The expression of congenital Shoc2 variants induces AKT-dependent crosstalk activation of the ERK1/2 pathway.

The Shoc2 scaffold protein is crucial in transmitting signals within the Epidermal Growth Factor Receptor (EGFR)-mediated Extracellular signal-Regulated Kinase (ERK1/2) pathway. While the significance of Shoc2 in this pathway is well-established, the precise mechanisms through which Shoc2 governs signal transmission remain to be fully elucidated. Hereditary variants in Shoc2 are responsible for Noonan Syndrome with Loose anagen Hair (NSLH). However, due to the absence of known enzymatic activity in Shoc2, directly assessing how these variants affect its function is challenging. ERK1/2 phosphorylation is used as a primary parameter of Shoc2 function, but the impact of Shoc2 mutants on the pathway activation is unclear. This study investigates how the NSLH-associated Shoc2 variants influence EGFR signals in the context of the ERK1/2 and AKT downstream signaling pathways. We show that when the ERK1/2 pathway is a primary signaling pathway activated downstream of EGFR, Shoc2 variants cannot upregulate ERK1/2 phosphorylation to the level of the WT Shoc2. Yet, when the AKT and ERK1/2 pathways were activated, in cells expressing Shoc2 variants, ERK1/2 phosphorylation was higher than in cells expressing WT Shoc2. In cells expressing the Shoc2 NSLH mutants, we found that the AKT signaling pathway triggers the PAK activation, followed by phosphorylation of Raf-1/MEK1/2 and activation of the ERK1/2 signaling axis. Hence, our studies reveal a previously unrecognized feedback regulation downstream of the EGFR and provide additional evidence for the role of Shoc2 as a "gatekeeper" in controlling the selection of downstream effectors within the EGFR signaling network.

Serum Amyloid A Is an Exchangeable Apolipoprotein.

Objective- SAA (serum amyloid A) is a family of acute-phase reactants that have proinflammatory and proatherogenic activities. SAA is more lipophilic than apoA-I (apolipoprotein A-I), and during an acute-phase response, <10% of plasma SAA is found lipid-free. In most reports, SAA is found exclusively associated with high-density lipoprotein; however, we and others have reported SAA on apoB (apolipoprotein B)-containing lipoproteins in both mice and humans. The goal of this study was to determine whether SAA is an exchangeable apolipoprotein. Approach and Results- Delipidated human SAA was incubated with SAA-free human lipoproteins; then, samples were reisolated by fast protein liquid chromatography, and SAA analyzed by ELISA and immunoblot. Both in vitro and in vivo, we show that SAA associates with any lipoprotein and does not remain in a lipid-free form. Although SAA is preferentially found on high-density lipoprotein, it can exchange between lipoproteins. In the presence of CETP (cholesterol ester transfer protein), there is greater exchange of SAA between lipoproteins. Subjects with diabetes mellitus, but not those with metabolic syndrome, showed altered SAA lipoprotein distribution postprandially. Proteoglycan-mediated lipoprotein retention is thought to be an underlying mechanism for atherosclerosis development. SAA has a proteoglycan-binding domain. Lipoproteins containing SAA had increased proteoglycan binding compared with SAA-free lipoproteins. Conclusions- Thus, SAA is an exchangeable apolipoprotein and increases apoB-containing lipoproteins' proteoglycan binding. We and others have previously reported the presence of SAA on low-density lipoprotein in individuals with obesity, diabetes mellitus, and metabolic syndrome. We propose that the presence of SAA on apoB-containing lipoproteins may contribute to cardiovascular disease development in these populations.

Prevention of renal apoB retention is protective against diabetic nephropathy: role of TGF-ß inhibition.

Animal studies demonstrate that hyperlipidemia and renal lipid accumulation contribute to the pathogenesis of diabetic nephropathy (DN). We previously demonstrated that renal lipoproteins colocalize with biglycan, a renal proteoglycan. The purpose of this study was to determine whether prevention of renal lipid (apoB) accumulation attenuates DN. Biglycan-deficient and biglycan wild-type Ldlr-/- mice were made diabetic via streptozotocin and fed a high cholesterol diet. As biglycan deficiency is associated with elevated transforming growth factor-ß (TGF-ß), in some experiments mice were injected with either the TGF-ß-neutralizing antibody, 1D11, or with 13C4, an irrelevant control antibody. Biglycan deficiency had no significant effect on renal apoB accumulation, but led to modest attenuation of DN with ∼30% reduction in albuminuria; however, biglycan deficiency caused a striking elevation in TGF-ß. Use of 1D11 led to sustained suppression of TGF-ß for approximately 8 weeks at a time. The 1D11 treatment caused decreased renal apoB accumulation, decreased albuminuria, decreased renal hypertrophy, and improved survival, compared with the 13C4 treatment. Thus, prevention of renal apoB accumulation is protective against development of DN. Furthermore, this study demonstrates that prevention of renal apoB accumulation is a mechanism by which TGF-ß inhibition is nephroprotective.

Depression in Young People: More than Just a Bad Day-A Concept Analysis.

AIM: Suicide from depression is the second leading cause of death in young people. To better understand depression, a concept analysis was conducted using the Lorraine Walker and Kay Avant method. SOURCE OF DATA: Three electronic databases searched using keywords such as depress*, stigma, and feeling depressed yielded 40 articles in English from 2006 through 2016. RESULTS: Primary attribute was depressed mood; stress was the primary antecedent found in young people. Consequences included health, emotional, and financial well-being. CONCLUSION: A better understanding of depression by healthcare providers can foster quicker assessment and treatment in young people and impact final outcome-suicide.

A brief elevation of serum amyloid A is sufficient to increase atherosclerosis.

Serum amyloid A (SAA) has a number of proatherogenic effects including induction of vascular proteoglycans. Chronically elevated SAA was recently shown to increase atherosclerosis in mice. The purpose of this study was to determine whether a brief increase in SAA similarly increased atherosclerosis in a murine model. The recombination activating gene 1-deficient (rag1(-/-)) × apolipoprotein E-deficient (apoe(-/-)) and apoe(-/-) male mice were injected, multiple times or just once respectively, with an adenoviral vector encoding human SAA1 (ad-SAA); the injected mice and controls were maintained on chow for 12-16 weeks. Mice receiving multiple injections of ad-SAA, in which SAA elevation was sustained, had increased atherosclerosis compared with controls. Strikingly, mice receiving only a single injection of ad-SAA, in which SAA was only briefly elevated, also had increased atherosclerosis compared with controls. Using in vitro studies, we demonstrate that SAA treatment leads to increased LDL retention, and that prevention of transforming growth factor beta (TGF-ß) signaling prevents SAA-induced increases in LDL retention and SAA-induced increases in vascular biglycan content. We propose that SAA increases atherosclerosis development via induction of TGF-ß, increased vascular biglycan content, and increased LDL retention. These data suggest that even short-term inflammation with concomitant increase in SAA may increase the risk of developing CVD.

Biglycan deficiency: increased aortic aneurysm formation and lack of atheroprotection.

Proteoglycans of the arterial wall play a critical role in vascular integrity and the development of atherosclerosis owing to their ability to organize extracellular matrix molecules and to bind and retain atherogenic apolipoprotein (apo)-B containing lipoproteins. Prior studies have suggested a role for biglycan in aneurysms and in atherosclerosis. Angiotensin II (angII) infusions into mice have been shown to induce abdominal aortic aneurysm development, increase vascular biglycan content, increase arterial retention of lipoproteins, and accelerate atherosclerosis. The goal of this study was to determine the role of biglycan in angII-induced vascular diseases. Biglycan-deficient or biglycan wildtype mice crossed to LDL receptor deficient (Ldlr-/-) mice (C57BL/6 background) were infused with angII (500 or 1000ng/kg/min) or saline for 28days while fed on normal chow, then pumps were removed, and mice were switched to an atherogenic Western diet for 6weeks. During angII infusions, biglycan-deficient mice developed abdominal aortic aneurysms, unusual descending thoracic aneurysms, and a striking mortality caused by aortic rupture (76% for males and 48% for females at angII 1000ng/kg/min). Histological analyses of non-aneurysmal aortic segments from biglycan-deficient mice revealed a deficiency of dense collagen fibers and the aneurysms demonstrated conspicuous elastin breaks. AngII infusion increased subsequent atherosclerotic lesion development in both biglycan-deficient and biglycan wildtype mice. However, the biglycan genotype did not affect the atherosclerotic lesion area induced by the Western diet after treatment with angII. Biglycan-deficient mice exhibited significantly increased vascular perlecan content compared to biglycan wildtype mice. Analyses of the atherosclerotic lesions demonstrated that vascular perlecan co-localized with apoB, suggesting that increased perlecan compensated for biglycan deficiency in terms of lipoprotein retention. Biglycan deficiency increases aortic aneurysm development and is not protective against the development of atherosclerosis. Biglycan deficiency leads to loosely packed aortic collagen fibers, increased susceptibility of aortic elastin fibers to angII-induced stress, and up-regulation of vascular perlecan content.

Prevention of TGFß induction attenuates angII-stimulated vascular biglycan and atherosclerosis in Ldlr-/- mice.

Angiotensin II (angII) accelerates atherosclerosis, but the mechanisms are not fully understood. The aim of this study was to determine whether TGFß is required for angII-induced atherosclerosis. Ldlr-null mice fed a normal chow diet were infused with angII or saline for 28 days. A single injection of TGFß neutralizing antibody 1D11 (2 mg/kg) prevented angII-induction of TGFß1 levels, and strikingly attenuated angII-induced accumulation of aortic biglycan content. To study atherosclerosis, mice were infused with angII or saline for 4 weeks, and then fed Western diet for a further 6 weeks. 1D11 had no effect on systolic blood pressure or plasma cholesterol; however, angII-infused mice that received 1D11 had reduced atherosclerotic lesion area by 30% (P < 0.05). Immunohistochemical analyses demonstrated that angII induced both lipid retention and accumulation of biglycan and perlecan which colocalized with apoB. 1D11 strikingly reduced the effect of angII on biglycan but not perlecan. 1D11 decreased total collagen content (P < 0.05) in the lesion area without changing plaque inflammation markers (CD68 and CD45). Thus, this study demonstrates that neutralization of TGFß attenuated angII stimulation of biglycan accumulation and atherogenesis in mice, suggesting that TGFß-mediated biglycan induction is one of the mechanisms underlying angII-promoted atherosclerosis.

Decreased body fat, elevated plasma transforming growth factor-ß levels, and impaired BMP4-like signaling in biglycan-deficient mice.

Biglycan (BGN), a small leucine-rich proteoglycan, binds the pro-fibrotic cytokine transforming growth factor ß (TGFß) and inhibits its bioactivity in vitro. Nevertheless, it is controversial whether BGN plays an inhibitory role in vivo. Therefore, the purpose of this study was to evaluate the effect of BGN deficiency on TGFß activity in vivo by studying 1-year-old Bgn null and wild-type (WT) mice on an Ldlr-null background. Phenotypic and metabolic characterization showed that the Bgn null mice had lower body weight, shorter body length, and shorter femur length (all p < 0.05). Surprisingly, the Bgn null mice also exhibited a striking reduction in percent body fat compared to WT mice (p == 0.006), but no changes were observed in plasma triglycerides, total cholesterol, or glycohemoglobin. Both total and bioactive TGFß1 concentrations in plasma were markedly elevated in Bgn null mice compared to WT mice (4-fold and 11-fold increase, respectively, both p < 0.001), but no changes were found in hepatic levels of mRNA for Tgfß1 or its receptors. Bgn null mice exhibited elevated expression of hepatic fibronectin protein (p = 0.034) without changes in hepatic or renal histology, and Bgn null mice had decreased urinary albumin/creatinine ratio (p = 0.01). Two key downstream targets of bone morphogenetic protein 4-like signaling, SMAD1/3/5 phosphorylation and Id2 gene expression, were found dramatically reduced in Bgn null livers (p = 0.034). Thus, BGN deficiency decreases body fat in this hyperlipidemic mouse model without changing liver or kidney histology. Overall, we propose that this unexpected phenotype arises from the effects of BGN deficiency in vivo to elevate TGFß levels while decreasing bone morphogenetic protein 4-like signaling.

Single-domain antibodies for functional targeting of the signaling scaffold Shoc2.

The accurate transmission of signals by the canonical ERK1/2 kinase pathway critically relies on the proper assembly of an intricate multiprotein complex by the scaffold protein Shoc2. However, the details of the mechanism by which Shoc2 guides ERK1/2 signals are not clear, in part, due to the lack of research tools targeting specific protein binding moieties of Shoc2. We report generation and characterization of single domain antibodies against human Shoc2 using a universal synthetic library of humanized nanobodies. Our results identify eight synthetic single-domain antibodies and show that two evaluated antibodies have binding affinities to Shoc2 in the nanomolar range. High affinity antibodies were uniquely suited for the analysis of the Shoc2 complex assembly. Selected single-domain antibodies were also functional in intracellular assays. This study illustrates that Shoc2 single-domain antibodies can be used to understand functional mechanisms governing complex multiprotein signaling modules and have promise in application for therapies that require modulation of the ERK1/2-associated diseases.

Serum amyloid A, but not C-reactive protein, stimulates vascular proteoglycan synthesis in a pro-atherogenic manner.

Inflammatory markers serum amyloid A (SAA) and C-reactive protein (CRP) are predictive of cardiac disease and are proposed to play causal roles in the development of atherosclerosis, in which the retention of lipoproteins by vascular wall proteoglycans is critical. The purpose of this study was to determine whether SAA and/or CRP alters vascular proteoglycan synthesis and lipoprotein retention in a pro-atherogenic manner. Vascular smooth muscle cells were stimulated with either SAA or CRP (1 to 100 mg/L) and proteoglycans were then isolated and characterized. SAA, but not CRP, increased proteoglycan sulfate incorporation by 50 to 100% in a dose-dependent manner (P < 0.0001), increased glycosaminoglycan chain length, and increased low-density lipoprotein (LDL) binding affinity (K(d), 29 microg/ml LDL versus 90 microg/ml LDL for SAA versus control proteoglycans; P < 0.005). Furthermore, SAA up-regulated biglycan via the induction of endogenous transforming growth factor (TGF)-beta. To determine whether SAA stimulated proteoglycan synthesis in vivo, ApoE(-/-) mice were injected with an adenovirus expressing human SAA-1, a null virus, or saline. Mice that received adenovirus expressing SAA had increased TGF-beta concentrations in plasma and increased aortic biglycan content compared with mice that received either null virus or saline. Thus, SAA alters vascular proteoglycans in a pro-atherogenic manner via the stimulation of TGF-beta and may play a causal role in the development of atherosclerosis.

VCP/p97 controls signals of the ERK1/2 pathway transmitted via the Shoc2 scaffolding complex: novel insights into IBMPFD pathology.

Valosin-containing protein (VCP), also named p97, is an essential hexameric AAA+ ATPase with diverse functions in the ubiquitin system. Here we demonstrate that VCP is critical in controlling signals transmitted via the essential Shoc2-ERK1/2 signaling axis. The ATPase activity of VCP modulates the stoichiometry of HUWE1 in the Shoc2 complex as well as HUWE1-mediated allosteric ubiquitination of the Shoc2 scaffold and the RAF-1 kinase. Abrogated ATPase activity leads to augmented ubiquitination of Shoc2/RAF-1 and altered phosphorylation of RAF-1. We found that in fibroblasts from patients with inclusion body myopathy with Paget's disease of bone and frontotemporal dementia (IBMPFD) that harbor germline mutations in VCP, the levels of Shoc2 ubiquitination and ERK1/2 phosphorylation are imbalanced. This study provides a mechanistic basis for the critical role of VCP in the regulation of the ERK1/2 pathway and reveals a previously unrecognized function of the ERK1/2 pathway in the pathogenesis of IBMPFD.

Elevated circulating TGF-ß is not the cause of increased atherosclerosis development in biglycan deficient mice.

BACKGROUND AND AIMS: Vascular biglycan contributes to atherosclerosis development and increased biglycan expression correlates with increased atherosclerosis. However, mice deficient in biglycan have either no reduction in atherosclerosis or an unexpected increase in atherosclerosis. Biglycan deficient mice have systemically elevated TGF-ß, likely due to lack of sequestration of TGF-ß in the extracellular matrix. The purpose of this study was to determine if prevention of TGF-ß elevations in biglycan deficient mice affected atherosclerosis development. METHODS: Biglycan deficient mice were crossed to Ldlr deficient mice. Diabetes was induced via streptozotocin and all mice were fed a high cholesterol diet. Diabetic biglycan wild type and biglycan deficient Ldlr deficient mice were injected with the TGF-ß neutralizing antibody 1D11 or the irrelevant control antibody 13C4. RESULTS: Biglycan deficient mice had significantly elevated plasma TGF-ß levels, which was further increased by diabetes, and significantly increased atherosclerosis. There was a significant correlation between TGF-ß concentrations and atherosclerosis. However, despite nearly complete suppression of plasma TGF-ß levels in mice treated with the TGF-ß neutralizing antibody 1D11, there was no significant difference in atherosclerosis between mice with elevated TGF-ß levels and mice with suppressed TGF-ß levels. CONCLUSIONS: The increased atherosclerosis in biglycan deficient mice does not appear to be due to elevations in TGF-ß.

Serum amyloid A3 is pro-atherogenic.

BACKGROUND AND AIMS: Serum amyloid A (SAA) predicts cardiovascular events. Overexpression of SAA increases atherosclerosis development; however, deficiency of two of the murine acute phase isoforms, SAA1.1 and SAA2.1, has no effect on atherosclerosis. SAA3 is a pseudogene in humans, but is an expressed acute phase isoform in mice. The goal of this study was to determine if SAA3 affects atherosclerosis in mice. METHODS: ApoE-/- mice were used as the model for all studies. SAA3 was overexpressed by an adeno-associated virus or suppressed using an anti-sense oligonucleotide approach. RESULTS: Over-expression of SAA3 led to a 4-fold increase in atherosclerosis lesion area compared to control mice (p = 0.01). Suppression of SAA3 decreased atherosclerosis in mice genetically deficient in SAA1.1 and SAA2.1 (p < 0.0001). CONCLUSIONS: SAA3 augments atherosclerosis in mice. Our results resolve a previous paradox in the literature and support extensive epidemiological data that SAA is pro-atherogenic.

An SMA project report: neural cell-based assays derived from human embryonic stem cells.

Human embryonic stem (ES) cells are promising resources for developing new treatments for neurodegenerative diseases. Spinal muscular atrophy (SMA) is one of the leading causes of childhood paralysis and infant mortality. SMA is caused by inactivation of the survival motor neuron-1 (SMN1) gene. The nearly identical SMN2 gene contains a silent polymorphism that disrupts splicing and as a result cannot compensate for loss of SMN1. The SMA Project was established by the National Institute of Neurological Disorders and Stroke (NINDS) as a pilot effort to establish a fully transparent coalition between academics, industry, and government to create a centralized network of shared resources and information to identify and test new SMA therapeutics. As one of the funded projects, the work described here tested the feasibility of generating a SMA cell-based assay using neural lineages derived from human ES cells approved for National Institutes of Health (NIH)-funded research. Minigene cassettes were constructed, employing firefly luciferase or green fluorescent protein (GFP) as reporters for splicing efficiency of SMN1 and/or SMN2 under the control of the SMN1, SMN2, or cytomegalovirus (CMV) promoters. Transient transfection of proliferating neuroprogenitors in a 96-well format with plasmid DNA or adenoviral vectors showed differential levels that correlated with the splicing minigene and the promoter used; luciferase activities with SMN1 splicing minigenes were higher than SMN2, and the CMV promoter generated higher levels of activity than the SMN1 and SMN2 promoters. Our results indicate that human ES cell-derived neuroprogenitors provide a promising new primary cell source for assays of new therapeutics for neurodegenerative diseases.

Development and differentiation of neural rosettes derived from human embryonic stem cells.

Neurons and glia are important targets of human embryonic stem cell research, promising a renewable source of these differentiated cells for biomedical research and regenerative medicine. Neurons and glia are derived in vivo from the neuroepithelium of the neural tube. Concomitant to development along the anterior to posterior axis, gradients of morphogens across the dorsal and ventral axis of the neural tube establish positional codes that generate distinct progenitor domains and ultimately specify subtype identity. The neural rosette is the developmental signature of neuroprogenitors in cultures of differentiating embryonic stem cells; rosettes are radial arrangements of columnar cells that express many of the proteins expressed in neuroepithelial cells in the neural tube. In addition to similar morphology, neuroprogenitors within neural rosettes differentiate into the main classes of progeny of neuroepithelial cells in vivo: neurons, oligodendrocytes, and astrocytes. Despite these similarities, important differences exist and the extent to which neural rosettes can model neurogenesis in vivo is not yet clear. Here, the authors review the recent studies on the development and differentiation of neural rosettes from human embryonic stem cells. The authors focus on efforts to generate motor neurons and oligodendrocytes in vitro as representative of the challenges to obtaining the progeny of a single progenitor domain with in vitro methods. Opportunities for further progress are discussed.

Genetic reprogramming of human amniotic cells with episomal vectors: neural rosettes as sentinels in candidate selection for validation assays.

The promise of genetic reprogramming has prompted initiatives to develop banks of induced pluripotent stem cells (iPSCs) from diverse sources. Sentinel assays for pluripotency could maximize available resources for generating iPSCs. Neural rosettes represent a primitive neural tissue that is unique to differentiating PSCs and commonly used to identify derivative neural/stem progenitors. Here, neural rosettes were used as a sentinel assay for pluripotency in selection of candidates to advance to validation assays. Candidate iPSCs were generated from independent populations of amniotic cells with episomal vectors. Phase imaging of living back up cultures showed neural rosettes in 2 of the 5 candidate populations. Rosettes were immunopositive for the Sox1, Sox2, Pax6 and Pax7 transcription factors that govern neural development in the earliest stage of development and for the Isl1/2 and Otx2 transcription factors that are expressed in the dorsal and ventral domains, respectively, of the neural tube in vivo. Dissociation of rosettes produced cultures of differentiation competent neural/stem progenitors that generated immature neurons that were immunopositive for ßIII-tubulin and glia that were immunopositive for GFAP. Subsequent validation assays of selected candidates showed induced expression of endogenous pluripotency genes, epigenetic modification of chromatin and formation of teratomas in immunodeficient mice that contained derivatives of the 3 embryonic germ layers. Validated lines were vector-free and maintained a normal karyotype for more than 60 passages. The credibility of rosette assembly as a sentinel assay for PSCs is supported by coordinate loss of nuclear-localized pluripotency factors Oct4 and Nanog in neural rosettes that emerge spontaneously in cultures of self-renewing validated lines. Taken together, these findings demonstrate value in neural rosettes as sentinels for pluripotency and selection of promising candidates for advance to validation assays.

Increased atherosclerosis in mice with increased vascular biglycan content.

OBJECTIVE: The response to retention hypothesis of atherogenesis proposes that atherosclerosis is initiated via the retention of atherogenic lipoproteins by vascular proteoglycans. Co-localization studies suggest that of all the vascular proteoglycans, biglycan is the one most closely co-localized with LDL. The goal of this study was to determine if over-expression of biglycan in hyperlipidemic mice would increase atherosclerosis development. METHODS: Transgenic mice were developed by expressing biglycan under control of the smooth muscle actin promoter, and were crossed to the LDL receptor deficient (C57BL/6 background) atherosclerotic mouse model. Biglycan transgenic and non-transgenic control mice were fed an atherogenic Western diet for 4-12 weeks. RESULTS: LDL receptor deficient mice overexpressing biglycan under control of the smooth muscle alpha actin promoter had increased atherosclerosis development that correlated with vascular biglycan content. CONCLUSION: Increased vascular biglycan content predisposes to increased lipid retention and increased atherosclerosis development.

Serum amyloid A is found on ApoB-containing lipoproteins in obese humans with diabetes.

OBJECTIVE: In murine models of obesity/diabetes, there is an increase in plasma serum amyloid A (SAA) levels along with redistribution of SAA from high-density lipoprotein (HDL) to apolipoprotein B (apoB)-containing lipoprotein particles, namely, low-density lipoprotein and very low-density lipoprotein. The goal of this study was to determine if obesity is associated with similar SAA lipoprotein redistribution in humans. DESIGN AND METHODS: Three groups of obese individuals were recruited from a weight loss clinic: healthy obese (n = 14), metabolic syndrome (MetS) obese (n = 8), and obese with type 2 diabetes (n = 6). Plasma was separated into lipoprotein fractions by fast protein liquid chromatography, and SAA was measured in lipid fractions using enzyme-linked immunosorbent assay and Western blotting. RESULTS: Only the obese diabetic group had SAA detectable in apoB-containing lipoproteins, and SAA reverted back to HDL with active weight loss. CONCLUSIONS: In human subjects, SAA is found in apoB-containing lipoprotein particles only in obese subjects with type 2 diabetes, but not in healthy obese or obese subjects with MetS.

Clonal populations of amniotic cells by dilution and direct plating: evidence for hidden diversity.

Fetal cells are widely considered a superior cell source for regenerative medicine; fetal cells show higher proliferative capacity and have undergone fewer replicative cycles that could generate spontaneous mutations. Fetal cells in amniotic fluid were among the first normal primary cells to be cultured ex vivo, but the undefined composition of amniotic fluid has hindered advance for regenerative applications. We first developed a highly efficient method to generate clonal populations by dilution of amniocentesis samples in media and direct plating without intervening refrigeration, centrifugation, or exposure of cells to the paracrine effects in mixed cell cultures. More than 40 clonal populations were recovered from 4 amniocentesis samples and representative clones were characterized by flow cytometry, conventional assays for differentiation potential, immunofluorescence imaging, and transcript analysis. The results revealed previously unreported diversity among stromal and epithelial cell types and identified unique cell types that could be lost or undetected in mixed cell populations. The differentiation potential of amniotic cells proved to be uncoupled from expression of definitive cell surface or cytoplasmic markers for stromal and epithelial cells. Evidence for diversity among stromal and epithelial cells in amniotic fluid bears on interpretations applied to molecular and functional tests of amniotic cell populations.

Centriole inheritance.

Early cell biologists perceived centrosomes to be permanent cellular structures. Centrosomes were observed to reproduce once each cycle and to orchestrate assembly a transient mitotic apparatus that segregated chromosomes and a centrosome to each daughter at the completion of cell division. Centrosomes are composed of a pair of centrioles buried in a complex pericentriolar matrix. The bulk of microtubules in cells lie with one end buried in the pericentriolar matrix and the other extending outward into the cytoplasm. Centrioles recruit and organize pericentriolar material. As a result, centrioles dominate microtubule organization and spindle assembly in cells born with centrosomes. Centrioles duplicate in concert with chromosomes during the cell cycle. At the onset of mitosis, sibling centrosomes separate and establish a bipolar spindle that partitions a set of chromosomes and a centrosome to each daughter cell at the completion of mitosis and cell division. Centriole inheritance has historically been ascribed to a template mechanism in which the parental centriole contributed to, if not directed, assembly of a single new centriole once each cell cycle. It is now clear that neither centrioles nor centrosomes are essential to cell proliferation. This review examines the recent literature on inheritance of centrioles in animal cells.