Discovery and preclinical development of a therapeutically active nanobody-based chimeric antigen receptor targeting human CD22.

Chimeric antigen receptor (CAR) T cell therapies targeting B cell-restricted antigens CD19, CD20, or CD22 can produce potent clinical responses for some B cell malignancies, but relapse remains common. Camelid single-domain antibodies (sdAbs or nanobodies) are smaller, simpler, and easier to recombine than single-chain variable fragments (scFvs) used in most CARs, but fewer sdAb-CARs have been reported. Thus, we sought to identify a therapeutically active sdAb-CAR targeting human CD22. Immunization of an adult Llama glama with CD22 protein, sdAb-cDNA library construction, and phage panning yielded >20 sdAbs with diverse epitope and binding properties. Expressing CD22-sdAb-CAR in Jurkat cells drove varying CD22-specific reactivity not correlated with antibody affinity. Changing CD28- to CD8-transmembrane design increased CAR persistence and expression in vitro. CD22-sdAb-CAR candidates showed similar CD22-dependent CAR-T expansion in vitro, although only membrane-proximal epitope targeting CD22-sdAb-CARs activated direct cytolytic killing and extended survival in a lymphoma xenograft model. Based on enhanced survival in blinded xenograft studies, a lead CD22sdCAR-T was selected, achieving comparable complete responses to a benchmark short linker m971-scFv CAR-T in high-dose experiments. Finally, immunohistochemistry and flow cytometry confirm tissue and cellular-level specificity of the lead CD22-sdAb. This presents a complete report on preclinical development of a novel CD22sdCAR therapeutic.

A large-scale, targeted metabolomics method for the analysis and quantification of metabolites in human plasma via liquid chromatography-mass spectrometry.

Metabolomics is the study of small molecules, primarily metabolites, that are produced during metabolic processes. Analysis of the composition of an organism's metabolome can yield useful information about an individual's health status at any given time. In recent years, the development of large-scale, targeted metabolomic methods has allowed for the analysis of biological samples using analytical techniques such as LC-MS/MS. This paper presents a large-scale metabolomics method for analysis of biological samples, with a focus on quantification of metabolites found in blood plasma. The method comprises a 10-min chromatographic separation using HILIC and RP stationary phases combined with positive and negative electrospray ionization in order to maximize metabolome coverage. Complete analysis of a single sample can be achieved in as little as 40 min using the two columns and dual modes of ionization. With 540 metabolites and the inclusion of over 200 analytical standards, this method is comprehensive and quantitatively robust when compared to current targeted metabolomics methods. This study uses a large-scale evaluation of metabolite recovery from plasma that enables absolute quantification of metabolites by correcting for analyte loss throughout processes such as extraction, handling, or storage. In addition, the method was applied to plasma collected from adjuvant breast cancer patients to confirm the suitability of the method to clinical samples.

Analysis of the Circulating Metabolome of Patients with Cutaneous, Mucosal and Uveal Melanoma Reveals Distinct Metabolic Profiles with Implications for Response to Immunotherapy.

Cutaneous melanoma (CM) patients respond better to immune checkpoint inhibitors (ICI) than mucosal and uveal melanoma patients (MM/UM). Aiming to explore these differences and understand the distinct response to ICI, we evaluated the serum metabolome of advanced CM, MM, and UM patients. Levels of 115 metabolites were analyzed in samples collected before ICI, using a targeted metabolomics platform. In our analysis, molecules involved in the tryptophan-kynurenine axis distinguished UM/MM from CM. UM/MM patients had higher levels of 3-hydroxykynurenine (3-HKyn), whilst patients with CM were found to have higher levels of kynurenic acid (KA). The KA/3-HKyn ratio was significantly higher in CM versus the other subtypes. UM, the most ICI-resistant subtype, was also associated with higher levels of sphingomyelin-d18:1/22:1 and the polyamine spermine (SPM). Overall survival was prolonged in a cohort of CM patients with lower SPM levels, suggesting there are also conserved metabolic factors promoting ICI resistance across melanoma subtypes. Our study revealed a distinct metabolomic profile between the most resistant melanoma subtypes, UM and MM, compared to CM. Alterations within the kynurenine pathway, polyamine metabolism, and sphingolipid metabolic pathway may contribute to the poor response to ICI. Understanding the different metabolomic profiles introduces opportunities for novel therapies with potential synergic activity to ICI, to improve responses of UM/MM.

A targeted isotope dilution mass spectrometry assay for osteopontin quantification in plasma of metastatic breast cancer patients.

Osteopontin (OPN) is a secreted glycophosphoprotein that derives its name from its high abundance in bone and secretion by osteoblasts. It is also secreted by a number of immune cells and, therefore, is present in human plasma at nanogram per millilitre levels where it affects cell adhesion and motility. OPN is involved in several normal physiological processes; however, OPN dyregulation leads to overexpression by tumor cells leading to immune evasion and increased metastasis. Plasma OPN is primarily measured by enzyme-linked immunosorbent assay (ELISA). However, due to the complexity of the various OPN isoforms, conflicting results have been obtained on the use of OPN as a biomarker even in the same disease condition. These discrepant results may result from the difficulty in comparing ELISA results obtained with different antibodies that target unique OPN epitopes. Mass spectrometry can be used to quantify proteins in plasma and, by targeting OPN regions that do not bear post-translational modifications, may provide more consistent quantification. However, the low (ng/mL) levels in plasma present a significant analytical challenge. In order to develop a sensitive assay for plasma OPN, we explored a single-step precipitation method using a recently developed spin-tube format. Quantification was performed using isotope-dilution mass spectrometry. The concentration detection limit of this assay was 39 ± 15 ng/mL. The assay was applied to the analysis of plasma OPN in metastatic breast cancer patients, where levels from 17 to 53 ng/mL were detected. The sensitivity of the method is higher than previously published methods and sufficient for OPN detection in large, high grade tumors but still requires improvement in sensitivity to be widely applicable.

Successful Dietary Therapy in Paediatric Crohn's Disease is Associated with Shifts in Bacterial Dysbiosis and Inflammatory Metabotype Towards Healthy Controls.

BACKGROUND AND AIMS: Nutritional therapy with the Crohn's Disease Exclusion Diet + Partial Enteral Nutrition [CDED+PEN] or Exclusive Enteral Nutrition [EEN] induces remission and reduces inflammation in mild-to-moderate paediatric Crohn's disease [CD]. We aimed to assess if reaching remission with nutritional therapy is mediated by correcting compositional or functional dysbiosis. METHODS: We assessed metagenome sequences, short chain fatty acids [SCFA] and bile acids [BA] in 54 paediatric CD patients reaching remission after nutritional therapy [with CDED + PEN or EEN] [NCT01728870], compared to 26 paediatric healthy controls. RESULTS: Successful dietary therapy decreased the relative abundance of Proteobacteria and increased Firmicutes towards healthy controls. CD patients possessed a mixture of two metabotypes [M1 and M2], whereas all healthy controls had metabotype M1. M1 was characterised by high Bacteroidetes and Firmicutes, low Proteobacteria, and higher SCFA synthesis pathways, and M2 was associated with high Proteobacteria and genes involved in SCFA degradation. M1 contribution increased during diet: 48%, 63%, up to 74% [Weeks 0, 6, 12, respectively.]. By Week 12, genera from Proteobacteria reached relative abundance levels of healthy controls with the exception of E. coli. Despite an increase in SCFA synthesis pathways, remission was not associated with increased SCFAs. Primary BA decreased with EEN but not with CDED+PEN, and secondary BA did not change during diet. CONCLUSION: Successful dietary therapy induced correction of both compositional and functional dysbiosis. However, 12 weeks of diet was not enough to achieve complete correction of dysbiosis. Our data suggests that composition and metabotype are important and change quickly during the early clinical response to dietary intervention. Correction of dysbiosis may therefore be an important future treatment goal for CD.

Metabolome Changes With Diet-Induced Remission in Pediatric Crohn's Disease.

BACKGROUND & AIMS: The Crohn's disease (CD) exclusion diet (CDED) plus partial enteral nutrition (PEN) and exclusive enteral nutrition (EEN) both induce remission in pediatric CD. CDED+PEN is better tolerated and able to sustain remission. We characterized the changes in fecal metabolites induced by CDED+PEN and EEN and their relationship with remission. METHODS: A total of 216 fecal metabolites were measured in 80 fecal samples at week (W) 0, W6, and W12, of children with mild to moderate CD in a prospective randomized trial comparing CDED+PEN vs EEN. The metabolites were measured using liquid chromatography coupled to mass spectrometry. Metagenome Kyoto Encyclopedia of Genes and Genomes Orthology analysis was performed to investigate the differential functional gene abundance involved in specific metabolic pathways. Data were analyzed according to clinical outcome of remission (W6_rem), no remission (W6_nr), sustained remission (W12_sr), and nonsustained (W12_nsr) remission. RESULTS: A decrease in kynurenine and succinate synthesis and an increase in N-α-acetyl-arginine characterized CDED+PEN W6_rem, whereas changes in lipid metabolism characterized EEN W6_rem, especially reflected by lower levels in ceramides. In contrast, fecal metabolites in EEN W6_nr were comparable to baseline/W0 samples. CDED+PEN W6_rem children maintained metabolome changes through W12. In contrast, W12_nsr children in the EEN group, who resumed a free diet after week 6, did not. The metabolome of CDED+PEN differed from EEN in the purine, pyrimidine, and sphingolipid pathways. A significant differential abundance in several genes involved in these pathways was detected. CONCLUSION: CDED+PEN- and EEN-induced remission are associated with significant changes in inflammatory bowel disease-associated metabolites such as kynurenine, ceramides, amino acids, and others. Sustained remission with CDED+PEN, but not EEN, was associated with persistent changes in metabolites. CLINICALTRIALS: gov, Number NCT01728870.

Multiplexed Quantitative Proteomic Profiling of Cancer Cells and Tissues Using Isobaric Labeling-Based Tags.

Comparing cancer proteomes across many samples offers a window into cancer cell biology and may reveal new treatment options for specific subsets of cancer. Here we describe a method using tandem mass tag (TMT) technology to multiplex up to 18 samples in a single analysis, paving the way for the analysis of large cohorts of tumors, cell lines, and perturbations thereof. The procedure we describe will result in samples ready for in-depth LC-MS/MS analysis in 3-4 days.

Tryptophan-derived microbial metabolites activate the aryl hydrocarbon receptor in tumor-associated macrophages to suppress anti-tumor immunity.

The aryl hydrocarbon receptor (AhR) is a sensor of products of tryptophan metabolism and a potent modulator of immunity. Here, we examined the impact of AhR in tumor-associated macrophage (TAM) function in pancreatic ductal adenocarcinoma (PDAC). TAMs exhibited high AhR activity and Ahr-deficient macrophages developed an inflammatory phenotype. Deletion of Ahr in myeloid cells or pharmacologic inhibition of AhR reduced PDAC growth, improved efficacy of immune checkpoint blockade, and increased intra-tumoral frequencies of IFNγ+CD8+ T cells. Macrophage tryptophan metabolism was not required for this effect. Rather, macrophage AhR activity was dependent on Lactobacillus metabolization of dietary tryptophan to indoles. Removal of dietary tryptophan reduced TAM AhR activity and promoted intra-tumoral accumulation of TNFα+IFNγ+CD8+ T cells; provision of dietary indoles blocked this effect. In patients with PDAC, high AHR expression associated with rapid disease progression and mortality, as well as with an immune-suppressive TAM phenotype, suggesting conservation of this regulatory axis in human disease.

Metabolite profiling reveals a connection between aldehyde dehydrogenase 1A3 and GABA metabolism in breast cancer metastasis.

INTRODUCTION: Aldehyde dehydrogenase 1A3 (ALDH1A3) is a cancer stem cell (CSC) marker and in breast cancer it is associated with triple-negative/basal-like subtypes and aggressive disease. Studies on the mechanisms of ALDH1A3 in cancer have primarily focused on gene expression changes induced by the enzyme; however, its effects on metabolism have thus far been unstudied and may reveal novel mechanisms of pathogenesis. OBJECTIVE: Determine how ALDH1A3 alters the metabolite profile in breast cancer cells and assess potential impacts. METHOD: Triple-negative MDA-MB-231 tumors and cells with manipulated ALDH1A3 levels were assessed by HPLC-MS metabolomics and metabolite data was integrated with transcriptome data. Mice harboring MDA-MB-231 tumors with or without altered ALDH1A3 expression were treated with γ-aminobutyric acid (GABA) or placebo. Effects on tumor growth, and lungs and brain metastasis were quantified by staining of fixed thin sections and quantitative PCR. Breast cancer patient datasets from TCGA, METABRIC and GEO were used to assess the co-expression of GABA pathway genes with ALDH1A3. RESULTS: Integrated metabolomic and transcriptome data identified GABA metabolism as a primary dysregulated pathway in ALDH1A3 expressing breast tumors. Both ALDH1A3 and GABA treatment enhanced metastasis. Patient dataset analyses revealed expression association between ALDH1A3 and GABA pathway genes and corresponding increased risk of metastasis. CONCLUSION: This study revealed a novel pathway affected by ALDH1A3, GABA metabolism. Like ALDH1A3 expression, GABA treatment promotes metastasis. Given the clinical use of GABA mimics to relieve chemotherapy-induced peripheral nerve pain, further study of the effects of GABA in breast cancer progression is warranted.

Coenzyme A fuels T cell anti-tumor immunity.

Metabolic programming is intricately linked to the anti-tumor properties of T cells. To study the metabolic pathways associated with increased anti-tumor T cell function, we utilized a metabolomics approach to characterize three different CD8+ T cell subsets with varying degrees of anti-tumor activity in murine models, of which IL-22-producing Tc22 cells displayed the most robust anti-tumor activity. Tc22s demonstrated upregulation of the pantothenate/coenzyme A (CoA) pathway and a requirement for oxidative phosphorylation (OXPHOS) for differentiation. Exogenous administration of CoA reprogrammed T cells to increase OXPHOS and adopt the CD8+ Tc22 phenotype independent of polarizing conditions via the transcription factors HIF-1α and the aryl hydrocarbon receptor (AhR). In murine tumor models, treatment of mice with the CoA precursor pantothenate enhanced the efficacy of anti-PDL1 antibody therapy. In patients with melanoma, pre-treatment plasma pantothenic acid levels were positively correlated with the response to anti-PD1 therapy. Collectively, our data demonstrate that pantothenate and its metabolite CoA drive T cell polarization, bioenergetics, and anti-tumor immunity.

Comparative performance of different methods for circulating tumor cell enrichment in metastatic breast cancer patients.

The isolation and analysis of circulating tumor cells (CTC) has the potential to provide minimally invasive diagnostic, prognostic and predictive information. Widespread clinical implementation of CTC analysis has been hampered by a lack of comparative investigation between different analytic methodologies in clinically relevant settings. The objective of this study was to evaluate four different CTC isolation techniques-those that rely on surface antigen expression (EpCAM or CD45 using DynaBeads® or EasySep™ systems) or the biophysical properties (RosetteSep™ or ScreenCell®) of CTCs. These were evaluated using cultured cells in order to calculate isolation efficiency at various levels including; inter-assay and inter-operator variability, protocol complexity and turn-around time. All four techniques were adequate at levels above 100 cells/mL which is commonly used for the evaluation of new isolation techniques. Only the RosetteSep™ and ScreenCell® techniques were found to provide adequate sensitivity at a level of 10 cells/mL. These techniques were then applied to the isolation and analysis of circulating tumor cells blood drawn from metastatic breast cancer patients where CTCs were detected in 54% (15/28) of MBC patients using the RosetteSep™ and 75% (6/8) with ScreenCell®. Overall, the ScreenCell® method had better sensitivity.

Heat stress response in the closest algal relatives of land plants reveals conserved stress signaling circuits.

All land plants (embryophytes) share a common ancestor that likely evolved from a filamentous freshwater alga. Elucidating the transition from algae to embryophytes - and the eventual conquering of Earth's surface - is one of the most fundamental questions in plant evolutionary biology. Here, we investigated one of the organismal properties that might have enabled this transition: resistance to drastic temperature shifts. We explored the effect of heat stress in Mougeotia and Spirogyra, two representatives of Zygnematophyceae - the closest known algal sister lineage to land plants. Heat stress induced pronounced phenotypic alterations in their plastids, and high-performance liquid chromatography-tandem mass spectroscopy-based profiling of 565 transitions for the analysis of main central metabolites revealed significant shifts in 43 compounds. We also analyzed the global differential gene expression responses triggered by heat, generating 92.8 Gbp of sequence data and assembling a combined set of 8905 well-expressed genes. Each organism had its own distinct gene expression profile; less than one-half of their shared genes showed concordant gene expression trends. We nevertheless detected common signature responses to heat such as elevated transcript levels for molecular chaperones, thylakoid components, and - corroborating our metabolomic data - amino acid metabolism. We also uncovered the heat-stress responsiveness of genes for phosphorelay-based signal transduction that links environmental cues, calcium signatures and plastid biology. Our data allow us to infer the molecular heat stress response that the earliest land plants might have used when facing the rapidly shifting temperature conditions of the terrestrial habitat.

Immunoproteomic Biomarkers: From Publication to Personalized Medicine.

As the number of immunotherapies increases, so does the need for biomarkers that can aid in identifying an optimal therapy or combination therapy for patients. These predictive biomarkers are of enormous value to patients but present unique challenges to researchers due to the complexity of the immune system and the variability of individual patient molecular profiles. This chapter draws on recent examples of the use of biomarkers to explore the range of phenotypes encountered in immunotherapy trials for the treatment of neoplastic disease. These examples are discussed in the context of immunoproteomic analysis with a particular focus on the unique challenges that are presented when a high dimensionality technique such as immunoproteomics is applied to study a complex system, the immune system in this case. In order to overcome these challenges, immunoproteomic researchers must pay close attention to study design in order to ensure that the results are not only statistically valid but also that the biomarker strategy as a whole is compatible with the standard of care. We propose that, in spite of its limitations, the use of immunoproteomic analysis of liquid biopsies may present a unique opportunity for translation of immunoproteomic biomarkers to the clinic.

Enhanced killing of breast cancer cells by a d-amino acid analog of the winter flounder-derived pleurocidin NRC-03.

Cationic antimicrobial peptides (CAPs) defend against pathogens and, in some cases, exhibit potent anticancer activities. We previously reported that the pleurocidin NRC-03 causes lysis of breast cancer and multiple myeloma cells. NRC-03 also reduces the EC50 of other cytotoxic compounds and prevents tumor growth in vivo. However, the therapeutic utility of NRC-03 may be limited by its susceptibility to degradation by proteases. The goal of this study was to characterize the anticancer activities of a d-amino acid analog of NRC-03 ([D]-NRC-03) that was predicted to be resistant to proteolytic degradation. Unlike NRC-03, [D]-NRC-03 was not degraded by human serum or trypsin and, in comparison to NRC-03, showed increased killing of breast cancer cells, including multidrug-resistant cells; however, [D]-NRC-03 was somewhat more cytotoxic than NRC-03 for several types of normal cells. Importantly, [D]-NRC-03 was more effective than NRC-03 in vivo since 4-fold less peptide was required for an equivalent inhibitory effect on the growth of breast cancer cell xenografts in immune-deficient mice. These findings demonstrate that a d-amino acid analog of NRC-03 overcomes a major limitation to the therapeutic use of NRC-03, namely peptide stability. Further modification of [D]-NRC-03 is required to improve its selectivity for cancer cells.

Ras transformation results in cleavage of reticulon protein Nogo-B that is associated with impairment of IFN response.

Dysregulation of Ras signaling is the major cause of various cancers. Aberrant Ras signaling, however, provides a favorable environment for many viruses, making them suitable candidates as cancer-killing therapeutic agents. Susceptibility of cancer cells to such viruses is mainly due to impaired type I interferon (IFN) response, often as a result of activated Ras/ERK signaling in these cells. In this study, we searched for cellular factors modulated by Ras signaling and their potential involvement in promoting viral oncolysis. We found that upon Ras transformation of NIH-3T3 cells, the N-terminus of Nogo-B (reticulon 4) was proteolytically cleaved. Interestingly, Nogo knockdown (KD) in non-transformed and Ras-transformed cells both enhanced virus-induced IFN response, suggesting that both cleaved and uncleaved Nogo can suppress IFN response. However, pharmacological blockade of Nogo cleavage in Ras-transformed cells significantly enhanced virus-induced IFN response, suggesting that cleaved Nogo contributes to enhanced IFN suppression in these cells. We further showed that IFN suppression associated with Ras-induced Nogo-B cleavage was distinct from but synergistic with that associated with an activated Ras/ERK pathway. Our study therefore reveals an important and novel role of Nogo-B and its cleavage in the suppression of anti-viral immune responses by oncogenic Ras transformation.

Motoneurons derived from induced pluripotent stem cells develop mature phenotypes typical of endogenous spinal motoneurons.

Induced pluripotent cell-derived motoneurons (iPSCMNs) are sought for use in cell replacement therapies and treatment strategies for motoneuron diseases such as amyotrophic lateral sclerosis (ALS). However, much remains unknown about the physiological properties of iPSCMNs and how they compare with endogenous spinal motoneurons or embryonic stem cell-derived motoneurons (ESCMNs). In the present study, we first used a proteomic approach and compared protein expression profiles between iPSCMNs and ESCMNs to show that <4% of the proteins identified were differentially regulated. Like ESCs, we found that mouse iPSCs treated with retinoic acid and a smoothened agonist differentiated into motoneurons expressing the LIM homeodomain protein Lhx3. When transplanted into the neural tube of developing chick embryos, iPSCMNs selectively targeted muscles normally innervated by Lhx3 motoneurons. In vitro studies showed that iPSCMNs form anatomically mature and functional neuromuscular junctions (NMJs) when cocultured with chick myofibers for several weeks. Electrophysiologically, iPSCMNs developed passive membrane and firing characteristic typical of postnatal motoneurons after several weeks in culture. Finally, iPSCMNs grafted into transected mouse tibial nerve projected axons to denervated gastrocnemius muscle fibers, where they formed functional NMJs, restored contractile force. and attenuated denervation atrophy. Together, iPSCMNs possess many of the same cellular and physiological characteristics as ESCMNs and endogenous spinal motoneurons. These results further justify using iPSCMNs as a source of motoneurons for cell replacement therapies and to study motoneuron diseases such as ALS.

Compositional complexity of the mitochondrial proteome of a unicellular eukaryote (Acanthamoeba castellanii, supergroup Amoebozoa) rivals that of animals, fungi, and plants.

We present a combined proteomic and bioinformatic investigation of mitochondrial proteins from the amoeboid protist Acanthamoeba castellanii, the first such comprehensive investigation in a free-living member of the supergroup Amoebozoa. This protist was chosen both for its phylogenetic position (as a sister to animals and fungi) and its ecological ubiquity and physiological flexibility. We report 1033 A. castellanii mitochondrial protein sequences, 709 supported by mass spectrometry data (676 nucleus-encoded and 33 mitochondrion-encoded), including two previously unannotated mtDNA-encoded proteins, which we identify as highly divergent mitochondrial ribosomal proteins. Other notable findings include duplicate proteins for all of the enzymes of the tricarboxylic acid (TCA) cycle-which, along with the identification of a mitochondrial malate synthase-isocitrate lyase fusion protein, suggests the interesting possibility that the glyoxylate cycle operates in A. castellanii mitochondria. Additionally, the A. castellanii genome encodes an unusually high number (at least 29) of mitochondrion-targeted pentatricopeptide repeat (PPR) proteins, organellar RNA metabolism factors in other organisms. We discuss several key mitochondrial pathways, including DNA replication, transcription and translation, protein degradation, protein import and Fe-S cluster biosynthesis, highlighting similarities and differences in these pathways in other eukaryotes. In compositional and functional complexity, the mitochondrial proteome of A. castellanii rivals that of multicellular eukaryotes. BIOLOGICAL SIGNIFICANCE: Comprehensive proteomic surveys of mitochondria have been undertaken in a limited number of predominantly multicellular eukaryotes. This phylogenetically narrow perspective constrains and biases our insights into mitochondrial function and evolution, as it neglects protists, which account for most of the evolutionary and functional diversity within eukaryotes. We report here the first comprehensive investigation of the mitochondrial proteome in a member (A. castellanii) of the eukaryotic supergroup Amoebozoa. Through a combination of tandem mass spectrometry (MS/MS) and in silico data mining, we have retrieved 1033 candidate mitochondrial protein sequences, 709 having MS support. These data were used to reconstruct the metabolic pathways and protein complexes of A. castellanii mitochondria, and were integrated with data from other characterized mitochondrial proteomes to augment our understanding of mitochondrial proteome evolution. Our results demonstrate the power of combining direct proteomic and bioinformatic approaches in the discovery of novel mitochondrial proteins, both nucleus-encoded and mitochondrion-encoded, and highlight the compositional complexity of the A. castellanii mitochondrial proteome, which rivals that of animals, fungi and plants.

Data showing the compositional complexity of the mitochondrial proteome of a unicellular eukaryote (Acanthamoeba castellanii, supergroup Amoebozoa).

This article describes and directly links to 1033 Acanthamoeba castellanii mitochondrial protein sequences. Of these, 709 are supported by Mass Spectrometry (MS) data (676 nucleus-encoded and 33 mitochondrion-encoded). Two of these entries are previously unannotated mtDNA-encoded proteins, which we identify as highly divergent mitochondrial ribosomal proteins. Our analysis corrects many A. castellanii protein sequences that were incorrectly inferred previously from genomic data deposited in NCBI.

Serum biomarkers of papillary thyroid cancer.

OBJECTIVE: To identify serum biomarkers of papillary thyroid cancer. METHODS: Prospective analysis was performed of banked tumor and serum specimens from 99 patients with thyroid masses. Enzyme-linked immunosorbent assay (ELISA) was employed to measure levels of five serum proteins previously demonstrated to be up-regulated in papillary thyroid cancer (PTC): angiopoietin-1 (Ang-1), cytokeratin 19 (CK-19), tissue inhibitor of metalloproteinase-1 (TIMP-1), chitinase 3 like-1 (YKL-40), and galectin-3 (GAL-3). Serum levels were compared between patients with PTC and those with benign tumors. RESULTS: A total of 99 patients were enrolled in the study (27 men, 72 women), with a median age of 54 years. Forty-three patients had PTC and 58 cases were benign tumors. There were no statistically significant differences when comparing all five different biomarkers between PTC and other benign thyroid tumors. The p-values were 0.94, 0.48, 0.72, 0.48, and 0.90 for YKL-40, Gal-3, CK19, TIMP-1, and Ang-1, respectively. CONCLUSION: Serum levels of four of the five proteins were elevated in patients with thyroid masses relative to normal values. However, the difference between benign and PTC was not significant. Two of the markers (Gal-3 & TIMP-1) displayed a greater potential difference, which may warrant further investigation. This study suggests that other serum markers should be sought. This is the first study to investigate potential serum biomarkers based on over-expressed proteins in thyroid cancer versus benign pathology.

Proteomics analysis suggests broad functional changes in potato leaves triggered by phosphites and a complex indirect mode of action against Phytophthora infestans.

Phosphite (salts of phosphorous acid; Phi)-based fungicides are increasingly used in controlling oomycete pathogens, such as the late blight agent Phytophthora infestans. In plants, low amounts of Phi induce pathogen resistance through an indirect mode of action. We used iTRAQ-based quantitative proteomics to investigate the effects of phosphite on potato plants before and after infection with P. infestans. Ninety-three (62 up-regulated and 31 down-regulated) differentially regulated proteins, from a total of 1172 reproducibly identified proteins, were identified in the leaf proteome of Phi-treated potato plants. Four days post-inoculation with P. infestans, 16 of the 31 down-regulated proteins remained down-regulated and 42 of the 62 up-regulated proteins remained up-regulated, including 90% of the defense proteins. This group includes pathogenesis-related, stress-responsive, and detoxification-related proteins. Callose deposition and ultrastructural analyses of leaf tissues after infection were used to complement the proteomics approach. This study represents the first comprehensive proteomics analysis of the indirect mode of action of Phi, demonstrating broad effects on plant defense and plant metabolism. The proteomics data and the microscopy study suggest that Phi triggers a hypersensitive response that is responsible for induced resistance of potato leaves against P. infestans. BIOLOGICAL SIGNIFICANCE: Phosphie triggers complex functional changes in potato leaves that are responsible for the induced resistance against Phytophthora infestans. This article is part of a Special Issue entitled: Translational Plant Proteomics.