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Periostin is a non-structural matricellular protein. Little is known about periostin in human limbal stem cells (LSCs). This study was to explore the unique expression pattern and functional role of periostin in maintaining the properties of human LSCs. Fresh donor corneal tissues were used to make cryosections for evaluation of periostin expression on ex vivo tissues. Primary human limbal epithelial cells (HLECs) were generated from limbal explant culture. In vitro culture models for proliferation and epithelial regeneration were performed to explore functional role of periostin in LSCs. The mRNA expression was determined by reverse transcription and quantitative real-time PCR (RT-qPCR), and the protein production and localization were detected by immunofluorescent staining and Western blot analysis. Periostin protein was found to be exclusively immunolocalized in the basal layer of human limbal epithelium. Periostin localization was well matched with nuclear factor p63, but not with corneal epithelial differentiation marker Keratin 3. Periostin transcripts was also highly expressed in limbal than corneal epithelium. In primary HLECs, periostin expression at mRNA and protein levels was significantly higher in 50% and 70% confluent cultures at exponential growth stage than in 100% confluent cultures at slow growth or quiescent condition. This expression pattern was similar to other stem/progenitor cell markers (p63, integrin β1 and TCF4). Periostin expression at transcripts, protein and immunoreactivity levels increased significantly during epithelial regeneration in wound healing process, especially in 16-24 hours at wound edge, which was accompanied by similar upregulation and activation of p63, integrin β1 and TCF4. Our findings demonstrated that periostin is exclusively produced by limbal basal epithelium and co-localized with p63, where limbal stem cells reside. Periostin promotes HLEC proliferation and regeneration with accompanied activation of stem/progenitor cell markers p63, integrin β1 and TCF4, suggesting its novel role in maintaining the phenotype and functional properties of LSC.

Oxidative stress has been known to be involved in pathogenesis of dry eye disease. However, few studies have comprehensively investigated the relationship between hyperosmolarity and oxidative damage in human ocular surface. This study was to explore whether and how hyperosmolarity induces oxidative stress markers in primary human corneal epithelial cells (HCECs). Primary HCECs were established from donor limbal explants. The hyperosmolarity model was made in HCECs cultured in isosmolar (312 mOsM) or hyperosmotic (350, 400, 450 mOsM) media. Production of reactive oxygen species (ROS), oxidative damage markers, oxygenases and anti-oxidative enzymes were analyzed by DCFDA kit, RT-qPCR, immunofluorescent and immunohistochemical staining and Western blotting. Compared to isosmolar medium, ROS production significantly increased at time- and osmolarity-dependent manner in HCECs exposed to media with increasing osmolarities (350-450 mOsM). Hyperosmolarity significantly induced oxidative damage markers in cell membrane with increased toxic products of lipid peroxidation, 4-hydroxynonenal (4-HNE) and malondialdehyde (MDA), and in nuclear and mitochondria DNA with increased aconitase-2 and 8-OHdG. Hyperosmotic stress also increased the mRNA expression and protein production of heme oxygenase-1 (HMOX1) and cyclooxygenase-2 (COX2), but reduced the levels of antioxidant enzymes, superoxide dismutase-1 (SOD1), and glutathione peroxidase-1 (GPX1). In conclusion, our comprehensive findings demonstrate that hyperosmolarity induces oxidative stress in HCECs by stimulating ROS production and disrupting the balance of oxygenases and antioxidant enzymes, which in turn cause cell damage with increased oxidative markers in membrane lipid peroxidation and mitochondrial DNA damage.

LEPA is one of the most conserved translation factors and is found from bacteria to higher plants. However, the physiological function of the chloroplast LEPA homolog in higher plants remains unknown. Herein, we demonstrate the physiological role of cpLEPA in enabling efficient photosynthesis in higher plants. The cplepa-1 mutant displays slightly high chlorophyll fluorescence and pale green phenotypes under normal growth conditions. The growth of the cplepa-1 mutant is reduced when grown on soil, and greater reduction is observed under intense light illumination. Photosynthetic activity is impaired in the cplepa-1 mutants, which is reflected in the decreased steady-state levels of chloroplast proteins. In vivo protein labeling experiments explained the decrease in the steady-state levels of chloroplast proteins. An abnormal association of the chloroplast-encoded mRNAs with ribosomes suggests that the protein synthesis deficiencies in cplepa-1 are due to defects in translation initiation in the chloroplasts. The cpLEPA protein appears to be an essential translation factor that promotes the efficiency of chloroplast protein synthesis.

MicroRNAs (miRNAs) are small non-coding RNA molecules that are processed from large 'hairpin' precursors and function as post-transcriptional regulators of target genes. Although many individual miRNAs have recently been extensively studied, there has been very little research on miRNA transcriptomes in teleost fishes. By using high throughput sequencing technology, we have identified 167 and 166 conserved miRNAs (belonging to 108 families) in bighead carp (Hypophthalmichthys nobilis) and silver carp (Hypophthalmichthys molitrix), respectively. We compared the expression patterns of conserved miRNAs by means of hierarchical clustering analysis and log2 ratio. Results indicated that there is not a strong correlation between sequence conservation and expression conservation, most of these miRNAs have similar expression patterns. However, high expression differences were also identified for several individual miRNAs. Several miRNA* sequences were also found in our dataset and some of them may have regulatory functions. Two computational strategies were used to identify novel miRNAs from un-annotated data in the two carps. A first strategy based on zebrafish genome, identified 8 and 22 novel miRNAs in bighead carp and silver carp, respectively. We postulate that these miRNAs should also exist in the zebrafish, but the methodologies used have not allowed for their detection. In the second strategy we obtained several carp-specific miRNAs, 31 in bighead carp and 32 in silver carp, which showed low expression. Gain and loss of family members were observed in several miRNA families, which suggests that duplication of animal miRNA genes may occur through evolutionary processes which are similar to the protein-coding genes.