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PubMed2004

A modular approach to the ECVAM principles on test validity.

The European Centre for the Validation of Alternative Methods (ECVAM) proposes to make the validation process more flexible, while maintaining its high standards. The various aspects of validation are broken down into independent modules, and the information necessary to complete each module is defined. The data required to assess test validity in an independent peer review, not the process, are thus emphasised. Once the information to satisfy all the modules is complete, the test can enter the peer-review process. In this way, the between-laboratory variability and predictive capacity of a test can be assessed independently. Thinking in terms of validity principles will broaden the applicability of the validation process to a variety of tests and procedures, including the generation of new tests, new technologies (for example, genomics, proteomics), computer-based models (for example, quantitative structure-activity relationship models), and expert systems. This proposal also aims to take into account existing information, defining this as retrospective validation, in contrast to a prospective validation study, which has been the predominant approach to date. This will permit the assessment of test validity by completing the missing information via the relevant validation procedure: prospective validation, retrospective validation, catch-up validation, or a combination of these procedures.

Animal Testing Alternatives↗
PubMed2025

Widespread release of translational repression across Plasmodium's host-to-vector transmission event.

Malaria parasites must respond quickly to environmental changes, including during their transmission between mammalian and mosquito hosts. Therefore, female gametocytes proactively produce and translationally repress mRNAs that encode essential proteins that the zygote requires to establish a new infection. While the release of translational repression of individual mRNAs has been documented, the details of the global release of translational repression have not. Moreover, changes in the spatial arrangement and composition of the DOZI/CITH/ALBA complex that contribute to translational control are also not known. Therefore, we have conducted the first quantitative, comparative transcriptomics and DIA-MS proteomics of Plasmodium parasites across the host-to-vector transmission event to document the global release of translational repression. Using female gametocytes and zygotes of P. yoelii, we found that ~200 transcripts are released for translation soon after fertilization, including those encoding essential functions. Moreover, we identified that many transcripts remain repressed beyond this point. TurboID-based proximity proteomics of the DOZI/CITH/ALBA regulatory complex revealed substantial spatial and/or compositional changes across this transmission event, which are consistent with recent, paradigm-shifting models of translational control. Together, these data provide a model for the essential translational control mechanisms that promote Plasmodium's efficient transmission from mammalian host to mosquito vector.

Animals↗
PubMed2003

Proteome analysis of Escherichia coli W3110 expressing an heterologous sigma factor.

The recombinant plasmid pASK18 carries a Streptomyces DNA fragment which includes an open reading frame, designated psfS (putative sigma factor, Streptomyces), as its putative product showed a high degree of similarity with RNA polymerase sigma factors. Previous results showed that PsfS causes transcription initiation within the bgl operon promoter-silencer region in Escherichia coli K12. In this study a proteomic approach has been applied in order to perform a comparative analysis of E. coli K12 W3110 wild-type, W3110 (pASK18) and a W3110 Bgl(+) spontaneous mutant. Either by qualitative or quantitative analysis, no significant difference was observed between the proteomes of W3110 and its Bgl+ derivative, while W3110 (pASK18) showed an altered profile by both analyses. Fourteen out of the 37 protein spots showing a different expression level in E. coli W3110 harboring pASK18 were identified by peptide mass fingerprinting. Among the proteins identified, thiol peroxidase (Tpx) was the only one up-regulated. The possible involvement of bgl and tpx in the survival of the pathogen E. coli during infection is discussed.

Amino Acid Sequence↗
PubMed2006

Proteomic analysis of malignant ovarian cancer effusions as a tool for biologic and prognostic profiling.

PURPOSE: Malignant epithelial ovarian cancer effusions are important in disease dissemination and clinical outcome. The identification of biochemical events active in effusions may improve our identification and application of targeted therapeutics. EXPERIMENTAL DESIGN: Archival effusion samples for which outcome information was known were studied. Clinical variables were comparable between these groups. Two cohorts of patients with malignant effusion were assessed: those with effusion at presentation (Tap1) or at first recurrence (Tap2). Expression and activated fraction of selected signaling proteins were quantitated on serial protein microarrays using validated antibodies. Proteomic results and clinical variables were analyzed by univariate analysis followed by Cox proportional hazards model analysis. RESULTS: Malignant effusions (>80% malignant cells) were distinguished from benign effusions by higher expression of AKT, activated extracellular signal-regulated kinase, activated (P < or = 0.001) and total cAMP-responsive element binding protein (P = 0.01), and JNK (P = 0.03). Malignant pleural effusions could not be differentiated from ascites by signaling profiles. Both had signal expression clusters for survival, proliferation and metastasis, and injury pathways. Cox proportional hazards model analysis revealed high p38 and pEGFR/EGFR ratio as jointly associated with poor survival in Tap1 cases (both P < or = 0.002). Phospho-JNK quantity was associated with worse outcome in Tap2 patients (P = 0.004), when taking other factors into consideration. CONCLUSIONS: Proliferation, survival, and apoptosis signaling dysregulation can be identified in ovarian cancer effusion samples. Biochemical characterization of clinical effusions may provide either predictive and/or correlative information on patient outcome from which to further understand the mechanisms of effusion development and target clinical intervention.

Apoptosis↗
PubMed2026

Systematic Identification of Therapeutic Targets and Repurposed Drugs for Stroke: From Genome Causal Analysis to Multilevel Validation.

BACKGROUND: Stroke is a severe cerebrovascular disease characterized by narrow time windows and complications. This study aimed to identify novel drug targets and repurposed drugs for stroke. METHODS: This study used expression quantitative trait loci data from druggable genes in brain and blood as instrumental variables. Mendelian randomization, colocalization, and phenome-wide Mendelian randomization were applied to evaluate causal relationships and potential side effects, with stroke and ischemic stroke as primary outcomes. Preclinical validation used oxygen-glucose deprivation/reperfusion and middle cerebral artery occlusion/reperfusion models. Pharmacological and behavioral assessments evaluated the therapeutic potential of candidate targets and drugs. Additionally, proteomic sequencing was performed following GGCX (&#x3b3;-glutamyl carboxylase) overexpression to explore its biological functions. RESULTS: Elevated GGCX expression in brain and blood was potentially causally associated with reduced risk of stroke and ischemic stroke, supported by colocalization evidence, although potential cardiovascular risks could not be excluded. Drug repositioning identified ifenprodil as a candidate agent that reduced infarction volume, improved motor and cognitive functions, and reversed GGCX downregulation in mice. Ifenprodil treatment and GGCX overexpression alleviated oxygen-glucose deprivation/reperfusion-induced injury and upregulated GGCX expression. Mechanistically, GGCX conferred neuroprotection by regulating protein homeostasis, suppressing inflammation, promoting metabolic recovery, and modulating nuclear transcriptional regulation. CONCLUSIONS: This study established a potential causal link between GGCX and stroke risk, particularly ischemic stroke. GGCX represents a promising therapeutic target for ischemic stroke. Targeted GGCX expression upregulation and drug repurposing, particularly ifenprodil, may offer novel therapeutic avenues. Further validation is warranted to assess clinical efficacy and safety.

Animals↗
PubMed2001

Proteomic analysis reveals that 14-3-3sigma is down-regulated in human breast cancer cells.

The class of molecular chaperones known as 14-3-3 is involved in the control of cellular growth by virtue of its apparent regulation of various signaling pathways, including the Raf/mitogen-activated protein kinase pathway. In breast cancer cells, the sigma form of 14-3-3 has been shown to interact with cyclin-dependent kinases and to control the rate of entry into mitosis. To test for a direct role for 14-3-3 in breast epithelial cell neoplasia, we have quantitated 14-3-3 protein levels using a proteomic approach based on two-dimensional electrophoresis and matrix-assisted laser desorption/ionization mass spectrometry (MALDI-TOF). We show here that 14-3-3sigma protein is strongly down-regulated in the prototypic breast cancer cell lines MCF-7 and MDA-MB-231 and in primary breast carcinomas as compared with normal breast epithelial cells. In contrast, levels of the alpha, beta, delta, or zeta isoforms of 14-3-3 were the same in both normal and transformed cells. The data support the idea that 14-3-3sigma is involved in the neoplastic transition of breast epithelial cells by virtue of its role as a tumor suppressor; as such, it may constitute a robust marker with clinical efficacy for this pathology.

14-3-3 Proteins↗
PubMed2002

Contributions of proteome profiling to the molecular analysis of cancer.

The proteome is the most functional compartment encoded for in the genome. Technologies for protein separation and quantitation, coupled with mass spectrometry for protein identification, have provided the means for proteome profiling of tumor cell lines and tissues that complement genomic and transcriptomic profiling. The application of established and novel proteomic technologies to the molecular analysis of cancer is reviewed.

Electrophoresis, Gel, Two-Dimensional↗
PubMed2003

Chromosome mapping and identification of amphiphilic proteins of hexaploid wheat kernels.

Amphiphilic proteomic analysis was carried out on the ITMI (International Triticae Mapping Population) population resulting from a cross between "Synthetic", i.e.: "W7984" and "Opata". Out of a total of 446 spots, 170 were specific to either of the two parents, and 276 were common to both. Preliminary analysis, which was performed on 80 progenies (Amiour et al. 2002a), was completed here using a total of 101 selfed lines. Seventy two Loci of amphiphilic spots placed at LOD = 5 were conclusively assigned to 15 chromosomes. Some spots mapped during the first analysis were eliminated because of the significant distortion segregation observed in the second analysis. Group-1 chromosomes had by far the greatest number of mapped spots (51). Using the Quantitative Trait Loci (QTLs) approach, analysis of the quantitative variation of each spot revealed that 96 spots out of the 170 specific ones showed at least one Protein Quantity Locus (PQL). These PQLs were distributed throughout the genome. With Matrix Laser Desorption Ionisation Time Of Flight (MALDI-TOF) spectrometry and Database interrogation, a total of 93 specific and 41 common spots were identified. This enabled us to show that the majority of these proteins are associated with membranes and/or play a role in plant defence against external invasions. Using multiple-regression analysis, other amphiphilic proteins, in addition to puroindolines, were shown to be involved in variation in kernel hardness in the ITMI population.

Chromosome Mapping↗
PubMed2026

Trans-omics integration underscores distinct roles of polyunsaturated phospholipids in bidirectional offspring birth weight deviations.

BACKGROUND: Abnormal birth weights are associated with adverse pregnancy outcomes and future metabolic consequences. We aimed to examine cord blood lipidomes from low, normal and high birth weight (LBW, NBW, HBW) infants to identify core lipid signatures associated with non-optimum birth weight, and to derive biological insights through trans-omics data integration with placental proteome, maternal plasma lipidome and clinical phenome. METHODS: We conducted quantitative lipidomics of cord blood samples from two independent cohorts: a retrospective discovery cohort (n = 147) and a prospective validation cohort (n = 73). Integration with placental proteomics, maternal plasma lipidomics and clinical phenomics was conducted to elucidate potential biological implications. FINDINGS: We identified substantial reductions in cord blood polyunsaturated phospholipids (PUFA-PLs) (FDR <0.05) associated with placental vesicle trafficking and formation in LBW, and altered neutrophil degranulation in HBW. Combinatorial analyses of paired maternal plasma and cord blood samples indicated that cord blood PUFA-PL reductions were not attributable to deficient maternal supply, but rather to impeded assimilation (LBW) and increased utilisation (HBW). INTERPRETATION: Our findings provide biological insights that may inform targetable, lipid-oriented nutritional and/or pharmacological strategies to modulate foetal growth and development, with the goal of optimising clinical outcomes for both mother and child. FUNDING: This work was supported by the National Natural Science Foundation of China (82170854, 81870579, 81870545, 82571043, 2357308); National High Level Hospital Clinical Research Funding (2022-PUMCH-C-019); Noncommunicable Chronic Diseases-National Science and Technology Major Project (2024ZD0530200 and 2024ZD0530204); Beijing Municipal Science & Technology Commission (Z201100005520011); Peking University Clinical Scientist Training Program (No. BMU2023PYJH022); Beijing Municipal Natural Science Foundation (7202163, 7184252).

Humans↗
PubMed2007

Depletion of high-abundance proteins in plasma by immunoaffinity subtraction for two-dimensional difference gel electrophoresis analysis.

Blood plasma is believed the most complex human-derived proteome, containing other tissue proteome subsets. Almost all body cells communicate with the plasma, either directly or through tissues or biological fluids, and many of these cells release at least a part of their content into the plasma upon damage or death. A comprehensive, systematic characterization of the plasma proteome in the healthy and diseased states will greatly facilitate the development of biomarkers for early disease detection, clinical diagnosis, and therapy. However, the characterization of human plasma proteome is a very complicated task, owing to the wide dynamic range of concentration that separates the most abundant proteins and the less common ones (10-12 orders of magnitude). The removal of its predominant proteins by affinity chromatography using an FPLC system improves the presence of low-abundance proteins in two-dimensional gel electrophoresis (2DE). The "Multiple Affinity Removal System" (Agilent Technologies) retains albumin, IgG, IgA, haptoglobin, transferrin, and antitrypsin with high specificity and reproducibility. After depletion, we have independently analyzed the flow-through (low-abundance proteins), and the retained fractions, by 2DE (4.0-7.0 pH range). Image analysis of the stained gels revealed that more than 300 spots appeared in the retained fraction and about 1800 spots appeared in the nonretained fraction. This methodology is a valuable tool for clinical proteomics, because its reproducibility allows comparative studies and quantitative analysis by 2DE or two-dimensional differential gel electrophoresis of plasma or sera samples from subjects with different pathological or physiological conditions. In addition, the method allows the comparison of experimental results from different laboratories.

Biomarkers↗
PubMed2006

From genetical genomics to systems genetics: potential applications in quantitative genomics and animal breeding.

This article reviews methods of integration of transcriptomics (and equally proteomics and metabolomics), genetics, and genomics in the form of systems genetics into existing genome analyses and their potential use in animal breeding and quantitative genomic modeling of complex traits. Genetical genomics or the expression quantitative trait loci (eQTL) mapping method and key findings in this research are reviewed. Various procedures and potential uses of eQTL mapping, global linkage clustering, and systems genetics are illustrated using actual analysis on recombinant inbred lines of mice with data on gene expression (for diabetes- and obesity-related genes), pathway, and single nucleotide polymorphism (SNP) linkage maps. Experimental and bioinformatics difficulties and possible solutions are discussed. The main uses of this systems genetics approach in quantitative genomics were shown to be in refinement of the identified QTL, candidate gene and SNP discovery, understanding gene-environment and gene-gene interactions, detection of candidate regulator genes/eQTL, discriminating multiple QTL/eQTL, and detection of pleiotropic QTL/eQTL, in addition to its use in reconstructing regulatory networks. The potential uses in animal breeding are direct selection on heritable gene expression measures, termed "expression assisted selection," and genetical genomic selection of both QTL and eQTL based on breeding values of the respective genes, termed "expression-assisted evaluation."

Animals↗
PubMed2006

The cassava (Manihot esculenta Crantz) root proteome: protein identification and differential expression.

Using high-resolution 2-DE, we resolved proteins extracted from fibrous and tuberous root tissues of 3-month-old cassava plants. Gel image analysis revealed an average of 1467 electrophoretically resolved spots on the fibrous gels and 1595 spots on the tuberous gels in pH 3-10 range. Protein spots from both sets of gels were digested with trypsin. The digests were subjected to nanoelectrospray quadrupole TOF tandem mass analysis. Currently, we have obtained 299 protein identifications for 292 gel spots corresponding to 237 proteins. The proteins span various functional categories from energy, primary and secondary metabolism, disease and defense, destination and storage, transport, signal transduction, protein synthesis, cell structure, and transcription to cell growth and division. Gel image analysis has shown unique, as well as up- and down-regulated proteins, present in the tuberous and the fibrous tissues. Quantitative and qualitative analysis of the cassava root proteome is an important step towards further characterization of differentially expressed proteins and the elucidation of the mechanisms underlying the development and biological functions of the two types of roots.

Electrophoresis, Gel, Two-Dimensional↗
PubMed2006

A proteomic approach for dissecting H-Ras signaling networks in NIH/3T3 mouse embryonic fibroblast cells.

To elucidate an understanding into H-Ras protein network, we have established various oncogene H-Ras-expressing NIH/3T3 mouse embryonic fibroblast cell clones, which are expressing G12V H-Ras, G12R H-Ras, and G12V/T35S H-Ras proteins under the tight control of expression by an antibiotic doxycycline. Here we provide a catalog of proteome profiles in total cell lysate derived from the oncogenic and partial loss of function H-Ras-expressing NIH/3T3 cells. In this biological context, we compared total proteome changes by the combined methods of 2-DE, quantitative image analysis and MALDI-TOF-MS analysis both commonly in oncogenic and partial loss of function H-Ras expression system. Thus, we tried to dissect H-Ras signaling pathway, especially a downstream effector molecule, Raf in NIH/3T3 cells using proteomics tools. In this study, we centralized upon the proteome profile changes as common targets for oncogenic H-Ras and a partial loss of function H-Ras in the H-Ras-expressing cells. Thirteen protein spots were selected as what the staining intensities on the gels for 2-DE images from both kinds of cells were consistently changed in their protein expression level. Differentially regulated expression was further confirmed for some subsets of candidates by semiquantitative RT-PCR and Western blot analysis using specific antibodies. Taken together, our results obtained and present here show that the comparative analysis of proteome from oncogenic and partial loss of function H-Ras-expressing cells has yielded interpretable data to elucidate the protein network directly and/or indirectly.

Animals↗
PubMed2003

Protein microarrays: meeting analytical challenges for clinical applications.

Protein microarrays, one emerging class of proteomic technologies, have broad applications for discovery and quantitative analysis. A rapidly expanding use of this technology is the acquisition of information about the posttranslational modifications of proteins reflecting the activity state of signal pathways and networks, and is now employed for the analysis of biopsy samples in clinical trial research.

Animals↗
PubMed2004

Multiplexed fluorescence detection of phosphorylation, glycosylation, and total protein in the proteomic analysis of breast cancer refractoriness.

The Multiplexed Proteomics (MP) technology is a new approach that permits quantitative, multicolor fluorescence detection of proteins in one-dimensional or two-dimensional gels. This methodology allows for multiplexed identification and differential analysis of phosphoproteins, glycoproteins, and total proteins within a single gel electrophoresis experiment. Here the MP system was applied to the differential proteomic analysis of pregnancy-induced refractoriness to breast cancer using a rat model system. Differential analyses identified multiple proteins with altered phosphorylation, glycosylation, or protein expression patterns.

Animals↗
PubMed2005

High-resolution functional proteomics by active-site peptide profiling.

Characterization and functional annotation of the large number of proteins predicted from genome sequencing projects poses a major scientific challenge. Whereas several proteomics techniques have been developed to quantify the abundance of proteins, these methods provide little information regarding protein function. Here, we present a gel-free platform that permits ultrasensitive, quantitative, and high-resolution analyses of protein activities in proteomes, including highly problematic samples such as undiluted plasma. We demonstrate the value of this platform for the discovery of both disease-related enzyme activities and specific inhibitors that target these proteins.

Animals↗
PubMed2005

Proteohistography--direct analysis of tissue with high sensitivity and high spatial resolution using ProteinChip technology.

On the proteomic level, all tissues, tissue constituents, or even single cells are heterogeneous, but the biological relevance of this cannot be adequately investigated with any currently available technique. The analysis of proteins of small tissue areas by any proteomic approach is limited by the number of required cells. Increasing the number of cells only serves to lower the spatial resolution of expressed proteins. To enhance sensitivity and spatial resolution we developed Proteohistography. Laser microdissection was used to mark special areas of interest on tissue sections attached to glass slides. These areas were positioned under microscopic control directly on an affinity chromatographic ProteinChip Array so that cells were lysed and their released proteins bound on a spatially defined point. The ProteinChip System, surface enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI-TOF-MS), allows the laser to be steered to up to 215 distinct positions across the surface of the spot, enabling a high spatial resolution of measured protein profiles for the analyzed tissue area. Protein profiles of the single positions were visually plotted over the used tissue section to visualize distribution proteohistologically. Results show that the spatial distribution of detectable proteins could be used as a Proteohistogram for a given tissue area. Consequently, this procedure can provide additional information to both a matrix-assisted laser desorption/ionization (MALDI)-based approach and immunohistochemistry, as it is more sensitive, highly quantitative, and no specific antibody is needed. Hence, proteomic heterogeneity can be visualized even if proteins are not known or identified.

Carcinoma, Hepatocellular↗
PubMed2006

Proteomic analysis of effect of hyperthermia on spermatogenesis in adult male mice.

We characterized cellular and molecular mechanisms involved in spermatogenesis following short-term heat exposure of murine testis. For these studies, we utilized a proteomic approach with two-dimensional gel electrophoresis (2DE) analyses and mass spectroscopic identification of proteins with altered expression in mouse testes at different times after heat shock. We established a proteome reference map from 7-wk-old mouse testis linked to a federated proteome database. We used these tools to analyze quantitative variations in the tissue over a time course of 0.5, 2, 6, and 12 h following heat exposure. We separated 108 protein spots expressed differentially between the heat shock tissues and the control mouse testes. Of these spots, we identified 36 by comparing with the control reference map. We then focused on the heterogeneous nuclear ribonucleoproteins (hnRNPs) and the chaperonins containing t-complex polypeptide-1 (CCT). Further analysis in this heat-shocked model suggests numerous potential mechanisms for heat shock-induced spermatogenic disorder.

Animals↗