Robustness of dead Cas9 activators in human pluripotent and mesenchymal stem cells

Human pluripotent stem cells (hPSCs) and mesenchymal stromal/stem cells (hMSCs) are clinically relevant sources for cellular therapies and for modeling human development and disease. Many stem cell-based applications rely on the ability to activate several endogenous genes simultaneously to modify cell fate. However, genetic intervention of these cells remains challenging. Several catalytically dead Cas9 (dCas9) proteins fused to distinct activation domains can modulate gene expression when directed to their regulatory regions by a specific single-guide RNA (sgRNA). In this study, we have compared the ability of the first-generation dCas9-VP64 activator and the second-generation systems, dCas9-SAM and dCas9-SunTag, to induce gene expression in hPSCs and hMSCs. Several stem cell lines were tested for single and multiplexed gene activation. When the activation of several genes was compared, all three systems induced specific and potent gene expression in both single and multiplexed settings, but the dCas9-SAM and dCas9-SunTag systems resulted in the highest and most consistent level of gene expression. Simultaneous targeting of the same gene with multiple sgRNAs did not result in additive levels of gene expression in hPSCs nor hMSCs. We demonstrate the robustness and specificity of second-generation dCas9 activators as tools to simultaneously activate several endogenous genes in clinically relevant human stem cells.We thank CERCA/Generalitat de Catalunya and Fundació Josep Carreras-Obra Social la Caixa for their institutional support. We thank Jose Luis Sardina (IJC, Barcelona) for technical assistance with the teratoma assays. Financial support for this work was obtained from the Catalunya Goverment (SGR330 and PERIS 2017-2019), the Spanish Ministry of Economy and Competitiveness (SAF2016-80481-R), the European Research Council (CoG-2014-646903), and the Fundación Leo Messi to P.M.; the Spanish Association against Cancer (AECC-CI-2015) and the Health Institute Carlos III (ISCIII/FEDER, PI17/01028) to C.B.; the Biotechnology and Biological Sciences Research Council (BBRSC) to L.M.F. and A.F.; and the Spanish National Research and Development Plan (ISCIII/FEDER, PI17/02303) and the AEI/MICIU EXPLORA Project (BIO2017-91272-EXP) to S.R.-P. P.M. is an investigator of the Spanish Cell Therapy Cooperative Network (TERCEL). R.T.-R. is supported by a postdoctoral fellowship from the Asociación Española Contra el Cáncer (AECC).S

The Loss of the p53 Activator HIPK2 Is Responsible for Galectin-3 Overexpression in Well Differentiated Thyroid Carcinomas

Background: Galectin-3 (Gal-3) is an anti-apoptotic molecule involved in thyroid cells transformation. It is specifically overexpressed in thyroid tumour cells and is currently used as a preoperative diagnostic marker of thyroid malignancy. Gal-3 expression is downregulated by wt-p53 at the transcriptional level. In well-differentiated thyroid carcinomas (WDTCs) there is an unexplained paradoxical concomitant expression of Gal-3 and wt-p53. HIPK2 is a co-regulator of different transcription factors, and modulates basic cellular processes mainly through the activation of wt-p53. Since we demonstrated that HIPK2 is involved in p53-mediated Gal-3 downregulation, we asked whether HIPK2 deficiency might be responsible for such paradoxical Gal-3 overexpression in WDTC. Methodology/Principal Findings: We analyzed HIPK2 protein and mRNA levels, as well as loss of heterozygosity (LOH) at the HIPK2 locus (7q32-34), in thyroid tissue samples. HIPK2 protein levels were high in all follicular hyperplasias (FHs) analyzed. Conversely, HIPK2 was undetectable in 91.7% of papillary thyroid carcinomas (PTCs) and in 60.0% of follicular thyroid carcinomas (FTCs). HIPK2 mRNA levels were upregulated in FH compared to normal thyroid tissue (NTT), while PTC showed mean HIPK2 mRNA levels lower than FH and, in 61.5% of cases, also lower than NTT. We found LOH at HIPK-2 gene locus in 37.5% of PTCs, 14.3% of FTCs and 18.2% of follicular adenomas. To causally link these data with Gal-3 upregulation, we performed in vitro experiments, using the PTC-derived K1 cells, in which HIPK2 expression was manipulated by RNA interference (RNAi) or plasmid-mediated overexpression. HIPK2 RNAi was associated with Gal-3 upregulation, while HIPK2 overexpression with Gal-3 downregulation. Conclusions/Significance: Our results indicate that HIPK2 expression and function are impaired in WDTCs, in particular in PTCs, and that this event explains Gal-3 overexpression typically observed in these types of tumours. Therefore, HIPK2 can be considered as a new tumour suppressor gene for thyroid cancers

Thyroid hormones downregulate the expression of the antiapoptotic factor Galectin-3 via p53 in human thyroid cancer cells.

P-077

Cardiovascular risk factors predict survival in middle-aged men during 50years.

OBJECTIVES: We aimed at studying the expectancy of life in middle-aged men as a function of several personal characteristics and risk factors. MATERIAL AND METHODS: A sample of 1712 Italian men aged 40-59, first examined in 1960, was followed-up for mortality for 50years. The length of survival was estimated as a function of 48 personal characteristics and risk factors using the multiple linear regression. RESULTS: In 50years 1672 men died (97.7\%) and 40 survived (2.3\%). Twenty risk factors, most of which were never measured in previous studies of such duration, proved to be significant, for the estimation of survival with an overall adjusted R(2) of 0.3236. They were: age, 4 anthropometric measurements (body mass index, and its squared term, laterality-linearity index, shoulder/pelvis shape), mean blood pressure, father and mother history of premature (<65-year) death, marital status, arm circumference, 2 respiratory measurements (vital capacity and forced expiratory volume), serum cholesterol, corneal arcus, xantelasma, cancer, cardiovascular diseases, diabetes and chronic bronchitis. Coefficients of 5 suitable risk factors became definitely larger after adjustment for regression dilution bias with 5year data. All 40 cases of survival were located in the higher 5 deciles of estimated survival and 25 (62.5\%) were in the upper decile. CONCLUSION: A small number of risk factors and personal characteristics, mainly known as cardiovascular risk factors and measured once in middle-aged men, are strongly associated with the length of survival in a 50-year follow-up

Lifestyle habits and mortality from all and specific causes of death: 40-year follow-up in the italian rural areas of the seven countries study.

Objectives: Three lifestyle factors were investigated in a population study to explore their relationships with a long-term mortality. Material and Methods: In a cohort of 1564 men aged 45-64 and examined in 1965 within the Italian Rural Areas of the Seven Countries Study, smoking habits, physical activity at work and eating habits (as derived from factor analysis) were determined. During the follow-up 693 men died in 20 years and 1441 in 40 years. Results: In Cox proportional hazards models men smoking cigarettes (versus never smokers), those having a sedentary activity (versus the very active) and those following the Diet Score 1, indexing an unhealthy Diet (versus men with a Diet close to the healthy Mediterranean style) had highly significant hazards ratios (HR) in relations with 20- and 40-year mortality from all causes, coronary heart disease (CHD), cardiovascular disease (CVD) and cancer. HR for all causes in 40 years were 1.44 (95\% confidence intervals, CI, 1.27 and 1.64) for smokers, 1.43 (CI 1.23 and 1.67) for sedentary people, and 1.31 (CI 1.15 and 1.50) for men with unhealthy diet. Larger HR were found for CHD, CVD and cancers deaths. Combination of 3 unhealthy risk factors versus their absence was associated with 4.8-year life loss in the 20-year follow-up and 10.7-year in the 40-year follow-up. Conclusions: Lifestyle behavior linked to physical activity and smoking and eating habits is strongly associated with mortality and survival in middle aged men during long-term follow-up

Baseline fatty acids, food groups, a diet score and 50-year all-cause mortality rates. an ecological analysis of the seven countries study

Objectives: This analysis deals with the ecologic relationships of dietary fatty acids, food groups and the Mediterranean Adequacy Index (MAI, derived from 15 food groups) with 50-year all-cause mortality rates in 16 cohorts of the Seven Countries Study. Material and methods: A dietary survey was conducted at baseline in cohorts subsamples including chemical analysis of food samples representing average consumptions. Ecologic correlations of dietary variables were computed across cohorts with 50-year all-cause mortality rates, where 97% of men had died. Results: There was a 12-year average age at death population difference between extreme cohorts. In the 1960s the average population intake of saturated (S) and trans (T) fatty acids and hard fats was high in the northern European cohorts while monounsaturated (M), polyunsaturated (P) fatty acids and vegetable oils were high in the Mediterranean areas and total fat was low in Japan. The 50-year all-cause mortality rates correlated (r= -0.51 to -0.64) ecologically inversely with the ratios M/S, (M + P)/(S + T) and vegetable foods and the ratio hard fats/vegetable oils. Adjustment for high socio-economic status strengthened (r= -0.62 to -0.77) these associations including MAI diet score. Conclusion: The protective fatty acids and vegetable oils are indicators of the low risk traditional Mediterranean style diets. KEY MESSAGES We aimed at studying the ecologic relationships of dietary fatty acids, food groups and the Mediterranean Adequacy Index (MAI, derived from 15 food groups) with 50-year all-cause mortality rates in the Seven Countries Study. The 50-year all-cause mortality rates correlated (r = -0.51 to -0.64) ecologically inversely with the ratios M/S [monounsaturated (M) + polyunsaturated (P)]/[saturated (S) + trans (T)] fatty acids and vegetable foods and the ratio hard fats/vegetable oils. After adjustment for high socio-economic status, associations with the ratios strengthened (r = -0.62 to -0.77) including also the MAI diet score. The protective fatty acids and vegetable oils are indicators of the low risk traditional Mediterranean style diets