First identification of multidrug-resistant Acinetobacter bereziniae isolates harboring bla NDM-1 from hospitals in South China.

This study is a first report on the identification of multidrug-resistant (MDR) Acinetobacter bereziniae among non-baumannii acinetobacters that had previously escaped automated laboratory detection, and characterize their clinical courses of infection at two tertiary-care hospitals in Shenzhen city, China (2015-2017). Herein, definitive identification by PCR was performed with universal and species-specific primers targeting 16S rDNA and rpoB genes, respectively, followed by Sanger sequencing and blast analysis. Antimicrobial susceptibility of A. bereziniae isolates was assessed accordingly. Three of the five identified A. bereziniae isolates exhibited carbapenem-resistance and were subjected to a multiplex PCR assay to detect drug-resistance genes. Sequences of the rpoB amplicon were aligned with curated sequences from global databases for phylogenetic analysis on evolutionary relations. Five clinical isolates of A. bereziniae were thereby re-identified, whose infections were primarily nosocomial. Automated identification and susceptibility testing systems (Phoenix-100 and VITEK 2) proved insufficient for discriminating A. bereziniae from other acinetobacters such as Acinetobacter baumannii and Acinetobacter guillouiae. Among these isolates, three exhibited carbapenem-resistant phenotypes indistinguishable from that of carbapenem-resistant A. baumannii. The carbapenem-resistant A. bereziniae isolates were subsequently confirmed to carry a bla NDM-1 (New Delhi metallo-ß-lactamase-1) gene downstream of ISAba125. Phylogenetic analysis revealed that A. bereziniae isolates evolved slowly but independently in local habitats. A. bereziniae isolates are difficult to distinguish by traditional automated detection systems. PCR-based identification via amplification and sequencing of selected house-keeping genes provides sufficient resolution for discriminating the isolates.

IFI44L as a novel epigenetic silencing tumor suppressor promotes apoptosis through JAK/STAT1 pathway during lung carcinogenesis.

Numerous evidence showed that the occurrence and development of lung cancer is closely related to environmental pollution. Therefore, new environmental response predictive markers are urgently needed for early diagnosis and screening of lung cancer. Interferon-induced protein 44-like (IFI44L) has been shown to be related in a variety of tumors, but its function and mechanism during lung carcinogenesis still have remained largely unknown. In this study, gene expression and methylation status were analyzed through online tools and malignant transformation models. Differentially expressed cell models and xenograft tumor models were established and used to clarify the gene function. RT-qPCR, western blotting, immunohistochemistry, and co-immunoprecipitation (Co-IP) were used to explore the mechanism. Results showed that IFI44L was dramatically downexpressed during lung carcinogenesis, and its low expression may be attributed to DNA methylation. Overexpression of IFI44L obviously inhibited cell growth and promoted apoptosis. After knockdown of IFI44L expression, the proliferation ability was remarkably increased and the apoptosis was significantly reduced. Functional enrichment showed that IFI44L was involved in apoptosis and JAK/STAT1 signaling pathway, and was highly correlated with downstream molecules. After overexpression of IFI44L, the expression of P-STAT1 and downstream molecules XAF1, OAS1, OAS2 and OAS3 were significantly increased. After knockdown of STAT1 expression, the pro-apoptotic effect of IFI44L was reduced. Co-IP results showed that IFI44L had protein interaction with STAT1. Results proved that IFI44L promoted STAT1 phosphorylation and activated the JAK/STAT1 signaling pathway by directly binding to STAT1 protein, thereby leading to cell apoptosis. Our study revealed that IFI44L promotes cell apoptosis and exerts tumor suppressors by activating the JAK/STAT1 signaling pathway. It further suggests that IFI44L has clinical therapeutic potential and may be a promising biomarker during lung carcinogenesis.

Computational elucidation of the binding mechanisms of curcumin analogues as bacterial RecA inhibitors.

Antimicrobial resistance (AMR) presents as a serious threat to global public health, which urgently demands action to develop alternative antimicrobial strategies with minimized selective pressure. The bacterial SOS response regulator RecA has emerged as a promising target in the exploration of new classes of antibiotic adjuvants, as RecA has been implicated in bacterial mutagenesis and thus AMR development through its critical roles in error-prone DNA repair. The natural product curcumin has been reported to be an effective RecA inhibitor in several Gram-negative bacteria, but details on the underlying mechanisms are wanting. In order to bridge the gap in how curcumin operates as a RecA inhibitor, we used computational approaches to model interactions between RecA protein and curcumin analogues. We first identified potential binding sites on E. coli RecA protein and classified them into four major binding pockets based on biological literature and computational findings from multiple in silico calculations. In docking analysis, curcumin-thalidomide hybrids were predicted to be superior binders of RecA compared with bis-(arylmethylidene)acetone curcumin analogues, which was further confirmed by MMGBSA calculations. Overall, this work provides mechanistic insights into bacterial RecA protein as a target for curcumin-like compounds and offers a theoretical basis for rational design and development of future antibiotic adjuvants.

Solvent-Controlled Synthesis of Highly Luminescent Carbon Dots with a Wide Color Gamut and Narrowed Emission Peak Widths.

Carbon dots (CDs) have tremendous potential applications in bioimaging, biomedicine, and optoelectronics. By far, it is still difficult to produce photoluminescence (PL) tunable CDs with high quantum yield (QY) across the entire visible spectrum and narrow the emission peak widths of CDs close to those of typical quantum dots. In this work, a series of CDs with tunable emission from 443 to 745 nm, quantum yield within 13-54%, and narrowed full width at half maximum (FWHM) from 108 to 55 nm, are obtained by only adjusting the reaction solvents in a one-pot solvothermal route. The distinct optical features of these CDs are based on their differences in the particle size, and the content of graphitic nitrogen and oxygen-containing functional groups, which can be modulated by controlling the dehydration and carbonization processes during solvothermal reactions. Blue, green, yellow, red, and even pure white light emitting films (Commission Internationale de L'Eclairage (CIE)= 0.33, 0.33, QY = 39%) are prepared by dispersing one or three kinds of CDs into polyvinyl alcohol with appropriate ratios. The near-infrared emissive CDs are excellent fluorescent probes for both in vitro and in vivo bioimaging because of their high QY in water, long-term stability, and low cytotoxicity.

Design, synthesis and insecticidal activity of novel hydrocarbylidene nitrohydrazinecarboximidamides.

BACKGROUND: Neonicotinoids, among the most important pesticides in recent decades, have played pivotal roles in controlling agricultural pests. However, the toxicity to some environmentally beneficial insects is a cause of concern. The development of novel insecticides safer to these insects is increasingly urgent. RESULTS: A novel series of hydrocarbylidene nitrohydrazinecarboximidamides were designed and synthesised, starting from S-methylisothiourea sulphate. Preliminary bioassays showed that the target molecules exhibited good activities against Lipaphis erysimi (turnip aphid) and Myzus persicae. As shown by initial insecticidal activity data, most of the target compounds had moderate to excellent activities at a concentration of 600 mg L-1 against L. erysimi, and the lethal rate of most compounds exceeded 90%. They were also highly effective against M. persicae. Some of them have shown excellent insecticidal activities, for example, the LC50 values of compounds Ie-02 to Ie-07 were found to be 3.8, 3.0, 2.5, 3.1, 4.1 and 4.0 mg L-1 respectively. CONCLUSION: Structure-activity relationship analysis indicated that a suitable flexible alkyl chain at the imine point and a Cl-substituted pyridine ring are the most crucial factors affecting the activity. © 2017 Society of Chemical Industry.

Facile synthesis of red-emitting carbon dots from pulp-free lemon juice for bioimaging.

In this work, red-emitting carbon dots (R-CDs) with a high quantum yield (QY) of 28% in water were synthesized for the first time by heating an ethanol solution of pulp-free lemon juice. The obtained R-CDs were mono-dispersed with an average diameter of 4.6 nm, and exhibited excitation-independent emission at 631 nm. Meanwhile, these R-CDs featured low cytotoxicity and good photostability, which allow R-CDs to be employed as luminescent probes for in vitro/in vivo bioimaging. In addition, a detailed study on the physical properties and structural compositions of the sodium borohydride (NaBH4) reduced R-CDs with orange emission suggested that surface states on the R-CD surfaces and nitrogen-derived structures in the R-CD cores synergistically caused their intense red luminescence. The low-cost and eco-friendly synthesis method and favorable optical properties of R-CDs make these carbon dots promising for further applications, such as bioimaging and light-emitting diodes.

Enhancement of Gleditsia sinensis gum rheological properties with pressure cell treatment in semi-solid state.

The apparent viscosity, molecular weight, and molecular weight distribution are important physical properties that determine the functional properties of galactomannan gum. Gleditsia sinensis gum (GSG) in semi-solid state was pressure cell treated over a range of temperature (30-110 °C) under nitrogen maintained at a pressure of 1.0-4.0 MPa. Physicochemical properties of GSG samples both before and after the pressure cell treatment were characterized. These include measurements of rheological properties by LVDV-III Ultra Rheometer, molecular weight and radius of gyration by light scattering, and changes in surface morphology by scanning electron microscopy. GSG had the highest apparent viscosity at a treatment temperature of 30 °C; further increase in temperature led to decrease in apparent viscosity. The apparent viscosity of GSG can be efficiently improved at room temperature and low pressure. The process of pressure cell treatment of GSG in semi-solid state could be industrialized for enhancement of rheological properties of galactomannan gum.

Molecular analysis of DNA repair gene methylation and protein expression during chemical-induced rat lung carcinogenesis.

A defective ratio between DNA damage and repair may result in the occurrence of a malignant phenotype. Previous studies have found that many genetic alterations in DNA repair genes occur frequently in lung cancer. However, the epigenetic mechanisms underlying this tumorigenesis are not clear. Herein, we have used a chemical-induced rat lung carcinogenesis model to study the evolution of methylation alterations of DNA repair genes BRCA1, ERCC1, XRCC1, and MLH1. Methylation-specific PCR and immunohistochemistry were used to analyze gene methylation status and protein expression during the progression of lung carcinogenesis. Promoter hypermethylation of BRCA1 was only detected in three samples of infiltrating carcinoma. CpG island hypermethylation of ERCC1, XRCC1, and MLH1 was found to increase gradually throughout lung carcinogenesis progression. Both the prevalence of at least one methylated gene and the average number of methylated genes were heightened in squamous metaplasia and dysplasia compared with normal tissue and hyperplasia, and was further increased in carcinoma in situ (CIS) and infiltrating carcinoma. Immunohistochemical analysis showed that BRCA1 and MLH1 protein expression decreased progressively during the stages of lung carcinogenesis, whereas ERCC1 and XRCC1 expression were only found in later stages. Although methylation levels were elevated for ERCC1 and XRCC1 during carcinogenesis, an inverse correlation with protein expression was found only for BRCA1 and MLH1. These results suggest that a continuous accumulation of DNA repair gene hypermethylation and the consequent protein alterations might be a vital molecular mechanism during the process of multistep chemical-induced rat lung carcinogenesis.

Aberrant methylation accounts for cell adhesion-related gene silencing during 3-methylcholanthrene and diethylnitrosamine induced multistep rat lung carcinogenesis associated with overexpression of DNA methyltransferases 1 and 3a.

To evaluate the significance of alterations in cell adhesion-related genes methylation during lung multistep carcinogenesis induced by the genotoxic carcinogens 3-methylcholanthrene (MCA) and diethylnitrosamine (DEN), tissue samples microdissected from MCA/DEN-induced rat lung carcinogenesis model were subjected to methylation-specific PCR to evaluate the DNA methylation status of CADM1, TIMP3, E-cadherin and N-cadherin. Immunohistochemistry was used to determine protein expression of CADM1, TIMP3, N-cadherin and the DNA methyltransferases (DNMTs) 1, 3a and 3b. E-cadherin hypermethylation was not detected in any tissue. CADM1, TIMP3 and N-cadherin hypermethylation was correlated with the loss of their protein expression during the progression of pathologic lesions. The prevalence of DNA methylation of at least one gene and the average number of methylated genes increased with the histological progression. DNMT1 and DNMT3a protein expression increased progressively during the stages of lung carcinogenesis, whereas DNMT3b overexpression was only found in several samples. Furthermore, DNMT1 protein expression levels were correlated with CADM1 methylation, and DNMT3a protein expression levels were correlated with CADM1, TIMP3 and N-cadherin methylation. The average number of methylated genes during carcinogenesis was significantly correlated with DNMT1 and DNMT3a protein expression levels. Moreover, mRNA expression of CADM1 significantly increased after treatment with DNMT inhibitor 5-aza-2'-deoxycytidine in CADM1-methylated primary tumor cell lines. Our findings suggest that an accumulation of hypermethylation accounts for cell adhesion-related gene silencing is associated with dynamic changes in the progression of MCA/DEN-induced rat lung carcinogenesis. We suggest that DNMT1 and DNMT3a protein overexpression may be responsible for this aberrant DNA methylation.

CpG island hypermethylation of multiple tumor suppressor genes associated with loss of their protein expression during rat lung carcinogenesis induced by 3-methylcholanthrene and diethylnitrosamine.

The epigenetic mechanisms underlying the tumorigenesis caused by polycyclic aromatic hydrocarbons and nitrosamine compounds such as 3-methylcholanthrene (MCA) and diethylnitrosamine (DEN) are currently unknown. We reported previously that dynamic changes in DNA methylation occurred during MCA/DEN-induced rat lung carcinogenesis. Here, we used the same animal model to further study the evolution of methylation alterations in tumor suppressor genes (TSGs) DAPK1, FHIT, RASSF1A, and SOCS-3. We found that none of these genes were methylated in either normal or hyperplasia tissue. However, as the severity of the cancer progressed through squamous metaplasia and dysplasia to carcinoma in situ (CIS) and infiltrating carcinoma, so methylation became more prevalent. Particularly dramatic increases in the level of methylation, the average number of methylated genes, and the incidence of concurrent methylation in three genes were observed in CIS and infiltrating carcinoma. Similar but less profound changes were seen in squamous metaplasia and dysplasia. Furthermore, methylation status was closely correlated to loss of protein expression for these genes, with protein levels markedly declining along the continuum of carcinogenesis. These results suggest that progressive CpG island hypermethylation leading to inactivation of TSGs might be a vital molecular mechanism in the pathogenesis of MCA/DEN-induced multistep rat lung carcinogenesis.

Epigenetic silencing of cell cycle regulatory genes during 3-methylcholanthrene and diethylnitrosamine-induced multistep rat lung cancer.

The rat lung cancers induced by 3-methylcholanthrene (MCA) and diethylnitrosamine (DEN) are considered to be a good model for illustrating genetic alterations in human lung precancerous and cancerous lesions. Recently, we had reported that the model can also be used to investigate the step-by-step dynamic changes in DNA methylation during lung carcinogenesis. In this study, we have used the same animal model to further study the evolution of methylation alterations of cell cycle regulatory genes CDKN1B (p27) and CDKN1C (p57). Our results showed epigenetic alterations in p27 and p57. Promoter hypermethylation of p27 was detected in one sample of carcinoma in situ (CIS) and two samples of infiltrating carcinoma, all three of which lacked expression of the p27 protein. Methylation of the p57 promoter correlated with the loss of protein expression in lung pathologic lesions, with a gradual increase in methylation frequency from 0 sample in the normal epithelium and hyperplasia, to 11.1% in squamous metaplasia, 18.9% in dysplasia, 26.7% in CIS, and finally 36.0% in infiltrating carcinoma samples. Immunohistochemical analysis showed that p27 and p57 protein expression decreased as lung carcinogenesis progressed. Moreover, weak expression of p27 and p57 in methylated primary tumor cell lines increased markedly after treatment with 5-aza-2'-deoxycytidine (5-aza-dC), confirming that methylation was indeed responsible for the gene downregulation. These results suggest that the progression of rat lung carcinogenesis induced by MCA/DEN is associated with dynamic changes in promoter hypermethylation of cell cycle regulatory genes, including p27 and p57, accounting for their defective expression.

Dynamic changes in DNA methylation during multistep rat lung carcinogenesis induced by 3-methylcholanthrene and diethylnitrosamine.

3-methylcholanthrene (MCA) and diethylnitrosamine (DEN) are typical genotoxic carcinogens that can induce tumors in a variety of human and rodent tissues. However, the epigenetic mechanisms underlying their tumorigenesis are unclear. In this study we used a MCA/DEN-induced multistep lung carcinogenesis rat model to study the evolution of alterations in DNA methylation. Rats were treated with a single dose of MCA and DEN in iodized oil by left intra-bronchial instillation. The animals were killed on days 15, 35, 55, 65 and 75 and samples of various pathological phases during carcinogenesis were obtained on these days. The status of global methylation was analyzed for each sample using a monoclonal antibody specific for 5-methycytosine (5-mC) and quantified by image analysis software. We found that the degree of global methylation was, in general, higher in basal cells compared to luminal cells of normal, precancerous and tumor tissues. The combined 5-mC scores of different types of tissues decreased gradually during the progression of carcinogenesis. We also used methylation-sensitive arbitrarily primed PCR (MS-AP-PCR) to screen a total of eight differentially methylated DNA fragments in both precancerous and tumor tissues isolated using laser capture microdissection (LCM), and observed that both unique hypomethylation and hypermethylation fragments coexist after exposure to genotoxic carcinogens. Remarkably, epigenetic alterations in p16 (CDKN2A), but not in p15 (CDKN2B), were observed, and these correlated with the presence of pathologic lung lesions and loss of p16 protein expression. Moreover, defective expression of p16 in methylated primary tumor cell lines recovered markedly after treated with 5-aza-2'-deoxycytidine (5-aza-dC). These results suggest that DNA methylation alterations are an early event in tumorigenesis and play an important role during MCA/DEN-induced multistep rat lung carcinogenesis.

Development of a semi-quantitative food frequency questionnaire to determine variation in nutrient intakes between urban and rural areas of Chongqing, China.

Nationwide surveys of food and nutrient intake in China have revealed geographical variation between urban and rural areas. This study developed a semi-quantitative food frequency questionnaire (SQFFQ) for cancer risk assessment suitable for both urban and rural populations by conducting a survey of food intake in Chongqing, China. We recruited 100 urban and 104 rural healthy residents aged from 35 to 55 years in Chongqing, and collected dietary data with 3-day weighed records to assist in the development of the SQFFQ. The intake of 35 nutrients was calculated according to Standard Food Composition Tables for China and Japan. For each nutrient estimated by percentage contribution analysis (CA) and multiple regression analysis (MRA), foods with up to a 90% contribution or a 0.90 cumulative R(2) were selected as items for SQFFQs. The food items of the combined SQFFQ were selected from all items listed in either urban or rural SQFFQs. Mean intake of energy, protein and carbohydrate did not differ between the urban and rural residents. The latter consumed more fat than their urban counterparts. We selected 119 food items for the combined SQFFQ, comprising 22 specific items for the urban SQFFQ, 6 for the rural, and 78 common and 13 additional items. The combined SQFFQ covered 33 nutrients with up to a 90% contribution in each area. We were able to develop a data-based SQFFQ that can estimate nutrient intake of both urban and rural populations, with suitable coverage rates. Further reliability and reproducibility tests are now needed to assess its applicability.