聚多曲霉菌原生质体和液泡的制备及优化

原生质体的大量制备是研究原生质体转化、诱变、融合等技术的关键,液泡的制备对研究液泡中分解、转运有机物的特性具有重要意义,但目前分离技术还不够成熟.本研究从聚多曲霉菌菌丝中分离原生质体和液泡并对分离条件进行优化,以聚多曲霉菌DJ515-2菌丝为材料,探索不同因素对原生质体和液泡制备分离效果的影响.结果表明,聚多曲霉菌DJ515-2在菌龄42 h,以3％纤维素酶、1％蜗牛酶和3％的溶壁酶组成复合酶液,25℃下酶解4 h,原生质体达到最大产量,为5.167×105个/mL.同时,在此基础上裂解液泡的最优条件为pH7.5、1.0mol/L KCl、0.020％Triton X-10,其产量达2.63×105个/mL,产率为原生质体的50.8％,相比采用多元碱化合物诱导真菌原生质体裂解释放液泡的产率增加了 40％～45％.本研究为真菌和植物的各种原生质体技术及基于亚细胞层面的研究提供了材料基础.     

Implementing Extended Producer Responsibility Legislation

The goal of this article is to contribute to the understanding of how the multiple, and sometimes conflicting, stakeholder perspectives and prevailing conditions (economic, geographic, etc.) in the implementation locality shape extended producer responsibility (EPR) “on the ground.” We provide an in‐depth examination of the implementation dimension of EPR in a specific case study by examining concrete activities at the operational front of the collection and recycling system, and probing the varying stakeholder preferences that have driven a specific system to its status quo. To this end, we conduct a detailed case study of the Washington State EPR implementation for electronic waste. We provide an overview of various stakeholder perspectives and their implications for the attainment of EPR policy objectives in practice. These findings shed light on the intrinsic complexity of EPR implementation. We conclude with recommendations on how to achieve effective and efficient EPR implementation, including improving design incentives, incorporating reuse and refurbishing, expanding product scope, managing downstream material flows, and promoting operational efficiency via fair cost allocation design.     

MicroRNA-21 in Pancreatic Ductal Adenocarcinoma Tumor-Associated Fibroblasts Promotes Metastasis

Introduction

Pancreatic ductal adenocarcinoma (PDAC) is projected to rise to the second leading cause of U.S. cancer-related deaths by 2020. Novel therapeutic targets are desperately needed. MicroRNAs (miRs) are small noncoding RNAs that function by suppressing gene expression and are dysregulated in cancer. miR-21 is overexpressed in PDAC tumor cells (TC) and is associated with decreased survival, chemoresistance and invasion. Dysregulation of miR regulatory networks in PDAC tumor-associated fibroblasts (TAFs) have not been previously described. In this study, we show that miR-21 expression in TAFs promotes TC invasion.

Methods

In-situ hybridization for miR-21 was performed on the 153 PDAC patient UCLA tissue microarray and 23 patient-matched lymph node metastases. Stromal and TC histoscores were correlated with clinicopathologic parameters by univariate and multivariate Cox regression. miR-21 positive cells were further characterized by immunofluorescence for mesenchymal/epithelial markers. For in vitro studies, TAFs were isolated from freshly resected human PDAC tumors by the outgrowth method. miR-21 was overexpressed/inhibited in fibroblasts and then co-cultured with GFP-MiaPaCa TCs to assess TC invasion in modified Boyden chambers.

Results

miR-21 was upregulated in TAFs of 78% of tumors, and high miR-21 significantly correlated with decreased overall survival (P = 0.04). Stromal miR-21 expression was also significantly associated with lymph node invasion (P = 0.004), suggesting that it is driving TC spread. Co-immunofluorescence revealed that miR-21 colocalized with peritumoral fibroblasts expressing α-smooth muscle actin. Moreover, expression of miR-21 in primary TAFs correlated with miR-21 in TAFs from patient-matched LN metastases; evidence that PDAC tumor cells induce TAFs to express miR-21. miR-21 expression in TAFs and TCs promotes invasion of TCs and is inhibited with anti-miR-21.

Conclusions

miR-21 expression in PDAC TAFs is associated with decreased overall survival and promotes TC invasion. Anti-miR-21 may represent a novel therapeutic strategy for dual targeting of both tumor and stroma in PDAC.     

Roles of Apolipoprotein E (ApoE) and Inducible Nitric Oxide Synthase (iNOS) in Inflammation and Apoptosis in Preeclampsia Pathogenesis and Progression

Objectives

To investigate potential roles of inducible nitric oxide synthase (iNOS) and apolipoprotein (apoE) in inflammation and apoptosis promoting pathological changes in preeclampsia in pregnant mice with apoE and/or iNOS knock out.

Methods

B6.129 mice were crossed to produce WT, apoE^−/−, apoE^+/−, iNOS^−/−, iNOS^+/− and apoE^−/−iNOS^−/− groups. Variants were confirmed by PCR. Serum lipid parameters (triglycerides, TG; total cholesterol, TC; high density lipoprotein, HDL; and low density lipoprotein, LDL), NO levels and placental electronic microscopic ultrastructures were evaluated, and blood pressure (BP), 24-hour urine protein and pregnancy outcomes were recorded for pregnant F1 generation mice. Placental expressions of inflammatory (tumor necrosis factor-α, TNF-α; interleukin-6, IL-6; nuclear factor-κB, NF-κb) and apoptotic markers (Bcl-2 associated X protein, Bax, B-cell lymphoma/leukemia-2, Bcl-2, and Caspase-3) were evaluated via Western blot.

Results

Serum lipids, BP and 24-hour urine protein levels were shown to be significantly higher and parturition and placenta weights were lower in apoE^−/− and apoE^−/−iNOS^−/− groups (p<0.05). NO levels were lower in the apoE^−/−iNOS^−/− group. In addition, inflammatory/apoptosis parameters, including TNF-α, IL-6, NF-κb, Bax, Bcl-2 and Caspase-3 in the apoE^−/−iNOS^−/− group (p<0.01), as well as in the apoE^−/− group (p<0.05), and NF-κB, Bax in iNOS^−/− group (p<0.05) were higher compared with WT group. However, most of the inflammatory/apoptosis parameters in the iNOS^+/− and the apoE^+/− groups (p>0.05) showed no differences. In addition, placenta vascular endothelial and trophoblast cell morphological changes were demonstrated in both the apoE^−/−iNOS^−/− and apoE^−/− groups.

Conclusion

Elevated lipid metabolism and inflammatory/apoptosis parameters suggest a potentially significant role of apoE in preeclampsia pathology, as well as a relationship between iNOS and preeclampsia progression.     

Cooling Induced Surface Reconstruction during Synthesis of High‐Ni Layered Oxides

Transition metal layered oxides have been the dominant cathodes in lithium‐ion batteries, and among them, high‐Ni ones (LiNi_xMn_yCo_zO₂; x ≥ 0.7) with greatly boosted capacity and reduced cost are of particular interest for large‐scale applications. The high Ni loading, on the other hand, raises the critical issues of surface instability and poor rate performance. The rational design of synthesis leading to layered LiNi_0.7Mn_0.15Co_0.15O₂ with greatly enhanced rate capability is demonstrated, by implementing a quenching process alternative to the general slow cooling. In situ synchrotron X‐ray diffraction, coupled with surface analysis, is applied to studies of the synthesis process, revealing cooling‐induced surface reconstruction involving Li₂CO₃ accumulation, formation of a Li‐deficient layer and Ni reduction at the particle surface. The reconstruction process occurs predominantly at high temperatures (above 350 °C) and is highly cooling‐rate dependent, implying that surface reconstruction can be suppressed through synthetic control, i.e., quenching to improve the surface stability and rate performance of the synthesized materials. These findings may provide guidance to rational synthesis of high‐Ni cathode materials.     

Balanced expression of mitochondrial apoptosis regulatory proteins correlates with long-term survival of cardiac allografts

Abnormal regulation of apoptosis is observed in ischemic injury and may contribute to the pathogenesis of atherosclerosis. However, its role in cardiac allograft vasculopathy (CAV), the fundamental lesion of chronic rejection (CR) in heart transplantation, remains uncertain. To clarify this issue, apoptosis was quantitated in myocardium and coronary arteries from 5 cardiac allograft donors (NL) and explanted hearts of 24 patients with ischemic cardiomyopathy (IsCM) and 15 patients with CR. Tissue samples were analyzed via end-labeling fragmented DNA [via deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL)] and immunoblotting for activated caspase-3 and -9. Myocyte apoptosis assessed by TUNEL was similarly increased over NL (0.21%) in both the CR (0.88%; P < 0.01) and IsCM (0.88%; P < 0.01) groups. Activated caspase-9 levels were significantly higher in CR (14.7%) compared with IsCM (6.9%; P < 0.01) and NL (0%) groups, whereas activated caspase-3 levels were similarly elevated in both CR and IsCM (7.8 and 6.5% vs. 0% in NL; P < 0.01 and P < 0.05) groups. Expression of myocardial Bcl-2 and Bax was increased in CR compared with both NL (Bax, 4.3-fold; P < 0.01; Bcl-2, 5.9-fold; P < 0.01) and IsCM (IsCM: Bax, 2.2-fold; P < 0.05; Bcl-2, 3.2-fold; P < 0.01) groups. The rate of apoptosis and the Bcl-2/Bax ratio independently correlated to graft survival in CR (activation of caspase-9: r = 0.87; P < 0.01; Bcl-2/Bax: r = 0.57; P = 0.05). Compared with native atherosclerosis, coronary arteries with CAV showed more medial apoptosis (7.8-fold; P < 0.01) and higher Bcl-2 levels (5.1-fold; P < 0.01) with lower Bax levels (threefold; P < 0.05) in the intima. These results indicate that abnormal Bcl-2 and Bax expression in myocardium and coronary arteries of cardiac allografts with CR is distinct from that in IsCM and suggest that balancing Bcl-2 to Bax in transplanted hearts promotes long-term graft survival.     

Cardiac matrix metalloproteinase-2 expression independently induces marked ventricular remodeling and systolic dysfunction

Although enhanced cardiac matrix metalloproteinase (MMP)-2 synthesis has been associated with ventricular remodeling and failure, whether MMP-2 expression is a direct mediator of this process is unknown. We generated transgenic mice expressing active MMP-2 driven by the alpha-myosin heavy chain promoter. At 4 mo MMP-2 transgenic hearts demonstrated expression of the MMP-2 transgene, myocyte hypertrophy, breakdown of Z-band registration, lysis of myofilaments, disruption of sarcomere and mitochondrial architecture, and cardiac fibroblast proliferation. Hearts from 8-mo-old transgenic mice displayed extensive myocyte disorganization and dropout with replacement fibrosis and perivascular fibrosis. Older transgenic mice also exhibited a massive increase in cardiac MMP-2 expression, representing recruitment of endogenous MMP-2 synthesis, with associated expression of MMP-9 and membrane type 1 MMP. Increases in diastolic [control (C) 33 +/- 3 vs. MMP 51 +/- 12 microl; P = 0.003] and systolic (C 7 +/- 2 vs. MMP 28 +/- 14 microl; P = 0.003) left ventricular (LV) volumes and relatively preserved stroke volume (C 26 +/- 4 vs. MMP 23 +/- 3 microl; P = 0.16) resulted in markedly decreased LV ejection fraction (C 78 +/- 7% vs. MMP 48 +/- 16%; P = 0.0006). Markedly impaired systolic function in the MMP transgenic mice was demonstrated in the reduced preload-adjusted maximal power (C 240 +/- 84 vs. MMP 78 +/- 49 mW/microl(2); P = 0.0003) and decreased end-systolic pressure-volume relation (C 7.5 +/- 1.5 vs. MMP 4.7 +/- 2.0; P = 0.016). Expression of active MMP-2 is sufficient to induce severe ventricular remodeling and systolic dysfunction in the absence of superimposed injury.     

Salicylic Acid Regulates Sugar Metabolism that Confers Freezing Tolerance in Magnolia wufengensis During Natural Cold Acclimation

Journal of Plant Growth Regulation - To improving the understand of the accumulation pattern of soluble sugars in Magnolia wufengensis during natural cold acclimation, the dynamics of freezing...     

Interface Welding via Thermal Pulse Sintering to Enable 4.6 V Solid-State Batteries

NASICON-type Li_1.3Al_0.3Ti_1.7(PO4)₃ (LATP) is one of the most promising solid-state electrolytes (SSEs) to achieve high-energy-density solid-state batteries (SSBs) due to its high ionic conductivity, high-voltage stability, and low cost. However, its practical application is constrained by inadequate interfacial compatibility with cathode materials and significant incompatibility with lithium metal. In this work, a cost-effective interface welding approach is reported, utilizing an innovative thermal pulse sintering (TPS) to fabricate LATP-based solid-state batteries. Initially, the rapid thermal pulses enhance the ionic conductivity of LATP SSE by inducing selective growth of LATP nanowires, effectively occupying interparticle voids. Additionally, this process results in the formation of a dense layer (GCM) comprising graphene oxide, carbon nanotubes, and MXene with a controlled Li⁺ transport pathway, facilitating lithium stripping and plating processes. Moreover, these thermal pulses facilitate the interfacial fusion between LATP and cathode materials, while avoiding undesired phase diffusion. As a result, SSBs with a LiCoO₂ cathode deliver favorable cycle stability at 4.6 V, marking significant progress. This facile interface welding strategy represents a substantial step toward high-energy-density SSB development.