[Drug resistance and genomic characteristics of a strain of O139 Vibrio cholerae isolated from human bloodstream infection].

Objective: To analyze the drug resistance and genomic characteristics of a strain of serogroup O139 Vibrio cholerae producing cholera toxin isolated from the bloodstream of a person with bacteremia. Methods: The broth dilution method and automatic drug sensitivity analyzer were used to determine the antibiotic sensitivity of the strain. The complete genome sequence of the strain was obtained by using second-generation gene sequencing and nanopore sequencing. BLAST software was used for comparison and analysis with CARD, Resfinder, ISfinder, VFDB, and other databases. The drug-resistant genes, insertion sequences and virulence genes carried by the strain were identified. MEGA 5.1 software was used to construct a genetic phylogenetic tree based on the core genomic single nucleotide polymorphisms. Results: V. cholerae SH400, as the toxigenic strain, carried multiple virulence-related genes and four virulence islands. The strain was resistant to streptomycin, tetracycline and cotrimoxazole, carrying corresponding drug-resistant genes. The strain also carried IncA/C plasmid with the size of 172914 bp and contained 10 drug-resistant genes. Combined with the genomic evolutionary relationship, this study found that the drug-resistant genes and drug-resistant plasmids carried among strains showed certain aggregation. The traditional ST type of strain SH400 was ST69, and the cgMLST type was a new type highly similar to cgST-252. Conclusion: This strain of serogroup O139 V. cholerae carries the ctxAB gene, multiple drug-resistant genes and IncA/C plasmid, and there are multiple drug-resistant islands.

[Application and reflection of laboratory-based monitoring in early warning of infectious diseases].

Despite the fact that our cognition towards infectious disease prevention, the advanced technology and the economic status of the whole society has made a great progress in the last decade, the outbreak of COVID-19 pneumonia has again enabled the public to acquire more about super-challenges of infectious diseases, epidemics and the relevant preventive measurements. In order to identify the epidemic signals in early stage or even before the onset of epidemic, the data research and utilization of a series of factors related to the occurrence and transmission of infectious diseases have played a significant role in research of prevention and control during the whole period of surveillance and early warning. Laboratory-based monitoring for the etiology has always been an important part of infectious disease warning system due to pathogens as the direct cause of such diseases. China has initially established a laboratory-based monitoring and early warning system for bacterial infectious diseases based on the Chinese Pathogen Identification Network with an aim to identify pathogens, outbreaks and sources. This network has played an essential role in early detection, tracking and precise prevention and control of bacterial infectious diseases, such as plague, cholera, and epidemic cerebrospinal meningitis. This issue focuses on the function of laboratory-based monitoring during the period of early warning, prevention, and control of bacterial infectious diseases, and conducted a wide range of researches based on the analysis of the epidemic and outbreak isolates, together with field epidemiological studies and normal monitoring systems. All of these could illustrate the effect of laboratory surveillance in the infectious disease risk assessment and epidemic investigation. At the same time, we have put forward our review and expectation of scenarios about laboratory-based monitoring and early warning technologies to provide innovative thoughts for promoting a leapfrog development of infectious disease monitoring and early warning system in China.

Mechanistic Study of Macranthoside B Effects on Apoptotic Cell Death in Human Cervical Adenocarcinoma Cells.

Macranthoside B (MB) is a triterpenoid saponin extracted from Lonicera macranthoides, a traditional Chinese medicine. In the current study, we investigated the anticancer potential of MB in various cancer cells and elucidated its underlying mechanisms. MB exposure inhibited cell proliferation, induced mitochondrial membrane potential (MMP) loss, increased sub-G1 accumulation, and resulted in cleavage of caspase-3 and PARP, which are reflective of apoptosis. In HeLa cells, MB induced down-regulation of SOD2 and GPx1, phosphorylation of Akt and PDK1, and thus promoted ROS-mediated apoptosis. This was further supported by the protection of sub-G1 accumulation, MMP loss, cleavage of caspase-3 and PARP in the presence of N-acetylcysteine (NAC). Additionally, MB induced cell death via down-regulation of ubiquitin-like with PHD and ringfinger domains 1 (UHRF1) and Bcl-xL. Taken together, this study provides a new insight into the apoptosis- inducing potential of MB, and its molecular mechanisms are associated with an increase in oxidative stress and inhibition of the PDK1/Akt pathway.

[Genomic subtyping of nosocomial transmission of carbapenem resistant Klebsiella pneumoniae].

Objective: To analyze the genomic epidemiological subtyping of carbapenem resistant Klebsiella pneumoniae (CRKP) isolated from a Third-class A hospital in Zhengzhou. Methods: From December 4, 2019 to January 10, 2020, 67 strains of CRKP were isolated from the samples submitted by the clinical departments of a Third-class A teaching hospital in Zhengzhou for microbiological testing. Multi-locus sequence typing (MLST) and carbapenem resistance genes were identified by whole genome sequencing and sequence analysis. Based on the whole genome SNP, the phylogenetic tree was constructed, and 67 CRKP strains were divided into clonal groups. The isolation ward and date of each clone group were analyzed. Results: Sixty-seven CRKP strains were classified into four MLST types (STs), of which 64 were ST11. There were 62 ST11 strains carrying blaKPC-2 gene. Based on genome-wide SNP phylogenetic tree, 64 ST11 strains were divided into four clone groups, two of which were dominant clone groups, including 33 and 27 strains respectively; the other two clone groups only contained 2 strains respectively. There was no aggregation of the dominant clones in the isolation department and date. Conclusion: Multiple clonal groups of ST11 strain carrying blaKPC-2 gene are differentiated during spreading, and they can spread in parallel and independently in the same hospital.

Excessive Oxidative Stress in the Synergistic Effects of Shikonin on the Hyperthermia-Induced Apoptosis.

BACKGROUND: Hyperthermia (HT) has been used widely for cancer therapy, and the development of modern devices has made it more efficient. Shikonin (SHK) is a natural naphthoquinone derivative from a Chinese herb. Although the anticancer properties of SHK are evident, the underlying molecular mechanisms are not fully understood. OBJECTIVE: In this study, the effects of combining low doses of SHK with mild HT were investigated in the U937 cell line. METHODS: The cells were subjected to HT at 44°C for 10 min with or without SHK pretreatment, and parameters reflecting apoptosis, ROS generation and intracellular calcium elevation were evaluated by using DNA fragmentation, flow cytometry, and western blot analyses. RESULTS: SHK 0.5 µM significantly enhanced HT-induced apoptosis as indicated by DNA fragmentation and caspase-3 activation with increased generation of ROS and elevation of intracellular calcium. The combined treatment also synergistically activated proapoptotic proteins and inactivated anti-apoptotic proteins. Furthermore, the phosphorylation of JNK and PKC- δ and the dephosphorylation of ERK and AKT were the upstream effects that may have compounded the induction of apoptosis. The modulatory effects of HT and SHK were abrogated with the employment of NAC and JNK-IN-8 by inactivating the MAPK pathway and cleavage of caspase-3. Intracellular calcium was also elevated and was found to be responsible for the induction of cell death evident by the DNA fragmentation with or without the employment of BAPTA-AM. CONCLUSION: Conclusively, this study provides persuasive evidence that SHK in combination with HT is a propitious therapeutic way for augmentation of apoptosis and hence suggest a novel strategy for treating cancers.

[Study on antibiotic resistance of Escherichia coli and Enterococcus colonized in intestine of neonates from neonatal intensive care unit].

Objective: To understand the antibiotic resistance of bacteria colonized in intestine of the neonates from neonatal intensive care unit (NICU) and provide evidence to guide clinical antibiotic treatment. Methods: From May, 2014 to May, 2015, a total of 572 stool samples were collected from the neonates of NICU in our hospital. Escherichia coli and Enterococcus were detected with VITEK-2 system. Results: A total of 328 strains of E. coli and 243 strains of Enterococcus were isolated respectively in this study. The 199 strains of E. coli selected for drug susceptibility test showed lower resistant rate to imipenem, ertapenem, amikacin, nitrofurantoin, ranging from 0.50% to 3.52% and showed higher resistant rate to ampicillin, tetracycline, trimethoprim/sulfamethoxazole and cefazolin, ranging from 54.27% to 84.92%. No meropenem resistant strainsere were found. The percentage of ESBLs production strains was 45%. The multi drug resistance test showed that 34.6% of the strains were resistant to four antibiotics. Three strains were resistant to seven antibiotics. The 243 strains of Enterococcus showed lower resistant rate to quinupristin/dalfopristin, nitrofurantoin, streptomycin, ranging from 0.41% to 4.53% and showed higher resistant rate to ampicillin, benzylpenicillin, ciprofloxacin, tetracycline, gentamicin and erythromycin, ranging from 70.78% to 91.77%. No strains which were resistant to tigecycline, vancomycin, rina thiazole amine/ketone were found. The multi drug-resistance test showed that 86.5% of the strains were resistant to five antibiotics. Conclusions: According to the analysis of the 199 strains of E. coli and 243 strains of Enterococcus isolated from the neonates, we found that the resistance of intestinal bacteria in the neonates was very serious, showing multi drug resistance. It is necessary to use antibiotics according to the drug susceptibility test results in clinical treatment.

[Study on identification of Cronobacter spp. species in 11 areas in China].

OBJECTIVE: To compare different methods on the identification of Cronobacter (C.) spp. species and to choose an optimum one. METHODS: Biochemical test, 16S rRNA and fusA sequencing methods were carried out. RESULTS: When using the biochemical test, 105 strains showed six different conditions but C. turicensis and C. universalis could not be effectively identified. Under the 16S rRNA sequencing analysis, all the strains were divided into 5 groups but C. sakazakii and C. malonaticus were tangled. Finally, all the strains were identified into 58 C. sakazakii, 30 C. malonaticus, 11 C. dublinensis, 5 C. turicensis, 1 C. muytjensii, under the fusA sequencing analysis. CONCLUSION: Currently, fusA sequencing analysis seemed an effective method for identifying the species of Cronobacter. Since fusA sequencing analysis method was less intuitive, another method for rapid testing should be developed.

MIR-150 promotes prostate cancer stem cell development via suppressing p27Kip1.

OBJECTIVE: Our previous study found that high miR-150 expression was positively correlated with prostate tumor recurrence or metastasis. In this work, we investigated the expression of miR-150 in prostate cancer stem cells (CSCs) and explored its regulation over p27 in the development of CSCs. MATERIALS AND METHODS: MiR-150 expression in CD144 or CD44 positive primary prostate cells and in DU145 cell line was measured. It regulation over CSCs was measured using tumor sphere assay and qRT-PCR analysis of CSC related Oct4, Nestin and Nanog genes. The direct binding between miR-150 and 3'UTR of p27 mRNA was verified using dual luciferase, qRT-PCR and western blot assay. The influence of miR-150-p27 axis on prostate CSC properties was further investigated. RESULTS: Findings of this study found miR-150 expression was significantly upregulated in CD44+ or CD133+ subgroups of prostate cancer cells. MiR-150 could directly target 3'UTR of p27 and decrease its expression, through which it increased the number and volume of tumor sphere formed by DU145 cells, as well as the expression of CSC related Oct4, Nestin and Nanog genes. CONCLUSIONS: Increased miR-150 expression might participate in the development and progression of human prostate CSC by suppressing p27. This supported our previous study which found miR-150 was positively correlated with prostate tumor recurrence or metastasis.

Fish consumption and depressive symptoms in undergraduate students: A cross-sectional analysis.

BACKGROUND: Emerging evidence suggests that fish consumption may have beneficial effects on mood disorders. However, no study has been reported on this issue in young adults to date. The aim of this study was to investigate the relationship between fish consumption and depressive symptoms in Japanese undergraduate students. METHODS: The 20-item Center for Epidemiologic Studies Depression Scale was used to measure depressive symptoms with a cut-off score of 16. A total of 4190 completed questionnaires (from 2124 men and 2066 women) were received for analysis. RESULTS: Multivariate logistic analysis showed that fish intake was inversely associated with risk of depressive symptoms in undergraduate students. After adjustment for possible confounders, the odds-ratios (95% confidence intervals) for fish intake 1-2 times/month, 1-2 times/week, 3-4 times/week, and almost every day (compared with "almost never") were 0.78 (0.62-0.99), 0.70 (0.56-0.87), 0.67 (0.53-0.85) and 0.65 (0.46-0.92), respectively. This association tended to be stronger in women than in men. CONCLUSIONS: Frequent fish consumption in undergraduate students seems to moderate depressive symptoms. Further research is warranted to clarify the causality.

An artificially constructed radiation-responsive promoter is activated by doxorubicin.

We previously developed an artificially constructed promoter that was activated in response to X-ray irradiation in LNCap, a prostate cancer cell line. Anticancer drugs were examined to see whether some of them could stimulate the activity of the promoter. It was found that doxorubicin (Dox) treatment to LNCap transfected with a gene cassette of the luciferase gene under control of the promoter-enhanced luciferase activity in a dose-dependent manner, indicating that the promoter could be controlled by Dox. When the luciferase gene was replaced with the fcy::fur gene whose product facilitates conversion of 5-fluorocytosine into 5-fluorouracil that is highly toxic, Dox stimulated the expression of the gene product, resulting in facilitation of cell killing effect in the presence of 5-fluorocytosine. These results suggest that therapeutic gene expression controlled with an anticancer drug may lead to a more effective cancer therapy with less hazardous side effects.

Multiple phase inversion of emulsions stabilized by in situ surface activation of CaCO3 nanoparticles via adsorption of fatty acids.

The in situ surface activation of raw CaCO(3) nanoparticles by interaction with a series of sodium carboxylates of chain length between 6 and 12 as well as sodium 2-ethylhexylsulfosuccinate (AOT) was studied, and the impact of this on the stabilization and phase inversion of toluene-water emulsions was assessed. By using complementary experiments including measurement of particle zeta potentials, adsorption isotherms of amphiphile, and relevant contact angles, the mechanism of this activation was revealed. The results show that hydrophilic CaCO(3) nanoparticles can be surface activated by interaction with sodium carboxylates and AOT even if they are not surface-active themselves. Both the electrostatic interaction between the positive charges on particle surfaces and the negative charges of anionic amphiphile headgroups and the chain-chain interactions of the amphiphile result in monolayer adsorption of the amphiphile at the particle-water interface. This transforms the particles from hydrophilic to partially hydrophobic such that they become surface-active and stabilize oil-in-water O/W(1) emulsions and induce O/W(1) → water-in-oil W/O phase inversion, depending on the chain length of the carboxylate molecules. At high amphiphile concentration, bilayer or hemimicelle adsorption may occur at the particle-water surface, rendering particles hydrophilic again and causing their desorption from the oil-water interface. A second phase inversion, W/O → O/W(2), may occur depending on the surface activity of the amphiphile. CaCO(3) nanoparticles can therefore be made good stabilizers of both O/W and W/O emulsions once surface activated by mixing with traces of suitable anionic amphiphile.

The α-isoform of class II phosphoinositide 3-kinase is necessary for the activation of ERK but not Akt/PKB.

Phosphoinositide 3-kinases (PI3Ks) are key enzymes that activate intracellular signaling molecules when a number of different growth factors bind to cell surface receptors. PI3Ks are divided into three classes (I, II, III), and enzymes of each class have different tissue specificities and physiological functions. The α-isoform (PI3K-C2α) of class II PI3Ks is considered ubiquitous and preferentially activated by insulin. Our previous study showed that suppression of PI3K-C2α leads to apoptotic cell death. The aim of this study is to determine whether depletion of PI3K-C2α affects ERK or PKB/Akt activity following stimulation with serum and insulin growth factors in Chinese hamster ovary cells expressing human insulin receptors (CHO-IR) and human HepG2 liver cells. Different antisense oligonucleotides (ODNs), which were designed based on the sequence of the C2 domain of the human PI3K-C2α gene, were transfected into cells to inhibit PI3K-C2α expression. Insulin- or serum-induced stimulation of ERK was significantly suppressed by depletion of PI3K-C2α, whereas phosphorylation of IRS-1 and the stimulation of PKB/Akt by insulin were not affected. The number of apoptotic cells was also increased by depletion of PI3K-C2α protein levels. Taken together, our data indicate that PI3K-C2α may be a crucial factor in the stimulation of ERK activity in response to serum or insulin, whereas it is less important for the stimulation of PKB/Akt activity in response to insulin.

Aqueous foams stabilized by in situ surface activation of CaCO3 nanoparticles via adsorption of anionic surfactant.

The in situ surface activation of unmodified CaCO(3) nanoparticles by interaction with surfactant in aqueous media has been studied, and the impact of this on the foamability and foam stability of aqueous dispersions was assessed. Using complementary experiments including measurement of particle zeta potentials, adsorption isotherms of surfactant, air-water surface tensions, and relevant contact angles, the mechanism of this activation was revealed. The results show that the non-surface-active CaCO(3) nanoparticles cannot be surface activated by interaction with cationic or nonionic surfactants but can be surface activated by interaction with anionic surfactants such as SDS and AOT, leading to a synergistic effect in both foamability and foam stability. The electrostatic interaction between the positive charges on particle surfaces and the negative charges of anionic surfactant headgroups results in monolayer adsorption of the surfactant at the particle-water interface and transforms the particles from hydrophilic to partially hydrophobic such that particles become surface active and stabilize bubbles. SDS is a more efficient surfactant for this surface activation than AOT. Possible reasons for this difference are suggested.

Effects of surfactant structure on the phase inversion of emulsions stabilized by mixtures of silica nanoparticles and cationic surfactant.

Silica nanoparticles without any surface modification are not surface active at the toluene-water interface due to their extreme hydrophilicity but can be surface activated in situ by adsorbing cationic surfactant from water. This work investigates the effects of the molecular structure of water-soluble cationic surfactant on the surface activation of the nanoparticles by emulsion characterization, adsorption and zeta potential measurements, dispersion stability experiments, and determination of relevant contact angles. The results show that an adsorbed cationic surfactant monolayer on particle surfaces is responsible for the wettability modification of the particles. In the presence of a trace amount of cationic surfactant, the hydrophobicity of the particles increases, leading to the formation of stable oil-in-water O/W(1) emulsions. At high surfactant concentration (>cmc) the particle surface is retransformed to hydrophilic due to double-layer or hemimicelle formation, and the concentration of the free surfactant in the aqueous phase is high enough to stabilize emulsions alone. O/W(2) emulsions, probably costabilized by free surfactant and particles, are then formed. The monolayer adsorption seems to be charge-site dependent. Thus, using single-chain trimethylammonium bromide surfactants or a double-head gemini cationic surfactant, the hydrophobicity of the particles achieved is not sufficient to stabilize water-in-oil (W/O) emulsions, and no phase inversion is induced. However, using a double-chain cationic surfactant, the chain density on the particle surfaces endows them with a hydrophobicity high enough to stabilize W/O emulsions, and double phase inversion, O/W(1) --> W/O --> O/W(2), can then be achieved by increasing the surfactant concentration.

Effects of Asparagus officinalis extracts on liver cell toxicity and ethanol metabolism.

Asparagus officinalis is a vegetable that is widely consumed worldwide and has also long been used as a herbal medicine for the treatment of several diseases. Although A. officinalis is generally regarded as a supplement for the alleviation of alcohol hangover, little is known about its effects on cell metabolism. Therefore, this study was conducted to analyze the constituents of the young shoots and the leaves of asparagus and to compare their biochemical properties. The amino acid and inorganic mineral contents were found to be much higher in the leaves than the shoots. In addition, treatment of HepG2 human hepatoma cells with the leaf extract suppressed more than 70% of the intensity of hydrogen peroxide (1 mM)-stimulated DCF fluorescence, a marker of reactive oxygen species (ROS). Cellular toxicities induced by treatment with hydrogen peroxide, ethanol, or tetrachloride carbon (CCl(4)) were also significantly alleviated in response to treatment with the extracts of A. officinalis leaves and shoots. Additionally, the activities of 2 key enzymes that metabolize ethanol, alcohol dehydrogenase and aldehyde dehydrogenase, were upregulated by more than 2-fold in response to treatment with the leaf- and shoot extracts. Taken together, these results provide biochemical evidence of the method by which A. officinalis exerts its biological functions, including the alleviation of alcohol hangover and the protection of liver cells against toxic insults. Moreover, the results of this study indicate that portions of asparagus that are typically discarded, such as the leaves, have therapeutic use.

Comparison of automated ribotyping and pulsed-field gel electrophoresis for subtyping of Vibrio cholerae.

AIMS: To compare the discriminatory power of an automated ribotyping method for Vibrio cholerae subtyping with the pulsed-field gel electrophoresis (PFGE), to evaluate the possibility of automated ribotyping in use of outbreak investigations and surveillance of cholera. METHODS AND RESULTS: Eight-one epidemiologically unrelated isolates of V. cholerae, and 19 isolates from seven cholera outbreaks were used as the panels. When comparing the two methods using the epidemiologically unrelated isolates, automated ribotyping using PvuII distinguished 38 different ribotypes with a D-value of 0.8956. When combined with serotyping, the D-value is 0.9466. However, PFGE with NotI and SfiI digestions had higher D-values of 0.9951 and 0.9948, respectively. PFGE could cluster the isolates from each outbreak into the same pattern, and distinguish different patterns from different outbreaks, whereas automated ribotyping had lower discriminatory ability. CONCLUSIONS: The automated ribotyping has lower discriminatory ability compared to PFGE, and is limited to application in V. cholerae subtyping and outbreak investigation. SIGNIFICANCE AND IMPACT OF THE STUDY: The study evaluated the limitation in subtyping of automated ribotyping for V. cholerae, and raise the question of improvement for the automated ribotyping in subtyping.

Upregulated EMMPRIN/CD147 might contribute to growth and angiogenesis of gastric carcinoma: a good marker for local invasion and prognosis.

Tumour growth depends on angiogenesis, which is closely associated with vascular endothelial growth factor (VEGF) and matrix metalloproteinases (MMPs). Extracellular MMP inducer (EMMPRIN) was reported to involve in the progression of malignancies by regulating expression of VEGF and MMPs in stromal cells. To clarify the role of EMMPRIN in progression and angiogenesis of gastric carcinoma, expression of EMMPRIN, ki-67, MMP-2, MMP-9 and VEGF was examined on tissue microarray containing gastric carcinomas (n=234) and non-cancerous mucosa adjacent to carcinoma (n=85) by immunohistochemistry. Additionally, microvessel density (MVD) was assessed after labelling with anti-CD34 antibody. Extracellular MMP inducer expression was compared with clinicopathological parameters of tumours, including levels of ki-67, MMP-2, MMP-9 and vascular endothelial growth factor (VEGF), MVD as well as survival time of carcinoma patients. Gastric carcinoma cell lines (HGC-27, MKN28 and MKN45) were studied for EMMPRIN expression by immunohistochemistry and Western blot. Extracellular MMP inducer expression was gradually increased from normal mucosa to carcinomas through hyperplastic or metaplastic mucosa of the stomach (P<0.05). There was strong EMMPRIN expression in all gastric carcinoma cell lines despite different levels of glycosylation. Extracellular MMP inducer expression was positively correlated with tumour size, depth of invasion, lymphatic invasion, expression of ki-67, MMP-2, MMP-9 and VEGF of tumours (P<0.05), but not with lymph node metastasis, UICC staging or differentiation (P>0.05). Interestingly, there was a significantly positive relationship between EMMPRIN expression and MVD in gastric carcinomas (P<0.05). Survival analysis indicated EMMPRIN expression to be negatively linked to favourable prognosis (P<0.05), but not be independent factor for prognosis (P>0.05). Further analysis showed three independent prognostic factors, depth of invasion, lymphatic and venous invasion, to influence the relationship between EMMPRIN expression and prognosis. Upregulated expression of EMMPRIN possibly contributes to genesis, growth and local invasion of gastric carcinomas. Altered EMMPRIN expression might enhance growth, invasion and angiogenesis of gastric carcinoma via upregulating MMP expression of both stromal fibroblasts and gastric cancer cells and could be considered as an objective and effective marker to predict invasion and prognosis.