Knockout of miR-184 in zebrafish leads to ocular abnormalities by elevating p21 levels.

miR-184 is one of the most abundant miRNAs expressed in the lens and corneal tissue. Mutations in the seed region of miR-184 are responsible for inherited anterior segment dysgenesis. Animal models recapitulating miR-184-related anterior segment dysgenesis are still lacking, and the molecular basis of ocular abnormalities caused by miR-184 dysfunction has not been well elucidated in vivo. In the present study, we constructed a miR-184-/- zebrafish line by destroying both two dre-mir-184 paralogs with CRISPR-Cas9 technology. Although there were no gross developmental defects, the miR-184-/- zebrafish displayed microphthalmia and cataract phenotypes. Cytoskeletal abnormalities, aggregation of γ-crystallin, and lens fibrosis were induced in miR-184-/- lenses. However, no obvious corneal abnormalities were observed in miR-184-/- zebrafish. Instead of apoptosis, deficiency of miR-184 led to aberrant cell proliferation and a robust increase in p21 levels in zebrafish eyes. Inhibition of p21 by UC2288 compromised the elevation of lens fibrosis markers in miR-184-/- lenses. RNA-seq demonstrated that levels of four transcriptional factors HSF4, Sox9a, CTCF, and Smad6a, all of which could suppress p21 expression, were reduced in miR-184-/- eyes. The predicted zebrafish miR-184 direct target genes (e.g., atp1a3a and nck2a) were identified and verified in miR-184-/- eye tissues. The miR-184-/- zebrafish is the first animal model mimicking miR-184-related anterior segment dysgenesis and could broaden our understanding of the roles of miR-184 in eye development.

Extracellular HSP90 promotes differentiation of lens epithelial cells to fiber cells by activating LRP1-YAP-PROX1 axis.

Capsular residual lens epithelial cells (CRLEC) undergo differentiation to fiber cells for lens regeneration or tansdifferentiation to myofibroblasts leading to posterior capsular opacification (PCO) after cataract surgery. The underlying regulatory mechanism remains unclear. Using human lens epithelial cell lines and the ex vivo cultured rat lens capsular bag model, we found that the lens epithelial cells secrete HSP90α extracellularly (eHSP90) through an autophagy-associated pathway. Administration of recombinant GST-HSP90α protein or its M-domain induces the elongation of rat CRLEC cells with concomitant upregulation of the crucial fiber cell transcriptional factor PROX1and its downstream targets, ß- and γ-crystallins and structure proteins. This regulation is abolished by PROX1 siRNA. GST-HSP90α upregulates PROX1 by binding to LRP1 and activating LRP1-AKT mediated YAP degradation. The upregulation of GST-HSP90α on PROX1 expression and CRLEC cell elongation is inhibited by LRP1 and AKT inhibitors, but activated by YAP-1 inhibitor (VP). These data demonstrated that the capsular residue epithelial cells upregulate and secrete eHSP90α, which in turn drive the differentiation of lens epithelial cell to fiber cells. The recombinant HSP90α protein is a potential novel differentiation regulator during lens regeneration.

Deinococcus arenicola sp. nov., a novel radiation-resistant bacterium isolated from sandy soil in Antarctica.

A Gram-stain-positive, aerobic, non-mobile and spherical strain, designated ZS9-10T, belonging to the genus Deinococcus was isolated from soil sampled at the Chinese Zhong Shan Station, Antarctica. Growth was observed in the presence of 0-4 % (w/v) NaCl, at pH 7.0-8.0 and at 4-25 °C. Phylogenetic analysis based on 16S rRNA gene sequences showed that strain ZS9-10T formed a lineage in the genus Deinococcus. It exhibited highest sequence similarity (97.4 %) to Deinococcus marmoris DSM 12784T. The major phospholipids of ZS9-10T were unidentified phosphoglycolipid, unidentified glycolipids and unidentified lipids. The major fatty acids were summed feature 3 (C16 : 1 ω7c and/or C16 : 1 ω6c), C16 : 0 and C16 : 1 ω7c. MK-8 was the predominant respiratory quinone. The digital DNA-DNA hybridization and average nucleotide identity values between strain ZS9-10T and its close relative D. marmoris DSM 12784T were 27.4 and 83.9 %, respectively. Based on phenotypic, phylogenetic and genotypic data, a novel species, named Deinococcus arenicola sp. nov., is proposed. The type strain iis ZS9-10T (=CCTCC AB 2019392T=KCTC43192T).

Cryobacterium zhongshanensis sp. nov., an actinobacterium isolated from Antarctic soil.

A Gram-staining-positive, heterotrophic, non-spore-forming, non-motile, rod-shaped, strain ZS14-85T belonging to the genus Cryobacterium was isolated from soil in Antarctica. Growth was observed in the presence of 0-2% (w/v) NaCl, at pH 7.0-9.0 (optimum, pH 7.0) and 4-30 ℃ (optimum, 20 ℃). Phylogenetic analysis showed that strain ZS14-85T formed a lineage in the genus Cryobacterium. The digital DNA-DNA hybridization (dDDH) values between strain ZS14-85T and its close relatives Cryobacterium psychrotolerans CGMCC 1.5382T, Cryobacterium soli MCCC 1K03549T and Cryobacterium breve NBRC 113800T were 22.5, 22.3 and 22.2%, respectively. Orthologous Average Nucleotide Identity (OrthoANI) scores between strain ZS14-85T and C. psychrotolerans CGMCC 1.5382T, C. breve NBRC 113800T and C. soli MCCC 1K03549T were 78.7, 78.1 and 77.7%, respectively. The polar lipids of strain ZS14-85T were diphosphatidylglycerol (DPG), phosphatidylglycerol (PG), one unidentified glycolipid (GL) and two unidentified lipids (L). The major fatty acids were anteiso-C15:0 (60.7%), iso-C16:0 (17.0%) and anteiso-C17:0 (15.2%). MK-10, MK-11 and MK-9 were the predominant respiratory menaquinones. Based on phenotypic, phylogenetic and genotypic data, a novel species, Cryobacterium zhongshanensis sp. nov. is proposed. The type strain is ZS14-85T (= CCTCC AB 2019396T = KCTC 49384T).

Kaistella polysaccharea sp. nov., isolated from Antarctic intertidal sediment produces a novel extracellular polymeric substance.

An exopolysaccharide-producing bacterial strain GW4-15T, belonging to the genus Kaistella, was isolated from intertidal sediment from King George Island, Antarctic. The strain was Gram-stain-negative, aerobic, rod-shaped, non-motile and yellow-pigmented. The strain was able to grow in the presence of 0-2 % (w/v) NaCl (optimum, 0 %), at 4-30 °C (optimum, 20-28 °C) and at pH 5.0-10.0 (optimum, pH 8.0). A phylogenetic tree based on 16S rRNA gene sequences showed that strain GW4-15T formed a lineage within the genus Kaistella with the closest phylogenetic neighbours Kaistella carnis NCTC 13525T (98.3 %), Kaistella gelatinilytica G5-32T (97.7 %), Kaistella antarctica LMG 24720T (97.4 %) and Kaistella yonginensis HMD1043T (96.9 %). Digital DNA-DNA hybridization values of strain GW4-15T with K. carnis NCTC 13525T, K. antarctica LMG 24720T, K. gelatinilytica G5-32T and K. yonginensis HMD1043T were 22.8, 22.0, 21.7 and 21.6 %, respectively. The average nucleotide identity values between strain GW4-15T and K. carnis NCTC 13525T , K. antarctica LMG 24720T, K. gelatinilytica G5-32T and K. yonginensis HMD1043T were 79.3, 78.6, 77.5 and 77.2 %, respectively. The G+C content of the genome was 36.2 mol%. The major phospholipids were phosphatidylethanolamine and aminophospholipid. The predominant menaquinone was MK-6. The major fatty acids were anteiso-C15 : 0 (28.7 %), iso-C16 : 0 3-OH (15.7 %), iso-C16 : 0 H (10.0 %), iso-C16 : 0 (5.4 %), summed feature 9 (comprising iso-C17 : 1 ω9c and/or 10-methyl C16 : 0; 5.2 %) and iso-C15 : 0 (5.1 %). The monosaccharide composition of the new type of extracellular polymeric of GW4-15T was Glc, GalN, GlcN, Rha, Man and Gal with a molar ratio of 3.14 : 3.83 : 8.38 : 5.16 : 1 : 2.82. Based on phenotypic, phylogenetic and genotypic data, a novel species, Kaistella polysaccharea sp. nov., is proposed with the type strain GW4-15T (=CGMCC 1.19368T=KCTC 92753T).

Retinitis pigmentosa 2 pathogenic mutants degrade through BAG6/HUWE1 complex.

Retinitis pigmentosa (RP) is the most common inherited retinal degenerative disease which is the major cause of vision loss. X-linked RP patients account for 5%-15% of all inherited RP cases and mutations in RP2 (Retinitis pigmentosa 2) were responsible for about 20% X-linked RP families. A majority of RP2 pathogenic mutations displayed a vulnerable protein stability and degraded rapidly through ubiquitin-proteasome system (UPS). Though the RP2 protein could be readily recovered by proteasome inhibitors, e.g., MG132, their applications for RP2-related RP therapy were limited by their nonspecific characterization. In the present study, we aimed to identify UPS-related factors, such as E3 ligases, which are specifically involved in degradation of RP2 pathogenic mutants. We identified several E3 ligases, such as HUWE1, and the co-chaperon BAG6 specifically interacting with RP2 pathogenic mutants. Knockdown of HUWE1 and BAG6 could partially rescue the reduced protein levels of RP2 mutants. BAG6 is required for recruitment of HUWE1 to ubiquitinate RP2 mutants at the K268 site. The HUWE1 inhibitor BI8622 could restore the levels of RP2 mutant and then the binding to its partner ARL3 in retina cell lines. This study revealed the details of UPS-related degradation of RP2 mutants and possibly provided a potential treatment for RP2-related RP.

Differentially expressed microRNAs in the aqueous humor of patients with exfoliation glaucoma or primary open-angle glaucoma.

Both exfoliation glaucoma (XFG) and primary open-angle glaucoma (POAG) have been linked to decreased conventional outflow of aqueous humor (AH). To better understand the molecular changes in the AH content under such conditions, we analyzed the miRNA profiles of AH samples from patients with POAG and XFG compared to non-glaucoma controls. Individual AH samples (n = 76) were collected from POAG and XFG patients and age-matched controls during surgical procedure. After RNA extraction, the miRNA profiles were individually determined in 12 POAG, 12 XFG and 11 control samples. We identified 205, 295 and 195 miRNAs in the POAG, XFG and control samples, respectively. Our differential expression analysis identified three miRNAs (miR-125b-5p, miR-302d-3p and miR-451a) significantly different between POAG and controls, five miRNAs (miR-122-5p, miR-3144-3p, miR-320a, miR-320e and miR-630) between XFG and controls and one miRNA (miR-302d-3p) between POAG and XFG. While none of these miRNAs have been previously linked to glaucoma, miR-122-5p may target three glaucoma-associated genes: OPTN, TMCO1 and TGF-ß1. Pathway analysis revealed that these miRNAs are involved in potential glaucoma pathways, including focal adhesion, tight junctions, and TGF-ß signaling. Comparison of the miRNA profile in AH to unrelated human serum (n = 12) exposed potential relationships between these two fluids, although they were not significantly correlated. In summary, we have successfully profiled the miRNA expression without amplification in individual human AH samples and identified several POAG or XFG-associated miRNAs. These miRNAs may play a role in pathways previously implicated in glaucoma and act as biomarkers for disease pathogenesis.

HSP90 as a novel therapeutic target for posterior capsule opacification.

Posterior capsule opacification (PCO) is a common complication of cataract surgery, resulting from a combination of proliferation, migration, epithelial-mesenchymal transition (EMT) of residual capsular epithelial cells and fibrosis of myofibroblasts. HSP90 is known to regulate the proteostasis of cells under pathophysiological conditions. The role of HSP90 in PCO formation, however, is not clear. To do this, the lens epithelial cell lines and an ex vivo cultured rat capsular bag model were used to study the role of HSP90 in PCO formation. The expression of protein and mRNA was measured by immunoblotting and quantitative RT-PCR, and cell apoptosis was measured by TUNEL(TdT-mediated dUTP nick-end labeling). The cell proliferation was measured by cell viability assays. The results showed that 17-AAG (Tanespimycin), an inhibitor of HSP90, suppresses the proliferation of immortalized lens epithelial cell lines HLE-B3, SRA01/04, and mLEC, with IC50 values of 0.27, 0.27, and 0.49 µM, respectively. In an ex vivo cultured rat capsular model, the capsular residual epithelial cells resisted the stress of the capsulorhexis surgery and took 3-6 days to completely overlay the capsular posterior wall. During this process, heat shock factor 1 and its downstream targets HSP90, HSP25, αB-crystallin, and HSP40 were upregulated. Treatment with 17-AAG inhibited the viability of capsular residual epithelial cells and induced the cells apoptosis, characterized by increases in ROS levels, apoptotic DNA injury, and the activation of caspases 9 and 3. HSP90 participated in regulating both EGF receptor (EGFR) and TGF receptor (TGFR) signaling pathways. HSP90 was found to interact with the EGFR, such that inhibition of HSP90 by 17-AAG destabilized the EGFR protein and suppressed p-ERK1/2 and p-AKT levels. 17-AAG also inhibited the TGF-ß-induced phosphorylation of SMAD2/3 and ERK1/2 and the decrease in E-cadherin and ZO-1 expression. Accordingly, these data suggest that the induction of HSP90 protects capsular residual epithelial cells against capsulorhexis-induced stress and participates in regulating the processes of proliferation, EMT and migration of rat capsular residual epithelial cells, at least partly, through the EGFR and TGFR signaling pathways. Treatment with 17-AAG suppresses PCO formation and is therefore a potential therapeutic candidate for PCO prevention.

Efficacy of Nimodipine Plus Yufeng Ningxin Tablets for Patients with Frequent Migraine.

BACKGROUND/AIMS: To test the effects of Nimodipine plus Yufeng Ningxin tablets on frequent migraine. METHODS: Two hundred forty-two patients with frequent migraine were divided into the control group with those consuming Flunarizine (120 cases) and the treatment group with those consuming Nimodipine plus Yufeng Ningxin tablets (122 cases). The course of frequent migraine treatment lasted 7 weeks. The number of migraine days, visual analogue scale (VAS) score, and response rate were measured. RESULTS: There was significant difference in the cure rate as the Nimodipine plus Yufeng Ningxin tablets group compared with the Flunarizine group (78.7 vs. 21.7%; p < 0.001). Fewer migraine days and VAS score were observed in the treatment group when compared with the control group (p < 0.05). Nimodipine plus Yufeng Ningxin tablets were superior to Flunarizine in terms of the response rate at week 7 (p < 0.05). CONCLUSION: Due to its high cure rate, treatment with Nimodipine plus Yufeng Ningxin tablets is recommended to control frequent migraine, and this hypothesis needs to be confirmed through further studies conducted on a more extensive population.

Regulatory effect of caspase-11 on interleukin-1ß in the fungal keratitis.

Caused by fungus, fungal keratitis is a kind of infections corneal disease with high rate of blindness, which patients are mainly farmers in developing countries. Interleukin, as important proinflammatory cytokines, involve in immune defense process against fungal infection of cornea. The expression of interleukin in the pathogenesis of fungal keratitis, especially the main source of its cells, is not clear and the cell signaling pathways which regulate the synthesis and modification of interleukin is still unknown. Caspase-11 was obtained and cultured. And the ELISA and Western-blot methods were used to explore the regulatory effect of Caspse-11 on Interleukin-1ß in the fungal keratitis. neutrophils were the main cell lineage of IL-1ß to take part in the innate anti-fungi immunity in the cornea; IL-1ß generation induced by fungal infection might not be through the pre-excitation in the classical signal pathway; TLR4/TRIF pathway was not involved in pro-IL-1ß generation; while Dectin-1/syk pathway was involved in IL-1ß generation in the fungal keratitis; Caspase-l participated in the modification of IL-1ß to change from the precursor into the mature body; but NLRP3 inflammasome and ASC inflammasome were not involved in IL-1ß generation; Caspase-11 was involved in IL-1ß generation through regulating the modified process of Caspase-l to turning from precursor into mature body. TLR4/TRIF pathway and NLRP3 inflammasome and ASC inflammasome are not involved in the pro-IL-1ß generation, while Caspase-l, Caspase-11 and Dectin-1/syk pathway are involved in the IL-1ß generation.

Effect of therapeutic ultrasound on brain angiogenesis following intracerebral hemorrhage in rats.

Intracerebral hemorrhage (ICH) can produce severe neurological deficits in stroke survivors. However, few effective approaches are available to improve the recovery from ICH. Given that therapeutic ultrasound exposure can enhance on angiogenesis in peripheral tissues, the present study was designed to examine the effects of therapeutic ultrasound exposure on the brain angiogenesis following ICH. To this end, we applied once daily therapeutic ultrasound treatment to rats for 7 consecutive days after intracranial infusion of vehicle (Sham control) or collagenase (ICH). Repeated exposure to the low intensity of therapeutic ultrasound decreased behavioral scores in ICH rats, but not in sham control rats. Such an effect was correlated with an increased number of vessel-like structures and microvessels and PCNA positive cells in vWF-positive blood vessels in perihematomal brain tissues at post-ICH day 7. Furthermore, immunohistochemistry and western blotting results showed that ICH trigged the expression of extracellular matrix (ECM)-related molecules, including collagen Is, III, and IV, as well as integrins αvß3 and α5ß1, and exposure to therapeutic ultrasound increased the expression of these molecules. Therefore, our results indicated that repeated exposure to a low intensity of therapeutic ultrasound can increase the expression of collagen and integrins of ECM-related molecules, promote the formation of a large number of vessel-like structure and capillaries around the hematoma, and accelerate the recovery of neurological function impaired by ICH.

Glucose regulates heat shock factor 1 transcription activity via mTOR pathway in HCC cell lines.

HSF1-mediated heat shock response is activated in most tumors and plays important roles in regulating tumor homeostasis. However, the signals underlying HSF1 activation is still not completely understood. In this paper, we find that glucose, the dominant tumor energy supplement, participates in regulating HSF1's activation in HCC cell lines. The immunoblotting results indicate that the phosphorylation of HSF1/S326, a hallmark of HSF1 activation, varies between the HCC cell lines (e.g., SMMC7721, HapG2, plc/prf5, and Chang-liver). Glucose, but not 2D-glucose, can induce the phosphorylation of HSF1 at S326 and upregulate the expression of HSF1's downstream alpha B-crystallin and Hsp70 as well as the none-heat shock proteins CSK2 and RBM23 in two tested hepatocellular carcinoma cell lines (prl/prf5 and SMMC7721). Rapamycin, an inhibitor of mTOR, can suppress the glucose-induced phosphorylation of HSF1/S326 and the expression of alpha B-crystallin. Knockdown of HSF1 with shRNA enhances the glucose-depletion-mediated inhibition of plc/prf5 cell proliferation. Our data reveal that HSF1 can be activated by glucose-mTOR pathway, providing an alternative pathway for targeting HSF1 in tumor therapy.

Genome-based classification of Pedobacter polysacchareus sp. nov., isolated from Antarctic soil producing exopolysaccharide.

A psychrotolerant bacterial strain, designated ZS13-49T, with strong extracellular polysaccharide synthesis ability was isolated from soil collected in Antarctica and subjected to polyphasic taxonomic and comparative genomics. Chemotaxonomic features, including fatty acids, and polar lipid profiles, support the assignment of strain ZS13-49T to the genus Pedobacter. 16S rRNA gene phylogeny demonstrates that strain ZS13-49T forms a well-supported separate branch as a sister clade to Pedobacter gandavensis LMG 31462T and is clearly separated from Pedobacter steynii DSM 19110T and Pedobacter caeni DSM 16990T. Phylogenetic analysis showed strain ZS13-49T shared the highest 16S rRNA gene sequence similarity (99.9%) with P. gandavensis LMG 31462T. However, the digital DNA-DNA hybridization (dDDH), average nucleotide identity (ANI) value and average amino identity (AAI) value between strain ZS13-49T and P. gandavensis LMG 31462T were 26.5%, 83.3%, and 87.5%, respectively. Phylogenomic tree and a comparative genomic analysis indicated distinct characteristics to distinguish strain ZS13-49T from the closely related species. The complete genome sequence of strain ZS13-49T consists of 5 830 353 bp with 40.61% G + C content. Genomic features of strain ZS13-49T adapted to Antarctic environment were also revealed. Based on the phenotypic, chemotaxonomic, and genomic data, strain ZS13-49T could be assigned to a novel species within the genus Pedobacter for which the name Pedobacter polysacchareus sp. nov. is proposed. The type strain is ZS13-49T ( = CCTCC AB 2019394T = KCTC 72824T).

Knockout of DLIC1 leads to retinal cone degeneration via disturbing Rab8 transport in zebrafish.

Retinal photoreceptors execute phototransduction functions and require an efficient system for the transport of materials (e.g. proteins and lipids) from inner segments to outer segments. Cytoplasmic dynein 1 is a minus-end-directed microtubule motor and participates in cargo transport in the cytoplasm. However, the roles of dynein 1 motor in photoreceptor cargo transport and retinal development are still ambiguous. In our present study, the light intermediate chain protein DLIC1 (encoded by dync1li1), links activating adaptors to bind diverse cargos in the dynein 1 motor, was depleted using CRISPR-Cas9 technology in zebrafish. The dync1li1-/- zebrafish displayed progressive degeneration of retinal cone photoreceptors, especially blue cones. The retinal rods were not affected in dync1li1-/- zebrafish. Knockout of DLIC1 resulted in abnormal expression and localization of cone opsins in dync1li1-/- retinas. TUNEL staining suggested that apoptosis was induced after aberrant accumulation of cone opsins in photoreceptors of dync1li1-/- zebrafish. Instead of Rab11 transport, Rab8 transport was disturbed in dync1li1-/- retinas. Our data demonstrate that DLIC1 is required for function maintenance and survival of cone photoreceptors, and hint at an essential role of the cytoplasmic dynein 1 motor in photoreceptor cargo transport.

Biotin attenuates heat shock factor 4b transcriptional activity by lysine 444 biotinylation.

Genetic mutations in HSF4 cause congenital cataracts. HSF4 exhibits both positive and negative regulation on the transcription of heat shock and non-heat shock proteins during lens development, and its activity is regulated by posttranslational modifications. Biotin is an essential vitamin that regulates gene expression through protein biotinylation. In this paper, we report that HSF4b is negatively regulated by biotinylation. Administration of biotin or ectopic bacterial biotin ligase BirA increases HSF4b biotinylation at its C-terminal amino acids from 196 to 493. This attenuates the HSF4b-controlled expression of αB-crystallin in both lens epithelial cells and tested HEK293T cells. HSF4b interacts with holocarboxylase synthetase (HCS), a ubiquitous enzyme for catalyzing protein biotinylation in mammal. Ectopic HA-HCS expression downregulates HSF4b-controlled αB-crystallin expression. Lysine-mutation analyses indicate that HSF4b/K444 is a potential biotinylation site. Mutation K444R reduces the co-precipitation of HSF4b by streptavidin beads and biotin-induced reduction of αB-crystallin expression. Mutations of other lysine residues such as K207R/K209R, K225R, K288R, K294R and K355R in HSF4's C-terminal region do not affect HSF4's expression level and the interaction with streptavidin, but they exhibit distinct regulation on αB-crystallin expression through different mechanisms. HSF4/K294R leads to upregulation of αB-crystallin expression, while mutations K207R/K209R, K225R, K288R, K255R and K435R attenuate HSF4's regulation on αB-crystallin expression. K207R/K209R blocks HSF4 nuclear translocation, and K345R causes HSF4 destabilization. Taken together, the data reveal that biotin maybe a novel factor in modulating HSF4 activity through biotinylation.

[Analysis of metabolite differences in skin between Clapp's Favorite and its mutant Red Clapp's Favorite through non-targeted metabolomics].

Red Clapp's Favorite is the red mutation cultivar of the pear cultivar Clapp's Favorite. Fruit color is an important feature of pear fruits, with red skin generally attracting consumers. Anthocyanin, chlorophyll, and carotenoids are the most important pigments in the color formation of fruits. The red color of pear skin is mainly due to the concentration and composition of anthocyanin. Metabolomics is an emerging discipline that focuses on the qualitative and quantitative analysis of small metabolites with low molecular weight in biological cells and tissues. As an important part of systems biology, it is an effective means to solve many complex biological problems. Studies have analyzed pigment content, composition, and differentially expressed genes in the skin of green and red pears from various aspects. Anthocyanins are responsible for physiological activity on regulating pathways. The aim of this study was to discover differential metabolites in the skin of Clapp's Favorite and its red mutation cultivar Red Clapp's Favorite. The metabolic components were detected using high-performance liquid chromatography coupled with tandem mass spectrometry (UPLC-MS/MS). Chromatographic experiments were performed on an HSS T3 column (100 mm×2.1 mm, 1.8 µm) by using a mobile phase consisting of 0.1% (v/v) formic acid in acetonitrile and water, and mass spectrometry was conducted in the positive and negative modes by electrospray ionization (ESI). Red Clapp's Favorite and Clapp's Favorite were collected from the pear germplasm resource nursery of Yantai Institute of Agricultural Sciences in Shandong. The data were analyzed by principal component analysis (PCA) and orthogonal partial least squares discriminant analysis (OPLS-DA) as well as cluster analysis and heat map. The first two principal components exhibited 62.3% and 8% of the total variance in the positive and negative ion modes, respectively. PCA can generally reflect metabolite differences between the two groups of samples, and there are significant differences in metabolites between the two cultivars. The results showed that PLS-DA clearly distinguishes the two groups of samples, which can be used to analyze the subsequent difference in components. The compounds were identified based on data retrieved from the PMDB databases according to the accurate mass number, secondary fragment, and isotope distribution. The results showed that the metabolite content in the skin of Red Clapp's Favorite and Clapp's Favorite were significant. There were 83 different metabolites (P<0.05, variable importance in project (VIP)≥1), including phenols and amino acids, which are involved in flavonoid metabolism, amino acid metabolism, phenyl propanoid biosynthesis, and other metabolic pathways, including 5 polyphenols, 3 flavonoids, 1 amino acid and derivatives, 8 phenylpropanes, 2 anthocyanins, 5 proanthocyanidins, 6 flavanols, 14 flavonols, 2 isoflavones, 13 triterpenoids, 3 organic acids and derivatives, 1 vitamin, 3 organic acids and derivatives, 15 lipids, and 2 other compounds. The chlorogenic acid and crypto-chlorogenic acid in Red Clapp's Favorite are 2.40 and 3.46 times as much as those in Clapp's Favorite. The anthocyanins of cornulin 3-glucoside and cornulin 3-galactoside were 10.235 and 9.394 times, respectively. Phenolic epicatechin and catechin increased by 4.689 and 4.635, respectively. The content of phenylpropane 3, 4-dihydroxycinnamic acid in Red Clapp's Favorite increased by 3.13 times. Among the 83 differential metabolites, 23 metabolites were enriched in the pathway. To display the relationship between the samples and the differences in metabolites among the different samples intuitively, hierarchical clustering and heat map analysis were performed on the metabolite expression levels with significant differences in the enrichment pathways. The Kyoto Encyclopedia of Genes and Genomes database was used to further analyze the pathway enrichment of different metabolites. According to the results, there were 6 metabolic pathways (P<0.05): flavonoid biosynthesis, flavone and flavonol biosynthesis, phenylpropanoid biosynthesis, butanoate metabolism, phenylalanine metabolism, and tyrosine metabolism. Plant secondary metabolism shows a complex diversity. This study would screen out other pathways affecting the biosynthesis of flavonoids, which could provide reference for the further study of biosynthesis and biological function of flavonoids in red fruits. This study provides a useful reference for metabolomics of red pears, which could provide a theoretical reference for the quality analysis and biological function research of pears.

Deficiency of heat shock factor 4 promotes lens epithelial cell senescence through upregulating p21cip1 expression.

Genetic mutations in heat shock factor 4 (Hsf4) is associated with both congenital and age-related cataracts. Hsf4 regulates lens development through its ability to both activate and inhibit transcription. Previous studies suggested Hsf4 is involved in modulating cellular senescence depending on p21cip1 and p27 kip1 expression in MEF cells. Here, we found that Hsf4 acts as a suppressor of p21cip1 expression and plays an anti-senescence role during lens development. Knocking out Hsf4 facilitated UVB-induced cellular senescence in mouse lens epithelial cells (mLECs). p21cip1 was upregulated at both the mRNA and protein levels in HSF4-/- mLECs under control and UVB-treated conditions, and knockdown of p21cip1 by siRNA alleviated UVB-induced cellular senescence. HSF4 directly bound to the p21cip1 promoter and increased H3K27m3 levels at the p21cip1 proximal promoter region by recruiting the methyltransferase EZH2. In animal models, p21cip1 was gradually upregulated in wild-type mouse lenses with increasing age, while Hsf4 levels decreased. We generated a Hsf4 mutant mice line (Hsf4del-42) which displayed obvious congenital cataract phenotype. The expression of p21cip1 and senescence-associated cytokines were induced in the cataractous lenses of Hsf4del-42 mice. H3K27m3 and EZH2 levels decreased in p21cip1 promoters in the lenses of Hsf4del-42 mice. The SA-ß-Gal activities were positive in lens epithelia of aged Hsf4null zebrafish compared to wild-type lenses. p21cip1 and senescence-associated cytokines levels were also upregulated in lenses of Hsf4null zebrafish. Accordingly, we propose that HSF4 plays a protective role in lens epithelial cells against cellular senescence during lens development and aging, partly by fine-tuning p21cip1 expression.

HSP90 acts as a senomorphic target in senescent retinal pigmental epithelial cells.

The senescence of retinal pigment epithelial (RPE) cells is associated with age-related macular degeneration (AMD), a leading cause of blindness in the world. HSP90 is a predominant chaperone that regulates cellular homeostasis under divergent physio-pathological conditions including senescence. However, the role of HSP90 in senescent RPE cells still remains unclear. Here, we reported that HSP90 acts as a senomorphic target of senescent RPE cells in vitro. Using H2O2-induced senescent ARPE-19 cells and replicative senescent primary RPE cells from rhesus monkey, we found that HSP90 upregulates the expression of IKKα, and HIF1α in senescent ARPE-19 cells and subsequently controls the induction of distinct senescence-associated inflammatory factors. Senescent ARPE-19 cells are more resistant to the cytotoxic HSP90 inhibitor IPI504 (IC50 = 36.78 µM) when compared to normal ARPE-19 cells (IC50 = 6.16 µM). Administration of IPI504 at 0.5-5 µM can significantly inhibit the induction of IL-1ß, IL-6, IL-8, MCP-1 and VEGFA in senescent ARPE-19 and the senescence-mediated migration of retinal capillary endothelial cells in vitro. In addition, we found that inhibition of HSP90 by IPI504 reduces SA-ß-Gal's protein expression and enzyme activity in a dose-dependent manner. HSP90 interacts with and regulates SA-ß-Gal protein stabilization in senescent ARPE-19 cells. Taken together, these results suggest that HSP90 regulates the SASP and SA-ß-Gal activity in senescent RPE cells through associating with distinctive mechanism including NF-κB, HIF1α and lysosomal SA-ß-Gal. HSP90 inhibitors (e.g. IPI504) could be a promising senomorphic drug candidate for AMD intervention.

Alkaloid accumulation in different parts and ages of Lycoris chinensis.

The galanthamine, lycorine, and lycoramine content of Lycoris chinensis was researched during development from young to old plants, i.e. in seeds, ten-day-old seedlings, three-month-old seedlings, one-year-old seedlings, and perennial seedlings. Notably the alkaloid level reduced to its lowest content 10 days after seed germinating. Then the accumulation of galanthamine tended to increase with age, reaching a higher value in perennial seedlings. The production pattern of lycorine and lycoramine was found similar to that of galanthamine. Different plant organs were also evaluated for their galanthamine, lycorine, and lycoramine contents. Mature seeds had the highest content of galanthamine (671.33 microg/g DW). Kernels, seed capsules, and root-hairs were the main repository sites for galanthamine, lycorine, and lycoramine. The leaves were the least productive organs.

Expression of mRNAs, miRNAs, and lncRNAs in Human Trabecular Meshwork Cells Upon Mechanical Stretch.

Purpose: Intraocular pressure (IOP), the primary risk factor for primary open-angle glaucoma, is determined by resistance to aqueous outflow through the trabecular meshwork (TM). IOP homeostasis relies on TM responses to mechanical stretch. To model the effects of elevated IOP on the TM, this study sought to identify coding and non-coding RNAs differentially expressed in response to mechanical stretch. Methods: Monolayers of TM cells from non-glaucomatous donors (n = 5) were cultured in the presence or absence of 15% mechanical stretch, 1 cycle/second, for 24 hours using a computer-controlled Flexcell unit. We profiled mRNAs and lncRNAs with stranded total RNA sequencing and microRNA (miRNA) expression with NanoString-based miRNA assays. We used two-tailed paired t-tests for mRNAs and long non-coding RNAs (lncRNAs) and the Bioconductor limma package for miRNAs. Gene ontology and pathway analyses were performed with WebGestalt. miRNA-mRNA interactions were identified using Ingenuity Pathway Analysis Integrative miRNA Target Finder software. Validation of differential expression was conducted using droplet digital PCR. Results: We identified 219 mRNAs, 42 miRNAs, and 387 lncRNAs with differential expression in TM cells upon cyclic mechanical stretch. Pathway analysis indicated significant enrichment of genes involved in steroid biosynthesis, glycerolipid metabolism, and extracellular matrix-receptor interaction. We also identified several miRNA master regulators (miR-125a-5p, miR-30a-5p, and miR-1275) that regulate several mechanoresponsive genes. Conclusions: To our knowledge, this is the first demonstration of the differential expression of coding and non-coding RNAs in a single set of cells subjected to cyclic mechanical stretch. Our results validate previously identified, as well as novel, genes and pathways.