[LncRNA DRAIC regulates the proliferation, apoptosis, migration and invasion of lung adenocarcinoma cells by targeting let-7i-5p].

Objective: To investigate the effects of lncRNA DRAIC on proliferation, apoptosis, migration and invasion of lung adenocarcinoma cells and its mechanism. Methods: Reverse transcription-quantitative real-time polymerase chain reaction (RT-qPCR) was used to detect the expression of DRAIC in lung cancer tissues and corresponding adjacent normal tissues of 40 patients with lung adenocarcinoma who underwent surgery in Tangshan People's Hospital from 2019 to 2020. Lung adenocarcinoma cells A549 and H1299 were cultured in vitro and divided into si-NC group, si-DRAIC group, miR-NC group, let-7i-5p mimics group, si-DRAIC+ inhibitor-NC group, and si-DRAIC+ let-7i-5p inhibitor group. CCK-8 method and clone formation experiment were used to detect cell proliferation. Flow cytometry was used to detect cell apoptosis. Transwell array was used to detect the cell migration and invasion. Western blot was used to detect the protein expressions of Caspase-3, Caspase-9, Bcl-2 and Bax. The double luciferase reporter gene experiment was used to verify the regulatory relationship between DRAIC and let-7i-5p. Independent sample t test was used for comparison between two groups, one-way ANOVA was used for comparison between multiple groups, and Pearson correlation analysis was used for correlation analysis. Results: Compared with adjacent tissues, the expression level of DRAIC in lung adenocarcinoma tissues increased (P<0.05), but the expression level of let-7i-5p decreased (P<0.05). The expression levels of DRAIC and let-7i-5p in lung adenocarcinoma tissues were negatively correlated (r=-0.737, P<0.05). The absorbance value of A549 and H1299 cells in the si-DRAIC group at 48, 72 and 96 hours were lower than those in the si-NC group (P<0.05), the number of clones formed [(91.00±6.08 vs. 136.67±6.51); (50.67±1.53 vs. 76.67±4.51)], the number of migration [(606.67±31.34 vs. 960.00±33.06); (483.33±45.96 vs. 741.67±29.67)], the number of invasion [(185.00±8.19 vs. 447.33±22.05); (365.00±33.87 vs. 688.00±32.97)] were lower than those in the si-NC group (P<0.05). However, the apoptosis rates of cells [(13.43±2.79)% vs. (4.53±0.42)%; (23.77±1.04)% vs. (6.60±1.42)%] were higher than those in the si-NC group (P<0.05). The protein expressions of Caspase-3, Caspase-9 and Bax in si-DRAIC group were higher than those in si-NC group, and the protein expression of Bcl-2 was lower than that in si-NC group (P<0.05). DRAIC is located in the cytoplasm. DRAIC targeted and negatively regulated the expression of let-7i-5p. The absorbance values of A549 and H1299 cells in the let-7i-5p mimics group at 48, 72 and 96 hours were lower than those in the miR-NC group (P<0.05), the number of clones formed [(131.33±14.47 vs. 171.33±6.11); (59.33±4.93 vs. 80.33±7.09)], the number of migration [(137.67±3.06 vs. 579.33±82.03); (425.00±11.14 vs. 669.33±21.13)], the number of invasion [(54.00±4.36 vs. 112.67±11.59); (80.00±4.58 vs. 333.33±16.80)] were lower than those in the miR-NC group (P<0.05). However, the apoptosis rates of cells [(14.57±1.10)% vs. (6.97±1.11)%; (23.97±0.42)% vs. (7.07±1.21)%] were higher than those in the miR-NC group (P<0.05). The protein expressions of Caspase-3, Caspase-9 and Bax in let-7i-5p mimics group were higher than those in miR-NC group, and the protein expression of Bcl-2 was lower than that in miR-NC group (P<0.05). The absorbance values of A549 and H1299 cells in the si-DRAIC+ let-7i-5p inhibitor group at 48, 72 and 96 hours were higher than those in the si-DRAIC+ inhibitor-NC group (P<0.05), the number of clones formed [(82.00±5.29 vs. 59.00±5.57); (77.67±4.93 vs. 41.33±7.57)], the number of migration [(774.33±35.81 vs. 455.67±19.04); (569.67±18.72 vs. 433.67±16.77)], the number of invasion [(670.33±17.21 vs. 451.00±17.52); (263.67±3.06 vs. 182.33±11.93)] were higher than those in the si-DRAIC+ inhibitor-NC group (P<0.05). However, the apoptosis rates of cells [(7.73±0.45)% vs. (19.13±1.50)%; (8.00±0.53)% vs. (28.40±0.53)%] were lower than those in the si-NC group (P<0.05). The protein expressions of Caspase-3, Caspase-9 and Bax in si-DRAIC+ let-7i-5p inhibitor group were higher than those in si-DRAIC+ inhibitor-NC group, and the protein expression of Bcl-2 was lower than that in si-DRAIC+ inhibitor-NC group (P<0.05). Conclusion: DRAIC is highly expressed in lung adenocarcinoma, and DRAIC promotes the proliferation, migration and invasion of lung adenocarcinoma cells and inhibits apoptosis by targeting let-7i-5p.

[Modified posteromedial approach via lateral side of flexor hallucis longus for the treatment of posterior Pilon fracture].

Objective: To compare the clinical efficacy and the level of muscle and soft tissue damage between modified posteromedial approach via lateral side of flexor hallucis longus and modified posteromedial approach in the treatment of posterior Pilon fracture. Methods: Total of 43 patients (27 males and 16 females, aged from 19 to 71 years) diagnosed with posterior Pilon fracture from June 2016 to June 2018 in Foshan Hospital of Traditional Chinese Medicine were randomly divided into observation group (modified posteromedial approach via lateral side of flexor hallucis longus, 21 cases) and control group (modified posteromedial approach, 22 cases) according to the operation approach. The preoperative waiting time, intraoperative time, intraoperative blood loss, hospitalization time and the complications were recorded and compared between the two groups. The differences of blood creatine kinase (CK), myoglobin (Myo) and C-reactive protein (CRP) at different time points before and after operation were compared between the two groups to elevate the level of muscle and soft tissue damage. The fracture reduction qualities of the two groups were compared by Burwell-Charnley criteria. The differences of fracture healing time, range of motion of metatarsophalangeal joint of the great toe (MTP-ROM), ankle range of motion (Ankle-ROM), American Orthopaedic Foot & Ankle Society (AOFAS) score and visual analogue scale (VAS) score of pain were compared between the two groups at the last follow-up. Results: The observation group and the control group were followed-up for (19±6) months and (16±8) months, respectively; there was no significant difference between the two groups (P>0.05). There were no significant differences in preoperative waiting time, intraoperative blood loss, hospitalization time and fracture healing time between the two groups (all P>0.05). At the last follow-up, there was no significant difference in the MTP-ROM and Ankle-ROM between the two groups (both P>0.05); the AOFAS score of the observation group was 88.2±7.8 and it was 84.5±7.6 in the control group (P>0.05); the VAS score of the observation group was (0.9±1.0) and it was (1.3±0.8) in the control group(P>0.05). Anatomical reduction rate in observation group was higher than that in control group (90.5% vs 81.8%, P>0.05). The operation time in the observation group was (87±16) min and it was (98±11) min in the control group (P<0.05). CK, Myo and CRP were increased in both groups after surgery, but there was no statistical significance between groups at the same time point (all P>0.05). There was no nerve injury in the observation group, while 2 cases (9.0%) of nerve paralysis occurred in the control group. No incision infection and checkrein deformity of the Hallux was found in the two groups. Conclusion: The modified posteromedial approach via lateral side of flexor hallucis longus can obtain good operative field exposure, and does not increase muscle and soft tissue injury, with shorter operative time and fewer complications, without nerve injury and checkrein deformity, it is a safe approach for the treatment of posterior Pilon fracture.

[Clinical study of Youngswick versus distal oblique osteotomy in the treatment of grade â ¢ and â £ hallux rigidus].

Objective: To investigate the clinical outcomes and effects of distal oblique osteotomy and the Youngswick osteotomy in the treatment of grade Ⅲ and Ⅳ hallux rigidus. Methods: Totally 29 patients (33 feet) suffered from grade Ⅲ and Ⅳ hallux rigidus who received the distal first metatarsal osteotomy in Ningbo NO.6 Hospital from May 2013 to December 2018 were analyzed retrospectively. Among them, there were 10 males and 19 females. The average age was (56±6) years. Seventeen cases (18 feet) underwent distal oblique osteotomy of the first metatarsal, 12 cases (15 feet) underwent the Youngswick osteotomy. The overall clinical evaluations were investigated before the operation and at the final follow-up, included the visual analogue scale (VAS) of the pain, American Orthopedic Foot & Ankle Society (AOFAS) scores, the motionrange of the first metatarsophalangeal joint and the first metatarsophalangeal joint space. The data before and after operation were compared by paired t test and non-parametric rank sum test. Results: The operation time were (1.1±0.2) hours and (1.3±0.1) hours, and the intraoperative bleeding was 35 (25, 36) ml and 35 (30, 40) ml in the distal oblique osteotomy and Youngswick osteotomy respectively. All patients were followed up for 12 to 46 months. No nonunion or delayed union occurred in all patients. All patients did not need to be operated again, and no complications such as transferring metatarsalgia, infection and osteonecrosis occurred. In the two groups, the pain VAS score decreased significantly (Z=-3.8, -3.5, both P<0.01), the first metatarsophalangeal joint dorsal extension activity increased (Z=-3.7, -3.4, both P<0.01), the AOFAS score increased (t=28.0, 15.4, both P<0.05) and the first metatarsophalangeal joint space also improved significantly (t=17.7, 14.6, P<0.05) after the operation. There was no significant difference in VAS score, the first metatarsophalangeal joint activity and Horton index between the two groups(Z=-1.3, -0.3, -0.4, all P>0.05), and there was no statistical difference inAOFAS score (t=0.1, P>0.05). But compared with it in the first distal oblique metatarsal osteotomy, the joint space with the Youngswick osteotomy after the operation was larger ((2.4±0.3) mm vs (2.1±0.4) mm, t=2.2, P=0.04). Conclusions: The oblique metatarsal osteotomy and Youngswick osteotomy can effectively relieve the pain of the hallux rigidus, increase the dorsal extension activity and joint space of the first metatarsophalangeal joint, sink the first metatarsal head, and improve the living conditions of the patients. The results of the distal metatarsal osteotomy and the Youngswick osteotomy in the treatment of stage Ⅲ and Ⅳ patients with rigid are similar, which can delay the progress of the disease.

[Clinical outcomes of gastric cancer patients received capecitabine based adjuvant chemotherapy and the corresponding pharmacogenomics analysis].

Objective: To investigate the association between Thymidine phosphorylase(TYMP)genetic variation and clinical outcomes of postoperative gastric cancer (GC) patients received capecitabine based regimens. Methods: A total of 198 GC patients underwent surgical treatment and received capecitabine based adjuvant chemotherapy were included in this retrospective study. Peripheral blood and the postoperative tissue specimen of the GC patients were collected for the genotyping of polymorphism and TYMP mRNA expression, respectively. The correlation between polymorphism and clinical outcomes and safety of postoperative GC patients were analysed. Results: Located in the upstream, rs11479 was of clinical significance. The prevalence of rs11479 in TYMP among the GC patients were as follows: CC genotype 125 cases (63.13%), CT genotype 65 cases (32.83%), TT genotype 8 cases (4.04%), minor allele frequency of rs11479 is 0.20. The distribution of three genotypes were in accordance with Hardy-Weinberg Equilibrium (P=0.901). The analysis results of patients with different genotypes found that the 3-year disease free survival rate of the patients with CT/TT genotype and CC genotype were 73.97% and 65.60%, respectively, which was statistically significant (P=0.003). In terms of overall survival, the 3-year overall survival rate of the two genotypes were 83.56% and 72.80% (P=0.012), respectively. Adjusted in multivariate Cox regression analysis, CT/TT genotype was an independent favorable factor for disease free survival (OR=0.55, P=0.011). Safety analysis indicated that there was no significant association between genotypes and grade 2 adverse reaction. Additionally, of the 79 postoperative tissue specimens, the results showed that the expression of TYMP in cancer tissues of the patients with CT/TT genotypes were significantly higher than those of the wild type CC genotype patients (P<0.001). Conclusion: The polymorphism rs11479 of TYMP have favorable influence on the clinical outcomes of gastric cancer patients received capecitabine based adjuvant chemotherapy treatment through changing the mRNA expression of TYMP.

Value of pretherapeutic DWI in evaluating prognosis and therapeutic effect in immunocompetent patients with primary central nervous system lymphoma given high-dose methotrexate-based chemotherapy: ADC-based assessment.

AIM: To investigate apparent diffusion coefficient (ADC) as a prognostic indicator in primary central nervous system lymphoma (PCNSL) by analysing patient clinical characteristics and pretherapeutic diffusion-weighted imaging (DWI). MATERIALS AND METHODS: Clinical characteristics and pretherapeutic DWI were studied retrospectively in 28 patients receiving high-dose methotrexate (HD-MTX)-based chemotherapy. Mean (ADCmean), 95th percentile (ADC95%), and 5th percentile (ADC5%) ADC values of the enhancing tumour volume were measured. The influence of prognostic parameters on progression-free survival (PFS) was investigated by log-rank test and Cox regression analysis. Correlations between the variables and PFS or the level of Ki-67 expression were analysed. ADC and clinical features were analysed using an independent sample t-test between the complete response (CRi) and partial response (PRi) groups after initial four cycles of chemotherapy. Receiver operating characteristic (ROC) curves were constructed using ADC parameters. RESULTS: Patients with CRi, lower Ki-67 level, higher Karnofsky performance status (KPS), ADC5%, or ADCmean showed better PFS. The level of Ki-67 expression and ADC5% were independent risk factors. There was a positive correlation between KPS, ADC5%, and PFS, and negative correlation between ADC5%, PFS, and the level of Ki-67 expression. There was a significant difference for PFS, KPS, ADCmean, and ADC5% between CRi and non-CRi; however, ADC5% outperformed ADCmean because the area under the ROC curve (AUC) using ADC5% (0.983) was higher than the AUC using ADCmean (0.822). CONCLUSION: ADC measurements, especially ADC5%, are useful predictors for PFS and response to HD-MTX in PCNSL.

[Analysis of cardiac troponin C gene TNNC1 c. G175C mutation in a Chinese pedigree with familial hypertrophic cardiomyopathy and the correlation between genotype and phenotype].

Objective: To investigate the genotype-phenotype correlation in Chinese familial hypertrophic cardiomyopathy (HCM )focusing on the cardiac troponin C gene TNNC1 c. G175C mutation. Methods: All family members of a Chinese pedigree with hypertrophic cardiomyopathy admitted in Third People's Hospital of Qingdao in February 2005 and 200 healthy volunteers were included in this study. The coding exons of 30 hypertrophic cardiomyopathy associated genes were identified by whole exons amplification and high-throughput sequencing in the proband, and the identified mutation were further detected through bi-directional Sanger sequencing in all family members and 200 healthy volunteers. Pedigree analysis included clinical manifestation, physical examination, ECG and echocardiogram. Results: A missense mutation c. G175C was identified in the TNNC1 gene in 2 family members, which resulted in a glutamic acid (E) to glutamine (Q) exchange at amino acid residue 59. A mutation c. A1319G was identified in the MYLK2 gene in 1 family member, which resulted in a lysine (K) to arginine (R) exchange at amino acid residue 440. These mutations were absent in 200 healthy controls. The proband carried the two kinds of mutations and expressed various clinical manifestations of heart failure and had history of ventricular tachycardia, paraxial atrial fibrillation, pacemaker implantation, electrocardiogram showed right bundle branch block and echocardiography examination evidenced thickened interventricular septum (23.3 mm) and apex and reduced wall motion of these segments. The daughter of the proband carried the TNNC1 c. G175C mutation and was also diagnosed with asymptomatic HCM by echocardiography with thickened interventricular septum (19 mm) and apex (15 mm). Conclusion: The novel missense mutation of TNNC1 c. G175C might be the disease-causing gene mutation in this Chinese pedigree with familiar HCM.

Pedigrees of infertile Chinese men with Y chromosome microdeletions derived from natural transmission and de novo mutation.

Y chromosome microdeletions can cause male infertility and are classified as natural transmission and de novo mutations. To examine the source of these deletions in Chinese men and to provide a theoretical and laboratory basis for genetic counseling, patients from Northeast China with primary male infertility (N = 22) and their fathers were investigated. Karyotype analysis was performed on peripheral blood lymphocytes using standard G-banding. Multiplex polymerase chain reaction amplification using 18 specific sequence-tagged sites was selected to detect Y chromosome microdeletions. De novo mutations were observed in 17 father-son pairs, leading to a mutation rate of 77.27% (17/22), while the vertical transmission of Yq AZFc microdeletions was detected in 5 cases of the families investigated (29.41%, 5/17). There were no statistically significant differences between vertically transmitted and de novo mutations in men with AZFc deletions regarding age, testicular volume, and reproductive hormone levels. Most Y chromosome microdeletions in men from Northeast China are the result of de novo mutations via natural conception, and men with Yq AZFc deletions showed no clear differences between vertical transmission and de novo mutations.

First Report of Leaf Spot Disease on Walnut Caused by Colletotrichum fioriniae in China.

In May 2014, a severe leaf spot disease was observed on walnut tree (Juglans regia L.) in Hechi, Guangxi, China. Leaf spots were circular to semicircular in shape, water-soaked, later becoming grayish white in the center with a dark brown margin and bordered by a tan halo. Necrotic lesions were approximately 3 to 4 mm in diameter. Diseased leaves were collected from 10 trees in each of five commercial orchards. The diseased leaves were cut into 5 × 5 mm slices, dipped in 75% ethanol for 30 s, washed three times in sterilized water, sterilized with 0.1% (w/v) HgCl2 for 3 min, and then rinsed five times with sterile distilled water. These slices were placed on potato dextrose agar (PDA), followed by incubating at 28°C for about 3 to 4 days. Fungal isolates were obtained from these diseased tissues, transferred onto PDA plates, and incubated at 28°C. These isolates produced gray aerial mycelium and then became pinkish gray with age. Moreover, the reverse of the colony was pink. The growth rate was 8.21 to 8.41 mm per day (average = 8.29 ± 0.11, n = 3) at 28°C. The colonies produced pale orange conidial masses and were fusiform with acute ends, hyaline, sometimes guttulate, 4.02 to 5.25 × 13.71 to 15.72 µm (average = 4.56 ± 0.31 × 14.87 ± 1.14 µm, n = 25). The morphological characteristics and measurements of this fungal isolate matched the previous descriptions of Colletotrichum fioriniae (Marcelino & Gouli) R.G. Shivas & Y.P. Tan (2). Meanwhile, these characterizations were further confirmed by analysis of the partial sequence of five genes: the internal transcribed spacer (ITS) of the ribosomal DNA, beta-tubulin (ß-tub) gene, glyceraldehyde 3-phosphate dehydrogenase (GAPDH) gene, chitin synthase 3(CHS-1) gene, and actin (ACT) gene, with universal primers ITS4/ITS5, T1/ßt2b, GDF1/GDR1, CHS1-79F/CHS1-354R, and ACT-512F/ACT-783R, respectively (1). BLAST of these DNA sequences using the nucleotide database of GenBank showed a high identify (ITS, 99%; ß-tub, 99%; GAPDH, 99%; CHS-1, 99%; and ACT, 100%) with the previously deposited sequences of C. fioriniae (ITS, KF278459.1, NR111747.1; ß-tub, AB744079.1, AB690809.1; GAPDH, KF944355.1, KF944354.1; CHS-1, JQ948987.1, JQ949005.1; and ACT, JQ949625.1, JQ949626.1). Koch's postulates were fulfilled by inoculating six healthy 1-year-old walnut trees in July 2014 with maximum and minimum temperatures of 33 and 26°C. The 6-mm mycelial plug, which was cut from the margin of a 5-day-old colony of the fungus on PDA, was placed onto each pin-wounded leaf, ensuring good contact between the mycelium and the wound. Non-colonized PDA plugs were placed onto pin-wounds as negative controls. Following inoculation, both inoculated and control plants were covered with plastic bags. Leaf spots, similar to those on naturally infected plants, were observed on the leaves inoculated with C. fioriniae within 5 days. No symptoms were observed on the negative control leaves. Finally, C. fioriniae was re-isolated from symptomatic leaves; in contrast, no fungus was isolated from the control, which confirmed Koch's postulates. To our knowledge, this is the first report of leaf disease on walnut caused by C. fioriniae. References: (1) L. Cai et al. Fungal Divers. 39:183, 2009. (2) R. G. Shivas and Y. P. Tan. Fungal Divers. 39:111, 2009.

Effect of γ-Aminobutyric Acid-producing Lactobacillus Strain on Laying Performance, Egg Quality and Serum Enzyme Activity in Hy-Line Brown Hens under Heat Stress.

Heat-stress remains a costly issue for animal production, especially for poultry as they lack sweat glands, and alleviating heat-stress is necessary for ensuring animal production in hot environment. A high γ-aminobutyric acid (GABA)-producer Lactobacillus strain was used to investigate the effect of dietary GABA-producer on laying performance and egg quality in heat-stressed Hy-line brown hens. Hy-Line brown hens (n = 1,164) at 280 days of age were randomly divided into 4 groups based on the amount of freeze-dried GABA-producer added to the basal diet as follows: i) 0 mg/kg, ii) 25 mg/kg, iii) 50 mg/kg, and iv) 100 mg/kg. All hens were subjected to heat-stress treatment through maintaining the temperature and the relative humidity at 28.83±3.85°C and 37% to 53.9%, respectively. During the experiment, laying rate, egg weight and feed intake of hens were recorded daily. At the 30th and 60th day after the start of the experiment, biochemical parameters, enzyme activity and immune activity in serum were measured. Egg production, average egg weight, average daily feed intake, feed conversion ratio and percentage of speckled egg, soft shell egg and misshaped egg were significantly improved (p<0.05) by the increasing supplementation of the dietary GABA-producer. Shape index, eggshell thickness, strength and weight were increased linearly with increasing GABA-producer supplementation. The level of calcium, phosphorus, glucose, total protein and albumin in serum of the hens fed GABA-producing strain supplemented diet was significantly higher (p<0.05) than that of the hens fed the basal diet, whereas cholesterol level was decreased. Compared with the basal diet, GABA-producer strain supplementation increased serum level of glutathione peroxidase (p = 0.009) and superoxide dismutase. In conclusion, GABA-producer played an important role in alleviating heat-stress, the isolated GABA-producer strain might be a potential natural and safe probiotic to use to improve laying performance and egg quality in heat-stressed hens.

Proteolysis and cathepsin activities in the processing of dry-cured duck.

Changes in sarcoplasmic and myofibrillar proteins, free amino acids, and cathepsin activities were measured to evaluate the contribution of cathepsins to the proteolysis of muscle proteins in dry-cured duck processing. Thirty-six dry-cured ducks were processed with the traditional method, and samples were collected at different stages. Sarcoplasmic and myofibrillar proteins were found to be degraded to some degree at different stages, whereas content of free amino acids increased from 43.9 to 133.97 mg/100 g during the whole process. Cathepsin B, D, and L activities decreased significantly, and the activities in the end product were 22.4, 26.2, and 40.5% of those in the raw material, respectively. Statistical analysis showed there were significant correlations among changes in proteins, free amino acids, and cathepsin activities. The results indicated that cathepsins are involved in the proteolysis of muscle proteins in dry-cured duck processing.

Mutations in the alpha 1 subunit of the inhibitory glycine receptor cause the dominant neurologic disorder, hyperekplexia.

Hereditary hyperekplexia, or familial startle disease (STHE), is an autosomal dominant neurologic disorder characterized by marked muscle rigidity of central nervous system origin and an exaggerated startle response to unexpected acoustic or tactile stimuli. Linkage analyses in several large families provided evidence for locus homogeneity and showed the disease gene was linked to DNA markers on the long arm of chromosome 5. Here we describe the identification of point mutations in the gene encoding the alpha 1 subunit of the glycine receptor (GLRA1) in STHE patients from four different families. All mutations occur in the same base pair of exon 6 and result in the substitution of an uncharged amino acid (leucine or glutamine) for Arg271 in the mature protein.

Thanatophoric dysplasia (types I and II) caused by distinct mutations in fibroblast growth factor receptor 3.

Thanatophoric dysplasia (TD), the most common neonatal lethal skeletal dysplasia, affects one out of 20,000 live births. Affected individuals display features similar to those seen in homozygous achondroplasia. Mutations causing achondroplasia are in FGFR3, suggesting that mutations in this gene may cause TD. A sporadic mutation causing a Lys650Glu change in the tyrosine kinase domain of FGFR3 was found in 16 of 16 individuals with one type of TD. Of 39 individuals with a second type of TD, 22 had a mutation causing an Arg248Cys change and one had a Ser371Cys substitution, both in the extracellular region of the protein. None of these mutations were found in 50 controls showing that mutations affecting different functional domains of FGFR3 cause different forms of this lethal disorder.

Expression, Identification and Characterize of CD25-Binding Epitope Modified Human IL-2 in Pichia pastoris.

Interleukin-2 (IL-2) plays important roles in variety of immune functions and it is widely used in the medication. But in recent years it was reported that vascular leak syndrome (VLS) was induced by IL-2. Evidences showed that the interaction of IL-2 and IL-2Rαßγ (CD25) caused VLS. Thus, this experiment modified the CD25-binding epitope in human IL-2 (hIL-2) to minimize the side effect of IL-2 in the medication. In this study, a recombinant human interleukin 2 (rhIL-2) was expressed in Pichia (P.) pastoris. An effective strategy was established to express rhIL-2 protein in 120 L scale and the optimal purification procedure was investigated. The purity of rhIL-2 in final product was about 98 % and the concentration of the rhIL-2 was 0.45 mg/mL. Bioactivity analysis showed that the purified rhIL-2 protein displayed high activity on proliferation of CTLL-2 cells and increased the ratio of CD4(+)/CD8(+). It indicates that the target protein is expressed and the character of the rhIL-2 has high activity. This study provides a strategy for large-scale production of bioactive rhIL-2 protein using P. pastoris as an expression host.

The role of liver metabolism in compensatory-growth piglets induced by protein restriction and subsequent protein realimentation.

The aim of this work was to study the role of hepatic metabolism of compensatory growth in piglets induced by protein restriction and subsequent protein realimentation. Thirty-six weaned piglets were randomly distributed in a control group and a treatment group. The control group piglets were fed with a normal protein level diet (18.83% CP) for the entire experimental period (day 1-28). The treatment group piglets were fed with a protein-restriction diet (13.05% CP) for day 1 to day 14, and the diet was restored to normal protein level diet for day 15 to day 28. RNA-seq is used to analyze samples of liver metabolism on day 14 and day 28, respectively. Hepatic RNA-sequencing analysis revealed that some KEGG signaling pathways involved in glycolipid metabolism (eg, "AMPK signaling pathway," "insulin signaling pathway," and "glycolysis or gluconeogenesis") were significantly enriched on day 14 and day 28. On day 14, protein restriction promoted hepatic lipogenesis by increasing the genes expression level of ACACA, FASN, GAPM, and SREBP1C, decreasing protein phosphorylation levels of AMPKɑ and ACC in AMPK signaling pathway. In contrast, on day 28, protein realimentation promoted hepatic gluconeogenesis by increasing the concentration of G6Pase and PEPCK, decreasing protein phosphorylation levels of IRS1, Akt, and FoXO1 in insulin signaling pathway. In addition, protein realimentation activated the GH-IGF1 axis between the liver and skeletal muscle. Overall, these findings revealed the importance of liver metabolism in achieving compensatory growth.

Hepatitis B virus X protein induces hypermethylation of p16(INK4A) promoter via DNA methyltransferases in the early stage of HBV-associated hepatocarcinogenesis.

The aim of the present study was to authenticate the involvement of DNA methyltransferases (DNMTs) and methyl-CpG binding domain protein 2 (MBD2) in the process of HBx induced p16(INK4A) promoter hypermethylation in HBV-related hepatocellular carcinoma (HCC) and their corresponding noncancerous liver tissues. Eighty-eight fresh tissue specimens of surgically resected HBV-associated HCC and their corresponding noncancerous liver tissues were studied. The methylation status of the p16(INK4A) promoter was determined by methylation-specific polymerase chain reaction (MSP). Reverse transcription and real-time polymerase chain reaction (RT-PCR) showed the expression of DNMTs, MBD2 and HBx. Western blot and immunohistochemistry were used for the protein analysis of HBx, DNMT1, DNMT3A and P16. Tissue HBV-DNA levels were determined by RT-PCR. HBV genotype was examined by nested PCR and restriction fragment length polymorphism (RFLP). In the corresponding noncancerous liver tissues, higher HBx expression was associated with the hypermethylation of the p16(INK4A) promoter. HBx was positively correlated with the DNMT1 and DNMT3A at both the mRNA and protein level. Furthermore, HBx, DNMT1 and DNMT3A protein expression were negatively correlated with p16 protein expression. In HCC tissues, HBx was positively correlated with DNMT1 and DNMT3A at both mRNA and protein level, but HBx expression did not correlate with hypermethylation of the p16(INK4A) promoter or p16 protein expression. The methylation status of the p16(INK4A) promoter did not correlate with clinicopathological characteristics. DNMT1 and DNMT3A may play important roles in the process of HBx inducing hypermethylation of the p16(INK4A) promoter in the early stages of HBV-associated HCC. HBx-DNMTs-p16(INK4A) promoter hypermethylation may constitute a mechanism for tumorigenesis during HBV-associated hepatocarcinogenesis.

Chronic stimulation of corticotropin-releasing factor receptor 1 enhances the anxiogenic response of the cholecystokinin system.

Corticotropin-releasing factor (CRF) and cholecystokinin (CCK), two highly colocalized neuropeptides, have been linked to the etiology of stress-related anxiety disorders. Recent evidence points to the possibility that some of the anxiogenic effects of the central CCK system take place through interplay with the CRF system. The aim of the present study was to examine the effects of chronic, mild activation of CRF receptor 1 (CRF(1)) on the central CCK system of the C57BL/6J mouse. As shown by in situ hybridization, real-Time PCR and immunohistochemistry, 5 days of intracerebroventricular (i.c.v.) injections of a subeffective dose (2.3 pmol) of cortagine, a CRF(1)-selective agonist, resulted in an increase in CCK mRNA levels and CCK(2) receptor immunoreactivity in several brain regions, such as amygdala and hippocampus, known to be involved in the regulation of anxiety. Mice with elevated endogenous central CCK tone exhibited significantly higher anxiety-like behaviors in the open-field task and elevated plus maze, and enhanced conditioned fear. These behavioral changes were reversed by i.c.v. administration of the CCK(2)-selective antagonist LY225910, after 5 days of priming with cortagine. Under the same conditions, the intraperitoneal administration of the CRF(1) antagonist antalarmin was ineffective. This result indicated that once the CCK system was sensitized by prior CRF(1) activation, it exhibited its anxiogenic effects, without influence by CRF(1), possibly because of its observed downregulation. In sum, our results provide a novel model for the interaction of the CRF and CCK systems contributing to the development of hypersensitive emotional circuitry.

First Report of Southern Blight on Canadian Goldenrod (Solidago canadensis) Caused by Sclerotium rolfsii in China.

Canadian goldenrod (Solidago canadensis L., Asteraceae) is a rhizomatous perennial plant native to North America that has invaded eastern China and continues to spread northward and westward. It is quite common on field borders, roadsides, and in undeveloped areas, posing a serious threat to native ecosystems and their biodiversity. During the late summers of 2007 and 2008, wilted Canadian goldenrod plants were occasionally found in invasive populations in the suburb of Nanjing city. Wilted plants were transplanted and maintained in a greenhouse at Nanjing Agricultural University. A white mass of fungal hyphae, which grew on the soil surface around the stem of the symptomatic S. canadesis plants and eventually covered the stem, was observed. Initially, the base of the stem became yellow, turned brown, and the light brown discoloration extended up the stem to a height of 3 to 7 cm. The leaves then collapsed, starting from the top until the entire plant wilted. The fungus produced numerous, small, roundish sclerotia of uniform size (0.7 to 2.0 mm in diameter), which were white at first and then became brown to dark brown. The fungus grew into the stems and downward into the rhizome area, but no sclerotia were detected inside the stem or root. Diseased tissue with sclerotia was disinfested for 1 min in 1% NaOCl and plated on potato dextrose agar amended with 100 mg/liter of streptomycin sulfate. On the basis of sclerotia morphology and the presence of clamp connections at hyphal septa, the fungus was identified as Sclerotium rolfsii. Pathogenicity of the isolate was confirmed by inoculating 1-year-old S. canadensis plants (average 1.5 m high) grown in pots. The inoculum consisted of cottonseed hulls infested with mycelium and sclerotia of the pathogen and was placed on the soil surface around the base of each unwounded plant. Noninoculated plants served as controls. The pathogenicity test was conducted twice. After inoculation, the plants were maintained at high humidity and 30°C for 3 days and then transferred to a greenhouse. All inoculated plants developed symptoms of southern blight. Inoculated plants developed symptoms of wilting 5 to 7 days after inoculation and were completely wilted within 15 to 20 days. Symptoms of wilting were soon followed by the appearance of white-to-light brown sclerotia on the collar region. Control plants remained symptomless and Sclerotium rolfsii was reisolated from inoculated plants. To our knowledge, this is the first report of southern blight of Canadian goldenrod caused by Sclerotium rolfsii in China.

Composition of intramuscular phospholipids and free fatty acids in three kinds of traditional Chinese duck meat products.

Composition of intramuscular phospholipids and free fatty acids in 3 kinds of traditional Chinese duck meat products (dry-cured duck, roasted duck, and water-boiled salted duck) was investigated. Phospholipids were identified and quantified by HPLC combined with ultraviolet and evaporative light scattering detectors. The types and quantities of free fatty acids and fatty acids derived from phospholipids were analyzed by capillary gas chromatography. The results showed that raw duck meat had high quantities of phosphatidyl-ethanolamine and phosphatidylcholine (28.1 and 64.7% of total phospholipids, respectively), which contained high percentages of polyunsaturated fatty acids. The percentages of total phospholipids, phosphatidylethanolamine, and phosphatidylcholine decreased during processing, with a concomitant increase in quantities of free fatty acids. A selective degradation of fatty acids distributions of phospholipids was found through the dry-cured duck processing, but was not found during the roasted and water-boiled duck processing.