Technological Approaches in the Analysis of Extracellular Vesicle Nucleotide Sequences

Together with metabolites, proteins, and lipid components, the EV cargo consists of DNA and RNA nucleotide sequence species, which are part of the intracellular communication network regulating specific cellular processes and provoking distinct target cell responses. The extracellular vesicle (EV) nucleotide sequence cargo molecules are often investigated in association with a particular pathology and may provide an insight into the physiological and pathological processes in hard-to-access organs and tissues. The diversity and biological function of EV nucleotide sequences are distinct regarding EV subgroups and differ in tissue- and cell-released EVs. EV DNA is present mainly in apoptotic bodies, while there are different species of EV RNAs in all subgroups of EVs. A limited sample volume of unique human liquid biopsy provides a small amount of EVs with limited isolated DNA and RNA, which can be a challenging factor for EV nucleotide sequence analysis, while the additional difficulty is technical variability of molecular nucleotide detection. Every EV study is challenged with its first step of the EV isolation procedure, which determines the EV’s purity, yield, and diameter range and has an impact on the EV’s downstream analysis with a significant impact on the final result. The gold standard EV isolation procedure with ultracentrifugation provides a low output and not highly pure isolated EVs, while modern techniques increase EV’s yield and purity. Different EV DNA and RNA detection techniques include the PCR procedure for nucleotide sequence replication of the molecules of interest, which can undergo a small-input EV DNA or RNA material. The nucleotide sequence detection approaches with their advantages and disadvantages should be considered to appropriately address the study problem and to extract specific EV nucleotide sequence information with the detection using qPCR or next-generation sequencing. Advanced next-generation sequencing techniques allow the detection of total EV genomic or transcriptomic data even at the single-molecule resolution and thus, offering a sensitive and accurate EV DNA or RNA biomarker detection. Additionally, with the processes where the EV genomic or transcriptomic data profiles are compared to identify characteristic EV differences in specific conditions, novel biomarkers could be discovered. Therefore, a suitable differential expression analysis is crucial to define the EV DNA or RNA differences between conditions under investigation. Further bioinformatics analysis can predict molecular cell targets and identify targeted and affected cellular pathways. The prediction target tools with functional studies are essential to help specify the role of the investigated EV-targeted nucleotide sequences in health and disease and support further development of EV-related therapeutics. This review will discuss the biological diversity of human liquid biopsy–obtained EV nucleotide sequences DNA and RNA species reported as potential biomarkers in health and disease and methodological principles of their detection, from human liquid biopsy EV isolation, EV nucleotide sequence extraction, techniques for their detection, and their cell target prediction.

Isolation and Characterization of Quercetin from Bambusa arundinacea

Flavonoids are natural antioxidants that are formed from plants and found in meals such as fruits and vegetables. They have the capacity to bind to free radicals. Because of its abundant prevalence in foods, Quercetin, which belongs to the flavonol subclass of flavonoids, has gotten a lot of attention. Quercetin is also found in Bambusa arundinacea. The plant was obtained and authenticated. Further the isolation procedure was done and was analyzed via TLC, FT-IR, and UV, 1 H NMR, Mass and XRD Analysis. The results obtained from the above parameters showed the resemblance with standard quercetin. Thus it was concluded for the presence of quercetin from Bambusa arundinacea.

A chemically defined biomimetic surface for enhanced isolation efficiency of high-quality human mesenchymal stromal cells under xeno-/serum-free conditions

Mesenchymal stromal cells (MSCs) are one of the most frequently used cell types in regenerative medicine and cell therapy. Generating sufficient cell numbers for MSC-based therapies is constrained by: 1) their low abundance in tissues of origin, which imposes the need for significant ex vivo cell amplification, 2) donor-specific characteristics including MSC frequency/quality that decline with disease state and increasing age, 3) cellular senescence, which is promoted by extensive cell expansion and results in decreased therapeutic functionality. The final yield of a manufacturing process is therefore primarily determined by the applied isolation procedure and its efficiency in isolating therapeutically active cells from donor tissue. To date, MSCs are predominantly isolated using media supplemented with either serum or its derivatives, which pose safety and consistency issues. To overcome those limitations while enabling robust MSC production with constant high yield and quality, we developed a chemically defined biomimetic surface coating, called isoMATRIX, that facilitates the isolation of significantly higher numbers of MSCs in xeno-/serum-free and chemically defined conditions. The isolated cells display a smaller cell size and higher proliferation rate than those derived from a serum-containing isolation procedure and a strong immunomodulatory capacity. In sum, the isoMATRIX promotes enhanced xeno-, serum-free, or chemically defined isolation of human MSCs and supports consistent and reliable cell performance for improved stem cell-based therapies.

Protocol for the Isolation of Stratum Corneum from Pig Ear Skin: Evaluation of the Trypsin Digestion Conditions

Stratum corneum (SC) represents the outermost layer of the skin, being an effective barrier against the entry of molecules and pathogens. Skin research has given particular focus to SC as it hampers effective drug delivery for cosmetical and therapeutical purposes. Following recommendations to develop alternative models to animals, the SC isolated from skin obtained from medical procedures or from pigs has gained extensive attention. Yet, there is still missing a standard and simple procedure accepted within the scientific community to avoid application of different isolated SC methodologies, a fact that may hamper progress in skin research. Considering this challenge, the present study evaluated different experimental conditions aiming to establish a useful and sustainable solvent-free procedure for the obtention of a realistic SC model. The studied trypsin digestion parameters included concentration, incubation period and temperature. Isolated SC was characterized using histological analysis and calcein’s permeability, after the procedure and during a 6-week storage period. Data recommend trypsin digestion at 4 °C for 20 h as the most effective procedure to isolate SC from pig ear skin. This work contributes to standardize the SC isolation procedure, and to obtain a valuable and reliable SC mimetic model for skin drug development.

ISOlation Procedure vs. conventional procedure during Distal Pancreatectomy (ISOP-DP trial): study protocol for a randomized controlled trial

Background Radical antegrade modular pancreatosplenectomy (RAMPS) is an isolation procedure in pancreatosplenectomy for pancreatic body/tail cancer. Connective tissues around the bifurcation of the celiac axis are dissected, followed by median-to-left retroperitoneal dissection. This procedure has the potential to isolate blood and lymphatic flow to the area of the pancreatic body/tail and the spleen to be excised. This is achieved by division of the inflow artery, transection of the pancreas, and then division of the outflow vein in the early phases of surgery. In cases of pancreatic ductal adenocarcinoma (PDAC), the procedure has been shown to decrease intraoperative blood loss and increase R0 resection rate by complete clearance of the lymph nodes. This trial investigates whether the isolation procedure can prolong the survival of patients with pancreatic ductal adenocarcinoma who undergo distal pancreatosplenectomy (DPS) compared with those that undergo the conventional approach. Methods/design Patients with PDAC scheduled to undergo DPS are randomized before surgery to undergo either a conventional procedure (arm A) or to undergo the isolation procedure (arm B). In arm A, the pancreatic body, tail, and spleen are mobilized, followed by removal of the regional lymph nodes. The splenic vein is transected at the end of the procedure. The timing of division of the splenic artery (SA) is not restricted. In arm B, regional lymph nodes are dissected, then we transect the root of the SA, the pancreas, then the splenic vein. At the end of the procedure, the pancreatic body/tail and spleen are mobilized and removed. In total, 100 patients from multiple Japanese high-volume centers will be randomized. The primary endpoint is 2-year recurrence-free survival by intention-to-treat analysis. Secondary endpoints include intraoperative blood loss, R0 resection rate, and overall survival. Discussion If this trial shows that the isolation procedures can improve survival with a similar R0 rate and with a similar number of lymph node dissections to the conventional procedure, the isolation procedure is expected to become a standard procedure during DPS for PDAC. Conversely, if there were no significant differences in endpoints between the groups, it would demonstrate justification of either procedure from surgical and oncological points of view. Trial registration UMIN Clinical Trials Registry UMIN000041381. Registered on 10 August 2020. ClinicalTrials.gov NCT04600063. Registered on 22 October 2020.

Optimization of dye solutions for detecting damaged pancreatic tissues during islet isolation procedures

We previously reported that dye was effective to prevent the leakage of enzyme solutions from pancreatic glands during an islet isolation procedure. However, the dye used for islet isolation has not yet been optimized. In this study, we focused on pyoktanin blue (PB), diagnogreen (DG), and indigo carmine (IC) as potential candidates among clinically established dyes. A serial dilution assay was performed to determine minimal effective concentrations of each dye for detecting damaged pancreatic tissues. According to the outcome of serial dilution assays, double minimum effective concentrations of each dye were used for in vitro toxicity assays on islets and used in the isolation procedure to investigate whether they adversely affect islet isolation efficiency. The evaluations included islet yield, ADP/ATP, ATP/DNA, glucose stimulation test, and insulin/DNA assays. Islet viability cultured with PB contained medium was significantly lower than the other dyes. DG and IC appeared to be non-toxic to the islets. In isolation experiments, the islet yield in the DG group was considerably lower than that in the Control group, suggesting that DG might inhibit enzyme activity. The present study demonstrates that IC could be a promising candidate for an effective dye to detect damaged pancreatic tissues without affecting the enzyme activity and islet quality.

Isolation Procedure for CP E. coli from Caeca Samples under Review Towards an Increased Sensitivity

Due to the increasing reports of carbapenemase-producing Enterobacteriaceae (CPE) from livestock in recent years, the European Reference Laboratory for Antimicrobial Resistances (EURL-AR) provided a protocol for their recovery from caecum and meat samples. This procedure exhibited limitations for the detection of CPE with low carbapenem MIC values. Therefore, it was modified by a second, selective enrichment in lysogeny broth with cefotaxime (CTX 1 mg/L) and with meropenem (MEM 0.125 mg/L) at 37 °C under microaerophilic conditions. By Real-time PCR, these enrichments are pre-screened for the most common carbapenemase genes. Another adaptation was the use of in-house prepared MacConkey agar with MEM and MEM+CTX instead of commercial selective agar. According to the EURL-method, we achieved 100% sensitivity and specificity using the in-house media instead of commercial agar, which decreased the sensitivity to ~75%. Comparing the method with and without the second enrichment, no substantial influence on sensitivity and specificity was detected. Nevertheless, this enrichment has simplified the CPE-isolation regarding the accompanying microbiota and the separation of putative colonies. In conclusion, the sensitivity of the method can be increased with slight modifications.

Target Accumulation of Selective Anticancer Depsipeptides by Reconstructing Precursor Supply in Neoantimycins Biosynthetic Pathway

BackgroundNeoantimycins are a group of 15-membered ring depsipeptides isolated from streptomycetes with a broad-spectrum of anticancer activities. Their biosynthesis is directed by the hybrid multimodular megaenzymes of non-ribosomal peptide synthetase and polyketide synthase. We have previously discovered a new neoantimycin analogue unantimycin B, which was demonstrated with selective anticancer activities and was produced from neoantimycins biosynthetic pathway with a starter unit of 3-hydroxybenzoate, instead of the 3-formamidosalicylate for neoantimycins. However, the low fermentation yield and tough isolation procedure have been hindering in-depth pharmacology investigation of unantimycin B as anticancer agents.ResultsIn the work, we genetically constructed two unantimycin B producer strains with neoantimycins production destroyed by removing natO and natJ-L genes essential for 3-formamidosalicylate biosynthesis and therefore facilitated chromatographic separation of unantimycin B from the complex fermentation extract. Based on the △natO mutant, we improved unantimycin Bproduction by two times, reaching to approximate 12.8 mg/L, by feeding 3-hydroxybenzoate in fermentation. Further, the production was improved by more than six times, reaching to approximate 40.0 mg/L, in the △natO strain introduced with a chorismatase gene highly expressed under a strong promoter for over-producing 3-hydroxybenzoate endogenously.ConclusionThe work gives a case of targeting accumulation and significant production improvement of medicinally interesting natural products via genetically manipulation of precursor biosynthesis in streptomycetes, the talented producers of pharmaceutical molecules.