Overexpression of miR-96 leads to retinal degeneration in mice.

Double knockout of miR-183 and miR-96 results in retinal degeneration in mice; however, single knockout of miR-96 leads to developmental delay but not substantial retinal degeneration. To further explore the role of miR-96, we overexpressed this miRNA in mouse retinas. Interestingly, we found that overexpression of miR-96 at a safe dose results in retinal degeneration in the mouse retina. The retinal photoreceptors dramatically degenerated in the miR-96-overexpressing group, as shown by OCT, ERG and cryosectioning at one month after subretinal injection. Degenerative features such as TUNEL signals and reactive gliosis were observed in the miR-96-overexpressing retina. RNA-seq data revealed that immune responses and microglial activation occurred in the degenerating retina. Further qRT‒PCR and immunostaining experiments verified the microglial activation. Moreover, the number of microglia in the miR-96-overexpressing retinas was significantly increased. Our findings demonstrate that appropriate miR-96 expression is required for mouse retinal homeostasis.

Identification of a novel ferroptosis-related gene signature associated with retinal degeneration induced by light damage in mice.

Background: Neurodegenerative retinal diseases such as retinitis pigmentosa are serious disorders that may cause irreversible visual impairment. Ferroptosis is a novel type of programmed cell death, and the involvement of ferroptosis in retinal degeneration is still unclear. This study aimed to investigate the related ferroptosis genes in a mice model of retinal degeneration induced by light damage. Methods: A public dataset of GSE10528 deriving from the Gene Expression Omnibus database was analyzed to identify the differentially expressed genes (DEGs). Gene set enrichment analysis between light damage and control group was conducted. The differentially expressed ferroptosis-related genes (DE-FRGs) were subsequently identified by intersecting the DEGs with a ferroptosis genes dataset retrieved from the FerrDb database. The Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) were further performed using the DE-FRGs. A protein-protein interaction (PPI) network was constructed to identify hub ferroptosis-related genes (HFRGs). The microRNAs (miRNAs)-HFRGs, transcription factors (TFs)-HFRGs networks as well as target drugs potentially interacting with HFRGs were analyzed utilizing bioinformatics algorithms. Results: A total of 932 DEGs were identified between the light damage and control group. Among these, 25 genes were associated with ferroptosis. GO and KEGG analyses revealed that these DE-FRGs were mainly enriched in apoptotic signaling pathway, response to oxidative stress and autophagy, ferroptosis, necroptosis and cytosolic DNA-sensing pathway. Through PPI network analysis, six hub ferroptosis-related genes (Jun, Stat3, Hmox1, Atf3, Hspa5 and Ripk1) were ultimately identified. All of them were upregulated in light damage retinas, as verified by the GSE146176 dataset. Bioinformatics analyses predicated that 116 miRNAs, 23 TFs and several potential therapeutic compounds might interact with the identified HFRGs. Conclusion: Our study may provide novel potential biomarkers, therapeutic targets and new insights into the ferroptosis landscape in retinal neurodegenerative diseases.

Dysregulated Arginine Metabolism Is Linked to Retinal Degeneration in Cep250 Knockout Mice.

Purpose: Degeneration of retinal photoreceptors is frequently observed in diverse ciliopathy disorders, and photoreceptor cilium gates the molecular trafficking between the inner and the outer segment (OS). This study aims to generate a homozygous global Cep250 knockout (KO) mouse and study the resulting phenotype. Methods: We used Cep250 KO mice and untargeted metabolomics to uncover potential mechanisms underlying retinal degeneration. Long-term follow-up studies using optical coherence tomography (OCT) and electroretinography (ERG) were performed. Results: OCT and ERG results demonstrated gradual thinning of the outer nuclear layer (ONL) and progressive attenuation of the scotopic ERG responses in Cep250-/- mice. More TUNEL signal was observed in the ONL of these mice. Immunostaining of selected OS proteins revealed mislocalization of these proteins in the ONL of Cep250-/- mice. Interestingly, untargeted metabolomics analysis revealed arginine-related metabolic pathways were altered and enriched in Cep250-/- mice. Mis-localization of a key protein in the arginine metabolism pathway, arginase 1 (ARG1), in the ONL of KO mice further supports this model. Moreover, adeno-associated virus (AAV)-based retinal knockdown of Arg1 led to similar architectural and functional alterations in wild-type retinas. Conclusions: Altogether, these results suggest that dysregulated arginine metabolism contributes to retinal degeneration in Cep250-/- mice. Our findings provide novel insights that increase understanding of retinal degeneration in ciliopathy disorders.

Causal effects of serum lipid biomarkers on early age-related macular degeneration using Mendelian randomization.

BACKGROUND: Age-related macular degeneration (AMD) is one of the major causes of vision loss. Early AMD needs to be taken seriously, but the causal effects of lipid biomarkers on early AMD remain unclear. METHODS: In this study, two-sample Mendelian randomization (MR) analysis was performed to systematically assess the causal relationships between seven serum lipid biomarkers (apolipoprotein A (ApoA), apolipoprotein B (ApoB), total cholesterol (CHOL), high-density lipoprotein cholesterol (HDL-C), direct low-density lipoprotein cholesterol (LDL-C), lipoprotein A [Lp(a)], and triglycerides (TG)) and risk of early AMD. In total, 14,034 cases and 91,214 controls of European ancestry were included in the analysis (number of SNPs = 11,304,110). RESULTS: MR estimates revealed that a higher HDL-C level is strongly associated with increased risk of early AMD (OR = 1.25, 95% CI: 1.15-1.35, P = 2.61 × 10-8). In addition, level of ApoA is also positively associated with risk of early AMD (OR = 2.04, 95% CI: 1.50-2.77, P = 6.27 × 10-6). Conversely, higher levels of TG significantly decrease the risk of early AMD (OR = 0.77, 95% CI: 0.71-0.84, P = 5.02 × 10-10). Sensitivity analyses further supported these associations. Moreover, multivariable MR analyses, adjusted for the effects of correlated lipid biomarkers, yielded similar results. CONCLUSION: This study identifies causal relationships between elevated circulating HDL-C/ApoA levels and increased risk of early AMD, in addition to finding that TG specifically reduces the risk of early AMD. These findings contribute to a better understanding of the role of lipid metabolism in drusen formation, particularly in early AMD development.

Image-Guided Optical Coherence Tomography to Assess Structural Changes in Rodent Retinas.

Ocular diseases, such as age-related macular degeneration, glaucoma, retinitis pigmentosa, and uveitis, are always accompanied by retinal structural changes. These diseases affecting the fundus always exhibit typical abnormalities in certain cell types in the retina, including photoreceptor cells, retinal ganglion cells, cells in the retinal blood vessels, and cells in the choroidal vascular cells. Noninvasive, highly efficient, and adaptable imaging techniques are required for both clinical practice and basic research. Image-guided optical coherence tomography (OCT) satisfies these requirements because it combines fundus photography and high-resolution OCT, providing an accurate diagnosis of tiny lesions as well as important changes in the retinal architecture. This study details the procedures of data collection and data analysis for image-guided OCT and demonstrates its application in rodent models of choroidal neovascularization (CNV), optic nerve crush (ONC), light-induced retinal degeneration, and experimental autoimmune uveitis (EAU). This technique helps researchers in the eye field to identify rodent retinal structural changes conveniently, reliably, and tractably.

Progress on the application of growth factor-related drugs in ophthalmology.

Vascular endothelial growth factor（VEGF）, fibroblast growth factor（FGF）, nerve growth factor（NGF）, epidermal growth factor and interferon are important endogenous proteins that regulate cell proliferation, differentiation and regeneration. Biological products targeting growth factors are used in the treatment of ocular diseases such as wet age-related macular degeneration, corneal injury and neurotrophic keratitis. Anti-VEGF drugs can regulate the proliferation of vascular endothelia, reduce the edema and exudation of retinal tissue，which are the main therapeutic agents for wet age-related macular degeneration and diabetic retinopathy. The basic FGF (b-FGF) can promote the proliferation, differentiation, and migration of corneal epithelial cells, accelerating the healing of the corneal injury and reduces corneal inflammation；and bovine b-FGF has been approved for the treatment of corneal injuries. The NGF promotes the growth, development, and differentiation of central and peripheral neurons, thus accelerating the repair of nerve damage；and the European Medicines Agency approved the use of nerve growth factor for the treatment of neurotrophic keratitis in 2017. Recent clinical studies show that patients with moderate or severe neurotrophic keratitis achieved complete corneal healing following 8 weeks of NGF therapy. Epidermal growth factor derivative eye drops have been approved for the treatment of corneal epithelial injuries. Recombinant human interferon has been clinically used in the treatment of ocular viral infections. This article reviews the research progress in the development of new cell growth factor drugs for the treatment of ophthalmic diseases, to provide insights for expanding the application of cell growth factors in ophthalmology.

Roles of growth factors in eye development and ophthalmic diseases.

Growth factors are active substances secreted by a variety of cells, which act as messengers to regulate cell migration, proliferation and differentiation. Many growth factors are involved in the eye development or the pathophysiological processes of eye diseases. Growth factors such as vascular endothelial growth factor and basic fibroblast growth factor mediate the occurrence and development of diabetic retinopathy, choroidal neovascularization, cataract, diabetic macular edema, and other retinal diseases. On the other hand, growth factors like nerve growth factor, ciliary neurotrophic factor, glial cell line-derived neurotrophic factor, pigment epithelial-derived factor and granulocyte colony-stimulating factor are known to promote optic nerve injury repair. Growth factors are also related to the pathogenesis of myopia. Fibroblast growth factor, transforming growth factor-ß, and insulin-like growth factor regulate scleral thickness and influence the occurrence and development of myopia. This article reviews growth factors involved in ocular development and ocular pathophysiology, discusses the relationship between growth factors and ocular diseases, to provide reference for the application of growth factors in ophthalmology.

Depletion of miR-96 Delays, But Does Not Arrest, Photoreceptor Development in Mice.

Purpose: Abundant retinal microRNA-183 cluster (miR-183C) has been reported to be a key player in photoreceptor development and functionality in mice. However, whether there is a protagonist in this cluster remains unclear. Here, we used a mutant mouse model to study the role of miR-96, a member of miR-183C, in photoreceptor development and functionality. Methods: The mature miR-96 sequence was removed using the CRISPR/Cas9 genome-editing system. Electroretinogram (ERG) and optical coherence tomography (OCT) investigated the changes in structure and function in mouse retinas. Immunostaining determined the localization and morphology of the retinal cells. RNA sequencing was conducted to observe retinal transcription alterations. Results: The miR-96 mutant mice exhibited cone developmental delay, as occurs in miR-183/96 double knockout mice. Immunostaining of cone-specific marker genes revealed cone nucleus mislocalization and exiguous Opn1mw/Opn1sw in the mutant (MT) mouse outer segments at postnatal day 10. Interestingly, this phenomenon could be relieved in the adult stages. Transcriptome analysis revealed activation of microtubule-, actin filament-, and cilia-related pathways, further supporting the findings. Based on ERG and OCT results at different ages, the MT mice displayed developmental delay not only in cones but also in rods. In addition, a group of miR-96 potential direct and indirect target genes was summarized for interpretation and further studies of miR-96-related retinal developmental defects. Conclusions: Depletion of miR-96 delayed but did not arrest photoreceptor development in mice. This miRNA is indispensable for mouse photoreceptor maturation, especially for cones.

The cwp66 Gene Affects Cell Adhesion, Stress Tolerance, and Antibiotic Resistance in Clostridioides difficile.

Clostridioides difficile is a Gram-positive, spore-forming anaerobic bacteria that is one of the leading causes of antibiotic-associated diarrhea. The cell wall protein 66 gene (cwp66) encodes a cell wall protein, which is the second major cell surface antigen of C. difficile. Although immunological approaches, such as antibodies and purified recombinant proteins, have been implemented to study the role of Cwp66 in cell adhesion, no deletion mutant of the cwp66 gene has yet been characterized. We constructed a cwp66 gene deletion mutant using Clustered Regularly Interspaced Short Palindromic Repeats Cpf1 (CRISPR-Cpf1) system. The phenotypic and transcriptomic changes of the Δcwp66 mutant compared with the wild-type (WT) strain were studied. The deletion of the cwp66 gene led to the decrease of cell adhesive capacity, cell motility, and stresses tolerance (to Triton X-100, acidic environment, and oxidative stress). Interestingly, the Δcwp66 mutant is more sensitive than the WT strain to clindamycin, ampicillin, and erythromycin but more resistant than the latter to vancomycin and metronidazole. Moreover, mannitol utilization capability in the Δcwp66 mutant was lost. Comparative transcriptomic analyses indicated that (i) 22.90-fold upregulation of cwpV gene and unable to express gpr gene were prominent in the Δcwp66 mutant; (ii) the cwp66 gene was involved in vancomycin resistance of C. difficile by influencing the expression of d-Alanine-d-Alanine ligase; and (iii) the mannose/fructose/sorbose IIC and IID components were upregulated in Δcwp66 mutant. The present work deepens our understanding of the contribution of the cwp66 gene to cell adhesion, stress tolerance, antibiotic resistance, and mannitol transportation of C. difficile. IMPORTANCE The cell wall protein 66 gene (cwp66) encodes a cell wall protein, which is the second major cell surface antigen of C. difficile. Although immunological approaches, such as antibodies and purified recombinant proteins, have been implemented to study the role of Cwp66 in cell adhesion, no deletion mutant of the cwp66 gene has yet been characterized. The current study provides direct evidence that the cwp66 gene serves as a major adhesion in C. difficile, and also suggested that deletion of the cwp66 gene led to the decrease of cell adhesive capacity, cell motility, and stresses tolerance (to Triton X-100, acidic environment, and oxidative stress). Interestingly, the antibiotic resistance and carbon source utilization profiles of the Δcwp66 mutant were significantly changed. These phenotypes were detrimental to the survival and pathogenesis of C. difficile in the human gut and may shed light on preventing C. difficile infection.

An inexpensive anaerobic chamber for the genetic manipulation of strictly anaerobic bacteria.

Strictly anaerobic bacteria are important to both human health and industrial usage. These bacteria are sensitive to oxygen, therefore, it is preferable to manipulate these microbes in an anaerobic chamber. However, commercial anaerobic chambers (CACs) are expensive, making them less accessible to scientists with a limited budget, especially to those in developing countries. The high price of commercial chambers has hindered, at least partially, the progress of research on anaerobes in developing countries. In the research presented here, we developed an inexpensive and reliable anaerobic chamber and successfully achieved routine maintenance of eleven strictly anaerobic bacterial strains. Furthermore, genetic manipulation examples have been set for both Clostridioidesdifficile 630 and Clostridiumbeijerinckii NCIMB 8052 strains to validate that the chamber could applied to advanced genetic engineering of strictly anaerobes. C. difficile and C. beijerinckii were both genetically manipulated in this chamber, showing it's utility for the genetic engineering of anaerobes. Most importantly, the anaerobic chamber was 76% - 88% less expensive than a CACs and has similar functionality with regards to the cultivation and manipulation of strictly anaerobic bacteria. The anaerobic chamber described in this study will promote the research of anaerobes in developing counties and scientists who have limited research budgets.

[A temperature-inducible Targetron system for efficient gene inactivation in Escherichia coli].

To construct TeI3c/4c-based and temperature-inducible gene inactivation system (Thermotargetron) and to apply it to gene inactivation of mesophilic bacteria. The subunit of flagellum (fliC) and C4 dicarboxylate orotate:H⁺ symporter (dctA) genes were chosen as targets in the genome of Escherichia coli HMS174 (DE3) strain. According to recognition roles of TeI3c/4c intron, the fliC489a, fliC828s, fliC1038s and dctA2a sites were chosen as target sites. Gene-targeting plasmids were constructed based on pHK-TT1A by using overlap PCR method and transformed into HMS174 cells. An aliquot mid-log phase cultures of the transformants were shocked at 48 °C and plated on LB plate (containing chloramphenicol). Afterwards, gene mutants were screened by using colony PCR and DNA sequencing. After the mutants were obtained, the phenotypes of ΔfliC and ΔdctA gene mutants were characterized by using agar puncture and carbon metabolism experiments. Colony PCR and sequencing results show that TeI3c/4c intron was inserted in the designed sites of fliC and dctA genes. The gene-targeting efficiency of Thermotargetron system was 100%. Phenotype verification experiments of the mutants demonstrated that the cell motility of all ΔfliC mutants was damaged and the malate assimilation ability of ΔdctA mutant was deprived comparing to wild-type HMS174 strain. In our study, a temperature-inducible and high-efficiency gene inactivation system was established for mesophilic bacteria. This system could achieve high efficiency and precise gene inactivation by modulation of the incubation duration of the transformants at 48 °C.

Bacteria Co-colonizing with Clostridioides Difficile in Two Asymptomatic Patients.

BACKGROUND: Clostridium difficile infection (CDI) is the leading cause of nosocomial diarrhea. Co-colonization of key bacterial taxa may prevent the transition from asymptomatic C. difficile colonization to CDI. However, little is known about the composition of key bacterial taxa in asymptomatic patients. METHODS: In the present study, the culture method was used to examine the composition of stool microbiota in two asymptomatic patients from Guizhou, China. RESULTS: A total of 111 strains were isolated and phylogenetic relationships were determined by 16S ribosomal gene sequencing and Molecular Evolutionary Genetics Analysis version 7. The results demonstrated that Escherichia (33.3%, 37/111), Clostridium (24.3%, 27/111) and Enterococcus (11.7%, 13/111) exhibited a high ratio in asymptomatic patients. These isolates derived from two phyla: Firmicutes (51.3%, 57/111) and Proteobacteria (44.1%, 49/111). In addition, co-colonization of human pathogens Fusobacterium nucleatum, Ralstonia pickettii, Klebsiella pneumoniae, Klebsiella quasipneumoniae and Clostridium tertium with C. difficile was identified. To the best of our knowledge, these pathogens have not been co-isolated with C. difficile previously. CONCLUSIONS: In summary, the present study identified the composition of fecal microbiota in two asymptomatic patients in Guizhou, China. These results suggested that co-infection with human pathogens may be ubiquitous during CDI progression.

Co-infection of Clostridioides (Clostridium) difficile GMU1 and Bacillus cereus GMU2 in one patient in Guizhou, China.

Clostridioides (Clostridium) difficile and Bacillus cereus infections are frequently reported in human individually. However, co-infection of both pathogens in human is extremely rare. In the present study, we reported a case of human enteric disease caused by co-infection of C. difficile and B. cereus in Guizhou, China. The 16S rDNA sequencing result showed that C. difficile GMU1 and B. cereus GMU2 were most related to C. difficile ATCC 9689 and B. cereus ATCC 14579. The toxin genotype of C. difficile GMU1 and B. cereus GMU2 were tcdA+tcdB+tcdC+ and bceT+nheA+nheB+nheC+, respectively. Cytotoxicity assay demonstrated that C. difficile GMU1 produced significantly higher toxin B compare to C. difficile 630 stain. In contrast, B. cereus GMU2 has comparable NheA toxin productivity compare to previous report. The antimicrobial susceptibility test showed that the combination of ampicillin and vancomycin was most efficient to inhibit both C. difficile GMU1 and B. cereus GMU2.

Mutations in LRP5,FZD4, TSPAN12, NDP, ZNF408, or KIF11 Genes Account for 38.7% of Chinese Patients With Familial Exudative Vitreoretinopathy.

Purpose: Familial exudative vitreoretinopathy (FEVR) is a severe hereditary retinal disorder characterized by defects in retinal vascular development. To date, six genes have been reported to be responsible for this disease, including LRP5, FZD4, TSPAN12, NDP, ZNF408, and KIF11. The purpose of our study was to investigate the genetic defects in Chinese patients with FEVR through mutational analyses of 31 pedigrees. Methods: Clinical data and peripheral blood were collected from 31 pedigrees with FEVR. All coding sequences and intron/exon junctions were amplified and sequenced comprehensively, followed by cosegregation testing to verify suspected variants in the family members. Finally, we assessed clinical relevance of the identified mutations, according to the standards and guidelines from the American College of Medical Genetics and Genomics. Results: Twelve index cases (12/31, 38.7%) were confirmed to harbor mutations in the known genes, including one previously reported mutation and 11 novel mutations. Among the detected mutations, LRP5 accounted for the largest proportion with a mean mutation rate of 16.1% (5/31, 16.1%), followed by NDP (3/31, 9.7%), FZD4 (2/31, 6.5%), TSPAN12 (1/31, 3.2%), and KIF11 (1/31, 3.2%). All the novel changes were predicted to be pathogenic by a series of bioinformatics analyses. Conclusions: We comprehensively screened six known disease-causing genes in 31 pedigrees with FEVR and achieved a clear picture of the mutation spectrum in Chinese patients with FEVR, which highlights the importance and utility of clinical genetic diagnosis.

Genome-Wide Detection of Copy Number Variations in Unsolved Inherited Retinal Disease.

Purpose: Inherited retinal diseases (IRDs) are a clinically and genetically heterogeneous group of Mendelian disorders that plays a crucial role in the etiology of blindness across the world. Molecular genetic diagnosis of IRD remains extremely complex and challenging because mutations are only detected in 40% to 60% of cases. In this study, we aimed to dissect the contributions of copy number variations (CNVs) in IRD patients. Methods: A total of 50 patients were diagnosed with IRD, all of whom previously tested negative for pathogenic mutations in known disease genes. Single-nucleotide polymorphism array analysis was performed by using the HumanCoreExome BeadChip. Analyses of CNVs were carried out by using GenomeStudio, KaryoStudio, and cnvPartition. The putative pathogenic CNVs were further confirmed by real-time quantitative PCR. Results: We identified four novel CNVs in three different genes (one duplication in USH2A gene, two duplications in CEP290 gene, and one duplication in RIMS2 gene) in total four families, at a detection rate of 8% (4/50). All of these CNVs are currently absent in all databases. Three variations are located in genes that are already known to cause inherited retinal disease: USH2A and CEP290, while the association between mutation in the RIMS2 gene and IRD is reported for the first time. Conclusions: We performed whole-genome-wide CNV analyses in a large cohort as an alternative approach to molecular diagnosis of IRDs. This study dissected the contributions of CNVs of IRDs, not only increasing the yield in genetic testing but also suggesting the CNVs should be analyzed in the patients with IRDs.