The Biological Action and Structural Characterization of Eryngitin 3 and 4, Ribotoxin-like Proteins from Pleurotus eryngii Fruiting Bodies.

Ribotoxin-like proteins (RL-Ps) are specific ribonucleases found in mushrooms that are able to cleave a single phosphodiester bond located in the sarcin-ricin loop (SRL) of the large rRNA. The cleaved SRL interacts differently with some ribosomal proteins (P-stalk). This action blocks protein synthesis because the damaged ribosomes are unable to interact with elongation factors. Here, the amino acid sequences of eryngitin 3 and 4, RL-Ps isolated from Pleurotus eryngii fruiting bodies, were determined to (i) obtain structural information on this specific ribonuclease family from edible mushrooms and (ii) explore the structural determinants which justify their different biological and antipathogenic activities. Indeed, eryngitin 3 exhibited higher toxicity with respect to eryngitin 4 against tumoral cell lines and model fungi. Structurally, eryngitin 3 and 4 consist of 132 amino acids, most of them identical and exhibiting a single free cysteinyl residue. The amino acidic differences between the two toxins are (i) an additional phenylalanyl residue at the N-terminus of eryngitin 3, not retrieved in eryngitin 4, and (ii) an additional arginyl residue at the C-terminus of eryngitin 4, not retrieved in eryngitin 3. The 3D models of eryngitins show slight differences at the N- and C-terminal regions. In particular, the positive electrostatic surface at the C-terminal of eryngitin 4 is due to the additional arginyl residue not retrieved in eryngitin 3. This additional positive charge could interfere with the binding to the SRL (substrate) or with some ribosomal proteins (P-stalk structure) during substrate recognition.

MRPL15 is a novel prognostic biomarker and therapeutic target for epithelial ovarian cancer.

PURPOSE: To analyze the role of six human epididymis protein 4 (HE4)-related mitochondrial ribosomal proteins (MRPs) in ovarian cancer and selected MRPL15, which is most closely related to the tumorigenesis and prognosis of ovarian cancer, for further analyses. METHODS: Using STRING database and MCODE plugin in Cytoscape, six MRPs were identified among genes that are upregulated in response to HE4 overexpression in epithelial ovarian cancer cells. The Cancer Genome Atlas (TCGA) ovarian cancer, GTEX, Oncomine, and TISIDB were used to analyze the expression of the six MRPs. The prognostic impact and genetic variation of these six MRPs in ovarian cancer were evaluated using Kaplan-Meier Plotter and cBioPortal, respectively. MRPL15 was selected for immunohistochemistry and GEO verification. TCGA ovarian cancer data, gene set enrichment analysis, and Enrichr were used to explore the mechanism of MRPL15 in ovarian cancer. Finally, the relationship between MRPL15 expression and immune subtype, tumor-infiltrating lymphocytes, and immune regulatory factors was analyzed using TCGA ovarian cancer data and TISIDB. RESULTS: Six MRPs (MRPL10, MRPL15, MRPL36, MRPL39, MRPS16, and MRPS31) related to HE4 in ovarian cancer were selected. MRPL15 was highly expressed and amplified in ovarian cancer and was related to the poor prognosis of patients. Mechanism analysis indicated that MRPL15 plays a role in ovarian cancer through pathways such as the cell cycle, DNA repair, and mTOR 1 signaling. High expression of MRPL15 in ovarian cancer may be associated with its amplification and hypomethylation. Additionally, MRPL15 showed the lowest expression in C3 ovarian cancer and was correlated with proliferation of CD8+ T cells and dendritic cells as well as TGFßR1 and IDO1 expression. CONCLUSION: MRPL15 may be a prognostic indicator and therapeutic target for ovarian cancer. Because of its close correlation with HE4, this study provides insights into the mechanism of HE4 in ovarian cancer.

MRPL27 contributes to unfavorable overall survival and disease-free survival from cholangiocarcinoma patients.

Objective: This study aimed to investigate the roles of MRPL27 in survival from cholangiocarcinoma patients in The Cancer Genome Atlas (TCGA) database. Methods: In TCGA-CHOL profile, MRPL27 gene expression and clinical data were obtained. Cox regression models were used to evaluate the potential links between MRPL27 and cholangiocarcinoma survival. Enrichment analysis of MRPL27 was conducted in Metascape and Gene Set Enrichment Analysis (GSEA) databases. Results: 36 cholangiocarcinoma patients were included in this analysis. MRPL27 mRNA was significantly upregulated in tumor tissues in cholangiocarcinoma patients including intrahepatic, distal and hilar/perihilar cholangiocarcinoma cases (all p < 0.01). Cholangiocarcinoma patients with high MRPL27 had worse overall survival (OS) and disease-free survival (DFS) compared to those with low MRPL27 (all p < 0.05). Univariate and multivariate Cox models indicated that MRPL27 should be a risk factor for the OS and DFS in cholangiocarcinoma patients (both p < 0.01). Bioinformatic analysis revealed that MRPL27 mainly involved in the processes of mitochondrial translation elongation, respiratory electron transport, ATP synthesis, and inner mitochondrial membrane organization. No mutations of MRPL27 were screened in cholangiocarcinoma patients. Conclusion: Upregulated in tumors, MRPL27 contributes to unfavorable survival in cholangiocarcinoma patients.

First-in-class candidate therapeutics that target mitochondria and effectively prevent cancer cell metastasis: mitoriboscins and TPP compounds.

Cancer stem cells (CSCs) have been proposed to be responsible for tumor recurrence, distant metastasis and drug-resistance, in the vast majority of cancer patients. Therefore, there is an urgent need to identify new drugs that can target and eradicate CSCs. To identify new molecular targets that are unique to CSCs, we previously compared MCF7 2D-monolayers with 3D-mammospheres, which are enriched in CSCs. We observed that 25 mitochondrial-related proteins were >100-fold over-expressed in 3D-mammospheres. Here, we used these 25 proteins to derive short gene signatures to predict distant metastasis (in N=1,395 patients) and tumor recurrence (in N=3,082 patients), by employing a large collection of transcriptional profiling data from ER(+) breast cancer patients. This analysis resulted in a 4-gene signature for predicting distant metastasis, with a hazard ratio of 1.91-fold (P=2.2e-08). This provides clinical evidence to support a role for CSC mitochondria in metastatic dissemination. Next, we employed a panel of mitochondrial inhibitors, previously shown to target mitochondria and selectively inhibit 3D-mammosphere formation in MCF7 cells and cell migration in MDA-MB-231 cells. Remarkably, these five mitochondrial inhibitors had only minor effects or no effect on MDA-MB-231 tumor formation, but preferentially and selectively inhibited tumor cell metastasis, without causing significant toxicity. Mechanistically, all five mitochondrial inhibitors have been previously shown to induce ATP-depletion in cancer cells. Since 3 of these 5 inhibitors were designed to target the large mitochondrial ribosome, we next interrogated whether genes encoding the large mitochondrial ribosomal proteins (MRPL) also show prognostic value in the prediction of distant metastasis in both ER(+) and ER(-) breast cancer patients. Interestingly, gene signatures composed of 6 to 9 MRPL mRNA-transcripts were indeed sufficient to predict distant metastasis, tumor recurrence and Tamoxifen resistance. These gene signatures could be useful as companion diagnostics to assess which patients may benefit most from anti-mito-ribosome therapy. Overall, our studies provide the necessary proof-of-concept, and in vivo functional evidence, that mitochondrial inhibitors can successfully and selectively target the biological process of cancer cell metastasis. Ultimately, we envision that mitochondrial inhibitors could be employed to develop new treatment protocols, for clinically providing metastasis prophylaxis, to help prevent poor clinical outcomes in cancer patients.

Reliable identification of lactic acid bacteria by targeted and untargeted high-resolution tandem mass spectrometry.

Probiotic lactic acid bacteria (LAB) are generally employed in food industry because they contribute to nutritional value of fermented foods. Although knowledge of LAB composition is of high relevance for various industrial and biotechnological applications, the comprehensive identification of LAB species is sometimes technically challenging. Recently, MALDI-TOF MS-based methodologies for bacteria detection/identification in clinical diagnostics and agri-food proved to be an attractive strategy, complementary to traditional techniques for their sensitivity and specificity. In this study, we propose, for the first time, a novel methodology based on high resolution nano-LC-ESI-MS/MS for LAB identification at genus, species and sub-species level by using the sequence regions 33-52 and 72-82 of the S16 ribosomal protein as proteotypic peptide markers. The developed methodology was then applied to the analyses of buffalo and bovine whey starter cultures, thus assessing the applicability of the approach for the detection of LAB also in complex matrices.

The MRPS18-2 protein levels correlate with prostate tumor progression and it induces CXCR4-dependent migration of cancer cells.

We have earlier found abnormal expression of the mitochondrial ribosomal protein S18-2 (MRPS18-2, S18-2) in endometrial cancer, compared to the expression in hyperplasia and in normal endometrium. Here we report that expression of S18-2 was increased with disease progression in clinical specimens of prostate cancer (PCa). The level of induction of epithelial to mesenchymal cell transition (EMT) correlated with the expression level of S18-2 in PCa cell lines. Moreover, cells acquired increased ability of migration upon S18-2 overexpression, as was evaluated in zebrafish embryo model and in trans-well assay. We found that this is due to increased CXCR4 cell surface expression. Neutralizing CXCR4 protein or abrogating S18-2 expression in cells significantly reduced their migratory ability directed toward CXCL12. The mRNA expression of TWIST2, encoding one of transcription factors that induce EMT upon CXCR4 increase, positively correlated with the S18-2 protein level. Together, these data suggest that the S18-2 protein induces EMT through the TWIST2/E-cadherin signalling and, consequently, CXCR4-mediated migration of PCa cells.

High-resolution structures of mitochondrial ribosomes and their functional implications.

Mitochondrial ribosomes (mitoribosomes) almost exclusively synthesize essential components of the oxidative phosphorylation machinery. Dysfunction of mitochondrial protein biosynthesis leads to human diseases and plays an important role in the altered metabolism of cancer cells. Recent developments in cryo-electron microscopy enabled the structural characterization of complete yeast and mammalian mitoribosomes at near-atomic resolution. Despite originating from ancestral bacterial ribosomes, mitoribosomes have diverged in their composition and architecture. Mitoribosomal proteins are larger and more numerous, forming an extended network around the ribosomal RNA, which is expanded in yeast and highly reduced in mammals. Novel protein elements at the entrance or exit of the mRNA channel imply a different mechanism of mRNA recruitment. The polypeptide tunnel is optimized for the synthesis of hydrophobic proteins and their co-translational membrane insertion.

The Expanding Toolkit of Translating Ribosome Affinity Purification.

Translating ribosome affinity purification is a method initially developed for profiling mRNA from genetically defined cell types in complex tissues. It has been applied both to identify target molecules in cell types that are important for controlling a variety of behaviors in the brain, and to understand the molecular consequences on those cells due to experimental manipulations, ranging from drugs of abuse to disease-causing mutations. Since its inception, a variety of methodological advances are opening new avenues of investigation. These advances include a variety of new methods for targeting cells for translating ribosome affinity purification by features such as their projections or activity, additional tags and mouse reagents increasing the flexibility of the system, and new modifications of the method specifically focused on studying the regulation of translation. The latter includes methods to assess cell type-specific regulation of translation in specific subcellular compartments. Here, I provide a summary of these recent advances and resources, highlighting both new experimental opportunities and areas for future technical development.

Macromolecular biosynthetic parameters and metabolic profile in different life stages of Leishmania braziliensis: Amastigotes as a functionally less active stage.

It was recently hypothesized that Leishmania amastigotes could constitute a semi-quiescent stage characterized by low replication and reduced metabolic activity. This concept developed with Leishmania (Leishmania) mexicana and Leishmania (Leishmania) major models might explain numerous clinical and sub-clinical features of Leishmania (Viannia) braziliensis infections, like reactivation of the disease, non-response to chemotherapy or asymptomatic infections. We compared here in vitro the proliferative capability of L. (V.) braziliensis amastigotes and promastigotes, assessed the expression of key molecular parameters and performed metabolomic analysis. We found that contrary to the highly proliferative promastigotes, amastigotes (axenic and intracellular) do not show evidence of extensive proliferation. In parallel, amastigotes showed a significant decrease of (i) the kDNA mini-circle abundance, (ii) the intracellular ATP level, (iii) the ribosomal components: rRNA subunits 18S and 28S α and ribosomal proteins RPS15 and RPL19, (iv) total RNA and protein levels. An untargeted metabolomic study identified clear differences between the different life stages: in comparison to logarithmic promastigotes, axenic amastigotes showed (a) a strong decrease of 14 essential and non-essential amino acids and eight metabolites involved in polyamine synthesis, (b) extensive changes in the phospholipids composition and (c) increased levels of several endogenous and exogenous sterols. Altogether, our results show that L. (V.) braziliensis amastigotes can show a phenotype with negligible rate of proliferation, a lower capacity of biosynthesis, a reduced bio-energetic level and a strongly altered metabolism. Our results pave the way for further exploration of quiescence among amastigotes of this species.

Cancer-associated fibroblasts promote bone invasion in oral squamous cell carcinoma.

BACKGROUND: The molecular mechanisms involved in the invasion of bone by oral squamous cell carcinomas (OSCC) are poorly understood, and little is known about the role of cancer-associated fibroblasts (CAF), the presence of which confers a poor prognosis. METHODS: Clinicopathological data from 277 OSCC cases involving bone resections were reviewed, and 32 cases thoroughly analysed histologically. Immunohistochemistry was used to examine αSMA, RANKL and OPG. Western blotting and qPCR were used to assess myofibroblast (CAF-like) differentiation, RANKL and OPG expression in vitro, and RANKL secretion was analysed by ELISA. Osteoclastogenesis was examined using TRAP staining, multinucleation and pit forming assays. RESULTS: Fibrous stroma intervened between tumour and bone in the majority of cases, with no direct contact between cancer cells and bone. RANKL and OPG, two proteins key to regulating bone resorption, were expressed in tumour cells as well as fibrous stroma adjacent to bone and αSMA-positive myofibroblastic CAF were consistently seen infiltrating into bone ahead of tumour cells. Human primary osteoblasts cultured with conditioned media from human OSCC-derived cells and human primary CAF showed a significant increase in RANKL and a decline in OPG mRNA expression. RANKL secretion was significantly increased in primary oral fibroblasts induced to differentiate into a CAF-like phenotype by transforming growth factor-ß1 (TGF-ß1) treatment and in primary CAF. Indirect co-culture of murine macrophages with conditioned media from CAF (experimentally derived and isolated from OSCCs) resulted in a marked increase in osteoclastogenesis (in excess of that provoked by cancer cells) determined by tartrate-resistant acid phosphatase activity, multinucleation and resorption pit formation. CONCLUSIONS: This study is the first to describe a functional role for CAFs in bone invasion and turnover, identifying a novel potential therapeutic target and diagnostic indicator in this difficult to treat bone invasive malignancy.

Upregulation of Mrps18a in breast cancer identified by selecting phage antibody libraries on breast tissue sections.

BACKGROUND: One of the hallmarks of cancer is an altered energy metabolism, and here, mitochondria play a central role. Previous studies have indicated that some mitochondrial ribosomal proteins change their expression patterns upon transformation. METHOD: In this study, we have used the selection of recombinant antibody libraries displayed on the surface of filamentous bacteriophage as a proteomics discovery tool for the identification of breast cancer biomarkers. A small subpopulation of breast cells expressing both cytokeratin 19 and cytokeratin 14 was targeted using a novel selection procedure. RESULTS: We identified the mitochondrial ribosomal protein s18a (Mrps18a) as a protein which is upregulated in breast cancer. However, Mrps18a was not homogeneously upregulated in all cancer cells, suggesting the existence of sub-populations within the tumor. The upregulation was not confined to cytokeratin 19 and cytokeratin 14 double positive cells. CONCLUSION: This study illustrates how phage display can be applied towards the discovery of proteins which exhibit changes in their expression patterns. We identified the mitochondrial protein Mrps18a as being upregulated in human breast cancer cells compared to normal breast cells.

Neutrophil proteomic analysis reveals the participation of antioxidant enzymes, motility and ribosomal proteins in the prevention of ischemic effects by preconditioning.

Intestinal ischemia and reperfusion injury are widely used models, which result into tissue injury and multiple organ failure also observed after trauma and surgery. Ischemic preconditioning (IPC) preceding ischemia and reperfusion (IR) was shown to attenuate this injury and has a potential therapeutic application; however the exact underlying mechanism is not clear. Neutrophils play an important role in the mechanism of injuries caused by ischemia and reperfusion while IPC led to a decrease in neutrophil stimulation and activation. The effect of preconditioning on the neutrophil proteome is unclear. Proteomic analysis has been ratified as an appropriate tool for studying complex systems. In order to evaluate the effect of IPC preceding 45min of ischemia on the proteome of neutrophils we used Wistar rats divided in four experimental groups: Control, sham laparotomy, intestinal ischemia reperfusion and ischemic preconditioning. After neutrophil separation, proteins were extracted, trypsin digested and the resulting peptides were iTRAQ labeled followed by HILIC fractionation and nLC-MS/MS analysis. After database searches, normalization and statistical analysis our proteomic analysis resulted in the identification of 2437 protein groups that were assigned to five different clusters based on the relative abundance profiles among the experimental groups. The clustering followed by statistical analysis led to the identification of significantly up and downregulated proteins in IR and IPC. Cluster based KEGG pathways analysis revealed up- regulation of actin cytoskeleton, metabolism, Fc gamma R mediated phagocytosis, chemokine signaling, focal adhesion and leukocyte transendothelial migration whereas downregulation in ribosome, spliceosome, RNA transport, protein processing in endoplasmic reticulum and proteasome, after intestinal ischemic preconditioning. Furthermore, enzyme prediction analysis revealed the regulation of some important antioxidant enzymes and having their role in reactive oxygen species production. To our knowledge, this work describes the most comprehensive and detailed quantitative proteomic study of the neutrophil showing the beneficial role of ischemic preconditioning and its effects on the neutrophil proteome. This data will be helpful to understand the effect of underlying protective mechanisms modulating the role of PMNs after IPC and provide a trustworthy basis for future studies. BIOLOGICAL SIGNIFICANCE: Preconditioning is a relevant strategy to overcome clinical implications from ischemia and reperfusion. Such implications have the neutrophil as a major player. Although many publications describe specific biochemical and physiological roles of the neutrophil in such conditions, there is no report of a proteomic study providing a broader view of this scenario. Here we describe a group of proteins significantly regulated by ischemia and reperfusion being such regulation prevented by preconditioning. Such finding may provide relevant information for a deeper understanding of the mechanisms involved, as well as serve as basis for future biomarker or drug target assays.

A new in-frame deletion in ribosomal protein S19 in a Chinese infant with Diamond-Blackfan anemia.

Diamond-Blackfan anemia (DBA) is a congenital erythroid aplasia that usually presents as macrocytic anemia during infancy. Ribosomal protein S19 (RPS19) is identified as the first gene associated with DBA. RPS19 is mutated in 25% of DBA patients, but its role in DBA pathogenesis remains to be elucidated. We have identified a novel heterozygous frameshift mutation in RPS19 gene in a DBA child presenting with profound anemia after birth. A single nucleotide heterozygous deletion (C.251delG) results in frameshift in RPS19 gene in exon 4 at codon 84 with possible premature stop codon (p.Arg84LysfsX21). The mutant allele was not detected in her parents, indicating de novo mutation. Both alleles were expressed at the same level. Using an immunofluorescence technique, the mutated-type RPS19 expressions were mostly localized to entire nuclei with little staining for nucleoli and its intracellular localization significantly differed from the wild-type RPS19, which was localized to both nuclei and nucleoli. This type of a mutation could be very helpful in further understanding the role of the RPS19 protein in DBA pathogenesis.

Estrogen deficiency in ovariectomized rats: can resistance training re-establish angiogenesis in visceral adipose tissue?

OBJECTIVE: The purpose of this study was to investigate the effects of resistance training on angiogenesis markers of visceral adipose tissue in ovariectomized rats. METHOD: Adult Sprague-Dawley female rats were divided into four groups (n=6 per group): sham-sedentary, ovariectomized sedentary, sham-resistance training and ovariectomized resistance training. The rats were allowed to climb a 1.1-m vertical ladder with weights attached to their tails and the weights were progressively increased. Sessions were performed three times per week for 10 weeks. Visceral adipose tissue angiogenesis and morphology were analyzed by histology. VEGF-A mRNA and protein levels were analyzed by real-time PCR and ELISA, respectively. RESULTS: Ovariectomy resulted in higher body mass (p=0.0003), adipocyte hypertrophy (p=0.0003), decreased VEGF-A mRNA (p=0.0004) and protein levels (p=0.0009), and decreased micro-vascular density (p=0.0181) in the visceral adipose tissue of the rats. Resistance training for 10 weeks was not able to attenuate the reduced angiogenesis in the visceral adipose tissue of the ovariectomized rats. CONCLUSION: Our findings indicate that the resistance training program used in this study could not ameliorate low angiogenesis in the visceral adipose tissue of ovariectomized rats.

Estrogen deficiency in ovariectomized rats: can resistance training re-establish angiogenesis in visceral adipose tissue?

OBJECTIVE: The purpose of this study was to investigate the effects of resistance training on angiogenesis markers of visceral adipose tissue in ovariectomized rats. METHOD: Adult Sprague-Dawley female rats were divided into four groups (n=6 per group): sham-sedentary, ovariectomized sedentary, sham-resistance training and ovariectomized resistance training. The rats were allowed to climb a 1.1-m vertical ladder with weights attached to their tails and the weights were progressively increased. Sessions were performed three times per week for 10 weeks. Visceral adipose tissue angiogenesis and morphology were analyzed by histology. VEGF-A mRNA and protein levels were analyzed by real-time PCR and ELISA, respectively. RESULTS: Ovariectomy resulted in higher body mass (p=0.0003), adipocyte hypertrophy (p=0.0003), decreased VEGF-A mRNA (p=0.0004) and protein levels (p=0.0009), and decreased micro-vascular density (p=0.0181) in the visceral adipose tissue of the rats. Resistance training for 10 weeks was not able to attenuate the reduced angiogenesis in the visceral adipose tissue of the ovariectomized rats. CONCLUSION: Our findings indicate that the resistance training program used in this study could not ameliorate low angiogenesis in the visceral adipose tissue of ovariectomized rats.

Differential expression of ribosomal proteins in myelodysplastic syndromes.

Aberrations of ribosomal biogenesis have been implicated in several congenital bone marrow failure syndromes, such as Diamond-Blackfan anaemia, Shwachman-Diamond syndrome and Dyskeratosis Congenita. Recent studies have identified haploinsufficiency of RPS14 in the acquired bone marrow disease isolated 5q minus syndrome, a subtype of myelodysplastic syndromes (MDS). However, the expression of various proteins comprising the ribosomal subunits and other proteins enzymatically involved in the synthesis of the ribosome has not been explored in non-5q minus MDS. Furthermore, differences in the effects of these expression alterations among myeloid, erythroid and megakaryocyte lineages have not been well elucidated. We examined the expression of several proteins related to ribosomal biogenesis in bone marrow biopsy specimens from patients with MDS (5q minus patients excluded) and controls with no known myeloid disease. Specifically, we found that there is overexpression of RPS24, DKC1 and SBDS in MDS. This overexpression is in contrast to the haploinsufficiency identified in the congenital bone marrow failure syndromes and in acquired 5q minus MDS. Potential mechanisms for these differences and aetiology for these findings in MDS are discussed.

Expression, tissue localization and serodiagnostic potential of Taenia multiceps acidic ribosomal protein P2.

BACKGROUND: The larval stage of Taenia multiceps, also known as coenurus, is the causative agent of coenurosis, which results in severe health problems in sheep, goats, cattle and other animals that negatively impact on animal husbandry. There is no reliable method to identify coenurus infected goats in the early period of infection. METHODS: We identified a full-length cDNA that encodes acidic ribosomal protein P2 from the transcriptome of T. multiceps (TmP2). Following cloning, sequencing and structural analyses were performed using bioinformatics tools. Recombinant TmP2 (rTmP2) was prokaryotically expressed and then used to test immunoreactivity and immunogenicity in immunoblotting assays. The native proteins in adult stage and coenurus were located via immunofluorescence assays, while the potential of rTmP2 for indirect ELISA-based serodiagnostics was assessed using native goat sera. In addition, 20 goats were randomly divided into a drug treatment group and a control group. Each goat was orally given mature, viable T. multiceps eggs. The drug treatment group was given 10% praziquantel by intramuscular injection 45 days post-infection (p.i), and all goats were screened for anti-TmP2 antibodies with the indirect ELISA method established here, once a week for 17 weeks p.i. RESULTS: The open reading frame (366 bp) of the target gene encodes a 12.62 kDa protein, which showed high homology to that from Taenia solium (93% identity) and lacked a signal peptide. Immunofluorescence staining showed that TmP2 was highly localized to the parenchymatous zone of both the adult parasite and the coenurus; besides, it was widely distributed in cystic wall of coenurus. Building on good immunogenic properties, rTmP2-based ELISA exhibited a sensitivity of 95.0% (19/20) and a specificity of 96.3% (26/27) in detecting anti-P2 antibodies in the sera of naturally infected goats and sheep. In goats experimentally infected with T. multiceps, anti-TmP2 antibody was detectable in the control group from 3 to 10 weeks and 15 to 17 weeks p.i. In the drug-treated group, the anti-TmP2 antibody dropped below the cut-off value about 2 weeks after treatment with praziquantel and remained below this critical value until the end of the experiment. CONCLUSION: The indirect ELISA method developed in this study has the potential for detection of T. multiceps infections in hosts.

Proteomic profiling reveals autoimmune targets in sarcoidosis.

RATIONALE: There is a need to further characterize the antibody repertoire in relation to sarcoidosis and potentially related autoantigens. OBJECTIVES: We investigated bronchoalveolar lavage (BAL) and serum samples from patients with sarcoidosis and healthy and diseased control subjects to discover sarcoidosis-associated autoantigens. METHODS: Antigen microarrays built on 3,072 protein fragments were used to screen for IgG reactivity in 73 BAL samples from subjects with sarcoidosis, subjects with asthma, and healthy subjects. A set of 131 targets were selected for subsequent verification on suspension bead arrays using 272 additional BAL samples and 141 paired sera. Reactivity to four antigens was furthermore analyzed in 22 unprocessed BAL samples from patients with fibrosis and 269 plasma samples from patients diagnosed with myositis. MEASUREMENTS AND MAIN RESULTS: Reactivity toward zinc finger protein 688 and mitochondrial ribosomal protein L43 were discovered with higher frequencies in patients with sarcoidosis, for mitochondrial ribosomal protein L43 especially in patients with non-Löfgren syndrome. Increased reactivity toward nuclear receptor coactivator 2 was also observed in patients with non-Löfgren syndrome as compared with patients with Löfgren syndrome. The antigen representing adenosine diphosphate-ribosylation factor GTPase activating protein 1 revealed high reactivity frequency in all sample groups but with significantly higher level of IgG reactivities in patients with sarcoidosis. CONCLUSIONS: Autoantigen reactivity was present in most BAL and serum samples analyzed, and the results revealed high interindividual heterogeneity, with most of the reactivities observed in single individuals only. Four proteins are here proposed as sarcoidosis-associated autoimmune targets and of interest for further validation in independent cohorts.

Comparison of mesenchymal stem cells isolated from pulp and periodontal ligament.

BACKGROUND: Cell-based therapy using mesenchymal stem cells (MSCs) seems promising to obtain regeneration of dental tissues. A comparison of tissue sources, including periodontal ligament (PDL) versus pulp (P), could provide critical information to select an appropriate MSC population for designing predictable regenerative therapies. The purpose of this study is to compare the proliferation and stemness and the MSC-specific and mineralized tissue-specific gene expression of P-MSCs and PDL-MSCs. METHODS: MSCs were obtained from PDL and P tissue of premolars (n = 3) extracted for orthodontic reasons. MSC proliferation was evaluated using a real-time cell analyzer for 160 hours. Telomerase activity was evaluated by a telomeric repeat amplification protocol assay based on enzyme-linked immunosorbent assay. Total RNA was isolated from the MSCs on day 3. A polymerase chain reaction (PCR) array was used to compare the expression of MSC-specific genes. The expression of mineralized tissue-associated genes, including Type I collagen (COL I), runt-related transcription factor 2 (RunX2), bone sialoprotein (BSP), and osteocalcin (OCN) messenger RNA (mRNA), was evaluated using quantitative real-time PCR. RESULTS: Higher proliferation potential and telomerase activity were observed in the P-MSCs compared to PDL-MSCs of premolar teeth. Fourteen of 84 genes related to MSCs were expressed differently in the PDL-MSCs versus the P-MSCs. The expressions of bone morphogenetic protein 2 (BMP2) and BMP6; sex-determining region Y-box 9 (SOX9); integrin, alpha 6 (ITGA6); melanoma cell adhesion molecule (MCAM); phosphatidylinositol glycan anchor biosynthesis, class S (PIGS); prominin 1 (PROM1); ribosomal protein L13A (RPL13A); and microphthalmia-associated transcription factor (MITF) were higher in the P-MSCs compared to the PDL-MSCs, and higher expression of matrix metalloproteinase 2 (MMP2), interleukin (IL)-6, insulin (INS), alanyl (membrane) aminopeptidase (ANPEP), and IL-10 were observed in the PDL-MSCs. However, there was no statistically significant difference in the expression of mineralized tissue-associated genes, including BSP and RunX2, between the P-MSCs and the PDL-MSCs. Higher expression of COL I and lower expression of OCN mRNA transcripts were noted in the PDL-MSCs compared to the P-MSCs. CONCLUSIONS: The results of this study suggest that MSCs isolated from P and PDL tissues show different cellular behavior. To increase the predictability of MSC-based regenerative treatment, differences in dental tissue-derived MSCs and favorable aspects of cell sources should be further clarified.