In this study, the prevalence of vitamin D deficiency was assessed in a group of children and adolescent patients with recent-onset type 1 diabetes mellitus (T1DM).Fifty-three patients with age 8–18 years and duration of T1DM less than 8 weeks were recruited. A food frequency questionnaire (FFQ) was used to assess dietary vitamin D and calcium intake. Sunshine exposure was measured using a questionnaire to quantify the amount of time children spent in the sun and other sun-related habits, and a sun index score was generated. Serum 25(OH)D < 20 ng/ml was considered as vitamin D deficiency. Logistic regression was used to assess predictors of vitamin D deficiency.P=0.029). In addition, vitamin D deficiency in boys was lower than girls in this model .All patients were vitamin D deficient (77%) or insufficient (23%). In a logistic regression model, it was shown that the risk of being vitamin D deficient was significantly decreased by sunlight exposure ≥ 15 minutes during the weekends versus < 15 minutes (OR: 0.06, 95% CI: 0.01–0.75; Vitamin D deficiency is highly prevalent among children and adolescents with T1DM in Iran. Boys and children with ≥ 15 minutes sunlight exposure in weekends were less likely to be vitamin D deficient than girls and those with < 15 minutes sunlight exposure. Type 1 diabetes mellitus (T1DM) is a complex disease characterized by autoimmune and progressive destruction of insulin secreting pancreatic β-cells. Both genetic and environmental factors are main agents participating in this autoimmune process .The incidence is very different according to geographic variation, with a child in Finland being about 400 times more likely than a child in Venezuela to acquire the disease . Season One of the environmental factors thought to be protective against the development of T1DM is early supplementation with vitamin D . VitaminThe marker of vitamin D status is 25(OH)D since it is the major circulating metabolite of vitamin D . StudiesBefore conducting a clinical trial of supplementing patients with T1DM, it is required to assess the existing status. In this cross-sectional study, the prevalence of vitamin D deficiency was assessed in children and adolescents with new-onset T1DM. Factors such as BMI, sex, sunlight exposure, and dietary vitamin D and calcium were assessed in relation to vitamin D deficiency. In addition, serum iPTH (intact parathyroid hormone) level was assessed to identify patients with secondary hyperparathyroidism.The research was approved by the Ethics Committee of Endocrinology and Metabolism Research Center (EMRC)/Tehran University of Medical Sciences. Fifty three patients with newly diagnosed T1DM were recruited from outpatient diabetes clinic in Shariati Hospital and Children Medical Center of Tehran University of Medical Sciences.Subjects included if they were 8–18 year, consecutively diagnosed as having T1DM , duratioA 49-item semi-quantitative food frequency questionnaire (FFQ) on calcium and vitamin D intake was administered by the nutritionist. Frequencies of food intakes over the previous 3 months were reported in day, week, or month. Calcium content of foods was derived from Nutritionist III software modified for Iranian foods. Vitamin D was calculated according to the “Provisional Table on the Vitamin D Content of Foods” released by United States Department of Agriculture (USDA) .Average sunshine exposure was measured using a questionnaire to quantify the amount of time children spent in the sun and other sun-related habits during past 3 months since patients’ recruitment. The questions asked about time spent outdoors, time of day when outdoors, use of sunscreen, sun protection factor (SPF), type of clothing, and the skin pigment. It was a modified version of the sun exposure questionnaire designed by Dr Glanz , and theSun index score was generated as a measure combining hours spent in sun exposure during weekdays and weekends, and sun protective behaviors (i.e. clothing and sunscreen use) and patients’ skin color. Possible scores ranged from 4–14. Higher scores represented higher sunlight exposure. The sun scores were examined with respect to their relationship to serum vitamin D levels. Fasting serum 25(OH)D and intact PTH level was measured using immunoradiometric assay with the IDS kit .Data was analyzed using the statistical software SPSS version 16.0 for windows . P-values < 0.05 were considered statistically significant. Logistic regression was used to assess predictors of vitamin D deficiency. Serum 25(OH)D as a dichotomous variable  was used to examine the associations of vitamin D deficiency with sex , time duration in sun exposure in weekdays , time duration in sun exposure during weekend , color of skin , and type of clothing .Patients were recruited at the end of summer and during autumn of Iran (September to December). The mean age of the patients was 10.3 ± 2.0 (8–14 years) and the mean duration since diagnosis of T1DM was 43 ± 15 days. Seventy-four percent of subjects were girls and 26% were boys.P = 0.860). Vitamin D intake was higher in boys than girls, but the difference was only borderline significant (P = 0.076).Mean daily dietary calcium and vitamin D intake among patients was 931 ± 506 mg/day (range 197–2164 mg/day) and 38 ± 17 IU/day (range 13–76 IU). Daily vitamin D intakes in all patients were less than the recommended 200 IU per day for 9–13 year old children . Calcium intake was not different between boys and girls (P < 0.001). Figure vs. 2.22 ± 0.28, P = 0.004), “use of sunscreen” , and “type of clothing” . In fact, the boys spent more time outside in the sun. In addition, their clothing was less covered than girls, and they never used sunscreen . The score of skin color did not differ between the boys and girls (P > 0.05).Mean baseline sun index score among patients was 9.8 ± 1.5. The sun index score was significantly higher in boys than girls .Mean serum levels of 25(OH)D was 13.2 ± 6.1 ng/ml (range 4.1–27.4 ng/ml). According to these baseline values, 77% of patients were vitamin D deficient and the remaining 23% were vitamin D insufficient. No patient had sufficient level of serum 25(OH)D. There was a negative correlation between 25(OH)D concentration and iPTH level . In addition, there was a negative significant correlation between serum 25(OH)D and BMI . However, the difference of BMI values between vitamin D deficient and vitamin D insufficient subgroups did not reach a significant level (P = 0.132). Serum levels of 25(OH)D were not correlated with the age of the patients .Serum vitamin D levels were significantly higher in boys compared to the girls  and calcium intake . Likewise, there was no significant difference in dietary vitamin D intake among vitamin D deficient and vitamin D insufficient patients (P = 0.294). Calcium intake was higher in vitamin D deficient patients compared to vitamin D insufficient ones, but the difference was not significant .Furthermore, serum 25(OH)D levels were not correlated with dietary vitamin D intake . The sunlight index score was significantly lower in subjects with vitamin D deficiency compared to those with vitamin D ≥ 20 ng/ml .There was a positive significant correlation between sun scores and serum levels of vitamin D . The effect of sex on vitamin D status in this model was borderline significant, meaning that after adjustment for sun exposure-related behaviors, boys were less likely to be vitamin D deficient than girls . The likelihood of being vitamin D deficient was not associated with the duration of sun exposure during weekdays, skin color, and type of clothing.In a logistic regression model, the impact of sex and sun exposure behaviors was assessed on the likelihood of vitamin D deficiency. As shown in the Table An important finding of this study was the high prevalence of vitamin D deficiency (77%) compared to those in an Australian, US, Swiss, and Italian study -13, demoVitamin D deficiency is a common problem even in healthy children and adolescents at a variety of different latitudes -23. Few Significant difference in 25(OH)D concentration between girls and boys in the present study was in agreement with previous research . This fiA significant negative correlation was found between 25(OH)D levels and BMI as previously reported ,28,29. HVitamin D intake in this study included only intakes from foods, since we did not enroll patients with a history of vitamin D supplementation during the previous 3 months. In addition, food sources of vitamin D are very limited, unless they are fortified. There are some vitamin D fortified foods in Iran; however they are rare and expensive. Children participating in this study did not consume any kind of foods fortified with vitamin D. As a result, low intakes of vitamin D were expected. All participants reported a daily intake of vitamin D less than the recent RDA (600 IU/day) and even lower than previous DRI (200 IU/day) . SimilarDietary vitamin D intake can be an important contributor to vitamin D status, but the current study did not find any association between vitamin D intake and serum levels of 25(OH)D. There are similar findings which show only poor correlation between vitamin D intake and serum vitamin D ,33. In cLow intake of calcium induces high serum PTH level and increases catabolism of 25(OH)D, therefore low intake of calcium causes decreased 25(OH)D . The aveExposure to sunlight or UVB (Ultraviolet B) radiation is the primary source of vitamin D in the body . In thisIt was found that participants with more time exposed to sun during weekend had higher levels of serum vitamin D, suggesting that as a predictor of vitamin D status in this study. Several studies have also correlated outdoor activity with vitamin D status -42. UVB In summary, we observed a high prevalence of vitamin D deficiency or insufficiency among children and adolescents with T1DM. However, lack of a comparison group is the main limitation of this study, since it has been shown that vitamin D deficiency is highly prevalent in healthy children in Iran. Time duration of sunlight exposure during weekends and sex were the main predictors of serum vitamin D deficiency. Boys and children with ≥ 15 minutes exposed to sunlight in weekends were less likely to be vitamin D deficient than girls and those with < 15 minutes exposing to sunlight. Dietary vitamin D and calcium intake did not have any effect on serum vitamin D.The authors declare that they have no competing interests.AA-J, design, data analysis, and interpretation, AR, design, FA, design, SCL, interpretation, MQ, interpretation, BL, design and revision. All authors read and approved the final manuscript.


Nyssomyia trapidoi is considered to be its main vector. Dujardin et al. [1] recorded some differences in body pigmentation and isoenzymatic profiles in sympatric populations of Ny. trapidoi from the Pacific coast of Ecuador and suggested the existence of two cryptic species.Cutaneous leishmaniasis is endemic to the Pacific coast of Ecuador, and b and cytochrome c oxidase 1 of the mitochondrial DNA) analyses, such as detection of Leishmania DNA and phlebovirus RNA in some females, were performed.Entomological collections were performed in November 2008 and March 2011 in the locality of Paraíso Escondido using CDC miniature light traps and human bait. Morphological, isoenzymatical and molecular (sequencing of cytochome Ny. trapidoi (including the two color variants) in one cluster, except for two specimens which clustered separately in both genes. Isoenzymatic characterization confirmed that the color variants belong to the same population. Additionally, 11.5% of females were found by PCR to contain Endotrypanum monterogeii kinetoplastid DNA. All pools of Ny. trapidoi were negative for phlebovirus RNA.Neighbor-joining trees from mitochondrial sequences grouped all of Ecuadorian Ny. trapidoi, which is a probable vector of Endotrypanum monterogeii.Analysis of mitochondrial gene sequences and isoenzymes was unable to support the existence of two sibling species within  Nyssomyia trapidoi, Lutzomyia hartmanni, Lutzomyia gomezi and Lutzomyia ayacuchensis are proven vectors of Leishmania parasites [Currently, 76 species of phlebotomine sand flies belonging to 17 genera have been reported for Ecuador ; of thesarasites . Leishmaarasites  and is narasites   panamensis[Ny. trapidoi is highly anthropophilic [Ny. trapidoi by Fairchild & Hertig [Leishmania naiffi in Ny. trapidoi[In the Pacific region of Ecuador, the sand fly species anamensis,8-10. Nypophilic -13 and i& Hertig  who anal trapidoi.Ny. trapidoi showing a slight color variation. Additionally, isoenzymatic analysis revealed a possible presence of two species living in sympatry [Ny. trapidoi at these locations. This report describes the results of an isoenzymatic and mitochondrial DNA analysis carried out on specimens of Ny. trapidoi collected in 2008 and 2011 at one of the locations described previously by Dujardin et al.[Ny. trapidoi for Trypanosomatids and phleboviruses.In 1996, an entomological collection carried out in two communities on the north coast of Ecuador, obtained specimens of Sand flies were captured in November 2008 and March 2011 in the locality of Paraíso Escondido , Pichincha Province, using CDC miniature light traps. Moreover, female sand flies were collected manually on human bait (captured on the skin of the authors). An out-group was analyzed consisting of specimens from Nicaragua (Musun), a place close to the type-locality of the species. Ecuadorian specimens were identified based on color phenotypes: light (B), dark (G) and indistinguishable (T). Specimens were killed using carbon dioxide and immediately stored in 96% ethanol for molecular studies and liquid nitrogen for isoenzymatic analysis. Once in the laboratory, the thorax of each specimen was separated and stored at −20°C for subsequent DNA extraction. The specimens used for isoenzymatic study and virus detection were processed as described below. The head, wings and genitalia of each specimen processed for morphology, molecular biology or isoenzyme analysis were cleared in boiling Marc-André  solutionTM II Electrophoresis system, GE Healthcare Life Sciences) with the ampholyte at pH 4.0-6.5 in accordance with the protocols described by Pesson et al.[Isoelectrofocusing was carried out in ultrathin agarose gels  and cytochrome b genes from sand fly mitochondrial DNA. Amplification conditions for COI were as follows: an initial denaturation step at 94°C for 3 min followed by 5 cycles of 94°C denaturation for 30 s, 45°C annealing for 90 s, and 68°C extension for 60 s followed by 35 cycles of 94°C denaturation for 30 s, 51°C annealing for 30 s, 68°C extension for 60 s and, finally, a 68°C extension for 10 min [b, an initial denaturation step at 94°C for 3 min was followed by 5 cycles  then 35 cycles , with a final extension at 68°C for 10 min [DNA was extracted using the QIAmp®DNA Mini Kit  and following the protocol used by Depaquit et al.. Polymerand LepR  and C3B-and LepR , as descr 10 min ,25. For r 10 min .PCR products were analyzed by electrophoresis in 1.5% agarose gels stained with 0.1% ethidium bromide. Amplicons were sequenced in both directions using the primers for DNA amplification.DNA sequences were edited using the Pregap and Gap programs from the Staden Package software . SequencLeishmania reference strains belonging to L. panamensis, L. guyanensis, L. braziliensis and L. peruviana were used. The conditions for these PCR reactions were the same as those described by Nicolas et al.[et al.[Detection of trypanosomatid DNA was carried out by PCR using two segments: a 120 bp kDNA corresponding to the conserved region of kinetoplast minicircles   and the as et al. and Clarl.[et al..Ny. trapidoi sand flies  were ground using a Mixer Mill MM300 (Qiagen) with a 3-mm tungsten bead at a frequency of 30 cycles s−1 for 3 min in the presence of 600 μl Eagle's minimal essential medium supplemented with 5% decomplemented calf serum, 1% L-Glutamine and 100 IU penicillin G ml−1, 100 mg kanamycin ml−1, 100 mg streptomycin ml−1 and 7.5 μg amphotericin B ml−1. The resulting mixture was clarified by centrifugation at 5800 g for 10 min and the supernatant fluid aliquoted  and stored at −80°C. One 200 μl aliquot was used for viral DNA and RNA purification using a BioRobot EZ1XL and the EZ1 Virus Mini kit (Qiagen), eluted into 90 μl and stored at −80°C. 10 μl was subsequently used in each RT-PCR reaction. Two sets of primers targeting different genes were used in independent reactions: (i) phlebovirus consensus primers targeting the polymerase gene in the L RNA segment [Twelve pools, each containing 50  segment , (ii) pr segment . The RT-−1, 100 mg streptomycin ml−1, 100 mg kanamycin ml−1 and 7.5 μg amphotericin B ml−1) and used to seed Vero monolayers in a 12.5-cm2 flask. After incubation at room temperature for 1 hr, 4 ml of fresh 5% FBS medium was added. The flask was incubated at 37°C in an atmosphere containing 5% CO2. Flasks were examined daily for the presence of a cytopathic effect, and 400 μl supernatant was removed and tested by RT-PCR after viral RNA/DNA extraction as aforementioned.Pooled sandfly homogenates stored at −80°C were used to inoculate Vero cells. Briefly, 100 μl each homogenate was diluted with 900 μl Eagle's minimal essential medium without fetal bovine serum (FBS), but enriched with antibiotics  hartmanni. Captures with CDC light traps yielded, in order of abundance: Ny. trapidoi, Trichophoromyia reburra, Pressatia dysponeta, Psathyromyia aragoi, Psychodopygus panamensis, Lutzomyia hartmanni, Psathyromyia abunaensis and Psychodopygus carrerai thula respectively . Nucleotide sequences from COI amplicons (683 bp) revealed 31 variable sites, 10 of which were informative; cytochrome b (511 bp) contained 16 variable sites, two of them informative. The nucleotide composition was 37% T, 19% C, 28% A and 17% G for COI and 38% T, 15% C, 39% A and 9% G for cytochrome b. The alignment showed no indel in either sequence. COI sequences showed an average base substitution per site of 0.6% and cytochrome b sequences showed an average of 0.25%. There are more COI haplotypes than cyt b haplotypes. .Both populations of light and dark sand flies were in Hardy-Weinberg equilibrium (P > 0.05). When combined as one group, only PGM deviated (P = 0.033); this disequilibrium was due to a deficiency of heterozygotes. There was no significant linkage disequilibrium for either populations tested alone or grouped together (1 > P >0.180). Nei’s genetic distance value was 0.004 and there was no genotypic differentiation between light and dark Ny. trapidoi females tested by PCR for the presence of trypanosomatid kDNA, 9 (11.5%) were positive. BLAST analysis of the 120 bp PCR products failed to reveal any significant score. However, 8 out of 78 kDNA PCR positive females were also PCR positive for the SSU rDNA locus. All the 2,135 bp sequences obtained from our samples were similar and showed 100% identity to the Endotrypanum monterogeii sequence (X53911) described by Fernandes et al.[Out of 78 Ny. trapidoi is the most common species captured [Psychodopygus panamensis and Lutzomyia hartmanni, were recorded at this locality. Other non-anthropophilic species, i.e. Trichophoromyia reburra, Pressatia dysponeta, Psathyromyia aragoi, Lutzomyia hartmanni, Psathyromyia abunaensis and Psychodopygus carrerai thula were also collected there . The Ny. trapidoi from Nicaragua differed from the Ecuadorian specimens in 12 nucleotide positions  and isoenzymatic analyses did not support the existence of two populations (possible cryptic species) at Paraíso Escondido as previously hypothesized by Dujardin et al.. The tops Figure  grouped b haplotype. Another branch includes the B001 and B010 specimens, which grouped exactly as in the cytochrome b NJ clustering, thus perhaps warranting additional studies. These two specimens differ from the main haplotype by two nucleotide positions: 35 and 642. Specimens B007, B033 and G309 make up a third cluster that is supported mainly by one mutation. The fourth cluster  is strongly supported by seven positions  [Ny. trapidoi in the locality of Paraíso Escondido.lovirus) ,37. AccoNy. trapidoi females, 2 engorged on human blood and the rest unfed. Nine (11.5%) of the unfed sand flies had identical trypanosomatid kDNA sequences and showed no homology to Leishmania panamensis and L. guyanensis, species commonly found in N. trapidoi[Endotrypanum monterogeii sequence (GenBank X53911) obtained from a sloth (Choloepus hoffmanni) and described by Fernandes et al.[Endotrypanum sp. by Ferreira et al.[Endotrypanum isolated from Lutzomyia gomezi (EU21238) and 99.8-99.9% to isolates from Psathyromyia dendrophyla . If we consider this variability as intraspecific, it might suggest that E. monterogeii has a wide spectrum of vectors, including the genera LutzomyiaPsathyromyia and Nyssomyia. Although sloths were common in study sites some time ago, they are now rare, and other vertebrates may play a role as reservoirs. Further studies are needed to learn more about the vertebrate hosts of E. monterrogeii, since to date our knowledge is limited to C. hoffmanni[In this work, we processed 78  trapidoi. Howeveres et al.. Moreovera et al.: 99.7%, hoffmanni,39.The authors declare that they have no competing interests.SZ, FLP, RL & JD were responsible for the conception and design of the work and have been involved in drafting the manuscript. SZ & BP carried out the isoenzymatic analysis and have given final approval of the version to be published. SZ, JD and CR did the parasitologic study and have given final approval of the version to be published. CC did the sequencing of the samples through the project @speed-ID and have given final approval of the version to be published. LM & GT helped with collecting and processing the samples, revising the manuscript critically and have given final approval of the version to be published. LB and RC carried out the viral study, critically reviewed the manuscript and have given final approval of this version to be submitted for publication.


Bifidobacterium animalis subsp. lactis BB-12, Lactobacillus rhamnosus RO011, or Lactobacillus helveticus RO052) or a mixed culture containing B. animalis subsp. lactis BB-12 and L. helveticus RO052 (MC1), both lactobacilli strains (MC2), or all three strains (MC3). DNA extractions were then carried out on PMA-treated and non-treated cell pellets in order to assess PMA treatment efficiency, followed by quantification using the 16S rRNA gene, the elongation factor Tu gene (tuf) or the transaldolase gene . Results with intact/dead ratios of bacteria showed that PMA-treated cheese samples had a significantly lower bacterial count than non-treated DNA samples (P < 0.005), confirming that PMA did eliminate dead bacteria from PCR quantification. For both quantification methods, the addition of probiotic strains seemed to accelerate the loss of lactococci viability in comparison to control cheese samples, especially when L. helveticus RO052 was added. Viability of all three probiotic strains was also significantly reduced in mixed culture cheese samples (P < 0.0001), B. animalis subsp. lactis BB-12 being the most sensitive to the presence of other strains. However, all probiotic strains did retain their viability (log 9 cfu/g of cheese) throughout ripening. This study was successful in monitoring living probiotic species in Cheddar cheese samples through PMA-qPCR.The use of a suitable food carrier such as cheese could significantly enhance probiotic viability during storage. The main goal of this study was to assess viability of commercial probiotic strains during Cheddar cheesemaking and ripening (4–6 months) by comparing the efficiency of microbiological and molecular approaches. Molecular methods such as quantitative PCR (qPCR) allow bacterial quantification, and DNA-blocking molecules such as propidium monoazide (PMA) select only the living cells’ DNA. Cheese samples were manufactured with a lactococci starter and with one of three probiotic strains ( PMA-qPCR is optimized for cheese and its efficiency is compared with traditional culture media selective for each species.The aim of this study was to analyze probiotic viability during Cheddar cheese manufacture and ripening using a combination of qPCR and PMA-qPCR. Two strains of lactobacilli . Toxicity studies for these two strains have been completed and no toxicity was observed  in frozen form. This culture, which has been used for over 25 years in fermented dairy products, has been classified as GRAS  by the FDA  was added, as was veal rennet , when the milk acidity had increased by 4.0°Dornic. When coagulation was achieved, the curd was cut and heated to 38°C, followed by whey draining and cheddaring. The resulting cheese was cut, salted (2.0% w/w) and pressed overnight to produce 1 kg blocks, which were then vacuum-packed in plastic bags and stored at 4°C. Microbiological and molecular analyses were conducted on samples taken after the initial probiotic inoculation, after cooking, after cheddaring, after salting, and after pressing. During ripening, new cheese samples were analyzed every month for a total of 4  to 6 months .Experimental Cheddar cheese samples were manufactured following standard procedures  and a 0.4 mL/min flow rate for chromatographic separation.Total titratable acidity was measured at all cheesemaking stages using an automatic titrator  along with pH, and final pH of the cheese samples was also measured. All other chemical analyses were conducted on cheese samples after 7 days. Fat content was analyzed following the Mojonnier extraction method  for 5 min at 230 rpm, two 1 mL samples of the resulting suspension were used, one for microbiological counts and the other for DNA extraction after PMA treatment.Ten grams of cheese were mixed with 90 mL of sterile 2% trisodium citrate (2% w/v) and homogenized with the StomacherOne milliliter of homogenized cheese suspension was serially diluted in 9 mL of sterile peptone water (0.1%) acidified with cysteine (0.05%) and adjusted to a final pH of 6.8. For all three probiotic strains, the MRS culture medium  was used in combination with a specific antibiotic for each strain. RO052 was grown on MRS agar containing 5 mg/L of ciprofloxacin (Fluka) and incubated in anaerobic jars for 72 h at 37°C. For RO011, 100 mg/L of vancomycin  was used, and plates were incubated aerobically for 72 h at 43°C. Finally, 50 mg/L of mupirocin  was used for BB-12 selection, and all plates were incubated at 37°C for 72 h in anaerobic jars. The M17 culture medium  was used for lactococci enumeration and incubated aerobically at 30°C for 48 h. Additionally, routine quality controls for total mesophilic and psychrotrophic bacteria , coliforms , and staphylococci  were carried out on raw and pasteurized milk and on the fresh cheese samples .g for 20 min, and the fat layer was removed with sterile swabs before the cell pellet was suspended in 500 μL of a TE 2X solution . The PMA powder  propyl]-6-phenyl dichloride, Biotium Inc., Hayward, CA, USA) was initially dissolved in 20% dimethyl sulfoxide (DMSO) to reach a final concentration of 20 mM and stored at −20°C. For each bacterial suspension, 1.25 μL of the PMA was added , and the tubes were shaken in the dark for 5 min to allow for maximal PMA contact with DNA. Then, all tubes were placed in a PMA lamp apparatus  for 15 min. The PMA-treated cell suspensions were then centrifuged at 10,000×g for 15 min to recover cell pellets for DNA extraction and stored at −20°C.Bacterial suspensions were treated with PMA according to the procedure developed by Nocker and Camper  with theg for 10 min at room temperature. Finally, the nucleic acids were precipitated from the supernatant by adding 200 μL of ice-cold absolute ethanol. DNA purification was performed as specified in Qiagen’s instructions. Residual RNA was removed using DNAse free RNase  according to the manufacturer’s instructions. DNA samples were stored at −20°C.Total genomic DNA from cheese samples was obtained following the protocol from Licitra et al. . Brieflytuf) for L. helveticus and L. rhamnosus and for the transaldolase gene  for B. animalis subsp. lactis , 1 μL of DNA (50 ng/μL), and an optimized concentration of each primer .In order to confirm that PMA-qPCR only quantified viable cells, each bacterial species in defined dead to live cell ratio were quantified in pure culture and spiked into 1 g of sterilized cheese sample. Dead cells were obtained following the protocol by Taskin et al. , either 10 to 104 cfu/mL) of each bacterial species were spiked into 1 g of sterilized cheese sample and treated with PMA. DNA was then extracted following the same protocol as the experimental cheese samples. For the standard curves, the DNA from the highest bacterial concentration was serially diluted in nuclease-free water. qPCR amplifications were carried out in triplicate, and Ct results were then plotted against the corresponding microbiological count (cfu/g).To evaluate the detection limit of the PMA-qPCR protocol, serial dilutions . Each 20 μL PCR amplification with SYBR Green primers contained 10 μL of Fast SYBR Green PCR Master Mix (Applied Biosystems), 1 μL of DNA, and an optimized concentration for each primer . The Mixed procedure was conducted with a full factorial design (variance components covariance structure) with cheesemaking step, ripening time and single or mixed culture as fixed effects, and replicate cheese blocks as the random classification variable. When results were significantly different, a pairwise comparison of all means was done using the Tukey–Kramer HSD test.Lactococcus sp., L. helveticus RO052, L. rhamnosus RO011, and B. animalis subsp. lactis BB-12), Ct values for targeted DNA were between 17 and 21, and non-targeted DNA Ct values were always above 30 with no cross amplification  and the highest for L. rhamnosus , and quantification results decreased significantly with lower viable to killed cell ratios for PMA-treated samples . For unblocked DNA samples (results obtained with no PMA treatment), quantification remained stable for all ratios with a slight but non-significant decrease for Lactococcus sp. and RO011 with lower viable to killed cell ratios.In order to assess PMA efficiency in detecting only living cells and for blocking the quantification of dead cells, viable to dead cell ratios were obtained from pure cultures or spiked cheese samples and were submitted to qPCR with or without prior PMA treatment Table . No signLactococcus sp. (Table P < 0.0001) with significantly different results between cheese samples (P < 0.0001), the lowest viable counts being obtained in samples containing RO052 in single culture or combined with RO011 (MC2) or all three probiotic strains (MC3). PMA-qPCR results for all species produced significantly higher viable counts than cultivable counts obtained with culture media (P < 0.0001) as they reached log 12 cfu/g of cheese for lactococci during manufacture (Table P < 0.0001), as only a two to three log cfu/g of cheese decrease was observed, particularly when RO052 was added in single culture, but not when it was added in mixed culture.Cultivable and viable cells were quantified with selective culture media and qPCR coupled with PMA Tables  and 6. Fp. Table , cultivaP < 0.0001 for RO011, P = 0.0007 for RO052, and P < 0.0001 for BB-12), but reached higher levels afterward and were up to one log cfu/g cheese higher at the end of ripening , and acetic acid and galactose concentrations were significantly different between samples (P < 0.0001).Final pH, moisture level, fat and protein content, as well as salt, glucose and lactic acid concentration were not significantly different between cheese samples of the same batch  on probiotic strains must also be verified  might augment the quantification of strains with health benefits.Results obtained by PMA-qPCR were lower than those obtained by traditional microbiological methods for the first data point, indicating that culture media may lack specificity and detect non-targeted probiotic strains or non-starter lactic acid bacteria naturally found in Cheddar cheese, especially with lower numbers of viable probiotic cells at the beginning of cheesemaking. However, the viable counts obtained by PMA-qPCR reached higher levels of log 10 cfu/g during cheesemaking. As results obtained by molecular methods are more accurate than those obtained by traditional microbiological methods, acceptance of PMA-qPCR for viable bacteria quantification could be helpful for cheese manufacturers in adjusting inoculation level and reducing production cost, as less viable cells would actually be required during the cheesemaking process in order to achieve the legislated level of log 9 cfu/30 g portion during storage. PMA-qPCR quantification for viability assessment showed greater accuracy for probiotic quantification in yogurt as well (García-Cayuela et al., L. rhamnosus RO011 and L. helveticus RO052 has already been tested in capsule form, demonstrating their basic synergy (Foster et al., B. animalis subsp. lactis BB-12 remain to be studied. The use of multiple probiotic strains as adjunct could also contribute to accelerated cheese ripening and enhance flavor through their effect on lactococci as well as through their own metabolic activity (Ong et al., For all probiotic strains used in our study, there was no significant impact of strain interaction on their viable counts with either quantification method, indicating no ill-effect on viability when the strains were used together in the Cheddar cheese matrix. These strains could therefore be combined in a probiotic cheese, which should widen the health benefits of such a product (Matto et al., To our knowledge, this study was the first to optimize PMA-qPCR to monitor probiotic viability in Cheddar cheese. The results demonstrate that PMA-qPCR is a specific and powerful approach compared with traditional quantification methods for the three probiotic species as well as for lactococci in cheese. The technique can be used with mixtures of species and provides an accurate estimation of viability in cheese. This approach could now be used to further assess bacterial survival in other types of cheese and to determine the impact of probiotic strain interactions on such food matrices.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The effect of sodium benzoate on the quality attributes of improved tamarind beverage during storage was investigated. Tamarind beverages were produced according to a previously reported improved method, with or without chemical preservatives (100 mg/100 mL sodium benzoate). Tamarind beverage produced according to traditional processing method served as the control. The tamarind beverages were stored for 4 months at room (29 ± 2°C) and refrigerated (4–10°C) temperatures. Samples were analyzed, at regular intervals, for chemical, sensory, and microbiological qualities. Appearance of coliforms or overall acceptability score of 5.9 was used as deterioration index. The control beverages deteriorated by 2nd and 10th days at room and refrigerated temperatures, respectively. Improved tamarind beverage produced without the inclusion of sodium benzoate was stable for 3 and 5 weeks at room and refrigerated temperatures, respectively. Sodium benzoate extended the shelf life of the improved tamarind beverage to 6 and 13 weeks, respectively, at room and refrigerated temperatures. Tamarindus indica L.), a tropical fruit found in Africa and Asia, is highly valued for its pulp  or “tsamiya” (Hausa tribe). While tamarind-based drinks are produced by industries in many countries where tamarinds grow, none exists in Nigeria. Tamarind fruits are only processed into beverage by indigenous people located in Northern Nigeria, despite the fact that tamarinds grow in some parts of southwest Nigeria. The production of tamarind beverage by the indigenous method is characterized by poor hygiene, labor-intensive manual operations, low aesthetic value, short shelf life and nonstandardization of ingredients, factors which have restricted the consumption of tamarind beverage to very few indigenous people in Northern Nigeria.Adeola and Aworh  had prevSyzgium aromaticum L. and Zingiber officinale) on the chemical, sensory, and microbial attributes during storage of tamarind beverage produced by the improved method.This study therefore aimed at investigating the comparative effects of sodium benzoate and local spices (T. indica L.) fruits were obtained from Saki in Oyo State. Clove (S. aromaticum L.), ginger , and granulated sugar were procured from a local market in Oyo town. All the chemicals used were produced by BDH .Mature tamarind . Paired preference test was used to determine the acceptable concentration of sodium benzoate, which was found to be 100 mg/100 mL. Consequently, improved tamarind beverage preserved with sodium benzoate was prepared according to Figure Tamarind beverage produced according to the traditional method Fig.  was usedTamarind beverages were stored at room (28 ± 2°C) and refrigerated (4–10°C) temperatures for 4 months. The beverage was assumed stable until the appearance of coliform or when the overall acceptance score was less than 5.9.dislike extremely, 5 = neither like nor dislike, and 9 = like extremely.Tamarind beverages were evaluated by a panel of 20 assessors comprising of staff and students of Emmanuel Alayande College of Education, Oyo, using a 9-point hedonic scale, where 1 = The pH, total acidity, soluble solids, and total solids of tamarind beverages were determined according to A.O.A.C. , ascorbi−1 to 10−4 for microbial counts. About 1.0 mL was transferred aseptically to petri dishes, poured with plate count agar (PCA) for total viable counts, McConkey agar (MCC) for coliform counts, and acidified potato dextrose agar (PDA) for total molds and yeasts counts. PCA and MCC were incubated at 35°C for 48 h while PDA was incubated at 25°C for 3–5 days.Samples were serially diluted in one-fourth strength Ringer's solution. Aliquots were further diluted to obtain 1010 CFU/mL. One-way analysis of variance (ANOVA) was used for the analysis of the microbial logarithmic values and hedonic scores. Duncan's multiple range tests and least significant difference (LSD) were, respectively, used to separate means that were significantly different (P < 0.05) for microbial logarithmic values and hedonic scores.Data obtained from chemical analyses were averaged and standard deviation calculated. Microbial counts were converted to logTable The shelf life of the tamarind beverage produced by the improved processing method was 3 and 5 weeks at room and refrigerated temperatures, respectively Table . There wThe shelf life of the beverage preserved with 100 mg/100 mL sodium benzoate was 6 and 13 weeks at room and refrigerated temperatures, respectively Table  advocateTables β-carotene and lycopene in tamarind pulp could have decomposed to affect the color of beverage during heating in the traditional processing method. Gomez-Palomares et al. (The high total solids recorded for the traditional beverage may be attributed to the increased sugar addition owing to its high sourness requiring more sweetener than the improved beverage which is less sour. The improved processing method resulted in an improvement in the color and ascorbic acid contents of beverages, probably due to the less severe effect of pasteurization on these quality attributes since carotenoids, s et al.  had earls et al.  reporteds et al. . The loss et al.  that Mais et al. . The lows et al. . Beverags et al.  and Okols et al.  also rep3 to 6.9 × 107 CFU/mL and 1.5 × 103 to 6.6 × 106 CFU/mL for traditionally manufactured tamarind beverage stored at room and refrigerated temperatures, respectively. Osundahunsi (1 CFU/mL for commercial samples of soborodo. This discrepancy may be due to differences in the initial microbial load and source of raw materials, processing methods, and levels of hygiene observed during production.Table ndahunsi  reportedThe microbial load of tamarind beverage produced by the improved processing method is as shown in Table Generally, tamarind beverage produced by the traditional processing method had higher microbial load than the ones produced by the improved processing method. This may be due to the fact that tamarind beverage, like many other traditional Nigerian beverages, is produced in homes and little attention is given to hygienic rules in the selection of processing materials and activities. The beverage is therefore highly susceptible to contamination by the microflora of the raw materials, utensils, and environment. The processing and storage conditions associated with traditional processing methods can encourage the presence of pathogenic or spoilage microorganisms in the beverage. Adeyemi and Umar , OnuorahTables The inclusion of sodium benzoate at 100 mg/100 mL in the processing of tamarind beverage by the previously improved method enhanced the sensory qualities and shelf life of the beverage. The total acidity, color, and browning index of beverages increase while the pH, cloudiness, ash, total solids, soluble solids, ascorbic acid, and total sugar decrease during storage.None declared.


In the article “Trends in Financial Barriers to Medical Care for Women Veterans, 2003–2004 and 2009–2010,” a sentence in the Acknowledgments was omitted. The additional sentence should read, “The authors thank Dr Diane C. Cowper Ripley for her review and Nichole Snyder for manuscript assistance.”http://www.cdc.gov/pcd/issues/2013/13_0071.htm.The correction was made to our website on November 6, 2013, and appears online at


To evaluate the influence of corneal astigmatism (CA) on retinal nerve fiber layer (RNFL) thickness and optic nerve head(ONH) parameters measured with spectral-domain optical coherence tomography (OCT) in high myopes patients before refractive surgery.Seventy eyes of 35 consecutive refractive surgery candidates were included in this study. The mean age of the subjects was 26.42 ± 6.95 years, the average CA was −1.17 diopters , All subjects in this study were WTR CA. 34 eyes were in the normal CA group with a mean CA was −0.67 ± 0.28D, 36 eyes were in the high CA group with an average CA of −1.65 ± 0.49D. All subjects underwent ophthalmic examination and imaging with the Cirrus HD OCT.P values > 0.05). Compared with the normal CA group, the high CA group had a larger disc area and rim area, thinner RNFL thickness in the temporal quadrant, and the superotemporal and inferotemporal peaks were farther to the temporal horizon . There were no significant differences between the two groups in global average RNFL thickness, as well as superior, nasal and inferior quadrant RNFL thickness .No significant difference was noted in the average cup-to-disk ratio, vertical cup-to-disk ratio and cup volume  for this article can be found here: Astigmatism is a worldwide common ocular disorder. Total astigmatism is mainly driven by corneal astigmatism(CA), which occurs due to an irregular shape of the cornea. In eyes with astigmatism, retinal images can be distorted. Langenbucher et al. reportedOptical coherence tomography (OCT) can provide imaging of ocular structures by a noninvasive method, It is widely used in clinical and scientific ophthalmology to obtain high-resolution cross-sections of the retina images. The thickness of the retinal nerve fiber layer (RNFL) and optic nerve head (ONH) parameters can be measured by OCT. Evaluation of these parameters is essential, since the thickness of the RNFL may be effected in various diseases. For example, the RNFL becomes thinner in glaucoma and optic atrophy, whereas it is thicker in papilledema.Many studies have reported the effect of refractive error changes induced by refractive surgery or contact lenses on RNFL thickness measured by OCT-4, while70 eyes of 35 consecutive refractive surgery candidates with spherical equivalent ≥ −6 diopters (D) were recruited for the study. Ethical approval for the study was obtained from the local medical ethics committee. All subjects were volunteers and informed consents were obtained.Each subject underwent a full ophthalmic examination, which included measures of visual acuity, refraction, intraocular pressure (IOP) by a noncontact tonometer. Axial length measurements were obtained in each eye with the IOL Master , CA measurements were obtained by a Topolyzer , optic nerve head evaluation was performed with a 90-D lens, and peripapillary RNFL thickness and ONH parameters were measured with the Cirrus HD OCT . The peak locations of the superotemporal and inferotemporal areas were evaluated by the RNFL TSNIT curve of the Cirrus HD OCT. The peak locations, which were measured by the RNFL TSNIT curve, were translated to units of degrees by multiplying 360/256. For example, the superior peak location of 40 in the TSNIT curve was translated to 56.25 degrees (40 × 360/256 degrees). This means that the thickest superior RNFL was located at the point 56.25 degrees away from the temporal horizontal meridian. We defined α angle as the angle between the horizontal meridian and superotemporal peak location by clockwise rotation, and the angle between the horizontal meridian and inferotemporal peak locations by counterclockwise rotation were defined as β angle Figure .The individuals were included if they had the following: best corrected visual acuity of 20/20 or better, an intraocular pressure (IOP) lower than 21 mmHg in either eye, CA as a with-the-rule (WTR) astigmatism, a healthy ONH without glaucomatous damage .Those with a history of severe ocular trauma, intraocular or refractive surgery or any ocular or neurological disease that could have affected the ONH or RNFL were excluded from the study. Subjects with evidence of macular disease or peripapillary atrophy extending more than 1.73 mm from the center of the optic disc or with glaucoma or an IOP higher than 21 mmHg in either eye were also excluded. In addition, participants with a history of systemic diseases including hypertension and diabetes were excluded.We assigned astigmatic types as defined in Katz and Kruger: with-thCA was measured with Topolyzer  and the total astigmatism was measured by a refractor keratometer (Topcon KR-8800 Auto Refractor).After pupillary dilation to a minimum diameter of 5 mm, the eyes of the subjects that satisfied the study criteria were scanned using the Cirrus HD-OCT system with software version 5.0. All the scans had signal strength of at least 6 and all measurements were taken by a single, well-trained examiner. The superior clock hour was 12 o’clock and the others were assigned accordingly in a clockwise manner in the right eye and counterclockwise in the left eye.t-test. Correlations between RNFL parameters and astigmatism were examined by linear regression analysis and expressed as the Pearson coefficient of correlation (r). A p value <0.05 was considered statistically significant.Statistical analyses were performed with commercially available software . The total average and mean clock hour RNFL measurements were compared between the two groups with an independent Seventy high myopic eyes of 35 subjects were analyzed. The mean age was 26.42 ± 6.95 years (range 18 to 39 years). The mean spherical equivalent, axial length, CA, and total astigmatism were −8.08 ± 1.77 D (range-6.00 to-15.00D), 26.86 ± 1.04 mm (range 24.24 to 29.84 mm), -1.17 ± 0.64D (range −0.20 to −3.30D), and −0.83 ± 0.65D (range 0.00 to −2.50 D), respectively.Of the 70 subjects, 36 eyes were classified as high astigmatism , and 34 eyes were classified as normal astigmatism . Characteristics of the two groups are listed in Table Table r = −0.316 (P = 0.008), r = −0.353 (P = 0.003), r = 0.452 (P = 0.000), r = −0.471 (P = 0.000), respectively, while correlation coefficients for high CA were r = −0.121 (P = 0.317), r = −0.102 (P =0.400), r = 0.250 (P = 0.037), r = −0.261 (P = 0.029), respectively .These findings are ascribable to CA induced ocular magnification. The relationship between the measurement of the OCT image and the size of the actual fundus dimension can be expressed as  formula, Where tl length. Althougth[et al suggesteth[et al. There wIn this study, the sample size may be inadequate to reveal a statistically significant correlation between total astigmatism and the temporal / nasal quadrant average RNFL thickness. Further studies are needed to clarify this point.In conclusion, we found that high corneal astigmatism with the rule influences the measurements of both RNFL thickness and ONH parameters by the Cirrus HD OCT. Eyes with higher corneal astigmatism had a larger disc area and rim area, thinner temporal RNFL thickness and farther temporally positioned superotemporal and inferotemporal peak locations of the RNFL in high myopes. Therefore, the degree of corneal astigmatism with the rule influences should be considered when interpreting the ONH parameters and peripapillary RNFL thickness measured by the Cirrus HD OCT in high myopes.The authors declare that they have no competing interests.LL participated in the study design, reviewed the literature, collected the clinical data, and drafted the manuscript. JZ provided the conception and design of the study and reviewing the manuscript. HH collected the clinical data and selected the material. J-gY took part in the study design and performed the statistical analysis. S-rC participated in collected the clinical data. All authors have read and approved the manuscript.


As the molecular weight of the fractions decreased, the TTHM yield coefficients increased. The NOM fractions with AMW values less than 1 K had lower SUVA values (<3 L/mg·m) for all treatment stages and also they had higher yield of TTHM per unit of DOC. The NOM fraction with AMW < 1 K for chlorinated raw water samples has the highest yield coefficient (42 μg-TTHM/mg-DOC).This study investigates the fractions of natural organic matter (NOM) and trihalomethane (THM) formation after chlorination in samples of raw water and the outputs from ozonation, coagulation-flocculation, and conventional filtration treatment units. All the water samples are passed through various ultrafiltration (UF) membranes. UF membranes with different molecular size ranges based on apparent molecular weight (AMW), such as 1000, 3000, 10,000, and 30,000 Daltons (Da), are commonly used. The NOM fraction with AMW < 1000 Da (1 K) is the dominant fraction within all the fractionated water samples. Its maximum percentage is 85.86% after the filtration process and the minimum percentage is 65.01% in raw water samples. The total THM (TTHM) yield coefficients range from 22.5 to 42  On the other hand, the EC regulation limit for total THM concentration in drinking water is 100 μg/L [μg/L [Chlorination has been used for disinfection to eradicate pathogenic microorganisms from drinking water –3. Never/L [μg/L .NOM has been recognized as the most important source of DBPs precursors , 13. TheUltrafiltration (UF) is a simple fractionation technique used to separate NOM into different molecular size ranges based on apparent molecular weight (AMW) , 20. UF The main objectives of this research were (i) to determine THMs formation from different molecular weight fractions of NOM in the disinfection process using chlorine and (ii) identify the main precursor of the disinfection byproducts among the different fractions of NOM.3/day of raw water to KWTP. KWTP is a common conventional treatment plant including prechlorination, ozonation, coagulation-flocculation, and filtration process units. The oxidation of NOM in raw water is performed with chlorine and ozone gases. The applied optimal ozone and chlorine doses in raw water were nearly 2.5 mg/L and 3 mg/L, respectively. Raw waters were coagulated using alum, for which the average applied dose was 60 mg/L. Finally, coagulated waters were passed through the rapid sand filters and then the filtered waters were disinfected with chlorine. The physicochemical characteristics of raw water quality parameters are shown in μm membrane filter papers within 24 h and stored in a refrigerator at +4°C to retard microbial activity prior to use.Raw and processed water samples were collected from the Kağıthane drinking water treatment plant (KWTP) in İstanbul, within a summer season in 2010. Raw water is transferred from Terkos Lake to KWTP, in İstanbul, Turkey. Terkos Lake is one of the most important water reservoirs in İstanbul and provides a maximum of 700,000 mThe ultrafiltration (UF) process was used to fractionate the molecular size of NOM in water samples taken from each treatment stage: raw water, ozonation, coagulation-flocculation, and filtration. In this study, UF was carried out by using a stirred UF cell (Millipore 8200) with YM disc membrane  and molecular weight cut-off (MWCOs) membranes including 1, 3, 5, 10, and 30 K. The scheme of the UF process is illustrated in Fractionated water samples were chlorinated following the procedure described in Standard Methods 5710 B . The chl254 absorbance divided by the DOC concentrations. THM analyses were conducted as liquid-liquid extraction (LLE) with pentane. THM samples were pipetted to 40 mL EPA vials, after that 3 mL of pentane was added to each vial. The samples were shaken vigorously by hand from one to minutes to ensure phase separation. The pentane extract from each vial was measured by an Agilent Gas Chromatography (GC) instrument equipped with a microelectron capture detector (GC-μECD), autosampler, and a fitted capillary column, (J&W Science DB-1), 30 m∗0.32 mi.d. ∗ 1 μm film thickness. The sum of four THM compounds  was reported as total THM (TTHM), in μg/L.After all the water samples had been fractionated by the UF process, they were analyzed for DOC, UV absorbance, and THM measurements. DOC analyses were performed with a Shimadzu TOC-5000 Analyzer equipped with autosampler, using the persulphate oxidation method as described in Standard Methods 3510 C. A UV −1608 Shimadzu spectrophotometer was used for measurements of UV absorbance at 254 nm wavelength. Specific UV absorbance (SUVA) was calculated as the UVUF plays a significant role in conventional drinking water treatment processes. Further, the UF technique is widely used for the determination of molecular weight distributions of NOM in water treatment and membrane technologies. This investigation includes two main goals. The first one is to fractionate water samples containing NOM taken from the KWTP processing units and establish the carbon mass balance of the UF processes according to the DOC measurements. The other goal is to determine the formation potential of THMs produced by the chlorination of the different NOM fractions in each water treatment stage. Prior to UF processes, a mass balance should be performed for each NOM fraction as regards DOC analysis. In other words, any loss or contamination was evaluated with mass balance based on the DOC concentrations of NOM fractions. The results of the mass balance calculations for each NOM fraction which are related to the KWTP stages  are given in 254 recoveries is better than (96 ± 3.2%) for raw water samples. The distribution of NOM fractions for each water sample with regard to DOC concentrations is presented in According to According to Zhao et al.  reported254, with respect to AMW distributions from water samples at different treatment steps in KWTP, are presented in 254 values in 254 values; for example, the NOM fraction with AMW < 1 K represented as 55–60% of UV254 values in the raw water and ozonation stages and was determined to be about 74% and 76% in coagulation and filtration processes, respectively. Nonetheless, the UV254 percentages of the other NOM fractions (AMW > 3 K and 1 K−3 K) ranged from 10% to 20% at all treatment units  was around maximum of 20% of DOC and UV254 values in all water samples  for each chlorinated NOM fraction. Comparing the TTHM formation for all NOM fractions in the KWTP processing units, about 65–90% TTHM is generated from chlorinating the fraction of AMW < 1 K in all water samples. As its value was 66% of the total in raw water, it reached about 90% of that in filtrated water. Besides, the TTHM percentages of the other fractions varied from 9% to 2% at all treatment steps. These findings therefore show that the low-molecular-weight fractions (<1 K), defined as hydrophilic compounds, play a greater role in the formation of THMs. Besides, Zhao et al. [As compared to all the water samples taken from each treatment unit in KWTP, the highest DOC and UVo et al.  suggeste24 h (generated TTHM per unit of DOC for the reaction time of 24 hours) and SUVA with regard to the AMW fractions from the different treatment stages.SUVA is a good surrogate parameter for understanding the humic content and a good predictor parameter for representing the aromatic carbon content of NOM in water as well. Meanwhile, it can be related to THM reactivity, described as generated TTHM per unit of DOC or specific TTHM (STTHM). 24 h values for NOM fractions at all water samples were 18 μg-TTHM/mg-DOC to 42 μg-TTHM/mg-DOC While the SUVA values were lower than 2 L/mg·m for AMW < 1 K, they were higher than 3 L/mg·m for the other fractions. Although the fraction of AMW < 1 K had the lowest SUVA value (<2 L/mg·m), it had the highest reactivity among all the fractions. Nonetheless, the lowest yield coefficient (22.5 μg-TTHM/mg-DOC) was observed for the fractions with AMW greater than 3 K. This result also demonstrated that as the molecular weight of the fractions decreased, STTHM24 h values increased. Similar results were obtained by some researches [STTHMsearches , 27 in t254 values were obtained with the fraction of AMW < 1 K among the other fractions in raw water samples, as 4.1 mg/L and 0.06 cm−1. One of the most important findings is that the lowest molecular weight fractions (<1 K), known as hydrophilic compounds, play a greater role in the formation of THMs. TTHM yield coefficients ranged from 18 to 42 μg-TTHM/mg-DOC. Although the fraction of AMW < 1 K had the lowest SUVA values (<2 L/mg·m), it had the highest THM reactivity among all the fractions. In addition, as the molecular weight of the fractions decreased, STTHM24 h values increased. This result also shows that the NOM fraction with AMW less than 1 K, consisting of hydrophilic compounds, is the major THM precursor.In this study, the water samples collected from the treatment stages in KWTP, a main conventional treatment plant, were separated according to molecular weight cut-off using various UF membranes. The fraction with AMW < 1 K was the predominant organic matter fraction among all NOM fractions, in accordance with the results of mass balance on DOC determinations. The results of DOC concentration related to the fractions of AMW > 30 K, 30−10 K, 10−5 K, and 5−3 K were not reported because DOC measurements of these fractions were <0.1 mg/L in all water samples. The highest DOC and UVThe determination of NOM fractions with the UF technique may be a good alternative approach for operating conventional treatment plants with respect to applied coagulants such as alum or disinfectant dose using chlorine.


Gadus morhua) stock. Further, we show that recruitment is strengthened during cold years and weakened during warm years. Our combined model explained 45 per cent of the total variance in cod recruitment, while the traditional Ricker and Beverton–Holt models only explained about 10 per cent. Specifically, our approach predicts that a full recovery of the North Sea cod stock might not be expected until the environment becomes more favourable.In order to provide better fisheries management and conservation decisions, there is a need to discern the underlying relationship between the spawning stock and recruitment of marine fishes, a relationship which is influenced by the environmental conditions. Here, we demonstrate how the environmental conditions (temperature and the food availability for fish larvae) influence the stock–recruitment relationship and indeed what kind of stock–recruitment relationship we might see under different environmental conditions. Using unique zooplankton data from the Continuous Plankton Recorder, we find that food availability (i.e. zooplankton) in essence determines which model applies for the once large North Sea cod ( For iIn their pioneering study, Cushing & Horwood  found unGadus morhua L.) in the North Sea, where excellent long-term data on zooplankton (i.e. food for fish larvae) are available [Here, we test this expectation by developing a modelling approach linking the traditional Ricker and Beverton–Holt stock–recruitment models so that the Beverton–Holt model will have most weight when food availability for fish larvae is good and the Ricker model will have most weight when food availability is sparse . We apply this model to long-term data on cod  and climate change will not be independent.2.(a)Calanus finmarchicus [The Atlantic cod is an important food fish found along both the western and eastern parts of the North Atlantic Ocean. Many of the historically large populations have been severely depleted by harvesting, including cod in the North Sea basin . The Atlarchicus .(b)www.ices.dk, following the procedure described by Beaugrand et al. [et al. [C. finmarchicus and euphausiids and low abundance of Calanus helgolandicus. The plankton index was scaled to range between zero and one. Sea surface temperatures (SSTs) were obtained from the International Comprehensive Ocean–Atmosphere DataSet  provided by the NOAA-ESRL Physical Sciences Division, Boulder CO, USA (http://www.cdc.noaa.gov/). Annual averaged values (°C) were calculated for the North Sea–Skagerrak area (from longitude −3.5–11.5° E and latitude 51.5–61.5° N). SST is standardized to a mean of zero and a standard deviation of one.We used data series from the available period of overlap 1958–2002 Z) and climate  to the available stock (S) and recruitment (R) data on North Sea cod (γ = exp(c)/max S, and max S is the maximum observed spawning biomass. The reproductive rate equals exp (a0 −a1T)((1−Z) exp(−bS) + Z(1 + γS)−1), with exp (a0 −a1T) as the maximum reproductive rate at temperature T. The rescaling of the spawning biomass by its observed maximum value makes the variables comparable in their scales, which avoid multi-collinearity [c). The parameterization exp(c) ensures that the coefficient γ is positive, which prevents numerical problems in fitting the model. While the parameter b is also postulated to be positive, it does not create numerical problems in fitting by not constraining it to be positive, and hence no such constraint was imposed. Note that the term ((1 − Z) exp(−bS) + Z(1 + γS)−1) represents the density dependence, which is now a function of the zooplankton index Z. As Z tends to 1, the density dependence approaches the form of Ricker model with an exponential decay in the density, namely exp(−bS), whereas as Z tends to 0, the density dependence is asymptotically Beverton–Holt, namely (1 + γS)−1. For a fractional Z, the density dependence is a weighted mean of these two types of density dependence with weight Z for the Beverton–Holt density dependence. In particular, when Z = 0, the model becomes the Ricker model but it reduces to the Beverton–Holt model when Z = 1.We fitted a combined Ricker–Beverton–Holt model incorporating food availability  is not moved to the right side of the equation for the subtle technical reason that the nls function does not have an offset option.A model formulation on the logarithmic scale , model 1rogram R . Here, aa priori defined models needed for testing our hypotheses. Model selection was based on the Akaike Information Criterion , where 3.a0 = 2.06, s.e. = 0.40, p < 0.001; a1 = 0.18, s.e. = 0.09, p = 0.05; b = 0.018, s.e. = 0.007, p = 0.01; c = 0.96, s.e. = 0.58, p = 0.10). This model fitted the data reasonably well . When the zooplankton index was low, the model indicated a positive relationship between spawning stock biomass and recruitment until the stock reached about 50 000 tonnes; above this level, the model predicted a negative relationship between spawning stock and recruitment  study by Van der Meeren & Naess  showed tOur results of North Sea cod recruitment being weak during warm periods finds support in earlier work. Evidence suggests that cod stocks towards the northern limit of the species geographical range tend to produce strong year classes during anomalously warm years while southerly stocks are favoured by temperatures below average ,52. The Recruitment modelling is a vital component of the assessment and management of marine fish populations, such as the North Sea Atlantic cod. Nevertheless, our understanding of what regulates the number of young fishes has remained opaque since the birth of fisheries science itself . We cert


Pseudomonas aeruginosa, Klebsiella pneumoniae and Acinetobacter baumannii), three Gram-positive bacterial strains , and seven clinically isolated yeasts ; their antioxidant activity was tested using a DPPH free radical assay. No activity against Gram-negative bacteria was observed with any extract up to the maximum concentration tested, 1000 μg ml−1. We report here for the first time activity of Ceanothus coeruleus against S. aureus  125 μg ml−1), C. glabrata (MICs 31.25 μg ml−1) and C. parapsilosis (MICs between 31.25 and 125 μg ml−1); Chrysanctinia mexicana against C. glabrata (MICs 31.25 μg ml−1); Colubrina greggii against E. faecalis (MICs 250 μg ml−1) and Cordia boissieri against C. glabrata (MIC 125 μg ml−1). Furthermore, this is the first report about antioxidant activity of extracts from Ceanothus coeruleus, Chrysanctinia mexicana, Colubrina greggii and Cyperus alternifolius. Some correlation could exist between antioxidant activity and antiyeast activity against yeasts in the species Ceanothus coeruleus, Schinus molle, Colubrina greggii and Cordia boissieri.Traditional medicine has a key role in health care worldwide. Obtaining scientific information about the efficacy and safety of the plants from our region is one of the goals of our research group. In this report, 17 plants were selected and collected in different localities from northeast Mexico. The dried plants were separated into leaves, flowers, fruit, stems, roots and bark. Each part was extracted with methanol, and 39 crude extracts were prepared. The extracts were tested for their antimicrobial activity using three Gram-negative bacterial strains ( The plant kingdom has been the best source of remedies for curing a variety of disease and pain. This is why medicinal plants have played a key role in the worldwide maintenance of health. Traditional herbal medicine is intimately related to the Mexican popular culture; its use has origins based on ancestral knowledge. Natural products of higher plants are an important source of therapeutic agents; therefore, many research groups are currently screening the different biological activities of plants –3.∼30 000 not yet described [Mexico has an extensive variety of plants; it is the fourth richest country worldwide in this aspect. Some 25 000 species are registered, and it is thought that there are escribed . In partescribed . It is wIn the course of our investigations, we have found that several plants from northwest Mexico possess interesting biological activities and could be a source of interesting new secondary metabolites –10.The aim of this work was to investigate the antimicrobial and antioxidant properties of some wild plants grown in northeast Mexico used in the traditional medicine to treat infections or general ailments. In the current study, 39 extracts prepared from 17 plants belonging to different genera were submitted to antibacterial and antifungal assays using a microdilution method and to a test of antioxidant activity using the free radical scavenging activity of 1,1-diphenyl-2-picrylhydrazyl (DPPH) using both thin-layer chromatography (TLC) and spectrophotometric assay methods.The activities have been selected because of their great medicinal relevance. It is well known that in past years, infection rates have increased and antibiotic resistance has become an increasing therapeutic problem , 12. In Wild plants were selected based upon their reported traditional use for the treatment of diseases including tuberculosis, fever, cough, hepatitis, rheumatism, skin diseases and respiratory or gastrointestinal infections. Collections were conducted between October 2000 and July 2002 from different regions in the states of Nuevo León and Coahuila . VoucherThe air-dried plants were separated into leaves, stems, roots, flowers, fruit and bark; each part was treated independently and immediately extracted. Powdered plant material (100 g) was extracted by direct maceration with methanol (3 × 600 ml) for 2 hours at room temperature. The extracts were filtered and evaporated to dryness under low pressure at 38°C. The different extracts were stored at 4°C until tested.Candida albicans (HU501), C. albicans (HU498), C. albicans (HU53), C. krusei (HU168), C. tropicalis (HU166), C. parapsilosis (HU96), C. glabrata (HU84), Pseudomonas aeruginosa, Klebsiella pneumoniae, Acinetobacter baumannii and Enterococcus faecalis . In addition, two Staphylococcus aureus strains: a wild type (ATCC 12598) and an oxacillin-resistant strain (IMSS-NL/HE22:01) were also used.The following were used as test organisms: −1 in 5% DMSO in the appropriate fresh liquid medium. Concentrations for each extract ranged from 1000 to 0.5 μg ml−1. A 100 μl volume of the extract sample was transferred to the first well of each row, and serial 2-fold dilutions were performed; the remaining 100 μl was discarded. A 100 μl volume of working suspension was added to each well. Seven samples and one antimicrobial drug control, cephalothin or fluconazole , were included in each plate. Three additional wells were used as growth controls where no drug was added, and culture medium was added to two wells. Plates were incubated at 37°C for 48 hours, and the growth was visually examined.Both bacteria and yeast strains were tested using a microdilution assay following the National Committee for Clinical Laboratory Standards (NCCLS) , 25, andμl of each extract (1 mg ml−1 in ethanol) was applied to a chromatographic plate. Chromatography was conducted using ethyl acetate : acetic acid : formic acid : water (100 : 11 : 11 : 27) as eluent. The plate was developed using a DPPH solution (2 mg ml−1 in ethanol); 30 min later, the yellow spots from reduced DPPH were clearly observed against a purple background.To measure antioxidant activity, DPPH free radical scavenging assay by TLC was used as previously reported . Briefly−1), and different concentrations (200–0.234 μg ml−1) of each extract were used. In a total volume of 1 mL, the assay mixture contained 500 μl of the extract and 500 μl of DPPH (125 μM in ethanol). The assay mixture was shaken and allowed to stand at room temperature in darkness for 30 min. The absorbance was then measured at 517 nm in a DU 7500 spectrophotometer (Beckman Coulter). Quercetin was used as a positive control. The capacity to scavenge the DPPH radical was calculated as follows:A is the absorbance of the negative control (DPPH plus ethanol) and B is the absorbance of the sample . The correlation between each concentration and its percentage of scavenging was plotted, and the EC50 was calculated by interpolation. The activity was expressed as EC50 .Assay of DPPH scavenging activity by spectrophotometry was conducted according to Leu et al. , with soThis article describes the antimicrobial and antioxidant activities of plants used in traditional medicine in northeast Mexico. A total of 39 extracts from 17 different plants species belonging to 11 families were tested. In P. aeruginosa, K. pneumoniae and A. baumannii) was observed. The extracts from Ceanothus coeruleus (flowers), C. boissieri (flowers), Cyperus alternifolius  and Schinus molle  displayed the strongest activity against both sensitive and resistant S. aureus strains (μg ml−1), the flower extracts from C. boissieri and Schinus molle were the most active. Five extracts belonging to Colubrine greggii (leaves and flowers), Cyperus alternifolius (root) and Schinus molle (bark) displayed the strongest activity against E. faecalis (MIC between 125 and 250 μg ml−1). The results of the extracts displaying antimicrobial activity are shown in C. glabrata (MIC ranged between 31.25 and 1000 μg ml−1). All test microorganisms were more sensitive to the extracts obtained from Ceanothus coeruleus (leaves and root), Colubrina greggii  and Schinus molle (leaves and flowers) (Most of the extracts tested exhibited some activity against flowers) .Ceanothus coeruleus , Chrysanctinia mexicana (leaves), Colubrina greggii , Cyperus alternifolius (root), Heliotropium angiospermum (flowers and leaves-stem), Phyla nodiflora (leaves) and Schinus molle (bark). Smaller spots or spots with less intensity were displayed by three other extracts: Cordia boissieri (flowers), Chrysanctinia mexicana (flowers) and Schinus molle (flowers). The 15 active extracts were further tested for their scavenging activity using a DPPH spectrophotometric assay. The percentage reduction of DPPH radical exhibited by the different concentrations of each extract was calculated and subsequently its EC50 was determined (Ceanothus coeruleus (leaves and root), Chrysanctinia mexicana (flowers), Colubrina greggii (root), Cyperus alternifolius (root) and Schinus molle (bark), exhibited remarkable antioxidant activity with EC50s less than 10 μg ml−1, while eight extracts displayed good antioxidant activities between 10.5 and 35.2 μg ml−1. Quercetin was used only as a positive control for the antioxidant activity assay. The EC50 for quercetin was 3.0 μg ml−1 (8.9 μM), similar to the result reported by Torres et al. [To test their antioxidant activity, all the extracts were analyzed by a DPPH free radical assay using TLC. Only 12 extracts displayed a strong antioxidant activity on the chromatographic plate: termined . Methanos et al. . The results of our investigation confirmed the rationale for the medicinal use of some of the studied plants. The plants were selected according to their popular use, and in few cases, chemotaxonomic criteria were used .The first remarkable aspect of the results obtained, was that no one of the extracts inhibited the growth of Gram-negative bacteria at the highest concentration tested. This lack of activity against Gram-negative bacteria is consistent with results discussed by other research groups . In contSchinus molle [H. angiospermum [Some of the results obtained are consistent with those reported by other authors such as the antibacterial activity of us molle , 27, 28 ospermum .Ceanothus coeruleus are active against an antibiotic-sensitive S. aureus strain, as well as against C. glabrata and C. parapsilosis; the leaves and root extracts had significant activity against all tested yeasts; moreover, all the extracts from this plant exhibited a remarkable antioxidant activity (EC50 between 5.6 and 28 μg ml−1). We could not find any previous report regarding antioxidant activity of this species.In the present study, we demonstrated for the first time that the flowers from Chrysanctinia mexicana displayed a very potent antioxidant activity , as well as antifungal activity against C. glabrata (MIC 31.25 μg ml−1). We could not find any report regarding activity against yeast or antioxidant activity from Chrysanctinia mexicana or other species belonging to the same species.Leaves and flowers extracts from Colubrina greggii showed inhibitory activity against E. faecalis (MIC 250 μg ml−1). The DPPH assay demonstrated highly potent antioxidant activity of leaves, flowers and roots from this plant (EC50 between 7.9 and 29 μg ml−1). There were hitherto no reports regarding the antioxidant activity of any species from the Colubrina genus.The leaves and flowers extracts from Cyperus alternifolius showed a moderate activity against all yeasts tested, while the root extract produced a clear antioxidant activity (EC50 9.3 μg ml−1).The root and leaf-stem extracts from C. glabrata. It appears that this isolate is sensitive to all the extracts used. Nine extracts showed activity against the rest of the yeasts tested; from these, eight extracts resulted with antioxidant activity  of the 15 extracts that showed antioxidant activity, displayed antibacterial activity as well, and nine (80%) resulted active against yeast. In summary, we conclude that most of the results of this study are in good agreement with the traditional uses of the investigated plants. All the extracts were active against activity . The rooBelofsky et al.  demonstrGrant 103.5/06/0135 from PROMEP-México; U.A.N.L. (PAICYT) SA-1425-06.


Attention deficit hyperactivity disorder (ADHD) is understood as a developmental disorder which shares common characteristics between childhood, adolescence and adulthood. However, ADHD is widely associated with misconceptions and misbeliefs which can lead to stigmatization. Teachers have an important role for the individual development as they accompany students for a long period of time. The aim of the present study was to explore stigmatizing attitudes in teachers towards adults with ADHD, thereby focusing on the developmental trajectory of the condition. Furthermore, it was aimed to identify factors contributing to prevention and intervention of stigmatization in ADHD.Stigma responses of 170 teachers and 170 comparison participants were measured and compared with a recently developed tool for the assessment of stigmatization towards adults with ADHD. Furthermore, the contribution of knowledge about ADHD and the frequency of contact with adults with ADHD to stigmatization were explored.Reliability and Social Functioning, Malingering and Misuse of Medication and the total scale. With regard to teachers, frequency of contact with adults with ADHD was not related to stigma. However, knowledge about the disorder was negatively correlated with stigma in teachers, indicating lower expressed stigma with increasing knowledge about adult ADHD.Teachers showed significantly less stigmatizing attitudes than comparison participants in various dimensions, including Teachers demonstrated more sensitized attitudes towards stigma in adults with ADHD than comparison participants. Since the present results indicate that knowledge about ADHD increase the sensitivity towards the disorder, special education programs for the community may have the potential to reduce stigmatization towards adults with ADHD. Possibilities for intervention strategies of stigmatization in educational settings were discussed. The association of a group of people with unfavorable, negative beliefs can be described as stigmatization. In this context, it is not relevant whether the assumption underlying these associations is correct or wrong. Research on stigmatization demonstrated that not only physical deviances can set individuals apart and trigger stigmatization but also intrinsic characteristics of the individual such as behavioral deviances have also been found to provoke stigma  in both teachers and education students  = 1.804, p = .179) and educational level (t(338) = 0.974, p = .331).A total of 170 teachers and 170 comparison participants took part in the study and completed the disease specific questionnaire on stigmatization towards adults with ADHD. All teachers were recruited from local secondary schools in Germany. The teachers’ age ranged from 24 to 64 years with a mean age of 43.0 years (SD = 12.4 years). The sample of teachers consisted of 112 (65.9%) female and 58 (34.1%) male participants. All teachers achieved a university degree and completed an applied training in school education. Mean education of teachers was 19.0 years (SD = 2.5 years). Comparison participants were recruited via public announcements, word-of-mouth and through contacts of the researchers involved in Germany. The sample’s age ranged from 22 to 65 years with a mean age of 42.7 years (SD = 13.9 years). The comparison group consisted of 100 (58.8%) female and 70 (41.2%) male participants. Corresponding to the sample of teachers, all participants in the comparison group achieved a university degree with a mean education of 18.6 years in total (SD = 3.8 years). Comparison participants had a variety of professions. However, none of the comparison participants completed a study program for teachers or completed an applied training in school education and none of the comparison participants was currently working in the educational setting in a teaching position. Teachers and comparison participants did not differ in age (t(338) = 0.185, p = .853), gender , exploratory factor analysis (EFA) suggested a 6-factor structure in a principal component analysis (PCA). The use of a PCA was justified by adequate sample size (KMO = .86) and by sufficient large correlations between the items indicated by Bartlett’s test of sphericity (χ2(2016) = 8258.3, p < .001). The scale demonstrated adequate internal consistency (Cronbach’s α = .91) with internal consistencies of the six subscales ranging from .61 to .87. In a second sample (n2 = 517), confirmatory factor analysis (CFA) confirmed the proposed 6-factor model with satisfying model fit (χ2 (614) = 1763.68; p < .01; χ2/df = 2.87; RMSEA = .06; 90%-CI for RMSEA = ; SRMR = .07; CFI = .93). The questionnaire yields a total stigma score and the six subscales Reliability and Social Functioning, Malingering and Misuse of Medication, Ability to Take Responsibility, Norm-violating and Externalizing Behavior, Consequences of Diagnostic Disclosure and Etiology. The six subscales and included items are presented in Table As reported in a previous study  affiliated to the University of Groningen, the Netherlands.2, Cohen’s d) were calculated for all comparisons. As described by Cohen  to an η2 = .0588 and a large effect size (f = .40) to an η2 = .1379. For pairwise comparisons of means, negligible effects (d < 0.20), small effects (d = 0.20), medium effects (d = 0.50) and large effects (d = 0.80) were distinguished . Effect sizes  for teachers and -1.08 (SD = 0.65) for comparison participants. Teachers showed highest stigma responses on subscale 5, followed by subscale 4, subscale 3, subscale 6 and subscale 1, with the lowest observed stigma score on subscale 2. Comparison participants showed a very similar pattern of stigmatization compared to teachers, with highest stigma scores on subscale 5, followed by subscale 4, subscale 1, subscale 3, subscale 6 and subscale . Teachers expressed significant lower stigma than comparison participants on the total scale  = 4.35, p = .038, d = 0.23). For pairwise group comparisons of stigma scores on the six subscales, a Bonferroni adjusted significance level of α = .0083 was applied because of multiple comparisons. Teachers revealed significant lower stigma scores than comparison participants on the two subscales Reliability and Social Functioning  = 12.90, p < .001, d = 0.39) and Malingering and Misuse of Medication F = 16.34, p < .001, d = 0.43). Effects were of small size. Group comparisons with regard to the other subscales revealed only non-significant differences of negligible size  = 1.17, p = .281, d = 0.12; Norm-violating and Externalizing Behavior: F = 3.11, p = .079, d = 0.19; Consequences of Diagnostic Disclosure: F = 0.02, p = .902, d < 0.01; Etiology: F = 0.44, p = .509, d = 0.07). Stigma scores of teachers and comparison participants are presented in Figure Multivariate analysis of variance (MANOVA) revealed a significant small difference of stigmatization between teachers and comparison participants  = 5.604, p < .001, ηThe contribution of self-rated knowledge about ADHD and frequency of contact with adults with ADHD on stigma responses were examined by applying exploratory correlation analyses. Knowledge about ADHD was rated on a scale from 0 (no knowledge) to 10 (expert knowledge) and resulted in a mean score of 4.16 (SD = 1.96) for teachers and 3.77 (SD = 2.25) for comparison participants. Frequency of contact with adults with ADHD was rated on a scale from 0 (never) to 5  and resulted in mean score of 1.56 (SD = 1.41) for teachers and 1.39 (SD = 1.42) for comparison participants. Differences between teachers and comparison participants were non-significant and of negligible size for both knowledge about ADHD (t (311) = 1.443, p = .150, d = 0.18) and frequency of contact with adults with ADHD (t (306) = 1.042, p = .298; d = 0.12).With regard to teachers, significant negative correlations were found between knowledge about ADHD and stigma scores on the total scale , on subscale 1  and on subscale 3 . Associations were of small to medium size. Non-significant negligible to small correlations were found between self-rated knowledge and stigma responses on subscale 2 , subscale 4 , subscale 5  and subscale 6 . Moreover, with regard to the group of teachers, no significant correlations were obtained between frequency of contact with adults with ADHD and stigma responses  and on subscale 4 . The remaining subscales of the stigma questionnaire did not correlate significantly with knowledge about ADHD . Furthermore, the observed correlations were of small size. Significant negative correlations were found for comparison participants between frequency of contact with adults with ADHD and stigma responses on subscale 1 , on subscale 2 , on subscale 3 , on subscale 6  and on the total scale . Correlations were of small to medium size. The remaining subscales  did not correlate significantly with frequency of contact. The observed correlations were of negligible to small size  resulted in a significant lower total stigma score for teachers . Furthermore, these group differences support the sensitivity of the questionnaire in measuring stigma towards adults with ADHD and thereby underline the usefulness of the stigma questionnaire for future research.Comparison of stigma responses between teachers and comparison participants revealed significantly lower stigmatization of teachers on dimensions of To measure the teachers’ personal experiences with adult ADHD, participants were asked to quantify the frequency of contact with an adult suffering from ADHD on a scale ranging from 0 (never) to 5 . Teachers’ personal experiences have not been assessed via their general teaching experience. This approach has been chosen as it can be assumed that most contact teachers have had with individuals with ADHD within school was related to children with ADHD. Since the present study focuses on adults with ADHD, the general teaching experience would therefore not lead to a realistic estimation of teachers’ experiences with adults with ADHD. Moreover, even though it is widely assumed that the actual number of ADHD students taught in the past is directly related to years of teaching experience, it has been emphasized that this relationship is not significant (Kos et al. Reliability and Social Functioning, Ability to Take Responsibility and on the total stigma scale. The more teachers knew about ADHD the less they stigmatized on particular dimensions as indicated by small to medium effects. The present results on teachers’ knowledge about ADHD and frequency of contact to adults with ADHD are in line with the literature on educational approaches to the reduction of stigmatization. This literature reported positive effects of special trainings and education certifications on stigmatization of individuals with ADHD, whereas increased personal experiences with persons with ADHD have been shown to be ineffective (Bell et al. Data analysis revealed that the frequency of contact to adults with ADHD was not significantly related to stigma scores of teachers, while small to medium significant relationships were found on four dimensions and the total score for comparison participants, indicating decreased stigmatization with an increased frequency of contact. The lack of significant associations in teachers might have resulted from a rather frequent contact of teachers with children with ADHD and their parents who might partly also have suffered from the condition. Furthermore, teachers might have engaged with the topic of ADHD from an educational perspective via professional books, education programs as well as discussions with colleagues (Bell et al. In conclusion, even though previous research showed that teachers frequently present with incomplete knowledge about ADHD Bekle , the preThe present study must be viewed in the context of some limitations. Knowledge about ADHD was assessed with a self-rated scale rather than an objective measure of knowledge. The validity of self-evaluations can of course be questioned. A response bias in teachers’ self-evaluations about ADHD is reported by Kos et al.  who demo


Lysionotus serratus, has been used in traditional Chinese medicine. It also has a great development potential as an ornamental plant with its attractive foliage and beautiful flowers. An efficient propagation and regeneration system via direct shoot organogenesis from leaf explant was established in this study. High active cytokinin (6-benzyladenine (BA) or thidiazuron (TDZ)) was effective for direct organogenesis of initial induction. Murashige and Skoog (MS) growth media containing 0.5 mg L−1 BA alone or with combination of 0.1 mg L−1  α-Naphthaleneacetic acid (NAA) were the most effective for shoot proliferation. High BA concentration (1.0 mg L−1) in the media caused high percentage of vitrified shoots though they introduced high shoot proliferation rate. Histological observation indicated that adventitious shoot regeneration on the medium containing 0.5 mg L−1 BA alone occurred directly from leaf epidermal cells without callus formation. Regenerated shoots rooted well on medium containing half-strength MS medium with 0.5 mg L−1 indole-3-butyric acid (IBA) and indole-3-acetic acid (IAA), and the plantlets successfully acclimatized and grew vigorously in the greenhouse with a 94.2% and 92.1% survival rate.The gesneriaceous perennial plant,  Saintpaulia), lipstick plant (Aeschynanthus), and gloxinias (Sinningia) are widely cultivated as houseplants. The appeal of gesneriads lies not only in their abundant, colorful flowers, but also in their attractive and colorful foliage. More and more people are starting to know and grow gesneriads as ornamental plants [Saintpaulia [Sinningia [Chirita [, Metabriggsia ovalifolia [, Titanotrichum oldhamii [Primulina tabacum [in vitro both for conservation and breeding purposes.The plant family Gesneriaceae contains about 150 genera and 3700 species, which distributes worldwide primarily in tropical to temperate zones of eastern and southern Asia, Africa, Oceania, and South America to Mexico . Many spntpaulia  and Sinninningia . Even prinningia . Other s[Chirita , 10, Metalifolia , Titanotoldhamii , and Pri tabacum  were alsLysionotus serratus D. Don is a perennial herb under evergreen forests, with distribution ranging from western China to the eastern Himalayas [Lysionotus, micropropagation has been reported on Lysionotus pauciflorus [in vitro plant regeneration system for L. serratus through leaf explants. It will be useful for mass production and also for future breeding by biotechnological methods such as protoplast fusion and genetic transformation.imalayas . This heimalayas . The funimalayas , 17. Harciflorus , 19. HowL. serratus were collected from 1,850–2,000 m elevation, in Zhengyuan county, in the central part of Yunnan province, China. The plants were transplanted in plastic pots (μmol m−2 s−1), where the day/night temperatures ranged from 15–32°C/10–25°C, depending on the season and local weather conditions.Plants of tic pots  and grow−1 HgCl2 solution and then rinsing 3 times with sterilized distilled water. The leaves were then cut into explants about 0.5 cm2 in size and inoculated onto MS basal media [−1 BA, TDZ, KIN, or without any plant growth regulators (PGRs) for initial induction . After 4 weeks of culture, induction of shoot organogenesis was investigated and compared. Each treatment was replicated 3 times. Healthy fully expanded young leaves, ranging from 30 to 40 mm in length, were harvested from potted mother plants and were used as initial explants. The leaves were soaked in 10x diluted liquid soap and rubbed gently by fingers prior to washing them under running tap water for 1 hour. The leaf surfaces were disinfected by 75% (v/v) ethanol for 10 s, followed by soaking for 10 min in 1 g Lal media  supplemenduction . Each tr−1 BA to let shoots grow for another 4 weeks. These first generation in vitro shoots from the same media will be used as aseptic mother plants for PGR effects study.After counting the numbers of adventitious buds of each explant, the clumps with adventitious buds were divided into smaller clumps with 3–5 buds and then transferred onto the same MS media with 0.5 mg Lin vitro shoots from the same media were then used as explants. They were cut into explants about 1.0 cm2 in area and inoculated onto MS basal media supplemented with different concentrations  of BA or TDZ, with or without the combination of 0.1 mg L−1 NAA . The plastic film was kept closed to maintain the inside relative air humidity above 90% in the first 10 days, and then the plastic film was rolled up at night but kept partly closed during the daytime for another 5 days; after that the plastic film was fully removed. The survival rates of plantlets were recorded one month after transplanting.When the second generation adventitious shoots reached over 10 mm in height, they were transferred to half-strength MS media supplemented with 0.5 mg Lμm thick, were stained with Safranin/Fast Green and mounted on neutral balsam. All the sections were observed under a light microscope. Samples collected from different culture batches of leaf explants were taken weekly after culture initiation and were fixed in FAA . After dehydration through an ethanol-xylol series, the samples were embedded in a paraffin wax (58°C melting point). The sections, 8 P = 0.05).All the experiments of initial induction, shoot proliferation, and root formation were repeated 3 times. Data were analyzed by one-way ANOVA using the SPSS Statistics 19 and means were separated using Duncan's new multiple range test  occurred in PGR-free medium  was observed on the plantlets from medium containing 0.5 mg Le medium . The sure medium  grew vig−1 BA, cell divisions in the adaxial leaf epidermis and close to the vascular bundles (arrowheads) led to the formation of meristem cells  and 0.25–0.5 μM NAA [Titanotrichum oldhamii, adventitious shoots were formed efficiently on medium containing 0.1 mg L−1 BA [Aeschynanthus radicans, 40% of leaf explants produced somatic embryos on 6.81 μM TDZ and 2.68 μM 2,4-D medium [ Primulina tabacum, somatic embryogenesis and shoot organogenesis could be switched simply by changing the order of TDZ and BAP (Benzylaminopurine) in the culture media [Because a large number of plants in the family Gesneriaceae are rosette-shape herbaceous, most tissue culture regeneration systems have been established from leaf blade explants in this family. Type and concentration of PGRs were critical factors influencing organogenesis and callus formation from leaf explants in members of the Gesneriaceae. Both shoot organogenesis or/and somatic embryogenesis were successfully induced in some species. For example, in A and BA . In Meta5 μM NAA . In Titag L−1 BA . In AescD medium . In Primre media . These sSaintpaulia) [Ochna, for example [Chirita, when the medium contained high concentration of BA (>0.5 mg L−1) and lower NAA, adventitious shoots were easily induced, but when BA concentration was reduced to 0.1 mg L−1, both somatic embryos and adventitious shoots were induced [Different concentrations of TDZ and BA can lead to different morphogenesis response by explants, low concentration of TDZ has been shown to be beneficial for shoot organogenesis  , and hig example , 23. In  induced .L. serratus, even with high concentration TDZ. The media containing 0.5 mg L−1 BA alone or 0.5 mg L−1 BA with 0.1 mg L−1 NAA resulted in a similar shoot induction ratio, but indirect shoots were found only on media containing NAA. Although NAA and IBA, as well as IAA, could all successfully induce the adventitious roots, NAA could prompt the formation of callus on the leaf explant. However, the roots induced by NAA were brown within 4 weeks with illness symptoms present on the plantlets, which indicated that NAA not only induced adventitious roots, but also prompted the accumulation of harmful secondary metabolites in the roots, which may directly inhibit the development of the roots and plant growth. On the contrary, IBA and IAA had no obvious differences on root induction. The roots grew vigorously and consistently in the media containing IBA and IAA. Therefore, both IAA and IBA were suitable for root induction for L. serratus. In the present study, somatic embryogenesis was not induced in leaf explants of L. serratus leaf explants. Although high concentration BA could induce more shoots, about half of the shoots were vitrified which made them hard to develop into mature shoots or to induce roots on rooting medium. In conclusion, 0.5 mg L−1 BA concentration was ideal for adventitious shoot induction and 0.5 mg L−1 IBA or IAA was ideal for adventitious root induction for the rapid propagation of L. serratus. BA had better effects than the same concentration of TDZ on the induction of adventitious shoots in