Efficacy in pigs of a new inactivated vaccine combining porcine circovirus type 2 and Mycoplasma hyorhinis.

Coinfection with porcine circovirus type 2 (PCV2) and Mycoplasma hyorhinis (Mhr) can induce more-severe disease than a single infection with either. We evaluated the efficacy of a new vaccine combining inactivated PCV2 and Mhr, in a model of PCV2 and Mhr infection. Twenty-five 35-day-old PCV2- and Mhr-free pigs were randomly divided into five groups, with five pigs in each group. The pigs in groups 1 and 2 were vaccinated with the combined vaccine and then challenged with Mhr or PCV2, respectively. The pigs in groups 3 and 4 were not vaccinated and then challenged with PCV2 or Mhr, respectively, and group 5 was used as the unvaccinated unchallenged control. Two weeks after booster immunization via the intramuscular route, all the pigs except those in control group 5 were challenged with PCV2 or Mhr. All the pigs were euthanized 28 days after challenge. The pigs in vaccinated groups 1 and 2 showed a significant increase in weight after challenge with PCV2 or Mhr (P < 0.001), with an average daily gain (ADG) of 0.315 kg compared with unvaccinated groups 3 and 4 (0.279 kg). Mhr was isolated from the unvaccinated pig lungs after Mhr challenge, whereas it was not isolated from the vaccinated pigs. No PCV2 or Mhr was detected with PCR or histochemical staining in vaccinated groups 1 and 2. A statistical analysis showed that the PCV2 and Mhr combined vaccine providing protected against PCV2 infection causing viremia and inguinal lymphadenopathy (5 pigs protected out 5) or against Mhr infection causing fiber inflammation (4 pigs out 5). Thus, we have developed an effective combined vaccine for the prevention and control of PCV2 or Mhr infections in swine herds, this will help reduce prevalence of PCV2 and Mhr coinfections.

A group of serum proteins as potential diagnostic biomarkers for Yin-deficiency-heat syndrome.

Yin-deficiency-heat (YDH) syndrome is a very common subhealth status in Traditional Chinese Medicine. However, currently, there is no unified standard for diagnosing YDH syndrome. We applied the iTRAQ-2D LC-MS/MS method to explore the potential of serum protein profiles as biomarker for YDH syndrome. A total of 120 differentially expressed proteins (79 downregulated and 41 upregulated) were identified by the proteomic profiling. The results of KEGG pathway analysis showed that the functions of the differentially expressed proteins were mainly involved in complement and coagulation cascades. The clinical data showed that YDH syndrome was closely related to inflammation and coagulation, compared with the healthy controls. The ELISA validation results indicated that the expression levels of ALB, CFI, and KLKB1 were downregulated in the YDH syndrome group (p < .05). Moreover, we established a decision tree model based on the combination of these three proteins and achieved a sensitivity of 87.5%, a specificity of 84.4%, and AUC of 0.891. The results indicated that the combination of ALB, CFI, and KLKB1 may serve as potential biomarkers for diagnosing YDH syndrome. Our study can provide a new method for YDH syndrome diagnosis, and may also provide an experimental basis to understand the molecular mechanism of YDH syndrome.

Screening of potential biomarkers for Yin-deficiency-heat syndrome based on UHPLC-MS method and the mechanism of Zhibai Dihuang granule therapeutic effect.

BACKGROUND: Yin-deficiency-heat (YDH) syndrome is a subhealth state of the individual, mainly manifested as oral ulcers, dry mouth, constipation, and other symptoms. Zhibai Dihuang granule (ZDG), as a classic traditional Chinese medicine, is effective in treating YDH syndrome. We screened the potential biomarkers for diagnosing YDH syndrome, and explored the mechanisms of the therapeutic effect of ZDG. METHODS: Plasma samples from the Pinghe (PH, healthy control) group, the Shanghuo (SH, YDH syndrome) group, and the ZDG treated group (therapeutic group) were analyzed by using metabolomics profiling. The data were analyzed by multivariate statistical and bioinformatics analyses. RESULTS: We screened four differential metabolites such as, decanoylcarnitine, dodecanoylcarnitine, phosphatidylcholine (PC), and Aspartate (Asp) Arginine (Arg) Proline (Pro) in the SH group and the PH group. The results showed that the combination of above four metabolites could serve as a potential biomarker for the early diagnosis of YDH syndrome. The metabolites decanoylcarnitine and glucose were found to be differentially expressed in the YDH syndrome group and tended to be normalized after ZDG treatment. CONCLUSION: The increased levels of four differential metabolites (decanoylcarnitine, dodecanoylcarnitine, PC, and Asp Arg Pro) revealed that individuals with YDH syndrome may have increased energy metabolism in the body, which could lead to disorders of fatty acids ß-oxidation and immune function. The levels of two differential metabolites including decanoylcarnitine and glucose returned to normal after ZDG treatment, indicating that ZDG could treat YDH syndrome by regulating glucose metabolism and fatty acids ß-oxidation. Our study provides a new method for the diagnosis of YDH syndrome, and may provide theoretical basis for novel therapeutic strategies of YDH syndrome.

Serum proteins TGFBI, PCSK9, and CCL14 are potential biomarkers for different traditional Chinese medicine syndromes of multidrug-resistant tuberculosis.

Patients with multidrug-resistant tuberculosis (MDR-TB) tend to have a long course of anti-TB treatment and severe side effects. Traditional Chinese Medicine (TCM) has a synergistic effect in attenuation of MDR-TB. However, the lack of objective biological standards to classify and diagnose MDR-TB TCM syndromes could result in less effective TCM treatment. Therefore, in this study, we identified differentially expressed proteins (DEPs) in serum of individuals with MDR-TB TCM syndromes by applying isobaric tags for relative and absolute quantification coupled with two-dimensional liquid chromatography-tandem mass spectrometry (iTRAQ-2DLC-MS/MS) method and bioinformatics analysis. The functional analysis of DEPs was also performed. Additionally, DEPs among three different TCM syndromes of MDR-TB were validated by enzyme-linked immunosorbent assay (ELISA). Finally, a receiver operating characteristic (ROC) curve was performed to estimate the diagnostic ability of DEPs. A total of 71 DEPs were identified in the three different MDR-TB TCM syndrome groups such as the pulmonary Yin deficiency (PYD) syndrome group, the Hyperactivity of Fire due to Yin deficiency (HFYD) syndrome group, and the deficiency of Qi and Yin (DQY) syndrome group. The results showed that the expression level of transforming growth factor-beta-induced protein ig-h3 (TGFBI) was lower in the PYD syndrome group (p = .002), the proprotein convertase subtilisin/kexin type 9 (PCSK9) was overexpressed in the HFYD syndrome group (p < .0001), and the C-C motif chemokine ligand 14 (CCL14) expression level was reduced in the DQY syndrome group (p = .004). Our study demonstrated that serum TGFBI, PCSK9, and CCL14 may serve as potential novel biomarkers for PYD syndrome, HFYD syndrome and DQY syndrome of MDR-TB, respectively. The study provides a biological basis for MDR-TB TCM syndromes classification and can be of great significance for the treatment of different TCM syndromes.

Study on potential biomarkers of energy metabolism-related to early-stage Yin-deficiency-heat syndrome based on metabolomics and transcriptomics.

Yin-deficiency-heat (YDH) syndrome is a common sub-health state of the human body in traditional Chinese medicine (TCM). However, due to the lack of objective quantitative diagnostic indicators, patients with early-stage YDH syndrome cannot be treated in time and can develop a pathological (disease) state. Therefore, it is necessary to apply modern diagnostic techniques in order to identify the biological markers for the diagnosis of early-stage YDH syndrome. In the present study, we performed Solexa sequencing and non-targeted metabolomics analysis using high-performance liquid chromatography coupled with mass spectrometry to screen differentially expressed mRNAs and differential metabolites in individuals with early-stage YDH syndrome and healthy controls. Bioinformatics methods were used to perform enrichment analysis of differentially expressed mRNAs and differential metabolites for biological functions and signaling pathways. Furthermore, we found that differentially expressed mRNAs and differential metabolites were related to energy metabolism. Real-time PCR was used to validate the mRNA expression in the serum of subjects with early-stage YDH syndrome. We found that the mitochondrially encoded NADH dehydrogenase 2 (MT-ND2) mRNA was differentially expressed in the serum of individuals with early-stage YDH syndrome. Receiver operating characteristic (ROC) curve and logistic regression analysis were used to evaluate the efficacy of the diagnostic model based on eight differential metabolites. We combined the three metabolites such as Glycine, Sphingomyelin, and Isocitrate to establish the diagnostic model with a sensitivity of 0.853 and a specificity of 0.800. The combination of the above three metabolites may serve as a potential biomarker for the diagnosis of early-stage YDH syndrome. Our study reveals potential biomarker for the diagnosis of early-stage YDH syndrome and also provides a new method for the quantification and objectification of TCM syndromes.

Identification of potential metabolic biomarkers in Yin deficiency syndrome using LC-MS.

Yin and Yang are the two counter-balancing aspects in ancient Chinese philosophy. In traditional Chinese medicine, Yin deficiency syndrome (YDS) is a common sub-health state with complex causes. While the syndrome may be treated to various degrees of effectiveness with traditional Chinese medicine, efficient modern methods are yet to be developed for diagnosing and treating the YDS. Here we performed a metabolomics study on YDS in rats. Serum metabolites in rats were analyzed using ultra-performance liquid chromatography-mass spectrometry (UPLC-MS) method to identify potential biomarkers for YDS. The rats were divided randomly into the healthy control group, the untreated YDS group, and the anemarrhena treated YDS group. Compared with the control group, significant increase in the metabolites such as dihydrotestosterone (DHT) and 5ß-DHT, 4-imidazolone-5-propanoate, 4-(L-alanin-3-yl)-2-hydroxy-cis,cis-muconate 6-semialdehyde, and 5-(L-alanin-3-yl)-2-hydroxy-cis,cis-muconate 6-semialdehyde were observed in the serum of untreated YDS group, which returned to normal in the anemarrhena treated group. Therefore, these metabolites may serve as potential biomarkers for YDS, and may facilitate the diagnosis and treatment of YDS.

Cancer cell membrane-cloaked mesoporous silica nanoparticles with a pH-sensitive gatekeeper for cancer treatment.

Nanoparticular drug delivery system (NDDS) has great potential for enhancing the efficacy of traditional chemotherapeutic drugs. However, it is still a great challenge to fabricate a biocompatible NDDS with simple structure capable of optimizing therapeutic efficacy, such as high tumor accumulation, suitable drug release profile (e.g. no premature drug leakage in normal physiological conditions while having a rapid release in cancer cells), low immunogenicity, as well as good biocompatibility. In this work, a simple core/shell structured nanoparticle was fabricated for prostate cancer treatment, in which a mesoporous silica nanoparticle core was applied as a container to high-efficiently encapsulate drugs (doxorubicin, DOX), CaCO3 interlayer was designed to act as sheddable pH-sensitive gatekeepers for controlling drug release, and cancer cell membrane wrapped outlayer could improve the colloid stability and tumor accumulation capacity. In vitro cell experiments demonstrated that the as-prepared nanovehicles (denoted as DOX/MSN@CaCO3@CM) could be efficiently uptaken by LNCaP-AI prostate cancer cells and even exhibited a better anti-tumor efficiency than free DOX. In addition, Live/Dead cell detection and apoptosis experiment demonstrated that MSN/DOX@CaCO3@CM could effectively induce apoptosis-related death in prostate cancer cells. In vivo antitumor results demonstrated that DOX/MSN@CaCO3@CM administration could remarkably suppress the tumor growth. Compared with other tedious approaches to optimize the therapeutic efficacy, this study provides an effective drug targeting system only using naturally biomaterials for the treatment of prostate cancer, which might have great potential in clinic usage.

Screening and identification of potential protein biomarkers for evaluating the efficacy of intensive therapy in pulmonary tuberculosis.

This research aimed to discover potential biomarkers for evaluating the therapeutic efficacy of intensive therapy in pulmonary tuberculosis (TB). Protein profiles in 2-months intensively treated TB patients, untreated TB patients, and healthy controls were investigated with iTRAQ-2DLC-MS/MS technique. 71 differential proteins were identified in 2-months intensively treated TB patients. Significant differences in complement component C7 (CO7), apolipoprotein A-IV (APOA4), apolipoprotein C-II (APOC2), and angiotensinogen (ANGT) were found by ELISA validation. CO7 and ANGT were also found significantly different in sputum negative patients, compared with sputum positive patients after intensive treatment. Clinical analysis showed that after 2-months intensive treatment several indicators were significantly changed, and the one-year cure rate of sputum negative patients were significantly higher than sputum positive patients. Diagnostic models consisting of APOC2, CO7 and APOA4 were established to distinguish intensively treated TB patients from untreated TB patients and healthy controls with the AUC value of 0.910 and 0.935. Meanwhile, ANGT and CO7 were combined to identify sputum negative and sputum positive TB patients after intensive treatment with 89.36% sensitivity, 71.43% specificity, and the AUC value of 0.853. The results showed that APOC2, CO7, APOA4, and ANGT may be potential biomarkers for evaluating the efficacy of intensive anti-TB therapy.

The Chinese herbal formula Zhibai Dihuang Granule treat Yin-deficiency-heat syndrome rats by regulating the immune responses.

ETHNOPHARMACOLOGICAL RELEVANCE: Zhibai Dihuang Granule (ZDG), a traditional Chinese medicine (TCM) made from eight Chinese herbs, has been classically used to treat Yin-deficiency-heat (YDH) syndrome. ZDG is well known with the therapeutic efficacy of nourishing Yin and decreasing internal heat in clinic, but the mechanism of ZDG's therapeutic effect is still not clear. MATERIALS AND METHODS: High doses of triiodothyronine (T3) were given intraperitoneally to induce Hyperthyroid YDH syndrome in SD rats. The animals were then treated with ZDG for one week. The iTRAQ-coupled with two-dimensional liquid chromatography-tandem mass spectrometry (2D LC-MS/MS) technique was used to screen the differentially expressed serum proteins between ZDG treated rats and YDH syndrome rats. The differentially expressed proteins were analyzed by bioinformatics method and were verified by enzyme-linked immunosorbent assay (ELISA). RESULTS: A total of 55 differentially expressed proteins were identified, including 23 up-regulated proteins (>1.25 fold, p < 0.05) and 32 down-regulated proteins (<0.80 fold, p < 0.05). Among the differentially expressed proteins, 26 proteins returned to normal after ZDG treatment. Bioinformatics analysis showed that these proteins were mainly involved in immune response, including regulation of immune system process, complement activation, and humoral immune response mediated by circulating immunoglobulin. ELISA revealed significantly increased levels of Zinc-alpha-2-glycoprotein (Azgp1), L-selectin, C-reactive protein (Crp), Plasminogen (Plg), Kininogen 1 (Kng1), and significantly decreased levels of Mannose binding lectin 2 (Mbl2) and Complement C1qb chain (C1qb) in ZDG treated rats compared with YDH syndrome rats. Bioinformatics analyses indicated that Azgp1 participated in antigen processing and presentation, Crp, C1qb, and Mbl2 were involved in complement activation, while L-selectin, Plg, and Kng1 were involved in regulating the inflammatory response. CONCLUSIONS: Our study provides experimental evidence to understand the therapeutic mechanism of ZDG in YDH syndrome. The results suggested that ZDG may regulate the complement activation and inflammatory response, and promote the ability to recognize antigens to alleviate YDH syndrome.

[Stochastic characteristics of daily precipitation and its spatiotemporal difference over China based on information entropy]. / 基于信息熵的我国日降水量随机性和时空差异性.

Daily precipitation process in China showed obvious randomness and spatiotemporal variation. It is important to accurately understand the influence of precipitation changes on control of flood and waterlogging disaster. Using the daily precipitation data measured at 520 stations in China during 1961-2013, we quantified the stochastic characteristics of daily precipitation over China based on the index of information entropy. Results showed that the randomness of daily precipitation in the southeast region were larger than that in the northwest region. Moreover, the spatial distribution of stochastic characteristics of precipitation was different at various grades. Stochastic characteri-stics of P0(precipitation at 0.1-10 mm) was large, but the spatial variation was not obvious. The stochastic characteristics of P10(precipitation at 10-25 mm) and P25(precipitation at 25-50 mm) were the largest and their spatial difference was obvious. P50(precipitation ≥50 mm) had the smallest stochastic characteristics and the most obviously spatial difference. Generally, the entropy values of precipitation obviously increased over the last five decades, indicating more significantly stochastic characteristics of precipitation (especially the obvious increase of heavy precipitation events) in most region over China under the scenarios of global climate change. Given that the spatial distribution and long-term trend of entropy values of daily precipitation could reflect thespatial distribution of stochastic characteristics of precipitation, our results could provide scientific basis for the control of flood and waterlogging disaster, the layout of agricultural planning, and the planning of ecological environment.

Screening and identification of four serum miRNAs as novel potential biomarkers for cured pulmonary tuberculosis.

Rapid and efficient methods for the determination of cured pulmonary tuberculosis (TB) are lacking. We screened serum miRNAs using the Solexa sequencing method among untreated TB patients, two-month treated TB patients, cured TB patients, and healthy controls. A total of 100 differentially expressed miRNAs were identified in cured TB patients, including 37 up-regulated (fold change >1.50, P < 0.05) and 63 down-regulated (fold change <0.60, P < 0.05) miRNAs. Gene ontology (GO) enrichment analysis revealed that most of the predicted genes were present in the nucleus with a strong protein binding function. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis strongly suggested alterations in the metabolic pathways. Following quantitative real time chain reaction (qRT-PCR), significantly reduced expression levels of miR-21-5p (0.30, P < 0.001), miR-92a-3p (0.63, P < 0.001), and miR-148b-3p (0.17, P < 0.001) were found in the cured TB patients compared with the untreated TB patients, while significantly increased expression levels of miR-21-5p (2.09, P = 0.001), miR-92a-3p (1.40, P = 0.005), and miR-148b-3p (4.80, P = 0.003) were found in the untreated TB patients compared with the healthy controls. And significantly increased level of miR-125a-5p was found between two-month treated TB patients and untreated TB patients (1.81, P = 0.004). We established a cured TB model with 83.96% accuracy by four miRNAs (miR-21-5p, miR-92a-3p, miR-148b-3p, and miR-125a-5p), and also established a diagnostic model with 70.09% accuracy. Our study provides experimental data for establishing objective indicators of cured TB, and also provides a new experimental basis to understand the pathogenesis and prognosis of TB.

iTRAQ-based proteomic analysis to identify the molecular mechanism of Zhibai Dihuang Granule in the Yin-deficiency-heat syndrome rats.

BACKGROUND: Zhibai Dihuang Granule (ZDG) is a traditional Chinese medicine which has been used to treat Yin-deficiency-heat (YDH) syndrome for thousands of years in China. However, little work has been conducted to explore the molecular mechanism of ZDG in YDH syndrome, and the processes of YDH syndrome prevention and treatment have been developed slowly. The present study was aimed to explore the therapeutic mechanism of ZDG on YDH syndrome. METHODS: The YDH syndrome rats were induced by hot Chinese herbs, then treated by ZDG orally for 1 week. Body weight was measured every 2 days. After sacrifice, blood samples were collected and the thymus, adrenal glands, spleen, and liver were immediately removed and weighed. iTRAQ-based proteomics approach was applied to explore the serum protein alterations with the treatment of ZDG, and to investigate the underlying mechanism of ZDG in treating YDH syndrome. RESULTS: The body weights of YDH syndrome rats were significantly decreased compared with control group, and increased in ZDG treated rats. The relative weights of thymus in YDH syndrome rats were increased compared with the control rats, and significantly decreased in after ZDG treatment. In the proteomic analyses, seventy-one proteins were differentially expressed in the YDH syndrome group and the ZDG treated group, including 10 up-regulated and 61 down-regulated proteins. Gene ontology analysis revealed that the differentially expressed proteins were mostly related to immune response, and pathway enrichment analysis showed that these proteins were enriched in coagulation and complement cascades. Enzyme-linked immunosorbent assay was performed to detect the protein levels in coagulation and complement cascades, and the results showed that complement component 5 levels were significantly increased, while fibrinogen gamma chain levels were significantly decreased in the ZDG treated group. CONCLUSIONS: We found that ZDG treatment could lead to proteins alteration in immune response, especially in coagulation and complement cascades. ZDG can up-regulate the proteins in the complement cascade to eliminate pathogens, and down-regulate the proteins in the coagulation cascade to suppress inflammation. Our study provides experimental basis to understand the therapeutic mechanism of ZDG and revealed that ZDG can regulate coagulation and complement cascades in treating YDH syndrome.

Screening and identification of lncRNAs as potential biomarkers for pulmonary tuberculosis.

Pulmonary tuberculosis (TB) is among the diseases with the highest morbidity and mortality worldwide. Effective diagnostic methods for TB are lacking. In this study, we investigated long non-coding RNAs (lncRNAs) in plasma using microarray and the potential diagnostic value of lncRNAs for TB. We found a total of 163 up-regulated lncRNAs and 348 down-regulated lncRNAs. Gene ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG) and coding-noncoding co-expression (CNC) analyses showed that functions of differentially expressed lncRNAs were mainly enriched in the regulation of alpha-beta T cell activation and the T cell receptor signalling pathway. Four differentially expressed lncRNAs, NR_038221 (fold change = 3.79, P < 0.01), NR_003142 (fold change = 1.69, P < 0.05), ENST00000570366 (fold change = 3.04, P < 0.05), and ENST00000422183 (fold change = 2.11, P < 0.001), were verified using RT-qPCR. Among those, NR_038221, NR_003142, and ENST00000570366 were found to be up-regulated, while ENST00000422183 was down-regulated. The value of the area under the curve (AUC) for the diagnostic model consisting of the four lncRNAs was 0.845 (sensitivity = 79.2%, specificity = 75%). We further predicted 85 mRNAs and 404 miRNAs that potentially interact with these lncRNAs. Our study revealed the potential value of lncRNAs as biomarkers for early diagnosis of TB and the underlying mechanisms of these abnormally expressed lncRNAs in the pathogenesis of TB.

Cytoreductive cryosurgery in patients with bone metastatic prostate cancer: A retrospective analysis.

The current study is a retrospective analysis of 49 patients with bone metastatic prostate cancer: 26 receiving androgen deprivation therapy (ADT) alone versus 23 receiving cytoreductive cryosurgery of the primary tumor plus ADT treatment. Progression-free survival (PFS) was the primary outcome variable, and Cox proportional hazards regression analysis was used to identify predictors for PFS. The baseline characteristics were generally comparable between the 2 groups. Median follow-up time was 41 months (range 24-56) and 37 months (range 19-53) in ADT alone group and cryosurgery groups, respectively. Patients receiving cryosurgery had significantly longer PFS (35 vs 25 months, P = 0.0027) and time to castration resistance (36 vs 25 months, P = 0.0011). Cox multivariate analysis associated longer PFS with the following factors: cryosurgery (HR0.207, 95% CI 0.094-0.456), lower prostate specific antigen at diagnosis (≤100 ng/ml, HR0.235, 95% CI 0.072-0.763) and lower Gleason score (≤7, HR0.195, 95% CI 0.077-0.496). Cryosurgery reduced the risk of progression by 79.3%. In conclusion, cytoreductive cryosurgery of the primary tumor in patients with bone metastatic prostate cancer could reduce the risk of progression and delay time to castration-resistant prostate cancer.

Association of genetic variations in IL-6/IL-6R pathway genes with gastric cancer risk in a Chinese population.

Interleukin-6 (IL-6) and its receptor (IL-6R) were regarded to be responsible for the occurrence of gastric cancer for their regulation roles in the inflammation. The genetic variations in these two genes (IL-6: rs6949149, rs1800796, rs10499563 and IL-6R: rs2228145) have been suggested to be associated with gastric cancer risk. However, the published results were inconsistent among subjects of different ethnicity. To evaluate such an association in Chinese population, we carried out this case-control study based on 473 patients with gastric cancer and 474 healthy controls, whose genotypes were detected by the Sequenom MassARRAY platform, and Helicobacter pylori infection was assessed by immunogold testing kit. This study showed that rs1800796 CG genotype was associated with decreased risk of gastric cancer (adjusted OR=0.75, 95% CI: 0.57-0.99, p=0.043). The stratified analysis revealed that, in the Helicobacter pylori negative infection subgroup, rs2228145 AC (adjusted OR=0.64, 95% CI: 0.42-0.97) and AC/CC (adjusted OR=0.66, 95% CI: 0.45-0.99) genotypes were associated with decreased risk of gastric cancer, respectively. In contrast, in the Helicobacter pylori positive infection subgroup, rs10499563 TC (adjusted OR=0.64, 95% CI: 0.43-0.95), CC (adjusted OR=0.35, 95% CI: 0.14-0.90), TC/CC (adjusted OR=0.59, 95% CI: 0.40-0.87) genotype were associated with decreased gastric cancer risk, respectively. Moreover, in the male subgroup, rs1800796 CG (adjusted OR=0.61, 95% CI: 0.44-0.84) and CG/GG (adjusted OR=0.67, 95% CI: 0.49-0.91) genotype were associated with decreased risk of gastric cancer, respectively. In short, this study suggested that IL-6R rs2228145 and IL-6 rs10499563 genotype were associated with decreased risk of gastric cancer for the individuals with negative and positive Helicobacter pylori infection.

Serum amyloid A, protein Z, and C4b-binding protein ß chain as new potential biomarkers for pulmonary tuberculosis.

The aim of this study was to discover novel biomarkers for pulmonary tuberculosis (TB). Differentially expressed proteins in the serum of patients with TB were screened and identified by iTRAQ-two dimensional liquid chromatography tandem mass spectrometry analysis. A total of 79 abnormal proteins were discovered in patients with TB compared with healthy controls. Of these, significant differences were observed in 47 abnormally expressed proteins between patients with TB or pneumonia and chronic obstructive pulmonary disease (COPD). Patients with TB (n = 136) exhibited significantly higher levels of serum amyloid A (SAA), vitamin K-dependent protein Z (PROZ), and C4b-binding protein ß chain (C4BPB) than those in healthy controls (n = 66) (P<0.0001 for each) albeit significantly lower levels compared with those in patients with pneumonia (n = 72) (P<0.0001 for each) or COPD (n = 72) (P<0.0001, P<0.0001, P = 0.0016, respectively). After 6 months of treatment, the levels of SAA and PROZ were significantly increased (P = 0.022, P<0.0001, respectively), whereas the level of C4BPB was significantly decreased (P = 0.0038) in treated TB cases (n = 72). Clinical analysis showed that there were significant differences in blood clotting and lipid indices in patients with TB compared with healthy controls, patients with pneumonia or COPD, and treated TB cases (P<0.05). Correlation analysis revealed significant correlations between PROZ and INR (rs = 0.414, P = 0.044), and between C4BPB and FIB (rs = 0.617, P = 0.0002) in patients with TB. Receiver operating characteristic curve analysis revealed that the area under the curve value of the diagnostic model combining SAA, PROZ, and C4BPB to discriminate the TB group from the healthy control, pneumonia, COPD, and cured TB groups was 0.972, 0.928, 0.957, and 0.969, respectively. Together, these results suggested that SAA, PROZ, and C4BPB may serve as new potential biomarkers for TB. Our study may thus provide experimental data for the differential diagnosis of TB.

Microarray expression profile analysis of mRNAs and long non-coding RNAs in pulmonary tuberculosis with different traditional Chinese medicine syndromes.

BACKGROUND: Combination chemotherapy with Western anti-tuberculosis (TB) drugs is the mainstay of TB treatment. Chinese herbal medicines with either heat clearing and detoxifying effects or nourishing Yin and reducing fire effects have been used to treat TB based on the Traditional Chinese Medicine (TCM) syndromes of TB patients. This study analyzed the expression profiles of long non-coding RNAs (lncRNAs) and mRNAs in TB patients with different TCM syndromes. METHODS: TB patients were classified as pulmonary Yin deficiency (PYD) syndrome, hyperactivity of fire due to Yin deficiency (HFYD) syndrome, and deficiency of Qi and Yin (DQY) syndrome. Total RNA from 44 TB patients and healthy controls was extracted and hybridized with a human lncRNA microarray containing 30586 lncRNAs and 26109 mRNAs probes. Bioinformatics analyses, including gene ontology (GO) and pathways, were performed. Related clinical data were also analyzed. RESULTS: Differentially expressed mRNAs and lncRNAs were identified (fold change >2, and P < 0.05) in PYD (634 mRNAs and 566 lncRNAs), HFYD (47 mRNAs and 55 lncRNAs), and DQY (63 mRNAs and 60 lncRNAs) patients. The most enriched pathways were the hippo signaling pathway (P = 0.000164) and the protein digestion and absorption pathway (P = 5.89017E-05). Clinical analyses revealed that the lipid indexes of TB patients were abnormal and that the triglyceride concentration was significantly higher in DQY patients (P = 0.0252). Our study is the first to acquire the microarray expression profiles of lncRNAs and mRNAs and analyze pathway enrichment in PYD, HFYD, and DQY patients with TB. CONCLUSIONS: Our analyses of the expression profiles of lncRNAs and mRNAs may represent a novel method to explore the biological essence of TCM syndromes of TB.

Targeting the pseudorabies virus DNA polymerase processivity factor UL42 by RNA interference efficiently inhibits viral replication.

RNA interference (RNAi) is a conserved gene-silencing mechanism in which small interfering RNAs (siRNAs) induce the sequence-specific degradation of homologous RNAs. It has been shown to be a novel and effective antiviral therapy against a wide range of viruses. The pseudorabies virus (PRV) processivity factor UL42 can enhance the catalytic activity of the DNA polymerase and is essential for viral replication, thus it may represent a potential drug target of antiviral therapy against PRV infection. Here, we synthesized three siRNAs (siR-386, siR-517, and siR-849) directed against UL42 and determined their antiviral activities in cell culture. We first examined the kinetics of UL42 expression and found it was expressed with early kinetics during PRV replication. We verified that siR-386, siR-517, and siR-849 efficiently inhibited UL42 expression in an in vitro transfection system, thereby validating their inhibitory effects. Furthermore, we confirmed that these three siRNAs induced potent inhibitory effects on UL42 expression after PRV infection, comparable to the positive control siRNA, siR-1046, directed against the PRV DNA polymerase, the UL30 gene product, which is essential for virus replication. In addition, PRV replication was markedly reduced upon downregulation of UL42 expression. These results indicate that UL42-targeted RNAi efficiently inhibits target gene expression and impairs viral replication. This study provides a new clue for the design of an intervention strategy against herpesviruses by targeting their processivity factors.

The Pseudorabies Virus DNA Polymerase Accessory Subunit UL42 Directs Nuclear Transport of the Holoenzyme.

Pseudorabies virus (PRV) DNA replication occurs in the nuclei of infected cells and requires the viral DNA polymerase. The PRV DNA polymerase comprises a catalytic subunit, UL30, and an accessory subunit, UL42, that confers processivity to the enzyme. Its nuclear localization is a prerequisite for its enzymatic function in the initiation of viral DNA replication. However, the mechanisms by which the PRV DNA polymerase holoenzyme enters the nucleus have not been determined. In this study, we characterized the nuclear import pathways of the PRV DNA polymerase catalytic and accessory subunits. Immunofluorescence analysis showed that UL42 localizes independently in the nucleus, whereas UL30 alone predominantly localizes in the cytoplasm. Intriguingly, the localization of UL30 was completely shifted to the nucleus when it was coexpressed with UL42, demonstrating that nuclear transport of UL30 occurs in an UL42-dependent manner. Deletion analysis and site-directed mutagenesis of the two proteins showed that UL42 contains a functional and transferable bipartite nuclear localization signal (NLS) at amino acids 354-370 and that K(354), R(355), and K(367) are important for the NLS function, whereas UL30 has no NLS. Coimmunoprecipitation assays verified that UL42 interacts with importins α3 and α4 through its NLS. In vitro nuclear import assays demonstrated that nuclear accumulation of UL42 is a temperature- and energy-dependent process and requires both importins α and ß, confirming that UL42 utilizes the importin α/ß-mediated pathway for nuclear entry. In an UL42 NLS-null mutant, the UL42/UL30 heterodimer was completely confined to the cytoplasm when UL42 was coexpressed with UL30, indicating that UL30 utilizes the NLS function of UL42 for its translocation into the nucleus. Collectively, these findings suggest that UL42 contains an importin α/ß-mediated bipartite NLS that transports the viral DNA polymerase holoenzyme into the nucleus in an in vitro expression system.

A Group of Novel Serum Diagnostic Biomarkers for Multidrug-Resistant Tuberculosis by iTRAQ-2D LC-MS/MS and Solexa Sequencing.

The epidemic of pulmonary tuberculosis (TB), especially multidrug-resistance tuberculosis (MDR-TB) presented a major challenge for TB treatment today. We performed iTRAQ labeling coupled with two-dimensional liquid chromatography-tandem mass spectrometry (2D LC-MS/MS) and Solexa sequencing among MDR-TB patients, drug-sensitive tuberculosis (DS-TB) patients, and healthy controls. A total of 50 differentially expressed proteins and 43 differentially expressed miRNAs (fold change >1.50 or <0.60, P<0.05) were identified in the MDR-TB patients compared to both DS-TB patients and healthy controls. We found that 22.00% of differentially expressed proteins and 32.56% of differentially expressed miRNAs were related, and could construct a network mainly in complement and coagulation cascades. Significant differences in CD44 antigen (CD44), coagulation factor XI (F11), kininogen-1 (KNG1), miR-4433b-5p, miR-424-5p, and miR-199b-5p were found among MDR-TB patients, DS-TB patients and healthy controls (P<0.05) by enzyme-linked immunosorbent assay (ELISA) and SYBR green qRT-PCR validation. A strong negative correlation, consistent with the target gene prediction, was found between miR-199b-5p and KNG1 (r=-0.232, P=0.017). Moreover, we established the MDR-TB diagnostic model based on five biomarkers (CD44, KNG1, miR-4433b-5p, miR-424-5p, and miR-199b-5p). Our study proposes potential biomarkers for MDR-TB diagnosis, and also provides a new experimental basis to understand the pathogenesis of MDR-TB.