Evaluation of HA negatively charged membranes in the recovery of human adenoviruses and hepatitis A virus in different water matrices

Human adenoviruses (HAdV) and hepatitis A virus (HAV) are shed in the faeces and consequently may be present in environmental waters, resulting in an increase in pathogen concentration that can affect water quality and human health. The aim of this study was to evaluate an adsorption-elution method which utilizes negatively charged membrane HA to determine the efficient recovery of HAdV and HAV from different water matrices and to combine this procedure with a qualitative molecular method (nested RT-PCR and nested PCR). The best efficiency recovery was achieved in distilled water and treated wastewater effluent (100%) for both viruses and in recreational lagoon water for HAV (100%). The efficiency recovery was 10% for HAdV and HAV in seawater and 10% for HAdV in lagoon water. The viral detection limit by nested PCR for HAV in water samples ranged between 20-0.2 FFU/mL and 250 and 25 TCID50/mL for HAdV. In conclusion, these results suggest that the HA negatively charged membranes vary their efficiency for recovery of viral concentration depending upon the types of both enteric viruses and water matrices.

An improved method for concentrating rotavirus from water samples

A modified adsorption-elution method for the concentration of seeded rotavirus from water samples was used to determine various factors which affected the virus recovery. An enzyme-linked immunosorbent assay was used to detect the rotavirus antigen after concentration. Of the various eluents compared, 0.05M glycine, pH 11.5 gave the highest rotavirus antigen recovery using negatively charged membrane filtration whereas 2.9% tryptose phosphate broth containing 6% glycine; pH 9.0 was found to give the greatest elution efficiency when a positively charged membrane was used. Reconcentration of water samples by a speedVac concentrator showed significantly higher rotavirus recovery than polyethylene glycol precipitation through both negatively and positively charged filters (p-value <0.001). In addition, speedVac concentration using negatively charged filtration resulted in greater rotavirus recovery than that using positively charged filtration (p-value = 0.004). Thirty eight environmental water samples were collected from river, domestic sewage, canals receiving raw sewage drains, and tap water collected in containers for domestic use, all from congested areas of Bangkok. In addition, several samples of commercial drinking water were analyzed. All samples were concentrated and examined for rotavirus antigen. Coliforms and fecal coliforms (0->1,800 MPN/100 ml) were observed but rotavirus was not detected in any sample. This study suggests that the speedVac reconcentration method gives the most efficient rotavirus recovery from water samples.

Molecular detection of human astrovirus in an urban sewage treatment plant in Rio de Janeiro, Brazil

The objective of this study was to evaluate the prevalence and dissemination of human astroviruses (HAstV) in the environment by analyzing urban sewage samples from a wastewater treatment plant in the city of Rio de Janeiro, Brazil. A one-year study was performed with a total of 48 raw and treated sewage composite samples, which were collected biweekly from an activated sludge plant. Virus particles were concentrated by the adsorption-elution method using negatively charged membranes associated to a Centriprep Concentrator® 50 (Nihon Millipore). HAstV were detected in 16.7% of the samples in raw and treated sewage by using both qualitative and quantitative reverse transcriptase-polymerase chain reactions (RT-PCR and qPCR, respectively). Positive untreated sewage sample exhibited mean values of 1.1 x 10(4) gEq/mL. The qPCR sensitivity was 18 gEq/reaction. Through utilization of qPCR, a HAstV recovery efficiency of 4.2% and 4.3% was demonstrated for raw and treated sewage samples, respectively. The presence of HAstV in both the raw and treated sewage samples demonstrated the dissemination of these viruses in the environment as well as viral permanence after sewage treatment. There was a reduction in the total and faecal coliform levels, indicating efficiency of the wastewater treatment plant.

Detection of enteric viruses in recreational waters of an urban lagoon in the city of Rio de Janeiro, Brazil

The aim of this study was to determine the occurrences of the group A rotavirus (RVA), norovirus (NoV) and human adenovirus (HAdV) in the surface waters of an urban lagoon (Rodrigo de Freitas Lagoon) in the city of Rio de Janeiro, Brazil. During one year of surveillance, water samples were obtained from the lagoon and other interconnected ecosystems (river and beach). The samples were concentrated using an adsorption-elution method with a negatively charged membrane and tested by qualitative and quantitative polymerase chain reaction assays. RVA was the most prevalent virus detected (24.3%) with a viral load ranging from 3.0 x 10¹-5.6 x 10(4) genome copies/L, followed by NoV (18.8%) and HAdV (16.7%). Considering water samples suitable for bathing, according to Escherichia coli criterion (< 2,000 most probable number/100 mL), viruses were detected in 50% (57/114) of them. Physicochemical parameters were also measured and showed possible correlations between turbidity and RVA presence and between pH and NoV presence. These data demonstrate the importance of considering viral parameters to ensure water quality and the utilisation of these parameters as additional tools for the characterisation of environmental contamination.

Hidrólise seletiva de carboxiamidas de resíduos de asparagina e glutamina em colágeno: preparação e caracterização de matrizes aniônicas para uso como biomateriais

This work describes the selective hydrolysis of carboxyamide groups of asparagine and glutamine of collagen matrices for the preparation of negatively charged collagen biomaterials. The reaction was performed in the presence of chloride and sulfate salts of alkaline and alkaline earth metals in aqueous dimethylsulfoxide solution and, selectively hydrolysis of carboxyamide groups of collagen matrices was promoted without cleavage of the peptide bond. The result is a new collagen material with controlled increase in negative charge content. Although triple helix secondary structure of tropocollagen was preserved, significative changes in thermal stabilities were observed in association with a new pattern of tropocollagen macromolecular association, particularly in respect microfibril assembly, thus providing at physiological pH a new type of collagen structure for biomaterial preparation, characterized by different charge and structural contents .

Influência das características das substâncias húmicas aquáticas na eficiência da coagulação com o cloreto férrico

The aim of this study was to verify the influence of the apparent molecular size of aquatic humic substances on the effectiveness of coagulation with ferric chloride. Coagulation-filtration tests using jar test and bench-scale sand filters were carried out on samples of water with true color of approximately 100 Hazen units, prepared with aquatic humic substances of different molecular sizes (F1: < 0.45 µm, F2: 100 kDa - 0.45 µm, F3: 30 - 100 kDa and F4': < 30 kDa). For the water samples with lower apparent molecular size fractions, greater dosages of coagulant was needed to remove the color around 5.0 Hanzen units, mainly because these water samples contain higher concentrations of fulvic acids, which exhibited a larger number of negatively-charged groups.

The effect of porphyrins on normal and transformed mouse cell lines in the presence of visible light

Photodynamic therapy consists of the uptake of a photosensitizing dye, often a porphyrin, by tumor tissue and subsequent irradiation of the tumor with visible light of an appropriate wavelength matched to the absorption spectrum of the photosensitizing dye. This class of molecules produces reactive oxygen species when activated by light, resulting in a direct or indirect cytotoxic effect on the target cells. Photodynamic therapy has been used in the treatment of cancer but the technology has a potential for the treatment of several disease conditions mainly because of its selectivity. However, it is not clear why the porphyrins are retained preferentially by abnormal tissue. This paper describes a study of the effect of the association of porphyrin and visible light on two mouse fibroblast cell lines: A31, normal cells and B61, an EJ-ras transformed variant of A31. Two water-soluble porphyrins were used, a positively charged one, tetra(N-methyl-4-pyridyl)porphyrin chloride, and a negatively charged one, tetra(4-sulfonatophenyl)porphyrin-Na salt (TPPS4) in order to assess the effect on cell survival. The results suggest that the B61 cell line is more sensitive to incubation with the anionic porphyrin (TPPS4) followed by light irradiation and that the anionic porphyrin is more efficient in killing the cells than the cationic porphyrin.

Genomics and X-ray microanalysis indicate that Ca2+ and thiols mediate the aggregation and adhesion of Xylella fastidiosa

The availability of the genome sequence of the bacterial plant pathogen Xylella fastidiosa, the causal agent of citrus variegated chlorosis, is accelerating important investigations concerning its pathogenicity. Plant vessel occlusion is critical for symptom development. The objective of the present study was to search for information that would help to explain the adhesion of X. fastidiosa cells to the xylem. Scanning electron microscopy revealed that adhesion may occur without the fastidium gum, an exopolysaccharide produced by X. fastidiosa, and X-ray microanalysis demonstrated the presence of elemental sulfur both in cells grown in vitro and in cells found inside plant vessels, indicating that the sulfur signal is generated by the pathogen surface. Calcium and magnesium peaks were detected in association with sulfur in occluded vessels. We propose an explanation for the adhesion and aggregation process. Thiol groups, maintained by the enzyme peptide methionine sulfoxide reductase, could be active on the surface of the bacteria and appear to promote cell-cell aggregation by forming disulfide bonds with thiol groups on the surface of adjacent cells. The enzyme methionine sulfoxide reductase has been shown to be an auxiliary component in the adhesiveness of some human pathogens. The negative charge conferred by the ionized thiol group could of itself constitute a mechanism of adhesion by allowing the formation of divalent cation bridges between the negatively charged bacteria and predominantly negatively charged xylem walls.

Biochemical characterization of the GM2 gangliosidosis B1 variant

The deficiency of the A isoenzyme of ß-hexosaminidase (Hex) produced by different mutations of the gene that codes for the alpha subunit (Tay-Sachs disease) has two variants with enzymological differences: the B variant consists of the absence of Hex A isoenzyme and the B1 variant produces an inactive Hex A isoenzyme for the hydrolysis of the GM2 ganglioside and synthetic substrates with negative charge. In contrast to the early childhood form of the B variant, the B1 variant appears at a later clinical stage (3 to 7 years of age) with neurodegenerative symptoms leading to the death of the patient in the second decade of life. The most frequent mutation responsible for the GM2 gangliosidosis B1 variant is R178H, which has a widespread geographic and ethnic distribution. The highest incidence has been described in Portugal, which has been suggested as the point of origin of this mutation. Biochemical characterization of this lysosomal disease is carried out using negatively charged synthetic alpha subunit-specific sulfated substrates, since Hex A isoenzyme heat-inactivation assays are not applicable. However, the determination of the apparent activation energy of Hex using the neutral substrate 3,3'-dichlorophenolsulfonphthaleinyl N-acetyl-ß-D-glucosaminide, may offer a valid alternative. The presence of an alpha subunit in the alphaß heterodimer Hex A means that its activation energy (41.8 kJ/mol) is significantly lower than that of the ßß homodimer Hex B (75.1 kJ/mol); however, as mutation inactivates the alpha subunit, the Hex A of the B1 variant presents an activation energy that is similar to that of the Hex B isoenzyme.

Cloning, purification and comparative characterization of two digestive lysozymes from Musca domestica larvae

cDNA coding for two digestive lysozymes (MdL1 and MdL2) of the Musca domestica housefly was cloned and sequenced. MdL2 is a novel minor lysozyme, whereas MdL1 is the major lysozyme thus far purified from M. domestica midgut. MdL1 and MdL2 were expressed as recombinant proteins in Pichia pastoris, purified and characterized. The lytic activities of MdL1 and MdL2 upon Micrococcus lysodeikticus have an acidic pH optimum (4.8) at low ionic strength (μ = 0.02), which shifts towards an even more acidic value, pH 3.8, at a high ionic strength (μ = 0.2). However, the pH optimum of their activities upon 4-methylumbelliferyl N-acetylchitotrioside (4.9) is not affected by ionic strength. These results suggest that the acidic pH optimum is an intrinsic property of MdL1 and MdL2, whereas pH optimum shifts are an effect of the ionic strength on the negatively charged bacterial wall. MdL2 affinity for bacterial cell wall is lower than that of MdL1. Differences in isoelectric point (pI) indicate that MdL2 (pI = 6.7) is less positively charged than MdL1 (pI = 7.7) at their pH optima, which suggests that electrostatic interactions might be involved in substrate binding. In agreement with that finding, MdL1 and MdL2 affinities for bacterial cell wall decrease as ionic strength increases.

Perimicrovillar membrane assembly: the fate of phospholipids synthesised by the midgut of Rhodnius prolixus

In this study, we describe the fate of fatty acids that are incorporated from the lumen by the posterior midgut epithelium of Rhodnius prolixus and the biosynthesis of lipids. We also demonstrate that neutral lipids (NL) are transferred to the haemolymphatic lipophorin (Lp) and that phospholipids remain in the tissue in which they are organised into perimicrovillar membranes (PMMs). 3H-palmitic acid added at the luminal side of isolated midguts of R. prolixus females was readily absorbed and was used to synthesise phospholipids (80%) and NL (20%). The highest incorporation of 3H-palmitic acid was on the first day after a blood meal. The amounts of diacylglycerol (DG) and triacylglycerol synthesised by the tissue decreased in the presence of Lp in the incubation medium. The metabolic fates of 3H-lipids synthesised by the posterior midgut were followed and it was observed that DG was the major lipid released to Lp particles. However, the majority of phospholipids were not transferred to Lp, but remained in the tissue. The phospholipids that were synthesised and accumulated in the posterior midgut were found to be associated with Rhodnius luminal contents as structural components of PMMs.

Mir-190b negatively contributes to the Trypanosoma cruzi- infected cell survival by repressing PTEN protein expression

Chagas disease, which is caused by the intracellular protozoanTrypanosoma cruzi, is a serious health problem in Latin America. The heart is one of the major organs affected by this parasitic infection. The pathogenesis of tissue remodelling, particularly regarding cardiomyocyte behaviour after parasite infection, and the molecular mechanisms that occur immediately following parasite entry into host cells are not yet completely understood. Previous studies have reported that the establishment of parasitism is connected to the activation of the phosphatidylinositol-3 kinase (PI3K), which controls important steps in cellular metabolism by regulating the production of the second messenger phosphatidylinositol-3,4,5-trisphosphate. Particularly, the tumour suppressor PTEN is a negative regulator of PI3K signalling. However, mechanistic details of the modulatory activity of PTEN on Chagas disease have not been elucidated. To address this question, H9c2 cells were infected with T. cruzi Berenice 62 strain and the expression of a specific set of microRNAs (miRNAs) were investigated. Our cellular model demonstrated that miRNA-190b is correlated to the decrease of cellular viability rates by negatively modulating PTEN protein expression in T. cruzi-infected cells.

SIRT1 negatively regulates amyloid-beta-induced inflammation via the NF-κB pathway

Chronic inflammation induced by amyloid-beta (Aβ) plays a key role in the development of age-related macular degeneration (AMD), and matrix metalloproteinase-9 (MMP-9), interleukin (IL)-6, and IL-8 may be associated with chronic inflammation in AMD. Sirtuin 1 (SIRT1) regulates inflammation via inhibition of nuclear factor-kappa B (NF-κB) signaling, and resveratrol has been reported to prevent Aβ-induced retinal degeneration; therefore, we investigated whether this action was mediated via activation of SIRT1 signaling. Human adult retinal pigment epithelial (RPE) cells were exposed to Aβ, and overactivation and knockdown of SIRT1 were performed to investigate whether SIRT1 is required for abrogating Aβ-induced inflammation. We found that Aβ-induced RPE barrier disruption and expression of IL-6, IL-8, and MMP-9 were abrogated by the SIRT1 activator SRT1720, whereas alterations induced by Aβ in SIRT1-silenced RPE cells were not attenuated by SRT1720. In addition, SRT1720 inhibited Aβ-mediated NF-κB activation and decrease of the NF-κB inhibitor, IκBα. Our findings suggest a protective role for SIRT1 signaling in Aβ-dependent retinal degeneration and inflammation in AMD.