BACKGROUND: Recently, a plethora of novel systemic agents have been incorporated into the therapeutic armamentarium of advanced urothelial carcinoma (aUC). The antibody-drug conjugate (ADC), enfortumab vedotin (EV), has demonstrated relevant clinical benefit in patients with aUC refractory to platinum and immune-checkpoint inhibitor (ICI) therapy. Our study provides a retrospective, international, real-world analysis comparing the effectiveness of EV to chemotherapy in this setting. METHODS: The data were extracted from the medical records of patients treated with EV or chemotherapy following pembrolizumab for recurrent or progressive aUC after platinum-based chemotherapy. Patients were assessed for overall survival (OS), progression-free survival (PFS), overall response rate (ORR) and duration of response (DoR). RESULTS: Our analysis included 247 patients treated with EV (88, 36%) or chemotherapy (159, 64%). Median OS was 9.1 months (95%CI 7.2-10.7) in the overall study population, 13.6 months (95%CI 10.0-31.0) in patients receiving EV and 6.8 months (95%CI 6.0-8.9) in patients receiving chemotherapy (p < 0.001). The OS benefit of EV was not affected by primary tumour site and histology, metastatic sites, type of first platinum-based chemotherapy or response to pembrolizumab. In the EV cohort, the median PFS was significantly longer (8.8 months [95%CI 6.5-17.0] vs. 3.0 months [95%CI 2.6-3.7]) and the ORR was significantly higher (56% vs. 23%) than in the chemotherapy cohort. CONCLUSIONS: The results of our international analysis of real-world data confirm the effectiveness of EV in the sequential strategy of aUC patients who have received prior platinum-based chemotherapy and anti-PD-1 pembrolizumab, regardless of commonly considered prognostic factors. TRIAL REGISTRATION: ClinicalTrials.gov identifier: NCT05290038.
- MeSH
- Progression-Free Survival MeSH
- Adult MeSH
- Antibodies, Monoclonal, Humanized * therapeutic use administration & dosage MeSH
- Immune Checkpoint Inhibitors therapeutic use MeSH
- Carcinoma, Transitional Cell drug therapy mortality pathology MeSH
- Middle Aged MeSH
- Humans MeSH
- Antibodies, Monoclonal therapeutic use administration & dosage MeSH
- Antineoplastic Combined Chemotherapy Protocols * therapeutic use MeSH
- Retrospective Studies MeSH
- Aged, 80 and over MeSH
- Aged MeSH
- Urologic Neoplasms drug therapy pathology mortality MeSH
- Treatment Outcome MeSH
- Check Tag
- Adult MeSH
- Middle Aged MeSH
- Humans MeSH
- Male MeSH
- Aged, 80 and over MeSH
- Aged MeSH
- Female MeSH
- Publication type
- Journal Article MeSH
- Multicenter Study MeSH
OBJECTIVES: Small extracellular vesicles (EVs) contain various signaling molecules, thus playing a crucial role in cell-to-cell communication and emerging as a promising source of biomarkers. However, the lack of standardized procedures impedes their translation to clinical practice. Thus, we compared different approaches for high-throughput analysis of small EVs transcriptome. METHODS: Small EVs were isolated from 150 μL of serum. Quality and quantity were assessed by dynamic light scattering, transmission electron microscopy, and Western blot. Comparison of RNA extraction efficiency was performed, and expression of selected genes was analyzed by RT-qPCR. Whole transcriptome analysis was done using microarrays. RESULTS: Obtained data confirmed the suitability of size exclusion chromatography for isolation of small EVs. Analyses of gene expression showed the best results in case of samples isolated by Monarch Total RNA Miniprep Kit. Totally, 7,182 transcripts were identified to be deregulated between colorectal cancer patients and healthy controls. The majority of them were non-coding RNAs with more than 70 % being lncRNAs, while protein-coding genes represented the second most common gene biotype. CONCLUSIONS: We have optimized the protocol for isolation of small EVs and their RNA from low volume of sera and confirmed the suitability of Clariom D Pico Assays for transcriptome profiling.
- MeSH
- Extracellular Vesicles * genetics metabolism MeSH
- Chromatography, Gel MeSH
- Humans MeSH
- RNA MeSH
- Gene Expression Profiling * methods MeSH
- Check Tag
- Humans MeSH
- Publication type
- Journal Article MeSH
Forty different sample preparation methods were tested to obtain the most informative MALDI-TOF MS protein profiles of pork meat. Extraction by 25% formic acid with the assistance of zirconia-silica beads followed by defatting by methanol:chloroform mixture (1:1, v/v) and deposition by using the layer-by-layer method was determined as the optimum sample preparation protocol. The discriminatory power of the method was then examined on samples of pork meat and meat products. The method was able to discriminate between selected salami based on the production method and brand and was able to monitor the ripening process in salami. However, it was not able to differentiate between different brands of pork ham or closely located parts of pork meat. In the latter case, a more comprehensive analysis using LC-MS/MS was used to assess the differences in protein abundance and their relation to the outputs of MALDI - TOF MS profiling.
UNLABELLED: Several mutations in this gene for the α subunit of the cardiac sodium channel have been identified in a heterogeneous subset of cardiac rhythm syndromes, including Brugada syndrome, progressive cardiac conduction defect, sick sinus node syndrome, atrial fibrillation and dilated cardiomyopathy. The aim of our study was to associate some SCN5A polymorphic variants directly with confirmed coronary stenoses in patients with non-LQTS ventricular fibrillation/flutter treated by an implantable cardioverter defibrillator. MATERIALS AND METHODS: A group of 32 unrelated individuals, aged 63 ± 12 years, was included in the study. All the patients were examined, diagnosed and treated with an implantable cardioverter defibrillator at the Department of Internal Cardiology Medicine, Faculty Hospital Brno. The control group included 87 persons of similar age without afflicted coronary circulation, which was confirmed coronagraphically. Genomic DNA was extracted from samples of peripheral blood according to the standard protocol. Two SCN5A polymorphisms-IVS9-3C/A (rs41312433) and A1673G (rs1805124, H558R)-were examined in association with coronary artery stenosis in the patients. RESULTS: In the case-control study, no significant differences in genotype distribution/allelic frequencies were observed for IVS9-3c>a and A1673G gene polymorphisms between patients with severe arrhythmias and healthy persons. The distribution of SCN5A double genotypes was not significantly different among different types of arrhythmias according to their ejection fraction in arrhythmic patients (p = 0.396). The ventricular arrhythmias with an ejection fraction below 40% were found to be 10.67 times more frequent in patients with multiple coronary stenosis with clinically valid sensitivity, specificity and power tests. In the genotype-phenotype study, we observed a significant association of both SCN5A polymorphisms with the stenosis of coronary vessels in the patients with severe arrhythmia. The double genotype of polymorphisms IVS9-3C/A together with A1673G (CCAA) as well as their simple genotypes were associated with significant multiple stenosis of coronary arteries (MVS) with high sensitivity and specificity (p = 0.05; OR = 5 (95% CI 0.99-23.34); sensitivity 0.70; specificity 0.682; power test 0.359) Moreover, when a concrete stenotic coronary artery was associated with SCN5A genotypes, the CCAA double genotype was observed to be five times more frequent in patients with significant stenosis in the right coronary artery (RCA) compared to those without affliction of this coronary artery (p = 0.05; OR = 5 (95% CI 0.99-23.34); sensitivity 0.682; specificity 0.700; power test 0.359). The CCAA genotype was also more frequent in patients without RCA affliction with MVS (p = 0.008); in patients with ACD affliction but without MVS (p = 0.008); and in patients with both ACD affliction and MVS compared to those without ACD affliction and MVS (p = 0.005). CONCLUSIONS: Our study presents a highly sensitive and specific association of two polymorphisms in SCN5A with significant coronary artery stenoses in patients with potentially fatal ventricular arrhythmias. At the same time, these polymorphisms were not associated with arrhythmias themselves. Thus, SCN5A gene polymorphic variants may form a part of germ cell gene predisposition to ischemia.
Uric acid and its oxidation product allantoin are excellent biomarkers of oxidative stress in humans. Currently, there are high requirements not only for tests monitoring oxidative stress but also for screening laboratory tests in general. The highest demand is imposed on the simplest sampling, easy transport of the sample, and the shortest possible analysis time. The possible solution how to fulfil the requirements is sampling by dried blood spot technique with subsequent HPLC-MS/MS analysis. A fast, sensitive, and reliable HPLC-MS/MS method for the simultaneous determination of uric acid and allantoin from dried blood spots using stable isotopically labelled analogs as internal standards was developed. The separation took place in the reversed phase within 3 min, with protein precipitation and extraction in a one-step procedure. The analytical parameters of the method were satisfactory with an excellent linear range. The presented method was used to determine allantoin and uric acid levels in dried blood spot samples from 100 healthy volunteer donors. The median uric acid concentration in the cohort was 239.3 μmol/L and the median allantoin concentration was 5.6 μmol/L. The presented analytical protocol and method are suitable for screening and monitoring allantoin and uric acid levels as biomarkers of oxidative stress in clinical practice.
The success of bottom-up proteomic analysis frequently depends on the efficient removal of contaminants from protein or peptide samples before LC-MS/MS. For a peptide clean-up workflow, single-pot solid-phase-enhanced peptide sample preparation on carboxylate-modified paramagnetic beads (termed SP2) was evaluated for sodium dodecyl sulfate or polyethylene glycol removal from Arabidopsis thaliana tryptic peptides. The robust and efficient 40-min SP2 protocol, tested for 10-ng, 250-ng, and 10-μg peptide samples, was proposed and benchmarked thoroughly against the ethyl acetate extraction protocol. The SP2 protocol on carboxylated magnetic beads proved to be the most robust approach, even for the simultaneous removal of massive sodium dodecyl sulfate (SDS) and polyethylene glycol (PEG) contaminations from AT peptide samples in respect of the LC-MS/MS data outperforming ethyl acetate extraction.
Healing of dense regular connective tissue, due to a high fiber-to-cell ratio and low metabolic activity and regeneration potential, frequently requires surgical implantation or reconstruction with high risk of reinjury. An alternative to synthetic implants is using bioscaffolds obtained through decellularization, a process where the aim is to extract cells from the tissue while preserving the tissue-specific native molecular structure of the ECM. Proteins, lipids, nucleic acids and other various extracellular molecules are largely involved in differentiation, proliferation, vascularization and collagen fibers deposit, making them the crucial processes in tissue regeneration. Because of the multiple possible forms of cell extraction, there is no standardized protocol in dense regular connective tissue (DRCT). Many modifications of the structure, shape and composition of the bioscaffold have also been described to improve the therapeutic result following the implantation of decellularized connective tissue. The available data provide a valuable source of crucial information. However, the wide spectrum of decellularization makes it important to understand the key aspects of bioscaffolds relative to their potential use in tissue regeneration.
INTRODUCTION: Surgical site infection (SSI) is a potential complication of surgical procedure. SSI after implant surgery is a disaster both for patients and surgeons. Although predictive tools for SSI are available, none of them estimate early infection based on inflammatory blood parameters. The inflammatory process can be measured using several parameters including interleukin-6, C reactive protein, neutrophil to lymphocyte ratio, white cell count, erythrocyte sedimentation rate or procalcitonin. This systematic review aims to determine whether inflammatory blood parameters could be used as significant predictive factors for SSI after primary hip or knee arthroplasty. METHODS AND ANALYSIS: A systematic review of randomised controlled trials, cross-sectional studies, case-control studies and cohort studies, published in English, will be searched in the following electronic bibliographic databases: MEDLINE, Embase, PubMed, Cochrane Central Register of Controlled Trials and Web of Science. Studies performed in adult patients of all ages who underwent knee or hip arthroplasty, studies containing data on the risk/prognostic factors for preknee or postknee or hip arthroplasty SSI and studies with a minimum follow-up of 30 days after surgery will be included. A standardised form will be used to extract data from the included studies comprising study characteristics, participant characteristics, details of the intervention, study methodology and outcomes. Quality Assessment of Diagnostic Accuracy tool, second version, and Standards for Reporting of Diagnostic Accuracy Studies checklist will be used to assess risk of bias. Heterogeneity will be assessed using Cochran χ² statistic and I2 statistics where applicable. Grading of Recommendations Assessment, Development and Evaluation and Preferred Reporting Items for Systematic Reviews and Meta-Analyses guidance will be used to report findings. ETHICS AND DISSEMINATION: No ethics approval is required. The findings will be disseminated at national and international scientific sessions, also to be published in a peer-reviewed journal. PROSPERO REGISTRATION NUMBER: CRD42020147925.
- MeSH
- Surgical Wound Infection diagnosis epidemiology etiology MeSH
- Humans MeSH
- Arthroplasty, Replacement, Hip * adverse effects MeSH
- Prognosis MeSH
- Cross-Sectional Studies MeSH
- Systematic Reviews as Topic MeSH
- Arthroplasty, Replacement, Knee * adverse effects MeSH
- Check Tag
- Humans MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
An aberrant immune response developed early in life may trigger inflammatory bowel disease (IBD) and food allergies (e.g., celiac disease). Fecal levels of immune markers categorize an inflammatory response (e.g., food allergy, autoimmune) paralleled with the initial microbial colonization. The immunoaffinity assays are routinely applied to quantify circulating immune protein markers in blood/serum. However, a reliable, multiplex assay to quantify fecal levels of immune proteins is unavailable. We developed mass spectrometry assays to simultaneously quantify fecal calprotectin, myeloperoxidase, eosinophil-derived neurotoxin, eosinophil cationic protein, alpha-1-antitrypsin 1, and adaptive immunity effectors in 134 neonatal stool swabs. We optimized extraction and proteolytic protocol and validated the multiplex assay in terms of linearity of response (> 100; typically 0.04 to 14.77 µg/mg of total protein), coefficient of determination (R2; > 0.99), the limit of detection (LOD; 0.003 to 0.04 µg/mg of total protein), the limit of quantification (LOQ; 0.009 to 0.122 µg/mg of total protein) and robustness. The median CV of intra- and interday precision was 9.8% and 14.1%, respectively. We quantified breast milk-derived IGHA2 to differentiate meconium from feces samples and to detect the first food intake. An early life profiling of immune markers reflects disrupted intestinal homeostasis, and it is perhaps suitable for pre-symptomatic interception of IBD and food allergies.
- MeSH
- Biomarkers metabolism MeSH
- Chemistry Techniques, Analytical methods MeSH
- Feces chemistry MeSH
- Mass Spectrometry methods MeSH
- Inflammatory Bowel Diseases etiology metabolism MeSH
- Humans MeSH
- Infant, Newborn MeSH
- Specimen Handling methods MeSH
- Food Hypersensitivity etiology metabolism MeSH
- Inflammation diagnosis microbiology MeSH
- Check Tag
- Humans MeSH
- Male MeSH
- Infant, Newborn MeSH
- Female MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
Brown adipose tissue (BAT) is responsible for non-shivering thermogenesis in mammals, and brown adipocytes (BAs) are the functional units of BAT. BAs contain both multilocular lipid droplets and abundant mitochondria, and they express uncoupling protein 1 (UCP1). BAs are categorized into two sub-types based on their origin: embryo derived classical BAs (cBAs) and white adipocytes derived BAs. Due to their relatively low density, BAs cannot be isolated from BAT with traditional centrifugation method. In this study, a new method was developed to isolate BAs from mice for gene and protein expression analysis. In this protocol, interscapular BAT from adult mice was digested with Collagenase and Dispase solution, and the dissociated BAs were enriched with 6% iodixanol solution. Isolated BAs were then lysed with Trizol reagent for simultaneous isolation of RNA, DNA, and protein. After RNA isolation, the organic phase of the lysate was used for protein extraction. Our data showed that 6% iodixanol solution efficiently enriched BAs without interfering with follow-up gene and protein expression studies. Platelet-derived growth factor (PDGF) is a growth factor that regulates the growth and proliferation of mesenchymal cells. Compared to the brown adipose tissue, isolated BAs had significantly higher expression of Pdgfa. In summary, this new method provides a platform for studying the biology of brown adipocytes at a single cell-type level.
- MeSH
- Adipose Tissue, Brown cytology metabolism MeSH
- Adipocytes, Brown cytology metabolism MeSH
- Scapula cytology metabolism MeSH
- Mice MeSH
- Proteins genetics metabolism MeSH
- Gene Expression Regulation * MeSH
- Cell Separation methods MeSH
- Animals MeSH
- Check Tag
- Mice MeSH
- Animals MeSH
- Publication type
- Video-Audio Media MeSH
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
- Research Support, N.I.H., Extramural MeSH
