Decolorization of Synthetic Textile Dyes by Fungal Endophytes Isolated from the Leaves of Philippine Mangrove (Avicennia marina)

Textile dyes in wastewater can be harmful pollutants when released into the environment without treatment. Biodegradation of textile dye effluents by different microbes, including fungi, has become popular as an alternative to physicochemical methods. The mangrove Avicennia marina is known to harbor endophytic fungi which have the potential to carry out dye degradation. Therefore, this study assessed the ability to decolorize synthetic dyes of endophytic fungi isolated from the leaves of A. marina. Of the nine fungal endophytes, Aspergillus niger, Syncephalastrum racemosum and Penicillium citrinum exhibited the highest mycelial growths in solid media, while all endophytes adsorbed Congo red. Through liquid decolorization assay, four isolates decolorized Congo red at greater than 89% decolorization rates. P. citrinum (55.45%), Mycelia sterilia (85.19%), A. flavus (44.91%) showed the highest decolorization rates of Methylene blue, Malachite green and Rhodamine B, respectively. The ligninolytic enzymes produced by the endophytic fungi, laccase exhibited the highest activity with values higher than the positive control

Dengue Virus Activates Polyreactive, Natural IgG B Cells after Primary and Secondary Infection

BACKGROUND: Dengue virus is transmitted by mosquitoes and has four serotypes. Cross-protection to other serotypes lasting for a few months is observed following infection with one serotype. There is evidence that low-affinity T and/or B cells from primary infections contribute to the severe syndromes often associated with secondary dengue infections. such pronounced immune-mediated enhancement suggests a dengue-specific pattern of immune cell activation. This study investigates the acute and early convalescent B cell response leading to the generation of cross-reactive and neutralizing antibodies following dengue infection. METHODOLOGY/PRINCIPAL FINDINGS: We assayed blood samples taken from dengue patients with primary or secondary infection during acute disease and convalescence and compared them to samples from patients presenting with non-dengue related fever. Dengue induced massive early plasmablast formation, which correlated with the appearance of polyclonal, cross-reactive IgG for both primary and secondary infection. Surprisingly, the contribution of IgG to the neutralizing titer 4-7 days after fever onset was more than 50% even after primary infection. CONCLUSIONS/SIGNIFICANCE: Poly-reactive and virus serotype cross-reactive IgG are an important component of the innate response in humans during both primary and secondary dengue infection, and "innate specificities" seem to constitute part of the adaptive response in dengue. While of potential importance for protection during secondary infection, cross-reactive B cells will also compete with highly neutralizing B cells and possibly interfere with their development

Molecular detection of whitefly-transmissible geminiviruses (family Geminiviridae, genus Begomovirus) in the Philippines

Whitefly-transmissible geminiviruses (family Geminiviridae, genus Begomovirus) were detected by the polymerase chain reaction (PCR) in total nucleic acid preparations of tomato and squash leaf samples from different areas in the country. Begomovirus DNA fragments were detected by PCR using 3 sets of degenerate primers that amplify different regions of the genomic A DNA component of begomoviruses. The core coat protein primer pair gave the diagnostic ~560 bp amplicons; the top half primers produced ~1.6 kb PCR products; and the bottom half primers yielded ~1.2 kb amplicons. Not all samples produced amplicons of the expected sizes in PCR. Some samples produced bands in all 3 primer sets, some in only 2 of the 3 sets of primers, while some produced PCR fragments in only 1 of the 3 primer pairs, which suggests variation in the virus DNA sequence in terms of the presence or absence of primer annealing sites. Southern blot analysis using as probe the PCR product amplified from a top half clone of the genomic A DNA component of the Philippine isolate of Tomato leaf curl geminivirus (ToLCV- Ph), confirmed the Begomovirus identity of the top half primer pair-generated amplicons. Results confirmed that begomoviruses are responsible for some tomato and squash leaf curl diseases and the prevalence of these diseases threatens sustainable production of tomato and squash in the country

Evaluation of the potential anti-viral activity of microRNAs in rainbow trout

Background: Microribonucleic acids (miRNAs) are small (18-22 nucleotides) endogenous RNAs that potently mediate post-transcriptional silencing of a wide range of genes. They are emerging as critical regulators of cellular processes and some miRNAs have been demonstrated to possess direct antiviral effects. We have previously observed and validated that the fish-specific miRNAs, miR-462 and miR- 731, were among the most highly expressed miRNAs in rainbow trout liver following Viral hemorrhagic septicemia virus (VHSV) infection. These miRNAs were also upregulated in the liver and muscle (vaccination site) of fish vaccinated with a DNA vaccine encoding the glycoprotein gene of VHSV. Recent studies further suggest that the expression of these miRNAs is induced by type I interferons (IFN). Here, we analyzed if miR-462 and miR-731 have antiviral effect contributing to the potent antiviral activity of type I IFNs

Putative targets of virus-induced teleost fish homologues of mammalian immune-relevant microribonucleic acids

Background: Microribonucleic acids or microRNAs (miRNAs) are short (18-22 nucleotides) endogenous RNAs that potently regulate the expression of a wide spectrum of genes through the RNA interference mechanism (RNAi). In RNAi, miRNAs repress the translation of target mRNAs or mediate mRNA degradation following interaction with the target 3’-untranslated region (UTR). RNAi has been implicated in the regulation of almost all cellular processes, including host-pathogen interactions. Our previous microarray-based miRNA expression profiling revealed that infection of a salmonid fish with a rhabdovirus induced the expression of several miRNAs in the liver. These miRNAs are homologous to mammalian miRNAs shown to regulate genes involved in immune responses, whereas the roles of these fish miRNAs are yet unclear. Here, we aimed to further investigate the potential interactions of these virus- induced fish miRNAs with mRNAs that may be relevant to host-virus cross-talk. Methodology: To identify potential target mRNAs of 8 of the most highly expressed rhabdovirus-induced miRNAs, we analysed microarray-based mRNA profiles (obtained simultaneously with miRNA expression profiles of the same liver samples). FASTA nucleotide sequences of negatively regulated miRNAs were obtained through NCBI Batch BLAST. The nucleotide sequences were aligned against the Salmon protein database (GCF_000233375.1 version 2) using Local BLASTx (BLAST 2.2.6) to determine identities of presumptive target expressed sequence tags-mRNA. Resulting XML files were further processed for annotation, gene ontology, and inclusive pathways using BLAST2GO 3.1. Additionally, putative targets of the upregulated miRNAs in genomes of salmonid-infecting viruses were predicted using RNA hybrid. Major findings: Analysis of the co-expressed mRNA profiles in the same samples and comparison with miRNA expression data revealed negatively correlated miRNA-mRNA pairs, suggesting miRNA-target relationship. The downregulated mRNAs mapped to more than 80% of the EST-mRNAs in the salmon database. Subsequently, we investigated the potential roles during virus infection of the downregulated mRNAs (and that of their protein products) by analyzing the signaling pathways enriched by the target genes of upregulated miRNAs. Gene-ontology-based annotation and functional/pathway enrichment analysis of inversely correlated mRNAs showed that they are encoded by genes whose protein products were most frequently associated with the cell cycle, immune-relevant pathways, and metabolic pathways known to be altered by virus infections in humans. Finally, analysis of genomes of some salmonid-infecting viruses showed potential targets for rhabdovirus- induced miRNAs, suggesting direct antiviral activities. Conclusions/Perspectives: Our results highlight pathways essential to antiviral defense, besides key miRNAs that modulate these processes. Collectively, our data provide a snapshot of host gene expression dynamics induced by fish rhabdovirus infection and mediated by miRNAs that orchestrate the activities of key regulators of host-virus interactions by their convergent action on multiple target genes involved in these processes. Results will further our understanding of cellular processes that are altered in response to rhabdovirus infection in fish, providing a context for future experimental analyses and functional studies, while miRNA signatures during infection may be used as biomarkers of immune responses in fish and generally, in vertebrates

Isolation and characterization of the oil bodies and oleosin of coconut (Cocos nucifera L.)

Oil bodies were isolated from the solid endosperm of 11–12 month old coconut and purified by sucrose density gradient washing, urea washing and floatation centrifugation. Under light microscope, purified oil bodies showed two types of small and large oil bodies: about 90% with diameter of 0.5–4.0 μ μm and 10%, 10–40 m, respectively. The oil bodies consisted of 97% triacylglycerols, phospholipids (phosphatidylethanoloamine and phosphatidylcholine) and the oil body storage protein oleosin. Puri- fied oleosin showed one band of 14400 on SDS-PAGE. Two-dimensional electrophoresis resolved one band of 14400 with an isoelectric pH between 9 and 10. The 14400 band had an N-terminal amino acid sequence of GEERR or GEEER. Additional urea washings of the coconut oil bodies followed by ether delipidation revealed two oleosin bands of 14400 and 11000 Mr on SDS-PAGE

Oil bodies and oil-associated proteins from coconut (Cocos nucifera L.): Characterization and a modified method for isolation

Background: Oil bodies are small spherical organelles which are deposition sites of plant storage lipids composed mainly of triacylglycerols (TAGs) covered by a protein- phospholipid complex. They are synthesized during the early to middle stages of seed development and are used as energy reserves for germination. Oleosins are a unique class of proteins found in the oil bodies of diverse plant species. They are small, largely hydrophobic proteins embedded in the phospholipid coat of oil bodies. They comprise up to 2–8% of the total seed proteins and are responsible for the oil body size and stability. Oleosins are suggested to play roles including the stabilization of oil bodies preventing their aggregation and as receptor for the binding of lipase to mobilize TAGs during germination and post-germinative growth. The solid endosperm of the seed of coconut (Cocos nucifera L.) consists of about 45–55% oil with high lauric acid content and medium chain triacylglycerol profile. Here, we explored the presence of coconut oil bodies and oleosins, which, by far, have not been explored in coconut. We aimed to improve existing methods for their isolation, determine oil body composition, and characterize oil body components. Materials and Methods: Coconut oil bodies were isolated from 11-12 month old coconut samples (Laguna Tall variety) using both liquid nitrogen-powdered solid endosperm and coconut milk extracted from the solid endosperm as starting material. The oil bodies were purified using two methods. The first method consisted of sucrose density gradient washing, collection of the oil body pad, and floatation centrifugation. An alternative method involved urea washing and recovery by centrifugation. Purified oil bodies were observed and measured under a light microscope. Thin layer chromatography was employed to determine oil body lipid composition. Oil body-associated proteins were separated from lipids by two washes of diethyl ether, removing the ether layer while leaving the protein layer in the middle each time. Alternatively, the aqueous and interfacial layers were washed with chloroform:methanol (2:1 v/v) and centrifuged, followed by collection of protein-containing precipitate. Purified proteins were characterized using SDS-PAGE and two-dimensional electrophoresis. SDS-PAGE-resolved proteins were electroblotted onto PVDF membrane and the protein of the desired molecular weight was subjected to N-terminal sequence analysis. Results: Of 1055 oil bodies measured, up to 76% had a diameter in the range of 1-4 μm (Figure 1a,b). The average diameter of these oil bodies is 2.54 μm consistent with published sizes of plant seed oil bodies. Up to 10 percent of the oil bodies had diameter several times larger (10 μm) than the normal size (1-2 μm). The oil bodies consisted of 97% TAGs, the rest being phospholipids (phosphophatidylethanolamine and phosphatidylcholine) and the oil body storage protein, oleosin. The purified oleosin showed a single 14400 Mr band on SDS-PAGE (Figure 1c). Two-dimensional electrophoresis also resolved one band of 14400 with an isoelectric point of 9-10 (not shown). N-terminal amino acid sequence analysis of the 14.4 kDa band gave GEERR or GEEER, indicating the presence of isoforms. Additional urea washings of the coconut oil bodies followed by ether delipidation showed two oleosin bands of 14400 and 11000 Mr on SDS-PAGE (Figure 1c). While smaller than the reported size of oleosins from other organisms (15–26 kDa), we believe these to be oleosins as they withstood the stringent purification steps. Conclusion/Perspectives: Coconut oil bodies were successfully purified from powdered coconut meat and milk by floatation centrifugation, buffer washing with and without KCl, and buffer with urea. Our results will contribute to the understanding of the biochemistry of the accumulation and storage of proteins and oils in coconut. The discovery of oil bodies and oleosins in coconut intensifies the possibilities of improving the quality of the coconut oil to make it more competitive in the world market. Oleosins are also potential materials in the pharmaceutical industry. Knowing the characteristics of oil body-associated proteins will lay the ground for its applications in the genetic improvement of coconut through biotechnology. Our work is the first report on the isolation and characterization of oil bodies and oleosins of the oil-rich solid endosperm of coconut

Rapid induction of cross-reactive IgG antibodies after dengue infection.

<p>Longitudinal plasma samples of dengue patients were tested by ELISA for dengue-specific IgM (A) and IgG antibodies (B). A and B) Four plasma dilutions from 1∶200 to 1∶25'000 were measured to exclude non-specific binding. For the combined illustration of all samples that were analysed the OD450 of one dilution (1∶5000 for IgG and 1∶1000 for IgM) was divided by the OD450 of a standard serum that was included on each plate. Time points: 1 = 1–3 days after fever, 2 = 4–7 days after fever, 3 = 15–25 days after fever. The same patient samples were analyzed for DENV1–4. Binding intensity correlated for all four serotypes, i.e. high binding to DENV1 would also apply to DENV2, 3 and 4. Primary versus secondary infection status was confirmed with the commercially available Panbio ELISA kit (Inverness Medical, Australia). The experiment was repeated for selected samples, confirming the original results. C) Concentrations of BAFF were measured in the plasma of dengue and flu patients at the indicated time points. D) moDCs were infected with DENV, heat-inactivated (HI) DENV, polyI:C or medium and BAFF was measured in the supernatant at different time points after infection. Data are presented as % of medium control and are the means±SEM of two independent experiments, done in triplicates. TSV01∶medium compared to polyIC∶medium is significantly different (p>0.05, Two-way ANOVA). The source of BAFF in DENV-infected patients is therefore unlikely to be DCs.</p

Polyclonal B cell activation after dengue infection.

<p>Fresh blood samples of dengue and control fever patients were analyzed by flow cytometry at 1–3, 4–7 and 15–25 days after onset of fever. A) CD19⁺CD20⁻CD27⁺CD138⁻ plasmablasts and CD27⁺CD138⁺ plasma cells (B) as % of lymphocytes. squares: other febrile illness (OFI), triangles: primary or secondary dengue infection. Each symbol represents one patient, lines indicate the mean. A Mann-Whitney test was used for statistical analysis. C) Gating strategy for plasmacells and plasmablasts. One secondary dengue patient with high plasma blast formation is shown. Blood was taken 3, 4 and 20 days after onset of fever.</p

Contribution of IgM and IgG to virus neutralization.

<p>A) Plasma samples of primary patients 2 and 3 (same patients as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0029430#pone-0029430-g003" target="_blank">Fig. 3</a>) collected during acute disease and early convalescence were treated with 0.1 M 2-Mercaptoethanol (2-ME) or medium before serial dilution and incubation with virus. 1∶200 to 1∶437'400 diluted plasma samples were incubated with a constant amount of virus and the percentage of infected cells for each plasma dilution was determined by flow cytometry. B) Plasma samples from two primary patients were IgG-depleted and analyzed as in (A). Lower panels show ELISA results to confirm IgG-depletion. 50% neutralizing titers NT50 are summarized in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0029430#pone-0029430-t003" target="_blank">table 3</a>. C) IgM and IgG binding to DENV3 (ELISA; ODsample/ODstandard of plasma samples) were correlated with % plasmacells amongst lymphocytes at day 4–7 after fever onset (refer to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0029430#pone-0029430-g001" target="_blank">Fig. 1</a>). Each symbol represents one patient, n = 9 (6 secondary and 3 primary infections). SR: Spearman R. The correlation with IgG is significant, p = 0.03.</p