A Novel Co-polymer Based on Hydroxypropyl ��-Cyclodextrin Conjugated to Low Molecular Weight Polyethylenimine as an in Vitro Gene Delivery Vector

A novel co-polymer based on 2-hydroxypropyl-α-cyclodextrin cross-linked by low molecular weight polyethylenimine was synthesized as a gene delivery vector. The copolymer could bind and condense DNA tightly. It showed lower cytotoxicity than PEI 25kDa in SK-BR-3 cells. Transfection efficiency was increased over 5.5-fold higher than PEI 25 kDa in SK-BR-3 cells in complete serum medium. It is a potential candidate vector for gene therapy.     

我国手性液晶的研究进展与应用

阐述了手性液晶的结构和分类,介绍了近年来国内手性液晶的合成与研究进展情况,并着重讨论了胆甾相手性液晶的热色效应及其作为手性添加剂在显示领域的应用以及铁电液晶的非线性光学效应.     

Surface Anchoring and Chevron Structure in Ferroelectric Liquid Crystals

Applying a sufficiently high electric field to a ferroelectric smectic-C liquid crystal deforms the so-called chevron structure into one in which the layers are flat. This transition happens in three different ways depending on the type of liquid crystal phase. In all three cases, the flattening has been believed to begin from the middle of the smectic layers. We show that a previously proposed model can predict the possibility of forming a new type of partially flattened layer in which the flattening begins at the edges of the layer. This interesting behavior takes place when the surface anchoring is strong.     

Liposome Formulation and In Vitro Testing in Non-Physiological Conditions Addressed to Ex Vivo Kidney Perfusion

This work focuses on formulating liposomes to be used in isolated kidney dynamic machine perfusion in hypothermic conditions as drug delivery systems to improve preservation of transplantable organs. The need mainly arises from use of kidneys from marginal donors for transplantation that are more exposed to ischemic/reperfusion injury compared to those from standard donors. Two liposome preparation techniques, thin film hydration and microfluidic techniques, are explored for formulating liposomes loaded with two model proteins, myoglobin and bovine serum albumin. The protein-loaded liposomes are characterized for their size by DLS and morphology by TEM. Protein releases from the liposomes are tested in PERF-GEN perfusion fluid, 4 °C, and compared to the in vitro protein release in PBS, 37 °C. Fluorescent liposome uptake is analyzed by fluorescent microscope in vitro on epithelial tubular renal cell cultures and ex vivo on isolated pig kidney in hypothermic perfusion conditions. The results show that microfluidics are a superior technique for obtaining reproducible spherical liposomes with suitable size below 200 nm. Protein encapsulation efficiency is affected by its molecular weight and isoelectric point. Lowering incubation temperature slows down the proteins release; the perfusion fluid significantly affects the release of proteins sensitive to ionic media (such as BSA). Liposomes are taken up by epithelial tubular renal cells in two hours’ incubation time.     

人幽门螺杆菌尿素通道蛋白UreI基因穿梭质粒的构建及表达

目的构建人幽门螺杆菌尿素通道蛋白UreI基因的大肠杆菌-分枝杆菌穿梭表达质粒,并在耻垢分枝杆菌中进行表达。方法PCR扩增幽门螺杆菌尿素通道蛋白UreI编码基因全长片段,克隆入pET32a(+)质粒,鉴定正确后,再亚克隆入大肠杆菌-分枝杆菌穿梭质粒pJHSP70,构建分枝杆菌穿梭表达质粒pJHSP70-UreI,转化耻垢分枝杆菌,诱导表达后,进行SDS-PAGE和Western blot检测。结果从幽门螺杆菌基因组中扩增出585bp的UreI基因片段。重组质粒pJHSP70-UreI经酶切和PCR鉴定,与预期结果一致。目的蛋白表达量约占菌体总蛋白的18.5%,且具有良好的反应原性。结论已成功构建了幽门螺杆菌尿素通道蛋白UreI基因大肠杆菌-分枝杆菌穿梭表达质粒,并在耻垢分枝杆菌中获得表达。     

Cationic DOPC–Detergent Conjugates for Safe and Efficient in Vitro and in Vivo Nucleic Acid Delivery

The ability of a nonviral nucleic acid carrier to deliver its cargo to cells with low associated toxicity is a critical issue for clinical applications of gene therapy. We describe biodegradable cationic DOPC–C₁₂E₄ conjugates in which transfection efficiency is based on a Trojan horse strategy. In situ production of the detergent compound C₁₂E₄ through conjugate hydrolysis within the acidic endosome compartment was expected to promote endosome membrane destabilization and subsequent release of the lipoplexes into cytosol. The transfection efficiency of the conjugates has been assessed in vitro, and associated cytotoxicity was determined. Cellular uptake and intracellular distribution of the lipoplexes have been investigated. The results show that direct conjugation of DOPC with C₁₂E₄ produces a versatile carrier that can deliver both DNA and siRNA to cells in vitro with high efficiency and low cytotoxicity. SAR studies suggest that this compound might represent a reasonable compromise between the membrane activity of the released detergent and susceptibility of the conjugate to degradation enzymes in vitro. Although biodegradability of the conjugates had low impact on carrier efficiency in vitro, it proved critical in vivo. Significant improvement of transgene expression was obtained in the mouse lung tuning biodegradability of the carrier. Importantly, this also allowed reduction of the inflammatory response that invariably characterizes cationic‐lipid‐mediated gene transfer in animals.     

A Nanochannel Platform for Single DNA Studies: From Crowding,Protein DNA Interaction,to Sequencing of Genomic Information

The study of nanochannel-confined DNA is important from biotechnological and biophysical points of view. We produce nanochannels in elastomer with soft lithography and proton beam writing. Issues concerning DNA confined in such quasi one-dimensional channels are discussed. We describe DNA stretching via the control of channel diameter and buffer conditions and how the extension can be interpreted with theory and computer simulation. We then discuss the conformation of nano-confined DNA crowded by neutral polymers and like-charged proteins. As an example of a protein that has an affinity to DNA, the effect of heat-stable nucleoid-structuring protein, H-NS, on the folding and compaction of DNA is reviewed. Compaction of DNA by eukaryotic protamine and unpacking of pre-compacted DNA through an increase in salt concentration are discussed. We review results obtained with a novel, cross-channel device that allows the monitoring of the dynamic, conformational response of DNA after exposure to a ligand or protein and/or a change in buffer conditions in situ. As a biotechnological application, linearization of DNA by bottlebrush coating with a polypeptide copolymer is discussed. It is demonstrated that large-scale genomic organization can be sequenced using single DNA molecules on an array of elastomeric nanochannels. Overall, our results show that the effects of ligands and proteins on the conformation, folding, and condensation of DNA are not only related to classical controlling factors, such as osmotic pressure, charge, and binding, but that the interplay with confinement in a nanospace is of paramount importance.     

A pH and Redox Dual Responsive 4-Arm Poly(ethylene glycol)-block-poly(disulfide histamine) Copolymer for Non-Viral Gene Transfection in Vitro and in Vivo

A novel 4-arm poly(ethylene glycol)-b-poly(disulfide histamine) copolymer was synthesized by Michael addition reaction of poly(ethylene glycol) (PEG) vinyl sulfone and amine-capped poly(disulfide histamine) oligomer, being denoted as 4-arm PEG-SSPHIS. This copolymer was able to condense DNA into nanoscale polyplexes (<200 nm in average diameter) with almost neutral surface charge (+(5–10) mV). Besides, these polyplexes were colloidal stable within 4 h in HEPES buffer saline at pH 7.4 (physiological environment), but rapidly dissociated to liberate DNA in the presence of 10 mM glutathione (intracellular reducing environment). The polyplexes also revealed pH-responsive surface charges which markedly increased with reducing pH values from 7.4–6.3 (tumor microenvironment). In vitro transfection experiments showed that polyplexes of 4-arm PEG-SSPHIS were capable of exerting enhanced transfection efficacy in MCF-7 and HepG2 cancer cells under acidic conditions (pH 6.3–7.0). Moreover, intravenous administration of the polyplexes to nude mice bearing HepG2-tumor yielded high transgene expression largely in tumor rather other normal organs. Importantly, this copolymer and its polyplexes had low cytotoxicity against the cells in vitro and caused no death of the mice. The results of this study indicate that 4-arm PEG-SSPHIS has high potential as a dual responsive gene delivery vector for cancer gene therapy.     

口蹄疫病毒2B基因绿色荧光蛋白融合表达质粒的构建及表达

目的构建口蹄疫病毒(FMDV)2B基因绿色荧光蛋白(GFP)融合表达质粒,并在BHK-21细胞中表达。方法RT-PCR扩增O型口蹄疫病毒WFL株的2B基因,克隆入表达载体pEGFP-C1,并进行双酶切、PCR及测序鉴定。将阳性重组质粒转染BHK-21细胞,检测绿色荧光蛋白的表达和2B基因转录水平。结果经双酶切及PCR鉴定,目的基因片段大小与预期相符,测序结果与WFL株相应序列一致。荧光显微镜和流式细胞仪均检测到细胞内绿色荧光蛋白的表达,荧光定量PCR检测到细胞内有2B基因的转录。结论已成功构建了FMDV2B基因GFP融合表达质粒,并在BHK-21细胞中获得了表达。     

Nanoparticles in Liquid Crystals: Synthesis,Self-Assembly,Defect Formation and Potential Applications

Revolutionary developments in the fabrication of nanosized particles have created enormous expectations in the last few years for the use of such materials in areas such as medical diagnostics and drug-delivery, and in high-tech devices. By its very nature, nanotechnology is of immense academic and industrial interest as it involves the creation and exploitation of materials with structural features in between those of atoms and bulk materials, with at least one dimension limited to between 1 and 100 nm. Most importantly, the properties of materials with nanometric dimensions are, in most instances, significantly different from those of atoms or bulk materials. Research efforts geared towards new synthetic procedures for shape and size-uniform nanoscale building blocks as well as efficient self-assembly protocols for manipulation of these building blocks into functional materials has created enormous excitement in the field of liquid crystal research. Liquid crystals (LCs) by their very nature are suitable candidates for matrix-guided synthesis and self-assembly of nanoscale materials, since the liquid crystalline state combines order and mobility at the molecular (nanoscale) level. Based on selected relevant examples, this review attempts to give a short overview of current research efforts in LC-nanoscience. The areas addressed in this review include the synthesis of nanomaterials using LCs as templates, the design of LC nanomaterials, self-assembly of nanomaterials using LC phases, defect formation in LC-nanoparticle suspensions, and potential applications. Despite the seeming diversity of these research topics, this review will make an effort to establish logical links between these different research areas.     

Preparation of monomethyl poly(ethylene glycol)-g-chitosan copolymers with various degrees of substitution: Their ability to encapsulate and condense plasmid DNA

Chitosan (CS) has great potential as a nonvirus gene delivery vector, but its application is limited because of poor water solubility. Monomethyl poly(ethylene glycol) (mPEG)-graft-CS copolymers were synthesized by the reaction of mPEG–aldehyde (oxidized mPEG) with amino groups on CS chains; they showed enhanced solubility in water. Copolymers with various mPEG degrees of substitution (DS) and CS molecular weights were obtained, and their capabilities of DNA encapsulation were compared through gel retardation assay and particle size and ζ potential measurements. The effects of different ratios of primary amines on CS to the phosphate groups on DNA (N/P ratios), DS, and molecular weights on particle size and encapsulation efficiency were investigated. The results show that high N/P ratios and proper DS were necessary for the formation of well-distributed complex particles. Among all of these samples, mPEG (3.55)–CS (50 kDa)/DNA complexes [where the parentheses following mPEG indicate DS (%), and the parentheses following CS indicate the molecular weight of CS] raised the ζ potential from negative to positive most quickly, yielded the smallest particle size, and were retarded in agarose gel at the lowest N/P ratio; this indicated the best efficiency of DNA encapsulation. On the contrary, mPEG (0.80)–CS (50 kDa)/DNA complexes raised the ζ potential to positive most slowly, fluctuated around the value 0 from N/P ratios of 15 : 1 to 30 : 1, and were retarded in agarose gel at the highest N/P ratio; this indicated the lowest efficiency of encapsulating plasmids. Copolymers with desirable efficiencies of DNA encapsulation could be promising gene carriers. © 2008 Wiley Periodicals, Inc. J Appl Polym Sci, 2008     

Evaluation of a Novel Plasmid for Simultaneous Gene Electrotransfer-Mediated Silencing of CD105 and CD146 in Combination with Irradiation

Targeting tumor vasculature through specific endothelial cell markers represents a promising approach for cancer treatment. Here our aim was to construct an antibiotic resistance gene-free plasmid encoding shRNAs to simultaneously target two endothelial cell markers, CD105 and CD146, and to test its functionality and therapeutic potential in vitro when delivered by gene electrotransfer (GET) and combined with irradiation (IR). Functionality of the plasmid was evaluated by determining the silencing of the targeted genes using qRT-PCR. Antiproliferative and antiangiogenic effects were determined by the cytotoxicity assay tube formation assay and wound healing assay in murine endothelial cells 2H-11. The functionality of the plasmid construct was also evaluated in malignant melanoma tumor cell line B16F10. Additionally, potential activation of immune response was measured by induction of DNA sensor STING and proinflammatory cytokines by qRT-PCR in endothelial cells 2H-11. We demonstrated that the plasmid construction was successful and can efficiently silence the expression of the two targeted genes. As a consequence of silencing, reduced migration rate and angiogenic potential was confirmed in 2H-11 endothelial cells. Furthermore, induction of DNA sensor STING and proinflammatory cytokines were determined, which could add to the therapeutic effectiveness when used in vivo. To conclude, we successfully constructed a novel plasmid DNA with two shRNAs, which holds a great promise for further in vivo testing.     

Cartilage Oligomeric Matrix Protein Gene Multilayers Inhibit Osteogenic Differentiation and Promote Chondrogenic Differentiation of Mesenchymal Stem Cells

There are still many challenges to acquire the optimal integration of biomedical materials with the surrounding tissues. Gene coatings on the surface of biomaterials may offer an effective approach to solve the problem. In order to investigate the gene multilayers mediated differentiation of mesenchymal stem cells (MSCs), gene functionalized films of hyaluronic acid (HA) and lipid-DNA complex (LDc) encoding cartilage oligomeric matrix protein (COMP) were constructed in this study via the layer-by-layer self-assembly technique. Characterizations of the HA/DNA multilayered films indicated the successful build-up process. Cells could be directly transfected by gene films and a higher expression could be obtained with the increasing bilayer number. The multilayered films were stable for a long period and DNA could be easily released in an enzymatic condition. Real-time polymerase chain reaction (RT-PCR) assay presented significantly higher (p < 0.01) COMP expression of MSCs cultured with HA/COMP multilayered films. Compared with control groups, the osteogenic gene expression levels of MSCs with HA/COMP multilayered films were down-regulated while the chondrogenic gene expression levels were up-regulated. Similarly, the alkaline phosphatase (ALP) staining and Alizarin red S staining of MSCs with HA/COMP films were weakened while the alcian blue staining was enhanced. These results demonstrated that HA/COMP multilayered films could inhibit osteogenic differentiation and promote chondrogenic differentiation of MSCs, which might provide new insight for physiological ligament-bone healing.     

Ipr1和GFP基因真核共表达穿梭质粒的构建及其在人肺癌细胞中的表达

目的构建胞内病原体抗性基因1(Ipr1)和绿色荧光蛋白(GFP)基因真核共表达穿梭质粒,并在人肺腺癌细胞A549中表达。方法采用PCR方法,分别从质粒pEGFP-C1-Ipr1和pEGFP-C1中扩增Ipr1和GFP基因,将GFP基因、分枝杆菌复制子OriM和Ipr1基因同时克隆入多启动子真核共表达载体pBudCE4.1中,构建pBud-GFP-OriM-Ipr1穿梭质粒,脂质体法转染A549细胞,荧光显微镜观察GFP的表达,免疫组化方法检测Ipr1蛋白的表达。结果酶切和测序分析表明,pBud-GFP-OriM-Ipr1真核共表达穿梭质粒构建正确。转染A549细胞后,荧光显微镜下可观察到转染细胞中有GFP表达,免疫组化法可检测到Ipr1蛋白的表达,且定位于细胞核内。结论已成功构建Ipr1和GFP基因真核共表达穿梭质粒,为进一步研究Ipr1抗结核的功能奠定了基础。     

Abiotic Stresses: Insight into Gene Regulation and Protein Expression in Photosynthetic Pathways of Plants

Global warming and climate change intensified the occurrence and severity of abiotic stresses that seriously affect the growth and development of plants, especially, plant photosynthesis. The direct impact of abiotic stress on the activity of photosynthesis is disruption of all photosynthesis components such as photosystem I and II, electron transport, carbon fixation, ATP generating system and stomatal conductance. The photosynthetic system of plants reacts to the stress differently, according to the plant type, photosynthetic systems (C₃ or C₄), type of the stress, time and duration of the occurrence and several other factors. The plant responds to the stresses by a coordinate chloroplast and nuclear gene expression. Chloroplast, thylakoid membrane, and nucleus are the main targets of regulated proteins and metabolites associated with photosynthetic pathways. Rapid responses of plant cell metabolism and adaptation to photosynthetic machinery are key factors for survival of plants in a fluctuating environment. This review gives a comprehensive view of photosynthesis-related alterations at the gene and protein levels for plant adaptation or reaction in response to abiotic stress.     

Genome-Wide Identification of the AGC Protein Kinase Gene Family Related to Photosynthesis in Rice (Oryza sativa)

The cAMP-dependent protein kinase A, cGMP-dependent protein kinase G and phospholipid-dependent protein kinase C (AGC) perform various functions in plants, involving growth, immunity, apoptosis and stress response. AGC gene family is well described in Arabidopsis, however, limited information is provided about AGC genes in rice, an important cereal crop. This research studied the AGC gene family in the AA genome species: Oryza sativa ssp. japonica, Oryza sativa ssp. indica, Oryza nivara, Oryza rufipogon, Oryza glaberrima, Oryza meridionalis, Oryza barthii, Oryza glumaepatula and Oryza longistaminata were searched and classified into six subfamilies, and it was found that these species have similar numbers of members. The analysis of gene duplication and selection pressure indicated that the AGC gene family expanded mainly by segmental or whole genome duplication (WGD), with purifying selection during the long evolutionary period. RNA-seq analysis revealed that OsAGCs of subfamily V were specifically highly expressed in leaves, and the expression patterns of these genes were compared with that of photosynthesis-related genes using qRT-PCR, discovered that OsAGC9, OsAGC20, and OsAGC22 might participate in photosynthesis. These results provide an informative perspective for exploring the evolutionary of AGC gene family and its practical application in rice.     

The Investigations of Construction and in vitro Release of Curcumin Lyotropic Liquid Crystals: Phase Diagrams,Small Angle X-ray Scattering,Rheological Technology,and Release Kinetics

In this study, hexagonal and cubic lyotropic liquid crystals (LLC) were constructed in Brij 97-Tween 40 (MS₈₂, MS₆₄ and MS₄₆)/OLA/H₂O systems to encapsulate curcumin. MS₈₂, MS₆₄, and MS₄₆ indicated that the mass ratio of Brij 97/Tween 40 was 8/2, 6/4, and 4/6. The microstructure of curcumin LLC was studied using small angle X-ray scattering (SAXS). Phase diagrams showed that the increase of MS reduced the phase transition temperature (T_C). Particularly, the T_C of sample C₁Cur [Brij 97-Tween 40 (MS₄₆)/OLA/H₂O = 50.0/2.8/47.2] and C₂Cur [Brij 97-Tween 40 (MS₄₆)/OLA/H₂O = 50.0/25.0/25.0] was 37.6 and 35.4 °C, respectively, close to the temperature of the human body. Thus, the shear rheology and SAXS were used to study the structural changes of samples C₁Cur and C₂Cur with temperature. The moduli values of samples C₁Cur and C₂Cur decreased with the increase of temperature, showing various structural strengths. in vitro release experiment was used to study the drug release kinetics. The release of curcumin from LLC conformed to the concentration diffusion model. Due to a similar a_S, the release of curcumin from samples A₁Cur, B₁Cur, and C₁Cur (Brij 97-Tween 40/OLA/H₂O = 50.0/2.8/47.2 and the MS is MS₈₂, MS₆₄, and MS₄₆) showed a similar release behavior under different MS. The release behavior of curcumin was related to the structure of samples C₁Cur and C₂Cur at different temperatures. Curcumin exhibited the fastest release rate when the samples behaved as the micellar phase.