CD226 implicated in Akt-dependent apoptosis of CD4+ T cell contributes to asthmatic pathogenesis.

Asthma is a chronic airway inflammatory disease in which CD4+ T cell dysregulation occurs. Here, we investigated the molecular role and clinical significance of CD226, a costimulatory molecule of T lymphocytes, in the development of allergic asthma. Our results revealed that the expression of CD226 was significantly increased in CD4+ effector T cells, especially in T helper (Th) 2 cells and Th17 cells in patients with asthma. Moreover, CD4+ T cell-specific Cd226-knockout mice were generated and together with littermates were challenged with ovalbumin (OVA) to establish a model of allergic asthma. We found that CD226 deficiency in CD4+ T cells mitigated lung inflammation, IgE production, and eosinophil infiltration and reduced airway remodeling in experimental allergic asthma. However, the impact of CD226 on asthma was independent of Treg cell modulation. Through RNA-seq data analysis, the apoptosis pathway was screened. Mechanistically, CD226 deletion promoted CD4+ T cell late apoptosis via the activation of Caspase-3 in an Akt-dependent manner. Furthermore, blocking CD226 signaling with a recombinant fusion protein attenuated asthma features in mice and achieved a good therapeutic effect. Overall, this study revealed a unique role of CD226 in CD4+ T cell regulation in asthma pathogenesis. Therefore, targeting CD226 may provide new insights into the clinical treatment of asthma.

Organocatalytic enantioselective construction of Si-stereocenters: recent advances and perspectives.

Silicon-stereogenic chiral organosilanes have found increasing applications in synthetic chemistry, medicinal chemistry, and materials science. In this context, various asymmetric catalytic methods have been established for the diverse synthesis of silicon-stereogenic silanes. In particular, asymmetric organocatalysis is emerging as an important and complementary synthetic tool for the enantioselective construction of silicon-stereocenters, along with the rapid development of chiral-metal catalyzed protocols. Its advent provides a powerful platform to achieve functionalized silicon-stereogenic organosilanes with structural diversity, and should lead to great development in chiral organosilicon chemistry. In this Tutorial Review, we highlight these latest achievements from two aspects: desymmetrizations of prochiral tetraorganosilanes and dynamic kinetic asymmetric transformations of racemic organosilanes by employing five organocatalytic activation modes. The advantages, limitations and synthetic value of each protocol, as well as the synthetic opportunities still open for further exploration, are also discussed.

Polyimide Films Based on ß-Cyclodextrin Polyrotaxane with Low Dielectric and Excellent Comprehensive Performance.

In order to prepare polyimide (PI) films with a low dielectric constant and excellent comprehensive performance, a two-step method was employed in this study to integrate ß-cyclodextrin into a semi-aromatic fluorine-containing polyimide ternary system. By introducing trifluoromethyl groups to reduce the dielectric constant, the dielectric constant was further reduced to 2.55 at 10 MHz. Simultaneously, the film exhibited noteworthy thermal stability (a glass transition temperature exceeding 300 °C) and a high coefficient of thermal expansion. The material also demonstrated outstanding mechanical properties, boasting a strength of 122 MPa and a modulus of 2.2 GPa, along with high optical transparency (transmittance reaching up to 89% at 450 nm). Moreover, the inherent high transparency of colorless polyimide (CPI) combined with good stretchability contributed to the attainment of a low dielectric constant. This strategic approach not only opens up new opportunities for novel electroactive polymers but also holds potential applications in flexible displays, circuit printing, and chip packaging.

Differential and substrate-specific inhibition of γ-secretase by the C-terminal region of ApoE2, ApoE3, and ApoE4.

Aberrant low γ-secretase activity is associated with most of the presenilin mutations that underlie familial Alzheimer's disease (fAD). However, the role of γ-secretase in the more prevalent sporadic AD (sAD) remains unaddressed. Here, we report that human apolipoprotein E (ApoE), the most important genetic risk factor of sAD, interacts with γ-secretase and inhibits it with substrate specificity in cell-autonomous manners through its conserved C-terminal region (CT). This ApoE CT-mediated inhibitory activity is differentially compromised in different ApoE isoforms, resulting in an ApoE2 > ApoE3 > ApoE4 potency rank order inversely correlating to their associated AD risk. Interestingly, in an AD mouse model, neuronal ApoE CT migrates to amyloid plaques in the subiculum from other regions and alleviates the plaque burden. Together, our data reveal a hidden role of ApoE as a γ-secretase inhibitor with substrate specificity and suggest that this precision γ-inhibition by ApoE may protect against the risk of sAD.

Orai3 and Orai1 mediate CRAC channel function and metabolic reprogramming in B cells.

The essential role of store-operated Ca2+ entry (SOCE) through Ca2+ release-activated Ca2+ (CRAC) channels in T cells is well established. In contrast, the contribution of individual Orai isoforms to SOCE and their downstream signaling functions in B cells are poorly understood. Here, we demonstrate changes in the expression of Orai isoforms in response to B cell activation. We show that both Orai3 and Orai1 mediate native CRAC channels in B cells. The combined loss of Orai1 and Orai3, but not Orai3 alone, impairs SOCE, proliferation and survival, nuclear factor of activated T cells (NFAT) activation, mitochondrial respiration, glycolysis, and the metabolic reprogramming of primary B cells in response to antigenic stimulation. Nevertheless, the combined deletion of Orai1 and Orai3 in B cells did not compromise humoral immunity to influenza A virus infection in mice, suggesting that other in vivo co-stimulatory signals can overcome the requirement of BCR-mediated CRAC channel function in B cells. Our results shed important new light on the physiological roles of Orai1 and Orai3 proteins in SOCE and the effector functions of B lymphocytes.

Highly Enantioselective Construction of Multifunctional Silicon-Stereogenic Silacycles by Asymmetric Enamine Catalysis.

We report the first highly enantioselective construction of silicon-stereocenters by asymmetric enamine catalysis. An unprecedented desymmetric intramolecular aldolization of prochiral siladials was thus developed for the facile access of multifunctional silicon-stereogenic silacycles in high to excellent enantioselectivity. With an enal moiety, these adducts could be readily elaborated for the diverse synthesis of silicon-stereogenic compounds, and for late-stage modification.

Nickel-catalyzed regio- and enantio-selective Markovnikov hydromonofluoroalkylation of 1,3-dienes.

A highly enantio- and regio-selective Markovnikov hydromonofluoro(methyl)alkylation of 1,3-dienes was developed using redox-neutral nickel catalysis. It provided a facile strategy to construct diverse monofluoromethyl- or monofluoroalkyl-containing chiral allylic molecules. Notably, this represents the first catalytic asymmetric Markovnikov hydrofluoroalkylation of olefins. The practicability of this methodology is further highlighted by its broad substrate scope, mild base-free conditions, excellent enantio- and regio-selectivity, and diversified product elaborations to access useful fluorinated building blocks.

The mitochondrial sodium/calcium exchanger NCLX (Slc8b1) in B lymphocytes.

Antigen receptor stimulation triggers cytosolic Ca2+ signals, which activate transcriptional and metabolic programs critical for immune function. B-cell receptor (BCR) engagement causes rapid cytosolic Ca2+ rise through the ubiquitous store-operated calcium entry (SOCE) pathway. Slc8b1, which encodes the mitochondrial Na+/Ca2+ exchanger (NCLX), extrudes Ca2+ out of the mitochondria and maintains optimal SOCE activity. Inhibition of NCLX in DT40 and A20 B lymphocyte lines was recently shown to impair cytosolic Ca2+ transients in response to antigen-receptor stimulation, however the downstream functional consequences of this impairment remain unclear. Here, we generated Slc8b1 knockout A20 B-cell lines using CRISPR/Cas9 technology and B-cell specific Slc8b1 knockout mice. Surprisingly, while loss of Slc8b1 in B lymphocytes led to reduction in SOCE, it had a marginal effect on mitochondrial Ca2+ extrusion, suggesting that NCLX is not the major mitochondrial Ca2+ extrusion mechanism in B cells. Furthermore, endoplasmic reticulum (ER) Ca2+ content and rates of ER depletion and refilling remained unaltered in Slc8b1 knockout B cells. Slc8b1 deficiency increased mitochondrial production of oxidants, reduced mitochondrial bioenergetics and altered mitochondrial ultrastructure. B-cell specific Slc8b1 knockout mice showed reduced germinal center B cell responses following foreign antigen and pathogen driven immune responses. Our studies provide novel insights into the function of Slc8b1 in germinal center B cells and its contribution to B-cell signaling and effector function.

The effect of the nucleotides immediately upstream of the AUG start codon on the efficiency of translation initiation in sperm cells.

It is widely known that an optimal nucleotide sequence context immediately upstream of the AUG start codon greatly improves the efficiency of translation initiation of mRNA in mammalian and plant somatic cells, which in turn increases protein levels. However, it is still unclear whether a similar regulatory mechanism is also present in highly differentiated cells. Here, we surveyed this issue in Arabidopsis thaliana sperm cells and found that the sequence context-mediated regulation of translation initiation in sperm cells is generally similar to that in somatic cells. A simple motif of four adenine nucleotides at positions - 1 to - 4 greatly improved the efficiency of translation initiation, and when the motif was present there, translation was even initiated at some non-AUG codons in sperm cells. However, unlike that in mammalian cells, a mainly effective nucleotide site to regulate the efficiency of translation initiation was not present at positions - 1 to - 4 in sperm cells. Meanwhile, different from somatic cells, sperm cells did not use eukaryotic translation initiation factor 1 to regulate the efficiency in a poor context consisting of the lowest frequency nucleotides. All these results contribute to our understanding of the cytoplasmic event of translation initiation in highly differentiated sperm cells.

Curcumin and NCLX inhibitors share anti-tumoral mechanisms in microsatellite-instability-driven colorectal cancer.

BACKGROUND AND AIMS: Recent evidences highlight a role of the mitochondria calcium homeostasis in the development of colorectal cancer (CRC). To overcome treatment resistance, we aimed to evaluate the role of the mitochondrial sodium-calcium-lithium exchanger (NCLX) and its targeting in CRC. We also identified curcumin as a new inhibitor of NCLX. METHODS: We examined whether curcumin and pharmacological compounds induced the inhibition of NCLX-mediated mitochondrial calcium (mtCa2+) extrusion, the role of redox metabolism in this process. We evaluated their anti-tumorigenic activity in vitro and in a xenograft mouse model. We analyzed NCLX expression and associations with survival in The Cancer Genome Atlas (TCGA) dataset and in tissue microarrays from 381 patients with microsatellite instability (MSI)-driven CRC. RESULTS: In vitro, curcumin exerted strong anti-tumoral activity through its action on NCLX with mtCa2+ and reactive oxygen species overload associated with a mitochondrial membrane depolarization, leading to reduced ATP production and apoptosis. NCLX inhibition with pharmacological and molecular approaches reproduced the effects of curcumin. NCLX inhibitors decreased CRC tumor growth in vivo. Both transcriptomic analysis of TCGA dataset and immunohistochemical analysis of tissue microarrays demonstrated that higher NCLX expression was associated with MSI status, and for the first time, NCLX expression was significantly associated with recurrence-free survival. CONCLUSIONS: Our findings highlight a novel anti-tumoral mechanism of curcumin through its action on NCLX and mitochondria calcium overload that could benefit for therapeutic schedule of patients with MSI CRC.

STIM1 is a core trigger of airway smooth muscle remodeling and hyperresponsiveness in asthma.

Airway remodeling and airway hyperresponsiveness are central drivers of asthma severity. Airway remodeling is a structural change involving the dedifferentiation of airway smooth muscle (ASM) cells from a quiescent to a proliferative and secretory phenotype. Here, we show up-regulation of the endoplasmic reticulum Ca2+ sensor stromal-interacting molecule 1 (STIM1) in ASM of asthmatic mice. STIM1 is required for metabolic and transcriptional reprogramming that supports airway remodeling, including ASM proliferation, migration, secretion of cytokines and extracellular matrix, enhanced mitochondrial mass, and increased oxidative phosphorylation and glycolytic flux. Mechanistically, STIM1-mediated Ca2+ influx is critical for the activation of nuclear factor of activated T cells 4 and subsequent interleukin-6 secretion and transcription of pro-remodeling transcription factors, growth factors, surface receptors, and asthma-associated proteins. STIM1 drives airway hyperresponsiveness in asthmatic mice through enhanced frequency and amplitude of ASM cytosolic Ca2+ oscillations. Our data advocates for ASM STIM1 as a target for asthma therapy.

Singular perturbation-based saturated adaptive control for underactuated Euler-Lagrange systems.

This paper proposes a saturated smooth adaptive controller for regulating a certain type of underactuated Euler-Lagrange systems (UELSs) with modeling uncertainties and control saturations based on a singular perturbation approach. Compared with relevent literature, the advantages of the proposed controller include: (1) it renders the UELS semiglobally asymptotically track the desired position without the violation of control input constraints; (2) high-order derivatives of positions are not required in its implementation. The Hoppensteadt's Theorem is employed to show that the proposed saturated controller renders the UELS semiglobally asymptotically stable about the desired set point with the satisfaction of control input constraints. The control effectiveness is validated by simulations on a two-link compliant robot arm.

PKC-ε regulates vesicle delivery and focal exocytosis for efficient IgG-mediated phagocytosis.

Protein kinase C (PKC)-ε is required for membrane addition during IgG-mediated phagocytosis, but its role in this process is ill defined. Here, we performed high-resolution imaging, which reveals that PKC-ε exits the Golgi and enters phagosomes on vesicles that then fuse. TNF and PKC-ε colocalize at the Golgi and on vesicles that enter the phagosome. Loss of PKC-ε and TNF delivery upon nocodazole treatment confirmed vesicular transport on microtubules. That TNF+ vesicles were not delivered in macrophages from PKC-ε null mice, or upon dissociation of the Golgi-associated pool of PKC-ε, implies that Golgi-tethered PKC-ε is a driver of Golgi-to-phagosome trafficking. Finally, we established that the regulatory domain of PKC-ε is sufficient for delivery of TNF+ vesicles to the phagosome. These studies reveal a novel role for PKC-ε in focal exocytosis - its regulatory domain drives Golgi-derived vesicles to the phagosome, whereas catalytic activity is required for their fusion. This is one of the first examples of a PKC requirement for vesicular trafficking and describes a novel function for a PKC regulatory domain. This article has an associated First Person interview with the first author of the paper.

The Mitochondrial Ca2+ uniporter is a central regulator of interorganellar Ca2+ transfer and NFAT activation.

Mitochondrial Ca2+ uptake tailors the strength of stimulation of plasma membrane phospholipase C-coupled receptors to that of cellular bioenergetics. However, how Ca2+ uptake by the mitochondrial Ca2+ uniporter (MCU) shapes receptor-evoked interorganellar Ca2+ signaling is unknown. Here, we used CRISPR/Cas9 gene knockout, subcellular Ca2+ imaging, and mathematical modeling to show that MCU is a universal regulator of intracellular Ca2+ signaling across mammalian cell types. MCU activity sustains cytosolic Ca2+ signaling by preventing Ca2+-dependent inactivation of store-operated Ca2+ release-activated Ca2+ channels and by inhibiting Ca2+ extrusion. Paradoxically, MCU knockout (MCU-KO) enhanced cytosolic Ca2+ responses to store depletion. Physiological agonist stimulation in MCU-KO cells led to enhanced frequency of cytosolic Ca2+ oscillations, endoplasmic reticulum Ca2+ refilling, nuclear translocation of nuclear factor for activated T cells transcription factors, and cell proliferation, without altering inositol-1,4,5-trisphosphate receptor activity. Our data show that MCU has dual counterbalancing functions at the cytosol-mitochondria interface, whereby the cell-specific MCU-dependent cytosolic Ca2+ clearance and buffering capacity of mitochondria reciprocally regulate interorganellar Ca2+ transfer and nuclear factor for activated T cells nuclear translocation during receptor-evoked signaling. These findings highlight the critical dual function of the MCU not only in the acute Ca2+ buffering by mitochondria but also in shaping endoplasmic reticulum and cytosolic Ca2+ signals that regulate cellular transcription and function.

Palladium/Zinc Co-Catalyzed Asymmetric Hydrogenation of γ-Keto Carboxylic Acids.

A palladium-catalyzed asymmetric hydrogenation of levulinic acid has been successful developed by using Zn(OTf)2 as co-catalyst. The present method not only has provided a strategy in the palladium-catalyzed asymmetric hydrogenation of ketone, but also allowed the preparation of a wide range of chiral γ-valerolactones in good yields with excellent enantioselectivities.

MicroRNA-221 promotes tumor progression by targeting HHIP in human glioblastoma.

BACKGROUND: MicroRNAs are found to be aberrantly expressed in multiple cancers, including glioblastoma (GBM), and microRNA-221 (miR-221) has been verified as an oncogene in various human cancers. Nevertheless, the role of miR-221 in GBM is unclear. This study aimed to investigate the miR-221 expression level in GBM and to evaluate its function and underlying mechanisms. METHODS: Western blotting and qPCR were used to determine the expression of human hedgehog-interacting protein (HHIP) and miR-221 levels. MiR-221-inhibited cell models were constructed, and siRNA was used for HHIP silencing. Cell proliferation was analyzed by MTT and colony formation assays and a subcutaneous xenograft model. Cell migration and invasion was analyzed by wound healing and Transwell invasion assays. A dual luciferase reporter assay system was used to clarify the relationship between miR-221 and HHIP. RESULTS: The results of this study revealed that miR-221 expression was upregulated in GBM tissues and A172, U251, as well as T98G cells, as detected by real-time PCR analysis. MTT, Transwell, and colony formation assays revealed that miR-221 knockdown could suppress GBM cells from proliferating, migrating, and invading in vitro. Moreover, animal experiments showed that tumor growth in vivo was inhibited when miR-221 expression decreased. Furthermore, HHIP was predicted and verified to be a target of miR-221 by bioinformatics analysis, and luciferase and western blot assays. In addition, HHIP silencing rescued the suppressive effect of a miR-221 inhibitor on the proliferation, migration, and invasion of GBM cells. CONCLUSIONS: Our results indicated that miR-221 is upregulated in GBM and enhances tumor progression by targeting HHIP, which suggests this may be a potential therapeutic target for GBM.

[Preparation and application of mouse-derived monoclonal antibodies against mouse CD226].

Objective To prepare and preliminarily identify anti-mouse CD226 monoclonal antibodies (mAbs) using CD226 knockout (CD226 KO) mice as immunized animals. Methods Animals were immunized by recombinant mouse CD226 protein expressed by eukaryotic cells. Anti-mouse CD226 mAbs were prepared by conventional B-cell hybridoma technology. The application of the generated mAbs for flow cytometry and Western blotting was tested. A sandwich ELISA system was established for the detection of soluble CD226 in mice. And the concentrations of plasma soluble CD226 was determined by this sandwich ELISA system in an LPS-induced sepsis mouse model. Results Two hybridoma cell lines secreting mouse anti-mouse CD226 mAbs were successfully prepared. The clones were named mA1.1 and mA1.3, respectively. The antibody subclasses were both IgG1, and the light chains were κ. The obtained mAbs could be applied for flow cytometry to detect exogenous transfected CD226 on the cell surface and natural CD226 on the mouse platelet membrane. In Western blot assay, the mAb could bind to lymphocyte membrane proteins with a relative molecular mass (Mr) of 67 000 that was consistent with the Mr of CD226. In the sandwich ELISA system, the purified mAbs of mA1.3 were coated on ELISA plates as capture antibody, and the mAbs of mA1.1 were labeled with horseradish peroxidase or biotin as detection antibody. The detection sensitivities were 3.0 and 0.25 ng/mL, respectively. The concentration of plasma soluble CD226 of the septic mice was lower than that of the normal mice in the control group. Conclusion The mouse mAbs for identifying mouse CD226 have been prepared successfully and can be applied in various detection techniques.

Distinct pharmacological profiles of ORAI1, ORAI2, and ORAI3 channels.

The ubiquitous Ca2+ release-activated Ca2+ (CRAC) channel is crucial to many physiological functions. Both gain and loss of CRAC function is linked to disease. While ORAI1 is a crucial subunit of CRAC channels, recent evidence suggests that ORAI2 and ORAI3 heteromerize with ORAI1 to form native CRAC channels. Furthermore, ORAI2 and ORAI3 can form CRAC channels independently of ORAI1, suggesting diverse native CRAC stoichiometries. Yet, most available CRAC modifiers are presumed to target ORAI1 with little knowledge of their effects on ORAI2/3 or heteromers of ORAIs. Here, we used ORAI1/2/3 triple-null cells to express individual ORAI1, ORAI2, ORAI3 or ORAI1/2/3 concatemers. We reveal that GSK-7975A and BTP2 essentially abrogate ORAI1 and ORAI2 activity while causing only a partial inhibition of ORAI3. Interestingly, Synta66 abrogated ORAI1 channel function, while potentiating ORAI2 with no effect on ORAI3. CRAC channel activities mediated by concatenated ORAI1-1, ORAI1-2 and ORAI1-3 dimers were inhibited by Synta66, while ORAI2-3 dimers were unaffected. The CRAC enhancer IA65 significantly potentiated ORAI1 and ORAI1-1 activity with marginal effects on other ORAIs. Further, we characterized the profiles of individual ORAI isoforms in the presence of Gd3+ (5µM), 2-APB (5 µM and 50 µM), as well as changes in intracellular and extracellular pH. Our data reveal unique pharmacological features of ORAI isoforms expressed in an ORAI-null background and provide new insights into ORAI isoform selectivity of widely used CRAC pharmacological compounds.