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Microscale thermophoresis (MST) is a technique used to measure the strength of molecular interactions. MST is a thermophoretic-based technique that monitors the change in fluorescence associated with the movement of fluorescent-labeled molecules in response to a temperature gradient triggered by an IR LASER. MST has advantages over other approaches for examining molecular interactions, such as isothermal titration calorimetry, nuclear magnetic resonance, biolayer interferometry, and surface plasmon resonance, requiring a small sample size that does not need to be immobilized and a high-sensitivity fluorescence detection. In addition, since the approach involves the loading of samples into capillaries that can be easily sealed, it can be adapted to analyze oxygen-sensitive samples. In this Bio-protocol, we describe the troubleshooting and optimization we have done to enable the use of MST to examine protein–protein interactions, protein–ligand interactions, and protein–nanocrystal interactions. The salient elements in the developed procedures include 1) loading and sealing capabilities in an anaerobic chamber for analysis using a NanoTemper MST located on the benchtop in air, 2) identification of the optimal reducing agents compatible with data acquisition with effective protection against trace oxygen, and 3) the optimization of data acquisition and analysis procedures. The procedures lay the groundwork to define the determinants of molecular interactions in these technically demanding systems.

Objective: Liver disease is a potential complication from using dietary supplements. This study investigated an outbreak of non-viral liver disease associated with the use of OxyELITE Pro(TM), a dietary supplement used for weight loss and/or muscle building., Methods: Illness details were ascertained from MedWatch reports submitted to the U.S. Food and Drug Administration (FDA) describing consumers who ingested OxyELITE Pro alone or in combination with other dietary supplements. FDA's Forensic Chemistry Center analyzed samples of OxyELITE Pro., Results: From February 2012 to February 2014, FDA received 114 reports of adverse events of all kinds involving consumers who ingested OxyELITE Pro. The onset of illness for the first report was December 2010 and for the last report was January 2014. Thirty-three states, two foreign nations, and Puerto Rico submitted reports. Fifty-five of the reports (48%) described liver disease in the absence of viral infection, gallbladder disease, autoimmune disease, or other known causes of liver damage. A total of 33 (60%) of these patients were hospitalized, and three underwent liver transplantation. In early 2013, OxyELITE Pro products entered the market with a formulation distinct from products sold previously. The new formulation replaced 1,3-dimethylamylamine with aegeline. However, the manufacturer failed to submit to FDA a required "new dietary ingredient" notice for the use of aegeline in OxyELITE Pro products. Laboratory analysis identified no drugs, poisons, pharmaceuticals, toxic metals, usnic acid, N-Nitroso-fenfluramine, pyrrolizidine alkaloids, aristocholic acid, or phenethylamines in the products., Conclusions: Vigilant surveillance is required for adverse events linked to the use of dietary supplements.

Background: In May, 2013, an outbreak of symptomatic hepatitis A virus infections occurred in the USA. Federal, state, and local public health officials investigated the cause of the outbreak and instituted actions to control its spread. We investigated the source of the outbreak and assessed the public health measures used., Methods: We interviewed patients, obtained their shopping information, and did genetic analysis of hepatitis A virus recovered from patients' serum and stool samples. We tested products for the virus and traced supply chains., Findings: Of 165 patients identified from ten states, 69 (42%) were admitted to hospital, two developed fulminant hepatitis, and one needed a liver transplant; none died. Illness onset occurred from March 31 to Aug 12, 2013. The median age of patients was 47 years (IQR 35-58) and 91 (55%) were women. 153 patients (93%) reported consuming product B from retailer A. 40 patients (24%) had product B in their freezers, and 113 (68%) bought it according to data from retailer A. Hepatitis A virus genotype IB, uncommon in the Americas, was recovered from specimens from 117 people with hepatitis A virus illness. Pomegranate arils that were imported from Turkey--where genotype IB is common--were identified in product B. No hepatitis A virus was detected in product B., Interpretation: Imported frozen pomegranate arils were identified as the vehicle early in the investigation by combining epidemiology--with data from several sources--genetic analysis of patient samples, and product tracing. Product B was removed from store shelves, the public were warned not to eat product B, product recalls took place, and postexposure prophylaxis with both hepatitis A virus vaccine and immunoglobulin was provided. Our findings show that modern public health actions can help rapidly detect and control hepatitis A virus illness caused by imported food. Our findings show that postexposure prophylaxis can successfully prevent hepatitis A illness when a specific product is identified. Imported food products combined with waning immunity in some adult populations might make this type of intervention necessary in the future., Funding: US Centers for Disease Control and Prevention, US Food and Drug Administration, and US state and local public health departments., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

We report three cases of digoxin toxicity presenting with decreased visual acuity, unaccompanied by the classic visual symptom of xanthopsia. Visual function improved and symptoms diminished upon lowering of blood digoxin level. Electroretinographic (ERG) abnormalities in the first case were suggestive of a cone deficit, which supports recent electrodiagnostic and labeled tracer studies implicating retinal dysfunction in the pathogenesis of digoxin toxicity. The second patient presented with visual symptoms exclusively. Evaluation of the third case was hampered by advanced retinitis pigmentosa and the presentation was complicated by a syncopal episode. Digoxin toxicity is a potentially life-threatening condition. Ophthalmologists and neurologists may be the first physicians consulted. It must be considered in the differential diagnosis in patients with otherwise unexplained visual loss.

A stratified random sample of 50 Ohio dairy herds, monitored for 1 year between March 1988 and May 1989, was used to estimate the component costs of clinical mastitis per cow-year overall and by organism, the component costs of an episode of clinical mastitis overall and by organism, and the incidence of clinical mastitis by organism. Each herd was visited monthly by a veterinarian who conducted on-farm interviews and completed standardized data-collection forms designed to elicit economic information about the on-farm costs of clinical mastitis and mastitis prevention. Producers collected milk samples prior to treatment of clinical mastitis cases. Culturing methods allowed identification of 18 specific mastitis pathogen classifications. Annual costs estimated were on a per cow-year and clinical episode basis. The monthly mean population of cows monitored was 4,068. Mastitis prevention cost $14.50/cow-year, whereas the cost incurred by producers because of clinical cases of mastitis was $37.91. Organisms prevalent in the cows' environment caused the most costly types of mastitis. Disregarding contaminated samples and episodes for which no milk samples were taken, mastitis for which 2 organisms were isolated accounted for 35.5% of costs of clinical mastitis, followed by cases for which Escherichia coli (21.3%) was isolated, cases for which culturing yielded no growth (8.6%), and cases for which esculin-positive Streptococcus spp (6.4%), Klebsiella spp (5.7%), esculin-negative CAMP-negative Streptococcus spp (5.1%), Enterobacter spp (4.8%), coagulase-negative Staphylococcus spp (4.1%), coagulase-positive Staphylococcus spp (3.0%), S agalactiae (2.5%), and Bacillus spp (1.2%) were isolated.(ABSTRACT TRUNCATED AT 250 WORDS)

Forty-eight herds participating in the 1988/1989 Ohio National Animal Health Monitoring System dairy project were monitored for 1 year to determine the effects of environment and management on mortality in preweaned calves. Environmental factors were evaluated by veterinarians during monthly visits to the herds. Management procedures were measured through the use of a questionnaire administered near the end of the project. Mortality in preweaned calves was calculated for each herd by using data from project records on calf mortality and animal inventory, which were collected monthly by veterinarians. Relationships between the management/environment variables and calf mortality were examined by use of analysis of covariance. Herd size, days on a nipple feeder, navel disinfection, type of housing, and whether each calf observed with diarrhea was treated with antibiotics were the variables that had an impact on herd mortality. These variables explained approximately 39% of the variation in mortality among herds.

To estimate herd prevalence of Salmonella spp, fecal specimens were obtained for culture from neonatal calves of 47 Ohio dairy herds. Of the 452 calves tested, 10 calves from 7 farms were culture-positive. Salmonella serotypes isolated were S dublin, S typhimurium, S enteritidis, S agona, S mbandaka, and S montevideo. Bulk tank milk filters from these dairies were also submitted for culture. Salmonella sp was isolated from 1 of the 50 filters, and 2 calves from this herd were found to be shedding Salmonella sp of the same serotype.

Dairy herds in Ohio were selected by stratified random sampling for participation in a disease-monitoring study to relate Streptococcus agalactiae intramammary prevalence to herd management and environmental conditions. Of 48 herds studied, 27 herds had at least 1 cow infected with this pathogen. Management and environmental conditions were assessed by direct observation as well as by an interview with the dairy producers. One-way ANOVA or chi 2 analysis, with presence or absence of Streptococcus agalactiae as the dependent variable, was used to test each of 70 independent variables. Variables found significant at P less than 0.20 were further evaluated by use of logistic regression. Our sample size permitted only 4 independent variables to be simultaneously evaluated by logistic regression. The most predictive risk factors were identified as poor teat and udder hygiene, poor environmental sanitation, large herd population, and use of a shared washcloth for premilking cleaning of teats and udders.

Over a period of 3 summers, 21 colostrum-fed Holstein bull calves, 1 to 3 days old, were assigned to 7 replicates, each consisting of 3 calves. Within each replicate of 3 calves, 2 were selected at random, to be given 100,000 to 146,000 sporulated coccidia oocysts (principally Eimeria bovis) orally 60 hours after arrival at the college research farm. On the thirteenth day after coccidia inoculation, 1 of the 2 calves that had been given coccidia and the third calf that had not been inoculated, were given coronavirus by intranasal and oral routes. Calves were observed daily, and consistency of feces was scored visually. Nasal swab specimens for indirect immunofluorescent antibody testing for coronavirus and fecal samples for oocyst determination were obtained approximately every third day. Of 7 calves that were given only coronavirus, 3 developed diarrhea of short duration. Of 7 calves that were given only coccidia oocysts, 6 developed diarrhea. All 7 calves inoculated initially with coccidia and subsequently with coronavirus developed diarrhea. For 5 of 7 replicates, calves that were given coccidia and coronavirus developed diarrhea first. When overall severity, measured by fecal score and by blood in the feces, was compared, calves inoculated with coccidia followed by coronavirus were more severely affected (P less than 0.05) than were calves that were given only coronavirus. Calves that were given only coccidia oocysts appeared more severely affected than calves that were given only coronavirus, but differences were not significant.(ABSTRACT TRUNCATED AT 250 WORDS)

Forty-eight dairy herds in Ohio were selected as a stratified random sample for participation in a disease monitoring study to relate the prevalence of IMI with coliform and environmental streptococci to herd management and environmental conditions. Management and environmental conditions were assessed by farm inspection and by an interview with the dairy producers. A separate analysis for each independent variable identified many potential disease determinants. A multivariable analysis of a covariance model to predict the prevalence of coliforms had 6 model df (R2 = .47). Increased prevalence of coliform infection was associated with an increased amount of milk remaining in the udder after milking, use of free stalls, regular use of a running water wash, increased person hours per cow spent milking, and poor sanitation. The multivariable model for environmental streptococci used 5 model df (R2 = .51). Increased prevalence of environmental streptococci was associated with poor sanitation, increased number of days dry, use of tie stalls, no use of a shared wash cloth, and no use of an individual dry cloth.

Fecal samples were collected from 450 neonatal calves, ranging from 1 to 30 days old, between May, 1988 and May, 1989 to estimate the prevalence of bovine group A rotavirus in a stratified random sample of Ohio dairy herds. Calves were from 47 dairy herds chosen to be representative of Ohio herds. Bovine group A rotavirus was detected in fecal samples by a cell culture immunofluorescence test (CCIF) and ELISA. Of 450 samples tested, 46 (10%) were positive by CCIF and 67 (15%) were positive by ELISA. The agreement beyond chance between the 2 assays was good (kappa = 0.65). The overall prevalence rate of rotavirus shedding was 16.4% (74/450). Forty-three percent (29/67) of the samples positive by ELISA were subgroup 1, none were subgroup 2, and the remaining 57% (38/67) could not be assigned to either subgroups 1 or 2. Thirty herds (62.5%) had at least 1 group A rotavirus-positive calf (mean number of samples per positive herd = 12.4), and 17 herds (37.5%) had no rotavirus-positive calves (mean number of samples per negative herd = 6.0). A live oral rotacoronavirus vaccine was used in neonatal calves of only 1 herd and 3 of 17 (17.6%) calves from this herd were positive for group A rotavirus. The percentage of the rotavirus-positive fecal samples from all calves (n = 450) when stratified by fecal consistency was as follows: 28.3% (13/46) had liquid feces; 25.6% (10/39) had semiliquid feces; 23.4% (22/94) had pasty feces; and 10.7% (29/271) had firm feces.(ABSTRACT TRUNCATED AT 250 WORDS)

The use of a single bulk tank milk culture and a single milk filter culture was studied for their suitability as screening tests for coagulase-positive staphylococci and Streptococcus agalactiae . Bulk tank and bulk tank milk filter cultures were compared to quarter milk cultures taken from individual cows at 49 Ohio dairy herds selected from all Ohio dairy herds by a stratified random sampling scheme. Individual cow quarter milk samples were collected from a sample of all milking cows using a sampling scheme designed to detect an organism present in 2% of quarters, with 95% confidence intervals between 1 and 3%. Seventeen (35%) herds had one or more cows positive for S. agalactiae and 34 (69%) had one or more cows positive for coagulase-positive staphylococci. Using the results of individual cow sampling as the standard, the sensitivity for S. agalactiae was estimated as 23.5% for a single milk filter sample and 35.3% for a single bulk tank milk sample. The sensitivity for coagulase-positive staphylococci was estimated as 52.9% for a single milk filter culture and 41.2% for a single bulk tank milk culture. Based on these results and those of others, it appears that a single bulk tank or milk filter sample has a relatively low sensitivity for both coagulase-positive staphylococci and S. agalactiae , making these poor screening tests for the presence of these pathogens within a dairy herd.

The surgical management of 95 cases of involutional entropion were reviewed to form this retrospective study. The 66 cases treated with the Wies procedure alone had a recurrence rate of 11%. The 29 cases treated with a combined procedure consisting of a Wies procedure with a lateral canthal horizontal shortening had no recurrences. All patients had a minimum of 6 months postoperative follow-up. The etiologic factors as they relate to the appropriate surgical procedures are discussed.

We evaluated the dose relationship between the antiproliferative and toxic effects of 5-fluorouracil (5-FU) on the ocular surface epithelium following experimental corneal epithelial wounding in rabbits. Central corneal epithelial defects 8 mm in diameter were made using n-heptanol. 5-FU (0.05 mg, 0.5 mg, or 5.0 mg per day in divided doses) or saline was applied topically for up to 18 days beginning on the day of wounding. The animals were sacrificed at 1, 7 or 14 days after wound closure. The effect on the ocular surface epithelium was assessed by observation of the clinical and histological appearance, and determination of the rate of corneal epithelial defect closure, corneal epithelial mitotic rate and conjunctival goblet cell frequency. A daily dose of 0.05 mg of topical 5-FU for 18 days had no discernable clinical or histopathological effect compared to wounded, saline treated controls. Treatment with 0.5 mg daily prevented the high mitotic rate typically noted 1 day immediately following defect closure, yet had no significant effect on clinical appearance, histological appearance, or healing rate. Daily topical application of 5.0 mg of 5-FU reduced the corneal epithelial mitotic rate to approximately 1% of the wounded controls, with persistent epithelial defects occurring in 22% of the eyes in this group. In those eyes which did heal, the corneal epithelium was markedly thinner than controls 1 day after defect closure. Fourteen days after healing, epithelial thickness in this group varied from 2 to 13 cells across each cornea, with the thickest area occurring centrally and tapering gradually to the limbus.(ABSTRACT TRUNCATED AT 250 WORDS)

We compared the effects of diamond burring keratectomy (DBK) and surgical superficial lamellar keratectomy (SLK) on the rabbit corneal epithelial healing rate and corneal morphology 1, 4, 7, and 14 days after wounding. Epithelial defects healed significantly more slowly following 10-mm-diameter SLK than following DBK. Restoration of normal epithelial morphology occurred within two weeks in the DBK corneas. During the same interval, SLK corneas showed poor transition of basal to superficial cell morphology. Four days after surgery, the DBK corneas all exhibited marked keratocyte depletion in the anterior stroma but fibrocytes uniformly repopulated this area by 14 days after wounding. Conversely, the SLK corneas all had a hypercellular band of fibrocytes in the anterior stroma by four days following surgery and continued to increase in cellularity through 14 days after wounding. These data indicate that DBK is the preferred procedure for creation of a recipient bed for epithelial lenticles in keratoepithelioplasty and conjunctival transplantation.

Endogenous Aspergillus flavus endophthalmitis with layering of inflammatory cells in the subretinal space and vitreous infiltration developed in a 35-year-old female intravenous drug abuser. The patient underwent a diagnostic and therapeutic vitrectomy within 48 hours of presentation, receiving intravitreal amphotericin B. Intravenous amphotericin also was administered several days later. Despite extensive retinal and vitreous involvement, the patient recovered useful vision because of the aggressive diagnostic and therapeutic approach.