A novel, low-volume method for organ culture of embryonic kidneys that allows development of cortico-medullary anatomical organization

Here, we present a novel method for culturing kidneys in low volumes of medium that offers more organotypic development compared to conventional methods. Organ culture is a powerful technique for studying renal development. It recapitulates many aspects of early development very well, but the established techniques have some disadvantages: in particular, they require relatively large volumes (1-3 mls) of culture medium, which can make high-throughput screens expensive, they require porous (filter) substrates which are difficult to modify chemically, and the organs produced do not achieve good cortico-medullary zonation. Here, we present a technique of growing kidney rudiments in very low volumes of medium-around 85 microliters-using silicone chambers. In this system, kidneys grow directly on glass, grow larger than in conventional culture and develop a clear anatomical cortico-medullary zonation with extended loops of Henle. © 2010 Sebinger et al

A role for intracellular calcium downstream of G-protein signaling in undifferentiated human embryonic stem cell culture

AbstractMultiple signalling pathways maintain human embryonic stem cells (hESC) in an undifferentiated state. Here we sought to define the significance of G protein signal transduction in the preservation of this state distinct from other cellular processes. Continuous treatment with drugs targeting Gαs-, Gα-i/o- and Gα-q/11-subunit signalling mediators were assessed in independent hESC lines after 7days to discern effects on normalised alkaline phosphatase positive colony frequency vs total cell content. This identified PLCβ, intracellular free calcium and CAMKII kinase activity downstream of Gα-q/11 as of particular importance to the former. To confirm the significance of this finding we generated an agonist-responsive hESC line transgenic for a Gα-q/11 subunit-coupled receptor and demonstrated that an undifferentiated state could be promoted in the presence of an agonist without exogenously supplied bFGF and that this correlated with elevated intracellular calcium. Similarly, treatment of unmodified hESCs with a range of intracellular free calcium-modulating drugs in biologically defined mTESR culture system lacking exogenous bFGF promoted an hESC phenotype after 1week of continuous culture as defined by co-expression of OCT4 and NANOG. At least one of these drugs, lysophosphatidic acid significantly elevates phosphorylation of calmodulin and STAT3 in this culture system (p<0.05). These findings substantiate a role for G-protein and calcium signalling in undifferentiated hESC culture

The Impact of Gender on Mid-Career Labour Income: The Case of Bulgaria

The impact assessment of education and gender on mid-career labour income in a transitional economy could provide for better understanding of the influence of the labour market dynamics over individuals with different characteristics. Here, we attempt to find an answer to the question: How education and gender determine mid-career labour income? We estimate the returns to education depending on gender using Mincerian equations and regressions. The data set we use is from the Structure of Earnings Survey conducted by the National Statistical Institute in 2002 and 2006. The analysis covers over 130,000 employees between 35 and 49 years old. The impact assessment allows conclusions about the wage gap between men and women, working in different economic sectors incl. the division of public and private sector, services and industry. The access to managerial position and gender differences in the type of the labour contract have been investigated for their contribution to the persistence of a gender pay gap among the individuals with a tertiary education

Identification of tomato accessions as source of new genes for improving heat tolerance: from controlled experiments to field

Background: Due to global warming, the search for new sources for heat tolerance and the identification of genes involved in this process has become an important challenge as of today. The main objective of the current research was to verify whether the heat tolerance determined in controlled greenhouse experiments could be a good predictor of the agronomic performance in field cultivation under climatic high temperature stress. Results: Tomato accessions were grown in greenhouse under three temperature regimes: control (T1), moderate (T2) and extreme heat stress (T3). Reproductive traits (flower and fruit number and fruit set) were used to define heat tolerance. In a first screening, heat tolerance was evaluated in 219 tomato accessions. A total of 51 accessions were identified as being potentially heat tolerant. Among those, 28 accessions, together with 10 accessions from Italy (7) and Bulgaria (3), selected for their heat tolerance in the field in parallel experiments, were re-evaluated at three temperature treatments. Sixteen tomato accessions showed a significant heat tolerance at T3, including five wild species, two traditional cultivars and four commercial varieties, one accession from Bulgaria and four from Italy. The 15 most promising accessions for heat tolerance were assayed in field trials in Italy and Bulgaria, confirming the good performance of most of them at high temperatures. Finally, a differential gene expression analysis in pre-anthesis (ovary) and post-anthesis (developing fruit) under heat stress among pairs of contrasting genotypes (tolerant and sensitive from traditional and modern groups) showed that the major differential responses were produced in post-anthesis fruit. The response of the sensitive genotypes included the induction of HSP genes, whereas the tolerant genotype response included the induction of genes involved in the regulation of hormones or enzymes such as abscisic acid and transferases. Conclusions: The high temperature tolerance of fifteen tomato accessions observed in controlled greenhouse experiments were confirmed in agronomic field experiments providing new sources of heat tolerance that could be incorporated into breeding programs. A DEG analysis showed the complex response of tomato to heat and deciphered the different mechanisms activated in sensitive and tolerant tomato accessions under heat stress

Identification of tomato accessions as source of new genes for improving heat tolerance: from controlled experiments to field

Background: Due to global warming, the search for new sources for heat tolerance and the identification of genes involved in this process has become an important challenge as of today. The main objective of the current research was to verify whether the heat tolerance determined in controlled greenhouse experiments could be a good predictor of the agronomic performance in field cultivation under climatic high temperature stress. Results: Tomato accessions were grown in greenhouse under three temperature regimes: control (T1), moderate (T2) and extreme heat stress (T3). Reproductive traits (flower and fruit number and fruit set) were used to define heat tolerance. In a first screening, heat tolerance was evaluated in 219 tomato accessions. A total of 51 accessions were identified as being potentially heat tolerant. Among those, 28 accessions, together with 10 accessions from Italy (7) and Bulgaria (3), selected for their heat tolerance in the field in parallel experiments, were re-evaluated at three temperature treatments. Sixteen tomato accessions showed a significant heat tolerance at T3, including five wild species, two traditional cultivars and four commercial varieties, one accession from Bulgaria and four from Italy. The 15 most promising accessions for heat tolerance were assayed in field trials in Italy and Bulgaria, confirming the good performance of most of them at high temperatures. Finally, a differential gene expression analysis in pre-anthesis (ovary) and post-anthesis (developing fruit) under heat stress among pairs of contrasting genotypes (tolerant and sensitive from traditional and modern groups) showed that the major differential responses were produced in post-anthesis fruit. The response of the sensitive genotypes included the induction of HSP genes, whereas the tolerant genotype response included the induction of genes involved in the regulation of hormones or enzymes such as abscisic acid and transferases. Conclusions: The high temperature tolerance of fifteen tomato accessions observed in controlled greenhouse experiments were confirmed in agronomic field experiments providing new sources of heat tolerance that could be incorporated into breeding programs. A DEG analysis showed the complex response of tomato to heat and deciphered the different mechanisms activated in sensitive and tolerant tomato accessions under heat stress

Electron cryo-tomography reveals the subcellular architecture of growing axons in human brain organoids.

Funder: Natural Sciences and Engineering Research Council of Canada; FundRef: http://dx.doi.org/10.13039/501100000038During brain development, axons must extend over great distances in a relatively short amount of time. How the subcellular architecture of the growing axon sustains the requirements for such rapid build-up of cellular constituents has remained elusive. Human axons have been particularly poorly accessible to imaging at high resolution in a near-native context. Here, we present a method that combines cryo-correlative light microscopy and electron tomography with human cerebral organoid technology to visualize growing axon tracts. Our data reveal a wealth of structural details on the arrangement of macromolecules, cytoskeletal components, and organelles in elongating axon shafts. In particular, the intricate shape of the endoplasmic reticulum is consistent with its role in fulfilling the high demand for lipid biosynthesis to support growth. Furthermore, the scarcity of ribosomes within the growing shaft suggests limited translational competence during expansion of this compartment. These findings establish our approach as a powerful resource for investigating the ultrastructure of defined neuronal compartments

A Phase 3 Trial of Luspatercept in Patients with Transfusion-Dependent β-Thalassemia

BACKGROUND: Patients with transfusion-dependent β-thalassemia need regular red-cell transfusions. Luspatercept, a recombinant fusion protein that binds to select transforming growth factor β superfamily ligands, may enhance erythroid maturation and reduce the transfusion burden (the total number of red-cell units transfused) in such patients. METHODS: In this randomized, double-blind, phase 3 trial, we assigned, in a 2:1 ratio, adults with transfusion-dependent β-thalassemia to receive best supportive care plus luspatercept (at a dose of 1.00 to 1.25 mg per kilogram of body weight) or placebo for at least 48 weeks. The primary end point was the percentage of patients who had a reduction in the transfusion burden of at least 33% from baseline during weeks 13 through 24 plus a reduction of at least 2 red-cell units over this 12-week interval. Other efficacy end points included reductions in the transfusion burden during any 12-week interval and results of iron studies. RESULTS: A total of 224 patients were assigned to the luspatercept group and 112 to the placebo group. Luspatercept or placebo was administered for a median of approximately 64 weeks in both groups. The percentage of patients who had a reduction in the transfusion burden of at least 33% from baseline during weeks 13 through 24 plus a reduction of at least 2 red-cell units over this 12-week interval was significantly greater in the luspatercept group than in the placebo group (21.4% vs. 4.5%, P<0.001). During any 12-week interval, the percentage of patients who had a reduction in transfusion burden of at least 33% was greater in the luspatercept group than in the placebo group (70.5% vs. 29.5%), as was the percentage of those who had a reduction of at least 50% (40.2% vs. 6.3%). The least-squares mean difference between the groups in serum ferritin levels at week 48 was -348 μg per liter (95% confidence interval, -517 to -179) in favor of luspatercept. Adverse events of transient bone pain, arthralgia, dizziness, hypertension, and hyperuricemia were more common with luspatercept than placebo. CONCLUSIONS: The percentage of patients with transfusion-dependent β-thalassemia who had a reduction in transfusion burden was significantly greater in the luspatercept group than in the placebo group, and few adverse events led to the discontinuation of treatment. (Funded by Celgene and Acceleron Pharma; BELIEVE ClinicalTrials.gov number, NCT02604433; EudraCT number, 2015-003224-31.)

End-group ionisation enables the use of poly(N-(2-methacryloyloxy)ethyl pyrrolidone) as an electrosteric stabiliser block for polymerisation-induced self-assembly in aqueous media

A series of near-monodisperse poly(N-2-(methacryloyloxy)ethyl pyrrolidone) (PNMEP) homopolymers was prepared via reversible addition-fragmentation chain transfer (RAFT) solution polymerisation of NMEP in ethanol at 70 °C using a carboxylic acid-functional RAFT agent. The mean degree of polymerisation (DP) was varied from 19 to 89 and acid titration indicated end-group pK a values of 5.07-5.44. Turbidimetry studies indicated that homopolymer cloud points were significantly higher at pH 7 (anionic carboxylate) than at pH 3 (neutral carboxylic acid). Moreover, this enhanced hydrophilic character enabled PNMEP to be used as a steric stabiliser for aqueous polymerisation-induced self-assembly (PISA) syntheses. Thus, a PNMEP 42 precursor was chain-extended via RAFT aqueous dispersion polymerisation of 2-hydroxypropyl methacrylate (HPMA) at 44 °C. A series of PNMEP 42 -PHPMA x diblock copolymers were synthesised using this protocol, with target PHPMA DPs of 150 to 400. High conversions were achieved and a linear evolution in M n with increasing PHPMA DP was observed. Dynamic light scattering (DLS) and transmission electron microscopy (TEM) studies confirmed a spherical morphology in all cases. The nanoparticles flocculated either below pH 4.5 (owing to protonation) or on addition of 60 mM KCl (as a result of charge screening). Thus the anionic end-groups on the PNMEP stabiliser chains make an important contribution to the overall colloidal stability. Similarly, a PNMEP 53 macro-CTA was chain-extended via RAFT aqueous emulsion polymerisation of 2-ethoxyethyl methacrylate (EEMA) at 44 °C. Again, a neutral solution pH was critical for the synthesis of colloidally stable nanoparticles. High conversions were achieved as the target PEEMA DP was varied between 100 and 600 and a linear evolution in molecular weight with PEEMA DP was confirmed by chloroform GPC studies. DLS experiments indicated a monotonic increase in nanoparticle diameter with PEEMA DP and TEM studies confirmed a spherical morphology in each case. In summary, PNMEP can be used as a water-soluble steric stabiliser for aqueous PISA syntheses provided that it contains an anionic carboxylate end-group to enhance its hydrophilic character