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In this study, we evaluated the performance of the flow cytometer-based Sysmex UF-5000 automated urine analyzer as a screening tool for ruling out urinary tract infections in elderly patients presenting at the emergency department. A total of 1119 unselected patient samples (including 544 samples from elderly patients) submitted for urine culture were included in this study. Samples were measured on UF-5000 and dipsticks and the results were compared with interpretation of culture results, which is the gold standard. We obtained a diagnostic sensitivity of 99% and specificity of 51% with a low rate of false negatives (0.2%) and a negative predictive value of 99% at 108 colony forming bacteria/L (CFB/L). A bacterial count ≥ 50x106/L or yeast like cells ≥ 25x106/L was used as the cutoff value. At this cutoff value, 30% of the urine cultures would have been redundant. This resulted in 35% false positive samples, mainly due to particle contamination or nongrowing bacteria. In comparison, at best, the dipsticks have a diagnostic sensitivity of 89%, a specificity of 52% and a negative predictive value of 92% at 108 CFB/L. [ABSTRACT FROM AUTHOR]

Introduction. This study aimed to relations between the expression of the Twist transcription factor, the content of tumour-associated macrophages (TAMs), and clinicopathological indicators of tumour progression in patients with stages I-II and III endometrial cancer (EC). Material and methods. Surgical specimen from 45 patients with endometrioid carcinoma of the endometrium (ECE) (average age -- 60.1 ± 2.3 y.o.) were investigated using morphological, immunohistochemical, flow cytofluorometry and statistical methods. Results. Nuclear expression of Twist was determined in 47.1% of ECE samples with individual fluctuations in the range of 6.3-43.0%, which was 16.6 ± 2.9% on average. Twist expression in G3 endometrial tumours and those with deep invasion into the myometrium tended to increase (21.4 ± 4.3 and 18.0 ± 3.5%, respectively) as compared with the expression of this marker in G2-tumors and the ones, invading < 1/2 of the myometrium (13.2 ± 3.3 and 16.7 ± 3.9%, respectively). Positive expression of Twist in ECE was associated with reduced expression of E-cadherin (44.3 ± 3.8%) and increased expression of vimentin (33.9 ± 3.4%), the content of TAMs in the stromal component of the tumour (30.2 ± 3.7 cells/f.v.), and microvessels density (MVD) (46.5 ± 5.4 vessels/mm²) as compared with the same indices for ECE with negative expression of Twist (61.4 ± 4.7%, p < 0.05; 14.6 ± 3.1%, p < 0.05; 18.0 ± 2.4 cells/f.v., p < 0.05 and 34.3 ± 4.7 vessels/mm2, respectively). Conclusions. Higher content of stromal TAMs and higher MVD are observed in Twist-positive endometrial carcinomas as compared with the same indices in Twist-negative neoplasms which are associated with different morphological specificities of invasive processes in the endometrium. [ABSTRACT FROM AUTHOR]

The tooth is mainly composed of dentin and enamel. Identification of dentin-producing odontoblasts and enamel-producing ameloblasts using reporter techniques is useful to study tooth development and regeneration with tissue engineering. Ameloblasts express Amelogenin, Ameloblastin, Enamelin, and Amelotin, whereas odontoblasts express Dentin sialophosphoprotein (Dspp) and Dentin matrix protein1 (Dmp1). Although there are several transgenic lines using promoter elements or bacterial artificial chromosomes (BACs) to label odontoblasts and ameloblasts, there is a possibility that the expression patterns vary from the endogenous genes. Here, we established 2 lines of mice where tdTomato was knocked into the second exon of X-chromosomal Amelogenin (Amelx), and green fluorescent protein (GFP) was knocked into the second exon of Dspp. tdTomato and GFP were highly expressed on secretory ameloblasts and secretory and fully differentiated odontoblasts, respectively. In addition, DSPP and AMELX were not produced in the dentin matrix and enamel matrix of Dspp GFP/GFP and Amelx tdTomato male mice (as representative of Amelx tdTomato/Y hemizygous male mice), respectively. Moreover, micro–computed tomography analysis of Amelx tdTomato male mice revealed a notable reduction in enamel volume but increased dentin mineral density. Dspp GFP/GFP mice had reduced dentin mineral density. To identify odontoblasts and ameloblasts from developing tooth, we examined the expression of mesenchymal cell surface molecules CD90, CD166 and epithelial cell surface molecules CD49f, Epcam1 with fluorescence on odontoblasts and ameloblasts in these mice. We found that GFP+ odontoblasts and tdTomato+ ameloblasts in tooth germ from 0.5-d-old Dspp GFP/+ mice and Amelx tdTomato male mice were enriched in CD45−/Ter119−/Epcam1−/CD90+/Integrin α4+cell fractions and CD45−/Ter119−/Epcam1+/CD49f+/CD147+ cell fractions, respectively. By using antibodies against mesenchymal and epithelial cell surface molecules and fluorescence, we can easily distinguish odontoblasts from ameloblasts and isolate each cell for further studies. These mice would serve as useful models for tooth development and regeneration as well as provide concurrent observation for the differentiation processes of odontoblasts and ameloblasts in vivo and in vitro. [ABSTRACT FROM AUTHOR]

The purpose of the study was to compare cytokines (CK) and chemokines concentrations in blood and cervico-vaginal samples between human papillomavirus (HPV)-positive and HPV-negative women, who had no previous history of HPV infection. A case-control study compares the activity and the concentration of CK/chemokines between 19 HPV-positive and 22 HPV-negative women matched by age. Plasma and cervico-vaginal levels of CK and chemokines were measured using cytofluorimetric analysis and expressed as mean of percentages. Plasma rates of interleukin (IL)-6 were significantly greater in HPVnegative women (mean value of 5.20±4.79 pg/ml) in comparison with HPV-positive women (mean value of 2.57±3.09 pg/ml) (p = 0.001). On the contrary, plasma levels of Eotaxin and hMCP-1 were significantly higher in HPV-positive women, with a mean value of 13.87±4.54 pg/ml (p = 0.022) and 53.53±19.51 pg/ml (p = 0.005), respectively. Differences in cervico-vaginal CK/chemokines concentrations were statistically not significant. Difference in plasma concentrations of IL-6, Eotaxin, IL-1ß and hMCP-1 was statistically significant even by analyzing HPV-16/18 and multiple HPV genotypes infections. Primary HPV infection shows a characteristic pattern of plasma CK/chemokines concentration as opposed to HPV-negative subjects and persistent HPV infection. [ABSTRACT FROM AUTHOR]

We have designed and fabricated a set of AlGaAs multilayer waveguides, which can serve as a source of entangled photons at 1.55 μm through parametric fluorescence. In our scheme two counterpropagating, orthogonally polarized signal/idler modes are nonlinearly generated by a pump wave impinging on the upper surface of the waveguide. To check the compliance with design specifications on phase-matching wavelength and parametric gain, we have systematically measured effective indices and surface-emitting second-harmonic generation, respectively. This characterization allowed us to single out a nominal sample with optimum performances, which we numerically modeled for counterpropagating parametric fluorescence. We predict a pair generation efficiency ηPF=4×10-13 (signal photons per pump photon). For a 1 W (peak), 100 ns pump pulse at normal incidence, this corresponds to about 14 photons per dark count with state-of-the-art avalanche photodiodes. [ABSTRACT FROM AUTHOR]

This work presents a dual-junction, single-photon avalanche diode (SPAD) with electrical μ-lens designed and simulated in 90 nm standard complementary metal oxide semiconductor (CMOS) technology. The evaluated structure can collect the photons impinging beneath the pixel guard ring, as well as the pixel active area. The fill factor of the SPAD increases from 12.5% to 42% in comparison with similar works on the same technology, according to new charge collections. Although the designed SPAD suffers from high dark count rate (DCR of 300kHz at 0.17V excess bias at room temperature) due to high amount of tunneling which was predicted in previous similar works, it still can be used in different applications such as random number generators and charged particle positioning pixels. [ABSTRACT FROM AUTHOR]

Flow cytometry is a widely applied approach for exploratory immune profiling and biomarker discovery in cancer and other diseases. However, flow cytometry is limited by the number of parameters that can be simultaneously analyzed, severely restricting its utility. Recently, the advent of mass cytometry (CyTOF) has enabled high dimensional and unbiased examination of the immune system, allowing simultaneous interrogation of a large number of parameters. This is important for deep interrogation of immune responses and particularly when sample sizes are limited (such as in tumors). Our goal was to compare the accuracy and reproducibility of CyTOF against flow cytometry as a reliable analytic tool for human PBMC and tumor tissues for cancer clinical trials. We developed a 40+ parameter CyTOF panel and demonstrate that compared to flow cytometry, CyTOF yields analogous quantification of cell lineages in conjunction with markers of cell differentiation, function, activation, and exhaustion for use with fresh and viably frozen PBMC or tumor tissues. Further, we provide a protocol that enables reliable quantification by CyTOF down to low numbers of input human cells, an approach that is particularly important when cell numbers are limiting. Thus, we validate CyTOF as an accurate approach to perform high dimensional analysis in human tumor tissue and to utilize low cell numbers for subsequent immunologic studies and cancer clinical trials. [ABSTRACT FROM AUTHOR]

Abstract CD25 deficiency is a very rare autosomal recessive disorder that shows a clinical phenotype highly overlapping IPEX syndrome with an increased susceptibility to viral, bacterial, and fungal infections. It is due to mutations in the IL2Rα gene that codes for the α subunit of the IL2 receptor complex. Here we report the characterization of a novel IL2Rα gene mutation leading to a severe protein conformational alteration that abrogates its cell surface expression in a child presenting with early-onset IPEX-like disorder. Cytofluorimetric analysis revealed the total absence of CD25 cell surface expression and addressed IL2Rα molecular investigation. The early clinical and molecular diagnosis of CD25 deficiency in this patient promptly led to hematopoietic stem cell transplantation (HSCT), allowing complete resolution of the symptoms and definitive cure of the disease. Highlights • Novel homozygous missense IL2Rα gene mutation. • Cytofluorimetric analysis as diagnostic tool of CD25 deficiency. • Homology Modeling as method to confirm IL2Rα mutated protein instability. [ABSTRACT FROM AUTHOR]

Graphical abstract Highlights • Novel and highly sensitive colorimetric and fluorimetric probe FLRHYDDFP designed and synthesized to identify the Cu2+ and PPi ions in invitro studies with revsersibility. • By the aid of PPi sensing strategy, alkaline phosphatase activities (ALP) were monitored with lowest detection limit of 0.012 U/mL. • For the first time endogenous PPi productions aided ALP recognition demonstrated using fluorescence confocal image guided technique in the presence of ALP inhibitor. Abstract A doubly armed hydrazone-based FLRHYDDFP probe selectively detects Cu2+ and pyrophosphate (PPi) ions through an colorimetric response- "colorless → yellow → colorless "- as well as " on-off-on " photonic switching response under physiological conditions in a sequential manner. The binding stoichiometries of the analytes Cu2+ and PPi were 1:2 and 2:4 for FLRHYDDFP -Cu2+ and Cu2+/PPi, respectively. The sequential sensing ability of FLRHYDDFP toward Cu2+ and PPi, attributed to effective complexation-aided d→π* electron transfer (ET) from Cu2+ to FLRHYDDFP and intramolecular charge/electron transfer from FLRHYDDFP to FLRHYDDFP+ , resulted in the formation of a non-symmetric Cu2+ chelate that provided a yellow-colored solution with a significant bathochromic shift from 376 to 446 nm in the UV–vis spectrum and quenching in the emission spectrum. Upon addition of PPi, Cu2+ was extruded from the complex, resulting in a revival of the fluorescence centered at 572 nm. Thus, sequential addition of Cu2+ and PPi yielded a colorless–yellow–colorless transition under visible light and on-off-on switching under 365-nm light (fluorescence). The lowest detection limits for Cu2+ and PPi, when using colorimetric and fluorimetric methods, were in the sub-micromolar and nanomolar levels, respectively. By exploiting this PPi sensing strategy, invitro as well as endogenous alkaline phosphatase activity could be monitored effectively, as demonstrated by exploiting the intracellular production or residual PPi in human salivary glands (normal) and cancer cell lines. [ABSTRACT FROM AUTHOR]

Detection of heavy metals with high sensitivity and selectivity is of vital importance. In this study, we report the synthesis of small molecular chemosensors and the fabrication of corresponding macromolecular fluorescent chemosensors. Boron dipyrrole methane (BODIPY)-derived chemosensors, BE and BB, were designed and synthesized first. Then, they were incorporated into polymeric derivatives to prepare macromolecular fluorescent chemosensors. The small molecular sensors were highly sensitive and selective to Hg2+. Other ions and albumin did not exhibit clear interference to the sensing of Hg2+ ions. Once the macromolecular chemosensors were formed, the recognition ability was preserved; moreover, the sensitivity was even enhanced by the “molecular wire” mechanism. The minimum detection limits of four macromolecular chemosensors, PBEP, PBEB, PBBP and PBBB, for Hg2+ reached 2.05 μM, 0.23 μM, 4.23 μM, and 0.15 μM, respectively. Under natural light, the addition of Hg2+ led to significant color changes; also, they can be used as Hg2+-sensitive “naked eye” indicators in the natural environment. [ABSTRACT FROM AUTHOR]

Morphological manifestations of acute colitis and subpopulation composition of colon lymphocytes were studied in male and female C57Bl/6 mice with acute dextran-induced colitis. We evaluated the severity of colitis symptoms, morphological changes in the colon, and prevalence of epithelialized and non-epithelialized ulcers. The subpopulation composition of lymphocytes (CD3—CD19+ B cells, CD3+CD4+ T helpers, CD3+CD8+ cytotoxic T cells, and CD4+CD25+FOXP3+ regulatory T cells) was assessed by flow cytofluorometry in suspension of colon cells prepared by enzymatic disintegration. In males, clinical manifestations of acute colitis and morphological changes were more severe and the prevalence of non-epithelialized ulcers was higher than in females. In females, the content of T, B, and regulatory T cells in the colon wall was higher, while the content of cytotoxic T cells was lower than in males. In females with acute colitis, the absolute lymphocyte count and the content of B cells and regulatory T cells decreased, while the percentage of cytotoxic T cells increased in comparison with intact animals. In males with acute colitis, the levels of regulatory T and B cells increased in comparison with the corresponding parameter in intact animals. Morphological changes and changes in the lymphocyte subpopulations, detected in males and females with acute colitis, were determined by different levels of sex steroid hormones. [ABSTRACT FROM AUTHOR]

Although the etiology of leiomyoma is unclear, a progenitor/undifferentiated cell population has been described whose dysregulation may be involved in the onset of uterine conditions. Moreover, inflammation is involved in the development of several tumors. The aim of this work was to understand if progenitor cells sustain a chronic inflammatory microenvironment that enhances leiomyoma development. Cells from 12 human leiomyoma and 12 normal myometrium samples of the same patients were in vitro isolated and exhaustively characterized (morphology, proliferation, cytofluorometry, differentiation, RT-PCR, immunofluorescence, immunohistochemistry, and Western blotting assays). Selected cytokines (ELISA) and inflammation-related genes (RT-PCR) were analyzed to identify healthy myometrium progenitor cells (MPCs) and leiomyoma progenitor cells (LPCs). Results show that (i) MPCs and LPCs share stemness features, such as immunophenotype and multidifferentiation assay, (ii) LPCs have a significantly shorter doubling time and a significantly higher expression of stemness genes (p<0.05), and (iii) LPCs secreted significantly higher levels (p<0.05) of cytokines related to chronic inflammation and significantly lower amounts (p<0.05) of cytokines related to acute inflammation. Despite the limited sample size, comparisons between leiomyoma and normal myometrium tissue from each patient allowed normalization of patient-specific differences. The evidenced cytokine expression pattern related to chronic inflammation in LPCs may play a role in the increased risk of adverse obstetric outcomes (infertility, spontaneous miscarriage, and preterm birth) in women affected by leiomyomas. These women should be recognized as “high risk” and subjected to specialized management both before and during pregnancy. [ABSTRACT FROM AUTHOR]

Hepatic insulin resistance and increased gluconeogenesis are known therapeutic targets of metformin, but the role of hepatic glycogen in the pathogenesis of diabetes is less clear. Mouse model of neuropeptide Y (NPY) overexpression in noradrenergic neurons (OE-NPYDβH) with a phenotype of late onset obesity, hepatosteatosis, and prediabetes was used to study early changes in glycogen structure and metabolism preceding prediabetes. Furthermore, the effect of the anti-hyperglycemic agent, metformin (300 mg/kg/day/4 weeks in drinking water), was assessed on changes in glycogen metabolism, body weight, fat mass, and glucose tolerance. Glycogen structure was characterized by cytofluorometric analysis in isolated hepatocytes and mRNA expression of key enzymes by qPCR. OE-NPYDβH mice displayed decreased labile glycogen fraction relative to stabile fraction (the intermediate form of glycogen) suggesting enhanced glycogen cycling. This was supported by decreased filling of glucose residues in the 10th outer tier of the glycogen molecule, which suggests accelerated glycogen phosphorylation. Metformin reduced fat mass gain in both genotypes, but glucose tolerance was improved mostly in wild-type mice. However, metformin inhibited glycogen accumulation and normalized the ratio between glycogen structures in OE-NPYDβH mice indicating decreased glycogen synthesis. Furthermore, the presence of glucose residues in the 11th tier together with decreased glycogen phosphorylase expression suggested inhibition of glycogen degradation. In conclusion, structural changes in glycogen of OE-NPYDβH mice point to increased glycogen metabolism, which may predispose them to prediabetes. Metformin treatment normalizes these changes and suppresses both glycogen synthesis and phosphorylation, which may contribute to its preventive effect on the onset of diabetes. [ABSTRACT FROM AUTHOR]

Glucose-6-phosphate dehydrogenase (G6PD) deficiency is the most common enzyme deficiency worldwide. Detection of heterozygously deficient females can be difficult as residual activity in G6PD-sufficient red blood cells (RBCs) can mask deficiency. In this study, we compared accuracy of 4 methods for detection of G6PD deficiency in females. Blood samples from females more than 3 months of age were used for spectrophotometric measurement of G6PD activity and for determination of the percentage G6PD-negative RBCs by cytofluorometry. An additional sample from females suspected to have G6PD deficiency based on the spectrophotometric G6PD activity was used for measuring chromate inhibition and sequencing of the G6PD gene. Of 165 included females, 114 were suspected to have heterozygous deficiency. From 75 females, an extra sample was obtained. In this group, mutation analysis detected 27 heterozygously deficient females. The sensitivity of spectrophotometry, cytofluorometry, and chromate inhibition was calculated to be 0.52 (confidence interval [CI]: 0.32–0.71), 0.85 (CI: 0.66–0.96), and 0.96 (CI: 0.71–1.00, respectively, and the specificity was 1.00 (CI: 0.93–1.00), 0.88 (CI: 0.75–0.95), and 0.98 (CI: 0.89–1.00), respectively. Heterozygously G6PD-deficient females with a larger percentage of G6PD-sufficient RBCs are missed by routine methods measuring total G6PD activity. However, the majority of these females can be detected with both chromate inhibition and cytofluorometry. [ABSTRACT FROM AUTHOR]

Based on previous assessments on stallions, 40 ejaculates of 20 Duroc boars were split and evenly frozen with a conventional vapour freezing method and two directional, drum and directional, prototype methods using commercial extenders and relative standard procedures. The directional prototype was provided with a double internal setup that allowed the positioning of experimental 2ml flat straws with 1 billion sperm (Flat) in a fixed support, or both classical 0.5 ml paillettes with 250 million spermatozoa and flats in a rotating drum designed so as to ensure a more uniform heat exchange. Preliminary tests for individuation of the most appropriate thawing rate showed beneficial effects (P≤0.05) of thawing the sperm at 50°C for 13 s when compared to 42°C for 20 s, in terms of total motility (42.8±8.4% and 35.6±6.8%, respectively). With regard to freezing/packaging methods, major improvements (P≤0.05) were shown for the drum method with paillettes for total motility (38.6±14.2%) assessed immediately after thawing, when compared with the conventional (29.4±13.3%) and the directional methods with flats (30.2±12.8%), and for total motility (P≤0.01) assessed following incubation for 120 min at 37°C after thawing (24.8±11.6%) with respect to the conventional method (15.6±10.9%). Despite the statistical non-significance of results, both the prototype freezing approaches using the experimental flat straw showed some improvements in functional parameters assessed by cytofluorometry when compared to the conventional method. [ABSTRACT FROM AUTHOR]

Multiple sclerosis is an inflammatory T cell-mediated autoimmune disease. In a Phase II clinical trial, high-dose immunosuppressive therapy combined with autologous CD34+ haematopoietic stem cell transplant resulted in 69·2% of subjects remaining disease-free without evidence of relapse, loss of neurological function or new magnetic resonance imaging (MRI) lesions to year 5 post-treatment. A combination of CyTOF mass cytometry and multi-parameter flow cytometry was used to explore the reconstitution kinetics of immune cell subsets in the periphery post-haematopoietic cell transplant (HSCT) and the impact of treatment on the phenotype of circulating T cells in this study population. Repopulation of immune cell subsets progressed similarly for all patients studied 2 years post-therapy, regardless of clinical outcome. At month 2, monocytes and natural killer (NK) cells were proportionally more abundant, while CD4 T cells and B cells were reduced, relative to baseline. In contrast to the changes observed at earlier time-points in the T cell compartment, B cells were proportionally more abundant and expansion in the proportion of naive B cells was observed 1 and 2 years post-therapy. Within the T cell compartment, the proportion of effector memory and late effector subsets of CD4 and CD8 T cells was increased, together with transient increases in proportions of CD45RA-regulatory T cells (Tregs) and T helper type 1 (Th1 cells) and a decrease in Th17·1 cells. While none of the treatment effects studied correlated with clinical outcome, patients who remained healthy throughout the 5-year study had significantly higher absolute numbers of memory CD4 and CD8 T cells in the periphery prior to stem cell transplantation. [ABSTRACT FROM AUTHOR]

The aim of this study was to investigate the value of pirarubicin (THP) photosensitizer fluorescence cystoscopy for the early diagnosis of bladder cancer. From January 2012 to June 2015, 25 patients with painless gross hematuria, were injected with THP 15 min prior to surgery and subsequently underwent fluorescence cystoscopy examination. Locations of tumors were recorded and biopsies were performed (total of 109 biopsies) under white and fluorescence light guidance. Biopsies were conducted at the THP-positive and -negative areas under white light and THP-positive areas under fluorescence light. Positive rate of bladder tumor tissue at the THP-positive areas detected under fluorescence light was 92.86%. The sensitivity and specificity were 100 and 96.15%, respectively. The positive rate of tumor tissue at the THP-positive areas detected under white light was 70.97%. The sensitivity and specificity were 100 and 84.74%, respectively. Fifty biopsies taken at the THP-negative areas under white light were found to be non-urothelial carcinoma tumor lesions. Thus, applying (THP) photosensitizer fluorescence cystoscopy for early-period bladder cancer diagnosis is safe, effective and practical. It showed a high degree of specificity and a low rate of false positives. It was a convenient visual diagnostic method for the early detection of non-muscular invasive bladder cancer. [ABSTRACT FROM AUTHOR]

Morphological changes in the thymus and composition of its cells and peripheral blood lymphocytes were studied in experiments on C57Bl/6 mice with sodium dextran sulfate-induced acute ulcerative colitis. Severe acute ulcerative colitis in rats was accompanied by stage III-IV accidental involution of the thymus. This state was characterized by inversion of the layers, death of thymocytes, and increase in the number and area of thymic corpuscles from CK19 epithelial cells. Flow cytofluorometry revealed an increase in the relative number of F4/80 macrophages in the thymus stroma and CD4CD8CD45CD11c dendritic cells and CD326UEACD205 epithelial cells in the medulla. By contrast, the count of CD326UEACD205 epithelial cells remained unchanged in the cortex. Accidental involution of the thymus was accompanied by an increase in the number of apoptotic AnnVPI cells, but decrease in the count of lymphocytes, CD3CD19 B lymphocytes, CD3CD8 cytotoxic T lymphocytes, immature CD4CD8 lymphocytes, and CD3CD4 T helpers. The level of peripheral blood endotoxin in adult male C57Bl/6 mice with fibrinous ulcerative colitis was 10-fold lower than in the control. Moreover, we observed a decrease in the absolute number of leukocytes, lymphocytes, CD3CD4 T helpers, CD3CD8 cytotoxic T lymphocytes, CD4CD25FOXP3 regulatory T lymphocytes, and CD3CD19 B lymphocytes in the peripheral blood of animals. [ABSTRACT FROM AUTHOR]

Background: Flow cytometry is an important technique for understanding multiple aspects of blood platelet biology. Despite the widespread use of the platform for assessing platelet function, the optimization and careful consideration of preanalytical conditions, sample processing techniques, and data analysis strategies should be regularly assessed. When set up and designed with optimal conditions, it can ensure the acquisition of robust and reproducible flow cytometry data. However, these parameters are rarely described despite their importance., Objectives: We aimed to characterize the effects of several preanalytical variables on the analysis of blood platelets by multiparameter fluorescent flow cytometry., Methods: We assessed anticoagulant choice, sample material, sample processing, and storage times on 4 distinct and commonly used markers of platelet activation, including fibrinogen binding, expression of CD62P and CD42b, and phosphatidylserine exposure., Results: The use of suboptimal conditions led to increases in basal platelet activity and reduced sensitivities to stimulation; however, the use of optimal conditions protected the platelets from artifactual stimulation and preserved basal activity and sensitivity to activation., Conclusion: The optimal preanalytical conditions identified here for the measurement of platelet phenotype by flow cytometry suggest a framework for future development of multiparameter platelet assays for high-quality data sets and advanced analysis., (© 2023 The Authors.)

Este estudo visou a determinar os valores eritroleucométricos e quantificar as subpopulações linfocitárias no sangue do cordão umbilical (SCU) e no sangue jugular de eqüinos neonatos. Foi realizada a colheita de SCU e do sangue jugular de 20 potros ao nascimento. As amostras foram submetidas às determinações dos valores eritroleucométricos e à quantificação de subpopulações de linfócitos-T, pela técnica citofluorométrica. Não foram verificadas diferenças significativas (PThe objective of the present study was to determine erythometric and leukometic values and quantify subpopulations of lymphocytes in umbilical cord blood (UCB) and jugular blood of neonate foals. UCB and jugular blood were collected from 20 foals at birth and processed for determination of erythometric and leukometric values and quantification of subpopulations of T lymphocytes by cytofluorometry. No significant difference (P

We obtained the morphologically, cytofluorometrically, and functionally mature dendritic cells from rats that were pulsed with antigens of the C6 glioma tissue extract. The concentrations of angiogenesis antigens (VEGF, VEGFR-1, and VEGFR-2) and periglioma zone proteins (GFAP, connexin 43, and BSAT1) in the pulsing extract were measured by ELISA. Our results drove us to a conclusion that despite mature phenotype of pulsed dendritic cell, the antigenic composition of glioma tissue extracts should be modified. [ABSTRACT FROM AUTHOR]

Background: We have previously reported the design, pharmacological properties and imaging application of bradykinin (BK) B2 receptor (B2R) ligands conjugated with fluorophores such as fluorescein derivatives at their N-terminus. To take advantage of the high penetration of infrared light into living tissues and their low autofluorescence in this region of the spectrum, additional probes conjugated with cyanine dye 7 (Cy7) were synthesized and characterized. Results: The antagonist B-9430 (D-Arg-[Hyp3,Igl5,D-Igl7,Oic8]-BK) and the agonist B-9972 (D-Arg-[Hyp³,Igl5,Oic7,Igl8]- BK) were N-terminally extended with the infrared fluorophore Cy7, producing the peptides B-10665 and B-10666, respectively. Pharmacological studies indicated that the agonist B-10666 lost much affinity for the B2R vs. the parent peptide, whereas the antagonist B-10665 better retained its potency vs. B-9430 (competition of [3H]BK binding to human B2R, contractility of the human isolated umbilical vein for which potency losses were more important in each case). Both probes stained HEK 293 cells that expressed the B2R-green fluorescent protein (GFP) construction in a specific manner (confocal microscopy) and with very extensive co-localization of the green and infrared fluorescence in either case. The agonist B-10666 at 100 nM promoted the endocytosis of B2R-GFP in live cells, but not the antagonist version at 10-25 nM. The Cy7-labeled peptides did not label cells expressing the β2-adrenoceptor-GFP construction. B-10665 at low nanomolar concentrations was an effective probe for the recombinant B2Rs in cytofluorometry and macroscopic imaging of cell wells (IVIS imaging system operated for infrared fluorescence detection). Conclusions: Despite a propensity for non-specific binding when used at high concentrations and limited sensitivity, Cy7-conjugated peptidase-resistant B2R ligands support original imaging and cytofluorometric applications. [ABSTRACT FROM AUTHOR]

Background: Implant infections are a major complication in the field of orthopaedics. Bacteria attach to the implant-surface and form biofilm-colonies which makes them difficult to treat. Not only immune cells exclusively respond to bacterial challenges, but also local tissue cells are capable of participating in defense mechanisms. The aim of this study was to evaluate the role of osteoblasts in the context of implant infections.Methods: Primary osteoblasts were cultivated and stimulated with free-swimming bacteria at 4 °C and 37 °C. Supernatants were harvested for ELISA and expression of pro-inflammatory cytokines evaluated by RT-PCR. Bacterial binding to osteoblasts was evaluated using cytofluorometry and uptake was investigated by (3)H thymidine-labelling of bacteria. Osteoblasts were additionally stimulated with the extracellular polymeric substance (EPS) of Staphylococcus epidermidis biofilms, as well as components of the EPS; the bacterial heat shock protein GroEL in particular.Results: We demonstrated that binding of bacteria to the osteoblast cell surface leads to an increased production of pro-inflammatory cytokines. Bacteria are capable of surviving intracellular. Furthermore, osteoblasts do not only respond to free-swimming, planktonic bacteria, but also to components of the EPS, including lipoteichoic acid and the heat shock protein GroEL.Conclusion: In conclusion, local tissue cells, specifically osteoblasts, might contribute to the persistence of the inflammatory response associated with implant-infections. [ABSTRACT FROM AUTHOR]

In vitrotissue culture represents a useful technique for advancingCitrusbreeding and propagation. Amongin vitroregeneration systems, anther culture is commonly used to produce haploids and doubled haploids for a fast-track producing homozygous lines, in comparison with the traditional self-pollination approach, which involves several generations of selfing. In addition, anthers culture can produce somatic embryos that can also be used for clonal propagation. In this study, two thermal shocks were applied to the anthers of sixCitrusgenotypes (two clementine and four sweet oranges), just after they were put in culture. The response obtained was different depending on the genotype: both clementines, namely Hernandina and Corsica, produced homozygous and triploid regenerants (microspore-derived embryos), whereas all of the analyzed regenerants from sweet oranges, three cultivars of Tarocco and Moro, produced heterozygous and diploid regenerants similar to the parental genotypes (somatic embryos). [ABSTRACT FROM AUTHOR]

Activity of serum antibodies in vitro binding to endothelial cells in women with normal pregnancy and preeclampsia was studied. Flow cytometry detected peculiarities of antibody binding to endothelial cells in health and disease. Detection of antiendothelial antibodies in trimesters II and III can be diagnostically important in preeclampsia. [ABSTRACT FROM AUTHOR]

Substitution of the oxygen atom in the glycosidic linkage by a disulfide bond or by selenium makes the resulting glycoside resistant to hydrolysis. To clarify the consequences for affinity to lectins we prepared benzene-based mono- to trivalent dithiogalactosides. Inhibitory capacity increased with valency for a plant toxin, the synthetic compounds potently blocking its binding to a lactose-presenting matrix and to cells. Human galectins were much less sensitive to the disulfides than the toxin. This differential response constitutes a beneficial effect to avoid cross-reactivity in vivo. Symmetrical selenodigalactoside and diselenodigalactoside were prepared and similarly tested. Both compounds proved rather equally bioactive for the toxin, graded activity was measured for human galectins. This result directs attention to further studies to relate Se-dependent alterations in bond angle and length as well as van der Waals radius to binding properties of selenoglycosides to biomedically relevant lectins. [ABSTRACT FROM AUTHOR]

Multifunctional biomimetic nanoparticles(NPs) are acquiring increasinginterest as carriers in medicine and basic research since they canefficiently combine labels for subsequent tracking, moieties for specificcell targeting, and bioactive molecules, e.g., drugs. In particular,because of their easy synthesis, low cost, good biocompatibility,high resorbability, easy surface functionalization, and pH-dependentsolubility, nanocrystalline apatites are promising candidates as nanocarriers.This work describes the synthesis and characterization of bioinspiredapatite nanoparticles to be used as fluorescent nanocarriers targetedagainst the Met/hepatocyte growth factor receptor, which is considereda tumor associated cell surface marker of many cancers. To this aimthe nanoparticles have been labeled with Fluorescein-5-isothiocyanate(FITC) by simple isothermal adsorption, in the absence of organic,possibly toxic, molecules, and then functionalized with a monoclonalantibody (mAb) directed against such a receptor. Direct labeling ofthe nanoparticles allowed tracking the moieties with spatiotemporalresolution and thus following their interaction with cells, expressingor not the targeted receptor, as well as their fate in vitro. Cytofluorometry and confocal microscopy experiments showed thatthe functionalized nanocarriers, which emitted a strong fluorescentsignal, were rapidly and specifically internalized in cells expressingthe receptor. Indeed, we found that, once inside the cells expressingthe receptor, mAb-functionalized FITC nanoparticles partially dissociatedin their two components, with some mAbs being recycled to the cellsurface and the FITC-labeled nanoparticles remaining in the cytosol.This work thus shows that FITC-labeled nanoapatites are very promisingprobes for targeted cell imaging applications. [ABSTRACT FROM AUTHOR]

Dental implants are one of the most frequently used treatment options for tooth replacement. Approximately 30% of patients with dental implants develop peri-implantitis, which is an oral inflammatory disease that leads to loss of the supporting tissues, predominately the bone. For the development of future therapeutic strategies, it is essential to understand the molecular pathophysiology of human dental peri-implant infections. Here, we describe the gene and protein expression patterns of peri-implantitis bone tissue compared with healthy peri-implant bone tissue. Furthermore, cells from the osteoblastic lineage derived from peri-implantitis samples were immortalized and characterized. We applied microarray, quantitative reverse transcription polymerase chain reaction, fluorescence-activated cell sorting, and Western blot analyses. The levels of typical bone matrix molecules, including SPP1, BGLAP, and COL9A1, in patients with peri-implantitis were reduced, while the inflammation marker interleukin 8 (IL8) was highly expressed. RUNX2, one of the transcription factors of mature osteoblasts, was also decreased in peri-implantitis. Finally, the human telomerase reverse transcriptase immortalized cell line from peri-implantitis exhibited a more fibro-osteoblastic character than did the healthy control. [ABSTRACT FROM PUBLISHER]

Purpose: Loosening of implants occurs mainly for two reasons: bacterial infection of the implant or 'aseptic loosening' presumably due to wear particles derived from the implant. To gain further insight into the pathomechanism, we analysed activation of the T cell response in these patients. Methods: Activation of peripheral T lymphocytes was determined by cytofluorometry as down-regulation of CD28 and up-regulation of CD11b. In addition, tissue samples obtained during surgery were analysed by quantitative RT-PCR for gene expression of CD3, CD14 and cathepsin K, as markers for T cells, monocytes/macrophages or osteoclasts, respectively. Results: Activated T lymphocytes were detected in patients with infection but not in patients with aseptic loosening. Gene expression of CD3 was significantly enhanced in tissues of patients with infection compared to those with aseptic loosening. Expression of CD14 and of cathepsin K did not differ between the two groups. Conclusion: Implant-associated infection and aseptic loosening are associated with a local inflammatory response, which eventually results in osteoclastogenesis and bone resorption. Systemic T cell activation, in contrast, occurs only in patients with implant-associated infection, and hence analysis of T cell activation markers could serve as a diagnostic tool to differentiate between the two entities. [ABSTRACT FROM AUTHOR]

The impact of intermittent fasting versus ad libitum feeding during Salmonella typhimurium infection was evaluated in terms of duodenum Ig A levels, bacterial clearance and intestinal and extra-intestinal infection susceptibility. Mice that were intermittently fasted for 12 weeks or fed ad libitum were infected with S. typhimurium and assessed at 7 and 14 days post-infection. Next, we evaluated bacterial load in the faeces, Peyer's patches, spleen and liver by plate counting, as well as total and specific intestinal Ig A and plasmatic corticosterone levels (by immunoenzymatic assay) and lamina propria Ig A levels in plasma cells (by cytofluorometry). Polymeric immunoglobulin receptor, α- and J-chains, Pax-5 factor, pro-inflammatory cytokine (tumour necrosis factor- α and interferon- γ) and anti-inflammatory cytokine (transforming growth factor- β) mRNA levels were assessed in mucosal and liver samples (by real-time PCR). Compared with the infected ad libitum mice, the intermittently fasted infected animals had (1) lower intestinal and systemic bacterial loads; (2) higher SIg A and Ig A plasma cell levels; (3) higher mRNA expression of most intestinal parameters; and (4) increased or decreased corticosterone levels on day 7 and 14 post-infection, respectively. No contribution of liver IgA was observed at the intestinal level. Apparently, the changes following metabolic stress induced by intermittent fasting during food deprivation days increased the resistance to S. typhimurium infection by triggering intestinal Ig A production and presumably, pathogen elimination by phagocytic inflammatory cells. [ABSTRACT FROM AUTHOR]

Background: We evaluated the performance of Sysmex UF- 1000i for cell counting and differential cell count, as well as for assessment of bacteria load in cerebrospinal fluid (CSF), as a potential approach for the rapid screening of meningitis or bacterial encephalitis. Methods: We analyzed 77 consecutive CSF samples, 34 of which (44%) displayed leukocyte count >5 white blood cell (WBQ/^iL with optical microscopy. Results on the UF-1000i were compared with those obtained by microscopic analysis. Imprecision was evaluated by testing three CSF samples with leukocyte values between 3.5 and 28.8 WBC/nL in 10 repli- cates. Carry-over was evaluated with the Broughton formula on three CSF pools with leukocyte counts between 93.5 and 132.5 WBC/^L. Linearity was assessed according to CLSI document EP6-A. In the presence of bacteria, identification and antibiogram were performed with Vitex (Biomerieux), except for Neisserie meningitidis (ApiNH, Biomerieux). Sensitivity tests were performed with Vitex and disc diffusion. Results: Optimal correlation was found between UF-1000i and optical microscopy, displaying Pearson's correlation of 0.99 and mean bias of-3.5 WBC/^L (95% Cl, from -7.0 to 0.0 WBC/nL). Imprecision varied between 12 and 16%. Li- nearity was excellent, 4-278 WBC/nL. Carry-over was neg- ligible. ROC analysis yielded AUC of 0.99 for both WBC and bacterial counts. The agreement at threshold >4 WBC/nL was 0.91, with sensitivity and specificity of 1.00 and 0.84. At S19 bacteria^nL cut-off, accuracy was 0.98, sensitivity 1.00 and specificity 0.97. Conclusions: According to these results, CSF screening with UF-I000i seems a reliable approach in terms of instrument performance, turnaround time and overall laboratory effi- ciency. [ABSTRACT FROM AUTHOR]

Objective(s): Cefixime (Cfx), is a semi-synthetic third-generation oral cephalosporin antibiotic that is prescribed for the treatment of susceptible infections. There are some procedures for the determination of Cfx in pharmaceutical formulations and biological samples. Herein a spectrofluorimetric method was proposed for Cfx determination based on the fluorescence quenching of terbium-danofloxacin (Tb3+-Dano) in the presence of Cfx. Materials and Methods: Cfx was detected based on fluorescence quenching of terbiumdanofloxacin (Tb3+-Dano) in the presence of Cfx with maximum excitation and emission wavelengths at 347 nm and 545 nm, respectively. The quenched fluorescence intensity of Tb3+- Dano system is proportional to the concentration of Cfx. The optimum conditions for the determination of Cfx were studied. Results: The maximum response was achieved under optimum conditions of [Tris buffer]= 0.008 mol/l (pH 6.5), [Tb3+]=1×10-4 mol/l and [Dano]=1×10-4 mol/l. The developed method was evaluated in terms of accuracy, precision and limit of detection. The linear concentration ranges for quantification of Cfx were 8.8×10-8-8.8×10-7 mol/l and 1.1×10-7-8.8×10-7 mol/l in standard and human serum samples with the detection limits (S/N=3) of 2.8×10-8 mol/l and 3.9×10-8 mol/l, respectively. The Cfx was determined in pharmaceutical tablets and spiked serum samples and the results were satisfactory. Conclusion: This method is simple, practical and relatively interference-free for determination of Cfx in pharmaceutical tablets and serum samples. [ABSTRACT FROM AUTHOR]

Abstract: Evaluation of radiation dose is very important for the detection of radiation damage. γ-H2AX is a popular biological dosimeter to evaluate the radiation effect. Typically, bulky and expensive commercial flow cytometers are used to detect γ-H2AX. This paper presents a miniaturized and high sensitive cytometer using a microfluidic chip for evaluating the radiation dose by detecting the mean immunofluorescence intensity of γ-H2AX. A compact optical focusing system and a shift-phase differential amplifier are designed to improve the detection sensitivity. Sample lymphocyte cells are stained by FITC fluorescent dye after being irradiated by UVC. Comparison experiments between the developed miniature cytometer and a commercial flow cytometer were conducted under different radiation doses. The developed microfluidic cytometer also demonstrates a good linear correlation between the measured fluorescence intensity and the irradiation dose with a detection limit similar to that of the commercial flow cytometer. The developed cytometer can evaluate quantitatively the radiation dose by the mean fluorescence intensity of γ-H2AX with a significantly smaller amount of blood samples than a commercial flow cytometer. [Copyright &y& Elsevier]

One of the research lines developed in our laboratory is focused on the study of the bioactivity of natural substances. Resveratrol (RV) is a polyphenol nonflavonoid compound present in a number of plant species but mainly in the berries of the red grape Vitis vinifera. The powerful antioxidant action of this molecule is well documented. In this work we evaluated the effects of this substance by adopting diverse experimental strategies. In particular, we studied the effects on cell vitality and cycle by MTT and cytofluorimetric assays. In addition, we explored the action of RV on the cell membrane by a well-consolidated biophysical approach: electrorotation. This technique allows assessment of the structure/function of the cell membrane. The results presented here demonstrate that RV shows a modest effect on the biological properties of the cell in terms of cytotoxicity and cell cycle alterations. On the contrary, a significant effect on the membrane structure/function was observed, consisting of an enhanced intramembrane ion transport. The implications and interpretation of these membrane alterations are discussed. Graphical Abstract: [Figure not available: see fulltext.] [ABSTRACT FROM AUTHOR]

The extraction of fluorescence intensity profiles of single cells from image data is a common challenge in cell biology. The manual segmentation of cells, the extraction of cell orientation and finally the extraction of intensity profiles are time-consuming tasks. This article proposes a routine for the segmentation of single rod-shaped cells (i.e. without neighboring cells in a distance of the cell length) from image data combined with an extraction of intensity dis-tributions along the longitudinal cell axis under the aggravated conditions of (i) a low spatial resolution and (ii) lacking information on the imaging system i.e. the point spread function and signal-to-noise ratio. The algorithm named cipsa transfers a new approach from particle streak velocimetry to cell classification interpreting the rod-shaped as streak-like structures. An au-tomatic reduction of systematic errors such as photobleaching and defocusing is included to guarantee robustness of the proposed approach under the described conditions and to the convenience of end-users unfamiliar with image processing. Performance of the algorithm has been tested on image sequences with high noise level produced by an overlay of different error sources. The developed algorithm provides a user-friendly, stand-alone procedure. [ABSTRACT FROM AUTHOR]

Contents Flow cytometry is a useful tool that provides an accurate, objective and rapid evaluation of semen quality. The use of this technique could significantly improve the quality of buffalo semen samples used in artificial insemination. This study was carried out to evaluate, by flow cytometry, frozen-thawed buffalo spermatozoa quality parameters such as sperm viability by SYBR-14/propidium iodide staining; mitochondrial function by JC-1 potentiometric probe; sperm chromatin stability (SCSA) by acridine orange; and acrosome reaction (AR) by FITC-PNA staining. Semen samples from five Italian Mediterranean buffalo bulls were used. Sperm viability was not different between bulls and ranged from 33.4% to 43.6%. A consistent rate (55.1 ± 10.8%) of sperm cells showed high mitochondrial membrane potential (Δψhigh), with no significant differences between subjects. Sperm chromatin structure assay differed significantly between the five buffalo bulls; moreover, data showed high stability within each buffalo. DNA fragmentation indexes (DFI), such as %-DFI, -DFI, SD-DFI, were 11.2 ± 8.6, 153.3 ± 24.6 and 81.6 ± 21.2, respectively. Regarding AR, the percentage of acrosome-reacted live (ARL) and acrosome-reacted dead (ARD) spermatozoa was 0.3 ± 0.2 and 15.3 ± 5.5, respectively. This functional parameter differed significantly between buffalo bulls and showed high stability. Following to Ca2+ ionophore A23187 for 3 h, AR significantly differed between subjects and was characterized by an increase in both ARL (10.8%) and ARD population (22.0%). This study indicates that flow cytometry could be a useful tool for a quick multiparametric evaluation of sperm quality in buffalo. In particular, SCSA and AR resulted in sperm functional parameters sensitive enough for the diagnosis of frozen-thawed semen fertilizing potential. [ABSTRACT FROM AUTHOR]

Screening and isolation of high expression mammalian cell lines for production of recombinant proteins for the clinic is a resource-intensive and time-consuming procedure due to the substantial variation in expression levels of recombinant protein expression amongst transfected cells. Several investigators have reported instability in expression titers early in cell line development and in cell banks. However, in most cases the exact molecular mechanisms of instability remain unknown. In this study we used a fluorescence-activated cell sorting (FACS) based mAb staining method to enable the detection and selective gating of cells with vastly different recombinant expression levels present in transfected pools. Expression diversity and changes within transfected populations were detected and isolated in real time during cell line development. Molecular genetic analysis on the isolated clones revealed an unsuspected rearrangement of the heavy chain in the non-expressing clones. Implications of the genetic rearrangements as well as the use of the FACS method as a tool to improve cell line development to detect expression heterogeneity in pools and to investigate root cause for the molecular genetics of expression instability will be discussed. [ABSTRACT FROM AUTHOR]

Abstract: Advanced glycation end products (AGEs) seem to be involved in aging as well as in the development of cardiovascular diseases. During aging, AGEs accumulate in extracellular matrix proteins like collagen and contribute to vessel stiffness. Whether non-invasive measurement of AGE accumulation in the skin may reflect vessel function and vessel protein modification is unknown. Herein we set out to analyze the AGE-modifications in the collagens extracted from residual bypass graft material, the skin autofluorescence reflecting the accumulation of AGEs in the body as well as the pulse wave velocity reflecting vessel stiffness. Collagen types I and III (pepsin digestible collagen fraction) were isolated from the veins of 52 patients by proteolysis. The residual collagen fraction was further extracted by collagenase digestion. Collagen was quantified by hydroxyproline assay and AGEs by the AGE intrinsic fluorescence. Skin autofluorescence was measured with an autofluorescence reader; pulse wave velocity with the VICORDER®. The collagen AGE autofluorescence in patient vein graft material increased with patient age. The pepsin digestible collagen fraction was significantly less modified in comparison to the collagenase digestible fraction. Decreasing amounts of extracted collagenase digestible collagen correspond with increasing AGE autofluorescence. Skin autofluorescence and vessel stiffness were significantly linked to the AGE autofluorescence of the collagenase digestible collagen fraction from graft material. In conclusion we have found that skin autofluorescence and pulse wave velocity as non-invasive parameters significantly correlate with the AGE contained in graft material and therefore are strong predictors of vessel AGE modifications in patients with coronary heart disease. Whether the analysis of the skin autofluorescence leads to an improvement of the risk stratification in patients suffering from cardiovascular disease has to be further tested. [Copyright &y& Elsevier]

High-throughput multiparallel activity profiling for oxygen consuming cell layers has been recently developed for extracellular flux analysis. This technology has great potential for determining the enzymatic activity of oxidoreductases (i.e., laccase) both in vivo and in vitro, which is usually measured using photometrical tests monitoring the colored oxidation products. Improvements in terms of sample throughput, comparability, and gain of information (i.e., stoichiometry, electron transfer rate) can be achieved by means of a multiwell plate-based fluorimetric oxygen sensor. In the present study, various laccases have been applied to develop protocols that allow the multiparallel measurement of O2-consumption by enzymatic reactions. The developed and validated method enables the comparative quantitation of laccase characteristics (i.e., profiles of activity at various pH values) and minimizes the time it usually takes to collect respiratory data of oxygen-consuming enzymes. Furthermore, the possibility to assess differences between single and multisubstrate kinetics of laccases has been demonstrated. [ABSTRACT FROM AUTHOR]

In this study we report for the first time a rapid, efficient and cost-effective method for the enumeration of lactic acid bacteria (LAB) in wine. Indeed, up to now, detection of LAB in wine, especially red wine, was not possible. Wines contain debris that cannot be separated from bacteria using flow cytometry (FCM). Furthermore, the dyes tested in previous reports did not allow an efficient staining of bacteria. Using FCM and a combination of BOX/PI dyes, we were able to count bacteria in wines. The study was performed in wine inoculated with Oenococcus oeni (10 CFU ml) stained with either FDA or BOX/PI and analyzed by FCM during the malolactic fermentation (MLF). The analysis show a strong correlation between the numbers of BOX/PI-stained cells determined by FCM and the cell numbers determined by plate counts (red wine: R ≥ 0.97, white wine R ≥ 0.965). On the other hand, we found that the enumeration of O. oeni labeled with FDA was only possible in white wine ( R ≥ 0.97). Viable yeast and LAB populations can be rapidly discriminated and quantified in simultaneous malolactic-alcoholic wine fermentations using BOX/PI and scatter parameters in a one single measurement. This rapid procedure is therefore a suitable method for monitoring O. oeni populations during winemaking, offers a detection limit of <10 CFU ml and can be considered a useful method for investigating the dynamics of microbial growth in wine and applied for microbiological quality control in wineries. [ABSTRACT FROM AUTHOR]

Markers of insulin resistance such as the adiponectin:leptin ratio (A:L) and the homeostasis model assessment ratio (HOMA-IR) are associated with obesity and hyperinsulinemia, both established risk factors for endometrial cancer, and may therefore be informative regarding endometrial cancer risk. This study investigated the association between endometrial cancer risk and markers of insulin resistance, namely adiponectin, leptin, the A:L ratio, insulin, fasting glucose, and the HOMA-IR. We analyzed data from 541 incident endometrial cancer cases and 961 frequency agematched controls in a population-based case-control study in Alberta, Canada from 2002 to 2006. Participants completed interview-administered questionnaires were assessed for anthropometric measures, and provided 8-h fasting blood samples either pre- or postoperatively. Blood was analyzed for concentrations of leptin, adiponectin, and insulin by immunoassay, and fasting plasma glucose levels were determined by fluorimetric quantitative determination. Compared with the lowest quartile, the highest quartile of insulin and HOMA-IR was associated with 64% (95% confidence intervals (CI): 1.12-2.40) and 72% (95% CI: 1.17-2.53) increased risks of endometrial cancer, respectively, and the highest quartile of adiponectin was associated with a 45% (95% CI: 0.37-0.80) decreased risk after multivariable adjustments. Null associations were observed between fasting glucose, leptin and A:L, and endometrial cancer risk. This population-based study provides evidence for a role of insulin resistance in endometrial cancer etiology and may provide one possible pathway whereby obesity increases the risk of this common cancer. Interventions aimed at decreasing both obesity and insulin resistance may decrease endometrial cancer risk. [ABSTRACT FROM AUTHOR]

Abstract: Current methods to measure physiological properties of cardiomyocytes and predict fatal arrhythmias that can cause sudden death, such as Torsade de Pointes, lack either the automation and throughput needed for early-stage drug discovery and/or have poor predictive value. To increase throughput and predictive power of in vitro assays, we developed kinetic imaging cytometry (KIC) for automated cell-by-cell analyses via intracellular fluorescence Ca2+ indicators. The KIC instrument simultaneously records and analyzes intracellular calcium concentration [Ca2+]i at 30-ms resolution from hundreds of individual cells/well of 96-well plates in seconds, providing kinetic details not previously possible with well averaging technologies such as plate readers. Analyses of human embryonic stem cell and induced pluripotent stem cell-derived cardiomyocytes revealed effects of known cardiotoxic and arrhythmogenic drugs on kinetic parameters of Ca2+ dynamics, suggesting that KIC will aid in the assessment of cardiotoxic risk and in the elucidation of pathogenic mechanisms of heart disease associated with drugs treatment and/or genetic background. [Copyright &y& Elsevier]

We compared the clinical value of minimal residual disease (MRD) monitoring by cytofluorimetric methods, Wilms tumor gene 1 (WT1) expression and the study of nucleophosmin gene (NPM) mutations in a series of 26 patients with NPM-mutated de novo acute myeloid leukemia (NPM-AML) who achieved complete hematological remission after conventional chemotherapy. The relapse risk was significantly lower only in patients achieving a NPM molecular complete response (NPM mol-CR) and confirmed NPM mol-CR (non-detectable NPM mutations in two consecutive marrow samples). The disease-free survival (DFS) of patients achieving a < 4-log or ≥ 4-log reduction in NPM value after induction therapy was 12.6 % and 50%, respectively, at 36 months ( p = 0.009). The attainment of a confirmed NPM-CR had a significant influence on overall survival (OS at 36 months was 64.3% and 11.9% in patients obtaining or not obtaining confirmed NPM-CR, respectively, p < 0.03). We confirm that NPM-molecular relapse (NPM-rel) is always followed by hematological relapse (H-rel), but longitudinal studies of NPM mutations may predict an impending H-rel earlier than flow cytometric- or WT1-based methods. [ABSTRACT FROM AUTHOR]

Abstract: Background: Abnormally high activity of protein kinase CK2 is linked to various diseases including cancer. Therefore, the inhibition of CK2 is a promising therapeutic strategy to fight this disease. Methods: We screened a library of synthetic molecules concerning their capacity to inhibit CK2. The activity of CK2 and their IC50 and Ki values were determined by a capillary electrophoresis assay. The effects of the inhibitor in a cell culture model were analyzed by cell counting, a viability assay, cytofluorimetry and Western blot. Results: The best CK2 inhibitor found in this screen was 6,7-dichloro-1,4-dihydro-8-hydroxy-4-[(4-methylphenylamino)methylen]dibenzo [b,d]furan-3(2H)-one, which we refer to as “TF”. TF showed tight binding to CK2 with low IC50 (29nM) and Ki (15nM) values. TF inhibited only seven out of 61 human kinases tested (>70% inhibition). Incubation of LNCaP cells with 50μM TF for 48h decreased the intracellular CK2 activity by 50%, confirming that the inhibitor is membrane permeable. The decrease in activity was correlated with a severe reduction in cell viability. The reduction in cell viability is at least partly due to the induction of apoptosis. General significance: In many cancers the protein kinase CK2 is significantly up-regulated and supports the neoplastic phenotype. New therapeutic strategies should be based on diverse reliable inhibitors to reverse the abnormal high levels to normal settings. [Copyright &y& Elsevier]

Abstract: We demonstrated an intriguing method to discriminate adenine by incident appearance of an intense new emission via exciplex formation in hybridization of target DNA with newly designed fluorescent 8-arylethynylated deoxyguanosine derivatives. We described the synthesis of such highly electron donating fluorescent guanosine derivatives and their incorporation into DNA oligomers which may be used for the structural study and the fluorometric analysis of nucleic acids. [Copyright &y& Elsevier]

The TRPV4 Ca2+-permeable channel is sensitive to mechanical stimuli. In the current study we have employed immunocytochemical staining in kidney slices and functional assessments (Ca2+ imaging) in isolated, split-opened, tubule segments to define TRPV4 sites of expression and flow-dependent function in the collecting duct system. Staining patterns revealed strong expression of TRPV4 along the entire collecting duct system with highest levels at the apical (luminal)/subapical region of the principal cells (PCs), the dominant cell type, with more diffuse staining in intercalated cells (ICs). Using fluorescence Ca2+ imaging and the selective TRPV4 agonist, GSK1016790A, we demonstrated functional TRPV4 channels in PCs and ICs of split-opened cortical collecting ducts and connecting tubules. The agonist was ineffective in inducing a rise in [Ca2+]i in the absence of extracellular Ca2+ or in tubules from TRPV4-deficient animals. Most importantly, a 10-fold elevation in luminal (apical) fluid flow induced a rapid and sustained influx of Ca2+ that was abolished by the TRPV channel inhibitor, ruthenium red, or in tubules isolated from TRPV4 deficient animals. We concluded that TRPV4 is highly expressed along the entire collecting duct system where it appears to function as a sensor/transducer of flow-induce mechanical stresses. [ABSTRACT FROM AUTHOR]