Rational Design of a Potent Antimicrobial Peptide Based on the Active Region of a Gecko Cathelicidin.

The emergence of multidrug-resistant (MDR) bacteria presents a significant challenge to public health, increasing the risk of infections that are resistant to current antibiotic treatment. Antimicrobial peptides (AMPs) offer a promising alternative to conventional antibiotics in the prevention of MDR bacterial infections. In the present study, we identified a novel cathelicidin AMP from Gekko japonicus, which exhibited broad-spectrum antibacterial activity against both Gram-negative and Gram-positive bacteria, with minimal inhibitory concentrations ranging from 2.34 to 4.69 µg/mL. To improve its potential therapeutic application, a series of peptides was synthesized based on the active region of the gecko-derived cathelicidin. The lead peptide (RH-16) showed an antimicrobial activity comparable to that of the parent peptide. Structural characterization revealed that RH-16 adopted an amphipathic α-helical conformation. Furthermore, RH-16 demonstrated neither hemolytic nor cytotoxic activity but effectively killed a wide range of clinically isolated, drug-resistant bacteria. The antimicrobial activity of RH-16 was attributed to the nonspecific targeting of bacterial membranes, leading to rapid bacterial membrane permeabilization and rupture. RH-16 also retained its antibacterial activity in plasma and exhibited mild toxicity in vivo. Notably, RH-16 offered robust protection against skin infection in a murine model. Therefore, this newly identified cathelicidin AMP may be a strong candidate for future pharmacological development targeting multidrug resistance. The use of a rational design approach for isolating the minimal antimicrobial unit may accelerate the transition of natural AMPs to clinically applicable antibacterial agents.

Dual blockages of a broad and potent neutralizing IgM antibody targeting GH loop of EV-As.

The reported enterovirus A 71 (EVA71) vaccines and immunoglobin G (IgG) antibodies have no cross-antiviral efficacy against other enterovirus A (EV-A) which caused hand, foot and mouth disease (HFMD). Here we constructed an IgM antibody (20-IgM) based on our previous discovery to address the resistance encountered by IgG-based immunotherapy. Although binding to the same conserved neutralizing epitope within the GH loop of EV-As VP1, the antiviral breath and potency of 20-IgM are still higher than its parental 20-IgG1. The 20-IgM blocks the interaction between the EV-As and its receptors, scavenger receptor class B, member 2 (SCARB2) and Kringle-containing transmembrane protein 1(KREMEN1) of the host cell. The 20-IgM also neutralizes the EV-As at the post-attachment stages, including postattachment neutralization, uncoating and RNA release inhibition after internalization. Mechanistically, the dual blockage effect of 20-IgM is dependent on both a conserved site targeting and high affinity binding. Meanwhile, 20-IgM provides cross-antiviral efficacy in EV-As orally infected neonatal ICR mice. Collectively, 20-IgM and its property exhibit excellent antiviral activity with a dual-blockage inhibitory effect at both the pre- and post-attachment stages. The finding enhances our understanding of IgM-mediated immunity and highlights the potential of IgM subtype antibodies against enterovirus infections.

A broad and potent IgM antibody against tetra-EV-As induced by EVA71 and CVA16 co-immunization.

OBJECTIVE: To determine the potent and broad neutralizing monoclonal antibody (mAb) against enterovirus A (EV-A) in vitro and in vivo induced by enterovirus A71(EVA71) and coxsackievirus 16 (CVA16) co-immunization. METHODS: The mAb was Generated by co-immunization with EVA71 and CVA16 through hybridomas technology. The characteristics and neutralizing ability of mAb were analysed in vitro and in mice. RESULTS: We screened three mAb, the IgM antibody M20 and IgG antibody B1 and C31. All three antibodies showed cross-reactivity against tetra-EV-As. However, M20 showed potent and broad neutralizing ability against tetra-EV-As than B1 and C31. Meanwhile, M20 provided cross-antiviral efficacy in tetra-EV-As orally infected mice. Moreover, M20 binds to a conserved neutralizing epitope within the GH loop of tetra-EV-As VP1. CONCLUSIONS: M20 and its property exhibited potent and broad antiviral activity against tetra-EV-As, and that is expected to be a potential preventive and therapeutic candidate against EV-As.

Qualitative analysis of the roots of Salvia miltiorrhiza and Salvia yunnanensis based on NIR, UHPLC and LC-MS-MS.

Near infrared spectroscopy (NIR) was applied to discriminate the roots of salvia miltiorhiza Bunge (Danshen for short) and Salvia yunnanensis C. H. Wright (Zidanshen for short) by means of principal component analysis (PCA), improved and simplified K nearest neighbors (IS-KNN). Furthermore, an ultra-high performance liquid chromatographic (UHPLC) coupled with photodiode-array detector was developed for building fingerprints of lipophilic components of Danshen and Zidanshen, respectively. Basing on NIR information, both PCA and IS-KNN method classified the two kinds of Chinese medical herbs with 100% accuracy. The chromatographic fingerprints of the lipophilic components of Danshen and Zidanshen have 10 and 12 common peaks, respectively. Liquid chromatography coupled with mass spectroscopy (LC-MS-MS) was applied to identify these peaks. Among these, three small peaks in the fingerprints of Zidanshen are not found in Danshen, one of which was identified as α-lapachone, and the other two compounds were not yet identified; a small peak after tanshinone IIA in the fingerprints of Danshen was not found in Zidanshen, which was identified as miltirone. The two herbs have 10 common lipophilic components. The similarity between the two reference chromatograms of Zidanshen and Danshen is 0.902, but the mean similaritie between Zidanshen (or Danshen) fingerprints and its own reference chromatogram is 0.973 (or 0.976). The contents of main lipophilic components are significantly lower in Zidanshen than in Danshen (P < 0.01 or P < 0.05). The results indicate that the two Chinese medical materials are not only different in NIR spectra, but also different in species and quantities of lipophilic components. NIR spectra analysis can identify Danshen and Zidanshen rapidly and accurately. UHPLC coupled with MS analysis demonstrates the detail differences between the two herbs both in species and contents of their lipophilic components.

[Analysis of lipophilic components of Salvia miltiorrhiza roots and S. yunnanensis roots by UPLC and LC-MS/MS].

Fingerprints of lipophilic components in the roots of Salvia miltiorrhiza and S.yunnanensis were analyzed by UPLC-DADand UPLC coupled with mass spectroscopy to evaluate the differences and similarities of the lipophilic components in the two kinds of herbs.The UPLC analysis of 18 batches of S.miltiorrhiza and 16 batches of S.yunnanensis was performed on a 25℃Thermo Accucore C_(18)column(2.1 mm×100 mm,2.6µm)by Shimadzu LC-20AD;mobile phase was 0.026%phosphoric acid(A)-acetonitrile(B)with gradient elution;flow rate was 0.4 m L·min~(-1);detection wavelength was set at 270 nm;injection volume was 2µL.The molecular structures of the lipophilic components were analyzed on a 25℃Thermo Accucore C_(18)column(2.1 mm×100 mm,2.6µm)by Thermo U3000 UPLC Q Exactive Orbitrap LC-MS/MS with a mobile phaseconsisting of 0.1%formic acid water(A)and 0.1%formic acidacetonitrile(B).The mass spectrometry was acquired in positive modes using ESI.There are 10 common peaks in the lipophilic components of S.miltiorrhiza.The similarity between the 16 batches of S.miltiorrhiza and their own reference spectra was greater than 0.942,and the average similarity was 0.973.There are 12 common peaks in the lipophilic components of S.yunnanensis.The similarity between the 18 batches of S.yunnanensis and their own reference spectra was greater than 0.937,and the average similarity was 0.976.The similarity between the reference chromatograms of S.miltiorrhiza and S.yunnanensis was only 0.900.There are three lipophilic components in S.yunnanensis,which are not found in S.miltiorrhiza,and one of which isα-lapachone.There is a lipophilic component in S.miltiorrhiza not found in S.yunnanensis,which may be miltirone.The two herbs contain 8 common lipophilic components including dihydrotanshinoneⅠ,cryptotanshinone,tanshinoneⅠ,tanshinoneⅡ_A,nortanshinone in which the content of tanshinoneⅡ_A,dihydrotanshinoneⅠand cryptotanshinone of S.yunnanensisis significantly lower than that of S.miltiorrhiza(P<0.01),and the contents of tanshinoneⅠand nortanshinone are significantly lower than that of S.miltiorrhiza too(P<0.05).There are significant differences in the types and contents of lipophilic components between the roots of S.miltiorrhiza and S.yunnanensis,and the similarity between the fingerprints of interspecies is much lower than that between the same species.Therefore,the roots of S.miltiorrhiza and S.yunnanensis are two kinds of herbs which are quite different in chemical compounds and compositions.

Profiling of novel microRNAs elicited by EV71 and CA16 infection in human bronchial epithelial cells using high-throughput sequencing.

Enterovirus 71 (EV71) and coxsackievirus A16 (CA16) are two major etiologic agents associated with hand, foot, and mouth disease (HFMD) worldwide. Despite that they both belong to the Enterovirus genus of the Picornaviridae family, there are many differences in the infection process of these viruses. However, the underlying mechanisms have not been elucidated. Multiple studies indicated that microRNAs (miRNAs) can play critical roles in the host-pathogen interaction. Our previous study reported that EV71 and CA16 infection leads to differential expression of miRNAs in human bronchial epithelial (16HBE) cells. Herein, we aimed to further explore the expression profile and possible roles of other differentially expressed miRNAs in 16HBE cells following EV71 and CA16 infections using high-throughput sequencing. We describe 44 novel differentially expressed miRNAs in all samples. Among these miRNAs, 7 novel differentially expressed miRNAs show an opposite expression trend during the progression of EV71 and CA16 infections. Subsequently, bioinformatics analyses, including Gene Oncology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) databases, were used to identify the biological processes, molecular functions, cellular components, and pathways involved. The top 10 significant GO and Pathway annotations indicated that 849 target genes are involved in cell development, such as nervous system development, multicellular organism development, and developmental biology. Finally, the genes identified in both the GO and Pathway analysis were used to construct a co-expression network to further identify the potential function of these co-expressed genes. Thus, our data may be beneficial in guiding further studies on the molecular mechanism of developmental regulation in HFMD pathogenesis caused by EV71 and CA16. In addition, it provided new candidate biomarkers or therapeutic targets for HFMD.

Development of a tree shrew-specific interferon-gamma assay.

Tree shrews (Tupaia belangeri) are small squirrel-like mammals closely related to primates. Due to their susceptibility to several human viruses, tree shrews have been proposed as potential animal models for the study of human viral infections. However, there are no standardized assays currently available for the detection of tree shrew-specific interferon (IFN)-γ, a major cytokine secreted during the antiviral immune response. Herein, we developed a novel enzyme-linked immunosorbent assay (ELISA) for the quantification of IFN-γ in tree shrew serum samples. Tree shrew-specific IFN-γ was expressed in Escherichia coli via fusion with glutathione S-transferase (GST-TS-IFN-γ) to obtain recombinant IFN-γ. To generate anti-IFN-γ monoclonal antibodies, mice were immunized with the GST-TS-IFN-γ recombinant fusion protein, and hybridoma cell lines were established. Similarly, anti-IFN-γ polyclonal antibodies were obtained from immunized rabbits, purified, and conjugated to horseradish peroxidase (HRP). Based on the results obtained from the antibody matching test, we optimized the monoclonal antibody (1:2000) and the HRP-conjugated polyclonal antibody (1:8000) as coating and detection antibodies, respectively. Titration curves were generated with recombinant IFN-γ to develop a sensitive sandwich ELISA; the lowest detection limit of the assay was 20 ng/mL. We also tested mitogen-stimulated tree shrew blood samples in this ELISA, and found significantly higher levels of IFN-γ in the stimulated versus the unstimulated samples. Most importantly, our ELISA system detected native IFN-γ in serum samples from 50 healthy tree shrews. We have thus developed a novel ELISA, and have demonstrated the first ELISA-based measurement of IFN-γ in tree shrew serum samples.

[Expression and purification of rabies virus glycoprotein and analysis of its specific binding capacity to memory B cells].

We aimed to express and purify three rabies virus glycoproteins with different tags and sizes. After analyzing their binding function, we wish to obtain a rabies virus glycoprotein with higher affinity and ability to specifically bind memory B cells. Experiments were carried out to express full length, as well as the ectodomain RVG by gene engineering method. Combined with the antibody of CD19 and CD27, the candidate protein labeling with fluorescence was used to analyze its binding function. Flow cytometry was used to detect the anti-rabies virus specific memory B cells in PBMCs, and confirm the binding ability between the candidate proteins and anti-rabies virus-specific memory B cells. We successfully constructed three expression vectors pGEX-5X-1-RVG, pET28a-RVG and pET30a-G. Three glycoproteins GST-RVG, His-RVG and His-G were obtained by optimized expression and purification conditions. The antigen specificity of purified GST-RVG, His-RVG and His-G were identified by Western blotting and ELISA. The affinity of these three purified glycoproteins to anti-rabies virus antibody were detected by competitive ELISA. Anti-rabies virus specific memory B cells in positive PBMCs gained from people who had ever been injected with the vaccine can be detected by flow cytometry. Thus, we got a recombinant rabies virus glycoprotein that had high-affinity and could sort antigen specific memory B cells.

Hepatitis C virus genotype diversity among intravenous drug users in Yunnan Province, Southwestern China.

BACKGROUND: Recently, high proportions (15.6%-98.7%) of intravenous drug users (IDUs) in China were found to be positive for hepatitis C virus (HCV). Yunnan Province is located in southwestern China and borders one of the world's most important opium-producing regions, thus it is an important drug trafficking route to other regions of China. METHODOLOGY/PRINCIPAL FINDINGS: Here, we assessed 100 HCV-positive plasma samples from IDUs who were enrolled through the Kunming Center for Disease Control and Prevention in 2012. HCV C/E1 fragments were PCR-amplified and sequenced. We identified eight HCV subtypes (1a, 1b, 3a, 3b, 6a, 6n, 6u and 6v), of which genotype 6 was most predominant (frequency, 47%) followed by genotypes 3 (41%) and 1 (12%). HCV subtypes 6n (30%) and 3b (29%) were most common and were identified in 59% of the IDUs. We compared HCV genotypes among IDUs in Yunnan Province with those from other regions and found that the distribution patterns of HCV genotypes in Yunnan Province were similar to those in southern China, but different from those in eastern China. However, the distribution patterns of HCV subtypes varied among Yunnan Province and southern China, despite the shared similar genotypes. A comparison of the current data with those previously reported showed that the frequency of HCV genotype 6 increased from 25% to 47% within 5 years, especially subtypes 6a (5% to 15%) and 6n (11.2% to 30%). In contrast, the frequencies of subtypes 3b and 1b decreased by almost 50% within 5 years. CONCLUSION/SIGNIFICANCE: Our results provided further information to support the assertion that drug trafficking routes influence HCV transmission patterns among IDUs in Yunnan Province. The frequency of HCV genotypes and subtypes changed rapidly among IDUs in Yunnan Province and subtypes 6a and 6n may have originated in Vietnam and Myanmar, respectively.

Passive air sampling of organochlorine pesticides and polychlorinated biphenyls in the Yangtze River Delta, China: concentrations, distributions, and cancer risk assessment.

The Yangtze River Delta (YRD) has been quickly industrialized and urbanized. Passive air sampling of organochlorine pesticides (OCPs) and polychlorinated biphenyls (PCBs) was carried out in the YRD in 2010-2011 to investigate their spatiotemporal distributions and estimate the risk of cancer from their inhalation. Annual concentrations were 151, 168, 18.8, 110, 17.9, and 35.0 pg m(-3) for HCB, ∑DDTs, ∑HCHs, ∑chlordane, mirex, and PCBs, respectively. The highest OCP and PCB concentrations were generally detected in the autumn and winter. The average concentrations of OCPs and PCBs for the different site groups followed the order urban ≈ urban-rural transition > rural. The lifetime excess cancer risks from the inhalation of OCPs and PCBs were <1.0 × 10(-6). The predicted cancer cases per lifetime associated with the inhalation of OCPs and PCBs are 12, 7, and 4 per ten thousand people for urban, urban-rural transition, and rural areas, respectively.

[Determination of organochlorine pesticide residues in soil/sediment using isotope dilution gas chromatography-mass spectrometry].

A method was developed for the determination of organochlorine pesticide (OCP) residues in soil/sediment using high resolution gas chromatography coupled with low resolution mass spectrometry. The analytical procedures consisted of Soxhlet extraction, sulfur removal with copper powder, clean-up with gel permeation chromatography (GPC) and a florisil column of solid phase extraction (SPE). The analytes were separated on an HP-5MS capillary column, detected in selected ion monitoring ( SIM) mode and quantified using internal standard calibration curves of isotope dilution technique. The linear correlations of calibration standard solutions were good for all the OCPs. The recoveries and relative standard deviations of labeled compound solutions ranged from 60% to 110% and from 1.5% to 18%, respectively. The limits of detection ranged from 0.20 to 10.3 microg/kg were established for the 23 OCPs. The method showed satisfactory clean-up effect and precision quantification. It is suitable for the determination and confirmation of pesticides in complex matrices such as soil, sediment.

[Preparation of soil candidate reference materials for the analysis of organochlorine pesticides in soils].

The preparation method of new soil candidate certified reference materials (CRM) for the analysis of organochlorine pesticides in soils has been developed. The soil sample was dried, ground, homogenized and packed. After Soxhlet extraction and Florisil purification, the organochlorine pesticides in soil candidate were further determined using gas chromatography-mass spectrometry. The results showed that the soil collected from Shenyang was an ideal soil candidate material for organochlorine pesticide analysis. This method established a foundation for the development of soil reference materials for the analysis of organochlorine pesticides.

[Development of standard gas mixture of chlorinated hydrocarbons in nitrogen].

A method for the preparation of the standard gas mixture of 6 chlorinated hydrocarbons, containing dichloromethane, 1,1-dichloroethane, 1,2-dichloroethane, chloroform, 1,1, 1-trichloroethane, and 1,1,2-trichloroethane, at the concentration of 5 micromol/mol in nitrogen was developed. The reproducibility of this method and the homogeneity and long-term stability of the standard mixture were evaluated. The results showed that all 6 chlorinated hydrocarbons have shown stability as long as 12 months and the expanded relative uncertainty of 5%. The certified value of developed standard gas agreed with the similar standard gas from Scott Specialty Gases. This research established a foundation for the analysis of volatile chlorinated hydrocarbon gases.