Population analysis of heavy metal and biocide resistance genes in Salmonella enterica from human clinical cases in New Hampshire, United States.

Microbes frequently encounter heavy metals and other toxic compounds generated from natural biogeochemical processes and anthropogenic activities. Here, we analyzed the prevalence and association of genes conferring resistance to heavy metals, biocides, and antimicrobial compounds in 394 genome sequences of clinical human-derived S. enterica from New Hampshire, USA. The most prevalent was the gold operon (gesABC-golTSB), which was present in 99.2% of the genomes. In contrast, the other five heavy metal operons (arsenic, copper, mercury, silver, tellurite) were present in 0.76% (3/394)-5.58% (22/394) of the total population. The heavy metal operons and three biocide resistance genes were differentially distributed across 15 sequence types (STs) and 16 serotypes. The number of heavy metal operons and biocide resistance genes per genome was significantly associated with high number of antimicrobial resistance (AMR) genes per genome. Notable is the mercury operon which exhibited significant association with genes conferring resistance to aminoglycosides, cephalosporins, diaminopyrimidine, sulfonamide, and fosfomycin. The mercury operon was co-located with the AMR genes aac(3)-IV, ant(3")-IIa, aph(3')-Ia, and aph(4)-Ia, CTX-M-65, dfrA14, sul1, and fosA3 genes within the same plasmid types. Lastly, we found evidence for negative selection of individual genes of each heavy metal operon and the biocide resistance genes (dN/dS < 1). Our study highlights the need for continued surveillance of S. enterica serotypes that carry those genes that confer resistance to heavy metals and biocides that are often associated with mobile AMR genes. The selective pressures imposed by heavy metals and biocides on S. enterica may contribute to the co-selection and spread of AMR in human infections.

Diastereoselective Synthesis of Chromeno[3,2-d]isoxazoles via Brønsted Acid Catalyzed Tandem 1,6-Addition/Double Annulations of o-Hydroxyl Propargylic Alcohols.

A Brønsted acid catalyzed tandem process to access densely functionalized chromeno[3,2-d]isoxazoles with good to excellent yields and diastereoselectivities was disclosed. The procedure is proposed to involve a 1,6-conjugate addition/electrophilic addition/double annulations process of alkynyl o-quinone methides (o-AQMs) in situ generated from o-hydroxyl propargylic alcohols with nitrones. Mild conditions, good functional group compatibility, easy scale-up of the reaction, and further product transformation demonstrated its potential application.

Genome characteristics of clinical Salmonella enterica population from a state public health laboratory, New Hampshire, USA, 2017-2020.

BACKGROUND: The implementation of whole genome sequencing (WGS) by PulseNet, the molecular subtyping network for foodborne diseases, has transformed surveillance, outbreak detection, and public health laboratory practices in the United States. In 2017, the New Hampshire Public Health Laboratories, a member of PulseNet, commenced the use of WGS in tracking foodborne pathogens across the state. We present some of the initial results of New Hampshire's initiative to transition to WGS in tracking Salmonella enterica, a bacterial pathogen that is responsible for non-typhoidal foodborne infections and enteric fever. We characterize the population structure and evolutionary history of 394 genomes of isolates recovered from human clinical cases in New Hampshire from 2017 to 2020. RESULTS: The New Hampshire S. enterica population is phylogenetically diverse, consisting of 78 sequence types (ST) and 67 serotypes. Six lineages dominate the population: ST 11 serotype Enteritidis, ST 19 Typhimurium, ST 32 Infantis, ST 118 Newport, ST 22 Braenderup, and ST 26 Thompson. Each lineage is derived from long ancestral branches in the phylogeny, suggesting their extended presence in the region and recent clonal expansion. We detected 61 genes associated with resistance to 14 antimicrobial classes. Of these, unique genes of five antimicrobial classes (aminocoumarins, aminoglycosides, fluoroquinolones, nitroimidazoles, and peptides) were detected in all genomes. Rather than a single clone carrying multiple resistance genes expanding in the state, we found multiple lineages carrying different combinations of independently acquired resistance determinants. We estimate the time to the most recent common ancestor of the predominant lineage ST 11 serotype Enteritidis (126 genomes) to be 1965 (95% highest posterior density intervals: 1927-1982). Its population size expanded until 1978, followed by a population decline until 1990. This lineage has been expanding since then. Comparison with genomes from other states reveal lack of geographical clustering indicative of long-distance dissemination. CONCLUSIONS: WGS studies of standing pathogen diversity provide critical insights into the population and evolutionary dynamics of lineages and antimicrobial resistance, which can be translated to effective public health action and decision-making. We highlight the need to strengthen efforts to implement WGS-based surveillance and genomic data analyses in state public health laboratories.

Diverse lineages of multidrug resistant clinical Salmonella enterica and a cryptic outbreak in New Hampshire, USA revealed from a year-long genomic surveillance.

Salmonella enterica, the causative agent of gastrointestinal diseases and typhoid fever, is a human and animal pathogen that causes significant mortality and morbidity worldwide. In this study, we examine the genomic diversity and phylogenetic relationships of 63 S. enterica isolates from human-derived clinical specimens submitted to the Department of Health and Human Services (DHHS) in the state of New Hampshire, USA in 2017. We found a remarkably large genomic, phylogenetic and serotype variation among the S. enterica isolates, dominated by serotypes Enteritidis (sequence type [ST] 11), Heidelberg (ST 15) and Typhimurium (ST 19). Analysis of the distribution of single nucleotide polymorphisms in the core genome suggests that the ST 15 cluster is likely a previously undetected or cryptic outbreak event that occurred in the south/southeastern part of New Hampshire in August-September. We found that nearly all of the clinical S. enterica isolates carried horizontally acquired genes that confer resistance to multiple classes of antimicrobials, most notably aminoglycosides, fluoroquinolones and macrolides. Majority of the isolates (76.2%) carry at least four resistance determinants per genome. We also detected the genes mdtK and mdsABC that encode multidrug efflux pumps and the gene sdiA that encodes a regulator for a third multidrug resistance pump. Our results indicate rapid microevolution and geographical dissemination of multidrug resistant lineages over a short time span. These findings are critical to aid the DHHS and similar public health laboratories in the development of effective disease control measures, epidemiological studies and treatment options for serious Salmonella infections.

Genomic Epidemiology and Evolution of Diverse Lineages of Clinical Campylobacter jejuni Cocirculating in New Hampshire, USA, 2017.

Campylobacter jejuni is one of the leading causes of bacterial gastroenteritis worldwide. In the United States, New Hampshire was one of the 18 states that reported cases in the 2016 to 2018 multistate outbreak of multidrug-resistant C. jejuni Here, we aimed to elucidate the baseline diversity of the wider New Hampshire C. jejuni population during the outbreak. We used genome sequences of 52 clinical isolates sampled in New Hampshire in 2017, including 1 of the 2 isolates from the outbreak. Results revealed a remarkably diverse population composed of at least 28 sequence types, which are mostly represented by 1 or a few strains. A comparison of our isolates with 249 clinical C. jejuni from other states showed frequent phylogenetic intermingling, suggesting a lack of geographical structure and minimal local diversification within the state. Multiple independent acquisitions of resistance genes from 5 classes of antibiotics characterize the population, with 47/52 (90.4%) of the genomes carrying at least 1 horizontally acquired resistance gene. Frequently recombining genes include those associated with heptose biosynthesis, colonization, and stress resistance. We conclude that the diversity of clinical C. jejuni in New Hampshire in 2017 was driven mainly by the coexistence of phylogenetically diverse antibiotic-resistant lineages, widespread geographical mixing, and frequent recombination. This study provides an important baseline census of the standing pangenomic variation and drug resistance to aid the development of a statewide database for epidemiological studies and clinical decision making. Continued genomic surveillance will be necessary to accurately assess how the population of C. jejuni changes over the long term.

Application of Narrow-Band Imaging thoracoscopy in diagnosis of pleural diseases.

BACKGROUND: Patients with undiagnostic pleural effusions are routinely examined by conventional medical thoracoscopy under the white light (WL). The endoscopic appearance of pleural diseases under WL could be misleading. Narrow-Band Imaging (NBI) has been applied as an interesting and effective diagnostic tool for endoscopy. However, there is also controversy about its value in the application of thoracoscopy. OBJECTIVE: The objective of this study was to investigate the diagnostic value of NBI technology during thoracoscopy. METHODS: Patients with undiagnosed pleural effusions admitted to our hospital between September 2017 and September 2019 were enrolled. During the thoracoscopy, we performed WL mode first and then NBI. Pictures of endoscopic real-time lesions were recorded under two modes, and at least five pieces of tissue were taken, respectively, on pleura lesions. Biopsy specimens were respectively taken for pathologic analysis. Diagnostic sensitivity, specificity were calculated to compare with pathologic results. RESULTS: 100 eligible patients were enrolled, including 63 with malignancy, 23 with tuberculous pleurisy, 3 with systemic disease and 11 with the negative condition. Compared with pathological results, the sensitivity of WL was 91.01%, and NBI 84.27%; while the specificity of WL was 27.27%, and NBI 81.82%. Compared NBI with WL, the former's specificity is superior to the latter's, which is statistically significant (P < 0.05). CONCLUSIONS: The advantage of NBI lies in its high specificity. It's useful to diagnose unknown pleural effusions in clinical practice. With better visualization of blood vessels, we can enhance the accuracy of biopsy and reduce the risk of unexpected bleeding arose from the biopsy.

Tibetan sheep grazing modifies rodent density and their interactions effect on GHG emissions of alpine meadow.

Digging and mound-building by rodents lead to considerable disturbances in the topsoil and may affect plant composition, soil properties. However, little is known about the effects of these activities on GHG emissions, especially under different grazing management. This paper aimed to measure changes in CO2 and CH4 efflux with varying grazing management during the warm and cold seasons and to relate CO2 and CH4 efflux to pika burrow density and zokor mound density with different grazing management. Results of this study showed that CO2 efflux was significantly affected by the grazing season, whereas CH4 efflux was significantly affected by the grazing system. There were significant relationships between GHG efflux and rodent population density which were regulated by grazing management. CO2 efflux increased linearly with rodent density under seasonal continuous grazing in warm season. CO2 and CH4 efflux and rodent population density showed a significant quadratic convex relationship under rotational grazing at 24 SM/ha in warm and cold seasons and rotational grazing at 48 SM/ha in cold season. Under rotational grazing at light stocking rate (24 SM/ha), appropriate populations of rodents were beneficial for decreasing GHG emissions. This results also used to help drive a best-practices model for grazing practices of local herders.

Isocyanide-Based Multicomponent Bicyclization with Substituted Allenoate and Isatin: Synthesis of Unusual Spirooxindole Containing [5.5]-Fused Heterocycle.

A three-component bicyclization reaction of isocyanide, substituted allenoate, and isatin has been disclosed. This protocol is proposed to proceed through Michael addition, double cyclization, and [1,5]-hydride shift sequence, thus leading to the formation of two new rings and five new chemical bonds, including C-C, C-O, and C-N bonds.

Exploiting the Reactivity of Isocyanide: Coupling Reaction between Isocyanide and Toluene Derivatives Using the Isocyano Group as an N1 Synthon.

An unusual oxidative coupling reaction of isocyanide and toluene derivatives using tetrabutylammonium iodide (TBAI) as a catalyst is disclosed. The experimental results and mechanistic study show that the isocyano group acts formally as an N1 synthon during the transformation, thus expanding the reactivity profile of isocyanide.

Bu4NI-Catalyzed α-Oxyacylation of Carbonyl Compounds with Toluene Derivatives.

A TBAI (tetrabutylammonium iodide)-catalyzed direct α-oxyacylation of carbonyl compounds from readily available toluene derivatives has been developed. The distinguished features of this metal-free protocol include the employment of simple starting material, a wide carbonyl compound scope, and mild reaction conditions.

Nitric oxide production upregulates Wnt/ß-catenin signaling by inhibiting Dickkopf-1.

Nitric oxide signaling plays complex roles in carcinogenesis, in part, due to incomplete mechanistic understanding. In this study, we investigated our discovery of an inverse correlation in the expression of the inducible nitric oxide synthase (iNOS) and the Wnt/ß-catenin regulator Dickkopf-1 (DKK1) in human cancer. In human tumors and animal models, induced nitric oxide synthesis increased Wnt/ß-catenin signaling by negatively regulating DKK1 gene expression. Human iNOS (hiNOS) and DKK1 gene expression were inversely correlated in primary human colon and breast cancers, and in intestinal adenomas from Min (Apc(min/+)) mice. Nitric oxide production by various routes was sufficient to decrease constitutive DKK1 expression, increasing Wnt/ß-catenin signaling in colon and breast cancer cells and primary human hepatocytes, thereby activating the transcription of Wnt target genes. This effect could be reversed by RNA interference-mediated silencing of iNOS or treatment with iNOS inhibitors, which restored DKK1 expression and its inhibitory effect on Wnt signaling. Taken together, our results identify a previously unrecognized mechanism through which the nitric oxide pathway promotes cancer by unleashing Wnt/ß-catenin signaling. These findings further the evidence that nitric oxide promotes human cancer and deepens insights in the complex control Wnt/ß-catenin signaling during carcinogenesis.

Cytoplasmic overexpression of CD95L in esophageal adenocarcinoma cells overcomes resistance to CD95-mediated apoptosis.

INTRODUCTION: The CD95/CD95L pathway plays a critical role in tissue homeostasis and immune system regulation; however, the function of this pathway in malignancy remains poorly understood. We hypothesized that CD95L expression in esophageal adenocarcinoma confers advantages to the neoplasm other than immune privilege. METHODS: CD95L expression was characterized in immortalized squamous esophagus (HET-1A) and Barrett esophagus (BAR-T) cells; adenocarcinoma cell lines FLO-1, SEG-1, and BIC-1, and MDA468 (- control); and KFL cells (+ control). Analyses included reverse transcription-polymerase chain reaction, immunoblots of whole cell and secretory vesicle lysates, FACScan analysis, laser scanning confocal microscopy of native proteins and fluorescent constructs, and assessment of apoptosis and ERK1/2 pathways. RESULTS: Cleaved, soluble CD95L is expressed at both the RNA and protein levels in these cell lines derived from esophageal adenocarcinoma and other human tissues. CD95L was neither trafficked to the cell membrane nor secreted into the media or within vesicles, rather the protein seems to be sequestered in the cytoplasm. CD95 and CD95L colocalize by immunofluorescence, but an interaction was not proven by immunoprecipitation. Overexpression of CD95L in the adenocarcinoma cell lines induced robust apoptosis and, under conditions of pan-caspase inhibition, resulted in activation of ERK signaling. CONCLUSIONS: CD95L localization in EA cells is inconsistent with the conference of immune privilege and is more consistent with a function that promotes tumor growth through alternative CD95 signaling. Reduced cell surface expression of CD95 affects cell sensitivity to extracellular apoptotic signals more significantly than alterations in downstream modulators of apoptosis.

Wnt/beta-catenin signaling regulates cytokine-induced human inducible nitric oxide synthase expression by inhibiting nuclear factor-kappaB activation in cancer cells.

The human inducible nitric oxide synthase (hiNOS) gene is regulated by nuclear factor kappaB (NF-kappaB) and has recently been shown to be a target of the Wnt/beta-catenin pathway. In this study, we tested the hypothesis that Wnt/beta-catenin signaling might regulate cytokine- or tumor necrosis factor alpha (TNFalpha)-induced hiNOS expression through interaction with NF-kappaB. A cytokine mixture of TNFalpha + interleukin (IL)-1beta + IFNgamma induced a 2- to 3-fold increase in hiNOS promoter activity in HCT116 and DLD1 colon cells, but produced a 2-fold decrease in SW480 colon cancer cells. A similar differential activity was seen in liver cancer cells (HepG2, Huh7, and Hep3B). Overexpression of beta-catenin produced a dose-dependent decrease in NF-kappaB reporter activity and decreased cytokine mixture-induced hiNOS promoter activity. Gel shift for TNFalpha-induced hiNOS NF-kappaB activation showed decreased p50 binding and decreased NF-kappaB reporter activity in the beta-catenin-mutant HAbeta18 cells. Conversely, enhanced p50 binding and increased NF-kappaB reporter activity were seen in HAbeta85 cells, which lack beta-catenin signaling. Coimmunoprecipitation confirmed that beta-catenin complexed with both p65 and p50 NF-kappaB proteins. NF-kappaB-dependent Traf1 protein expression also inversely correlated with the level of beta-catenin. Furthermore, SW480 cells stably transformed with wild-type adenomatous polyposis coli showed decreased beta-catenin protein and increased TNFalpha-induced p65 NF-kappaB binding as well as iNOS and Traf1 expression. Finally, beta-catenin inversely correlated with iNOS and Fas expression in vivo in hepatocellular carcinoma tumor samples. Our in vitro and in vivo data show that beta-catenin signaling inversely correlates with cytokine-induced hiNOS and other NF-kappaB-dependent gene expression. These findings underscore the complex role of Wnt/beta-catenin, NF-kappaB, and iNOS signaling in the pathophysiology of inflammation-associated carcinogenesis.

Inhibition of HGF/MET as therapy for malignancy.

BACKGROUND: Inhibition of inappropriate tyrosine kinase activity by neoplasms is an attractive strategy for the treatment of malignancy. OBJECTIVE: We aimed to produce a concise review of the potential role of hepatocyte growth factor (HGF)/Mesenchymal-epithelial transition factor (MET) tyrosine kinase pathway inhibition in the treatment of cancer. METHODS: The current literature, abstracts and internet resources related to HGF/MET structure, function and inhibition are summarized. The potential of inhibiting this pathway as a therapy for cancer and remaining hurdles prior to routine clinical use of MET inhibition are discussed. RESULTS/CONCLUSIONS: Current knowledge suggests that the inhibition of the HGF/MET pathway has significant potential for the treatment of cancer. A number of MET inhibitor molecules are nearing completion of their development for clinical use.

Inhibition of c-Met as a therapeutic strategy for esophageal adenocarcinoma.

The hepatocyte growth factor (HGF) receptor c-Met is a tyrosine kinase receptor with established oncogenic properties. We have previously shown that c-Met is usually overexpressed in esophageal adenocarcinoma (EA), yet the implications of c-Met inhibition in EA remain unknown. Three c-Met-overexpressing EA cell lines (Seg-1, Bic-1, and Flo-1) were used to examine the effects of a c-Met-specific small molecule inhibitor (PHA665752) on cell viability, apoptosis, motility, invasion, and downstream signaling pathways. PHA665752 demonstrated dose-dependent inhibition of constitutive and/or HGF-induced phosphorylation of c-Met, which correlated with reduced cell viability and inhibition of extracellular regulated kinase 1/2 phosphorylation in all three EA cell lines. In contrast, PHA665752 induced apoptosis and reduced motility and invasion in only one EA cell line, Flo-1. Interestingly, Flo-1 was the only cell line in which phosphatidylinositol 3-kinase (PI3K)/Akt was induced following HGF stimulation. The PI3K inhibitor LY294002 produced effects equivalent to those of PHA665752 in these cells. We conclude that inhibition of c-Met may be a useful therapeutic strategy for EA. Factors other than receptor overexpression, such as c-Met-dependent PI3K/Akt signaling, may be predictive of an individual tumor's response to c-Met inhibition.