Soluble Prokaryotic Overexpression and Purification of Human GM-CSF Using the Protein Disulfide Isomerase b′a′ Domain

Granulocyte-macrophage colony-stimulating factor (GM-CSF) is a member of the colony-stimulating factor (CSF) family, which functions to enhance the proliferation and differentiation of hematopoietic stem cells and other hematopoietic lineages such as neutrophils, dendritic cells, or macrophages. These proteins have thus generated considerable interest in clinical therapy research. A current obstacle to the prokaryotic production of human GM-CSF (hGM-CSF) is its low solubility when overexpressed and subsequent complex refolding processes. In our present study, the solubility of hGM-CSF was examined when combined with three N-terminal fusion tags in five E. coli strains at three different expression temperatures. In the five E. coli strains BL21 (DE3), ClearColi BL21 (DE3), LOBSTR, SHuffle T7 and Origami2 (DE3), the hexahistidine-tagged hGM-CSF showed the best expression but was insoluble in all cases at each examined temperature. Tagging with the maltose-binding protein (MBP) and the b′a′ domain of protein disulfide isomerase (PDIb′a′) greatly improved the soluble overexpression of hGM-CSF at 30 °C and 18 °C. The solubility was not improved using the Origami2 (DE3) and SHuffle T7 strains that have been engineered for disulfide bond formation. Two conventional chromatographic steps were used to purify hGM-CSF from the overexpressed PDIb′a′-hGM-CSF produced in ClearColi BL21 (DE3). In the experiment, 0.65 mg of hGM-CSF was isolated from a 0.5 L flask culture of these E. coli and showed a 98% purity by SDS-PAGE analysis and silver staining. The bioactivity of this purified hGM-CSF was measured at an EC₅₀ of 16.4 ± 2 pM by a CCK8 assay in TF-1 human erythroleukemia cells.     

An Engineered Biliverdin-Compatible Cyanobacteriochrome Enables a Unique Ultrafast Reversible Photoswitching Pathway

Cyanobacteriochromes (CBCRs) are promising optogenetic tools for their diverse absorption properties with a single compact cofactor-binding domain. We previously uncovered the ultrafast reversible photoswitching dynamics of a red/green photoreceptor AnPixJg2, which binds phycocyanobilin (PCB) that is unavailable in mammalian cells. Biliverdin (BV) is a mammalian cofactor with a similar structure to PCB but exhibits redder absorption. To improve the AnPixJg2 feasibility in mammalian applications, AnPixJg2_BV4 with only four mutations has been engineered to incorporate BV. Herein, we implemented femtosecond transient absorption (fs-TA) and ground state femtosecond stimulated Raman spectroscopy (GS-FSRS) to uncover transient electronic dynamics on molecular time scales and key structural motions responsible for the photoconversion of AnPixJg2_BV4 with PCB (Bpcb) and BV (Bbv) cofactors in comparison with the parent AnPixJg2 (Apcb). Bpcb adopts the same photoconversion scheme as Apcb, while BV4 mutations create a less bulky environment around the cofactor D ring that promotes a faster twist. The engineered Bbv employs a reversible clockwise/counterclockwise photoswitching that requires a two-step twist on ~5 and 35 picosecond (ps) time scales. The primary forward P_fr → P_o transition displays equal amplitude weights between the two processes before reaching a conical intersection. In contrast, the primary reverse P_o → P_fr transition shows a 2:1 weight ratio of the ~35 ps over 5 ps component, implying notable changes to the D-ring-twisting pathway. Moreover, we performed pre-resonance GS-FSRS and quantum calculations to identify the Bbv vibrational marker bands at ~659,797, and 1225 cm⁻¹. These modes reveal a stronger H-bonding network around the BV cofactor A ring with BV4 mutations, corroborating the D-ring-dominant reversible photoswitching pathway in the excited state. Implementation of BV4 mutations in other PCB-binding GAF domains like AnPixJg4, AM1_1870g3, and NpF2164g5 could promote similar efficient reversible photoswitching for more directional bioimaging and optogenetic applications, and inspire other bioengineering advances.     

鞍钢鲅鱼圈4038m3高炉高比例使用中块焦炭生产实践

针对鞍钢股份有限公司鲅鱼圈钢铁分公司4038 m3高炉高比例使用中块焦炭后出现的风量萎缩、顺行变差,尤其是高炉休送风恢复过程中炉缸活跃性差、煤气流中心通道打不开、频繁崩滑料现象,提出采取合理匹配上下部调剂、组织好炉前渣铁排放、小焦布料制度以及改善休风料结构等措施.实践后,高炉实现了中块焦比为90 kg/t情况下的长期稳定顺行,休送风恢复未出现超时现象,适应了高比例使用中块焦炭的生产.     

LM339在数控电路中的应用

本文介绍了LM339在数控电路中的应用，并对其电路进行了分析。     

ON THE RAPIDLY SOLIDIFIED Cu-Zn-Al SHAPE MEMORY ALLOY

采用快速凝固工艺制取了不同规格的Cu-24.9wt-％Zn-4.5wt-％AJ合金带材、并通过应力-应变曲线、电阻-温度曲线、光学金相、X射线衍射、扫描电镜和透射电镜等方法研究了该合金的形状记忆性能、组织结构特征及热处理的影响。结果表明:合金喷带原始组织是单一的β细晶(晶粒尺寸约20μm);经720℃10min水淬处理后晶粒长大到100μm左右,室温下为(β+M)组织时,可以获得最佳的形状记忆性能,且避免了粗晶脆化;在应变值ε≤8％时能实现完全的记忆回复;加载变形过程中,样品表面形成的马氏体和晶粒状浮雕具有可逆性。     

复杂零件半固态压铸充型过程的计算机仿真

使用二次开发的ANSYS有限元软件，对铝合金半固态压铸复杂件的充型与模具内浇道形貌之间关系进行详细的计算机仿真。首先对仿真模型进行了合理的简化处理，然后分别模拟和分析了压铸速度及浆料粘度等主要工艺参数对压铸件质量的影响，进而对半固态模具的内浇道进行改进。X射线探伤证实，铝合金半固态压铸造模具的内浇道是影响其性能的关键因素，同时也表明，文中提出的简化仿真模型可以较好地仿真铝合金复杂件半固态压铸造过程。     

Stage-efficiency of centrifugal extractor used in nuclear industry

1　INTRODUCTIONComparedwithamixer settlerorpulsedcolumnusedinthenuclearfuelreprocessingextraction pro cesses,ashort residence timecentrifugalextractorcatchesresearcher seyesbecauseofthe goodphaseseparationandasmallhold upvolume .Itshowsthefollowingadvantagesinthenuclearindustry[1] :1)Reducingsolventdegradationduetoradiation ;2 )Reducinginventoryofaqueousandorganicphases ,whichgivesrapidstart upandshut down ;3)Com pactdesignthatcontributestothereductionofplantcost .Manydifferenttypesofcen…     

Strengthening technology and mechanism for semi-solid die casting of aluminum alloy

Combined with theoretical evaluation,an optimized strengthening process for the semi-solid die castings of A356 aluminum alloy was obtained by studying the mechanical properties of castings solution treated and aged under different conditions in detail,then,the semi-solid die castings and liquid die castings were heat treated with the optimized process.The results show that the mechanical properties of semi-solid die castings of aluminum alloy are superior to those of the liquid die castings,especially the strengthening degree of heat treated semi-solid die castingsis much greater than that of liquid die castings with the tensile strength more than 330 MPa and the elongation more than 10％,and this is mainly contributed to the non-dendritic and more compact microstructure of semi-solid die castings.The strengthening mechanism of heat treatment for the semi-solid die castings of A356 aluminum alloy is due to the dispersive precipitation of the second phase(Mg2 Si) and formation of GP Zone.     

Co和Cu薄膜电学特性和连续性特征的判据

采用离子束溅射沉积了不同厚度的Co膜和Cu膜,利用四电极法测量了薄膜的电阻率,从而得到了Co膜和Cu膜的电导率随薄膜厚度的变化关系。实验结果表明,Co膜和Cu膜的电学特性都具有明显的尺寸效应。比较了同时考虑表面散射和晶界散射的电导理论得到的电导率公式与实验结果,不同薄膜厚度电导率的理论结果与实验结果符合较好。提出了厚度作为金属薄膜生长从不连续膜进入连续膜的一个特征判据,并利用原子力显微镜(AFM)观测了膜厚在特征厚度附近的Co膜和Cu膜的表面形貌。     

取代水杨醛Schiff碱金属配合物催化氧化脱硫性能

以水杨醛类Schiff碱为配体,与Co(NO_3)_2、Cu(NO_3)_2反应合成6种Salen(M)型配合物Ⅰ～Ⅵ。以1-己硫醇、二丁基硫醚和2-甲基噻吩为模型化合物配制模拟油体系,考察了配合物Ⅰ～Ⅵ的催化氧化脱硫性能,并分析了配合物结构与氧化脱硫性能的关系。结果表明,6种配合物在75 min时的总脱硫效果为Ⅵ>Ⅴ>Ⅳ>Ⅰ>Ⅲ>Ⅱ,Ⅵ的总脱硫率为31.9%。对1-己硫醇及二丁基硫醚脱除效果最佳的是Ⅴ,脱除率分别为74.2%和65.1%;对2-甲基噻吩脱除效果最好的是Ⅰ,脱除率为26.8%。构效关系研究表明,中心金属离子与O_2的配位能力越强,配体的共轭体系越大、电子云密度越高,配合物的脱硫性能越好;通过IR和离子色谱对单一硫化物模拟油体系氧化前后的产物进行分析发现,硫化物氧化后皆生成相应的砜类或亚砜类,且1-己硫醇和二丁基硫醚被进一步氧化生成SO_3~(2–)或SO_4~(2–)。