The ATM serine/threonine kinase (HGNC: ATM) is involved in initiation of repair of DNA double-stranded breaks, and ATM inhibitors are currently being tested as anti-cancer agents in clinical trials, ...where pharmacodynamic (PD) assays are crucial to help guide dose and scheduling and support mechanism of action studies. To identify and quantify PD biomarkers of ATM inhibition, we developed and analytically validated a 51-plex assay (DDR-2) quantifying protein expression and DNA damage-responsive phosphorylation. The median lower limit of quantification was 1.28 fmol, the linear range was over 3 orders of magnitude, the median inter-assay variability was 11% CV, and 86% of peptides were stable for storage prior to analysis. Use of the assay was demonstrated to quantify signaling following ionizing radiation-induced DNA damage in both immortalized lymphoblast cell lines and primary human peripheral blood mononuclear cells, identifying PD biomarkers for ATM inhibition to support preclinical and clinical studies.
There is growing interest in proteomic analyses of tissue biopsies to reveal pathophysiology and identify biomarkers. The current gold standard for collecting tissue biopsies for preserving the ...proteome and post-translational modifications is flash freezing in liquid nitrogen (LN2). However, in many clinical settings, this is not an option due to unavailability of LN2 nor trained personnel for rapid biospecimen processing. To address this need, we developed a proof-of-concept quick-freeze prototype device to rapidly freeze biospecimens at the point-of-care to preserve the phosphoproteome without the need for LN2. Our objectives were to develop the device, demonstrate the ease of use, confirm the ability to ship through existing cold chain logistics, and evaluate the cooling performance (i.e., cool a tissue sample to <0°C in <60 seconds, below -8°C in <120 seconds, and maintain temperature <0°C for >60 minutes) in the context of preserving the proteome in a tissue biospecimen. To demonstrate feasibility, the performance of the prototype was benchmarked against flash freezing in LN2 using a murine melanoma patient-derived xenograft model subjected to total body irradiation to elicit phosphosignaling in the DNA damage response network. Tumors were harvested and quadrisected, with two parts of the tumor being snap frozen in LN2, and the remaining two parts being rapidly cooled in the prototype quick-freeze biospecimen containers. Phosphoproteins were profiled by liquid chromatography tandem mass spectrometry and quantified by targeted multiple reaction monitoring MS. Overall, the phosphoproteome was equivalent in biospecimens processed using the quick-freeze containers to those using the LN2 gold standard, although the measurements of a subset of phosphopeptides in the device-frozen specimens were more variable than LN2-frozen specimens. The prototype device forms the framework for development of a commercial device that will improve tissue biopsy preservation for measurement of important phosphosignaling molecules.
In response to replication stress, a phospho-signaling cascade is activated and required for coordination of DNA repair and replication of damaged templates (intra-S-phase checkpoint) . How ...phospho-signaling coordinates the DNA replication stress response is largely unknown. We employed state-of-the-art liquid chromatography tandem-mass spectrometry (LC-MS/MS) approaches to generate high-coverage and quantitative proteomic and phospho-proteomic profiles during replication stress in yeast, induced by continuous exposure to the DNA alkylating agent methyl methanesulfonate (MMS) . We identified 32,057 unique peptides representing the products of 4296 genes and 22,061 unique phosphopeptides representing the products of 3183 genes. A total of 542 phosphopeptides (mapping to 339 genes) demonstrated an abundance change of greater than or equal to twofold in response to MMS. The screen enabled detection of nearly all of the proteins known to be involved in the DNA damage response, as well as many novel MMS-induced phosphorylations. We assessed the functional importance of a subset of key phosphosites by engineering a panel of phosphosite mutants in which an amino acid substitution prevents phosphorylation. In total, we successfully mutated 15 MMS-responsive phosphorylation sites in seven representative genes including APN1 (base excision repair); CTF4 and TOF1 (checkpoint and sister-chromatid cohesion); MPH1 (resolution of homologous recombination intermediates); RAD50 and XRS2 (MRX complex); and RAD18 (PRR). All of these phosphorylation site mutants exhibited MMS sensitivity, indicating an important role in protecting cells from DNA damage. In particular, we identified MMS-induced phosphorylation sites on Xrs2 that are required for MMS resistance in the absence of the MRX activator, Sae2, and that affect telomere maintenance.
A lead-glazed porcelain teapot with an aluminous-silicic body recovered from an 18th-century privy in Philadelphia developed a partly crystallized (alkali feldspar) integrated body-glaze layer (IBGL) ...during kiln firing. Experiments on compositional analogs of the teapot show that a mineralogically and microstructurally similar ware can be created by firing for 48 hours at a peak kiln temperature of 1370°C. An IBGL, also with alkali feldspars, formed between the glaze and its ceramic substrate during >5 hours firing at 1000°C. The IBGL formed as alkalis and lead fluxed the substrate. These results confirm that very high temperatures were required to vitrify aluminous-silicic (“true” or “hard paste”) porcelain pastes, and indicate that heat was not invariably dumped from the glost kiln as soon as lead-rich glazes melted. Although relatively aluminous, the uptake of alumina by the glaze was less than that shown by lead-rich glazes on phosphatic porcelain fired at lower temperatures (~1250°C), evidently because this component is largely sequestered in a relatively refractory mineral (mullite) in true porcelain.
Una tetera de porcelana esmaltada con plomo y con cuerpo de silicio aluminoso recuperada de un retrete del siglo XVIII, en Filadelfia, desarrolló una capa integral de esmalte corporal (IBGL por sus siglas en inglés) (feldespato alcalino) parcialmente cristalizada durante el horneado. Los experimentos con análogos de composición de la tetera muestran que se puede crear una cerámica mineralógica y microestructuralmente similar horneando durante 48 horas (h) a una temperatura de horno máxima (T) de 1370°C. Se formó una capa IBGL, también con feldespatos alcalinos, entre el esmalte y su sustrato cerámico durante un horneado de >5 h a 1000°C. La capa IBGL se formó como álcalis y el plomo actuó como fundente del sustrato. Estos resultados confirman que para esmaltar las pastas de porcelana aluminosa-silícica ("verdadera" o "pasta dura") se requerían temperaturas muy altas, e indican que el calor no siempre se redujo rápidamente en el horno glost tan pronto como se fundieron los esmaltes ricos en plomo. Aunque relativamente aluminoso, la absorción de alúmina por el esmalte fue menor que la mostrada por esmaltes ricos en plomo en porcelana fosfática horneada a una T menor (~1250°C), evidentemente porque en porcelana verdadera este componente es secuestrado en gran parte en un mineral relativamente refractario (mullita).
Une théière en porcelaine à glaçure plombifère avec corps alumineux siliceux provenant de toilettes du 18e siècle de Philadelphie a développé une couche de glaçure intégrée (integrated body-glaze layer ou IBGL en anglais) partiellement cristallisée (feldspath alcalin) durant sa cuisson au four. Des expériences menées sur des répliques compositionnelles de la théière démontrent qu’un article similaire aux points de vue minéralogique et microstructurel peut être créé à une température de pointe de 1 370°C après 48 heures (h) de cuisson. Une IBGL, comportant aussi des feldspaths alcalins, s’est formée entre la glaçure et son substrat céramique durant >5 h de cuisson à 1 000°C. L’IBLG s’est formée en tant qu’alcalis et le plomb a fluxé le substrat. Ces résultats confirment que des températures très élevées étaient requises pour vitrifier les pâtes de porcelaine alumineuse siliceuse (« dures » ou « pâtes dures »), et indiquent que la chaleur n’était pas invariablement évacuée du four de deuxième cuisson dès que les glaçures riches en plomb fondaient. Quoique relativement alumineuse, l’absorption d’oxyde d’aluminium par la glaçure était inférieure à celle notée pour les glaçures riches en plomb appliquées sur de la porcelaine phosphatée cuite à des températures inférieures (~1 250°C), évidemment puisque cet élément est largement séquestré dans un minerai relativement réfractaire (mullite) à l’intérieur de la porcelaine dure.
Summary
Virulence of the opportunistic pathogen Pseudomonas aeruginosa involves the coordinate expression of a wide range of virulence factors including type IV pili which are required for ...colonization of host tissues and are associated with a form of surface translocation termed twitching motility. Twitching motility in P. aeruginosa is controlled by a complex signal transduction pathway which shares many modules in common with chemosensory systems controlling flagella rotation in bacteria and which is composed, in part, of the previously described proteins PilG, PilH, PilI, PilJ and PilK. Here we describe another three components of this pathway: ChpA, ChpB and ChpC, as well as two downstream genes, ChpD and ChpE, which may also be involved. The central component of the pathway, ChpA, possesses nine potential sites of phosphorylation: six histidine‐containing phosphotransfer (HPt) domains, two novel serine‐ and threonine‐containing phosphotransfer (SPt, TPt) domains and a CheY‐like receiver domain at its C‐terminus, and as such represents one of the most complex signalling proteins yet described in nature. We show that the Chp chemosensory system controls twitching motility and type IV pili biogenesis through control of pili assembly and/or retraction as well as expression of the pilin subunit gene pilA. The Chp system is also required for full virulence in a mouse model of acute pneumonia.
RAS genes are frequently mutated in cancer and have for decades eluded effective therapeutic attack. The National Cancer Institute's RAS Initiative has a focus on understanding pathways and ...discovering therapies for RAS-driven cancers. Part of these efforts is the generation of novel reagents to enable the quantification of RAS network proteins. Here we present a dataset describing the development, validation (following consensus principles developed by the broader research community), and distribution of 104 monoclonal antibodies (mAbs) enabling detection of 27 phosphopeptides and 69 unmodified peptides from 20 proteins in the RAS network. The dataset characterizes the utility of the antibodies in a variety of applications, including Western blotting, immunoprecipitation, protein array, immunohistochemistry, and targeted mass spectrometry. All antibodies and characterization data are publicly available through the CPTAC Antibody Portal, Panorama Public Repository, and/or PRIDE databases. These reagents will aid researchers in discerning pathways and measuring expression changes in the RAS signaling network.
Addition of ferrocene to solutions of alkali metal hexamethyldisilazides M(HMDS) in arenes (in which M=Na, K, Rb, Cs) allows the subsequent crystallization of the homologous series of compounds ...{(Me3Si)2NM}2⋅ (Cp2Fe)∞ (1–4). Similar reactions using LiHMDS led to the recrystallization of the starting materials. The crystal structures of 1–4 reveal the formation of one‐dimensional chains composed of dimeric {M(HMDS)}2 aggregates, which are bridged through neutral ferrocene molecules by η5‐cation–π interactions. In addition, compounds 3 and 4 also contain interchain agostic MC interactions, producing two‐dimensional 44‐nets. Whereas 1 and 2 were prepared from toluene, the syntheses of 3 and 4 required the use of tert‐butylbenzene as the reaction media. The attempted crystallization of 3 and 4 from toluene resulted in formation of the mixed toluene/ferrocene solvated complexes {(Me3Si)2NM)2}2⋅ (Cp2Fe)x⋅(Tol)y∞ (in which M=Rb, x=0.6, y=0.8, 5; M=Cs, x=0.5, y=1, 6). The extended solid‐state structures of 5 and 6 are closely related to the 44‐sheets 3 and 4, but are now assembled from a combination of cation–π, agostic, and π–π interactions. The charge‐separated complex K{(C6H6)2Cr}1.5(Mes)Mg(HMDS)3 (15) was also structurally characterized and found to adopt an anionic two‐dimensional 63‐network through doubly η3‐coordinated bis(benzene)chromium molecules. DFT calculations at the B3 LYP/6–31G* level of theory indicate that the binding energies of both ferrocene and toluene to the M(HMDS) dimers increases in the sequence Li<Na<K. This pattern is a consequence of the larger metals allowing more open coordination spheres to support cation–π contacts. By comparison, binding of the isolated metal cations to the aromatic groups follow the reverse order K<Na<Li. A combined analysis of theoretical and experimental data suggest that ferrocene is a stronger cation–π donor than toluene for the lighter metals, but that this difference is eliminated on descending the group.
Directional cation–π interactions: Metallocenes, such as ferrocene and bis(benzene)chromium, can be used in association with pre‐assembled alkali metal aggregates to control the assembly of polymeric materials (an example of which is illustrated here) through directional cation–π interactions.
Regional gray matter (GM) alterations have been reported in early-onset psychosis (EOP, onset before age 18), but previous studies have yielded conflicting results, likely due to small sample sizes ...and the different brain regions examined. In this study, we conducted a whole brain voxel-based morphometry (VBM) analysis in a large sample of individuals with EOP, using the newly developed ENIGMA-VBM tool.
15 independent cohorts from the ENIGMA-EOP working group participated in the study. The overall sample comprised T1-weighted MRI data from 482 individuals with EOP and 469 healthy controls. Each site performed the VBM analysis locally using the standardized ENIGMA-VBM tool. Statistical parametric T-maps were generated from each cohort and meta-analyzed to reveal voxel-wise differences between EOP and healthy controls as well as the individual-based association between GM volume and age of onset, chlorpromazine (CPZ) equivalent dose, and other clinical variables.
Compared with healthy controls, individuals with EOP showed widespread lower GM volume encompassing most of the cortex, with the most marked effect in the left median cingulate (Hedges' g = 0.55, p = 0.001 corrected), as well as small clusters of lower white matter (WM), whereas no regional GM or WM volumes were higher in EOP. Lower GM volume in the cerebellum, thalamus and left inferior parietal gyrus was associated with older age of onset. Deficits in GM in the left inferior frontal gyrus, right insula, right precentral gyrus and right superior frontal gyrus were also associated with higher CPZ equivalent doses.
EOP is associated with widespread reductions in cortical GM volume, while WM is affected to a smaller extent. GM volume alterations are associated with age of onset and CPZ equivalent dose but these effects are small compared to case-control differences. Mapping anatomical abnormalities in EOP may lead to a better understanding of the role of psychosis in brain development during childhood and adolescence.
Abstract
Although high-grade serous ovarian cancers (HGSOC) are highly chemosensitive with an 85% initial response rate to platinum-based chemotherapy, 15% of patients are “exceptional ...nonresponders,” with platinum-refractory tumors that remain stable or progress during treatment. Unfortunately, we have no predictive biomarkers to identify refractory patients up front, and they receive futile chemotherapy through which most patients become too ill to be eligible for clinical trials. Hence, no progress has been made in treating these deadly tumors. The goal of this study is to identify mechanisms of platinum refractoriness to: i) predict refractory HGSOCs up front and ii) identify potential new drug targets in refractory disease to point to desperately needed new therapeutic approaches. Of note, 80-90% of patients who are initially platinum responsive will relapse and develop platinum-resistant disease, and it is possible that findings in platinum-refractory tumors might also provide insights into platinum-resistant tumors. Our NCI Clinical Proteomic Tumor Analysis Consortium (CPTAC)-funded approach combines genomic and proteomic (“proteogenomic”) analyses of both preclinical models (0, 8, and 24 hours post-platinum exposure) and treatment-naïve human tumors. For preclinical models, we studied a well-characterized collection of patient-derived xenograft (PDX) models (10 sensitive, 10 refractory), as well as intrapatient HGSOC cell line pairs derived from patients before and after the development of platinum resistance. For the PDX models, proteogenomic profiling included RNASeq, WES, global proteomics, and phosphoproteomics at all 3 time points (0, 8, 24 hours). For cell line models (3 sensitive, 3 resistant), proteogenomic profiling was performed at all 3 timepoints (0, 8, 24 hours), and experiments were performed in complete biologic triplicate. Analyses included RNASeq, WES, global proteomics, phosphoproteomics, ubiquitin proteome, acetylated proteome, and pTyr. A large collection of 275 human HGSOCs (an approximate equal balance of platinum sensitive and refractory tumors) is currently undergoing proteomic profiling, and genomic profiles (WGS, RNASeq) will be performed on a subset. In parallel, we have performed a comprehensive review of 31 years of published work on platinum responses of human cancers, identifying ~700 genes implicated in the response and scoring each gene with respect to strength of the published evidence. Using a Bayesian approach, we are integrating the curated candidates from the literature with our empirical proteogenomic datasets to identify a candidate signature for detecting platinum-refractory disease prior to chemotherapy. We are also performing gene-regulatory network analysis to identify potential drivers of chemo response. NextGen, targeted, multiplex, multiple reaction monitoring mass spectrometry-based assays are being developed to quantify proteins in the signature for validation studies, using independent patient cohorts.
This abstract is also being presented as Poster A62.
Citation Format: Jacob J. Kennedy, Shrabanti Chowdhury, Sara R. Savage, Xiaonan Hou, Catherine J. Huntoon, Richard G. Ivey, Qing Yu, Chenwei Lin, Dongqing Huang, Lei Zhao, Uliana J. Voytovich, Regine M. Schoenherr, Zahra Shire, Steven J. Skates, Jeffrey R. Whiteaker, Andrew N. Hoofnagle, Samuel C. Mok, Bing Zhang, Larry M. Karnitz, S. John Weroha, Steven P. Gygi, Scott H. Kaufmann, Pei Wang, Michael J. Birrer, Amanda G. Paulovich. Proteogenomic approach to identify mechanisms of platinum refractoriness in high-grade serous ovarian cancers abstract. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research; 2019 Sep 13-16, 2019; Atlanta, GA. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(13_Suppl):Abstract nr PR02.
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