Involvement of C4 Protein of Beet Severe Curly Top Virus (Family Geminiviridae) in Virus Movement

Background: Beet severe curly top virus (BSCTV) is a leafhopper transmitted geminivirus with a monopartite genome. C4 proteins encoded by geminivirus play an important role in virus/plant interaction. Methods and Findings: To understand the function of C4 encoded by BSCTV, two BSCTV mutants were constructed by introducing termination codons in ORF C4 without affecting the amino acids encoded by overlapping ORF Rep. BSCTV mutants containing disrupted ORF C4 retained the ability to replicate in Arabidopsis protoplasts and in the agro-inoculated leaf discs of N. benthamiana, suggesting C4 is not required for virus DNA replication. However, both mutants did not accumulate viral DNA in newly emerged leaves of inoculated N. benthamiana and Arabidopsis, and the inoculated plants were asymptomatic. We also showed that C4 expression in plant could help C4 deficient BSCTV mutants to move systemically. C4 was localized in the cytosol and the nucleus in both Arabidopsis protoplasts and N. benthamiana leaves and the protein appeared to bind viral DNA and ds/ssDNA nonspecifically, displaying novel DNA binding properties. Conclusions: Our results suggest that C4 protein in BSCTV is involved in symptom production and may facilitate virus movement instead of virus replication

A Novel Two-Component Bacteriocin, Acidicin P, and Its Key Residues for Inhibiting Listeria monocytogenes by Targeting the Cell Membrane

ABSTRACT Listeria monocytogenes is an important pathogen which easily contaminates food and causes fatal systemic infections in human. Bacteriocins have received much attention regarding their natural methods of controlling health-related pathogens. Here, we investigated and characterized a novel two-component bacteriocin named acidicin P from Pediococcus acidilactici LAC5-17. Acidicin P showed obvious antimicrobial activity to L. monocytogenes. Through a sequence similarity network analysis for two-component bacteriocin precursors mined in the RefSeq database, acidicin P was observed to belong to an unusual group of two-component bacteriocins. Acidicin P contains two peptides designated Adpα and Adpβ which are assessed to interact with each other and form a helical dimer structure which can be inserted into the lipid bilayer of target cell membrane. We demonstrate that A5, N7, and G9 in the A5xxxG9 motif of Adpα and S16, R19, and G20 in the S16xxxG20 motif of Adpβ played crucial roles in stabilizing the helix-helix interaction of Adpα and Adpβ and were essential for the antilisterial activity of acidicin P by site-directed mutagenesis. A positive residue, R14, in Adpα and a negative residue, D12, in Adpβ are also important for acidicin P to fight against L. monocytogenes. These key residues are supposed to form hydrogen bonding, which is crucial for the interaction of Adpα and Adpβ. Furthermore, acidicin P induces severe permeabilization and depolarization of the cytoplasmic membrane and causes dramatic changes in L. monocytogenes cell morphology and ultrastructure. Acidicin P has the potential to be applied to inhibit L. monocytogenes efficiently both in the food industry and medical treatments. IMPORTANCE L. monocytogenes can cause widespread food contamination and severe human listeriosis, which amount to a large proportion of the public health and economic burdens. Today, L. monocytogenes is usually treated with chemical compounds in the food industry or antibiotics for human listeriosis. Natural and safe antilisterial agents are urgently required. Bacteriocins are natural antimicrobial peptides that have comparable narrow antimicrobial spectra and are attractive potentials for precision therapy for pathogen infection. In this work, we discover a novel two-component bacteriocin designated acidicin P, which shows obvious antilisterial activity. We also identify the key residues in both peptides of acidicin P and demonstrate that acidicin P is inserted into the target cell membrane and disrupts the cell envelop to inhibit the growth of L. monocytogenes. We believe that acidicin P is a promising lead for further development as an antilisterial drug

Analysis of Pharmaceutical Processes in Chinese Medicine Based on Quantity and Measure Value Transfer: A Case of <i>Salvia miltiorrhiza</i> Purified Extract Preparation

Quantity and measure value transfer is widely used to examine the correlation between the quality of Chinese herbs, Chinese herbal intermediates, and Chinese patent medicines. This study performed a quantity and measure value transfer analysis to assess the total solids yield, phenolic component yield, and phenolic component purity in the Salvia miltiorrhiza purified extract (SMPE) preparation process. The amount of extracted total solids was between 45–250 mg/g following the processes of reflux extraction, vacuum concentration, lime–sulfuric acid refining, first ethanol precipitation, second ethanol precipitation, first acidification, alkalization, thermal hydrolysis, and second acidification. Regarding yield and purity, Danshensu ranked first among all phenolic components. Additionally, a quantitative index defined as the total variation (TV) value was proposed to describe the consistency of the SMPE preparation process. The batch-to-batch variation in the SMPE came from the variable in herb quality and the preparation process, and the latter contributed more. The contribution of individual processes to the total variation (TVP) was proposed as an index to measure the impact of processes on batch-to-batch consistency. According to the TVP value, the lime–sulfuric acid refining process, the first ethanol precipitation process, and the second acidification process were all deemed crucial. When the similarity algorithms for the composition of the intermediates and SMPE were examined, the Euclidean distance outperformed the Pearson correlation coefficient, Spearman correlation coefficient, and cosine of the pinch angles. Based on the variation in the average Euclidean distance (ΔDj¯) during the process, the second ethanol precipitation, alkalization, and thermal hydrolysis processes were determined to be critical. This study clarified the primary causes of extract quality fluctuation, identified the critical processes, and examined the pharmaceutical process of traditional Chinese medicine (TCM) from the standpoint of quantity and measure value transfer. The method can be used as a reference for the analysis of other TCM pharmaceutical processes

Etiology and prognosis of acute viral encephalitis and meningitis in Chinese children: a multicentre prospective study

Abstract Background In China, there were few studies about the pathogens of acute viral encephalitis and meningitis in children in recent years. The aims of this study were to characterize the etiology and prognosis of acute viral encephalitis and meningitis in Chinese children. Methods This was a multicentre prospective study. Two hundred and sixty one viral encephalitis patients and 285 viral meningitis patients were enrolled. The mean age of viral encephalitis and meningitis were 5.88 ± 3.60 years and 6.39 ± 3.57 years, respectively. Real-time reverse transcription PCR and multiplex PCR were used to detect human enteroviruses and herpes viruses in cerebrospinal fluid (CSF) of patients with encephalitis or meningitis. The enzyme-linked immune absorbent assay (ELISA) was used for detecting IgM antibody against Japanese encephalitis virus (JEV) in CSF and against mumps virus, tick-borne encephalitis virus (TBEV), dengue virus and rubella virus in acute serum. The clinical and outcome data were collected during patients’ hospitalization. Results The etiology of viral encephalitis was confirmed in 52.5% patients. The primary pathogen was human enteroviruses (27.7%) in viral encephalitis. The incidence of sequelae and the fatality rate of viral encephalitis with confirmed etiology were 7.5% and 0.8%, respectively. The etiology of viral meningitis was identified in 42.8% cases. The leading pathogen was also human enteroviruses (37.7%) in viral meningitis. The prognosis of viral meningitis was favorable with only 0.7% patients had neurological sequelae. Conclusions Human enteroviruses were the leading cause both in acute viral encephalitis and viral meningitis in children. The incidence of sequelae and fatality rate of viral encephalitis with confirmed etiology were 7.5% and 0.8%, respectively. The prognosis of viral meningitis was favorable compared to viral encephalitis

New Insights into the Cellular Toxicity of Carbon Quantum Dots to Escherichia coli

In this study, the cytotoxicity and toxic mechanism of carbon quantum dots (CQDs) to E. coli were evaluated in vitro. The synthetic CQDs were extremely small in size (~2.08 nm) and displayed strong fluorescence. The results demonstrated that CQDs showed good biocompatibility with E. coli within a short culture time. However, when the exposure time exceeded 24 h, the toxicity of CQDs became apparent, and the contents of reactive oxygen species, lactate dehydrogenase, and the crystal violet absorption rate increased significantly. To further explore the cytotoxic mechanism, approaches including confocal laser scanning microscopy, scanning electron microscopy, and biological transmission electron microscopy combined with zeta potential tests, osmotic pressure measurement, and comet assays were performed. On the one hand, the CQDs altered the surface charges of cells and induced lipid peroxidation by adhesion on the surface of E. coli, leading to an increase in the permeability of the cell wall. On the other hand, when the concentration of CQDs reached 200 &micro;g/mL, the osmotic pressure of the extracellular environment was significantly reduced. These are the main factors that lead to cell edema and death. Finally, the comet assays confirmed that CQDs could induce DNA damage, which could inhibit the proliferation of E. coli

Viral DNA analysis in <i>N. benthamiana</i> leaf discs and Arabidopsis protoplasts infected with wild-type and mutated BSCTV.

<p>(A) Southern blot analysis of total nucleic acids extracted from leaf discs at 0, 5, 10, 15d after agro-inoculated with wild-type and mutated BSCTV. (B) Southern blot analysis of total nucleic acids extracted from Arabidopsis protoplasts at 0 and 4d after transfection of plasmids containing wild-type and mutated BSCTV.</p