hosts for expression of pectin lyase (PNL) and the fusion protein streptavidin–PNL, respectively. The transformation efficiency of the ku80 ^? mutants was higher than the respective parental strains (HP108 and JA6). In addition, PNL secretion was detected by PNL assay in both of the K. lactis ku80 ^? strains. In HP108ku80^?/cStpPlg1 and JA6ku80^?/Plg1 cultures, the PNL extracellular specific activity was 551.48 (±38.66) and 369.04 (±66.33) U/mg protein. This study shows that disruption of the KU80 gene is an effective strategy to increase the efficiency of homologous recombination with pKLAC1 vectors and the production and secretion of recombinant proteins in K. lactis transformants.     

Common opossum (<Emphasis Type="Italic">Didelphis marsupialis</Emphasis>Linnaeus, 1758): food and medicine for people in the Amazon

Background

In the Amazon rainforest, biodiversity is a significant resource for traditional communities, as it can be used as a relevant source of protein and it has a promising zootherapeutic potential. Studies on knowledge and ways how local peoples use the fauna are still incipient. This paper presents both the knowledge on and food and medicinal uses of common opossum (Didelphis marsupialis) by riverine communities in an Amazon floodplain region.

Methods

The study was conducted with riverine communities in the municipality of Abaetetuba, Pará, Brazil. The main methods used were structured and semi-structured interviews, the “snowball” technique, and participant observation.

Results

The study showed that D. marsupialis has an undeniable cultural significance for the local community, both in terms of food and medicine. Its meat is prized by inhabitants as it is classified as tasty, soft and, in some cases, it is designated as the best bushmeat in the region. The interviewees have demonstrated a thorough knowledge on various aspects of the animal’s biology, such as its diet, behavior, and places of occurrence. The hunting activity is practiced by men, but the preparation of meat and medicinal oil are tasks mainly performed by women. In medical terms, common opossum is used in the treatment of various diseases, such as rheumatism, asthma, sore throat, and inflammation. Given the importance of this species, its meat or live individuals are often sold in the city fair at prices that can reach R$ 40.00 (U$D 18,00) per individual.

Conclusions

D. marsupialis is an important source of protein for riverine communities in the region studied. Its fat is used as a traditional medicine and it is indicated for many types of diseases. Although the species concerned is treated with hostility in various Brazilian regions, in the case of Abaetetuba this animal is strongly prized due to the good quality of its meat. However, despite the value assigned to the species, its consumption should be the subject of further studies, as this marsupial species has been described as a reservoir for parasites that cause severe diseases.

     

Differential sRNA Regulation in Leaves and Roots of Sugarcane under Water Depletion

Plants have developed multiple regulatory mechanisms to respond and adapt to stress. Drought stress is one of the major constraints to agricultural productivity worldwide and recent reports have highlighted the importance of plant sRNA in the response and adaptation to water availability. In order to increase our understanding of the roles of sRNA in response to water depletion, cultivars of sugarcane were submitted to treatment of ceasing drip irrigation for 24 hours. Deep sequencing analysis was carried out to identify the sRNA regulated in leaves and roots of sugarcane cultivars with different drought sensitivities. The pool of sRNA selected allowed the analysis of different sRNA classes (miRNA and siRNA). Twenty-eight and 36 families of conserved miRNA were identified in leaf and root libraries, respectively. Dynamic regulation of miRNA was observed and the expression profiles of eight miRNA were verified in leaf samples from three biological replicates by stem-loop qRT-PCR assay using the cultivars: SP90–1638 - sensitive cultivar- and; SP83–2847 and SP83–5073 - tolerant cultivars. Altered miRNA regulation was correlated with changes in mRNA levels of specific targets. Two leaf libraries from individual sugarcane cultivars with contrasting drought-tolerance properties were also analyzed. An enrichment of 22-nt sRNA species was observed in leaf libraries. 22-nt miRNA triggered siRNA production by cleavage of their targets in response to water depletion. A number of genes of the sRNA biogenesis pathway were down-regulated in tolerant genotypes and up-regulated in sensitive in response to water depletion treatment. Our analysis contributes to increase the knowledge on the roles of sRNA in sugarcane submitted to water depletion.     

Comparing the Relative Importance of Water-Borne Cues and Direct Grazing for the Induction of Defenses in the Brown Seaweed Fucus vesiculosus

Some seaweed species have been shown to release water-borne cues after herbivore attack, for example, to attract natural enemies of the herbivore. These cues may also be sensed by neighboring seaweeds and used to adjust their defenses in anticipation of a possible herbivore attack. Several studies indicated information transfer between seaweed individuals in the past, including the brown seaweed Fucus vesiculosus. Previous work showed induction of defenses in F. vesiculosus in response to water-borne cues released by isopod-grazed conspecifics. In contrast, another study on induced responses after exposure to cues from isopod-grazed neighbors using the same seaweed species yielded contradictory results. This study reassessed the ability of F. vesiculosus individuals to sense water-borne cues released by isopod-grazed neighbors in a series of experiments that monitored F. vesiculosus palatability in response to direct grazing by Idotea baltica and water-borne cues from isopod-grazed neighbors relative to unmanipulated seaweed pieces. Two-choice feeding assays were conducted with both fresh and reconstituted seaweed pieces. Direct grazing by I. baltica induced a chemical defense in F. vesiculosus, confirming results of previous studies. In contrast, evidence for increased herbivore resistance in seaweed pieces that were located downstream of isopod-grazed F. vesiculosus could not be provided. The lack of defense induction in response to grazing of conspecific neighbors may be explained by the environmental conditions and the scattered distribution of F. vesiculosus individuals in the intertidal zone of Helgoland, which may render resource investment in the emission and/or response to water-borne cues at this site unprofitable.     

Therapeutic Administration of Recombinant Paracoccin Confers Protection against Paracoccidioides brasiliensis Infection: Involvement of TLRs

Background

Paracoccin (PCN) is an N-acetylglucosamine-binding lectin from the human pathogenic fungus Paracoccidioides brasiliensis. Recombinant PCN (rPCN) induces a T helper (Th) 1 immune response when prophylactically administered to BALB/c mice, protecting them against subsequent challenge with P. brasiliensis. In this study, we investigated the therapeutic effect of rPCN in experimental paracoccidioidomycosis (PCM) and the mechanism accounting for its beneficial action.

Methodology/Principal Findings

Four distinct regimens of rPCN administration were assayed to identify which was the most protective, relative to vehicle administration. In all rPCN-treated mice, pulmonary granulomas were less numerous and more compact. Moreover, fewer colony-forming units were recovered from the lungs of rPCN-treated mice. Although all therapeutic regimens of rPCN were protective, maximal efficacy was obtained with two subcutaneous injections of 0.5 µg rPCN at 3 and 10 days after infection. The rPCN treatment was also associated with higher pulmonary levels of IL-12, IFN-γ, TNF-α, nitric oxide (NO), and IL-10, without IL-4 augmentation. Encouraged by the pulmonary cytokine profile of treated mice and by the fact that in vitro rPCN-stimulated macrophages released high levels of IL-12, we investigated the interaction of rPCN with Toll-like receptors (TLRs). Using a reporter assay in transfected HEK293T cells, we verified that rPCN activated TLR2 and TLR4. The activation occurred independently of TLR2 heterodimerization with TLR1 or TLR6 and did not require the presence of the CD14 or CD36 co-receptors. The interaction between rPCN and TLR2 depended on carbohydrate recognition because it was affected by mutation of the receptor''s N-glycosylation sites. The fourth TLR2 N-glycan was especially critical for the rPCN-TLR2 interaction.

Conclusions/Significance

Based on our results, we propose that PCN acts as a TLR agonist. PCN binds to N-glycans on TLRs, triggers regulated Th1 immunity, and exerts a therapeutic effect against P. brasiliensis infection.     

Reprogramming of myeloid angiogenic cells by Bartonella henselae leads to microenvironmental regulation of pathological angiogenesis

The contribution of myeloid cells to tumour microenvironments is a decisive factor in cancer progression. Tumour‐associated macrophages (TAMs) mediate tumour invasion and angiogenesis through matrix remodelling, immune modulation and release of pro‐angiogenic cytokines. Nothing is known about how pathogenic bacteria affect myeloid cells in these processes. Here we show that Bartonella henselae, a bacterial pathogen causing vasculoproliferative diseases (bacillary angiomatosis), reprogrammes human myeloid angiogenic cells (MACs), a pro‐angiogenic subset of circulating progenitor cells, towards a TAM‐like phenotype with increased pro‐angiogenic capacity. B. henselae infection resulted in inhibition of cell death, activation of angiogenic cellular programmes and induction of M2 macrophage polarization. MACs infected with B. henselae incorporated into endothelial sprouts and increased angiogenic growth. Infected MACs developed a vascular mimicry phenotype in vitro, and expression of B. henselae adhesin A was essential in inducing these angiogenic effects. Secretome analysis revealed that increased pro‐angiogenic activities were associated with the creation of a tumour‐like microenvironment dominated by angiogenic inflammatory cytokines and matrix remodelling compounds. Our results demonstrate that manipulation of myeloid cells by pathogenic bacteria can contribute to microenvironmental regulation of pathological tissue growth and suggest parallels underlying both bacterial infections and cancer.     

Can the Liebowitz Social Anxiety Scale - Self-Report Version Be Used to Differentiate Clinical and Non-Clinical SAD Groups among Brazilians?

Background

The Liebowitz Social Anxiety Scale (LSAS) was the first evaluation instrument developed for screening for the signs and symptoms of Social Anxiety Disorder (SAD) and is currently still the most used worldwide. The aim of this study is to evaluate the ability of the LSAS - self-report version (LSAS-SR) to discriminate different Social Anxiety Disorder (SAD) clinical groups.

Method

The sample was composed of Brazilians university students, allocated into three different groups, i.e., cases (C=118), non-cases (NC=95) and subclinical cases (SC=39). To achieve the aim, calculations of the ROC Curve and ANOVA were performed.

Results

The results found were excellent regardless of the technique used, highlighting the discriminatory capacity of the LSAS-SR. The score equal to or greater than 32 is suggested as a cutoff score for the Brazilian population, since this presented balance between the standards evaluated and the ability to differentiate both clinical and subclinical SAD cases from non-cases.

Conclusion

Despite the specific sample used in this study being composed only of university students, the use of the LSAS-SR can be indicated, in the Brazilian setting, for SAD screening in both clinical and research contexts.     

In Vitro Comparison of Raypex 6 and Endopilot Using a Novel,Computer-Aided Measurement System,for Determining the Working Length

Introduction

Experimental evaluation of endometric devices usually relies on visual, subjective detection of the apical constriction to determine the accuracy of measurements. The aim of the present study was to analyze the accuracy of measurements of Raypex 6 and EndoPilot using a novel, objective image-analysis system.

Methods

Onehundred and twenty teeth were randomized and allocated to three groups: After coronal flaring, either Raypex 6 or EndoPilot were used to determine the endodontic working length during instrumentation using manual files (RPM and EPM group respectively). In addition, EndoPilot was used for continuous, automatic measurement during rotating instrumentation (EPA group). If the working length had been reached according to endometric results, the files were fixed in place. Tooth and file were then embedded and prepared for analysis. Subsequently, the distance between the tip of the file and the apical constriction (D_AC) or the apical foramen (D_AF) was calculated using trigonometric analysis and the position of the file relative to AC and AF was analyzed.

Results

Both inter- and intra-examiner-reliability of the trigonometric analysis were nearly perfect (ICC = 0.999, p<0.001). D_AC was not significantly different between groups (p>0.05, t-test). D_AF was significantly decreased when EPA had been used compared to EPM (p<0.05, Exact-test). EPA resulted in files being positioned beyond AF significantly more often than the other two methods (p<0.01).

Conclusions

All methods allowed reliable detection of AC. However, EPA significantly increased the risk of overpreparation. Objective, digital assessment based on image analysis was suitable to compare the accuracy of different endometric devices.     

PyNTTTTGT and CpG Immunostimulatory Oligonucleotides: Effect on Granulocyte/Monocyte Colony-Stimulating Factor (GM-CSF) Secretion by Human CD56+ (NK and NKT) Cells

CD56⁺ cells have been recognized as being involved in bridging the innate and acquired immune systems. Herein, we assessed the effect of two major classes of immunostimulatory oligonucleotides (ODNs), PyNTTTTGT and CpG, on CD56⁺ cells. Incubation of human peripheral blood mononuclear cells (hPBMC) with some of these ODNs led to secretion of significant amounts of interferon gamma (IFN-γ), tumor necrosis factor alpha (TNF-α) and granulocyte/monocyte colony-stimulating factor (GM-CSF), but only if interleukin 2 (IL2) was present. IMT504, the prototype of the PyNTTTTGT ODN class, was the most active. GM-CSF secretion was very efficient when non-CpG ODNs with high T content and PyNTTTTGT motifs lacking CpGs were used. On the other hand, CpG ODNs and IFNα inhibited this GM-CSF secretion. Selective cell type removal from hPBMC indicated that CD56⁺ cells were responsible for GM-CSF secretion and that plasmacytoid dendritic cells (PDCs) regulate this process. In addition, PyNTTTTGT ODNs inhibited the IFNα secretion induced by CpG ODNs in PDCs by interference with the TLR9 signaling pathway. Since IFNα is essential for CD56⁺ stimulation by CpG ODNs, there is a reciprocal interference of CpG and PyNTTTTGT ODNs when acting on this cell population. This suggests that these synthetic ODNs mimic different natural alarm signals for activation of the immune system.