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CRISPR/Cas9-based therapeutics hold the possibility for permanent treatment of genetic disease. The potency and specificity of this system has been used to target dominantly inherited conditions caused by heterozygous missense mutations through inclusion of the mutated base in the short-guide RNA (sgRNA) sequence. This research evaluates a novel approach for targeting heterozygous single-nucleotide polymorphisms (SNPs) using CRISPR/Cas9. We determined that a mutation within KRT12, which causes Meesmann's epithelial corneal dystrophy (MECD), leads to the occurrence of a novel protospacer adjacent motif (PAM). We designed an sgRNA complementary to the sequence adjacent to this SNP-derived PAM and evaluated its potency and allele specificity both in vitro and in vivo. This sgRNA was found to be highly effective at reducing the expression of mutant KRT12 mRNA and protein in vitro. To assess its activity in vivo we injected a combined Cas9/sgRNA expression construct into the corneal stroma of a humanized MECD mouse model. Sequence analysis of corneal genomic DNA revealed non-homologous end-joining repair resulting in frame-shifting deletions within the mutant KRT12 allele. This study is the first to demonstrate in vivo gene editing of a heterozygous disease-causing SNP that results in a novel PAM, further highlighting the potential for CRISPR/Cas9-based therapeutics.

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Current pesticide health risk assessments in the United States require the characterization of aggregate exposure and cumulative risk in the setting of food tolerances. Biologic monitoring can aggregate exposures from all sources and routes, and can integrate exposures for chemicals with a common mechanism of action. Its value was demonstrated in a recent study of organophosphorus (OP) pesticide exposure among 109 children in an agricultural community in Washington State; 91 of the children had parents working in agriculture. We estimated individual OP pesticide doses from urinary metabolite concentrations with a deterministic steady state model, and compared them to toxicologic reference values. We evaluated doses by assuming that metabolites were attributable entirely to either azinphos-methyl or phosmet, the two OP pesticides used most frequently in the region. Creatinine-adjusted average dose estimates during the 6- to 8-week spraying season ranged from 0 to 36 microg/kg/day. For children whose parents worked in agriculture as either orchard applicators or as fieldworkers, 56% of the doses estimated for the spray season exceeded the U.S. Environmental Protection Agency (EPA) chronic dietary reference dose, and 19% exceeded the World Health Organization acceptable daily intake values for azinphos-methyl. The corresponding values for children whose parents did not work in agriculture were 44 and 22%, respectively. The percentage of children exceeding the relevant reference values for phosmet was substantially lower (< 10%). Single-day dose estimates ranged from 0 to 72 microg/kg/day, and 26% of these exceeded the EPA acute reference dose for azinphos-methyl. We also generated dose estimates by adjustment for total daily urine volume, and these estimates were consistently higher than the creatinine-adjusted estimates. None of the dose estimates exceeded the empirically derived no-observable-adverse-effect levels for these compounds. The study took place in an agricultural region during a period of active spraying, so the dose estimates for this population should not be considered representative of exposures in the general population. The findings indicate that children living in agricultural regions represent an important subpopulation for public health evaluation, and that their exposures fall within a range of regulatory concern. They also demonstrate that biologically based exposure measures can provide data for health risk evaluations in such populations.

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Children can be exposed to pesticides from multiple sources and through multiple pathways. In addition to the standard pathways of diet, drinking water and residential pesticide use, children in agricultural communities can be exposed to pesticides used in agricultural production. A research program on children and pesticides was established at the University of Washington (UW) in 1991 and has focused on two major exposure pathway issues: residential proximity to pesticide-treated farmland and transfer of pesticides from the workplace to the home (paraoccupational or take-home exposure). The UW program selected preschool children of agricultural producers and farm workers in the tree fruit region of Washington state as a population that was likely to have elevated exposures from these pathways. The organophosphorus (OP) pesticides were selected as a common class of chemicals for analysis so that issues of aggregate exposure and cumulative risk could be addressed. This paper provides an overview of key findings of our research group over the past 8 years and describes current studies in this field. Soil and housedust concentrations of OP pesticides were elevated in homes of agricultural families (household members engaged in agricultural production) when compared to non-agricultural reference homes in the same community. Dialkyl phosphate metabolites of OP pesticides measured in children's urine were also elevated for agricultural children when compared to reference children and when compared to children in the Seattle metropolitan area. Proximity to farmland was associated with increased OP pesticide concentrations in housedust and OP pesticide metabolites in urine. Current studies include a community-based intervention to reduce parental transfer of pesticides from the workplace, and a systematic investigation of the role of agricultural spray drift in children's exposure to pesticides.

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This paper presents mass spectral data of eleven aminoglycoside antibiotics by using californium-252 plasma desorption (252Cf PD) mass spectrometry. This mass spectral data could be used to develop a confirmatory method for monitoring aminoglycoside antibiotic residues isolated from food products of animal origin. Mass spectra were determined by applying time-of-flight 252Cf PD mass spectrometry to eleven aminoglycoside antibiotics, namely: neomycin, kanamycin, paromomycin, tobramycin, apramycin, streptomycin, dihydrostreptomycin, amikacin, netilmicin, sisomicin and gentamicins. All eleven antibiotics yielded positive ion spectra. These hydrophilic antibiotics were derivitized to extractable chromopheric compounds. All but two antibiotics (streptomycin and dihydrostreptomycin) yielded nitrophenyl derivatives and spectra were obtained in both negative and positive ion modes. Derivatized aminoglycosides produced cation and anion spectra with quasimolecular ions corresponding to [M + H]+., M+, [M - H]-., [M + Na]+, [M + K]+ and M-. or [M - H]- and M-. or [M - H]-. Underivatized antibiotics were best examined in the positive ion mode. 252Cf PD mass spectrometry consistently produced very strong molecular or quasimolecular ions for all aminoglycoside antibiotics.

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This survey determines the dental care needs of hospitalized adults with psychiatric and mental disorders. Comprehensive clinical and radiographic oral examinations, conducted on 33% of the patients in a large state mental hospital, found that extensive unmet needs existed in this population. The major requirements were for prophylaxis, calculus removal, and periodontal therapy. The patients' needs varied depending on several demographic factors, including length of hospitalization and psychiatric diagnoses.

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Extraction efficiency was monitored, using 14C-neomycin and liquid scintillation counting, during development of a procedure to isolate neomycin from fortified kidney tissue. Chemiluminescence caused 119% false positive recovery at 0.16 ppm neomycin (1260 disintegrations/min (dpm) activity). However, the contribution of chemiluminescence to false positive recovery was negligible above 8 ppm neomycin (63,000 dpm activity). To reduce chemiluminescence, fortified kidney tissue was extracted with saline by homogenization, mixed with Protosol at 70 degrees C for 1 h, cooled 5 min at 4 degrees C and neutralized with acetic acid. The digest was mixed with Aquasol and incubated 1 h at 47 degrees C, followed by equilibration for 2 h in the counter. The procedure allows reliable monitoring of extraction efficiency down to about 407 dpm activity from 14C-neomycin (0.05 ppm) with maximum chemiluminescence of about 4%.

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A survey of literature is presented dealing with physical-chemical methods for the detection and quantitation of aminoglycoside antibiotics (gentamicin, streptomycin, dihydrostreptomycin, and neomycin) that are used in food-producing animals. Recent developments in cleanup and determinative procedures, particularly liquid chromatography, for these compounds in fluids and tissues are emphasized. Little research has been done on residues in tissues compared with other biological matrices. This review also covers the chemistry, general characteristics, tolerances, and withdrawal times for the approved uses of these antibiotics in animals that are used for food.

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Although chloramphenicol is not approved for use in food-producing animals in the United States, this broad spectrum antibiotic has been widely used to treat diseases in such animals including the lactating dairy cow. Extremely low ophthalmologic doses of chloramphenicol are known to cause aplastic anemia in humans. The residues in meat, milk, and eggs intended for human consumption cause particular public health concern because the bone marrow aplasia is not dose dependent. Furthermore, chloramphenicol, a known inhibitor of protein synthesis, also retards erythropoiesis, a condition that is dose dependent and may cause allergic hypersensitivity reactions. This paper is a review of sensitive methods that use gas, liquid, thin layer, and simple column chromatography as both determinative and cleanup steps for detecting and quantitating chloramphenicol in edible animal tissues, milk, and eggs.

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A liquid chromatographic (LC) method is described for the determination of neomycin in animal tissues. Tissues are homogenized in 0.2M potassium phosphate buffer (pH 8.0); the homogenate is centrifuged, and the supernate is heated to precipitate the protein. The heat-deproteinated extract is acidified to pH 3.5-4 and directly analyzed by LC. The LC method consists of an ion-pairing mobile phase, a reverse phase ODS column, post-column derivatization with o-phthalaldehyde reagent, and fluorometric detection. The LC method uses paromomycin as an internal standard, and separates neomycin from streptomycin or dihydrostreptomycin because they have different retention times. The LC column separates neomycin in 25 min; the detection limit is about 3.5 ng neomycin. The overall recovery of neomycin from kidney tissues spiked at 1-30 ppm was 96% with a 9.0% coefficient of variation. The method was also applied to muscle tissue.

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Serum samples from 27 yearling heifers and 50 lactating cows were analyzed for pepsinogen concentration. Concentrations were 44 +/- 12 (SD) ng of pepsinogen/ml (2,200 mU of tyrosine/ml) of serum for the yearling heifers and 20 +/- 8 ng/ml (1,400 mU of tyrosine/ml) for the lactating cows. These initial data indicate that a yearling calf, grazing on pasture, with a serum pepsinogen concentration greater than 68 ng/ml (2,900 mU of tyrosine/ml) should be examined for ostertagiasis.

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Plasma and tissue concentration and pharmacokinetics of chlortetracycline (CTC) was determined in milk-fed and conventionally fed Holstein calves. A two-compartment open model was used after a single intravenous dose (11 mg CTC/kg body weight). There were no significant differences between dietary treatments. The drug was rapidly distributed from plasma into the peripheral compartment but was slowly eliminated, with detectable concentration of CTC continuing for 72 h after dosing. A single-compartment model was used after a single oral dose (22 mg CTC/kg body weight). All but four of the kinetic parameters were significantly different for the two dietary treatments. Milk-fed calves had a larger area under the plasma level curve, a larger fraction of the dose absorbed, a smaller volume of distribution and a smaller overall body clearance rate. Estimated recovery of CTC in the urine of the milk-fed calves was greater, regardless of route of administration. The concentration of CTC in tissues following an oral dose was greatest in kidney, followed by liver, heart, skeletal muscle, spleen and brain. Tissue depletion of CTC closely paralleled the decline in plasma concentration.

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A simplification of the traditional hemoglobin methods for determining serum pepsinogen concentration was developed. In this method, 10% trichloroacetic acid solution was added to control samples, and hemoglobin substrate was added to controls and active enzyme samples; standards and samples were incubated for 18 hours, the proteins in the active tubes were precipitated with trichloroacetic acid and removed by filtration, and the absorbances of the supernatant of each standard and sample at 280 nm were measured. The major differences between this method and other methods for determining pepsinogen values are that the preacidification of serum with hydrochloric acid was eliminated, the incubation period was reduced to 18 hours (down from 24 hours), the relative pepsinogen concentration was determined by measuring the concentration of hydrolysis products, using ultraviolet, rather than visible absorbance, and a pepsin standard curve was used to determine the serum pepsinogen concentration. Comparison of freshly prepared pepsinogen and pepsin standard curves indicated that the pepsinogen preparations were slightly more active than the pepsin preparations (on a weight-to-weight basis) on the same substrate. Pepsin standards are used because they are more stable than pepsinogen standards. Three linear standard curve ranges were used: O 10 to 100, 50 to 300, and 100 to 500 ng of pepsinogen/ml of serum. The use of pepsin standard curves permits some variability of the incubation conditions without altering the results. For best results, the hemoglobin substrate solution should be prepared daily. This method may be useful in diagnosing ostertagiasis.

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Effects of diet and age on selected blood chemistry and hematologic characteristics were studied in 20 Holstein bull calves. Milk-fed calves (treatment 1) were maintained exclusively on a milk-replacer diet. Conventionally fed calves (treatment 2) were provided a calf-starter diet and alfalfa hay beginning at 2 wk of age and were weaned from milk replacer at 7 wk of age. Blood samples were collected by jugular puncture from all animals just before the morning feeding at 6, 7, 9, and 11 wk of age. Milk-fed calves had greater glucose concentration in blood serum. Urea nitrogen of blood increased in conventionally fed calves from 6 through 11 wk and decreased in milk-fed calves. Hemoglobin concentration and packed cell volume decreased in calves receiving milk-replacer diet. Total protein and albumin concentrations were greater in the conventionally fed group. Treatment differences also existed for calcium and cholesterol concentrations and in alkaline phosphatase activity.

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This study describes the early radiological changes which occur in infections of the spine, by examination of the initial radiographs in 45 patients with proven spinal infection. The commonest radiological change in infection of the spine was a symmetrical lesion involving the adjacent surfaces of two vertebrae, with a variable degree of reduction in height of the disc space. A paravertebral abscess was a common associated finding. No radiological pattern was completely reliable in distinguishing tuberculous from non-tuberculous infections, but in white patients the formation of new bone strongly suggested a pyogenic lesion. The presence or absence of a paravertebral abscess was not helpful in distinguishing the variety of infection.

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