Quality control (QC) analysis is an important component in maize breeding and seed systems. Genotyping by next-generation sequencing (GBS) is an emerging method of SNP genotyping, which is being increasingly adopted for discovery applications, but its suitability for QC analysis has not been explored. The objectives of our study were 1) to evaluate the level of genetic purity and identity among two to nine seed sources of 16 inbred lines using 191 Kompetitive Allele Specific PCR (KASP) and 257,268 GBS markers, and 2) compare the correlation between the KASP-based low and the GBS-based high marker density on QC analysis.Genetic purity within each seed source varied from 49 to 100 % for KASP and from 74 to 100 % for GBS. All except one of the inbred lines obtained from CIMMYT showed 98 to 100 % homogeneity irrespective of the marker type. On the contrary, only 16 and 21 % of the samples obtained from EIAR and partners showed ≥95 % purity for KASP and GBS, respectively. The genetic distance among multiple sources of the same line designation varied from 0.000 to 0.295 for KASP and from 0.004 to 0.230 for GBS. Five lines from CIMMYT showed ≤ 0.05 distance among multiple sources of the same line designation; the remaining eleven inbred lines, including two from CIMMYT and nine from Ethiopia showed higher than expected genetic distances for two or more seed sources. The correlation between the 191 KASP and 257,268 GBS markers was 0.88 for purity and 0.93 for identity. A reduction in the number of GBS markers to 1,343 decreased the correlation coefficient only by 0.03.Our results clearly showed high discrepancy both in genetic purity and identity by the origin of the seed sources (institutions) irrespective of the type of genotyping platform and number of markers used for analyses. Although there were some numerical differences between KASP and GBS, the overall conclusions reached from both methods was basically similar, which clearly suggests that smaller subset of preselected and high quality markers are sufficient for QC analysis that can easily be done using low marker density genotyping platforms, such as KASP. Results from this study would be highly relevant for plant breeders and seed system specialists.The online version of this article (doi:10.1186/s12864-015-2180-2) contains supplementary material, which is available to authorized users. Zea mays L.) is a staple food for more than 300 million people and is commonly grown by small-scale and resource-poor farmers in rural areas [http://faostat3.fao.org). During the same period, (i) maize yield in the country doubled from 1.6 t ha−1 in 2000 to 3.2 t ha−1 in 2013; and (ii) after eight years of erratic production, grain yield showed a rapid increase since 2007 [In sub Saharan Africa (SSA), maize . After nearly four decades, the breeding program released its first top cross hybrid, BH140, in 1988  SubsequeInitial adoption of hybrids by resource poor farmers was very slow for a number of reasons, including (i) high cost of hybrid seed relative to OPVs  (ii) limited or no access to improved hybrid seed in some regions; (iii) inadequate knowledge on agronomic management; (iv) insufficient seed companies and seed regulations in the country; (v) inadequate seed production infrastructure; and (vi) high cost of fertilizers . The demInbred lines are assumed to be genetically pure and possess all the genetic qualities that a breeder has selected for. Small changes in allele frequencies may occur during seed regeneration and maintenance breeding, and possible contamination with seeds or pollen of other samples , 6 Signihttp://www.biotech.cornell.edu/brc/genomic-diversity-facility/services/gbs-project-design-and-optimization). To our knowledge, however, the correlation between GBS and KASP markers for QC analyses is not known. The objectives of our study were to 1) evaluate the level of genetic purity within each inbred line and understand genetic identity among different seed sources of the same line designation; and 2) compare the correlation between the KASP-based low density and the GBS-based high density information for QC analysis.Single nucleotide polymorphic (SNP) markers have emerged as powerful tools for many genetic applications, including germplasm characterization , QC analysis , quantitative trait loci (QTL) mapping, allele mining, marker-assisted backcrossing, marker-assisted recurrent selection, and genomic selection. SNP data can be obtained using one of the numerous uniplex or multiplex SNP genotyping platforms that combine a variety of chemistries, detection methods, and reaction formats. Kompetitive Allele Specific PCR (KASP) is a uniplex SNP genotyping platform, and has developed into a global benchmark technology. CIMMYT uses a subset of 100 to 200 SNPs for routine QC genotyping using the KASP platform at LGC Genomics in the UK. The subset of SNPs for QC genotyping were selected out of the 1536 Illumina GoldenGate random chip using the following criteria: (1) ease of scoring with unambiguous separation of the two homozygous and heterozygous genotypes; (2) a minor allele frequency (MAF) and polymorphism information content (PIC) of at least 0.20 and 0.25, respectively; (3) good distribution across chromosomes based on physical position, and (4) good polymorphism across a wide range of mapping populations. The genotyping cost for KASP depends on the number of data points (1 data point = 1 sample genotyped by 1 SNP) and data turnaround, which is 4 to 6 weeks for normal turnaround and 2 to 3 weeks for rapid turnaround. The current KASP genotyping cost for normal and rapid data turnaround ranges from US$0.064 to US$0.242 and from US$0.100 to US$0.360, respectively . Genotyphttp://www.lgcgroup.com) and the Genomic Diversity facility at Cornell University (http://www.biotech.cornell.edu/brc/genomic-diversity-facility). Samples were genotyped with 200 SNPs , were used in this study , Cornell University using an algorithm that searches for the closest neighbor in small SNP windows across the maize database [Eight datasets were used for all statistical analyses. Dataset 1 and dataset 2 consisted of the 191 KASP SNPs and the 257,268 GBS markers after filtering using a MAF of 0.05, respectively. Six additional input files were later created for correlation analyses: (a) dataset 3 consisted of a subset of 100 out of 191 SNPs routinely used by CIMMYT for QC genotyping; and (b) data set 4 to dataset 8 were created from dataset 2 using a MAF of 0.10, 0.20, 0.30, 0.40 and 0.50. For QC analysis, the best SNPs would be those which amplify the two alleles equally . The proportion of heterogeneity  in each sample was calculated from all datasets using TASSEL version 4.3.2. Genetic purity was calculated from all datasets in Excel as the difference between 100-h, where h refers to heterogeneity in percentage obtained from TASSEL. For all eight datasets, genetic distance was calculated between each pair of samples using the identity by descent (IBS) method implemented in TASSEL. Dendrograms were constructed from the genetic distance matrices of both dataset 1 and dataset 2 using the Unweighted Pair Group Method with Arithmetic Mean (UPGMA) algorithm implemented in molecular evolutionary genetics analysis (MEGA), version 6 . Mantel 4 or later generations, but previously, lines were often derived at earlier generations. An inbred line may be considered pure or homogenous if the proportion of heterozygous or heterogeneous loci does not exceed 5 % [We first computed the proportion of homozygous SNPs on each of the 80 samples as an estimate of genetic purity or homogeneity. The results were highly variable across samples, with homogeneity varying from 49 to 100 % for KASP and from 74 to 100 % for GBS . They are now developing inbred lines after four or later generations of selfing and more recently released hybrids developed using inbred lines with higher genetic purity. This will assist to maintain homogeneous parental inbred lines in the next generation hybrids in Ethiopia.The use of parental inbred lines with high heterogeneity in breeding programs can have multifold negative effects, including the use of wrong donors in new breeding starts for line development, hybrid formation as well as genetic and molecular studies. Genetic purity of a given parental line also has major impact on the production and distribution of certified seed to farmers. Failure to undertake regular quality control analysis could result in generation and dissemination of incorrect products to the end user. Maize breeders commonly exchange seed of the most widely used inbred lines; therefore, unexpected level of genetic heterogeneity in a given seed lot or high mismatch across different seed sources of a given inbred line can quickly spread across different programs with consequent negative effects. Obtaining seed from a reliable source and undertaking routine quality control, will be useful for minimizing errors associated with purity and identity. Our results showed that a subset of 100 to 200 SNP (KASP) markers would be sufficient for routine QC analysis. In cases where there is no reference genotype data for inbred lines that will be used for comparison purposes, it is recommended to grow out multiple sources of different sources of the same line designation in nurseries, generate SNP data, compare SNP data with field notes, and discard those sources that show deviation from expectation in terms of purity and identity. CIMMYT has carried out this exercise to resolve genetic purity and identity issues from 280 seed sources involving 40 inbred lines and strongly recommends this regular activity for other breeders . The information presented in this paper would be highly useful for maize breeders that are involved in new pedigree start, developing populations for QTL mapping and marker-assisted breeding, and the seed companies.Our results clearly showed high discrepancy both in genetic purity and identity by the origin of the seed sources (institutions) irrespective of the type of genotyping platform and number of markers used for analyses. Overall, most seed sources from CIMMYT were considered genetically pure, which was not the case for the majority of samples originated from EIAR. One of the reasons for such discrepancy in genetic purity and identity was the level of inbreeding prior to releasing the parental lines for hybrid formation. Although there were some differences between KASP and GBS results, the overall conclusions reached from both methods was basically similar, which clearly suggests that smaller subset of preselected high quality markers are sufficient for QC analysis that can easily be done using low marker density genotyping platforms, such as KASP. GBS data would be highly useful for establishing reference marker database at the time of releasing an inbred parental line for used in heterotic grouping and planning hybrid combinations and new pedigree starts. Results from this study would be highly relevant for plant breeders and seed system specialists.


Objective(s): Skin extracellular matrix, which contains type I and type III collagens, is involved in skin development. The aim of this study was to investigate type III collagen distribution pattern as well as its changes during pre and post-natal skin morphogenesis in rats.Materials and Methods: Ventral skins of Wistar rat embryos at different stages from 10 to 20 gestational day (E10-E20) and also one month and one year post natal rat pups were fixed in normalin, embedded in paraffin and 5 µm thick sections were incubated with Anti type III collagen antibody. In order to detect staining intensity, the reactions were observed and graded by three examiners separately. Kruskal-Wallis non-parametric statistical test and SPSS software version 11.5 were used to compare differences between samples. Results: Immunoreactivity of type III collagen was distributed weakly in the mesenchymal connective tissue on day 10 (E10). The observed reaction was increased onE12 and E14. This reaction was clear in basement membrane, relatively intensive in dermal papillae and moderate in dermal reticularis on E14. This immunoreactivity pattern was increased afterward on E16, not changed on E18 and decreased in dermal reticularis on E20. The density of collagen type III in dermal papillae and dermal reticularis in skin of one year old rats were decreased comparing to one month old rats. Conclusion: Our results showed that type III collagen is expressed and timely regulated during pre and post natal rat skin morphogenesis. Collagen, an important post-translationally modified protein, in cell-cell interactions during embryonic development, plays a critical role in morphogenesis. Collagen is a family of proteins in all vertebrates which varies in size, function and tissue distribution . All memMoreover, collagens as the most predominant matrix components regarding tissue distribution and quantity are involved in the developmental processes such as angiogenesis, intramembranous ossification as well as tooth germ development , 8. It hLittle information is available about the distribution and function of type III collagen during embryonic skin morphogenesis and its content changes in skin after birth and aging. Several studies have been done about of collagen amount and changes during aging; we nevertheless do not have exact information about changes of type III collagen during aging. So, here we investigated the distribution of collagen type III during embryonic skin morphogenesis and after birth.This research was done in Mashhad University of Medical Sciences according to ethics committee guide lines and all protocols of animal experiments have been approved by the Institution's Animal Care Committee.ad libitum. Thirty virgin Wistar rats (10-week-old and 180–200 g weight) were purchased from Laboratory Animal house of Mashhad University of Medical Sciences, Mashhad, Iran. The animals were maintained in animal house under controlled condition  with laboratory chow and water provided 0). The gestational period of this strain of rats is approximately 21 days. At various gestational days from E10 to E20, pregnant rats (three rats at each stage) were anesthetized with ether inhalation and fetuses were removed and sacrificed. The skin of the fetuses and the ventral skin of one month and one year old rats were taken immediately, washed in normal saline and fixed in 4% formaldehyde in 0.01 M phosphate buffer  overnight at room temperature. After fixation, the tissue specimen were dehydrated with increasing concentrations of alcohol, cleared in xylene and then embedded in paraffin. The paraffin blocks were cut using microtome  into 5µm thick sections and put on poly L- lysine coated slides  and incubated overnight at 4°C. After washing the sections in PBS containing 0.025% Trition X-100 for 10 min, the HRP- conjugated secondary antibody (goat anti rabbit IgG) was diluted at the ratio of 1:1000 in PBS with 1% BSA and applied to sections for 2 hr at room temperature. The antigenic sites were demonstrated via reaction of sections with a mixture of 0.03% DAB in 0.05 M PBS buffer, pH 7.6, containing 0.01% HIn order to detect staining intensity, the reactions of the tested antibody (five slides for each stage) were observed by three examiners blindly with Olympus microscope . On the basis of their staining intensity, the sections were graded as very weak (+), weak (++), moderate (+++) and strong (++++) .The immunoreactive specificity was checked by negative and positive controls as follows:Negative control: a control incubated with normal mouse serum instead of the primary antibodyPositive control: adult rat skin with the corresponding antigen was incubated with antibodies overnight at 4°C.Statistical analysisP≤0.05 whereas non significance is indicated (NS).SPSS software version 11.5 and Kroskal-Wallis non-parametric statistical test were used to compare differences between samples taken from different gestational days, one month and one year old rat offspring. Significance was determined at 10 to E20. In fetuses on E10, immunoreactivity specific for type III collagen was weakly and sparsely distributed in the mesenchymal connective tissue (1.125±0.6292) and it was relatively distinct on a fairly wide region of mesenchymal connective tissue just beneath the epithelium of the developing skin, where will be formed dermal papillae (12 comparing to E10 (1.25±0.5000) but it was not significant. At this embryonic stage, the embryos were covered with an epithelium composed of one layer of cuboidal cells (ectoderm) which reacted with hematoxilin and could be clearly distinguished but showed no immunoreactivity . The reaction of the mesenchymal connective tissue just beneath the epithelium, Corium, which will form the dermal papillae, was observed (2.75±0.5000) but the intensity of the reaction was not significant comparing to pervious stage. At this embryonic stage, the above mentioned immunoreactivity was appeared on the developing basement membrane region. At the same time, specific immunoreactivity for type III collagen appeared very slightly and weakly in the mesenchymal tissue under the corium, which is called subcorium that will be developed to hypodermis. At this stage, total thickness of the epithelium was increased and developed into two layers including a cuboidal basal cell layer and a flattened superficial cell layer, known as epitrichium or periderm. Each epithelial cell still had a large round nucleus. On their free surface side above these epithelial cells, peridermal cells, in a single layer, became to some extent swollen and elliptical  was more distinct than dermal reticularis (1.75±0.5000). Also, epithelium had gradually become stratified that the cells nuclei were stained deeply with hematoxilin . However, this reaction was not so distinct in the region under the dermal papillae, known as dermal reticularis (2.25±0.5000) and variation in intensity of reaction between this stage, E10 and E12 was significantly (P<0.05). The surface layer of squamous stratified epithelium, which was not stained deeply by hematoxilin, appeared to be parakeratin-ized, but the nuclei were still visible in the surface layer of epithelial cells , immunoreactivity specific for type III collagen was observed like to E18 in dermal papillae (3±0.8165) and there was not any difference in staining intensity comparing to the pervious embryonic day (E18) but the intensity of immunoreactivity in dermal reticularis was decreased compared to the previous stages (0.875±0.2500) and the immunoreactivity varied significantly between this stage and E18 (P<0.05). In addition, intensity of this reaction was detected mainly around the developing hair follicles located in dermal reticularis. On embryonic day 20  and reticular dermis (2.25±0.5000) but immunoreactivity was detected to be lower in dermal reticularis . Additio6 to E9 (th week of pregnancy the dermis was divided into dermal papillae and reticularis (8 to E12. Moreover, in other studies results were shown that collagen type III expression was decreased before the birth but after the birth its expression has been continued at a lower level compared to collagen type I. Shuttleworth and Forrest represented that collagen type III is essential skin collagen for normal dermal development in embryo that is necessary for normal ectodermal development (Our results suggested that type III of collagen existed in extra cellular matrix in primary stage of development and during the skin development, the amount of this type of collagen gradually increased in skin. During rat skin development, two parts was formed: 1- dermal papillae and 2- dermal reticularis that expression of collagen type III, but this type of collagen in dermal papillae was more than dermal reticularis. Additionally, type III of collagen plays important role in basal membrane development. In one study, this type of collagen was observed close to basal lamina, skin appendix, blood vessels endothelium, nerve and muscular fibers . Also co6 to E9 . In paraicularis . Our reselopment . Also, oOn the other hand, our result showed abundant expression of collagen type III in dermal papillary in one month old rats but this fibrils were less in dermal reticularis compared to dermal papillae. collagen type III were decreased during aging as this fibril's density were decreased in dermis. In addition, other studies have shown that during the aging, structurally epidermis probably becomes thinner, adherence of corneocytes was declined and dermoepidermal interface was flattening. Also, it was observed that the number of melanocytes and Langerhans cells was decreased and the dermis became atrophic which was comparatively acellular and avascular. Furthermore, dermal collagen, elastin and glycosaminoglycans were altered resulting in wrinkles, laxity and fragility of aged skin , 18, 19.Our results showed that type III collagen is expressed and timely regulated during pre and post natal rat skin morphogenesis. Also, at the latest stage of development, expression of collagen type III decreased in dermal reticularis but in dermal papillae its expression was ascending until birth. After birth, the expression of collagen type III in dermal papillae and dermal reticularis was seen but during the aging, its amount decreased.


In Switzerland, as in other developed countries, the prevalence of overweight and obesity has increased substantially since the early 1990s. Most of the analyses so far have been based on sporadic surveys or self-reported data and did not offer potential for small-area analyses. The goal of this study was to investigate spatial variation and determinants of obesity among young Swiss men using recent conscription data.A complete, anonymized dataset of conscription records for the 2010–2012 period were provided by Swiss Armed Forces. We used a series of Bayesian hierarchical logistic regression models to investigate the spatial pattern of obesity across 3,187 postcodes, varying them by type of random effects , level of adjustment by individual  and area-based [urbanicity and index of socio-economic position (SEP)] characteristics.The analysed dataset consisted of 100,919 conscripts, out of which 5,892 (5.8 %) were obese. Crude obesity prevalence increased with age among conscripts of lower individual and area-based SEP and varied greatly over postcodes. Best model’s estimates of adjusted odds ratios of obesity on postcode level ranged from 0.61 to 1.93 and showed a strong spatial pattern of obesity risk across the country. Odds ratios above 1 concentrated in central and north Switzerland. Smaller pockets of elevated obesity risk also emerged around cities of Geneva, Fribourg and Lausanne. Lower estimates were observed in North-East and East as well as south of the Alps. Importantly, small regional outliers were observed and patterning did not follow administrative boundaries. Similarly as with crude obesity prevalence, the best fitting model confirmed increasing risk of obesity with age and among conscripts of lower professional status. The risk decreased with higher area-based SEP and, to a lesser degree – in rural areas.In Switzerland, there is a substantial spatial variation in obesity risk among young Swiss men. Small-area estimates of obesity risk derived from conscripts records contribute to its understanding and could be used to design further studies and interventions.The online version of this article (doi:10.1186/s40608-016-0092-6) contains supplementary material, which is available to authorized users. The prevalence of overweight and obesity (OWOB), with steady increase over three decades, has reached global pandemic proportions in the developed world –3, with The negative associations of overweight and obesity with chronic disease such as diabetes type II, hypertension, cardiovascular disease and some cancers is well documented –8. CurreFrom the early 1990s an increase in the prevalence of OWOB in many developed countries including Switzerland –15has imResearch into OWOB in Switzerland comprises an important component for public health research and planning. However longitudinal, nationally measured data on the prevalence of OWOB in Switzerland is lacking . A numbeThese results indicate early signs of a slowdown in the increase of OWOB among both adults and children in recent years, implying some success in increased awareness of the condition and public health programmes targeting physical activity and healthy eating , 28–32. The complex pathogenesis of obesity has yet to be completely understood . In addiThe main goal of this study is to investigate small-area variation and determinants of obesity among young Swiss men using georeferenced conscription data from 2010–2012 in order to better understand spatial and socio-economic pattering of obesity in Switzerland and to assist planning interventions on a small scale.Nations in Europe which continue to conscript their male citizens into military infrastructure include Finland, Norway, Denmark, Austria and Switzerland. Conscription is mandatory for all young males and entails a medical examination with recording of standardised anthropometric measurements. The process is repeated annually and provides valuable anthropometric data on young men of a directive age . Though Bundesgesetz über die Armee und die Militärverwaltung, Militärgesetz MG, 510.10, Art. 2; MG Art. 9, Verordnung über die Rekrutierung VREK, 511.11, Art. 3 and Art. 9). Measurements are continuously entered in the medical-information database of the Swiss Armed Forces (MEDISA) and are accessible to Army personnel. The resulting data reflect over 90 % of the yearly male birth cohorts, allowing analyses of variation across temporal, regional and socioeconomic strata . INLA implements the integrated nested Laplace approximation approach for latent Gaussian models  and would be missed or distorted if they were used for the analysis. Lastly, results indicated age and socioeconomic gradients in obesity risk, with smaller influence of urbanicity.The aim of this study was to describe small-area variation and socioeconomic determinants of obesity among young men in Switzerland using recent waves of conscription data. Using records from 2010–12 period, we found a strong spatial structure of risk of obesity across postcodes. A clear concentration of high and low values of ORs emerged, but also small, regional outliers were observed. Importantly, spatial patterning did not always follow administrative boundaries of large administrative units [cantons and districts  and because of its ease of use and availability it is the most commonly utilised measure for large-scale investigations and convenient assessment in clinical practice [MedStat regions [Medizinische Statistik der Krankenhäuser).The main strength of this study is its reliance on representative, large and objectively measured georeferenced data. The sample size allowed high-resolution analysis on the level of individual postcodes. Such findings could contribute to the understanding of the small-area obesity risk that not necessarily follows administrative boundaries (cantons) and thus help to design further studies and target interventions. The study however has several limitations and any generalisation of the results should take them into account. First of all, this study focusses on young, Swiss men only and the associations cannot be directly applied to women, other age groups and non-Swiss individuals. BMI driven obesity indicator was the only body shape measure available for this study. Because BMI cannot accurately differentiate between weight associated with fat and lean muscle mass, it not an ideal measure of body composition –78. Thispractice , 80. By practice . Additiopractice , 82. We practice . Further regions  and can 2) of 1,503 young men aged 15-24 participating in SHS in 2012 was 3.2 % and thus also lower compared with the conscripts, possibly due to the fact that SHS data were self-reported [2) were slightly lower in Switzerland (5.8 %) compared to other European countries with mandatory conscription in 2010/2011  [Obesity prevalence among Swiss conscripts 2010-2012 (5.8 %) was slightly higher compared to the prevalence (3.6 %) detected among the small group of 165 men aged 15 to 29 years examined for the 2011 Swiss salt survey  , 35. Obereported . The leva 8.4 %) , 84. In a 8.4 %) . To our a 8.4 %) , 55. Lasa 8.4 %) –20.Future studies could offer even higher spatial resolution of the analyses and use the main city of residence or even full residential addresses geocoded to point data instead of the data aggregated to postcodes. Furthermore, more conscription years could be included, which would allow use of spatio-temporal models , 85. FutThe present study contributes to the understanding of spatial and socio-economic pattering of obesity in Switzerland. The results indicate large discrepancies in the risk of obesity among Swiss conscripts and could assist planning interventions on a small scale, irrespectively of administrative boundaries. Research on obesity and other branches of public health in Switzerland might benefit from leaving the usage of the common administrative boundaries behind.


Raw eggs are a potential health hazard and a new federally approved process uses ozone to maintain freshness while ensuring safety. The impact of an ozone process on the flavor, color, and shell integrity of eggs must be known for market acceptance. The visual perception and consumer acceptance of full commercial scale ozone-pasteurized eggs are reported, using a degree of liking test and a Just-About-Right analysis. Instrumental analysis of albumen turbidity, yolk color, and Haugh Units correlate with human perception. Visual tests reveal that ozone-pasteurized eggs were equivalent to thermally pasteurized eggs in attributes such as appearance, spread, and cloudiness. They were equivalent to untreated eggs in yolk height, yellowness, and appeal. There were no differences in taste among all egg treatments for measures of aroma, flavor, texture and overall liking. Ozone-pasteurized eggs have the same appeal as raw eggs, and can be cooked without flavor loss. This promising new ozone process maintains good sensory quality relative to thermal processing. Salmonella enteritidis is the illness caused by eating raw eggs. About 200,000 illnesses per year may be caused by Salmonella enteritidis contaminated eggs  and ozone-pasteurized eggs (ozone eggs) laid by hens between 32 and 45 weeks of age were shipped immediately after laying from the same Ohio farm and same flock by standard surface transport to a nearby egg distribution site. Thermally pasteurized eggs (heated eggs) were purchased, as it is commercially available, from a local grocery with a Julian date of 110 and evaluated 1 week later. All eggs were stored on standard fiber board egg trays inside a cardboard and held at a refrigerated temperature of 4°C until further use. Eggs from all sources were industrially washed, sorted, and any with cracks or checks discarded.Unprocessed control eggs (raw eggs) received no further treatments; thus, they are identical to what is now most commonly purchased in groceries throughout the world. Heat–ozone-pasteurized eggs (ozone eggs) were processed at a commercial egg facility in Coldwater, Ohio following an FDA validated process, where gaseous ozone under pressure ensured a 5-log reduction of pathogens in the geometric center of each egg. Process conditions remain proprietary but were validated by a certified process control specialist. Thermally processed eggs (heated eggs) are treated with heat only up to 63°C for 40 min, as specified by Davidson . The heaSensory studies were done by the Ohio State University Food Science Sensory Group in a standardized test facility . Ten segregated booths were illuminated by 3000°K white fluorescent light and the temperature was kept constant at 21°C. All responses were collected using Compusense® version 5.2 software  on a color LCD monitor interface controlled via mouse and keyboard by each panelist. Tests were conducted in one morning session in a complete balanced design, serial monadic approach for all three different egg treatments. Eggs were presented on black plastic plates, coded with three random digits, and mineral water provided between samples for mouth rinsing. Ninety-eight untrained panelists  were recruited from a roster of consumers maintained by the OSU Sensory Analysis Laboratory. Over 90% of the panelists reported eating eggs regularly, 25% ate two per week, 12% ate three per week, and 13% ate four per week. Prior to testing, it was verified that participants had no egg allergies or aversions. Ethnicity was 4% Asian, 7% Black, 3% Hispanic, 81% White, 2% other, and 3% preferred not to disclose.cloudiness of the thick albumen, 2 = amount of spreading of thick albumen, 3 = cloudiness of the egg yolk, 4 = height of the yolk, 5 = color of the yolk, 6 = yellowness of the yolk, 7 = visual appeal of the yolk, 8 = visual appeal of the albumen, and 9 = visual appeal of the whole egg. Panelists viewed the freshly cracked uncooked eggs in random order within 1 h of removing the shell. The endpoint anchors at rating 1 and 10 for each attribute were labeled as shown in Figure Randomly selected eggs from each of three treatments  were cracked into a clear 15.9-cm plate , labeled with three-digit random numbers, and placed over a black matte paper background to test visual acceptance. Each panelist was asked to individually rate samples on a 10-point linear scale for these nine different attributes: 1 = 2) using a #1905 rubber spatula  and cooked for an additional 1.0 min. Finally, the eggs were cut into smaller pieces (∼2 cm2) using the spatula and covered with cling wrap  for 30 sec. Temperature was measured with a handheld thermometer (Rubbermaid) until a consistent internal temperature of 73.9°C was reached for all treatments. Cooking across all treatments was consistent. Each batch was placed in equal portion (about 45 g) onto ten 15.2 cm disposable white foam plates  using a 46.1 g scoop . Filtered spring water was provided to the panelists as their palate cleanser between samples.In a consumer acceptance test, six eggs for each treatment were premixed a day before the test using a hand blender with a whisk attachment  on high for 45 sec. Each premixed egg sample was placed into labeled Ziploc bags and stored in a commercial refrigerator  at 4°C until further use. On test day, each bag of egg mix was placed into a microwave safe glass bowl and mixed on high for 10 sec. The egg mixes were cooked in a microwave  for 1.0 min on high at 1100 W. The eggs were then mixed on low for 10 sec and cooked for an additional 1.0 min. The eggs were cut into large pieces  scale to rate the amount of scrambled egg color, moistness, and texture. The descriptors for the JAR analysis across all three egg treatments are listed in Figure Instrumental analyses measured differences between ozone eggs versus raw eggs, both from the same flock at the same time. Albumen turbidity measurements were based on 12 replicates. Haugh units and yolk color measurements were based on 10 replicates. Albumen turbidity measurement was performed by separating the albumen from the yolk using an egg separator . Three milliliter of the albumen were pipetted into disposable cuvettes. Absorbance was measured at 600 nm with a Shimadzu UV-Vis 2450 spectrophotometer  and samples were read against a blank reference of distilled water , where HU is the Haugh units, h is the thick albumen height, and w is the weight of the unbroken egg  were measured using a HU meter  and is based on the following formula HU = 100 logThe yolk color was measured using a Colorquest XE Hunter Lab colorimeter  with Illuminant D65/2 and 2.54-cm-diameter viewing aperture .Significant differences within consumer acceptance tests were determined using repeated measures analysis of variance and Tukey's HSD post hoc analysis at P < 0.05). This result is similar to an earlier pilot-scale study conducted by Kamotani et al. (P < 0.05), confirming the consumer panel observations.Cloudiness of the albumen is a visual indication of egg freshness. Fresh eggs are cloudier than older eggs because carbon dioxide inside eggs escapes over time, thus creating albumen that is more translucent AEB . As showi et al. . These rPerry et al.  reportedA common egg-grading and consumer acceptance criterion is the spreading of the thick albumen. Eggs with the least spreading of the thick albumen earn higher grades than those with more spread AEB . The sprThe standard for grading of egg quality is the Haugh Unit, devised by Raymond Haugh in 1937 and in continuous industry and regulatory use. HU measures the height of the thick albumen. Because spreading of the albumen directly correlates with the height, a higher HU indicates thicker albumen and less spread. Eggs that have greater than 72.0 HU are grade AA USDA . Table 1Yolk color is not a grading criterion but affects consumer perception. Ozone eggs were perceived by consumers to be darker in color than raw eggs shown by a 6.18 mean rating score compared to 5.65 mean rating score. All three egg treatments were perceived to have no significance difference in their yellowness, as shown in P < 0.05) as the treatment slightly weakens the eggshell. These data are not shown as the eggshell has no effect on the sensory acceptance of the egg contents when presented visually or as cooked scrambled eggs.The effect of ozone processing on the strength of the eggshell was measured by Instron analyses across seven different batches of eggs laid over a period of 6 months and measured as a function of storage at 0, 6, and 12 days. There was no significant difference among batches of ozone-pasteurized eggs regardless of storage days or treatment batches. As expected, significantly more force was required to crack untreated versus ozone eggs  in the height of the yolk among ozone, heated, and raw eggs. Conversely, ozone eggs were perceived to have a shorter yolk when compared to other treatments  results are presented in Figure When panelists were asked to rate the amount of moistness on a JAR scale, heated eggs were perceived as slightly drier than the ozone or raw treatments. In the attribute of moistness, both heated and ozone eggs received more “way too dry” rankings than raw eggs. When panelists were asked to rate the egg texture on the JAR scale, heated eggs were slightly tougher than the other two treatments, while raw eggs received the most “just about right” ratings in the texture attribute among all eggs. Heated eggs had the highest number of responses in the “slightly” and “much too dry” category as shown in Figure In correlation with the liking results, the dryness and toughness were attributable to the microwave method of cooking. A conventional stovetop yields less dry and less tough scrambled eggs, but the large variation in the conventional cooking method would mask differences between treatments. These results are consistent with prior work where there were no differences in JAR ratings for moistness and texture among treatments. Both methods of pasteurization of eggs were perceived to be slightly too dry and too tough (Kamotani et al. Commercially pasteurized and regulatory approved ozone eggs have the same or better visual appeal as heated pasteurized eggs. In some cases, including height, yellowness, and visual appeal of the yolk, ozone eggs were perceived equivalent to raw eggs. In taste tests, there were no significant differences in score among all egg treatments for the visual, aroma, flavor, texture, and overall liking when presented and tasted as scrambled eggs. Sensory test results indicate that yolk quality has improved in the commercial process relative to a pilot-scale process, where the yolk quality of the commercially processed ozone eggs is perceived equivalent to that of heated eggs. Ozone eggs had an average of 92.2 HU, well above that needed to qualify this process as grade AA. Commercially processed ozone eggs achieve the safety required by the US Egg Safety Action Plan. These sensory tests show that they achieve equivalent consumer acceptance to the traditional raw shell egg and are appropriate for in-home or institutional cooked egg recipes.


Objective. Postoperative hyperalgesia is very frequent and hard to treat. Dezocine is widely used and has a modulatory effect for thermal hyperalgesia in animal models. So, this study was designed to investigate the potential role of dezocine in decreasing postoperative hyperalgesia for patients undergoing open abdominal surgery. Methods. This is a randomized, double-blinded, and placebo-controlled trial. 50 patients for elective open gastrectomy were randomly allocated to either a true treatment group (0.15 mg/kg intravenous dezocine at the end of surgery) or a sham treatment group  in a 1 : 1 ratio. Patients were followed up for 48 hours postoperatively and pain threshold to Von Frey filaments, pain scores, PCIA consumption, rescue analgesics use, sedation score, and occurrence of postoperative nausea and vomiting were recorded. Results. Patients in the true treatment group experienced statistically significantly higher pain threshold on forearm and smaller extent of peri-incisional hyperalgesia than the sham treatment group. Rescue analgesic use, cumulative PCIA consumption, and pain scores were statistically significantly decreased in the true treatment group compared to the sham treatment group. Conclusions. Dezocine offers a significant antihyperalgesic and analgesic effect in patients undergoing elective open gastrectomy for up to 48 hours postoperatively. Hyperalgesia frequently occurs after surgery, especially in the early postoperative period . The occμ opioid receptor agonist. Its short context sensitive half-life and short elimination half time make it suitable for anesthetic maintenance during surgery. After intravenous administration, remifentanil exhibits a stable plasma concentration [Remifentanil is a potent ntration , organ intration , 7, minintration , and attntration . Neverthntration , 11. Posntration . Postopeμ receptor partial agonist/antagonist [κ receptor profile. Although initially identified as a κ receptor agonist, recent studies suggest that dezocine might be a κ receptor antagonist [κ receptor antagonist, it can probably have a modulatory effect on spinal dynorphin, preventing it from binding to κ receptor, which plays a crucial role in the spinal effect of hyperalgesia [Dezocine, first developed in 1970s, is an opioid tagonist . Dezocintagonist . There htagonist , 15. As ralgesia . Studiesralgesia . HoweverTherefore, the present study aimed at investigating the effect of dezocine in preventing postoperative hyperalgesia using a random double-blinded study.ChiCTR-TRC-14004723). Patients scheduled to have open gastrectomy from August 2012 to February 2014 were included in this study.This was a double-blinded, placebo-controlled randomized trial. This study was approved by the Ethic Committee of Shanghai Renji Hospital affiliated to Shanghai Jiaotong University of Medicine (document number 2012032) and was also registered in the Chinese Clinical Trials Registry (Patients were included if they were (1) aged 18~64 years and (2) with American Society Anesthesiologists physical statuses I-II.Patients were excluded when (1) they had chronic use of analgesics or had used opioids within 24 h of surgery; (2) immediate extubation was not planned after surgery; (3) patients had neurological or psychiatric disorders; (4) there was end-stage cancer in preoperative evaluation; (5) there was diabetes or severe hypertension; (6) there were either abnormal findings in laboratory tests including complete blood count, liver and renal function, electrolytic analysis, and thrombin time; (7) they were unable to understand the usage of the patient-controlled intravenous analgesia (PCIA) device; (8) body mass index (BMI) was > 30 or < 17; (9) there was alcoholic abuse or coffee drinking > 2 cups/day, and (10) there was rejection either by the surgeon or by the patient. Moreover, after initial enrollment, if any of the following activities occurred, the patients were also excluded: (1) ICU hospitalization after surgery; (2) transient perioperative hypothermia; (3) blood loss > 400 mL or hemoglobin decreasing > 30% in the last perioperative ABG; (4) perioperative acid-alkaline or electrolytes disorders; (5) advanced gastric cancer found on the surgical table. After initial inclusion and exclusion, we had 93 patients enrolled in the study and 43 patients were finally excluded because of perioperative findings as listed above.2, North Coast Medical, Inc.), the visual analogue scale , and a four-point verbal rating scale (VRS) for pain evaluation . The static hyperalgesia was also assessed proximally to the surgical wound and on the forearm. Tactile pain thresholds were measured 2 to 3 cm away from the potential incision at 3 levels , separated by about 5 cm on the right side. It is defined as the smallest force that was just perceived as painful [During the preoperative evaluation on the day before surgery, after informed consent was signed, patients were instructed in the use of the PCIA pump, the quantitative sensory tests with calibrated Von Frey filaments .In our preliminary of 20 patients, we concluded that an estimated sample size of 22 patients per group would give us a μg/kg sufentanil was given intravenously for postoperative analgesia. After incision closure, sevoflurane and remifentanil were discontinued, a dose of 0.15 mg/kg dezocine  was administered to dezocine group, and the same volume of normal saline was administered to group control. The patients and the investigators involved in patient management or data collection were all unaware of the group assignment.These 50 patients were randomly assigned to one of the two groups: dezocine group and control group. Each group had a random-number generated by computer precising the group assignment and envelopes containing the results were prepared. In the morning of surgery, a person not involved in the evaluation procedure opened the envelope and prepared the drugs. Thirty minutes before the end of the surgery, a titration of 0.1 μg/h with a 15 min lockout and continuous infusion of 3μg/h. In general ward this PCIA regimen was maintained and rescue analgesics were administered if patient required extra pain control or VAS > 5.Inspired sevoflurane concentration was increased stepwise by 0.5%–1% when insufficient anesthesia was considered which was defined as an accelerated heart rate by 15% or a systolic arterial blood pressure exceeding baseline values by 20% with or without clinical signs of inadequate anesthesia such as patient movement, coughing, and tearing. Inadequate anesthesia was also treated with propofol. Atropine and ephedrine were prepared to treat bradycardia and hypotension perioperatively. After recovery of adequate spontaneous ventilation and the obeisance to verbal commands (eye opening and limb moving), the tracheal tube was removed and the patients were kept in the postanesthesia care unit (PACU) for an hour, where standard monitoring was recorded every 15 min. Additional analgesics were given to the patients who had VAS > 5. Background infusion of PCIA was started just before leaving PACU and the postoperative pain was controlled by PCIA, which was programmed to deliver demand doses of sufentanil 3Baseline MAP and HR were defined as the mean of the two lowest measurements recorded during a 5 min interval just before the induction of anesthesia. Values from all routine anesthetic monitors were recorded at a 5 min interval perioperatively. Duration of anesthesia and surgery, the total doses of remifentanil, and the use of atropine or ephedrine were also recorded.Rescue analgesics, VAS score evaluating the pain intensity, and sedation score monitored by Ramsay scale  were recorded during the PACU stay. The cumulative consumption of sufentanil given by PCIA and pain scores (VAS and VRS) were also recorded at 6, 24, and 48 h after surgery. The primary outcome was the consumption of sufentanil during the first 24 h after surgery. The incidences of postoperative nausea and vomiting were recorded during the visit at 48 h postoperatively. Subjects who experienced severe postoperative nausea and vomiting (PONV) were treated with ondansetron 4 mg intravenously. Other side effects such as respiratory depression, muscle rigidity, pruritus, and dysphoria were also recorded.2) at 6, 24, and 48 h after surgery. The extent of mechanical static hyperalgesia to punctuate stimulation proximal to the wound was assessed with Von Frey filament number 17 (60 g/mm2) as previously described [The pain threshold for mechanical static stimuli was evaluated both at 2 cm proximal to the surgical wound and on the forearm by calibrated Von Frey filaments . P values less than 0.05 were considered to be statistical significance.Age, weight, height, BMI, duration of surgery and anesthesia, intraoperative remifentanil use, haemodynamic variables, cumulative sufentanil consumption, and VAS scale were analysed by Student's P > 0.05).A total of 50 patients (25 patients in each group) were recruited in our study. P > 0.05). P > 0.05).During the surgical procedure, hemodynamic status and drug consumption were noted. P < 0.05) and lower VAS pain score at the first hour after the surgery compared with the patients in the control group (P < 0.05) (P < 0.05). P < 0.05). The extent of peri-incisional hyperalgesia was statistically significantly smaller in patients of the treatment group than the control group (P < 0.05) (P > 0.05).During postoperative follow-up, patients treated with dezocine showed statistically significantly decreased PCIA consumption at 6 postoperative hours ( < 0.05) .  Table  < 0.05) , whereas < 0.05) , and theTo the best of our knowledge, this study firstly showed that intravenous injection of 0.15 mg/kg dezocine at the end of surgery leads to a better postoperative pain control manifesting as indicated by a fewer PACU rescue analgesics demands, a decreased cumulative postoperative PCIA consumption during the first 6 postoperative hours, and a more satisfactory VAS score at 1 hour postoperatively. Dezocine can also provide antihyperalgesia effects, including a higher pain threshold on the forearm and a smaller extent of hyperalgesia proximal to the surgical wound.μg/kg/min and then adjusted to the hemodynamic changes or the anesthetic depth for the maintenance of anesthesia, which is widely considered dose-dependently [Pain after surgery is a major management challenge in clinical practice and postoperative hyperalgesia is usually complicated in such situations. A considerable number of surgical patients suffer from moderate to severe acute postoperative pain and a large number of patients experience inadequate pain relief with available pain managements. Postoperative hyperalgesia manifesting as an exaggerated original underlying painful condition or a more generalized state mainly involves both surgical nociception and opioid administrations and it is considered as a very important contributive cause of inadequate postoperative pain control . Postopeendently  to generendently . Surgicaendently . Here baμ receptor antagonist. A recently published study identified that dezocine is a κ opioid antagonist and also inhibits norepinephrine and serotonin reuptake in vitro [Dezocine, as a combined opioid agonist/antagonist, is of great interest because of its ability to produce analgesia, decreased liability to addiction, and limited depression of respiration compared with the classical opioid . Clinicain vitro , which mμg/kg/min and 0.4 μg/kg/min is the most widely reported concentration inducing significant postoperative RIH in the literature. In the clinic, we found that patients with high perioperative dose of remifentanil (0.4 μg/kg/min) had frequently unstable haemodynamics and usually required repeated administration of ephedrine and atropine. Thus, in terms of patients' safety and as required by both of the anesthesiologists and the surgeons, we decreased the dose and hyperalgesia was still induced as proven by the quantitative pain test, calibrated Von Frey filaments. Secondly, we did not note the delay of the first demand of PACU rescue analgesics and the dosage of the analgesics used during PACU stay. The length of PACU stay was not assumed, which should be dependent on the patient status. But in our institute it might be more dependent on the department policy which usually requires one-hour PACU stay. Thirdly, our study was powered on the postoperative PCIA consumption. A larger study powered on the pain threshold change for about several weeks warrants being conducted to see the long term hyperalgesia relief effects of dezocine since evidences have showed that there might be a link between postoperative hyperalgesia and the risk of chronic pain development.Several limitations of the present study should be highlighted. Firstly, the dosage of remifentanil used in our study was less than 0.4 In conclusion, our study shows that, for patients undergoing open gastrectomy, intravenous injection of dezocine at the end of surgery decreases PCIA consumption and helps achieve a better postoperative pain control as well as a decreased generalized and peri-incisional extent of hyperalgesia level. Dezocine may be a potentially useful adjunctive agent for the management of postoperative hyperalgesia and further studies are needed to investigate the optimal dose and to explain the mechanisms.This prospective, double-blinded, and randomised study has confirmed that dezocine provides analgesic and antihyperalgesic effects for patients undergoing elective open gastrectomy. No adverse effects were observed for up to 48 postoperative hours.


The only susceptible murine models have involved intracerebral inoculations or juvenile animals, which do not replicate natural infection. Our report has studied the effect of ZIKV infection in type-I interferon receptor deficient (A129) mice and the parent strain (129Sv/Ev) after subcutaneous challenge in the lower leg to mimic a mosquito bite. A129 mice developed severe symptoms with widespread viral RNA detection in the blood, brain, spleen, liver and ovaries. Histological changes were also striking in these animals. 129Sv/Ev mice developed no clinical symptoms or histological changes, despite viral RNA being detectable in the blood, spleen and ovaries, albeit at lower levels than those seen in A129 mice. Our results identify A129 mice as being highly susceptible to ZIKV and thus A129 mice represent a suitable, and urgently required, small animal model for the testing of vaccines and antivirals.Zika virus (ZIKV) is a mosquito-borne pathogen which has recently spread beyond Africa and into Pacific and South American regions. Despite first being detected in 1947, very little information is known about the virus, and its spread has been associated with increases in Guillain-Barre syndrome and microcephaly. There are currently no known vaccines or antivirals against ZIKV infection. Progress in assessing interventions will require the development of animal models to test efficacies; however, there are only limited reports on  Since first being recognised in 1947, Zika virus (ZIKV) has mainly been associated with a mild illness with symptoms including a limited fever and rash. In 2007 the virus spread from Africa into French Polynesia and then onwards across Pacific regions and into South America. In these new regions, ZIKV has been associated with more severe clinical conditions including Gullain-Barre syndrome (GBS) and microcephaly. There are no currently approved antivirals or vaccines available with proven activity against ZIKV, and the World Health Organisation declared the spread of ZIKV as a Public Health Emergency of International Concern. Here, we have used a mouse strain with a deficiency in the type-I interferon receptor (A129) and shown that these mice are susceptible to ZIKV infection after an inoculation that closely resembles the natural route of infection via mosquito bite. Although A129 mice are deficient in the innate interferon response, they retain their adaptive immunity and thus have successfully been used as suitable models for the testing of vaccinations and antivirals. Our study provides details on a suitable small animal model for the testing of future interventions against ZIKV. Aedes africanus mosquitoes from the same forested area [Zika virus (ZIKV) is a mosquito-borne flavivirus first isolated from a sentinel rhesus macaque placed in the Zika forest in Uganda in 1947; thereafter, ZIKV was isolated from a pool of ted area , Pakistated area , Malaysited area . Until ated area . No furtted area . The virted area . In 2015ted area . Whilst ted area ; anotherted area . The latted area  and alsost February, 2016, the World Health Organisation declared ZIKV to be a “Public Health Emergency of International Concern” [The expansion of ZIKV endemicity follows closely that of chikungunya virus, which now circulates in most inhabited continents where competent mosquito vectors are extant, and has become a global public health problem in the past decade . At the Concern” .Recently, ZIKV infection has been associated with GBS, an autoimmune disease that causes acute or subacute flaccid paralysis , 20. In in vivo.Recent widespread attention has been given to increases in the cases of microcephaly and its coincidence with the ZIKV epidemic in South America. Over the past five years, the incidence of microcephaly has been between 130 and 170 cases annually, but in the first nine months of 2015 this figure roughly doubled . In the A small animal disease model would advance knowledge about this pathogen, and open avenues for further research including studies of ZIKV vertical transmission, and any direct or indirect effects of infecton on neural development . It woulAedes africanus [ZIKV  was obtAll procedures with animals were undertaken according to the United Kingdom Animals (Scientific Procedures) Act 1986. These studies were approved by the ethical review process of Public Health England, Porton Down, UK and the Home Office, UK via an Establishment Licence (PEL PCD 70/1707) and project licence (30/3147).Mice deficient in the IFN-α/β receptor (A129) and congenic control mice (129Sv/Ev) were obtained from an established breeding colony approved by the UK Home Office . Female mice aged 5–6 weeks were used for all studies.6 plaque-forming units (pfu) of ZIKV diluted in phosphate buffered saline (PBS) were administered subcutaneously in volumes of 40μL into each of the right and left hind legs just above the ankle of 12 A129 and 12 129Sv/Ev mice. Control groups of a further three animals of each mouse strain were inoculated with 40 μL PBS in each of the hind legs. Mice were monitored three times each day for clinical signs of disease and a numerical score was assigned at each observation . Temperatures were recorded by an indwelling temperature chips and weights were also recorded daily. A set of humane clinical end points were defined by veterinary staff as a 20% weight loss, or 10% weight loss and a clinical symptom, which mandated euthanasia. At 3 and 7 days post-challenge, a group of four animals from each of the A129 and 129Sv/Ev ZIKV-challenged groups were scheduled to be culled to assess local responses. At necropsy, samples of spleen, liver, brain and ovary were collected and immediately frozen at -80°C for virological analysis or inserted into pots containing 10% neutral buffered saline for microscopic analysis. Blood was collected into RNAprotect tubes  for viral load testing.Virus inocula containing 10Tissue samples were weighed and homogenised into PBS using ceramic beads and an automated homogeniser  using six 5 second cycles of 6500 rpm with a 30 second gap. 200 μL of tissue homogenate or blood solution was transferred to 600 μL RLT buffer  for RNA extraction using the RNeasy Mini extraction kit ; samples were passed through a QIAshredder  as an initial step.4, 1μl of each primer (ZIKV 1086 and ZIKV 1162c both at 18μM working concentration), 0.8μL of probe (ZIKV 1107-FAM at 25μM working concentration) and 0.8μL of SSIII enzyme mix. 5μL of template RNA was added to the mastermix in order to give a final reaction volume of 20μL. The cycling conditions used were 50°C for 10 minutes, 95°C for 2 minutes, followed by 45 cycles of 95°C for 10 seconds and 60°C for 40 seconds and a final cooling step of 40°C for 30 seconds. Quantification analysis using fluorescence was performed at the end of each 60°C step. Reactions were run and analysed on the 7500 Fast platform  using 7500 software version 2.0.6.A ZIKV specific real-time RT-PCR assay was utilised for the detection of viral RNA from subject animals. The primer and probe sequences were adopted from a published method  with in-Quantification of viral load in samples was performed using a dilution series of quantified RNA oligonucleotide (Integrated DNA Technologies). The oligonucleotide comprised the 77 bases of ZIKV RNA targeted by the assay, based on GenBank accession AY632535.2 and was synthesised to a scale of 250 nmole with HPLC purification. This RNA oligonucleotide was quantified using Nanodrop technology  with a 10-fold dilution series of quantified material used to assess viral load in subject samples. Eight dilutions of quantified ZIKV RNA oligonucleotide control material were used to assess the limit of detection ranging from 1,000,000 copies per reaction to 0.1 copies per reaction; all dilutions were run in triplicate. The limit of detection for this assay was assessed to be 10 genome copies per PCR reaction with 100% detection for this and all higher concentrations. All samples for 1 copy per reaction and 0.1 copies per reaction were negative. In-run analysis indicated the reaction efficiency to be 101% with a slope of -3.29, a Y intercept of 40.17 cycles and an R2 value of 0.998.Tissue samples were fixed in 10% neutral buffered formalin for 48 hours and processed routinely to paraffin wax. Sections were cut at 3–5 μm, stained with haematoxylin and eosin (H&E) and examined microscopically. Lesions referable to infection were scored subjectively using the following scale: within normal limits (WNL), minimal, mild, moderate and marked. The pathologist was blinded to the groups in order to prevent bias.6 pfu ZIKV in the lower leg. Survival analysis was compared between the two ZIKV-infected mouse strains and mock-infected animals which received PBS only, and demonstrated that all A129 animals met humane clinical endpoints 6 days after challenge with ZIKV (Groups of 5–6 week old mice either lacking receptors for IFN-α/β (A129) or from the wild-type strain (129Sv/Ev) were subcutaneously inoculated with 10ith ZIKV . Wild-tyith ZIKV . Similarith ZIKV . The A12ith ZIKV . None ofViral RNA levels were quantified in the blood and tissues  of ZIKV-challenged A129 and 129Sv/Ev mice culled at day 3 post-challenge and days 6 (A129) or 7 (129Sv/Ev) post-challenge . ResultsInflammatory and degenerative changes were observed in the brains of A129 mice challenged with ZIKV. These comprised widespread nuclear fragments, scattered diffusely throughout the grey and white matter . PerivasNumerous small well-defined germinal centres with apoptotic bodies and mitotic figures were noted in the splenic white pulp of control animals. In the spleen of challenged A129 mice, large, poorly defined germinal centres were observed in the white pulp, together with numerous apoptotic bodies and a prominent depletion in the number of mature lymphocytes . PromineLesions referable to Zika virus infection were not observed in the brain, spleen, liver or ovaries of the wild-type 129Sv/Ev mice or the control animals .in vivo passaging of virus was needed to strengthen effects [6 pfu used for inoculation was within the limits of studies with another mosquito-borne flavivirus, West Nile virus, which demonstrated a viral dose 104−106 pfu per mosquito bite [At the time of writing, our studies provide the first evidence for a susceptible adult mouse model for ZIKV infection after shallow inoculation of virus below the skin surface at one of the body’s extremities, mimicking natural infection via mosquito bite . Additio effects . The conito bite .Throughout the course of infection, animals were monitored regularly for clinical signs of disease, including changes in weight and temperature fluctuations. Only the A129 mice with a knockout in their type-I interferon receptor showed evidence of disease. Although fever and general malaise are typical of human ZIKV infection , similarHistological analysis showed that microscopic lesions attributable to infection with ZIKV were observed in A129 mice and these focused primarily in the brain. The lesions observed share similarities with those reported in previous studies ; necrosiWhilst the main focus of attention for ZIKV is its possible links with microcephaly, as with other intrauterine infections, it is possible that the reported cases of microcephaly represent only the more severely affected children. It may be that newborns with less severe disease, affecting not only the brain but also other organs, have not yet been identified ; a rangeThe most significant lesions referable to infection with ZIKV in the mice were those observed in the brain. Nuclear fragmentation, perivascular cuffing by inflammatory cells, and degenerative cells in the hippocampus have been reported previously , 37. It When local viral loads were analysed by real-time RT-PCR, ZIKV RNA was detected in the blood, brain, ovary, spleen and liver of all A129 mice. Interestingly, viral loads in the blood, ovary, spleen and liver decreased by approximately 1–2 logs between days three and six post infection despite adverse changes in clinical symptoms, temperature and weight; however, the mean viral load in brain samples increased by over 3 logs during this period. Whilst we cannot discount very low levels of vRNA contamination in the organs from the viremic blood, the assays are quantitative and so the levels we report demonstrate viral RNA levels which are specific to the time points and tissues. Whilst no histological changes were observed in 129Sv/Ev mice challenged with ZIKV, viral RNA was detected in the blood, ovary and spleen at day 3 post-challenge and just in the ovary and spleen on day 7 post-challenge indicating that the virus entered the circulation and seeded into some of the organs. These findings suggest that despite showing no overt clinical disease, the wild-type 129Sv/Ev mice subclinically harbour ZIKV. The short timeframe of detectable viral RNA is in line with observations in humans with transient viraemia even during the acute phase of clinical disease , 61. DueThe model system reported was assessed with respect to challenge with ZIKV strain MP1751, an African isolate from 1962 . This stin vivo model for the testing of vaccines and therapeutics. The challenge route and dose are similar to those found in natural infection from mosquito bite. This is the first report of a suitable murine model and can accelerate the testing of novel interventions against this pathogen declared as public health emergency of international concern.In summary, the results we report demonstrate that A129 mice are a susceptible mouse strain to ZIKV infection which we propose as a suitable and informative


Several respiratory diseases are associated with specific respiratory sounds. In contrast to auscultation, computerized lung sound analysis is objective and can be performed continuously over an extended period. Moreover, audio recordings can be stored. Computerized lung sounds have rarely been assessed in neonates during the first year of life. This study was designed to determine and validate optimal cut-off values for computerized wheeze detection, based on the assessment by trained clinicians of stored records of lung sounds, in infants aged <1 year.Lung sounds in 120 sleeping infants, of median (interquartile range) postmenstrual age of 51 (44.5–67.5) weeks, were recorded on 144 test occasions by an automatic wheeze detection device (PulmoTrack®). The records were retrospectively evaluated by three trained clinicians blinded to the results. Optimal cut-off values for the automatically determined relative durations of inspiratory and expiratory wheezing were determined by receiver operating curve analysis, and sensitivity and specificity were calculated.The optimal cut-off values for the automatically detected durations of inspiratory and expiratory wheezing were 2% and 3%, respectively. These cutoffs had a sensitivity and specificity of 85.7% and 80.7%, respectively, for inspiratory wheezing and 84.6% and 82.5%, respectively, for expiratory wheezing. Inter-observer reliability among the experts was moderate, with a Fleiss’ Kappa  of 0.59 (0.57-0.62) for inspiratory and 0.54 (0.52 - 0.57) for expiratory wheezing.Computerized wheeze detection is feasible during the first year of life. This method is more objective and can be more readily standardized than subjective auscultation, providing quantitative and noninvasive information about the extent of wheezing. Wheezes consisting of continuous musical sounds of one or more tonal components , 2 amongWheezing is most frequently diagnosed by auscultation using a stethoscope or is based on parental reports of wheezes. However, parents often differ in their understanding of wheeze , 15 and To date, few studies have used computerized methods to detect wheezes during the first year of life , 23. TheComputerized wheeze detection and subjective lung sound assessment were performed in 120 infants, of median age 51 postmenstrual weeks, on 144 test occasions. Lung sounds were recorded during lung function testing (LFT) as part of our routine follow-up care of infants requiring intensive care . PatientAll parents provided written informed consent before each LFT, and the study protocol was approved by our Institutional Data Safety Committee.Wheezes were detected using the PulmoTrack® Model 2020 , an instrument developed for the continuous tracking and recording of breathing sounds and the detection of wheezing. Lung sounds were analyzed using a fast Fourier transform (FFT)-based algorithm for lung sound analysis and two phonopneumographic contact sensors, one applied to the region of the manubrium and the other over the left axillary line Figure . The senThe PulmoTrack® calculates the relative inspiratory and expiratory wheeze rates as.w in/ex is the breathing time with wheeze during inspiration/expiration and Tin/ex is the total inspiratory/expiratory breathing time.where TRecorded sounds coded for each infant were retrospectively evaluated by three medical doctors working in the neonatal intensive care unit and trained before the study using a computer aided instruction on respiratory sounds (R.A.L.E.® Lung Sounds 3.2). Using headphones that minimized surrounding noise, each observer listened to the sound of each infant in a blinded fashion and assessed if wheezing was present or absent, independent of the strength and duration of sounds.−1), since sedation was necessary for subsequent more complex LFT [Lung sounds were recorded in clinically stable and sleeping infants who had no respiratory infections during the 3 weeks preceding the tests. Sleep was induced 15–30 min before LFT by oral administration of chloral hydrate  or as medians and interquartile ranges (IQR). Incidences of wheezing were compared using Fisher’s exact test. The Kruskal-Wallis rank test was used to investigate the influence of birth weight, mechanical ventilation and BPD on wheeze rates. Inter-observer reliability of lung sound assessment was assessed using Fleiss’ kappa, which is a generalization of Cohen’s kappa to multiple raters that provides a conservative measure of agreement. The 95% confidence interval of Fleiss’ kappa was calculated as described , with FlLung sounds were recorded in 120 infants on 144 test occasions, with 98 infants (82%) tested on one occasion, 20 (17%) on two occasions and 2 (1.7%) on three occasions. Patient characteristics are shown in Table The distributions of inspiratory and expiratory wheeze rates are shown in Figure Evaluation of wheezing in the 144 recordings by’ the three observers is shown in Table Because the PulmoTrack® detected wheezing in almost all infants, cut-off values for the duration of wheezing were needed to compare computerized and subjective assessments of wheezing. For this purpose, the results of the three observers were classified into three groups: no wheezing detected by all three (group 1), lack of agreement on the presence of wheezing (group 2) and wheezing detected by all three group 3). Classifications of inspiratory and expiratory wheezing into these three groups are shown in Figure . ClassifThe study showed that the PulmoTrack® can reliably detect wheezing in neonates, with sensitivities of 85.7% for inspiratory and 84.6% for expiratory wheezes and specificities of 80.7% and 82.5%, respectively, using appropriate cut-off values. Computerized wheeze detection reliably detects even short periods of wheezing, as reflected by the low cut-off values of 2% for inspiratory and 3% for expiratory wheezing.The equipment used in this study differs from that used in previous studies in older children, in which sound was recorded by five sensors , 23. DueThe inter-observer reliability for wheeze detection, expressed as the Fleiss Kappa coefficient, was moderate in our study, reflecting a higher inter-observer reliability than reported in most previous studies , 28–30. The disparity in lung sound nomenclature has been cited as contributing to disagreements among observers , 5, 31. This study has several strengths and limitations. The main strengths include the use of a larger sample size than in previous studies on wheeze detection in infants , 20 and One study limitation was that all sound recordings were performed in a quiet lung function testing unit. Thus, we cannot determine the quality of computerized wheeze detection in noisier clinical settings. Moreover, all infants included in our study were sedated for LFT, preventing a determination of the quality of computerized wheeze detection in awake and possibly restless infants.Computerized wheeze detection using PulmoTrack® is feasible and reliable in neonates <1 year when using appropriate cut-off values for inspiratory and expiratory wheeze rate. This method provided quantitative and noninvasive information about the extent of wheezing, in contrast to the assessment by trained clinicians, which was subjective and only moderate in the inter-observer agreement. Since this included only infants indicated for LFT due to pulmonary impairment, further studies are needed to evaluate lung sounds in healthy infants.


A rapid expansion of HFMD with enterovirus 71 infection outbreaks has occurred and caused deaths in recent years in China, but no vaccine or antiviral drug is currently available for EV71 infection. This study aims to provide treatment programs for HFMD patients. We conducted a randomized, double-blind, controlled trial and evaluated clinical efficacy of therapy with rHuIFN-α1b in HFMD patients with EV71 infection. There were statistical differences in outcomes including the fever clearance time, healing time of typical skin or oral mucosa lesions, and EV71 viral load of the HFMD patients among ultrasonic aerosol inhalation group, intramuscular injection group and control group. rHuIFN-α1b therapy reduced the fever clearance time, healing time of typical skin or oral mucosa lesions, and EV71 viral load in children with HFMD.Trial Registration: Chinese Clinical Trial Registry ChiCTR-TRC-14005153 Hand, foot, and mouth disease (HFMD) is an important infectious disease in young children, particularly in those less than 5 years old. HFMD epidemics have affected various countries in the past 40 years . Large-sE. coli to yield an approximately 19 kDa single non-glycosylated polypeptide chain containing 166 amino acids [Interferons (IFNs) are natural glycoproteins belonging to the cytokine superfamily, and are produced in response to foreign agents such as viruses, parasites and tumor cells. They are important modulators of the immune response, particularly in inhibiting viral replication within host cells, activating natural killer cells and macrophages, increasing antigen presentation to lymphocytes, and inducing the resistance of host cells to viral infection , 8. Inteno acids –13.We undertook a clinical trial in HFMD patients with EV71 infection to examine clinical efficacy of therapy with rHuIFN-α1b. Our studies intended to provide treatment programs for HFMD patients.The protocol for this clinical trial and supporting CONSORT checklist are available as supporting information; see E. coli, purified and processed into a freeze-dried powder including human serum albumin, sodium chloride. The freeze-dried powder is stable when stored and transported at 2–8°C from light. 20 μg of the freeze-dried powder per vial was used for this study (Authorization number: Guo Yao zhun Zi S20033034).Recombinant human interferon α1b (rHuIFN-α1b) for injection  was expressed in The incidence of severe cases is about 15% among hospitalized patients with HFMD in Henan province . To baseHFMD was clinically diagnosed based on the guidelines for control and prevention of HFMD issued by the National Health and Family Planning Commission of the People’s Republic of China (2010). Briefly this includes patients from HFMD endemic regions presenting during the epidemic season; fever; and, skin eruptions on the hands, feet, and mouth, or only in the oral cavity.Criteria for inclusion were: age between 0.5 to 5 years; according to the clinical diagnostic criteria of HFMD, subject patients with one of following clinical manifestations: 1) fever (axillary temperature ≥ 38.5C); 2) neurological complications including mental fatigue, irritability, headache, vomiting, convulsions, limb weakness; and 3) respiratory frequency (RF) ≥30 under resting conditions, heart rate (HR) ≥ 140; and stool specimen was positive for EV71 by real-time RT-PCR.Exclusions from participation included: 1) critically ill HFMD patients whose treatment could be complicated by central respiratory failure or who had brain-stem encephalitis, acute flaccid paralysis, neurogenic pulmonary edema, neurogenic shock or other severe symptoms, or required endotracheal intubation and mechanical ventilation; 2) allergic constitution, especially allergy to multiple antibiotics or interferon products; 3) congenital heart disease; and 4) epilepsy and other central nervous system dysfunction.Between August 2014 and July 2015, 317 patients with HFMD were registered at Children’s Hospital of Zhengzhou and Children’s Hospital of Kaifeng. The patients were randomized into a control group, an ultrasonic aerosol inhalation group, and an intramuscular injection group. The randomization codes were prepared by the Department of Epidemiology and Statistics, College of Public Health, Zhengzhou University with the use of STATA 8.0 software. The study was conducted in an inpatient setting according to a randomized, double-blind, comparative study design, in which the purpose was to evaluate efficacy of rHuIFN-α1b in patients with EV71 infection .The patients who met the eligibility criteria were randomized to an ultrasonic aerosol inhalation group, an intramuscular injection group and a control group. All HFMD patients received supportive and symptomatic treatment. Patients randomized to the ultrasonic aerosol inhalation group were administered inhaled ultrasonically-nebulized rHuIFN-α1b  once daily over 15–20 minutes for 5 days. The intramuscular injection group received rHuIFN-α1b  by intramuscular injection for 5 days. The control group only received supportive and symptomatic treatment as per current standard of care. Stool specimens for viral load measurements were collected on days 0 and 6.The patients were observed from 0 to 5 days and data was recorded in case report forms (CRF) by the investigators, including the following: temperature, skin rashes, oral ulcers, heart rate, respiratory rate, and nervous system signs/symptoms . During the treatment period, safety was assessed by clinical examination and laboratory investigations, and adverse events were recorded by asking the HFMD subjects’ parents. Adverse events were graded according to the degree of correlation between those events and rHuIFN-α1b.Termination criteria for the study or for individual patients included: 1) development of critical illness in HFMD subjects; 2) development of intolerable side effects from rHuIFN-α1b in the HFMD subjects; 3) requests from parents for withdrawal from the trial; 4) protocol treatment discontinuation after physician evaluation.Total RNA was extracted from clinical samples using the QIAamp viral RNA mini Kit , following the manufacturer’s instructions. The RNA was eluted in a final volume of 60 μL of elution buffer and used immediately or stored at -80°C.6 to 2.5 copies per μL. Since the primers and probe were designed based to amplify the 226 bp cloned fragment, the RNA panels were used as a standard for real-time quantitative RT-PCR and EV71 viral load testing as in our previous studies [In order to establish copy number as a unit of viral load, a 226 bp fragment of the VP1 gene of EV71 was amplified by RT-PCR then cloned into vector pGEM-T . Next, the recombinant plasmids extracted were linearized by the SpeI digestion . The digestion products were both transcribed in vitro into RNAs and purified by a RioMAX large-scale RNA production system-T7 (Promega). Finally, the RNA panels were serially diluted from 2.5 × 10 studies , 16. The2 test or Fisher’s exact test. A significant difference was considered with a P value less than 0.05.All data was double-entered with the EPIDATA 3.1 software to prevent transcription errors. All statistical analyses were performed using SAS v9.13 . Measurement data was analyzed by single factor variance analysis or Student’s t-test. Count data was analyzed by χA total of 317 clinically diagnosed HFMD subjects were enrolled in our study; 9 did not meet inclusion criteria and 3 met exclusion criteria. 305 HFMD subjects were then enrolled into the rHuIFN-α1b treatment trial after obtaining written informed consent. The patients were randomized, 106 cases to control group, 118 cases to ultrasonic aerosol inhalation group and 81 cases to intramuscular injection group. 10 HFMD patients withdrew from the treatment trial because of parental request, and 21 HFMD patients withdrew because they developed critical illness requiring transfer to the intensive care unit (ICU). 274 HFMD subjects completed the rHuIFN-α1b treatment trial .Of the 274 subjects who completed treatment trial patients, 172 (62.77%) were male and 102 (37.23%) were female. The mean age of the HFMD patients was 1.98 years . Baseline demographics and clinical characteristics were well matched among the randomized groups .P<0.001), and between the ultrasonic aerosol inhalation and the control groups , but no significant differences between the ultrasonic aerosol inhalation and the intramuscular injection groups .There were statistical differences in outcomes including the fever clearance time, and the healing time for typical skin or oral mucosal lesions. The 3 groups had similar mean scores and no statistical differences for initial body temperature, but the improvement was greater for the ultrasonic aerosol inhalation and intramuscular injection groups compared with the control group at follow up. The mean time to fever clearance was 80.64 hours  for the control group, 66.00 hours  for the ultrasonic aerosol inhalation group, and 65.52 hours  for the intramuscular injection group, respectively. There were statistically significantly differences between the intramuscular injection and the control group , and between the ultrasonic aerosol inhalation group  and the control group , but no significant differences were observed between the ultrasonic aerosol inhalation group and the intramuscular injection group .The healing time for typical skin or oral mucosal lesions was significantly different between the intramuscular injection group  and the control group   and interact time . When compared by pairwise comparison methods, there was a statistical difference between the control group and other groups or between the ultrasonic aerosol inhalation group and the intramuscular injection group. In interact time, the respiratory rates of first day and second day were not statistical difference, the fourth day and fifth day were also not statistical difference, but the first day and second day were statistically significant difference between the third day, fourth day or fifth day, respectively (P<0.05). The mean respiratory rates decreased gradually from first day to fifth day. The heart rates were not statistical difference among the groups  and interact time  by GEE. There were a statistical difference between the first day and third day (P<0.05). The mean heart rates increased on second day, but began to decrease from third day , but was significantly different at day 6 . The mean viral load decrease  were significantly different among groups , but not significantly different between the ultrasonic aerosol inhalation and the intramuscular injection group   .EV71 is a small, non-enveloped, positive-stranded RNA virus with a genome of approximately 7,400 bases, and it is a member of the genus Enterovirus in the family Picornaviridae . Large EThis randomized, double-blind, controlled trial demonstrates that rHuIFN-α1b combined with supportive and symptomatic therapy greatly decreases viral load, significantly accelerates fever clearance time and the healing time for shin or oral mucosal lesions. No deaths or severe adverse events were observed in rHuIFN-α1b therapy group. Some previous studies showed that ribavirin, several kinds of herbal injections  could reduce the time of healing, time of fever clearance, and time for rash to subside. However, the evidence supporting the use of herbal medicine for HMFD is of insufficient quality, and the available data indicate that herbal medicine, especially herbal injections or combinations of herbal and Western medications might improve symptoms of HMFD .When used in the systemic therapy, IFNs are mostly administered by an intramuscular injection. The most frequent adverse effects are flu-like symptoms, especially increased body temperature. EV71 infection causes HFMD, a common exanthema of young children that is characterized by a fever, rashes on the palms and the bottoms of the feet, and ulcers in the oral cavity . As a reWe observed that the mean respiratory rates and heart rates were significantly different in the groups on the second and third days, respectively. The mean respiratory rates for the intramuscular injection group was higher than for other groups on day 2, and the mean heart rates of intramuscular injection group was higher than that of other groups on third day. This phenomenon could be considered a potential adverse effect of intramuscular rHuIFN-α1b.There were a number of limitations related to this study, including: lack of long-term follow up after the treatment; and outcome measures during rHuIFN-α1b treatment were only collected for HFMD patients with EV71 infection. Thus, it is premature to conclude that treatment with rHuIFN-α1b for HFMD is safe owing to the lack of long-term follow up. Furthermore, we only investigated hospitalized patients, and these results might not be applicable to the primary care setting.S1 CONSORT Checklist(PDF)Click here for additional data file.S1 Consent(PDF)Click here for additional data file.S1 Protocol(PDF)Click here for additional data file.S1 Table(PDF)Click here for additional data file.