Adenosine-mono-phosphate-activated protein kinase-independent effects of metformin in T cells

The anti-diabetic drug metformin regulates T-cell responses to immune activation and is proposed to function by regulating the energy-stress-sensing adenosine-monophosphate-activated protein kinase (AMPK). However, the molecular details of how metformin controls T cell immune responses have not been studied nor is there any direct evidence that metformin acts on T cells via AMPK. Here, we report that metformin regulates cell growth and proliferation of antigen-activated T cells by modulating the metabolic reprogramming that is required for effector T cell differentiation. Metformin thus inhibits the mammalian target of rapamycin complex I signalling pathway and prevents the expression of the transcription factors c-Myc and hypoxia-inducible factor 1 alpha. However, the inhibitory effects of metformin on T cells did not depend on the expression of AMPK in T cells. Accordingly, experiments with metformin inform about the importance of metabolic reprogramming for T cell immune responses but do not inform about the importance of AMPK

Plasma sphingosine-1-phosphate is elevated in obesity

Background: Dysfunctional lipid metabolism is a hallmark of obesity and insulin resistance and a risk factor for various cardiovascular and metabolic complications. In addition to the well known increase in plasma triglycerides and free fatty acids, recent work in humans and rodents has shown that obesity is associated with elevations in the bioactive class of sphingolipids known as ceramides. However, in obesity little is known about the plasma concentrations of sphinogsine-1-phosphate (S1P), the breakdown product of ceramide, which is an important signaling molecule in mammalian biology. Therefore, the purpose of this study was to examine the impact of obesity on circulating S1P concentration and its relationship with markers of glucose metabolism and insulin sensitivity. Methodology/Principal Findings: Plasma S1P levels were determined in high-fat diet (HFD)-induced and genetically obese (ob/ob) mice along with obese humans. Circulating S1P was elevated in both obese mouse models and in obese humans compared with lean healthy controls. Furthermore, in humans, plasma S1P positively correlated with total body fat percentage, body mass index (BMI), waist circumference, fasting insulin, HOMA-IR, HbA1c (%), total and LDL cholesterol. In addition, fasting increased plasma S1P levels in lean healthy mice. Conclusion: We show that elevations in plasma S1P are a feature of both human and rodent obesity and correlate with metabolic abnormalities such as adiposity and insulin resistance

HIV-1 Nef Employs Two Distinct Mechanisms to Modulate Lck Subcellular Localization and TCR Induced Actin Remodeling

The Nef protein acts as critical factor during HIV pathogenesis by increasing HIV replication in vivo via the modulation of host cell vesicle transport and signal transduction processes. Recent studies suggested that Nef alters formation and function of immunological synapses (IS), thereby modulating exogenous T-cell receptor (TCR) stimulation to balance between partial T cell activation required for HIV-1 spread and prevention of activation induced cell death. Alterations of IS function by Nef include interference with cell spreading and actin polymerization upon TCR engagement, a pronounced intracellular accumulation of the Src kinase Lck and its reduced IS recruitment. Here we use a combination of Nef mutagenesis and pharmacological inhibition to analyze the relative contribution of these effects to Nef mediated alterations of IS organization and function on TCR stimulatory surfaces. Inhibition of actin polymerization and IS recruitment of Lck were governed by identical Nef determinants and correlated well with Nef's association with Pak2 kinase activity. In contrast, Nef mediated Lck endosomal accumulation was separable from these effects, occurred independently of Pak2, required integrity of the microtubule rather than the actin filament system and thus represents a distinct Nef activity. Finally, reduction of TCR signal transmission by Nef was linked to altered actin remodeling and Lck IS recruitment but did not require endosomal Lck rerouting. Thus, Nef affects IS function via multiple independent mechanisms to optimize virus replication in the infected host

Fine mapping of the 9q31 Hirschsprung’s disease locus

Hirschsprung’s disease (HSCR) is a congenital disorder characterised by the absence of ganglia along variable lengths of the intestine. The RET gene is the major HSCR gene. Reduced penetrance of RET mutations and phenotypic variability suggest the involvement of additional modifying genes in the disease. A RET-dependent modifier locus was mapped to 9q31 in families bearing no coding sequence (CDS) RET mutations. Yet, the 9q31 causative locus is to be identified. To fine-map the 9q31 region, we genotyped 301 tag-SNPs spanning 7 Mb on 137 HSCR Dutch trios. This revealed two HSCR-associated regions that were further investigated in 173 Chinese HSCR patients and 436 controls using the genotype data obtained from a genome-wide association study recently conducted. Within one of the two identified regions SVEP1 SNPs were found associated with Dutch HSCR patients in the absence of RET mutations. This ratifies the reported linkage to the 9q31 region in HSCR families with no RET CDS mutations. However, this finding could not be replicated. In Chinese, HSCR was found associated with IKBKAP. In contrast, this association was stronger in patients carrying RET CDS mutations with p = 5.10 × 10−6 [OR = 3.32 (1.99, 5.59)] after replication. The HSCR-association found for IKBKAP in Chinese suggests population specificity and implies that RET mutation carriers may have an additional risk. Our finding is supported by the role of IKBKAP in the development of the nervous system

Differential responses to doxorubicin-induced phosphorylation and activation of Akt in human breast cancer cells

INTRODUCTION: We have shown previously that overexpression of constitutively active Akt or activation of Akt caused by constitutively active Ras or human epidermal growth factor receptor-2 (HER2) confers on breast cancer cells resistance to chemotherapy or radiotherapy. As an expanded study we here report differential responses in terms of phosphorylation and activation of Akt as a result of treatment with doxorubicin in a panel of breast cancer cell lines. METHODS: The levels of Akt phosphorylation and activity were measured by Western blot analysis with an anti-Ser473-phosphorylated Akt antibody and by in vitro Akt kinase assay using glycogen synthase kinase-3 as a substrate. RESULTS: Within 24 hours after exposure to doxorubicin, MCF7, MDA468 and T47D cells showed a drug-dose-dependent increase in the levels of phosphorylated Akt; in contrast, SKBR3 and MDA231 cells showed a decrease in the levels of phosphorylated Akt, and minimal or no changes were detected in MDA361, MDA157 and BT474 cells. The doxorubicin-induced Akt phosphorylation was correlated with increased kinase activity and was dependent on phosphoinositide 3-kinase (PI3-K). An increased baseline level of Akt was also found in MCF7 cells treated with ionizing radiation. The cellular responses to doxorubicin-induced Akt phosphorylation were potentiated after the expression of Akt upstream activators including HER2, HER3 and focal adhesion kinase. CONCLUSION: Taken together with our recent published results showing that constitutive Akt mediates resistance to chemotherapy or radiotherapy, our present data suggest that the doxorubicin-induced phosphorylation and activation of Akt might reflect a cellular defensive mechanism of cancer cells to overcome doxorubicin-induced cytotoxic effects, which further supports the current efforts of targeting PI3-K/Akt for enhancing the therapeutic responses of breast cancer cells to chemotherapy and radiotherapy

Non-Centered Spike-Triggered Covariance Analysis Reveals Neurotrophin-3 as a Developmental Regulator of Receptive Field Properties of ON-OFF Retinal Ganglion Cells

The functional separation of ON and OFF pathways, one of the fundamental features of the visual system, starts in the retina. During postnatal development, some retinal ganglion cells (RGCs) whose dendrites arborize in both ON and OFF sublaminae of the inner plexiform layer transform into RGCs with dendrites that monostratify in either the ON or OFF sublamina, acquiring final dendritic morphology in a subtype-dependent manner. Little is known about how the receptive field (RF) properties of ON, OFF, and ON-OFF RGCs mature during this time because of the lack of a reliable and efficient method to classify RGCs into these subtypes. To address this deficiency, we developed an innovative variant of Spike Triggered Covariance (STC) analysis, which we term Spike Triggered Covariance – Non-Centered (STC-NC) analysis. Using a multi-electrode array (MEA), we recorded the responses of a large population of mouse RGCs to a Gaussian white noise stimulus. As expected, the Spike-Triggered Average (STA) fails to identify responses driven by symmetric static nonlinearities such as those that underlie ON-OFF center RGC behavior. The STC-NC technique, in contrast, provides an efficient means to identify ON-OFF responses and quantify their RF center sizes accurately. Using this new tool, we find that RGCs gradually develop sensitivity to focal stimulation after eye opening, that the percentage of ON-OFF center cells decreases with age, and that RF centers of ON and ON-OFF cells become smaller. Importantly, we demonstrate for the first time that neurotrophin-3 (NT-3) regulates the development of physiological properties of ON-OFF center RGCs. Overexpression of NT-3 leads to the precocious maturation of RGC responsiveness and accelerates the developmental decrease of RF center size in ON-OFF cells. In summary, our study introduces STC-NC analysis which successfully identifies subtype RGCs and demonstrates how RF development relates to a neurotrophic driver in the retina

Translational Up-Regulation and High-Level Protein Expression from Plasmid Vectors by mTOR Activation via Different Pathways in PC3 and 293T Cells

BACKGROUND: Though 293T cells are widely used for expression of proteins from transfected plasmid vectors, the molecular basis for the high-level expression is yet to be understood. We recently identified the prostate carcinoma cell line PC3 to be as efficient as 293T in protein expression. This study was undertaken to decipher the molecular basis of high-level expression in these two cell lines. METHODOLOGY/PRINCIPAL FINDINGS: In a survey of different cell lines for efficient expression of platelet-derived growth factor-B (PDGF-B), β-galactosidase (β-gal) and green fluorescent protein (GFP) from plasmid vectors, PC3 was found to express at 5-50-fold higher levels compared to the bone metastatic prostate carcinoma cell line PC3BM and many other cell lines. Further, the efficiency of transfection and level of expression of the reporters in PC3 were comparable to that in 293T. Comparative analyses revealed that the high level expression of the reporters in the two cell lines was due to increased translational efficiency. While phosphatidic acid (PA)-mediated activation of mTOR, as revealed by drastic reduction in reporter expression by n-butanol, primarily contributed to the high level expression in PC3, multiple pathways involving PA, PI3K/Akt and ERK1/2 appear to contribute to the abundant reporter expression in 293T. Thus the extent of translational up-regulation attained through the concerted activation of mTOR by multiple pathways in 293T could be achieved through its activation primarily by the PA pathway in PC3. CONCLUSIONS/SIGNIFICANCE: Our studies reveal that the high-level expression of proteins from plasmid vectors is effected by translational up-regulation through mTOR activation via different signaling pathways in the two cell lines and that PC3 is as efficient as 293T for recombinant protein expression. Further, PC3 offers an advantage in that the level of expression of the protein can be regulated by simple addition of n-butanol to the culture medium

SOSORT consensus paper: school screening for scoliosis. Where are we today?

This report is the SOSORT Consensus Paper on School Screening for Scoliosis discussed at the 4th International Conference on Conservative Management of Spinal Deformities, presented by SOSORT, on May 2007. The objectives were numerous, 1) the inclusion of the existing information on the issue, 2) the analysis and discussion of the responses by the meeting attendees to the twenty six questions of the questionnaire, 3) the impact of screening on frequency of surgical treatment and of its discontinuation, 4) the reasons why these programs must be continued, 5) the evolving aim of School Screening for Scoliosis and 6) recommendations for improvement of the procedure