Siobhan A. Corbett

Integrins are required for the differentiation of visceral endoderm.

Integrins of the β1 subfamily are highly expressed in the early mouse embryo and are essential for the formation of primitive germ layers from the inner cell mass (ICM). We investigated the mechanisms by which αβ1 integrins regulate ICM morphogenesis by using the embryonic-stem-cell-derived embryoid body (EB), a model for peri-implantation development. Ablation of integrin β1 in EBs resulted in endoderm detachment and in maturation defects, which were manifested by the mislocalization of GATA4 in the cytoplasm and the markedly reduced synthesis of basement membrane (BM) components and the lineage marker disabled homolog 2. The mutant endoderm cells failed to spread on BM substrates, but could spread on vitronectin, which induced upregulation of αvβ3 integrin and integrin-dependent GATA4 nuclear translocation. Forced expression of integrin β3 in the mutant EBs completely rescued endoderm morphogenesis, suggesting that integrin β3 can substitute for integrin β1 in the endoderm. Furthermore, the mitogen-activated protein kinases (MAPKs) ERK1 and ERK2 (ERK1/2) and p38 were activated in endoderm in an integrin-dependent fashion. Pharmacological inhibition of ERK1/2 or p38 MAPK blocked vitronectin-induced GATA4 nuclear translocation and endoderm maturation, whereas expression of a constitutively active ERK kinase (MEK1) or p38 MAPK in the mutant cells rescued endoderm maturation in integrin-β1-null endoderm cells. Collectively, these results suggest that integrins are required for both the stable adhesion and maturation of visceral endoderm, the latter being mediated through the activation of ERK1/2 and p38 MAPK.

Temporal metabolic profiling of plasma during endotoxemia in humans.

ABSTRACT Endotoxemia induced by the administration of low-dose lipopolysaccharide (LPS) to healthy human volunteers is a well-established experimental protocol and has served as a reproducible platform for investigating the responses to systemic inflammation. Because metabolic composition of a tissue or body fluid is uniquely altered by stimuli and provides information about the dominant regulatory mechanisms at various cellular processes, understanding the global metabolic response to systemic inflammation constitutes a major part in this investigation complementing the studies undertaken so far in both clinical and systems biology fields. This article communicates the first proof-of-principle metabonomic analysis, which comprised global biochemical profiles in human plasma samples from healthy subjects given intravenous endotoxin at 2 ng/kg. Concentrations of a total of 366 plasma biochemicals were determined in archived blood samples collected from 15 endotoxin-treated subjects at five time points within 24 h after treatment and compared with control samples collected from four saline-treated subjects. Principal component analysis within this data set determined the sixth hour as a critical time point separating development and recovery phases of the LPS-induced metabolic changes. Consensus clustering of the differential metabolites identified two distinct subsets of metabolites that displayed common coherent profiles with opposing directionality. The first group of metabolites, which were mostly associated with pathways related to lipid metabolism, was upregulated within the first 6 h and downregulated by the 24th hour following LPS administration. The second group of metabolites, in contrast, was first downregulated until the sixth hour, then upregulated. Metabolites in this group were predominantly amino acids or their derivatives. In summary, nontargeted biochemical profiling and unsupervised multivariate analyses highlighted the prominent roles of lipid and protein metabolism in regulating the response to systemic inflammation while also revealing their dynamics in opposite directions.

Effect of educational debt on career and quality of life among academic surgeons

Objective:The purpose of this study was to assess indebtedness among academic surgeons and its repercussions on personal finances, quality of life, and career choices. Summary Background Data:The influence of educational debt on academic surgical career choices and quality of life is unknown. We hypothesized that educational debt affects professional choices and quality of life. Methods:A web-based survey was designed to assess respondent demographics, educational and consumer indebtedness, and the influence of educational debt on career choices and quality of life among academic surgeons. Results:Five hundred fifty-five surgeons responded (20.6% response rate). Two hundred seventy-four (66%) respondents finished postgraduate training with educational debt, 139 (34%) reported no debt, and 142 (26%) did not respond. Among those with educational debt, mean educational debt was

Cdc42 Controls Vascular Network Assembly Through Protein Kinase Cι During Embryonic Vasculogenesis

90,801 and mean noneducational consumer debt was

Integrated transcriptional and metabolic profiling in human endotoxemia.

32,319. Individuals without educational debt reported a mean of

Dexamethasone-Mediated Upregulation of Calreticulin Inhibits Primary Human Glioblastoma Dispersal Ex Vivo

15,104 of noneducational consumer debt (P < 0.001) and had higher mean salaries (P = 0.017) versus those with educational debt. Eighty-seven percent of respondents with educational debt would make the same career choice again. However, 35% acknowledged it placed a strain on their relationship with their significant other, 48% felt it influenced the type of living accommodations they could afford, and 29% reported it forced their significant other to work. Alarmingly, 32% of academic surgeons would not recommend their career choice to their children or medical students. Conclusions:Many academic surgeons reported that their educational debt affected their academic productivity, career choices, and quality of life. Consequently, efforts to mitigate the impact of educational debt on academic surgeons are required to ensure medical students continue to pursue academic surgical careers.

Instantaneous frequency analysis shows greater sensitivity to parasympathetic components of heart rate than spectral analysis

Objective—The goal of this study was to determine the role of Cdc42 in embryonic vasculogenesis and the underlying mechanisms. Methods and Results—By using genetically modified mouse embryonic stem (ES) cells, we demonstrate that ablation of the Rho GTPase Cdc42 blocks vascular network assembly during embryoid body (EB) vasculogenesis without affecting endothelial lineage differentiation. Reexpression of Cdc42 in mutant EBs rescues the mutant phenotype, establishing an essential role for Cdc42 in vasculogenesis. Chimeric analysis revealed that the vascular phenotype is caused by inactivation of Cdc42 in endothelial cells rather than surrounding cells. Endothelial cells isolated from Cdc42-null EBs are defective in directional migration and network assembly. In addition, activation of atypical protein kinase C&igr; (PKC&igr;) is abolished in Cdc42-null endothelial cells, and PKC&igr; ablation phenocopies the vascular abnormalities of the Cdc42-null EBs. Moreover, the inhibitory phosphorylation of glycogen synthase kinase-3&bgr; (GSK-3&bgr;) at Ser9 depends on Cdc42 and PKC&igr;, and expression of kinase-dead GSK-3&bgr; in Cdc42-null EBs promotes the formation of linear endothelial segments without branches. These results suggest that PKC&igr; and GSK-3&bgr; are downstream effectors of Cdc42 during vascular morphogenesis. Conclusion—Cdc42 controls vascular network assembly but not endothelial lineage differentiation by activating PKC&igr; during embryonic vasculogenesis.

University of Medicine and Dentistry of New Jersey Robert Wood Johnson Medical School.

Abstract In this meta-study, we aimed to integrate biological insights gained from two levels of -omics analyses on the response to systemic inflammation induced by lipopolysaccharide in humans. We characterized the interplay between plasma metabolite compositions and transcriptional response of leukocytes through integration of transcriptomics with plasma metabonomics. We hypothesized that the drastic changes in the immediate environment of the leukocytes might have an adaptive effect on shaping their transcriptional response in conjunction with the initial inflammatory stimuli. Indeed, we observed that leukocytes, most notably, tune the activity of lipid- and protein-associated processes at the transcriptional level in accordance with the fluctuations in metabolite compositions of surrounding plasma. A closer look into the transcriptional control of only metabolic pathways uncovered alterations in bioenergetics and defenses against oxidative stress closely associated with mitochondrial dysfunction and shifts in energy production observed during inflammatory processes.

Cell Structure, Function, and Genetics

Dispersal of Glioblastoma (GBM) renders localized therapy ineffective and is a major cause of recurrence. Previous studies have demonstrated that Dexamethasone (Dex), a drug currently used to treat brain tumor–related edema, can also significantly reduce dispersal of human primary GBM cells from neurospheres. It does so by triggering α5 integrin activity, leading to restoration of fibronectin matrix assembly (FNMA), increased neurosphere cohesion, and reduction of neurosphere dispersal velocity (DV). How Dex specifically activates α5 integrin in these GBM lines is unknown. Several chaperone proteins are known to activate integrins, including calreticulin (CALR). We explore the role of CALR as a potential mediator of Dex-dependent induction of α5 integrin activity in primary human GBM cells. We use CALR knock-down and knock-in strategies to explore the effects on FNMA, aggregate compaction, and dispersal velocity in vitro, as well as dispersal ex vivo on extirpated mouse retina and brain slices. We show that Dex increases CALR expression and that siRNA knockdown suppresses Dex-mediated FNMA. Overexpression of CALR in GBM cells activates FNMA, increases compaction, and decreases DV in vitro and on explants of mouse retina and brain slices. Our results define a novel interaction between Dex, CALR, and FNMA as inhibitors of GBM dispersal.

Normal Cell Structure and Function

Septic shock is a leading cause of hospital deaths in the United States. A system to detect initial immune response to an infection could allow for treatment before septic shock occurs, which would save many lives. Here, we demonstrate the analysis of beat-to-beat heart rate intervals using the Hilbert-Huang transform (HHT) as a method of detection of the immune/inflammatory response. The HHT is ideal for nonlinear nonstationary signals. This works by detecting differences in the heart rate variability (HRV), a marker of autonomic signaling to the heart. The instantaneous frequency (IF) as computed via the HHT was compared with spectral analysis and pNN50, Our data set were beat-to-beat intervals measured from subjects administered bacterial endotoxin ranging in dose from 0.1-2.0 ng/kg. Measurements were compared with placebo. While all measurements accurately differentiated the 2.0 ng/kg dosage and placebo groups, the IF showed improved discrimination at lower dosages. The first mode was found to be most significant for discrimination, though each mode could classify subjects with at least 77% accuracy, indicating that information is contained at many scales within the beat-to-beat intervals. The IF appears to have superior sensitivity to parasympathetic signaling reflected in the beat-to-beat interval.