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Automated behavior analysis are promising tools to overcome current assessment limitations in psychiatry. At 9 months of age, we recorded 32 infants with West syndrome (WS) and 19 typically developing (TD) controls during a standardized mother–infant interaction. We computed infant hand movements (HM), speech turn taking of both partners (vocalization, pause, silences, overlap) and motherese. Then, we assessed whether multimodal social signals and interactional synchrony at 9 months could predict outcomes (autism spectrum disorder (ASD) and intellectual disability (ID)) of infants with WS at 4 years. At follow-up, 10 infants developed ASD/ID (WS+). The best machine learning reached 76.47% accuracy classifying WS vs. TD and 81.25% accuracy classifying WS+ vs. WS−. The 10 best features to distinguish WS+ and WS− included a combination of infant vocalizations and HM features combined with synchrony vocalization features. These data indicate that behavioral and interaction imaging was able to predict ASD/ID in high-risk children with WS.

Introduction/Background Clinical trials in Epithelial Ovarian Cancer (EOC) frequently examine treatment at a particular phase of the patient’s disease trajectory. Few major studies have examined the management of patients from diagnosis to death. This international network was developed to compare treatment sequencing and outcomes from six centres across Europe and South Korea. Methodology This retrospective cohort study used longitudinal data collected from electronic medical records from 6 European and South Korean treatment centres. A standard protocol & common data model was developed to capture consistent data for patients diagnosed between January 2012 and December 2018, with a minimum of 12 months follow-up. Full treatment data was collected and categorized in programmes of care based on exemplar patient narratives. An extensive data harmonization process was implemented to ensure different country and site medical records were interpreted in a common manner. Overall survival (OS) and time to next treatment (TTNT) were estimated using Kaplan Meier methodology and outcomes stratified by categories of interest. Each site analysed their own EMR data and shared aggregated results for comparison. Results Table 1 demonstrates patient characteristics from six sites. In total the overall study cohort includes 2889 patients. Median age for each centre ranged from 53 to 67 years. The majority of patients were FIGO stage III (range 31% to 66%) and high-grade serous morphology (52% to 69.9%) Additional data on treatment pathways and outcomes for each centre will be presented. Conclusion Preliminary analysis from this network suggests a consistent profile of adults treated for EOC across most contributing treatment centres in Europe, but with some substantial differences compared to patients treated at centres in South Korea and Romania. The establishment of a common data model between sites across five different countries allows for detailed exploration of the factors influencing differences in patient management and treatment outcomes in ovarian cancer patients. Disclosures Sue Cheeseman: I receive consultancy fees from IQVIA Bethany Levick: I am an employee of IQVIA Eunji Nam: I have no conflict of interest to disclose Dongkyu Kim: I have no conflict of interest to disclose Roman Rouzier: I have no conflict of interest to disclose Claire Bonneau: I have no conflict of interest to disclose Paul Kubelac: served on a speaker’s bureau for Roche, Bristol-Myers Squibb, AstraZeneca, Novartis. Patriciu Achimas-Cadariu: I have no conflict of interest to disclose Jean-Marc Classe: I have no conflict of interest to disclose Francois Bocquet: I have no conflict of interest to disclose Sven Becker: I have no conflict of interest to disclose Mariana Guergova-Kuras: I am an employee of IQVIA Geoff Hall: I receive grant support and consultancy fees from IQVIA

e13554 Background: The value of real-world evidence derived from the care of patients managed outside the context of clinical trials is well recognised. However, the ability to link data from multiple centres, especially those from different countries, is complicated by complex legal and information governance differences. The Oncology Evidence Network is a collaboration of large hospital centres, with strong clinical informatics capabilities in six countries in Europe and Asia working with the support of an industrial partner to provide high quality, real world data reflecting routine clinical care. We have developed an efficient workflow based on a study-specific common data model (CDM) clinically validated at each site and analysed with a single analysis script, which embeds a set of data quality rules. Local implementation allows each centre to generate analytical outputs aligned across the different sites without the need for any patient level data to leave the participating site. This approach has been designed and tested in Epithelial Ovarian Cancer (EOC) patients. Methods: A CDM was agreed using expert advisors from each centre. Clinical alignment was achieved through iterative assessment of clinical vignettes, to ensure common definitions of clinical assessment, prognosis, and treatment algorithms in EOC patients. A data guide detailing variable level derivations and validation rules, general data coding principles, and conversions/codes from international coding systems was developed. The analysis scripts were implemented as a bespoke package (OpenOvary) in R. The package includes functions to validate the data against the CDM, and generate a standard output including tables, numerical summaries and Kaplan-Meier analysis of progression and overall survival. Results: 2,925 patient records from 6 centres across 6 countries were included in the study with 27 key data items curated by each centre. Treatment data is available detailing relevant surgical procedures and their outcomes, and regimens of SACT throughout patients’ care from diagnosis to death. Data completeness was generally high for key data items, with missing data ranging from 0-16% for FIGO stage at diagnosis and 0-14% for tumour morphology. The CDM and R script will be made publicly available for other centres to adopt and facilitate analysis of their local data. Conclusions: This collaboration has brought together a substantial body of data describing the care and outcomes for EOC patients. A CDM and flexible shared analysis approach enabled unified analysis and reporting whilst avoiding the transfer of patient level data and its pooling into a common database. The process of clinical and data alignment has generated a replicable model for rapid extension to other study centres to join the EOC study, or application to other disease areas.

5531 Background: Few major studies have examined and compared the management and outcomes of patients from diagnosis to death between countries. We have established an international collaboration across Europe and South Korea to compare treatment and outcomes in Epithelial Ovarian Cancer (EOC). Methods: Patients diagnosed with EOC between January 2012 and December 2018 (age ≥18), were included for analysis. Data from medical records from five European and a single South Korean treatment centre were extracted, standardised to a common data model and analysed at each centre using a common script developed in R. Time to each progression/recurrence event (defined as time to next treatment) and overall survival have been estimated using Kaplan Meier methodology and outcomes stratified by categories of interest. Changes in the use of anti-cancer therapies over time and the incidence of BRCA mutations and incidence/timing of second breast cancers have also been examined. Results: A total cohort of 2925 patients was identified with a median age at each centre of 53 to 67 years. Advanced disease (FIGO stage III - IV) (range 57% to 84%) and high-grade serous morphology (38% to 70%) were most common at each centre. The timing of surgery (primary, interval debulking or delayed) and the proportion of patients undergoing surgery varied with stage. Patients with stage I disease where most likely to undergo surgery (range 73% to 100%) and stage IV the least (range 39% to 84%). Median overall survival for high grade serous cancers ranged from 1.9 to 4.9 years, and for the whole cohort from 2.1 to 5.5 years. Median time to next treatment at first relapse for the whole cohort ranged from 14 to 22 months. Second breast cancers were noted in 6 to 17% of patients and the majority of these occurred before the diagnosis of EOC at a median time of 96 to 118 months prior to diagnosis of EOC. Additional data on treatment pathways, BRCA status and outcomes by line of therapy for each centre will be presented. Conclusions: Preliminary analysis of results across this network suggests a variation in patient populations between sites and substantial differences in both treatments and outcomes. The establishment of a common data model and the use of a common analytic script between sites across six different countries allows for detailed exploration of the factors influencing differences in patient management and treatment outcomes in ovarian cancer patients.

Background: Gastric cancer (GC) is the third leading cause of cancer death. Early detection is a key factor to reduce its mortality. Methods: We retrospectively collected pre- and postoperative serum samples as well as tumor tissues and adjacent normal tissues from 100 GC patients. Serum samples from non-cancerous patients were served as controls (n=50). A high-throughput protein detection technology, multiplex proximity extension assays (PEA), was applied to measure levels of over 300 proteins. Alteration of each protein was analyzed by univariate analysis. Elastic-net logistic regression was performed to select serum proteins into the diagnostic model. Findings: We identified 19 serum proteins (CEA, CA9, MSLN, CCL20, SCF, TGF-alpha, MMP-1, MMP-10, IGF-1, CDCP1, PPIA, DDAH-1, HMOX-1, FLI1, IL-7, ZBTB-17, APBB1IP, KAZALD-1, and ADAMTS-15) that together distinguish the GC cases from controls with a diagnostic sensitivity of 93%, specificity of 100%, and area under receiver operating characteristic curve (AUC) of 0·99 (95% CI: 0·98-1). Moreover, the 19-serum protein signature provided a satisfactory diagnostic capacity in patients at TNM I-II stage (sensitivity 89%, specificity 100%, AUC 0·988) and in patients with high microsatellite instability (91%, 98%, and 0·990). A large-scale independent cohort of cases and controls will be necessary for validating the proposed protein signature. Interpretation: Based on targeted proteomics and elastic-net logistic regression, we identified a 19-serum protein signature which could contribute to clinical GC diagnosis. Funding Statement: This study was supported by a Marie Curie ITN grant (316929, GastricGlycoExplorer). Funder had no influence on trial design, data evaluation, and interpretation. Declaration of Interests: The authors declare that they have no competing interests. Ethics Approval Statement: This study was conducted following the Declaration of Helsinki. All patients were provided with the informed consent and ethical approval was granted by the Institutional Ethics Committee of University of Siena.

International audience; Introduction: The identification of frequent acquired mutations shows that patients with oligodendrogliomas have divergent biology with differing prognoses regardless of histological classification. A better understanding of molecular features as well as their metabolic pathways is essential.Objectives: The aim of this study was to examine the relationship between the tumor metabolome, six genomic aberrations (isocitrate dehydrogenase1 [IDH1] mutation, 1p/19q codeletion, tumor protein p53 [TP53] mutation, O6-methylguanin-DNA methyltransferase [MGMT] promoter methylation, epidermal growth factor receptor [EGFR] amplification, phosphate and tensin homolog [PTEN] methylation), and the patients' survival time.Methods: We applied 1H high-resolution magic-angle spinning (HRMAS) nuclear magnetic resonance (NMR) spectroscopy to 72 resected oligodendrogliomas.Results: The presence of IDH1, TP53, 1p19q codeletion, MGMT promoter methylation reduced the relative risk of death, whereas PTEN methylation and EGFR amplification were associated with poor prognosis. Increased concentration of 2-hydroxyglutarate (2HG), N-acetyl-aspartate (NAA), myo-inositol and the glycerophosphocholine/phosphocholine (GPC/PC) ratio were good prognostic factors. Increasing the concentration of serine, glycine, glutamate and alanine led to an increased relative risk of death.Conclusion: HRMAS NMR spectroscopy provides accurate information on the metabolomics of oligodendrogliomas, making it possible to find new biomarkers indicative of survival. It enables rapid characterization of intact tissue and could be used as an intraoperative method.

Microsatellite instability (MSI) is a distinct molecular subtype of gastric cancer. In recent years, the clinical consequences of MSI and the therapeutic opportunities to target this peculiar cancer subtype became evident. However, despite the importance of MSI for the stratification of patients, the time and resources required for diagnosis still present an obstacle. In an attempt to identify a new marker for MSI in gastric cancer, we evaluated the expression of five cancer-associated glycan epitopes in a cohort of 13 MSI and 17 microsatellite stable (MSS) cases. Our analysis revealed a highly significant (p < 0.001) association between the expression of the Thomsen-Friedenreich (TF) antigen and MSI status. Hence, we present here the identification of the first single marker for MSI in gastric cancer, excelling with a specificity of 94% (16/17), sensitivity of 69.2% (9/13), negative predictive value of 80% (16/20), and positive predictive value of 90% (9/10). The TF antigen, detected by simple antibody-based assays, is highly specific for carcinoma being undetectable in gastric healthy and premalignant epithelia. This finding lays the basis for new studies and holds promise in improving the rapid identification of MSI in the clinical setting.

Dengue remains an unmet public health burden. We determined risk factors for dengue in-hospital mortality in Brazil. Of 326,380 hospitalised dengue cases in 9-45-year-old individuals, there were 971 deaths. Risk of dying was 11-times higher in the presence of underlying common comorbidities (renal, infectious, pulmonary disease and diabetes), similar to the risk of dying from severe dengue and much higher with the combination. Ensuring access to integrated dengue preventative measures in individuals aged ≥ 9 years including those with comorbidities may help achieve the WHO objective of 50% reduction in mortality and 25% reduction in morbidity due to dengue by 2020.

Recent progress in understanding the Q-cycle mechanism of the bc(1) complex is reviewed. The data strongly support a mechanism in which the Q(o)-site operates through a reaction in which the first electron transfer from ubiquinol to the oxidized iron-sulfur protein is the rate-determining step for the overall process. The reaction involves a proton-coupled electron transfer down a hydrogen bond between the ubiquinol and a histidine ligand of the [2Fe-2S] cluster, in which the unfavorable protonic configuration contributes a substantial part of the activation barrier. The reaction is endergonic, and the products are an unstable ubisemiquinone at the Q(o)-site, and the reduced iron-sulfur protein, the extrinsic mobile domain of which is now free to dissociate and move away from the site to deliver an electron to cyt c(1) and liberate the H(+). When oxidation of the semiquinone is prevented, it participates in bypass reactions, including superoxide generation if O(2) is available. When the b-heme chain is available as an acceptor, the semiquinone is oxidized in a process in which the proton is passed to the glutamate of the conserved -PEWY- sequence, and the semiquinone anion passes its electron to heme b(L) to form the product ubiquinone. The rate is rapid compared to the limiting reaction, and would require movement of the semiquinone closer to heme b(L) to enhance the rate constant. The acceptor reactions at the Q(i)-site are still controversial, but likely involve a "two-electron gate" in which a stable semiquinone stores an electron. Possible mechanisms to explain the cyt b(150) phenomenon are discussed, and the information from pulsed-EPR studies about the structure of the intermediate state is reviewed. The mechanism discussed is applicable to a monomeric bc(1) complex. We discuss evidence in the literature that has been interpreted as shown that the dimeric structure participates in a more complicated mechanism involving electron transfer across the dimer interface. We show from myxothiazol titrations and mutational analysis of Tyr-199, which is at the interface between monomers, that no such inter-monomer electron transfer is detected at the level of the b(L) hemes. We show from analysis of strains with mutations at Asn-221 that there are coulombic interactions between the b-hemes in a monomer. The data can also be interpreted as showing similar coulombic interaction across the dimer interface, and we discuss mechanistic implications.

In photosystem I, oxidation of reduced acceptor A1– through iron-sulfur cluster FX is biphasic with half-times of ∼5–30 ns (“fast” phase) and ∼150–300 ns (“slow” phase). Whether these biphasic kinetics reflect unidirectional electron transfer, involving only the PsaA-side phylloquinone or bi-directional electron transfer, involving both the PsaA- and PsaB-side phylloquinones, has been the source of some controversy. Brettel (Brettel, K. (1988) FEBS Lett. 239, 93–98) and Joliot and Joliot (Joliot, P., and Joliot, A. (1999) Biochemistry 38, 11130–11136) have attributed to nearby carotenoids electrochromic band shifts, accompanying A1 reduction, centered at ∼450 and 500–510 nm. As a test of these assignments, we separately deleted in Synechocystis sp. PCC 6803 the genes that encode phytoene desaturase (encoded by crtP (pds)) and ζ-carotene desaturase (encoded by crtQ (zds)). The pds– and zds– strains synthesize phytoene and ζ-carotene, respectively, both of which absorb to shorter wavelength than β-carotene. Compared with wild type, the mutant A1– (FeS) – A1(FeS)– difference spectra, measured in cells and photosystem I complexes, retain the electrochromic band shift centered at 450 nm but show a complete loss of the electrochromic band shifts centered at 500–510 nm. Thus, the latter clearly arise from β-carotene. In the wild type, the electrochromic band shift of the slow phase (centered at 500 nm) is shifted by 6 nm to the blue compared with the fast phase (centered at 506 nm). Thus, the carotenoid pigments acting as electrochromic markers during the fast and slow phases of A1– oxidation are different, indicating the involvement of both the PsaA- and the PsaB-side phylloquinones in photosystem I electron transport.

The directionality of electron transfer in Photosystem I (PS I) is investigated using site-directed mutations in the phylloquinone (QK) and FX binding regions of Synnechocystis sp. PCC 6803. The kinetics of forward electron transfer from the secondary acceptor A1 (phylloquinone) were measured in mutants using time-resolved optical difference spectroscopy and transient EPR spectroscopy. In whole cells and PS I complexes of the wild-type both techniques reveal a major, slow kinetic component of tau approximately 300 ns while optical data resolve an additional minor kinetic component of tau approximately 10 ns. Whole cells and PS I complexes from the W697FPsaA and S692CPsaA mutants show a significant slowing of the slow kinetic component, whereas the W677FPsaB and S672CPsaB mutants show a less significant slowing of the fast kinetic component. Transient EPR measurements at 260 K show that the slow phase is approximately 3 times slower than at room temperature. Simulations of the early time behavior of the spin polarization pattern of P700+A1-, in which the decay rate of the pattern is assumed to be negligibly small, reproduce the observed EPR spectra at 260 K during the first 100 ns following laser excitation. Thus any spin polarization from P700+FX- in this time window is very weak. From this it is concluded that the relative amplitude of the fast phase is negligible at 260 K or its rate is much less temperature-dependent than that of the slow component. Together, the results demonstrate that the slow kinetic phase results from electron transfer from QK-A to FX and that this accounts for at least 70% of the electrons. Although the assignment of the fast kinetic phase remains uncertain, it is not strongly temperature dependent and it represents a minor fraction of the electrons being transferred. All of the results point toward asymmetry in electron transfer, and indicate that forward transfer in cyanobacterial PS I is predominantly along the PsaA branch.

In Rhodobacter sphaeroides, transfer of the first electron in quinol oxidation by the bc(1) complex shows kinetic features (a slow rate (approx. 1.5 x 10(3)/s), high activation energy (approx. 65 kJ/mol) and reorganization energy, lambda (2.5 V)) that are unexpected from Marcus theory and the distances shown by the structures. Reduction of the oxidized iron-sulfur protein occurs after formation of the enzyme-substrate complex, and involves a H-transfer in which the electron transfer occurs through the approx. 7 A of a bridging histidine forming a H-bond with quinol and a ligand to 2Fe-2S. The anomalous kinetic features can be explained by a mechanism in which the electron transfer is constrained by coupled transfer of the proton. We discuss this in the context of mutant strains with modified E(m,7) and pK for the iron-sulfur protein, and Marcus theory for proton-coupled electron transfer. We suggest that transfer of the second proton and electron involve movement of semiquinone in the Q(o) site, and rotation of the Glu of the conserved -PEWY- sequence. Mutational studies show a key role for the domain proximal to heme b(L). The effects of mutation at Tyr-302 (Tyr-279 in bovine sequence) point to a possible linkage between conformational changes in the proximal domain, and changes leading to closure of the iron-sulfur protein access channel at the distal domain.

In the Rieske iron-sulfur protein (ISP) of the ubiquinol:cytochrome c(2) oxidoreductase (bc(1) complex) of Rhodobacter sphaeroides, residue Tyr 156 is located close to the iron-sulfur cluster. Previous studies of the equivalent residue in both Saccharomyces cerevisiae [Denke, E., Merbitz-Zahradnik, T., Hatzfeld, O. M., Snyder, C. H., Link, T. A., and Trumpower, B. L. (1998) J. Biol. Chem. 273, 9085-9093] and Paracoccus denitrificans [Schroter, T., Hatzfeld, O. M., Gemeinhardt, S., Korn, M., Friedrich, T., Ludwig, B. , and Link, T. A. (1998) Eur. J. Biochem. 255, 100-106] have indicated that mutations at this site can lead to modifications in the redox potential of the ISP. To study the effect of similar modifications on the thermodynamic behavior and kinetics of partial reactions of the bc(1) complex upon flash activation, we have constructed four mutant strains of Rb. sphaeroides where Tyr 156 was mutated to His, Leu, Phe, or Trp. The bc(1) complex was assembled and able to support photosynthetic growth in all mutants. Three substitutions (Leu, Phe, Trp) led to alteration of the midpoint potential (E(m)) of the ISP and a slowing in rate of quinol oxidation, suggesting that electron transfer from quinol to the oxidized ISP controls the overall rate and that this step includes the high activation barrier. The Trp mutation led to an increase of approximately 1 pH unit in the pK value of the oxidized ISP. The pH dependence of the rate of quinol oxidation in this mutant was also shifted up by approximately 1 pH unit, showing the importance of the protonation state of the ISP for quinol oxidation. This provides support for a model in which the dissociated form of the oxidized ISP is required for formation of the enzyme-substrate complex [Ugulava, N., and Crofts, A. R. (1998) FEBS Lett. 440, 409-413].

Structures of mitochondrial ubihydroquinone:cytochrome c oxidoreductase (bc(1) complex) from several animal sources have provided a basis for understanding the functional mechanism at the molecular level. Using structures of the chicken complex with and without inhibitors, we analyze the effects of mutation on quinol oxidation at the Q(o) site of the complex. We suggest a mechanism for the reaction that incorporates two features revealed by the structures, a movement of the iron sulfur protein between two separate reaction domains on cytochrome c(1) and cytochrome b and a bifurcated volume for the Q(o) site. The volume identified by inhibitor binding as the Q(o) site has two domains in which inhibitors of different classes bind differentially; a domain proximal to heme b(L), where myxothiazole and beta-methoxyacrylate- (MOA-) type inhibitors bind (class II), and a distal domain close to the iron sulfur protein docking interface, where stigmatellin and 5-n-undecyl-6-hydroxy-4,7-dioxobenzothiaole (UHDBT) bind (class I). Displacement of one class of inhibitor by another is accounted for by the overlap of their volumes, since the exit tunnel to the lipid phase forces the hydrophobic "tails" to occupy common space. We conclude that the site can contain only one "tailed" occupant, either an inhibitor or a quinol or one of their reaction products. The differential sensitivity of strains with mutations in the different domains is explained by the proximity of the affected residues to the binding domains of the inhibitors. New insights into mechanism are provided by analysis of mutations that affect changes in the electron paramagnetic resonance (EPR) spectrum of the iron sulfur protein, associated with its interactions with the Q(o)-site occupant. The structures show that all interactions with the iron sulfur protein must occur at the distal position. These include interactions between quinone, or class I inhibitors, and the reduced iron sulfur protein and formation of a reaction complex between quinol and oxidized iron sulfur protein. The step with high activation energy is after formation of the reaction complex, likely in formation of the semiquinone and subsequent dissociation of the complex into products. We suggest that further progress of the reaction requires a movement of semiquinone to the proximal position, thus mapping the bifurcated reaction to the bifurcated volume. We suggest that such a movement, together with a change in conformation of the site, would remove any semiquinone formed from further interaction with the oxidized [2Fe-2S] center and also from reaction with O(2) to form superoxide anion. We also identify two separate reaction paths for exit of the two protons released in quinol oxidation.

Activation energies for partial reactions involved in oxidation of quinol by the bc(1) complex were independent of pH in the range 5. 5-8.9. Formation of enzyme-substrate complex required two substrates, ubihydroquinone binding from the lipid phase and the extrinsic domain of the iron-sulfur protein. The activation energy for ubihydroquinone oxidation was independent of the concentration of either substrate, showing that the activated step was in a reaction after formation of the enzyme-substrate complex. At all pH values, the partial reaction with the limiting rate and the highest activation energy was oxidation of bound ubihydroquinone. The pH dependence of the rate of ubihydroquinone oxidation reflected the pK on the oxidized iron-sulfur protein and requirement for the deprotonated form in formation of the enzyme-substrate complex. We discuss different mechanisms to explain the properties of the bifurcated reaction, and we preclude models in which the high activation barrier is in the second electron transfer or is caused by deprotonation of QH(2). Separation to products after the first electron transfer and movement of semiquinone formed in the Q(o) site would allow rapid electron transfer to heme b(L). This would also insulate the semiquinone from oxidation by the iron-sulfur protein, explaining the efficiency of bifurcation.

It is generally considered that metabolic reactions are well described by homogeneous kinetic models in which the reaction phase is statistically uniform. In membranes, especially in photosynthetic systems where the protein complement is high, it has recently been recognized that effects of local heterogeneity might contribute additional factors that perturb the kinetic behavior, and require more extensive treatment. We show in this paper that statistical heterogeneity in vesicular systems can also contribute to quite marked discrepancies from the behavior expected from standard kinetic and thermodynamic models, for reactions involving free diffusion in the aqueous phase. We explain the kinetic and thermodynamic effects observed in studies of photosynthetic electron transfer in cells and chromatophores from Rhodobacter sphaeroides previously attributed to supercomplexes, in terms of a model based on heterogeneity in distribution of electron transfer components among the chromatophore population. We discuss examples of data inconsistent with the supercomplex model, but well explained by the heterogeneity model.

A challenge in the treatment of lung cancer is the lack of early diagnostics. Here, we describe the application of monoclonal antibody proteomics for discovery of a panel of biomarkers for early detection (stage I) of non-small cell lung cancer (NSCLC). We produced large monoclonal antibody libraries directed against the natural form of protein antigens present in the plasma of NSCLC patients. Plasma biomarkers associated with the presence of lung cancer were detected via high throughput ELISA. Differential profiling of plasma proteomes of four clinical cohorts, totaling 301 patients with lung cancer and 235 healthy controls, identified 13 lung cancer-associated (p < 0.05) monoclonal antibodies. The monoclonal antibodies recognize five different cognate proteins identified using immunoprecipitation followed by mass spectrometry. Four of the five antigens were present in non-small cell lung cancer cells in situ. The approach is capable of generating independent antibodies against different epitopes of the same proteins, allowing fast translation to multiplexed sandwich assays. Based on these results, we have verified in two independent clinical collections a panel of five biomarkers for classifying patient disease status with a diagnostics performance of 77% sensitivity and 87% specificity. Combining CYFRA, an established cancer marker, with the panel resulted in a performance of 83% sensitivity at 95% specificity for stage I NSCLC.

A mass spectrometric (MS)-based strategy for antigen (Ag) identification and characterization of globally produced monoclonal antibodies (mAbs) is described. Mice were immunized with a mixture of native glycoproteins, isolated from the pooled plasma of patients with non-small cell lung cancer (NSCLC), to generate a library of IgG-secreting hybridomas. Prior to immunization, the pooled NSCLC plasma was subjected to 3-sequential steps of affinity fractionation, including high abundant plasma protein depletion, glycoprotein enrichment and polyclonal antibody affinity chromatography normalization. In this paper, in order to demonstrate the high quality of the globally produced mAbs, we selected 3 mAbs of high differentiating power against a matched, pooled normal plasma sample. After production of large quantities of the mAbs from ascites fluids, Ag identification was achieved by immunoaffinity purification, SDS-PAGE, Western blotting and MS analysis of in-gel digest products. One antigen was found to be complement factor H, and the other two were mapped to different subunits of haptoglobin (Hpt). The 2 Hpt mAbs were characterized in detail in order to assess the quality of the mAbs produced by the global strategy. The affinity of one of the mAbs to the Hpt native tetramer form was found to have a KD of roughly 10−9 M and to be 2 orders of magnitude lower than the reduced form, demonstrating the power of the mAb proteomics technology in generating mAbs to the natural form of the proteins in blood. The binding of this mAb to the β-chain of haptoglobin was also dependent on glycosylation on this chain. The characterization of mAbs in this work reveals that the global mAb proteomics process can generate high-quality lung cancer specific mAbs capable of recognizing proteins in their native state.

In photosystem I, oxidation of reduced acceptor A(1)(-) through iron-sulfur cluster F(X) is biphasic with half-times of approximately 5-30 ns ("fast" phase) and approximately 150-300 ns ("slow" phase). Whether these biphasic kinetics reflect unidirectional electron transfer, involving only the PsaA-side phylloquinone or bi-directional electron transfer, involving both the PsaA- and PsaB-side phylloquinones, has been the source of some controversy. Brettel (Brettel, K. (1988) FEBS Lett. 239, 93-98) and Joliot and Joliot (Joliot, P., and Joliot, A. (1999) Biochemistry 38, 11130-11136) have attributed to nearby carotenoids electrochromic band shifts, accompanying A(1) reduction, centered at approximately 450 and 500-510 nm. As a test of these assignments, we separately deleted in Synechocystis sp. PCC 6803 the genes that encode phytoene desaturase (encoded by crtP (pds)) and zeta-carotene desaturase (encoded by crtQ (zds)). The pds(-) and zds(-) strains synthesize phytoene and zeta-carotene, respectively, both of which absorb to shorter wavelength than beta-carotene. Compared with wild type, the mutant A(1)(-) (FeS) - A(1)(FeS)(-) difference spectra, measured in cells and photosystem I complexes, retain the electrochromic band shift centered at 450 nm but show a complete loss of the electrochromic band shifts centered at 500-510 nm. Thus, the latter clearly arise from beta-carotene. In the wild type, the electrochromic band shift of the slow phase (centered at 500 nm) is shifted by 6 nm to the blue compared with the fast phase (centered at 506 nm). Thus, the carotenoid pigments acting as electrochromic markers during the fast and slow phases of A(1)(-) oxidation are different, indicating the involvement of both the PsaA- and the PsaB-side phylloquinones in photosystem I electron transport.

The directionality of electron transfer in Photosystem I (PS I) is investigated using site-directed mutations in the phylloquinone (QK) and FX binding regions of Synnechocystis sp. PCC 6803. The kinetics of forward electron transfer from the secondary acceptor A1 (phylloquinone) were measured in mutants using time-resolved optical difference spectroscopy and transient EPR spectroscopy. In whole cells and PS I complexes of the wild-type both techniques reveal a major, slow kinetic component of tau approximately 300 ns while optical data resolve an additional minor kinetic component of tau approximately 10 ns. Whole cells and PS I complexes from the W697FPsaA and S692CPsaA mutants show a significant slowing of the slow kinetic component, whereas the W677FPsaB and S672CPsaB mutants show a less significant slowing of the fast kinetic component. Transient EPR measurements at 260 K show that the slow phase is approximately 3 times slower than at room temperature. Simulations of the early time behavior of the spin polarization pattern of P700+A1-, in which the decay rate of the pattern is assumed to be negligibly small, reproduce the observed EPR spectra at 260 K during the first 100 ns following laser excitation. Thus any spin polarization from P700+FX- in this time window is very weak. From this it is concluded that the relative amplitude of the fast phase is negligible at 260 K or its rate is much less temperature-dependent than that of the slow component. Together, the results demonstrate that the slow kinetic phase results from electron transfer from QK-A to FX and that this accounts for at least 70% of the electrons. Although the assignment of the fast kinetic phase remains uncertain, it is not strongly temperature dependent and it represents a minor fraction of the electrons being transferred. All of the results point toward asymmetry in electron transfer, and indicate that forward transfer in cyanobacterial PS I is predominantly along the PsaA branch.

The dependence of the P(700)(+)/P(700) midpoint potential on kinetics of reduction of P(700)(+) in vivo has been examined in a series of site-directed mutants of Chlamydomonas reinhardtii in which the histidyl axial ligand to the Mg(2+) of the P(700) chlorophyll a has been changed to several different amino acids. In wild-type photosystem I, the potential of P(700)(+)/P(700) is 447 mV and the in vivo half-time of P(700)(+) reduction by its natural donor, plastocyanin, is 4 micros. Substitution of the axial histidine ligand with cysteine increases the potential of P(700)(+)/P(700) to 583 mV and changes the rate of P(700)(+) reduction to 0.8 micros. Mutants with a range of potentials between 447 and 583 mV show a strong correlation of the P(700)(+)/P(700) potential to the rate of reduction of P(700)(+) by plastocyanin. There is also an increase in the rate of photosystem I-mediated electron transfer from the artificial electron donor DCPIP to methyl viologen in thylakoid membranes. The results indicate that the overall rate constant of P(700)(+) reduction is determined by the rate of electron transfer between the copper and P(700)(+) and confirmed that in vivo there is a preformed complex between plastocyanin and photosystem I. Using approximations of the Marcus electron transfer theory, it is possible to estimate that the distance between the copper of plastocyanin and P(700)(+) is approximately 15 A. On the basis of this distance, the plastocyanin docking site should lie in a 10 A hollow formed by the lumenal exposed loops between transmembrane helices i and j of PsaA and PsaB.

The mechanism of charge recombination of the S(2)Q(A)(-) state in photosystem II was investigated by modifying the free energy gap between the quinone acceptor Q(A) and the primary pheophytin acceptor Ph. This was done either by changing the midpoint potential of Ph (using mutants of the cyanobacterium Synechocystis with a modified hydrogen bond to this cofactor), or that of Q(A) (using different inhibitors of the Q(B) pocket). The results show that the recombination rate is dependent on the free energy gap between Ph and Q(A), which confirms that the indirect recombination pathway involving formation of Ph(-) has a significant contribution. In the mutant with the largest free energy gap, direct electron transfer from Q(A)(-) to P(+) predominates. The temperature dependence of the recombination rate was investigated, showing a lower activation enthalpy in this mutant compared with the WT. The data allow the determination of the rate of the direct route and of its relative weight in the various strains. The set of currently accepted values for the midpoint potentials of the Q(A)/Q(A)(-), Ph/Ph(-), and P(+)/P* couples is not consistent with the relatively rapid rate of the indirect recombination pathway found here, nor with the 3% yield of delayed fluorescence as previously estimated by de Grooth and van Gorkom (1981, Biochim. Biophys. Acta 635, 445-456). It is argued that a likely explanation is that the midpoint potentials of the two latter couples are more positive than believed due to electrostatic interactions. If such is the case, the estimation of the midpoint potential of the P(+)/P and S(2)/S(1) couples must also be revised upward, with values of 1260 and 1020 mV, respectively.

▪ Abstract The cytochrome bc complexes represent a phylogenetically diverse group of complexes of electron-transferring membrane proteins, most familiarly represented by the mitochondrial and bacterial bc1complexes and the chloroplast and cyanobacterial b6f complex. All these complexes couple electron transfer to proton translocation across a closed lipid bilayer membrane, conserving the free energy released by the oxidation-reduction process in the form of an electrochemical proton gradient across the membrane. Recent exciting developments include the application of site-directed mutagenesis to define the role of conserved residues, and the emergence over the past five years of X-ray structures for several mitochondrial complexes, and for two important domains of the b6f complex.

Native structures of ubihydroquinone:cytochrome c oxidoreductase (bc(1) complex) from different sources, and structures with inhibitors in place, show a 16-22 A displacement of the [2Fe-2S] cluster and the position of the C-terminal extrinsic domain of the iron sulfur protein. None of the structures shows a static configuration that would allow catalysis of all partial reactions of quinol oxidation. We have suggested that the different conformations reflect a movement of the subunit necessary for catalysis. The displacement from an interface with cytochrome c(1) in native crystals to an interface with cytochrome b is induced by stigmatellin or 5-n-undecyl-6-hydroxy-4,7-dioxobenzothiazole (UHDBT) and involves ligand formation between His-161 of the [2Fe-2S] binding cluster and the inhibitor. The movement is a rotational displacement, so that the same conserved docking surface on the iron sulfur protein interacts with cytochrome c(1) and with cytochrome b. The mobile extrinsic domain retains essentially the same tertiary structure, and the anchoring N-terminal tail remains in the same position. The movement occurs through an extension of a helical segment in the short linking span. We report details of the protein structure for the two main configurations in the chicken heart mitochondrial complex and discuss insights into mechanism provided by the structures and by mutant strains in which the docking at the cytochrome b interface is impaired. The movement of the iron sulfur protein represents a novel mechanism of electron transfer, in which a tethered mobile head allows electron transfer through a distance without the entropic loss from free diffusion.

Quinol oxidation by the bc 1 complex of Rhodobacter sphaeroides occurs from an enzyme–substrate complex formed between quinol bound at the Q o site and the iron–sulfur protein (ISP) docked at an interface on cytochrome b . From the structure of the stigmatellin-containing mitochondrial complex, we suggest that hydrogen bonds to the two quinol hydroxyl groups, from Glu-272 of cytochrome b and His-161 of the ISP, help to stabilize the enzyme–substrate complex and aid proton release. Reduction of the oxidized ISP involves H transfer from quinol. Release of the proton occurs when the acceptor chain reoxidizes the reduced ISP, after domain movement to an interface on cytochrome c 1 . Effects of mutations to the ISP that change the redox potential and/or the pK on the oxidized form support this mechanism. Structures for the complex in the presence of inhibitors show two different orientations of Glu-272. In stigmatellin-containing crystals, the side chain points into the site, to hydrogen bond with a ring hydroxyl, while His-161 hydrogen bonds to the carbonyl group. In the native structure, or crystals containing myxothiazol or β-methoxyacrylate-type inhibitors, the Glu-272 side chain is rotated to point out of the site, to the surface of an external aqueous channel. Effects of mutation at this residue suggest that this group is involved in ligation of stigmatellin and quinol, but not quinone, and that the carboxylate function is essential for rapid turnover. H + transfer from semiquinone to the carboxylate side chain and rotation to the position found in the myxothiazol structure provide a pathway for release of the second proton.

The fbcB and fbcC genes encoding cytochromes b and c1 of the bc1 complex were extended with a segment to encode a polyhistidine tag linked to their C-terminal sequence allowing a one-step affinity purification of the complex. Constructions were made in vitro in a pUC-derived background using PCR amplification. The modified fbc operons were transferred to a pRK derivative plasmid, and this was used to transform the fbc- strain of Rhodobacter sphaeroides, BC17. The transformants showed normal rates of growth. Chromatophores prepared from these cells showed kinetics of turnover of the bc1 complex on flash activation which were essentially the same as those from wild-type strains, and analysis of the cytochrome complement and spectral and thermodynamic properties by redox potentiometry showed no marked difference from the wild type. Chromatophores were solubilized and mixed with Ni-NTA-Sepharose resin. A modification of the standard elution protocol in which histidine replaced imidazole increased the activity 20-fold. Imidazole modified the redox properties of heme c1, suggesting ligand displacement and inactivation when this reagent is used at high concentration. The purified enzyme contained all four subunits in an active dimeric complex. This construction provides a facile method for preparation of wild-type or mutant bc1 complex, for spectroscopy and structural studies.

Ubiquinolxytochrome c oxidoreductase (bc1 -complex) is a central component of the electron transfer system in almost all organisms, occurring ubiquitously in respiratory and photosynthetic chains of mitochondria and bacteria, and (as the b6 f-complex) in the photo-synthetic chain of oxygenic photosynthesis (see refs. 1-3 for recent reviews). A modified Q-cycle (4—9) accounts well for a large body of experimental data from studies in which the function has been explored. The mechanism involves two catalytic sites for oxidation or reduction of the quinone couple, and a third site for electron transfer to cytochrome c. The function of the two quinone reactive sites has been explored biochemically, and characterized using kinetic spectroscopy, and their activities differentiated through use of inhibitors that act specifically at one site or the other (1-13). The quinol oxidizing site (Qo -site) catalyzes a unique reaction in which the electrons from QH2 are passed to separate electron transport chains in the complex through a bifurcated reaction. Further information on the occupancy of the Qo -site has been obtained though study of the interaction of the occupant with the 2Fe2S center of the reduced ISP, observed through changes in the EPR spectrum of this center (33-40). In addition to the effects observed with stigmatellin and UHDBT (10-13, 33), a well-defined band at gx = 1. 800 has been observed when quinone occupies the pocket. The dependence of these spectral changes on ambient redox potential, and on the extent of extraction of quinone, has lead Ding (34, 35) and colleagues to suggest a double occupancy of the site by weakly (Qow) and strongly (Qos) binding quinone species.

We have modified the cytochrome b subunit of the cytochrome bc1 complex from the purple bacterium Rhodobacter sphaeroides to introduce two distinctive features of cytochrome b6 f complexes. In the first one, we have split cyt b into two polypeptides thus mimicking the organization of cyt b6 and subunit IV in the b6 f complexes. In the second, an extra residue was added between His198 and Phe199, thus extending the span between the histidine ligands for the two b-hemes in helix D. The properties of the mutant strains were determined using thermodynamic and kinetic analysis. The two mutant enzymes were assembled and functioned so as to allow the photosynthetic growth of the mutant strains. For the split enzyme, we show that two independently translated fragments of cyt b are inserted in the membrane. Our results indicate a decrease in the stability of the semiquinone formed at the quinone reduction (Qi) site in this mutant. This property, characteristic for b6 f complexes, indicates the functional importance of the connecting span between helices D and E. The presence of the inserted threonine in helix D modified the spectrum and redox potential of the bL-heme, shifting the potential difference between the two b-hemes from 140 mV in the wild-type to 55 mV in the mutant strain. This change in the driving force of electron transfer through the membrane was reflected in an inability of the mutant strain to accumulate a large transmembrane electrical potential on successive flashes.

Structures for the bc1 complex (1) show several features of interest to catalysis at the Qo-site. The iron sulfur protein (ISP) occurs in several different positions: at a catalytic interface on cytochrome (cyt) b close to the Qo-site, in H-bond contact with a heme propionate of cyt c1, or in several intermediate positions (1–3). The Qo-site is a bifurcated volume, in which myxothiazol and MOA-inhibitors bind in a proximal domain near heme bL, but stigmatellin and UHDBT bind in a distal domain, where they interact with the ISP docked on cyt b. The structures also allow for the first time insights into the functional significance of mutations that modify turn-over or inhibitor binding. These structural observations have led us to suggest several new mechanistic features for the operation of the Qo-site, and have suggested experiments to test these, some of which we report here.

10561 Background: A challenge in the treatment of lung cancer is the lack of early, pre-symptomatic diagnosis. Here, we describe the application of monoclonal antibody proteomics approach to profil...