Nicholas J. Hand

Circulating microRNA is a biomarker of pediatric Crohn disease.

Objective: The gold standard for the diagnosis and evaluation of Crohn disease (CD) is endoscopy/colonoscopy, although this is invasive, costly, and associated with risks to the patient. Recently, circulating microRNAs (miRNAs) have emerged as promising noninvasive biomarkers. Here, we examined the utility of serum miRNAs as biomarkers of CD in children. Patients and Methods: Studies were conducted using sera samples from patients with pediatric CD, healthy controls, and a comparison group of patients with pediatric celiac disease. Serum miRNA levels were explored initially using a microfluidic quantitative reverse transcription-polymerase chain reaction array platform. Findings were subsequently validated using quantitative reverse transcription-polymerase chain reaction in larger validation sample sets. The diagnostic utility of CD-associated serum miRNA was examined using receiver operating characteristic analysis. Results: A survey of miRNA levels in the sera of control and patients with CD detected significant elevation of 24 miRNAs, 11 of which were chosen for further validation. All of the candidate biomarker miRNAs were confirmed in an independent CD sample set (n = 46). To explore the specificity of the CD-associated miRNAs, they were measured in the sera of patients with celiac disease (n = 12); none were changed compared with healthy controls. Receiver operating characteristic analyses revealed that serum miRNAs have promising diagnostic utility, with sensitivities for CD above 80%. Significant decreases in serum miRNAs were observed in 24 incident patients with pediatric CD after 6 months of treatment. Conclusions: The present study identifies 11 CD-associated serum miRNA with encouraging diagnostic potential. Our findings suggest serum miRNAs may prove useful as noninvasive biomarkers in CD.

The microRNA-30 family is required for vertebrate hepatobiliary development

BACKGROUND & AIMS The function of microRNA (miRNA) in liver development is unknown. To address this issue, we characterized miRNA expression in the embryonic mouse liver, performed functional miRNA analysis in zebrafish larvae, and identified novel hepatic miRNA targets. METHODS Hepatic RNA isolated from mice at embryonic days 15.5, 18.5, and postnatal day 2 was hybridized to a mouse miRNA microarray. The microarray results were confirmed by Northern blot hybridization and quantitative reverse-transcription polymerase chain reaction. The spatial distribution of selected miRNAs was determined by in situ hybridization. Functional analysis of miR-30a was performed in zebrafish using antisense-mediated miRNA knockdown. Targets of miR-30a were identified by microarray analysis of gene expression following knockdown in cultured cells. RESULTS A set of 38 differentially expressed fetal hepatic miRNAs was identified. Several of these miRNAs were found to exhibit distinct temporal and spatial patterns of expression in hepatocytes, cholangiocytes, and nonepithelial cells within the liver. Two (miR-30a and miR-30c) are the first examples of ductal plate and bile duct-specific hepatic miRNAs. Knockdown of miR-30a in the zebrafish larva results in defective biliary morphogenesis. Several newly identified targets of miR-30a are known regulators of liver development and function. CONCLUSIONS We have identified miRNAs whose spatial and temporal patterns of expression are suggestive of functional roles in hepatic development and/or function. One of these, the biliary miRNA miR-30a, is required for biliary development in zebrafish. This is the first demonstration of a functional role for miRNA in hepatic organogenesis.

Hepatic function is preserved in the absence of mature microRNAs

MicroRNAs (miRNAs) are small noncoding RNA molecules that regulate gene expression through partial or complete complementarity with target messenger RNAs. The function of miRNAs in normal liver physiology is largely unknown. We address the role of Dicer1 in the differentiated liver. We derived mice lacking Dicer1 function in hepatocytes and assessed the loss of mature miRNA via quantitative polymerase chain reaction. Gene expression microarray analysis was performed on liver RNA from mutant and control mice. Liver sections from mutant and control mice were examined and liver function tests were performed. Mice lacking Dicer1 function in hepatocytes appeared and behaved normally. Despite the loss of mature miRNAs, hepatic function was maintained, as reflected by normal blood glucose, albumin, cholesterol, and bilirubin. However, mutant mice between 2 and 4 months of age exhibited progressive hepatocyte damage with elevated serum alanine aminotransferase and aspartate aminotransferase. Liver mass was increased in mutant mice, as were cellular markers of both proliferation and apoptosis. Microarray analysis indicated large‐scale changes in gene expression, with increased expression of many miRNA targets, particularly imprinted genes. Conclusions: Loss of miRNA processing in the liver at late gestation has a remarkably mild phenotype, suggesting that miRNAs do not play an essential role in hepatic function. However, miRNA deficiency results in hepatocyte apoptosis, hepatocyte regeneration, and portal inflammation. Finally, microarray analysis of gene expression in the mutant liver supports a previously hypothesized role for Dicer1 in the repression of imprinted genes. (HEPATOLOGY 2008.)

MicroRNA profiling identifies miR-29 as a regulator of disease-associated pathways in experimental biliary atresia

ABSTRACT Biliary atresia (BA) is a pediatric liver disease of unknown underlying etiology, in which fibroinflammatory destruction of the extrahepatic biliary system leads to obstructive cholestasis. MicroRNAs are a class of short (18–23 nucleotide), noncoding RNA molecules, which act as negative regulators of target mRNA stability and translation. The importance of these molecules in normal and diseased liver has been demonstrated, but their potential role in the pathogenesis of BA has not been addressed. We have profiled changes in liver microRNA levels in an established mouse model of the disease, identified significantly altered transcripts, and defined the spatial expression patterns of selected microRNAs. Two of these, miR-29a/29b1, are upregulated in experimental BA. Using antisense oligonucleotide-mediated inhibition in mice, we have delineated the full set of hepatic genes regulated by miR-29 and identified 2 mRNA targets of potential pathological relevance in experimental BA, Igf1 and Il1RAP. We have used reporter assays to confirm that Igf1 and Il1RAP are direct targets of miR-29.

Circulating MicroRNA is a Biomarker of Biliary Atresia

Objective: The lack of reliable noninvasive diagnostic biomarkers of biliary atresia (BA) results in delayed diagnosis and worsened patient outcome. Circulating microRNAs (miRNAs) are a new class of noninvasive biomarkers with encouraging diagnostic utility. Methods: We examined the ability of serum miRNAs to distinguish BA from other forms of neonatal hyperbilirubinemia. BA-specific serum miRNAs were identified using a microfluidic array platform and validated in a larger, independent sample set. Results: The miR-200b/429 cluster was significantly increased in the sera of patients with BA relative to infants with non-BA cholestatic disorders. Conclusions: Circulating levels of the miR-200b/429 cluster are elevated in infants with BA and have promising diagnostic clinical performance.

Dynamic recruitment of microRNAs to their mRNA targets in the regenerating liver.

BackgroundValidation of physiologic miRNA targets has been met with significant challenges. We employed HITS-CLIP to identify which miRNAs participate in liver regeneration, and to identify their target mRNAs.ResultsmiRNA recruitment to the RISC is highly dynamic, changing more than five-fold for several miRNAs. miRNA recruitment to the RISC did not correlate with changes in overall miRNA expression for these dynamically recruited miRNAs, emphasizing the necessity to determine miRNA recruitment to the RISC in order to fully assess the impact of miRNA regulation. We incorporated RNA-seq quantification of total mRNA to identify expression-weighted Ago footprints, and developed a microRNA regulatory element (MRE) prediction algorithm that represents a greater than 20-fold refinement over computational methods alone. These high confidence MREs were used to generate candidate ‘competing endogenous RNA’ (ceRNA) networks.ConclusionHITS-CLIP analysis provide novel insights into global miRNA:mRNA relationships in the regenerating liver.

Archaeal and Bacterial SecD and SecF Homologs Exhibit Striking Structural and Functional Conservation

The majority of secretory proteins are translocated into and across hydrophobic membranes via the universally conserved Sec pore. Accessory proteins, including the SecDF-YajC Escherichia coli membrane complex, are required for efficient protein secretion. E. coli SecDF-YajC has been proposed to be involved in the membrane cycling of SecA, the cytoplasmic bacterial translocation ATPase, and in the stabilizing of SecG, a subunit of the Sec pore. While there are no identified archaeal homologs of either SecA or SecG, many archaea possess homologs of SecD and SecF. Here, we present the first study that addresses the function of archaeal SecD and SecF homologs. We show that the SecD and SecF components in the model archaeon Haloferax volcanii form a cytoplasmic membrane complex in the native host. Furthermore, as in E. coli, an H. volcanii deltasecFD mutant strain exhibits both severe cold sensitivity and a Sec-specific protein translocation defect. Taken together, these results demonstrate significant functional conservation among the prokaryotic SecD and SecF homologs despite the distinct composition of their translocation machineries.

Extrahepatic cholangiocyte cilia are abnormal in biliary atresia

Objectives: Biliary atresia (BA) is a rapidly progressive form of biliary fibrosis affecting neonates. We previously reported that primary cilia on the intrahepatic cholangiocytes of patients with both syndromic and nonsyndromic BA were structurally abnormal. Our objective was to determine whether extrahepatic cholangiocytes in human biliary atesia, intrahepatic and extrahepatic cholangiocytes of rhesus rotavirus (RRV)-infected neonatal mice, and RRV-infected primary neonatal extrahepatic cholangiocytes also demonstrate ciliary abnormalities. Methods: The livers of neonatal BALB/c mice injected with RRV that developed jaundice, human extrahepatic bile duct samples obtained at time of hepatoportoenterostomy, and RRV-infected primary neonatal cholangiocytes were stained with antibodies against acetylated &agr; tubulin to identify primary cilia. Results: Extrahepatic cholangiocytes from RRV-treated mice demonstrated minimal loss of primary cilia at day 3 but almost complete loss at day 8 and partial loss at day 12. No changes were seen in mouse intrahepatic bile ducts at any of the time points. In the human BA samples, primary cilia were almost completely absent from extrahepatic duct cholangiocytes. There were, however, abundant cilia in the peribiliary glands adjacent to extrahepatic ducts in the BA sample. Cilia in RRV-infected primary neonatal cholangiocytes were significantly decreased compared with controls. Conclusions: Primary cilia are selectively lost from neonatal extrahepatic but not intrahepatic cholangiocytes after RRV infection in BALB/c mice. The cilia are also decreased in RRV-infected primary cholangiocytes and the extrahepatic ducts from human patients with BA. This suggests that ciliary abnormalities are part of the pathophysiology of BA.

Null Mutations in a Nudix Gene, ygdP, Implicate an Alarmone Response in a Novel Suppression of Hybrid Jamming

Induction of the toxic LamB-LacZ protein fusion, Hyb42-1, leads to a lethal generalized protein export defect. The prlF1 suppressor causes hyperactivation of the cytoplasmic Lon protease and relieves the inducer sensitivity of Hyb42-1. Since prlF1 does not cause a detectable change in the stability or level of the hybrid protein, we conducted a suppressor screen, seeking factors genetically downstream of lon with prlF1-like phenotypes. Two independent insertions in the ygdP open reading frame relieve the toxicity of the fusion protein and share two additional properties with prlF1: cold sensitivity and the ability to suppress the temperature sensitivity of a degP null mutation. Despite these similarities, ygdP does not appear to act in the same genetic pathway as prlF1 and lon, suggesting a fundamental link between the phenotypes. We speculate that the common properties of the suppressors relate to secretion defects. The ygdP gene (also known as nudH) has been shown to encode a Nudix protein that acts as a dinucleotide oligophosphate (alarmone) hydrolase. Our results suggest that loss of ygdP function leads to the induction of an alarmone-mediated response that affects secretion. Using an epitope-tagged ygdP construct, we present evidence that this response is sensitive to secretion-related stress and is regulated by differential proteolysis of YgdP in a self-limiting manner.

Zinc transporter Slc39a8 is essential for cardiac ventricular compaction

Isolated left ventricular noncompaction (LVNC) results from excessive trabeculation and impaired myocardial compaction during heart development. The extracellular matrix (ECM) that separates endocardium from myocardium plays a critical but poorly understood role in ventricular trabeculation and compaction. In an attempt to characterize solute carrier family 39 member 8–null (Slc39a8-null) mice, we discovered that homozygous null embryos do not survive embryogenesis and exhibit a cardiac phenotype similar to human LVNC. Slc39a8 encodes a divalent metal cation importer that has been implicated in ECM degradation through the zinc/metal regulatory transcription factor 1 (Zn/MTF1) axis, which promotes the expression of ECM-degrading enzymes, including Adamts metalloproteinases. Here, we have shown that Slc39a8 is expressed by endothelial cells in the developing mouse heart, where it serves to maintain cellular Zn levels. Furthermore, Slc39a8-null hearts exhibited marked ECM accumulation and reduction of several Adamts metalloproteinases. Consistent with the in vivo observations, knockdown of SLC39A8 in HUVECs decreased ADAMTS1 transcription by decreasing cellular Zn uptake and, as a result, MTF1 transcriptional activity. Our study thus identifies a gene underlying ventricular trabeculation and compaction development, and a pathway regulating ECM during myocardial morphogenesis.