Wet Gram Research Articles

Persistent Ascaris Transmission Is Possible in Urban Areas Even Where Sanitation Coverage Is High.

In low-income, urban, informal communities lacking sewerage and solid waste services, onsite sanitation (sludges, aqueous effluent) and child feces are potential sources of human fecal contamination in living environments. Working in informal communities of urban Maputo, Mozambique, we developed a quantitative, stochastic, mass-balance approach to evaluate plausible scenarios of localized contamination that could explain why the soil-transmitted helminth Ascaris remains endemic despite nearly universal coverage of latrines that sequester most fecal wastes. We used microscopy to enumerate presumptively viable Ascaris ova in feces, fecal sludges, and soils from compounds (i.e., household clusters) and then constructed a steady-state mass-balance model to evaluate possible contamination scenarios capable of explaining observed ova counts in soils. Observed Ascaris counts (mean = -0.01 log10 ova per wet gram of soil, sd = 0.71 log10) could be explained by deposits of 1.9 grams per day (10th percentile 0.04 grams, 90th percentile 84 grams) of child feces on average, rare fecal sludge contamination events that transport 17 kg every three years (10th percentile 1.0 kg, 90th percentile 260 kg), or a daily discharge of 2.7 kg aqueous effluent from an onsite system (10th percentile 0.09 kg, 90th percentile 82 kg). Results suggest that even limited intermittent flows of fecal wastes in this setting can result in a steady-state density of Ascaris ova in soils capable of sustaining transmission, given the high prevalence of Ascaris shedding by children (prevalence = 25%; mean = 3.7 log10 per wet gram, sd = 1.1 log10), the high Ascaris ova counts in fecal sludges (prevalence = 88%; mean = 1.8 log10 per wet gram, sd = 0.95 log10), and the extended persistence and viability of Ascaris ova in soils. Even near-universal coverage of onsite sanitation may allow for sustained transmission of Ascaris under these conditions.

Bioengineering of bacteria to assemble custom-made polyester affinity resins.

Proof of concept for the in vivo bacterial production of a polyester resin displaying various customizable affinity protein binding domains is provided. This was achieved by engineering various protein binding domains into a bacterial polyester-synthesizing enzyme. Affinity binding domains based on various structural folds and derived from molecular libraries were used to demonstrate the potential of this technique. Designed ankyrin repeat proteins (DARPins), engineered OB-fold domains (OBodies), and VHH domains from camelid antibodies (nanobodies) were employed. The respective resins were produced in a single bacterial fermentation step, and a simple purification protocol was developed. Purified resins were suitable for most lab-scale affinity chromatography purposes. All of the affinity domains tested produced polyester beads with specific affinity for the target protein. The binding capacity of these affinity resins ranged from 90 to 600 nmol of protein per wet gram of polyester affinity resin, enabling purification of a recombinant protein target from a complex bacterial cell lysate up to a purity level of 96% in one step. The polyester resin was efficiently produced by conventional lab-scale shake flask fermentation, resulting in bacteria accumulating up to 55% of their cellular dry weight as polyester. A further proof of concept demonstrating the practicality of this technique was obtained through the intracellular coproduction of a specific affinity resin and its target. This enables in vivo binding and purification of the coproduced "target protein." Overall, this study provides evidence for the use of molecular engineering of polyester synthases toward the microbial production of specific bioseparation resins implementing previously selected binding domains.

Improvement of the enantioselectivity of lipase (fraction B) from Candida antarctica via adsorpiton on polyethylenimine-agarose under different experimental conditions

Agarose gels coated with a dense layer of polyethylenimine (PEI-agarose containing 1000 μmol of ionised groups per wet gram of support) are able to adsorb proteins under a very wide range of experimental conditions (different temperatures and pH ranging from pH 5.0 to 9.0). Candida antarctica lipase (fraction B) (CAL-B) was adsorbed on PEI-agarose under very different experimental conditions. The different CAL-B preparations were evaluated as catalysts of the enantioselective hydrolysis of R, S-mandelic acid methyl ester under identical conditions. Interestingly, the best enantioselectivity was achieved with lipase adsorbed at pH 9.0 and 4 °C, conditions where the enzyme exhibited the best enantioslectivity when the reaction was carried out under those conditions. Even more interestingly, these properties were even improved if this preparation was used at pH 5 and 4 °C ( E = 25). On the contrary, CAL-B adsorbed at pH 5.0 and 25 °C exhibits a much lower enantioselectivity ( E = 3.5) under the same experimental conditions. That is, the same lipase (CAL-B) adsorbed on the same support (PEI-agarose) and used under the same conditions exhibits very different activity–selectivity properties just by using different adsorption conditions. It seems that different conformations of CAL-B can be fixed by intense multipoint anion-exchange involving very large regions of the enzyme surface interacting with these dense layers of polyethyleneimine.

Purification and very strong reversible immobilization of large proteins on anionic exchangers by controlling the support and the immobilization conditions

The interaction of two large beta-galactosidases (from Escherichia coli and from Thermus sp.) with tailor-made anion exchangers was studied. Using lowly activated supports (e.g., containing 2–3 μmol of ionised groups per wet gram of support), large proteins selectively adsorbed and easily desorbed (e.g., using 200 mM of NaCl), giving highly purified proteins. However, these supports cannot be used to immobilize the enzymes for industrial use, because the weak adsorption. On the other hand, these large proteins strongly adsorb on very highly activated supports (e.g., containing 40 μmol of ionic groups per wet gram of 4 BCL agarose). Thus, these supports may be not valid for large protein purification, but may be very suitable for immobilization of these proteins. Using high ionic strength (e.g., 300 mM NaCl), large proteins still may be adsorbed on these supports, while only around 20% of total proteins adsorb, permitting some purification of the large proteins but not a total one. Moreover, adsorption under these conditions increase the adsorption strength (now there are not desorption even using 800 mM NaCl). Thus, the purification and the strong reversible immobilization of both beta-galactosidases were performed in a very simple two-step process. The large proteins can be directly adsorbed on these supports after desorption (at 200 mM of NaCl) from poorly activated supports. Furthermore, adsorption on very highly activated supports promotes a significant thermal stabilization of both enzymes, mainly in dissociations conditions.

Overproduction and One-Step Purification ofEscherichia coliUDP-N-Acetylglucosamine Enolpyruvyl Reductase

TheEscherichia coligenemurB,encoding the enzyme uridine-5′-diphospho-N-acetyl-2-amino-2-deoxy-3-O-lactylglucose:nicotinamide adenine dinucleotide phosphate oxidoreductase (EC 1.1.1.158) (EP-reductase), the second enzyme in the peptidoglycan biosynthetic pathway, has been amplified using PCR technology with the Kohara recombinant λ phage E11C11 (534) as template. The synthetic gene was subcloned into theNdeI andBamHI restriction sites of the expression vector pT7-7, designed to utilize T7 RNA polymerase to direct transcription of the target gene, in a two-step procedure. The first step involved the directional insertion of the 590-bpNdeI toBamHI restriction fragment ofmurBinto the pT7-7 vector to give the plasmid pT7-7-murB-590. The construction of the desired overproducing plasmid was completed by the bidirectional insertion of the 442-bpBamHI toBamHI restriction fragment ofmurBinto a similarly restricted pT7-7-murB-590 plasmid followed by restriction digestion to select the properly oriented insert, pT7-7-murB.Overexpression of EP-reductase from theE. colistrain BL 21 (DE 3) containing the pT7-7-murBgene, after induction, allowed the production of 36 mg of target protein per 3 wet grams ofE. colicells. The EP-reductase was purified in a single step utilizing dye–ligand chromatography to yield 30 mg of pure protein. The availability of these levels of reductase will allow the mechanism of this pivotal enzyme to be thoroughly studied as a potential target for the design of a new generation of antibiotics. In addition, the EP-reductase generated in this study has been utilized as a coupling enzyme to assay the first enzyme in the peptidoglycan biosynthetic pathway, UDP-N-acetylglucosamine enolpyruvyl transferase, and these results are also presented.

Separation, phenotyping and limiting dilution analysis of T-lymphocytes infiltrating human solid tumors.

Tumor-infiltrating lymphocytes (TIL) were obtained by a combination of mechanical release and enzymatic disaggregation from 35 human solid tumors. The number of lymphocytes in TIL-enriched suspensions varied from 1 X 10(4) to 7.6 X 10(6) per wet gram of tumor. The TIL preparations separated by differential centrifugation on Ficoll-Hypaque gradients contained 10-95% of T11+ cells (mean 50%), and tumor cells accounted for the other major cellular component. Macrophages, NK cells, B cells and granulocytes were infrequently seen. Morphologically, TIL-T were small non-activated cells. They expressed the T11 and T3 antigens but not the receptor for IL-2 (IL-2R) or HLA-DR antigens as determined by double immunofluorescence staining. Rare T11+/IL-2R+ cells were recovered only from colon and lung carcinomas. The mean T4/T8 ratio in 12 TIL preparations was 1.1 +/- 0.8. Immunohistology with monoclonal antibodies (MAbs) performed in 31/35 tumors confirmed that the T11+ cells infiltrating solid tumors rarely expressed the IL-2R and that the cell content of suspensions enriched in TIL was comparable to that determined in situ. The recovered TIL were cloned in a microculture system that permits proliferation of nearly all normal peripheral blood T lymphocytes (PBL-T). Under these culture conditions, frequencies of the proliferating T lymphocyte precursors (PTL-P) were depressed in both the TIL preparations (less than 0.01 to 0.39) and patients' PBL-T (0.05 to 0.5). These low frequencies of PTL-P were seen in patients with all tumor types, both primary and metastatic.

Solid Substrate Fermentation of Feedlot Waste Combined with Feed Grains

A new process is being developed for recycling animal wastes based on a solid substrate fermentation with cracked grains. To date, the work has been done mainly with corn and the liquid fraction of cattle feedlot waste. This liquid, separated by an oscillating 30 mesh screen, contains 10% solids in the form of microbial cells and either fine or soluble waste components; it has a nitrogen content of 3-4 mg/ml. When added to cracked corn at a ratio of 1:2, the thick liquid adheres to grain surfaces. Grain waste mixtures at about 40% moisture, incubated aerobically by tumbling in a slowly revolving (0.5 rpm) vessel, such as a small cement mixer, rapidly undergo an acid fermentation caused by the selective growth of lactic acid bacteria originally present in the waste; other waste organisms die. The number of bacterial cells increases 100 fold with 24 hr; yeasts emerge after extended incubation. The fetid waste odor quickly disappears as 0.1 meq of acid per wet gram of fermented solids is generated while nitrogen is conserved. The acids formed are primarily lactic and acetic, with lesser amounts of propionic and butyric. Exploratory tests with milo and wheat, as well as with wastes from hogs and cattle on different rations, indicate that the new process may be widely applicable. Diluted whole waste may be used when inclusion of fiber in the fermented feed product is of no concern. The solid state system provides an easily established selective environment for microbial growth. Specific organisms and added inorganic nitrogen may be used for a continuous operation to generate higher protein content in grain based rations.

ION EXCHANGE CHROMATOGRAPHIC STUDIES ON ACID-SOLUBLE BRAIN NUCLEOTIDES AND INCORPORATION OF P32INTO THEM

Summary By the modified method of ion exchange resin chromatography of Hurlbert et al, which is designed to perform fractionation in a short time with relatively simple instruments, the brain acid-soluble nucleotide of albino rats were seperated and measured. Under the condition of freezing animals with dropping of liquid air, adenine nucleotide accounted for about 70 % of all nucleotide, and ATP, ADP, AMP were each 0.55, 0.37, 0.36/xM per wet gram of the brain, ATP/ADP being 1.49. nucleotide polyphosphate, especially ATP, were decomposed soon after the death of animals to get vanished at about 30 minutes, while the decrease of ADP was slower than that of ATP. As the result of investigation on the cerebral phosphorus metabolism utilizing P32 in ion exchange resin chromatography, it proved that P32 was rapidly incorporated into the labile-P such as ATP and ADP in case of subaraehnoideal P32 injection with about the same speed as in case of the liver. The main reason why the turn-over from inorganic phosphorus to ATP and ADP was delayed apparently in the case of intraperitoneal P32 injection consists presumably in the unavoidable blood interfusion into the brain in cases of freezing and extraction.