Biofilms are mono- or multispecies microbial communities enclosed in an extracellular matrix (EPS). They have high potential for dissemination and are difficult to remove. In addition, biofilms ...formed by multidrug-resistant strains (MDRs) are even more aggravated if we consider antimicrobial resistance (AMR) as an important public health issue. Quorum sensing (QS) and horizontal gene transfer (HGT) are mechanisms that significantly contribute to the recalcitrance (resistance and tolerance) of biofilms, making them more robust and resistant to conventional sanitation methods. These mechanisms coordinate different strategies involved in AMR, such as activation of a quiescent state of the cells, moderate increase in the expression of the efflux pump, decrease in the membrane potential, antimicrobial inactivation, and modification of the antimicrobial target and the architecture of the EPS matrix itself. There are few studies investigating the impact of the use of inhibitors on the mechanisms of recalcitrance and its impact on the microbiome. Therefore, more studies to elucidate the effect and applications of these methods in the food production chain and the possible combination with antimicrobials to establish new strategies to control MDR biofilms are needed.
Trypan blue exclusion assay by flow cytometry Avelar-Freitas, B A; Almeida, V G; Pinto, M C X ...
Brazilian journal of medical and biological research,
04/2014, Letnik:
47, Številka:
4
Journal Article
Recenzirano
Odprti dostop
Dye exclusion tests are used to determine the number of live and dead cells. These assays are based on the principle that intact plasma membranes in live cells exclude specific dyes, whereas dead ...cells do not. Although widely used, the trypan blue (TB) exclusion assay has limitations. The dye can be incorporated by live cells after a short exposure time, and personal reliability, related to the expertise of the analyst, can affect the results. We propose an alternative assay for evaluating cell viability that combines the TB exclusion test and the high sensitivity of the flow cytometry technique. Previous studies have demonstrated the ability of TB to emit fluorescence when complexed with proteins. According to our results, TB/bovine serum albumin and TB/cytoplasmic protein complexes emit fluorescence at 660 nm, which is detectable by flow cytometry using a 650-nm low-pass band filter. TB at 0.002% (w/v) was defined as the optimum concentration for distinguishing unstained living cells from fluorescent dead cells, and fluorescence emission was stable for 30 min after cell treatment. Although previous studies have shown that TB promotes green fluorescence quenching, TB at 0.002% did not interfere with green fluorescence in human live T-cells stained with anti-CD3/fluorescein isothiocyanate (FITC) monoclonal antibody. We observed a high correlation between the percentage of propidium iodide+CD3/FITC+ and TB+CD3/FITC+ cells, as well as similar double-stained cell profiles in flow cytometry dot-plot graphs. Taken together, the results indicate that a TB exclusion assay by flow cytometry can be employed as an alternative tool for quick and reliable cell viability analysis.
It is well known in the literature that vacuum fluctuations can induce a random motion of particles which is sometimes called quantum Brownian motion or quantum stochastic motion. In this paper, we ...consider Lorentz Invariance Violation (LIV) in an acoustic spatially flat Friedman–Robertson–Walker (FRW) geometry. In particular, we are looking for the LIV effects in the stochastic motion of scalar and massive test particles. This motion is induced by a massless quantized scalar field on this geometry, which in turn is derived from an Abelian Higgs model with LIV. Deviations in the velocity dispersion of the particles proportional to the LIV parameter are found.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
•52 volatile compounds and 177 PC was tentatively identified in PCF and CSF.•Chlorogenic, p-coumaric, ferulic and caffeic acids were the most abundant compounds.•Phytol, linalool and α-terpineol were ...identified by GC-MS.•Pineapple crown flour exhibited the highest antioxidant capacity in bound extract.•CSF and PCF presented great bioactive potential and discrete antimicrobial activity.
Unconventional parts of vegetables represent a rich source of health-promoting phytochemicals. The phenolic profile of cabbage-stalk flour (CSF), pineapple-crown flour (PCF), and their essential oils were characterized via UPLC-ESI-QTOF-MSE and GC-FID/MS. Antimicrobial activity was tested against five strains, and antioxidant activities were determined in free and bound extracts. Globally, 177 phenolics were tentatively identified in PCF (major p-coumaric acid, ferulic acid, and 4-hydroxybenzaldehyde) and 56 in CSF (major chlorogenicacid, quercetin 3-O-glucuronide, and p-coumaric acid). PCF exhibited a distinguished profile (lignans, stilbenes) and antioxidant capacity, especially in bound extracts (1.3 g GAE.100 g−1; 0.6 g catechin eq.100 g−1; DPPH: 244.7; ABTS: 467.8; FRAP: 762.6 µg TE.g−1, ORAC: 40.9 mg TE.g−1). The main classes of volatile compounds were fatty acids, their esters, and terpenes in CSF (30) and PCF (41). A comprehensive metabolomic approach revealed CSF and PCF as a promising source of PC, showing great antioxidant and discrete antimicrobial activities.
Major abdominal oncology surgery is associated with substantial postoperative loss of functional capacity, and exercise may be an effective intervention to improve outcomes. The aim of this study was ...to assess efficacy, feasibility and safety of a supervised postoperative exercise programme.
We performed a single-blind, parallel-arm, randomized trial in patients who underwent major abdominal oncology surgery in a tertiary university hospital. Patients were randomized to an early mobilization postoperative programme based on supervised aerobic exercise, resistance and flexibility training or to standard rehabilitation care. The primary outcome was inability to walk without human assistance at postoperative day 5 or hospital discharge.
A total of 108 patients were enrolled, 54 into the early mobilization programme group and 54 into the standard rehabilitation care group. The incidence of the primary outcome was nine (16.7%) and 21 (38.9%), respectively (P=0.01), with an absolute risk reduction of 22.2% 95% confidence interval (CI) 5.9–38.6 and a number needed to treat of 5 (95% CI 3–17). All patients in the intervention group were able to follow at least partially the exercise programme, although the performance among them was rather heterogeneous. There were no differences between groups regarding clinical outcomes or complications related to the exercises.
An early postoperative mobilization programme based on supervised exercises seems to be safe and feasible and improves functional capacity in patients undergoing major elective abdominal oncology surgery. However, its impact on clinical outcomes is still unclear.
NCT01693172.
Deciphering Tacrolimus‐Induced Toxicity in Pancreatic β Cells Triñanes, J.; Rodriguez‐Rodriguez, A. E.; Brito‐Casillas, Y. ...
American journal of transplantation,
November 2017, 2017-Nov, 2017-11-00, 20171101, Letnik:
17, Številka:
11
Journal Article
Recenzirano
Odprti dostop
β Cell transcription factors such as forkhead box protein O1 (FoxO1), v‐maf musculoaponeurotic fibrosarcoma oncogene homolog A (MafA), pancreatic and duodenal homeobox 1, and neuronal differentiation ...1, are dysfunctional in type 2 diabetes mellitus (T2DM). Posttransplant diabetes mellitus resembles T2DM and reflects interaction between pretransplant insulin resistance and immunosuppressants, mainly calcineurin inhibitors (CNIs). We evaluated the effect of tacrolimus (TAC), cyclosporine A (CsA), and metabolic stressors (glucose plus palmitate) on insulinoma β cells in vitro and in pancreata of obese and lean Zucker rats. Cells were cultured for 5 days with 100 μM palmitate and 22 mM glucose; CsA (250 ng/mL) or TAC (15 ng/mL) were added in the last 48 h. Glucose plus palmitate increased nuclear FoxO1 and decreased nuclear MafA. TAC in addition to glucose plus palmitate magnified these changes in nuclear factors, whereas CsA did not. In addition to glucose plus palmitate, both drugs reduced insulin content, and TAC also affected insulin secretion. TAC withdrawal or conversion to CsA restored these changes. Similar results were observed in pancreata of obese animals on CNIs. TAC and CsA, in addition to glucose plus palmitate, induced comparable inhibition of calcineurin and nuclear factor of activated T cells (NFAT); therefore, TAC potentiates glucolipotoxicity in β cells, possibly by sharing common pathways of β cell dysfunction. TAC‐induced β cell dysfunction is potentially reversible. Inhibition of the calcineurin–NFAT pathway may contribute to the diabetogenic effect of CNIs but does not explain the stronger effect of TAC compared with CsA.
Tacrolimus, but not cyclosporine, potentiates glucolipotoxicity in β cells by sharing common pathways of β cell dysfunction, while the inhibition of the calcineurin–NFAT pathway does not explain the greater diabetogenic effect of tacrolimus compared to cyclosporine.
1 Institute for Cancer Research, Section of Haemato-Oncology, London
2 Department of Medical and Molecular Genetics, Division of Genetics and Molecular Medicine, Kings College London, School of ...Medicine
3 Leukaemia Research Fund UK Myeloma Forum Cytogenetics Group, Wessex Regional Genetics Laboratory, Salisbury
4 Northern Institute for Cancer Research, Newcastle University, UK
Correspondence: Gareth Morgan, Brookes Lawley Building, Institute of Cancer Research, 15 Cotswold Road, Sutton, Surrey, SM2 5NG, UK., E-mail: Gareth.morgan{at}icr.ac.uk
Background: The recurrent immunoglobulin translocation, t(4;14)(p16;q32) occurs in 15% of multiple myeloma patients and is associated with poor prognosis, through an unknown mechanism. The t(4;14) up-regulates fibroblast growth factor receptor 3 ( FGFR3 ) and multiple myeloma SET domain ( MMSET ) genes. The involvement of MMSET in the pathogenesis of t(4;14) multiple myeloma and the mechanism or genes deregulated by MMSET upregulation are still unclear.
Design and Methods: The expression of MMSET was analyzed using a novel antibody. The involvement of MMSET in t(4;14) myelomagenesis was assessed by small interfering RNA mediated knockdown combined with several biological assays. In addition, the differential gene expression of MMSET-induced knockdown was analyzed with expression microarrays. MMSET gene targets in primary patient material was analyzed by expression microarrays.
Results: We found that MMSET isoforms are expressed in multiple myeloma cell lines, being exclusively up-regulated in t(4;14)-positive cells. Suppression of MMSET expression affected cell proliferation by both decreasing cell viability and cell cycle progression of cells with the t(4;14) translocation. These findings were associated with reduced expression of genes involved in the regulation of cell cycle progression (e.g. CCND2 , CCNG1 , BRCA1 , AURKA and CHEK1 ), apoptosis ( CASP1 , CASP4 and FOXO3A ) and cell adhesion ( ADAM9 and DSG2 ). Furthermore, we identified genes involved in the latter processes that were differentially expressed in t(4;14) multiple myeloma patient samples.
Conclusions: In conclusion, dysregulation of MMSET affects the expression of several genes involved in the regulation of cell cycle progression, cell adhesion and survival.
Key words: MMSET , WHSC1 , myeloma.
Gastric cancer (GC) is a highly heterogeneous, complex disease and the fifth most common cancer worldwide (about 1 million cases and 784,000 deaths worldwide in 2018). GC has a poor prognosis (the ...5-year survival rate is less than 20%), but there is an effort to find genes highly expressed during tumor establishment and use the related proteins as targets to find new anticancer molecules. Data were collected from the Gene Expression Omnibus (GEO) bank to obtain three dataset matrices analyzing gastric tumor tissue versus normal gastric tissue and involving microarray analysis performed using the GPL570 platform and different sources. The data were analyzed using the GEPIA tool for differential expression and KMPlot for survival analysis. For more robustness, GC data from the TCGA database were used to corroborate the analysis of data from GEO. The genes found in in silico analysis in both GEO and TCGA were confirmed in several lines of GC cells by RT-qPCR. The AlphaFold Protein Structure Database was used to find the corresponding proteins. Then, a structure-based virtual screening was performed to find molecules, and docking analysis was performed using the DockThor server. Our in silico and RT-qPCR analysis results confirmed the high expression of the AJUBA, CD80 and NOLC1 genes in GC lines. Thus, the corresponding proteins were used in SBVS analysis. There were three molecules, one molecule for each target, MCULE-2386589557-0-6, MCULE-9178344200-0-1 and MCULE-5881513100-0-29. All molecules had favorable pharmacokinetic, pharmacodynamic and toxicological properties. Molecular docking analysis revealed that the molecules interact with proteins in critical sites for their activity. Using a virtual screening approach, a molecular docking study was performed for proteins encoded by genes that play important roles in cellular functions for carcinogenesis. Combining a systematic collection of public microarray data with a comparative meta-profiling, RT-qPCR, SBVS and molecular docking analysis provided a suitable approach for finding genes involved in GC and working with the corresponding proteins to search for new molecules with anticancer properties.
Ongoing pollution of the marine environment requires quick and reasonably priced analytical techniques that allow routine checking of the concentrations of persistent organic pollutants (POPs) found ...in different samples and, in particular, coastal samples. Miniaturized extraction techniques enable us to analyze samples more quickly and cheaply. These methodologies are highly effective for analysis of liquid samples, but not for solid samples, and even less so for microplastic pollutant analyses. A miniaturized solid-liquid-liquid extraction technique (μSLLE) has been developed to extract, pre-concentrate, and analyze up to 27 POPs from marine microplastics, using a quick methodology which does not require drying of the extract for its preconcentration. Target pollutants were analyzed using single quadrupole gas chromatography with mass spectrometry (GC-MS). The method has been optimized and validated, and was applied to different marine microplastic samples collected on the islands of Gran Canaria and Fuerteventura (Canary Islands, Spain).
Graphical abstract
mRNA vaccines are rapidly emerging as a powerful platform for infectious diseases because they are well tolerated, immunogenic, and scalable and are built on precise but adaptable antigen design. We ...show that two immunizations of modified non-replicating mRNA encoding influenza H10 hemagglutinin (HA) and encapsulated in lipid nanoparticles (LNP) induce protective HA inhibition titers and H10-specific CD4+ T cell responses after intramuscular or intradermal delivery in rhesus macaques. Administration of LNP/mRNA induced rapid and local infiltration of neutrophils, monocytes, and dendritic cells (DCs) to the site of administration and the draining lymph nodes (LNs). While these cells efficiently internalized LNP, mainly monocytes and DCs translated the mRNA and upregulated key co-stimulatory receptors (CD80 and CD86). This coincided with upregulation of type I IFN-inducible genes, including MX1 and CXCL10. The innate immune activation was transient and resulted in priming of H10-specific CD4+ T cells exclusively in the vaccine-draining LNs. Collectively, this demonstrates that mRNA-based vaccines induce type-I IFN-polarized innate immunity and, when combined with antigen production by antigen-presenting cells, lead to generation of potent vaccine-specific responses.
mRNA vaccines have been proven to be suitable and efficient against pandemic pathogens. However, the immune processes after mRNA vaccination that lead to robust responses remain elusive. Now in Molecular Therapy, Liang et al. (2017) define the target cells and immune responses at the vaccination sites and in the lymph nodes that result in vaccine immunity.
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