Evolutionary roles of polyploidization-derived structural variations in the phenotypic diversification of Panax species.

Genomic structural variations (SVs) are widespread in plant and animal genomes and play important roles in phenotypic novelty and species adaptation. Frequent whole genome duplications followed by (re)diploidizations have resulted in high diversity of genome architecture among extant species. In this study, we identified abundant genomic SVs in the Panax genus that are hypothesized to have occurred through during the repeated polyploidizations/(re)diploidizations. Our genome-wide comparisons demonstrated that although these polyploidization-derived SVs have evolved at distinct evolutionary stages, a large number of SV-intersecting genes showed enrichment in functionally important pathways related to secondary metabolites, photosynthesis and basic cellular activities. In line with these observations, our metabolic analyses of these Panax species revealed high diversity of primary and secondary metabolites both at the tissue and interspecific levels. In particular, genomic SVs identified at ginsenoside biosynthesis genes, including copy number variation and large fragment deletion, appear to have played important roles in the evolution and diversification of ginsenosides. A further herbivore deterrence experiment demonstrated that, as major triterpenoidal saponins found exclusively in Panax, ginsenosides provide protection against insect herbivores. Our study provides new insights on how polyploidization-derived SVs have contributed to phenotypic novelty and plant adaptation.

Phylogenomic analyses of Camellia support reticulate evolution among major clades.

Camellia (Theaceae) is a morphologically highly diverse genus of flowering plants and includes many famous species with high economic value, and the phylogeny of this genus is not fully resolved. We used 95 transcriptomes from 87 Camellia species and identified 1481 low-copy genes to conduct a detailed analysis of the phylogeny of this genus according to various data-screening criteria. The results show that, very different from the two existing classification systems of Camellia, 87 species are grouped into 8 main clades and two independent species, and that all 8 clades except Clade 8 were strongly supported by almost all the coalescent or concatenated trees using different gene subsets. However, the relationships among these clades were weakly supported and different from analyses using different gene subsets; furthermore, they do not agree with the phylogeny from chloroplast genomes of Camellia. Additional analyses support reticulate evolution (probably resulting from introgression or hybridization) among some major Camellia lineages, providing explanation for extensive gene tree conflicts. Furthermore, we inferred that together with the formation of East Asian subtropical evergreen broad-leaved forests, Camellia underwent a radiative divergence of major clades at 23 âˆ¼ 19 Ma in the late Miocene then had a subsequent species burst at 10 âˆ¼ 5 Ma. Principal component and cluster analyses provides new insights into morphological changes underlying the evolution of Camellia and a reference to further clarify subgenus and sections of this genus. The comprehensive study here including a nuclear phylogeny and other analyses reveal the rapid evolutionary history of Camellia.

GBP2 as a potential prognostic predictor with immune-related characteristics in glioma.

Guanylate binding protein 2 (GBP2) is a member of the guanine binding protein family, and its relationship with prognostic outcomes and tumor immune microenvironments in glioma remains elusive. We found GBP2 were increased in glioma tissues at both mRNA and protein levels. Kaplan-Meier curves revealed that high GBP2 expression was linked with worse survival of glioma patients, and multivariate Cox regression analysis indicated that high GBP2 expression was an independent prognostic factor for glioma. Combined analysis in immune database revealed that the expression of GBP2 was significantly related to the level of immune infiltration and immunomodulators. Single-cell analysis illustrated the high expression of GBP2 in malignant glioma cells showed the high antigen presentation capability, which were confirmed by real-time polymerase chain reaction (qRT-PCR) data. Additionally, the hsa-mir-26b-5p and hsa-mir-335-5p were predicted as GBP2 regulators and were validated in U87 and U251 cells. Our results first decipher immune-related characteristics and noncoding regulators of GBP2 in glioma, which may provide insights into associated immunotherapies and prognostic predictor.

Phenotypic and transcriptomic responses of the shade-grown species Panax ginseng to variable light conditions.

BACKGROUND AND AIMS: Elucidating how plant species respond to variable light conditions is important to understand the ecological adaptation to heterogeneous natural habitats. Plant performance and its underlying gene regulatory network have been well documented in sun-grown plants. However, the phenotypic and molecular responses of shade-grown plants under variable light conditions have remained largely unclear. METHODS: We assessed the differences in phenotypic performance between Panax ginseng (shade-grown) and Arabidopsis thaliana (sun-grown) under sunlight, shade and deep-shade conditions. To further address the molecular bases underpinning the phenotypic responses, we compared time-course transcriptomic expression profiling and candidate gene structures between the two species. KEY RESULTS: Our results show that, compared with arabidopsis, ginseng plants not only possess a lower degree of phenotypic plasticity among the three light conditions, but also exhibit higher photosynthetic efficiency under shade and deep-shade conditions. Further comparisons of the gene expression and structure reveal that differential transcriptional regulation together with increased copy number of photosynthesis-related genes (e.g. electron transfer and carbon fixation) may improve the photosynthetic efficiency of ginseng plants under the two shade conditions. In contrast, the inactivation of phytochrome-interacting factors (i.e. absent and no upregulation of the PIF genes) are potentially associated with the observed low degree of phenotypic plasticity of ginseng plants under variable light conditions. CONCLUSIONS: Our study provides new insights into how shade-grown plants respond to variable light conditions. Candidate genes related to shade adaptation in ginseng provide valuable genetic resources for future molecular breeding of high-density planting crops.

[Impacts of Fertilization on Soil Antibiotic Resistance Genes Across Croplands: A Meta-Analysis].

The objective of this study was to clarify and quantify the impact of fertilizer applications on antibiotic resistance genes (ARGs) in cropland soil. The target was to provide scientific basis for a better understanding of the source and accumulation and transportation characteristics of ARGs in soil and adaptive management strategy-making to secure the ecological environment and human health safety. By collecting data from literature published within the last 20 years (2000-2020), we established a database with 215 and 201 groups of a paired data-set consisting of the quantity and relative abundance of ARGs under independent experimental conditions. Compared to that with no fertilizer, the combined application of organic fertilizer significantly increased the quantity and relative abundance of soil ARGs by 110.0% and 91.0%, respectively. However, chemical fertilization had no significant effect on soil ARGs. The increment of relative abundance of soil ARGs by the combined application of organic fertilizer in the subtropical region was equivalent to 2.6 times that in the warm temperate zone. Compared with that in black soil and dark brown soil, the combined application of organic fertilizer significantly increased the relative abundance of ARGs in red soil and paddy soil in the subtropical region. The increment for the quantity of ARGs (147.6%) by the combined application of organic fertilizer in soil with pH<7 was significantly higher than that in soil with pH>7(110.4%). Compared to poultry manure, livestock manure application significantly increased the quantity and relative abundance of ARGs. The increment of the relative abundance of organic fertilizer to sulfonamide, multidrug, and macrolide ARGs (170.5%-201.2%) was significantly higher than that of quinolone, tetracycline, and aminoglycoside ARGs (61.5%-115.6%). After more than 10 years of applying organic fertilizer, the quantity of soil ARGs significantly increased by 104.2%-112.3%, whereas the effect on the relative abundance was uncertain. Climate, soil spatial properties, and source and amount of organic fertilizer were the main factors affecting the accumulation of ARGs in farmland soil. Management strategies and solutions should pay more attention to effectively minimizing the accumulation and spread of ARGs in agro-ecosystems for high-quality agricultural development in the future.

Alternate Partial Root-Zone Drip Nitrogen Fertigation Reduces Residual Nitrate Loss While Improving the Water Use but Not Nitrogen Use Efficiency.

The efficient utilization of irrigation water and nitrogen is of great importance for sustainable agricultural production. Alternate partial root-zone drip irrigation (APRD) is an innovative water-saving drip irrigation technology. However, the coupling effects of water and nitrogen (N) supply under APRD on crop growth, water and N use efficiency, as well as the utilization and fate of residual nitrates accumulated in the soil profile are not clear. A simulated soil column experiment where 30-40 cm soil layer was 15NO3-labeled as residual nitrate was conducted to investigate the coupling effects of different water [sufficient irrigation (W1), two-thirds of the W1(W2)] and N [high level (N1), 50% of N1 (N2)] supplies under different irrigation modes [conventional irrigation (C), APRD (A)] on tomato growth, irrigation water (IWUE) and N use efficiencies (NUE), and the fate of residual N. The results showed that, compared with CW1N1, AW1N1 promoted root growth and nitrogen absorption, and increased tomato yield, while the N absorption and yield did not vary significantly in AW2N1. The N absorption in AW2N2 decreased by 16.1%, while the tomato yield decreased by only 8.8% compared with CW1N1. The highest IWUE appeared in AW2N1, whereas the highest NUE was observed in AW2N2, with no significant difference in NUE between AW2N1 and CW1N1 at the same N supply level. The 15N accumulation peak layer was almost the same as the originally labeled layer under APRD, whereas it moved 10-20 cm downwards under CW1N1. The amount of 15N accumulated in the 0-40 cm layer increased with the decreasing irrigation water and nitrogen supply, with an increase of 82.9-141.1% in APRD compared with that in CW1N1. The utilization of the 15N labeled soil profile by the tomato plants increased by 9-20.5%, whereas the loss rate of 15N from the plant-soil column system decreased by 21.3-50.1% in APRD compared with the CW1N1 treatment. Thus, APRD has great potential in saving irrigation water, facilitating water use while reducing the loss of residual nitrate accumulated in the soil profile, but has no significant effect on the NUE absorbed.

Effects of Clostridium butyricum on growth performance, metabonomics and intestinal microbial differences of weaned piglets.

BACKGROUND: Weaning stress of piglets causes a huge economic loss to the pig industry. Balance and stability of the intestinal microenvironment is an effective way to reduce the occurance of stress during the weaning process. Clostridium butyricum, as a new microecological preparation, is resistant to high temperature, acid, bile salts and some antibiotics. The aim of present study is to investigate the effects of C. butyricum on the intestinal microbiota and their metabolites in weaned piglets. RESULTS: There was no statistical significance in the growth performance and the incidence of diarrhoea among the weaned piglets treated with C. butyricum during 0-21 days experimental period. Analysis of 16S rRNA gene sequencing results showed that the operational taxonomic units (OTUs), abundance-based coverage estimator (ACE) and Chao index of the CB group were found to be significantly increased compared with the NC group (P < 0.05). Bacteroidetes, Firmicutes and Tenericutes were the predominant bacterial phyla in the weaned piglets. A marked increase in the relative abundance of Megasphaera, Ruminococcaceae_NK4A214_group and Prevotellaceae_UCG-003, along with a decreased relative abundance of Ruminococcaceae_UCG-005 was observed in the CB group, when compared with the NC group (P < 0.05). With the addition of C. butyricum, a total of twenty-two significantly altered metabolites were obtained in the feces of piglets. The integrated pathway analysis by MetaboAnalyst indicated that arginine and proline metabolism; valine, leucine and isoleucine biosynthesis; and phenylalanine metabolism were the main three altered pathways, based on the topology. Furthermore, Spearman's analysis revealed some altered gut microbiota genus such as Oscillospira, Ruminococcaceae_NK4A214_group, Megasphaera, Ruminococcaceae_UCG-005, Prevotella_2, Ruminococcaceae_UCG-002, Rikenellaceae_RC9_gut_group and Prevotellaceae_UCG-003 were associated with the alterations in the fecal metabolites (P < 0.05), indicating that C. butyricum presented a potential protective impact through gut microbiota. The intestinal metabolites changed by C. butyricum mainly involved the variation of citrulline, dicarboxylic acids, branched-chain amino acid and tryptophan metabolic pathways. CONCLUSIONS: Overall, this study strengthens the idea that the dietary C. butyricum treatment can significantly alter the intestinal microbiota and metabolite profiles of the weaned piglets, and C. butyricum can offer potential benefits for the gut health.

Concerted and birth-and-death evolution of 26S ribosomal DNA in Camellia L.

BACKGROUND AND AIMS: The ribosomal DNA (rDNA) gene family, encoding ribosomal RNA (rRNA), has long been regarded as an archetypal example illustrating the model of concerted evolution. However, controversy is arising, as rDNA in many eukaryotic species has been proved to be polymorphic. Here, a metagenomic strategy was applied to detect the intragenomic polymorphism as well as the evolutionary patterns of 26S rDNA across the genus Camellia. METHODS: Degenerate primer pairs were designed to amplify the 26S rDNA fragments from different Camellia species. The amplicons were then paired-end sequenced on the Illumina MiSeq platform. KEY RESULTS: An extremely high level of rDNA polymorphism existed universally in Camellia. However, functional rDNA was still the major component of the family, and was relatively conserved among different Camellia species. Sequence variations mainly came from rRNA pseudogenes and favoured regions that are rich in GC. Specifically, some rRNA pseudogenes have existed in the genome for a long time, and have even experienced several expansion events, which has greatly enriched the abundance of rDNA polymorphism. CONCLUSIONS: Camellia represents a group in which rDNA is subjected to a mixture of concerted and birth-and-death evolution. Some rRNA pseudogenes may still have potential functions. Conversely, when released from selection constraint, they can evolve in the direction of decreasing GC content and structural stability through a methylation-induced process, and finally be eliminated from the genome.

Fermented cottonseed meal improves production performance and reduces fat deposition in broiler chickens.

OBJECTIVE: This study was conducted to investigate the effect of fermented cottonseed meal (FCSM) on growth performance, carcass traits, and fat deposition in white-feather broiler chickens. METHODS: A total of 480 male one-day-old white-feather broiler chickens were selected randomly and divided into four groups with six replicates of 20 chickens in each. The experimental chickens were fed diets including 3%, 6%, or 9% FCSM fermented by Candida tropicalis until 42 days old. In the experiment, the chickens of the control group were fed soybean meal. RESULTS: FCSM supplementation linearly decreased the feed conversion ratio from d 15 to 21 and d 36 to 42, respectively (p<0.05). The percentage of carcass and semi-eviscerate increased in response to dietary FCSM supplementation at d 21 (p<0.05). The percentage of eviscerated and semi-eviscerate of 3FCSM was higher than that in other groups at d 35 (p<0.05). At the age of 42 d, the percentage of carcass increased in a quadratic way among increasing FCSM in diets (p<0.05). The subcutaneous fat thickness linearly decreased with the increasing levels of FCSM at d 21 (p<0.05). Gompertz and Logistic functions provided a better fit on abdominal fat and subcutaneous fat, respectively. The best fitted equation predicted that the maximum growth rate of abdominal fat weight and subcutaneous fat thickness occurred at d 28. FCSM had no significant effects on the shape of growth curve of abdominal fat weight and subcutaneous fat thickness, but reduced the height of the curve. Birds receiving the 6FCSM diet for 21 d had smaller adipocyte surface and lower serum glucose as well as triglyceride concentration. CONCLUSION: FCSM is beneficial for broiler chickens as it positively affects their growth and carcass in addition to altering their fat deposition.

[Clinical study of staging operation for large gouty stone on the first metatarsophalangeal joint].

OBJECTIVE: To explore short-term curative effect of staging operation for large gouty stone on the first metatarsophalangeal joint. METHODS: From January 2015 to December 2016, 12 patients with giant gout stone on the first metatarsophalangeal joint were treated with staging operation, including 12 males, aged from 45 to 73 years old, 6 cases on the right side and 6 cases on the left side. After conservative treatment for more than 2 years, the size and location of ventilator stone were detected by dual energy CT. The bone destruction of the first metatarsophalangeal joint was observed on X-ray examination. All patients underwent stageⅠgout radical removal and temporary fixation with Kirschner wire, and metatarsophalangeal joint fusion and internal fixation were performed after local soft tissue conditions were stabilized. The level of blood uric acid before and after hand were compared, and correction and complications of limb deformity were observed, VAS score was applied to evaluate pain relieved degree. RESULTS: All patients were successfully completed two stages ' operation. Twelve patients were followed up for 9 to 13 months. VAS sco re and uric acid content at different time points were observed and recorded. VAS score before operation ranged from 6 to 9, and decreased to 0 to 1 at 7 weeks after operation; the level of blood uric acid ranged from 443 to 501 µmol/L before operation, and decreased to 307 to 330 µmol/L at 7 weeks after operation. The first metatarsophalangeal joint deformity of foot was corrected and the shape was recovered at 5 to 7 months after operation. One patient occurred incision infection and flap edgenecrosis, and the wound healed by debridement and dressing change. CONCLUSION: Staged operation for the treatment of giant gout stone on the first metatarsophalangeal joint of foot could correct joint deformity, restore shape of the first metatarsophalangeal joint, relieve pain of the affected foot, and beneficial for control content of serum uric acid, and has less complications.

[Four kinds of operative methods for subtalar arthrodesis in treating old calcaneal fracture].

OBJECTIVE: To explore clinical efficacy of four surgical methods for subtalar joint fusion and internal fixation in treating old calcaneal fractures. METHODS: From March 2014 to November 2017, 25 patients (26 feet) with old calcaneal fractures treated with four surgical methods of subtalar arthrodesis were retrospectively analyzed, including subtalar joint fusion in situ for 2 patients (2 feet), subtalar articular strut bone graft fusion for 6 patients (6 feet), subtalar joint fusion with calcaneus V-shaped osteotomy for 6 patients(6 feet), and subtalar bone graft fusion with calcaneus cuneiform osteotomy for 11 patients (11 feet). They aged from 23 to 70 years old with an average of (36.7±5.8) years old, and the courses of disease ranged from 3 to 35 months with an average of (9.5±5.1) months. Postoperative complications were observed, talar tilt angle, calcaneus Böhler angle, Gissane angle, and the heel height were compared before and after surgery at 12 months. VAS score and American Orthopaedic Foot and Ankle Society(AOFAS) score were employed to evaluate clinical efficacy at 12 months after surgery. RESULTS: Twenty-one patients (21 feet) were followed up for 13 to 34 months with an average of (20.1±3.7) months, four patients(5 feet) were lost to follow-up. Two patients were underwent surgical incision rupture, 1 patient occurred fracture nonunion, and no complications such as fracture nonunion, loosening or fracture of internal fixation occurred in the other patients. There were significant differences between preoperative and postoperative in talar tilt angle[(5.3±2.4)° vs (11.2±4.6)°, t=7.24, P<0.05], calcaneus Böhler angle[(5.4±2.7)° vs(25.5±5.3)°, t=11.2, P<0.05], Gissane angle[(89.4±9.6)° vs (122.0±5.2)°, t=8.13, P<0.05], and heel height[(28.5±5.1) mm vs(47.1±3.7) mm, t=6.45, P<0.05]. VAS score was decreased from 5.2±1.0 before operation to 1.6±0.7 at 12 months after operation(t=5.12, P<0.05); AOFAS score was improved from 52.4±6.4 to 86.2±5.2 at 12 months after operation(t=6.41, P<0.05); 14 feet got excellent results, 4 good , 2 moderate and 1 poor. CONCLUSIONS: Subtalar joint fusion in situ, subtalar articular strut bone graft fusion, subtalar joint fusion with calcaneus V-shaped osteotomy, and subtalar bone graft fusion with calcaneus cuneiform osteotomy are effective operation methods for old calcaneal fractures, which have advantages of relieving pain, correcting deformity of calcaneal, improving foot function. Reasonable selection and treatment is the key to ensure the efficacy.

Development of a Direct Competitive ELISA Kit for Detecting Deoxynivalenol Contamination in Wheat.

This study was conducted to develop a self-assembled direct competitive enzyme-linked immunosorbent assay (dcELISA) kit for the detection of deoxynivalenol (DON) in food and feed grains. Based on the preparation of anti-DON monoclonal antibodies, we established a standard curve with dcELISA and optimized the detection conditions. The performance of the kit was evaluated by comparison with high-performance liquid chromatography (HPLC). The minimum detection limit of DON with the kit was 0.62 ng/mL, the linear range was from 1.0 to 113.24 ng/mL and the half-maximal inhibition concentration (IC50) was 6.61 ng/mL in the working buffer; there was a limit of detection (LOD) of 62 ng/g, and the detection range was from 100 to 11324 ng/g in authentic agricultural samples. We examined four samples of wheat bran, wheat flour, corn flour and corn for DON recovery. The average recovery was in the range of 77.1% to 107.0%, and the relative standard deviation (RSD) ranged from 4.2% to 11.9%. In addition, the kit has the advantages of high specificity, good stability, a long effective life and negligible sample matrix interference. Finally, wheat samples from farms in the six provinces of Henan, Anhui, Hebei, Shandong, Jiangsu and Gansu in China were analyzed by the kit. A total of 30 samples were randomly checked (five samples in each province), and the results were in good agreement with the standardized HPLC method. These tests showed that the dcELISA kit had good performance and met relevant technical requirements, and it had the characteristics of accuracy, reliability, convenience and high-throughput screening for DON detection. Therefore, the developed kit is suitable for rapid screening of DON in marketed products.

A Prospective Randomized Multicenter Controlled Trial on Salvianolate for Treatment of Unstable Angina Pectoris in A Chinese Elderly Population.

OBJECTIVE: To evaluate the efficacy and safety of salvianolate in elderly patients with unstable angina pectoris (UAP). METHODS: A prospective double-blind randomized placebo-controlled multicenter trial in elderly patients with UAP from 13 third-grade class-A hospitals in China was performed. A total of 318 patients were randomly allocated in a 1:1 ratio to an experimental group (160 patients) and a control group (158 patients). The experimental group was treated with salvianolate for 14 days on the basis of conventional medicine, and the control group was given a placebo for 14 days with the same criteria. Follow-up was lasted 28 days in both groups. The primary endpoint was biweekly frequency of angina pectoris attacks. The secondary endpoints included biweekly dosage of nitroglycerin, the Seattle Angina Questionnaire, angina pectoris severity and duration, myocardial injury markers, high-sensitivity C-reactive protein (hs-CRP) and N-terminal pro-B-type natriuretic peptide (NT-proBNP), as well as major adverse cardiovascular events (MACEs). Safety was assessed according to adverse events and serious adverse events. RESULTS: Baseline characteristics were similar between treatment groups. Compared with those in the control group, the frequency of biweekly angina attacks (2.92 vs . 4.08, P=0.025), the biweekly dosage of nitroglycerin, as well as the severity and duration of angina attacks (P<0.01) were reduced by salvianolate. The Seattle Angina Questionnaire score was also significantly improved in the experimental group than in the control group (P<0.05). No significant differences were observed between the two groups with respect to the incidence of MACEs. Salvianolate was well tolerated. CONCLUSIONS: Salvianolate appear to have efficacy and well tolerated for elderly patients with UAP. [ClinicalTrials.gov identifier: NCT03037047].

Effect of Fermented Cottonseed Meal on the Lipid-Related Indices and Serum Metabolic Profiles in Broiler Chickens.

This study aimed to investigate the changes of lipid-related gene and serum metabolites in broiler chickens fed with fermented cottonseed meal (FCSM) diet, through quantitative real-time PCR and metabolomics analysis. Totally, 180 1-day-old Cobb broilers were randomly assigned to two groups with six replicates of 15 birds in each. The two diets consisted of a control diet supplemented with 0% FCSM (CON group) and an experimental diet with 6% FCSM (fermented by Candida tropicalis) replacing the soybean meal (FCSM group). The results showed that both abdominal fat content and subcutaneous fat thickness significantly reduced (p < 0.05) in response to dietary FCSM supplementation at the age of 21 d. Serum concentrations of glucose, triglyceride, and low-density lipoprotein cholesterol decreased (p < 0.05) in FCSM fed broilers compared with CON fed broilers, while the levels of epinephrine and growth hormone in serum, liver and abdominal fat tissue were higher (p < 0.05) in FCSM than in CON fed broilers. The activity of hormone-sensitive esterase and lipoprotein lipase (LPL) in the liver and abdominal fat were higher (p < 0.05) in FCSM than CON group. Additionally, compared with the CON group (p < 0.05), the expression of peroxisome proliferator-activated receptor alpha and LPL genes were upregulated in the livers of FCSM group broilers. Gene expressions of hormone-sensitive lipase and LPL in the abdominal fat tissue were also upregulated (p < 0.05) with the broilers fed with FCSM diets. A total of 20 significantly different metabolites were obtained in the serum of different dietary FCSM supplemented fed broilers. The mainly altered pathways were clustered into organic acid metabolism, fatty acid metabolism, and amino acid metabolism. These results not only provide a better understanding of broilers' lipid metabolism with FCSM but also can be helpful in further improvement of the broilers' healthy production and utilization of FCSM.

Effects of parental genetic divergence on gene expression patterns in interspecific hybrids of Camellia.

BACKGROUND: The merging of two divergent genomes during hybridization can result in the remodeling of parental gene expression in hybrids. A molecular basis underling expression change in hybrid is regulatory divergence, which may change with the parental genetic divergence. However, there still no unanimous conclusion for this hypothesis. RESULTS: Three species of Camellia with a range of genetic divergence and their F1 hybrids were used to study the effect of parental genetic divergence on gene expression and regulatory patterns in hybrids by RNA-sequencing and allelic expression analysis. We found that though the proportion of differentially expressed genes (DEGs) between the hybrids and their parents did not increase, a greater proportion of DEGs would be non-additively (especially transgressively) expressed in the hybrids as genomes between the parents become more divergent. In addition, the proportion of genes with significant evidence of cis-regulatory divergence increased, whereas with trans-regulatory divergence decreased with parental genetic divergence. CONCLUSIONS: The discordance within hybrid would intensify as the parents become more divergent, manifesting as more DEGs would be non-additively expressed. Trans-regulatory divergence contributed more to the additively inherited genes than cis, however, its contribution to expression difference would be weakened as cis mutations accumulated over time; and this might be an important reason for that the more divergent the parents are, the greater proportion of DEGs would be non-additively expressed in hybrid.

Optimization of the process parameters for reduction of gossypol levels in cottonseed meal by functional recombinant NADPH-cytochrome P450 reductase and cytochrome P450 CYP9A12 of Helicoverpa armigera.

Gossypol is a toxic polyphenolic product that is derived from cotton plants. The toxicity of gossypol has limited the utilization of cottonseed meal (CSM) in the feed industry. The gene, Helicoverpa armigera CYP9A12, is a gossypol-inducible cytochrome P450 gene. The objective of our study was to obtain the functional recombinant H. armigera CYP9A12 enzyme in Pichia pastoris and to verify whether this candidate enzyme could decrease gossypol in vitro. Free and total gossypol contents were detected in the enzyme solution and in CSM. The H. armigera CYP9A12 enzyme degraded free concentration of gossypol. After optimization of the single-test and response surface method, free gossypol content could be decreased to 40.91 mg/kg in CSM by the H. armigera CYP9A12 enzyme when the initial temperature was 35 °C, the enzymatic hydrolysis time lasted 2.5 h, the enzyme addition was 2.5 mL, and the substrate moisture was 39%.

[Bone and soft tissue combined with surgery for the treatment soft flatfoot combined with painful navicular bone].

OBJECTIVE: To explore short-term clinical effects of bone and soft tissue combined with surgery for the treatment of soft flatfoot accompanied with painful navicular bone. METHODS: From May 2015 to August 2017, 16 patients (16 feet) with navicular bone soft flatfoot accompanied with painful navicular bone were treated with bone and soft tissues operation (gastrocnemius release, medial displacement calcaneal osteotomy, and excision of accessory navicular with reconstruction of posterior tibial tendon). Among them, there were 9 males (9 feet) and 7 females (7 feet), aged from 22 to 48 years old with an average of (32.0±3.4) years old. The duration of diseases ranged from 6 months to 5 years with an average of (2.4±1.7) years. The postoperative complications were observed, talocalcaneal angle, the first metatarsal horn of the talus, arch height, angle of inclination and calcaneal valgus before and after operation at 12 months were compared. VAS score and AOFAS score after operation at 12 months were applied to evaluate pain relief and function. RESULTS: All patients were followed up for an average of (18.4±3.5) months(13~25 months). The incisions of patients were healed at grade A without wound infection, nonunion or delayed union, internal fixation fracture or loosening and other complications. Medial pain of foot was disappeared and motor ability was restored at 12 months after operation. Arch height, angle of inclination and the first metatarsal horn of the talus of lateral X-ray before operation and after operation at 12 months on weight-bearing foot were(21.51±1.20)°vs(31.01±1.62)°, (10.71±1.52)°vs(22.12±2.11)°, (15.61±1.41)°vs(5.10±1.20)°; talocalcaneal angle, the first metatarsal horn of the talus of AP X-ray before operation and after operation at 12 months on weight-bearing foot were (36.12±2.21) ° vs (22.12±2.61)°, (13.41±1.51)°vs(4.30±0.91)°; calcaneal valgus of axial X-ray before operation and after operation at 12 months on weight-bearing foot were (10.80±1.21)°vs(3.92±1.81)°; there were statistical difference in imaging indicators between preoperation and 12 months after operation. VAS score was significantly decreased from (6.21±2.31) before operation to (1.82±0.56) at 12 months after operation (t=2.64, P<0.05). AOFAS score was obviously increased from (51.2±5.6)before operation to (87.1±4.7)at 12 months after operation (t=3.43, P<0.05). CONCLUSIONS: Bone and soft tissue operation (namely, gastrocnemius release, medial displacement calcaneal osteotomy, and excision of accessory navicular with reconstruction of posterior tibial tendon) could obviously relieve foot pain, improve foot appearance and function in patients with navicular bone soft flatfoot complicated with painful navicular bone, and has certain clinical efficacy.

Effect of a functional recombinant cytochrome P450 enzyme of Helicoverpa armigera on gossypol metabolism co-expressed with NADPH-cytochrome P450 reductase in Pichia pastoris.

Gossypol is a polyphonic toxic compound that is present in cotton plants. The P450 cytochromes CYP6AE14 and CYP9A12 of Helicoverpa armigera are highly induced by gossypol and have been reported to be possibly involved in gossypol degradation. To determine whether the candidate H. armigera CYP6AE14 and CYP9A12 enzymes could metabolize gossypol in vitro, functional recombinant H. armigera CYP6AE14 and CPR (CYP9A12 and CPR) enzymes were successfully expressed in Pichia pastoris (P. pastoris). UPLC-QTOF/MS demonstrated the following results: (1) Free gossypol was spontaneously degraded to the gossypol metabolites G1 (m/z 265) and G2 (m/z 293) without the addition of any enzyme. (2) Free gossypol was observed following the addition of the endogenous or recombinant H. armigera P450 cytochrome CYP6AE14/CYP9A12 enzyme: in the first pathway, free gossypol was dehydroxylated and decarboxylated to G3 (m/z 453), and in the second pathway, the aldehyde group of gossypol and its metabolite were covalently bound with the amine products to form G4 (m/z 437) and G5 (m/z 783). (3) In addition to the gossypol binding pathways, the recombinant H. armigera CPR and CYP9A12 enzymes was found that could further decarboxylate the gossypol intermediate demethylated reduction of gossypolonic acid (m/z 294) and demethylated gossic acid (m/z 265) to G0 (m/z 209) and G0' (m/z 249) respectively.

[Treatment of old calcaneal fractures with calcaneus V-shape osteotomy for the subtalar joint preservation].

OBJECTIVE: To observe the clinical effect of calcaneus V-shape osteotomy for the subtalar joint preservation in the treatment of old calcaneal fractures. METHODS: A retrospective study was conducted on 18 patients (18 feet) with old calcaneal fractures treated by calcaneus V-shape osteotomy from March 2014 to November 2017. Included 13 males and 5 females, aged from 23 to 48 years old with an average of (34.3±4.5) years and the course of disease was from 2 to 9 months with an average of (4.5±4.1) months. According to the classification of Sanders, 14 cases of type II and 4 cases of type III. The postoperative complications were observed. The changes of Böhler angle, Gissane angle, heel height and the tilt angle of talus were compared between before operation and 12 months after operation. Maryland foot score was used to evaluate the curative effect. RESULTS: Fourteen patients were followed up, 4 patients were loss to follow-up. And the follow-up time was 13 to 35 months, with an average of (21.2±4.1) months. The fracture healing time was 10 to 12 weeks with an average of (12.4±2.1) weeks. One patient had skin necrosis of incision after operation, and no complications such as nonunion of fracture and loosening or rupture of internal fixation were found. The tilt angle of talus, Böhler angle, Gissane angle, heel height at 12 months after operation were (11.4±5.1)°, (24.7±4.3)°, (124±3.1)°, (46.1±2. 8) mm, respectively, while before operation were (9.1±2.9)°, (6.8±3.1)°, (93.4±11.7)°, (34.5±5.3) mm, there were significant difference between before and after operation. Maryland score was significantly increased from 59.21±7.21 preoperatively to 86.34 ±4.14 postoperatively 12 months(t=43.1, P<0.05), and 8 cases got excellent results, 4 good, 1 fair, 1 poor. CONCLUSIONS: Calcaneus V-shape osteotomy for subtalar joint preservation is one of the effective methods in the treatment of old calcaneal fractures, which has the advantages of preserving subtalar joint, correcting calcaneal deformity, inproving foot appearance, alleviating foot pain and raising foot function.

[Treatment of distal radius fracture with manual reduction and external fixation with small splint steel support external fixation].

OBJECTIVE: To evaluate the short-term result for the treatment of distal radius fracture with manual resetting and fixing with small splint. METHODS: From January 2015 to December 2016, 60 cases of distal radial fractures were treated by manual resetting and small splint fixation including 39 males and 21 females with an average age of (43.50±10.56) years old ranging from 18 to 65 years old. According to AO classification, 38 cases were type A, 19 cases were type B, 3 cases were type C. The anteroposterior and lateral X-ray of the wrist joint was performed before reduction, 1, 2, 4 weeks after reduction, and the removal of the small splint, the palm inclination angle, ulnar deviation angle and radius height were measured. At 8, 24 and 48 weeks after reduction, the wrist extensor, palmar flexion, pronation, supination, ulnar deviation and radial deviation were measured. Wrist strength was measured at 4, 8, 24 and 48 weeks after fracture reduction. After the distal radius fracture healed, the splint support was removed. After dismantling small splint steel support, Mayo score of wrist joint was performed. RESULTS: All cases of distal radius fractures were followed up, the healing time of fracture was 6 to 8 (7.1±0.9) weeks. The difference of palmar obliquity, ulnar deviation angle and radius shortening between before reduction and 2 weeks after reduction was statistically significant. There was no significant difference in palmar inclination, ulnar deviation and radius shortening between 2 and 4 weeks after reduction. After 24 weeks and 8 weeks, 48 weeks and 24 weeks after reduction, there was significant difference in wrist extensor, palmar flexion, pronation, supination, ulnar deviation and radial deviation. The grip strength was statistically significant between after reduction and 4 weeks after reduction, 8 weeks and 4 weeks after reduction, 16 weeks and 8 weeks after reduction, 24 and 16 weeks after reduction. There was no significant difference in grip strength between 48 weeks and 24 weeks after reduction. The Mayo wrist function scoring system was used to evaluate the curative effect, the results were excellent in 50 cases, good in 8 cases, fair in 1 case and poor in 1 case. CONCLUSIONS: The treatment of distal radius fracture with the method of manual resetting and fixing with plywood is simple and the clinical efficacy is accurate.