Large Structural Alterations Research Articles

Prognostic value of ultra-low-pass whole-genome sequencing of circulating tumor DNA in hepatocellular carcinoma under systemic treatment.

New prognostic markers are needed to identify patients with hepatocellular carcinoma (HCC) who carry a worse prognosis. Ultra-low-pass whole-genome sequencing (ULP-WGS) (≤0.5× coverage) of cell-free DNA (cfDNA) has emerged as a low-cost promising tool to assess both circulating tumor DNA (ctDNA) fraction and large structural genomic alterations. Here, we studied the performance of ULP-WGS of plasma cfDNA to infer prognosis in patients with HCC. Plasma samples were obtained from patients with HCC prior to surgery, locoregional or systemic therapy, and were analyzed by ULP-WGS of cfDNA to an average genome-wide fold coverage of 0.3x. ctDNA and copy number alterations (CNA) were estimated using the software package ichorCNA. Samples were obtained from 73 HCC patients at different BCLC stages (BCLC 0/A: n=37, 50.7%; BCLC B/C: n=36, 49.3%). ctDNA was detected in 18 out of 31 patients who received systemic treatment. Patients with detectable ctDNA showed significantly worse overall survival (median, 13.96 months vs not reached). ctDNA remained an independent predictor of prognosis after adjustment by clinical-pathologic features and type of systemic treatment (hazard ratio 7.69; 95%, CI 2.09-28.27). Among ctDNA-positive patients under systemic treatments, the loss of large genomic regions in 5q and 16q arms was associated with worse prognosis after multivariate analysis. ULP-WGS of cfDNA provides clinically relevant information about the tumor biology. The presence of ctDNA and the loss of 5q and 16q arms in ctDNA-positive patients are independent predictors of worse prognosis in patients with advanced HCC receiving systemic therapy.

Association of the interaction between mosaic chromosomal alterations and polygenic risk score with the risk of lung cancer: an array-based case-control association and prospective cohort study.

Mosaic chromosomal alterations (mCAs) detected from blood-derived DNA are large structural alterations of clonal haematopoietic origin and are associated with various diseases, such as haematological malignancies, infections, and solid cancers. We aimed to investigate whether mCAs contribute to the risk of lung cancer and modify the effect of polygenic risk score (PRS) on lung cancer risk prediction. The blood-derived DNA of patients with lung cancer and cancer-free controls with Chinese ancestry from the Nanjing Lung Cancer Cohort (NJLCC) study were genotyped with a Global Screening Array, and mCAs were detected with the Mosaic Chromosomal Alterations (MoChA) pipeline. mCA call sets of individuals with European ancestry were obtained from the prospective cohort UK Biobank (UKB) study, including documented incident lung cancer. All patients with lung cancer from the NJLCC study (aged 15 years or older at diagnosis) were histopathologically confirmed as new lung cancer cases by at least two pathologists and were free of chemotherapy or radiotherapy before diagnosis. Participants with incident lung cancer (aged 37-73 years at assessment) diagnosed after recruitment to the UKB were identified through linkage to national cancer registries. Logistic regression and Cox proportional hazard models were applied to evaluate associations between mCAs and risk of lung cancer in the NJLCC (logistic regression) and UKB (Cox proportional hazard model) studies. The NJLCC study included 10 248 individuals (6445 [62·89%] men and 3803 [37·11%] women; median age 60·0 years [IQR 53·0-66·0]) with lung cancer and 9298 individuals (5871 [63·14%] men and 3427 [36·86%] women; median age 60·0 years [52·0-65·0]) without lung cancer recruited from three sub-regions (north, central, and south) across China between April 15, 2003, and Aug 18, 2017. The UKB included 450 821 individuals recruited from 22 centres across the UK between March 13, 2006, and Nov 1, 2010, including 2088 individuals with lung cancer (1075 [51·48%] men and 1013 [48·52%] women; median age 63·0 years [IQR 59·0-66·0]), and 448 733 participants were free of lung cancer (204 713 [45·62%] men and 244 020 [54·38%] women; median age 58·0 years [IQR 50·0-63·0]). Compared with non-carriers of mosaic losses, carriers had a significantly increased risk of lung cancer in the NJLCC (odds ratio [OR] 1·81, 95% CI 1·43-2·28; p=6·69 × 10-7) and UKB (hazard ratio [HR] 1·40, 95% CI 1·00-1·95; p=0·048) studies. This increased risk was even higher in patients with expanded cell fractions of mCAs (ie, cell fractions ≥10% vs cell fractions <10%) in the NJLCC (OR 1·61 [95% CI 1·26-2·08] vs 1·03 [0·83-1·26]; p for heterogeneity test=6·41 × 10-3). A significant multiplicative interaction was observed between PRS and mosaic losses on the risk of lung cancer in both the NJLCC (interaction p value=0·030) and UKB (p=0·043). Compared with non-carriers of mosaic loss abnormalities with low genetic risk, participants with expanded mosaic losses (cell fractions ≥10%) and high genetic risk had around a six-times increased risk of lung cancer in the NJLCC study (OR 6·40 [95% CI 3·22-12·69]), and an almost four-times increased risk of lung cancer (HR 3·75 [95% CI 1·86-7·55]) in the UKB study. The additive interaction also contributed a 3·67 (95% CI 0·49-6·85) relative excess risk of developing lung cancer in the NJLCC study, and a 2·15 (0·12-4·19) relative excess risk in the UKB study. mCAs act as a new endogenous indicator for the risk of lung cancer and might be jointly used with PRS to optimise personalised risk stratification for lung cancer. National Natural Science Foundation of China, Outstanding Youth Foundation of Jiangsu Province, Natural Science Foundation of Jiangsu Province, and Postdoctoral Science Foundation of China. For the Chinese translation of the abstract see Supplementary Materials section.

In-utero exposure to zidovudine-containing antiretroviral therapy and clonal hematopoiesis in HIV-exposed uninfected newborns.

Zidovudine (ZDV) has been extensively used in pregnant women to prevent vertical transmission of HIV but few studies have evaluated potential mutagenic effects of ZDV during fetal development. Our study investigated clonal hematopoiesis in HIV-exposed uninfected (HEU) newborns, 94 of whom were ZDV-exposed and 91 antiretroviral therapy (ART)-unexposed and matched for potential confounding factors. Utilizing high depth sequencing and genotyping arrays, we comprehensively examined blood samples collected during the first week after birth for potential clonal hematopoiesis associated with fetal ZDV exposure, including clonal single nucleotide variants (SNVs), small insertions and deletions (indels), and large structural copy number or copy neutral alterations. We observed no statistically significant difference in the number of SNVs and indels per person in ZDV-exposed children (adjusted ratio [95% confidence interval, CI] for expected number of mutations = 0.79 [0.50--1.22], P = 0.3), and no difference in the number of large structural alterations. Mutations in common clonal hematopoiesis driver genes were not found in the study population. Mutational signature analyses on SNVs detected no novel signatures unique to the ZDV-exposed children and the mutational profiles were similar between the two groups. Our results suggest that clonal hematopoiesis at levels detectable in our study is not strongly influenced by in-utero ZDV exposure; however, additional follow-up studies are needed to further evaluate the safety and potential long-term impacts of in-utero ZDV exposure in HEU children as well as better investigate genomic aberrations occurring late in pregnancy.

Cerebral cortical thickness and surface area in adolescent anorexia nervosa: Separate and joint analyses with a permutation-based nonparametric method.

Reduction in cerebral volume is often found in underweight patients with anorexia nervosa (AN), but few studies have investigated other morphological measures. Cortical thickness (CTh) and surface area (CSA), often used to produce the measure of cortical volume, are developmentally distinct measures that may be differentially affected in AN, particularly in the developing brain. In the present study, we investigated CTh and CSA both separately and jointly to gain further insight into structural alterations in adolescent AN patients. Thirty female AN inpatients 12-18 years of age, and 27 age-matched healthy controls (HC) underwent structural magnetic resonance imaging. Group differences in CTh and CSA were investigated separately and jointly with a permutation-based nonparametric combination method (NPC) which may be more sensitive in detecting group differences compared to traditional volumetric methods. Results showed significant reduction in in both CTh and CSA in several cortical regions in AN compared to HC and the reduction was related to BMI. Different results for the two morphological measures were found in a small number of cortical regions. The joint NPC analyses showed significant group differences across most of the cortical mantle. Results from this study give novel insight to areal reduction in adolescent AN patients and indicate that both CTh and CSA reduction is related to BMI. The study is the first to use the NPC method to reveal large structural alterations covering most of the brain in adolescent AN.

Abstract 5893: Comprehensive proteogenomic analysis and classification of lung adenocarcinoma

Abstract Lung adenocarcinoma is a highly lethal tumor that displays extensive molecular heterogeneity of which deep characterization may drive therapeutic development and improve clinical outcomes. Through the Applied Proteogenomics Organizational Learning and Outcomes (APOLLO) research network, we utilized five molecular profiling technologies (DNA whole genome sequencing, RNA sequencing, total and phospho-proteomics by mass spectrometry, and reverse phase protein arrays [RPPA]) to characterize a longitudinally-annotated cohort of 87 lung adenocarcinomas. Through whole genome sequencing, we identified molecular signatures from patterns of somatic SNVs, indels, and large structural alterations that stratified tumors into three groups associated with patient smoke exposures. We also identified TP53, EGFR, KRAS, and STK11 as recurrently mutated genes, which together represent 80% of the cohort, in addition to genes mutated in smaller cohort subsets (e.g. RBM10), fusion genes, and pathogenic germline variants. To characterize tumor proteomes, we quantified &amp;gt;7,000 proteins and &amp;gt;10,000 phosphopeptides by mass spectrometry and &amp;gt;300 species by RPPA. Matched RNAs and proteins were typically positively correlated across samples (median ρ = 0.49). Through quantitative trait loci analyses, we identified genes whose RNA and protein expression levels were significantly modified by somatic mutations. We then classified tumors into RNA expression subtypes and found coordinated proteogenomic alterations and distinct clinical associations: terminal respiratory unit subtype – EGFR mutations and RNA/protein overexpression, acinar histology, non-smokers; proximal-proliferative subtype – STK11 mutations and RNA/protein underexpression, high smoking signature; proximal-inflammatory subtype – high tumor mutational burden. We also identified phospho-peptide signatures associated with these subtypes, including downregulation of CDK1/2 targets in terminal respiratory unit tumors. Protein co-expression network analysis discovered biologically-diverse pathway activities of the RNA expression subtypes. To interrogate somatic mutations in the context of molecular pathways, we projected DNA alterations onto known interaction networks and identified four subtypes with markedly distinct proteomic and microenvironment characteristics. Finally, several molecular characteristics were found to significantly predict patient outcomes, including RNA expression subtype classification against metastasis-free survival. Thus, our integrative, proteogenomic characterization of lung adenocarcinoma uncovered novel tumor biology and identified potential molecular markers for predicting patient outcomes. The views expressed in this abstract are solely of the authors and do not reflect the official policy of the Departments of Army/Navy/Air Force, Department of Defense, USUHS, HJF, or U.S. Government. Citation Format: Anthony R. Soltis, Nicholas W. Bateman, Thomas P. Conrads, Clifton L. Dalgard, Hai Hu, Teri J. Franks, Jianfang Liu, Daoud Meerzaman, Emanuel F. Petricoin, Qingrong Chen, Chunhua Yan, Xijun Zhang, Clesson E. Turner, The APOLLO Research Network, Craig D. Shriver, Christopher A. Moskaluk, Robert F. Browning, Matthew D. Wilkerson. Comprehensive proteogenomic analysis and classification of lung adenocarcinoma [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5893.

Importance of gastric cancer for the diagnosis and surveillance of Japanese Lynch syndrome patients.

Lynch syndrome (LS) is an autosomal dominantly inherited disease predisposed to not only colorectal cancer but also other LS-related tumors. Although the clinical and genetic characteristics of LS in Western countries have been well characterized, the information of Japanese LS is limited. As a collaborative study of Japanese Society for Cancer of the Colon and Rectum (JSCCR), we registered colorectal cancer (CRC) patients who fulfilled the modified Amsterdam II criteria including gastric cancer as an LS-related tumor. Among 4030 CRC patients initially registered in this project, 85 patients (2.1%) fulfilled the modified criteria. An additional 26 patients who met the same criteria were enrolled in the analysis. We analyzed three major responsible genes, MLH1, MSH2, and MSH6 by direct sequencing, and further performed multiplex ligation-dependent probe amplification for MLH1 and MSH2. Consequently, we identified pathogenic variants in 64 of the 111 patients comprising of 34 patients in MLH1, 28 in MSH2, and 2 in MSH6. It is of note that large structural alterations were found in 17 patients. Among the 64 patients, 11 patients would not have been enrolled in the analysis if gastric cancer were not included in the modified criteria. In addition, 10 of the 64 variant carriers (15.6%) had medical history of gastric cancer. Furthermore, the standardized incidence ratio of gastric cancer in the LS patients to the Japanese population is estimated to be as high as 20.2. These data underscore the importance of gastric cancer in the diagnosis and healthcare of Japanese LS patients.

Multiplex SERS Chemosensing of Metal Ions via DNA-Mediated Recognition.

The combination of molecular sensors and plasmonic materials is emerging as one of the most promising approaches for ultrasensitive SERS-based detection of metal ions in complex fluids. However, only a very small fraction of the large pool of potential chemosensors described in classical analytical chemistry has been successfully implemented into viable SERS platforms for metal ion determination. This is due to the molecular restrictions that require the chemosensor to adhere onto the plasmonic surface while retaining the capability to undergo large structural alterations upon metal ion binding. In this work, we demonstrate that the structural and functional plasticity of DNA for interacting with small aromatic molecules can be exploited to this end. DNA coating of silver nanoparticles modulates the interaction of the commercially available alizarin red S (ARS) chemosensor with the nanomaterial, translating its recognition capabilities from bulk solution onto the plasmonic surface, while simultaneously directing the particle assembling into highly efficient SERS clusters. The sensing approach was successfully applied to the multiplex, quantitative determination of Al(III) and Fe(III) in tap water in the subppb level.

Double-Edged Sword of Tumour Suppressor Genes in Schizophrenia.

Schizophrenia (SCZ) is a common psychiatric disorder with polygenetic pathogenesis. Among the many identified candidate genes and loci, the group of tumour suppressor genes has drawn our interest. In this mini-review article, we describe evidence of a correlation between major tumour suppressor genes and SCZ development. Genetic mutations ranging from single nucleotide polymorphisms to large structural alterations have been found in tumour-related genes in patients with SCZ. Epigenetic mechanisms, including DNA methylation/acetylation and microRNA regulation of tumour suppressor genes, have also been implicated in SCZ. Beyond genetic correlations, we hope to establish causal relationships between tumour suppressor gene function and SCZ risk. Accumulating evidence shows that tumour suppressor genes may mediate cell survival and neural development, both of which contribute to SCZ aetiology. Moreover, converging intracellular signalling pathways indicate a role of tumour suppressor genes in SCZ pathogenesis. Tumour suppressor gene function may mediate a direct link between neural development and function and psychiatric disorders, including SCZ. A deeper understanding of how neural cell development is affected by tumour suppressors may lead to improved anti-psychotic drugs.

Oligomeric states of microbial rhodopsins determined by high-speed atomic force microscopy and circular dichroic spectroscopy

Oligomeric assembly is a common feature of membrane proteins and often relevant to their physiological functions. Determining the stoichiometry and the oligomeric state of membrane proteins in a lipid bilayer is generally challenging because of their large size, complexity, and structural alterations under experimental conditions. Here, we use high-speed atomic force microscopy (HS-AFM) to directly observe the oligomeric states in the lipid membrane of various microbial rhodopsins found within eubacteria to archaea. HS-AFM images show that eubacterial rhodopsins predominantly exist as pentamer forms, while archaeal rhodopsins are trimers in the lipid membrane. In addition, circular dichroism (CD) spectroscopy reveals that pentameric rhodopsins display inverted CD couplets compared to those of trimeric rhodopsins, indicating different types of exciton coupling of the retinal chromophore in each oligomer. The results clearly demonstrate that the stoichiometry of the fundamental oligomer of microbial rhodopsins strongly correlate with the phylogenetic tree, providing a new insight into the relationship between the oligomeric structure and function-structural evolution of microbial rhodopsins.

Abstract 3756: PARP inhibitor sensitivity in small cell lung cancer cell lines and patient-derived xenografts correlates with SLFN11 expression but not with structural homologous recombination deficiency

Abstract Poly-(ADP)-ribose polymerase (PARP) was identified as a potential novel therapeutic target for small cell lung cancer (SCLC) in two key proteomic studies. These findings led to the inclusion of a SCLC cohort in a Phase I study of talazoparib (BMN 673) - currently the most potent PARP inhibitor available - with promising efficacy signals. Identifying predictors of response to PARP inhibition would bring a needed therapeutic advance in this recalcitrant disease. PARP inhibition was first demonstrated to have marked efficacy in BRCA1/2 mutant cancers that are deficient in homologous recombination (HR). Large chromosomal structural alterations characteristic of these tumors can be quantitated by three correlated HR deficiency (HRD) metrics. We sought to determine if HRD would be a relevant predictive biomarker in SCLC. Thirty-six SCLC cell lines represented in the Cancer Cell Line Encyclopedia (CCLE) were interrogated for sensitivity to talazoparib, olaparib and cisplatin. We found significant correlation between these three pharmacologic agents and sought to determine response predictors. Three different HRD metrics were computed in these 36 SCLC cell lines using publically available CCLE Affymetrix SNP 6.0 array datasets. Surprisingly, none of these three measures of HRD were discriminatory for response to talazoparib in vitro. However, we found that after controlling for HRD and PARP1 target expression, expression of a single gene, SLFN11, was significantly correlated with talazoparib sensitivity as measured by the CCLE Affymetrix U133+2 arrays (q value = 0.008). After deriving SLFN11 knock-out SCLC cell lines by CRISPR/Cas9 gene editing, we found that loss of SLFN11 conferred resistance to talazoparib (IC50 &amp;gt;log difference) as compared to parental SLFN11 proficient cells. We then treated several SCLC patient-derived xenograft (PDX) models with talazoparib daily and found that SLFN11 expression in vivo was necessary but not sufficient for response. None of the SLFN11 low expressing PDXs responded to therapy, whereas 3/5 (60%) of SLFN11 high expressing PDXs demonstrated significant tumor growth inhibition of 54-75% after 21-28 days of daily treatment with talazoparib 0.3mg/kg. In conclusion, we demonstrate that SLFN11 expression is correlated with response to PARP inhibition in SCLC in vitro and in vivo. HRD structural aberration scores do not predict for in vitro PARP inhibitor sensitivity, however functional HR interrogation (such as RAD51 foci recruitment) may still be a relevant discriminator in addition to ongoing in vivo analysis. SLFN11 may be a pertinent predictive biomarker in SCLC for therapeutic response to PARP inhibition with considerable clinical implications. Citation Format: Benjamin H. Lok, Ying Feng, G. Karen Yu, Eric E. Gardner, Nadeem Riaz, Yuanbin (Kevin) Ru, Elisa de Stanchina, Simon N. Powell, Jerry Shen, John T. Poirier, Charles M. Rudin. PARP inhibitor sensitivity in small cell lung cancer cell lines and patient-derived xenografts correlates with SLFN11 expression but not with structural homologous recombination deficiency. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3756.

ELF-EMF exposure decreases the peroxidase catalytic efficiency in vitro

AbstractThe activity of purified Fe3+ containing horseradish peroxidase (HRP) was recorded while the enzyme was exposed to two low frequency pulsed electromagnetic fields (ELF-EMFs) with different frequencies: 50 Hz/2.7 mT and 100 Hz/5.5 mT. No statistically significant difference in the Michaelis constant (Km) values between the control enzyme and the enzyme exposed to any of the two ELF-EMF used could be detected (53 ± 11 μM vs 67 ± 10 μM vs 42 ± 11 μM respectively), indicating that upon short exposure times, there are no large structural alterations of the enzyme that affect the substrate affinity. A 50 Hz/2.7 mT EMF did however, cause a significant decrease in the maximum rate (Vmax) value (from 3.31 ± 0.17 μmoles/min to 1.6 ± 0.19 μmoles/min, p&lt;0.03) and a drop of the catalytic efficiency to less than half (from 0.66 ± 0.03 s-1 to 0.32 ± 0.04 s-1, p&lt;0.05). These observations are the first experimental proofs that support the previously postulated mechanism of coupling between ELF-EMF and living systems based on a resonant frequency specific for a given ion molecule.

Analysis of the self-discharge process in LiCoPO4 electrodes: bulks

LiCoPO4 (LCP) is a promising high voltage cathode for next-generation high energy Li-ion batteries. However, once electrochemically de-lithiated, it suffers from a spontaneous self-discharge process in open circuit conditions. Here we present our systematic study about the phase composition and structural changes of LCP electrodes upon charging and self-discharging processes by ex situ synchrotron X-ray diffraction and Fast-Fourier Transform Infrared Spectroscopy (FT-IR) analyses. Various initial charging states and different current rates have been studied. Our investigation highlights large structural alterations of the de-lithiated CoPO4 phase upon self-discharge. However, after 160hours with open circuit the pristine LCP structure is recovered. The electrochemically de-lithiated phase reincorporates lithium by a spontaneous reduction reaction releasing gaseous CO2 and likely degrading the electrolyte molecules.

Characterization of Large Structural Genetic Mosaicism in Human Autosomes

Analyses of genome-wide association study (GWAS) data have revealed that detectable genetic mosaicism involving large (>2 Mb) structural autosomal alterations occurs in a fraction of individuals. We present results for a set of 24,849 genotyped individuals (total GWAS set II [TGSII]) in whom 341 large autosomal abnormalities were observed in 168 (0.68%) individuals. Merging data from the new TGSII set with data from two prior reports (the Gene-Environment Association Studies and the total GWAS set I) generated a large dataset of 127,179 individuals; we then conducted a meta-analysis to investigate the patterns of detectable autosomal mosaicism (n = 1,315 events in 925 [0.73%] individuals). Restricting to events >2 Mb in size, we observed an increase in event frequency as event size decreased. The combined results underscore that the rate of detectable mosaicism increases with age (p value = 5.5 × 10(-31)) and is higher in men (p value = 0.002) but lower in participants of African ancestry (p value = 0.003). In a subset of 47 individuals from whom serial samples were collected up to 6 years apart, complex changes were noted over time and showed an overall increase in the proportion of mosaic cells as age increased. Our large combined sample allowed for a unique ability to characterize detectable genetic mosaicism involving large structural events and strengthens the emerging evidence of non-random erosion of the genome in the aging population.

Detectable Clonal Mosaicism in the Human Genome

Human genetic mosaicism is the presence of two or more cellular populations with distinct genotypes in an individual who developed from a single fertilized ovum. While initially observed across a spectrum of rare genetic disorders, detailed assessment of data from genome-wide association studies now reveal that detectable clonal mosaicism involving large structural alterations (>2 Mb) can also be seen in populations of apparently healthy individuals. The first generation of descriptive studies has generated new interest in understanding the molecular basis of the affected genomic regions, percent of the cellular subpopulation involved, and developmental timing of the underlying mutational event, which could reveal new insights into the initiation, clonal expansion, and phenotypic manifestations of mosaic events. Early evidence indicates detectable clonal mosaicism increases in frequency with age and could preferentially occur in males. The observed pattern of recurrent events affecting specific chromosomal regions indicates some regions are more susceptible to these events, which could reflect inter-individual differences in genomic stability. Moreover, it is also plausible that the presence of large structural events could be associated with cancer risk. The characterization of detectable genetic mosaicism reveals that there could be important dynamic changes in the human genome associated with the aging process, which could be associated with risk for common disorders, such as cancer, cardiovascular disease, diabetes, and neurological disorders.

Intermediates of the Channel Rhodopsin Photocycle

Channelrhodopsins are sensory photoreceptors that mediate phototaxis in green algae (1, 2). Their function as ion channels renders them useful tools in the field of neuroscience. Due to the prolonged lifetime and light-sensitivity of the conducting state, mutantants of cysteine 128 are especially important. They can be switched on and off by light, and are therefore called step-function rhodopsins (3). Recent photocycle models have been proposed on the basis of visual and vibrational spectroscopy (4, 5), however, details of the photoreactions are still widely unknown. Here we present spectroscopical data on the slow cycling mutant C128T, with 200 fold extended lifetime of the conducting state. During the photocycle, a fraction of the proteins branches off into a sideway consisting of blue-shifted species which accumulate during prolonged illumination and play an important role in dark state regeneration (5). We will show that - depending on the illumination conditions - several structurally different dark states are regenerated. However, although such large structural alterations occur during the photocycle, they are not necessarily connected with on- and off-switching of the channel. A new model that connects chromophore isomerization and structural alterations of the protein will be discussed.(1) Nagel, G. et al. (2002) Science 296, 2395-8.(2) Nagel, G. et al. (2003) Proceedings of the National Academy of Sciences of the United States of America 100, 13940-5.(3) Berndt, A. et al. (2009) Nature neuroscience 12, 229-34.(4) Radu, I. et al. (2009) Journal of the American Chemical Society 131, 7313-9.(5) Stehfest, K. et al. (2010) Journal of molecular biology 398, 690-702.

In Search of a Vaccine for Mouse Allergy: Significant Reduction of Mus m 1 Allergenicity by Structure-Guided Single-Point Mutations

Background: Mouse urinary proteins are relevant allergens from mice urine. We used the recombinant protein Mus m 1 as an allergen model to identify if, by altering Mus m 1 architecture via single-point mutations, we could effectively modify its allergenicity. Methods: Based on structural considerations, we synthesized two single-point mutants, Mus m 1-Y120A and Mus m 1-Y120F, which were expected to harbor large structural alterations. Circular dichroism and fluorescence analysis showed significant conformational rearrangements of the aromatic side chains in the internal cavity of Mus m 1-Y120A when compared to Mus m 1-Y120F and Mus m 1. Evaluation of the allergenic potential of the recombinant molecules was performed in vitro with both immunochemical approaches and assays based on the measurement of basophil degranulation. Moreover, to assess the integrity of the T cell epitopes and as an in vitro measure of immunogenicity, we tested the reactivity of T lymphocytes from subjects allergic to mouse urine against proteins and synthetic peptides encompassing the immunodominant linear epitope containing the mutation. Results: We found that the selected point mutation was able to modulate the protein allergenicity, and to severely impair the recognition of Mus m 1 by IgE, while T cell reactivity was fully maintained. Conclusions: In silico predicted, minimum selected structural modifications allowed to design one protein with reduced allergenicity and preserved immunogenicity. Structurally guided mutations can direct the design of proteins with reduced allergenicity which can be used as vaccines for a safer and more effective immunotherapy of allergic disorders.

Mutations Targeting the C-Terminal Domain of FliG Can Disrupt Motor Assembly in the Na +-Driven Flagella of Vibrio alginolyticus

The torque of the bacterial flagellar motor is generated by the rotor–stator interaction coupled with specific ion translocation through the stator channel. To produce a fully functional motor, multiple stator units must be properly incorporated around the rotor by an as yet unknown mechanism to engage the rotor–stator interactions. Here, we investigated stator assembly using a mutational approach of the Na +-driven polar flagellar motor of Vibrio alginolyticus, whose stator is localized at the flagellated cell pole. We mutated a rotor protein, FliG, which is located at the C ring of the basal body and closely participates in torque generation, and found that point mutation L259Q, L270R or L271P completely abolishes both motility and polar localization of the stator without affecting flagellation. Likewise, mutations V274E and L279P severely affected motility and stator assembly. Those residues are localized at the core of the globular C-terminal domain of FliG when mapped onto the crystal structure of FliG from Thermotoga maritima, which suggests that those mutations induce quite large structural alterations at the interface responsible for the rotor–stator interaction. These results show that the C-terminal domain of FliG is critical for the proper assembly of PomA/PomB stator complexes around the rotor and probably functions as the target of the stator at the rotor side.

A glimpse at tumor genome evolution

Next-generation DNA sequencing has dramatically affected cancer genomics efforts in several important ways. Although whole genome sequencing remains an analytical challenge, such efforts are yielding data that elucidate the myriad ways in which a genome can be influenced by single point mutations, focused insertions or deletions, and large structural alterations. In addition to cataloguing somatic alterations, various correlation analyses are indicating the genes whose alterations most profoundly determine patient outcomes, patient responses to therapeutics and other important aspects of disease biology. We have recently begun exploring how the digital nature of next-generation sequencing reads allows important information about tumor cell genomic heterogeneity to be inferred, revealing the earliest mutations and how the composition of the tumor cell mass changes over time under the influence of stressors such as chemotherapy.

Analysis of next-generation genomic data in cancer: accomplishments and challenges

The application of next-generation sequencing technology has produced a transformation in cancer genomics, generating large data sets that can be analyzed in different ways to answer a multitude of questions about the genomic alterations associated with the disease. Analytical approaches can discover focused mutations such as substitutions and small insertion/deletions, large structural alterations and copy number events. As our capacity to produce such data for multiple cancers of the same type is improving, so are the demands to analyze multiple tumor genomes simultaneously growing. For example, pathway-based analyses that provide the full mutational impact on cellular protein networks and correlation analyses aimed at revealing causal relationships between genomic alterations and clinical presentations are both enabled. As the repertoire of data grows to include mRNA-seq, non-coding RNA-seq and methylation for multiple genomes, our challenge will be to intelligently integrate data types and genomes to produce a coherent picture of the genetic basis of cancer.

Characterization of Recombinant Human Prolyl 3-Hydroxylase Isoenzyme 2, an Enzyme Modifying the Basement Membrane Collagen IV

The single 3-hydroxyproline residue in the collagen I polypeptides is essential for proper fibril formation and bone development as its deficiency leads to recessive osteogenesis imperfecta. The vertebrate prolyl 3-hydroxylase (P3H) family consists of three members, P3H1 being responsible for the hydroxylation of collagen I. We expressed human P3H2 as an active recombinant protein in insect cells. Most of the recombinant polypeptide was insoluble, but small amounts were also present in the soluble fraction. P3H1 forms a complex with the cartilage-associated protein (CRTAP) that is required for prolyl 3-hydroxylation of fibrillar collagens. However, coexpression with CRTAP did not enhance the solubility or activity of the recombinant P3H2. A novel assay for P3H activity was developed based on that used for collagen prolyl 4-hydroxylases (C-P4H) and lysyl hydroxylases (LH). A large amount of P3H activity was found in the P3H2 samples with (Gly-Pro-4Hyp)5 as a substrate. The Km and Ki values of P3H2 for 2-oxoglutarate and its certain analogues resembled those of the LHs rather than the C-P4Hs. Unlike P3H1, P3H2 was strongly expressed in tissues rich in basement membranes, such as the kidney. P3H2 hydroxylated more effectively two synthetic peptides corresponding to sequences that are hydroxylated in collagen IV than a peptide corresponding to the 3-hydroxylation site in collagen I. These findings suggest that P3H2 is responsible for the hydroxylation of collagen IV, which has the highest 3-hydroxyproline content of all collagens. It is thus possible that P3H2 mutations may lead to a disease with changes in basement membranes.