Respiratory syncytial virus (RSV) is a significant cause of severe respiratory illness worldwide, particularly in infants, young children, and the elderly. Although no licensed vaccine is currently available, an engineered version of the metastable RSV fusion (F) surface glycoprotein-stabilized in the pre-fusion (pre-F) conformation by "DS-Cav1" mutations-elicits high titer RSV-neutralizing responses. Moreover, pre-F-specific antibodies, often against the neutralization-sensitive antigenic site Ø in the membrane-distal head region of trimeric F glycoprotein, comprise a substantial portion of the human response to natural RSV infection. To focus the vaccine-elicited response to antigenic site Ø, we designed a series of RSV F immunogens that comprised the membrane-distal head of the F glycoprotein in its pre-F conformation. These "head-only" immunogens formed monomers, dimers, and trimers. Antigenic analysis revealed that a majority of the 70 engineered head-only immunogens displayed reactivity to site Ø-targeting antibodies, which was similar to that of the parent RSV F DS-Cav1 trimers, often with increased thermostability. We evaluated four of these head-only immunogens in detail, probing their recognition by antibodies, their physical stability, structure, and immunogenicity. When tested in naïve mice, a head-only trimer, half the size of the parent RSV F trimer, induced RSV titers, which were statistically comparable to those induced by DS-Cav1. When used to boost DS-Cav1-primed mice, two head-only RSV F immunogens, a dimer and a trimer, boosted RSV-neutralizing titers to levels that were comparable to those boosted by DS-Cav1, although with higher site Ø-directed responses. Our results provide proof-of-concept for the ability of the smaller head-only RSV F immunogens to focus the vaccine-elicited response to antigenic site Ø. Decent primary immunogenicity, enhanced physical stability, potential ease of manufacture, and potent immunogenicity upon boosting suggest these head-only RSV F immunogens, engineered to retain the pre-fusion conformation, may have advantages as candidate RSV vaccines.

A very small proportion of people remain negative for HIV infection after repeated HIV-1 viral exposure, which is called HIV-1 resistance. Understanding the mechanism of HIV-1 resistance is important for the development of HIV-1 vaccines and Acquired Immune Deficiency Syndrome (AIDS) therapies. In this study, we analyzed the gene expression profiles of CD4+ T cells from HIV-1-resistant individuals and HIV-susceptible individuals. One hundred eighty-five discriminative HIV-1 resistance genes were identified using the Minimum Redundancy-Maximum Relevance (mRMR) and Incremental Feature Selection (IFS) methods. The virus protein target enrichment analysis of the 185 HIV-1 resistance genes suggested that the HIV-1 protein nef might play an important role in HIV-1 infection. Moreover, we identified 29 infection information exchanger genes from the 185 HIV-1 resistance genes based on a virus-host interaction network analysis. The infection information exchanger genes are located on the shortest paths between virus-targeted proteins and are important for the coordination of virus infection. These proteins may be useful targets for AIDS prevention or therapy, as intervention in these pathways could disrupt communication with virus-targeted proteins and HIV-1 infection.

The risk that the plant pathogen Phytophthora melonis develops resistance to carboxylic acid amide (CAA) fungicides was determined by measuring baseline sensitivities of field isolates, generating resistant mutants, and measuring the fitness of the resistant mutants. The baseline sensitivities of 80 isolates to flumorph, dimethomorph and iprovalicarb were described by unimodal curves, with mean EC(50) values of 0.986 (±0.245), 0.284 (±0.060) and 0.327 (±0.068) µg/ml, respectively. Seven isolates with different genetic background (as indicated by RAPD markers) were selected to generate CAA-resistance. Fifty-five resistant mutants were obtained from three out of seven isolates by spontaneous selection and UV-mutagenesis with frequencies of 1×10(-7) and 1×10(-6), respectively. CAA-resistance was stable for all mutants. The resistance factors of these mutants ranged from 7 to 601. The compound fitness index (CFI  =  mycelial growth × zoospore production × pathogenicity) was often lower for the CAA-resistant isolates than for wild-type isolates, suggesting that the risk of P. melonis developing resistance to CAA fungicides is low to moderate. Among the CAA-resistant isolates, a negative correlation between EC(50) values was found for iprovalicarb vs. flumorph and for iprovalicarb vs. dimethomorph. Comparison of the full-length cellulose synthase 3 (CesA3) between wild-type and CAA-resistant isolates revealed only one point mutation at codon position 1109: a valine residue (codon GTG in wild-type isolates) was converted to leucine (codon CTG in resistant mutants). This represents a novel point mutation with respect to mutations in CesA3 conferring resistance to CAA fungicides. Based on this mutation, an efficient allelic-specific PCR (AS-PCR) method was developed for rapid detection of CAA-resistance in P. melonis populations.

Recent findings have revealed that gut microbiota plays a substantial role in modulating diseases such as autism, rheumatoid arthritis, allergies, and cancer that occur at sites distant to the gut. Athymic nude mice have been employed for tumorigenic research for decades; however, the relationships between the gut microbiome and host's response in drug treatment to the grafted tumors have not been explored. In this study, we analyzed the fecal microbiome of nonxenograft and xenograft nude mice treated with phytosaponins from a popular medicinal plant, Gynostemma pentaphyllum (Gp). Analysis of enterobacterial repetitive intergenic consensus (ERIC)-PCR data showed that the microbiota profile of xenograft mice departed from that of the nonxenograft mice. After ten days of treatment with Gp saponins (GpS), the microbiota of the treated mice was closer to the microbiota at Day 0 before the implantation of the tumor. Data obtained from 16S pyrosequencing of fecal samples reiterates the differences in microbiome between the nonxenograft and xenograft mice. GpS markedly increased the relative abundance of Clostridium cocleatum and Bacteroides acidifaciens, for which the beneficial effects on the host have been well documented. This study, for the first time, characterizes the properties of gut microbiome in nude mice responding to tumor implant and drug treatment. We also demonstrate that dietary saponins such as GpS can potentially regulate the gut microbial ecosystem by increasing the number of symbionts. Interestingly, this regulation of the gut ecosystem might, at least in part, be responsible for or contribute to the anticancer effect of GpS.

Recombinant subunit vaccines should contain minimal non-pathogen motifs to reduce potential off-target reactivity. We recently developed a vaccine antigen against respiratory syncytial virus (RSV), which comprised the fusion (F) glycoprotein stabilized in its pre-fusion trimeric conformation by "DS-Cav1" mutations and by an appended C-terminal trimerization motif or "foldon" from T4-bacteriophage fibritin. Here we investigate the creation of a cysteine zipper to allow for the removal of the phage foldon, while maintaining the immunogenicity of the parent DS-Cav1+foldon antigen. Constructs without foldon yielded RSV F monomers, and enzymatic removal of the phage foldon from pre-fusion F trimers resulted in their dissociation into monomers. Because the native C terminus of the pre-fusion RSV F ectodomain encompasses a viral trimeric coiled-coil, we explored whether introduction of cysteine residues capable of forming inter-protomer disulfides might allow for stable trimers. Structural modeling indicated the introduced cysteines to form disulfide "rings", with each ring comprising a different set of inward facing residues of the coiled-coil. Three sets of rings could be placed within the native RSV F coiled-coil, and additional rings could be added by duplicating portions of the coiled-coil. High levels of neutralizing activity in mice, equivalent to that of the parent DS-Cav1+foldon antigen, were elicited by a 4-ring stabilized RSV F trimer with no foldon. Structure-based alteration of a viral coiled-coil to create a cysteine zipper thus allows a phage trimerization motif to be removed from a candidate vaccine antigen.

Real-time reverse transcription PCR (RT-qPCR) is a preferred method for rapid and accurate quantification of gene expression studies. Appropriate application of RT-qPCR requires accurate normalization though the use of reference genes. As no single reference gene is universally suitable for all experiments, thus reference gene(s) validation under different experimental conditions is crucial for RT-qPCR analysis. To date, only a few studies on reference genes have been done in other plants but none in papaya. In the present work, we selected 21 candidate reference genes, and evaluated their expression stability in 246 papaya fruit samples using three algorithms, geNorm, NormFinder and RefFinder. The samples consisted of 13 sets collected under different experimental conditions, including various tissues, different storage temperatures, different cultivars, developmental stages, postharvest ripening, modified atmosphere packaging, 1-methylcyclopropene (1-MCP) treatment, hot water treatment, biotic stress and hormone treatment. Our results demonstrated that expression stability varied greatly between reference genes and that different suitable reference gene(s) or combination of reference genes for normalization should be validated according to the experimental conditions. In general, the internal reference genes EIF (Eukaryotic initiation factor 4A), TBP1 (TATA binding protein 1) and TBP2 (TATA binding protein 2) genes had a good performance under most experimental conditions, whereas the most widely present used reference genes, ACTIN (Actin 2), 18S rRNA (18S ribosomal RNA) and GAPDH (Glyceraldehyde-3-phosphate dehydrogenase) were not suitable in many experimental conditions. In addition, two commonly used programs, geNorm and Normfinder, were proved sufficient for the validation. This work provides the first systematic analysis for the selection of superior reference genes for accurate transcript normalization in papaya under different experimental conditions.

Cigarette smoking is a major pathogenic factor in lung cancer. Macrophages play an important role in host defense and adaptive immunity. These cells display diverse phenotypes for performing different functions. M2 type macrophages usually exhibit immunosuppressive and tumor-promoting characteristics. Although macrophage polarization toward the M2 phenotype has been observed in the lungs of cigarette smokers, the molecular basis of the process remains unclear. In this study, we evaluated the possible mechanisms for the polarization of mouse macrophages that are induced by cigarette smoking (CS) or cigarette smoke extract (CSE). The results showed that exposure to CSE suppressed the production of reactive oxygen species (ROS) and nitric oxide (NO) and down-regulated the phagocytic ability of Ana-1 cells. The CD163 expressions on the surface of macrophages from different sources were significantly increased in in vivo and in vitro studies. The M1 macrophage cytokines TNF-α, IL-12p40 and enzyme iNOS decreased in the culture supernatant, and their mRNA levels decreased depending on the time and concentration of CSE. In contrast, the M2 phenotype macrophage cytokines IL-10, IL-6, TGF-β1 and TGF-β2 were up-regulated. Moreover, phosphorylation of JAK2 and STAT3 was observed after the Ana-1 cells were treated with CSE. In addition, pretreating the Ana-1 cells with the STAT3 phosphorylation inhibitor WP1066 inhibited the CSE-induced CD163 expression, increased the mRNA level of IL-10 and significantly decreased the mRNA level of IL-12. In conclusion, we demonstrated that the M2 polarization of macrophages induced by CS could be mediated through JAK2/STAT3 pathway activation.

This study was designed to investigate the anti-inflammatory and anti-nociceptive activity of the methanol extract from the aerial part of Phlomis younghusbandii (MEAP) and to explore the possible related mechanisms. Anti-inflammatory effects of MEAP were evaluated by using the ear edema test induced by dimethylbenzene and vascular permeability test induced by acetic acid. Anti-nociceptive activities of MEAP were evaluated by the chemical nociception in models of acetic acid-induced writhing and formalin-induced hind paw licking, and by the thermal nociception in hot plate tests. Mechanisms of MEAP activities also were explored by evaluating expression levels of TNF-α, IL-6 and iNOS induced by LPS using real-time fluorogenic PCR and expression of COX-2 using Western blotting and an open-field test. The results indicated that the MEAP administered orally could significantly decrease ear edema induced by dimethylbenzene and increase vascular permeability induced by acetic acid. Additionally, the nociceptions induced by acetic acid and formalin were significantly inhibited. The anti-nociceptive effect could not be decreased by naloxone in the formalin test, and MEAP did not affect the normal autonomic activities of mice. Expression levels of pro-inflammatory cytokines (TNF-α, IL-6, iNOS) induced by LPS were decreased obviously by treatment with MEAP. Furthermore, COX-2 expression in the spinal dorsal horns of the pain model mice induced by formalin was significantly down-regulated by MEAP. In conclusion, MEAP has significant anti-inflammatory and antinociceptive activities, and the mechanisms may be related to the down-regulated expression of TNF-α, IL-6, iNOS and COX-2.

Highly pathogenic porcine reproductive and respiratory syndrome virus (HP-PRRSV) had caused catastrophic losses in swine industry in China. The current inactivated vaccine provided only limited protection, and the attenuated live vaccine could protect piglets against the HP-PRRSV but there was a possibility that the attenuated virus returned to high virulence. In this study, the eukaryotic expression vector pVAX1© was modified under the control of rabbit β-globin intron II gene and the modified vector pMVAX1© was constructed. Porcine interleukin-2 (IL-2) and GP3-GP5 fusion protein of HP-PRRSV strain SD-JN were highly expressed by pMVAX1©. Mice inoculated with pMVAX1©-GP35 developed significantly higher PRRSV-specific antibody responses and T cell proliferation than those vaccinated with pVAX1©-GP35. pMVAX1©-GP35 was selected as PRRS DNA vaccine candidate and co-administrated with pVAX1©-IL-2 or pMVAX1©-IL-2 in pigs. pMVAX1©-IL-2+pMVAX1©-GP35 could provide enhanced PRRSV-specific antibody responses, T cell proliferation, Th1-type and Th2-type cytokine responses and CTL responses than pMVAX1©-GP35 and pVAX1©-IL-2+pMVAX1©-GP35. Following homologous challenge with HP-PRRSV strain SD-JN, similar with attenuated PRRS vaccine group, pigs inoculated with pMVAX1©-IL-2+pMVAX1©-GP35 showed no clinical signs, almost no lung lesions and no viremia, as compared to those in pMVAX1©-GP35 and pVAX1©-IL-2+pMVAX1©-GP35 groups. It indicated that pMVAX1©-IL-2 effectively increases humoral and cell mediated immune responses of pMVAX1©-GP35. Co-administration of pMVAX1©-IL-2 and pMVAX1©-GP35 might be attractive candidate vaccines for preventing HP-PRRSV infections.

Prediction of disease-specific survival (DSS) for individual patient with gastric cancer after R0 resection remains a clinical concern. Since the clinicopathologic characteristics of gastric cancer vary widely between China and western countries, this study is to evaluate a nomogram from Memorial Sloan-Kettering Cancer Center (MSKCC) for predicting the probability of DSS in patients with gastric cancer from a Chinese cohort.

Although the number of available protein sequences is growing exponentially, functional protein annotations lag far behind. Therefore, accurate identification of protein functions remains one of the major challenges in molecular biology. In this study, we presented a novel approach to predict mouse protein functions. The approach was a sequential combination of a similarity-based approach, an interaction-based approach and a pseudo amino acid composition-based approach. The method achieved an accuracy of about 0.8450 for the 1st-order predictions in the leave-one-out and ten-fold cross-validations. For the results yielded by the leave-one-out cross-validation, although the similarity-based approach alone achieved an accuracy of 0.8756, it was unable to predict the functions of proteins with no homologues. Comparatively, the pseudo amino acid composition-based approach alone reached an accuracy of 0.6786. Although the accuracy was lower than that of the previous approach, it could predict the functions of almost all proteins, even proteins with no homologues. Therefore, the combined method balanced the advantages and disadvantages of both approaches to achieve efficient performance. Furthermore, the results yielded by the ten-fold cross-validation indicate that the combined method is still effective and stable when there are no close homologs are available. However, the accuracy of the predicted functions can only be determined according to known protein functions based on current knowledge. Many protein functions remain unknown. By exploring the functions of proteins for which the 1st-order predicted functions are wrong but the 2nd-order predicted functions are correct, the 1st-order wrongly predicted functions were shown to be closely associated with the genes encoding the proteins. The so-called wrongly predicted functions could also potentially be correct upon future experimental verification. Therefore, the accuracy of the presented method may be much higher in reality.

Hydroxylation is an important post-translational modification and closely related to various diseases. Besides the biotechnology experiments, in silico prediction methods are alternative ways to identify the potential hydroxylation sites.

Most of pyruvoyl-dependent proteins observed in prokaryotes and eukaryotes are critical regulatory enzymes, which are primary targets of inhibitors for anti-cancer and anti-parasitic therapy. These proteins undergo an autocatalytic, intramolecular self-cleavage reaction in which a covalently bound pyruvoyl group is generated on a conserved serine residue. Traditional detections of the modified serine sites are performed by experimental approaches, which are often labor-intensive and time-consuming. In this study, we initiated in an attempt for the computational predictions of such serine sites with Feature Selection based on a Random Forest. Since only a small number of experimentally verified pyruvoyl-modified proteins are collected in the protein database at its current version, we only used a small dataset in this study. After removing proteins with sequence identities >60%, a non-redundant dataset was generated and was used, which contained only 46 proteins, with one pyruvoyl serine site for each protein. Several types of features were considered in our method including PSSM conservation scores, disorders, secondary structures, solvent accessibilities, amino acid factors and amino acid occurrence frequencies. As a result, a pretty good performance was achieved in our dataset. The best 100.00% accuracy and 1.0000 MCC value were obtained from the training dataset, and 93.75% accuracy and 0.8441 MCC value from the testing dataset. The optimal feature set contained 9 features. Analysis of the optimal feature set indicated the important roles of some specific features in determining the pyruvoyl-group-serine sites, which were consistent with several results of earlier experimental studies. These selected features may shed some light on the in-depth understanding of the mechanism of the post-translational self-maturation process, providing guidelines for experimental validation. Future work should be made as more pyruvoyl-modified proteins are found and the method should be evaluated on larger datasets. At last, the predicting software can be downloaded from http://www.nkbiox.com/sub/pyrupred/index.html.

Given a compound, how can we effectively predict its biological function? It is a fundamentally important problem because the information thus obtained may benefit the understanding of many basic biological processes and provide useful clues for drug design. In this study, based on the information of chemical-chemical interactions, a novel method was developed that can be used to identify which of the following eleven metabolic pathway classes a query compound may be involved with: (1) Carbohydrate Metabolism, (2) Energy Metabolism, (3) Lipid Metabolism, (4) Nucleotide Metabolism, (5) Amino Acid Metabolism, (6) Metabolism of Other Amino Acids, (7) Glycan Biosynthesis and Metabolism, (8) Metabolism of Cofactors and Vitamins, (9) Metabolism of Terpenoids and Polyketides, (10) Biosynthesis of Other Secondary Metabolites, (11) Xenobiotics Biodegradation and Metabolism. It was observed that the overall success rate obtained by the method via the 5-fold cross-validation test on a benchmark dataset consisting of 3,137 compounds was 77.97%, which is much higher than 10.45%, the corresponding success rate obtained by the random guesses. Besides, to deal with the situation that some compounds may be involved with more than one metabolic pathway class, the method presented here is featured by the capacity able to provide a series of potential metabolic pathway classes ranked according to the descending order of their likelihood for each of the query compounds concerned. Furthermore, our method was also applied to predict 5,549 compounds whose metabolic pathway classes are unknown. Interestingly, the results thus obtained are quite consistent with the deductions from the reports by other investigators. It is anticipated that, with the continuous increase of the chemical-chemical interaction data, the current method will be further enhanced in its power and accuracy, so as to become a useful complementary vehicle in annotating uncharacterized compounds for their biological functions.

The DNA of all eukaryotic organisms is packaged into nucleosomes, the basic repeating units of chromatin. The nucleosome consists of a histone octamer around which a DNA core is wrapped and the linker histone H1, which is associated with linker DNA. By altering the accessibility of DNA sequences, the nucleosome has profound effects on all DNA-dependent processes. Understanding the factors that influence nucleosome positioning is of great importance for the study of genomic control mechanisms. Transcription factors (TFs) have been suggested to play a role in nucleosome positioning in vivo.

Cancer is a significant public health problem worldwide. Complete identification of genes related to one type of cancer facilitates earlier diagnosis and effective treatments. In this study, two widely used algorithms, the random walk with restart algorithm and the shortest path algorithm, were adopted to construct two parameterized computational methods, namely, an RWR-based method and an SP-based method; based on these methods, an integrated method was constructed for identifying novel disease genes. To validate the utility of the integrated method, data for oral cancer were used, on which the RWR-based and SP-based methods were trained, thereby building two optimal methods. The integrated method combining these optimal methods was further adopted to identify the novel genes of oral cancer. As a result, 85 novel genes were inferred, among which eleven genes (e.g., MYD88, FGFR2, NF-κBIA) were identified by both the RWR-based and SP-based methods, 70 genes (e.g., BMP4, IFNG, KITLG) were discovered only by the RWR-based method and four genes (L1R1, MCM6, NOG and CXCR3) were predicted only by the SP-based method. Extensive analyses indicate that several novel genes have strong associations with cancers, indicating the effectiveness of the integrated method for identifying disease genes.

Nuclear factor erythroid 2-related factor 2 (NRF2) functions as a transcription factor and regulates a wide array of antioxidant and stress-responsive genes. NRF2 has been widely implicated in different types of cancers, but only limited studies concerning the relationship between NRF2 expression and tumour invasion or prognosis in lung cancer. Therefore, we conducted a meta-analysis to determine the prognostic value of NRF2 in patients with non-small cell lung cancer (NSCLC). The relationship between NRF2 expression in NSCLC patients and clinicopathological features was also investigated. Overall survival (OS) and treatment response rate were evaluated using STATA software. Twenty eligible articles with 2530 lung cancer patients were included in this meta-analysis. The results revealed that high expression level of NRF2 was associated with pathologic distant metastasis (odds ratio (OR) = 2.64, 95% confidence interval (CI) 1.62-4.31; P < 0.001), lymph node metastasis (OR = 2.14, 95% CI: 1.53-3.00; P < 0.001), and tumour node metastasis (TNM) stage (OR = 1.95, 95% CI: 1.52-2.49, P < 0.001). High NRF2 expression was associated with low treatment response rate in platinum-based chemotherapy (HR = 0.11, 95% CI 0.02-0.51; P = 0.005). High expression level of NRF2 is predictive for poor overall survival rate (HR = 1.86, 95% CI 1.44-2.41, P < 0.001) and poor progression-free survival (PFS) (HR = 2.27, 95% CI 1.26-4.09, P = 0.006). Compared to patients with a low level of NRF2 expression, patients with high NRF2 expression levels were associated with worse OS and PFS when given the chemotherapy or EGFR-TKI. Together, our meta-analysis results suggest that NRF2 can act as a potential indicator of NSCLC tumour aggressiveness and help the prognosis and design of a better treatment strategy for NSCLC patients.

The toxicity of doxorubicin to the cardiovascular system often limits its benefits and widespread use as chemotherapy. The mechanisms involved in doxorubicin-induced cardiovascular damage and possible protective interventions are not well-explored. Using human aortic endothelial cells, we show vitamin D3 strongly attenuates doxorubicin-induced senescence and cell cycle arrest. We further show the protective effects of vitamin D3 are mediated by the upregulation of IL-10 and FOXO3a expression through fine modulation of pAMPKα/SIRT1/FOXO3a complex activity. These results have great significance in finding a target for mitigating doxorubicin-induced cardiovascular toxicity.

Doxorubicin, an anthracycline chemotherapeutic known to incur heart damage, decreases heart function in up to 11% of patients. Recent investigations have implicated the Wnt signaling cascade as a key modulator of cardiac tissue repair after myocardial infarction. Wnt upregulation in murine models resulted in stimulation of angiogenesis and suppression of fibrosis after ischemic insult. However, the molecular mechanisms of Wnt in mitigating doxorubicin-induced cardiac insult require further investigation. Identifying cardioprotective mechanisms of Wnt is imperative to reducing debilitating cardiovascular adverse events in oncologic patients undergoing treatment.

Mammalian target of rapamycin (mTOR) is a central controller of cell growth, proliferation, metabolism, and angiogenesis. Thus, there is a great deal of interest in developing clinical drugs based on mTOR. In this paper, in silico models based on multi-scaffolds were developed to predict mTOR inhibitors or non-inhibitors.