TIMP3/Wnt axis regulates gliosis of Müller glia.

BACKGROUND: Previous studies have confirmed the expression of tissue inhibitor of metalloproteinase-3 (TIMP3) in Müller glia (MG). However, the role of TIMP3 in MG remains unknown. METHODS: A mouse model of laser-induced retinal damage and gliosis was generated using wild-type C57BL/6 mice. TIMP3 and associated proteins were detected using Western blotting and immunofluorescence microscopy. RNA sequencing (GSE132140) of mouse laser-induced gliosis was utilized for pathway analysis. TIMP3 overexpression was induced in human MG. Human vitreous samples were obtained from patients with proliferative diabetic retinopathy (PDR) and healthy controls for protein analysis. RESULTS: TIMP3 levels increased in mouse eyes after laser damage. Morphology and spatial location of TIMP3 indicated its presence in MG. TIMP3-overexpressing MG showed increased cellular proliferation, migration, and cell nuclei size, suggesting TIMP3-induced gliosis for retinal repair. Glial fibrillary acidic protein (GFAP) and vimentin levels were elevated in TIMP3-overexpressing MG and laser-damaged mouse retinas. RNA sequencing and Western blotting suggested a role for ß-catenin in mediating TIMP3 effects on the retina. Human vitreous samples from patients with PDR showed a positive correlation between TIMP3 and GFAP levels, both of which were elevated in patients with PDR. CONCLUSIONS: TIMP3 is associated with MG gliosis to enhance the repair ability of damaged retinas and is mediated by the canonical Wnt/ß-catenin. Changes in TIMP3 could potentially be used to control gliosis in a range of retinal diseases However, given the multifaceted nature of TIMP3, care must be taken when developing treatments that aim solely to boost the function of TIMP3. FUNDING: National Cheng Kung University Hospital, Taiwan (NCKUH-10604009 and NCKUH-11202007); the Ministry of Science and Technology (MOST 110-2314-B-006-086-MY3).

A systematic approach to reduce intraocular pressure for the treatment of glaucoma.

Glaucoma is the leading cause of irreversible blindness due to increased intraocular pressure (IOP) in the eye. We have developed a novel treatment option for glaucoma based on a real-time IOP-dependent nitric oxide synthase (NOS) and packed in a therapeutic contact lens to reduce the IOP. First, 1.6 nmole nitric oxide was produced from the genetic chassis, which was optimized for isopropyl ß-d-1-thiogalactopyranoside (IPTG) induction in a T7 expression system. For biosafety concerns to human being, the csgAD genes responsible for curli biofilm formation in Escherichia coli were co-expressed with NOS in the designated NOSAD strain to strengthen the adherence of cells to the contact lens, thereby preventing the contamination into the eyes. Moreover, NOSAD is a diaminopimelic acid (DAP) auxotrophic strain, which cannot survive without supplementation of DAP and reached the critical consideration of biosafety to the environment. We also demonstrated that the nitric oxide diffusion was 3.6-times enhanced from penetration into the aqueous humor of porcine eyes. The deformation ratio of the contact lens was correlated to the change of IOP by using a digital image correlation (DIC) system in a porcine eye model. The novel systematic approach provides an alternative treatment for glaucoma patients in the future.

Purification and biofabrication of 5-aminolevulinic acid for photodynamic therapy against pathogens and cancer cells.

5-Aminolevulinic acid (5-ALA) is a non-proteinogenic amino acid which has involved in heme metabolism of organisms, and has been widely applied in agriculture, and medical fields nowadays. 5-ALA is used in the elimination of pathogens or cancer cells by photodynamic therapy (PDT) owing to the photosensitizer reaction which releases the reactive oxygen species (ROS). Currently, biofabrication of 5-ALA is regarded as the most efficient and eco-friendly approach, but the complicated ingredient of medium causes the nuisance process of purification, resulting in low recovery and high producing cost. In this study, hydrogen chloride, sodium acetate, and ammonia were examined to maximize the recovery of 5-ALA from ion-exchange chromatography (IEC), thus a 92% recovery in 1 M ammonia at pH 9.5 was obtained. Afterward, the activated carbon was used for decolorization to further remove the pigments from the eluent. Four organic solvents, i.e., diethyl ether, methanol, ethanol, and acetone were compared to extract and form 5-ALA precipitation. The purified 5-ALA was verified to eliminate 74% of A549 human lung cancer and 83% of A375 melanoma skin cancer cell. Moreover, Proteus hauseri, Aeromonas hydrophila, Bacillus cereus, and Staphylococcus aureus were killed via anti-microbial PDT with 1% 5-ALA and reached 100% killing rate at optimal condition. With the addition of 0.05% 5-ALA during the culture, the growth of microalgae Chlorella sorokiniana was improved to against a common aquatic pathogen, A. hydrophila. The broad application of 5-ALA was demonstrated in this study for the first time.

Resveratrol induced premature senescence and inhibited epithelial-mesenchymal transition of cancer cells via induction of tumor suppressor Rad9.

Resveratrol (RSV) has been reported to influence many biological processes, including the stimulation of cellular senescence and inhibition of epithelial-mesenchymal transition (EMT). In this research, we explored the mechanisms of RSV on EMT and cellular senescence through the expression of a DNA damage response (DDR) protein, Rad9, in breast and lung cancer cell lines. Upon treating breast and lung cancer cell lines with RSV at the concentrations of 10-50 µM, Rad9 expression was increased at both transcriptional and translational levels. The results indicated that RSV-induced Rad9 expression, mediated by DNA damage and ROS, can significantly suppress proliferation by activating cellular senescence, and diminishing the expression of EMT markers with concomitant downregulation of Slug in breast and lung cancer cell lines. By using a siRNA approach, RSV was shown to mediate cellular senescence and EMT through a Rad9-dependent mechanism. The treatment with RSV can inhibit the proliferation, EMT, and increase cellular senescence of breast and lung cancer cell lines by activating Rad9. Our results suggest that the breast and lung tumor suppressive activities of RSV are, at least in part, mediated by the upregulation of Rad9.

Upregulation of RPA2 promotes NF-κB activation in breast cancer by relieving the antagonistic function of menin on NF-κB-regulated transcription.

RPA2, a subunit of the heterotrimeric replication protein A (RPA) complex, is overexpressed in various cancers. In this study, we showed a significant RPA2 upregulation in breast cancer tissues and cell lines. Ectopic expression of RPA2 in MCF7 and MDA-MB-231 cells promoted cell proliferation, adhesion, migration and invasion, and induced epithelial-mesenchymal transition (EMT) of MCF7 cells. Ablation of RPA2 in MDA-MB-231 cells induced apoptosis and suppressed colony formation, EMT and invasion. Binding assays indicated that menin, the multiple endocrine neoplasia type 1 (MEN1) tumor suppressor gene product, interacted with RPA2. Ectopic expression of RPA2 inhibited the formation of the menin-NK-κB p65 complex and repressed the inhibitory effect of menin on expression of NF-κB-regulated genes that contribute to tumor progression. Conversely, knockdown of RPA2 promoted formation of the menin-p65 complex and repressed the expression of NF-κB-mediated genes. RPA2 expression was induced via an E2F1-dependent mechanism in MCF7 and MDA-MB-231 cells treated with NF-κB activators, TNF-alpha or lipopolysaccharide (LPS). These results suggested that RPA2-dependent tumorigenicity was mediated via enhancement of NF-κB activity by relieving the antagonistic function of menin on NF-κB-regulated transcription in breast cancer cells.

GCIP functions as a tumor suppressor in non-small cell lung cancer by suppressing Id1-mediated tumor promotion.

Grap2 and cyclin D1 interacting protein (GCIP) has been recognized as a putative tumor suppressor, but the molecular mechanisms underlying its anti-tumor properties remain undefined. Here, we report that GCIP is frequently downregulated in non-small cell lung cancer (NSCLC) tissues. Binding assays indicated that inhibitor of DNA binding/differentiation 1 (Id1) interacts with GCIP in the nucleus. Ectopic GCIP expression in the highly invasive NSCLC cell line, H1299, inhibited proliferation, colony formation, invasion and migration, and increased susceptibility to anticancer drugs. Conversely, silencing GCIP expression in the minimally invasive NSCLS cell line, A549, increased proliferation, colony formation, invasion, and migration in vitro, and increased survival and resistance to anticancer drugs. GCIP also suppresses tumorigenicity of NSCLC cells in vivo and GCIP suppresses NSCLC progression is mediated in part by interfering with Id1 signaling, which was confirmed in conditionally induced stable cell lines. In addition, GCIP downregulates the expression of Id1, and GCIP and Id1 are inversely expressed in NSCLC cell lines and specimens. Taken together, these results suggest that GCIP is a potential tumor suppressor in NSCLC and that suppression of Id1-mediated oncogenic properties may be a key mechanism by which GCIP can potently suppress NSCLC tumor progression.

4E-BP3 regulates eIF4E-mediated nuclear mRNA export and interacts with replication protein A2.

In nucleus, eIF4E regulates the nucleus export of specific mRNA. In this study, altered 4E-BP3 (eIF4E-binding protein 3) expression resulted in profoundly affected cyclin D1 protein levels, partially due to changes in the cytoplasmic cyclin D1 mRNA levels in both U2OS and MCF7 cells, whereas altered 4E-BP1 expression did not affect eIF4E-mediated cyclin D1 mRNA export. 4E-BP3 also affected a subset of growth promoting mRNAs exported in an eIF4-dependent manner. Furthermore, 4E-BP3 interacted with dephosphorylated RPA2 (replication protein A2). The results indicated 4E-BP3 acts as an inhibitor of eIF4E-mediated mRNA export in the examined cells, and 4E-BP3 inhibition of eIF4E-mediated mRNA export is regulated by the phosphorylation state of RPA2.

Is Duhuo Jisheng Tang containing Xixin safe? A four-week safety study.

BACKGROUND: Though the nephrotoxicity and carcinogenicity of aristolochic acid (AA) are known, its safety in clinical usage is not clear. This study aims to evaluate the safety of Duhuo Jisheng Tang (DJT) in a four-week study to treat osteoarthritis (OA) of the knee. METHODS: A qualitative and quantitative investigations on DJT were conducted. A list of adverse events (AEs), complete blood counts, and liver and kidney function tests were measured for participants with knee OA at their scheduled hospital visits. Each detected AEs was independently assessed for severity and causality by site investigators (Chinese medical doctors) and study nurses. RESULTS: A total of 71 eligible subjects were included in the clinical study where 287 AEs were reported. DJT did not contain detectable aristolochic acid (AA) under thin-layer chromatography (TLC) analysis and gas chromatography coupled with mass spectrometry (GC-MS). There were no significant changes in liver or kidney functions. CONCLUSION: In four-week use of DJT, no renal tubular damage, no severe incidences of AEs and adverse drug reactions (ADRs) were observed. The present study obtained safety data from active surveillance of DJT.

Molecular cloning and characterization of Taiwan macaque lactoferrin.

BACKGROUND: Lactoferrin (LF) is an iron-binding glycoprotein that plays an important role in combating a wide range of pathogens and contributes innate protective defenses of mammals. METHODS: We cloned and sequenced the full-length cDNA for LF from Taiwan macaque. The antimicrobial activity and iron-binding ability of the purified recombinant macaque LF (rmLF) were determined and compared with those of human LF (hLF). RESULTS AND CONCLUSIONS: The complete mLF cDNA (GenBank: EU523857) encoded a 710-aa precursor with a 19-aa signal peptide. The nucleotide sequence of mLF showed the highest identity to the rhesus monkey LF (98%), whereas the putative amino acid sequence of mLF showed the highest identity to the hLF (90%). The rmLF and natural hLF showed almost equivalent antibacterial activities against Klebsiella pneumoniae and mLF presented a slightly lower activity against Listeria monocytogenes than natural hLF. In addition, the patterns of iron release from mLF and hLF were nearly identical.

Duhuo jisheng tang for treating osteoarthritis of the knee: a prospective clinical observation.

BACKGROUND: Little scientific evidence supports the efficacy of herbal medicines in the treatment of degenerative arthritis of the knee. The purpose of this study is to evaluate both the efficacy and safety of a finished Chinese herbal preparation duhuo jisheng tang (DJT) in reducing symptoms of degenerative osteoarthritis of the knee. METHODS: A prospective follow-up study was carried out in two hospitals in Taipei between April and October 2005. Sixty-eight osteoarthritis patients, with symptoms diagnosed by radiologists, received DJT at a rate of 2.5 g, twice daily for four weeks. Baseline scores were measured on the Western Ontario and McMaster Universities Osteoarthritis (WOMAC) index, followed by further measures at the end of weeks 1, 2 and 4. The World Health Organization Quality of Life (WHOQOL) assessment was undertaken as a secondary outcome, with pattern identification questionnaires being adopted. Regression models were constructed to explore the score differences between the baseline and at weeks 2 and 4 by various determinants including age, gender, body mass index (BMI), severity at baseline, use of rescue medication, aversion to cold and flaccidity of the lower back and knees. RESULTS: Among the 68 participants, there were statistically significant reductions in the WOMAC index scores for pain, stiffness and physical functioning in the second and fourth weeks, with effects first appearing during week 2. By week 4, the mean WOMAC index scores had fallen from 22.2 (+/- 19.2) to 16.1 (+/- 16.2) for pain, from 28.1 (+/- 24.9) to 18.5 (+/- 20.3) for stiffness, and from 22.6 (+/- 18.0) to 18.2 (+/- 17.8) for physical functioning, while the global score for pain under the visual analogue scale (VAS) was reduced from 38.7 (+/- 21.5) to 27.8 (+/- 19.8). CONCLUSION: In the treatment of degenerative osteoarthritis of the knee, a 4-week therapy with the Chinese herbal preparation DJT reduced pain and stiffness and improved physical functioning, but it was less effective in treating flaccidity and aversion to cold.