BACKGROUND & AIMS: :A novel lectin was purified from the fruiting bodies of king bolete mushrooms (Boletus edulis, also called porcino, cep or penny bun). The lectin was structurally characterized i.e its amino acid sequence and three-dimensional structure were determined. The new protein is a homodimer and each protomer folds as β-trefoil domain and therefore we propose the name Boletus edulis lectin (BEL) β-trefoil to distinguish it from the other lectin that has been described in these mushrooms. The lectin has potent anti-proliferative effects on human cancer cells, which confers to it an interesting therapeutic potential as an antineoplastic agent. Several crystal forms of the apoprotein and of complexes with different carbohydrates were studied by X-ray diffraction. The structure of the apoprotein was solved at 1.12 Å resolution. The interaction of the lectin with lactose, galactose, N-acetylgalactosamine and T-antigen disaccharide, Galβ1-3GalNAc, was examined in detail. All the three potential binding sites present in the β-trefoil fold are occupied in at least one crystal form and are described in detail in this paper. No important conformational changes are observed in the lectin when comparing its co-crystals with carbohydrates with those of the ligand-free protein.

背景与目标： ：从牛肝菌王菇（Boletus edulis，也称为牛肝菌，cep或便士面包）的子实体中纯化出一种新型凝集素。对凝集素进行结构表征，即确定其氨基酸序列和三维结构。新蛋白是同源二聚体，每个前体折叠成β-三叶结构域，因此我们提出了牛肝菌凝集素（BEL）β-三叶的名称，以将其与这些蘑菇中描述的其他凝集素区分开。所述凝集素对人癌细胞具有有效的抗增殖作用，使其具有作为抗肿瘤剂的令人感兴趣的治疗潜力。通过X射线衍射研究了载脂蛋白的几种晶体形式以及与不同碳水化合物的复合物。载脂蛋白的结构以1.12Å的分辨率解析。详细检查了凝集素与乳糖，半乳糖，N-乙酰半乳糖胺和T抗原二糖Galβ1-3GalNAc的相互作用。 β-三叶折叠中存在的所有三个潜在结合位点均以至少一种晶体形式占据，并在本文中进行了详细描述。当将其与碳水化合物的共晶体与不含配体的蛋白质的共晶体进行比较时，在凝集素中未观察到重要的构象变化。

BACKGROUND & AIMS: :A series of twenty-five derivatives of tetrahydro-β-carbolines 1-3 was synthesized and assayed on FAAH and TRPV1 and TRPA1 channels. Four carbamates, that is, 5a,c,e, and 9b inhibited FAAH with significant potency and interacted also effectively with TRPV1 and TRPA1 nociceptive receptors, while ureas 7b,d,f, and 8a,b were endowed with specific submicromolar TRPV1 modulating activities.

背景与目标： ：合成了一系列的二十五个四氢-β-咔啉1-3衍生物，并在FAAH和TRPV1和TRPA1通道上进行了测定。四种氨基甲酸酯5a，c，e和9b显着抑制FAAH，并且还与TRPV1和TRPA1伤害感受器有效相互作用，而尿素7b，d，f和8a，b具有特定的亚微摩尔TRPV1调节活性。 。

BACKGROUND & AIMS: :Propolis is a glue, prepared by honeybees from plant materials to stick their hives on the beehive wall. It has gained popularity in Japan as a healthy drink and people believe that propolis can cure inflammation, heart diseases and even diabetes and cancer. We have evaluated the β-cell protective effect of propolis against the toxicity of streptozotocin (STZ) in rats. The water extract of propolis (PWE) completely protected β-cell destruction against STZ toxicity. The protective effect of PWE was found to be almost equal to that of nicotinamide. PWE also inhibited the interleukin-1 β (IL-1 β) generation from human leukocytes. The free radical scavenging activity together with IL-1 β and nitric oxide (NO) synthase inhibitory activities are thought to be the prime factors for the protective effect of PWE against STZ toxicity.

背景与目标： ：蜂胶是一种胶水，是由蜜蜂从植物材料中制备的，用以将其蜂巢粘贴在蜂巢壁上。它在日本作为一种健康的饮料而受到欢迎，人们相信蜂胶可以治愈炎症，心脏病甚至糖尿病和癌症。我们评估了蜂胶对大鼠链脲佐菌素（STZ）毒性的β-细胞保护作用。蜂胶（PWE）的水提取物完全保护β细胞不受STZ毒性的破坏。发现PWE的保护作用几乎等于烟酰胺的保护作用。 PWE还抑制了人类白细胞产生的白介素-1β（IL-1β）。自由基清除活性以及IL-1β和一氧化氮（NO）合酶抑制活性被认为是PWE对抗STZ毒性的保护作用的主要因素。

BACKGROUND & AIMS: :Extended-spectrum β-lactamase (ESBL)-producing-Enterobacteriaceae strains were detected in 12% (6 out of 50) of fecal samples collected from the inpatients of a Japanese pediatric hospital. All the ESBLs belonged to the CTX-M-1 group. The proportion of carriage of ESBL producers was higher among patients who had received antibiotics within the past 3 months and among those who had cardiologic diseases.

背景与目标： ：从日本儿科医院住院患者收集的粪便样本中，有12％（50份中有6份）检测到产生超广谱β-内酰胺酶（ESBL）的肠杆菌科菌株。所有ESBL都属于CTX-M-1组。在过去3个月内接受过抗生素治疗的患者和患有心脏病的患者中，携带ESBL生产者的比例更高。

BACKGROUND & AIMS: OBJECTIVE:To evaluate the predictive value of gestational age and maternal serum β-hCG concentration for the determination of the depth of trophoblastic invasion into the tubal wall. METHODS:This is a retrospective trial conducted on women with a diagnosis of ampullary pregnancy (71) who were submitted to salpingectomy. Serum β-hCG measurements were obtained at the initial admission of hospital. Histological investigation was performed by a single well-experienced pathologist who was blind to the clinical and laboratory characteristics of the patients. Ampullary pregnancy was classified histologically according to the depth of trophoblastic infiltration into tubal wall: trophoblast limited to the tubal mucosa (stage I), extended to muscularis layer (stage II) and complete tubal wall infiltration up to serosal layer (stage III). RESULTS:There was a significant difference in maternal serum β-hCG concentrations regarding the histological stages of trophoblastic invasion. The serum β-hCG concentrations that the best predicted for stage III trophoblastic invasion was 6,475 mIU/ml, with a sensitivity of 100 %, a specificity of 92 %. CONCLUSION:The depth of trophoblastic tissue infiltration into tubal wall is correlated with serum β-hCG levels, but not with gestational age. These findings may explain the reason for conservative management failure of EP in women with high β-hCG concentrations.

背景与目标： 目的：评价孕龄和孕产妇血清β-hCG浓度对确定滋养细胞浸润输卵管壁的深度的预测价值。
方法：这是一项回顾性试验，针对接受壶腹部切除术的诊断为壶腹妊娠的妇女（71名）。初次入院时获得血清β-hCG测量值。组织学调查由一位经验丰富的病理学家进行，该病理学家对患者的临床和实验室特征视而不见。根据滋养细胞向输卵管壁的浸润深度在组织学上对壶腹妊娠进行分类：滋养细胞仅限于输卵管粘膜（I期），延伸至肌层（II期），完全输卵管壁浸润至浆膜层（III期）。
结果：就滋养细胞侵袭的组织学阶段而言，母体血清β-hCG浓度存在显着差异。对于第三阶段滋养细胞浸润最好的预测血清β-hCG浓度为6,475 mIU / ml，敏感性为100％，特异性为92％。
结论：滋养细胞组织渗入输卵管壁的深度与血清β-hCG水平有关，与胎龄无关。这些发现可能解释了高β-hCG浓度女性EP保守治疗失败的原因。

BACKGROUND & AIMS: BACKGROUND:There is a lack of information on stiffness parameter β, an index of arterial stiffness, in hemodialysis (HD) patients. The aim of the present study was to investigate whether stiffness parameter β is predictive of the long-term mortality of chronic HD patients. METHODS:We measured biochemical parameters and the stiffness parameter β of 80 patients on maintenance HD therapy and followed their course for 4 years, and we enrolled 70 of these 80 patients in the study. We divided the 70 patients into tertiles according to their stiffness parameter β values, and conducted multivariate analyses to examine the impact of the tertiles on 4-year mortality. RESULTS:Older age and the presence of diabetes mellitus were found to be independently associated with higher stiffness parameter β values. Fifteen patients (21.4 %) died and 16 (22.9 %) experienced a new cardiovascular event during the follow-up period. The results of a Kaplan-Meier analysis revealed a significantly higher risk of all-cause mortality in the HD patients with highest stiffness parameter β values (p = 0.0106). According to the ROC curve, the cut-off level that yielded maximal sensitivity and specificity for predicting all-cause mortality was 10.1, and the sensitivity and specificity using the cut-off value were 69.2 and 70.2 %, respectively. CONCLUSION:The results of this study suggest that stiffness parameter β is a predictor of all-cause mortality in chronic HD patients.

背景与目标： 背景：在血液透析（HD）患者中，缺乏关于刚度参数β（动脉刚度指标）的信息。本研究的目的是研究刚度参数β是否可预测慢性HD患者的长期死亡率。
方法：我们测量了维持HD治疗的80例患者的生化参数和刚度参数β，并对其病程进行了为期4年的研究，我们在这80名患者中招募了70名患者。我们根据其刚度参数β值将70例患者分为三分位数，并进行多变量分析以检查三分位数对4年死亡率的影响。
结果：老年人年龄和糖尿病的存在与高刚性参数β值独立相关。在随访期间，有15例患者（21.4％）死亡，而16例（22.9％）发生了新的心血管事件。 Kaplan-Meier分析的结果表明，刚度参数β值最高的HD患者全因死亡的风险显着更高（p = 0.0106）。根据ROC曲线，可产生最大预测全因死亡率的敏感性和特异性的临界值为10.1，使用该临界值的敏感性和特异性分别为69.2％和70.2％。
结论：这项研究的结果表明，刚度参数β是慢性HD患者全因死亡率的预测指标。

BACKGROUND & AIMS: :β-Lapachone is a naturally occurring quinine, originally isolated from the bark of the lapacho tree (Tabebuia avellanedae) which is currently being evaluated in clinical trials for the treatment of cancer. In addition, recent investigations suggest its potential application for treatment of inflammatory diseases. Multiple sclerosis (MS) is an autoimmune disorder characterized by CNS inflammation and demyelination. Reactive T cells including IL-17 and IFN-γ-secreting T cells are believed to initiate MS and the associated animal model system experimental autoimmune encephalomyelitis (EAE). IL-12 family cytokines secreted by peripheral dendritic cells (DCs) and CNS microglia are capable of modulating T-cell phenotypes. The present studies demonstrated that β-lapachone selectively inhibited the expression of IL-12 family cytokines including IL-12 and IL-23 by DCs and microglia, and reduced IL-17 production by CD4(+) T-cells indirectly through suppressing IL-23 expression by microglia. Importantly, our studies also demonstrated that β-lapachone ameliorated the development on EAE. β-Lapachone suppression of EAE was associated with decreased expression of mRNAs encoding IL-12 family cytokines, IL-23R and IL-17RA, and molecules important in Toll-like receptor signaling. Collectively, these studies suggest mechanisms by which β-lapachone suppresses EAE and suggest that β-lapachone may be effective in the treatment of inflammatory diseases such as MS.

背景与目标： ：-Lapachone是一种天然存在的奎宁，最初从lapacho树（Tabebuia avellanedae）的树皮中分离出来，目前正在临床试验中评估其治疗癌症的能力。另外，最近的研究表明其在治疗炎性疾病中的潜在应用。多发性硬化症（MS）是一种以CNS炎症和脱髓鞘为特征的自身免疫性疾病。据信包括IL-17和分泌IFN-γ的T细胞在内的反应性T细胞可引发MS和相关的动物模型系统实验性自身免疫性脑脊髓炎（EAE）。外周树突状细胞（DC）和中枢神经系统小胶质细胞分泌的IL-12家族细胞因子能够调节T细胞表型。本研究表明，β-拉帕酮可选择性抑制DC和小胶质细胞的IL-12家族细胞因子（包括IL-12和IL-23）的表达，并通过抑制IL-23间接降低CD4（）T细胞的IL-17产生。小胶质细胞表达。重要的是，我们的研究还表明，β-拉帕酮可改善EAE的发展。 β-拉帕酮对EAE的抑制作用与编码IL-12家族细胞因子，IL-23R和IL-17RA以及在Toll样受体信号转导中重要的分子的mRNA表达降低有关。这些研究共同提出了β-拉帕酮抑制EAE的机制，并提出了β-拉帕酮可能有效治疗诸如MS的炎性疾病。

BACKGROUND & AIMS: :The synaptic toxicity of soluble amyloid-β (Aβ) oligomers plays a critical role in the pathophysiology of Alzheimer's disease (AD). Here we report that overexpressed α1-takusan, which we previously identified as a protein that enhances synaptic activity via interaction with PSD-95, mitigates oligomeric Aβ-induced synaptic loss. In contrast, takusan knockdown results in enhanced synaptic damage. α1-Takusan interacts with tau either directly or indirectly, and prevents Aβ-induced tau hyperphosphorylation and mitochondrial fragmentation. Deletion analysis identified the second domain (D2) within the takusan protein that is required for PSD-95 clustering and synaptic protection from Aβ. A 51 aa sequence linking D2 to the PDZ-binding C terminus was found to be as effective as full-length takusan in protecting synapses from Aβ-induced damage. Moreover, a sequence containing the D2 from the human protein discs large homolog 5, when linked to a C-terminal PDZ-binding motif, can also increase the clustering of PSD-95 in cortical dendrites. In summary, α1-takusan protects synapses from Aβ-induced insult via interaction with PSD-95 and tau. Thus, takusan-based protein sequences from either mouse or human may be of potential therapeutic benefit in AD.

背景与目标： ：可溶性淀粉样蛋白-β（Aβ）低聚物的突触毒性在阿尔茨海默氏病（AD）的病理生理中起关键作用。在这里，我们报道过表达的α1-takusan（我们先前鉴定为通过与PSD-95相互作用增强突触活性的蛋白）减轻了寡聚Aβ诱导的突触损失。相反，takusan敲低导致突触损伤增强。 α1-Takusan直接或间接与tau相互作用，并防止Aβ诱导的tau过度磷酸化和线粒体断裂。缺失分析鉴定了takusan蛋白内的第二个结构域（D2），这是PSD-95聚集和突触保护Aβ所必需的。发现将D2连接至PDZ结合C末端的51氨基酸序列与全长takusan一样有效，可保护突触免受Aβ诱导的损伤。此外，包含来自人类蛋白质的D2序列的大同系物5，当与C端PDZ结合基序连接时，也可以增加PSD-95在皮质树突中的簇集。总之，α1-takusan通过与PSD-95和tau的相互作用保护突触免受Aβ诱导的侵害。因此，来自小鼠或人的基于takusan的蛋白质序列可能在AD中具有潜在的治疗益处。

BACKGROUND & AIMS: AIMS:CAWS, Candida albicans water-soluble fraction, is an extracellular mannoprotein produced by C. albicans NBRC1385. It is a ligand of dectin-2, the C-type lectin receptor for innate immunity, and has strong potency for induction of vasculitis in DBA/2 mice. The structure of this mannoprotein is known to be modulated by the culture conditions. To clarify the structure required for vasculitis, CAWSs were prepared in the two culture conditions with or without pH control, and biological properties were compared. METHODS:CAWSs prepared by the standard protocol and pH controlled at 7.0 were designated as CAWS and CAWS727, respectively. The antigenicity was detected by the anti-Candida mannan IgG. These chemical structures were assessed by nuclear magnetic resonance analysis and the lectin array system. The in vitro activity of CAWSs was tested by tumor necrosis factor-α (TNF-α) induction using bone marrow-derived dendritic cells and spleen cell cultures. RESULTS:The antigenicity of CAWS727 was similar to CAWS but the nuclear magnetic resonance analysis showed a higher ratio of β-mannosyl linkages were detected in CAWS727. The lectin array showed relative affinities of CAWS727 to α-mannosyl specific lectins were weaker than those of CAWS. CAWS induced severe vasculitis in DBA/2 mice while CAWS727 did not. CAWS significantly induced TNF-α but CAWS727 did slightly. In addition, CAWS-induced TNF-α production was inhibited by mixing with CAWS727 in a concentration dependent manner. CONCLUSION:The α-mannosyl linkages of Candida mannan is a key molecule for the immunotoxicity. CAWS727, which conatins β-mannosyl linkages, competitively bound to lectin receptors, and resulted in reductions in the inflammatory response.

背景与目标： 目的：CAWS，白色念珠菌水溶性级分，是白色念珠菌NBRC1385产生的细胞外甘露糖蛋白。它是先天性免疫的C型凝集素受体dectin-2的配体，对DBA / 2小鼠的血管炎具有很强的诱导作用。已知该甘露糖蛋白的结构受培养条件调节。为了阐明血管炎所需的结构，在两种培养条件下（有或没有pH值控制）制备了CAWS，并比较了生物学特性。
方法：将标准方案制备的且pH控制在7.0的CAWS分别命名为CAWS和CAWS727。通过抗Candida甘露聚糖IgG检测抗原性。这些化学结构通过核磁共振分析和凝集素阵列系统进行评估。使用骨髓来源的树突状细胞和脾细胞培养物，通过肿瘤坏死因子-α（TNF-α）诱导来测试CAWS的体外活性。
结果：CAWS727的抗原性与CAWS相似，但核磁共振分析表明在CAWS727中检测到较高比例的β-甘露糖基键。凝集素阵列显示CAWS727对α-甘露糖基特异性凝集素的相对亲和力弱于CAWS。 CAWS诱发了DBA / 2小鼠的严重血管炎，而CAWS727并未诱发。 CAWS显着诱导TNF-α，但CAWS727略有诱导。另外，通过以浓度依赖性方式与CAWS727混合来抑制CAWS诱导的TNF-α的产生。
结论：甘露假丝酵母的α-甘露糖基键是免疫毒性的关键分子。 CAWS727具有β-甘露糖基键，与凝集素受体竞争性结合，并导致炎症反应降低。

BACKGROUND & AIMS: INTRODUCTION:The development of endometrial cancer is known to be affected by estrogens. Thus, genetic variations like single nucleotide polymorphisms (SNPs) in genes involved in estrogen biosynthesis, metabolism, and signal transduction might affect risk for endometrial cancer. In this study, we tested the hypothesis that polymorphisms in the promoter of ESR2 gene may be associated with susceptibility to this disease. METHODS:We compared the frequency of three SNPs in the promoter region of ESR2 gene (rs2987983, rs3020450, and rs3020449) in 135 women with endometrial cancer and 135 healthy women serving as controls by means of allele-specific tetra-primer PCR. RESULTS:Regarding allele frequency, allele positivity or genotype frequencies of these SNPs we did not observe any significant difference between healthy women and women with endometrial cancer. CONCLUSION:Our data clearly suggest that the tested SNPs in the promotor region of human ESR2 gene are not associated with the development of endometrial cancer.

背景与目标： 简介：子宫内膜癌的发展已知受雌激素的影响。因此，涉及雌激素生物合成，代谢和信号转导的基因中的遗传变异（如单核苷酸多态性（SNP））可能会影响子宫内膜癌的风险。在这项研究中，我们检验了ESR2基因启动子中的多态性可能与这种疾病的易感性有关的假设。
方法：我们通过等位基因特异性四引物PCR比较了135例子宫内膜癌女性和135例健康女性作为对照的ESR2基因启动子区域（rs2987983，rs3020450和rs3020449）中三个SNP的频率。
结果：关于这些SNP的等位基因频率，等位基因阳性或基因型频率，我们没有观察到健康女性和子宫内膜癌女性之间的任何显着差异。
结论：我们的数据清楚地表明，人类ESR2基因启动子区域中受测的SNP与子宫内膜癌的发生无关。

BACKGROUND & AIMS: :Inhibition of urease results in Helicobacter pylori growth arrest in the stomach, promoting urease as promising targets for gastrointestinal ulcer therapy. Twenty hybrid derivatives of flavonoid scaffold and hydroxamic acid, β-hydroxy-β-phenylpropionylhydroxamic acids, were therefore synthesized and evaluated against H. pylori urease. Biological evaluation of these compounds showed improved urease inhibition exhibiting micromolar to mid-nanomolar IC50 values. Most importantly, 3-(3-chlorophenyl)-3-hydroxypropionyl-hydroxamic acid (6g) exhibited high potency with IC50 of 0.083±0.004 μM and Ki of 0.014±0.003 μM, indicating that 6g is an excellent candidate to develop novel antiulcer agent. A mixture of competitive and uncompetitive mechanism was putatively proposed to understand the inconsistency between the crystallographic and kinetic studies for the first time, which is supported by our molecular docking studies.

背景与目标： ：脲酶的抑制导致幽门螺杆菌在胃中的生长停滞，促进脲酶成为胃肠道溃疡治疗的有希望的靶标。因此，合成了黄酮类支架和异羟肟酸的二十种杂合衍生物，β-羟基-β-苯基丙酰基异羟肟酸，并针对幽门螺杆菌脲酶进行了评估。这些化合物的生物学评估表明，尿素酶抑制作用得到改善，表现出微摩尔至中纳摩尔级的IC50值。最重要的是，3-（3-氯苯基）-3-羟基丙酰基-异羟肟酸（6g）表现出很高的效力，IC50为0.083±0.004μM，Ki为0.014±0.003μM，表明6g是开发新型抗溃疡药的极佳候选药物。为了首次了解晶体学和动力学研究之间的矛盾，提出了一种竞争机制和非竞争机制的混合物，这是我们的分子对接研究所支持的。

BACKGROUND & AIMS: :Plant pathogenic Pseudomonas syringae produce the hydroxy-β-lactam antimetabolite tabtoxinine-β-lactam (TβL) as a time-dependent inactivating glutamine analogue of plant glutamine synthetases. The producing pseudomonads use multiple modes of self-protection, two of which are characterized in this study. The first is the dipeptide ligase TblF which converts tabtoxinine-β-lactam to the TβL-Thr dipeptide known as tabtoxin. The dipeptide is not recognized by glutamine synthetase. This represents a Trojan Horse strategy: the dipeptide is secreted, taken up by dipeptide permeases in neighboring cells, and TβL is released by peptidase action. The second self-protection mode is elaboration by the acetyltransferase Ttr, which acetylates the α-amino group of the proximal inactivator TβL, but not the tabtoxin dipeptide.

背景与目标： 植物致病性丁香假单胞菌可产生羟基-β-内酰胺抗代谢物毒素-β-内酰胺（TβL），作为植物谷氨酰胺合成酶的时间依赖性灭活谷氨酰胺类似物。产生的假单胞菌使用多种自我保护模式，本研究表征了其中两种。第一个是二肽连接酶TblF，它将Tabtoxinine-β-lactam转化为称为Tabtoxin的TβL-Thr二肽。谷氨酰胺合成酶不能识别二肽。这代表了特洛伊木马策略：二肽被分泌，被邻近细胞中的二肽渗透酶吸收，而TβL通过肽酶的作用释放。第二种自我保护模式是通过乙酰基转移酶Ttr进行的，该酶可乙酰化近端灭活剂TβL的α-氨基，但不能使毒素二肽乙酰化。

BACKGROUND & AIMS: Background:To produce second-generation biofuels, enzymatic catalysis is required to convert cellulose from lignocellulosic biomass into fermentable sugars. β-Glucosidases finalize the process by hydrolyzing cellobiose into glucose, so the efficiency of cellulose hydrolysis largely depends on the quantity and quality of these enzymes used during saccharification. Accordingly, to reduce biofuel production costs, new microbial strains are needed that can produce highly efficient enzymes on a large scale. Results:We heterologously expressed the fungal β-glucosidase D2-BGL from a Taiwanese indigenous fungus Chaetomella raphigera in Pichia pastoris for constitutive production by fermentation. Recombinant D2-BGL presented significantly higher substrate affinity than the commercial β-glucosidase Novozyme 188 (N188; Km = 0.2 vs 2.14 mM for p-nitrophenyl β-d-glucopyranoside and 0.96 vs 2.38 mM for cellobiose). When combined with RUT-C30 cellulases, it hydrolyzed acid-pretreated lignocellulosic biomasses more efficiently than the commercial cellulase mixture CTec3. The extent of conversion from cellulose to glucose was 83% for sugarcane bagasse and 63% for rice straws. Compared to N188, use of D2-BGL halved the time necessary to produce maximal levels of ethanol by a semi-simultaneous saccharification and fermentation process. We upscaled production of recombinant D2-BGL to 33.6 U/mL within 15 days using a 1-ton bioreactor. Crystal structure analysis revealed that D2-BGL belongs to glycoside hydrolase (GH) family 3. Removing the N-glycosylation N68 or O-glycosylation T431 residues by site-directed mutagenesis negatively affected enzyme production in P. pastoris. The F256 substrate-binding residue in D2-BGL is located in a shorter loop surrounding the active site pocket relative to that of Aspergillus β-glucosidases, and this short loop is responsible for its high substrate affinity toward cellobiose. Conclusions:D2-BGL is an efficient supplement for lignocellulosic biomass saccharification, and we upscaled production of this enzyme using a 1-ton bioreactor. Enzyme production could be further improved using optimized fermentation, which could reduce biofuel production costs. Our structure analysis of D2-BGL offers new insights into GH3 β-glucosidases, which will be useful for strain improvements via a structure-based mutagenesis approach.

背景与目标： 背景：为生产第二代生物燃料，需要酶催化才能将纤维素从木质纤维素生物质转化为可发酵糖。 β-葡糖苷酶通过将纤维二糖水解为葡萄糖来完成这一过程，因此纤维素水解的效率很大程度上取决于糖化过程中所用这些酶的数量和质量。因此，为了降低生物燃料的生产成本，需要可以大规模生产高效酶的新微生物菌株。
结果：我们在巴斯德毕赤酵母中异源表达了台湾原产的一种真菌，即南美白对虾中的真菌β-葡萄糖苷酶D2-BGL，用于发酵生产。重组D2-BGL的底物亲和力明显高于商品β-葡萄糖苷酶Novozyme 188（N188;对硝基苯基β-d-吡喃葡萄糖苷Km = 0.2 vs 2.14mM，纤维二糖Km = 0.2 vs 2.14mM）。当与RUT-C30纤维素酶结合时，它比商业纤维素酶混合物CTec3更有效地水解酸预处理的木质纤维素生物质。对于甘蔗渣，从纤维素到葡萄糖的转化程度为83％，对于稻草为63％。与N188相比，D2-BGL的使用通过半同时糖化和发酵过程生产最大量乙醇所需的时间减少了一半。我们使用1吨生物反应器在15天内将重组D2-BGL的生产规模提升至33.6 U / mL。晶体结构分析表明D2-BGL属于糖苷水解酶（GH）家族3。通过定点诱变去除N-糖基化N68或O-糖基化T431残基会对巴斯德毕赤酵母中的酶产生产生负面影响。相对于曲霉β-葡萄糖苷酶，D2-BGL中的F256底物结合残基位于围绕活性位点袋的较短环中，并且该短环是其对纤维二糖的高底物亲和力的原因。
结论：D2-BGL是木质纤维素生物质糖化的有效补充剂，我们使用1吨生物反应器提高了该酶的生产规模。通过优化发酵可以进一步改善酶的生产，这可以降低生物燃料的生产成本。我们对D2-BGL的结构分析为GH3β-葡萄糖苷酶提供了新的见解，这将有助于通过基于结构的诱变方法改善菌株。

BACKGROUND & AIMS: :High-throughput TCR sequencing allows interrogation of the human TCR repertoire, potentially connecting TCR sequences to antigenic targets. Unlike the highly polymorphic MHC proteins, monomorphic Ag-presenting molecules such as MR1, CD1d, and CD1b present Ags to T cells with species-wide TCR motifs. CD1b tetramer studies and a survey of the 27 published CD1b-restricted TCRs demonstrated a TCR motif in humans defined by the TCR β-chain variable gene 4-1 (TRBV4-1) region. Unexpectedly, TRBV4-1 was involved in recognition of CD1b regardless of the chemical class of the carried lipid. Crystal structures of two CD1b-specific TRBV4-1+ TCRs show that germline-encoded residues in CDR1 and CDR3 regions of TRBV4-1-encoded sequences interact with each other and consolidate the surface of the TCR. Mutational studies identified a key positively charged residue in TRBV4-1 and a key negatively charged residue in CD1b that is shared with CD1c, which is also recognized by TRBV4-1 TCRs. These data show that one TCR V region can mediate a mechanism of recognition of two related monomorphic Ag-presenting molecules that does not rely on a defined lipid Ag.

背景与目标： ：高通量TCR测序可以询问人TCR的所有组成部分，从而可能将TCR序列连接到抗原靶标上。与高度多态的MHC蛋白不同，诸如MR1，CD1d和CD1b之类的单态Ag呈递分子将Ags呈递给具有全物种TCR图案的T细胞。 CD1b四聚体研究和对27种已发表的CD1b限制性TCR的调查表明，TCRβ链可变基因4-1（TRBV4-1）区域定义了人类的TCR基序。出乎意料的是，无论所携带脂质的化学类别如何，TRBV4-1都参与了对CD1b的识别。两个CD1b特异性TRBV4-1 TCR的晶体结构表明，TRBV4-1编码序列的CDR1和CDR3区中的种系编码残基彼此相互作用并巩固TCR的表面。突变研究确定了TRBV4-1中一个关键的带正电荷的残基和CD1b中一个关键的带负电荷的残基，与CD1c共享，TRBV4-1 TCR也意识到了这一点。这些数据表明，一个TCR V区可以介导识别不依赖于确定的脂质Ag的两个相关的单态Ag呈递分子的识别机制。

BACKGROUND & AIMS: Single-residue mutations have been made of the hydrophobic Ile or Val residue in position 8 of each of the four calcium-binding loop sequences (sites I-IV) of Drosophila calmodulin. These highly conserved residues are part of the hydrophobic core of either calmodulin domain and are involved in the structural link of two calcium-binding sites via a short antiparallel beta-sheet. In the apo-form, the replacement of Ile (or Val) by Gly causes a significant destabilization, shown by the unfolding of the secondary structure of the domain carrying the mutation. In the presence of calcium, the deficiency in alpha-helical structure at 20 degrees C is restored for the mutants at site I, II, or III but not at site IV, which requires the further binding of a high-affinity target peptide to re-establish the native conformation. The extent of the destabilization is seen in the depression of the melting temperature of individual domains, which can be as large as 80 degrees C in the case of Ca4-CaM(V136G). However, because of low values of the unfolding enthalpy for calmodulin domains, only relatively low values of <2 kcal/mol are implied for DeltaDeltaG, the free energy of destabilization due to mutation. Consistent with this, the secondary structure of any unfolded mutant domain is highly sensitive to solvent composition and is largely refolded in the presence of 12.5% (v/v) aqueous trifluoroethanol. Compared to wild-type calmodulin, the affinities of the mutants for calcium and target peptides from sk-MLCK at 20 degrees C are significantly reduced but the effects are relatively small. These results indicate that the conformation of calmodulin can be dramatically altered by mutation of a single highly conserved residue but that changes in solvent or the binding of a target sequence can readily compensate for this, restoring the wild-type properties. The results also suggest that the integrity of both the apo- and holo-forms of calmodulin is important for the maintenance of its biological function and confirm the importance of conserving the structural function of the residues involved in the beta-sheet interactions.

背景与目标： 果蝇钙调节蛋白的四个钙结合环序列（I-IV位）中每个位置8的疏水性Ile或Val残基均已形成单残基突变。这些高度保守的残基是任一钙调蛋白结构域的疏水核心的一部分，并通过短的反平行β-折叠参与两个钙结合位点的结构连接。在脱辅基形式中，Ily（或Val）被Gly取代引起明显的不稳定，这由携带突变的域的二级结构的展开显示。在钙的存在下，位点I，II或III但位点IV处的突变体在20℃时α-螺旋结构的缺陷得以恢复，这需要进一步结合高亲和力的靶肽才能重新结合。 -建立本地构象。不稳定的程度体现在单个域的熔化温度降低，对于Ca4-CaM（V136G）而言，可能高达80摄氏度。但是，由于钙调蛋白结构域的解折叠焓值较低，因此对于DeltaDeltaG仅隐含了相对较低的<2 kcal / mol值，这是由于突变引起的去稳定的自由能。与此一致的是，任何未折叠的突变域的二级结构对溶剂组成高度敏感，并在存在12.5％（v / v）的三氟乙醇水溶液的情况下大大重折叠。与野生型钙调蛋白相比，该突变体在20摄氏度下对sk-MLCK的钙和目标肽的亲和力显着降低，但影响相对较小。这些结果表明，钙调蛋白的构象可以通过单个高度保守残基的突变而显着改变，但是溶剂的改变或靶序列的结合可以很容易地弥补这一点，从而恢复野生型特性。该结果还表明钙调蛋白的载脂蛋白和全脂蛋白形式的完整性对于维持其生物学功能非常重要，并确认了保守参与β-sheet相互作用的残基的结构功能的重要性。