The crosstalk between efflux pump and resistance gene mutation in Helicobacter pylori.

Efflux pumps play a crucial role in the development of antibiotic resistance. The aim of this study was to investigate the relationship between efflux pump gene expression and resistance gene mutations in Helicobacter pylori. Twenty-six clinical strains with varying resistance characteristics were selected for further experiment. Seven susceptible strains were induced to become resistant, and the expression of efflux pump genes and point mutations were recorded. Four susceptible strains were selected to undergo candidate mutation construction, and changes in efflux pump gene expression were detected. Efflux pump knockout strains were constructed, and their effects on preventing and reversing antibiotic resistance gene mutations were assessed. Results showed that the expression of efflux pump genes hefA and hefD was significantly higher in the multidrug-resistant group compared to other groups. During the process of antibiotic-induced resistance, efflux pump gene expression did not exhibit a steady increase or decrease. Strains with the A2143G or A2142G point mutations in 23S rRNA exhibited lower hefA gene expression. Strains with mutations at 87K/91N, 87N/91 G, 87K/91D, or 87N/91Y in gyrA and the 194insertA mutation in rdxA showed higher hefA gene expression compared to the wild-type strain. During the process of antibiotic-induced resistance, the strain with the knockout of the efflux pump gene hefA developed mutations in the 23S rRNA, gyrA, or rdxA genes later compared to the wild-type strain. Knockout of the efflux pump gene could reverse the phenotypic resistance to clarithromycin or metronidazole in some strains but had no effect on reverse resistance gene mutation. This study suggested that different resistance gene point mutations may have varying effects on efflux pump gene expression. Knockout of the efflux pump gene can delay or prevent antibiotic resistance gene mutations to some extent and can reverse phenotypic resistance to clarithromycin and metronidazole in certain strains.

Identifying Qingkailing ingredients-dependent mesenchymal-epithelial transition factor-axiation "π" structuring module with angiogenesis and neurogenesis effects.

OBJECTIVE: To explore the functional role of the drug-dependent mesenchymal-epithelial transition (Met)-axiation "π" structural module of neurogenesis after processing by three components of Qingkailing injection in neurogenesis and angiogenesis in cerebral ischemia. METHODS: We used a Glutathione S-transferase (GST)-pull down assay, isothermal titration calorimetry assay, and other related methods to identify the relationships among Met, inositol polyphosphate phosphatase like 1 (Inppl1), and death associated protein kinase 3 (Dapk3) in this allosteric module. The biological effects of the modules of neurons generation composed of Met, Inppl1, and Dapk3 were measured through Western blot, apoptosis analysis, and double immunofluorescence labeling. RESULTS: The GST-pull down assay revealed that proline-serine-threonine rich domain of Met binds to the Src homology domain of Inppl1 to form a protein-protein complex; Dapk3 with a C-terminal domain interacts weakly with the protein kinase C domain of Met in the intracellular region. Thus, we obtained a "π" structuring module considered a neural regeneration module. The biological effects of angiogenesis and neurogenesis modules composed of Met, Inppl1, and Dapk3 were also verified. CONCLUSION: The study suggested that understanding the functional modules that contribute to pharmaceutics might provide novel signatures that can be used as endpoints to define disease processes under stroke or cerebral ischemia conditions.

[Evaluation of implementation effect of schistosomiasis control program in Jiangsu Province from 2010 to 2015].

OBJECTIVE: To evaluate the actual effect of the schistosomiasis control program in Jiangsu Province from 2010 to 2015. METHODS: A total of 67 schistosomiasis-endemic counties in 10 cities were selected, and a combination of retrospective investigation and on-site investigation was adopted to collect and record the epidemic data of the counties from 2010 to 2015, and a retrospective survey database of epidemic situation was established. The effects of integrated control strategies with both Oncomelania hupensis snail control and infection source control were evaluated. RESULTS: From 2010 to 2015, 2 465 911 persons who lived in endemic areas were detected for schistosomiasis, with 16 974 positive cases of blood examinations, and 8 positive cases of fecal examinations. Totally 5 145 people with advanced schistosomiasis were treated and 40 460 people with the history of schistosome cercarial-infested water contact received the expanded chemotherapy. A total of 127 636 cattle raised in the endemic areas were detected, and 51 619 cattle (head-times) with the history of cercarial-infested water contact also received the expanded chemotherapy. The area with snails control by molluscicides was 18 604.84 hm2. By the end of 2015, schistosomeinfected snails had not been found and there was no zoological schistosome infection for 5 consecutive years, and in addition, there had been no acute schistosome-infected persons for 6 consecutive years in the whole province. The area with snails dropped to 1 977.18 hm2, with a decreasing rate of 55.24% compared with that in 2010. CONCLUSIONS: After the implementation of the plan for the prevention and control of schistosomiasis in Jiangsu Province (2010-2015), the prevention and control of schistosomiasis has achieved remarkable effects and realized the goal of the plan.

[Investigation of Toxoplasma gondii infection among special populations in Nanjing City, Jiangsu Province].

OBJECTIVE: To understand the prevalence of Toxoplasma gondii infection among special populations in Nanjing City, so as to provide the reference for formulating the interventions for the prevention and control of T. gondii infections in this population. METHODS: The HIV/AIDS patients, tumor patients, pregnant women, and people with livestock and poultry breeding or processing works were selected as the study subjects from September to November 2015. The venous blood samples were collected from each participant for detecting IgG and IgM antibodies against T. gondii by ELISA. RESULTS: The overall prevalence of T. gondii infection was 10.2% in the study subjects in Nanjing City. The T. gondii infection rates were 12.2%, 11.3%, 4.0%, and 13.0% among the HIV/AIDS patients, tumor patients, pregnant women, and people with livestock and poultry breeding or processing works, respectively, and there was no statistically significant difference among the four groups (χ2 = 5.668, P = 0.130). The prevalence of T. gondii infection was higher in men than in women (15.3% vs. 5.8%; χ2 = 10.213, P = 0.001), and there were significant differences in the prevalence of T. gondii infection in terms of gender (χ2 = 9.501, P = 0.023), education levels (χ2 = 9.850, P = 0.043) or occupations (χ2 = 8.983, P = 0.062). CONCLUSIONS: The infection rate of T. gondii among the special population in Nanjing City is high. Therefore, the health education intervention should be strengthened in the follow-up work for the special population.

[Analysis of human intestinal nematode infections in Nanjing City from 2006 to 2015].

OBJECTIVE: To analyze the epidemiological situation of human intestinal nematode infections in Nanjing City from 2006 to 2015, so as to provide the reference for formulating prevention and control measures. METHODS: The surveillance data of human intestinal nematode infections in Nanjing City from 2006 to 2015 were collected and analyzed statistically. RESULTS: From 2006 to 2015, 98 804 person-times of residents were surveyed in Nanjing City, and 465 person-times of residents were detected with intestinal nematode infections. The highest infection rate was in 2006 (1.97%), and the lowest in 2013 and 2015 (both 0.05%). Moreover, the positive rate of human intestinal nematode infections showed a significantly declining trend in total ( χ2 = 552.19, P < 0.001). Meanwhile, the numbers of Ascaris lumbricoides, hookworm and Trichuris trichura cases were 329, 98 and 25 respectively, and the infection rates were 0.33%, 0.10% and 0.03% respectively. Among them, 443 cases had mild infection intensity (98.66%). There were 462 cases of single-infection (99.35%), and 3 of co-infection of two parasites (0.65%). From 2006 to 2015, 92 539 person-times of children under 12 years old were surveyed for Enterobius vermicularis infection and 352 cases were detected with E. vermicularis infection. Moreover, the positive rate showed a significantly decreasing trend in total (χ2 = 147.94, P <0.001). CONCLUSIONS: The control effect of human intestinal nematode infections in Nanjing City is remarkable. However, the surveillance and health education in key groups still should be strengthened, and the prevention and control programs should be adjusted promptly to further consolidating the effectiveness of intestinal nematode disease prevention and control.

[Epidemiological analysis of malaria prevalence in Nanjing City from 2010 to 2015].

OBJECTIVE: To analyze the epidemiological characteristics of malaria in Nanjing City, so as to provide the evidence for further formulating and adjusting the malaria prevention and control strategy. METHODS: The data of malaria situation, malaria cases and epidemiological investigations were collected from the Internet Reporting System in Nanjing City from 2010 to 2015 and analyzed statistically. RESULTS: A total of 137 confirmed malaria cases were reported in Nanjing City from 2010 to 2015, including 102 falciparum malaria cases (74.45%), 33 vivax malaria cases (24.09%), one ovale malaria case (0.73%) and one quartan malaria case (0.73%). Among the 137 malaria cases, 126 cases (91.97%) were imported from foreign countries, 2 cases (1.46%) were infected locally, and nine cases (6.57%) were imported from other provinces in China. Among the 126 overseas imported cases, 117 cases were imported from African countries and 9 from Asian countries. These malaria cases were majorly young men working as migrant workers, laborers and technical persons. About 19.30% of the cases went to hospital on onset day, and 55.65% were confirmed by medical institutions as malaria in the same day. The majority of diagnosis institutions were municipal hospitals (74.45%). CONCLUSIONS: The number of malaria cases in Nanjing City is declining year by year. The local infections are eliminating gradually. However, the situation of imported malaria from overseas is still serious. Therefore, the surveillance work and health education still should be strengthened, so as to reduce the risk of imported malaria.

[Comparison of Schistosoma japonicum development between single sexual infection and double sexual infection in mice and rabbits].

OBJECTIVE: To observe the development of Schistosoma japonicum after single sexual infection and double sexual infection respectively. METHODS: A single Oncomelania hupensis snail was infected by a single schistosome miracidium. The larva were induced and released from the snail after 60-day incubation at 26 ℃. The mice were infected by the larva (single sexual infection) and dissected 40 days after the infection. All the worms were collected and the sex of the larvae was determined by the sex of the adult worms. Then, the mice and rabbits were infected by single sex of larvae (single sexual infection) and double sex of larvae (double sexual infection) respectively. The mice and the rabbits were dissected 40 days after the infection. All the worms were collected and measured under a microscope. RESULTS: All the male or female worms were collected from the mice and rabbits after single sexual infection. There were three main forms of worms after dissection of double sexual infection of mice and rabbits: folded mature male and female, male or female. Few folded male and immature female were found. Only the double sexual larva infected mice or rabbits had schistosome eggs in the liver and the liver had typical schistosome egg nodules. The single sexual larva infected mice or rabbits had no schistosome eggs or schistosome egg nodules in the liver tissues. The single male larva could develop to worms with the testis, and with a little smaller size compared to the mature folded male, while the single sexual infection female worm could not develop to the mature stage with much thinner and smaller compared to the mature folded female. CONCLUSIONS: The male or female worms from single sexual infection are smaller than those from double sexual infection (mature worms - folded male and female). So it is necessary to check single sex worms in vessels of intestinal mucosa thoroughly in the sentinel mice when no schistosome eggs were found in the liver.

[Endemic situation of schistosomiasis at national surveillance sites in Nanjing City, 2015].

OBJECTIVE: To understand the dynamic endemic situation of schistosomiasis in Nanjing City, 2015, so as to provide evidences for policy-making of schistosomiasis control. METHODS: According to The National Scheme of Schistosomiasis Surveillance (2014), the endemic situation of schistosomiasis was monitored in 11 national surveillance sites in Nanjing. RESULTS: The positive rates of serological (IHA) and stool examinations were 2.97% (97/3 269) and 0 for local residents, and 0.52％ (12/2 298) and 0 for migrant people, respectively. No schistosome-infected livestock was found. Totally 147.295 3 hm2 area with Oncomelania hupensis snails were found, but no schistosome-infected snails were discovered. CONCLUSIONS: The endemic situation of schistosomiasis declines greatly in Nanjing City in 2015. However, the control work still should be strengthened to consolidate the achievements of schistosomiasis control.

Dynamic behavior analysis of fractional-order Hindmarsh-Rose neuronal model.

Previous experimental work has shown that the firing rate of multiple time-scales of adaptation for single rat neocortical pyramidal neurons is consistent with fractional-order differentiation, and the fractional-order neuronal models depict the firing rate of neurons more verifiably than other models do. For this reason, the dynamic characteristics of the fractional-order Hindmarsh-Rose (HR) neuronal model were here investigated. The results showed several obvious differences in dynamic characteristic between the fractional-order HR neuronal model and an integer-ordered model. First, the fractional-order HR neuronal model displayed different firing modes (chaotic firing and periodic firing) as the fractional order changed when other parameters remained the same as in the integer-order model. However, only one firing mode is displayed in integer-order models with the same parameters. The fractional order is the key to determining the firing mode. Second, the Hopf bifurcation point of this fractional-order model, from the resting state to periodic firing, was found to be larger than that of the integer-order model. Third, for the state of periodically firing of fractional-order and integer-order HR neuron model, the firing frequency of the fractional-order neuronal model was greater than that of the integer-order model, and when the fractional order of the model decreased, the firing frequency increased.

Characterization of 23S rRNA gene mutation in primary and secondary clarithromycin-resistant Helicobacter pylori strains from East China.

BACKGROUND/AIMS: Clarithromycin is an effective antibiotic for treating Helicobacter pylori; however, the development clarithromycin- resistance by multiple strains prevents the eradication of Helicobacter pylori. We aimed to characterize mutations in the 23S rRNA gene of primary clarithromycin-sensitive, primary clarithromycin-resistant and secondary clarithromycin-resistant Helicobacter pylori strains that were collected in East China and elucidate the mechanisms of clarithromycin resistance. MATERIALS AND METHODS: The disk diffusion test and E-test method were used to determine the clarithromycin susceptibility of clinical Helicobacter pylori strains. The 23S rRNA gene fragments were amplified by polymerase chain reaction from 18 primary clarithromycin- resistant strains, 15 primary sensitive strains and 8 secondary clarithromycin-resistant strains. Polymerase chain reaction-products were sequenced to determine mutations of the 23S rRNA gene. RESULTS: We found an A2143G (8 strains) mutation in primary clarithromycin-resistant strains, an A2143T (5 strains) mutation in secondary clarithromycin-resistant strains; but no mutations were found in position 2143 of sensitive strains. A T2182C mutation in primary clarithromycin-sensitive, primary clarithromycinresistant and secondary clarithromycin-resistant strains was found with a prevalence of 86.7% (13 strains), 72.2% (13 strains) or 87.5% (7 strains), respectively. In addition, we found a G2254T (8 strains) and a G2172T (7 strains) mutation in secondary clarithromycin- resistant strains. These point mutations were absent in primary clarithromycin-resistant and -sensitive strains. CONCLUSION: The gene mutation in position 2143 was associated with resistance to clarithromycin, but the mutation was different between primary and secondary clarithromycin-resistant strains. The T2182C mutation was not associated with clarithromycin resistance. Two new hotspot mutations: G2254T and G2172T, in 23S rRNA were discovered in secondary clarithromycin-resistant strains.

Reversal of cardiomyopathy in patients with congestive heart failure secondary to tachycardia.

OBJECTIVES: Tachycardia-induced cardiomyopathy (TCM) is a reversible cause of heart failure. Little is known of the characteristics of tachycardia associated with the development of left ventricular (LV) dysfunction and the reversal of cardiomyopathy after cure of tachycardia. This study aimed to examine the reversal of cardiomyopathy in patients undergoing ablation with congestive heart failure secondary to tachycardia. METHODS: A total of 625 patients underwent radiofrequency ablation for tachycardiarrhymias between January 2009 and July 2011. Echocardiography analysis was performed to identify patients with depressed LV function, defined as a left ventricular ejection fraction <50 %. Patients with preexisting structural heart disease (n = 10) were excluded. NT-pro-B-type natriuretic peptide (NT-proBNP) assessment was performed before ablation in patients considered to have TCM (n = 17). Repeated echocardiography study and NT-proBNP assessment were measured after a mean follow-up of 3 months. Levels of NT-proBNP before and after ablation were compared. Reversal of cardiomyopathy was also assessed. RESULTS: The incidence of TCM was 2.7 % (12 males; age, 35.8 ± 17.1 years). Successful ablation was performed in 16 of 17 patients (94.1 %). There was a significant improvement in left ventricular ejection fraction (36.7 ± 7.5 vs. 59.4 ± 9.7 %; P < 0.001). The mean left ventricular end-diastolic diameter before treatment was 59.5 ± 8.3 mm (range, 43 to 70), compared with 51.9 ± 7.4 mm (range, 40 to 67) (P = 0.009) after 3 months follow-up. The levels of NT-proBNP decreased after ablation procedure, from 4,092.6 ± 3,916.6 to 478.9 ± 881.9 pg/ml (P < 0.001). After successful ablation, ventricular function normalized in 15 of 17 (88.2 %) patients at a mean of 3 months. CONCLUSIONS: Restoration of LV function and reversal of LV remodeling can be achieved with successful elimination of tachycardia in the majority of patients. NT-proBNP level elevates in subjects with TCM and decreases sharply after ablation.

Helicobacter pylori vaccine: mucosal adjuvant & delivery systems.

Vaccination, especially mucosal vaccination, is considered to be effective in the management of Helicobacter pylori infections. However, most antigens alone cannot induce immune responses when administered mucosally and need to be co-administered with adjuvants or delivery systems. The current research on the mucosal adjuvant and delivery systems of vaccine against H. pylori, including advantages and disadvantages, mechanisms and applications is discussed in this review. Mutants of cholera toxin (CT) and the heat labile enterotoxin of Escherichia coli (LT), CpG oligodeoxynucleotides, biocompatible and biodegradable polymers, and live attenuated bacterial vectors may be promising adjuvant and delivery systems for H. pylori vaccine.

A DNA vaccine constructed with human papillomavirus type 16 (HPV16) E7 and E6 genes induced specific immune responses.

OBJECTIVE: Cervical cancer is found highly associated with human papillomaviruses type 16 (HPV16). HPV16 E6 and E7 oncogenes are important transforming genes which have become the main focus of anti-cervical cancer therapy. In this study, a recombinant DNA vaccine candidate, termed HPV16-DNA-E6E7, constructed with HPV16 E7 and E6 genes was generated and used to against HPV16-induced tumors. METHODS: We inserted an E7 DNA fragment into E6 gene to produce a recombinant gene (E6E7-DNA). The E6E7-DNA gene was inserted into a mammalian expression vector, pcDNA 3.1+, to construct the DNA vaccine candidate. Animals (C57BL/6 mice) were immunized with the vaccine candidate with various concentrations (50 microg, 100 microg or 200 microg, respectively), and cytotoxicity measurement and tumor protection assay were carried out to examine the immunological effects of the vaccine candidate. RESULTS: Immunization of with HPV16-E6E7-DNA induced HPV16-specific immune response and also conveyed protection against TC-1 induced tumor in vivo. A survival rate (90%) after 45 days of tumor challenge was observed. The animals injected with a higher dosage of the vaccine (200 microg) exhibited prolonged survival duration of more than 55 days. No transforming activity of the vaccine candidate was detected, as determined by focus formation and degradation of endogenous p53. CONCLUSION: Our results demonstrated that the HPV16-E6E7-DNA compound might become a candidate for HPV16 precautionary and immunotherapy.

The significance of the flexible loop in the azurin (Az-iso2) from the obligate methylotroph Methylomonas sp. strain J.

The obligate methylotroph Methylomonas sp. strain J produces two azurins (Az-iso1 and Az-iso2) as candidates for electron acceptor from methylamine dehydrogenase (MADH) in the electron-transfer process involving the oxidation of methylamine to formaldehyde and ammonia. The X-ray crystallographic study indicated that Az-iso2 gives two types of crystals (form I and form II) with polyethylene glycol (PEG4000) and ammonium sulfate as the precipitants, respectively. Comparison between the two Az-iso2 structures in forms I and II reveals the remarkable structural changes at the top surface of the molecule around the copper atom. Az-iso2 possesses Gly43 instead of Val43 or Ala43, which is unique among all other azurins around the copper ligand His46, inducing the remarkable structural change in the loop region from Gly37 to Gly43. When the structure of Az-iso2 is superimposed on that of amicyanin in the ternary complex composed of MADH, amicyanin, and cytochrome c(551), the loop of Az-iso2 deeply overlaps with the light subunit of MADH. However, the Az-iso2 molecule is probably able to avoid any steric hindrance with the cognate MADH to form the complex for intermolecular electron-transfer reaction, since the loop containing Gly43 is flexible. We discuss why the electron-transfer activity of Az-iso2 is fivefold higher than that of Az-iso1.