CRB1-associated retinal degeneration is dependent on bacterial translocation from the gut.

The Crumbs homolog 1 (CRB1) gene is associated with retinal degeneration, most commonly Leber congenital amaurosis (LCA) and retinitis pigmentosa (RP). Here, we demonstrate that murine retinas bearing the Rd8 mutation of Crb1 are characterized by the presence of intralesional bacteria. While normal CRB1 expression was enriched in the apical junctional complexes of retinal pigment epithelium and colonic enterocytes, Crb1 mutations dampened its expression at both sites. Consequent impairment of the outer blood retinal barrier and colonic intestinal epithelial barrier in Rd8 mice led to the translocation of intestinal bacteria from the lower gastrointestinal (GI) tract to the retina, resulting in secondary retinal degeneration. Either the depletion of bacteria systemically or the reintroduction of normal Crb1 expression colonically rescued Rd8-mutation-associated retinal degeneration without reversing the retinal barrier breach. Our data elucidate the pathogenesis of Crb1-mutation-associated retinal degenerations and suggest that antimicrobial agents have the potential to treat this devastating blinding disease.

Resveratrol improves palmitic acid­induced insulin resistance via the DDIT4/mTOR pathway in C2C12 cells.

The present study aimed to establish a model of palmitic acid (PA)­induced insulin resistance (IR) in C2C12 cells and to determine the mechanism underlying how resveratrol (RSV) improves IR. C2C12 cells were divided into the control (CON), PA, PA + RSV, PA + RSV + DNA damage­inducible transcript 4 (DDIT4)­small interfering (si)RNA and PA + RSV + MHY1485 (mTOR agonist) groups. Glucose contents in culture medium and triglyceride contents in cells were determined. Oil red O staining was performed to observe the pathological changes in the cells. Reverse transcription­quantitative PCR and western blotting were conducted to evaluate the mRNA and protein expression levels, respectively, of DDIT4, mTOR, p70 ribosomal protein S6 kinase (p70S6K), insulin receptor substrate (IRS)­1, PI3K, AKT and glucose transporter 4 (GLUT4). Compared with in the CON group, glucose uptake was decreased, cellular lipid deposition was increased, phosphorylated (p)­IRS­1, p­mTOR and p­p70S6K protein expression levels were increased, and p­PI3K, p­AKT, GLUT4 and DDIT4 protein expression levels were decreased in the PA group. By contrast, compared with in the PA group, culture medium glucose content and cellular lipid deposition were decreased, p­PI3K, p­AKT, GLUT4 and DDIT4 protein expression levels were increased, p­IRS­1 protein expression levels were decreased, and mTOR and p70S6K mRNA and protein expression levels were decreased in the PA + RSV group. Compared with in the PA + RSV group, DDIT4 protein and mRNA expression levels were reduced in the PA + RSV + DDIT4­siRNA group, but showed no change in the PA + RSV + MHY1485 group. Following transfection with DDIT4­siRNA or treatment with MHY1485, the effects of RSV on improving IR and lipid metabolism were weakened, mTOR and p70S6K protein expression levels were upregulated, p­PI3K, p­AKT and GLUT4 protein expression levels were down­regulated, p­IRS­1 protein expression levels were upregulated, and culture medium glucose content and cellular lipid deposition were increased. In conclusion, RSV may improve PA­induced IR in C2C12 cells through the DDIT4/mTOR/IRS­1/PI3K/AKT/GLUT4 signaling pathway, as well as via improvements in glucose and lipid metabolism.

Obstruction of the Tear Drainage Altered Lacrimal Gland Structure and Function.

Purpose: Orbital glands and drainage conduits are two distinct entities that constitute the lacrimal apparatus system, the malfunction of which leads to a range of ocular surface disorders. Despite the close functional relationship, how the two parts interact under pathophysiological conditions has not been directly tested. The study aims to investigate the lacrimal gland (LG) structural and functional changes upon the drainage system obstruction, thus, testing their function link. Methods: Dacryocystectomy was performed in C57BL/6 mice to create a surgical model for tear duct (TD) obstruction (STDOB). Prickle1 mutant line with congenital nasolacrimal duct dysplasia serves as a genetic model for TD obstruction (GTDOB). Alterations of the LG and the ocular surface in tear duct obstruction mice were examined. Results: STDOB and GTDOB mice showed similar ocular surface phenotypes, including epiphora, corneal epithelial defects, and conjunctival goblet cell abnormalities. At the molecular and cellular levels, aberrant secretory vesicle fusion of the LG acinar cells was observed with altered expression and localization of Rab3d, Vamp8, and Snap23, which function in membrane fusion. LG secretion was also altered in that lactoferrin, lipocalin2, and lysozyme expression were increased in both LG and tears. Furthermore, STDOB and GTDOB mice exhibited similar LG transcription profiles. Conclusions: Physical obstruction of tear drainage in STDOB or GTDOB mice leads to LG dysfunction, suggesting a long-distance interaction between the tear drainage conduits and the LG. We propose that various components of the lacrimal apparatus should be considered an integral unit in diagnosing and treating ocular surface diseases.

Single-cell characterization of malignant phenotypes and microenvironment alteration in retinoblastoma.

Retinoblastoma (RB) is the most common primary intraocular malignancy of childhood. It is known that the tumor microenvironment (TME) regulates tumorigenesis and metastasis. However, how the malignant progression in RB is determined by the heterogeneity of tumor cells and TME remains uncharacterized. Here, we conducted integrative single-cell transcriptome and whole-exome sequencing analysis of RB patients with detailed pathological and clinical measurements. By single-cell transcriptomic sequencing, we profiled around 70,000 cells from tumor samples of seven RB patients. We identified that the major cell types in RB were cone precursor-like (CP-like) and MKI67+ cone precursor (MKI67+ CP) cells. By integrating copy number variation (CNV) analysis, we found that RB samples had large clonal heterogeneity, where the malignant MKI67+ CP cells had significantly larger copy number changes. Enrichment analysis revealed that the conversion of CP-like to MKI67+ CP resulted in the loss of photoreceptor function and increased cell proliferation ability. The TME in RB was composed of tumor-associated macrophages (TAMs), astrocyte-like, and cancer-associated fibroblasts (CAFs). Particularly, during the invasion process, TAMs created an immunosuppressive environment, in which the proportion of TAMs decreased, M1-type macrophage was lost, and the TAMs-related immune functions were depressed. Finally, we identified that TAMs regulated tumor cells through GRN and MIF signaling pathways, while TAMs self-regulated through inhibition of CCL and GALECTIN signaling pathways during the invasion process. Altogether, our study creates a detailed transcriptomic map of RB with single-cell characterization of malignant phenotypes and provides novel molecular insights into the occurrence and progression of RB.

dsRNA Induced IFNß-MMP13 Axis Drives Corneal Wound Healing.

Purpose: Cornea, the outermost transparent layer of the eye, is the first line of defense against external threats. Following injury, the wound healing response is crucial to corneal repair and regeneration, yet its underlying mechanism is poorly understood. Our study was designed to investigate the role of dsRNA and its regulatory network in corneal wound healing. Methods: A corneal wound healing model was established via the surgical removal of half of the corneal surface and adjoining limbus. RNase III was then used to clarify the role of dsRNA in corneal wound closure and RNA-seq was performed to investigate the mechanism of dsRNA in the healing process. Related gene expression was assessed using immunofluorescence staining, qPCR, and Western blot. Flow cytometry and scratch assay were used to analyze the proliferation and migration of limbal stem/progenitor cells (LSCs) in vitro and functional analysis of the target genes was completed using the corneal wound healing model. Results: Corneal wound healing was delayed and impaired when the dsRNAs were removed or damaged following RNase III digestion. The dsRNAs released following corneal damage activate type I interferon (IFN-I) signaling, primarily IFNß, via the corneal epithelium and neutralizing IFNß or blocking IFN-I signaling delays corneal wound closure. Moreover, our data identified MMP13 as a downstream effector of IFNß where its expression promotes LSC proliferation and enhances corneal epithelial reconstruction in vivo. Conclusions: The dsRNA induced IFNß-MMP13 axis plays a key role in corneal wound healing.

Loss of FOXC1 contributes to the corneal epithelial fate switch and pathogenesis.

Forkhead box C1 (FOXC1) is required for neural crest and ocular development, and mutations in FOXC1 lead to inherited Axenfeld-Rieger syndrome. Here, we find that FOXC1 and paired box 6 (PAX6) are co-expressed in the human limbus and central corneal epithelium. Deficiency of FOXC1 and alternation in epithelial features occur in patients with corneal ulcers. FOXC1 governs the fate of the corneal epithelium by directly binding to lineage-specific open promoters or enhancers marked by H3K4me2. FOXC1 depletion not only activates the keratinization pathway and reprograms corneal epithelial cells into skin-like epithelial cells, but also disrupts the collagen metabolic process and interferon signaling pathways. Loss of interferon regulatory factor 1 and PAX6 induced by FOXC1 dysfunction is linked to the corneal ulcer. Collectively, our results reveal a FOXC1-mediated regulatory network responsible for corneal epithelial homeostasis and provide a potential therapeutic target for corneal ulcer.

Ocular surface pathogenesis associated with precocious eyelid opening and necrotic autologous tissue in mouse with disruption of Prickle 1 gene.

Ocular surface disease is one major type of eye diseases. Different etiologies trigger distinct pathological responses of the ocular surface. We previously reported that genetically engineered mice with ablation of Prickle 1 manifested precocious eyelid opening with ensuing cornea dysplasia. The current study aimed to characterize the molecular traits and the direct cause of ocular pathology associated with precocious eyelid opening in the Prickle 1 mutant mouse. Prickle 1 mutant mice exhibited a slew of ocular surface pathology including cell proliferation, cell fate transformation and inflammatory infiltration coinciding with the timing of the precocious eyelid opening. Forced eyelid opening in wild type mice did not induce cornea pathology comparable to that of the Prickle 1 mutants. Necrotic tissue debris was found associated with the lesioned cornea. RNAseq analysis of the mutant cornea revealed an expression profile shared by a range of dermatological diseases involving immune responses and cancer. Taken together, the data suggest that the necrotic eyelid debris plays an important role in ocular pathogenesis of the Prickle 1 mutant mouse, which may represent a type of non-infectious keratoconjunctivitis caused by damaged autologous tissues. Additionally, Prickle 1 mutant cornea pathogenesis may offer molecular insights into other types of epithelial pathogenesis.

A secreted WNT-ligand-binding domain of FZD5 generated by a frameshift mutation causes autosomal dominant coloboma.

Ocular coloboma is a common eye malformation resulting from incomplete fusion of the optic fissure during development. Coloboma is often associated with microphthalmia and/or contralateral anophthalmia. Coloboma shows extensive locus heterogeneity associated with causative mutations identified in genes encoding developmental transcription factors or components of signaling pathways. We report an ultra-rare, heterozygous frameshift mutation in FZD5 (p.Ala219Glufs*49) that was identified independently in two branches of a large family with autosomal dominant non-syndromic coloboma. FZD5 has a single-coding exon and consequently a transcript with this frameshift variant is not a canonical substrate for nonsense-mediated decay. FZD5 encodes a transmembrane receptor with a conserved extracellular cysteine rich domain for ligand binding. The frameshift mutation results in the production of a truncated protein, which retains the Wingless-type MMTV integration site family member-ligand-binding domain, but lacks the transmembrane domain. The truncated protein was secreted from cells, and behaved as a dominant-negative FZD5 receptor, antagonizing both canonical and non-canonical WNT signaling. Expression of the resultant mutant protein caused coloboma and microphthalmia in zebrafish, and disruption of the apical junction of the retinal neural epithelium in mouse, mimicking the phenotype of Fz5/Fz8 compound conditional knockout mutants. Our studies have revealed a conserved role of Wnt-Frizzled (FZD) signaling in ocular development and directly implicate WNT-FZD signaling both in normal closure of the human optic fissure and pathogenesis of coloboma.

Loss of lysophosphatidylcholine acyltransferase 1 leads to photoreceptor degeneration in rd11 mice.

Retinal degenerative diseases, such as retinitis pigmentosa and Leber congenital amaurosis, are a leading cause of untreatable blindness with substantive impact on the quality of life of affected individuals and their families. Mouse mutants with retinal dystrophies have provided a valuable resource to discover human disease genes and helped uncover pathways critical for photoreceptor function. Here we show that the rd11 mouse mutant and its allelic strain, B6-JR2845, exhibit rapid photoreceptor dysfunction, followed by degeneration of both rods and cones. Using linkage analysis, we mapped the rd11 locus to mouse chromosome 13. We then identified a one-nucleotide insertion (c.420-421insG) in exon 3 of the Lpcat1 gene. Subsequent screening of this gene in the B6-JR2845 strain revealed a seven-nucleotide deletion (c.14-20delGCCGCGG) in exon 1. Both sequence changes are predicted to result in a frame-shift, leading to premature truncation of the lysophosphatidylcholine acyltransferase-1 (LPCAT1) protein. LPCAT1 (also called AYTL2) is a phospholipid biosynthesis/remodeling enzyme that facilitates the conversion of palmitoyl-lysophosphatidylcholine to dipalmitoylphosphatidylcholine (DPPC). The analysis of retinal lipids from rd11 and B6-JR2845 mice showed substantially reduced DPPC levels compared with C57BL/6J control mice, suggesting a causal link to photoreceptor dysfunction. A follow-up screening of LPCAT1 in retinitis pigmentosa and Leber congenital amaurosis patients did not reveal any obvious disease-causing mutations. Previously, LPCAT1 has been suggested to be critical for the production of lung surfactant phospholipids and biosynthesis of platelet-activating factor in noninflammatory remodeling pathway. Our studies add another dimension to an essential role for LPCAT1 in retinal photoreceptor homeostasis.

A novel way to enhance the oil recovery ratio by Streptococcus sp. BT-003.

Degrading hydrocarbon by Streptococcus sp. BT-003 as a kind of microbe for oil recovery was analyzed in this paper. The Streptococcus sp. BT-003 showed that it could use crude oil as the sole source of carbon and produce organic acid, bio-gas and polysaccharide which were propitious to emulsify and reduce the viscosity of crude oil. After cultivating 8-14 h, the viscosity of crude oil reduced from 8000-15000 mPa . s to 50-250 mPa . s. The content of organic acid increased 8-15 times, and carbon dioxide and polysaccharide reached 55 ml/l and 8 g/l respectively. Paraffin and resin reduced by 60-95%, and light components increased obviously. Fluid rheology was better than before, the interfacial tension between the crude oil and water were reduced effectively.

An essential role for Frizzled5 in neuronal survival in the parafascicular nucleus of the thalamus.

Frizzled5 (Fz5), a putative Wnt receptor, is expressed in the retina, hypothalamus, and the parafascicular nucleus (PFN) of the thalamus. By constructing Fz5 alleles in which beta-galactosidase replaces Fz5 or in which Cre-mediated recombination replaces Fz5 with alkaline phosphatase, we observe that Fz5 is required continuously and in a cell autonomous manner for the survival of adult PFN neurons, but is not required for proliferation, migration, or axonal growth and targeting of developing PFN neurons. A motor phenotype associated with loss of Fz5 establishes a role for the PFN in sensorimotor coordination. Transcripts coding for Wnt9b, the likely Fz5 ligand in vivo, and beta-catenin, a mediator of canonical Wnt signaling, are both downregulated in the Fz5(-/-) PFN, implying a positive feedback mechanism in which Wnt signaling is required to maintain the expression of Wnt signaling components. These data suggest that defects in Wnt-Frizzled signaling could be the cause of neuronal loss in degenerative CNS diseases.

Identification of the downstream targets of SIM1 and ARNT2, a pair of transcription factors essential for neuroendocrine cell differentiation.

SIM1 and ARNT2 are two basic helix-loop-helix/PAS (Per-Arnt-Sim) transcription factors that control the differentiation of neuroendocrine lineages in the mouse hypothalamus. Heterozygous Sim1 mice also develop early onset obesity, possibly due to hypodevelopment of the hypothalamus. Although SIM1 and ARNT2 form heterodimers to direct the same molecular pathway, knowledge of this pathway is limited. To facilitate the identification of their downstream genes, we combined an inducible gene expression system in a neuronal cell line with microarray analysis to screen for their transcriptional targets. This method identified 268 potential target genes of SIM1/ARNT2 that displayed >1.7-fold induced expression. 15 of these genes were subjected to Northern analysis, and a high percentage of them were confirmed to be up-regulated. In vivo, several of these genes showed neuroendocrine hypothalamic expression correlating with that of Sim1. Furthermore, we found that expression of two of these potential targets, the Jak2 and thyroid hormone receptor beta2 genes, was lost in the neuroendocrine hypothalamus of the Sim1 mutant. The expression and predicted functions of many of these genes provide new insight into both the Sim1/Arnt2 action in neuroendocrine hypothalamus development and the molecular basis for the Sim1 haplo-insufficient obesity phenotype.

A role for the mesenchymal T-box gene Brachyury in AER formation during limb development.

During limb development, several signaling centers organize limb pattern. One of these, the apical ectodermal ridge (AER), is critical for proximodistal limb outgrowth mediated by FGFs. Signals from the underlying mesoderm, including WNTs and FGFs, regulate early steps of AER induction. Ectodermal factors, particularly En1, play a critical role in regulating morphogenesis of a mature, compact AER along the distal limb apex, from a broad ventral ectodermal precursor domain. Contribution of mesodermal factors to the morphogenesis of a mature AER is less clear. We previously noted that the chick T gene (Brachyury), the prototypical T-box transcription factor, is expressed in the limb bud as well as axial mesoderm and primitive streak. Here we show that T is expressed in lateral plate mesoderm at the onset of limb bud formation and subsequently in the subridge mesoderm beneath the AER. Retroviral misexpression of T in chick results in anterior extension of the AER and subsequent limb phenotypes consistent with augmented AER extent and function. Analysis of markers for functional AER in mouse T(-/-) null mutant limb buds reveals disrupted AER morphogenesis. Our data also suggest that FGF and WNT signals may operate both upstream and downstream of T. Taken together, the results show that T plays a role in the regulation of AER formation, particularly maturation, and suggest that T may also be a component of the epithelialmesenchymal regulatory loop involved in maintenance of a mature functioning AER.