Thermal expansion coefficients in Invar processed by selective laser melting.

This work investigates whether the unique low thermal expansion property of Invar (64Fe-36Ni) is retained after processing using the additive manufacturing process selective laser melting (SLM). Using this process, near-full-density components (99.96%) were formed by melting thin (20 μm) layers of powdered Invar (15-45 μm particle size).

Culture adaptation alters transcriptional hierarchies among single human embryonic stem cells reflecting altered patterns of differentiation.

We have used single cell transcriptome analysis to re-examine the substates of early passage, karyotypically Normal, and late passage, karyotypically Abnormal ('Culture Adapted') human embryonic stem cells characterized by differential expression of the cell surface marker antigen, SSEA3. The results confirmed that culture adaptation is associated with alterations to the dynamics of the SSEA3(+) and SSEA3(-) substates of these cells, with SSEA3(-) Adapted cells remaining within the stem cell compartment whereas the SSEA3(-) Normal cells appear to have differentiated. However, the single cell data reveal that these substates are characterized by further heterogeneity that changes on culture adaptation.

Rediscovering pluripotency: from teratocarcinomas to embryonic stem cells. Cardiff, 10-12 October 2011.

The pluripotent potential of embryonic stem cells has often seen them touted as the future of regenerative medicine. The road to any therapeutic success however, must stretch back to teratocarcinoma, the tumour from which pluripotent stem cells (embryonal carcinoma cells) were first derived. This 2011 meeting in Cardiff acted as a historical perspective from which the impact of embryonal carcinoma cell research on the present pluripotent stem cell landscape could be observed, with many of the early luminaries in this field still very active.

Multiple Tyrosine Residues Contribute to GABA Binding in the GABA(C) Receptor Binding Pocket.

The ligand binding site of Cys-loop receptors is dominated by aromatic amino acids. In GABA(C) receptors, these are predominantly tyrosine residues, with a number of other aromatic residues located in or close to the binding pocket. Here we examine the roles of these residues using substitution with both natural and unnatural amino acids followed by functional characterization.

Genetic instability in neural stem cells: an inconvenient truth?

The evolutionary struggles from which mutants arise have been documented in almost every living system. In this issue of the JCI, Varela and colleagues extend this list of systems to include neural derivatives of human embryonic stem cells, which they show exhibit a repeated gain of material from chromosome 1q. Although this raises safety issues for therapeutic use of such cells, the frequent observation of a particular change may direct screening strategies for detection and removal of these unwanted cellular variants.

Modeling the evolution of culture-adapted human embryonic stem cells.

The long-term culture of human embryonic stem (ES) cells is inevitably subject to evolution, since any mutant that arises with a growth advantage will be selectively amplified. However, the evolutionary influences of population size, mutation rate, and selection pressure are frequently overlooked. We have constructed a Monte Carlo simulation model to predict how changes in these factors can influence the appearance and spread of mutant ES cells, and verified its applicability by comparison with in vitro data.

Analysis of gene expression profiles of microdissected cell populations indicates that testicular carcinoma in situ is an arrested gonocyte.

Testicular germ cell cancers in young adult men derive from a precursor lesion called carcinoma in situ (CIS) of the testis. CIS cells were suggested to arise from primordial germ cells or gonocytes. However, direct studies on purified samples of CIS cells are lacking.

CD30 expression reveals that culture adaptation of human embryonic stem cells can occur through differing routes.

Human embryonic stem cells undergo adaptive changes that can increase their growth capacity upon prolonged culture in vitro. This is frequently associated with nonrandom karyotypic changes, commonly involving amplification of genetic material from chromosomes 12, 17, and X. A recent study suggested that the karyotypically abnormal cells can be identified by their expression of CD30, which confers resistance to apoptosis.

Cancer genes hypermethylated in human embryonic stem cells.

Developmental genes are silenced in embryonic stem cells by a bivalent histone-based chromatin mark. It has been proposed that this mark also confers a predisposition to aberrant DNA promoter hypermethylation of tumor suppressor genes (TSGs) in cancer. We report here that silencing of a significant proportion of these TSGs in human embryonic and adult stem cells is associated with promoter DNA hypermethylation.

Culture adaptation of embryonic stem cells echoes germ cell malignancy.

Teratocarcinomas are a subset of tumours that result from the neoplastic transformation of primordial germ cells. Such germ cell tumours (GCT) are histologically heterogeneous, reflecting a capacity for differentiation (pluripotency) of their embryonal carcinoma (EC) stem cells. However, malignant evolution of these tumours may ultimately correlate with a decrease in pluripotency, because this would tend to increase the propensity of EC cells for self-renewal.

Nucleus accumbens NMDA receptor subunit expression and function is enhanced in morphine-dependent rats.

We have previously shown, using radioligand binding studies, that N-methyl-d-aspartate (NMDA) NR1 and NR2A receptor subunits density was decreased in the forebrain of morphine-dependent rats. We have now determined if morphine-dependent rats display regional differences in NMDA receptor expression and whether such changes are functionally relevant. In morphine-dependent rats, the expression of NR1 and NR2A subunits protein, as determined by Western blotting with NMDA receptor subunit antibodies, were decreased in frontal cortex and hippocampus but significantly increased in the nucleus accumbens.

Adaptation to culture of human embryonic stem cells and oncogenesis in vivo.

The application of human embryonic stem cells (HESCs) to provide differentiated cells for regenerative medicine will require the continuous maintenance of the undifferentiated stem cells for long periods in culture. However, chromosomal stability during extended passaging cannot be guaranteed, as recent cytogenetic studies of HESCs have shown karyotypic aberrations. The observed karyotypic aberrations probably reflect the progressive adaptation of self-renewing cells to their culture conditions.

Locating the carboxylate group of GABA in the homomeric rho GABA(A) receptor ligand-binding pocket.

gamma-Aminobutyric acid, type A (GABA(A)) receptors, of which the GABA(C) receptor family is a subgroup, are members of the Cys loop family of neurotransmitter receptors. Homology modeling of the extracellular domain of these proteins has revealed many molecular details, but it is not yet clear how GABA is orientated in the binding pocket. Here we have examined the role of arginine residues that the homology model locates in or close to the binding site of the GABA(C) receptor (Arg-104, Arg-170, Arg-158, and Arg-249) using mutagenesis and functional studies.

Molecular modeling of the GABA(C) receptor ligand-binding domain.

We have constructed a molecular model of the ligand-binding domain of the GABA(C) receptor, which is a member of the Cys-loop ligand-gated ion channel family. The extracellular domains of these receptors share similar sequence homology (20%) with Limnaea acetylcholine-binding protein for which an X-ray crystal structure is available. We used this structure as a template for homology modeling of the GABA(C) receptor extracellular domain using FUGUE and MODELLER software.

A cation-pi binding interaction with a tyrosine in the binding site of the GABAC receptor.

GABA(C) (rho) receptors are members of the Cys-loop superfamily of neurotransmitter receptors, which includes nicotinic acetylcholine (nACh), 5-HT(3), and glycine receptors. As in other members of this family, the agonist binding site of GABA(C) receptors is rich in aromatic amino acids, but while other receptors bind agonist through a cation-pi interaction to a tryptophan, the GABA(C) binding site has tyrosine at the aligning positions. Incorporating a series of tyrosine derivatives at position 198 using unnatural amino acid mutagenesis reveals a clear correlation between the cation-pi binding ability of the side chain and EC(50) for receptor activation, thus demonstrating a cation-pi interaction between a tyrosine side chain and a neurotransmitter.