Gender, living area, Se intake without supplements, Se-enriched salt, oil source and protein intake were identified as significant factors of hair Se contents. Cereals, meat and milk were commonly included as significant food categories that mainly contributed to Se intake without supplement of the whole population. Balanced dietary structure without Se supplement could effectively enhance and maintain children’s Se nutrition. It may be the time to stop applying Se-enriched salt in KBD areas in Shaanxi Province.We aimed to identify significant factors of selenium (Se) nutrition of children in Kashin-Beck disease (KBD) endemic areas and non-KBD area in Shaanxi Province for providing evidence of whether it is the time to stop applying Se-enriched salt in KBD areas. A cross-sectional study contained 368 stratified randomly selected children aged 4–14 years was conducted with 24-h retrospective questionnaire based on a pre-investigation. Food and hair samples were collected and had Se contents determined with hydride generation atomic fluorescence spectrometry. Average hair Se content of 349.0 ± 60.2 ng/g in KBD-endemic counties was significantly lower than 374.1 ± 47.0 ng/g in non-KBD counties. It was significantly higher in the male children (365.2 ± 52.3 ng/g) than in the female (345.0 ± 62.2 ng/g,  Until 1957, scientists have discovered the essential positive effects of Se on animals breaking the limitation of only studying its biologically toxic effects . Since tKashin-Beck disease (KBD) is a serious kind of endemically deformed osteoarthropathy with unclear etiology and pathology. It mainly is distributed from the northeast to southwest China, where the environment is Se-deficient . For decNonetheless, apart from Se-enriched salt, some other dietary and/or non-dietary factors may also play positive roles in controlling KBD, according to our recent findings that are based on an analysis of a three-year cohort study . To provAccording to the results of prior investigation, we adjusted the contents of questionnaires, most of which were about the food items, such as deleting the unusual foods and adding the ones that the residents often consume. Then, we conducted a 2:1:1 matched cross-sectional investigation in the same counties in Shaanxi Province where the prior investigation was performed from June to September in 2012 and 2013, avoiding summer vacations  and versus one child in the non-KBD area  separately. All the subjects were aged 4–14 years and Han nationals, which eliminated the interferences of genetic background and racial bias. The diagnosis criterion of KBD [WS/T 207-2010] in China was used to distinguish patients and healthy children. Subjects with any other osteoarthropathy would be excluded. Based on the results of the pre-investigation, sample size was calculated according to Equation (1).A stratified random sampling method was applied to recruit subjects. According to the design, 2:1:1 matching means that two children in the Se-supplemented KBD area (Linyou and Bin County) were selected k is the county number, Α = 0.05, β = 0.10, ν1 = k−1,ν2 = ∞, k = 4,n = 78.To ensure the response rate, 15% more subjects than calculated were required, thus sample size increased to 90 per county with 30 subjects in each mild , medium  and severe  endemic area which divided according to the primary prevalence; meanwhile, an equal amount of 90 subjects were required in a non-KBD county. Eventually, 368 eligible subjects were included.All the subjects and guardians  were informed and gave their consents about the questionnaire and sample collection. The study was approved by the ethnic committee of Xi’an Jiaotong University .Height and weight measurements were carried out in children in light clothing and without shoes with traditional scales for medical use . All results were recorded by a mean value which calculated from two times of measurements, except for a median which would be used instead if a third measurement was taken when the difference between the former two measurements was more than 10%. Then Children’s BMIs were calculated according to Equation (2).Semi-quantitative questionnaires with a series of dietary and non-dietary items  were applied when the data was not normally distributed. Pearson’s chi-square test was used to assess the difference in categorical data between the KBD endemic area and non-KBD area. The possible dietary and non-dietary factors thought to be associated with hair Se content and the main food groups contributing to the population’s Se intake without supplement were examined by univariate regression analysis. Only the significant variables with All the measurement results are displayed as mean ± standard deviation (X ± SD).p = 0.86) whilst the subjects in each county above were slightly but significantly younger than those in Ningshan County (p < 0.01). However, no such difference was found among mild, medium and severe areas within each endemic county (p > 0.05). As for the gender ratio, significant differences were found between Linyou and Bin County (p = 0.021), Bin and Ningshan County (p = 0.013), Ningshan and Liquan County (p = 0.035), but no significant difference was analyzed among different areas within each endemic county (p > 0.05). The details are displayed in No significant difference of age composition was found among Linyou, Bin and Liquan County . Within each endemic county, only slight significant differences of Children’s BMIs were found in Ningshan County .Children’s BMIs, calculated according to Equation (2), were 16.8 ± 1.7 for Ningshan, 15.8 ± 1.9 for Liquan, 15.2 ± 2.0 for Linyou and 14.6 ± 1.7 for Bin in descending order. The difference was significant . In Ningshan County, it was 296.8 ± 26.7 ng/g in severe areas, 289.8 ± 30.0 ng/g in mild areas and 269.0 ± 9.4 ng/g in medium areas in descending order . The differences are displayed in Average hair Se contents were 399.7 ± 38.8 ng/g in Linyou, 374.1 ± 47.0 ng/g in Liquan, 357.7 ± 45.7 ng/g in Bin and 284.8 ± 26.3 ng/g in Ningshan in descending order and the differences were significant  and females  were significantly different among the four counties. Besides, it was 365.2 ± 52.3 ng/g in all the male children and significantly higher than 345.0 ± 62.2 ng/g in all the female . Except for Linyou County , significant differences of hair Se content between the male and the female were found within each of the rest of the counties. In Bin County, 373.7 ± 40.8 ng/g of hair Se content for the male was significantly higher than 334.5 ± 42.8 ng/g for the female . In Ningshan County, it was 293.0 ± 27.0 ng/g for male and significantly higher than 279.3 ± 24.5 ng/g for female . In Liquan County, it was 383.3 ± 44.5 ng/g for male and significantly higher than 362.0 ± 48.2 ng/g for female . More intuitive results of comparisons are shown in Hair Se content of males . Except for Bin County , such differences were found within the rest of the counties. In Linyou County, hair Se content was 418.7 ± 42.0 ng/g for 4.0–6.9 years and significantly higher than 391.2 ± 34.2 ng/g for 7.0–14.0 years ; in Ningshan County, it was 270.3 ± 23.7 ng/g for 4.0–6.9 years and significantly higher than 287.4 ± 26.0 ng/g for 7.0–14.0 years ; in Liquan County, it was 387.4 ± 31.7 ng/g for 4.0–6.9 years old, which was significantly higher than 366.7 ± 52.5 ng/g for 7.0–14.0 years old , see In each age group, hair Se content was significantly different among the four counties  which was significantly lower than 38.6 ± 5.4 ng/g in Liquan, the non-endemic County . In Bin County, it significantly increased from 7.8 ± 0.9 ng/g in the mild area, 8.2 ± 0.4 ng/g in the medium area to 11.0 ± 1.3 ng/g in the severe area , see Corn is now neither often consumed in Ningshan nor in Liquan and it accounts for only a small part of the staple food in Linyou and Bin County. Corn Se content of 8.7 ± 4.9 ng/g in Linyou was slightly but significantly lower than 9.1 ± 1.6 ng/g in Bin , see figure 3E. There was no significant difference between each of the measurements in Linyou, Bin and Liquan (p > 0.05) but all of them were significantly lower than Ningshan . Among mild, medium and severe areas, it was 29.1 ± 5.9 µg/day, 30.4 ± 8.0 µg/day and 29.4 ± 7.0 µg/day separately in Linyou , 29.2 ± 6.8 µg/day, 29.3 ± 5.5 µg/day and 29.4 ± 6.4 µg/day separately in Bin , 35.7 ± 7.4 µg/day, 29.8 ± 5.8 µg/day and 33.2 ± 9.3 µg/day separately in Ningshan , see In this study, we can tell that without Se-enriched salt, children’s daily Se intake was 29.6 ± 7.0 µg/day in Linyou, 29.3 ± 6.2 µg/day in Bin, 32.9 ± 7.9 µg/day in Ningshan and 27.8 ± 7.9 µg/day in Liquan , meat mainly livestock and poultry, and milk were included in every county by both methods, details are displayed in in vivo via the food chain. Naturally, various methods of Se supplement such as Se-enriched salt, yeast, egg, tea, milk and other food, oral sodium selenite [KBD could be traced back to the 16th century, and hundreds of years of research have been applied worldwide. It was found to be tightly related to Se-deficiency in the 1970s in China. Environmentally low Se leads to insufficiency selenite , sprayinselenite ,27 had bselenite .Although Se deficiency was not a direct cause of KBD, it was demonstrated to be a major risk factor. Se supplements remarkably improved the pathological changes of KBD and efficiently contributed to the control of the incidence ,28. As tIn this study, hair Se content of children from KBD supplemented counties (Linyou and Bin), KBD non-Se-supplemented county (Ningshan) and non-KBD county (Liquan) were much higher than 200 ng/g which was considered as the critical value of non-endemic level . Gender,et al. [et al. [About the non-dietary factors, hair Se level was significantly higher in male than that in female which was similar with S. Letsiou’s finding  whilst aet al.  and Vero [et al.  pointed  [et al. . This exIn spite of Se-enriched salt still playing a role in maintaining the Se nutrition of children, dietary Se intake without supplements also considerably contributes to the Se nutrition of children. It is well known that Se can only be used after being incorporated into proteins and then being further transformed into selenocysteine, the active form of Se ,34. ProtDisregarding the area division, the food categories majorly contributing to the Se intake without supplement were wheat, rice, livestock and poultry meat, green and solanaceous vegetables, tuber and bulb vegetables, nuts, milk and beverages. Except for the uncertainty of some beverages, Se gained from the rest of the food are mainly in organic form , which has much higher bioavailability and longer half-life than the inorganic form . In addition, the organic form can increase blood Se level more rapidly and to a greater extent than the inorganic form ,34. DietAlthough Se-enriched salt still plays a role in keeping children’s Se nutritional status, the better source of food and improved dietary structure may enhance and maintain the children’s Se nutrition in a better and safer way, which indicates that more attention should be paid to the quality and diversity of food for better comprehensive nutrition of children. Is it now the time to stop applying selenium supplementation in KBD endemic areas in Shaanxi Province, China? Based on the pros and cons, the answer may be yes. However, so far, no cohort study longer than three years has been conducted to demonstrate whether ceasing Se-enriched salt in KBD endemic areas would create no risk of a resurgence of KBD. In addition, no clear evidence could estimate how long it takes to get KBD and there are occasionally a few new suspect cases in Qinghai Province and Tibet (data unshown) in China. Therefore, for safety’s sake, we suggest ceasing the supplement from one county for a start while continuing to monitor the Se status of children and KBD incidence twice a year for at least three years to prevent increase in incidence, and then expand the program to the rest of the endemic areas in or even beyond Shaanxi Province.


Hamigera clade were analysed to assess their correlations to a multi-gene species-level phylogeny. High-pressure-liquid-chromatography-based chemical analysis revealed three distinctive patterns of secondary metabolite production: (1) the nine species could be divided into two groups on the basis of production of the sesquiterpene tricinonoic acid; (2) the tricinonoic acid-producing group produced two cyclic peptides avellanins A and B; (3) the tricinonoic acid-non-producing group could be further divided into two groups according to the production of avellanins A and B. The chemical phenotype was consistent with the phylogeny of the species, although metabolite patterns were not diagnostic at the species level. In addition, the taxonomy of the Hamigera clade was updated with the new combination Hamigera ingelheimensis proposed for Merimbla ingelheimensis, so that all species in the clade are now in the same genus.Secondary metabolite phenotypes in nine species of the  Penicillium, Aspergillus, Talaromyces, Monascus, Paecilomyces and other lesser known genera, e.g., Hamigera. Species of Hamigera are widespread and common soil fungi, and one species is associated with beetles , polyketide synthases (PKS), terpene cyclases and prenyl transferases  separation methods linked to UV and mass spectrometer (MS) detectors to generate profiles of metabolites characteristic of species and species complexes   afforded an opportunity to test the hypothesis that these newly described taxa would contribute significant new variations in secondary metabolism beyond those previously reported compounds. In this report, representative strains of the nine species (one undescribed) were grown under a set of fermentation conditions, extracted with an organic solvent, and their extracts profiled by HPLC-diode array detector (DAD) to detect major UV-absorbing metabolites. We therefore describe the first genus-wide assessment of chemical diversity in 2. 2.1. Mcm7, RPB2 and Tsr1  and incubating flasks for 4-day growth at 30°C with agitation at 200 rpm. Aliquots of inoculum (3 mL) were transferred to 100-mL flasks containing six different production media including A-3M composed of glucose 0.5 g, glycerol 2 g, soluble starch 2 g, Pharmamedia (Archer-Daniels-Midland Co.) 1.5 g and yeast extract 0.3 g, per litre distilled water; super malt composed of malt extract 50 g, yeast extract 10 g, FeSO4·7H2O 2 mg and ZnSO4·7H2O 0.7 mg, per litre distilled water; A-11M composed of glucose 0.5 g, soluble starch 2.5 g, yeast extract 0.5 g, polypeptone 0.5 g, NZ-amine 0.5 g and CaCO3 0.3 g, per litre distilled water; A-16 composed of glucose 2 g, Pharmamedia 1 g and CaCO3 0.5 g, per litre distilled water; MGTY composed of maltose 15 g, glycerol 10 g, tryptone 10 g, yeast extract 10 g, KH2PO4 1 g, MgSO4·7H2O 0.2 g and CaCl2 0.5 g, per litre distilled water; SMY+A composed of maltose 40 g, peptone 10 g, yeast extract 10 g and agar 4 g, per litre distilled water. Production media were incubated 4 days at 30°C with agitation at 200 rpm.Liquid inoculum was grown by inoculating agar plugs from PDA plates into 100 mL of medium V-22  5 g, K2.3. TM C-18 , solvent was MeCN:0.15% KH2PO4 (pH 3.5) with the elution program as follows: 0–3 min (15% MeCN), 3–6 min (15–40% MeCN), 6–12 min (40% MeCN), 12–19 min (40–45% MeCN), 19–22 min (45–85% MeCN), 22–29 min (85% MeCN), 29–32 min (85–15% MeCN) with a flow rate of 1.2 mL/min, monitoring at 254 nm; HPLC condition B: XTerraTM RP18, , solvent was MeCN:0.1% HCO2H at 40:60 ratio with a flow rate of 1.0 mL/min, monitoring at 254 nm.Equal volumes of 1-butanol were added to fermentations, and flasks were agitated for 60 min and then contents were centrifuged to separate fungal cells from the supernatant. A portion (1 mL) of the organic layer was concentrated under reduced pressure to give a crude extract that was then dissolved in dimethlysulfoxide  for HPLC analysis on an Agilent HP1200 system equipped with a photodiode array detector. Spectral data were collected in the range from 200 to 600 nm. A measure of 100 μL of the DMSO solution was injected into an octadecylsilyl (ODS) column: HPLC condition A: Microsorb-MV2.4. The crude culture extract was fractionated by silica gel and ODS column chromatography and was further purified by preparative HPLC. Isolated compounds were subjected to spectroscopic analysis including nuclear magnetic resonance (NMR) and MS for structure determination.3. 3.1. Hamigera ingelheimensis S.W. Peterson, comb. nov., MB no. 807715Penicillium ingelheimensis J.F.H. Beyma, Antonie van Leuuwenhoek 8:109, 1942; MB no. 289090.basionym: Merimbla ingelheimensis (J.F.H. Beyma) J.I. Pitt, Canadian Journal of Botany 57:2395, 1979; MB no. 317567.≡ Raperia ingelheimensis (J.F.H. Beyma) Arx, Mycotaxon 26:121, 1986; MB no. 103783.≡ Holotype IMI 234977M. ingelheimensis and H. avellanea are sister taxa, and concordance analysis of DNA sequences from multiple loci  in A-3M, A-11M and A-16 to establish the fermentation period for chemical phenotype pattern comparison. These strains produced tricinonoic acid and avellanins A and B as the major metabolites (Metabolite production was analysed by HPLC after 4, 6 or 10 days of cultivation with three strains (abolites . Productabolites . TherefoHamigera clade (Nineteen strains from the ra clade  were ferra clade : tricinoH. ingelheimensis did not. The second most common metabolite was tricinonoic acid, a sesquiterpene first isolated from Fusarium tricinctum (Bashyal and Leslie Gunatilaka Hamigera or Merimbla. The production pattern of this terpene compound divided nine Hamigera species into two groups. The producer group contains H. insecticola, H. inflata, H. terricola, H. pallida and H. fusca, and the non-producer group containing Hamigera sp., H. avellanea, H. ingelheimensis and H. paravellanea. Interestingly, all three H. ingelheimensis strains produced neither avellanins A and B or tricinonoic acid. Avellanins A and B, first reported from H. avellanea, consist of five amino acid components: alanine, phenylalanine, proline and anthranillic acid as common monomers and isoleucine for avellanin A and valine for avellanin B. PF1171B, the leucine version of the pentapeptide avellanin A was produced only by three species, H. avellanea, H. paravellanea and H. fusca. PF1171A and PF1171C are the cyclic hexapeptides first isolated from H. avellanea. The major difference of these peptides from avellanins is the substitution of pipecolic acid for proline. Production of PF1171C was limited to three species, H. ingelheimensis, H. paravellanea and H. fusca. H. paravellanea NRRL35714 produced another hexapeptide PF1171A and a new pentapeptide avellanin C in which the isoleucine residue of avellanin A was replaced by phenylalanine (H. ingelheimensis NRRL29060 and was the only compound detected from this strain (max spectral profile with the absorption maximum around 445 nm (H. paravellanea NRRL35714 although their structures were not fully elucidated yet. Strain NRRL35714 was the most productive strain tested in this study, which produced the full range of cyclic pentapeptides and hexapeptides and aromatic polyketides.The most commonly produced metabolites were avellanins A and B . Fifteenlalanine . Detailss strain . Productd 445 nm  was notiHamigera species to date; therefore, data on genome size and numbers and distribution of secondary metabolite gene clusters in these fungi remain unknown. Based on secondary metabolome studies of other species of the Eurotiales, it is likely that we have only sampled a portion of the secondary metabolites encoded in the genomes of these fungi. Our assessment is preliminary because we have not exhaustively probed media and fermentation techniques for stimulating metabolite expression, and our primary detection method was based on UV absorbance. Perhaps, mass-based detection methods or sensitive biological assays would have revealed additional metabolite complexity.Genomic sequencing has not been completed for any H. terricola, H. pallida, H. fusca, H. inflata and H. insecticola (Hamigera. This pattern could be explained by presence of their pathways in the most recent common ancestor followed by loss of function in some descendent species or simply lack of expression in some of the strains and species during our study. Most studies (e.g. Frisvad and Samson Hamigera also. Statistically convincing chemotaxonomy must await a more thorough mining of the secondary metabolites produced by the species and isolates and use of more chemical markers.Our data did not provide species-level diagnostic patterns. Nonetheless, the major metabolites reported here were consistent with the phylogeny of the species; tricinonoic acid production was a definitive character for the clade containing ecticola . Other m


The deposition and distribution of body fat play a vital role in the quality of ruminant carcasses and are of great commercial value since they influence the profitability and consumer acceptability of ruminant meat. The current study examined the effects of dietary blend of 80 % canola oil and 20 % palm oil (BCPO) on carcass characteristics, meat yield and accretion of fatty acid (FA) in subcutaneous, omental, perirenal, and mesentery adipose depots and Twenty four Boer crossbred bucks (BW 20.54 ± 0.47 kg) were randomly assigned to diets containing on DM basis 0, 4 and 8 % BCPO, fed for 100 d and harvested.P > 0.05) on slaughter weight, dressing percentage, carcass and non-carcass components, meat yield, color, moisture and carotenoid contents and weight of adipose tissues in goats. The proportion of C18:1n-9 and cis-9 trans-11 CLA in the omental, perirenal and SS was higher (P < 0.05) in goats fed 4 and 8 % BCPO compared with the control goats. Dietary BCPO reduced (P < 0.05) the proportion of C14:0 in the omental, perirenal and mesentery depots, C18:0 in the perirenal depot, C16:0 in the SS and C16:1n-7 in the SS, omental and perirenal tissues. Dietary BCPO enhanced the proportion of C18:1 trans-11 Vaccenic and C18:3n-3 in SS and C20:5n-3 in SS and mesentery depot. No significant changes were found in the FA composition of subcutaneous depot.Diet had no effect (Results indicate that dietary BCPO can be utilized to alter the FA composition of adipose tissues without detrimental effects on carcass characteristics in goats. Nonetheless, dietary BCPO is not an effective repartitioning agent for body fats in goats. Dietary fat is a popular means of increasing the energy density of diet in ruminant nutrition and can influence adipose depots and carcass composition –3. DietaGoats deposit more visceral fat and less subcutaneous, inter and intra muscular fat compared with sheep and cattle , 8. The trans-vaccenic acid  and it has been shown that the blend of 80 % canola oil and 20 % palm oil (BCPO) had no detrimental effect on in vitro [in vivo rumen fermentation and growth performance in goats [supraspinatus muscle in goats.Although, there are large discrepancies, dietary fat can influence deposition of body fats and fatty acid composition of adipose tissue in ruminants , 3, 10. nic acid , 14. TheC18:3n-3 , 14. ThuC18:3n-3 . Canola C18:3n-3 . Palm oiC18:3n-3 . Given tin vitro  and in vThe study was conducted following the guidelines of research policy of the Universiti Putra Malaysia on animal welfare and ethics.Twenty four Boer bucks  were used in the study. Each animal was individually housed in wooden slatted floor pens (1.20 m × 0.80 m × 0.70 m) equipped with feeding and drinking facilities. The animals were randomly assigned to dietary treatments containing on dry matter basis 0, 4 or 8 % BCPO and fed for 100 d following two weeks of adaptation. All diets were formulated to meet the nutritional requirements of growing goats based on NRC  recommenomentum majus), perirenal fat was sampled near the kidney on its great curve , and mesentery fat was sampled between the ileum and jejunum while pelvic fat was sampled at the left pelvic region. The Supraprinatus muscle was sampled from the right forelimb. The weight of gut fill was estimated by subtracting the weight of empty gut from the weight of full gut. Empty body weight was estimated as the difference between the slaughter weight and weight of gut fill. Dressing percentage (based on full live body weight and empty body weight) was estimated as described by Kadim et al. [Longissimus muscle area (rib eye area) was measured between the 12th and 13th ribs using tracing paper and estimated using two-dimension polygons area calculator software as described by Branscome and Jesseman [The animals were fasted for 12 h with free access to water, weighed and slaughtered in a commercial abattoir according to the Halal procedure as outlined in MS1500:2009 . After sm et al. . DressedJesseman . CarcassJesseman  and LaviJesseman . These mJesseman . SaleablJesseman .The color coordinates of adipose tissues were measured with a ColorFlex spectrophotometer  based on the International Commission on Illumination (CIE) Lab-values  with D65 illuminant and 10° standard observer. The device was calibrated against black and white reference tiles prior to use. The moisture content of the adipose tissue was measured in accordance to the method of AOAC .Total carotenoid in feed and adipose tissues was determined in accordance to the method described by Okonkwo . One gra3) in accordance with the method of AOAC [The total lipids in feed and tissue samples was extracted in chloroform:methanol  mixture following the method of Folch et al.  modified of AOAC . Heneico of AOAC  as folloThe experiment followed a completely randomized design. Data obtained for all parameters were subjected to the generalized linear model (GLM) procedure of SAS . SlaughtElaeis guinensis) tree and constitute one of the main by-products of the oil palm industry in Malaysia [The ingredients and chemical composition of the experimental diets are shown in Table Malaysia . The OPFMalaysia , sheep [Malaysia , 37 and Malaysia , 38, 39.Malaysia . As expeP > 0.05) live, empty body and carcass weights, dressing percentage and meat yield in goats. Similarly, the rib eye area and other carcass linear measurements (Table P > 0.05). The back fat thickness, intramuscular fat  by diets. The carotenoid and moisture contents and color coordinates of all adipose tissues (Table P > 0.05) across dietary treatments.The carcass traits of goats fed varying levels of BCPO are presented in Table ts Table , non-carts Table  and propat Table  of suprats Table  were notes Table  were simSupraspinatus (SS) muscle from goats fed varying level of BCPO is shown in Table P < 0.05) while that of C18:1n-9, C18:1 trans-11, CLA cis-9 trans-11, C18:3n-3 and C20:5n-3n increased (P < 0.05) as the level of BCPO increased in diet. The proportion of C18:0, C18:2n-6 and C20:4n-6 was not influenced (P > 0.05) by diet. The total saturated fatty acids (SFA), the n-6/n-3 ratio and ID 16 decreased (P < 0.05) while the total monounsaturated fatty acids (MUFA), PUFA/SFA ratio, ID 18 and elongation index increased (P < 0.05) with increase in the level of BCPO in diet.The FA composition of The FA composition of omental, perirenal, mesentery and subcutaneous adipose tissues from goats fed graded levels of BCPO is shown in Tables trans-11 decreased (P < 0.05) as the level of BCPO increased in diet. Goats fed 4 and 8 % BCPO had similar (P > 0.05) proportion of cis-9 trans-11 CLA which was greater (P < 0.05) than those fed the control diet. The proportion of C18:3n-3 was greater (P < 0.05) in goats fed 4 % BCPO compared with those fed 0 and 8 % BCPO. The proportion of C20:5n-3 increased (P < 0.05) while the n-6/n-3 decreased (P < 0.05) with increasing level of BCPO in diet. Proportions of C16:0, C18:0, C18:2n-6, C20:4n-6, trans-10 cis-12 CLA, C22:5n-3 and C22:6n-3, total FA, sums and ratios of FA and the estimated elongase and desaturase indices were not affected (P > 0.05) by diet.In the omental depot, the proportion of C14:0, C16:1n-7 and C18:1 P < 0.05) as the level of BCPO increased in diet. The proportion of C18:1n-9 and CLA cis-9 trans-11, total MUFA and UFA:SFA increased (P < 0.05) with increasing level of BCPO in diet. The control goats had higher (P < 0.05) ID 16 compared to those fed 4 and 8 % BCPO. The ID 18 increased linearly (P < 0.05) as the level of BCPO increased in diet. No significant (P > 0.05) changes were observed for total FA, proportion of C16:0, C18:2n-6, C18:3n-3, C20:4n-6, C22:5n-3, C22:6n-3 and trans-10 cis-12 CLA and the estimated elongase activity across the treatments.In the perirenal depot, the proportion of C14:0, C16:1n-7, C18:0 and total SFA decreased (P < 0.05) as the level of BCPO increased in diet. The proportion of C18:1n-9 in goats fed 8 % BCPO was significantly higher (P < 0.05) compared with those fed 0 and 4 % BCPO. Goats fed 4 and 8 % BCPO had greater (P < 0.05) proportion of C20:5n-3 than the control goats. No significant differences (P > 0.05) were observed between diets with respect to the proportions of C16:0, C16:1n-7, C18:0, C18:2n-6, C20:4n-6, C18:3n-3, C22:5n-3, C22:6n-3, C18:1 trans-11, cis-9 trans-11 CLA and trans-10 cis-12 CLA and total FA. Similarly, estimated elongase and desaturase indices were not affected (P > 0.05) by diet.In the mesentery depot, the proportion of C14:0 decreased (P > 0.05) influencing the proportions of all FA, sums and ratios of FA and the estimated elongase and desaturase indices. However, the proportions of C14:0 and C16:1n-7 tended (P = 0.07) to decrease while that of C22:5n-3 tended (P = 0.07) to increase with increase in the level of BCPO in diet.In the subcutaneous depot, dietary BCPO was not a significant source of variation  for healthy diet . Dietarytrans-11 and total MUFA in perirenal fat. In omental fat, dietary BCPO increased the concentration of CLA cis-9 trans-11, C20:5n-3 and total PUFA. In the mesentery fat, goats fed 8 % BCPO had higher C18:1n-9 and Δ 9 desaturase activity (ID 18) compared to other diets. Also, BCPO caused a linear increase in the proportion of C20:5n-3. The current observation is consistent with those of Stanford et al. [The major fatty acids in all adipose tissues were C16:0, C18:0 and C18:1n-9. This observation is in line with those of Bas et al. . The decd et al.  who obsed et al. .Contrary to the findings in perirenal, mesentery and omental adipose tissues, dietary BCPO did not affect the FA composition of subcutaneous adipose tissue. This finding concurs with those of Potkański et al.  who obsesupraspinatus muscle, mesentery, omental and perirenal adipose tissues in goats.The results of this study showed that dietary supplementation of blend of 80 % canola oil and 20 % palm oil in diet did not affect carcass traits, fat color, and fat deposition and distribution in goats. Thus, dietary BCPO is not an effective repartitioning agent for fat in goats. However, BCPO altered the fatty acid composition of


Background. The anaerobic gut fungi  represent a promising source of novel lignocellulolytic enzymes. Here, we report on the cloning, expression, and characterization of a glycoside hydrolase family 39 (GH39) enzyme (Bgxg1) that is highly transcribed by the anaerobic fungus Orpinomycessp. strain C1A under different growth conditions. This represents the first study of a GH39-family enzyme from the anaerobic fungi.Methods. Using enzyme activity assays, we performed a biochemical characterization of Bgxg1 on a variety of substrates over a wide range of pH and temperature values to identify the optimal enzyme conditions and the specificity of the enzyme. In addition, substrate competition studies and comparative modeling efforts were completed.Results. Contrary to the narrow range of activities (β-xylosidase or α-L-iduronidase) observed in previously characterized GH39 enzymes, Bgxg1 is unique in that it is multifunctional, exhibiting strong β-xylosidase, β-glucosidase, β-galactosidase activities  and a weak xylanase activity (10.8 ± 1.25 U/mg), as compared to previously characterized enzymes. Further, Bgxg1 possesses extremely high affinity , compared to all previously characterized β-glucosidases, β-galactosidases, and xylanases. Physiological characterization revealed that Bgxg1 is active over a wide range of pH  and temperatures , and possesses excellent temperature and thermal stability. Substrate competition assays suggest that all observed activities occur at a single active site. Using comparative modeling and bioinformatics approaches, we putatively identified ten amino acid differences between Bgxg1 and previously biochemically characterized GH39 β-xylosidases that we speculate could impact active site architecture, size, charge, and/or polarity.Discussion. Collectively, the unique capabilities and multi-functionality of Bgxg1 render it an excellent candidate for inclusion in enzyme cocktails mediating cellulose and hemicellulose saccharification from lignocellulosic biomass. The production of biofuels from lignocellulosic biomass is a global priority, necessitated by the continuous depletion of recoverable fossil fuel reserves, the deleterious impact of fossil fuels on air quality, as well as their contribution to global climate change . Lignoceβ-1,4-linked xylose units  signify essential thrusts in biofuel research. Members of the anaerobic gut fungi  represent a promising, and largely untapped, source of biomass-degrading enzymes . Membersergillus , Trichodchoderma ), anaerostridium  and Theracterium ) and metacterium ; in compacterium .Orpinomyces sp. strain C1A (henceforth referred to as C1A) as a novel source of lignocellulolytic enzymes. C1A is an isolate from the feces of an angus steer on cellobiose-switchgrass media  was annotated as member of the GH39 CAZyme family based on the presence of the conserved protein domain pfam01229 (Glyco_hydro_39) family. When strain C1A was grown on different substrates , m.21910 constituted 58–84% of the transcriptional activity  of all GH39 transcripts (n = 9), and 5.7–18.2% of the transcriptional activities of all C1A genes putatively annotated as β-xylosidases  . The ctg7180000059688.1 gene consists of 1,115 bp and no introns . The protein product is predicted to be extracellular and non-cellulosomal, based on the presence of a signal peptide, and the absence of a CBM fungal dockerin domain, respectively.As a part of an extensive transcriptomic analysis of lignocellulosic biomass degradation by the anaerobic fungal isolate rain C1A , the mos n = 41) . The genβ-xylosidase sequences available in CAZY database  , in addition to Bgxg1, were aligned using Clustal Omega  plasmid . The plasmid, pET28a(+)-bgxg1, harbors kanamycin resistance (kan) and NdeI and XhoI restriction sites for selection and cloning. The pET28a(+)-bgxg1 plasmid was first transformed into One-Shot Chemically Competent Top10 E. coli cells , and the transformants were grown overnight on LB-kanamycin agar (15 µg/mL) for selection. The purified plasmid was electroporated into a protease-deficient BL21(DE3)pLysS E. coli strain , possessing an additional chloramphenicol resistance (cm) marker, using a single pulse of 1.8 kV in 0.1 cm electrocuvettes. Transformants were grown on LB agar using both kanamycin (15 µg/mL) and chloramphenicol (34 µg/mL) for selection and screened for the presence of correctly sized inserts via colony PCR using T7 forward and reverse primers.A fraction  of m.21910 transcript was codon optimized for ideal expression in E. coli cells transformed with pET28a(+)-bgxg1 were used to inoculate 1 L LB broth, containing kanamycin (15 µg/mL) and chloramphenicol (34 µg/mL). The culture was incubated at 37 °C with shaking at 200 rpm until an OD600 = 0.6 was reached. Isopropyl-β-D-thiogalactopyranoside  was then added to induce protein production, and the culture was gently shaken at room temperature overnight. Cells were then pelleted by centrifugation  and the pellets were collected and stored at −20 °C.Ten milliliters of overnight cultures of BL21(DE3)pLysS Preliminary small-scale experiments indicated that the protein is expressed in the inclusion body fraction . Inclusiβ-mercaptoethanol  for 2 h at room temperature with gentle shaking, transferred to dialysis tubing , and placed for 3 h into inclusion body exchange buffer  for removal of the β-mercaptoethanol. The buffer was refreshed and dialyzed for an additional 3 h. The dialysis tubing was then placed into a low-urea refolding buffer  and dialyzed overnight, followed by a no-urea refolding buffer  for 36 h.Recombinant protein refolding was achieved using slow dialysis as previously described . In brieFollowing dialysis, the contents of the tubing were centrifuged to remove insoluble, precipitated proteins . The supernatant, containing refolded soluble protein, was then exposed to a nickel-nitriloacetic acid  slurry , packed in a glass frit column , and allowed to incubate at 4 °C for 1 h on an orbital shaker. Protein purification followed as detailed previously . Samplesβ-xylosidase activity (described below) at pH 3, 4, 5, 6, 7, 8, 9, and 10, using the following buffer systems: sodium acetate buffer (pH 3.0–6.0), sodium phosphate buffer (pH 7.0–8.0), and glycine buffer (pH 9.0–10). Similarly, the temperature range and subsequent thermal optimum for Bgxg1 was determined by assaying its β-xylosidase activity at 25, 30, 39, 50, and 60 °C. In a second and separate study, the stability of Bgxg1 after exposure to pH extremes was determined by assaying its β-xylosidase activity following a one-hour incubation at pH 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, and 13 at 4 °C. The following pH buffering systems were used for pH adjustment: sodium acetate buffer (pH 3.0–6.0), sodium phosphate buffer (pH 7.0–8.0), glycine buffer (pH 9.0–10), sodium bicarbonate (pH 11.0), and KCl–NaOH (pH 12–13). Similarly, in a separate study, the thermal stability of Bgxg1 was determined by assaying its β-xylosidase activity following a one-hour incubation at 4, 25, 30, 37, 39, 50, 60, and 70 °C. In all cases, 2.2 µg of pure Bgxg1 was used, since this concentration was determined to be optimal in initial testing. Following the one-hour long exposure at the above-described extremes, enzymatic activity was tested at 39 °C and pH 6.0, the optimal conditions as determined for this enzyme. All experiments were completed in triplicate, and relative specific activities in relation to the best performing condition (100% activity) were reported.The pH range and subsequent pH optimum for Bgxg1 was determined by assaying its All enzyme assays with Bgxg1 were conducted in pH 6.0 buffer and at 39 °C, as these conditions were determined to be optimal for Bgxg1. All reagents were purchased from Sigma Aldrich  unless noted otherwise.Endoglucanase, exoglucanase, xylanase, and mannanase activities were determined using a DNS -based assay , with caβ-xylosidase, arabinosidase, mannosidase, β-glucosidase, β-galactosidase, and acetyl xylan esterase activities were determined using (10 mM) of the p-nitrophenol-based (PNP) substrates: p-nitrophenyl-β-d-cellobioside (PNPC), p-nitrophenyl-β-d-xylopyranoside (PNPX), p-nitrophenyl-β-d-arabinofuranoside (PNPA) p-nitrophenyl-β-d-mannoside (PNPM), p-nitrophenyl-β-d-glucopyranoside (PNPG), p-nitrophenyl-β-d-galactopyranoside , and p-nitrophenyl-acetate (PNPAc), respectively .Cellobiohydrolase, ectively . Assays U) was defined as one µmol of products  released from the substrate per minute. Specific activity was calculated by determining the units released per mg of enzyme.All experiments were conducted in triplicate. One unit of enzymatic activity .Standard procedures were used to determine the ed above . Km and β-xylosidase activity of Bgxg1 was measured by conducting the β-xylosidase assay, using 10 mM of PNPX as the substrate, in the presence of different concentrations of cellobiose  and evaluating the impact of cellobiose presence on the release of PNP. Conversely, the effect of xylobiose (as a competitive inhibitor) on the β-glucosidase activity of Bgxg1 was measured by conducting the β-glucosidase assay (using 10 mM of PNPG as the substrate) in the presence of different concentrations of xylobiose , and evaluating the impact of xylobiose presence on the release of PNP. In both experiments, the effect of inhibitor concentration on Km and Vmax was evaluated using Competitive inhibition experiments were conducted to determine whether the observed multiple oligosaccharide hydrolase activities are catalyzed via a single or multiple active sites. In such experiments, the effect of cellobiose (as a competitive inhibitor) on the Substrate preferences of Bgxg1 were determined by conducting a substrate competition assay, where Bgxg1 (2.2 µg of pure enzyme preparation) was challenged by a mixture of xylobiose (10 mM) and cellobiose (10 mM). The kinetics of xylose and glucose release were compared to the results obtained in control experiments where only one substrate (xylobiose or cellobiose) was utilized. Samples were taken at 0, 1, 5, 10, 15, 30, and 60 min for the determination of the glucose and xylose concentrations. Glucose was assayed using PGO Enzyme Preparation Capsules  and xylose was assayed using Megazyme Xylose Kit . All experiments were conducted in triplicate.Thermoanaerobacterium saccharolyticumβ-xylosidase (PBD entry 1UHV) as a template. PyMOL was used to align the Bgxg1 structural prediction to that of Thermoanaerobacterium saccharolyticum (PBD entry 1UHV) to examine and speculate the impact of variations in amino acids residue on the enzyme’s active site topology and putative substrate binding capacities (Homology modeling by Iterative Threading ASSEmbly Refinement (I-TASSER) , was conpacities .Orpinomyces sp. strain C1A Bgxg1 protein belonged to Class III, forming a well-supported cluster with GH39 proteins from the anaerobic fungus Piromyces sp. strain E2, as well as GH39 proteins from the bacterial genera Clostridium and Teredinibacter (70–74% sequence identities)  . To our SDS-PAGE results show that the Bgxg1 protein is consistent with the predicted size of 42.7 kDa (protein predicted molecular weight is 39.6 KDa + 0.996 kDa linker + 2.101 kDa double histidine tag) .p-value > 0.05, 95% confidence interval, The thermal and pH ranges and optima were determined by conducting assays at a range of temperatures and pH’s, as described above. Bgxg1 exhibited activity in a wide range of pH (3–8) and temperatures (25–60 °C), with optimal activity at pH 6 and 39 °C  and 2B. β-xylosidase or α-L-iduronidase) , β-galactosidase (54.6 ± 2.26 U/mg), and weak xylanase (10.8 ± 1.25 U/mg) activities  . As predtivities . While h16 U/mg, , Neocall28 U/mg, , and Sph27 U/mg,   in the low nM range for PNPG and PNPGal, the low µM range for PNPX . This pattern strongly indicates that a single active site is responsible for the observed activities (Substrate competition studies were conducted using a variable concentration of an unlabeled substrate (acting as an inhibitor) and a fixed concentration of a chromophore (PNP-based) substrate . The restivities , a conclKm value) of Bgxg1 for PNPG (12.5 nM) over PNPX (4.85 µM)  .β-xylosidase originating from Thermoanaerobacterium saccharolyticum (PBD entry 1UHV) as a template for model creation (α∕β)8 barrel fold domain (position 26–307), a small α-helical domain (position 1–25), and a β sandwich domain (position 308–344) (β-sheets (8 in (α∕β)8-barrel, 3 in β-sandwich), and 10 α-helices (8 in (α∕β)8-barrel, 2 in α-domain). The catalytic (α∕β)8-barrel fold domain is predicted to consist of eight parallel β-sheets (β1–β8), and eight parallel α-helices (α1–α8). Consistent with β-xylosidases of Thermoanaerobacterium saccharolyticum (1UHV) and Geobacillus stearothermophilus (1PX8), the active site pocket of Bgxg1 is predicted to be located on the upper side of the (α∕β)8-barrel  . Overall8-barrel  , or to the entire Class III  changes  within citutions . Howeveror III-D . CollectOrpinomyces sp. strain C1A. Our results demonstrate that the expressed protein is multifunctional, possessing strong β-xylosidase (11.5 U/mg), β-glucosidase (73.4 U/mg), and β-galactosidase (54.6 U/mg) activities, as well as a weak xylanase activity (10.8 U/mg)  from the anaerobic gut fungus .8 U/mg)  and 2, a.8 U/mg) –S4. This.8 U/mg) , and the.8 U/mg)  and pH v.8 U/mg) . Though .8 U/mg) . It is iKm and Vmax values calculated via extrapolation through Lineweaver–Burke plot). As a β-xylosidase, Bgxg1 has one of the highest β-xylosidase specific activity among all reported ambient (<50 °C) β-xylosidases, but lower than other anaerobic fungi Neocallimastix frontalis towards its multiple substrates , regardand GH30 . Finallyand GH42 ) regardland GH42 .Orpinomyces sp. strain C1A was isolated from the feces of an angus steer . The hius steer . We hencα∕β)8-barrel fold structure typically observed in GH39-family enzymes    and S5 iletions) . The impletions) . GH39 enstigated . Such st bicolor ) interac bicolor . On the mediates , the preβ-xylosidases remain to be conclusively determined. Phylogenetic analysis demonstrated the occurrence of nine out of ten amino acids substitutions/deletions in all sequenced members of Class III-C, residues which we speculate to be of importance to the observed multi-functionality of Bgxg1, but as Bgxg1 is the only biochemically-characterized enzyme within Class III, this analysis is purely speculative (β-xylosidases contain sequences from the genera Clostridium and Teredinibacter (β-xylosidases, has been acquired from bacteria via horizontal gene transfer (β-xylosidases within the domain Bacteria, prior to the acquisition of GH39 β-xylosidases by the anaerobic fungi and that the acquired capability is speculated to be retained in all anaerobic fungal GH39 β-xylosidases.The ecological relevance, global distribution, and evolutionary patterns of multi-functionality within GH39 culative . In addinibacter . Since itransfer , and speβ-xylosidase that represents the first GH39-family enzyme cloned and expressed from anaerobic fungi. The enzyme is multi-functional, capable of hydrolyzing cellobiose, xylobiose, as well as several PNP-glycosides. It also displays high affinity towards various substrates, retains activity over a wide range of temperatures and pHs, and possesses excellent temperature and thermal stability. Structurally predictive modeling identified putative differences which potentially could account for the observed relaxed specificity. Collectively, these capabilities render Bgxg1 an excellent candidate for inclusion in enzyme cocktails mediating cellulose and hemicellulose saccharification from lignocellulosic biomass (In conclusion, we have characterized a novel  biomass .10.7717/peerj.2289/supp-1Supplemental Information 1Click here for additional data file.10.7717/peerj.2289/supp-2Data S1Click here for additional data file.


These findings indicated the D-ribose production performance was significantly improved compared to that under original conditions.D-Ribose is a five-carbon sugar and generally used as an energy source to improve athletic performance and the ability. The culture conditions for maximum D-ribose production performance from cheap raw material corn starch hydrolysate were improved by using one-factor-at-a-time experiments and a three-level Box-Behnken factorial design. The optimal fermentation parameters were obtained as 36°C culture temperature, 10% inoculum volume, and 7.0 initial pH. The mathematical model was then developed to show the effect of each medium composition and their interactions on the production of D-ribose and estimated that the optimized D-ribose production performance with the concentration of 62.13 g/L, yield of 0.40 g/g, and volumetric productivity of 0.86 g/L·h could be obtained when the medium compositions were set as 157 g/L glucose, 21 g/L corn steep liquor, 3.2 g/L (NH D-Ribose has been used to improve athletic performance/ability as an energy source [2), animal feed additives, cosmetics and foods [D-Ribose C5H0O5 is a nd foods . Bacillus are the main D-ribose producers including B. subtilis and B. pumilus. Most of those strains, however, restricted their usefulness for commercial production due to certain disadvantages such as long fermentation time and lower ribose concentration and productivity. For example, about 40 g/L of D-ribose was produced from 200 g/L of glucose by Bacillus subtilis ATCC 21951 after 7-d fermentation [ Bacillus strains deficient in the transketolase and/or D-ribulose-5-phosphate-3-epimerase have the ability to accumulate ribose in the fermented broth since these enzymes will further catalyze the produced ribose to the aromatic amino acids [ Bacillus stains with industrial application potency [ Bacillus subtilis B941, was screened to produce 41.8 g/L of D-ribose from 180 g/L of glucose. After mutation with UV irradiation, the mutant strain Bacillus subtilis Buvp-24 produced the maximum D-ribose concentration of 55 g/L [Two methods including yeast RNA hydrolysis and chemical synthesis from gluconic acids, glucose, arabinose, and xylose are previously used for preparing D-ribose , 5. For no acids . Our gro potency –10. One Starchy biomass is a promising feedstock for chemical bioproduction due to its abundant availability. Commercial scale for converting starchy biomass to chemicals such as ethanol, lactic acid, and 2-keto-gluconic acid has been realized . Corn st Bacillus subtilis UJS0717 using one-factor-at-a-time experiments followed by a three-level Box-Behnken factorial design combining with response surface methodology (RSM).Statistical techniques have been used for optimizing the culture media to produce microbial metabolites –16. Resp B. subtilis UJS0717 was a mutant from Industrial Microbiology Laboratory in Shanxi Institute of Biology and maintained at 4°C in ampoule tube and kept in our laboratory. Stock medium contained 5 g/L of D-sorbitol, 10 g/L of peptone, 2 g/L of NaCl, 2 g of yeast extract, and 20 g/L of agar. A loopful of the stock culture was diluted with sterilized water and inoculated into 20 mL of seed medium containing glucose 20 g/L, yeast extract 3 g/L, K2HPO4 3 g/L, and KH2PO4 1 g/L, in a 250 mL Erlenmeyer flask, incubated at 36°C, 240 rpm, for 20 h, and used as a seed culture.The D-ribose producer4)2SO4 7.5 g/L, yeast extract 1 g/L, and MnSO4·H2O 0.05 g/L. Glucose and other nutrients were sterilized separately at 121°C for 20 min. 20.0 g/L of CaCO3 was added to the media for balancing the broth pH.Corn starch hydrolysate (CSH) was obtained from Shandong Depu Chemical Technology Co., Ltd. , produced by liquefaction and saccharification processes with amylases and glucoamylase. The corn starch hydrolysate contains approximately 30% (w/v) of glucose and 0.6% of protein . CSH was diluted with deionized water to obtain various concentrations of glucose. Other analytical chemicals were obtained from Sigma Aldrich . The basic fermentation medium contained glucose 120 g/L, corn steep liquor 15 g/L, (NH4)2SO4 concentration on D-ribose production, the glucose, corn steep liquor, and (NH4)2SO4 concentrations in the basic fermentation medium were adjusted ranging from 100 g/L to 210 g/L, 5 g/L to 25 g/L, and 1.5 g/L to 9 g/L, respectively. Four fermentation temperatures from 30°C to 39°C, four inoculum volumes from 5% to 20% (v/v), and five initial pH values from 6.0 to 8.0 were used for selecting the optimal temperature, inoculum volume, and initial pH using one-factor-at-a-time experimental design. The fermentation time was 72 hours if not further specified.For one-factor-at-a-time experiments investigating effect of glucose, corn steep liquor, and (NH4)2SO4 significantly influencing D-ribose production, Box-Behnken design for 3 variables at three levels  was further employed to optimize their concentrations to maximize the D-ribose production by B. subtilis UJS0717 based on the one-factor-at-a-time experiments [To find the interactions and give the precise levels of media compositions including glucose, corn steep liquor, and  and the medium component contents in second-order equation, was developed with the least squares method as follows:ficients . The acc3 with 1 M HCl solution, centrifuging at 10000 ×g for 5 min to obtain B. subtilis cells, and drying the cells at 80°C to the constant weight.The cell growth was represented with dry cell weight  by neutralizing the residual CaCO2SO4 at a flow rate of 0.6 mL/min. The column temperature was maintained at 50°C. About 3 mL samples were taken and filtered with Whatman 0.45 μm syringe filter to obtain about 1 mL permeate for HPLC analysis.Glucose and D-ribose were measured using high performance liquid chromatography  equipped with a SUGAR SH1011 column  and a differential refractometer (Agilent 1100 series). The mobile phase was 0.005 M HThe performance of D-ribose production was evaluated based on D-ribose concentration, D-ribose productivity, glucose conversion ratio, and D-ribose yield. D-Ribose productivity was defined as the amount of D-ribose produced per hour liter. D-Ribose yield was calculated by dividing the amount of D-ribose produced by the amount of glucose consumed. All fermentation tests were run in duplicate. Data analysis including analysis of variance was conducted using the SAS System .P < 0.05). The maximum D-ribose productivity of 0.50 g/L·h, glucose utilization ratio of 97.05%, and highest cell concentration of 8.70 g/L were obtained at 36°C after 72 h of fermentation. D-Ribose productivity of 0.44 g/L·h and 0.48 g/L·h and glucose utilization ratio of 90.49% and 93.75% were obtained at 33°C and 39°C, respectively. At 30°C, the ribose productivity and glucose utilization ratio decreased to 0.42 g/L·h and 89.04%, respectively. Kishimoto et al. (1990) found that temperature about 37°C was suitable for D-ribose production by B. pumilus NO.716 [ B. subtilis UJS0717.In order to understand the influence of temperature on the production of D-ribose, fermentation with an initial concentration of 10% (v/v), pH 7.0, and basic fermentation medium was conducted at four temperatures ranging from 30°C to 39°C . D-Riboss NO.716 . TherefoP < 0.05). Further increases in inoculum volume (beyond 10%) had no significant effect on the D-ribose production (P > 0.05). Similar results were also observed by Ren et al. that high inoculum volume resulted in a negative impact on D-ribose production by B. subtilis ptn15-1 [The influence of inoculum volume, ranging from 5 to 20% (v/v), on D-ribose production with conditions of 36°C, pH 7.0, and basic fermentation medium was investigated. As shown in  ptn15-1 . For the B. subtilis UJS0717 was investigated at 36°C, inoculum volume of 10% (v/v) with basic fermentation medium. As shown in P < 0.05). Further increases in initial pH (beyond 7.0) had a negative impact on D-ribose production. Park and Seo also found that initial pH about 7.0 was suitable for D-ribose production by B. subtilis JY1 [ B. subtilis UJS0717.The effect of initial pH, ranging from 6.0 to 8.0, on the performance of D-ribose production byilis JY1 . Therefo B. subtilis UJS0717 was investigated at 36°C, pH 7.0, and inoculum volume of 10% (v/v). As shown in Figures The effect of glucose concentration ranging from 100 g/L to 210 g/L on the performance of D-ribose production by B. subtilis cells were in the exponential phase with the lower D-ribose production in the 48 h of fermentation and then entered the stationary phase at the 48–96 h with the higher D-ribose production. Glucose concentration of 150 g/L benefited the cell growth and reached maximum concentration of 10.59 g/L.Similar trends of cell growth could be observed . B. subt B. subtilis UJS0717 during the overall fermentation process. B. subtilis UJS0717 gave the total D-ribose yield of 0.32 g/g with the medium composed of 150 g/L glucose, followed by 0.30 g/g with 120 g/L and 0.24 g/g with 180 g/L of glucose. High concentrations of glucose (over 180 g/L) had the lower D-ribose yield of 0.17 g/g. The highest volumetric productivity (0.67 g/L·h) was reached at 72 h with the 150 g/L of glucose. With 100, 120, 180, and 210 g/L of glucose, their productivity reached 0.37 g/L·h, 0.49 g/L·h, 0.57 g/L·h, and 0.35 g/L·h at 48 h, 48 h, 72 h, and 72 h, respectively. Therefore, glucose concentration of about 150 g/L appeared to be optimal for D-ribose production by B. subtilis UJS0717.Figures Corn steep liquor is a major byproduct of the corn wet milling industry and is a low-cost nutrient source available on a large scale . It is t4)2SO4 have been shown to be useful for large-scale D-ribose production [4)2SO4 concentration on the production of D-ribose, fermentation with 36°C, pH 7.0, inoculum volume of 10% (v/v), glucose concentration of about 150 g/L, and corn steep liquor of 20 g/L was conducted at six (NH4)2SO4 concentrations ranging from 1.5 g/L to 9.0 g/L (Nitrogen substrates such as (NHoduction . In orde 9.0 g/L .4)2SO4 concentrations (P < 0.05). The maximum D-ribose productivity and yield of 0.84 g/L·h and 0.40 g/g were obtained at (NH4)2SO4 concentration of 3.0 g/L. After 72 h fermentation, cell concentrations increased from 9.40 g/L to 10.77 g/L with the increase of (NH4)2SO4 concentration from 1.5 g/L to 3.0 g/L. Higher (NH4)2SO4 concentrations (>4.5 g/L) resulted in the residual glucose. With (NH4)2SO4 concentration of 6.0 g/L, D-ribose production reached the minimum level of 42.89 g/L with the lowest productivity of 0.60 g/L·h and yield of 0.29 g/g. Too high levels of (NH4)2SO4 concentration (>3.0 g/L) seemed not to benefit the D-ribose production and cell growth, while Srivastava and Wangikar found that 5.0 g/L of (NH4)2SO4 was optimum for D-ribose yield and too high concentrations of (NH4)2SO4 resulted in lower quantities of D-ribose and large quantities of acetic acid and acetoin of 20 g/L and 30 g/L, respectively [4)2SO4 of 3.0 g/L appeared to be optimal for D-ribose production from glucose by B. subtilis UJS0717.D-Ribose productivity was significantly affected by the , pH 7.0, glucose concentration of 150 g/L, corn steep liquor concentration of 20 g/L, and (NH4)2SO4 concentration of 3.0 g/L. However, one-factor-at-a-time experiments are incapable of reaching the true optimum especially due to interactions among various factors while RSM statistically designs and builds models, evaluates the effects of factors, and searches optimum conditions of factors for the desirable responses. Hence, following response surface methodology combined Box-Behnken design was applied to find the precise levels and interactions among significant factors.Based on one-factor-at-one-time experimental results, it could be concluded that the optimal culture conditions for D-ribose production from glucose byF value of 83.91 and low P value (P < 0.01) indicated that the model was highly significant and yielded good predictions of the experimental results. The value of the coefficient of determination (R2 = 0.9934) also reflected the good fit of the response model [In this work, the actual levels of the variables for each of the experiments in the design matrix were calculated and experimental results obtained as given in se model , which sY stands for the response variable (D-ribose concentration) and X1, X2, and X3 are the actual values of D-glucose, corn steep liquor, and (NH4)2SO4 concentrations, respectively. In this equation, the signs of the linear coefficients of X1, X2, and X3 were positive. This result indicated that glucose, corn steep liquor, and (NH4)2SO4 had a synergistic effect on the production of D-ribose. The signs of the coefficients of X1X2, X1X3, X2X3, X12,  X22, and X32 were negative, indicating that they had an inverse effect on D-ribose concentration [P < 0.01) according to the P value of the model. This result indicated that the factor had significant effect on the production of D-ribose. Therefore, the different ratio of the factor in fermentation would affect the production of D-ribose, and small variations in the factor would produce large changes in the results.The following second-order polynomial equation based on the multiple regression analysis explained the relationship between variables and D-ribose concentration:ntration . The firThe three-dimensional (3D) response surface plots drawn by Design-Expert 8.0.6.1 software based on the model equations were used to explain the interactions among the variables and to determine the optimal ratios of each component for the production of D-ribose. The response surface shapes reflected the nature and range of different components, and the peaks suggested that the optimum points were within the design limits. In this experiment, each plot was generated for the interactive effects of two variables on the production of D-ribose while holding the other factor at “zero” levels. 4)2SO4. To confirm the predicted results and verify the model, the above-calculated critical levels of the three variables were used to produce D-ribose, and the mean value of D-ribose production was 62.13 ± 1.16 g/L, which was in agreement with the predicted value (61.01 g/L).The maximum D-ribose concentration of 61.01 g/L was obtained by solving the model regression equation with the medium composed of 157 g/L glucose, 21 g/L corn steep liquor, and 3.2 g/L (NH B. subtilis UJS0717, which were higher than those under the original conditions (nditions . Bacillus is the main D-ribose producer which utilizes glucose, gluconic acid, or xylose as substrate.  B. subtilis UJS0717 used in this work is a comparable D-ribose producing bacterium that is able to produce D-ribose with the concentration of 62.13 g/L from 157 g/L glucose and high efficiency of converting glucose to D-ribose.Genus B. subtilis UJS0717 produced D-ribose at level of 62.13 g/L with yield of 0.40 g/g and volumetric productivity of 0.86 g/L·h, which was therefore potentially useful as an industrial D-ribose producer from cheap raw material corn starch hydrolysate. Semicontinuous/continuous fermentation and scale-up experiments for D-ribose production are ongoing in our lab for further improving the D-ribose production performance and evaluating the technical feasibility.The transketolase-deficient strain


Dinutuximab , a monoclonal antibody against disialoganglioside, improved survival as part of post-consolidation therapy for high-risk neuroblastoma. United Therapeutics Corporation (UTC) assumed ch14.18 production from the National Cancer Institute (NCI); this study evaluates pharmacokinetic comparability, safety, and tolerability of UTC and NCI products.2/day (ch14.18-UTC) and 25 mg/m2/day (ch14.18-NCI). Patients received one product during therapy cycles 1 and 2, the other during cycles 3–5. Ch14.18 pharmacokinetic profile characterization used population modeling (NONMEM® version 7.2). A two-compartment model with first-order distribution and elimination processes described pharmacokinetic data. Estimated product parameters were normalized to UTC nominal dose. For pharmacokinetic comparability, the final model was used to estimate exposure ratios (UTC/NCI) and associated 90 % confidence intervals (CIs) for area under the curve from time zero to infinity (AUCinf) and maximum concentration (Cmax). All comparisons were based on a standardized single-dose regimen (17.5 mg/m2 over 10 h).In this randomized, two-sequence crossover study, 28 patients aged ≤8 years with high-risk neuroblastoma received equivalent ch14.18-UTC or ch14.18-NCI doses. Despite comparable protein content, nominal doses differed: 17.5 mg/minf  and Cmax  within standard bioequivalence bounds (90 % CI 0.80–1.25). Products’ adverse events were similar and consistent with those previously reported.Final-model pharmacokinetic parameters were similar to previously published ch14.18-NCI parameters and comparable for UTC and NCI products. Products’ systemic exposures were comparable, with 90 % CIs around ratios for AUCEquivalent actual ch14.18-UTC and ch14.18-NCI doses produced comparable exposures, with no notable safety or tolerability differences.The online version of this article (doi:10.1007/s00280-015-2955-9) contains supplementary material, which is available to authorized users. MYCN oncogene) [Neuroblastoma, which is a tumor of the autonomic nervous system, accounts for approximately 7 % of cancers in children <15 years of age . At diagncogene) . High-rincogene) . Despitencogene) , 7.D2) is a surface glycolipid antigen that is strongly expressed on neuroblastoma tumor cells, with limited expression in normal human tissues [D2 is an important molecular target for immunotherapeutic approaches to treating neuroblastoma, and anti-GD2 monoclonal antibodies are efficacious in patients with high-risk neuroblastoma.Disialoganglioside  and overall survival (p = 0.02) at 2 years on the immunotherapy arm [Dinutuximab (Unituxin™), formerly called ch14.18, is a murine–human chimeric anti-Gantibody . Initialesleukin . Subsequesleukin . The trirapy arm . Based o2) differs from the dose of the prior NCI product (25 mg/m2) because of a difference in the extinction coefficient used to determine the protein concentration during the manufacturing process. Despite the change in nominal dosing, the amount of antibody delivered per dose is equivalent for the two products. Corrections for this difference in the products’ nominal dose must be made when comparing dose-dependent pharmacokinetic parameters, such as clearance and volumes of distribution.As part of a collaborative research agreement and development agreement (CRADA) with the National Cancer Institute (NCI), United Therapeutics Corporation (UTC) has licensed ch14.18 and assumed production. The nominal  dose of the UTC product . The secondary objective was to compare the products’ safety and tolerability profiles.Study DIV-NB-201 was a phase 2 randomized, open-label, two-sequence crossover trial evaluating ch14.18 in patients with high-risk neuroblastoma scheduled to receive immunotherapy. The clinical trial was conducted in accordance with the ethical principles of the Declaration of Helsinki and the International Conference on Harmonization E6 Good Clinical Practice Guideline. The protocol was approved by the Institutional Review Board at each participating site, and the parents or guardians provided written informed consent with patient assent, as appropriate. Patients were randomized 1:1 to receive either ch14.18-UTC or ch14.18-NCI during cycles 1 and 2, followed by ch14.18 from the other product during cycles 3–5. Eligible patients were randomized between 56 and 105 days after ASCT. Randomization must have occurred after the completion of tumor assessments post-ASCT and radiotherapy, if applicable.D2 antibody therapy or had prior vaccine therapy for the treatment of neuroblastoma. Patients were also excluded if they had received or planned to receive anticancer therapies, cytokines, or growth factors not included in the prescribed protocol therapy during the study or immunosuppressive drugs other than for acute allergic reactions and anaphylaxis during the study.Eligible patients were ≤8 years of age, had a diagnosis of high-risk neuroblastoma, and had completed standard induction therapy, surgery, myeloablative therapy and ASCT, and local radiotherapy to the primary tumor if indicated. Patients must have achieved a partial response or better per International Neuroblastoma Response Criteria (INRC)  at the p2 dose of ch14.18-NCI contains the same amount of ch14.18 as a 17.5-mg/m2 dose of ch14.18-UTC, making the respective dosing equivalent despite differences in the nominal doses. The dosing schema in this study is summarized in Table 2/day for 14 days) on cycles 1, 3, and 5 prior to, during, and following ch14.18, and aldesleukin IV  during cycles 2 and 4. In addition, all patients received six cycles of isotretinoin over 14 days  after completion of ch14.18 therapy.The study material manufactured by NCI used a theoretical extinction coefficient of 1.00 to calculate the concentration of antibody, whereas UTC material used an actual extinction coefficient of 1.41 to determine the antibody concentration. Thus, each 25 mg/mIn addition to study treatment, supportive care measures, including the use of narcotics and other concomitant medications, were required for the treatment of anticipated toxicities.Pharmacokinetic blood samples for the determination of ch14.18 plasma concentrations were obtained at 22 time points over the course of the treatment (Table Q), volume of the central compartment (V1), volume of the peripheral compartment (V2), steady-state volume of distribution (Vss), first-order elimination rate constant (Kel), and first-order distribution rate constants . The final structural model for the data from the prior Desai study was then used to estimate pharmacokinetic parameters for ch14.18-UTC and ch14.18-NCI in the formal comparability study (DIV-NB-201). This staged analysis approach was taken to avoid the potential for inflated alpha error associated with iterative model development using the formal comparability data from DIV-NB-201.Because of the study complexity and data limitations, a model-based approach, rather than a traditional noncompartmental bioequivalence analysis, was used to assess the pharmacokinetic comparability of ch14.18-UTC and ch14.18-NCI. Detailed pharmacokinetic data from nine patients with high-risk neuroblastoma enrolled in a previous pharmacokinetic study of ch14.18-NCI at the Children’s Hospital of Philadelphia by Desai [Because calculations of some pharmacokinetic parameters are dependent on the nominal dose of drug, differences in the nominal doses of ch14.18-UTC and ch14.18-NCI will affect pharmacokinetic parameter estimates. In the Desai study [Cmax) and area under the curve from time zero to infinity (AUCinf) were calculated using the population pharmacokinetic parameter estimates of CL and V from DIV-NB-201 and appropriate closed-form equations for Cmax and AUCinf. Variability in exposure estimates was captured by sampling the posterior distribution of pharmacokinetic parameter estimates using the NONMEM® Markov Chain Monte Carlo methodology. To allow for valid comparisons, all calculations were based on a standardized single dose of 17.5 mg/m2 infused over 10 h, using nominal UTC doses. Pharmacokinetic bioequivalence was assessed by calculation of ratios (UTC/NCI) for AUCinf and Cmax with 90 % confidence interval (CI) of the ratios. AUCinf was the primary comparability end point. Bioequivalence was established if the 90 % CIs for the exposure ratios were completely contained within accepted bioequivalence bounds (0.80–1.25).To formally assess the pharmacokinetic bioequivalence of the UTC and NCI products, maximum concentration . Safety analyses were performed on all patients receiving at least one study drug dose. No inferential statistical analyses of safety data were planned.n = 2), adverse events (n = 2), or withdrawal of consent (n = 1). Two patients received cycles 1–5 in the USA and returned to their home country to complete cycle 6. Ch14.18 was administered over a median duration of 11 h (range 10–20 h).Twenty-eight patients were enrolled, and 14 patients were randomized to each treatment sequence. Patient characteristics are summarized in Table Six of 27 patients had detectable HACA during the study. Only one patient (17 %) had a pharmacokinetic-neutralizing response (detected in cycle 3) and was therefore excluded from the pharmacokinetic analysis.Representative concentration time profiles for ch14.18-UTC and ch14.18-NCI from a single patient are presented in Fig. inf were 431 µg·h/mL for ch14.18-UTC and 413 µg·h/mL for ch14.18-NCI . Population pharmacokinetic estimates for Cmax were 6.57 µg/mL and 6.88 µg/mL, respectively . The 90 % CIs for exposure ratios of AUCinf and Cmax were contained within the standard bioequivalence bounds (0.80–1.25), consistent with comparable exposure between products.Following standardized single-dose regimens, population pharmacokinetic estimates for AUCAll 28 randomized patients were included in the safety analyses and had at least one treatment-related adverse event (TRAE) overall and at least one TRAE attributed to ch14.18. Overall, a total of 1945 TRAEs were reported, with most being grades 1–3. The most commonly reported TRAEs included pyrexia (100 %), hypoalbuminemia (96 %), hypokalemia (96 %), hyponatremia (82 %), cough (75 %), increased alanine aminotransferase (ALT) (68 %), anemia (68 %), hypocalcemia (68 %), pain (68 %), pruritus (68 %), increased aspartate aminotransferase (AST) (64 %), hypertriglyceridemia (64 %), and abdominal pain (61 %). Although differences between products were seen for individual adverse events, evaluation over the entire study showed no notable differences in TRAE incidence by manufacturer, either overall or attributable to ch14.18. Pain-related TRAEs were generally similar between treatment sequences; the most commonly reported pain-related events in the ch14.18-UTC and ch14.18-NCI groups were pain (59 and 44 %), abdominal pain (48 and 41 %), and pain in extremity (33 and 41 %), respectively. Pain-related TRAEs were most commonly reported during cycles 1, 2, and 4 . Allergic-type adverse events were also generally similar between ch14.18-UTC and ch14.18-NCI groups, with the most commonly reported events  including urticaria (30 vs. 26 %), peripheral edema (15 vs. 11 %), pruritus (63 vs. 52 %), and rash (26 vs. 26 %), respectively, and occurred most frequently in cycles 1, 2, and 4.Table 2) and ch14.18-NCI (25 mg/m2) was conducted to confirm equivalence of the pharmacokinetic and safety profiles of the two products.The NCI-manufactured ch14.18 was used in the pivotal randomized phase 3 trial that demonstrated the efficacy of ch14.18 combined with sargramostim, aldesleukin, and isotretinoin administered as continuation therapy for high-risk neuroblastoma . This rainf for ch14.18-UTC and ch14.18-NCI, and 90 % CIs about the geometric least-squares mean ratios for AUCinf and Cmax within standard bioequivalence bounds (90 % CI 0.80–1.25). Clearance, volume of distribution, and rate constants were also equivalent between the UTC and NCI products.Pharmacokinetic parameters were estimated by fitting a two-compartment pharmacokinetic model to individual concentration–time data. The products were formally compared using the results of a model-based bioequivalence analysis, which showed equivalent systemic exposures as measured by the AUC2. Historical parameters, such as CL and volumes of distribution, for the ch14.18-NCI material were derived using a 25-mg/m2 dose and require a correction factor of 0.7 for comparison with ch14.18-UTC material.For this analysis, dose-dependent pharmacokinetic parameters were derived using the ch14.18-UTC nominal dose of 17.5 mg/m2; Vl, 2.2 vs. 1.9 L; Kel, 0.026 vs. 0.027 1/h; Kcp, 0.021 vs. 0.023 1/h; and Kpc, 0.010 vs. 0.015 1/h).Data from an independent study (CHP1002) were used to develop a pharmacokinetic model for ch14.18. This model was then used to estimate pharmacokinetic parameters for the comparability study (DIV-NB-201). This approach was taken to avoid the potential for inflated alpha error associated with iterative model development based on the formal comparability data (DIV-NB-201). A model-based approach  was chosen to assess the pharmacokinetic comparability of ch14.18-UTC and ch14.18-NCI because of data limitations and the complexity of DIV-NB-201. The pharmacokinetic parameters derived from the population model for the CHP1002 study were comparable to those previously published by Desai et al. [Overall, the TRAEs  observed in this study were consistent with those reported in other studies in which ch14.18 was administered with growth factors and cytokines , 15. TheThe ANBL0032 study demonstrated that approximately 17 % of patients treated with ch14.18 develop HACA, with <5 % having a neutralizing antibody response in a biological assay . In this study, 6/27 (22 %) of patients had confirmed HACA, and only one patient was excluded from the pharmacokinetic analysis due to a neutralizing antibody response.2 is comparable to the ch14.18-NCI dose of 25 mg/m2 in terms of systemic exposure and with no notable safety and tolerability differences.In summary, the current analysis confirms that the ch14.18-UTC dose of 17.5 mg/mSupplementary material 1 (DOCX 148 kb)


AbstractEvemphyron Alonso-Zarazaga, Lv & Wang, gen. n., belonging to AttelabidaeRhynchitinae, is described. Its single species, Evemphyronsinense Alonso-Zarazaga, Lv & Wang, sp. n., was reared from larvae found inside seed pods of the legume Calleryadielsiana  in Sichuan Province (China). The species is figured and placed in the Deporaini because of the presence of minute labial palpi, the strongly crescentic apex of the postmentum, and the apodemes of male IX sternite and female VIII sternite curved sinistro-anterially near their cephalic end. It shows 3-segmented labial palpi and male sex patches on the procoxae, characters that suggest a basal position in the tribe.A new genus  Rhynchitini (Attelabidae: Rhynchitinae). However, an in depth-study of the available material by another author (MAAZ) revealed that the new species belonged in fact to Deporaini. This weevil species was found to infest seedpods of Calleryadielsiana (Harms) P.K.Lôc ex Z.Wei & Pedley (Fabaceae)  have been investigating the diversity of insect seed predators of woody trees in a subtropical forest of Dujiangyan City  since 2002. The Dujiangyan region is in the northern part of the Hengduan Mountains, a biodiversity hotspot and priority area for biodiversity conservation in China. Located in the mountains on the western border of the Sichuan Basin, it is in an ecotone between two biogeographical regions, the Qinghai-Tibetan Plateau and the Chengdu Plain. Climatically, it lies in the middle subtropical zone, characterized by evergreen broad-leaved forests. After checking the weevil specimens obtained, a new species belonging to a new genus was identified. It was thought at first sight to belong to the tribe ae) Figs . In thisCalleryadielsiana near Dujiangyan City (Sichuan province). All larvae were placed for adult emergence in a PVC tube  filled with local soil (30 cm in depth). We checked the emergence of adults once every week from March 2014, and collected adult specimens every day after the emergence of the first adult. In total, nine adult specimens emerged during June, July and September 2014. They were stored in 96% ethanol, and later seven were mounted for the morphological study, leaving two for molecular analysis.On 27 October 2013, 262 weevil larvae were collected from seedpods of the plant The dry specimens show different degrees of immaturity, mainly affecting their abdomens. Only the male preserved in ethanol was mature enough to allow the extraction of moderately sclerotized genitalia and terminalia. This extraction was done directly in the conserving medium. The abdomen was then soaked overnight in lukewarm 10% sodium hydroxide for digestion of soft tissues. Genitalia and terminalia were photographed in glycerine and later mounted in DMHF  on an acetate card, and pinned together with the tergites and sternites. These have been cross labelled with the specimen in ethanol from which they were extracted.PageBreakmicroscope, Figures Descriptions were made using a binocular Nikon SMZ 1500. Photographs Figures  were takOriginal label data have been written below in Chinese script. Added transliterations into pinyin or translations are placed between square brackets. Data from different labels are separated by two slashes (//) and lines within a label by one slash (/).Nomenclature follows Taxon classificationAnimaliaColeopteraAttelabidaeAlonso-Zarazaga, Lv & Wanggen. n.http://zoobank.org/747DBE94-0E6E-4E94-8A24-D0C927515082Evemphyronsinense Alonso-Zarazaga, Lv & Wang, sp. n.Deporaini Voss, 1929 as currently understood  keel (te) keel , this alte) keel ; protibiMale genitalia and terminalia. Penis  and the present active participle of the verb ἐμφύρω (to confuse), ἐμφῦρον (the confusing one). Gender neuter. Stem is Evemphyront-.The genus name is based on the classical Greek prefix εὖ (well), latinized as 豆毛象属 [dòu máo xiàng shǔ].Taxon classificationAnimaliaColeopteraAttelabidaeAlonso-Zarazaga, Lv & Wangsp. n.http://zoobank.org/57EF4590-5C0D-4B43-83F5-C2A7D4317ABDCharacters as given for the genus. In addition:Measurements (in mm) : Body length : 6.76–7.33. Rostrum: length: 2.83–3.06 (♂), 3.43–3.46 (♀); width : 0.60–0.67. Distance from antennal insertion to base: 1.13–1.33 (♂), 1.47 (♀). Forehead width: 0.53–0.67. Eye length: 0.67–0.73. Scape: 0.28 × 0.16 (♂), 0.28 × 0.12 (♀). Desmomeres: 1: 0.22 × 0.14 (♂), 0.24 × 0.13 (♀); 2: 0.32 × 0.14 (♂), 0.40 × 0.12 (♀); 3: 0.28 × 0.12 (♂), 0.34 × 0.12 (♀); 4: 0.34 × 0.14 (♂), 0.40 × 0.14 (♀); 5: 0.26 × 0.14 (♂), 0.24 × 0.14 (♀); 6: 0.24 × 0.14 (♂), 0.22 × 0.14 (♀); 7: 0.18 × 0.18 (♂), 0.20 × 0.16 (♀). Club: 1: 0.30 × 0.32 (♂), 0.30 × 0.30 (♀); 2: 0.28 × 0.34 ; 3: 0.54 × 0.30 (♂), 0.58 × 0.28 (♀). Pronotum: length: 2.33–2.43 (♂), 2.26–2.30 (♀); maximum width: 2.26–2.37. Elytra: length: 4.43–4.90; maximum width: 3.30–3.46.Rostrum 1.17–1.27 × as long as pronotum in male .N31°03'45.07" / E103°43'0.52" / Alt. 709 m // printed: 寄主 香花鸡血藤 [jìzhǔ [host] xiānghuā jī xuè téng] / Calleryadielsiana (Harms) // handwritten: A037 / 1(in red) ♂ / H.7.31.Holotype: 1 male, labelled: printed: 2014-VII-31 / 四川 都江堰 浦阳镇 花 / 溪村 , 肖治术 [Xiāo Zhìshù] leg. // printed: Paratypes: 1 male, same data as holotype, except dated 2014-VII-12 and a handwritten label: A037, 14-7-12 / 山胡豆[Shānhúdòu], 左后[zuǒhòu] / 足断[zúduàn] ♂; 1 male, same data as holotype, except dated 2014-VII-7 and a handwritten label: A037 / 1(in red) ♂ H.7.7 ; 1 male, same data as holotype, except dated 2014-VI-24, and a handwritten label: A037 / H / ♂ 6.24; 1 male, same data as holotype, except dated 2014-VII-7, and a handwritten label: A037 / 2 (in red) / ♂ H.7.7.; 1 female, same data as holotype, except dated 2014-VI-20, and a handwritten label: A037 / ♀ H.6.20; 1 female, same data as holotype, with a handwritten label: A037 / 2 (in red) / ♀ H.7.31 ; 2 males, same data as holotype, but dated 2014-VI-12 and 2014-IX-17 respectively, conserved in pure ethanol vials for DNA extraction. .PageBreaksinensis, -e: Chinese). It is an adjective, in neuter form to agree with the gender of the genus.The species is named after the country where it has been found, China  P.K.Lôc ex Z.Wei & Pedley . Larvae develop in seeds inside the pods. This weevil species was not found on other plant species despite long-term collecting of several Fabaceae species and other possible host plants carried out at the same study site.This species is known only from the type locality in Sichuan Province (China).Cyllorhynchites Voss, 1930, mainly in the head not being constricted behind the eyes and the presence of dense yellowish piliform vestiture, somewhat reminiscent of that of Cyllorhynchites (Cyllorhynchites) ursulusrostralis , a common weevil in China. This led to the initial placement in the tribe “Rhynchitini”, sensuDeporaini . Rhynchitini as well. No mention is made of the genitalic structures. Depasophilina are reddish, thinly pubescent, tropical animals of unknown habits. A close relationship of the new genus with them seems improbable.The new genus should belong, according to the keys proposed by Deporaina. However, this is also a very disparate group regarding its contents. The combination of 3-segmented labial palpi, PageBreakpropygidium practically covered by the elytra and lacking definite wing folding patches, tibiae neither spurred nor mucronate in both sexes, long rostrum without long, dense patches of long hairs in female and male procoxae with sex patch does not indicate any included genus as an obvious close relative. This set of character states suggests that the new genus could be very primitive (or even the most primitive) in the subtribe, since the palpi segment number, the long rostrum and the male sex patch on the procoxae are clearly symplesiomorphies shared with genera of Byctiscini, Rhynchitini and Auletini, but they are absent from advanced members of Deporaini.If the character of the covered propygidium is not taken into consideration, the only possible placement is within the subtribe Eusproda shares with Evemphyron the male sex patch on the procoxae  and the trophic link to Fabaceae, although Eusproda behaves as a shoot cutter of kudzu (Puerariamontana (Lour.) Merr.) and Japanese clover (Lespedezacyrtobotrya Miq.) , and not as an ovary or young fruit driller, as is the case for Evemphyronsinense. They also share the long rostrum, the very weak basal constriction of the head, the antennae inserted just behind middle of the rostrum, the non-contiguous hypomera, the subisodiametric pronotum, the absence of a scutellar striole, the tibiae without mucrones or spurs, overall similarity of the male genitalia and the ovipositor divided into two regions and with styli. Table PageBreakEvemphyron could be close to any of the genera related to Deporaus Samouelle, 1819. However, the same combination of characters precludes the finding of another genus sharing putative synapomorphic features, as no known genus matches the combination found in Evemphyron. Caenorhinus C.G. Thomson, 1859 shares with Evemphyron the confluence of 9th and 10th elytral stria at metacoxal level and the absence of wing folding patches on the propygidium, but differs clearly by the presence of spurs in at least one pair of tibiae in both sexes and of mucrones at least in one pair of tibiae in males, the shorter, apically widened rostrum and the presence of defined endophallic sclerites.DeporainiDeporaina on the basis of the minute labial palpi, the strongly crescentic apex of postmentum, the absence of scutellar striole, and the apodemes of male sternite IX and female sternite VIII curved sinistro-anteriad near their cephalic end.In summary, this new genus is placed in Rhynchitinae as a whole) is performed.The definitive placement of this new genus will have to wait until a molecular phylogeny of the tribe (and the subfamily