Stepwise synthesis of RNA conjugates carrying peptide sequences for RNA interference studies

Oligoribonucleotide conjugates carrying nuclear localization peptide sequences at the 3′-end were prepared stepwise on a single support. The siRNA duplex carrying the nuclear localization peptide sequence at the 3′-end of the passenger strand has similar inhibitory properties as those of unmodified or cholesterol-modified RNA duplexes.     

Synthesis and properties of small interfering RNA duplexes carrying 5-ethyluridine residues

Oligoribonucleotides carrying 5-ethyluridine units were prepared using solid-phase phosphoramidite chemistry. The introduction of the tert-butyldimethylsilyl group at the 2′-OH position proceeded in good yield and very high 2′-regioselectivity. RNA duplexes carrying 5-ethyluridine either at the sense or the guide strands display RNAi activity comparable to or slightly better than that of unmodified RNA duplexes. Gene suppression experiments using luciferase targets in SH-SY5Y cells show that the ethyl group is generally well accepted at all positions although a small decrease in RNA interference activity is observed when one 5-ethylU residue is incorporated in the 3′ overhangs.     

Nebulization of siRNA for inhalation therapy based on a microfluidic surface acoustic wave platform

The local delivery of therapeutic small interfering RNA or siRNA to the lungs has the potential to improve the prognosis for patients suffering debilitating lung diseases. Recent advances in materials science have been aimed at addressing delivery challenges including biodistribution, bioavailability and cell internalization, but an equally important challenge to overcome is the development of an inhalation device that can deliver the siRNA effectively to the lung, without degrading the therapeutic itself. Here, we report the nebulization of siRNA, either naked siRNA or complexed with polyethyleneimine (PEI) or a commercial transfection agent, using a miniaturizable acoustomicrofluidic nebulization device. The siRNA solution could be nebulised without significant degradation into an aerosol mist with tunable mean aerodynamic diameters of approximately 3 µm, which is appropriate for deep lung deposition via inhalation. The nebulized siRNA was tested for its stability, as well as its toxicity and gene silencing properties using the mammalian lung carcinoma cell line A549, which demonstrated that the gene silencing capability of siRNA is retained after nebulization. This highlights the potential application of the acoustomicrofluidic device for the delivery of efficacious siRNA via inhalation, either for systemic delivery via the alveolar epithelium or local therapeutic delivery to the lung.     

Mesoporous silica nanorods toward efficient loading and intracellular delivery of siRNA

The technology of RNA interference (RNAi) that uses small interfering RNA (siRNA) to silence the gene expression with complementary messenger RNA (mRNA) sequence has great potential for the treatment of cancer in which certain genes were usually found overexpressed. However, the carry and delivery of siRNA to the target site in the human body can be challenging for this technology to be used clinically to silence the cancer-related gene expression. In this work, rod shaped mesoporous silica nanoparticles (MSNs) were developed as siRNA delivery system for specific intracellular delivery. The rod MSNs with an aspect ratio of 1.5 had a high surface area of 934.28 m²/g and achieved a siRNA loading of more than 80 mg/g. With the epidermal growth factor (EGF) grafted on the surface of the MSNs, siRNA can be delivered to the epidermal growth factor receptor (EGFR) overexpressed colorectal cancer cells with high intracellular concentration compared to MSNs without EGF and lead to survivin gene knocking down to less than 30%.     

Layer-by-Layer-Assembled Capsule Size Affects the Efficiency of Packaging and Delivery of Different Genetic Cargo

The lack of an efficient and versatile intracellular nucleic acids delivery platform impedes the clinical implementation of gene therapy. Advances in layer-by-layer (LbL) technology have led to the production of LbL polymer capsules, a promising universal delivery tool. The biocompatibility, sufficient packaging capacity, safety, low cost, and high variability of structure and composition of the LbL capsules make it possible to meet the requirements for clinical-grade nonviral gene transfer. Here, the possibility of polymeric LbL capsules of different sizes (micrometer and sub-micrometer-sized) to serve as universal nonviral carriers for messenger RNA (mRNA) and small interfering RNA (siRNA) is considered. In particular, the internalization of capsules into human mesenchymal stem cells (hMSCs, as an example of adult primary stem cells), capsule uptake, and intracellular delivery of mRNA and siRNA is studied. Importantly, the use of micrometer- or sub-micrometer-sized polymer capsules (MicCaps and SubCaps) allows the mRNA or siRNA to be packaged and transferred into hMSCs with high efficiency. While the uptake efficiency is comparable between MicCaps and SubCaps, the latter are significantly more efficient than MicCap when transferring siRNAs. These results demonstrate the potential of the LbL capsules as a universal gene delivery platform, which can be tuned according to the properties of genetic cargo.     

Magnetic nanoparticle formulations for DNA and siRNA delivery

Newly synthesized magnetic nanomaterials possess high DNA binding capacity either itself or in the presence of a positively charged lipid-based Metafectene™ reagent or branched polyethylene imine 25 kDa. Polyethylene imine (PEI)-modified nanomaterials are able to deliver nucleic acids in cell culture in duplexes. Magnetofection with triplexes of nanomaterials results in higher transduction efficiencies compared to optimal PEI or Metafectene formulations. 90% transient down-regulation of the target protein in HeLa-green fluorescence protein cells was achieved at short interfering RNA concentrations as low as 8 nM with a formulation of PEI-modified nanoparticles.     

Glycosylated Quantum Dots for the Selective Labelling of <Emphasis Type="Italic">Kluyveromyces bulgaricus</Emphasis> and <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> Yeast Strains

Highly fluorescent CdTe quantum dots (QDs) stabilized by thioglycolic acid (TGA) were prepared by an aqueous solution approach and used as fluorescent labels in detecting yeast cells. Sugars (mannose, galactose or glucose) were adsorbed on CdTe@TGA QDs and the interaction of these nanoparticles with yeast cells was studied by fluorescence microscopy. Results obtained demonstrate that galactose and mannose functionalized QDs associate respectively with Kluyveromyces bulgaricus and Saccharomyces cerevisiae yeast strains due to saccharide/lectin specific recognition. Glucose-functionalized CdTe QDs, which are not recognized by cell lectins, preferentially localize in the bud scars of S. cerevisiae.     

Ultrasound activates ataxia telangiectasia mutated- and rad3-related (ATR)-checkpoint kinase 1 (Chk1) pathway in human leukemia Jurkat cells

Low-intensity ultrasound (US) has been shown to induce death of cancer cells; however, the underlying mechanism remains unclarified. Here, we provide novel evidence that the inhibition of checkpoint kinase 1 (Chk1) by a selective inhibitor or small interfering RNA (siRNA) enhances US-induced apoptosis in Jurkat cells. Jurkat cells showed insignificant lysis immediately after US at any applied intensity, whereas approximately 70% of the cells were γH2AX-positive 30min after US at 0.4W/cm(2). Regarding DNA damage response (DDR), Chk1, known as a target of ataxia telangiectasia mutated (ATM) and rad3-related (ATR), was phosphorylated in cells after US exposure. An ATM inhibitor showed nearly no effect on Chk1 phosphorylation, whereas chemicals showing the ATR inhibitory effect markedly abrogated the phosphorylation, indicating that Chk1 phosphorylation is preferentially more dependent on ATR than on ATM in cells exposed to US. The pharmacological inhibition of Chk1 promoted caspase-3 cleavage and increased the percentage of cells in SubG1 after US exposure. siRNA targeting Chk1 abrogated approximately 55% of Chk1 expression and also promoted apoptosis, suggesting that Chk1 plays anti-apoptotic roles in response to US. These findings revealed, for the first time, that US activates Chk1 dependently on ATR and the activated Chk1 is involved in apoptosis of cells exposed to US. Moreover, we propose that Chk1 may be a promising target in US-aided therapy.     

Therapeutic siRNA targeting the cancer cell stemness regulator PRDI-BF1 and RIZ domain zinc finger protein 14

PRDI-BF1 and RIZ (PR) domain zinc finger protein 14 (PRDM14), first reported in 2007 to be overexpressed in breast cancer, plays an important role in breast cancer proliferation. Subsequent studies reported that PRDM14 is expressed in embryonic stem cells, primordial germ cells, and various cancers. PRDM14 was reported to confer stemness properties to cancer cells. These properties induce cancer initiation, cancer progression, therapeutic resistance, distant metastasis, and recurrence in refractory tumors. Therefore, PRDM14 may be an ideal therapeutic target for various types of tumors. Silencing PRDM14 expression using PRDM14-specific siRNA delivered through an innovative intravenous drug delivery system reduced the size of inoculated tumors, incidence of distant metastases, and increased overall survival in nude mice without causing adverse effects. Therapeutic siRNA targeting PRDM14 is now being evaluated in a human phase I clinical trial for patients with refractory breast cancer, including triple-negative breast cancer.     

Glycosaminoglycans and glycoconjugates in the adult anuran integument (Lithobates catesbeianus)

Glycosaminoglycans (GAGs) from the integument of Lithobates catesbeianus were biochemically characterized and histochemically localized. Moreover, carbohydrate distribution was investigated using conventional and lectin histochemistry at light microscopy. Hyaluronan (HA), dermatan sulfate (DS) and a heparanoid were found in the integument. Sulfated and carboxylated GAGs were visualized in the Eberth-Katschenko (EK) layer, in the mucous glands, in the hypodermis as well as in the mast cells. Furthermore, glucose and galactose were identified in the integument through thin layer chromatography (TLC) assays. N-Acetyl-β-glucosamine residues were identified in the mucous glandular cells, between the corneum and spinosum strata, in the subepidermal region, and in the EK layer. N-Acetyl-galactosamine residues were evident in the EK layer, corresponding to a residue of the dermatan sulfate chain, which may be related to the collagenous fiber arrangement. These glycoconjugates occurred as secretory glandular products and as dermal structural elements. Moreover, HA and DS are the predominant GAGs in the L. catesbeianus integument. Considering the importance of glycoconjugates, they play a significant role to the integrity of the skin, providing mechanical support for integument cells. In addition, they are important to the water regulation mechanisms, since L. catesbeianus is preferably aquatic.     

Application of high-resolution ESI and MALDI mass spectrometry to metabolite profiling of small interfering RNA duplex

We investigated the application of a high-resolution Orbitrap mass spectrometer equipped with an electrospray ionization (ESI) source and a matrix-assisted laser desorption/ionization-time-of-flight (MALDI-TOF) mass spectrometer to the metabolite profiling of a model small interfering RNA (siRNA) duplex TSR#34 and compared their functions and capabilities. TSR#34 duplex was incubated in human serum in vitro, and the duplex and its metabolites were then purified by ion exchange chromatography in order to remove the biological matrices. The fraction containing the siRNA duplex and its metabolites was collected and desalted and then subjected to high-performance liquid chromatography (HPLC) equipped with a reversed phase column. The siRNA and its metabolites were separated into single strands by elevated chromatographic temperature and analyzed using the ESI-Orbitrap or the MALDI-TOF mass spectrometer. Using this method, the 5' and/or 3' truncated metabolites of each strand were detected in the human serum samples. The ESI-Orbitrap mass spectrometer enabled differentiation between two possible RNA-based sequences, a monoisotopic molecular mass difference which was less than 2 Da, with an intrinsic mass resolving power. In-source decay (ISD) analysis using a MALDI-TOF mass spectrometer allowed the sequencing of the RNA metabolite with characteristic fragment ions, using 2,4-dihydroxyacetophenone (2,4-DHAP) as a matrix. The ESI-Orbitrap mass spectrometer provided the highest mass accuracy and the benefit of on-line coupling with HPLC for metabolite profiling. Meanwhile, the MALDI-TOF mass spectrometer, in combination with 2,4-DHAP, has the potential for the sequencing of RNA by ISD analysis. The combined use of these methods will be beneficial to characterize the metabolites of therapeutic siRNA compounds. Copyright ? 2012 John Wiley & Sons, Ltd.     

不同糖类化合物的太赫兹光谱特性

选取对人体有重要作用的三种糖类化合物,采用高分辨率太赫兹时域光谱系统与傅里叶变换红外光谱系统,在较宽的频谱范围内,对样品进行测谱分析;实验发现无水葡萄糖在1.10,1.30,1.45,1.79,1.88,1.97,2.08,2.40,2.55,2.70,2.84 ,2.96,3.24,3.64和4.23 THz频率处存在特征吸收,无水果糖在1.09,1.33,1.65,2.14,2.62,2.97,3.24,4.75,6.97,7.35,7.98,8.36,9.16,9.32,9.53和9.73 THz频率处存在特征吸收,无水半乳糖在2.21,2.33,2.70,2.82,3.17,3.42,3.93,4.51,5.07,5.96,6.60,6.91,8.03,8.71和9.01 THz频率处存在特征吸收。对掺杂不同比例葡萄糖、果糖、半乳糖与聚乙烯样品的实验结果做定量分析,发现在测得的上述特征吸收频率处,随化合物样品质量分数的增加,样品的吸收系数或吸光度呈线性递增。实验进一步得到无水葡萄糖与无水果糖在2.96 THz存在共同的特征吸收,无水葡萄糖与无水半乳糖在2.33,2.70和2.82 THz三处存在共同的特征吸收,无水果糖与无水半乳糖在8.00 THz处存在共同的特征吸收,而无水葡萄糖、果糖、半乳糖三者在3.20 THz处存在共同的特征吸收。三种化合物具有相同的分子式,所以三者都具有的3.20 THz特征吸收频率主要源于分子内相互作用,反应同分异构体相同的化学键或者基团。三者特征吸收频率的差异主要源于分子结构以及分子间相互作用的不同,代表同分异构体结构以及分子间振动模式的差异。通过分析三种糖类样品的实验测量结果,预测了葡萄糖在4.70,5.30,5.60,5.98,7.03,7.85,8.26,8.71和9.01 THz处存在特征吸收。基于密度泛函理论,采用CASTEP量子化学计算软件对三种化合物进行理论模拟,对样品在THz波段的特征吸收进行指认,计算得到的结果与实验结果吻合,这表明了CASTEP晶体模拟软件在化合物的THz光谱模拟方面应用的可行性。     

Flexible for Multiple Equations about GHZ States and a Prototype Case

According to the peculiar entanglement and measurement properties of the three-particle GHZ state, we have systematically analyzed that two GHZ states and three GHZ states satisfy some expressions after exchanging one or two groups of particles respectively, which are described as four interesting and flexible equations. The four equations can deduce that four GHZ states or even m GHZ states still satisfy some expressions after exchanging one group and two groups of particles, and they can be summarized as two general flexible equations. Furthermore, we also investigate their application in the field of quantum key agreement based on these equations. In particular, we combine with decoy photons to propose a novel session key sharing protocol, which can guarantee the unconditional security of the protocol. It is feasible to use the existing quantum processing technology to realize the proposed protocol.

     

Hybridization potential of oligonucleotides comprising 3′-O-methylated altritol nucleosides

A series of 3??-O-methylated-d-altrohexitol nucleoside analogs (MANA) was synthesized comprising all four base moieties, adenine, cytosine, uracil, and guanine. These monomers were incorporated into oligonucleotides (ONs) by automated solid phase synthesis and the thermal and thermodynamic stability of all new modified constructs were evaluated. Data were compared with results obtained for both anhydrohexitol (HNAs) and 3??-O-altrohexitol-modified ONs (ANAs). We hereby demonstrate that ONs modified with MANA monomers have an improved thermal and thermodynamic stability compared to RNA, ANA, or HNA containing ONs of which the extent depends on the number of incorporated moieties and their position in the sequence. Thermodynamic analysis afforded comparable or even improved results in comparison with the incorporation of locked nucleic acids. While the specificity of these new synthons is slightly lower compared to mismatches within RNA double strands, it is similar to the discrimination potential of other hexitol modifications (HNA and ANA) which already proved their biologic interest, highlighting the potential of MANA constructs in antisense and in siRNA applications.     

固相果糖的太赫兹及红外特征吸收谱研究

果糖是一种常见于蜂蜜和多种植物中的简单酮糖，属于三种可食用单糖之一，可与葡萄糖结合形成双糖蔗糖。纯净的果糖常温呈白色晶体状，具有甜度高、升糖指数低等优点，广泛应用于食品行业。截止目前，由于针对固相果糖的太赫兹和红外特征吸收谱研究大多局限于单纯的实验测试或者基于单分子理论计算，缺乏较为系统完善的研究，因此从理论和实验上系统研究了固相果糖的太赫兹特征吸收谱及红外特征吸收谱，首次报道了固相果糖在频谱大于3.0 THz以外的特征吸收峰的实验值，采用基于单分子的MP2和B3LYP泛函以及基于晶胞的PBE和PW91交换相关泛函计算获得了固相果糖太赫兹及红外特征吸收谱，并与实验获得的固相果糖太赫兹及红外特征吸收谱进行了比对分析，发现基于晶胞的PBE和PW91交换相关泛函计算结果与实验获得的果糖太赫兹特征吸收峰更相符，表明固相果糖在0.1～4.0 THz的大多数太赫兹特征吸收峰源自分子间相互作用而非分子内相互作用，揭示了果糖分子周围环境对果糖振动模式的显著影响。     

Insights into UV-induced apoptosis: ultrastructure, trichrome stain and spectral imaging

Nuclear substructures associated with apoptosis in HeLa cells have been examined using light-microscopic morphometry, trichrome staining, spectral imaging and transmission electron microscopy. This detailed analysis reveals several sites where alterations in the normal cellular ultrastructure occur during apoptotic progression. To correlate these ultrastructural changes with the underlying molecular processes, we have characterized and quantified apoptotic cell morphology with and without inhibition of two caspases, which are key effectors of the apoptotic program. Using this analysis, early apoptotic events included: (a) the segregation of nucleolar components, a diminished granular component, and a reduction in number but increase in size of fibrillar centers, (b) an increase in the number of cytoplasmic ribosomes and (c) a very minimal increase in the amount of peripherally condensed DNA. Apoptosis progressed with: (a) an increase in the number of perichromatin granules and perichromatin fibrils, (b) a reduction in number of interchromatin granule centers concomitant with an increase in their size, (c) partial digestion and circumferential condensation of the DNA at the nuclear membrane and (d) rounding of the cytoplasm with an increase in organellar density and shrinkage in cell size. Endstage apoptotic cells showed: (a) one (or two) very large pools of incompletely digested DNA, (b) one (or two) very large interchromatin granule centers, (c) an increased number of perichromatin granules which were distanced from DNA and often closely apposed to the nucleolus, (d) formation of unusually condensed, highly coiled perinucleolar bodies and (e) blebbing of highly dense cytoplasm.In HeLa cells treated with UV and inhibitors of caspase 1 and 3, the length of time from early apoptosis to the formation of apoptotic bodies was greatly extended. Inhibiting caspase activity: (a) prevented the pooling of DNA, (b) retarded the formation of large interchromatin granule centers, (c) increased the number of perichromatin granules, (d) produced disassembly of the nucleolus, (e) prevented the formation of highly coiled perinucleolar bodies, and (f) caused vacuolization in the cell center and a unipolar blebbing of the cytoplasm.Spectral imaging in conjunction with serial section electron microscopy confirmed the staining specificities of the condensed DNA, of the large condensed interchromatin granule centers, and of the nucleoli. The results indicate that the interface between the components of the chromatin domain and the interchromatin space is a critical site of caspase activity in apoptosis, and precedes other events such as internucleosomal DNA degradation.     

Dielectric microsphere mediated transfection using a femtosecond laser

We demonstrate the permeabilization of cell membranes by an enhanced optical field generated under polystyrene microspheres of 1000 nm diameter excited by a femtosecond laser pulse. Fluorescent molecules and short interfering RNA (siRNA) have been successfully delivered to many cells in the irradiated area by a single 80 fs laser pulse at 800 nm wavelength in the presence of antibody-conjugated polystyrene spheres. The ratios of the cells showing permeabilization were 38% and 21% for Fluorescein isothiocyanate-dextran and siRNA, respectively, at the laser fluence of 1.06 J/cm(2). The present method has advantages both in high throughput of many cell treatments and precise processing of minute areas on cell membranes.     

Functional distribution of Ca<Superscript>2+</Superscript>-coupled P2 purinergic receptors among adrenergic and noradrenergic bovine adrenal chromaffin cells

Background  

Adrenal chromaffin cells mediate acute responses to stress through the release of epinephrine. Chromaffin cell function is regulated by several receptors, present both in adrenergic (AD) and noradrenergic (NA) cells. Extracellular ATP exerts excitatory and inhibitory actions on chromaffin cells via ionotropic (P2X) and metabotropic (P2Y) receptors. We have taken advantage of the actions of the purinergic agonists ATP and UTP on cytosolic free Ca²⁺ concentration ([Ca²⁺]_i) to determine whether P2X and P2Y receptors might be asymmetrically distributed among AD and NA chromaffin cells.     

Live Imaging of Glucose Homeostasis in Nuclei of COS-7 Cells

Measuring subcellular glucose levels deep in tissues can provide new insights into compartmentalization and specialization of glucose metabolism among different cells. As shown previously, a FRET-based glucose-sensor consisting of two GFP-variants and the Escherichia coli periplasmic glucose/galactose binding protein was successfully expressed in the cytosol of COS7-cells and used to determine cytosolic glucose levels. Recording cytosolic fluorescence intensities in cells located in deeper layers of tissues is often difficult due to loss of signal intensity caused by effects of other cell layers on excitation and emission light. These interfering effects may be reduced by restricting fluorophores to occupy only a fraction of the assayed tissue volume. This can be accomplished by confining fluorophores to a sub-compartment of each cell in the tissue, such as the nucleus. The glucose-sensor was targeted to nuclei of COS7-cells. To determine, whether nuclear glucose levels can be used to track cytosolic changes, nuclear glucose concentrations were quantified as the cells were challenged with external glucose over a range of 0.5 to 10 mM and compared to cytosolic levels. Internal glucose concentrations in both compartments were similar, corresponding to approximately 50% of the external concentration. Taken together, these results indicate that nuclear glucose levels can be used to determine cytosolic levels indirectly, permitting more reliable quantification of fluorescence intensities and providing a tool for measurements not only in cell cultures but also in tissues.