Small RNA sequencing analysis of peptide-affinity isolated plasma extracellular vesicles distinguishes pancreatic cancer patients from non-affected individuals.

Pancreatic ductal adenocarcinoma (PDAC) has a high fatality rate, mainly due to its asymptomatic nature until late-stage disease and therefore delayed diagnosis that leads to a lack of timely treatment intervention. Consequently, there is a significant need for better methods to screen populations that are at high risk of developing PDAC. Such advances would result in earlier diagnosis, more treatment options, and ultimately better outcomes for patients. Several recent studies have applied the concept of liquid biopsy, which is the sampling of a biofluid (such as blood plasma) for the presence of disease biomarkers, to develop screening approaches for PDAC; several of these studies have focused on analysis of extracellular vesicles (EVs) and their cargoes. While these studies have identified many potential biomarkers for PDAC that are present within EVs, their application to clinical practice is hindered by the lack of a robust, reproducible method for EV isolation and analysis that is amenable to a clinical setting. Our previous research has shown that the Vn96 synthetic peptide is indeed a robust and reproducible method for EV isolation that has the potential to be used in a clinical setting. We have therefore chosen to investigate the utility of the Vn96 synthetic peptide for this isolation of EVs from human plasma and the subsequent detection of small RNA biomarkers of PDAC by Next-generation sequencing (NGS) analysis. We find that analysis of small RNA from Vn96-isolated EVs permits the discrimination of PDAC patients from non-affected individuals. Moreover, analyses of all small RNA species, miRNAs, and lncRNA fragments are most effective at segregating PDAC patients from non-affected individuals. Several of the identified small RNA biomarkers have been previously associated with and/or characterized in PDAC, indicating the validity of our findings, whereas other identified small RNA biomarkers may have novel roles in PDAC or cancer in general. Overall, our results provide a basis for a clinically-amendable detection and/or screening strategy for PDAC using a liquid biopsy approach that relies on Vn96-mediated isolation of EVs from plasma.

Application of annotation-agnostic RNA sequencing data analysis tools for biomarker discovery in liquid biopsy.

RNA sequencing analysis is an important field in the study of extracellular vesicles (EVs), as these particles contain a variety of RNA species that may have diagnostic, prognostic and predictive value. Many of the bioinformatics tools currently used to analyze EV cargo rely on third-party annotations. Recently, analysis of unannotated expressed RNAs has become of interest, since these may provide complementary information to traditional annotated biomarkers or may help refine biological signatures used in machine learning by including unknown regions. Here we perform a comparative analysis of annotation-free and classical read-summarization tools for the analysis of RNA sequencing data generated for EVs isolated from persons with amyotrophic lateral sclerosis (ALS) and healthy donors. Differential expression analysis and digital-droplet PCR validation of unannotated RNAs also confirmed their existence and demonstrates the usefulness of including such potential biomarkers in transcriptome analysis. We show that find-then-annotate methods perform similarly to standard tools for the analysis of known features, and can also identify unannotated expressed RNAs, two of which were validated as overexpressed in ALS samples. We demonstrate that these tools can therefore be used for a stand-alone analysis or easily integrated into current workflows and may be useful for re-analysis as annotations can be integrated post hoc.

Peptide-Affinity Isolation of Extracellular Vesicles and Cell-Free DNA From Human Plasma.

Extracellular vesicles (EVs) are membrane-bound nanoparticles that carry DNA, RNA, and protein cargoes and are found in a variety of biofluids. EVs, along with cell-free DNA (cfDNA), have attracted interest as a source of biomarker material for liquid biopsy, a process in which a sample of body fluid is used for the detection or monitoring of disease. The Vn96 synthetic peptide facilitates the isolation of both EVs and cfDNA from multiple body fluids, including human plasma, placing it as a versatile tool for the capture of multiple biomarker materials for disease detection and/or treatment monitoring. In this chapter, we describe an optimized protocol for Vn96-mediated isolation of EVs and cfDNA from human plasma samples, as well as downstream methods for EV enumeration and DNA, RNA, and protein extraction from the material captured by Vn96 for use in biomarker discovery or detection.

The polysaccharide chitosan facilitates the isolation of small extracellular vesicles from multiple biofluids.

Several studies have demonstrated the potential uses of extracellular vesicles (EVs) for liquid biopsy-based diagnostic tests and therapeutic applications; however, clinical use of EVs presents a challenge as many currently-available EV isolation methods have limitations related to efficiency, purity, and complexity of the methods. Moreover, many EV isolation methods do not perform efficiently in all biofluids due to their differential physicochemical properties. Thus, there continues to be a need for novel EV isolation methods that are simple, robust, non-toxic, and/or clinically-amenable. Here we demonstrate a rapid and efficient method for small extracellular vesicle (sEV) isolation that uses chitosan, a linear cationic polyelectrolyte polysaccharide that exhibits biocompatibility, non-immunogenicity, biodegradability, and low toxicity. Chitosan-precipitated material was characterized using Western blotting, nanoparticle tracking analysis (NTA), transmission electron microscopy (TEM), and relevant proteomic-based gene ontology analyses. We find that chitosan facilitates the isolation of sEVs from multiple biofluids, including cell culture-conditioned media, human urine, plasma and saliva. Overall, our data support the potential for chitosan to isolate a population of sEVs from a variety of biofluids and may have the potential to be a clinically amenable sEV isolation method.

A multiparametric extraction method for Vn96-isolated plasma extracellular vesicles and cell-free DNA that enables multi-omic profiling.

Extracellular vesicles (EVs) have been recognized as a rich material for the analysis of DNA, RNA, and protein biomarkers. A remaining challenge for the deployment of EV-based diagnostic and prognostic assays in liquid biopsy testing is the development of an EV isolation method that is amenable to a clinical diagnostic lab setting and is compatible with multiple types of biomarker analyses. We have previously designed a synthetic peptide, known as Vn96 (ME kit), which efficiently isolates EVs from multiple biofluids in a short timeframe without the use of specialized lab equipment. Moreover, it has recently been shown that Vn96 also facilitates the co-isolation of cell-free DNA (cfDNA) along with EVs. Herein we describe an optimized method for Vn96 affinity-based EV and cfDNA isolation from plasma samples and have developed a multiparametric extraction protocol for the sequential isolation of DNA, RNA, and protein from the same plasma EV and cfDNA sample. We are able to isolate sufficient material by the multiparametric extraction protocol for use in downstream analyses, including ddPCR (DNA) and 'omic profiling by both small RNA sequencing (RNA) and mass spectrometry (protein), from a minimum volume (4 mL) of plasma. This multiparametric extraction protocol should improve the ability to analyse multiple biomarker materials (DNA, RNA and protein) from the same limited starting material, which may improve the sensitivity and specificity of liquid biopsy tests that exploit EV-based and cfDNA biomarkers for disease detection and monitoring.

Extracellular vesicles and their diagnostic potential in amyotrophic lateral sclerosis.

Extracellular vesicles, small reservoirs that carry various biomolecules, have gained significant interest from the clinical field in recent years based on the diagnostic, therapeutic and prognostic possibilities they offer. While information abound regarding the clinical potential of such vesicles in diverse conditions, the information demonstrating their likely importance in amyotrophic lateral sclerosis (ALS) is more limited. This review will thus provide a brief introduction to extracellular vesicles, highlight their diagnostic significance in various diseases with a focus on ALS and explore additional applications of extracellular vesicles in the medical field. Overall, this work sheds further light on the clinical importance of extracellular vesicles in diagnostic applications as well as supports the need to better characterize their roles and signatures in patients diagnosed with ALS.

Identification of a circulating miRNA signature in extracellular vesicles collected from amyotrophic lateral sclerosis patients.

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disorder associated with the progressive death of motor neurons. Mean survival for a patient diagnosed with ALS is between 2 and 5 years. Early and efficient diagnosis of the various forms of ALS remains a significant challenge, resulting in a need to identify clinically-relevant biomarkers in readily accessible body fluids. microRNAs (miRNAs) are short, evolutionarily conserved non-coding RNA molecules involved in post-transcriptional regulation of gene expression that have received interest as disease biomarkers. This study was undertaken to identify an ALS-associated miRNA signature in extracellular vesicles (EVs), which can cross the blood-brain barrier and enter the circulatory system, obtained from plasma samples of persons diagnosed and living with ALS (PALS). Next-generation sequencing was used to identify differentially expressed miRNAs recovered from EVs of PALS and healthy controls. High-throughput sequencing data for select miRNA targets was subsequently validated by droplet digital PCR (ddPCR). This approach revealed elevated levels of 5 miRNAs and reduced levels of 22 miRNAs in EVs collected from PALS as compared with healthy controls subjects. miRNAs with relevance to ALS were found to be deregulated, including miR-9-5p, miR-183-5p, miR-338-3p and miR-1246. MiR-15a-5p and miR-193a-5p were identified for their diagnostic potential of ALS and association with disability progression, respectively. Functional assessment of transcripts targeted by select ALS-associated miRNAs revealed processes such as transcriptional regulation and protein ubiquitination. These data identify an ALS-associated miRNAs signature in EVs of PALS and further strengthen the potential diagnostic relevance of these small molecules for this condition.

Tyrosine kinase-mediated axial motility of basal cells revealed by intravital imaging.

Epithelial cells are generally considered to be static relative to their neighbours. Basal cells in pseudostratified epithelia display a single long cytoplasmic process that can cross the tight junction barrier to reach the lumen. Using in vivo microscopy to visualize the epididymis, a model system for the study of pseudostratified epithelia, we report here the surprising discovery that these basal cell projections--which we call axiopodia--periodically extend and retract over time. We found that axiopodia extensions and retractions follow an oscillatory pattern. This movement, which we refer to as periodic axial motility (PAM), is controlled by c-Src and MEK1/2-ERK1/2. Therapeutic inhibition of tyrosine kinase activity induces a retraction of these projections. Such unexpected cell motility may reflect a novel mechanism by which specialized epithelial cells sample the luminal environment.

Role of testicular luminal factors on Basal cell elongation and proliferation in the mouse epididymis.

A subset of basal cells (BCs) in the initial segment (IS) of the mouse epididymis has a slender body projection between adjacent epithelial cells. We show here that these projections occasionally cross the apical tight junctions and are in contact with the luminal environment. Luminal testicular factors are critical for the establishment of the IS epithelium, and we investigated their role in the regulation of this luminal sensing property. Efferent duct ligation (EDL) was performed to block luminal flow from the testis without affecting blood flow. Cytokeratin 5 (KRT5) labeling showed a time-dependent reduction of the percentage of BCs with intercellular projections from 1 to 5 days after EDL, compared to controls. Double labeling for caspase-3 and KRT5 showed that a subset of BCs undergoes apoptosis 1 day after EDL. Ki67/KRT5 double labeling showed a low rate of BC proliferation under basal conditions. However, EDL induced a marked increase in the proliferation rate of a subset of BCs 2 days after EDL. A 2-wk treatment with the androgen receptor antagonist flutamide did not affect the number of BCs with intercellular projections, but reduced BC proliferation. Flutamide treatment also reduced the increase in BC proliferation induced 2 days after EDL. We conclude that, in the adult mouse IS, 1) luminal testicular factors play an important role in the ability of BCs to extend their body projection towards the lumen, and are essential for the survival of a subset of BCs; 2) androgens play an important role in the proliferation of some of the BCs that survive the initial insult induced by EDL; and 3) the formation and elongation of BC intercellular projections do not depend on androgens.

ROS1 signaling regulates epithelial differentiation in the epididymis.

The initial segment (IS) of the epididymis plays an essential role in male fertility. The IS epithelium is undifferentiated and nonfunctional at birth. Prior to puberty, the epithelium undergoes differentiation that leads to the formation of a fully functional organ. However, the mechanistic details of this program are not well understood. To explore this further, we used genetic engineering to create a kinase dead allele of the ROS1 receptor tyrosine kinase in mice and studied the effects of ROS1 tyrosine kinase activity on the differentiation of the IS epithelium. We show that the expression and activation of ROS1 coincides with the onset of differentiation and is exclusively located in the IS of the maturing and adult mouse epididymides. Here we demonstrate that the differentiation of the IS is dependent on the kinase activity of ROS1 and its downstream effector MEK1/2-ERK1/2 signaling axis. Using genetic engineering, we show that germ line ablation of ROS1 kinase activity leads to a failure of the IS epithelium to differentiate, and as a consequence sperm maturation and infertility were dramatically perturbed. Pharmacological inhibition of ROS1 kinase activity in the developing epididymis, however, only delayed differentiation transiently and did not result in infertility. Our results demonstrate that ROS1 kinase activity and the ensuing MEK1/2-ERK1/2 signaling are necessary for the postnatal development of the IS epithelium and that a sustained ablation of ROS1 kinase activity within the critical window of terminal differentiation abrogate the function of the epididymis and leads to sterility.

Epithelial basal cells are distinct from dendritic cells and macrophages in the mouse epididymis.

The epithelium that lines the epididymal duct establishes the optimal milieu in which spermatozoa mature, acquire motility, and are stored. This finely tuned environment also protects antigenic sperm against pathogens and autoimmunity, which are potential causes of transient or permanent infertility. The epididymal epithelium is pseudostratified and contains basal cells (BCs) that are located beneath other epithelial cells. Previous studies showed that in the mouse epididymis, BCs possess macrophage-like characteristics. However, we previously identified a dense population of cells belonging to the mononuclear phagocyte (MP) system (comprised of macrophages and dendritic cells) in the basal compartment of the mouse epididymis and showed that a subset of MPs express the macrophage marker F4/80. In the present study, we evaluate the distribution of BCs and MPs in the epididymis of transgenic CD11c-EYFP mice, in which EYFP is expressed exclusively in MPs, using antibodies against the BC marker keratin 5 (KRT5) and the macrophage marker F4/80. Immunofluorescence labeling for laminin, a basement membrane marker, showed that BCs and most MPs are located in the basal region of the epithelium. Confocal microscopy showed that in the initial segment, both BCs and MPs project intraepithelial extensions and establish a very intricate network. Flow cytometry experiments demonstrated that epididymal MPs and BCs are phenotypically distinct. BCs do not express F4/80, and MPs do not express KRT5. Therefore, despite their proximity and some morphological similarities with peritubular macrophages and dendritic cells, BCs do not belong to the MP system.

Circulating aldosterone induces the apical accumulation of the proton pumping V-ATPase and increases proton secretion in clear cells in the caput epididymis.

Clear cells express the vacuolar proton-pumping H(+)-ATPase (V-ATPase) and acidify the lumen of the epididymis, a process that is essential for male fertility. The renin-angiotensin-aldosterone system (RAAS) regulates fluid and electrolyte balance in the epididymis, and a previous study showed binding of aldosterone exclusively to epididymal clear cells (Hinton BT, Keefer DA. Steroid Biochem 23: 231-233, 1985). We examined here the role of aldosterone in the regulation of V-ATPase in the epididymis. RT-PCR showed expression of the mineralocorticoid receptor [MR; nuclear receptor subfamily 3, group C member 2 (NR3C2)] and 11-ß-dehydrogenase isozyme 2 (HSD11ß2) mRNAs specifically in clear cells, isolated by fluorescence-activated cell sorting from B1-enhanced green fluorescent protein (EGFP) mice. Tail vein injection of adult rats with aldosterone, 1,2-dioctanoyl-sn-glycerol (DOG), or 8-(4-chlorophenylthio)-cAMP (cpt-cAMP) induced V-ATPase apical membrane accumulation and extension of V-ATPase-labeled microvilli in clear cells in the caput epididymis but not in the cauda. V-ATPase activity was measured in EGFP-expressing clear cells using the intracellular pH (pHi)-sensing dye seminaphthorhodafluor-5F-5-(and 6)-carboxylic acid, acetoxymethyl ester acetate (SNARF-5F). Aldosterone induced a rapid increase in the rate of Na(+)- and bicarbonate-independent pHi recovery following an NH4Cl-induced acid load in clear cells isolated from the caput but not the cauda. This effect was abolished by concanamycin A, spironolactone, and chelerythrine but not myristoylated-protein kinase inhibitor (mPKI) or mifepristone. Thus aldosterone increases V-ATPase-dependent proton secretion in clear cells in the caput epididymis via MR/NR3C2 and PKC activation. This study, therefore, identifies aldosterone as an active member of the RAAS for the regulation of luminal acidification in the proximal epididymis.

Pharmacological separation of hEAG and hERG K+ channel function in the human mammary carcinoma cell line MCF-7.

Pharmacological inhibitors of the human ether-a-go-go (hEAG) potassium channel, astemizole and imipramine, have been used to demonstrate that hEAG plays a role in cancer cell proliferation. Astemizole and imipramine are, however, relatively non-specific ion channel blockers, as astemizole can also block the related potassium channel, human ether-a-go-go-related (hERG). Therefore, we aimed to determine the molecular target of astemizole, in the human mammary carcinoma cell line MCF-7. We initially confirmed the expression of KCNH1 and KCNH2 mRNA and hEAG and hERG channel protein in MCF-7 cells. Using a [3H]-thymidine incorporation assay we determined that astemizole inhibited MCF-7 cell proliferation, whereas the hERG-specific channel blocker E-4031 had no effect. We then determined that E-4031 inhibited the regulatory volume decrease (RVD) observed in these cells following exposure to hypotonic solutions, confirming that functional hERG channels are present and may be important for cell volume regulation in MCF-7 cells. Our results suggest, for the first time, that hERG is involved in cell volume regulation. In addition, the function of hEAG and hERG in MCF-7 cell proliferation can be separated pharmacologically by utilizing the channel inhibitors astemizole and E-4031. The hEAG channel function in MCF-7 cells appears to be involved in the regulation of cell proliferation, whereas hERG is involved in cell volume regulation.

Exposure to sodium butyrate leads to functional downregulation of calcium-activated potassium channels in human airway epithelial cells.

Cystic fibrosis (CF) is caused by genetic mutations that lead to dysfunction of the cystic fibrosis transmembrane conductance regulator (CFTR) Cl(-) channel. The most common mutation, DeltaF508, causes inefficient trafficking of mutant CFTR protein from the endoplasmic reticulum to the cell membrane. Therapeutic efforts have been aimed at increasing the level of DeltaF508-CFTR protein in the membrane using agents such as sodium butyrate. In this study, we investigated the effects of culturing a human airway epithelial cell line, Calu-3, in the presence of 5 mM sodium butyrate. Within 24 h, butyrate exposure caused a significant decrease in the basal, as well as Ca(2+)-activated, anion secretion by Calu-3 cell monolayers, determined by the change in transepithelial short-circuit current in response to the Ca(2+)-elevating agent thapsigargin. The secretory response to 1-ethyl-2-benzimidazolinone, an activator of the basolateral Ca(2+)-activated K(+) channel KCNN4, was similarly reduced by butyrate treatment. Quantitative PCR revealed that these functional effects were associated with dramatic decreases in mRNA for both KCNN4 and CFTR. Furthermore, the KCNQ1 K(+) channel was upregulated after butyrate treatment. We suggest that prolonged exposure to sodium butyrate downregulates the expression of both KCNN4 and CFTR, leading to a functional loss of Ca(2+)-activated anion secretion. Thus, butyrate may inhibit, rather than stimulate, the anion secretory capacity of human epithelial cells that express wild-type CFTR, particularly in tissues that normally exhibit robust Ca(2+)-activated secretion.

Pituitary resistin gene expression: effects of age, gender and obesity.

Resistin is a new adipocytokine which is expressed in rat, mouse and possibly human adipose tissue. Its putative role as a mediator of insulin resistance is controversial. We hypothesized that resistin, like leptin, would have multiple roles in non-adipose tissues and we reported that resistin is expressed in mouse brain and pituitary. Moreover, resistin expression in female mouse pituitary is developmentally regulated and maximal expression occurs peripubertally. Although the role of endogenous resistin in mouse brain and pituitary has not been determined, our data suggest that resistin could be important in the postnatal maturation of the hypothalamic-pituitary system. In the present study we compared the ontogeny of resistin gene expression in the pituitary of male and female mice using semi-quantitative RT-PCR analysis. We show that resistin expression is developmentally regulated in the pituitary of male and female CD1 mice. However, significant gender differences were evident (male > female at postnatal day 28 and 42) and this was not modified by neonatal treatment of female pups with testosterone. Since resistin expression in adipose tissue is also influenced by obesity, we evaluated resistin expression in fat, brain and pituitary of the obese ob/ob mouse. Resistin mRNA was significantly increased in both visceral and subcutaneous adipose depots in postnatal day 28 ob/ob mice compared to controls, but pituitary resistin expression was significantly reduced. In contrast to the prepubertal levels, and in agreement with other reports, adipose resistin expression was reduced in adult ob/ob mice. In a third set of experiments we examined the influence of food deprivation on pituitary and fat resistin mRNA. Resistin gene expression was severely down-regulated by a 24-hour fast in adipose and pituitary tissue but not in hypothalamus. In conclusion, pituitary resistin expression is age- and gender-dependent. In ob/ob mice, and in fasted mice, resistin is regulated in a tissue-specific manner. Thus in visceral fat obesity increases but starvation decreases resistin mRNA. In contrast, pituitary levels are decreased in the presence of both high (ob/ob) and low (fasting) adipose stores. Further studies are required to define the unexpected hormonal regulation of resistin gene expression in the pituitary.