Supernatant Samples Research Articles

Bioactive Effect of Plasma-Rich in Growth Factors (PRGFs) on Cell-Based In Vitro Models of Skin Inflammation in Relation to Inflammatory Skin Disorders.

Plasma rich in growth factors (PRGFs) has proven potentially beneficial as a bioregenerator in patients with chronic skin disorders due to its anti-inflammatory effect. However, its therapeutic potential may be limited by soluble autoimmune components associated with inflammatory dermatoses in blood plasma. To evaluate the impact of skin health status on cell bioactivity, PRGF was prepared from healthy (H) donors as well as from individuals with atopic dermatitis (AD), psoriasis (PS), or lichen sclerosus (LS). Leukocyte exclusion and heat inactivation (Immunosafe treatment) were evaluated as potential methods to reduce the inflammatory components of the samples under study. The biological effect of PRGF-derived formulations was investigated using cell-based in vitro skin inflammation models, including human dermal fibroblasts (HDFs) and human epidermal keratinocytes (HEKs) exposed to a pro-inflammatory environment. The data confirmed that viability, proliferation, and migration rates were enhanced in inflamed cell cultures supplemented with PRGF formulations compared to those maintained in standard culture media. Nevertheless, significant differences have been identified. About the healthy control, inflamed epidermal keratinocytes supplemented with most PRGF-based formulations obtained from pathological donors (PS/LS) showed lower viability. Heat inactivation significantly promoted cell proliferation in epidermal keratinocytes supplemented with SP (PS/LS) and L-PRP supernatant (LSP) samples (AD), and also cell migration in inflamed HDF (AD/H/LS) and HEK (AD/LS) models supplemented with LSP. Leukocyte exclusion improved cell behavior in terms of migration with the only exception of LSP from individuals with AD added to inflamed HEK cultures. In conclusion, PRGF derived from pathological patients contains autoimmune components that could compromise its effectiveness as a therapy for treating individuals with chronic inflammatory disorders. However, heat inactivation (Immunosafe treatment) or leukocyte exclusion could minimize local adverse effects.

Non-targeted Metabolomics-based Exploration of Radiation-induced Metabolic Alterations in Mouse Lung Epithelial Cells

Metabolic change is one of the important characteristics of radiation pneumonitis. Radiotherapy, as a conventional method for the treatment of thoracic tumors, can not only effectively kill tumor cells, but also cause adverse reactions such as local inflammation and fibrosis, which leads to limited therapeutic effect and profound impact on the quality of life of patients. Therefore, it is of great significance to explore the metabolic changes caused by radiotherapy. The aim of this study was to investigate the effects of X-ray irradiation on the metabolism of a mouse lung epithelial cell line (murine lung epithelial-12, MLE12). MLE12 cells were' cultured in vitro and randomly divided into radiation group (IR) and control group (NC). Cells in the IR group were irradiated at a dose of 10 Gy using a Hitachi X-ray irradiator. Cell supernatant samples were collected at 48 h after irradiation. Metabolomic analysis of the samples was performed by liquid chromatograph mass spectrometer (LC/MS). LC/MS metabolomics analysis revealed the metabolic changes of MLE12 cells at 48 h after irradiation. A total of 38 secretory metabolites were altered in the IR group compared with the NC group. According to the annotation of Kyoto Encyclopedia of Genes and Genomes (KEGG) database, the differential metabolites are mainly involved in nucleotide metabolism, amino acid metabolism and lipid metabolism, among which the difference in nucleotide metabolism is the most significant. The metabolism of MLE12 cells was significantly affected by X-ray irradiation, mainly affecting the nucleotide metabolic pathways, including purine and pyrimidine metabolites and related metabolic pathways.

Assessment of amino acids and metabolites in the supernatant of stored concentrates blood from sickle cell trait (SCT) and reference (non-SCT) donors.

Sickle cell trait (SCT) persons are significant donors, and discarding these blood units reduces their supplies, mainly in the third-world countries. This work focused on 12 metabolites associated with the red blood cell (RBC) storage lesion and 23 amino acids in the supernatants of packed RBC units from SCT and reference (non-SCT) donors stored in the same conditions. All samples of RBC concentrates were collected and separated from the storage of Colsan (Beneficient Association of Blood Collection), where they were routinely processed and separated as packed RBC units and stored in the refrigerator (2°-6°C). The supernatant samples of each packed RBC bag were separated by centrifugation at days 1, 7, 14, 21, 28 and 35 of storage and kept at -80°C till the metabolite analysis together. The quantitation of metabolites and amino acids examined in the supernatant of SCT and reference donors showed no statistical differences along the cold storage. Lactic acid and malic acid releases occur in three phases during RBC storage. Basic and acid amino acids and corresponding amides have low and stable values during the first 14 days of storage, followed by a steep increase. Our metabolomic results give elements that seem not to contraindicate the transfusion of RBC with SCT, besides its more structural fragility.

Effect of Dietary Sucrose on Body Weight and Antioxidant Status (Vitamin C and Uric Acid) of Wister Strain of Albino Rats

Sucrose has been reported to be the most widely consumed carbohydrate in affluent countries, with 40% of carbohydrate consumed coming from sugar sources majorly glucose and fructose. Although claims have it that sucrose in diet aggravates asthma, muster illness, nourish nervous disorders, hasten hypertension; leads to diabetes, hurry heart diseases and add arteries. The present study aimed at the investigating the effects of dietary sucrose on the body weight and antioxidant status (vitamin c and uric acid) of wister strain of albino rats Thirty six (36) wister strain albino rats were randomly divided into 6 groups namely: Super High (SH), High (H), Medium (M), Low (L), Control (C) and Base-line (B) group containing basically weanling rats of (4 weeks of age). They were raised together in plastic cages and fed ad libitum with palletized growers feed. The rats were fasted overnight after the end of the 18 weeks and anaesthetized with diethyl ether in a kilner jar on the following morning between 8 and 10 0’clock. They were opened up and their blood samples were collected by cardiopunture method using a syringe and needle. Extraction of Serum from the whole blood samples involved centrifuging the blood at 1,500 r.p.m (6,000 X g) for 15 minutes. After centrifugation, the serum (supernatant) samples were decanted and stored for further Analysis. Vitamin C concentration, uric acid concentration, and the body weight of the rats were determined. The results of this study revealed that there was no significant difference (P < 0.05) observed for the terminal body weights of the groups: Control group (226.38 ± 15.31): Low group (229.42 ± 11.28) and Super high group (239.30 ± 11.01) For serum uric acid level,a significant difference (P < 0.05) was observed between the control group (7.55 ± 0.11) and Super High group (16.30 ± 0.09) High (15.07 ± 0.08).Medium (12.88 ± 0.12) Low (8.90 ± 0.17). However, a downward trend was observed between the groups. Dietary sucrose was observed to significantly affect uric acid concentration .A significant difference (P < 0.05) was observed between the serum vitamin C levels of control group (184.8 ± 5.57) and low group. There was significant difference observed in the uric acid concentration and vitamin C concentration of the experimental rats used. However, no significant difference was observed in the body weight of the rats used for this study when sucrose consumption was increased. Yet care must be taken because the results clearly indicated a strong detrimental effect of sucrose on the cardiovascular well-being of the subjects.

99 The Effects of β-caryophyllene on butyrate utilization and metabolism in Caco 2 cells

Abstract Butyrate is important for regulating energy status and cellular metabolism. Beta-caryophyllene (BCP) is a plant compound which may exert potentially beneficial effects on intestinal epithelial cells, such as barrier integrity and modulation of nutrient utilization. The goal of this preliminary study was to investigate the effects of BCP on butyrate utilization and metabolism in intestinal epithelial cells. Caco 2 cells were used as a model for the intestinal barrier. Cells were seeded in multi-well plates with hanging inserts (n = 4) and grown for 18 d. On d 19, 1 of 4 treatments was added to triplicate wells per plate. Treatments included vehicle control (VC), 40 µM BCP, 2 mM butyrate (BTR), and butyrate plus BCP (BB). Treatments were added to the apical side and incubated for 24 and 48 h. Transepithelial electrical resistance (TEER; ohms/cm2) readings and supernatant samples from both the apical and basolateral sides of the cell layer were taken at each time point, and butyrate and β- hydroxybutyrate (BHB) concentrations measured. Butyrate utilization was calculated as absolute nmol lost. Statistical analyses were performed using lm and emmeans packages of R, with treatment, time and side (of cell layer) as fixed effects. All wells started with the same statistical TEER values on h 0. After 24 and 48 h BCP showed no changes in TEER (P > 0.05). Compared with VC, the mean TEER for BTR and BB was greater at 24 h (579 vs 878 and 1,008 ± 35, respectively; P < 0.001). After 48 h, all groups returned to baseline, except BB which had a greater TEER (883 ± 35; P < 0.001) compared with other treatments. After 24 and 48 h BCP showed no changes for both sides of the cell layer (P > 0.05). Overall, absolute BHB concentrations increased from 24 to 48 h in all treatments (P < 0.05). Production of BHB in each of the VC and BC groups was less than 15 nmol at each time point. For BB and BTR groups, more nmol BHB was detected on the basolateral side (31.9 and 22.2 ± 1.3) vs apical (14.0 and 10.4 ± 1.3, respectively; P < 0.001) after 24 h. After 48 h, BHB was also greater on the basolateral side (58.2 and 45.2 ± 1.3 nmol for BB and BUT, respectively) vs apical side (30.2 and 24.0 ± 1.3 nmol; P < 0.001). When looking at BTR utilization, the BB treatment had greater butyrate disappearance than BTR in 24 h (254 vs 169 ± 12.6 nmol; P = 0.0005) and 48h (707 vs 573 ± 12.6 nmol; P < 0.0001). BCP may promote the metabolism of butyrate by intestinal epithelial cells. More research is needed to understand the mechanism of how BCP increases cellular butyrate utilization.

Improving Vaccine Response through Probiotics and Micronutrient Supplementation: Evaluating the Role of TLR5 in Adult Female BALB/c Mice.

The role of probiotics and micronutrients in improving immune system function and response to vaccination has been proven. Hence, this study aimed to investigate the effects of probiotics enriched with micronutrients on the immunogenicity of PastoCovac® vaccine. The probiotic supplement BioBoost® and PastoCovac® vaccine, which contain six expressed receptor-binding domains (RBD) and conjugated with tetanus toxin, were administered concurrently. The safety and efficacy were assessed by determining Immunoglobulin G (IgG) antibody titers to RBD and cytokines, mRNA expression of Toll-like Receptors (TLRs) 5, and clinical symptoms. Results revealed that the administration of the probiotics enriched with micronutrients and vitamins for 14 days before the first vaccine dose, followed by continued supplementation for 14 days after the first dose, and in conjunction with the second vaccine dose, yielded the most significant elevation in interleukin 4 (IL-4), Tumor Necrosis Factor-alpha (TNF alpha), Interferon-gamma (IFN-gamma), and anti-SARS-CoV-2 RBD IgG levels within the supernatant samples collected from spleen cultures with the highest expression of TLR5 genes in intestinal samples, compared to the control group. Our results indicated that the inclusion of probiotics enriched with micronutrients and vitamins significantly enhanced the immunogenicity of the PastoCovac® vaccine. Based on the recommendation to administer third and fourth vaccine doses, particularly for vulnerable and elderly individuals, the utilization of supplements containing probiotics is expected to favorably influence immune responses.

Investigation of SARS-CoV-2 release in fecal specimens of discharge COVID-19 patients

Abstract Objectives The presence of live SARS-CoV-2 viruses in the fecal specimens and the positive results for SARS-CoV-2 RNA in gastrointestinal samples after respiratory specimens had become negative indicate that there may be a risk of transmission of the disease not only through the respiratory tract but also through the fecal-oral route. The study aim is to determine the time period that the SARS-CoV-2 virus remains in the fecal specimens of discharged COVID-19 patients to reveal the time interval in which the risk of transmission continues. Methods In 65 patients hospitalized with a COVID-19 diagnosis, the viral RNA was isolated from the supernatant of the stool sample using CVXTM viral RNA extraction kit. The extracted RNAs from the stool samples were detected using a commercial RT-PCR method. Results Positive results were obtained in the stool samples in eight of sixty-five patients and the presence of SARS-CoV-2 in these patients was detected for up to three weeks. Conclusions By determining the residence time of SARS-CoV-2 virus in stool samples of discharged COVID-19 patients, the time interval during which the possibility of transmission risk continues has been revealed. The findings of the study could prove beneficial in comprehending the risks of transmission and translating them into preventative measures.

The First Isolation of Insect-Specific Alphavirus (Agua Salud alphavirus) in Culex (Melanoconion) Mosquitoes in the Brazilian Amazon.

Advances in diagnostic techniques coupled with ongoing environmental changes have resulted in intensified surveillance and monitoring of arbovirus circulation in the Amazon. This increased effort has resulted in increased detection of insect-specific viruses among hematophagous arthropods collected in the field. This study aimed to document the first isolation of Agua Salud alphavirus in mosquitoes collected within the Brazilian Amazon. Arthropods belonging to the family Culicidae were collected within a forest fragment located in the Environmental Protection Area of the metropolitan region of Belem. Subsequently, these specimens were meticulously identified to the species level. Afterward, the collected batches were macerated, and the resulting supernatant was then inoculated into C6/36 and Vero cell cultures to facilitate viral isolation. The presence of arboviruses within the inoculated cell cultures was determined through indirect immunofluorescence analysis. Furthermore, positive supernatant samples underwent nucleotide sequencing to precisely identify the viral strains present. Notably, a batch containing Culex (Melanoconion) mosquitoes was identified to be positive for the genus Alphavirus via indirect immunofluorescence. This study is the first report on insect-specific alphavirus isolation in Brazil and the first-ever description of Agua Salud alphavirus isolation within Amazon Forest remnants.

Microscopic ferning in human pre-ejaculate is highly correlated with the absence of sperm

ObjectiveWe characterize microscopic ferning in pre-ejaculate samples with and without sperm. Study designHealthy, male, withdrawal-experienced participants provided up to three paired pre-ejaculate and ejaculate samples. We centrifuged ejaculate samples to obtain a supernatant without sperm. After sperm analysis, we dried and evaluated pre-ejaculate, ejaculate, and supernatants for microscopic ferning. ResultsOf 57 pre-ejaculate samples (N = 24 men), seven (12.3%) contained sperm, none of which exhibited ferning. Sixty-six percent (33/50) of pre-ejaculate samples without sperm exhibited ferning. Neither ejaculate nor supernatant samples exhibited ferning. ConclusionFerning may distinguish clinical pre-ejaculate with and without sperm. Ferning exhibited 100% specificity for pre-ejaculate without sperm.

Biolayer interferometry for adeno-associated virus capsid titer measurement and applications to upstream and downstream process development

Faster and more accurate analytical methods are needed to support the advancement of recombinant adeno-associated virus (rAAV) production systems. Recently, bio-layer interferometry (BLI) has been developed for high-throughput AAV capsid titer measurement by functionalization of the AAVX ligand onto biosensor probes (AAVX-BLI). In this work, an AAVX-BLI method was evaluated using Octet® AAVX biosensors across four rAAV serotypes (rAAV2, 5, 8, and 9) and applied in an upstream and downstream processing context. AAVX-BLI measured capsid titer across a wide concentration range (1×1010 – 1×1012 capsids/mL) for different rAAV serotypes and sample backgrounds with reduced measurement variance and error compared to an enzyme-linked immunosorbent assay (ELISA) method. Biosensors were regenerated for repeated use, with lysate samples showing reduced regeneration capacity compared to purified and supernatant samples. The AAVX-BLI method was applied in a transfection optimization study where direct capsid titer measurement of culture supernatants generated a representative response surface for total vector genome (VG) titer. For rAAV purification, AAVX-BLI was used to measure dynamic binding capacity with POROS™ CaptureSelect™ AAVX affinity chromatography, showing resin breakthrough dependence on operating flow rate. Measurement accuracy, serotype and sample background flexibility, and high sample throughput make AAVX-BLI an attractive alternative to other capsid titer measurement techniques.

Measuring Concentration of Nanoparticles in Polydisperse Mixtures Using Interferometric Nanoparticle Tracking Analysis.

Quantitative measurements of nanoparticle concentration in liquid suspensions are in high demand, for example, in the medical and food industries. Conventional methods remain unsatisfactory, especially for polydisperse samples with overlapping size ranges. Recently, we introduced interferometric nanoparticle tracking analysis (iNTA) for high-precision measurement of nanoparticle size and refractive index. Here, we show that by counting the number of trajectories that cross the focal plane, iNTA can measure concentrations of subpopulations in a polydisperse mixture in a quantitative manner and without the need for a calibration sample. We evaluate our method on both monodisperse samples and mixtures of known concentrations. Furthermore, we assess the concentration of SARS-CoV-2 in supernatant samples obtained from infected cells.

Comparing nitric acid treatment and microwave digestion for efficiency of metal extraction from bioprocess samples

Metal ions may act as enzyme cofactors and influence the kinetics of biochemical reactions that may also influence the biological production of therapeutic proteins and quality attributes such as glycosylation. Because sample preparation is a significant step in the reliable analysis of metals, we compared two sample preparation procedures for metal analysis of bioreactor culture media samples by ICP-MS: (i) samples were diluted in 2 % nitric acid (treatment with nitric acid, TNA); and (ii) samples were mixed with equal volume of 5 % nitric acid and closed vessel digestion was performed in a microwave (closed vessel digestion, CVD). In the comparison of extraction efficiencies between TNA and CVD procedures, CVD showed better extraction for Ca and Cu among bulk metals (∼30 %) and for Ni among the trace metals (∼65 %) for the bioreactor broth supernatant samples. For the cell pellet samples, the CVD procedure was found to be better for extraction of Fe (∼65 % more) among bulk metals, Zn (∼20 % more) among minor metals and Co (∼60 % more) and Ni (∼45 % more) among trace metals. Differences between the two procedures were less than 10 % and TNA was better for all other metals quantified from both supernatant samples and cell pellet samples. The current study helps bring more clarity to the methodology on comprehensive metal analysis to monitor and maintain trace metal content for biologics production.

Peripheral blood mononuclear cells isolated from e-cigarette users have a greater inflammatory cytokine response to LPS stimulation compared to controls

Chronically elevated inflammation is a contributing factor in the development and progression of chronic disease, including cardiovascular disease. Biomarker studies suggest that chronic e-cigarette use is associated with elevated circulating inflammatory cytokines and overall vascular inflammation. However, few studies have examined measures of immune cell activity in e-cigarette users. We hypothesized that healthy young adults (18-24 years) who chronically (history of use ≥6 months) and exclusively use e-cigarettes (EC) would have higher plasma concentrations of the inflammatory cytokine TNFα compared to healthy controls (HC), and that peripheral blood mononuclear cells (PBMC) isolated from EC would release more TNFα when stimulated with lipopolysaccharide (LPS) compared to PBMC isolated from HC. 9 HC (20±2 years) and 10 EC (20±1 years; 4±2 years of e-cigarette use) participated in 1 study visit in which we collected 20mL of whole blood via venipuncture and isolated plasma and PBMC. PBMC were incubated in RPMI and 10% FBS with or without 1mg/mL LPS (37°C, 5% CO2). PBMC supernatants were collected at 4 and 24 hours and TNFα concentrations were quantified in plasma and supernatant samples via ELISA. EC had greater plasma TNFα concentrations compared to HC (HC: 1.7±1.5 vs. EC: 3.1±2.7 pg/mL; p=0.03). PBMC supernatants from EC had elevated TNFα following 4 (HC: 28.0±16.9 vs. EC: 52.1±46.5 pg/mL; p=0.04) and 24 (HC: 87.7±71.6 vs. EC: 178.9±121.0 pg/mL; p=0.04) hours of LPS stimulation compared to HC. There were no group of time differences in TNFα in supernatants from unstimulated PBMC (all p>0.05). These data agree with prior findings that chronic e-cigarette use is associated with elevated circulating inflammatory cytokines and suggest that mononuclear cells of e-cigarette users have a greater cytokine response to inflammatory stimuli. NIEHS/NIH P30 ES005605. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Ranked abundance of 115 proteins following simultaneous measurement by multiplex immunoassay in serum, plasma, and cell culture supernatant samples.

Abstract MILLIPLEX™ PLEXpedition, a customizable multiplex immunoassay kit, was developed to simultaneously screen up to 115 targets including cytokines, chemokines, growth factors, matrix metalloproteinases, and biomarkers of bone health, metabolism, and cardiovascular disease. Two Luminex™ instruments, the xMAP™ INTELLIFLEX™ and the FLEXMAP 3D™, can accommodate all 115 bead regions with comparable sensitivity and sample correlation observed between them. The analyte selection incorporates many biomarkers critical to the field of immunology, using assays developed for two MILLIPLEX™ 48-plex human cytokine, chemokine, and growth factor kits (Cat. No. HCYTA-60K and Cat. No. HCYTB-60K). Sample correlation in serum, plasma, and cell culture supernatants demonstrated that this discovery biomarker panel with a matrix-free format can be used to transition seamlessly to our highly qualified panels, MILLIPLEX™ Human Cytokine Panel A and B, which have optimized serum matrix and lot-to-lot verification for absolute quantification of protein concentration. To probe the relative abundance of each analyte in typical sample matrices, the 115-plex assay was run with a set of 8 cell culture supernatants and 24 healthy and disease serum and plasma samples. Additionally, analytes were ranked by their response to stimulation by LPS or ConA in PBMCs. Our results highlight the utility of the novel PLEXpedition screening kit for evaluating relative abundance in biological samples.

Measuring fecal neurotransmitter concentrations using Dried Fecal Spot (DFS)-based LC-MS/MS bioanalysis

Background: Guthrie implemented dried blood spot (DBS)-based bioanalytical techniques in the 1960s in order to screen for phenylketonuria (PKU) disease in neonates. Historically, the dominant matrix analyzed using the “Guthrie card” platform has been whole blood, but recent advances in device technology now allow for whole blood-to-plasma sample processing prior to drying and subsequent bioanalysis. This project aimed to use this dried matrix technologies in an entirely new direction by performing dried fecal spot (DFS)-based bioanalysis for analytical microbiology and microbiome applications. Homogenized fecal sample extracts were loaded onto quantitative DBS (qDBS) devices and were allowed to dry overnight, then were shipped at ambient shipping temperature. The DBS spots were processed and subjected to LC-MS/MS-based targeted metabolomics using our Glutamate Cycle method (Glu, Gln, and GABA). Methods & Results: Fecal samples were collected from each of the five human subjects enlisted in the following cohorts: 1) healthy controls; 2) patients with diarrhea; and, 3) Clostridioides diffcile-infected (CDI) patients. A volume of ice-cold methanol:water (1:1, v:v) equivalent to a fecal density of 50 mg/mL was added into individual homogenization tubes that each contained 100 mg of 1.4 mm silica beads. Fecal samples were homogenized using 5 bead-beating cycles (4 m/s for each 20 s pulse). After centrifugation (@500 g), a 20-μL sample supernatant volume was loaded onto qDBS devices that yielded an exact 10-μL DFS spot volume on the embedded cards. Dried DFS spots were removed from the device and homogenized in a 200-μL volume of an IS solution. The Glu, Gln, and GABA concentration of each sample was compared against the concentrations measured in the matched frozen fecal extracts. We were able to successfully detect GABA in both the standard extraction method and in our processed DFS samples. Interestingly, we observed large variability between patient samples and there were no statistically significant differences between the cohorts. Conclusion: Collectively, these data demonstrate the utility of our method for measuring the concentrations of Glu, Gln and GABA contained in our processed DFS-based sample sets. This study was supported by the NIH K01K123195-01 (MAE). The Texas Children’s Hospital Department of Pathology provides salary support to Texas Children’s Microbiome Center-Metabolomics Lab staff, and purchased all of the reagents, the consumables and durable supplies, and the liquid chromatography-tandem mass spectrometry (LC-MS/MS) equipment described. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Automated sample preparation for electrospray ionization mass spectrometry based on CLOCK-controlled autonomous centrifugal microfluidics

We report a centrifugal microfluidic device that automatically performs sample preparation under steady-state rotation for clinical applications using mass spectrometry. The autonomous microfluidic device was designed for the control of liquid operation on centrifugal hydrokinetics (CLOCK) paradigm. The reported device was highly stable, with less than 7% variation with respect to the time of each unit operation (sample extraction, mixing, and supernatant extraction) in the preparation process. An agitation mechanism with bubbling was used to mix the sample and organic solvent in this device. We confirmed that the device effectively removed the protein aggregates from the sample, and the performance was comparable to those of conventional manual sample preparation procedures that use high-speed centrifugation. In addition, probe electrospray ionization mass spectrometry (PESI-MS) was performed to compare the device-treated and manually treated samples. The obtained PESI-MS spectra were analyzed by partial least squares discriminant analysis, and the preparation capability of the device was found to be equivalent to that of the conventional method.

High-efficiency EGFR genotyping using cell-free DNA in bronchial washing fluid.

EGFR mutation testing is required for treatment of lung adenocarcinoma using epidermal growth factor receptor-tyrosine kinase inhibitor. However, the amounts of tumor tissue or tumor cells obtained by bronchoscopy are often insufficient. Bronchial washing fluid, obtained by lavage with saline after tumor biopsy or brushing, and the supernatant of bronchial washing fluid are thought to contain cell-free DNA that would be potentially applicable for EGFR testing. From among patients with suspected adenocarcinoma or non-small cell lung carcinoma diagnosed from biopsy or surgical specimens at the University of Tsukuba Hospital between 2015 and 2019, cell-free DNAs from 80 specimens of supernatant of bronchial washing fluid (50 with EGFR mutation and 30 with wild type EGFR) and 8 blood serum samples were examined for EGFR mutation using droplet digital PCR. Among the 50 patients harboring EGFR mutation, the rate of positivity for cell-free DNA extracted from supernatant of bronchial washing fluid was 80% (40/50). In nine of the EGFR mutation-positive cases, tumor cells were not detected by either biopsy or cytology, but the mutation was detected in four cases (4/9, 44%). Comparison of the cell-free DNA mutation detection rate between supernatant of bronchial washing fluid and blood serum in six cases showed that mutations were detected from the former in all cases (6/6, 100%), but from the latter in only one case (1/6, 17%). Using supernatant of bronchial washing fluid samples, the detection rate of EGFR mutation was high, and EGFR mutations were detectable even when no tumor cells had been detectable by biopsy or cytology. Supernatant of bronchial washing fluid might be an effective sample source for EGFR mutation testing.

Small extracellular vesicle microRNAs in pediatric myasthenia gravis plasma and skeletal muscle.

The diagnosis of myasthenia gravis (MG) in children remains difficult. Circulating small extracellular vesicle (sEV)-derived miRNAs (sEV-miRNAs) have been recognized as biomarkers of various diseases and can be excreted by different cell types. These biomarker candidates also play a vital role in autoimmune diseases via intercellular communication. In the present study, we used sEV isolation and purification methods to extract the plasma-derived sEV-miRNAs from children with MG and healthy controls. A small RNA sequencing analysis confirmed the miRNA expression features in plasma-derived sEVs from MG patients. The miRNA expression analysis in vitro was determined using microarray analysis. The enrichment and network analyses of altered sEV-miRNAs were performed using miRNA databases and Database for Annotation, Visualization, and Integrated Discovery website. Quantitative real-time polymerase chain reaction was performed for validation of sEV-miRNA. The diagnostic power of altered sEV-miRNAs was evaluated using receiver operating characteristic curve analyses. Twenty-four sEV-miRNAs with altered expression level were identified between groups by DESeq2 method. The miRNAs were extracted from the sEVs, which were isolated from human primary skeletal muscle cell culture treated with mAb198. The target genes and enriched pathways of sEV-miRNAs partially overlapped between cell supernatant and plasma samples. The significantly downregulated miR-143-3p was validated in quantitative real-time polymerase chain reaction analysis. For the first time, we report that plasma-derived sEV-miRNAs may act as novel circulating biomarkers and therapeutic targets in pediatric MG.

Increased Proteolytic Activity of Serratia marcescens Clinical Isolate HU1848 Is Associated with Higher eepR Expression.

Serratia marcescens is a global opportunistic pathogen. In vitro cytotoxicity of this bacterium is mainly related to metalloprotease serralysin (PrtS) activity. Proteolytic capability varies among the different isolates. Here, we characterized protease production and transcriptional regulators at 37°C of two S. marcescens isolates from bronchial expectorations, HU1848 and SmUNAM836. As a reference strain the insect pathogen S. marcescens Db10 was included. Zymography of supernatant cultures revealed a single (SmUNAM836) or double proteolytic zones (HU1848 and Db10). Mass spectrometry confirmed the identity of PrtS and the serralysin-like protease SlpB from supernatant samples. Elevated proteolytic activity and prtS expression were evidenced in the HU1848 strain through azocasein degradation and qRT-PCR, respectively. Evaluation of transcriptional regulators revealed higher eepR expression in HU1848, whereas cpxR and hexS transcriptional levels were similar between studied strains. Higher eepR expression in HU1848 was further confirmed through an in vivo transcriptional assay. Moreover, two putative CpxR binding motifs were identified within the eepR regulatory region. EMSA validated the interaction of CpxR with both motifs. The evaluation of eepR transcription in a cpxR deletion strain indicated that CpxR negatively regulates eepR. Sequence conservation suggests that regulation of eepR by CpxR is common along S. marcescens species. Overall, our data incorporates CpxR to the complex regulatory mechanisms governing eepR expression and associates the increased proteolytic activity of the HU1848 strain with higher eepR transcription. Based on the global impact of EepR in secondary metabolites production, our work contributes to understanding virulence factors variances across S. marcescens isolates.

Relationship between the cytokine profle of supernatants of invasive breast carcinoma and its molecular and histopathological characteristics

The aim of the study was to analyze the correlation between the cytokine profile of supernatants of invasive breast carcinoma of a nonspecific type (IBC-NST) samples, histopathological and molecular genetic parameters of IBC-NST, expression of the CD34 as a marker of angiogenesis and metastasis to regional lymph nodes (RLN).Material and Methods. The production of 14 cytokines in IBC-NST biopsy samples from 28 patients aged 37–60 years was studied. The concentration of cytokines in the supernatants of biopsies (CCSB) was determined (in pg/ml) using enzyme immunoassay (ELISA). The expression of CD34 and markers of IBC-NST molecular subtypes (HER2/neu, ER, PR, Ki67) in IBC-NST biopsy samples was evaluated by immunohistochemical method. The relative content of tumor cells of different differentiation grade in the IBC- NST samples was evaluated by histopathological analysis.Results. The assessment of CCSB showed statistically significant differences in IFN-γ, G-CSF, IL-2, IL-10 and MCP-1 between patients of group I (with metastases in RLNs) and group II (without metastases in RLNs). In group I, the correlations between histopathological parameters (Her2/neu, CD34 and Ki67 expressions, % of mitoses and poorly-differentiated cancer cells) and CCSB (MCP-1, IL-18) were revealed. In group II, the correlations between CCSB (IL-2, VEGF-A, G-CSF, IL-1Ra) and histopathological parameters, such as expression of Her2/neu, CD34, PR, % of mitoses and well-differentiated cancer cells, were revealed. The ROC analysis showed that the presence or absence of metastases in RLNs can be predicted on the basis of CD34 expression levels and concentrations of IL-10, G-CSF, and MCP-1 in supernatants of IBC-NST biopsy samples. The quality of the model for stratifying patients into groups with and without RLN metastases, based on the assessment of the concentration of MCP-1 in the supernatants of IBC-NST biopsies, reached maximum values (AUC=1.000) with relatively high CD34 expression.Conclusion. The analysis of the data obtained showed that the assessment of CD34 expression and production of cytokines in IBC-NST biopsies is important for predicting the presence or absence of metastases in RLNs.