MiR-378a-5p inhibits angiogenesis of oral squamous cell carcinoma by targeting KLK4.

Oral squamous cell carcinoma (OSCC) is still a leading cause of cancer death owing to distant metastasis, which is largely facilitated by tumor angiogenesis. MicroRNA (miR)-378a-5p and Kallikrein-related peptidase 4 (KLK4) participate in tumorigenesis and tumor metastasis according to previous studies, yet the exact role they play in tumor angiogenesis remains poorly understood. The aim of the present study is to investigate the effect of miR-378a-5p and KLK4 on angiogenesis of OSCC. MTT assay showed that the expression level of miR-378a-5p was negatively correlated with the proliferation of OSCC cells. ELISA and Western blot assay showed that down-regulation of miR-378a-5p promotes VEGF expression. Tube formation and in vivo chicken chorioallantoic membrane (CAM) assay showed that inhibition of miR-378a-5p reduced tube formation of human umbilical vein endothelial cells (HUVECs) and newly formed microvessel. On the contrary, over-expression of KLK4 enhanced angiogenesis of OSCC cells with increased VEGF expression, tube formation activity of HUVECs and newly formed microvessel. Moreover, the dual-luciferase assay validated that KLK4 was a target gene of miR-378a-5p. MiR-378a-5p silencing induced tube formation was suppressed by the downregulation of KLK4. Besides, the activation of Wnt/ß-catenin signaling pathway in miR-378a-5p antagomir transfected cells was also blocked by the KLK4 shRNA. To sum up, our study suggests that miR-378a-5p suppressed angiogenesis of OSCC at least partly by the regulation of KLK4.

[Correlation between the new lung adenocarcinoma classification and epidermal growth factor receptor mutation].

OBJECTIVE: To evaluate the association of the histological subtype of lung adenocarcinoma with epidermal growth factor receptor (EGFR) mutation. METHODS: A total of 94 patients with resected lung adenocarcinoma in the Department of Thoracic Surgery of China-Japan Friendship Hospital from January 2010 to December 2014 were enrolled in the study. All specimens were tested for EGFR mutation by a company. In the 94 patients, histological subtypes were classified according to the 2011 International Association for the Study of Lung Cancer and American Thoracic Society and European Respiratory Society classification. We compared the association with the histological subtype of lung adenocarcinoma with EGFR mutation frequency by the χ2 test, with SPSS 20.0. RESULTS: The 94 patients of surgically resected lung adenocarcinomas were included in this analysis, of whom, 47 were male and 47 female (male:female=1:1). The median age was 61 (range: 24-79) years, and 48 of the 94 patients were 60 years and above. Regarding the pathological staging, 34 patients were diagnosed as Stage I of the disease, 17 as Stage II,24 as Stage III, and 19 as Stage IV. Among the 51 patients with EGFR mutation, exon 19 mutation was 22, exon 20 mutation was 2, exon 21 mutation was 26, exon 20 and 21 mutation were 1, and the total EGFR mutation rate was 54.3% (51/94). The cases of EGFR gene mutation of acinar predominant lung adenocarcinoma, lepidic predominant lung adenocarcinoma, papillary predominant lung adenocarcinoma, solid predominant lung adenocarcinoma, micropapillary predominant lung adenocarcinoma and mucious adenocarcinoma were 24, 14, 5, 5, 3, and 0, respectively. The rate of EGFR gene mutation of acinar predominant lung adenocarcinoma was higher than that of non-acinar predominant lung adenocarcinom, but there was not statistically significant (66.7% vs. 46.6%, P=0.057). The rate of EGFR gene mutation of solid predominant lung adenocarcinoma was lower than that of non-solid predominant lung adenocarcinom (26.3% vs. 61.3%, P=0.005). The rate of EGFR gene mutation of mucious adenocarcinoma was lower than that of non-mucious adenocarcinom (0 vs. 57.3%, P=0.018). CONCLUSION: There is heterogeneity of EGFR mutation in lung adenocarcinoma. The presence of lung adenocarcinoma with acinar indicates a higher EGFR mutation rate, while the solid and mucinous component indicates a lower EGFR mutation rate.

Apelin-12 exerts neuroprotective effect against ischemia-reperfusion injury by inhibiting JNK and P38MAPK signaling pathway in mouse.

OBJECTIVE: Cerebral ischemia is a common neurological disease, and its pathological process remains elusive. This study focused on the protective mechanism of Apelin-12 protein on the nervous system of mice during cerebral ischemia-reperfusion injury through JNK and P38MAPK signaling pathway. MATERIALS AND METHODS: The mouse model with an ischemia-reperfusion injury in middle cerebral artery was prepared by the modified thread-occlusion method and divided into 4 groups randomly. Before implantation of the mice, we assessed the neurological function and evaluated the cerebral edema by the wet-dry weight method. Lactate dehydrogenase (LDH) kit was used to assess the degree of cell injury. Malondialdehyde (MDA) kit was used to measure the level of neuron MDA. Immunohistochemistry was performed to evaluate the neuronal cell in the ischemic brain. Protein expressions of JNK and P38MAPK and apoptosis-related molecules, including Bax, Bcl-2, caspase-3, and cleaved caspase-3, were measured by Western blot assay. RESULTS: After focal cerebral ischemia-reperfusion, a significant decrease in neurobehavioral score, brain edema and neuron injury in mice occurred. Apelin-12 significantly improved the neurobehavioral score of the mice with ischemia-reperfusion injury, alleviated brain edema and the damage to neurons. In addition, Apelin-12 inhibited the morphological changes and apoptosis of neuronal cells in the ischemic penumbra of mice. Apelin-12 could downregulate the expression of Bax and caspase-3, inhibit the activity of caspase-3 and upregulate the expression of Bcl-2, an anti-apoptotic protein. A significant reduction in the protein expression of p-JNK and p-p38 was observed in the Apelin-12 group compared with that in the I/R or Vehicle group (p<0.05). CONCLUSIONS: When an ischemia-reperfusion injury occurred, Apelin-12 can inhibit the JNK and P38MAPK signaling pathway of the apoptosis-related MAPKs family, thus offering protection to neurons.

[Antimicrobial susceptibility and drug-resistance genes of Yersinia spp. of retailed poultry in 4 provinces of China].

Objective: To monitor the antimicrobial resistance and drug-resistance genes of Yersinia enterocolitis, Y. intermedia and Y. frederiksenii recovered from retailed fresh poultry of 4 provinces of China. Methods: The susceptibility of 25 isolated Yersinia spp. to 14 classes and 25 kinds of antibiotics was determined by broth microdilution method according to CLSI (Clinical and Laboratory Standards Institute). The antibiotic resistance genes were predicted with antibiotic resistance genes database (ARDB) using whole genome sequences of Yersinia spp. Results: In all 22 Y. enterocolitis tested, 63.7% (14 isolates), 22.8% (5 isolates), 4.6% and 4.6% of 1 isolates exhibited the resistance to cefoxitin, ampicillin-sulbactam, nitrofurantoin and trimethoprim-sulfamethoxazole, respectively. All the 25 isolates were multi-drug resistant to more than 3 antibiotics, while 64.0% of isolates were resistant to more than 4 antibiotics. A few Y. enterocolitis isolates of this study were intermediate to ceftriaxone and ciprofloxacin. Most Yersinia spp. isolates contained antibiotic resistance genes mdtG, ksgA, bacA, blaA, rosAB and acrB, and 5 isolates recovered from fresh chicken also contained dfrA1, catB2 and ant3ia. Conclusion: The multi-drug resistant Yersinia spp. isolated from retailed fresh poultry is very serious in the 4 provinces of China, and their contained many kinds of drug-resistance genes.

[Clinical characteristics of human recombination activating gene 1 mutations in 8 immunodeficiency patients with diverse phenotypes].

Objective: To investigate the clinical characteristics of 8 immunodeficiency cases caused by human recombination activating gene 1 (RAG1) mutations, and to explore the relationship among genotypes, clinical manifestations and immunophenotypes. Methods: Clinical data were collected and analyzed from patients with RAG1 mutations who visited the Department of Clinical Immunology, Children's Hospital of Fudan University between October 2013 and June 2017. The data included clinical manifestations, immunophenotypes and genotypes. Results: A total of 8 patients were diagnosed with RAG1 deficiency (6 boys and 2 girls). The minimum age of onset was 2 months, and the maximum age was 4 months. The minimum age of diagnosis was 2 months, and the maximum age was 13 years. Four patients had a family history of infant death due to severe infections. Two cases were born to the same consanguineous parents. All cases had recurrent infections, including involvement of respiratory tract (8 cases), digestive tract (6 cases), urinary tract (1 case), and central nervous system (1 case). The pathogens of infection included bacteria, viruses and fungi. Rotavirus was found in 3 cases, cytomegalovirus (CMV) in 5 cases, bacillus Calmette-Guérin adverse reaction in 2 cases (1 of whom had a positive acid-fast smear from lymph node puncture fluid), fungal infection in 3 cases. One case had multiple nodular space-occupying lesions in lungs and abdominal cavity complicated with multiple bone destruction. The peripheral blood lymphocyte counts of all patients ranged between 0.1 ×10(9)/L and 3.3×10(9)/L (median, 0.65×10(9)/L). Eosinophilia was found in 3 cases (range, (0.48-1.69) ×10(9)/L). The patients were classified according to immunophenotype as severe combined immunodeficiency phenotype (4 cases), leaky severe combined immunodeficiency (2 cases), Omenn syndrome (1 case) and combined immunodeficiency (1 case) . Decreased serum IgG levels were found in 3 cases, increased serum IgM levels in 3 cases, increased serum IgE levels in 5 cases. RAG1 homozygous mutations were detected in 5 cases and RAG1 compound heterozygous mutations in 3 cases. Two novel mutations and six previously reported mutations were identified. Three cases were successfully treated with hematopoietic stem cell transplantation. Four cases died due to infections, and the 13 year-old patient was still under follow-up in the outpatient clinic. Conclusions: Different RAG1 gene mutations can lead to diverse clinical presentations and immune phenotypes. Clinicians should pay attention to the family history of infant death with severe infection. In that situation, immunological evaluation and gene detection should be performed as early as possible.

[Laboratory diagnosis of chronic granulomatous disease].

OBJECTIVE: To evaluate the methods of flow cytometric-dihydrorhodamine 123 (DHR) analysis, gp91 protein detection, gene mutation analysis for the precise diagnosis of chronic granulomatous disease (CGD). METHOD: Clinical and laboratory data of patients with CGD confirmed by gene mutation analysis from 2008 to 2015 in Children's Hospital of Fudan University were retrospectively reviewed.The results of respiratory burst, gp91 protein level, and gene mutations were analyzed.The relationships among these three methods were explored. RESULT: A total of 138 patients of CGD with confirmed gene mutation were included in this study, of them, 123 cases(89.1%) had CYBB gene mutation, 4 cases(2.9%) had CYBA mutation, 5 cases(3.6%) had NCF1 mutation and 6 cases(4.4%) had NCF2 mutation.The range of stimulatory index (SI) was 0.8-60.5, the 25 th, 50 th, 75th percent was 1.7, 2.7, 4.7; 112 cases had the results of gp91, of them, 100 with gp91(0,) 2 with gp91(-), and 10 with gp91(+) . Six mutations, which were not reported before, were c. 76-77delTT, c. 343-344delCA, c. 481A>T, c. 1152G>C, c. 1613G>A for CYBB gene, and c. 137T>G for NCF2 gene. Among CGD patients with CYBB mutation, SI of patients with gp91(+) was higher than patients with gp91(0) 14.6 vs. 2.5(t=44.21, P=0.004). Patients of NCF1 mutation had higher SI than patients with CYBB mutation, 17.7 vs. 2.5 (t=60.8, P=0.003). CONCLUSION: Flow cytometric-DHR analysis and gp91 protein detection are important diagnostic methods for CGD, they could help the precise diagnosis of CGD.Different mutation types, different mutation genes could have impact on the results of respiratory burst and gp91 level.The application of diagnostic technology from function, protein to gene analysis could help precise diagnosis of CGD.

Molecular characterization, tissue distribution, and immune reaction expression of karyopherins in the domestic silkworm (Bombyx mori).

Karyopherins, including alpha and beta types, are transport proteins in the eukaryotic cell that carry cargoes across nuclear pore complexes into or out of the nucleus. In this study, full open reading frames of one beta and three alpha types of karyopherin were cloned from cDNA of the domestic silkworm (Bombyx mori). The one beta and three alpha types' open reading frames were 2661, 1563, 1515, and 1551 base pairs long, respectively, and coded 886, 520, 504, and 516 amino acids, respectively. The alphas all had one importin-beta-binding (IBB) domain, and eight, four, or seven armadillo/beta-catenin-like repeats. The beta had 19 HEAT repeat domains, which constructed one importin-beta-N-terminal domain and one IBB domain. The recombinant proteins were expressed in Escherichia coli cells. The molecular weight of the beta type was approximately 100 kDa, and the alphas weighed approximately 60 kDa. Phylogenic tree construction revealed that the alphas could be classified into three known karyopherin-alpha subfamilies. We detected mRNA of the four karyopherins in normal 3rd day of 5th instar larvae, and in larvae injected with Gram-positive bacteria, Gram-negative bacteria, viruses, and fungi using real-time fluorescence quantitative reverse transcriptase-polymerase chain reaction, and found that the four karyopherins were widely distributed, but their expression levels were related to tissues type, the microbe injected, and the time point.

Giant nonlinear optical properties of bismuth thin films grown by pulsed laser deposition.

Thin films of Bi were grown by pulsed laser deposition on glass substrates at room temperature. The thickness and roughness of the films were characterized by grazing-incidence x-ray reflectivity, and the complex refractive indices were measured in the range from 1.5 to 4 eV by spectroscopic ellipsometry. We performed Z-scan measurements to study the third-order optical nonlinearity of the films. It was found that the Bi films exhibited an unusually large nonlinear refractive coefficient, n(I)~1.24x10(-1) cm(2)/kW and nonlinear absorption coefficient, alpha(I)~-3.97 cm/W , at low laser intensity, ~60 kW/cm(2) . This anomaly is believed to have an origin related to melting of the Bi films at the focus spot by the laser beam.

[Clinical observation on effect of electroacupuncture therapy in treating superficial tumor].

OBJECTIVE: To investigate the clinical effect of electroacupuncture therapy (EAT) in treating superficial tumors. METHODS: The healthy tissue was protected by insulation sleeve, and the platinum electrodes served as needles was inserted into the tumor and connected to an EAT instrument using galvanic current. The electric voltage applied was 6-8 V, the electric current was in a range of 40-80 mA, and 80-100 coulomb electricity for 1 cm diameter of tumor mass was administered. RESULTS: In the 320 cases, 123 were complete remission (CR), 129 partial remission (PR), 36 with their tumor shrinked by 1/4 and 32 with size of tumor unchanged. The total effective rate (CR + PR) was 78.7%. CONCLUSION: EAT shows good effect in treating superficial tumor and provides a new therapeutic means for the patients with tumor of unresectable or relapsed. It is a simple, convenient, safe and effective method with less injury and quick recovery.

Progress toward the evolution of an organism with an expanded genetic code.

Several significant steps have been completed toward a general method for the site-specific incorporation of unnatural amino acids into proteins in vivo. An "orthogonal" suppressor tRNA was derived from Saccharomyces cerevisiae tRNA2Gln. This yeast orthogonal tRNA is not a substrate in vitro or in vivo for any Escherichia coli aminoacyl-tRNA synthetase, including E. coli glutaminyl-tRNA synthetase (GlnRS), yet functions with the E. coli translational machinery. Importantly, S. cerevisiae GlnRS aminoacylates the yeast orthogonal tRNA in vitro and in E. coli, but does not charge E. coli tRNAGln. This yeast-derived suppressor tRNA together with yeast GlnRS thus represents a completely orthogonal tRNA/synthetase pair in E. coli suitable for the delivery of unnatural amino acids into proteins in vivo. A general method was developed to select for mutant aminoacyl-tRNA synthetases capable of charging any ribosomally accepted molecule onto an orthogonal suppressor tRNA. Finally, a rapid nonradioactive screen for unnatural amino acid uptake was developed and applied to a collection of 138 amino acids. The majority of glutamine and glutamic acid analogs under examination were found to be uptaken by E. coli. Implications of these results are discussed.

Characterization of an 'orthogonal' suppressor tRNA derived from E. coli tRNA2(Gln).

BACKGROUND: In an effort to expand further our ability to manipulate protein structure, we have completed the first step towards a general method that allows the site-specific incorporation of unnatural amino acids into proteins in vivo. Our approach involves the construction of an 'orthogonal' suppressor tRNA that is uniquely acylated in vivo, by an engineered aminoacyl-tRNA synthetase, with the desired unnatural amino acid. The Escherichia coli tRNA2(Gln)-glutaminyl-tRNA synthetase (GlnRS) pair provides a biochemically and structurally well-characterized starting point for developing this methodology. To generate the orthogonal tRNA, mutations were introduced into the acceptor stem, D-loop/stem, and anticodon loop of tRNA2(Gln). We report here the characterization of the properties of the resulting tRNAs and their suitability to severe as an orthogonal suppressor. Our efforts to generate an engineered synthetase are described elsewhere. RESULTS: Mutant tRNAs were generated by runoff transcription and assayed for their ability to be aminoacylated by purified E. coli GlnRS and to suppress an amber codon in an in vitro transcription/translation reaction. One tRNA bearing eight mutations satisfies the minimal requirements for the delivery of an unnatural amino acid: it is not acylated by any endogenous E. coli aminoacyl-tRNA synthetase, including GlnRS, yet functions efficiently during protein translation. Mutations in the acceptor stem and D-loop/stem, when introduced in combination, had very different effects on the properties of the resulting tRNAs compared with the effects of the individual mutations. CONCLUSIONS: Mutations at sites within tRNA2(Gln) separated by 23-31 A interact strongly with each other, often in a nonadditive fashion, to modulate both aminoacylation activities and translational efficiencies. The observed correlation between the effects of mutations at very distinct regions of the GlnRS-tRNA and possibly the ribosomal/tRNA complexes may contribute in part to the fidelity of protein biosynthesis.

Engineering a tRNA and aminoacyl-tRNA synthetase for the site-specific incorporation of unnatural amino acids into proteins in vivo.

In an effort to expand the scope of protein mutagenesis, we have completed the first steps toward a general method to allow the site-specific incorporation of unnatural amino acids into proteins in vivo. Our approach involves the generation of an "orthogonal" suppressor tRNA that is uniquely acylated in Escherichia coli by an engineered aminoacyl-tRNA synthetase with the desired unnatural amino acid. To this end, eight mutations were introduced into tRNA2Gln based on an analysis of the x-ray crystal structure of the glutaminyl-tRNA aminoacyl synthetase (GlnRS)-tRNA2Gln complex and on previous biochemical data. The resulting tRNA satisfies the minimal requirements for the delivery of an unnatural amino acid: it is not acylated by any endogenous E. coli aminoacyl-tRNA synthetase including GlnRS, and it functions efficiently in protein translation. Repeated rounds of DNA shuffling and oligonucleotide-directed mutagenesis followed by genetic selection resulted in mutant GlnRS enzymes that efficiently acylate the engineered tRNA with glutamine in vitro. The mutant GlnRS and engineered tRNA also constitute a functional synthetase-tRNA pair in vivo. The nature of the GlnRS mutations, which occur both at the protein-tRNA interface and at sites further away, is discussed.

Development of improved tRNAs for in vitro biosynthesis of proteins containing unnatural amino acids.

BACKGROUND: Chemically aminoacylated suppressor tRNAs have previously been used in vitro to generate mutant proteins in which unnatural amino acids are incorporated site-specifically. Although the existing methodology often provides adequate quantities of mutant proteins, the suppression efficiencies of some unnatural amino acids are not high enough to yield useful amounts of protein. In an effort to make this useful mutagenesis strategy more general, we report here the results of a search to find alternative tRNAs as a way of increasing suppression efficiencies. RESULTS: Three suppressor tRNAs have been generated by runoff transcription and their ability to deliver unnatural amino acids site-specifically into proteins has been assessed in an E. coli-derived in vitro transcription/translation system. Analysis of their ability to insert both polar and nonpolar residues in response to an amber codon in two proteins suggests that an E. coli tRNAAsn-derived suppressor offers a significant improvement in suppression efficiency over other previously used tRNAs. CONCLUSIONS: Use of an E. coli tRNAAsn-derived suppressor may provide substantially higher yields of proteins containing unnatural amino acids, in addition to offering a broader tolerance for polar amino acids. A comparison of suppressor tRNAs derived from tRNAAsn, tRNAGln or tRNAAsp with that derived from tRNAPhe supports emerging evidence that the identity of an amino acid may be important in message recognition.

Molecular cloning of the human gene encoding lanosterol synthase from a liver cDNA library.

Lanosterol synthase [(S)-2,3-epoxysqualene mutase (cyclizing, lanosterol forming), EC 5.4.99.7] catalyzes the cyclization of (S)-2,3-oxidosqualene to lanosterol in the reaction that forms the sterol nucleus. We report herein the cloning and characterization of the human gene (OSC) encoding lanosterol synthase, a predicted 83 kDa protein of 732 amino acids. The deduced amino acid sequence is 36-40% identical to known yeast and plant homologues and 83% identical to Rattus norvegicus lanosterol synthase. The new gene was shown to encode lanosterol synthase. The yeast lanosterol synthase deficient mutant SMY8 was complemented by the human gene, and a cell-free homogenate of SMY8 expressing the human gene was shown to convert 2,3-oxidosqualene to lanosterol.

Accumulation of wound-inducible ACC synthase transcript in tomato fruit is inhibited by salicylic acid and polyamines.

Regulation of wound-inducible 1-aminocyclopropane-1-carboxylic acid (ACC) synthase expression was studied in tomato fruit (Lycopersicon esculentum cv. Pik-Red). A 70 base oligonucleotide probe homologous to published ACC synthase cDNA sequences was successfully used to identify and analyze regulation of a wound-inducible transcript. The 1.8 kb ACC synthase transcript increased upon wounding the fruit as well as during fruit ripening. Salicylic acid, an inhibitor of wound-responsive genes in tomato, inhibited the wound-induced accumulation of the ACC synthase transcript. Further, polyamines (putrescine, spermidine and spermine) that have anti-senescence properties and have been shown to inhibit the development of ACC synthase activity, inhibited the accumulation of the wound-inducible ACC synthase transcript. The inhibition by spermine was greater than that caused by putrescine or spermidine. The transcript level of a wound-repressible glycine-rich protein gene and that of the constitutively expressed rRNA were not affected as markedly by either salicylic acid or polyamines. These data suggest that salicylic acid and polyamines may specifically regulate ethylene biosynthesis at the level of ACC synthase transcript accumulation.

Thyroid autoimmunity in endemic goitre caused by excessive iodine intake.

The pathophysiology of endemic goitre caused by excessive iodine intake is not well defined. By interacting with the immune system, iodine excess may trigger the development of autoimmune thyroid disease such as lymphocytic Hashimoto's thyroiditis (LT). In an attempt to examine this further, we compared the presence of thyroid autoantibodies in 29 goitrous children, from an iodine excess area, and in 26 healthy children, from an iodine sufficient area, of north central China. Serum was tested for antimicrosomal (MAb), anti-thyroglobulin (TgAb), second colloid antigen antibodies (CA2-Ab) and TSH binding inhibitory immunoglobulins (TBII). Affinity chromatographically purified IgG was tested for thyroid growth-stimulating activity (TGI) by two different methods: a sensitive cytochemical bioassay (CBA) using guinea-pig thyroid explants and a mitotic arrest assay (MAA) employing a continuous rat thyroid cell line (FRTL-5). We found no increased prevalence of LT in patients with endemic iodine goitre. The levels of MAb, TgAb and CA2-Ab did not differ significantly between the two groups of children. Further, TBII were not present in either group. Thyroid growth-stimulating immunoglobulins (TGI) were the major autoantibodies found in children with goitres caused by iodine excess. In the CBA, 12 of 20 (60%) goitrous children and 0 of 12 (0% P less than 0.05) healthy children were positive for TGI. Similar results were found in the MAA, and a good correlation between results of the CBA and MAA was found (P = 0.003). Maximal TGI activity in dose-response CBA showed a good relation with clinical goitre size (r = 0.63; P less than 0.05) indicating a possible pathophysiological role for these antibodies. We conclude that endemic iodine goitre is not associated with Hashimoto's lymphocytic thyroiditis. Nevertheless, autoimmune growth factors such as TGI may play a primary role in the pathogenesis of thyroid growth in this condition.