Ethanol promotes differentiation of embryonic stem cells through retinoic acid receptor-γ.

Ethanol (EtOH) is a teratogen, but its teratogenic mechanisms are not fully understood. The alcohol form of vitamin A (retinol/ROL) can be oxidized to all-trans-retinoic acid (RA), which plays a critical role in stem cell differentiation and development. Using an embryonic stem cell (ESC) model to analyze EtOH's effects on differentiation, we show here that EtOH and acetaldehyde, but not acetate, increase differentiation-associated mRNA levels, and that EtOH decreases pluripotency-related mRNAs. Using reporter assays, ChIP assays, and retinoic acid receptor-γ (RARγ) knockout ESC lines generated by CRISPR/Cas9 and homologous recombination, we demonstrate that EtOH signals via RARγ binding to RA response elements (RAREs) in differentiation-associated gene promoters or enhancers. We also report that EtOH-mediated increases in homeobox A1 (Hoxa1) and cytochrome P450 family 26 subfamily A member 1 (Cyp26a1) transcripts, direct RA target genes, require the expression of the RA-synthesizing enzyme, aldehyde dehydrogenase 1 family member A2 (Aldh1a2), suggesting that EtOH-mediated induction of Hoxa1 and Cyp26a1 requires ROL from the serum. As shown with CRISPR/Cas9 knockout lines, the retinol dehydrogenase gene Rdh10 and a functional RARE in the ROL transporter stimulated by retinoic acid 6 (Stra6) gene are required for EtOH induction of Hoxa1 and Cyp26a1 We conclude that EtOH stimulates stem cell differentiation by increasing the influx and metabolism of ROL for downstream RARγ-dependent transcription. In stem cells, EtOH may shift cell fate decisions to alter developmental outcomes by increasing endogenous ROL/RA signaling via increased Stra6 expression and ROL oxidation.

Different Effects of Knockouts in ALDH2 and ACSS2 on Embryonic Stem Cell Differentiation.

BACKGROUND: Ethanol (EtOH) is a teratogen that causes severe birth defects, but the mechanisms by which EtOH affects stem cell differentiation are unclear. Our goal here is to examine the effects of EtOH and its metabolites, acetaldehyde (AcH) and acetate, on embryonic stem cell (ESC) differentiation. METHODS: We designed ESC lines in which aldehyde dehydrogenase (ALDH2, NCBI#11669) and acyl-CoA synthetase short-chain family member 2 (ACSS2, NCBI#60525) were knocked out by CRISPR-Cas9 technology. We selected these genes because of their key roles in EtOH oxidation in order to dissect the effects of EtOH metabolism on differentiation. RESULTS: By using kinetic assays, we confirmed that AcH is primarily oxidized by ALDH2 rather than ALDH1A2. We found increases in mRNAs of differentiation-associated genes (Hoxa1, Cyp26a1, and RARß2) upon EtOH treatment of WT and Acss2-/- ESCs, but not Aldh2-/- ESCs. The absence of ALDH2 reduced mRNAs of some pluripotency factors (Nanog, Sox2, and Klf4). Treatment of WT ESCs with AcH or 4-hydroxynonenal (4-HNE), another substrate of ALDH2, increased differentiation-associated transcripts compared to levels in untreated cells. mRNAs of genes involved in retinoic acid (RA) synthesis (Stra6 and Rdh10) were also increased by EtOH, AcH, and 4-HNE treatment. Retinoic acid receptor-γ (RARγ) is required for both EtOH- and AcH-mediated increases in Hoxa1 and Stra6, demonstrating the critical role of RA:RARγ signaling in AcH-induced ESC differentiation. CONCLUSIONS: ACSS2 knockouts showed no changes in differentiation phenotype, while pluripotency-related transcripts were decreased in ALDH2 knockout ESCs. We demonstrate that AcH increases differentiation-associated mRNAs in ESCs via RARγ.

Clonal Lineage Tracing with Somatic Delivery of Recordable Barcodes Reveals Migration Histories of Metastatic Prostate Cancer.

The patterns by which primary tumors spread to metastatic sites remain poorly understood. Here, we define patterns of metastatic seeding in prostate cancer using a novel injection-based mouse model-EvoCaP (Evolution in Cancer of the Prostate), featuring aggressive metastatic cancer to bone, liver, lungs, and lymph nodes. To define migration histories between primary and metastatic sites, we used our EvoTraceR pipeline to track distinct tumor clones containing recordable barcodes. We detected widespread intratumoral heterogeneity from the primary tumor in metastatic seeding, with few clonal populations instigating most migration. Metastasis-to-metastasis seeding was uncommon, as most cells remained confined within the tissue. Migration patterns in our model were congruent with human prostate cancer seeding topologies. Our findings support the view of metastatic prostate cancer as a systemic disease driven by waves of aggressive clones expanding their niche, infrequently overcoming constraints that otherwise keep them confined in the primary or metastatic site. Significance: Defining the kinetics of prostate cancer metastasis is critical for developing novel therapeutic strategies. This study uses CRISPR/Cas9-based barcoding technology to accurately define tumor clonal patterns and routes of migration in a novel somatically engineered mouse model (EvoCaP) that recapitulates human prostate cancer using an in-house developed analytical pipeline (EvoTraceR).

Modification of stem cell states by alcohol and acetaldehyde.

Ethanol (EtOH) is a recreationally ingested compound that is both teratogenic and carcinogenic in humans. Because of its abundant consumption worldwide and the vital role of stem cells in the formation of birth defects and cancers, delineating the effects of EtOH on stem cell function is currently an active and urgent pursuit of scientific investigation to explicate some of the mechanisms contributing to EtOH toxicity. Stem cells represent a primordial, undifferentiated phase of development; thus encroachment on normal physiologic processes of differentiation into terminal lineages by EtOH can greatly alter the function of progenitors and terminally differentiated cells, leading to pathological consequences that manifest as fetal alcohol spectrum disorders and cancers. In this review we explore the disruptive role of EtOH in differentiation of stem cells. Our primary objective is to elucidate the mechanisms by which EtOH alters differentiation-related gene expression and lineage specifications, thus modifying stem cells to promote pathological outcomes. We additionally review the effects of a reactive metabolite of EtOH, acetaldehyde (AcH), in causing both differentiation defects in stem cells as well as genomic damage that incites cellular aging and carcinogenesis.

Wnt of the two horizons: putting stem cell self-renewal and cell fate determination into context.

The canonical Wnt/ß-catenin pathway has long been associated with self-renewal and expansion of embryonic stem cells (ESCs). Recent studies have brought into question some earlier assumptions concerning the functional role that canonical Wnt signaling plays in self-renewal mechanisms by demonstrating clear effects on differentiation. In addition, Wnt is crucial for cell fate determination during embryogenesis. The seemingly contradictory data compiled over several years now point to a more complex system of organismal development by which the downstream effects of Wnt activity are largely determined by the context in which signaling occurs. This review will assess seemingly contradictory findings regarding Wnt signaling in either self-renewal or differentiation in ESCs and then explicate cellular scenarios that determine the context by which Wnt ligands exert their differential effects. Both physiological embryonic development and pathogenic adult carcinogenesis provide opportunities to gleam illuminating insights from stem or stem-like progenitor cells that place Wnt signaling at the center of important cellular decisions to expand or differentiate. The impact of heterogeneity, concentration, metabolic state, and the array of accessible interacting cofactors from various signaling pathways that regulate these functions in ESCs will be critically examined, and implications for early embryonic development and cancer biology will be explored.

Coupled ion binding and structural transitions along the transport cycle of glutamate transporters.

Membrane transporters that clear the neurotransmitter glutamate from synapses are driven by symport of sodium ions and counter-transport of a potassium ion. Previous crystal structures of a homologous archaeal sodium and aspartate symporter showed that a dedicated transport domain carries the substrate and ions across the membrane. Here, we report new crystal structures of this homologue in ligand-free and ions-only bound outward- and inward-facing conformations. We show that after ligand release, the apo transport domain adopts a compact and occluded conformation that can traverse the membrane, completing the transport cycle. Sodium binding primes the transport domain to accept its substrate and triggers extracellular gate opening, which prevents inward domain translocation until substrate binding takes place. Furthermore, we describe a new cation-binding site ideally suited to bind a counter-transported ion. We suggest that potassium binding at this site stabilizes the translocation-competent conformation of the unloaded transport domain in mammalian homologues.DOI: http://dx.doi.org/10.7554/eLife.02283.001.

Toward an integrative analysis of the tumor microenvironment in ovarian epithelial carcinoma.

Ovarian epithelial carcinomas are heterogeneous malignancies exhibiting great diversity in histological phenotypes as well as genetic and epigenetic aberrations. A general early event in tumorigenesis is regional dissemination into the peritoneal cavity. Initial spread to the peritoneum is made possible by cooperative signaling between a wide array of molecules constituting the tissue microenvironment in the coelomic epithelium. Changes in the activity of key microenvironmental components not constitutively expressed in normal tissue, including several disclosed adhesion molecules, growth factors, proteases, and G-protein coupled receptors (GPCRs), coordinate the transition. Remodeling of the extracellular matrix (ECM) and subsequent cell surface interactions enable transformation by promoting chromosomal instability (CIN) and stimulating several common signal transduction cascades to prepare the tissue for harboring and facilitating growth, angiogenesis and metastasis of the developing tumor.

Unraveling the mysteries of aging through a Hutchinson-Gilford progeria syndrome model.

Hutchinson-Gilford progeria syndrome (HGPS) is a rare, deadly laminopathy. Research into its nature has provided valuable insights into understanding molecular mechanisms underlying cell senescence. Phenotypic changes in nuclear structure and heterochromatin, resulting from increased progerin production following overuse of a cryptic splice site in the LMNA gene have profound effects on cell cycle progression and DNA repair mechanisms. A high degree of genomic instability occurs as DNA double-strand breaks are introduced but not repaired, despite appropriate machinery. Damage-response mechanisms become deregulated as a result of failed recruitment of elongation factors and possibly ancillary to disruption of chromatin organization. Key regulatory regions on mRNA transcripts governing splicing activity have been exposed using antisense technology, and identification of novel molecular targets, such as xeroderma pigmentosum group A, have generated optimism for the possibility of finally yielding an effective therapy for slowing the aging process in HGPS patients while concomitantly providing valuable insights into physiological aging in general.

Acute delirium associated with combined diphenhydramine and linezolid use.

OBJECTIVE: To report a case of delirium with hallucinations presumably caused by the combination of diphenhydramine and linezolid. CASE SUMMARY: A 56-year-old white man was receiving diphenhydramine 300 mg/d for 2 days to treat pruritus caused by a bullous rash possibly induced by vancomycin. He subsequently developed visual and auditory hallucinations, with erratic, aggressive behavior persisting for 3 days. Central anticholinergic syndrome was first suspected, but the long duration and exaggerated response by a patient not prone to anticholinergic toxicity suggest that a second agent may have enhanced the reaction. DISCUSSION: The pharmacodynamic properties of linezolid make this drug a likely contributor to the marked, prolonged effects experienced by this patient. The Naranjo probability scale suggests a possible relationship between the reaction and the combination of diphenhydramine and linezolid. CONCLUSIONS: Drug-induced delirium can occur with several drugs, including diphenhydramine. Linezolid has dopaminergic properties that may enhance the central nervous system effects of anticholinergics. Precautionary monitoring of mental status should be advised when concomitantly administering linezolid with drugs in this class.

Infliximab treatment of sarcoidosis.

OBJECTIVE: To determine whether there is sufficient evidence in the literature to support the use of infliximab in the treatment of sarcoidosis. DATA SOURCES AND SELECTION: Literature was accessed through MEDLINE (1966-August 2002), OVID (2001-January 2003), and bibliographic searches. Additional databases were also searched. Published literature relevant to the use of infliximab in the treatment of sarcoidosis was evaluated. DATA SYNTHESIS: In theory, tumor-necrosis factor-alpha blockade appears to be an optimal strategy for treating sarcoidosis. Case series reporting the use of infliximab in refractory sarcoidosis was reviewed to evaluate its potential role as a treatment option. CONCLUSIONS: Although there is insufficient evidence suggesting that infliximab is an appropriate alternative to conventional treatment options as first-line therapy for sarcoidosis, the preliminary outlook on its use in treatment-refractory cases is promising.