Transport and survival of bacterial and viral tracers through submerged-flow constructed wetland and sand-filter system

Untreated or improperly treated wastewater has often been cited as the primary contamination source of groundwater. The use of decentralized wastewater treatment systems has applicability around the world since it obviates the need for extensive infrastructure development and expenditures. The use of a submerged flow constructed wetland (CW) and a sand filter to remove bacterial and viral pathogens from wastewater streams was evaluated in this study Salmonella sp. and a bacteriophages tracer were used in conjunction with the conservative bromide tracer to understand the fate and transport of these organisms in these treatment systems. Viral breakthrough numbers in the sand filter and CW were similar with a Spearman Rank correlation of 0.8 (P<0.05). In the CW, the virus exhibited almost a 3-log reduction, while in the sand filter, the viruses exhibited a 2-log reduction. The bacterial tracers, however, did not exhibit similar reductions. Low numbers of bacteria and viruses were still detectable in the effluent streams suggesting that disinfection of the effluent is critical. The survival of the tracer bacteria and viruses was as expected dependent on the biotic and abiotic conditions existing within the wastewater. The results suggest that the microbial removal characteristics of decentralized wastewater treatment systems can vary and depend on factors such as adsorption, desorption and inactivation which in turn depend on the design specifics such as filter media characteristics and local climatic conditions.     

Delineating the Specific Influence of Virus Isoelectric Point and Size on Virus Adsorption and Transport through Sandy Soils

Many of the factors controlling viral transport and survival within the subsurface are still poorly understood. In order to identify the precise influence of viral isoelectric point on viral adsorption onto aquifer sediment material, we employed five different spherical bacteriophages (MS2, PRD1, Qβ, X174, and PM2) having differing isoelectric points (pI 3.9, 4.2, 5.3, 6.6, and 7.3 respectively) in laboratory viral transport studies. We employed conventional batch flowthrough columns, as well as a novel continuously recirculating column, in these studies. In a 0.78-m batch flowthrough column, the smaller phages (MS2, X174, and Qβ), which had similar diameters, exhibited maximum effluent concentration/initial concentration values that correlated exactly with their isoelectric points. In the continuously recirculating column, viral adsorption was negatively correlated with the isoelectric points of the viruses. A model of virus migration in the soil columns was created by using a one-dimensional transport model in which kinetic sorption was used. The data suggest that the isoelectric point of a virus is the predetermining factor controlling viral adsorption within aquifers. The data also suggest that when virus particles are more than 60 nm in diameter, viral dimensions become the overriding factor.     

Agrobacterium-mediated transient GUS gene expression in buffel grass (Cenchrus ciliaris L.)

The study was conducted to standardize a protocol for Agrobacterium-mediated genetic transformation of buffel grass (Cenchrus ciliaris L.). Embryogenic calli, produced from one-year-old mature seeds of buffel grass, were used as target cells for Agrobacterium-mediated transformation. A. tumefaciens strain LBA4404, harbouring pCAMBIA-1301 or pCAMBIA-2301, was used for co-cultivation with embryogenic calli from three genotypes (IG-3108, IG-9757 and IG-97101). Co-culturing of calli with Agrobacterium for 30 minutes, followed by co-cultivation with 0.1 mM acetosyringone for 3 days was found to be optimum for maximum transformation efficiency. Presence of acetosyringone during co-cultivation was found to be necessary for transformation. Transient GUS (beta-glucuronidase) gene expression was used to monitor T-DNA delivery into the target cells. Significant genotypic variations in response to transformation were observed among the tested genotypes. A very high frequency (63.3%) of GUS gene expression was obtained following Agrobacterium-mediated gene transfer into embryogenic calli. The standardized protocol would be useful for Agrobacterium-mediated genetic transformation of buffel grass with genes of agronomic importance.     

Studies on the production and application of cellulase from Trichoderma reesei QM-9414

High yielding mutant strain, Trichoderma reesei QM-9414, was employed for the cellulase enzyme production. Enzyme production conditions (pH, inoculum age and concentration, and organic supplements) were optimized. The ability of partially purified enzyme to hydrolyze various regionally abundant lignocellulosic raw materials was studied. Enzymatic hydrolysis conditions (temperature, pH, enzyme and substrate concentrations) were optimized. Temperature 50v°C, pH 4.5, enzyme concentration 40 FPU/g substrate and substrate concentration 2.5% were found to be optimum for the maximum yields of sugars. #-glucosidase supplementation was found to increase both the sugar yield and hydrolysis rate, and shorten the reaction time significantly.     

Production of Recombinant Human Bile Salt Stimulated Lipase and Its Variant inPichia pastoris

hBSSL and its truncated variant hBSSL-C cDNA clones were expressed inPichia pastorisusing two different signal peptides, native signal peptide and invertase signal peptide, respectively, to facilitate secretion of the recombinant proteins into the culture medium. Both recombinant proteins were secreted into the culture medium to a level of 45–50 mg/liter in shake flask cultures. Native signal peptide of hBSSL was recognized inP. pastorisand was cleaved at the same site as in humans. The level of expression of the hBSSL gene was found to be dependent on the number of its copies integrated into the host chromosome. The multicopy transformant clone was found to be very stable. When grown and induced in a fermentor, the level of accumulation of the recombinant hBSSL in the culture medium improved from 50 mg/liter in shake flask cultures to 300 mg/liter. The recombinant hBSSL purified from the culture supernatant was found to be similar to the native hBSSL in its biochemical properties except for the lectin-binding profile.     

Mimetic ligand-based affinity purification of immune complexes and immunoconjugates

We developed a simple purification method to purify alkaline phosphatase/anti-alkaline phosphatase IgG as immune complexes using mimetic affinity chromatography wherein the antibody was either a monospecific antibody, a bispecific antibody or a commercial polyclonal IgG conjugated with alkaline phosphatase (AP–IgG) covalently. The immune complexes or conjugates were efficiently bound on the mimetic Blue A6XL column and eluted under mild conditions (5–20 mM phosphate buffer). A similar strategy of purifying peroxidase/anti-peroxidase antibody complexes was also successfully demonstrated using the mimetic Red 3 column. Mimetic affinity chromatography thus appears to be a simple method to purify the desired monospecific or bispecific antibodies from the respective hybridomas and quadromas.     

Arrestin‐Domain Containing Protein 1 (Arrdc1) Regulates the Protein Cargo and Release of Extracellular Vesicles

Extracellular vesicles (EVs) are lipid‐bilayered vesicles that are released by multiple cell types and contain nucleic acids and proteins. Very little is known about how the cargo is packaged into EVs. Ubiquitination of proteins is a key posttranslational modification that regulates protein stability and trafficking to subcellular compartments including EVs. Recently, arrestin‐domain containing protein 1 (Arrdc1), an adaptor for the Nedd4 family of ubiquitin ligases, has been implicated in the release of ectosomes, a subtype of EV that buds from the plasma membrane. However, it is currently unknown whether Arrdc1 can regulate the release of exosomes, a class of EVs that are derived endocytically. Furthermore, it is unclear whether Arrdc1 can regulate the sorting of protein cargo into the EVs. Exosomes and ectosomes are isolated from mouse embryonic fibroblasts isolated from wild type and Arrdc1‐deficient (Arrdc1^?/?) mice. Nanoparticle tracking analysis–based EV quantitation shows that Arrdc1 regulates the release of both exosomes and ectosomes. Proteomic analysis highlights the change in protein cargo in EVs upon deletion of Arrdc1. Functional enrichment analysis reveals the enrichment of mitochondrial proteins in ectosomes, while proteins implicated in apoptotic cleavage of cell adhesion proteins and formation of cornified envelope are significantly depleted in exosomes upon knockout of Arrdc1.