Volatile metabolomics and transcriptomics analyses provide insights into the mechanism of volatile changes during fruit development of 'Ehime 38' (Citrus reticulata) and its bud mutant.

Volatile compounds are important determinants affecting fruit flavor. Previous study has identified a bud mutant of 'Ehime 38' (Citrus reticulata) with different volatile profile. However, the volatile changes between WT and MT during fruit development and underlying mechanism remain elusive. In this study, a total of 35 volatile compounds were identified in the pulps of WT and MT at five developmental stages. Both varieties accumulated similar and the highest levels of volatiles at stage S1, and showed a downward trend as the fruit develops. However, the total volatile contents in the pulps of MT were 1.4-2.5 folds higher than those in WT at stages S2-S5, which was mainly due to the increase in the content of d-limonene. Transcriptomic and RT-qPCR analysis revealed that most genes in MEP pathway were positively correlated with the volatile contents, of which DXS1 might mainly contribute to the elevated volatiles accumulation in MT by increasing the flux into the MEP pathway. Moreover, temporal expression analysis indicated that these MEP pathway genes functioned at different developmental stages. This study provided comprehensive volatile metabolomics and transcriptomics characterizations of a citrus mutant during fruit development, which is valuable for fruit flavor improvement in citrus.

Nickel Nanowires Combined with Surface-Enhanced Raman Spectroscopy: Application in Label-Free Detection of Cytochrome c-Mediated Apoptosis.

Intrinsic properties of nickel have enabled its wide applications as an effective catalyst. In this study, nickel nanowires (Ni NWs) as electron donors for oxidized cytochrome c (Cyt c) are investigated, which are NW diameter, temperature, and pH value-dependent. The reductive and magnetic properties facilitate the Ni NWs to rapidly and conveniently reduce Cyt c in complicated biological samples. Moreover, we find that the Ni NWs combined with resonance Raman spectroscopy have specificity toward Cyt c detection in real biological samples, which is successfully used to distinguish the redox state of the released Cyt c from isolated mitochondria in apoptotic Hela cells. Moreover, rapid label-free Cyt c quantification can be achieved by surface-enhanced Raman spectroscopy with a limit of detection of 1 nM and long concentration linear range (1 nM-1 µM). The proposed Ni NWs-based reduction approach will significantly simplify the traditional biological methods and has great potential in the application of Cyt c-related apoptotic studies.

Comparison of immunogenicity, efficacy and transcriptome changes of inactivated rabies virus vaccine with different adjuvants.

Adjuvants have been proven to be very effective in enhancement of immune response of many antigens. However, few studies involved head-to-head comparison of their potentials in inactive rabies virus vaccine. In this study, we investigated two types of aluminum adjuvants and five other adjuvants (BLPs, AS02, AS03, MF59 and Poly I:C) on their capacity in enhancing the efficacy of rabies vaccine. The differences in immunogenicity and potency of rabies vaccines with different adjuvants were evaluated by immunizations in ICR mice. Compared with other adjuvants, nano-sized aluminum induced earlier and more vigorous production of rabies virus neutralizing antibodies and facilitated a more effective protection in the challenge test. Based on these results, to comprehensively and systematically explore the role of adjuvants in rabies vaccine immunization, blood samples from four groups were chosen to perform mRNA sequencing. The differentially expressed genes (DEGs) of groups were identified, both gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses were conducted for these DEGs. The results showed that there were significant differences in mRNA expression between the mice after immunization with different adjuvants, but the two aluminum adjuvant vaccines induced similar gene expression. Moreover, the data of enrichment analysis indicated that adjuvants were more advantageous in activating the pathways associated with antigen processing, presentation and initial immunity. These results revealed that adjuvants can be used as an enhancer in rabies vaccination, and nano-sized aluminum may be a candidate adjuvant for the development of more effective rabies vaccines. And these data also provide a basis for understanding the mechanisms underlying adjuvants enhancement of the immune response.

Performance of Homologous and Heterologous Prime-Boost Immunization Regimens of Recombinant Adenovirus and Modified Vaccinia Virus Ankara Expressing an Ag85B-TB10.4 Fusion Protein against Mycobacterium tuberculosis.

Tuberculosis (TB) remains a serious health issue around the word. Adenovirus (Ad)-based vaccine and modified vaccinia virus Ankara (MVA)-based vaccine have emerged as two of the most promising immunization candidates over the past few years. However, the performance of the homologous and heterologous prime-boost immunization regimens of these two viral vector-based vaccines remains unclear. In the present study, we constructed recombinant Ad and MVA expressing an Ag85B-TB10.4 fusion protein (AdH4 and MVAH4) and evaluated the impact of their different immunization regimens on the humoral and cellular immune responses. We found that the viral vector-based vaccines could generate significantly higher levels of antigen-specific antibodies, IFN-γ-producing splenocytes, CD69⁺CD8⁺ T cells, and IFN-γ secretion when compared with bacillus Calmette-Guérin (BCG) in a mouse model. AdH4-containing immunization regimens (AdH4-AdH4, AdH4-MVAH4, and MVAH4-AdH4) induced significantly stronger antibody responses, much more IFN-γ-producing splenocytes and CD69⁺CD8⁺ T cells, and higher levels of IFN-γ secretion when compared with the MVAH4-MVAH4 immunization regimen. The number of IFN-γ-producing splenocytes sensitive to CD8⁺ T-cell restricted peptides of Ag85B (9-1p and 9-2p) and Th1-related cytokines (IFN-γ and TNF-α) in the AdH4-MVAH4 heterologous prime-boost regimen immunization group was significantly higher than that in the other viral vector-based vaccine- and BCG-immunized groups, respectively. These results indicate that an immunization regimen involving AdH4 may have a higher capacity to induce humoral and cellular immune responses against TB in mice than that by regimens containing BCG or MVAH4 alone, and the AdH4-MVAH4 prime-boost regimen may generate an ideal protective effect.

Novel intranasal pertussis vaccine based on bacterium-like particles as a mucosal adjuvant.

Pertussis, or whooping cough, has recently reemerged as a major public health threat despite high levels of vaccination. The development of a novel pertussis vaccine, especially an intranasal (i.n.) vaccine is undoubtedly necessary, and mucosal adjuvants have been explored to enhance the immune response. In the present study, bacterium-like particles (BLPs) were adopted as a mucosal adjuvant for an i.n. pertussis vaccine and evaluated on the ability to induce serum and mucosal antibodies as well as potency against i.n. challenge in mice. Groups with or without aluminum adjuvant were also evaluated through both i.n. and intraperitoneal inoculations. Vaccination with BLPs via the i.n. route led to rapid IgG and IgA production and provided strong protection against inflammation induced by infection. The results support an i.n. pertussis vaccine with BLPs adjuvant as a promising candidate to elicit protective immunity against whooping cough.

Tissue plasminogen activator (tPA) signal sequence enhances immunogenicity of MVA-based vaccine against tuberculosis.

Tuberculosis (TB) remains a serious health problem worldwide, and the only available vaccine, bacillus Calmette-Guérin (BCG), has shown highly variable efficacy in adults against TB. New vaccines are urgently needed, and the modified vaccinia virus Ankara (MVA)-based vaccine has emerged as one of the most promising candidates based on many preclinical and early clinical studies over the past few years. However, the maximum tolerable dose and strength of induced immune responses have limited the protective effect of MVA-based prophylactic vaccines. To improve the immunogenicity of MVA-based vaccines, we introduced the tPA signal sequence in order to increase the antigen expression and secretion. Two recombinant MVA vectors expressing the Ag85B-TB10.4 fusion protein with or without tPA signal sequence were constructed and verified. Following the homologous prime-boost administration regimen in mice, levels of antigen-specific antibodies and cytokines (e.g., IFN-γ, TNF-α, IL-5, IL-6) and the percent of activated T cells were found to be significantly increased by the tPA signal sequence. However, the mean IgG2a/IgG1 ratios in the two recombinant MVA immunization groups were similar. Our present study demonstrated that the tPA signal sequence could enhance the immunogenicity of an MVA-based vaccine against TB without changing the balance of Th1 and Th2 immune responses. Thus, the tPA signal sequence may be applied to MVA-vector based vaccines for providing a better immune effect.