The impact of the non-invasive follicular thyroid neoplasm with papillary-like nuclear feature terminology in the routine diagnosis of thyroid tumours.

BACKGROUND: Due to the recent proposal of the non-invasive follicular thyroid neoplasm with papillary-like nuclear feature (NIFTP) category, the authors analyse the state of the art in the challenging diagnosis of follicular thyroid neoplasms in routine practice. METHODS AND RESULTS: A consecutive series of 200 histological diagnoses, with complete cytological correlation, was analysed following the introduction of the NIFTP definition. The study was conducted in a general hospital with a high prevalence of thyroid benign nodules that accounted for approximately 60% of surgically-treated nodules. The significant incidence of the new NIFTP category was 7%. Concurrently, a gradual decrease of the follicular variant of papillary thyroid carcinoma (fvPTC) was observed (3.5%). When evaluating the FNA biopsies within the NIFTP group, despite the systematic evaluation of nuclear crowding, enlargement, irregularities and clearing, the final cytological class was often indeterminate for malignancy (Thy3/III-IV, 71%). At histology, the application of the semiquantitative NIFTP score for the evaluation of the PTC-like nuclear features was able to discriminate benign lesions (score 0/1) from fvPTC (score 2/3). A certain degree of overlapping still persisted between NIFTP and fvPTC (score 2) or between NIFTP and benign lesions (score 1). CONCLUSIONS: In the routine evaluation of FNA biopsies, the presence of subtle and questionable PTC-like nuclear features still remains a controversial aspect of the diagnostic workflow. Given that the NIFTP category was introduced to stratify the low-risk group of thyroid tumours more precisely, pathologists should force themselves to apply the nuclear score rigorously and to classify cases assigned a score of 1 as benign proliferations.

NANOG priming before full reprogramming may generate germ cell tumours.

Reprogramming somatic cells into a pluripotent state brings patient-tailored, ethical controversy-free cellular therapy closer to reality. However, stem cells and cancer cells share many common characteristics; therefore, it is crucial to be able to discriminate between them. We generated two induced pluripotent stem cell (iPSC) lines, with NANOG pre-transduction followed by OCT3/4, SOX2, and LIN28 overexpression. One of the cell lines, CHiPS W, showed normal pluripotent stem cell characteristics, while the other, CHiPS A, though expressing pluripotency markers, failed to differentiate and gave rise to germ cell-like tumours in vivo. Comparative genomic hybridisation analysis of the generated iPS lines revealed that they were genetically more stable than human embryonic stem cell counterparts. This analysis proved to be predictive for the differentiation potential of analysed cells. Moreover, the CHiPS A line expressed a lower ratio of p53/p21 when compared to CHiPS W. NANOG pre-induction followed by OCT3/4, SOX2, MYC, and KLF4 induction resulted in the same tumour-inducing phenotype. These results underline the importance of a re-examination of the role of NANOG during reprogramming. Moreover, this reprogramming method may provide insights into primordial cell tumour formation and cancer stem cell transformation.

Isolation of rat embryonic stem-like cells: a tool for stem cell research and drug discovery.

The establishment of rat embryonic stem cells constitutes a precious tool since rat has been extensively used in biomedical research, in particular for the generation of human neurodisease animal models. Up to now only a few studies have described the isolation of rat embryonic stem-like cells. One out of 9 isolated rat embryonic stem-like cell lines (B1-RESC) obtained from a 4.5-day post-coitum blastocyst were extensively characterized and kept in culture for up to 80 passages on feeders with LIF. The stable growth of these cells and the expression of pluripotent markers were confirmed up to a high number of passages in culture, also in the absence of feeders and LIF. B1-RESC expresses the three germ layers markers both in vitro, within differentiating embryoid bodies, and in vivo through teratoma formation. Collectively, the B1-RESC line with a stable near-diploid karyotype can be used as a highly sensitive tool for testing anti-proliferative molecules.

Stem cell science: current ethical and policy issues.

There is increasing support for embryonic stem (ES) cell research on both sides of the Atlantic. In the United States, the outcome is more funding from non-federal sources, despite the current administration's opposing views. In Europe, a similar pragmatic turn is in the making, but the future is still uncertain. Acceptance of ES cells is mitigated by the uncritical belief that their use is ethically more suspect than is the case for adult cells.

Embryonic and adult stem cell-derived cardiomyocytes: lessons from in vitro models.

For years, research has focused on how to treat heart failure by sustaining the overloaded remaining cardiomyocytes. Recently, the concept of cell replacement therapy as a treatment of heart diseases has opened a new area of investigation. In vitro-generated cardiomyocytes could be injected into the heart to rescue the function of a damaged myocardium. Embryonic and/or adult stem cells could provide cardiac cells for this purpose. Knowledge of fundamental cardiac differentiation mechanisms unraveled by studies on animal models has been improved using in vitro models of cardiogenesis such as mouse embryonal carcinoma cells, mouse embryonic stem cells and, recently, human embryonic stem cells. On the other hand, studies suggesting the existence of cardiac stem cells and the potential of adult stem cells from bone marrow or skeletal muscle to differentiate toward unexpected phenotypes raise hope and questions about their potential use for cardiac cell therapy. In this review, we compare the specificities of embryonic vs adult stem cell populations regarding their cardiac differentiation potential, and we give an overview of what in vitro models have taught us about cardiogenesis.

Current status of cardiac MRI in small animals.

Cardiac magnetic resonance imaging (MRI) on small animals is possible but remains challenging and not well standardized. This publication aims to provide an overview of the current techniques, applications and challenges of cardiac MRI in small animals for researchers interested in moving into this field. Solutions have been developed to obtain a reliable cardiac trigger in both the rat and the mouse. Techniques to measure ventricular function and mass have been well validated and are used by several research groups. More advanced techniques like perfusion imaging, delayed enhancement or tag imaging are emerging. Regarding cardiac applications, not only coronary ischemic disease but several other pathologies or conditions including cardiopathies in transgenic animals have already benefited from these new developments. Therefore, cardiac MRI has a bright future for research in small animals.

[Embryonic stem cells: new possible therapy of degenerative diseases]. / Embryonale Stammzellen: Neues Therapiepotential für degenerative Erkrankungen.

The capacity of embryonic stem (ES) cells for virtually unlimited self-renewal and differentiation has opened up the prospect of widespread applications in biomedical research and regenerative medicine. The use of these cells would allow to overcome the problems of donor tissue shortage and also implant rejection if the cells are made immunocompatible with the recipient. Since the derivation in 1998 of human ES cell lines from pre-implantation embryos, considerable research is centered on their biology, on how differentiation can be encouraged towards particular cell lineages and also on means to enrich and purify derivative cell types. In addition, ES cells may be used as an in vitro system not only to study cell differentiation but also to evaluate the effects of new drugs and the identification of genes as potential therapeutic targets. This review will summarize what is known about animal and human ES cells with particular emphasis on their application in four animal models of human diseases. Present studies of mouse ES cell transplantation reveal encouraging results but also technical barriers that have to be overcome before clinical trials can be considered.

Directed inhibition of nuclear import in cellular hypertrophy.

Each nuclear pore is responsible for both nuclear import and export with a finite capacity for bidirectional transport across the nuclear envelope. It remains poorly understood how the nuclear transport pathway responds to increased demands for nucleocytoplasmic communication. A case in point is cellular hypertrophy in which increased amounts of genetic material need to be transported from the nucleus to the cytosol. Here, we report an adaptive down-regulation of nuclear import supporting such an increased demand for nuclear export. The induction of cardiac cell hypertrophy by phenylephrine or angiotensin II inhibited the nuclear translocation of H1 histones. The removal of hypertrophic stimuli reversed the hypertrophic phenotype and restored nuclear import. Moreover, the inhibition of nuclear export by leptomycin B rescued import. Hypertrophic reprogramming increased the intracellular GTP/GDP ratio and promoted the nuclear redistribution of the GTP-binding transport factor Ran, favoring export over import. Further, in hypertrophy, the reduced creatine kinase and adenylate kinase activities limited energy delivery to the nuclear pore. The reduction of activities was associated with the closure of the cytoplasmic phase of the nuclear pore preventing import at the translocation step. Thus, to overcome the limited capacity for nucleocytoplasmic transport, cells requiring increased nuclear export regulate the nuclear transport pathway by undergoing a metabolic and structural restriction of nuclear import.

Identification of BARD1 as mediator between proapoptotic stress and p53-dependent apoptosis.

The BRCA1-associated protein BARD1 is a putative tumor suppressor. We suggest that BARD1 is a mediator of apoptosis since (1) cell death in vivo (ischemic stroke) and in vitro is accompanied by increased levels of BARD1 protein and mRNA; (2) overexpression of BARD1 induces cell death with all features of apoptosis; and (3) BARD1-repressed cells are defective for the apoptotic response to genotoxic stress. The proapoptotic activity of BARD1 involves binding to and elevations of p53. BRCA1 is not required for but partially counteracts apoptosis induction by BARD1. A tumor-associated mutation Q564H of BARD1 is defective in apoptosis induction, thus suggesting a role of BARD1 in tumor suppression by mediating the signaling from proapoptotic stress toward induction of apoptosis.

A fluorescent reporter gene as a marker for ventricular specification in ES-derived cardiac cells.

We have established a CGR8 embryonic stem (ES) cell clone (MLC2ECFP) which expresses the enhanced cyan variant of Aequorea victoria green fluorescent protein (ECFP) under the transcriptional control of the ventricular myosin light chain 2 (MLC2v) promoter. Using epifluorescence imaging of vital embryoid bodies (EB) and reverse transcription-polymerase chain reaction (RT-PCR), we found that the MLC2v promoter is switched on as early as day 7 and is accompanied by formation of cell clusters featuring a bright ECFP blue fluorescence. The fluorescent areas within the EBs were all beating on day 8. MLC2ECFP ES cells showed the same time course of cardiac differentiation as mock ES cells as assessed by RT-PCR of genes encoding cardiac-specific transcription factors and contractile proteins. The MLC2v promoter conferred ventricular specificity to ECFP expression within the EB as revealed by MLC2v co-staining of ECFP fluorescent cells. MLC2ECFP-derived cardiac cells still undergo cell division on day 12 after isolation from EBs but withdraw from the cell cycle on day 16. This ES cell clone provides a powerful cell model to study the signalling roads of factors regulating cardiac cell proliferation and terminal differentiation with a view to using them for experimental cell therapy.

Inositol 1,4,5-trisphosphate directs Ca(2+) flow between mitochondria and the Endoplasmic/Sarcoplasmic reticulum: a role in regulating cardiac autonomic Ca(2+) spiking.

The signaling role of the Ca(2+) releaser inositol 1,4, 5-trisphosphate (IP(3)) has been associated with diverse cell functions. Yet, the physiological significance of IP(3) in tissues that feature a ryanodine-sensitive sarcoplasmic reticulum has remained elusive. IP(3) generated by photolysis of caged IP(3) or by purinergic activation of phospholipase Cgamma slowed down or abolished autonomic Ca(2+) spiking in neonatal rat cardiomyocytes. Microinjection of heparin, blocking dominant-negative fusion protein, or anti-phospholipase Cgamma antibody prevented the IP(3)-mediated purinergic effect. IP(3) triggered a ryanodine- and caffeine-insensitive Ca(2+) release restricted to the perinuclear region. In cells loaded with Rhod2 or expressing a mitochondria-targeted cameleon and TMRM to monitor mitochondrial Ca(2+) and potential, IP(3) induced transient Ca(2+) loading and depolarization of the organelles. These mitochondrial changes were associated with Ca(2+) depletion of the sarcoplasmic reticulum and preceded the arrest of cellular Ca(2+) spiking. Thus, IP(3) acting within a restricted cellular region regulates the dynamic of calcium flow between mitochondria and the endoplasmic/sarcoplasmic reticulum. We have thus uncovered a novel role for IP(3) in excitable cells, the regulation of cardiac autonomic activity.

Structural plasticity of the cardiac nuclear pore complex in response to regulators of nuclear import.

Communication between the cytoplasm and nucleoplasm of cardiac cells occurs by molecular transport through nuclear pores. In lower eukaryotes, nuclear transport requires the maintenance of cellular energetics and ion homeostasis. Although heart muscle is particularly sensitive to metabolic stress, the regulation of nuclear transport through nuclear pores in cardiomyocytes has not yet been characterized. With the use of laser confocal and atomic force microscopy, we observed nuclear transport in cardiomyocytes and the structure of individual nuclear pores under different cellular conditions. In response to the depletion of Ca2+ stores or ATP/GTP pools, the cardiac nuclear pore complex adopted 2 distinct conformations that led to different patterns of nuclear import regulation. Depletion of Ca2+ indiscriminately prevented the nuclear import of macromolecules through closure of the nuclear pore opening. Depletion of ATP/GTP only blocked facilitated transport through a simultaneous closure of the pore and relaxation of the entire complex, which allowed other molecules to pass into the nucleus through peripheral routes. The current study of the structural plasticity of the cardiac nuclear pore complex, which was observed in response to changes in cellular conditions, identifies a gating mechanism for molecular translocation across the nuclear envelope of cardiac cells. The cardiac nuclear pore complex serves as a conduit that differentially regulates nuclear transport of macromolecules and provides a mechanism for the control of nucleocytoplasmic communication in cardiac cells, in particular under stress conditions associated with disturbances in cellular bioenergetics and Ca2+ homeostasis.

A spliced variant of AE1 gene encodes a truncated form of Band 3 in heart: the predominant anion exchanger in ventricular myocytes.

The anion exchangers (AE) are encoded by a multigenic family that comprises at least three genes, AE1, AE2 and AE3, and numerous splicoforms. Besides regulating intracellular pH (pHi) via the Cl-/HCO3- exchange, the AEs exert various cellular functions including generation of a senescent antigen, anchorage of the cytoskeleton to the membrane and regulation of metabolism. Most cells express several AE isoforms. Despite the key role of this family of proteins, little is known about the function of specific AE isoforms in any tissue, including the heart. We therefore chose isolated cardiac cells, in which a tight control of pHi is mandatory for the excitation-contraction coupling process, to thoroughly investigate the expression of the AE genes at both the mRNA and protein levels. RT-PCR revealed the presence of AE1, AE2 and AE3 mRNAs in both neonatal and adult rat cardiomyocytes. AE1 is expressed both as the erythroid form (Band 3 or eAE1) and a novel alternate transcript (nAE1), which was more specifically characterized using a PCR mapping strategy. Two variants of AE2 (AE2a and AE2c) were found at the mRNA level. Cardiac as well as brain AE3 mRNAs were expressed in both neonatal and adult rat cardiomyocytes. Several AE protein isoforms were found, including a truncated form of AE1 and two AE3s, but there was no evidence of AE2 protein in adult rat cardiomyocytes. In cardiomyocytes transfected with an AE3 oligodeoxynucleotide antisense, AE3 immunoreactivity was dramatically decreased but the activity of the Cl-/HCO3- exchange was unchanged. In contrast, intracellular microinjection of blocking anti-AE1 antibodies inhibited the AE activity. Altogether, our findings suggest that a specific and novel AE1 splicoform (nAE1) mediates the cardiac Cl-/HCO3- exchange. The multiple gene and protein expression within the same cell type suggest numerous functions for this protein family.

Specific activation of adenylyl cyclase V by a purinergic agonist.

The present study was designed to investigate whether and how the purinergic stimulation of rat ventricular myocytes modulates the cAMP-dependent pathway. Stimulation of cardiomyocytes with ATPgammaS in the presence of the phosphodiesterase inhibitor IBMX increases by 3-fold intracellular cAMP content. In contrast to beta-adrenergic stimulation, the purinergic stimulation of adenylyl cyclase was not inhibited by activation or enhanced by inhibition of a Gi protein. Forskolin did not potentiate the effect of extracellular ATPgammaS on intracellular cAMP content but the effect of isoproterenol did. Like isoproterenol, the purinergic agonist decreased subsequent ADP-ribosylation of a 45 kDa G(alpha s) by cholera toxin. ATPgammaS also increased cAMP content in neonatal rat cardiomyocytes, a cell type that expresses a long form of Gs protein (alpha(s), 52 kDa) in contrast to adult rat cardiomyocytes that express mostly a short form of Gs protein (alpha(s), 45 kDa). Both purinergic and beta-adrenergic agonists increased cAMP in HEK 293 cells expressing type V adenylyl cyclase while cAMP was only increased by beta-adrenergic stimulation of HEK expressing type IV or VI adenylyl cyclases. Thus, we propose that the purinergic and beta-adrenergic stimulations differentially activate adenylyl cyclase isoforms in rat cardiomyocytes and that adenylyl cyclase V is the specific target of the purinergic stimulation.

Nuclear calcium and the regulation of the nuclear pore complex.

In eukaryotic cells the nucleus and its contents are separated from the cytoplasm by the nuclear envelope. Macromolecules, as well as smaller molecules and ions, can cross the nuclear envelope through the nuclear pore complex. Molecules greater than approx. 60 kDa and containing a nuclear localization signal are actively transported across the nuclear membranes, but there has been little evidence for regulatory mechanisms for smaller molecules and ions. Recently, diffusion across the nuclear envelope has been observed to be regulated by nuclear cisternal Ca2+ concentrations. Following depletion of Ca2+ from the nuclear store by inositol 1,4,5-trisphosphate or Ca2+ chelators, a fluorescent 10 kDa marker molecule was no longer able to enter the nucleus. Distinct conformational states of the nuclear pore complexes depended on the Ca2+ filling state of the nuclear envelope, supporting the assumption that a switch in the conformation of the nuclear pore complex may control the transport of intermediate-sized molecules across the nuclear envelope. Thus nuclear Ca2+ stores may regulate the conformational state of the nuclear pore complex, and thereby passive diffusion of molecules between the cytosol and the nucleoplasm. The physiological significance of this finding is currently unknown.

Calcium release and influx colocalize to the endoplasmic reticulum.

Intracellular Ca2+ is released from intracellular stores in the endoplasmic reticulum (ER) in response to the second messenger inositol (1,4,5) trisphosphate (InsP3) [1,2]. Then, a poorly understood cellular mechanism, termed capacitative Ca2+ entry, is activated [3,4]; this permits Ca2+ to enter cells through Ca(2+)-selective Ca(2+)-release-activated ion channels [5,6] as well as through less selective store-operated channels [7]. The level of stored Ca2+ is sensed by Ca(2+)-permeant channels in the plasma membrane, but the identity of these channels, and the link between them and Ca2+ stores, remain unknown. It has been argued that either a diffusible second messenger (Ca2+ influx factor; CIF) [8] or a physical link [9,10] connects the ER Ca(2+)-release channel and store-operated channels; strong evidence for either mechanism is lacking, however [7,10]. Petersen and Berridge [11] showed that activation of the lysophosphatidic acid receptor in a restricted region of the oocyte membrane results in stimulation of Ca2+ influx only in that region, and concluded that a diffusible messenger was unlikely. To investigate the relationship between ER stores and Ca2+ influx, we used centrifugation to redistribute into specific layers the organelles inside intact Xenopus laevis oocytes, and used laser scanning confocal microscopy with the two-photon technique to 'uncage' InsP3 while recording intracellular Ca2+ concentration. Ca2+ release was localized to the stratified ER layer and Ca2+ entry to regions of the membrane directly adjacent to this layer. We conclude that Ca2+ depletion and entry colocalize to the ER and that the mechanism linking Ca2+ stores to Ca2+ entry is similarly locally constrained.