Development of a duplex real-time PCR for differentiation between E. coli and Shigella spp

Aims: The aim of this study was to develop a real‐time PCR test for differentiation between Shigella spp. and E. coli, in particular enteroinvasive Escherichia coli (EIEC). Methods and Results: A duplex real‐time PCR specific for the genes encoding for β‐glucuronidase (uidA) and lactose permease (lacY) was developed. Ninety‐six isolates including 11 EIEC isolates of different serotypes and at least three representatives of each Shigella species were used for selectivity testing. All isolates tested were positive for the uidA gene. Additionally, all E. coli isolates were positive for the lacY gene, whereas no Shigella isolate tested harboured lacY. Conclusions: The duplex real‐time PCR assay was found to be simple, rapid, reliable and specific. Significance and Impact of the Study: If possible at all, delineation of so‐called inactive EIEC from Shigella spp. is cumbersome. Biochemical and serological methods are limited to specific pheno‐ and serotypes. This assay clearly simplifies the differentiation of both.     

Genotypes with the apolipoprotein ε4 allele are predictors of coronary heart disease mortality in a longitudinal study of elderly Finnish men

Earlier we reported that allelic variation in the gene coding for apolipoprotein (apoE) is a significant predictor of variation in the risk of coronary heart disease (CHD) death in a longitudinal study of elderly Finnish men. Here we address the question: which of the apoE genotypes confers the risk information in these men, and whether such information persists after other CHD risk factors are considered? We followed two cohorts of elderly Finnish men aged 65 to 84 years, one in Eastern (n = 281) and the other in the Southwestern (n = 344) Finland for 5 years during which 26 (9.3%) of the men from the Eastern cohort and 40 (11.6%) of the men in the Southwestern cohort died from CHD. Baseline high density lipoprotein (HDL) cholesterol and (HDL cholesterol)² in the Eastern cohort and age, and total and HDL cholesterol and smoking status in the Southwestern cohort were significant predictors of CHD death (P < 0.05). The apoE genotypes were significant predictors in the Southwestern cohort atP = 0.02 and in the Eastern cohort atP = 0.18. In multivariable models, information about apoE genotypes improved the prediction atP = 0.10 level of statistical significance in both cohorts. When genotypes were considered separately, the ε2/4 combined with the ε4/4 in the Eastern cohort (odds ratio = 7.69, 95% CI = 1.67-35.52) and the ε3/4 in the Southwestern cohort (odds ratio = 2.44, 95% CI = 1.16–5.10) had sigificanctly greater odds of CHD death compared to the common ε3/3 genotype. We conclude that apoE genotypes confer risk information about CHD death in two cohorts of elderly Finnish men in a longitudinal study, and this information persists after adjustment for other CHD risk factors. Because different genotypes were predictors in these two cohorts, we further conclude that the utility of a particular genotype as a predictor of CHD death in other populations may depend on the distribution of risk factor profiles at baseline, geographically defined environmental exposures, the CHD mortality history, and the evolutionary history of background genotypes in the population considered.     

Durable engraftment of genetically modified FVIII‐secreting autologous bone marrow stromal cells in the intramedullary microenvironment

Genetically modified FVIII‐expressing autologous bone marrow‐derived mesenchymal stromal cells (BMSCs) could cure haemophilia A. However, culture‐expanded BMSCs engraft poorly in extramedullary sites. Here, we compared the intramedullary cavity, skeletal muscle, subcutaneous tissue and systemic circulation as tissue microenvironments that could support durable engraftment of FVIII‐secreting BMSC in vivo. A zinc finger nuclease integrated human FVIII transgene into PPP1R12C (intron 1) of culture‐expanded primary canine BMSCs. FVIII‐secretory capacity of implanted BMSCs in each dog was expressed as an individualized therapy index (number of viable BMSCs implanted × FVIII activity secreted/million BMSCs/24 hours). Plasma samples before and after implantation were assayed for transgenic FVIII protein using an anti‐human FVIII antibody having negligible cross‐reactivity with canine FVIII. Plasma transgenic FVIII persisted for at least 48 weeks after implantation in the intramedullary cavity. Transgenic FVIII protein levels were low after intramuscular implantation and undetectable after both intravenous infusion and subcutaneous implantation. All plasma samples were negative for anti‐human FVIII antibodies. Plasma concentrations and durability of transgenic FVIII secretion showed no correlation with the therapy index. Thus, the implantation site microenvironment is crucial. The intramedullary microenvironment, but not extramedullary tissues, supported durable engraftment of genetically modified autologous FVIII‐secreting BMSCs.     

Discovery of a potent and selective COX-2 inhibitor in the alkoxy lactone series with optimized metabolic profile

The COX-2 inhibitor DFP [5,5-dimethyl-3-(2-propoxy)-4-methanesulfonylphenyl)-2(5H)-furanone] was found to have a long half-life in humans. Analogues have been characterized in order to optimize pharmacokinetics. This has lead to the discovery of 5(S)-(5-ethyl-5-methyl-3-(2-propoxy)-4-methanesulfonylphenyl)-2(5H)-furanone analogue 11 a potent and selective COX-2 inhibitor which is metabolized to a greater extent than DFP upon incubation with rat and human hepatocytes, suggesting a shorter half-life in humans.     

Pyridazinones as selective cyclooxygenase-2 inhibitors

Pyridazinone was found to be an excellent core template for selective COX-2 inhibitors. Two potent, selective and orally active COX-2 inhibitors, which were highly efficacious in rat paw edema and rat pyresis models, have been obtained.     

A Higher Activation Threshold of Memory CD8+ T Cells Has a Fitness Cost That Is Modified by TCR Affinity during Tuberculosis

T cell vaccines against Mycobacterium tuberculosis (Mtb) and other pathogens are based on the principle that memory T cells rapidly generate effector responses upon challenge, leading to pathogen clearance. Despite eliciting a robust memory CD8⁺ T cell response to the immunodominant Mtb antigen TB10.4 (EsxH), we find the increased frequency of TB10.4-specific CD8⁺ T cells conferred by vaccination to be short-lived after Mtb challenge. To compare memory and naïve CD8⁺ T cell function during their response to Mtb, we track their expansions using TB10.4-specific retrogenic CD8⁺ T cells. We find that the primary (naïve) response outnumbers the secondary (memory) response during Mtb challenge, an effect moderated by increased TCR affinity. To determine whether the expansion of polyclonal memory T cells is restrained following Mtb challenge, we used TCRβ deep sequencing to track TB10.4-specific CD8⁺ T cells after vaccination and subsequent challenge in intact mice. Successful memory T cells, defined by their clonal expansion after Mtb challenge, express similar CDR3β sequences suggesting TCR selection by antigen. Thus, both TCR-dependent and -independent factors affect the fitness of memory CD8⁺ responses. The impaired expansion of the majority of memory T cell clonotypes may explain why some TB vaccines have not provided better protection.     

The Effects of Empagliflozin,an SGLT2 Inhibitor,on Pancreatic β-Cell Mass and Glucose Homeostasis in Type 1 Diabetes

The novel sodium glucose co-transporter 2 (SGLT2) inhibitor empagliflozin has recently been reported to improve glycemic control in streptozotocin-induced type 1 diabetic rats in an insulin-independent manner, via an increase in urinary glucose output. We investigated the potential of empagliflozin to recover insulin pathways in type 1 diabetes by improving pancreatic β-cell mass. Blood glucose homeostasis was assessed by an intraperitoneal glucose tolerance test. Serum insulin levels and insulin mRNA expression were determined using commercial insulin ELISA kits and real-time quantitative polymerase chain reaction, respectively. Immunohistochemistry was used to investigate β-cell areas, β-cell proliferation, apoptosis of pancreatic β-cells, and reactive oxygen species production in the pancreatic β-cells. Results showed that glucose tolerance was significantly improved in streptozotocin-induced type 1 diabetic mice treated with empagliflozin. Empagliflozin-treated mice also showed an increase in insulin mRNA expression. Higher serum insulin levels were detected in mice treated with empagliflozin compared with the vehicle group. Immunohistochemistry indicated that β-cell area/total pancreatic area and the expression of cell proliferation marker Ki-67 (co-stained with insulin) were significantly enhanced by empagliflozin treatment. These effects were due, probably, to a reduction in apoptosis and reactive oxygen species in the pancreatic β-cells. Taken together, the results of this study indicate that empagliflozin may have a beneficial effect on preserving β-cell regeneration, thus improving blood glucose homeostasis in type 1 diabetes mellitus, probably via the protection of pancreatic β-cell from glucotoxicity-induced oxidative stress.     

On-site rapid detection of toxic <Emphasis Type="Italic">Alexandrium tamiyavanichii</Emphasis>: integrating the species-specific hydrolysis probe in insulated isothermal polymerase chain reaction (iiPCR)

On-site investigation of phytoplankton samples is important for rapid detection of harmful algal species and for early warning of harmful algal bloom. Molecular detection method by DNA amplification in a portable insulated isothermal PCR (iiPCR) device provides a simple and rapid detection based on fluorescent probe within an hour of reaction time. The assay was developed for a paralytic shellfish toxin-producing dinoflagellate Alexandrium tamiyavanichii. The assay presents the data as positive or negative on the presence or absence of A. tamiyavanichii cells, with a limit of detection (LOD) at five target cells per reaction. While the assay is incapable to accurately quantify cell density, it exhibits high detection accuracy and strongly correlated with quantitative PCR (qPCR) data. The user repeatability of iiPCR assay was evaluated; the results showed that no significant differences in the assay run by different operators. Field applicability of the assay was further validated by environmental samples. Despite the shortcoming of the assay, the overall performance of the assay to detect cells, its low-cost effectiveness, and portability for on-site detection, iiPCR has proven its potential as an early screening tool for harmful algae monitoring.     

Interactions of the Anticancer Drug Tamoxifen with Lipid Membranes

Interactions of the hydrophobic anticancer drug tamoxifen (TAM) with lipid model membranes were studied using calcein-encapsulated vesicle leakage, attenuated total reflection Fourier transform infrared (FTIR) spectroscopy, small-angle neutron scattering (SANS), atomic force microscopy (AFM) based force spectroscopy, and all-atom molecular dynamics (MD) simulations. The addition of TAM enhances membrane permeability, inducing calcein to translocate from the interior to the exterior of lipid vesicles. A large decrease in the FTIR absorption band’s magnitude was observed in the hydrocarbon chain region, suggesting suppressed bond vibrational dynamics. Bilayer thickening was determined from SANS data. Force spectroscopy measurements indicate that the lipid bilayer area compressibility modulus K_A is increased by a large amount after the incorporation of TAM. MD simulations show that TAM decreases the lipid area and increases chain order parameters. Moreover, orientational and positional analyses show that TAM exhibits a highly dynamic conformation within the lipid bilayer. Our detailed experimental and computational studies of TAM interacting with model lipid membranes shed new light on membrane modulation by TAM.