17ß-Estradiol promotes angiogenesis of stria vascular in cochlea of C57BL/6J mice.

It is widely accepted that the stria vascularis (SV) in cochlea plays a critical role in the generation of endocochlear potential (EP) and the secretion of the endolymph. 17ß-estradiol (E2) is the most potent and abundant endogenous estrogen during the premenopausal period, thus, considered as the reference estrogen. This study aimd to investigate the protective effect of E2 by promoting the expression of vascular endothelial growth factor (VEGF) and thus promoting the vascular regeneration of the SV in elderly mice. After being treated with E2 either in vivo or in vitro, the hearing threshold changes of C57BL/6J elder mice continuously reduced, endothelial cell morphology improved, the number of endothelial cells (ECs) tubular nodes increased significantly, the ability of tubular formation enhanced significantly and the expression of VEGF increased. In vitro, cell model in conjunction with in vivo ovariectomized model was established to demonstrate for the first time that E2 promotes angiogenesis by promoting the secretion of VEGF through the phosphatidylinositol 3-kinase (PI3K)/AKT pathway (PI3K/AKT). In conclusion, E2 demonstrated potent angiogenesis properties with significant protection against Age-Related Hearing Loss (ARHL), which provides a new idea for the improvement of ARHL.

[Effects of estrogen on apoptosis of spiral ganglion cells in cochlea of aged C57BL/6J mice].

Objective: To observe the effects of estrogen on cochlear spiral ganglia cell apoptosis in aged C57BL/6J mice, and to explore the possible mechanism of estrogen's protective effects on senile deafness. Methods: Forty C57BL/6J mice were divided into the following four groups (10 mice/group): 3 m group (3 months old group), 12 m group (12 months old sham operation group); In the 12 m OVX group (ovariectomized at 12 months), bilateral oophorectomy was performed at the age of 9 months and normal feeding was performed until the age of 12 months.The 12m OVX+E2 group (estrogen intervention group) underwent bilateral oophorectomy at 9 months of age. After the one-month washout period, mice in the other groups were treated with estrogen at the dose of 100 µg/(kg·d) by subcutaneous injection, lasting 2 months to 12 months old. Mice in the other groups were fed normally.Blood samples were collected from the tail vein at the end of the treatment in 12 m OVX+E2 group. Enzyme-linked immunosorbent assays (ELISAs) was used to determine the serum estrogen levels. Auditory brainstem response (ABR) was used to detect the changes of hearing threshold in each group.Mice were anesthetized with 2% pentobarbital sodium. Bilateral cochlea was extracted after neck amputation and paraffin-embedded sections were performed.Hematoxylin eosin (HE) staining was used to observe the morphological changes in the cochlea spiral ganglion neurons (SGN), and TUNEL staining was used to observe the apoptosis of SGN. The expression levels of Caspase-3, Bax and Bcl-2 mRNA of the apoptotic proteins in cochlear spiral ganglion were measured by real-time fluorescence quantitative PCR (QRT-PCR). Results: Compared with the 3 m group, the hearing threshold of the 12 m group was improved, the loss of spiral ganglion cells was aggravated, and the apoptosis of the cells was increased(P＜0.01). After removal of the ovaries, the hearing threshold of the mice in the 12 m OVX group was higher than that in the 12 m control group (P＜0.01), and this increased threshold was accompanied by an increased loss of spiral ganglion cells, and increased apoptosis (P＜0.01). Meanwhile, the mRNA levels of apoptotic protein Caspase-3 and Bax were increased (P＜0.01), while the mRNA level of anti-apoptotic protein Bcl-2 was decreased (P＜0.01). After exogenous estrogen was given to the 12 m OVX+E2 group, the hearing threshold was lower than that in 12 m OVX group(P＜0.01). At the same time, the apoptosis of helical ganglion cells was reduced, the mRNA levels of Caspase-3 and Bax were decreased (P＜0.01), and the Bcl-2 mRNA level was increased (P＜0.01). Conclusion: Estrogen inhibited apoptosis of cochlear spiral ganglion cells in aged C57BL/6J mice ,thus achieving a protective effect on presbycusis.

[Effects of T16Ainh-A01 on the apoptosis and senescence of endothelial cells in the cochlea stria vascularis].

Objective: Primary cultured cochlear stria vascularis endothelial cells (ECs) of guinea pig were used to investigate the expression changes of TMEM16A and its effect on apoptosis and senescence of ECs in the cochlear stria vascularis. Methods: Primary cultured ECs in the cochlear stria vascularis were used to establish aging models according to CCK-8 and SA-ß-galactosidase. Senescent cells were randomly divided into senescent group (P12), DMSO group (P12+DMSO), T16Ainh-A01 group (P12+T16Ainh-A01). Immunofluorescence and Western blot were used to detect the expression of TMEM16A in ECs. Flow cytometry was used to detect the apoptotic rate. Western blot was used to detect the protein expressions of Bax, Bcl-2 and cleaved casepase-3 in each group. Results: The positive rate of primary cultured cochlear stria vascularis ECs was above 95%, and the 12th generation cochlear stria vascularis ECs were determined as the senescence group, and the expression of TMEM16A in protein and fluorescence was increased (P＜0.05). After intervention with T16Ainh-A01 for 24 h, the protein expressions of Bax and cleaved casepase-3 were down-regulated (P＜0.01), the protein expression of Bcl-2 was increased (P＜0.05), the apoptotic rate and the positive rate of SA-ß-gal were down-regulated (P＜0.01). Conclusion: It was found that apoptosis and TMEM16A expression were increased in cochlear stria vascularis senescent ECs, TMEM16A specific blocker T16Ainh-A01 could reduce the apoptosis and senescence in ECs of the cochlear stria vascularis. These results suggest that TMEM16A may participate in apoptosis and senescence of ECs in the cochlear stria vascularis.

The genetic divergences of codon usage shed new lights on transmission of hepatitis E virus from swine to human.

Hepatitis E virus (HEV) is an important pathogen causing public health burden. Swine has been recognized as a main reservoir. Interestingly, genotype 1 HEV only infects human; whereas genotype 3 and 4 are zoonotic. However, there is a lack of in-depth understanding in respect to the transmission from swine to human. Codon usage patterns generally participate in viral survival and fitness towards its hosts. We have analyzed codon usage patterns of the three open reading frames (ORFs) for 243 full-length genomes of HEV genotypes 1, 3 and 4. The divergence of synonymous codon usage patterns is different in each ORF for genotypes 1, 3 and 4, but the genotype-specific codon usage bias in genotype 1 is stronger than those of genotypes 3 and 4. In respect to genotypes 3 and 4, compared with strains isolated from human, HEV isolated from swine shows appreciable variation in adaptation of codon usages to human or swine. These results may help to understand the transmission and host adaptation of HEV genotypes 3 and 4 from swine to human.

The IMPDH inhibitors, ribavirin and mycophenolic acid, inhibit peste des petits ruminants virus infection.

Peste des petits ruminants virus (PPRV) causes highly contagious diseases in domestic and particular wild small ruminants, leading to substantial economic loss. The development of effective and cheap antiviral medications shall help to circumvent this emerging burden. In this study, we found that ribavirin, a competitive inosine-5'-monophosphate dehydrogenase (IMPDH) inhibitor, significantly inhibits the replication of PPRV. As IMPDH is a key enzyme in purine nucleotide synthesis, supplementation of exogenous guanosine attenuate the anti-PPRV effect of ribavirin. Interestingly, an uncompetitive IMPDH inhibitor, mycophenolic acid (MPA), exerted more potent antiviral effect again PPRV. Similarly, this effect was largely restored upon supplementation of guanosine. Thus, we have demonstrated that the IMPDH inhibitors ribavirin and MPA combat PPRV infection through purine nucleotide depletion. Because both regimens have been widely used in the clinic for treating viral infection or organ rejection in transplantation patients for decades, respectively, repurposing these existing safe and cheap medications may provide a new avenue for combating PPRV infection.

Analyses of nucleotide, synonymous codon and amino acid usages at gene levels of Brucella melitensis strain QY1.

Brucella melitensis is the causative pathogen of the zoonotic disease brucellosis in China. This work focused on analyses of genetic features represented by nucleotide, synonymous codon and amino acid usages at gene levels of B. melitensis strain QY1 isolated from China. Although nucleotide usage biases at different codon positions all work on synonymous codon usage bias, nucleotide usage biases at the 1st and 3rd positions play more important roles in codon usages. Mutation pressure caused by nucleotide composition constraint influences the formation of over-representative synonymous codons, but neighboring nucleotides surrounding a codon strongly influence synonymous codon usage bias for B. melitensis strain QY1. There is significant correlation between amino acid usage bias and hydropathicity of proteins for B. melitensis strain QY1. Compared with different Brucella species about synonymous codon usage patterns, synonymous codon usages are not obviously influenced by hosts. Due to nucleotide usage bias at the 1st codon position influencing synonymous codon and amino acid usages, good interactions among nucleotide, synonymous codon and amino acid usages exist in the evolutionary process of B. melitensis.

The effects of codon usage on the formation of secondary structures of nucleocapsid protein of peste des petits ruminants virus.

The nucleocapsid (N) protein of peste des petits ruminants virus (PPRV) with a conserved amino acid usage pattern plays an important role in viral replication. The primary objective of this study was to estimate roles of synonymous codon usages of PPRV N gene and tRNA abundances of host in the formation of secondary structure of N protein. The potential effects of synonymous codon usages of N gene and tRNA abundances of host on shaping different folding units (α-helix, ß-strand and the coil) in N protein were estimated, based on the information about the modeling secondary structure of PPRV N protein. The synonymous codon usage bias was found in different folding units in PPRV N protein. To better understand the role of translation speed caused by variant tRNA abundances in shaping the specific folding unit in N protein, we modeled the changing trends of tRNA abundance at the transition boundaries from one folding unit to another folding unit (ß-strand â†’ coil, coil â†’ ß-strand, α-helix â†’ coil, coil â†’ α-helix). The obvious fluctuations of tRNA abundance were identified at the two transition boundaries (ß-strand â†’ coil and coil â†’ ß-strand) in PPRV N protein. Our findings suggested that viral synonymous codon usage bias and cellular tRNA abundance variation might have potential effects on the formation of secondary structure of PPRV N protein.

Unique features of nucleotide and codon usage patterns in mycoplasmas revealed by information entropy.

Currently, the comparison between GC usage pattern at the 3rd codon position and codon usage index is commonly used to estimate the roles of evolutionary forces in shaping synonymous codon usages, however, this kind of analysis often losses the information about the role of A/T usage bias in shaping synonymous codon usage bias. To overcome this limitation and better understand the interplay between nucleotide and codon usages for the evolution of bacteria at gene levels, in this study, we employed the information entropy method with some modification to estimate roles of nucleotide compositions in the overall codon usage bias for 18 mycoplasma species in combination with Davies-Bouldin index. At gene levels, the overall nucleotide usage bias represents A content as the highest, followed by T, G and C for mycoplasmas, resulting in a low GC content. This feature is universal across these species derived from different hosts, suggesting that the hosts have the limited impact on nucleotide usage bias of mycoplasmas. Information entropy and Davies-Bouldin index can better reveal that the nucleotide usage bias at the 3rd codon position is essential in shaping the overall nucleotide bias for all given mycoplasmas except M. pneumoniae M129. Davies-Bouldin index revealed that the 1st and 2nd codon position play more important role in synonymous codon usage bias than that of the 3rd position at gene levels. To our knowledge, this is the first comprehensive investigation into cooperation between nucleotide and codon usages for mycoplasma and extends our knowledge of the mechanisms that contribute to codon usage and evolution of this microorganism.

The effect of multiple evolutionary selections on synonymous codon usage of genes in the Mycoplasma bovis genome.

Mycoplasma bovis is a major pathogen causing arthritis, respiratory disease and mastitis in cattle. A better understanding of its genetic features and evolution might represent evidences of surviving host environments. In this study, multiple factors influencing synonymous codon usage patterns in M. bovis (three strains' genomes) were analyzed. The overall nucleotide content of genes in the M. bovis genome is AT-rich. Although the G and C contents at the third codon position of genes in the leading strand differ from those in the lagging strand (p<0.05), the 59 synonymous codon usage patterns of genes in the leading strand are highly similar to those in the lagging strand. The over-represented codons and the under-represented codons were identified. A comparison of the synonymous codon usage pattern of M. bovis and cattle (susceptible host) indicated the independent formation of synonymous codon usage of M. bovis. Principal component analysis revealed that (i) strand-specific mutational bias fails to affect the synonymous codon usage pattern in the leading and lagging strands, (ii) mutation pressure from nucleotide content plays a role in shaping the overall codon usage, and (iii) the major trend of synonymous codon usage has a significant correlation with the gene expression level that is estimated by the codon adaptation index. The plot of the effective number of codons against the G+C content at the third codon position also reveals that mutation pressure undoubtedly contributes to the synonymous codon usage pattern of M. bovis. Additionally, the formation of the overall codon usage is determined by certain evolutionary selections for gene function classification (30S protein, 50S protein, transposase, membrane protein, and lipoprotein) and translation elongation region of genes in M. bovis. The information could be helpful in further investigations of evolutionary mechanisms of the Mycoplasma family and heterologous expression of its functionally important proteins.

Tau expression correlated with breast cancer sensitivity to taxanes-based neoadjuvant chemotherapy.

The purpose of this study was to investigate the correlation between tau expression in primary breast cancer and sensitivity to taxanes during neoadjuvant chemotherapy in patients with breast cancer. We used immunohistochemistry to examine tau expression in breast cancer biopsies from 113 primary breast cancer patients and evaluated the correlation between tau expression and taxane sensitivity. Twenty-eight (24.78 %, 28/113) patients were positive for tau expression. After taxanes-based neoadjuvant chemotherapy, 40 patients achieved pathological complete response (pCR) (35.4 %). Among the 40 patients with pCR, five (12.5 %) were positive for tau expression. In univariate analysis, estrogen receptor (ER), progesterone receptor, human epidermal growth factor receptor 2 (HER2), and tau were found to be significantly predictive of a pCR (P = 0.001, 0.030, 0.002, and 0.025, respectively). Tau, ER, and HER2 status were significant for pCR on multivariate analysis (P = 0.025, 0.005, and 0.043, respectively). Tau expression was positively related to ER (P = 0.007) and progestin receptor (P = 0.008). In conclusion, tau protein expression correlated with breast cancer sensitivity to taxanes-based neoadjuvant chemotherapy; patients negative for tau expression were more likely to achieve pCR.

Accuracy and axilla sparing potentials of sentinel lymph node biopsy with methylene blue alone performed before versus after neoadjuvant chemotherapy in breast cancer: a single institution experience.

INTRODUCTION: The timing of sentinel lymph node biopsy (SLNB) of breast cancer in the neoadjuvant setting is still controversial. We retrospectively analyzed a Chinese patient cohort with neoadjuvant chemotherapy (NAC) to evaluate the accuracy and axilla sparing potentials of different SLNB timings with methylene blue alone for lymphatic mapping. MATERIALS AND METHODS: Patients with NAC and axillary lymph node dissection (ALND) and either pre- or post-NAC SLNB were eligible. Clinicopathological characteristics, identification rate (IR), false-negative rate (FNR), accuracy, and positive-predictive value were calculated and compared between the pre- and post-NAC SLNB group using appropriate statistical methods. Axilla sparing potentials of different SLNB timings were evaluated and compared. RESULTS: One hundred and fifteen eligible cases were included, and 58 had pre-NAC SLNB while the other 57 had post-NAC SLNB. Both groups were comparable in clinicopathological characteristics, neoadjuvant treatments and pathologic complete response rate. IR, FNR, and accuracy of SLNB, as pre-NAC versus post-NAC, were 100 versus 98.2 % (P = 0.496), 0 versus 8.0 % (P = 0.181), and 100 versus 96.4 % (P = 0.239), respectively. Post-NAC SLNB had significantly higher positive-predictive value for ALNs than pre-NAC SLNB (70.0 vs. 36.4 %, P = 0.014), suggesting as high as 63.6 % of ALND performed in the pre-NAC group could have been avoided while only 30 % of ALND in the post-NAC group were theoretically unnecessary. CONCLUSIONS: Both SLNB timings of breast cancer patients with NAC were feasible and accurate. Although pre-NAC SLNB tends to be better in accuracy, post-NAC SLNB is significantly superior in terms of axilla sparing.

Pre-treatment hormonal receptor status and Ki67 index predict pathologic complete response to neoadjuvant trastuzumab/taxanes but not disease-free survival in HER2-positive breast cancer patients.

Trastuzumab-containing neoadjuvant chemotherapy achieves a pathologic complete response (pCR) rate of about 40 % in HER2-positive breast cancers, and pCR predicts better survival. A cohort of 102 consecutive Chinese HER2-positive stage II/III patients with neoadjuvant trastuzumab/taxanes were retrospectively analyzed, to evaluate the role of hormonal receptor (HR) status and Ki67 index, along with other parameters, in pCR and survival prediction. pCR rate of the cohort was 44.1 % (45/102). Fifty-three patients were HR-positive and 49 were HR-negative. Median Ki67 index was 40 %, and 49 patients had a high Ki67 index (>40 %) whereas 53 had a low Ki67 index (≤40 %). HR status and Ki67 index were confirmed as the only two parameters associated with pCR in multivariate analysis (hazard ratio = 2.952; 95 % CI, 1.227-7.105; P = 0.016 for HR status and hazard ratio = 2.583, 95 % CI 1.107-6.026, P = 0.028 for Ki67 index). Patients with coexisting HR-negative and high Ki67 index had higher pCR rate (69.2 %), compared to those with either HR-negative alone or high Ki67 alone (hazard ratio = 3.038; 95 % CI, 1.102-8.372; P = 0.029), and to those with coexisting HR-positive and low Ki67 index as well (hazard ratio = 7.071; 95 % CI, 2.150-23.253; P = 0.001). In a median follow-up duration of 25.9 months, 11 disease-free survival events (DFS) were recorded. pCR predicted better DFS (log rank P = 0.018) and was the only significant factor in Cox regression analysis (hazard ratio = 0.184; 95 % CI, 0.038-0.893; P = 0.036). Our study indicates that HR status and Ki67 index are predictors for pCR but not for DFS in HER2-positive patients with neoadjuvant trastuzumab/taxanes, which deserves further investigations.

Rescue of Newcastle disease virus from cloned cDNA using an RNA polymerase II promoter.

A new system was developed to improve the efficiency and simplify the procedure of recovery of Newcastle disease virus (NDV) from cloned cDNA. A full-length cDNA clone of mesogenic NDV vaccine strain Mukteswar was assembled from five subgenomic cDNA fragments and cloned into a plasmid allowing transcription driven by cellular RNA polymerase II. The full-length viral cDNA was flanked by hammerhead ribozyme (HamRz) and hepatitis delta virus ribozyme (HdvRz) sequences, resulted in the synthesis of antigenomic RNA with exact termini. Without supplying T7 RNA polymerase, infectious NDV could be generated efficiently in some eukaryotic cell lines by simultaneous transcription of antigenomic RNA from the full-length plasmid and expression of NP, P and L proteins from helper plasmids introduced by cotransfection. The efficiency of recovery with the conventional T7 promoter system based on BRS-T7 cells and the cytomegalovirus (CMV) promoter system was compared, and the results demonstrate that the new system facilitates the generation of recombinant NDV and more efficient than the T7 rescue system using BRS-T7.

HER2-positive status is an independent predictor for coexisting invasion of ductal carcinoma in situ of the breast presenting extensive DCIS component.

DCIS of the breast with coexisting invasion is commonly seen, and no consensus on any biomarker capable of discriminating this subgroup has been reached yet. We retrospectively examined the receptor status and the histological grade in Chinese DCIS patients to identify any independent predictor in order to discriminate a subgroup with coexisting invasion from pure DCIS patients. A consecutive Chinese DCIS patient cohort registered at a single institution was included for ER, PR, and HER2 status, as well as for evaluation of the histological grade. Patients with invasion foci >1cm in diameter were excluded. The HER2 gene amplification status was further examined by FISH when the IHC result was HER2 (2+). Molecular subtypes were also profiled. Age, histological grade, ER, PR, and HER2 status were included in association analyses. In total, 183 patients were included. A hundred and forty patients had pure DCIS, and 43 patients had DCIS with invasion. The luminal A subtype accounted for 49.7% of all cases, the HER2-positive subtype for 27.9%, and only 10.4% and 12.0% represented the luminal B and basal-like subtypes, respectively. Univariate analyses showed that histological Grade 2, Grade 3, and HER2-positive status were associated with DCIS with invasion, odds ratios 5.1 (P = 0.017), 5.2 (P = 0.01) and 3.34 (P = 0.001), respectively. However, only the HER2-positive status was of statistical significance in the multivariate logistic regression analyses after adjustment for other markers, odds ratio 3.8 (95%CI 1.4-10, P = 0.008). The 43 cases with invasion were further stratified into extensive or small DCIS components according to the percentage of DCIS to total tumor area using 25% as the cutoff point. Multinomial logistic regression with pure DCIS cases as reference showed that the HER2-positive status was associated only with the group showing an extensive DCIS component, odds ratio 6.2 (95%CI 1.8-21, P = 0.003), but not with the group having a small DCIS component. Our study demonstrates that HER2-positive status is an independent predictor for DCIS, with invasion presenting an extensive DCIS component, and favors the hypothesis that HER2 overexpression or gene amplification is involved in the transition from DCIS to invasive disease.

Development of an immunochromatographic strip for the rapid detection of Toxoplasma gondii circulating antigens.

We developed a rapid immunochromatographic strip (ICS) procedure that can detect circulating antigens in the blood of animals during the acute stage of toxoplasmosis. The aim of this study was to evaluate this test using sera from field samples and from experimentally infected animals. The sensitivity and specificity of the ICS were compared with those of an enzyme-linked immunosorbent assay (ELISA). Both assays detected circulating antigens in the sera of animals experimentally infected with the Gansu Jingtai strain of Toxoplasma gondii, and the agreement between the two assays was 100%. In the infected animals, circulating antigens could be detected as early as the second day post-infection (PI) and in all animals by the fourth day. In the 381 field serum samples, the positive rates of the ICS and ELISA were 5.2% and 5.8%, respectively. In addition, there was no cross-reactivity of the antigens with Neospora caninum. The results presented here suggest that the ICS is a feasible, convenient, rapid and effective method to detect infection by T. gondii. This test could be a powerful supplement to the current diagnostic methods. Taken together, the results of this study encourage further research toward the production of commercial diagnostic tests for detecting T. gondii in animals.

[Efficacy and safety assessment of neoadjuvant paclitaxel and trastuzumab in Chinese women with Her-2 positive operable breast cancer].

OBJECTIVE: To assess the efficacy and safety of neoadjuvant 3-weekly paclitaxel plus trastuzumab (TH) in Chinese women with Her-2 overexpressing operable breast cancer. METHODS: This is a single center open-label phase II clinical trial. The included patients underwent 4 cycles of neoadjuvant 3-weekly TH before surgery. The primary endpoint was pathologic complete response rate (pCR rate) and the secondary endpoint was overall response rate (OR rate). Patients were also stratified according to hormone receptor status, and pCR rate and OR rate were compared between subgroups. Adverse events were graded according to CTCAE v3.0. RESULTS: There were 40 eligible patients entering this study with median age of 49 years. All patients completed 4 cycles of neoadjuvant treatment. pCR rate was 52.5% and OR rate was 87.5%. The differences of pCR and OR rates between subgroups were of no statistical significance. No cardiac toxicity event severer than grade 2 was recorded. CONCLUSION: 3-weekly TH regimen has satisfactory pCR rate and OR rate in Chinese patients with Her-2 overexpressing operable breast cancer and reliable safety.

Complete genomic sequence of transmissible gastroenteritis virus TS and 3' end sequence characterization following cell culture.

The complete genome sequence of transmissible Gastroenteritis virus (TGEV) strain TS, previously isolated from Gansu province, was cloned and compared with published sequence data from other TGEV strains. Phylogenetic tree analysis based on the amino acid and nucleotide sequences of the S gene showed that the TGEV strains were divided into 3 clusters. TGEV TS showed a close evolutionary relationship to the American Miller cluster but had a 5' non-translated region (NTR) sequence closely related to the American Purdue cluster. Continued culture in different cell types indicated that TGEV TS virulence could be attenuated after fifty passages in Porcine kidney (PK-15) cells, and that the Porcine kidney cell line IB-RS-2 (IBRS) was not suitable for culture of the TGEV strain TS.

Prokaryotic expression and potential application of the truncated PCV-2 capsid protein.

Three pairs of specific primers were designed to amplify the F2-1, F2-2 and XF2-2 truncated sequences of ORF2 which encodes the capsid protein of porcine circovirus type 2 (PCV-2). The F2-1 sequence had most of the NLS region of ORF2, but the F2-2 and XF2-2 genes had the NLS region deleted. Truncated genes were subcloned into pET-32a(+) vectors to construct recombinant fusion expression vectors. The vectors were then transformed into Rosetta(DE3) E. coli and expressed by induction of IPTG. Expressed proteins were detected by western blotting and ELISA. The protein with best immunoreactivity was confirmed and selected, then utilized to inoculate SPF rabbits to prepare polyclonal antibodies. The protein and prepared polyclonal antibody were utilized to detect sera samples against PCV-2 from Shandong province and PCV-2 particles in PK-15 cells. In our study, three recombinant fusion proteins were successfully obtained, and the molecular weights of fusion proteins were 35.9 kDa, 33.6 kDa and 38.6 kDa respectively detected by SDS-PAGE. All of the proteins showed positive reaction with anti-PCV-2 antisera, and His-XF2-2 showed better immunoreactivity than the others. The protein of His-XF2-2 was coated as antigen in ELISA to detect the seroprevalence of PCV-2 in certain districts of Shandong province, the seropositivity rate was 27.7 % (73/264). Specific fluorescence and positive signals for PCV-2 could be detected in PK-15 cells inoculated with PCV-2 with the participation of prepared antibodies against His-XF2-2 in IFA and IPMA. Experimental results indicated that the truncated PCV-2 ORF2 gene containing most of the NLS region was successfully expressed in E. coli, and His-XF2-2 was demonstrated to have better immunoreactivity with anti-PCV-2 antisera than the other two fusion proteins. His-XF2-2 and prepared polyclonal antibodies against it had a satisfactory capability in detecting PCV-2 infection.

Reverse transcription-loop-mediated isothermal amplification assay for rapid detection of hepatitis E virus.

The one-step single-tube betaine-free reverse transcription (RT)-loop-mediated isothermal amplification assay was developed for rapid diagnosis of hepatitis E virus. This assay amplified the target gene in less than 45 min (even as short as 20 min) under isothermal conditions at 63 degrees C, and the sensitivity of this assay was 100-fold greater than that of RT-PCR. This assay demonstrated a detection limit of 0.045 fg (nine copies/reaction).

[A meta-analysis of neoadjuvant chemotherapy combined with trastuzumab for HER2-positive breast cancers].

OBJECTIVE: To evaluate the efficacy and safety of neoadjuvant chemotherapy combined with trastuzumab for HER2 positive breast cancers. METHODS: PubMed online database, ASCO abstract database, SABCS abstract database, ESMO abstract database and CBMdisc database were searched for literatures related to trastuzumab in neoadjuvant chemotherapy for breast cancers. A meta-analysis was performed for retrieved literatures meeting the inclusion criteria. RESULTS: Three clinical trials were included. Meta-analysis showed that compared to chemotherapy only, regimens combined with trastuzumab could significantly improved the pCR rate of HER2-positive breast cancers (RR=1.65, 95% CI 1.28-2.13, P<0.0001) without increasing the frequencies of cardiac toxicity (RR=1.16, 95% CI 0.82-1.64, P=0.41). CONCLUSION: In neoadjuvant chemotherapy for HER2-positive breast cancers, chemotherapy combined with trastuzumab is superior to exclusive chemotherapy.