P contributes to type 2 diabetes development in mice. Here we phylogenetically and physiologically characterized this unique mouse gut bacterium. The 16S rRNA gene analysis revealed that the strain belongs to the family Lachnospiraceae but shows low sequence similarities ( < 92.5%) to valid species, and rather formed a distinct cluster with uncultured mouse gut bacteria clones. In metagenomic database survey, the 16S sequence of AJ110941P also matched with mouse gut-derived datasets  with > 99% similarity, suggesting that AJ110941P-related bacteria mainly reside in mouse digestive tracts. Strain AJ110941P shared common physiological traits  with relative species of the Lachnospiraceae. Notably, the biofilm-forming capacity was found in both AJ110941P and relative species. However, AJ110941P possessed far more strong ability to produce biofilm than relative species and formed unique structure of extracellular polymeric substances. Furthermore, AJ110941P cells are markedly long fusiform-shaped rods (9.0–62.5 µm) with multiple flagella that have never been observed in any other Lachnospiraceae members. Based on the phenotypic and phylogenetic features, we propose a new genus and species, Fusimonas intestini gen. nov., sp. nov. for strain AJ110941P (FERM BP-11443).Our previous study shows that an anaerobic intestinal bacterium strain AJ110941 Recent extensive studies based on next generation sequencing approach enabled to characterize composition, diversity, and spatial distribution of gut microbial communities3, and suggested that changes in the gut microbiota might be associated with human health and diseases. This attracts a worldwide interest for its potential impact in the field of medical science as well as microbial ecology4.The mammalian digestive tract is one of the largest microbial habitats: more than 10Bacteroidetes and Firmicutes represent the most dominant and prevalent bacterial groups in the human gut ecosystem, and the Firmicutes is in particular the main component accounting for >50% of all the 16S rRNA gene sequences7. These intestinal Firmicutes bacteria are known to have an influence on the human health. For instance, several Lactobacillus spp. in the family Lactobacillaceae are widely recognized as beneficial bacteria (lactic acid bacteria) for maintaining the intestinal environment8, whereas some Clostridium spp. in the family Clostridiaceae are known as pathogens causing a bowel inflammation and food poisoning9.Culture-independent metagenomic studies revealed that members of the phyla Lachnospiraceae constitute the abundant taxa within the phylum Firmicutes in human gut microbiota7. Over half of human intestinal bacteria are not cultivated yet and many of those belong to the family Lachnospiraceae10. Recently, new bacterial strains representing novel genera in the family Lachnospiraceae were isolated from not only digestive tract but also microbial mat and biogas reactor, i.e., Eisenbergiella tayi strain B086562T11, Fusicatenibacter saccharivorans strain HT03–11T12, Herbinix hemicellulosilytica strain T3/55T13, Mobilitalea sibirica strain P3M-3T14, Murimonas intestini strain SRB-530-5-HT15 and Anaerobium acetethylicum GluBS11T 16. The family Lachnospiraceae currently consists of 31 genera according to the LPSN database (http://www.bacterio.net/-news.html), Bergey’s Manual of Systematic Bacteriology (second edition), and recent study by Patil et al.16.Members of the family et al. reported that the abundance of Lachnospiraceae bacteria in the gut was positively correlated with type 2 diabetes, one of the major public health concerns17, implying that the Lachnospiraceae might be associated with occurrence of the disease. However, no clear evidence was shown due to the lack of axenic cultures of the possible causing agent. Very recently, we succeeded in isolating a new member of the family Lachnospiraceae, designated strain AJ110941P, from the feces of hyperglycemic obese mouse18, and further demonstrated that the new isolate is obviously involved in development of obesity and diabetes in germ-free (GF) ob/ob mice18. In fact, the intestinal colonization of strain AJ110941P in GF mice induced the typical symptoms such as significant increases in fasting blood glucose levels together with liver and mesenteric adipose tissue weights, and decrease in plasma insulin levels and HOMA-β values18. In this work, we phylogenetically and physiologically characterized the new strain AJ110941P, compared its phenotypic characteristics with other Lachnospiraceae species, and consequently propose the novel genus and species, Fusimonas intestini gen. nov., sp. nov., for this strain.Importantly, based on metagenomics, Qin P with those of validly described species indicated that the strain is moderately related to members of the family Lachnospiraceae with relatively low sequence similarities ( < 92.5%). The most closely related species to strain AJ110941P was Ruminococcus gauvreauii strain CCRI-16110T isolated from the human faecal specimen (92.5% sequence similarity)19. Other close relatives were Clostridium amygdalinum strain BR-10T (92.3%)20, Clostridium citroniae strain RAM16102T (92.1%)21, Eisenbergiella tayi strain B086562T (92.1%)11, Anaerostipes caccae strain L1-92T (92.0%)22, Clostridium lavalense strain CCRI-9842 (92.0%)23, and Desulfotomaculum guttoideum strain DSM 4024 T (92.0%)24. Strain AJ110941P formed a monophyletic cluster with those relatives and was a neighbor of Eisenbergiella tayi strain B086562T  to the close relatives.Comparison of 16S rRNA gene sequence of strain AJ110941ties  < 9.5%. The P showed high 16S rRNA gene sequence similarities (99–100%) to clones retrieved from the mouse digestive tracts: uncultured bacterial clones WT ctrl D2 F09, NOD dss A5 D06 and NOD dss A2 C04 from mice colon tissues25, clones 16saw23-2g06.p1k and 16saw23-1b01.p1k from mice cecal contents26, clone F09 from wild-type mouse’s colon tissue, clones D06 and C04 from colon tissue samples from Nod2 KO mice25, and clones 16saw23-2g06.p1k and 16saw23-1b01.p1k from avirulent pathogen infected mouse26. Based on the IMNGS27 search (metagenome-derived 16S rRNA gene sequence database search), we further found that the 16S rRNA gene sequence of strain AJ110941P matched with 3699 and 826 datasets , 56% of which were derived from mouse guts . No growth was observed at 10 °C and 45 °C. The strain grew at pH 6.5–8.0, with an optimum at pH 8.0, and no growth occurred at pH 6.0 and 8.5. The strain did not require NaCl for growth and tolerated up to 0.5% (w/v) NaCl. Brown disk-like colonies (0.1–1.4 mm in diameter and <0.2 mm height) were formed in the GAM agar medium after 4 days of incubation at 37 °C. Cells were fusiform-shaped rods, 9.0–62.5 µm in length and 0.55–0.9 µm in width (mean size 25.05 × 0.63 µm), which occurred mainly as single cells : glucose, lactose, maltose, raffinose, xylose, sucrose, trehalose (0.1%), cellobiose (0.1%), galactose, xylan (5 g l−1), starch (5 g l−1) and melibiose as a sole carbon source. The following substrates  were not utilized: arabinose, rhamnose (0.1%), ribose, mannose, fructose, glycerol (5 mM), cellulose (0.1%), pectin (5 g l−1), soytone (5 g l−1), pyruvate, crotonate (10 mM), tryptone (0.1%), casamino acids (0.1%), yeast extract (0.1%), H2/CO2 , inositol (10 g l−1), mannitol (0.1%), melezitose (0.1%) and sorbitol (0.1%). The results suggests that strain AJ110941P prefers monosaccharides and polysaccharides as carbon source.The biochemical tests indicated that strain AJ110941P were determined. The cellular fatty acid profiles were as follows: C18:1cis9 (44.3%), C16:0 (22.5%), C18:1trans9 or cis6 (11.4%), C14:0 (9.8%), C18:0 (8.5%), C17:1 or cycloC17:0 (3.5%). The major respiratory quinone was MK-5 (H4). The G + C content of genomic DNA was 41.1 mol%.Whole-cell fatty acid compositions, the major respiratory quinones, and G + C content of strain AJ110941P was determined by crystal violet (CV) method, and was compared with those of other Lachnospiraceae species. Strain AJ110941P produced the highest amount of biofilm among the all Lachnospiraceae species tested  by scanning electron microscopy , while all the other relatives are rod21, oval20 or coccus-shaped cells19 (0.5–10.0 μm in length). This new isolate also possesses multiple flagella  are commonly utilized. Besides, strain AJ110941P produced large amounts of EPS, and showed the highest biofilm-forming capacity among Lachnospiraceae species tested in the present study ).18:1cis9, C16:0, and C18:1trans9 or cis6. The major respiratory quinone is MK-5 (H4). The type species of the genus is Fusimanas intestini.Cells are Gram-positive, non-motile, non-spore-forming and long fusiform-shaped. The strain is mesophilic and strictly anaerobic. The DNA G + C content is 41.1 mol%. The main fatty acids are CFusimonas intestini (in.tes.ti′ni. L. gen. n. intestini of the gut).N-acetyl-β-D-glucosaminidase are detected. No activity is found for alkaline phosphatase, esterase, lipase, arylamidase, trypsin, chymotrypsin, acid phosphatase, β-galactosidase, α-mannosidase, α-fucosidase, urease, arginine dihydrolase, glutamate decarboxylase, and protease. The following substrates serve as a sole carbon source for the isolate: cellobiose, glucose, lactose, maltose, raffinose, xylose, sucrose, trehalose, galactose, xylan, starch and melibiose. Arabinose, rhamnose, ribose, mannose, fructose, glycerol, cellulose, pectin, soytone, pyruvate, crotonate, tryptone, casamino acids, yeast extract, H2/CO2, inositol, mannitol, melezitose and sorbitol do not support growth. The end-products of glucose metabolism are common short chain fatty acids and CO2. The cellular fatty acids profile includes C18:1cis9 (44.3%), C16:0 (22.5%), C18:1trans9 or cis6 (11.4%), C14:0 (9.8%), C18:0 (8.5%), C17:1 or cycloC17:0 (3.5%).The species displays the following characteristics in addition to those given in the genus description. Cells are long fusiform-shaped, approximately 9.0–62.5 µm in length and 0.55–0.9 µm in width. Growth occurs at 15–40 °C (optimum temperature at 37 °C), at pH 6.5–8.0 (optimum growth at pH 8.0), and at 0.3–0.5% (w/v) NaCl. In GAM agar, colonies (0.1–1.4 mm in diameter and <0.2 mm height) are brown-color and disk like form. The strain is catalase-negative. Based on testing with the API ZYM, API ID32A and API 20 A system, naphthol-AS-BI-phosphohydrolase, α-galactosidase, β-glucuronidase, β-glucosidase, α-glucosidase, α-arabinosidase and P isolated from the feces of five weeks-old hyperglycemic obesity model mouse. Strain AJ110941P has been deposited in the International Patent Organism Depositary (IPOD), National Institute of Technology and Evaluation (NITE) as a patent strain (FEMR BP-11443).The type strain is AJ110941P (P = patent strain) was isolated from the feces of five weeks-old db/db mouse (obesity model mouse) as described previously18. In brief, the cultivation and isolation were performed using Eggerth-Gagnon medium (pH 7.7) with headspace gas of H2/N2/CO2  at 37 °C under anaerobic conditions28. Since strain AJ110941P was also able to grow in Gifu anaerobic medium  with headspace gas of N2/CO2 , liquid and solid (containing 1.6% agar) GAM media were used for further morphological and physiological characterizations.Strain AJ110941P were observed by phase-contrast microscopy , transmission electron microscopy , and scanning electron microscopy  as described previously30. Gram staining was performed using a Gram-stain kit  according to the manufacturer’s instructions, and stained-cells were observed by microscopy. Temperature range for growth were investigated on GAM liquid cultures incubated at 5, 10, 15, 20, 25, 30, 35, 37, 40, 45, and 50 °C, respectively. The pH ranges were tested at pH 5.0, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, and 10.0. The NaCl concentration range for growth was determined at 0.3, 0.5, 1.0, 1.5, 2.0, and 2.5%. The biochemical features were characterized using API ZYM, API ID32A and API 20 A  as described previously31. Sole carbon source utilization test was performed using the basal medium supplemented with one of 32 different carbon sources as described previously32. The basal medium contained (per liter): KH2PO4, 0.136 g; NaHCO3, 2.52 g; NH4Cl, 0.535 g; MgCl2.6H2H, 0.204 g; CaCl2.2H2O, 0.147 g; Na2S.9H2O, 0.3 g; cysteine·HCl, 0.3 g; trace elements solution32, 1 ml; vitamin solution32, 1 ml; and resazurin solution . All cultures were incubated anaerobically at 37 °C. Growth and substrate utilization were determined by monitoring increase in OD600. Catalase activity was determined using a 3% hydrogen peroxide solution. Main end-products of glucose metabolism were determined by using LC20 HPLC system with a Shim-pack SPR-H column  and GC-2014 gas chromatography (Shimadzu) after 8 days of cultivation.Cells of strain AJ110941P were performed according to the previously described methods33. Briefly, the genomic DNA G + C content and major respiratory quinones were analyzed using LC10 HPLC system with a Shim-pack CLC-ODS (Shimadzu) and Zorbax SB-C18 , respectively. Cellular fatty acid compositions were determined using a GCMS-QP2010 system (Shimadzu).Chemotaxonomic analyses of strain AJ110941P was previously determined18. Comparative 16S rRNA gene sequence analysis was performed using BLAST program against the nucleotide collection (nr/nt) database at NCBI. Multiple alignments of the 16S rRNA gene sequences of strain AJ110941P were performed with its closest relatives within family Lachnospiraceae using CLUSTAL W program. The phylogenetic tree was constructed by neighbor-joining method. The percentage nucleotide similarity was calculated by using p-distance available in MEGA software34. Bootstrap values were estimated using neighbour-joining and maximum-likelihood methods .Nearly complete 16S rRNA gene sequence  of strain AJ110941P and other eight close relatives (type strains) of the family Lachnospiraceae; Anaerostipes caccae JCM 13470 T22; Blautia hydrogenotrophica DSM 10507 T35; Clostridium citroniae DSM 19261 T21; Desulfotomaculum guttoideum DSM 4024 T24; Eisenbergiella tayi DSM 26961 T11; Eubacterium fissicatena DSM 3598 T36; Murimonas intestini DSM 27577 T15; Ruminococcus gauvreauii JCM 14987 T19. Biofilm formation assay was performed by crystal violet method as described previous study37. In brief, full-grown cultures of strain AJ110941P and other relative spices were inoculated to fresh GAM broth (1% inoculation) in 96-well polystyrene tissue culture plates  in anaerobic glove box. After static incubation for 48 h and 96 h at 37 °C under anaerobic condition, 20 μl of 1% crystal violet solution  was added. After static incubation for 30 min at room temperature, all crystal violet solutions were removed and each wells was carefully washed twice with sterilized distilled water. The crystal violet stained substances were solved in 95% ethanol, and measured the absorbance of the crystal violet solution on a SPARK 10 M multimode microplate reader  at 595 nm. For microscopic observation, biofilm-like aggregate of strain AJ110941P grown in GAM medium for 96 h was transferred to the glass surface , dried, washed with sterilized distilled water, and stained with crystal violet solution. Phase-contrast image was collected by using an Olympus PROVIS microscope.We determined the biofilm formation activity for strain AJ110941P was investigated using IMNGS web platform (https://www.imngs.org/)27, which is the biggest and most detailed 16S rRNA gene amplicon datasets available to date. The current size of the IMNGS database includes 88,579 of 16S rRNA gene amplicon datasets from 96 different environments27. In the present study, we used the 16S rRNA gene sequence of strain AJ110941P (AB861470) as a query sequence, and selected the all 16S rRNA sequences available from the NCBI sequence reads archive (SRA) with 97% sequence similarity threshold. The results of metagenomic database search were categorized based on the level of sequence similarities by BLAST search above 99 and 97% similarities, respectively.The potential habitability of strain AJ110941Supplementary Information


Jenni et al, Heart 2001) and MRI with Petersen's criteria . Jacquier  has proposed a 3 D evaluation of the non compacted mass with a threshold of 20% for the non compacted (NC)/compacted (C) mass ratio. They manually delineate the trabeculation and do not suppress blood. The aim of this study was to assess the effect of blood suppression with a semi-automatic software on this threshold.There is no gold standard for the diagnosis of patients with Left ventricular non compaction (LVNC). There are 2D criteria in sonography with Jenni's criteria (p < 0.05.Segmentation of compacted (C) and non-compacted (NC) mass was performed on short axis SSFP CMR from 60 healthy participants and 15 patients with LVNC from the study by Jacquier et al, with a semi-automatic software. Papillary muscles were segmented using semi-automatic thresholding and included in the compacted mass. Blood was removed from trabeculae using the same threshold tool. The 15 patients with LVNC were also manually segmented for the calculation of NC mass. Inter-observer reproducibility was assessed by using Bland-Altman analysis (BA) and by computing the correlation coefficient in LVNC patients segmented with our software. Differences between manual and software segmentation were assessed using a Mann whitney U test. We compared the area under the 20% receiver operating characteristics (ROC) curve for NC/C mass ratio. We used ROC curves to determine the optimal cut off values for NC mass. Results were considered significant with a Healthy subjects:NC mass was 10.57 ± 3.89 g, C mass was 113.07 ± 27.27 g, NC/C was 9.51 ± 3.12 %.LVNC patients segmented manually:NC mass was 52.43 ± 24.47 g, C mass was 149,3 +/- 46,37 g, NC/C was 37.58 ± 15.68 %.Area under ROC curve NC/C=20%: 0.98Area under ROC curve NC=15g: 0.99LVNC patients segmented with our software including blood suppression:NC mass was 35.46 ± 17.02 g (p=0.03), C mass was 113.07 ± 27.27 g, NC/C was 24.97 ± 10.41 % (p=0.74). Volume of suppressed blood: 16.97 ± 9.56 mL. BA NC 0.96, BA C 0.98, BA NC/C 0.97.Area under ROC curve NC/C=20%: 0.92 (p=0.03)Area under ROC curve NC=15g: 0.98 (p=0.12)The method that we propose is highly reproducible and allows to calculate the exact mass of trabeculation. It could help for the diagnosis of LVNC with high area under curve but seems to be more discriminating with NC mass than with NC/C mass ratio.


Human papillomavirus (HPV) bivalent and quadrivalent vaccines have been widely implemented in worldwide organized immunization programs. A nonavalent HPV vaccine is now available in several countries. The objective was to describe the fraction of squamous non-invasive high-grade cervical intraepithelial lesions attributable to genotypes targeted by bi-quadrivalent vaccines and by nonavalent vaccine according to age and diagnosis in women living in the city of Vigo .n = 145) and grade 3-carcinoma in situ  were tested with Linear Array HPV Genotyping test . Hierarchical estimation of the fraction attributable to HPV 16/18 or HPV 31/33/45/52/58 detected alone or in combination was calculated. Absolute additional fraction attributable to genotypes targeted by nonavalent vaccine compared to genotypes targeted by bi-quadrivalent vaccines was calculated as the increment of attributable cases with respect to all studied cases. Age group 1, 2 and 3 included women 18 to 34, 35–44 and ≥45 years old, respectively. EPIDAT 3.1 was used.Cervical scrapings (2009–2014) of women with histological diagnosis of cervical intraepithelial neoplasia grade 2 . It was 63/51/50% of CIN2 and 78/66/45% of CIN3-CIS in age group 1, 2, 3, respectively. Fraction attributable to genotypes targeted by nonavalent vaccine was 86% CIN2 and 86% CIN3-CIS. It was 87/91/75% of CIN2 and 90/86/76% of CIN3-CIS in age group 1, 2, 3, respectively. Fraction attributable to genotypes targeted by these vaccines tended to decrease as age increased (p-trend <0.05). Globally, absolute additional attributable fraction was 16%, 26% and 29% in age group 1, 2 and 3, respectively (p < 0.005).Fraction attributable to genotypes targeted by bi-quadrivalent vaccines was 59% CIN2 vs. 69% CIN3-CIS (Absolute additional fraction of CIN2 and CIN3-CIS attributable to genotypes targeted by nonavalent vaccine was observed in women of any age, especially in those over 35 years old. Human papillomavirus (HPV) vaccines have demonstrated their preventive potential for different HPV-related diseases , 2. InvaNo HPV vaccine protects against all HR HPV genotypes responsible for ICC. HPV 16 and HPV 18 cause the majority -around 70%- of ICC. HPV genotypes targeted by 9-valent vaccine are responsible for approximately 90% of ICC. The process for making a decision about introducing HPV vaccine into an immunization programme or about changing to a second generation vaccine has to be systematic and transparent  and theyThe objective of this study was to describe the fraction of squamous non-invasive high-grade cervical intraepithelial lesions attributable to HPV genotypes targeted by a 9-valent vaccine and by 2/4-valent vaccines according to age and diagnosis in women living in the city of Vigo .n = 123) or grade 3-carcinoma in situ  were prospectively recruited between the years 2011–2014 in the University Hospital of Vigo, Spain. Women with CIN2 (n = 22) and CIN3-CIS (n = 51) lesions histologically diagnosed from 2009 to 2010 in the same hospital were also included in this study. Characteristics of these 2009–2010 cases were previously described in a retrospective study  or 2.7 of these 3 multiple type infected lesions would be attributed to HPV 16 and [3 × 1/ , only if there were not detected HPV 16 and/or HPV 18 . For example, a lesion was attributed to HPV 35/39/51/56/59  only if there were not detected HPV 16/18/31/33/45/52/58.Absolute additional fraction of cervical lesions attributable to genotypes targeted by 9-valent vaccine compared to 2/4-valent vaccines was calculated as the increment of attributable cases with respect to all studied cases. Three age groups were considered: Group 1 (18–34 years), 2 (35–44 years) and 3 (≥45 years).p-value <0.05 or p-trend value <0.05 were considered statistically significant (EPIDAT software version 3.1) [For means, standard deviation (SD) and 95% confidence interval (95%CI) were calculated. Quantitative values were compared by Chi-square test. A two-sided ion 3.1) .Ethics Committee of Clinical Investigation of Galicia approved this study (CEIC reference number 2008/190). Women signed a consent form before their participation.In this study, 389 cases  were included. Global mean age was 35.3 years . Mean age of CIN2 cases was 33.5 years . Mean age of CIN3-CIS cases was 36.4 years  (Table HR HPV was detected in 386/389 lesions. Single genotype infection was detected in 66 CIN2 and 135 CIN3-CIS lesions. Multiple genotype infection was detected in 77 CIN2 and 108 CIN3-CIS lesions.n = 201). The most frequently detected genotypes were HPV 16 among 2/4-valent vaccine genotypes, HPV 31/33 among 9-valent vaccine genotypes other than HPV 16/18, HPV 35/51 among HR non vaccine genotypes and HPV 82 among probable/possible HR genotypes. A linear trend was found for HPV 16 prevalence  in age group 1 , 2  and 3  (p = 0.01). Prevalence of HPV 31 was 15.1% (10/66) in CIN2 vs 3.0% (4/135) in CIN3-CIS (p = 0.003).Prevalence of individual HPV genotypes is shown in Fig. n = 386). Data of 2/4-valent vaccine genotypes converged for all estimate methods except for hierarchical attribution and crude prevalence in CIN2 (6% difference with respect to minimum estimate). Fraction attributable to 2/4-valent vaccine genotypes was 53.0% (35/66) of CIN2 vs. 65.9% (89/135) of CIN3-CIS (p < 0.001) (minimum estimate) and 58.6% (85/145) of CIN2 vs. 69.3% (169/244) of CIN3-CIS (p < 0.001) . Data of 9-valent vaccine genotypes converged for all estimate methods. Fraction attributable to 9-valent vaccine genotypes was 84.8% (56/66) of CIN2 vs. 81.5% (110/135) of CIN3-CIS (minimum estimate) and 86.2% (125/145) of CIN2 vs. 86.5% (211/244) of CIN3-CIS .Figure p-trend <0.05). Considering hierarchical attribution, fraction of CIN2–3 attributable to 9-valent vaccine genotypes also tended to decrease with increasing age (p-trend <0.05).Age specific attributable fraction according to lesion grade is shown in Table p < 0.05) (Table p < 0.005), in comparison with 2/4-valent vaccine genotypes.For 9-valent vaccine genotypes, absolute additional attributable fraction of CIN2–3 was observed for all age groups , Joste et al.  and Riethmuller et al. 90%) [0% [9, 16The lowest oncogenic HPV genotypes were found mainly in CIN2–3 lesions diagnosed in the oldest women. This influenced the analysis of the vaccine genotype attributable fraction in each specific age group. First, vaccine genotype attributable fraction decreased as age increased. Secondly, the greatest absolute additional protective effect would be observed when vaccinated preadolescent women were more than 35 years old.Half cases were infected by only one genotype. These single infections could be useful for testing if global results obtained after mathematical attribution of HPV genotypes were reasonable. Among these single genotype lesions, HPV 16 was more prevalent in the youngest than in the oldest women, as previously reported , 11; HPVThe introduction of 2/4-valent vaccines has demonstrated to reduce cervical abnormalities, genital warts and HPV prevalence as well as the appearance of herd immunity after immunization programmes –20. TherLimitations: Quadrivalent and bivalent vaccine genotype attributable fraction of lesions was considered to be the same. The possible oncogenic effect of HPV 6/11  has not been taken into account. Cross protection against other related genotypes was not considered. Nonavalent vaccine antigens might probably produce a much stronger and longer lasting immunogenic response than cross protection. It would not be simple to establish whether the immunity directly induced by a vaccine and the cross-protection should be quantified in the same way. In this study, influence of vaccination coverage and vaccine efficacy were not taken into account. Current Spanish vaccine coverage is high , exceediIn comparison with bivalent and quadrivalent HPV vaccine, additional fraction of CIN2 and CIN3-CIS attributable to genotypes targeted by nonavalent HPV vaccine was observed in women of any age, especially on women more than 35 years old. Age-specific potential impact of nonavalent HPV vaccine should be taken into account in cost-effectiveness evaluations of HPV immunization programmes and in the organization of screening of vaccinated populations.


Our study was aimed to explore the clinical implication of chromosome microarray analysis (CMA) in genetically etiological diagnosis of children with congenital heart disease (CHD).A total of 104 children with CHD with or without multiple congenital anomalies (MCA) or intellectual disabilities/developmental delay (ID/DD) but normal karyotype were investigated using Affymetrix CytoScan HD array.DVL1, SKI, STIM1, CTNNA3 and PLN were identified as candidate genes associated with CHD phenotypes.Pathogenic copy number variations (PCNVs) were identified in 29 children (27.9%). The detection rates in children with simple CHD and complex CHD were 31.1% (19/61) and 23.2% (10/43), respectively. The detection rates of PCNVs were 17.9% (7/39), 20% (5/25), 63.2% (12/19) and 23.8% (5/21) in isolated CHD, CHD plus MCA, CHD plus ID/DD, CHD plus MCA and ID/DD, respectively. The PCNVs rate of CHD plus ID/DD was significantly higher than that of isolated CHD. Two genomic loci including 15q11.2 deletion and 1q43-q44 deletion were considered as CHD locus. The CMA can increase the diagnostic rate and improve the etiological diagnosis in children with CHD. We suggest CMA as a first-tier test in children with CHD, especially in children with CHD plus ID/DD. Congenital heart disease (CHD) is one of the most common birth defects. The incidence of CHD in the neonate is 8-9/1000, and nearly 1.35 million CHD neonates were born every year in the whole world [The causes of CHD include non-genetic factors and genetic factors. Non-genetic factors include: environmental factors, maternal exposure and infection. Chromosomal causes of CHD include chromosome aneuploidies, like trisomy 21, and copy number variations (CNVs). Chromosomal aneuploidies represent 12.5% of CHD causes –5.The resolution of conventional karyotype analysis is limited to 5 Mb or larger genomic imbalances . The draIn this study, we present the results of genome-wide high resolution SNP array analysis in 106 children with CHD in the Chinese cohort, to explore the clinical implication of CMA in genetically etiological diagnosis of CHD.In this study, we collected children diagnosed with CHD with or without MCA or ID/DD and normal karyotype at Guangzhou Women and Children’s Medical Center from January 2012 to December 2014. All the children were investigated by the complete cardiac evaluation, echocardiogram, medical history, physical examination, and/or operative reports. Considering patent ductus arteriosus (PDA) was common in children with CHD, the child with PDA was selected only if he/she was born at >37 weeks’ gestational age and the PDA was unclosed after 6 weeks of life [According to the National Birth Defects Prevention Study , 21, CHDAccording to the complexity of CHD, CHD was divided into two groups: simple CHD and complex CHD . Simple According to whether the children with CHD have ID/DD and/or MCA, CHD was divided into two types: isolated CHD and syndromic CHD. The syndromic CHD contained CHD plus MCA, CHD plus ID/DD, and CHD plus MCA and ID/DD.n > 3 studies or documented in >1% of the normal population) or no gene included (not close to known CHD genes within 1 Mb) [http://www.ncbi.nlm.nih.gov/projects/dbvar/clingen/). Rare CNVs  that do not meet the criteria above, should be considered as variant of uncertain clinical significance (VOUS), until proven otherwise  [http://projects.tcag.ca/variation/), Cytogenomics Array Group CNV Database (https://www.cagdb.org), Database of Chromosomal Imbalance and Phenotype in Humans using Ensembl Resources database , Online Mendelian Inheritance in Man , University of California Santa Cruz , ClinVar (http://www.clinvar.com/) and CHD wiki (http://homes.esat.kuleuven.be/~bioiuser/chdwiki/index.php/Main_Page). All CNVs were further confirmed by Real-Time Polymerase Chain Reaction. Parental analysis were performed to interpret VOUS.CMA was performed using Affymetrix CytoScan HD arrays according to manufacturer’s instructions. The procedure included genomic DNA extraction, digestion and ligation, PCR amplification, PCR product purification, quantification and fragmentation, labeling, array hybridization, washing and scanning. Data was analyzed with Chromosome Analysis Suite software version 1.2 (Affymetrix). The reporting threshold was set at 100 kb with marker count ≥50. CNVs were interpreted as benign listed in Database of Genomic Variants (DGV) database (in 1 Mb) . CNVs wastudies) , 23. ForP value <0.05 was statistically significant. The PCNVs detection rates in isolated CHD, CHD plus MCA, CHD plus ID/DD, and CHD plus MCA and ID/DD were pairwise compared using chi square test or fisher’s exact test, p value <0.008 was statistically significant.Statistical analyses were performed using the Statistic Package for Social Science (SPSS) 13.0 statistical software package (SPSS Inc.). The PCNVs detection rates in children with simple CHD and complex CHD were compared using chi square test. Frequencies of recurrent CNVs in this study and the control cohorts were compared using fisher’s exact test. CMA was performed in 104 children  with CHD aged from 5 days to 8 years old. CNVs were identified in 96.2% (100/104) of the children. The size of CNVs ranged from 102 kb to 13.8 Mb. CNVs were interpreted as benign or likely benign in 69.2% (72/104) children. The detection rate for PCNVs was 27.9% (29/104), and the VOUS rate was 2.9% (3/104) after parental analysis.P > 0.05). PCNVs were detected in 23.3% (10/43) of septal defect, 0 (0/3) of AVSD, 12.5% (2/16) of conotruncal defects, 35.7% (5/14) of LVOTO, 45.5% (5/11) of RVOTO, 75% (3/4) of LVOTO + RVOTO, 0 (0/1) of SV, and 33.3% (4/12) of PDA.Detailed CHD classification and demographic data of the children were listed in Table P > 0.05). The detection rates for PCNVs were 20% (5/25) in CHD plus MCA, 63.2% (12/19) in CHD plus ID/DD, and 23.8% (5/21) in CHD plus MCA and ID/DD, respectively (Table P = 0.001) and CHD plus MCA .The detection rates of PCNVs in isolated CHD and syndromic CHD were 17.9% (7/39) and 33.8% 22/65), respectively. There was no significant difference between these two groups , 22q11 duplication syndrome (n = 1), Williams-Beuren syndrome (n = 6), Angleman/Prader-Willi syndrome (n = 1), Wolf-Hirschhorn syndrome (n = 2), Schinzel-Giedion midface retraction syndrome (n = 1), 15q24 recurrent microdeletion syndrome (n = 1), 1p36 microdeletion syndrome (n = 1), Cornelia de Lange syndrome 4(n = 1), Marfan syndrome (n = 1), Opitz G/BBB syndrome (n = 1), 6q24 LOH (n = 1), and CNVs overlapped with DECIPHER patient entries (n = 6).PCNVs were identified in 29 children (Table TBX1 (22q11 deletion syndrome), ELN (Williams-Beuren syndrome), EVC2 and EVC (Wolf-Hirschhorn syndrome), STRA6 (15q24 recurrent microdeletion syndrome), FBN1 (Marfan syndrome), MID1 (Opitz G/BBB syndrome), RAD21 (Cornelia de Lange syndrome 4) and SETBP1 (Schinzel-Giedion midface retraction). In addition, the genes contributing to non-syndromic CHD included CRELD1, RAF1 and CITED2. DVL1 and SKI were identified as candidate genes for CHD in the current study.In this study, PCNVs in 79.3% (23/29) of the children contained genes contributing to CHD  Table  9, 11, , 16–19. P = 0.001) and CHD plus MCA (P = 0.004). Our data demonstrated that CMA is the most useful for genetic diagnosis in children with CHD plus ID/DD. In addition, we also recommended CMA investigation for children with isolated CHD.CMA has also been applied in children with isolated CHD previously –12, 15. P > 0.05). Detection rates in various types of CHD were different. Shaffer et al. reviewed 580 fetuses with CHD and normal karyotype by aCGH [n = 2) and SV (n = 1), this may be due to the small sample size in our study.In this study, PCNVs were detected in 31.1% (19/61) children with simple CHD and 23.2% (10/43) children with complex CHD by CMA. There was no significant difference between the two groups  was detected in child 6 (Table P = 0.002) [P < 0.001, Table In the present study, we identified two children with 1q43-q44 deletion. Child 1 manifested atrial septal defect (ASD) and ID. Child 21 manifested TOF and absence of corpus callosum. Our study showed significantly higher frequency of 1q43-q44 deletion (2/104) than that of the 9170 control cases (4/9170) (= 0.002) , 32. The= 0.002) . SummariSETBP1 gene located in the fragment which was responsible for Schinzel-Giedion midface retraction syndrome, and point mutation is the most common type. The clinical features included severe mental retardation, distinctive facial features, and multiple congenital malformations such as skeletal abnormalities and cardiac defects. A dominant-negative or gain-of-function mechanism was proposed to underlie this syndrome [SETBP1 gene, and no mutation was detected. The SETBP1 gene duplication was in accordance with gain-of-function mechanism, and the child’s manifestations were similar with the phenotypes of Schinzel-Giedion midface retraction syndrome, Therefore, SETBP1 should be the main responsible gene for this patient.The child 11, a girl with ASD and cleft palate which had been corrected surgically. She was diagnosed with mental retardation at 2 years old by pediatric doctor. CMA test revealed chromosome 18q12.3 duplication . syndrome . We sequPLAG1 and HYMAI genes, which were associated with imprinting disorder intrauterine growth retardation and neonatal hyperglycemia [CITED2 also located in the LOH, which has been identified associated with cardiac malformations, including atrial and ventricular septal defects, overriding aorta, double-outlet right ventricle and right-side aortic arches [The child 22 had coarctation of aorta, VSD, ASD (cardiac surgery was performed when he was 1 month old) and ID. Brain MRI showed normal result. CMA test revealed a 5.2 Mb LOH on chromosome 6q24.1-q24.2 . This LOH fragment included glycemia . Anotherc arches . Therefon = 6) or duplication (n = 1) syndrome, which was the most common type. The incidence of 22q11 deletion/duplication was 6.7% (7/104) in the children with CHD in the present study. These data indicate that in addition to CMA it could be more cost-effective to exclude 22q11 deletion/duplication firstly by targeted technique such as MLPA in children with CHD.Of the 29 children with PCNVs, 7 (24.1%) were with 22q11 deletion (STIM1, CTNNA3 and PLN. STIM1 gene mutation could cause cardiomyocyte hypertrophy [PLN gene mutation was associated with cardiomyopathy [CTNNA3 gene mutation could cause arrhythmogenic right ventricular dysplasia, compound heterozygous deletion was related to ASD [In the 3 children with VOUS, we identified CHD candidate genes such as ertrophy . PLN genmyopathy . CTNNA3 d to ASD , 40. HowCMA has been recommended as a first-tier clinical diagnosis test in patients with DD, ID and/or MCA , 41. Stu


Salvia hispanica L.) is known as power house of omega fatty acids which has great health benefits. It contains up to 78% linolenic acid (ω-3) and 18% linoleic acid (ω-6), which could be a great source of omega-3 fatty acids for functional foods. Therefore, in this study, margarines were prepared with supplementation of different concentrations of chia oil to enhance omega-3 fatty acids, antioxidant characteristics and oxidative stability of the product.Chia , respectively. Margarine without any addition of chia oil was kept as control. Margarine samples were stored at 5 °C for a period of 90 days. Physico-chemical  and sensory characteristics were studied at the interval of 45 days.Margarines were formulated from non-hydrogenated palm oil, palm kernel and butter. Margarines were supplemented with 5, 10, 15 and 20% chia oil . Sensory characteristics of treatments were not different from the control.The melting point of TMargarines supplemented with chia oil showed enhanced level of omega-3 fatty acids and antioxidant characteristics. These results suggest that chia oil can be used for formulation of margarine with increased level of omega-3 fatty acids and acceptable sensory characteristics. On the basis of scientific information, it has been concluded that trans fatty acids do not perform any beneficial biochemical function in the human body [Trans fatty acids increase the harmful LDL cholesterol and reduce the beneficial HDL cholesterol. The excessive intake of trans fatty acids may lead to cardiovascular diseases, cancer and inflammation [Margarines are characterized on the basis of melting point and hardness . Margariman body . Trans fammation . Blendinammation , 5. On tammation .Salvia hispanica L.) has its place from the family Labiatae and chia seeds have been the part of human nutrition for about 1500 years BC as staple food in Mexican region [Chia .1 was comprised of 70% RBD palm oil, 25% non-hydrogenated palm kernel oil and 5% chia oil. T2 was comprised of 65% RBD palm oil, 25% non-hydrogenated palm kernel oil, 10% chia oil. T3 was comprised of 60% RBD palm oil, 25% non-hydrogenated palm kernel oil and 15% chia oil and T4 was comprised of 55% RBD palm oil, 25% non-hydrogenated palm kernel oil and 20% chia oil. Margarine without any addition of chia oil was kept as control. Margarine samples were stored at 5 °C for a period of 90 days. Chemical and sensory characteristics were studied at the interval of 45 days. Experiment was planned in a completely randomized design and each treatment was replicated three times.In the present Investigation, TFormulation of margarine was comprised of 82% oil phase, 1% lecithin, 0.6% salt and 16.4% aqueous phase. Ingredients were mixed in respective oil and aqueous phases. Emulsion was formulated by mixing the both phases in electrical blender . For the solidification of margarine, emulsion was cooled in 1 l beaker, in ice bath, containing 10% NaCl .Fat, moisture, refractive index, melting point, solid fat index, free fatty acids and peroxide value in margarine samples was determined by following the standard methods , 19.n-hexane and 2 mL deionized water were added. Vortex process was performed at 2200 rpm for exactly 2 min followed by the resting of 15 min and supernatant was dried over anhydrous sodium sulphate. Prepared methyl esters were transferred to GC vials and injected into GC-MS  fitted with fused silica capillary Column  and Mass Selective Detector. Helium was used as carrier gas at the flow rate of 2 mL/min. Fatty acids were identified and quantified by FAME 37 internal standards  [Fatty acid methyl esters were prepared by acid transesterification technique using Methanolic HCl (15%) as transesterifying agent. Briefly, 50 mg samples were taken in screw capped test tubes and 2 mL methanolic HCl was added. Tubes were put in a heating block at 100 °C for 60 min and contents of tubes were shaken after every 10 min. After 60 min tubes were cooled to room temperature and 2 mL Total phenolic contents were determined according to the method prescribed by Velioglu et al. . Two hunDPPH free radical scavenging activity was determined by following the method of Brand-Williams et al. . 60 mg sChlorogenic acid, caffeic acid, quercetin, phenolic glycoside-K and phenolic glycoside-Q were determined on HPLC fitted with quaternary pump and Diode Array Detector. Light sources were deuterium and tungsten lamps on 190-195 nm through reverse phase elution while column specifications were 250 × 4.6 mm, 5 μm (LC-18 column) and 250 mm and 4.6 i.d. (Symmetry C18 column). Mobile phase was comprised of 6% acetic acid prepared in 2 mM sodium acetate and acetonitrile. 10 μL was injected, flow rate was maintained at 1 mL/min and total run time was 75 min. Internal standards of chlorogenic acid, caffeic acid, quercetin, phenolic glycoside-K and phenolic glycoside-Q were prepared in ethanol .Margarines were stored at 5 °C for 90 days and were sampled at 0, 45 and 90 days. Peroxide value and free fatty acids value was determined according to the standard methods of American Oil Chemists Society . ThriobaSensory evaluation of margarines supplemented with chia oil was performed by a panel of ten trained judges. Sensory evaluation was performed in well illuminated laboratory at 20 ± 2 °C. Samples of margarines were coded with three digit random number and all the servings were fully randomized. Margarine samples were evaluated for colour, flavour and texture on 9 point scale .The average of the three samples was reported as the measured value with standard deviation. Significant difference among the treatments was determined by Tuckey’s Test. The sample analysis for storage stability and consumer acceptability was carried out in triplicate and the significant differences were calculated among means at a probability level of 5% .p > 0.05). In our preliminary investigations, chemical composition of control was determined. The ratios of fat, moisture and salt content were adjusted according to the control, which was the reason for non-variation in compositional attributes of margarine. Chemical characteristics of control and different types of margarine are presented in Table p > 0.05). Free fatty acids of RBD palm oil, palm kernel oil, butter and mechanically extracted crude oil from non-roasted chia seeds were 0.10, 0.11, 0.13 and 0.14%, respectively. Lower free fatty acids content of substrate oil led to the lower fatty acids in all the treatments. Free fatty acids are produced as a result of hydrolysis of triglycerides, moisture, lipases, metal ions and temperature. These are considered as the important factors which influence the generation of free fatty acids. Studies of Zhang et al. [p < 0.05). Melting point is an important parameter during the development of table margarine as it helps to determine the spreadibility of margarine after taking out from refrigerator. Melting point provides a temperature indication at which margarine ought to be smooth in the palette. Table (soft) margarine should be immediately spreadable, after taken out from the refrigerator, with no oiling out [1, T2, T3 and T4 developed in current investigation were 34.2, 33.8, 33.1 and 32.5 °C, which were within the range of international standards. Solid fat index indicates the percentage of triglycerides solidified at a particular temperature. It is an important indicator of many features of foods together with appearance; spreadability and mouth feel of margarine. It also measures the degree of crystallization of fats [1, T2, T3 and T4 were 47.21, 22.71, 20.33, 18.12 and 16.58%, respectively .The quality control analysis of substrate oils have been given in Table g et al.  revealedling out . Interna of fats . Solid f of fats . Concent than 6% . In curr than 6% . Formulay et al.  describetrans fatty acids. Partially hydrogenated fats are the major carrier of trans fatty acids in human body. Harmful impacts of trans fatty acids on serum cholesterol level and cardiovascular diseases have been scientifically proven [trans fatty acids in control was 18.72% while experimental samples did not reveal trans fatty acids. Nadeem et al. [trans fatty acids in partially hydrogenated vegetable fat available in Pakistan. The concentration of trans fatty acids was greater than 20%. Dollahs et al. [Moringa oleifera oil through interesterification. The blends thus formulated were free of trans fatty acids.Results of fatty acid profile of margarines supplemented with chia oil are given in Table y proven . Concentm et al.  analyzeds et al.  improved1, T2, T3 and T4 were 2.92, 5.85, 9.22, 12.29%, respectively. Concentration of eicosanoic acid in T2, T3 and T4 were 1.82, 3.52, 6.43 and 9.81%, respectively. While content of docosahexanoic acid in in T2, T3 and T4 were 1.26%, 2.64, 3.49 and 5.19%, respectively. Omega-3 fatty acids were not detected in the control. Health benefits associated with the intake of foods containing omega-3 fatty has led the researchers to formulate the functional containing omega-3 fatty acids. Concentration of omega-3 fatty acids in chia oil and its olein fraction was greater than 60 and 80% [trans-11; CLA) in T1, T2, T3 and T4 were 0.038, 0.035, 0.037 and 0.034%, whereas, CLA was not found in control. Anticarcinogenic, anti-diabetic, immunomodulating, antiatherogenic perspectives of CLA have been published in literature [trans fatty acids.Concentrations of α-linolenic acid in T and 80% . Chia se and 80% . Scienti and 80% . Chia oi and 80% . Concentterature . Fatty a1, T2, T3 and T4 were 0.04, 0.14, 0.24 and 0.21%, respectively, from the initial values. After 90 days of storage at 5 °C, losses of eicosanoic acid in T1, T2, T3 and T4 were 0.07, 0.11, 0.15 and 0.18%, respectively, from the initial values. Chlorogenic acid, caffeic acid, quercetein, phenolic glycoside-k and phenolic glycoside-Q are the major phenolic compounds present in chia oil. Although the concentration of polyunsaturated fatty acids was higher in margarines, however, phenolic compounds of chia oil strongly inhibited the lipid peroxidation in supplemented margarines. Among the food products, margarine is a typical example, which is susceptible to oxidation, because of 82% fat content [p > 0.05). Fatty composition of blends of butter oil and mango kernel oil in ambient and accelerated oxidation (25 and 55 °C) was different from the fresh samples [Transition in fatty acid profile of fats and oils during the storage is a good indication of oxidative stability . Concent content . Oxidati content . For the content  studied  content  monitore samples .Table 5T1, T2, T3 and T4 were 0.27, 2.22, 4.15, 7.23 and 11.42 mg GAE/mL, respectively. The higher phenolic contents of experimental margarines are in line with earlier investigations on chia seed. Total phenolic contents of chia seed extract and chia oil were 35 and 7.6 mg GAE/mL, respectively [Moringa oleifera oil and sesame cake extract, 7.1 and 1.84%, respectively [Total phenolic contents of control, Tectively . Total pectively , 44. Higectively . Antioxiectively . Supplemectively .1, T2, T3 and T4 were 65.8, 5.37, 17.82, 24.95 and 62.80%, respectively. EC50 for T4 was 1.25 mg/mL as compared to EC50 of BHT 1.13 mg/mL. Phenolic substances of chia oil efficiently inhibited the lipid peroxidation, which is also evident form the lower peroxide value while flavour score and peroxide value were strongly correlated (R2 = 0.9982). Supplementation of margarine and butter with chokeberry polyphenols extract enhanced the antioxidant activity of fat matrix in cookies and altered the fat oxidation during the storage period of 9 weeks [2 value of 37.35 L/moL, vitamin E is regarded as one of the most antioxidant in butter and margarine. Earlier investigation has shown that k2 value of Camu-camu (Myrciaria dubia) was 69.24 ± 5.72 L/moL, which indicate the possibility of using phytochemicals for the preservation of fats and oils [Determination of DPPH free radical scavenging activity is one of the most effective and widely used methods for the assessment of antioxidant activity of antioxidants of plant origin. In current investigation, DPPH free radical scavenging activity of 100 ppm BHT, control, T 9 weeks . With a and oils .1 were 0.33, 0.22, 0.42, 1.07 and 0.62 mg/mL, respectively. Concentrations of chlorogenic acid, caffeic acid, quercetin, phenolic glycoside k and phenolic glycoside Q, in T2 were, 0.49, 0.25, 0.69, 1.42 and 1.32 mg/mL. Concentrations of chlorogenic acid, caffeic acid, quercetin, phenolic glycoside k and phenolic glycoside Q, in T3 were 0.78, 0.73, 1.82, 4.12 and 4.49 mg/mL. Concentrations of chlorogenic acid, caffeic acid, quercetin, phenolic glycoside k and phenolic glycoside Q, in T4 were, 1.21, 0.83, 2.91, 5.23 and 5.58 mg/mL. Whereas, these phenolic compounds were not detected in control samples. During orthodox processing of oils and fats, they are exposed to higher temperature while phenolic compounds are usually lost during the commercial processing of oils and fats. HPLC characterization of ethanolic chia seed extract revealed the existence of chlorogenic acid, caffeic acid, quercetin, phenolic glycoside-k and phenolic glycoside-Q [HPLC characterization of margarines supplemented with chia oil revealed that the chlorogenic acid, caffeic acid, quercetin, phenolic glycoside k and phenolic glycoside Q were the major phenolic compounds Fig. . Concentcoside-Q . Health coside-Q . Earliercoside-Q . Chia secoside-Q .Fig. 2Ph4 (1.14 MeqO2/kg), which is much lower than the maximum limit . Addition of chia oil in margarine not only improved the concentration of omega-3 fatty acids but also altered the lipid peroxidation in supplemented margarines [Results of oxidative stability of margarine supplemented with chia oil have been presented in Fig. rgarines . After 6rgarines . During rgarines . Due to rgarines . Use of rgarines . Additiorgarines . In currp > 0.05). Storage period up to 45 days was non-significant for all the treatments and control. Color, flavor and texture score of 90 days stored T4 was less than other treatments and control (p < 0.05). After 45 days of storage period, decline in flavor score of T4 may be connected to the generation of peroxides that lead to the formation of odoriferous compounds, such as, aldehydes, ketones and alcohols. Nadeem et al. [trans free margarine from palm mid fraction and canola oil and sensory characteristics of trans free margarine were not different from the reference margarine.Results of sensory evaluation of margarines supplemented with chia oil are presented in Fig. m et al.  recordedm et al. . Lumor em et al.  preparedtrans free margarine can be enhanced by chia oil supplementation.Chia oil at all levels enhanced the concentration of beneficial omega-3 & omega-6 fatty acids antioxidant characteristics of margarine. Supplemented margarines revealed low degree of changes in fatty acid profile, yielded the lower concentration of primary and secondary oxidation products. Sensory characteristics of margarine supplemented with 15% chia oil were not different from the control. Overall, omega fatty acids and antioxidant characteristics of


Enzymatic degradation of chitin has attracted substantial attention because chitin is an abundant renewable natural resource, second only to lignocellulose, and because of the promising applications of N-acetylglucosamine in the bioethanol, food and pharmaceutical industries. However, the low activity and poor tolerance to salts and N-acetylglucosamine of most reported β-N-acetylglucosaminidases limit their applications. Mining for novel enzymes from new microorganisms is one way to address this problem.Microbacterium sp. HJ5 harboured in the saline soil of an abandoned salt mine and was expressed in Escherichia coli. The purified recombinant enzyme showed specific activities of 1773.1 ± 1.1 and 481.4 ± 2.3 μmol min−1 mg−1 towards p-nitrophenyl β-N-acetylglucosaminide and N,N'-diacetyl chitobiose, respectively, a Vmax of 3097 ± 124 μmol min−1 mg−1 towards p-nitrophenyl β-N-acetylglucosaminide and a Ki of 14.59 mM for N-acetylglucosamine inhibition. Most metal ions and chemical reagents at final concentrations of 1.0 and 10.0 mM or 0.5 and 1.0% (v/v) had little or no effect (retaining 84.5 − 131.5% activity) on the enzyme activity. The enzyme can retain more than 53.6% activity and good stability in 3.0–20.0% (w/v) NaCl. Compared with most GlcNAcases, the activity of the enzyme is considerably higher and the tolerance to salts and N-acetylglucosamine is much better. Furthermore, the enzyme had higher proportions of aspartic acid, glutamic acid, alanine, glycine, random coils and negatively charged surfaces but lower proportions of cysteine, lysine, α-helices and positively charged surfaces than its homologs. These molecular characteristics were hypothesised as potential factors in the adaptation for salt tolerance and high activity of the GH 20 GlcNAcase.A glycoside hydrolase family 20 (GH 20) β-N-acetylglucosaminidase (GlcNAcase) was identified from Biochemical characterization revealed that the GlcNAcase had novel salt–GlcNAc tolerance and high activity. These characteristics suggest that the enzyme has versatile potential in biotechnological applications, such as bioconversion of chitin waste and the processing of marine materials and saline foods. Molecular characterization provided an understanding of the molecular–function relationships for the salt tolerance and high activity of the GH 20 GlcNAcase.The online version of this article (doi:10.1186/s12896-017-0358-1) contains supplementary material, which is available to authorized users. With the rapid development of genome sequencing technology, many GlcNAcases have been identified in the genomes of animal tissues, insects, plants, bacteria and fungi in recent years. GH 20 GlcNAcases are mainly found in microorganisms, especially bacteria (http://www.cazy.org/GH20.html). However, the molecular and biochemical characteristics of the majority of these GH 20 GlcNAcases have not been reported (http://www.cazy.org/GH20.html).According to a homology comparison of amino acid sequences, GlcNAcases have been classified into GH 3, 20, 73 and 84 , which was expressed in E. coli, showed better tolerance to salts and GlcNAc and higher activity than most GlcNAcases reported in the literature, including the GH 3 GlcNAcase we previously reported [In this study, a novel GH 20 GlcNAcase, called HJ5Nag, was mined from the genome of reported . PotentiE. coli BL21 (DE3) was purchased from TransGen  and was used for gene expression.Strain HJ5 was isolated from the saline soil of an old salt mine located in Heijing Town. The details of the strain isolation and identification were described in our previous study . The purd-1-thiogalactopyranoside ; Ni2+-NTA agarose ; Qubit protein assay kit ; pEASY-E2 vector (TransGen); N,N'-diacetyl chitobiose (GlcNAc2), N,N',N'',N'''-tetraacetyl chitotetraose (GlcNAc4), p-nitrophenyl β-N-acetylgalactosaminide , and p-nitrophenyl β-d-glucopyranoside (pNPGlc) ; peptidoglycan from Bacillus subtilis ; p-nitrophenol (pNP), p-nitrophenyl-β-d-xylopyranoside (pNPXyl), p-nitrophenyl α-d-galactopyranoside , p-nitrophenyl-α-l-arabinofuranoside (pNPAra), p-nitrophenyl β-N-acetylglucosaminide (pNPGlcNAc), mutanolysin from Streptomyces globisporus ATCC 21553, chitin and chitosan ; and silica gel G plates . All other chemicals were of analytical grade.The following vectors and reagents were used: plasmid isolation and genomic DNA kits ; dNTPs and DNA polymerases ; isopropyl-β-hj5Nag, was obtained by genome sequencing performed on a Miseq sequencer (Illumina) in our lab. The HJ5 genome sequencing details were described in our previous study [The GlcNAcase-encoding gene, designated us study .http://www.ncbi.nlm.nih.gov/BLAST/) online tools were used to search homologous sequences and to calculate the identity values. SignalP  and InterPro (http://www.ebi.ac.uk/interpro/) online tools were used to predict the signal peptide and protein domain, respectively. Vector NTI 10.3  software was used to calculate frequencies of the amino acid residues. Multiple sequences and structures alignments were generated by ESPript [Open reading frames from the draft genome of HJ5 were predicted using the tools described in our previous study . BLASTN  ESPript .http://swissmodel.expasy.org/). The proportions of secondary structures were analysed by VADAR [The homology model of HJ5Nag was predicted using the SwissModel platform (by VADAR . The chahj5NagEF (5'-GGGTGCAGCCCCGCCGC-3') and rhj5NagER (5'-CTCGGTGGCCCAGTCGATCTCGC-3'). The resulting PCR product was ligated to pEASY-E2 vector, which has a single 3′-T overhang at the insertion site according to the manufacturer. E. coli BL21 (DE3) competent cells were transformed with the plasmid for recombinant enzyme expression. A positive transformant harbouring the recombinant plasmid was confirmed by DNA sequencing performed by Tsingke . A seed culture of the positive transformant was grown overnight at 37 °C and then inoculated in 1:100 dilutions into fresh Luria–Bertani medium with the addition of 100 μg mL−1 ampicillin. Upon reaching an OD600 nm of approximately 0.7, IPTG at a final concentration of 0.25 mM was added to the culture to induce enzyme expression at 20 °C for approximately 20 h.The coding sequence of HJ5Nag was amplified by PCR using the primer set r2+-NTA agarose gel columns to bond the recombinant enzyme. The target recombinant enzyme was eluted with a linear imidazole gradient of 20–500 mM in buffer A. The protein concentration was determined using a Qubit protein assay kit using a Qubit 2.0 fluorometer (Invitrogen).Cultures containing positive transformant cells were centrifuged and resuspended in ice-cold buffer A containing 20 mM Tris–HCl, 0.5 M NaCl, and 10% (w/v) glycerol (pH 7.2). The cells were disrupted by ultrasonication on ice with 100 short bursts of 4 s each at a power output of 150 W. After removing cell debris by centrifugation, the supernatant was loaded onto NiSodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS–PAGE) was performed to analyse the expression of the recombinant enzyme and the purity of the eluted fractions. The molecular masses of internal peptides from the single band present in the SDS–PAGE gel were analysed via matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI–TOF MS) and were compared with the molecular masses of the internal peptides from rHJ5Nag.pNP and 3,5-dinitrosalicylic acid (DNS) methods, as described in our previous study [pNP method was applied to determine the activity of rHJ5Nag towards substrates pNPXyl, pNPGal, pNPGlc, pNPAra, pNPGlcNAc and pNPGalNAc. The DNS method was used for the substrates GlcNAc2, GlcNAc4, colloidal chitin, chitosan, peptidoglycan and muropeptides (degradation products of peptidoglycan by mutanolysin treatment).An enzyme assay of purified rHJ5Nag towards various substrates was performed spectrophotometrically using the us study . The pNPpNP method using pNPGlcNAc as substrate was applied to determine the biochemical characteristics of purified rHJ5Nag, unless otherwise noted. McIlvaine buffer was used for pH 5.0–8.0, and 0.1 M glycine–NaOH was used for pH 9.0–12.0.The The pH-dependent activity of rHJ5Nag was determined at 37 °C, with pH values ranging from 5.0 to 9.0. The pH-dependent stability of rHJ5Nag was determined by measuring the residual enzyme activity after incubating the enzyme at pH 5.0–12.0 for 1 h.The temperature-dependent activity of rHJ5Nag was examined at pH 6.0 and temperatures from 0 to 60 °C. The thermostability of rHJ5Nag was determined by measuring the residual activity of the enzyme after pre-incubation at 30, 45 and 50 °C and pH 6.0 for 1–60 min.3, CaCl2, CoCl2, HgCl2, Pb(CH3COO)2, MgSO4, NiSO4, CuSO4, MnSO4, ZnSO4, FeCl3, EDTA, β-mercaptoethanol and SDS, and 0.5 or 1.0% (v/v) Tween 80 and Triton X-100. The absorption was measured after removing the precipitate by centrifugation when AgNO3, HgCl2 or SDS was added to the reaction mixture. Furthermore, the enzyme activity in 1.0–30.0% (w/v) NaCl was also examined, and the enzyme stability in NaCl was determined by measuring the residual enzyme activity after pre-incubating the enzyme in 3.0–30.0% (w/v) NaCl for 1 h.To determine the effects of various metal ions and chemical reagents on purified rHJ5Nag, the enzyme activities were measured after the addition of different metal ions and chemical reagents to the reaction mixture. The metal ions and chemical reagents included 1.0 or 10.0 mM  KCl, AgNOKm, Vmax and kcat values of purified rHJ5Nag were analysed using 0.1–1.2 mM pNPGlcNAc as substrate at 45 °C and pH 6.0. A nonlinear regression Michaelis − Menten fit was applied to analyse the data using GraphPad Prism . The extent of GlcNAc inhibition on rHJ5Nag activity was determined adding 5.0 and 10.0 mM GlcNAc to the reaction solution. Then, the β-N-acetylglucosaminidase activity was determined according to the aforementioned kinetic assay. The inhibition constant (Ki) was calculated using GraphPad Prism using the nonlinear mixed-model inhibition equation described detailedly in a previous paper [The us paper .2 or GlcNAc4 and approximately 0.1 units of purified rHJ5Nag. After incubating the mixture in McIlvaine buffer (pH 6.0) at 37 °C for 6 h, the hydrolysis products were analysed by thin-layer chromatography (TLC), as previously described [The hydrolysis mixture included 160 μL of 0.5% (w/v) GlcNAcescribed .Streptomyces griseus, designated CtnSg, was purchased from YuanYe Bio-Technology . The reaction mixture contained colloidal chitin  and either CtnSg (0.01 U mL−1 reaction system), rHJ5Nag (0.5 U mL−1 reaction system) or both enzymes and was incubated for 120 min. In the time-course study, the reaction mixture containing 0.5% (w/v) colloidal chitin and CtnSg alone was incubated for 30, 60 or 90 min, rHJ5Nag was then added to the reaction and incubated for 90, 60 or 30 min, respectively. According to the manufacturer’s instructions for CtnSg and the biochemical characteristics of rHJ5Nag, degradation of colloidal chitin was performed at 25 °C and pH 6.0 (McIlvaine buffer). The DNS method was used to measure the amount of reducing sugars released from the colloidal chitin. A reaction containing heat-inactivated enzyme (incubating in a boiling water bath for 10 min) was used as a control. The degree of synergy was calculated using the following equations:DS is the degree of synergy, RSrHJ5NagCtnSg is the amount of reducing sugars released from simultaneous or sequential enzyme reactions, and RSrHJ5Nag and RSCtnSg are the amounts of reducing sugars released from individual enzyme reactions.Commercial chitinase from hj5Nag and Microbacterium sp. HJ5 16S rDNA in GenBank are KX400857 and KX400858, respectively.The accession numbers of Microbacterium flavescens (accession no. HQ530520), Microbacterium takaoensis (AB201047) and Microbacterium trichothecenolyticum (KU179351). Phylogenetic analysis also placed HJ5 in the Microbacterium cluster but not in the clusters grouped by the species of other genera of Microbacteriaceae  and the 16S rDNA sequences from hj5Nag. hj5Nag has a length of 1608 bp and encodes the 535-residue GlcNAcase HJ5Nag, with a predicted molecular mass of 55.9 kDa.Sequence data of approximately 606.6 Mbp was generated by genome sequencing for HJ5, and these data yielded a draft genomic DNA sequence of approximately 3.6 Mbp after sequence assembly. The analysis of the draft genome predicted an open reading frame designated gene The alanine (A) and glycine (G) frequencies of the GlcNAcase were 16.3 and 10.8%, respectively, both of which are the highest values among the GlcNAcases shown in Table Microbacterium sp. Root180 (accession no. WP_056119055) shared the highest identity of 78.9% with HJ5Nag. Based on the alignment of HJ5Nag with other experimentally characterized GlcNAcases  and the catalytic domain of GH 20 GlcNAcases . The homology model of HJ5Nag and the crystal structures of 7 GlcNAcases were used to calculate the proportions of secondary structures and the charge distributions on the surfaces. Among these GlcNAcases, the proportions of amino acids used to build the α-helix and β-sheet structures of HJ5Nag were the second lowest, while the proportion of amino acids used to build random coil structures and the ratio of random coils to α-helices of HJ5Nag were both the highest  and purified by Ni2+-NTA affinity chromatography. The MALDI–TOF MS spectrum of the purified band further confirmed that the purified product was rHJ5Nag  showed little to no effect (retaining 84.5 − 131.5% activity) on the enzyme activity  [Paenibacillus sp. (57.5 kDa) [S. thermoviolaceus (58.3 kDa) [S. thermophilum (55.9 kDa) [Drastic inhibition by Agbacillus  and V. furnissii , the GH . miehei  and Sphirium sp. , the GH trificum  and the adoensis . Ag+ and. miehei , Sphingorium sp.  and L. cL. casei , are strnd 50 °C , the GH nd 50 °C , and thend 50 °C . Furthernd 50 °C , C. fimi7.5 kDa)  and S. t5.9 kDa) .4 and NaCl. Most GlcNAcases reported to date are strongly inhibited by Cu2+, such as the GH 20 GlcNAcases from Paenibacillus [A. caviae [L. casei [R. miehei [C. paraputrificum [S. cerradoensis [Paenibacillus are strongly inhibited by Ni2+ [A. caviae is strongly inhibited by Mn2+ [T. reesei is inhibited by Fe3+ [C. paraputrificum is inhibited by Ni2+, Co2+, Mn2+, Zn2+ and Fe3+ [R. miehei is inhibited by Ni2+, Co2+, Zn2+ and EDTA [Purified rHJ5Nag exhibited good salt tolerance, as the addition of most salts had little to no effect on the enzyme activity, especially CuSObacillus , A. cavi. caviae  and L. cL. casei , the GH . miehei , the GH trificum  and the adoensis . Further by Ni2+ ; the GH2 by Mn2+ ; the GH2 by Fe3+ ; the GH and Fe3+ ; and theand EDTA . Salt toand EDTA , 50–60, and EDTA , 18. In and EDTA .To reveal the potential catalytic adaptations of rHJ5Nag to high concentrations of salt, sequence and structure comparisons to its homologs were performed. (I). Similar to the results from other salt-tolerant enzymes , 53–59, Vmax of lower than 1000 μmol min−1 mg−1 towards pNPGlcNAc, as summarized in Table −1 mg−1 and a Vmax of 3097 ± 124 μmol min−1 mg−1 towards pNPGlcNAc. Furthermore, rHJ5Nag showed a specificity activity of 481.4 ± 2.3 μmol min−1 mg−1 towards GlcNAc2, which is much higher than the values from other GlcNAcases recorded in the literature, such as the values from Sphingobacterium sp. (0.3 μmol min−1 mg−1) [T. spiralis (0.79 nmol min−1 mg−1) [R. miehei (1.06 μmol min−1 mg−1) [V. alginolyticus (18.1 μmol min−1 mg−1) [S. thermophilum (76.8 μmol min−1 mg−1) [Most characterized GlcNAcases, including GH 3, 20 and 84 members, show specific activities or −1 mg−1) , T. spir−1 mg−1) , R. mieh−1 mg−1) , V. algi−1 mg−1)  and S. t−1 mg−1) . As discV. furnissii, Trichoderma harzianum, A. oryzae, S. frugiperda, R. miehei and T. spiralis were competitively or noncompetitively inhibited by GlcNAc, with Ki values of 0.21 [Ki of 14.59 mM.Although the synergistic action of chitinases and GlcNAcases for complete enzymatic degradation of chitin is attractive, there are few reports , 66, 67. of 0.21 , 0.21 [6 of 0.21 , 1.6 [13 of 0.21 , 9.68 [3 of 0.21  and 15.7 of 0.21 , respectMicrobacterium sp. HJ5 harboured in the saline soil of an abandoned salt mine. Biochemical characterization revealed that the GlcNAcase had novel salt–GlcNAc tolerance and high activity. These characteristics suggest that the enzyme has versatile potential in biotechnological applications, such as bioconversion of chitin waste and processing of marine materials and saline foods. Molecular characterization provides an understanding of the molecular–function relationships for the salt tolerance and the high activity of the GH 20 GlcNAcase.A GH 20 GlcNAcase was isolated from


Blend membranes, nHBP-PAZ-SiO/EC and nHBP-PAZ-SiO/PS were prepared by blending the THF solution of nHBP-PAZ-SiO with ethanol solution of EC and dichloromethane solution of PS, respectively. Surprisingly, the permeabilities for CO2 of the blend membranes were more than 15–16 times higher than those of pure EC and PS membranes without any drop of pemselectivity to N2. This unusual improvement has been achieved by both enhancement of diffusivity for carbon dioxide and nitrogen by the oligosiloxane groups and enhancement of affinity of the amino groups with carbon dioxide at the end groups of nHBP-PAZ-SiO.Three soluble hyperbranched polyazomethines containing oligosiloxane end group  Amonflue gas ,13–17.PA such as PCO2), 2) a high gas permselectivity of Gas A to Gas B (PA/PB such as PCO2/PN2), and 3) good membrane-forming ability giving an ability to yield a thin membrane. However, trade-off relationships between the permeability and permselectivity have been often observed [PAZ) are known for their excellent thermal stability, good mechanical strength, environmental resistance, and optoelectronic property [PAZs are usually synthesized from diamine and dialdehyde and therefore they have amine end groups. Since amine groups have strong interaction with CO2, they are useful for CO2 separation [PAZ is very low. Hyperbranched polymers (HBP) have been reported, and they have high solubility compared with the corresponding linear ones. [PAZ (= HBP-PAZ).In general, requirements for polymer materials as a practical gas permselective membrane are the following three: 1) a high gas permeability coefficient for a Gas A  and polysulfone (PS).In this paper, in order to enhance solubility of 2.2.1.All the solvents used for monomer synthesis and polymerization were distilled as usual. Melamine and isophthalaldehyde were purchased from Aladdin Industrial Corporation. The silicon containing reagents purchased from TCI chemical Co., Inc., were used as received.2.2.2.2.1.PCO2 and PN2: cm3(STP)·cm·cm−2·s−1·cmHg−1) and the carbon dioxide separation factor (PCO2/PN2) were measured by a gas chromatographic method by using YANACO GTR-11 MH according to our previous report. [Carbon dioxide and nitrogen permeability coefﬁcients ) was used for the feed gas. The l, A, Δp, and t are the amount of the permeated gas, the thickness of the membrane, the permeation area of the membrane, the pressure difference across the membrane and the permeation time, respectively. Disc-type membranes were used. The A and l of the membranes were 1.77 cm2 and around 120–310 μm, respectively. The Δwhere Q, 2/N2 test gas was feed to membrane cell through ‘In’. After the retention time and wait for steady state, the permeate gas in the ‘permeate storage tube’ was measured by gas chromatography through the time lag. The residue gas in ‘Membrane cell’ was released through ‘Retentate’. The amount of the permeated gas  spectra were recorded on an AVANCE III spectrometer. The average molecular weights (Mn and Mw) were evaluated by gel permeation chromatography (GPC) by using Polymer Laboratories (Varoam) liquid chromatography instruments . The infrared spectra were recorded on Spotlight 400.2.3.nHBP-PAZ-SiO were synthesized according to Soluble oligosiloxane-end-capped polyazomethines 2.3.1.1 was purified by vacuum drying.n-Butyllithium  was added dropwise to the tetrahydrofuran (THF) (50.0mL) solution of hexamethyldisiloxane  at 0℃. After refluxing for 24h, THF was removed by evaporation and the crude product 1  was add to the mixture, and after 1h stirring at room temperature, THF(25.0mL) was add to the mixture followed by stirring at room temperature for 24h. Finally, the dimethylchlorosilane  was injected into the mixture and stirred for another 3h at room temperature. The mixture was filtered, THF was removed by evaporation. The crude product was purified by vacuum drying to give 2 as a yellowish transparent liquid.The mixture of hexamethylcyclotrisiloxane (D3)  and cyclohexane (50.0mL) was stirred for 30min at room temperature. Then, compound n = 9: Yield: 65.3% (5.52 g). 1H NMR : δ = 4.70 2SiH), 0.05–0.09 2SiO).n = 18: Yield: 70.7% (11.1 g). 1H NMR : δ = 4.70 2SiH), 0.05–0.09 2SiO).n = 39: Yield: 49.2% (9.23 g). 1H NMR : δ = 4.70 2SiH), 0.05–0.09 2SiO).2.3.2.2 (1.71mmol) was added to the toluene (8.10mL) solution of allylamine  and 3-divinyl-1,1,3,3,3,3-tetramethyldisiloxane platinum (0) complexes . The mixture was stirred at 45℃ for 24 h, and then, the mixture was filtered, solvent was removed by evaporation. The crude product was purified by vacuum drying to give 3 as a brown transparent liquid.The resulting compound n = 9: Yield: 79.8% (1.42 g). 1H NMR : δ = 2.66 , 1.45 , 0.53 , 0.05–0.09 2SiO).n = 18: Yield: 88.0% (2.32 g). 1H NMR : δ = 2.66 , 1.45 , 0.53 , 0.05–0.09 2SiO).n = 39: Yield: 84.3% (4.53 g). 1H NMR : δ = 2.66 , 1.45 , 0.53 , 0.05–0.09 2SiO).2.3.3.HBP-PAZ was as follows: A solution of melamine (A3)  and isophthalaldehyde (B2)  (A3/B2 = 0.65) in 1,3-dimethyl-2-imidazolidinone was stirred for 2h at 65℃. Then, the mixture was poured into a 100mL beaker containing 50.0mL ethyl acetate. After precipitation, the liquid was filtered. The crud solid product was purified by vacuum drying to give a white solid.A typical procedure for synthesis of Other polymerizations of melamine and isophthalaldehyde were carried out similarly. The results are shown in HBP-PAZ: IR(KBr): 3469&3133cm−1 (NH2), 1671cm−1 (C = O), 1543cm−1 (C = N), 1438cm−1(C-N).2.3.4.9HBP-PAZ-SiO was as follows: To a mixture of HBP-PAZ (5.00mg) and THF(1.20mL), oligosiloxane end capping reagent 3 (n = 9)  was added and refluxed for 48h. The mixture was filtered, solvent was removed by evaporation. The crude product was purified by vacuum drying to give 9HBP-PAZ-SiO as a brown viscous liquid.A typical procedure for synthesis of HBP-PAZ with 3 were carried out similarly. The yields of nHBP-PAZ-SiO  were 94.5%, 94.2% and 93%, respectively.Other end capping reactions of nHBP-PAZ-SiO: IR(KBr): 3469&3133cm−1 (NH2), 1671cm−1(C = O), 1543cm−1 (C = N), 1438cm−1(C-N), 1263cm−1(Si-C), 1091cm−1(Si-O), 788cm−1 (Si-C).2.4.n/HBP-PAZ-SiOEC and n/HBP-PAZ-SiOPS blend membranes, were fabricated as follows: a solution of nHBP-PAZ-SiO in THF (1.5 mg/mL) and a solution of the substrate EC in ethanol (30 mg/mL)  were blended together, and then the resulting blend solution was cast on a Teflon sheet. After evaporating the solvent for 24h at room temperature, the membrane was detached from the sheet and dried in vacuo for 24 h. And then the carbon dioxide and nitrogen permeability were measured by a gas chromatographic method by using YANACO GTR-11 MH [2 and the thickness of the membranes were 120-310μm.TR-11 MH ,36. The 3.3.1.nSiO  were synthesized by living polymerization in the different feed ratio of hexamethylcyclotrisiloxane/lithium trimethylsilanolate with yield of 52.1, 34.7 and 34.4%, respectively. The n of nSiO were 9, 18 and 39 which were confirmed by the integral ratio of OSi((CH3)2) to the terminal SiH in the 1H NMR spectra of 2. The observed values were consistent with the theoretical calculation values.Three kinds of oligosiloxane end-capping reagents 3.2.HPB-PAZ) were successfully synthesized by condensation polymerization of melamine (A3) and isophthalaldehyde (B2) with yields higher than 56.6%. The chemical structures were confirmed by FT-IR spectra  were changed, the results are shown in 3/B2 feed ratio reaches to 0.65, the HPB-PAZ shows the highest solubility of 19.5% in THF and 20.9% in DMSO. The Mn and Mw values of the soluble part were also the highest among the four HPB-PAZs .By changing the A3.3.nHBP-PAZ-SiO) were synthesized by reaction of the aldehyde end groups of HBP-PAZ with the amine group of the oligosiloxane end capping reagent nSiO . The yields of the resulting nHBP-PAZ-SiO were higher than 94%. From the FT-IR spectra of 39HBP-PAZ-SiO  was blended with EC and PS to give a self-standing membrane. The CO2/N2 permeation experiments were carried out and the results are shown in 9HBP-PAZ-SiO, the permeability (PCO2) of EC and PS were enhanced and reached more than 9 times higher values without any drops of the pemselectivity (PCO2/PN2)  were synthesized by reaction of aldehyde end group of HBP-PAZ with the amine group of oligosiloxane end capping reagent nSiO . The permeability of EC and PS substrate membranes were enhanced more than 15 times by using the 39HBP-PAZ-SiO as modifiers without any drop on pemselectivity. In the three n, HBP-PAZ-SiO39HBP-PAZ-SiO showed the highest performance due to the longer oligosiloxane chain end. This unusual improvement has been achieved by both enhancement of diffusivity for carbon dioxide and nitrogen by the oligosiloxane groups and enhancement of affinity of the amino groups with carbon dioxide at the end groups of nHBP-PAZ-SiO.Three soluble oligosiloxane-end-capped hyperbranched polyazomethines


Saccharosydne procerus (Matsumura) is the main pest of water bamboo (Zizania latifolia). This insect has two color morphs. The present study explored the influence of photoperiod and its interaction with temperature in nymph stage on adult melanism. Additionally, the longevity, fecundity, mating rate, and hatching rate of S. procerus were examined to determine whether the fitness of the insect was influenced by melanism under different temperature and photoperiod. The results showed that a greater number of melanic morphs occurred if the photoperiod was extremely long. A two-factor ANOVA showed that temperature and photoperiod both have a significant influence on melanism. The percentages of variation explained by these factors were 45.53 and 48.71%, respectively. Moreover, melanic morphs had greater advantages than non-melanic morphs under an environmental regime of high temperatures and a long photoperiod, whereas non-melanic morphs were better adapted to cold temperatures and a short photoperiod. These results cannot be explained by the thermal melanism hypothesis. Thus, it may be unavailable to seek to explain melanism in terms of only one hypothesis.Melanization reflects not only body color variation but also environmental plasticity. It is a strategy that helps insects adapt to environmental change. Different color morphs may have distinct life history traits, e.g., development time, growth rate, and body weight. The green slender planthopper  Saccharosydne procerus (Matsumura) is the principal pest of water bamboo (Zizania latifolia) and has also been shown to damage rice (Oryza sativa) in some East Asian countries such as China and Vietnam. Some green slender planthoppers have a black spot on the terminus of the forewing , China. To provide living conditions similar to the natural habitat, the greenhouse was only covered by a gauze net. The S. procerus in the present study were not exposed to any pesticides. Hence, they did not face any selection pressure from pesticides.In this study, Leaves of water bamboo with spawning marks were collected in the greenhouse. These eggs were placed in an artificial climate box at 26°C with L:D 16:8 h photoperiod. Four photoperiod treatments  were employed to explore the effect of photoperiod on melanism. Five nymphs were placed on one water bamboo leaf in a glass tube (14 × 1.5 cm). Each replicate contained 25 nymphs and each treatment contained four replicates. Therefore, 100 individuals were used in one treatment.To compare the relative influence of photoperiod and temperature on melanism, four treatments  were employed. The egg collection and rearing methods used were stated above.Water bamboo leaves with spawning marks were gathered from the field to facilitate the collection of eggs of green slender planthoppers. These eggs were placed in an artificial climate box at 26°C and L:D = 16:8 photoperiod to feed for one generation.S. procerus  with one water bamboo leaf. Each experimental tube received a selected combination of temperature and photoperiod; three temperatures  and three photoperiods  were used in this experiment. The experiment was performed to compare selected life history traits of adults, namely, adult longevity, fecundity, mating rate, hatching rate, pre-oviposition period, and egg stage duration. All the data were recorded daily. The leaves were changed once a day, and 100 melanic adults and 100 non-melanic adults were employed for each treatment. The rationale for selecting these temperatures was that under these treatments, the melanism proportion was found to differ significantly; moreover, a previous study showed that these conditions were suitable for s Kuang, .In the present study, one-way ANOVA and pairwise assessment using the Multiple Range Test were applied to analyze the effect of photoperiod on melanism and the influence of the studied factors on life history traits. A two-factor ANOVA was employed to analyze the contributions of photoperiod and temperature to variation in melanism. All analyses were conducted with SPSS Statistics 17.0.P = 0.0341) and 8:16 (P = 0.0279). The differences between the melanism proportions under 20:04 and 16:08 were not significant (P > 0.05). The proportion of melanism under 12:12 did not differ significantly from that under 8:16 (P > 0.05).The proportion of melanic morphs increased from 0.56 ± 0.03 under short photoperiod (L:D = 8:16) to 0.7 ± 0.04 under long photoperiod L:D = 20:04) . The percentages of variation in melanism explained by photoperiod and temperature were 45.53% and 48.71%, respectively. The interaction between photoperiod and temperature explained 3.57% of the total variation in melanism  the melanism proportion was 0.61 ± 0.01 while this proportion decreased to 0.25 ± 0.03 under 22°C at L:D = 8:16. A two-factor ANOVA showed that temperature and photoperiod both have significant influences on melanism  and 18.8 ± 1.79 (non-melanic morphs), respectively, while, when the temperature increased to 30°C, it was 13.8 ± 0.84 (melanic morphs) and 10.2 ± 3.03 (non-melanic morphs), respectively. The male adult longevity of two morphs varied from 5.4 ± 1.34 (melanic morphs) and 13.8 ± 1.92 (non-melanic morphs) under 12:12 to 14.7 ± 1.51 (melanic morphs) and 10.0 ± 1.84 (non-melanic morphs) under 20:04.Similarly under 22°C the female adult longevity of two color morphs was respectively 10.0 ± 1.06 (melanic morphs) and 16.2 ± 2.17 (non-melanic morphs), while when temperature increased to 30°C, it was 12.0 ± 1.87 (melanic morphs) and 8.8 ± 0.45 (non-melanic morphs), respectively. With the change of photoperiod, the female adult longevity of two morphs also changed from 5.25 ± 0.90 (melanic morphs) and 15.4 ± 1.52 (non-melanic morphs) under 12:12 to 14.1 ± 0.89 (melanic morphs) and 8.45 ± 1.23 (non-melanic morphs) under 20:04.P > 0.05). The longevity of non-melanic male and female adults was significantly longer than that of melanic male adults (P = 0.0006) and female adults (P = 0.0004) at 22°C. At 26°C, the longevity of non-melanic adults did not differ significantly from that of melanic morphs (P > 0.05). The longevity of melanic male and female adults was significantly longer than that of non-melanic male adults (P = 0.0337) and female adults (P = 0.0059) at 30°C.One-way ANOVA showed that under all temperature and photoperiod treatments, there were no significant differences between the longevity of male adults and that of female adults (P < 0.0001) and females (P < 0.0001). Melanic males and females had a longer longevity than non-melanic males (P = 0.0013) and females (P < 0.0001) under the 20:04 photoperiod. There were no significant differences among the treatment groups under the 16:08 photoperiod (P > 0.05) , 80.9 ± 16.98 (12:12) to 68.2 ± 5.67 (30°C), 35.5 ± 7.07 (20:04), respectively.P = 0.0158) and 20:04 (P = 0.0081), and lower than that of non-melanic morphs at 22°C (P = 0.0003) and 12:12 (P = 0.0036). There were no statistically significant differences between the two morphs at 26°C or under 16:08 (P > 0.05) % to (60.0 ± 7.07)%, similarly, it was (92.0 ± 8.37)% under 22°C and decreased to (44.0 ± 5.48)% under 30°C for non-melanic morphs. Under 12:12, it was (52.502 ± 2.28)% (melanic morphs) and (84.446 ± 6.09)% (non-melanic morphs), when the daytime became longer it was (95.0 ± 6.09)% (melanic morphs) and (70.0 ± 6.85)% (non-melanic morphs), respectively, under 20:04.P = 0.0039) and under 20:04 (P = 0.0002) and lower than that of non-melanic morphs at 22°C (P = 0.0004) and under 12:12 (P < 0.0001). There were no statistically significant differences between the two morphs at 26°C or under 16:08. (P > 0.05) % and (46.778 ± 7.44)% to (79.464 ± 7.61)% and (78.037 ± 4.42)%, while it decreased from (85.45 ± 4.54)% and (71.689 ± 4.97)% to (56.618 ± 8.95)% and (46.416 ± 5.79)% for non-melanic morphs, respectively.P = 0.0003) and under 12:12 (P = 0.0003). The hatching rate of eggs produced by melanic adults was significantly higher than that of eggs produced by non-melanic adults at 30°C (P = 0.0025) and under 20:04 (P < 0.0001). No significant differences between two morphs were found at 26°C or under 16:08 (P > 0.05)  d and (5.0 ± 1.7070) d to (4.0 ± 0.7071) d and (3.8 ± 0.4472). Egg period of melanic and non-melanic morphs varied from (15.0 ± 0.7701) d and (16.4 ± 1.3416) d to (10.2 ± 2.2804) d and (11.8 ± 1.7889) d. Pre-oviposition and egg period of two morphs both shortened when the temperature increased.P > 0.05).When the photoperiod increased from 12:12 to 20:04, the pre-oviposition of melanic and non-melanic morphs varied from (5.2 ± 0.7583) d and (5.0 ± 1.0000) d to (5.0 ± 1.5811) d and (5.1 ± 0.4472) d. Egg period of melanic and non-melanic morphs varied from (11.8 ± 1.3038) d and (10.7 ± 0.6708) d to (12.0 ± 0.7071) d and (11.1 ± 0.7416) d. Pre-oviposition and egg period of two morphs did not varied significantly under different photoperiod. One-way ANOVA showed that the preoviposition period and egg period had no significant differences between the two morphs , China from June to October 2012 also proved that high temperature improve the melanic proportion  production in China should still be explained by the effect of temperature.A previous study has shown that the proportion of melanism in S. procerus may change in the same way. According to previous study, increased temperature is usually associated with the increase in infectious diseases (Roulin, S. procerus appeared, which may be due to the higher PO activity and melanin synthesis caused by greater infectious risk under high temperature (Wilson et al., S. procerus induced by extremely long photoperiods serves to facilitate UV protection. Moreover, we infer that the multiple function of melanin may not only change the body color of S. procerus, but also the environmental fitness.Several previous studies showed that the host-pathogen interaction can be influenced by the thermal environment (Cotter et al.,  Roulin, . Hence, S. procerus. We found that melanic morphs enjoyed a greater advantage than non-melanic morphs in a hot environment and under extremely long photoperiod, whereas, non-melanic morphs were better adapted to cold conditions and relatively short photoperiods. These results showed that the melanism of S. procerus is expressed not only as variation in body color but also in fitness differences in the studied environments. Given that melanism can be induced in hot environments and under extremely long photoperiod, we think that melanism may be a strategy by which S. procerus adapts to environmental alterations. However, the thermal plasticity of S. procerus cannot be explained by the TMH. Insects face complex selection pressures. Hence, it may be inadvisable to explain melanism in terms of only one hypothesis (Wilson et al., In this study, differences between the life history traits of the two studied morphs under different conditions were compared to explore the influence of melanism on the fitness of Several previous studies have shown that the TMH can fail to explain relationships between melanism and environmental adaptations. In some cases, melanic morphs could be induced at relatively high temperatures or under a high level of incident radiation and were advantageous under warm conditions (Välimäki et al., Spodoptera exempta showed a significantly higher resistance to baculovirus than non-melanic morphs (Reeson et al., Tenebrio molitor showed lower mortality when exposed to a generalist entomopathogenic fungus (Barnes and Siva-Jothy, Ephestia kuhuiella were better able to inhibit the oviposition and larval development of parasitic wasps (Verhoog et al., According to previous studies, the immunocompetence of darker individuals is usually greater than that of lighter ones (Armitage and Siva-Jothy, Although melanic morphs have multiple advantages, disadvantages still exist when the selection pressure changes. Melanin-based coloration is costly in terms of energy and other resources because it requires investment in production and maintenance Roulin, . A tradeHarmonia axyridis prefer succinea-form males to melanic males in the spring generation due to the disadvantages experienced by melanic morphs during the summer (Su et al., S. procerus will also have their own mating preferences based on fitness variation in different environments although the melanic pattern found in the species is opposite to that expected from the THM. We consider that this preference may influence the rate of successful mating in S. procerus under different temperatures and photoperiods.The studies cited above identified potential selective disadvantages affecting melanic morphs under certain conditions. For this reason, melanism can at times be a cue for mate choice because it signals environment-dependent fitness to potential mates. For example, females of S. procerus. Additionally, the adult longevity, fecundity, mating rate, and hatching rate of S. procerus were examined in the present study. Our results suggest that melanic morphs enjoy advantages in hot environments and under long photoperiods, whereas non-melanic morphs can adapt more successfully to low temperatures and short photoperiods. These results did not support the TMH. In view of the demonstrated differences in fitness between the two studied color morphs, we infer that the frequency of melanic morphs will increase as a result of continued increase in temperature associated with future global warming. The melanism of S. procerus appears in the adult stage but is a response to the environmental pressure experienced by the nymph. However, the mechanisms by which stressors in the nymphal or larval stage affect the adult stage remain unclear (Debecker et al., S. procerus.In summary, we found that photoperiod, in addition to temperature, is an environmental factor that influences melanism in Our work conforms to the legal requirements of the country in which it was carried out.HY and JL: conception and design of research; HY and QS: performed experiments; HY: analyzed data; HY, QS, MS, JK, and JL: interpreted results of experiments; HY, MS: prepared figures; HY, MS: drafted manuscript; HY, MS, and JL: edited and revised manuscript; HY, QS, MS, JK, and JL: approved final version of manuscript.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.