Undetectable off-target effects induced by FokI catalytic domain in mouse embryos.

The FokI catalytic domain can be fused to various DNA binding architectures to improve the precision of genome editing tools. However, evaluation of off-target effects is essential for developing these tools. We use Genome-wide Off-target analysis by Two-cell embryo Injection (GOTI) to detect low-frequency off-target editing events in mouse embryos injected with FokI-based architectures. Specifically, we test FokI-heterodimers fused with TALENs, FokI homodimers fused with RYdCas9, or FokI catalytic domains alone resulting in no significant off-target effects. These FokI genome editing systems exhibit undetectable off-target effects in mouse embryos, supporting the further development of these systems for clinical applications.

Protease inhibitor ASP enhances freezing tolerance by inhibiting protein degradation in kumquat.

Cold acclimation is a complex biological process leading to the development of freezing tolerance in plants. In this study, we demonstrated that cold-induced expression of protease inhibitor FmASP in a Citrus-relative species kumquat [Fortunella margarita (Lour.) Swingle] contributes to its freezing tolerance by minimizing protein degradation. Firstly, we found that only cold-acclimated kumquat plants, despite extensive leaf cellular damage during freezing, were able to resume their normal growth upon stress relief. To dissect the impact of cold acclimation on this anti-freezing performance, we conducted protein abundance assays and quantitative proteomic analysis of kumquat leaves subjected to cold acclimation (4°C), freezing treatment (-10°C) and post-freezing recovery (25°C). FmASP (Against Serine Protease) and several non-specific proteases were identified as differentially expressed proteins induced by cold acclimation and associated with stable protein abundance throughout the course of low-temperature treatment. FmASP was further characterized as a robust inhibitor of multiple proteases. In addition, heterogeneous expression of FmASP in Arabidopsis confirmed its positive role in freezing tolerance. Finally, we proposed a working model of FmASP and illustrated how this extracellular-localized protease inhibitor protects proteins from degradation, thereby maintaining essential cellular function for post-freezing recovery. These findings revealed the important role of protease inhibition in freezing response and provide insights on how this role may help develop new strategies to enhance plant freezing tolerance.

[Effects of organic solvents on proliferation of Hela cells line].

OBJECTIVE: To research the effects of organic solvents on proliferation inhibition of Hela cells line. METHODS: The apoptosis of Hela cells induced by ethanol and dimethylsulfoxide (DMSO) were studied to analyse the error analyse from background organic solvents. The apoptosis of Hela cells induced by ethanol, DMSO and the combination of these two organic solvents with different concentrations were observed by MTT test and Giemsa staining. RESULTS: The Hela cells proliferation were significantly inhibited by ethanol with the concentrations ranged from 30% to 100%, and DMSO with the concentrations ranged from 50% to 100% (P < 0.01). There were no significantly difference between the two combination of ethanol 40% + DMSO 60%, ethanol 30% + DMSO 70% and control group (P > 0. 05) respectively. CONCLUSION: The cells proliferation were inhibited by organic solvents itself and were in a time and dose dependent. The background experimental error can be reduced remarkably by choosing the two organic solvents combination of ethanol and DMSO.

Phytosterols in hull-less pumpkin seed oil, rich in ∆7-phytosterols, ameliorate benign prostatic hyperplasia by lowing 5α-reductase and regulating balance between cell proliferation and apoptosis in rats.

BACKGROUND: Pumpkin seed oil is widely used to treat benign prostatic hyperplasia (BPH), a common disease in elder men. However, its active components and mechanism have remained to be elucidated. OBJECTIVE: The objective of the present study was to investigate the active components of pumpkin seed oil and its mechanism against BPH. DESIGN: Total phytosterol (TPS) was isolated from hull-less pumpkin (Cucurbita pepo L. var. Styriaca) seed oil and analyzed by gas chromatography/mass spectrometry (GC/MS). Three phytosterols were purified by preparative HPLC (high performance liquid chromatography) and confirmed by NMR (nuclear magnetic resonance). TPS (3.3 mg/kg body weight, 1 mL/day/rat) was administered intragastrically to the testosterone propionate-induced BPH rats for 4 weeks. The structure changes of prostate tissues were assessed by hematoxylin & eosin (H&E) staining. The expression of androgen receptor (AR) and steroid receptor coactivator 1 (SRC-1) was analyzed by immunohistochemistry, while that of 5α-reductase (5AR), apoptosis, or proliferation-related growth factors/proteins was detected by real-time quantitative polymerase chain reaction or western blotting. RESULTS: The ∆7-phytosterols in TPS reached up to 87.64%. Among them, 24ß-ethylcholesta-7,22,25-trienol, 24ß-ethylcholesta-7,25(27)-dien-3-ol, and ∆7-avenasterol were confirmed by NMR. TPS treatment significantly ameliorated the pathological prostate enlargement and restored histopathological alterations of prostate in BPH rats. It effectively suppressed the expressions of 5AR, AR, and coactivator SRC-1. TPS inhibited the expression of proliferation-related growth factor epidermal growth factor, whereas it increased the expressions of apoptosis-related growth factor/gene transforming growth factor-ß1. The proliferation-inhibiting effect was achieved by decreasing the ERK (extracellular signal-regulated kinase) phosphorylation, while apoptosis was induced by Caspase 3 activation through JNK (c-Jun N-terminal kinase) and p38 phosphorylation. CONCLUSION: TPS from hull-less pumpkin seed oil, with ∆7-phytosterols as its main ingredients, is a potential nutraceutical for BPH prevention.

A protocol of homozygous haploid callus induction from endosperm of Taxus chinensis Rehd. var. mairei.

Obtainment and characterization of the novel endosperm callus of Taxus chinensis Rehd. var. mairei are valuable for haploid breeding, genome, and functional genome in Taxus. Callus was obtained by hydropriming with sterile water for 3 days and suitable medium composition. The highest callus induction (70.89 %) and lower browning ratio (7.95 %) were obtained from Gamborg (B5) medium supplemented with 30 g l(-1) of sucrose, 2.5 mg l(-1) of 2,4-dichlorophenoxyacetic (2,4-D), 0.5 mg l(-1) of 6-benzylademine (6-BA) and 7 g l(-1) of agar under dark conditions. The auxin of 2,4-D had a better efficiency of callus induction than naphthylacetic acid, and over 1 mg l(-1) of 6-BA was inhibitory to the callogensis of endosperm. The endosperm callus was haploid which was detectable by the flow cytometry. The genome block of homozygosity of callus was homozygous which was indicated by PCR-based SNP marks. The homozygous haploid of endosperm callus in vitro culture may be useful tools for taxoid-metabolism of gene engineering and bio-fermentation engineering.

Inhibition of migration and induction of apoptosis in LoVo human colon cancer cells by polysaccharides from Ganoderma lucidum.

Ganoderma lucidum polysaccharides (GLPs), which were purified from the medicinal herb G. lucidum followed by ethanol precipitation, protein depletion using the Sevage assay, purification using DEAE­cellulose (DE-52), dialysis and the use of ultrafiltration membranes, are used as an ingredient in traditional anticancer treatments in China. The aim of the current study was to evaluate the anticancer effects and investigate the underlying molecular mechanisms of GLPs on LoVo human colon cancer cells. The results demonstrated that the GLP­mediated anticancer effect in LoVo cells was characterized by cytotoxicity, migration inhibition, enhanced DNA fragmentation, morphological alterations and increased lactate dehydrogenase release. Furthermore, the activation of caspases­3, ­8 and ­9 was involved in GLP­stimulated apoptosis. Additionally, treatment with GLPs promoted the expression of Fas and caspase­3 proteins, whilst reducing the expression of cleaved poly(ADP­ribose) polymerase. These data indicate that GLPs demonstrate potential antitumor activity in human colon cancer cells, predominantly through the inhibition of migration and induction of apoptosis. Furthermore, activation of the Fas/caspase-dependent apoptosis pathway is involved in the cytotoxicity of GLPs.

Ganoderma lucidum polysaccharides target a Fas/caspase dependent pathway to induce apoptosis in human colon cancer cells.

Ganoderma lucidum polysaccharides (GLP) extracted from Ganoderma lucidum have been shown to induce cell death in some kinds of cancer cells. This study investigated the cytotoxic and apoptotic effect of GLP on HCT-116 human colon cancer cells and the molecular mechanisms involved. Cell proliferation, cell migration, lactate dehydrogenase (LDH) levels and intracellular free calcium levels ([Ca(2+)]i) were determined by MTT, wound-healing, LDH release and fluorescence assays, respectively. Cell apoptosis was observed by scanning and transmission electron microscopy. For the mechanism studies, caspase-8 activation, and Fas and caspase-3 expression were evaluated. Treatment of HCT-116 cells with various concentrations of GLP (0.625-5 mg/mL) resulted in a significant decrease in cell viability (P< 0.01). This study showed that the antitumor activity of GLP was related to cell migration inhibition, cell morphology changes, intracellular Ca(2+) elevation and LDH release. Also, increase in the levels of caspase-8 activity was involved in GLP-induced apoptosis. Western blotting indicated that Fas and caspase-3 protein expression was up-regulated after exposure to GLP. This investigation demonstrated for the first time that GLP shows prominent anticancer activities against the HCT-116 human colon cancer cell line through triggering intracellular calcium release and the death receptor pathway.

PAPP2C interacts with the atypical disease resistance protein RPW8.2 and negatively regulates salicylic acid-dependent defense responses in Arabidopsis.

Many fungal and oomycete pathogens differentiate a feeding structure named the haustorium to extract nutrition from the plant epidermal cell. The atypical resistance (R) protein RPW8.2 activates salicylic acid (SA)-dependent, haustorium-targeted defenses against Golovinomyces spp., the causal agents of powdery mildew diseases on multiple plant species. How RPW8.2 activates defense remains uncharacterized. Here, we report that RPW8.2 interacts with the phytochrome-associated protein phosphatase type 2C (PAPP2C) in yeast and in planta as evidenced by co-immunoprecipitation and bimolecular fluorescence complementation assays. Down-regulation of PAPP2C by RNA interference (RNAi) in Col-0 plants lacking RPW8.2 leads to leaf spontaneous cell death and enhanced disease resistance to powdery mildew via the SA-dependent signaling pathway. Moreover, down-regulation of PAPP2C by RNAi in the RPW8.2 background results in strong HR-like cell death, which correlates with elevated RPW8.2 expression. We further demonstrate that hemagglutinin (HA)-tagged PAPP2C prepared from tobacco leaf cells transiently transformed with HA-PAPP2C possesses phosphatase activity. In addition, silencing a rice gene (Os04g0452000) homologous to PAPP2C also results in spontaneous cell death in rice. Combined, our results suggest that RPW8.2 is functionally connected with PAPP2C and that PAPP2C negatively regulates SA-dependent basal defense against powdery mildew in Arabidopsis.

Construction of tandem repeats of DNA fragments by a polymerase chain reaction-based method.

We describe a new application of megaprimer polymerase chain reaction (PCR) for constructing a tandemly repeated DNA sequence using the drought responsive element (DRE) from Arabidopsis thaliana as an example. The key feature in the procedure was PCR primers with partial complementarity but differing melting temperatures (T(m)). The reverse primer had a higher T(m), a 3' end complementary to the DRE sequence and a 5' region complementary to the forward primer. The initial cycles of the PCR were conducted at a lower primer annealing temperature to generate products that served as megaprimers in the later cycles conducted at a higher temperature to prevent annealing of the forward primer. The region of overlap between the megaprimers was extended for generating products with a variable copy number (one to four copies) of tandem DRE sequence repeats (71 bp). The PCR product with four tandem repeats (4× DRE) was used as a template to generate tandem repeats with higher copies (copy number large than four) or demonstrated to bind DRE-binding protein in an yeast one-hybrid assay using promotorless reporter genes (HIS and lacZ). This PCR protocol has numerous applications for generating DNA fragments of repeated sequences.