The AGC kinase SGK1 regulates TH1 and TH2 differentiation downstream of the mTORC2 complex.

SGK1 is an AGC kinase that regulates the expression of membrane sodium channels in renal tubular cells in a manner dependent on the metabolic checkpoint kinase complex mTORC2. We hypothesized that SGK1 might represent an additional mTORC2-dependent regulator of the differentiation and function of T cells. Here we found that after activation by mTORC2, SGK1 promoted T helper type 2 (TH2) differentiation by negatively regulating degradation of the transcription factor JunB mediated by the E3 ligase Nedd4-2. Simultaneously, SGK1 repressed the production of interferon-γ (IFN-γ) by controlling expression of the long isoform of the transcription factor TCF-1. Consistent with those findings, mice with selective deletion of SGK1 in T cells were resistant to experimentally induced asthma, generated substantial IFN-γ in response to viral infection and more readily rejected tumors.

T cell factor 1 initiates the T helper type 2 fate by inducing the transcription factor GATA-3 and repressing interferon-gamma.

The differentiation of activated CD4(+) T cells into the T helper type 1 (T(H)1) or T(H)2 fate is regulated by cytokines and the transcription factors T-bet and GATA-3. Whereas interleukin 12 (IL-12) produced by antigen-presenting cells initiates the T(H)1 fate, signals that initiate the T(H)2 fate are not completely characterized. Here we show that early GATA-3 expression, required for T(H)2 differentiation, was induced by T cell factor 1 (TCF-1) and its cofactor beta-catenin, mainly from the proximal Gata3 promoter upstream of exon 1b. This activity was induced after T cell antigen receptor (TCR) stimulation and was independent of IL-4 receptor signaling through the transcription factor STAT6. Furthermore, TCF-1 blocked T(H)1 fate by negatively regulating interferon-gamma (IFN-gamma) expression independently of beta-catenin. Thus, TCF-1 initiates T(H)2 differentiation of activated CD4(+) T cells by promoting GATA-3 expression and suppressing IFN-gamma expression.

Vibrationally Induced Conformational Isomerization and Tunneling in Pyrrole-2-Carboxylic Acid.

The conformational behavior of carboxylic acids has attracted considerable attention, as it can be used as a gateway for the study of more complex phenomena. Here, we present an experimental and computational study of pyrrole-2-carboxylic acid (PCA) conformational space and the vibrational characterization of the compound by infrared spectroscopy. The possibility of promoting conformational transformations using selective vibrational excitation of the 2ν(OH) and 2ν(NH) stretching overtones is explored. Two conformers, exhibiting the cis configuration of the COOH group (O═C-O-H dihedral angle near 0°) and differing by the orientation of the carboxylic group with respect to the pyrrole ring (i.e., showing either a cis or a trans NCC═O arrangement), were found to coexist initially for the compound isolated in a cryogenic nitrogen matrix, in an 86:14 ratio, and were characterized by infrared spectroscopy. A third conformer, with the COOH group in the trans configuration, was produced, in situ, by narrowband near-infrared (NIR) excitation of the most stable PCA form (with a cis NCC═O moiety). The photogenerated PCA conformer was found to decay back to the most stable PCA form, by H-atom quantum mechanical tunneling, with a characteristic half-life time of ∼10 min in the nitrogen matrix at 10 K. Tunneling rates were theoretically estimated and compared for the observed isomerization of pyrrole-2-carboxylic acid and for the structurally similar furan-2-carboxylic acid. This comparison showcases the effect of small modifications in the potential energy surface and the implications of quantum tunneling for the stability of short-living species.

DSP30 and interleukin-2 as a mitotic stimulant in B-cell disorders including those with a low disease burden.

Chromosome abnormalities detected during cytogenetic investigations for B-cell malignancy offer prognostic information that can have wide ranging clinical impacts on patients. These impacts may include monitoring frequency, treatment type, and disease staging level. The use of the synthetic oligonucleotide DSP30 combined with interleukin 2 (IL2) has been described as an effective mitotic stimulant in B-cell disorders, not only in chronic lymphocytic leukemia (CLL) but also in a range of other B-cell malignancies. Here, we describe the comparison of two B-cell mitogens, lipopolysaccharide (LPS), and DSP30 combined with IL2 as mitogens in a range of common B-cell disorders excluding CLL. The results showed that DSP30/IL2 was an effective mitogen in mature B-cell disorders, revealing abnormal cytogenetic results in a range of B-cell malignancies. The abnormality rate increased when compared to the use of LPS to 64% (DSP30/IL2) from 14% (LPS). In a number of cases the disease burden was proportionally very low, less than 10% of white cells. In 37% of these cases, the DSP30 culture revealed abnormal results. Importantly, we also obtained abnormal conventional cytogenetics results in 3 bone marrow cases in which immunophenotyping showed an absence of an abnormal B-cell clone. In these cases, the cytogenetics results correlated with the provisional diagnosis and altered their staging level. The use of DSP30 and IL2 is recommended for use in many B-cell malignancies as an effective mitogen and their use has been shown to enable successful culture of the malignant clone, even at very low levels of disease.

Upregulation of GRAIL is associated with impaired CD4 T cell proliferation in sepsis.

The loss of numbers and functionality of CD4 T cells is observed in sepsis; however, the mechanism remains elusive. Gene related to anergy in lymphocytes (GRAIL) is critical for the impairment of CD4 T cell proliferation. We therefore sought to examine the role of GRAIL in CD4 T cell proliferation during sepsis. Sepsis was induced in 10-wk-old male C57BL/6 mice by cecal ligation and puncture. Splenocytes were isolated and subjected to flow cytometry to determine CD4 T cell contents. CD4 T cell proliferation was assessed by CFSE staining, and the expression of GRAIL in splenocytes was measured by immunohistochemistry, real-time PCR, and flow cytometry. The expressions of IL-2 and early growth response-2 were determined by real-time PCR. As compared with shams, the numbers of CD4 T cells were significantly reduced in spleens. Septic CD4 T cells were less efficient in proliferation than shams. The IL-2 expression was significantly reduced, whereas the GRAIL expression was significantly increased in septic mice splenocytes as compared with shams. The small interfering RNA-mediated knockdown of GRAIL expression re-established the CD4 T cell proliferation ability ex vivo. Similarly, the treatment with recombinant murine IL-2 to the septic CD4 T cells restored their proliferation ability by downregulating GRAIL expression. Our findings reveal a novel association of the increased GRAIL expression with impaired CD4 T cell proliferation, implicating an emerging therapeutic tool in sepsis.

ß-Catenin is required for the differentiation of iNKT2 and iNKT17 cells that augment IL-25-dependent lung inflammation.

BACKGROUND: Invariant Natural Killer T (iNKT) cells have been implicated in lung inflammation in humans and also shown to be a key cell type in inducing allergic lung inflammation in mouse models. iNKT cells differentiate and acquire functional characteristics during development in the thymus. However, the correlation between development of iNKT cells in the thymus and role in lung inflammation remains unknown. In addition, transcriptional control of differentiation of iNKT cells into iNKT cell effector subsets in the thymus during development is also unclear. In this report we show that ß-catenin dependent mechanisms direct differentiation of iNKT2 and iNKT17 subsets but not iNKT1 cells. METHODS: To study the role for ß-catenin in lung inflammation we utilize mice with conditional deletion and enforced expression of ß-catenin in a well-established mouse model for IL-25-dependen lung inflammation. RESULTS: Specifically, we demonstrate that conditional deletion of ß-catenin permitted development of mature iNKT1 cells while impeding maturation of iNKT2 and 17 cells. A role for ß-catenin expression in promoting iNKT2 and iNKT17 subsets was confirmed when we noted that enforced transgenic expression of ß-catenin in iNKT cell precursors enhanced the frequency and number of iNKT2 and iNKT17 cells at the cost of iNKT1 cells. This effect of expression of ß-catenin in iNKT cell precursors was cell autonomous. Furthermore, iNKT2 cells acquired greater capability to produce type-2 cytokines when ß-catenin expression was enhanced. DISCUSSION: This report shows that ß-catenin deficiency resulted in a profound decrease in iNKT2 and iNKT17 subsets of iNKT cells whereas iNKT1 cells developed normally. By contrast, enforced expression of ß-catenin promoted the development of iNKT2 and iNKT17 cells. It was important to note that the majority of iNKT cells in the thymus of C57BL/6 mice were iNKT1 cells and enforced expression of ß-catenin altered the pattern to iNKT2 and iNKT17 cells suggesting that ß-catenin may be a major factor in the distinct pathways that critically direct differentiation of iNKT effector subsets. CONCLUSIONS: Thus, we demonstrate that ß-catenin expression in iNKT cell precursors promotes differentiation toward iNKT2 and iNKT17 effector subsets and supports enhanced capacity to produce type 2 and 17 cytokines which in turn augment lung inflammation in mice.

Milk fat globule epidermal growth factor-factor 8-derived peptide attenuates organ injury and improves survival in sepsis.

INTRODUCTION: Sepsis involves overwhelming inflammatory responses with subsequent immune-suppression that can lead to multiple organ dysfunction and ultimately death. Milk fat globule epidermal growth factor-factor 8 (MFG-E8) is a secretory protein found to have multiple biological activities against autoimmune and inflammatory diseases. MFG-E8 contains an Arg-Gly-Asp (RGD) motif involved in cell-cell and cell-matrix interactions. In sepsis, excessive neutrophils migration through endothelial cells and matrix to sites of inflammation results in organ damage. We hypothesized that MFG-E8-derived short peptides (MSP) flanking its RGD motif could provide protection against organ injury in sepsis. METHODS: The differentiated human neutrophil-like HL-60 cells (dHL60) were incubated with a series of peptides flanking the RGD motif of human MFG-E8 for a cell adhesion assay to fibronectin or human pulmonary artery endothelial cells (PAECs). For the induction of sepsis, male C57BL/6 mice (20-25 g) were subjected to cecal ligation and puncture (CLP). Peptide MSP68 (1 mg/kg body weight) or normal saline (vehicle) was injected intravenously at 2 h after CLP. Blood and tissue samples were collected at 20 h after CLP for various measurements. RESULTS: After screening, peptide MSP68 (VRGDV) had the highest inhibition of dHL-60 cell adhesion to fibronectin by 55.8 % and to PAEC by 67.7 %. MSP68 treatment significantly decreased plasma levels of organ injury marker AST by 37.1 % and the proinflammatory cytokines IL-6 and TNF-α by 61.9 % and 22.1 %, respectively after CLP. MSP68 improved the integrity of microscopic architectures, decreased IL-6 levels in the lungs by 85.1 %, and reduced apoptosis. MSP68 treatment also significantly reduced the total number of neutrophil infiltration by 61.9 % and 48.3 % as well as MPO activity by 40.8 % and 47.3 % in the lungs and liver, respectively, after CLP. Moreover, the number of bacteria translocated to mesenteric lymph nodes was decreased by 57 % with MSP68 treatment. Finally, the 10-day survival rate was increased from 26 % in the vehicle group to 58 % in the MSP68-treated group. CONCLUSIONS: MSP68 effectively inhibits excessive neutrophils infiltrating to organs, leading to moderate attenuation of organ injury and significantly improved survival in septic mice. Thus, MSP68 may be a potential therapeutic agent for treating sepsis.

T cell factor-1 and ß-catenin control the development of memory-like CD8 thymocytes.

Innate memory-like CD8 thymocytes develop and acquire effector function during maturation in the absence of encounter with Ags. In this study, we demonstrate that enhanced function of transcription factors T cell factor (TCF)-1 and ß-catenin regulate the frequency of promyelocytic leukemia zinc finger (PLZF)-expressing, IL-4-producing thymocytes that promote the generation of eomesodermin-expressing memory-like CD8 thymocytes in trans. In contrast, TCF1-deficient mice do not have PLZF-expressing thymocytes and eomesodermin-expressing memory-like CD8 thymocytes. Generation of TCF1 and ß-catenin-dependent memory-like CD8 thymocytes is non-cell-intrinsic and requires the expression of IL-4 and IL-4R. CD8 memory-like thymocytes migrate to the peripheral lymphoid organs, and the memory-like CD8 T cells rapidly produce IFN-γ. Thus, TCF1 and ß-catenin regulate the generation of PLZF-expressing thymocytes and thereby facilitate the generation of memory-like CD8 T cells in the thymus.

Receptor-interacting protein kinase 3 deficiency inhibits immune cell infiltration and attenuates organ injury in sepsis.

INTRODUCTION: Sepsis is defined as a systemic hyper-inflammatory immune response, with a subsequent immune-suppressive phase, which leads to multiple organ dysfunction and late lethality. Receptor-interacting protein kinase 3 (RIPK3)-dependent necrosis is implicated in driving tumor necrosis factor alpha (TNF-α)- and sepsis-induced mortality in mice. However, it is unknown if RIPK3 deficiency has any impact on immune cell trafficking, which contributes to organ damage in sepsis. METHODS: To study this, male wild-type (WT) and RIPK3-deficient (Ripk3-/-) mice on C57BL/6 background were subjected to sham operation or cecal ligation and puncture (CLP)-induced sepsis. Blood and tissue samples were collected 20 hours post-CLP for various measurements. RESULTS: In our severe sepsis model, the mean survival time of Ripk3-/- mice was significantly extended to 68 hours compared to 41 hours for WT mice. Ripk3-/- mice had significantly decreased plasma levels of TNF-α and IL-6 and organ injury markers compared to WT mice post-CLP. In the lungs, Ripk3-/- mice preserved better integrity of microscopic structure with reduced apoptosis, and decreased levels of IL-6, macrophage inflammatory protein (MIP)-2 and keratinocyte-derived chemokine (KC), compared to WT. In the liver, the levels of MIP-1, MIP-2 and KC were also decreased in septic Ripk3-/- mice. Particularly, the total number of neutrophils in the lungs and liver of Ripk3-/- mice decreased by 59.9% and 66.7%, respectively, compared to WT mice post-CLP. In addition, the number of natural killer (NK) and CD8T cells in the liver decreased by 64.8% and 53.4%, respectively, in Ripk3-/- mice compared to WT mice post-sepsis. CONCLUSIONS: Our data suggest that RIPK3 deficiency modestly protected from CLP-induced severe sepsis and altered the immune cell trafficking in an organ-specific manner attenuating organ injury. Thus, RIPK3 acts as a detrimental factor in contributing to the organ deterioration in sepsis.

Early markers of occult megaloblastosis for low-cost detection of hyperhomocysteinemia in patients with ischaemic stroke: preventive approach for primary health care.

Recent studies have focussed on the association between elevated homocysteine levels with megaloblastic changes and thromboembolic events, but the relationship between occult megaloblastosis (with normal haemoglobin levels) and ischaemic stroke has not been widely explored. The objective of this study is to establish a simple and economical marker for the detection of occult megaloblastosis at the community health care level in developing countries. A hundred patients who met the inclusion criteria were studied. At the 5% level of significance, the levels of cobalamin and folate were significantly lower, while the number of hypersegmented neutrophils on the peripheral smear was higher in patients from Group A (70 patients with high homocysteine) compared with the patients in Group B (30 patients with normal homocysteine). Forty-five (64.2%) of the 70 patients in Group A showed hypersegmentation of neutrophils in the peripheral smear. The high cost and difficulty in performing the vitamin assays limit their use as early markers of megaloblastosis. Hence, we conclude that in developing countries, the detection of hypersegmented neutrophils can be used at the primary healthcare level for early diagnosis of occult megaloblastosis, so that early therapeutic interventions with vitamins can prevent attacks of hyperhomocysteinemia-induced ischaemic stroke.

Methoxyacetone revisited.

Conformational space of methoxyacetone (MA) was studied at the MP2/6-311++G(d,p) and DFT(B3LYP)/6-311++G(d,p) levels of theory. Computations predict MA to adopt four conformations, resulting from internal rotations around the O=C-C-O (Trans, Cis) and C-C-O-C (trans, gauche) dihedral angles. The Tt (Trans-trans) conformer is the most stable. The computed energies of two gauche (Tg and Cg) conformers fall in the 3-8 kJ mol-1 range above Tt and should account for 1/3 of the room-temperature gas-phase equilibrium. The energy of Ct form is 11 kJ mol-1 above Tt, and its expected population is negligible (below 1 %). In our earlier work, MA monomers were isolated in cryogenic argon matrices and characterized by infrared spectroscopy. In the experiment, only the most stable Tt conformer was detected in the sample. Signatures of the other conformers were not detected, either in freshly deposited samples, or in samples subjected to different UV irradiations. We rationalize those observations in terms of computed barriers for intramolecular torsions, indicating occurrence of conformational cooling during deposition. The experimental infrared spectrum of the Tt form is now assigned with the aid of anharmonic DFT computations. Exposure of MA to UV irradiation in the 300-260 nm range led to photolysis, according to the Norrish type II mechanism, resulting in dimer between enol acetone and formaldehyde observed as a cage-confined intermediate photoproduct. The subsequent photolysis resulted in the formation of carbon monoxide as the dominating photoproduct, formed in the Norrish type I photoreaction. Mechanistic interpretation of this photo decarbonylation reaction is presented.

T cell factor-1 negatively regulates expression of IL-17 family of cytokines and protects mice from experimental autoimmune encephalomyelitis.

Activated CD4 T cells are associated with protective immunity and autoimmunity. The manner in which the inflammatory potential of T cells and resultant autoimmunity is restrained is poorly understood. In this article, we demonstrate that T cell factor-1 (TCF1) negatively regulates the expression of IL-17 and related cytokines in activated CD4 T cells. We show that TCF1 does not affect cytokine signals and expression of transcription factors that have been shown to regulate Th17 differentiation. Instead, TCF1 regulates IL-17 expression, in part, by binding to the regulatory regions of the Il17 gene. Moreover, TCF1-deficient Th17 CD4 T cells express higher levels of IL-7Rα, which potentially promotes their survival and expansion in vivo. Accordingly, TCF1-deficient mice are hyperresponsive to experimental autoimmune encephalomyelitis. Thus, TCF1, a constitutively expressed T cell-specific transcription factor, is a critical negative regulator of the inflammatory potential of TCR-activated T cells and autoimmunity.

Prevalence of specific developmental disorder of scholastic skill in school students in Chandigarh, India.

BACKGROUND & OBJECTIVES: Several studies have been conducted in India to determine the prevalence of learning disabilities in school children which has been reported to be 3-10 per cent among students population. The present study was conducted to find out prevalence of specific developmental disorder of scholastic skills in students of classes VII to XII and to find out feasibility of screening tool in Chandigarh, India. METHODS: A cross-sectional study on school students was carried out in two phases. The students were drawn from classes VII to XII from 10 schools of Chandigarh, India. Details of academic performance of all the students was taken, subjectively from class teachers and objectively from the marks obtained in the last academic session. In phase I, 2402 students were assessed. In phase II, 108 students were randomly selected for evaluation for assessing sensitivity and specificity of screening proforma for teachers. A total of 124 students from phase I and all students in phase II were assessed in detail. Tests of intelligence (Malin's Intelligence Scale for Indian Children and Standard Progressive Matrices), and NIMHANS Index for specific learning disability (SLD) battery were administered. RESULTS: A total of 38 students were found to be having specific developmental disorder of scholastic skills in phase I, that gave a prevalence of 1.58 per cent. Majority had mixed type of errors on SLD battery. There were more boys diagnosed with specific learning disability. Teacher's screening instrument had high sensitivity (90.385) and specificity (94.68). INTERPRETATION & CONCLUSIONS: The findings of our study conducted in community, showed that specific learning disability was not identified even till later age. The screening instrument thus could be used by teachers to suspect students with specific learning disability.

Extracellular CIRP Induces Calpain Activation in Neurons via PLC-IP3-Dependent Calcium Pathway.

Abnormal calcium homeostasis, activation of protease calpain, generation of p25 and hyperactivation of cyclin-dependent kinase 5 (Cdk5) have all been implicated in the pathogenesis of neurogenerative diseases including Alzheimer's disease. We have recently shown that extracellular cold-inducible RNA-binding protein (eCIRP) induces Cdk5 activation via p25. However, the precise molecular mechanism by which eCIRP regulates calcium signaling and calpain remains to be addressed. We hypothesized that eCIRP regulates p25 via Ca2+-dependent calpain activation. eCIRP increased calpain activity and decreased the endogenous calpain inhibitor calpastatin in Neuro 2a (N2a) cells. Calpain inhibition with calpeptin attenuated eCIRP-induced calpain activity and p25. eCIRP specifically upregulated cytosolic calpain 1, and calpain 1 silencing attenuated the eCIRP-induced increase in p25. eCIRP stimulation increased cytosolic free Ca2+, especially in hippocampal neuronal HT22 cells, which was attenuated by the eCIRP inhibitor Compound 23 (C23). Endoplasmic reticulum (ER) inositol 1,4,5-trisphosphate receptor (IP3R) inhibition using 2-aminoethoxy-diphenyl-borate or xestospongin-C (X-C), interleukin-6 receptor alpha (IL-6Rα)-neutralization, and phospholipase C (PLC) inhibition with U73122 attenuated eCIRP-induced Ca2+ increase, while Ca2+ influx across the plasma membrane remained unaffected by eCIRP. Finally, C23, IL-6Rα antibody, U73122 and X-C attenuated eCIRP-induced p25 in HT-22 cells. In conclusion, the current study uncovers eCIRP-triggered Ca2+ release from ER stores in an IL-6Rα/PLC/IP3-dependent manner as a novel molecular mechanism underlying eCIRP's induction of Cdk5 activity and potential involvement in neurodegeneration.

Using heavy atom rare gas matrix to control the reactivity of 4-methoxybenzaldehyde: a comparison with benzaldehyde.

Different patterns of photochemical behavior were observed for 4-methoxybenzaldehyde (p-anisaldehyde) isolated in xenon and in argon matrices. Monomers of the compound isolated in solid Xe decarbonylate upon middle ultraviolet irradiation, yielding methoxybenzene (anisole), and CO. On the other hand, p-anisaldehyde isolated in an Ar matrix and subjected to identical irradiation, predominantly isomerizes to the closed-ring isomeric ketene (4-methoxycyclohexa-2,4-dien-1-ylidene) methanone. Experimental detection of a closed-ring ketene photoproduct, generated from an aromatic aldehyde, constitutes a rare observation. The difference between the patterns of photochemical transformations of p-anisaldehyde isolated in argon and xenon environments can be attributed to the external heavy-atom effect, where xenon enhances the rate of intersystem crossing from the singlet to the triplet manifold in which decarbonylation (via p-methoxybenzoyl radical) takes place. The parent compound, benzaldehyde, decarbonylates (to benzene + CO) when subjected to middle ultraviolet irradiation in both argon and xenon matrices. This demonstrates the role of the methoxy p-anisaldehyde substituent in activation of the reaction channel leading to the formation of the ketene photoproduct.

Prospective cohort study on the use of low molecular weight heparin calibrated anti-Xa assay for measurement of direct oral Xa inhibitors in ex vivo patient samples.

Drug-specific anti-Xa chromogenic assays are recommended for measurement of direct anti-Xa inhibitor levels but are not routinely available in many institutions. We performed a prospective study to determine: (1) the relationship between low molecular weight heparin (LMWH) calibrated anti-Xa measurements and apixaban or rivaroxaban levels measured using drug-specific anti-Xa assays and, (2) if a LMWH calibrated anti-Xa assay can be used to detect clinically significant apixaban or rivaroxaban levels. Haematology outpatients on rivaroxaban or apixaban for at least 72 h were recruited for this study. Anti-Xa LMWH assay was performed using the Innovance Heparin Anti-Xa kit/calibrator. Drug-specific levels were determined using STA-Liquid anti-Xa kit/STA-Apixaban or STA-Rivaroxaban calibrators. Serial dilutions with pooled normal plasma were performed for specimens with anti-Xa LMWH activity greater than 1.50 ng/mL to obtain anti-Xa levels within the reportable range (0.10-1.50 ng/mL) and multiplied by the dilution factor to determine actual anti-Xa level. Seventy-five (39 rivaroxaban, 36 apixaban) specimens from 67 patients (mean age 60.3 years; 53.3% males) were available for analysis. Rivaroxaban levels ranged from <25 to 500 ng/mL while apixaban levels ranged from <20 to 236.1 ng/mL. For both rivaroxaban and apixaban, there was linear and good correlation (R2 = 0.96) between direct oral anticoagulants and anti-Xa LMWH levels. Using the correlation equation from our data, a rivaroxaban concentration of 50 ng/mL [International Society on Thrombosis and Haemostasis (ISTH) threshold for consideration of antidotes in bleeding patients] and 30 ng/mL (ISTH threshold for consideration of reversal agents prior to interventions), corresponds to anti-Xa LMWH levels of 0.50 and 0.35 IU/mL, respectively. For apixaban the corresponding anti-Xa LMWH levels were 0.35 and 0.20 IU/mL, respectively. In conclusion, LWWH calibrated anti-Xa assay can be used in emergency situations to screen for clinically significant apixaban or rivaroxaban levels when drug-specific calibrators are not available.

Conformation-changing aggregation in hydroxyacetone: a combined low-temperature FTIR, jet, and crystallographic study.

Aggregation in hydroxyacetone (HA) is studied using low-temperature FTIR, supersonic jet expansion, and X-ray crystallographic (in situ cryocrystallization) techniques. Along with quantum chemical methods (MP2 and DFT), the experiments unravel the conformational preferences of HA upon aggregation to dimers and oligomers. The O-H···O═C intramolecular hydrogen bond present in the gas-phase monomer partially opens upon aggregation in supersonic expansions, giving rise to intermolecular cooperatively enhanced O-H···O-H hydrogen bonds in competition with isolated O-H···O═C hydrogen bonds. On the other hand, low-temperature IR studies on the neat solid and X-ray crystallographic data reveal that HA undergoes profound conformational changes upon crystallization, with the HOCC dihedral angle changing from ~0° in the gas phase to ~180° in the crystalline phase, hence giving rise to a completely new conformation. These conclusions are supported by theoretical calculations performed on the geometry derived from the crystalline phase.

Pre-TCR-induced beta-catenin facilitates traversal through beta-selection.

Pre-TCR induced signals regulate development of the alphabeta TCR lineage cells at the beta-selection checkpoint. We have previously shown that conditional deletion of beta-catenin, a central mediator of Wnt-beta-catenin-T cell factor signaling pathway, impairs traversal through the beta-selection checkpoint. We now provide a molecular basis for the impairment. We demonstrate that pre-TCR signals specifically stabilize beta-catenin in CD4-CD8- double negative thymocytes during beta-selection. Pre-TCR induced Erk activity was required to stabilize beta-catenin. Enforced expression of stabilized beta-catenin was sufficient to mediate aspects of beta-selection including sustained expression of early growth response (Egr) genes. Consistently, deletion of beta-catenin reduced induction of Egr gene expression by the pre-TCR signal and blocked efficient beta-selection. Thus, we demonstrate that pre-TCR induced beta-catenin sustains expression of Egr genes that facilitate traversal through the beta-selection checkpoint.

Sustained expression of pre-TCR induced beta-catenin in post-beta-selection thymocytes blocks T cell development.

Pre-TCR and IL-7R signals regulate beta-selection of thymocytes and then must be down-regulated for further development. However, the molecular events that control down-regulation remain unknown. We and others have previously shown that beta-catenin in cooperation with TCF regulates beta-selection. In this paper, we demonstrate that beta-catenin expression is stringently regulated by intrathymic signals, it is expressed at the highest levels in the pre-TCR signaled thymocytes, and is down-regulated in post-beta-selection thymocytes. Pre-TCR-induced beta-catenin regulates initial stages of pre-TCR signaling including expression of early growth response (Egr) genes but must be down-regulated to express RORgammat, which is essential for maturation to the CD4+CD8+ double positive (DP) stage. Sustained expression of beta-catenin results in the generation of IL-7R-, Egr-, and TGFbeta-expressing pre-DP thymocytes that are blocked in development. These data are consistent with a model in which post-beta-selection, pre-TCR-induced beta-catenin expression must return to background levels for efficient transition to the DP stage.