The chloroplast localization of protease mediated by the potato rbcS signal peptide and its improvement for construction of photorespiratory bypasses

Location of the proteases would affect on protease stability and photorespiratory bypass pathway, while it is unsolved. Potato rbcS signal peptide was analyzed and constructed into the protease for study of their localization site. The tartronate semialdehyde reductase (EcTSR) proteins could be accurately and efficiently located in chloroplast only when this signal peptide was extended to 80 amino acids. The signal peptide would help malate synthase (CmMS) locate to the surface of chloroplast, to form granules on the outer membrane of chloroplast. The whole spectrum scanning showed that these proteins could enter chloroplast. A signal peptide named PCS1 (Peptide of self-cleavage site 1) carrying a self-cleavage site was designed, and sixteen amino acids from the blue pigment precursor protein of chloroplast positioning signal of Silene pratensis were added to the C-terminal of PCS1. Transient expression, Western blot analysis and full-spectrum scanning showed that PCS1 could locate the EcTSR to the chloroplast, after the removal of the signal peptide

Development of a sensitive nested-polymerase chain reaction (PCR) assay for the detection of Ustilago scitaminea

A species-specific polymerase chain reaction (PCR) assay was developed for rapid and accurate detection of Ustilago scitaminea, the causal agent of sugarcane smut disease. Based on nucleotide differences in the internal transcribed spacer (ITS) sequences of U. scitaminea, a pair of species-specific primers, SL1 (5`-CAGTGCACGAAAGTACCTGTGG-3`) and SR2 (5`-CTAGGGCGGTGTTCAGAAGCAC-3`) was designed by using a panel of fungal and bacterial species as controls. The primers SL1/SR2 specifically amplified a unique PCR product about 530 bp in length from U. scitaminea strains with a detecting sensitivity at 200 fg of the fungal genomic DNA in a 25 μl reaction solution. To increase sensitivity, a nested-PCR protocol was further established, which used ITS4/ITS5 as the first-round primers followed by the primer pair SL1/SR2. This protocol increased the detection sensitivity by 10,000-fold compared to the PCR method and could detect the fungal DNA as low as 20 ag. The nested-PCR detected U. scitaminea from young sugarcane leaves with no visible smut disease symptoms. The findings from this study provide a sensitive and reliable technique for the early detection of U. scitaminea, which would be useful for sugarcane quarantine and production of germ-free seedcanes.Keywords: Sugarcane, Ustilago scitaminea, nested-polymerase chain reaction (PCR), molecular detectio

Genetic diversity of Ustilago scitaminea Syd. in Southern China revealed by combined ISSR and RAPD analysis

The polymorphism and similarity relationships among 35 mating-type isolates of Ustilago scitaminea collected from Southern China were determined with random amplified polymorphic DNA (RAPD) and inter-simple  sequence repeat (ISSR) analyses. These fungal isolates were collected from 16 sugarcane cultivars including F134 that is resistant to the physiological race 1 but susceptible to the race 2 of U. scitaminea, and N: Co376  that is immune to both races 1 and 2. Unweighted pair group method with arithmetic mean (UPGMA) cluster  analysis revealed that the U. scitaminea isolates could be divided into 2 groups with a coefficient of 0.74. The  first group comprises two isolates collected from the sugarcane cultivar F134, while the remaining 33 isolates were clustered into the second group. The second group was further divided into two subgroups with most of  the isolates from Guangdong Province which clustered in the same subgroup, and all the isolates from Guangxi  and Yunnan Provinces were clustered in another subgroup. Given that the member of the second group could  infect the cultivar N:Co376, which is immune to the races 1 and 2, our results suggest that majority of U.  scitaminea in sugarcane-producing regions of Southern China may belong to or genetically similar to race 3.Key words: Ustilago scitaminea, sugarcane, inter-simple sequence repeat (ISSR), random amplified  polymorphic deoxyribonucleic acid (DNA) (RAPD), genetic diversity

RNA Helicase DDX17 Inhibits Hepatitis B Virus Replication by Blocking Viral Pregenomic RNA Encapsidation

DDX17 is a member of the DEAD-box helicase family proteins involved in cellular RNA folding, splicing, and translation. It has been reported that DDX17 serves as a cofactor of host zinc finger antiviral protein (ZAP)-mediated retroviral RNA degradation and exerts direct antiviral function against Raft Valley fever virus through binding to specific stem-loop structures of viral RNA. Intriguingly, we have previously shown that ZAP inhibits hepatitis B virus (HBV) replication through promoting viral RNA decay, and the ZAP-responsive element (ZRE) of HBV pregenomic RNA (pgRNA) contains a stem-loop structure, specifically epsilon, which serves as the packaging signal for pgRNA encapsidation. In this study, we demonstrated that the endogenous DDX17 is constitutively expressed in human hepatocyte-derived cells but dispensable for ZAP-mediated HBV RNA degradation. However, DDX17 was found to inhibit HBV replication primarily by reducing the level of cytoplasmic encapsidated pgRNA in a helicase-dependent manner. Immunofluorescence assay revealed that DDX17 could gain access to cytoplasm from nucleus in the presence of HBV RNA. In addition, RNA immunoprecipitation and electrophoretic mobility shift assays demonstrated that the enzymatically active DDX17 competes with HBV polymerase to bind to pgRNA at the 5' epsilon motif. In summary, our study suggests that DDX17 serves as an intrinsic host restriction factor against HBV through interfering with pgRNA encapsidation. IMPORTANCE Hepatitis B virus (HBV) chronic infection, a long-studied but yet incurable disease, remains a major public health concern worldwide. Given that HBV replication cycle highly depends on host factors, deepening our understanding of the host-virus interaction is thus of great significance in the journey of finding a cure. In eukaryotic cells, RNA helicases of the DEAD box family are highly conserved enzymes involved in diverse processes of cellular RNA metabolism. Emerging data have shown that DDX17, a typical member of the DEAD box family, functions as an antiviral factor through interacting with viral RNA. In this study, we, for the first time, demonstrate that DDX17 inhibits HBV through blocking the formation of viral replication complex, which not only broadens the antiviral spectrum of DDX17 but also provides new insight into the molecular mechanism of DDX17-mediated virus-host interaction

Galectin-9 contributes to the pathogenesis of atopic dermatitis via T cell immunoglobulin mucin-3

BackgroundAtopic dermatitis (AD), a common type 2 inflammatory disease, is driven by T helper (TH) 2/TH22polarization and cytokines.Galectin-9 (Gal-9), via its receptor T cell immunoglobulin- and mucin-domain-containing molecule-3 (TIM-3), can promote TH2/TH22 immunity. The relevance of this in AD is largely unclear.ObjectivesTo characterize the role of TIM-3 and Gal-9 in the pathogenesis of AD and underlying mechanisms.MethodsWe assessed the expression of Gal-9 and TIM-3 in 30 AD patients, to compare them with those of 30 healthy controls (HC) and to explore possible links with disease features including AD activity (SCORAD), IgE levels, and circulating eosinophils and B cells. We also determined the effects of Gal-9 on T cells from the AD patients.ResultsOur AD patients had markedly higher levels of serum Gal-9 and circulating TIM-3-expressing TH1 and TH17 cells than HC. Gal-9 and TIM-3 were linked to high disease activity, IgE levels, and circulating eosinophils and/or B cells. The rates of circulating TIM-3-positive CD4+ cells were positively correlated with rates of TH2/TH22 cells and negatively correlated with rates of TH1/TH17 cells. Gal-9 inhibited the proliferation and induced the apoptosis of T cells in patients with AD, especially in those with severe AD.ConclusionOur findings suggest thatGal-9, via TIM-3, contributes to the pathogenesis of AD by augmenting TH2/TH22 polarization through the downregulation of TH1/TH17immunity. This makes Gal-9 and TIM-3 interesting to explore further, as possible drivers of disease and targets of novel AD treatment

Comparison between transsylvian-transinsular and transcortical-transtemporal approach for evacuation of intracerebral hematoma

PURPOSE: Hypertensive cerebral hemorrhage in the basal ganglia is a potentially life-threatening cerebrovascular disease with high mortality. Surgical evacuation is an important treatment for intracerebral hemorrhage. However, little is reported about the comparison on the efficacy of various approaches on the prognosis. METHODS: Clinical data of 80 cases of intracerebral hemorrhage which surgically treated via transsylvian-transinsular approach or transcortical-transtemporal approach were collected. The proportion of post-surgery tracheostomy, incidence of digestive tract hemorrhage, revision surgery, the average length of hospital stay, and the six-month efficacy (defined by an ADL score) rate between these two groups were compared. RESULTS: The six-month efficacy rates were 75% and 50% in patients receiving transsylvian-transinsular and transcortical-transtemporal surgery, respectively (p<0.05). Compared to the transcortical-transtemporal group, the proportion of revision surgery was statistically significantly lower in the transsylvian-transinsular group, (p<0.05). The proportion of post-surgery tracheostomy, the incidence of digestive tract hemorrhage, and the average length of hospital stay were lower in the transsylvian-transinsular group, compared to the transcortical-transtemporal group, but no statistically significant differences were noted in them between the two groups. CONCLUSION: The transsylvian-transinsular approach for evacuation of intracerebral hematoma demonstrates limited complications, shorter length of hospital stay, and improved long-term efficacy and prognosis. These findings suggest this operative approach has potential for wider application