Small RNA Sequencing across Diverse Biofluids Identifies Optimal Methods for exRNA Isolation.

Poor reproducibility within and across studies arising from lack of knowledge regarding the performance of extracellular RNA (exRNA) isolation methods has hindered progress in the exRNA field. A systematic comparison of 10 exRNA isolation methods across 5 biofluids revealed marked differences in the complexity and reproducibility of the resulting small RNA-seq profiles. The relative efficiency with which each method accessed different exRNA carrier subclasses was determined by estimating the proportions of extracellular vesicle (EV)-, ribonucleoprotein (RNP)-, and high-density lipoprotein (HDL)-specific miRNA signatures in each profile. An interactive web-based application (miRDaR) was developed to help investigators select the optimal exRNA isolation method for their studies. miRDar provides comparative statistics for all expressed miRNAs or a selected subset of miRNAs in the desired biofluid for each exRNA isolation method and returns a ranked list of exRNA isolation methods prioritized by complexity, expression level, and reproducibility. These results will improve reproducibility and stimulate further progress in exRNA biomarker development.

exRNA Atlas Analysis Reveals Distinct Extracellular RNA Cargo Types and Their Carriers Present across Human Biofluids.

To develop a map of cell-cell communication mediated by extracellular RNA (exRNA), the NIH Extracellular RNA Communication Consortium created the exRNA Atlas resource (https://exrna-atlas.org). The Atlas version 4P1 hosts 5,309 exRNA-seq and exRNA qPCR profiles from 19 studies and a suite of analysis and visualization tools. To analyze variation between profiles, we apply computational deconvolution. The analysis leads to a model with six exRNA cargo types (CT1, CT2, CT3A, CT3B, CT3C, CT4), each detectable in multiple biofluids (serum, plasma, CSF, saliva, urine). Five of the cargo types associate with known vesicular and non-vesicular (lipoprotein and ribonucleoprotein) exRNA carriers. To validate utility of this model, we re-analyze an exercise response study by deconvolution to identify physiologically relevant response pathways that were not detected previously. To enable wide application of this model, as part of the exRNA Atlas resource, we provide tools for deconvolution and analysis of user-provided case-control studies.

CITED4 Protects Against Adverse Remodeling in Response to Physiological and Pathological Stress.

RATIONALE: Cardiac CITED4 (CBP/p300-interacting transactivators with E [glutamic acid]/D [aspartic acid]-rich-carboxylterminal domain4) is induced by exercise and is sufficient to cause physiological hypertrophy and mitigate adverse ventricular remodeling after ischemic injury. However, the role of endogenous CITED4 in response to physiological or pathological stress is unknown. OBJECTIVE: To investigate the role of CITED4 in murine models of exercise and pressure overload. METHODS AND RESULTS: We generated cardiomyocyte-specific CITED4 knockout mice (C4KO) and subjected them to an intensive swim exercise protocol as well as transverse aortic constriction (TAC). Echocardiography, Western blotting, qPCR, immunohistochemistry, immunofluorescence, and transcriptional profiling for mRNA and miRNA (microRNA) expression were performed. Cellular crosstalk was investigated in vitro. CITED4 deletion in cardiomyocytes did not affect baseline cardiac size or function in young adult mice. C4KO mice developed modest cardiac dysfunction and dilation in response to exercise. After TAC, C4KOs developed severe heart failure with left ventricular dilation, impaired cardiomyocyte growth accompanied by reduced mTOR (mammalian target of rapamycin) activity and maladaptive cardiac remodeling with increased apoptosis, autophagy, and impaired mitochondrial signaling. Interstitial fibrosis was markedly increased in C4KO hearts after TAC. RNAseq revealed induction of a profibrotic miRNA network. miR30d was decreased in C4KO hearts after TAC and mediated crosstalk between cardiomyocytes and fibroblasts to modulate fibrosis. miR30d inhibition was sufficient to increase cardiac dysfunction and fibrosis after TAC. CONCLUSIONS: CITED4 protects against pathological cardiac remodeling by regulating mTOR activity and a network of miRNAs mediating cardiomyocyte to fibroblast crosstalk. Our findings highlight the importance of CITED4 in response to both physiological and pathological stimuli.

Evaluation of commercially available small RNASeq library preparation kits using low input RNA.

BACKGROUND: Evolving interest in comprehensively profiling the full range of small RNAs present in small tissue biopsies and in circulating biofluids, and how the profile differs with disease, has launched small RNA sequencing (RNASeq) into more frequent use. However, known biases associated with small RNASeq, compounded by low RNA inputs, have been both a significant concern and a hurdle to widespread adoption. As RNASeq is becoming a viable choice for the discovery of small RNAs in low input samples and more labs are employing it, there should be benchmark datasets to test and evaluate the performance of new sequencing protocols and operators. In a recent publication from the National Institute of Standards and Technology, Pine et al., 2018, the investigators used a commercially available set of three tissues and tested performance across labs and platforms. RESULTS: In this paper, we further tested the performance of low RNA input in three commonly used and commercially available RNASeq library preparation kits; NEB Next, NEXTFlex, and TruSeq small RNA library preparation. We evaluated the performance of the kits at two different sites, using three different tissues (brain, liver, and placenta) with high (1 µg) and low RNA (10 ng) input from tissue samples, or 5.0, 3.0, 2.0, 1.0, 0.5, and 0.2 ml starting volumes of plasma. As there has been a lack of robust validation platforms for differentially expressed miRNAs, we also compared low input RNASeq data with their expression profiles on three different platforms (Abcam Fireplex, HTG EdgeSeq, and Qiagen miRNome). CONCLUSIONS: The concordance of RNASeq results on these three platforms was dependent on the RNA expression level; the higher the expression, the better the reproducibility. The results provide an extensive analysis of small RNASeq kit performance using low RNA input, and replication of these data on three downstream technologies.

The immunomodulatory role of tumor-derived extracellular vesicles in colorectal cancer.

Colorectal cancer (CRC) is one of the most prevalent cancers worldwide with rising mortality rates predicted in the coming decades. In light of this, there is a continued need for improvement in our understanding of CRC biology and the development of novel treatment options. Tumor-derived extracellular vesicles (tEVs) have emerged as both novel cancer biomarkers and functional mediators of carcinogenesis. tEVs are released by tumor cells in abundance and play an important role in mediating tumor cell-immune cell interactions in the tumor microenvironment. Furthermore, tEVs are released into the circulation in humans where they could also interact with circulating immune cells. This review aims to describe CRC-specific tEVs and what is currently known about their role in immunomodulation. In particular, we discuss the ability of CRC-derived tEVs to affect monocyte differentiation into macrophages and dendritic cells, and their effects on T-cell viability and activity. Finally, the potential for tEVs in the development of immunotherapies will be discussed.

Circulating MicroRNA-30d Is Associated With Response to Cardiac Resynchronization Therapy in Heart Failure and Regulates Cardiomyocyte Apoptosis: A Translational Pilot Study.

BACKGROUND: Biomarkers that predict response to cardiac resynchronization therapy (CRT) in heart failure patients with dyssynchrony (HFDYS) would be clinically important. Circulating extracellular microRNAs (miRNAs) have emerged as novel biomarkers that may also play important functional roles, but their relevance as markers for CRT response has not been examined. METHODS AND RESULTS: Comprehensive miRNA polymerase chain reaction arrays were used to assess baseline levels of 766 plasma miRNAs in patients undergoing clinically indicated CRT in an initial discovery set (n=12) with and without subsequent echocardiographic improvement at 6 months after CRT. Validation of candidate miRNAs in 61 additional patients confirmed that baseline plasma miR-30d was associated with CRT response (defined as an increase in left ventricular ejection fraction ≥10%). MiR-30d was enriched in coronary sinus blood and increased in late-contracting myocardium in a canine model of HFDYS, indicating cardiac origin with maximal expression in areas of high mechanical stress. We examined the functional effects of miR-30d in cultured cardiomyocytes and determined that miR-30d is expressed in cardiomyocytes and released in vesicles in response to mechanical stress. Overexpression of miR-30d in cultured cardiomyocytes led to cardiomyocyte growth and protected against apoptosis by targeting the mitogen-associated kinase 4, a downstream effector of tumor necrosis factor. In HFDYS patients, miR-30d plasma levels inversely correlated with high-sensitivity troponin T, a marker of myocardial necrosis. CONCLUSIONS: Baseline plasma miR-30d level is associated with response to CRT in HFDYS in this translational pilot study. MiR-30d increase in cardiomyocytes correlates with areas of increased wall stress in HFDYS and is protective against deleterious tumor necrosis factor signaling.

The effects of nicotine and tobacco particulate matter on dopamine uptake in the rat brain.

Cigarette smoking is the leading cause of preventable death worldwide. Recently, tobacco extracts have been shown to have a different pharmacological profile to nicotine alone and there is increasing evidence of a role for non-nicotinic components of cigarette smoke in smoking addiction. Nicotine is known to affect the uptake of dopamine in the brain of laboratory animals, but studies in the literature are often contradictory and little is known of the effects on non-nicotinic tobacco components on dopamine uptake. This study has examined the acute and chronic effects of nicotine and a tobacco extract (TPM) on dopamine uptake by the dopamine and norepinephrine transporters (DAT and NET) ex vivo using rotating disk electrode voltammetry, and quantified DAT and NET protein and mRNA expression in key brain regions. Nicotine (0.35 mg/kg) significantly decreased DAT function in the nucleus accumbens (NAc) at 30 min with no change in protein expression. This effect was sensitive to mecamylamine and DHßE but not MLA, indicating that it is dependent on α4 subunit containing nicotinic receptors. Furthermore, TPM, but not nicotine, increased DAT function in the dorsal striatum at 1 h in a nicotinic receptor independent manner with no change in DAT protein expression. At 1 h DAT mRNA in the ventral tegmental area was decreased by both acute and chronic TPM treatments.

Potential role of volatile organic compound breath testing in the Australasian colorectal cancer pathway.

With colonoscopy resources under pressure and inequitable participation rates in our screening programmes, there is an urgent need to consider trialling new testing technology for the detection of colorectal cancer (CRC) in Australasia. Research has shown that volatile organic compounds (VOCs) emitted from the human body can act as biomarkers for CRC, indicating high sensitivity and specificity for early and late-stage CRC and for adenomatous polyps. Breath-based VOC testing shows promise due to acceptability and ease of sampling via simple hand-held devices. Analysis can occur via mass spectrometry, or via 'e-nose' or sensor techniques. This review summarizes the current state of knowledge in using VOC-based testing for CRC. Adoption of this technique has the potential to improve CRC survival, reduce incidence and reduce colonoscopy burden in Australasia, and positively impact on ethnic disparities in cancer outcomes. Future multicentre trials should be conducted using standardized processes and protocols. This will ensure accuracy and reproducibility under different environmental and physiological conditions, and for different ethnic groups. Studies should be explicitly targeted to various points along the CRC patient pathway.

Adipose derived stem cell extracellular vesicles modulate primary human macrophages to an anti-inflammatory phenotype in vitro.

EVs released by adipose derived stem cells (ADSCs) have shown promise as a therapeutic for tissue repair because of their purported immune-regulatory properties. Extracellular vesicles (EVs) from ADSCs could be beneficial in improving graft retention rates for autologous fat grafting (AFG) post-mastectomy as, currently, grafted tissue rates are variable. Enriching grafted tissue with ADSC-EVs may improve retention rates by modulating macrophages resident within both the breast and lipoaspirate. We aimed to identify key macrophage phenotypes that are modulated by ADSC-EVs in vitro. ADSCs were isolated from lipoaspirates of women undergoing AFG and characterised by flow cytometry and differentiation potential. ADSC-EVs were isolated from culture media and characterised by tuneable resistive pulse sensing, transmission electron microscopy and Western blot. Primary monocyte-derived macrophages were polarized to an M1-like (GM-CSF, IFNγ), M2-like phenotype (M-CSF, IL-4) or maintained (M0-like; M-CSF) and ADSC-EVs were co-cultured with macrophages for 48 h. Flow cytometry and high-dimensional analysis clustered macrophages post co-culture. A manual gating strategy was generated to recapitulate these clusters and was applied to a repeat experimental run. Both runs were analysed to examine the prevalence of each cluster, representing a unique macrophage phenotype, with and without ADSC-EVs. Following the addition of ADSC-EVs, M0-like macrophages demonstrated a reciprocal shift of cell distribution from a cluster with a 'high inflammatory profile' (CD36+++CD206+++CD86+++; 16.5 ± 7.0%; p < 0.0001) to a cluster with a 'lower inflammatory profile' (CD36+CD206+CD86+; 35  ± 21.5%; p < 0.05). M1-like macrophages shifted from a cluster with a 'high inflammatory profile' (CD206++CD11b++CD36++CD163++; 26.1 ± 9.4%; p = 0.0024) to a 'lower inflammatory profile' (CD206+CD11b+CD36+CD163+; 72.8  ± 8.7%; p = 0.0007). There was no shift in M2-like clusters following ADSC-EV treatment. ADSC-EVs are complex regulators of macrophage phenotype that can shift macrophages away from a heightened pro-inflammatory state.

Recommendations for extracellular vesicle miRNA biomarker research in the endometrial cancer context.

Endometrial cancer (EC) is the most common gynaecological malignancy in the developed world, and concerningly incidence is rising, particularly in younger people. Therefore, there is increased interest in novel diagnostic and prognostic biomarkers. Extracellular vesicles (EVs) are membrane-bound particles present in bodily fluids that have the potential to facilitate non-invasive, early diagnosis of EC and could aid with monitoring of recurrence and treatment response. EV cargo provides molecular insight into the tumor, with the lipid bilayer providing stability for RNA species usually prone to degradation. miRNAs have recently become a focus for EV biomarker research due to their ability to regulate cancer related pathways and influence cancer development and progression. This review evaluates the current literature on EV miRNA biomarkers with a focus on EC, and discusses the challenges facing this research. This review finally highlights areas of focus for EV miRNA biomarker research going forward, such as standardization of normalization approaches, sample storage and processing, extensive reporting of methodologies and moving away from single miRNA biomarkers.

The effects of nicotine and cigarette smoke on the monoamine transporters.

Cigarette smoking is the leading cause of preventable illness worldwide; however, smoking addiction remains poorly understood and cessation therapies based on nicotine replacement have limited success. The monoamine transporters are the primary mechanism for regulating the levels of dopamine, serotonin and norepinephrine in the synapse, and have been implicated in addiction and associated behaviors. Furthermore, the non-nicotinic smoking cessation therapy bupropion acts at least in part by blocking the dopamine and norepinephrine transporters. Despite this, little work has been conducted into the effects of nicotine and cigarette smoke on the monoamine transporters. This review will outline research that has been conducted to date on cigarette smoke, nicotine and the monoamine transporters. This will include monoamine transporter regulation by nicotine and cigarette smoke, genetic associations of the transporters with smoking behavior, and the potential for monoamine transporters to be targets in the development of smoking cessation pharmacotherapies.

Characterisation of Levonorgestrel-Resistant Endometrial Cancer Cells.

BACKGROUND: Endometrial cancer (EC) is the most common gynaecologic malignancy in the developed world, and incidence is increasing in premenopausal women. The levonorgestrel intrauterine system (LNG-IUS) is gaining traction as an alternative treatment for hyperplasia and early-stage EC for women who are unable to undergo surgery. Thirty to 60% of the women do not respond to this treatment, making the unknown mechanisms of levonorgestrel (LNG) resistance a critical obstacle for the conservative management of EC. This study aimed to characterise LNG-IUS treatment resistance in early-stage endometrial cancer in cell-line models. METHODS: LNG-resistant endometrial cancer cell lines (MFE296R and MFE319R) and cultures from three early stage endometrial cancer patients were developed. The behavioural profile of MFE296R and MFE319R was analysed using proliferation, adhesion, migration (wound healing and transwell) and invasion (spheroid) assays. LNG-sensitive cell lines (MFE296S and MFE319S) were compared to LNGR cell lines (MFE296R and MFE319R). A literature search was conducted to identify possible candidate biomarkers of LNG resistance. RT-qPCR was used to analyse the mRNA expression of 17 candidate biomarkers in MFE296R and MFE319R. mRNA expression of the top differentially expressed genes was measured using RT-qPCR in primary cultures. RESULTS: LNG resistance did not affect proliferation or invasion in immortalised endometrial cancer cells. Transwell migration was significantly increased in MFE319R cells (p=0.03). Cellular adhesion significantly decreased in both MFE296R cells (p=0.012) and MFE319R cells (p=0.04). mRNA expression of KLF4 and SATB2 was significantly amplified in MFE296R and MFE319R cells. mRNA expression of KLF4 was significantly upregulated LNG-resistant primary cell lines. CONCLUSION: LNG-resistant cells may have more oncogenic potential than their LNG-sensitive counterparts. Significant changes in the mRNA expression of KLF4 and SATB2 of LNG-resistant cells is a promising preliminary result in biomarker discovery for guiding LNG-IUS treatment of early stage endometrial cancer.

Perceptions of competency with evidence-based medicine among medical students: changes through training and alignment with objective measures.

AIMS: To identify whether medical students' self-perception of competence with evidence-based medicine (EBM) increases throughout their senior years of medical training. Furthermore, to identify whether their self-perception aligns with their true competence measured using a validated tool. This investigation also outlines whether students report observation of and participation in the process of EBM in clinical practice. METHODS: A cross-sectional survey was undertaken with a convenience sample of medical students in their fourth, fifth and sixth years of training at one campus site of Otago Medical School between February and April 2018. Self-perceived competence with EBM was measured using a 10-item questionnaire. True competence was measured using the Assessing Competency in Evidence-Based Medicine (ACE) tool. Students were asked to self-report their observation of and participation in the process of EBM in clinical settings. RESULTS: Out of 99 students invited to participate, we received a response rate of 97%. Participants included 37 fourth-year, 32 fifth-year and 27 sixth-year students. Mean self-perceived EBM competence was higher in sixth-year compared to fourth-year students. True competence was not significantly different between year groups. Medical students reported little observation of EBM in clinical settings, and few students reported to have participated in the process of EBM during clinical encounters. CONCLUSION: The lack of explicit role modelling of EBM in clinical environments may be a barrier to students improving EBM competence in the senior years of medical training.

Efficacy of the LNG-IUS for treatment of endometrial hyperplasia and early stage endometrial cancer: Can biomarkers predict response?

Endometrial Cancer (EC) is the most common gynaecologic malignancy in the developed world, and is increasing in premenopausal women. The surgical standard of care for early-stage EC is not possible in women with concurrent comorbidities or women who desire a fertility sparing approach. The Levonorgestrel Intrauterine System (LNG-IUS) is gaining traction as an alternative treatment for endometrial hyperplasia and early stage EC in inoperable women. Whilst early evidence appears promising, predictive biomarkers need to be established to determine non-responders, which make up one in three women. This timely review discusses the current literature around the identification of clinical, molecular and novel biomarkers that show potential to predict response to progesterone treatment, including the LNG-IUS.

Brief report: Lymph node morphology in stage II colorectal cancer.

BACKGROUND: Colorectal cancer is one of the leading causes of cancer-associated morbidity and mortality worldwide. The local anti-tumour immune response is particularly important for patients with stage II where the tumour-draining lymph nodes have not yet succumbed to tumour spread. The lymph nodes allow for the expansion and release of B cell compartments such as primary follicles and germinal centres. A variation in this anti-tumour immune response may influence the observed clinical heterogeneity in stage II patients. AIM: The aim of this study was to explore tumour-draining lymph node histomorphological changes and tumour pathological risk factors including the immunomodulatory microRNA-21 (miR-21) in a small cohort of stage II CRC. METHODS: A total of 23 stage II colorectal cancer patients were included. Tumour and normal mucosa samples were analysed for miR-21 expression levels and B-cell compartments were quantified from Haematoxylin and Eosin slides of lymph nodes. These measures were compared to clinicopathological risk factors such as perforation, bowel obstruction, T4 stage and high-grade. RESULTS: We observed greater Follicle density in patients with a lower tumour T stage and higher germinal centre density in patients with higher pre-operative carcinoembryonic antigen levels. Trends were also detected between tumours with deficiency in mismatch repair proteins, lymphatic invasion and both the density and size of B-cell compartments. Lastly, elevated tumour miR-21 was associated with decreased Follicle and germinal centre size. CONCLUSION: Variation in B-cell compartments of tumour-draining lymph nodes is associated with clinicopathological risk factors in stage II CRC patients.

MicroRNA Profiling in Adipose Before and After Weight Loss Highlights the Role of miR-223-3p and the NLRP3 Inflammasome.

OBJECTIVE: Adipose tissue plays a key role in obesity-related metabolic dysfunction. MicroRNA (miRNA) are gene regulatory molecules involved in intercellular and inter-organ communication. It was hypothesized that miRNA levels in adipose tissue would change after gastric bypass surgery and that this would provide insights into their role in obesity-induced metabolic dysregulation. METHODS: miRNA profiling (Affymetrix GeneChip miRNA 2.0 Array) of omental and subcutaneous adipose (n = 15 females) before and after gastric bypass surgery was performed. RESULTS: One omental and thirteen subcutaneous adipose miRNAs were significantly differentially expressed after gastric bypass, including downregulation of miR-223-3p and its antisense relative miR-223-5p in both adipose tissues. mRNA levels of miR-223-3p targets NLRP3 and GLUT4 were decreased and increased, respectively, following gastric bypass in both adipose tissues. Significantly more NLRP3 protein was observed in omental adipose after gastric bypass (P = 0.02). Significant hypomethlyation of NLRP3 and hypermethylation of miR-223 were observed in both adipose tissues after gastric bypass. In subcutaneous adipose, significant correlations were observed between both miR-223-3p and miR-223-5p and glucose and between NLRP3 mRNA and protein levels and blood lipids. CONCLUSIONS: This is the first report detailing genome-wide miRNA profiling of omental adipose before and after gastric bypass, and it further highlights the association of miR-223-3p and the NLRP3 inflammasome with obesity.

Circulating Extracellular Vesicle MicroRNA as Diagnostic Biomarkers in Early Colorectal Cancer-A Review.

Colorectal cancer (CRC) is one of the most common malignancies in the developed world, with global deaths expected to double in the next decade. Disease stage at diagnosis is the single greatest prognostic indicator for long-term survival. Unfortunately, early stage CRC is often asymptomatic and diagnosis frequently occurs at an advanced stage, where long-term survival can be as low as 14%. Circulating microRNAs encapsulated in extracellular vesicles (EVs) have recently come to prominence as novel diagnostic markers for cancer. EV-miRNAs are dysregulated in the circulation of CRC patients compared to healthy controls, and several specific miRNA candidates have been posited as diagnostic markers, including miR-21, miR-23a, miR-1246, and miR-92a. This review outlines the current landscape of EV-miRNAs as potential diagnostic markers for CRC, with a specific focus on those able to detect early stage disease.

Extracellular RNA Isolation from Cell Culture Supernatant.

Extracellular RNAs are emerging as novel biomarkers and mediators of intercellular communication. Various methods to isolate RNA from biofluids and cell culture supernatants have been previously used by investigators. Here, we describe several standardized protocols for the isolation of RNAs from cell culture supernatants that utilize commercially available kits and reagents.

Plasma Circulating Extracellular RNAs in Left Ventricular Remodeling Post-Myocardial Infarction.

Despite substantial declines in mortality following myocardial infarction (MI), subsequent left ventricular remodeling (LVRm) remains a significant long-term complication. Extracellular small non-coding RNAs (exRNAs) have been associated with cardiac inflammation and fibrosis and we hypothesized that they are associated with post-MI LVRm phenotypes. RNA sequencing of exRNAs was performed on plasma samples from patients with "beneficial" (decrease LVESVI ≥ 20%, n = 11) and "adverse" (increase LVESVI ≥ 15%, n = 11) LVRm. Selected differentially expressed exRNAs were validated by RT-qPCR (n = 331) and analyzed for their association with LVRm determined by cardiac MRI. Principal components of exRNAs were associated with LVRm phenotypes post-MI; specifically, LV mass, LV ejection fraction, LV end systolic volume index, and fibrosis. We then investigated the temporal regulation and cellular origin of exRNAs in murine and cell models and found that: 1) plasma and tissue miRNA expression was temporally regulated; 2) the majority of the miRNAs were increased acutely in tissue and at sub-acute or chronic time-points in plasma; 3) miRNA expression was cell-specific; and 4) cardiomyocytes release a subset of the identified miRNAs packaged in exosomes into culture media in response to hypoxia/reoxygenation. In conclusion, we find that plasma exRNAs are temporally regulated and are associated with measures of post-MI LVRm.