Recent patents on oligonucleotide synthesis and gene synthesis

Gene synthesis is an emerging field which has widespread implications in synthetic biology and molecular biology. The field is constantly evolving which has led to key advances in oligonucleotide synthesis and gene synthesis technologies, with simplicity, cost effectiveness and high throughput. The miniaturization, multiplexing, microfluidic processing and the integrated microchip engineering will drive down cost and increase productivity without compromising DNA synthesis fidelity, whereas the gigantic amount of genome information provides infinite source of DNA elements and genes as raw material for synthetic biology. This article describes some of the recent patents on oligonucleotide synthesis and gene synthesis.     

葡萄糖淀粉酶在高压静电纺PEI/PVA纳米纤维膜上的离子吸附固定

旨在应用离子结合法将葡萄糖淀粉酶固定在PEI/PVA纳米纤维膜上并对其理化性质进行研究。采用高压静电纺丝技术制备聚乙烯亚胺(PEI)/聚乙烯醇(PVA)纳米纤维膜,采用热交联方法使其具备水稳定性后,再利用离子吸附法固定葡萄糖淀粉酶。结果显示,利用红外光谱(FT-IR)表征固定有葡萄糖淀粉酶的PEI/PVA纳米纤维膜,表明葡萄糖淀粉酶可成功固定在静电纺丝形成的PEI/PVA纳米纤维膜表面。通过固定化葡萄糖淀粉酶的酶学性质鉴定,发现固定化葡萄糖淀粉酶的最适反应温度为65℃,比游离的葡萄糖淀粉酶提高了6℃;固定化葡萄糖淀粉酶的适用p H值范围明显变宽;热稳定性和存贮稳定性显著增强且可以重复使用。利用离子吸附法能简便地将蛋白质分子固定于纳米纤维膜上,具有一定的应用前景。     

Rapid, large-scale generation of Ds transposant lines and analysis of the Ds insertion sites in rice

Rapid, large-scale generation of a Ds transposant population was achieved using a regeneration procedure involving tissue culture of seed-derived calli carrying Ac and inactive Ds elements. In the F(2) progeny from genetic crosses between the same Ds and Ac starter lines, most of the crosses produced an independent germinal transposition frequency of 10-20%. Also, many Ds elements underwent immobilization even though Ac was expressed. By comparison, in a callus-derived regenerated population, over 70% of plants carried independent Ds insertions, indicating transposition early in callus formation. In the remaining population, the majority of plants carried only Ac. Most of the new Ds insertions were stably transmitted to a subsequent generation. An exceptionally high proportion of independent transposants in the regenerated population means that selection markers for transposed Ds and continual monitoring of Ac/Ds activities may not necessarily be required. By analyzing 1297 Ds-flanking DNA sequences, a genetic map of 1072 Ds insertion sites was developed. The map showed that Ds elements were transposed onto all of the rice chromosomes, with preference not only near donor sites (36%) but also on certain physically unlinked arms. Populations from both genetic crossing and tissue culture showed the same distribution patterns of Ds insertion sites. The information of these mapped Ds insertion sites was deposited in GenBank. Among them, 55% of Ds elements were on predicted open-reading frame (ORF) regions. Thus, we propose an optimal strategy for the rapid generation of a large population of Ds transposants in rice.     

Tissue Binding Patterns and In Vitro Effects of <Emphasis Type=Italic>Campylobacter jejuni</Emphasis> DNA-Binding Protein from Starved Cells

Campylobacter jejuni (C. jejuni) is frequently associated with axonal Guillain-Barré syndrome (GBS). We reported that C. jejuni DNA-binding protein from starved cells (C-Dps) binds to and damages myelinated nerves in vivo. We studied the binding patterns of C-Dps to nervous tissues and its in vitro effects on neural cells. Immunohistochemically, C-Dps labeled the nodes of Ranvier, the outermost parts of internodal myelin and the basement membrane in the peripheral nerves, and neurons and myelin in the central nervous tissues. Its binding was blocked by sulfatide. C-Dps bound to the cell surfaces of nerve growth factor (NGF)-treated PC12 cells leading to dose-dependent LDH release, which was inhibited by either heat-denaturation of C-Dps or coincubation with an anti-C-Dps mAb. However, its binding to the surfaces of cultured NSC34 cells, S16 cells, or dorsal root ganglion cells, did not induce cytotoxicity. These findings suggest a possible involvement of C-Dps in C. jejuni-related GBS.     

Benzylisoquinoline alkaloids from the tubers of Corydalis ternata and their cytotoxicity

Chemical investigation of the tubers of Corydalis ternata resulted in the isolation and characterization of four new benzylisoquinoline alkaloids, epi-coryximine (1) and coryternatines A–C (2–4), along with 10 known alkaloids (5–14). Their structures were established on the basis of extensive spectroscopic data analyses and comparison with spectroscopic data reported. In addition, the cytotoxicities of the alkaloids (1–14) were evaluated by determining their inhibitory effects on several human tumor cell lines (A549, SK-OV-3, SK-MEL-2, and HCT-15) using the SRB assay. Compound 8 showed significant cytotoxicity against A549, SK-OV-3, SK-MEL-2, and HCT-15 cell lines (IC₅₀ = 8.34, 5.14, 7.87, and 2.86 μM, respectively). The four new compounds (1–4) exhibited selective cytotoxicity against the HCT-15 cell line.     

Change in terrestrial ecosystem water‐use efficiency over the last three decades

Defined as the ratio between gross primary productivity (GPP) and evapotranspiration (ET), ecosystem‐scale water‐use efficiency (EWUE) is an indicator of the adjustment of vegetation photosynthesis to water loss. The processes controlling EWUE are complex and reflect both a slow evolution of plants and plant communities as well as fast adjustments of ecosystem functioning to changes of limiting resources. In this study, we investigated EWUE trends from 1982 to 2008 using data‐driven models derived from satellite observations and process‐oriented carbon cycle models. Our findings suggest positive EWUE trends of 0.0056, 0.0007 and 0.0001 g C m^?2 mm^?1 yr^?1 under the single effect of rising CO₂ (‘CO₂’), climate change (‘CLIM’) and nitrogen deposition (‘NDEP’), respectively. Global patterns of EWUE trends under different scenarios suggest that (i) EWUE‐CO₂ shows global increases, (ii) EWUE‐CLIM increases in mainly high latitudes and decreases at middle and low latitudes, (iii) EWUE‐NDEP displays slight increasing trends except in west Siberia, eastern Europe, parts of North America and central Amazonia. The data‐driven MTE model, however, shows a slight decline of EWUE during the same period (?0.0005 g C m^?2 mm^?1 yr^?1), which differs from process‐model (0.0064 g C m^?2 mm^?1 yr^?1) simulations with all drivers taken into account. We attribute this discrepancy to the fact that the nonmodeled physiological effects of elevated CO₂ reducing stomatal conductance and transpiration (TR) in the MTE model. Partial correlation analysis between EWUE and climate drivers shows similar responses to climatic variables with the data‐driven model and the process‐oriented models across different ecosystems. Change in water‐use efficiency defined from transpiration‐based WUE_t (GPP/TR) and inherent water‐use efficiency (IWUE_t, GPP×VPD/TR) in response to rising CO₂, climate change, and nitrogen deposition are also discussed. Our analyses will facilitate mechanistic understanding of the carbon–water interactions over terrestrial ecosystems under global change.     

Phosphocreatine protects against LPS-induced human umbilical vein endothelial cell apoptosis by regulating mitochondrial oxidative phosphorylation

Phosphocreatine (PCr) is an exogenous energy substance, which provides phosphate groups for adenosine triphosphate (ATP) cycle and promotes energy metabolism in cells. However, it is still unclear whether PCr has influenced on mitochondrial energy metabolism as well as oxidative phosphorylation (OXPHO) in previous studies. Therefore, the aim of the present study was to investigate the regulation of PCr on lipopolsaccharide (LPS)-induced human umbilical vein endothelial cells (HUVECs) and mitochondrial OXPHO pathway. PCr protected HUVECs against LPS-induced apoptosis by suppressing the mitochondrial permeability transition, cytosolic release of cytochrome c (Cyt C), Ca²⁺, reactive oxygen species and subsequent activation of caspases, and increasing Bcl₂ expression, while suppressing Bax expression. More importantly, PCr significantly improved mitochondrial swelling and membrane potential, enhanced the activities of ATP synthase and mitochondrial creatine kinase (CK_mt) in creatine shuttle, influenced on respiratory chain enzymes, respiratory control ratio, phosphorus/oxygen ratio and ATP production of OXPHO. Above PCr-mediated mitochondrial events were effectively more favorable to reduced form of flavin adenine dinucleotide (FADH₂) pathway than reduced form of nicotinamide-adenine dinucleotid pathway in the mitochondrial respiratory chain. Our results revealed that PCr protects against LPS-induced HUVECs apoptosis, which probably related to stabilization of intracellular energy metabolism, especially for FADH₂ pathway in mitochondrial respiratory chain, ATP synthase and CK_mt. Our findings suggest that PCr may play a certain role in the treatment of atherosclerosis via protecting endothelial cell function.     

Zn‐doped CaTiO3:Eu3+ red phosphors for enhanced photoluminescence in white LEDs by solid‐state reaction

Zn‐doped CaTiO₃:Eu³⁺ red phosphors for enhanced photoluminescence in white light‐emitting diodes (LEDs) were synthesized by a solid‐state method. The structure and morphology of the obtained phosphor samples were observed by X‐ray diffraction (XRD) and scanning electron microscopy (SEM), and the impact of Ca, Zn and Eu content on their photoluminescence properties was studied. The results indicated that Zn not only participates in the formation of defects in suitable lattice matrices but also has a role in flux in the transformation from ZnO to Zn₂TiO₄, which is beneficial for the enhancement of photoluminescence properties. Photoluminescence test data showed that the Zn‐doped phosphor is excited efficiently by near‐ultraviolet (NUV) light at wavelengths around 398 nm and emits an intense red light with a broad peak around 616 nm corresponding to the ⁵D₀→⁷F₂ transition of Eu³⁺. The intensity of this phosphor emission is three times stronger than that without Zn‐doping. Furthermore, this phosphor has very good thermal stability, high color purity and a low sintered temperature, all of which suggest its potential as a promising red phosphor for white LEDs. Copyright © 2015 John Wiley & Sons, Ltd.     

Cytoplasmic Fragment of Alcadein α Generated by Regulated Intramembrane Proteolysis Enhances Amyloid β-Protein Precursor (APP) Transport into the Late Secretory Pathway and Facilitates APP Cleavage

The neural type I membrane protein Alcadein α (Alcα), is primarily cleaved by amyloid β-protein precursor (APP) α-secretase to generate a membrane-associated carboxyl-terminal fragment (Alcα CTF), which is further cleaved by γ-secretase to secrete p3-Alcα peptides and generate an intracellular cytoplasmic domain fragment (Alcα ICD) in the late secretory pathway. By association with the neural adaptor protein X11L (X11-like), Alcα and APP form a ternary complex that suppresses the cleavage of both Alcα and APP by regulating the transport of these membrane proteins into the late secretory pathway where secretases are active. However, it has not been revealed how Alcα and APP are directed from the ternary complex formed largely in the Golgi into the late secretory pathway to reach a nerve terminus. Using a novel transgenic mouse line expressing excess amounts of human Alcα CTF (hAlcα CTF) in neurons, we found that expression of hAlcα CTF induced excess production of hAlcα ICD, which facilitated APP transport into the nerve terminus and enhanced APP metabolism, including Aβ generation. In vitro cell studies also demonstrated that excess expression of Alcα ICD released both APP and Alcα from the ternary complex. These results indicate that regulated intramembrane proteolysis of Alcα by γ-secretase regulates APP trafficking and the production of Aβ in vivo.