Modelling the tumour microenvironment in long-term microencapsulated 3D co-cultures recapitulates phenotypic features of disease progression

3D cell tumour models are generated mainly in non-scalable culture systems, using bioactive scaffolds. Many of these models fail to reflect the complex tumour microenvironment and do not allow long-term monitoring of tumour progression. To overcome these limitations, we have combined alginate microencapsulation with agitation-based culture systems, to recapitulate and monitor key aspects of the tumour microenvironment and disease progression. Aggregates of MCF-7 breast cancer cells were microencapsulated in alginate, either alone or in combination with human fibroblasts, then cultured for 15 days. In co-cultures, the fibroblasts arranged themselves around the tumour aggregates creating distinct epithelial and stromal compartments. The presence of fibroblasts resulted in secretion of pro-inflammatory cytokines and deposition of collagen in the stromal compartment. Tumour cells established cell–cell contacts and polarised around small lumina in the interior of the aggregates. Over the culture period, there was a reduction in oestrogen receptor and membranous E-cadherin alongside loss of cell polarity, increased collective cell migration and enhanced angiogenic potential in co-cultures. These phenotypic alterations, typical of advanced stages of cancer, were not observed in the mono-cultures of MCF-7 cells. The proposed model system constitutes a new tool to study tumour-stroma crosstalk, disease progression and drug resistance mechanisms

Phytoplankton across Tropical and Subtropical Regions of the Atlantic, Indian and Pacific Oceans

Postprint4,411

Protocols and characterization data for 2D, 3D, and slice-based tumor models from the PREDECT project

Two-dimensional (2D) culture of cancer cells in vitro does not recapitulate the three-dimensional (3D) architecture, heterogeneity and complexity of human tumors. More representative models are required that better reflect key aspects of tumor biology. These are essential studies of cancer biology and immunology as well as for target validation and drug discovery. The Innovative Medicines Initiative (IMI) consortium PREDECT (www.predect.eu) characterized in vitro models of three solid tumor types with the goal to capture elements of tumor complexity and heterogeneity. 2D culture and 3D mono-and stromal cocultures of increasing complexity, and precision-cut tumor slice models were established. Robust protocols for the generation of these platforms are described. Tissue microarrays were prepared from all the models, permitting immunohistochemical analysis of individual cells, capturing heterogeneity. 3D cultures were also characterized using image analysis. Detailed step-by-step protocols, exemplary datasets from the 2D, 3D, and slice models, and refined analytical methods were established and are presented.Peer reviewe

Maternal haemoglobin levels in pregnancy and child DNA methylation : a study in the pregnancy and childhood epigenetics consortium

Altered maternal haemoglobin levels during pregnancy are associated with pre-clinical and clinical conditions affecting the fetus. Evidence from animal models suggests that these associations may be partially explained by differential DNA methylation in the newborn with possible long-term consequences. To test this in humans, we meta-analyzed the epigenome-wide associations of maternal haemoglobin levels during pregnancy with offspring DNA methylation in 3,967 newborn cord blood and 1,534 children and 1,962 adolescent whole-blood samples derived from 10 cohorts. DNA methylation was measured using Illumina Infinium Methylation 450K or MethylationEPIC arrays covering 450,000 and 850,000 methylation sites, respectively. There was no statistical support for the association of maternal haemoglobin levels with offspring DNA methylation either at individual methylation sites or clustered in regions. For most participants, maternal haemoglobin levels were within the normal range in the current study, whereas adverse perinatal outcomes often arise at the extremes. Thus, this study does not rule out the possibility that associations with offspring DNA methylation might be seen in studies with more extreme maternal haemoglobin levels.Peer reviewe

Oligomerization of ZFYVE27 (Protrudin) Is Necessary to Promote Neurite Extension

ZFYVE27 (Protrudin) was originally identified as an interacting partner of spastin, which is most frequently mutated in hereditary spastic paraplegia. ZFYVE27 is a novel member of FYVE family, which is implicated in the formation of neurite extensions by promoting directional membrane trafficking in neurons. Now, through a yeast two-hybrid screen, we have identified that ZFYVE27 interacts with itself and the core interaction region resides within the third hydrophobic region (HR3) of the protein. We confirmed the ZFYVE27's self-interaction in the mammalian cells by co-immunoprecipitation and co-localization studies. To decipher the oligomeric nature of ZFYVE27, we performed sucrose gradient centrifugation and showed that ZFYVE27 oligomerizes into dimer/tetramer forms. Sub-cellular fractionation and Triton X-114 membrane phase separation analysis indicated that ZFYVE27 is a peripheral membrane protein. Furthermore, ZFYVE27 also binds to phosphatidylinositol 3-phosphate lipid moiety. Interestingly, cells expressing ZFYVE27ΔHR3 failed to produce protrusions instead caused swelling of cell soma. When ZFYVE27ΔHR3 was co-expressed with wild-type ZFYVE27 (ZFYVE27WT), it exerted a dominant negative effect on ZFYVE27WT as the cells co-expressing both proteins were also unable to induce protrusions and showed cytoplasmic swelling. Altogether, it is evident that a functionally active form of oligomer is crucial for ZFYVE27 ability to promote neurite extensions

Capturing tumor complexity in vitro : Comparative analysis of 2D and 3D tumor models for drug discovery

Two-dimensional (2D) cell cultures growing on plastic do not recapitulate the three dimensional (3D) architecture and complexity of human tumors. More representative models are required for drug discovery and validation. Here, 2D culture and 3D mono-and stromal co-culture models of increasing complexity have been established and cross-comparisons made using three standard cell carcinoma lines: MCF7, LNCaP, NCI-H1437. Fluorescence-based growth curves, 3D image analysis, immunohistochemistry and treatment responses showed that end points differed according to cell type, stromal co-culture and culture format. The adaptable methodologies described here should guide the choice of appropriate simple and complex in vitro models.Peer reviewe

The Biodiversity of the Mediterranean Sea: Estimates, Patterns, and Threats

The Mediterranean Sea is a marine biodiversity hot spot. Here we combined an extensive literature analysis with expert opinions to update publicly available estimates of major taxa in this marine ecosystem and to revise and update several species lists. We also assessed overall spatial and temporal patterns of species diversity and identified major changes and threats. Our results listed approximately 17,000 marine species occurring in the Mediterranean Sea. However, our estimates of marine diversity are still incomplete as yet—undescribed species will be added in the future. Diversity for microbes is substantially underestimated, and the deep-sea areas and portions of the southern and eastern region are still poorly known. In addition, the invasion of alien species is a crucial factor that will continue to change the biodiversity of the Mediterranean, mainly in its eastern basin that can spread rapidly northwards and westwards due to the warming of the Mediterranean Sea. Spatial patterns showed a general decrease in biodiversity from northwestern to southeastern regions following a gradient of production, with some exceptions and caution due to gaps in our knowledge of the biota along the southern and eastern rims. Biodiversity was also generally higher in coastal areas and continental shelves, and decreases with depth. Temporal trends indicated that overexploitation and habitat loss have been the main human drivers of historical changes in biodiversity. At present, habitat loss and degradation, followed by fishing impacts, pollution, climate change, eutrophication, and the establishment of alien species are the most important threats and affect the greatest number of taxonomic groups. All these impacts are expected to grow in importance in the future, especially climate change and habitat degradation. The spatial identification of hot spots highlighted the ecological importance of most of the western Mediterranean shelves (and in particular, the Strait of Gibraltar and the adjacent Alboran Sea), western African coast, the Adriatic, and the Aegean Sea, which show high concentrations of endangered, threatened, or vulnerable species. The Levantine Basin, severely impacted by the invasion of species, is endangered as well

SARS-CoV-2 viral load in nasopharyngeal swabs is not an independent predictor of unfavorable outcome

The aim was to assess the ability of nasopharyngeal SARS-CoV-2 viral load at first patient’s hospital evaluation to predict unfavorable outcomes. We conducted a prospective cohort study including 321 adult patients with confirmed COVID-19 through RT-PCR in nasopharyngeal swabs. Quantitative Synthetic SARS-CoV-2 RNA cycle threshold values were used to calculate the viral load in log10 copies/mL. Disease severity at the end of follow up was categorized into mild, moderate, and severe. Primary endpoint was a composite of intensive care unit (ICU) admission and/or death (n = 85, 26.4%). Univariable and multivariable logistic regression analyses were performed. Nasopharyngeal SARS-CoV-2 viral load over the second quartile (≥ 7.35 log10 copies/mL, p = 0.003) and second tertile (≥ 8.27 log10 copies/mL, p = 0.01) were associated to unfavorable outcome in the unadjusted logistic regression analysis. However, in the final multivariable analysis, viral load was not independently associated with an unfavorable outcome. Five predictors were independently associated with increased odds of ICU admission and/or death: age ≥ 70 years, SpO2, neutrophils > 7.5 × 103/µL, lactate dehydrogenase ≥ 300 U/L, and C-reactive protein ≥ 100 mg/L. In summary, nasopharyngeal SARS-CoV-2 viral load on admission is generally high in patients with COVID-19, regardless of illness severity, but it cannot be used as an independent predictor of unfavorable clinical outcome

Epidemiological trends of HIV/HCV coinfection in Spain, 2015-2019

Altres ajuts: Spanish AIDS Research Network; European Funding for Regional Development (FEDER).Objectives: We assessed the prevalence of anti-hepatitis C virus (HCV) antibodies and active HCV infection (HCV-RNA-positive) in people living with HIV (PLWH) in Spain in 2019 and compared the results with those of four similar studies performed during 2015-2018. Methods: The study was performed in 41 centres. Sample size was estimated for an accuracy of 1%. Patients were selected by random sampling with proportional allocation. Results: The reference population comprised 41 973 PLWH, and the sample size was 1325. HCV serostatus was known in 1316 PLWH (99.3%), of whom 376 (28.6%) were HCV antibody (Ab)-positive (78.7% were prior injection drug users); 29 were HCV-RNA-positive (2.2%). Of the 29 HCV-RNA-positive PLWH, infection was chronic in 24, it was acute/recent in one, and it was of unknown duration in four. Cirrhosis was present in 71 (5.4%) PLWH overall, three (10.3%) HCV-RNA-positive patients and 68 (23.4%) of those who cleared HCV after anti-HCV therapy (p = 0.04). The prevalence of anti-HCV antibodies decreased steadily from 37.7% in 2015 to 28.6% in 2019 (p < 0.001); the prevalence of active HCV infection decreased from 22.1% in 2015 to 2.2% in 2019 (p < 0.001). Uptake of anti-HCV treatment increased from 53.9% in 2015 to 95.0% in 2019 (p < 0.001). Conclusions: In Spain, the prevalence of active HCV infection among PLWH at the end of 2019 was 2.2%, i.e. 90.0% lower than in 2015. Increased exposure to DAAs was probably the main reason for this sharp reduction. Despite the high coverage of treatment with direct-acting antiviral agents, HCV-related cirrhosis remains significant in this population