SRY-Box transcription factor 9 triggers YAP nuclear entry via direct interaction in tumors.

The translocation of YAP from the cytoplasm to the nucleus is critical for its activation and plays a key role in tumor progression. However, the precise molecular mechanisms governing the nuclear import of YAP are not fully understood. In this study, we have uncovered a crucial role of SOX9 in the activation of YAP. SOX9 promotes the nuclear translocation of YAP by direct interaction. Importantly, we have identified that the binding between Asp-125 of SOX9 and Arg-124 of YAP is essential for SOX9-YAP interaction and subsequent nuclear entry of YAP. Additionally, we have discovered a novel asymmetrical dimethylation of YAP at Arg-124 (YAP-R124me2a) catalyzed by PRMT1. YAP-R124me2a enhances the interaction between YAP and SOX9 and is associated with poor prognosis in multiple cancers. Furthermore, we disrupted the interaction between SOX9 and YAP using a competitive peptide, S-A1, which mimics an α-helix of SOX9 containing Asp-125. S-A1 significantly inhibits YAP nuclear translocation and effectively suppresses tumor growth. This study provides the first evidence of SOX9 as a pivotal regulator driving YAP nuclear translocation and presents a potential therapeutic strategy for YAP-driven human cancers by targeting SOX9-YAP interaction.

Discovery of GPX4 inhibitors through FP-based high-throughput screening.

Ferroptosis is a form of non-apoptotic cell death, regulated by phospholipid hydroperoxide glutathione peroxidase 4 (GPX4), a selenoprotein with a selenocysteine residue (sec) in the active site. GPX4 is a promising target for cancer cells in therapy-resistant conditions via ferroptosis, which can reduce the level of lipid reactive oxygen species (ROS). So far, all existing GPX4 inhibitors covalently bind to GPX4 via a reactive alkyl chloride moiety or masked nitrile-oxide electrophiles with poor selectivity and pharmacokinetic properties and most were obtained by cell phenotype-based screening. Lacking of effective high-throughput screening methods for GPX4 protein limits the discovery of GPX4 inhibitors. Here, we report a fluorescence polarization (FP)-based high throughput screening (HTS) assay for GPX4-U46C-C10A-C66A in vitro, and found Metamizole sodium from our in-house compound library inhibits GPX4-U46C-C10A-C66A enzyme activity. Structure-activity relationships (SAR) demonstrated the importance of sulfonyl group on interaction between Metamizole sodium and GPX4-U46C-C10A-C66A. Our FP assay could be an effective tool for discovery of GPX4 inhibitors and Metamizole sodium was a potential inhibitor for GPX4 in vitro.

Discovery of a novel OGT inhibitor through high-throughput screening based on Homogeneous Time-Resolved Fluorescence (HTRF).

O-GlcNAcylation is a specific type of post-translational glycosylation modification, which is regulated by two enzymes, O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA). Aberrant overexpression of OGT is associated with the development of many solid tumors. In this study, we have developed and optimized a sensitive Homogeneous Time-Resolved Fluorescence (HTRF) assay then identified a novel OGT inhibitor CDDO (also called Bardoxolone) through a high-throughput screening (HTS) based on HTRF assay. Further characterization suggested that CDDO is an effective OGT inhibitor with an IC50 value of 6.56 ± 1.69 µM. CPMG-NMR analysis confirmed that CDDO is a direct binder of OGT with a binding affinity (Kd) of approximately 1.7 µM determined by the MST analysis. Moreover, HDX-MS analysis indicated that CDDO binds to the TPR domain and N-Terminal domain of OGT, which was further confirmed by the enzymatic competition experiments as the binding of CDDO to OGT was not affected by the catalytic site binding inhibitor OSMI-4. Our docking modeling analysis further predicted the possible interactions between CDDO and OGT, providing informative molecular basis for further optimization of the inhibitor in the future. Together, our results suggested CDDO is a new inhibitor of OGT with a distinct binding pocket from the reported OGT inhibitors. Our work paved a new direction for developing OGT inhibitors driven by novel mechanisms.

Spermine is a natural suppressor of AR signaling in castration-resistant prostate cancer.

In castration-resistant prostate cancer (CRPC), clinical response to androgen receptor (AR) antagonists is limited mainly due to AR-variants expression and restored AR signaling. The metabolite spermine is most abundant in prostate and it decreases as prostate cancer progresses, but its functions remain poorly understood. Here, we show spermine inhibits full-length androgen receptor (AR-FL) and androgen receptor splice variant 7 (AR-V7) signaling and suppresses CRPC cell proliferation by directly binding and inhibiting protein arginine methyltransferase PRMT1. Spermine reduces H4R3me2a modification at the AR locus and suppresses AR binding as well as H3K27ac modification levels at AR target genes. Spermine supplementation restrains CRPC growth in vivo. PRMT1 inhibition also suppresses AR-FL and AR-V7 signaling and reduces CRPC growth. Collectively, we demonstrate spermine as an anticancer metabolite by inhibiting PRMT1 to transcriptionally inhibit AR-FL and AR-V7 signaling in CRPC, and we indicate spermine and PRMT1 inhibition as powerful strategies overcoming limitations of current AR-based therapies in CRPC.

Salinimicrobium profundisediminis sp. nov., isolated from deep-sea sediment of the Mariana Trench.

A Gram-stain-negative, aerobic, rod-shaped, non-gliding bacterial strain, designated as MT39T, was isolated from a deep-sea sediment sample collected from the Mariana Trench. Strain MT39T grew optimally at 35°C and pH 7.0, and could tolerate up to 10% (w/v) NaCl. The strain was positive for catalase and negative for oxidase. The genome of strain MT39T was 4 033 307 bp, with a 41.1 mol % genomic G+C content and 3514 coding sequences. Phylogenetic analysis based on 16S rRNA gene sequences placed strain MT39T within the genus Salinimicrobium, showing the highest 16S rRNA gene sequence similarity to Salinimicrobium terrea CGMCC 1.6308T (98.1%). The average nucleotide identity and in silico DNA-DNA hybridization values between strain MT39T and the type strains of seven Salinimicrobium species were all less than the threshold values to discriminate bacterial species, indicating that strain MT39T is affiliated with a novel species within the genus. The major cellular fatty acids of strain MT39T were iso-C15 : 0, anteiso-C15 : 0 and iso-C17 : 0 3-OH. Polar lipids of strain MT39T included phosphatidylethanolamine, one unidentified aminolipid and four unidentified lipids. Menaquinone-6 was the only respiratory quinone in strain MT39T. On the basis of the polyphasic data present in this study, strain MT39T represents a novel species of the genus Salinimicrobium, for which the name Salinimicrobium profundisediminis sp. nov. is proposed, with type strain being MT39T (=MCCC 1K07832T=KCTC 92381T).

Discovery of new small molecule inhibitors of the BPTF bromodomain.

Chromatin remodeling regulates many basic cellular processes, such as gene transcription, DNA repair, and programmed cell death. As the largest member of nucleosome remodeling factor (NURF), BPTF plays a vital role in the occurrence and development of cancer. Currently, BPTF bromodomain inhibitors are still in development. In this study, by conducting homogenous time-resolved fluorescence resonance energy transfer (HTRF) assay, we identified a potential, novel BPTF inhibitor scaffold Sanguinarine chloride with the IC50 value of 344.2 ± 25.1 nM. Biochemical analysis revealed that compound Sanguinarine chloride exhibited high binding affinity to the BPTF bromodomain. Molecular docking predicted the binding mode of Sanguinarine chloride and elucidated the activities of its derivatives. Moreover, Sanguinarine chloride showed a potent anti-proliferative effect in MIAPaCa-2 cells and inhibited the expression of BPTF target gene c-Myc. Taken together, Sanguinarine chloride provides a qualified chemical tool for developing potent BPTF bromodomain inhibitors.

A potent PGK1 antagonist reveals PGK1 regulates the production of IL-1ß and IL-6.

Glycolytic metabolism enzymes have been implicated in the immunometabolism field through changes in metabolic status. PGK1 is a catalytic enzyme in the glycolytic pathway. Here, we set up a high-throughput screen platform to identify PGK1 inhibitors. DC-PGKI is an ATP-competitive inhibitor of PGK1 with an affinity of K d = 99.08 nmol/L. DC-PGKI stabilizes PGK1 in vitro and in vivo, and suppresses both glycolytic activity and the kinase function of PGK1. In addition, DC-PGKI unveils that PGK1 regulates production of IL-1ß and IL-6 in LPS-stimulated macrophages. Mechanistically, inhibition of PGK1 with DC-PGKI results in NRF2 (nuclear factor-erythroid factor 2-related factor 2, NFE2L2) accumulation, then NRF2 translocates to the nucleus and binds to the proximity region of Il-1ß and Il-6 genes, and inhibits LPS-induced expression of these genes. DC-PGKI ameliorates colitis in the dextran sulfate sodium (DSS)-induced colitis mouse model. These data support PGK1 as a regulator of macrophages and suggest potential utility of PGK1 inhibitors in the treatment of inflammatory bowel disease.

Asymptomatic uterine metastasis of breast cancer: Case report and literature review.

RATIONALE: Uterine metastasis from breast cancer is extremely rare. Asymptomatic patients with cervical metastases from breast cancer are rarer and more likely to be missed. We present an asymptomatic patient with breast cancer metastasized to the uterus and share opinions on diagnosing and treating for this kind of cases. PATIENT CONCERNS: We present the case of a 64-year-old woman who was diagnosed with both breast cancer and uterine fibroids after examination. She had no symptoms of gynecological disease during breast cancer treatment. A positron emission tomography/computed tomography (PET/CT) scan was performed during reexamination, revealing multiple metastases of the bone throughout the body and an abnormal hypermetabolic mass in the uterus. It was later confirmed as uterine metastasis by pathology. DIAGNOSIS: A diagnosis of metastatic breast invasive lobular carcinoma was established after a uterine curettage. INTERVENTIONS AND OUTCOMES: Treatment of the uterine metastasis included systemic chemotherapy, total abdominal hysterectomy and bilateral salpingo-oophorectomy (TAH and BSO), postoperative radiotherapy, and postoperative chemotherapy. The patient eventually refused further treatment for personal reasons and died at home. LESSONS: Breast cancer metastases to the uterus are very rare and further research is needed for their diagnosis and treatment. During reexamination of breast cancer patients, clinicians must be alert to metastasis to gynecologic organs. This is particularly important in hormone receptor-positive patients with asymptomatic distant metastasis.

Design, synthesis and biological evaluation of 2-aminopyridine derivatives as USP7 inhibitors.

A series of novel 2-aminopyridine derivatives 1-26 have been designed and synthesized by structural modifications on a lead USP7 inhibitor, GNE6640. All the compounds were evaluated for their USP7 inhibitory activities. The results showed that most of the compounds have good USP7 inhibitory activities at the concentration of 50 µM. Among them, compounds 7, 14 and 21 are the most potential ones from each category with the IC50 values of 7.6 ± 0.1 µM, 17.0 ± 0.2 µM and 11.6 ± 0.5 µM, respectively. Compounds 7 and 21 expressed significant binding interactions with USP7 by surface plasmon resonance (SPR)-based binding assay, but both of them presented moderate antiproliferative activities against HCT116 cells. They could effectively promote MDM2 degradation, p53 stabilization and p21 gene expression in the western blot analysis.

Design and synthesis of novel benzimidazole-iminosugars linked a substituted phenyl group and their inhibitory activities against ß-glucosidase.

A series of novel benzimidazole-iminosugars linked a (substuituted) phenyl group on benzene ring of benzimidazole 5(a-p) and 6(a-p) have been rationally designed and conveniently synthesized through Suzuki coupling reaction in high yields. All compounds have been evaluated for their inhibitory activities against ß-glucosidase (almond). Six compounds 5d, 6d, 6e, 6i, 6n, and 6p showed more significant inhibitory activities with IC50 values in the range of 0.03-0.08 µM, almost 10-fold improved than that of the parent analogue 4, and much higher than that of the positive control castanospermine. The additional phenyl ring and the electron donating groups on it would be beneficial for the activity. Compounds 6d, 6n, and 4 had been chosen to be tested for their inhibition types against ß-glucosidase. Interestingly, three compounds have different inhibition types although they had very similar structure. Their Ki values were calculated to be 0.02 ± 0.01 µM, 0.02 ± 0.01 µM, and 0.66 ± 0.14 µM, respectively. The equilibrium dissociation constant (KD) for 6d, 6n, and 4 and ß-glucosidase was 0.04 µM, 0.03 µM and 0.45 µM by the ITC-based assay, respectively. Molecular docking work suggests that such benzimidazole-iminosugars derivatives might bind to the active site of ß-glucosidase mainly through hydrogen bonds, the additional phenyl ring towards the solvent-exposed region played an important effect on their inhibitory activity against ß-glucosidase.

High-Throughput-Methyl-Reading (HTMR) assay: a solution based on nucleotide methyl-binding proteins enables large-scale screening for DNA/RNA methyltransferases and demethylases.

Epigenetic therapy has significant potential for cancer treatment. However, few small potent molecules have been identified against DNA or RNA modification regulatory proteins. Current approaches for activity detection of DNA/RNA methyltransferases and demethylases are time-consuming and labor-intensive, making it difficult to subject them to high-throughput screening. Here, we developed a fluorescence polarization-based 'High-Throughput Methyl Reading' (HTMR) assay to implement large-scale compound screening for DNA/RNA methyltransferases and demethylases-DNMTs, TETs, ALKBH5 and METTL3/METTL14. This assay is simple to perform in a mix-and-read manner by adding the methyl-binding proteins MBD1 or YTHDF1. The proteins can be used to distinguish FAM-labelled substrates or product oligonucleotides with different methylation statuses catalyzed by enzymes. Therefore, the extent of the enzymatic reactions can be coupled with the variation of FP binding signals. Furthermore, this assay can be effectively used to conduct a cofactor competition study. Based on the assay, we identified two natural products as candidate compounds for DNMT1 and ALKBH5. In summary, this study outlines a powerful homogeneous approach for high-throughput screening and evaluating enzymatic activity for DNA/RNA methyltransferases and demethylases that is cheap, easy, quick, and highly sensitive.

A Potential Predictive Biomarker for Miller/Payne Grading: PD-L1 Expression before Neoadjuvant Chemotherapy in Breast Cancer.

BACKGROUND AND OBJECTIVE: The aim of this study was to investigate the value of programmed death ligand 1 (PD-L1) expression as a predictive biomarker for Miller/Payne grading before neoadjuvant chemotherapy (NACT) in breast cancer. PATIENTS AND METHODS: The expression of PD-L1 in pretreatment biopsies of breast cancer was assessed by immunohistochemistry in tissue microarrays. The results were analyzed using SPSS 22.0 statistical software. RESULTS: Of 53 female patients, 10 (18.9%) patients had a grade 5 (G5) response, and 12 (22.6%) patients showed PD-L1 expression, including 7 (13.2%) patients with staining in tumor cells (TCs) and 8 (15.1%) patients with staining in peritumoral lymphocytes (PTLCs). Logistic regression analysis revealed that G5 response to NACT was significantly associated with TCs or PTLCs PD-L1 positivity, whether with univariate analysis (TCs PD-L1: p = 0.00, OR 20.50, 95% CI 3.11-134.94; PTLCs PD-L1: p = 0.02, OR 6.50, 95% CI 1.27-33.20) or with multivariate analysis (TCs PD-L1: p = 0.00, OR 42.23, 95% CI 3.36-530.90; PTLCs PD-L1: p = 0.02, OR 9.07, 95% CI 1.37-60.02). The same trend was found in the luminal subgroup analysis (TCs PD-L1: p = 0.02, OR 23.43, 95% CI 1.66-331.58; PTLCs PD-L1: p = 0.01, OR 47.89, 95% CI 2.47-927.41). CONCLUSION: G5 response to NACT in breast cancer was significantly associated with TCs or PTLCs PD-L1-positive expression in pretreatment biopsies; it can be expected that PD-L1 will become a new independent biomarker of response to NACT in breast cancer.

Phylogenetic and biological characterizations of a GI.3 norovirus.

Noroviruses (NoVs) are a major cause of acute non-bacterial gastroenteritis worldwide. In this study, we report the isolation, near-complete genome sequencing, and expression and biological characterization of the major capsid protein (VP1) of a GI.3 NoV isolated from a child presenting acute gastroenteritis. The genome of the GI.3 NoV is 7746 bp in length, not including the poly-adenylation tail. Phylogenetic analysis based on the complete VP1 nucleotide sequences indicates that GI.3 NoVs could be divided into four clusters, with 4.6%, 5.3%, 6.6%, 1.9% intracluster variations in nucleotide and 4.8%, 3.8%, 6.1%, 1.7% intracluster variations in amino acid sequences, respectively. A Bayesian evolutionary analysis showed that GI.3 NoVs evolved at 2.44 × 10-3, 2.78 × 10-3, and 3.04 × 10-3 nucleotide substitutions/site/year using a strict clock model, an uncorrelated log-normal model (UCLN), and an uncorrelated exponential derivation model (UCED), respectively. VP1 protein expression using a recombinant baculovirus expression system leads to the successful assembly of virus-like particles (VLPs). In vitro VLP-Histo-blood group antigen (HBGA) binding assay indicates that GI.3 NoV VLPs strongly bind to blood type A salivary HBGAs, moderately bind to blood type O salivary HBGAs, and weakly bind or do not bind to blood type B and AB salivary HBGAs. In vitro VLP-HBGA binding blockade assay indicated that the binding of GI.3 NoV VLPs to blood type A salivary HBGAs could only be blocked by anti-GI.3 NoV VLPs serum but not non-GI.3 NoV genotype-specific hyperimmune sera (GI.2, GI.7, GII.4, GII.6, GII.7, and GII.17). The detailed characterization of GI.3 NoV in this study provides evidence that GI.3 NoV undergoes rapid evolution and exhibits no cross-blocking effects, suggesting that GI.3 NoV may potentially be utilized in the development of multivalent NoV vaccines.

The Prognostic Value of Deficient Mismatch Repair in Stage II-IVa Nasopharyngeal Carcinoma in the Era of IMRT.

In the era of intensity-modulated radiotherapy (IMRT), it is important to analyse the prognostic value of deficient mismatch repair (dMMR) in nasopharyngeal carcinoma (NPC). In this study, in pretreatment biopsies of 69 patients with stage II-IVa NPC, the expression levels of MMR proteins, including MLH1, MSH2, MSH6 and PMS2, were assessed by immunohistochemistry (IHC). The median follow-up time was 37.5 months (3.1-87.4 months). 50.7% of cases (35/69) showed preserved expression of all 4 MMR proteins, which was interpreted as proficient mismatch repair (pMMR). Only 1.5% of cases (1/69) lost expression of all 4 MMR proteins, 26.1% of cases (18/69) have PMS2 loss alone and 21.7% of cases (15/69) lost expression of both PMS2 and MLH1. Thus, 49.3% of cases (34/69) lost expression of one or more MMR proteins, which was interpreted as dMMR. There was no significant difference (P > 0.05) in terms of sex, age, clinical stage, T category, N category or therapy regimens between the dMMR and pMMR groups. The multivariate Cox regression analysis revealed that dMMR was an independent significant prognostic factor for distant metastasis-free survival (DMFS) (dMMR vs pMMR: P = 0.01, HR = 0.25, 95% CI: 0.09~0.75). Therefore, NPC patients with dMMR had significantly superior DMFS compared with patients with pMMR. It can be expected that dMMR will become a new independent prognostic factor for NPC.

Production and characterization of monoclonal antibodies against GII.6 norovirus virus-like particles.

In this study we generated and characterized a series of monoclonal antibodies (mAbs) against GII.6 norovirus (NoV) virus like particles (VLPs). Mice were immunized with purified GII.6 NoV VLPs and peptide-bovine serum albumin (BSA) conjugates with the peptide sequence (31 aa) derived from the trypsin cleavage region. An indirect enzyme-linked immunosorbent assay was used to identify positive cell clones during cloning and subcloning, and an in vitro VLP-histo-blood group antigens (HBGAs) binding blockade assay was used to identify mAbs with blocking ability. A total of seven mAbs comprising five (1F7, 1F11, 2B6, 2C4, and 2E10) reactive with major capsid proteins (VP1) and two (1E5 and 2B2) reactive with both VP1 proteins and the peptide were identified. mAb 1F7, 1F11, and 2B6 were identified as blocking antibodies. Sandwich ELISA indicated that all these mAbs recognized soluble GII.6 NoV VLPs. Cross-reactivities with GI.7, GII.3, and GII.4 NoV VLPs were observed in indirect and sandwich ELISA. Western blot analysis indicated that all non-blocking mAbs recognized denatured GII.6 VP1 proteins and blocking mAbs only recognized non-denatured proteins. The in vitro VLP-HBGA binding blockade assay indicated that the three blocking antibodies exhibited blocking effects against GII.6 NoV VLPs, but not GI.7, GII.3, and GII.4 NoV VLPs. Epitope mapping and HBGA blocking assay indicated that mAbs targeting the predicted surface-exposed loop region did not have blocking effects, suggesting a possible important role of this region in regulating NoV-HBGA interactions. This is the first report regarding the characterization of mAbs with blocking ability against GII.6 NoV VLPs. These mAbs might be useful in facilitating our understanding of this group of viruses.

Linear epitope binding antibodies against GII.3 Norovirus exhibit no histo-blood group antigens (HBGAs) blocking effects.

Noroviruses are leading cause of acute gastroenteritis worldwide. In our previous study, we established an in vitro histo-blood group antigens (HBGAs) binding blockade assay against GII.3 Norovirus virus like particles (VLPs) with trypsin digestion. In this study, we characterized the blocking antibody binding site and epitope type (linear or conformational) by using hyperimmune sera produced against different antigens. VP1 from Jingzhou402 (GII.3, JZ402) strain was expressed by using pGEX-6p-1 expression vector and the insoluble proteins were purified for immunization in rabbit. Previously characterized chimeric VP1-assembled VLPs (GII.4-VP1/GII.3-P2) were used to immunize guinea pig. Peptides reactive with hyperimmune serum against VLPs derived from the VP1 of JZ402 strain were conjugated with BSA and used to immunize rabbits. Hyperimmune sera against above antigens and JZ402 and JZ403 strain-derived VLPs were used to compare their HBGAs blocking effects. Rabbit anti-GST-VP1 and BSA-peptide conjugated hyperimmune sera demonstrated no blocking effects against the binding of GII.3 and GII.4 NoV VLPs to salivary HBGAs. Guinea pig anti-GII.4-VP1/GII.3-P2 hyperimmune serum blocked the binding of trypsin cleaved GII.3 VLPs to salivary HBGAs with no or very weak blocking effects against the binding of GII.4 VLPs to salivary HBGAs. Our data indicated that HBGAs blocking antibodies primarily bound the P2 domain of GII.3 NoV VP1 and their binding epitopes were most probably conformation-dependent.

Long non­coding RNA DLX6­AS1 is associated with malignant progression and promotes proliferation and invasion in esophageal squamous cell carcinoma.

Despite being one of the most prevalent and fatal types of cancer worldwide, the biological details of esophageal squamous cell carcinoma (ESCC) remain unknown. Recent studies have demonstrated the crucial roles of long non­coding RNAs (lncRNAs) in diverse biological processes including cancer initiation, progression and metastasis. The aim of the present study was to assess the expression profile of distal­less homeobox 6 antisense RNA 1 (DLX6­AS1) in ESCC tissues and its contributions to ESCC cell proliferation, apoptosis and invasion. The expression of DLX6­AS1 in a series of ESCC samples and paired adjacent noncancerous tissues was evaluated by reverse transcription­quantitative polymerase chain reaction. Cell proliferation, apoptosis, wound healing and Transwell invasion assays were performed to evaluate the roles of DLX6­AS1 in the ESCC cell lines EC109 and KYSE30 transfected with DLX6­AS1 small interfering RNA (siRNA). Compared with the paired adjacent noncancerous tissues, DLX6­AS1 expression was upregulated in the ESCC tissues and significantly associated with differentiation status, Tumor­Node­Metastasis stage, distant metastasis, and lymph node metastasis. Knockdown of DLX6­AS1 significantly suppressed cell proliferation, invasion and migration abilities, and enhanced the apoptotic rate in the two ESCC cell lines. Furthermore, western blot assays revealed that silencing DLX6­AS1 partly influenced the epithelial­mesenchymal transition process in ESCC cells. These results imply that the oncogenic function of DLX6­AS1 may be a novel candidate target for treating human ESCC.

Gelatin-epigallocatechin gallate nanoparticles with hyaluronic acid decoration as eye drops can treat rabbit dry-eye syndrome effectively via inflammatory relief.

INTRODUCTION: Dry-eye syndrome (DES) is a general eye disease. Eye drops are the common ophthalmological medication. However, the ocular barrier makes it difficult to attain high drug bioavailability. Nanomedicine is a promising alternative treatment for ocular diseases and may increase drug content in the affected eye. METHODS: To explore this potential, we constructed nanoparticles (NPs) containing an anti-inflammatory agent for DES treatment. The NPs were made of gelatin-epigallocatechin gallate (EGCG) with surface decoration by hyaluronic acid (HA) and designated "GEH". The particle size, surface charge, and morphology were evaluated. The in vitro biocompatibility and anti-inflammation effect of nanoparticles were assayed via culturing with human corneal epithelium cells (HCECs) and in vivo therapeutic effect was examined in a DES rabbit's model. RESULTS: The synthesized GEH NPs had a diameter of approximately 250 nm and were positively charged. A coculture experiment revealed that 20 µg/mL GEH was not cytotoxic to HCECs and that an EGCG concentration of 0.2 µg/mL downregulated the gene expression of IL1B and IL6 in inflamed HCECs. Large amounts of GEH NPs accumulated in the cytoplasm of HCECs and the ocular surfaces of rats and rabbits, indicating the advantage of GEH NPs for ocular delivery of medication. Twice-daily topical treatment with GEH NPs was performed in a rabbit model of DES. The ocular surface of GEH-treated rabbits displayed normal corneal architecture with no notable changes in inflammatory cytokine levels in the cornea lysate. The treatment improved associated clinical signs, such as tear secretion, and fluorescein staining recovered. CONCLUSION: We successfully produced GEH NPs with high affinity for HCECs and animal eyes. The treatment can be delivered as eye drops, which retain the drug on the ocular surface for a longer time. Ocular inflammation was effectively inhibited in DES rabbits. Therefore, GEH NPs are potentially valuable as a new therapeutic agent delivered in eye drops for treating DES.

Chimeric GII.3/GII.6 norovirus capsid (VP1) proteins: characterization by electron microscopy, trypsin sensitivity and binding to histo-blood group antigens.

GII.3 and GII.6 noroviruses (NoVs) are similar in several aspects, including the presence of a short sequence insertion in the P2 domain of the major capsid protein (VP1) and trypsin susceptibility of VP1-containing virus-like particles (VLPs). In this study, we generated two constructs with the S or P domains of VP1 from GII.3 and GII.6 NoV strains exchanged (GII.3S/GII.6P and GII.6S/GII.3P), and the resultant chimeric capsid proteins were expressed from recombinant baculoviruses. The assembly of VLPs was confirmed by electron microscopy, and the susceptibility of assembled VLPs to trypsin digestion was analyzed by SDS-PAGE. Salivary histo-blood group antigen (HBGA) binding and binding blockade assays were performed to determine the binding characteristics of chimeric VP1-containing VLPs with and without trypsin digestion. Our results indicated that both expressed GII.3S/GII.6P and GII.6S/GII.3P chimeric proteins successfully assembled into VLPs. Trypsin digestion of VLPs assembled from both chimeric proteins led to the generation of two fragments with molecular sizes similar to those of wild-type VP1-containing VLPs. An in vitro salivary HBGA binding assay demonstrated that VLPs assembled from both chimeric proteins exhibited enhanced binding after trypsin cleavage. An HBGA binding blockade assay indicated that the binding of GII.3S/GII.6P and GII.6S/GII.3P VLPs against salivary HBGAs could only be blocked by GII.3 and GII.6 NoV VLP-specific hyperimmune sera, respectively. For GII.6 and GII.3S/GII.6P VLPs, a difference in binding enhancement after trypsin cleavage was observed. Our results demonstrate that the S domains of GII.3 and GII.6 NoV VP1 are interchangeable and that the S domain affects the binding of the P domain to HBGAs.

Lutein inhibits proliferation, invasion and migration of hypoxic breast cancer cells via downregulation of HES1.

An intratumoral hypoxic microenvironment is frequently observed in solid tumors, including breast cancer. Lutein, a plant-derived compound and non-vitamin A carotenoid, has been demonstrated to possess multiple protective properties including anti-inflammation, anti-oxidative stress and antitumor effects. The main objective of the present research was to elucidate the involvement of lutein in the production of reactive oxygen species (ROS) under hypoxia, the activation of hairy and enhancer of split 1 (HES1), and the proliferation, invasion and migration of breast cancer cells. The human breast cancer cell lines MDA­MB­157 and MCF­7 were exposed to hypoxic conditions and various concentrations of lutein. An MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay was performed to examine cell proliferation, and Annexin V-fluorescein isothiocyanate/propidium iodide staining was performed to analyze the apoptosis ratio. The levels of hypoxia inducible factor-1α (HIF­1α), NOTCH signaling molecules, HES1 and epithelial-mesenchymal transition (EMT)-associated factors were examined by reverse transcription-quantitative polymerase chain reaction and western blot analysis. Wound healing and Transwell invasion assays were used to detect the invasion and migration of breast cancer cells. Intracellular ROS levels were examined using 2,7-dichlorodihydrofluorescein-diacetate and flow cytometry. The results revealed that cell proliferation was inhibited by lutein in a dose-dependent manner, and the apoptosis ratio gradually increased with lutein treatment under hypoxia as evident from flow cytometry-based analysis. Exposure to lutein inhibited hypoxia-mediated activation of HIF­1α, NOTCH signaling and HES1 expression, and suppressed the hypoxia-induced expression of EMT-associated factors. Lutein markedly inhibited the invasion and migration of breast cancer cells under hypoxia. Hypoxia-induced production of ROS was also decreased by lutein. Furthermore, the ROS scavenger N­acetylcysteine also suppressed hypoxia inducible factor 1α and HES1 expression in breast cancer cells during hypoxia, but hydrogen peroxide (H2O2) levels were increased. Taken together, the results of the present study suggested that lutein may be a novel candidate for the chemoprevention of breast cancer. Furthermore, HES1 may be crucial in mediating the involvement of lutein in the suppression of hypoxia-driven ROS-induced breast cancer progression.