Blood trace metals in a sporadic amyotrophic lateral sclerosis geographical cluster

Amyotrophic lateral sclerosis (ALS) is a fatal disorder with unknown etiology, in which genetic and environmental factors interplay to determine the onset and the course of the disease. Exposure to toxic metals has been proposed to be involved in the etiology of the disease either through a direct damage or by promoting oxidative stress. In this study we evaluated the concentration of a panel of metals in serum and whole blood of a small group of sporadic patients, all living in a defined geographical area, for which acid mine drainage has been reported. ALS prevalence in this area is higher than in the rest of Italy. Results were analyzed with software based on artificial neural networks. High concentrations of metals (in particular Se, Mn and Al) were associated with the disease group. Arsenic serum concentration resulted lower in ALS patients, but it positively correlated with disease duration. Comet assay was performed to evaluate endogenous DNA damage that resulted not different between patients and controls. Up to now only few studies considered geographically well-defined clusters of ALS patients. Common geographical origin among patients and controls gave us the chance to perform metallomic investigations under comparable conditions of environmental exposure. Elaboration of these data with software based on machine learning processes has the potential to be extremely useful to gain a comprehensive view of the complex interactions eventually leading to disease, even in a small number of subjects.     

<Emphasis Type="Italic">Lecanicillium attenuatum</Emphasis> isolates affecting the invasive cypress aphid (<Emphasis Type="Italic">Cinara cupressi</Emphasis>) in Chile

The cypress aphid (Cinara cupressi) is listed among the hundred most important invasive pests in the world. In Chile, it was first detected in 2003 and currently is present throughout the country. In the course of a survey of their natural enemies in Chile, three strains of entomopathogenic fungi were isolated. The isolates were identified and tested against the aphid in laboratory experiments. Two further entomopathogenic fungi (ARSEF 5126 and 5128), formulated in the mycoinsecticides Vertalec^® and Mycotal^®, were used as reference strains. The three Chilean isolates were identified genomically as Lecanicillium attenuatum and were pathogenic to third-instar nymphs. The isolate ARSEF 13279 yielded the lowest overall lethal concentration (LC₅₀), 3 × 10⁵ conidia ml^?1 at four days post-inoculation, and the shortest lethal time (LT₅₀), 3.7 days after inoculation with 10⁶ conidia ml^?1. The results indicate that the isolates have considerable potential as microbial control agents of the invasive cypress aphid.     

Enteroparasitoses and Toxocarosis Affecting Children from Mar del Plata City,Argentina

This study evaluated the existence of enteroparasitoses and toxocarosis in children of peripheral (PC) and urban communities (UC) from Mar del Plata city (Argentina) and their associations with socio-environmental conditions. A Parasite Vulnerability Index (PVI) was elaborated using variables such as overcrowding, floor type, drinking water source, wastewater disposal, solid waste disposal, presence of animals and schooling level. The PC evidenced statistically significant higher frequencies of families with high (38.9%) and medium (55.5%) PVI, while in the UC low PVI (93%) was the most frequent. A statistically significant higher frequency of PC children was parasitized (30.2 vs. 14.5%; χ ² Pearson = 5.21; P < 0.05), presented higher parasite frequencies, specific richness, parasitic loads, and they also evidenced polyparasitism. The Multiple Correspondence Analysis (MCA) showed associations between PC-parasitized children, overcrowding and contact with pets and farm animals. The ELISA test to the specified determination of Toxocara canis IgG was reactive in a statistically significant higher proportion of PC children than the UC (55 vs. 8.5%; χ ² = 30.5; P < 0.01). The MCA associated PC reactive children, not adequate hand washing, moderate and hypereosinophilia and contact with pets and farm animals. Deficient socio-environmental conditions became children more vulnerable to get enteroparasitoses and toxocarosis in the PC than in the UC.     

Alternating flow filtration as an alternative to internal spin filter based perfusion process: Impact on productivity and product quality

This article reports the results obtained from comparison of internal spin filter (ISF) and alternating flow filtration (ATF) as cell retention systems, regarding cell growth, volumetric perfusion rate, cell specific perfusion rate and cell productivity in the fermentation process. As expected we were able to reach higher cell densities and to achieve longer runs since ATF systems are known to be less affected by fouling. Volumetric production of the reactor using the ATF system was 50‐70% higher than the production achieved using the ISF due to higher cell density and a two‐fold increase in the perfusion rate. On the other hand, downstream processing performances were evaluated regarding chromatographic steps yields and productivity and quality attributes of the purified materials. Similar results were obtained for all evaluated systems. The fact that we were able to achieve a 2 working volumes (WV)/day perfusion rate using an ATF system as cell retention device allowed us to virtually double the WV of a 25 L reactor. These results constitute valuable data for the optimization of recombinant protein production in perfusion processes since a two‐fold increase in the average production of a manufacturing facility could be easily achieved as long as downstream scale up is possible. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:1010–1014, 2017     

Tunicates Illuminate the Enigmatic Evolution of Chordate Metallothioneins by Gene Gains and Losses,Independent Modular Expansions,and Functional Convergences

To investigate novel patterns and processes of protein evolution, we have focused in the metallothioneins (MTs), a singular group of metal-binding, cysteine-rich proteins that, due to their high degree of sequence diversity, still represents a “black hole” in Evolutionary Biology. We have identified and analyzed more than 160 new MTs in nonvertebrate chordates (especially in 37 species of ascidians, 4 thaliaceans, and 3 appendicularians) showing that prototypic tunicate MTs are mono-modular proteins with a pervasive preference for cadmium ions, whereas vertebrate and cephalochordate MTs are bimodular proteins with diverse metal preferences. These structural and functional differences imply a complex evolutionary history of chordate MTs—including de novo emergence of genes and domains, processes of convergent evolution, events of gene gains and losses, and recurrent amplifications of functional domains—that would stand for an unprecedented case in the field of protein evolution.     

Mechanism of thioflavin T binding to amyloid fibrils

Thioflavin T is a benzothiazole dye that exhibits enhanced fluorescence upon binding to amyloid fibrils and is commonly used to diagnose amyloid fibrils, both ex vivo and in vitro. In aqueous solutions, thioflavin T was found to exist as micelles at concentrations commonly used to monitor fibrils by fluorescence assay ( approximately 10-20 microM). Specific conductivity changes were measured at varying concentration of thioflavin T and the critical micellar concentration was calculated to be 4.0+/-0.5 microM. Interestingly, changes in the fluorescence excitation and emission of thioflavin T were also dependent on the micelle formation. The thioflavin T micelles of 3 nm diameter were directly visualized using atomic force microscopy, and bound thioflavin T micelles were observed along the fibril length for representative fibrils. Increasing concentration of thioflavin T above the critical micellar concentration shows increased numbers of micelles bound along the length of the amyloid fibrils. Thioflavin T micelles were disrupted at low pH as observed by atomic force microscopy and fluorescence enhancement upon binding of thioflavin T to amyloid fibrils also reduced by several-fold upon decreasing the pH to below 3. This suggests that positive charge on the thioflavin T molecule has a role in its micelle formation that then bind the amyloid fibrils. Our data suggests that the micelles of thioflavin T bind amyloid fibrils leading to enhancement of fluorescence emission.     

High-throughput screening with HyperCyt flow cytometry to detect small molecule formylpeptide receptor ligands

High-throughput flow cytometry (HTFC), enabled by faster automated sample processing, represents a promising high- content approach for compound library screening. HyperCyt is a recently developed automated HTFC analysis system by which cell samples are rapidly aspirated from microplate wells and delivered to the flow cytometer. The formylpeptide receptor (FPR) family of G protein-coupled receptors contributes to the localization and activation of tissue-damaging leukocytes at sites of chronic inflammation. Here, the authors describe development and application of an HTFC screening approach to detect potential anti-inflammatory compounds that block ligand binding to FPR. Using a homogeneous no-wash assay, samples were routinely processed at 1.5 s/well (approximately 2500 cells analyzed/sample), allowing a 96-well plate to be processed in less than 2.5 min. Assay sensitivity and accuracy were validated by detection of a previously documented active compound with relatively low FPR affinity (sulfinpyrazone, inhibition constant [K(i)]=14 microM) from among a collection of 880 compounds in the Prestwick Chemical Library. The HyperCyt system was therefore demonstrated to be a robust, sensitive, and highly quantitative method with which to screen lead compound libraries in a 96-well format.     

Robust estimation of protein expression ratios with lysate microarray technology

MOTIVATION: The protein lysate microarray is a developing proteomic technology for measuring protein expression levels in a large number of biological samples simultaneously. A challenge for accurate quantification is the relatively narrow dynamic range associated with the commonly used chromogenic signal detection system. To facilitate accurate measurement of the relative expression levels, each sample is serially diluted and each diluted version is spotted on a nitrocellulose-coated slide in triplicate. Thus, each sample yields multiple measurements in different dynamic ranges of the detection system. This study aims to develop suitable algorithms that yield accurate representations of the relative expression levels in different samples from multiple data points. RESULTS: We evaluated two algorithms for estimating relative protein expression in different samples on the lysate microarray by means of a cross-validation procedure. For this purpose as well as for quality control we designed a 1440-spot lysate microarray containing 80 identical samples of purified bovine serum albumin, printed in triplicate with six 2-fold dilutions. Our analysis showed that the algorithm based on a robust least squares estimator provided the most accurate quantification of the protein lysate microarray data. We also demonstrated our methods by estimating relative expression levels of p53 and p21 in either p53(+/+) or p53(-/-) HCT116 colon cancer cells after two drug treatments and their combinations on another lysate microarray. AVAILABILITY: http://www.cs.tut.fi/~mirceanc/lysate_array_bioinformatics.htm     

A framework for multidimensional modelling of activity and structure of multispecies biofilms

Concepts from previous biofilm models were integrated to create a framework for the implementation of multidimensional (2D and 3D) multispecies biofilm models. The framework is here described at three levels: (i) mathematical representation of the processes involved in biofilm formation, (ii) numerical implementation into a computer program (freely available from our website http://www.biofilms.bt.tudelft.nl/frameworkMaterial) and (iii) using the program for the creation of biofilm models with multiple bacterial and solute species. An improved version of the individual-based modelling (IbM) that allows structured biomass was used. In this approach biomass composition may be discriminated into any number of particulate species, including extracellular polymeric substances (EPS) for which specific functionality was included. Detachment is also included, described as occurring at the biofilm surface with variable local rates derived from functions of state variables. The application of this modelling framework to a multispecies system with structured biomass is illustrated in a case study where the competition between an organism capable of accumulating polyhydroxybutyrate (PHB, an internal storage compound) and an EPS-producing organism in a two-species biofilm is analysed. Results illustrate that biofilms enriched in PHB-producing organisms may be obtained by supplying substrate intermittently in feast/famine cycles.