Experimental Analysis of Viral–Host Interactions

Viral and pathogen protein complexity is often limited by their relatively small genomes, thus critical functions are often accomplished by complexes of host and pathogen proteins. This requirement makes the study of host–pathogen interactions critical for the understanding of pathogenicity and virology. This review article discusses proteomic methods that offer an opportunity to experimentally identify and analyze the binding partners of a target protein and presents the representative studies performed with these methods. These methods divide into two classes: ex situ and in situ. Ex situ assays depend on bindings that occur outside of the normal cellular environment and include yeast two hybrids, pull-downs, and nucleic acid-programmable protein arrays (NAPPA). In situ assays depend on bindings that occur inside of host cells and include affinity purification (AP) and proximity dependent labeling (PDL). Either ex or in situ methods can be reliably used for generating protein–protein interactions networks but it is important to understand and recognize the limitations of the chosen methods when developing an interactomic network. In summary, proteomic methods can be extremely useful for interactomics but it is important to recognize the nature of the method when designing and analyzing an experiment

N-glycosylation status of E-cadherin controls cytoskeletal dynamics through the organization of distinct β-catenin- and γ-catenin-containing AJs.

N-glycosylation of E-cadherin has been shown to inhibit cell-cell adhesion. Specifically, our recent studies have provided evidence that the reduction of E-cadherin N-glycosylation promoted the recruitment of stabilizing components, vinculin and serine/threonine protein phosphatase 2A (PP2A), to adherens junctions (AJs) and enhanced the association of AJs with the actin cytoskeleton. Here, we examined the details of how N-glycosylation of E-cadherin affected the molecular organization of AJs and their cytoskeletal interactions. Using the hypoglycosylated E-cadherin variant, V13, we show that V13/β-catenin complexes preferentially interacted with PP2A and with the microtubule motor protein dynein. This correlated with dephosphorylation of the microtubule-associated protein tau, suggesting that increased association of PP2A with V13-containing AJs promoted their tethering to microtubules. On the other hand V13/γ-catenin complexes associated more with vinculin, suggesting that they mediated the interaction of AJs with the actin cytoskeleton. N-glycosylation driven changes in the molecular organization of AJs were physiologically significant because transfection of V13 into A253 cancer cells, lacking both mature AJs and tight junctions (TJs), promoted the formation of stable AJs and enhanced the function of TJs to a greater extent than wild-type E-cadherin. These studies provide the first mechanistic insights into how N-glycosylation of E-cadherin drives changes in AJ composition through the assembly of distinct β-catenin- and γ-catenin-containing scaffolds that impact the interaction with different cytoskeletal components

Metals Toxic Effects in Aquatic Ecosystems: Modulators of Water Quality

The topic of this work was based on the assessment of aquatic systems quality related to the persistent metal pollution. The use of aquatic organisms as bioindicators of metal pollution allowed the obtaining of valuable information about the acute and chronic toxicity on common Romanian aquatic species and the estimation of the environment quality. Laboratory toxicity results showed that Cd, As, Cu, Zn, Pb, Ni, Zr, and Ti have toxic to very toxic effects on Cyprinus carpio, and this observation could raise concerns because of its importance as a fishery resource. The benthic invertebrates’ analysis showed that bioaccumulation level depends on species, type of metals, and sampling sites. The metal analysis from the shells of three mollusk species showed that the metals involved in the metabolic processes (Fe, Mn, Zn, Cu, and Mg) were more accumulated than the toxic ones (Pb, Cd). The bioaccumulation factors of metals in benthic invertebrates were subunitary, which indicated a slow bioaccumulation process in the studied aquatic ecosystems. The preliminary aquatic risk assessment of Ni, Cd, Cr, Cu, Pb, As, and Zn on C. carpio revealed insignificant to moderate risk considering the measured environmental concentrations, acute and long-term effects and environmental compartment

Microbiological efficiency tests of the cosmetic tools disinfection procedures

In the last decade, the cosmetic industry has experienced a massive development, but there have also been issues related to their influence on the health of the population. The application methods of the cosmetic products could trigger the appearance of the skin infectious, which raise the need for efficient disinfection processes of the cosmetic products. At the present, the economic operators in the field of cosmetics are guided by medical regulations, but, unfortunately there are not standardized procedure for the application and control of disinfection. The aim of this study was to determine an efficient procedure of disinfection for instruments used in the application of cosmetics such as the beauty blender. There were performed two different disinfection procedures, a chemical disinfection using 70% ethanol and a physical disinfection with UV radiation. The influence of foundation on disinfection procedures was also tested. A standardized S. aureus and a S. haemolyticus bacterium from a human abscess were tested and an antibiotic resistance pattern was also analyzed. The disinfection efficiency tests showed that the ethanol solution was effective after 5 minutes, decreasing the S. haemolyticus bacterial density by 50% in the absence of foundation. In the presence of the foundation, this process was no longer efficient, foundation having a possible protection and nutritional role for bacteria. The radiation with UV at 265 nm showed a complete eradication of both bacterial strains after 1 minute, regardless of foundation presence or not. The antibiotic susceptibility tested showed that both strains had the natural penicillin resistance

Evaluating the ecotoxicity of different pharmaceuticals using Aliivibrio fischeri bioassays

An endless list of companies have produced a large amount of pharmaceutical compounds in a year-on-year growth trend. Due to the excessive consumption of these substances and the inappropriate disposal, the environment was contaminated, especially aquatic ecosystems, with quantities of pharmaceuticals (PHACs) so that they have affected the living organisms, leading to decreased biodiversity and ecological degradation. Many studies on PHACs environmental presence and toxic effects were performed, but unfortunately, no limit was establish for discharging into environment, especially into the aquatic systems. The aim of this study was to use the bioluminescence of Aliivibrio fischeri bacteria as an indicator of toxically effect of different PHACs in simulated marine medium. The Microtox® bioassay is based on the PHACs inhibitory effect on the metabolism of bacteria which induced changes in their bacterial bioluminescence. The test organisms were exposed to analgesics and anti-inflammatories such as Diclofenac, Ketoprofen, Naproxen and Ibuprofen. The results showed that based on EC50 values, Naproxen had a very low toxicity but Diclofenac, Ketoprofen and Ibuprofen had a harmful effect on the aquatic organisms

A novel ladder-like lectin relates to sites of mucosal immunity in Atlantic Halibut (Hippoglossus hippoglossus L.)

A novel 27 kDa ladder-lectin-like protein, showing a multimeric structure under non-reducing conditions, was isolated from halibut serum by binding to N-acetyl glucosamine. Mass-spectrometry analysis did not show significant homology with known proteins. Specific antibodies were produced and used in immunohistochemistry on tissue sections of early halibut ontogeny from 119 until 1050 °d post hatching. A strong positive response was detected in the mucosal cells of the skin, gills and gut, indicating a role in the mucosal immune defence at these sites. Further immunopositivity was detected in liver, myeloma of kidney and the brain at different developmental stages but predominant expression was found in mucosal surfaces at later stages of development tested (1050 °d). It is still uncertain whether this ladder-like lectin forms part of the complement pathway, as a lectin or ficolin, or if it belongs to galectins. A strong detection in mucosal surfaces on skin, gills and gut, show similar patterns of expression as both mucosal lectins and galectins in other fish. Detection in neuronal tissue may indicate putative roles in tissue remodelling of brain and in ongoing neurogenesis in the fish eye

Proteomics approaches to fibrotic disorders

This review provides an introduction to mass spectrometry based proteomics and discusses several proteomics approaches that are relevant in understanding the pathophysiology of fibrotic disorders and the approaches that are frequently used in biomarker discovery

In vitro evaluation of a novel bioreactor based on an integral oxygenator and a spirally wound nonwoven polyester matrix for hepatocyte culture as small aggregates

BACKGROUND/AIMS: The development of custom-made bioreactors for use as a bioartificial liver (BAL) is considered to be one of the last challenges on the road to successful temporary extracorporeal liver support therapy. We devised a novel bioreactor (patent pending) which allows individual perfusion of high density cultured hepatocytes with low diffusional gradients, thereby more closely resembling the conditions in the intact liver lobuli. METHODS: The bioreactor consists of a spirally wound nonwoven polyester matrix, i.e. a sheet-shaped, three-dimensional framework for hepatocyte immobilization and aggregation, and of integrated hydrophobic hollow-fiber membranes for decentralized oxygen supply and CO2 removal. Medium (plasma in vivo) was perfused through the extrafiber space and therefore in direct hepatocyte contact. Various parameters were assessed over a period of 4 days including galactose elimination, urea synthesis, lidocaine elimination, lactate/pyruvate ratios, amino acid metabolism, pH, the last day being reserved exclusively for determination of protein secretion. RESULTS: Microscopic examination of the hepatocytes revealed cytoarchitectural characteristics as found in vivo. The biochemical performance of the bioreactor remained stable over the investigated period. The urea synthesizing capacity of hepatocytes in the bioreactor was twice that of hepatocytes in monolayer cultures. Flow sensitive magnetic resonance imaging (MRI) revealed that the bioreactor construction ensured medium flow through all parts of the device irrespective of its size. CONCLUSIONS: The novel bioreactor showed encouraging efficiency. The device is easy to manufacture with scale-up to the liver mass required for possible short-term support of patients in hepatic failur

The active oxygen response of raspberry protoplasts to O-glycans of Fusarium sp M7-1

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