Functional assessment of cryopreserved clinical grade hESC-RPE cells as a qualified cell source for stem cell therapy of retinal degenerative diseases.

The biosafety and efficiency of transplanting retinal pigment epithelial (RPE) cells derived from both human embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSCs) have been evaluated in phase I and phase II clinical trials. For further large-scale application, cryopreserved RPE cells must be used; thus, it is highly important to investigate the influence of cryopreservation and thawing on the biological characteristics of hESC-RPE cells and their post-transplantation vision-restoring function. Here, via immunofluorescence, qPCR, transmission electron microscopy, transepithelial electrical resistance, and enzyme-linked immunosorbent assays (ELISAs), we showed that cryopreserved hESC-RPE cells retained the specific gene expression profile, morphology, ultrastructure, and maturity-related functions of induced RPE cells. Additionally, cryopreserved hESC-RPE cells exhibited a polarized monolayer, tight junction, and gap junction structure and an in vitro nanoparticle phagocytosis capability similar to those of induced hESC-RPE cells. However, the level of pigment epithelium-derived factor (PEDF) secretion was significantly decreased in cryopreserved hESC-RPE cells. Royal College of Surgeons rats with cryopreserved hESC-RPE cells engrafted into the subretinal space exhibited a significant decrease in the b-wave amplitude compared with rats engrafted with induced hESC-RPE cells at 4 weeks post transplantation. However, the difference disappeared at 8 weeks and 12 weeks post operation. No significant difference in the outer nuclear layer (ONL) thickness was observed between the two groups. Our data showed that even after cryopreservation and thawing, cryopreserved hESC-RPE cells are still qualified as a donor cell source for cell-based therapy of retinal degenerative diseases.

Edaravone alleviates Alzheimer's disease-type pathologies and cognitive deficits.

Alzheimer's disease (AD) is one of most devastating diseases affecting elderly people. Amyloid-ß (Aß) accumulation and the downstream pathological events such as oxidative stress play critical roles in pathogenesis of AD. Lessons from failures of current clinical trials suggest that targeting multiple key pathways of the AD pathogenesis is necessary to halt the disease progression. Here we show that Edaravone, a free radical scavenger that is marketed for acute ischemic stroke, has a potent capacity of inhibiting Aß aggregation and attenuating Aß-induced oxidation in vitro. When given before or after the onset of Aß deposition via i.p. injection, Edaravone substantially reduces Aß deposition, alleviates oxidative stress, attenuates the downstream pathologies including Tau hyperphosphorylation, glial activation, neuroinflammation, neuronal loss, synaptic dysfunction, and rescues the behavioral deficits of APPswe/PS1 mice. Oral administration of Edaravone also ameliorates the AD-like pathologies and memory deficits of the mice. These findings suggest that Edaravone holds a promise as a therapeutic agent for AD by targeting multiple key pathways of the disease pathogenesis.

Detecting novel genetic mutations in Chinese Usher syndrome families using next-generation sequencing technology.

Usher syndrome (USH) is the most common cause of combined blindness and deafness inherited in an autosomal recessive mode. Molecular diagnosis is of great significance in revealing the molecular pathogenesis and aiding the clinical diagnosis of this disease. However, molecular diagnosis remains a challenge due to high phenotypic and genetic heterogeneity in USH. This study explored an approach for detecting disease-causing genetic mutations in candidate genes in five index cases from unrelated USH families based on targeted next-generation sequencing (NGS) technology. Through systematic data analysis using an established bioinformatics pipeline and segregation analysis, 10 pathogenic mutations in the USH disease genes were identified in the five USH families. Six of these mutations were novel: c.4398G > A and EX38-49del in MYO7A, c.988_989delAT in USH1C, c.15104_15105delCA and c.6875_6876insG in USH2A. All novel variations segregated with the disease phenotypes in their respective families and were absent from ethnically matched control individuals. This study expanded the mutation spectrum of USH and revealed the genotype-phenotype relationships of the novel USH mutations in Chinese patients. Moreover, this study proved that targeted NGS is an accurate and effective method for detecting genetic mutations related to USH. The identification of pathogenic mutations is of great significance for elucidating the underlying pathophysiology of USH.

Base-promoted transannulation of heterocyclic enamines and 2,3-epoxypropan-1-ones: regio- and stereoselective synthesis of fused pyridines and pyrroles.

Base-promoted transannulation of heterocyclic enamines and 2,3-epoxypropan-1-ones has been successfully achieved, providing a new access to structurally diverse fused pyridines and pyrroles with excellent regio- and stereoselectivity. Treatment with N-aryl 4-aminofuran-2(5H)-ones and 2,3-epoxypropan-1-ones under microwave heating resulted in functional furo[3,2-b]pyridines in good yields. The N-aryl 4-aminopyrrol-2(5H)-ones bearing an electron-withdrawing group engaged in the reaction afforded pyrrolo[3,2-b]pyridines, whereas their counterparts with an electron-neutral or an electron-donating group underwent a different reaction pathway to form pyrrolo[3,2-b]pyrroles through C-C bond cleavage.

Effect of optogenetic stimulus on the proliferation and cell cycle progression of neural stem cells.

Modulation of stem cell proliferation is a crucial aspect of neural developmental biology and regenerative medicine. To investigate the effect of optical stimulation on neural stem cell proliferation, cells transduced with channelrhodopsin-2 (ChR2) were used to analyze changes in cell proliferation and cell cycle distribution after light stimulation. Blue light significantly inhibited cell proliferation and affected the cell cycle, which increased the percentage of cells in G1 phase and reduced the percentage in S phase. It is likely that the influence of blue light on cell proliferation and the cell cycle was mediated by membrane depolarization, which induced accumulation of p21 and p27 proteins. Our data provide additional specific evidence that membrane depolarization may inhibit neural stem cell proliferation.

Detecting genetic variations in hereditary retinal dystrophies with next-generation sequencing technology.

PURPOSE: To identify pathogenic mutations responsible for retinal dystrophies (RDs) in three unrelated Chinese families. METHODS: Three probands from unrelated families with RDs were recruited. Genomic DNA prepared from leukocytes was analyzed using gene chip-based next-generation sequencing (NGS) to capture and sequence all of the exons of 100 known RD-associated genes. Candidate variants were validated with PCR and Sanger sequencing in the respective families. Thorough ophthalmic examinations including best-corrected visual acuity, funduscopic examination, and full-field electroretinograms were performed in the affected individuals. RESULTS: We successfully identified causative mutations in patients from the Chinese families with RDS: the known mutation IMPDH1 c.942_944delGAA in a family with retinitis pigmentosa, the novel mutation ABCA4 c.1924T>A in a family with Stargardt disease, and the novel mutation NMNAT1 c.272A>G and known mutation NMNAT1 c.196C>T in a family with Leber congenital amaurosis. All variations segregated with the disease phenotypes in the respective families and were absent from ethnically matched control chromosomes. Prediction analysis demonstrated the two novel missense mutations might be damaging. CONCLUSIONS: The results strongly suggested these mutations were responsible for different RD phenotypes in the Chinese families. NGS technology provides an accurate and economic method for identifying causative genes for RDs.

Identification of novel CYP4V2 gene mutations in 92 Chinese families with Bietti's crystalline corneoretinal dystrophy.

PURPOSE: To characterize the spectrum of CYP4V2 gene mutations in 92 unrelated Chinese probands with Bietti's crystalline dystrophy (BCD) and to describe the molecular and clinical characteristics of four novel CYP4V2 mutations associated with BCD. METHODS: All study participants underwent a complete ophthalmological examination. Mutational screening of CYP4V2 coding regions and flanking intron sequences was examined via directional Sanger sequencing, with allele separation confirmed by screening other family members. Subsequent in silico analysis of the mutational consequence on protein function was undertaken, with the impact of the novel mutation on pre-mRNA splicing examined via RT-PCR. RESULTS: Fifteen disease-causing variants were identified in 92 probands with BCD, including four novel mutations and eleven previously reported mutations. The most prevalent mutation was c.802_810del17insGC, which was detected in 69 unrelated families, with an allele frequency of 52.7% (97/184). Homozygosity was revealed in 35 unrelated families, and compound heterozygosity was observed in 43 subjects. Four patients harbored four novel variants, with these mutations cosegregated within all affected individuals and were not found in unaffected family members and 100 unrelated controls. Transcriptional analysis of a novel splice mutation revealed altered RNA splicing. In silico analysis predicted that the missense variant, p.Tyr343Asp, disrupted the CYP4V2 surface electrostatic potential distribution and spatial conformation. Among the patients with four novel mutations, genotype did not always correlate with age at onset, disease course, or electroretinogram (ERG) changes, with phenotypic variations even noted within the same genotype. CONCLUSIONS: The c.802_810del17insCG mutation was the most common mutation in the 92 Chinese probands with BCD examined. Four novel mutations were identified, contributing to the spectrum of CYP4V2 mutations associated with BCD, with no clear link established between disease phenotype and genotype.

Synthesis and anti-BVDV activity of novel δ-sultones in vitro: implications for HCV therapies.

In this study we report the synthesis and activity against bovine viral diarrhea virus (BVDV) of a novel series of bicycle δ-sultones containing γ-lactones. BVDV is responsible for major losses in cattle. Some of the synthesized δ-sultones showed pronounced anti-BVDV activity with EC50 values of 0.12-1.0µM and no significant cytotoxicity. Among them, the ortho bromosubstituted derivative 4f (EC50=0.12µM) showed better antiviral activity than other derivatives and was 10 fold more that of than positive control ribavirin (EC50=1.3µM). BVDV is also considered to be a valuable surrogate for the hepatitis C virus (HCV) in antiviral drug studies. The above results provided a novel candidate for the development of anti-HCV agents.

A facile synthesis, antibacterial activity of pulvinone and its derivatives.

Pulvinone and several 3-fluoro-4-morpholino substituted pulvinone derivatives were synthesized in five steps from a common precursor, phenyl acetic acid. Most of synthetic morpholine substituted pulvinones showed inhibitory activity against Esherichia coli. For the first time, the inhibition of pulvinone and its derivatives against Gram-negative bacteria was reported.

Improved inhibitory activities against tumor-cell migration and invasion by 15-benzylidene substitution derivatives of andrographolide.

In the present study, andrographolide (Andro, 1) derivatives were screened to identify potent inhibitors against tumor-cell migration and invasion, and associated structure-activity relationships were studied. Compared to 1, compounds 8a-8d exhibited more potent activities against migration in SGC-7901, PC-3, A549, HT-29 and Ec109 cell lines. Improved activities against tumor-cell migration and invasion were proved to be associated with the down-regulation of MMPs.

A novel three-component [5 + 1] heterocyclization leading to 2-azafluorenone synthesis and its polyfunctionalizations.

An efficient methodology for the synthesis of new and highly functionalized 2-azafluorenones via a three-component domino reaction involving C1-aryl acylation, C3-thiolation, and C4-cyanation has been developed. This domino reaction enables successful assembly of three new sigma bonds including a C-S bond and a C-N bond in a one-pot operation. Features of this strategy include the mild condition, convenient one-pot operation, and short reaction periods (15-30 min).

A novel domino strategy for forming poly-substituted quaternary imidazoles through a Cs2CO3-promoted aryl migration process.

A new domino strategy for the synthesis of highly functionalized quaternary imidazole derivatives via [3 + 2] heterocyclization, involving aryl migration and ring-opening of oxirane, has been developed. This domino reaction enables the successful assembly of three new sigma bonds including two C-N bonds in a simple operation. Features of this strategy include the mild conditions, convenient operation, and short reaction periods (15-20 min).

A domino synthetic strategy leading to two-carbon-tethered fused acridine/indole pairs and fused acridine derivatives.

A series of new poly-functionalized two-carbon-tethered fused acridine/indole pairs were synthesized via Brønsted acid-promoted domino reactions between indoline-2,3-dione and C(2)-tethered indol-3-yl enaminones. The reactions were further expanded to prepare C-tethered fused acridine/pyridine pairs, N-substituted amino acids, N-cyclopropyl and N-aryl substituted fused acridine derivatives, as well as bis-furan-3-yl-substituted indoles. During these reaction processes, the domino construction of a fused acridine skeleton with concomitant formation of two new rings was readily achieved in a one-pot operation. The procedures are facile, avoiding time-consuming and costly syntheses, tedious work-up and purification of precursors.

Ablation of Liver X receptor ß in mice leads to overactive macrophages and death of spiral ganglion neurons.

Age-related hearing loss is the most common type of hearing impairment, and is typically characterized by the loss of spiral ganglion neurons (SGNs). The two Liver X receptors (LXRs) are oxysterol-activated nuclear receptors which in adults, regulate genes involved in cholesterol homeostasis and modulation of macrophage activity. LXRß plays a key role in maintenance of health of dopaminergic neurons in the substantia nigra, large motor neurons in the spinal cord, and retinal ganglion cells in adult mice. We now report that LXRß is expressed in the SGNs of the cochlea and that loss of LXRß leads to age-related cochlea degeneration. We found that in the cochlea of LXRß-/- mice, there is loss of SGNs, activation of macrophages, demyelination in the spiral ganglion, decrease in glutamine synthetase (GS) expression and increase in glutamate accumulation in the cochlea. Part of the cause of damage to the SGNs might be glutamate toxicity which is known to be very toxic to these cells. Our study provides a so far unreported role of LXRß in maintenance of SGNs whose loss is a very common cause of hearing impairment.

Synthesis and in vitro antitumor activity of new butenolide-containing dithiocarbamates.

Three series of butenolide-containing dithiocarbamates were designed and synthesized. Their anti-tumor activity in vitro was evaluated. Among them compound I-14 exhibited broad spectrum anti-cancer activity against five human cancer cell lines with IC(50) <30 µM. Structure-activity relationship analysis showed that the introduction of dithiocarbamate side chains on the C-3 position of butenolide was crucial for anti-tumor activity.

A phase I clinical trial of human embryonic stem cell-derived retinal pigment epithelial cells for early-stage Stargardt macular degeneration: 5-years' follow-up.

OBJECTIVES: To evaluate the long-term biosafety and efficacy of transplantation of human embryonic stem cells-derived retinal pigment epithelial (hESC-RPE) cells in early-stage of Stargardt macular degeneration (STGD1). MATERIALS AND METHODS: Seven patients participated in this prospective clinical study, where they underwent a single subretinal transplantation of 1 × 105 hESC-RPE cells in one eye, whereas the fellow eye served as control. These patients were reassessed for a 60-month follow-up through systemic and ophthalmic examinations. RESULTS: None of the patients experienced adverse reactions systemically or locally, except for two who had transiently high intraocular pressure post-operation. Functional assessments demonstrated that all of the seven operated eyes had transiently increased or stable visual function 1-4 months after transplantation. At the last follow-up visit, two of the seven eyes showed visual function loss than the baseline; however, one of them showed a stable visual acuity when compared with the change of fellow eye. Obvious small high reflective foci in the RPE layer were displayed after the transplantation, and maintained until the last visit. Interestingly, three categories of patients who were classified based on autofluorescence, exhibited distinctive patterns of morphological and functional change. CONCLUSIONS: Subretinal transplantation of hESC-RPE in early-stage STGD1 is safe and tolerated in the long term. Further investigation is needed for choosing proper subjects according to the multi-model image and function assessments.

LCT-3d Induces Oxidative Stress-Mediated Apoptosis by Upregulating Death Receptor 5 in Gastric Cancer Cells.

Gastric cancer is a global health problem. In this study, we investigate the role of a novel Indole derivative, named LCT-3d, in inhibiting the growth of gastric cancer cells by MTT assay. The Western blotting results showed that LCT-3d modulated the mitochondrial-related proteins and Cleaved-Caspases 3/9, to induce cell apoptosis. The up-regulation of Death receptor 5 (DR5) in MGC803 cells was observed with LCT-3d treatment. Knockdown of DR5 on MGC803 cells partially reversed the LCT-3d-induced mitochondrial apoptosis. The level of Reactive Oxygen Species (ROS) in MGC803 cells was increased with LCT-3d treatment and could be blocked with the pretreatment of the ROS inhibitor N-Acetylcysteine (NAC). The results demonstrate that the elevating ROS can up-regulate the expression of DR5, resulting in apoptosis via mitochondrial pathway. Although the nuclear factor erythroid-2 related factor 2 (Nrf2) pathway served an important role in protecting gastric cancer cells against the injury of ROS, it can't reverse LCT-3d-induced cell apoptosis. Taken together, our study showed that LCT-3d induced apoptosis via DR5-mediated mitochondrial apoptotic pathway in gastric cancer cells. LCT-3d could be a novel lead compound for development of anti-cancer activity in gastric cancer.

Synthesis and vasodilative activity of tanshinone IIA derivatives.

A series of 2,2'-(substituted methylene)bis-(1,6,6-trimethyl-6,7,8,9-tetrahydrophenanthro[1,2-b]furan-10,11-dione) derivatives were synthesized by the reaction of tanshinone IIA (D(1)) and aromatic aldehyde in the presence of p-TsOH. Bromination derivative of D(1) and hydrolysis product of cryptotanshinone (D(2)) were also prepared in this work. Vasodilation activity in vitro of them was valuated on the contractile response of vascular thoracic aorta smooth muscle from Wistar rats for the first time. Most of them exhibited a concentration-dependent inhibition on the contractile response of norepinephrine.

Stereoselective synthesis of desloratadine derivatives as antagonist of histamine.

A series of desloratadine derivatives were stereoselectively synthesized and evaluated for H(1) antihistamine activity. For the evaluation of H(1) antihistamine activity, the in vitro histamine-induced contraction of the guinea-pig ileum assay (HC) was used. The synthesized desloratadine derivatives 7, 8 and 9 are structurally related to rupatadine and were generated by replacement of the 5-methyl-3-pyridine group of rupatadine with gamma-alkylidene butenolide. Their H(1) antihistamine activities have shown a high dependence on the exact nature of the substituent in the lactone ring. Optimum structures 7, 8a and 8g display potent activity inhibiting histamine-induced effects.

A low toxic CRM1 degrader: Synthesis and anti-proliferation on MGC803 and HGC27.

Chromosome region maintenance 1 (CRM1) is the sole nuclear exporter of several tumor suppressor, a growth regulatory protein as an attractive cancer drug target. In the present work, a novel CRM1 degrader was discovered from newly synthesized α, ß-unsaturated-δ-lactone based on a natural product Goniothalamin. It induces apoptosis of both MGC803 and HGC27 cell lines via degrading CRM1. Selective inhibition was observed for the proliferation of gastric cancer cell lines MGC803, HGC27 comparing to Human Gastric Mucosal Epithelial Cell Line (GES1). For the first time, CRM1 inhibitor or degrader inducing apoptosis in gastric carcinoma was investigated.