The Kinase PDK1 Is Essential for B-Cell Receptor Mediated Survival Signaling

Phosphoinositide-dependent kinase 1 (PDK1) plays an important role in integrating the T cell antigen receptor (TCR) and CD28 signals to achieve efficient NF-κB activation. PDK1 is also an important regulator of T cell development, mediating pre-TCR induced proliferation signals. However, the role of PDK1 in B cell antigen receptor (BCR) signaling and B cell development remains largely unknown. In this study we provide genetic evidence supporting the role of PDK1 in B cell survival. We found PDK1 is required for BCR mediated survival in resting B cells, likely through regulation of Foxo activation. PDK1-dependent signaling to NF-κB is not crucial to resting B cell viability. However, PDK1 is necessary for triggering NF-κB during B cell activation and is required for activated B cell survival. Together these studies demonstrate that PDK1 is essential for BCR-induced signal transduction to Foxo and NF-κB and is indispensable for both resting and activated B cell survival

Ribosomal S6 Kinase 2 (RSK2) Maintains Genomic Stability by Activating the Atm/p53-Dependent DNA Damage Pathway

10.1371/journal.pone.0074334PLoS ONE89-POLN

STRUCTURAL TEST AND ANALYSIS OF RC SLAB AFTER FIRE LOADING

In the present study the behavior of fire and the residual strength of fire-ignited RC slabs are investigated by experimental tests and numerical simulations. The fire tests of RC slabs were carried out in a furnace using the ISO 834 standard fire. The load capacity of the cooled RC slabs that were not loaded during the fire tests was evaluated by additional 3 point bending tests. The influence of the proportion of PP (polypropylene) fibers in the RC slabs on the structural behavior of the RC slabs after the fire loading was investigated. The results of the fire tests showed that the maximum temperature of concrete with PP fiber was lower than that of concrete without PP fiber. As the concrete was heated, the ultimate compressive strength decreased and the ultimate strain increased. The load-deflection relations of RC slabs after fire loading were compared by using existing stress-strain-temperature models. The comparison between the numerical analysis and the experimental tests showed that some numerical analyses were reliable and therefore, can be applied to evaluate the ultimate load of RC slabs after fire loading. The ultimate load capacity after cooling down the RC slabs without PP fiber showed a considerable reduction from that of the RC slabs with PP fiber

Co-culture-based biological carbon monoxide conversion by Citrobacter amalonaticus Y19 and Sporomusa ovata via a reducing-equivalent transfer mediator

The biological conversion of carbon monoxide (CO) has been highlighted for the development of a C1 gas biorefinery process. Despite this, the toxicity and low reducing equivalent of CO uptake make biological conversion difficult. The use of synthetic co-cultures is an alternative way of enhancing the performance of CO bioconversion. This study evaluated a synthetic co-culture consisting of Citrobacter amalonaticus Y19 and Sporomusa ovata for acetate production from CO. In this consortium, the CO2 and H-2 produced by the water-gas shift reaction of C. amalonaticus Y19, were utilized further by S. ovata. Higher acetate production was achieved in the co-culture system compared to the monoculture counterparts. Furthermore, syntrophic cooperation via various reducing equivalent carriers provided new insights into the synergistic metabolic benefits with a toxic and refractory substrate, such as CO. This study also suggests an appropriate model for examining the syntrophic interaction between microbial species in a mixed community

PDK1 is essential for B cell survival after bone marrow immature B cell stage.

<p>(<b>A</b>) Flow cytometric analyses of apoptosis (as indicated by Annexin V staining) were performed with bone marrow cells of CD19-Cre⁺<i>PDK1⁺</i>^/+ and CD19-Cre⁺<i>PDK1</i>^flox/flox mice. The histograms shown were gated as indicated on the right of each plot. Apoptotic percentage of each B cell lineage in bone marrow of CD19-Cre⁺<i>PDK1⁺</i>^/+ and CD19-Cre⁺<i>PDK1</i>^flox/flox mice (n = 4 mice) presented as mean ± SD. Numbers indicate the percentage of 7AAD and annexin V–double positive cells (top), annexin V–positive cells (right bottom) or 7AAD–annexin V–negative cells (left bottom). (<b>B</b>) Foxo target genes (<i>Aicda</i>, <i>Rag1</i>, <i>Bcl2l11</i> and <i>p27/Kip</i>) and NF-κB target genes (<i>Bcl-2</i> and <i>Bcl-xl</i>) expression levels in IgM-positive bone marrow B cells were analyzed by quantitative RT-PCR analysis as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0055378#s2" target="_blank">Materials and Methods</a>. Data are presented as mean ± SD (*<i>p</i><0.05, **<i>p</i><0.01). (<b>C</b>) Flow cytometric analyses of apoptosis markers on B220⁺ B cells were performed with spleen and PLN cells of CD19-Cre⁺<i>PDK1⁺</i>^/+ and CD19-Cre⁺<i>PDK1</i>^flox/flox mice. Data are representative of two independent experiments. (<b>D</b>) Primary B cells were stimulated with LPS and were then infected with Cre expressing retrovirus (MSCV-Cre IRES GFP). The ratio of GFP-positive to GFP-negative B cell one day after infection was set to 1. Each following day the ratios were checked by flow-cytometry. The experiment was done in triplicate and data are presented as mean ± SD. Data are representative of three independent experiments.</p

Peripheral B cell numbers are dramatically reduced in B cell specific <i>Pdk1</i> knockout mice.

<p>(<b>A</b>) Flow cytometric analyses were performed with lymphocytes from spleen and lymph nodes of CD19-Cre⁺<i>PDK1⁺</i>^/+(+/+) and CD19-Cre⁺<i>PDK1</i>^flox/flox (flox/flox) mice. The FACS plots shown are representative of five different experiments. (<b>B</b>) The number of B220⁺ cells in spleen and lymphnode of CD19-Cre⁺<i>PDK1⁺</i>^/+ and CD19-Cre⁺<i>PDK1</i>^flox/flox mice (n = 5 mice) presented as mean ± SD. (<b>C</b>) Spleen and lymph nodes from CD19-Cre⁺<i>PDK1^+/+</i> and CD19-Cre⁺<i>PDK1</i>^flox/flox mice. (<b>D</b>) Structure of Spleen from CD19-Cre⁺<i>PDK1^+/+</i> and CD19-Cre⁺<i>PDK1</i>^flox/flox mice. Paraffin-embedded spleen sections from CD19-Cre⁺<i>PDK1^+/+</i> and CD19-Cre⁺<i>PDK1</i>^flox/flox mice were stained by hematoxylin and eosin (top panel) or anti-B220 (bottom panel).</p

3-HAA (PDK1 inhibitor) inhibits B cell receptor-meditated NF-κB activation.

<p>(<b>A</b>) HEK293 cell were transfected with plasmids for expression of Myc-PDK1 and HA-PKCβ or HA-PKCθ, the cell lysates were then analyzed for the binding between PDK1 and PKCβ or PKCθ by immunoprecipitation and immunoblotting. In the construct for expression of HA-PKCβ or HA-PKCθ, internal ribosome entry site (IRES) sequence and GFP open reading frame (ORF) is integrated after the PKC ORF. Thus, GFP expression level was used for internal control. (<b>B</b>) The B cells stimulated with anti-IgM were analyzed for the binding between PDK1 and PKCβ by immunoprecipitation and immunoblotting. (<b>C</b>) Luciferase assay with reporter plasmids containing NF-κB binding sites. Expression plasmids of PDK1, PKCβ, or CARMA1-Bcl10-Malt1 (CBM) were cotransfected as indicated. Data are presented as mean ± SD. (<b>D</b>) <i>Bcl-xl</i> gene expression was analyzed through quantitative RT-PCR analysis. Data are presented as mean ± SD. (<b>E</b>) Flow cytometric analyses of apoptosis markers were performed with primary B cells stimulated with or without anti-IgM antibody and with 3-HAA, NF-κB inhibitor peptide (NBD peptide) or DMSO. Numbers indicate the percentage of 7AAD and annexin V–double positive cells (top), annexin V–positive cells (right bottom) or 7AAD–annexin V–negative cells (left bottom). (<b>A</b>), (<b>B</b>), (<b>C</b>), (<b>D</b>) and (<b>E</b>) were representative of two to three experiments.</p

The defect of B cell development in CD19-Cre⁺<i>PDK1</i>^flox/flox mice is caused by B cell intrinsic defects.

<p>Bone marrow cells of CD19-Cre⁺<i>PDK1⁺</i>^/+ and CD19-Cre⁺<i>PDK1</i>^flox/flox mice were transferred into unirradiated Rag1 deficient recipients. Six weeks after transfer, flow cytometric analyses of B cell surface markers were performed with bone marrow cells, spleen cells and lymph node cells from the recipient mice. This is representative of two independent experiments.</p

PDK1 is essential for B cell activation.

<p>(<b>A</b>) Phosphorylation of AKT at T308 and S473 in primary B cells stimulated with AffiniPure F(ab′)2 fragment goat anti-Mouse IgM and with or without 3-HAA were analyzed by immunoblot anlaysis. (<b>B</b>) Phosphorylation of JNK and p38 MAPK and IκBα degradation in primary B cells stimulated with anti-IgM antibody and with or without 3-HAA were analyzed by immunobloting analysis. (<b>C</b>) Flow cytometric analyses of B cell activation markers (CD69 and CD86) were performed with primary B cells stimulated with anti-IgM antibody for 24 hours and with or without 3-HAA. Numbers indicate the percentage of CD69 or CD86 positive cells. DMSO was used for the vehicle control. (<b>A</b>), (<b>B</b>) and (<b>C</b>) are representative of three independent experiments.</p