Effect of terahertz pulse on gene expression in human eye cells

In recent years, the advances in terahertz applications have stimulated interest in the biological effects associated with this frequency range. We study the gene expression profile in three types of cells exposed to terahertz radiation,i.e., human ARPE-19 retinal pigment epithelial cells, simian virus 40-transformed human corneal epithelial cells, and human MIO-M1 Müller cells. We find that the gene expression in response to heat shock is unaffected, indicating that the minimum temperature increases under controlled environment. The transcriptome sequencing survey demonstrates that 6-hour irradiation with a broadband terahertz source results in specific change in gene expression and also the biological functions that are closely related to these genes. Our results imply that the effect of terahertz radiation on gene expression can last over 15 hours and depends on the type of cell.

Eyes of diff ering colors in Alvinocaris longirostris from deep-sea chemosynthetic ecosystems: genetic and molecular evidence of its formation mechanism

Coloration is an important phenotypic trait for multiple adaptive functions.It is interesting to fi nd white-eye(AW)and orange-eye(AO)phenotypes in the shrimp Alvinocaris longirostris inhabiting the deep-sea cold seep and hydrothermal vent areas of the northwestern Pacifi c.By comparative transcriptome analyses,1491 diff erentially expressed genes(DEGs)were identified between AW and AO.Among them,many DEGs were associated with immunity,antioxidation,and detoxifi cation.Two signifi cant enzyme encoding genes,xanthine dehydrogenase,and tryptophan oxidase involved in pigment biosynthesis pathways were up-regulated in AW and AO,respectively,which might be related to the diff erences of white and orange eye phenotypes.Moreover,single nucleotide polymorphism(SNP)calling detected that genotypes of 28 SNP distributing in 14 unigenes were completely diff erent between AW and AO.Particularly,there were three and two non-synonymous mutations in immune genes crustin Pm5 and antimicrobial peptide,respectively.Results indicate that the diff erence in eye color is probably resulted from immune response to variable micro-environmental stressors encountered in the dispersal process of the shrimps,such as symbiotic microbes,pathogens,and toxic substances,and might be genetically fi xed at last.The suggested pathway preliminarily explained the formation mechanism of diff erent eye phenotypes in Alvinocaridid shrimps,providing a basis for further study on adaptive evolution of eyes in deep-sea chemosynthetic faunas.

Whole transcriptome analysis on blue light-induced eye damage

AIM:To analyze abnormal gene expressions of mice eyes exposed to blue light using RNA-seq and analyze the related signaling pathways.METHODS:Kunming mice were divided into an experimental group that was exposed to blue light and a control group that was exposed to natural light.After 14 d,the mice were euthanized and their eyeballs were collected.Whole transcriptome analysis was attempted to analyze the gene expression of the eyeballs using RNA-seq to reconstruct genetic networks.Gene Ontology(GO)and Kyoto Encyclopedia of Genes and Genomes(KEGG)analysis were used to reveal the related signaling pathways.RESULTS:The 737 differentially expressed genes were identified,including 430 up and 307 down regulated genes,by calculating the gene FPKM in each sample and conducting differential gene analysis.GO and KEGG pathway enrichment analysis showed that blue light damage may associated with the visual perception,sensory perception of light stimulus,phototransduction,and JAKSTAT signaling pathways.Differential lnc RNA,circ RNA and mi RNA analysis showed that blue light exposure affected pathways for retinal cone cell development and phototransduction,among others.CONCLUSION:Exposure to blue light can cause a certain degree of abnormal gene expression and modulate signaling pathways in the eye.

Simultaneous expression of two pathogenic genes in four Chinese patients affected with inherited retinal dystrophy

●AIM:To describe the complex,overlapping phenotype of four Chinese patients with inherited retinal dystrophies(IRDs)who harbored two pathogenic genes simultaneously.●METHODS:This retrospective study included 4 patients affected with IRDs.Medical and ophthalmic histories were obtained,and clinical examinations were performed.A specific Hereditary Eye Disease Enrichment Panel(HEDEP)based on exome capture technology was used for genetic screening.●RESULTS:Four patients were identified to harbor disease-causing variants in two different genes.Patient retinitis pigmentosa(RP)01-II:1 exhibited both classical ABCA4-induced Stargardt disease(STGD)1 and USH2 Aassociated RP,patient RP02-III:2 exhibited both classical ABCA4-induced STGD1 and CDH23-associated RP,patient RP03-II:1 exhibited both USH2 A-induced autosomal recessive retinitis pigmentosa(arRP)syndrome and SNRNP200-induced autosomal dominant retinitis pigmentosa(adRP),and patient RP04-II:2 exhibited USH2 Ainduced arRP syndrome and EYS-induced arRP at the same time.●CONCLUSION:Our study demonstrates that genotype–phenotype correlations and comprehensive genetic screening is crucial for diagnosing IRDs and helping family planning for patients suffering from the disease.

Epigenetic Silencing of Eyes Absent 4 Gene by Acute Myeloid Leukemia 1-Eight-twenty-one Oncoprotein Contributes to Leukemogenesis in t（8;21） Acute Myeloid Leukemia

Background： The acute myeloid leukemia 1 （AML1）-eight-twenty-one （ETO） fusion protein generated by the t（8;2 1）（q22;q22） translocation is considered to display a crucial role in leukemogenesis in AML. By focusing on the anti-leukemia effects of eyes absent 4 （EYA4） gene on AML cells, we investigated the biologic and molecular mechanism associated with AML 1 -ETO expressed in t（8;21） AML. Methods： Qualitative polymerase chain reaction （PCR）, quantitative reverse transcription PCR （RT-PCR）, and Western blotting analysis were used to observe the mRNA and protein expression levels of EYA4 in cell lines. Different plasmids （including mutant plasmids） of dual luciferase reporter vector were built to study the binding status of AML1-ETO to the promoter region of EYA4. Chromatin immunoprecipitation assay was used to study the epigenetic silencing mechanism of EYA4. Bisulfite sequencing was applied to detect the methylation status in EYA4 promoter region. The influence ofEYA4 gene in the cell proliferation, apoptosis, and cell clone-forming ability was detected by the technique of Cell Counting Kit-8, flow cytometry, and clonogenic assay. Results： EYA4 gene was hyperrnethylated in AMLI-ETO＋ patients and its expression was down-regulated by 6-fold in Kasumi-1 and SKNO-1 cells, compared to HL-60 and SKNO-1-siA/E cells, respectively. We demonstrated that AML1-ETO triggered the epigenetic silencing of EYA 4 gene by binding at AML 1-binding sites and recruiting histone deacetylase 1 and DNA methyltransferases. Enhanced EYA4 expression levels inhibited cellular proliferation and suppressed cell colony formation in AMLI-ETO cell lines. We also found EYA4 transfection increased apoptosis of Kasumi- 1 and SKNO-1 cells by 1.6-fold and 1.4-fold compared to negative control, respectively. Conclusions： Our study identified EYA4 gene as targets for AML1-ETO and indicated it as a novel tumor suppressor gene. In addition, we provided evidence that EYA4 gene might be a novel therapeutic target and a potential candidate for treating AML 1-ETO＋ t （8;21 ） AML.

Phylogenetic analysis and expression differences of eye-related genes in cavefish genus Sinocyclocheilus

The adaptive evolution of visual systems has been observed in many cavefish.However,little is known about the molecular mechanisms underlying these adaptations,which include regressive changes such as eye degeneration.Here,we analyzed phylogenetic and expression patterns of 6 eye-related genes(crx,foxg1b,opn1sw2,otx2,rho and sox2)in 12 Sinocyclocheilus species from China,including 8 stygobionts and 4 stygophiles,and examined photoreceptor cell morphology of these species.Those eye-degenerated species of Sinocyclocheilus were polyphyletic and showed different degrees of photoreceptor defects in responses to cave environments.The eye loss and degeneration are the result of convergent evolution.Although S.anophthalmus grouped with the eye-normal species,it displayed not only a high degree of eye degeneration but also significant expression differences in eyerelated genes compared with the eye-normal species.The gene foxg1b,which was determined to be under positive selection,might play an important role in the process of eye degeneration in S.anophthalmus based on differential expression.Eye-related gene expression and selection may have contributed to the polyphyly of the cave species.We examined gene expression and duplication in 6 eye-related genes and revealed that these genes displayed considerable diversity in relative expression in Sinocyclocheilus fishes.Otx2 and sox2 were significantly up-regulated in individual cave species,while the other 4 genes(crx,foxg1b,opn1sw2 and rho)were significantly down-regulated.These findings provide a valuable resource for elucidating molecular mechanisms associated with visual system evolution in cavefish.

Highly efficient retinal gene delivery with helper-dependent adenoviral vectors

There have been significant advancements in the field of retinal gene therapy in the past several years.In particular,therapeutic efficacy has been achieved in three separate human clinical trials conducted to assess the ability of adeno-associated viruses(AAV)to treat of a type of Leber’s congenital amaurosis caused by RPE65 mutations.However,despite the success of retinal gene therapy with AAV,challenges remain for delivering large therapeutic genes or genes requiring long DNA regulatory elements for controlling their expression.For example,Stargardt’s disease,a form of juvenile macular degeneration,is caused by defects in ABCA4,a gene that is too large to be packaged in AAV.Therefore,we investigated the ability of helper dependent adenovirus(HD-Ad)to deliver genes to the retina as it has a much larger transgene capacity.Using an EGFP reporter,our results showed that HD-Ad can transduce the entire retinal epithelium of a mouse using a dose of only 1
105 infectious units and maintain transgene expression for at least 4 months.The results demonstrate that HD-Ad has the potential to be an effective vector for the gene therapy of the retina.

Differentially expressed genes identified by cross-species microarray in the blind cavefish Astyanax

Changes in gene expression were examined by microarray analysis during development of the eyed surface dwelling(surface fish)and blind cave-dwelling(cavefish)forms of the teleost Astyanax mexicanus De Filippi,1853.The cross-species microarray used surface and cavefish RNA hybridized to a DNA chip prepared from a closely related species,the zebrafish Danio rerio Hamilton,1822.We identified a total of 67 differentially ex-pressed probe sets at three days post-fertilization:six upregulated and 61 downregulated in cavefish relative to surface fish.Many of these genes function either in eye development and/or maintenance,or in programmed cell death.The upregulated probe set showing the highest mean fold change was similar to the human ubiquitin specific protease 53 gene.The downregulated probe sets showing some of the highest fold changes corresponded to genes with roles in eye development,including those encoding gamma crystallins,the guanine nucleotide binding pro-teins Gnat1 and Gant2,a BarH-like homeodomain transcription factor,and rhodopsin.Downregulation of gam-ma-crystallin and rhodopsin was confirmed by in situ hybridization and immunostaining with specific antibodies.Additional downregulated genes encode molecules that inhibit or activate programmed cell death.The results suggest that cross-species microarray can be used for identifying differentially expressed genes in cavefish,that many of these genes might be involved in eye degeneration via apoptotic processes,and that more genes are downregulated than upregulated in cavefish,consistent with the predominance of morphological losses over gains during regressive evolution.