The effectiveness of schemes that refine referrals between primary and secondary care - the UK experience with glaucoma referrals: the Health Innovation & Education Cluster (HIEC) Glaucoma Pathways Project

Objectives: A comparison of glaucoma referral refinement schemes (GRRS) in the UK during a time period of considerable change in national policy and guidance. Design: Retrospective multisite review. Setting: The outcomes of clinical examinations by optometrists with a specialist interest in glaucoma (OSIs) were compared with optometrists with no specialist interest in glaucoma (non-OSIs). Data from Huntingdon and Nottingham assessed non-OSI findings, while Manchester and Gloucestershire reviewed OSI findings. Participants: 1086 patients. 434 patients were from Huntingdon, 179 from Manchester, 204 from Gloucestershire and 269 from Nottingham. Results: The first-visit discharge rate (FVDR) for all time periods for OSIs was 14.1% compared with 36.1% from non-OSIs (difference 22%, CI 16.9% to 26.7%; p<0.001). The FVDR increased after the April 2009 National Institute for Health and Clinical Excellence (NICE) glaucoma guidelines compared with pre-NICE, which was particularly evident when pre-NICE was compared with the current practice time period (OSIs 6.2–17.2%, difference 11%, CI −24.7% to 4.3%; p=0.18, non-OSIs 29.2–43.9%, difference 14.7%, CI −27.8% to −0.30%; p=0.03). Elevated intraocular pressure (IOP) was the commonest reason for referral for OSIs and non-OSIs, 28.7% and 36.1%, respectively, of total referrals. The proportion of referrals for elevated IOP increased from 10.9% pre-NICE to 28.0% post-NICE for OSIs, and from 19% to 45.1% for non-OSIs. Conclusions: In terms of ‘demand management’, OSIs can reduce FVDR of patients reviewed in secondary care; however, in terms of ‘patient safety’ this study also shows that overemphasis on IOP as a criterion for referral is having an adverse effect on both the non-OSIs and indeed the OSIs ability to detect glaucomatous optic nerve features. It is recommended that referral letters from non-OSIs be stratified for risk, directing high-risk patients straight to secondary care, and low-risk patients to OSIs

Meta-analysis of radiofrequency ablation versus hepatic resection for small hepatocellular carcinoma

<p>Abstract</p> <p>Background</p> <p>There is no clear consensus on the better therapy [radiofrequency ablation (RFA) versus hepatic resection (HR)] for small hepatocellular carcinoma (HCC) eligible for surgical treatments. This study is a meta-analysis of the available evidence.</p> <p>Methods</p> <p>Systematic review and meta-analysis of trials comparing RFA with HR for small HCC published from 1997 to 2009 in PubMed and Medline. Pooled odds ratios (OR) with 95% confidence intervals (95% CI) were calculated using either the fixed effects model or random effects model.</p> <p>Results</p> <p>One randomized controlled trial, and 9 nonrandomized controlled trials studies were included in this analysis. These studies included a total of 1411 patients: 744 treated with RFA and 667 treated with HR. The overall survival was significantly higher in patients treated with HR than in those treated with RFA at 3 years (OR: 0.56, 95% CI: 0.44-0.71), and at 5 year (OR: 0.60, 95% CI: 0.36-1.01). RFA has a higher rates of local intrahepatic recurrence compared to HR (OR: 4.50, 95% CI: 2.45-8.27). In the HR group the 1, 3, and 5 years disease -free survival rates were significantly better than in the HR-treated patients (respectively: OR: 0.54, 95% CI: 0.35-0.84; OR: 0.44, 95% CI: 0.28-0.68; OR: 0.64, 95% CI: 0.42-0.99). The postoperative morbidity was higher with HR (OR: 0.29, 95% CI: 0.13-0.65), but no significant differences were found concerning mortality. For tumors ≤ 3 cm HR did not differ significantly from RFA for survival, as reported in three NRCTs .</p> <p>Conclusions</p> <p>HR was superior to RFA in the treatment of patients with small HCC eligible for surgical treatments, particularly for tumors > 3 cm. However, the findings have to be carefully interpreted due to the lower level of evidence.</p

De Novo Transcriptome of Safflower and the Identification of Putative Genes for Oleosin and the Biosynthesis of Flavonoids

Safflower (Carthamus tinctorius L.) is one of the most extensively used oil crops in the world. However, little is known about how its compounds are synthesized at the genetic level. In this study, Solexa-based deep sequencing on seed, leaf and petal of safflower produced a de novo transcriptome consisting of 153,769 unigenes. We annotated 82,916 of the unigenes with gene annotation and assigned functional terms and specific pathways to a subset of them. Metabolic pathway analysis revealed that 23 unigenes were predicted to be responsible for the biosynthesis of flavonoids and 8 were characterized as seed-specific oleosins. In addition, a large number of differentially expressed unigenes, for example, those annotated as participating in anthocyanin and chalcone synthesis, were predicted to be involved in flavonoid biosynthesis pathways. In conclusion, the de novo transcriptome investigation of the unique transcripts provided candidate gene resources for studying oleosin-coding genes and for investigating genes related to flavonoid biosynthesis and metabolism in safflower

De novo assembly of Euphorbia fischeriana root transcriptome identifies prostratin pathway related genes

Background Euphorbia fischeriana is an important medicinal plant found in Northeast China. The plant roots contain many medicinal compounds including 12-deoxyphorbol-13-acetate, commonly known as prostratin that is a phorbol ester from the tigliane diterpene series. Prostratin is a protein kinase C activator and is effective in the treatment of Human Immunodeficiency Virus (HIV) by acting as a latent HIV activator. Latent HIV is currently the biggest limitation for viral eradication. The aim of this study was to sequence, assemble and annotate the E. fischeriana transcriptome to better understand the potential biochemical pathways leading to the synthesis of prostratin and other related diterpene compounds. Results In this study we conducted a high throughput RNA-seq approach to sequence the root transcriptome of E. fischeriana. We assembled 18,180 transcripts, of these the majority encoded protein-coding genes and only 17 transcripts corresponded to known RNA genes. Interestingly, we identified 5,956 protein-coding transcripts with high similarity (>=75%) to Ricinus communis, a close relative to E. fischeriana. We also evaluated the conservation of E. fischeriana genes against EST datasets from the Euphorbeacea family, which included R. communis, Hevea brasiliensis and Euphorbia esula. We identified a core set of 1,145 gene clusters conserved in all four species and 1,487 E. fischeriana paralogous genes. Furthermore, we screened E. fischeriana transcripts against an in-house reference database for genes implicated in the biosynthesis of upstream precursors to prostratin. This identified 24 and 9 candidate transcripts involved in the terpenoid and diterpenoid biosyntehsis pathways, respectively. The majority of the candidate genes in these pathways presented relatively low expression levels except for 1-hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate synthase (HDS) and isopentenyl diphosphate/dimethylallyl diphosphate synthase (IDS), which are required for multiple downstream pathways including synthesis of casbene, a proposed precursor to prostratin. Conclusion The resources generated in this study provide new insights into the upstream pathways to the synthesis of prostratin and will likely facilitate functional studies aiming to produce larger quantities of this compound for HIV research and/or treatment of patients

An efficient approach to finding Siraitia grosvenorii triterpene biosynthetic genes by RNA-seq and digital gene expression analysis

<p>Abstract</p> <p>Background</p> <p><it>Siraitia grosvenorii </it>(Luohanguo) is an herbaceous perennial plant native to southern China and most prevalent in Guilin city. Its fruit contains a sweet, fleshy, edible pulp that is widely used in traditional Chinese medicine. The major bioactive constituents in the fruit extract are the cucurbitane-type triterpene saponins known as mogrosides. Among them, mogroside V is nearly 300 times sweeter than sucrose. However, little is known about mogrosides biosynthesis in <it>S. grosvenorii</it>, especially the late steps of the pathway.</p> <p>Results</p> <p>In this study, a cDNA library generated from of equal amount of RNA taken from <it>S. grosvenorii </it>fruit at 50 days after flowering (DAF) and 70 DAF were sequenced using Illumina/Solexa platform. More than 48,755,516 high-quality reads from a cDNA library were generated that was assembled into 43,891 unigenes. De novo assembly and gap-filling generated 43,891 unigenes with an average sequence length of 668 base pairs. A total of 26,308 (59.9%) unique sequences were annotated and 11,476 of the unique sequences were assigned to specific metabolic pathways by the Kyoto Encyclopedia of Genes and Genomes. cDNA sequences for all of the known enzymes involved in mogrosides backbone synthesis were identified from our library. Additionally, a total of eighty-five cytochrome P450 (CYP450) and ninety UDP-glucosyltransferase (UDPG) unigenes were identified, some of which appear to encode enzymes responsible for the conversion of the mogroside backbone into the various mogrosides. Digital gene expression profile (DGE) analysis using Solexa sequencing was performed on three important stages of fruit development, and based on their expression pattern, seven <it>CYP450</it>s and five <it>UDPG</it>s were selected as the candidates most likely to be involved in mogrosides biosynthesis.</p> <p>Conclusion</p> <p>A combination of RNA-seq and DGE analysis based on the next generation sequencing technology was shown to be a powerful method for identifying candidate genes encoding enzymes responsible for the biosynthesis of novel secondary metabolites in a non-model plant. Seven <it>CYP450</it>s and five <it>UDPG</it>s were selected as potential candidates involved in mogrosides biosynthesis. The transcriptome data from this study provides an important resource for understanding the formation of major bioactive constituents in the fruit extract from <it>S. grosvenorii</it>.</p

Identification of Sequence Variants in Genetic Disease-Causing Genes Using Targeted Next-Generation Sequencing

Identification of gene variants plays an important role in research on and diagnosis of genetic diseases. A combination of enrichment of targeted genes and next-generation sequencing (targeted DNA-HiSeq) results in both high efficiency and low cost for targeted sequencing of genes of interest.To identify mutations associated with genetic diseases, we designed an array-based gene chip to capture all of the exons of 193 genes involved in 103 genetic diseases. To evaluate this technology, we selected 7 samples from seven patients with six different genetic diseases resulting from six disease-causing genes and 100 samples from normal human adults as controls. The data obtained showed that on average, 99.14% of 3,382 exons with more than 30-fold coverage were successfully detected using Targeted DNA-HiSeq technology, and we found six known variants in four disease-causing genes and two novel mutations in two other disease-causing genes (the STS gene for XLI and the FBN1 gene for MFS) as well as one exon deletion mutation in the DMD gene. These results were confirmed in their entirety using either the Sanger sequencing method or real-time PCR.Targeted DNA-HiSeq combines next-generation sequencing with the capture of sequences from a relevant subset of high-interest genes. This method was tested by capturing sequences from a DNA library through hybridization to oligonucleotide probes specific for genetic disorder-related genes and was found to show high selectivity, improve the detection of mutations, enabling the discovery of novel variants, and provide additional indel data. Thus, targeted DNA-HiSeq can be used to analyze the gene variant profiles of monogenic diseases with high sensitivity, fidelity, throughput and speed

RNF185, a Novel Mitochondrial Ubiquitin E3 Ligase, Regulates Autophagy through Interaction with BNIP1

Autophagy is an evolutionarily conserved catabolic process that allows recycling of cytoplasmic organelles, such as mitochondria, to offer a bioenergetically efficient pathway for cell survival. Considerable progress has been made in characterizing mitochondrial autophagy. However, the dedicated ubiquitin E3 ligases targeting mitochondria for autophagy have not been revealed. Here we show that human RNF185 is a mitochondrial ubiquitin E3 ligase that regulates selective mitochondrial autophagy in cultured cells. The two C-terminal transmembrane domains of human RNF185 mediate its localization to mitochondrial outer membrane. RNF185 stimulates LC3II accumulation and the formation of autophagolysosomes in human cell lines. We further identified the Bcl-2 family protein BNIP1 as one of the substrates for RNF185. Human BNIP1 colocalizes with RNF185 at mitochondria and is polyubiquitinated by RNF185 through K63-based ubiquitin linkage in vivo. The polyubiquitinated BNIP1 is capable of recruiting autophagy receptor p62, which simultaneously binds both ubiquitin and LC3 to link ubiquitination and autophagy. Our study might reveal a novel RNF185-mediated mechanism for modulating mitochondrial homeostasis through autophagy

Loss of Guanylyl Cyclase C (GCC) Signaling Leads to Dysfunctional Intestinal Barrier

Guanylyl Cyclase C (GCC) signaling via uroguanylin (UGN) and guanylin activation is a critical mediator of intestinal fluid homeostasis, intestinal cell proliferation/apoptosis, and tumorigenesis. As a mechanism for some of these effects, we hypothesized that GCC signaling mediates regulation of intestinal barrier function.Paracellular permeability of intestinal segments was assessed in wild type (WT) and GCC deficient (GCC-/-) mice with and without lipopolysaccharide (LPS) challenge, as well as in UGN deficient (UGN-/-) mice. IFNγ and myosin light chain kinase (MLCK) levels were determined by real time PCR. Expression of tight junction proteins (TJPs), phosphorylation of myosin II regulatory light chain (MLC), and STAT1 activation were examined in intestinal epithelial cells (IECs) and intestinal mucosa. The permeability of Caco-2 and HT-29 IEC monolayers, grown on Transwell filters was determined in the absence and presence of GCC RNA interference (RNAi). We found that intestinal permeability was increased in GCC-/- and UGN-/- mice compared to WT, accompanied by increased IFNγ levels, MLCK and STAT1 activation in IECs. LPS challenge promotes greater IFNγ and STAT1 activation in IECs of GCC-/- mice compared to WT mice. Claudin-2 and JAM-A expression were reduced in GCC deficient intestine; the level of phosphorylated MLC in IECs was significantly increased in GCC-/- and UGN-/- mice compared to WT. GCC knockdown induced MLC phosphorylation, increased permeability in IEC monolayers under basal conditions, and enhanced TNFα and IFNγ-induced monolayer hyperpermeability.GCC signaling plays a protective role in the integrity of the intestinal mucosal barrier by regulating MLCK activation and TJ disassembly. GCC signaling activation may therefore represent a novel mechanism in maintaining the small bowel barrier in response to injury

A population-based survey of the epidemiology of symptom-defined gastroesophageal reflux disease: the Systematic Investigation of Gastrointestinal Diseases in China

<p>Abstract</p> <p>Background</p> <p>The epidemiology of gastroesophageal reflux disease (GERD) has yet to be investigated using the symptomatic threshold criteria recommended by the Montreal Definition. This study aimed to determine the prevalence of symptom-defined GERD across five regions of China, and to investigate variables associated with GERD.</p> <p>Methods</p> <p>A representative sample of 18 000 adults (aged 18-80 years) were selected equally from rural and urban areas in each region (n = 1800). According to the Montreal Definition, GERD is present when mild symptoms of heartburn and/or regurgitation occur on ≥2 days a week, or moderate-to-severe symptoms of heartburn and/or regurgitation occur on ≥1 day a week.</p> <p>Results</p> <p>In total, 16 091 participants completed the survey (response rate: 89.4%) and 16 078 responses were suitable for analysis. Applying the Montreal criteria, the prevalence of symptom-defined GERD was 3.1% and varied significantly (<it>p </it>< 0.001) among the five regions (from 1.7% in Guangzhou to 5.1% in Wuhan) and between rural and urban populations (3.8% vs 2.4%). Factors significantly associated with GERD included living in a rural area and a family history of gastrointestinal diseases.</p> <p>Conclusions</p> <p>This population-based survey found that the prevalence of symptom-defined GERD in China was 3.1%, which is lower than that found in Western countries.</p