Persistence of a chimerical phenotype after hepatocyte differentiation of human bone marrow mesenchymal stem cells

Abstract.  Objectives : Recent studies have suggested the potential of mesenchymal stem cells (MSCs) to differentiate into a hepatocyte-like lineage. Here, we evaluate the efficacy of hepatocyte differentiation of MSCs by studying acquisition of hepatocyte-like features together with alteration of the native mesenchymal phenotype. Material and methods : In vitro , we have investigated protein and mRNA level expression of hepatocyte and mesenchymal markers of mesenchymal-derived hepatocyte-like cells (MDHLCs) and we have evaluated their functionality using metabolic assays. In vivo , we investigated co-expression of hepatocyte (albumin, α-foetoprotein, cytokeratin 18) and mesenchymal (fibronectin, vimentin) markers after transplantation of MSCs or MDHLCs into severe combined immune deficiency mice. Results : We observed that while in vitro these cells acquired some phenotypic and functional features of mature hepatocytes, they partially preserved their mesenchymal phenotype. After intrasplenic transplantation, engrafted MSCs with isolated expression of fibronectin and α-foetoprotein were observed. When these cells were injected into the liver, they expressed all analysed markers, confirming the chimaeric co-expression observed in vitro . Conversely, liver-engrafted MDHLCs conserved their hepatocyte-lineage markers but lost their chimaeric phenotype. Conclusions : Hepatocyte differentiation of MSCs predominantly allows the acquisition of phenotypic hallmarks and provides chimaeric cells that maintain expression of initial lineage markers. However, advanced maturation to the hepatocyte-like phenotype could be obtained in vivo by conditioning MSCs prior to transplantation or by infusing cells into the liver micro-environment.     

Effect of Environmental Conditions on Hydrophobicity of Marine Bacteria Adapted to Textile Effluent Treatment

Summary Bioflocculation of bacteria isolated from the marine biomass acclimatized to textile wastewater has been achieved. Pseudomonas was the main genus found in the marine biomass. This bacterium showed an important hydrophobicity but slightly weaker than that of pilot plant genera adapted to the conventional sewage. This hydrophobicity provided the bacteria with hydrophobic characters, especially those of the polymeric extracellular substances (ECS) released in the sewage. Whereas some factors such as the divalent cations, calcium and magnesium had an effect on this bacterial hydrophobicity, glucose concentration did not have any impact. Furthermore, the physiological state of the cells hatched in the sewage had an impact on the hydrophobicity. Indeed, during the logarithmic growth phase, cell hydrophobicity increase enhanced floc formation, thus improving wastewater treatment.     

Fibromodulin gene is expressed in human epidermal keratinocytes in culture and in human epidermis in vivo

Fibromodulin is a small leucine-rich proteoglycan that has a central role in the maintenance of collagen fibrils structure, and in regulation of TGF-β biological activity. Although, it is mainly found in cartilage and tendon, little is known regarding the expression of the fibromodulin gene in other cell types. By RT-PCR, real time PCR and immunohistochemistry, we describe the expression of the fibromodulin gene and the presence of the protein in human epidermal keratinocytes (HEK), both in culture and in normal human epidermis. Our results show, for the first time, that fibromodulin gene is constantly expressed in HEK during culture time. Immunostaining showed that fibromodulin is located intracytoplasmically in basal and stratified keratinocytes of the growing colonies, confluent cultures, and epidermis in vivo. The expression and intracellular localization of fibromodulin in HEK is a new finding and opens new possible biological roles for the SLRP family.     

Adipogenic genes on induction and stabilization of commitment to adipose conversion

Most late events of adipose conversion are known, but those early events that lead to cell commitment, and important aspects of its mechanism remain unknown. We recently described that, in the absence of any other adipogenic factor, 4 h incubation with staurosporine promotes commitment of 3T3-F442A cells to adipogenesis. This commitment consists of two stages; a first stage of 4 h induction by staurosporine, and, in the absence of this drug, a second stage of stabilization which becomes completed after 40-48 h from staurosporine treatment. Here, we demonstrate that pparg2 gene is expressed early after induction stage but before commitment is stabilized, whereas cebpa is highly expressed during the last part of stabilization stage. A decrease of dlk1 expression, whose down-regulation is indispensable for adipogenesis, began to take place between 24 and 48 h of St-Dex incubation started, reaching the lowest levels well into the end of stabilization stage.     

Extracellular matrix production by mouse 3T3-F442A cells during adipose differentiation in culture

When cultured 3T3-F442A cells undergo adipose differentiation, they produce extracellular matrix (ECM) that is not present in undifferentiated cells. This ECM stains strongly with ruthenium red, tannic acid and with Alcian blue at both pH 1 and 2.5, showing histochemical characteristics similar to sulphated and non-sulphated glycosaminoglycans. Under the electron microscope, ECM was observed bound to the cell surface and in the intercellular space; it was composed of fibrils of several thicknesses with attached granules and fibrous long-spacing forms of collagen. In addition, adipocytes were observed as rounded cells interconnected with the ECM fibrils, thus giving rise to fat cell clusters similar to the adipocyte lobules found in adipose tissue. Since fat cell clusters in culture emerge by clonal expansion of one adipose precursor cell, we suggest that this ECM can keep daughter adipocytes interconnected during differentiation. ECM production by adipocytes might have some significance for the formation of fat cell lobules in vivo.     

Intragenic recombination in the CSR1 locus of Arabidopsis

Four classes of herbicides are known to inhibit plant acetolactate synthase (ALS). In Arabidopsis, ALS is encoded by a single gene, CSR1. The dominant csr1-1 allele encodes an ALS resistant to chlorsulfuron and triazolopyrimidine sulfonamide while the dominant csr1-2 allele encodes an ALS resistant to imazapyr and pyrimidyl-oxy-benzoate. The molecular distance between the point mutations in csr1-1 and csr1-2 is 1369 bp. Here we used multiherbicide resistance as a stringent selection to measure the intragenic recombination frequency between these two point mutations. We found this frequency to be 0.008 ± 0.0028. The recombinant multiherbicide-resistant allele, csr1-4, provides an ideal marker for plant genetic transformation.     

Increased contribution of wheat nocturnal transpiration to daily water use under drought

Increasing evidence suggests that in crops, nocturnal water use could represent 30% of daytime water consumption, particularly in semi‐arid and arid areas. This raises the questions of whether nocturnal transpiration rates (TR_N) are (1) less influenced by drought than daytime TR (TR_D), (2) increased by higher nocturnal vapor pressure deficit (VPD_N), which prevails in such environments and (3) involved in crop drought tolerance. In this investigation, we addressed those questions by subjecting two wheat genotypes differing in drought tolerance to progressive soil drying under two long‐term VPD_N regimes imposed under naturally fluctuating conditions. A first goal was to characterize the response curves of whole‐plant TR_N and TR_N/TR_D ratios to progressive soil drying. A second goal was to examine the effect of VPD_N increase on TR_N response to soil drying and on 13 other developmental traits. The study revealed that under drought, TR_N was not responsive to progressive soil drying and – intriguingly – that TR_N seemingly increased with drought under high VPD_N consistently for the drought‐sensitive genotype. Because TR_D was concomitantly decreasing with progressive drought, this resulted in TR_N representing up to 70% of TR_D at the end of the drydown. In addition, under drought, VPD_N increase was found not to influence traits such as leaf area or stomata density. Overall, those findings indicate that TR_N contribution to daily water use under drought might be much higher than previously thought, that it is controlled by specific mechanisms and that decreasing TR_N under drought might be a valuable trait for improving drought tolerance.     

Overexpressed recombinant quorum quenching lactonase reduces the virulence,motility and biofilm formation of multidrug-resistant Pseudomonas aeruginosa clinical isolates

The increasing occurrence of resistance among Pseudomonas aeruginosa clinical isolates necessitates finding alternatives to antibiotics for controlling the infection of such pathogenic bacteria. In this study, lactonase gene ahl-1 from Bacillus weihenstephanensis isolate-P65 was successfully cloned and expressed in Escherichia coli BL21 (DE3) under the control of T7 promoter for utilizing its quorum quenching activity against three multidrug-resistant (MDR) P. aeruginosa clinical isolates. The biological activity of the overexpressed lactonase enzyme (Ahl-1), tested using a synthetic signal and Chromobacterium violaceum CV026 as a biosensor, displayed good catalytic activity using hexanoyl homoserine lactone (HHL) as a substrate and Chromobacterium violaceum (CV026) as a biosensor (77.2 and 133 nm min⁻¹ for the crude and the purified Ahl-lactonase enzymes, respectively). Upon challenging its ability to inhibit the virulence of three MDR P. aeruginosa clinical isolates, recombinant Ahl-1 successfully prevented the accumulation of acylhomoserine lactone signals resulting in a significant reduction in the investigated virulence determinants; protease (from 40 up to 75.5%), pyocyanin (48–75.9%), and rhamnolipids (52.7–63.4%) (P value < 0.05). Ahl-1 also displayed significant inhibitory activities on the swarming motility and biofilm formation of the three tested MDR P. aeruginosa clinical isolates (P value < 0.05). Consequently, Ahl-1 lactonase enzyme in this study is considered a promising therapeutic agent to inhibit P. aeruginosa pathogenicity with no fear of emergence of resistance.