Long noncoding RNA UCA1 promotes carboplatin resistance in retinoblastoma cells by acting as a ceRNA of miR-206.

Chemoresistance has become a major obstacle to effective retinoblastoma treatment. The urothelial cancer-associated gene 1 (UCA1) is commonly considered an oncogene in certain types of cancer and is related to drug resistance. Nonetheless, the molecular mechanism and effect of UCA1 in carboplatin resistance in retinoblastoma are unclear. In this study, UCA1 expression was determined by sequential screening and lncRNA profile analysis, which is highly abundant in carboplatin-resistant retinoblastoma cells. Functional analyses revealed that UCA1 promoted carboplatin resistance by promoting c-Met and AXL expression. Mechanistic studies revealed that UCA1 facilitated c-Met and AXL expression as a ceRNA of miR-206. Importantly, retinoblastoma nude mouse model experiments revealed that targeting UCA1 or c-Met and AXL can restore drug sensitivity in carboplatin-resistant retinoblastoma. Collectively, we found that UCA1 is a mediator of carboplatin resistance in retinoblastoma cells. It competes with others as the endogenous RNA of miR-206, thus upregulating its targets, c-MET and AXL expression.

Dickkopf-1 exerts protective effects by inhibiting PANoptosis and retinal neovascularization in diabetic retinopathy.

Diabetic retinopathy (DR) is a key reason for legal blindness worldwide. Currently, it is urgently necessary to determine the etiology and pathological molecular mechanism of DR to search for resultful therapies. Dickkopf-1 (DKK1) is inhibitive for canonical Wnt signaling via negative feedback, and has been reported as a biomarker for DR. However, the related mechanisms are still unclear. In this work, our data showed that DKK1 was decreased in the vitreous tissues at an early stage of diabetes triggered by streptozotocin (STZ) injection in rats. We subsequently found that DKK1 intravitreal injection significantly ameliorated the physiological function of retina in STZ-challenged rats, accompanied by improved retinal structure. Surprisingly, our results indicated that DKK1 injection remarkably suppressed PANoptosis in retinal tissues of STZ-challenged rats with DR, as proved by ameliorated pyroptosis, apoptosis and necroptosis, which were mainly through the blockage of cleaved Gasdermin-D (GSDMD), Caspase-3 and receptor-interacting protein kinase-3 (RIPK3). Additionally, Wnt signaling including the expression of Wnt, ß-catenin and LDL receptor-related protein 5/6 (LRP5/6) was also highly prohibited in retina of DKK1-injected rats with DR. Furthermore, retinal neovascularization and acellular vessel in DR rats were also considerably abolished after DKK1 injection, accompanied by reduced expression levels of retinal vascular endothelial growth factor (VEGF), matrix metalloproteinase-2 (MMP2) and matrix metalloproteinase-9 (MMP9). More in vitro experiments showed that DKK1 treatment markedly repressed the proliferative and migratory ability of endothelial cells via inhibiting angiogenesis-related molecules. Together, all our results broaden the knowledge of the correlation between DKK1 and DR, and then provide a novel therapeutic strategy for the suppression of management of DR.

A genome-wide CRISPR-Cas9 knockout screen identifies essential and growth-restricting genes in human trophoblast stem cells.

The recent derivation of human trophoblast stem cells (hTSCs) provides a scalable in vitro model system of human placental development, but the molecular regulators of hTSC identity have not been systematically explored thus far. Here, we utilize a genome-wide CRISPR-Cas9 knockout screen to comprehensively identify essential and growth-restricting genes in hTSCs. By cross-referencing our data to those from similar genetic screens performed in other cell types, as well as gene expression data from early human embryos, we define hTSC-specific and -enriched regulators. These include both well-established and previously uncharacterized trophoblast regulators, such as ARID3A, GATA2, and TEAD1 (essential), and GCM1, PTPN14, and TET2 (growth-restricting). Integrated analysis of chromatin accessibility, gene expression, and genome-wide location data reveals that the transcription factor TEAD1 regulates the expression of many trophoblast regulators in hTSCs. In the absence of TEAD1, hTSCs fail to complete faithful differentiation into extravillous trophoblast (EVT) cells and instead show a bias towards syncytiotrophoblast (STB) differentiation, thus indicating that this transcription factor safeguards the bipotent lineage potential of hTSCs. Overall, our study provides a valuable resource for dissecting the molecular regulation of human placental development and diseases.

The Association of Axial Length with Macular Microvascular Changes in Chinese Diabetic Retinopathy Patients.

Objective: To use optical coherence tomography angiography (OCTA) to compare macular blood flow density, subfoveal choroidal thickness (SFCT) and outer retina thickness (ORT) in non-proliferative diabetic retinopathy (NPDR) patients with different axial length (AL). Methods: Total 42 patients with NPDR with different eye axis were divided into three groups: group A: 22 mm≤AL<24 mm; group B: 24 mm≤AL<26 mm; group C: AL≥26 mm. Superficial capillary plexus (SCP) in the macular area, vascular length density (VLD) and vascular perfusion density (VPD) in the foveal region, the parafoveal region, the perifoveal region and whole macular region were analyzed. The correlations among axial length, macular microvascular density, SFCT and outer retinal thickness (ORT) were analyzed. Results: Compared with group A and B, VLD and VPD in group C were significantly lower except the foveal region, and VLD and VPD were negatively correlated with AL. The difference in SFCT among group A, B and C was significant, and SFCT was negatively correlated with AL. Compared with group A, parafoveal ORT in group C was significantly lower than that in group A, and parafoveal ORT was negatively correlated with AL. Conclusion: In NPDR patients with different AL, macular microvascular density, SFCT, and parafoveal ORT decreased with the increase of AL.

MiR-195 inhibits the ubiquitination and degradation of YY1 by Smurf2, and induces EMT and cell permeability of retinal pigment epithelial cells.

The dysregulated microRNAs (miRNAs) are involved in diabetic retinopathy progression. Epithelial mesenchymal transition (EMT) and cell permeability are important events in diabetic retinopathy. However, the function and mechanism of miR-195 in EMT and cell permeability in diabetic retinopathy remain largely unclear. Diabetic retinopathy models were established using streptozotocin (STZ)-induced diabetic mice and high glucose (HG)-stimulated ARPE-19 cells. Retina injury was investigated by hematoxylin-eosin (HE) staining. EMT and cell permeability were analyzed by western blotting, immunofluorescence, wound healing, and FITC-dextran assays. MiR-195 expression was detected via qRT-PCR. YY1, VEGFA, Snail1, and Smurf2 levels were detected via western blotting. The interaction relationship was analyzed via ChIP, Co-IP, or dual-luciferase reporter assay. The retina injury, EMT, and cell permeability were induced in STZ-induced diabetic mice. HG induced EMT and cell permeability in ARPE-19 cells. MiR-195, YY1, VEGFA, and Snail1 levels were enhanced, but Smurf2 abundance was reduced in STZ-induced diabetic mice and HG-stimulated ARPE-19 cells. VEGFA knockdown decreased Snail1 expression and attenuated HG-induced EMT and cell permeability. YY1 silence reduced VEGFA and Snail1 expression, and mitigated HG-induced EMT and cell permeability. YY1 could bind with VEGFA and Snail1, and it was degraded via Smurf2-mediated ubiquitination. MiR-195 knockdown upregulated Smurf2 to decrease YY1 expression and inhibited HG-induced EMT and cell permeability. MiR-195 targeted Smurf2, increased expression of YY1, VEGFA, and Snail1, and promoted HG-induced EMT and cell permeability. MiR-195 promotes EMT and cell permeability of HG-stimulated ARPE-19 cells by increasing VEGFA/Snail1 via inhibiting the Smurf2-mediated ubiquitination of YY1.

Suppressing long noncoding RNA OGRU ameliorates diabetic retinopathy by inhibition of oxidative stress and inflammation via miR-320/USP14 axis.

Long noncoding RNAs (lncRNAs) are important regulators in various diseases including diabetic retinopathy (DR). In this study, DR patients exhibited significantly increased expression of serum LncRNA-OGRU compared with normal individuals. Streptozotocin (STZ)-challenged rats with DR also had higher OGRU expression in retinas than that of the control group, which was confirmed in Müller cells upon high glucose (HG) stimulation. OGRU knockdown remarkably decreased vascular endothelial growth factor (VEGF) and transforming growth factor-ß1 (TGF-ß1) expression in HG-incubated Müller cells. HG-induced inflammatory response and oxidative stress in vitro were markedly mitigated by OGRU knockdown through restraining IκBɑ/nuclear factor kappa beta (NF-κB) and improving nuclear factor erythroid 2-related factor 2 (Nrf2) signaling pathways, respectively. Further studies indicated that OGRU suppression greatly restored miR-320 expression, and a negative correlation between them was detected in DR patients. We also found that miR-320 over-expression considerably restrained TGF-ß1 signaling, and hindered inflammation and reactive oxygen species (ROS) production in HG-stimulated Müller cells. Additionally, OGRU knockdown or miR-320 over-expression could dramatically down-regulate ubiquitin-specific peptidase 14 (USP14) expression levels in HG-incubated Müller cells, and miR-320 could directly target USP14. Notably, OGRU/miR-320 axis-mediated TGF-ß1 signaling, inflammation and ROS were largely dependent on USP14. Intriguingly, our results showed that USP14 directly interacted with transforming growth factor-beta type 1 receptor (TßR1), and impeded TßR1 ubiquitination and degradation. Furthermore, USP14 could also facilitate IκBɑ deubiquitination and degradation, exacerbating IκBɑ phosphorylation and NF-κB activation. Finally, our in vivo studies confirmed that OGRU knockdown considerably ameliorated DR progression in STZ-challenged rats through mediating the mechanisms observed in vitro. Collectively, these findings implicated that LncRNA-OGRU mediated DR progression through competing for miR-320 to regulate USP14 expression, and thus LncRNA-OGRU/miR-320/USP14 axis may be considered as a therapeutic target for DR treatment.

miR-132 mediates cell permeability and migration by targeting occludin in high-glucose -induced ARPE-19 cells.

This study investigated the effects and mechanisms of miR-132 related to the permeability and mobility of human retinal pigment epithelium ARPE-19 cells in high-glucose (HG) condition. ARPE-19 cells were cultured in normal and HG condition and identified by immunofluorescence staining. Cell viability was assessed by the MTT assay, cell permeability was assessed by the FITC-dextran assay and cell mobility was assessed by the wound healing assay. Different miRNA and mRNA expression levels were determined by quantitative real-time polymerase chain reaction (RT-qPCR). The expression of tight junction-related proteins was determined by Western blot assay and immunofluorescence. The interaction between occludin and miR-132 was confirmed by a dual-luciferase reporter assay. We revealed that HG-treated ARPE-19 cells exhibited significantly increased miR-132 expression, decreased expression of the tight-junction markers including occludin and E-cadherin, and increased cell mobility and permeability. Occludin is a direct target of miR-132, which could regulate cell viability, mobility and permeability under HG condition through the JAK/STAT3 signaling pathway. These are the first data to suggest that miR-132 may contribute to the progression of diabetic retinopathy (DR) and that targeting the effect of miR-132 on occudin and the JAK/STAT3 pathway could represent a novel effective DR-treatment strategy.

Endothelial Dysfunction in Diabetic Retinopathy.

Diabetic retinopathy (DR) is a diabetic complication which affects retinal function and results in severe loss of vision and relevant retinal diseases. Retinal vascular dysfunction caused by multifactors, such as advanced glycosylation end products and receptors, pro-inflammatory cytokines and chemokines, proliferator-activated receptor-γ disruption, growth factors, oxidative stress, and microRNA. These factors promote retinal endothelial dysfunction, which results in the development of DR. In this review, we summarize the contributors in the pathophysiology of DR for a better understanding of the molecular and cellular mechanism in the development of DR with a special emphasis on retinal endothelial dysfunction.

Tissue-specific usage of transposable element-derived promoters in mouse development.

BACKGROUND: Transposable elements (TEs) are a significant component of eukaryotic genomes and play essential roles in genome evolution. Mounting evidence indicates that TEs are highly transcribed in early embryo development and contribute to distinct biological functions and tissue morphology. RESULTS: We examine the epigenetic dynamics of mouse TEs during the development of five tissues: intestine, liver, lung, stomach, and kidney. We found that TEs are associated with over 20% of open chromatin regions during development. Close to half of these accessible TEs are only activated in a single tissue and a specific developmental stage. Most accessible TEs are rodent-specific. Across these five tissues, 453 accessible TEs are found to create the transcription start sites of downstream genes in mouse, including 117 protein-coding genes and 144 lincRNA genes, 93.7% of which are mouse-specific. Species-specific TE-derived transcription start sites are found to drive the expression of tissue-specific genes and change their tissue-specific expression patterns during evolution. CONCLUSION: Our results suggest that TE insertions increase the regulatory potential of the genome, and some TEs have been domesticated to become a crucial component of gene and regulate tissue-specific expression during mouse tissue development.

Comparison of differential accessibility analysis strategies for ATAC-seq data.

ATAC-seq is widely used to measure chromatin accessibility and identify open chromatin regions (OCRs). OCRs usually indicate active regulatory elements in the genome and are directly associated with the gene regulatory network. The identification of differential accessibility regions (DARs) between different biological conditions is critical in determining the differential activity of regulatory elements. Differential analysis of ATAC-seq shares many similarities with differential expression analysis of RNA-seq data. However, the distribution of ATAC-seq signal intensity is different from that of RNA-seq data, and higher sensitivity is required for DARs identification. Many different tools can be used to perform differential analysis of ATAC-seq data, but a comprehensive comparison and benchmarking of these methods is still lacking. Here, we used simulated datasets to systematically measure the sensitivity and specificity of six different methods. We further discussed the statistical and signal density cut-offs in the differential analysis of ATAC-seq by applying them to real data. Batch effects are very common in high-throughput sequencing experiments. We illustrated that batch-effect correction can dramatically improve sensitivity in the differential analysis of ATAC-seq data. Finally, we developed a user-friendly package, BeCorrect, to perform batch effect correction and visualization of corrected ATAC-seq signals in a genome browser.

A study of dry eye after cataract surgery in MGD patients.

OBJECTIVE: To investigate the dry eye symptoms after cataract surgery in MGD patients and their relationships METHODS: The study included 115 patients (115 eyes) with age-related cataract that underwent uncomplicated cataract surgery, and the patients were divided into two groups according to the MGD diagnostic criteria: group A (MGD group) and group B (control group). Schirmer I test (ST-I), tear breakup time (TBUT), and corneal fluorescein staining (CFS) were performed preoperatively and at 3 days, 7 days, 14 days, and 30 days postoperatively. We also measured eyelid meibomian gland morphology, meibomian gland expression, and meibum character scores before and after the cataract surgery. RESULTS: Postoperatively, in group A, TBUT decreased and CFS scores increased significantly. ST-I increased in the early postoperative course but decreased later. The eyelid margin morphology scores and meibomian gland expression scores of group A significantly increased after the cataract operation. Thus, patients with MGD may have a greater chance of developing the dry eye disease after cataract surgery. Cataract surgery may aggravate the signs of MGD, and the severity of MGD may positively correlate with TBUT, CFS, and corneal lesions after surgery. CONCLUSIONS: The characteristics of dry eye after cataract surgery in patients with MGD are different from common cataract patients, changes in the early postoperative phase to the ocular surface were caused by surgical factors, and the damages to epithelial function in the later postoperative phase were mainly associated with the inflammation of the meibomian gland and eyelid.

Gene expression correlates of facultative predation in the blow fly Chrysomya rufifacies (Diptera: Calliphoridae).

Effects of intraguild predation (IGP) on omnivores and detritivores are relatively understudied when compared to work on predator guilds. Functional genetic work in IGP is even more limited, but its application can help answer a range of questions related to ultimate and proximate causes of this behavior. Here, we integrate behavioral assays and transcriptomic analysis of facultative predation in a blow fly (Diptera: Calliphoridae) to evaluate the prevalence, effect, and correlated gene expression of facultative predation by the invasive species Chrysomya rufifacies. Field work observing donated human cadavers indicated facultative predation by C. rufifacies on the native blow fly Cochliomyia macellaria was rare under undisturbed conditions, owing in part to spatial segregation between species. Laboratory assays under conditions of starvation showed predation had a direct fitness benefit (i.e., survival) to the predator. As a genome is not available for C. rufifacies, a de novo transcriptome was developed and annotated using sequence similarity to Drosophila melanogaster. Under a variety of assembly parameters, several genes were identified as being differentially expressed between predators and nonpredators of this species, including genes involved in cell-to-cell signaling, osmotic regulation, starvation responses, and dopamine regulation. Results of this work were integrated to develop a model of the processes and genetic regulation controlling facultative predation.

Identifying similar transcripts in a related organism from de Bruijn graphs of RNA-Seq data, with applications to the study of salt and waterlogging tolerance in Melilotus.

BACKGROUND: A popular strategy to study alternative splicing in non-model organisms starts from sequencing the entire transcriptome, then assembling the reads by using de novo transcriptome assembly algorithms to obtain predicted transcripts. A similarity search algorithm is then applied to a related organism to infer possible function of these predicted transcripts. While some of these predictions may be inaccurate and transcripts with low coverage are often missed, we observe that it is possible to obtain a more complete set of transcripts to facilitate possible functional assignments by starting the search from the intermediate de Bruijn graph that contains all branching possibilities. RESULTS: We develop an algorithm to extract similar transcripts in a related organism by starting the search from the de Bruijn graph that represents the transcriptome instead of from predicted transcripts. We show that our algorithm is able to recover more similar transcripts than existing algorithms, with large improvements in obtaining longer transcripts and a finer resolution of isoforms. We apply our algorithm to study salt and waterlogging tolerance in two Melilotus species by constructing new RNA-Seq libraries. CONCLUSIONS: We have developed an algorithm to identify paths in the de Bruijn graph that correspond to similar transcripts in a related organism directly. Our strategy bypasses the transcript prediction step in RNA-Seq data and makes use of support from evolutionary information.

A comparative evaluation of hybrid error correction methods for error-prone long reads.

BACKGROUND: Third-generation sequencing technologies have advanced the progress of the biological research by generating reads that are substantially longer than second-generation sequencing technologies. However, their notorious high error rate impedes straightforward data analysis and limits their application. A handful of error correction methods for these error-prone long reads have been developed to date. The output data quality is very important for downstream analysis, whereas computing resources could limit the utility of some computing-intense tools. There is a lack of standardized assessments for these long-read error-correction methods. RESULTS: Here, we present a comparative performance assessment of ten state-of-the-art error-correction methods for long reads. We established a common set of benchmarks for performance assessment, including sensitivity, accuracy, output rate, alignment rate, output read length, run time, and memory usage, as well as the effects of error correction on two downstream applications of long reads: de novo assembly and resolving haplotype sequences. CONCLUSIONS: Taking into account all of these metrics, we provide a suggestive guideline for method choice based on available data size, computing resources, and individual research goals.

Emerging roles of transforming growth factor ß signaling in wet age-related macular degeneration.

Age-related macular degeneration (AMD) is one of the major causes of irreversible blindness among aging populations in developed countries and can be classified as dry or wet according to its progression. Wet AMD, which is characterized by angiogenesis on the choroidal membrane, is uncommonly seen but more severe. Controlling or completely inhibiting the factors that contribute to the progression of events that lead to angiogenesis may be an effective strategy for treating wet AMD. Emerging evidence has shown that transforming growth factor-ß (TGF-ß) signaling plays a significant role in the progression of wet AMD. In this review, we described the roles of and changes in TGF-ß signaling in the development of AMD and discussed the mechanisms of the TGF-ß superfamily in choroidal neovascularization (CNV) and wet AMD, including the modulation of angiogenesis-related factors, inflammation, vascular fibrosis, and immune responses, as well as cross-talk with other signaling pathways. These remarkable findings indicate that TGF-ß signaling is a potential target for wet AMD treatment.

GEP100/ARF6 regulates VEGFR2 signaling to facilitate high-glucose-induced epithelial-mesenchymal transition and cell permeability in retinal pigment epithelial cells.

Cell permeability and epithelial-mesenchymal transition (EMT) were found to be enhanced in diabetic retinopathy, and the aim of this study was to investigate the underlying mechanism. ARPE-19 cell line or primary retinal pigment epithelial (RPE) cells were cultured under high or normal glucose conditions. Specific shRNAs were employed to knock down ADP-ribosylation factor 6 (ARF6), GEP100, or VEGF receptor 2 (VEGFR2) in ARPE-19 or primary RPE cells. Cell migration ability was measured using Transwell assay. Western blotting was used to measure indicated protein levels. RPE cells treated with high glucose showed increased cell migration, paracellular permeability, EMT, and expression of VEGF. Knockdown of VEGFR2 inhibited the high-glucose-induced effects on RPE cells via inactivation of ARF6 and MAPK pathways. Knockdown ARF6 or GEP100 led to inhibition of high-glucose-induced effects via inactivation of VEGFR2 pathway. Knockdown of ARF6, but not GEP100, decreased high-glucose-induced internalization of VEGFR2. High-glucose enhances EMT and cell permeability of RPE cells through activation of VEGFR2 and ARF6/GEP100 pathways, which form a positive feedback loop to maximize the activation of VEGF/VEGFR2 signaling.

Down-regulation of microRNA-216a confers protection against yttrium aluminium garnet laser-induced retinal injury via the GDNF-mediated GDNF/GFRα1/RET signalling pathway.

Retinal injury plays a leading role in the onset of visual impairment. Current forms of treatment are not able to ameliorate scarring, cell death and tissue and axon regeneration. Recently, microRNA-216a (miR-216a) has been reported to regulate snx5, a novel notch signalling pathway component during retinal development. This study aims to elucidate the role of miR-216a in yttrium aluminium garnet (YAG) laser-induced retinal injury by targeting glial cell line-derived neurotrophic factor (GDNF) via GDNF/GDNF family neurotrophic factor receptor α1 (GFRα1)/rearranged during transfection (RET) signalling pathway. Wistar male rats were first randomly assigned into control and model groups. Immunohistochemistry was performed to detect the GDNF positive expression rate and terminal deoxynucleotidyl transferase-mediated dUTP nick end labelling (TUNEL) staining for apoptotic index (AI) of retinal tissue. Retinal neurons were divided into normal, blank, negative control (NC), miR-216a mimic, miR-216a inhibitor, siRNA-GDNF and miR-216a inhibitor?siRNA-GDNF groups. Dual luciferase reporter assay was conducted in order to identify the targeting relationship between GDNF and miR-216a. Reverse transcription quantitative polymerase chain reaction (RT-qPCR) and western blot were used for the analysis of mRNA and protein levels of miR-216a and related genes. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was used to determine cell proliferation and flow cytometry was used to observe cell cycle and apoptosis. Results show that the model group had an increased GDNF positive rate, AI of retinal tissue and mRNA and protein levels of cellular oncogene fos (c-fos), vascular endothelial growth factor (VEGF), brain-derived neurotrophic factor (BDNF), GDNF, GFRα1 and bcl-2-associated X protein (bax), declined miR-216a level and mRNA and protein levels of RET and bcl-2 compared with the control group. GDNF was verified as the target gene for miR-216a. Compared with the blank and NC groups, the miR-216a mimic and siRNA-GDNF groups had higher mRNA and protein levels of c-fos, VEGF and bax, cell number in the G1 phase and increased cell apoptosis but reduced BDNF, GDNF, GFRα1, RET and bcl-2 expression, cell proliferation and cell numbers in the S phase, while the opposite trend was observed in the miR-216a inhibitor group. Taken together, our findings demonstrate that elevated GDNF levels can reduce the retinal injury, whereby down-regulated miR-216a aggravates the YAG laser-induced retinal injury by targeting the GDNF level through the GDNF/ GFRα1/RET signalling pathway.

IDP-denovo: de novo transcriptome assembly and isoform annotation by hybrid sequencing.

Motivation: In the past years, the long read (LR) sequencing technologies, such as Pacific Biosciences and Oxford Nanopore Technologies, have been demonstrated to substantially improve the quality of genome assembly and transcriptome characterization. Compared to the high cost of genome assembly by LR sequencing, it is more affordable to generate LRs for transcriptome characterization. That is, when informative transcriptome LR data are available without a high-quality genome, a method for de novo transcriptome assembly and annotation is of high demand. Results: Without a reference genome, IDP-denovo performs de novo transcriptome assembly, isoform annotation and quantification by integrating the strengths of LRs and short reads. Using the GM12878 human data as a gold standard, we demonstrated that IDP-denovo had superior sensitivity of transcript assembly and high accuracy of isoform annotation. In addition, IDP-denovo outputs two abundance indices to provide a comprehensive expression profile of genes/isoforms. IDP-denovo represents a robust approach for transcriptome assembly, isoform annotation and quantification for non-model organism studies. Applying IDP-denovo to a non-model organism, Dendrobium officinale, we discovered a number of novel genes and novel isoforms that were not reported by the existing annotation library. These results reveal the high diversity of gene isoforms in D.officinale, which was not reported in the existing annotation library. Availability and implementation: The dataset of Dendrobium officinale used/analyzed during the current study has been deposited in SRA, with accession code SRP094520. IDP-denovo is available for download at www.healthcare.uiowa.edu/labs/au/IDP-denovo/. Supplementary information: Supplementary data are available at Bioinformatics online.

VEGF as a Trophic Factor for Müller Glia in Hypoxic Retinal Diseases.

Age-related macular degeneration (AMD) and diabetic retinopathy (DR), leading causes of blindness, share a common retinal environment: hypoxia which is a major stimulator for the upregulation of vascular endothelial growth factor (VEGF), a cardinal pathogenic factor for the breakdown of blood-retina barrier (BRB). As a result of intensive studies on VEGF pathobiology, anti-VEGF strategy has become a major therapeutics for wet AMD and DR. To investigate the potential impact of anti-VEGF strategy on major retinal supporting cells, Müller glia (MG), we disrupted VEGF receptor-2 (VEGFR2) in MG with conditional knockout (CKO) and examined the effect of VEGFR2-null on MG viability and neuronal integrity in mice. VEGFR2 CKO mice demonstrated a significant loss of MG density in diabetes/hypoxia, which in turn resulted in accelerated retinal degeneration. These defects appear similar to the clinical characteristics in a significant portion of wet-AMD patients with long-term anti-VEGF therapies. In this article, we will discuss the potential relevance of these clinical characteristics to the critical role of VEGF signaling in MG viability and neuronal integrity in hypoxia.

Hybrid Sequencing of Full-Length cDNA Transcripts of Stems and Leaves in Dendrobium officinale.

Dendrobium officinale is an extremely valuable orchid used in traditional Chinese medicine, so sought after that it has a higher market value than gold. Although the expression profiles of some genes involved in the polysaccharide synthesis have previously been investigated, little research has been carried out on their alternatively spliced isoforms in D. officinale. In addition, information regarding the translocation of sugars from leaves to stems in D. officinale also remains limited. We analyzed the polysaccharide content of D. officinale leaves and stems, and completed in-depth transcriptome sequencing of these two diverse tissue types using second-generation sequencing (SGS) and single-molecule real-time (SMRT) sequencing technology. The results of this study yielded a digital inventory of gene and mRNA isoform expressions. A comparative analysis of both transcriptomes uncovered a total of 1414 differentially expressed genes, including 844 that were up-regulated and 570 that were down-regulated in stems. Of these genes, one sugars will eventually be exported transporter (SWEET) and one sucrose transporter (SUT) are expressed to a greater extent in D. officinale stems than in leaves. Two glycosyltransferase (GT) and four cellulose synthase (Ces) genes undergo a distinct degree of alternative splicing. In the stems, the content of polysaccharides is twice as much as that in the leaves. The differentially expressed GT and transcription factor (TF) genes will be the focus of further study. The genes DoSWEET4 and DoSUT1 are significantly expressed in the stem, and are likely to be involved in sugar loading in the phloem.