Development of extracellular matrix supported 3D culture of renal cancer cells and renal cancer stem cells

Novel experimental conditions of cancer cell line culture have evolved throughout the recent years, with significantly growing interest in xeno-free, serum-free and three-dimensional culture variants. The choice of proper culture media may enable to mimic tumor microenvironment and promotion of cancer stem cells proliferation. To assess whether stem-like phenotype inducing media may be applied in renal cancer stem cell research, we performed a widespread screening of 13 cell culture media dedicated for mesenchymal cells, stem cells as well as mesenchymal stem cells. We have also screened extracellular matrix compounds and selected optimal RCC 3D—ECM supported culture model. Our results revealed that 786-O as well as HKCSCs cell line cultures in xeno-free media (NutriStem/StemXvivo) and laminin coated plates provide a useful tool in RCC cancer biology research and at the same time enable effective drug toxicity screening. We propose bio-mimic 3D RCC cell culture model with specific low-serum and xeno-free media that promote RCC cell viability and stem-like phenotype according to the tested genes encoding stemness factors including E-cadherin, N-cadherin, HIF1, HIF2, VEGF, SOX2, PAX2 and NESTIN

Reactivity of pulmonary circulation and right ventricle function to inhaled nitric oxide in systemic sclerosis patients

Systemic sclerosis (SSc) is complicated by pulmonary hypertension and right ventricle (RV) failure in approximately 10% of the patients. Factors influencing the reactivity of pulmonary circulation to vasodilators are not established, while the examination of vasoreactivity is important in determining the treatment, because systemic administration of oral vasodilators can induce severe adverse events in nonresponders. The mechanism of RV failure in SSc is unclear and may result either from increased RV afterload or intrinsic myocardial disease. The aim of the study was to assess the reactivity of pulmonary circulation to inhaled nitric oxide (iNO) and to evaluate its influence on RV function in SSc patients with elevated right ventricle systolic pressure (RVSP). In 60 SSc patients aged 24–73 (58 females, two males; 33 patients with limited SSc and 27 with diffuse SSc), echocardiographic examination with tissue Doppler echocardiography (TDE) was performed. RV function was measured by systolic (S) and early diastolic (E) velocity of tricuspid annulus by TDE. In patients with RVSP >45 mmHg, the reactivity of pulmonary circulation was assessed by iNO test. High-resolution computerized tomography (HRCT) was performed to assess the extent of pulmonary fibrosis. Of 14 SSc subjects with elevated RVSP (13 females, one male; RVSP 47–62 mmHg), positive reaction to iNO was observed in five (RVSP decreased from 51.6 ± 3.7 to 32.24 ± 2.3 mmHg); nine patients were not reactive (RVSP 53.5 ± 5.7 mmHg before iNO vs. 49.6 ± 6.7 mmHg). RV systolic function was decreased in patients with elevated RVSP as compared to the patients with normal pulmonary pressure (S velocity 13.2 ± 1.3 vs. 14.4 ± 1.6 cm/s, respectively, p < 0.05). Significant increase of RV systolic function during iNO test was found in reactive patients only (S velocity before iNO 12.8 ± 1.2 cm/s, during iNO 14.5 ± 1.5 cm/s, p < 0.01). RVSP decrease strongly correlated with S velocity increase (r = 0.95, p < 0.0001). Response to iNO was found only in limited form of SSc; diffuse SSc patients showed no response. Pulmonary fibrosis on HRCT was more frequent in subjects nonreactive to iNO (67% of patients) than in the reactive group (40% of patients). The reactivity of pulmonary circulation to iNO in SSc patients with elevated RVSP was found predominantly in limited form of the disease. Pulmonary fibrosis typical for diffuse SSc was more frequent in nonreactive subjects. Elevated pulmonary pressure plays an important role in RV systolic dysfunction. Pulmonary pressure decrease during iNO test leads to the improvement of RV systolic function. Therapy for right-heart failure in reactive SSc patients should be directed, if possible, at the decrease in pulmonary resistance

Fluorescent RNA cytosine analogue - an internal probe for detailed structure and dynamics investigations

The bright fluorescent cytosine analogue tCO stands out among fluorescent bases due to its virtually unquenched fluorescence emission in duplex DNA. However, like most reported base analogues, it has not been thoroughly characterized in RNA. We here report on the first synthesis and RNA-incorporation of tCO, and characterize its base-mimicking and fluorescence properties in RNA. As in DNA, we find a high quantum yield inside RNA duplexes (&lt;?F&gt; = 0.22) that is virtually unaffected by the neighbouring bases (?F = 0.20-0.25), resulting in an average brightness of 1900 M-1 cm-1. The average fluorescence lifetime in RNA duplexes is 4.3 ns and generally two lifetimes are required to fit the exponential decays. Fluorescence properties in ssRNA are defined by a small increase in average quantum yield (&lt;?F &gt; = 0.24) compared to dsRNA, with a broader distribution (?F = 0.17-0.34) and slightly shorter average lifetimes. Using circular dichroism, we find that the tCO-modified RNA duplexes form regular A-form helices and in UV-melting experiments the stability of the duplexes is only slightly higher than that of the corresponding natural RNA (&lt;?T m&gt; = + 2.3 °C). These properties make tCO a highly interesting fluorescent RNA base analogue for detailed FRET-based structural measurements, as a bright internal label in microscopy, and for fluorescence anisotropy measurements of RNA dynamics

Nuclear localised more sulphur accumulation1 epigenetically regulates sulphur homeostasis in Arabidopsis thaliana

Sulphur (S) is an essential element for all living organisms. The uptake, assimilation and metabolism of S in plants are well studied. However, the regulation of S homeostasis remains largely unknown. Here, we report on the identification and characterisation of the more sulphur accumulation1 (msa1-1) mutant. The MSA1 protein is localized to the nucleus and is required for both S adenosylmethionine (SAM) production and DNA methylation. Loss of function of the nuclear localised MSA1 leads to a reduction in SAM in roots and a strong S-deficiency response even at ample S supply, causing an over- accumulation of sulphate, sulphite, cysteine and glutathione. Supplementation with SAM suppresses this high S phenotype. Furthermore, mutation of MSA1 affects genome-wide DNA methylation, including the methylation of S-deficiency responsive genes. Elevated S accumulation in msa1-1 requires the increased expression of the sulphate transporter genes SULTR1;1 and SULTR1;2 which are also differentially methylated in msa1-1. Our results suggest a novel function for MSA1 in the nucleus in regulating SAM biosynthesis and maintaining S homeostasis epigenetically via DNA methylation

Treatment outcome in early diffuse cutaneous systemic sclerosis: the European Scleroderma Observational Study (ESOS).

OBJECTIVES: The rarity of early diffuse cutaneous systemic sclerosis (dcSSc) makes randomised controlled trials very difficult. We aimed to use an observational approach to compare effectiveness of currently used treatment approaches. METHODS: This was a prospective, observational cohort study of early dcSSc (within three years of onset of skin thickening). Clinicians selected one of four protocols for each patient: methotrexate, mycophenolate mofetil (MMF), cyclophosphamide or 'no immunosuppressant'. Patients were assessed three-monthly for up to 24 months. The primary outcome was the change in modified Rodnan skin score (mRSS). Confounding by indication at baseline was accounted for using inverse probability of treatment (IPT) weights. As a secondary outcome, an IPT-weighted Cox model was used to test for differences in survival. RESULTS: Of 326 patients recruited from 50 centres, 65 were prescribed methotrexate, 118 MMF, 87 cyclophosphamide and 56 no immunosuppressant. 276 (84.7%) patients completed 12 and 234 (71.7%) 24 months follow-up (or reached last visit date). There were statistically significant reductions in mRSS at 12 months in all groups: -4.0 (-5.2 to -2.7) units for methotrexate, -4.1 (-5.3 to -2.9) for MMF, -3.3 (-4.9 to -1.7) for cyclophosphamide and -2.2 (-4.0 to -0.3) for no immunosuppressant (p value for between-group differences=0.346). There were no statistically significant differences in survival between protocols before (p=0.389) or after weighting (p=0.440), but survival was poorest in the no immunosuppressant group (84.0%) at 24 months. CONCLUSIONS: These findings may support using immunosuppressants for early dcSSc but suggest that overall benefit is modest over 12 months and that better treatments are needed. TRIAL REGISTRATION NUMBER: NCT02339441

Genome-Wide Scan Identifies TNIP1, PSORS1C1, and RHOB as Novel Risk Loci for Systemic Sclerosis

Systemic sclerosis (SSc) is an orphan, complex, inflammatory disease affecting the immune system and connective tissue. SSc stands out as a severely incapacitating and life-threatening inflammatory rheumatic disease, with a largely unknown pathogenesis. We have designed a two-stage genome-wide association study of SSc using case-control samples from France, Italy, Germany, and Northern Europe. The initial genome-wide scan was conducted in a French post quality-control sample of 564 cases and 1,776 controls, using almost 500 K SNPs. Two SNPs from the MHC region, together with the 6 loci outside MHC having at least one SNP with a P<10−5 were selected for follow-up analysis. These markers were genotyped in a post-QC replication sample of 1,682 SSc cases and 3,926 controls. The three top SNPs are in strong linkage disequilibrium and located on 6p21, in the HLA-DQB1 gene: rs9275224, P = 9.18×10−8, OR = 0.69, 95% CI [0.60–0.79]; rs6457617, P = 1.14×10−7 and rs9275245, P = 1.39×10−7. Within the MHC region, the next most associated SNP (rs3130573, P = 1.86×10−5, OR = 1.36 [1.18–1.56]) is located in the PSORS1C1 gene. Outside the MHC region, our GWAS analysis revealed 7 top SNPs (P<10−5) that spanned 6 independent genomic regions. Follow-up of the 17 top SNPs in an independent sample of 1,682 SSc and 3,926 controls showed associations at PSORS1C1 (overall P = 5.70×10−10, OR:1.25), TNIP1 (P = 4.68×10−9, OR:1.31), and RHOB loci (P = 3.17×10−6, OR:1.21). Because of its biological relevance, and previous reports of genetic association at this locus with connective tissue disorders, we investigated TNIP1 expression. A markedly reduced expression of the TNIP1 gene and also its protein product were observed both in lesional skin tissue and in cultured dermal fibroblasts from SSc patients. Furthermore, TNIP1 showed in vitro inhibitory effects on inflammatory cytokine-induced collagen production. The genetic signal of association with TNIP1 variants, together with tissular and cellular investigations, suggests that this pathway has a critical role in regulating autoimmunity and SSc pathogenesis

Key mechanisms governing resolution of lung inflammation

Innate immunity normally provides excellent defence against invading microorganisms. Acute inflammation is a form of innate immune defence and represents one of the primary responses to injury, infection and irritation, largely mediated by granulocyte effector cells such as neutrophils and eosinophils. Failure to remove an inflammatory stimulus (often resulting in failed resolution of inflammation) can lead to chronic inflammation resulting in tissue injury caused by high numbers of infiltrating activated granulocytes. Successful resolution of inflammation is dependent upon the removal of these cells. Under normal physiological conditions, apoptosis (programmed cell death) precedes phagocytic recognition and clearance of these cells by, for example, macrophages, dendritic and epithelial cells (a process known as efferocytosis). Inflammation contributes to immune defence within the respiratory mucosa (responsible for gas exchange) because lung epithelia are continuously exposed to a multiplicity of airborne pathogens, allergens and foreign particles. Failure to resolve inflammation within the respiratory mucosa is a major contributor of numerous lung diseases. This review will summarise the major mechanisms regulating lung inflammation, including key cellular interplays such as apoptotic cell clearance by alveolar macrophages and macrophage/neutrophil/epithelial cell interactions. The different acute and chronic inflammatory disease states caused by dysregulated/impaired resolution of lung inflammation will be discussed. Furthermore, the resolution of lung inflammation during neutrophil/eosinophil-dominant lung injury or enhanced resolution driven via pharmacological manipulation will also be considered