Recent Advances in the Heterologous Expression of Biosynthetic Gene Clusters for Marine Natural Products.

Marine natural products (MNPs) are an important source of biologically active metabolites, particularly for therapeutic agent development after terrestrial plants and nonmarine microorganisms. Sequencing technologies have revealed that the number of biosynthetic gene clusters (BGCs) in marine microorganisms and the marine environment is much higher than expected. Unfortunately, the majority of them are silent or only weakly expressed under traditional laboratory culture conditions. Furthermore, the large proportion of marine microorganisms are either uncultivable or cannot be genetically manipulated. Efficient heterologous expression systems can activate cryptic BGCs and increase target compound yield, allowing researchers to explore more unknown MNPs. When developing heterologous expression of MNPs, it is critical to consider heterologous host selection as well as genetic manipulations for BGCs. In this review, we summarize current progress on the heterologous expression of MNPs as a reference for future research.

Next-generation Sequencing Reveals Age-dependent Genetic Underpinnings in Lung adenocarcinoma.

Background: More than 40% of lung cancer patients are diagnosed at ages over 70. However, the genomic and clinical characteristics among them remain elusive. Here, we performed targeted capture sequence to characterize the mutational spectrum of Chinese lung adenocarcinoma (LUAD) patients across ages. Patients and Methods: 2025 LUAD patients were divided into three groups: young (≤50 years old) (n=416, 20.54%), intermediate (51~69 years old) (n=1271, 62.77%), and aged (≥70 years old) (n=338, 16.69%). 1,021-gene panel and 59-gene panel were used for sequencing with tissue samples. Genetic alterations and tumor mutation burden (TMB) in LUAD patients were investigated. Results: The frequency of mutations affecting 20 genes were significantly higher in aged group than in young group, and fourteen of them were not reported before, including involved in cell cycle/apoptosis signaling (FAT1, FAT2), DNA damage repair (FANCA and FANCM), chromatin histone modification (KDM6A), RTK/RAS/PI3K signaling (FLT4 and MTOR), NOTCH signaling (NOTCH1, NOTCH2 and NOTCH4) and other signaling pathway or cellular regulatory factor (KEAP1, ASXL1, EPHB1 and ABCB1). Six previously reported mutated genes (RBM10, KRAS, LRP1B, CDKN2A and KMT2C/D) were also significantly more frequent in aged group. Among clinical actionable mutation sites, KRAS mutation was presented more common in aged group; both MET exon 14 skipping and MET amplification were significantly positively correlated with old age; the fusions of ALK, ROS1, RET and ERBB2 exon 20 insertion were less frequent in aged group. Furthermore, a higher level of TMB was found in aged group compared with young group. Conclusion: In this study, we revealed the differences of somatic genetic mutations and TMB between young and aged LUAD patients, which may provide directions of targeted therapy and advantages of immunotherapy for the elderly in the future.

Liquid biopsy in diffuse large B-cell lymphoma: utility in cell origin determination and survival prediction in Chinese patients.

The utility of circulating tumor DNA (ctDNA) in classifying the cell origin of diffuse large B-cell lymphoma (DLBCL) has not been explored in the Chinese population. In this study, we aimed to investigate the genetic characteristics of DLBCL based on both tumor and ctDNA sequencing and to assess the predictive value of ctDNA in DLBCL. A targeted sequencing panel of 413 genes was applied to tumor biopsies and paired plasma samples obtained from 30 patients with DLBCL before therapeutic intervention (pretreatment). The concordance between plasma genotyping classification and traditional cell-of-origin classification using tumor tissue was 80.0% (20/25). Patients with higher baseline plasma ctDNA levels had poorer survival compared to those with lower ctDNA levels (2-year progression survival rate: 40.0% vs. 80.0%, p = 0.011; 5-year overall survival rate: 30.5% vs. 70.0%, p = 0.004). Collectively, our results demonstrated that pretreatment ctDNA analysis could assist origin determination and prognosis prediction clinically.

HDAC6 regulates primordial follicle activation through mTOR signaling pathway.

Primordial follicle pool established perinatally is a non-renewable resource which determines the female fecundity in mammals. While the majority of primordial follicles in the primordial follicle pool maintain dormant state, only a few of them are activated into growing follicles in adults in each cycle. Excessive activation of the primordial follicles accelerates follicle pool consumption and leads to premature ovarian failure. Although previous studies including ours have emphasized the importance of keeping the balance between primordial follicle activation and dormancy via molecules within the primordial follicles, such as TGF-ß, E-Cadherin, mTOR, and AKT through different mechanisms, the homeostasis regulatory mechanisms of primordial follicle activation remain unclear. Here, we reported that HDAC6 acts as a key negative regulator of mTOR in dormant primordial follicles. In the cytoplasm of both oocytes and granulosa cells of primordial follicles, HDAC6 expressed strong, however in those activated primordial follicles, its expression level is relatively weaker. Inhibition or knockdown of HDAC6 significantly promoted the activation of limited primordial follicles while the size of follicle pool was not affected profoundly in vitro. Importantly, the expression level of mTOR in the follicle and the activity of PI3K in the oocyte of the follicle were simultaneously up-regulated after inhibiting of HDAC6. The up-regulated mTOR leads to not only the growth and differentiation of primordial follicles granulosa cells (pfGCs) into granulosa cells (GCs), but the increased secretion of KITL in these somatic cells. As a result, inhibition of HDAC6 awaked the dormant primordial follicles of mice in vitro. In conclusion, HDAC6 may play an indispensable role in balancing the maintenance and activation of primordial follicles through mTOR signaling in mice. These findings shed new lights on uncovering the epigenetic factors involved physiology of sustaining female reproduction.

Characteristics and Management of TP53-Mutated Diffuse Large B-Cell Lymphoma Patients.

BACKGROUND/AIM: TP53 mutation is recognized as a negative prognostic factor for patients with diffuse large B-cell lymphoma (DLBCL). Here, we present the characteristics of TP53 mut DLBCL patients following investigation of the effect of a treatment approach on survival of TP53 mut DLBCL patients. METHODS: A total of 44 DLBCL patients with TP53 mut and treated with an R-CHOP regimen were included for analysis. Patients who failed to achieve a complete response (CR) to initial treatment or relapsed in the first 6 months after initial CR were deemed to have primary refractory disease. RESULTS: Among 44 patients harboring TP53 mutations who underwent upfront R-CHOP or R-CHOP-like treatment, 21 (47.7%) had limited-stage and 23 (52.3%) presented advanced-stage disease. Apart from the seven patients receiving upfront surgical resection, 37 had measurable disease under the R-CHOP regimen, with 59.1% (n=26) developing primary refractory disease. Seven limited-stage patients after early complete resection and one with residue resection remained event-free at median follow-up of 37 months. Multivariate analysis revealed that elevated baseline lactate dehydrogenase (LDH), extranodal involvement (two or more), Ann Arbor stage, and locoregional treatment (surgery or radiation therapy) were independent indicators for progression-free survival (PFS). After adjustment for baseline LDH and extranodal involvement, adding locoregional treatment including surgery and radiation to the R-CHOP regimen significantly improved PFS (p=0.008) and overall survival (p=0.017) in limited-stage TP53 mut DLBCL patients compared to R-CHOP-only treatment. CONCLUSION: This study presents the characteristics of TP53-mutated DLBCL and implies a potential benefit of locoregional treatment in limited-stage DLBCL patients with TP53 mutations.

Circulating tumor DNA predicts response in Chinese patients with relapsed or refractory classical hodgkin lymphoma treated with sintilimab.

BACKGROUND: Blood-based biomarker such as circulating tumor DNA (ctDNA) has emerged as a promising tool for assessment of response to immunotherapy in solid tumors; But in hematological malignances, evidences are still lacking to support its clinical utility. In current study the feasibility of ctDNA for prediction and monitoring of response to anti-PD-1 therapy in Chinese patients with relapsed or refractory classical Hodgkin lymphoma (r/r cHL) was assessed. METHODS: A total of 192 plasma samples from 75 patients with r/r cHL were collected at baseline and upon therapeutic evaluation. ctDNA were sequenced by targeting panels capturing frequently mutated genes in cHL and other hematological malignancies and then quantified. Analysis on: 1) Gene mutation profile and association of the gene mutations with progression-free survival; 2) Association of pre- and post-treatment ctDNA variant allelic frequencies with clinical outcome; (3) Correlation of the mutated genes with treatment resistance; were performed. FINDINGS: Somatic mutations were detected in 50 out of 61 patients by ctDNA genotyping. The mutations of CHD8 was significantly higher in patients with PFS ≥ 12 months. Baseline ctDNA was significantly higher in responders and a decrease of ctDNA ≥ 40% from baseline indicated superior clinical outcome. Strong agreement between ctDNA dynamic and radiographic response change during therapy was observed in majority of the patients. Furthermore, the mutations of B2M, TNFRSF14 and KDM2B were found to be associated with acquired resistance. INTERPRETATION: ctDNA could be an informative biomarker for anti-PD-1 immunotherapy in r/r cHL. FUNDING: This work was supported by Innovent Biologics, Eli Lilly and Companyhttps://doi.org/10.13039/501100002852, China National New Drug Innovation Program (2014ZX09201041-001 and 2017ZX09304015), Chinese Academy of Medical Sciences (CAMS) Innovation Fund for Medical Sciences (CIFMS) (2016-I2M-1-001) and National Key Scientific Program Precision Medicine Research Fund of China (2017YFC0909801). The funders had no role in study design, data collection, data analysis, interpretation or writing.

Genomic Alterations and MYD88MUT Variant Mapping in Patients with Diffuse Large B-Cell Lymphoma and Response to Ibrutinib.

BACKGROUND: Diffuse large B-cell lymphoma (DLBCL) is a clinically heterogeneous malignancy. Following front-line immunochemotherapy, 30-40% of DLBCL patients develop relapsed or refractory (r/r) disease, which can be treated with ibrutinib. It has been previously reported that MYD88MUT affects the response to ibrutinib in patients with r/r DLBCL. OBJECTIVE: Here, we aimed to gather understanding of MYD88MUT in r/r DLBCL patients and to evaluate its influence on response to ibrutinib. PATIENTS AND METHODS: In this study, tissue samples from DLBCL patients (n = 212) were retrospectively collected and sequenced by target-capturing panels of either 413 or 112 genes that are frequently mutated in non-Hodgkin's lymphoma. Sixty patients with MYD88 mutations and available clinical information were included for further analysis. RESULTS: Seven MYD88MUT variants were identified, L265P (65.0%, n = 39), S219C (13.3%, n = 8), S243N (8.3%, n = 5), P258L (6.7%, n = 4), F283V (1.7%, n = 1), P141R (1.7%, n = 1), and V217F (1.7%, n = 1). One patient had MYD88 amplification. In addition, mutations in PIM1 (67%, n = 40), IGH fusion (48%, n = 29), CD79B (43%, n = 26), KMT2D (30%, n = 18), and TP53 (27%, n = 17) were identified. For patients with L265P, IRF4 (p = 0.011) was frequently mutated. Otherwise, TET2 (p = 0.016), NOTCH2 (p = 0.044), MET (p = 0.037), SOCS1 (p = 0.011), TNFRSF14 (p = 0.011), EZH2 (p = 0.037), and BCL6 (p < 0.001) mutations were associated with MYD88MUT non-L265P variants. The incidence rate of MYD88MUT L265P was significantly higher with central nervous system involvement (p = 0.034). Four out of nine MYD88MUT patients responded to ibrutinib containing treatment, and this included those with MYD88MUT/CD79BWT. CONCLUSIONS: This study adds clinical observations with MYD88MUT patients, further helping to understand the genetic features and possible correlation of MYD88MUT with response to ibrutinib.

SIRT1 facilitates primordial follicle recruitment independent of deacetylase activity through directly modulating Akt1 and mTOR transcription.

In female mammals, the majority of primordial follicles (PFs) are physiologically quiescent, and only a few of them are activated and enter the growing follicle pool. Specific molecules, such as mammalian target of rapamycin (mTOR) and the serine/threonine kinase Akt (AKT), have been proven to be important for PF activation. However, how the transcription of these genes is regulated is not clear. Although activators of mTOR or AKT have been successfully used to rescue the fertility of patients with premature ovarian insufficiency, the low efficacy and unclear safety profile of these drugs hinder their clinical use in the in vitro activation (IVA) of PFs. Here, sirtuin 1 (SIRT1), an NAD-dependent deacetylase, was demonstrated to activate mouse PFs independent of its deacetylase activity. SIRT1 was prominently expressed in pregranulosa cells (pGCs) and oocytes, and its expression was increased during PF activation. PF activation was achieved by either up-regulating SIRT1 with a specific activator or overexpressing SIRT1. Moreover, SIRT1 knockdown in oocytes or pGCs could significantly suppress PF activation. Further studies demonstrated that SIRT1 enhanced both Akt1 and mTOR expression by acting more as a transcription cofactor, directly binding to the respective gene promoters, than as a deacetylase. Importantly, we explored the potential clinical applications of targeting SIRT1 in IVA via short-term treatment of cultured ovaries from mice and human ovarian tissues to activate PFs by applying the SIRT1 activator resveratrol. RSV-induced IVA could be a candidate strategy to develop more efficient procedures for future clinical treatment of infertility.-Zhang, T., Du, X., Zhao, L., He, M., Lin, L., Guo, C., Zhang, X., Han, J., Yan, H., Huang, K., Sun, G., Yan, L., Zhou, B., Xia, G., Qin, Y., Wang, C. SIRT1 facilitates primordial follicle recruitment independent of deacetylase activity through directly modulating Akt1 and mTOR transcription.

Clinical application of multicolour scanning laser imaging in diabetic retinopathy.

To compare the visualization of the lesions of diabetic retinopathy (DR) using multicolour scanning laser imaging (MSLI) and conventional colour fundus photography (CFP). The paired images of diabetic patients who underwent same-day MSLI and CFP examinations were reviewed. Combined multicolour (MC) images were acquired simultaneously using three laser wavelengths: blue reflectance (BR, λ = 488 nm), green reflectance (GR, λ = 518 nm) and infrared reflectance (IR, λ = 820 nm). The number of positive DR lesions was calculated using fundus fluorescein angiography as the reference standard. The visibility of the microaneurysms (Mas) was graded using a scale, and the number of Mas for each method was counted by two masked readers. Eighty eyes of 42 diabetic patients were included. The average grading score for Mas visualization was significantly higher with MC (1.50 ± 0.71) and GR (1.55 ± 0.69) than with CFP (0.95 ± 0.81). The average number of Mas was also significantly higher with MC (11.41 ± 14.02) and GR (11.93 ± 13.43) than with CFP (6.43 ± 9.39). The number of positive Mas, diabetic macular edema (DME) and epiretinal membranes (ERM) were significantly higher with MC than CFP (P < 0.05), while the numbers of cotton wool spots, haemorrhages, hard exudates, venous beading and abnormal new vessels were not significantly different (P > 0.05). Mas and ERM were most effectively detected on GR images, and an elevated greenish shift was clearly visualized in patients with DME on the MC images. MSLI can effectively visualize Mas and other pathological lesions of DR compared with CFP. MSLI with superior resolution may be a useful complement for DME and ERM detection.

G protein-coupled receptor 91 signaling in diabetic retinopathy and hypoxic retinal diseases.

G protein-coupled receptor 91 (GPR91) is a succinate-specific receptor and activation of GPR91 could initiate a complex signal transduction cascade and upregulate inflammatory and pro-angiogenic cytokines. In the retina, GPR91 is predominately expressed in ganglion cells, a major cellular entity involved in the pathogenesis of diabetic retinopathy (DR) and other hypoxic retinal diseases. During the development of DR and retinopathy of prematurity (ROP), chronic hypoxia causes an increase in the levels of local succinate. Succinate-mediated GPR91 activation upregulates vascular endothelial growth factor (VEGF) through ERK1/2-C/EBP ß (c-Fos) and/or ERK1/2-COX-2/PGE2 signaling pathways, which in turn, leads to the breakdown of blood-retina barriers in these disorders. In this review, we will have a brief introduction of GPR91 and its biological functions and a more detailed discussion about the role and mechanisms of GPR91 in DR and ROP. A better understanding of GPR91 regulation may be of great significance in identifying new biomarkers and drug targets for the prediction and treatment of DR, ROP, and hypoxic retinal diseases.

Comparison of spectral-domain optical coherence tomography for intra-retinal layers thickness measurements between healthy and diabetic eyes among Chinese adults.

PURPOSE: To compare intra-retinal layer thickness measurements between eyes with no or mild diabetic retinopathy (DR) and age-matched controls using Spectralis spectral-domain optical coherence tomography (SD-OCT). METHODS: Cross-sectional observational analysis study. High-resolution macular volume scans (30° * 25°) were obtained for 133 type 2 diabetes mellitus (T2DM) patients with no DR, 42 T2DM patients with mild DR and 115 healthy controls. The mean thickness was measured in all 9 Early Treatment Diabetic Retinopathy Study (ETDRS) sectors for 8 separate layers, inner retinal layer (IRL), outer retinal layer (ORL) and total retina (TR), after automated segmentation. The ETDRS grid consisted of three concentric circles of 1-, 3-, and 6-mm diameter. The superior, inferior, temporal, and nasal sectors of the 3- and 6-mm circles were respectively designated as S3, I3, T3, and N3 and S6, I6, T6, and N6. Linear regression analyses were conducted to evaluate the associations between the intra-retinal layer thicknesses, age, diabetes duration, fasting blood glucose and HbA1c. RESULTS: The mean age and duration of T2DM were 61.1 and 13.7 years, respectively. Although no significant differences in the average TR and ORL volumes were observed among the groups, significant differences were found in the volume and sectorial thicknesses of the inner plexiform layer (IPL), outer plexiform layer (OPL) and IRL among the groups. In particular, the thicknesses of the IPL (S3, T3, S6, I6 and T6 sectors) and the IRL (S6 sector) were decreased in the no-DR group compared with the controls (P < 0.05). The thickness of the OPL (S3, N3, S6 and N6 sectors) was thinner in the no-DR group than in mild DR (P < 0.05). The average IPL thickness was significantly negatively correlated with age and the duration of diabetes. CONCLUSION: The assessment of the intra-retinal layer thickness showed a significant decrease in the IPL and IRL thicknesses in Chinese adults with T2DM, even in the absence of visible microvascular signs of DR.

Transcription factors regulate GPR91-mediated expression of VEGF in hypoxia-induced retinopathy.

Hypoxia is the most important factor in the pathogenesis of diabetic retinopathy (DR). Our previous studies demonstrated that G protein-coupled receptor 91(GPR91) participated in the regulation of vascular endothelial growth factor (VEGF) secretion in DR. The present study induced OIR model in newborn rats using exposure to alternating 24-hour episodes of 50% and 12% oxygen for 14 days. Treatment with GPR91 shRNA attenuated the retinal avascular area, abnormal neovascularization and pericyte loss. Western blot and qRT-PCR demonstrated that CoCl2 exposure promoted VEGF expression and secretion, activated the ERK1/2 signaling pathways and upregulated C/EBP and AP-1. Knockdown of GPR91 inhibited ERK1/2 activity. GPR91 siRNA transduction and the ERK1/2 inhibitor U0126 inhibited the increases in C/EBP ß, C/EBP δ, c-Fos and HIF-1α. Luciferase reporter assays and a chromatin immunoprecipitation (ChIP) assay demonstrated that C/EBP ß and c-Fos bound the functional transcriptional factor binding site in the region of the VEGF promoter, but not C/EBP δ. Knockdown of C/EBP ß and c-Fos using RNAi reduced VEGF expression. Our data suggest that activation of the GPR91-ERK1/2-C/EBP ß (c-Fos, HIF-1α) signaling pathway plays a tonic role in regulating VEGF transcription in rat retinal ganglion cells.

Population-based survey of prevalence, causes, and risk factors for blindness and visual impairment in an aging Chinese metropolitan population.

AIM: To assess the prevalence, causes, and risk factors for blindness and visual impairment among elderly (≥60 years of age) Chinese people in a metropolitan area of Shanghai, China. METHODS: Random cluster sampling was conducted to identify participants among residents ≥60 years of age living in the Xietu Block, Xuhui District, Shanghai, China. Presenting visual acuity (PVA) and best-corrected visual acuity (BCVA) were checked by the Early Treatment Diabetic Retinopathy Study (ETDRS) visual chart. All eligible participants underwent a comprehensive eye examination. Blindness and visual impairment were defined according to World Health Organization (WHO) criteria. RESULTS: A total of 4190 persons (1688 men and 2502 women) participated in the study, and the response rate was 91.1%. Based on PVA, the prevalence of blindness was 1.1% and that of visual impairment was 7.6%. Based on BCVA, the prevalence of blindness and visual impairment decreased to 0.9% and 3.9%, respectively. Older (≥80 years of age) women, with low educational levels and smoking habits, exhibited a significantly greater chance for blindness and visual impairment than did those with high educational levels and no smoking habits (P<0.05). Based on PVA and BCVA, the main causes of blindness were cataract, myopic maculopathy, and age-related macular degeneration (AMD). CONCLUSION: Our findings help to identify the population in need of intervention, to highlight the need for additional eye healthcare services in urban China.

Rac1 modulates the formation of primordial follicles by facilitating STAT3-directed Jagged1, GDF9 and BMP15 transcription in mice.

The size of the primordial follicle pool determines the reproductive potential of mammalian females, and establishment of the pool is highly dependent on specific genes expression. However, the molecular mechanisms by which the essential genes are regulated coordinately to ensure primordial follicle assembly remain a mystery. Here, we show that the small GTPase Rac1 plays an indispensable role in controlling the formation of primordial follicles in mouse ovary. Employing fetal mouse ovary organ culture system, we demonstrate that disruption of Rac1 retarded the breakdown of germline cell cysts while Rac1 overexpression accelerated the formation of primordial follicles. In addition, in vivo inhibitor injection resulted in the formation of multi-oocyte follicles. Subsequent investigation showed that Rac1 induced nuclear import of STAT3 by physical binding. In turn, nuclear STAT3 directly activated the transcription of essential oocyte-specific genes, including Jagged1, GDF9, BMP15 and Nobox. Further, GDF9 and BMP15 regulated the translation of Notch2 via mTORC1 activation in pregranulosa cells. Overexression or addition of Jagged1, GDF9 and BMP15 not only reversed the effect of Rac1 disruption, but also accelerated primordial follicle formation via Notch2 signaling activation. Collectively, these results indicate that Rac1 plays important roles as a key regulator in follicular assembly.

Minocycline inhibits PARP­1 expression and decreases apoptosis in diabetic retinopathy.

The present study aimed to investigate the mechanism underlying the effects of minocycline on diabetic retinopathy­associated cellular apoptosis. A total of 40 Sprague Dawley (SD) rats were used as a diabetic retinopathy model following injection with streptozotocin. Among the 34 rats in which the diabetes model was successfully established, 24 rats were divided into two experimental groups: I and II (T1 and T2, respectively), and orally administered with various doses of minocycline. The remaining 10 rats served as the diabetic retinopathy control group. An additional group of 10 healthy SD rats with comparable weight served as normal controls. The rats in T1 and T2 groups were treated daily for eight consecutive weeks with minocycline at a dose of 2.5 mg/kg and 5 mg/kg, respectively. The mRNA expression levels of poly (ADP­ribose) polymerase­1 (PARP­1) were subsequently measured by reverse transcription­quantitative polymerase chain reaction, and the protein expression levels of poly­ADP­ribose were measured by western blot analysis and immunohistochemistry. Retinal morphology was observed following hematoxylin and eosin staining, and retinal cell apoptosis was measured by terminal deoxynucleotidyl transferase dUTP nick end labeling and caspase­3 activity assays. The amplitudes of the electroretinogram (ERG) b­wave and oscillary potentials (OPs) were measured using visual electrophysiology, and compared among the four groups. The results of the present study demonstrated that in the diabetic rats, retinal PARP­1 gene expression was markedly upregulated, the number of apoptotic cells and the activity levels of caspase­3 were increased, and the amplitude of the ERG b­wave and the OPs were markedly lower as compared with the normal rats. Following treatment with minocycline, the abnormal expression of PARP­1 in the retina was inhibited, and cellular apoptosis was decreased. In conclusion, the results of the present study suggest that PARP­1 is involved in the development of diabetic retinopathy, and minocycline is able to inhibit PARP­1 expression and decrease cellular apoptosis, suggesting that minocycline may prove to be a promising drug for the treatment of diabetic retinopathy.

Altered retinal microRNA expression profiles in early diabetic retinopathy: an in silico analysis.

PURPOSE: MicroRNAs (miRNAs) - as negative regulators of target genes - are associated with various human diseases, but their precise role(s) in diabetic retinopathy (DR) remains to be elucidated. The aim of this study was to elucidate the involvement of miRNAs in early DR using in silico analysis to explore their gene expression patterns. METHODS: We used the streptozotocin (STZ)-induced diabetic rat to investigate the roles of miRNAs in early DR. Retinal miRNA expression profiles from diabetic versus healthy control rats were examined by miRNA array analysis. Based on several bioinformatic systems, specifically, gene ontology (GO) and the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways, we identified signatures of the potential pathological processes, gene functions, and signaling pathways that are influenced by dysregulated miRNAs. We used quantitative real-time polymerase chain reaction (qRT-PCR) to validate six (i.e. those with significant changes in expression levels) of the 17 miRNAs that were detected in the miRNA array. We also describe the significant role of the miRNA-gene network, which is based on the interactions between miRNAs and target genes. RESULTS: GO analysis of the 17 miRNAs detected in the miRNA array analysis revealed the most prevalent miRNAs to be those related to biological processes, olfactory bulb development and axonogenesis. These miRNAs also exert significant influence on additional pathways, including the mitogen-activated protein and calcium signaling pathways. Six of the seventeen miRNAs were chosen for qRT-PCR validation. With the exception of a slight difference in miRNA-350, our results are in close agreement with the differential expressions detected by array analysis. CONCLUSIONS: This study, which describes miRNA expression during the early developmental phases of DR, revealed extensive miRNA interactions. Based on both their target genes and signaling pathways, we suggest that miRNAs perform critical regulatory functions during the early stages of DR evolution.

[Integration sites and their characteristic analysis of piggyBac transposon in cattle genome].

As a useful tool for genetic engineering, piggyBac (PB) transposons have been widely used in more than one species of transgenosis or generating mutation studies. At present, the studies about PB transposons in cattle were few. In order to get the PB transposon integration sites and summarize its characteristics in bovine genome, donor plasmid of PB[CMV-EGFP] and helper-dependent plasmid of pcDNA-PBase were constructed and transferred into bovine fibroblasts by Amaxa basic nucleofector kit for primary mammalian fibroblasts. Cell clones stably transfected were obtained after screening by G-418. Genomic DNA of transgenic cells was extracted and the integration sites of PB transposon were detected by genome walking technology. Eight integration sites were obtained in bovine genome, although only 5 sites were mapped on chromosomes 1, 2, 11, and X chromosome. We found that PB transposon was inserted into the "TTAA" location and integrated into the intergenic non-regulatory sites between two genes. Analysis of the composition of the five bases, which was close to the side of the PB integration sites "TTAA", showed that PB 5' tended to be inserted into region rich in GC (62.5%). From the study, we got that transposition occurred in cattle genome by PB transposons and the integration site information acquired from the research will provide theoretical references for bovine study by PB transposon.

Evaluating subconjunctival bleb function after trabeculectomy using slit-lamp optical coherence tomography and ultrasound biomicroscopy.

BACKGROUND: The existing classifications for evaluating glaucoma filtering blebs rely mostly on external bleb characteristics and the postoperative control of intraocular pressure (IOP). Internal bleb structures are not carefully observed. This study aimed to analyze and compare glaucoma filtering bleb morphology using slit-lamp-adapted optical coherence tomography (SL-OCT) and ultrasound biomicroscopy (UBM), and to classify blebs according to results and intraocular pressure. METHODS: We followed 29 eyes of 21 male patients and 40 eyes of 32 female patients who underwent glaucoma filtering surgery in Sixth People's Hospital of Shanghai, between 2002 and 2006. The blebs were imaged using SL-OCT and UBM and classified according to the intrableb morphology and control of IOP after surgery. A Fisher's exact test was used to compare the sensitivity for predicting a functioning bleb differed significantly between SL-OCT and UBM. A Fisher's exact test was also used for morphological analysis of the trabeculectomy blebs based on SL-OCT. RESULTS: In the 69 eyes, there were 45 (65.2%) functioning blebs and 24 (34.8%) non-functioning blebs. We classified the blebs into four categories on the basis of SL-OCT images: diffuse, cystic, encapsulated and flat. Diffuse and cystic blebs were typically functional, whereas the other two types were always non-functional. The sensitivity of SL-OCT for predicting a functioning bleb was 92.7% (38/41 eyes) and specificity of predicting a non-functioning bleb was 83.3% (20/24 eyes). By contrast, sensitivity of UBM was 66.7% (30/45 eyes) and specificity was 75.0% (18/24 eyes). The sensitivity for predicting a functioning bleb differed significantly between the two techniques (P = 0.003). CONCLUSIONS: SL-OCT provides high-axial-resolution images of anterior segment structures. The non-contact approach of SL-OCT enables visualization of intrableb structures at any time after surgery. SL-OCT has greater sensitivity and specificity than UBM in evaluating filtering bleb function. The morphological classification supported the assessment of bleb function and could provide objective data for evaluating the outcome of antiglaucoma surgery or the need for a second procedure.

[Evaluation of photodynamic therapy in idiopathic choroidal neovascularization].

OBJECTIVE: To evaluate the effectiveness of photodynamic therapy (PDT) in the treatment of idiopathic choroidal neovascularization (CNV). METHODS: Sixty-one cases (61 eyes) of CNV were treated with PDT and the fundus appearance, visual acuity, retina thickness as well as the fundus angiographic imaging were observed before and after the therapy. PDT was performed 1.2 times in average and the follow-up period was 6 - 36 months (mean 19 months). RESULTS: At the last follow up, the visual acuity was improved in 41 eyes (67.2%), unchanged in 15 eyes (24.6%) and slightly decreased in 5 eyes (8.2%). Macular hemorrhage and exudation reduced in all cases after PDT. Fundus angiography showed complete closure of CNV in 38 eyes (62.3%), partial closure in 4 eyes (6.6%), incomplete closure in 14 eyes (23.0%) and recurrence in 5 eyes (8.2%). In 6 eyes CNV was complete closed after single PDT with diminish of macular edema and neuronal retinal epithelial detachment. No recurrent CNV was observed during three years' follow-up and the visual acuity remained stable. Our results also demonstrated that the therapeutic effect decreased with patient's age (t = 0.476, P = 0.016). The decrease of visual acuity averaged 0.008 per year. CONCLUSION: Photodynamic therapy is a potential treatment for idiopathic CNV and better outcomes are achieved in younger patients.

[Clinical research of the implantation of phakic anterior chamber intraocular lens for high myopia].

OBJECTIVE: To investigate the efficacy and safety of the implantation of phakic anterior chamber intraocular lens (IOL) for high myopia. METHODS: A consecutive group of 73 eyes in 41 patients with -7.00 to -30.00 diopters (D) of myopia were implanted. RESULTS: All of 73 eyes were implanted successfully and have been followed-up for 3 m (months). The uncorrected visual acuity was from FC/33 cm to 0.2 pre-operatively and 0.1 to 1.0 3 m post-operatively. The best corrected visual acuity (BCVA) was from 0.05 to 1.0 pre-operatively and 0.1 to 1.0 3 m post-operatively. The post-operative BCVA of every eye was not worse than the pre-operative one. The refractive diopters were from -7.00 to -30.00 D pre-operatively and -6.00 to +2.50 D 3 m post-operatively. There were no significant differences between pre- and 3 m post-operative mean corneal astigmatism (t = 1.751, P = 0.082) and mean intraocular pressure (IOP) (t = 1.181, P = 0.240), respectively. The mean counts of endothelial cells was (2 680 +/- 538)/mm(2) pre-operatively and (2 514 +/- 420)/mm(2) 3 m post-operatively. There was no significant difference (t = 1.182, P = 0.242) though it decreased 6.19%. No severe complications occurred. CONCLUSIONS: The implantation of phakic anterior chamber IOL for high myopia is predictable, reversible and controllable with simple manipulation. No severe complication occurred in 3 m post-operatively, and long-time follow-up is still required.