Structure-Based High-Efficiency Homogeneous Antibody Platform by Endoglycosidase Sz Provides Insights into Its Transglycosylation Mechanism.

Monoclonal antibodies (mAbs) have gradually dominated the drug markets for various diseases. Improvement of the therapeutic activities of mAbs has become a critical issue in the pharmaceutical industry. A novel endo-ß-N-acetylglucosaminidase, EndoSz, from Streptococcus equisubsp. zooepidemicus Sz105 is discovered and applied to enhance the activities of mAbs. Our studies demonstrate that the mutant EndoSz-D234M possesses an excellent transglycosylation activity to generate diverse glycoconjugates on mAbs. We prove that EndoSz-D234M can be applied to various marketed therapeutic antibodies and those in development for antibody remodeling. The remodeled homogeneous antibodies (mAb-G2S2) produced by EndoSz-D234M increase the relative ADCC activities by 3-26-fold. We further report the high-resolution crystal structures of EndoSz-D234M in the apo-form at 2.15 Å and the complex form with a bound G2S2-oxazoline intermediate at 2.25 Å. A novel pH-jump method was utilized to obtain the complex structure with a high resolution. The detailed interactions of EndoSz-D234M and the carried G2S2-oxazoline are hence delineated. The oxazoline sits in a hole, named the oxa-hole, which stabilizes the G2S2-oxazoline in transit and catalyzes the further transglycosylation reaction while targeting Asn-GlcNAc (+1) of Fc. In the oxa-hole, the H-bonding network involved with oxazoline dominates the transglycosylation activity. A mobile loop2 (a.a. 152-159) of EndoSz-D234M reshapes the binding grooves for the accommodation of G2S2-oxazoline upon binding, at which Trp154 forms a hydrogen bond with Man (-2). The long loop4 (a.a. 236-248) followed by helix3 is capable of dominating the substrate selectivity of EndoSz-D234M. In addition, the stepwise transglycosylation behavior of EndoSz-D234M is elucidated. Based on the high-resolution structures of the apo-form and the bound form with G2S2-oxazoline as well as a systematic mutagenesis study of the relative transglycosylation activity, the transglycosylation mechanism of EndoSz-D234M is revealed.

Exploring the Molecular Characteristics and Role of PDGFB in Testis and Epididymis Development of Tibetan Sheep.

Platelet-derived growth factor B (PDGFB), as an important cellular growth factor, is widely involved in the regulation of cellular events such as cell growth, proliferation, and differentiation. Although important, the expression characteristics and biological functions in the mammalian reproductive system remain poorly understood. In this study, the PDGFB gene of Tibetan sheep was cloned by RT-PCR, and its molecular characteristics were analyzed. Subsequently, the expression of the PDGFB gene in the testes and epididymides (caput, corpus, and cauda) of Tibetan sheep at different developmental stages (3 months, 1 year, and 3 years) was examined by qRT-PCR and immunofluorescence staining. A bioinformatic analysis of the cloned sequences revealed that the CDS region of the Tibetan sheep PDGFB gene is 726 bp in length and encodes 241 amino acids with high homology to other mammals, particularly goats and antelopes. With the increase in age, PDGFB expression showed an overall trend of first decreasing and then increasing in the testis and epididymis tissues of Tibetan sheep, and the PDGFB mRNA expression at 3 months of age was extremely significantly higher than that at 1 and 3 years of age (p < 0.05). The PDGFB protein is mainly distributed in testicular red blood cells and Leydig cells in Tibetan sheep at all stages of development, as well as red blood cells in the blood vessel, principal cells, and the pseudostratified columnar ciliated epithelial cells of each epididymal duct epithelium. In addition, PDGFB protein expression was also detected in the spermatocytes of the 3-month-old group, spermatids of the 1-year-old group, spermatozoa and interstitial cells of the 3-year-old group, and loose connective tissue in the epididymal duct space in each developmental period. The above results suggest that the PDGFB gene, as an evolutionarily conserved gene, may play multiple roles in the development and functional maintenance of testicular cells (such as red blood cells, Leydig cells, and germ cells) and epididymal cells (such as red blood cells, principal cells, and ciliated epithelial cells) during testicular and epididymal development, which lays a foundation for the further exploration of the mechanisms by which the PDGFB gene influences spermatogenesis in Tibetan sheep.

Evolution of H6N6 viruses in China between 2014 and 2019 involves multiple reassortment events.

H6N6 avian influenza viruses (AIVs) have been widely detected in wild birds, poultry, and even mammals. Recently, H6N6 viruses were reported to be involved in the generation of H5 and H7 subtype viruses. To investigate the emergence, evolutionary pattern, and potential for an epidemic of H6N6 viruses, the complete genomes of 198 H6N6 viruses were analyzed, including 168 H6N6 viruses deposited in the NCBI and GISAID databases from inception to January 2019 and 30 isolates collected from China between November 2014 and January 2019. Using phylogenetic analysis, the 198 strains of H6N6 viruses were identified as 98 genotypes. Molecular clock analysis indicated that the evolution of H6N6 viruses in China was constant and not interrupted by selective pressure. Notably, the laboratory isolates reassorted with six subtype viruses: H6N2, H5N6, H7N9, H5N2, H4N2, and H6N8, resulting in nine novel H6N6 reassortment events. These results suggested that H6N6 viruses can act as an intermediary in the evolution of H5N6, H6N6, and H7N9 viruses. Animal experiments demonstrated that the 10 representative H6N6 viruses showed low pathogenicity in chickens and were capable of infecting mice without prior adaptation. Our findings suggest that H6N6 viruses play an important role in the evolution of AIVs, and it is necessary to continuously monitor and evaluate the potential epidemic of the H6N6 subtype viruses.

Isoquercitrin attenuates the osteoclast-mediated bone loss in rheumatoid arthritis via the Nrf2/ROS/NF-κB pathway.

An excess of osteoclastogenesis significantly contributes to the development of rheumatoid arthritis (RA). Activation of the nuclear factor erythroid-2 related factor 2 (Nrf2) and nuclear factor kappa B (NF-κB) ligand (RANKL)-induced reactive oxygen species (ROS)-to-NF-κB signaling cascade are important mechanisms regulating osteoclastogenesis; however, whether Nrf2 is involved in RANKL-induced NF-κB activation is controversial. Isoquercitrin, a natural flavonoid compound, has been shown to have Nrf2-dependent antioxidant effects inprevious studies. We sought to verify whether isoquercitrin could modulate RANKL-induced NF-κB activation by activating Nrf2, thereby affecting osteoclastogenesis. Tartrate-resistant acid phosphatase staining, F-actin ring staining and resorption pit assay suggested that isoquercitrin significantly inhibited osteoclastogenesis and osteolytic function. Mitosox staining showed that RANKL-induced ROS generation was significantly inhibited by isoquercitrin from day 3 of the osteoclast differentiation cycle. Quantitative real-time PCR, Western blot, and immunofluorescence indicated that isoquercitrin activated the Nrf2 signaling pathway and inhibited NF-κB expression. And when we used the Nrf2-specific inhibitor ML385, the inhibition of NF-κB by isoquercitrin disappeared. Moreover, we found that Nrf2 is not uninvolved in RANKL-induced NF-κB activation and may be related to the timing of ROS regulation. When we limited isoquercitrin administration to 2 days, Nrf2 remained activated and the inhibition of NF-κB disappeared. In vivo experiments suggested that isoquercitrin attenuated RA modeling-induced bone loss. Overall, isoquercitrin-activated Nrf2 blocked the RANKL-induced ROS-to-NF-κB signaling cascade response, thereby inhibiting osteoclastogenesis and bone loss. These findings provide new ideas for the treatment of RA.

Universal Water Disinfection by the Introduction of Fe-N3 Traps between g-C3N4 Layers under Visible Light.

Efficient inactivation of bacteria in the sewage via a photocatalytic process represents a promising strategy for the efficient chemical utilization of solar energy. Herein, uniformly dispersed Fe atoms were embedded between layers of g-C3N4 photocatalysts (CNFx), which were facilely prepared by thermal treatment. The optimized photocatalyst (CNF100) first showed excellent photoactivity for killing a variety of bacteria (93.0% for E. coli, 93.9% for Salmonella, and 96.2% for S. aureus) under visible light irradiation. The superior activity can be attributed to the formation of shallow electron traps (Fe-N3) that can capture excitons of excited states, which promote the charge transfer and energy transfer process of activated adsorbed molecular oxygen, respectively, forming reactive oxygen species, improving separation efficiency of photoexcited electrons and holes, and the Fe-N3 traps can also be used as photosensitive sites to broaden the absorption range of visible light. This strategy of constructing shallow electronic traps lays a theoretical foundation for the design of new environmentally friendly and efficient water disinfectants.

An online survey on clinical characteristics of otologic symptoms linked to COVID-19 infection.

Objective: To report the otologic symptoms that present in patients with COVID-19 infection and investigate the pathogenic characteristics during the period of the pandemic. Materials and methods: This cross-sectional descriptive study included participants with COVID-19 infection. COVID-19 infection was verified in these patients by nucleic acid test or antigen test. An online questionnaire was developed to analyze the association between the COVID-19 pandemic and the characteristics of otologic symptoms. Results: This study included 2,247 participants, of which nearly half had one or more otologic symptoms. The presents of otologic symptoms were associated with gender (OR = 1.575, p < 0.0001), age (OR = 0.972, p < 0.0001), and occupation (healthcare worker: p < 0.0001; personnel of enterprises or institutions: OR = 1.792, p < 0.0001; student: OR = 0.712, p < 0.044). The otologic symptoms following COVID-19 infection in order were vertigo (25.95%), tinnitus (19.05%), otalgia (19.00%), aural fullness (17.18%), hearing loss (11.62%), otorrhea (1.25%), and facial paralysis (0.27%). Conclusion: The present study shows that otologic symptoms are common among the COVID-19 infected participants and that these symptoms mostly recover spontaneously. During the corona-virus pandemic, the involvement of the cochleovestibular system and facial nerve should not be overlooked while treating the COVID-19 infected individuals.

[Construction and evaluation of a gradient stress model of PC12 cells induced by corticosterone].

Objective: A gradient stress model of PC12 cells induced by corticosterone was established to provide a basis for the evaluation and regulation of cell stress. Methods: The effect of corticosterone on cell viability was observed by measuring PC12 cell viability at different concentrations of corticosterone (0~1 000 µmol/L) after different intervention times (8~48 h) to screen the cell models for optimal intervention conditions. Key stress indicators (MDA, SOD, NADH, LDH) were measured spectrophotometrically and microscopically to evaluate the models. Results: When the concentration of corticosterone was below 200 µmol/L and the intervention time was 12 h, the cell viability was below half inactivation rate, which could reduce the confounding factors due to the decrease of cell viability in each group. Compared with the blank control group, corticosterone increased the levels of MDA, NADH and LDH,and decreased the levels of SOD in the model group in a concentration-dependent manner (P＜0.01), which was consistent with the construction of the gradient stress model. Conclusion: A gradient stress injury model of PC12 cells was successfully established, with intervention concentrations of 0 µmol/L, 25 µmol/L, 50 µmol/L, 100 µmol/L, 150 µmol/L and 200 µmol/L corticosterone at an intervention time of 12 h. The degree of stress injury of the cell model was increased gradually, which could be used as a basis and object for conducting cell stress injury assessment and regulation experiments.

Simultaneous chirality separation of amino acids and their derivative by natamycin based on mobility measurements.

The separation of chiral amino acids (AAs) and their derivatives has always been a research difficulty in the field of biochemistry due to the high similarity of enantiomeric structures. In this work, a simple and quick method using natamycin (Nat) as chiral selector has been developed to simultaneously separate chiral AAs and their derivatives of carbobenzoxy/benzyl-AAs (Cbz/Bzl-AAs) by trapped ion mobility spectrometry-mass spectrometry (TIMS-MS). Specifically, 12 groups of the Cbz-AAs and Bzl-AAs can get baseline mobility separation by simple mixing with Nat to form binary diastereomeric complex ions [Nat+(Cbz-D/L-AA)+H]+ and [Nat+(Bzl-D/L-AA)+H]+. While for the remained 5 groups of Bzl-D/L-AAs and 16 groups of D/L-AAs with unsatisfying separation, by further adding P-toluenesulfonic acid (PTS), the formed ternary complexes can allow their baseline chiral separation. Specifically, Bzl-D-AAs and Bzl-L-AAs get much improved separation effect by the formed diastereomeric complexes of [Nat+(Bzl-AA)2+PTS2+H]+, which the Rp-p was improved from 0 to 2.40; while the D/L-AAs can get baseline separation by the formed diastereomeric complexes of [Nat + AA + PTS + H]+, [Nat + AA+(PTS)2+H]+, and [Nat+(AA)2+(PTS)2+H]+, with the Rp-p ranged from 0.44 to 3.53. Definitely, PTS is the first time reported as the ligand to improve the separation effect for the enantiomers, and with the higher assembly of chiral analyte, Nat, and PTS might enable better chiral separation for the chiral amino acid and their derivatives. Moreover, method validation of relative quantification and accuracy for the D/L-AA and their derivatives were measured in a mixture, yielding R2 greater than 0.99 and RSD% ≤ 2.68%. Overall, Nat and PTS as chiral selector and ligand can be widely used for chiral AAs and their derivatives mobility separation, and potentially for the separation of other AA-related chiral molecules.

Genetic context diversity of plasmid-borne blaCTX-M-55 in Escherichia coli isolated from waterfowl.

OBJECTIVES: The aim of this study was to explore the prevalence of blaCTX-M and elucidate the characteristics of transferable blaCTX-M-55 plasmids. METHODS: Escherichia coli were isolated from 10 large-scale duck farms from nine counties and cities in Guizhou Province. They were identified, subjected to antimicrobial susceptibility testing and screened for antibiotic resistance genes. blaCTX-M-positive strains were subtyped, and blaCTX-M-55-containing strains were further analysed by multilocus sequence typing, conjugation experiments and polymerase chain reaction-based replicon typing. Three transferable plasmids were selected for whole genome sequencing sequencing and further analysis of genetic context. RESULTS: In total, 128 E. coli strains were identified, and 23 were blaCTX-M-positive. Subtype analysis of blaCTX-M-positive strains revealed that 23 were blaCTX-M-55 and one of the strains contained both blaCTX-M-65 and blaCTX-M-55. They were divided into 12 multilocus sequence typing types, and 6 were undetermined. blaCTX-M-55-bearing plasmids in seven were transferred to E. coli C600 at frequencies between 10-6 and 10-9, and blaCTX-M-55 was found to be located on IncN, IncFⅡ and IncFII-N fusion type plasmids. Three plasmids (IncFⅡ plasmids pEC99_Plas3 and pS1_Plas2 and IncFII-N plasmid pD2_plas2) shared a homologous region of IS26-ARGs-IS26-blaTEM-orf477-blaCTX-M-55-ISEcp1△-IS26. CONCLUSION: Our study demonstrates the environmental presence of blaCTX-M-55 harbouring E. coli in Guizhou. Analysis of the genetic context of blaCTX-M-55 aids in understanding possible dissemination routes of ESBL genes and controlling widespread antibiotic resistance.

[Resveratrol attenuates cardiac function impairment in plateau hypobaric hypoxia rats].

Objective: To study the protective effects of resveratrol (RSV) on cardiac function in rats with high altitude hypobaric hypoxia and its mechanisms. Methods: Thirty-six rats were randomly divided into control group, hypobaric hypoxia group (HH) and hypobaric hypoxia + RSV group (HH+RSV) according to the random number, 12 rats in each group. Rats in the HH and HH+RSV groups were subjected to chronic long-term high altitude hypobaric hypoxia intervention for 8 weeks in a hypobaric chamber at a simulated altitude of 6 000 m for 20 h / d. The rats of HH + RSV were fed with RSV at a dose of 400 mg/(kg·d). The rats were tested once a week for body weight and twice a week for food intake. Before execution, the rats were tested by blood cell analyzer for routine blood parameters and echocardiogram for cardiac function parameters in each group. The routine blood indexes of each group were measured by blood cell analyzer, the cardiac function indexes of each group were measured by echocardiography, myocardial hypertrophy was evaluated by HE staining, myocardial tissue reactive oxygen levels were evaluated by dihydroethidium (DHE) staining. Oxidative stress was evaluated by serum and myocardial tissue total antioxidant capacity (T-AOC), superoxide dismutase activity (SOD) and malondialdehyde (MDA) content. Results: Compared with the C group, the body mass and food intake of rats were decreased significantly (P＜0.05) in HH group, while compared with the C group, RSV had no significant effects on the body mass and food intake of rats in the HH+RSV group (P＞0.05). Compared with the C group, the levels of erythrocytes and hemoglobin of rats in the HH group were increased significantly (P＜0.05), while the platelet concentration was decreased significantly(P＜0.05); compared with the HH group, the erythrocyte and hemoglobin levels were decreased significantly (P＜0.05) and platelet concentration was increased significantly(P＜0.05) in rats of the HH+RSV group. Compared with the C group, the cardiac coefficient, myocardial fiber diameter and thickness were significantly increased in the HH group (P＜0.05); compared with the HH group, the cardiac coefficient and myocardial fiber thickness were significantly decreased in the HH+RSV group (P＜0.05). Echocardiographic analysis showed a significant increase in ventricular wall thickness (P＜0.05) and a significant decrease in ejection fraction and cardiac output (P＜0.05) in the HH group compared with the C group, and a significant decrease in ventricular wall thickness and a significant improvement in cardiac function (P＜0.05) in the HH+RSV group compared with the HH group. The results of DHE staining showed that myocardial tissue reactive oxygen levels were increased significantly in the HH group compared with the C group (P＜0.05); myocardial tissue reactive oxygen levels were significantly restored in the HH+RSV group compared with the HH group (P＜0.05). The oxidative/antioxidant results showed that the serum and myocardial T-AOC and SOD activities were decreased significantly (P＜0.05) and the MDA level was increased significantly (P＜0.05) in the HH group compared with the C group; the serum and myocardial T-AOC and SOD activities were increased significantly (P＜0.05) and the MDA level was decreased significantly(P＜0.05) in the HH+RSV group compared with the HH group. Conclusion: Long-term plateau hypobaric hypoxia exposure leads to myocardial hypertrophy and reduced cardiac function in rats. Resveratrol intervention significantly improves myocardial hypertrophy and cardiac function in rats caused by altitude hypobaric hypoxia exposure, which is closely related to reducing of reactive oxygen species and improving myocardial oxidative stress levels.

Alterations of mRNA and lncRNA profiles associated with the extracellular matrix and spermatogenesis in goats.

OBJECTIVE: Spermatozoa are produced within the seminiferous tubules after sexual maturity. The expression levels of mRNAs and lncRNAs in testicular tissues are different at each stage of testicular development and are closely related to formation of the extracellular matrix (ECM) and spermatogenesis. Therefore, we set out to study the expression of lncRNAs and mRNAs during the different developmental stages of the goat testis. METHODS: We constructed 12 RNA libraries using testicular tissues from goats aged 3, 6, and 12 months, and studied the functions of mRNAs and lncRNAs using the gene ontogeny (GO) and Kyoto encyclopedia of genes and genomes (KEGG) databases. Relationships between differentially expressed genes (DEGs) were analyzed by lncRNA-mRNA coexpression network and protein-protein interaction network (PPI). Finally, the protein expression levels of matrix metalloproteinase 2 (MMP2), insulin-like growth factor 2 (IGF2), and insulin-like growth factor-binding protein 6 (IGFBP6) were detected by western blotting. RESULTS: We found 23, 8, and 135 differentially expressed lncRNAs and 161, 12, and 665 differentially expressed mRNAs that were identified between 3 vs 6, 6 vs 12, and 3 vs 12 months, respectively. GO, KEGG, and PPI analyses showed that the differential genes were mainly related to the ECM. Moreover, MMP2 was a hub gene and co-expressed with the lncRNA TCONS-0002139 and TCONS-00093342. The results of quantitative reverse-transcription polymerase chain reaction verification were consistent with those of RNA-seq sequencing. The expression trends of MMP2, IGF2, and IGFBP6 protein were the same as that of mRNA, which all decreased with age. IGF2 and MMP2 were significantly different in the 3 vs 6-month-old group (p<0.05). CONCLUSION: These results improve our understanding of the molecular mechanisms involved in sexual maturation of the goat testis.

Comparative genomic analysis and multi-drug resistance differences of Acinetobacter baumannii in Chongqing, China.

INTRODUCTION: Multidrug-resistance in Acinetobacter baumannii has emerged as a serious problem to public health. There is still a significant gap in the understanding of the multidrug-resistance and the genome diversity evolutionary process of A. baumannii in China, especially in the central and western regions. METHODS: Ten A. baumannii strains were collected from three hospitals in Chongqing, China. Whole-genome re-sequencing was used to obtain differences in genomic levels among strains. The diversity were determined by multi-locus sequence typing method, and investigate the genetic relationship between the ten strains and others by phylogenetic analysis. Comparative analysis focused on resistance genes related to insertions and deletions (InDels) and single-nucleotide polymorphisms (SNPs) was performed. RESULTS: The overall G+C% content was 39.05%~39.43%, the average sequencing depth was 273.95~428.99, and the alignment ratio of the sequencing data was 92.93%~99.27%. A total of 42 InDels and 11,387 SNPs were detected in the coding sequence region of the isolates. Phylogenetic tree shows that the 10 A. baumannii isolates were divided into four relative groups, and there exist the possibility of cross-regional spread pattern. A total number of 19 drug resistance genes had been found in each strain, and efflux pump-related genes accounted for the most. Only AacA4 underwent a change in InDel. Six types of drug resistance genes were found in the SNPs resistance gene-related loci, among which gene ANT(3'')-II and QacE mutations were found in each strain. CONCLUSION: In this study, the main mechanism of A. baumannii multi-drug resistance is due to the multi-drug efflux pump related genes. The point mutations at the SNPs sites of the six types of resistance genes are the main differences in A. baumannii between Chongqing and the eastern coastal areas of China.

Rapid Analysis of Components in Coptis chinensis Franch by Ultra-Performance Liquid Chromatography with Quadrupole Time-of-Flight Mass Spectrometry.

BACKGROUND: Coptis chinensis Franch is a traditional Chinese medical herb. OBJECTIVE: In this article, ultra-performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry was used to rapidly, qualitatively, and comprehensively identify the components in Coptis chinensis Franch. MATERIALS AND METHODS: Chromatographic separation was achieved on an Agilent Zorbax RRHD Eclipse Plus C18 column. The mobile phase consisted of 0.1% formic acid water (A) and 0.1% formic acid acetonitrile (B) with a gradient program. Qualitative analysis was performed on an Agilent 6540 quadrupole time-of-flight mass spectrometer, which was equipped with a Dual AJS ESI source operating in negative mode. RESULTS: A total of 30 alkaloid and non-alkaloid components of Coptis chinensis Franch were identified in only 14 min. CONCLUSION: This study helped to provide a basis for the quality control of Coptis chinensis Franch. SUMMARY: Qualitative analysis method of chlorogenic alkaloids and non-alkaloids in Coptis chinensis Franch is developed by Ultra-performance liquid chromatography with quadrupole time-of-flight mass spectrometry (UPLC-Q-TOF-MS/MS) method.Established UPLC-Q-TOF-MS/MS analysis method is validated with rapidness and accuracy.The developed method was successfully applied for qualitative analysis of Coptis chinensis Franch sample collected from cultivation place in China. Abbreviations used: Q-TOF-MS: quadrupole time-of-flight mass spectrometry, UPLC: ultra-performance liquid chromatography, pos: positive, neg: negative. Q-TOF-MS: quadrupole time-of-flight mass spectrometry, UPLC: ultra-performance liquid chromatography, pos: positive, neg: negative. UPLC: ultra-performance liquid chromatography, pos: positive, neg: negative. pos: positive, neg: negative. neg: negative.

[Effects of MAPKs inhibitors on GSH metabolic enzymes in rat hepatocytes].

OBJECTIVE: To investigate the effects of mitogen-activated protein kinases (MAPKs) inhibitors on glutathione (GSH) metabolism, and to explore the pathway related to GSH metabolism. METHODS: BRL rat hepatocytes were treated by c-Jun NH2-terminal kinase (JNK),p38, and extracellular signal-regulated kinase 1/2 (ERK1/2) inhibitors:SP600125, SB203580 and PD98659, respectively, for 24 h. MTT method was used to measure hepatocytes viability. The content of GSH was determined by high performance liquid chromatography. The protein expressions of JNK and phosphorylated JNK (p-JNK) was tested by Luminex method. Activities of GSH metabolic enzymes were detected by commercial kits. RESULTS: Hepatocytes vitality was inhibited when the concentrations of SP600125, SB203580 and PD98659 were higher than 10 µmol/L, 20 µmol/L, and 40 µmol/L, respectively; SP600125 decreased the content of GSH in hepatocytes, while SB203580 and PD98659 had no effect. SP600125 reduced p-JNK protein expression, and enhanced GSH-Px activity significantly. CONCLUSIONS: JNK MAPK pathway takes part in the GSH metabolism in hepatocytes.

[Analysis of volatile ingredients in Gardeniae Fructus and its processed products by GC-MS].

Gardeniae Fructus contains volatile ingredients, however, the species and proportions in different processed products of Gardeniae Fructus are different. In this experiment, volatile ingredients were separated by steam distillation with content of 1.2, 1.0, 0.9, 0.7 µL · g(-1) in Gardeniae Fructus, fried Gardeniae Fructus, stir-baked Gardeniae Fructus, Gardeniae Fructus fried into carbon respectively. One hundred and twenty-four kinds of volatile components were identified by GC-MS. Fifty-three kinds of volatile ingredients consisted in Gardeniae Fructus accounting for 93.85%, 54 kinds in fried Cardeniae Fructus accounting for 92.01%, 32 kinds in stir-baked Cardeniae Fructus accounting for 91.59% and 43 kinds in Gardeniae Fructus fried into carbon accounting for 90.81%. In this paper, analysis of Gardeniae Fructus by GC-MS provides a scientific basis for elucidating the mechanism of different processed products.

Development of a Rapid Immunochromatographic Lateral Flow Device Capable of Differentiating Phytase Expressed from Recombinant Aspergillus niger phyA2 and Genetically Modified Corn.

Phytase is a phosphohydrolase considered highly specific for the degradation of phytate to release bound phosphorus for animal consumption and aid in the reduction of environmental nutrient loading. New sources of phytase have been sought that are economically and efficiently productive including the construction of genetically modified (GM) phytase products designed to bypass the costs associated with feed processing. Four monoclonal antibodies (EH10a, FA7, AF9a, and CC1) raised against recombinant Aspergillus niger phyA2 were used to develop a highly specific and sensitive immunochromatographic lateral flow device for rapid detection of transgenic phytase, such as in GM corn. Antibodies sequentially paired and tested along lateral flow strips showed that the EH10a-FA7 antibody pair was able to detect the recombinant yeast-phytase at 5 ng/mL, whereas the AF9a-CC1 antibody pair to GM phytase corn was able to detect at 2 ng/mL. Concurrent to this development, evidence was revealed which suggests that antibody binding sites may be glycosylated.

A massive expansion of effector genes underlies gall-formation in the wheat pest Mayetiola destructor.

Gall-forming arthropods are highly specialized herbivores that, in combination with their hosts, produce extended phenotypes with unique morphologies [1]. Many are economically important, and others have improved our understanding of ecology and adaptive radiation [2]. However, the mechanisms that these arthropods use to induce plant galls are poorly understood. We sequenced the genome of the Hessian fly (Mayetiola destructor; Diptera: Cecidomyiidae), a plant parasitic gall midge and a pest of wheat (Triticum spp.), with the aim of identifying genic modifications that contribute to its plant-parasitic lifestyle. Among several adaptive modifications, we discovered an expansive reservoir of potential effector proteins. Nearly 5% of the 20,163 predicted gene models matched putative effector gene transcripts present in the M. destructor larval salivary gland. Another 466 putative effectors were discovered among the genes that have no sequence similarities in other organisms. The largest known arthropod gene family (family SSGP-71) was also discovered within the effector reservoir. SSGP-71 proteins lack sequence homologies to other proteins, but their structures resemble both ubiquitin E3 ligases in plants and E3-ligase-mimicking effectors in plant pathogenic bacteria. SSGP-71 proteins and wheat Skp proteins interact in vivo. Mutations in different SSGP-71 genes avoid the effector-triggered immunity that is directed by the wheat resistance genes H6 and H9. Results point to effectors as the agents responsible for arthropod-induced plant gall formation.

Microwave-assisted one-pot synthesis of 1,6-anhydrosugars and orthogonally protected thioglycosides.

Living organisms employ glycans as recognition elements because of their large structural information density. Well-defined sugar structures are needed to fully understand and take advantage of glycan functions, but sufficient quantities of these compounds cannot be readily obtained from natural sources and have to be synthesized. Among the bottlenecks in the chemical synthesis of complex glycans is the preparation of suitably protected monosaccharide building blocks. Thus, easy, rapid, and efficient methods for building-block acquisition are desirable. Herein, we describe routes directly starting from the free sugars toward notable monosaccharide derivatives through microwave-assisted one-pot synthesis. The procedure followed the in situ generation of per-O-trimethylsilylated monosaccharide intermediates, which provided 1,6-anhydrosugars or thioglycosides upon treatment with either trimethylsilyl trifluoromethanesulfonate or trimethyl(4-methylphenylthio)silane and ZnI2, respectively, under microwave irradiation. We successfully extended the methodology to regioselective protecting group installation and manipulation toward a number of thioglucosides and the glycosylation of persilylated derivatives, all of which were conducted in a single vessel. These developed approaches open the possibility for generating arrays of suitably protected building blocks for oligosaccharide assembly in a short period with minimal number of purification stages.

Comparative validation of the D. melanogaster modENCODE transcriptome annotation.

Accurate gene model annotation of reference genomes is critical for making them useful. The modENCODE project has improved the D. melanogaster genome annotation by using deep and diverse high-throughput data. Since transcriptional activity that has been evolutionarily conserved is likely to have an advantageous function, we have performed large-scale interspecific comparisons to increase confidence in predicted annotations. To support comparative genomics, we filled in divergence gaps in the Drosophila phylogeny by generating draft genomes for eight new species. For comparative transcriptome analysis, we generated mRNA expression profiles on 81 samples from multiple tissues and developmental stages of 15 Drosophila species, and we performed cap analysis of gene expression in D. melanogaster and D. pseudoobscura. We also describe conservation of four distinct core promoter structures composed of combinations of elements at three positions. Overall, each type of genomic feature shows a characteristic divergence rate relative to neutral models, highlighting the value of multispecies alignment in annotating a target genome that should prove useful in the annotation of other high priority genomes, especially human and other mammalian genomes that are rich in noncoding sequences. We report that the vast majority of elements in the annotation are evolutionarily conserved, indicating that the annotation will be an important springboard for functional genetic testing by the Drosophila community.

Does Traditional Chinese Medicine pattern affect acupoint specific effect? Analysis of data from a multicenter, randomized, controlled trial for primary dysmenorrhea.

OBJECTIVES: This study assessed the importance of the Traditional Chinese Medicine (TCM) pattern on an acupoint-specific effect. DESIGN: This was a TCM pattern subdivision analysis of the first intervention data from a multicenter, randomized, controlled trial (ISRCTN24863192) (the main trial). SETTINGS: The main trial recruited participants from six hospitals in three provinces in China. SUBJECTS: Five hundred and one (501) participants diagnosed with primary dysmenorrhea (PD) were enrolled in the main trial. INTERVENTIONS: The main trial randomly and equally divided participants into three treatment groups with bilateral electroacupuncture at three sites, respectively: Sanyinjiao (SP6), Xuanzhong (GB39), and an adjacent nonacupoint. Participants were diagnosed with TCM patterns before the treatment. The intervention was carried out when the visual analogue scale (VAS) score of participant's menstrual pain was ≥ 40 mm on the first day of menstruation and lasted for 30 minutes. OUTCOME MEASURES: The immediate improvement of pain was measured with a 100-mm VAS before the intervention, at 5 minutes, 10 minutes, and 30 minutes during the intervention, and at 30 minutes after the completion of this intervention. RESULTS: Three (3) TCM patterns (n=320) were eligible for analysis, including Cold and Dampness Stagnation pattern (n=184), Qi and Blood Stagnation pattern (n=84), and Qi and Blood Deficiency pattern (n=52). In Cold and Dampness Stagnation pattern, the SP6 group had a significant reduction in VAS scores compared with the GB39 group (mean difference -7.6 mm) and the nonacupoint group (mean difference -8.2 mm), respectively. There was no difference between the latter two groups. There were no group differences in VAS scores in the other two patterns. CONCLUSIONS: It suggested that TCM pattern might affect acupoint specific effect on the immediate pain relief obtained for participants with PD.