高分辨率熔解曲线及其应用

饱和荧光染料、未标记探针与实时荧光PCR结合产生的高分辨率熔解曲线(HRM)是一种新的实时定量技术,在检测速度、灵敏度和准确性上具有突出的优点,近几年来在突变扫描、DNA甲基化和基因分型等医学检测中发展迅速.就HRM的原理、应用以及在HRM基础上发展起来的未标记探针(unlabled probe)HMR、弹回探针(snap probe)HMR技术作一介绍.     

Oligomeric Structure Diversity within the GIY-YIG Nuclease Family

The GIY-YIG nuclease domain has been identified in homing endonucleases, DNA repair and recombination enzymes, and restriction endonucleases. The Type II restriction enzyme Eco29kI belongs to the GIY-YIG nuclease superfamily and, like most of other family members, including the homing endonuclease I-TevI, is a monomer. It recognizes the palindromic sequence 5′-CCGC/GG-3′ (“/” marks the cleavage position) and cuts it to generate 3′-staggered ends. The Eco29kI monomer, which contains a single active site, either has to nick sequentially individual DNA strands or has to form dimers or even higher-order oligomers upon DNA binding to make a double-strand break at its target site. Here, we provide experimental evidence that Eco29kI monomers dimerize on a single cognate DNA molecule forming the catalytically active complex. The mechanism described here for Eco29kI differs from that of Cfr42I isoschisomer, which also belongs to the GIY-YIG family but is functional as a tetramer. This novel mechanism may have implications for the function of homing endonucleases and other enzymes of the GIY-YIG family.     

miR‐29a suppresses tristetraprolin,which is a regulator of epithelial polarity and metastasis

Several microRNAs (miRNAs) have recently been described as crucial regulators of epithelial‐to‐mesenchymal transition (EMT) and metastasis. By comparing the expression profiles of miRNAs, we found upregulation of miR‐29a in mesenchymal, metastatic RasXT cells relative to epithelial EpRas cells. Overexpression of miR‐29a suppressed the expression of tristetraprolin (TTP), a protein involved in the degradation of messenger RNAs with AU‐rich 3′‐untranslated regions, and led to EMT and metastasis in cooperation with oncogenic Ras signalling. We also observed enhanced miR‐29a and reduced TTP levels in breast cancer patient samples, indicating relevance for human disease. Previously, miR‐29 family members were shown to have tumour‐suppressive effects in haematopoietic, cholangiocytic and lung tumours. Therefore, miRNAs can act as either oncogenes or tumour suppressors, depending on the context.     

A Targeted siRNA Screen to Identify SNAREs Required for Constitutive Secretion in Mammalian Cells

The role of SNAREs in mammalian constitutive secretion remains poorly defined. To address this, we have developed a novel flow cytometry‐based assay for measuring constitutive secretion and have performed a targeted SNARE and Sec1/Munc18 (SM) protein‐specific siRNA screen (38 SNAREs, 4 SNARE‐like proteins and 7 SM proteins). We have identified the endoplasmic reticulum (ER)/Golgi SNAREs syntaxin 5, syntaxin 17, syntaxin 18, GS27, SLT1, Sec20, Sec22b, Ykt6 and the SM protein Sly1, along with the post‐Golgi SNAREs SNAP‐29 and syntaxin 19, as being required for constitutive secretion. Depletion of SNAP‐29 or syntaxin 19 causes a decrease in the number of fusion events at the cell surface and in SNAP‐29‐depleted cells causes an increase in the number of docked vesicles at the plasma membrane as determined by total internal reflection fluorescence (TIRF) microscopy. Analysis of syntaxin 19‐interacting partners by mass spectrometry indicates that syntaxin 19 can form SNARE complexes with SNAP‐23, SNAP‐25, SNAP‐29, VAMP3 and VAMP8, supporting its role in Golgi to plasma membrane transport or fusion. Surprisingly, we have failed to detect any requirement for a post‐Golgi‐specific R‐SNARE in this process.     

Dynamic motions of free and bound O29 scaffolding protein identified by hydrogen deuterium exchange mass spectrometry

In the double-stranded DNA containing bacteriophages, hundreds of copies of capsid protein subunits polymerize to form icosahedral shells, called procapsids, into which the viral genome is subsequently packaged to form infectious virions. High assembly fidelity requires the assistance of scaffolding protein molecules, which interact with the capsid proteins to insure proper geometrical incorporation of subunits into the growing icosahedral lattices. The interactions between the scaffolding and capsid proteins are transient and are subsequently disrupted during DNA packaging. Removal of scaffolding protein is achieved either by proteolysis or alternatively by some form of conformational switch that allows it to dissociate from the capsid. To identify the switch controlling scaffolding protein association and release, hydrogen deuterium exchange was applied to Bacillus subtilis phage Ø29 scaffolding protein gp7 in both free and procapsid-bound forms. The H/D exchange experiments revealed highly dynamic and cooperative opening motions of scaffolding molecules in the N-terminal helix-loop-helix (H-L-H) region. The motions can be promoted by destabilizing the hydrophobic contact between two helices. At low temperature where high energy motions were damped, or in a mutant in which the helices were tethered through the introduction of a disulfide bond, this region displayed restricted cooperative opening motions as demonstrated by a switch in the exchange kinetics from correlated EX1 exchange to uncorrelated EX2 exchange. The cooperative opening rate was increased in the procapsid-bound form, suggesting this region might interact with the capsid protein. Its dynamic nature might play a role in the assembly and release mechanism.     

Calpains are involved in Entamoeba histolytica-induced death of HT-29 colonic epithelial cells

Entamoeba histolytica is an enteric tissue-invading protozoan parasite that can cause amebic colitis and liver abscess in humans. E. histolytica has the capability to kill colon epithelial cells in vitro; however, information regarding the role of calpain in colon cell death induced by ameba is limited. In this study, we investigated whether calpains are involved in the E. histolytica-induced cell death of HT-29 colonic epithelial cells. When HT-29 cells were co-incubated with E. histolytica, the propidium iodide stained dead cells markedly increased compared to that in HT-29 cells incubated with medium alone. This pro-death effect induced by ameba was effectively blocked by pretreatment of HT-29 cells with the calpain inhibitor, calpeptin. Moreover, knockdown of m- and μ-calpain by siRNA significantly reduced E. histolytica-induced HT-29 cell death. These results suggest that m- and μ-calpain may be involved in colon epithelial cell death induced by E. histolytica.     

Combinatorial anticancer effects of curcumin and 5-fluorouracil loaded thiolated chitosan nanoparticles towards colon cancer treatment

Background

Evaluation of the combinatorial anticancer effects of curcumin/5-fluorouracil loaded thiolated chitosan nanoparticles (CRC-TCS-NPs/5-FU-TCS-NPs) on colon cancer cells and the analysis of pharmacokinetics and biodistribution of CRC-TCS-NPs/5-FU-TCS-NPs in a mouse model.

Methods

CRC-TCS-NPs/5-FU-TCS-NPs were developed by ionic cross-linking. The in vitro combinatorial anticancer effect of the nanomedicine was proven by different assays. Further the pharmacokinetics and biodistribution analyses were performed in Swiss Albino mouse using HPLC.

Results

The 5-FU-TCS-NPs (size: 150 ± 40 nm, zeta potential: + 48.2 ± 5 mV) and CRC-TCS-NPs (size: 150 ± 20 nm, zeta potential: + 35.7 ± 3 mV) were proven to be compatible with blood. The in vitro drug release studies at pH 4.5 and 7.4 showed a sustained release profile over a period of 4 days, where both the systems exhibited a higher release in acidic pH. The in vitro combinatorial anticancer effects in colon cancer (HT29) cells using MTT, live/dead, mitochondrial membrane potential and cell cycle analysis measurements confirmed the enhanced anticancer effects (2.5 to 3 fold). The pharmacokinetic studies confirmed the improved plasma concentrations of 5-FU and CRC up to 72 h, unlike bare CRC and 5-FU.

Conclusions

To conclude, the combination of 5-FU-TCS-NPs and CRC-TCS-NPs showed enhanced anticancer effects on colon cancer cells in vitro and improved the bioavailability of the drugs in vivo.

General significance

The enhanced anticancer effects of combinatorial nanomedicine are advantageous in terms of reduction in the dosage of 5-FU, thereby improving the chemotherapeutic efficacy and patient compliance of colorectal cancer cases.     

MiR-29a/b/c regulate human circadian gene hPER1 expression by targeting its 3/UTR

Several essential biological progresses in mammals are regulated by circadian rhythms. Though the molecular mechanisms of oscillating these circadian rhythms have been uncovered, the specific functions of the circadian genes are not very clear. It has been reported that knocking down circadian genes by microRNA is a useful strategy to explore the function of the circadian rhythms. In this study, through a forward bioinformatics screening ap- proach, we identified miR-29a/b/c as potent inhibitors for the human circadian gene hPER1. We further found that miR-29a/b/c could directly target hPER1 3/untranslated region （UTR） and down-regulate hPER1 at both mRNA and protein expression levels in human A549 cells. Thus, our findings suggested that the expression of hPER1 is regulated by miR-29a/b/c, which may also provide a new clue for the function ofhPER1.     

MicroRNA-29b Regulates Ethanol-induced Neuronal Apoptosis in the Developing Cerebellum through SP1/RAX/PKR Cascade

Neuronal loss is a prominent etiological factor for fetal alcohol spectrum disorders. The cerebellum is one of the areas in the developing central nervous system that is most sensitive to ethanol, especially during the temporal window of ethanol vulnerability. MicroRNAs are small, non-coding RNAs capable of regulating diverse cellular functions including apoptosis. Ethanol exposure has been shown to interfere with the expression of microRNAs. However, the role of microRNAs in ethanol neurotoxicity is still not clear. In the present study, we identified a particular microRNA, miR-29b, as a novel target of ethanol in the developing cerebellar granule neurons. We discovered that ethanol exposure suppressed miR-29b and induced neuronal apoptosis. Overexpression of miR-29b rendered neurons protection against ethanol-induced apoptosis. Furthermore, our data indicated that miR-29b mediated ethanol neurotoxicity through the SP1/RAX/PKR cascade. More importantly, the expression of miR-29b is developmentally regulated, which may account for, at least partially, the temporal window of ethanol sensitivity in the developing cerebellum.     

C-terminal COOH of Integrin β1 Is Necessary for β1 Association with the Kindlin-2 Adapter Protein

Protein-protein interactions are driving forces in cellular processes. As a prime example, transmembrane integrins link extracellular matrix and intracellular proteins, resulting in bidirectional signaling that regulates cell migration, proliferation, differentiation, and survival. Here we provide the first evidence that interaction between the integrin β1 cytoplasmic tail and kindlin-2, a member of a family of adapters implicated in human disease pathogenesis, is mainly governed by the β1 C-terminal carboxylate moiety and is required for laterality organ development in zebrafish. Affinity measurements indicate that this unusual protein-protein interaction mode is coordinated by a putative carboxylate-binding motif in the kindlin-2 FERM subdomain F3. Contrary to the C terminus of proteins that engage PDZ domains, the C-terminal three residues of β1, per se, do not contribute to kindlin-2 binding or to laterality organ development. Thus, by employing zebrafish as an in situ physiological tool to correlate protein structure and function, we have discovered an unexpected association chemistry between an integrin and a key adapter involved in integrin signaling.