Quantitative 3D comparison of biofilm imaged by X-ray microtomography and two-photon laser scanning microscopy

Optical imaging techniques for biofilm observation, like laser scanning microscopy, are not applicable when investigating biofilm formation in opaque porous media. X-ray micro-tomography (X-ray CMT) might be an alternative but it finds limitations in similarity of X-ray absorption coefficients for the biofilm and aqueous phases. To overcome this difficulty, barium sulphate was used in Davit et al. (2011) to enable high-resolution 3D imaging of biofilm via X-ray CMT. However, this approach lacks comparison with well-established imaging methods, which are known to capture the fine structures of biofilms, as well as uncertainty quantification. Here, we compare two-photon laser scanning microscopy (TPLSM) images of Pseudomonas Aeruginosa biofilm grown in glass capillaries against X-ray CMT using an improved protocol where barium sulphate is combined with low-gelling temperature agarose to avoid sedimentation. Calibrated phantoms consisting of mono-dispersed fluorescent and X-ray absorbent beads were used to evaluate the uncertainty associated with our protocol along with three different segmentation techniques, namely hysteresis, watershed and region growing, to determine the bias relative to image binarization. Metrics such as volume, 3D surface area and thickness were measured and comparison of both imaging modalities shows that X-ray CMT of biofilm using our protocol yields an accuracy that is comparable and even better in certain respects than TPLSM, even in a nonporous system that is largely favourable to TPLSM

Positive Regulation of DNA Double Strand Break Repair Activity during Differentiation of Long Life Span Cells: The Example of Adipogenesis

Little information is available on the ability of terminally differentiated cells to efficiently repair DNA double strand breaks (DSBs), and one might reasonably speculate that efficient DNA repair of these threatening DNA lesions, is needed in cells of long life span with no or limited regeneration from precursor. Few tissues are available besides neurons that allow the study of DNA DSBs repair activity in very long-lived cells. Adipocytes represent a suitable model since it is generally admitted that there is a very slow turnover of adipocytes in adult. Using both Pulse Field Gel Electrophoresis (PFGE) and the disappearance of the phosphorylated form of the histone variant H2AX, we demonstrated that the ability to repair DSBs is increased during adipocyte differentiation using the murine pre-adipocyte cell line, 3T3F442A. In mammalian cells, DSBs are mainly repaired by the non-homologous end-joining pathway (NHEJ) that relies on the DNA dependent protein kinase (DNA-PK) activity. During the first 24 h following the commitment into adipogenesis, we show an increase in the expression and activity of the catalytic sub-unit of the DNA-PK complex, DNA-PKcs. The increased in DNA DSBs repair activity observed in adipocytes was due to the increase in DNA-PK activity as shown by the use of DNA-PK inhibitor or sub-clones of 3T3F442A deficient in DNA-PKcs using long term RNA interference. Interestingly, the up-regulation of DNA-PK does not regulate the differentiation program itself. Finally, similar positive regulation of DNA-PKcs expression and activity was observed during differentiation of primary culture of pre-adipocytes isolated from human sub-cutaneous adipose tissue

Caffeine reduces TNFalpha up-regulation in human adipose tissue primary culture.

International audienceAdipose tissue secretions play an important role in the development of obesity-related pathologies such as diabetes. Through inflammatory cytokines production, adipose tissue stromavascular fraction cells (SVF), and essentially macrophages, promote adipocyte insulin resistance by a paracrine way. Since xanthine family compounds such as caffeine were shown to decrease inflammatory production by human blood cells, we investigated the possible effect of caffeine on Tumor Necrosis Factor alpha (TNFalpha) and Interleukin-6 (IL-6) expression by human adipose tissue primary culture. For that purpose, human subcutaneous adipose tissue obtained from healthy non-obese women (BMI: 26.7 +/- 2.2 kg/m2) after abdominal dermolipectomy, was split into explants and cultured for 6 hours with or without caffeine. Three different concentrations of caffeine were tested (0.5 microg/mL, 5 microg/mL and 50 microg/mL). After 6 hours of treatment, explants were subjected to collagenase digestion in order to isolate adipocytes and SVF cells. Then, TNFalpha and IL-6 mRNA were analysed by real-time PCR alternatively in adipocytes and SVF cells. In parallel, we checked gene expression of markers involved in adipocyte differenciation and in SVF cells inflammation and proliferation. Our findings show a strong and dose dependent down-regulation of TNF-alpha gene expression in both adipocyte and SVF cells whereas IL-6 was only down regulated in SVF cells. No effect of caffeine was noticed on the other genes studied. Thus, caffeine, by decreasing TNFalpha expression, could improve adipose tissue inflammation during obesity

The transcriptional co-activator PGC-1alpha up regulates apelin in human and mouse adipocytes.

International audienceBy using pangenomic microarray, we identified apelin as a unique adipokine up regulated by the transcriptional co-activator peroxisome proliferator-activated receptor gamma (PPARgamma) co-activator 1alpha (PGC-1alpha) in human white adipocytes. We investigated its regulation in vitro and in vivo. Overexpression of PGC-1alpha by adenovirus in human adipocytes induces apelin expression and secretion. Pharmacological induction of cAMP, an upstream regulator of endogenous PGC-1alpha expression, up regulates apelin gene expression and also apelin secretion in human and mice adipocytes. Moreover, during cold exposure in mice, a physiological situation known to induce both cAMP and PGC-1alpha, apelin expression in adipocytes and plasma levels were increased. This is the first demonstration that PGC-1alpha is involved in the regulation of an adipokine gene expression and release

Endothelial differentiation gene-2 receptor is involved in lysophosphatidic acid-dependent control of 3T3F442A preadipocyte proliferation and spreading.

EDG-2, EDG-4, EDG-7, and PSP24 genes encode distinct lysophosphatidic acid (LPA) receptors. The aim of the present study was to determine which receptor subtype is involved in the biological responses generated by LPA in preadipocytes. Growing 3T3F442A preadipocytes express EDG-2 and EDG-4 mRNAs, with no expression of EDG-7 or PSP24 mRNAs. Quantitative reverse transcriptase-polymerase chain reaction revealed that EDG-2 transcripts were 10-fold more abundant than that of EDG-4. To determine the involvement of the EDG-2 receptor in the responses of growing preadipocytes to LPA, stable transfection of antisense EDG-2 cDNA was performed in growing 3T3F442A preadipocytes. This procedure, led to a significant and specific reduction in EDG-2 mRNA and protein. This was associated with a significant alteration in the effect of LPA on both cell proliferation and cell spreading. Finally, the differentiation of growing preadipocytes into quiescent adipocytes led to a strong reduction in the level of EDG-2 transcripts. Results demonstrate the significant contribution of the EDG-2 receptor in the biological responses generated by LPA in 3T3F442A preadipocytes

Gbeta gamma-independent coupling of alpha2-adrenergic receptor to p21(rhoA) in preadipocytes.

In preadipocytes, alpha2-adrenergic receptor (alpha2-AR) stimulation leads to a Gi/Go-dependent rearrangement of actin cytoskeleton. This is characterized by a rapid cell spreading, the formation of actin stress fibers, and the increase in tyrosyl phosphorylation of the focal adhesion kinase (pp125(FAK)). These cellular events being tightly controlled by the small GTPase p21(rhoA), the existence of a Gi/Go-dependent coupling of alpha2-AR to p21(rhoA) in preadipocytes was proposed. In alpha2AF2 preadipocytes (a cell clone derived from the 3T3F442A preadipose cell line and which stably expresses the human alpha2C10-adrenergic receptor) alpha2-adrenergic-dependent induction of cell spreading, formation of actin stress fibers, and increase in tyrosyl phosphorylation of pp125(FAK) were abolished by pretreatment of the preadipocytes with the C3 exoenzyme, a toxin which impairs p21(rhoA) activity by ADP-ribosylation. Conversely, C3 exoenzyme had no effect on the alpha2-adrenergic-dependent increase in tyrosyl phosphorylation and shift of ERK2 mitogen-activated protein kinase. alpha2-Adrenergic stimulation also led to an increase in GDP/GTP exchange on p21(rhoA), as well as to an increase in the amount of p21(rhoA) in the particulate fraction of alpha2AF2 preadipocytes. Stable transfection of alpha2AF2 preadipocytes with the COOH-terminal domain of betaARK1 (betaARK-CT) (a blocker of Gbeta gamma-action), strongly inhibited the alpha2-adrenergic-dependent increase in tyrosyl phos- phorylation and shift of ERK2, without modification of the tyrosyl phosphorylation of pp125(FAK) and spreading of preadipocytes. These results show that alpha2-adrenergic-dependent reorganization of actin cytoskeleton requires the activation of p21(rhoA) in preadipocytes. Conversely to the activation of the p21(ras)/mitogen-activated protein kinase pathway, the alpha2-adrenergic activation of p21(rhoA)-dependent pathways are independent of the beta gamma-subunits of heterotrimeric G proteins