Light harvesting, energy transfer and electron cycling of a native photosynthetic membrane adsorbed onto a gold surface

Photosynthetic membranes comprise a network of light harvesting and reaction center pigment-protein complexes responsible for the primary photoconversion reactions: light absorption, energy transfer and electron cycling. The structural organization of membranes of the purple bacterial species Rb. sphaeroides has been elucidated in most detail by means of polarized light spectroscopy and atomic force microscopy. Here we report a functional characterization of native and untreated membranes of the same species adsorbed onto a gold surface. Employing fluorescence confocal spectroscopy and light-induced electrochemistry we show that adsorbed membranes maintain their energy and electron transferring functionality. Gold-adsorbed membranes are shown to generate a steady high photocurrent of 10 μA/cm² for several minutes and to maintain activity for up to three days while continuously illuminated. The surface-adsorbed membranes exhibit a remarkable functionality under aerobic conditions, even when exposed to light intensities well above that of direct solar irradiation. The component at the interface of light harvesting and electron cycling, the LH1 complex, displays exceptional stability, likely contributing to the robustness of the membranes. Peripheral light harvesting LH2 complexes show a light intensity dependent decoupling from photoconversion. LH2 can act as a reversible switch at low-light, an increased emitter at medium light and photobleaches at high light.     

Evaluation of BM-573, a novel TXA2 synthase inhibitor and receptor antagonist, in a porcine model of myocardial ischemia-reperfusion

AIMS: To investigate whether BM-573 (N-tert-butyl-N'-[2-(4'-methylphenylamino)-5-nitro-benzenesulfonyl]urea), an original combined thromboxane A2 synthase inhibitor and receptor antagonist, prevents reperfusion injury in acutely ischemic pigs. METHODS: Twelve animals were randomly divided in two groups: a control group (n = 6) intravenously infused with vehicle, and a BM-573-treated group (n = 6) infused with BM-573 (10 mg kg(-1) h(-1)). In both groups, the left anterior descending (LAD) coronary artery was occluded for 60 min and reperfused for 240 min. Either vehicle or BM-573 was infused 30 min before LAD occlusion and throughout the experiment. Platelet aggregation induced by arachidonic acid ex vivo measured was prevented by BM-573. RESULTS: In both groups, LAD occlusion decreased cardiac output, ejection fraction, slope of stroke work--end-diastolic volume relationship, and induced end-systolic pressure-volume relationship (ESPVR) rightward shift, while left ventricular afterload increased. Ventriculo-arterial coupling and mechanical efficiency decreased. In both groups, reperfusion further decreased cardiac output and ejection fraction, while ESPVR displayed a further rightward shift. Ventriculo-arterial coupling and mechanical efficiency remained impaired. Area at risk, evidenced with Evans blue, was 33.2+/-3.4% of the LV mass (LVM) in both groups, and mean infarct size, revealed by triphenyltetrazolium chloride (TTC), was 27.3+/-2.6% of the LVM in the BM-573-treated group (NS). Histological examination and immunohistochemical identification of desmin revealed necrosis in the anteroseptal region similar in both groups, while myocardial ATP dosages and electron microscopy also showed that BM-573 had no cardioprotective effect. CONCLUSIONS: These data suggest that BM-573 failed to prevent reperfusion injury in acutely ischemic pigs.     

Structure-Function Correlation of G6, a Novel Small Molecule Inhibitor of Jak2: INDISPENSABILITY OF THE STILBENOID CORE*

Somatic mutations in the Jak2 protein, such as V617F, cause aberrant Jak/STAT signaling and can lead to the development of myeloproliferative neoplasms. This discovery has led to the search for small molecule inhibitors that target Jak2. Using structure-based virtual screening, our group recently identified a novel small molecule inhibitor of Jak2 named G6. Here, we identified a structure-function correlation of this compound. Specifically, five derivative compounds of G6 having structural similarity to the original lead compound were obtained and analyzed for their ability to (i) inhibit Jak2-V617F-mediated cell growth, (ii) inhibit the levels of phospho-Jak2, phospho-STAT3, and phospho-STAT5; (iii) induce apoptosis in human erythroleukemia cells; and (iv) suppress pathologic cell growth of Jak2-V617F-expressing human bone marrow cells ex vivo. Additionally, we computationally examined the interactions of these compounds with the ATP-binding pocket of the Jak2 kinase domain. We found that the stilbenoid core-containing derivatives of G6 significantly inhibited Jak2-V617F-mediated cell proliferation in a time- and dose-dependent manner. They also inhibited phosphorylation of Jak2, STAT3, and STAT5 proteins within cells, resulting in higher levels of apoptosis via the intrinsic apoptotic pathway. Finally, the stilbenoid derivatives inhibited the pathologic growth of Jak2-V617F-expressing human bone marrow cells ex vivo. Collectively, our data demonstrate that G6 has a stilbenoid core that is indispensable for maintaining its Jak2 inhibitory potential.     

Effective induction of anti-melanoma immunity following genetic vaccination with synthetic mRNA coding for the fusion protein EGFP.TRP2

RNA-based genetic immunization represents an alternative novel strategy for antigen-specific cancer vaccines. In the present paper we investigate the use of synthetic messenger RNA in an experimental melanoma model. We show that gene gun-based immunization using synthetic RNA mediates gene expression in the epidermis and effectively induces antigen-specific cellular and humoral immunity in mice in vivo. Importantly, bombardment of the skin with RNA coding for the melanocytic self-antigen TRP2 linked to the immunogenic protein EGFP was associated with protection against experimentally induced B16 melanoma lung metastases and vitiligo-like fur depigmentation. Our results provide a scientific basis for clinical trials using synthetic mRNA encoding melanocytic antigens linked to immunogenic helper proteins for vaccination of patients with melanoma. Julia Steitz and Cedrik M. Britten contributed equally to this work.     

Confidence-based Somatic Mutation Evaluation and Prioritization

Next generation sequencing (NGS) has enabled high throughput discovery of somatic mutations. Detection depends on experimental design, lab platforms, parameters and analysis algorithms. However, NGS-based somatic mutation detection is prone to erroneous calls, with reported validation rates near 54% and congruence between algorithms less than 50%. Here, we developed an algorithm to assign a single statistic, a false discovery rate (FDR), to each somatic mutation identified by NGS. This FDR confidence value accurately discriminates true mutations from erroneous calls. Using sequencing data generated from triplicate exome profiling of C57BL/6 mice and B16-F10 melanoma cells, we used the existing algorithms GATK, SAMtools and SomaticSNiPer to identify somatic mutations. For each identified mutation, our algorithm assigned an FDR. We selected 139 mutations for validation, including 50 somatic mutations assigned a low FDR (high confidence) and 44 mutations assigned a high FDR (low confidence). All of the high confidence somatic mutations validated (50 of 50), none of the 44 low confidence somatic mutations validated, and 15 of 45 mutations with an intermediate FDR validated. Furthermore, the assignment of a single FDR to individual mutations enables statistical comparisons of lab and computation methodologies, including ROC curves and AUC metrics. Using the HiSeq 2000, single end 50 nt reads from replicates generate the highest confidence somatic mutation call set.