In vitro co-cultures of Pinus pinaster with Bursaphelenchus xylophilus: a biotechnological approach to study pine wilt disease

Abstract Main conclusion Co-cultures of Pinus pinaster with Bursaphelenchus xylophilus were established as a biotechnological tool to evaluate the effect of nematotoxics addition in a host/parasite culture system. The pinewood nematode (PWN), Bursaphelenchus xylophilus, the causal agent of pine wilt disease (PWD), was detected for the first time in Europe in 1999 spreading throughout the pine forests in Portugal and recently in Spain. Plant in vitro cultures may be a useful experimental system to investigate the plant/nematode relationships in loco, thus avoiding the difficulties of field assays. In this study, Pinus pinaster in vitro cultures were established and compared to in vivo 1 year-old plantlets by analyzing shoot structure and volatiles production. In vitro co-cultures were established with the PWN and the effect of the phytoparasite on in vitro shoot structure, water content and volatiles production was evaluated. In vitro shoots showed similar structure and volatiles production to in vivo maritime pine plantlets. The first macroscopic symptoms of PWD were observed about 4 weeks after in vitro co-culture establishment. Nematode population in the culture medium increased and PWNs were detected in gaps of the callus tissue and in cavities developed from the degradation of cambial cells. In terms of volatiles main components, plantlets, P. pinaster cultures, and P. pinaster with B. xylophilus co-cultures were all b- and a-pinene rich. Cocultures may be an easy-to-handle biotechnological approach to study this pathology, envisioning the understanding of and finding ways to restrain this highly devastating nematode. Keywords Maritime pine ! Monoxenic culture ! Pinewood nematode ! Relative water content ! Shoots structure ! Volatiles Abbreviations BAP 6-Benzylaminopurine DAI Days after inoculation EPPO European and Mediterranean Plant Protectio

Functional and structural characterization of a novel putative cysteine protease cell wall-modifying multi-domain enzyme selected from a microbial metagenome

A current metagenomics focus is to interpret and transform collected genomic data into biological information. By combining structural, functional and genomic data we have assessed a novel bacterial protein selected from a carbohydrate-related activity screen in a microbial metagenomic library from Capra hircus (domestic goat) gut. This uncharacterized protein was predicted as a bacterial cell wall-modifying enzyme (CWME) and shown to contain four domains: an N-terminal, a cysteine protease, a peptidoglycan-binding and an SH3 bacterial domain. We successfully cloned, expressed and purified this putative cysteine protease (PCP), which presented autoproteolytic activity and inhibition by protease inhibitors. We observed cell wall hydrolytic activity and ampicillin binding capacity, a characteristic of most bacterial CWME. Fluorimetric binding analysis yielded a Kb of 1.8 × 10(5) M(-1) for ampicillin. Small-angle X-ray scattering (SAXS) showed a maximum particle dimension of 95 Å with a real-space Rg of 28.35 Å. The elongated molecular envelope corroborates the dynamic light scattering (DLS) estimated size. Furthermore, homology modeling and SAXS allowed the construction of a model that explains the stability and secondary structural changes observed by circular dichroism (CD). In short, we report a novel cell wall-modifying autoproteolytic PCP with insight into its biochemical, biophysical and structural features

Functional and structural characterization of a novel putative cysteine protease cell wall-modifying multi-domain enzyme selected from a microbial metagenome

A current metagenomics focus is to interpret and transform collected genomic data into biological information. By combining structural, functional and genomic data we have assessed a novel bacterial protein selected from a carbohydrate-related activity screen in a microbial metagenomic library from Capra hircus (domestic goat) gut. This uncharacterized protein was predicted as a bacterial cell wall-modifying enzyme (CWME) and shown to contain four domains: an N-terminal, a cysteine protease, a peptidoglycan-binding and an SH3 bacterial domain. We successfully cloned, expressed and purified this putative cysteine protease (PCP), which presented autoproteolytic activity and inhibition by protease inhibitors. We observed cell wall hydrolytic activity and ampicillin binding capacity, a characteristic of most bacterial CWME. Fluorimetric binding analysis yielded a Kb of 1.8 × 10(5) M(-1) for ampicillin. Small-angle X-ray scattering (SAXS) showed a maximum particle dimension of 95 Å with a real-space Rg of 28.35 Å. The elongated molecular envelope corroborates the dynamic light scattering (DLS) estimated size. Furthermore, homology modeling and SAXS allowed the construction of a model that explains the stability and secondary structural changes observed by circular dichroism (CD). In short, we report a novel cell wall-modifying autoproteolytic PCP with insight into its biochemical, biophysical and structural features

Nutritional assessment in patients with cirrhosis

CONTEXT: Malnutrition in cirrhotic patients with end-stage disease is common, and the degree of nutritional debilitation can play an important role in the pathogenesis of complications and cause a negative impact on prognosis. However, it involves difficulties and controversies regarding the identification of the best nutritional assessment method. OBJECTIVE: To identify a method that provides a safe and effective nutritional diagnosis. METHODS: Cross-sectional study with 129 cirrhotic patients. Anthropometric measurements, subjective global assessment, hand grip strength and bioelectrical impedance. RESULTS: Through phase angle of bioelectrical impedance analysis (BIA) method, significant associations with Child-Pugh (P = 0.008), age group and gender were observed. The ROC (receiver operator characteristic) curve was generated to determine the best cutoff point of the phase angle of cirrhotic patients, serving as one of the reference parameters for the nutritional assessment with bioimpedance in this study, considering the classification through Child-Pugh score as the reference standard for the clinical conditions of patients with cirrhosis. CONCLUSIONS: The assessment through bioelectrical impedance presented a statistically significant correlation with Child-Pugh score. The identification of phase angle of 5.44º is the new parameter suggested for the classification of the nutritional conditions of cirrhotic patients

Surface roughness and hardness of a composite resin: influence of finishing and polishing and immersion methods

This study evaluated the finishing and polishing effect on the surface roughness and hardness of the Filtek Supreme XT, in fluoride solutions. Specimens were prepared (n = 140) with half of the samples finished and polished with Super-Snap® disks. The experimental groups were divided according to the presence or absence of finishing and polishing and immersion solutions (artificial saliva, sodium fluoride solution at 0.05% - manipulated, Fluordent Reach, Oral B, Fluorgard). The specimens remained immersed in artificial saliva for 24 hours and were then subjected to initial analysis (baseline) of surface roughness and Vickers microhardness. Next, they were immersed in different fluoride solutions for 1 min/day, for 60 days. Afterwards, a new surface roughness and microhardness reading was conducted. The data were submitted to a two-way ANOVA and Tukey's test (5% significance level). For the comparison of mean roughness and hardness at baseline and after 60 days, the paired Student t test was used. The results showed that the surface roughness and microhardness of the Filtek Supreme XT were influenced by the finishing and polishing procedure, independently of the immersion methods