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Objective: To investigate the role of an inflammatory microenvironment induced by Porphyromonasgingivalis ( P. gingivalis ) in the occurrence of esophageal squamous cell carcinoma (ESCC) in mice. Methods: A total of 180 C57BL/6 mice were randomly divided into 6 groups, i.e. control group, P. gingivalis group, 4NQO group, 4NQO + P. gingivalis group, 4NQO + P. gingivalis + celecoxib group, and 4NQO + P. gingivalis + antibiotic cocktail (ABC, including metronidazole, neomycin, ampicillin, and vancomycin) group, with 30 mice in each group, using the random number table. All mice were normalized by treatment with ABC in drinking water for 2 weeks. In the following 2 weeks, the mice in the control group and the P. gingivalis group were given drinking water, while the other 4 groups were treated with 30 μg/ml 4NQO in the drinking water. In weeks 11-12, the mice in the P. gingivalis group, the 4NQO + P. gingivalis group, the 4NQO + P. gingivalis + celecoxib group, and the 4NQO + P. gingivalis + ABC group were subjected to ligation of the second molar in oral cavity followed by oral P. gingivalis infection thrice weekly for 24 weeks in weeks 11-34. In weeks 13-34, the mice in 4NQO + P. gingivalis +celecoxib group and 4NQO + P. gingivalis + ABC group were administered with celecoxib and ABC for 22 weeks, respectively. At the end of 34 weeks, gross and microscopic alterations were examined followed by RT-qPCR and immunohistochemistry to examine the expression profiles of inflammatory- and tumor-molecules in esophagi of mice. Results: At 34 weeks, 4NQO treatment alone did not affect the foci of papillary hyperproliferation, diseased area, and the thickness of the esophageal wall, but significantly enhanced the foci of hyperproliferation (median 1.00, P ＜0.05) and mild/moderate dysplasia (median 2.00, P ＜0.01). In addition, the expression levels of IL-6 [8.35(3.45,8.99)], IL-1β [6.90(2.01,9.72)], TNF-α [12.04(3.31,14.08)], c-myc [2.21(1.80,3.04)], pSTAT3, Ki-67, and pH2AX were higher than those in the control group. The pathological changes of the esophageal mucosa were significantly more overt in the 4NQO + P. gingivalis group in terms of the foci of papillary hyperproliferation (median 2.00), diseased area (median 2.51 mm 2 ), the thickness of the esophageal wall (median 172.52 μm), the foci of hyperproliferation (median 1.00, P ＜0.05), and mild/moderate dysplasia (median 1.00, P ＜0.01). In mice of the 4NQO + P. gingivalis group, the expression levels of IL-6 [12.27(5.35,22.08)], IL-1β [13.89(10.04,15.96)], TNF-α [19.56(6.07,20.36)], IFN-γ [11.37(8.23,20.07)], c-myc [2.62(1.51,4.25)], cyclin D1 [4.52(2.68,7.83)], nuclear pSTAT3, COX-2, Ki-67, and pH2AX were significantly increased compared with the mice in the control group. In mice of the 4NQO + P. gingivalis group, the diseased area, invasive malignant foci as well as pSTAT3 and pH2AX expression were significantly blunted by celecoxib. Treatment with ABC markedly reduced the papillary hyperproliferative foci, invasive malignant foci, and pSTAT3 expression but not pH2AX. Conclusions: P. gingivalis promotes the occurrence of esophageal squamous cell carcinoma in mice by inducing an inflammatory microenvironment primed with 4NQO induced DNA damage. Clearance of P. gingivalis with ABC or anti-inflammatory intervention holds promise for prevention of esophageal squamous cell malignant pathogenesis via blockage of IL-6/STAT3 signaling and amelioration of inflammation.

Background: In view of the low accuracy of the prognosis model of esophageal squamous cell carcinoma (ESCC), this study aimed to optimize the least squares support vector machine (LSSVM) algorithm to determine the uncertain prognostic factors using a Cloud model, and consequently, to establish a new high-precision prognosis model of ESCC., Methods: We studied 4,771 ESCC patients(training samples) from the Surveillance, Epidemiology, and End Results (SEER) database and 635 ESCC patients(validation samples) from the Henan Provincial Center for Disease Control and Prevention (HCDC) database, with the same exclusion criteria and inclusion criteria for both databases, and obtained permission to obtain a research data file in the SEER database from the National Cancer Institute. The independent risk factors were analyzed using the log-rank method, survival curves, univariate and multivariate Cox analysis. Finally, the independent prognostic factors were used to construct the nomogram, random forest and Cloud-LSSVM prognostic models were utilized for validation., Results: The overall median survival time of the SEER database was 14 months (HCDC samples was 46 months), the mean survival time was 26.5 months (HCDC samples was 36.8 months), and the 3-year survival rate was 65.8%. This is because most of the patients with Henan samples are early ESCC, and most of the Seer patients are T3 and T4 people. The multivariate Cox analysis showed that age at diagnosis (P<0.001), sex (P=0.001), race (P=0.002), differentiation grade (P<0.001), pathologic T category (P<0.001), and pathologic M category (P<0.001) were the factors affecting the prognosis of ESCC patients. The SEER data and HCDC database results showed that the accuracy of the Cloud-LSSVM (C-index =0.71, 0.689) model is higher than the differentiation grade (C-index =0.548, 0.506), random forest (C-index =0.649, 0.498), and nomogram (C-index =0.659, 0.563). This new model can realize the unity of the randomness and fuzziness of the Cloud model and utilize the powerful learning and non-linear mapping abilities of LSSVM., Conclusions: Due to the difference of clans between training samples and test samples, the accuracy of prediction is generally not high, but the accuracy of Cloud-LSSVM model is much higher than other models. The new model provides a clear prognostic superiority over the random forest, nomogram, and other models., Competing Interests: Conflicts of Interest: All authors have completed the ICMJE uniform disclosure form (available at https://jtd.amegroups.com/article/view/10.21037/jtd-23-1058/coif). The authors have no conflicts of interest to declare., (2023 Journal of Thoracic Disease. All rights reserved.)

Arginase has shown promising potential in treating cancers by arginine deprivation therapy; however, low enzymatic activity and stability of arginase are impeding its development. This study was aimed to improve the enzymological properties of a marine bacterial arginase by carboxymethyl chitosan (CMCS) conjugation. An arginase producing marine bacterium Priestia megaterium strain P6 was isolated and identified. The novel arginase PMA from the strain was heterologously expressed, purified, and then conjugated to CMCS by ionic gelation with calcium chloride as the crosslinking agent. Enzymological properties of both PMA and CMCS-PMA conjugate were determined. The optimum temperature for PMA and CMCS-PMA at pH 7 were 60 °C and 55 °C, respectively. The optimum pH for PMA and CMCS-PMA at 37 °C were pH 10 and 9, respectively. CMCS-PMA showed higher thermostability than PMA over 55-70 °C and higher pH stability over pH 4-11 with the highest pH stability at pH 7. At 37 °C and pH of 7, i.e., around the human blood temperature and pH, CMCS-PMA was higher than the free PMA in enzymatic activity and stability by 24% and 21%, respectively. CMCS conjugation not only changed the optimum temperature, optimum pH, and enzymatic activity of PMA, but also improved its pH stability and temperature stability, and thus made it more favorable for medical application., (© 2023. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Background: Mass spectrometry-based proteomics and glycomics reveal post-translational modifications providing significant biological insights beyond the scope of genomic sequencing., Aim: To characterize the N-linked glycoproteomic profile in esophageal squamous cell carcinoma (ESCC) via two complementary approaches., Methods: Using tandem multilectin affinity chromatography for enrichment of N-linked glycoproteins, we performed N-linked glycoproteomic profiling in ESCC tissues by two-dimensional gel electrophoresis (2-DE)-based and isobaric tags for relative and absolute quantification (iTRAQ) labeling-based mass spectrometry quantitation in parallel, followed by validation of candidate glycoprotein biomarkers by Western blot., Results: 2-DE-based and iTRAQ labeling-based quantitation identified 24 and 402 differentially expressed N-linked glycoproteins, respectively, with 15 in common, demonstrating the outperformance of iTRAQ labeling-based quantitation over 2-DE and complementarity of these two approaches. Proteomaps showed the distinct compositions of functional categories between proteins and glycoproteins with differential expression associated with ESCC. Western blot analysis validated the up-regulation of total procathepsin D and high-mannose procathepsin D, and the down-regulation of total haptoglobin, high-mannose clusterin, and GlcNAc/sialic acid-containing fraction of 14-3-3ζ in ESCC tissues. The serum levels of glycosylated fractions of clusterin, proline-arginine-rich end leucine-rich repeat protein, and haptoglobin in patients with ESCC were remarkably higher than those in healthy controls., Conclusion: Our study provides insights into the aberrant N-linked glycoproteome associated with ESCC, which will be a valuable resource for future investigations., Competing Interests: Conflict-of-interest statement: All authors report no relevant conflicts of interest for this article., (©The Author(s) 2022. Published by Baishideng Publishing Group Inc. All rights reserved.)

Background: The aim of this study was to identify prognostic markers for esophageal squamous cell carcinoma (ESCC) and build an effective prognostic nomogram for ESCC. Methods: A total of 365 patients with ESCC from three medical centers were divided into four cohorts. In the discovery phase of the study, we analyzed transcriptional data from 179 cancer tissue samples and identified nine marker genes using edgeR and rbsurv packages. In the training phase, penalized Cox regression was used to select the best marker genes and clinical characteristics in the 179 samples. In the verification phase, these marker genes and clinical characteristics were verified by internal validation cohort (n = 58) and two external cohorts ( n = 81, n = 105). Results: We constructed and verified a nomogram model based on multiple clinicopathologic characteristics and gene expression of a patient cohort undergoing esophagectomy and adjuvant radiochemotherapy. The predictive accuracy for 4-year overall survival (OS) indicated by the C-index was 0.75 (95% CI, 0.72-0.78), which was statistically significantly higher than that of the American Joint Committee on Cancer (AJCC) seventh edition (0.65). Furthermore, we found two marker genes (TM9SF1, PDZK1IP) directly related to the OS of esophageal cancer. Conclusion: The nomogram presented in this study can accurately and impersonally predict the prognosis of ESCC patients after partial resection of the esophagus. More research is required to determine whether it can be applied to other patient populations. Moreover, we found two marker genes directly related to the prognosis of ESCC, which will provide a basis for future research., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Liu, Jiao, Shen, Chen, Zhao, Li, Gu, Lan, Ruan, Liu, Xu, Yuan, Qi and Gao.)

Microbial dysbiosis in the upper digestive tract is linked to an increased risk of esophageal squamous cell carcinoma (ESCC). Overabundance of Porphyromonas gingivalis is associated with shorter survival of ESCC patients. We investigated the molecular mechanisms driving aggressive progression of ESCC by P. gingivalis. Intracellular invasion of P. gingivalis potentiated proliferation, migration, invasion, and metastasis abilities of ESCC cells via transforming growth factor-β (TGFβ)-dependent Drosophila mothers against decapentaplegic homologs (Smads)/Yes-associated protein (YAP)/Transcriptional coactivator with PDZ-binding motif (TAZ) activation. Smads/YAP/TAZ/TEA domain transcription factor1 (TEAD1) complex formation was essential to initiate downstream target gene expression, inducing an epithelial-mesenchymal transition (EMT) and stemness features. Furthermore, P. gingivalis augmented secretion and bioactivity of TGFβ through glycoprotein A repetitions predominant (GARP) up-regulation. Accordingly, disruption of either the GARP/TGFβ axis or its activated Smads/YAP/TAZ complex abrogated the tumor-promoting role of P. gingivalis. P. gingivalis signature genes based on its activated effector molecules can efficiently distinguish ESCC patients into low- and high-risk groups. Targeting P. gingivalis or its activated effectors may provide novel insights into clinical management of ESCC., Competing Interests: The authors have declared that no competing interests exist.

Objective: Given that FK506 binding protein 51 (FKBP51) is upregulated in multiple cancers, we designed the present study to characterize its role as well as underlying regulatory mechanisms in glioma in the presence and absence of the chemotherapeutic carmustine (BCNU)., Materials and Methods: Through lentiviral overexpression and shRNA knockdown of FKBP51, we examined the effects on BT325 glioma cell proliferation, migration and invasion using quantitative reverse transcription PCR (qRT-PCR), CCK-8 assay, flow cytometry, and transwell assay., Results: The upregulation of FKBP51 resulted in significantly decreased BT325 cell proliferation and cell viability, cell cycle arrest, reduced BCNU chemosensitivity and AKT pathway inactivation. However, FKBP51-overexpressed BT325 cells showed enhanced migration and invasion, which was supported by corresponding increase in phosphorylated IKKα (p-IKKα), MMP-2, and MMP-9 levels, as well as increased NF-κB p65 nuclear translocation. By contrast, FKBP51-suppressed BT325 cells showed excessive proliferation and BCNU resistance due to increased p-AKT activation and attenuated migration and invasion., Conclusions: We demonstrated that the effects of FKBP51 on BT325 glioma cell proliferation, migration, invasion and BCNU chemosensitization are modulated via the AKT and NF-κB pathways. Furthermore, our findings suggest the potential of FKBP51 as a prognostic glioma biomarker and an indicator of patient response to chemotherapy.

This study aimed to investigate the effect and possible mechanism of Bidens pilosa decoction on non-alcoholic fatty liver disease(NAFLD) induced by high fat and high glucose in mice. Bald/c mice were randomly divided into normal group, model group, metformin(200 mg·kg~(-1)) treatment group, Bidens pilosa decoction(10 g·kg~(-1)) treatment group, metformin and B. pilosa decoction(100 mg·kg~(-1)+5 g·kg~(-1)) treatment group. Except for the normal group, mice in the other four groups were fed with high-fat and high-glucose diet for 8 weeks to establish the non-alcoholic fatty liver model. After 4 weeks of treatment, blood was collected from the eyeballs, the mice were sacrificed, and relevant indicators were detected. The results showed that compared with the model group, blood lipid and blood glucose levels of each treatment group were significantly lower(P&lt;0.05); HE staining results showed that liver pathological damage in each treatment group was significantly improved; oil red O staining results showed fat distribution in each treatment group significantly reduced(P&lt;0.01); immunohistochemical staining showed that glucose regulated the protein expression of protein 78(GRP78) in liver tissues of each treatment group was also significantly reduced(P&lt;0.01); Western blot results showed that endoplasmic reticulum stress signal pathway-related factors GRP78, phosphorylated-protein kinase R-like ER kinase(p-PERK), eukaryotic translation-initiation factor 2α(eIF2α), activating transcription factor 4(ATF4), C/EBP homologous protein(Chop), inositol requiring 1α(IRE1α), and cleaved-cysteinyl aspartate specific proteinase 12(cleaved-caspase-12) were significantly reduced(P&lt;0.01). The results of the combined drug treatment group were better than those of the single drug treatment group. These results showed that B. pilosa decoction had the effect in improving non-alcoholic fatty liver, and its mechanism may be related to the down-regulation of the expression of endoplasmic reticulum stress(ERS)-related factors, and the reduction of the apoptosis of hepatocytes caused by ERS and the down-regulation of blood lipid and blood glucose levels.

The laurel family within the Magnoliids has attracted attentions owing to its scents, variable inflorescences, and controversial phylogenetic position. Here, we present a chromosome-level assembly of the Litsea cubeba genome, together with low-coverage genomic and transcriptomic data for many other Lauraceae. Phylogenomic analyses show phylogenetic discordance at the position of Magnoliids, suggesting incomplete lineage sorting during the divergence of monocots, eudicots, and Magnoliids. An ancient whole-genome duplication (WGD) event occurred just before the divergence of Laurales and Magnoliales; subsequently, independent WGDs occurred almost simultaneously in the three Lauralean lineages. The phylogenetic relationships within Lauraceae correspond to the divergence of inflorescences, as evidenced by the phylogeny of FUWA, a conserved gene involved in determining panicle architecture in Lauraceae. Monoterpene synthases responsible for production of specific volatile compounds in Lauraceae are functionally verified. Our work sheds light on the evolution of the Lauraceae, the genetic basis for floral evolution and specific scents.

Objective: To explore the relationship between physiological parameters changes and severe heatstroke induced by 5-km armed cross-country training., Methods: A total of 521 male officers and soldiers from a special team who participated in the summer training of 5-km armed cross-country training from year 2016 to 2017 were enrolled. All trainees participated in 5-km armed cross-country training in high temperature and humidity environment of ambient temperature > 32 centigradeand (or) relative humidity > 65%. The trainees were divided into two groups according to the incidence of severe heatstroke in the course of training. The age, enlistment time, constitution score, body mass index (BMI), external environment (ambient temperature, relative humidity, wind speed, heat index) of trainees of the two groups, and the change rates of arterial blood oxygen saturation (SaO 2 ), body temperature, pulse and blood pressure within 5 minutes after the 5-km armed cross-country training were compared between the two groups. The risk factors of severe heatstroke were screened by two classified Logistic regression analysis, and the predictive value of various risk factors of severe heatstroke was analyzed by the receiver operator characteristic curve (ROC)., Results: In 521 trainees of 5-km armed cross-country training, 29 trainees suffered from severe heatstroke accounting for 5.57%. There was no significant difference in the age, enlistment time, constitution score, BMI, or external environment during 5-km armed cross-country training between severe heatstroke group and non-severe heatstroke group. Compared with those without severe heatstroke, the descending rates of body temperature, pulse, blood pressure and SaO 2 increased rate within 5 minutes after 5-km armed cross-country training of severe heatstroke trainees were significantly decreased [temperature descending rate: (0.67±0.30)% vs. (1.43±1.28)%, pulse descending rate: (7.53±5.21)% vs. (13.48±8.07)%, blood pressure descending rate: (9.28±6.84)% vs. (19.42±7.73)%, SaO 2 increased rate: (0.51±0.39)% vs. (1.50±1.43)%, all P < 0.01]. Two classification Logistic regression analysis showed that the temperature descending rate [odds ratio (OR) = 0.485, 95% confidence interval (95%CI) = 0.289-0.817], pulse descending rate (OR = 0.903, 95%CI = 0.845-0.965), blood pressure descending rate (OR = 0.841, 95%CI = 0.790-0.896), and SaO2 increased rate (OR = 0.421, 95%CI = 0.250-0.711) were the risk factors for severe heatstroke during 5-km armed cross-country training (all P < 0.01). ROC curve analysis showed that temperature descending rate [area under ROC curve (AUC) = 0.659, 95%CI = 0.604-0.714], pulse descending rate (AUC = 0.730, 95%CI = 0.762-0.900), blood pressure descending rate (AUC = 0.831, 95%CI = 0.659-0.801), SaO 2 increased rate (AUC = 0.711, 95%CI = 0.655-0.767) could be used for the incidence of severe heatstroke prediction during 5-km armed cross-country training (all P < 0.01), and the predicted value was the same., Conclusions: Under the same conditions, the severe heatstroke during 5-km cross-country training is closely related to the descending rates of body temperature, pulse, and blood pressure as well as SaO 2 increased rate within 5 minutes after the training, whose predictive values for severe heatstroke were the same.

Objective: To explore the clinical characteristics and treatments of patients with idiopathic sudden sensorineural hearing loss（ISSHL） with or without vertigo. Method: One hundred and twenty ISSHL cases were divided into vertigo group ( n =36) , without vertigo group ( n =84) , and with benign paroxysmal positional vertigo group ( n =15). All patients were in regular treatment. Besides, according to the types of BPPV, patients do the Epley maneuver or Barbecue roll maneuver. We summarized the result and treatment of the patients. Result: The audiometric curve of ISSHL with vertigo were mainly at flat type. After treatment of the ISSHL patients were better than the patients with vertigo in the degrees of hearing loss . Furthermore, the rate of the patients of marked efficiency, efficiency and total efficiency of ISSHL was lower than the ones without.The patients with BPPV, including 12 cases of posterior semicircular canal and the 3 cases of lateral semicircular canal, were all ipsilateral. Conclusion: ISSHL with vertigo group lost hearing is severer than ISSHL without vertigo. Thus the hearing and the treatment effect were worse.The symptoms without vertigo in ISSHL were better than the patients with vertigo., Competing Interests: The authors of this article and the planning committee members and staff have no relevant financial relationships with commercial interests to disclose., (Copyright© by the Editorial Department of Journal of Clinical Otorhinolaryngology Head and Neck Surgery.)

The pleiotropic pro-inflammatory cytokine, macrophage migration inhibitory factor (MIF), represents an important link between chronic inflammation and tumorigenesis. Although accumulating evidence demonstrates that MIF overexpression is implicated in the development and progression of multiple cancers, including esophageal squamous cell carcinoma (ESCC), the molecular mechanisms underlying its tumor-promoting roles in ESCC remain unclear. In the present study, we observed that MIF is overexpressed in ESCC and correlated significantly with lymph node metastasis, advanced clinical stage, and poor survival of ESCC. MIF knockdown attenuated the proliferation, migration, and invasion of ESCC cells in vitro and in vivo. Moreover, blockage of MIF expression decreased the activation of the Akt, MEK/ERK, and NF-κB pathways and enhanced sensitivity to apoptosis. Meanwhile, repression of MIF expression resulted in activation of glycogen synthase kinase 3 beta (GSK3β) and subsequent decrease of active β-catenin, as well as its downstream targets including cyclin D1, matrix metalloproteinase (MMP)-7, c-myc, and c-Jun. Collectively, our results provided mechanistic insights into the tumor-promoting role of MIF in ESCC, and suggested that MIF represents a potential therapeutic target for treatment of ESCC., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Objective: This study investigated the effects of immune complexes (ICs) on tumor necrosis factor α (TNF-α) and B cell-activating factor (BAFF) production from U937 cells and further explored the mechanism., Materials and Methods: U937 cells were incubated with necrosis supernatant or systemic lupus erythematosus (SLE) sera alone, or their combination. The expression of TNF-α and BAFF was determined by Real-time polymerase chain reaction and enzyme-linked immunosorbent assay. High mobility group box protein 1(HMGB1) A-box was produced by gene recombination. HMGB1 A-box and anti-receptor for advanced glycation end products (RAGE) antibody were adopted in the blocking experiments. The importance of DNA for cytokine induction was investigated by DNase treatment., Results: The combination of necrosis supernatant and SLE sera induced the expression of TNF-α and BAFF significantly increased compared to necrosis supernatant or SLE sera alone. Recombinant HMGB1 A-box protein was purified, and TNF-α and BAFF production, which were induced by this combination, was blocked via HMGB1 A-box and anti-RAGE antibody. Moreover, we found that DNA component is important for the immunostimulatory activity of this combination., Conclusions: ICs containing DNA can promote TNF-α and BAFF production in U937 cells, and this process can be mediated by HMGB1 and RAGE. One possible mechanism of increasing BAFF production in SLE is proposed in this study whereby B cell activation, antibody production and ICs stimulated monocytes may create a vicious cycle that leads to B cell hyperactivity, which can be of importance for SLE etiopathogenesis.

Objective: To investigate the expression of serum TM and MPO in adult patients with obstructive sleep apnea hypopnea syndrome(OSAHS). Method: Ninety OSAHS patients who confirmed by PSG as OSAHS group. According to AHI, the patients were divided into 3 groups include heavy, medium and light, and 30 healthy outpatients were control group. The serum TM and MPO were determined by ELISA; TM and MPO were measured after comprehensive treatment in patients with severe OSAHS, and the correlation between TM, MPO and PSG were analyzed. Result: ①With the severity of OSAHS patients increased, the serum levels of TM and MPO increased gradually（F=20.761,21.433； P <0.01). There was no significant difference about the concentration of TM and MPO between light and control group（ P >0.05）；The concentration of TM and MPO was significantly higher in heavy and medium group compared with light and control group（ P <0.01）.②There was no correlation between serum levels of TM, MPO, BMI, age and sex in OSAHS patients( P >0.05). The serum concentration of TM was positively related to MPO. The serum concentrations of TM and MPO were positively correlated with AHI, but negatively with LSaO₂ ( P <0.05). ③LSaO₂ was significantly increased, AHI and peripheral blood TM, MPO levels were significantly reduced in thirty severe OSAHS cases received the combined treatment( P <0.01). Conclusion: The increase of TM and MPO concentration in peripheral blood is one of characteristics of cardiovascular damage in patients with OSAHS. Combined detection of serum TM and MPO concentrations in patients with the disease is helpful for evaluation and risk assessment of cardiovascular disease., Competing Interests: The authors of this article and the planning committee members and staff have no relevant financial relationships with commercial interests to disclose., (Copyright© by the Editorial Department of Journal of Clinical Otorhinolaryngology Head and Neck Surgery.)

Objective: Natural killer (NK) cells serve as primary immune surveillance and are partially regulated by combinations of killer immunoglobulin-like receptors (KIR) and their human leukocyte antigen-C (HLA-C) ligands. Alterations in NK cell activity have been associated with Hashimoto thyroiditis (HT). The aim of this study was to determine whether certain KIR/HLA-C genotype combinations play a role in HT pathogenesis., Methods: The present study enrolled 107 unrelated HT patients and 108 random healthy individuals in a case-control study. Blood was collected for DNA extraction; typing of KIR genes and HLA-C alleles was performed by polymerase chain reaction with sequence specific primers (PCR-SSP), followed by electrophoresis on agarose gels., Results: Among a panel of KIR2D/HLA-C genotype combinations, the frequency of KIR2DS2/HLA-C1 was significantly increased in HT patients compared to controls (33.64% vs. 12.96%, P<.001). To further analyze the precise genotype, we investigated inhibitory or activating KIR/HLA-C gene pairs when their corresponding activating or inhibitory KIR genes were absent in the 2 groups. Only the frequency of KIR2DS2(-)2DL2/3(+)HLA-C1(+) was significantly decreased in HT patients compared to controls (48.60% vs. 70.37%, P = .001)., Conclusion: Our data suggest that KIR2DS2/HLA-C1 may correlate with HT pathogenesis. On the contrary, the predominance of KIR2DL2/3/HLA-C1 in the absence of KIR2DS2 suggests a potential inhibitory role in HT pathogenesis. In conclusion, our findings may further elucidate the mechanisms underlying the pathogenesis of HT and other autoimmune diseases., Abbreviations: HLA-C = human leukocyte antigen-C HT = Hashimoto thyroiditis KIR = killer immunoglobulin-like receptor NK = natural killer PCR = polymerase chain reaction.

Subcutaneous injection of BmK I could be adopted to well establish a novel pain model. Moreover, 5-hydroxytryptamine (serotonin, 5-HT) receptor is involved in regulating animal pain-related behaviors. However, the underlying mechanism of 5-HT3R on BmK I-induced pain remains unclear. Animal behavioral testing, RT-PCR and Western blotting were used to yield the following results: first, intraplantar (i.pl.) injection of BmK I (10 μg) induced elevated mRNA and protein levels of 5-HT3AR in bilateral L4-L5 spinal cord; Second, intrathecal (i.t.) injection of ondansetron (a specific antagonist of 5-HT3AR) reduced spontaneous pain responses, attenuated unilateral thermal and bilateral mechanical hypersensitivity elicited by BmK I; Microglia could be activated by BmK I (i.pl.) in both sides of L4-L5 spinal cord, and this effect was reversed by intrathecal pre-treatment with 5-HT3AR antagonist. Meanwhile, the 5-HT3AR in L4-L5 spinal cord was almost co-localized with NeuN (a marker of nerve cell), but not co-expressed with Iba-1 (a marker of microglia). Finally, the expression level of CX3CL1 and CX3CR1 was reduced by intrathecal pre-treatment with ondansetron. Our results indicate that both 5-HT3AR signaling pathway and microglia are activated in the process of induction and maintenance of BmK I-induced pain nociception. Meanwhile, our results suggest that the neuronal 5-HT3AR may communicate with microglia indirectly via CX3CL1 which is involved in regulating the BmK I-induced hyperalgesia and sensitization.

Background: Prostate cancer is one of the main causes of cancer death, and drug resistance is the leading reason for therapy failure. However, how this occurs is largely unknown. We therrfore aimed to study the response of DU145 cells to cisplatin., Materials and Methods: Du145 prostate cancer cells were treated with a low dose of cisplatin for 24 h and cell viability and number were determined by MTT assay and trypan blue exclusion assay, respectively. The real time polymerase chain reaction (PCR) was used to assess responses to cisplatin treatment., Results: After 24h 2 μg/ml treatment did not result in significant reduction in cell viability or number. However, it led to enhanced cancer cell invasiveness. E-cadherin mRNA was reduced, and vimentin, Snail, Slug, metalloproteinase 9 (MMP9) mRNA expression increased significantly, a feature of epithelial-mesenchymal transition (EMT)., Conclusions: Short time low concentration cisplatin treatment leads to elevated invasiveness of DU145 cancer cells and this is possibly due to EMT.

The mammalian target of rapamycin (mTOR) pathway is essential for maintenance of the sensitivity of certain adult sensory neurons. Here, we investigated whether the mTOR cascade is involved in scorpion envenomation-induced pain hypersensitivity in rats. The results showed that intraplantar injection of a neurotoxin from Buthus martensii Karsch, BmK I (10 μg), induced the activation of mTOR, as well as its downstream molecules p70 ribosomal S6 protein kinase (p70 S6K) and eukaryotic initiation factor 4E-binding protein 1 (4E-BP1), in lumbar 5-6 dorsal root ganglia neurons on both sides in rats. The activation peaked at 2 h and recovered 1 day after injection. Compared with the control group, the ratios of p-mTOR/p-p70 S6K/p-4EBP1 in three types of neurons changed significantly. The cell typology of p-mTOR/p-p70 S6K/p-4E-BP1 immuno-reactive neurons also changed. Intrathecal administration of deforolimus, a specific inhibitor of mTOR, attenuated BmK I-induced pain responses (spontaneous flinching, paroxysmal pain-like behavior, and mechanical hypersensitivity). Together, these results imply that the mTOR signaling pathway is mobilized by and contributes to experimental scorpion sting-induced pain.

The dextranase added in current commercial dextranase-containing mouthwashes is largely from fungi. However, fungal dextranase has shown much higher optimum temperature than bacterial dextranase and relatively low activity when used in human oral cavities. Bacterial dextranase has been considered to be more effective and suitable for dental caries prevention. In this study, a dextranase (Dex410) from marine Arthrobacter sp. was purified and characterized. Dex410 is a 64-kDa endoglycosidase. The specific activity of Dex410 was 11.9 U/mg at optimum pH 5.5 and 45 °C. The main end-product of Dex410 was isomaltotriose, isomaltoteraose, and isomaltopentaose by hydrolyzing dextran T2000. In vitro studies showed that Dex410 effectively inhibited the Streptococcus mutans biofilm growth in coverage, biomass, and water-soluble glucan (WSG) by more than 80, 90, and 95 %, respectively. The animal experiment revealed that for short-term use (1.5 months), both Dex410 and the commercial mouthwash Biotene (Laclede Professional Products, Gardena, CA, USA) had a significant inhibitory effect on caries (p = 0.0008 and 0.0001, respectively), while for long-term use (3 months), only Dex410 showed significant inhibitory effect on dental caries (p = 0.005). The dextranase Dex410 from a marine-derived Arthrobacter sp. strain possessed the enzyme properties suitable to human oral environment and applicable to oral hygiene products.

Intraplantar (i.pl.) injection of BmK I, a receptor site 3-specific modulator of voltage-gated sodium channels (VGSCs) from the venom of scorpion Buthus martensi Karsch (BmK), was shown to induce long-lasting and spontaneous nociceptive responses as demonstrated through experiments utilizing primary thermal and mirror-imaged mechanical hypersensitivity with different time course of development in rats. In this study, microglia was activated on both sides of L4-L5 spinal cord by i.pl. injection of BmK I. Meanwhile, the activation of p38/MAPK in L4-L5 spinal cord was found to be co-expressed with OX-42, the cell marker of microglia. The unilateral thermal and bilateral mechanical pain hypersensitivity of rat induced by BmK I was suppressed in a dose-dependent manner following pretreatment with SB203580 (a specific inhibitor of p-p38). Interestingly, microglia activity was also reduced in the presence of SB203580, which suggests that BmK I-induced microglial activation is mediated by p38/MAPK pathway. Combined with previously published literature, the results of this study demonstrate that p38-dependent microglial activation plays a role in scorpion envenomation-induced pain-related behaviors., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Thermostable amylopullulanase (TAPU) is valuable in starch saccharification industry for its capability to catalyze both α-1,4 and α-1,6 glucosidic bonds under the industrial starch liquefication condition. The majority of TAPUs belong to glycoside hydrolase family 57 (GH57). In this study, we performed a phylogenetic analysis of GH57 amylopullulanase (APU) based on the highly conserved DOMON&#95;glucodextranase&#95;like (DDL) domain and classified APUs according to their multidomain architectures, phylogenetic analysis and enzymatic characters. This study revealed that amylopullulanase, pullulanase, andα-amylase had passed through a long joint evolution process, in which DDL played an important role. The phylogenetic analysis of DDL domain showed that the GH57 APU is directly sharing a common ancestor with pullulanase, and the DDL domains in some species undergo evolution scenarios such as domain duplication and recombination., (© 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Objective: This study aimed to express a fusion protein of diphtheria toxin and human B cell-activating factor (DT388sBAFF) in Escherichia coli (E. coli) and investigate its activity in human B-lineage acute lymphoblastic leukemia 1 cells (BALL-1)., Methods: A fragment of DT388sBAFF fusion gene was separated from plasmid pUC57-DT388sBAFF digested with Nde I and Xho I, and inserted into the expression vector pcold II digested with the same enzymes. Recombinants were screened by the colony polymerase chain reaction (PCR) and restriction map. The recombinant expression vector was transformed into BL21 and its expression was induced by isopropyl β-D-1-thiogalactopyranoside (IPTG). The recombinant protein was identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot, and then purified by Ni(2+)-NTA affinity chromatography. The expression level of B cell-activating factor receptor (BAFF-R) on BALL-1 cells was assessed by real-time PCR. The receptor binding capacity of recombinant protein was determined by cell fluorescent assay. The specific cytotoxicity of recombinant protein on BALL-1 cells was detected by 3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide (MTT) assay., Results: The expression level of recombinant protein was 50% of total bacterial proteins in E. coli, and the recombinant protein could bind to BAFF-R-positive BALL-1 cells and thereby produce a cytotoxic effect on the cells., Conclusion: The fusion protein expression vector DT388sBAFF was successfully constructed and the recombinant protein with selective cytotoxicity against BALL-1 cells was obtained, providing foundation for further study of the therapy of human B-lineage acute lymphoblastic leukemia.

In recent years, the role of high mobility group box-1 (HMGB1) protein and its receptors in autoimmune diseases has received increasing attention. It has been documented that HMGB1 is associated with disease activity in patients with systemic lupus erythematosus (SLE). This study was undertaken to determine the potential role of receptor for advanced glycation end products (RAGE), one receptor for HMGB1, in the pathogenesis of SLE. Plasma levels of soluble RAGE (sRAGE) from 105 patients with clinical diagnosis of SLE and 43 healthy controls were determined by ELISA. Associations between sRAGE levels and clinical, laboratory characteristics were assessed. The data showed that plasma levels of sRAGE in patients with SLE were significantly lower than those in healthy controls (HC) (P = 0.003). Plasma sRAGE in patients receiving short-period treatment showed an immediate decrease compared with the untreated patients (P = 0.023). In contrast, plasma sRAGE in patients receiving long-period treatment were significantly increased compared to those with short-period treatment (P = 0.000) and comparable with those in HC (P = 0.305). The significant decreased levels of sRAGE in patients with SLE suggest the potential association of RAGE signalling and SLE clinical pathology, whereas, long-period antilupus treatment may counteract the decreased sRAGE levels in patients with SLE., (© 2012 The Authors. Scandinavian Journal of Immunology © 2012 Blackwell Publishing Ltd.)

Recent studies indicate that high-mobility group box protein 1 (HMGB1) contributes to the pathogenesis of diverse autoimmune disorders. It induces the production of interferon-alpha (IFN-alpha) and tumor necrosis factor alpha (TNF-alpha) in vitro. In the present study, plasma HMGB1, TNF-alpha, and IFN-alpha were determined with ELISA in 37 patients with systemic lupus erythematosus (SLE) and 39 age- and sex-matched healthy controls (HC). The possible associations of these cytokines with disease activities, autoantibodies, and certain laboratory parameters were also explored. The plasma levels of HMGB1, TNF-alpha, and IFN-alpha were increased in SLE patients compared with those of HC (P < 0.05). Moreover, the levels of HMGB1 and TNF-alpha in the active SLE patients were elevated compared with those in inactive patients and HC. Additionally, plasma HMGB1 was positively correlated with peripheral neutrophils, and plasma TNF-alpha was positively correlated with anti-Sm, ESR and CRP, while plasma IFN-alpha was inversely correlated with the age and platelet level in SLE patients. Our data indicated that increased plasma HMGB1 was associated with disease activity in SLE, which was similar to TNF-alpha. High level of plasma IFN-alpha may be related to nephritis and thrombocytopenia in SLE.

The gene encoding a new extracellular amylopullulanase (type II pullulanase) was cloned from an extremely thermophilic anaerobic archaeon Thermococcus siculi strain HJ21 isolated previously from a deep-sea hydrothermal vent. The functional hydrolytic domain of the amylopullulanase (TsiApuN) and its MalE fusion protein (MTsiApuN) were expressed heterologously. The complete amylopullulanase (TsiApu) was also purified from fermentation broth of the strain. The pullulanase and amylase activities of the three enzymes were characterized. TsiApu had optimum temperature of 95°C for the both activities, while MTsiApuN and TsiApuN had a higher optimum temperature of 100°C. The residual total activities of MTsiApuN and TsiApuN were both 89% after incubation at 100°C for 1 h, while that of TsiApu was 70%. For all the three enzymes the optimum pHs for amylase and pullulanase activities were 5.0 and 6.0, respectively. By analyzing enzymatic properties of the three enzymes, this study suggests that the carboxy terminal region of TsiApu might interfere with the thermoactivity. The acidic thermoactive amylopullulanases MTsiApuN and TsiApuN could be further employed for industrial saccharification of starch.

Accumulating evidences indicate that killer cell immunoglobulin-like receptors (KIRs) and their corresponding specific HLA-C ligands contribute to the pathogenesis of multiple autoimmune diseases via the modulation of natural killer (NK) cell and T cell functions. The present study was performed to investigate whether the polymorphism of KIR genes and HLA ligands associates with the susceptibility of ankylosing spondylitis (AS). Previous studies have demonstrated a strong association between HLA-B27 gene and the pathogenesis of AS. In this study, 115 unrelated HLA-B27-positive AS patients and 119 HLA-B27-positive healthy controls were recruited. Polymerase chain reaction using sequence-specific primers was used to determine the genotypes of KIR genes and HLA-C alleles. The results showed that the frequencies of KIR2DL1 and KIR2DL5 were significantly higher in the AS patient group than those in the control group (p = 0.012 and p = 0.009, respectively). Meanwhile, individuals with AS showed an increased frequency of HLA-Cw*08 (p = 0.001, p (c)  = 0.008) compared with that in controls. Our findings indicate that with the genetic background of HLA-B27, variation at the KIRs and their corresponding specific HLA-C ligands may influence the ability of NK cells and T cells to recognize and lyse targets in immune responses, which thereby contributes to pathogenesis of AS.

Aim: To clone human high mobility guoup box1 A box (HMGB1 A box) and express it in escherichia coli effectly, investigate the inhibit effection of the purpose protern to the activation of monocytes stimulated by immunocomplex., Methods: According to human HMGB1 gene order which was optimized by our laboratory the PCR primer was designed which containing restriction enzyme cutting site. The HMGB1 A box gene was cloned following the whole gene synthesis template of human HMGB1, then the PCR product was inserted into clone vector pMD19-T. The positive colone was identified by colony PCR, zymography analysis and DNA sequencing. Recombinant colne vector was digested by restriction enzymes Nde I and Xho I and separated by agarose gel electrophoresis, then the fragment was inserted into the corresponding sites of expression vector pQE-T7-2. The positive recombinant expression vector was identified by colony PCR and the recombinant strains was induced by IPTG, then the purpose protein was identified by SDS-PAGE and Western blot. The recombinant protein of human HMGB1 A box was purificated by Ni(2+)-NTA chromatography and the inhibit effection of the purpose protern to the activation of monocyte stimulated by immunocomplex was identified by RT-PCR., Results: We acquired expression strains of recombinant human HMGB1 A box, the target protein account for up to 40% of the whole protein of E.coli. Western blot showed recombinant protein can specificly reacted with anti-human HMGB1 polyclonal antibody and anti-His-Tag polyclonal antibody.The purpose protein was found more than 90% after purified, and can effectively inhibit the production of BAFF, IFN-gamma and TNF-alpha in monocyte which were induced by IC., Conclusion: A recombinant bacterial strain for expressing human HMGB1A box with biological activities was constructed successfully.

The function of a new starter unit acyltransferase (SAT) domain SAT-EF080951 (GenBank accession number) encoded in a new type I polyketide synthase (PKS) gene cluster EF568935 (GenBank accession number) isolated for this study was analyzed by domain replacement with an extender unit AT (EAT) domain of avermectin PKS. It was shown that the SAT-EF080951 incorporated malonyl-CoA specifically in vivo, which contradicted the specificity that we had previously determined by substrate binding test in vitro. The result of this study indicates that type I PKS-SAT can alter its specificity in vivo and functions well in extender units and proved the feasibility of the SAT-EAT domain replacement in type I PKS. We propose that SAT-EAT replacement strategy could be a novel route for increasing the diversity of new polyketides combinatorially biosynthesized. The new type I PKS-SAT-EF080951 studied herein may be further employed for related studies on enzymology or combinatorial biosynthesis of polyketides.

The genes of killer cell immunoglobulin-like receptors (KIRs), which are involved in the activation of T cells and natural killer cells, are highly variable. In recent years, the role of KIRs in autoimmune diseases has received increasing attention. The present study was undertaken to determine the association of the polymorphism of KIR genes with the susceptibility to systemic lupus erythematosus (SLE). The polymorphism of KIR genes of 93 patients with SLE together with 123 healthy donors as the control group was determined by polymerase chain reaction with sequence-specific primers. Twenty-seven novel gene combinations were found. Genotypic frequencies of KIR2DL2 (p < 0.001) and KIR2DS1 (p < 0.001) were much higher in patients with SLE than in control subjects. Individuals with two and more than two activating KIR genes were found more frequently in patients than in control subjects (80.7% versus 66.7%, p = 0.022). The results suggest that a genetic disturbance between activating and inhibitory KIR genes may be one of the key factors underlying the pathogenesis of SLE.

Background: The narrow host range of Mycobacterium leprae and the fact that it is refractory to growth in culture has limited research on and the biologic understanding of leprosy. Host genetic factors are thought to influence susceptibility to infection as well as disease progression., Methods: We performed a two-stage genomewide association study by genotyping 706 patients and 1225 controls using the Human610-Quad BeadChip (Illumina). We then tested three independent replication sets for an association between the presence of leprosy and 93 single-nucleotide polymorphisms (SNPs) that were most strongly associated with the disease in the genomewide association study. Together, these replication sets comprised 3254 patients and 5955 controls. We also carried out tests of heterogeneity of the associations (or lack thereof) between these 93 SNPs and disease, stratified according to clinical subtype (multibacillary vs. paucibacillary)., Results: We observed a significant association (P<1.00x10(-10)) between SNPs in the genes CCDC122, C13orf31, NOD2, TNFSF15, HLA-DR, and RIPK2 and a trend toward an association (P=5.10x10(-5)) with a SNP in LRRK2. The associations between the SNPs in C13orf31, LRRK2, NOD2, and RIPK2 and multibacillary leprosy were stronger than the associations between these SNPs and paucibacillary leprosy., Conclusions: Variants of genes in the NOD2-mediated signaling pathway (which regulates the innate immune response) are associated with susceptibility to infection with M. leprae., (2009 Massachusetts Medical Society)

Aim: To analyze the association of estrogen receptor alpha (OR alpha) gene polymorphisms with cytokine genes expression in patients with systemic lupus erythematosus (SLE) and controls., Methods: Genomic DNA was extracted and polymorphisms of XbaI, Ukrainian (XX, Xx, or xx genotype) and PvuII (PP, Pp, or pp) in intron 1 of OR alpha gene were detected by polymerase chain reaction-restriction fragment length polymorphisms (PCR-RFLP) method. The messenger RNA (mRNA) levels of interleukin (IL)-10, IL-4, interferon (IFN)-gamma, and IL-2 were assessed by semiquantitative reverse transcription polymerase chain reaction (RT-PCR)., Results: In patients with SLE with PpXx genotype, IL-10 and IL-4 mRNA expression was higher (P < 0.001 and P = 0.013, respectively), while in patients with SLE with Ppxx genotype IFN-gamma and IL-2 mRNA expression was lower than in controls (P < 0.001). There was no significant difference in mRNA expression of 4 cytokines among controls with various genotypes., Conclusion: OR alpha gene polymorphism may be associated with the expression of IL-10, IL-4, IL-2, and IFN-gamma in patients with SLE.

Osteoclast inhibitory lectin (OCIL) is a novel regulator of bone remodeling, however, little is known concerning how OCIL is regulated to date. In this study, approximately 4.4 kb of the 5'-flanking sequence of rat OCIL gene was cloned into the promoter-less reporter vector pGL3-basic and transiently transfected into three different cell lines. The differences in the levels of luciferase activity paralleled well with the levels of OCIL mRNA expression in these cells, suggesting that the regulation of rat OCIL gene expression occurs mainly at the transcriptional level. Additional luciferase assays using a series of constructs containing unidirectionally deleted fragments showed that the construct-1819/pGL3 (-1819 to +118) exhibited the highest luciferase activity, suggesting the presence of functional promoter in this region. The region from -4370 to -2805 might contain negative regulatory elements, while the region from -1819 to -1336 might have important positive regulatory elements that enhance OCIL transcription. Sequence analysis of the promoter revealed the absence of both TATA and CAAT boxes. However, in the proximal promoter region (-81 to +118), several potential transcription factor binding sites that may be responsible for the basal transcriptional activity of rat OCIL promoter were observed. The promoter contains several potential Sp1 binding sites, and cotransfection of a shRNA expression plasmid that knockdowns Sp1 significantly reduced OCIL promoter activity and endogenous gene expression and moreover, overexpressing Sp7, a Sp1 family member that also binds to Sp1 binding sequence, increased OCIL promoter activity and gene expression, suggesting a role of Sp1 family proteins in regulation of OCIL transcription.

Objective: To investigate the association of killer cell immunoglobulin-like receptors (KIR) genotype and haplotype with ankylosing spondylitis (AS)., Methods: Peripheral blood samples were collected from 105 AS patients, 62 patients of osteoarthritis (OA), and 412 randomly selected healthy controls. Polymerase chain reaction with sequence-specific primers (PCR-SSP) was used to detect the KIR genotype and haplotype., Results: The genotype frequency of 3DL3-2DL3-2DL1-2DP1-2DL4-3DL1-2DS4-3DL2 (6.67%) was significantly lower in the AS patients than in the control subjects (20.15%) and OA patients (17.74%, P = 0.001, 0.037 respectively). The genotype frequency of 3DL3-2DL3-2DL2-2DL1-2DP1-2DLA-3DL1-2DL5-2DS1-2DS2-2DS3-2DS4-2DS5-3DS1-3DL2 and 3DL3-2DL3-2DL2- 2DL1-2DP1-2DL4-3DL1-2DL5-2DS1-2DS4-3DL2 of the AS patients (9.52%, 5.71%)was significantly higher than that of the controls(2.18%, 0.49%; P = 0.001, 0.001), and these two genotypes were not detected in the OA patients. There were not significant differences in the haplotypes A and B among the AS patients, OA patients, and healthy controls., Conclusion: KIR genotypes may be associated with the susceptibility to AS.

We have developed a novel protocol for directional cloning of PCR products into any vector. The target sequence is amplified in two parallel asymmetric PCRs using specially but simply designed primers. Two single-stranded products are produced and they are annealed to form a double-stranded DNA fragment bearing overhangs at both ends that correspond to the restriction overhangs of certain restriction enzymes. The fragment can then be cloned into a certain vector previously treated with the corresponding enzymes without restriction of the inserted fragment. Compared with previously published protocols, the procedure described in this paper is highly efficient and it is independent of the restriction sites of the insert and is therefore applicable to molecular biology and biotechnology studies.

Traditional strategies for establishing shRNA expression constructs are inefficient, error-prone, or costly. We describe a simple approach that overcomes these drawbacks. Briefly, the sense and antisense strands of the short hairpin RNA coding sequence are segmented into two parts, respectively, at asymmetric sites. The four resulting short oligonucleotides are synthesized. Each oligonucleotide is annealed with its opposite, resulting in a double-stranded fragment with sticky termini at both ends. The two fragments so generated can be easily spliced by simple ligation to reconstitute the full-length short hairpin RNA coding sequence which can then be cloned into an appropriately restricted vector.

We have developed a novel protocol for site-directed mutagenesis of double-stranded DNA. The procedure, termed SORS (named because it undergoes the sequential procedure of segmentation-overhang creating PCR-reannealing-splicing) mutagenesis, is exemplified by a substitution, a deletion, and an insertion of nucleotide(s) in target genes. The template DNA is PCR-amplified into two separate segments divided at the prospective mutation site, and each segment is amplified in two parallel PCRs using primers introducing the mutation. The primers are designed to be able to create protruding bases upon pooling, denaturing, and reannealing the two parallel reactions. The protruding bases at the prospective junction of the two segments are mutually complementary; therefore, the two segments can be re-spliced together to generate the mutated gene. Compared to previously published protocols, this procedure is rapid, restriction-independent and ensures higher success rate and lower potential to produce second-site mutations.

Lipoteichoic acid (LTA), as a primary immunostimulus, triggers the systematic inflammatory responses. Our hypothesis is that ApoA-I can neutralize LTA toxicity, like its effect on LPS. BALB/c mice were challenged with LTA, followed by human ApoA-I administration. We found that ApoA-I could attenuate LTA-induced acute lung injury and inflammation and significantly inhibit LTA-induced IL-1beta and TNF-alpha accumulation in the serum (P<0.01 and P<0.05, respectively), as well as in bronchoalveolar lavage (BAL) fluid (P<0.01 and P<0.05, respectively). Moreover, ApoA-I could significantly reduce the L-929 cell mortality caused by LTA-activated macrophages in a dose-dependent fashion. Furthermore, ApoA-I treatment could diminish LTA-mediated NFkappaB nuclear translocation in macrophages. An in vitro binding assay indicated that ApoA-I can bind LTA. These results clearly indicated that ApoA-I can effectively protect against LTA-induced sepsis and acute lung damage. The mechanism might be related to the binding and neutralization of LTA.

Introduction: An emerging body of evidence is accumulating to suggest that killer cell immunoglobulin-like receptors (KIRs) and human leukocyte antigen (HLA) class I ligands contribute to the pathogenesis of diverse kinds of autoimmune diseases. However, the functional effects of their polymorphism remain largely unknown to date. Thus, the present study was undertaken to determine the association of the polymorphisms KIRs gene and HLA-C alleles with the susceptibility to ankylosing spondylitis (AS) by means of polymerase chain reaction/sequence-specific primers for genotyping KIRs from genomic DNA of 119 patients with AS together with 128 healthy donors as a control group., Results and Discussion: We found that the frequencies of KIR3DS1 and KIR2DL5 were statistically significantly higher in the patient group than those in the control group (P = 0.016 and P = 0.003, respectively). Meanwhile, the percentage of patients, who were carrying two or more of the activating KIRs, was higher than that of control group. With respect to HLA-C alleles, individuals with AS showed an increased frequency of HLA-Cw02. If HLA-C was divided into group 1 or group 2 based on whether there was an asparagine or lysine present at position 80 of the alpha-chain, HLA-C group 2 was more common in subjects with AS compared to control subjects. The genotype 2DS1+/HLA-C lys(80)+ was more common in subjects with AS. Moreover, the CD69 expression, a NK activation marker, remarkably increased in patient with AS., Conclusion: In conclusions, this study suggests that KIR3DS1 may severe as AS susceptive genes to trigger continuous injury of arthrosis. The imbalance of activating and inhibitory KIR as well as HLA-C group 1 and group 2 may be the key factor, which influences the pathogenesis of AS. Moreover, KIR2DS1 might associate with the susceptibility of AS by influencing NK cell activity once group 2 HLA-C ligands are present.

cDNAs of certain target genes are difficult to obtain by traditional reverse transcription. Herein we describe a novel method to synthesize cDNA based upon the use of the class IIS restriction enzymes. Briefly, the exons of a certain gene are separately PCR-amplified, each using the primers containing a recognition sequence of a certain class IIS restriction enzyme. All the fragments are restricted using the enzyme(s), resulting in the cohesive end of each exon that is complementary to the one in its adjacent exon. Then the fragments can be assembled together in their naturally occurring order. We successfully applied this method to acquire the coding sequence of Hoxa7 gene. This approach is simple, highly efficient, less error prone and cost-effective, and can also be used to fuse different PCR-fragments from distinct genes to create a chimeric gene or to perform site-directed mutagenesis.

The petroleum ether fraction of MeOH extract from Paederia scandens was evaluated on anti-nociceptive activity in mice using chemical and thermal models of nociception. Given orally, the petroleum ether fraction (PEF) at doses of 20, 40 and 80mg/kg produced significant inhibitions on chemical nociception induced by intraperitoneal acetic acid and subplantar formalin or capsaicin injections and on thermal nociception in the tail-flick test and in the hot plate test. More significant inhibition of nociception was observed at dose of 80mg/kg of the petroleum ether fraction. In the pentobarbital sodium-induced sleeping time test and the open-field test, the petroleum ether fraction neither significantly enhanced the pentobarbital sodium-induced sleeping time nor impaired the motor performance, indicating that the observed anti-nociception was unlikely due to sedation or motor abnormality. Moreover, the petroleum ether fraction-induced anti-nociception in both capsaicin and formalin tests was insensitive to naloxone, but was significantly antagonized by glibenclamide. These results suggested that the petroleum ether fraction produced anti-nociception possibly related to glibenclamide-sensitive K(+)-ATP channels, which merited further studies regarding the precise site and mechanism of action. The major constituents of the petroleum ether fraction (PEF) determined by GC/MS analysis, are linoleic acid, the sterols and vitamin E. Therefore it can be suggested that they exert synergetic effects and are together responsible for the antinociceptive activity of the PEF-fraction.

Using the consensus-degenerate hybrid oligonucleotide primer polymerase chain reaction method, 26 new ketoacyl synthase (KS) fragments were isolated from a marine sediment sample in the East China Sea (ECS) and analyzed by construction of a phylogenetic tree. With a digoxigenin-labeled KS gene fragment used as a probe, a partial polyketide synthase (PKS) gene cluster was isolated and identified by hybridization screening of a marine sediment sample metagenome fosmid library constructed for this study. A new acyltransferase (AT) gene was cloned from the PKS gene cluster and heterogeneously expressed as a protein fused to maltose-binding protein (MBP). Ultraviolet spectrophotometry was used to study the binding of the MBP-AT fusion protein and single AT domain to substrates using MBP and bovine serum albumin as control proteins. Binding constants (Ka, per micromolar) were calculated and used to analyze the substrate specificity of the acyltransferase. We concluded that there are many unrevealed new PKS gene clusters in marine sediments in the ECS. The acyltransferase is presumably an acetyltransferase from a new PKS gene cluster.

Accumulating evidence indicates natural killer (NK) cells play crucial roles in successful pregnancy. To investigate whether the killer cell immunoglobulin-like receptor (KIR) gene polymorphism and the corresponding specific HLA ligands in parent couples possessing a susceptibility to unexplained recurrent spontaneous abortion (RSA), we searched 73 pairs of childless couples with three or more abortions characterized as unexplained RSA and 68 pairs of healthy control couples. Peripheral blood was drawn to obtain genomic DNA which was used for a polymerase chain reaction using sequence-specific primers (PCR-SSP) in order to determine whether 15 selected KIR genes and two groups of HLA-C alleles were present. Our result showed that gene frequency of KIR2DS1 was higher in patients with RSA compared to that of control subjects (P =0.029). Increased numbers of activating KIR genes was observed in patients (P =0.041). Women who possessed more than two activating KIR genes were found more frequently in patients than those in control subjects (P =0.018). From a cohort of husband and wife couples, the women with a KIR2DS1 gene, and with a decreased group 2 HLA-C allele for the homologous inhibitory receptor KIR2DL1, had a tendency to fall into the RSA group (P =0.004). The results suggest that a genetic variation at the KIR locus influences the susceptibility to unexplained RSA in the Chinese Han population. Moreover, decreased ligands for inhibitory KIRs could potentially lower the threshold for NK cell activation, mediated through activating receptors, thereby contributing to pathogenesis of RSA.

Taking two peanut varieties with different drought-resistance Luhua 11 and Nongda 818 as test crops, the effects of different irrigation amount at seedling stage on their physiological characteristics and yield were studied from 2003 to 2004. The results showed that with decreasing irrigation amount, the leaf photosynthesis rate of test varieties decreased, while malondialdeyde (MDA) content increased. Watering 60-80 mm ( suitable drought) enhanced the activities of superoxide dismutase (SOD) , peroxidase (POD) and catlase (CAT) , and increased the content of soluble protein. After re-watering by the end of treatments, the activities of SOD, POD and CAT and the contents of soluble protein and MDA decreased significantly, while photosynthesis rate increased obviously. The pod and kernel yield decreased with decreasing irrigation amount, and Luhua 11 had a greater loss than Nongda 818, indicating that Nongda 818 was more drought-tolerant than Luhua 11. It was suggested that under water-saving culture, the irrigation amount at seedling stage could not be less than 80 mm for Luhua 11, and less than 60 mm for Nongda 818.

To clone human interleukin-26 (hIL-26) and express it in E. coli efficiently. Two pairs of primers were synthesized according to the hIL-26 gene reported on GenBank. The hIL-26 gene was cloned by nest PCR following the first round RT-PCR from human peripherial blood monocytes total RNA, and then the PCR product was cloned into pMD18-T vector. Colony PCR, restriction analysis and sequence analysis showed that the gene cloned was the same as the reported hIL-26. The recombinant was cut with BamHI and EcoR I to obtain the hIL-26 fragment, and then the fragment was inserted into pBV220 which was cut with the same enzymes. The recombinant expression vector was induced to express hIL-26 at 42 degrees C, SDS-PAGE analysis showed that the recombinant protein accounted for up to 20% of the whole protein of E. coli, and the protein was also confirmed by Western blotting. Purity of the protein was found to be above 90% after purified with molecular sieve. After renaturalized with glutathione buffer, the promoting effect of it on the production of IFN-y in PBMC was detected by RT-PCR. A recombinant bacterial strain for expressing hIL-26 with biological activity was constructed successfully.

Aim: To construct an eukaryotic co-expression plasmid pSLC-IRES-IL-2, and to express it in COS-7 cells., Methods: Human IL-2 and SLC genes were cloned by RT-PCR and PCR, respectively, and then the eukaryotic expression plasmid pSLC-IRES-IL-2 was constructed.The constructed plasmid was transfected into COS-7 cells by electroporation method. The expression of SLC and IL-2 was detected by Western blot., Results: SLC and IL-2 in the culture supernatant and lysate of transfected COS-7 cells were detected by Western blot. The relative molecular masses of the expressed products were consistent with the theoretical values., Conclusion: The SLC and IL-2 co-expression plasmid is successfully constructed and expressed in COS-7 cells, which offers a pathway for research on gene therapy of tumors.

Objective: To study the curative effect of grafting great auricular nerve with pediculated fascial tube in defective damage of facial nerve., Methods: All the 7 patients in this study were treated by grafting great auricular nerve covered by pediculated fascial tube near facial nerve trunk., Results: Four cases with otogenic facial paralysis had grade III - IV recovery of facial nerve function from two to two and half years after the nerve grafting operation. Three patients post-traumatic facial paralysis had grade III recovery of facial nerve function 2 years after the nerve grafting operation., Conclusions: The grafting of pediculated fascial tube surrounded great auricular nerve can provide a biological environment with better blood supply for the plerosis and regeneration of nerve and can accelerate the functional recovery of nerves after the nerve grafting.