Study of curtaining effect reduction methods in Inconel 718 using a plasma focused ion beam.

The curtaining effect is a common challenge in focused ion beam (FIB) surface preparation. This study investigates methods to reduce this effect during plasma FIB milling of Inconel 718 (nickel-based superalloy). Platinum deposition, silicon mask and XeF2 gas injection were explored as potential solutions. These methods were evaluated for two ion beam current conditions; a high ion beam intensity condition (30 kV-1 µA) and a medium one (30 kV-100 nA) and their impact on curtaining reduction and resulting cross-section quality was assessed quantitatively thanks to topographic measurements done by atomic force microscopy (AFM). XeF2 assistance notably improved cross-section quality at medium current level. Pt deposition and Si mask individually mitigated the curtaining effect, with greater efficacy at 100 nA. Both methods also contributed to reducing cross-section curvature, with the Si mask outperforming Pt deposition. However, combining Pt deposition and Si mask with XeF2 injection led to deterioration of these protective layers and the reappearance of the curtaining effect after a quite short exposure time.

Does Background Matter? A Comparative Characterization of Mouse Models of Autosomal Retinitis Pigmentosa rd1 and Pde6b-KO.

Many retinal degenerative diseases result in vision impairment or permanent blindness due to photoreceptor loss or dysfunction. It has been observed that Pde6brd1 mice (rd1), which carry a spontaneous nonsense mutation in the pde6b gene, have a strong phenotypic similarity to patients suffering from autosomal recessive retinitis pigmentosa. In this study, we present a novel mouse model of retinitis pigmentosa generated through pde6b gene knockout using CRISPR/Cas9 technology. We compare this Pde6b-KO mouse model to the rd1 mouse model to gain insights into the progression of retinal degeneration. The functional assessment of the mouse retina and the tracking of degeneration dynamics were performed using electrophysiological methods, while retinal morphology was analyzed through histology techniques. Interestingly, the Pde6b-KO mouse model demonstrated a higher amplitude of photoresponse than the rd1 model of the same age. At postnatal day 12, the thickness of the photoreceptor layer in both mouse models did not significantly differ from that of control animals; however, by day 15, a substantial reduction was observed. Notably, the decline in the number of photoreceptors in the rd1 model occurred at a significantly faster rate. These findings suggest that the C3H background may play a significant role in the early stages of retinal degeneration.

Arterial Thrombosis and Acute Limb Ischemia as a Complication of COVID-19 Infection.

COVID-19 refers to viral respiratory infections and is the predisposing factor for the development of venous and arterial thrombotic events due to a pronounced inflammatory response, platelet activation, endothelial dysfunction, and stasis. Recent studies have confirmed a high incidence of thromboembolic events, especially in the group of patients with severe coronavirus pneumonia. There have been an increasing number of reports of peripheral arterial thrombosis as well. Most cases of arterial thrombosis are noted in critical ill patients in intensive care setting. However, an increase of adverse arterial events was also noted in cases of asymptomatic or mild forms of COVID-19. Herein, we report a case of patient with asymptomatic SARS-CoV-2 infection, who developed a threatening lower limb ischemia. Our own clinical observation suggests that COVID-19-associated arterial thrombosis can be successfully treated by embolectomy, administration of in-hospital parenteral anticoagulation, and continuation of antithrombotic therapy with a "vascular" dose of rivaroxaban after discharge.

The Dynamics of Mycoplasma gallisepticum Nucleoid Structure at the Exponential and Stationary Growth Phases.

The structure and dynamics of bacterial nucleoids play important roles in regulating gene expression. Bacteria of class Mollicutes and, in particular, mycoplasmas feature extremely reduced genomes. They lack multiple structural proteins of the nucleoid, as well as regulators of gene expression. We studied the organization of Mycoplasma gallisepticum nucleoids in the stationary and exponential growth phases at the structural and protein levels. The growth phase transition results in the structural reorganization of M. gallisepticum nucleoid. In particular, it undergoes condensation and changes in the protein content. The observed changes corroborate with the previously identified global rearrangement of the transcriptional landscape in this bacterium during the growth phase transition. In addition, we identified that the glycolytic enzyme enolase functions as a nucleoid structural protein in this bacterium. It is capable of non-specific DNA binding and can form fibril-like complexes with DNA.

Discrimination of dynamically and post-dynamically recrystallized grains based on EBSD data: application to Inconel 718.

The importance of angular resolution in EBSD analyses is discussed based on an Inconel 718 sample containing several populations of recrystallized grains, with subtle differences in dislocation contents. Classical EBSD analyses (with angular resolution in the range of 0.5-1°) do not allow for distinguishing recrystallized grains grown dynamically or post-dynamically. The angular resolution of EBSD orientation and misorientation data can be significantly improved (down to about 0.1-0.2°) either using more sophisticated Kikuchi pattern indexing methods and/or using the recently proposed LLASS denoising filter (Local Linear Automatic Smoothing Splines). Then the coexistence of both dynamically and post-dynamically recrystallized grains in the sample can be confirmed and quantified. ECCI images unambiguously confirm the conclusions drawn from the analysis of improved angular resolution EBSD data, and furthermore reveal the presence of thermal stress induced dislocations with typical patterns in water quenched Inconel 718 recrystallized grains. LAY DESCRIPTION: EBSD is widely used to study recrystallization phenomena. Conventional EBSD is nevertheless not able to distinguish dynamic recrystallized grains from post-dynamic recrystallized grains which differ by subtitle differences in dislocation contents. In this paper, we show that improving the orientation precision of EBSD data by means of different methods allows distinguishing these two recrystallized grains populations. Analyses and discussion are based on an Inconel 718, a famous Nickel-based superalloy in aeronautic.

Detection of renal tissue and urinary tract proteins in the human urine after space flight.

The urine protein composition samples of ten Russian cosmonauts (male, aged of 35 up to 51) performed long flight missions and varied from 169 up to 199 days on the International Space Station (ISS) were analyzed. As a control group, urine samples of six back-up cosmonauts were analyzed. We used proteomic techniques to obtain data and contemporary bioinformatics approaches to perform the analysis. From the total number of identified proteins (238) in our data set, 129 were associated with a known tissue origin. Preflight samples contained 92 tissue-specific proteins, samples obtained on Day 1 after landing had 90 such proteins, while Day 7 samples offered 95 tissue-specific proteins. Analysis showed that consistently present proteins in urine (under physiological conditions and after space flight) are cubilin, epidermal growth factor, kallikrein-1, kininogen-1, megalin, osteopontin, vitamin K-dependent protein Z, uromodulin. Variably present proteins consists of: Na(+)/K(+) ATPase subunit gamma, ß-defensin-1, dipeptidyl peptidase 4, maltasa-glucoamilasa, cadherin-like protein, neutral endopeptidase and vascular cell adhesion protein 1. And only three renal proteins were related to the space flight factors. They were not found in the pre-flight samples and in the back-up cosmonaut urine, but were found in the urine samples after space flight: AFAM (afamin), AMPE (aminopeptidase A) and AQP2 (aquaporin-2). This data related with physiological readaptation of water-salt balance. The proteomic analysis of urine samples in different phases of space missions with bioinformation approach to protein identification provides new data relative to biomechemical mechanism of kidney functioning after space flight.

Core proteome of the minimal cell: comparative proteomics of three mollicute species.

Mollicutes (mycoplasmas) have been recognized as highly evolved prokaryotes with an extremely small genome size and very limited coding capacity. Thus, they may serve as a model of a 'minimal cell': a cell with the lowest possible number of genes yet capable of autonomous self-replication. We present the results of a comparative analysis of proteomes of three mycoplasma species: A. laidlawii, M. gallisepticum, and M. mobile. The core proteome components found in the three mycoplasma species are involved in fundamental cellular processes which are necessary for the free living of cells. They include replication, transcription, translation, and minimal metabolism. The members of the proteome core seem to be tightly interconnected with a number of interactions forming core interactome whether or not additional species-specific proteins are located on the periphery. We also obtained a genome core of the respective organisms and compared it with the proteome core. It was found that the genome core encodes 73 more proteins than the proteome core. Apart of proteins which may not be identified due to technical limitations, there are 24 proteins that seem to not be expressed under the optimal conditions.

Specific adipocytokines profiles in patients with hyperactive and/or binge/purge form of anorexia nervosa.

OBJECTIVE: The aim of our study was to determine whether eating behaviors and/or physical activity level may explain contradicting results in adipocytokines levels in anorexia nervosa (AN). SUBJECTS/METHODS: Fasting levels of circulating adipocytokines (adiponectin, resistin and leptin), insulin, glucose, C-reactive protein, cytokines (tumor necrosis factor-alpha and interleukin (IL)-1beta), body composition and resting energy expenditure were measured in 24 women AN patients and 14 women controls. These parameters were compared according to AN subtypes: 15 patients with restrictive (R-AN) form versus 9 patients with binge/purge (BP-AN) form; 15 patients with hyperactive (H-AN) form versus 9 patients with nonhyperactive (NH-AN) form. RESULTS: BP-AN patients had significantly higher serum adiponectin levels compared with R-AN patients (P<0.05), and H-AN patients had higher serum leptin and lower serum resistin levels compared with NH-AN patients (P<0.05 for both). CONCLUSIONS: Our study shows specific adipocytokines profiles depending on the subtype of AN: restrictive versus binge/purge and hyperactive versus Nonhyperactive forms. We suggest that these biological signatures could interfere with the outcome of the disease.

N-terminal amphipathic helix as a trigger of hemolytic activity in antimicrobial peptides: a case study in latarcins.

In silico structural analyses of sets of alpha-helical antimicrobial peptides (AMPs) are performed. Differences between hemolytic and non-hemolytic AMPs are revealed in organization of their N-terminal region. A parameter related to hydrophobicity of the N-terminal part is proposed as a measure of the peptide propensity to exhibit hemolytic and other unwanted cytotoxic activities. Based on the information acquired, a rational approach for selective removal of these properties in AMPs is suggested. A proof of concept is gained through engineering specific mutations that resulted in elimination of the hemolytic activity of AMPs (latarcins) while leaving the beneficial antimicrobial effect intact.

Parallel increase of plasma apoproteins C-II and C-III in Type 2 diabetic patients.

AIMS: To determine plasma levels of apoprotein (apo) C-II and apoprotein C-III in Type 2 diabetic patients and to examine the clinical and biological factors that are associated with elevated apoC concentrations. METHODS: We measured apoC-II and apoC-III in total plasma and in non-high-density lipoprotein fractions by an immunoturbidimetric assay in 88 Caucasian Type 2 diabetic patients and in 138 healthy control subjects. RESULTS: Plasma levels of both apoC-II and apoC-III were increased in Type 2 diabetic patients. The clinical conditions associated with an increase of plasma apoC-II and apoC-III were abdominal obesity, body mass index, poor glycaemic control and lack of insulin treatment. However, when multivariate analysis was used, plasma apoCs levels correlated with triglyceride levels only. The apoC-III/apoC-II ratio was similar in the Type 2 diabetic and control subjects. CONCLUSIONS: Our study shows the parallel increase of apoC-II and C-III in Type 2 diabetic patients. This parallel increase is related to hypertriglyceridaemia only.

Development and validation of HPLC method for the determination of Cyclosporin A and its impurities in Neoral capsules and its generic versions.

Cyclosporin A (CyA) is a cornerstone immunosuppressant for the prophylaxis against allograft rejection after organ transplantation. The most widely prescribed CyA formulation is Neoral soft gelatine capsules (Novartis Pharmaceuticals, Basel, Switzerland). After Novartis patent expiration, several generic formulations have been developed. In this paper, a simple and reliable HPLC method was developed and validated for the evaluation of four CyA degradation products (ID-005-95, CyH, IsoCyH and IsoCyA) and two related compounds (CyB and CyG) aimed for the quality control of Neoral capsules and its generic formulations. In a second step, the validated method was then compared to the USP assay method for capsules, where some of the mentioned impurities were not adequately resolved from the CyA peak. Isocratic elution at a flow rate of 1.0mLmin(-1) was employed on a Lichrospher RP-18 (4mmx250mm; 5microm) analytical column maintained at 75 degrees C with a tetrahydrofuran:phosphoric acid (0.05M) (44:56, v/v) as mobile phase. The chromatograms were recorded using a Hewlett Packard 1100 chromatographic system. The UV detection wavelength was performed at 220nm and 10microL of sample was injected. The developed method was validated in terms of selectivity, linearity, precision, accuracy, limit of detection and limit of quantitation. The validate method was successfully applied to commercial capsules, Neoral and generic versions. Therefore, the proposed method is suitable for the simultaneous determination of CyA as well as its major impurities.

Multivariate optimization of a derivatisation procedure for the simultaneous determination of nine anabolic steroids by gas chromatography coupled with mass spectrometry.

The medical commission of the International Olympic Committee forbids the use of anabolic androgenic steroids to improve sporting performances. Nine anabolic steroids (androsterone (A), nandrolone, estradiol, testosterone propionate, nandrolone-17 propionate, dydrogesterone, testosterone, epitestosterone, boldenone) and alpha-cholestane as internal standard were studied by gas chromatography coupled with mass spectrometry (GC/MS). The derivatisation reagent employed for the derivatisation of anabolic steroids was a mixture of N-methyl-N-trimethylsilyltrifluoroacetamide (MSTFA), ammonium iodide and 2-mercaptoethanol (1000:2:6, v/w/v). Trimethylsilyl (TMS) derivatives were obtained. Anabolic steroids can be derivatised into one or two forms, mainly for androsterone into A-monoTMS and A-diTMS. The aim of this study was to research the optimization conditions of the derivatisation process (maximum yield of silylation reaction) of each anabolic steroid into only one form. A two-level factorial Doelhert design was used to determine the influence of different parameters and their interactions on each compound, thanks to response surface methodology. The parameters to be optimized were the reaction time and the temperature. The interaction "temperature-reaction time" is significant and has a positive effect on the improvement of the effectiveness of the derivatisation. Considering the large amount of information, often not convergent, a global desirability function was applied for multi-responses optimization. Thus, the optimized temperature and the reaction time of silylation were 85 degrees C and 24 min, respectively. Several GC/MS analytical parameters were also studied: linearity (regression coefficient upper than 0.99 for each compound, sensibility (range of concentration 0.05-0.30 microg/ml). Confirmatory experiments were applied to check the predicted values and to validate the model. The confirmatory assay responses are relatively close to the responses predicted. We observed satisfactory resolutions by GC/MS and a run lower than 12 min.

Progression of atherosclerosis in ApoE-deficient mice that express distinct molecular forms of TNF-alpha.

TNFalpha (TNF) critically regulates inflammation-driven atherosclerosis. Because the transmembrane (tmTNF) and soluble (sTNF) forms of TNF possess distinct immuno-modulatory properties, we hypothesized that they might differentially regulate atherosclerosis progression. Three groups of male ApoE(-/-) mice were studied: one expressing wild-type TNF (WT-TNF); one expressing exclusively a mutated non-cleavable form of TNF (KI-TNF); and one deficient in TNF (KO-TNF). Mice aged 5 weeks were fed the high-fat diet for 5 (T5) and 15 weeks (T15) or a standard chow diet for 15 weeks. At T5, in mice fed the high-fat diet, no significant differences in lesion area were observed among the three groups, either in valves or in aortas. At T15, lesion areas in valves were significantly lower in KO-TNF mice compared with those in WT-TNF mice, whereas in KI-TNF mice, they were intermediate between KO- and WT-TNF mice but not significantly different from these two groups. In aortas, lesions in KI-TNF were comparable to those of KO-TNF, both being significantly lower than those in WT-TNF. Theses differences were not linked to circulating lipids, or to macrophage, actin, and collagen contents of lesions. At T15, in mice fed the chow diet, lesion areas in valves and the aortic arch were not significantly different between the three groups. Levels of IL-6, IFNgamma, IL-10, and Foxp3 mRNAs in spleens and production of IL-6, IL-10, MCP-1, RANTES, and TNFR-2 by peritoneal macrophages at T15 of the high-fat diet showed a decrease in pro-inflammatory status, more marked in KO-TNF than in KI-TNF mice. Apoptosis was reduced only in KO-TNF mice. In conclusion, these data show that TNF effects on atherosclerosis development are detectable at stages succeeding fatty streaks and that wild-type TNF is superior to tmTNF alone in promoting atherosclerosis. TNF-dependent progression of atherosclerosis is probably linked to the differential production of pro-inflammatory mediators whether tmTNF is preponderant or essentially cleaved.

A new approach to overcome natural cholesterol interference during simultaneous determination of two stable isotope-enriched cholesterol tracers in human plasma.

We have developed a validated gas chromatography/mass spectrometry (GC/MS) method with two labelled cholesterol tracers, i.e. (2)H(4) ([2H4]-Chol) and (2)H(7) ([2H7]-Chol) enriched moieties, with a new way of calculating the abundance of labelled cholesterol in plasma without natural cholesterol interference. The isotopomers of the analytes could interfere during analysis. Elimination of these interferences can be performed by the blank or mathematical subtraction method. Validation was performed with the two interference elimination methods. For both methods, linearity was obtained in the range 5 x 10(-4) to 10(-2) mM for both labelled cholesterol moieties. In the same range, repeatability and reproducibility were less than 6.5% and 7.5% for [2H4]-Chol and [2H7]-Chol, respectively. Accuracy was about 100% and recoveries always included 100% for the two labelled cholesterols. We demonstrate that measurement of blank plasma is not necessary when using the validated abundance isotope calculation method. This saves time, reagent and samples. This calculation strategy can be extrapolated to comparable tracer approaches.

Effect of lithium on norepinephrine metabolic pathways.

We investigated lithium-induced changes in norepinephrine (NE) catabolism. NE and its major metabolites 3-methoxy-4-hydroxyphenylglycol (MHPG) and 3,4-dihydroxyphenyl glycol (DHPG), ions such as lithium (Li(+)), magnesium (Mg(2+)), and potassium (K(+)) were measured in rat plasma and cerebral cortex using an HPLC method with electrochemical detection for amines. The results obtained with a group of rats treated by lithium chloride (2 mmol/kg/IP) were compared with a control group receiving sodium chloride (2 mmol/kg/IP). Animals were killed at different times over a period of six hours in the morning following salt administration to minimize possible chronobiological effects. There are two pathways leading to MHPG formation: way A, without DHPG, and way B, with DHPG. In plasma and cerebral cortex of lithium treated rats, way A catabolism seems to be preferential. Lithium increases Mg(2+) and K(+) plasma levels. These results suggest that lithium may increase inactivation of NE and decrease NE available for adrenergic receptors.

Synthesis, immunomodulating activity and (1)H NMR studies of 7-oxo-9,11-ethano-13-azaprostanoids.

Novel 9,11-ethano analogues of prostaglandin endoperoxides with a nitrogen in position 13 were synthesized. (1)H NMR spectra of the obtained compounds were studied. All prostanoids administered perorally at doses of 2.5-10.0 microg x kg(-1) had specific dose-dependent effects on the B-cellular immunity estimated under in vivo conditions on the model of the B-cellular immune response. In terms of the direction of their activities, eight of the studied compounds were found to be immunostimulators, whereas other three compounds displayed immunosuppressing effect. Two of the compounds increased the amount of antibody-forming cells (AFC) per 10(6) spleen cells by 1.9 times in comparison with the respective parameter of control group.

Transepithelial transport of bepridil in the human intestinal cell line, Caco-2, using a "dynamic model".

The purpose of the study was to go further into the transepithetial transport of bepridil, an anticalcic agent, through monolayer cells Caco-2, using a "dynamic model" including a transfer of inserts with Caco-2 cells into new wells, free of drug, at regular intervals, in order to simulate the blood flux. The state of cells was evaluated by measuring the transepithelial electrical resistance and the transport of bepridil was followed using a gas chromatography/mass spectrometry determination. This study exhibits the importance of the basolateral renewal both on the transport of bepridil and the maintenance of cells in a satisfactory state. Two elimination phases from the cell compartment seem to occur, with basolateral half lives respectively of 12.2 and 25.6 hours, probably linked with two kinds of cellular binding sites. This dynamic model permits the reflection and simulation of the slowness of the in vivo absorption of bepridil in the small intestine.

Pregnancy-Associated Plasma Protein-A, Alpha-2-Macroglobulin, Pregnancy Zone Protein and Their Complexes with IgG in Sera of Healthy Non-Pregnant and Pregnant Woman, and Patients with Breast Cancer.

The purpose of this study was to measure alpha-2-macroglobulin-IgG (alpha(2)-MG-IgG), pregnancy zone protein-IgG (PZP-IgG) and pregnancy-associated plasma protein-A-IgG (PAPP-A-IgG) complex concentrations in serum in comparison with total alpha(2)-MG, PZP and PAPP-A concentration, and to determine the possible role of such complexes. Serum samples were obtained from 20 healthy women, 30 patients with verified metastatic breast cancer (stage III-IV) before the treatment and 40 healthy women with normal pregnancy (I-III trimester in dynamics). The serum concentrations of alpha(2)-MG-IgG, PZP-IgG and PAPP-A-IgG complexes were measured by ELISA. It has been shown that the immune complexes of the major proteinase inhibitors such as alpha(2)-MG, PZP and PAPP-A with IgG may be found at minor concentration in blood of both the healthy women and patients with breast cancer. On the other hand, their physiological functions in organism are absolutely different. We suggest that alpha(2)-MG-IgG and PZP-IgG complexes can serve as immunoregulatory signal molecules. Thus, after calculation of a molar ratio, less than 1% of alpha(2)-MG and PZP were marked by IgG. On the contrary, one PAPP-A molecule can bind to 10-15 IgG molecules in samples of control non-pregnant women and breast cancer patients. Such a kind of binding gives an evidence in favor of alternative way of utilization and degradation of PAPP-A by monocyte-macrophages through a Fc-receptor.

Another use of silica gel and aqueous eluent for HPLC analysis of clozapine and desmethylclozapine.

A procedure that involves a high-performance liquid chromatographic system with silica-bonded columns and reversed-phase eluents was fitted from a previously described method to measure clozapine and desmethylclozapine plasma levels. Clozapine and its demethylated metabolite were extracted from alkalinized serum by a liquid-liquid extraction, separated in 10 min, then quantitated at 254 nm at a minimum concentration of 20 ng/mL. The standard curves were linear over the range of 50-3000 ng/mL (r > 0.99) both for clozapine and desmethylclozapine and the assay had good sensitivity and recovery. Intra- and interday coefficients of variation for 200 and 800 ng/mL controls were less than 11.5% for clozapine and desmethylclozapine. This simple and efficient assay was used to monitor clozapine and desmethylclozapine levels from some treatment-refractory schizophrenic patients.

Simultaneous analysis of ketamine and bupivacaine in plasma by high-performance liquid chromatography.

A reversed-phase HPLC technique for the simultaneous measurement of both bupivacaine and ketamine in plasma is described. Plasma samples (0.5 ml) were prepared using a rapid and simple back-extraction technique. Resolution of both analytes and the internal standard, desipramine, from medicines coadministered to surgical paediatric patients was obtained using a 5 microm cyano (CN) (250x4.6 mm) column and a mobile phase comprising methanol-acetonitrile-orthophosphoric acid-0.01 M sodium dihydrogenphosphate (200:80:2:718). Good sensitivity for both analytes was observed using UV detection at a wavelength of 215 nm. The method has been validated according to the criteria established by the Journal of Chromatography B.