Cytotoxic and Pathogenic Properties of Klebsiella oxytoca Isolated from Laboratory Animals

Klebsiella oxytoca is an opportunistic pathogen implicated in various clinical diseases in animals and humans. Studies suggest that in humans K. oxytoca exerts its pathogenicity in part through a cytotoxin. However, cytotoxin production in animal isolates of K. oxytoca and its pathogenic properties have not been characterized. Furthermore, neither the identity of the toxin nor a complete repertoire of genes involved in K. oxytoca pathogenesis have been fully elucidated. Here, we showed that several animal isolates of K. oxytoca, including the clinical isolates, produced secreted products in bacterial culture supernatant that display cytotoxicity on HEp-2 and HeLa cells, indicating the ability to produce cytotoxin. Cytotoxin production appears to be regulated by the environment, and soy based product was found to have a strong toxin induction property. The toxin was identified, by liquid chromatography-mass spectrometry and NMR spectroscopy, as low molecular weight heat labile benzodiazepine, tilivalline, previously shown to cause cytotoxicity in several cell lines, including mouse L1210 leukemic cells. Genome sequencing and analyses of a cytotoxin positive K. oxytoca strain isolated from an abscess of a mouse, identified genes previously shown to promote pathogenesis in other enteric bacterial pathogens including ecotin, several genes encoding for type IV and type VI secretion systems, and proteins that show sequence similarity to known bacterial toxins including cholera toxin. To our knowledge, these results demonstrate for the first time, that animal isolates of K. oxytoca, produces a cytotoxin, and that cytotoxin production is under strict environmental regulation. We also confirmed tilivalline as the cytotoxin present in animal K. oxytoca strains. These findings, along with the discovery of a repertoire of genes with virulence potential, provide important insights into the pathogenesis of K. oxytoca. As a novel diagnostic tool, tilivalline may serve as a biomarker for K oxytoca-induced cytotoxicity in humans and animals through detection in various samples from food to diseased samples using LC-MS/MS. Induction of K. oxytoca cytotoxin by consumption of soy may be in part involved in the pathogenesis of gastrointestinal disease.     

Gastric Pepsin in an Anuran Larva

Larval stomach development was studied in the obligate carnivorous larva of the frog Lepidobatrachus laevis . Pepsin producing cells of the larval stomach were identified using rabbit anti-porcine pepsin and immunohistochemical techniques. Pepsin production was detected at a very early stage of development (stage 24: during opercular development) when the larvae were first competent for feeding. Peptic activity in isolated larval stomachs was demonstrated in a microassay using acid denatured hemoglobin at pH 1.7. The total activity per stomach increased 5,400 fold through the beginning of metamorphosis and the specific activity increased 345 fold through the same period. Electrophoretic analysis of the larval pepsinogens, using a caseinolytic assay revealed the presence of one major pepsinogen at stage 24; two additional isozymes were observed during later larval development. The molecular weight of the isopepsinogens was 34,800.     

A spatially explicit representation of conservation agriculture for application in global change studies

Conservation agriculture (CA) is widely promoted as a sustainable agricultural management strategy with the potential to alleviate some of the adverse effects of modern, industrial agriculture such as large‐scale soil erosion, nutrient leaching and overexploitation of water resources. Moreover, agricultural land managed under CA is proposed to contribute to climate change mitigation and adaptation through reduced emission of greenhouse gases, increased solar radiation reflection, and the sustainable use of soil and water resources. Due to the lack of official reporting schemes, the amount of agricultural land managed under CA systems is uncertain and spatially explicit information about the distribution of CA required for various modeling studies is missing. Here, we present an approach to downscale present‐day national‐level estimates of CA to a 5 arcminute regular grid, based on multicriteria analysis. We provide a best estimate of CA distribution and an uncertainty range in the form of a low and high estimate of CA distribution, reflecting the inconsistency in CA definitions. We also design two scenarios of the potential future development of CA combining present‐day data and an assessment of the potential for implementation using biophysical and socioeconomic factors. By our estimates, 122–215 Mha or 9%–15% of global arable land is currently managed under CA systems. The lower end of the range represents CA as an integrated system of permanent no‐tillage, crop residue management and crop rotations, while the high estimate includes a wider range of areas primarily devoted to temporary no‐tillage or reduced tillage operations. Our scenario analysis suggests a future potential of CA in the range of 533–1130 Mha (38%–81% of global arable land). Our estimates can be used in various ecosystem modeling applications and are expected to help identifying more realistic climate mitigation and adaptation potentials of agricultural practices.     

Effects of climate extremes on the terrestrial carbon cycle: concepts,processes and potential future impacts

Extreme droughts, heat waves, frosts, precipitation, wind storms and other climate extremes may impact the structure, composition and functioning of terrestrial ecosystems, and thus carbon cycling and its feedbacks to the climate system. Yet, the interconnected avenues through which climate extremes drive ecological and physiological processes and alter the carbon balance are poorly understood. Here, we review the literature on carbon cycle relevant responses of ecosystems to extreme climatic events. Given that impacts of climate extremes are considered disturbances, we assume the respective general disturbance‐induced mechanisms and processes to also operate in an extreme context. The paucity of well‐defined studies currently renders a quantitative meta‐analysis impossible, but permits us to develop a deductive framework for identifying the main mechanisms (and coupling thereof) through which climate extremes may act on the carbon cycle. We find that ecosystem responses can exceed the duration of the climate impacts via lagged effects on the carbon cycle. The expected regional impacts of future climate extremes will depend on changes in the probability and severity of their occurrence, on the compound effects and timing of different climate extremes, and on the vulnerability of each land‐cover type modulated by management. Although processes and sensitivities differ among biomes, based on expert opinion, we expect forests to exhibit the largest net effect of extremes due to their large carbon pools and fluxes, potentially large indirect and lagged impacts, and long recovery time to regain previous stocks. At the global scale, we presume that droughts have the strongest and most widespread effects on terrestrial carbon cycling. Comparing impacts of climate extremes identified via remote sensing vs. ground‐based observational case studies reveals that many regions in the (sub‐)tropics are understudied. Hence, regional investigations are needed to allow a global upscaling of the impacts of climate extremes on global carbon–climate feedbacks.     

Association, interaction, and replication analysis of genes encoding serotonin transporter and 5-HT3 receptor subunits A and B in alcohol dependence

On the basis of the converging evidence showing regulation of drinking behavior by 5-HT_3AB receptors and the serotonin transporter, we hypothesized that the interactive effects of genetic variations in the genes HTR3A, HTR3B, and SLC6A4 confer greater susceptibility to alcohol dependence (AD) than do their effects individually. We examined the associations of AD with 22 SNPs across HTR3A, HTR3B, and two functional variants in SLC6A4 in 500 AD and 280 healthy control individuals of European descent. We found that the alleles of the low-frequency SNPs rs33940208:T in HTR3A and rs2276305:A in HTR3B were inversely and nominally significantly associated with AD with odds ratio (OR) and 95 % confidence interval of 0.212 and 0.073, 0.616 (P = 0.004) and 0.261 and 0.088, 0.777 (P = 0.016), respectively. Further, our gene-by-gene interaction analysis revealed that two four-variant models that differed by only one SNP carried a risk for AD (empirical P < 1 × 10^?6 for prediction accuracy of the two models based on 10⁶ permutations). Subsequent analysis of these two interaction models revealed an OR of 2.71 and 2.80, respectively, for AD (P < 0.001) in carriers of genotype combinations 5′-HTTLPR:LL/LS(SLC6A4)–rs1042173:TT/TG(SLC6A4)–rs1176744:AC(HTR3B)–rs3782025:AG(HTR3B) and 5′-HTTLPR:LL/LS(SLC6A4)–rs10160548:GT/TT(HTR3A)–rs1176744:AC(HTR3B)–rs3782025:AG(HTR3B). Combining all five genotypes resulted in an OR of 3.095 (P = 2.0 × 10^?4) for AD. Inspired by these findings, we conducted the analysis in an independent sample, OZ-ALC-GWAS (N = 6699), obtained from the NIH dbGAP database, which confirmed the findings, not only for all three risk genotype combinations (Z = 4.384, P = 1.0 × 10^?5; Z = 3.155, P = 1.6 × 10^?3; and Z = 3.389, P = 7.0 × 10^?4, respectively), but also protective effects for rs33940208:T (χ ² = 3.316, P = 0.0686) and rs2276305:A (χ ² = 7.224, P = 0.007). These findings reveal significant interactive effects among variants in SLC6A4–HTR3A–HTR3B affecting AD. Further studies are needed to confirm these findings and characterize the molecular mechanisms underlying these effects.     

P2X7 receptor antagonism inhibits tumour growth in human high-grade gliomas

Gliomas, the most common primary brain cancer, are highly infiltrative and extremely difficult to treat. Despite advancements, current treatment is limited, with patients surviving for a median of 14–15 months post-diagnosis. Previous research has demonstrated the upregulation of a purinergic receptor, P2X7R, in human gliomas. P2X7R is expressed on both glioma cells and microglia within the glioma microenvironment. It is hypothesized that P2X7R contributes to tumour growth and proliferation via immune-mediated mechanisms involving tumour cells and surrounding microglia. We sought to elucidate the role of P2X7R in a human glioblastoma cell line (U251) and on surgically resected human glioma samples. We treated U251 and human glioma cultures for 72 h with P2X7R antagonists, Brilliant Blue G (BBG), oxidized ATP (oATP) and AZ10606120. Cell counting via fluorescence confocal microscopy was conducted to assess tumour proliferation. We observed no significant reductions in tumour cell numbers following P2X7R antagonism with BBG (20 μM) and oATP (250 μM) in both U251 cells and human glioma samples. Interestingly, there was a significant reduction in tumour cell number in both U251 cells (p =?0.0156) and human glioma samples (p =?0.0476) treated with varying concentrations of AZ10606120. When compared with the conventional chemotherapeutic agent, temozolomide, AZ10606120 was also found to more effectively inhibit tumour proliferation in U251 cells (p <?0.0001). Our pilot results demonstrate a potential trophic role of P2X7R where its inhibition by AZ10606120, a potent antagonist, hinders glioma growth directly or through the inactivation of microglia. This sheds new light on P2X7R as a therapeutic target for human gliomas.

     

Prevalence and antifungal drug sensitivity of non‐albicans Candida in oral rinse samples of self‐caring elderly

doi: 10.1111/j.1741‐2358.2010.00407.x
Prevalence and antifungal drug sensitivity of non‐albicans Candida in oral rinse samples of self‐caring elderly Aim: To assess the prevalence and antifungal drug sensitivity of non‐albicans Candida (NAC) species in elderly outpatients. Materials and methods: We investigated oral rinse samples of 194 self‐caring elderly population (mean age 83 years) with emphasis on background factors for harbouring NAC. Susceptibility of Candida species to antifungal drugs was determined using standard methodology. Multiple logistic regression analysis was performed taking positive NAC count as the dependent variable and a number of known Candida risk factors as independent variables. Results: Prevalence of candidal carriage of the population was 78.4%, of which 0.5% of the subjects were NAC positive. Candida dubliniensis was the most prevalent NAC species, followed by Candida glabrata and Candida parapsilosis. The NAC positive elderly were more often edentulous with dental prostheses or had fewer teeth than Candida albicans‐positive or yeast‐negative subjects. Dental caries slightly increased the risk for having NAC strains (odds ratio 1.08), whilst greater age appeared to lower the risk (odds ratio 0.77). Candida species were susceptible to the commonly used antifungal agents in general, but with considerable variation among species. Occasionally, some NAC exhibited lower antifungal susceptibility. Conclusion: The possibility of oral reservoirs of NAC strains which are resistant to common antifungals should be noted in elderly outpatients.     

Investigating Microenvironmental Regulation of Human Chordoma Cell Behaviour

The tumour microenvironment is complex and composed of many different constituents, including matricellular proteins such as connective tissue growth factor (CCN2), and is characterized by gradients in oxygen levels. In various cancers, hypoxia and CCN2 promote stem and progenitor cell properties, and regulate the proliferation, migration and phenotype of cancer cells. Our study was aimed at investigating the effects of hypoxia and CCN2 on chordoma cells, using the human U-CH1 cell line. We demonstrate that under basal conditions, U-CH1 cells express multiple CCN family members including CCN1, CCN2, CCN3 and CCN5. Culture of U-CH1 cells in either hypoxia or in the presence of recombinant CCN2 peptide promoted progenitor cell-like characteristics specific to the notochordal tissue of origin. Specifically, hypoxia induced the most robust increase in progenitor-like characteristics in U-CH1 cells, including increased expression of the notochord-associated markers T, CD24, FOXA1, ACAN and CA12, increased cell growth and tumour-sphere formation, and a decrease in the percentage of vacuolated cells present in the heterogeneous population. Interestingly, the effects of recombinant CCN2 peptide on U-CH1 cells were more pronounced under normoxia than hypoxia, promoting increased expression of CCN1, CCN2, CCN3 and CCN5, the notochord-associated markers SOX5, SOX6, T, CD24, and FOXA1 as well as increased tumour-sphere formation. Overall, this study highlights the importance of multiple factors within the tumour microenvironment and how hypoxia and CCN2 may regulate human chordoma cell behaviour.