Genetic and molecular characterization of the human Osteosarcoma 3AB‐OS cancer stem cell line: A possible model for studying osteosarcoma origin and stemness

Finding new treatments targeting cancer stem cells (CSCs) within a tumor seems to be critical to halt cancer and improve patient survival. Osteosarcoma is an aggressive tumor affecting adolescents, for which there is no second‐line chemotherapy. Uncovering new molecular mechanisms underlying the development of osteosarcoma and origin of CSCs is crucial to identify new possible therapeutic strategies. Here, we aimed to characterize genetically and molecularly the human osteosarcoma 3AB‐OS CSC line, previously selected from MG63 cells and which proved to have both in vitro and in vivo features of CSCs. Classic cytogenetic studies demonstrated that 3AB‐OS cells have hypertriploid karyotype with 71–82 chromosomes. By comparing 3AB‐OS CSCs to the parental cells, array CGH, Affymetrix microarray, and TaqMan® Human MicroRNA array analyses identified 49 copy number variations (CNV), 3,512 dysregulated genes and 189 differentially expressed miRNAs. Some of the chromosomal abnormalities and mRNA/miRNA expression profiles appeared to be congruent with those reported in human osteosarcomas. Bioinformatic analyses selected 196 genes and 46 anticorrelated miRNAs involved in carcinogenesis and stemness. For the first time, a predictive network is also described for two miRNA family (let‐7/98 and miR‐29a,b,c) and their anticorrelated mRNAs (MSTN, CCND2, Lin28B, MEST, HMGA2, and GHR), which may represent new biomarkers for osteosarcoma and may pave the way for the identification of new potential therapeutic targets. J. Cell. Physiol. 228: 1189–1201, 2013. © 2012 Wiley Periodicals, Inc.     

Ehrlich cell plasma membrane redox system is modulated through signal transduction pathways involvingcGMP and Ca2+ as second messengers

Ehrlich cell plasma membrane ferricyanide reductase activity increased in the presence of mastoparan, a generic activator of G proteins, using either whole cells or isolated plasma membrane fractions. Agents that increase intracellularcAMP also increased the rate of ferricyanide reduction by Ehrlich cells. For the first time, evidence is shown on a modulation of plasma membrane redox system bycGMP. In fact, permeant analogs ofcGMP, dibutyrylcGMP, and 8-bromo-cGMP increased the rate of ferricyanide reduction by the Ehrlich cell plasma membrane redox system. Furthermore, specific inhibition ofcGMP-phosphodiesterases by dipyridamole was also accompanied by an enhancement in the rate of ferricyanide reduction. On the other hand, treatments expected to increase cytoplasmic Ca²⁺ concentrations were accompanied by a remarkable stimulation of the reductase activity. Taking all these data together, it seems that the Ehrlich cell plasma membrane redox system is under a multiple and complex regulation by different signal transduction pathways involving G proteins, cyclic nucleotides, and Ca²⁺ ions.     

Cellulase immobilization on magnetic nanoparticles encapsulated in polymer nanospheres

Immobilization of cellulases on magnetic nanoparticles, especially magnetite nanoparticles, has been the main approach studied to make this enzyme, economically and industrially, more attractive. However, magnetite nanoparticles tend to agglomerate, are very reactive and easily oxidized in air, which has strong impact on their useful life. Thus, it is very important to provide proper surface coating to avoid the mentioned problems. This study aimed to investigate the immobilization of cellulase on magnetic nanoparticles encapsulated in polymeric nanospheres. The support was characterized in terms of morphology, average diameter, magnetic behavior and thermal decomposition analyses. The polymer nanospheres containing encapsulated magnetic nanoparticles showed superparamagnetic behavior and intensity average diameter about 150 nm. Immobilized cellulase exhibited broader temperature stability than in the free form and great reusability capacity, 69% of the initial enzyme activity was maintained after eight cycles of use. The magnetic support showed potential for cellulase immobilization and allowed fast and easy biocatalyst recovery through a single magnet.

     

A systematic investigation of multiheme c-type cytochromes in prokaryotes

Multiheme c-type cytochromes (MHCs) are metalloproteins that can play various biochemical roles, including enzymatic activity and electron transfer. As electron transfer proteins, the presence of multiple heme cofactors in the vicinity allows electrons to rapidly travel relatively long distances. MHCs are often characterized by relatively low structural complexity, with the heme cofactors being largely responsible for maintaining the structure in place, owing to the protein–heme covalent linkages. In this work, we analyzed an extensive ensemble of 594 complete prokaryotic proteomes, amounting to more than 1.9 million sequences, to characterize their content in MHCs. We identified 1,659 MHCs in 258 organisms. The presence of MHCs was found to correlate quite well with the capability of an organism to synthesize or take up heme. For two organisms, the presence of MHCs in the proteome could be taken as a hint to the presence of divergent heme uptake pathways. The most common numbers of heme-binding motifs in a sequence were four (25%) and two (23%), followed by five (13%) and ten (9.8%). The average protein-to-heme ratio was relatively similar for all MHCs, except diheme proteins, regardless of the number of motifs at around 60 ± 30. The latter ratio could in favorable cases be a useful indicator for functional assignments of novel MHCs. Finally, we showed that the amount of structural information currently available for MHCs is limited with respect to the diversity of this broad class of metalloproteins. Experimental efforts in the structural investigation of MHCs are thus warranted.     

Change in conformation with reduction of alpha-helix content causes loss of neutrophil binding activity in fully cytotoxic Shiga toxin 1

Shiga toxins (Stx) play an important role in the pathogenesis of hemolytic uremic syndrome, a life-threatening renal sequela of human intestinal infection caused by specific Escherichia coli strains. Stx target a restricted subset of human endothelial cells that possess the globotriaosylceramide receptor, like that in renal glomeruli. The toxins, composed of five B chains and a single enzymatic A chain, by removing adenines from ribosomes and DNA, trigger apoptosis and the production of pro-inflammatory cytokines in target cells. Because bacteria are confined to the gut, the toxins move to the kidney through the circulation. Polymorphonuclear leukocytes (PMN) have been indicated as the carriers that "piggyback" shuttle toxins to the kidney. However, there is no consensus on this topic, because not all laboratories have been able to reproduce the Stx/PMN interaction. Here, we demonstrate that conformational changes of Shiga toxin 1, with reduction of α-helix content and exposition to solvent of hydrophobic tryptophan residues, cause a loss of PMN binding activity. The partially unfolded toxin was found to express both enzymatic and globotriaosylceramide binding activities being fully active in intoxicating human endothelial cells; this suggests the presence of a distinct PMN-binding domain. By reviewing functional and structural data, we suggest that A chain moieties close to Trp-203 are recognized by PMN. Our findings could help explain the conflicting results regarding Stx/PMN interactions, especially as the groups reporting positive results obtained Stx by single-step affinity chromatography, which could have preserved the correct folding of Stx with respect to more complicated multi-step purification methods.     

Hybrid physics-based and data-driven modeling for bioprocess online simulation and optimization

Model-based online optimization has not been widely applied to bioprocesses due to the challenges of modeling complex biological behaviors, low-quality industrial measurements, and lack of visualization techniques for ongoing processes. This study proposes an innovative hybrid modeling framework which takes advantages of both physics-based and data-driven modeling for bioprocess online monitoring, prediction, and optimization. The framework initially generates high-quality data by correcting raw process measurements via a physics-based noise filter (a generally available simple kinetic model with high fitting but low predictive performance); then constructs a predictive data-driven model to identify optimal control actions and predict discrete future bioprocess behaviors. Continuous future process trajectories are subsequently visualized by re-fitting the simple kinetic model (soft sensor) using the data-driven model predicted discrete future data points, enabling the accurate monitoring of ongoing processes at any operating time. This framework was tested to maximize fed-batch microalgal lutein production by combining with different online optimization schemes and compared against the conventional open-loop optimization technique. The optimal results using the proposed framework were found to be comparable to the theoretically best production, demonstrating its high predictive and flexible capabilities as well as its potential for industrial application.