Pluripotent embryonic stem cells (ESCs) maintain self-renewal while ensuring a rapid response to differentiation cues. The identification of genes maintaining ESC identity is important to develop ...these cells for their potential therapeutic use. Here we report a genome-scale RNAi screen for a global survey of genes affecting ESC identity via alteration of Oct4 expression. Factors with the strongest effect on Oct4 expression included components of the Paf1 complex, a protein complex associated with RNA polymerase II. Using a combination of proteomics, expression profiling, and chromatin immunoprecipitation, we demonstrate that the Paf1C binds to promoters of key pluripotency genes, where it is required to maintain a transcriptionally active chromatin structure. The Paf1C is developmentally regulated and blocks ESC differentiation upon overexpression, and the knockdown in ESCs causes expression changes similar to Oct4 or Nanog depletions. We propose that the Paf1C plays an important role in maintaining ESC identity.
Generating human podocytes in vitro could offer a unique opportunity to study human diseases. Here, we describe a simple and efficient protocol for obtaining functional podocytes in vitro from human ...induced pluripotent stem cells. Cells were exposed to a three-step protocol, which induced their differentiation into intermediate mesoderm, then into nephron progenitors and, finally, into mature podocytes. After differentiation, cells expressed the main podocyte markers, such as synaptopodin, WT1, α-Actinin-4, P-cadherin and nephrin at the protein and mRNA level, and showed the low proliferation rate typical of mature podocytes. Exposure to Angiotensin II significantly decreased the expression of podocyte genes and cells underwent cytoskeleton rearrangement. Cells were able to internalize albumin and self-assembled into chimeric 3D structures in combination with dissociated embryonic mouse kidney cells. Overall, these findings demonstrate the establishment of a robust protocol that, mimicking developmental stages, makes it possible to derive functional podocytes in vitro.
•Human iPSC differentiation into podocytes recapitulates kidney developmental stages.•The differentiation protocol is reproducible and highly efficient.•The generated podocytes reflect primary cell behaviour and are functional.
The human protease family HtrA is responsible for preventing protein misfolding and mislocalization, and a key player in several cellular processes. Among these, HtrA1 is implicated in several ...cancers, cerebrovascular disease and age-related macular degeneration. Currently, HtrA1 activation is not fully characterized and relevant for drug-targeting this protease. Our work provides a mechanistic step-by-step description of HtrA1 activation and regulation. We report that the HtrA1 trimer is regulated by an allosteric mechanism by which monomers relay the activation signal to each other, in a PDZ-domain independent fashion. Notably, we show that inhibitor binding is precluded if HtrA1 monomers cannot communicate with each other. Our study establishes how HtrA1 trimerization plays a fundamental role in proteolytic activity. Moreover, it offers a structural explanation for HtrA1-defective pathologies as well as mechanistic insights into the degradation of complex extracellular fibrils such as tubulin, amyloid beta and tau that belong to the repertoire of HtrA1.
Seawater is rich in minerals which may help confer good palatability to foods, favouring the use of smaller amounts of salt, a recognized measure of cardiovascular prevention. The aim of this study ...was to investigate the nutritional properties, sensory characteristics and metabolic effects of a typical Neapolitan pizza prepared with seawater (SWP) in place of common salt, in comparison with Standard traditional Pizza (StP). The nutritional characteristics and the chemical profile of the SWP and StP were assessed by chemical analyses and the use of Food Composition Tables. Twelve healthy volunteers were recruited for a Randomized Controlled Trial, with the consumption of one StP and one SWP using a balanced crossover design. The satiating power and palatability of the two pizzas were tested by the administration of Visual Analogue Scales. Serum glucose, insulin and sodium were measured every 30 min and 3 h urines were collected after each meal. SWP contained nearly 50% less NaCl and a larger amount of micronutrients compared with StP. No significant differences were detected between the two pizzas with regard to satiating power, pleasantness and glycemic and insulinemic response. However, a significant difference was found in the urine volume collected over the 3 h after the two meals (194 mL after StP vs. 292 mL after SWP,
= 0.018) and in the 3 h sodium balance (+1.6 g after StP vs. +0.5 g after SWP,
= 0.002). Conclusions: SWP appears to be a food with favourable nutritional characteristics, very good acceptability and healthy metabolic properties: these results warrant confirmation by a larger intervention trial.
Renal tubular sodium (Na) handling plays a key role in blood pressure (BP) regulation. Several cross-sectional studies reported a positive association between higher proximal tubule fractional ...reabsorption of Na and BP, but no prospective investigation has been reported of this possible association. Hence, the purpose of this study was to estimate the predictive role of renal Na handling on the risk of incident hypertension and the changes in BP occurring in the 8-year follow-up observation of a sample of initially normotensive men (The Olivetti Heart Study). The study included 294 untreated normotensive non-diabetic men with normal renal function examined twice (1994-95 and 2002-04). Renal tubular Na handling was estimated by exogenous lithium clearance. Fractional reabsorption of Na in proximal and distal tubules was calculated and included in the analysis. At baseline, there was no association between BP and either proximal or distal fractional reabsorption of Na. At the end of the 8-year follow-up, direct associations were observed between baseline proximal (but not distal) Na fractional reabsorption and the changes occurred in systolic and diastolic BP over time (+2.79 and +1.53 mmHg, respectively, per 1SD difference in proximal Na-FR; p<0.01). Also multivariable analysis showed a direct association between baseline proximal Na fractional reabsorption and risk of incident hypertension, independently of potential confounders (OR: 1.34, 95%CI:1.06-1.70). The results of this prospective investigation strongly suggest a causal relationship between an enhanced rate of Na reabsorption in the proximal tubule and the risk of incident hypertension in initially normotensive men.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Monitoring the population iodine status is essential for iodine deficiency eradication. This study assessed the average dietary iodine intake and the iodine status of a random sample of the Italian ...general adult population. The study population included 2378 adults aged 35–79 years (1229 men and 1149 women) from all 20 Italian regions, participating in the Osservatorio Epidemiologico Cardiovascolare/Health Examination Survey 2008–2012 (OEC/HES), and were examined for iodine intake in the framework of the MINISAL-GIRCSI Programme. Dietary iodine intake was assessed by the measurement of 24 h urinary iodine excretion. The median daily iodine intake of the whole population was lower (96 µg/d, interquartile range 51–165) than the daily adequate iodine intake according to both EFSA and WHO recommendation (150 µg/d), with a significantly lower value among women (85 µg/d) compared with men (111 µg/d). Iodine intake diminished with age and increased with BMI (body mass index) in male but not in female participants, without achieving the adequate intake in any sex, age, or BMI category. In this random sample of Italian general adult population examined in 2008–2012, iodine intake still remained lower than the recommended values despite the implementation of a strategy of iodoprophylaxis based on salt iodization in 2005. These data represent a valuable reference for future monitoring of iodine status in our country.
Background/Aims: Association between cigarette smoke and albuminuria (UA) was already demonstrated in cross-sectional studies and in selected population samples (i.e diabetic patients). This study ...aims to evaluate, prospectively, the relationship between cigarette smoke and UA in a male adult population sample, with basal normal kidney function, participating in the Olivetti Heart Study (OHS). Methods: Among 994 participants, examined in both 1994-95 and 2002-04, were selected those resulted in both visits smokers (n=221) and non-smokers (n=416) and with basal normal kidney function (GFR> 60 mL/min) and basal albumin/creatinine ratio (ACR< 30 mg/g). Results: At baseline, the prevalence of hypertension was 41%, diabetes affected 6.3% and obesity 17% of the whole sample. Smokers showed statistically significant lower levels of systolic (SBP) and diastolic blood pressure (DBP) and BMI (p< 0.001) compared to non-smokers. There were not basal differences in UA, GFR and metabolic profile. However, at follow-up examination, smokers showed a statistically significant increase in SBP and DBP (p< 0.05), but not in GFR and BMI. Moreover, smokers showed a higher risk compared to non-smokers to be in the higher median levels group of UA (OR: 2.17, C.I.95%: 1.51-3.13; p < 0.001), even after correction for major confounding factors. Further adjustment for basal antihypertensive and hypoglycemic treatment did not change these patterns of association. Conclusion: In a selected male adult population sample, cigarette smoke was independently associated with the development of higher levels of albuminuria over time.
Since the Italian iodoprophylaxis strategy is based on the use of iodized salt, we assessed the relationship between dietary salt consumption and iodine intake in the Italian adult population. We ...estimated the relative contribution given by the use of iodized salt and by the iodine introduced by foods to the total iodine intake. The study population included 2219 adults aged 25–79 years (1138 men and 1081 women) from all Italian regions, participating to the Osservatorio Epidemiologico Cardiovascolare/Health Examination Survey 2008–2012 (OEC/HES), and examined for sodium and iodine intake in the framework of the MINISAL-GIRCSI Programme. Dietary sodium and total iodine intake were assessed by the measurement of 24 h urinary excretion, while the EPIC questionnaire was used to evaluate the iodine intake from food. Sodium and iodine intake were significantly and directly associated, upon accounting for age, sex, and BMI (Spearman rho = 0.298; p < 0.001). The iodine intake increased gradually across quintiles of salt consumption in both men and women (p < 0.001). The European Food Safety Authority (EFSA) adequacy level for iodine intake was met by men, but not women, only in the highest quintile of salt consumption. We estimated that approximately 57% of the iodine intake is derived from food and 43% from salt. Iodized salt contributed 24% of the total salt intake, including both discretionary and non-discretionary salt consumption. In conclusion, in this random sample of the Italian general adult population examined in 2008–2012, the total iodine intake secured by iodized salt and the iodine provision by food was insufficient to meet the EFSA adequate iodine intake.
Direct transdifferentiation of somatic cells is a promising approach to obtain patient-specific cells for numerous applications. However, conversion across germ-layer borders often requires ectopic ...gene expression with unpredictable side effects. Here, we present a gene-free approach that allows efficient conversion of human fibroblasts via a transient progenitor stage into Schwann cells, the major glial cell type of peripheral nerves. Using a multikinase inhibitor, we transdifferentiated fibroblasts into transient neural precursors that were subsequently further differentiated into Schwann cells. The resulting induced Schwann cells (iSCs) expressed numerous Schwann cell-specific proteins and displayed neurosupportive and myelination capacity in vitro. Thus, we established a strategy to obtain mature Schwann cells from human postnatal fibroblasts under chemically defined conditions without the introduction of ectopic genes.
Display omitted
•Direct conversion of human fibroblasts into Schwann cells•No introduction of ectopic genes•Induction of conversion by multikinase inhibitor
Here, Thoma and colleagues developed a strategy to convert human fibroblasts into Schwann cells solely by chemical treatment. Using a multikinase inhibitor, fibroblasts are transdifferentiated into transient neural precursors differentiating into cells with molecular and functional characteristics of Schwann cells. This study shows that cellular conversion can be achieved solely by small molecules and presents an approach to generate human Schwann cells without genetic modifications.
The study compares the essential amino acid (EAA) composition of different parenteral nutrition (PN) mixtures with whey protein EAA profile and the theoretical daily EAA requirements (set by ...WHO/FAO/UNU or IAAO method). According to the individual EAA profile, the potential effect of several PN mixtures was evaluated on the skeletal muscle mass (SMM) of patients on home PN.
Eight AA solutions and fifteen complete PN mixtures were considered. Twenty-nine clinically stable patients with short bowel syndrome on home total PN were retrospectively evaluated. SMM was estimated by bioelectrical impedance analysis.
The prescribed doses of EAA that showed a significant increase in home PN patients muscle mass were considerably greater than the theoretical ones, showing an EAA profile similar to whey protein. At the daily dose of 1 g of total AA s/kg body weight (BW), the considered PN mixtures mostly failed to improve SMM. Only prescribed doses which included more than 0.25 g/kg BW of total BCAA with at least 0.10 g/kg BW leucine, 0.08 g/kg BW isoleucine, and 0.06 g/kg BW methionine showed a significant increase in SMM.
The theoretical daily requirement for each EAA was met by all considered PN solutions when the prescribed daily dose of total AAs was set at 1 g/kg BW. Nevertheless, our data suggest that only an increase in total BCAA, also richer in single AA leucine, isoleucine, and methionine, is associated with the maintenance and/or increase of SMM. According to these preliminary observations, we support the prescription of an EAA composition of PN mixtures close to that of whey protein for the preservation of SMM in patients on long-term total PN.
PDF
