Vitamin C Mitigates Oxidative Stress and Tumor Necrosis Factor-Alpha in Severe Community-Acquired Pneumonia and LPS-Induced Macrophages

Oxidative stress is an important part of host innate immune response to foreign pathogens. However, the impact of vitamin C on oxidative stress and inflammation remains unclear in community-acquired pneumonia (CAP). We aimed to determine the effect of vitamin C on oxidative stress and inflammation. CAP patients were enrolled. Reactive oxygen species (ROS), DNA damage, superoxide dismutases (SOD) activity, tumor necrosis factor-alpha (TNF-α), and IL-6 were analyzed in CAP patients and LPS-stimulated macrophages cells. MH-S cells were transfected with RFP-LC3 plasmids. Autophagy was measured in LPS-stimulated macrophages cells. Severe CAP patients showed significantly increased ROS, DNA damage, TNF-α, and IL-6. SOD was significantly decreased in severe CAP. Vitamin C significantly decreased ROS, DNA damage, TNF-α, and IL-6. Vitamin C inhibited LPS-induced ROS, DNA damage, TNF-α, IL-6, and p38 in macrophages cells. Vitamin C inhibited autophagy in LPS-induced macrophages cells. These findings indicated that severe CAP exhibited significantly increased oxidative stress, DNA damage, and proinflammatory mediator. Vitamin C mitigated oxidative stress and proinflammatory mediator suggesting a possible mechanism for vitamin C in severe CAP

Electrical Transport Spectroscopy (ETS) for In Situ Probing Electrode-Electrolyte Interfaces

Electrochemistry of metallic nanostructures has played an essential role in a wide range of energy-related technologies. A fundamental understanding of the electrocatalytic surface is crucial for developing future generations of electrocatalysts with improved catalytic activity. Various spectroscopy-based methods have been applied in electrochemical surface studies, but most of these techniques are limited to ex-situ studies. In situ monitoring of catalytic interface during the electrochemical process is considered most informative yet extremely challenging. The fact that electrochemical interfaces are buried between the solid electrode and liquid electrolyte makes them difficult to access by traditional spectroscopic method. Methods that are able to circumvent these challenges often require unusual device design or highly complicated facilities.Our group specifically designed Electrical transport spectroscopy (ETS) for in situ monitor of electrochemical interfaces. When the dimension of a metallic structure decreases to the mean free path of the electrons within it, the diffusive scattering induced by the surface adsorbates can produce a significant change in resistivity. During an electrochemical cycle, the specifixally adsorbed molecules on the nanocatalysts surface could produce a detectable conductance change, providing an effective signaling pathway for in situ probing different molecular species at the electrode-electrolyte interface during the electrochemical process. With this novel nanoelectronic measurement approach, we can conduct a series of systematic investigations of nano- electrocatalysts surface chemistry for a wide range of fundamentally or practically important electrocatalytic reactions. In this thesis, I will present three examples utilizing ETS for the in-situ characterization of the electrode-electrolyte interfaces. First, we investigated competitive anionic chemisorption on the platinum surface and its relationship with ORR kinetics on Pt (Chapter 2). Nest, we acquired ETS results under controlled potential range in a serials of pH conditions, which allows to determine the pKa of hydronium adsorbed on the platinum surface for interpreting distinct hydrogen evolution reaction kinetics at different pH (Chapter 3). Lastly, we explored the ETS approach sensing applciations including their potential for the detection of hydrogen peroxide and DNA molecules (Chapter 4)

Electrical Transport Spectroscopy (ETS) for In Situ Probing Electrode-Electrolyte Interfaces

Electrochemistry of metallic nanostructures has played an essential role in a wide range of energy-related technologies. A fundamental understanding of the electrocatalytic surface is crucial for developing future generations of electrocatalysts with improved catalytic activity. Various spectroscopy-based methods have been applied in electrochemical surface studies, but most of these techniques are limited to ex-situ studies. In situ monitoring of catalytic interface during the electrochemical process is considered most informative yet extremely challenging. The fact that electrochemical interfaces are buried between the solid electrode and liquid electrolyte makes them difficult to access by traditional spectroscopic method. Methods that are able to circumvent these challenges often require unusual device design or highly complicated facilities.Our group specifically designed Electrical transport spectroscopy (ETS) for in situ monitor of electrochemical interfaces. When the dimension of a metallic structure decreases to the mean free path of the electrons within it, the diffusive scattering induced by the surface adsorbates can produce a significant change in resistivity. During an electrochemical cycle, the specifixally adsorbed molecules on the nanocatalysts surface could produce a detectable conductance change, providing an effective signaling pathway for in situ probing different molecular species at the electrode-electrolyte interface during the electrochemical process. With this novel nanoelectronic measurement approach, we can conduct a series of systematic investigations of nano- electrocatalysts surface chemistry for a wide range of fundamentally or practically important electrocatalytic reactions. In this thesis, I will present three examples utilizing ETS for the in-situ characterization of the electrode-electrolyte interfaces. First, we investigated competitive anionic chemisorption on the platinum surface and its relationship with ORR kinetics on Pt (Chapter 2). Nest, we acquired ETS results under controlled potential range in a serials of pH conditions, which allows to determine the pKa of hydronium adsorbed on the platinum surface for interpreting distinct hydrogen evolution reaction kinetics at different pH (Chapter 3). Lastly, we explored the ETS approach sensing applciations including their potential for the detection of hydrogen peroxide and DNA molecules (Chapter 4)

Having a Same Type IIS Enzyme’s Restriction Site on Guide RNA Sequence Does Not Affect Golden Gate (GG) Cloning and Subsequent CRISPR/Cas Mutagenesis

Golden gate/modular cloning facilitates faster and more efficient cloning by utilizing the unique features of the type IIS restriction enzymes. However, it is known that targeted insertion of DNA fragment(s) must not include internal type IIS restriction recognition sites. In the case of cloning CRISPR constructs by using golden gate (GG) cloning, this narrows down the scope of guide RNA (gRNA) picks because the selection of a good gRNA for successful genome editing requires some obligation of fulfillment, and it is unwanted if a good gRNA candidate cannot be picked only because it has an internal type IIS restriction recognition site. In this article, we have shown that the presence of a type IIS restriction recognition site in a gRNA does not affect cloning and subsequent genome editing. After each step of GG reactions, correct insertions of gRNAs were verified by colony color and restriction digestion and were further confirmed by sequencing. Finally, the final vector containing a Cas12a nuclease and four gRNAs was used for Agrobacterium-mediated citrus cell transformation. Sequencing of PCR amplicons flanking gRNA-2 showed a substitution (C to T) mutation in transgenic plants. The knowledge derived from this study could widen the scope of GG cloning, particularly of gRNAs selection for GG-mediated cloning into CRISPR vectors

Genome-wide Expression Profiling in Ponkan Infected by Candidatus Liberibacter asiaticus

Huanglongbing (HLB) is an economical and destructive disease of citrus in South China, such as in Guangdong, Guangxi that is caused by the bacterium Candidatus Liberibacter asiaticus. The interaction at mRNA level between pathogen and citrus (Ponkan, Citrus reticulata Blanco ) was primarily researched by Digital Gene Expression Tag Profiling. Ponkan leaves at 13 weeks and 26 weeks after HLB inoculation were used for analysis. The numbers of up-regulated genes were increased from 37% in 13 wpi (weeks post inoculation) to 64% in 26 wpi. The differentially expressed genes (DEGs) fold change increased more than 8 times from 16.7% to 87.3%. Gene ontology (GO) process molecular function enrichment analysis showed that the DEGs with oxidation reduction function increased from 4.41% to 8.48% and that DEGs responsive to stresses increased from 1.10% to 2.08%, but those related to defense responses decreased from 0.74% to 0.64%. However, those related to defense responses of down-regulated genes increased from 0.55% to 0.79%. Apparently, the expression level of resistance genes strengthened, while the defense ability of host declined along with enhanced stresses caused by HLB infection. Photosynthesis-related genes were down-regulated at both 13 wpi and 26 wpi, which indicated that HLB infection greatly reduced the citrus photosynthesis, perhaps via feedback regulation of the accumulated starches resulted from blockage of sieve tubes by the bacteria in the phloem tissue. RIN4,a negative regulator of plant immunity, was also found up-regulated by approximately 9-fold

Genome-wide Expression Profiling in Ponkan Infected by Candidatus Liberibacter asiaticus

Huanglongbing (HLB) is an economical and destructive disease of citrus in South China, such as in Guangdong, Guangxi that is caused by the bacterium Candidatus Liberibacter asiaticus. The interaction at mRNA level between pathogen and citrus (Ponkan, Citrus reticulata Blanco ) was primarily researched by Digital Gene Expression Tag Profiling. Ponkan leaves at 13 weeks and 26 weeks after HLB inoculation were used for analysis. The numbers of up-regulated genes were increased from 37% in 13 wpi (weeks post inoculation) to 64% in 26 wpi. The differentially expressed genes (DEGs) fold change increased more than 8 times from 16.7% to 87.3%. Gene ontology (GO) process molecular function enrichment analysis showed that the DEGs with oxidation reduction function increased from 4.41% to 8.48% and that DEGs responsive to stresses increased from 1.10% to 2.08%, but those related to defense responses decreased from 0.74% to 0.64%. However, those related to defense responses of down-regulated genes increased from 0.55% to 0.79%. Apparently, the expression level of resistance genes strengthened, while the defense ability of host declined along with enhanced stresses caused by HLB infection. Photosynthesis-related genes were down-regulated at both 13 wpi and 26 wpi, which indicated that HLB infection greatly reduced the citrus photosynthesis, perhaps via feedback regulation of the accumulated starches resulted from blockage of sieve tubes by the bacteria in the phloem tissue. RIN4,a negative regulator of plant immunity, was also found up-regulated by approximately 9-fold

Comparison of micro RNA (miRNA) Profiles and Some miRNA Target Gene Expression levels in Roots of Non-infected and Huanglongbing-infected Tangerine (Citrus reticulata Blanco cv.‘Sanhu’ ) Trees

Solexa sequencing was used to reveal the changes in small RNAome profile in roots of mock-inoculated (CK) and Huanglongbing bacteria-inoculated (HLB) ‘Sanhu’ tangerine (Citrus reticulata Blanco) trees. Results showed that the number of reads of both unique and total sRNAs decreased apparently in roots following infection with HLB. Distribution in length of sRNAs changed also remarkably, showing an increase in 22 nt and 21 nt small RNAs (sRNAs) and a decrease in 24 nt sRNAs in HLB-infected samples. A total of 42 known micro RNAs (miRNAs) belonging to 27 highly conserved miRNA families were identified. Comparisons showed that 33 known miRNAs exhibited a significant expression difference between CK and HLB-inoculated samples. In addition, 34 novel miRNAs, among which 24 were differentially expressed, were also identified, and their expression levels were analyzed by qRT-PCR. Three hundred and eighty five potential target genes were predicted for most of the 57 differentially expressed miRNAs. GO and KEGG annotation analysis revealed that most miRNA-target genes were those implicated in developmental process, response to stress and stimulus, transcription and protein metabolism. The characterization of the miRNAomes between the healthy and HLB infected Sanhu tangerine roots provided new insight into the involvement of miRNAs in HLB infection of citrus

Digital Gene Expression Analysis of Ponkan Mandarin (Citrus reticulata Blanco) in Response to Asia Citrus Psyllid-Vectored Huanglongbing Infection

Citrus Huanglongbing (HLB), the most destructive citrus disease, can be transmitted by psyllids and diseased budwoods. Although the final symptoms of the two main HLB transmission ways were similar and hard to distinguish, the host responses might be different. In this study, the global gene changes in leaves of ponkan (Citrus reticulata) mandarin trees following psyllid-transmission of HLB were analyzed at the early symptomatic stage (13 weeks post inoculation, wpi) and late symptomatic stage (26 wpi) using digital gene expression (DGE) profiling. At 13 wpi, 2452 genes were downregulated while only 604 genes were upregulated in HLB infected ponkan leaves but no pathway enrichment was identified. Gene function analysis showed impairment in defense at the early stage of infection. At late stage of 26 wpi, however, differentially expressed genes (DEGs) involved in carbohydrate metabolism, plant defense, hormone signaling, secondary metabolism, transcription regulation were overwhelmingly upregulated, indicating that the defense reactions were eventually activated. The results indicated that HLB bacterial infection significantly influenced ponkan gene expression, and a delayed response of the host to the fast growing bacteria might be responsible for its failure in fighting against the bacteria

Transcriptomic and physiological comparison of Shatangju (Citrus reticulata) and its late-maturing mutant provides insights into auxin regulation of citrus fruit maturation

Previous studies have shown that abscisic acid (ABA) and ethylene are involved in pulp maturation and peel coloration in the nonclimacteric citrus fruits. There are also signs indicating that other plant hormones may play some roles in citrus fruit ripening. In this study, we compared profiles of genome-wide gene expression and changes in hormones and peel pigments between fruits of Shatangju mandarin (Citrus reticulata Blanco, designated WT) and its natural mutant, Yuenongwanju (designated MT). The MT fruit matures ~2 months later than the WT fruit. Significant differences in fruit diameter, total soluble solids, titratable acid content, chlorophylls and carotenoids were detected between the fruits of the two genotypes at the sampled time points. Genome-wide transcriptome profiling showed that many genes involved in auxin and ABA metabolism and/or signaling pathways were differentially expressed between the MT and the WT fruits. Importantly, the expression of CrYUCCA8 was significantly lower and the expression of CrNCED5 was significantly higher in WT than in MT fruits at 230 and 250 DPA, respectively. In addition, the indole-3-acetic acid (IAA) level in the MT fruit was significantly higher than that in the WT counterpart, whereas a significantly lower level of ABA was detected in the mutant. Treatment of the WT fruit with exogenous IAA significantly delayed fruit maturation. Our results provide experimental evidence supporting the notion that auxin is a negative regulator of fruit maturation in citrus