The E3 ligase TaGW2 mediates transcription factor TaARR12 degradation to promote drought resistance in wheat.

Drought stress limits crop yield, but the molecular modulators and their mechanisms underlying the trade-off between drought resistance and crop growth and development remain elusive. Here, a grain width and weight2 (GW2)-like really interesting new gene finger E3 ligase, TaGW2, was identified as a pivotal regulator of both kernel development and drought responses in wheat (Triticum aestivum). TaGW2 overexpression enhances drought resistance but leads to yield drag under full irrigation conditions. In contrast, TaGW2 knockdown or knockout attenuates drought resistance but remarkably increases kernel size and weight. Furthermore, TaGW2 directly interacts with and ubiquitinates the type-B Arabidopsis response regulator TaARR12, promoting its degradation via the 26S proteasome. Analysis of TaARR12 overexpression and knockdown lines indicated that TaARR12 represses the drought response but does not influence grain yield in wheat. Further DNA affinity purification sequencing combined with transcriptome analysis revealed that TaARR12 downregulates stress-responsive genes, especially group-A basic leucine zipper (bZIP) genes, resulting in impaired drought resistance. Notably, TaARR12 knockdown in the clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated nuclease 9 (Cas9)-mediated tagw2 knockout mutant leads to significantly higher drought resistance and grain yield compared to wild-type plants. Collectively, these findings show that the TaGW2-TaARR12 regulatory module is essential for drought responses, providing a strategy for improving stress resistance in high-yield wheat varieties.

Influence of hydrophilic amino acids and GC-content on expression of recombinant proteins used in vaccines against foot-and-mouth disease virus in Escherichia coli.

Epitope-based protein expression in Escherichia coli can be improved by adjusting its amino acid composition and encoding genes. To that end, we analyzed 24 recombinant epitope proteins (rEPs) that carry multiple epitopes derived from VP1 protein of foot-and-mouth disease virus. High level expression of the rEPs was attributed to a high content of Arg, Asn, Asp and Thr, a low content of Gln, Pro and Lys, a high content of hydrophilic amino acids and a higher isoelectric point value resulting from abundant Arg. It is also attributed to the appropriate guanine and cytosine content in the encoding genes. The data provide a reference for adjusting the amino acid composition in designing epitope-based proteins used in vaccines and for adjusting the synonymous codons to improve their expressions in E. coli.

Immunization with a HSP65-HER2 fusion peptide selectively eliminates HER2(+) B16 melanoma cells in a xenograft tumor mouse model.

HER2/neu peptide-based vaccines can eliminate human tumors overexpressing the human epidermal growth factor receptor 2 (HER2/neu), but the efficacy of this therapeutic strategy is suboptimal. Heat shock proteins (HSPs) are capable of eliciting efficient cytotoxic T lymphocyte (CTL) responses by cross-presentation. To evaluate whether immunization with a HSP65-HER2 fusion peptide could selectively eliminate HER2(+) B16 melanoma cells in a xenograft tumor mouse model, a HSP65-HER2 fusion peptide was incubated with immature dendritic cells (iDCs) in vitro to determine whether loading of iDCs with HSP65-HER2 could induce the expression of the immunomodulatory cell surface molecule, CD86. In vivo mouse immunizations with HSP65-HER2 or PBS (control) were performed to determine the antitumor effects by longitudinally monitoring changes in tumor volume, weight, and incidence. The effects on percentages of HER2(+) B16 cells in tumors were assessed by confocal microscopy and flow cytometry. The results indicated that loading of iDCs with HSP65-HER2 induced the expression of CD86 in vitro, suggesting that the hybrid antigen was able to stimulate an immune response. Immunization with HSP65-HER2 had no significant influence on tumor weight or volume but significantly reduced tumor incidence (62.5 % in mice injected with 25 µg of HSP65-HER2 vs. 100 % in PBS-injected controls; P < 0.05). Confocal microscopy and flow cytometry analyses revealed that HSP65-HER2 immunization significantly reduced the percentages of HER2(+) B16 cells in xenografted tumors (1.86 % vs. 30.56 % in PBS-injected controls; P = 0.01). Our findings suggest that immunization with the HSP65-HER2 fusion peptide selectively eliminates HER2(+) B16 melanoma cells in a xenograft tumor mouse model and may represent a novel and efficacious targeted therapy of HER2/neu(+) tumors.

Vaccination with TCL plus MHSP65 induces anti-lung cancer immunity in mice.

To develop effective anti-lung cancer vaccines, we directly mixed mycobacterial heat shock protein 65 (MHSP65) and tumor cell lysate (TCL) from Lewis lung cancer cells in vitro and tested its efficacy on stimulating anti-tumor immunity. Our results showed that MHSP65-TCL immunization significantly inhibited the growth of lung cancer in mice and prolonged the survival of lung cancer bearing mice. In vivo and in vitro data suggest that MHSP65-TCL could induce specific CTL responses and non-specific immunity, both of which could contribute to the tumor inhibition. Thus, this report provides an easy approach to prepare an efficient TCL based tumor vaccine.

Therapeutic injection of C-class CpG ODN in draining lymph node area induces potent activation of immune cells and rejection of established breast cancer in mice.

In order to develop novel CpG ODNs for the treatment of breast cancer, we have designed a series of CpG ODNs and evaluated their anti-tumor activity in a breast cancer mouse model. Interestingly, a C-class CpG ODN, designated as YW002, showed a vigorous activity on the inhibition of tumor growth in mice and completely cured some of the tumor-bearing mice through injection at tumor draining lymph node (TDLN) area. The expansion of immune cells in the TDLN and tumor and the generation of tumor specific immune memory were found associated with YW002-induced anti-tumor activity in mice. These results indicate that C-class CpG ODN could be developed into a medicament in a monotherapeutic regimen for the treatment of breast cancer through injection at TDLN area in clinic.

Breastfeeding initiation: impact of obesity in a large Canadian perinatal cohort study.

OBJECTIVE: To evaluate incidence of breastfeeding initiation according to maternal pre-pregnancy body mass index (BMI) in "Grossesse en Santé", a large prospective birth cohort in Quebec City. METHODS: Breastfeeding initiation in the post-partum period, pre-pregnancy BMI, sociodemographic determinants and obstetrical and neonatal factors were collected from years 2005 to 2010 in 6592 women with single pregnancies. Prenatal non-intention to breastfeed was documented in a subgroup of the cohort (years 2009-2010). Log-binomial regression analyses were performed to assess relative risk (RR) of non-initiation of breastfeeding between maternal BMI categories in models including pre- and post-natal determinants, after exclusion of variables with a mediating effect. RESULTS: Twenty percent (20%) of obese women did not initiate breastfeeding in the post-natal period at hospital compared to 12% for normal weight women. Compared with those having a normal pre-pregnancy BMI, obese women had a higher risk of non-initiation of breastfeeding (RRunadj 1.69, 95% CI 1.44-1.98), even after adjustment for prenatal and sociodemographic factors (RRadj 1.26, 95% CI 1.08-1.46). Furthermore, the risk of non-initiation of breastfeeding in obese women still remained higher after introduction of per- and post-natal factors (RR 1.22, 95% CI 1.04-1.42). The prenatal non-intention to breastfeed was strongly associated with the non-initiation of breastfeeding for all categories of BMI. CONCLUSION: Maternal obesity is associated with a two-fold rate of non-initiation of breastfeeding. Considering the benefits of breastfeeding and the increasing obesity rate, adapted interventions and specialized support should target both pre- and immediate post-natal periods in this population.

Omega-3 long-chain polyunsaturated fatty acids for extremely preterm infants: a systematic review.

BACKGROUND AND OBJECTIVE: Omega-3 long chain polyunsaturated fatty acid (LCPUFA) exposure can be associated with reduced neonatal morbidities. We systematically review the evidence for the benefits of omega-3 LCPUFAs for reducing neonatal morbidities in extremely preterm infants. METHODS: Data sources were PubMed, Embase, Center for Reviews and Dissemination, and the Cochrane Register of Controlled Trials. Original studies were selected that included infants born at <29 weeks' gestation, those published until May 2013, and those that evaluated the relationship between omega-3 LCPUFA supplementation and major adverse neonatal outcomes. Data were extracted on study design and outcome. Effect estimates were pooled. RESULTS: Of the 1876 studies identified, 18 randomized controlled trials (RCTs) and 6 observational studies met the defined criteria. No RCT specifically targeted a population of extremely preterm infants. Based on RCTs, omega-3 LCPUFA was not associated with a decreased risk of bronchopulmonary dysplasia in infants overall (pooled risk ratio [RR] 0.97, 95% confidence interval [CI] 0.82-1.13], 12 studies, n = 2809 infants); however, when considering RCTs that include only infants born at ≤32 weeks' gestation, a trend toward a reduction in the risk of bronchopulmonary dysplasia (pooled RR 0.88, 95% CI 0.74-1.05, 7 studies, n = 1156 infants) and a reduction in the risk of necrotizing enterocolitis (pooled RR 0.50, 95% CI 0.23-1.10, 5 studies, n = 900 infants) was observed with LCPUFA. CONCLUSIONS: Large-scale interventional studies are required to determine the clinical benefits of omega-3 LCPUFA, specifically in extremely preterm infants, during the neonatal period.

Effect of prophylactically applied CpG ODN on the development of myocarditis in mice infected with Coxsackievirus B3.

Coxsackievirus B3 was one of the major pathogens causing viral myocarditis. Toll-like receptor 9 activation contributed to the innate immune response in the process of CVB3-induced myocarditis. In order to find out how CpG oligodeoxynucleotide, known as a TLR-9 agonist, would affect the CVB3-induced myocarditis, we chose a C-type CpG oligodeoxynucleotide (YW002) injected to the mice one day before CVB3 challenge. On day 4 post CVB3 infection, 3 mice in each group were randomly sacrificed and their hearts were isolated to detect CVB3 replication. On day 10, the CVB3 neutralizing antibody and inflammatory change of the hearts were detected. The results indicated that the CVB3-induced myocarditis was aggravated with the declining body weight of mice, decreasing neutralizing antibody, and uncontrolling virus replication by injecting 20 µg YW002 per mouse. When adjusted the amount at 10 µg YW002 per mouse, there were no signs of aggravation in myocarditis. Plus, the mortality of the infected mice was reduced, the neutralizing antibody level was raised and the replication of virus was restrained. These results suggested that a proper amount of CpG oligodeoxynucleotide application could help to inhibit CVB3 infection.

Recombinant mycobacterial HSP65 in combination with incomplete Freund's adjuvant induced rat arthritis comparable with that induced by complete Freund's adjuvant.

Complete Freund's adjuvant (CFA) containing the inactivated mycobacterium has been conventionally used to induce typical arthritis in rats. In comparison, incomplete Freund's adjuvant (IFA), lacking the mycobacterium, can only induce less severe arthritis. However, the key components responsible for the arthritogenic effect of the whole mycobacterium are rarely known. Although mycobacterial heat-shock protein 65 (MHSP65) specific humoral and cellular immune responses were detected in rats with arthritis induced by CFA, MHSP65 alone cannot induce arthritis. In this study, we replaced the whole mycobacterium in CFA with recombinant MHSP65 (rMHSP65), prepared rMHSP65-IFA emulsion by mixing rMHSP65 and IFA, and investigated whether rMHSP65-IFA could induce arthritis in rats as CFA did. We found that intradermal injection of the rMHSP65-IFA emulsion induced arthritic lesions in testing animals to the same degree as those induced by CFA, manifested by severe swelling in hind paws, and synovial thickening, cartilage erosion and lymphocytes infiltration in ankle joints. Notably, the rMHSP65-IFA recipe also induced the production of anti-dsDNA and -rMHSP65 antibodies in rats. These results thus demonstrate that rMHSP65 can be used to substitute the inactivated mycobacteria in CFA to induce typical arthritis in rats.

Recombinant heat shock protein 65 carrying PADRE and HBV epitopes activates dendritic cells and elicits HBV-specific CTL responses.

BCG Hsp65 and PADRE have been shown to be potent to enhance antigen specific immunity. In order to explore the possibility to utilize them for the development of HBV therapeutic vaccine, a chimeric protein, Hsp65-HBV, was created by fusing PADRE and epitopes from HBV to the carboxyl-terminus of BCG Hsp65 and expressed in E. coli. We evaluated its effects on human dendritic cell maturation and specific CTL induction in vitro. Results showed that Hsp65-HBV could activate human dendritic cells by up-regulating the expressions of HLA-A2, HLA-DR and CD86, companioning with high level of IL-12 secretion. Furthermore, Hsp65-HBV matured DCs could significantly stimulate human autologous CD8(+) T cell proliferation and induce HBV-specific CTLs. Hsp65-HBV was also shown to generate HBsAg-specific CTLs in vivo in mice. These results indicated that Hsp65-HBV might be a candidate for the treatment of chronic HBV infection.

Detection of circulating anti-mucin 1 (MUC1) antibodies in breast tumor patients by indirect enzyme-linked immunosorbent assay using a recombinant MUC1 protein containing six tandem repeats and expressed in Escherichia coli.

Mucin 1 (MUC1), a tumor-associated antigen, is a transmembrane glycoprotein expressed by normal epithelial cells and overexpressed by carcinomas of epithelial origin. Autoantibodies against MUC1 are often found in circulation, either free or bound to immune complexes, which might contribute to limit tumor outgrowth and dissemination by antibody-dependent cell-mediated cytotoxicity, and were found favorably predictive of survival in early breast cancer patients. There is no commercial enzyme-linked immunosorbent assay (ELISA) kit for detecting the anti-MUC1 antibodies in human serum thus far. To detect circulating anti-MUC1 antibodies, we established an indirect ELISA (I-ELISA) using a recombinant MUC1 protein containing six tandem repeat sequences of MUC1 after the antigenicity and specificity of the protein were confirmed. The I-ELISA had a sensitivity of 91.3% and a specificity of 94.1% when a competitive I-ELISA was used as a reference test. The results showed that more patients with benign breast tumors (P = 0.001) and breast cancer patients before primary treatment (P = 0.010) were found to have anti-MUC1 IgG than healthy women; anti-MUC1 IgG before primary treatment was found more than after primary treatment (P = 0.016) in breast cancer patients. Interestingly, the anti-MUC1 IgG serum level was reversely correlated to that of CA15-3 antigen in advanced-stage patients (r = -0.4294, P = 0.046). Our study has demonstrated the suitability of the established I-ELISA for detecting circulating anti-MUC1 antibodies in human serum. Furthermore, we found that circulating anti-MUC1 antibodies may still bind MUC1 shed into blood in stage IV breast cancer, which can support the use of MUC1-target immune therapy strategies.

A novel canine favored CpG oligodeoxynucleotide capable of enhancing the efficacy of an inactivated aluminum-adjuvanted rabies vaccine of dog use.

In order to develop novel canine CpG ODNs as adjuvant for rabies vaccine of dog use, a panel of CpG ODNs containing different CpG motifs was designed and screened for their ability to induce the proliferation of canine splenocytes. Three AACGTT motif-containing CpG ODNs, designated as YW07, YW08 and YW09, respectively, were outshined with stronger ability to activate canine immune cells. The CpG ODNs were tested for their adjuvant activity for rabies vaccine in mice and dogs. It was found that YW07 could facilitate the rabies vaccine to induce more vigorous and long-lasting specific antibody response in mice and dogs, respectively. These findings suggest that YW07, a canine favored CpG ODN, could be used as a novel adjuvant for developing more efficient rabies vaccine of dog use.

Vaccination with B16 tumor cell lysate plus recombinant Mycobacterium tuberculosis Hsp70 induces antimelanoma effect in mice.

Tumor cell lysate (TCL) has an advantage of containing an extensive repertoire of tumor antigens but requires proper adjuvants to enhance its immunogenicity when used as an efficient tumor vaccine. Mycobacterium tuberculosis-derived heat shock protein 70 (TBHsp70) has been shown to assist crosspresentation of exogenously applied tumor antigens and activate innate immunity against tumor cells. In this study, TBHsp70-B16TCL, a preparation generated by mixing recombinant TBHsp70 and TCL of B16 melanoma cells directly, was tested for its immunogenicity as a tumor vaccine. The TBHsp70-B16TCL induced a significant inhibition of the growth and metastasis of B16 melanoma in mice and prolonged the survival of B16 melanoma-bearing mice. The inhibition was correlated with the specific immune responses induced by TBHsp70-B16TCL. The data suggest that recombinant TBHsp70-adjuvanted TCL might be developed into effective tumor vaccines for melanomas and possibly for other tumors.

[Expression, purification and activity analysis of BCG HSP70.].

AIM: To get BCG HSP70 protein with excellent biologic activity through E.coli. expression and purification. METHODS: BCG HSP70 gene was amplified by PCR and inserted into vector pMD18-T. After confirmed by sequencing, the gene was subcloned into expression vector pET28a. Recombinant pET28a/HSP70 was transformed into E.coli. BL21(DE3). Recombinant BCG HSP70 protein was expressed with IPTG induction and the purified protein was then identified by SDS-PAGE and Western blot. And its effect on the proliferation of mouse splenocytes was observed. RESULTS: Gene encoding BCG HSP70 which was identical with that published in GenBank was successfully obtained by PCR. SDS-PAGE analysis showed a protein with relative molecular mass of 70 000 was expressed. When the purified protein was detected by Western blot analysis, a specific protein with a molecular mass of 70 000 could be visualized. The purity of the purified protein was about 96.5%. The purified protein could stimulate the proliferation of mouse splenocytes significantly. CONCLUSION: BCG HSP70 is expressed and purified successfully, which would lay a foundation for further research on BCG HSP70 and BCG.

[Expression of predicted B cell epitope peptide in S2 subunit of SARS coronavirus spike protein in E.coli and identification of its mimic antigenicity].

AIM: To study the expression of predicted B cell epitope peptide in S2 subunit of SARS coronavirus spike protein in E.coli and its mimic antigenicity to S2 protein. METHODS: B cell epitopes in S2 subunit of SARS coronavirus spike protein was predicted using DNAStar software. The cDNA sequence encoding the B cell epitope peptide was constructed artificially by PCR and then cloned into the downstream of chaperone 10 gene in vector pET28a(+) to construct pET28-chap10-S2epi plasmid. The fusion protein, chap10-S2epi, was expressed in E.coli BL21(DE3) and identified by SDS-PAGE and Western blot. The rabbit was immunized by purified Chap10-S2epi for the preparation of antiserum, which was used to identify the mimic antigenicity of Chap10-S2epi to S2 protein by ELISA. RESULTS: Chap10-S2epi fusion protein was successfully constructed and expressed in E.coli. The antiserum from the animal immunized by Chap10-S2epi recognized full length of SARS coronavirus S2 spike protein. CONCLUSION: The predicted B cell epitope peptide of SARS coronavirus S2 spike protein can induce the antigenicity of S2 protein, which provides some fundamental data for developing engineering vaccine against SARS coronavirus infection.

Heat shock fusion protein induces both specific and nonspecific anti-tumor immunity.

Mucin 1 (MUC1) is a tumor antigen, and the most important epitopes that can induce cytotoxic T lymphocytes (CTL) reside in the variable-number tandem repeats (VNTR). Heat shock protein (HSP) complexes isolated from tumors have been shown to induce specific anti-tumor immunity. HSP alone can also induce nonspecific immunity. To explore the possibility to utilize the specific anti-tumor immunity induced by MUC1 VNTR and the nonspecific immunity induced by HSP, we constructed a recombinant protein (HSP65-MUC1) by fusing Bacillus Calmette-Guérin-derived HSP65 with the MUC1 VNTR peptide and tested its ability to induce anti-tumor activities in a tumor challenge model. The growth of MUC1-expressing tumors was significantly inhibited in mice immunized with HSP65-MUC1, both before and after tumor challenge. A much larger percentage of immunized mice survived the tumor challenge than non-immunized mice. Correlating with the anti-tumor activity, HSP65-MUC1 was shown to induce MUC1-specific CTL as well as nonspecific anti-tumor immunity. In the human system, HSP65-MUC1-loaded human DC induced the generation of autologous MUC1-specific CTL in vitro. These results suggest that exogenously applied HSP65-MUC1 may be used to treat MUC1 tumors by inducing the epitope-specific CTL as well as nonspecific anti-tumor responses mediated by the HSP part of the fusion protein.