LEO1 Is Required for Efficient Entry into Quiescence, Control of H3K9 Methylation and Gene Expression in Human Fibroblasts.

(1) Background: The LEO1 (Left open reading frame 1) protein is a conserved subunit of the PAF1C complex (RNA polymerase II-associated factor 1 complex). PAF1C has well-established mechanistic functions in elongation of transcription and RNA processing. We previously showed, in fission yeast, that LEO1 controls histone H3K9 methylation levels by affecting the turnover of histone H3 in chromatin, and that it is essential for the proper regulation of gene expression during cellular quiescence. Human fibroblasts enter a reversible quiescence state upon serum deprivation in the growth media. Here we investigate the function of LEO1 in human fibroblasts. (2) Methods: We knocked out the LEO1 gene using CRISPR/Cas9 methodology in human fibroblasts and verified that the LEO1 protein was undetectable by Western blot. We characterized the phenotype of the ΔLEO1 knockout cells with FACS analysis and cell growth assays. We used RNA-sequencing using spike-in controls to measure gene expression and spike-in controlled ChIP-sequencing experiments to measure the histone modification H3K9me2 genome-wide. (3) Results: Gene expression levels are altered in quiescent cells, however factors controlling chromatin and gene expression changes in quiescent human cells are largely unknown. The ΔLEO1 knockout fibroblasts are viable but have reduced metabolic activity compared to wild-type cells. ΔLEO1 cells showed a slower entry into quiescence and a different morphology compared to wild-type cells. Gene expression was generally reduced in quiescent wild-type cells. The downregulated genes included genes involved in cell proliferation. A small number of genes were upregulated in quiescent wild-type cells including several genes involved in ERK1/ERK2 and Wnt signaling. In quiescent ΔLEO1 cells, many genes were mis-regulated compared to wild-type cells. This included genes involved in Calcium ion transport and cell morphogenesis. Finally, spike-in controlled ChIP-sequencing experiments demonstrated that the histone modification H3K9me2 levels are globally increased in quiescent ΔLEO1 cells. (4) Conclusions: Thus, LEO1 is important for proper entry into cellular quiescence, control of H3K9me2 levels, and gene expression in human fibroblasts.

Dimeric G-quadruplex motifs-induced NFRs determine strong replication origins in vertebrates.

Replication of vertebrate genomes is tightly regulated to ensure accurate duplication, but our understanding of the interplay between genetic and epigenetic factors in this regulation remains incomplete. Here, we investigated the involvement of three elements enriched at gene promoters and replication origins: guanine-rich motifs potentially forming G-quadruplexes (pG4s), nucleosome-free regions (NFRs), and the histone variant H2A.Z, in the firing of origins of replication in vertebrates. We show that two pG4s on the same DNA strand (dimeric pG4s) are sufficient to induce the assembly of an efficient minimal replication origin without inducing transcription in avian DT40 cells. Dimeric pG4s in replication origins are associated with formation of an NFR next to precisely-positioned nucleosomes enriched in H2A.Z on this minimal origin and genome-wide. Thus, our data suggest that dimeric pG4s are important for the organization and duplication of vertebrate genomes. It supports the hypothesis that a nucleosome close to an NFR is a shared signal for the formation of replication origins in eukaryotes.

SARS-CoV-2 variant Delta rapidly displaced variant Alpha in the United States and led to higher viral loads.

We report on the sequencing of 74,348 SARS-CoV-2 positive samples collected across the United States and show that the Delta variant, first detected in the United States in March 2021, made up the majority of SARS-CoV-2 infections by July 1, 2021 and accounted for >99.9% of the infections by September 2021. Not only did Delta displace variant Alpha, which was the dominant variant at the time, it also displaced the Gamma, Iota, and Mu variants. Through an analysis of quantification cycle (Cq) values, we demonstrate that Delta infections tend to have a 1.7× higher viral load compared to Alpha infections (a decrease of 0.8 Cq) on average. Our results are consistent with the hypothesis that the increased transmissibility of the Delta variant could be due to the ability of the Delta variant to establish a higher viral load earlier in the infection as compared to the Alpha variant.

Transcriptome and Methylome Analysis Reveal Complex Cross-Talks between Thyroid Hormone and Glucocorticoid Signaling at Xenopus Metamorphosis.

BACKGROUND: Most work in endocrinology focus on the action of a single hormone, and very little on the cross-talks between two hormones. Here we characterize the nature of interactions between thyroid hormone and glucocorticoid signaling during Xenopus tropicalis metamorphosis. METHODS: We used functional genomics to derive genome wide profiles of methylated DNA and measured changes of gene expression after hormonal treatments of a highly responsive tissue, tailfin. Clustering classified the data into four types of biological responses, and biological networks were modeled by system biology. RESULTS: We found that gene expression is mostly regulated by either T3 or CORT, or their additive effect when they both regulate the same genes. A small but non-negligible fraction of genes (12%) displayed non-trivial regulations indicative of complex interactions between the signaling pathways. Strikingly, DNA methylation changes display the opposite and are dominated by cross-talks. CONCLUSION: Cross-talks between thyroid hormones and glucocorticoids are more complex than initially envisioned and are not limited to the simple addition of their individual effects, a statement that can be summarized with the pseudo-equation: TH ∙ GC > TH + GC. DNA methylation changes are highly dynamic and buffered from genome expression.

Emergence and rapid transmission of SARS-CoV-2 B.1.1.7 in the United States.

The highly transmissible B.1.1.7 variant of SARS-CoV-2, first identified in the United Kingdom, has gained a foothold across the world. Using S gene target failure (SGTF) and SARS-CoV-2 genomic sequencing, we investigated the prevalence and dynamics of this variant in the United States (US), tracking it back to its early emergence. We found that, while the fraction of B.1.1.7 varied by state, the variant increased at a logistic rate with a roughly weekly doubling rate and an increased transmission of 40%-50%. We revealed several independent introductions of B.1.1.7 into the US as early as late November 2020, with community transmission spreading it to most states within months. We show that the US is on a similar trajectory as other countries where B.1.1.7 became dominant, requiring immediate and decisive action to minimize COVID-19 morbidity and mortality.

Genomic epidemiology identifies emergence and rapid transmission of SARS-CoV-2 B.1.1.7 in the United States.

As of January of 2021, the highly transmissible B.1.1.7 variant of SARS-CoV-2, which was first identified in the United Kingdom (U.K.), has gained a strong foothold across the world. Because of the sudden and rapid rise of B.1.1.7, we investigated the prevalence and growth dynamics of this variant in the United States (U.S.), tracking it back to its early emergence and onward local transmission. We found that the RT-qPCR testing anomaly of S gene target failure (SGTF), first observed in the U.K., was a reliable proxy for B.1.1.7 detection. We sequenced 212 B.1.1.7 SARS-CoV-2 genomes collected from testing facilities in the U.S. from December 2020 to January 2021. We found that while the fraction of B.1.1.7 among SGTF samples varied by state, detection of the variant increased at a logistic rate similar to those observed elsewhere, with a doubling rate of a little over a week and an increased transmission rate of 35-45%. By performing time-aware Bayesian phylodynamic analyses, we revealed several independent introductions of B.1.1.7 into the U.S. as early as late November 2020, with onward community transmission enabling the variant to spread to at least 30 states as of January 2021. Our study shows that the U.S. is on a similar trajectory as other countries where B.1.1.7 rapidly became the dominant SARS-CoV-2 variant, requiring immediate and decisive action to minimize COVID-19 morbidity and mortality.

Clustering of strong replicators associated with active promoters is sufficient to establish an early-replicating domain.

Vertebrate genomes replicate according to a precise temporal program strongly correlated with their organization into A/B compartments. Until now, the molecular mechanisms underlying the establishment of early-replicating domains remain largely unknown. We defined two minimal cis-element modules containing a strong replication origin and chromatin modifier binding sites capable of shifting a targeted mid-late-replicating region for earlier replication. The two origins overlap with a constitutive or a silent tissue-specific promoter. When inserted side-by-side, these modules advance replication timing over a 250 kb region through the cooperation with one endogenous origin located 30 kb away. Moreover, when inserted at two chromosomal sites separated by 30 kb, these two modules come into close physical proximity and form an early-replicating domain establishing more contacts with active A compartments. The synergy depends on the presence of the active promoter/origin. Our results show that clustering of strong origins located at active promoters can establish early-replicating domains.

Medico-economic impact of robot-assisted lung segmentectomy: what is the cost of the learning curve?

OBJECTIVES: The objective of this study was to assess the learning curve (LC) of robot-assisted lung segmentectomy and to evaluate hospital-related costs. METHODS: We conducted a retrospective study of Robot-assisted thoracic surgery (RATS) segmentectomies performed by 1 surgeon during 5 years. Perioperative and medical device data were collected. The LC, based on operating time, was assessed by Cumulative SUM analysis and an exponential model. Cost of care was estimated using the French National Cost Study method. RESULTS: One hundred and two RATS segmentectomies were included. The LC was completed at ∼30 procedures according to both models without significant difference in patients' characteristics before or after the LC. Mean operative time decreased from 136 min [95% confidence intervals (CI) 124-149] for the first 30 procedures to 97 min (95% CI 88-107) for the last 30 procedures. Mean length of stay decreased non-significantly (P = 0.10 for linear trend) from 8.1 days (95% CI 6.1-11.0) to 6.2 days (95% CI 4.9-7.9). The overall costs for the last 30 procedures as compared with the first 30 did not significantly decrease in the primary economic analysis but significantly decreased (P = 0.02) by €1271 (95% CI -2688 to +108, P = 0.02 for linear trend) after exclusion of 1 outlier (hospitalization-related costs > €10 000). After exclusion of this outlier, costs related to EndoWrist® instruments significantly decreased by €-135 (95% CI -220 to -35, P = 0.004), whereas costs related to clips decreased non-significantly (P = 0.28). CONCLUSIONS: The LC was completed at ∼30 procedures. Inexperienced surgeons may have higher procedure costs, related to consumable medical devices and operating time.

Feedback on the use of three surgical sealants for preventing prolonged air leak after robot-assisted anatomical lung resection.

BACKGROUND: Prolonged air leak (PAL) is the most common complication after lung resection. Several surgical sealants have been developed to reduce PAL, including fibrin-based (FS), polyethylene glycol-based (PEGS) and polyglycolic acid-based (PGAS) sealants. In this work we report our experience of surgical sealant use after robot-assisted lung resection. METHODS: A 7-year retrospective study was conducted, including patients who had robot-assisted lobectomy or segmentectomy. Data were collected using a prospective national database. The use of surgical sealants was recorded in traceability sheets. RESULTS: PAL occurred in 60 of the 299 patients included. American Society of Anesthesiologists score (ASA) and index of prolonged air leak (IPAL) were higher for patients with sealants. In this group, operative time, chest drain duration and length of stay were significantly longer. PAL occurrence was significantly associated to sealant in univariate analysis (odds ratio =1.88, 95% CI: 1.07 to 3.36, P=0.03) but the association was slightly decreased when adjusting on IPAL and ASA score (Odds ratio =1.70, 95% CI: 0.94 to 3.10, P=0.08). Comparing sealants, more segmentectomies were performed in patients with PGAS (P=0.0013) and their operative time was shorter (P=0.0002). PAL occurrences were not different. Length of stay (P=0.0045) and operative time (P=0.0002) were longer in patients with PEGS who had more postoperative complications (P=0.024). CONCLUSIONS: This study did not identify a positive effect of surgical sealants for preventing PAL. However it highlighted the need to rationalize their use.

Evolution of replication origins in vertebrate genomes: rapid turnover despite selective constraints.

The replication program of vertebrate genomes is driven by the chromosomal distribution and timing of activation of tens of thousands of replication origins. Genome-wide studies have shown the association of origins with promoters and CpG islands, and their enrichment in G-quadruplex motifs (G4). However, the genetic determinants driving their activity remain poorly understood. To gain insight on the constraints operating on origins, we conducted the first evolutionary comparison of origins across vertebrates. We generated a genome-wide map of chicken origins (the first of a bird genome), and performed a comparison with human and mouse maps. The analysis of intra-species polymorphism revealed a strong depletion of genetic diversity at the core of replication initiation loci. This depletion is not linked to the presence of G4 motifs, promoters or CpG islands. In contrast, we show that origins experienced a rapid turnover during vertebrate evolution, since pairwise comparisons of origin maps revealed that <24% of them are conserved among vertebrates. This study unravels the existence of a novel determinant of origins, the precise functional role of which remains to be determined. Despite the importance of replication initiation for the fitness of organisms, the distribution of origins along vertebrate chromosomes is highly flexible.

Submucosal hematoma: a new distinctive sign during emergency upper digestive endoscopy for ammonia ingestion.

BACKGROUND: Submucosal hematoma has never been associated with caustic injuries. Long-term follow-up of patients who ingested ammonia is not well known and ammonia ingestion is rare. METHODS: In a Single-center observational study, prospective data were collected from 2009 to 2013, in patients over the age of 14 years old referred for ammonia ingestion. The emergency and follow-up endoscopic data and the outcome were reported. RESULTS: Ammonia ingestion occurred in 43 patients. Submucosal hematoma of the gastric wall was a distinctive endoscopic sign observed in 15 (34.8%) cases. Oropharyngeal lesions were present in 30 (69.8%) patients, which was associated with ingestion with suicidal intent in 18 cases. Mild and severe endoscopic lesions (grade IIB to IIIB) were found in 16 (37.2%) cases with 10 (23.3%) cases presenting submucosal hematoma at initial endoscopy. A complete spontaneous gastric healing was frequently observed in 36 (83.7%) cases. In 11 cases with submucosal hematoma, a favourable outcome was observed with a medical treatment, however 6 of these patients had severe endoscopic lesions initially. CONCLUSIONS: Submucosal hematoma of the gastric wall is an endoscopic sign occurring frequently in ammonia ingestion. Submucosal hematoma should be distinguished from necrosis in order to avoid false misclassification in favour of more severe lesions, which would lead to an abusive surgery.

The affordability of minimally invasive procedures in major lung resection: a prospective study.

OBJECTIVES: Minimally invasive procedures are used for the surgical treatment of lung cancer. Two techniques are proposed: video-assisted thoracic surgery or robotic-assisted thoracic surgery. Our goal was to study the economic impact of our long-standing program for minimally invasive procedures for major lung resection. METHODS: We conducted a single-centre, 1-year prospective cost study. Patients who underwent lobectomy or segmentectomy were included. Patient characteristics and perioperative outcomes were collected. Medical supply expenses based on the microcosting method and capital depreciation were estimated. Total cost was evaluated using a national French database. RESULTS: One hundred twelve patients were included, 57 with and 55 without robotic assistance. More segmentectomies were performed with robotic assistance. The median length of stay was 5 days for robotic-assisted and 6 days for video-assisted procedures (P = 0.13). The duration of median chest drains (4 days, P = 0.36) and of operating room time (255 min, P = 0.55) was not significantly different between the groups. The overall conversion rate to thoracotomy was 9%, significantly higher in the video-assisted group than in the robotic group (16% vs 2%, P = 0.008). No difference was observed in postoperative complications. The cost of most robotic-assisted procedures ranged from €10 000 to €12 000 (median €10 972) and that of most video-assisted procedures ranged from €8 000 to €10 000 (median €9 637) (P = 0.007); median medical supply expenses were €3 236 and €2 818, respectively (P = 0.004). The overall mean cost of minimally invasive techniques (€11 759) was significantly lower than the mean French cost of lung resection surgical procedures (€13 424) (P = 0.001). CONCLUSIONS: The cost at our centre of performing minimally invasive surgical procedures appeared lower than the cost nationwide. Robotic-assisted thoracic surgery demonstrated acceptable additional costs for a long-standing program.

Finite element based nonlinear normalization of human lumbar intervertebral disc stiffness to account for its morphology.

Disc degeneration, usually associated with low back pain and changes of intervertebral stiffness, represents a major health issue. As the intervertebral disc (IVD) morphology influences its stiffness, the link between mechanical properties and degenerative grade is partially lost without an efficient normalization of the stiffness with respect to the morphology. Moreover, although the behavior of soft tissues is highly nonlinear, only linear normalization protocols have been defined so far for the disc stiffness. Thus, the aim of this work is to propose a nonlinear normalization based on finite elements (FE) simulations and evaluate its impact on the stiffness of human anatomical specimens of lumbar IVD. First, a parameter study involving simulations of biomechanical tests (compression, flexion/extension, bilateral torsion and bending) on 20 FE models of IVDs with various dimensions was carried out to evaluate the effect of the disc's geometry on its compliance and establish stiffness/morphology relations necessary to the nonlinear normalization. The computed stiffness was then normalized by height (H), cross-sectional area (CSA), polar moment of inertia (J) or moments of inertia (Ixx, Iyy) to quantify the effect of both linear and nonlinear normalizations. In the second part of the study, T1-weighted MRI images were acquired to determine H, CSA, J, Ixx and Iyy of 14 human lumbar IVDs. Based on the measured morphology and pre-established relation with stiffness, linear and nonlinear normalization routines were then applied to the compliance of the specimens for each quasi-static biomechanical test. The variability of the stiffness prior to and after normalization was assessed via coefficient of variation (CV). The FE study confirmed that larger and thinner IVDs were stiffer while the normalization strongly attenuated the effect of the disc geometry on its stiffness. Yet, notwithstanding the results of the FE study, the experimental stiffness showed consistently higher CV after normalization. Assuming that geometry and material properties affect the mechanical response, they can also compensate for one another. Therefore, the larger CV after normalization can be interpreted as a strong variability of the material properties, previously hidden by the geometry's own influence. In conclusion, a new normalization protocol for the intervertebral disc stiffness in compression, flexion, extension, bilateral torsion and bending was proposed, with the possible use of MRI and FE to acquire the discs' anatomy and determine the nonlinear relations between stiffness and morphology. Such protocol may be useful to relate the disc's mechanical properties to its degree of degeneration.

Seroepidemiology of chronic hepatitis B and C in the French Caribbean Island of Guadeloupe.

BACKGROUND: The prevalence of chronic hepatitis B and C was evaluated some twenty years ago among specific populations in Guadeloupe. The present study was designed to update these data and determine epidemiological features of chronic hepatitis B and C infections in the French Caribbean island of Guadeloupe. FINDINGS: The present study was carried out at the Sainte Genevieve Health and Prevention Center (Guadeloupe), between May 2006 and July 2007. This is a medical center where patients can attend a free medical check-up paid for by the Social Security system. Data on hepatitis B (HBV) and C (HCV) status and epidemiological factors were collected for this study.A total of 2,200 patients were included in the study. The prevalence of HBV surface antigen was 1.41% (95% CI: 1.0-2.0), and 0.55% (95% CI: 0.28-0.96) for HCV. The vaccination rate against HBV was 42.0%. HBV transmission was associated with piercing (12.9%, p = 0.014) and familial exposure (6.4%, p < 0.001) and HCV transmission with gynecological surgery (50.0%, p = 0.01). The HBV profile was generally hepatitis B e antigen-negative (94.5%). No hepatitis delta was found. For HCV, genotype 1 was predominant (80%). CONCLUSIONS: This is the first study on the prevalence of HBV and HCV among a general clinic based population in Guadeloupe and the Caribbean islands. This study reveals that Guadeloupe is an area of low endemicity for HBV and low HCV prevalence. The reasons for these low prevalence rates are mainly related to the vaccination campaigns carried out during the past twenty years for HBV and the decrease of nosocomial transmission for HCV.

Comparative study of the effect of rivaroxaban and fondaparinux on monocyte's coagulant activity and cytokine release.

OBJECTIVES: Tissue factor (TF) exposed on activated monocytes and macrophages is involved in thrombosis through activation of factor X and cytokine release, responsible for inflammation and thrombosis. We investigated the effect of two anti-factor Xa drugs: rivaroxaban, a direct anti-Xa inhibitor, and fondaparinux, an antithrombin dependent anti-Xa inhibitor, on monocyte/macrophage procoagulant activity and cytokine release. METHODS: Rivaroxaban and fondaparinux were tested at pharmacological concentrations on LPS-activated monocytes and on THP-1 cells, a human monocytic cell line, to assess 1) TF expression by flow cytometry 2) prothrombinase activity by its coagulant activity and 3) cytokine release in cell supernatants by antibody based cytokine array and ELISA for IL-8 and TNFα. RESULTS AND CONCLUSION: Rivaroxaban and fondaparinux did not modify TF expression level on activated cells. In contrast procoagulant activity associated to monocytes and macrophages was dose dependently inhibited by rivaroxaban, but not significantly by fondaparinux. These results could explain why patients undergoing major orthopedic surgery with rivaroxaban prophylaxis were able to achieve significant reductions in venous thromboembolism, compared with drugs commonly used, i.e. fondaparinux and low molecular weight heparin. In addition, rivaroxaban and fondaparinux suppressed some chemokine secretion produced by activated macrophages. This may also contribute to their antithrombotic effect in clinic.

Rac3 induces a molecular pathway triggering breast cancer cell aggressiveness: differences in MDA-MB-231 and MCF-7 breast cancer cell lines.

BACKGROUND: Rho GTPases are involved in cellular functions relevant to cancer. The roles of RhoA and Rac1 have already been established. However, the role of Rac3 in cancer aggressiveness is less well understood. METHODS: This work was conducted to analyze the implication of Rac3 in the aggressiveness of two breast cancer cell lines, MDA-MB-231 and MCF-7: both express Rac3, but MDA-MB-231 expresses more activated RhoA. The effect of Rac3 in cancer cells was also compared with its effect on the non-tumorigenic mammary epithelial cells MCF-10A. We analyzed the consequences of Rac3 depletion by anti-Rac3 siRNA. RESULTS: Firstly, we analyzed the effects of Rac3 depletion on the breast cancer cells' aggressiveness. In the invasive MDA-MB-231 cells, Rac3 inhibition caused a marked reduction of both invasion (40%) and cell adhesion to collagen (84%), accompanied by an increase in TNF-induced apoptosis (72%). This indicates that Rac3 is involved in the cancer cells' aggressiveness. Secondly, we investigated the effects of Rac3 inhibition on the expression and activation of related signaling molecules, including NF-κB and ERK. Cytokine secretion profiles were also analyzed. In the non-invasive MCF-7 line; Rac3 did not influence any of the parameters of aggressiveness. CONCLUSIONS: This discrepancy between the effects of Rac3 knockdown in the two cell lines could be explained as follows: in the MDA-MB-231 line, the Rac3-dependent aggressiveness of the cancer cells is due to the Rac3/ERK-2/NF-κB signaling pathway, which is responsible for MMP-9, interleukin-6, -8 and GRO secretion, as well as the resistance to TNF-induced apoptosis, whereas in the MCF-7 line, this pathway is not functional because of the low expression of NF-κB subunits in these cells. Rac3 may be a potent target for inhibiting aggressive breast cancer.

BAP1 loss defines a new class of renal cell carcinoma.

The molecular pathogenesis of renal cell carcinoma (RCC) is poorly understood. Whole-genome and exome sequencing followed by innovative tumorgraft analyses (to accurately determine mutant allele ratios) identified several putative two-hit tumor suppressor genes, including BAP1. The BAP1 protein, a nuclear deubiquitinase, is inactivated in 15% of clear cell RCCs. BAP1 cofractionates with and binds to HCF-1 in tumorgrafts. Mutations disrupting the HCF-1 binding motif impair BAP1-mediated suppression of cell proliferation but not deubiquitination of monoubiquitinated histone 2A lysine 119 (H2AK119ub1). BAP1 loss sensitizes RCC cells in vitro to genotoxic stress. Notably, mutations in BAP1 and PBRM1 anticorrelate in tumors (P = 3 × 10(-5)), [corrected] and combined loss of BAP1 and PBRM1 in a few RCCs was associated with rhabdoid features (q = 0.0007). BAP1 and PBRM1 regulate seemingly different gene expression programs, and BAP1 loss was associated with high tumor grade (q = 0.0005). Our results establish the foundation for an integrated pathological and molecular genetic classification of RCC, paving the way for subtype-specific treatments exploiting genetic vulnerabilities.

Stimulation of angiogenesis resulting from cooperation between macrophages and MDA-MB-231 breast cancer cells: proposed molecular mechanism and effect of tetrathiomolybdate.

BACKGROUND: Infiltration by macrophages (Mphi) indicates a poor prognosis in breast cancers, in particular by inducing angiogenesis. Our study aimed 1) to investigate the mechanism by which cooperation between Mphi and aggressive breast cancer cells (MDA-MB-231) induces angiogenesis; 2) to examine the effect of tetrathiomolybdate (TM) on this angiogenic activity. METHODS: Mphi coincubated with MDA-MB-231 were used as a model to mimic the inflammatory microenvironment. Angiogenesis induced by the culture media was tested in the chick chorioallantoic membrane (CAM). Mphi phenotype was evaluated by 1) expression of the M1 marker CD80, and secretion of interleukin 10 (IL-10), an M2 marker; 2) capacity to secrete Tumour Necrosis Factor alpha (TNFalpha) when stimulated by lipopolysaccharide/interferon gamma (LPS/IFNgamma); 3) ability to induce MDA-MB-231 apoptosis. To explore the molecular mechanisms involved, cytokine profiles of conditioned media from MDA-MB-231, Mphi and the coculture were characterised by an antibody cytokine array. All experiments were carried out both in presence and in absence of TM. RESULTS: Incubation of Mphi with MDA-MB-231 induced a pro-angiogenic effect in the CAM. It emerged that the angiogenic activity of the coculture is due to the capacity of Mphi to switch from M1 Mphi towards M2, probably due to an increase in Macrophage Colony Stimulating Factor. This M1-M2 switch was shown by a decreased expression of CD80 upon LPS/IFNgamma stimulation, an increased secretion of IL-10, a decreased secretion of TNFalpha in response to LPS/IFNgamma and an inability to potentiate apoptosis. At the molecular level, the angiogenic activity of the coculture medium can be explained by the secretion of CXC chemokines/ELR+ and CC chemokines. Although TM did not modify either the M2 phenotype in the coculture or the profile of the secreted chemokines, it did decrease the angiogenic activity of the coculture medium, suggesting that TM inhibited angiogenic activity by interfering with the endothelial cell signalling induced by these chemokines. CONCLUSIONS: Cooperation between Mphi and MDA-MB-231 transformed M1 Mphi to an angiogenic, M2 phenotype, attested by secretion of CXC chemokines/ELR+ and CC chemokines. TM inhibited this coculture-induced increase in angiogenic activity, without affecting either Mphi phenotype or cytokine secretion profiles.

Illumina universal bead arrays.

This chapter describes an accurate, scalable, and flexible microarray technology. It includes a miniaturized array platform where each individual feature is quality controlled and a versatile assay that can be adapted for various genetic analyses, such as single nucleotide polymorphism genotyping, DNA methylation detection, and gene expression profiling. This chapter describes the concept of the BeadArray technology, two different Array of Arrays formats, the assay scheme and protocol, the performance of the system, and its use in large-scale genetic, epigenetic, and expression studies.