Affinity recovery of Moloney Murine Leukaemia Virus

Lipid enveloped retroviruses such as Moloney Murine Leukaemia Virus (MoMuLV) are commonly used gene therapy vectors. Downstream processing protocols used for their purification are time consuming and a potentially generic, single step capture method for the recovery of retroviral particles is proposed that exploits streptavidin-biotin affinity chromatography. The ability of four conventional adsorbent solid phases, Fractogel, Sepharose, Magnespheres and STREAMLINE immobilised with streptavidin, to capture and recover biotinylated Moloney Murine Leukaemia Virus was studied. MoMuLV can be biotinylated whilst retaining infectivity and the biotinylated virus can be adsorbed to Streptavidin Magnespheres yielding a 2298-fold increase in titre. For optimal virus biotinylation purification using Fractogel streptavidin can yield a 1896-fold increase in cfu/mg of protein and a 1191-fold decrease in DNA/cfu. Infectious virus can be recovered from Fractogel streptavidin with a maximum recovery of 16.7%.     

Caloric restriction reduces cell loss and maintains estrogen receptor-alpha immunoreactivity in the pre-optic hypothalamus of female B6D2F1 mice

Life-long calorie restriction (CR) remains the most robust and reliable means of extending life span in mammals. Among the several theories to explain CR actions, one variant of the neuroendocrine theories of aging postulates that changing hypothalamic sensitivity to endocrine feedback is the clock that times phenotypic change over the life span. If the feedback sensitivity hypothesis is correct, CR animals should display a significantly different pattern of hormone-sensitive cell density and distribution in the hypothalamus. Of the many endocrine signal receptors that may be involved in maintaining hypothalamic feedback sensitivity, our study has selected to begin mapping those for estrogen (E). Altered hypothalamic sensitivity to E is known to schedule reproductive maturation and influence reproductive senescence. Taking estrogen receptor-alpha (ERalpha) immunoreactivity as an index of sensitivity to E, we counted ERalpha immunoreactive and non-immunoreactive cells in the pre-optic hypothalamus of young (6 weeks), ad-libitum (Old-AL) fed old (22 months), and calorie restricted (Old-CR) old (22 months) female B6D2F1 mice. An automated imaging microscopy system (AIMS) was used to generate cell counts for each sampled section of pre-optic hypothalamus. Results show a 38% reduction in ERalpha immunoreactive cells and an 18% reduction in total cell numbers in AL-old mice in comparison to young mice. However, CR mice only show a 19% reduction in ERalpha immunoreactive cells and a 13% reduction in total cell numbers in comparison to young mice. This indicates CR prevents age-related cell loss and maintains estrogen sensitivity in the pre-optic hypothalamus of old female B6D2F1 mice.     

Conjugation of lentivirus to paramagnetic particles via nonviral proteins allows efficient concentration and infection of primary acute myeloid leukemia cells

Nonviral producer cell proteins incorporated into retroviral vector surfaces profoundly influence infectivity and in vivo half-life. We report the purification and concentration of lentiviral vectors using these surface proteins as an efficient gene transduction strategy. Biotinylation of these proteins and streptavidin paramagnetic particle concentration enhances titer 400- to 2,500-fold (to 10(9) CFU/ml for vesicular stomatitis virus G protein and 5 x 10(8) for amphotropic murine leukemia virus envelope). This method also uses newly introduced membrane proteins (B7.1 and DeltaLNGFR) directed to lentiviral surfaces, allowing up to 17,000-fold concentrations. Particle conjugation of lentivirus allows facile manipulation in vitro, resulting in the transduction of 48 to 94% of human acute myeloid leukemia blasts.     

Mechanism of inhibition of fatty acid amide hydrolase by sulfonamide-containing benzothiazoles: long residence time derived from increased kinetic barrier and not exclusively from thermodynamic potency

Fatty acid amide hydrolase (FAAH) has emerged as a potential target for developing analgesic, anxiolytic, antidepressant, sleep-enhancing, and anti-inflammatory drugs, and tremendous efforts have been made to discover potent and selective inhibitors of FAAH. Most known potent FAAH inhibitors described to date employ covalent mechanisms, inhibiting the enzyme either reversibly or irreversibly. Recently, a benzothiazole-based analogue (1) has been described possessing a high potency against FAAH yet lacking a structural feature previously known to interact with FAAH covalently. However, covalent inhibition of FAAH by 1 has not been fully ruled out, and the issue of reversibility has not been addressed. Confirming previous reports, 1 inhibited recombinant human FAAH (rhFAAH) with high potency with IC(50) ~2 nM. It displayed an apparently noncompetitive and irreversible inhibition, titrating rhFAAH stoichiometrically within normal assay times. The inhibition appeared to be time dependent, but the time dependence only improved potency by a small degree (from ~8 to ~2 nM). However, mass spectrometric analyses of the reaction mixture failed to reveal any cleavage product or covalent adduct and showed full recovery of the parent compound, ruling out covalent, irreversible inhibition. Dialysis revealed recovery of enzyme activity from enzyme-inhibitor complex over a prolonged time (>10 h), demonstrating that 1 is indeed a reversible, albeit slowly dissociating inhibitor of FAAH. Molecular docking indicated that the sulfonamide group of 1 could form hydrogen bonds with several residues involved in catalysis, thereby mimicking the transition state. The long residence time displayed by 1 does not appear to derive exclusively from great thermodynamic potency and is consistent with an increased kinetic energy barrier that prevents dissociation from happening quickly.     

The effect of hydraulic retention time on the performance and fouling characteristics of membrane sequencing batch reactors used for the treatment of synthetic petroleum refinery wastewater

The use of membrane sequencing batch reactors, operated at HRT of 8, 16 and 24 h, was considered for the treatment of a synthetic petroleum wastewater. Increase in HRT resulted in statistically significant decrease in MLSS. Removal efficiencies higher than 97% were found for the three model hydrocarbon pollutants at all HRTs, with air stripping making a small contribution to overall removal. Particle size distribution (PSD) and microscopic analysis showed reduction in the protozoan populations in the activated sludge with decreasing HRT. PSD analysis also showed a higher proportion of larger and smaller sized particles at the lowest HRT. The rate of membrane fouling was found to increase with decreasing HRT; SMP, especially carbohydrate SMP, and mixed liquor apparent viscosity also showed a pronounced increase with decreasing HRT, whereas the concentration of EPS and its components decreased. FTIR analysis identified organic compounds as the main component of membrane pore fouling.     

Stimulation of Native Microorganisms for Improving Loose Salty Sand

Prolonged droughts and excessive water harvesting in western Asia has accelerated desertification and caused longer dry seasons of salt lakes. The Aral Sea experience has proven that dust from saline soil is a serious health issue. Various stabilization techniques to reduce wind erosion have been used in the past. However, in recent years, a potentially viable method has been developed; microbial induced calcite precipitation (MICP) has been introduced as a method of soil stabilization, though its effectiveness in saline soils remains to be examined. The effect of salt content in loose sandy soil on calcite precipitation of calcite through stimulation of native bacteria is investigated in this article. Samples with salinity up to 30% salt content were prepared and treated with different culture medium compounds. A number of tests were used to evaluate the effect of the mentioned parameters. The results show that improvement increases with increasing salinity up to 5% salt, and further increase in salinity reduces the effectiveness of improvement. It is also shown that the addition of urea in the culture medium has a significant effect on the urea hydrolysis which resulted in a five-fold increase in compressive strength. Four native strains of halotolerant urease-positive bacteria were also identified.     

Colonization and Biodegradation of Photo-Oxidized Low-Density Polyethylene (LDPE) by New Strains of Aspergillus sp. and Lysinibacillus sp.

The primary objective of this study was the isolation of low-density polyethylene (LDPE)-degrading microorganisms. Soil samples were obtained from an aged municipal landfill in Tehran, Iran, and enrichment culture procedures were performed using LDPE films and powder. Screening steps were conducted using linear paraffin, liquid ethylene oligomer, and LDPE powder as the sole source of carbon. Two landfill-source isolates, identified as Lysinibacillus xylanilyticus XDB9 (T) strain S7-10F and Aspergillus niger strain F1-16S, were selected as super strains. Photo-oxidation (25 days under ultraviolet [UV] irradiation) was used as a pretreatment of the LDPE samples without pro-oxidant additives. The PE biodegradation process was performed for 56 days in a liquid mineral medium using UV-irradiated pure LDPE films without pro-oxidant additives in the presence of the bacterial isolate, the fungal isolate, and the mixture of the two isolates. The process was monitored by measuring the fungal biomass, the bacterial growth, and the pH of the medium. During the process, the fungal biomass and the bacterial growth increased, and the pH of the medium decreased, which suggests the utilization of the preoxidized PE by the selected isolates as the sole source of carbon. Carbonyl and double bond indices exhibited the highest amount of decrement and increment, respectively, in the presence of the fungal isolate, and the lowest indices were obtained from the treatment of a mixture of both fungal and bacterial isolates. Fourier transform infrared (FT-IR), x-ray diffraction (XRD), and scanning electron microscopy (SEM) analyses showed that the selected isolates modified and colonized preoxidized pure LDPE films without pro-oxidant additives.     

Role of channel lysines and the "push through a one-way valve" mechanism of the viral DNA packaging motor

Linear double-stranded DNA (dsDNA) viruses package their genomes into preformed protein shells via nanomotors using ATP as an energy source. The central hub of the bacteriophage φ29 DNA-packaging motor contains a 3.6-nm channel for dsDNA to enter during packaging and to exit during infection. The negatively charged interior channel wall is decorated with a total of 48 positively charged lysine residues displayed as four 12-lysine rings from the 12 gp10 subunits that enclose the channel. The standard notion derived from many models is that these uniquely arranged, positively charged rings play active roles in DNA translocation through the channel. In this study, we tested this prevailing view by examining the effect of mutating these basic lysines to alanines, and assessing the impact of altering the pH environment. Unexpectedly, mutating these basic lysine residues or changing the pH to 4 or 10, which could alter the charge of lysines, did not measurably impair DNA translocation or affect the one-way traffic property of the channel. The results support our recent findings regarding the dsDNA packaging mechanism known as the "push through a one-way valve".