Gene Regulatory Networks in Coronary Artery Disease.

PURPOSE OF REVIEW: Coronary artery disease is a complex disorder and the leading cause of mortality worldwide. As technologies for the generation of high-throughput multiomics data have advanced, gene regulatory network modeling has become an increasingly powerful tool in understanding coronary artery disease. This review summarizes recent and novel gene regulatory network tools for bulk tissue and single cell data, existing databases for network construction, and applications of gene regulatory networks in coronary artery disease. RECENT FINDINGS: New gene regulatory network tools can integrate multiomics data to elucidate complex disease mechanisms at unprecedented cellular and spatial resolutions. At the same time, updates to coronary artery disease expression data in existing databases have enabled researchers to build gene regulatory networks to study novel disease mechanisms. Gene regulatory networks have proven extremely useful in understanding CAD heritability beyond what is explained by GWAS loci and in identifying mechanisms and key driver genes underlying disease onset and progression. Gene regulatory networks can holistically and comprehensively address the complex nature of coronary artery disease. In this review, we discuss key algorithmic approaches to construct gene regulatory networks and highlight state-of-the-art methods that model specific modes of gene regulation. We also explore recent applications of these tools in coronary artery disease patient data repositories to understand disease heritability and shared and distinct disease mechanisms and key driver genes across tissues, between sexes, and between species.

Simultaneous quantitation of urine aldosterone and tetrahydroaldosterone in healthy Chinese subjects using a validated LC-MS/MS method.

Aldosterone (ALD) is excreted in urine mainly as glucuronide conjugates of ALD and tetrahydroaldosterone. Measuring these urinary metabolites might be an alternative screening test to plasma ALD for primary aldosteronism. We report a validated LC-MS/MS method to measure both analytes simultaneously. Urine samples underwent enzymatic hydrolysis to release the analytes from their glucuronide conjugates followed by organic solvent extraction and LC-MS/MS. The analytical performance of this method was evaluated. The within-batch and between-batch coefficients of variation for urine ALD and urine THA were all ≤5.2 and ≤3.7%. The lower limit of quantification was 0.5 nmol/L, and the linearity was up to at least 2770 nmol/L for both analytes. No significant matrix interference and carryover were observed. Both analytes in urine were stable for at least 48 h at 10°C and at least 18 months at -80°C. Local reference intervals were established from 126 healthy normotensive Chinese subjects (53% women, age: 20-65 years). Reference intervals for urine ALD and tetrahydroaldosterone were 2-38 and 9-139 nmol/day, respectively. This validated method can be applied to screening and diagnosing primary aldosteronism.

Chinese normotensive and essential hypertensive reference intervals for plasma aldosterone and renin activity by liquid chromatography-tandem mass spectrometry.

OBJECTIVES: The renin-angiotensin-aldosterone system (RAAS) regulates blood pressure. Plasma renin activities (PRA) and plasma aldosterone concentrations (PAC) are biomarkers related to RAAS. Liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based measurements for PRA and PAC have become popular. Method-specific reference intervals (RIs) are required. METHODS: Routine PRA and PAC services in a Hong Kong teaching hospital were based on LC-MS/MS methods. PRA and PAC RIs were developed for normotensive subjects and essential hypertensive (EH) patients. Healthy volunteers were recruited to establish normotensive RIs. PRA and PAC results of hypertensive patients with urine aldosterone tests for primary aldosteronism (PA) screening were retrieved from the laboratory information system. Patients without PA were included. Patients with secondary hypertension and patients on medications affecting the RAAS were excluded. The central 95% RIs were established based on the recommendations of the Clinical and Laboratory Standards Institute guideline C28-A3. RESULTS: PRA and PAC of 170 normotensive volunteers and 362 EH patients were analysed. There was no sex-specific difference in PRA and PAC for normotensive and EH reference subjects. Differences for PRA and PAC were noted between normotensive subjects aged below 45 and their older counterparts. However, such a difference was only identified for PRA but not PAC in EH patients. Age-specific RIs were established accordingly. CONCLUSIONS: This study presented age-specific LC-MS/MS RIs of PRA and PAC for both normotensive and EH populations for local Chinese in Hong Kong.

Can the same controlled attenuation parameter cut-offs be used for M and XL probes for diagnosing hepatic steatosis?

BACKGROUND AND AIM: There are limited studies on controlled attenuation parameter (CAP) using Fibroscan XL probe for the diagnosis of hepatic steatosis grade. The aim of this study was to determine whether previously defined optimal cut-offs for CAP using the M probe could be applied for the XL probe. METHODS: Adult patients with chronic liver disease who had a liver biopsy and examination with both the M and XL probes were included. Previously defined optimal cut-offs for CAP using the M probe were used for the diagnosis of steatosis grades ≥S1, ≥S2, and S3 (248, 268, and 280 dB/m, respectively). RESULTS: Data for 180 patients were analyzed (mean age 53.7 ± 10.8 years; central obesity 84.5%; non-alcoholic fatty liver disease 86.7%). The distribution of steatosis grades was S0, 9.4%; S1, 28.3%; S2, 43.9%, and S3, 18.3%. The sensitivity, specificity, positive predictive value, and negative predictive value of CAP using the M/XL probe for the diagnosis of steatosis grade ≥S1 was 93.9%/93.3%, 58.8%/58.8%, 95.6%/95.6%, and 50.0%/47.6%, respectively. These values were 94.6%/94.6%, 41.2%/44.1%, 72.6%/73.6%, and 82.4%/83.3%, respectively, for ≥S2, and 87.9%/87.9%, 27.2%/27.9%, 21.3%/21.5%, and 90.9%/91.1%, respectively, for S3. CONCLUSION: The same cut-off values for CAP may be used for the M and XL probes for the diagnosis of hepatic steatosis grade.

Metabolic syndrome increases cardiovascular events but not hepatic events and death in patients with chronic hepatitis B.

Metabolic syndrome is an independent risk factor of liver cirrhosis in chronic hepatitis B (CHB). Whether metabolic syndrome affects the long-term prognosis of CHB patients in terms of hepatic events, cardiovascular events, and death remains unknown. We aimed to determine the incidences of hepatic events, cardiovascular events, and death in CHB patients with or without metabolic syndrome. From 2006 to 2008, we prospectively recruited 1,466 CHB patients for liver stiffness measurement (LSM) with transient elastography together with detailed metabolic profiling as baseline assessment. Patients were prospectively followed for any clinical events. The impact of LSM and metabolic syndrome on hepatic events, cardiovascular events, and death was evaluated. At baseline visit, the mean age was 46 ± 12 years, LSM value was 8.4 ± 6.3 kPa, and 188 patients (12.8%) had metabolic syndrome. At a mean follow-up of 88 ± 20 months, 93 and 44 patients developed hepatic and cardiovascular events, respectively; 70 patients died. Patients with baseline LSM >8.0 kPa had higher cumulative probability of hepatic events than those with LSM ≤8.0kPa at 8 years (12.3% versus 3.1%, P < 0.001). Patients with metabolic syndrome had higher cumulative probability of cardiovascular events than those without (8.0% versus 2.1%, P < 0.001). High LSM had no impact on cardiovascular events; neither did metabolic syndrome on hepatic events. LSM >8.0 kPa but not metabolic syndrome was an independent risk factor of death, with adjusted hazard ratios of 1.9 (95% confidence interval 1.1-3.2, P = 0.023) and 1.3 (95% confidence interval 0.8-2.4, P = 0.310), respectively. CONCLUSIONS: Metabolic syndrome increased the risk of cardiovascular events but not hepatic events and death; LSM was the important risk factor of hepatic events and death in CHB patients. (Hepatology 2016;64:1507-1517).

Hepatitis C virus induced up-regulation of microRNA-27: a novel mechanism for hepatic steatosis.

UNLABELLED: MicroRNAs (miRNAs) are small RNAs that posttranscriptionally regulate gene expression. Their aberrant expression is commonly linked with diseased states, including hepatitis C virus (HCV) infection. Herein, we demonstrate that HCV replication induces the expression of miR-27 in cell culture and in vivo HCV infectious models. Overexpression of the HCV proteins core and NS4B independently activates miR-27 expression. Furthermore, we establish that miR-27 overexpression in hepatocytes results in larger and more abundant lipid droplets, as observed by coherent anti-Stokes Raman scattering (CARS) microscopy. This hepatic lipid droplet accumulation coincides with miR-27b's repression of peroxisome proliferator-activated receptor (PPAR)-α and angiopoietin-like protein 3 (ANGPTL3), known regulators of triglyceride homeostasis. We further demonstrate that treatment with a PPAR-α agonist, bezafibrate, is able to reverse the miR-27b-induced lipid accumulation in Huh7 cells. This miR-27b-mediated repression of PPAR-α signaling represents a novel mechanism of HCV-induced hepatic steatosis. This link was further demonstrated in vivo through the correlation between miR-27b expression levels and hepatic lipid accumulation in HCV-infected SCID-beige/Alb-uPa mice. CONCLUSION: Collectively, our results highlight HCV's up-regulation of miR-27 expression as a novel mechanism contributing to the development of hepatic steatosis.

Population Pharmacokinetic Modeling of Cotadutide: A Dual Agonist Peptide of Glucagon-Like Peptide and Glucagon Receptors Administered to Participants with Type II Diabetes Mellitus, Chronic Kidney Disease, Obesity and Non-Alcoholic Steatohepatitis.

BACKGROUND: Cotadutide is a dual glucagon-like peptide-1 (GLP-1) and glucagon (GCG) receptor agonist peptide. The objective of this analysis was to develop a population pharmacokinetic (popPK) model of cotadutide, and to identify any potential effect on the PK from intrinsic and extrinsic covariates. METHODS: The popPK analysis utilized a non-linear mixed-effects modeling approach using the data from 10 clinical studies in different participant categories following once-daily subcutaneous dose administration ranging from 20 to 600 µg. Additionally, the covariates affecting cotadutide exposure were quantified, and the model performance was evaluated through the prediction-corrected visual predictive checks. RESULTS: A one-compartment model with first-order absorption and elimination adequately described the data as confirmed via visual predictive check plots and parameter plausibility. The mean values for cotadutide apparent clearance (CL/F), apparent volume of distribution (V/F), absorption rate constant (Ka), and half-life were 1.05 L/h, 20.0 L, 0.38 h-1, and 13.3 hours, respectively. Covariate modeling identified body weight, alanine transaminase, albumin, anti-drug antibody (ADA) titer values, formulation strength and injection device, and participant categories as significant covariates on PK parameters, where ADAs have been identified to decrease cotadutide clearance. The model demonstrated that a 150-kg participant was estimated to have 30% lower for both AUC and Cmax and a 66 kg participant was estimated to have 35% higher for both AUC and Cmax relative to a reference individual with a median weight of 96 kg. CONCLUSIONS: A popPK model was developed for cotadutide with cotadutide clinical data, and the impact of the statistically significant covariates identified was not considered clinically meaningful. The popPK model will be used to evaluate exposure-response relationships for cotadutide clinical data.

Dopamine infusion at typical infusion rates does not cause interference on plasma creatinine assays.

a Objectives: Dopamine is known to cause negative interference on enzymatic creatinine measurement. However, its effect on the Jaffe reaction, and its concentration required to interfere with enzymatic reactions, remain uncertain. This study was designed to study the interference of stable dopamine infusion on Jaffe and enzymatic creatinine assays, as well as the effect of dopamine infusion drip arm contamination on both creatinine assays. b Design and Methods: For the first part of the study, dopamine was spiked into pooled plasma samples at different concentrations to mimic the scenario of patients on dopamine infusion at an infusion rate between 2 and 20 µg/kg/min. For the second part, dopamine preparation of 2 g/L (same as the preparation used clinically) was mixed with pooled plasma samples at different proportions to mimic drip arm contamination. Creatinine concentrations were measured using Jaffe and enzymatic reactions. c Results: The first part showed that creatinine measurements were not interfered by dopamine infusion at an infusion rate between 2 and 20 µg/kg/min. The second part showed that dopamine could negatively interfere with enzymatic creatinine assays, even with minute drip arm contamination. The effect on the Jaffe reaction was less significant. d Discussion: Creatinine concentration could be reliably measured by Jaffe or enzymatic reactions if samples are from venous access sites other than the site of dopamine infusion. When dopamine interference on enzymatic creatinine assays is suspected, using the Jaffe reaction to cross-check may provide additional useful information.

THRB Gene Mosaicism Confirmed by Next-Generation Sequencing in a Clinically Symptomatic Infant.

A 4-day-old infant was admitted for neonatal jaundice. He had persistent tachycardia and tachypnea. Initial workup showed a serum free T4 of 75.6 pmol/L (5.87 ng/dL) (reference range: 11.5-28.3 pmol/L; 0.89-2.20 ng/dL) and a nonsuppressed TSH 3.76 mIU/L (reference range: 0.72-11.0 mIU/L). A TRH stimulation test showed an exaggerated TSH response with a peak of 92.1 mIU/L at 30 minutes after TRH injection, which suggested the diagnosis of resistance to thyroid hormone ß syndrome. Sanger sequencing showed a questionable pathogenic variant in the THRB gene with low signal amplitude. Restriction fragment length polymorphism was consistent with its presence. The variant was originally reported as heterozygous. Next-generation sequencing was performed on blood and buccal swab samples of the patient and his parents, which confirmed this de novo mosaic variant NM_000461.5:c.1352T > C p.(Phe451Ser) in the patient but not in his asymptomatic parents. As it was in a mosaic state, only the offspring, but not other first-degree relatives, of the patient would have the risk of inheriting that variant.

Thyroid hormone T4 mitigates traumatic brain injury in mice by dynamically remodeling cell type specific genes, pathways, and networks in hippocampus and frontal cortex.

The complex pathology of mild traumatic brain injury (mTBI) is a main contributor to the difficulties in achieving a successful therapeutic regimen. Thyroxine (T4) administration has been shown to prevent the cognitive impairments induced by mTBI in mice but the mechanism is poorly understood. To understand the underlying mechanism, we carried out a single cell transcriptomic study to investigate the spatiotemporal effects of T4 on individual cell types in the hippocampus and frontal cortex at three post-injury stages in a mouse model of mTBI. We found that T4 treatment altered the proportions and transcriptomes of numerous cell types across tissues and timepoints, particularly oligodendrocytes, astrocytes, and microglia, which are crucial for injury repair. T4 also reversed the expression of mTBI-affected genes such as Ttr, mt-Rnr2, Ggn12, Malat1, Gnaq, and Myo3a, as well as numerous pathways such as cell/energy/iron metabolism, immune response, nervous system, and cytoskeleton-related pathways. Cell-type specific network modeling revealed that T4 mitigated select mTBI-perturbed dynamic shifts in subnetworks related to cell cycle, stress response, and RNA processing in oligodendrocytes. Cross cell-type ligand-receptor networks revealed the roles of App, Hmgb1, Fn1, and Tnf in mTBI, with the latter two ligands having been previously identified as TBI network hubs. mTBI and/or T4 signature genes were enriched for human genome-wide association study (GWAS) candidate genes for cognitive, psychiatric and neurodegenerative disorders related to mTBI. Our systems-level single cell analysis elucidated the temporal and spatial dynamic reprogramming of cell-type specific genes, pathways, and networks, as well as cell-cell communications as the mechanisms through which T4 mitigates cognitive dysfunction induced by mTBI.

Three-mode electrochemical sensing of ultralow microRNA levels.

MicroRNAs (miRNAs) are an emerging class of biomarkers that are frequently deregulated in cancer cells and have shown great promise for cancer classification and prognosis. In this work, we developed a three-mode electrochemical sensor for detection and quantitation of ultralow levels of miRNAs in a wide dynamic range of measured concentrations. The sensor facilitates three detection modalities based on hybridization (H-SENS), p19 protein binding (P-SENS), and protein displacement (D-SENS). The combined three-mode sensor (HPD-SENS) identifies as low as 5 aM or 90 molecules of miRNA per 30 µL of sample without PCR amplification, and can be operated within the dynamic range from 10 aM to 1 µM. The HPD sensor is made on a commercially available gold nanoparticles-modified electrode and is suitable for analyzing multiple miRNAs on a single electrode. This three-mode sensor exhibits high selectivity and specificity and was used for sequential analysis of miR-32 and miR-122 on one electrode. In addition, the H-SENS can recognize miRNAs with different A/U and G/C content and distinguish between a fully matched miRNA and a miRNA comprising either a terminal or a middle single base mutation. Furthermore, the H- and P-SENS were successfully employed for direct detection and profiling of three endogenous miRNAs, including hsa-miR-21, hsa-miR-32, and hsa-miR-122 in human serum, and the sensor results were validated by qPCR.

Metabolic memory of Δ9-tetrahydrocannabinol exposure in pluripotent stem cells and primordial germ cells-like cells.

Cannabis, the most consumed illicit psychoactive drug in the world, is increasingly used by pregnant women. However, while cannabinoid receptors are expressed in the early embryo, the impact of phytocannabinoids exposure on early embryonic processes is lacking. Here, we leverage a stepwise in vitro differentiation system that captures early embryonic developmental cascade to investigate the impact of exposure to the most abundant phytocannabinoid, Δ9-tetrahydrocannabinol (Δ9-THC). We demonstrate that Δ9-THC increases the proliferation of naïve mouse embryonic stem cells (ESCs) but not of their primed counterpart. Surprisingly, this increased proliferation, dependent on the CB1 receptor binding, is only associated with moderate transcriptomic changes. Instead, Δ9-THC capitalizes on ESCs' metabolic bivalence by increasing their glycolytic rates and anabolic capabilities. A memory of this metabolic rewiring is retained throughout differentiation to Primordial Germ Cell-Like Cells in the absence of direct exposure and is associated with an alteration of their transcriptional profile. These results represent the first in-depth molecular characterization of the impact of Δ9-THC exposure on early stages of germline development.

Metabolic memory of Δ9-tetrahydrocannabinol exposure in pluripotent stem cells and primordial germ cells-like cells.

Cannabis, the most consumed illicit psychoactive drug in the world, is increasingly used by pregnant women. However, while cannabinoid receptors are expressed in the early embryo, the impact of phytocannabinoids exposure on early embryonic processes is lacking. Here, we leverage a stepwise in vitro differentiation system that captures the early embryonic developmental cascade to investigate the impact of exposure to the most abundant phytocannabinoid, Δ9-tetrahydrocannabinol (Δ9-THC). We demonstrate that Δ9-THC increases the proliferation of naive mouse embryonic stem cells (ESCs) but not of their primed counterpart. Surprisingly, this increased proliferation, dependent on the CB1 receptor binding, is only associated with moderate transcriptomic changes. Instead, Δ9-THC capitalizes on ESCs' metabolic bivalence by increasing their glycolytic rates and anabolic capabilities. A memory of this metabolic rewiring is retained throughout differentiation to Primordial Germ Cell-Like Cells in the absence of direct exposure and is associated with an alteration of their transcriptional profile. These results represent the first in-depth molecular characterization of the impact of Δ9-THC exposure on early stages of germline development.

Cannabis is the most widely used illicit drug in the world, with 4.3% of the global adult population estimated to have used it in the previous year. In particular, the consumption of cannabis by pregnant women has almost doubled in recent years and is particularly increased in those aged under 18. The main psychoactive component in cannabis, known as Δ9-THC, activates cannabinoid receptors in the brain, including the receptor CB1. Recent research has shown that CB1 is also active in the mouse embryo, but it remained unclear if Δ9-THC could also affect the development of an embryo. To better understand the potential effects of this exposure, scientists can study stem cells that develop into germ cells (which go on to form egg and sperm), which have been grown in the laboratory. Emerging research has shown that germ cells are particularly sensitive to changes in their environment and due to their role in reproduction, changes can have knock-on effects for embryos. Verdikt et al. studied the effects of Δ9-THC on mouse embryonic stem cells, finding that it caused them to multiply more quickly. This was dependent on both Δ9-THC binding to the CB1 receptor that causes the psychoactive effects of cannabis in the brain and an increased energy metabolism. Blocking an important metabolic pathway called glycolysis caused the Δ9-THC-treated cells to return to a normal multiplication rate. The exposed stem cells also gave rise to germ cells with abnormal metabolism and altered gene expression, suggesting that this metabolic 'memory' can be passed on to cells in the next developmental stage. Overall, the findings indicate that exposure to Δ9-THC alters the metabolism in early embryonic cells of mice and that these effects can be lasting. This emphasises the need for further research on the impact of cannabis use during pregnancy, particularly as the drug's availability is expected to increase significantly with changes in regulation. The work also contributes to research highlighting the inheritance of metabolism.

Shared and distinct pathways and networks genetically linked to coronary artery disease between human and mouse.

Mouse models have been used extensively to study human coronary artery disease (CAD) or atherosclerosis and to test therapeutic targets. However, whether mouse and human share similar genetic factors and pathogenic mechanisms of atherosclerosis has not been thoroughly investigated in a data-driven manner. We conducted a cross-species comparison study to better understand atherosclerosis pathogenesis between species by leveraging multiomics data. Specifically, we compared genetically driven and thus CAD-causal gene networks and pathways, by using human GWAS of CAD from the CARDIoGRAMplusC4D consortium and mouse GWAS of atherosclerosis from the Hybrid Mouse Diversity Panel (HMDP) followed by integration with functional multiomics human (STARNET and GTEx) and mouse (HMDP) databases. We found that mouse and human shared >75% of CAD causal pathways. Based on network topology, we then predicted key regulatory genes for both the shared pathways and species-specific pathways, which were further validated through the use of single cell data and the latest CAD GWAS. In sum, our results should serve as a much-needed guidance for which human CAD-causal pathways can or cannot be further evaluated for novel CAD therapies using mouse models.

Shared and distinct pathways and networks genetically linked to coronary artery disease between human and mouse.

Mouse models have been used extensively to study human coronary artery disease (CAD) or atherosclerosis and to test therapeutic targets. However, whether mouse and human share similar genetic factors and pathogenic mechanisms of atherosclerosis has not been thoroughly investigated in a data-driven manner. We conducted a cross-species comparison study to better understand atherosclerosis pathogenesis between species by leveraging multiomics data. Specifically, we compared genetically driven and thus CAD-causal gene networks and pathways, by using human GWAS of CAD from the CARDIoGRAMplusC4D consortium and mouse GWAS of atherosclerosis from the Hybrid Mouse Diversity Panel (HMDP) followed by integration with functional multiomics human (STARNET and GTEx) and mouse (HMDP) databases. We found that mouse and human shared >75% of CAD causal pathways. Based on network topology, we then predicted key regulatory genes for both the shared pathways and species-specific pathways, which were further validated through the use of single cell data and the latest CAD GWAS. In sum, our results should serve as a much-needed guidance for which human CAD-causal pathways can or cannot be further evaluated for novel CAD therapies using mouse models.

Genome-wide association meta-analysis of spontaneous coronary artery dissection identifies risk variants and genes related to artery integrity and tissue-mediated coagulation.

Spontaneous coronary artery dissection (SCAD) is an understudied cause of myocardial infarction primarily affecting women. It is not known to what extent SCAD is genetically distinct from other cardiovascular diseases, including atherosclerotic coronary artery disease (CAD). Here we present a genome-wide association meta-analysis (1,917 cases and 9,292 controls) identifying 16 risk loci for SCAD. Integrative functional annotations prioritized genes that are likely to be regulated in vascular smooth muscle cells and artery fibroblasts and implicated in extracellular matrix biology. One locus containing the tissue factor gene F3, which is involved in blood coagulation cascade initiation, appears to be specific for SCAD risk. Several associated variants have diametrically opposite associations with CAD, suggesting that shared biological processes contribute to both diseases, but through different mechanisms. We also infer a causal role for high blood pressure in SCAD. Our findings provide novel pathophysiological insights involving arterial integrity and tissue-mediated coagulation in SCAD and set the stage for future specific therapeutics and preventions.

GCK-MODY in pregnancy: A pregnant woman with diabetes and a small-for-gestational-age fetus.

Glucokinase-maturity-onset diabetes of the young (GCK-MODY) is often misdiagnosed as other forms of diabetes. A 42-year-old pregnant lady with pre-existing diabetes was treated with insulin during first trimester. Fetal growth restriction was noted since mid-second trimester. Genetic testing suggested the diagnosis of GCK-MODY.

Enhanced specificity of the viral suppressor of RNA silencing protein p19 toward sequestering of human microRNA-122.

Tombusviruses express a 19 kDa protein (p19) that, as a dimeric protein, suppresses the RNAs silencing pathway during infection by binding short-interfering RNA (siRNA) and preventing their association with the RNA-induced silencing complex (RISC). The p19 protein can bind to both endogenous and synthetic siRNAs with a high degree of size selectivity but with little sequence dependence. It also binds to other endogenous small RNAs such as microRNAs (miRNAs) but with lower affinity than to canonical siRNAs. It has become apparent, however, that miRNAs play a large role in gene regulation; their influence extends to expression and processing that affects virtually all eukaryotic processes. In order to develop new tools to study endogenous small RNAs, proteins that suppress specific miRNAs are required. Herein we describe mutational analysis of the p19 binding surface with the aim of creating p19 mutants with increased affinity for miR-122. By site-directed mutagenesis of a single residue, we describe p19 mutants with a nearly 50-fold increased affinity for miR-122 without altering the affinity for siRNA. Upon further mutational analysis of this site, we postulate that the higher affinity relies on hydrogen-bonding interactions but can be sterically hindered by residues with bulky side chains. Finally, we demonstrate the effectiveness of a mutant p19, p19-T111S, at sequestering miR-122 in human hepatoma cell lines, as compared to wild-type p19. Overall, our results suggest that p19 can be engineered to enhance its affinity toward specific small RNA molecules, particularly noncanonical miRNAs that are distinguishable based on locations of base-pair mismatches. The p19-T111S mutant also represents a new tool for the study of the function of miR-122 in post-transcriptional silencing in the human liver.

Quantitative analysis of microRNA in blood serum with protein-facilitated affinity capillary electrophoresis.

MicroRNAs (miRNAs) are small (∼22 nt) regulatory RNAs that are frequently deregulated in cancer and have shown promise as tissue- and blood-based biomarkers for cancer classification and prognostication. Here we present a protein-facilitated affinity capillary electrophoresis (ProFACE) assay for rapid quantification of miRNA levels in blood serum using single-stranded DNA binding protein (SSB) and double-stranded RNA binding protein (p19) as separation enhancers. The method utilizes either the selective binding of SSB to a single-stranded DNA/RNA probe or the binding of p19 to miRNA-RNA probe duplex. For the detection of ultralow amounts of miRNA without polymerase chain reaction (PCR) amplification in blood samples we apply off-line preconcentration of synthetic miRNA-122 from serum by p19-coated magnetic beads followed by online sample stacking in the ProFACE assay. The detection limit is 0.5 fM or 30 000 miRNA molecules in 1 mL of serum as a potential source of naïve miRNAs.