Immobilization of microalgae cells in alginate facilitates isolation of DNA and RNA.

PubMed

Lopez, Blanca R; Hernandez, Juan-Pablo; Bashan, Yoav; de-Bashan, Luz E

2017-04-01

Isolation of nucleic acids from Chlorella is difficult, given the chemically complex nature of their cell walls and variable production of metabolites. Immobilization of microalgae in polymers adds additional difficulty. Here, we modified, amended, and standardized methods for isolation of nucleic acids and compared the yield of DNA and RNA from free-living and encapsulated microalgae C. sorokiniana. Isolation of nucleic acids from immobilized cells required two steps in dissolving the alginate matrix, releasing the cells, and mechanical disruption with glass beads. For DNA extraction, we used modified versions of a commercial kit along with the hexadecyltrimethylammonium bromide (CTAB) method. For RNA extraction, we used the commercial TRI reagent procedure and the CTAB-dithiotreitol method. Quantity and quality of nucleic acids in extracts varied with growth conditions, isolation procedures, and time of incubation of the original culture. There were consistently higher amounts of DNA and RNA in extracts from immobilized cells. Quantitatively, the modified procedure with the commercial Promega kit was the most reliable procedure for isolating DNA and a modified commercial TRI reagent procedure was the choice for isolating RNA. All four procedures eliminated proteins efficiently and had low levels of contamination from residual polysaccharides from the matrices and/or metabolites naturally produced by the microalgae. All DNA extracts under both growth conditions, time of incubation, and two isolation methods successfully amplified the 18S ribosomal RNA by PCR and quantitative reverse transcription (RT-qPCR). Copyright © 2017 Elsevier B.V. All rights reserved.

Isolation of Individual Egg Cells and Zygotes in Alstroemeria Followed by Manual Selection with a Microcapillary-connected Micropump

PubMed Central

HOSHINO, YOICHIRO; MURATA, NAHO; SHINODA, KOICHI

2006-01-01

• Aims To develop a procedure for isolating living egg cells and zygotes from Alstroemeria ovules. • Scope An attempt was made to isolate egg cells and zygotes from the ovules of Alstroemeria aurea. The ovules were histologically observed using a clearing procedure which revealed the localization and sizes of the embryo sacs and egg apparatus within the ovules. For the isolation of egg cells, ovules were cut into sections with a surgical blade and treated with an enzyme solution. Subsequently, these ovule sections were dissected using a glass needle under an inverted microscope. Egg cells successfully isolated by this procedure were collected using microcapillaries connected to a micropump. For zygote isolation, ovules were excised from ovaries 24 h after self-pollination. By treating excised ovules with an enzyme solution and subsequently dissecting them using a glass needle, zygotes were successfully isolated from the ovules and collected with a microcapillary. The isolated zygotes were associated with pollen tubes and one of the synergids. Egg cells and zygotes were viable for up to 2 h following isolation, as determined by fluorescein diacetate staining. • Conclusions The procedures for isolating egg cells and zygotes in Alstroemeria were established, and each egg cell and zygote was captured with a microcapillary. PMID:16621859

Remote magnetic navigation for circumferential pulmonary vein ablation: single-catheter technique or additional use of a circular mapping catheter?

PubMed

Vollmann, Dirk; Lüthje, Lars; Seegers, Joachim; Sohns, Christian; Sossalla, Samuel; Sohns, Jan; Röver, Christian; Hasenfuß, Gerd; Zabel, Markus

2014-10-01

Remote magnetic navigation (RMN) is utilized for catheter guidance during pulmonary vein ablation (PVA). We aimed to determine whether the additional use of a circular mapping catheter (CMC) influences efficacy and outcome of RMN-guided PVA. A total of 80 consecutive subjects (65 % male, age 62 ± 9 years) underwent circumferential PVA with a 3D mapping system and an RMN-guided irrigated catheter. Procedural endpoint was complete PV isolation (PVI), total radiofrequency (RF) time >60 min, or procedure duration >5 h. PVI was defined as an entrance and/or exit block, diagnosed with a CMC within the PV ostium or by pacing via the roving RMN-guided catheter (single-catheter technique). Prolonged Holter monitoring after 3 and 6 months was used to detect atrial tachyarrhythmia (AT/AF) recurrences. Complete PVI was achieved in 56 % (45/80) of all subjects (isolated PVs per patient, 3.1 ± 1.2; RF time, 56.3 ± 17.2 min; procedure duration, 3.8 ± 0.8 h). Prospective validation of the single-catheter technique for diagnosing PVI demonstrated high concordance (94 %) with blinded CMC results. CMC use in first-time PVA was associated with similar total RF and procedure times but higher PV isolation rate. Upon multivariate analysis, CMC use, female gender, left PV, smaller PV ostium and repeat PVA predicted PVI during RMN-guided ablation. Persistent AF and mitral regurgitation at baseline and the number of non-isolated PVs predicted AT/AF recurrence during follow-up. Concomitant CMC use for first-time, RMN-guided PVA is associated with similar procedure duration but higher PV isolation rates as compared to a single-catheter approach. Since the number of isolated PVs predicts freedom from AT/AF, CMC utilization appears advisable for first-time, RMN-guided PVA.

Optimization of Unnicked β2-Glycoprotein I and High Avidity Anti-β2-Glycoprotein I Antibodies Isolation

PubMed Central

Artenjak, Andrej; Leonardi, Adrijana; Križaj, Igor; Ambrožič, Aleš; Sodin-Semrl, Snezna; Božič, Borut; Čučnik, Saša

2014-01-01

Patient biological material for isolation of β2-glycoprotein I (β2GPI) and high avidity IgG anti-β2-glycoprotein I antibodies (HAv anti-β2GPI) dictates its full utilization. The aim of our study was to evaluate/improve procedures for isolation of unnicked β2GPI and HAv aβ2GPI to gain unmodified proteins in higher yields/purity. Isolation of β2GPI from plasma was a stepwise procedure combining nonspecific and specific methods. For isolation of polyclonal HAv aβ2GPI affinity chromatographies with immobilized protein G and human β2GPI were used. The unknown protein found during isolation was identified by liquid chromatography electrospray ionization mass spectrometry and the nonredundant National Center for Biotechnology Information database. The average mass of the isolated unnicked purified β2GPI increased from 6.56 mg to 9.94 mg. In the optimized isolation procedure the high molecular weight protein (proteoglycan 4) was successfully separated from β2GPI in the 1st peaks with size exclusion chromatography. The average efficiency of the isolation procedure for polyclonal HAv anti-β2GPI from different matrixes was 13.8%, as determined by our in-house anti-β2GPI ELISA. We modified the in-house isolation and purification procedures of unnicked β2GPI and HAv anti-β2GPI, improving the purity of antigen and antibodies as well as increasing the number of tests routinely performed with the in-house ELISA by ~50%. PMID:24741579

Evaluation of molecular markers for Phytophthora ramorum detection and identification: testing for specificity using a standardized library of isolates.

PubMed

Martin, F N; Coffey, M D; Zeller, K; Hamelin, R C; Tooley, P; Garbelotto, M; Hughes, K J D; Kubisiak, T; Bilodeau, G J; Levy, L; Blomquist, C; Berger, P H

2009-04-01

Given the importance of Phytophthora ramorum from a regulatory standpoint, it is imperative that molecular markers for pathogen detection are fully tested to evaluate their specificity in detection of the pathogen. In an effort to evaluate 11 reported diagnostic techniques, we assembled a standardized DNA library using accessions from the World Phytophthora Genetic Resource Collection for 315 isolates representing 60 described Phytophthora spp. as well as 11 taxonomically unclassified isolates. These were sent blind to collaborators in seven laboratories to evaluate published diagnostic procedures using conventional (based on internal transcribed spacer [ITS] and cytochrome oxidase gene [cox]1 and 2 spacer regions) and real-time polymerase chain reaction (based on ITS and cox1 and 2 spacer regions as well as beta-tubulin and elicitin genes). Single-strand conformation polymorphism (SSCP) analysis using an automated sequencer for data collection was also evaluated for identification of all species tested. In general, the procedures worked well, with varying levels of specificity observed among the different techniques. With few exceptions, all assays correctly identified all isolates of P. ramorum and low levels of false positives were observed for the mitochondrial cox spacer markers and most of the real-time assays based on nuclear markers (diagnostic specificity between 96.9 and 100%). The highest level of false positives was obtained with the conventional nested ITS procedure; however, this technique is not stand-alone and is used in conjunction with two other assays for diagnostic purposes. The results indicated that using multiple assays improved the accuracy of the results compared with looking at a single assay alone, in particular when the markers represented different genetic loci. The SSCP procedure accurately identified P. ramorum and was helpful in classification of a number of isolates to a species level. With one exception, all procedures accurately identified P. ramorum in blind evaluations of 60 field samples that included examples of plant infection by 11 other Phytophthora spp. The SSCP analysis identified eight of these species, with three identified to a species group.

Methods of determination of periods in the motion of asteroids

NASA Astrophysics Data System (ADS)

Bien, R.; Schubart, J.

Numerical techniques for the analysis of fundamental periods in asteroidal motion are evaluated. The specific techniques evaluated were: the periodogram analysis procedure of Wundt (1980); Stumpff's (1937) system of algebraic transformations; and Labrouste's procedure. It is shown that the Labrouste procedure permitted sufficient isolation of single oscillations from the quasi-periodic process of asteroidal motion. The procedure was applied to the analysis of resonance in the motion of Trojan-type and Hilda-type asteroids, and some preliminary results are discussed.

Diphoton production at the LHC: a QCD study up to NNLO

NASA Astrophysics Data System (ADS)

Catani, Stefano; Cieri, Leandro; de Florian, Daniel; Ferrera, Giancarlo; Grazzini, Massimiliano

2018-04-01

We consider the production of prompt-photon pairs at the LHC and we report on a study of QCD radiative corrections up to the next-to-next-to-leading order (NNLO). We present a detailed comparison of next-to-leading order (NLO) results obtained within the standard and smooth cone isolation criteria, by studying the dependence on the isolation parameters. We highlight the role of different partonic subprocesses within the two isolation criteria, and we show that they produce large radiative corrections for both criteria. Smooth cone isolation is a consistent procedure to compute QCD radiative corrections at NLO and beyond. If photon isolation is sufficiently tight, we show that the NLO results for the two isolation procedures are consistent with each other within their perturbative uncertainties. We then extend our study to NNLO by using smooth cone isolation. We discuss the impact of the NNLO corrections and the corresponding perturbative uncertainties for both fiducial cross sections and distributions, and we comment on the comparison with some LHC data. Throughout our study we remark on the main features that are produced by the kinematical selection cuts that are applied to the photons. In particular, we examine soft-gluon singularities that appear in the perturbative computations of the invariant mass distribution of the photon pair, the transverse-momentum spectra of the photons, and the fiducial cross section with asymmetric and symmetric photon transverse-momentum cuts, and we present their behaviour in analytic form.

Integrated HPTLC-based Methodology for the Tracing of Bioactive Compounds in Herbal Extracts Employing Multivariate Chemometrics. A Case Study on Morus alba.

PubMed

Chaita, Eliza; Gikas, Evagelos; Aligiannis, Nektarios

2017-03-01

In drug discovery, bioassay-guided isolation is a well-established procedure, and still the basic approach for the discovery of natural products with desired biological properties. However, in these procedures, the most laborious and time-consuming step is the isolation of the bioactive constituents. A prior identification of the compounds that contribute to the demonstrated activity of the fractions would enable the selection of proper chromatographic techniques and lead to targeted isolation. The development of an integrated HPTLC-based methodology for the rapid tracing of the bioactive compounds during bioassay-guided processes, using multivariate statistics. Materials and Methods - The methanol extract of Morus alba was fractionated employing CPC. Subsequently, fractions were assayed for tyrosinase inhibition and analyzed with HPTLC. PLS-R algorithm was performed in order to correlate the analytical data with the biological response of the fractions and identify the compounds with the highest contribution. Two methodologies were developed for the generation of the dataset; one based on manual peak picking and the second based on chromatogram binning. Results and Discussion - Both methodologies afforded comparable results and were able to trace the bioactive constituents (e.g. oxyresveratrol, trans-dihydromorin, 2,4,3'-trihydroxydihydrostilbene). The suggested compounds were compared in terms of R f values and UV spectra with compounds isolated from M. alba using typical bioassay-guided process. Chemometric tools supported the development of a novel HPTLC-based methodology for the tracing of tyrosinase inhibitors in M. alba extract. All steps of the experimental procedure implemented techniques that afford essential key elements for application in high-throughput screening procedures for drug discovery purposes. Copyright © 2017 John Wiley & Sons, Ltd. Copyright © 2017 John Wiley & Sons, Ltd.

Coagulase gene typing of Staphylococcus aureus isolated from cows with mastitis in southeastern Brazil

PubMed Central

2005-01-01

Abstract A typing procedure based on polymorphism of the coagulase gene (coa) was used to discriminate Staphylococcus aureus isolated from Minas Gerais dairy cows with mastitis. Amplification of the gene from the 64 S. aureus isolates produced 27 different polymerase chain reaction (PCR) products; 60 isolates showed only 1 amplicon, and 4 showed 2 amplicons. The isolates were grouped into 49 types by analyzing the restriction fragment length polymorphism (RFLP) of the coa gene; the 10 most common types accounted for 39% of the isolates. The results demonstrate that many variants of the coa gene are present in the studied region, although only a few predominate. PMID:16479723

Mutant Enrichment in the Colonial Alga, EUDORINA ELEGANS

PubMed Central

Toby, A. L.; Kemp, C. L.

1975-01-01

An enrichment procedure has been developed that results in at least a 200x increase in mutation frequency in the colonial alga, Eudorina elegans. A period of nitrogen starvation followed by treatment with 8-azaguanine results in the death of wild-type cells and the maintenance of mutants. N'-nitro-N-nitro-soguanidine-induced acetate, p-aminobenzoic acid and reduced nitrogen requiring mutants have been isolated by this procedure. PMID:1205128

Predictive value of the "clue cells" investigation and the amine volatilization test in vaginal infections caused by Gardnerella vaginalis.

PubMed Central

Marquez-Davila, G; Martinez-Barreda, C E

1985-01-01

Although still controversial, an etiologic role of Gardnerella vaginalis is imputed in vaginitis. Besides isolation of the organism by culture, two alternative diagnostic procedures have been claimed to be useful: the investigation of "clue cells" in clinical specimens and the amine volatilization test or fishy odor perception in genital secretions. Herein we report on the findings of the simultaneous use of G. vaginalis isolation, the clue cell test and amine volatilization perception in specimens from 1,263 consecutive female patients referred to our clinic. Our results show that the simultaneous use of both alternative tests is very useful as a screening procedure. A negative result of both tests predicts a negative culture result in 99% of the cases. However, a positive result of either or both should be considered as an indication to proceed to culture and not as diagnostic of infection. PMID:3878365

Trends in Aortic Valve Replacement Procedures Between 2009 and 2015: Has Transcatheter Aortic Valve Replacement Made a Difference?

PubMed

Culler, Steven D; Cohen, David J; Brown, Phillip P; Kugelmass, Aaron D; Reynolds, Matthew R; Ambrose, Karen; Schlosser, Michael L; Simon, April W; Katz, Marc R

2018-04-01

This study reports trends in volume and adverse events associated with isolated aortic valve procedures performed in Medicare beneficiaries between 2009 and 2015. This retrospective study used the annual fiscal year Medicare Provider Analysis and Review file to identify all Medicare beneficiaries undergoing an isolated aortic valve procedure. Outcome measures included three mortality rates and nine in-hospital adverse events. The final study population consisted of 233,660 hospitalizations. During the study period, Medicare beneficiaries undergoing an aortic valve procedure increased from 22,076 to 49,362, for an average annual growth rate of 14.45%. Transcatheter aortic valve replacement (TAVR) procedures per 100,000 Medicare beneficiaries grew from 10.7 in 2012 to 41.1 in 2015. Overall, in-hospital mortality rates, cumulative 30-day mortality rates, and 90-day postdischarge mortality rates declined annually during the study period. However, the 90-day mortality rate for TAVR was nearly double the rate for the tissue surgical aortic valve replacement group. Nearly 68% of Medicare beneficiaries experienced at least one in-hospital adverse event during their index hospitalization. Medicare beneficiaries undergoing TAVR had the lowest observed adverse events rates among the aortic valve procedures in 2015. The total number of Medicare beneficiaries undergoing isolated aortic valve procedures increased from 47.5 to 88.9 per 100,000 Medicare beneficiaries during the study period. Aortic valve procedures increased significantly during this study period primarily due to the increase in TAVR, with clinical outcomes improving as well. Although long-term outcomes of TAVR are still under investigation, these results are promising. Copyright © 2018 The Society of Thoracic Surgeons. Published by Elsevier Inc. All rights reserved.

[The isolated perfused porcine kidney model for investigations concerning surgical therapy procedures].

PubMed

Peters, Kristina; Michel, Maurice Stephan; Matis, Ulrike; Häcker, Axel

2006-01-01

Experiments to develop innovative surgical therapy procedures are conventionally conducted on animals, as crucial aspects like tissue removal and bleeding disposition cannot be investigated in vitro. Extracorporeal organ models however reflect these aspects and could thus reduce the use of animals for this purpose fundamentally in the future. The aim of this work was to validate the isolated perfused porcine kidney model with regard to its use for surgical purposes on the basis of histological and radiological procedures. The results show that neither storage nor artificial perfusion led to any structural or functional damage which would affect the quality of the organ. The kidney model is highly suitable for simulating the main aspects of renal physiology and allows a constant calibration of perfusion pressure and tissue temperature. Thus, with only a moderate amount of work involved, the kidney model provides a cheap and readily available alternative to conventional animal experiments; it allows standardised experimental settings and provides valid results.

An efficient protocol for tissue sampling and DNA isolation from the stem bark of Leguminosae trees.

PubMed

Novaes, R M L; Rodrigues, J G; Lovato, M B

2009-02-03

Traditionally, molecular studies of plant species have used leaves as the source of DNA. However, sampling leaves from tall tree species can be quite difficult and expensive. We developed a sequence of procedures for using stem bark as a source of DNA from Leguminosae trees of the Atlantic Forest and the Cerrado. Leguminosae is an important species-rich family in these two highly diverse and endangered biomes. A modified CTAB protocol for DNA isolation is described, and details of the procedures for sampling and storage of the bark are given. The procedures were initially developed for three species, and then their applicability for 15 other species was evaluated. DNA of satisfactory quality was obtained from the bark of all species. The amounts of DNA obtained from leaves were slightly higher than from bark samples, while its purity was the same. Storing the bark frozen or by drying in silica gel yielded similar results. Polymerase chain reaction amplification worked for both plastid and nuclear genomes. This alternative for isolating DNA from bark samples of trees facilitates field work with these tree species.

[Cryoballon isolation of the pulmonary veins in atrial fibrillation: mid-term results after the first 55 patients].

PubMed

Tóth, Zsuzsanna; Nagy-Baló, Edina; Kertész, Attila; Clemens, Marcell; Herczku, Csaba; Tint, Diana; Kun, Csaba; Edes, István; Csanádi, Zoltán

2010-01-31

Several transcatheter techniques based on radiofrequency energy were elaborated for the treatment of atrial fibrillation through the last decade. Recently, similar success rates with a better safety profile concerning life threatening complications were reported with the novel methode of cryoballon isolation of the pulmonary veins. This paper summarizes our initial experience with cryoballon ablation after the first 55 patients. [corrected] Symptomatic patients refractory to aniarrhythmic medication mostly with paroxysmal atrial fibrillation without significant structural heart disease were enrolled. Cannulation and isolation of all pulmonary veins were attempted using a 28 mm double-wall cryoballon inflated at the ostium of the vein and abolishing eletrical activity of atrial tissue around its perimeter by freezing to -70 C. Intravenous heparin during and oral anticoagulant after the procedure was administered. Conventional ECGs, Holter ECGs and transtelephonic ECG recordings were used through 6 months follow-up for rhythm monitoring. In 55 patients enrolled (18 female; age: 56 + or - 33,64 years) 165 out ot 192 (86%) pulmonary veins were successfully isolated. All pulmonary veins were isolated in 37 patients (67%). Procedure time was 155.67 + or - 100.66 min, while fluoroscopy time was 34.04 + or - 31.89 min. In 34 patients with 6 months follow-up 24 (70%) either remained free of arrhythmia (17 patients) or had a significant decrease in arrhythmia burden (7 patients). Based on our initial experience, cryoballon isolation of pulmonary veins appears to be a more simple procedure with similar efficacy to radiofrequency ablation in the treatment of atrial fibrillation.

Mouse Liver Mitochondria Isolation, Size Fractionation, and Real-time MOMP Measurement.

PubMed

Renault, Thibaud T; Luna-Vargas, Mark P A; Chipuk, Jerry E

2016-08-05

The mitochondrial pathway of apoptosis involves a complex interplay between dozens of proteins and lipids, and is also dependent on the shape and size of mitochondria. The use of cellular models in past studies has not been ideal for investigating how the complex multi-factor interplay regulates the molecular mechanisms of mitochondrial outer membrane permeabilization (MOMP). Isolated systems have proven to be a paradigm to deconstruct MOMP into individual steps and to study the behavior of each subset of MOMP regulators. In particular, isolated mitochondria are key to in vitro studies of the BCL-2 family proteins, a complex family of pro-survival and pro-apoptotic proteins that directly control the mitochondrial pathway of apoptosis (Renault et al ., 2013). In this protocol, we describe three complementary procedures for investigating in real-time the effects of MOMP regulators using isolated mitochondria. The first procedure is "Liver mitochondria isolation" in which the liver is dissected from mice to obtain mitochondria. "Mitochondria labeling with JC-1 and size fractionation" is the second procedure that describes a method to label, fractionate by size and standardize subpopulations of mitochondria. Finally, the "Real-time MOMP measurements" protocol allows to follow MOMP in real-time on isolated mitochondria. The aforementioned procedures were used to determine in vitro the role of mitochondrial membrane shape at the level of isolated cells and isolated mitochondria (Renault et al ., 2015).

Comparison of various molecular methods for rapid differentiation of intestinal bifidobacteria at the species, subspecies and strain level.

PubMed

Jarocki, Piotr; Podleśny, Marcin; Komoń-Janczara, Elwira; Kucharska, Jagoda; Glibowska, Agnieszka; Targoński, Zdzisław

2016-07-22

Members of the genus Bifidobacterium are anaerobic Gram-positive Actinobacteria, which are natural inhabitants of human and animal gastrointestinal tract. Certain bifidobacteria are frequently used as food additives and probiotic pharmaceuticals, because of their various health-promoting properties. Due to the enormous demand on probiotic bacteria, manufacture of high-quality products containing living microorganisms requires rapid and accurate identification of specific bacteria. Additionally, isolation of new industrial bacteria from various environments may lead to multiple isolations of the same strain, therefore, it is important to apply rapid, low-cost and effective procedures differentiating bifidobacteria at the intra-species level. The identification of new isolates using microbiological and biochemical methods is difficult, but the accurate characterization of isolated strains may be achieved using a polyphasic approach that includes classical phenotypic methods and molecular procedures. However, some of these procedures are time-consuming and cumbersome, particularly when a large group of new isolates is typed, while some other approaches may have too low discriminatory power to distinguish closely related isolates obtained from similar sources. This work presents the evaluation of the discriminatory power of four molecular methods (ARDRA, RAPD-PCR, rep-PCR and SDS-PAGE fingerprinting) that are extensively used for fast differentiation of bifidobacteria up to the strain level. Our experiments included 17 reference strains and showed that in comparison to ARDRA, genotypic fingerprinting procedures (RAPD and rep-PCR) seemed to be less reproducible, however, they allowed to differentiate the tested microorganisms even at the intra-species level. In general, RAPD and rep-PCR have similar discriminatory power, though, in some instances more than one oligonucleotide needs to be used in random amplified polymorphic DNA analysis. Moreover, the results also demonstrated a high discriminatory power of SDS-PAGE fingerprinting of whole-cell proteins. On the other hand, the protein profiles obtained were rather complex, and therefore, difficult to analyze. Among the tested procedures, rep-PCR proved to be the most effective and reliable method allowing rapid differentiation of Bifidobacterium strains. Additionally, the use of the BOXA1R primer in the differentiation of 21 Bifidobacterium strains, newly isolated from infant feces, demonstrated slightly better discriminatory power in comparison to PCR reactions with the (GTG)5 oligonucleotide. Thus, BOX-PCR turned out to be the most appropriate and convenient molecular technique in differentiating Bifidobacterium strains at all taxonomic levels.

Legionella pneumophila Toxin, Isolation and Purification

DTIC Science & Technology

1981-01-01

which dis- plays an in vivo lethality. The purification procedures involve acid precipitation, gel chromatography, and preparative isotachophoresis. The...Chymotrypsinogen A, Ribonuclease A, and Apoprotinin as markers. Preparation of antiserum One milliqram amounts of protein from Le jonella acid ...RESULTS Toxin isolation Step 1: Acid precipitation of crude toxin. 1.0 N HCl acid was slowly added to rapidly stirred crude toxin until pH 3.5 was

Isolating cells from female/male blood mixtures using florescence in situ hybridization combined with low volume PCR and its application in forensic science.

PubMed

Feng, Lei; Li, Cai-Xia; Han, Jun-Ping; Xu, Cheng; Hu, Lan

2015-11-01

To obtain single-source short tandem repeat (STR) profiles in trace female/male blood mixture samples, we combined florescence in situ hybridization (FISH), laser microdissection, and low volume PCR (LV-PCR) to isolate male/female cells and improve sensitivity. The results showed that isolation of as few as 10 leukocytes was sufficient to yield full STR profiles in fresh female or male blood samples for 32 independent tests with a low additional alleles rate (3.91%) and drop-out alleles rate (5.01%). Moreover, this procedure was tested in two fresh blood mixture series at three ratios (1:5, 1:10, and 1:20), two mock female/male blood mixture casework samples, and one practical casework sample. Male and female STR profiles were successfully detected in all of these samples, showing that this procedure could be used in forensic casework in the future.

German Heart Surgery Report 2016: The Annual Updated Registry of the German Society for Thoracic and Cardiovascular Surgery.

PubMed

Beckmann, Andreas; Funkat, Anne-Katrin; Lewandowski, Jana; Frie, Michael; Ernst, Markus; Hekmat, Khosro; Schiller, Wolfgang; Gummert, Jan F; Harringer, Wolfgang

2017-10-01

Based on a long-standing voluntary registry founded by the German Society for Thoracic and Cardiovascular Surgery (GSTCVS), well-defined data of all cardiac, thoracic, and vascular surgery procedures performed in 78 German heart surgery departments during the year 2016 are analyzed. In 2016, a total of 103,128 heart surgery procedures (implantable defibrillator, pacemaker, and extracardiac procedures excluded) were submitted to the registry. Approximately 15.7% of the patients were at least 80 years of age, resulting in an increase of 0.9% compared with the data of 2015. For 37,614 isolated coronary artery bypass grafting procedures (relationship on-/off-pump 4.4:1), an unadjusted in-hospital mortality of 2.9% was observed. Concerning the 33,451 isolated heart valve procedures (including 11,701 catheter-based procedures), the unadjusted in-hospital mortality was 4.3%. This annual updated registry of the GSTCVS represents voluntary public reporting by accumulating actual information for nearly all heart surgical procedures in Germany, describes advancements in heart medicine, and is a basis for internal and external quality assurances for all participants. In addition, the registry demonstrates that the provision of cardiac surgery in Germany is appropriate and patients are treated nationwide at all times.

Isolation of xanthone and benzophenone derivatives from Cyclopia genistoides (L.) Vent. (honeybush) and their pro-apoptotic activity on synoviocytes from patients with rheumatoid arthritis.

PubMed

Kokotkiewicz, Adam; Luczkiewicz, Maria; Pawlowska, Justyna; Luczkiewicz, Piotr; Sowinski, Pawel; Witkowski, Jacek; Bryl, Ewa; Bucinski, Adam

2013-10-01

A fast and efficient method for the isolation of the C-glucosidated xanthones mangiferin and isomangiferin from the South-African plant Cyclopia genistoides was developed for the first time. The procedure involved extraction, liquid-liquid partitioning with ethyl acetate and subsequent precipitation of mangiferin and isomangiferin from methanol and acetonitrile-water fractions, respectively. Additionally, two benzophenone derivatives: 3-C-β-glucosides of maclurin and iriflophenone, were isolated from C. genistoides extracts using semi-preparative HPLC. Apart from the above, the isolation procedure also yielded hesperidin and small amounts of luteolin. The structures of the compounds were determined by 1D and 2D NMR experiments and/or LC-DAD-ESI-MS. The selected Cyclopia constituents were screened for pro-apoptotic activity on TNF-α-stimulated synovial cells isolated from rheumatoid arthritis patients. The strongest effect, measured as percent of apoptotic cells, was recorded for isomangiferin (75%), followed by iriflophenone 3-C-β-glucoside (71%), hesperidin (67%) and mangiferin (65%). The results are encouraging for further studies on the use of the above compounds in the treatment of rheumatoid arthritis. © 2013.

Procedure for the Isolation of Endothelial Cells from Human Cerebral Arteriovenous Malformation (cAVM) Tissues.

PubMed

Hao, Qiang; Chen, Xiao-Lin; Ma, Li; Wang, Tong-Tong; Hu, Yue; Zhao, Yuan-Li

2018-01-01

In this study, we successfully established a stable method for the isolation of endothelial cells (ECs) from human cerebral arteriovenous malformation (cAVM) tissues. Despite human cAVM tissues having a minor population of ECs, they play an important role in the manifestation and development of cAVM as well as in hemorrhagic stroke and thrombogenesis. To characterize and understand the biology of ECs in human cAVM (cAVM-ECs), methods for the isolation and purification of these cells are necessary. We have developed this method to reliably obtain pure populations of ECs from cAVMs. To obtain pure cell populations, cAVM tissues were mechanically and enzymatically digested and the resulting single cAVM-ECs suspensions were then labeled with antibodies of specific cell antigens and selected by flow cytometry. Purified ECs were detected using specific makers of ECs by immunostaining and used to study different cellular mechanisms. Compared to the different methods of isolating ECs from tissues, we could isolate ECs from cAVMs confidently, and the numbers of cAVM-ECs harvested were almost similar to the amounts present in vessel components. In addition to optimizing the protocol for isolation of ECs from human cAVM tissues, the protocol could also be applied to isolate ECs from other human neurovascular-diseased tissues. Depending on the tissues, the whole procedure could be completed in about 20 days.

Response of selected microorganisms to experimental planetary environments

NASA Technical Reports Server (NTRS)

Foster, T. L.; Helms, S. J. S.; Kirschner, L. E.; Stevens, W. C.; Talley, L. K.; Winans, L., Jr.

1976-01-01

The anaerobic utilization of phosphite or phosphine and the significance of this conversion to potential contamination of Jupiter were investigated. A sporeforming organism was isolated from Cape Canaveral soil which anaerobically converts hypophosphite to phosphate. This conversion coincides with an increase in turbidity of the culture and with phosphate accumulation in the medium. Investigations of omnitherms (organisms which grow over a broad temperature range, i.e. 3 -55 C were also conducted. The cellular morphology of 28 of these isolates was investigated, and all were demonstrated to be sporeformers. Biochemical characterizations are also presented. Procedures for replicate plating were evaluated, and those results are also presented. The procedures for different replicate-plating techniques are presented, and these are evaluated on the basis of reproducibility, percentage of viable transfer, and ease of use. Standardized procedures for the enumeration of microbial populations from ocean-dredge samples from Cape Canaveral are also presented.

Rubber dam isolation of cervical lesions. Part 1: Alternative techniques which avoid injury to the periodontium.

PubMed

Liebenberg, W H

1994-01-01

While it is arguable that the operative procedures depicted in this article could have been done equally well with alternative forms of isolation, it is indisputable that rubber dam provides for the most thorough 'isolation' in the comprehensive sense of the term. It allows for the finite control over peripheral variables such as access and controlled gingival retraction. This in turn permits meticulous execution of restorative procedures within the luxury of 'true four-handed dentistry'. Most operative procedures are done within the limitations of single handed dentistry as the other 'three hands' are involved with retraction, fluid evacuation and access control. The rubber dam is indeed indispensable and with the increased awareness of infection control it is conceivable that rubber dam isolation will continue to be associated with quality patient care. Part 2 of this article will detail the application of the 'Modified Gingival Retractor' in the isolation of cervical lesions.

Mouse Liver Mitochondria Isolation, Size Fractionation, and Real-time MOMP Measurement

PubMed Central

Renault, Thibaud T.; Luna-Vargas, Mark P.A.; Chipuk, Jerry E.

2016-01-01

The mitochondrial pathway of apoptosis involves a complex interplay between dozens of proteins and lipids, and is also dependent on the shape and size of mitochondria. The use of cellular models in past studies has not been ideal for investigating how the complex multi-factor interplay regulates the molecular mechanisms of mitochondrial outer membrane permeabilization (MOMP). Isolated systems have proven to be a paradigm to deconstruct MOMP into individual steps and to study the behavior of each subset of MOMP regulators. In particular, isolated mitochondria are key to in vitro studies of the BCL-2 family proteins, a complex family of pro-survival and pro-apoptotic proteins that directly control the mitochondrial pathway of apoptosis (Renault et al., 2013). In this protocol, we describe three complementary procedures for investigating in real-time the effects of MOMP regulators using isolated mitochondria. The first procedure is “Liver mitochondria isolation” in which the liver is dissected from mice to obtain mitochondria. “Mitochondria labeling with JC-1 and size fractionation” is the second procedure that describes a method to label, fractionate by size and standardize subpopulations of mitochondria. Finally, the “Real-time MOMP measurements” protocol allows to follow MOMP in real-time on isolated mitochondria. The aforementioned procedures were used to determine in vitro the role of mitochondrial membrane shape at the level of isolated cells and isolated mitochondria (Renault et al., 2015). PMID:28093578

Isolation and characterization of mammalian eumelanins from hair and irides.

PubMed

Novellino, L; Napolitano, A; Prota, G

2000-07-26

A new enzymatic procedure was developed for isolation of eumelanin from black human hair which might provide a substantially intact pigment for structural characterization. Sequential digestion with protease, proteinase K and papaine in the presence of dithiothreitol afforded a pigment with a 6% w/w protein content. HPLC analysis of pyrrole acids resulting from alkaline H(2)O(2) degradation, carboxyl content determination, and ferricyanide titration showed that the isolated pigment is made up of 5,6-dihydroxyindole (DHI)- and 5, 6-dihydroxyindole-2-carboxylic acid (DHICA)-derived units at a 6:1 ratio, exhibiting a significant degree of oxidative degradation. For comparison, a different eumelanin isolated from black bovine irides by a similar enzymatic procedure was analyzed. Matrix-assisted laser desorption ionization (MALDI) mass spectrometry of the final pigment provided evidence for homologous series of DHICA oligomers, while chemical analysis allowed an estimate of 2:1 DHICA/DHI-derived units in the polymer, with a substantial proportion of intact o-diphenolic functions. Iris melanin proved able to promote the Fenton oxidation of deoxyribose while hair melanin was ineffective. Overall, these results provide, for the first time, unambiguous evidence for marked structural differences of mammalian eumelanins which may be directly related to the diversity of the sites of biosynthesis and storage, as well as to functional role of these pigments.

The simultaneous isolation of multiple high and low frequent T-cell populations from donor peripheral blood mononuclear cells using the major histocompatibility complex I-Streptamer isolation technology.

PubMed

Roex, Marthe C J; Hageman, Lois; Heemskerk, Matthias T; Veld, Sabrina A J; van Liempt, Ellis; Kester, Michel G D; Germeroth, Lothar; Stemberger, Christian; Falkenburg, J H Frederik; Jedema, Inge

2018-04-01

Adoptive transfer of donor-derived T cells can be applied to improve immune reconstitution in immune-compromised patients after allogeneic stem cell transplantation. The separation of beneficial T cells from potentially harmful T cells can be achieved by using the major histocompatibility complex (MHC) I-Streptamer isolation technology, which has proven its feasibility for the fast and pure isolation of T-cell populations with a single specificity. We have analyzed the feasibility of the simultaneous isolation of multiple antigen-specific T-cell populations in one procedure by combining different MHC I-Streptamers. First, the effect of combining different amounts of MHC I-Streptamers used in the isolation procedure on the isolation efficacy of target antigen-specific T cells and on the number of off-target co-isolated contaminating cells was assessed. The feasibility of this approach was demonstrated in large-scale validation procedures targeting both high and low frequent T-cell populations using the Good Manufacturing Practice (GMP)-compliant CliniMACS Plus device. T-cell products targeting up to 24 different T-cell populations could be isolated in one, simultaneous MHC I-Streptamer procedure, by adjusting the amount of MHC I- Streptamers per target antigen-specific T-cell population. Concurrently, the co-isolation of potentially harmful contaminating T cells remained below our safety limit. This technology allows the reproducible isolation of high and low frequent T-cell populations. However, the expected therapeutic relevance of direct clinical application without in vitro expansion of these low frequent T-cell populations is questionable. This study provides a feasible, fast and safe method for the generation of highly personalized MHC I-Streptamer isolated T-cell products for adoptive immunotherapy. Copyright © 2018 International Society for Cellular Therapy. Published by Elsevier Inc. All rights reserved.

Implementation of transcatheter aortic valve replacement in California: Influence on aortic valve surgery.

PubMed

Maximus, Steven; Milliken, Jeffrey C; Danielsen, Beate; Shemin, Richard; Khan, Junaid; Carey, Joseph S

2018-04-01

Transcatheter aortic valve replacement (TAVR) procedures were introduced in 2011. Initially, procedures were limited to patients who were not surgical candidates, but subsequently high-risk surgical candidates were considered for TAVR. The influence on aortic valve surgery in California is unknown. The California Office of Statewide Health Planning and Development hospitalized patient discharge database was queried for the years 2009 through 2014. isolated surgical aortic valve and aortic valve/coronary artery bypass graft (SAVR) and TAVR procedures were identified by International Classification of Diseases-9th revision clinical modification procedure codes. Seven TAVR programs were introduced in 2011, 12 in 2012, 3 in 2013, and 6 in 2014. SAVR procedure volumes were compared from the 2 years before institution with SAVR volumes during the year(s) after institution of the TAVR program in these 28 hospitals. Overall, surgical volumes increased during the first, second, and third years after implementation of TAVR procedures. Among 7 hospitals with 4-year programs, surgical volumes increased to a maximum of 15.5% during the third year, then began to decrease. The hospital performing the largest number of TAVR procedures showed a marked decrease in SAVR volume by the fourth year, suggesting a shift of SAVR candidates to TAVR. Among all hospitals with 4-year programs, TAVR exceeded SAVR procedures by the fourth year. In California overall, SAVR increased during 2011 through 2013, due primarily to increasing volume of isolated SAVR procedures. Statewide, isolated SAVR increased from a yearly average of 3111 procedures during 2009-2010 to 3592 (+15.5%) in 2013, then decreased slightly in 2014. SAVR plus coronary artery bypass graft procedures decreased during the same time period. After implementation of TAVR, hospital SAVR volumes increased moderately, then began to decrease by the fourth year, when TAVR volume exceeded SAVR. Surgical candidates may be identified during evaluation for TAVR, resulting in increased SAVR volume. Increasing SAVR volume may also be related to improved patient and provider awareness of aortic valve disease. Copyright © 2017 The American Association for Thoracic Surgery. Published by Elsevier Inc. All rights reserved.

How-to-Do-It: A Simple DNA Isolation Technique Using Halophilic Bacteria.

ERIC Educational Resources Information Center

Guilfoile, Patrick

1989-01-01

Described is a simple technique for isolating DNA from halophilic bacteria. Materials, procedure, and additional experiments are outlined. It is stated that the DNA obtained will be somewhat contaminated with cellular proteins and RNA. Offers a procedure for greater purification. (RT)

Quantification, morphology, and viability of equine preantral follicles obtained via the Biopsy Pick-Up method.

PubMed

Haag, K T; Magalhães-Padilha, D M; Fonseca, G R; Wischral, A; Gastal, M O; King, S S; Jones, K L; Figueiredo, J R; Gastal, E L

2013-03-01

A Biopsy Pick-Up (BPU) method was tested to determine the feasibility of retrieving preantral follicles from mare ovaries in vivo. A total of 33 ovarian biopsy procedures were performed on 18 mares during the breeding season. Mares were 5 to 21 years old and biopsies were performed during the estrous and/or diestrous phase, as confirmed by transrectal ultrasonography. Follicles were mechanically isolated using a tissue chopper, counted, and classified as normal or abnormal and primordial or primary. Viability of isolated follicles was determined by Trypan Blue dye. A total of 256 biopsy attempts were made resulting in 185 successful tissue sample collections (72% success rate). The mean weight of ovarian tissue collected per procedure was 25.0 ± 1.6 mg. Overall, 620 preantral follicles were collected and isolated (95% primordial and 5% primary). The mean (±SEM) number of follicles isolated per biopsy procedure was 18.8 ± 1.9. Primordial and primary follicles had an average diameter of 31.3 ± 6.2 and 41.1 ± 6.6 μm, respectively. Viability rate was higher (P < 0.001) for primordial follicles (91%) compared with primary follicles (50%). Primordial follicles tended (P < 0.06) to have a higher rate of morphological normality (96%) compared with primary follicles (80%). The total number of follicles isolated, amount of tissue harvested, and number of follicles per mg of tissue did not differ (P > 0.05) according to phase of the estrous cycle. Younger mares (5 to 7 years old) had more (P < 0.05) follicles isolated per procedure than older mares (14 to 21 years old). The length of the interovulatory interval was not affected (P > 0.05) by any biopsy procedure, and there were no adverse effects on cyclicity or general reproductive health. In conclusion, the BPU method provided large numbers of normal and viable preantral follicles for the study of early follicular development in mares. The BPU method might be used in the future to obtain preantral follicles for in vitro culture to enable the use of numerous oocytes present within the equine ovary. This could allow for the preservation of genetic material or large-scale embryo production. Copyright © 2013 Elsevier Inc. All rights reserved.

Approaches to catheter ablation for persistent atrial fibrillation.

PubMed

Verma, Atul; Jiang, Chen-yang; Betts, Timothy R; Chen, Jian; Deisenhofer, Isabel; Mantovan, Roberto; Macle, Laurent; Morillo, Carlos A; Haverkamp, Wilhelm; Weerasooriya, Rukshen; Albenque, Jean-Paul; Nardi, Stefano; Menardi, Endrj; Novak, Paul; Sanders, Prashanthan

2015-05-07

Catheter ablation is less successful for persistent atrial fibrillation than for paroxysmal atrial fibrillation. Guidelines suggest that adjuvant substrate modification in addition to pulmonary-vein isolation is required in persistent atrial fibrillation. We randomly assigned 589 patients with persistent atrial fibrillation in a 1:4:4 ratio to ablation with pulmonary-vein isolation alone (67 patients), pulmonary-vein isolation plus ablation of electrograms showing complex fractionated activity (263 patients), or pulmonary-vein isolation plus additional linear ablation across the left atrial roof and mitral valve isthmus (259 patients). The duration of follow-up was 18 months. The primary end point was freedom from any documented recurrence of atrial fibrillation lasting longer than 30 seconds after a single ablation procedure. Procedure time was significantly shorter for pulmonary-vein isolation alone than for the other two procedures (P<0.001). After 18 months, 59% of patients assigned to pulmonary-vein isolation alone were free from recurrent atrial fibrillation, as compared with 49% of patients assigned to pulmonary-vein isolation plus complex electrogram ablation and 46% of patients assigned to pulmonary-vein isolation plus linear ablation (P=0.15). There were also no significant differences among the three groups for the secondary end points, including freedom from atrial fibrillation after two ablation procedures and freedom from any atrial arrhythmia. Complications included tamponade (three patients), stroke or transient ischemic attack (three patients), and atrioesophageal fistula (one patient). Among patients with persistent atrial fibrillation, we found no reduction in the rate of recurrent atrial fibrillation when either linear ablation or ablation of complex fractionated electrograms was performed in addition to pulmonary-vein isolation. (Funded by St. Jude Medical; ClinicalTrials.gov number, NCT01203748.).

Antimicrobial susceptibility of Brachyspira hyodysenteriae isolated from 21 Polish farms.

PubMed

Zmudzki, J; Szczotka, A; Nowak, A; Strzelecka, H; Grzesiak, A; Pejsak, Z

2012-01-01

Swine dysentery (SD) is a common disease among pigs worldwide, which contributes to major production losses. Antimicrobial susceptibility testing of B. hyodysenteriae, the etiological agent of SD, is mainly performed by the agar dilution method. This method has certain limitations due to difficulties in interpretation of results. The aim of this study was the analysis of antimicrobial susceptibility of Brachyspira hyodysenteriae (B. hyodysenteriae) Polish field isolates by broth microdilution procedure. The study was performed on 21 isolates of B. hyodysenteriae, collected between January 2006 to December 2010 from cases of swine dysentery. VetMIC Brachyspira panels with antimicrobial agents (tiamulin, valnemulin, doxycycline, lincomycin, tylosin and ampicillin) were used for susceptibility testing of B. hyodysenteriae. The minimal inhibitory concentration (MIC) was determined by the broth dilution procedure. The lowest antimicrobial activity was demonstrated for tylosin and lincomycin, with inhibition of bacterial growth using concentrations > 128 microg/ml and 32 microg/ml, respectively. In the case of doxycycline, the MIC values were < or = 2.0 microg/ml. No decreased susceptibility to tiamulin was found among the Polish isolates and MIC values for this antibiotic did not exceed 1.0 microg/ml. The results of the present study confirmed that Polish B. hyodysenteriae isolates were susceptible to the main antibiotics (tiamulin and valnemulin) used in treatment of swine dysentery. Further studies are necessary to evaluate a possible slow decrease in susceptibility to tiamulin and valnemulin of B. hyodysenteriae strains in Poland.

A MEMBRANE FILTER PROCEDURE FOR ASSAYING CYTOTOXIC ACTIVITY IN HETEROTROPHIC BACTERIA ISOLATED FROM DRINKING WATER

EPA Science Inventory

Cytotoxic activity assays of Gram-negative, heterotrophic bacteria are often laborious and time consuming. The objective of this study was to develop in situ procedures for testing potential cytotoxic activities of heterotrophic bacteria isolated from drinking water systems. Wate...

Evaluation of Procedures for Isolation of Nontuberculous Mycobacteria from Soil and Water

PubMed Central

Kamala, T.; Paramasivan, C. N.; Herbert, Daniel; Venkatesan, P.; Prabhakar, R.

1994-01-01

Six methods of decontamination each for the isolation of mycobacteria from soil and water were compared. On the basis of the results obtained, three of the six methods for soil and two of the six methods for water were further evaluated. For both soil and water samples, the method using 3% sodium lauryl sulfate in combination with 1% NaOH yielded more positives than the other methods. PMID:16349208

Double-label immunofluorescence method for simultaneous detection of adenovirus and herpes simplex virus from the eye.

PubMed

Walpita, P; Darougar, S

1989-07-01

The development and application of a double-label immunofluorescence method which has the potential to screen for single or dual infections from any site, in single shell vial cultures, is described. In this study, a total of 1,141 ocular specimens were inoculated in shell vials, centrifuged at 15,000 X g for 1 h, incubated at 37 degrees C for 48 h, and fixed in methanol at room temperature for 15 min. The virus inclusions were detected by staining with a double-label indirect immunofluorescence procedure using mixtures of appropriate first antibodies, followed by fluorescein- and rhodamine-conjugated second antibodies. Each specimen was also inoculated in parallel by the conventional virus isolation method. The sensitivity and specificity of the double-label shell vial procedure were comparable to those with the conventional method, and the former test took only 48 h to complete. The test offers a rapid and simple single-vial procedure which allows for individual or simultaneous detection of multiple pathogens. It results in savings in time and cost over the conventional virus isolation method and other shell vial procedures.

A rare case of large isolated internal iliac artery aneurysm with ureteral obstruction and hydronephrosis: Compression symptoms are limitation for endovascular procedures.

PubMed

Nenezic, Dragoslav; Tanaskovic, Slobodan; Gajin, Predrag; Ilijevski, Nenad; Novakovic, Aleksandra; Radak, Djordje

2015-04-01

In this report, we aim to present a rare case of isolated internal iliac artery aneurysm with associated left ureteric obstruction and consequent hydronephrosis. A 66-year-old male patient was admitted for occasional pain in the lower back that appeared one month earlier. CT arteriography revealed isolated internal iliac artery (diameter 99 mm) with ureteral obstruction, hydroureter and left kidney hydronephrosis occurrence. Aneurysm was resected, after six months the patient was doing well. Bearing in mind that 77% of the patients with isolated internal iliac artery have symptoms caused by aneurysmal compression on adjacent organs, we wanted to highlight that despite the amazing expansion of endovascular procedures in the last decades, its therapeutic effect in isolated internal iliac artery's treatment is to a great extent limited since compression symptoms cannot be solved. Open surgery remains the gold standard for isolated internal iliac artery's treatment considering significant limitations of endovascular procedures due to the inability to eliminate problems caused by compression. © The Author(s) 2014 Reprints and permissions: sagepub.co.uk/journalsPermissions.nav.

The feasibility of a Box isolation strategy for non-paroxysmal atrial fibrillation in elderly patients.

PubMed

Higuchi, Satoshi; Sohara, Hiroshi; Nakamura, Yoshinori; Ihara, Minoru; Yamaguchi, Yoshio; Shoda, Morio; Hagiwara, Nobuhisa; Satake, Shutaro

2016-06-01

Catheter ablation of non-paroxysmal atrial fibrillation (non-PAF) is a therapeutic challenge especially in elderly patients. This study describes the feasibility of a posterior left atrium isolation as a substrate modification in addition to pulmonary vein isolation, the so-called Box isolation, for elderly patients with non-PAF. Two hundred twenty-nine consecutive patients who underwent Box isolations for drug-refractory non-PAF were divided into two groups according to their age; younger group comprising 175 patients aged <75 years and elderly group comprising 54 patients aged ≥75 years. During 23.7±12.0 months of follow-up, the arrhythmia-free rates after one procedure were 53.1% in younger group versus 48.1% in elderly group (p=0.50). Following the second procedure, all patients had electrical conduction recoveries along the initial Box lesion. However, a complete Box re-isolation was highly established in both age groups (87.1% vs. 92.9%, respectively; p=1.00). Recurrence of macro-reentrant atrial tachycardia was mainly associated with the gaps through the initial Box lesion in both age groups (25.8% vs. 21.4%, p=1.00), but typical cavo-tricuspid isthmus (CTI) dependent atrial flutter was significantly observed in the elderly patients' group only (all events were observed within 6 months after the initial procedure; 3.2% vs. 28.6%, p=0.009). After two procedures, the arrhythmia-free rates increased to 73.1% in younger group versus 66.7% in elderly group (p=0.38). The occurrence rate of procedural-related complications did not differ between the two age groups, and there were no life-threatening complications even in elderly patients. Box isolation of non-PAF is effective and safe even in elderly patients. A prophylactic CTI ablation combined with Box isolation might be feasible to improve the long-term outcome.

Microplate-based high throughput screening procedure for the isolation of lipid-rich marine microalgae

PubMed Central

2011-01-01

We describe a new selection method based on BODIPY (4,4-difluoro-1,3,5,7-tetramethyl-4-bora-3a,4a-diaza-s-indacene) staining, fluorescence activated cell sorting (FACS) and microplate-based isolation of lipid-rich microalgae from an environmental sample. Our results show that direct sorting onto solid medium upon FACS can save about 3 weeks during the scale-up process as compared with the growth of the same cultures in liquid medium. This approach enabled us to isolate a biodiverse collection of several axenic and unialgal cultures of different phyla. PMID:22192119

Microplate-based high throughput screening procedure for the isolation of lipid-rich marine microalgae.

PubMed

Pereira, Hugo; Barreira, Luísa; Mozes, André; Florindo, Cláudia; Polo, Cristina; Duarte, Catarina V; Custódio, Luísa; Varela, João

2011-12-22

We describe a new selection method based on BODIPY (4,4-difluoro-1,3,5,7-tetramethyl-4-bora-3a,4a-diaza-s-indacene) staining, fluorescence activated cell sorting (FACS) and microplate-based isolation of lipid-rich microalgae from an environmental sample. Our results show that direct sorting onto solid medium upon FACS can save about 3 weeks during the scale-up process as compared with the growth of the same cultures in liquid medium. This approach enabled us to isolate a biodiverse collection of several axenic and unialgal cultures of different phyla.

Comparison of protocols and RNA carriers for plasma miRNA isolation. Unraveling RNA carrier influence on miRNA isolation

PubMed Central

Martos, Laura; Fernández-Pardo, Álvaro; Oto, Julia; Medina, Pilar; España, Francisco; Navarro, Silvia

2017-01-01

microRNAs are promising biomarkers in biological fluids in several diseases. Different plasma RNA isolation protocols and carriers are available, but their efficiencies have been scarcely compared. Plasma microRNAs were isolated using a phenol and column-based procedure and a column-based procedure, in the presence or absence of two RNA carriers (yeast RNA and MS2 RNA). We evaluated the presence of PCR inhibitors and the relative abundance of certain microRNAs by qRT-PCR. Furthermore, we analyzed the association between different isolation protocols, the relative abundance of the miRNAs in the sample, the GC content and the free energy of microRNAs. In all microRNAs analyzed, the addition of yeast RNA as a carrier in the different isolation protocols used gave lower raw Cq values, indicating higher microRNA recovery. Moreover, this increase in microRNAs recovery was dependent on their own relative abundance in the sample, their GC content and the free-energy of their own most stable secondary structure. Furthermore, the normalization of microRNA levels by an endogenous microRNA is more reliable than the normalization by plasma volume, as it reduced the difference in microRNA fold abundance between the different isolation protocols evaluated. Our thorough study indicates that a standardization of pre- and analytical conditions is necessary to obtain reproducible inter-laboratory results in plasma microRNA studies. PMID:29077772

Reply to Effect of concentration of organic matter on optical maturity parameters. Interlaboratory results of the organic matter concentration working group of the ICCP. Discussion by Vinay K. Sahay

USGS Publications Warehouse

Mendonca, Filho J.G.; Araujo, C.V.; Borrego, A.G.; Cook, A.; Flores, D.; Hackley, P.; Hower, J.C.; Kern, M.L.; Kommeren, K.; Kus, J.; Mastalerz, Maria; Mendonca, J.O.; Menezes, T.R.; Newman, J.; Ranasinghe, P.; Souza, I.V.A.F.; Suarez-Ruiz, I.; Ujiie, Y.

2011-01-01

This reply is motivated by Sahay's comments on the paper published by Mendon??a Filho et al. (2010) dealing with the effect of concentration of an organic matter on optical maturity parameters. Four points were raised by Sahay: suggestion to use of chemical parameters to assess the effect of isolation, indication that suppression of vitrinite reflectance in liptinite-rich rocks was insufficiently addressed, discussion on the way to deal with the existence of multiple vitrinite populations in a dispersed organic matter, and contradictory explanation of results involving the influence of isolation procedure on fluorescence properties but no effect on vitrinite reflectance. The four points were separately addressed being the two first ones out of the scope of the paper. The existence of multiple vitrinite populations is a well-recognized problem whose importance in the results could be addressed because the participants provided individual records of vitrinite reflectance. These results indicated that election of different populations was not a major problem in the results. The influence of isolation procedure on the fluorescence spectra of alginite while the vitrinite reflectance remains unaltered is not considered contradictory because both parameters are measured on different components which may have a different response to the acid treatment. ?? 2011 Elsevier B.V.

Bonded-phase extraction column isolation of organic compounds in groundwater at a hazardous waste site

USGS Publications Warehouse

Rostad, C.E.; Pereira, W.E.; Ratcliff, S.M.

1984-01-01

A procedure for isolation of hazardous organic compounds from water for gas chromatography/mass spectrometry analysis Is presented and applied to creosote- and pentachlorophenol-contaminated groundwater resulting from wood-treatment processes. This simple procedure involved passing a 50-100-mL sample through a bonded-phase extraction column, eluting the trapped organic compounds from the column with 2-4 mL of solvent, and evaporating the sample to 100 ??L with a stream of dry nitrogen, after which the sample was ready for gas chromatography/mass spectrometry analysis. Representative compounds indicative of creosote contamination were used for recovery and precision studies from the cyclohexyl-bonded phase. Recovery of these compounds from n-octyl-, n-octadecyl-, cyclohexyl-, and phenyl-bonded phases was compared. The bonded phase that exhibited the best recovery and least bias toward acidic or basic cmpounds was the n-octadecyl phase. Detailed compound Identification Is given for compounds Isolated from creosote- and pentachlorophenol-contaminated groundwater using the cyclohexyl-bonded phase.

(GTG)5 MSP-PCR fingerprinting as a technique for discrimination of wine associated yeasts?

PubMed

Ramírez-Castrillón, Mauricio; Mendes, Sandra Denise Camargo; Inostroza-Ponta, Mario; Valente, Patricia

2014-01-01

In microbiology, identification of all isolates by sequencing is still unfeasible in small research laboratories. Therefore, many yeast diversity studies follow a screening procedure consisting of clustering the yeast isolates using MSP-PCR fingerprinting, followed by identification of one or a few selected representatives of each cluster by sequencing. Although this procedure has been widely applied in the literature, it has not been properly validated. We evaluated a standardized protocol using MSP-PCR fingerprinting with the primers (GTG)5 and M13 for the discrimination of wine associated yeasts in South Brazil. Two datasets were used: yeasts isolated from bottled wines and vineyard environments. We compared the discriminatory power of both primers in a subset of 16 strains, choosing the primer (GTG)5 for further evaluation. Afterwards, we applied this technique to 245 strains, and compared the results with the identification obtained by partial sequencing of the LSU rRNA gene, considered as the gold standard. An array matrix was constructed for each dataset and used as input for clustering with two methods (hierarchical dendrograms and QAPGrid layout). For both yeast datasets, unrelated species were clustered in the same group. The sensitivity score of (GTG)5 MSP-PCR fingerprinting was high, but specificity was low. As a conclusion, the yeast diversity inferred in several previous studies may have been underestimated and some isolates were probably misidentified due to the compliance to this screening procedure.

(GTG)5 MSP-PCR Fingerprinting as a Technique for Discrimination of Wine Associated Yeasts?

PubMed Central

Inostroza-Ponta, Mario; Valente, Patricia

2014-01-01

In microbiology, identification of all isolates by sequencing is still unfeasible in small research laboratories. Therefore, many yeast diversity studies follow a screening procedure consisting of clustering the yeast isolates using MSP-PCR fingerprinting, followed by identification of one or a few selected representatives of each cluster by sequencing. Although this procedure has been widely applied in the literature, it has not been properly validated. We evaluated a standardized protocol using MSP-PCR fingerprinting with the primers (GTG)5 and M13 for the discrimination of wine associated yeasts in South Brazil. Two datasets were used: yeasts isolated from bottled wines and vineyard environments. We compared the discriminatory power of both primers in a subset of 16 strains, choosing the primer (GTG)5 for further evaluation. Afterwards, we applied this technique to 245 strains, and compared the results with the identification obtained by partial sequencing of the LSU rRNA gene, considered as the gold standard. An array matrix was constructed for each dataset and used as input for clustering with two methods (hierarchical dendrograms and QAPGrid layout). For both yeast datasets, unrelated species were clustered in the same group. The sensitivity score of (GTG)5 MSP-PCR fingerprinting was high, but specificity was low. As a conclusion, the yeast diversity inferred in several previous studies may have been underestimated and some isolates were probably misidentified due to the compliance to this screening procedure. PMID:25171185

Total main rotor isolation system analysis

NASA Technical Reports Server (NTRS)

Sankewitsch, V.

1981-01-01

Requirements, preliminary design, and verification procedures for a total main rotor isolation system at n/rev are presented. The fuselage is isolated from the vibration inducing main rotor at one frequency in all degrees of freedom by four antiresonant isolation units. Effects of parametric variations on isolation system performance are evaluated.

Evaluating the influence of goal setting on intravenous catheterization skill acquisition and transfer in a hybrid simulation training context.

PubMed

Brydges, Ryan; Mallette, Claire; Pollex, Heather; Carnahan, Heather; Dubrowski, Adam

2012-08-01

Educators often simplify complex tasks by setting learning objectives that focus trainees on isolated skills rather than the holistic task. We designed 2 sets of learning objectives for intravenous catheterization using goal setting theory. We hypothesized that setting holistic goals related to technical, cognitive, and communication skills would result in superior holistic performance, whereas setting isolated goals related to technical skills would result in superior technical performance. We randomly assigned practicing health care professionals to set holistic (n = 14) or isolated (n = 15) goals. All watched an instructional video and studied a list of 9 goals specific to their group. Participants practiced independently in a hybrid simulation (standardized patient combined with an arm simulator). The first and the last practice trials were videotaped for analysis. One-week later, participants completed a transfer test in another hybrid simulation scenario. Blinded experts evaluated performance on all 3 trials using the Direct Observation of Procedural Skills tool. The holistic group scored higher than the isolated group on the holistic Direct Observation of Procedural Skills score for all 3 trials [mean (SD), 45.0 (9.16) vs. 38.4 (9.17); P = 0.01]. The isolated group did not perform better than the holistic group on the technical skills score [10.3 (2.73) vs. 11.6 (3.01); P = 0.11]. Our results suggest that asking learners to set holistic goals did not interfere with their attaining competent holistic and technical skills during hybrid simulation training. This exploratory trial provides preliminary evidence for how to consider integrating hybrid simulation into medical curricula and for the design of learning goals in simulation-based education.

Comparison of various RNA extraction methods, cDNA preparation and isolation of calmodulin gene from a highly melanized isolate of apple leaf blotch fungus Marssonina coronaria.

PubMed

Chauhan, Arjun; Sharma, J N; Modgil, Manju; Siddappa, Sundaresha

2018-05-29

Marssonina coronaria causes apple blotch disease resulting in severe premature defoliation, and is distributed in many leading apple-growing areas in the world. Effective, reliable and high-quality RNA extraction is an indispensable procedure in any molecular biology study. No method currently exists for RNA extraction from M. coronaria that produces a high quantity of melanin-free RNA. Therefore, we evaluated eight RNA extraction methods including manual and commercial kits, to yield a sufficient quantity of high-quality and melanin-free RNA. Manual methods used here resulted in low quality and black colored RNA pellets showing the presence of melanin, despite all the modifications employed to original procedures. However, these methods when coupled with clean up resulted in melanin-free RNA. On the other hand, all commercial kits used were able to yield high-quality melanin-free RNA having variable yields. TRIzol™ Reagent + RNA Clean & Concentrator™-5 and Ambion-PureLink® RNA Mini Kit were found to be the best methods as the RNA extracted with these methods from 15 day old fungal culture grown on solid medium were free of melanin with good yield. RNA extracted by this improved methodology was applied for RT-PCR, subsequent PCR amplification, and isolation of calmodulin gene sequences from M. coronaria and infected apple leaf pieces. These methods are more time effective than traditional methods and take only an hour to complete. To our knowledge, this is the first report on the method of isolation of high-quality RNA for cDNA synthesis as well as isolation of the calmodulin gene sequence from this fungus. Copyright © 2018 Elsevier B.V. All rights reserved.

[Bacteriological evaluation of a procedure for disinfecting the Olympus GIF-D2 panendoscope].

PubMed

Ramírez Ramos, A; Domínguez, N; Makino, R; Barrera, C

1980-01-01

We have performed a total of 107 cultures from three critical areas of an Olympus Panendoscope Model GIF-D2 in order to evaluate bacteriologically cur system of desinfection of this endoscope. Samples were taken from the distal end, external surface and biopsy canal before and after an endoscopic examination was performed. The procedure of desinfection employed was as follows: washing of the distal end, external surface and biopsy canal with Hexaclorophel (Phisohex) diluted 50% with water and a second washing with tap water. In the middle of the study, we added a second washing of the biopsy canal with ten ml. of ether alcohol to allow for better drying. As a result of the present study we observed that in the distal end in 50% of the samples we encountered bacteria. Cultures of the external surface were positive in 20% of samples. The biopsy canal should be washed with ether alcohol to allow for complete drying, because when we did not use this method, Pseudomonas Aeruginosa was isolated. After this modification we did not isolate bacteria. The most frequent types of isolated bacteria were from the normal oropharyngeal flora. From the present study we can conclude that desinfection of the Panendespe with Hexaclorophen gives satisfactory results on the external surface of the endoscope. Biopsy canal requires additional washing with ether alcohol. However, both procedures do not assure a satisfactory desinfection of the distal end.

Rapid Ovary Mass-Isolation (ROMi) to Obtain Large Quantities of Drosophila Egg Chambers for Fluorescent In Situ Hybridization.

PubMed

Jambor, Helena; Mejstrik, Pavel; Tomancak, Pavel

2016-01-01

Isolation of large quantities of tissue from organisms is essential for many techniques such as genome-wide screens and biochemistry. However, obtaining large quantities of tissues or cells is often the rate-limiting step when working in vivo. Here, we present a rapid method that allows the isolation of intact, single egg chambers at various developmental stages from ovaries of adult female Drosophila flies. The isolated egg chambers are amenable for a variety of procedures such as fluorescent in situ hybridization, RNA isolation, extract preparation, or immunostaining. Isolation of egg chambers from adult flies can be completed in 5 min and results, depending on the input amount of flies, in several milliliters of material. The isolated egg chambers are then further processed depending on the exact requirements of the subsequent application. We describe high-throughput in situ hybridization in 96-well plates as example application for the mass-isolated egg chambers.

NHEXAS PHASE I ARIZONA STUDY--STANDARD OPERATING PROCEDURE FOR ISOLATION OF MALFUNCTIONING OR DAMAGED EQUIPMENT (UA-G-2.0)

EPA Science Inventory

The purpose of this SOP is to identify and isolate malfunctioning or damaged equipment for the NHEXAS Arizona research project. This procedure was followed to ensure consistent data custody, storage, transfer and analysis of a high quality during the Arizona NHEXAS project and t...

No-migration variance petition. Appendices C--J: Volume 5, Revision 1

DOE Office of Scientific and Technical Information (OSTI.GOV)

Not Available

1990-03-01

Volume V contains the appendices for: closure and post-closure plans; RCRA ground water monitoring waver; Waste Isolation Division Quality Program Manual; water quality sampling plan; WIPP Environmental Procedures Manual; sample handling and laboratory procedures; data analysis; and Annual Site Environmental Monitoring Report for the Waste Isolation Pilot Plant.

9 CFR 147.26 - Procedures for establishing isolation and maintaining sanitation and good management practices...

Code of Federal Regulations, 2010 CFR

2010-01-01

... 9 Animals and Animal Products 1 2010-01-01 2010-01-01 false Procedures for establishing isolation... infections. 147.26 Section 147.26 Animals and Animal Products ANIMAL AND PLANT HEALTH INSPECTION SERVICE, DEPARTMENT OF AGRICULTURE LIVESTOCK IMPROVEMENT AUXILIARY PROVISIONS ON NATIONAL POULTRY IMPROVEMENT PLAN...

Methods for Isolation and Purification of Murine Liver Sinusoidal Endothelial Cells: A Systematic Review.

PubMed

Meyer, Jeremy; Gonelle-Gispert, Carmen; Morel, Philippe; Bühler, Léo

2016-01-01

To study the biological functions of liver sinusoidal endothelial cells (LSEC) and to identify their interplay with blood or liver cells, techniques allowing for the isolation and purification of LSEC have been developed over the last decades. The objective of the present review is to summarize and to compare the efficiency of existing methods for isolating murine LSEC. Toward this end, the MEDLINE database was searched for all original articles describing LSEC isolation from rat and mouse livers. Out of the 489 publications identified, 23 reported the main steps and outcomes of the procedure and were included in our review. Here, we report and analyse the technical details of the essential steps of the techniques used for LSEC isolation. The correlations between the prevalence of some steps and the efficiency of LSEC isolation were also identified. We found that centrifugal elutriation, selective adherence and, more recently, magnetic-activated cell sorting were used for LSEC purification. Centrifugal elutriation procured high yields of pure LSEC (for rats 30-141.9 million cells for 85-98% purities; for mice 9-9.25 million cells for >95% purities), but the use of this method remained limited due to its high technical requirements. Selective adherence showed inconsistent results in terms of cell yields and purities in rats (5-100 million cells for 73.7-95% purities). In contrast, magnetic-activated cell sorting allowed for the isolation of highly pure LSEC, but overall lower cell yields were reported (for rats 10.7 million cells with 97.6% purity; for mice 0.5-9 million cells with 90-98% purities). Notably, the controversies regarding the accuracy of several phenotypic markers for LSEC should be considered and their use for both magnetic sorting and characterization remain doubtful. It appears that more effort is needed to refine and standardize the procedure for LSEC isolation, with a focus on the identification of specific antigens. Such a procedure is required to identify the molecular mechanisms regulating the function of LSEC and to improve our understanding of their role in complex cellular processes in the liver.

Methods for Isolation and Purification of Murine Liver Sinusoidal Endothelial Cells: A Systematic Review

PubMed Central

Meyer, Jeremy; Gonelle-Gispert, Carmen; Morel, Philippe; Bühler, Léo

2016-01-01

To study the biological functions of liver sinusoidal endothelial cells (LSEC) and to identify their interplay with blood or liver cells, techniques allowing for the isolation and purification of LSEC have been developed over the last decades. The objective of the present review is to summarize and to compare the efficiency of existing methods for isolating murine LSEC. Toward this end, the MEDLINE database was searched for all original articles describing LSEC isolation from rat and mouse livers. Out of the 489 publications identified, 23 reported the main steps and outcomes of the procedure and were included in our review. Here, we report and analyse the technical details of the essential steps of the techniques used for LSEC isolation. The correlations between the prevalence of some steps and the efficiency of LSEC isolation were also identified. We found that centrifugal elutriation, selective adherence and, more recently, magnetic-activated cell sorting were used for LSEC purification. Centrifugal elutriation procured high yields of pure LSEC (for rats 30–141.9 million cells for 85–98% purities; for mice 9–9.25 million cells for >95% purities), but the use of this method remained limited due to its high technical requirements. Selective adherence showed inconsistent results in terms of cell yields and purities in rats (5–100 million cells for 73.7–95% purities). In contrast, magnetic-activated cell sorting allowed for the isolation of highly pure LSEC, but overall lower cell yields were reported (for rats 10.7 million cells with 97.6% purity; for mice 0.5–9 million cells with 90–98% purities). Notably, the controversies regarding the accuracy of several phenotypic markers for LSEC should be considered and their use for both magnetic sorting and characterization remain doubtful. It appears that more effort is needed to refine and standardize the procedure for LSEC isolation, with a focus on the identification of specific antigens. Such a procedure is required to identify the molecular mechanisms regulating the function of LSEC and to improve our understanding of their role in complex cellular processes in the liver. PMID:26992171

A combined dynamic analysis method for geometrically nonlinear vibration isolators with elastic rings

NASA Astrophysics Data System (ADS)

Hu, Zhan; Zheng, Gangtie

2016-08-01

A combined analysis method is developed in the present paper for studying the dynamic properties of a type of geometrically nonlinear vibration isolator, which is composed of push-pull configuration rings. This method combines the geometrically nonlinear theory of curved beams and the Harmonic Balance Method to overcome the difficulty in calculating the vibration and vibration transmissibility under large deformations of the ring structure. Using the proposed method, nonlinear dynamic behaviors of this isolator, such as the lock situation due to the coulomb damping and the usual jump resulting from the nonlinear stiffness, can be investigated. Numerical solutions based on the primary harmonic balance are first verified by direct integration results. Then, the whole procedure of this combined analysis method is demonstrated and validated by slowly sinusoidal sweeping experiments with different amplitudes of the base excitation. Both numerical and experimental results indicate that this type of isolator behaves as a hardening spring with increasing amplitude of the base excitation, which makes it suitable for isolating both steady-state vibrations and transient shocks.

Pulmonary vein isolation using a pace-capture-guided versus an adenosine-guided approach: effect on dormant conduction and long-term freedom from recurrent atrial fibrillation--a prospective study.

PubMed

Andrade, Jason G; Pollak, Scott J; Monir, George; Khairy, Paul; Dubuc, Marc; Roy, Denis; Talajic, Mario; Deyell, Marc; Rivard, Léna; Thibault, Bernard; Guerra, Peter G; Nattel, Stanley; Macle, Laurent

2013-12-01

Atrial fibrillation recurrence after pulmonary vein (PV) isolation is associated with PV to left atrium reconduction. We prospectively studied the use of 2 procedural techniques designed to facilitate identification of residual gaps within the index ablation line. After wide circumferential PV isolation, 40 patients received additional ablation targeted at locations of left atrial capture during high-output pacing (pace-capture group), while 40 patients underwent adenosine testing with targeted ablation at sites of dormant conduction (adenosine group). Patients were followed up at 3, 6, and 12 months. After PV isolation, high-output pace-capture was documented in 39 PVs (25%; 50% of patients) in the pace-capture group. Dormant conduction was unmasked in 34 PVs (22%; 53% of patients) in the adenosine group. A subset of 25 patients in the pace-capture group underwent adenosine testing without targeted ablation of dormant conduction. In these patients, only 10 out of 86 PVs (11.6%; 24% of patients) demonstrated dormant conduction after the elimination of local pace-capture. At a follow-up of 329±124 days, the single procedure off antiarrhythmic drug freedom from recurrent atrial fibrillation was 67.5% in the adenosine group and 65.0% in the pace-capture group (P=0.814). Procedure duration and fluoroscopy time were significantly longer in the pace-capture group (P=0.002 and P<0.001), whereas radiofrequency ablation time was comparable (P=0.192). The use of high-output pacing post-PV isolation results in a significant reduction in the incidence of dormant conduction with a comparable long-term freedom from recurrent atrial fibrillation (versus adenosine-guided ablation). The use of these approaches requires evaluation in a long-term prospective randomized study. [corrected].

Isolation of Microarray-Grade Total RNA, MicroRNA, and DNA from a Single PAXgene Blood RNA Tube

PubMed Central

Kruhøffer, Mogens; Dyrskjøt, Lars; Voss, Thorsten; Lindberg, Raija L.P.; Wyrich, Ralf; Thykjaer, Thomas; Orntoft, Torben F.

2007-01-01

We have developed a procedure for isolation of microRNA and genomic DNA in addition to total RNA from whole blood stabilized in PAXgene Blood RNA tubes. The procedure is based on automatic extraction on a BioRobot MDx and includes isolation of DNA from a fraction of the stabilized blood and recovery of small RNA species that are otherwise lost. The procedure presented here is suitable for large-scale experiments and is amenable to further automation. Procured total RNA and DNA was tested using Affymetrix Expression and single-nucleotide polymorphism GeneChips, respectively, and isolated microRNA was tested using spotted locked nucleic acid-based microarrays. We conclude that the yield and quality of total RNA, microRNA, and DNA from a single PAXgene blood RNA tube is sufficient for downstream microarray analysis. PMID:17690207

Structure Elucidation of a Natural Product.

ERIC Educational Resources Information Center

Letcher, Roy M.

1983-01-01

Describes an experiment simulating a real-life structure elucidation problem through isolation, characterization, and chemical transformation of an "unknown," naturally occurring monoterpene, with extensive use being made of spectroscopy and aided by biogenetic considerations. Information given to students, procedures, results, and discussion of…

Comparative proteome analysis of cryopreserved flagella and head plasma membrane proteins from sea bream spermatozoa: effect of antifreeze proteins.

PubMed

Zilli, Loredana; Beirão, José; Schiavone, Roberta; Herraez, Maria Paz; Gnoni, Antonio; Vilella, Sebastiano

2014-01-01

Cryopreservation induces injuries to fish spermatozoa that in turn affect sperm quality in terms of fertilization ability, motility, DNA and protein integrity and larval survival. To reduce the loss of sperm quality due to freezing-thawing, it is necessary to improve these procedures. In the present study we investigated the ability of two antifreeze proteins (AFPI and AFPIII) to reduce the loss of quality of sea bream spermatozoa due to cryopreservation. To do so, we compared viability, motility, straight-line velocity and curvilinear velocity of fresh and (AFPs)-cryopreserved spermatozoa. AFPIII addition to cryopreservation medium improved viability, motility and straight-line velocity with respect to DMSO or DMSO plus AFPI. To clarify the molecular mechanism(s) underlying these findings, the protein profile of two different cryopreserved sperm domains, flagella and head plasma membranes, was analysed. The protein profiles differed between fresh and frozen-thawed semen and results of the image analysis demonstrated that, after cryopreservation, out of 270 proteins 12 were decreased and 7 were increased in isolated flagella, and out of 150 proteins 6 showed a significant decrease and 4 showed a significant increase in head membranes. Mass spectrometry analysis identified 6 proteins (4 from isolated flagella and 2 present both in flagella and head plasma membranes) within the protein spots affected by the freezing-thawing procedure. 3 out of 4 proteins from isolated flagella were involved in the sperm bioenergetic system. Our results indicate that the ability of AFPIII to protect sea bream sperm quality can be, at least in part, ascribed to reducing changes in the sperm protein profile occurring during the freezing-thawing procedure. Our results clearly demonstrated that AFPIII addition to cryopreservation medium improved the protection against freezing respect to DMSO or DMSO plus AFPI. In addition we propose specific proteins of spermatozoa as markers related to the procedures of fish sperm cryopreservation.

Isolation and quantification of Quillaja saponaria Molina saponins and lipids in iscom-matrix and iscoms.

PubMed

Behboudi, S; Morein, B; Rönnberg, B

1995-12-01

In the iscom, multiple copies of antigen are attached by hydrophobic interaction to a matrix which is built up by Quillaja triterpenoid saponins and lipids. Thus, the iscom presents antigen in multimeric form in a small particle with a built-in adjuvant resulting in a highly immunogenic antigen formulation. We have designed a chloroform-methanol-water extraction procedure to isolate the triterpenoid saponins and lipids incorporated into iscom-matrix and iscoms. The triterpenoids in the triterpenoid phase were quantitated using orcinol sulfuric acid detecting their carbohydrate chains and by HPLC. The cholesterol and phosphatidylcholine in the lipid phase were quantitated by HPLC and a commercial colorimetric method for the cholesterol. The quantitative methods showed an almost total separation and recovery of triterpenoids and lipids in their respective phases, while protein was detected in all phases after extraction. The protein content was determined by the method of Lowry and by amino acid analysis. Amino acid analysis was shown to be the reliable method of the two to quantitate proteins in iscoms. In conclusion, simple, reproducible and efficient procedures have been designed to isolate and quantitate the triterpenoids and lipids added for preparation of iscom-matrix and iscoms. The procedures described should also be useful to adequately define constituents in prospective vaccines.

Maze Procedures for Atrial Fibrillation, From History to Practice.

PubMed

Kik, Charles; Bogers, Ad J J C

2011-10-01

Atrial fibrillation may result in significant symptoms, (systemic) thrombo-embolism, as well as tachycardia-induced cardiomyopathy with cardiac failure, and consequently be associated with significant morbidity and mortality. Nowadays symptomatic atrial fibrillation can be treated with catheter-based ablation, surgical ablation or hybrid approaches. In this setting a fairly large number of surgical approaches and procedures are described and being practised. It should be clear that the Cox-maze procedure resulted from building up evidence and experience in different steps, while some of the present surgical approaches and techniques are being based only on technical feasibility with limited experience, rather than on a process of consequent methodology. Some of the issues still under debate are whether or not the maze procedure can be limited to the left atrium or even to isolation of the pulmonary veins or that bi-atrial procedures are indicated, whether or not cardiopulmonary bypass is to be applied and which route of exposure facilitates an optimal result. In addition, maze procedures are not procedures guide by electrophysiological mapping. At least in theory not in all patients all lesions of the maze procedures are necessary. A history and aspects of current practise in surgical treatment of atrial fibrillation is presented.

Maze Procedures for Atrial Fibrillation, From History to Practice

PubMed Central

Kik, Charles; Bogers, Ad J.J.C.

2011-01-01

Atrial fibrillation may result in significant symptoms, (systemic) thrombo-embolism, as well as tachycardia-induced cardiomyopathy with cardiac failure, and consequently be associated with significant morbidity and mortality. Nowadays symptomatic atrial fibrillation can be treated with catheter-based ablation, surgical ablation or hybrid approaches. In this setting a fairly large number of surgical approaches and procedures are described and being practised. It should be clear that the Cox-maze procedure resulted from building up evidence and experience in different steps, while some of the present surgical approaches and techniques are being based only on technical feasibility with limited experience, rather than on a process of consequent methodology. Some of the issues still under debate are whether or not the maze procedure can be limited to the left atrium or even to isolation of the pulmonary veins or that bi-atrial procedures are indicated, whether or not cardiopulmonary bypass is to be applied and which route of exposure facilitates an optimal result. In addition, maze procedures are not procedures guide by electrophysiological mapping. At least in theory not in all patients all lesions of the maze procedures are necessary. A history and aspects of current practise in surgical treatment of atrial fibrillation is presented. PMID:28357007

Single-ring ablation compared with standard circumferential pulmonary vein isolation using remote magnetic catheter navigation.

PubMed

Sohns, Christian; Bergau, Leonard; Seegers, Joachim; Lüthje, Lars; Vollmann, Dirk; Zabel, Markus

2014-10-01

In ablation of atrial fibrillation, the single-ring method aims for isolation of the posterior wall of the left atrium (LA) including the pulmonary veins (PVs) but avoiding posterior LA lesions. The aim of this randomized prospective study was to evaluate safety and efficacy of remote magnetic navigation (RMN)-guided single-ring ablation strategy as compared to standard RMN-guided circumferential PV ablation (PVA). Eighty consecutive patients undergoing PVA were enrolled prospectively and randomized equally into two study groups. RMN using the Stereotaxis system and open-irrigated 3.5-mm ablation catheters were used with a 3D mapping system in all procedures. Forty patients underwent RMN-guided single-ring ablation, and 40 patients received RMN-guided circumferential PVA. In the circumferential group, 3.3 ± 1.1 PVs were successfully isolated at the end of the procedure as compared to 3.1 ± 1.3 in the single-ring (box) group (p=0.38). All patients in the box group required additional posterior lesions in order to achieve electrical isolation of the PVs. Single-ring ablation was associated with longer procedure duration (p=0.01) and ablation time (p=0.001). After a single procedure, the proportion of patients free of any atrial tachycardia (AT)/atrial fibrillation (AF) episode at 12-month follow-up was 57 % in the box group and 58 % in the circ group. Using RMN, only minor complications have been observed. RMN-guided single-ring PVA provides comparable acute and long-term success rates as compared to RMN-guided circumferential PVA but requires additional posterior lesions to achieve PV isolation and increased procedure and ablation time. Procedural complication rates are low when using RMN.

INCIDENCE AND DETECTION OF PLEUROPNEUMONIA-LIKE ORGANISMS IN CELL CULTURES BY FLUORESCENT ANTIBODY AND CULTURAL PROCEDURES1

PubMed Central

Barile, Michael F.; Malizia, Walter F.; Riggs, Donald B.

1962-01-01

Barile, Michael F. (National Institutes of Health, Bethesda, Md.), Walter F. Malizia, and Donald B. Riggs. Incidence and detection of pleuropneumonia-like organisms in cell cultures by fluorescent antibody and cultural procedures. J. Bacteriol. 84:130–136. 1962—A total of 102 tissue-cell cultures from 17 separate laboratories was examined for pleuropneumonia-like organisms (PPLO) by the fluorescent antibody and cultural procedures. PPLO were isolated from 48 of the 49 tissue-cell cultures found positive for PPLO by the fluorescent antibody procedure, and results of the two procedures agreed in 101 of the 102 (99%) cases. PPLO were isolated from none of 10 primary-cell cultures prepared from six animal species and from 48 of 92 (52%) continuous-cell cultures prepared from eight animal species. Cells grown in media containing antibiotics were more frequently contaminated with PPLO (72%) than cells grown in antibiotic-free media (7%). Cultures (91%) from tissue-culture-producing laboratories and cultures (76%) used for propagation of microorganisms were contaminated with PPLO, although none used for tissue-culture metabolic studies was contaminated. In addition, our findings support the view that PPLO contamination of cell cultures is probably owing to bacterial contaminants which revert to L forms in the presence of antibiotics. Images PMID:13865001

Actinomycetes in Karstic caves of northern Spain (Altamira and Tito Bustillo).

PubMed

Groth, I; Vettermann, R; Schuetze, B; Schumann, P; Saiz-Jimenez, C

1999-05-01

A variety of isolation procedures were carried out to study the involvement of bacteria in the colonisation and biodeterioration of Spanish caves with paleolithic rock art (Altamira and Tito Bustillo). The applied techniques mainly aimed to isolate heterotrophic bacteria such as streptomycetes, nocardioform and coryneform actinomycetes, and other gram-positive and gram-negative bacteria. The results demonstrated that actinomycetes were the most abundant gram-positive bacteria in the caves. Actinomycetes revealed a great taxonomic diversity with the predominant isolates belonging to the genus Streptomyces. Members of the genera Nocardia, Rhodococcus, Nocardioides, Amycolatopsis, Saccharothrix, Brevibacterium, Microbacterium, and coccoid actinomycetes (family Micrococcaceae) were also found.

An Isolator System for minimally invasive surgery: the new design

PubMed Central

Jansen, Frank-Willem; Dankelman, Jenny

2010-01-01

Background The risk of obtaining a postsurgical infection depends highly on the air quality surrounding the exposed tissue, surgical instruments, and materials. Many isolators for open surgery have been invented to create a contained sterile volume around the exposed tissue. With the use of an isolator, a surgical procedure can be performed outside sterile environments. The goal of this study was to design an Isolator System (IS) for standard laparoscopic instruments while instrument movements are not restricted. Methods The developed IS consists of a sleeve to protect the instrument shaft and tip and a special balloon to protect the incision and trocar tube. A coupling mechanism connected at the sleeve allows instrument changes without contamination of the isolated parts. Smoke tests were performed to show that outside air does not enter the new IS during a simulated laparoscopic procedure. Eight test runs and one baseline run inside a contained volume filled with thick smoke were performed to investigate whether smoke particles entered the Isolator System. Filters were used to identify smoke entering the Isolator System. Results Seven filters showed no trace of smoke particles. In one test run, a part of the IS loosened and a small brown spot was visible. The filter from the baseline run was completely covered with a thick layer of particles, proving the effectiveness of the test. During all test runs, the isolated instrument was successfully locked on and unlocked from the isolated trocar. Instrument movements gave no complications. After removal of the isolated instrument, it took three novices an average of 3.1 (standard deviation (SD), 0.7) seconds to replace it correctly on the isolated trocar. Conclusions The designed IS for laparoscopy can increase sterility in environments where sterility cannot be guaranteed. The current design is developed for laparoscopy, but it can easily be adapted for other fields in minimally invasive surgery. PMID:20108141

U.S.-MEXICO BORDER PROGRAM ARIZONA BORDER STUDY--STANDARD OPERATING PROCEDURE FOR ISOLATION OF MALFUNCTIONING OR DAMAGED EQUIPMENT (UA-G-2.0)

EPA Science Inventory

The purpose of this SOP is to identify and isolate malfunctioning or damaged equipment for the Arizona Border Study. This procedure was followed to ensure consistent data custody, storage, transfer and analysis of a high quality during the Arizona NHEXAS project and the Border s...

Separation and Analysis of Citral Isomers.

ERIC Educational Resources Information Center

Sacks, Jeff; And Others

1983-01-01

Provides background information, procedures, and results of an experiments designed to introduce undergraduates to the technique of steam distillation as a means of isolating thermally sensitive compounds. Chromatographic techniques (HPLC) and mass spectrometric analysis are used in the experiment which requires three laboratory periods. (JN)

One-pot, two-step desymmetrization of symmetrical benzils catalyzed by the methylsulfinyl (dimsyl) anion.

PubMed

Ragno, Daniele; Bortolini, Olga; Giovannini, Pier Paolo; Massi, Alessandro; Pacifico, Salvatore; Zaghi, Anna

2014-08-14

An operationally simple one-pot, two-step procedure for the desymmetrization of benzils is herein described. This consists in the chemoselective cross-benzoin reaction of symmetrical benzils with aromatic aldehydes catalyzed by the methyl sulfinyl (dimsyl) anion, followed by microwave-assisted oxidation of the resulting benzoylated benzoins with nitrate, avoiding the costly isolation procedure. Both electron-withdrawing and electron-donating substituents may be accommodated on the aromatic rings of the final unsymmetrical benzil.

Plasma membrane isolation using immobilized concanavalin A magnetic beads.

PubMed

Lee, Yu-Chen; Srajer Gajdosik, Martina; Josic, Djuro; Lin, Sue-Hwa

2012-01-01

Isolation of highly purified plasma membranes is the key step in constructing the plasma membrane proteome. Traditional plasma membrane isolation method takes advantage of the differential density of organelles. While differential centrifugation methods are sufficient to enrich for plasma membranes, the procedure is lengthy and results in low recovery of the membrane fraction. Importantly, there is significant contamination of the plasma membranes with other organelles. The traditional agarose affinity matrix is suitable for isolating proteins but has limitation in separating organelles due to the density of agarose. Immobilization of affinity ligands to magnetic beads allows separation of affinity matrix from organelles through magnets and could be developed for the isolation of organelles. We have developed a simple method for isolating plasma membranes using lectin concanavalin A (ConA) magnetic beads. ConA is immobilized onto magnetic beads by binding biotinylated ConA to streptavidin magnetic beads. The ConA magnetic beads are used to bind glycosylated proteins present in the membranes. The bound membranes are solubilized from the magnetic beads with a detergent containing the competing sugar alpha methyl mannoside. In this study, we describe the procedure of isolating rat liver plasma membranes using sucrose density gradient centrifugation as described by Neville. We then further purify the membrane fraction by using ConA magnetic beads. After this purification step, main liver plasma membrane proteins, especially the highly glycosylated ones and proteins containing transmembrane domains could be identified by LC-ESI-MS/MS. While not described here, the magnetic bead method can also be used to isolate plasma membranes from cell lysates. This membrane purification method should expedite the cataloging of plasma membrane proteome.

Total main rotor isolation system analysis

NASA Technical Reports Server (NTRS)

Halwes, D. R.

1981-01-01

The requirements for a preliminary design study and verification procedure for a total main rotor isolation system at n/rev are established. The system is developed and analyzed, and predesign drawings are created for an isolation system that achieves over 95 percent isolation of all six degrees of freedom.

Increasing verbal interaction among elderly socially isolated mentally retarded adults: a group language training procedure.

PubMed Central

Kleitsch, E C; Whitman, T L; Santos, J

1983-01-01

The present study examined the effectiveness of a group language training procedure for directly increasing and generalizing the rate of verbal interaction among four elderly, socially isolated, moderately mentally retarded men. A withdrawal of treatment design was used to examine the effect of the procedure that used verbal prompts. behavioral rehearsal, and contingent social praise. Changes in behavior were examined in two generalization settings, one similar to the training environment (Generalization I) and the other arranged as part of the subjects' daily routine (Generalization II). Baseline data indicated no verbal interaction among the subjects. During treatment the training procedure increased the rate of subjects' verbal interactions not only in the training situation, but also in the two generalization settings. An analysis of the data obtained during the Generalization II situation indicated that subjects' verbal interaction increased not only among themselves, but with nonsubject peers present in this setting. Follow-up data showed that increases in rates of verbal interaction were maintained four months after the cessation of training. The implications of the results for program generalization and work with the language deficient individual is discussed. PMID:6885671

LAPAROSCOPIC UNCINATECTOMY: A MORE CONSERVATIVE APPROACH TO THE UNCINATE PROCESS OF THE PANCREAS.

PubMed

Surjan, Rodrigo Cañada; Basseres, Tiago; Makdissi, Fabio Ferrari; Machado, Marcel Autran Cesar; Ardengh, José Celso

2017-01-01

The isolate resection of the uncinate process of the pancreas is a rarely described procedure but is an adequate surgery to treat benign and low grade malignancies of the uncinate process of the pancreas. To detail laparoscopic uncinatectomy technique and present the initial results. Patient is placed in supine position with the surgeon between legs. Three 5-mm, one 10-mm and one 12-mm trocars were used to perform the isolated resection of the uncinate process of the pancreas. Parenchymal transection is performed with harmonic scalpel. A hemostatic absorbable tissue is deployed over the area previously occupied by the uncinate process. A Waterman drain is placed. This procedure was applied to an asymptomatic 62-year-old male with biopsy proven low grade neuroendocrine tumor of the pancreatic uncinate process. A laparoscopic pancreaticoduodenectomy was proposed. During the initial surgical evaluation, intraoperative sonography was performed and disclosed that the lesion was a few millimeters away from the Wirsung. The option was to perform a laparoscopic uncinatectomy. Postoperative period until full recovery was swift and uneventful. Laparoscopic uncinatectomy is a safe and efficient procedure when performed by surgical teams with large experience in minimally invasive biliopancreatic procedures.

Rapid preparation of nuclei-depleted detergent-resistant membrane fractions suitable for proteomics analysis.

PubMed

Adam, Rosalyn M; Yang, Wei; Di Vizio, Dolores; Mukhopadhyay, Nishit K; Steen, Hanno

2008-06-05

Cholesterol-rich membrane microdomains known as lipid rafts have been implicated in diverse physiologic processes including lipid transport and signal transduction. Lipid rafts were originally defined as detergent-resistant membranes (DRMs) due to their relative insolubility in cold non-ionic detergents. Recent findings suggest that, although DRMs are not equivalent to lipid rafts, the presence of a given protein within DRMs strongly suggests its potential for raft association in vivo. Therefore, isolation of DRMs represents a useful starting point for biochemical analysis of lipid rafts. The physicochemical properties of DRMs present unique challenges to analysis of their protein composition. Existing methods of isolating DRM-enriched fractions involve flotation of cell extracts in a sucrose density gradient, which, although successful, can be labor intensive, time consuming and results in dilute sucrose-containing fractions with limited utility for direct proteomic analysis. In addition, several studies describing the proteomic characterization of DRMs using this and other approaches have reported the presence of nuclear proteins in such fractions. It is unclear whether these results reflect trafficking of nuclear proteins to DRMs or whether they arise from nuclear contamination during isolation. To address these issues, we have modified a published differential detergent extraction method to enable rapid DRM isolation that minimizes nuclear contamination and yields fractions compatible with mass spectrometry. DRM-enriched fractions isolated using the conventional or modified extraction methods displayed comparable profiles of known DRM-associated proteins, including flotillins, GPI-anchored proteins and heterotrimeric G-protein subunits. Thus, the modified procedure yielded fractions consistent with those isolated by existing methods. However, we observed a marked reduction in the percentage of nuclear proteins identified in DRM fractions isolated with the modified method (15%) compared to DRMs isolated by conventional means (36%). Furthermore, of the 21 nuclear proteins identified exclusively in modified DRM fractions, 16 have been reported to exist in other subcellular sites, with evidence to suggest shuttling of these species between the nucleus and other organelles. We describe a modified DRM isolation procedure that generates DRMs that are largely free of nuclear contamination and that is compatible with downstream proteomic analyses with minimal additional processing. Our findings also imply that identification of nuclear proteins in DRMs is likely to reflect legitimate movement of proteins between compartments, and is not a result of contamination during extraction.

Isolation of Tonoplast Vesicles from Tomato Fruit Pericarp

PubMed Central

Snowden, Christopher J.; Thomas, Benjamin; Baxter, Charles J.; Smith, J. Andrew C.; Sweetlove, Lee J.

2017-01-01

This protocol describes the isolation of tonoplast vesicles from tomato fruit. The vesicles isolated using this procedure are of sufficiently high purity for downstream proteomic analysis whilst remaining transport competent for functional assays. The methodology was used to study the transport of amino acids during tomato fruit ripening (Snowden et al., 2015) and based on the procedure used by Betty and Smith (Bettey and Smith, 1993). Such vesicles may be useful in further studies into the dynamic transfer of metabolites across the tonoplast for storage and metabolism during tomato fruit development. PMID:29085859

Isolation of plasmodesmata from Arabidopsis suspension culture cells.

PubMed

Grison, Magali S; Fernandez-Calvino, Lourdes; Mongrand, Sébastien; Bayer, Emmanuelle M F

2015-01-01

Due to their position firmly anchored within the plant cell wall, plasmodesmata (PD) are notoriously difficult to isolate from plant tissue. Yet, getting access to isolated PD represents the most straightforward strategy for the identification of their molecular components. Proteomic and lipidomic analyses of such PD fractions have provided and will continue to provide critical information on the functional and structural elements that define these membranous nano-pores. Here, we describe a two-step simple purification procedure that allows isolation of pure PD-derived membranes from Arabidopsis suspension cells. The first step of this procedure consists in isolating cell wall fragments containing intact PD while free of contamination from other cellular compartments. The second step relies on an enzymatic degradation of the wall matrix and the subsequent release of "free" PD. Isolated PD membranes provide a suitable starting material for the analysis of PD-associated proteins and lipids.

Preparation and Characterization of Myosin Proteins.

ERIC Educational Resources Information Center

Caldwell, Elizabeth; Eftink, Maurice R.

1985-01-01

Students complete five experimental projects at the end of a senior-level biochemistry course which involves the isolation and characterization of myosin and its water-soluble subfragments. Procedures used and results obtained are provided for such projects as viscosity and ATPase measurements and gel electrophoresis experiments. (JN)

Preparative isolation of oligomeric procyanidins from Hawthorn (Crataegus spp.).

PubMed

Zumdick, S; Petereit, F; Luftmann, H; Hensel, A

2009-04-01

The oligomeric procyanidins (OPC) from Hawthorn leaves and flowers (Crataegi folium cum flore) are considered to be in part responsible for the cardiotonic clinical activity of the herbal material. Effective methods for rapid isolation of these heterogenous oligomeric clusters with defined molecular weight as reference compounds are not published until now. Therefore the water soluble fraction of an acetone/water (7 + 3) extract of Hawthorn leaves and flowers was fractionated by a combination of MPLC on RP-18 material and preparative HPLC using a diol stationary phase. This procedure resulted in the effective isolation of procyanidins with a distinct degree of polymerization (DP) from dimers DP2 up to tridecamers DP13. Exact mass measurements with negative ESI-TOF/MS were employed to confirm the respective structures of the isolated procyanidins.

Tumour-cytolytic human monocyte-derived macrophages: a simple and efficient method for the generation and long-term cultivation as non-adherent cells in a serum-free medium.

PubMed

Streck, R J; Hurley, E L; Epstein, D A; Pauly, J L

1992-01-01

We report a simple and efficient culture procedure for the generation of tumour-cytolytic human monocyte-derived macrophages (MAC). In this method, normal human peripheral blood mononuclear cells, isolated using a conventional Ficoll-Hypaque density gradient procedure, are cultured as a heterogenous leukocyte population in Teflon or other hydrophobic cultureware, in a commercially available serum-free culture medium (M-SFM) that has been formulated specifically for the cultivation and ex vivo stimulation of human monocytes and MAC, and in the absence of exogenous mitogens, antigens, cytokines or other stimulants. This procedure features a negative-selection technique that takes advantage of the differential survival of blood leukocytes. Using the prescribed in vitro conditions, lymphocytes survived relatively poorly, whereas monocytes differentiated in the absence of exogenous stimulants into mature tumour-cytolytic MAC. The MAC were present as non-adherent, single cells that expressed good viability (greater than 95%) for a prolonged period (greater than 60 days). When compared to conventional procedures for generating MAC, the prescribed technique is thought to offer several important advantages in that it: (a) eliminates the tedious and cumbersome monocyte isolation procedures, thus providing a significant savings not only in time and money but also in eliminating repetitive cell manipulations that have often been associated with damage to monocyte morphology and/or function; (b) reduces the loss of monocyte subsets that are not recovered during specific isolation procedures; (c) facilitates harvesting a single cell, non-adherent suspension of immunocompetent MAC suitable for various examinations including analyses defining MAC morphology, cytochemistry, phenotype and function; and (d) eliminates variability and artifacts associated with different sera that are utilised frequently as medium supplements. The utility of the prescribed method is illustrated by the results of ongoing studies in which scanning electron microscopy and confocal laser scanning microscopy are being used to define MAC function in different immunological reactions, and examples of these observations are presented herein.

[Method and procedures in bacteriological study of necrotic teeth].

PubMed

Rodríguez-Ponce, A; López Campos, A; López Paz, J; Pazos Sierra, R

1991-01-01

Research was conducted of 160 radicular canals with necrotic pulp. Results of different bacteriological analyses are presented. Culture analyses in aerobic and anaerobic media, resulted in the isolation of Staphylococcus Epidermidis, Streptococcus Viridans and Corynebacterium sp in the group studied, as the most frequent bacteria. There was no evidence of a specific germ linked with the pulp necrosis.

Phycobilisomes from blue-green and red algae: isolation criteria and dissociation characteristics

DOE Office of Scientific and Technical Information (OSTI.GOV)

Gantt, E.; Lipschultz, C.A.; Grabowski, J.

1979-04-01

A general procedure for the isolation of functionally intact phycobilisomes was devised, based on modifications of previously used procedures. It has been successful with numerous species of red and blue-green algae (Anabaena variabilis, Anacystis nidulans, Agmenellum quadruplicatum, Fremyella diplosiphon, Glaucosphaera vacuolata, Griffithsia pacifica, Nemalion multifidum, Nostoc sp., Phormidium persicinum, Porphyridium cruentum, P. sordidum, P. aerugineum, Rhodosorus marinus). Isolation was carried out in 0.75 molar K-phosphate (pH 6.8 to 7.0) at 20 to 23 C on sucrose step gradients. Lower temperature (4 to 10 C) was usually unfavorable resulting in uncoupling of energy transfer and partial dissociation of the phycobilisomes, sometimesmore » with complete loss of allophycocyanin. Intact phycobilisomes were characterized by fluorescence emission peaks of 670 to 675 nanometers at room temperature, and 678 to 685 nanometers at liquid nitrogen temperature. Uncoupling and subsequent dissociation of phycobilisomes, in lowered ionic conditions, varied with the species and the degree of dissociation but occurred preferentially between phycocyanin and allophycocyanin, or between phycocyanin and phycoerythrin.« less

Isolation of Mouse Pancreatic Islets of Langerhans.

PubMed

Ramírez-Domínguez, Miriam

The aim of any pancreatic islet isolation is obtaining pure, viable and functional pancreatic islets, either for in vitro or in vivo purposes. The islets of Langerhans are complex microorgans with the important role of regulating glucose homeostasis. Imbalances in glucose homeostasis lead to diabetes, which is defined by the American Diabetes Association as a "group of metabolic diseases characterized by hyperglycemia resulting from defects in insulin secretion, insulin action or both" (American Diabetes Association 2011). Currently, the rising demand of human islets is provoking a shortage of this tissue, limiting research and clinical practice on this field. In this scenario, it is essential to investigate and improve islet isolation procedures in animal models, while keeping in mind the anatomical and functional differences between species. This chapter discusses the main aspects of mouse islet isolation research, highlighting the critical factors and shortcomings to take into account for the selection and/or optimization of a mouse islet isolation protocol.

Application of zinc chloride precipitation method for rapid isolation and concentration of infectious Pectobacterium spp. and Dickeya spp. lytic bacteriophages from surface water and plant and soil extracts.

PubMed

Czajkowski, Robert; Ozymko, Zofia; Lojkowska, Ewa

2016-01-01

This is the first report describing precipitation of bacteriophage particles with zinc chloride as a method of choice to isolate infectious lytic bacteriophages against Pectobacterium spp. and Dickeya spp. from environmental samples. The isolated bacteriophages are ready to use to study various (ecological) aspects of bacteria-bacteriophage interactions. The method comprises the well-known precipitation of phages from aqueous extracts of the test material by addition of ZnCl2, resuscitation of bacteriophage particles in Ringer's buffer to remove the ZnCl2 excess and a soft agar overlay assay with the host bacterium to isolate infectious individual phage plaques. The method requires neither an enrichment step nor other steps (e. g., PEG precipitation, ultrafiltration, or ultracentrifugation) commonly used in other procedures and results in isolation of active viable bacteriophage particles.

Transcatheter Treatment of Tricuspid Regurgitation Using Edge-to-Edge Repair: Procedural Results, Clinical Implications and Predictors for Success.

PubMed

Lurz, Philipp; Besler, Christian; Noack, Thilo; Forner, Anna Flo; Bevilacqua, Carmine; Seeburger, Joerg; Rommel, Karl-Philipp; Blazek, Stephan; Hartung, Philipp; Zimmer, Marion; Mohr, Friedrich; Schuler, Gerhard; Linke, Axel; Ender, Joerg; Thiele, Holger

2018-04-10

To analyze the feasibility, safety and effectiveness of Tricuspid valve (TV) repair using the MitraClip system in patients at high surgical risk. Forty-two elderly high-risk patients (76.8±7.3 years, EuroScore II 8.1±5.7) with isolated TR or combined TR and mitral regurgitation (MR) underwent edge-to-edge repair of the TV (n=11) or combined edge-to-edge repair of the TV and mitral valve (n=31). Procedural details, success rate, impact on TR severity and predictors for success at 30 day follow-up were analyzed. Successful edge-to-edge repair of TR was achieved in 35/42 patients (83%, 68 clips in total, 94% in the anteroseptal commissure, 6% in the posteroseptal commissure). In 5 patients, grasping of the leaflets was impossible and two patients had no decrease in TR after clipping. In those with procedural success, clipping of the TV led to a reduction in effective regurgitant orifice area by -62,5 % (from 0.8±0.4 to 0.3±0.2 cm2; p<0.0001). In both, patients with isolated TV and combined procedures, 6 minute walking distance improved (from 285±118 to 344±81 and 225±113 to 261±130 m, p=0.02 and 0.03, respectively). Predominant anteroseptal or central TR was identified as predictor of procedural success (p=0.025). Edge-to-edge repair of the TV is feasible with promising reduction in TR, which could result in clinical improvement.

Derivation of Functional Human Astrocytes from Cerebral Organoids

PubMed Central

Dezonne, Rômulo Sperduto; Sartore, Rafaela Costa; Nascimento, Juliana Minardi; Saia-Cereda, Verônica M.; Romão, Luciana Ferreira; Alves-Leon, Soniza Vieira; de Souza, Jorge Marcondes; Martins-de-Souza, Daniel; Rehen, Stevens Kastrup; Gomes, Flávia Carvalho Alcantara

2017-01-01

Astrocytes play a critical role in the development and homeostasis of the central nervous system (CNS). Astrocyte dysfunction results in several neurological and degenerative diseases. However, a major challenge to our understanding of astrocyte physiology and pathology is the restriction of studies to animal models, human post-mortem brain tissues, or samples obtained from invasive surgical procedures. Here, we report a protocol to generate human functional astrocytes from cerebral organoids derived from human pluripotent stem cells. The cellular isolation of cerebral organoids yielded cells that were morphologically and functionally like astrocytes. Immunolabelling and proteomic assays revealed that human organoid-derived astrocytes express the main astrocytic molecular markers, including glutamate transporters, specific enzymes and cytoskeletal proteins. We found that organoid-derived astrocytes strongly supported neuronal survival and neurite outgrowth and responded to ATP through transient calcium wave elevations, which are hallmarks of astrocyte physiology. Additionally, these astrocytes presented similar functional pathways to those isolated from adult human cortex by surgical procedures. This is the first study to provide proteomic and functional analyses of astrocytes isolated from human cerebral organoids. The isolation of these astrocytes holds great potential for the investigation of developmental and evolutionary features of the human brain and provides a useful approach to drug screening and neurodegenerative disease modelling. PMID:28345587

Biochemistry of Cells.

ERIC Educational Resources Information Center

McIntosh, Elizabeth; Moss, Robert

1995-01-01

While other lab exercises allow the student to isolate and study one component of the cell, the purpose of this lab is to break down the cell into several components and perform simultaneous assays to determine the constituents. Centrifugation is used as a separation technique. Provides procedure and expected results. (LZ)

Clinical outcome after Chevron-Akin double osteotomy versus isolated Chevron procedure: a prospective matched group analysis.

PubMed

Lechler, Philipp; Feldmann, Christine; Köck, Franz Xaver; Schaumburger, Jens; Grifka, Joachim; Handel, Martin

2012-01-01

Hallux valgus is a frequent condition of the forefoot, resulting in cosmetic deformity and pain. Chevron osteotomy (CO) is widely employed for the treatment of hallux valgus. Chevron-Akin double osteotomy (CAO) was previously described and superiority over an isolated Chevron procedure was assumed. The objective of this study was to compare the short-to-middle term outcomes of CO and CAO. This study included 72 patients with established diagnosis of mild-to-moderate hallux valgus, treated by either isolated CO or CAO. The CO group included 46 patients (mean 51.5 years) with a mean hallux valgus angle of 27°, while the CAO group included 26 subjects (mean 53.1 years) and a mean hallux valgus angle of 32° preoperatively. All patients were reviewed by physical examination, and standardized questionnaire, and pre- and postoperative standing X-rays were performed. Matched group analysis was carried out to analyze statistical differences of both techniques. The patients were assessed and group matched at a mean of 1.37 years for the CO group and 1.04 years for the CAO group, postoperatively. Mean improvement of HVA (hallux valgus angle) was 10.6° in the CO group and the CAO group improved by 17.5°. DMAA (distal metatarsal articular angle) improved in the CO group by 5.4° and in the CAO by 13.7°. Mean AOFAS score improved by 27.9 (CO) and 21.5 (CAO). Patient satisfaction was high in both groups, with a tendency towards higher values within the CAO group. These findings indicate that Chevron-Akin double osteotomy is a save and practicable procedure for the treatment of mild-to-moderate hallux valgus. Superiority of combined Chevron-Akin procedure over an isolated Chevron osteotomy might be limited to distinct clinical settings, but should not be generally assumed.

Evaluation of signal timing and coordination procedures.

DOT National Transportation Integrated Search

1985-01-01

Based on a review of available literature, recommended procedures for timing the various types of signals are provided. Specifically, procedures are included for both pretimed and vehicle-actuated controllers located at isolated intersections and at ...

The Kjeldahl method as a primary reference procedure for total protein in certified reference materials used in clinical chemistry. II. Selection of direct Kjeldahl analysis and its preliminary performance parameters.

PubMed

Vinklárková, Bára; Chromý, Vratislav; Šprongl, Luděk; Bittová, Miroslava; Rikanová, Milena; Ohnútková, Ivana; Žaludová, Lenka

2015-01-01

To select a Kjeldahl procedure suitable for the determination of total protein in reference materials used in laboratory medicine, we reviewed in our previous article Kjeldahl methods adopted by clinical chemistry and found an indirect two-step analysis by total Kjeldahl nitrogen corrected for its nonprotein nitrogen and a direct analysis made on isolated protein precipitates. In this article, we compare both procedures on various reference materials. An indirect Kjeldahl method gave falsely lower results than a direct analysis. Preliminary performance parameters qualify the direct Kjeldahl analysis as a suitable primary reference procedure for the certification of total protein in reference laboratories.

Destruction-free procedure for the isolation of bacteria from sputum samples for Raman spectroscopic analysis.

PubMed

Kloß, Sandra; Lorenz, Björn; Dees, Stefan; Labugger, Ines; Rösch, Petra; Popp, Jürgen

2015-11-01

Lower respiratory tract infections are the fourth leading cause of death worldwide. Here, a timely identification of the causing pathogens is crucial to the success of the treatment. Raman spectroscopy allows for quick identification of bacterial cells without the need for time-consuming cultivation steps, which is the current gold standard to detect pathogens. However, before Raman spectroscopy can be used to identify pathogens, they have to be isolated from the sample matrix, i.e., sputum in case of lower respiratory tract infections. In this study, we report an isolation protocol for single bacterial cells from sputum samples for Raman spectroscopic identification. Prior to the isolation, a liquefaction step using the proteolytic enzyme mixture Pronase E is required in order to deal with the high viscosity of sputum. The extraction of the bacteria was subsequently performed via different filtration and centrifugation steps, whereby isolation ratios between 46 and 57 % were achieved for sputa spiked with 6·10(7) to 6·10(4) CFU/mL of Staphylococcus aureus. The compatibility of such a liquefaction and isolation procedure towards a Raman spectroscopic classification was shown for five different model species, namely S. aureus, Staphylococcus epidermidis, Streptococcus pneumoniae, Klebsiella pneumoniae, and Pseudomonas aeruginosa. A classification of single-cell Raman spectra of these five species with an accuracy of 98.5 % could be achieved on the basis of a principal component analysis (PCA) followed by a linear discriminant analysis (LDA). These classification results could be validated with an independent test dataset, where 97.4 % of all spectra were identified correctly. Graphical Abstract Development of an isolation protocol of bacterial cells out of sputum samples followed by Raman spectroscopic measurement and species identification using chemometrical models.

Safe and rapid isolation of pulmonary veins using a novel circular ablation catheter and duty-cycled RF generator.

PubMed

Fredersdorf, Sabine; Weber, Stefan; Jilek, Clemens; Heinicke, Norbert; VON Bary, Christian; Jungbauer, Carsten; Riegger, Günter A; Hamer, Okka W; Jeron, Andreas

2009-10-01

Ablation of atrial fibrillation (AF) has been one of the most difficult and time-consuming electrophysiological procedures. Due to the rapidly increasing demand for ablation procedures, technical advances would be helpful to reduce complexity and procedure time in AF ablation. Therefore, we investigated the feasibility of a single-catheter technique for pulmonary vein (PV) isolation utilizing a decapolar catheter combined with a duty-cycled, unipolar-bipolar radiofrequency (RF) generator. AF mapping and ablation was performed in 21 consecutive patients (mean age 59 +/- 12 years, 9 males) with paroxysmal AF (n = 17) and persistent AF (n = 4). The ablation catheter was forwarded to the LA via single-transseptal puncture. All electrodes were energized in 2 to 5 applications per vein, followed by segmental RF applications, as needed, to achieve electrical isolation. To assess left atrial anatomy for purposes of catheter manipulation, and later evaluate the possibility of asymptomatic PV-stenosis, CT or MR imaging was performed both prior to ablation and at 6-month follow-up. Isolation could be achieved in 85/86 veins (99%). Procedure time for ablation was 81 +/- 13 minutes, and fluoroscopy time was 30 +/- 11 minutes. There were no procedural complications. Success rate at 6 months was 86% (18/21). MR or CT imaging excluded asymptomatic PV-stenosis. Mapping and ablation of PVs can be performed in a safe and efficient manner using a single-catheter technique, with short procedure times and minimal learning curve. Thus, this system may be of high interest not only for high volume but all centers performing AF ablation.

Biochemical and technological studies on the production of isolated guar protein.

PubMed

Khalil, M M

2001-02-01

Guar seeds contain 32% crude protein. Therefore, attempts were made to prepare protein isolates from guar seed flour (GSF) by extraction in different media (distilled water, salt solution, alkali solution alone or in combination) followed by a precipitation at acid pH. From the four technologies adopted, mixed salt-alkali solution was found to be the most satisfactory for extraction of protein from GSF. The highest amount of product was obtained in the mixed technology along with the highest amount of protein (87.5%). Protein isolates were also nutritionally evaluated following well-established rat bioassay procedures in a comparative study with casein as standard. The protein isolates are rich in lysine but poor in sulphur-containing amino acids such as methionine and cysteine. Protein isolates obtained by mixed salt-alkali solution showed high water and oil absorption as well as good emulsifying and foaming stability. The results indicate that protein isolates can be used as a supplementary source of protein in different food industries.

Systematic internal transcribed spacer sequence analysis for identification of clinical mold isolates in diagnostic mycology: a 5-year study.

PubMed

Ciardo, Diana E; Lucke, Katja; Imhof, Alex; Bloemberg, Guido V; Böttger, Erik C

2010-08-01

The implementation of internal transcribed spacer (ITS) sequencing for routine identification of molds in the diagnostic mycology laboratory was analyzed in a 5-year study. All mold isolates (n = 6,900) recovered in our laboratory from 2005 to 2009 were included in this study. According to a defined work flow, which in addition to troublesome phenotypic identification takes clinical relevance into account, 233 isolates were subjected to ITS sequence analysis. Sequencing resulted in successful identification for 78.6% of the analyzed isolates (57.1% at species level, 21.5% at genus level). In comparison, extended in-depth phenotypic characterization of the isolates subjected to sequencing achieved taxonomic assignment for 47.6% of these, with a mere 13.3% at species level. Optimization of DNA extraction further improved the efficacy of molecular identification. This study is the first of its kind to testify to the systematic implementation of sequence-based identification procedures in the routine workup of mold isolates in the diagnostic mycology laboratory.

Magnetic techniques for the isolation and purification of proteins and peptides

PubMed Central

Safarik, Ivo; Safarikova, Mirka

2004-01-01

Isolation and separation of specific molecules is used in almost all areas of biosciences and biotechnology. Diverse procedures can be used to achieve this goal. Recently, increased attention has been paid to the development and application of magnetic separation techniques, which employ small magnetic particles. The purpose of this review paper is to summarize various methodologies, strategies and materials which can be used for the isolation and purification of target proteins and peptides with the help of magnetic field. An extensive list of realised purification procedures documents the efficiency of magnetic separation techniques. PMID:15566570

Anomalous bodily experiences and perceived social isolation in schizophrenia: An extension of the Social Deafferentation Hypothesis.

PubMed

Michael, Jamie; Park, Sohee

2016-10-01

Disturbances of the bodily self are fundamental to the phenomenological experience of individuals with schizophrenia, a population at risk for social isolation. Both proprioception and exteroception contribute to a sense of consistent body boundary that contains the self across time and space, and this process is influenced by self-other (social) interactions. However, the relationship between social isolation, exteroception, and in-the-moment changes in body representation has not been elucidated. We investigated susceptibility to anomalous bodily experiences with a phantom nose induction procedure that elicits a sensation that one's nose is changing (Pinocchio Illusion: PI) in relation to exteroceptive awareness and social isolation. 25 individuals with schizophrenia (SZ) and 15 matched controls (CO) participated in a PI induction procedure to quantify susceptibility to bodily aberrations and a tactile discrimination task to assess exteroception. Clinical symptoms in SZ and schizotypy in CO were assessed, in addition to a self-report measure of perceived social isolation. Compared to CO, SZ showed increased PI and impaired tactile discriminability. SZ reported greater loneliness than CO. PI scores were correlated with increased loneliness and decreased tactile discriminability. Greater susceptibility to anomalous bodily experiences, together with reduced exteroceptive awareness and increased loneliness, is compatible with the framework of Hoffman's Social Deafferentation Hypothesis, which posits that a functional "amputation" from one's social environment could lead to a reorganization of the social brain network, resulting in hallucinations and delusions. These findings underscore the importance of the relationship between social isolation and self-disturbances in schizophrenia. Copyright © 2016 Elsevier B.V. All rights reserved.

Nursing Procedures. NAVMED P-5066.

ERIC Educational Resources Information Center

Bureau of Medicine and Surgery (Navy), Washington, DC.

The revised manual of nursing procedures covers fundamental nursing care, admission and discharge of the patient, assisting with therapeutic measures, pre- and postoperative care, diagnostic tests and procedures, and isolation technique. Each of the over 300 topics includes the purpose, equipment, and procedure to be used and, where relevant, such…

5-Year Outcome of Pulmonary Vein Isolation by Loss of Pace Capture on the Ablation Line Versus Electrical Circumferential Pulmonary Vein Isolation.

PubMed

Moser, Julia; Sultan, Arian; Lüker, Jakob; Servatius, Helge; Salzbrunn, Tim; Altenburg, Manuel; Schäffer, Benjamin; Schreiber, Doreen; Akbulak, Ruken Ö; Vogler, Julia; Hoffmann, Boris A; Willems, Stephan; Steven, Daniel

2017-11-01

This study sought to compare long-term arrhythmia-free survival between electrical circumferential pulmonary vein isolation (PVI) and PVI with the endpoint of unexcitability along the ablation line. PVI is the standard ablation strategy of paroxysmal atrial fibrillation, although arrhythmia recurrence in long-term follow-up (FU) is high. The endpoint of unexcitability along the ablation line results in decreased arrhythmia recurrence compared to electrical PVI in 1-year FU. Seventy-four consecutive patients (age 62.5 ± 10.6 years; 70.3% male) with de novo paroxysmal atrial fibrillation who were initially included in our randomized trial and underwent catheter ablation at our institution were analyzed. Patients who were randomized to either a conventional group (PVI, guided by circumferential catheter signals) or a pace-guided group (PG, anatomical ablation line encircling, ablation until loss of pace capture at 10 V, 2-ms pulse width on the ablation line) underwent long-term FU. The primary endpoint was recurrence of any atrial fibrillation or atrial tachycardia after a blanking period of 3 months. Sixty-nine patients completed a mean FU period of 5.14 ± 0.98 years. Arrhythmia-free survival without antiarrhythmic drug therapy was significantly higher in the PG group (71.05% vs. 25.81%, p = 0.002). Furthermore, multiple procedure success (1.29 ± 0.61 procedures in PG vs. 1.97 ± 1.06 procedures in conventional group, p < 0.001) was higher in the PG group compared to the conventional group (89.47% vs. 58.06%, p = 0.005). The endpoint of unexcitability along the PVI line improves success rates, resulting in a significant reduction of exposure to invasive procedures in 5-year FU. Copyright © 2017 American College of Cardiology Foundation. Published by Elsevier Inc. All rights reserved.

Isolated Capsulorhexis Flap Technique in Femtosecond Laser-Assisted Cataract Surgery to Protect the Corneal Endothelial Cells.

PubMed

Li, Shaowei; Chen, Xu; Zhao, Jun; Xu, Man; Yu, Zhouxing

2017-12-18

To present a novel technique that using capsulorhexis flap to protect corneal endothelial cells during femtosecond laser-assisted cataract surgery. During the procedure, an isolated capsulorhexis flap was used to lift it up carefully by using OVD until the flap attached to the corneal inner layer. It kept steady during the phaco phase, and been removed by I/A tip after intraocular lens implantation. In our pilot study, the damage to the corneal endothelial cells from the phacoemulsification procedure were no more than the traditional phaco procedure, even femtosecond laser assisted cataract surgery. Isolated capsulorhexis flap technique initiate a new approach to protect the endothelial cell. It is a simple technique with a short learning curve, but still need a further investigation to prove the protection of corneal endothelial cells.

In Vitro Acquisition of Secondary Azole Resistance in Aspergillus fumigatus Isolates after Prolonged Exposure to Itraconazole: Presence of Heteroresistant Populations

PubMed Central

Escribano, Pilar; Recio, Sandra; Peláez, Teresa; González-Rivera, Milagros; Bouza, Emilio

2012-01-01

Secondary resistance to azoles in Aspergillus fumigatus isolates from patients taking long-term itraconazole therapy has been described. We studied the acquisition of secondary azole resistance in 20 A. fumigatus isolates with no mutations at codon 54, 98, 138, 220, 432, or 448 in the cyp51A gene. Adjusted conidium inocula (3 × 107 CFU/ml) of each isolate were prepared and progressively or directly exposed to increasing itraconazole concentrations, ranging from 0.5 μg/ml to 16 μg/ml. Itraconazole, voriconazole, and posaconazole MICs were determined using the CLSI M38-A2 procedure before (MICinitial) and after (MICfinal) exposure to itraconazole. In both procedures, the MICfinal was significantly higher than the MICinitial. However, after progressive exposure to itraconazole, the MICs of the three azoles were higher than after direct exposure. No mutations were found at codon 54, 98, 138, 220, 432, or 448 in the cyp51A gene of isolates growing at the highest concentration of itraconazole. More concentrated conidium inocula (2 × 109 CFU/ml) plated in itraconazole at 4 μg/ml revealed the presence of heteroresistant populations in two initially wild-type isolates. These isolates became resistant to itraconazole and posaconazole only after use of the concentrated inoculum. These heteroresistant isolates harbored a mutation at codon G54, and the MICs of itraconazole and posaconazole were >16 μg/ml. In all procedures, A. fumigatus short tandem repeat (STRAf) typing was used to demonstrate that the genotype did not change before or after exposure to itraconazole. PMID:22006000

Effect of Manufacturing Procedures on Human Islet Isolation from Donor Pancreata Standardized by the North American Islet Donor Score

PubMed Central

Yeh, Chun-Chieh; Wang, Ling-Jia; Mcgarrigle, James J.; Wang, Yong; Liao, Chien-Chang; Omami, Mustafa; Khan, Arshad; Nourmohammadzadeh, Mohammad; Mendoza-Elias, Joshua; Mccracken, Benjamin; Marchese, Enza; Barbaro, Barbara; Oberholzer, Jose

2017-01-01

This study investigates manufacturing procedures that affect islet isolation outcomes from donor pancreata standardized by the North American Islet Donor Score (NAIDS). Islet isolations performed at the University of Illinois, Chicago, from pancreata with NAIDS ≥65 were investigated. The research cohort was categorized into two groups based on a postpurification yield either greater than (group A) or less than (group B) 400,000 IEQ. Associations between manufacturing procedures and islet isolation outcomes were analyzed using multivariate logistic or linear regressions. A total of 119 cases were retrieved from 630 islet isolations performed since 2003. Group A is composed of 40 cases with an average postpurified yield of 570,098 IEQ, whereas group B comprised 79 cases with an average yield of 235,987 IEQ. One third of 119 cases were considered successful islet isolations that yielded >400,000 IEQ. The prepurified and postpurified islet product outcome parameters were detailed for future reference. The NAIDS (>80 vs. 65–80) [odds ratio (OR): 2.91, 95% confidence interval (CI): 1.27–6.70], cold ischemic time (≤10 vs. >10 h) (OR: 3.68, 95% CI: 1.61–8.39), and enzyme perfusion method (mechanical vs. manual) (OR: 2.38, 95% CI: 1.01–5.56) were independent determinants for postpurified islet yield ≥400,000 IEQ. The NAIDS (>80, p < 0.001), cold ischemic time (≤10 h, p < 0.05), increased unit of collagenase (p < 0.01), and pancreatic duct cannulation time (<30 min, p < 0.01) all independently correlated with better islet quantity parameters. Furthermore, cold ischemic time (≤10 h, p < 0.05), liberase MTF (p < 0.001), increased unit of collagenase (p < 0.05), duct cannulation time (<30 min, p < 0.05), and mechanical enzyme perfusion (p < 0.05) were independently associated with better islet morphology score. Analysis of islet manufacturing procedures from the pancreata with standardized quality is essential in identifying technical issues within islet isolation. Adequate processing duration in each step of islet isolation, using liberase MTF, and mechanical enzyme perfusion all affect isolation outcomes. PMID:27524672

Anti-microbial resistance stratified by risk factor among Escherichia coli strains isolated from the urinary tract at a rural clinic in Central India.

PubMed

Chatterjee, B; Kulathinal, S; Bhargava, A; Jain, Y; Kataria, R

2009-01-01

The failure of empirical therapy is frequently observed, even in community-acquired urinary tract infections. We, therefore, conducted a prospective, clinic-based study in 2004-2005 to document anti-microbial resistance rates and correlate them with possible risk factors to assist empirical decision-making. Symptomatic patients with pyuria underwent urine culture. Isolates were identified using standard methods and anti-microbial resistance was determined by disk-diffusion. Ultrasonography was used to detect complicating factors. Patients were stratified by the presence of complicating factors and history of invasive procedures for comparison of resistance rates. STATISTICAL METHOD USED: Chi-square or Fisher exact tests, as appropriate. There were 156 E. coli isolates, of which 105 were community-acquired. Twenty-three community-acquired isolates were from patients with complicating factors while 82 were from patients without any. Fifty-one isolates were from patients who had recently undergone invasive procedures on the urinary tract. Thirty-two community-acquired isolates from reproductive-age women without apparent complicating factors had resistance rates of 50% or above against tetracyclines, Co-trimoxazole, aminopenicillins, Nalidixic acid, Ciprofloxacin and 1 st generation cephalosporins. Resistance rates were significantly higher among isolates from patients subjected to invasive procedures, except against Co-trimoxazole, tetracyclines and Amikacin. High rates of anti-microbial resistance in community-acquired uropathogens have made antimicrobial sensitivity testing necessary even in a rural, primary-care setting.

Early results of patellofemoral inlay resurfacing arthroplasty using the HemiCap Wave prosthesis.

PubMed

Patel, Akash; Haider, Zakir; Anand, Amarjit; Spicer, Dominic

2017-01-01

Common surgical treatment options for isolated patellofemoral osteoarthritis include arthroscopic procedures, total knee replacement and patellofemoral replacement. The HemiCap Wave patellofemoral resurfacing prosthesis is a novel inlay design introduced in 2009 with scarce published data on its functional outcomes. We aim to prospectively evaluate early functional outcomes and complications, for patients undergoing a novel inlay resurfacing arthroplasty for isolated patellofemoral arthrosis in an independent centre. From 2010 to 2013, 16 consecutive patients underwent patellofemoral resurfacing procedures using HemiCap Wave (Arthrosurface Inc., Franklin, Massachusetts, USA) for anterior knee pain with confirmed radiologically and/or arthroscopically isolated severe patellofemoral arthrosis. Standardized surgical technique, as recommended by the implant manufacturer, was followed. Outcome measures included range of movement, functional knee scores (Oxford Knee Score (OKS), Knee Injury and Osteoarthritis Outcome Score (KOOS) and Short Form-36 (SF-36)), radiographic disease progression, revision rates and complications. Eight men and eight women underwent patellofemoral HemiCap Wave resurfacing, with an average age of 63 years (range: 46-83). Average follow-up was 24.1 months (6-34). Overall, post-operative scores were excellent. There was a statistically significant improvement in the post-operative OKS, KOOS and SF-36 scores ( p < 0.01). One patient had radiological disease progression. One patient underwent revision for deep infection. Two other minor complications were observed and treated conservatively. The HemiCap Wave patellofemoral resurfacing prosthesis has excellent early results in terms of functional outcomes, radiological outcomes and low complication rates. At the very least, early results show that the HemiCap Wave is comparable to more established onlay prostheses. The HemiCap Wave thus provides a safe and effective surgical option in the treatment of isolated patellofemoral osteoarthritis in selected patients.

Methods for detection, identification and specification of listerias

DOEpatents

Bochner, Barry

1992-01-01

The present invention relates generally to differential carbon source metabolism in the genus Listeria, metabolic, biochemical, immunological and genetic procedures to measure said differential carbon source metabolism and the use of these produces to detect, isolate and/or distinguish species of the genus Listeria as well as detect, isolate and/or distinguish strains of species of Listeria. The present invention also contemplates test kits and enrichment media to facilitate these procedures.

A simple method for isolating chicken egg yolk immunoglobulin using effective delipidation solution and ammonium sulfate.

PubMed

Tong, Chenyao; Geng, Fang; He, Zhenjiao; Cai, Zhaoxia; Ma, Meihu

2015-01-01

Chicken egg yolk immunoglobulin (IgY) is a superior alternative to mammalian immunoglobulin. However, the practical application of IgY in research, diagnostics, and functional food is limited due to complex or time-consuming purification procedures. The objective of this study was to develop a simple, safe, large-scale separation method for IgY from egg yolk. Egg yolk was diluted with 6-fold delipidation solutions made of different types (pectin, λ-carrageenan, carboxymethylcellulose, methylcellulose, and dextran sulfate) and concentrations (0.01, 0.05, 0.1, 0.15, and 0.2%) of polysaccharides, respectively. The yolk solution was adjusted to pH 5.0, and then kept overnight at 4°C before being centrifuged at 4°C. The resulting supernatant was added to 35% (w/v) (NH4)2SO4 and then centrifuged. The precipitant, which contained IgY, was dissolved in distilled water and then dialyzed. SDS-PAGE and Western blotting were utilized to conduct qualitative analysis of IgY; high-performance liquid chromatography (HPLC) was used for quantitative analysis. The immunoreactivity of IgY was measured by ELISA. The results showed that yield, purity, and immunoreactivity varied with types and concentrations of polysaccharides. The optimal isolation of IgY for pectin, λ-carrageenan, dextran sulfate, and carboxymethylcellulose was at the concentration of 0.1%; for methylcellulose, optimal isolation was at 0.15%. The best results were obtained in the presence of 0.1% pectin. In this condition, yield and purity can reach 8.36 mg/mL egg yolk and 83.3%, respectively, and the negative effect of IgY on immunoreactivity can be minimized. The procedure of isolation was simplified to 2 steps with a higher yield of IgY, avoiding energy- and time-consuming methods. Therefore, the isolation condition under study has a great potential for food industry production of IgY on a large scale. © 2014 Poultry Science Association Inc.

Fault detection and isolation in the challenging Tennessee Eastman process by using image processing techniques.

PubMed

Hajihosseini, Payman; Anzehaee, Mohammad Mousavi; Behnam, Behzad

2018-05-22

The early fault detection and isolation in industrial systems is a critical factor in preventing equipment damage. In the proposed method, instead of using the time signals of sensors, the 2D image obtained by placing these signals next to each other in a matrix has been used; and then a novel fault detection and isolation procedure has been carried out based on image processing techniques. Different features including texture, wavelet transform, mean and standard deviation of the image accompanied with MLP and RBF neural networks based classifiers have been used for this purpose. Obtained results indicate the notable efficacy and success of the proposed method in detecting and isolating faults of the Tennessee Eastman benchmark process and its superiority over previous techniques. Copyright © 2018 ISA. Published by Elsevier Ltd. All rights reserved.

Lipreading in the prelingually deaf: what makes a skilled speechreader?

PubMed

Rodríguez Ortiz, Isabel de los Reyes

2008-11-01

Lipreading proficiency was investigated in a group of hearing-impaired people, all of them knowing Spanish Sign Language (SSL). The aim of this study was to establish the relationships between lipreading and some other variables (gender, intelligence, audiological variables, participants' education, parents' education, communication practices, intelligibility, use of SSL). The 32 participants were between 14 and 47 years of age. They all had sensorineural hearing losses (from severe to profound). The lipreading procedures comprised identification of words in isolation. The words selected for presentation in isolation were spoken by the same talker. Identification of words required participants to select their responses from set of four pictures appropriately labelled. Lipreading was significantly correlated with intelligence and intelligibility. Multiple regression analyses were used to obtain a prediction equation for the lipreading measures. As a result of this procedure, it is concluded that proficient deaf lipreaders are more intelligent and their oral speech was more comprehensible for others.

Experimental and computational studies on molecularly imprinted solid-phase extraction for gonyautoxins 2,3 from dinoflagellate Alexandrium minutum.

PubMed

Lian, Ziru; Li, Hai-Bei; Wang, Jiangtao

2016-08-01

An innovative and effective extraction procedure based on molecularly imprinted solid-phase extraction (MISPE) was developed for the isolation of gonyautoxins 2,3 (GTX2,3) from Alexandrium minutum sample. Molecularly imprinted polymer microspheres were prepared by suspension polymerization and and were employed as sorbents for the solid-phase extraction of GTX2,3. An off-line MISPE protocol was optimized. Subsequently, the extract samples from A. minutum were analyzed. The results showed that the interference matrices in the extract were obviously cleaned up by MISPE procedures. This outcome enabled the direct extraction of GTX2,3 in A. minutum samples with extraction efficiency as high as 83 %, rather significantly, without any need for a cleanup step prior to the extraction. Furthermore, computational approach also provided direct evidences of the high selective isolation of GTX2,3 from the microalgal extracts.

Production and isolation of homologs of flerovium and element 115 at the Lawrence Livermore National Laboratory Center for Accelerator Mass Spectrometry

DOE PAGES

Despotopulos, John D.; Kmak, Kelly N.; Gharibyan, Narek; ...

2015-10-01

Here, new procedures have been developed to isolate no-carrier-added (NCA) radionuclides of the homologs and pseudo-homologs of flerovium (Hg, Sn) and element 115 (Sb), produced by 12–15 MeV proton irradiation of foil stacks with the tandem Van-de-Graaff accelerator at the Lawrence Livermore National Laboratory Center for Accelerator Mass Spectrometry (CAMS) facility. The separation of 113Sn from natIn foil was performed with anion-exchange chromatography from hydrochloric and nitric acid matrices. A cation-exchange chromatography method based on hydrochloric and mixed hydrochloric/hydroiodic acids was used to separate 124Sb from natSn foil. A procedure using Eichrom TEVA resin was developed to separate 197Hg frommore » Au foil. These results demonstrate the suitability of using the CAMS facility to produce NCA radioisotopes for studies of transactinide homologs.« less

DNA ISOLATION FROM SMALL TISSUE SAMPLES USING SALT AND SPERMINE

EPA Science Inventory

Common DNA isolation methods rely upon protein denaturation by organic solvents such as phenol and chloroform. hese solvents pose some risk to the user and require special disposal procedures. e have previously reported a method for isolating DNA from peripheral blood lymphocytes...

Selective Osmotic Shock (SOS)-Based Islet Isolation for Microencapsulation.

PubMed

Enck, Kevin; McQuilling, John Patrick; Orlando, Giuseppe; Tamburrini, Riccardo; Sivanandane, Sittadjody; Opara, Emmanuel C

2017-01-01

Islet transplantation (IT) has recently been shown to be a promising alternative to pancreas transplantation for reversing diabetes. IT requires the isolation of the islets from the pancreas, and these islets can be used to fabricate a bio-artificial pancreas. Enzymatic digestion is the current gold standard procedure for islet isolation but has lingering concerns. One such concern is that it has been shown to damage the islets due to nonselective tissue digestion. This chapter provides a detailed description of a nonenzymatic method that we are exploring in our lab as an alternative to current enzymatic digestion procedures for islet isolation from human and nonhuman pancreatic tissues. This method is based on selective destruction and protection of specific cell types and has been shown to leave the extracellular matrix (ECM) of islets intact, which may thus enhance islet viability and functionality. We also show that these SOS-isolated islets can be microencapsulated for transplantation.

Exploring MALDI-TOF MS approach for a rapid identification of Mycobacterium avium ssp. paratuberculosis field isolates.

PubMed

Ricchi, M; Mazzarelli, A; Piscini, A; Di Caro, A; Cannas, A; Leo, S; Russo, S; Arrigoni, N

2017-03-01

The aim of the study was to explore the suitability of matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOF MS) for a rapid and correct identification of Mycobacterium avium ssp. paratuberculosis (MAP) field isolates. MALDI-TOF MS approach is becoming one of the most popular tests for the identification of intact bacterial cells which has been shown to be fast and reliable. For this purpose, 36 MAP field isolates were analysed through MALDI-TOF MS and the spectra compared with two different databases: one provided by the vendor of the system employed (Biotyper ver. 3·0; Bruker Daltonics) and a homemade database containing spectra from both tuberculous and nontuberculous Mycobacteria. Moreover, principal component analysis procedure was employed to confirm the ability of MALDI-TOF MS to discriminate between very closely related subspecies. Our results suggest MAP can be differentiated from other Mycobacterium species, both when the species are very close (M. intracellulare) and when belonging to different subspecies (M. avium ssp. avium and M. avium ssp. silvaticum). The procedure applied is fast, easy to perform, and achieves an earlier accurate species identification of MAP and nontuberculous Mycobacteria in comparison to other procedures. The gold standard test for the diagnosis of paratuberculosis is still isolation of MAP by cultural methods, but additional assays, such as qPCR and subculturing for determination of mycobactin dependency are required to confirm its identification. We have provided here evidence pertaining to the usefulness of MALDI-TOF MS approach for a rapid identification of this mycobacterium among other members of M. avium complex. © 2016 The Society for Applied Microbiology.

Bed isolation in experimental flap studies in rats: a dispensable procedure.

PubMed

Heimer, Sina; Schaefer, Amelia; Mueller, Wolf; Lass, Ulrike; Gebhard, Martha M; Germann, Günter; Leimer, Uwe; Köllensperger, Eva; Reichenberger, Matthias A

2013-03-01

Review of the literature regarding rodent experimental flap models reveals fundamental differences in applied surgical procedures. Although some authors isolate the flap from its wound bed, others do not. This study was planned to investigate to what extent the insertion of a silicone sheet affects physiological wound healing in experimental flap surgery. An extended epigastric adipocutaneous flap (6 × 10 cm) was raised in 16 male Lewis rats. In the control group (group C), flaps were immediately inset without any intervention. In the experimental group (group M), a silicone sheet barrier was placed between the flap and the wound bed. Mean flap survival area and flap perfusion were evaluated. Microvessel density was visualized by immunohistochemistry, and semiquantitative real-time polymerase chain reaction addressed differential gene expression. All animals were investigated on postoperative day 5. Flap survival area and flap perfusion were found to be similar. Immunohistochemistry, however, demonstrated a significantly increased number of CD31-positive small vessels in group C. The insertion of the silicone sheet barrier (group M) was accompanied by a significantly enhanced expression of proinflammatory genes and a suppression of proangiogenic genes. Our results show that although the silicone membrane has no influence on the surgical outcome in terms of flap survival and perfusion, it does lead to significant molecular alterations in pathways involved in physiological wound healing. These alterations are artificially induced by the foreign body material and conceal the true driving forces of the healing process. As the latter might include relevant therapeutic targets to ameliorate surgical results, we regard wound bed isolation as a dispensable procedure in the study of rodent flap models.

Preparation of Cytokine-activated NK Cells for Use in Adoptive Cell Therapy in Cancer Patients: Protocol Optimization and Therapeutic Potential.

PubMed

van Ostaijen-ten Dam, Monique M; Prins, Henk-Jan; Boerman, Gerharda H; Vervat, Carly; Pende, Daniela; Putter, Hein; Lankester, Arjan; van Tol, Maarten J D; Zwaginga, Jaap J; Schilham, Marco W

2016-01-01

Cell-based immunotherapy using donor-derived natural killer (NK) cells after allogeneic hematopoietic stem cell transplantation may be an attractive treatment of residual leukemia. This study aimed to optimize clinical grade production of a cytokine-activated NK-cell product. NK cells were isolated either by double depletion (CD3(-), CD19(-)) or by sequential depletion and enrichment (CD3(-,) CD56(+)) via CliniMACS from leukapheresis material and cultured in vitro with interleukin (IL)-2 or IL-15. Both NK cell isolation procedures yielded comparable recovery of NK cells and levels of T-cell contamination. After culture with cytokines, the CD3(-)CD56(+) procedure resulted in NK cells of higher purity, that is, less T cells and monocytes, higher viability, and a slightly higher yield than the CD3(-)CD19- procedure. CD69, NKp44, and NKG2A expression were higher on CD3(-)CD56(+) products, whereas lysis of Daudi cells was comparable. Five days of culture led to higher expression of CD69, NKp44, and NKp30 and lysis of K562 and Daudi cell lines. Although CD69 expression and lysis of Daudi cells were slightly higher in cultures with IL-2, T-cell contamination was lower with IL-15. Therefore, further experiments were performed with CD3(-)CD56(+) products cultured with IL-15. Cryopreservation of IL-15-activated NK cells resulted in a loss of cytotoxicity (>92%), whereas thawing of isolated, uncultured NK cells followed by culture with IL-15 yielded cells with about 43% of the original lytic activity. Five-day IL-15-activated NK cells lysed tumor target cell lines and primary leukemic blasts, providing the basis for NK cell–based immunotherapeutic strategies in a clinical setting.

Comparison of the Vitek 2 Antifungal Susceptibility System with the Clinical and Laboratory Standards Institute (CLSI) and European Committee on Antimicrobial Susceptibility Testing (EUCAST) Broth Microdilution Reference Methods and with the Sensititre YeastOne and Etest Techniques for In Vitro Detection of Antifungal Resistance in Yeast Isolates ▿ ‖

PubMed Central

Cuenca-Estrella, Manuel; Gomez-Lopez, Alicia; Alastruey-Izquierdo, Ana; Bernal-Martinez, Leticia; Cuesta, Isabel; Buitrago, Maria J.; Rodriguez-Tudela, Juan L.

2010-01-01

The commercial technique Vitek 2 system for antifungal susceptibility testing of yeast species was evaluated. A collection of 154 clinical yeast isolates, including amphotericin B- and azole-resistant organisms, was tested. Results were compared with those obtained by the reference procedures of both the CLSI and the European Committee on Antimicrobial Susceptibility Testing (EUCAST). Two other commercial techniques approved for clinical use, the Etest and the Sensititre YeastOne, were included in the comparative exercise as well. The average essential agreement (EA) between the Vitek 2 system and the reference procedures was >95%, comparable with the average EAs observed between the reference procedures and the Sensititre YeastOne and Etest. The EA values were >97% for Candida spp. and stood at 92% for Cryptococcus neoformans. Intraclass correlation coefficients (ICC) between the commercial techniques and the reference procedures were statistically significant (P < 0.01). Percentages of very major errors were 2.6% between Vitek 2 and the EUCAST technique and 1.6% between Vitek 2 and the CLSI technique. The Vitek 2 MIC results were available after 14 to 18 h of incubation for all Candida spp. (average time to reading, 15.5 h). The Vitek 2 system was shown to be a reliable technique to determine antifungal susceptibility testing of yeast species and a more rapid and easier alternative for clinical laboratories than the procedures developed by either the CLSI or EUCAST. PMID:20220169

Intraoperative culture positive allograft bone and subsequent postoperative infections: a retrospective review

PubMed Central

Sims, Laura; Kulyk, Paul; Woo, Allan

2017-01-01

Background Obtaining intraoperative cultures of allograft bone just before use in orthopedic procedures is standard practice in many centres; however, the association between positive cultures and subsequent surgical infections is unknown. Our study had 3 goals: to determine the prevalence of positive intraoperative allograft culture and subsequent infection; to determine if, in cases of subsequent infection, organisms isolated at reoperation were the same as those cultured from the allograft at the time of the index procedure; and to assess the costs associated with performing intraoperative allograft cultures. Methods In this retrospective case series, we obtained data on patients receiving allograft bone between 2009 and 2012. Patients receiving allograft with positive cultures were reviewed to identify cases of significant infection. Organisms isolated at reoperation were compared with the allograft culture taken at the time of implantation, and we performed a cost assessment. Results Of the 996 allograft bone grafts used, 43 (4.3%) had positive intraoperative cultures and significant postoperative infections developed in 2, requiring reoperation. Antibiotics based on culture results were prescribed in 24% of cases. Organisms cultured at the time of reoperation differed from those isolated initially. The cost of performing 996 allograft cultures was $169 320. Conclusion This series suggests that rates of positive intraoperative bone allograft culture are low, and subsequent infection is rare. In cases of postoperative infection, primary allograft culture and secondary tissue cultures isolated different organisms. Costs associated with performing cultures are high. Eliminating initial culture testing could save $42 500 per year in our health region. PMID:28234217

Use of a rep-PCR system to predict species in the Aspergillus section Nigri.

PubMed

Palencia, Edwin R; Klich, Maren A; Glenn, Anthony E; Bacon, Charles W

2009-10-01

The Aspergillus niger aggregate within the A. section Nigri is a group of black-spored aspergilli of great agro-economic importance whose well defined taxonomy has been elusive. Rep-PCR has become a rapid and cost-effective method for genotyping fungi and bacteria. In the present study, we evaluated the discriminatory power of a semi-automated rep-PCR barcoding system to distinguish morphotypic species and compare the results with the data obtained from ITS and partial calmodulin regions. For this purpose, 20 morphotyped black-spored Aspergillus species were used to create the A. section Nigri library in this barcoding system that served to identify 34 field isolates. A pair-wise similarity matrix was calculated using the cone-based Pearson correlation method and the dendrogram was generated by the unweighted pair group method with arithmetic mean (UPGMA), illustrating four different clustered groups: the uniseriate cluster (I), the Aspergillus carbonarius cluster (II), and. the two A. niger aggregate clusters (named III.A and III.B). Rep-PCR showed higher resolution than the ITS and the partial calmodulin gene analytical procedures. The data of the 34 unknown field isolates, collected from different locations in the United States, indicated that only 12% of the field isolates were >95% similar to one of the genotypes included in the A. section Nigri library. However, 64% of the field isolates matched genotypes with the reference library (similarity values >90%). Based on these results, this barcoding procedure has the potential for use as a reproducible tool for identifying the black-spored aspergilli.

Rapid isolation of bone marrow mesenchymal stromal cells using integrated centrifuge-based technology.

PubMed

Meppelink, Amanda M; Wang, Xing-Hua; Bradica, Gino; Barron, Kathryn; Hiltz, Kathleen; Liu, Xiang-Hong; Goldman, Scott M; Vacanti, Joseph P; Keating, Armand; Hoganson, David M

2016-06-01

The use of bone marrow-derived mesenchymal stromal cells (MSCs) in cell-based therapies is currently being developed for a number of diseases. Thus far, the clinical results have been inconclusive and variable, in part because of the variety of cell isolation procedures and culture conditions used in each study. A new isolation technique that streamlines the method of concentration and demands less time and attention could provide clinical and economic advantages compared with current methodologies. In this study, we evaluated the concentrating capability of an integrated centrifuge-based technology compared with standard Ficoll isolation. MSCs were concentrated from bone marrow aspirate using the new device and the Ficoll method. The isolation capabilities of the device and the growth characteristics, secretome production, and differentiation capacity of the derived cells were determined. The new MSC isolation device concentrated the bone marrow in 90 seconds and resulted in a mononuclear cell yield 10-fold higher and with a twofold increase in cell retention compared with Ficoll. The cells isolated using the device were shown to exhibit similar morphology and functional activity as assessed by growth curves and secretome production compared to the Ficoll-isolated cells. The surface marker and trilineage differentiation profile of the device-isolated cells was consistent with the known profile of MSCs. The faster time to isolation and greater cell yield of the integrated centrifuge-based technology may make this an improved approach for MSC isolation from bone marrow aspirates. Copyright © 2016 International Society for Cellular Therapy. Published by Elsevier Inc. All rights reserved.

[Effects of surgery on muscles on clinical and radiographic findings in the hip joint region in cerebral palsy patients].

PubMed

Schejbalová, A; Havlas, V

2008-10-01

PURPOSE OF THE STUDY Isolated or combined surgical procedures on muscles around the hip joint are currently indicated by many authors. In cerebral palsy patients they are regarded as essential intervention. MATERIAL In the years 2005-2007, surgery in the hip joint region was essential for 150 children between 3 and 18 years of age. At the time of surgery, the patients' locomotion ranged from stage 1 to stage 7 of the Vojta system. METHODS The outcome was evaluated by clinical and radiographic examination at 2 and 6 months post-operatively and hip migration percentage and Wiberg's CE angle were measured. RESULTS The best clinical and radiographic outcomes were achieved in children younger than 6 years of age. On the other hand, isolated transfer of the distal rectus femoris muscle significantly affected pelvis anteflexion in adolescent patients. The most marked decrease in migration percentage was found after adductor tenotomy combined with surgery on the iliopsoas muscle (55.6 %) or when the two procedures were combined with distal rectus femoris transfer. DISCUSSION Combined surigical procedures, i.e., adductor tenotomy, surgery on the iliopsoas muscle or rectus femoris muscle and medial hamstrings, with fixation using an abduction modified Atlanta brace, are effective in patients with marked lateral hip migration who are younger that 6 years. Isolated adductor tenotomy and distal transfer of the rectus femoris muscle markedly improve standing position in walking patients. CONCLUSION An appropriate combination of surgical procedures on muscles in the hip region and on medial hamstrings can significantly improve the patient's locomotion and, if lateral migration is present, help to avoid surgery on bones.

Surgical approaches for atrial fibrillation.

PubMed

Schouchoff, Barbara

2007-01-01

The Cox Maze procedure, a cardiac intervention that was developed by James Cox, MD, was first performed in 1988 to surgically cure atrial fibrillation. Over the years, changes in techniques of the classic maze were made, culminating in the Cox Maze III procedure, the Gold Standard. Modifications in the original procedure included simplifying the procedure to a minimally invasive approach. As a result of some of these modifications, the initial maze-like series of surgical atrial incisions has been reduced with the use of alternate energy sources that create hyperthermic lesion lines of conduction block that isolate and interrupt the abnormal impulses. The minimize, a minimally invasive thorascopic approach, can be performed off pump, thus avoiding a median sternotomy and cardiopulmonary bypass and cardioplegic arrest intraoperatively and ensuring a shorter, less painful recovery.

[Cellular transplantation laboratory: a new field of action for nurses].

PubMed

Corradi, Maria Inês; da Silva, Sandra Honorato

2008-01-01

This article presents the experience of a nurse at a cellular transplantation laboratory. This laboratory goal is to isolate insulin producing cells for human transplantation. The nurse, as a member of an interdisciplinary team, took part in the planning of all work processes: working procedures and team training. The main activities under the nurse responsibilities include contamination control, on-the-job training and evaluation of the Quality of the procedures developed by the interdisciplinary team. Results have shown the effectiveness of the nurses' work in this new field.

Multilocus sequence typing scheme versus pulsed-field gel electrophoresis for typing Mycobacterium abscessus isolates.

PubMed

Machado, Gabriel Esquitini; Matsumoto, Cristianne Kayoko; Chimara, Erica; Duarte, Rafael da Silva; de Freitas, Denise; Palaci, Moises; Hadad, David Jamil; Lima, Karla Valéria Batista; Lopes, Maria Luiza; Ramos, Jesus Pais; Campos, Carlos Eduardo; Caldas, Paulo César; Heym, Beate; Leão, Sylvia Cardoso

2014-08-01

Outbreaks of infections by rapidly growing mycobacteria following invasive procedures, such as ophthalmological, laparoscopic, arthroscopic, plastic, and cardiac surgeries, mesotherapy, and vaccination, have been detected in Brazil since 1998. Members of the Mycobacterium chelonae-Mycobacterium abscessus group have caused most of these outbreaks. As part of an epidemiological investigation, the isolates were typed by pulsed-field gel electrophoresis (PFGE). In this project, we performed a large-scale comparison of PFGE profiles with the results of a recently developed multilocus sequence typing (MLST) scheme for M. abscessus. Ninety-three isolates were analyzed, with 40 M. abscessus subsp. abscessus isolates, 47 M. abscessus subsp. bolletii isolates, and six isolates with no assigned subspecies. Forty-five isolates were obtained during five outbreaks, and 48 were sporadic isolates that were not associated with outbreaks. For MLST, seven housekeeping genes (argH, cya, glpK, gnd, murC, pta, and purH) were sequenced, and each isolate was assigned a sequence type (ST) from the combination of obtained alleles. The PFGE patterns of DraI-digested DNA were compared with the MLST results. All isolates were analyzable by both methods. Isolates from monoclonal outbreaks showed unique STs and indistinguishable or very similar PFGE patterns. Thirty-three STs and 49 unique PFGE patterns were identified among the 93 isolates. The Simpson's index of diversity values for MLST and PFGE were 0.69 and 0.93, respectively, for M. abscessus subsp. abscessus and 0.96 and 0.97, respectively, for M. abscessus subsp. bolletii. In conclusion, the MLST scheme showed 100% typeability and grouped monoclonal outbreak isolates in agreement with PFGE, but it was less discriminative than PFGE for M. abscessus. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

Antimicrobial resistance trends among Salmonella isolates obtained from horses in the northeastern United States (2001-2013).

PubMed

Cummings, Kevin J; Perkins, Gillian A; Khatibzadeh, Sarah M; Warnick, Lorin D; Aprea, Victor A; Altier, Craig

2016-05-01

OBJECTIVE To describe the antimicrobial resistance patterns of Salmonella isolates obtained from horses in the northeastern United States and to identify trends in resistance to select antimicrobials over time. SAMPLE 462 Salmonella isolates from horses. PROCEDURES Retrospective data were collected for all Salmonella isolates obtained from equine specimens that were submitted to the Cornell University Animal Health Diagnostic Center between January 1, 2001, and December 31, 2013. Temporal trends in the prevalence of resistant Salmonella isolates were investigated for each of 13 antimicrobials by use of the Cochran-Armitage trend test. RESULTS The prevalence of resistant isolates varied among antimicrobials and ranged from 0% (imipenem) to 51.5% (chloramphenicol). During the observation period, the prevalence of resistant isolates decreased significantly for amoxicillin-clavulanic acid, ampicillin, cefazolin, cefoxitin, ceftiofur, chloramphenicol, and tetracycline and remained negligible for amikacin and enrofloxacin. Of the 337 isolates for which the susceptibility to all 13 antimicrobials was determined, 138 (40.9%) were pansusceptible and 192 (57.0%) were multidrug resistant (resistant to ≥ 3 antimicrobial classes). The most common serovar isolated was Salmonella Newport, and although the annual prevalence of that serovar decreased significantly over time, that decrease had only a minimal effect on the observed antimicrobial resistance trends. CONCLUSIONS AND CLINICAL RELEVANCE Results suggested that current antimicrobial use in horses is not promoting the emergence and dissemination of antimicrobial-resistant Salmonella strains in the region served by the laboratory.

Analysis of 16S rRNA gene lactic acid bacteria (LAB) isolate from Markisa fruit (Passiflora sp.) as a producer of protease enzyme and probiotics

NASA Astrophysics Data System (ADS)

Hidayat, Habibi

2017-03-01

16S rRNA gene analysis of bacteria lactic acid (LAB) isolate from Markisa Kuning Fruit (Passiflora edulis var. flavicarpa) as a producer of protease enzyme and probiotics has been done. The aim of the study is to determine the protease enzyme activity and 16S rRNA gene amplification using PCR. The calculation procedure was done to M4 isolate bacteria lactic acid (LAB) Isolate which has been resistant to acids with pH 2.0 in the manner of screening protease enzyme activity test result 6.5 to clear zone is 13 mm againts colony diametre is 2 mm. The results of study enzyme activity used spectrophotometer UV-Vis obtainable the regression equation Y=0.02983+0.001312X, with levels of protein M4 isolate is 0.6594 mg/mL and enzyme activity of obtainable is 0.8626 unit/ml while the spesific enzyme activity produced is 1.308 unit/mg. Then, 16S rRNA gene amplificatiom and DNA sequencing has been done. The results of study showed that the bacteria species contained from M4 bacteria lactic acid (LAB) isolate is Weisella cibiria strain II-I-59. Weisella cibiria strain II-I-59 is one of bacteria could be utilized in the digestive tract.

Biolog(TM) ID as compared to 16S ribosomal RNA ID for environmental isolates from the deep subsurface

DOE Office of Scientific and Technical Information (OSTI.GOV)

McKinsey, P.C.

2000-05-05

The U.S. Dept of Energy (DOE) Subsurface Microbial Culture Collection (SMCC) contains nearly 10,000 strains of microorganisms isolated from terrestrial subsurface environments. Many of the aerobic, gram-negative, chemoheterotrophs isolated from the DOE Savannah River Site (SRS) have previously been identified by phylogenetic analysis of 16S ribosomal RNA (rRNA) gene nucleotide sequences. These SMCC isolates are currently being examined using Biolog GN Microplates and the Biolog Microstation System in order to gain knowledge of their metabolic capabilities and to compare Biolog IDs with 16S IDs. To accommodate the particular needs of these subsurface isolates, which are often incapable of growing undermore » high-nutrient conditions, Biolog's recommendations for inoculating isolates into Biolog GN Microplates have been altered. The isolates are grown on low nutrient media, sodium thioglycolate (3mM) is added to the culture media to inhibit capsule formation, and a low density of bacteria is inoculated into the microplate. Using these altered inoculation criteria, 60 percent of these SMCC isolates have a Biolog genus ID that matches the 16S rRNA ID. These results indicate that the Biolog System can be a good means of identifying unusual environmental isolates, even when recommended inoculation procedures are altered to accommodate particular isolate needs.« less

Matrix solid phase dispersion assisted enzymatic hydrolysis as a novel approach for cocaine and opiates isolation from human hair.

PubMed

Míguez-Framil, Martha; Cabarcos, Pamela; Tabernero, María Jesús; Bermejo, Ana María; Bermejo-Barrera, Pilar; Moreda-Piñeiro, Antonio

2013-11-05

The possibility of assisting enzymatic hydrolysis (EH) procedures by sample disruption mechanisms inherent to matrix solid phase dispersion (MSPD) has been explored in the current study. EH of hair specimens from poly-drug abusers was assisted by dispersing/blending the sample (0.05 g) with alumina (2.25 g) before loading the dissolved enzyme (6 mL of 1 mg mL(-1) Pronase E in 1.4 M/1.4 M Tris/HCl, pH 7.3) through the hair-alumina solid phase packaged inside a disposable MSPD syringe. The MSPD-EH method was developed, and it proved to offer quantitative results when isolating cocaine, benzoylecgonine (BZE), codeine, morphine and 6-monoacethylmorphine (6-MAM) from human hair samples. The procedure allows an on column clean-up/pre-concentration procedure of the isolated targets by attaching a previously conditioned Oasis HLB cartridge to the end of the MSPD syringe. The EH procedure of human hair with Pronase E can therefore be shortened to approximately 30 min. Within this time, sample blending/dispersion, MSPD syringe package, elution (EH when dissolved Pronase E is passing through the sample-dispersant bed), and extract clean-up and target pre-concentration stages are achieved. Gas chromatography-mass spectrometry (GC-MS) was used for determining each target after elution from the Oasis HLB cartridges with 2 mL of 2% (v/v) acetic acid in methanol, concentration by N2 stream evaporation, and dried extract derivatization with N-methyl-tert-butylsilyltrifluoroacetamide (BSTFA) and chlorotrimethylsilane (TMCS). The method was validated according to the guidance for bioanalytical method validation of the US Department of Health and Human Services, Food and Drug Administration. The simplicity of the proposed approach makes it a useful procedure for screening/quantifying drugs of abuse in hair specimens from poly-drug abusers. Copyright © 2013 Elsevier B.V. All rights reserved.

The Secure Distributed Operating System Design Project

DTIC Science & Technology

1988-06-01

a di- verse group of people . Its organization isolates different aspects of the project, such as expected results, preliminary results, and technical...modeled after these procedures. " Automation: computers are commonly used to automate tasks previously performed by people ; many of these tasks are... people commonly con- sidered the threats anticipated to the system and mechanisms that are used to prevent those threats. Both hardware and software

Environmental and biological monitoring of platinum-containing drugs in two hospital pharmacies using positive air pressure isolators.

PubMed

Kopp, Bettina; Crauste-Manciet, Sylvie; Guibert, Agnès; Mourier, Wilhelmine; Guerrault-Moro, Marie-Noelle; Ferrari, Sylvie; Jomier, Jean-Yves; Brossard, Denis; Schierl, Rudolf

2013-04-01

Environmental and biological monitoring of platinum containing drugs was implemented in two French hospital pharmacies using positive air pressure isolators and having similar working procedures when preparing antineoplastic drugs. Wipe sampling of surfaces, gloves, and vials was performed in the preparation room and in storage areas. All employees involved in the preparation of antineoplastic drugs were tested for urinary platinum on Monday before work and Friday after shift. Only traces of platinum were detected on surfaces in the preparation room outside the isolators (less than 1.61 pg cm(-2)). However, in one center, significant contamination was found in the storage area of the drug vials, which can most likely be linked to the rupture of a platinum vial and due to inefficient cleaning procedures. Surfaces inside the isolators were found to be contaminated (maximum: 198.4 pg cm(-2)). A higher level of contamination was detected in one pharmacy and could be explained by the lack of overgloving with regular changes during the preparation process. Nitrile gloves used during drug handling outside the isolator showed the highest platinum concentration (maximum: 5.86 ng per pair). With regards to platinum urine concentration, no significant difference was found between exposed and unexposed pharmacy personnel. Isolator technology combined with individual protective measures seems to be efficient to protect workers from occupational exposure to antineoplastic drugs, whereas specific individual protective procedures implemented were focussing on the risk of handling vials outside the isolator (e.g. high frequency of glove changing). Moreover, overgloving inside the isolator would contribute to substantially decrease inner surface contamination and should be recommended in order to limit the transfer of chemical contamination to the end products.

Handicapped English: The Language of the Socially Disadvantaged.

ERIC Educational Resources Information Center

Nist, John

This book deals with the isolation and alienation of native-language speaking minority groups resulting from the teaching strategies and procedures used in the public school system. Chapters include: "The Problem Defined," which states the problem of handicapped English and argues that it is a cultural rather than a psychological phenomenon; "The…

Defining the identity of human adipose-derived mesenchymal stem cells.

PubMed

Montelatici, Elisa; Baluce, Barbara; Ragni, Enrico; Lavazza, Cristiana; Parazzi, Valentina; Mazzola, Riccardo; Cantarella, Giovanna; Brambilla, Massimiliano; Giordano, Rosaria; Lazzari, Lorenza

2015-02-01

Adipose-derived mesenchymal stem cells (ADMSCs) are an ideal population for regenerative medical application. Both the isolation procedure and the culturing conditions are crucial steps, since low yield can limit further cell therapies, especially when minimal adipose tissue harvests are available for cell expansion. To date, a standardized procedure encompassing both isolation sites and expansion methods is missing, thus making the choice of the most appropriate conditions for the preparation of ADMSCs controversial, especially in view of the different applications needed. In this study, we compared the effects of three different commercial media (DMEM, aMEM, and EGM2), routinely used for ADMSCs expansion, and two supplements, FBS and human platelet lysate, recently proven to be an effective alternative to prevent xenogeneic antibody transfer and immune alloresponse in the host. Notably, all the conditions resulted in being safe for ADMSCs isolation and expansion with platelet lysate supplementation giving the highest isolation and proliferation rates, together with a commitment for osteogenic lineage. Then, we proved that the high ADMSC hematopoietic supportive potential is performed through a constant and abundant secretion of both GCSF and SCF. In conclusion, this study further expands the knowledge on ADMSCs, defining their identity definition and offers potential options for in vitro protocols for clinical production, especially related to HSC expansion without use of exogenous cytokines or genetic modifications.

Isolation of adherent polycyclic aromatic hydrocarbon (PAH)-degrading bacteria using PAH-sorbing carriers

DOE Office of Scientific and Technical Information (OSTI.GOV)

Bastiaens, L.; Springael, D.; Wattiau, P.

Two different procedures were compared to isolate polycyclic aromatic hydrocarbon (PAH)-utilizing bacteria from PAH-contaminated soil and sludge samples, i.e., (i) shaken enrichment cultures in liquid mineral medium in which PAHs were supplied as crystals and (ii) a new method in which PAH degraders were enriched on and recovered from hydrophobic membranes containing sorbed PAHs. Both techniques were successful, but selected from the same source different bacterial strains able to grow on PAHs as the sole source of carbon and energy. The liquid enrichment mainly selected for Sphingomonas spp., whereas the membrane method exclusively led to the selection of Mycobacterium spp.more » Furthermore, in separate membrane enrichment set-ups with different membrane types, three repetitive extragenic palindromic PCR-related Mycobacterium strains were recovered. The new Mycobactereium isolates were strongly hydrophobic and displayed the capacity to adhere strongly to different surfaces. One strain, Mycobacterium sp. LB501T, displayed an unusual combination of high adhesion efficiency and an extremely high negative charge. This strain may represent a new bacterial species as suggested by 16S rRNA gene sequence analysis. These results indicate that the provision of hydrophobic sorbents containing sorbed PAHs in the enrichment procedure discriminated in favor of certain bacterial characteristics. The new isolation method is appropriate to select for adherent PAH-degrading bacteria, which might be useful to biodegrade sorbed PAHs in soils and sludge.« less

Isolation of Adherent Polycyclic Aromatic Hydrocarbon (PAH)-Degrading Bacteria Using PAH-Sorbing Carriers

PubMed Central

Bastiaens, Leen; Springael, Dirk; Wattiau, Pierre; Harms, Hauke; deWachter, Rupert; Verachtert, Hubert; Diels, Ludo

2000-01-01

Two different procedures were compared to isolate polycyclic aromatic hydrocarbon (PAH)-utilizing bacteria from PAH-contaminated soil and sludge samples, i.e., (i) shaken enrichment cultures in liquid mineral medium in which PAHs were supplied as crystals and (ii) a new method in which PAH degraders were enriched on and recovered from hydrophobic membranes containing sorbed PAHs. Both techniques were successful, but selected from the same source different bacterial strains able to grow on PAHs as the sole source of carbon and energy. The liquid enrichment mainly selected for Sphingomonas spp., whereas the membrane method exclusively led to the selection of Mycobacterium spp. Furthermore, in separate membrane enrichment set-ups with different membrane types, three repetitive extragenic palindromic PCR-related Mycobacterium strains were recovered. The new Mycobacterium isolates were strongly hydrophobic and displayed the capacity to adhere strongly to different surfaces. One strain, Mycobacterium sp. LB501T, displayed an unusual combination of high adhesion efficiency and an extremely high negative charge. This strain may represent a new bacterial species as suggested by 16S rRNA gene sequence analysis. These results indicate that the provision of hydrophobic sorbents containing sorbed PAHs in the enrichment procedure discriminated in favor of certain bacterial characteristics. The new isolation method is appropriate to select for adherent PAH-degrading bacteria, which might be useful to biodegrade sorbed PAHs in soils and sludge. PMID:10788347

Resource Conservation and Recovery Act, Part B Permit Application [for the Waste Isolation Pilot Plant (WIPP)]. Volume 5, Chapter D, Appendix D1 (conclusion), Revision 3

DOE Office of Scientific and Technical Information (OSTI.GOV)

Cook, Neville G.W.; Heuze, Francois E.; Miller, Hamish D.S.

1993-03-01

The reference design for the underground facilities at the Waste Isolation Pilot Plant was developed using the best criteria available at initiation of the detailed design effort. These design criteria are contained in the US Department of Energy document titled Design Criteria, Waste Isolation Pilot Plant (WIPP). Revised Mission Concept-IIA (RMC-IIA), Rev. 4, dated February 1984. The validation process described in the Design Validation Final Report has resulted in validation of the reference design of the underground openings based on these criteria. Future changes may necessitate modification of the Design Criteria document and/or the reference design. Validation of the referencemore » design as presented in this report permits the consideration of future design or design criteria modifications necessitated by these changes or by experience gained at the WIPP. Any future modifications to the design criteria and/or the reference design will be governed by a DOE Standard Operation Procedure (SOP) covering underground design changes. This procedure will explain the process to be followed in describing, evaluating and approving the change.« less

[Description of a staphylococcal alimentary poisoning outbreak in Las Rosas, Santa Fe Province, Argentina].

PubMed

Brizzio, Aníbal A; Tedeschi, Fabián A; Zalazar, Fabián E

2011-01-01

On February 2008, a suspected foodborne outbreak was reported in Las Rosas (Santa Fe Province, Argentina). The formal procedures indicated that an undetermined number of individuals had experienced food poisoning following consumption of vegetable cannelloni bought at a local shop. The manufacturer establishment was audited. Samples from the suspected food, as well as environmental samples and swabs from food handlers were obtained and involved subjects were interviewed. Remnants of ingested food were also obtained. Routine microbiological procedures of the foodborne outbreak revealed the presence of coagulase positive S. aureus subspecies aureus in samples from ingested and raw food, and from manipulators. Indicator microorganisms did not show significant levels and no other foodborne pathogen was isolated. Presence of staphylococcal enterotoxin-producing genes was subsequently investigated, and a positive result for enterotoxin B was shown in S. aureus strains isolated from a food handler as well as from food linked to the outbreak Moreover, these isolates showed 100% similarity by SmaI-PFGE. Timely notification together with coordinated sanitary measures and the availability of appropriate laboratory tools allowed to interrupt the chain of disease transmission by identifying risk and protective factors.

Submental liposuction versus formal cervicoplasty: which one to choose?

PubMed

Fattahi, Tirbod

2012-12-01

Esthetic rejuvenation of the submental area is a fairly common concern of patients seeking cosmetic surgery. There are several techniques used to obtain esthetic results. A common dilemma is the proper determination as to which procedure, liposuction versus formal cervicoplasty, is more appropriate. This manuscript describes the factors involved in the aging process of the submental area, as well as the inherent advantages of formal cervicoplasty over liposuction. A comprehensive review of the intrinsic and extrinsic aging process is described, and advantages and disadvantages of liposuction as well as cervicoplasty are detailed. On the basis of the specific factors leading to the fullness of the anterior neck/submental area, proper rejuvenation technique must include platysmaplasty, in addition to liposuction. Isolated liposuction is only beneficial in an isolated group of cosmetic patients. Formal cervicoplasty, including open liposuction and platysmaplasty, is a superior operation compared with isolated liposuction of the submental area. Whereas liposuction does have a role in cosmetic surgery of the submental area, it is not a comprehensive procedure and does not address all of the anatomic components leading to submental fullness. Copyright © 2012 American Association of Oral and Maxillofacial Surgeons. Published by Elsevier Inc. All rights reserved.

Umbilical Stiffness Matrix Characterization and Testing for Microgravity Science Payloads

NASA Technical Reports Server (NTRS)

Engberg, Robert C.

2003-01-01

This paper describes efforts of testing and analysis of various candidate cables and umbilicals for International Space Station microgravity science payloads. The effects of looping, large vs. small displacements, and umbilical mounting configurations were assessed. A 3-DOF stepper motor driven fixture was used to excite the umbilicals. Forces and moments were directly measured in all three axes with a 6-DOF load cell in order to derive suitable stiffness matrices for design and analysis of vibration isolation controllers. Data obtained from these tests were used to help determine the optimum type and configuration of umbilical cables for the International Space Station microgravity science glovebox (MSG) vibration isolation platform. The data and procedures can also be implemented into control algorithm simulations to assist in validation of actively controlled vibration isolation systems. The experimental results of this work are specific in support of the Glovebox Integrated Microgravity Isolation Technology (g-LIMIT) isolation platform, to be located in the microgravity science glovebox aboard the U.S. Destiny Laboratory Module.

Fluorescent Amplified-Fragment Length Polymorphism Genotyping of Neisseria meningitidis Identifies Clones Associated with Invasive Disease

PubMed Central

Goulding, Jonathan N.; Hookey, John V.; Stanley, John; Olver, Will; Neal, Keith R.; Ala'Aldeen, Dlawer A. A.; Arnold, Catherine

2000-01-01

Fluorescent amplified-fragment length polymorphism (FAFLP), a genotyping technique with phylogenetic significance, was applied to 123 isolates of Neisseria meningitidis. Nine of these were from an outbreak in a British university; 9 were from a recent outbreak in Pontypridd, Glamorgan; 15 were from sporadic cases of meningococcal disease; 26 were from the National Collection of Type Cultures; 58 were carrier isolates from Ironville, Derbyshire; 1 was a disease isolate from Ironville; and five were representatives of invasive clones of N. meningitidis. FAFLP analysis results were compared with previously published multilocus sequence typing (MLST) and pulsed-field gel electrophoresis (PFGE) results. FAFLP was able to identify hypervirulent, hyperendemic lineages (invasive clones) of N. meningitidis as well as did MLST. PFGE did not discriminate between two strains from the outbreak that were classified as similar but distinct by FAFLP. The results suggest that high resolution of N. meningitidis for outbreak and other epidemiological analyses is more cost efficient by FAFLP than by sequencing procedures. PMID:11101599

Collection, Isolation and Culture of Marine Algae.

ERIC Educational Resources Information Center

James, Daniel E.

1984-01-01

Methods of collecting, isolating, and culturing microscopic and macroscopic marine algae are described. Three different culture media list of chemicals needed and procedures for preparing Erdschreiber's and Provasoli's E. S. media. (BC)

Microbial Safari.

ERIC Educational Resources Information Center

Wagner, Stephen C.; Stewart, Robert S., Jr.

2000-01-01

Introduces an investigative microbiology laboratory activity emphasizing critical thinking and experimental design in which students isolate and characterize a bacterium from a specific habitat. Explains the procedures of the laboratory including safety, sample collection, and isolation. (YDS)

The influence of extraction procedure on ion concentrations in sediment pore water

USGS Publications Warehouse

Winger, P.V.; Lasier, P.J.; Jackson, B.P.

1998-01-01

Sediment pore water has the potential to yield important information on sediment quality, but the influence of isolation procedures on the chemistry and toxicity are not completely known and consensus on methods used for the isolation from sediment has not been reached. To provide additional insight into the influence of collection procedures on pore water chemistry, anion (filtered only) and cation concentrations were measured in filtered and unfiltered pore water isolated from four sediments using three different procedures: dialysis, centrifugation and vacuum. Peepers were constructed using 24-cell culture plates and cellulose membranes, and vacuum extractors consisted of fused-glass air stones attached with airline tubing to 60cc syringes. Centrifugation was accomplished at two speeds (2,500 and 10,000 x g) for 30 min in a refrigerated centrifuge maintained at 4?C. Only minor differences in chemical characteristics and cation and anion concentrations were found among the different collecting methods with differences being sediment specific. Filtering of the pore water did not appreciably reduce major cation concentrations, but trace metals (Cu and Pb) were markedly reduced. Although the extraction methods evaluated produced pore waters of similar chemistries, the vacuum extractor provided the following advantages over the other methods: (1) ease of extraction, (2) volumes of pore water isolated, (3) minimal preparation time and (4) least time required for extraction of pore water from multiple samples at one time.

Systematic Internal Transcribed Spacer Sequence Analysis for Identification of Clinical Mold Isolates in Diagnostic Mycology: a 5-Year Study▿ †

PubMed Central

Ciardo, Diana E.; Lucke, Katja; Imhof, Alex; Bloemberg, Guido V.; Böttger, Erik C.

2010-01-01

The implementation of internal transcribed spacer (ITS) sequencing for routine identification of molds in the diagnostic mycology laboratory was analyzed in a 5-year study. All mold isolates (n = 6,900) recovered in our laboratory from 2005 to 2009 were included in this study. According to a defined work flow, which in addition to troublesome phenotypic identification takes clinical relevance into account, 233 isolates were subjected to ITS sequence analysis. Sequencing resulted in successful identification for 78.6% of the analyzed isolates (57.1% at species level, 21.5% at genus level). In comparison, extended in-depth phenotypic characterization of the isolates subjected to sequencing achieved taxonomic assignment for 47.6% of these, with a mere 13.3% at species level. Optimization of DNA extraction further improved the efficacy of molecular identification. This study is the first of its kind to testify to the systematic implementation of sequence-based identification procedures in the routine workup of mold isolates in the diagnostic mycology laboratory. PMID:20573873

Development and worldwide use of non-lethal, and minimal population-level impact, protocols for the isolation of amphibian chytrid fungi.

PubMed

Fisher, Matthew C; Ghosh, Pria; Shelton, Jennifer M G; Bates, Kieran; Brookes, Lola; Wierzbicki, Claudia; Rosa, Gonçalo M; Farrer, Rhys A; Aanensen, David M; Alvarado-Rybak, Mario; Bataille, Arnaud; Berger, Lee; Böll, Susanne; Bosch, Jaime; Clare, Frances C; A Courtois, Elodie; Crottini, Angelica; Cunningham, Andrew A; Doherty-Bone, Thomas M; Gebresenbet, Fikirte; Gower, David J; Höglund, Jacob; James, Timothy Y; Jenkinson, Thomas S; Kosch, Tiffany A; Lambertini, Carolina; Laurila, Anssi; Lin, Chun-Fu; Loyau, Adeline; Martel, An; Meurling, Sara; Miaud, Claude; Minting, Pete; Ndriantsoa, Serge; O'Hanlon, Simon J; Pasmans, Frank; Rakotonanahary, Tsanta; Rabemananjara, Falitiana C E; Ribeiro, Luisa P; Schmeller, Dirk S; Schmidt, Benedikt R; Skerratt, Lee; Smith, Freya; Soto-Azat, Claudio; Tessa, Giulia; Toledo, Luís Felipe; Valenzuela-Sánchez, Andrés; Verster, Ruhan; Vörös, Judit; Waldman, Bruce; Webb, Rebecca J; Weldon, Che; Wombwell, Emma; Zamudio, Kelly R; Longcore, Joyce E; Garner, Trenton W J

2018-05-17

Parasitic chytrid fungi have emerged as a significant threat to amphibian species worldwide, necessitating the development of techniques to isolate these pathogens into culture for research purposes. However, early methods of isolating chytrids from their hosts relied on killing amphibians. We modified a pre-existing protocol for isolating chytrids from infected animals to use toe clips and biopsies from toe webbing rather than euthanizing hosts, and distributed the protocol to researchers as part of the BiodivERsA project RACE; here called the RML protocol. In tandem, we developed a lethal procedure for isolating chytrids from tadpole mouthparts. Reviewing a database of use a decade after their inception, we find that these methods have been applied across 5 continents, 23 countries and in 62 amphibian species. Isolation of chytrids by the non-lethal RML protocol occured in 18% of attempts with 207 fungal isolates and three species of chytrid being recovered. Isolation of chytrids from tadpoles occured in 43% of attempts with 334 fungal isolates of one species (Batrachochytrium dendrobatidis) being recovered. Together, these methods have resulted in a significant reduction and refinement of our use of threatened amphibian species and have improved our ability to work with this group of emerging pathogens.

An EThcD-Based Method for Discrimination of Leucine and Isoleucine Residues in Tryptic Peptides

NASA Astrophysics Data System (ADS)

Zhokhov, Sergey S.; Kovalyov, Sergey V.; Samgina, Tatiana Yu.; Lebedev, Albert T.

2017-08-01

An EThcD-based approach for the reliable discrimination of isomeric leucine and isoleucine residues in peptide de novo sequencing procedure has been proposed. A multistage fragmentation of peptide ions was performed with Orbitrap Elite mass spectrometer in electrospray ionization mode. At the first stage, z-ions were produced by ETD or ETcaD fragmentation of doubly or triply charged peptide precursor ions. These primary ions were further fragmented by HCD with broad-band ion isolation, and the resulting w-ions showed different mass for leucine and isoleucine residues. The procedure did not require manual isolation of specific z-ions prior to HCD stage. Forty-three tryptic peptides (3 to 27 residues) obtained by trypsinolysis of human serum albumin (HSA) and gp188 protein were analyzed. To demonstrate a proper solution for radical site migration problem, three non-tryptic peptides were also analyzed. A total of 93 leucine and isoleucine residues were considered and 83 of them were correctly identified. The developed approach can be a reasonable substitution for additional Edman degradation procedure, which is still used in peptide sequencing for leucine and isoleucine discrimination.

Isolating USB connections in medical equipment.

PubMed

Broeders, Jan-Hein

2009-01-01

Although offering several benefits, the universal serial bus (USB) port has not been rapidly adopted for connecting medical equipment. This is because it could affect safety procedures, with equipment not operating isolated from the mains. To overcome this, a single package isolation device has been developed that can be inserted directly into the USB signal path.

A new rapid method for isolating nucleoli.

PubMed

Li, Zhou Fang; Lam, Yun Wah

2015-01-01

The nucleolus was one of the first subcellular organelles to be isolated from the cell. The advent of modern proteomic techniques has resulted in the identification of thousands of proteins in this organelle, and live cell imaging technology has allowed the study of the dynamics of these proteins. However, the limitations of current nucleolar isolation methods hinder the further exploration of this structure. In particular, these methods require the use of a large number of cells and tedious procedures. In this chapter we describe a new and improved nucleolar isolation method for cultured adherent cells. In this method cells are snap-frozen before direct sonication and centrifugation onto a sucrose cushion. The nucleoli can be obtained within a time as short as 20 min, and the high yield allows the use of less starting material. As a result, this method can capture rapid biochemical changes in nucleoli by freezing the cells at a precise time, hence faithfully reflecting the protein composition of nucleoli at the specified time point. This protocol will be useful for proteomic studies of dynamic events in the nucleolus and for better understanding of the biology of mammalian cells.

A decade of improvements in Mimiviridae and Marseilleviridae isolation from amoeba.

PubMed

Pagnier, Isabelle; Reteno, Dorine-Gaelle Ikanga; Saadi, Hanene; Boughalmi, Mondher; Gaia, Morgan; Slimani, Meriem; Ngounga, Tatsiana; Bekliz, Meriem; Colson, Philippe; Raoult, Didier; La Scola, Bernard

2013-01-01

Since the isolation of the first giant virus, the Mimivirus, by T.J. Rowbotham in a cooling tower in Bradford, UK, and after its characterisation by our group in 2003, we have continued to develop novel strategies to isolate additional strains. By first focusing on cooling towers using our original time-consuming procedure, we were able to isolate a new lineage of giant virus called Marseillevirus and a new Mimivirus strain called Mamavirus. In the following years, we have accumulated the world's largest unique collection of giant viruses by improving the use of antibiotic combinations to avoid bacterial contamination of amoeba, developing strategies of preliminary screening of samples by molecular methods, and using a high-throughput isolation method developed by our group. Based on the inoculation of nearly 7,000 samples, our collection currently contains 43 strains of Mimiviridae (14 in lineage A, 6 in lineage B, and 23 in lineage C) and 17 strains of Marseilleviridae isolated from various environments, including 3 of human origin. This study details the procedures used to build this collection and paves the way for the high-throughput isolation of new isolates to improve the record of giant virus distribution in the environment and the determination of their pangenome.

Quality evaluation of LC-MS/MS-based E. coli H antigen typing (MS-H) through label-free quantitative data analysis in a clinical sample setup.

PubMed

Cheng, Keding; Sloan, Angela; McCorrister, Stuart; Peterson, Lorea; Chui, Huixia; Drebot, Mike; Nadon, Celine; Knox, J David; Wang, Gehua

2014-12-01

The need for rapid and accurate H typing is evident during Escherichia coli outbreak situations. This study explores the transition of MS-H, a method originally developed for rapid H antigen typing of E. coli using LC-MS/MS of flagella digest of reference strains and some clinical strains, to E. coli isolates in clinical scenario through quantitative analysis and method validation. Motile and nonmotile strains were examined in batches to simulate clinical sample scenario. Various LC-MS/MS batch run procedures and MS-H typing rules were compared and summarized through quantitative analysis of MS-H data output for a standard method development. Label-free quantitative data analysis of MS-H typing was proven very useful for examining the quality of MS-H result and the effects of some sample carryovers from motile E. coli isolates. Based on this, a refined procedure and protein identification rule specific for clinical MS-H typing was established and validated. With LC-MS/MS batch run procedure and database search parameter unique for E. coli MS-H typing, the standard procedure maintained high accuracy and specificity in clinical situations, and its potential to be used in a clinical setting was clearly established. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Isolated hepatic perfusion as a treatment for liver metastases of uveal melanoma.

PubMed

Ben-Shabat, Ilan; Hansson, Christoffer; Sternby Eilard, Malin; Cahlin, Christian; Rizell, Magnus; Lindnér, Per; Mattsson, Jan; Olofsson Bagge, Roger

2015-01-25

Isolated hepatic perfusion (IHP) is a procedure where the liver is surgically isolated and perfused with a high concentration of the chemotherapeutic agent melphalan. Briefly, the procedure starts with the setup of a percutaneous veno-venous bypass from the femoral vein to the external jugular vein. Via a laparotomy, catheters are then inserted into the proper hepatic artery and the caval vein. The portal vein and the caval vein, both supra- and infrahepatically, are then clamped. The arterial and venous catheters are connected to a heart lung machine and the liver is perfused with melphalan (1 mg/kg body weight) for 60 min. This way it is possible to locally perfuse the liver with a high dose of a chemotherapeutic agent, without leakage to the systemic circulation. In previous studies including patients with isolated liver metastases of uveal melanoma, an overall response rate of 33-100% and a median survival between 9 and 13 months, have been reported. The aim of this protocol is to give a clear description of how to perform the procedure and to discuss IHP as a treatment option for liver metastases of uveal melanoma.

An improved synthesis of 1-methylcyclopropanol using the Kulinkovich reaction.

PubMed

Delsarte, Christine; Etuin, Guillaume; Petit, Laurent

2018-02-15

An improved process for the preparation of 1-methylcyclopropanol using the Kulinkovich reaction is described. The use of titanium tetramethoxide as catalyst resulted in minimal side product formation. The reaction, isolation and purification procedures were optimized so they can be easily implemented in multi-purpose equipment. Copyright © 2017 Elsevier Ltd. All rights reserved.

Isolated and combined medial patellofemoral ligament reconstruction in revision surgery for patellofemoral instability: a prospective study.

PubMed

Kohn, Ludwig M; Meidinger, Gebhart; Beitzel, Knut; Banke, Ingo J; Hensler, Daniel; Imhoff, Andreas B; Schöttle, Philip B

2013-09-01

Persistent pain and redislocations after surgical treatment of patellofemoral instability are described in up to 40% of patients. However, prospective outcome data about revision surgery are missing. To evaluate the clinical outcome after revision medial patellofemoral ligament (MPFL) reconstruction using isolated and combined procedures, with a follow-up of 24 months. Case series; Level of evidence, 4. Study participants were 42 patients (median age, 22 years; range, 13-46 years) who underwent revision surgery between January 2007 and December 2009 because of persistent patellofemoral instability after a mean of 1.8 previous failed surgical interventions (lateral release, medial imbrication/vastus medialis obliquus distalization, medialization of the tuberosity). An isolated MPFL reconstruction was performed in 15 cases, while a combination procedure was performed in 27 cases. The clinical results were evaluated preoperatively and 24 months postoperatively using the International Knee Documentation Committee (IKDC), Kujala, and Tegner scores as well as a subjective questionnaire. Patellar shift, tilt, and height, as well as level of degeneration, were defined preoperatively and at the latest follow-up on plain radiographs and magnetic resonance imaging. At 24-month follow-up, 87% of the patients were satisfied or very satisfied with the treatment. No apprehension or redislocation was reported at follow-up, and there was a significant decrease in pain during daily activities. There were significant improvements (P < .001) in IKDC (from 50 to 80), Kujala (from 51 to 85), and Tegner scores (from 2.4 to 4.9). Patellar shift, tilt, and height decreased significantly (P < .05) to anatomic values, and there was no aggravation to the level of pre-existing degeneration. No significant difference was noticed between the isolated and combined procedures. As patellofemoral instability is a multifactorial problem, revision surgery should be indicated only after a comprehensive examination. The results of this study show that MPFL reconstruction, alone or in combination, seems to be an effective treatment for recurrent patellar dislocations after a failed previous surgery, leading to significant increases in stability and functionality as well as a reduction in pain.

Environmental manipulations alter age differences in attribution of incentive salience to reward-paired cues

PubMed Central

Anderson, Rachel I.; Bush, Peter C.; Spear, Linda P.

2013-01-01

Cues repeatedly paired with rewards often themselves become imbued with enhanced motivational value, or incentive salience. During Pavlovian conditioned approach procedures, a cue repeatedly preceding reward delivery often elicits conditioned responses at either the reward delivery location (“goal-tracking”) or the cue itself (“sign-tracking”). Sign-tracking behavior is thought to reflect the individual differences in attribution of incentive salience to reward-paired cues that may contribute to addiction vulnerability. Adolescent rats typically demonstrate less sign-tracking behavior than adult rats, a surprising finding given that adolescence is hypothesized to be a time of heightened addiction vulnerability. Given evidence that adult sign-tracking behavior can be influenced by environmental conditions, the present study compared the effects of isolate housing and food deprivation on expression of sign-tacking and goal-tracking behavior in adolescent and adult male rats across eight days of a Pavlovian conditioned approach procedure. Pair-housed adults exhibited more sign-tracking behavior than pair-housed adolescents; however, this age difference was not apparent in isolate-housed subjects. Adolescents often appeared more sensitive than adults to both food restriction- and isolate housing-induced changes in behavior, with food restriction promoting an increase in sign-tracking among isolate-housed adolescents and an increase in goal-tracking among pair-housed adolescents. For adults, food restriction resulted in a modest increase in overall expression of both sign-and goal-tracking behavior. To the extent that sign-tracking behavior reflects attribution of incentive salience to reward-paired cues, results from the present study provide evidence that reactivity to rewards during adolescence is strongly related to the nature of the surrounding environment. PMID:24050888

Environmental manipulations alter age differences in attribution of incentive salience to reward-paired cues.

PubMed

Anderson, Rachel I; Bush, Peter C; Spear, Linda P

2013-11-15

Cues repeatedly paired with rewards often themselves become imbued with enhanced motivational value, or incentive salience. During Pavlovian conditioned approach procedures, a cue repeatedly preceding reward delivery often elicits conditioned responses at either the reward delivery location ("goal-tracking") or the cue itself ("sign-tracking"). Sign-tracking behavior is thought to reflect the individual differences in attribution of incentive salience to reward-paired cues that may contribute to addiction vulnerability. Adolescent rats typically demonstrate less sign-tracking behavior than adult rats, a surprising finding given that adolescence is hypothesized to be a time of heightened addiction vulnerability. Given evidence that adult sign-tracking behavior can be influenced by environmental conditions, the present study compared the effects of isolate housing and food deprivation on expression of sign-tacking and goal-tracking behavior in adolescent and adult male rats across eight days of a Pavlovian conditioned approach procedure. Pair-housed adults exhibited more sign-tracking behavior than pair-housed adolescents; however, this age difference was not apparent in isolate-housed subjects. Adolescents often appeared more sensitive than adults to both food restriction- and isolate housing-induced changes in behavior, with food restriction promoting an increase in sign-tracking among isolate-housed adolescents and an increase in goal-tracking among pair-housed adolescents. For adults, food restriction resulted in a modest increase in overall expression of both sign- and goal-tracking behavior. To the extent that sign-tracking behavior reflects attribution of incentive salience to reward-paired cues, results from the present study provide evidence that reactivity to rewards during adolescence is strongly related to the nature of the surrounding environment. Copyright © 2013 Elsevier B.V. All rights reserved.

Successes and Short Comings in Four Years of an International External Quality Assurance Program for Animal Influenza Surveillance

PubMed Central

Spackman, Erica; Cardona, Carol; Muñoz-Aguayo, Jeannette; Fleming, Susan

2016-01-01

The US National institutes of Health-Centers of Excellence for Influenza Research and Surveillance is a research consortium that funds numerous labs worldwide to conduct influenza A surveillance in diverse animal species. There is no harmonization of testing procedures among these labs; therefore an external quality assurance (EQA) program was implemented to evaluate testing accuracy among labs in the program in 2012. Accurate detection of novel influenza A variants is crucial because of the broad host range and potentially high virulence of the virus in diverse species. Two molecular detection sample sets and 2 serology sample sets (one with avian origin isolates, and one with mammalian origin isolates each) were made available at approximately six month intervals. Participating labs tested the material in accordance with their own protocols. During a five year period a total of 41 labs from 23 countries ordered a total of 132 avian molecular, 121 mammalian molecular and 90 serology sample sets. Testing was completed by 111 individuals. Detection of type A influenza by RT-PCR was reliable with a pass rate (80% or greater agreement with expected results) of 86.6% for avian and 86.2% for mammalian origin isolates. However, identification of subtype by RT-PCR was relatively poor with 54.1% and 75.9% accuracy for avian and mammalian influenza isolates respectively. Serological testing had an overall pass rate of 86.9% and 22/23 labs used commercial ELISA kits. Based on the results of this EQA program six labs modified their procedures to improve accuracy and one lab identified an unknown equipment problem. These data represent the successful implementation of an international EQA program for an infectious disease; insights into the logistics and test design are also discussed. PMID:27788155

Successes and Short Comings in Four Years of an International External Quality Assurance Program for Animal Influenza Surveillance.

PubMed

Spackman, Erica; Cardona, Carol; Muñoz-Aguayo, Jeannette; Fleming, Susan

2016-01-01

The US National institutes of Health-Centers of Excellence for Influenza Research and Surveillance is a research consortium that funds numerous labs worldwide to conduct influenza A surveillance in diverse animal species. There is no harmonization of testing procedures among these labs; therefore an external quality assurance (EQA) program was implemented to evaluate testing accuracy among labs in the program in 2012. Accurate detection of novel influenza A variants is crucial because of the broad host range and potentially high virulence of the virus in diverse species. Two molecular detection sample sets and 2 serology sample sets (one with avian origin isolates, and one with mammalian origin isolates each) were made available at approximately six month intervals. Participating labs tested the material in accordance with their own protocols. During a five year period a total of 41 labs from 23 countries ordered a total of 132 avian molecular, 121 mammalian molecular and 90 serology sample sets. Testing was completed by 111 individuals. Detection of type A influenza by RT-PCR was reliable with a pass rate (80% or greater agreement with expected results) of 86.6% for avian and 86.2% for mammalian origin isolates. However, identification of subtype by RT-PCR was relatively poor with 54.1% and 75.9% accuracy for avian and mammalian influenza isolates respectively. Serological testing had an overall pass rate of 86.9% and 22/23 labs used commercial ELISA kits. Based on the results of this EQA program six labs modified their procedures to improve accuracy and one lab identified an unknown equipment problem. These data represent the successful implementation of an international EQA program for an infectious disease; insights into the logistics and test design are also discussed.

Isolation of GMP Grade Human Hepatocytes from Remnant Liver Tissue of Living Donor Liver Transplantation.

PubMed

Enosawa, Shin

2017-01-01

For the purpose of clinical research of hepatocyte transplantation, procedures for isolation, cryopreservation, thawing, and functional assessment of hepatocytes are described. Although demands for human hepatocytes are increasing in not only cell therapy but also drug development, it is highly difficult to obtain good lots of hepatocytes from human liver tissue. This chapter describes essential issues such as alleviation of warm ischemia, prevention of shear stress, optimization of cryopreservation, and functional assessment, along with securement of quality. All procedures described here are compliant with good manufacturing procedure (GMP) in cell processing facility, approved by the act on measures to ensure safety of regenerative medicine and ethical regulations in Japan.

Oxidative potential of some endophytic fungi using 1-indanone as a substrate.

PubMed

Fill, Taicia Pacheco; da Silva, Jose Vinicius; de Oliveira, Kleber Thiago; da Silva, Bianca Ferreira; Rodrigues-Fo, Edson

2012-06-01

The oxidative potential of the fungus Penicillium brasilianum, a strain isolated as an endophyte from a Meliaceae plant (Melia azedarach), was investigated using 1-indanone as a substrate to track the production of monooxygenases. The fungus produced the dihydrocoumarin from 1-indanone with the classical Baeyer-Villiger reaction regiochemistry, and (-)-(R)-3-hydroxy-1-indanone with 78% ee. Minor compounds resulting from lipase and SAM activities were also detected. The biotransformation procedures were also applied to a collection of Penicillium and Aspergillus fungi obtained from M. azedarach and Murraya paniculata. The results showed that Baeyer-Villiger were mostly active in fungi isolated from M. azedarach. Almost all of the fungi tested produced 3-hydroxy-1-indanone..

Isolation and purification of Cu-free methanobactin from Methylosinus trichosporium OB3b

PubMed Central

2011-01-01

Background The isolation of highly pure copper-free methanobactin is a prerequisite for the investigation of the biogeochemical functions of this chalkophore molecule produced by methane oxidizing bacteria. Here, we report a purification method for methanobactin from Methylosinus trichosporium OB3b cultures based on reversed-phase HPLC fractionation used in combination with a previously reported resin extraction. HPLC eluent fractions of the resin extracted product were collected and characterized with UV-vis, FT-IR, and C-1s NEXAFS spectroscopy, as well as with elemental analysis and ESI-MS. Results The results showed that numerous compounds other than methanobactin were present in the isolate obtained with resin extraction. Molar C/N ratios, mass spectrometry measurements, and UV-vis spectra indicated that methanobactin was only present in one of the HPLC fractions. On a mass basis, methanobactin carbon contributed only 32% to the total organic carbon isolated with resin extraction. Our spectroscopic results implied that besides methanobactin, the organic compounds in the resin extract comprised breakdown products of methanobactin as well as polysaccharide-like substances. Conclusion Our results demonstrate that a purification step is indispensable in addition to resin extraction in order to obtain pure methanobactin. The proposed HPLC purification procedure is suitable for semi-preparative work and provides copper-free methanobactin. PMID:21299876

Evaluation of Bacto TB hydrolysis reagent (Tween 80) for the identification of Branhamella catarrhalis.

PubMed Central

Weiner, M; Penha, P D

1990-01-01

An investigation of the hydrolysis of Tween 80 reagent by Branhamella catarrhalis and related organisms (Neisseria and Moraxella species) revealed that only B. catarrhalis gave a positive result. A total of 226 strains, including reference organisms and clinical isolates, were studied. B. catarrhalis changed the color of the reagent from amber to pink-red after overnight incubation. We recommend this simple and cost-effective test as an alternative procedure to DNase testing or tributyrin hydrolysis or as a supplemental procedure for the identification of B. catarrhalis in clinical specimens. PMID:2105335

Response of Selected Microorganisms to Experimental Planetary Environments

NASA Technical Reports Server (NTRS)

Foster, T. L.; Winans, L., Jr.

1977-01-01

Results of studies in anaerobic phosphorus metabolism are presented. Specific topics discussed include: (1) anaerobic utilization of PH3; (2) reduction of phosphate or phosphite; (3) isolation of organisms which utilize phosphite or phosphate anaerobically as a final hydrogen acceptor; and (4) the toxicity of PH3 to the organisms. Techniques of anaerobic microbiology associated with space hardware were also studied. These include: (1) the Brewer anaerobe jar/GasPak system; (2) a new procedure to grow aerobes and anaerobes simultaneously; (3) a culture medium to differentiate oblagate from facultative anaerobes; and (4) a procedure to quantitate O2 sensitivity of anaerobes.

Rapid step-gradient purification of mitochondrial DNA.

PubMed

Welter, C; Meese, E; Blin, N

1988-01-01

A convenient modification of the step gradient (CsCl/ethidium bomide) procedure is described. This rapid method allows isolation of covalently closed circular DNA separated from contaminating proteins, RNA and chromosomal DNA in ca. 5 h. Large scale preparations can be performed for circular DNA from eukaryotic organelles (mitochondria). The protocol uses organelle pelleting/NaCl-sarcosyl incubation steps for mitochondria followed by a CsCl step gradient and exhibits yields equal to the conventional procedures. It results in DNA sufficiently pure to be used for restriction endonuclease analysis, subcloning, 5'-end labeling, gel retention assays, and various types of hybridization.

Optimization of EGFR high positive cell isolation procedure by design of experiments methodology.

PubMed

Levi, Ofer; Tal, Baruch; Hileli, Sagi; Shapira, Assaf; Benhar, Itai; Grabov, Pavel; Eliaz, Noam

2015-01-01

Circulating tumor cells (CTCs) in blood circulation may play a role in monitoring and even in early detection of metastasis patients. Due to the limited presence of CTCs in blood circulation, viable CTCs isolation technology must supply a very high recovery rate. Here, we implement design of experiments (DOE) methodology in order to optimize the Bio-Ferrography (BF) immunomagnetic isolation (IMI) procedure for the EGFR high positive CTCs application. All consequent DOE phases such as screening design, optimization experiments and validation experiments were used. A significant recovery rate of more than 95% was achieved while isolating 100 EGFR high positive CTCs from 1 mL human whole blood. The recovery achievement in this research positions BF technology as one of the most efficient IMI technologies, which is ready to be challenged with patients' blood samples. © 2015 International Clinical Cytometry Society.

New methods for isolation of keratolytic bacteria inducing intractable hoof wall cavity (Gidoh) in a horse; double screening procedures of the horn powder agar-translucency test and horn zymography

PubMed Central

KUWANO, Atsutoshi; NIWA, Hidekazu; ARAI, Katsuhiko

2017-01-01

ABSTRACT To establish a new system to isolate keratolytic bacteria from the hoof wall cavity (Gidoh) of a racehorse, we invented the horn powder agar-translucency (HoPAT) test and horn zymography (HZ). Using routine bacteriological techniques and these methods, we isolated five strains of keratolytic soil bacteria, which were then identified by means of 16S ribosomal RNA (rRNA) gene sequencing analysis. The findings from the study on the horse suggested that Brevibacterium luteolum played the main role in the local fragility of the hoof, eventually forming a Gidoh in coordination with four other strains of keratolytic bacteria. The double screening procedures of the HoPAT test and HZ were useful and easy techniques for isolating the keratolytic bacteria from the horn lesions. PMID:28400703

Efficient harvesting methods for early-stage snake and turtle embryos.

PubMed

Matsubara, Yoshiyuki; Kuroiwa, Atsushi; Suzuki, Takayuki

2016-04-01

Reptile development is an intriguing research target for understating the unique morphogenesis of reptiles as well as the evolution of vertebrates. However, there are numerous difficulties associated with studying development in reptiles. The number of available reptile eggs is usually quite limited. In addition, the reptile embryo is tightly adhered to the eggshell, making it a challenge to isolate reptile embryos intact. Furthermore, there have been few reports describing efficient procedures for isolating intact embryos especially prior to pharyngula stage. Thus, the aim of this review is to present efficient procedures for obtaining early-stage reptilian embryos intact. We first describe the method for isolating early-stage embryos of the Japanese striped snake. This is the first detailed method for obtaining embryos prior to oviposition in oviparous snake species. Second, we describe an efficient strategy for isolating early-stage embryos of the soft-shelled turtle. © 2016 Japanese Society of Developmental Biologists.

Isolation and purification of all-trans diadinoxanthin and all-trans diatoxanthin from diatom Phaeodactylum tricornutum.

PubMed

Kuczynska, Paulina; Jemiola-Rzeminska, Malgorzata

2017-01-01

Two diatom-specific carotenoids are engaged in the diadinoxanthin cycle, an important mechanism which protects these organisms against photoinhibition caused by absorption of excessive light energy. A high-performance and economical procedure of isolation and purification of diadinoxanthin and diatoxanthin from the marine diatom Phaeodactylum tricornutum using a four-step procedure has been developed. It is based on the use of commonly available materials and does not require advanced technology. Extraction of pigments, saponification, separation by partition and then open column chromatography, which comprise the complete experimental procedure, can be performed within 2 days. This method allows HPLC grade diadinoxanthin and diatoxanthin of a purity of 99 % or more to be obtained, and the efficiency was estimated to be 63 % for diadinoxanthin and 73 % for diatoxanthin. Carefully selected diatom culture conditions as well as analytical ones ensure highly reproducible performance. A protocol can be used to isolate and purify the diadinoxanthin cycle pigments both on analytical and preparative scale.

[Evaluation of mass spectrometry: MALDI-TOF MS for fast and reliable yeast identification].

PubMed

Relloso, María S; Nievas, Jimena; Fares Taie, Santiago; Farquharson, Victoria; Mujica, María T; Romano, Vanesa; Zarate, Mariela S; Smayevsky, Jorgelina

2015-01-01

The matrix-assisted laser desorption/ionization time-of-flight mass spectrometry technique known as MALDI-TOF MS is a tool used for the identification of clinical pathogens by generating a protein spectrum that is unique for a given species. In this study we assessed the identification of clinical yeast isolates by MALDI-TOF MS in a university hospital from Argentina and compared two procedures for protein extraction: a rapid method and a procedure based on the manufacturer's recommendations. A short protein extraction procedure was applied in 100 isolates and the rate of correct identification at genus and species level was 98.0%. In addition, we analyzed 201 isolates, previously identified by conventional methods, using the methodology recommended by the manufacturer and there was 95.38% coincidence in the identification at species level. MALDI TOF MS showed to be a fast, simple and reliable tool for yeast identification. Copyright © 2014 Asociación Argentina de Microbiología. Publicado por Elsevier España, S.L.U. All rights reserved.

Yersinia enterocolitica in slaughter pig tonsils: enumeration and detection by enrichment versus direct plating culture.

PubMed

Van Damme, Inge; Habib, Ihab; De Zutter, Lieven

2010-02-01

Tonsil samples from 139 slaughter pigs were examined for the presence of pathogenic Yersinia enterocolitica by enrichment procedures based on the standard method ISO 10273:2003. In addition, samples were tested by direct plating method to evaluate its efficiency compared to the enrichment culture methods and to quantify the level of contamination in porcine tonsils. In total, 52 samples (37.4%) were positive for pathogenic Y. enterocolitica, all belonging to bioserotype 4/O:3. Fifty out of the 52 positive samples (96.2%) were detected by direct plating. Enumeration showed an average concentration of 4.5 log(10) CFU g(-1) and 4.4 log(10) CFU g(-1) tonsil on Salmonella-Shigella-desoxycholate-calcium chloride (SSDC) and cefsulodin-irgasan-novobiocin (CIN) agar plates, respectively. The enrichment procedures recommended by the ISO 10273:2003 method were not optimal for the isolation of pathogenic Y. enterocolitica from pig tonsils: two days enrichment in irgasan-ticarcillin-potassium chlorate (ITC) broth resulted in an isolation rate of 84.6%, while 5 days enrichment in peptone-sorbitol-bile (PSB) broth recovered only 59.6% of positive samples. Reducing the enrichment time in PSB from 5 to 2 days resulted in a significantly higher recovery rate (94.2%) and might serve as an appropriate enrichment protocol for the isolation of pathogenic Y. enterocolitica from pig tonsils. Compared to enrichment culture methods, results based on direct plating can be obtained in a shorter time course and provide quantitative data that might be needed for further risk assessment studies.

Antimicrobial activity of yeasts against some pathogenic bacteria

PubMed Central

Younis, Gamal; Awad, Amal; Dawod, Rehab E.; Yousef, Nehal E.

2017-01-01

Aim: This study was designed to isolate and identify yeast species from milk and meat products, and to test their antimicrobial activity against some bacterial species. Materials and Methods: A total of 160 milk and meat products samples were collected from random sellers and super markets in New Damietta city, Damietta, Egypt. Samples were subjected to yeast isolation procedures and tested for its antimicrobial activity against Staphylococcus aureus, Pseudomonas aeruginosa, and Escherichia coli. In addition, all yeast species isolates were subjected to polymerase chain reaction (PCR) for detection of khs (kievitone hydratase) and pelA (pectate degrading enzyme)genes. Results: The recovery rate of yeasts from sausage was 20% (2/10) followed by kareish cheese, processed cheese, and butter 10% (1/10) each as well as raw milk 9% (9/100), and fruit yoghurt 30% (6/20). Different yeast species were recovered, namely, Candida kefyr (5 isolates), Saccharomyces cerevisiae (4 isolates), Candida intermedia (3 isolates), Candida tropicalis (2 isolates), Candida lusitaniae (2 isolates), and Candida krusei (1 isolate). khs gene was detected in all S. cerevisiae isolates, however, pelA gene was not detected in all identified yeast species. Antimicrobial activity of recovered yeasts against the selected bacterial species showed high activity with C. intermedia against S. aureus and E. coli, C. kefyr against E. coli, and C. lusitaniae against S. aureus. Moderate activities were obtained with C. tropicalis, C. lusitaniae, and S. cerevisiae against E. coli; meanwhile, all the tested yeasts revealed a very low antimicrobial activity against P. aeruginosa. Conclusion: The obtained results confirmed that some kinds of yeasts have the ability to produce antimicrobial compounds that could inhibit some pathogenic and spoilage bacteria and these antimicrobial activity of yeasts enables them to be one of the novel agents in controlling spoilage of food. PMID:28919693

Aseptic laboratory techniques: plating methods.

PubMed

Sanders, Erin R

2012-05-11

Microorganisms are present on all inanimate surfaces creating ubiquitous sources of possible contamination in the laboratory. Experimental success relies on the ability of a scientist to sterilize work surfaces and equipment as well as prevent contact of sterile instruments and solutions with non-sterile surfaces. Here we present the steps for several plating methods routinely used in the laboratory to isolate, propagate, or enumerate microorganisms such as bacteria and phage. All five methods incorporate aseptic technique, or procedures that maintain the sterility of experimental materials. Procedures described include (1) streak-plating bacterial cultures to isolate single colonies, (2) pour-plating and (3) spread-plating to enumerate viable bacterial colonies, (4) soft agar overlays to isolate phage and enumerate plaques, and (5) replica-plating to transfer cells from one plate to another in an identical spatial pattern. These procedures can be performed at the laboratory bench, provided they involve non-pathogenic strains of microorganisms (Biosafety Level 1, BSL-1). If working with BSL-2 organisms, then these manipulations must take place in a biosafety cabinet. Consult the most current edition of the Biosafety in Microbiological and Biomedical Laboratories (BMBL) as well as Material Safety Data Sheets (MSDS) for Infectious Substances to determine the biohazard classification as well as the safety precautions and containment facilities required for the microorganism in question. Bacterial strains and phage stocks can be obtained from research investigators, companies, and collections maintained by particular organizations such as the American Type Culture Collection (ATCC). It is recommended that non-pathogenic strains be used when learning the various plating methods. By following the procedures described in this protocol, students should be able to: Perform plating procedures without contaminating media. Isolate single bacterial colonies by the streak-plating method. Use pour-plating and spread-plating methods to determine the concentration of bacteria. Perform soft agar overlays when working with phage. Transfer bacterial cells from one plate to another using the replica-plating procedure. Given an experimental task, select the appropriate plating method.

Intraoperative culture positive allograft bone and subsequent postoperative infections: a retrospective review.

PubMed

Sims, Laura; Kulyk, Paul; Woo, Allan

2017-04-01

Obtaining intraoperative cultures of allograft bone just before use in orthopedic procedures is standard practice in many centres; however, the association between positive cultures and subsequent surgical infections is unknown. Our study had 3 goals: to determine the prevalence of positive intraoperative allograft culture and subsequent infection; to determine if, in cases of subsequent infection, organisms isolated at reoperation were the same as those cultured from the allograft at the time of the index procedure; and to assess the costs associated with performing intraoperative allograft cultures. In this retrospective case series, we obtained data on patients receiving allograft bone between 2009 and 2012. Patients receiving allograft with positive cultures were reviewed to identify cases of significant infection. Organisms isolated at reoperation were compared with the allograft culture taken at the time of implantation, and we performed a cost assessment. Of the 996 allograft bone grafts used, 43 (4.3%) had positive intraoperative cultures and significant postoperative infections developed in 2, requiring reoperation. Antibiotics based on culture results were prescribed in 24% of cases. Organisms cultured at the time of reoperation differed from those isolated initially. The cost of performing 996 allograft cultures was $169 320. This series suggests that rates of positive intraoperative bone allograft culture are low, and subsequent infection is rare. In cases of postoperative infection, primary allograft culture and secondary tissue cultures isolated different organisms. Costs associated with performing cultures are high. Eliminating initial culture testing could save $42 500 per year in our health region.

Occurrence of bla genes encoding carbapenem-resistant Pseudomonas aeruginosa and Acinetobacter baumannii from Intensive Care Unit in a tertiary care hospital

PubMed Central

Subramaniyan, Jayanthi Siva; Sundaram, Jeya Meenakshi

2018-01-01

CONTEXT: ICU shows increasing incidence of infection associated with the use of invasive procedures for the diagnostic purpose as well as the indiscriminate use of antibiotics. Pseudomonas aeruginosa and Acinetobacter species are “very successful” pathogen and the emergence of the Metallo-β-Lactamases (MBL) is becoming a therapeutic challenge. AIMS: To isolate the Nonfermenting Gram negative bacilli from the ICU samples. To identify the metallo betalactamase producers and to detect the bla gene presence among the Pseudomonas aeruginosa and Acinetobacter baumannii. SETTINGS AND DESIGN: The Nonfermenting Gram negative bacilli isolates from the ICU samples were taken over for 5 years (2009-2014) in a tertiary care hospital. METHODS AND MATERIALS: The isolates of Pseudomonas species and Acinetobacter species were confirmed by API analyser and processed according to standard procedures. Detection of the MBL producers were done by E strip method and subjected for bla gene detection by PCR method. RESULTS: In our study a total of 195 isolates of NFGNB were obtained from various ICU. Of these MBL producers, 26 % were Pseudomonas aeruginosa and 25 % were Acinetobacter baumannii. The subtypes of blaVIM MBL producing P.aeruginosa were 26%. The predominant gene coding for MBL activity in A.baumannii were found to be blaOXA gene 11.9%. The gene accession numbers were KF975367, KF975372. CONCLUSIONS: We have to control the development and dissemination of these superbugs among the ICU's. PMID:29692589

On the isolation of elemental carbon for micro-molar 14C accelerator mass spectrometry; evaluation of alternative isolation procedures, and accuracy assurance using a hybrid isotopic particulate carbon reference material

NASA Astrophysics Data System (ADS)

Currie, L. A.; Kessler, J. D.

2005-05-01

The primary objective of the research reported here has been the development of an hybrid reference material (RM) to serve as a test of accuracy for elemental carbon (EC) isotopic (14C) speciation measurements. Such measurements are critically important for the quantitative apportionment of fossil and biomass sources of ''soot'' (EC), the tracer of fire that has profound effects on health, atmospheric visibility, and climate. Previous studies of 14C-EC measurement quality, carried out with NIST SRM 1649a (Urban Dust), showed a range of results, but since the ''truth'' was not known for this natural matrix RM, one had to rely on isotopic-chemical consistency evidence (14C in PAH, EC) of measurement validity (Currie et al., 2002). Components of the new Hybrid RM (DiesApple), however, have known 14C and EC composition, and they are nearly orthogonal (isotopically and chemically). NIST SRM 2975 (Forklift Diesel Soot) has little or no 14C, and its major compositional component is EC. SRM 1515 (Apple Leaves) has the 14C content of biomass-C, and it has little or no EC. Thus, the hybrid RM can serve as an absolute isotopic test for the absence of EC-mimicking pyrolysis-C from SRM 1515 in the EC isolate of the hybrid RM, together with testing for conservation of its dominant soot fraction through the isolation procedure.

Analysis of gene expression in small numbers of purified hemopoietic progenitor cells by RT-PCR.

PubMed

Ziegler, B L; Lamping, C P; Thoma, S J; Fliedner, T M

1995-05-01

Primitive hemopoietic stem cells represent the most probable targets for genetic alterations due to exposure to ionizing irradiation or chemical carcinogens. We have applied a two-step protocol for the purification of CD34+HLA-DR-/low hemopoietic progenitor cells from cord blood (CB). CD34+ cells were isolated by monoclonal antibody (mAb) against CD34 (My10) and immunomagnetic beads. Beads were cleaved off the CD34+ cells by enzymatic treatment with chymopapain. Due to chymopapain-resistance of epitopes recognized by the used mAbs purity control of CD34+ cells and separation into CD34+HLA-DR-/low and CD34+HLA-DR+ subsets could be performed by using flow cytometry. Two miniaturized procedures were applied to isolate poly(A)+ mRNA for the reverse transcription polymerase chain reaction (RT-PCR) from small numbers of CD34+HLA-DR-/low cells. In five experiments, the mean purity of immunomagnetically isolated CD34+ cells was 93.8% +/- 3.9. Flow cytometry sorting of CD34+ cells resulted in pure CD34+HLA-DR-/low populations (purity > 98.8%; range 98.8% to 99.9%; viability > 96%) with an average yield of 2600 +/- 800 cells/5 x 10(7) low density CB cells. By RT-PCR using both poly(A)+ mRNA isolation procedures, sequences corresponding to CD34 and beta 2-microglobulin were amplified from as few as 20 cells. Furthermore, a sequence-independent RT-PCR (SIP-RT-PCR) was applied to amplify the cDNA derived from five erythroblasts isolated from a burst-forming unit-erythroid (BFU-E). Upon hybridization, full-length c-fos message was detected in the SIP-RT-PCR amplified material. Our data demonstrate that gene expression can be detected at the transcriptional level in small numbers of hemopoietic progenitor cells. In addition, the SIP-RT-PCR may allow the amplification of unique mRNA species when subtractive hybridization procedures are performed. The presented data should be useful to analyze gene expression in rare subsets of radiation-exposed immature hemopoietic stem/progenitor cells.

Surgeon-Directed Cost Variation in Isolated Rotator Cuff Repair.

PubMed

Terhune, E Bailey; Cannamela, Peter C; Johnson, Jared S; Saad, Charles D; Barnes, John; Silbernagel, Janette; Faciszewski, Thomas; Shea, Kevin G

2016-12-01

As value becomes a larger component of heath care decision making, cost data can be evaluated for regional and physician variation. Value is determined by outcome divided by cost, and reducing cost increases value for patients. "Third-party spend" items are individual selections by surgeons used to perform procedures. Cost data for third-party spend items provide surgeons and hospitals with important information regarding care value, potential cost-saving opportunities, and the total cost of ownership of specific clinical decisions. To perform a cost review of isolated rotator cuff repair within a regional 7-hospital system and to document procedure cost variation among operating surgeons. Economic and decision analysis; Level of evidence, 4. Current Procedural Terminology (CPT) codes were used to retrospectively identify subjects who received an isolated rotator cuff repair within a 7-hospital system. Cost data were collected for clinically sensitive third-party spend items and divided into 4 cost groups: (1) suture anchors, (2) suture-passing devices and needles, (3) sutures used for cuff repair, and (4) disposable tools or instruments. A total of 62 isolated rotator cuff repairs were performed by 17 surgeons over a 13-month period. The total cost per case for clinically sensitive third-party spend items (in 2015 US dollars) ranged from $293 to $3752 (mean, $1826). Four surgeons had a mean procedure cost that was higher than the data set mean procedure cost. The cost of an individual suture anchor ranged from $75 to $1775 (mean, $403). One disposable suture passer was used, which cost $140. The cost of passing needles ranged from $140 to $995 (mean, $468). The cost per repair suture (used to repair cuff tears) varied from $18 to $298 (mean, $61). The mean suture (used to close wounds) cost per case was $81 (range, $0-$454). A total of 316 tools or disposable instruments were used, costing $1 to $1573 per case (mean, $624). This study demonstrates significant cost variation with respect to cost per case and cost of individual items used during isolated rotator cuff repair. Suture anchors represent the most expensive and variable surgeon-directed cost. The wide cost variation seen in all cost categories illustrates both the effect of surgeon choice in procedure cost and the opportunity for significant cost savings in cases of isolated rotator cuff repair. Engaging surgeons in discussion on cost can positively influence the value of care provided to patients if costs can be reduced without affecting the quality of patient outcomes.

A prospective study on the risk of glove fingertip contamination during draping in joint replacement surgery

PubMed Central

Deierl, K; Pandit, A; Trakru, S

2014-01-01

Introduction The aim of this prospective study was to investigate the risk of contamination of surgical gloves during preparation and draping in joint replacement surgery. Methods During 46 hip and knee replacement procedures, the gloves of orthopaedic consultants (n=5) and registrars (n=3) were assessed for contamination immediately after draping by impression of gloved fingers on blood agar. Contamination was evaluated by the surgeon’s grade, the type of procedure, the role of the assistant and the dominance of the hand. Results A total of 125 pairs of top gloves were examined (79 pairs from registrars and 46 pairs from consultants). Bacterial contamination was isolated on 19 pairs (15.2%) (16 pairs from registrars and 3 pairs from consultants, p=0.04). Coagulase negative staphylococci were the main isolates and contamination was considered low in all cases (1–5 colonies). Contamination was seen more on the dominant hand (16 gloves from dominant hands and 6 from non-dominant hands, p=0.04), on the index finger and thumb. More contaminated gloves were seen in hip arthroplasty procedures (16 pairs from total hip replacements vs 3 pairs from total knee replacements, p=0.02). Conclusions Contamination of glove fingertips during draping in joint replacement procedures is more likely to occur among junior surgeons, in hip rather than knee arthroplasty procedures and on the dominant hand. It is therefore essential that surgeons of different grades replace gloves used in draping to avoid exposing patients to the risk of infection. PMID:25198974

Development and Evaluation of a Multiplex Real-Time Polymerase Chain Reaction Procedure to Clinically Type Prevalent Salmonella enterica Serovars

PubMed Central

Muñoz, Nélida; Diaz-Osorio, Miguel; Moreno, Jaime; Sánchez-Jiménez, Miryan; Cardona-Castro, Nora

2010-01-01

A multiplex real-time polymerase chain reaction procedure was developed to identify the most prevalent clinical isolates of Salmonella enterica subsp. enterica. Genes from the rfb, fliC, fljB, and viaB groups that encode the O, H, and Vi antigens were used to design 15 primer pairs and TaqMan probes specific for the genes rfbJ, wzx, fliC, fljB, wcdB, the sdf-l sequence, and invA, which was used as an internal amplification control. The primers and probes were variously combined into six sets. The first round of reactions used two of these sets to detect Salmonella O:4, O:9, O:7, O:8, and O:3,10 serogroups. Once the serogroups were identified, the results of a second round of reactions that used primers and probes for the flagellar antigen l genes, 1,2; e,h; g,m; d; e,n,x; and z10, and the Vi gene were used to identify individual serovars. The procedure was standardized using 18 Salmonella reference strains and other enterobacteria. The procedure's reliability and sensitivity was evaluated using 267 randomly chosen serotyped Salmonella clinical isolates. The procedure had a sensitivity of 95.5% and was 100% specific. Thus, our technique is a quick, sensitive, reliable, and specific means of identifying S. enterica serovars and can be used in conjunction with traditional serotyping. Other primer and probe combinations could be used to increase the number of identifiable serovars. PMID:20110454

Indication for Co-evolution of Lactobacillus johnsonii with its hosts

PubMed Central

2012-01-01

Background The intestinal microbiota, composed of complex bacterial populations, is host-specific and affected by environmental factors as well as host genetics. One important bacterial group is the lactic acid bacteria (LAB), which include many health-promoting strains. Here, we studied the genetic variation within a potentially probiotic LAB species, Lactobacillus johnsonii, isolated from various hosts. Results A wide survey of 104 fecal samples was carried out for the isolation of L. johnsonii. As part of the isolation procedure, terminal restriction fragment length polymorphism (tRFLP) was performed to identify L. johnsonii within a selected narrow spectrum of fecal LAB. The tRFLP results showed host specificity of two bacterial species, the Enterococcus faecium species cluster and Lactobacillus intestinalis, to different host taxonomic groups while the appearance of L. johnsonii and E. faecalis was not correlated with any taxonomic group. The survey ultimately resulted in the isolation of L. johnsonii from few host species. The genetic variation among the 47 L. johnsonii strains isolated from the various hosts was analyzed based on variation at simple sequence repeats (SSR) loci and multi-locus sequence typing (MLST) of conserved hypothetical genes. The genetic relationships among the strains inferred by each of the methods were similar, revealing three different clusters of L. johnsonii strains, each cluster consisting of strains from a different host, i.e. chickens, humans or mice. Conclusions Our typing results support phylogenetic separation of L. johnsonii strains isolated from different animal hosts, suggesting specificity of L. johnsonii strains to their hosts. Taken together with the tRFLP results, that indicated the association of specific LAB species with the host taxonomy, our study supports co-evolution of the host and its intestinal lactic acid bacteria. PMID:22827843

Grass material as process standard for compound-specific radiocarbon analysis

NASA Astrophysics Data System (ADS)

Cisneros-Dozal, Malu; Xu, Xiaomei; Bryant, Charlotte; Pearson, Emma; Dungait, Jennifer

2015-04-01

Compound-specific radiocarbon analysis (CSRA) is a powerful tool to study the carbon cycle and/or as a dating technique in paleoclimate reconstructions. The radiocarbon value of individual compounds can provide insight into turnover times, organic matter sources and in specific cases can be used to establish chronologies when traditional dating materials (e.g. macrofossils, pollen, charcoal) are not available. The isolation of compounds (or group of compounds) from parent material (e.g. soil, plant) for radiocarbon analysis can, however, introduce carbon contamination through chemical separation steps and preparative capillary gas chromatography (PCGC). In addition, the compounds of interest are often in low abundance which amplifies the contamination effect. The extraneous carbon can be of modern 14C age and/or 14C -free and its amount and 14C value must be determined for a given system/isolation procedure in order to report accurate 14C values. This can be achieved by using adequate standard materials but, by contrast with traditional radiocarbon dating, there are not established reference standards for CSRA work, in part because the type of standard material depends on the compounds of interest and the isolation procedure. Here we evaluate the use of n-alkanes extracted from single-year growth grass as modern process standard material for CSRA using PCGC isolation. The grass material has a known 14C value of 1.224 ± 0.006 fraction modern (FM) and the individual n-alkanes are expected to have a similar 14C value. In order to correct for the addition of extraneous carbon during PCGC isolation of the n-alkanes, we used commercially available compounds of modern 14C content and 14C -free (adipic acid, FM= 0.0015 ± 0.0001 and docosane, FM=1.059 ± 0.003) to evaluate our PCGC procedure. The corrected 14C values of the isolated n-alkanes extracted from the modern grass are within one sigma of the grass bulk 14C value for n-C29 and within two sigma for n-C23-C27, C31 and C33. Our results show that single-year growth grass can be a process standard suitable for quality control of extraction of n-alkanes (and potentially other compounds) from soil or plant material for CSRA.

Diagnostic procedures for Trend Monitoring System (TMS) communications. [coaxial cable bus system

NASA Technical Reports Server (NTRS)

Brown, J. S.; Lenker, M. D.

1979-01-01

A prototype coaxial cable bus communications sytem was developed to support the trend monitoring system (TMS). Troubleshooting procedures are described at the system level. The procedures can be used by repair personnel to isolate a fault in the TMS and to restore the system to operation by swapping out failed components.

Identification of multiple mRNA and DNA sequences from small tissue samples isolated by laser-assisted microdissection.

PubMed

Bernsen, M R; Dijkman, H B; de Vries, E; Figdor, C G; Ruiter, D J; Adema, G J; van Muijen, G N

1998-10-01

Molecular analysis of small tissue samples has become increasingly important in biomedical studies. Using a laser dissection microscope and modified nucleic acid isolation protocols, we demonstrate that multiple mRNA as well as DNA sequences can be identified from a single-cell sample. In addition, we show that the specificity of procurement of tissue samples is not compromised by smear contamination resulting from scraping of the microtome knife during sectioning of lesions. The procedures described herein thus allow for efficient RT-PCR or PCR analysis of multiple nucleic acid sequences from small tissue samples obtained by laser-assisted microdissection.

Molecular analysis of a mutant defective in photosynthetic oxygen evolution and isolation of a complementing clone by a novel screening procedure.

PubMed Central

Dzelzkalns, V A; Bogorad, L

1988-01-01

Photosynthesis-defective mutants of the transformable cyanobacterium Synechocystis 6803 have been isolated following nitrosoguanidine mutagenesis. The photosystem II- phenotype of one of these mutants is shown by DNA sequencing to be attributable to a short deletion in psbC, the gene encoding the 44-kd, chlorophyll-binding protein of photosystem II. Although not a component of the reaction center of photosystem II, the 44-kd protein is none the less shown to be essential in vivo for photosystem II activity. The deletion in psbC also results in greatly diminished levels of D-2 (a component of the reaction center of photosystem II) indicating that the loss of the product of the psbC gene affects the assembly or stability of the photosystem II reaction center. The isolation of a clone capable of restoring both photosystem II activity and photoautotrophy to the mutant cells was aided by the observation that restriction fragments or cloned Synechocystis 6803 DNA applied in liquid or in melted agarose directly onto a lawn of Synechocystis 6803 will lead to the transformation of the cells. This in situ 'dot' transformation procedure provides a convenient method for the rapid identification of fractions or clones containing complementing Synechocystis 6803 DNA. Images PMID:3130247

Circulating tumor cell detection: A direct comparison between negative and unbiased enrichment in lung cancer.

PubMed

Xu, Yan; Liu, Biao; Ding, Fengan; Zhou, Xiaodie; Tu, Pin; Yu, Bo; He, Yan; Huang, Peilin

2017-06-01

Circulating tumor cells (CTCs), isolated as a 'liquid biopsy', may provide important diagnostic and prognostic information. Therefore, rapid, reliable and unbiased detection of CTCs are required for routine clinical analyses. It was demonstrated that negative enrichment, an epithelial marker-independent technique for isolating CTCs, exhibits a better efficiency in the detection of CTCs compared with positive enrichment techniques that only use specific anti-epithelial cell adhesion molecules. However, negative enrichment techniques incur significant cell loss during the isolation procedure, and as it is a method that uses only one type of antibody, it is inherently biased. The detection procedure and identification of cell types also relies on skilled and experienced technicians. In the present study, the detection sensitivity of using negative enrichment and a previously described unbiased detection method was compared. The results revealed that unbiased detection methods may efficiently detect >90% of cancer cells in blood samples containing CTCs. By contrast, only 40-60% of CTCs were detected by negative enrichment. Additionally, CTCs were identified in >65% of patients with stage I/II lung cancer. This simple yet efficient approach may achieve a high level of sensitivity. It demonstrates a potential for the large-scale clinical implementation of CTC-based diagnostic and prognostic strategies.

Routine use of PCR-restriction fragment length polymorphism analysis for identification of mycobacteria growing in liquid media.

PubMed Central

Taylor, T B; Patterson, C; Hale, Y; Safranek, W W

1997-01-01

A PCR-restriction fragment length polymorphism (PCR-RFLP) procedure capable of rapidly identifying 28 species of clinically encountered mycobacteria was evaluated for use in the routine identification of acid-fast isolates growing in BACTEC 12B and 13A liquid media. PCR-RFLP identified 100 of 103 acid-fast isolates recovered from 610 patient specimens submitted for culture during the study. The three isolates unidentifiable by PCR-RFLP produced restriction patterns not included in the PCR-RFLP algorithm and could therefore not be assigned to a species. These isolates were characterized by their morphologic and biochemical characteristics. Two of the isolates were identified as M. terrae complex and M. gordonae. The third isolate could not be definitively identified and could only be characterized as a Mycobacterium sp. most closely resembling M. chelonae. PCR-RFLP identifications agreed with the conventional identifications for 96 of the 100 isolates identified by PCR-RFLP. Subsequent identification of the four discordant isolates by gas chromatography analysis supported the PCR-RFLP identification of each isolate. Amplification products were also obtained from isolates of Streptococcus albus and Rhodococcus equi recovered from patient specimens; however, the restriction patterns of these nonmycobacterial species did not resemble the patterns of any mycobacterial species included in the PCR-RFLP algorithm. PCR-RFLP seems to be a reliable procedure for the routine identification of mycobacteria and has the potential for providing identifications of mycobacterial isolates which are more accurate than conventional identification techniques based on morphologic and biochemical characteristics. PMID:8968884

Combining phylogenetic and demographic inferences to assess the origin of the genetic diversity in an isolated wolf population

PubMed Central

Fabbri, Elena; Ahmed, Atidje; Bolfíková, Barbora Černá; Czarnomska, Sylwia D.; Galov, Ana; Godinho, Raquel; Hindrikson, Maris; Hulva, Pavel; Jędrzejewska, Bogumiła; Jelenčič, Maja; Kutal, Miroslav; Saarma, Urmas; Skrbinšek, Tomaž; Randi, Ettore

2017-01-01

The survival of isolated small populations is threatened by both demographic and genetic factors. Large carnivores declined for centuries in most of Europe due to habitat changes, overhunting of their natural prey and direct persecution. However, the current rewilding trends are driving many carnivore populations to expand again, possibly reverting the erosion of their genetic diversity. In this study we reassessed the extent and origin of the genetic variation of the Italian wolf population, which is expanding after centuries of decline and isolation. We genotyped wolves from Italy and other nine populations at four mtDNA regions (control-region, ATP6, COIII and ND4) and 39 autosomal microsatellites. Results of phylogenetic analyses and assignment procedures confirmed in the Italian wolves a second private mtDNA haplotype, which belongs to a haplogroup distributed mostly in southern Europe. Coalescent analyses showed that the unique mtDNA haplotypes in the Italian wolves likely originated during the late Pleistocene. ABC simulations concordantly showed that the extant wolf populations in Italy and in south-western Europe started to be isolated and declined right after the last glacial maximum. Thus, the standing genetic variation in the Italian wolves principally results from the historical isolation south of the Alps. PMID:28489863

Epidemiological analysis of structural alterations of the nasal cavity associated with obstructive sleep apnea syndrome (OSA).

PubMed

Mekhitarian Neto, Levon; Fava, Antonio Sérgio; Lopes, Hugo Canhete; Stamm, Aldo

2005-01-01

The objective of this paper is to demonstrate that structural alterations of the nasal cavity, e.g. septal deviation and conchal hypertrophy have high incidence in patients with sleep apnea and hypopnea syndrome and must be addressed with associated specific procedures of the syndrome. Clinical retrospective. A retrospective study of 200 patients was performed, with 196 male and 4 female, attended at the otorhinolaryngology ambulatory of Hospital Prof. Edmundo Vasconcelos and Unidade Paulista de Otorrinolaringologia, all of them subjected to polysomnography, otorhinolaryngological physical exam, endoscopy exam, and surgical treatment with nasal and pharyngeal procedures. All of them were subjected to pharyngeal procedure: uvulopalatopharyngoplasty or uvulopalatoplasty and nose procedure: 176 septoplasty with partial turbinectomy (88%) and 24 isolated turbinectomy, with satisfactory results. We can see that structural alterations of the nasal cavity have high incidence in patients with OSA.

Training a Network of Electronic Neurons for Control of a Mobile Robot

NASA Astrophysics Data System (ADS)

Vromen, T. G. M.; Steur, E.; Nijmeijer, H.

An adaptive training procedure is developed for a network of electronic neurons, which controls a mobile robot driving around in an unknown environment while avoiding obstacles. The neuronal network controls the angular velocity of the wheels of the robot based on the sensor readings. The nodes in the neuronal network controller are clusters of neurons rather than single neurons. The adaptive training procedure ensures that the input-output behavior of the clusters is identical, even though the constituting neurons are nonidentical and have, in isolation, nonidentical responses to the same input. In particular, we let the neurons interact via a diffusive coupling, and the proposed training procedure modifies the diffusion interaction weights such that the neurons behave synchronously with a predefined response. The working principle of the training procedure is experimentally validated and results of an experiment with a mobile robot that is completely autonomously driving in an unknown environment with obstacles are presented.

Pesticide-sampling equipment, sample-collection and processing procedures, and water-quality data at Chicod Creek, North Carolina, 1992

USGS Publications Warehouse

Manning, T.K.; Smith, K.E.; Wood, C.D.; Williams, J.B.

1994-01-01

Water-quality samples were collected from Chicod Creek in the Coastal Plain Province of North Carolina during the summer of 1992 as part of the U.S. Geological Survey's National Water-Quality Assessment Program. Chicod Creek is in the Albemarle-Pamlico drainage area, one of four study units designated to test equipment and procedures for collecting and processing samples for the solid-phase extraction of selected pesticides, The equipment and procedures were used to isolate 47 pesticides, including organonitrogen, carbamate, organochlorine, organophosphate, and other compounds, targeted to be analyzed by gas chromatography/mass spectrometry. Sample-collection and processing equipment equipment cleaning and set-up procedures, methods pertaining to collecting, splitting, and solid-phase extraction of samples, and water-quality data resulting from the field test are presented in this report Most problems encountered during this intensive sampling exercise were operational difficulties relating to equipment used to process samples.

Evaluation of molecular markers for Phytophthora ramorum detection and identification using a standardized library of isolates

Treesearch

F.N. Martin; M. Coffey; R. Hamelin; P. Tooley; M. Garbelotto; K. Hughes; T. Kubisiak

2008-01-01

A number of molecular diagnostic procedures for detection of Phytophthora ramorum have been reported in the literature. In an effort to evaluate the specificity of 10 of these techniques a standardized DNA library for 317 isolates was assembled that included 60 described species as well as 22 taxonomically unclassified isolates. These were sent blind...

Antibiotic resistance profiles of coagulase-positive and coagulase-negative staphylococci from pit latrine fecal sludge in a peri-urban South African community.

PubMed

Beukes, Lorika S; Schmidt, Stefan

2018-04-16

The aim of this study was to assess pit latrine samples from a peri-urban community in KwaZulu-Natal (South Africa) for the presence of multidrug-resistant (MDR) Staphylococcus spp. Standard procedures were used to isolate Staphylococcus spp. from pit latrine fecal sludge samples, with confirmation at genus level by polymerase chain reaction (PCR). Sixty-eight randomly selected pit latrine Staphylococcus spp. isolates were further characterized by using established disk diffusion procedures. An average Staphylococcus spp. count of 2.1 × 10 5  CFU per g fecal material was established using two randomly selected pit latrine samples. Of the 68-selected Staphylococcus spp. pit latrine isolates, 49% were identified as coagulase positive, 51% as coagulase negative and 65% (12 coagulase positive, 32 coagulase negative isolates) were categorized as MDR. The majority (66/68) of Staphylococcus spp. isolates displayed resistance to fusidic acid while only 5/68 isolates displayed resistance to chloramphenicol. The pit latrine samples analyzed in this study are a source of MDR Staphylococcus spp., highlighting the need for proper hygiene and sanitation regimes in rural communities using these facilities.

Influence of Prior Coronary Stenting on the Immediate and Mid-term Outcome of Isolated Coronary Artery Bypass Surgery.

PubMed

Pliam, Michael B; Zapolanski, Alex; Anastassiou, Peter; Ryan, Colman J; Manila, Louis L; Shaw, Richard E; Pira, Bob-Kenneth

2007-09-01

: There has been little emphasis on the possible consequences of prior stent placement on the outcome of coronary bypass surgery (CABG). We compared the results of isolated CABG patients who had prior stents with those who had not with respect to preoperative status, operative procedure, and postoperative immediate and long-term outcome. : Records of 1471 patients undergoing isolated CABG at our institution between January 1, 2000, and March 31, 2005, were reviewed. Patients were divided into three groups. Group I had no stents (n = 1317). Group II had one to three stents (n = 137). Group III had more than three stents (n = 17). Groups were compared with respect to preoperative risk factors, operative procedures, and postoperative results. Long-term survival data were obtained on 97.6% of patients with a mean follow-up, 4.1 ± 2.3 years. : Stented patients were younger (66.1 ± 10.8 vs. 69.1 ± 10.8 years, P = 0.006), had more unstable angina (68.2% vs. 58.9%, P = 0.02), hypercholesterolemia (83.8% vs. 61.2%, P = 0.00), chronic obstructive pulmonary disease (13.6% vs. 8.4%, P = 0.03), peripheral vascular disease (15.2% vs. 8.4%, P = 0.00), and previous CABG (10.1% vs. 4.2%, P = 0.00), fewer low ejection fractions (1.3% vs. 5.2%, P = 0.02), left main disease (25.3% vs. 32.6%, P = 0.04), diabetes (31.2% vs. 40.8%, P = 0.01), or diffuse disease (19.5 ± 10.5 vs. 22.5 ± 10.9, P = 0.00), had more off pump procedures (53.2% vs. 45.3%, P = 0.03), fewer internal thoracic artery grafts (80.5% vs. 86.6%, P = 0.03), fewer grafts placed (>3: 52.6% vs. 61.8%, P = 0.02), more complications (76.5% vs. 42.6%, P = 0.005), atrial fibrillation (47.1% vs. 19.7%, P = 0.011), longer hospital stays (12.2 vs. 8.3 days, P = 0.019). Percentage survival for groups I, II, and III at 60 months was 82.1%, 84.7%, and 72.6%, respectively. : Stents placed before surgery in isolated CABG patients may be associated with higher preoperative risk, altered operative procedures, more postoperative complications, longer hospitalizations, and more readmissions. Overall, stented patients experienced more preoperative hospitalizations, catheterizations, and percutaneous coronary interventions (PCIs) than nonstented patients. Survival for those with more than three stents may be diminished.

Resistance to Biocides in Listeria monocytogenes Collected in Meat-Processing Environments

PubMed Central

Conficoni, Daniele; Losasso, Carmen; Cortini, Enzo; Di Cesare, Andrea; Cibin, Veronica; Giaccone, Valerio; Corno, Gianluca; Ricci, Antonia

2016-01-01

The emergence of microorganisms exerting resistance to biocides is a challenge to meat-processing environments. Bacteria can be intrinsically resistant to biocides but resistance can also be acquired by adaptation to their sub-lethal concentrations. Moreover, the presence of biocide resistance determinants, which is closely linked to antibiotic resistance determinants, could lead to co-selection during disinfection practices along the food chain, and select cross-resistant foodborne pathogens. The purpose of this work was to test the resistance of wild strains of Listeria monocytogenes, isolated from pork meat processing plants, toward benzalkonium chloride (BC), used as proxy of quaternary ammonium compounds. Furthermore, the expression of two non-specific efflux pumps genes (lde and mdrL) under biocide exposure was evaluated. L. monocytogenes were isolated from five processing plants located in the Veneto region (northeast of Italy) before and after cleaning and disinfection (C&D) procedures. A total of 45 strains were collected: 36 strains before and nine after the C&D procedures. Collected strains were typed according to MLST and ERIC profiles. Strains sampled in the same site, isolated before, and after the C&D procedures and displaying the same MLST and ERIC profiles were tested for their sensitivity to different concentrations of BC, in a time course assay. The expression of non-specific efflux pumps was evaluated at each time point by qPCR using tufA gene as housekeeping. A differential expression of the two investigated genes was observed: lde was found to be more expressed by the strains isolated before C&D procedures while its expression was dose-dependent in the case of the post C&D procedures strain. On the contrary, the expression of mdrL was inhibited under low biocidal stress (10 ppm BC) and enhanced in the presence of high stress (100 ppm BC). These findings suggests a possible role for C&D procedures to select L. monocytogenes persisters, pointing out the importance of dealing with the identification of risk factors in food plants sanification procedures that might select more tolerant strains. PMID:27807430

Real-Time Whole-Genome Sequencing for Routine Typing, Surveillance, and Outbreak Detection of Verotoxigenic Escherichia coli

PubMed Central

Scheutz, Flemming; Lund, Ole; Hasman, Henrik; Kaas, Rolf S.; Nielsen, Eva M.; Aarestrup, Frank M.

2014-01-01

Fast and accurate identification and typing of pathogens are essential for effective surveillance and outbreak detection. The current routine procedure is based on a variety of techniques, making the procedure laborious, time-consuming, and expensive. With whole-genome sequencing (WGS) becoming cheaper, it has huge potential in both diagnostics and routine surveillance. The aim of this study was to perform a real-time evaluation of WGS for routine typing and surveillance of verocytotoxin-producing Escherichia coli (VTEC). In Denmark, the Statens Serum Institut (SSI) routinely receives all suspected VTEC isolates. During a 7-week period in the fall of 2012, all incoming isolates were concurrently subjected to WGS using IonTorrent PGM. Real-time bioinformatics analysis was performed using web-tools (www.genomicepidemiology.org) for species determination, multilocus sequence type (MLST) typing, and determination of phylogenetic relationship, and a specific VirulenceFinder for detection of E. coli virulence genes was developed as part of this study. In total, 46 suspected VTEC isolates were characterized in parallel during the study. VirulenceFinder proved successful in detecting virulence genes included in routine typing, explicitly verocytotoxin 1 (vtx1), verocytotoxin 2 (vtx2), and intimin (eae), and also detected additional virulence genes. VirulenceFinder is also a robust method for assigning verocytotoxin (vtx) subtypes. A real-time clustering of isolates in agreement with the epidemiology was established from WGS, enabling discrimination between sporadic and outbreak isolates. Overall, WGS typing produced results faster and at a lower cost than the current routine. Therefore, WGS typing is a superior alternative to conventional typing strategies. This approach may also be applied to typing and surveillance of other pathogens. PMID:24574290

The Path of Carbon in Photosynthesis V. Paper Chromatography and Radioautography of the Products

DOE R&D Accomplishments Database

Benson, A. A.; Bassham, J. A.; Calvin, M.; Goodale, T. C.; Haas, V. A.; Stepka, W.

1949-06-13

Detailed procedure and results for the separation and identification of labeled carboxylic acids and phosphate esters, formed during photosynthesis in C{sup 14}O{sub 2}; the first observed product of CO{sub 2} assimilation during photosynthesis was isolated and shown to be phosphoglyceric acid; tracer use of P{sup 32} and C{sup 14}.

Isolation and Evaluation of Bacillus Strains for Industrial Production of 2,3-Butanediol.

PubMed

Song, Chan Woo; Rathnasingh, Chelladurai; Park, Jong Myoung; Lee, Julia; Song, Hyohak

2018-03-28

Biologically produced 2,3-butanediol (2,3-BDO) has diverse industrial applications. In this study, schematic isolation and screening procedures were designed to obtain generally regarded as safe (GRAS) and efficient 2,3-BDO producers. Over 4,000 candidate strains were isolated by pretreatment and enrichment, and the isolated Bacillus strains were further screened by morphological, biochemical, and genomic analyses. The screened strains were then used to test the utilization of the most common carbon (glucose, xylose, fructose, sucrose) and nitrogen (yeast extract, corn steep liquor) sources for the economical production of 2,3-BDO. Two-stage fed-batch fermentation was finally carried out to enhance 2,3-BDO production. In consequence, a newly isolated Bacillus licheniformis GSC3102 strain produced 92.0 g/l of total 2,3-BDO with an overall productivity and yield of 1.40 g/l/h and 0.423 g/g glucose, respectively, using a cheap and abundant nitrogen source. These results strongly suggest that B. licheniformis , which is found widely in nature, can be used as a host strain for the industrial fermentative production of 2,3-BDO.

General method for rapid purification of native chromatin fragments.

PubMed

Kuznetsov, Vyacheslav I; Haws, Spencer A; Fox, Catherine A; Denu, John M

2018-05-24

Biochemical, proteomic and epigenetic studies of chromatin rely on the efficient ability to isolate native nucleosomes in high yield and purity. However, isolation of native chromatin suitable for many downstream experiments remains a challenging task. This is especially true for the budding yeast Saccharomyces cerevisiae, which continues to serve as an important model organism for the study of chromatin structure and function. Here, we developed a time- and cost-efficient universal protocol for isolation of native chromatin fragments from yeast, insect, and mammalian cells. The resulting protocol preserves histone posttranslational modification in the native chromatin state, and is applicable for both parallel multi-sample spin-column purification and large scale isolation. This protocol is based on the efficient and stable purification of polynucleosomes, features a combination of optimized cell lysis and purification conditions, three options for chromatin fragmentation, and a novel ion-exchange chromatographic purification strategy.  The procedure will aid chromatin researchers interested in isolating native chromatin material for biochemical studies, and as a mild, acid- and detergent-free sample preparation method for mass-spectrometry analysis. Published under license by The American Society for Biochemistry and Molecular Biology, Inc.

[Microbiological surveillance in an intensive care unit of a large Roman hospital].

PubMed

Franchi, Cristiana; Venditti, Mario; Rocco, Monica; Spadetta, Gustavo; Vullo, Vincenzo; Raponi, Massimiliano; Orsi, Giovanni Battista

2002-06-01

Infection surveillance in ICU is fundamental to monitor endemic rates, to identify outbreaks on-time in order to activate control procedures implementing a correct empirical antibiotic treatment. The data collection surveillance software CIN-20002 was established in the Teaching Hospital Umberto I ICU to monitor the following site-specific infection rates: urinary tract infections (UTI), pneumonia (PNE), blood stream infections (BSI), surgical site infections (SSI). According to CDC definitions all patients developing infection 48 hours or more after ward admission were included. Furthermore risk factors (i.e. age, sex, SAPS II), invasive procedures (i.e. endotracheal intubation, vascular and urinary catheterisation), microbiological isolates and their antibiotic susceptibility were screened. Overall 279 patients (183 men and 96 women) were admitted; age 54,8 20,3 years (mean), SAPS II 44,4 17,1 (mean) and average ward stay 13,3 17,8 days. Results showed a total of 121 infection episodes (56 PNE, 51 BSI, 10 UTI e 4 SSI) in 80 patients (28,7%). Standardized infection rates associated to invasive procedures were: Urinary catheter-associated UTI rate (2.9/1000), Ventilator-associated PNE rate (20.4/1000), Vascular catheter-associated BSI rate (19.1/1000). Among the infected patients the most common microrganisms isolated were P. aeruginosa (31,8%), MRSA (14,8%), A. baumanni (12,5%) e S. maltophilia (8,5%). Considering site-specific infections: PNE (P. aeruginosa 38,3% and MRSA 18,1%), BSI (MR CNS 21,9% and P. aeruginosa 17,2%), UTI (P. aeruginosa 70,0%). The surveillance software CIN-2000 proved to be very accurate, usefull and easy to use. The results showed a high incidence of infections associated to invasive procedures and the presence of multiresistant bacteria

Outcome after procedures for retained blood syndrome in coronary surgery.

PubMed

Tauriainen, Tuomas; Kinnunen, Eeva-Maija; Koski-Vähälä, Joni; Mosorin, Matti-Aleksi; Airaksinen, Juhani; Biancari, Fausto

2017-06-01

Incomplete drainage of blood from around the heart and lungs can lead to retained blood syndrome (RBS) after cardiac surgery. The aim of this study was to assess the incidence of and the outcome after procedures for RBS in patients undergoing isolated coronary artery bypass grafting (CABG). A total of 2764 consecutive patients who underwent isolated CABG from 2006 to 2013 were investigated retrospectively. Patients undergoing any procedure for RBS were compared with patients who did not undergo any procedure for RBS. Multivariate analyses were performed to assess the impact of procedures for RBS on the early outcome. A total of 254 patients (9.2%) required at least one procedure for RBS. Multivariate analysis showed that RBS requiring a procedure for blood removal was associated with significantly increased 30-day mortality [8.3% vs 2.7%, odds ratio (OR) 2.11, 95% confidence interval (95% CI) 1.15-3.86] rates. Procedures for RBS were independent predictors of the need for postoperative antibiotics (51.6% vs 32.1%, OR 2.08, 95% CI 1.58-2.74), deep sternal wound infection/mediastinitis (6.7% vs 2.2%, OR 3.12, 95% CI 1.72-5.66), Kidney Disease: Improving Global Outcomes acute kidney injury (32.7% vs 15.3%, OR 2.50, 95% CI 1.81-3.46), length of stay in the intensive care unit (mean 8.3 vs 2.0 days, beta 1.74, 95% CI 1.45-2.04) and composite major adverse events (21.3% vs 6.9%, OR 3.24, 95% CI 2.24-4.64). These findings were also confirmed in a subgroup of patients with no pre- or postoperative unstable haemodynamic conditions. RBS requiring any procedure for blood removal from pericardial and pleural spaces is associated with an increased risk of severe complications after isolated CABG. © The Author 2017. Published by Oxford University Press on behalf of the European Association for Cardio-Thoracic Surgery. All rights reserved.

Comparison of carbon dioxide laser-assisted versus stapler-assisted endoscopic cricopharyngeal myotomy.

PubMed

Pollei, Taylor R; Hinni, Michael L; Hayden, Richard E; Lott, David G; Mors, Matthew B

2013-09-01

We directly compared endoscopic carbon dioxide (CO2) laser and stapler treatment methods for both cricopharyngeal hypertrophy (CPH) and Zenker's diverticulum (ZD). We performed a single-institution retrospective chart review of 153 patients who underwent either CO2 laser-assisted or stapler-assisted endoscopic cricopharyngeal myotomy (CPM). Isolated CPH was more likely to be treated with the CO2 laser than by stapler techniques. The ZD pouch size decreased significantly after surgery in both laser (p = 0.04) and stapler (p = 0.008) groups. The average duration of the procedure for CPM was longer for the laser than for the stapler (p = 0.01). Both techniques were successful when used in revision procedures. The overall complication rates were not statistically significantly different. Laser surgery trended toward a higher rate of major complications (2.4% versus 0%). Symptomatic recurrence was more likely after stapler surgery (p = 0.002). The rates of revision surgery were similar in the two groups (3.3% for laser and 4.3% for stapler). In the treatment of isolated CPH or ZD, stapler-assisted endoscopic surgery results in a shorter operative time, whereas laser-assisted CPM results in a decreased incidence of symptomatic recurrence.

Preparation, isolation, and characterization of cutin monomers and oligomers from tomato peels.

PubMed

Osman, S F; Irwin, P; Fett, W F; O'Connor, J V; Parris, N

1999-02-01

Cutin in tomato peels was depolymerized in methanolic base to yield cutin monomers or a mixture of cutin oligomers. These products were isolated by typical solvent extraction methods or by precipitation, and the isolates were characterized by chromatographic and spectroscopic analyses. It was determined that the compositions of the isolates from both isolation procedures were similar, although solvent extraction gave higher yields. However, the precipitation method, which is easy to carry out and avoids the use of undesirable organic solvents, may be preferable in commercial processes for recovering these compounds.

A simple and effective protocol for fast isolation of human Tenon's fibroblasts from a single trabeculectomy biopsy - a comparison of cell behaviour in different culture media.

PubMed

Przekora, Agata; Zarnowski, Tomasz; Ginalska, Grazyna

2017-01-01

Human Tenon's fibroblasts (HTFs) play a crucial role in wound healing. They cause postoperative scarring of the filtering bleb and are thus responsible for trabeculectomy failure. This study aimed to find an effective and fast protocol for HTF isolation from trabeculectomy biopsies. The protocol was compared with the commonly recommended HTF isolation procedure, which uses Dulbecco's modified Eagle's medium (DMEM). We used Eagle's minimum essential medium (EMEM) enriched with fibroblast growth factor (FGF), which selectively promoted the proliferation of HTF cells. A secondary goal was to compare HTF morphology, metabolism and growth during parallel cultivation of the isolated cells in FGF-enriched EMEM and DMEM. Standard procedures for HTF isolation from tissue biopsies require a 20- to 30-day culture of the explants to obtain the first monolayer. Our protocol yielded the first monolayer after approx. 15 days. More importantly, the majority of the cells were fibroblasts with only individual epithelium-derived cells present. Using FGF-enriched EMEM allowed 1.3 × 10 6 vimentin-positive fibroblasts to be obtained from a single biopsy within approx. 25 days. Using DMEM resulted in isolation failure and required exchange to FGF-enriched medium to recover the fibroblast culture. HTFs maintained in FGF-enriched EMEM also showed faster proliferation and a different type I collagen production ability compared to HTFs cultured in DMEM. Thus, FGF-enriched EMEM is recommended for fast propagation of HTFs unless the aim of the study is to assess the effect of a tested agent on proliferation ability or type I collagen production. Our fast protocol for HTF isolation allows easy setup of cell banks by researchers under laboratory conditions and could be very useful during testing of novel ophthalmologic anti-fibrotic agents in vitro. Molecular analysis of HTFs isolated from patients with known treatment histories may provide valuable information on the effects of some medications taken before glaucoma surgery on the subsequent wound-healing process and potential for trabeculectomy failure.

Distribution of Yeast-Like Fungi at a University Hospital in Turkey

PubMed Central

Ece, Gulfem

2014-01-01

Background: The increased life span has led to application of more invasive procedures for diagnosis and treatment of particularly immunosuppressed individuals. This situation drew more attention to fungal infections due to existence of yeast-like fungi. Candida infections have increased due to transplant in patients, prolonged intensive care unit (ICU) stays, and invasive procedures. Recently, identification of yeast-like fungi as well as antifungal susceptibility test has been gaining more importance. Objectives: In our study, we aimed to evaluate the distribution of yeast-like fungi strains isolated from blood, urine, wound and respiratory specimens, which were sent from various departments of Izmir University School of Medicine University Hospital. Materials and Methods: The 262 yeast strains (of 13860 clinical specimens), isolated during 30.05.2012-20.05.2013, which were sent from various departments of Izmir University School of Medicine to Medical Microbiology Laboratory, were included in this study. Blood, wound, respiratory (sputum, tracheal secretion), and urine specimens were cultivated on blood agar and Sabouraud dextrose agar and incubated for 24-48 hours at 37°C. The isolates were cultivated on CHROMagar Candida and Cornmeal Tween 80 medium for identification. Besides, the automatized Vitek version 2.0 system was used for identification of the yeast strains as well as the antifungal susceptibility of blood culture strains. Results: A total of 262 strains, isolated from the Anesthesiology and Reanimation Unit, as well as from the departments of Hematology, Urology, Infectious Diseases, Gynecology and Obstetrics, and Ear Nose and Throat, were included in this study. The most common isolated yeast-like species was Candida albicans. C. parapsilosis was the most common yeast-like fungus isolated from blood cultures. All the blood culture strains were susceptible to amphotericin B, flucytosine, fluconazole and voriconazole. Conclusions: Candida strains isolated from newborns, elderly patients, and intensive care patients, identified and isolates from blood cultures, should be studied for antifungal susceptibility for management of the treatment. Our University Hospital is a recently opened center and these are the first data of our center. Gradually, as the number of patients increases, this data will be evaluated further. PMID:25741432

Use of muscovy duck embryo fibroblasts for the isolation of viruses from wild birds

USGS Publications Warehouse

Docherty, D.E.; Slota, Paul G.

1988-01-01

Techniques are described for the preparation, cryopreservation, and inoculation of Muscovy duck embryo cell cultures. The procedure yields a susceptible reproducible cell culture system for the isolation and cultivation of viruses from wild birds.

An adapted isolation procedure reveals Photobacterium spp. as common spoilers on modified atmosphere packaged meats.

PubMed

Hilgarth, M; Fuertes-Pèrez, S; Ehrmann, M; Vogel, R F

2018-04-01

The genus Photobacterium comprises species of marine bacteria, commonly found in open-ocean and deep-sea environments. Some species (e.g. Photobacterium phosphoreum) are associated with fish spoilage. Recently, culture-independent studies have drawn attention to the presence of photobacteria on meat. This study employed a comparative isolation approach of Photobacterium spp. and aimed to develop an adapted isolation procedure for recovery from food samples, as demonstrated for different meats: Marine broth is used for resuspending and dilution of food samples, followed by aerobic cultivation on marine broth agar supplemented with meat extract and vancomycin at 15°C for 72 h. Identification of spoilage-associated microbiota was carried out via Matrix Assisted Laser Desorption/Ionization Time of Flight Mass Spectrometry using a database supplemented with additional mass spectrometry profiles of Photobacterium spp. This study provides evidence for the common abundance of multiple Photobacterium species in relevant quantities on various modified atmosphere packaged meats. Photobacterium carnosum was predominant on beef and chicken, while Photobacterium iliopiscarium represented the major species on pork and Photobacterium phosphoreum on salmon, respectively. This study demonstrates highly frequent isolation of multiple photobacteria (Photobacterium carnosum, Photobacterium phosphoreum, and Photobacterium iliopiscarium) from different modified-atmosphere packaged spoiled and unspoiled meats using an adapted isolation procedure. The abundance of photobacteria in high numbers provides evidence for the hitherto neglected importance and relevance of Photobacterium spp. to meat spoilage. © 2018 The Society for Applied Microbiology.

Surgical ablation for atrial fibrillation for two decades: are the results of new techniques equivalent to the Cox maze III procedure?

PubMed

Stulak, John M; Suri, Rakesh M; Burkhart, Harold M; Daly, Richard C; Dearani, Joseph A; Greason, Kevin L; Joyce, Lyle D; Park, Soon J; Schaff, Hartzell V

2014-05-01

A significant evolution has occurred in surgical ablation for atrial fibrillation (AF) toward alternate energy sources, lesion sets, and approaches, with the intent of simplifying the Cox maze III operation and maintaining similar outcomes. Because no large comparative studies with long-term follow-up exist, we have reviewed our experience. From January 1993 to January 2011, 1540 patients underwent surgical ablation for AF. The operations were performed in conjunction with repair of congenital heart disease in 351 (30%) and adult-acquired disease in 1189 patients (70%). In the 1189 patients, preoperative AF was paroxysmal in 598 (50%) and persistent in 591 (50%). The energy sources included cut and sew in 521 (44%), cryothermy in 267 (22%), radiofrequency in 262 (22%), and a combination in 139 patients (12%). The lesion sets included biatrial in 810 (68%), isolated pulmonary vein isolation in 269 (23%), and isolated left atrial in 110 (9%). AF ablation was performed during isolated mitral valve surgery in 516 patients (43%). The median follow-up was 33 months (maximum, 20.3 years), and late rhythm follow-up was available for 80%. The cut and sew Cox maze III procedure was superior at each follow-up interval (P = .01, P = .03, and P < .001). On multivariate analysis, the cut and sew maze procedure was independently associated with less risk of recurrent AF at a follow-up period of 1 to 5 years (hazard ratio, 0.4; 95% confidence interval, 0.24-0.69; P < .001) and >5 years (hazard ratio, 0.23; 95% confidence interval, 0.12-0.42; P < .001) for all patients. When performed during isolated mitral valve surgery, the cut and sew Cox maze III was also independently associated with less risk of recurrent AF at >5 years (hazard ratio, 0.23; 95% confidence interval, 0.08-0.66; P = .007). The cut and sew Cox maze III procedure appears to offer significantly greater freedom from AF without antiarrhythmic medications compared with alternate energy sources and lesion sets. Although alternate energy sources offer the advantage of quicker application and the possibility of minimally invasive applications, the Cox maze III operation arguably remains the reference standard for the surgical treatment of AF and should still be considered, especially for patients for whom AF ablation is of critical importance. Copyright © 2014 The American Association for Thoracic Surgery. Published by Mosby, Inc. All rights reserved.

[Amanitine determination as an example of peptide analysis in the biological samples with HPLC-MS technique].

PubMed

Janus, Tomasz; Jasionowicz, Ewa; Potocka-Banaś, Barbara; Borowiak, Krzysztof

Routine toxicological analysis is mostly focused on the identification of non-organic and organic, chemically different compounds, but generally with low mass, usually not greater than 500–600 Da. Peptide compounds with atomic mass higher than 900 Da are a specific analytical group. Several dozen of them are highly-toxic substances well known in toxicological practice, for example mushroom toxin and animal venoms. In the paper the authors present an example of alpha-amanitin to explain the analytical problems and different original solutions in identifying peptides in urine samples with the use of the universal LC MS/MS procedure. The analyzed material was urine samples collected from patients with potential mushroom intoxication, routinely diagnosed for amanitin determination. Ultra filtration with centrifuge filter tubes (limited mass cutoff 3 kDa) was used. Filtrate fluid was directly injected on the chromatographic column and analyzed with a mass detector (MS/MS). The separation of peptides as organic, amphoteric compounds from biological material with the use of the SPE technique is well known but requires dedicated, specific columns. The presented paper proved that with the fast and simple ultra filtration technique amanitin can be effectively isolated from urine, and the procedure offers satisfactory sensitivity of detection and eliminates the influence of the biological matrix on analytical results. Another problem which had to be solved was the non-characteristic fragmentation of peptides in the MS/MS procedure providing non-selective chromatograms. It is possible to use higher collision energies in the analytical procedure, which results in more characteristic mass spectres, although it offers lower sensitivity. The ultra filtration technique as a procedure of sample preparation is effective for the isolation of amanitin from the biological matrix. The monitoring of selected mass corresponding to transition with the loss of water molecule offers satisfactory sensitivity of determination.

Isolation and Purification of Cu-free Methanobactin from Methylosinus trichosporium OB3b

DOE Office of Scientific and Technical Information (OSTI.GOV)

M Pesch; I Christl; K Barmettler

The isolation of highly pure copper-free methanobactin is a prerequisite for the investigation of the biogeochemical functions of this chalkophore molecule produced by methane oxidizing bacteria. Here, we report a purification method for methanobactin from Methylosinus trichosporium OB3b cultures based on reversed-phase HPLC fractionation used in combination with a previously reported resin extraction. HPLC eluent fractions of the resin extracted product were collected and characterized with UV-vis, FT-IR, and C-1s NEXAFS spectroscopy, as well as with elemental analysis and ESI-MS. The results showed that numerous compounds other than methanobactin were present in the isolate obtained with resin extraction. Molar C/Nmore » ratios, mass spectrometry measurements, and UV-vis spectra indicated that methanobactin was only present in one of the HPLC fractions. On a mass basis, methanobactin carbon contributed only 32% to the total organic carbon isolated with resin extraction. Our spectroscopic results implied that besides methanobactin, the organic compounds in the resin extract comprised breakdown products of methanobactin as well as polysaccharide-like substances. Our results demonstrate that a purification step is indispensable in addition to resin extraction in order to obtain pure methanobactin. The proposed HPLC purification procedure is suitable for semi-preparative work and provides copper-free methanobactin.« less

GMP-based CD133+ cells isolation maintains progenitor angiogenic properties and enhances standardization in cardiovascular cell therapy

PubMed Central

Gaipa, Giuseppe; Tilenni, Manuela; Straino, Stefania; Burba, Ilaria; Zaccagnini, Germana; Belotti, Daniela; Biagi, Ettore; Valentini, Marco; Perseghin, Paolo; Parma, Matteo; Campli, Cristiana Di; Biondi, Andrea; Capogrossi, Maurizio C; Pompilio, Giulio; Pesce, Maurizio

2010-01-01

Abstract The aim of the present study was to develop and validate a good manufacturing practice (GMP) compliant procedure for the preparation of bone marrow (BM) derived CD133+ cells for cardiovascular repair. Starting from available laboratory protocols to purify CD133+ cells from human cord blood, we implemented these procedures in a GMP facility and applied quality control conditions defining purity, microbiological safety and vitality of CD133+ cells. Validation of CD133+ cells isolation and release process were performed according to a two-step experimental program comprising release quality checking (step 1) as well as ‘proofs of principle’ of their phenotypic integrity and biological function (step 2). This testing program was accomplished using in vitro culture assays and in vivo testing in an immunosuppressed mouse model of hindlimb ischemia. These criteria and procedures were successfully applied to GMP production of CD133+ cells from the BM for an ongoing clinical trial of autologous stem cells administration into patients with ischemic cardiomyopathy. Our results show that GMP implementation of currently available protocols for CD133+ cells selection is feasible and reproducible, and enables the production of cells having a full biological potential according to the most recent quality requirements by European Regulatory Agencies. PMID:19627397

Performance assessment of conventional and base-isolated nuclear power plants for earthquake and blast loadings

NASA Astrophysics Data System (ADS)

Huang, Yin-Nan

Nuclear power plants (NPPs) and spent nuclear fuel (SNF) are required by code and regulations to be designed for a family of extreme events, including very rare earthquake shaking, loss of coolant accidents, and tornado-borne missile impacts. Blast loading due to malevolent attack became a design consideration for NPPs and SNF after the terrorist attacks of September 11, 2001. The studies presented in this dissertation assess the performance of sample conventional and base isolated NPP reactor buildings subjected to seismic effects and blast loadings. The response of the sample reactor building to tornado-borne missile impacts and internal events (e.g., loss of coolant accidents) will not change if the building is base isolated and so these hazards were not considered. The sample NPP reactor building studied in this dissertation is composed of containment and internal structures with a total weight of approximately 75,000 tons. Four configurations of the reactor building are studied, including one conventional fixed-base reactor building and three base-isolated reactor buildings using Friction Pendulum(TM), lead rubber and low damping rubber bearings. The seismic assessment of the sample reactor building is performed using a new procedure proposed in this dissertation that builds on the methodology presented in the draft ATC-58 Guidelines and the widely used Zion method, which uses fragility curves defined in terms of ground-motion parameters for NPP seismic probabilistic risk assessment. The new procedure improves the Zion method by using fragility curves that are defined in terms of structural response parameters since damage and failure of NPP components are more closely tied to structural response parameters than to ground motion parameters. Alternate ground motion scaling methods are studied to help establish an optimal procedure for scaling ground motions for the purpose of seismic performance assessment. The proposed performance assessment procedure is used to evaluate the vulnerability of the conventional and base-isolated NPP reactor buildings. The seismic performance assessment confirms the utility of seismic isolation at reducing spectral demands on secondary systems. Procedures to reduce the construction cost of secondary systems in isolated reactor buildings are presented. A blast assessment of the sample reactor building is performed for an assumed threat of 2000 kg of TNT explosive detonated on the surface with a closest distance to the reactor building of 10 m. The air and ground shock waves produced by the design threat are generated and used for performance assessment. The air blast loading to the sample reactor building is computed using a Computational Fluid Dynamics code Air3D and the ground shock time series is generated using an attenuation model for soil/rock response. Response-history analysis of the sample conventional and base isolated reactor buildings to external blast loadings is performed using the hydrocode LS-DYNA. The spectral demands on the secondary systems in the isolated reactor building due to air blast loading are greater than those for the conventional reactor building but much smaller than those spectral demands associated with Safe Shutdown Earthquake shaking. The isolators are extremely effective at filtering out high acceleration, high frequency ground shock loading.

Limitations of the Current Microbial Identification System for Identification of Clinical Yeast Isolates

PubMed Central

Kellogg, James A.; Bankert, David A.; Chaturvedi, Vishnu

1998-01-01

The ability of the rapid, computerized Microbial Identification System (MIS; Microbial ID, Inc.) to identify a variety of clinical isolates of yeast species was compared to the abilities of a combination of tests including the Yeast Biochemical Card (bioMerieux Vitek), determination of microscopic morphology on cornmeal agar with Tween 80, and when necessary, conventional biochemical tests and/or the API 20C Aux system (bioMerieux Vitek) to identify the same yeast isolates. The MIS chromatographically analyzes cellular fatty acids and compares the results with the fatty acid profiles in its database. Yeast isolates were subcultured onto Sabouraud dextrose agar and were incubated at 28°C for 24 h. The resulting colonies were saponified, methylated, extracted, and chromatographically analyzed (by version 3.8 of the MIS YSTCLN database) according to the manufacturer’s instructions. Of 477 isolates of 23 species tested, 448 (94%) were given species names by the MIS and 29 (6%) were unidentified (specified as “no match” by the MIS). Of the 448 isolates given names by the MIS, only 335 (75%) of the identifications were correct to the species level. While the MIS correctly identified only 102 (82%) of 124 isolates of Candida glabrata, the predictive value of an MIS identification of unknown isolates as C. glabrata was 100% (102 of 102) because no isolates of other species were misidentified as C. glabrata. In contrast, while the MIS correctly identified 100% (15 of 15) of the isolates of Saccharomyces cerevisiae, the predictive value of an MIS identification of unknown isolates as S. cerevisiae was only 47% (15 of 32), because 17 isolates of C. glabrata were misidentified as S. cerevisiae. The low predictive values for accuracy associated with MIS identifications for most of the remaining yeast species indicate that the procedure and/or database for the system need to be improved. PMID:9574676

Fatigue and stress studies : an improved semi-automated procedure for flurometric determination of plasma catecholamines.

DOT National Transportation Integrated Search

1966-04-01

A semiautomated technique is described for the estimation of total catecholamines in plasma by the trihydroxyindole procedure. The method utilizes conventional alumina-column chromatography for isolation of the amines. Catecholamine oxidation, tautom...

An Examination of Disciplinary Procedures with Children

ERIC Educational Resources Information Center

Forehand, Rex; And Others

1976-01-01

Experiment 1 examined the effect of negative attention and repeated demands on the noncompliant behavior of children. Experiment 2 studied the relative effectiveness of negative attention, isolation, ignoring, and a combination of procedures in reducing noncompliance in children. (GO)

Panning for sperm gold: Isolation and purification of apyrene and eupyrene sperm from lepidopterans.

PubMed

Karr, Timothy L; Walters, James R

2015-08-01

We describe a simple and straightforward procedure for the purification and separation of apyrene and eupyrene forms of lepidopteran sperm. The procedure is generally applicable to both butterfly and moth species with results varying according to the relative amounts of sperm produced and size of sperm storage organs. The technique relies upon inherent differences between eupyene sperm bundles and free apyrene sperm morphology. These differences allow for separation of the sperm morphs by repeated "panning" of sperm bundles into the center of a plastic dish. The purified eupyrene sperm bundles can then be removed and apyrene sperm collected from the supernatant by centrifugation. Efficacy of the purification process was confirmed by light microscopy and gel electrophoresis of the resulting fractions. Both one- and two-dimensional gel electrophoresis identified significant protein differences between the fractions further suggesting that the panning procedure effectively separated eurpyrene from apyrene sperm. The panning procedure should provide a convenient and accessible technique for further studies of sperm biology in lepidopterans. Copyright © 2015 Elsevier Ltd. All rights reserved.

Laparoscopic sentinel node procedure using a combination of patent blue and radiocolloid in women with endometrial cancer.

PubMed

Barranger, Emmanuel; Cortez, Annie; Grahek, Dany; Callard, Patrice; Uzan, Serge; Darai, Emile

2004-03-01

We assessed the feasibility of a laparoscopic sentinel node (SN) procedure based on the combined use of radiocolloid and patent blue labeling in patients with endometrial cancer. Seventeen patients (median age, 69 years) with endometrial cancer of stage I (16 patients) or stage II (1 patient) underwent a laparoscopic SN procedure based on combined radiocolloid and patent blue injected pericervically. After the SN procedure, all patients underwent complete laparoscopic pelvic lymphadenectomy and either laparoscopically assisted vaginal hysterectomy (16 patients) or laparoscopic radical hysterectomy (1 patient). SNs (mean number per patient, 2.6; range, 1-4) were identified in 16 (94.1%) of the 17 patients. Macrometastases were detected in three SNs from two patients by hematoxylin and eosin staining. In three other patients, immunohistochemical analysis identified six micrometastatic SNs and one SN containing isolated tumor cells. No false-negative SN results were observed. An SN procedure based on a combination of radiocolloid and patent blue is feasible in patients with early endometrial cancer. Combined use of laparoscopy and this SN procedure permits minimally invasive management of endometrial cancer.

Isolation, purification and chemical composition of insoluble collagen from Guerin epithelioma.

PubMed

Bańkowski, E; Galasiński, W; Gindzieẃski, A; Rzeczycki, W

1975-01-01

1. The insoluble collagen from Guerin epithelioma was isolated and its chemical composition was determined. The unusually high histidine content is accompanied in tumour collagen by a relatively small amount of lysine and arginine. 2. The isolated protein was strongly bound to glycoprotein, which could not be removed by EDTA treatment unless this procedure was preceded by digestion of the complex with trypsin.

Antimicrobial Treatment Options for Granulomatous Mastitis Caused by Corynebacterium Species

PubMed Central

Dobinson, Hazel C.; Anderson, Trevor P.; Chambers, Stephen T.; Doogue, Matthew P.; Seaward, Lois

2015-01-01

Corynebacterium species are increasingly recognized as important pathogens in granulomatous mastitis. Currently, there are no published treatment protocols for Corynebacterium breast infections. This study describes antimicrobial treatment options in the context of other management strategies used for granulomatous mastitis. Corynebacterium spp. isolated from breast tissue and aspirate samples stored from 2002 to 2013 were identified and determined to the species level using matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS), 16S RNA sequencing, and rpoB gene targets. The MICs for 12 antimicrobials were performed using Etest for each isolate. Correlations of these with antimicrobial characteristics, choice of antimicrobial, and disease outcome were evaluated. Corynebacterium spp. from breast tissue and aspirate samples were confirmed in 17 isolates from 16 patients. Based on EUCAST breakpoints, Corynebacterium kroppenstedtii isolates (n = 11) were susceptible to seven antibiotic classes but resistant to β-lactam antibiotics. Corynebacterium tuberculostearicum isolates (n = 4) were multidrug resistant. Two nonlipophilic species were isolated, Corynebacterium glucuronolyticum and Corynebacterium freneyi, both of which have various susceptibilities to antimicrobial agents. Short-course antimicrobial therapy was common (median, 6 courses per subject; range, 1 to 9 courses). Patients with C. kroppenstedtii presented with a hot painful breast mass and underwent multiple surgical procedures (median, 4 procedures; range, 2 to 6 procedures). The management of Corynebacterium breast infections requires a multidisciplinary approach and includes culture and appropriate sensitivity testing to guide antimicrobial therapy. Established infections have a poor outcome, possibly because adequate concentrations of some drugs will be difficult to achieve in lipophilic granulomata. Lipophilic antimicrobial therapy may offer a therapeutic advantage. The role of immunotherapy has not been defined. PMID:26135858

Indication for Co-evolution of Lactobacillus johnsonii with its hosts.

PubMed

Buhnik-Rosenblau, Keren; Matsko-Efimov, Vera; Jung, Minju; Shin, Heuynkil; Danin-Poleg, Yael; Kashi, Yechezkel

2012-07-25

The intestinal microbiota, composed of complex bacterial populations, is host-specific and affected by environmental factors as well as host genetics. One important bacterial group is the lactic acid bacteria (LAB), which include many health-promoting strains. Here, we studied the genetic variation within a potentially probiotic LAB species, Lactobacillus johnsonii, isolated from various hosts. A wide survey of 104 fecal samples was carried out for the isolation of L. johnsonii. As part of the isolation procedure, terminal restriction fragment length polymorphism (tRFLP) was performed to identify L. johnsonii within a selected narrow spectrum of fecal LAB. The tRFLP results showed host specificity of two bacterial species, the Enterococcus faecium species cluster and Lactobacillus intestinalis, to different host taxonomic groups while the appearance of L. johnsonii and E. faecalis was not correlated with any taxonomic group. The survey ultimately resulted in the isolation of L. johnsonii from few host species. The genetic variation among the 47 L. johnsonii strains isolated from the various hosts was analyzed based on variation at simple sequence repeats (SSR) loci and multi-locus sequence typing (MLST) of conserved hypothetical genes. The genetic relationships among the strains inferred by each of the methods were similar, revealing three different clusters of L. johnsonii strains, each cluster consisting of strains from a different host, i.e. chickens, humans or mice. Our typing results support phylogenetic separation of L. johnsonii strains isolated from different animal hosts, suggesting specificity of L. johnsonii strains to their hosts. Taken together with the tRFLP results, that indicated the association of specific LAB species with the host taxonomy, our study supports co-evolution of the host and its intestinal lactic acid bacteria.

Isolation and Structural Studies of Mitochondria from Pea Roots.

PubMed

Vishwakarma, Abhaypratap; Gupta, Kapuganti Jagadis

2017-01-01

For structural and respiratory studies, isolation of intact and active mitochondria is essential. Here, we describe an isolation method which gave good yield and intact mitochondria from 2-week-old pea (Pisum sativum) roots grown hydroponically under standard growth conditions. We used Percoll gradient centrifugation for this isolation procedure. The yield of purified mitochondria was 50 μg/g FW. Isolated mitochondria maintained their structure which was observed by using MitoTracker green in confocal microscope and scanning electron microscopy (SEM). Intact mitochondria are clearly visible in SCM images. Taken together this isolation method can be used for physiological and microscopic studies on mitochondria.

Chronic social isolation and chronic variable stress during early development induce later elevated ethanol intake in adult C57BL/6J mice.

PubMed

Lopez, Marcelo F; Doremus-Fitzwater, Tamara L; Becker, Howard C

2011-06-01

Experience with stress situations during early development can have long-lasting effects on stress- and anxiety-related behaviors. Importantly, this can also favor drug self-administration. These studies examined the effects of chronic social isolation and/or variable stress experiences during early development on subsequent voluntary ethanol intake in adult male and female C57BL/6J mice. The experiments were conducted to evaluate the effect of chronic isolation between weaning and adulthood (Experiment 1), chronic isolation during adulthood (Experiment 2), and chronic variable stress (CVS) alone or in combination with chronic social isolation between weaning and adulthood (Experiment 3) on subsequent voluntary ethanol intake. Mice were born in our facility and were separated into two housing conditions: isolate housed (one mouse/cage) or group housed (four mice/cage) according to sex. Separate groups were isolated for 40 days starting either at time of weaning postnatal day 21 (PD 21) (early isolation, Experiments 1 and 3) or at adulthood (PD 60: late isolation, Experiment 2). The effects of housing condition on subsequent ethanol intake were assessed starting at around PD 65 in Experiments 1 and 3 or PD 105 days in Experiment 2. In Experiment 3, starting at PD 32, isolate-housed and group-housed mice were either subjected to CVS or left undisturbed. CVS groups experienced random presentations of mild stressors for 14 days, including exposure to an unfamiliar open field, restraint, physical shaking, and forced swim, among others. All mice were tested for ethanol intake for 14 days using a two-bottle choice (ethanol 15% vol/vol vs. water) for a 2-h limited access procedure. Early social isolation resulted in greater ethanol intake compared with the corresponding group-housed mice (Experiment 1). In contrast, social isolation during adulthood (late isolation) did not increase subsequent ethanol intake compared with the corresponding group-housed mice (Experiment 2). For mice that did not experience CVS, early social isolation resulted in greater ethanol intake compared with group-housed mice (Experiment 3). CVS subsequently resulted in a significant increase in ethanol intake in group-housed mice, but CVS failed to further increase ethanol intake in mice that experienced chronic social isolation early in life (Experiment 3). Overall, female mice consumed more ethanol than males, whether isolated (early or late) or group housed. These results indicate that early but not late social isolation can subsequently influence ethanol consumption in C57BL/6J mice. Thus, the developmental timing of chronic social isolation appears to be an important factor in defining later effects on ethanol self-administration behavior. In addition, experience with CVS early in life results in elevated ethanol intake later in adulthood. Taken together, these results emphasize the important role of early stress experiences that modulate later voluntary ethanol intake during adulthood. Copyright © 2011 Elsevier Inc. All rights reserved.

Intense Pulsed Light (IPL) in Aesthetic Dermatology

NASA Astrophysics Data System (ADS)

Pytras, B.; Drozdowski, P.; Zub, K.

2011-08-01

Introduction. Newer and newer technologies have been widely developed in recent years due to increasing need for aesthetic medicine procedures. Less invasive methods of skin imperfection and time-related lesions removal, IPL (Intense Pulse Light) being one of them, are gaining more and more interest. The shorter the "downtime" for the patient is and the more efficient the procedure results, the more popular the method becomes. Materials and methods_Authors analyse the results of treatment of a 571 patients-group (501 women and 70 men) aged 5-72 years in the period: October 2006-August 2010. IPL™ Quantum (Lumenis Ltd.) device with 560 nm. cut-off filter was used. Results. The results were regarded as: very good, good or satisfying (%):Skin photoaging symptomes 37/40/23, Isolated facial dyschromia 30/55/25, Isolated facial erythema 62/34/4, Lower limbs teleangiectasia 12/36/52, Keratosis solaris on hands 100/-/-. Approximately half of the patients developed transitory erythema and 25%- transitory, mild, circumscribed oedema. Following undesirable effects were noted: skin thermal irritation (6,1% of the patients) and skin hypopigmentation (2% of the patients). Discussion. Results and post-treatment management proposed by authors are similar to those reported by other authors. Conclusions. Treatment results of the 571-patients group prove IPL to be a very efficient method of non-ablative skin rejuvenation. It turned out effective also in lower limbs teleangiectasia treatment. It presents low risk of transitory and mild side effects. Futhermore, with short or no downtime, it is well-tolerated by the patients.

Frequency of Resistance and Susceptible Bacteria Isolated from Houseflies

PubMed Central

Davari, B; Kalantar, E; Zahirnia, A; Moosa-Kazemi, SH

2010-01-01

Background: In this study, we determine the vector competence of Musca domestica with reference to the transmission of susceptible and resistance bacterial strains in hospitals and slaughter house in Sanandaj City, west Iran. Methods: Totally 908 houseflies were collected to isolate bacteria from their external body based on standard procedures.Antibiotic susceptibility testing was performed by Kirby-Bauer disc diffusion method on Mueller Hinton agar based on recommendations of CLSI (formerly the National Committee for Clinical Laboratory Standards). Results: From collected houseflies, 366 bacteria species were isolated. The most common isolated bacterium at hospitals was Klebsiella pneumoniae 43.3% (n= 90) followed by Pseudomonas aeruginosa 37% (n= 77), while that of slaughterhouse was Proteus mirabilis. 29.1% (n= 46) followed by Citrobacter freundii 28.4% (n= 45). Among all the isolates from hospitals, cephalexin, chloramphenicol, ampicillin, and tetracycline, resistance rates were above 32.5% and gentamicin expressed the highest susceptibility among all the isolates from hospitals. It is worth to note that K. pneumoniae showed 61% and 44.5% resistance to cephalexin and chloramphenicol respectively. Similarly, all isolates from slaughterhouse were more than 28% and 30% resistant to cephalexin and chloramphenicol respectively. Surprisingly, among all the isolates, Citrobacter freundii were highly resistant to gentamicin. Conclusion: Houseflies collected from hospitals and slaughterhouse may be involved in the spread of drug resistant bacteria and may increase the potential of human exposure to drug resistant bacteria. PMID:22808400

Methods for the extraction and RNA profiling of exosomes

PubMed Central

Zeringer, Emily; Li, Mu; Barta, Tim; Schageman, Jeoffrey; Pedersen, Ketil Winther; Neurauter, Axl; Magdaleno, Susan; Setterquist, Robert; Vlassov, Alexander V

2013-01-01

AIM: To develop protocols for isolation of exosomes and characterization of their RNA content. METHODS: Exosomes were extracted from HeLa cell culture media and human blood serum using the Total exosome isolation (from cell culture media) reagent, and Total exosome isolation (from serum) reagent respectively. Identity and purity of the exosomes was confirmed by Nanosight® analysis, electron microscopy, and Western blots for CD63 marker. Exosomal RNA cargo was recovered with the Total exosome RNA and protein isolation kit. Finally, RNA was profiled using Bioanalyzer and quantitative reverse transcription-polymerase chain reaction (qRT-PCR) methodology. RESULTS: Here we describe a novel approach for robust and scalable isolation of exosomes from cell culture media and serum, with subsequent isolation and analysis of RNA residing within these vesicles. The isolation procedure is completed in a fraction of the time, compared to the current standard protocols utilizing ultracentrifugation, and allows to recover fully intact exosomes in higher yields. Exosomes were found to contain a very diverse RNA cargo, primarily short sequences 20-200 nt (such as miRNA and fragments of mRNA), however longer RNA species were detected as well, including full-length 18S and 28S rRNA. CONCLUSION: We have successfully developed a set of reagents and a workflow allowing fast and efficient extraction of exosomes, followed by isolation of RNA and its analysis by qRT-PCR and other techniques. PMID:25237619

[Anesthetic care for fibrobronchoscopy in patients with chronic terminal respiratory insufficiency undergoing evaluation for isolated lung transplantation].

PubMed

Solca, M; Elena, A; Croci, M; Damia, G

1993-01-01

During the first 18 month operation of the isolated lung transplantation program at or Institution, eight patients with terminal chronic respiratory failure underwent fiberoptic bronchoscopy and broncho-alveolar lavage as part of their evaluation for isolated lung transplantation. Four patients had severe obstructive, three restrictive, and one mixed, obstructive and restrictive, disease; all of them were on continuous supplemental oxygen. Procedures were performed under topical anaesthesia, with either light sedation or simple monitored anaesthesia care. Monitoring included non-invasive blood pressure measurement, pulse oximeter and precordial stethoscope. No adverse events were recorded, except in one case, when pulse oximeter reading precipitously dropped below 80%, to a minimum of 68-69%. The procedures was terminated short of its completion, and the patient was briefly assisted with manual bag ventilation on oxygen 100%. Pulse oximeter quickly returned to normal levels (above 90%), and the patient promptly recovered, without complications. The importance of monitored anaesthesia care during fiberoptic bronchoscopy (a usually benign procedure) in critically ill patients is greatly emphasized.

Growth in liquid or solid media.

PubMed

Elbing, K L; Brent, R

2001-05-01

This appendix presents basic procedures for growing overnight (and larger) cultures, monitoring growth, and titering and isolating bacterial cultures by serial dilution. In addition, protocols are provided for isolating single colonies by streaking and spreading a plate. Replica plating and strain storage/retrieval is also detailed.

Revisiting susceptibility testing in MDR-TB by a standardized quantitative phenotypic assessment in a European multicentre study.

PubMed

Cambau, E; Viveiros, M; Machado, D; Raskine, L; Ritter, C; Tortoli, E; Matthys, V; Hoffner, S; Richter, E; Perez Del Molino, M L; Cirillo, D M; van Soolingen, D; Böttger, E C

2015-03-01

Treatment outcome of MDR-TB is critically dependent on the proper use of second-line drugs as per the result of in vitro drug susceptibility testing (DST). We aimed to establish a standardized DST procedure based on quantitative determination of drug resistance and compared the results with those of genotypes associated with drug resistance. The protocol, based on MGIT 960 and the TB eXiST software, was evaluated in nine European reference laboratories. Resistance detection at a screening drug concentration was followed by determination of resistance levels and estimation of the resistance proportion. Mutations in 14 gene regions were investigated using established techniques. A total of 139 Mycobacterium tuberculosis isolates from patients with MDR-TB and resistance beyond MDR-TB were tested for 13 antituberculous drugs: isoniazid, rifampicin, rifabutin, ethambutol, pyrazinamide, streptomycin, para-aminosalicylic acid, ethionamide, amikacin, capreomycin, ofloxacin, moxifloxacin and linezolid. Concordance between phenotypic and genotypic resistance was >80%, except for ethambutol. Time to results was short (median 10 days). High-level resistance, which precludes the therapeutic use of an antituberculous drug, was observed in 49% of the isolates. The finding of a low or intermediate resistance level in 16% and 35% of the isolates, respectively, may help in designing an efficient personalized regimen for the treatment of MDR-TB patients. The automated DST procedure permits accurate and rapid quantitative resistance profiling of first- and second-line antituberculous drugs. Prospective validation is warranted to determine the impact on patient care. © The Author 2014. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

Modified salting-out method: high-yield, high-quality genomic DNA extraction from whole blood using laundry detergent.

PubMed

Nasiri, H; Forouzandeh, M; Rasaee, M J; Rahbarizadeh, F

2005-01-01

Different approaches have been used to extract DNA from whole blood. In most of these methods enzymes (such as proteinase K and RNAse A) or toxic organic solvents (such as phenol or guanidine isothiocyanate) are used. Since these enzymes are expensive, and most of the materials that are used routinely are toxic, it is desirable to apply an efficient DNA extraction procedure that does not require the use of such materials. In this study, genomic DNA was extracted by the salting-out method, but instead of using an analytical-grade enzyme and chemical detergents, as normally used for DNA isolation, a common laundry powder was used. Different concentrations of the powder were tested, and proteins were precipitated by NaCl-saturated distilled water. Finally, DNA precipitation was performed with the use of 96% ethanol. From the results, we conclude that the optimum concentration of laundry powder for the highest yield and purity of isolated DNA is 30 mg/mL. The procedure was optimized, and a final protocol is suggested. Following the same protocol, DNA was extracted from 100 blood samples, and their amounts were found to be >50 microg/mL of whole blood. The integrity of the DNA fragments was confirmed by agarose gel electrophoresis. Furthermore, the extracted DNA was used as a template for PCR reaction. The results obtained from PCR showed that the final solutions of extracted DNA did not contain any inhibitory material for the enzyme used in the PCR reaction, and indicated that the isolated DNA was of good quality. These results show that this method is simple, fast, safe, and cost-effective, and can be used in medical laboratories and research centers. Copyright 2005 Wiley-Liss, Inc.

Iron acquisition in Pasteurella haemolytica: expression and identification of a bovine-specific transferrin receptor.

PubMed Central

Ogunnariwo, J A; Schryvers, A B

1990-01-01

Seven type 1 field isolates of Pasteurella haemolytica were screened for their ability to use different transferrins as a source of iron for growth. All seven strains were capable of using bovine but not human, porcine, avian, or equine transferrin. A screening assay failed to detect siderophore production in any of the strains tested. Iron-deficient cells from these strains expressed a binding activity, specific for bovine transferrin, that was regulated by the level of iron in the medium. Inhibition of expression by translation and transcription inhibitors suggested that iron regulation was occurring at the gene level. Affinity isolation of receptor proteins from all seven strains with biotinylated bovine transferrin identified a 100-kilodalton iron-regulated outer membrane protein as the bovine transferrin receptor. Iron-regulated outer membrane proteins of 71 and 77 kilodaltons were isolated along with the 100-kilodalton protein when less stringent washing procedures were employed in the affinity isolation procedure. Images PMID:2365453

Improved production of isomaltulose by a newly isolated mutant of Serratia sp. cells immobilized in calcium alginate.

PubMed

Kim, Yonghwan; Koo, Bong-Seong; Lee, Hyeon-Cheol; Yoon, Youngdae

2015-03-01

Isomaltulose, also known as palatinose, is produced by sucrose isomerase and has been highlighted as a sugar substitute due to a number of advantageous properties. For the massive production of isomaltulose, high resistance to sucrose and stability of sucrose isomerase as well as sucrose conversion yields would be critical factors. We describe a series of screening procedures to isolate the mutant strain of Serratia sp. possessing enhanced isomaltulose production with improved stability. The new Serratia sp. isolated from a series of screening procedures allowed us to produce isomaltulose from 60% sucrose solution, with over 90% conversion yield. Moreover, when this strain was immobilized in calcium alginate beads and placed in a medium containing 60% sucrose, it showed over 70% sucrose conversion yields for 30 cycles of repeated-batch reactions. Thus, improved conversion activity and stability of the newly isolated Serratia sp. strain in the present study would be highly valuable for industries related to isomaltulose production.

Isolation of nonvolatile, organic solutes from natural waters by zeotrophic distillation of water from N,N-dimethylformamide

USGS Publications Warehouse

Leenheer, J.A.; Brown, P.A.; Stiles, E.A.

1987-01-01

Nonvolatile, organic solutes that comprise the dissolved organic carbon (DOC) in saline waters were isolated by removal of the water by distillation from a N,N-dimethylformamideformic acid-acetonitrile mixture. Salts isolated with the DOC were removed by crystallization of sodium chloride and sodium sulfate from the solvent mixture, removal of silicic acid by acidification and precipitation, removal of boric acid by methylation and volatilization, and removal of phosphate by zinc acetate precipitation. Chemical alteration of the organic solutes was minimized during evaporative concentration steps by careful control of acid concentrations in the solvent mixture and was minimized during drying by conversion of the samples to pyridinium and sodium salts. Recoveries of various hydrophilic organic standards from aqueous salt solutions and recoveries of natural organic solutes from various water samples varied from 60 to 100%. Losses of organic solutes during the isolation procedure were nonselective and related to the number of salt- and precipitate-washing cycles in the procedure.

Chemotherapy and Biochemistry of Leishmania.

DTIC Science & Technology

1986-12-01

product of this reaction, could reverse Inhibition but methionine, the precursor, could not. This enzyme catalyzes the following reaction L-methionine...because SAM, the product * is necessary for the methylation of DNA. The isolation procedures described eliminated 99% of the starting protein and resulted...sinefungin was slightly inhibitory (14%) at concentrations up to 1 mM, depending on temperatures ATP concentration and the addition of the product of the

Current Status of Islet Cell Transplantation

PubMed Central

Ichii, Hirohito; Ricordi, Camillo

2013-01-01

Despite substantial advances in islet isolation methods and immunosuppressive protocol, pancreatic islet cell transplantation remains an experimental procedure currently limited to the most severe cases of type 1 diabetes mellitus (T1DM). The objectives of this treatment are to prevent severe hypoglycemic episodes in patients with hypoglycemia unawareness, and to achieve a more physiological metabolic control. Insulin independence and long term-graft function with improvement of quality of life have been obtained in several international islet transplant centers. However, experimental trials of islet transplantation clearly highlighted several obstacles that remain to be overcome before the procedure could be proposed to a much larger patient population. This review provides a brief historical perspective of islet transplantation, islet isolation techniques, the transplant procedure, immunosuppressive therapy, and outlines current challenges and future directions in clinical islet transplantation. PMID:19110649

Subsequent Shoulder Surgery After Isolated Arthroscopic SLAP Repair.

PubMed

Mollon, Brent; Mahure, Siddharth A; Ensor, Kelsey L; Zuckerman, Joseph D; Kwon, Young W; Rokito, Andrew S

2016-10-01

To quantify the incidence of and identify the risk factors for subsequent shoulder procedures after isolated SLAP repair. New York's Statewide Planning and Research Cooperative System database was searched between 2003 and 2014 to identify individuals with the sole diagnosis of a SLAP lesion who underwent isolated arthroscopic SLAP repair. Patients were longitudinally followed up for a minimum of 3 years to analyze for subsequent ipsilateral shoulder procedures. Between 2003 and 2014, 2,524 patients met our inclusion criteria. After 3 to 11 years of follow-up, 10.1% of patients (254 of 2,524) underwent repeat surgical intervention on the same shoulder as the initial SLAP repair. The mean time to repeat shoulder surgery was 2.3 ± 2.1 years. Subsequent procedures included subacromial decompression (35%), debridement (26.7%). repeat SLAP repair (19.7%), and biceps tenodesis or tenotomy (13.0%). After isolated SLAP repair, patients aged 20 years or younger were more likely to undergo arthroscopic Bankart repair (odds ratio [OR], 2.91; 95% confidence interval [CI], 1.36-6.21; P = .005), whereas age older than 30 years was an independent risk factor for subsequent acromioplasty (OR, 2.3; 95% CI, 1.4-3.7; P < .001) and distal clavicle resection (OR, 2.5; 95% CI, 1.1-5.5; P = .030). The need for a subsequent procedure was significantly associated with Workers' Compensation cases (OR, 2.4; 95% CI, 1.7-3.2; P < .001). We identified a 10.1% incidence of subsequent surgery after isolated SLAP repair, often related to an additional diagnosis, suggesting that clinicians should consider other potential causes of shoulder pain when considering surgery for patients with SLAP lesions. In addition, the number of isolated SLAP repairs performed has decreased over time, and management of failed SLAP repair has shifted toward biceps tenodesis or tenotomy over revision SLAP repair in more recent years. Level III, case-control study. Copyright © 2016 Arthroscopy Association of North America. Published by Elsevier Inc. All rights reserved.

Isolation of the posterior left atrium for patients with persistent atrial fibrillation: routine adenosine challenge for dormant posterior left atrial conduction improves long-term outcome.

PubMed

McLellan, Alex J A; Prabhu, Sandeep; Voskoboinik, Alex; Wong, Michael C G; Walters, Tomos E; Pathik, Bhupesh; Morris, Gwilym M; Nisbet, Ashley; Lee, Geoffrey; Morton, Joseph B; Kalman, Jonathan M; Kistler, Peter M

2017-12-01

Catheter ablation to achieve posterior left atrial wall (PW) isolation may be performed as an adjunct to pulmonary vein isolation (PVI) in patients with persistent atrial fibrillation (AF). We aimed to determine whether routine adenosine challenge for dormant posterior wall conduction improved long-term outcome. A total of 161 patients with persistent AF (mean age 59 ± 9 years, AF duration 6 ± 5 years) underwent catheter ablation involving circumferential PVI followed by PW isolation. Posterior left atrial wall isolation was performed with a roof and inferior wall line with the endpoint of bidirectional block. In 54 patients, adenosine 15 mg was sequentially administered to assess reconnection of the pulmonary veins and PW. Sites of transient reconnection were ablated and adenosine was repeated until no further reconnection was present. Holter monitoring was performed at 6 and 12 months to assess for arrhythmia recurrence. Posterior left atrial wall isolation was successfully achieved in 91% of 161 patients (procedure duration 191 ± 49 min, mean RF time 40 ± 19 min). Adenosine-induced reconnection of the PW was demonstrated in 17%. The single procedure freedom from recurrent atrial arrhythmia was superior in the adenosine challenge group (65%) vs. no adenosine challenge (40%, P < 0.01) at a mean follow-up of 19 ± 8 months. After multiple procedures, there was significantly improved freedom from AF between patients with vs. without adenosine PW challenge (85 vs. 65%, P = 0.01). Posterior left atrial wall isolation in addition to PVI is a readily achievable ablation strategy in patients with persistent AF. Routine adenosine challenge for dormant posterior wall conduction was associated with an improvement in the success of catheter ablation for persistent AF. Published on behalf of the European Society of Cardiology. All rights reserved. © The Author 2017. For permissions please email: journals.permissions@oup.com.

Bioassay-guided isolation of wound healing active compounds from Echium species growing in Turkey.

PubMed

Eruygur, Nuraniye; Yılmaz, Gülderen; Kutsal, Osman; Yücel, Gözde; Üstün, Osman

2016-06-05

The roots and root barks of Echium sp. have been used to treat ulcers, burns and wounds in traditional Turkish medicine. On the basis of them traditional use and literature references, four Echium species were selected for evaluation of them wound healing potential. Isolation of active component(s) from the active extracts through the bioassay guided fractionation procedures. In vivo the wound healing activity of the plants was evaluated by linear incision experimental models. The chloroform extract of Echium italicum L. was fractionated by successive chromatographic techniques. Wound healing activity of each fraction was investigated following the bioassay-guided fractionation procedures. Moreover, the tissue samples of isolated compounds were examined histopathologically. The healing potential was comparatively assessed with a reference ointment Madecassol®, which contains 1% extract of Centella asiatica. Significant wound healing activity was observed from the ointment prepared with ethanol extract at 1% concentration. The ethanol root extract treated in groups of animals showed a significant increase (37.38%, 40.97% and 35.29% separately for E. italicum L, Echium vulgare L. and Echium angustifolium Miller) wound tensile strength in the incision wound model. Subfractions showed significant but reduced wound healing activity on in vivo wound models. Shikonin derivatives "Acetylshikonin", "Deoxyshikonin" and "2-methyl-n-butyrylshikonin+Isovalerylshikonin", were isolated and determined as active components of active final subfraction from E. italicum L. roots. The results of histopathological examination supported the outcome of linear incision wound models. The experimental study revealed that Echium species display remarkable wound healing activity. Copyright © 2016. Published by Elsevier Ireland Ltd.

An Investigation of the Cryogenic Freezing of Water in Non-Metallic Pipelines

NASA Astrophysics Data System (ADS)

Martin, C. I.; Richardson, R. N.; Bowen, R. J.

2004-06-01

Pipe freezing is increasingly used in a range of industries to solve otherwise intractable pipe line maintenance and servicing problems. This paper presents the interim results from an experimental study on deliberate freezing of polymeric pipelines. Previous and contemporary works are reviewed. The object of the current research is to confirm the feasibility of ice plug formation within a polymeric pipe as a method of isolation. Tests have been conducted on a range of polymeric pipes of various sizes. The results reported here all relate to freezing of horizontal pipelines. In each case the process of plug formation was photographed, the frozen plug pressure tested and the pipe inspected for signs of damage resulting from the freeze procedure. The time to freeze was recorded and various temperatures logged. These tests have demonstrated that despite the poor thermal and mechanical properties of the polymers, freezing offers a viable alternative method of isolation in polymeric pipelines.

Purification of Proteins Fused to Maltose-Binding Protein.

PubMed

Lebendiker, Mario; Danieli, Tsafi

2017-01-01

Maltose-Binding Protein (MBP) is one of the most popular fusion partners being used for producing recombinant proteins in bacterial cells. MBP allows the use of a simple capture affinity step on Amylose-Agarose or Dextrin-Sepharose columns, resulting in a protein that is often 70-90 % pure in a single step. In addition to protein isolation applications, MBP provides a high degree of translation, and facilitates the proper folding and solubility of the target protein. This paper describes efficient procedures for isolating highly purified MBP target proteins. Special attention is given to considerations for downstream applications such as structural determination studies, protein activity assays, and assessing the chemical characteristics of the target protein.

An analytical procedure for evaluating shuttle abort staging aerodynamic characteristics

NASA Technical Reports Server (NTRS)

Meyer, R.

1973-01-01

An engineering analysis and computer code (AERSEP) for predicting Space Shuttle Orbiter - HO Tank longitudinal aerodynamic characteristics during abort separation has been developed. Computed results are applicable at Mach numbers above 2 for angle-of-attack between plus or minus 10 degrees. No practical restrictions on orbiter-tank relative positioning are indicated for tank-under-orbiter configurations. Input data requirements and computer running times are minimal facilitating program use for parametric studies, test planning, and trajectory analysis. In a majority of cases AERSEP Orbiter-Tank interference predictions are as accurate as state-of-the-art estimates for interference-free or isolated-vehicle configurations. AERSEP isolated-orbiter predictions also show excellent correlation with data.

Quantitative Field Testing Rotylenchulus reniformis DNA from Metagenomic Samples Isolated Directly from Soil

PubMed Central

Showmaker, Kurt; Lawrence, Gary W.; Lu, Shien; Balbalian, Clarissa; Klink, Vincent P.

2011-01-01

A quantitative PCR procedure targeting the β-tubulin gene determined the number of Rotylenchulus reniformis Linford & Oliveira 1940 in metagenomic DNA samples isolated from soil. Of note, this outcome was in the presence of other soil-dwelling plant parasitic nematodes including its sister genus Helicotylenchus Steiner, 1945. The methodology provides a framework for molecular diagnostics of nematodes from metagenomic DNA isolated directly from soil. PMID:22194958

Isolation of monomeric photosystem II that retains the subunit PsbS.

PubMed

Haniewicz, Patrycja; De Sanctis, Daniele; Büchel, Claudia; Schröder, Wolfgang P; Loi, Maria Cecilia; Kieselbach, Thomas; Bochtler, Matthias; Piano, Dario

2013-12-01

Photosystem II has been purified from a transplastomic strain of Nicotiana tabacum according to two different protocols. Using the procedure described in Piano et al. (Photosynth Res 106:221-226, 2010) it was possible to isolate highly active PSII composed of monomers and dimers but depleted in their PsbS protein content. A "milder" procedure than the protocol reported by Fey et al. (Biochim Biophys Acta 1777:1501-1509, 2008) led to almost exclusively monomeric PSII complexes which in part still bind the PsbS protein. This finding might support a role for PSII monomers in higher plants.

Electrical neuromodulation for disabling angina pectoris related to isolated stenoses of small epicardial coronary arteries.

PubMed

Jessurun, G A; Hautvast, R W; DeJongste, M J; Meyler, W J; van Boven AJ; Crijns, H J

1999-07-01

Patients with symptomatic small vessel coronary artery disease may be inadequate candidates for revascularization procedures. They may suffer from refractory angina, which does not respond to maximal anti-anginal drug therapy. In addition to patients with end stage coronary artery disease and syndrome X, this newly defined group of subjects with an isolated stenosis of a small coronary artery may benefit from electrical neurostimulation. We describe two patients with intractable angina caused by a significant narrowing of a diagonal branch. This treatment modality should be considered as an alternative method for unsatisfactory revascularization procedures.

[Cytostatic hyperthermic isolated limb perfusion (HILP) in VFN (General Faculty Hospital in Prague)].

PubMed

Spacek, M; Mitás, P; Lacina, L; Krajsová, I; Hodková, G; Salmay, M; Spunda, R; Brlicová, L; Lindner, J

2011-01-01

Hyperthermic isolated limb perfusion (HILP) is a standardized method of treatment in selected patients with in-transient locoregional recurrence/methastasis of melanoma or, some other soft tissue tumors (incl. sarcoma etc.) Authors present history and current status of this treatment modality in General University Hospital in Prague. During one year period (7/2009-6/2010) 10 patients were indicated for this procedure. We performed 13 procedures (3x redo), 11 in lower extremity and 2 in upper extremity. There was no serious complication in this cohort of patiens. Multidisciplinar approach is indicated in melanoma patients care.

[Differentiation of Staphylococcus aureus isolates based on phenotypical characters].

PubMed

Miedzobrodzki, Jacek; Małachowa, Natalia; Markiewski, Tomasz; Białecka, Anna; Kasprowicz, Andrzej

2008-06-30

Typing of Staphylococcus aureus isolates is a necessary procedure for monitoring the transmission of S. aureus among carriers and in epidemiology. Evaluation of the range of relationship among isolates rely on epidemiological markers and is possible because of the clonal character of S. aureus species. Effective typing shows the scheme of transmission of infection in a selected area, enables identifying the reservoir of the microorganism, and may enhance effective eradication. A set of typing methods for use in analyses of epidemiological correlations and the identification of S. aureus isolates is presented. The following methods of typing are described: biotyping, serotyping, antibiogram, protein electrophoresis, cell protein profiles (proteom), immunoblotting, multilocus enzyme electrophoresis (MLEE), zymotyping, and standard species identification of S. aureus in the diagnostic laboratory. Phenotyping methods for S. aureus isolates used in the past and today in epidemiological investigations and in analyses of correlations among S. aureus isolates are presented in this review. The presented methods use morphological characteristics, physiological properties, and chemical structures of the bacteria as criteria for typing. The precision of these standard methods is not always satisfactory as S. aureus strains with atypical biochemical characters have evolved recently. Therefore it is essential to introduce additional typing procedures using molecular biology methods without neglecting phenotypic methods.

Impact of different isolation procedures on the functionality of zein and kafirin

USDA-ARS?s Scientific Manuscript database

Commercial corn prolamin (zein) aggregates in water above room temperature into an extensible, viscoelastic gluten-like substance. This specific functionality of zein can be used in the production of superior gluten-free bread. The present study examined laboratory-scale isolation of such functional...

9 CFR 3.110 - Veterinary care.

Code of Federal Regulations, 2013 CFR

2013-01-01

... Mammals Animal Health and Husbandry Standards § 3.110 Veterinary care. (a) Newly acquired marine mammals must be isolated from resident marine mammals. Animals with a known medical history must be isolated... sanitizing procedures. Any marine mammal exposed to a contagious animal must be evaluated by the attending...

Isolation of the most immunoreactive antigenes of echinococcus granulosus from sheep hydatid fluid.

PubMed

Pozzuoli, R; Piantelli, M; Perucci, C; Arru, E; Musiani, P

1975-11-01

This paper describes a simplified procedure for obtaining purified Echinococcus granulosus antigens from sheep hydatid fluid by using affinity chromatography on concanavalin A-Sepharose. The presence of two "major" antigens (4 and 5) was confirmed. Antigen 5 was isolated by preparative polyacrylamide gel electrophoresis. Antigen 4, eluted by diffusion from the gel, was seen to be "contaminated" by antigen 5 and was isolated by using anti-5 Sepharose-linked serum. These two major antigens were then tested separately against the sera of hydatidosis patients by using very simple immunolgic tests. The best results were obtained in passive hemagglutination with antigen 4. Antigen 4 is the most immunoreactive parasitic antigen; antibodies against it were found in the sera of all hydatidosis patients showing positive reaction. Apart from the direct use of this antigen in serologic tests, it appears possible to standarize the most frequently used and commerically available antigenic materials by titrating this component.

Isolation of bacterial metabolites as natural inducers for larval settlement in the marine polychaete Hydroides elegans (Haswell).

PubMed

Harder, Tilmann; Lau, Stanley Chun Kwan; Dahms, Hans-Uwe; Qian, Pei-Yuan

2002-10-01

The bacterial component of marine biofilms plays an important role in the induction of larval settlement in the polychaete Hydroides elegans. In this study, we provide experimental evidence that bacterial metabolites comprise the chemical signal for larval settlement. Bacteria were isolated from biofilms, purified and cultured according to standard procedures. Bacterial metabolites were isolated from spent culture broth by chloroform extraction as well as by closed-loop stripping and adsorption of volatile components on surface-modified silica gel. A pronounced biological activity was exclusively observed when concentrated metabolites were adsorbed on activated charcoal. Larvae did not respond to waterbome metabolites when prevented from contacting the bacterial film surface. These results indicate that an association of the chemical signal with a sorbent-like substratum may be an essential cofactor for the expression of biological activity. The functional role of bacterial exopolymers as an adsorptive matrix for larval settlement signals is discussed.

Facile mechanical shaking method is an improved isolation approach for islet preparation and transplantation.

PubMed

Yin, Nina; Chen, Tao; Yu, Yuling; Han, Yongming; Yan, Fei; Zheng, Zhou; Chen, Zebin

2016-12-01

Successful islet isolation is crucial for islet transplantation and cell treatment for type 1 diabetes. Current isolation methods are able to obtain 500-1,000 islets per rat, which results in a waste of ≥50% of total islets. In the present study, a facile mechanical shaking method for improving islet yield (up to 1,500 per rat) was developed and summarized, which was demonstrated to be more effective than the existing well-established stationary method. The present results showed that isolated islets have a maximum yield of 1,326±152 when shaking for 15 min for the fully-cannulated pancreas. For both fully-cannulated and half-cannulated pancreas in the presence of rat DNAse inhibitor, the optimal shaking time was amended to 20 min with a further increased yield of 1,344±134 and 1,286±124 islets, respectively. Furthermore, the majority of the isolated islets were morphologically intact with a well-defined surface and almost no central necrotic zone, which suggested that the condition of islets obtained via the mechanical shaking method was consistent with the stationary method. Islet size distribution was also calculated and it was demonstrated that islets from the stationary method exhibited the same size distribution as the non-cannulated group, which had more larger islets than the fully-cannulated and half-cannulated groups isolated via the shaking method. In addition, the results of glucose challenge showed that the refraction index of each group was >2.5, which indicated the well-preserved function of isolated islets. Furthermore, the transplanted islets exhibited a therapeutic effect after 1 day of transplantation; however, they failed to control blood glucose levels after ~7 days of transplantation. In conclusion, these results demonstrated that the facile mechanical shaking method may markedly improve the yield of rat islet isolation, and in vitro and in vivo investigation demonstrated the well-preserved function of isolated islets in the control of blood glucose. Therefore, the facile mechanical shaking method may be an alternative improved procedure to obtain higher islet yield for islet preparation and transplantation in the treatment of type 1 diabetes.

Preanalytical blood sample workup for cell-free DNA analysis using Droplet Digital PCR for future molecular cancer diagnostics.

PubMed

van Ginkel, Joost H; van den Broek, Daan A; van Kuik, Joyce; Linders, Dorothé; de Weger, Roel; Willems, Stefan M; Huibers, Manon M H

2017-10-01

In current molecular cancer diagnostics, using blood samples of cancer patients for the detection of genetic alterations in plasma (cell-free) circulating tumor DNA (ctDNA) is an emerging practice. Since ctDNA levels in blood are low, highly sensitive Droplet Digital PCR (ddPCR) can be used for detecting rare mutational targets. In order to perform ddPCR on blood samples, a standardized procedure for processing and analyzing blood samples is necessary to facilitate implementation into clinical practice. Therefore, we assessed the technical sample workup procedure for ddPCR on blood plasma samples. Blood samples from healthy individuals, as well as lung cancer patients were analyzed. We compared different methods and protocols for sample collection, storage, centrifugation, isolation, and quantification. Cell-free DNA (cfDNA) concentrations of several wild-type targets and BRAF and EGFR-mutant ctDNA concentrations quantified by ddPCR were primary outcome measurements. Highest cfDNA concentrations were measured in blood collected in serum tubes. No significant differences in cfDNA concentrations were detected between various time points of up to 24 h until centrifugation. Highest cfDNA concentrations were detected after DNA isolation with the Quick cfDNA Serum & Plasma Kit, while plasma isolation using the QIAamp Circulating Nucleic Acid Kit yielded the most consistent results. DdPCR results on cfDNA are highly dependent on multiple factors during preanalytical sample workup, which need to be addressed during the development of this diagnostic tool for cancer diagnostics in the future. © 2017 The Authors. Cancer Medicine published by John Wiley & Sons Ltd.

Environmental and human monitoring of Americium-241 utilizing extraction chromatography and alpha-spectrometry.

PubMed

Goldstein, S J; Hensley, C A; Armenta, C E; Peters, R J

1997-03-01

Recent developments in extraction chromatography have simplified the separation of americium from complex matrices in preparation for alpha-spectroscopy relative to traditional methods. Here we present results of procedures developed/adapted for water, air, and bioassay samples with less than 1 g of inorganic residue. Prior analytical methods required the use of a complex, multistage procedure for separation of americium from these matrices. The newer, simplified procedure requires only a single 2 mL extraction chromatographic separation for isolation of Am and lanthanides from other components of the sample. This method has been implemented on an extensive variety of "real" environmental and bioassay samples from the Los Alamos area, and consistently reliable and accurate results with appropriate detection limits have been obtained. The new method increases analytical throughput by a factor of approximately 2 and decreases environmental hazards from acid and mixed-waste generation relative to the prior technique. Analytical accuracy, reproducibility, and reliability are also significantly improved over the more complex and laborious method used previously.

Development of a multiple-parameter nonlinear perturbation procedure for transonic turbomachinery flows: Preliminary application to design/optimization problems

NASA Technical Reports Server (NTRS)

Stahara, S. S.; Elliott, J. P.; Spreiter, J. R.

1983-01-01

An investigation was conducted to continue the development of perturbation procedures and associated computational codes for rapidly determining approximations to nonlinear flow solutions, with the purpose of establishing a method for minimizing computational requirements associated with parametric design studies of transonic flows in turbomachines. The results reported here concern the extension of the previously developed successful method for single parameter perturbations to simultaneous multiple-parameter perturbations, and the preliminary application of the multiple-parameter procedure in combination with an optimization method to blade design/optimization problem. In order to provide as severe a test as possible of the method, attention is focused in particular on transonic flows which are highly supercritical. Flows past both isolated blades and compressor cascades, involving simultaneous changes in both flow and geometric parameters, are considered. Comparisons with the corresponding exact nonlinear solutions display remarkable accuracy and range of validity, in direct correspondence with previous results for single-parameter perturbations.

Quantitative Analysis of Localized Sources Identified by Focal Impulse and Rotor Modulation Mapping in Atrial Fibrillation

PubMed Central

Benharash, Peyman; Buch, Eric; Frank, Paul; Share, Michael; Tung, Roderick; Shivkumar, Kalyanam; Mandapati, Ravi

2015-01-01

Background New approaches to ablation of atrial fibrillation (AF) include focal impulse and rotor modulation (FIRM) mapping, and initial results reported with this technique have been favorable. We sought to independently evaluate the approach by analyzing quantitative characteristics of atrial electrograms used to identify rotors and describe acute procedural outcomes of FIRM-guided ablation. Methods and Results All FIRM-guided ablation procedures (n=24; 50% paroxysmal) at University of California, Los Angeles Medical Center were included for analysis. During AF, unipolar atrial electrograms collected from a 64-pole basket catheter were used to construct phase maps and identify putative AF sources. These sites were targeted for ablation, in conjunction with pulmonary vein isolation in most patients (n=19; 79%). All patients had rotors identified (mean, 2.3±0.9 per patient; 72% in left atrium). Prespecified acute procedural end point was achieved in 12 of 24 (50%) patients: AF termination (n=1), organization (n=3), or >10% slowing of AF cycle length (n=8). Basket electrodes were within 1 cm of 54% of left atrial surface area, and a mean of 31 electrodes per patient showed interpretable atrial electrograms. Offline analysis revealed no differences between rotor and distant sites in dominant frequency or Shannon entropy. Electroanatomic mapping showed no rotational activation at FIRM-identified rotor sites in 23 of 24 patients (96%). Conclusions FIRM-identified rotor sites did not exhibit quantitative atrial electrogram characteristics expected from rotors and did not differ quantitatively from surrounding tissue. Catheter ablation at these sites, in conjunction with pulmonary vein isolation, resulted in AF termination or organization in a minority of patients (4/24; 17%). Further validation of this approach is necessary. PMID:25873718

Mould routine identification in the clinical laboratory by matrix-assisted laser desorption ionization time-of-flight mass spectrometry.

PubMed

Cassagne, Carole; Ranque, Stéphane; Normand, Anne-Cécile; Fourquet, Patrick; Thiebault, Sandrine; Planard, Chantal; Hendrickx, Marijke; Piarroux, Renaud

2011-01-01

MALDI-TOF MS recently emerged as a valuable identification tool for bacteria and yeasts and revolutionized the daily clinical laboratory routine. But it has not been established for routine mould identification. This study aimed to validate a standardized procedure for MALDI-TOF MS-based mould identification in clinical laboratory. First, pre-extraction and extraction procedures were optimized. With this standardized procedure, a 143 mould strains reference spectra library was built. Then, the mould isolates cultured from sequential clinical samples were prospectively subjected to this MALDI-TOF MS based-identification assay. MALDI-TOF MS-based identification was considered correct if it was concordant with the phenotypic identification; otherwise, the gold standard was DNA sequence comparison-based identification. The optimized procedure comprised a culture on sabouraud-gentamicin-chloramphenicol agar followed by a chemical extraction of the fungal colonies with formic acid and acetonitril. The identification was done using a reference database built with references from at least four culture replicates. For five months, 197 clinical isolates were analyzed; 20 were excluded because they were not identified at the species level. MALDI-TOF MS-based approach correctly identified 87% (154/177) of the isolates analyzed in a routine clinical laboratory activity. It failed in 12% (21/177), whose species were not represented in the reference library. MALDI-TOF MS-based identification was correct in 154 out of the remaining 156 isolates. One Beauveria bassiana was not identified and one Rhizopus oryzae was misidentified as Mucor circinelloides. This work's seminal finding is that a standardized procedure can also be used for MALDI-TOF MS-based identification of a wide array of clinically relevant mould species. It thus makes it possible to identify moulds in the routine clinical laboratory setting and opens new avenues for the development of an integrated MALDI-TOF MS-based solution for the identification of any clinically relevant microorganism.

A simple, convenient method to synthesize cobalamins: synthesis of homocysteinylcobalamin, N -acetylcysteinylcobalamin, 2-N -acetylamino-2-carbomethoxyethanethiolatocobalamin, sulfitocobalamin and nitrocobalamin†

PubMed Central

Suarez-Moreira, Edward; Hannibal, Luciana; Smith, Clyde A.; Chavez, Roberto A.; Jacobsen, Donald W.

2009-01-01

Glutathionylcobalamin, nitrocobalamin and sulfitocobalamin are important cobalamin metabolites isolable from human tissues. Herein we demonstrate that a procedure used to synthesize and isolate γ-glutamylcysteinylcobalamin and glutathionylcobalamin in aqueous solution in high yield and purity can be used to synthesize other novel, biologically relevant thiolatocobalamins, including D,L-homocysteinylcobalamin, N-acetyl-L-cysteinylcobalamin (Na+ salt) and 2-N-acetylamino-2-carbomethoxy-L-ethanethiolatocobalamin, as well as other non-alkylcobalamins, such as sulfitocobalamin (Na+ salt) and nitrocobalamin. This uncomplicated, general procedure will assist researchers in identifying unknown cobalamin metabolites isolated from biological samples, and researchers interested in studying the uptake and intracellular cobalamin processing mechanisms utilizing non-alkylcobalamin derivatives that are not yet commercially available. The X-ray structure and XAS spectrum of N-acetyl-L-cysteinylcobalamin are also presented. PMID:17088966

Aseptic Laboratory Techniques: Plating Methods

PubMed Central

Sanders, Erin R.

2012-01-01

Microorganisms are present on all inanimate surfaces creating ubiquitous sources of possible contamination in the laboratory. Experimental success relies on the ability of a scientist to sterilize work surfaces and equipment as well as prevent contact of sterile instruments and solutions with non-sterile surfaces. Here we present the steps for several plating methods routinely used in the laboratory to isolate, propagate, or enumerate microorganisms such as bacteria and phage. All five methods incorporate aseptic technique, or procedures that maintain the sterility of experimental materials. Procedures described include (1) streak-plating bacterial cultures to isolate single colonies, (2) pour-plating and (3) spread-plating to enumerate viable bacterial colonies, (4) soft agar overlays to isolate phage and enumerate plaques, and (5) replica-plating to transfer cells from one plate to another in an identical spatial pattern. These procedures can be performed at the laboratory bench, provided they involve non-pathogenic strains of microorganisms (Biosafety Level 1, BSL-1). If working with BSL-2 organisms, then these manipulations must take place in a biosafety cabinet. Consult the most current edition of the Biosafety in Microbiological and Biomedical Laboratories (BMBL) as well as Material Safety Data Sheets (MSDS) for Infectious Substances to determine the biohazard classification as well as the safety precautions and containment facilities required for the microorganism in question. Bacterial strains and phage stocks can be obtained from research investigators, companies, and collections maintained by particular organizations such as the American Type Culture Collection (ATCC). It is recommended that non-pathogenic strains be used when learning the various plating methods. By following the procedures described in this protocol, students should be able to: ● Perform plating procedures without contaminating media. ● Isolate single bacterial colonies by the streak-plating method. ● Use pour-plating and spread-plating methods to determine the concentration of bacteria. ● Perform soft agar overlays when working with phage. ● Transfer bacterial cells from one plate to another using the replica-plating procedure. ● Given an experimental task, select the appropriate plating method. PMID:22617405

A Laccase with HIV-1 Reverse Transcriptase Inhibitory Activity from the Broth of Mycelial Culture of the Mushroom Lentinus tigrinus

PubMed Central

Xu, LiJing; Wang, HeXiang; Ng, TziBun

2012-01-01

A 59 kDa laccase with inhibitory activity against HIV-1 reverse transcriptase (IC50 = 2.4 μM) was isolated from the broth of mycelial culture of the mushroom Lentinus tigrinus. The isolation procedure involved ion exchange chromatography on DEAE-cellulose and CM-cellulose, and gel filtration by fast protein liquid chromatography on Superdex 75. The laccase was adsorbed on both types of ion exchangers. About 95-fold purification was achieved with a 25.9% yield of the enzyme. The procedure resulted in a specific enzyme activity of 76.6 U/mg. Its N-terminal amino acid sequence was GIPDLHDLTV, which showed little similarity to other mushroom laccase and other Lentinus tigrinus strain laccase. Its characteristics were different from previously reported laccase of other Lentinus tigrinus strain. Maximal laccase activity was observed at a pH of 4 and at a temperature of 60°C, respectively. This study yielded the information about the potentially exploitable activities of Lentinus tigrinus laccase. PMID:22536022

Contingency contracting with school problems

PubMed Central

Cantrell, Robert P.; Cantrell, Mary Lynn; Huddleston, Clifton M.; Wooldridge, Ralph L.

1969-01-01

Contingency contracting procedures used in managing problems with school-age children involved analyzing teacher and/or parental reports of behavior problem situations, isolating the most probable contingencies then in effect, the range of reinforcers presently available, and the ways in which they were obtained. The authors prepared written contracts delineating remediative changes in reinforcement contingencies. These contracts specified ways in which the child could obtain existing individualized reinforcers contingent upon approximations to desired appropriate behaviors chosen as incompatible with the referral problem behaviors. Contract procedures were administered by the natural contingency managers, parents and/or teachers, who kept daily records of contracted behaviors and reinforcers. These records were sent to the authors and provided feedback on the progress of the case. Initial results of this procedure have been sufficiently encouraging to warrant recommending an experimental analysis of contingency contracting as a clinical method. PMID:16795222

Isolation and Characterization of Poliovirus in Cell Culture Systems.

PubMed

Thorley, Bruce R; Roberts, Jason A

2016-01-01

The isolation and characterization of enteroviruses by cell culture was accepted as the "gold standard" by clinical virology laboratories. Methods for the direct detection of all enteroviruses by reverse transcription polymerase chain reaction, targeting a conserved region of the genome, have largely supplanted cell culture as the principal diagnostic procedure. However, the World Health Organization's Global Polio Eradication Initiative continues to rely upon cell culture to isolate poliovirus due to the lack of a reliable sensitive genetic test for direct typing of enteroviruses from clinical specimens. Poliovirus is able to infect a wide range of mammalian cell lines, with CD155 identified as the primary human receptor for all three seroytpes, and virus replication leads to an observable cytopathic effect. Inoculation of cell lines with extracts of clinical specimens and subsequent passaging of the cells leads to an increased virus titre. Cultured isolates of poliovirus are suitable for testing by a variety of methods and remain viable for years when stored at low temperature.This chapter describes general procedures for establishing a cell bank and routine passaging of cell lines. While the sections on specimen preparation and virus isolation focus on poliovirus, the protocols are suitable for other enteroviruses.

Rescue of Targeted Regions of Mammalian Chromosomes by in Vivo Recombination in Yeast

PubMed Central

Kouprina, Natalya; Kawamoto, Kensaku; Barrett, J. Carl; Larionov, Vladimir; Koi, Minoru

1998-01-01

In contrast to other animal cell lines, the chicken pre-B cell lymphoma line, DT40, exhibits a high level of homologous recombination, which can be exploited to generate site-specific alterations in defined target genes or regions. In addition, the ability to generate human/chicken monochromosomal hybrids in the DT40 cell line opens a way for specific targeting of human genes. Here we describe a new strategy for direct isolation of a human chromosomal region that is based on targeting of the chromosome with a vector containing a yeast selectable marker, centromere, and an ARS element. This procedure allows rescue of the targeted region by transfection of total genomic DNA into yeast spheroplasts. Selection for the yeast marker results in isolation of chromosome sequences in the form of large circular yeast artificial chromosomes (YACs) up to 170 kb in size containing the targeted region. These YACs are generated by homologous recombination in yeast between common repeated sequences in the targeted chromosomal fragment. Alternatively, the targeted region can be rescued as a linear YACs when a YAC fragmentation vector is included in the yeast transformation mixture. Because the entire isolation procedure of the chromosomal region, once a target insertion is obtained, can be accomplished in ∼1 week, the new method greatly expands the utility of the homologous recombinationproficient DT40 chicken cell system. PMID:9647640

Direct isolation of labeled low density lipoproteins for the determination of cholesteryl ester transfer protein activity.

PubMed

Sich, D; Saïdi, Y; Egloff, M; Giral, P; Gautier, V; Federspiel, M C; Turpin, G; Beucler, I

1997-10-31

The measurement of the activity of cholesteryl ester transfer protein (CETP), is of high clinical interest and this study reports the use of a direct LDL isolation (d-LDL) technique to determine in one step the amount of radiolabeled cholesteryls esters ([3H]-CE) transferred from exogenous HDL3 to LDL, avoiding the conveniences of the usually used ultracentrifugation or precipitation of apo-B containing lipoproteins in the CETP methodologies. The d-LDL technique providing a specific immunoprecipitation of VLDL, IDL and HDL allowed to directly determine the [3H]-CE transferred on LDL (d-[3H]-CE-LDL). Two methodologies were assayed for the CETP activity using either exogenous or endogenous lipoproteins, and the results with the d-LDL technique were compared with those obtained using the ultracentrifugation (u-[3H]-CE-LDL) considered as the reference method. The intra- and inter-assays were similar in both techniques for the two CETP activity assays. Strong positive correlations were established between values obtained with d-[3H]-CE-LDL and u-[3H]-CE-LDL isolation procedures for CETP activities with exogenous or endogenous lipoproteins (r = 0.972; p = 0.0001 and r = 0.965; p = 0.0001 respectively). In conclusion, the d-LDL technique represents an easy and accurate procedure to measure directly, in normotriglyceridemic plasmas, the amount of [3H]-CE transferred from HDL to LDL by the CETP.

Unpaired Shocks during Extinction Weaken the Contextual Renewal of a Conditioned Discrimination

ERIC Educational Resources Information Center

Vervliet, Bram; Vansteenwegen, Deb; Hermans, Dirk

2010-01-01

Extinction is generally more fragile than conditioning, as illustrated by the contextual renewal effect. The traditional extinction procedure entails isolated presentations of the conditioned stimulus. Extinction may be boosted by adding isolated presentations of the unconditioned stimulus, as this should augment breaking the contingency between…

Isolation of noninhibitory strains of Zymomonas mobilis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Haffie, T.L.; Louie, P.W.; Khachatourians, G.G.

1985-04-01

Wild-type Zymomonas mobilis strains inhibit the growth of Escherichia coli. The authors report the first isolation of noninhibitory strains, called Zymomonas inhibition negative (Zin/sup -/), after treatment with N-methyl-N'-nitro-N-nitrosoguanidine. A standardized soft-agar overlay procedure for detecting E. coli growth inhibition was also developed.

TOXICITY CHARACTERIZATION PROCEDURES FOR ORGANIC TOXICANTS IN BULK SEDIMENTS

EPA Science Inventory

We have been pursuing development of toxicant characterization, isolation, and identification procedures for organic toxicants that can be applied in the context of 10-d solid-phase sediment tests measuring survival and growth of freshwater in the context of 10-d solid-phase sedi...

Exosome-like vesicles in uterine aspirates: a comparison of ultracentrifugation-based isolation protocols.

PubMed

Campoy, Irene; Lanau, Lucia; Altadill, Tatiana; Sequeiros, Tamara; Cabrera, Silvia; Cubo-Abert, Montserrat; Pérez-Benavente, Assumpción; Garcia, Angel; Borrós, Salvador; Santamaria, Anna; Ponce, Jordi; Matias-Guiu, Xavier; Reventós, Jaume; Gil-Moreno, Antonio; Rigau, Marina; Colas, Eva

2016-06-18

Uterine aspirates are used in the diagnostic process of endometrial disorders, yet further applications could emerge if its complex milieu was simplified. Exosome-like vesicles isolated from uterine aspirates could become an attractive source of biomarkers, but there is a need to standardize isolation protocols. The objective of the study was to determine whether exosome-like vesicles exist in the fluid fraction of uterine aspirates and to compare protocols for their isolation, characterization, and analysis. We collected uterine aspirates from 39 pre-menopausal women suffering from benign gynecological diseases. The fluid fraction of 27 of those aspirates were pooled and split into equal volumes to evaluate three differential centrifugation-based procedures: (1) a standard protocol, (2) a filtration protocol, and (3) a sucrose cushion protocol. Characterization of isolated vesicles was assessed by electron microscopy, nanoparticle tracking analysis and immunoblot. Specifically for RNA material, we evaluate the effect of sonication and RNase A treatment at different steps of the protocol. We finally confirmed the efficiency of the selected methods in non-pooled samples. All protocols were useful to isolate exosome-like vesicles. However, the Standard procedure was the best performing protocol to isolate exosome-like vesicles from uterine aspirates: nanoparticle tracking analysis revealed a higher concentration of vesicles with a mode of 135 ± 5 nm, and immunoblot showed a higher expression of exosome-related markers (CD9, CD63, and CD81) thus verifying an enrichment in this type of vesicles. RNA contained in exosome-like vesicles was successfully extracted with no sonication treatment and exogenous nucleic acids digestion with RNaseA, allowing the analysis of the specific inner cargo by Real-Time qPCR. We confirmed the existence of exosome-like vesicles in the fluid fraction of uterine aspirates. They were successfully isolated by differential centrifugation giving sufficient proteomic and transcriptomic material for further analyses. The Standard protocol was the best performing procedure since the other two tested protocols did not ameliorate neither yield nor purity of exosome-like vesicles. This study contributes to establishing the basis for future comparative studies to foster the field of biomarker research in gynecology.

Chemical constituents of Aspergillus sp EJC08 isolated as endophyte from Bauhinia guianensis and their antimicrobial activity.

PubMed

Pinheiro, Eduardo A A; Carvalho, Josiwander M; Santos, Diellem C P dos; Feitosa, André O; Marinho, Patrícia S B; Guilhon, Giselle Maria S P; Santos, Lourivaldo S; Souza, Afonso L D de; Marinho, Andrey M R

2013-01-01

The present work reports the isolation of five compounds from Aspergillus sp EJC08 isolated as endophytic from Bauhinia guianensis, a tipical plant of the Amazon. The compounds ergosterol (1), ergosterol peroxide (2), mevalolactone (3), monomethylsulochrin (4) and trypacidin A (5) were isolated by chromatographic procedures and identified by spectral methods of 1D and 2D NMR and MS. Compounds 3, 4 and 5 were tested against Escherichia coli, Pseudomonas aeruginosa, Bacillus subtilis and Staphylococcus aureus and showed good activity.

Differentiation among isolates of prunus necrotic ringspot virus by transcript conformation polymorphism.

PubMed

Rosner, A; Maslenin, L; Spiegel, S

1998-09-01

A method based on differences in electrophoretic mobility of RNA transcripts made from polymerase chain reaction (PCR) products was used for differentiation among virus isolates. A T7 RNA polymerase promoter was attached to amplified prunus necrotic ringspot virus (PNRSV) sequences by PCR. The PCR products then served as a template for transcription. Single-stranded transcripts originated from different PNRSV isolates varied in electrophoretic mobility in polyacrylamide gels, presumably because of transcript conformation polymorphism (TCP). This procedure was applied for the differentiation of PNRSV isolates.

Selective isolation of hydrophobin SC3 by solid-phase extraction with polytetrafluoroethylene microparticles and subsequent mass spectrometric analysis.

PubMed

Kupčík, Rudolf; Zelená, Miroslava; Řehulka, Pavel; Bílková, Zuzana; Česlová, Lenka

2016-02-01

Hydrophobins are small proteins that play a role in a number of processes during the filamentous fungi growth and development. These proteins are characterized by the self-assembly of their molecules into an amphipathic membrane at hydrophilic-hydrophobic interfaces. Isolation and purification of hydrophobins generally present a challenge in their analysis. Hydrophobin SC3 from Schizophyllum commune was selected as a representative of class I hydrophobins in this work. A novel procedure for selective and effective isolation of hydrophobin SC3 based on solid-phase extraction with polytetrafluoroethylene microparticles loaded in a small self-made microcolumn is reported. The tailored binding of hydrophobins to polytetrafluoroethylene followed by harsh elution conditions resulted in a highly specific isolation of hydrophobin SC3 from the model mixture of ten proteins. The presented isolation protocol can have a positive impact on the analysis and utilization of these proteins including all class I hydrophobins. Hydrophobin SC3 was further subjected to reduction of its highly stable disulfide bonds and to chymotryptic digestion followed by mass spectrometric analysis. The isolation and digestion protocols presented in this work make the analysis of these highly hydrophobic and compact proteins possible. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Characterization of bacterial etiologic agents of biofilm formation in medical devices in critical care setup.

PubMed

Revdiwala, Sangita; Rajdev, Bhaumesh M; Mulla, Summaiya

2012-01-01

Background. Biofilms contaminate catheters, ventilators, and medical implants; they act as a source of disease for humans, animals, and plants. Aim. Critical care units of any healthcare institute follow various interventional strategies with use of medical devices for the management of critical cases. Bacteria contaminate medical devices and form biofilms. Material and Methods. The study was carried out on 100 positive bacteriological cultures of medical devices which were inserted in hospitalized patients. The bacterial isolates were processed as per microtitre plate. All the isolates were subjected to antibiotic susceptibility testing by VITEK 2 compact automated systems. Results. Out of the total 100 bacterial isolates tested, 88 of them were biofilm formers. A 16-20-hour incubation period was found to be optimum for biofilm development. 85% isolates were multidrug resistants and different mechanisms of bacterial drug resistance like ESBL, carbapenemase, and MRSA were found among isolates. Conclusion. Availability of nutrition in the form of glucose enhances the biofilm formation by bacteria. Time and availability of glucose are important factors for assessment of biofilm progress. It is an alarm for those who are associated with invasive procedures and indwelling medical devices especially in patients with low immunity.

Single-incision laparoscopic surgery and conventional laparoscopic treatment of varicocele in adolescents: Comparison between two techniques.

PubMed

Marte, Antonio; Pintozzi, Lucia; Cavaiuolo, Silvia; Parmeggiani, Pio

2014-01-01

Single-incision laparoscopic surgery (SILS) has gained great popularity in paediatric surgery due to its minimally invasive approach and improved cosmetic results. Notwithstanding, reports describing its adoption in children are still fragmentary and some perplexities have been raised by some surgeons. We reviewed our experience with the SILS Palomo varicocelectomy procedure (SIL-V) in children and adolescents, comparing this group with a similar series operated using conventional laparoscopic varicocelectomy (CL-V). A total of 69 Palomo laparoscopic varicocelectomies were performed in patients aged 11-17 years from January 2011 to January 2013. Indications for surgery included grades II-III varicocele or ipsilateral testicular hypotrophy. The SIL-V procedure was performed in 44 patients with roticulating and conventional 5 mm instruments. Testicular vessels were isolated "en bloc," clipped and cut. Operating time, visual analogue scale and post-operative results were compared to a similar group of 25 patients operated with CL-V. No patient of the SIL-V group required conversion to conventional laparoscopy, none to open surgery. Mean operative time was 22 min (range: 19-28) in the SIL-V group, not significantly different compared with CL-V (mean 21 min, range: 18-25). All patients experienced a smooth recovery from surgery without any complications, and were discharged on day 1. No difficulties were found in the SIL-V group. The post-operative pain score was significantly better in SIL-V. The SIL-V procedure is safe and effective and allows a fast and efficient isolation of the vascular bundle. The use of conventional instruments is technically feasible in SIL-V.

Comparison of a modified shell vial culture procedure with conventional mouse inoculation for rabies virus isolation.

PubMed

Ribas Antúnez, María de los Angeles; Girón, Blanca; Monsalvez, Iraima; Morier, Luis; Acosta, Gretel; Tejero, Yahisel; Cordero, Yanislet; Piedra, Dainelyd

2013-04-01

Rabies is a neurotropic disease that is often lethal. The early diagnosis of rabies infection is important and requires methods that allow for the isolation of the virus from animals and humans. The present study compared a modified shell vial (MSV) procedure using 24-well tissue culture plates with the mouse inoculation test (MIT), which is considered the gold standard for rabies virus isolation. Thirty brain samples (25 positive and 5 negative by the fluorescent antibody test) obtained from different animal species at the National Institute of Hygiene Rafael Rangel in Caracas, Venezuela, were studied by the MIT and MSV assays. Nine samples (36%) were positive at 24 h, 10 (40%) were positive at 48 h and six (24%) were positive at 72 h by the MSV assay. With the MIT assay, 76% were positive at six days post inoculation and 12% were positive at 12 and 18 days post inoculation. One sample that was negative according to the MSV assay was positive with MIT on the 12th day. The MSV procedure exhibited a sensitivity of 96.2%, a specificity of 100%, a positive predictive value of 100% and a negative predictive value 80%. This procedure allowed for rapid rabies virus detection. MIT can be employed as an alternative method in laboratories without tissue culture facilities.

A Signal Detection Model of Compound Decision Tasks

DTIC Science & Technology

2006-12-01

strict isolation (for many examples of such models see Egan, 1975; Macmillan & Creelman , 1991). The result has been twofold: A rich corpus of decision...Macmillan & Creelman , 1991). It is important to point out that SDT models are primarily decision models. They specify the rules and procedures for how...Broadbent, 1958; Macmillan & Creelman , 1991; Nolte & Jaarsma, 1967; Swensson & Judy, 1981; Tanner & Norman, 1954). To better understand how these two

A serological procedure for identifying strains of Germmeniella abietina.

Treesearch

Darroll D. Skilling; Mariann Kienzler

1983-01-01

This manual gives detailed laboratory serology procedures necessary to determine the identity of isolates of Gremmeniela abientina by the gel double diffusion method. The process is described from the arrival of the field sample to the reading of the precipitin bands on the diffusion plates.

Sensitive and Rapid Identification of Biological Threat Agents

DTIC Science & Technology

1999-12-01

plague, and Bruceila abortis , the etiologic agent of brucellosis, indicate that the Qiagen procedures will also be effective in preparing these agents for...procedure for two bacteria, Yersinia pestis and Bruceila abortis , and found that DNA isolated with this method yielded PCR detection limits at least

Balloon pulmonary valvotomy--not just a simple balloon dilatation.

PubMed

Mohanty, Subhendu; Pandit, Bhagya Narayan; Tyagi, Sanjay

2014-01-01

Balloon pulmonary valvotomy is the preferred mode of treatment in patients with isolated pulmonary valvar stenosis and has shown good long term results. It is generally considered a safe procedure with few complications. There have been however, case reports of potentially fatal acute severe pulmonary edema occurring after the procedure in some patients. The cause of this complication and its pathophysiology is still not clear. Its occurrence is also infrequent with less than 5 cases reported till now. We report a case of pulmonary valvar stenosis which developed acute severe refractory pulmonary edema immediately after balloon pulmonary valvotomy. Copyright © 2014 Cardiological Society of India. Published by Elsevier B.V. All rights reserved.

Study of dispersed small wind systems interconnected with a utility distribution system

NASA Astrophysics Data System (ADS)

Curtice, D.; Patton, J.; Bohn, J.; Sechan, N.

1980-03-01

Operating problems for various penetrations of small wind systems connected to the distribution system on a utility are defined. Protection equipment, safety hazards, feeder voltage regulation, line losses, and voltage flicker problems are studied, assuming different small wind systems connected to an existing distribution system. To identify hardware deficiencies, possible solutions provided by off-the-shelf hardware and equipment are assessed. Results of the study indicate that existing techniques are inadequate for detecting isolated operation of a small wind system. Potential safety hazards posed by small wind systems are adequately handled by present work procedures although these procedures require a disconnect device at synchronous generator and self commutated inverter small wind systems.

Reynolds-Averaged Turbulence Model Assessment for a Highly Back-Pressured Isolator Flowfield

NASA Technical Reports Server (NTRS)

Baurle, Robert A.; Middleton, Troy F.; Wilson, L. G.

2012-01-01

The use of computational fluid dynamics in scramjet engine component development is widespread in the existing literature. Unfortunately, the quantification of model-form uncertainties is rarely addressed with anything other than sensitivity studies, requiring that the computational results be intimately tied to and calibrated against existing test data. This practice must be replaced with a formal uncertainty quantification process for computational fluid dynamics to play an expanded role in the system design, development, and flight certification process. Due to ground test facility limitations, this expanded role is believed to be a requirement by some in the test and evaluation community if scramjet engines are to be given serious consideration as a viable propulsion device. An effort has been initiated at the NASA Langley Research Center to validate several turbulence closure models used for Reynolds-averaged simulations of scramjet isolator flows. The turbulence models considered were the Menter BSL, Menter SST, Wilcox 1998, Wilcox 2006, and the Gatski-Speziale explicit algebraic Reynolds stress models. The simulations were carried out using the VULCAN computational fluid dynamics package developed at the NASA Langley Research Center. A procedure to quantify the numerical errors was developed to account for discretization errors in the validation process. This procedure utilized the grid convergence index defined by Roache as a bounding estimate for the numerical error. The validation data was collected from a mechanically back-pressured constant area (1 2 inch) isolator model with an isolator entrance Mach number of 2.5. As expected, the model-form uncertainty was substantial for the shock-dominated, massively separated flowfield within the isolator as evidenced by a 6 duct height variation in shock train length depending on the turbulence model employed. Generally speaking, the turbulence models that did not include an explicit stress limiter more closely matched the measured surface pressures. This observation is somewhat surprising, given that stress-limiting models have generally been developed to better predict shock-separated flows. All of the models considered also failed to properly predict the shape and extent of the separated flow region caused by the shock boundary layer interactions. However, the best performing models were able to predict the isolator shock train length (an important metric for isolator operability margin) to within 1 isolator duct height.

Contamination of X-ray Cassettes with Methicillin-resistant Staphylococcus aureus and Methicillin-resistant Staphylococcus haemolyticus in a Radiology Department

PubMed Central

Kim, Han-Sung; Park, Ji-Young; Koo, Hyun-Sook; Choi, Chul-Sun; Song, Wonkeun; Cho, Hyoun Chan; Lee, Kyu Man

2012-01-01

Background We performed surveillance cultures of the surfaces of X-ray cassettes to assess contamination with methicillin-resistant Staphylococcus aureus (MRSA). Methods The surfaces of 37 X-ray cassettes stored in a radiology department were cultured using mannitol salt agar containing 6 µg/mL oxacillin. Suspected methicillin-resistant staphylococcal colonies were isolated and identified by biochemical testing. Pulsed-field gel electrophoresis (PFGE) analysis was performed to determine the clonal relationships of the contaminants. Results Six X-ray cassettes (16.2%) were contaminated with MRSA. During the isolation procedure, we also detected 19 X-ray cassettes (51.4%) contaminated with methicillin-resistant Staphylococcus haemolyticus (MRSH), identified as yellow colonies resembling MRSA on mannitol salt agar. PFGE analysis of the MRSA and MRSH isolates revealed that most isolates of each organism were identical or closely related to each other, suggesting a common source of contamination. Conclusions X-ray cassettes, which are commonly in direct contact with patients, were contaminated with MRSA and MRSH. In hospital environments, contaminated X-ray cassettes may serve as fomites for methicillin-resistant staphylococci. PMID:22563556

Identification of natural lactoylcholine in lactic acid bacteria-fermented food.

PubMed

Nakamura, Kozo; Okitsu, Sho; Ishida, Ryuya; Tian, Su; Igari, Naoki; Amano, Yoshihiko

2016-06-15

Acetylcholine (AcCh) is a major neurotransmitter and an agonist of nicotinic and muscarinic receptors in non-neuronal systems. Artificially synthesized lactoylcholine (LaCh) has potent nicotinic activity equal to that of AcCh. In this study, we report the isolation and purification of natural AcCh and LaCh from a lactic-fermented food known to reduce blood pressure. To our knowledge, we are the first to isolate natural LaCh. The choline esters were isolated using a novel purification procedure combining a weak cation-exchange cartridge with ODS and pentafluorophenyl HPLC columns, and the structure of LaCh was identified via various analyses. Assessment of D- and L-LaCh showed that the isolated LaCh was an enantiomer mixture with a D/L ratio of 1.6. D-LaCh induced vasorelaxation of thoracic aortas from spontaneously hypertensive rats (EC50=3.83×10(-7) M), while L-LaCh did not. Our results suggest that choline esters could be new functional ingredients in lactic-fermented foods. Copyright © 2016 Elsevier Ltd. All rights reserved.

Shape measurement of objects with large discontinuities and surface isolations using complementary grating projection

NASA Astrophysics Data System (ADS)

Hao, Yudong; Zhao, Yang; Li, Dacheng

1999-11-01

Grating projection 3D profilometry has three major problems that have to be handled with great care. They are local shadows, phase discontinuities and surface isolations. Carrying no information, shadow areas give us no clue about the profile there. Phase discontinuities often baffle phase unwrappers because they may be generated for several reasons difficult to distinguish. Spatial phase unwrapping will inevitably fail if the object under teste have surface isolations. In this paper, a complementary grating projection profilometry is reported, which attempts to tackle the three aforementioned problems simultaneously. This technique involves projecting two grating patterns form both sides of the CCD camera. Phase unwrapping is carried out pixel by pixel using the two phase maps based on the excess fraction method, which is immune to phase discontinuities or surface isolations. Complementary projection makes sure that no area in the visible volume of CCD is devoid of fringe information, although in some cases a small area of the reconstructed profile is of low accuracy compared with others. The system calibration procedures and measurement results are presented in detail, and possible improvement is discussed.

Passive sampler for dissolved organic matter in freshwater environments.

PubMed

Lam, Buuan; Simpson, André J

2006-12-15

A passive sampler for the isolation of dissolved organic matter (DOM) from freshwater environments is described. The sampler consists of a molecular weight selective membrane (1000 kDa) and an anion exchange resin (diethylaminoethylcellulose (DEAE-cellulose)). NMR indicates the samplers isolate DOM that is nearly indistinguishable from that isolated using the batch DEAE-cellulose procedure. In a comparative study DOM isolated from Lake Ontario cost approximately 0.30 dollars/mg to isolate using the passive samplers while DOM isolated using the traditional batch procedure cost approximately 8-10 dollars/mg. The samplers have been shown to be effective in a range of freshwater environments including a large inland lake (Lake Ontario), fast flowing tributary, and wetland. Large amounts (gram quantities of DOM) can be easily isolated by increasing the size or number of samplers deployed. Samplers are easy to construct, negate the need for pressure filtering, and also permit a range of temporal and spatial experiments that would be very difficult or impossible to perform using conventional approaches. For example, DOM can be monitored on a regular basis at numerous different locations, or samplers could be set at different depths in large lakes. Furthermore, they could potentially be deployed into hard to reach environments such as wells, groundwater aquifers, etc., and as they are easy to use, they can be mailed to colleagues or included with expeditions going to difficult to reach places such as the Arctic and Antarctic.

Physiological and biochemical characterization of Azospirillum brasilense strains commonly used as plant growth-promoting rhizobacteria.

PubMed

Di Salvo, Luciana P; Silva, Esdras; Teixeira, Kátia R S; Cote, Rosalba Esquivel; Pereyra, M Alejandra; García de Salamone, Inés E

2014-12-01

Azospirillum is a plant growth-promoting rhizobacteria (PGPR) genus vastly studied and utilized as agriculture inoculants. Isolation of new strains under different environmental conditions allows the access to the genetic diversity and improves the success of inoculation procedures. Historically, the isolation of this genus has been performed by the use of some traditional culture media. In this work we characterized the physiology and biochemistry of five different A. brasilense strains, commonly used as cereal inoculants. The aim of this work is to contribute to pose into revision some concepts concerning the most used protocols to isolate and characterize this bacterium. We characterized their growth in different traditional and non-traditional culture media, evaluated some PGPR mechanisms and characterized their profiles of fatty acid methyl esters and carbon-source utilization. This work shows, for the first time, differences in both profiles, and ACC deaminase activity of A. brasilense strains. Also, we show unexpected results obtained in some of the evaluated culture media. Results obtained here and an exhaustive knowledge revision revealed that it is not appropriate to conclude about bacterial species without analyzing several strains. Also, it is necessary to continue developing studies and laboratory techniques to improve the isolation and characterization protocols. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Isolation and characterization of mutants with lesions affecting pellicle formation and erythrocyte agglutination by type 1 piliated Escherichia coli.

PubMed Central

Harris, S L; Elliott, D A; Blake, M C; Must, L M; Messenger, M; Orndorff, P E

1990-01-01

The product of the pilE (also called fimH) gene is a minor component of type 1 pili in Escherichia coli. Mutants that have insertions in the pilE gene are fully piliated but unable to bind to and agglutinate guinea pig erythrocytes, a characteristic of wild-type type 1 piliated E. coli. In this paper we describe the isolation of 48 mutants with point lesions that map to the pilE gene. Such mutants were isolated by using mutT mutagenesis and an enrichment procedure devised to favor the growth of individuals that could form a pellicle in static broth containing alpha-methylmannoside, an inhibitor of erythrocyte binding and pellicle formation. Results indicated that the enrichment favored mutants expressing pilE gene products that were defective in mediating erythrocyte binding. Characterization of 12 of the mutants in greater detail revealed that certain lesions affected pilus number and length. In addition, a mutant that was temperature sensitive for erythrocyte binding was isolated and used to provide evidence that pellicle formation relies on the intercellular interaction of pilE gene products. Our results suggest a molecular explanation for the old and paradoxical observations connecting pellicle formation and erythrocyte agglutination by type 1 piliated E. coli. Images PMID:1977736

THE ALLEGED OCCURRENCE OF ADRENALIN IN THE MEALWORM

PubMed Central

Gregerman, Robert I.; Wald, George

1952-01-01

Wense has reported the isolation of crystalline adrenalin from larvae of the beetle Tenebrio molitor. An attempt to repeat his procedures failed to yield any evidence of adrenalin. Analysis of mealworm extracts by paper chromatography and colorimetric means also yielded no indication of adrenalin, though adrenalin added to mealworms can be detected by these procedures in amounts less than 1 mg. per 100 gm. Mealworm extracts reveal on the paper chromatogram the presence of two other orthodiphenols, neither of which appears to be dopa but which may be 3,4-dihydroxyphenyllactic acid and 3,4-dihydroxyphenylacetic acid, compounds recently isolated elsewhere from this organism. PMID:14898031

Triton X-100 as an effective surfactant for the isolation and purification of photosystem I from Arthrospira platensis.

PubMed

Yu, Daoyong; Huang, Guihong; Xu, Fengxi; Wang, Mengfei; Liu, Shuang; Huang, Fang

2014-06-01

Surfactants play important roles in the preparation, structural, and functional research of membrane proteins, and solubilizing and isolating membrane protein, while keeping their structural integrity and activity intact is complicated. The commercial n-Dodecyl-β-D-maltoside (DDM) and Triton X-100 (TX) were used as solubilizers to extract and purify trimeric photosystem I (PSI) complex, an important photosynthetic membrane protein complex attracting broad interests. With an optimized procedure, TX can be used as an effective surfactant to isolate and purify PSI, as a replace of the much more expensive DDM. A mechanism was proposed to interpret the solubilization process at surfactant concentrations lower than the critical solubilization concentration. PSI-TX and PSI-DDM had identical polypeptide bands, pigment compositions, oxygen consumption, and photocurrent activities. This provides an alternative procedure and paves a way for economical and large-scale trimeric PSI preparation.

Pancreas anatomy and surgical procedure for pancreatectomy in rhesus monkeys.

PubMed

Zhang, Yi; Fu, Lan; Lu, Yan-Rong; Guo, Zhi-Guang; Zhang, Zhao-Da; Cheng, Jing-Qiu; Hu, Wei-Ming; Liu, Xu-Bao; Mai, Gang; Zeng, Yong; Tian, Bo-Le

2011-12-01

The aim of this study was to investigate the pancreas anatomy and surgical procedure for harvesting pancreas for islet isolation while performing pancreatectomy to induce diabetes in rhesus monkeys. The necropsy was performed in three cadaveric monkeys. Two monkeys underwent the total pancreatectomy and four underwent partial pancreatectomy (70-75%). The greater omentum without ligament to transverse colon, the cystic artery arising from the proper hepatic artery and the branches supplying the paries posterior gastricus from the splenic artery were observed. For pancreatectomy, resected pancreas can be used for islet isolation. Diabetes was not induced in the monkeys undergoing partial pancreatectomy (70-75%). Pancreas anatomy in rhesus monkeys is not the same as in human. Diabetes can be induced in rhesus monkeys by total but not partial pancreatectomy (70-75%). Resected pancreas can be used for islet isolation while performing pancreatectomy to induce diabetes. © 2011 John Wiley & Sons A/S.

Isolation and characterization of a Ca/sup 2 +/ carrier candidate from calf heart inner mitochondrial membrane

DOE Office of Scientific and Technical Information (OSTI.GOV)

Jeng, A.Y.

1979-01-01

A protein was isolated from calf heart inner mitochondrial membrane with the aid of an electron paramagnetic resonance assay based on the relative binding properties of Ca/sup 2 +/, Mn/sup 2 +/, and Mg/sup 2 +/ to the protein. Partial delipidation of the protein was performed by using either the organic solvent extraction procedure or the silicic acid column chromatography. Control experiments indicated that the Ca/sup 2 +/ transport properties of the isolated protein were not due to the contaminating phospholipids. A complete delipidation procedure was developd by using Sephadex LH-20 column chromatography. Further characterization of the physical and chemicalmore » properties of the delipidated protein showed that delipidated protein becomes more hydrophobic in the presence of Ca/sup 2 +/ and alkaline pH in the organic solvent extraction experiments. Two possible models of calciphorin-mediated Ca/sup 2 +/ transport in mitochondria are proposed. (PCS)« less

[Means and methods of personal hygiene in the experiment with 520-day isolation].

PubMed

Shumilina, G A; Shumilina, I V; Solov'eva, S O

2013-01-01

Six volunteers (3 Russians, a Frenchman, an Italian and a Chinese) participated in assessment of the input of sanitation and housekeeping provisions to their wellbeing during 520-day isolation and confinement. Subject of the study was quality and sufficiency of housekeeping agents and procedures as well as more than 60 names of personal hygiene items. The sanitation and housekeeping monitoring involved the clinical, hygienic and microbiological methods, and also consideration of crew comments on the items at their disposal and recommended procedures. Based on the analysis of the functional condition of the integument and oral cavity and entries in the questionnaires, i.e. objective data and subjective feelings, all test subjects remained in the invariably good state. Owing to the application of the selected hygienic means and methods the microbial status of the crew was stable throughout 520-day isolation.

Reporter-Based Isolation of Developmental Myogenic Progenitors

PubMed Central

Kheir, Eyemen; Cusella, Gabriella; Messina, Graziella; Cossu, Giulio; Biressi, Stefano

2018-01-01

The formation and activity of mammalian tissues entail finely regulated processes, involving the concerted organization and interaction of multiple cell types. In recent years the prospective isolation of distinct progenitor and stem cell populations has become a powerful tool in the hands of developmental biologists and has rendered the investigation of their intrinsic properties possible. In this protocol, we describe how to purify progenitors with different lineage history and degree of differentiation from embryonic and fetal skeletal muscle by fluorescence-activated cell sorting (FACS). The approach takes advantage of a panel of murine strains expressing fluorescent reporter genes specifically in the myogenic progenitors. We provide a detailed description of the dissection procedures and of the enzymatic dissociation required to maximize the yield of mononucleated cells for subsequent FACS-based purification. The procedure takes ~6–7 h to complete and allows for the isolation and the subsequent molecular and phenotypic characterization of developmental myogenic progenitors. PMID:29674978

Identification of Malassezia species isolated from patients with extensive forms of pityriasis versicolor in Siena, Italy.

PubMed

Romano, Clara; Mancianti, Francesca; Nardoni, Simona; Ariti, Gaetano; Caposciutti, Paola; Fimiani, Michele

2013-01-01

Pityriasis versicolor (PV) is an infection caused by various species of Malassezia yeast. There is no agreement in the literature concerning the species of Malassezia and the demographic, clinical, and mycological data. To prospectively identify Malassezia species isolated from lesions of patients with extensive, long standing and recurrent forms of PV and to estimate the relationship between Malassezia species and the demographic and clinical data of the patients. All patients with PV were enrolled over a four-year period. Malassezia species were isolated in cultures and identified by morphological features and physiological tests. In the last 2 years a PCR-based technique was used to confirm the species' identification. A total of 74 patients (43 males and 31 females, mean age 39.5 years) were enrolled. Only one species was isolated in 45 patients, and more than one species were identified in the remaining 28 patients (38%). M. globosa was the most frequently isolated (60.3%) species. There was a significant association between the isolation of 2 or more species and the presence of at least one predisposing factor. In the last 29 cases, which were subjected to PCR, there were no differences in the identification of isolated species as compared to traditional methods. The isolation of more than one species in a single lesion is not infrequent in PV and is related to the presence of one predisposing factor. The isolated species isolated were not influenced by demographic and clinical features. The traditional and more recent (PCR) procedures gave the same results in the isolated species. Copyright © 2012 Revista Iberoamericana de Micología. Published by Elsevier Espana. All rights reserved.

Pellet-free isolation of human and bovine milk extracellular vesicles by size-exclusion chromatography.

PubMed

Blans, Kristine; Hansen, Maria S; Sørensen, Laila V; Hvam, Michael L; Howard, Kenneth A; Möller, Arne; Wiking, Lars; Larsen, Lotte B; Rasmussen, Jan T

2017-01-01

Studies have suggested that nanoscale extracellular vesicles (EV) in human and bovine milk carry immune modulatory properties which could provide beneficial health effects to infants. In order to assess the possible health effects of milk EV, it is essential to use isolates of high purity from other more abundant milk structures with well-documented bioactive properties. Furthermore, gentle isolation procedures are important for reducing the risk of generating vesicle artefacts, particularly when EV subpopulations are investigated. In this study, we present two isolation approaches accomplished in three steps based on size-exclusion chromatography (SEC) resulting in effective and reproducible EV isolation from raw milk. The approaches do not require any EV pelleting and can be applied to both human and bovine milk. We show that SEC effectively separates phospholipid membrane vesicles from the primary casein and whey protein components in two differently obtained casein reduced milk fractions, with one of the fractions obtained without the use of ultracentrifugation. Milk EV isolates were enriched in lactadherin, CD9, CD63 and CD81 compared to minimal levels of the EV-marker proteins in other relevant milk fractions such as milk fat globules. Nanoparticle tracking analysis and electron microscopy reveals the presence of heterogeneous sized vesicle structures in milk EV isolates. Lipid analysis by thin layer chromatography shows that EV isolates are devoid of triacylglycerides and presents a phospholipid profile differing from milk fat globules surrounded by epithelial cell plasma membrane. Moreover, the milk EV fractions are enriched in RNA with distinct and diverging profiles from milk fat globules. Collectively, our data supports that successful milk EV isolation can be accomplished in few steps without the use of ultracentrifugation, as the presented isolation approaches based on SEC effectively isolates EV in both human and bovine milk.

Pellet-free isolation of human and bovine milk extracellular vesicles by size-exclusion chromatography

PubMed Central

Blans, Kristine; Hansen, Maria S.; Sørensen, Laila V.; Hvam, Michael L.; Howard, Kenneth A.; Möller, Arne; Wiking, Lars; Larsen, Lotte B.; Rasmussen, Jan T.

2017-01-01

ABSTRACT Studies have suggested that nanoscale extracellular vesicles (EV) in human and bovine milk carry immune modulatory properties which could provide beneficial health effects to infants. In order to assess the possible health effects of milk EV, it is essential to use isolates of high purity from other more abundant milk structures with well-documented bioactive properties. Furthermore, gentle isolation procedures are important for reducing the risk of generating vesicle artefacts, particularly when EV subpopulations are investigated. In this study, we present two isolation approaches accomplished in three steps based on size-exclusion chromatography (SEC) resulting in effective and reproducible EV isolation from raw milk. The approaches do not require any EV pelleting and can be applied to both human and bovine milk. We show that SEC effectively separates phospholipid membrane vesicles from the primary casein and whey protein components in two differently obtained casein reduced milk fractions, with one of the fractions obtained without the use of ultracentrifugation. Milk EV isolates were enriched in lactadherin, CD9, CD63 and CD81 compared to minimal levels of the EV-marker proteins in other relevant milk fractions such as milk fat globules. Nanoparticle tracking analysis and electron microscopy reveals the presence of heterogeneous sized vesicle structures in milk EV isolates. Lipid analysis by thin layer chromatography shows that EV isolates are devoid of triacylglycerides and presents a phospholipid profile differing from milk fat globules surrounded by epithelial cell plasma membrane. Moreover, the milk EV fractions are enriched in RNA with distinct and diverging profiles from milk fat globules. Collectively, our data supports that successful milk EV isolation can be accomplished in few steps without the use of ultracentrifugation, as the presented isolation approaches based on SEC effectively isolates EV in both human and bovine milk. PMID:28386391

Isolation and Quantitative Estimation of Diesel Exhaust and Carbon Black Particles Ingested by Lung Epithelial Cells and Alveolar Macrophages In Vitro

EPA Science Inventory

A new procedure for isolating and estimating ingested carbonaceous diesel exhaust particles (DEP) or carbon black (CB) particles by lung epithelial cells and macrophages is described. Cells were incubated with DEP or CB to examine cell-particle interaction and ingestion. After va...

Isolation of Cholesterol from an Egg Yolk

ERIC Educational Resources Information Center

Taber, Douglass F.; Li, Rui; Anson, Cory M.

2011-01-01

A simple procedure for the isolation of the cholesterol, by hydrolysis and extraction followed by column chromatography, is described. The cholesterol can be further purified by complexation with oxalic acid. It can also be oxidized and conjugated to cholestenone. The source of the cholesterol is one egg yolk, which contains about 200 mg of…

Control of a Non-foodborne Outbreak of Salmonellosis: Day Care in Isolation.

ERIC Educational Resources Information Center

Chorba, Terence, L.; And Others

1987-01-01

In this study of a salmonellosis outbreak at a day care center an analysis of five variables revealed that the disease was transferred person to person or by a continuing common source. Successful preventive measures included instruction in proper handwashing and diaper changing procedures and isolation of the infected children. (VM)

Hepatic clearance of 6 polyaromatic aromatic hydrocarbons by isolated trout livers: Prediction from in vitro clearance by liver S9 fractions

EPA Science Inventory

An isolated perfused trout liver preparation was used to evaluate in vitro-to-in vivo metabolism extrapolation procedures for fish. Hepatic clearance (CLH) studies were conducted with six polyaromatic hydrocarbons (PAH) using an experimental design wherein each liver acted as it...

A rapid and easy method for the DNA extraction from Cryptococcus neoformans

PubMed Central

2011-01-01

DNA isolation from C. neoformans is difficult due to a thick and resistant capsule. We have optimized a new and rapid DNA isolation method for Cryptococcus using a short urea treatment followed by a rapid method using a chelex resin suspension. This procedure is simpler than previously reported methods. PMID:21777412

Regulatory considerations of occupational tuberculosis control.

PubMed

McDiarmid, M A; Gillen, N A; Hathon, L

1994-01-01

The authors argue that the classic hierarchy of industrial hygiene controls may be successfully used to control TB. Various elements of hygiene control programs reviewed here include TB exposure control programs, identification and isolation of patients, respiratory isolation, local source capture ventilation, laboratory procedures, employee surveillance programs, reporting of occupational illnesses, labeling requirements, and respiratory protection.

Preparative isolation of aquatic humic substances

USGS Publications Warehouse

Thurman, E.M.; Malcolm, R.L.

1981-01-01

A useful procedure has been developed which utilizes adsorption chromatography followed by size-exclusion chromatography, hydrogen saturation by ion exchange, and lypholization to obtain low-ash aqueous humic substances. The preparative concentration of aquatic humic substances is done by multiple reconcentration procedures even though initial concentrations of aqueous humus may be less than 25 ??g/L. The procedure yields concentration factors of 25 000 times for both humic and fulvic acid in water.

9 CFR 147.26 - Procedures for establishing isolation and maintaining sanitation and good management practices...

Code of Federal Regulations, 2012 CFR

2012-01-01

... rodent population and other pests under control; (6) Tailor vaccination programs to needs of farm and... and maintaining sanitation and good management practices for the control of Salmonella and Mycoplasma... management practices for the control of Salmonella and Mycoplasma infections. (a) The following procedures...

9 CFR 147.26 - Procedures for establishing isolation and maintaining sanitation and good management practices...

Code of Federal Regulations, 2014 CFR

2014-01-01

... rodent population and other pests under control; (6) Tailor vaccination programs to needs of farm and... and maintaining sanitation and good management practices for the control of Salmonella and Mycoplasma... management practices for the control of Salmonella and Mycoplasma infections. (a) The following procedures...

9 CFR 147.26 - Procedures for establishing isolation and maintaining sanitation and good management practices...

Code of Federal Regulations, 2013 CFR

2013-01-01

... rodent population and other pests under control; (6) Tailor vaccination programs to needs of farm and... and maintaining sanitation and good management practices for the control of Salmonella and Mycoplasma... management practices for the control of Salmonella and Mycoplasma infections. (a) The following procedures...

Decoding tree DNA

Treesearch

M. Thompson Conkle

1986-01-01

Check the laboratory reports after your next physical. You'll find information on a number of biochemical processes. Procedures like those used in the medical sciences are yielding valuable information about genetic differences among trees and tree pests. New procedures that provide ways to isolate and move genes are advancing progress in tree improvement. These...

9 CFR 147.26 - Procedures for establishing isolation and maintaining sanitation and good management practices...

Code of Federal Regulations, 2011 CFR

2011-01-01

... and maintaining sanitation and good management practices for the control of Salmonella and Mycoplasma... management practices for the control of Salmonella and Mycoplasma infections. (a) The following procedures...) Allow no visitors except under controlled conditions to minimize the introduction of Salmonella and...

Functional and Structural Analyses of Behavior: Approaches Leading to Reduced Use of Punishment Procedures?

ERIC Educational Resources Information Center

Axelrod, Saul

1987-01-01

Emerging approaches for dealing with inappropriate behaviors of the disabled involve conducting a functional or structural behavior analysis to isolate the factors responsible for the aberrant behavior and implementing corrective procedures (often alternatives to punishment) relevant to the function of the inappropriate behavior. (Author/DB)

45 CFR Appendix A to Subpart C of... - Security Standards: Matrix

Code of Federal Regulations, 2012 CFR

2012-10-01

... Procedure Termination Procedures (A) Information Access Management 164.308(a)(4) Isolating Health care... Protected Health Information Pt. 164, Subpt. C, App. A Appendix A to Subpart C of Part 164—Security...) Mechanism to Authenticate Electronic Protected Health Information (A) Person or Entity Authentication 164...

45 CFR Appendix A to Subpart C of... - Security Standards: Matrix

Code of Federal Regulations, 2014 CFR

2014-10-01

... Procedure Termination Procedures (A) Information Access Management 164.308(a)(4) Isolating Health care... Protected Health Information Pt. 164, Subpt. C, App. A Appendix A to Subpart C of Part 164—Security...) Mechanism to Authenticate Electronic Protected Health Information (A) Person or Entity Authentication 164...

45 CFR Appendix A to Subpart C of... - Security Standards: Matrix

Code of Federal Regulations, 2013 CFR

2013-10-01

... Procedure Termination Procedures (A) Information Access Management 164.308(a)(4) Isolating Health care... Protected Health Information Pt. 164, Subpt. C, App. A Appendix A to Subpart C of Part 164—Security...) Mechanism to Authenticate Electronic Protected Health Information (A) Person or Entity Authentication 164...

Methods for Isolation, Purification, and Propagation of Bacteriophages of Campylobacter jejuni.

PubMed

Gencay, Yilmaz Emre; Birk, Tina; Sørensen, Martine Camilla Holst; Brøndsted, Lone

2017-01-01

Here, we describe the methods for isolation, purification, and propagation of Campylobacter jejuni bacteriophages from samples expected to contain high number of phages such as chicken feces. The overall steps are (1) liberation of phages from the sample material; (2) observation of plaque-forming units on C. jejuni lawns using a spot assay; (3) isolation of single plaques; (4) consecutive purification procedures; and (5) propagation of purified phages from a plate lysate to prepare master stocks.

Microbiological evaluation of the mobile biological isolator system

NASA Technical Reports Server (NTRS)

Taylor, D. M.; Morelli, F.; Neiderheiser, W.; Tratz, W. M.

1979-01-01

Evaluations on critical components of the mobile biological isolation system were performed. High efficiency particulate air filter efficiency and suit integrity were found to withstand repeated ethylene oxide (ETO) sterilizations. The minimum ETO sterilization time required to inactivate all contaminant organisms was established at four hours. Two days of aerating at 120 F was found to dissipate all harmful ETO residuals from the suit. Donning and doffing procedures were clarified and written specifically for isolation rooms.

Explant culture: An advantageous method for isolation of mesenchymal stem cells from human tissues.

PubMed

Hendijani, Fatemeh

2017-04-01

Mesenchymal stem cell (MSC) research progressively moves towards clinical phases. Accordingly, a wide range of different procedures were presented in the literature for MSC isolation from human tissues; however, there is not yet any close focus on the details to offer precise information for best method selection. Choosing a proper isolation method is a critical step in obtaining cells with optimal quality and yield in companion with clinical and economical considerations. In this concern, current review widely discusses advantages of omitting proteolysis step in isolation process and presence of tissue pieces in primary culture of MSCs, including removal of lytic stress on cells, reduction of in vivo to in vitro transition stress for migrated/isolated cells, reduction of price, processing time and labour, removal of viral contamination risk, and addition of supporting functions of extracellular matrix and released growth factors from tissue explant. In next sections, it provides an overall report of technical highlights and molecular events of explant culture method for isolation of MSCs from human tissues including adipose tissue, bone marrow, dental pulp, hair follicle, cornea, umbilical cord and placenta. Focusing on informative collection of molecular and methodological data about explant methods can make it easy for researchers to choose an optimal method for their experiments/clinical studies and also stimulate them to investigate and optimize more efficient procedures according to clinical and economical benefits. © 2017 John Wiley & Sons Ltd.

Systematic extrahepatic Glissonean pedicle isolation for anatomical liver resection based on Laennec's capsule: proposal of a novel comprehensive surgical anatomy of the liver

PubMed Central

Sugioka, Atsushi; Kato, Yutaro; Tanahashi, Yoshinao

2017-01-01

Abstract Anatomical liver resection with the Glissonean pedicle isolation is widely approved as an essential procedure for safety and curability. Especially, the extrahepatic Glissonean pedicle isolation without parenchymal destruction should be an ideal procedure. However, the surgical technique has not been standardized due to a lack of anatomical understanding. Herein, we proposed a novel comprehensive surgical anatomy of the liver based on Laennec's capsule that would give a theoretical background to the extrahepatic Glissonean pedicle isolation. Laennec's capsule is the proper membrane that covers not only the entire surface of the liver including the bare area but also the intrahepatic parenchyma surrounding the Glissonean pedicles. Consequently, there exists a gap between the Glissonean pedicle and Laennec's capsule that could be reached extrahepatically and allows us to isolate the extrahepatic Glissonean pedicle without parenchymal destruction systematically. For standardization, it is essential to approach the “six gates” indicated by the “four anatomical landmarks”: the Arantius plate, the umbilical plate, the cystic plate and the Glissonean pedicle of the caudate process (G1c). This novel anatomy would contribute to standardize the surgical techniques of the systematic extrahepatic Glissonean pedicle isolation for anatomical liver resection including laparoscopic or robotic liver resection and to bring innovative changes in hepatobiliary surgery for spreading safe and curable liver resection. PMID:28156078

A Prospective Study in Comparison of Ambulatory Phlebectomy and Duplex Guided Foam Sclerotherapy in the Management of Varicosities with Isolated Perforator Incompetence.

PubMed

Kishore, R; Sankar, T Bavani; Anandi, A; Nedunchezhian, S; Murugan, Valarmathy

2016-10-01

Perforators are those which connect the superficial and deep venous system either directly to main veins or indirectly through the muscular and soleal venous plexus. The emergence of minimally invasive techniques like ambulatory phlebectomy (AP) and foam sclerotherapy (FS) has led to increasing interest about the appropriate therapy for the treatment of isolated perforator incompetence. There have been no studies which have compared the effectiveness of these in-office procedures in isolated perforator incompetence due to the low prevalence of cases. The primary goal of this study is to compare the clinical parameters (return to normal activity, primary symptom relief), functional parameters (procedure time, change in disease severity, course of venous ulcer), and duplex parameters (recurrence in treated veins, complete occlusion of treated veins) in the management of leg varicosities having isolated primary perforator incompetence by ambulatory phlebectomy and duplex guided foam sclerotherapy. Though the procedure time was shorter with FS than AP, the other parameters of primary symptom relief such as change in disease severity, faster healing of venous ulcer, complete occlusion of treated veins in follow-up duplex examination, and lower recurrence of treated veins are better with AP than FS. In conclusion, the interruption of perforators is effective in decreasing the symptoms of chronic venous insufficiency and for the rapid healing of ulcers. The interruption of the incompetent perforating veins appears to be essential to decrease ambulatory venous hypertension. It is apparent from this study that ambulatory phlebectomy stands distinct with enormous benefits and serves as a superior alternative to foam sclerotherapy in treating patients with isolated perforator incompetence.

Selection of appropriate isolation method based on morphology of blastocyst for efficient derivation of buffalo embryonic stem cells.

PubMed

Kumar, R; Ahlawat, S P S; Sharma, M; Verma, O P; Sai Kumar, G; Taru Sharma, G

2014-03-01

The efficiency of embryonic stem cell (ESC) derivation from all species except for rodents and primates is very low. There are however, multiple interests in obtaining pluripotent cells from these animals with main expectations in the fields of transgenesis, cloning, regenerative medicine and tissue engineering. Researches are being carried out in laboratories throughout the world to increase the efficiency of ESC isolation for their downstream applications. Thus, the present study was undertaken to study the effect of different isolation methods based on the morphology of blastocyst for efficient derivation of buffalo ESCs. Embryos were produced in vitro through the procedures of maturation, fertilization and culture. Hatched blastocysts or isolated inner cell masses (ICMs) were seeded on mitomycin-C inactivated buffalo fetal fibroblast monolayer for the development of ESC colonies. The ESCs were analyzed for alkaline phosphatase activity, expression of pluripotency markers and karyotypic stability. Primary ESC colonies were obtained after 2-5 days of seeding hatched blastocysts or isolated ICMs on mitomycin-C inactivated feeder layer. Mechanically isolated ICMs attached and formed primary cell colonies more efficiently than ICMs isolated enzymatically. For derivation of ESCs from poorly defined ICMs intact hatched blastocyst culture was the most successful method. Results of this study implied that although ESCs can be obtained using all three methods used in this study, efficiency varies depending upon the morphology of blastocyst and isolation method used. So, appropriate isolation method must be selected depending on the quality of blastocyst for efficient derivation of ESCs.

HPLC-Guided Isolation, Purification and Characterization of Phenylpropanoid and Phenolic Constituents of Nutmeg Kernel (Myristica fragrans).

PubMed

Chiu, Sharon; Wang, Thomas; Belski, Martin; Abourashed, Ehab A

2016-04-01

Many studies on the biological activities of nutmeg continue to appear in the literature. The most common targets include GIT, CNS, oxidative stress and inflammation. However, results obtained from most studies are often inconsistent due to the variability of utilized samples, lack of standardized nutmeg products or insufficient amounts of pure compounds for comprehensive follow-up investigation. To address the consistency and supply issue we utilized available technology to develop a reproducible procedure for preparation of specific extracts and isolation of the major phenolic constituents present in nutmeg kemel. A well-defined and reproducible sequence of extraction, fractionation and chromatographic purification was adopted and was guided by HPLC fingerprinting. Spectroscopic methods, mainly NMR, and mass spectrometry were utilized to identify each compound. Thirteen compounds were isolated in a pure form and identified as: elemicin (1), isoelemicin (2), myristicin (4), surinamensin (5), malabaricone C (6), 2-(3'-allyl-2',6'-dimethoxy-phenyloxy)-l- acetoxy-(3,4-dimethoxyphenyl)-propyl ester (7), methoxylicarin A (8), licarin A (9), malabaricone B (10), licarin C (11), 5'-methoxylicarin B (12), licarin B (13), and 2-(3'-allyl-2',6'-dimethoxy-phenyloxy)-l-methyl-5-methoxy-1,2-dihydrobenzofuran (3, a new compound). With repeated isolation runs, these pure compounds can be prepared in quantities sufficient for biological evaluation as needed. The availability of purified compounds will also allow the development of specific, accurate, and sensitive analytical procedures for pharmacokinetic studies and for quality control of nutmeg products. Both aspects are essential for nutmeg-focused drug discovery. The same approach can also be adapted to other medicinal plants of potential interest.

Fibulin-1 purification from human plasma using affinity chromatography on Factor H-Sepharose

PubMed Central

DiScipio, Richard G.; Liddington, Robert C.; Schraufstatter, Ingrid U.

2016-01-01

A method is reported to purify Fibulin-1 from human plasma resulting in a 36% recovery. The steps involve removal of the cryoglobulin and the vitamin K dependent proteins followed by polyethylene glycol and ammonium sulfate precipitations, DEAE-Sephadex column chromatography and finally Factor H-Sepharose affinity purification. The procedure is designed to be integrated into an overall scheme for the isolation of over 30 plasma proteins from a single batch of human plasma. Results from mass spectroscopy, SDS-PAGE, and Western blotting indicate that human plasma Fibulin-1 is a single chain of the largest isotype. Functional binding assays demonstrated calcium ion dependent interaction of Fibulin-1 for fibrinogen, fibronectin, and Factor H. The procedure described is the first to our knowledge that enables a large scale purification of Fibulin-1 from human plasma. PMID:26826315

Modal analysis and dynamic stresses for acoustically excited Shuttle insulation tiles

NASA Technical Reports Server (NTRS)

Ojalvo, I. U.; Ogilvie, P. I.

1976-01-01

The thermal protection system of the Space Shuttle consists of thousands of separate insulation tiles, of varying thicknesses, bonded to the orbiter's surface through a soft strain-isolation pad which is bonded, in turn, to the vehicle's stiffened metallic skin. A modal procedure for obtaining the acoustically induced RMS stress in these comparatively thick tiles is described. The modes employed are generated by a previously developed iterative procedure which converges rapidly for the combined system of tiles and primary structure considered. Each tile is idealized by several hundred three-dimensional finite elements and all tiles on a given panel interact dynamically. Acoustic response results from the present analyses are presented. Comparisons with other analytical results and measured modal data for a typical Shuttle panel, both with and without tiles, are made, and the agreement is good.

Nontuberculous Mycobacterial Infection after Fractionated CO2 Laser Resurfacing

PubMed Central

Culton, Donna A.; Miller, Becky A.; Miller, Melissa B.; MacKuen, Courteney; Groben, Pamela; White, Becky; Cox, Gary M.; Stout, Jason E.

2013-01-01

Nontuberculous mycobacteria are increasingly associated with cutaneous infections after cosmetic procedures. Fractionated CO2 resurfacing, a widely used technique for photorejuvenation, has been associated with a more favorable side effect profile than alternative procedures. We describe 2 cases of nontuberculous mycobacterial infection after treatment with a fractionated CO2 laser at a private clinic. Densely distributed erythematous papules and pustules developed within the treated area within 2 weeks of the laser procedure. Diagnosis was confirmed by histologic analysis and culture. Both infections responded to a 4-month course of a multidrug regimen. An environmental investigation of the clinic was performed, but no source of infection was found. The case isolates differed from each other and from isolates obtained from the clinic, suggesting that the infection was acquired by postprocedure exposure. Papules and pustules after fractionated CO2 resurfacing should raise the suspicion of nontuberculous mycobacterial infection. PMID:23628077

Past and present work practices of European interventional cardiologists in the context of radiation protection of the eye lens-results of the EURALOC study.

PubMed

Domienik-Andrzejewska, Joanna; Ciraj-Bjelac, Olivera; Askounis, Panagiotis; Covens, Peter; Dragusin, Octavian; Jacob, Sophie; Farah, Jad; Gianicolo, Emilio; Padovani, Renato; Teles, Pedro; Widmark, Anders; Struelens, Lara

2018-05-21

This paper investigates over five decades of work practices in interventional cardiology, with an emphasis on radiation protection. The analysis is based on data from more than 400 cardiologists from various European countries recruited for a EURALOC study and collected in the period from 2014 to 2016. Information on the types of procedures performed and their annual mean number, fluoroscopy time, access site choice, x-ray units and radiation protection means used was collected using an occupational questionnaire. Based on the specific European data, changes in each parameter have been analysed over decades, while country-specific data analysis has allowed us to determine the differences in local practices. In particular, based on the collected data, the typical workload of a European cardiologist working in a haemodynamic room and an electrophysiology room was specified for various types of procedures. The results showed that when working in a haemodynamic room, a transparent ceiling-suspended lead shield or lead glasses are necessary in order to remain below the recommended eye lens dose limit of 20 mSv. Moreover, the analysis revealed that new, more complex cardiac procedures such as chronic total occlusion, valvuloplasty and pulmonary vein isolation for atrial fibrillation ablation might contribute substantially to annual doses, although they are relatively rarely performed. The results revealed that considerable progress has been made in the use of radiation protection tools. While their use in electrophysiology procedures is not generic, the situation in haemodynamic procedures is rather encouraging, as ceiling-suspended shields are used in 90% of cases, while the combination of ceiling shield and lead glasses is noted in more than 40% of the procedures. However, we find that still 7% of haemodynamic procedures are performed without any radiation protection tools.

A technique to train new oculomotor behavior in patients with central macular scotomas during reading related tasks using scanning laser ophthalmoscopy: immediate functional benefits and gains retention.

PubMed

Déruaz, Anouk; Goldschmidt, Mira; Whatham, Andrew R; Mermoud, Christophe; Lorincz, Erika N; Schnider, Armin; Safran, Avinoam B

2006-11-23

Reading with a central scotoma involves the use of preferred retinal loci (PRLs) that enable both letter resolution and global viewing of word. Spontaneously developed PRLs however often privilege spatial resolution and, as a result, visual span is commonly limited by the position of the scotoma. In this study we designed and performed the pilot trial of a training procedure aimed at modifying oculomotor behavior in subjects with central field loss. We use an additional fixation point which, when combined with the initial PRL, allows the fulfillment of both letter resolution and global viewing of words. The training procedure comprises ten training sessions conducted with the scanning laser ophthalmoscope (SLO). Subjects have to read single letters and isolated words varying in length, by combining the use of their initial PRL with the one of an examiner's selected trained retinal locus (TRL). We enrolled five subjects to test for the feasibility of the training technique. They showed stable maculopathy and persisting major reading difficulties despite previous orthoptic rehabilitation. We evaluated ETDRS visual acuity, threshold character size for single letters and isolated words, accuracy for paragraphed text reading and reading strategies before, immediately after SLO training, and three months later. Training the use of multiple PRLs in patients with central field loss is feasible and contributes to adapt oculomotor strategies during reading related tasks. Immediately after SLO training subjects used in combination with their initial PRL the examiner's selected TRL and other newly self-selected PRLs. Training gains were also reflected in ETDRS acuity, threshold character size for words of different lengths and in paragraphed text reading. Interestingly, subjects benefited variously from the training procedure and gains were retained differently as a function of word length. We designed a new procedure for training patients with central field loss using scanning laser ophthalmoscopy. Our initial results on the acquisition of newly self-selected PRLs and the development of new oculomotor behaviors suggest that the procedure aiming primarily at developing an examiner's selected TRL might have initiated a more global functional adaptation process.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Vogl, Thomas J., E-mail: t.vogl@em.uni-frankfurt.de; Koch, Silvia A., E-mail: silvia.koch@web.de; Lotz, Gösta, E-mail: goesta.lotz@kgu.de

PurposePercutaneous isolated hepatic perfusion (PIHP) with Melphalan has been developed as a treatment for patients with isolated hepatic metastases of uveal melanoma. We discuss patient outcome and safety in a retrospective multi-centre study.Materials and MethodsBetween 2012 and 2016 18 patients with un-resectable isolated hepatic metastases of uveal melanoma received single or repeated PIHP with Melphalan (n = 35) at seven sites. Progression-free time, overall survival time (OS) and tumour response by means of RECIST 1.1 criteria were evaluated. Peri- and post-procedural adverse events (AE) were registered. Patients’ life quality was assessed using four-point scale questionnaires.ResultsOf 18 patients, initial PIHP treatment resulted inmore » partial response (PR) in eight, stable disease (SD) in seven and progressive disease (PD) in three cases. Nine patients underwent second PIHP with PR in eight cases and PD in one case. Six patients were evaluated after third PIHP with PR in five patients and SD in one patient. Two patients received fourth PIHP with PD in both cases. Median OS was 9.6 months (range 1.6–41.0 months). Median progression-free survival time was 12.4 months (range 0.9–41.0 months) with 1-year survival of 44%. Most common post-procedural AE grade 3 and 4 were temporary leukopenia (n = 11) and thrombocytopenia (n = 8). Patients’ self-assessments showed good ratings for overall health and quality of life with only slight changes after PIHP, and a high degree of satisfaction with PIHP treatment.ConclusionPIHP with Melphalan proved to be a relatively safe, minimal-invasive and repeatable treatment for patients with non-resectable hepatic metastases of uveal melanoma.« less

DOE Office of Scientific and Technical Information (OSTI.GOV)

Witherspoon Editor, P.A.; Bodvarsson Editor, G.S.

The broad range of activities on radioactive waste isolation that are summarized in Table 1.1 provides a comprehensive picture of the operations that must be carried out in working with this problem. A comparison of these activities with those published in the two previous reviews shows the important progress that is being made in developing and applying the various technologies that have evolved over the past 20 years. There are two basic challenges in perfecting a system of radioactive waste isolation: choosing an appropriate geologic barrier and designing an effective engineered barrier. One of the most important developments that ismore » evident in a large number of the reports in this review is the recognition that a URL provides an excellent facility for investigating and characterizing a rock mass. Moreover, a URL, once developed, provides a convenient facility for two or more countries to conduct joint investigations. This review describes a number of cooperative projects that have been organized in Europe to take advantage of this kind of a facility in conducting research underground. Another critical development is the design of the waste canister (and its accessory equipment) for the engineered barrier. This design problem has been given considerable attention in a number of countries for several years, and some impressive results are described and illustrated in this review. The role of the public as a stakeholder in radioactive waste isolation has not always been fully appreciated. Solutions to the technical problems in characterizing a specific site have generally been obtained without difficulty, but procedures in the past in some countries did not always keep the public and local officials informed of the results. It will be noted in the following chapters that this procedure has caused some problems, especially when approval for a major component in a project was needed. It has been learned that a better way to handle this problem is to keep all stakeholders fully informed of project plans and hold periodic meetings to brief the public, especially in the vicinity of the selected site. This procedure has now been widely adopted and represents one of the most important developments in the Third Worldwide Review.« less

The Isolation of Rubber from Milkweed Leaves. An Introductory Organic Chemistry Lab

NASA Astrophysics Data System (ADS)

Volaric, Lisa; Hagen, John P.

2002-01-01

We present an introductory organic chemistry lab in which students isolate rubber from the leaves of milkweed plants (Asclepias syriaca). Students isolated rubber with a recovery of 2.4 ± 1.8% and 1.8 ± 0.7% for the microscale and macroscale procedures, respectively. Infrared spectra of their products were compared with the spectrum of synthetic rubber, cis-polyisoprene. Students tested for elasticity of their product by twisting it on a spatula and pulling; all students found some degree of elasticity.

Undergraduate Separations Utilizing Flash Chromatography

NASA Astrophysics Data System (ADS)

Horowitz, G.

2000-02-01

This article describes the procedures used to carry out four flash chromatography experiments: the isolation of the carotenes, chlorophylls and xanthophylls from a spinach extract; the separation of ß-carotene from tetraphenyl cyclopentadienone; the isolation of (+) and (-) carvone from caraway and spearmint oil; and the purification of benzil from benzoin. Apparatus used is nonbreakable, easy to use, and inexpensive.

Preparative isolation, fast centrifugal partition chromatography purification and biological activity of cajaflavanone from Derris ferruginea stems.

PubMed

Morel, Sylvie; Landreau, Anne; Nguyen, Van Hung; Derbré, Séverine; Grellier, Philippe; Pape, Patrice Le; Pagniez, Fabrice; Litaudon, Marc; Richomme, Pascal

2012-01-01

The Derris genus is known to contain flavonoid derivatives, including prenylated flavanones and isoflavonoids such as rotenoids, which are generally associated with significant biological activity. To develop an efficient preparative isolation procedure for bioactive cajaflavanone. Fast centrifugal partition chromatography (FCPC) was optimised to purify cajaflavanone from Derris ferruginea stems in a single step as compared to fractionation from the cyclohexane extract by successive conventional solid-liquid chromatography procedures. The purification yield, purity, time and solvent consumption per procedure are described. The anti-fungal, anti-bacterial, anti-leishmanial, anti-plasmodial, anti-oxidant activities and the inhibition of advanced glycation end-products (AGEs) by cajaflavanone accumulation are described. FCPC enabled cajaflavanone purification in a single separation step, yielding sufficient quantities to perform in vitro biological screening. Interestingly, cajaflavanone had an inhibitory effect on the formation of AGEs, without displaying any in vitro anti-oxidant activity. A simple and efficient procedure, in comparison with other preparative methods, for bioactive cajaflavone purification has been developed using FCPC. Copyright © 2011 John Wiley & Sons, Ltd.

Effects of post-weaning social isolation and environment al enrichment on exploratory behavior and ankiety in Wistar rats.

PubMed

Tanaś, Łukasz; Ostaszewski, Paweł; Iwan, Anna

2015-01-01

Adverse early experience is generally regarded as a risk factor for both externalizing and internalizing behavioral disorders in humans. It can be modeled in rats by a post-weaning social isolation procedure. Effects of social isolation might possibly be ameliorated by environmental enrichment. In the current study, 24 male Wistar rats were divided post-weaning into four rearing conditions: control, environmental enrichment (EE), social isolation (SI) and a combination of the two experimental conditions; (EE+SI). Two observations of the effects of rearing conditions on the rate of social and object interactions were conducted during the juvenile and post-pubertal stages of development. The SI condition led to a marked increase of social interactions during the juvenile phase, but did not affect object interactions. The EE condition increased the level of social interactions during both the juvenile and post-pubertal measurements. The effects of early rearing conditions on adult exploratory behavior were less clear, with a significant difference between the groups obtained in one of three behavioral tests. Results suggest a general robustness in the development of adult exploratory behavior and anxiety when rats were exposed to early social isolation and provided brief opportunities for social play during the juvenile period. Further studies, aimed at distinguishing play-related protective factors serving against long-term adverse effects of juvenile social isolation, are suggested.

Antibacterial effect of roselle extracts (Hibiscus sabadariffa), sodium hypochlorite and acetic acid against multidrug-resistant Salmonella strains isolated from tomatoes.

PubMed

Gutiérrez-Alcántara, E J; Rangel-Vargas, E; Gómez-Aldapa, C A; Falfan-Cortes, R N; Rodríguez-Marín, M L; Godínez-Oviedo, A; Cortes-López, H; Castro-Rosas, J

2016-02-01

Antibiotic-resistant Salmonella strains were isolated from saladette and red round type tomatoes, and an analysis done of the antibacterial activity of roselle calyx extracts against any of the identified strains. One hundred saladette tomato samples and 100 red round tomato samples were collected from public markets. Each sample consisted of four whole tomatoes. Salmonella was isolated from the samples by conventional culture procedure. Susceptibility to 16 antibiotics was tested for the isolated Salmonella strains by standard test. The antibacterial effect of four roselle calyx extracts (water, methanol, acetone and ethyl acetate), sodium hypochlorite and acetic acid against antibiotic-resistant Salmonella isolates was evaluated on contaminated tomatoes. Twenty-four Salmonella strains were isolated from 12% of each tomato type. Identified Salmonella serotypes were Typhimurium and Typhi. All isolated strains exhibited resistance to at least three antibiotics and some to as many as 12. Over contaminated tomatoes, the roselle calyx extracts produced a greater reduction (2-2·6 log) in antibiotic-resistant Salmonella strain concentration than sodium hypochlorite and acetic acid. The presence of multidrug-resistant Salmonella in vegetables is a significant public health concern. Multidrug-resistant Salmonella strains were isolated from raw tomatoes purchased in public markets in Mexico and challenged with roselle Hibiscus sabdariffa calyx extracts, sodium hypochlorite and acetic acid. On tomatoes, the extracts caused a greater reduction in the concentration of antibiotic-resistant Salmonella strains than sodium hypochlorite and acetic acid. Roselle calyx extracts are a potentially useful addition to disinfection procedures of raw tomatoes in the field, processing plants, restaurants and homes. © 2015 The Society for Applied Microbiology.

Biotechnological procedures to select white rot fungi for the degradation of PAHs.

PubMed

Lee, Hwanhwi; Jang, Yeongseon; Choi, Yong-Seok; Kim, Min-Ji; Lee, Jaejung; Lee, Hanbyul; Hong, Joo-Hyun; Lee, Young Min; Kim, Gyu-Hyeok; Kim, Jae-Jin

2014-02-01

White rot fungi are essential in forest ecology and are deeply involved in wood decomposition and the biodegradation of various xenobiotics. The fungal ligninolytic enzymes involved in these processes have recently become the focus of much attention for their possible biotechnological applications. Successful bioremediation requires the selection of species with desirable characteristics. In this study, 150 taxonomically and physiologically diverse white rot fungi, including 55 species, were investigated for their performance in a variety of biotechnological procedures, such as dye decolorization, gallic acid reaction, ligninolytic enzymes, and tolerance to four PAHs, phenanthrene, anthracene, fluoranthene, and pyrene. Among these fungi, six isolates showed the highest (>90%) tolerance to both individual PAH and mixed PAHs. And six isolates oxidized gallic acid with dark brown color and they rapidly decolorized RBBR within ten days. These fungi revealed various profiles when evaluated for their biotechnological performance to compare the capability of degradation of PAHs between two groups selected. As the results demonstrated the six best species selected from gallic acid more greatly degraded four PAHs than the other isolates selected via tolerance test. It provided that gallic acid reaction test can be performed to rank the fungi by their ability to degrade the PAHs. Most of all, Peniophora incarnata KUC8836 and Phlebia brevispora KUC9033 significantly degraded the four PAHs and can be considered prime candidates for the degradation of xenobiotic compounds in environmental settings. Copyright © 2013 Elsevier B.V. All rights reserved.

Outbreak of Serratia marcescens postoperative infection traced to barbers and razors.

PubMed

Leng, P; Huang, W L; He, T; Wang, Y Z; Zhang, H N

2015-01-01

Fourteen postoperative infections caused by Serratia marcescens were detected in patients on the neurosurgical wards and spinal surgery ward of a 2640-bed hospital between 26th December 2012 and 5th June 2013. To investigate the source of the outbreak, identify risk factors and implement infection control measures. Cultures were collected from healthcare workers and potential environmental sources. S. marcescens isolates were characterized by antibiotic susceptibility testing and pulsed-field gel electrophoresis (PFGE). A retrospective case-control study was performed to identify the risk factors. The outbreak involved 14 patients, five of whom required more than one surgical procedure. S. marcescens was isolated from cerebrospinal fluid, brain tissue, sputum and other secretions. S. marcescens was also cultured from samples taken from the hands of two barbers and their razors. Exposure to the two barbers [odds ratio (OR) 78.0, P < 0.0001] and wound drainage (OR 4.889, P = 0.028) were risk factors. Pre-operative shaving by the barbers was the only independent risk factor (OR 78.0, P < 0.0001). Isolates of S. marcescens from patients, barbers and razors were indistinguishable by PFGE and antibiotic susceptibility pattern. The outbreak ended after removal of the implicated barbers, extensive re-inforcement of infection control procedures and re-education. These results underscore the risk of postoperative infection associated with pre-operative wet shaving. Copyright © 2014 The Healthcare Infection Society. Published by Elsevier Ltd. All rights reserved.

Dry column-thermal energy analyzer method for determining N-nitrosopyrrolidine in fried bacon: collaborative study.

PubMed

Fiddler, W; Pensabene, J W; Gates, R A; Phillips, J G

1984-01-01

A dry column method for isolating N-nitrosopyrrolidine (NPYR) from fried, cure-pumped bacon and detection by gas chromatography-thermal energy analyzer (TEA) was studied collaboratively. Testing the results obtained from 11 collaborators for homogeneous variances among samples resulted in splitting the nonzero samples into 2 groups of sample levels, each with similar variances. Outlying results were identified by AOAC-recommended procedures, and laboratories having outliers within a group were excluded. Results from the 9 collaborators remaining in the low group yielded coefficients of variation (CV) of 6.00% and 7.47% for repeatability and reproducibility, respectively, and the 8 collaborators remaining in the high group yielded CV values of 5.64% and 13.72%, respectively. An 85.2% overall average recovery of the N-nitrosoazetidine internal standard was obtained with an average laboratory CV of 10.5%. The method has been adopted official first action as an alternative to the mineral oil distillation-TEA screening procedure.

Contact lens disinfecting solutions antibacterial efficacy: comparison between clinical isolates and the standard ISO ATCC strains of Pseudomonas aeruginosa and Staphylococcus aureus

PubMed Central

Mohammadinia, M; Rahmani, S; Eslami, G; Ghassemi-Broumand, M; Aghazadh Amiri, M; Aghaie, Gh; Tabatabaee, S M; Taheri, S; Behgozin, A

2012-01-01

Purpose To evaluate the disinfectant properties of the three multipurpose contact lens disinfecting solutions available in Iran, against clinical isolates and the standard ISO ATCC strains of Pseudomonas aeruginosaand Staphylococcus aureus, based on the international organization for standardization (ISO) 14729 guidelines. Methods Three multipurpose solutions that were tested were ReNu Multiplus, Solo Care Aqua and All-Clean Soft. The test solutions were challenged with clinical isolates and the standard strains of P. aeruginosa(ATCC 9027) and S. aureus(ATCC 6538), based on the ISO Stand-alone procedure for disinfecting products. Solutions were sampled for surviving microorganisms at manufacturer's minimum recommended disinfection time. The number of viable organisms was determined and log reductions calculated. Results All of the three test solutions in this study provided a reduction greater than the required mean 3.0 logarithmic reduction against the recommended standard ATCC strains of P. aeruginosaand S. aureus. Antibacterial effectiveness of Solo Care Aqua and All-Clean Soft against clinical isolates of P. aeruginosaand S. aureuswere acceptable based on ISO 14729 Stand-alone test. ReNu MultiPlus showed a minimum acceptable efficacy against the clinical isolate of S. aureus, but did not reduce the clinical isolate by the same amount. Conclusions Although the contact lens disinfecting solutions meet/exceed the ISO 14729 Stand-alone primary acceptance criteria for standard strains of P. aeruginosaand S. aureus, their efficacy may be insufficient against clinical isolates of these organisms. PMID:22094301

Misidentification of methicillin-resistant Staphylococcus aureus (MRSA) in hospitals in Tripoli, Libya

PubMed Central

Ahmed, Mohamed O.; Abuzweda, Abdulbaset R.; Alghazali, Mohamed H.; Elramalli, Asma K.; Amri, Samira G.; Aghila, Ezzeddin Sh.; Abouzeed, Yousef M.

2010-01-01

Background Methicillin-resistant Staphylococcus aureus (MRSA) is a nosocomial (hospital-acquired) pathogen of exceptional concern. It is responsible for life-threatening infections in both the hospital and the community. Aims To determine the frequency of MRSA misidentification in hospitals in Tripoli, Libya using current testing methods. Methods One hundred and seventy S. aureus isolates previously identified as MRSA were obtained from three hospitals in Tripoli. All isolates were reidentified by culturing on mannitol salt agar, API 20 Staph System and retested for resistance to methicillin using the cefoxitin disk diffusion susceptibility test and PBP2a. D-tests and vancomycin E-tests (Van-E-tests) were also performed for vancomycin-resistant isolates. Results Of the 170 isolates examined, 86 (51%) were confirmed as MRSA (i.e. 49% were misidentified as MRSA). Fifteen (17%) of the confirmed MRSA strains exhibited inducible clindamycin resistance. Of the 86 confirmed MRSA isolates, 13 (15%) were resistant to mupirocin, 53 (62%) were resistant to ciprofloxacin, 41 (48%) were resistant to trimethoprim-sulfamethoxazole, and none were resistant to linezolid. Although disc-diffusion testing indicated that 23 (27%) of the isolates were resistant to vancomycin, none of the isolates were vancomycin-resistant by Van-E-test. Conclusions Misidentification of nosocomial S. aureus as MRSA is a serious problem in Libyan hospitals. There is an urgent need for the proper training of microbiology laboratory technicians in standard antimicrobial susceptibility procedures and the implementation of quality control programs in microbiology laboratories of Libyan hospitals. PMID:21483574

Mycobacterial RNA isolation optimized for non-coding RNA: high fidelity isolation of 5S rRNA from Mycobacterium bovis BCG reveals novel post-transcriptional processing and a complete spectrum of modified ribonucleosides.

PubMed

Hia, Fabian; Chionh, Yok Hian; Pang, Yan Ling Joy; DeMott, Michael S; McBee, Megan E; Dedon, Peter C

2015-03-11

A major challenge in the study of mycobacterial RNA biology is the lack of a comprehensive RNA isolation method that overcomes the unusual cell wall to faithfully yield the full spectrum of non-coding RNA (ncRNA) species. Here, we describe a simple and robust procedure optimized for the isolation of total ncRNA, including 5S, 16S and 23S ribosomal RNA (rRNA) and tRNA, from mycobacteria, using Mycobacterium bovis BCG to illustrate the method. Based on a combination of mechanical disruption and liquid and solid-phase technologies, the method produces all major species of ncRNA in high yield and with high integrity, enabling direct chemical and sequence analysis of the ncRNA species. The reproducibility of the method with BCG was evident in bioanalyzer electrophoretic analysis of isolated RNA, which revealed quantitatively significant differences in the ncRNA profiles of exponentially growing and non-replicating hypoxic bacilli. The method also overcame an historical inconsistency in 5S rRNA isolation, with direct sequencing revealing a novel post-transcriptional processing of 5S rRNA to its functional form and with chemical analysis revealing seven post-transcriptional ribonucleoside modifications in the 5S rRNA. This optimized RNA isolation procedure thus provides a means to more rigorously explore the biology of ncRNA species in mycobacteria. © The Author(s) 2014. Published by Oxford University Press on behalf of Nucleic Acids Research.

A Comparison of the Outcomes for Cartilage Defects of the Knee Treated With Biologic Resurfacing Versus Focal Metallic Implants.

PubMed

Pascual-Garrido, Cecilia; Daley, Erika; Verma, Nikhil N; Cole, Brian J

2017-02-01

To compare the results of focal metallic resurfacing with biologic procedures in patients more than 35 years of age with isolated, full thickness defects of the femoral condyle. A total of 61 patients met the selection criteria resulting in 30 patients treated with biological procedures, including debridement, microfracture, osteochondral autograft transplantation, osteochondral allograft, and autologous chondrocyte implantation (BIO group), and 32 patients treated with focal metallic resurfacing (CAP group). The BIO and CAP groups were matched according to treatment location, defect grade and size, and age profile. Outcomes included Western Ontario and McMaster Osteoarthritis Index (WOMAC), Short Form-12, and satisfaction. The primary combination endpoint was determined as a 20% improvement (minimum clinically important difference-20) on WOMAC pain and function at 2 years and no additional index lesion-related surgical intervention. Safety and effectiveness were also reported. Thirty patients in the BIO group (mean age of 44.6, range 35-64) had an average follow-up of 2.6 years and 32 patients in the CAP group (mean age 47.9, range 37-68) were followed for 2.0 years. Fifty-three percent in the BIO group and 75% in the CAP group achieved success per the endpoint definition. The mean total WOMAC score improved significantly for both groups (BIO: 57-78; P < .001) (CAP: 41-86; P < .001). The physical component score (Short Form-12 PCS) improved significantly in the CAP group only (30-36.4; P < .001). Good to excellent patient satisfaction was achieved by 80% in BIO and 91% in CAP. There were 4 secondary procedures on the index lesion in the BIO group and 2 in the CAP group. Careful patient selection can achieve high satisfaction rates with both biological and focal metal resurfacing procedures for the treatment of isolated focal chondral lesions of the femoral condyle in the knee. Focal metallic resurfacing results in similar clinical outcomes and provides excellent success rates at short-term follow-up. Level III comparative study. Copyright © 2016. Published by Elsevier Inc.

Molecular and epidemiological characterization of IMP-type metallo-β-lactamase-producing Enterobacter cloacae in a Large tertiary care hospital in Japan.

PubMed

Hayakawa, Kayoko; Miyoshi-Akiyama, Tohru; Kirikae, Teruo; Nagamatsu, Maki; Shimada, Kayo; Mezaki, Kazuhisa; Sugiki, Yuko; Kuroda, Emi; Kubota, Shiho; Takeshita, Nozomi; Kutsuna, Satoshi; Tojo, Masayoshi; Ohmagari, Norio

2014-06-01

IMP-type metallo-β-lactamase enzymes have been reported in different geographical areas and in various Gram-negative bacteria. However, the risk factors and epidemiology pertaining to IMP-type metallo-β-lactamase-producing Enterobacter cloacae (IMP-producing E. cloacae) have not been systematically evaluated. We conducted a retrospective, matched case-control study of patients from whom IMP-producing E. cloacae isolates were obtained, in addition to performing thorough molecular analyses of the clinically obtained IMP-producing E. cloacae isolates. Unique cases with IMP-producing E. cloacae isolation were included. Patients with IMP-producing E. cloacae were matched to uninfected controls at a ratio of 1 to 3. Fifteen IMP-producing E. cloacae cases were identified, with five of the isolates being obtained from blood, and they were matched to 45 uninfected controls. All (100%) patients from whom IMP-producing E. cloacae isolates were obtained had indwelling devices at the time of isolation, compared with one (2.2%) uninfected control. Independent predictors for isolation of IMP-producing E. cloacae were identified as cephalosporin exposure and invasive procedures within 3 months. Although in-hospital mortality rates were similar between cases and controls (14.3% versus 13.3%), the in-hospital mortality of patients with IMP-producing E. cloacae-caused bacteremia was significantly higher (40%) than the rate in controls. IMP-producing E. cloacae isolates were frequently positive for other resistance determinants. The MICs of meropenem and imipenem were not elevated; 10 (67%) and 12 (80%) of the 15 IMP-producing E. cloacae isolates had a MIC of ≤ 1 μg/ml. A phylogenetic tree showed a close relationship among the IMP-producing E. cloacae samples. Indwelling devices, exposure to cephalosporin, and a history of invasive procedures were associated with isolation of IMP-producing E. cloacae. Screening for carbapenemase production is important in order to apply appropriate clinical management and infection control measures. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

Stress during pregnancy alters the offspring hypothalamic, pituitary, adrenal, and testicular response to isolation on the day of weaning.

PubMed

Williams, M T; Davis, H N; McCrea, A E; Hennessy, M B

1999-01-01

Subjecting pregnant female rats to situations that activate the hypothalamic-pituitary-adrenal (HPA) axis can have long-term effects on the development of the offspring. Restraint under bright lights is a common method of stressing pregnant females that results in consistent behavioral changes in the offspring. We investigated the effects of gestationally administered restraint, bright lights, and heat on the HPA axis response of 21-day-old offspring following exposure to isolation in a novel environment or under resting conditions. Corticotropin-releasing factor titers in the hypothalamus were unaffected following isolation. Nonetheless, adrenocorticotropin hormone (ACTH) was found to be lower in the gestationally stressed offspring prior to or following the isolation period. Corticosterone was attenuated in gestationally stressed offspring following the postnatal stressor and there was also a tendency for the gestationally stressed females to have lower concentrations of aldosterone. Plasmatic testosterone levels were higher in the gestationally stressed males following the period of isolation. The present data suggest that the HPA axis of the offspring is differentially affected by the gestational stress procedure, that is, it is attenuated at the level of the pituitary and adrenal, but not at the level of the hypothalamus. These data have implications for behavioral differences observed in gestationally stressed animals.

Memory recall in arousing situations – an emotional von Restorff effect?

PubMed Central

Wiswede, Daniel; Rüsseler, Jascha; Hasselbach, Simone; Münte, Thomas F

2006-01-01

Background Previous research has demonstrated a relationship between memory recall and P300 amplitude in list learning tasks, but the variables mediating this P300-recall relationship are not well understood. In the present study, subjects were required to recall items from lists consisting of 12 words, which were presented in front of pictures taken from the IAPS collection. One word per list is made distinct either by font color or by a highly arousing background IAPS picture. This isolation procedure was first used by von Restorff. Brain potentials were recorded during list presentation. Results Recall performance was enhanced for color but not for emotional isolates. Event-related brain potentials (ERP) showed a more positive P300-component for recalled non-isolated words and color-isolated words, compared to the respective non-remembered words, but not for words isolated by arousing background. Conclusion Our findings indicate that it is crucial to take emotional mediator variables into account, when using the P300 to predict later recall. Highly arousing environments might force the cognitive system to interrupt rehearsal processes in working memory, which might benefit transfer into other, more stable memory systems. The impact of attention-capturing properties of arousing background stimuli is also discussed. PMID:16863589

Novel Technology for Enrichment of Biomolecules from Cell-Free Body Fluids and Subsequent DNA Sizing.

PubMed

Patel, Vipulkumar; Celec, Peter; Grunt, Magdalena; Schwarzenbach, Heidi; Jenneckens, Ingo; Hillebrand, Timo

2016-01-01

Circulating cell-free DNA (ccfDNA) is a promising diagnostic tool and its size fractionation is of interest. However, kits for isolation of ccfDNA available on the market are designed for small volumes hence processing large sample volumes is laborious. We have tested a new method that enables enrichment of ccfDNA from large volumes of plasma and subsequently allows size-fractionation of isolated ccfDNA into two fractions with individually established cut-off levels of ccfDNA length. This method allows isolation of low-abundant DNA as well as separation of long and short DNA molecules. This procedure may be important e.g., in prenatal diagnostics and cancer research that have been already confirmed by our primary experiments. Here, we report the results of selective separation of 200- and 500-bp long synthetic DNA fragments spiked in plasma samples. Furthermore, we size-fractionated ccfDNA from the plasma of pregnant women and verified the prevalence of fetal ccfDNA in all fractions.

Rapid molecular identification and characteristics of Lactobacillus strains.

PubMed

Markiewicz, L H; Biedrzycka, E; Wasilewska, E; Bielecka, M

2010-09-01

Eleven type strains and 24 Lactobacillus isolates, preliminarily classified to the species due to phenotypic features, were investigated. Standard methods of identification with species-specific PCRs and typing with PFGE (with ApaI, NotI and SmaI restriction enzymes) allowed us to distinguish 16 unique strains belonging to 5 species (L. acidophilus, L. delbrueckii ssp. bulgaricus, L. plantarum, L. rhamnosus, L. salivarius). Alternative approach with 16S-23S rDNA ARDRA identification (with merely two restrictases, BsuRI and TaqI) and PCR-based typing (RAPD with two random- and rep-PCR with (GTG)(5) primers) showed to be more discriminative, i.e. 21 unique strains were classified in the same species as above. As a result, 7 out of 24 phenotypically species-assigned isolates were reclassified. The alternative procedure of rapid identification and typing of Lactobacillus isolates appeared to be equally effective and shortened from 1 week to 2-3 d (in comparison to the standard methods).

Isolation of a hyperthermophilic archaeum predicted by in situ RNA analysis.

PubMed

Huber, R; Burggraf, S; Mayer, T; Barns, S M; Rossnagel, P; Stetter, K O

1995-07-06

A variety of hyperthermophilic bacteria and archaea have been isolated from high-temperature environments by plating and serial dilutions. However, these techniques allow only the small percentage of organisms able to form colonies, or those that are predominant within environmental samples, to be obtained in pure culture. Recently, in situ 16S ribosomal RNA analyses of samples from the Obsidian hot pool at Yellowstone National Park, Wyoming, revealed a variety of archaeal sequences, which were all different from those of previously isolated species. This suggests substantial diversity of archaea with so far unknown morphological, physiological and biochemical features, which may play an important part within high-temperature ecosystems. Here we describe a procedure to obtain pure cultures of unknown organisms harbouring specific 16S rRNA sequences identified previously within the environment. It combines visual recognition of single cells by phylogenetic staining and cloning by 'optical tweezers'. Our result validates polymerase chain reaction data on the existence of large archael communities.

Membrane filtration method for enumeration and isolation of Alicyclobacillus spp. from apple juice.

PubMed

Lee, S-Y; Chang, S-S; Shin, J-H; Kang, D-H

2007-11-01

To evaluate the applicability of filtration membranes for detecting Alicyclobacillus spp. spores in apple juice. Ten types of nitrocellulose membrane filters from five manufacturers were used to collect and enumerate five Alicyclobacillus spore isolates and results were compared to conventional K agar plating. Spore recovery differed among filters with an average recovery rate of 126.2%. Recovery levels also differed among spore isolates. Although significant difference (P < 0.05) in spore sizes existed, no correlation could be determined between spore size and membrane filter recovery rate. Recovery of spores using membrane filtration is dependent on the manufacturer and filter pore size. Correlations between spore recovery rate and spore size could not be determined. Low numbers of Alicyclobacillus spores in juice can be effectively detected using membrane filtration although recovery rate differences exist among different manufacturers. Use of membrane filtration is a simple, fast alternative to the week-long enrichment procedures currently employed in most quality assurance tests.

Lipase-catalysed esters synthesis of cafestol and kahweol.

PubMed

Novaes, Fábio Junior Moreira; Itabaiana Junior, Ivaldo; Sutili, Felipe Korbus; Marriott, Philip John; Bizzo, Humberto Ribeiro; Aquino Neto, Francisco Radler de; Souza, Rodrigo Octávio Mendonça Alves de; Rezende, Claudia Moraes

2018-09-01

Cafestol and kahweol (C&K), two coffee diterpene alcohols with structural similarity which exhibit anticarcinogenic effects, were isolated from green coffee Arabica beans, followed by their lipase-catalysed esterification and purification by preparative high-performance liquid chromatography (HPLC). The isolation and enzymatic synthesis parameters of C&K esters were studied, with the latter optimised by a Central Composite Design; both procedures were monitored by gas chromatography. Scale up and improved isolation conditions resulted in 1.29 g of C&K, with 98% purity from 300 g of green Arabica beans. The highest C&K ester yields were observed using an alcohol:fatty acid molar ratio of 1:5, 73.3 mg mL -1 of CAL-B enzyme, 70 °C and 240 rpm for 3 days in toluene, leading to 85-88% conversion among a variety of tested C&K esters, including n-C 14:0 -C 20:0 , C 18:1 , C 18:2 and C 18:3 . Copyright © 2018. Published by Elsevier Ltd.

Differentiation of closely related but biologically distinct cherry isolates of Prunus necrotic ringspot virus by polymerase chain reaction.

PubMed

Hammond, R W; Crosslin, J M; Pasini, R; Howell, W E; Mink, G I

1999-07-01

Prunus necrotic ringspot ilarvirus (PNRSV) exists as a number of biologically distinct variants which differ in host specificity, serology, and pathology. Previous nucleotide sequence alignment and phylogenetic analysis of cloned reverse transcription-polymerase chain reaction (RT-PCR) products of several biologically distinct sweet cherry isolates revealed correlations between symptom type and the nucleotide and amino acid sequences of the 3a (putative movement protein) and 3b (coat protein) open reading frames. Based upon this analysis, RT-PCR assays have been developed that can identify isolates displaying different symptoms and serotypes. The incorporation of primers in a multiplex PCR protocol permits rapid detection and discrimination among the strains. The results of PCR amplification using type-specific primers that amplify a portion of the coat protein gene demonstrate that the primer-selection procedure developed for PNRSV constitutes a reliable method of viral strain discrimination in cherry for disease control and will also be useful for examining biological diversity within the PNRSV virus group.

Environmental and human monitoring of Americium-241 utilizing extraction chromatography and {alpha}-Spectrometry

DOE Office of Scientific and Technical Information (OSTI.GOV)

Goldstein, S.J.; Hensley, C.A.; Armenta, C.E.

1997-03-01

Recent developments in extraction chromatography have simplified the separation of americium from complex matrices in preparation for {alpha}-spectroscopy relative to traditional methods. Here we present results of procedures developed/adapted for water, air, and bioassay samples with less than 1 g of inorganic residue. Prior analytical methods required the use of a complex, multistage procedure for separation of americium from these matrices. The newer, simplified procedure requires only a single 2 mL extraction chromatographic separation for isolation of Am and lanthanides from other components of the sample. This method has been implemented on an extensive variety of `real` environmental and bioassaymore » samples from the Los Alamos area, and consistently reliable and accurate results with appropriate detection limits have been obtained. The new method increases analytical throughput by a factor of {approx}2 and decreases environmental hazards from acid and mixed-waste generation relative to the prior technique. Analytical accuracy, reproducibility, and reliability are also significantly improved over the more complex and laborious method used previously. 24 refs., 2 figs., 2 tabs.« less

Comparative study of different procedures for the separation of peripheral blood mononuclear cells in cytokine-induced killer cell immunotherapy for hepatocarcinoma.

PubMed

Liu, Hui; Li, Jianyu; Wang, Fengmei; Gao, Yingtang; Luo, Ying; Wang, Peng; Li, Chenglong; Zhu, Zhengyan

2015-04-01

Cytokine-induced killer (CIK) cell immunotherapy exhibits significant advantages in the clinical treatment of tumors. This study was designed to compare the biological characteristics of autologous CIK cells from patients with hepatocarcinoma following different procedures for the separation of peripheral blood mononuclear cells (PBMCs). Forty-four hepatocarcinoma patients were enrolled and distributed into two groups. PBMCs were isolated either using a blood cell separator (apheresis method) or Ficoll lymphocyte separation medium (Ficoll method). The total amount, collection efficacy, and cell status of PBMCs in the two groups were determined. According to the number and status of collected PBMCs, different cultivation procedures were used for their amplification and activation and the proliferation ability, phenotype, and killing activity of CIK cells in the two groups were evaluated. Our results indicated that the number of collected PBMCs in the apheresis group was far more than that in the Ficoll group. However, the isolation rate was lower, and more cellular debris was observed in the apheresis group, which may be the cause of some untoward effects. Following in vitro culture, the enrichment time of CIK cells was longer in the Ficoll group, and the percentages of CD3(+)CD4(+) (Th) and CD4(+)CD25(+) (Treg) cells were higher. In the apheresis group, the percentages of CD3(-)CD56(+) (NK) and CD3(+)CD56(+) (NKT) cells were higher, and the CIK cells exhibited a higher cytolytic activity against HepG2 hepatoma cells. In conclusion, different procedures for PBMCs separation can influence the biological activities of CIK cells, and the apheresis method is more effective at enhancing the antitumor efficacy of CIK cells. However, significant attention should be paid to the possibility of adverse reactions in apheresis donors.

Multicenter Comparative Evaluation of Five Commercial Methods for Toxoplasma DNA Extraction from Amniotic Fluid▿

PubMed Central

Yera, H.; Filisetti, D.; Bastien, P.; Ancelle, T.; Thulliez, P.; Delhaes, L.

2009-01-01

Over the past few years, a number of new nucleic acid extraction methods and extraction platforms using chemistry combined with magnetic or silica particles have been developed, in combination with instruments to facilitate the extraction procedure. The objective of the present study was to investigate the suitability of these automated methods for the isolation of Toxoplasma gondii DNA from amniotic fluid (AF). Therefore, three automated procedures were compared to two commercialized manual extraction methods. The MagNA Pure Compact (Roche), BioRobot EZ1 (Qiagen), and easyMAG (bioMérieux) automated procedures were compared to two manual DNA extraction kits, the QIAamp DNA minikit (Qiagen) and the High Pure PCR template preparation kit (Roche). Evaluation was carried out with two specific Toxoplasma PCRs (targeting the 529-bp repeat element), inhibitor search PCRs, and human beta-globin PCRs. The samples each consisted of 4 ml of AF with or without a calibrated Toxoplasma gondii RH strain suspension (0, 1, 2.5, 5, and 25 tachyzoites/ml). All PCR assays were laboratory-developed real-time PCR assays, using either TaqMan or fluorescent resonance energy transfer probes. A total of 1,178 PCRs were performed, including 978 Toxoplasma PCRs. The automated and manual methods were similar in sensitivity for DNA extraction from T. gondii at the highest concentration (25 Toxoplasma gondii cells/ml). However, our results showed that the DNA extraction procedures led to variable efficacy in isolating low concentrations of tachyzoites in AF samples (<5 Toxoplasma gondii cells/ml), a difference that might have repercussions since low parasite concentrations in AF exist and can lead to congenital toxoplasmosis. PMID:19846633

Lymphocyte receptors for pertussis toxin

DOE Office of Scientific and Technical Information (OSTI.GOV)

Clark, C.G.; Armstrong, G.D.

1990-12-01

We have investigated human T-lymphocyte receptors for pertussis toxin by affinity isolation and photoaffinity labeling procedures. T lymphocytes were obtained from peripheral human blood, surface iodinated, and solubilized in Triton X-100. The iodinated mixture was then passed through pertussis toxin-agarose, and the fractions were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Autoradiography of the fixed, dried gels revealed several bands in the pertussis toxin-bound fraction that were not observed in fractions obtained from histone or fetuin-agarose. Further investigations employed a photoaffinity labeling reagent, sulfosuccinimidyl 2-(p-azido-salicylamido)-1,3'-dithiopropionate, to identify pertussis toxin receptors in freshly isolated peripheral blood monocytic cells, T lymphocytes, andmore » Jurkat cells. In all three cell systems, the pertussis toxin affinity probe specifically labeled a single protein species with an apparent molecular weight of 70,000 that was not observed when the procedure was performed in the presence of excess unmodified pertussis toxin. A protein comparable in molecular weight to the one detected by the photoaffinity labeling technique was also observed among the species that bound to pertussis toxin-agarose. The results suggest that pertussis toxin may bind to a 70,000-Da receptor in human T lymphocytes.« less

Structural characterization and antioxidant activity evaluation of lignins from rice husk.

PubMed

Salanti, Anika; Zoia, Luca; Orlandi, Marco; Zanini, Fabiana; Elegir, Graziano

2010-09-22

In recent years, lignin and extractives from herbaceous plants and crops are receiving increasing attention for their renewability and large annual biomass stock. It is worth noting that only a few studies deal with the chemical characterization of rice husk, a side product of one of the most important crops with regard to human nutrition. Thus, in this study lignin from rice husk was isolated and characterized. Two different extraction procedures were optimized and tested: acidolysis and alkaline enzymatic (AE). The different lignins isolated were fully characterized by means of gravimetric, chromatographic (GPC), and spectroscopic (31P NMR, 2D-HSQC-NMR) analyses with the aim to compare yields, sample purity, and chemical properties, recognized as key parameters for future development. Notwithstanding the extraction procedure, the results highlighted that rice husk lignin is mainly formed by guaiacyl and p-hydroxyphenyl units. The acidolytic approach showed an appreciable lignin recovery and high purity, whereas the AE lignin sample was found to be rich in residual polysaccharides and oxidized functionalities. Moreover, different rice husk extracts, along with acidolysis lignin and AE lignin specimens, were assayed for their antioxidant activity by means of a DPPH radical scavenging test.

The impact of lymphocyte isolation on induced DNA damage in human blood samples measured by the comet assay.

PubMed

Bausinger, Julia; Speit, Günter

2016-09-01

The comet assay is frequently used in human biomonitoring for the detection of exposure to genotoxic agents. Peripheral blood samples are most frequently used and tested either as whole blood or after isolation of lymphocytes (i.e. peripheral blood mononuclear cells, PBMC). To investigate a potential impact of lymphocyte isolation on induced DNA damage in human blood samples, we exposed blood ex vivo to mutagens with different modes of genotoxic action. The comet assay was performed either directly with whole blood at the end of the exposure period or with lymphocytes isolated directly after exposure. In addition to the recommended standard protocol for lymphocyte isolation, a shortened protocol was established to optimise the isolation procedure. The results indicate that the effects of induced DNA strand breaks and alkali-labile sites induced by ionising radiation and alkylants, respectively, are significantly reduced in isolated lymphocytes. In contrast, oxidative DNA base damage (induced by potassium bromate) and stable bulky adducts (induced by benzo[a]pyrene-7,8-dihydrodiol-9,10-epoxide; BPDE) seem to be less affected. Our findings suggest that in vivo-induced DNA damage might also be reduced in isolated lymphocytes in comparison with the whole blood depending of the types of DNA damage induced. Because only small genotoxic effects can generally be expected in human biomonitoring studies with the comet assay after occupational and environmental exposure to genotoxic agents, any loss might be relevant and should be avoided. The possibility of such effects and their potential impact on variability of comet assay results in human biomonitoring should be considered when performing or evaluating such kind of studies. © The Author 2016. Published by Oxford University Press on behalf of the UK Environmental Mutagen Society. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Systematic extrahepatic Glissonean pedicle isolation for anatomical liver resection based on Laennec's capsule: proposal of a novel comprehensive surgical anatomy of the liver.

PubMed

Sugioka, Atsushi; Kato, Yutaro; Tanahashi, Yoshinao

2017-01-01

Anatomical liver resection with the Glissonean pedicle isolation is widely approved as an essential procedure for safety and curability. Especially, the extrahepatic Glissonean pedicle isolation without parenchymal destruction should be an ideal procedure. However, the surgical technique has not been standardized due to a lack of anatomical understanding. Herein, we proposed a novel comprehensive surgical anatomy of the liver based on Laennec's capsule that would give a theoretical background to the extrahepatic Glissonean pedicle isolation. Laennec's capsule is the proper membrane that covers not only the entire surface of the liver including the bare area but also the intrahepatic parenchyma surrounding the Glissonean pedicles. Consequently, there exists a gap between the Glissonean pedicle and Laennec's capsule that could be reached extrahepatically and allows us to isolate the extrahepatic Glissonean pedicle without parenchymal destruction systematically. For standardization, it is essential to approach the "six gates" indicated by the "four anatomical landmarks": the Arantius plate, the umbilical plate, the cystic plate and the Glissonean pedicle of the caudate process (G1c). This novel anatomy would contribute to standardize the surgical techniques of the systematic extrahepatic Glissonean pedicle isolation for anatomical liver resection including laparoscopic or robotic liver resection and to bring innovative changes in hepatobiliary surgery for spreading safe and curable liver resection. © 2017 The Authors. Journal of Hepato-Biliary-Pancreatic Sciences published by John Wiley & Sons Australia, Ltd on behalf of Japanese Society of Hepato-Biliary-Pancreatic Surgery.

Design and test of aircraft engine isolators for reduced interior noise

NASA Technical Reports Server (NTRS)

Unruh, J. F.; Scheidt, D. C.

1982-01-01

Improved engine vibration isolation was proposed to be the most weight and cost efficient retrofit structure-borne noise control measure for single engine general aviation aircraft. A study was carried out the objectives: (1) to develop an engine isolator design specification for reduced interior noise transmission, (2) select/design candidate isolators to meet a 15 dB noise reduction design goal, and (3) carry out a proof of concept evaluation test. Analytical model of the engine, vibration isolators and engine mount structure were coupled to an empirical model of the fuselage for noise transmission evaluation. The model was used to develop engine isolator dynamic properties design specification for reduced noise transmission. Candidate isolators ere chosen from available product literature and retrofit to a test aircraft. A laboratory based test procedure was then developed to simulate engine induced noise transmission in the aircraft for a proof of concept evaluation test. Three candidate isolator configurations were evaluated for reduced structure-borne noise transmission relative to the original equipment isolators.

Characterization of Pasteurella multocida isolates from wetland ecosystems during 1996 to 1999

USGS Publications Warehouse

Samuel, M.D.; Shadduck, D.J.; Goldberg, Diana R.; Wilson, M.A.; Joly, D.O.; Lehr, M.A.

2003-01-01

We cultured 126 Pasteurella multocida isolates, 92 from water and 34 from sediment samples collected from wetlands in the Pacific and Central flyways of the United States between 1996 and 1999. Most (121) of the isolates were P. multocida serotype 1, but serotypes 3, 3/4, 10, and 11 were also found. Many (82) of the isolates were further characterized by DNA fingerprinting procedures and tested in Pekin ducks for virulence. Almost all the serotype 1 isolates we tested caused mortality in Pekin ducks. Serotype 1 isolates varied in virulence, but the most consistent pattern was higher mortality in male ducks than in females. We found no evidence that isolates found in sediment vs. water, between Pacific and Central flyways, or during El Nino years had consistently different virulence. We also found a number of non-serotype 1 isolates that were avirulent in Pekin ducks. Isolates had DNA fingerprint profiles similar to those found in birds that died during avian cholera outbreaks.

Fluorescence- and magnetic-activated cell sorting strategies to separate spermatozoa involving plural contributors from biological mixtures for human identification

PubMed Central

Xu, Yan; Xie, Jianhui; Chen, Ronghua; Cao, Yu; Ping, Yuan; Xu, Qingwen; Hu, Wei; Wu, Dan; Gu, Lihua; Zhou, Huaigu; Chen, Xin; Zhao, Ziqin; Zhong, Jiang; Li, Rui

2016-01-01

No effective method has been developed to distinguish sperm cells originating from different men in multi-suspect sexual assault cases. Here we combined MACS and FACS to isolate single donor sperm cells from forensic mixture samples including female vaginal epithelial cells and sperm cells from multiple contributors. Sperms from vaginal swab were isolated by MACS using FITC-conjugated A kinase anchor protein 3 (AKAP3) antibody; target individual sperm cells involving two or three donors were separated by FACS using FITC-labeled blood group A/B antigen antibody. This procedure was further tested in two mock multi-suspect sexual assault samples and one practical casework sample. Our results showed that complete single donor STR profiles could be successfully obtained from sperm/epithelial cell and sperm mixtures from two contributors. For unbalanced sperm/epithelial cells and sperm cells mixtures, sensitivity results revealed that target cells could be detected at as low as 1:32 and 1:8 mixed ratios, respectively. Although highly relies on cell number and blood types or secretor status of the individuals, this procedure would still be useful tools for forensic DNA analysis of multi-suspect sexual assault cases by the combined use of FACS and MACS based on sperm-specific AKAP3 antigen and human blood type antigen. PMID:27857155

Entamoeba histolytica. Phagocytosis as a virulence factor

PubMed Central

1983-01-01

In this paper, we attempted to define the role of phagocytosis in the virulence of Entamoeba histolytica. We have isolated, from a highly phagocytic and virulent strain, a clone deficient in phagocytosis. Trophozoites of wild-type strain HM1:IMSS were fed with Escherichia coli strain CR34-Thy- grown on 5-bromo,2'-deoxyuridine. The trophozoites that had incorporated the base analog through phagocytosis of the bacteria were killed by irradiation with 310 nm light. The survivors, presumably trophozoites defective in phagocytosis, were grown until log phase and submitted two more times to the selection procedure. Clone L-6, isolated from a subpopulation resulting from this selection procedure, showed 75-85% less erythrophagocytic activity than the wild-type strain. The virulence of clone L-6 and strain HM1:IMSS was measured. The inoculum required to induce liver abscesses in 50% of the newborn hamsters inoculated (AD50) of HM1:IMSS was 1.5 X 10(4) trophozoites. Clone L-6 trophozoites failed to induce liver abscesses in newborn hamsters even with inocula of 5 X 10(5) trophozoites. Virulence revertants were obtained by successive passage of L-6 trophozoites through the liver of young hamsters. The trophozoites that recovered the ability to produce liver abscesses simultaneously recuperate high erythrophagocytic rates. These results show that phagocytosis is involved in the aggressive mechanisms of E. histolytica. PMID:6313842

Environmentally Friendly Procedure Based on Supercritical Fluid Chromatography and Tandem Mass Spectrometry Molecular Networking for the Discovery of Potent Antiviral Compounds from Euphorbia semiperfoliata.

PubMed

Nothias, Louis-Félix; Boutet-Mercey, Stéphanie; Cachet, Xavier; De La Torre, Erick; Laboureur, Laurent; Gallard, Jean-François; Retailleau, Pascal; Brunelle, Alain; Dorrestein, Pieter C; Costa, Jean; Bedoya, Luis M; Roussi, Fanny; Leyssen, Pieter; Alcami, José; Paolini, Julien; Litaudon, Marc; Touboul, David

2017-10-27

A supercritical fluid chromatography-based targeted purification procedure using tandem mass spectrometry and molecular networking was developed to analyze, annotate, and isolate secondary metabolites from complex plant extract mixture. This approach was applied for the targeted isolation of new antiviral diterpene esters from Euphorbia semiperfoliata whole plant extract. The analysis of bioactive fractions revealed that unknown diterpene esters, including jatrophane esters and phorbol esters, were present in the samples. The purification procedure using semipreparative supercritical fluid chromatography led to the isolation and identification of two new jatrophane esters (13 and 14) and one known (15) and three new 4-deoxyphorbol esters (16-18). The structure and absolute configuration of compound 16 were confirmed by X-ray crystallography. This compound was found to display antiviral activity against Chikungunya virus (EC 50 = 0.45 μM), while compound 15 proved to be a potent and selective inhibitor of HIV-1 replication in a recombinant virus assay (EC 50 = 13 nM). This study showed that a supercritical fluid chromatography-based protocol and molecular networking can facilitate and accelerate the discovery of bioactive small molecules by targeting molecules of interest, while minimizing the use of toxic solvents.

Recovery of Renibacterium salmoninarum from naturally infected salmonine stocks in Michigan using a modified culture protocol

USGS Publications Warehouse

Faisal, M.; Eissa, A.E.; Starliper, C.E.

2010-01-01

Renibacterium salmoninarum, the causative agent of bacterial kidney disease (BKD), is a fastidious and slow-growing bacterium that is extremely difficult to grow in vitro. Herein, we describe a modified primary culture protocol that encompasses a modified bacteriological culture medium and a tissue processing procedure. In order to facilitate the release of R. salmoninarum from granulomatous tissues, kidneys of infected fish were homogenized in a high speed stomacher. The kidney disease medium (KDM2), routinely used for primary culture of R. salmoninarum was modified by the addition of antibiotics and metabolites. When a relatively large inoculum of diluted kidney homogenate was streak-plate inoculated onto the modified KDM2, colonial growth of R. salmoninarum was achieved within 5-7. days, compared to the standard of two weeks or more. The modified procedure was then used to determine the prevalence of R. salmoninarum among representative captive and feral salmonid stocks in Michigan. Prevalence and clinical manifestations varied among species, strains of fish, and locations; however, R. salmoninarum isolates were biochemically homogenous. The improved primary culture procedure described in this study enabled selective and quick isolation of R. salmoninarum. Also, the isolates retrieved in this study constitute a unique biological resource for future studies of R. salmoninarum in the Laurentian Great Lakes. ?? 2009 University of Cairo.

Metal shadowing for electron microscopy.

PubMed

Hendricks, Gregory M

2014-01-01

Metal shadowing of bacteria, viruses, isolated molecules, and macromolecular assemblies is another high-resolution method for observing the ultrastructure of biological specimens. The actual procedure for producing a metal shadow is relatively simple; a heavy metal is evaporated from a source at an oblique angle to the specimen. The metal atoms pile up on the surfaces that face the source, but the surfaces away from the source are shielded and receive little metal deposit, creating a "shadow." However, the process of producing biological specimens that are suitable for metal shadowing can be very complex. There are a whole host of specimen preparation techniques that can precede metal shadowing, and all provide superior preservation in comparison to air drying, a required step in negative staining procedures. The physical forces present during air drying (i.e., surface tension of the water-air interface) will literally crush most biological specimens as they dry. In this chapter I explain the development of and procedures for the production of biological specimens from macromolecular assemblies (e.g., DNA and RNA), purified isolated molecules (e.g., proteins), and isolated viruses and bacteria preparations suitable for metal shadowing. A variation on this basic technique is to rotate the specimen during the metal deposition to produce a high-resolution three-dimensional rendering of the specimen.

Modified arthroscopic Brostrom procedure.

PubMed

Lui, Tun Hing

2015-09-01

The open modified Brostrom anatomic repair technique is widely accepted as the reference standard for lateral ankle stabilization. However, there is high incidence of intra-articular pathologies associated with chronic lateral ankle instability which may not be addressed by an isolated open Brostrom procedure. Arthroscopic Brostrom procedure with suture anchor has been described for anatomic repair of chronic lateral ankle instability and management of intra-articular lesions. However, the complication rates seemed to be higher than open Brostrom procedure. Modification of the arthroscopic Brostrom procedure with the use of bone tunnel may reduce the risk of certain complications. Copyright © 2015 European Foot and Ankle Society. Published by Elsevier Ltd. All rights reserved.

Right atrial isolation associated with atrial septal closure in patients with atrial septal defect and chronic atrial fibrillation.

PubMed

Minzioni, G; Graffigna, A; Pagani, F; Vigano, M

1993-12-01

To restore sinus rhythm in the remaining heart chambers of six adult patients with atrial septal defect and chronic or paroxysmal atrial fibrillation, electrical, right atrial isolation associated with surgical correction of the defect was performed. All but one patient was free from atrial fibrillation without medication 2-25 months after operation. The isolated right atrial appendages showed intrinsic rhythmical activity in five patients and no electrical activity in one. Right atrial isolation is a safe and effective procedure that abolishes atrial fibrillation in patients with arrhythmia after surgical correction of atrial septal defect.

Entanglement in channel discrimination with restricted measurements

DOE Office of Scientific and Technical Information (OSTI.GOV)

Matthews, William; Piani, Marco; Watrous, John

2010-09-15

We study the power of measurements implementable with local quantum operations and classical communication (LOCC) measurements in the setting of quantum channel discrimination. More precisely, we consider discrimination procedures that attempt to identify an unknown channel, chosen uniformly from two known alternatives, that take the following form: (i) the input to the unknown channel is prepared in a possibly entangled state with an ancillary system, (ii) the unknown channel is applied to the input system, and (iii) an LOCC measurement is performed on the output and ancillary systems, resulting in a guess for which of the two channels was given.more » The restriction of the measurement in such a procedure to be an LOCC measurement is of interest because it isolates the entanglement in the initial input-ancillary systems as a resource in the setting of channel discrimination. We prove that there exist channel discrimination problems for which restricted procedures of this sort can be at either of the two extremes: they may be optimal within the set of all discrimination procedures (and simultaneously outperform all strategies that make no use of entanglement), or they may be no better than unentangled strategies (and simultaneously suboptimal within the set of all discrimination procedures).« less

Revealing hidden clonal complexity in Mycobacterium tuberculosis infection by qualitative and quantitative improvement of sampling.

PubMed

Pérez-Lago, L; Palacios, J J; Herranz, M; Ruiz Serrano, M J; Bouza, E; García-de-Viedma, D

2015-02-01

The analysis of microevolution events, its functional relevance and impact on molecular epidemiology strategies, constitutes one of the most challenging aspects of the study of clonal complexity in infection by Mycobacterium tuberculosis. In this study, we retrospectively evaluated whether two improved sampling schemes could provide access to the clonal complexity that is undetected by the current standards (analysis of one isolate from one sputum). We evaluated in 48 patients the analysis by mycobacterial interspersed repetitive unit-variable number tandem repeat of M. tuberculosis isolates cultured from bronchial aspirate (BAS) or bronchoalveolar lavage (BAL) and, in another 16 cases, the analysis of a higher number of isolates from independent sputum samples. Analysis of the isolates from BAS/BAL specimens revealed clonal complexity in a very high proportion of cases (5/48); in most of these cases, complexity was not detected when the isolates from sputum samples were analysed. Systematic analysis of isolates from multiple sputum samples also improved the detection of clonal complexity. We found coexisting clonal variants in two of 16 cases that would have gone undetected in the analysis of the isolate from a single sputum specimen. Our results suggest that analysis of isolates from BAS/BAL specimens is highly efficient for recording the true clonal composition of M. tuberculosis in the lungs. When these samples are not available, we recommend increasing the number of isolates from independent sputum specimens, because they might not harbour the same pool of bacteria. Our data suggest that the degree of clonal complexity in tuberculosis has been underestimated because of the deficiencies inherent in a simplified procedure. Copyright © 2014 European Society of Clinical Microbiology and Infectious Diseases. Published by Elsevier Ltd. All rights reserved.

Mapping and isolation of the pulmonary veins using the PVAC catheter.

PubMed

Duytschaever, Mattias; Anne, Wim; Papiashvili, Giorgi; Vandekerckhove, Yves; Tavernier, Rene

2010-02-01

We aimed to investigate the feasibility, efficacy, and safety of the pulmonary vein ablation catheter (PVAC) catheter (a novel multielectrode catheter using duty-cycled bipolar and unipolar radiofrequency energy, Medtronic, Minneapolis, MN, USA) to completely isolate the pulmonary veins (PVs). Twenty-seven patients (60 +/- 8 years) with paroxysmal atrial fibrillation (AF) underwent PV isolation with the PVAC catheter. PVAC was used for both mapping and isolation of the PVs (PVAC-guided ablation). After PVAC ablation, presence/absence of PV potentials (PVP) was verified using a conventional circular mapping catheter. In case of residual PVP on the circular catheter, PVAC ablation was continued. After PVAC-guided ablation 99 of 106 PVs (93%) and 21 of 27 patients (78%) were proven to be isolated. Failure to isolate was due to a mapping failure in four right-sided PVs and a true ablation failure in three right-sided PVs. After continued PVAC ablation, 103 of 106 PVs (97%) and 25 of 27 patients (93%) were shown to be isolated. The total procedural time from femoral vein access to complete catheter withdrawal was 176 +/- 25 minutes. The actual dwelling-time of the PVAC within the left atrium was 102 +/- 37 minutes. Esophageal T degrees rise to >38.5 degrees occurred in nine of 19 monitored patients (47%). (1) PVAC-guided ablation (i.e., mapping and ablation with a single catheter) results in isolation of all PVs in 73% of the patients. (2) An additional circular mapping catheter is required to increase complete isolation rate to 93% of the patients. (3) Given the esophageal T degrees rise in almost 50% of patients, safety precautions are needed.

Characterization of a novel swollenin from Penicillium oxalicum in facilitating enzymatic saccharification of cellulose

PubMed Central

2013-01-01

Background Plant expansins and fungal swollenin that can disrupt crystalline cellulose have great potential for applications in conversion of biomass. Recent studies have been mainly focused on Trichoderma reesei swollenin that show relatively low activity in the promotion of cellulosic hydrolysis. Our aim was to isolate a novel swollenin with greater disruptive activity, to establish an efficient way of producing recombinant swollenin, and to optimize the procedure using swollenin in facilitation of cellulosic hydrolysis. Results A novel gene encoding a swollenin-like protein, POSWOI, was isolated from the filamentous fungus Penicillium oxalicum by Thermal Asymmetric Interlaced PCR (TAIL-PCR). It consisted of a family 1 carbohydrate-binding module (CBM1) followed by a linker connected to a family 45 endoglucanase-like domain. Using the cellobiohydrolase I promoter, recombinant POSWOI was efficiently produced in T. reesei with a yield of 105 mg/L, and showed significant disruptive activity on crystalline cellulose. Simultaneous reaction with both POSWOI and cellulases enhanced the hydrolysis of crystalline cellulose Avicel by approximately 50%. Using a POSWOI-pretreatment procedure, cellulases can produce nearly twice as many reducing sugars as without pretreatment. The mechanism by which POSWOI facilitates the saccharification of cellulose was also studied using a cellulase binding assay. Conclusion We present a novel fungal swollenin with considerable disruptive activity on crystalline cellulose, and develop a better procedure for using swollenin in facilitating cellulosic hydrolysis. We thus provide a new approach for the effective bioconversion of cellulosic biomass. PMID:23688024

Measurement of sub-parts-per-billion levels of carbonyl compounds in marine air by a simple cartridge trapping procedure followed by liquid chromatography

DOE Office of Scientific and Technical Information (OSTI.GOV)

Xianliang, Zhou; Mopper, K.

1990-10-01

Carbonyl compounds in clean marine air were trapped onto 2,4-dinitrophenylhydrazine- (DNPH-) coated cartridges, and their hydrazone derivatives were separated by HPLC and detected by UV absorbance. More than 20 carbonyl compounds were isolated from marine air with >92% collection efficiency. The technique employs a highly effective reagent purification procedure, which results in much lower blanks compared to previously reported trapping techniques for carbonyl compounds. Blanks were routinely <0.07 ppb for formaldehyde and acetone and <0.02 ppb for the others. Humidity and reactive gases have no detectable effect on collection efficiencies. Carbonyl-DNPH derivatives eluted from the cartridges are stable in acetonitrilemore » for at least 2 weeks, which facilitates field studies. Several previously undetected unknown carbonyl compounds were found in marine air by this technique. Typical results for open ocean and coastal marine air are shown.« less

Outcomes of Cryoballoon Ablation in High- and Low-Volume Atrial Fibrillation Ablation Centres: A Russian Pilot Survey

PubMed Central

Mikhaylov, Evgeny N.; Lebedev, Dmitry S.; Pokushalov, Evgeny A.; Davtyan, Karapet V.; Ivanitskii, Eduard A.; Nechepurenko, Anatoly A.; Kosonogov, Alexey Ya.; Kolunin, Grigory V.; Morozov, Igor A.; Termosesov, Sergey A.; Maykov, Evgeny B.; Khomutinin, Dmitry N.; Eremin, Sergey A.; Mayorov, Igor M.; Romanov, Alexander B.; Shabanov, Vitaliy V.; Shatakhtsyan, Victoria; Tsivkovskii, Viktor; Revishvili, Amiran Sh.; Shlyakhto, Evgeny V.

2015-01-01

Purpose. The results of cryoballoon ablation (CBA) procedure have been mainly derived from studies conducted in experienced atrial fibrillation (AF) ablation centres. Here, we report on CBA efficacy and complications resulting from real practice of this procedure at both high- and low-volume centres. Methods. Among 62 Russian centres performing AF ablation, 15 (24%) used CBA technology for pulmonary vein isolation. The centres were asked to provide a detailed description of all CBA procedures performed and complications, if encountered. Results. Thirteen sites completed interviews on all CBAs in their centres (>95% of CBAs in Russia). Six sites were high-volume AF ablation (>100 AF cases/year) centres, and 7 were low-volume AF ablation. There was no statistical difference in arrhythmia-free rates between high- and low-volume centres (64.6 versus 60.8% at 6 months). Major complications developed in 1.5% of patients and were equally distributed between high- and low-volume centres. Minor procedure-related events were encountered in 8% of patients and were more prevalent in high-volume centres. Total event and vascular access site event rates were higher in women than in men. Conclusions. CBA has an acceptable efficacy profile in real practice. In less experienced AF ablation centres, the major complication rate is equal to that in high-volume centres. PMID:26640789

Impact of Corrections to the Spallings Volume Calculation on Waste Isolation Pilot Plant Performance Assessment

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kicker, Dwayne Curtis; Herrick, Courtney G; Zeitler, Todd

2015-11-01

The numerical code DRSPALL (from direct release spallings) is written to calculate the volume of Waste Isolation Pilot Plant solid waste subject to material failure and transport to the surface (i.e., spallings) as a result of a hypothetical future inadvertent drilling intrusion into the repository. An error in the implementation of the DRSPALL finite difference equations was discovered and documented in a software problem report in accordance with the quality assurance procedure for software requirements. This paper describes the corrections to DRSPALL and documents the impact of the new spallings data from the modified DRSPALL on previous performance assessment calculations.more » Updated performance assessments result in more simulations with spallings, which generally translates to an increase in spallings releases to the accessible environment. Total normalized radionuclide releases using the modified DRSPALL data were determined by forming the summation of releases across each potential release pathway, namely borehole cuttings and cavings releases, spallings releases, direct brine releases, and transport releases. Because spallings releases are not a major contributor to the total releases, the updated performance assessment calculations of overall mean complementary cumulative distribution functions for total releases are virtually unchanged. Therefore, the corrections to the spallings volume calculation did not impact Waste Isolation Pilot Plant performance assessment calculation results.« less

Phage selection of peptide "microantibodies".

PubMed

Fujiwara, Daisuke; Fujii, Ikuo

2013-01-01

A bioactive peptide capable of inhibiting protein-protein interactions has the potential to be a molecular tool for biological studies and a therapeutic by disrupting aberrant interactions involved in diseases. We have developed combinatorial libraries of peptides with helix-loop-helix structure, from which the isolated peptides have the constrained structure to reduce entropy costs in binding, resulting in high binding affinities for target molecules. Previously, we designed a de novo peptide of helix-loop-helix structure that we termed a "microantibody." Using the microantibody as a library scaffold, we have constructed a phage-display library to successfully isolate molecular-targeting peptides against a cytokine receptor (granulocyte colony-stimulating factor receptor), a protein kinase (Aurora-A), and a ganglioside (GM1). Protocols in this article describe a general procedure for the library construction and the library screening.

A novel atrial volume reduction technique to enhance the Cox maze procedure: initial results.

PubMed

Marui, Akira; Nishina, Takeshi; Tambara, Keiichi; Saji, Yoshiaki; Shimamoto, Takeshi; Nishioka, Masahiko; Ikeda, Tadashi; Komeda, Masashi

2006-11-01

Large left atrial diameter is reported to be a predictor for recurrent atrial fibrillation after the Cox maze procedure, and left atrial diameter by itself influences the chance of sinus rhythm recovery, as well as maintenance of sinus rhythm. However, additional cut-and-sew procedures to decrease left atrial diameter extend operative time and can cause bleeding. Thus we developed a no-bleeding, faster, and therefore less invasive left atrial volume reduction technique to enhance the Cox maze procedure. The modified Cox maze III procedure with cryoablation or the left atrial maze procedure in association with mitral valve surgery was performed in 80 patients with atrial fibrillation and enlarged left atria (> or =60 mm). Among them, 44 patients had the concomitant volume reduction technique (VR group); continuous horizontal mattress sutures for left atrial plication were placed on the left atrial wall along the pulmonary vein isolation line. Cryoablation was applied to the suture line so that the plicated left atrium is anatomically and electrically isolated. Another 36 patients did not have the volume reduction technique (control group). The VR group had preoperative left atrial diameters similar to those of the control group (67.1 +/- 7.8 vs 64.5 +/- 6.7 mm) and a longer preoperative duration of atrial fibrillation (14.1 +/- 5.4 vs 9.5 +/- 5.1 years, P < .05) but had smaller postoperative left atrial diameters (47.6 +/- 6.3 vs 62.1 +/- 7.9 mm, P < .01). There were no differences in mean crossclamp/bypass time and chest tube drainage for 12 hours between the groups. Twelve months after surgical intervention, the sinus rhythm recovery rate of the VR group was better than that of the control group (90% vs 69%, P < .05). Even in patients with long-standing atrial fibrillation and an enlarged left atrium, maze procedures concomitant with the novel left atrial volume reduction technique improved the sinus rhythm recovery rate without increasing complications. Although further study with a larger number of patients and a longer follow-up period is needed, this safe and thus far potent technique that catheter-based ablation cannot copy might extend indication of the Cox maze procedure for patients with tough atrial fibrillation.

9 CFR 147.30 - Laboratory procedure recommended for the polymerase chain reaction (PCR) test for Mycoplasma...

Code of Federal Regulations, 2012 CFR

2012-01-01

... for Mycoplasma gallisepticum and M. synoviae. (a) DNA isolation. Isolate DNA from 1 mL of eluate from... supernatant DNA to a nuclease-free tube. Estimate the DNA concentration and purity by spectrophotometric... primer (50 μM), 1 μl Forward primer (50 μM). (2) Perform DNA amplification in a Perkin-Elmer 9600...

9 CFR 147.30 - Laboratory procedure recommended for the polymerase chain reaction (PCR) test for Mycoplasma...

Code of Federal Regulations, 2014 CFR

2014-01-01

... for Mycoplasma gallisepticum and M. synoviae. (a) DNA isolation. Isolate DNA from 1 mL of eluate from... supernatant DNA to a nuclease-free tube. Estimate the DNA concentration and purity by spectrophotometric... primer (50 μM), 1 μl Forward primer (50 μM). (2) Perform DNA amplification in a Perkin-Elmer 9600...

9 CFR 147.30 - Laboratory procedure recommended for the polymerase chain reaction (PCR) test for Mycoplasma...

Code of Federal Regulations, 2013 CFR

2013-01-01

... for Mycoplasma gallisepticum and M. synoviae. (a) DNA isolation. Isolate DNA from 1 mL of eluate from... supernatant DNA to a nuclease-free tube. Estimate the DNA concentration and purity by spectrophotometric... primer (50 μM), 1 μl Forward primer (50 μM). (2) Perform DNA amplification in a Perkin-Elmer 9600...

Multi-User Domain Object Oriented (MOO) as a High School Procedure for Foreign Language Acquisition.

ERIC Educational Resources Information Center

Backer, James A.

Foreign language students experience added difficulty when they are isolated from native speakers and from the culture of the target language. It has been posited that MOO (Multi-User Domain Object Oriented) may help overcome the geographical isolation of these students. MOOs are Internet-based virtual worlds in which people from all over the real…

Experience of microbiological screening of human hepatocytes for clinical transplantation.

PubMed

Lehec, Sharon C; Hughes, Robin D; Mitry, Ragai R; Graver, Michelle A; Verma, Anita; Wade, Jim J; Dhawan, Anil

2009-01-01

Hepatocyte transplantation is being used in patients with liver-based metabolic disorders and acute liver failure. Hepatocytes are isolated from unused donor liver tissue under GMP conditions. Cells must be free of microbiological contamination to be safe for human use. The experience of microbiological screening during 72 hepatocyte isolation procedures at one center is reported. Samples were taken at different stages of the process and tested using a blood culture bottle system and Gram stain. Bacterial contamination was detected in 37.5% of the UW organ preservative solutions used to transport the liver tissue to the Cell Isolation Unit. After tissue processing the contamination was reduced to 7% overall in the final hepatocyte product, irrespective of the presence of initial contamination of the transport solution. The most common organisms recovered were coagulase-negative staphylococci, a skin commensal. A total of 41 preparations of fresh or cryopreserved hepatocytes were used for cell transplantation in children with liver-based metabolic disorders without any evidence of sepsis due to infusion of hepatocytes. In conclusion, the incidence of bacterial contamination of the final product was low, confirming the suitability of the organs used, hepatocyte isolation procedure, and the environmental conditions of the clean room.

The consequences of the economic crisis in radiology.

PubMed

2015-12-01

The effects of the economic crisis have led to complex problems in radiology. The crisis has led to a reduction in the turnover of imaging equipment. This reflects on the quantity and quality of output, an aspect which is worsened by the contraction of the radiology market, late payments on supplies, and competitive procurement of medical goods centralized on a regional or national level. Many local and national institutions have operated with significant reductions of reimbursement for procedures, forcing a reorganization of facilities, manpower, and equipment. The reduction in operating margins of the industry has resulted in a reduction of invested capital for projects of industrial R&D and direct or indirect sponsorship. The quality of care will be affected with less comfortable conditions, reduction of local availability of radiologists, and failure to invest in lower dose equipment to control population medical radiation exposure. The crisis resulted in a reduction in the number of graduates in medicine and scholarships for specialization induced by linear cuts will result in a drastic reduction of radiological specialists. This will favour the development of teleradiology services, with the risk of accelerating the demedicalisation of radiology departments, and isolation of the professionals. • The economic crisis has led to reduction in the turnover of imaging equipment. • The economic crisis has led to reductions of reimbursement for procedures. • The economic crisis has led to reductions in operating margins of the industry. • The economic crisis has led to contraction of quantity and quality of output. • The economic crisis resulted in demedicalisation of radiology departments and isolation of professionals.

Effect of different heat treatments and disinfectants on the survival of Prototheca zopfii.

PubMed

Lassa, Henryka; Jagielski, Tomasz; Malinowski, Edward

2011-03-01

Bovine mastitis caused by the yeast-like alga Prototheca zopfii represents a serious veterinary problem and may result in heavy economic losses to particular dairy farms. The purpose of this study was to evaluate the survival of 50 isolates of P. zopfii in milk subjected to different heat treatments and the survival of further 106 P. zopfii isolates after exposure to three classes of teat disinfectants: iodine (Dipal), quaternary ammonium compounds (Teat), and dodecylbenzenesulphonic acid (Blu-gard). Of the 50 isolates tested for thermal tolerance, 29 (58%) survived heat treatment at 62 °C for 30 s and 13 (26% of all isolates) of those survived after heat treatment at 72 °C for 15 s. None of the 106 isolates were able to withstand the in-use concentrations of the three disinfectants tested. The highest disinfectant concentrations that permitted survival of at least one isolate were dilutions: 1:1,000 for Dipal (survival rate of 52.8-57.5%), 1:100 for Teat (88.7-90.6%), and 1:10 for Blu-gard (100%). No differences in the survival rates of P. zopfii were observed with respect to the duration of exposure to disinfectant. The results of this study support the previous findings that P. zopfii may resist high-temperature treatments, including that applied in the high-temperature, short-time (HTST) pasteurization process. The obtained data also demonstrate the efficacy of the three classes of teat disinfectants against P. zopfii, with the efficacy of iodine being most pronounced. The study emphasizes the necessity of using higher temperatures in the pasteurization of raw milk to kill the Prototheca algae, as well as the particular suitability of the iodine for the control procedures of protothecal mastitis.

A short purification process for quantitative isolation of PrPSc from naturally occurring and experimental transmissible spongiform encephalopathies

PubMed Central

Polymenidou, Magdalini; Verghese-Nikolakaki, Susan; Groschup, Martin; Chaplin, Melanie J; Stack, Mick J; Plaitakis, Andreas; Sklaviadis, Theodoros

2002-01-01

Background Transmissible spongiform encephalopathies (TSEs) are neurodegenerative diseases affecting both humans and animals. They are associated with post-translational conversion of the normal cellular prion protein (PrPC) into a heat- and protease-resistant abnormal isoform (PrPSc). Detection of PrPSc in individuals is widely utilized for the diagnosis of prion diseases. Methods TSE brain tissue samples have been processed in order to quantitatively isolate PrPSc. The protocol includes an initial homogenization, digestion with proteinase K and salt precipitation. Results Here we show that over 97 percent of the PrPSc present can be precipitated from infected brain material using this simple salting-out procedure for proteins. No chemically harsh conditions are used during the process in order to conserve the native quality of the isolated protein. Conclusion The resulting PrPSc-enriched preparation should provide a suitable substrate for analyzing the structure of the prion agent and for scavenging for other molecules with which it may associate. In comparison with most methods that exist today, the one described in this study is rapid, cost-effective and does not demand expensive laboratory equipment. PMID:12370086

Phylogeny of lymphocyte heterogeneity: the cellular requirements for the mixed leucocyte reaction with channel catfish.

PubMed Central

Miller, N W; Deuter, A; Clem, L W

1986-01-01

Vigorous mixed leucocyte reactions (MLR) were obtained using channel catfish peripheral blood leucocytes (PBL) when equal numbers of responder and stimulator cells (5 X 10(5) cells each) were cocultured. The use of 2000 rads of X-irradiation was sufficient to block subsequent proliferative responses of the stimulator cells. The cellular requirements for channel catfish MLR responses were assessed by using three functionally distinct leucocyte subpopulations isolated from the PBL. B cells (sIg+ lymphocytes) and T cells (sIg- lymphocytes) were isolated by an indirect panning procedure employing a monoclonal antibody specific for channel catfish Ig. A third population, monocytes, was isolated or depleted by adherence to baby hamster kidney cell microexudate-coated surfaces or adherence to Sephadex G-10, respectively. The results indicated that only the T cells were able to respond in the fish MLR, with monocytes being required as accessory cells. In contrast, all three cell types could function as stimulator cells. In addition, it was observed that low in vitro culture temperatures inhibited the generation of channel catfish MLRs, thereby supporting the contention that low temperature immunosuppression in fish results from a preferential inhibition of the generation of primary T-cell responses. PMID:2944817

Effect of the conditions of isolation on the physicochemical properties of human serum albumin in the norm and with pathology

NASA Astrophysics Data System (ADS)

Ivanov, A. I.; Zhbankov, R. G.; Korolenko, E. A.; Korolik, E. V.; Meleshchenko, L. A.; Sarnatskaya, V. V.; Nikolaev, V. G.; Nikolaichik, V. V.; Yushko, L. A.

1997-01-01

Differential scanning calorimetry and IR spectrosocopy were used to investigate the effect of the procedure of isolation of human serum albumin on its physicochemical characteristics. It is shown that fractionation of blood plasma with ethylene glycol followed by ion exchange chromatography can be used to obtain albumin of normal donors that is similar to the albumin in the nonfractionated plasma according to melting thermograms. Endotherms of human serum albumin samples that were obtained by affinity chromatography and preparative electrophoresis are bimodal, unlike the monophasic for albumin obtained by polyethylene glycol precipitation. These changes result from a higher content of nonetherified fatty acids in the albumin samples obtained by affinity chromatography and from modification of the secondary protein structure in the samples obtained by electrophoresis. Analysis of melting thermograms of serum albumin from patients with uremia, chronic hepatitis, and peritonitis shows that fractionation of blood with polyethylene glycol preserves the thermodynamic characteristics of the various pathological serum albumins to the greatest extent. The present results demonstrate the advantage of polyethylene glycol fractionation for isolation of native preparations of normal and “pathological” human serum albumin.

Quartz crystal microbalance (QCM) affinity biosensor for genetically modified organisms (GMOs) detection.

PubMed

Mannelli, Ilaria; Minunni, Maria; Tombelli, Sara; Mascini, Marco

2003-03-01

A DNA piezoelectric sensor has been developed for the detection of genetically modified organisms (GMOs). Single stranded DNA (ssDNA) probes were immobilised on the sensor surface of a quartz crystal microbalance (QCM) device and the hybridisation between the immobilised probe and the target complementary sequence in solution was monitored. The probe sequences were internal to the sequence of the 35S promoter (P) and Nos terminator (T), which are inserted sequences in the genome of GMOs regulating the transgene expression. Two different probe immobilisation procedures were applied: (a) a thiol-dextran procedure and (b) a thiol-derivatised probe and blocking thiol procedure. The system has been optimised using synthetic oligonucleotides, which were then applied to samples of plasmidic and genomic DNA isolated from the pBI121 plasmid, certified reference materials (CRM), and real samples amplified by the polymerase chain reaction (PCR). The analytical parameters of the sensor have been investigated (sensitivity, reproducibility, lifetime etc.). The results obtained showed that both immobilisation procedures enabled sensitive and specific detection of GMOs, providing a useful tool for screening analysis in food samples.

Copper and tin isotopic analysis of ancient bronzes for archaeological investigation: development and validation of a suitable analytical methodology.

PubMed

Balliana, Eleonora; Aramendía, Maite; Resano, Martin; Barbante, Carlo; Vanhaecke, Frank

2013-03-01

Although in many cases Pb isotopic analysis can be relied on for provenance determination of ancient bronzes, sometimes the use of "non-traditional" isotopic systems, such as those of Cu and Sn, is required. The work reported on in this paper aimed at revising the methodology for Cu and Sn isotope ratio measurements in archaeological bronzes via optimization of the analytical procedures in terms of sample pre-treatment, measurement protocol, precision, and analytical uncertainty. For Cu isotopic analysis, both Zn and Ni were investigated for their merit as internal standard (IS) relied on for mass bias correction. The use of Ni as IS seems to be the most robust approach as Ni is less prone to contamination, has a lower abundance in bronzes and an ionization potential similar to that of Cu, and provides slightly better reproducibility values when applied to NIST SRM 976 Cu isotopic reference material. The possibility of carrying out direct isotopic analysis without prior Cu isolation (with AG-MP-1 anion exchange resin) was investigated by analysis of CRM IARM 91D bronze reference material, synthetic solutions, and archaeological bronzes. Both procedures (Cu isolation/no Cu isolation) provide similar δ (65)Cu results with similar uncertainty budgets in all cases (±0.02-0.04 per mil in delta units, k = 2, n = 4). Direct isotopic analysis of Cu therefore seems feasible, without evidence of spectral interference or matrix-induced effect on the extent of mass bias. For Sn, a separation protocol relying on TRU-Spec anion exchange resin was optimized, providing a recovery close to 100 % without on-column fractionation. Cu was recovered quantitatively together with the bronze matrix with this isolation protocol. Isotopic analysis of this Cu fraction provides δ (65)Cu results similar to those obtained upon isolation using AG-MP-1 resin. This means that Cu and Sn isotopic analysis of bronze alloys can therefore be carried out after a single chromatographic separation using TRU-Spec resin. Tin isotopic analysis was performed relying on Sb as an internal standard used for mass bias correction. The reproducibility over a period of 1 month (n = 42) for the mass bias-corrected Sn isotope ratios is in the range of 0.06-0.16 per mil (2 s), for all the ratios monitored.

Selective isolation of gonyautoxins 1,4 from the dinoflagellate Alexandrium minutum based on molecularly imprinted solid-phase extraction.

PubMed

Lian, Ziru; Wang, Jiangtao

2017-09-15

Gonyautoxins 1,4 (GTX1,4) from Alexandrium minutum samples were isolated selectively and recognized specifically by an innovative and effective extraction procedure based on molecular imprinting technology. Novel molecularly imprinted polymer microspheres (MIPMs) were prepared by double-templated imprinting strategy using caffeine and pentoxifylline as dummy templates. The synthesized polymers displayed good affinity to GTX1,4 and were applied as sorbents. Further, an off-line molecularly imprinted solid-phase extraction (MISPE) protocol was optimized and an effective approach based on the MISPE coupled with HPLC-FLD was developed for selective isolation of GTX1,4 from the cultured A. minutum samples. The separation method showed good extraction efficiency (73.2-81.5%) for GTX1,4 and efficient removal of interferences matrices was also achieved after the MISPE process for the microalgal samples. The outcome demonstrated the superiority and great potential of the MISPE procedure for direct separation of GTX1,4 from marine microalgal extracts. Copyright © 2017. Published by Elsevier Ltd.

Hofmeister series salts enhance purification of plasmid DNA by non-ionic detergents

PubMed Central

Lezin, George; Kuehn, Michael R.; Brunelli, Luca

2011-01-01

Ion-exchange chromatography is the standard technique used for plasmid DNA purification, an essential molecular biology procedure. Non-ionic detergents (NIDs) have been used for plasmid DNA purification, but it is unclear whether Hofmeister series salts (HSS) change the solubility and phase separation properties of specific NIDs, enhancing plasmid DNA purification. After scaling-up NID-mediated plasmid DNA isolation, we established that NIDs in HSS solutions minimize plasmid DNA contamination with protein. In addition, large-scale NID/HSS solutions eliminated LPS contamination of plasmid DNA more effectively than Qiagen ion-exchange columns. Large-scale NID isolation/NID purification generated increased yields of high quality DNA compared to alkali isolation/column purification. This work characterizes how HSS enhance NID-mediated plasmid DNA purification, and demonstrates that NID phase transition is not necessary for LPS removal from plasmid DNA. Specific NIDs such as IGEPAL CA-520 can be utilized for rapid, inexpensive and efficient laboratory-based large-scale plasmid DNA purification, outperforming Qiagen-based column procedures. PMID:21351074

STUDIES ON ISOLATED NUCLEI. II. ISOLATION AND CHEMICAL CHARACTERIZATION OF NUCLEOLAR AND NUCLEOPLASMIC SUBFRACTIONS.

PubMed

MAGGIO, R; SIEKEVITZ, P; PALADE, G E

1963-08-01

This paper describes the subfractionation of nuclei isolated from guinea pig liver by the procedure presented in the first article of the series (8). Centrifugation in a density gradient system of nuclear fractions disrupted by sonication permits the isolation of the following subfractions: (a) a nucleolar subfraction which consists mainly of nucleoli surrounded by a variable amount of nucleolus-associated chromatin and contaminated by chromatin blocks derived primarily from von Kupffer cell nuclei; (b) and (c), two nucleoplasmic subfractions (I and II) which consist mainly of chromatin threads in a coarser (I) or finer (II) degree of fragmentation. The protein, RNA, and DNA content of these subfractions was determined, and their RNA's characterized in terms of NaCl-solubility, nucleotide composition, and in vivo nucleotide turnover, using inorganic (32)P as a marker. The results indicate that there are at least three types of RNA in the nucleus (one in the nucleolus and two in the nucleoplasm or chromatin), which differ from one another in NaCl-solubility, nucleotide composition, turnover, and possibly sequence. Possible relations among these RNA's and those of the cytoplasm are discussed.

Isolation and quantification of oligomeric pyranoanthocyanin-flavanol pigments from red wines by combination of column chromatographic techniques.

PubMed

He, Jingren; Santos-Buelga, Celestino; Mateus, Nuno; de Freitas, Victor

2006-11-17

A combination of column chromatography on Toyopearl gel HW-40 (S) and polyamide resin has been developed for the preparative isolation and further determination of pyranoanthocyanins of oligomeric nature formed after reaction between anthocyanins and different flavanols in a complex wine matrix. Polyamide chromatography was found to be exceptionally useful to separate oligomeric pyanoanthocyanins from other classes of wine flavonoids and polymerized pigments into an advanced state of purity for further identification and quantification by HPLC-diode array detector coupled with electrospray ionization mass spectrometry (HPLC-DAD/ESI-MS). Fractionation on Toyopearl gel chromatography allowed the separation of pyranoanthocyanins bearing the same flavanols (catechin, epicatechin and procyanidin dimers) but with different anthocyanin moieties (either acylated or non-acylated in the glucose residue) in order to allow further isolation of individual oligomeric pigments on C18 chromatography. A quantitative procedure for analyzing the major pyranoanthocyanin-flavanol derivatives in different aged wines is proposed for the first time. Results obtained showed good reproducibility and recovery regarding sample pretreatment and quantitative method for all analyzed oligomeric pyranoanthocyanins. The combination of these two chromatographic separations is likely to be applicable to the preparative isolation of other anthocyanin-derived pigments.

Development of a novel automated cell isolation, expansion, and characterization platform.

PubMed

Franscini, Nicola; Wuertz, Karin; Patocchi-Tenzer, Isabel; Durner, Roland; Boos, Norbert; Graf-Hausner, Ursula

2011-06-01

Implementation of regenerative medicine in the clinical setting requires not only biological inventions, but also the development of reproducible and safe method for cell isolation and expansion. As the currently used manual techniques do not fulfill these requirements, there is a clear need to develop an adequate robotic platform for automated, large-scale production of cells or cell-based products. Here, we demonstrate an automated liquid-handling cell-culture platform that can be used to isolate, expand, and characterize human primary cells (e.g., from intervertebral disc tissue) with results that are comparable to the manual procedure. Specifically, no differences could be observed for cell yield, viability, aggregation rate, growth rate, and phenotype. Importantly, all steps-from the enzymatic isolation of cells through the biopsy to the final quality control-can be performed completely by the automated system because of novel tools that were incorporated into the platform. This automated cell-culture platform can therefore replace entirely manual processes in areas that require high throughput while maintaining stability and safety, such as clinical or industrial settings. Copyright © 2011 Society for Laboratory Automation and Screening. Published by Elsevier Inc. All rights reserved.

Secobatzellines A and B, two new enzyme inhibitors from a deep-water Caribbean sponge of the genus Batzella.

PubMed

Gunasekera, S P; McCarthy, P J; Longley, R E; Pomponi, S A; Wright, A E

1999-08-01

Secobatzelline A (1), a new batzelline natural analogue, and secobatzelline B (2), a likely artifact formed during the isolation procedure, have been isolated from a deep-water marine sponge of the genus Batzella. Secobatzellines A and B inhibited the phosphatase activity of calcineurin, and secobatzelline A inhibited the peptidase activity of CPP32. Both compounds showed in vitro cytotoxicity against P-388 and A-549 cell lines. The isolation and structure elucidation of secobatzellines A (1) and B (2) are described.

Isolation and characterization of trichothecin from corn cultures of Fusarium graminearum MRC 1125.

PubMed Central

Combrinck, S; Gelderblom, W C; Spies, H S; Burger, B V; Thiel, P G; Marasas, W F

1988-01-01

Trichothecin was isolated and purified from corn cultures of a toxic strain of Fusarium graminearum. This strain, designated MRC 1125, was obtained from corn in southern Africa. The brine shrimp toxicity assay was used throughout the isolation procedure to monitor the toxicity of the fractions. The compound was characterized by detailed 1H (500-MHz) and 13C (125-MHz) nuclear magnetic resonance spectroscopy and mass spectrometry. This is the first report of the production of trichothecin by a Fusarium species. PMID:3415233

Diversity and distribution of Listeria monocytogenes in meat processing plants.

PubMed

Martín, Belén; Perich, Adriana; Gómez, Diego; Yangüela, Javier; Rodríguez, Alicia; Garriga, Margarita; Aymerich, Teresa

2014-12-01

Listeria monocytogenes is a major concern for the meat processing industry because many listeriosis outbreaks have been linked to meat product consumption. The aim of this study was to elucidate L. monocytogenes diversity and distribution across different Spanish meat processing plants. L. monocytogenes isolates (N = 106) collected from food contact surfaces of meat processing plants and meat products were serotyped and then characterised by multilocus sequence typing (MLST). The isolates were serotyped as 1/2a (36.8%), 1/2c (34%), 1/2b (17.9%) and 4b (11.3%). MLST identified ST9 as the most predominant allelic profile (33% of isolates) followed by ST121 (16%), both of which were detected from several processing plants and meat products sampled in different years, suggesting that those STs are highly adapted to the meat processing environment. Food contact surfaces during processing were established as an important source of L. monocytogenes in meat products because the same STs were obtained in isolates recovered from surfaces and products. L. monocytogenes was recovered after cleaning and disinfection procedures in two processing plants, highlighting the importance of thorough cleaning and disinfection procedures. Epidemic clone (EC) marker ECI was identified in 8.5%, ECIII was identified in 2.8%, and ECV was identified in 7.5% of the 106 isolates. Furthermore, a selection of presumably unrelated ST9 isolates was analysed by multi-virulence-locus sequence typing (MVLST). Most ST9 isolates had the same virulence type (VT11), confirming the clonal origin of ST9 isolates; however, one ST9 isolate was assigned to a new VT (VT95). Consequently, MLST is a reliable tool for identification of contamination routes and niches in processing plants, and MVLST clearly differentiates EC strains, which both contribute to the improvement of L. monocytogenes control programs in the meat industry. Copyright © 2014 Elsevier Ltd. All rights reserved.

[Hepatocellular carcinoma originated in the caudate lobe. Surgical strategy for resection. A propos of a case].

PubMed

Martínez-Mier, Gustavo; Esquivel-Torres, Sergio; Calzada-Grijalva, José Francisco; Grube-Pagola, Peter

2015-01-01

Hepatocellular carcinoma originating from the caudate lobe has a worse prognosis than other hepatocellular carcinoma in another segment of the liver. An isolated caudate lobe resection of the liver represents a significant technical challenge. Caudate lobe resection can be performed along with a lobectomy or as an isolated liver resection. There are very few reports about isolated caudate lobe liver resection. We report a case of successful isolated resection of hepatocellular carcinoma in the caudate lobe with excellent long-term survival. A 74 years old female with 8cm mass lesion in the caudate lobe without clinical or biochemical evidence of liver cirrhosis, serum alpha-fetoprotein 3.7 U/l, and negative hepatitis serology was evaluated for surgery. Complete resection of the lesion in 270minutes with Pringle maneuver for 13minutes was satisfactorily performed. Patient was discharged ten days after surgery without complications. Patient is currently asymptomatic, without deterioration of liver function and 48 month tumor free survival after the procedure. Isolated caudate lobe resection is an uncommon but technically possible procedure. In order to achieve a successful resection, one must have a detailed knowledge of complete liver anatomy. Tumor free margins must be obtained to provide long survival for these patients who have a malignancy in this anatomic location. Copyright © 2015. Published by Masson Doyma México S.A.

[Breast asymmetry of the teenager and the young adult. Stability of the result in time. About 144 patients].

PubMed

Ellart, J; François, C; Calibre, C; Guerreschi, P; Duquennoy-Martinot, V

2016-10-01

Breast asymmetry is defined by a difference in breast shape and/or volume. The goal of this study was to assess the stability of the surgical results and to highlight predictive factors for deterioration in results. This retrospective and monocentric study included all patients presenting constitutional isolated asymmetry, Poland's syndrome, asymmetric tuberous breast or pectus excavatum treated between 1980 and 2015. The statistical analysis included 144 patients and contained two parts. The first analysis compared patients with or without breast implant. Results were significant only for symmetry of shape, with better outcomes without breast implant (P=0.0170). The second analysis compared the subgroups of patients with breast asymmetry, according to the etiology. Patients with tuberous breasts and constitutional isolated asymmetry have been compared. We found a significant difference only regarding the long-term results (P=0.0091). Patients with tuberous breasts had deteriorating in results over time. Patients with Poland's syndrome, compared to patients with constitutional isolated asymmetry, had significantly less good results for symmetry of volume, of shape, of areola, and for early results (P<0.025). Concerning patients with pectus excavatum, the early and long-term results and the stability were good for most patients. Better results have been obtained with similar surgical procedures on both sides. Predictive factors for instability of results were the use of unilateral breast implant, weight variations, pregnancies and breast-feeding, the use of hormonal treatments, ageing and tuberous malformation. Copyright © 2016 Elsevier Masson SAS. All rights reserved.

Wholly Patient-tailored Ablation of Atrial Fibrillation Guided by Spatio-Temporal Dispersion of Electrograms in the Absence of Pulmonary Veins Isolation

PubMed Central

Seitz, Julien; Bars, Clément; Théodore, Guillaume; Beurtheret, Sylvain; Lellouche, Nicolas; Bremondy, Michel; Ferracci, Ange; Faure, Jacques; Penaranda, Guillaume; Yamazaki, Masatoshi; Avula, Uma Mahesh R.; Curel, Laurence; Siame, Sabrina; Berenfeld, Omer; Pisapia, André; Kalifa, Jérôme

2017-01-01

Background The use of intra-cardiac electrograms to guide atrial fibrillation (AF) ablation has yielded conflicting results. We evaluated an electrogram marker of AF drivers: the clustering of electrograms exhibiting spatio-temporal dispersion — regardless of whether such electrograms were fractionated or not. Objective To evaluate the usefulness of spatio-temporal dispersion, a visually recognizable electric footprint of AF drivers, for the ablation of all forms of AF. Methods We prospectively enrolled 105 patients admitted for AF ablation. AF was sequentially mapped in both atria with a 20-pole PentaRay catheter. We tagged and ablated only regions displaying electrogram dispersion during AF. Results were compared to a validation set in which a conventional ablation approach was used (pulmonary vein isolation/stepwise approach). To establish the mechanism underlying spatio-temporal dispersion of AF electrograms, we conducted realistic numerical simulations of AF drivers in a 2-dimensional model and optical mapping of ovine atrial scar-related AF. Results Ablation at dispersion areas terminated AF in 95%. After ablation of 17±10% of the left atrial surface and 18 months of follow-up, the atrial arrhythmia recurrence rate was 15% after 1.4±0.5 procedure/patient vs 41% in the validation set after 1.5±0.5 procedure/patient (arrhythmia free-survival rates: 85% vs 59%, log rank P<0.001). In comparison with the validation set, radiofrequency times (49 ± 21 minutes vs 85 ± 34.5 minutes, p=0.001) and procedure times (168 ± 42 minutes vs. 230 ± 67 minutes, p<.0001) were shorter. In simulations and optical mapping experiments, virtual PentaRay recordings demonstrated that electrogram dispersion is mostly recorded in the vicinity of a driver. Conclusions The clustering of intra-cardiac electrograms exhibiting spatio-temporal dispersion is indicative of AF drivers. Their ablation allows for a non-extensive and patient-tailored approach to AF ablation. Clinical trial.gov number: NCT02093949 PMID:28104073

Unstable Expression of Commonly Used Reference Genes in Rat Pancreatic Islets Early after Isolation Affects Results of Gene Expression Studies.

PubMed

Kosinová, Lucie; Cahová, Monika; Fábryová, Eva; Týcová, Irena; Koblas, Tomáš; Leontovyč, Ivan; Saudek, František; Kříž, Jan

2016-01-01

The use of RT-qPCR provides a powerful tool for gene expression studies; however, the proper interpretation of the obtained data is crucially dependent on accurate normalization based on stable reference genes. Recently, strong evidence has been shown indicating that the expression of many commonly used reference genes may vary significantly due to diverse experimental conditions. The isolation of pancreatic islets is a complicated procedure which creates severe mechanical and metabolic stress leading possibly to cellular damage and alteration of gene expression. Despite of this, freshly isolated islets frequently serve as a control in various gene expression and intervention studies. The aim of our study was to determine expression of 16 candidate reference genes and one gene of interest (F3) in isolated rat pancreatic islets during short-term cultivation in order to find a suitable endogenous control for gene expression studies. We compared the expression stability of the most commonly used reference genes and evaluated the reliability of relative and absolute quantification using RT-qPCR during 0-120 hrs after isolation. In freshly isolated islets, the expression of all tested genes was markedly depressed and it increased several times throughout the first 48 hrs of cultivation. We observed significant variability among samples at 0 and 24 hrs but substantial stabilization from 48 hrs onwards. During the first 48 hrs, relative quantification failed to reflect the real changes in respective mRNA concentrations while in the interval 48-120 hrs, the relative expression generally paralleled the results determined by absolute quantification. Thus, our data call into question the suitability of relative quantification for gene expression analysis in pancreatic islets during the first 48 hrs of cultivation, as the results may be significantly affected by unstable expression of reference genes. However, this method could provide reliable information from 48 hrs onwards.

Measurement of Mitochondrial Respiration in Isolated Protoplasts: Cytochrome and Alternative Pathways.

PubMed

Sunil, Bobba; Raghavendra, Agepati S

2017-01-01

The electron partitioning between COX and AOX pathways of mitochondria and their coordination is necessary to meet the energy demands as well as to maintain optimized redox status in plants under varying environmental conditions. The relative contribution of these two pathways to total respiration is an important measure during a given stress condition. We describe in detail the procedure that allows the measurement of the parameters of COX and AOX pathway of respiration in mesophyll protoplasts using Clark-type O 2 electrode. This chapter also lists the steps for rapid isolation procedure for mesophyll protoplasts from pea leaves. The advantages and limitations of the use of metabolic inhibitors and the protoplasts for measuring the respiration are also briefly discussed.

Direct isolation of a functional violaxanthin cycle domain from thylakoid membranes of higher plants.

PubMed

Goss, Reimund; Greifenhagen, Anne; Bergner, Juliane; Volke, Daniela; Hoffmann, Ralf; Wilhelm, Christian; Schaller-Laudel, Susann

2017-04-01

A special domain of the thylakoid membrane of higher plants has been isolated which carries out the de-epoxidation of the xanthophyll cycle pigment violaxanthin to zeaxanthin. Recent models indicate that in the chloroplast of higher plants, the violaxanthin (V) cycle takes place within specialized domains in the thylakoid membrane. Here, we describe a new procedure to directly isolate such a domain in functional state. The procedure consists of a thylakoid membrane isolation at a pH value of 5.2 which realizes the binding of the enzyme V de-epoxidase (VDE) to the membrane throughout the preparation process. Isolated thylakoid membranes are then solubilized with the very mild detergent n-dodecyl α-D-maltoside and the pigment-protein complexes are separated by sucrose gradient ultracentrifugation. The upper main fraction of the sucrose gradient represents a V cycle domain which consists of the major light-harvesting complex of photosystem II (LHCII), a special lipid composition with an enrichment of the galactolipid monogalactosyldiacylglycerol (MGDG) and the VDE. The domain is isolated in functional state as evidenced by the ability to convert the LHCII-associated V to zeaxanthin. The direct isolation of a V cycle domain proves the most important hypotheses concerning the de-epoxidation reaction in intact thylakoid membranes. It shows that the VDE binds to the thylakoid membrane at low pH values of the thylakoid lumen, that it binds to membrane regions enriched in LHCII, and that the domain contains high amounts of MGDG. The last point is in line with the importance of the galactolipid for V solubilisation and, by providing inverted hexagonal lipid structures, for VDE activity.

Bone Marrow Biopsy: RNA Isolation with Expression Profiling in Men with Metastatic Castration-resistant Prostate Cancer—Factors Affecting Diagnostic Success

PubMed Central

Afonso, P. Diana; Vinson, Emily N.; Turnbull, James D.; Morris, Karla K.; Foye, Adam; Madden, John F.; Roy Choudhury, Kingshuk; Febbo, Phillip G.; George, Daniel J.

2013-01-01

Purpose To determine the rate at which computed tomographically guided pelvic percutaneous bone biopsy in men with metastatic castration-resistant prostate cancer (mCRPC) yields adequate tissue for genomic profiling and to identify issues likely to affect diagnostic yields. Materials and Methods This study was institutional review board approved, and written informed consent was obtained. In a phase II trial assessing response to everolimus, 31 men with mCRPC underwent 54 biopsy procedures (eight men before and 23 men both before and during treatment). Variables assessed were lesion location (iliac wing adjacent to sacroiliac joint, iliac wing anterior and/or superior to sacroiliac joint, sacrum, and remainder of pelvis), mean lesion attenuation, subjective lesion attenuation (purely sclerotic vs mixed), central versus peripheral lesion sampling, lesion size, core number, and use of zoledronic acid for more than 1 year. Results Of 54 biopsy procedures, 21 (39%) yielded adequate tissue for RNA isolation and genomic profiling. Three of four sacral biopsies were adequate. Biopsies of the ilium adjacent to the sacroiliac joints were more likely adequate than those from elsewhere in the ilium (48% vs 28%, respectively). All five biopsies performed in other pelvic locations yielded inadequate tissue for RNA isolation. Mean attenuation of lesions with inadequate tissue was 172 HU greater than those with adequate tissue (621.1 HU ± 166 vs 449 HU ± 221, respectively; P = .002). Use of zoledronic acid, peripheral sampling, core number, and lesion size affected yields, but the differences were not statistically significant. Histologic examination with hematoxylin-eosin staining showed that results of 36 (67%) biopsies were positive for cancer; only mean attenuation differences were significant (707 HU ± 144 vs 473 HU ± 191, negative vs positive, respectively; P < .001). Conclusion In men with mCRPC, percutaneous sampling of osseous metastases for genomic profiling is possible, but use of zoledronic acid for more than 1 year may reduce the yield of adequate tissue for RNA isolation. Sampling large low-attenuating lesions at their periphery maximizes yield. © RSNA, 2013 PMID:23925271

High-grade renal injuries are often isolated in sports-related trauma

PubMed Central

Patel, Darshan P.; Redshaw, Jeffrey D.; Breyer, Benjamin N.; Smith, Thomas G.; Erickson, Bradley A.; Majercik, Sarah D.; Gaither, Thomas W.; Craig, James R.; Gardner, Scott; Presson, Angela P.; Zhang, Chong; Hotaling, James M.; Brant, William O.; Myers, Jeremy B.

2016-01-01

Introduction Most high-grade renal injuries (American Association for Surgery of Trauma (AAST) grades III–V) result from motor vehicle collisions associated with numerous concomitant injuries. Sports-related blunt renal injury tends to have a different mechanism, a solitary blow to the flank. We hypothesized that high-grade renal injury is often isolated in sports-related renal trauma. Material and methods We identified patients with AAST grades III–V blunt renal injuries from four level 1 trauma centres across the United States between 1/2005 and 1/2014. Patients were divided into “Sport” or “Non-sport” related groups. Outcomes included rates of hypotension (systolic blood pressure <90 mm Hg), tachycardia (>110 bpm), concomitant abdominal injury, and procedural/surgical intervention between sports and non-sports related injury. Results 320 patients met study criteria. 18% (59) were sports-related injuries with the most common mechanisms being skiing, snowboarding and contact sports (25%, 25%, and 24%, respectively). Median age was 24 years for sports and 30 years for non-sports related renal injuries (p = 0.049). Males were more commonly involved in sports related injuries (85% vs. 72%, p = 0.011). Median injury severity score was lower for sports related injuries (10 vs. 27, p < 0.001). There was no difference in renal abbreviated injury scale scores. Sports related trauma was more likely to be isolated without other significant injury (69% vs. 39% (p < 0.001)). Haemodynamic instability was present in 40% and 51% of sports and non-sports renal injuries (p = 0.30). Sports injuries had lower transfusion (7% vs. 47%, p < 0.001) and lower mortality rates (0% vs. 6%, p = 0.004). There was no difference in renal-specific procedural interventions between the two groups (17% sports vs. 18% non-sports, p = 0.95). Conclusions High-grade sports-related blunt renal trauma is more likely to occur in isolation without other abdominal or thoracic injuries and clinicians must have a high suspicion of renal injury with significant blows to the flank during sports activities. PMID:25769197

The Payer and Patient Cost Burden of Open Breast Conserving Procedures Following Percutaneous Breast Biopsy.

PubMed

Kimball, Chloe C; Nichols, Christine I; Vose, Joshua G

2018-01-01

Percutaneous core-needle biopsy (PCNB) is the standard of care to biopsy and diagnose suspicious breast lesions. Dependent on histology, many patients require additional open procedures for definitive diagnosis and excision. This study estimated the payer and patient out-of-pocket (OOP) costs, and complication risk, among those requiring at least 1 open procedure following PCNB. This retrospective study used the Truven Commercial database (2009-2014). Women who underwent PCNB, with continuous insurance, and no history of cancer, chemotherapy, radiation, or breast surgery in the prior year were included. Open procedures were defined as open biopsy or lumpectomy. Study follow-up ended at chemotherapy, radiation, mastectomy, or 90 days-whichever occurred first. In total, 143 771 patients (mean age 48) met selection criteria; 85.1% underwent isolated PCNB, 12.4% one open procedure, and 2.5% re-excision. Incidence of complications was significantly lower among those with PCNB alone (9.2%) vs 1 open procedure (15.6%) or re-excision (25.3%, P  < .001). Mean incremental commercial payments were US $13 190 greater among patients with 1 open procedure vs PCNB alone (US $17 125 vs US $3935, P  < .001), and US $4767 greater with re-excision (US $21 892) relative to 1 procedure. Mean patient OOP cost was US $858 greater for 1 open procedure vs PCNB alone (US $1527 vs US $669), and US $247 greater for re-excision vs 1 procedure. A meaningful proportion of patients underwent open procedure(s) following PCNB which was associated with increased complication risk and costs to both the payer and the patient. These results suggest a need for technologies to reduce the proportion of cases requiring open surgery and, in some cases, re-excision.

Attitude control concepts for precision-pointing nonrigid spacecraft

NASA Technical Reports Server (NTRS)

Likins, P. W.

1974-01-01

Literal criteria are developed for the controllability and observability of general models of flexible spacecraft. Results are interpreted in special cases and in physical terms, permitting in some cases the identification of uncontrollable and unobservable states simply by examination of scalars composed of modal parameters and location matrices for sensors and actuators. A procedure is established for isolation of uncontrollable states, whereby sensor and actuator configurations assure that uncontrollable flexible mode states are also unobservable; in many applications such states can then be removed by coordinate truncation.

WIPP conceptual design report. Addendum A. Design calculations for Waste Isolation Pilot Plant (WIPP)

DOE Office of Scientific and Technical Information (OSTI.GOV)

Not Available

1977-04-01

The design calculations for the Waste Isolation Pilot Plant (WIPP) are presented. The following categories are discussed: general nuclear calculations; radwaste calculations; structural calculations; mechanical calculations; civil calculations; electrical calculations; TRU waste surface facility time and motion analysis; shaft sinking procedures; hoist time and motion studies; mining system analysis; mine ventilation calculations; mine structural analysis; and miscellaneous underground calculations.

Construction of a transfer vector for a clonal isolate of LdNPV

Treesearch

Shivanand T. Hiremath; Martha Fikes; Audrey Ichida

1991-01-01

Deoxyribonucleic acid from a clonal isolate of LdNPV (CI A2-1), obtained by in vivo cloning procedures, was used to construct genomic libraries in phage (lamda Gem 11) and cosmid (pHC79) vectors. Overlapping clones were selected to generate a restriction enzyme map. The restriction enzyme map, covering about 85% of the CI A2-1 genome, was determined...

Chromatographic method for the isolation of active 40S and 60S subunits from rat liver polyribosomes.

PubMed

Kopacz-Jodczyk, T; Paszkiewicz-Gadek, A; Lopaczyński, W; Gałasiński, W

1984-04-06

A rapid and simple procedure for isolation of 40S and 60S ribosomal subunits by ion-exchange column chromatography is described. The dissociated ribosomes can be separated and non-ribosomal proteins and low-molecular-weight substances removed. An assessment by physicochemical and functional criteria showed that the ribosomal subunits obtained are active and sufficiently homogeneous.

Protein distribution in lupin protein isolates from Lupinus angustifolius L. prepared by various isolation techniques.

PubMed

Muranyi, Isabel S; Volke, Daniela; Hoffmann, Ralf; Eisner, Peter; Herfellner, Thomas; Brunnbauer, Markus; Koehler, Peter; Schweiggert-Weisz, Ute

2016-09-15

Differences in the protein distribution of various protein isolates from Lupinus angustifolius L. Vitabor were identified as affected by the isolation procedure (alkaline and/or salt-induced extraction followed by isoelectric and/or dilutive precipitation). Protein isolates extracted in alkaline solution showed higher protein yields (26.4-31.7%) compared to salt-induced extraction (19.8-30.0%) or combined alkaline and salt-induced extraction (23.3-25.6%). Chemical variations among the protein isolates especially occurred within the albumins. Protein isolates precipitated isoelectrically showed the highest contents, whereas protein isolates precipitated by dilutive showed the lowest contents of conglutin δ. Furthermore, the alkaline subunits of conglutin α and conglutin γ decreased during alkaline extraction compared to salt-induced extraction. A decrease in protein-bound polar and basic amino acids was shown after protein isolation. In contrast, the amounts of nonpolar, aliphatic, aromatic, hydroxylated and sulfur-rich amino acids were higher in the lupin protein isolates compared to the lupin flakes. However, the functional side chains could not be related to the specific molecular arrangements of the protein isolates, as a similar amino acid composition was found among the protein isolates. Copyright © 2016 Elsevier Ltd. All rights reserved.

The Effect of Atrial Fibrillation Ablation Techniques on P Wave Duration and P Wave Dispersion.

PubMed

Furniss, Guy O; Panagopoulos, Dimitrios; Kanoun, Sadeek; Davies, Edward J; Tomlinson, David R; Haywood, Guy A

2018-02-14

A reduction in surface electrocardiogram (ECG) P wave duration and dispersion is associated with improved outcomes in atrial fibrillation ablation. We investigated the effects of different ablation strategies on P wave duration and dispersion, hypothesising that extensive left atrial (LA) ablation with left atrial posterior wall isolation would give a greater reduction in P wave duration than more limited ablation techniques. A retrospective analysis of ECGs from patients who have undergone atrial fibrillation (AF) ablation was performed and pre-procedural sinus rhythm ECGs were compared with the post procedure ECGs. Maximal P wave duration was measured in leads I or II, minimum P wave duration in any lead and values were calculated for P wave duration and dispersion. Left atrial dimensions and medications at the time of ECG were documented. Ablation strategies compared were; pulmonary vein isolation (PVI) for paroxysmal atrial fibrillation (PAF) and the persistent AF (PsAF) ablation strategies of pulmonary vein isolation plus additional linear lesions (Lines), left atrial posterior wall isolation via catheter (PWI) and left atrial posterior wall isolation via staged surgical and catheter ablation (Hybrid). Sixty-nine patients' ECGs were analysed: 19 PVI, 21 Lines, 14 PWI, 15 Hybrid. Little correlation was seen between pre-procedure left atrial size and P wave duration (r=0.24) but LA size and P wave duration was larger in PsAF patients. A significant difference was seen in P wave reduction driven by Hybrid AF ablation (p<0.005) and Lines (<0.02). There was no difference amongst P wave dispersion between groups but the largest reduction was seen in the Hybrid ablation group. P wave duration increased with duration of continuous atrial fibrillation. Hybrid AF ablation significantly reduced P wave duration and dispersion compared to other ablation strategies including posterior wall isolation via catheter despite this being the same lesion set. Copyright © 2018 Australian and New Zealand Society of Cardiac and Thoracic Surgeons (ANZSCTS) and the Cardiac Society of Australia and New Zealand (CSANZ). Published by Elsevier B.V. All rights reserved.

Usefulness of Doppler assessment of pulmonary vein and left atrial appendage flow following pulmonary vein isolation of chronic atrial fibrillation in predicting recovery of left atrial function.

PubMed

Donal, Erwan; Grimm, Richard A; Yamada, Hirotsugu; Kim, Yong Jin; Marrouche, Nassir; Natale, Andrea; Thomas, James D

2005-04-15

Atrial fibrillation (AF) is a widespread condition that causes significant morbidity and mortality. Recently, pulmonary venous (PV) isolation using radiofrequency ablation has been used successfully to exclude the pulmonary venous ostia, resulting in correction of AF. Further, miniaturized high-frequency ultrasound phased-array transducers currently provide Doppler and 2-dimensional imaging during the ablation procedure. We examined atrial function and its determinants using intracardiac echocardiography before and after PV isolation in 45 patients who had chronic AF (56 +/- 11 years old). PV, left atrial (LA) appendage, and mitral and tricuspid flows were recorded. Recovery of booster pump function (defined by the presence of mitral inflow A wave, LA appendage a-wave, and PV A-reversal wave velocities >10 cm/s) was observed in 39 of 45 patients (86.6%). PV flow systolic wave before and after ablation correlated with the degree of LA booster pump function after PV isolation. An early systolic PV flow peak velocity >57.47 cm/s predicted "good" LA booster pump function recovery with 96% specificity. Diastolic LA appendage emptying in AF correlated (p <0.001) and predicted good LA booster pump function with 92% specificity for velocities >46.4 cm/s. Thus, monitoring LA function during PV isolation for chronic AF is feasible. Most patients recovered LA booster pump function immediately after PV isolation, and the degree of recovery correlated with LA reservoir function. Preserved reservoir function during AF is predictive of satisfactory recovery of booster pump function after PV isolation.

Anaerobic infections in surgical wards: a two year study

PubMed Central

Ananth-Shenoy, Padmaja; Vishwanath, Shashidhar; Targain, Ryumzook; Shetty, Seema; Sunil-Rodrigues, Gabriel; Mukhopadhyay, Chiranjay; Chawla, Kiran

2016-01-01

Background and Objectives: Anaerobic bacteria are recognized as important pathogens in surgical infections. However, they are the most overlooked microorganisms by the clinic and the laboratory because of the tedious culture techniques with longer turn-around times. The study was aimed to analyze the frequency of anaerobic bacterial surgical infections and their predisposing factors. Materials and Methods: A retrospective study was conducted over a period of two years including patients with surgical infections. The specimens were processed by Gram staining, aerobic and anaerobic culture. The anaerobic bacteria were isolated using standard procedures. The predisposing factors and clinical presentation were studied in these patients. Results: A total of 261 specimens were received from patients with diverse infections from surgical wards. Ninety-one anaerobes were isolated from 64 (24.5%) surgical patients with a predominance of Gram-negative bacilli (37.4%). Anaerobic bacteria as monomicrobial isolates were seen in 21.9% isolates. Anaerobic bacterial isolation along with aerobic bacteria was seen in 71.9% of patients and polymicrobial anaerobic growth was detected in 6.3% of patients. Diabetes mellitus (28, 43.8%) was found to be the most frequent predisposing factor. Bacteroides fragilis group (20.9%) were the most frequent anaerobic Gram-negative bacilli followed by Prevotella spp. (12.1%). Peptostreptococcus anaerobius was the predominant anaerobic cocci isolated (14.3%). Necrotizing fascitis (34.4%) was the most common clinical presentation with anaerobic etiology followed by deep seated abscesses (23.4%). Conclusion: Anaerobic bacteria were isolated from a significant proportion of surgical infections. To avoid therapeutic failures, anaerobic bacteria in surgical infections need to be recognized by surgeons and laboratorians. PMID:27928485

Development of a method to extend by boron neutron capture process the therapeutic possibilities of a liver autograft

NASA Astrophysics Data System (ADS)

Pinelli, Tazio; Altieri, Saverio; Fossati, F.; Zonta, Aris; Prati, U.; Roveda, L.; Nano, Rosanna

1997-02-01

We present results on surgical technique, neutron filed and irradiation facility concerning the original treatment of the liver diffused metastases. Our method plans to irradiate the isolated organ at a thermal neutron field soon after having been explanted and boron enriched and before being grafted into the same donor. In particular the crucial point of boron uptake was investigated by a rat model with a relevant number of procedure. We give for the first time statistically significant results on the selective boron absorption by tumor tissues.

Autologous microtia reconstruction combined with ancillary procedures: a comprehensive reconstructive approach.

PubMed

Cugno, S; Farhadieh, R D; Bulstrode, N W

2013-11-01

Autologous microtia reconstruction is generally performed in two stages. The second stage presents a unique opportunity to carry out other complementary procedures. The present study describes our approach to microtia reconstruction, wherein the second stage of reconstruction is combined with final refinements to the ear construct and/or additional procedures to enhance facial contour and symmetry. Retrospective analysis of patients who underwent two-stage microtia reconstruction by a single surgeon (NWB) was conducted in order to ascertain those that had ancillary procedures at the time of the second stage. Patient and operative details were collected. Thirty-four patients (male, 15, median age and age range at second stage, 11 and 10-18 years, respectively) who had complementary procedures executed during the second stage of auricular reconstruction were identified. Collectively, these included centralizing genioplasty (n = 1), fat transfer (n = 22), ear piercing (n = 7), and contralateral prominauris correction (n = 7). Six patients had correction for unilateral isolated microtia and in the remaining 28 patients, auricular reconstruction for microtia associated with a named syndrome. All patients reported a high rate of satisfaction with the result achieved and the majority (85%) reported no perceived need for additional surgical refinements to the ear or procedure(s) to achieve further facial symmetry. No peri- or post-operative complications were noted. Combining the final stage of autologous microtia reconstruction with other ancillary procedures affords a superior aesthetic outcome and decreased patient morbidity. Copyright © 2013 British Association of Plastic, Reconstructive and Aesthetic Surgeons. All rights reserved.

Effect of Direct Ligament Repair and Tenodesis Reconstruction on Simulated Subtalar Joint Instability.

PubMed

Choisne, Julie; Hoch, Matthew C; Alexander, Ian; Ringleb, Stacie I

2017-03-01

Subtalar instability is associated with up to 80% of patients presenting with chronic ankle instability but is often not considered in the diagnosis or treatment. Operative procedures to repair ankle instability have shown good clinical results, but the effects of these reconstruction procedures on isolated subtalar instability are not well understood. The goal of this study was to investigate the effect of the Gould modification of the Broström procedure and a new tenodesis reconstruction procedure on ankle and subtalar joint kinematics after simulating a subtalar injury. Kinematic data were collected on 7 cadaveric ankles during inversion through the range of ankle flexion and during internal rotation. Testing was performed on the intact foot; after sectioning the calcaneofibular ligament, cervical ligament, and interosseous talocalcaneal ligament; after the Gould modification of the Broström procedure was performed; and after tenodesis was performed and sutures from the Gould modification removed. The Gould modification of the Broström procedure significantly decreased subtalar and ankle inversion motion and subtalar internal rotation compared to the unstable condition. The tenodesis method restricted internal rotation at the subtalar joint and ankle inversion compared to the intact state. Both operative procedures improved stability of the ankle complex, but tenodesis was unable to restore subtalar inversion and restricted ankle inversion in maximum plantarflexion. The Gould modification of Broström ligament repair may be a favorable operative procedure for the restoration of subtalar and ankle joint kinematics.

Ocular surface infections in northeastern state of malaysia: a 10-year review of bacterial isolates and antimicrobial susceptibility.

PubMed

Rahman, Zaidah A; Harun, Azian; Hasan, Habsah; Mohamed, Zeehaida; Noor, Siti S Md; Deris, Zakuan Z; Ismail, Nabilah; Hassan, Asma S; Ahmad, Fadzhilah; Yaakub, Azhany

2013-09-01

Ocular surface infections that include infections of conjunctiva, adnexa, and cornea have the potential risk of causing blindness within a given population. Empirical antibiotic therapy is usually initiated based on epidemiological data of common causative agents. Thus, the aims of this study were to determine the bacterial agents and their susceptibility patterns of isolates from ocular surface specimens in our hospital. This is a retrospective analysis and records of bacterial isolates from ocular surface specimens in Hospital Universiti Sains Malaysia from January 2001 to December 2010 were examined. Specimens were processed according to standard laboratory procedures. Antimicrobial susceptibility testing was conducted based on Clinical and Laboratory Standards Institute recommendations. Only single, nonrepetitive isolates were included in the analysis. A total of 1,267 isolates were obtained during the study period, which comprised Staphylococcus aureus (n = 299, 23.6%), Pseudomonas aeruginosa (n = 194, 15.3%), Streptococcus pneumoniae (n = 108, 8.5%), Haemophilus influenzae (n = 100, 7.9%), Haemophilus parainfluenzae (n = 84, 6.6%), and Enterobacter spp. (n = 81, 6.4%). Fungi contributed to 4.4% of the total isolates. The antimicrobial susceptibility testing demonstrated that gram-positive bacteria were generally resistant to gentamicin (19%-57%), whereas gram-negative bacteria were resistant to chloramphenicol (27%-58%). Based on the above results, knowledge of the initial Gram stain findings is imperative before the commencement of empirical antibiotic therapy. Therefore, a simple Gram staining for all eye specimens is highly recommended.

Postenrichment Population Differentials Using Buffered Listeria Enrichment Broth: Implications of the Presence of Listeria innocua on Listeria monocytogenes in Food Test Samples†

PubMed Central

KEYS, ASHLEY L.; DAILEY, RACHEL C.; HITCHINS, ANTHONY D.; SMILEY, R. DERIKE

2016-01-01

The recovery of low levels of Listeria monocytogenes from foods is complicated by the presence of competing microorganisms. Nonpathogenic species of Listeria pose a particular problem because variation in growth rate during the enrichment step can produce more colonies of these nontarget cells on selective and/or differential media, resulting in a preferential recovery of nonpathogens, especially Listeria innocua. To gauge the extent of this statistical barrier to pathogen recovery, 10 isolates each of L. monocytogenes and L. innocua were propagated together from approximately equal initial levels using the current U.S. Food and Drug Administration’s enrichment procedure. In the 100 isolate pairs, an average 1.3-log decrease was found in the 48-h enrichment L. monocytogenes population when L. innocua was present. In 98 of the 100 isolate pairs, L. innocua reached higher levels at 48 h than did L. monocytogenes, with a difference of 0.2 to 2.4 log CFU/ml. The significance of these population differences was apparent by an increase in the difficulty of isolating L. monocytogenes by the streak plating method. L. monocytogenes went completely undetected in 18 of 30 enrichment cultures even after colony isolation was attempted on Oxoid chromogenic Listeria agar. This finding suggests that although both Listeria species were present on the plate, the population differential between them restricted L. monocytogenes to areas of the plate with confluent growth and that isolated individual colonies were only L. innocua. PMID:24215687

Time-to-isolation guided titration of freeze duration in 3rd generation short-tip cryoballoon pulmonary vein isolation - Comparable clinical outcome and shorter procedure duration.

PubMed

Pott, Alexander; Kraft, Christoph; Stephan, Tilman; Petscher, Kerstin; Rottbauer, Wolfgang; Dahme, Tillman

2018-03-15

The optimal freeze duration in cryoballoon pulmonary vein isolation (PVI) is unknown. The 3rd generation cryoballoon facilitates observation of the time-to-isolation (TTI) and thereby enables individualized cryoenergy titration. To evaluate the efficacy of an individualized freeze duration we compared the clinical outcome of patients treated with a TTI-guided ablation protocol to the outcome of patients treated with a fixed ablation protocol. We compared 100 patients treated with the 3rd generation cryoballoon applying a TTI-based protocol (TTI group) to 100 patients treated by a fixed freeze protocol (fixed group). In the fixed group a 240s freeze cycle was followed by a 240s bonus freeze after acute PV isolation. In the TTI group freeze duration was 180s if TTI was ≥30s and reduced to only 120s, if TTI was <30s. In case of a TTI >60s a 180s bonus freeze was applied. Freedom from atrial arrhythmia recurrence off class I/III antiarrhythmic drugs after one year was not different between the TTI group (73.6%) and the fixed group (75.7%; p=0.75). Mean procedure duration was 85.8±27.3min in the TTI group compared to 115.7±27.1min in the fixed group (p<0.001). Mean fluoroscopy time was 17.5±6.6min in the TTI group and 22.5±9.8min in the fixed group (p<0.001). TTI-guided cryoenergy titration leads to reduced procedure duration and fluoroscopy time and appears to be as effective as a fixed ablation strategy. A single 2-minute freeze seems to be sufficient in case of short TTI. Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.

Avoidance of Maternal Cell Contamination and Overgrowth in Isolating Fetal Chorionic Villi Mesenchymal Stem Cells from Human Term Placenta

PubMed Central

Sardesai, Varda S.; Shafiee, Abbas; Fisk, Nicholas M.

2017-01-01

Abstract Human placenta is rich in mesenchymal stem/stromal cells (MSC), with their origin widely presumed fetal. Cultured placental MSCs are confounded by a high frequency of maternal cell contamination. Our recent systematic review concluded that only a small minority of placental MSC publications report fetal/maternal origin, and failed to discern a specific methodology for isolation of fetal MSC from term villi. We determined isolation conditions to yield fetal and separately maternal MSC during ex vivo expansion from human term placenta. MSCs were isolated via a range of methods in combination; selection from various chorionic regions, different commercial media, mononuclear cell digest and/or explant culture. Fetal and maternal cell identities were quantitated in gender‐discordant pregnancies by XY chromosome fluorescence in situ hybridization. We first demonstrated reproducible maternal cell contamination in MSC cultures from all chorionic anatomical locations tested. Cultures in standard media rapidly became composed entirely of maternal cells despite isolation from fetal villi. To isolate pure fetal cells, we validated a novel isolation procedure comprising focal dissection from the cotyledonary core, collagenase/dispase digestion and explant culture in endothelial growth media that selected, and provided a proliferative environment, for fetal MSC. Comparison of MSC populations within the same placenta confirmed fetal to be smaller, more osteogenic and proliferative than maternal MSC. We conclude that in standard media, fetal chorionic villi‐derived MSC (CV‐MSC) do not grow readily, whereas maternal MSC proliferate to result in maternal overgrowth during culture. Instead, fetal CV‐MSCs require isolation under specific conditions, which has implications for clinical trials using placental MSC. Stem Cells Translational Medicine 2017;6:1070–1084 PMID:28205414

Fate of Listeria monocytogenes in tissues of experimentally infected cattle and in hard salami.

PubMed Central

Johnson, J L; Doyle, M P; Cassens, R G; Schoeni, J L

1988-01-01

Muscle, organ, and lymphoid tissues of four Holstein cows experimentally inoculated (intravenously) with Listeria monocytogenes were examined 2, 6, or 54 days postinoculation for the presence of the organism by direct plating and cold enrichment procedures. L. monocytogenes was isolated from 66% of the tissues sampled; 38% of the isolations were attributed to the use of cold enrichment. Isolation of the organism from muscle tissue was possible only with animals inoculated 2 days before slaughter. The fate of L. monocytogenes during the manufacture and storage of fermented hard salami made from this meat also was determined. Three sausage treatments were evaluated: (i) uninoculated control sausage, (ii) "naturally" contaminated sausage (NC) made from meat of an experimentally inoculated cow, and (iii) sausage made from beef inoculated with a laboratory culture of L. monocytogenes (I). Initial Listeria levels in NC and I sausage were 10(3) CFU/g in trial 1 and 10(4) CFU/g in trial 2. Numbers of L. monocytogenes decreased by approximately 1 log10 CFU/g during fermentation and decreased further during drying and refrigerated storage. Small numbers (less than or equal to 20 CFU/g) of L. monocytogenes were present in I and NC sausage at the end of 12 weeks of refrigerated storage; recovery of these organisms generally depended on the use of an enrichment procedure. The results indicate that L. monocytogenes does not multiply during the fermentation and drying processes typical of hard salami manufacture but that survival may occur if the organism is initially present at greater than or equal to 10(3) CFU/g. PMID:3128165

Separation of periportal and perivenous rat hepatocytes by fluorescence-activated cell sorting: confirmation with colloidal gold as an exogenous marker.

PubMed

Braakman, I; Keij, J; Hardonk, M J; Meijer, D K; Groothuis, G M

1991-01-01

Periportal and perivenous hepatocytes are known to display various functional differences. In this study we present a new method to separate periportal and perivenous cells: after selectively loading zone 1 or zone 3 with the fluorescent label acridine orange in an antegrade or retrograde perfusion, respectively, we separated the isolated hepatocytes on a fluorescence-activated cell sorter. The common way to check on proper separation is to estimate activities of enzymes known to exhibit a heterogeneous acinar distribution. Using enzyme histochemistry, however, we found that already on short collagenase perfusion, some enzymes displayed a more shallow gradient than in vivo, making enzyme activities less suitable as zonal markers. We therefore used colloidal gold granules (17 nm) injected intravenously (2.5 mg) into the rat 2 to 3 hr before cell isolation. The gold is taken up predominantly by perivenous hepatocytes, probably because of the efficient removal of gold granules in zone 1 by competing Kupffer cells. We compared acridine orange fluorescence, presence of gold particles and activities of six marker enzymes, three biochemically and three histochemically determined. Acridine orange and gold both pointed to a high enrichment of the fractions, whereas most enzyme activities were more randomly distributed among the cells as a result of the isolation procedure. Our separation procedure yielded fractions highly enriched in either viable periportal or perivenous cells, both from one liver. The use of colloidal gold as a marker to monitor separation is a valuable alternative to the more risky estimation of enzyme activities.

Optimization of a wet microalgal lipid extraction procedure for improved lipid recovery for biofuel and bioproduct production.

PubMed

Sathish, Ashik; Marlar, Tyler; Sims, Ronald C

2015-10-01

Methods to convert microalgal biomass to bio based fuels and chemicals are limited by several processing and economic hurdles. Research conducted in this study modified/optimized a previously published procedure capable of extracting transesterifiable lipids from wet algal biomass. This optimization resulted in the extraction of 77% of the total transesterifiable lipids, while reducing the amount of materials and temperature required in the procedure. In addition, characterization of side streams generated demonstrated that: (1) the C/N ratio of the residual biomass or lipid extracted (LE) biomass increased to 54.6 versus 10.1 for the original biomass, (2) the aqueous phase generated contains nitrogen, phosphorous, and carbon, and (3) the solid precipitate phase was composed of up to 11.2 wt% nitrogen (70% protein). The ability to isolate algal lipids and the possibility of utilizing generated side streams as products and/or feedstock material for downstream processes helps promote the algal biorefinery concept. Copyright © 2015 Elsevier Ltd. All rights reserved.

A comparison of methods for teaching receptive labeling to children with autism spectrum disorders.

PubMed

Grow, Laura L; Carr, James E; Kodak, Tiffany M; Jostad, Candice M; Kisamore, April N

2011-01-01

Many early intervention curricular manuals recommend teaching auditory-visual conditional discriminations (i.e., receptive labeling) using the simple-conditional method in which component simple discriminations are taught in isolation and in the presence of a distracter stimulus before the learner is required to respond conditionally. Some have argued that this procedure might be susceptible to faulty stimulus control such as stimulus overselectivity (Green, 2001). Consequently, there has been a call for the use of alternative teaching procedures such as the conditional-only method, which involves conditional discrimination training from the onset of intervention. The purpose of the present study was to compare the simple-conditional and conditional-only methods for teaching receptive labeling to 3 young children diagnosed with autism spectrum disorders. The data indicated that the conditional-only method was a more reliable and efficient teaching procedure. In addition, several error patterns emerged during training using the simple-conditional method. The implications of the results with respect to current teaching practices in early intervention programs are discussed.

A surgical support system for Space Station Freedom

NASA Technical Reports Server (NTRS)

Campbell, M. R.; Billica, R. D.; Johnston, S. L.

1992-01-01

Surgical techniques in microgravity are being developed for the Health Maintenance Facility (HMF) on Space Station Freedom (SSF). This will be a presentation of the proposed surgical capabilities and ongoing hardware and procedural investigations. Methods: Procedures and prototype hardware, which include a medical restraint system, a surgical overhead isolation canopy, a suction device, and a regional laminar flow device were evaluated. This was accomplished by realistic sterile surgical simulations involving both mannequins and animals during KC-135 parabolic flight and in a high fidelity ground based HMF mockup. Results: Animal surgery in the environment of microgravity allowed the observation of unique arterial and venous bleeding characteristics for the first time. The ability to control bleeding and to prevent cabin atmosphere contamination was also demonstrated. Conclusions: The procedures and prototype hardware tested provided valuable information and should be investigated and developed further. The use of standard surgical techniques are possible in microgravity if the principles of personnel and supply restraint and operative field containment are adhered to.

Micro-organisms isolated from cadaveric samples of allograft musculoskeletal tissue.

PubMed

Varettas, Kerry

2013-12-01

Allograft musculoskeletal tissue is commonly used in orthopaedic surgical procedures. Cadaveric donors of musculoskeletal tissue supply multiple allografts such as tendons, ligaments and bone. The microbiology laboratory of the South Eastern Area Laboratory Services (SEALS, Australia) has cultured cadaveric allograft musculoskeletal tissue samples for bacterial and fungal isolates since 2006. This study will retrospectively review the micro-organisms isolated over a 6-year period, 2006-2011. Swab and tissue samples were received for bioburden testing and were inoculated onto agar and/or broth culture media. Growth was obtained from 25.1 % of cadaveric allograft musculoskeletal tissue samples received. The predominant organisms isolated were coagulase-negative staphylococci and coliforms, with the heaviest bioburden recovered from the hemipelvis. The rate of bacterial and fungal isolates from cadaveric allograft musculoskeletal tissue samples is higher than that from living donors. The type of organism isolated may influence the suitability of the allograft for transplant.

HIV-1 isolation from infected peripheral blood mononuclear cells.

PubMed

Dispinseri, Stefania; Saba, Elisa; Vicenzi, Elisa; Kootstra, Neeltje A; Schuitemaker, Hanneke; Scarlatti, Gabriella

2014-01-01

Human immunodeficiency virus 1 (HIV-1) isolation from peripheral blood mononuclear cells (PBMCs) allows retrieval of replication-competent viral variants. In order to impose the smallest possible selective pressure on the viral isolates, isolation must be carried out in primary cultures of cells and not in tumor derived cell lines. The procedure involves culture of PBMCs from an infected patient with phytohemagglutinin (PHA)-stimulated PBMC from seronegative donors, which provide susceptible target cells for HIV replication. HIV can be isolated from the bulk population of PBMCs or after cloning of the cells to obtain viral biological clones. Viral production is determined with p24 antigen (Ag) detection assays or with reverse transcriptase (RT) activity assay. Once isolated, HIV-1 can be propagated by infecting PHA-stimulated PBMCs from healthy donors. Aliquots from culture with a high production of virus are stored for later use.

Experimental evaluation of low-pass shock isolation performance of elastomers using frequency-based Kolsky bar analyses

DOE PAGES

Knight, Marlene E.; Sanborn, Brett; Song, Bo; ...

2017-01-26

Elastomeric materials are used as shock isolation materials in a variety of environments to dampen vibrations and/or absorb energy from external impact to minimize energy transfer between two objects or bodies. Some applications require the shock isolation materials to behave as a low-pass mechanical filter to mitigate the shock/impact at high frequencies but transmit the energy at low frequencies with minimal attenuation. To fulfill this requirement, a shock isolation material needs to be carefully evaluated and selected with proper experimental design, procedures, and analyses. In this study, a Kolsky bar was modified with precompression (up to 15.5 kN) and confinementmore » capabilities to evaluate low-pass shock isolation performance in terms of acceleration attenuation through a variety of elastomers. Also investigated were the effects of preload and specimen geometry on the low-pass shock isolation response.« less

Environmental considerations in the selection of isolation gowns: A life cycle assessment of reusable and disposable alternatives.

PubMed

Vozzola, Eric; Overcash, Michael; Griffing, Evan

2018-04-11

Isolation gowns serve a critical role in infection control by protecting healthcare workers, visitors, and patients from the transfer of microorganisms and body fluids. The decision of whether to use a reusable or disposable garment system is a selection process based on factors including sustainability, barrier effectiveness, cost, and comfort. Environmental sustainability is increasingly being used in the decision-making process. Life cycle assessment is the most comprehensive and widely used tool used to evaluate environmental performance. The environmental impacts of market-representative reusable and disposable isolation gown systems were compared using standard life cycle assessment procedures. The basis of comparison was 1,000 isolation gown uses in a healthcare setting. The scope included the manufacture, use, and end-of-life stages of the gown systems. At the healthcare facility, compared to the disposable gown system, the reusable gown system showed a 28% reduction in energy consumption, a 30% reduction in greenhouse gas emissions, a 41% reduction in blue water consumption, and a 93% reduction in solid waste generation. Selecting reusable garment systems may result in significant environmental benefits compared to selecting disposable garment systems. By selecting reusable isolation gowns, healthcare facilities can add these quantitative benefits directly to their sustainability scorecards. Copyright © 2018 Association for Professionals in Infection Control and Epidemiology, Inc. Published by Elsevier Inc. All rights reserved.

Molecular epidemiology of Candida auris in Colombia reveals a highly-related, country-wide colonization with regional patterns in Amphotericin B resistance.

PubMed

Escandón, Patricia; Chow, Nancy A; Caceres, Diego H; Gade, Lalitha; Berkow, Elizabeth L; Armstrong, Paige; Rivera, Sandra; Misas, Elizabeth; Duarte, Carolina; Moulton-Meissner, Heather; Welsh, Rory M; Parra, Claudia; Pescador, Luz Angela; Villalobos, Nohora; Salcedo, Soraya; Berrio, Indira; Varón, Carmen; Espinosa-Bode, Andrés; Lockhart, Shawn R; Jackson, Brendan R; Litvintseva, Anastasia P; Beltran, Mauricio; Chiller, Tom M

2018-05-16

Candida auris is a multidrug-resistant yeast associated with hospital outbreaks worldwide. During 2015-2016, multiple outbreaks were reported in Colombia. We aimed to understand the extent of contamination in healthcare settings and to characterize the molecular epidemiology of C. auris in Colombia. We sampled patients, patient contacts, healthcare workers, and the environment in four hospitals with recent C. auris outbreaks. Using standardized protocols, people were swabbed at different body sites. Patient and procedure rooms were sectioned into four zones and surfaces were swabbed. We performed whole-genome sequencing (WGS) and antifungal susceptibility testing (AFST) on all isolates. Seven (41%) of the 17 people swabbed were found to be colonized. C. auris was isolated from 37/322 (12%) environmental samples. These were collected from a variety of items in all four zones. WGS and AFST revealed that although isolates were similar throughout the country, isolates from the northern region were genetically distinct and more resistant to amphotericin B (AmB) than the isolates from central Colombia. Four novel non-synonymous mutations were found to be significantly associated with AmB resistance. Our results show that extensive C. auris contamination can occur and highlight the importance of adherence to appropriate infection control practices and disinfection strategies. Observed genetic diversity supports healthcare transmission and a recent expansion of C. auris within Colombia with divergent AmB susceptibility.

Laparoscopic sentinel lymph node procedure using a combination of patent blue and radioisotope in women with cervical carcinoma.

PubMed

Barranger, Emmanuel; Grahek, Dany; Cortez, Annie; Talbot, Jean Noel; Uzan, Serge; Darai, Emile

2003-06-15

The authors evaluated the feasibility of a laparoscopic sentinel lymph node (SN) procedure with combined radioisotopic and patent blue labeling in patients with cervical carcinoma. Thirteen women (median age, 52.5 years) with cervical carcinoma (Stage Ia2 in 1 patient, Stage Ib1 in 10 patients, Stage Ib2 in 1 patient, and Stage IIa in 1 patient) underwent a laparoscopic SN procedure using an endoscopic gamma probe after both radioactive isotope and patent blue injections. After the procedure, all patients underwent complete laparoscopic pelvic lymphadenectomy and either laparoscopic radical hysterectomy (eight patients) or the Schauta-Amreich operation (five patients). SNs (mean, 1.7 SNs per patient; range, 1-3 SNs per patient) were identified in 12 of 13 patients. A median of 10.5 pelvic lymph nodes per patient (range, 4-17 pelvic lymph nodes per patient) were removed. No lymph node involvement was detected in SNs with hematoxylin and eosin staining. Immunohistochemical studies identified four metastatic SNs in two patients, with micrometastases in two SNs from the first patient and isolated tumor cells in two SNs from the second patient. No false-negative SN results were obtained. The results of this study suggest that SN detection with a combination of radiocolloid and patent blue is feasible in patients with cervical carcinoma. The combination of laparoscopy and the SN procedure permitted minimally invasive management of early-stage disease. Copyright 2003 American Cancer Society.

Risk Factors Analysis for Occurrence of Asymptomatic Bacteriuria After Endourological Procedures

PubMed Central

Junuzovic, Dzelaludin; Hasanbegovic, Munira

2014-01-01

Introduction: Endourological procedures are performed according to the principles of aseptic techniques, jet still in certain number of patients urinary tract infections may occur. Considering the risk of urinary tract infection, there is no unique opinion about the prophylactic use of antibiotics in endourological procedures. Goal: The objective of this study was to determine the connection between endourological procedures and occurrence of urinary infections and to analyze the risk factors of urinary infection for patients who were hospitalized at the Urology Clinic of the Clinical Center University of Sarajevo CCUS. Materials and Methods: The research was conducted as a prospective study on a sample of 208 patients of both genders, who were hospitalized at the Urology Clinic of the CCUS and to whom some endourological procedure was indicated for diagnostic or therapeutic purposes. We analyzed data from patient’s histories of illness, laboratory tests taken at admission and after endourological procedures, also surgical programs for endoscopic procedures. All patients were clinically examined prior to endoscopic procedures while after the treatment attention was focused to the symptoms of urinary tract infections. Results: Statistical analysis of the tested patients indicates that there is no significant difference in the presence of postoperative, compared to preoperative bacteriuria, which implies that the endourological procedures are safe procedures in terms of urinary tract infections. Preoperatively, the most commonly isolated bacteria was Escherichia coli (30.9%) and postoperatively, Enterococcus faecalis (25%). Statistically significant effect on the occurrence of postoperative bacteriuria has preoperative bacteriuria, duration of postoperative catheterization, and duration of hospitalization. Conclusion: In everyday urological practice, it is very important to identify and control risk factors for the development of urinary infection after endourological procedures, with main objective to minimize occurrence of infectious complications. PMID:25568546

[Prompt control of an imported carbapenem-resistant Acinetobacter baumanii outbreak in a French intensive care unit].

PubMed

Guth, C; Cavalli, Z; Pernod, C; Lhopital, C; Wey, Pf; Gerome, P; Turc, J

2016-01-01

to describe the management and control of a limited outbreak of carbapenem-resistant Acinetobacter baumanii (CRAB) outbreak in a French intensive care unit. Careful review of the contact's and carrier's files and outbreak management procedures. An undiagnosed CRAB carrier was admitted to our intensive care unit after medical evacuation from Turkey. Despite preventive isolation and contact precautions, a secondary case was diagnosed 5 days after admission of the index case and resulted in the creation of a crisis unit. Prompt management included an epidemiologic investigation with contact screening and follow-up, environmental screening, and additional restrictive measures: isolation room, closure of adjacent rooms, patient cohorting with designated nurses, and reinforcement of contact precautions. restrictive management of CRAB outbreaks may allow prompt outbreak control and avoid prolonged room closures.

[Current knowledge on the strain typing of the pathogenic fungus Histoplasma capsulatum var. capsulatum: a review of the findings].

PubMed

Reyes-Montes, M del R; Taylor, M L; Curiel-Quesada, E; Mesa-Arango, A C

2000-12-01

The classification of microbial strains is currently based on different typing methods, which must meet certain criteria in order to be widely used. Phenotypic and genotypic methods are being employed in the epidemiology of several fungal diseases. However, some problems associated to the phenotypic methods have fostered genotyping procedures, from DNA polymorphic diversity to gene sequencing studies, all aiming to differentiate and to relate fungal isolates or strains. Through these studies, it is possible to identify outbreaks, to detect nosocomial infection transmission, and to determine the source of infection, as well as to recognize virulent isolates. This paper is aimed at analyzing the methods recently used to type Histoplasma capsulatum, causative agent of the systemic mycosis known as histoplasmosis, in order to recommend those that yield reproducible and accurate results.

Characterization of Radiation-Resistant Vegetative Bacteria in Beef1

PubMed Central

Welch, Ardyce B.; Maxcy, R. B.

1975-01-01

Ground beef contains numerous microorganisms of various types. The commonly recognized bacteria are associated with current problems of spoilage. Irradiation, however, contributes a new factor through selective destruction of the microflora. The residual microorganisms surviving a nonsterilizing dose are predominantly gram-negative coccobacilli. Various classifications have been given, e.g., Moraxella, Acinetobacter, Achromobacter, etc. For a more detailed study of these radiation-resistant bacteria occurring in ground beef, an enrichment procedure was used for isolation. By means of morphological and biochemical tests, most of the isolates were found to be Moraxella, based on current classifications. The range of growth temperatures was from 2 to 50 C. These bacteria were relatively heat sensitive, e.g., D10 of 5.4 min at 70 C or less. The radiation resistance ranged from D10 values of 273 to 2,039 krad. Thus, some were more resistant than any presently recognized spores. A reference culture of Moraxella osloensis was irradiated under conditions comparable to the enrichment procedure used with the ground beef. The only apparent changes were in morphology and penicillin sensitivity. However, after a few subcultures these bacteria reverted to the characteristics of the parent strain. Thus, it is apparent that these isolates are a part of the normal flora of ground beef and not aberrant forms arising from the irradiation procedure. The significance, if any, of these bacteria is not presently recognized. Images PMID:1164011

Characterization of radiation-resistant vegetative bacteria in beef.

PubMed

Welch, A B; Maxcy, R B

1975-08-01

Ground beef contains numerous microorganisms of various types. The commonly recognized bacteria are associated with current problems of spoilage. Irradiation, however, contributes a new factor through selective destruction of the microflora. The residual microorganisms surviving a nonsterilizing dose are predominantly gram-negative coccobacilli. Various classifications have been given, e.g., Moraxella, Acinetobacter, Achromobacter, etc. For a more detailed study of these radiation-resistant bacteria occurring in ground beef, an enrichment procedure was used for isolation. By means of morphological and biochemical tests, most of the isolates were found to be Moraxella, based on current classifications. The range of growth temperatures was from 2 to 50 C. These bacteria were relatively heat sensitive, e.g., D10 of 5.4 min at 70 C or less. The radiation resistance ranged from D10 values of 273 to 2,039 krad. Thus, some were more resistant than any presently recognized spores. A reference culture of Moraxella osloensis was irradiated under conditions comparable to the enrichment procedure used with the ground beef. The only apparent changes were in morphology and penicillin sensitivity. However, after a few subcultures these bacteria reverted to the characteristics of the parent strain. Thus, it is apparent that these isolates are a part of the normal flora of ground beef and not aberrant forms arising from the irradiation procedure. The significance, if any, of these bacteria is not presently recognized.

Biosafety evaluation of the DNA extraction protocol for Mycobacterium tuberculosis complex species, as implemented at the Instituto Nacional de Salud, Colombia.

PubMed

Castro, Claudia; González, Liliana; Rozo, Juan Carlos; Puerto, Gloria; Ribón, Wellman

2009-12-01

Manipulating Mycobacterium tuberculosis clinical specimens and cultures represents a risk factor for laboratory personnel. One of the processes that requires high concentrations of microorganisms is DNA extraction for molecular procedures. Pulmonary tuberculosis cases have occurred among professionals in charge of molecular procedures that require manipulation of massive quantities of microorganisms. This has prompted research studies on biosafety aspects of extraction protocols; however, as yet, no consensus has been reached regarding risks associated with the process. The biosafety was evaluated for the DNA extraction protocol of van Soolingen, et al. 2002 by determining M. tuberculosis viability at each process stage. Eight hundred eighty cultures were grown from 220 M. tuberculosis clinical isolates that had been processed through the first three DNA extraction stages. Molecular identifications of positive cultures used a PCR isolation of a fragment of the heat shock protein PRA-hsp65 and examination of its restriction enzyme profile (spoligotyping). Growth was seen in one culture with one of the procedures used. The molecular characterization did not correspond to the initially analyzed isolate, and therefore was deduced to be the product of a cross-contamination. The DNA extraction protocol, as described by van Soolingen, et al. 2002 and as implemented at the Instituto Nacional de Salud, was established to be safe for laboratory personnel as well as for the environment.

Phytochemistry of Cimicifugic Acids and Associated Bases in Cimicifuga racemosa Root Extracts

PubMed Central

GÖdecke, Tanja; Nikolic, Dejan; Lankin, David C.; Chen, Shao-Nong; Powell, Sharla L.; Dietz, Birgit; Bolton, Judy L.; Van Breemen, Richard B.; Farnsworth, Norman R.; Pauli, Guido F.

2009-01-01

Introduction Earlier studies reported serotonergic activity for cimicifugic acids (CA) isolated from Cimicifuga racemosa. The discovery of strongly basic alkaloids, cimipronidines, from the active extract partition and evaluation of previously employed work-up procedures has led to the hypothesis of strong acid/base association in the extract. Objective Re-isolation of the CAs was desired to permit further detailed studies. Based on the acid/base association hypothesis, a new separation scheme of the active partition was required, which separates acids from associated bases. Methodology A new 5-HT7 bioassay guided work-up procedure was developed that concentrates activity into one partition. The latter was subjected to a new 2-step centrifugal partitioning chromatography (CPC) method, which applies pH zone refinement gradient (pHZR CPC) to dissociate the acid/base complexes. The resulting CA fraction was subjected to a second CPC step. Fractions and compounds were monitored by 1H NMR using a structure based spin-pattern analysis facilitating dereplication of the known acids. Bioassay results were obtained for the pHZR CPC fractions and for purified CAs. Results A new CA was characterized. While none of the pure CAs was active, the serotonergic activity was concentrated in a single pHZR CPC fraction, which was subsequently shown to contain low levels of the potent 5-HT7 ligand, Nω–methylserotonin. Conclusion This study shows that CAs are not responsible for serotonergic activity in black cohosh. New phytochemical methodology (pHZR CPC) and a sensitive dereplication method (LC-MS) led to the identification of Nω–methylserotonin as serotonergic active principle. PMID:19140115

25 CFR 36.94 - What must a homeliving handbook contain?

Code of Federal Regulations, 2010 CFR

2010-04-01

...) Discipline Policy; (c) Parent/Student Rights and Responsibilities; (d) Confidentiality; (e) Sexual Harassment... Policy; (q) Medication Administration Policy and Procedure; and (r) Isolation/Separation Policy. ...

Unfavourable short-term outcomes of a poly-L/D-lactide scaffold for thumb trapeziometacarpal arthroplasty.

PubMed

Mattila, S; Waris, E

2016-03-01

The bioabsorbable poly-L-D-lactide joint scaffold arthroplasty is a recent attempt in the reconstruction of small joints in rheumatoid patients. In this study, we analysed the 1-year clinical, functional and radiologic results of partial trapeziectomy with the poly-L-D-lactide (96/4) joint scaffold in 23 patients with isolated trapeziometacarpal osteoarthritis. The results showed that the procedure provided pain relief and improvement in overall function according to the Quick Disabilities of the Arm, Shoulder and Hand score in most patients. However, radiographs demonstrated a high frequency of osteolysis around the implant. Seven patients developed clinically manifested foreign-body reactions 6 months to 1 year after surgery. The reason for the unexpected tissue reactions may relate to excessive mechanical cyclic loading of the implant. The outcomes of this implant in our patients have not been sufficiently beneficial and we have discontinued use of this implant in isolated trapeziometacarpal osteoarthritis. © The Author(s) 2015.

Fungal beta glucan protects radiation induced DNA damage in human lymphocytes

PubMed Central

Maurya, Dharmendra K.; Salvi, Veena P.; Janardhanan, Krishnankutty K; Nair, Cherupally K. K.

2014-01-01

Background Ganoderma lucidum (Ling Zhi), a basidiomycete white rot macrofungus has been used extensively for therapeutic use in China, Japan, Korea and other Asian countries for 2,000 years. The present study is an attempt to investigate its DNA protecting property in human lymphocytes. Materials and methods Beta glucan (BG) was isolated by standard procedure and the structure and composition were studied by infrared radiation (IR) and nuclear magnetic resonance (NMR) spectroscopy, gel filtration chromatography and paper chromatography. The radioprotective properties of BG isolated from the macro fungi Ganoderma lucidum was assessed by single cell gel electrophoresis (comet assay). Human lymphocytes were exposed to 0, 1, 2 and 4 Gy gamma radiation in the presence and absence of BG. Results The comet parameters were reduced by BG. The results indicate that the BG of G. lucidum possessed significant radioprotective activity with DNA repairing ability and antioxidant activity as the suggestive mechanism. Conclusions The findings suggest the potential use of this mushroom for the prevention of radiation induced cellular damages. PMID:25332989

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hahne, G.; Hoffmann, F.

A serious problem in the technology of plant cell culture is that isolated protoplasts from many species are reluctant to divide. We have succeeded in inducing consecutive divisions in a naturally arrested system i.e., protoplasts from a hibiscus cell line, which do not divide under standard conditions and in an artificially arrested system i.e., colchicine-inhibited callus protoplasts of Nicotiana glutinosa, which do readily divide in the absence of colchicine. In both cases, the reinstallation of a net of cortical microtubules, which had been affected either by colchicine or by the protoplast isolation procedure, resulted in continuous divisions of the formerlymore » arrested protoplasts. Several compounds known to support microtubule assembly in vitro were tested for their ability to promote microtubule assembly in vivo. Best results were obtained by addition of dimethyl sulfoxide to the culture medium. Unlimited amounts of callus could be produced with the dimethyl sulfoxide method from protoplasts which never developed a single callus in control experiments. 30 references, 3 figures.« less

3D Printed Abdominal Aortic Aneurysm Phantom for Image Guided Surgical Planning with a Patient Specific Fenestrated Endovascular Graft System

PubMed Central

Meess, Karen M.; Izzo, Richard L.; Dryjski, Maciej L.; Curl, Richard E.; Harris, Linda M.; Springer, Michael; Siddiqui, Adnan H.; Rudin, Stephen; Ionita, Ciprian N.

2017-01-01

Following new trends in precision medicine, Juxatarenal Abdominal Aortic Aneurysm (JAAA) treatment has been enabled by using patient-specific fenestrated endovascular grafts. The X-ray guided procedure requires precise orientation of multiple modular endografts within the arteries confirmed via radiopaque markers. Patient-specific 3D printed phantoms could familiarize physicians with complex procedures and new devices in a risk-free simulation environment to avoid periprocedural complications and improve training. Using the Vascular Modeling Toolkit (VMTK), 3D Data from a CTA imaging of a patient scheduled for Fenestrated EndoVascular Aortic Repair (FEVAR) was segmented to isolate the aortic lumen, thrombus, and calcifications. A stereolithographic mesh (STL) was generated and then modified in Autodesk MeshMixer for fabrication via a Stratasys Eden 260 printer in a flexible photopolymer to simulate arterial compliance. Fluoroscopic guided simulation of the patient-specific FEVAR procedure was performed by interventionists using all demonstration endografts and accessory devices. Analysis compared treatment strategy between the planned procedure, the simulation procedure, and the patient procedure using a derived scoring scheme. Results With training on the patient-specific 3D printed AAA phantom, the clinical team optimized their procedural strategy. Anatomical landmarks and all devices were visible under x-ray during the simulation mimicking the clinical environment. The actual patient procedure went without complications. Conclusions With advances in 3D printing, fabrication of patient specific AAA phantoms is possible. Simulation with 3D printed phantoms shows potential to inform clinical interventional procedures in addition to CTA diagnostic imaging. PMID:28638171

3D printed abdominal aortic aneurysm phantom for image guided surgical planning with a patient specific fenestrated endovascular graft system

NASA Astrophysics Data System (ADS)

Meess, Karen M.; Izzo, Richard L.; Dryjski, Maciej L.; Curl, Richard E.; Harris, Linda M.; Springer, Michael; Siddiqui, Adnan H.; Rudin, Stephen; Ionita, Ciprian N.

2017-03-01

Following new trends in precision medicine, Juxatarenal Abdominal Aortic Aneurysm (JAAA) treatment has been enabled by using patient-specific fenestrated endovascular grafts. The X-ray guided procedure requires precise orientation of multiple modular endografts within the arteries confirmed via radiopaque markers. Patient-specific 3D printed phantoms could familiarize physicians with complex procedures and new devices in a risk-free simulation environment to avoid periprocedural complications and improve training. Using the Vascular Modeling Toolkit (VMTK), 3D Data from a CTA imaging of a patient scheduled for Fenestrated EndoVascular Aortic Repair (FEVAR) was segmented to isolate the aortic lumen, thrombus, and calcifications. A stereolithographic mesh (STL) was generated and then modified in Autodesk MeshMixer for fabrication via a Stratasys Eden 260 printer in a flexible photopolymer to simulate arterial compliance. Fluoroscopic guided simulation of the patient-specific FEVAR procedure was performed by interventionists using all demonstration endografts and accessory devices. Analysis compared treatment strategy between the planned procedure, the simulation procedure, and the patient procedure using a derived scoring scheme. Results: With training on the patient-specific 3D printed AAA phantom, the clinical team optimized their procedural strategy. Anatomical landmarks and all devices were visible under x-ray during the simulation mimicking the clinical environment. The actual patient procedure went without complications. Conclusions: With advances in 3D printing, fabrication of patient specific AAA phantoms is possible. Simulation with 3D printed phantoms shows potential to inform clinical interventional procedures in addition to CTA diagnostic imaging.

Isolation of biologically-active exosomes from human plasma.

PubMed

Muller, Laurent; Hong, Chang-Sook; Stolz, Donna B; Watkins, Simon C; Whiteside, Theresa L

2014-09-01

Effects of exosomes present in human plasma on immune cells have not been examined in detail. Immunological studies with plasma-derived exosomes require their isolation by procedures involving ultracentrifugation. These procedures were largely developed using supernatants of cultured cells. To test biologic activities of plasma-derived exosomes, methods are necessary that ensure adequate recovery of exosome fractions free of contaminating larger vesicles, cell fragments and protein/nucleic acid aggregates. Here, an optimized method for exosome isolation from human plasma/serum specimens of normal controls (NC) or cancer patients and its advantages and pitfalls are described. To remove undesirable plasma-contaminating components, ultrafiltration of differentially-centrifuged plasma/serum followed by size-exclusion chromatography prior to ultracentrifugation facilitated the removal of contaminants. Plasma or serum was equally acceptable as a source of exosomes based on the recovered protein levels (in μg protein/mL plasma) and TEM image quality. Centrifugation on sucrose density gradients led to large exosome losses. Fresh plasma was the best source of morphologically-intact exosomes, while the use of frozen/thawed plasma decreased exosome purity but not their biologic activity. Treatments of frozen plasma with DNAse, RNAse or hyaluronidase did not improve exosome purity and are not recommended. Cancer patients' plasma consistently yielded more isolated exosomes than did NCs' plasma. Cancer patients' exosomes also mediated higher immune suppression as evidenced by decreased CD69 expression on responder CD4+ T effector cells. Thus, the described procedure yields biologically-active, morphologically-intact exosomes that have reasonably good purity without large protein losses and can be used for immunological, biomarker and other studies. Copyright © 2014 Elsevier B.V. All rights reserved.

Clinical experience with a novel electromyographic approach to preventing phrenic nerve injury during cryoballoon ablation in atrial fibrillation.

PubMed

Mondésert, Blandine; Andrade, Jason G; Khairy, Paul; Guerra, Peter G; Dyrda, Katia; Macle, Laurent; Rivard, Léna; Thibault, Bernard; Talajic, Mario; Roy, Denis; Dubuc, Marc; Shohoudi, Azadeh

2014-08-01

Phrenic nerve palsy remains the most frequent complication associated with cryoballoon-based pulmonary vein (PV) isolation. We sought to characterize our experience using a novel monitoring technique for the prevention of phrenic nerve palsy. Two hundred consecutive cryoballoon-based PV isolation procedures between October 2010 and October 2013 were studied. In addition to standard abdominal palpation during right phrenic nerve pacing from the superior vena cava, all patients underwent diaphragmatic electromyographic monitoring using surface electrodes. Cryoablation was terminated on any perceived reduction in diaphragmatic motion or a 30% decrease in the compound motor action potential (CMAP). During right-sided ablation, a ≥30% reduction in CMAP amplitude occurred in 49 patients (24.5%). Diaphragmatic motion decreased in 30 of 49 patients and was preceded by a 30% reduction in CMAP amplitude in all. In 82% of cases, this reduction in CMAP amplitude occurred during right superior PV isolation. The baseline CMAP amplitude was 946.5±609.2 mV and decreased by 13.8±13.8% at the end of application. This decrease was more marked in the 33 PVs with a reduction in diaphragmatic motion than in those without (40.9±15.3% versus 11.3±10.5%; P<0.001). In 3 cases, phrenic nerve palsy persisted beyond the end of the procedure, with all cases recovering within 6 months. Despite the shortened application all veins were isolated. At repeat procedure the right-sided PVs reconnected less frequently than the left-sided PVs in those with phrenic nerve palsy. Electromyographic phrenic nerve monitoring using the surface CMAP is reliable, easy to perform, and offers an early warning to impending phrenic nerve injury. © 2014 American Heart Association, Inc.

Box Isolation of Fibrotic Areas (BIFA): A Patient-Tailored Substrate Modification Approach for Ablation of Atrial Fibrillation.

PubMed

Kottkamp, Hans; Berg, Jan; Bender, Roderich; Rieger, Andreas; Schreiber, Doreen

2016-01-01

Catheter ablation strategies beyond pulmonary vein isolation (PVI) for treatment of atrial fibrillation (AF) are less well defined. Increasing clinical data indicate that atrial fibrosis is a critical common left atrial (LA) substrate in AF patients (pts). We applied a new substrate modification concept according to the individual fibrotic substrate as estimated from electroanatomic voltage mapping (EAVM) in 41 pts undergoing catheter ablation of AF. First, EAVM during sinus rhythm was done in redo cases of 10 pts with paroxysmal AF despite durable PVI. Confluent low-voltage areas (LVA) were found in all pts and were targeted with circumferential isolation, so-called box isolation of fibrotic areas (BIFA). This strategy led to stable sinus rhythm in 9/10 pts and was transferred prospectively to first procedures of 31 pts with nonparoxysmal AF. In 13 pts (42%), no LVA (<0.5 mV) were identified, and only PVI was performed. In 18 pts (58%), additional BIFA strategies were applied (posterior box in 5, anterior box in 7, posterior plus anterior box in 5, no box in 1 due to diffuse fibrosis). Mean follow-up was 12.5 ± 2.4 months. Single-procedure freedom from AF/atrial tachycardia was achieved in 72.2% of pts and in 83.3% of pts with 1.17 procedures/patient. In approximately 40% of pts with nonparoxysmal AF, no substantial LVA were identified, and PVI alone showed high success rate. In pts with paroxysmal AF despite durable PVI and in approximately 60% of pts with nonparoxysmal AF, individually localized LVA were identified and could be targeted successfully with the BIFA strategy. © 2015 Wiley Periodicals, Inc.

Evidence for function of the ferredoxin/thioredoxin system in the reductive activation of target enzymes of isolated intact chloroplasts.

PubMed

Crawford, N A; Droux, M; Kosower, N S; Buchanan, B B

1989-05-15

Results obtained with isolated intact chloroplasts maintained aerobically under light and dark conditions confirm earlier findings with reconstituted enzyme assays and indicate that the ferredoxin/thioredoxin system functions as a light-mediated regulatory thiol chain. The results were obtained by application of a newly devised procedure in which a membrane-permeable thiol labeling reagent, monobromobimane (mBBr), reacts with sulfhydryl groups and renders the derivatized protein fluorescent. The mBBr-labeled protein in question is isolated individually from chloroplasts by immunoprecipitation and its thiol redox status is determined quantitatively by combining sodium dodecyl sulfate-polyacrylamide gel electrophoresis and fluorescence measurements. The findings indicate that each member of the ferredoxin/thioredoxin system containing a catalytically active thiol group is reduced in isolated intact chloroplasts after a 2-min illumination. The extents of reduction were FTR, 38%; thioredoxin m, 75% (11-kDa form) and 87% (13-kDa form); thioredoxin f, 95%. Reduction of each of these components was negligible both in the dark and when chloroplasts were transferred from light to dark conditions. The target enzyme, NADP-malate dehydrogenase, also underwent net reduction in illuminated intact chloroplasts. Fructose-1,6-bisphosphatase showed increased mBBr labeling under these conditions, but due to interfering gamma globulin proteins it was not possible to determine whether this was a result of net reduction as is known to take place in reconstituted assays. Related experiments demonstrated that mBBr, as well as N-ethylmaleimide, stabilized photoactivated NADP-malate dehydrogenase and fructose-1,6-bisphosphatase so that they remained active in the dark. By contrast, phosphoribulokinase, another thioredoxin-linked enzyme, was immediately deactivated following mBBr addition. These latter results provide new information on the relation between the regulatory and active sites of these enzymes.

Comparison of resource utilization of pulmonary vein isolation: cryoablation versus RF ablation with three-dimensional mapping in the Value PVI Study.

PubMed

DeVille, J Brian; Svinarich, J Thomas; Dan, Dan; Wickliffe, Andrew; Kantipudi, Charan; Lim, Hae W; Plummer, Lisa; Baker, James; Kowalski, Marcin; Baydoun, Hassan; Jenkins, Mark; Chang-Sing, Peter

2014-06-01

Point-to-point focal radiofrequency (RF) catheter ablation for aberrant pulmonary vein triggers that manifest into atrial fibrillation (AF) is the traditional method for treating symptomatic drug-resistant paroxysmal AF (PAF) when an ablation procedure is warranted. More recently, pulmonary vein isolation (PVI) using the cryoballoon has been demonstrated to be safe and effective (STOP AF clinical trial). Currently, two small studies have reviewed the procedural efficiency when comparing cryoballoon to focal RF catheter ablation procedures; however, no multicenter study has yet reported on this comparison of the two types of ablation catheters. A multicenter retrospective chart extraction and evaluation was conducted at seven geographically mixed cardiac care centers. The study examined procedural variables during ablation for PVI in PAF patients. In several procedural measurements, the two modalities were comparable in efficiencies, including: acute PVI >96%; length of hospital stay at approximately 27 hours; and about 30% usage of adenosine after procedural testing. However, when compared to RF catheters, the cryoballoon procedure demonstrated a 13% reduction in laboratory occupancy time (247 min vs 283 min), a 13% reduction in procedure time (174 min vs 200 min), and a 21% reduction in fluoroscopy time (33 min vs 42 min). Additionally, when comparing the material usage of both cryoballoon and RF catheters, the cryoballoon used more radiopaque contrast agent (78 cc vs 29 cc) while using less intraprocedural saline (1234 cc vs 2386 cc), intracardiac echocardiography (88% vs 99%), three-dimensional electroanatomic mapping (30% vs 87%), and fewer transseptal punctures (1.5 vs 1.9). This study is the first United States multicenter examination to report the procedural comparisons between the cryoballoon and focal RF catheters when used for the treatment of PAF patients. In this hospital chart review study, potential advantages were found when operating the cryoballoon with regard to hospital resource allocation. There was no statistical difference between cryoballoon and RF catheters for acute PVI success during the ablation procedure.

[Perioperative complications after total hip revision surgery and their predictive factors. A series of 181 consecutive procedures].

PubMed

de Thomasson, E; Guingand, O; Terracher, R; Mazel, C

2001-09-01

We conducted a retrospective study to assess morbidity and mortality in patients undergoing revision total hip arthroplasty (THA) procedures. Perioperative complications were recorded in 181 revision procedures (162 patients) performed between January 1995 and March 1999 (117 bipolar revisions and 64 acetabular isolated revisions). There were 86 complications (68 patients) leading to 21 new revisions. About half (50/86) were related to the surgical procedure (dislocation, femoral fracture, infection.). Life-threatening complications (3.6%) ended in patient death in 1.6% of the cases. Complications were more frequent in patients with an ASA score=3 (p<0.01) or aged over 75 years (p<0.05). Age was also predictive of femoral misalignment and fracture (p<0.05). Dislocations (8.8%) were observed more frequently in patients who had undergone more than 2 procedures prior to the revision (p<0.05) (4.8% of the dislocations in patients undergoing a first revision procedure and 14.3% in the others). In addition, peroperative blood loss and duration of the procedure were significantly greater in case of bipolar replacement than for isolated acetabular replacement (sigma > 1.96). Our experience and data in the literature point to the important age factor in the development of complications. Preservation of a well-fixed femoral component does not appear to worsen prognosis and leads to fewer complications than bipolar changes. The decision to revise a THA must take into consideration the functional impairment but also the risks inherent in revision procedures, particularly in old patients who have undergone several procedures. Revising the acetabular component alone can be an interesting option if the femoral component remains well-fixed although our follow-up is insufficient to determine whether this attitude provides better long-term outcome than complete biopolar revision. Better patient selection and improved operative technique, in particular in femur preparation, should help reduce morbidity and mortality in this type of procedure.

HPLC-SPE-NMR identification of a novel metabolite containing the benzo[c]oxepin skeleton from the endophytic fungus Pestalotiopsis virgatula culture.

PubMed

Kesting, Julie R; Staerk, Dan; Tejesvi, Mysore V; Kini, Kukkundoor R; Prakash, Harishchandra S; Jaroszewski, Jerzy W

2009-08-01

HPLC-SPE-NMR analysis of a crude extract of fermentation broth of cultured PESTALOTIOPSIS VIRGATULA isolate TC-320 from TERMINALIA CHEBULA Retz. (Combretaceae) disclosed the presence of a simple but unprecedented low-molecular-weight metabolite, 9-hydroxybenzo[ C]oxepin-3[1 H]-one, subsequently isolated by a targeted purification procedure. Georg Thieme Verlag KG Stuttgart.New York.

105-KE Isolation Barrier Leak Rate Acceptance Test Report

DOE Office of Scientific and Technical Information (OSTI.GOV)

McCracken, K.J.

1995-06-14

This Acceptance Test Report (ATR) contains the completed and signed Acceptance Procedure (ATP) for the 105-KE Isolations Barrier Leak Rate Test. The Test Engineer`s log, the completed sections of the ATP in the Appendix for Repeat Testing (Appendix K), the approved WHC J-7s (Appendix H), the data logger files (Appendices T and U), and the post test calibration checks (Appendix V) are included.

Computer laboratory notification system via short message service to reduce health care delays in management of tuberculosis in Taiwan.

PubMed

Chen, Tun-Chieh; Lin, Wei-Ru; Lu, Po-Liang; Lin, Chun-Yu; Lin, Shu-Hui; Lin, Chuen-Ju; Feng, Ming-Chu; Chiang, Horn-Che; Chen, Yen-Hsu; Huang, Ming-Shyan

2011-06-01

We investigated the impacts of introducing an expedited acid-fast bacilli (AFB) smear laboratory procedure and an automatic, real-time laboratory notification system by short message with mobile phones on delays in prompt isolation of patients with pulmonary tuberculosis (TB). We analyzed the data for all patients with active pulmonary tuberculosis at a hospital in Kaohsiung, Taiwan, a 1,600-bed medical center, during baseline (January 2004 to February 2005) and intervention (July 2005 to August 2006) phases. A total of 96 and 127 patients with AFB-positive TB was reported during the baseline and intervention phases, respectively. There were significant decreases in health care system delays (ie, laboratory delays: reception of sputum to reporting, P < .001; response delays: reporting to patient isolation, P = .045; and interval from admission to patient isolation, P < .001) during the intervention phase. Significantly fewer nurses were exposed to each patient with active pulmonary TB during the intervention phase (P = .039). Implementation of expedited AFB smear laboratory procedures and an automatic, real-time laboratory mobile notification system significantly decreased delays in the diagnosis and isolation of patients with active TB. Copyright © 2011 Association for Professionals in Infection Control and Epidemiology, Inc. Published by Mosby, Inc. All rights reserved.

(4S)-4,8-dihydroxy-1-tetralone and other chemical constituents from Pestalotiopsis sp. EJC07, endophytic fromBauhinia guianensis.

PubMed

de Souza, Eleane M C; da Silva, Ellon L; Marinho, Andrey M R; Marinho, Patrícia S B

2016-03-01

The present work reports the isolation of eight compounds fromPestalotiopsis sp. EJC07 isolated as endophytic fromBauhinia guianensis, a tipical plant of the Amazon. The compounds (4S)-4,8-dihydroxy-1-tetralone (1), uracil (2), uridin (3), p-hydroxybenzoic acid (4), ergosterol (5), ergosterol peroxide (6), cerevisterol (7) and ducitol (8) were isolated by chromatographic procedures and identified by spectral methods of 1D and 2D NMR and MS. The compound 1 is being reported for the first time in the genusPestalotiopsis.

Isolation of genomic DNA using magnetic cobalt ferrite and silica particles.

PubMed

Prodelalová, Jana; Rittich, Bohuslav; Spanová, Alena; Petrová, Katerina; Benes, Milan J

2004-11-12

Adsorption separation techniques as an alternative to laborious traditional methods (e.g., based on phenol extraction procedure) have been applied for DNA purification. In this work we used two types of particles: silica and cobalt ferrite (unmodified or modified with a reagent containing weakly basic aminoethyl groups, aminophenyl groups, or alginic acid). DNA from chicken erythrocytes and DNA isolated from bacteria Lactococcus lactis were used for testing of adsorption/desorption properties of particles. The cobalt ferrite particles modified with different reagents were used for isolation of PCR-ready bacterial DNA from different dairy products.

Puupehenol, a potent antioxidant antimicrobial meroterpenoid from a Hawaiian deep-water Dactylospongia sp. sponge.

PubMed

Hagiwara, Kehau; Garcia Hernandez, Jaaziel E; Harper, Mary Kay; Carroll, Anthony; Motti, Cherie A; Awaya, Jonathan; Nguyen, Hoang-Yen; Wright, Anthony D

2015-02-27

From the organic extract of a deep-water Hawaiian sponge Dactylospongia sp., a new potent antioxidant and antimicrobial meroterpenoid, puupehenol (1), was isolated. The structure of 1 was determined using spectroscopic techniques ((1)H and (13)C NMR, MS, IR, UV, [α]D). The known compound puupehenone (2) was also isolated and suggested as a probable artifact of the isolation procedures. Complete unambiguous (1)H and (13)C NMR data are provided for compounds 1 and 2. Bioassays performed with 1 and 2 showed them both to be very effective antioxidants and to have antimicrobial properties.

Development of the Vibration Isolation System for the Advanced Resistive Exercise Device

NASA Technical Reports Server (NTRS)

Niebuhr, Jason H.; Hagen, Richard A.

2011-01-01

This paper describes the development of the Vibration Isolation System for the Advanced Resistive Exercise Device from conceptual design to lessons learned. Maintaining a micro-g environment on the International Space Station requires that experiment racks and major vibration sources be isolated. The challenge in characterizing exercise loads and testing the system in the presence of gravity led to a decision to qualify the system by analysis. Available data suggests that the system is successful in attenuating loads, yet there has been a major component failure and several procedural issues during its 3 years of operational use.

Rapid isolation of biomarkers for compound specific radiocarbon dating using high-performance liquid chromatography and flow injection analysis-atmospheric pressure chemical ionisation mass spectrometry.

PubMed

Smittenberg, Rienk H; Hopmans, Ellen C; Schouten, Stefan; Sinninghe Damsté, Jaap S

2002-11-29

Repeated semi-preparative normal-phase HPLC was performed to isolate selected biomarkers from sediment extracts for radiocarbon analysis. Flow injection analysis-mass spectrometry was used for rapid analysis of collected fractions to evaluate the separation procedure, taking only 1 min per fraction. In this way 100-1000 microg of glycerol dialkyl glycerol tetraethers, sterol fractions and chlorophyll-derived phytol were isolated from typically 100 g of marine sediment, i.e., in sufficient quantities for radiocarbon analysis, without significant carbon isotopic fractionation or contamination.

Lyme disease: a selective medium for isolation of the suspected etiological agent, a spirochete.

PubMed Central

Johnson, S E; Klein, G C; Schmid, G P; Bowen, G S; Feeley, J C; Schulze, T

1984-01-01

A simple procedure with a new selective culture medium for the isolation of the suspected etiological agent of Lyme disease from ticks is described. Live ticks (Ixodes dammini) were ground with a mortar and pestle, and the suspensions were inoculated into a selective and nonselective medium. The selective medium, which contained kanamycin and 5-fluorouracil, yielded positive spirochete cultures from 100% of the pooled ticks and from 79% of the single tick specimens. The isolation rate for the nonselective medium was 0% from the tick pools and 58% from the single tick specimens. PMID:6361065

Single Spore Isolation as a Simple and Efficient Technique to obtain fungal pure culture

NASA Astrophysics Data System (ADS)

Noman, E.; Al-Gheethi, AA; Rahman, N. K.; Talip, B.; Mohamed, R.; H, N.; Kadir, O. A.

2018-04-01

The successful identification of fungi by phenotypic methods or molecular technique depends mainly on the using an advanced technique for purifying the isolates. The most efficient is the single spore technique due to the simple requirements and the efficiency in preventing the contamination by yeast, mites or bacteria. The method described in the present work is depends on the using of a light microscope to transfer one spore into a new culture medium. The present work describes a simple and efficient procedure for single spore isolation to purify of fungi recovered from the clinical wastes.

Vertebral body clinico-morphological features following percutaneous vertebroplasty versus the conservatory approach.

PubMed

Constantin, Cristian; Albulescu, Dana Maria; Diţă, Daniel Răzvan; Georgescu, Claudia Valentina; Deaconu, Andrei Constantin

2018-01-01

Most percutaneous vertebroplasty procedures are being performed in order to relieve pain in patients with severe osteoporosis and associated stable fractures of one or more vertebral bodies. In addition, vertebroplasty is also recommended for patients suffering from post-traumatic symptoms associated with vertebral fractures, patients with large angiomas positioned inside the vertebral body, with an increased risk for collapse fracture and also patients presenting with pain associated with vertebral body metastatic disease. On another aspect, it is possible that in isolated cases, an orthopedic surgeon confronted with a vertebra plana presentation will recommend bone cement injection into the vertebral bodies adjacent to the fractured one, in order to have a better and more robust substrate for placement of screws or other fixation devices. The aim of our study is to compare results attained by the Department of Interventional Radiology, in performing this procedure, with results attained by following the classical orthopedic treatment procedure, involving non-operative treatment, using medication and bracing varying from simple extension orthoses in order to limit spinal flexion, light bracing for contiguous fractures, presenting either angulation or compression, and for severe cases standard thoracolumbosacral orthoses (TLSOs).

Parallel solution-phase synthesis of a 2-aminothiazole library including fully automated work-up.

PubMed

Buchstaller, Hans-Peter; Anlauf, Uwe

2011-02-01

A straightforward and effective procedure for the solution phase preparation of a 2-aminothiazole combinatorial library is described. Reaction, work-up and isolation of the title compounds as free bases was accomplished in a fully automated fashion using the Chemspeed ASW 2000 automated synthesizer. The compounds were obtained in good yields and excellent purities without any further purification procedure.

UTILIZATION OF CARBOHYDRATES AND POLYHYDRIC ALCOHOLS BY MYCOBACTERIUM TUBERCULOSIS

PubMed Central

Sweeney, Edward E.; Jann, Gregory J.

1962-01-01

Sweeney, Edward E. (University of California, Los Angeles) and Gregory J. Jann. Utilization of carbohydrates and polyhydric alcohols by Mycobacterium tuberculosis. J. Bacteriol. 84:459–465. 1962.—A new procedure, using a massive inoculum and nongrowth basal medium, was employed for testing carbohydrate and polyhydric alcohol utilization by human tubercle bacilli. A positive reaction was represented by acidification of the test medium rather than by growth, which was the criterion for carbohydrate utilization in studies by earlier workers. The new procedure was both more sensitive and more rapid than growth techniques; results were obtained within days, compared to weeks or months required for growth testing. The massive-inoculum technique may be applied to compounds other than carbohydrates and polyhydric alcohols, and is a sensitive means of detecting changes wrought by various chemical and physical agents upon the metabolism of tubercle bacilli. Three H37Rv strains and six strains of human tubercle bacilli freshly isolated from patients were tested with 21 carbohydrates and polyhydric alcohols. All nine strains gave strong positive reactions for glucose and glycerol, and usually weak positive reactions for ribose and sorbose. Five of the nine strains were trehalose positive, and six (all fresh patient isolates) of the nine were mannose positive. PMID:13979661

Western blotting revisited: critical perusal of underappreciated technical issues.

PubMed

Gorr, Thomas A; Vogel, Johannes

2015-04-01

The most commonly used semiquantitative analysis of protein expression still employs protein separation by denaturing SDS-PAGE with subsequent Western blotting and quantification of the resulting ODs of bands visualized with specific antibodies. However, many questions regarding this procedure are usually ignored, although still in need of answering: Does isolation or separation procedure harm the integrity or affect modifications (e.g., phosphorylation) of the protein of interest? Does denaturation reduce binding of antibodies used for detection? Should denaturation be performed or should a native gel be run? How can artificial degradations or aggregations be distinguished from biological relevant ones? If the antibody detects multiple bands (which is not uncommon), which one(s) should be taken into account for quantification and why? Which loading control protein should be chosen and is it really "housekeeping" and how can this be verified? Is the image acquisition system linear and does it come with a sufficient dynamic range? How to account and control for background staining? This article is intended to address these questions and raise the readers awareness to possible Western blot alternatives in the attempt of minimizing possible pitfalls that might loom anywhere from protein isolation to acquisition of final quantitative data. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Exploring target-specific primer extension in combination with a bead-based suspension array for multiplexed detection and typing using Streptococcus suis as a model pathogen

PubMed Central

van der Wal, Fimme J.; Achterberg, René P.; van Solt-Smits, Conny; Bergervoet, Jan H. W.; de Weerdt, Marjanne; Wisselink, Henk J.

2017-01-01

We investigated the feasibility of an assay based on target-specific primer extension, combined with a suspension array, for the multiplexed detection and typing of a veterinary pathogen in animal samples, using Streptococcus suis as a model pathogen. A procedure was established for simultaneous detection of 6 S. suis targets in pig tonsil samples (i.e., 4 genes associated with serotype 1, 2, 7, or 9, the generic S. suis glutamate dehydrogenase gene [gdh], and the gene encoding the extracellular protein factor [epf]). The procedure was set up as a combination of protocols: DNA isolation from porcine tonsils, a multiplex PCR, a multiplex target-specific primer extension, and finally a suspension array as the readout. The resulting assay was compared with a panel of conventional PCR assays. The proposed multiplex assay can correctly identify the serotype of isolates and is capable of simultaneous detection of multiple targets in porcine tonsillar samples. The assay is not as sensitive as the current conventional PCR assays, but with the correct sampling strategy, the assay can be useful for screening pig herds to establish which S. suis serotypes are circulating in a pig population. PMID:28980519

High Performance Liquid Chromatography Resolution of Ubiquitin Pathway Enzymes from Wheat Germ 1

PubMed Central

Sullivan, Michael L.; Callis, Judy; Vierstra, Richard D.

1990-01-01

The highly conserved protein ubiquitin is involved in several cellular processes in eukaryotes as a result of its covalent ligation to a variety of target proteins. Here, we describe the purification of several enzymatic activities involved in ubiquitin-protein conjugate formation and disassembly from wheat germ (Triticum vulgare) by a combination of ubiquitin affinity chromatography and anion-exchange high performance liquid chromatography. Using this procedure, ubiquitin activating enzyme (E1), several distinct ubiquitin carrier proteins (E2s) with molecular masses of 16, 20, 23, 23.5, and 25 kilodaltons, and a ubiquitin-protein hydrolase (isopeptidase) were isolated. Purified E1 formed a thiol ester linkage with 125I-ubiquitin in an ATP-dependent manner and transferred bound ubiquitin to the various purified E2s. The ubiquitin protein hydrolase fraction was sensitive to hemin, and in an ATP-independent reaction, was capable of removing the ubiquitin moiety from both ubiquitin 125I-lysozyme conjugates (ε-amino or isopeptide linkage) and the ubiquitin 52-amino acid extension protein fusion (α-amino or peptide linkage). Using this procedure, wheat germ represents an inexpensive source from which enzymes involved in the ubiquitin pathway may be isolated. Images Figure 1 Figure 2 Figure 3 Figure 4 PMID:16667769

Managing incidentally diagnosed isolated factor VII deficiency perioperatively: a brief expert consensus report.

PubMed

Sheth, Sujit; Soff, Gerald; Mitchell, Beau; Green, David; Kaicker, Shipra; Fireman, Fernando; Tugal, Oya; Guarini, Ludovico; Giardina, Patricia; Aledort, Louis

2012-02-01

While isolated factor VII (FVII) deficiency is being more frequently diagnosed owing to improved preoperative screening procedures, there is no specific guideline for perioperative management of such patients. To complicate the issue, FVII activity levels seem to correlate less well with the risk of hemorrhage than the patient's past and family bleeding history do. We have devised expert consensus recommendations for managing such patients perioperatively, taking into consideration the personal and family bleeding history, the FVII activity level and the inherent bleeding risk of the procedure itself. We hope that clinicians will find this a useful tool in the decision-making process, thereby limiting the use of recombinant factor VIIa to those who need it most, and preventing possible thrombotic complications in those without a strong indication for its use.

A comparison of the compositional differences between humic fractions isolated by the IHSS and exhaustive extraction procedures

NASA Astrophysics Data System (ADS)

Chang, R. R.; Mylotte, R.; Hayes, M. H. B.; Mclnerney, R.; Tzou, Y. M.

2014-03-01

Humic substances (HSs), consisting, on the basis of solubilities in aqueous acid and basic media, of humic acids (HAs), fulvic acids (FAs), and humin (Hu), are the major components of soil organic matter (SOM). Most studies of soil/natural organic matter (SOM/NOM) have been carried out on extracts of soils in dilute sodium hydroxide solutions, the solvent used to extract the Standards of the International Humic Substances Society (IHSS). However, Hu, the major component in the classical definition of HSs, is insoluble in aqueous base and is not isolated by the traditional IHSS method. Recently, a sequential exhaustive extraction (SEE) process has been shown to be capable of isolating and separating the major components of the classically defined HSs from the soils of the temperate and tropical regions. The SEE system was used in the present study to isolate the HA/FA and Hu fractions from a subtropical volcanic Taiwanese soil. Chemical and compositional properties of these extracts were then compared with similarly obtained isolates from soils from the different climatic regions. Increases in the aliphatic relative to aromatic carbon contents were observed for both the HA and FA fractions when the pH values of the extraction media were increased. HAs and FAs isolated using the SEE method have spectroscopic profiles similar to those from the IHSS isolate; however, the cumulative extraction efficiency (%) of the SEE method (65 %) for the volcanic soil was much higher than for the traditional IHSS method (33 %). When the residual volcanic soil, following extractions once, three, and eight times with 0.1 M NaOH were then extracted with dimethyl sulphoxide (DMSO) plus concentrated sulphuric acid (the final solvent in the SEE sequence) it was seen that the content of crystalline polymethylene hydrocarbon (33 ppm 13C-NMR resonance in the Hu (or DMSO/acid)) extract increased relative to the amorphous methylene (30 ppm). That highlights the difficulty in dissolving the more highly ordered hydrocarbon structures that would be expected to have closer associations with the mineral colloids. Although the SEE procedure isolated all of the HAs and FAs from the Yangmingshan soil, extractability of the Hu from the volcanic soil in the DMSO/acid solvent was low (21 %), and contrasted with the much higher yields from temperate and tropical regions. The decreased Hu extraction may arise from its associations with the extensive iron and aluminium hydroxide mineral colloids in the soil. The Hu from this sub-tropical soil was different from the Hus isolated from other soil types, indicating the need to isolate and characterise these recalcitrant organic material in order to understand the organic carbon components in soils in greater detail. Such results would indicate that more attention should be given to mineral colloids in soils, and to the organo/mineral associations that will have an important role in the stabilities of OM in the soil environment.

Adherence of Candida to complete denture surfaces in vitro: A comparison of conventional and CAD/CAM complete dentures

PubMed Central

Al-mejrad, Lamya A.; Albarrag, Ahmed M.

2017-01-01

PURPOSE The goal of this study was to compare the adhesion of Candida albicans to the surfaces of CAD/CAM and conventionally fabricated complete denture bases. MATERIALS AND METHODS Twenty discs of acrylic resin poly (methyl methacrylate) were fabricated with CAD/CAM and conventional procedures (heat-polymerized acrylic resin). The specimens were divided into two groups: 10 discs were fabricated using the CAD/CAM procedure (Wieland Digital Denture Ivoclar Vivadent), and 10 discs were fabricated using a conventional flasking and pressure-pack technique. Candida colonization was performed on all the specimens using four Candida albicans isolates. The difference in Candida albicans adhesion on the discs was evaluated. The number of adherent yeast cells was calculated by the colony-forming units (CFU) and by Fluorescence microscopy. RESULTS There was a significant difference in the adhesion of Candida albicans to the complete denture bases created with CAD/CAM and the adhesion to those created with the conventional procedure. The CAD/CAM denture bases exhibited less adhesion of Candida albicans than did the denture bases created with the conventional procedure (P<.05). CONCLUSION The CAD/CAM procedure for fabricating complete dentures showed promising potential for reducing the adherence of Candida to the denture base surface. Clinical Implications. Complete dentures made with the CAD/CAM procedure might decrease the incidence of denture stomatitis compared with conventional dentures. PMID:29142649

Study of non-syndromic thumb aplasia in six independent cases

PubMed Central

Riaz, Hafiza Fizzah; Lal, Karmoon; Ahmad, Bashir; Shuaib, Muhammad; Naqvi, Syeda Farwa; Malik, Sajid

2014-01-01

Objectives: To report on six independent and isolated cases demonstrating thumb aplasia as an essentially limb-specific phenotype. Methods: The subjects were ascertained during 2011-2013 from six different geographic regions of Pakistan, and underwent detailed clinical and phenotypic examination. Results: The affected arms of patients had complete absence of first digital rays, medial inclinations of second and fifth fingers, narrowing of palms, missing carpals, and shortening of zeugopod. All the subjects were presented with isolated and sporadic limb deficiencies, and five had no family history of limb or any other malformation. Parental consanguinity was denied in majority of the cases. We present detailed phenotypic manifestation of thumb apalsia in these subjects. Conclusion: Thumb aplasia markedly impairs the normal function of affected hand. Surgical procedures like pollicisation of the index finger should be employed to improve the quality of life of these subjects. There is so far no specific genetic factor known for isolated thumb aplasia, compromising an accurate genetic counseling. Collection of patients with similar phenotypic presentations could be useful in further molecular genetic investigations. PMID:24949004

Isolation of Ovicidal Fungi from Fecal Samples of Captive Animals Maintained in a Zoological Park.

PubMed

Hernández, José A; Vázquez-Ruiz, Rosa A; Cazapal-Monteiro, Cristiana F; Valderrábano, Esther; Arroyo, Fabián L; Francisco, Iván; Miguélez, Silvia; Sánchez-Andrade, Rita; Paz-Silva, Adolfo; Arias, María S

2017-06-02

Abstract : There are certain saprophytic fungi in the soil able to develop an antagonistic effect against eggs of parasites. Some of these fungal species are ingested by animals during grazing, and survive in their feces after passing through the digestive tract. To identify and isolate ovicidal fungi in the feces of wild captive animals, a total of 60 fecal samples were taken from different wild animals kept captive in the Marcelle Natureza Zoological Park (Lugo, Spain). After the serial culture of the feces onto Petri dishes with different media, their parasicitide activity was assayed against eggs of trematodes ( Calicophoron daubneyi ) and ascarids ( Parascaris equorum ). Seven fungal genera were identified in the feces. Isolates from Fusarium , Lecanicillium , Mucor , Trichoderma , and Verticillium showed an ovicidal effect classified as type 3, because of their ability to adhere to the eggshell, penetrate, and damage permanently the inner embryo. Penicillium and Gliocladium developed a type 1 effect (hyphae attach to the eggshell but morphological damage was not provoked). These results provide very interesting and useful information about fungi susceptible for being used in biological control procedures against parasites.

A Genuine TEAM Player

NASA Technical Reports Server (NTRS)

2001-01-01

Qualtech Systems, Inc. developed a complete software system with capabilities of multisignal modeling, diagnostic analysis, run-time diagnostic operations, and intelligent interactive reasoners. Commercially available as the TEAMS (Testability Engineering and Maintenance System) tool set, the software can be used to reveal unanticipated system failures. The TEAMS software package is broken down into four companion tools: TEAMS-RT, TEAMATE, TEAMS-KB, and TEAMS-RDS. TEAMS-RT identifies good, bad, and suspect components in the system in real-time. It reports system health results from onboard tests, and detects and isolates failures within the system, allowing for rapid fault isolation. TEAMATE takes over from where TEAMS-RT left off by intelligently guiding the maintenance technician through the troubleshooting procedure, repair actions, and operational checkout. TEAMS-KB serves as a model management and collection tool. TEAMS-RDS (TEAMS-Remote Diagnostic Server) has the ability to continuously assess a system and isolate any failure in that system or its components, in real time. RDS incorporates TEAMS-RT, TEAMATE, and TEAMS-KB in a large-scale server architecture capable of providing advanced diagnostic and maintenance functions over a network, such as the Internet, with a web browser user interface.

Antidiabetic complications and anti-Alzheimer activities of sophoflavescenol, a prenylated flavonol from Sophora flavescens, and its structure-activity relationship.

PubMed

Jung, Hyun Ah; Jin, Seong Eun; Park, Jun-Seong; Choi, Jae Sue

2011-05-01

It was previously reported that prenylated flavonols from Sophora flavescens are inhibitors of rat lens aldose reductase (RLAR), human recombinant aldose reductase (HRAR), advanced glycation endproducts (AGE), β-secretase (BACE1) and cholinesterases (ChE). Based upon structure-activity relationships, 3,4'-dihydroxy flavonols with a prenyl or lavandulyl group substitution at the C-8 position, and a hydroxy group at the C-5, are important for such inhibition. In our ongoing study to isolate active principles from S. flavescens by an activity-guided isolation procedure, further detailed phytochemical investigations of the CH(2)Cl(2) fraction were conducted via repeated chromatography over silica gel and Sephadex LH-20 columns. This ultimately resulted in the isolation of a promising active sophoflavescenol with higher inhibitory activities among the current prenylated flavonols isolated from S. flavescens against RLAR, HRAR, AGE, BACE1 and ChEs. The results further support that 3,4'-dihydroxy flavonols with a prenyl or lavandulyl substitution at the C-8 position and a methoxy group at C-5 represent a new class of RLAR, HRAR and AGE inhibitors. Nevertheless, the C-5 hydroxyl group of prenylated flavonoids is important for inhibition of BACE1 and ChEs, indicating that the hydroxyl group at C-5 might be the main contributor to the augmentation and/or modification of prenylated flavonol activity. Copyright © 2010 John Wiley & Sons, Ltd.

The procedure for isolation of neoplasms on the retina of the eye

NASA Astrophysics Data System (ADS)

Komkova, S. V.

2018-01-01

In operation, a computer diagnostic procedure on the human retina neoplasms. The use of this technique in medical institutions in the operation of the ophthalmic practitioner allows earlv detection of the disease, at periodic inspection of a pictorial pattern of disease progression. The test procedure is performed on a set of real human retinal photographs taken from international STARE database with known diagnoses. Given the numerous experiments which show the possibility of using this technique, developed on the basis of the diagnostic system in a doctor’s office-ophthalmic.

Contemporary bloodletting in cardiac surgical care.

PubMed

Koch, Colleen G; Reineks, Edmunds Z; Tang, Anne S; Hixson, Eric D; Phillips, Shannon; Sabik, Joseph F; Henderson, J Michael; Blackstone, Eugene H

2015-03-01

Health care providers are seldom aware of the frequency and volume of phlebotomy for laboratory testing, bloodletting that often leads to hospital-acquired anemia. Our objectives were to examine the frequency of laboratory testing in patients undergoing cardiac surgery, calculate cumulative phlebotomy volume from time of initial surgical consultation to hospital discharge, and propose strategies to reduce phlebotomy volume. From January 1, 2012 to June 30, 2012, 1,894 patients underwent cardiac surgery at Cleveland Clinic; 1,867 had 1 hospitalization and 27 had 2. Each laboratory test was associated with a test name and blood volume. Phlebotomy volume was estimated separately for the intensive care unit (ICU), hospital floors, and cumulatively. A total of 221,498 laboratory tests were performed, averaging 115 tests per patient. The most frequently performed tests were 88,068 blood gas analyses, 39,535 coagulation tests, 30,421 complete blood counts, and 29,374 metabolic panels. Phlebotomy volume differed between ICU and hospital floors, with median volumes of 332 mL and 118 mL, respectively. Cumulative median volume for the entire hospital stay was 454 mL. More complex procedures were associated with higher overall phlebotomy volume than isolated procedures; eg, combined coronary artery bypass grafting (CABG) and valve procedure median volume was 653 mL (25th/75th percentiles, 428 of 1,065 mL) versus 448 mL (284 of 658 mL) for isolated CABG and 338 mL (237 of 619) for isolated valve procedures. We were astonished by the extent of bloodletting, with total phlebotomy volumes approaching amounts equivalent to 1 to 2 red blood cell units. Implementation of process improvement initiatives can potentially reduce phlebotomy volumes and resource utilization. Copyright © 2015 The Society of Thoracic Surgeons. Published by Elsevier Inc. All rights reserved.

Arrhythmia Termination Versus Elimination of Dormant Pulmonary Vein Conduction as a Procedural End Point of Catheter Ablation for Paroxysmal Atrial Fibrillation: A Prospective Randomized Trial.

PubMed

Theis, Cathrin; Konrad, Torsten; Mollnau, Hanke; Sonnenschein, Sebastian; Kämpfner, Denise; Potstawa, Maik; Ocete, Blanca Quesada; Bock, Karsten; Himmrich, Ewald; Münzel, Thomas; Rostock, Thomas

2015-10-01

Pulmonary vein isolation (PVI) is still associated with a substantial number of arrhythmia recurrences in paroxysmal atrial fibrillation (AF). This prospective, randomized study aimed to compare 2 different procedural strategies. A total of 152 patients undergoing de novo ablation for paroxysmal AF were randomized to 2 different treatment arms. The procedure in group A consisted of PVI exclusively. In this group, all isolated PVs were challenged with adenosine to reveal and ablate dormant conduction. In group B, PVI was performed with the patient either in spontaneous or in induced AF. If AF did not terminate with PVI, ablation was continued by targeting extra-PV AF sources with the desired procedural end point of termination to sinus rhythm. Primary study end point was freedom from arrhythmia during 1-year follow-up. In group A, adenosine provoked dormant conduction in 31 (41%) patients with a mean of 1.6±0.8 transiently recovered PVs per patient. Termination of AF during PVI was observed in 31 (65%) patients, whereas AF persisted afterward in 17 (35%) patients. AF termination occurred in 13 (76%) patients by AF source ablation. After 1-year follow-up, significantly more group B patients were free of arrhythmia recurrences (87 versus 68%; P=0.006). During redo ablation, the rate of PV reconduction did not differ between both groups (group A: 55% versus group B: 61%; P=0.25). Elimination of extra-PV AF sources after PVI is superior to sole PV isolation with the adjunct of abolishing potential dormant conduction. URL: http://www.clinicaltrials.gov. Unique identifier: NCT02238392. © 2015 The Authors.

Remote magnetic versus manual catheter navigation for circumferential pulmonary vein ablation in patients with atrial fibrillation.

PubMed

Lüthje, Lars; Vollmann, Dirk; Seegers, Joachim; Dorenkamp, Marc; Sohns, Christian; Hasenfuss, Gerd; Zabel, Markus

2011-11-01

Only limited data exist on the clinical utility of remote magnetic navigation (RMN) for pulmonary vein (PV) ablation. Aim of this prospective study was to evaluate the safety and efficacy of RMN for PV isolation as compared to the manual (CON) approach. A total of 161 consecutive patients undergoing circumferential PV isolation were included. Open-irrigated 3.5 mm ablation catheters under the guidance of a mapping system were used. The catheter was navigated with the Stereotaxis Niobe II system in the RMN group (n = 107) and guided manually in the CON group (n = 54). Electrical isolation of all PVs was achieved in 90% of the patients in the RMN group and in 87% in the CON group (p = 0.6). All subjects were followed every 3 months by 7d Holter-ECG. At 12 months of follow-up, 53.5% (RMN) and 55.5% (CON) of the patients were free of any left atrial tachycardia/atrial fibrillation (AF) episode (p = 0.57). Free of symptomatic AF recurrence were 66.3% (RMN) and 62.1% (CON) of the subjects (p = 0.80). Use of RMN was associated with longer procedure duration (p < 0.0001), ablation times (p < 0.0001), and RF current application duration (p < 0.05). In contrast, fluoroscopy time was lower in the RMN group (p < 0.0001). Major complications occurred in 6 of 161 procedures (3.7%), with no significant difference between groups (p = 0.75). RMN-guided PV ablation provides comparable acute and long-term success rates as compared to manual navigation. Procedural complication rates are similar. The use of RMN is associated with markedly reduced fluoroscopy time, but prolonged ablation and procedure duration.

Microbial contamination of nonsterile pharmaceuticals in public hospital settings

PubMed Central

Mugoyela, Veronica; Mwambete, Kennedy D

2010-01-01

Purpose Contamination of pharmaceuticals with microorganisms irrespective whether they are harmful or nonpathogenic can bring about changes in physicochemical characteristics of the medicines. Although sterility is not a requirement in official compendia for nonsterile pharmaceuticals, bioburdens need to be within acceptable limits. Therefore, this study investigated microbial contamination of 10 nonsterile pharmaceuticals frequently delivered to outpatients by identifying and quantifying microbial contaminants and susceptibility pattern testing on the microbes isolated. Methods The study was carried out at Amana Municipal Hospital in Dar es Salaam, Tanzania. The protocol for the study involved structured selection of representative tablets, syrups, and capsules from the hospital’s outpatient pharmacy. Constitutive microorganisms were elaborated and enumerated using standard microbiologic procedures. Results Results showed that 50% of all tested products were heavily contaminated, and the predominant contaminants comprised Klebsiella, Bacillus, and Candida species. Furthermore, the results showed that the isolated Bacillus and Klebsiella species were resistant to Augmentin ® and cloxacillin. The differences in means for cfu/mL and zones of inhibition among the microorganisms isolated were considered significant at P < 0.05. Conclusion The nonsterile pharmaceuticals were presumably microbiologically contaminated due to poor handling during dispensing, repackaging, and/or nonadherence to good manufacturing practice. Therefore, training and educating the dispensers, as well as patients, on the proper handling and use of medicines cannot be overemphasized, because these are key aspects in controlling cross-contamination of medicines. PMID:20957135

DOE Office of Scientific and Technical Information (OSTI.GOV)

Knight, Marlene E.; Sanborn, Brett; Song, Bo

Elastomeric materials are used as shock isolation materials in a variety of environments to dampen vibrations and/or absorb energy from external impact to minimize energy transfer between two objects or bodies. Some applications require the shock isolation materials to behave as a low-pass mechanical filter to mitigate the shock/impact at high frequencies but transmit the energy at low frequencies with minimal attenuation. To fulfill this requirement, a shock isolation material needs to be carefully evaluated and selected with proper experimental design, procedures, and analyses. In this study, a Kolsky bar was modified with precompression (up to 15.5 kN) and confinementmore » capabilities to evaluate low-pass shock isolation performance in terms of acceleration attenuation through a variety of elastomers. Also investigated were the effects of preload and specimen geometry on the low-pass shock isolation response.« less

Simple & Safe Genomic DNA Isolation.

ERIC Educational Resources Information Center

Moss, Robert; Solomon, Sondra

1991-01-01

A procedure for purifying DNA using either bacteria or rat liver is presented. Directions for doing a qualitative DNA assay using diphenylamine and a quantitative DNA assay using spectroscopy are included. (KR)

Autonomous power expert system advanced development

NASA Technical Reports Server (NTRS)

Quinn, Todd M.; Walters, Jerry L.

1991-01-01

The autonomous power expert (APEX) system is being developed at Lewis Research Center to function as a fault diagnosis advisor for a space power distribution test bed. APEX is a rule-based system capable of detecting faults and isolating the probable causes. APEX also has a justification facility to provide natural language explanations about conclusions reached during fault isolation. To help maintain the health of the power distribution system, additional capabilities were added to APEX. These capabilities allow detection and isolation of incipient faults and enable the expert system to recommend actions/procedure to correct the suspected fault conditions. New capabilities for incipient fault detection consist of storage and analysis of historical data and new user interface displays. After the cause of a fault is determined, appropriate recommended actions are selected by rule-based inferencing which provides corrective/extended test procedures. Color graphics displays and improved mouse-selectable menus were also added to provide a friendlier user interface. A discussion of APEX in general and a more detailed description of the incipient detection, recommended actions, and user interface developments during the last year are presented.

Technical note: A method for isolating glycogen granules from ruminal protozoa for further characterization.

PubMed

Hall, Mary Beth

2016-03-01

Evaluation of physical, chemical, and enzymatic hydrolysis characteristics of protozoal glycogen is best performed on a pure substrate to avoid interference from other cell components. A method for isolating protozoal glycogen granules without use of detergents or other potentially contaminating chemicals was developed. Rumen inoculum was incubated anerobically in vitro with glucose. Glycogen-laden protozoa produced in the fermentation, primarily isotrichids, were allowed to sediment in a separatory funnel and were dispensed. The protozoa were processed through repeated centrifugations and sonication to isolate glycogen granules largely free of feed and cellular debris. The final water-insoluble lyophilized product analyzed as 98.3% α-glucan with very rare starch granules and 1.9% protein. Observed losses of glycogen granules during the clean-up process indicate that this procedure should not be used for quantitative assessment of protozoal glycogen from fermentations. Further optimization of this procedure to enhance the amount of glycogen obtained per fermentation may be possible. Copyright © 2016 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

Two self-test methods applied to an inertial system problem. [estimating gyroscope and accelerometer bias

NASA Technical Reports Server (NTRS)

Willsky, A. S.; Deyst, J. J.; Crawford, B. S.

1975-01-01

The paper describes two self-test procedures applied to the problem of estimating the biases in accelerometers and gyroscopes on an inertial platform. The first technique is the weighted sum-squared residual (WSSR) test, with which accelerator bias jumps are easily isolated, but gyro bias jumps are difficult to isolate. The WSSR method does not take full advantage of the knowledge of system dynamics. The other technique is a multiple hypothesis method developed by Buxbaum and Haddad (1969). It has the advantage of directly providing jump isolation information, but suffers from computational problems. It might be possible to use the WSSR to detect state jumps and then switch to the BH system for jump isolation and estimate compensation.

Genomic attributes of extended-spectrum β-lactamase-producing Escherichia coli isolated from patients in Lebanon.

PubMed

Tokajian, Sima; Salloum, Tamara; Eisen, Jonathan A; Jospin, Guillaume; Farra, Anna; Mokhbat, Jacques E; Coil, David A

2017-03-01

Extended-spectrum β-lactamase-producing (ESBL) Escherichia coli are a public threat worldwide. This study aimed at analyzing the genomic and functional attributes of nine ESBLs taken from rectal swabs. Samples were isolated from patients admitted for gastrointestinal and urological procedures at the University Medical Center-Rizk Hospital (UMCRH) in Lebanon. Illumina paired-end libraries were prepared and sequenced. The isolates were distributed into five lineages: ST131, ST648, ST405, ST73 and ST38, and harbored bla OXA-1 , bla TEM-1B , bla TEM-1C and aac(6')Ib-cr. ST131 isolates were carriers of stx2 converting I phage. This is the first comprehensive genomic analysis performed on ESBLs in Lebanon.

Rapid screening of pyogenic Staphylococcus aureus for confirmation of genus and species, methicillin resistance and virulence factors by using two novel multiplex PCR.

PubMed

Haque, Abdul; Haque, Asma; Saeed, Muhammad; Azhar, Aysha; Rasool, Samreen; Shan, Sidra; Ehsan, Beenish; Nisar, Zohaib

2017-01-01

Emergence of methicillin resistant Staphylococcus aureus (MRSA) is a major medical problem of current era. These bacteria are resistant to most drugs and rapid diagnosis can provide a clear guideline to clinicians. They possess specific virulence factors and relevant information can be very useful. We designed this study to develop multiplex PCRs to provide rapid information. We studied 60 Staphylococcus aureus isolates and detected methicillin resistance by cefoxitin sensitivity and targeting of mecA gene. After initial studies with uniplex PCRs we optimized two multiplex PCRs with highly reproducible results. The first multiplex PCR was developed to confirm genus, species and methicillin resistance simultaneously, and the second multiplex PCR was for screening of virulence factors. We found 38.33% isolates as methicillin resistant. α -toxin, the major cytotoxic factor, was detected in 40% whereas β-hemolysin was found in 25% cases. Panton Valentine leucocidin was detected in 8.33% and toxic shock syndrome toxin in5% cases. The results of uniplex and multiplex PCRs were highly compatible. These two multiplex PCRs when run simultaneously can provide vital information about methicillin resistance and virulence status of the isolate within a few hours as compared to several days needed by routine procedures.

Translational Profiles of Medullary Myofibroblasts during Kidney Fibrosis

PubMed Central

Grgic, Ivica; Krautzberger, A. Michaela; Hofmeister, Andreas; Lalli, Matthew; DiRocco, Derek P.; Fleig, Susanne V.; Liu, Jing; Duffield, Jeremy S.; McMahon, Andrew P.; Aronow, Bruce

2014-01-01

Myofibroblasts secrete matrix during chronic injury, and their ablation ameliorates fibrosis. Development of new biomarkers and therapies for CKD will be aided by a detailed analysis of myofibroblast gene expression during the early stages of fibrosis. However, dissociating myofibroblasts from fibrotic kidney is challenging. We therefore adapted translational ribosome affinity purification (TRAP) to isolate and profile mRNA from myofibroblasts and their precursors during kidney fibrosis. We generated and characterized a transgenic mouse expressing an enhanced green fluorescent protein (eGFP)–tagged L10a ribosomal subunit protein under control of the collagen1α1 promoter. We developed a one-step procedure for isolation of polysomal RNA from collagen1α1-eGFPL10a mice subject to unilateral ureteral obstruction and analyzed and validated the resulting transcriptional profiles. Pathway analysis revealed strong gene signatures for cell proliferation, migration, and shape change. Numerous novel genes and candidate biomarkers were upregulated during fibrosis, specifically in myofibroblasts, and we validated these results by quantitative PCR, in situ, and Western blot analysis. This study provides a comprehensive analysis of early myofibroblast gene expression during kidney fibrosis and introduces a new technique for cell-specific polysomal mRNA isolation in kidney injury models that is suited for RNA-sequencing technologies. PMID:24652793

Purification and characterization of an antifungal protein, C-FKBP, from Chinese cabbage.

PubMed

Park, Seong-Cheol; Lee, Jung Ro; Shin, Sun-Oh; Jung, Ji Hyun; Lee, Young Mee; Son, Hyosuk; Park, Yoonkyung; Lee, Sang Yeol; Hahm, Kyung-Soo

2007-06-27

An antifungal protein was isolated from Chinese cabbage (Brassica campestris L. ssp. pekinensis) by buffer-soluble extraction and two chromatographic procedures. The results of matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry revealed that the isolated Chinese cabbage protein was identical to human FK506-binding protein (FKBP). A cDNA encoding FKBP was isolated from a Chinese cabbage leaf cDNA library and named C-FKBP. The open reading frame of the gene encoded a 154-amino acid polypeptide. The amino acid sequence of C-FKBP exhibits striking degrees of identity with the corresponding mouse (61%), human (60%), and yeast (56%) proteins. Genomic Southern blot analyses using the full-length C-FKBP cDNA probe revealed a multigene family in the Chinese cabbage genome. The C-FKBP mRNA was highly expressed in vegetative tissues. We also analyzed the antifungal and peptidyl-prolyl cis-trans isomerase activity of recombinant C-FKBP protein expressed in Escherichia coli. This protein inhibited pathogenic fungal strains, including Candida albicans, Botrytis cinerea, Rhizoctonia solani, and Trichoderma viride, whereas it exhibited no activity against E. coli and Staphylococcus aureus. These results suggest that recombinant C-FKBP is an excellent candidate as a lead compound for the development of antifungal agents.

Epidemiological investigation of Candida species causing bloodstream infection in paediatric small bowel transplant recipients.

PubMed

Suhr, Mallory J; Gomes-Neto, João Carlos; Banjara, Nabaraj; Florescu, Diana F; Mercer, David F; Iwen, Peter C; Hallen-Adams, Heather E

2017-06-01

Small bowel transplantation (SBT) can be a life-saving medical procedure. However, these recipients experience high risk of bloodstream infections caused by Candida. This research aims to characterise the SBT recipient gut microbiota over time following transplantation and investigate the epidemiology of candidaemia in seven paediatric patients. Candida species from the recipients' ileum and bloodstream were identified by internal transcribed spacer sequence and distinguished to strain by multilocus sequence typing and randomly amplified polymorphic DNA. Antifungal susceptibility of bloodstream isolates was determined against nine antifungals. Twenty-two ileostomy samples harboured at least one Candida species. Fungaemia were caused by Candida parapsilosis, Candida albicans, Candida glabrata, Candida orthopsilosis and Candida pelliculosa. All but three bloodstream isolates showed susceptibility to all the antifungals tested. One C. glabrata isolate showed multidrug resistance to itraconazole, amphotericin B and posaconazole and intermediate resistance to caspofungin. Results are congruent with both endogenous (C. albicans, C. glabrata) and exogenous (C. parapsilosis) infections; results also suggest two patients were infected by the same strain of C. parapsilosis. Continuing to work towards a better understanding of sources of infection-particularly the exogenous sources-would lead to targeted prevention strategies. © 2017 Blackwell Verlag GmbH.

The contributions of eye movements to the efficacy of brief exposure treatment for reducing fear of public speaking.

PubMed

Carrigan, M H; Levis, D J

1999-01-01

The present study was designed to isolate the effects of the eye-movement component of the Eye Movement Desensitization and Reprocessing (EMDR) procedure in the treatment of fear of public speaking. Seventy-one undergraduate psychology students who responded in a fearful manner on the Fear Survey Schedule II and on a standardized, self-report measure of public speaking anxiety (Personal Report of Confidence as a Speaker; PRCS) were randomly assigned to one of four groups in a 2x2 factorial design. The two independent variables assessed were treatment condition (imagery plus eye movements vs. imagery alone) and type of imagery (fear-relevant vs. relaxing). Dependent variables assessed were self-reported and physiological anxiety during exposure and behavioral indices of anxiety while giving a speech. Although process measures indicated exposure to fear-relevant imagery increased anxiety during the procedure, no significant differences among groups were found on any of the outcome measures, except that participants who received eye movements were less likely to give a speech posttreatment than participants who did not receive eye movements. Addition of the eye movements to the experimental procedure did not result in enhancement of fear reduction. It was concluded, consistent with the results of past research, that previously reported positive effects of the EMDR procedure may be largely due to exposure to conditioned stimuli.

Isolation of Salmonella from alfalfa seed and demonstration of impaired growth of heat-injured cells in seed homogenates.

PubMed

Liao, Ching-Hsing; Fett, William F

2003-05-15

Three major foodborne outbreaks of salmonellosis in 1998 and 1999 were linked to the consumption of raw alfalfa sprouts. In this report, an improved method is described for isolation of Salmonella from alfalfa seed lots, which had been implicated in these outbreaks. From each seed lot, eight samples each containing 25 g of seed were tested for the presence of Salmonella by the US FDA Bacteriological Analytical Manual (BAM) procedure and by a modified method applying two successive pre-enrichment steps. Depending on the seed lot, one to four out of eight samples tested positive for Salmonella by the standard procedure and two to seven out of eight samples tested positive by the modified method. Thus, the use of two consecutive pre-enrichment steps led to a higher detection rate than a single pre-enrichment step. This result indirectly suggested that Salmonella cells on contaminated seeds might be injured and failed to fully resuscitate in pre-enrichment broth containing seed components during the first 24 h of incubation. Responses of heat-injured Salmonella cells grown in buffered peptone water (BPW) and in three alfalfa seed homogenates were investigated. For preparation of seed homogenates, 25 g of seeds were homogenized in 200 ml of BPW using a laboratory Stomacher and subsequently held at 37 degrees C for 24 h prior to centrifugation and filtration. While untreated cells grew at about the same rate in BPW and in seed homogenates, heat-injured cells (52 degrees C, 10 min) required approximately 0.5 to 4.0 h longer to resuscitate in seed homogenates than in BPW. This result suggests that the alfalfa seed components or fermented metabolites from native bacteria hinder the repair and growth of heat-injured cells. This study also shows that an additional pre-enrichment step increases the frequency of isolation of Salmonella from naturally contaminated seeds, possibly by alleviating the toxic effect of seed homogenates on repair or growth of injured cells.

Evaluation of cardiac surgery mortality rates: 30-day mortality or longer follow-up?

PubMed

Siregar, Sabrina; Groenwold, Rolf H H; de Mol, Bas A J M; Speekenbrink, Ron G H; Versteegh, Michel I M; Brandon Bravo Bruinsma, George J; Bots, Michiel L; van der Graaf, Yolanda; van Herwerden, Lex A

2013-11-01

The aim of our study was to investigate early mortality after cardiac surgery and to determine the most adequate follow-up period for the evaluation of mortality rates. Information on all adult cardiac surgery procedures in 10 of 16 cardiothoracic centres in Netherlands from 2007 until 2010 was extracted from the database of Netherlands Association for Cardio-Thoracic Surgery (n = 33 094). Survival up to 1 year after surgery was obtained from the national death registry. Survival analysis was performed using Kaplan-Meier and Cox regression analysis. Benchmarking was performed using logistic regression with mortality rates at different time points as dependent variables, the logistic EuroSCORE as covariate and a random intercept per centre. In-hospital mortality was 2.94% (n = 972), 30-day mortality 3.02% (n = 998), operative mortality 3.57% (n = 1181), 60-day mortality 3.84% (n = 1271), 6-month mortality 5.16% (n = 1707) and 1-year mortality 6.20% (n = 2052). The survival curves showed a steep initial decline followed by stabilization after ∼60-120 days, depending on the intervention performed, e.g. 60 days for isolated coronary artery bypass grafting (CABG) and 120 days for combined CABG and valve surgery. Benchmark results were affected by the choice of the follow-up period: four hospitals changed outlier status when the follow-up was increased from 30 days to 1 year. In the isolated CABG subgroup, benchmark results were unaffected: no outliers were found using either 30-day or 1-year follow-up. The course of early mortality after cardiac surgery differs across interventions and continues up to ∼120 days. Thirty-day mortality reflects only a part of early mortality after cardiac surgery and should only be used for benchmarking of isolated CABG procedures. The follow-up should be prolonged to capture early mortality of all types of interventions.

Unsaponifiable fraction isolated from grape (Vitis vinifera L.) seed oil attenuates oxidative and inflammatory responses in human primary monocytes.

PubMed

Millan-Linares, Maria C; Bermudez, Beatriz; Martin, Maria E; Muñoz, Ernesto; Abia, Rocio; Millan, Francisco; Muriana, Francisco J G; Montserrat-de la Paz, Sergio

2018-04-25

Grape (Vitis vinifera L.) seed has well-known potential for production of oil as a byproduct of winemaking and is a rich source of bioactive compounds. Herein, we report that the unsaponifiable fraction (UF) isolated from grape seed oil (GSO) possesses anti-oxidative and anti-inflammatory properties towards human primary monocytes. The UF isolated from GSO was phytochemically characterized by GC-MS and HPLC. Freshly obtained human monocytes were used to analyse the effects of GSOUF (10-100 μg mL-1) on oxidative and inflammatory responses using FACS analysis, RT-qPCR, and ELISA procedures. GSOUF skewed the monocyte plasticity towards the anti-inflammatory non-classical CD14+CD16++ monocytes and reduced the inflammatory competence of LPS-treated human primary monocytes diminishing TNF-α, IL-1β, and IL-6 gene expression and secretion. In addition, GSOUF showed a strong reactive oxygen species (ROS)-scavenging activity, reducing significantly nitrite levels with a significant decrease in Nos2 gene expression. Our results suggest that the UF isolated from GSO has significant potential for the management of inflammatory and oxidative conditions and offer novel benefits derived from the consumption of GSO in the prevention of inflammation-related diseases.

Emergence of Stenotrophomonas maltophilia nosocomial isolates in a Saudi children's hospital. Risk factors and clinical characteristics.

PubMed

Alqahtani, Jobran M

2017-05-01

  To describe the clinical characteristics of pediatric patients colonized or infected by Stenotrophomonas maltophilia (S. maltophilia) at a Saudi children's hospital, to identify risk factors associated with infection, and to investigate the antimicrobial resistance patterns of this emerging pathogen.  Methods: In this cross-sectional observational study, 64 non-duplicating S. maltophilia strains were isolated  in Najran Maternity and Children's Hospital, Najran,  Saudi Arabia between January 2015 to February 2016. Antimicrobial susceptibility testing was performed using the reference broth microdilution method.  Results: In this study, 48 (75%) isolates were identified in true infections and 16 (25%) isolates were considered colonization. The main types of S. maltophilia infection were pneumonia in 22 (45.8%) patients and bloodstream infection in 14 (29.2%) patients. The significant risk factors included exposure to invasive procedure (p=0.02), and presence of acute leukemia as an underlying disease (p=0.02). The most active antimicrobials were trimethoprim/sulfamethoxazole (100% sensitivity) and tigecycline (93.7% sensitivity). Conclusions: Stenotrophomonas maltophilia is an emerging nosocomial pathogen among pediatric patients. Accurate identification and susceptibility testing of this emerging pathogen are crucial for the management of infected patients and prevention of spread of this nosocomial pathogen.