AIM: To investigate the expression of CD73 and hypoxia-inducible factor-1α (HIF-1α) in human gastric carcinoma, and explore their clinical significance and prognostic value.
METHODS: CD73 and HIF-1α expressions were detected by immunohistochemistry in consecutive sections of tissue samples from 68 gastric carcinoma patients. The peritumor tissues 2 cm away from the tumor were obtained and served as controls. The presence of CD73 and HIF-1α was analyzed by immunohistochemistry using the Envision technique.
RESULTS: CD73 and HIF-1α expressions in gastric carcinoma were significantly higher than those in gastric mucosal tissues as control (P < 0.001) and showed a close correlation (Spearman r = 0.390, P = 0.001). Overexpression of CD73 was positively correlated with differentiation of tumor (P = 0.000), histopathology (P = 0.041), depth of invasion (P < 0.001), nodal status (P = 0.003), metastasis (P = 0.013), and the American Joint Committee on Cancer (AJCC) stage (P < 0.001). High expression of HIF-1α was positively correlated with tumor diameter (P = 0.031), depth of invasion (P = 0.022), and AJCC stage (P = 0.035). The overall survival rate was low in the patients with high expression of CD73 (P < 0.001). Moreover, CD73+/HIF-1α+ patients had the worst prognosis (P < 0.001). CD73 expression was proven to be an independent predictor for patients with gastric carcinoma by both multivariate Cox regression analysis (P = 0.021) and receiver operating characteristic curves (P = 0.001).
CONCLUSION: CD73 expression correlates closely with HIF-1α expression in gastric carcinoma. CD73 could be an independent prognostic indicator for gastric carcinoma.
CD73; Hypoxia-inducible factor-1α; Gastric carcinoma; Immunohistochemistry; Prognosis
AIM: To investigate the non-Helicobacter pylori (H. pylori) bacterial flora concurrent with H. pylori infection.
METHODS: A total of 103 gastric biopsy specimens from H. pylori positive patients were selected for bacterial culture. All the non-H. pylori bacterial isolates were identified by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS).
RESULTS: A total of 201 non-H. pylori bacterial isolates were cultivated from 67 (65.0%) of the 103 gastric samples, including 153 isolates identified successfully at species level and 48 at genus level by MALDI-TOF MS. The dominant species were Streptococcus, Neisseria, Rothia and Staphylococcus, which differed from the predominantly acid resistant species reported previously in healthy volunteers. The prevalence of non-H. pylori bacteria was higher in non-ulcer dyspepsia group than in gastric ulcer group (100% vs 42.9%, P < 0.001). Six bacterial species with urease activity (Staphylococcus epidermidis, Staphylococcus warneri, Staphylococcus capitis, Staphylococcus aureus, Brevibacterium spp. and Klebsiella pneumoniae) were also isolated.
CONCLUSION: There is a high prevalence of the non-H. pylori bacteria concurrent with H. pylori infection, and the non-H. pylori bacteria may also play important as-yet-undiscovered roles in the pathogenesis of stomach disorders.
Non-Helicobacter pylori; Bacterial flora; Gastrointestinal diseases; Matrix-assisted laser desorption ionization time-of-flight mass spectrometry
AIM: To determine the safety and effectiveness of transarterial embolization ablation (TEA) of hepatocellular carcinoma (HCC) with a lipiodol-ethanol mixture.
METHODS: Between January 1 and December 31, 2009, 15 patients with HCC (13 men/two women, aged 38-75 years) accepted TEA treatment and were enrolled in this study, including five newly diagnosed patients and 10 with refractory disease. Two months after TEA, angiography and contrast computed tomography (CT) were performed, and responses were assessed using a modified version of Response Evaluation Criteria in Solid Tumors (RECIST version 1.1). The follow-up period was to June 30, 2010.
RESULTS: Every new case was treated once. Angiography was performed immediately after TEA, and showed that the tumor-feeding vessels were completely embolized and that lipiodol was densely deposited inside tumors. Two months after treatment, contrast CT showed no enhanced lesions. Alpha fetoprotein levels returned to normal in four patients and markedly decreased in another. mean ± SD survival after treatment was 10.8 ± 4.5 mo. All five patients survived during the follow-up period. Ten patients with refractory disease were treated a total of 14 times. Angiography immediately after TEA showed that blood flow to the tumors was obviously decreased in all cases, and contrast CT showed obvious depositions of lipiodol. Two months after treatment, the tumors had shrunk (6/10) or were stable (3/10). One had progressed after 2 mo and died of tumor rupture 3 mo after TEA. mean ± SD survival after treatment was 8.6 ± 4.3 mo; two patients survived during the follow-up period. Adverse effects included reversible hepatic decompensation, upper abdominal pain, and fever.
CONCLUSION: TEA is an effective therapy for patients with HCC and might be more effective than transcather arterial chemoembolization for treating refractory disease.
Transarterial embolization ablation; Lipiodol-ethanol mixture; Hepatocellular carcinoma
AIM: To investigate the spatial distribution patterns of anorectal atresia/stenosis in China.
METHODS: Data were collected from the Chinese Birth Defects Monitoring Network (CBDMN), a hospital-based congenital malformations registry system. All fetuses more than 28 wk of gestation and neonates up to 7 d of age in hospitals within the monitoring sites of the CBDMN were monitored from 2001 to 2005. Two-dimensional graph-theoretical clustering was used to divide monitoring sites of the CBDMN into different clusters according to the average incidences of anorectal atresia/stenosis in the different monitoring sites.
RESULTS: The overall average incidence of anorectal atresia/stenosis in China was 3.17 per 10 000 from 2001 to 2005. The areas with the highest average incidences of anorectal atresia/stenosis were almost always focused in Eastern China. The monitoring sites were grouped into 6 clusters of areas. Cluster 1 comprised the monitoring sites in Heilongjiang Province, Jilin Province, and Liaoning Province; Cluster 2 was composed of those in Fujian Province, Guangdong Province, Hainan Province, Guangxi Zhuang Autonomous Region, south Hunan Province, and south Jiangxi Province; Cluster 3 consisted of those in Beijing Municipal City, Tianjin Municipal City, Hebei Province, Shandong Province, north Jiangsu Province, and north Anhui Province; Cluster 4 was made up of those in Zhejiang Province, Shanghai Municipal City, south Anhui Province, south Jiangsu Province, north Hunan Province, north Jiangxi Province, Hubei Province, Henan Province, Shanxi Province and Inner Mongolia Autonomous Region; Cluster 5 consisted of those in Ningxia Hui Autonomous Region, Gansu Province and Qinghai Province; and Cluster 6 included those in Shaanxi Province, Sichuan Province, Chongqing Municipal City, Yunnan Province, Guizhou Province, Xinjiang Uygur Autonomous Province and Tibet Autonomous Region.
CONCLUSION: The findings in this research allow the display of the spatial distribution patterns of anorectal atresia/stenosis in China. These will have important guiding significance for further analysis of relevant environmental factors regarding anorectal atresia/stenosis and for achieving regional monitoring for anorectal atresia/stenosis.
Spatial distribution; Anorectal atresia/stenosis; Two-dimensional graph-theoretical clustering; Incidence; Monitoring
Hyalophora gloveri gloverin is a glycine-rich and heat stable antimicrobial protein with activity mainly against Escherichia coli. However, Spodoptera exigua gloverin is active against a Gram-positive bacterium but inactive against E. coli. In this study, we investigated expression profile, binding ability and antimicrobial activity of Manduca sexta gloverin (MsGlv). MsGlv transcript was detected in several tissues of naïve larvae with higher levels in the midgut and testis. Expression of MsGlv mRNA in larvae was up-regulated by active Spätzle-C108 and peptidoglycans (PGs) of E. coli and Staphylococcus aureus, and the activation was blocked by pre-injection of antibody to M. sexta Toll, suggesting that MsGlv expression is regulated by the Toll-Spätzle pathway. Recombinant MsGlv bound to the O-specific antigen and outer core carbohydrate of lipopolysaccharide (LPS), Gram-positive lipoteichoic acid (LTA) and PG, and laminarin, but not to E. coli PG or mannan. MsGlv was active against Bacillus cereus, Saccharomyces cerevisiae and Cryptococcus neoformans, but was almost inactive against E. coli and S. aureus. Our results suggest that gloverins are active against some bacteria and fungi.
Gloverin; Toll-Spätzle pathway; lipopolysaccharide; lipoteichoic acid; peptidoglycan; antimicrobial activity
Enterovirus 71 (EV71) is a positive-stranded RNA virus which is capable of inhibiting innate immunity. Among virus-encoded proteins, the 3C protein compromises the type I interferon (IFN-I) response mediated by retinoid acid-inducible gene-I (RIG-I) or Toll-like receptor 3 that activates interferon regulatory 3 (IRF3) and IRF7. In the present study, we report that enterovirus 71 downregulates IRF7 through the 3C protein, which inhibits the function of IRF7. When expressed in mammalian cells, the 3C protein mediates cleavage of IRF7 rather than that of IRF3. This process is insensitive to inhibitors of caspase, proteasome, lysosome, and autophagy. H40D substitution in the 3C active site abolishes its activity, whereas R84Q or V154S substitution in the RNA binding motif has no effect. Furthermore, 3C-mediated cleavage occurs at the Q189-S190 junction within the constitutive activation domain of IRF7, resulting in two cleaved IRF7 fragments that are incapable of activating IFN expression. Ectopic expression of wild-type IRF7 limits EV71 replication. On the other hand, expression of the amino-terminal domain of IRF7 enhances EV71 infection, which correlates with its ability to interact with and inhibit IRF3. These results suggest that control of IRF7 by the 3C protein may represent a viral mechanism to escape cellular responses.
Epidermal growth factor receptor monoclonal antibody was approved for treatment of metastatic colorectal cancer patients carrying KRAS wild type DNA. However, recent studies showed that patients with KRAS G13D mutation may benefit from EGFR antibody therapy. In this study we tried to explore whether the abundance of KRAS mutation could affect the efficacy of EGFR antibody therapy. We firstly established a PNA-PCR method which could calculate the percentage of KRAS mutation in total DNA and proved its ability on 47 colorectal cancer samples bearing KRAS mutations. Then we analyzed the correlation between the abundance of KRAS mutations and efficacy of EGFR antibody therapy in another 35 metastatic colorectal cancer patients. We proved that PNA-PCR assay could calculate the abundance of KRAS mutation and the percentage of mutant DNA in tumor cells varied a lot (10.8%∼98.3%) on the 47 colorectal cancer patients. The efficacy of EGFR antibody correlated with the abundance of KRAS mutations: in the KRAS mutation less than 30% group, the disease control rate was 44.4% (4/9); the disease control rate of 30∼80% group was 5.6% (1/18) and the >80% group was 12.5% (1/8) (P = 0.038). In summary, our study showed that PNA-PCR method could easily detect the percentage of KRAS mutation in tumor cells and colorectal cancer patients with low abundance of KRAS mutation might benefit from EGFR antibody therapy.
We assess the progress in biomolecular modeling and simulation, focusing on structure prediction and dynamics, by presenting the field’s history, metrics for its rise in popularity, early expressed expectations, and current significant applications. The increases in computational power combined with improvements in algorithms and force fields have led to considerable success, especially in protein folding, specificity of ligand/biomolecule interactions, and interpretation of complex experimental phenomena (e.g. NMR relaxation, protein-folding kinetics and multiple conformational states) through the generation of structural hypotheses and pathway mechanisms. Although far from a general automated tool, structure prediction is notable for proteins and RNA that preceded the experiment, especially by knowledge-based approaches. Thus, despite early unrealistic expectations and the realization that computer technology alone will not quickly bridge the gap between experimental and theoretical time frames, ongoing improvements to enhance the accuracy and scope of modeling and simulation are propelling the field onto a productive trajectory to become full partner with experiment and a field on its own right.
Insects synthesize a battery of antimicrobial peptides (AMPs) and expression of AMP genes is regulated by the Toll and Imd (immune deficiency) pathways in Drosophila melanogaster. Drosophila Toll pathway is activated after Spätzle (Spz) is cleaved by Spätzle processing enzyme (SPE) to release the active C-terminal C106 domain (DmSpz-C106), which then binds to the Toll receptor to initiate the signaling pathway and regulate expression of AMP genes such as drosomycin. Toll and Spz genes have been identified in other insects, but interaction between Toll and Spz and direct evidence for a Toll-Spz pathway in other insect species have not been demonstrated. Our aim is to investigate a Toll-Spz pathway in Manduca sexta, and compare M. sexta and D. melanogaster Toll-Spz pathways. Co-immunoprecipitation (Co-IP) assays showed that MsTollecto (the ecto-domain of M. sexta Toll) could interact with MsSpz-C108 (the active C-terminal C108 domain of M. sexta Spz) but not with full-length MsSpz, and DmTollecto could interact with DmSpz-C106 but not DmSpz, suggesting that Toll receptor only binds to the active C-terminal domain of Spz. Co-expression of MsToll-MsSpz-C108, but not MsToll-MsSpz, could up-regulate expression of drosomycin gene in Drosophila S2 cells, indicating that MsToll-MsSpz-C108 complex can activate the Toll signaling pathway. In vivo assays showed that activation of AMP genes, including cecropin, attacin, moricin and lebocin, in M. sexta larvae by purified recombinant MsSpz-C108 could be blocked by pre-injection of antibody to MsToll, further confirming a Toll-Spz pathway in M. sexta, a lepidopteran insect.
Toll; Spätzle; antimicrobial peptide; drosomycin; moricin; antibody-blocking
Objectives: This study was designed to obtain the knowledge about TEG indexes distribution in Chinese aged people, as well as to test the hypothesis that previous TEG indexes are associated with the subsequent thromboembolic and bleeding events in the aged population. Methods: We conducted a two-year follow-up study in Chinese PLA General Hospital, Beijing, China. 403 aged people were enrolled in our study. They received TEG measurements at least once when they entered this study. We collected their demographical characteristics, clinical examination information and their outcome during their observational period. Structural equation modeling (SEM) was used to analyze the relationship between the four indexes from TEG and the outcome via a pathway of indicator. Results: We found that in the “model of bleeding” (adjusted by confounding of Anticoagulants), the model fit indices with chi-square/df = 9.555/7, CFI was 0.997, TLI was 0.994 and standardized root mean square residual (SRMR) was 0.034; while in the “model of thromboembolic events” (adjusted by confounding of Anticoagulants), the model fit indices with chi-square/df = 6.070/7, CFI was1.000, TLI was 1.002 and standardized root mean square residual (SRMR) was 0.000. The “model of thromboembolic events” showed that the four indexes (R, K, MA and ANGLE) were all significantly associated with thromboembolic events, while this significance was not found in the “model of bleeding”. Conclusions: Previous TEG indexes are significantly associated with the subsequent thromboembolic events in the aged population. Future study can test this association and provide more information for the clinical use.
Thrombelastography (TEG); thromboembolic event; bleeding; Chinese aged population; structural equation modeling (SEM)
Insects produce a group of antimicrobial peptides (AMPs) in response to microbial infections. Most AMPs are synthesized as inactive precursors/pro-proteins and require proteolytic processing to generate small active peptides. Here we report identification and functional analysis of two lebocin-related proteins (Leb-B and Leb-C) from the tobacco hornworm, Manduca sexta. The mRNA levels of Leb-B and Leb-C increased significantly in larval fat body and hemocytes after injection of Escherichia coli, Micrococcus luteus and Saccharomyces cerevisiae. Western blotting using rabbit polyclonal antibody to Leb-B showed accumulation of large protein(s) and small peptide(s) in larval hemolymph after microbial injection. This result and the presence of RXXR motifs in the deduced amino acid sequences led to our postulation that Leb-B/C may be inactive precursors that are processed in larval hemolymph to generate short active peptides. To test this hypothesis, we expressed and purified full-length and various fragments of Leb-B and Leb-C as thioredoxin (TRX) fusion proteins. We found that fusion proteins could be cleaved by induced larval plasma, and the cleavage sites were determined by protein sequencing. Antibacterial activity of peptide fragments was also verified using synthetic peptides, and active M. sexta lebocin peptides were located at the N-termini of Leb-B/C, which are different from Bombyx mori lebocins 1–4 that are located close to the C-termini. In addition, we found that synthetic Leb-B22–48 peptide not only had higher antibacterial activity but also caused agglutination of E. coli cells. Our results provide valuable information for studying processing of lebocin precursors in lepidopteran insects.
Antimicrobial peptide; Lebocin; Insect immunity; Manduca sexta
Abiotic stresses greatly influence plant growth and productivity. While glycosyltransferases are widely distributed in plant kingdom, their biological roles in response to abiotic stresses are largely unknown. In this study, a novel Arabidopsis glycosyltransferase gene UGT85A5 was identified as significantly induced by salt stress. Ectopic expression of UGT85A5 in tobacco enhanced the salt stress tolerance in the transgenic plants. There were higher seed germination rates, better plant growth and less chlorophyll loss in transgenic lines compared to wild type plants under salt stress. This enhanced tolerance of salt stress was correlated with increased accumulations of proline and soluble sugars, but with decreases in malondialdehyde accumulation and Na+/K+ ratio in UGT85A5-expressing tobacco. Furthermore, during salt stress, expression of several carbohydrate metabolism-related genes including those for sucrose synthase, sucrose-phosphate synthase, hexose transporter and a group2 LEA protein were obviously upregulated in UGT85A5-expressing transgenic plants compared with wild type controls. Thus, these findings suggest a specific protective role of this glycosyltransferase against salt stress and provide a genetic engineering strategy to improve salt tolerance of crops.
A street rabies virus (RV) isolate, GXHXN, was obtained from brain tissue of rabid cattle in the Guangxi Zhuang Autonomous Region of China in 2009. GXHXN is the first isolate from cattle in China with its entire genome sequenced and is closely related to BJ2011E from horse in Beijing, WH11 from donkey in the Hubei Province, and isolates from dogs in the Guangxi and Fujian Provinces, with homologies of 97.6% to 99.6%. It is more distantly related to isolates from domestic cat, pig, Chinese ferret badger, and vaccine strains, with homologies of 83.1% to 88.0%.
The secretory small GTPase Rab27b was recently identified as an oncogene in breast cancer (BC) in vivo and in vitro studies. This research was designed to further explore the clinical and prognostic significance of Rab27B in BC patients.
The mRNA/protein expression level of Rab27B was examined by performing Real-time PCR, western blot, and immunohistochemistry (IHC) assays in 12 paired BC tissues and matched adjacent noncancerous tissues (NAT). Then we carried out IHC assay in a large cohort of 221 invasive BC tissues, 22 normal breast tissues, 40 fibroadenoma (FA), 30 ductual carcinoma in situ (DCIS) and 40 metastatic lymph nodes (LNs). The receiver operating characteristic curve method was applied to obtain the optimal cutoff value for high Rab27B expression. Epithelial-mesenchymal transition (EMT) marker expression levels were detected in relation to Rab27B expression.
We observed that the increased expression of Rab27B was dependent upon the magnitude of cancer progression (P < 0.001). The elevated expression of Rab27B was closely correlated with lymph node metastasis, advanced clinical stage, ascending pathology classification, and positive ER status. Furthermore, patients with high expression of Rab27B had inferior survival outcomes. Multivariate Cox regression analysis proved that Rab27B was a significantly independent risk factor for patients’ survival (P < 0.001). Furthermore, a significant positive relationship was observed between Rab27B expression and elevated mesenchymal EMT markers.
Our findings suggest that overexpression of Rab27B in BC coincides with lymph node metastasis and acquisition of a poor prognostic phenotype.
Rab27B; Breast cancer; Prognosis; EMT; Metastasis
The interactions of C2C12 myoblasts and human bone marrow stem cells (hMSCs) with silk-tropoelastin biomaterials, and the capacity of each to promote attachment, proliferation, and either myogenic- or osteogenic-differentiation were investigated. Temperature-controlled water vapor annealing was used to control beta-sheet crystal formation to generate insoluble silk-tropoelastin biomaterial matrices at defined ratios of the two proteins. These ratios controlled surface roughness and micro/nano-scale topological patterns, and elastic modulus, stiffness, yield stress, and tensile strength. A combination of low surface roughness and high stiffness in the silk-tropoelastin materials promoted proliferation and myogenic-differentiation of C2C12 cells. In contrast, high surface roughness with micro/nano-scale surface patterns was favored by hMSCs. Increasing the content of human tropoelastin in the silk-tropoelastin materials enhanced the proliferation and osteogenic-differentiation of hMSCs. We conclude that the silk-tropoelastin composition facilitates fine tuning of the growth and differentiation of these cells.
Insects produce a variety of antimicrobial peptides (AMPs). Induction of insect AMP genes is regulated by the Toll and IMD (immune deficiency) pathways via NF-κB and GATA factors. Little is known about species-specific regulation of AMP genes. In this report, we showed that activities of most Manduca sexta and Drosophila melanogaster AMP gene promoters were regulated in a species-specific manner in Drosophila (Dipteran) S2 cells and Spodoptera frugiperda (Lepidopteran) Sf9 cells. A κB-GATA element (22bp) from M. sexta moricin (MsMoricin) promoter could significantly increase activities of Drosophila AMP gene promoters in S2 cells, and an MsMoricin promoter activating element (MPAE) (140bp) could increase activity of drosomycin promoter specifically in Sf9 cells. However, κB and GATA factors alone were not sufficient for MsMoricin gene activation, suggesting that other co-regulators may be required to fully activate AMP genes. Our results suggest that induction of insect AMP genes may require a transcription complex composed of common nuclear factors (such as NF-κB and GATA factors) and species-related co-regulators, and it is the co-regulators that may confer species-specific regulation of AMP genes. In addition, we showed that activity of Drosophila drosomycin promoter could be activated cooperatively by the inserted exogenous κB-GATA element and the endogenous κB element. These findings revealed an approach of engineering AMP genes with enhanced activities, which may lead to broad applications.
NF-κB; GATA; Drosophila melanogaster; Manduca sexta; Antimicrobial peptide; Promoter
Silk-elastin-like protein polymers (SELPs), consisting of the repeating units of silk and elastin blocks, combine a set of outstanding physical and biological properties of silk and elastin. Due to the unique properties, SELPs have been widely fabricated into various materials for the applications in drug delivery and tissue engineering. However, little is known about the fundamental self-assembly characteristics of these remarkable polymers. Here we propose a two-step self-assembly process of SELPs in aqueous solution for the first time and report the importance of the ratio of silk to elastin blocks in a SELP’s repeating unit on the assembly of the SELP. Through precise tuning of the ratio of silk to elastin, various structures including nanoparticles, hydrogels and nanofibers could be generated either reversibly or irreversibly. This assembly process might provide opportunities to generate innovative smart materials for biosensors, tissue engineering and drug delivery. Furthermore, the newly developed SELPs in this study may be potentially useful as biomaterials for controlled drug delivery and biomedical engineering.
SELPs; self-assembly; biosynthesis; nanostructures; protein polymer
This study describes the patterns and socioeconomic influences of tobacco use among adults in tobacco-cultivating regions of rural southwest China.
A cross-sectional survey was conducted in 8681 adults aged ≥18 years in rural areas of Yunnan Province, China from 2010 to 2011. A standardized questionnaire was administered to obtain data about participants’ demographic characteristics, individual socioeconomic status, ethnicity, self-reported smoking habits, and exposure to secondhand smoke (SHS). The socioeconomic predictors of current smoking, nicotine addiction, and SHS exposure were analyzed using multivariate logistic regression.
The prevalence rates of tobacco use were much higher in men compared with women (current smoking 68.5% vs. 1.3%; and nicotine dependence 85.2% vs. 72.7%). However, the rate of SHS exposure was higher in women compared with men (76.6% vs. 70.5%). Tobacco farmers had higher prevalence rates of current smoking, nicotine dependence, and SHS exposure compared with participants not engaged in tobacco farming (P<0.01). Most tobacco users (84.5%) reported initiating smoking during adolescence. A total of 81.1% of smokers smoked in public places, and 77.6% smoked in workplaces. Individuals belonging to an ethnic minority had a lower probability of SHS exposure and nicotine dependence. Individual educational level was found to be inversely associated with the prevalence of current smoking, exposure to SHS, and nicotine dependence. Higher annual household income was associated with a greater risk of nicotine dependence.
This study suggests that tobacco control efforts in rural southwest China must be tailored to address tobacco-cultivating status and socioeconomic factors.
Smoking; Exposure to secondhand smoke; Socioeconomic status; China
Several studies have shown that multidrug transporters, such as P-glycoprotein (PGP), are involved in cell resistance to chemotherapy and refractory epilepsy. The p38 mitogen-activated protein kinase (MAPK) signaling pathway may increase PGP activity. However, p38-mediated drug resistance associated with PGP is unclear. Here, we investigated p38-mediated doxorubicin-induced drug resistance in human leukemia K562 cells.
The expression of PGP was detected by RT-PCR, Western blot, and immunocytochemistry. Cell viability and half-inhibitory concentrations (IC50) were determined by CCK-8 assay. The intracellular concentration of drugs was measured by HPLC.
A doxorubicin-induced PGP overexpression cell line, K562/Dox, was generated. The p38 inhibitor SB202190 significantly decreased MDR1 mRNA expression, as well as PGP, in K562/Dox cells. The IC50 of phenytoin sodium and doxorubicin in K562/Dox cells was significantly higher than that in wild-type K562 cells, indicating the drug resistance of K562/Dox cells. During the blocking of p38 activity in the presence of SB202190, cell number was significantly reduced after the phenytoin sodium and doxorubicin treatment, and the IC50 of phenytoin sodium and doxorubicin was decreased in K562/Dox cells. HPLC showed that the intracellular levels of phenytoin sodium and doxorubicin were significantly lower in K562/Dox cells than those in K562 cells. The decrease of the intracellular level of these drugs was significantly abolished in the presence of SB202190.
Our study demonstrated that p38 is, at least in part, involved in doxorubicin-induced drug resistance. The mechanistic study of MAPK-mediated PGP and the action of SB202190 need further investigation.
p38 MAPK; drug resistance; P-glycoprotein; doxorubicin; cancer
Experimental evidence suggests that the overexpression of breast cancer-specific tumor suppressor protein 1 (BRCA1) gene enhances sensitivity to docetaxel and resistance to cisplatin and ribonucleotide reductase M1 (RRM1) gene overexpression enhances resistance to gemcitabine. To further examine the effect of BRCA1 and RRM1 mRNA levels on outcome in advanced non-small cell lung cancer (NSCLC), we performed this non-randomized phase II clinical trial which tested the hypothesis that customized therapy would confer improved outcome over non-customized therapy.
RNA was isolated from fresh tumor tissue. Patients received chemotherapy regimen based on their BRCA1 and RRM1 mRNA levels: both low–cisplatin plus gemcitabine (GP); both high–vinorelbine plus cisplatin (NP); BRCA1 low and RRM1 high–cisplatin plus docetaxel (TP); BRCA1 high and RRM1 low–vinorelbine plus gemcitabine (GN).
From Dec 2005 to Nov 2008, 94 metastatic and locally advanced NSCLC patients from our institute were enrolled in this study. The median age was 58 years old. Among them, 21 patients received GP, 30 patients received TP and 43 patients received NP chemotherapy. GP group had a higher response rate, and longer median time to progression (TTP) and median overall survival (OS) time than the other 2 groups. The response rates in the GP, TP and NP groups were 42.9%, 36.7% and 27.9%, respectively (P=0.568). The median TTP was 5.6, 5.0, 4.8 months (P=0.975), respectively, and the median OS time was 12.5, 11.0, 9.7 months (P=0.808), respectively.
Chemotherapy customized according to BRCA1 and RRM1 expression levels is associated with higher response rate and longer TTP and OS time in the GP group. This suggests that BRCA1 and RRM1 mRNA levels could be used as biomarkers in individual therapy in NSCLC.
Individualized chemotherapy; Non-small cell lung cancer; BRCA1; RRM1
Male sterile and seedless characters are highly desired for citrus cultivar improvement. In our breeding program, a male sterile cybrid pummelo, which could be considered as a variant of male fertile pummelo, was produced by protoplast fusion. Herein, ecotopic stamen primordia initiation and development were detected in this male sterile cybrid pummelo. Histological studies revealed that the cybrid showed reduced petal development in size and width, and retarded stamen primordia development. Additionally, disorganized cell proliferation was also detected in stamen-like structures (fused to petals and/or carpel). To gain new insight into the underlying mechanism, we compared, by RNA-Seq analysis, the nuclear gene expression profiles of floral buds of the cybrid with that of fertile pummelo. Gene expression profiles which identified a large number of differentially expressed genes (DEGs) between the two lines were captured at both petal primordia and stamen primordia distinguishable stages. For example, nuclear genes involved in nucleic acid binding and response to hormone synthesis and metabolism, genes required for floral bud identification and expressed in particular floral whorls. Furthermore, in accordance with flower morphology of the cybrid, expression of PISTILLATA (PI) was reduced in stamen-like structures, even though it was restricted to correct floral whorls. Down-regulated expression of APETALA3 (AP3) coincided with that of PI. These finding indicated that, due to their whorl specific effects in flower development, citrus class-B MADS-box genes likely constituted ‘perfect targets’ for CMS retrograde signaling, and that dysfunctional mitochondria seemed to cause male sterile phenotype in the cybrid pummelo.
Seedlessness is an important agronomic trait for citrus, and male sterility (MS) is one main cause of seedless citrus fruit. However, the molecular mechanism of citrus seedlessness remained not well explored.
An integrative strategy combining suppression subtractive hybridization (SSH) library with cDNA microarray was employed to study the underlying mechanism of seedlessness of a Ponkan mandarin seedless mutant (Citrus reticulata Blanco). Screening with custom microarray, a total of 279 differentially expressed clones were identified, and 133 unigenes (43 contigs and 90 singletons) were obtained after sequencing. Gene Ontology (GO) distribution based on biological process suggested that the majority of differential genes are involved in metabolic process and respond to stimulus and regulation of biology process; based on molecular function they function as DNA/RNA binding or have catalytic activity and oxidoreductase activity. A gene encoding male sterility-like protein was highly up-regulated in the seedless mutant compared with the wild type, while several transcription factors (TFs) such as AP2/EREBP, MYB, WRKY, NAC and C2C2-GATA zinc-finger domain TFs were down-regulated.
Our research highlighted some candidate pathways that participated in the citrus male gametophyte development and could be beneficial for seedless citrus breeding in the future.
Citrus; cDNA microarray; Differential transcript; Male sterility-like protein; Seedlessness
To investigate the cognitive enhancement effect of WNK, an extracts combination of P. ginseng, G. biloba, and C. sativus L. and possible mechanisms, 5-month-old APP/PS1 transgenic mice were used in this study. After 3 months of administration, all mice received Morris water maze (MWM) training and a probe test. Mouse brain sections were detected by immunohistochemistry, HE staining, and transmission electron microscopy. MWM results showed significant difference between transgenic mice and nontransgenic littermates (P < 0.05, P < 0.01). WNK-treated mice exhibited enhanced maze performance over the training progression, especially better spatial memory retention in probe test compared to transgenic mice (P < 0.05, P < 0.01) and better spatial learning and memory at the fourth day of MWM test compared to EGB761- (G. biloba extract-) treated ones (P < 0.05). Hippocampal Aβ plaque burden significantly differed between APP/PS1 and littermate mice (P < 0.001), while decreased Aβ plaque appeared in WNK- or EGB761-treated transgenic brains (P < 0.05). Neurodegenerative changes were evident from light microscopic and ultrastructural observations in transgenic brains, which were improved by WNK or EGB761 treatment. These data indicate WNK can reduce the decline in spatial cognition, which might be due to its effects on reducing Aβ plaque formation and ameliorating histopathology and ultrastructure in hippocampus of APP/PS1 mouse brain.
Elucidation of a genotype-phenotype relationship is critical to understand an organism at the whole-system level. Here, we demonstrate that comparative analyses of multi-omics data combined with a computational modeling approach provide a framework for elucidating the phenotypic characteristics of organisms whose genomes are sequenced.
We present a comprehensive analysis of genome-wide measurements incorporating multifaceted holistic data - genome, transcriptome, proteome, and phenome - to determine the differences between Escherichia coli B and K-12 strains. A genome-scale metabolic network of E. coli B was reconstructed and used to identify genetic bases of the phenotypes unique to B compared with K-12 through in silico complementation testing. This systems analysis revealed that E. coli B is well-suited for production of recombinant proteins due to a greater capacity for amino acid biosynthesis, fewer proteases, and lack of flagella. Furthermore, E. coli B has an additional type II secretion system and a different cell wall and outer membrane composition predicted to be more favorable for protein secretion. In contrast, E. coli K-12 showed a higher expression of heat shock genes and was less susceptible to certain stress conditions.
This integrative systems approach provides a high-resolution system-wide view and insights into why two closely related strains of E. coli, B and K-12, manifest distinct phenotypes. Therefore, systematic understanding of cellular physiology and metabolism of the strains is essential not only to determine culture conditions but also to design recombinant hosts.
In the title compound, C16H16ClNO2S, the benzene and thiophene rings make a dihedral angle of 72.60 (4)°. In the crystal, weak C—H⋯O interactions are observed.