Transfecting and Nucleofecting Human Induced Pluripotent Stem Cells

Genetic modification is continuing to be an essential tool in studying stem cell biology and in setting forth potential clinical applications of human embryonic stem cells (HESCs)¹. While improvements in several gene delivery methods have been described^2-9, transfection remains a capricious process for HESCs, and has not yet been reported in human induced pluripotent stem cells (iPSCs). In this video, we demonstrate how our lab routinely transfects and nucleofects human iPSCs using plasmid with an enhanced green fluorescence protein (eGFP) reporter. Human iPSCs are adapted and maintained as feeder-free cultures to eliminate the possibility of feeder cell transfection and to allow efficient selection of stable transgenic iPSC clones following transfection. For nucleofection, human iPSCs are pre-treated with ROCK inhibitor¹¹, trypsinized into small clumps of cells, nucleofected and replated on feeders in feeder cell-conditioned medium to enhance cell recovery. Transgene-expressing human iPSCs can be obtained after 6 hours. Antibiotic selection is applied after 24 hours and stable transgenic lines appear within 1 week. Our protocol is robust and reproducible for human iPSC lines without altering pluripotency of these cells.     

A unique latex protein, MLX56, defends mulberry trees from insects

The mulberry (Morus spp.)-silkworm (Bombyx mori) relationship has been a well-known plant-herbivore interaction for thousands of years. Recently, we found that mulberry leaves defend against insect herbivory by latex ingredients. Here we report that a 56-kDa (394 amino acid) defense protein in mulberry latex designated mulatexin (MLX56) with an extensin domain, two hevein-like chitin-binding domains, and an inactive chitinase-like domain provides mulberry trees with strong insect resistance. MLX56 is toxic to lepidopteran caterpillars, including the cabbage armyworm, Mamestra brassicae and the Eri silkworm, Samia ricini, at 0.01% concentration in a wet diet, suggesting that MLX56 is applicable for plant protection. MLX56 is highly resistant to protease digestion, and has a strong chitin-binding activity. Interestingly, MLX56 showed no toxicity to B. mori, suggesting that the mulberry specialist has developed adaptation to the mulberry defense. Our results show that defensive proteins in plant latex play key roles in mulberry-insect interactions, and probably also in other plant-insect interactions. Our results further suggest that plant latexes analogous to animal venom contain a treasury of applicable defense proteins and chemicals that has evolved through inter-specific interactions.     

Homology modeling and molecular dynamics simulations of the active state of the nociceptin receptor reveal new insights into agonist binding and activation

The opioid receptor-like receptor, also known as the nociceptin receptor (NOP), is a class A G protein-coupled receptor (GPCR) in the opioid receptor family. Although NOP shares a significant homology with the other opioid receptors, it does not bind known opioid ligands and has been shown to have a distinct mechanism of activation compared to the closely related opioid receptors mu, delta, and kappa. Previously reported homology models of the NOP receptor, based on the inactive-state GPCR crystal structures, give limited information on the activation and selectivity features of this fourth member of the opioid receptor family. We report here the first active-state homology model of the NOP receptor based on the opsin GPCR crystal structure. An inactive-state homology model of NOP was also built using a multiple template approach. Molecular dynamics simulation of the active-state NOP model and comparison to the inactive-state model suggest that NOP activation involves movements of transmembrane (TM)3 and TM6 and several activation microswitches, consistent with GPCR activation. Docking of the selective nonpeptidic NOP agonist ligand Ro 64-6198 into the active-state model reveals active-site residues in NOP that play a role in the high selectivity of this ligand for NOP over the other opioid receptors. Docking the shortest active fragment of endogenous agonist nociceptin/orphaninFQ (residues 1-13) shows that the NOP extracellular loop 2 (EL2) loop interacts with the positively charged residues (8-13) of N/OFQ. Both agonists show extensive polar interactions with residues at the extracellular end of the TM domain and EL2 loop, suggesting agonist-induced reorganization of polar networks, during receptor activation.     

Prokaryotic expression and immunogenicity of 56-kDa protein of Orientia tsutsugamushi strain Karp

To express the 56-kDa protein of O. tsutsugamushi strain Karp, this protein gene was cloned into pET30a(+) before transforming into host bacteria, E. coli Rossetta. Specificity of the recombinant protein was assessed by ELISA using rabbit sera against common members of the order Rickettsiae and 10 other pathogenic bacteria. After IPTG induction, SDS-PAGE analysis of isolated protein demonstrated a band at approximately 46-kDa. Western blot and mass spectrometry analysis proved that the recombinant protein was expressed successfully. Specificity analysis demonstrated that all sera were negative, except sera against O. tsutsugamushi strains TA763, TH1817 and Kato, B. quintana, A. phagocytophilum, E. chaffeensis and B. bacilliformis. The purified protein was used to immunize BALB/c mice and polyclonal antisera were harvested. By examination of IFA and ELISA, the highest titer of the polyclonal antibodies reaches 1:1600. The recombinant 56-kDa protein in the study is valuable for developing a simple and rapid diagnostic test and vaccine for O. tsutsugamushi.     

Production of 6-aminopenicillanic acid in aqueous two-phase systems by recombinant Escherichia coli with intracellular penicillin acylase

Bioconversion of penicillin G in PEG 20000/dextran T 70 aqueous two-phase systems was achieved using the recombinant Escherichia coli A56 (ppA22) with an intracellular penicillin acylase as catalyst. The best conversion conditions were attained for: 7% (w/v) substrate (penicillin G), enzyme activity in bottom phase 52 U ml(-1), pH 7.8, temperature 37 degrees C, reaction time 40 min. Five repeated batches could be performed in these conditions. Conversions ratios between 0.9-0.99 mol of 6-aminopenicillanic acid (6-APA) per mol of penicillin G, were obtained and volumetric productivity was 3.6-4.6 micromol min(-1) ml(-1). In addition the product 6-APA could be directly crystallized from the top phase with a purity of 96%.     

Activation of CMV promoter-controlled glycosyltransferase and β -galactosidase glycogenes by butyrate, tricostatin A, and 5-Aza-2′-deoxycytidine

Cytomegalovirus (CMV) immediate early promoter is a powerful promoter frequently used for driving the expression of transgenes in mammalian cells. However, this promoter gradually becomes silenced in stably transfected cells. We employed Chinese Hamster Ovary (CHO) and human pancreatic cancer (Panc 1) cells stably tansfected with three glycogenes driven by a CMV promoter to study the activation of silenced glycogenes. We found that butyrate, tricostatin A (TSA), and 5-aza-2-deoxycytidine (5-Aza-dC) can activate these CMV-driven glycogenes. The increase in mRNA and protein of a glycogene occurred 8–10 h after butyrate treatment, suggesting an indirect effect of butyrate in the activation of the transgene. The enhanced expression of the trangenes by butyrate and TSA, known inhibitors of histone deacetylase, was independent of the transgene or cell type. However, the transgene can be activated by these two agents in only a fraction of the cells derived from a single clone, suggesting that inactivation of histone deacetylase can only partially explain silencing of the transgenes. Combination treatment of one or both agents with 5-Aza-dC, a known inhibitor of DNA methylase, resulted in a synergistic activation of the transgene, suggesting a cross-talk between histone acetylation and DNA demethylation. Understanding the mechanisms of the inactivation and reactivation of CMV promoter-controlled transgenes should help develop an effective strategy to fully activate the CMV promoter-controlled therapeutic genes silenced by the host cells. Published in 2005.     

CD56和CD117浆细胞免疫表型与多发性骨髓瘤患者染色体核型和预后的研究

目的:探讨CD56和CD117浆细胞免疫表型与多发性骨髓瘤患者染色体核型和预后的关系。方法:选取2011年1月至2017年3月我院收治的66例多发性骨髓瘤患者,均采用以硼替佐米为基础的VTD化疗方法。采集患者新鲜骨髓液,采用流式细胞术(FCM)和荧光原位杂交技术(FISH)检测浆细胞免疫表型和细胞染色体核型。分析浆细胞免疫表型与患者染色体核型和预后的关系。结果:66例患者浆细胞CD19+、CD20+、CD45+、CD56+、CD117+表达频率分别为7.6%(5/66)、18.2%(12/66)、45.5%(30/66)、66.7%(44/66)和40.9%(27/66)。20例行FISH检测的患者,18例(90.0%)核型异常,其中12例(66.7%)IgH重排,9例(50.0%)1q21+扩增,8例(44.4%)del(13q14.3)缺失,10例(55.6%)del(13q14)缺失,3例(16.7%)del(17p)缺失。CD56+患者1q21+扩增和del(13q14.3)发生率显著低于CD56-患者(27.3%vs 85.7%,18.2%vs 85.7%,P0.05)。CD117+患者1q21+扩增和del(17p)发生率显著低于CD117-患者(12.5%vs 80.0%,12.5%vs 20.0%,P0.05)。CD56+患者的PFS和OS明显延长[23.4(2.0-91.4)月vs 19.8(3.0-85.1)月,34.5(8.9-96.5)月vs 30.1(6.7-84.3)月,P0.05]。CD117+患者PFS和OS明显延长[22.9(1.0-94.3)月vs 20.3(2.0-84.3)月,33.9(7.4-93.5)月vs 31.4(6.7-89.7)月,P0.05]。Kaplan-Meier分析CD56和CD117阳性与阴性患者的PFS曲线和OS曲线存在显著性差异(P0.05)。结论:CD56+和CD117+患者的预后明显优于CD56-和CD117-患者,CD56-和CD117-患者染色体异常核型的发生率明显增加。     

Geographical distribution of Orientia tsutsugamushi strains in chiggers from three provinces in Korea

Chiggers were collected from the central and southern parts of South Korea between April and November, 2009 with the aim of investigating the seasonal and geographical distribution of Or. A total of 1136 chiggers were identified. They included eight species belonging to four genera, as follows: Leptotrombidium scutellare (27.2%, n = 309), L. pallidum (54.6%, n = 621), L. orientale (6.25%, n = 71), L. palpale (1.59%, n = 18), L. zetum (2.0%, n = 23), Euschoengastia koreaensis (1.5%, n = 17), Cheladonta ikaoensis (0.08%, n = 1) and Neotrombicula japonica (1.05%, n = 12). The density of L. pallidum was high from April to May, whereas L. scutallare was not found in spring, being observed from October. Serotype‐specific nested PCR targeting the 56 kDa protein gene and sequencing analysis identified that the strains of 1136 O. tsutsugamushi in the chiggers as Boryong (6.8%), Kanda (0.4%), Oishi (0.3%), Jecheon (0.1%), Youngworl (0.1%) and Wonju (0.1%). Our findings indicate that L. pallidum and L. scutellare are dominant species in Korea and have geographical and seasonal variations.

     

Effect of heptane-extraction on the stability of subunit organization of photosystem I reaction center complexes

Treatment of lyophilized thylakoid membranes of the thermophilic cyanobacterium Synechococcus sp. with n-heptane for 6 h resulted in marked changes in the pattern of photosystem I reaction center complexes resolved by sodium dodecylsulfate-polyacrylamide gel electrophoresis. CP1-a, which consists of two large subunits and three small subunits, was a major chlorophyll-containing band resolved from the lyophilized thylakoid membranes, whereas the heptane-extracted membranes produced mainly CP1-e which totally lacks the small subunits. Electron transport from the primary donor P700 to the secondary acceptor P430 was not affected by the heptane-extraction of the membranes. The heptane-treatment removed 97% of -carotene present in the membranes, whereas all chlorophyll a, a major part of xanthophylls, more than a half of phylloquinone and one third of plastoquinone remained unextracted. The data suggest that -carotene has an important structural effect to stabilize the subunit organization of photosystem I reaction center complexes but is not essential for the early photochemical events of photosystem I.Abbreviations SDS sodium dodecylsulfate - PS photosystem