Locked nucleic acid aptamer based microfluidic devices for capturing tumor cells

Design and fabrication of novel microfluidic devices for sensitive and specific capture of circulating tumor cells using locked nucleic acid modified aptamers and antibodies targeting EpCAM/Nucleolin expression. These devices also allow re-usability, on-chip characterization of multiple markers and release of viable captured cells for further culture and in vitro characterization for cancer diagnosis, prognosis and therapeutic planning

Aerobic cometabolism of 1,1,1-trichloroethane and 1,1-dichloroethene by a bioaugmented butane-utilizing culture in a continuous flow column

The transformation of 1,1,1 -trichioroethane (1,1,1 -TCA) and 1,1 -dichioroethene (1,1 - DCE) was evaluated in a continuous flow column reactor using a mixed culture that grew on butane. The column was packed with aquifer materials and groundwater obtained from the in-situ bioremediation test site at Moffett Field, CA. The pore volume of the column was 38 ml and the dispersion coefficient was 1.93E-3 cm²/sec, based on the results of the bromide transport experiment. The 1,1,1 -TCA transport test prior to biostimulation showed a retardation coefficient of 3.2. The inoculum for bioaugmentation was a butane-utilizing Rhodococcus Sp. culture used in the Moffett Field experiments. The total mass of cells added was 0.9 mg on a dry mass basis. Three days after bioaugmentation, with the continuous addition of dissolved butane, dissolved oxygen, and l,1,l-TCA (200 ug/L), decreases in all three of these solutes began. A maximum removal of 1,1,1 -TCA of 84% was achieved 10 days after bioaugmentation and remained fairly constant for a period of 20 days. The ratio of dissolved oxygen to butane consumption during this period was 4.5 mg O₂: 1 mg Butane. The influent concentration of 1,1,1 -TCA was then doubled, while dissolved oxygen and butane addition was maintained constant. The transformation of 1,1,1 -TCA during this period fluctuated between 24%-84%. Upon restoring the 1,1,1 -TCA concentration back to 200 ug/L the transformation stabilized at 59% removal. The butane-utilizers were then tested for their dependence on butane by turning the butane pulse off. The dissolved oxygen concentration doubled during this period and a residual transformation activity of 22% was observed. On restarting butane addition, 1,1,1 -TCA transformation of 69% was achieved. In the final phase, l,1-DCE was injected at 130 ug/L along with l,1,l-TCA, dissolved butane and oxygen. The butane-utilizing culture transformed 70% of 1,1 -DCE; however, the presence of 1,1 -DCE inhibited 1,1,1 -TCA transformation and approximately 50% of the butane injected was not consumed. The concentration of dissolved oxygen in the column also increased, which also indicating that 1,1 -DCE transformation inhibited butane and dissolved oxygen utilization and 1,1,1 -TCA transformation. Real-time PCR analysis conducted by Li (2004) indicated that during periods of low biotransformation of 1,1,1 -TCA, bioaugmented cell densities observed in the column effluent was high. This corresponded to a period of anoxic conditions, which may have caused cell detachment from the aquifer solids. The column reactor results were simulated using a combined biotransformationtransport model that uses MonodlMichaelis-Menten kinetics along with first-order sorption kinetics, to predict substrate utilization and chlorinated solvent transformation (Semprini and McCarty, 1992). The culture parameter values used to simulate biotransformation in the model were obtained from laboratory culture experiments conducted by Kim et al (2002) and Mathias (2002). Transport parameters (dispersion coefficient, porosity) were determined from modeling breakthrough test data with the CXTFIT2 transport model prior to bioaugmentation and biostimulation. Simulations of the colunm data using the transport and biotransformation parameters demonstrated that the model was able to simulate biotransformation of 1,1,1 -TCA fairly well. The model also indicated that 1,l-DCE transformation was toxic to the butane-utilizing culture and predicted the decreases in consumption of butane, and dissolved oxygen and in 1,1,1 - TCA transformation. This study showed that column experiments conducted on a small scale in a laboratory could be used to study the biotransformation capabilities of bioaugmented microorganisms. On the whole, the results suggest that the butane-utilizing culture could be successfully used in-situ for bioremediation, however transformation of mixtures of 1,1 -DCE and 1,1,1 -TCA could prove difficult

Distinct exosomal mirna profiles from balf and lung tissue of copd and ipf patients

Chronic obstructive pulmonary disease (COPD) and idiopathic pulmonary fibrosis (IPF) are chronic, progressive lung ailments that are characterized by distinct pathologies. Early detection biomarkers and disease mechanisms for these debilitating diseases are lacking. Extracellular vesicles (EVs), including exosomes, are small, lipid-bound vesicles attributed to carry proteins, lipids, and RNA molecules to facilitate cell-to-cell communication under normal and diseased conditions. Exosomal miRNAs have been studied in relation to many diseases. However, there is little to no knowledge regarding the miRNA population of bronchoalveolar lavage fluid (BALF) or the lung-tissue-derived exosomes in COPD and IPF. Here, we determined and compared the miRNA profiles of BALF-and lung-tissue-derived exosomes of healthy non-smokers, smokers, and patients with COPD or IPF in independent cohorts. Results: Exosome characterization using NanoSight particle tracking and TEM demonstrated that the BALF-derived exosomes were ~89.85 nm in size with a yield of ~2.95 × 1010 particles/mL in concentration. Lung-derived exosomes were larger in size (~146.04 nm) with a higher yield of ~2.38 × 1011 particles/mL. NGS results identified three differentially expressed miRNAs in the BALF, while there was one in the lung-derived exosomes from COPD patients as compared to healthy non-smokers. Of these, miR-122-5p was three-or five-fold downregulated among the lung-tissue-derived exosomes of COPD patients as compared to healthy non-smokers and smokers, respectively. Interestingly, there were a large number (55) of differentially expressed miRNAs in the lung-tissue-derived exosomes of IPF patients compared to non-smoking controls. Conclusions: Overall, we identified lung-specific miRNAs associated with chronic lung diseases that can serve as potential biomarkers or therapeutic targets

Distinct Exosomal miRNA Profiles from BALF and Lung Tissue of COPD and IPF Patients

Chronic obstructive pulmonary disease (COPD) and idiopathic pulmonary fibrosis (IPF) are chronic, progressive lung ailments that are characterized by distinct pathologies. Early detection biomarkers and disease mechanisms for these debilitating diseases are lacking. Extracellular vesicles (EVs), including exosomes, are small, lipid-bound vesicles attributed to carry proteins, lipids, and RNA molecules to facilitate cell-to-cell communication under normal and diseased conditions. Exosomal miRNAs have been studied in relation to many diseases. However, there is little to no knowledge regarding the miRNA population of bronchoalveolar lavage fluid (BALF) or the lung-tissue-derived exosomes in COPD and IPF. Here, we determined and compared the miRNA profiles of BALF- and lung-tissue-derived exosomes of healthy non-smokers, smokers, and patients with COPD or IPF in independent cohorts. Results: Exosome characterization using NanoSight particle tracking and TEM demonstrated that the BALF-derived exosomes were ~89.85 nm in size with a yield of ~2.95 × 10(10) particles/mL in concentration. Lung-derived exosomes were larger in size (~146.04 nm) with a higher yield of ~2.38 × 10(11) particles/mL. NGS results identified three differentially expressed miRNAs in the BALF, while there was one in the lung-derived exosomes from COPD patients as compared to healthy non-smokers. Of these, miR-122-5p was three- or five-fold downregulated among the lung-tissue-derived exosomes of COPD patients as compared to healthy non-smokers and smokers, respectively. Interestingly, there were a large number (55) of differentially expressed miRNAs in the lung-tissue-derived exosomes of IPF patients compared to non-smoking controls. Conclusions: Overall, we identified lung-specific miRNAs associated with chronic lung diseases that can serve as potential biomarkers or therapeutic targets

Quick chip assay using locked nucleic acid modified epithelial cell adhesion molecule and nucleolin aptamers for the capture of circulating tumor cells

The role of circulating tumor cells (CTCs) in disease diagnosis, prognosis, monitoring of the therapeutic efficacy, and clinical decision making is immense and has attracted tremendous focus in the last decade. We designed and fabricated simple, flat channel microfluidic devices polydimethylsiloxane (PDMS based) functionalized with locked nucleic acid (LNA) modified aptamers (targeting epithelial cell adhesion molecule (EpCAM) and nucleolin expression) for quick and efficient capture of CTCs and cancer cells. With optimized flow rates (10 μl/min), it was revealed that the aptamer modified devices offered reusability for up to six times while retaining optimal capture efficiency (>90%) and specificity. High capture sensitivity (92%) and specificity (100%) was observed in whole blood samples spiked with Caco-2 cells (10-100 cells/ml). Analysis of blood samples obtained from 25 head and neck cancer patients on the EpCAM LNA aptamer functionalized chip revealed that an average count of 5 ± 3 CTCs/ml of blood were captured from 22/25 samples (88%). EpCAM intracellular domain (EpICD) immunohistochemistry on 9 oral squamous cell carcinomas showed the EpICD positivity in the tumor cells, confirming the EpCAM expression in CTCs from head and neck cancers. These microfluidic devices also maintained viability for in vitro culture and characterization. Use of LNA modified aptamers provided added benefits in terms of cost effectiveness due to increased reusability and sustainability of the devices. Our results present a robust, quick, and efficient CTC capture platform with the use of simple PDMS based devices that are easy to fabricate at low cost and have an immense potential in cancer diagnosis, prognosis, and therapeutic planning

Minimal information for studies of extracellular vesicles (MISEV2023): From basic to advanced approaches

© 2024 The Authors. Journal of Extracellular Vesicles, published by Wiley Periodicals, LLC on behalf of the International Society for Extracellular Vesicles. This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY), https://creativecommons.org/licenses/by/4.0/Extracellular vesicles (EVs), through their complex cargo, can reflect the state of their cell of origin and change the functions and phenotypes of other cells. These features indicate strong biomarker and therapeutic potential and have generated broad interest, as evidenced by the steady year-on-year increase in the numbers of scientific publications about EVs. Important advances have been made in EV metrology and in understanding and applying EV biology. However, hurdles remain to realising the potential of EVs in domains ranging from basic biology to clinical applications due to challenges in EV nomenclature, separation from non-vesicular extracellular particles, characterisation and functional studies. To address the challenges and opportunities in this rapidly evolving field, the International Society for Extracellular Vesicles (ISEV) updates its 'Minimal Information for Studies of Extracellular Vesicles', which was first published in 2014 and then in 2018 as MISEV2014 and MISEV2018, respectively. The goal of the current document, MISEV2023, is to provide researchers with an updated snapshot of available approaches and their advantages and limitations for production, separation and characterisation of EVs from multiple sources, including cell culture, body fluids and solid tissues. In addition to presenting the latest state of the art in basic principles of EV research, this document also covers advanced techniques and approaches that are currently expanding the boundaries of the field. MISEV2023 also includes new sections on EV release and uptake and a brief discussion of in vivo approaches to study EVs. Compiling feedback from ISEV expert task forces and more than 1000 researchers, this document conveys the current state of EV research to facilitate robust scientific discoveries and move the field forward even more rapidly.Peer reviewe

Minimal information for studies of extracellular vesicles (MISEV2023): From basic to advanced approaches

Extracellular vesicles (EVs), through their complex cargo, can reflect the state of their cell of origin and change the functions and phenotypes of other cells. These features indicate strong biomarker and therapeutic potential and have generated broad interest, as evidenced by the steady year-on-year increase in the numbers of scientific publications about EVs. Important advances have been made in EV metrology and in understanding and applying EV biology. However, hurdles remain to realising the potential of EVs in domains ranging from basic biology to clinical applications due to challenges in EV nomenclature, separation from non-vesicular extracellular particles, characterisation and functional studies. To address the challenges and opportunities in this rapidly evolving field, the International Society for Extracellular Vesicles (ISEV) updates its 'Minimal Information for Studies of Extracellular Vesicles', which was first published in 2014 and then in 2018 as MISEV2014 and MISEV2018, respectively. The goal of the current document, MISEV2023, is to provide researchers with an updated snapshot of available approaches and their advantages and limitations for production, separation and characterisation of EVs from multiple sources, including cell culture, body fluids and solid tissues. In addition to presenting the latest state of the art in basic principles of EV research, this document also covers advanced techniques and approaches that are currently expanding the boundaries of the field. MISEV2023 also includes new sections on EV release and uptake and a brief discussion of in vivo approaches to study EVs. Compiling feedback from ISEV expert task forces and more than 1000 researchers, this document conveys the current state of EV research to facilitate robust scientific discoveries and move the field forward even more rapidly

Human umbilical cord mesenchymal stem cell-derived extracellular vesicles ameliorate airway inflammation in a rat model of Chronic Obstructive Pulmonary Disease (COPD)

Background Chronic obstructive pulmonary disease (COPD) is an incurable and debilitating chronic disease characterized by progressive airflow limitation associated with abnormal levels of tissue inflammation. Therefore, stem cell-based approaches to tackle the condition are currently a focus of regenerative therapies for COPD. Extracellular vesicles (EVs) released by all cell types are crucially involved in paracrine, extracellular communication. Recent advances in the field suggest that stem cell-derived EVs possess a therapeutic potential which is comparable to the cells of their origin. Methods In this study, we assessed the potential anti-inflammatory effects of human umbilical cord mesenchymal stem cell (hUC-MSC)-derived EVs in a rat model of COPD. EVs were isolated from hUC-MSCs and characterized by the transmission electron microscope, western blotting, and nanoparticle tracking analysis. As a model of COPD, male Sprague-Dawley rats were exposed to cigarette smoke for up to 12 weeks, followed by transplantation of hUC-MSCs or application of hUC-MSC-derived EVs. Lung tissue was subjected to histological analysis using haematoxylin and eosin staining, Alcian blue-periodic acid-Schiff (AB-PAS) staining, and immunofluorescence staining. Gene expression in the lung tissue was assessed using microarray analysis. Statistical analyses were performed using GraphPad Prism 7 version 7.0 (GraphPad Software, USA). Student’s t test was used to compare between 2 groups. Comparison among more than 2 groups was done using one-way analysis of variance (ANOVA). Data presented as median ± standard deviation (SD). Results Both transplantation of hUC-MSCs and application of EVs resulted in a reduction of peribronchial and perivascular inflammation, alveolar septal thickening associated with mononuclear inflammation, and a decreased number of goblet cells. Moreover, hUC-MSCs and EVs ameliorated the loss of alveolar septa in the emphysematous lung of COPD rats and reduced the levels of NF-κB subunit p65 in the tissue. Subsequent microarray analysis revealed that both hUC-MSCs and EVs significantly regulate multiple pathways known to be associated with COPD. Conclusions In conclusion, we show that hUC-MSC-derived EVs effectively ameliorate by COPD-induced inflammation. Thus, EVs could serve as a new cell-free-based therapy for the treatment of COPD

Comprehensive review of genotoxicity data for diclofenac

Diclofenac is a non-steroidal anti-inflammatory drug (NSAID) discovered decades ago, which has since been used by an estimated one billion patients, and demonstrated a well acceptable safety profile. In support of its marketing approval, a comprehensive set of genotoxicity studies had been conducted in vitro and in vivo. Despite the fact that these studies preceded both GLP requirements and ICH guidelines on genotoxicity testing, they were conducted using the best scientific principles and are considered appropriate by contemporary standards. In addition to bacterial mutagenicity and mammalian in vitro assays, repeat-dose, germ cell and dominant lethal assays had been conducted. These data are made available for the first time to offer researchers an opportunity to review the existing data set which unequivocally demonstrates that diclofenac is not genotoxic. The lack of a genotoxic potential is further substantiated by long-term bioassay data demonstrating that diclofenac has no carcinogenic potential in rodents. However, more recently, new studies were published showing a genotoxic potential for diclofenac in novel or modified in vitro test systems. These new data are discussed in the context of the existing comprehensive data package

Differential plasma exosomal long non-coding RNAs expression profiles and their emerging role in E-cigarette users, cigarette, waterpipe, and dual smokers.

Long non-coding RNAs (lncRNAs) are the varied set of transcripts that play a critical role in biological processes like gene regulation, transcription, post-transcriptional modification, and chromatin remodeling. Recent studies have reported the presence of lncRNAs in the exosomes that are involved in regulating cell-to-cell communication in lung pathologies including lung cancer, chronic obstructive pulmonary disease (COPD), asthma, and idiopathic pulmonary fibrosis (IPF). In this study, we compared the lncRNA profiles in the plasma-derived exosomes amongst non-smokers (NS), cigarette smokers (CS), E-cig users (E-cig), waterpipe smokers (WP) and dual smokers (CSWP) using GeneChip™ WT Pico kit for transcriptional profiling. We found alterations in a distinct set of lncRNAs among subjects exposed to E-cig vapor, cigarette smoke, waterpipe smoke and dual smoke with some overlaps. Gene enrichment analyses of the differentially expressed lncRNAs demonstrated enrichment in the lncRNAs involved in crucial biological processes including steroid metabolism, cell differentiation and proliferation. Thus, the characterized lncRNA profiles of the plasma-derived exosomes from smokers, vapers, waterpipe users, and dual smokers will help identify the biomarkers relevant to chronic lung diseases such as COPD, asthma or IPF