Preparation of Cultured and Isolated Cells for X-Ray Microanalysis

Various electron microscopical preparation techniques are compared with regard to the preservation of the intracellular element distribution as determined by X-ray microanalysis in scanning and scanning transmission electron microscopy. By use of chemical agents for fixation and dehydration ions are redistributed and washed out. This is also true for freeze-substitution. Whole cells are prepared by cryofixation followed by freeze-drying. Interference of intracellular measurements by extracellular elements can be avoided by appropriate washing the cells before cryofixation. The washing medium has to be carefully selected in order to avoid distortions of the original intracellular element content. These problems are circumvented by the preparation of freeze-dried cryosections from cryofixed cells. This is demonstrated by data of the intracellular elemental composition in cultured cells (fibroblasts, Staphylococcus aureus bacteria) and in cells isolated from rat tissue (kidney papillary collecting duct and liver). Cryofixation of a single cell in a defined functional state is illustrated by results obtained from streaming Amoeba proteus cells, cryofixed under light microscopical control. The main conclusion is that X-ray microanalysis of cells in functional states requires cryofixation and cryopreparation techniques which have to be adapted to the particular cell biological problem to be investigated

Identification of Bone Marrow Cell Subpopulations Associated With Improved Functional Outcomes in Patients With Chronic Left Ventricular Dysfunction: An Embedded Cohort Evaluation of the FOCUS-CCTRN Trial

In the current study, we sought to identify bone marrow-derived mononuclear cell (BM-MNC) subpopulations associated with a combined improvement in left ventricular ejection fraction (LVEF), left ventricular end-systolic volume (LVESV), and maximal oxygen consumption (VO2 max) in patients with chronic ischemic cardiomyopathy 6 months after receiving transendocardial injections of autologous BM-MNCs or placebo. For this prospectively planned analysis, we conducted an embedded cohort study comprising 78 patients from the FOCUS-Cardiovascular Cell Therapy Research Network (CCTRN) trial. Baseline BM-MNC immunophenotypes and progenitor cell activity were determined by flow cytometry and colony-forming assays, respectively. Previously stable patients who demonstrated improvement in LVEF, LVESV, and VO2 max during the 6-month course of the FOCUS-CCTRN study (group 1, n = 17) were compared to those who showed no change or worsened in one to three of these endpoints (group 2, n = 61) and to a subset of patients from group 2 who declined in all three functional endpoints (group 2A, n = 11). Group 1 had higher frequencies of B-cell and CXCR4(+) BM-MNC subpopulations at study baseline than group 2 or 2A. Furthermore, patients in group 1 had fewer endothelial colony-forming cells and monocytes/macrophages in their bone marrow than those in group 2A. To our knowledge, this is the first study to show that in patients with ischemic cardiomyopathy, certain bone marrow-derived cell subsets are associated with improvement in LVEF, LVESV, and VO2 max at 6 months. These results suggest that the presence of both progenitor and immune cell populations in the bone marrow may influence the natural history of chronic ischemic cardiomyopathy-even in stable patients. Thus, it may be important to consider the bone marrow composition and associated regenerative capacity of patients when assigning them to treatment groups and evaluating the results of cell therapy trials

Clinical Performance of the Automated LIAISON® Meridian H. pylori SA Stool Antigen Test

Background. Antigens derived from Helicobacter pylori can be used as stool biomarkers to assist in the diagnosis of H. pylori infection. Since current assays have variable performance, we assessed the clinical performance of the automated LIAISON® Meridian H. pylori SA chemiluminescent immunoassay against more invasive biopsy tests that are considered to be the "gold standard" (Composite Reference Method). Methods. This prospective multisite study enrolled patients undergoing an esophagogastroduodenoscopy with collection of biopsy and stool specimens. Adult patients (≥22 years) participated in the study from February 2017 to August 2018. Specimens of the stomach were tested by three methods, known as the Composite Reference Method: (1) histological evaluation, (2) culture of the organism, and (3) rapid urease detection test. H. pylori in stool was detected using the automated LIAISON® Meridian H. pylori SA assay, a chemiluminescent immunoassay. Statistical analyses were performed using MedCalc 18.11.6. Results. 277 patients (63% female) were included in the study. The prevalence of infected subjects was 24.2% in this study cohort. Clinical performance assessed against the Composite Reference Method showed very good agreement (Cohen's kappa=0.922), with good sensitivity (95.5%) and specificity (97.6%). Reproducibility study results showed total imprecision ranging from 3.1% to 13.9% CV. Conclusion. The automated LIAISON® Meridian H. pylori SA assay brings reliable noninvasive testing for H. pylori to the laboratory that is in very good agreement with the current, more invasive biopsy-based methods such as histology, culture, or rapid urease test. The clinical trial identifiers are NCT03060746 (pretherapy) and NCT03060733 (posttherapy)

Mitochondrial DNA Regionalism and Historical Demography in the Extant Populations of Chirocephalus kerkyrensis (Branchiopoda: Anostraca)

Background: Mediterranean temporary water bodies are important reservoirs of biodiversity and host a unique assemblage of diapausing aquatic invertebrates. These environments are currently vanishing because of increasing human pressure. Chirocephalus kerkyrensis is a fairy shrimp typical of temporary water bodies in Mediterranean plain forests and has undergone a substantial decline in number of populations in recent years due to habitat loss. We assessed patterns of genetic connectivity and phylogeographic history in the seven extant populations of the species from Albania, Corfu Is. (Greece), Southern and Central Italy. Methodology/Principal Findings: We analyzed sequence variation at two mitochondrial DNA genes (Cytochrome Oxidase I and 16s rRNA) in all the known populations of C. kerkyrensis. We used multiple phylogenetic, phylogeographic and coalescence-based approaches to assess connectivity and historical demography across the whole distribution range of the species. C. kerkyrensis is genetically subdivided into three main mitochondrial lineages; two of them are geographically localized (Corfu Is. and Central Italy) and one encompasses a wide geographic area (Albania and Southern Italy). Most of the detected genetic variation (<81%) is apportioned among the aforementioned lineages. Conclusions/Significance: Multiple analyses of mismatch distributions consistently supported both past demographic and spatial expansions with the former predating the latter; demographic expansions were consistently placed during interglacial warm phases of the Pleistocene while spatial expansions were restricted to cold periods. Coalescence methods revealed a scenario of past isolation with low levels of gene flow in line with what is already known for other co-distributed fairy shrimps and suggest drift as the prevailing force in promoting local divergence. We recommend that these evolutionary trajectories should be taken in proper consideration in any effort aimed at protecting Mediterranean temporary water bodies

NISCO Cogeneration Facility

The NISCO Cogeneration facility utilizes two fluidized bed boilers to generate 200 MW of electricity and up to 80,000 LBS/HR of steam for process use. The partnership, of three industrial electricity users, Citgo, Conoco, and Vista Chemical, and the local utility, Gulf States utilities, was formed in the late 1980's. In August and September 1992 two fluidized bed boilers were brought into operation to repower existing turbine generating equipment. The fluidized bed units were designed to utilize 100 percent petroleum coke, a locally produced fuel. Petroleum coke is a high heating value, low volatile, high sulfur fuel which is difficult to utilize in conventional boilers. It is readily available in most areas throughout the world, including North and South America. Because of superior environmental performance, lower capital cost, and fuel versatility, circulating fluidized bed boilers were selected to repower the existing turbines. Fluidized bed boilers were ideally suited for a repowering application. Existing equipment matched or was modified for utilization in the project optimizing capital cost. The fluidized bed boilers, designed and fabricated by Foster Wheeler, are each capable of producing 825,000 LBS/HR of steam. This paper describes the results attained at NISCO during the first full year of operation. The design attributes of the project which enabled a successful and efficient unit startup are explained. Descriptions of design enhancements and modifications installed during the first year to improve the operability of the repowered facility are included. This paper describes technology and experiences of value to those considering steam generating unit repowering or construction

Diffusible ions in amebae studied by scanning electron microscopy and X-ray microanalysis

Calcium ions are involved in the regulation of ameboid movement but, so far, little is known about the distribution of calcium and other electrolyte ions in the amebae. This problem seems solvable by X-ray microanalysis with the scanning electron microscope (SEM). For this purpose Amoeba proteus was placed in the cultivation medium (Chalkey's solution) on the specimen stub covered with aluminum foil. Rapid freezing was done in liquid Freon 12 or propane precooled by liquid nitrogen. The specimens were either freeze-dried at -45° C for 15 hours and then coated with carbon or kept in the deep-frozen state throughout the preparation and investigation procedure by using a special cooling chain method. After coating ice was sublimated in the SEM under visual control until the ameboid structures became visible. Energy-dispersive microanalysis was done at 12.5 keV and 0.1 - 0.5 nA.</jats:p

X-ray microanalysis of cryofixed skeletal muscle in culture

X-ray microanalysis of electrolyte ions in cells and tissues involves the problem of preserving the distribution of diffusible substances throughout preparation and investigation in the scanning electron microscope. Cryofixation of objects thicker than about 200,um induces considerable freezing artifacts. The dissection of the specimen from the intact tissue also influences the distribution of diffusible ions. With respect to these problems, tissue culture cells seem advantageous, because they need not be dissected from their environment and are small enough to be frozen without ice crystal damage.Tissue cultures were grown from skeletal muscle of the hindleg of 20-day-old rat embryos on plastic coverslips. Small pieces of the specimens (diameter about 4 mm) were mounted on cupreous specimen stubs with conducting silver paint and frozen in liquid propane.</jats:p