Comparative studies on DNA-binding and in vitro antitumor activity of enantiomeric ruthenium(II) complexes.

A pair of ruthenium(II) complex enantiomers, Δ- and Λ-[Ru(bpy)2PBIP]2+ {bpy=2,2'-bipyridine, PBIP=2-(4-bromophenyl)imidazo[4,5-f]1,10-phenanthroline} have been synthesized and characterized. The systematic comparative studies between two enantiomers on their DNA binding-behaviors with calf thymus DNA (CT DNA) were carried out by viscosity measurements, spectrophotometric methods and molecular simulation technology. Additional assays were performed to explore the cytotoxicity of the ruthenium(II) enantiomers against tumor cell lines. DNA-binding studies show that both the enantiomers can bind to CT DNA via intercalative mode, and the Δ form binds to CT DNA more strongly than the Λ form does. Molecular simulation further shows that both the two enantiomers intercalate between base pairs of DNA in minor groove, and that the Δ form intercalates into DNA more deeply than the Λ form does. In addition, the cell proliferation assays show that the Δ form induces a greater cytotoxicity than the Λ form on human cervical cancer HeLa cells, which is positive correlated with the results in DNA binding studies and molecular docking, and implies that the DNA binding affinities of ruthenium(II) polypyridyl complexes might be constitute to the part of their anticancer mechanisms.

Exome sequencing identifies a novel mutation of the GDI1 gene in a Chinese non-syndromic X-linked intellectual disability family.

X-linked intellectual disability (XLID) has been associated with various genes. Diagnosis of XLID, especially for non-syndromic ones (NS-XLID), is often hampered by the heterogeneity of this disease. Here we report the case of a Chinese family in which three males suffer from intellectual disability (ID). The three patients shared the same phenotype: no typical clinical manifestation other than IQ score ≤ 70. For a genetic diagnosis for this family we carried out whole exome sequencing on the proband, and validated 16 variants of interest in the genomic DNA of all the family members. A missense mutation (c.710G > T), which mapped to exon 6 of the Rab GDP-Dissociation Inhibitor 1 (GDI1) gene, was found segregating with the ID phenotype, and this mutation changes the 237th position in the guanosine diphosphate dissociation inhibitor (GDI) protein from glycine to valine (p. Gly237Val). Through molecular dynamics simulations we found that this substitution results in a conformational change of GDI, possibly affecting the Rab-binding capacity of this protein. In conclusion, our study identified a novel GDI1 mutation that is possibly NS-XLID causative, and showed that whole exome sequencing provides advantages for detecting novel ID-associated variants and can greatly facilitate the genetic diagnosis of the disease.

Exome sequencing identifies a novel mutation of the GDI1 gene in a Chinese non-syndromic X-linked intellectual disability family

Abstract X-linked intellectual disability (XLID) has been associated with various genes. Diagnosis of XLID, especially for non-syndromic ones (NS-XLID), is often hampered by the heterogeneity of this disease. Here we report the case of a Chinese family in which three males suffer from intellectual disability (ID). The three patients shared the same phenotype: no typical clinical manifestation other than IQ score ≤ 70. For a genetic diagnosis for this family we carried out whole exome sequencing on the proband, and validated 16 variants of interest in the genomic DNA of all the family members. A missense mutation (c.710G > T), which mapped to exon 6 of the Rab GDP-Dissociation Inhibitor 1 (GDI1) gene, was found segregating with the ID phenotype, and this mutation changes the 237th position in the guanosine diphosphate dissociation inhibitor (GDI) protein from glycine to valine (p. Gly237Val). Through molecular dynamics simulations we found that this substitution results in a conformational change of GDI, possibly affecting the Rab-binding capacity of this protein. In conclusion, our study identified a novel GDI1 mutation that is possibly NS-XLID causative, and showed that whole exome sequencing provides advantages for detecting novel ID-associated variants and can greatly facilitate the genetic diagnosis of the disease.

A Novel Potentially Causative Variant of NDUFAF7 Revealed by Mutation Screening in a Chinese Family With Pathologic Myopia.

Purpose: Pathologic myopia described as myopia accompanied by severe deformation of the eye besides excessive elongation of eye, is usually a genetic heterogeneous disorder characterized by extreme, familial, early-onset vision loss. However, the exact pathogenesis of pathologic myopia remains unclear. In this study, we screened a Han Chinese family with pathologic myopia to identify the causative mutation and explore the possible pathogenic mechanism based on evaluation of the biological functions of the mutation. Methods: We identified the mutations in a family with pathologic myopia by single nucleotide polymorphism array combined with short tandem repeat microsatellite marker analysis and exome sequencing. Mutations were validated among family members by direct Sanger sequencing. The subcellular localization of the protein variant was investigated by immunofluorescence, and the stability of the mutant protein was determined by immunoblotting. Intracellular levels of adenosine triphosphate and reactive oxygen species and complex I activity were measured by traditional biochemical methods to determine the functional role of the disease-associated mutation. Results: The novel missense mutation: c.798C>G (p.Asp266Glu) in NDUFAF7, cosegregated with the disease and the resulting amino acid substitution affected a highly conserved residue in its protein. The mutation D266E in NDUFAF7 impaired complex I activity, which resulted in decreased ATP levels in cultured cells. Conclusions: We propose that the heterozygous mutation (c.798C>G) in NDUFAF7 may contribute to the pathogenesis of pathologic myopia, possibly by interfering with the phototransduction cascade. Mitochondrial dysfunction during eye development may lead to pathologic myopia.

Synthesis and biological activity evaluation of novel amino acid derivatives as potential elicitors against Tomato yellow leaf curl virus.

Disease caused by Tomato yellow leaf curl virus (TYLCV) brings serious production losses of cultivated tomato worldwide. In our previous study, two novel amino acid derivatives exerted satisfactory antiviral activities against TYLCV. In this study, the variation of TYLCV, the transcriptional expression level of Ty-1 and the enzyme activities of POD and PPO in tomato were monitored after treatment with two amino acid derivatives to illustrate the antiviral mechanism. The results showed the symptom severity caused by TYLCV was reduced significantly by two compounds and was associated with the inhibition of viral DNA level at the early stage. Among three levels of concentration, the highest inhibition rate of CNBF-His was 40.66% at 1000 mg/L, for CNBF-Asn, the highest inhibition rate was 36.26% at 2000 mg/L 30 days post-inoculation. Two compounds could also enhance the activities of PPO and POD and the transcriptional expression level of Ty-1 which correlates with plant resistance in tomato. In the field test, two compounds increased the yields of tomato and the maximum increase of yield was 37.66%. This is the first report of novel amino acid derivatives inducing resistance in tomato plant against TYLCV. It is suggested that amino acid derivatives have the potential to be an effective approach against TYLCV in tomato plant.

Synthesis, characterization, and application of microbe-triggered controlled-release kasugamycin-pectin conjugate.

The controlled and targeted release of pesticides with high water solubility has been a challenge for integrated pest management. In this paper, kasugamycin, an antibiotic broadly used in plant disease control, was covalently conjugated to pectin to form a kasugamycin-pectin conjugate by an amide bond. The conjugate was structurally characterized by Fourier transform infrared spectroscopy, ultraviolet spectrophotometry, and thermal gravimetric analysis. The results showed that the conjugate was stable over a wide range of pH and temperatures, as well as under UV irradiation. When incubated with Pseudomonas syringae pv. lachrymans, the conjugate could be activated, releasing the kasugamycin, which made it a promising controlled-release formulation of pesticide.

The bioaccumulation and biotransformation of synthetic estrogen quinestrol in crucian carp.

The occurrence and fate of endocrine disrupting chemicals (EDCs) in aquatic species have attracted close attention during the last decades. In this study, the bioaccumulation and biotransformation of synthetic estrogen quinestrol, one of the typical EDCs, in the plasma and liver of crucian carp, were investigated by a newly developed and validated reversed-phase high performance liquid chromatography with fluorescent detection method. Crucian carp were exposed to quinestrol in concentration of 2, 10, 50, 100 µg/L (5.49, 27.43, 137.17, 274.34 nmol/L) for 60 days. After 60 days' exposure, the concentrations of quinestrol found in liver and plasma were in the range of 0.25-0.69 mg/kg and 0.19-0.30 mg/L respectively, positively correlated with the exposure concentrations ranged 2-100 µg/L (5.49-274.34 nmol/L). There was a negative correlation between the bio-accumulation ratios and the exposure concentrations of quinestrol. 17α-Ethinylestradiol was also found in liver and plasma, and the concentrations were 0.02-0.19 mg/kg and 0.37-0.96 mg/L, respectively. The results indicated that quinestrol can be accumulated and transformed to 17α-ethinylestradiol in crucian carp. Moreover, exposure to quinestrol caused oxidative damages to crucian carp and the content of malondialdehyde increased in all treatment concentrations.

Simultaneous determination of glutathione, cysteine, homocysteine, and cysteinylglycine in biological fluids by ion-pairing high-performance liquid chromatography coupled with precolumn derivatization.

Biologically active low-molecular-mass thiols, mainly including glutathione (GSH), cysteine (Cys), homocysteine (Hcy), and cysteinylglycine (Cys-Gly), are important physiological components in biological fluids, and their analytical methods have gained continuous attention over recent years. We developed and validated a novel HPLC method for the quantification of GSH, Cys, Hcy, and Cys-Gly in human plasma, urine, and saliva using 4-chloro-3,5-dinitrobenzotrifluoride as the derivatization reagent. Analyses were linear from 0.15 to 500 µM with the coefficient regression range of 0.9987-0.9994. Detection limits ranged from 0.04 to 0.08 µM (S/N=3). The developed method was applied to quantification of four thiols in human biological fluids collected from five donors with the concentration range of 2.50-124.25 µM, 0-72.81 µM, and 0-4.25 µM for plasma, urine, and saliva, respectively. The present method seemed to be an attractive choice for the determination of thiols in plasma, urine, and saliva.

Solution-processible thieno-[3,4-b]-pyrazine derivatives with large Stokes shifts for non-doped red light-emitting diodes.

A group of novel thieno-[3,4-b]-pyrazine-cored molecules containing polyphenyl dendrons with or without arylamino or carbazolyl surface groups (DTP, N-DTP and C-DTP) are synthesized and investigated. They are characterized by extra large Stokes shifts of over 250 nm. In addition, to provide the site-isolation effect on the planar emissive core, the bulky dendrons enable these molecules to be solution processible. The peripheral carbazolyl or arylamino units facilitate the hole transporting ability in the neat films of these molecules. These dendritic materials are used as a non-doped emitting layer to fabricate organic light-emitting diodes (OLEDs) using a spin coating technique and saturated red emission is obtained. The dendritic molecules with arylamino or carbazolyl surface groups (N-DTP and C-DTP) exhibit a brightness of 1020 cd m(-2) and a luminous efficiency of 0.6 cd A(-1) , both higher than the dendritic analog without the surface functional groups (DTP), even superior to the small molecular reference compound which fails to transmit pure red emission under identical conditions. This performance is also comparable with that from vacuum deposited thieno-[3,4-b]-pyrazine-based counterparts and that for some other solution processible red fluorescent dendrimers. This is the first example of solution processible thieno-[3,4-b]-pyrazine derivatives for OLED applications.

Cationic nano-copolymers mediated IKKß targeting siRNA to modulate wound healing in a monkey model of glaucoma filtration surgery.

PURPOSE: To investigate the efficacy and safety of cationic nano-copolymers CS-g-(PEI-b-mPEG) mediated IκB kinase beta (IKKß) targeting siRNA in modulating wound healing in a monkey model of glaucoma filtration surgery. METHODS: The IKKß targeting siRNAs were chemically synthesized and screened in cultured monkey Tenon's fibroblasts in vitro. Fourteen monkeys underwent trabeculectomy and were randomly allocated to one of three treatment regimens: subconjunctival injection of either CS-g-(PEI-b-mPEG)/IKKß-siRNA (six eyes, 50nM, at the time of surgery and 7 days post surgery) or phosphate buffered saline (four eyes), or treated with mitomycin C (MMC; four eyes, 0.2 mg/ml). Bleb survival and characteristics, and intraocular pressure, were evaluated over a 60-day period. Histology of the surgical eyes was performed to evaluate ocular scarring and fibrosis in each group. RESULTS: Subconjunctival injection of CS-g-(PEI-b-mPEG)/IKKß-siRNA was well tolerated in this model. Both siRNA and MMC significantly prolonged bleb survival compared with the PBS group (the medians for survival days were 45.5, 60, and 29.5 in the siRNA, MMC, and PBS groups, respectively, p<0.01). Higher blebs were observed in the siRNA group than in the PBS group (p<0.01), while the MMC group showed the highest blebs among three groups (p<0.01). The surgical eyes in both the siRNA and MMC groups had significantly larger bleb area compared with the PBS group (p<0.01), but there was no significant difference between the siRNA and MMC groups (p=0.214). There were no significant differences in IOP readings among the three groups on the designated days after surgery (all p>0.05). The histologic examination demonstrated that the eyes treated with siRNA showed a marked reduction in subconjunctival scar tissue compared with the eyes in the PBS group. The conjunctival epithelium appeared healthy without the acellularity that was present in the MMC group. CONCLUSIONS: Subconjunctival injection of cationic nano-copolymers mediated IKKß targeting siRNA is associated with improved surgical outcome in a monkey model of trabeculectomy. This novel approach may potentially be a more controlled alternative as an anti-scarring agent in glaucoma filtration surgery.

Protective effect of bilberry (Vaccinium myrtillus L.) extracts on cultured human corneal limbal epithelial cells (HCLEC).

The use of bilberry (Vaccinium myrtillus L.) as a food and medicine for improving human vision has a long history all over the world. However, there is lack of convincing evidence from rigorous clinical trials or scientific research. This study investigated the effects of different concentrations of bilberry extracts on the cell viability, cell cycle and the expression of hyaluronic acid and glycosaminoglycans of cultured human corneal limbal epithelial cells. The data showed that bilberry extracts had no cytotoxicity to the corneal limbal epithelial cells at a wide range of concentrations (10(-9)-10(-4) M, equalized to the content of cyanidin-3-O-glucoside). Bilberry extract (10(-6), 10(-5) and 10(-4) M) increased cell viability after 48 h incubation. The number of cells decreased in G(0)/G(1) phase and increased prominently in S and G(2)/M phases after treatment with bilberry extracts at a high concentration (10(-4) M). The expression of glycosaminoglycans increased prominently after incubation with bilberry extracts (10(-7) and 10(-4) M) for 48 h while no significant changes were observed for the expression of hyaluronic acid. The results indicated that bilberry extract may be beneficial for the physiological renewal and homeostasis of corneal epithelial cells.

Dynamic alteration of low-density lipoprotein receptor after exposure to transforming growth factor-beta2 in human Tenon's capsule fibroblasts.

PURPOSE: The present study investigated dynamic alteration of low-density lipoprotein receptor and its binding and uptake of low-density lipoprotein (LDL) after exposure to transforming growth factor-beta(2) (TGF-beta(2)) in human Tenon's capsule fibroblasts. METHODS: Tenon's capsule fibroblasts obtained from elective cataract surgery patients were cultured and stimulated with different concentrations (0.1-10 ng/mL) of TGF-beta(2) for 24, 48, and 72 h. The LDLr mRNA and protein levels were analyzed by relative quantification real-time RT-PCR and Western blot analysis, respectively. The binding and uptake of DiO (3,3'-dioctadecyloxacarbocyanine)-labeled LDL was assessed by confocal microscopy. RESULTS: Real-time RT-PCR and Western blot analyses showed similar results revealing that after exposure to TGF-beta(2), the expression of protein and mRNA of LDLr occurred in a concentration-dependent and time-dependent manner with a peak at a concentration of 1.0 ng/mL at 72 h in Tenon's capsule fibroblasts. Confocal microscopy showed that DiO-LDL binding and uptake were time-dependent, reaching saturation at approximately 6 h. CONCLUSIONS: This study shows that LDLrs were overexpressed in the activated Tenon's capsule fibroblasts in a concentration-dependent and time-dependent manner after exposure to TGF-beta(2). The results suggest that LDLr in the activated Tenon's capsule fibroblasts may become a novel focus as a target receptor for controlled drug delivery, particularly in anti-scarring therapy during excessive conjunctival wound healing.

Upregulation of Copine1 in trabecular meshwork cells of POAG patients: a membrane proteomics approach.

PURPOSE: Primary open-angle glaucoma (POAG) is a leading cause of irreversible blindness worldwide, and its pathogenesis is still unknown. The purpose of this study was to determine molecular changes in membrane proteins in trabecular meshwork (TM) cells from POAG patients compared to those of age-matched normal controls. METHODS: Two-dimensional (2-D) gel electrophoresis profiles of membrane extracts from normal and glaucomatous TM cells were compared. The desired spots were identified after trypsin digestion and mass spectrometric analysis. Based on the results, a calcium-dependant membrane-binding protein, copine1, was further approached for a possible role in glaucomatous TM cells. The intracellular calcium concentration ([Ca(2+)]i) of TM cells was increased by incubating with calcium ionophore, A23187. Relative quantification real-time polymerase chain reaction (PCR) and western blot analysis measured copine1 expression and localization both in untreated and A23187-treated TM cells. RESULTS: Real-time PCR and western blot analysis confirmed that copine1 mRNA and protein expression were upregulated in glaucomatous TM cells when compared to normal ones. The cell distribution studies further showed that copine1 existed both in the membrane and cytoplasm fractions of glaucomatous TM cells but existed exclusively in cytoplasm fractions of their normal counterparts. More importantly, an influx of Ca(2+) markedly promoted the translocation of copine1 from the cytoplasm to membranes in glaucomatous TM cells. CONCLUSIONS: Copine1 is upregulated in plasma membranes of TM cells in individuals with POAG, which may be partly explained by its Ca(2+)-dependent translocation from the cytoplasm to the membranes. Investigation of the role and functions of copine1 in TM cells should offer new insight into the abnormal intracellular Ca(2+)-signaling pathway in glaucomatous TM and help to clarify the molecular mechanism of POAG.

Cationic nano-copolymers mediated IKKbeta targeting siRNA inhibit the proliferation of human Tenon's capsule fibroblasts in vitro.

PURPOSE: To synthesize a ternary cationic copolymer called CS-g-(PEI-b-mPEG) and characterize its features as a non-viral siRNA carrier; in turn, to investigate the influence of small interfering RNA (siRNA) targeting IkappaB kinase subunit beta (IKKbeta) on the proliferation of human Tenon's capsule fibroblasts (HTFs) in vitro. METHODS: First, a novel cationic copolymer composed of low molecular weight, linear poly(ethyleneimine) [PEI] blocked with polyethylene glycol (PEG) and grafted onto a chitosan (CS) molecule was synthesized. CS-g-(PEI-b-mPEG) was then compacted with 21nt siRNA at various copolymer/siRNA charge (N/P) ratios, and the resulting complexes were characterized by dynamic light scattering, gel electrophoresis, and serum incubation. Cell Titer 96 AQ(ueous) One Solution cell proliferation assay was used to investigate the cytotoxicity of this cationic copolymer. Second, siRNAs targeting IKKbeta (IKKBeta-siRNAs) were delivered into the HTFs using CS-g-(PEI-b-mPEG) as the vehicle. Real-time reverse transcription polymerase chain reaction (RT-PCR) subsequently assessed the mRNA level of IKKbeta, and western blot assay was used to determine protein expression. After IKKB-siRNA transfection, Cell Titer 96 AQ(ueous) One Solution cell proliferation assay was used to evaluate the proliferation of HTFs. RESULTS: The diameter of the CS-g-(PEI-b-mPEG)/siRNA complexes tended to decrease whereas their zeta potential tended to increase as the N/P ratio increased. The CS-g-(PEI-b-mPEG) copolymer showed good siRNA binding ability and high siRNA protection capacity. Furthermore, the copolymer presented remarkable transfection efficiency and showed much less cytotoxicity than 25 kDa PEI. IKKB-siRNAs were successfully delivered into HTFs using CS-g-(PEI-b-mPEG) as a vector. As a result, the expression of IKKbeta was downregulated at both the mRNA and protein levels, and the activation of nuclear factor-kappaB (NF-kappaB) in the HTFs was subsequently inhibited. Most impressively, the proliferation of HTFs was also effectively suppressed through the blocking of the NF-kappaB pathway. CONCLUSIONS: All the results demonstrate that CS-g-(PEI-b-mPEG) is a promising candidate for siRNA delivery, featuring excellent biocompatibility, biodegradability, and transfection efficiency. The RNA interference (RNAi) strategy using cationic copolymers as siRNA carriers will be a safe and efficient anti-scarring method following glaucoma filtration surgery.

[Induced differentiation of neural stem cells from subependymal zone into retinal cells in vitro].

OBJECTIVE: To study the cultivation and differentiation of neural stem cells (NSC) in vitro and observe the effects of retinal cell-conditioned medium on the differentiation of NSC. METHOD: Cells from subependymal zone of postnatal 0 - 3 days SD rat were isolated and cultured in vitro. Retinal cells of SD rat were cultured simultaneously. The supernatants (conditioned medium, SDR-CM) of cultured retinal cells were collected and used for the cultivation of the NSC from subependymal zone. Immunofluorescence method and anti-Thy1.1 antibodies were used to identify the cells derived from the NSC. RESULTS: Cultured NSC grew well in the serum-free culture medium. Cultured subependymal cells in the SDR-CM could be differentiated to the retinal cells. Some cells were stained positively with anti-Thy1.1 antibodies. CONCLUSION: These results showed that cultured NSC could survive well in vitro. SDR-CM can induce the NSC to differentiate to the retinal cells. The present study was designed to simulate the microenvironment of the eyes and to induce the differentiation of the NSC. Our in vitro model system may provide theoretical basis for the intraocular transplantation of retinal neurons.