Asynchronous mixing of kidney progenitor cells potentiates nephrogenesis in organoids.

A fundamental challenge in emulating kidney tissue formation through directed differentiation of human pluripotent stem cells is that kidney development is iterative, and to reproduce the asynchronous mix of differentiation states found in the fetal kidney we combined cells differentiated at different times in the same organoid. Asynchronous mixing promoted nephrogenesis, and heterochronic organoids were well vascularized when engrafted under the kidney capsule. Micro-CT and injection of a circulating vascular marker demonstrated that engrafted kidney tissue was connected to the systemic circulation by 2 weeks after engraftment. Proximal tubule glucose uptake was confirmed, but despite these promising measures of graft function, overgrowth of stromal cells prevented long-term study. We propose that this is a technical feature of the engraftment procedure rather than a specific shortcoming of the directed differentiation because kidney organoids derived from primary cells and whole embryonic kidneys develop similar stromal overgrowth when engrafted under the kidney capsule.

Myc cooperates with ß-catenin to drive gene expression in nephron progenitor cells.

For organs to achieve their proper size, the processes of stem cell renewal and differentiation must be tightly regulated. We previously showed that in the developing kidney, Wnt9b regulates distinct ß-catenin-dependent transcriptional programs in the renewing and differentiating populations of the nephron progenitor cells. How ß-catenin stimulated these two distinct programs was unclear. Here, we show that ß-catenin cooperates with the transcription factor Myc to activate the progenitor renewal program. Although in multiple contexts Myc is a target of ß-catenin, our characterization of a cell type-specific enhancer for the Wnt9b/ß-catenin target gene Fam19a5 shows that Myc and ß-catenin cooperate to activate gene expression controlled by this element. This appears to be a more general phenomenon as we find that Myc is required for the expression of every Wnt9b/ß-catenin progenitor renewal target assessed as well as for proper nephron endowment in vivo This study suggests that, within the developing kidney, tissue-specific ß-catenin activity is regulated by cooperation with cell type-specific transcription factors. This finding not only provides insight into the regulation of ß-catenin target genes in the developing kidney, but will also advance our understanding of progenitor cell renewal in other cell types/organ systems in which Myc and ß-catenin are co-expressed.

MYC activation cooperates with Vhl and Ink4a/Arf loss to induce clear cell renal cell carcinoma.

Renal carcinoma is a common and aggressive malignancy whose histopathogenesis is incompletely understood and that is largely resistant to cytotoxic chemotherapy. We present two mouse models of kidney cancer that recapitulate the genomic alterations found in human papillary (pRCC) and clear cell RCC (ccRCC), the most common RCC subtypes. MYC activation results in highly penetrant pRCC tumours (MYC), while MYC activation, when combined with Vhl and Cdkn2a (Ink4a/Arf) deletion (VIM), produce kidney tumours that approximate human ccRCC. RNAseq of the mouse tumours demonstrate that MYC tumours resemble Type 2 pRCC, which are known to harbour MYC activation. Furthermore, VIM tumours more closely simulate human ccRCC. Based on their high penetrance, short latency, and histologic fidelity, these models of papillary and clear cell RCC should be significant contributions to the field of kidney cancer research.

A Cre-inducible fluorescent reporter for observing apical membrane dynamics.

The ability to image living tissues with fluorescent proteins has revolutionized the fields of cell and developmental biology. Fusions between fluorescent proteins and various polypeptides are allowing scientists to image tissues with sub-cellular resolution. Here, we describe the generation and activity of a genetically engineered mouse line expressing a fusion between the green fluorescent protein (GFP) and the apically localized protein Crumbs3 (Crb3). This reporter drives Cre-inducible expression of Crb3-GFP under control of the EF1a regulatory domains. The fusion protein is broadly expressed in embryonic and adult tissues and shows apical restriction in the majority of epithelial cell types. It displays a variably penetrant gain of function activity in the neural tube. However, in several cell types, over-expression of Crb3 does not appear to have any effect on normal development or maintenance. Detailed analysis of kidneys expressing this reporter indicates normal morphology and function highlighting the utility for live imaging. Thus, the EF1a(Crb3-GFP) mouse line will be of broad use for studying membrane and/or tissue dynamics in living tissues.

Detoxifying PCDD/Fs and heavy metals in fly ash from medical waste incinerators with a DC double are plasma torch.

Medical waste incinerator (MWI) fly ash is regarded as a highly toxic waste because it contains high concentrations of heavy metals and dioxins, including polychlorinated dibenzo-p-dioxins (PCDDs) and polychlorinated dibenzofurans (PCDFs). Therefore fly ash from MWI must be appropriately treated before being discharged into the environment. A melting process based on a direct current thermal plasma torch has been developed to convert MWI fly ash into harmless slag. The leaching characteristics of heavy metals in fly ash and vitrified slag were investigated using the toxicity characteristic leaching procedure, while the content of PCDD/Fs in the fly ashes and slags was measured using method 1613 of the US EPA. The experimental results show that the decomposition rate of PCDD/Fs is over 99% in toxic equivalent quantity value and the leaching of heavy metals in the slag significantly decreases after the plasma melting process. The produced slag has a compact and homogeneous microstructure with density of up to 2.8 g/cm3.

Regeneration of activated carbon saturated with odors by non-thermal plasma.

The dielectric barrier discharge (DBD) regeneration process of an activated carbon (AC) saturated with dimethyl sulfide was studied on a laboratory scale. The results showed sustainable high regeneration efficiency (RE) (>90%) in successive regeneration cycles (10 cycles). Energy density, humidity and oxygen content were key factors for DBD system, with optimum conditions of 761JL(-1), 0-1vol% and 5%, respectively. The high efficiency was likely attributed to the improvement of structure and surface properties of AC by DBD. After the first regeneration, surface area, micropore volume and total pore volume of AC increased by 8%, 23% and 15% respectively, while average pore size decreased by 9.5%. The number of carboxylic groups doubled (from 0.22 to 0.48mmolg(-1)) while that of phenolic groups remarkably decreased (from 0.48 to 0.26mmolg(-1)) after successive regeneration cycles, which helped to maintain high RE. The results suggested DBD as a novel, efficient alternative process for odor-saturated AC regeneration.

The development of highly potent inhibitors for porcupine.

Porcupine is a member of the membrane-bound O-acyltransferase family of proteins. It catalyzes the palmitoylation of Wnt proteins, a process required for their secretion and activity. We recently disclosed a class of small molecules (IWPs) as the first reported Porcn inhibitors. We now describe the structure-activity relationship studies and the identification of subnanomolar inhibitors. We also report herein the effects of IWPs on Wnt-dependent developmental processes, including zebrafish posterior axis formation and kidney tubule formation.

Generation and characterization of KsprtTA and KsptTA transgenic mice.

The advent of technologies that allow tissue specific expression or ablation of genes has contributed enormously to our knowledge of the mechanism regulating organ development and maintenance in mice. The tetracycline inducible system allows reversible regulation of gene products upon administration of doxycycline. Here we describe the generation and activity of two transgenic lines expressing the cDNAs for the Tet responsive transcription factors rtTA and tTA (Tet-on and off) respectively under the control of an element that drives expression in the epithelium of the developing and adult kidney. Both lines show inducible and reversible activity in the embryonic and adult organ.

AMP-activated protein kinase is required for induction of apoptosis and epithelial-to-mesenchymal transition.

AMP-activated protein kinase (AMPK) is a serine/threonine protein kinase which has been implicated in the regulation of cellular energy homeostasis. Relatively very little is known about its role in other cellular processes. We observed that AMPK-alpha can be activated by transforming growth factor-beta1 (TGF-beta1) in mouse hepatocytes. Inhibition of AMPK by Compound C, a selective AMPK-alpha inhibitor, inhibited TGF-beta1-induced apoptosis and EMT in hepatocytes. In addition, overexpression of a dominant-negative form of AMPK-alpha subunit also suppressed TGF-beta1-induced EMT and apoptosis in AML12 cells. Furthermore, inhibition of AMPK suppressed TGF-beta1-induced Smad3 transcriptional activity. This study indicates that AMPK is able to modulate Smad3 transcriptional activity, which plays an important role in TGF-beta1-induced apoptosis and EMT.

Histone deacetylase 1 is required for transforming growth factor-beta1-induced epithelial-mesenchymal transition.

UNLABELLED: Epithelial-mesenchymal transition (EMT) has been implicated in embryonic development, fibrosis, and tumor metastasis. Histone deacetylases (HDACs) also play important roles in the control of various physiological and pathological events. However, whether HDACs are involved in the control of EMT in liver cells remains unidentified. Three structurally unrelated HDAC inhibitors completely suppress transforming growth factor-beta1 (TGF-beta1)-induced EMT in AML-12 murine hepatocytes and primary mouse hepatocytes. Expression of a dominant-negative mutant of HDAC1 but not HDAC2 or downregulation of HDAC1 but not HDAC2 by RNAi suppressed TGF-beta1-induced EMT. In addition, both HDAC inhibitor TSA and HDAC1 RNAi blocked cell migration. Overexpression of HDAC1 in invasive hepatocellular carcinoma (HCC) samples was detected. Further study showed that the mRNA levels of ZO-1 and E-cadherin were downregulated during TGF-beta1-induced EMT, and HDAC1 can downregulate the promoter activities of ZO-1 and E-cadherin. CONCLUSIONS: our results demonstrate that HDAC1 is required for TGF-beta1-induced EMT and cell migration in hepatocytes. Its high expression levels in majority of invasive HCC samples suggest that, by promoting EMT, HDAC1 can be related with the invasiveness of HCC. The data also suggest that the repression of transcription of ZO-1 and E-cadherin by HDAC1 may be involved in TGF-beta1-induced EMT.

Nitric oxide suppresses transforming growth factor-beta1-induced epithelial-to-mesenchymal transition and apoptosis in mouse hepatocytes.

UNLABELLED: Nitric oxide (NO) is a multifunctional regulator that is implicated in various physiological and pathological processes. Here we report that administration of NO donor S-nitroso-N-acetylpenicillamine (SNAP) inhibited transforming growth factor-beta1 (TGF-beta1)-induced epithelial-to-mesenchymal transition (EMT) and apoptosis in mouse hepatocytes. Overexpression of inducible NO synthase (iNOS) by transfection of the iNOS-expressing vector, which increased NO production, also inhibited the TGF-beta1-induced EMT and apoptosis in these cells. Treatment of cells with proinflammatory mediators, including tumor necrosis factor (TNF)-alpha, interleukin (IL)-1beta, and interferon (IFN)-gamma, which increased the endogenous NO production, produced the same inhibitory effect. Furthermore, exogenous NO donor SNAP treatment caused a decrease in the intracellular adenosine triphosphate (ATP) levels. Consistently, depletion of intracellular ATP by mitochondrial uncoupler carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP) inhibited the TGF-beta1-induced EMT and apoptosis, suggesting that an NO-induced decrease of ATP involved in the NO-mediated inhibition of TGF-beta1-induced EMT and apoptosis. NO and FCCP also inhibited TGF-beta1-induced STAT3 activation, suggesting that signal transducer and activator of transcription 3 inactivation is involved in the NO-induced effects on TGF-beta1-induced EMT and apoptosis. CONCLUSION: Our study indicates that NO plays an important role in the inhibition of TGF-beta1-induced EMT and apoptosis in mouse hepatocytes through the downregulation of intracellular ATP levels. The data provide an insight into the in vivo mechanisms on the function of NO during the processes of both EMT and apoptosis.

Ferritin heavy chain-mediated iron homeostasis and subsequent increased reactive oxygen species production are essential for epithelial-mesenchymal transition.

The epithelial-mesenchymal transition (EMT) plays a critical role in tumor progression. To obtain a broad view of the molecules involved in EMT, we carried out a comparative proteomic analysis of transforming growth factor-beta1 (TGF-beta1)-induced EMT in AML-12 murine hepatocytes. A total of 36 proteins with significant alterations in abundance were identified. Among these proteins, ferritin heavy chain (FHC), a cellular iron storage protein, was characterized as a novel modulator in TGF-beta1-induced EMT. In response to TGF-beta1, there was a dramatic decrease in the FHC levels, which caused iron release from FHC and, therefore, increased the intracellular labile iron pool (LIP). Abolishing the increase in LIP blocked TGF-beta1-induced EMT. In addition, increased LIP levels promoted the production of reactive oxygen species (ROS), which in turn activated p38 mitogen-activated protein kinase. The elimination of ROS inhibited EMT, whereas H2O2 treatment rescued TGF-beta1-induced EMT in cells in which the LIP increase was abrogated. Overexpression of exogenous FHC attenuated the increases in LIP and ROS production, leading to a suppression of EMT. We also showed that TGF-beta1-mediated down-regulation of FHC occurs via 3' untranslated region-dependent repression of the translation of FHC mRNA. Moreover, we found that FHC down-regulation is an event that occurs between the early and highly invasive advanced stages in esophageal adenocarcinoma and that depletion of LIP or ROS suppresses the migration of tumor cells. Our data show that cellular iron homeostasis regulated by FHC plays a critical role in TGF-beta1-induced EMT.

[Study on vitrification of fly ashes from municipal wastes incinerator with a plasma torch].

TCLP analysis (USEPA method 1311) was employed on fly ash in order to analyze the metals leachability and the concentration of cadmium was 0.3225 mg/L which exceeded state TCLP standard(0.3 mg/L). According to USEPA method 1613, I-TEQ of PCDD/Fs in fly ash was 0.45 ng/g. Then a double arcs DC plasma torch was developed to vitrified fly ash. And the results showed that heavy metals were mostly immobilized in the vitrified slag and also I-TEQ of PCDD/Fs in fly ash was destroyed near 91.6%. The morphology of vitrified slag was amorphous state which showed the glassy slag of SiO2 and the microstructure of slag was very compact.

[Temperature measurement of DC argon plasma jet].

The electron temperature of DC arc plasma jet is an important parameter, which determines the characteristics of plasma jet. The measurement of emission spectrum was performed to obtain the spectral intensities of some Ar lines and the method of diagrammatic view of Boltzmann was adopted to calculate the electron temperature. The results indicated that the electron temperature dropped at different speed along with the axes of the plasma jet and rose rapidly when the current was increased, and it also rose when the flowrate of argon was increased.

Regulation of transforming growth factor-beta 1-induced apoptosis and epithelial-to-mesenchymal transition by protein kinase A and signal transducers and activators of transcription 3.

Apoptosis and epithelial-to-mesenchymal transdifferentiation or transition (EMT) are crucial for normal development and body homeostasis. The alterations of these events are closely related to some pathologic processes, such as tumor formation and metastasis, fibrotic diseases of liver and kidney, and abnormal development of embryos. The mechanism that underlies the simultaneously occurring apoptosis and EMT induced by transforming growth factor-beta (TGF-beta) has not been well studied. In this report, we investigated the potential mechanism that underlies TGF-beta1-induced apoptosis and EMT. TGF-beta1-induced apoptosis and EMT were associated with the activation of protein kinase A (PKA) and signal transducers and activators of transcription 3 (STAT3). Inhibition of PKA by specific PKA inhibitor H89 or by PKA inhibitor peptide blocked STAT3 activation and suppressed TGF-beta1-induced apoptosis and EMT. Furthermore, overexpression of a phosphorylation-deficient form of STAT3, but not wild-type STAT3, produced an inhibitory effect on TGF-beta1-induced apoptosis and EMT. The results indicate that PKA is an upstream regulator for TGF-beta1-induced STAT3 activation and plays an important role in TGF-beta1-mediated apoptosis and EMT. These studies provided a new insight into the signaling mechanism underlying the apoptosis and EMT, which could be of importance in understanding some related physiologic and pathologic processes.

[Spectroscopic diagnostics of DC argon plasma at atmospheric pressure].

The optical emission spectra of DC argon plasma at atmospheric pressure were measured inside and outside the arc chamber. The electron temperature was determined from the Boltzmann plot, and the electron density was derived from Stark broadening of Ar I lines. The criteria for the existence of local thermodynamic equilibrium (LTE)in the plasma was discussed. The results indicate that the DC argon plasma at atmospheric pressure under our experimental conditions is in LTE.

[Measurement of rotational and vibrational temperatures in arc plasma based on the first negative system of N2+ (B(2) sigma --> X(2) sigma)].

The molecular emission spectra lines of the first negative system N2+ (B(2) sigma--> X(2) sigma ) are frequently observed in the plasma source containing nitrogen. (0-0) and (1--1) N2+ first negative system molecular bands around 391. 4 nm can be used to the measure the rotational and vibrational temperatures in a DC argon-nitrogen plasma at atmospheric pressure. The proposed method based on the comparison between this experimental emission spectrum and the computer simulated one is presented. The effect of the apparatus function, vibrational temperature and rotational temperatures on the line structure of numerical simulated spectrum is discussed. The results show that the electron temperature, rotational temperature, vibrational temperature and kinetic temperature of plasma arc are almost the same, which can be interpreted as that DC argon-nitrogen arc plasma at atmospheric pressure is in LTE under their experimental conditions.