Rapid host strain improvement by in vivo rearrangement of a synthetic yeast chromosome.

Synthetic biology tools, such as modular parts and combinatorial DNA assembly, are routinely used to optimise the productivity of heterologous metabolic pathways for biosynthesis or substrate utilisation, yet it is well established that host strain background is just as important for determining productivity. Here we report that in vivo combinatorial genomic rearrangement of Saccharomyces cerevisiae yeast with a synthetic chromosome V can rapidly generate new, improved host strains with genetic backgrounds favourable to diverse heterologous pathways, including those for violacein and penicillin biosynthesis and for xylose utilisation. We show how the modular rearrangement of synthetic chromosomes by SCRaMbLE can be easily determined using long-read nanopore sequencing and we explore experimental conditions that optimise diversification and screening. This synthetic genome approach to metabolic engineering provides productivity improvements in a fast, simple and accessible way, making it a valuable addition to existing strain improvement techniques.

Distinguishing Petroleum (Crude Oil and Fuel) From Smoke Exposure within Populations Based on the Relative Blood Levels of Benzene, Toluene, Ethylbenzene, and Xylenes (BTEX), Styrene and 2,5-Dimethylfuran by Pattern Recognition Using Artificial Neural Networks.

Studies of exposure to petroleum (crude oil/fuel) often involve monitoring benzene, toluene, ethylbenzene, xylenes (BTEX), and styrene (BTEXS) because of their toxicity and gas-phase prevalence, where exposure is typically by inhalation. However, BTEXS levels in the general U.S. population are primarily from exposure to tobacco smoke, where smokers have blood levels on average up to eight times higher than nonsmokers. This work describes a method using partition theory and artificial neural network (ANN) pattern recognition to classify exposure source based on relative BTEXS and 2,5-dimethylfuran blood levels. A method using surrogate signatures to train the ANN was validated by comparing blood levels among cigarette smokers from the National Health and Nutrition Examination Survey (NHANES) with BTEXS and 2,5-dimethylfuran signatures derived from the smoke of machine-smoked cigarettes. Classification agreement for an ANN model trained with relative VOC levels was up to 99.8% for nonsmokers and 100.0% for smokers. As such, because there is limited blood level data on individuals exposed to crude oil/fuel, only surrogate signatures derived from crude oil and fuel were used for training the ANN. For the 2007-2008 NHANES data, the ANN model assigned 7 out of 1998 specimens (0.35%) and for the 2013-2014 NHANES data 12 out of 2906 specimens (0.41%) to the crude oil/fuel signature category.

Marginal iodide deficiency and thyroid function: dose-response analysis for quantitative pharmacokinetic modeling.

Severe iodine deficiency (ID) results in adverse health outcomes and remains a benchmark for understanding the effects of developmental hypothyroidism. The implications of marginal ID, however, remain less well known. The current study examined the relationship between graded levels of ID in rats and serum thyroid hormones, thyroid iodine content, and urinary iodide excretion. The goals of this study were to provide parametric and dose-response information for development of a quantitative model of the thyroid axis. Female Long Evans rats were fed casein-based diets containing varying iodine (I) concentrations for 8 weeks. Diets were created by adding 975, 200, 125, 25, or 0 µg/kg I to the base diet (~25 µg I/kg chow) to produce 5 nominal I levels, ranging from excess (basal+added I, Treatment 1: 1000 µg I/kg chow) to deficient (Treatment 5: 25 µg I/kg chow). Food intake and body weight were monitored throughout and on 2 consecutive days each week over the 8-week exposure period, animals were placed in metabolism cages to capture urine. Food, water intake, and body weight gain did not differ among treatment groups. Serum T4 was dose-dependently reduced relative to Treatment 1 with significant declines (19 and 48%) at the two lowest I groups, and no significant changes in serum T3 or TSH were detected. Increases in thyroid weight and decreases in thyroidal and urinary iodide content were observed as a function of decreasing I in the diet. Data were compared with predictions from a recently published biologically based dose-response (BBDR) model for ID. Relative to model predictions, female Long Evans rats under the conditions of this study appeared more resilient to low I intake. These results challenge existing models and provide essential information for development of quantitative BBDR models for ID during pregnancy and lactation.

Evaluation of potassium iodide (KI) and ammonium perchlorate (NH4ClO4) to ameliorate 131I- exposure in the rat.

Nuclear reactor accidents and the threat of nuclear terrorism have heightened the concern for adverse health risks associated with radiation poisoning. Potassium iodide (KI) is the only pharmaceutical intervention that is currently approved by the Food and Drug Administration for treating (131)I(-) exposure, a common radioactive fission product. Though effective, KI administration needs to occur prior to or as soon as possible (within a few hours) after radioactive exposure to maximize the radioprotective benefits of KI. During the Chernobyl nuclear reactor accident, KI was not administered soon enough after radiation poisoning occurred to thousands of people. The delay in administration of KI resulted in an increased incidence of childhood thyroid cancer. Perchlorate (ClO(4)(-)) was suggested as another pharmaceutical radioprotectant for 131I- poisoning because of its ability to block thyroidal uptake of iodide and discharge free iodide from the thyroid gland. The objective of this study was to compare the ability of KI and ammonium perchlorate to reduce thyroid gland exposure to radioactive iodide (131I-). Rats were dosed with 131I- tracer and 0.5 and 3 h later dosed orally with 30 mg/kg of either ammonium perchlorate or KI. Compared to controls, both anion treatments reduced thyroid gland exposure to 131I- equally, with a reduction ranging from 65 to 77%. Ammonium perchlorate was more effective than stable iodide for whole-body radioprotectant effectiveness. KI-treated animals excreted only 30% of the (131)I(-) in urine after 15 h, compared to 47% in ammonium perchlorate-treated rats. Taken together, data suggest that KI and ammonium perchlorate are both able to reduce thyroid gland exposure to 131I- up to 3 h after exposure to 131I-. Ammonium perchlorate may offer an advantage over KI because of its ability to clear 131I- from the body.

Hypochlorite treatments are not a significant source of perchlorate exposure in lettuce.

Leafy vegetables, such as lettuce ( Lactuca sativa L), have been identified as a potential source of perchlorate exposure to humans. Perchlorate is of concern because excessive amounts may impair thyroid function by inhibiting iodide uptake by the sodium iodide symporter. Perchlorate has been identified as an oxidation product in sodium hypochlorite. Dilute hypochlorite solutions are widely used on lettuce as a preservative and as a treatment to reduce microbial food risks. However, the potential of hypochlorite to be a source of human perchlorate exposure from lettuce had not been evaluated. Studies were conducted with lettuce collected in the San Luis Valley of southern Colorado and in the lower Colorado River Valley of southwestern Arizona to represent conditions under which hypochlorite is applied to lettuce in the field and in salad processing facilities. We used spray and dipping solutions that were dilutions of concentrated sodium hypochlorite that would contain from 12000 and 120000 microg/L perchlorate. The perchlorate content of iceberg and romaine lettuce averaged 6.2 and 7.2 microg/kg fw in southern Colorado and 14.0 and 56.7 microg/kg fw in southwestern Arizona and there were no significant (P > 0.05) increases in the perchlorate content of lettuce due to hypochlorite treatments. Because of the relatively low concentrations of perchlorate present after dilution and the low volumes applied to lettuce, hypochlorite solutions do not appear to be a significant source of the perchlorate levels found in lettuce.

Perchlorate in the feed-dairy continuum of the southwestern United States.

Perchlorate has the potential to cause thyroid dysfunction by inhibiting iodide uptake by the sodium iodide symporter. Perchlorate-contaminated waters may lead to human exposure through drinking water and food chain transfer in crops by way of irrigation water. Perchlorate has been found in dairy milk collected nationally and internationally. This study was conducted to evaluate perchlorate in the feed-dairy continuum in the southwestern United States. All feed products collected at dairies in this study had detectable levels of perchlorate as analyzed by ion chromatography-tandem mass spectrometry. The calculated total perchlorate intake across dairies ranged from 1.9 to 12.7 mg/cow per day. The variation in total perchlorate intake across dairies was largely associated with variation in forage and silage products. Alfalfa products were the single most important source of perchlorate intake variability among dairies. The estimated perchlorate intake from drinking water ranged from 0.01 mg per cow per day and was generally less than 2% of the total perchlorate intake. The perchlorate content of milk ranged from 0.9 to 10.3 microg/L and was similar to levels reported by the Food and Drug Administration's Total Diet Study. The perchlorate content of milk was significantly related to the presence of perchlorate in feed but the variation of perchlorate in milk could not be explained by feed intake alone.

Disinfection by-product formation and mitigation strategies in point-of-use chlorination of turbid and non-turbid waters in western Kenya.

Over 1.1 billion people in the world lack access to improved drinking water. Diarrheal and other waterborne diseases cause an estimated 2.2 million deaths per year. The Safe Water System (SWS) is a proven household water treatment intervention that reduces diarrheal disease incidence in users in developing countries. Because the SWS recommends the addition of sodium hypochlorite to unfiltered water sources, concerns have been raised about the potential long-term health effects of disinfection by-products to SWS users. This study investigated the production of trihalomethanes (THMs) in water treated with sodium hypochlorite from six sources used for drinking water in western Kenya. The turbidity values of these sources ranged from 4.23 NTU to 305 NTU. THM concentrations were analysed at 1, 8, and 24 hours after addition of sodium hypochlorite. No sample exceeded the World Health Organization (WHO) guideline values for any of the four THMs: chloroform, bromodichloromethane, dibromochloromethane, or bromoform. In addition, no sample exceeded the WHO additive total THM guideline value. These results clearly show that point-of-use chlorination of a variety of realistic source waters used for drinking did not lead to THM concentrations that pose a significant health risk to SWS users.

Perchlorate, thiocyanate, and nitrate in edible cole crops (Brassica sp.) produced in the lower Colorado River region.

The Colorado River is contaminated with low levels of perchlorate. Perchlorate has the potential to disrupt thyroid function by inhibiting the uptake of iodide. Brassica are rich sources of thiocyanate and nitrate, also inhibitors of iodide uptake. This study was conducted to estimate potential human exposure to perchlorate, thiocyanate, and nitrate from Brassica sp. irrigated with Colorado River water. Results indicate that Brassica sp. irrigated with Colorado River water do accumulate trace levels of perchlorate. However, the levels of perchlorate observed are low relative to the nitrate and thiocyanate naturally present in these species and low relative to the reference dose recommended by the NAS and the USEPA.

Potential perchlorate exposure from Citrus sp. irrigated with contaminated water.

Citrus produced in the southwestern United States is often irrigated with perchlorate-contaminated water. This irrigation water includes Colorado River water which is contaminated with perchlorate from a manufacturing plant previously located near the Las Vegas Wash, and ground water from wells in Riverside and San Bernardino counties of California which are affected by a perchlorate plume associated with an aerospace facility once located near Redlands, California. Studies were conducted to evaluate the uptake and distribution of perchlorate in citrus irrigated with contaminated water, and estimate potential human exposure to perchlorate from the various citrus types including lemon (Citrus limon), grapefruit (Citrus paradise), and orange (Citrus sinensis) produced in the region. Perchlorate concentrations ranged from less than 2-9 microg/L for Colorado River water and from below detection to approximately 18 microg/L for water samples from wells used to irrigate citrus. Destructive sampling of lemon trees produced with Colorado River water show perchlorate concentrations larger in the leaves (1835 microg/kg dry weight (dw)) followed by the fruit (128 microg/kg dw). Mean perchlorate concentrations in roots, trunk, and branches were all less than 30 microg/kg dw. Fruit pulp analyzed in the survey show perchlorate concentrations ranged from below detection limit to 38 microg/kg fresh weight (fw), and were related to the perchlorate concentration of irrigation water. Mean hypothetical exposures (mug/person/day) of children and adults from lemons (0.005 and 0.009), grapefruit (0.03 and 0.24), and oranges (0.51 and 1.20) were estimated. These data show that potential perchlorate exposures from citrus in the southwestern United States are negligible relative to the reference dose recommended by the National Academy of Sciences.

Rosacea: a common, yet commonly overlooked, condition.

Rosacea is a common, but often overlooked, skin condition of uncertain etiology that can lead to significant facial disfigurement, ocular complications, and severe emotional distress. The progression of rosacea is variable; however, typical stages include: (1) facial flushing, (2) erythema and/or edema and ocular symptoms, (3) papules and pustules, and (4) rhinophyma. A history of exacerbation by sun exposure, stress, cold weather, hot beverages, alcohol consumption, or certain foods helps determine the diagnosis; the first line of treatment is avoidance of these triggering or exacerbating factors. Most patients respond well to long-term topical antibiotic treatment. Oral or topical retinoid therapy may also be effective. Laser treatment is an option for progressive telangiectasis or rhinophyma. Family physicians should be able to identify and effectively treat the majority of patients with rosacea. Consultation with subspecialists may be required for the management of rhinophyma, ocular complications, or severe disease. (Am Fam Physician 2002;66:442.)

Determination of aniline derivatives in oils related to the toxic oil syndrome by atmospheric pressure ionization-tandem mass spectrometry.

In 1981, an unknown disease appeared in Spain, the Spanish Toxic Oil Syndrome. Nowadays and despite all efforts, the etiological agent is still unknown. Early studies showed a link between this illness and the consumption of denatured rapeseed oil fraudulently processed and marketed as edible oil. Two families of aniline derivatives present in these oils (fatty acid anilides and acylated phenyl amino propanediol derivatives or PAPs) were found to be good chemical markers of toxic oils. In this work, a new method has been developed to analyze these aniline derivatives in oil samples by HPLC-MS and HPLC-MS/MS with an API source. For their quantification, three different internal standards were used, one for anilides and two for PAPs. Quantification limits were 8 ppm for anilides and 0.2 ppm for PAPs. Anilides and PAPs were found in marker-positive samples at levels up to 50,000 and 330 ppm, respectively. The relative abundance of the different fatty acid anilides and PAPs correlates with the fatty acid composition of the oils. More than 2,600 different samples were analyzed by this method in the most exhaustive screening of suspected toxic oils carried out to date.

Storage time and deodorization temperature influence the formation of aniline-derived compounds in denatured rapeseed oils.

In 1981 an epidemic, named Toxic Oil Syndrome, occurred in Spain as a result of ingestion of rapeseed oil denatured with 2% aniline, which had been imported for industrial use but was fraudulently diverted and processed for human consumption. Two groups of chemical compounds have been identified in the ingested toxic oil: fatty acid anilides and amino-propanediol derivatives. The objective of this work was to assess the effect of several refining process variables on the formation of 3-(N-phenylamino)-1,2-propanediol (PAP) esters. The amount of PAP esters in aniline-denatured oil increased dramatically when oil was heated from 250 degrees C to 300 degrees C. However, the ones formed when 300 degrees C was reached were lost during processing at that temperature. The level maintained during the operation time at 300 degrees C was higher in denatured samples stored for 3 weeks before refining than in denatured samples stored only for 1 week. Anilides were also analyzed. We found that anilides decreased very little with distillation time. In this paper we discuss the influence of storage time prior to refining and of elevated refining temperature, such as temperatures that might occur in close proximity to a deodorizer coil.

Levels of seven urinary phthalate metabolites in a human reference population.

Using a novel and highly selective technique, we measured monoester metabolites of seven commonly used phthalates in urine samples from a reference population of 289 adult humans. This analytical approach allowed us to directly measure the individual phthalate metabolites responsible for the animal reproductive and developmental toxicity while avoiding contamination from the ubiquitous parent compounds. The monoesters with the highest urinary levels found were monoethyl phthalate (95th percentile, 3,750 ppb, 2,610 microg/g creatinine), monobutyl phthalate (95th percentile, 294 ppb, 162 microg/g creatinine), and monobenzyl phthalate (95th percentile, 137 ppb, 92 microg/g creatinine), reflecting exposure to diethyl phthalate, dibutyl phthalate, and benzyl butyl phthalate. Women of reproductive age (20-40 years) were found to have significantly higher levels of monobutyl phthalate, a reproductive and developmental toxicant in rodents, than other age/gender groups (p < 0.005). Current scientific and regulatory attention on phthalates has focused almost exclusively on health risks from exposure to only two phthalates, di-(2-ethylhexyl) phthalate and di-isononyl phthalate. Our findings strongly suggest that health-risk assessments for phthalate exposure in humans should include diethyl, dibutyl, and benzyl butyl phthalates.

Quantitative detection of eight phthalate metabolites in human urine using HPLC-APCI-MS/MS.

Because of the ubiquity of phthalates and their potential role in increasing risk for cancer and reproductive dysfunction, the need for human exposure assessment studies is urgent. In response to this need, we developed a high-throughput, robust, sensitive, accurate, and precise assay for simultaneous measurement of trace levels of eight phthalate metabolites in human urine by HPLC-MS/MS. Human urine samples were processed using enzymatic deconjugation of the glucuronides followed by solid-phase extraction. The eluate was concentrated, and the phthalate metabolites were chromatographically resolved by reversed-phase HPLC, detected by APCI-tandem mass spectrometry, and quantified by isotope dilution. This selective analytical method permits rapid detection (7.7 min total run time) of eight urinary metabolites of the most commonly used phthalates with detection limits in the low nanagram per milliliter range. Assay precision was improved by incorporating 13C4-labeled internal standards for each of the eight analytes, as well as a conjugated internal standard to monitor deconjugation efficiency. This selective, sensitive, and rapid method will help elucidate potential associations (if any) between human exposure to phthalates and adverse health effects.

HPLC-MS/MS method for the measurement of seven phytoestrogens in human serum and urine.

The elevated exposure of children to hormonally active dietary phytoestrogens has led to the need for rapid, sensitive, and precise assays for phytoestrogen metabolites in physiological matrices. Here we report the development of a high-performance liquid chromatography (HPLC) MS/MS method for the quantitative detection of seven phytoestrogens in human serum and urine. The method uses enzymatic deconjugation of the phytoestrogen metabolites followed by solid phase extraction (SPE) and reverse-phase HPLC. The phytoestrogens are detected using a Sciex API III heated nebulizer atmospheric pressure chemical ionization (HN-APCI) interface coupled with tandem mass spectrometry. This method allows the detection of the primary dietary phytoestrogens (isoflavones and lignans) in human serum and urine with limits of detection (LODs) in the low parts per billion range. The combination of tandem mass spectrometry and chromatographic separation of the analytes helps ensure the selectivity of the method. Stable isotope-labeled internal standards for all seven analytes improve the precision of the assay, resulting in interday CV values of < 10% for most compounds studied. The accuracy and precision of the method were monitored over time using quality control (QC) samples containing known amounts of phytoestrogens. The majority of phytoestrogens in human sera and urine are present as their glucuronide and sulfate conjugates. Therefore, the thoroughness of deconjugation for each sample was monitored by the addition of a conjugated internal standard and subsequent detection of deconjugated compound. This method proves to be efficacious for measuring baseline urinary phytoestrogen levels in the American population and should prove useful for assessing the modulatory effects of dietary phytoestrogens on endocrine disrupter action in children.

Tuberculous cerebellar abscess.

BACKGROUND: Tuberculosis is an important illness with an increasing occurrence. Although considered primarily a pulmonary disease, tuberculosis can affect any organ system. Central nervous system involvement is potentially devastating and occurs with escalating frequency in both immunocompetent and immunologically incompetent populations. METHODS: This case report involved a patient admitted to the authors' inpatient service. Data were obtained from the patient's medical record. MEDLINE and Index Medicus literature searches were conducted for the years 1977 to the present, with cross-references for earlier articles. RESULTS AND CONCLUSIONS: A 36-year-old previously healthy, immunocompetent woman with a diagnosis of extrapulmonary tuberculosis and no determined primary focus was found to have a cerebellar abscess. Treatment included surgical excision of the abscess combined with 6 months of antituberculosis therapy. Diagnostic tools included the tuberculosis skin test, smears and culture of specimens, computed tomographic scans, and rapid assays based on nucleic acid amplification, ie, polymerase chain reaction. The polymerase chain reaction has great potential for rapid diagnosis of Mycobacterium tuberculosis, particularly when there might be few bacilli, as in pleural, peritoneal, or cerebrospinal fluid. Appropriate therapy for tuberculous cerebellar abscess includes standard antituberculosis medications for 6 to 9 months and surgical excision of the abscess.

Family practice house officer perceptions of faculty teaching behaviors.

In 1985, Wolverton and Bosworth published an often referenced study of family practice teaching behaviors. Their research identified 20 "most helpful" and 10 "least helpful" behaviors. In 1994 all United States Army family practice house officers were asked by survey to rate each of the original 38 Wolverton and Bosworth teaching behaviors as "not at all helpful," "somewhat helpful," "moderately helpful," "very helpful," or "most helpful" in aiding their learning. Mean values were calculated for each teaching behavior. Twenty most helpful and 10 least helpful faculty teaching behaviors were identified for U.S. Army family practice house officers; several differences were identified in comparison with Wolverton and Bosworth's original study. Additionally, three most helpful and four least helpful teaching behaviors were clearly illuminated. Incorporating the results of this new study into residency programs could improve the learning opportunities afforded family practice house officers during their internships and residencies.

Army family practice: does our training meet our needs?

OBJECTIVES: (1) To determine the perceived adequacy of residency training for current practice by Army family physicians; (2) to ascertain if differences exist by residency setting: medical center, medical activity, or civilian. METHODS: Surveys were mailed to the 334 family physicians in the Army in 1993. Training in various subject areas was rated as inadequate, adequate, or overly prepared. RESULTS: More than 75% of respondents felt prepared in 76% of general medical subjects (GM) but in only 39% of family medicine subjects (FM). There were no practice management subjects in which more than 75% felt adequately prepared. There were no differences in perceptions of GM or FM training between military- and civilian-trained respondents. CONCLUSIONS: Army and civilian residencies prepare family physicians for the medical aspects of practice. Early training in management subjects could be enhanced. Civilian and Army programs could improve training in family medicine subjects.

Peroxynitrite-mediated nitration of peptides: characterization of the products by electrospray and combined gas chromatography-mass spectrometry.

Peroxynitrite (ONOO-) can react with a wide range of biomolecules resulting in peroxidation, oxidation, and/or nitration and as a consequence cause their inactivation. In this study mass spectrometry (MS) combined with both liquid (LC) and gas chromatography (GC) has been employed to identify the products formed following ONOO- treatment of three peptides at physiological pH: leucine-enkephalin (YGGFL), V3 loop (GPGRAF), and LVV-hemorphin7 (LVVYPWTQRF). LC-MS analysis of leucine-enkephalin following ONOO treatment indicated the formation of products corresponding in mass to mono- and dinitrated forms of the starting material. LC-MS-MS and GC-MS analyses revealed no evidence for the formation of nitrophenylalanine; however, both 3-nitrotyrosine and 3,5-dinitrotyrosine were observed and characterized. GC-MS analysis of hydrolyzed peptides following ONOO- treatment confirmed the presence of nitrated and dinitrated tyrosine. However, when a 20-fold molar excess of ONOO- was reacted with leucine-enkephalin, only about half of the tyrosine originally present in the peptide could be accounted for in the acid hydrolysate. The main product was 3-nitrotyrosine which represented ca. 50% of the original tyrosine; traces of 3,5-dinitrotyrosine (ca. 3% of the original tyrosine) were also present.