Hydrogen and oxygen trapping at the H-cluster of [FeFe]-hydrogenase revealed by site-selective spectroscopy and QM/MM calculations

[FeFe]-hydrogenases are superior hydrogen conversion catalysts. They bind a cofactor (H-cluster) comprising a four-iron and a diiron unit with three carbon monoxide (CO) and two cyanide (CN^?) ligands. Hydrogen (H₂) and oxygen (O₂) binding at the H-cluster was studied in the C169A variant of [FeFe]-hydrogenase HYDA1, in comparison to the active oxidized (Hox) and CO-inhibited (Hox-CO) species in wildtype enzyme. ⁵⁷Fe labeling of the diiron site was achieved by in vitro maturation with a synthetic cofactor analogue. Site-selective X-ray absorption, emission, and nuclear inelastic/forward scattering methods and infrared spectroscopy were combined with quantum chemical calculations to determine the molecular and electronic structure and vibrational dynamics of detected cofactor species. Hox reveals an apical vacancy at Fe_d in a [4Fe4S-2Fe]^3 ? complex with the net spin on Fe_d whereas Hox-CO shows an apical CN^? at Fe_d in a [4Fe4S-2Fe(CO)]^3 ? complex with net spin sharing among Fe_p and Fe_d (proximal or distal iron ions in [2Fe]). At ambient O₂ pressure, a novel H-cluster species (Hox-O₂) accumulated in C169A, assigned to a [4Fe4S-2Fe(O₂)]^3 ? complex with an apical superoxide (O₂^?) carrying the net spin bound at Fe_d. H₂ exposure populated the two-electron reduced Hhyd species in C169A, assigned as a [(H)4Fe4S-2Fe(H)]^3 ? complex with the net spin on the reduced cubane, an apical hydride at Fe_d, and a proton at a cysteine ligand. Hox-O₂ and Hhyd are stabilized by impaired O₂^– protonation or proton release after H₂ cleavage due to interruption of the proton path towards and out of the active site.     

Environmental Factors Associated with Disease Progression after the First Demyelinating Event: Results from the Multi-Center SET Study

Objectives

To investigate the associations of environmental MS risk factors with clinical and MRI measures of progression in high-risk clinically isolated syndromes (CIS) after the first demyelinating event.

Methods

We analyzed 211 CIS patients (age: 28.9±7.8 years) enrolled in the SET study, a multi-center study of high-risk CIS patients. Pre-treatment samples were analyzed for IgG antibodies against cytomegalovirus (anti-CMV), Epstein Barr virus (EBV) early nuclear antigen-1 (EBNA-1), viral capsid antigen (VCA), early antigen-diffuse (EA-D), 25 hydroxy-vitamin D3 and cotinine levels and HLA DRB1*1501 status. The inclusion criteria required evaluation within 4 months of the initial demyelinating event, 2 or more brain MRI lesions and the presence of two or more oligoclonal bands in cerebrospinal fluid. All patients were treated with interferon-beta. Clinical and MRI assessments were obtained at baseline, 6, 12, and 24 months.

Results

The time to first relapse decreased and the number of relapses increased with anti-CMV IgG positivity. Smoking was associated with increased number and volume of contrast-enhancing lesions (CEL) during the 2-year period. The cumulative number of CEL and T2 lesions during the 2-year period was greater for individuals in the highest quartile of anti-EBV VCA IgG antibodies. The percent loss of brain volume was increased for those in the highest quartile of with anti-EBV VCA IgG antibodies.

Conclusions

Relapses in CIS patients were associated with CMV positivity whereas anti-EBV VCA positivity was associated with progression on MRI measures, including accumulation of CEL and T2 lesions and development of brain atrophy.     

Single-Cell Heterogeneity Analysis and CRISPR Screen Identify Key β-Cell-Specific Disease Genes

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[反]-β-法尼烯合成酶基因在植物抗蚜分子育种中的应用

蚜虫是重要的农业害虫,每年造成数以亿计的经济损失。[反]-β-法尼烯[(E)-β-farnesene,EβF]是绝大多数蚜虫报警信息素的主要成分,可使蚜虫产生骚动、从植株上脱落,并吸引蚜虫天敌,有效控制蚜虫危害。EβF合成酶是催化合成EβF的关键酶,目前该基因已从薄荷、香橙、花旗松、黄花蒿、洋甘菊等植物中得到分离鉴定。植物中表达EβF合成酶基因以催化法呢基焦磷酸(Farnesyl diphosphate,FPP)合成EβF是控制蚜虫危害的重要策略。文中概括了当前植物抗蚜转基因研究现状,综述了植物EβF合成酶基因及其在植物抗蚜分子育种中的应用。针对当前转基因植物的EβF生成量较低等问题,展望了EβF合成酶基因在植物抗蚜分子育种中的应用前景和研究策略。     

Intein-Mediated Protein trans-Splicing of the Recombinant Streptavidin on Magnetosomes

Molecular Biology - When expressing streptavidin recombinant polypeptide on magnetosomes (called bacterial magnetic nanoparticles, or BMPs), the presence of endogenous bacterial biotin might be...     

Microtubule-assisted mechanism for toxisome assembly in Fusarium graminearum

In Fusarium graminearum, a trichothecene biosynthetic complex known as the toxisome forms ovoid and spherical structures in the remodelled endoplasmic reticulum (ER) under mycotoxin-inducing conditions. Previous studies also demonstrated that disruption of actin and tubulin results in a significant decrease in deoxynivalenol (DON) biosynthesis in F. graminearum. However, the functional association between the toxisome and microtubule components has not been clearly defined. In this study we tested the hypothesis that the microtubule network provides key support for toxisome assembly and thus facilitates DON biosynthesis. Through fluorescent live cell imaging, knockout mutant generation, and protein–protein interaction assays, we determined that two of the four F. graminearum tubulins, α₁ and β₂ tubulins, are indispensable for DON production. We also showed that these two tubulins are directly associated. When the α₁–β₂ tubulin heterodimer is disrupted, the metabolic activity of the toxisome is significantly suppressed, which leads to significant DON biosynthesis impairment. Similar phenotypic outcomes were shown when F. graminearum wild type was treated with carbendazim, a fungicide that binds to microtubules and disrupts spindle formation. Based on our results, we propose a model where α₁–β₂ tubulin heterodimer serves as the scaffold for functional toxisome assembly in F. graminearum.     

Anti-cancer activities of tea epigallocatechin-3-gallate in breast cancer patients under radiotherapy

The purpose of this study was to test the hypothesis that administration of epigallocatechin-3-gallate (EGCG), a polyphenol present in abundance in widely consumed tea, inhibits cell proliferation, invasion, and angiogenesis in breast cancer patients. EGCG in 400 mg capsules was orally administered three times daily to breast cancer patients undergoing treatment with radiotherapy. Parameters related to cell proliferation, invasion, and angiogenesis were analyzed while blood samples were collected at different time points to determine efficacy of the EGCG treatment. Compared to patients who received radiotherapy alone, those given radiotherapy plus EGCG for an extended time period (two to eight weeks) showed significantly lower serum levels of vascular endothelial growth factor (VEGF), hepatocyte growth factor (HGF), and reduced activation of metalloproteinase-9 and metalloproteinase-2 (MMP9/MMP2). Addition of sera obtained from patients treated with combination of radiotherapy and EGCG feeding for 2-8 weeks to in vitro cultures of highly-metastatic human MDA-MB-231 breast cancer cells resulted in the following significant changes: (1) suppression of cell proliferation and invasion; (2) arrest of cell cycles at the G0/G1 phase; (3) reduction of activation of MMP9/MMP2, expressions of Bcl-2/Bax, c-Met receptor, NF-κB, and the phosphorylation of Akt. MDA-MB-231 cells exposed to 5-10 μM EGCG also showed significant augmentation of the apoptosis inducing effects of γ-radiation, concomitant with reduced NF-κB protein level and AKT phosphorylation. These results provide hitherto unreported evidence that EGCG potentiated efficacy of radiotherapy in breast cancer patients, and raise the possibility that this tea polyphenol has potential to be a therapeutic adjuvant against human metastatic breast cancer.     

Molecular weight dependence of the poly(L-lactide)/poly(D-lactide) Stereocomplex at the air-water interface

The molecular weight dependence of poly(L-lactide)/poly(D-lactide) (PLLA/PDLA) stereocomplex behavior at the air-water interface was studied by surface pressure-area (pi-A) isotherms and atomic force microscopy (AFM). It was found that the compression-induced sterecomplexation of a PDLA/PLLA equimolar blend with high molecular weight (M(w) = 1 x 10(6) and 9.8 x 10(5), respectively) could occur at the air-water interface. This result is in marked contrast with the stereocomplexation of PDLA/PLLA blends in the bulk from the melt or in solutions, where the homocrystallites of either PLLA or PDLA rather than stereocomplex crystallites will be formed preferentially when the molecular weights of both polymers are higher than 1 x 10(5). Unexpectedly, the Langmuir-Blodgett behavior of the PDLA/PLLA blend with lower molecular weight (M(w) = 4 x 10(3) and 3.2 x 10(3), respectively), which should be favored in the stereocomplex, was distinct from that of other higher molecular weight blends. AFM images clearly disclosed for the first time the morphological changes of the equimolar blends of PLLA and PDLA at the air-water interface induced by increasing the surface pressure of the monolayer. Of particular note, the bilayer mechanism for the plateau in the isotherm was directly verified by the AFM height images.     

Overexpression of BLCAP induces S phase arrest and apoptosis independent of p53 and NF-κB in human tongue carcinoma

Bladder cancer-associated protein gene (BLCAP) is a novel candidate tumor suppressor gene identified from the human bladder carcinoma. Our previous studies have shown that BLCAP overexpression could inhibit cell growth by inducing apoptosis in HeLa cells [Zuo Z, Zhao M, Liu J, Gao G, Wu X: Tumor Biol 27: 221–226, 2006]. Such evidence suggests the alterations in BLCAP may play an important role in tumorigenesis. To further study the biological function of the BLCAP gene, we constructed a recombinant retroviral vector encoding BLCAP cDNA. Overexpressed BLCAP, via stable infection of exogenous BLCAP, resulted in growth inhibition of the human tongue cancer cell line Tca8113 in vitro, accompanied by S phase cell cycle arrest and apoptosis. The growth inhibition was correlated with up-regulation of p21^WAF1/CIP1 expression and down-regulation of Bcl-XL and Bcl-2 expressions. However, p53 expression and NF-κB activity remained unchanged post infection. Furthermore, no changes in p53 phosphorylation at Ser46 and nuclear localization, which are critical to p53 function, were observed in BLCAP-overexpressed cells. Taken together, BLCAP may play a role not only in regulating cell proliferation but also in coordinating apoptosis and cell cycle via a novel way independent of p53 and NF-κB. Jun Yao and Li Duan contributed equally to this work.