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Onset of force development as a marker of thrombin generation in whole blood: the thrombin generation time (TGT).

Prothrombin activation requires the direct interplay of activated platelets and plasma clotting factors. Once formed, thrombin causes profound, irreversible activation of platelets and reinforces the platelet plug via fibrin formation. Delayed or deficient thrombin production increases bleeding risk. Commonly employed coagulation assays, the prothrombin and partial thromboplastin times, use clot formation as a surrogate marker of thrombin generation. These assays routinely utilize platelet-poor plasma and completely miss the effects of platelets. Other markers of thrombin generation, prothrombin fragment 1 + 2 (F1 + 2) and thrombin-antithrombin complex, are typically measured after the fact. We report a simple assay, which employs the onset of platelet contractile force (PCF) as a surrogate marker of thrombin generation. PCF generation occurs concomitant with the burst of F1 + 2 release. The time between assay start and PCF onset is termed the thrombin generation time (TGT). TGT is prolonged in clotting factor deficiencies and in the presence of direct and indirect thrombin inhibitors. TGT shortens to normal with clotting factor replacement and shortens with administration of recombinant factor VIIa. TGT is short in thrombophilic states such as coronary artery disease, diabetes and thromboangiitis obliterans and prolongs toward normal with oral and intravenous anticoagulants.

Biomechanical Phenomena↗

Low restenosis rate of the NIR coronary stent: results of the Danish multicenter stent study (DANSTENT)--a randomized trial comparing a first-generation stent with a second-generation stent.

BACKGROUND: Larger studies evaluating the angiographic results of second-generation stents are scarce. The objectives of this study were to assess current standards of angiographic and clinical outcomes after implantation of the second-generation stainless steel stent, NIR (Medinol Ltd, Tel Aviv, Israel), and to compare the outcomes with those of the first-generation Palmaz-Schatz (PS) stent (Johnson & Johnson, Warren, NJ). METHODS: Patients having coronary artery lesions that could be covered by a stent of 15 mm in length were randomly assigned to receive the NIR or the PS. Procedural success, 6-month angiographic findings, and 1-year clinical outcomes were determined. RESULTS: In 424 patients included in the study, the overall procedural success rate was high (NIR 98%, PS 99%, P =.90). Follow-up angiography was conducted in 91% of the patients. The overall rate of angiographic restenosis was low in both groups (NIR 9.9%, PS 12.6%, P =.35). We found a low restenosis rate in vessels with a minimal lumen diameter >3.1 mm after the procedure, particularly in the NIR group (<6%). The rate of target lesion revascularization after 1 year did not differ (NIR 12%, PS 10%, P =.47). CONCLUSIONS: The angiographic and clinical outcomes after implantation of the second-generation stainless steel stent were not significantly better than those of the first-generation stent. The low restenosis rates, particularly in patients with the largest minimal lumen diameters after stent implantation, warrants circumspection when planning the evaluation of newer stent technologies that aim to further reduce coronary restenosis.

Angina Pectoris↗

Prediction of genetic contributions and generation intervals in populations with overlapping generations under selection.

A method to predict long-term genetic contributions of ancestors to future generations is studied in detail for a population with overlapping generations under mass or sib index selection. An existing method provides insight into the mechanisms determining the flow of genes through selected populations, and takes account of selection by modeling the long-term genetic contribution as a linear regression on breeding value. Total genetic contributions of age classes are modeled using a modified gene flow approach and long-term predictions are obtained assuming equilibrium genetic parameters. Generation interval was defined as the time in which genetic contributions sum to unity, which is equal to the turnover time of genes. Accurate predictions of long-term genetic contributions of individual animals, as well as total contributions of age classes were obtained. Due to selection, offspring of young parents had an above-average breeding value. Long-term genetic contributions of youngest age classes were therefore higher than expected from the age class distribution of parents, and generation interval was shorter than the average age of parents at birth of their offspring. Due to an increased selective advantage of offspring of young parents, generation interval decreased with increasing heritability and selection intensity. The method was compared to conventional gene flow and showed more accurate predictions of long-term genetic contributions.

Animals↗

First-generation effects on second-generation fertility.

The number of children born to the second generation is shown to be inversely related to the predicted income of the first generation (father's father) and to the number of siblings in the second generation. This is true even when second-generation characteristics are taken account of. This long-term effect may be the result of parental influences on the tastes, opportunities or genes of their children. They do get an empirical expression because of the imperfect and partial measurement of second-generation characteristics.

Adult↗

Understanding and leading the quad matrix: four generations in the workplace: the Traditional Generation, Boomers, Gen-X, Nexters.

Generational diversity is not new, but 4 generations working side by side has not occurred before. This climate of 4 generational workers offers challenges and opportunities for leaders. Leaders must show the opportunities of a diverse workforce and provide the encouragement and enthusiasm of working in a 4-generation environment. This article will define the 4 generations, the perspectives, values and motivations of each and the opportunity to tap into this rich tapestry.

Adult↗

Heterogeneous mechanisms of human cytotoxic T lymphocyte generation. I. Differential helper cell requirement for the generation of cytotoxic effector cells from CD8+ precursor subpopulations.

The subpopulations of CD8+ T cells defined by CD45RA Ag expression have been hypothesized to represent cells varying in their relative maturation along a common, activation-dependent differentiation pathway. Previous studies have shown that both the CD8+CD45RA+ and CD8+CD45RA- subsets contain precursor cells capable of developing into alloreactive CTL. In the current study, we have examined the mechanisms involved in the generation of CTL effector cells from these two CD8+ subsets. Purified CD8+CD45RA+ or CD8+CD45RA- cells were stimulated with allogeneic non-T cells, either alone or in the presence of CD4+ Th cells. Although the generation of CTL from CD8+CD45RA- precursor cells consistently required the presence of CD4+ Th cells, cytotoxic effector cells could be generated from CD8+CD45RA+ precursor cells in the absence of CD4+ cells. Several lines of evidence indicated that the helper cell-independent generation of cytotoxic effector cells from CD8+CD45RA+ precursors resulted from the unique ability of this subset to produce and use IL-2 in an autocrine fashion: 1) exogenous IL-2 could replace the effects of CD4+ helper cells for either CD8+ subset; 2) the helper cell-independent functional maturation of CD8+CD45RA+ cells could be blocked by anti-CD25 or anti-IL-2 antibodies; and 3) CD8+CD45RA+ cells produced IL-2 after activation with allogeneic cells. The finding that precursors for helper cell-independent CTL generation are restricted to the CD8+CD45RA+ subset suggests that this capability may vary as a function of the maturation of CD8+ cells.

Antigens, CD↗

Reversible activation of the neutrophil superoxide generating system by hexachlorocyclohexane: correlation with effects on a subcellular superoxide-generating fraction.

gamma-Hexachlorocyclohexane was found to exert profound effects on the phosphatidylinositol cycle, cytosolic calcium level, and the respiratory burst of human neutrophils. Exposure of neutrophils prelabelled with 32P to 4 X 10(-4) M gamma-hexachlorocyclohexane almost tripled radioactivity in phosphatidic acid and correspondingly decreased radioactivity in phosphatidylinositol 4,5 bisphosphate. Under similar conditions, gamma-hexachlorocyclohexane evoked the generation of superoxide at a rate of over 11 nmol/min/10(6) cells and more than doubled cytosolic-free calcium concentration as monitored by Quin-2 fluorescence. Because intermediates of the phosphatidylinositol cycle, via increases in available calcium levels or activated protein kinase C, are considered potential second messengers for activation of the NADPH-dependent O-2-generating system, we compared neutrophil responses to gamma-hexachlorocyclohexane with responses to phorbol myristate acetate, an activator of protein kinase C with well known effects on neutrophils. Like phorbol myristate acetate, gamma-hexachlorocyclohexane induced neutrophil degranulation but was not an effective chemotactic stimulus. The ability of gamma-hexachlorocyclohexane to induce a pattern of oxidative activation in neutrophil cytoplasts similar to that in intact cells indicated that concurrent degranulation was not required for sustained O-2 generation in response to this agent. When neutrophils or neutrophil cytoplasts exposed to gamma-hexachlorocyclohexane were centrifuged and resuspended in stimulus-free medium, O-2 generation ceased entirely but could be reinitiated by addition of the same stimulus. This finding was in contrast to the continued O-2 production by phorbol myristate acetate-stimulated neutrophils similarly washed and resuspended in stimulus-free medium. Unlike subcellular fractions of phorbol myristate acetate-stimulated neutrophils, corresponding fractions prepared from gamma-hexachlorocyclohexane-stimulated neutrophils contained almost no detectable NADPH-dependent O-2-generating activity. Subcellular oxidase activity was not recovered when cells and membrane fractions were continuously exposed to gamma-hexachlorocyclohexane during disruption and fractionation after cell stimulation, nor could it be induced by the addition of the stimulus to the subcellular fractions. Thus, the stimulus dependence of continuous neutrophil superoxide release evoked by gamma-hexachlorocyclohexane does not merely reflect a physical interaction of the agonist with the enzyme system involved.(ABSTRACT TRUNCATED AT 400 WORDS)

Calcium↗

Generation of cytotoxic T lymphocytes from thymocyte precursors to trinitrophenyl-modified self antigens. I. Requirement of both killer-helper factor(s) and interleukin 2 for CTL generation from a subpopulation of thymocytes.

The requirement for signals in the induction of cytotoxic T lymphocytes (CTL) from thymocyte precursors has been investigated. Either unfractionated or peanut agglutinin-binding (PNA+) C3H/He thymocytes were stimulated with mitomycin C(MMC)-treated, 2,4,6-trinitrophenyl(TNP)-modified syngeneic spleen cells in the presence of a variety of lymphokine preparations. Cellfree supernatant (CFS) from purified protein derivatives(PPD-CFS) stimulated Mycobacterium tuberculosis (Tbc)-primed cells, or partially purified interleukin 2 (IL 2) mediated strong cytotoxic responses in unfractionated thymocytes, whereas only PPD-CFS at final concentrations beyond 30% was active for CTL generation in PNA+ thymocytes. Neither IL 2 at concentrations of below 60 U/ml nor a low concentration of PPD-CFS (at final below 10%) had such a capacity. The addition of monoclonal anti-IL 2 receptor antibody completely blocked CTL generation induced by PPD-CFS in PNA+ thymocytes. In contrast, anti-immune interferon (IFN-gamma) antibody showed a marginal effect. PPD-CFS (10%) and IL 2 (10 U/ml) could synergistically trigger PNA+ thymocytes to induce CTL generation. These results suggested that both IL 2 and "helper" factors other than IL 2 are required for CTL generation from PNA+ thymocytes. We refer to these kinds of helper factors as killer helper factors (KHF). Partially purified IL 2-free KHF show two peaks of activities at apparent m.w. 14,000 to 34,000 and 44,000 to 90,000, and are heterogeneous with respect to isoelectric point, which is between 4.5 and 5.1. Cultures that received TNP-modified syngeneic cells and KHF on day 0 and IL 2 on day 2 generated potent CTL responses, whereas the addition of IL 2 on day 0 followed by the addition of KHF on day 2 to the culture was ineffective, suggesting that KHF is required in the early phase of the culture to achieve optimal CTL responses.

Animals↗

Identification of oxidized histidine generated at the active site of Cu,Zn-superoxide dismutase exposed to H2O2. Selective generation of 2-oxo-histidine at the histidine 118.

Inactivation of Cu,Zn-superoxide dismutase (Cu,Zn-SOD) by its own reaction product H2O2 is a well-known phenomenon. Generation of the hydroxyl radical has been a matter of great concern, and the target molecule has been regarded as its own active site histidine residues, whose oxidized forms have not yet been identified (Hodgson, E.K., and Fridovich, I. (1975) Biochemistry 14, 5294-5299). Here we report on the identification of oxidized histidines generated at the active site of Cu,Zn-SOD by reaction with H2O2. When bovine erythrocyte Cu,Zn-SOD (0.5 mg/ml) was treated with 5 mM H2O2 in 50 mM sodium phosphate buffer (pH 7.2), histidine was significantly lost; however, except for a significant increase in aspartate and glutamate, nothing new appeared in the amino acid analysis of oxidized Cu,Zn-SOD. On the other hand, the hydrolysates of oxidized Cu,Zn-SOD involved an unknown product that was detectable by reverse-phase high performance liquid chromatography with electrochemical detection. The product was found to be identical to 2-oxo-histidine that had been discovered as the major oxidation product of histidine and its peptides treated with a copper/ascorbate-free radical generating system. The main product present in the hydrolysate of H2O2-treated Cu,Zn-SOD was 2-oxo-histidine. Approximately 0.66 mol/mol subunit was formed when Cu,Zn-SOD was treated with 5 mM H2O2 for 30 min. Both metal chelators and the hydroxyl radical scavengers only slightly inhibited the 2-oxo-histidine formation (10-39%), suggesting that the active species were produced mainly inside the ligands of the Cu2+ in the enzyme. Trypsin digestion of H2O2-treated Cu,Zn-SOD showed selective reactions at the sequences of Gly24-Lys67 and Thr114-Arg126, in that histidine residues locate at the active center. Two new products derived from those peptides appeared in the tryptic map. Amino acid analysis of both products demonstrated the loss of only histidine. One of them derived from Thr114-Arg126 contained an equimolar amount of 2-oxo-histidine, indicating that His-118 was converted selectively to 2-oxo-histidine; however, another product derived from Gly24-Lys67 contained only 0.085 mol of 2-oxo-histidine/mol of peptide, suggesting that the product is a mixture consisting of unidentified forms of oxidized histidine. Taken together, the present study provided direct evidence that 2-oxo-histidine was generated in the Cu,Zn-SOD exposed to H2O2 and that its generation was selective at histidine 118 of the active site of the enzyme.

Amino Acid Sequence↗

The relationship between microalbuminuria in first generation diabetic and non-diabetic subjects and microalbuminuria and hypertension in the second generation (a population based study).

BACKGROUND: Predisposition to hypertension has been proposed as a risk factor for development of diabetic nephropathy and hypertension. The aim of this study was to examine a possible relation between microalbuminuria (urinary albumin excretion (UAE) 20-200 micrograms/min) in diabetic and non-diabetic subjects and microalbuminuria as well as hypertension in the next generation. METHODS: We examined 280 non-diabetic subjects in a cross sectional study (mean age 47-48 years). 136 were first born offspring of non insulin dependent diabetic (NIDDM) patients and 144 were first born offspring of non-diabetic controls. Hypertension was defined as systolic blood pressure > 140 mmHg, and/or diastolic blood pressure > 90 mmHg, and/or the presence of antihypertensive medications. Data were analysed by multiple logistic regression. RESULTS: We found that parental microalbuminuria was not predictive for microalbuminuria in the second generation in this population at the time of follow-up. However, microalbuminuria was predictive for hypertension in the second generation of diabetic patients, Odds ratio (OR) = 2.5, P = 0.05 when adjusted for age, gender, smoking, and obesity. In offspring of non-diabetic persons parental microalbuminuria also increased the risk of hypertension in the offspring generation, OR = 3.7, P = 0.02. Obesity was the strongest predictor for microalbuminuria and for hypertension in offspring of diabetic patient and of non-diabetic persons. CONCLUSION: We found a significant relation between microalbuminuria in the parental generation and hypertension in the offspring both of a diabetic population and of a non-diabetic population.

Albuminuria↗

A second-generation cementless hip prosthesis: improved results over the first-generation prosthesis.

Forty-two patients who had a Porous Coated Anatomic (PCA) "E" series, second-generation, cementless hip arthroplasty (Howmedica, Rutherford, NJ) were compared with 42 patients who had a first-generation PCA prosthesis. Patients were directly matched for age, sex, diagnosis, weight, Charnley functional status, and duration of follow-up. All of the operations were done by the same two surgeons, who used the same operative approach and the same postoperative rehabilitation plan. All of the patients were followed up for at least 5 years (range, 60 to 76 months). In the "E" series group, there were 41 of 42 (98%) good and excellent clinical results with a mean Harris hip score of 94 points (range, 46 to 100 points); the first-generation group had 34 of 42 (81%) good and excellent clinical results and a mean Harris score of 81 points (range, 42 to 100 points) (P = 0.001). There was one acetabular component revision in the "E" series group (2%), which can be compared with eight revisions (19%) in the first-generation group (P = 0.012). The incidence of femoral radiolucencies was 19% (eight hips) for the "E" series group compared with 50% (21 hips) in the first-generation group (P = 0.009). The radiolucencies in the "E" series group were small, nonprogressive, and confined typically to zone I. We believe that the improvements in design of the "E" series component may account for these differences.

Adult↗

A mathematical model for the generation and control of a pH gradient in an immobilized enzyme system involving acid generation.

An optimal pH control technique has been developed for multistep enzymatic synthesis reactions where the optimal pH differs by several units for each step. This technique separates an acidic environment from a basic environment by the hydrolysis of urea within a thin layer of immobilized urease. With this technique, a two-step enzymatic reaction can take place simultaneously, in proximity to each other, and at their respective optimal pH. Because a reaction system involving an acid generation represents a more challenging test of this pH control technique, a number of factors that affect the generation of such a pH gradient are considered in this study. The mathematical model proposed is based on several simplifying assumptions and represents a first attempt to provide an analysis of this complex problem. The results show that, by choosing appropriate parameters, the pH control technique still can generate the desired pH gradient even if there is an acid-generating reaction in the system.

Acids↗

Absolute dependence of T cell receptor(hi) cell generation and relative dependence of T cell receptor(int) cell generation on the thymus.

Recent evidence indicates that conventional T cells are generated by the mainstream of T cell differentiation in the thymus and acquire a high density of T cell receptor expression (i.e. TCRhi). In contrast, primordial T cells (or NK1.1+ T cells) are generated by the extrathymic pathways or an alternative intrathymic pathway and express an intermediate density of TCR (i.e. TCRint). To obtain further evidence, it was examined how thymus grafting influenced the distribution of T cell populations in athymic nude mice. When BALB/c nu/nu mice were engrafted with thymocyte-depleted BALB/c+/+ fetal thymi, two changes emerged after grafting: nude mice generated TCRhi cells de novo in the periphery as well as in the grafted thymi, and the absolute number of interleukin-2 receptor beta chain+ TCRint cells increased prominently in number in the periphery. Among thymic hormones tested, the administration of thymosin alpha induced a slight expansion of CD3int cells in nude mice. To examine a possible interaction of TCRint cells with TCRhi cells in the periphery, B6 nu/nu mice (Ly5.2+) were injected with TCRhi cells purified from the spleen of B6 Ly5.1 congenic mice. In this case, TCRint (Ly5.2+) cells expanded well in all tested organs of nude mice. These results suggest that the generation of TCRhi cells is absolutely dependent on the thymus and that TCRint cells expand under the influence of the thymus (humoral) and due to interaction with thymus-derived conventional T cells.

Animals↗

Generation of a chemotactic lipid from a arachidonic acid by exposure to a superoxide-generating system.

Certain products of arachidonic acid have been demonstrated recently to possess chemotactic activity for human polymorphonuclear leukocytes (PMN). Enzymatic (lipoxygenase, cyclooxygenase) generation of these lipid chemotaxins proceeds through the formation of intermediate lipid peroxides. Since lipid peroxidation can be mediated by oxygen-derived free radicals, we have examined whether chemotactically active products of arachidonic acid could be produced by exposing this unsaturated fatty acid to a superoxide-generating system. A lipid with potent chemotactic activity for human PMN was produced by incubating arachidonic acid with xanthine oxidase and acetaldehyde. Generation of chemotactic activity was time-dependent and could be inhibited to the greatest extent by scavengers of singlet oxygen (i.e., histidine, uric acid, and 2,5-dimethylfuran). Inhibition was also observed with scavengers of superoxide anion radicals (i.e., superoxide dismutase), hydrogen peroxide (i.e., catalase), and hydroxyl radicals (i.e., mannitol). Silica gel thin-layer radiochromatography demonstrated a single peak with chemotactic activity (Rf = 0.33-0.38) distinct from unaltered arachidonic acid. The product of arachidonic acid was chemotactic at a concentration of 3.0 ng/ml and chemokinetic at concentrations of 0.75-1.5 ng/ml. Since PMN produce oxygen-derived free radicals and singlet oxygen upon stimulation of their plasma membrane, and since arachidonic acid is widely distributed in human tissues, free radical-mediated generations of chemotactic activity from arachidonic acid may play an important role in amplifying inflammatory responses.

Arachidonic Acids↗

Using Prime Editing Guide Generator (PEGG) for high-throughput generation of prime editing sensor libraries.

Prime editing enables the generation of nearly any small genetic variant. However, the process of prime editing guide RNA (pegRNA) design is challenging and requires automated computational design tools. We developed Prime Editing Guide Generator (PEGG), a fast, flexible, and user-friendly Python package that enables the rapid generation of pegRNA and pegRNA-sensor libraries. Here, we describe the installation and use of PEGG (https://pegg.readthedocs.io) to rapidly generate custom pegRNA-sensor libraries for use in high-throughput prime editing screens.

Gene Editing↗

Molecular basis for multiple sulfatase deficiency and mechanism for formylglycine generation of the human formylglycine-generating enzyme.

Sulfatases are enzymes essential for degradation and remodeling of sulfate esters. Formylglycine (FGly), the key catalytic residue in the active site, is unique to sulfatases. In higher eukaryotes, FGly is generated from a cysteine precursor by the FGly-generating enzyme (FGE). Inactivity of FGE results in multiple sulfatase deficiency (MSD), a fatal autosomal recessive syndrome. Based on the crystal structure, we report that FGE is a single-domain monomer with a surprising paucity of secondary structure and adopts a unique fold. The effect of all 18 missense mutations found in MSD patients is explained by the FGE structure, providing a molecular basis of MSD. The catalytic mechanism of FGly generation was elucidated by six high-resolution structures of FGE in different redox environments. The structures allow formulation of a novel oxygenase mechanism whereby FGE utilizes molecular oxygen to generate FGly via a cysteine sulfenic acid intermediate.

Alanine↗

Stability and characterization of perphenazine aerosols generated using the capillary aerosol generator.

Perphenazine (a potent antiemetic) was aerosolized using capillary aerosol generator to generate respirable condensation aerosols from drug in propylene glycol (PG) solutions, by pumping the liquids through a heated capillary tube. The study characterized the stability of perphenazine during and following aerosol generation. The stability-indicating HPLC method (C-8 column with a mobile phase of 52% 0.01 M pH 3.0 acetate buffer+48% acetonitrile) also enabled the study of perphenazine stability in solution under acidic, basic, oxidizing and photolysing conditions. An LC-MS (ESI+) method was used to characterize the degradation products. Perphenazine was found to be stable in acidic and basic conditions, while perphenazine sulfoxide was the major product formed in dilute peroxide solutions. Two photo-degradation products were formed in PG that were tentatively identified by LC-MS; one of these was synthesized and confirmed to be 2-[4-(3-phenothiazin-10-yl-propyl)-piperazino]-ethanol. Both photolysis products showed that aromatic dechlorination had occurred and one appeared to also result from interaction with the solvent. Within an aerosolization energy window of 84-95 J, fine particle aerosols were generated from perphenazine PG formulations with no significant degradation. Small amounts of degradation products were produced in all samples during aerosolization at elevated (non-optimal) energies. These were largely consistent with those seen to result from oxidation and photolysis in solution, showing that oxidation and dehalogenation appeared to be the main degradation pathways followed when the CAG system was overheated.

Aerosols↗

Common fears: a comparison of adolescents' self-generated and fear survey schedule generated fears.

Investigations into normative fear in adolescence have indicated that the most common fears are consistently death-related and danger-related. Assessments have most commonly been made from self-reports on fear survey schedules. The aim of the present study, therefore, was to determine whether adolescents would provide responses comparable to those generated through the use of a fear survey schedule when asked to list their 3 greatest fears. A total of 439 adolescents aged 11 to 18 years (237 girls, 201 boys, 1 sex not reported) listed their greatest fears; then they completed the 78-item Fear Survey Schedule for Children-II (E. Gullone & N. J. King, 1992). Consistent with past research, the 10 most common fears generated via the fear schedule related to death and danger. However, on the whole, the self-generated fears deviated from the death and danger theme, also including fear of failure, fear of animals, and fear of the unknown. A tendency toward global responses in self-generated fears appeared to encompass the majority of specific death-related fears included in the fear schedule, thus allowing for other predominant fears to be listed among the 3 most common.

Adolescent↗