Effect of epigallocatechin-3-gallate on the status of DNA methylation of E-cadherin promoter region on endometriosis mouse.

AIM: To evaluate whether epigallocatechin-3-gallate acts on endometriosis mouse, and changes the status of DNA methylation of E-cadherin promoter region. METHODS: According to our previous research, the tracing nude mouse model of endometriosis was built up and randomly divided into three groups: control group (group A), epigallocatechin-3-gallate group (group B) and decitabine group (group C). Normal saline, epigallocatechin-3-gallate and decitabine were isometrically intraperitoneally injected into each group once in 2 days. In this period, the growth situations of lesions were monitored by living image system. After 16 days, the lesions were taken out and the distribution of E-cadherin and its methylated situation of promoter region were analyzed. RESULTS: The region of interest of ectopic lesion increased from 4th to 16th day in group A (P < 0.01); in group B and C, the region of interest of ectopic lesion increased in the 0-8th day (P < 0.01), and decreased in the 8-16th day (P < 0.01). The positive expression rate of E-cadherin in group C was higher than group B, and group B was higher than group A (P < 0.01). The DNA methylation status of E-cadherin promoter region in group A was higher than group B, and group B was higher than group C (P < 0.01). CONCLUSION: Epigallocatechin-3-gallate may inhibit the growth of endometrial lesion, affect the expression of E-cadherin on the cell membrane and reduce the status of DNA methylation of E-cadherin promoter region.

Assessment of potential adjuvanticity of Cry proteins.

Genetically modified (GM) crops have achieved success in the marketplace and their benefits extend beyond the overall increase in harvest yields to include lowered use of insecticides and decreased carbon dioxide emissions. The most widely grown GM crops contain gene/s for targeted insect protection, herbicide tolerance, or both. Plant expression of Bacillus thuringiensis (Bt) crystal (Cry) insecticidal proteins have been the primary way to impart insect resistance in GM crops. Although deemed safe by regulatory agencies globally, previous studies have been the basis for discussions around the potential immuno-adjuvant effects of Cry proteins. These studies had limitations in study design. The studies used animal models with extremely high doses of Cry proteins, which when given using the ig route were co-administered with an adjuvant. Although the presumption exists that Cry proteins may have immunostimulatory activity and therefore an adjuvanticity risk, the evidence shows that Cry proteins are expressed at very low levels in GM crops and are unlikely to function as adjuvants. This conclusion is based on critical review of the published literature on the effects of immunomodulation by Cry proteins, the history of safe use of Cry proteins in foods, safety of the Bt donor organisms, and pre-market weight-of-evidence-based safety assessments for GM crops.

Evolution of the protease-activated receptor family in vertebrates.

Belonging to the G protein-coupled receptor (GPcr) family, the protease-activated receptors (Pars) consist of 4 members, PAR1-4. PARs mediate the activation of cells via thrombin, serine and other proteases. Such protease-triggered signaling events are thought to be critical for hemostasis, thrombosis and other normal pathological processes. In the present study, we examined the evolution of PARs by analyzing phylogenetic trees, chromosome location, selective pressure and functional divergence based on the 169 functional gene alignment sequences from 57 vertebrate gene sequences. We found that the 4 Pars originated from 4 invertebrate ancestors by phylogenetic trees analysis. The selective pressure results revealed that only PAR1 appeared by positive selection during its evolution, while the other PAR members did not. In addition, we noticed that although these PARs evolved separately, the results of functional divergence indicated that their evolutional rates were similar and their functions did not significantly diverge. The findings of our study provide valuable insight into the evolutionary history of the vertebrate PAR family.

Efficacy of Sublingual Immunotherapy with Dermatophagoides farinae Extract in Monosensitized and Polysensitized Patients with Allergic Rhinitis: Clinical Observation and Analysis.

AIM: To investigate differences in the efficacy of sublingual immunotherapy with Dermatophagoides farinae drops in monosensitized and polysensitized allergic rhinitis patients. METHODS: The patients enrolled in the study were treated for more than one year by sublingual immunotherapy (SLIT) using Dermatophagoides farinae drops and were divided into a monosensitized group (n = 20) and a polysensitized group (n = 30). Total nasal symptom scores of patients before and after SLIT were analyzed to evaluate the curative effect. The phylogenetic tree of dust mite allergens as well as other allergens that were tested by skin prick test was constructed to help the analysis. RESULTS: There was no significant difference in the efficacy of SLIT between dust mite monosensitized and polysensitized patients. CONCLUSIONS: Both dust mite monosensitized and polysensitized patients could be cured by SLIT using Dermatophagoides farinae drops. This study provides a reference for the selection of allergens to be used in immunotherapy for polysensitized AR patients.

In silico prediction of the T-cell and IgE-binding epitopes of Per a 6 and Bla g 6 allergens in cockroaches.

Per a 6 and Bla g 6 are cockroach allergens found in Periplaneta americana and Blattella germanica, respectively. The objective of the present study was to predict the B­ and T­cell epitopes of the Per a 6 and Bla g 6 allergens. Three immunoinformatics tools, the DNAStar Protean system, the Bioinformatics Predicted Antigenic Peptides system and the BepiPred 1.0 server, were used to predict the potential B­cell epitopes, while Net­MHCIIpan­2.0 and NetMHCII­2.2 were used to predict the T­cell epitopes of the two allergens. As a result, seven peptides were predicted in the Per a 6 allergen and seven peptides were predicted in the Bla g 6 allergen in the B­cell epitope predictions. In the T­cell prediction, the Per a 6 allergen was predicted to have nine strongly binding nonamer core epitope sequences (IC50<50 nm) and 28 weakly binding sequences (50 nm

Strengths and weaknesses of immunotherapy for advanced non-small-cell lung cancer: a meta-analysis of 12 randomized controlled trials.

BACKGROUND: Lung cancer is one of the leading causes of cancer death worldwide. Non-small-cell lung cancer (NSCLC) accounts for approximately 85% of all lung cancers. Immunotherapy has yielded no consistent benefit to date for those patients. Assessing the objective efficacy and safety of immunotherapy for advanced NSCLC patients will help to instruct the future development of immunotherapeutic drugs. METHODOLOGY AND PRINCIPAL FINDINGS: We performed a meta-analysis of 12 randomized controlled trials including 3134 patients (1570 patients in the immunotherapy group and 1564 patients in the control group) with histologically confirmed stage IIIA, IIIB, or IV NSCLC. The analysis was executed with efficacy end points regarding overall survival (OS), progression-free survival (PFS), complete response (CR), partial response (PR), and total effective rate. Overall unstratified OS, PFS, PR, and total effective rate were significantly improved in advanced NSCLC patients in the immunotherapy group (P = 0.0007, 0.0004, 0.002, 0.003, respectively), whereas CR was not improved (P = 0.97). Subgroup analysis showed that monoclonal antibody (mAb) immunotherapy significantly improved the PFS, PR, and total effective rate and showed a trend of improving OS of advanced NSCLC patients compared with the control group, with one kind of adverse event being significantly dominant. Compared with the control group, the vaccine subgroup showed no significant difference with regard to serious adverse events, whereas cytokine immunotherapy significantly induced three kinds of serious adverse events. CONCLUSIONS: Immunotherapy works efficiently on advanced NSCLC patients. Of several immunotherapies, mAb therapy may be a potential immunotherapy for advanced NSCLC patients, and become a standard complementary therapeutic approach in the future if the issues concerning toxicity and allergenicity of mAbs have been overcome.

Mutation analysis of p63 gene in the first Chinese family with ADULT syndrome.

BACKGROUND: ADULT syndrome (acro-dermato-ungual-lacrimal-tooth syndrome) is a rare ectodermal dysplasia disorder known as autosomal dominant inheritance. Recent studies have linked p63 gene mutation to the development of this disease. However, the genetic characteristics of ADULT syndrome were still not well understood. METHODS: Mutation analysis of p63 gene in the first Chinese ADULT syndrome family was performed using direct DNA sequencing. RESULTS: The sequence analysis of exon 8 of p63 gene disclosed a heterozygous G>A substitution at nucleotide 893 (R298Q) in the proband. In addition, a single nucleotide polymorphism (SNP) rs16864880 in the downstream flanking region (DFR) of p63 exon 8 was also identified in this family. The proband and the paternal side including her father exhibited the C/G genotype at this position. The C/G variant frequency in the paternal was significantly higher as compared with the maternal (6/10 vs 0/6, P = 0.034). CONCLUSIONS: ADULT syndrome may be caused by the p63 gene mutation, and it might have closer genetic association with the paternal side in this family.

[Detection of hearing threshold and polymorphic molecular marker analysis of guinea pigs of mimetic aging].

OBJECTIVE: To explore the establishment of the mimetic aging effect in guinea pigs induced by D-galactose, and to detect the biological indicatrix associated with hearing loss and provide a new tool for molecular pathogenesis of hearing loss. METHODS: Total of 51 guinea pigs were randomly divided into three groups: group A (model aging group, n = 25), which were injected with D-galactose (200 mgxkg(-1)xd(-1)) by intra peritoneum for 6 weeks, group B (model control group, n = 18), which were given the same amount of saline only, and group C (vacant group, n = 15) were not treated. Then, The guinea pigs in group A and B were exposed in noise for 8 days, 8 hours once a day. Auditory brainstem response (ABR) was used to test the hearing threshold of guinea pigs thrice, first before the drug administered, then after 6 weeks the drug used, third after noise exposure. And colorimetry was used to analyze the activity of superoxide dismutase (SOD) and malon dialdehyde (MDA) in brain and liver tissue. The DNA of inner ear tissue was harvested and amplified fragment length polymorphism (AFLP) was used to detect the differential polymorphic markers. RESULTS: After injection, there was no significant difference in elevation of ABR threshold between the group A and group B (t = 1.14, P > 0.05). However, exposure of noise later, elevation in ABR threshold of (22.97 +/- 10.56) dB peSPL was observed in group A, and (14.16 +/- 7.36) dB peSPL in group B. The was significant difference in variation of hearing threshold between group A and group B (t = 2.78, P < 0.05). The activity of SOD in brain and liver tissue in group A was lower than that in group B. the level of MDA was opposite between group A and group B. The difference between group A and group B was significant (P < 0.01). A differential polymorphic marker was observed by AFLP. CONCLUSIONS: The mimetic aging effect of the guinea pigs can be induced by D-galactose, and this model can not directly induce the hearing loss. The differential polymorphic marker possibly act as a predisposing factor which can greatly enhance the sensitivity of the ear to the noise.

Cloning, expression, and characterization of pollen allergens from Humulus scandens (Lour) Merr and Ambrosia artemisiifolia L.

AIM: To clone the pollen allergen genes in Humulus scandens (Lour) Merr (LvCao in Chinese) and short ragweed (Ambrosia artemisiifolia L) for recombinant allergen production and immunotherapy. METHODS: The allergen genes were selectively amplified in the weed pollen cDNA pool by using a special PCR profile, with the primers designed by a modeling procedure. Following truncated gene cloning and confirmation of the pollen source, unknown 3'cDNA ends were identified by using the 3'-RACE method. The gene function conferred by the full-length coding region was evaluated by a homologue search in the GenBank database. Recombinant proteins expressed in Escherichia coli pET-44 RosettaBlue cells were subsequently characterized by N-terminal end sequencing, IgE binding, and cross-reactivity. RESULTS: Three full-length cDNAs were obtained in each weed. Multiple alignment analysis revealed that the deduced amino acid sequences were 83% identical to each other and 56%-90% identical to panallergen profilins from other species. Five recombinant proteins were abundantly expressed in non-fusion forms and were confirmed by using the N-terminal end sequence identity. Sera from patients who were allergic to A artemisiifolia reacted not only with rAmb a 8(D03) derived from A artemisiifolia, but also with recombinant protein rHum s 1(LCM9) derived from H scandens, which confirmed the allergenicity and cross-reactivity of the recombinant proteins from the 2 sources. Comparison of the degenerate primers used for truncated gene cloning with the full-length cDNA demonstrated that alternative nucleotide degeneracy occurred. CONCLUSION: This study demonstrates a useful method for cloning homologous allergen genes across different species, particularly for little-studied species. The recombinant allergens obtained might be useful for the immunotherapeutic treatment of H scandens and/or A artemisiifolia pollen allergies.

Bridging PCR and partially overlapping primers for novel allergen gene cloning and expression insert decoration.

AIM: To obtain the entire gene open reading frame (ORF) and to construct the expression vectors for recombinant allergen production. METHODS: Gene fragments corresponding to the gene specific region and the cDNA ends of pollen allergens of short ragweed (Rg, Ambrosia artemisiifolia L.) were obtained by pan-degenerate primer-based PCR and rapid amplification of the cDNA ends (RACE), and the products were mixed to serve as the bridging PCR (BPCR) template. The full-length gene was then obtained. Partially overlapping primer-based PCR (POP-PCR) method was developed to overcome the other problem, i.e., the non-specific amplification of the ORF with routine long primers for expression insert decoration. Northern blot was conducted to confirm pollen sources of the gene. The full-length coding region was evaluated for its gene function by homologue search in GenBank database and Western blotting of the recombinant protein Amb a 8(D106) expressed in Escherichia coli pET-44 system. RESULTS: The full-length cDNA sequence of Amb a 8(D106) was obtained by using the above procedure and deduced to encode a 131 amino acid polypeptide. Multiple sequence alignment exhibited the gene D106 sharing a homology as high as 54-89% and 79-89% to profilin from pollen and food sources, respectively. The expression vector of the allergen gene D106 was successfully constructed by employing the combined method of BPCR and POP-PCR. Recombinant allergen rAmb a 8(D106) was then successfully generated. The allergenicity was hallmarked by immunoblotting with the allergic serum samples and its RNA source was confirmed by Northern blot. CONCLUSION: The combined procedure of POP-PCR and BPCR is a powerful method for full-length allergen gene retrieval and expression insert decoration, which would be useful for recombinant allergen production and subsequent diagnosis and immunotherapy of pollen and food allergy.

[Cloning full-length homologous cDNAs of pollen allergens in Humulus Scandens(Lour.) Merr by degenerate primer].

AIM: To establish a stable and reliable method for fast cloning homologous genes of pollen allergens in allergen-containing plants. METHODS: Degenerate primers were designed based on the bioinformatic analysis of numerous allergens available from the database. Subsequent amplification of the allergen genes was conducted in the weed pollen cDNA pool by a selective PCR profile. Following the truncated gene cloning, RACE method was used to isolate full-length cDNA. Gene function was deduced by sequence alignment in GenBank database. The degenerate ability of the primer was compared with the full-length cDNA sequences. RESULTS: Three full-length cDNAs were obtained. Sequence analysis showed that these new genes shared as high as 79%-85% homology with a large amount of known allergen profilins and were hence regarded as members of panallergen profilin family. Comparing these genes with the degenerate primers that were initially used in truncated gene cloning revealed that alternative nucleotide degeneracy occurred beyond the degenerate site predesigned, suggesting that further degeneracy was expanded by Touchdown-gradient PCR. CONCLUSION: Cloning of homologous genes or allergen genes can be efficiently achieved by using the combination of degenerate primer with Touchdown-gradient RT-PCR in the species such as Humulus scandens that has not yet been investigated.

[Effect of temperature on the fertility restoration of different two-line hybrid rice].

On the basis of the fertility observation of hybrids from Pei'ai 64S and Nongken 58S crossed with an indica variety Nanjing11 and japonica marker line FL235, respectively, The plant growth chambers were employed to expose the F2 plant individuals to such different day mean temperature as 24 degrees C, 27 degrees C and 30 degrees C during natural long hour daylight from July to August in Wuhan (30 degrees 27' N), with a view to analyzing the difference in the temperature sensitivity of fertility between the two kinds of different fertility-restoring genes for two years running. The results showed that, whether it was under long daylight and high temperature or under long daylight and middle temperature, the mean natural seed-set percent of F1 was higher than 68.75%, suggesting that Nanjing11 could completely restore the fertility of Pei'ai 64S. And however, under natural high temperature condition, N58S x FL235 F1 could set seed naturally with 21.93%-26.75%, the mean natural seed set percent of F1 was 46.36%-48.38% in natural middle temperature condition, ability of FL235 to restore the fertility of N58S was affected by high temperature. Further analysis proved that temperature could not alter the inheritance mode of F2 but affect the extent of fertility genes expression in Nanjing11. On the other hand, the expression of fertility-genes of FL235 was sensitive to high temperature, whose the putative critical temperature was 27 degrees C, and high temperature influenced not only the genetic interactions but also the segregation modes in F2 generations.

Genetic analysis of the low critical sterility temperature point in photoperiod-thermo sensitive genic male sterile rice.

It has been a long haul but photoperiod- and thermo-sensitive genic male sterile (PTGMS) rice has not been freely used in hybrid rice production because there are two perplexing problems corresponding to the critical sterility temperature point (CSTP): the uncertainty of the CSTP segregating pattern and the instability of CSTP for every originally useful line. N5088S, the most widely commercialized japonica-type PTGMS line in China, also saw that its CSTP variants have been isolated but with all other agronomic characteristics unchanged. In this report we analyzed the genetic basis of CSTP, by employing the iterated expectation and conditional maximization (IECM) algorithm on four tiller-splitting-formed sets of seven generations from N5088S and its CSTP-variant H5088S, each set treated with one temperature regime. The main results indicated that there are two dominant major genes and polygene, as well as their respective epistasis conditioning the CSTP in the 23.5 degrees C regime. Based on the results obtained, the strategy for breeding of PTGMS lines with stable low CSTP was outlined.