Semi-Rational Design of L-Isoleucine Dioxygenase Generated Its Activity for Aromatic Amino Acid Hydroxylation.

Fe (II)-and 2-ketoglutarate-dependent dioxygenases (Fe (II)/α-KG DOs) have been applied to catalyze hydroxylation of amino acids. However, the Fe (II)/α-KG DOs that have been developed and characterized are not sufficient. L-isoleucine dioxygenase (IDO) is an Fe (II)/α-KG DO that specifically catalyzes the formation of 4-hydroxyisoleucine (4-HIL) from L-isoleucine (L-Ile) and exhibits a substrate specificity toward L-aliphatic amino acids. To expand the substrate spectrum of IDO toward aromatic amino acids, in this study, we analyzed the regularity of the substrate spectrum of IDO using molecular dynamics (MD) simulation and found that the distance between Fe2+, C2 of α-KG and amino acid chain's C4 may be critical for regulating the substrate specificity of the enzyme. The mutation sites (Y143, S153 and R227) were also subjected to single point saturation mutations based on polarity pockets and residue free energy contributions. It was found that Y143D, Y143I and S153A mutants exhibited catalytic L-phenylalanine activity, while Y143I, S153A, S153Q and S153Y exhibited catalytic L-homophenylalanine activity. Consequently, this study extended the substrate spectrum of IDO with aromatic amino acids and enhanced its application property.

Declines in PDE4B activity promote myopia progression through downregulation of scleral collagen expression.

Myopia is the most common cause of a visual refractive error worldwide. Cyclic adenosine monophosphate (cAMP)-linked signaling pathways contribute to the regulation of myopia development, and increases in cAMP accumulation promote myopia progression. To pinpoint the underlying mechanisms by which cAMP modulates myopia progression, we performed scleral transcriptome sequencing analysis in form-deprived mice, a well-established model of myopia development. Form deprivation significantly inhibited the expression levels of genes in the cAMP catabolic pathway. Quantitative real-time polymerase chain reaction analysis validated that the gene expression level of phosphodiesterase 4B (PDE4B), a cAMP hydrolase, was downregulated in form-deprived mouse eyes. Under visually unobstructed conditions, loss of PDE4B function in Pde4b-knockout mice increased the myopic shift in refraction, -3.661 ± 1.071 diopters, more than that in the Pde4b-wildtype littermates (P < 0.05). This suggests that downregulation and inhibition of PDE4B gives rise to myopia. In guinea pigs, subconjunctival injection of rolipram, a selective inhibitor of PDE4, led to myopia in normal eyes, and it also enhanced form-deprivation myopia (FDM). Subconjunctival injection of dibutyryl-cyclic adenosine monophosphate, a cAMP analog, induced only a myopic shift in the normal visually unobstructed eyes, but it did not enhance FDM. As myopia developed, axial elongation occurred during scleral remodeling that was correlated with changes in collagen fibril thickness and distribution. The median collagen fibril diameter in the FDM + rolipram group, 55.09 ± 1.83 nm, was thinner than in the FDM + vehicle group, 59.33 ± 2.06 nm (P = 0.011). Thus, inhibition of PDE4 activity with rolipram thinned the collagen fibril diameter relative to the vehicle treatment in form-deprived eyes. Rolipram also inhibited increases in collagen synthesis induced by TGF-ß2 in cultured human scleral fibroblasts. The current results further support a role for PDE enzymes such as PDE4B in the regulation of normal refractive development and myopia because either loss or inhibition of PDE4B function increased myopia and FDM development through declines in the scleral collagen fibril diameter.

Structural insights into alcohol dehydrogenases catalyzing asymmetric reductions.

Alcohol dehydrogenases are a group of oxidoreductases that specifically use NAD(P)+ or NAD(P)H as cofactors for electron acceptance or donation and catalyze interconversion between alcohols and corresponding carbonyl compounds. In addition to their physiological roles in metabolizing alcohols and aldehydes or ketones, alcohol dehydrogenases have received considerable attention with respect to their symmetry-breaking traits in catalyzing asymmetric reactions and have Accordingly, they have become widely applied in fine chemical synthesis, particularly in the production of chiral alcohols and hydroxyl compounds that are key elements in the synthesis of active pharmaceutical ingredients (API) employed in the pharmaceutical industry. The application of structural bioinformatics to the study of functional enzymes and recent scientific breakthroughs in modern molecular biotechnology provide us with an effective alternative to gain an understanding of the molecular mechanisms involved in asymmetric bioreactions and in overcoming the limitations of enzyme availability. In this review, we discuss molecular mechanisms underlying alcohol dehydrogenase-mediated asymmetric reactions, based on protein structure-function relationships from domain structure to functional active sites. The molecular principles of the catalytic machinery involving stereochemical recognition and molecular interaction are also addressed. In addition, the diversity of enzymatic functions and properties, for example, enantioselectivity, substrate specificity, cofactor dependence, metal requirement, and stability in terms of organic solvent tolerance and thermostability, are also discussed and based on a comparative analysis of high-resolution 3 D structures of representative alcohol dehydrogenases.

Redox-dependent interactions between reduced/oxidized cytochrome c and cytochrome c oxidase evaluated by in-situ electrochemical surface plasmon resonance.

The interactions between the redox couple of cytochrome c (Cyt c) and cytochrome c oxidase (COX) were investigated at a mimic redox-modulated interface by using an electrochemical surface plasmon resonance (EC-SPR) system. Although early studies of the binding between COX and Cyt c have been conducted using several techniques in homogeneous solutions, a problem still inherent is that ferro-cytochrome c (Cyt c red), the reduced form of Cyt c, can be easily oxidized into ferri-cytochrome c (Cyt c ox) and adversely impact the accuracy and reproducibility of the binding measurements. In order to realize reliable redox-dependent binding tests, here the Cyt c red is quantitatively electro-generated from Cyt c ox by in situ cathodic polarization in a flow cell. Then the kinetic and dissociation constants of the bindings between COX and Cyt c red/Cyt c ox can be evaluated accurately. In this study, the values of association/dissociation rate constants (k a, k d) for both COX/Cyt c red and COX/Cyt c ox were obtained. The dissociation constants, K D, were finally calculated as 3.33 × 10(-8) mol · L(-1) for COX/Cyt c red and 4.25 × 10(-5) mol · L(-1) for COX/Cyt c ox, respectively. In-situ EC-SPR is promising for better mimicking the in vivo condition that COX is embedded in the inner mitochondrial membrane and Cyt c acts as an electron shuttle in the mobile phase. It is an effective method for the investigation of redox-dependent biomolecular interactions. Graphical Abstract Schematic representation of the experimental designs using EC-SPR system. (a) the Au-Cys-COX SPR chip with SAM layers. (b) redox-modulated Cyt c and its binding onto pre-immobilized COX.

Effect of High-Sucrose Diet on the Occurrence and Progression of Diabetic Retinopathy and Dietary Modification Strategies.

As the most serious of the many worse new pathological changes caused by diabetes, there are many risk factors for the occurrence and development of diabetic retinopathy (DR). They mainly include hyperglycemia, hypertension, hyperlipidemia and so on. Among them, hyperglycemia is the most critical cause, and plays a vital role in the pathological changes of DR. High-sucrose diets (HSDs) lead to elevated blood glucose levels in vivo, which, through oxidative stress, inflammation, the production of advanced glycation end products (AGEs) and vascular endothelial growth factor (VEGF), cause plenty of pathological damages to the retina and ultimately bring about loss of vision. The existing therapies for DR primarily target the terminal stage of the disease, when irreversible visual impairment has appeared. Therefore, early prevention is particularly critical. The early prevention of DR-related vision loss requires adjustments to dietary habits, mainly by reducing sugar intake. This article primarily discusses the risk factors, pathophysiological processes and molecular mechanisms associated with the development of DR caused by HSDs. It aims to raise awareness of the crucial role of diet in the occurrence and progression of DR, promote timely changes in dietary habits, prevent vision loss and improve the quality of life. The aim is to make people aware of the importance of diet in the occurrence and progression of DR. According to the dietary modification strategies that we give, patients can change their poor eating habits in a timely manner to avoid theoretically avoidable retinopathy and obtain an excellent prognosis.

Acinar cells and the development of pancreatic fibrosis.

Pancreatic fibrosis is caused by excessive deposition of extracellular matrixes of collagen and fibronectin in the pancreatic tissue as a result of repeated injury often seen in patients with chronic pancreatic diseases. The most common causative conditions include inborn errors of metabolism, chemical toxicity and autoimmune disorders. Its pathophysiology is highly complex, including acinar cell injury, acinar stress response, duct dysfunction, pancreatic stellate cell activation, and persistent inflammatory response. However, the specific mechanism remains to be fully clarified. Although the current therapeutic strategies targeting pancreatic stellate cells show good efficacy in cell culture and animal models, they are not satisfactory in the clinic. Without effective intervention, pancreatic fibrosis can promote the transformation from pancreatitis to pancreatic cancer, one of the most lethal malignancies. In the normal pancreas, the acinar component accounts for 82% of the exocrine tissue. Abnormal acinar cells may activate pancreatic stellate cells directly as cellular source of fibrosis or indirectly via releasing various substances and initiate pancreatic fibrosis. A comprehensive understanding of the role of acinar cells in pancreatic fibrosis is critical for designing effective intervention strategies. In this review, we focus on the role of and mechanisms underlying pancreatic acinar injury in pancreatic fibrosis and their potential clinical significance.

Synergy of 5-aminolevulinate supplement and CX3CR1 suppression promotes liver regeneration via elevated IGF-1 signaling.

Inadequate remnant volume and regenerative ability of the liver pose life-threatening risks to patients after partial liver transplantation (PLT) or partial hepatectomy (PHx), while few clinical treatments focus on safely accelerating regeneration. Recently, we discovered that supplementing 5-aminolevulinate (5-ALA) improves liver cold adaptation and functional recovery, leading us to uncover a correlation between 5-ALA metabolic activities and post-PLT recovery. In a mouse 2/3 PHx model, 5-ALA supplements enhanced liver regeneration, promoting infiltration and polarization of anti-inflammatory macrophages via P53 signaling. Intriguingly, chemokine receptor CX3CR1 functions to counterbalance these effects. Genetic ablation or pharmacological inhibition of CX3CR1 (AZD8797; phase II trial candidate) augmented the macrophagic production of insulin-like growth factor 1 (IGF-1) and subsequent hepatocyte growth factor (HGF) production by hepatic stellate cells. Thus, short-term treatments with both 5-ALA and AZD8797 demonstrated pro-regeneration outcomes superior to 5-ALA-only treatments in mice after PHx. Overall, our findings may inspire safe and effective strategies to better treat PLT and PHx patients.

The FGF2 gene in a myopia animal model and human subjects.

PURPOSE: Fibroblast growth factor-2 (FGF2) has been implied in the development of myopia according to previous studies investigating FGF2 in the sclera and retinal pigment epithelium. This study measured retinal FGF2 gene expression in an animal model and also tested for the association between single nucleotide polymorphisms (SNPs) in FGF2 and high myopia. METHODS: The guinea pigs were assigned to 2 groups: form deprivation myopia (FDM) for two weeks and normal control (free of form deprivation). Biometric measurement was performed and FGF2 expression levels were compared among the FDM eyes, the fellow eyes of the FDM group and the normal eyes in retina. We also enrolled 1,064 cases (≤-6.0 D) and 1,001 controls (≥-1.5 D) from a Chinese population residing in Taiwan. Six tagging SNPs were genotyped to test for an association between genotypes and high myopia. RESULTS: The FDM eyes had the most prominent changes of refraction and axial length. Compared with the mRNA levels of FGF2 in the normal eyes, the FDM eyes had the highest levels of mRNA (p=0.0004) followed by the fellow eyes (p=0.002). The FDM and normal eyes became more myopic compared with the fellow eyes, but the fellow eyes became more hyperopic (p=0.004) in the end of the experiment which may be due to its relatively short axial length when compared with normal eyes (p=0.05). The SNP genotypes were all in Hardy-Weinberg equilibrium. However, none of the SNPs were significantly associated with high myopia (all p values >0.1). CONCLUSIONS: We identified a significant change of FGF2 expression in the FDM eyes but FGF2 genetic variants are unlikely to influence susceptibility to myopia. There may be a systemic effect to influence gene expression and refraction on the fellow eyes, which may perturb emmetropization in the fellow eyes. Our data also suggest using normal eyes rather than the fellow eyes as the control eyes when study the form deprivation myopia.

Experimental murine myopia induces collagen type Iα1 (COL1A1) DNA methylation and altered COL1A1 messenger RNA expression in sclera.

PURPOSE: To investigate whether myopia development is associated with changes of scleral DNA methylation in cytosine-phosphate-guanine (CpG) sites in the collagen 1A1 (COL1A1) promoter and messenger RNA (mRNA) levels following murine form deprivation myopia. METHODS: Fifty-seven C57BL/6 mice (postnatal day 23) were randomly assigned to four groups: (1) monocular form deprivation (MD) in which a diffuser lens was placed over one eye for 28 days; (2) normal controls without MD; (3) MD recovery in which the diffuser lens was removed for seven days; and (4) MD recovery normal controls. The DNA methylation pattern in COL1A1 promoter and exon 1 was determined by bisulfite DNA sequencing, and the COL1A1 mRNA level in sclera was determined by quantitative PCR. RESULTS: MD was found to induce myopia in the treated eyes. Six CpG sites in the promoter and exon 1 region of COL1A1 were methylated with significantly higher frequency in the treated eyes than normal control eyes (p<0.05), with CpG island methylation in MD-contralateral eyes being intermediate. Consistent with the CpG methylation, scleral COL1A1 mRNA was reduced by 57% in the MD-treated eyes compared to normal controls (p<0.05). After seven days of MD recovery, CpG methylation was significantly reduced (p=0.01). The methylation patterns returned to near normal level in five CpG sites, but the sixth was hypomethylated compared to normal controls. CONCLUSIONS: In parallel with the development of myopia and the reduced COL1A1 mRNA, the frequency of methylation in CpG sites of the COL1A1 promoter/exon 1 increased during MD and returned to near normal during recovery. Thus, hypermethylation of CpG sites in the promoter/exon 1 of COL1A1 may underlie reduced collagen synthesis at the transcriptional level in myopic scleras.

Znhit1 Regulates p21Cip1 to Control Mouse Lens Differentiation.

Purpose: The transparency of the ocular lens is essential for refracting and focusing light onto the retina, and transparency is controlled by many factors and signaling pathways. Here we showed a critical role of chromatin remodeler zinc finger HIT-type containing 1 (Znhit1) in maintaining lens transparency. Methods: To explore the roles of Znhit1 in lens development, the cre-loxp system was used to generate lens-specific Znhit1 knockout mice (Znhit1Mlr10-Cre; Znhit1 cKO). Morphological changes in mice lenses were examined using hematoxylin and eosin staining. RNA sequencing (RNA-seq) and assay for transposase accessible chromatin using sequencing (ATAC-seq) were applied to screen transcriptome changes. Immunofluorescence staining were performed to assess proteins distribution and terminal deoxynucleotidyl transferase dUTP nick-end labeling staining were used for determining apoptosis. The mRNAs expression was examined by quantitative RT-PCR and proteins expression by Western blot. Results: Lens-specific conditional knockout mice had a severe cataract, microphthalmia phenotype, and seriously abnormal lens fiber cells differentiation. Deletion of Znhit1 in the lens resulted in decreased cell proliferation and increased cell apoptosis of the lens epithelia. ATAC-seq showed that Znhit1 deficiency increased chromatin accessibility of cyclin-dependent kinase inhibitors, including p57Kip2 and p21Cip1, and upregulated the expression of these genes in mRNA and protein levels. And we also showed that loss of Znhit1 lead to lens fibrosis by upregulating the expression of p21Cip1. Conclusions: Our findings suggested that Znhit1 is required for the survival of lens epithelial cells. The loss of Znhit1 leads to the overexpression of p21Cip1, further resulting in lens fibrosis, and impacted the establishment of lens transparency.

Local glutamate level dictates adenosine A2A receptor regulation of neuroinflammation and traumatic brain injury.

During brain injury, extracellular adenosine and glutamate levels increase rapidly and dramatically. We hypothesized that local glutamate levels in the brain dictates the adenosine-adenosine A(2A) receptor (A(2A)R) effects on neuroinflammation and brain damage outcome. Here, we showed that, in the presence of low concentrations of glutamate, the A(2A)R agonist 3-[4-[2-[[6-amino-9-[(2R,3R,4S,5S)-5-(ethylcarbamoyl)-3,4-dihydroxy-oxolan-2-yl]purin-2-yl]amino]ethyl]phenyl]propanoic acid (CGS21680) inhibited lipopolysaccharide (LPS)-induced nitric oxide synthase (NOS) activity of cultured microglial cells, an effect that was dependent on the protein kinase A (PKA) pathway. However, in high concentrations of glutamate, CGS21680 increased LPS-induced NOS activity in a protein kinase C (PKC)-dependent manner. Thus, increasing the local level of glutamate redirects A(2A)R signaling from the PKA to the PKC pathway, resulting in a switch in A(2A)R effects from antiinflammatory to proinflammatory. In a cortical impact model of traumatic brain injury (TBI) in mice, brain water contents, behavioral deficits, and expression of tumor necrosis factor-alpha, interleukin-1 mRNAs, and inducible NOS were attenuated by administering CGS21680 at post-TBI time when brain glutamate levels were low, or by administering the A(2A)R antagonist ZM241385 [4-(2-{[5-amino-2-(2-furyl)[1,2,4]triazolo[1,5-a][1,3,5]triazin-7-yl]amino}ethyl)phenol] at post-TBI time when brain glutamate levels were elevated. Furthermore, pre-TBI treatment with the glutamate release inhibitor (S)-4C3HPG [(S)-4-carboxy-3-hydroxyphenylglycine] converted the debilitating effect of CGS21680 administered at post-TBI time with high glutamate level to a neuroprotective effect. This further indicates that the switch in the effect of A(2A)R activation in intact animals from antiinflammatory to proinflammatory is dependent on glutamate concentration. These findings identify a novel role for glutamate in modulation of neuroinflammation and brain injury via the adenosine-A(2A)R system.

Normal Basal Epithelial Cells Stimulate the Migration and Invasion of Prostate Cancer Cell RM-1 by TGF-ß1/STAT3 Axis in vitro.

AIM: Basal epithelial cells are absent in distant prostate cancer. This study aimed to investigate whether basal epithelial cells could suppress migration and invasion of prostate cancer cells to become a new treatment strategy for prostate cancer. MAIN METHODS: Basal epithelial cells were identified by immunofluorescence with anti-p63. Wound healing assays or transwell assays were used to explore the effects of basal epithelial cells, TGF-ß1, SB431542 (inhibitor of TGF-ß type I receptor) or stattic (inhibitor of phosphorylated STAT3) on migration or invasion of mouse prostate cancer cell (RM-1). Concentration of TGF-ß1 was measured by ELISA assay. HE staining was used to investigate cell morphology. Immunocytochemistry with anti-p63 was used to identify basal epithelial cells. Levels of STAT3, p-STAT3 (Ser727) and proteins associated with EMT were measured with Western blot assay. Cell proliferation was measured with MTT or CCK8 assay. RESULTS: Normal basal epithelial cells acquired from mouse prostate were specific to anti-p63 and more than 90%. Basal epithelial cells and RM-1 could both secrete TGF-ß1. Basal epithelial cells and TGF-ß1 promoted the migration and invasion of RM-1 through changing the cell morphology and up-regulating expression of ZEB1, N-cadherin, vimentin, snail and p-STAT3 (Ser727), at the same time down-regulating E-cadherin of RM-1. SB431542 strongly suppressed migration, invasion as well as the expressions of EMT relevant proteins and p-STAT3 (Ser727) of co-cultured RM-1. In addition, stattic suppressed proliferation, migration and invasion of non-treated RM-1 and co-cultured RM-1. CONCLUSION: Our study suggests that normal basal epithelial cells might stimulate the migration and invasion of RM-1 by TGF-ß1/STAT3 axis which could be suppressed by inhibitor of TGF-ß receptor and inhibitor of p-STAT3. So, basal epithelial cells might not become a treatment strategy for prostate cancer, but our results could provide some researching references for other diseases which include basal epithelial cells such as prostatic intraepithelial neoplasia, prostatic hyperplasia, cervical cancer, or urinary bladder cancer.

Raloxifene Protects Cisplatin-Induced Renal Injury in Mice via Inhibiting Oxidative Stress.

PURPOSE: Cisplatin is one of the most widely used antineoplastic drugs but has limited therapeutic effects due to nephrotoxicity. The aim of this study was to determine the possible renoprotective effect of the antioxidant raloxifene on cisplatin-induced nephrotoxicity in mice. MATERIALS AND METHODS: Cisplatin-induced acute renal injury was established in female C57 mice that were treated with saline (normal control) or raloxifene over a 7-day period. The body weight of the mice was recorded. Histopathological examinations of the kidney tissues were performed using H&E, PAS staining and TEM. The histomorphology of liver and other organs was observed by H&E staining. The serum levels of creatinine, blood urea nitrogen (BUN), alanine transaminase (ALT) and glutamic oxalacetic transaminase (AST) were analyzed by specific kits. Superoxide dismutase (SOD) and glutathione (GSH) activity, and the content of malondialdehyde (MDA) in the kidney, liver homogenates and HK-2 cells were measured by WST-8 and thiobarbituric acid colorimetric methods. Moreover, the mitochondrial structures of HK-2 cells were performed using TEM. The viability and proliferation of HK-2 cells were examined by CCK-8 and EdU incorporation assays. The mitochondrial membrane potential was measured by JC-1 fluorescence. RESULTS: Raloxifene significantly reduced the levels of serum creatinine, urea, ALT and AST in the cisplatin-treated mice, and alleviated cisplatin-induced renal and hepatic tissue injury. Furthermore, raloxifene also increased the activity of GSH and SOD in the renal tissues and HK-2 cells, and reduced MDA levels, thereby limiting oxidative stress in the kidney. CONCLUSION: Raloxifene protected against cisplatin-induced nephrotoxicity by activating the antioxidant system, along with alleviating liver damage. It should be considered as a potential adjuvant in cisplatin-based chemotherapeutic protocols.

Gene Therapy for Rdh12-Associated Retinal Diseases Helps to Delay Retinal Degeneration and Vision Loss.

PURPOSE: The aim of study was to establish Rdh12-associated inherited retinal disease (Rdh12-IRD) mouse model and to identify the best timepoint for gene therapy. METHODS: We induced retinal degeneration in Rdh12-/- mice using a bright light. We clarified the establishment of Rdh12-IRD mouse model by analyzing the thickness of retinal layers and electroretinography (ERG). Rdh12-IRD mice received a subretinal injection of adeno-associated virus 2/8-packaged Rdh12 cDNA for treatment. We evaluated the visual function and retinal structure in the treated and untreated eyes to identify the best timepoint for gene therapy. RESULTS: Rdh12-IRD mice showed significant differences in ERG amplitudes and photoreceptor survival compared to Rdh12+/+ mice. Preventive gene therapy not only maintained normal visual function but also prevented photoreceptor loss. Salvage gene therapy could not reverse the retinal degeneration phenotype of Rdh12-IRD mice, but it could slow down the loss of visual function. CONCLUSION: The light-induced retinal degeneration in our Rdh12-/- mice indicated that a defect in Rdh12 alone was sufficient to cause visual dysfunction and photoreceptor degeneration, which reproduced the phenotypes observed in RDH12-IRD patients. This model is suitable for gene therapy studies. Early treatment of the primary Rdh12 defect helps to delay the later onset of photoreceptor degeneration and maintains visual function in Rdh12-IRD mice.

EMC3 Is Essential for Retinal Organization and Neurogenesis During Mouse Retinal Development.

Purpose: We used a mouse model to explore the role of the endoplasmic reticulum membrane protein complex subunit 3 (EMC3) in mammalian retinal development. Methods: The transcription pattern of Emc3 in C57BL/6 mice was analyzed by in situ hybridization. To explore the effects of EMC3 absence on retinal development, the Cre-loxP system was used to generate retina-specific Emc3 in knockout mice (Emc3flox/flox, Six3-cre+; CKO). Morphological changes in the retina of E13.5, E17.5, P0.5, and P7 mice were observed via hematoxylin and eosin staining. Immunofluorescence staining was used to assess protein distribution and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining to assess apoptosis changes. Proteins were identified and quantified by Western blotting and proteomic analysis. Electroretinogram (ERG), fundus color photography, and optical coherence tomography were performed on 5-week-old mice to evaluate retinal function and structure. Results: The Emc3 mRNA was widely distributed in the whole retina during development. Loss of retinal EMC3 led to retinal rosette degeneration with mislocalization of cell junction molecules (ß-catenin, N-cadherin, and zonula occludens-1) and polarity molecules (Par3 and PKCζ). Endoplasmic reticulum stress and TUNEL apoptosis signals were present in retinal rosette-forming cells. Although the absence of EMC3 promoted the production of photoreceptor cells, 5-week-old mice lost all visual function and had severe retinal morphological degeneration. Conclusions: EMC3 regulates retinal structure by maintaining the polarity of retinal progenitor cells and regulating retinal cell apoptosis.

Canonical Wnt Signaling Drives Myopia Development and Can Be Pharmacologically Modulated.

Purpose: The purpose of this study was to evaluate the role of the canonical Wnt signaling in the development of the myopia. Methods: Plasma from adult patients with myopia, myopic animal models including the adenomatous polyposis coli (APC) gene mutation mouse model, and the form deprivation (FD) induced mouse model of myopia were used. Niclosamide, a canonical Wnt pathway inhibitor, was orally administrated in animal models. Plasma levels of DKK-1 were determined by using enzyme-linked immunosorbent assay. Refraction, vitreous chamber depth (VCD), axial length (AL), and other parameters, were measured at the end of the FD treatment. Canonical Wnt signaling changes were evaluated by Western blot analysis and immunostaining analysis. Results: Plasma level of Wnt inhibitor DKK-1 was markedly decreased in patients with myopia. Meanwhile, the canonical Wnt pathway was progressively activated during myopia development in mice. Moreover, inhibition of canonical Wnt signaling by niclosamide in mouse models markedly reduced lens thickness (LT), VCD, and AL elongation, resulting in myopia inhibition. Conclusions: Dysregulation of canonical Wnt signaling is a characteristic of myopia and targeting Wnt signaling pathways has potential as a therapeutic strategy for myopia.

Adenosine A2A receptors in both bone marrow cells and non-bone marrow cells contribute to traumatic brain injury.

Adenosine A2A receptors (A(2A)Rs) in bone marrow-derived cells (BMDCs) are involved in regulation of inflammation and outcome in several CNS injuries; however their relative contribution to traumatic brain injury (TBI) is unknown. In this study, we created a mouse cortical impact model, and BMDC A(2A)Rs were selectively inactivated in wild-type (WT) mice or reconstituted in global A(2A)R knockout (KO) mice (i.e. inactivation of non-BMDC A(2A)Rs) by bone marrow transplantation. When compared with WT mice, selective inactivation of BMDC A(2A)Rs significantly attenuated the neurological deficits, brain water content and cell apoptosis at 24 h post-TBI as global A(2A)R KO did. However, compared with the A(2A)R KO mice, selective reconstitution of BMDC A(2A)Rs failed to reinstate brain injury, indicating the contribution of the non-BMDC A(2A)R to TBI. Furthermore, the protective outcome by selective inactivation of BMDC A(2A)R or broad inactivation of non-BMDC A(2A)Rs was accompanied with reduced CSF glutamate level and suppression of the inflammatory cytokines interleukin-1, or interleukin-1 and tumor necrosis factor-alpha. These findings demonstrate that inactivation of A(2A)Rs in either BMDCs or non-BMDCs is sufficient to confer the protective effect as global A(2A)R KO against TBI, indicating the A(2A)R involvement in TBI by multiple cellular mechanisms of A(2A)R involvement including inhibition of glutamate release and inflammatory cytokine expressions.

Reconstitution of TCA cycle involving l-isoleucine dioxygenase for hydroxylation of l-isoleucine in Escherichia coli using CRISPR-Cas9.

l-isoleucine dioxygenase (IDO) is an Fe (II)/α-ketoglutarate (α-KG)-dependent dioxygenase that specifically converts l-isoleucine (l-Ile) to (2S, 3R, 4S)-4-hydroxyisoleucine (4-HIL). 4-HIL is an important drug for the treatment and prevention of type 1 and type 2 diabetes but the yields using current methods are low. In this study, the CRISPR-Cas9 gene editing system was used to knockout sucAB and aceAK gene in the TCA cycle pathway of Escherichia coli (E. coli). For single-gene knockout, the whole process took approximately 7 days. However, the manipulation time was reduced by 2 days for each round of gene modification for multigene editing. Using the genome-edited recombinant strain E. coli BL21(DE3) ΔsucABΔaceAK/pET-28a(+)-ido (2Δ-ido), the bioconversion ratio of L-Ile to 4-HIL was enhanced by about 15% compared to E. coli BL21(DE3)/pET-28a(+)-ido [BL21(DE3)-ido] strain. The CRISPR-Cas9 editing strategy has the potential in modifying multiple genes more rapidly and in optimizing strains for industrial production.

[MicroRNA-200c-3p inhibits proliferation of nephroblastoma cells by targeting CCNE2].

OBJECTIVE: To predict and verify the target gene of miR-200c-3p and evaluate the inhibitory effect of miR-200c-3p on the proliferation of nephroblastoma cells. METHODS: The putative target genes of miR-200c-3p were predicted by bioinformatics approach. Nephroblastoma cell models with miR-200c-3p overexpression or knockdown were established in SK-NEP-1 and G401 cells with corresponding control groups. The expressions of CCNE2 in SK-NEP-1 and G401 cells in different groups were detected by RT-PCR and Western blotting. A luciferase reporter assay was used to determine the targeting relationship between miR-200c-3p and CCNE2. The effects of miR-200c-3p overexpression or knockdown on cell proliferation was detected by cell counting kit-8 (CCK-8) assay and soft agarose assay. RESULTS: CCNE2 was one of the target genes of miR-200c-3p as predicted by bioinformatics methods. Transfection of the two nephroblastoma cell lines with miR-200c-3p mimic resulted in significantly lowered CCNE2 mRNA and protein expressions (P < 0.05). The results of dual-luciferase assay confirmed that miR-200c-3p bound to the 3'UTR of CCNE2. CCK-8 assay and soft agarose assay demonstrated that overexpression of miR-200c-3p significantly inhibited the proliferation of the nephroblastoma cells (P < 0.01), and knocking down miR-200c-3p in the cells produced the opposite effects. CONCLUSIONS: miR-200c-3p overexpression inhibits the proliferation of nephroblastoma cells by down-regulating its target gene CCNE2.

Endometrial clear cell carcinoma invading the right oviduct with a cooccurring ipsilateral oviduct adenomatoid tumor: A case report.

BACKGROUND: To investigate the clinicopathological features of endometrial clear cell carcinoma that has invaded the right oviduct with a cooccurring ipsilateral oviduct adenomatoid tumor. CASE SUMMARY: A case of endometrial clear cell carcinoma invading the right oviduct with a cooccurring ipsilateral oviduct adenomatoid tumor was collected and analyzed using pathomorphology and immunohistochemistry. Endometrial clear cell carcinoma cells were distributed in a solid nest, papillary, shoe nail-like, and glandular tube-like distribution. There was infiltrative growth, and tumor cells had clear cytoplasm and obvious nuclear heteromorphism. The cancer tissue was necrotic and mitotic. The cancer tissue invaded the right oviduct. The ipsilateral oviduct also had an adenomatoid tumor. The adenomatoid tumor was arranged in microcapsules lined with flat or cubic cells that were surrounded by smooth muscle tissue. The adenomatoid tumor cells were round in shape. CONCLUSION: Clear cell carcinoma of the endometrium can invade the oviduct and occur simultaneously with tubal adenomatoid tumors. Upon pathological diagnosis, one should pay close attention to distinguishing whether an endometrial clear cell carcinoma is invading the oviduct or whether it is accompanied by an adenomatoid tumor of the oviduct. Immunohistochemistry is helpful to differentiate these two disease entities. Endometrial clear cell carcinomas express Napsin-A and P16 and are negative for estrogen receptor and progesterone receptor. The presence of endometrial clear cell carcinoma does not affect the expression of CK and calretinin in adenomatoid tumors.