The Interactive Effects of Transgenically Overexpressed 1Ax1 with Various HMW-GS Combinations on Dough Quality by Introgression of Exogenous Subunits into an Elite Chinese Wheat Variety

Seed storage proteins in wheat endosperm, particularly high-molecular-weight glutenin subunits (HMW-GS), are primary determinants of dough properties, and affect both end-use quality and grain utilization of wheat (Triticum aestivum L). In order to investigate the interactive effects between the transgenically overexpressed 1Ax1 subunit with different HMW-GS on dough quality traits, we developed a set of 8 introgression lines (ILs) overexpressing the transgenic HMW-glutenin subunit 1Ax1 by introgression of this transgene from transgenic line B102-1-2/1 into an elite Chinese wheat variety Chuanmai107 (C107), using conventional crossing and backcrossing breeding technique. The donor C107 strain lacks 1Ax1 but contains the HMW-GS pairs 1Dx2+1Dy12 and 1Bx7+1By9. The resultant ILs showed robust and stable expression of 1Ax1 even after five generations of self-pollination, and crossing/backcrossing three times. In addition, overexpression of 1Ax1 was compensated by the endogenous gluten proteins. All ILs exhibited superior agronomic performance when compared to the transgenic parent line, B102-1-2/1. Mixograph results demonstrated that overexpressed 1Ax1 significantly improved dough strength, resistance to extension and over-mixing tolerance, in the targeted wheat cultivar C107. Further, comparisons among the ILs showed the interactive effects of endogenous subunits on dough properties when 1Ax1 was overexpressed: subunit pair 17+18 contributed to increased over-mixing tolerance of the dough; expression of the Glu-D1 allele maintained an appropriate balance between x-type and y-type subunits and thereby improved dough quality. It is consistent with ILs C4 (HMW-GS are 1, 17+18, 2+12) had the highest gluten index and Zeleny sedimentation value. This study demonstrates that wheat quality could be improved by using transgenic wheat overexpressing HMW-GS and the feasibility of using such transgenic lines in wheat quality breeding programs.     

Dissection of the TssB-TssC Interface during Type VI Secretion Sheath Complex Formation

The Type VI secretion system (T6SS) is a versatile machine that delivers toxins into either eukaryotic or bacterial cells. At a molecular level, the T6SS is composed of a membrane complex that anchors a long cytoplasmic tubular structure to the cell envelope. This structure is thought to resemble the tail of contractile bacteriophages. It is composed of the Hcp protein that assembles into hexameric rings stacked onto each other to form a tube similar to the phage tail tube. This tube is proposed to be wrapped by a structure called the sheath, composed of two proteins, TssB and TssC. It has been shown using fluorescence microscopy that the TssB and TssC proteins assemble into a tubular structure that cycles between long and short conformations suggesting that, similarly to the bacteriophage sheath, the T6SS sheath undergoes elongation and contraction events. The TssB and TssC proteins have been shown to interact and a specific α-helix of TssB is required for this interaction. Here, we confirm that the TssB and TssC proteins interact in enteroaggregative E. coli. We further show that this interaction requires the N-terminal region of TssC and the conserved α-helix of TssB. Using site-directed mutagenesis coupled to phenotypic analyses, we demonstrate that an hydrophobic motif located in the N-terminal region of this helix is required for interaction with TssC, sheath assembly and T6SS function.     

Acid Ceramidase Promotes Nuclear Export of PTEN through Sphingosine 1-Phosphate Mediated Akt Signaling

The tumor suppressor PTEN is now understood to regulate cellular processes at the cytoplasmic membrane, where it classically regulates PI3K signaling, as well as in the nucleus where multiple roles in controlling cell cycle and genome stability have been elucidated. Mechanisms that dictate nuclear import and, less extensively, nuclear export of PTEN have been described, however the relevance of these processes in disease states, particularly cancer, remain largely unknown. We investigated the impact of acid ceramidase on the nuclear-cytoplasmic trafficking of PTEN. Immunohistochemical analysis of a human prostate tissue microarray revealed that nuclear PTEN was lost in patients whose tumors had elevated acid ceramidase. We found that acid ceramidase promotes a reduction in nuclear PTEN that is dependent upon sphingosine 1-phosphate-mediated activation of Akt. We were further able to show that sphingosine 1-phosphate promotes formation of a complex between Crm1 and PTEN, and that leptomycin B prevents acid ceramidase and sphingosine 1-phosphate mediated loss of nuclear PTEN, suggesting an active exportin-mediated event. To investigate whether the tumor promoting aspects of acid ceramidase in prostate cancer depend upon its ability to export PTEN from the nucleus, we used enforced nuclear expression of PTEN to study docetaxel-induced apoptosis and cell killing, proliferation, and xenoengraftment. Interestingly, while acid ceramidase was able to protect cells expressing wild type PTEN from docetaxel, promote proliferation and xenoengraftment, acid ceramidase had no impact in cells expressing PTEN-NLS. These findings suggest that acid ceramidase, through sphingosine 1-phosphate, promotes nuclear export of PTEN as a means of promoting tumor formation, cell proliferation, and resistance to therapy.     

Mechanistic insights into RNase L through use of an MDMX-derived multi-functional protein domain

RNase L is part of the innate immune response to viral infection. It is activated by a small oligonucleotide (2–5A) whose synthesis is initiated as part of the interferon response. Binding of 2–5A to the N-terminal regulatory region, the ANK domain, of RNase L activates its ribonuclease activity and results in cleavage of RNA in the cell, which ultimately leads to apoptosis of the infected cell. The mechanism by which 2–5A activates the ribonuclease activity of RNase L is currently unclear but 2–5A has been shown to induce dimerization of RNase L. To investigate the importance of dimerization of RNase L, we developed a 15 kDa dimerization-inducing protein domain that was fused to the N-terminus of RNase L. From these studies we provide direct evidence that dimerization of RNase L occurs at physiologically relevant protein concentrations and correlates with activation of ribonuclease activity. We also show that the binding of 2–5A to RNase L promotes dimerization of the ANK domain and suggest how this could transmit a signal to the rest of the protein to activate ribonuclease activity. Finally, we show that the dimerization-inducing domain can be used as a general fusion partner to aid in protein expression and purification.     

Overexpression of sema3a in myocardial infarction border zone decreases vulnerability of ventricular tachycardia post‐myocardial infarction in rats

The expression of the chemorepellent Sema3a is inversely related to sympathetic innervation. We investigated whether overexpression of Sema3a in the myocardial infarction (MI) border zone could attenuate sympathetic hyper‐innervation and decrease the vulnerability to malignant ventricular tachyarrhythmia (VT) in rats. Survived MI rats were randomized to phosphate buffered saline (PBS, n = 12); mock lentivirus (MLV, n = 13) and lentivirus‐mediated overexpression of Sema3a (SLV, n = 13) groups. Sham‐operated rats served as control group (CON, n = 20). Cardiac function and electrophysiological study (PES) were performed at 1 week later. Blood and tissue samples were collected for histological analysis, epinephrine (EPI), growth‐associated factor 43 (GAP43) and tyrosine hydroxylase (TH) measurements. QTc intervals were significantly shorter in SLV group than in PBS and MLV groups (168.6 ± 7.8 vs. 178.1 ± 9.5 and 180.9 ± 8.2 ms, all P < 0.01). Inducibility of VT by PES was significantly lower in the SLV group [30.8% (4/13)] than in PBS [66.7% (8/12)] and MLV [61.5% (8/13)] groups (P < 0.05). mRNA and protein expressions of Sema3a were significantly higher and the protein expression of GAP43 and TH was significantly lower at 7 days after transduction in SLV group compared with PBS, MLV and CON groups. Myocardial EPI in the border zone was also significantly lower in SLV group than in PBS and MLV group (8.73 ± 1.30 vs. 11.94 ± 1.71 and 12.24 ± 1.54 μg/g protein, P < 0.001). Overexpression of Sema3a in MI border zone could reduce the inducibility of ventricular arrhythmias by reducing sympathetic hyper‐reinnervation after infarction.     

Epigenetic silencing of the WNT antagonist Dickkopf 3 disrupts normal Wnt/β‐catenin signalling and apoptosis regulation in breast cancer cells

Dickkopf‐related protein 3 (DKK3) is an antagonist of Wnt ligand activity. Reduced DKK3 expression has been reported in various types of cancers, but its functions and related molecular mechanisms in breast tumorigenesis remain unclear. We examined the expression and promoter methylation of DKK3 in 10 breast cancer cell lines, 96 primary breast tumours, 43 paired surgical margin tissues and 16 normal breast tissues. DKK3 was frequently silenced in breast cell lines (5/10) by promoter methylation, compared with human normal mammary epithelial cells and tissues. DKK3 methylation was detected in 78% of breast tumour samples, whereas only rarely methylated in normal breast and surgical margin tissues, suggesting tumour‐specific methylation of DKK3 in breast cancer. Ectopic expression of DKK3 suppressed cell colony formation through inducing G0/G1 cell cycle arrest and apoptosis of breast tumour cells. DKK3 also induced changes of cell morphology, and inhibited breast tumour cell migration through reversing epithelial‐mesenchymal transition (EMT) and down‐regulating stem cell markers. DKK3 inhibited canonical Wnt/β‐catenin signalling through mediating β‐catenin translocation from nucleus to cytoplasm and membrane, along with reduced active‐β‐catenin, further activating non‐canonical JNK signalling. Thus, our findings demonstrate that DKK3 could function as a tumour suppressor through inducing apoptosis and regulating Wnt signalling during breast tumorigenesis.     

First-principles study of the structural transformation, electronic structure, and optical properties of crystalline 2,6-diamino-3,5-dinitropyrazine-1-oxide under high pressure

Periodic first-principles calculations have been performed to study the effect of high pressure on the geometric, electronic, and absorption properties of 2,6-diamino-3,5-dinitropyrazine-1-oxide (LLM-105) under hydrostatic pressures of 0–50 GPa. Obvious irregular changes in lattice constants, unit-cell angles, bond lengths, bond angles, and band gaps showed that crystalline LLM-105 undergoes four structural transformations at 8, 17, 25, and 42 GPa, respectively. The intramolecular H-bonds were strong at pressures of 0–41 GPa but weakened in the range 42–50 GPa. The lengths of the intermolecular H-bonds (<1.47 Å) indicated that these H-bonds have covalent character and tend to induce the formation of a new twelve-membered ring. Analysis of the DOS showed that the interactions between electrons, especially the valence electrons, strengthen under the influence of pressure. The p states play a very important role in chemical reactions of LLM-105. The absorption spectrum of LLM-105 displayed more bands—as well as stronger bands—in the fundamental absorption region when the pressure was high rather than low. A new absorption peak due to O–H stretching appeared at 18.3 eV above 40 GPa, indicating that covalent O–H bonds and a new twelve-membered ring are present in LLM-105.     

Naphtho‐γ‐pyrones from Endophyte Aspergillus niger Occurring in the Liverwort Heteroscyphus tener (Steph.) Schiffn.

Bioactivity‐guided fractionation of the cytotoxic extract of Aspergillus niger, an endophytic fungus from the Chinese liverwort Heteroscyphus tener (Steph .) Schiffn ., afforded five new naphtho‐γ‐pyrones, rubrofusarin‐6‐O‐α‐D ‐ribofuranoside ( 1 ), (R)‐10‐(3‐succinimidyl)‐TMC‐256A1 ( 2 ), asperpyrone E ( 3 ), isoaurasperone A ( 4 ), and isoaurasperone F ( 5 ), as well as four known ones, dianhydroaurasperone C ( 6 ), aurasperone D ( 7 ), asperpyrone D ( 8 ), and asperpyrone A ( 9 ), together with a cytotoxic cyclic pentapeptide, malformin A₁ ( 10 ). Their structures were determined by extensive spectroscopic analysis. The absolute configurations of dimeric naphtho‐γ‐pyrones 3 – 9 were also determined by analysis of their respective CD spectra.     

Sandwich‐Type Microporous Carbon Nanosheets for Enhanced Supercapacitor Performance

Sandwich‐type microporous hybrid carbon nanosheets (MHCN) consisting of graphene and microporous carbon layers are fabricated using graphene oxides as shape‐directing agent and the in‐situ formed poly(benzoxazine‐co‐resol) as carbon precursor. The reaction and condensation can be readily completed within 45 min. The obtained MHCN has a high density of accessible micropores that reside in the porous carbon with controlled thickness (e.g., 17 nm), a high surface area of 1293 m² g^?1 and a narrow pore size distribution of ca. 0.8 nm. These features allow an easy access, a rapid diffusion and a high loading of charged ions, which outperform the diffusion rate in bulk carbon and are highly efficient for an increased double‐layer capacitance. Meanwhile, the uniform graphene percolating in the interconnected MHCN forms the bulk conductive networks and their electrical conductivity can be up to 120 S m^?1 at the graphene percolation threshold of 2.0 wt.%. The best‐practice two‐electrode test demonstrates that the MHCN show a gravimetric capacitance of high up to 103 F g^?1 and a good energy density of ca. 22.4 Wh kg^?1 at a high current density of 5 A g^?1. These advanced properties ensure the MHCN a great promise as an electrode material for supercapacitors.     

5-羟色胺受体基因多态性位点与海洛因依赖的关联性研究

目的：5-HT（5-hydroxytryptamine,5-HT）参与了多种中枢神经活动的生理过程,其功能异常可以影响很多行为障碍,已有研究显示,5-HT水平与多种精神疾病密切相关。5-HT受体及其转运体基因在海洛因依赖发生发展中起到了重要的作用,是海洛因依赖的主要候选基因。探讨5羟色胺2A受体（Serotonin 2A receptor,HTR2A）基因启动子区-1438A/G（rs6311）、外显子区102T/C（rs6313）与5羟色胺1B受体（Serotonin 1B receptor,HTR1B）基因外显子区861G/C（rs6296）3个单核苷酸多态性和海洛因依赖的关联性分析。方法：严格按照诊断标准,选取无亲缘关系的海洛因依赖个体616例及健康个体600例提取基因组DNA,采用PCR-RFLP方法检测rs6311、rs6313和rs6296 3个SNPs位点的基因型频率,采用SPSS16.0软件分析各位点等位基因、基因型频率在病例-对照组间差异。结果：HTR2A基因rs6311和HTR1B基因rs6296位点的等位基因及基因型频率分布在2组间存在统计学差异（P〈0.05）,病例组rs6311位点的等位基因A频率显著高于对照组（X2=5.436,P=0.020,OR=1.208,CI=1.031~1.417）,rs6296位点的等位基因C频率显著高于对照组（X2=12.116,P=0.000,OR=1.329,CI=1.132~1.560）。连锁不平衡检验结果显示,HTR2A基因rs6311、rs6313位点处于不连锁状态,D＇〈0.5。结论：HTR2A基因rs6311和HTR1B基因rs6296多态性可能与海洛因成瘾有关,携带有rs6311 A等位基因与rs6296 C等位基因的人可能更容易对海洛因产生依赖。我们的研究为海洛因依赖易感人群筛选及药物靶向治疗提供了理论依据。