The Perils of Pathogen Discovery: Origin of a Novel Parvovirus-Like Hybrid Genome Traced to Nucleic Acid Extraction Spin Columns

Next-generation sequencing was used for discovery and de novo assembly of a novel, highly divergent DNA virus at the interface between the Parvoviridae and Circoviridae. The virus, provisionally named parvovirus-like hybrid virus (PHV), is nearly identical by sequence to another DNA virus, NIH-CQV, previously detected in Chinese patients with seronegative (non-A-E) hepatitis. Although we initially detected PHV in a wide range of clinical samples, with all strains sharing ∼99% nucleotide and amino acid identity with each other and with NIH-CQV, the exact origin of the virus was eventually traced to contaminated silica-binding spin columns used for nucleic acid extraction. Definitive confirmation of the origin of PHV, and presumably NIH-CQV, was obtained by in-depth analyses of water eluted through contaminated spin columns. Analysis of environmental metagenome libraries detected PHV sequences in coastal marine waters of North America, suggesting that a potential association between PHV and diatoms (algae) that generate the silica matrix used in the spin columns may have resulted in inadvertent viral contamination during manufacture. The confirmation of PHV/NIH-CQV as laboratory reagent contaminants and not bona fide infectious agents of humans underscores the rigorous approach needed to establish the validity of new viral genomes discovered by next-generation sequencing.     

Molecular Diagnostic and Prognostic Subtyping of Gliomas in Tunisian Population

It has become increasingly evident that morphologically similar gliomas may have distinct clinical phenotypes arising from diverse genetic signatures. To date, glial tumours from the Tunisian population have not been investigated. To address this, we correlated the clinico-pathology with molecular data of 110 gliomas by a combination of HM450K array, MLPA and TMA-IHC. PTEN loss and EGFR amplification were distributed in different glioma histological groups. However, 1p19q co-deletion and KIAA1549:BRAF fusion were, respectively, restricted to Oligodendroglioma and Pilocytic Astrocytoma. CDKN2A loss and EGFR overexpression were more common within high-grade gliomas. Furthermore, survival statistical correlations led us to identify Glioblastoma (GB) prognosis subtypes. In fact, significant lower overall survival (OS) was detected within GB that overexpressed EGFR and Cox2. In addition, IDH1R132H mutation seemed to provide a markedly survival advantage. Interestingly, the association of IDHR132H mutation and EGFR normal status, as well as the association of differentiation markers, defined GB subtypes with good prognosis. By contrast, poor survival GB subtypes were defined by the combination of PTEN loss with PDGFRa expression and/or EGFR amplification. Additionally, GB presenting p53-negative staining associated with CDKN2A loss or p21 positivity represented a subtype with short survival. Thus, distinct molecular subtypes with individualised prognosis were identified. Interestingly, we found a unique histone mutation in a poor survival young adult GB case. This tumour exceptionally associated the H3F3A G34R mutation and MYCN amplification as well as 1p36 loss and 10q loss. Furthermore, by exhibiting a remarkable methylation profile, it emphasised the oncogenic power of G34R mutation connecting gliomagenesis and chromatin regulation.     

Purification and characterization of hydroxycinnamoyl-coenzyme A: ω-hydroxypalmitic acid O-hydroxycinnamoyltransferase from tobacco (Nicotiana tabacum L.) cell-suspension cultures

An acyltransferase hydroxycinnamoyl-Coenzyme A: -hydroxypalmitic acid O-hydroxycinnamoyltransferase (HHT; EC 2.3.1.-), which transfers hydroxycinnamic acids from hydroxycinnamoyl-CoA thioesters to several hydroxylated fatty acid derivatives, was characterized from tobacco (Nicotiana tabacum L. cv. Xanthi nc) cell-suspension cultures. It exhibited the same properties as the enzyme previously detected in wound-healing potato tuber discs (Lotfy et al., 1994, Phytochemistry 35: 1419–1424), and especially a marked specificity for -hydroxypalmitic acid and feruloyl-CoA. It was purified 300-fold to near homogeneity from late logarithmic-phase cell suspensions. The apparent molecular mass of the native protein was 55 kDa and its isoelectric point, estimated by electrofocusing, was 4.6. The purified enzyme conjugated ferulic acid to -hydroxypalmitic acid and to 1-tetradecanol, its main lipidic substrates, suggesting that the same enzyme probably synthesizes the different esters of 1-alkanols and of -hydroxy fatty acids which are formed in vitro.Abbreviations ABA abscisic acid - IEF isoelectric focusing - HHT hydroxycinnamoyl-Coenzyme A: -hydroxypalmitic acid O-hydroxycinnamoyltransferase - pI isoelectric point     

EMMPRIN/CD147-encriched membrane vesicles released from malignant human testicular germ cells increase MMP production through tumor–stroma interaction

Background

Elevated levels of EMMPRIN/CD147 in cancer tissues have been correlated with tumor progression but the regulation of its expression is not yet understood. Here, the regulation of EMMPRIN expression was investigated in testicular germ cell tumor (TGCTs) cell lines.

Methods

EMMPRIN expression in seminoma JKT-1 and embryonal carcinoma NT2/D1 cell lines was determined by Western blot, immunofluorescence and qRT-PCR. Membrane vesicles (MVs) secreted from these cells, treated or not with EMMPRIN siRNA, were isolated by differential centrifugations of their conditioned medium. MMP-2 was analyzed by zymography and qRT-PCR.

Results

The more aggressive embryonic carcinoma NT2/D1 cells expressed more EMMPRIN mRNA than the seminoma JKT-1 cells, but surprisingly contained less EMMPRIN protein, as determined by immunoblotting and immunostaining. The protein/mRNA discrepancy was not due to accelerated protein degradation in NT2/D1 cells, but by the secretion of EMMPRIN within MVs, as the vesicles released from NT2/D1 contained considerably more EMMPRIN than those released from JKT-1. EMMPRIN-containing MVs obtained from NT2/D1, but not from EMMPRIN-siRNA treated NT2/D1, increased MMP-2 production in fibroblasts to a greater extent than those from JKT-1 cells.

Conclusion and general significance

The data presented show that the more aggressive embryonic carcinoma cells synthesize more EMMPRIN than seminoma cells, but which they preferentially target to secreted MVs, unlike seminoma cells which retain EMMPRIN within the cell membrane. This cellular event points to a mechanism by which EMMPRIN expressed by malignant testicular cells can exert its MMP inducing effect on distant cells within the tumor microenvironment to promote tumor invasion. This article is part of a Special Issue entitled Matrix-mediated cell behaviour and properties.     

Effect of Pedoclimatic Conditions on the Chemical Composition of the Sigoise Olive Cultivar

The present work focused on the quality and the chemical composition of monovarietal virgin olive oil from the Sigoise variety grown in two different locations in Tunisia, viz., a sub‐humid zone (Béjaoua, Tunis) and an arid zone (Boughrara, Sfax). In addition to the quality characteristics (acidity, peroxide value, and the spectrophotometric indices K232 and K270) and the chemical composition (content of fatty acids, antioxidants, and volatile compounds) of the oil, the fruit characteristics of the olives were studied. Except for the content of the majority of the fatty acids, there were significant differences observed in the oil composition of olives that were cultivated in different locations. The content of total phenols and lipoxygenase (LOX) oxidation products was higher for olives grown at the higher altitude, whereas that of α‐tocopherol, carotenes, and chlorophylls was higher for olives from the Boughrara region (lower altitude). Moreover, olives produced at the higher altitude showed a higher ripeness index and oil content than those cultivated at the lower altitude.     

Visualization of the molecular dynamics of lipopolysaccharide on the plasma membrane of murine macrophages by total internal reflection fluorescence microscopy

The molecular action of Alexa 594-labeled lipopolysaccharide (LPS) from Escherichia coli was examined on living peritoneal macrophages of C57BL/6 mice by total internal reflection fluorescence microscope (TIRFM), and the molecular kinetics of LPS was analyzed. TIRFM visualization of the action of fluorescence-labeled LPS revealed an increase in the mean fluorescence intensity of LPS on the plasma membrane of wild type macrophages at 60 min after administration, indicating the oligomerization of LPS after binding to the macrophages. Additionally, a time-dependent sharp decrease in the mean diffusion coefficient of LPS was observed. On the other hand, both mean fluorescence intensity and diffusion coefficient of LPS in cases of TLR4(-/-), MD2(-/-), MyD88(-/-), and TRIF(-/-) macrophages were significantly different from the corresponding values of wild type macrophage, whereas differences were also noticed among these molecule-deficient macrophages. Furthermore, statistical analysis indicated the major role of receptors (TLR4 and MD2) and intracellular signaling molecules (MyD88 and TRIF) in oligomerization and lowering of the diffusion rate of LPS on the plasma membrane of murine macrophages, respectively.     

Comparing native and irradiated E. coli lactose repressor-operator complex by molecular dynamics simulation

The function of the E. coli lactose operon requires the binding of the tetrameric repressor protein to the operator DNA. We have previously shown that γ-irradiation destabilises the repressor-operator complex because the repressor gradually loses its DNA-binding ability (Radiat Res 170:604–612, 2008). It was suggested that the observed oxidation of tyrosine residues and the concomitant structural changes of irradiated headpieces (DNA-binding domains of repressor monomers) could be responsible for the inactivation. To unravel the mechanisms that lead to repressor-operator complex destabilisation when tyrosine oxidation occurs, we have compared by molecular dynamic simulations two complexes: (1) the native complex formed by two headpieces and the operator DNA, and (2) the damaged complex, in which all tyrosines are replaced by their oxidation product 3,4-dihydroxyphenylalanine (DOPA). On a 20 ns time scale, MD results show effects consistent with complex destabilisation: increased flexibility, increased DNA bending, modification of the hydrogen bond network, and decrease of the positive electrostatic potential at the protein surface and of the global energy of DNA-protein interactions.     

Carnitine and acylcarnitine profiles in dried blood spots of patients with acute myocardial infarction

Earlier studies have suggested an important role of carnitine pathway in cardiovascular pathology. However, the redistribution of carnitine and acylcarnitine pools, as a result of altered carnitine metabolism, is not clearly known in patients with acute myocardial infarction (AMI). We compared the carnitine and acylcarnitine profiles of 65 AMI patients, including 26 ST-elevated myocardial infarction (STEMI) and 39 non-ST-elevated myocardial infarction (NSTEMI), 28 patients with chest pain and 154 normal controls. The levels of carnitine and acylcarnitines in the blood spots were determined using LC-MS/MS. Total and free carnitine levels were significantly higher in all the patient groups in the following order: STEMI > NSTEMI > chest pain. The levels of short- and medium-chain acylcarnitines were significantly higher in patient groups. Among the long-chain acylcarnitines, C14:2 and C16:1 levels were significantly increased in STEMI and NSTEMI. The ratio of free carnitine to short-chain or medium-chain acylcarnitines was significantly decreased in STEMI, NSTEMI and chest pain patients however a significant increase was observed in the ratio of carnitine to long-chain acylcarnitines in all the patient groups as compared to normal controls. In conclusion, alterations in carnitine and acylcarnitine levels in the blood of AMI patients indicate the possibility of impaired carnitine homeostasis in ischemic myocardium. The clinical implications of these findings for the risk screening or diagnosis and prognosis of AMI require additional follow-up studies on large number of patients. We also suggest that a dual-marker strategy using carnitine (longer plasma half-life) in combination with troponin (shorter plasma half-life) could be a more promising biomarker strategy in risk stratification of patients.     

Atypical recognition of particular DNA sequences by the archaeal chromosomal MC1 protein

The MC1 protein is a chromosomal protein likely involved in the DNA compaction of some methanogenic archaea. This small and monomeric protein, structurally unrelated to other DNA binding proteins, bends DNA sharply. By studying the protein binding to various kinds of kinked DNA, we have previously shown that MC1 is able to discriminate between different deformations of the DNA helix. Here we investigate its capacity to recognize particular DNA sequences by using a SELEX procedure. We find that MC1 is able to preferentially bind to a 15 base pair motif [AAAAACACAC(A/C)CCCC]. The structural parameters of this sequence are characterized by molecular dynamics simulation experiments, and the binding mode of the protein to the DNA is studied by footprinting experiments. Our results strongly suggest that the protein realizes an indirect readout of the DNA sequence by binding to the DNA minor groove.