The development and evaluation of a nested PCR assay for detection of Neospora caninum and Hammondia heydorni in feral mouse tissues

The development of a novel nested polymerase chain reaction is described and used for detecting the presence of Neospora caninum and Hammondia heydorni DNA in DNA extracted from feral rodent tissues. A unique strategy was used for design of an assay that could be adapted for detecting DNA from more than one member of Toxoplasmatinae simultaneously with a minimal number of additional steps. The level of sensitivity described for this assay is comparable to real time-PCR and other nested PCR assays. Twenty-eight of 104 feral mice tested positive for N. caninum in at least one tissue (the brain, heart or liver) studied. In this study, eight instances are reported where the brain tested negative to N. caninum while at least one other tissue was positive. This suggests that prior studies, which screened only the brain, describe prevalence levels that are under-represented. None of 54 mouse brains tested positive for H. heydorni DNA. This suggests that mice are rarely infected by H. heydorni although this hypothesis needs to be explored further. Data obtained in the current study suggest that N. caninum is a common parasite of feral rodents which may be important in the epidemiology of the disease.     

Evaluation of cytotoxic, oxidative stress, proinflammatory and genotoxic responses of micro- and nano-particles of dolomite on human lung epithelial cells A(549)

Dolomite is a natural mineral of great industrial importance and used worldwide, thus millions of workers are at risk of occupational exposure. Its toxicity is however, meagerly documented. In the present investigation, a dolomite powder obtained from its milling unit was analyzed by some standard methods namely, optical microscopy, transmission electron microscopy and dynamic light scattering. Results showed that dolomite powder contained particles of different shapes and size both microparticles (MPs) and nanoparticles (NPs), suggesting potential occupational exposure of these particles. An attempt was therefore, made to investigate dolomite toxicity in a particle size-dependent manner in human lung epithelial cells A(549). The comparative toxicity evaluation of MPs and NPs was carried out by assessing their effects on cell viability, membrane damage, glutathione, reactive oxygen species (ROS), lipid peroxidation (LPO), micronucleus (MN) and proinflammatory cytokines, namely tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β) and interleukin-6 (IL-6). These markers of cytotoxicity, genotoxicity and inflammation were assayed in cells exposed to MPs and NPs in a dose-and time-dependent manner. Invariably, their toxic effects were dose-and time-dependent while NPs in general were significantly more toxic. Notably, NPs caused oxidative stress, genotoxicity and inflammatory responses, as seen by significant induction of ROS, LPO, MN, TNF-α, IL-1β and IL-6. Thus, the study tends to suggest that separate health safety standards would be required for micrometer and nanometer scale particles of dolomite.     

Altered sensitivity to nitric oxide donors,induced by intravascular infusion of quantum dots,in murine mesenteric arteries

Quantum dots (QDs) are utilised in imaging diagnostics, tissue engineering and medical therapeutics, however, their influence on vascular function is not ascertained. Here, we examined small mesenteric arterial responses after acute intravascular exposure to QDs. Incubation in mercaptoundecanoic acid (MUA)-coated QDs (at 15 μg/mL) had no influence on endothelial-dependent dilator responses (Acetylcholine; Ach) but led to an attenuated relaxation to the nitric oxide donor, sodium nitroprusside (SNP). Conversely, incubation in POSS-PCU coated QDs (at 15 μg/mL) led to attenuated Ach responses (10^? 11–10^? 3 M; n = 5, P < 0.05), but had no influence on SNP-induced relaxation. At lower concentrations of POSS-PCU coated QDs (5 μg/mL), Ach responses were preserved. We demonstrate that acute exposure to QDs, can attenuate vasodilation but not vasoconstriction, and is dependent on their surface coatings. Our findings have implications in QD use for imaging diagnostics in disease states, where SNP based drugs are used in therapeutic intervention.From the Clinical EditorIn this paper, the influence of quantum dots on vascular function is investigated---an important aspect to consider with the growing utility of quantum dots in imaging diagnostics, tissue engineering and medical therapeutics.     

Key associations for hepatitis C virus genotypes in the Middle East and North Africa

This study aimed to investigate the epidemiology of hepatitis C virus (HCV) genotypes in the Middle East and North Africa (MENA) through an analytical and quantitative meta-regression methodology. For the most common genotypes 1, 3, and 4, country/subregion explained more than 77% of the variation in the distribution of each genotype. Genotype 1 was common across MENA, and was more present in high-risk clinical populations than in the general population. Genotype 3 was much more present in Afghanistan, Iran, and Pakistan than the rest of countries, and was associated with transmission through injecting drug use. Genotype 4 was broadly disseminated in Egypt in all populations, with overall limited presence elsewhere. While genotype 2 was more present in high-risk clinical populations and people who inject drugs, most of the variation in its distribution remained unexplained. Genotypes 5, 6, and 7 had low or no presence in MENA, limiting the epidemiological inferences that could be drawn. To sum up, geography is the principal determinant of HCV genotype distribution. Genotype 1 is associated with transmission through high-risk clinical procedures, while genotype 3 is associated with injecting drug use. These findings demonstrate the power of such analytical approach, which if extended to other regions and globally, can yield relevant epidemiological inferences.     

Bleeding-avoidance strategies and outcomes in patients ≥80 years of age with ST-elevation myocardial infarction undergoing primary percutaneous coronary intervention (from the NCDR CathPCI Registry)

The purpose of our study was to evaluate the use of bleeding-avoidance strategies (BAS) and risk-adjusted bleeding over time in patients ≥80 years of age undergoing primary percutaneous coronary intervention (PCI) for ST-segment elevation myocardial infarction. We analyzed data from the CathPCI Registry from July 1, 2006 through June 30, 2009. Patients were included if they were ≥80 years old, presented with ST-segment elevation myocardial infarction, and underwent primary PCI. We evaluated trends in use of BAS (direct thrombin inhibitors, vascular closure devices, and radial access) and risk-adjusted bleeding over time. Of 10,469 patients ≥80 years old undergoing primary PCI, 1,002, (9.6%) developed a bleeding complication. Use of direct thrombin inhibitors and vascular closure devices increased over time (12.8% to 24.9% and 29.2% to 32.7%, p <0.01 and <0.05 for trends, respectively). Radial access was extremely uncommon (<1%) and did not change over the course of the study. In multivariable analyses, use of BAS was associated with lower bleeding. However, over the course of the study period, overall risk-adjusted bleeding did not decrease significantly (9.9% to 9.4%, p = 0.14 for trend). In conclusion, patients ≥80 years old undergoing primary PCI are at high risk of bleeding, and despite significant increases in use of BAS, the overall rate of bleeding complications remains high.     

“They Need to Have an Understanding of Why They're Coming Here and What the Outcomes Might Be.” Clinician Perspectives on Goals of Care for Patients Discharged From Hospitals to Skilled Nursing Facilities

Context

The number of patients discharged from acute care hospitals to skilled nursing facilities (SNFs) is rising. These patients have increasingly complex needs and many experience poor outcomes while under SNF care, including hospital readmissions. Patients' goals of care (GoC) are viewed as a factor contributing to unplanned hospital readmissions from SNFs. However, clinicians' perspectives of GoC for hospitalized patients discharged to SNFs are not well-described.

Involvement of epigenetics and microRNA-29b in the urethane induced inception and establishment of mouse lung tumors

Epigenetic changes are correlated with tumor development showing aberrations in DNA methylation and histone modifications. To find the early changes, we evaluated the epigenetic events from early to late stage of the urethane induced lung tumor development in mouse model and tried to correlate the molecular events with the progression of tumor. We addressed the hypothesis by examining the tumor development, status of DNMTs, HDACs and MBDs, DNA methylation and expression of microRNA-29b during 1 to 36 weeks after urethane exposure that included the period before and after the tumor appearance. Tumors did not appear after 1 or 4 weeks but well defined tumors appeared after 12 weeks and larger tumors appeared at 36 weeks which was prevented by IP6. DNMT1, DNMT3a and DNMT3b were upregulated after urethane exposure at the time of no tumor till the tumor developed and showed its upregulated functional activity. DNMTs are shown to be the targets of microRNA-29b and we showed that microRNA-29b was downregulated in the line of DNMT upregulation. HDAC, the histone modifier, also showed progressive upregulation. Periodic increase in methyl binding proteins, MBD2, supported the expression of gene silencing pathways in terms of the downregulation of tumor suppressor genes, p16 and MLH1. All these molecular alterations were protected in the presence of IP6. Our results showed that the key steps of epigenetics, DNMTs, mir29b, and HDAC1, are altered both before and after the development of tumors.     

Analysis of Human Bone Sialoprotein in Normal and Pathological Tissues using a Monoclonal Antibody (BSP 1.2 mab)

Bone sialoprotein (BSP), a phosphorylated and sulphated glycoprotein that is expressed by mineralized connective tissues is also produced in tumors that metastasize to bone. To facilitate studies of BSP expression in normal and pathological human tissues a monoclonal antibody (BSP 1.2 mab) was raised against human bone BSP. BSP 1.2 mab was shown by ELISA assays to recognize the epitope “DEYSY” (amino acids 279–283) that is conserved in mammalian BSP sequences. However, whereas the antibody recognized recombinant BSPs expressed in bacteria, it did not recognize native forms of rat or pig BSP in which the first tyrosine of the DEYSY peptide sequence appears to be modified. Immunostaining of embryonic human tibiae and calvariae with BSP 1.2 mab showed strong reaction in osteoblasts and osteocytes with relatively weak staining of the bone matrix, suggesting that the BSP 1.2 mab epitope is partially masked in the bone matrix. BSP 1.2 mab also stained osteosarcoma cells and normal trophoblastic cells in the placenta in areas of microcrystalline deposits. Cancer cells in primary breast tumors, lymph nodes, and secondary bone metastases from individual patients were stained strongly by BSP 1.2 mab. Although BSP 1.2 mab also stained breast cancer carcinoma cell lines and SaOS2 osteosarcoma cells, biosynthesis of radiolabelled BSP could not be demonstrated in breast cancer cells. Notably, the staining of BSP in the breast cancer cells was diffuse contrasting the punctate staining, typical of secreted proteins, in SaOS2 cells. These studies, therefore, have identified a unique epitope in human BSP recognized by a monoclonal antibody, BSP 1.2 mab, which can be used for the unequivocal identification of BSP in normal and pathological human tissues.