A new bivalent fluorescent fusion protein for differential Cu(II) and Zn(II) ion detection in aqueous solution.

Simple and easy to engineer metal-sensing molecules that are capable of differentiating metal ions and producing metal-specific signals are highly desirable. Metal ions affect the thermal stability of proteins by increasing or decreasing their resistance to unfolding. This work illustrates a new strategy for designing bivalent fluorescent fusion proteins capable of differentiating metal ions in solution through their distinct effects on a protein's thermal stability. A new dual purpose metal sensor was developed consisting of biotin protein ligase (BirA) from B. pseudomallei (Bp) fused to green fluorescent protein (GFP). When coupled with differential scanning fluorimetry of GFP-tagged proteins (DSF-GTP) for signal-transduction detection, Bp BirA-GFP yields distinct protein unfolding signatures with Zn(II) and Cu(II) ions in aqueous solutions. The limit of detection of the system is ∼1 µM for both metal species. The system can be used in a variety of high-throughput assay formats including for the screening of metal-binding proteins and chelators. Bp BirA-GFP has also the additional benefit of being useful in Cu(II) ion field-testing applications through simple visual observation of a temperature-dependent loss of fluorescence. Bp BirA-GFP is the first example of a 2protein-based dual purpose Cu(II) and Zn(II) ion sensor compatible with two different yet complementary signal-transduction detection systems.

Multistate outbreak of Salmonella Paratyphi B variant L(+) tartrate(+) and Salmonella Weltevreden infections linked to imported frozen raw tuna: USA, March-July 2015.

Foodborne non-typhoidal salmonellosis causes approximately 1 million illnesses annually in the USA. In April 2015, we investigated a multistate outbreak of 65 Salmonella Paratyphi B variant L(+) tartrate(+) infections associated with frozen raw tuna imported from Indonesia, which was consumed raw in sushi. Forty-six (92%) of 50 case-patients interviewed ate sushi during the week before illness onset, and 44 (98%) of 45 who specified ate sushi containing raw tuna. Two outbreak strains were isolated from the samples of frozen raw tuna. Traceback identified a single importer as a common source of tuna consumed by case-patients; this importer issued three voluntary recalls of tuna sourced from one Indonesian processor. Four Salmonella Weltevreden infections were also linked to this outbreak. Whole-genome sequencing was useful in establishing a link between Salmonella isolated from ill people and tuna. This outbreak highlights the continuing foodborne illness risk associated with raw seafood consumption, the importance of processing seafood in a manner that minimises contamination with pathogenic microorganisms and the continuing need to ensure imported foods are safe to eat. People at higher risk for foodborne illness should not consume undercooked animal products, such as raw seafood.

Multistate outbreak of Listeria monocytogenes infections linked to whole apples used in commercially produced, prepackaged caramel apples: United States, 2014-2015.

Whole apples have not been previously implicated in outbreaks of foodborne bacterial illness. We investigated a nationwide listeriosis outbreak associated with caramel apples. We defined an outbreak-associated case as an infection with one or both of two outbreak strains of Listeria monocytogenes highly related by whole-genome multilocus sequence typing (wgMLST) from 1 October 2014 to 1 February 2015. Single-interviewer open-ended interviews identified the source. Outbreak-associated cases were compared with non-outbreak-associated cases and traceback and environmental investigations were performed. We identified 35 outbreak-associated cases in 12 states; 34 (97%) were hospitalized and seven (20%) died. Outbreak-associated ill persons were more likely to have eaten commercially produced, prepackaged caramel apples (odds ratio 326·7, 95% confidence interval 32·2-3314). Environmental samples from the grower's packing facility and distribution-chain whole apples yielded isolates highly related to outbreak isolates by wgMLST. This outbreak highlights the importance of minimizing produce contamination with L. monocytogenes. Investigators should perform single-interviewer open-ended interviews when a food is not readily identified.

Virginia agricultural health and safety survey.

This comprehensive study was conducted primarily to identify the common causes of agricultural injuries on active Virginia farms and to identify hazardous agricultural operations, exposure duration, and injuries associated with each hazardous operation. In addition, the influences of factors such as general health status of farmers, age, weight, and alcohol and tobacco use on injury were examined. This information will be used for the development of educational programs that will improve the safety of agricultural operations. The sample selected for the study included farms of 28 ha or more, operating on a full- or part-time basis. This stipulation was to ensure that all farms in the sample are active and that participants generated a major portion of their income from the farm. Of the 26,000 farms meeting this requirement, 1,650 were selected to participate in the study. A survey instrument was mailed to the farmers selected to collect the information needed for meeting the established objectives of the study. Approximately 19% of the surveys were returned. In terms of percentage injuries, livestock handling was the primary cause. This was followed by working in elevated locations, operating and repairing agricultural machinery, and heavy lifting. The activities carried out most frequently by the participants were: operating farm tractors, operating trucks/automobiles, using hand and power tools, and working with agricultural chemicals. The overall injury rate was 5.6 injuries per 100,000 h. The exposure to agricultural hazards appeared to have minimal or no effect on the health status of Virginia farmers. Farm workers in the 45 to 64 age group sustained the most injuries. Older, more experienced farmers reported fewer injuries because of limited exposure to hazards and work experience. The average age of Virginia farmers surveyed was 60. This is expected to rise because most respondents reported no plans to retire during the next five years. Based on the results, educational programs for improving the agricultural safety in Virginia should focus on aging farmers, hazardous agricultural activities, and weight control.

Myelinated, synapsing cultures of murine spinal cord--validation as an in vitro model of the central nervous system.

Research in central nervous system (CNS) biology and pathology requires in vitro models, which, to recapitulate the CNS in vivo, must have extensive myelin and synapse formation under serum-free (defined) conditions. However, finding such a model has proven difficult. The technique described here produces dense cultures of myelinated axons, with abundant synapses and nodes of Ranvier, that are suitable for both morphological and biochemical analysis. Cellular and molecular events were easily visualised using conventional microscopy. Ultrastructurally, myelin sheaths were of the appropriate thickness relative to axonal diameter (G-ratio). Production of myelinated axons in these cultures was consistent and repeatable, as shown by statistical analysis of multiple experimental repeats. Myelinated axons were so abundant that from one litter of embryonic mice, myelin was produced in amounts sufficient for bulk biochemical analysis. This culture method was assessed for its ability to generate an in vitro model of the CNS that could be used for both neurobiological and neuropathological research. Myelin protein kinetics were investigated using a myelin fraction isolated from the cultures. This fraction was found to be superior, quantitatively and qualitatively, to the fraction recovered from standard cultures of dissociated oligodendrocytes, or from brain slices. The model was also used to investigate the roles of specific molecules in the pathogenesis of inflammatory CNS diseases. Using the defined conditions offered by this culture system, dose-specific, inhibitory effects of inflammatory cytokines on myelin formation were demonstrated, unequivocally. The method is technically quick, easy and reliable, and should have wide application to CNS research.

Impact of 'Mad Cow Disease' publicity on trends in meat and total vitamin A consumption in Geneva between 1993 and 2000.

BACKGROUND: In March 1996, revelations about the possible risk for humans of the 'mad cow disease' epidemic had a sudden impact on the diets of European populations. OBJECTIVE: To assess changes in meat and nutrient intakes in adults living in Geneva, Switzerland from 1993 to 2000. DESIGN: Independent annual cross-sectional surveys (4047 women and 4092 men total). MAIN OUTCOME MEASURE: Dietary habits assessed and compared to baseline (January 1993-April 1996) via validated semi-quantitative food-frequency questionnaire. RESULTS: Women beef abstainers increased from 8.9 to 14.9% in late 1996 (P<0.001) and 13.3% in 1997 (P<0.05); among meat consumers, in late 1996 meat/beef intakes declined -10/-12% (both P<0.05). From 1997 to 2000 most intake levels drifted back toward those at baseline, but chicken intakes were significantly (all P<0.05) greater each year (+19% in 2000 (P<0.001). Consistent but less dramatic changes were observed among men. From late 1996 until 2000, liver abstention was significantly (all P<0.05) greater (women from 60 to 78%; men from 61 to 73% in 2000; (both P<0.001). The only nutrient intakes that decreased significantly (all P<0.05) each year from 1997 through 2000 were retinol and total vitamin A women: -22% (P<0.001); -11% (P<0.05) respectively; men: -16% (P<0.001); -10% (P<0.05) respectively, in 2000). Total vitamin A intakes exceeded the dietary reference intake (DRI) for liver eaters (women 185%, men 153%), but were below the DRI for liver abstainers (women 83%; men 66%) in 2000. CONCLUSION: The decreases in beef and liver consumption since late 1996 led to the discovery of a long-term disparity in the retinol and total vitamin A intakes of liver eaters vs abstainers.

Acute corneal endothelial changes after laser in situ keratomileusis.

PURPOSE: To report the acute effects of laser in situ keratomileusis (LASIK) on the corneal endothelium. METHODS: Twenty eyes of 10 consecutive patients (mean age, 38.1 +/- 10.84 years) underwent bilateral simultaneous LASIK for myopic astigmatism (spherical equivalent ranging from -1.75 to -7.13 diopters) without any complications. Each eye was evaluated by slit-lamp biomicroscopy and noncontact specular microscopy preoperatively, within 15 minutes after LASIK and 1 day after surgery. Specular microscopy images were then analyzed to calculate endothelial cell density (ECD), coefficient of variation (CV) of cell size, and percentage of hexagonal cells. RESULTS: All corneas demonstrated marked alterations in endothelial cell morphology by slit-lamp biomicroscopy within 15 minutes after surgery that resolved by the first postoperative day. Central corneal endothelial analysis by noncontact specular microscopy confirmed pleomorphism with definite loss of hexagonality. Mean ECD was calculated to be 2,816.3 +/- 286.02 cells/mm(2) preoperatively, 2,750.85 +/- 327.95 cells/mm(2) on day 0 (p = 0.395), and 2,810.55 +/- 218.48 cells/mm(2) on day 1 (p = 0.461). Mean CV was 32.65 +/- 7.29 preoperatively, 34.4 +/- 6.19 on day 0 (p = 0.412), and 30.9 +/- 5.54 on day 1 (p = 0.067). Mean percentage of hexagonal cells was 63.35 +/- 10.76 preoperatively, 47.55 +/- 9.69 on day 0 (p = 0.000009), and 60 +/- 9.3 on day 1 (p = 0.00003). CONCLUSION: Qualitative and quantitative changes in endothelial cell morphology (i.e., decreased endothelial cell hexagonality) demonstrate that LASIK does induce an acute effect on the corneal endothelium that may represent transient endothelial cell edema.

Bone-related mineral content of water samples collected on the Navajo reservation.

Although dairy food intake is low among the Navajo people, hip fracture rates are lower than in Caucasians. Genetic differences in bone density have been cited as the reasons for low fracture rates among Native Americans and other segments of the population. However, more detailed examination of mineral intakes suggests that environmental factors may provide part of the explanation for the lower fracture rates. Cultural practices such as the addition of ash to traditional foods and the high mineral content of water may provide much higher intakes of bone-related minerals than food intake surveys have previously reported. As part of a larger study to assess overall intake of minerals related to bone health and other conditions, water samples were collected from the Navajo reservation. Duplicates were collected at least one week apart from 53 sites including wells, springs, taps, and storage barrels and analyzed by atomic absorption and inductively coupled plasma spectrometry for a number of minerals. For average intakes of 2 l/day, water could provide up to 212 mg of calcium, 150 mg of magnesium and 8 mg of zinc. The combined contribution of mineral intakes provided by the addition of juniper ash to traditional foods, not genetic differences, may partially explain the lower fracture rates of the Navajo people. Further research in this area is required to confirm this hypothesis.

Ultrasonographic measurement of induced myopia associated with capsular bag distention syndrome.

OBJECTIVE: To determine the causes of postoperative myopia associated with capsular bag distention syndrome (CBDS) and characterize the associated findings. DESIGN: Prospective, multicenter, nonrandomized, comparative (self-controlled) trial. PARTICIPANTS: Six eyes from six patients had CBDS develop after phacoemulsification and posterior chamber intraocular lens implantation. METHODS: Keratometric values, axial length, visual acuity, and manifest refraction were assessed in each eye. Using 20-MHz (I3SYSTEM-ABD, Innovative Imaging Inc, Sacramento, CA) and 50-MHz (Ultrasound Biomicroscope, Zeiss Humphrey Systems, Dublin, CA) ultrasonographic probes, images and measurements of the anterior segment were obtained. After neodymium:yttrium-aluminum-garnet (Nd:YAG) capsulotomy, these measures were repeated and correlated with predicted and actual refractive changes. MAIN OUTCOME MEASURES: Uncorrected visual acuity (UCVA), best-corrected visual acuity (BCVA), manifest refraction, and anterior chamber depth before and after treatment of CBDS. RESULTS: Although intended postoperative refraction averaged -0.58 diopters (D) (range, -0.12 to -1.63 D), eyes with CBDS had an average spherical equivalent refraction of -2.35 D (range, +0.13 to -4.50 D), P < 0.05 (one-tailed, paired t test). BCVA averaged 20/24 (range, 20/15-20/40-1), but UCVA averaged 20/133 (range, 20/60 to 20/400). Average distance from the corneal surface to the anterior intraocular lens (IOL) optic surface was 3.55 mm in eyes with CBDS, and 4.30 mm after Nd:YAG capsulotomy. Posterior movement of the IOL optic after capsulotomy accounted for 1.23 D of hyperopic shift or 82% of the CBDS-induced myopia. Treatment of CBDS resulted in both improved UCVA and BCVA. Nd:YAG capsulotomy also released the colloidal suspension within the capsular bag posterior to the IOL optic. The incidence of CBDS was 0.3% in one of the practices reviewed. CONCLUSIONS: Capsular bag distention syndrome includes unexpected myopia and poor UCVA after cataract surgery with lens implantation in cases involving a continuous capsulorhexis. A dramatic posterior distention of the posterior capsule is observed, as well as anterior chamber shallowing, tight apposition of the iris to the IOL, and anterior bowing of the iris. A slightly turbid colloidal suspension behind the IOL implant and late posterior capsular fibrosis are also observed. Timely treatment of CBDS can correct unwanted myopia, improve UCVA and BCVA, and restore normal anatomic relationships in the eye.

Effects of essential oil of Croton zehntneri, and of anethole and estragole on skeletal muscles.

Extracts of the essential oil of Croton zehntneri (CzEO) yield a mixture of substances in which the major components are anethole and estragole. The action of CzEO and of pure anethole and estragole (at 0.05-1.0 mg/ml) were studied in muscle preparations of the toad and rat. All three oils blocked twitches evoked by nerve stimulation. In the rat diaphragm direct stimulation resulted in enhanced twitches (CzEO), partially depressed twitches (estragole) and anethole did not induce a significant change. The response of toad rectus to acetylcholine was reduced by all three oils. Caffeine contractions of the rectus abdominis and sartorius muscles were enhanced by all oils. This enhancement in the case of the sartorius was blocked by procaine. Lowering the temperature to 4 degrees C in the presence of CzEO, anethole or estragole provoked a contraction which could be blocked by procaine. CzEO, anethole or estragole alone sometimes caused contractions. The occurrence of this response was increased by elevation of calcium (to 22 mM). Other divalent cations and La3+ were also effective, in order: La3+ > Ca2+ > Mn2+ > Ba2+ > Mg2+ except in the case of anethole where the order of Ca2+ and Mn2+ are interchanged. CzEO- and estragole-induced contractions were blocked by procaine. The data suggest that CzEO, anethole and estragole may have two sites of action on muscle fibers: the post junctional membrane, by blocking neuromuscular transmission, and on the sarcoplasmic reticulum (SR), by increasing myoplasmic calcium.

Age and risk factors for colon cancer (United States and Australia): are there implications for understanding differences in case-control and cohort studies?

Data from two population-based case-control studies were used to investigate the effect of age on colon cancer risk. Dietary intake data were assessed from a study conducted in Utah (United States) between 1979 and 1983; reproductive data were assessed from a study conducted in Adelaide (Australia) between 1979 and 1980. Data from both studies were assessed for their impact on those less than 65 years of age and those 65 or more years of age. Intake of energy, fat, and protein had a greater impact on risk among older men than among younger men. Risk estimates for the upper quartile of intake relative to the lowest quartile of intake were 8.5 (95 percent confidence interval [CI] = 1.7-43.0) for energy, 8.2 (CI = 1.6-41.3) for protein, and 7.2 (CI = 1.6-31.4) for total fat for older men, while comparable risk estimates were 2.4 (CI = 0.6-9.1) for energy, 3.0 (CI = 0.7-13.6) for protein, and 1.9 (CI = 0.5-7.1) for total fat among younger men. Similar trends were seen for older women for energy and protein. beta-carotene decreased colon cancer risk among younger men (odds ratio [OR] = 0.4, CI = 0.1-1.2) and women (OR = 0.1, CI = 0.1-0.5), although not among older men (OR = 1.2, CI = 0.3-4.9) and women (OR = 1.9, CI = 0.6-64). Calcium decreased risk of colon cancer among older men (OR = 0.1, CI = < 0.1-0.8) and younger women (OR = 0.2, CI = < 0.1-0.7).(ABSTRACT TRUNCATED AT 250 WORDS)

Pharmacokinetics of FK506 after intravenous and oral administration in patients awaiting renal transplantation.

The authors examined the safety and pharmacokinetics of FK506, a new hepatically metabolized immunosuppressant, after single-dose intravenous (i.v.) infusion (20 micrograms.kg(-1) x 4 hours-1) and oral (80 micrograms/kg) administration in six nondialysis patients, aged 27 to 53 years, with chronic renal failure awaiting transplantation. A two-period, randomized, crossover study protocol was used with blood samples drawn for 72 hours after each dose and a washout period of 4 days. Whole-blood FK506 levels were determined using a standard, two-step, nonspecific enzyme immunoassay. There were no significant changes in vital signs, EKG, or complete laboratory test battery for any patient during the entire study period. No side effects were noted after i.v. or oral FK506 dosing. Mean +/- SD distribution half life was 0.9 +/- 0.2 hours, elimination half life (t1/2 beta) 33 +/- 8 hours, total body clearance (CL) 2.4 +/- 1.1 L/hour, and bioavailability 14 +/- 12%. There was no significant correlation between serum creatinine (Cr) and CL (r = 0.36) or between Cr and t1/2 beta (r = -0.30). It was found that FK506 is incompletely and erratically absorbed after oral administration and is rapidly distributed outside the blood compartment after IV dosing. An extended sampling period seems necessary to accurately characterize the slow elimination phase of FK506.