Hepatic neddylation targets and stabilizes electron transfer flavoproteins to facilitate fatty acid ß-oxidation.

Neddylation is a ubiquitination-like pathway that controls cell survival and proliferation by covalently conjugating NEDD8 to lysines in specific substrate proteins. However, the physiological role of neddylation in mammalian metabolism remains elusive, and no mitochondrial targets have been identified. Here, we report that mouse models with liver-specific deficiency of NEDD8 or ubiquitin-like modifier activating enzyme 3 (UBA3), the catalytic subunit of the NEDD8-activating enzyme, exhibit neonatal death with spontaneous fatty liver as well as hepatic cellular senescence. In particular, liver-specific UBA3 deficiency leads to systemic abnormalities similar to glutaric aciduria type II (GA-II), a rare autosomal recessive inherited fatty acid oxidation disorder resulting from defects in mitochondrial electron transfer flavoproteins (ETFs: ETFA and ETFB) or the corresponding ubiquinone oxidoreductase. Neddylation inhibition by various strategies results in decreased protein levels of ETFs in neonatal livers and embryonic hepatocytes. Hepatic neddylation also enhances ETF expression in adult mice and prevents fasting-induced steatosis and mortality. Interestingly, neddylation is active in hepatic mitochondria. ETFs are neddylation substrates, and neddylation stabilizes ETFs by inhibiting their ubiquitination and degradation. Moreover, certain mutations of ETFs found in GA-II patients hinder the neddylation of these substrates. Taken together, our results reveal substrates for neddylation and add insight into GA-II.

CD74 regulates cellularity and maturation of medullary thymic epithelial cells partially by activating the canonical NF-κB signaling pathway.

Thymic epithelial cells (TECs) are indispensable for T cell development, T cell receptor (TCR) repertoire selection, and specific lineage differentiation. Medullary thymic epithelial cells (mTECs), which account for the majority of TECs in adults, are critical for thymocyte selection and self-tolerance. CD74 is a nonpolymorphic transmembrane glycoprotein of major histocompatibility complex class II (MHCII) that is expressed in TECs. However, the exact role of CD74 in regulating the development of mTEC is poorly defined. In this research, we found that loss of CD74 resulted in a significant diminution in the medulla, a selective reduction in the cell number of mature mTECs expressing CD80 molecules, which eventually led to impaired thymic CD4+ T cell development. Moreover, RNA-sequence analysis showed that CD74 deficiency obviously downregulated the canonical nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) signaling pathway in mTECs. Our results suggest that CD74 positively controls mTEC cellularity and maturation partially by activating the canonical NF-κB signaling pathway.

Transplantation of rat frozen-thawed testicular tissues: Does fragment size matter?

Cryopreservation of testicular tissue from pre-pubertal boys before gonadotoxic treatment is an important step in fertility preservation. Yet, this approach remains experimental, and there is still few study measuring the effect of tissue size on the graft after cryopreservation and transplantation. The objective of this study is to detect the effect of varying tissue sizes on the efficacy of rat testicular tissue cryopreservation and transplantation. Varying sizes of rat testicular tissues were frozen-thawed and autografted. At the 30th day after grafting, the grafts were collected for histology assessment and immunohistochemistry assay for MAGE-A4 (germ cell marker) and CD34 (blood vessel marker). The transplant recovery, seminiferous tubule integrity, tubular diameter, spermatogonia number, and microsvessel density in testicular fragments sizing in 3 mm in length, 3 mm wide, and 3 mm in thickness were significantly lower than other groups. Whereas, the absorption rate of graft sizing in 1 mm in length, 1 mm in wide, and 1 mm in thickness was significantly higher than other groups. Testicular fragment sizing in 2-3 mm in length, 2-3 mm in wide, and 2 mm in thickness (8 mm3-18 mm3) is suitable for rat testicular tissue cryopreservation and transplantation.

Dynamic Changes in Plasma Urotensin II and Its Correlation With Plaque Stability.

ABSTRACT: Urotensin II (UII) is involved in the formation of atherosclerosis, but its role in the stability of atherosclerotic plaques is unknown. The purpose of this study was to observe the dynamic changes in plasma UII and analyze its relationship to the stability of atherosclerotic plaques. One hundred thirty-five consecutive patients with acute coronary syndrome (ACS) were enrolled. The plasma UII levels were measured immediately after admission and during three-month follow-up. A vulnerable plaque model was established using local transfection of a recombinant P53 adenovirus into plaques in rabbits fed with a high-cholesterol diet and subjected to balloon arterial injury. The levels of plasma UII were measured weekly. The changes in plasma UII during the formation of atherosclerotic plaques and before and after plaque transfection were observed. The morphology of the plaques and the expression, distribution, and quantitative expression of UII in the plaques also were observed. Our results showed that the levels of plasma UII in patients with ACS at admission were lower than levels observed at the three-month follow-up. UII dynamic changes and its correlation with plaque stabilities were further verified in rabbits with atherosclerotic vulnerable plaques. The UII levels in rabbits were significantly decreased immediately after the P53 gene transfection, which led to plaque instability and rupture. These results suggested that UII expression was down-regulated in ACS, which may be related to its ability to modulate mechanisms involved in plaque stability and instability.

Ablation and Inhibition of the Immunoproteasome Catalytic Subunit LMP7 Attenuate Experimental Abdominal Aortic Aneurysm Formation in Mice.

Low-molecular mass protein 7 (LMP7) is a proteolytic subunit of the immunoproteasome that is involved in regulating inflammatory responses. However, the role of LMP7 in the pathogenesis of abdominal aortic aneurysm (AAA) remains unknown. In this study, ApoE knockout (KO) or LMP7/ApoE double KO (dKO) mice were infused with angiotensin II (Ang II, 1000 ng/kg per minute) for up to 28 d. We found that LMP7 expression was significantly upregulated in AAA tissues from ApoE KO mice and human patients. Moreover, Ang II infusion markedly increased the incidence and severity of AAA in ApoE KO mice, which was considerably reduced in LMP7/ApoE dKO mice. Histological alterations, including aortic wall thickening, collagen deposition, elastin fragmentation, and vascular smooth muscle cell apoptosis in AAA tissue of ApoE KO mice, were also significantly attenuated in LMP7/ApoE dKO mice. Interestingly, LMP7/ApoE dKO mice showed a marked reduction of infiltration of CD3+ T cells, especially CD4+ T cells in AAA tissues compared with ApoE KO mice. Moreover, ablation of LMP7 substantially inhibited the differentiation of CD4+ T cells into Th1 and Th17 cells by reducing the activation of multiple transcriptional factors. We also investigated the effects of an LMP7-specific inhibitor PR-957 (also known as ONX 0914) on AAA formation in ApoE KO mice. PR-957 treatment could reduce the AAA incidence and severity. In conclusion, our results provide, to our knowledge, novel evidence that ablation or pharmacological inhibition of LMP7 attenuates Ang II-induced AAA formation, and LMP7 might be a novel therapeutic target for treating AAA in humans.

Nonviral gene editing via CRISPR/Cas9 delivery by membrane-disruptive and endosomolytic helical polypeptide.

Effective and safe delivery of the CRISPR/Cas9 gene-editing elements remains a challenge. Here we report the development of PEGylated nanoparticles (named P-HNPs) based on the cationic α-helical polypeptide poly(γ-4-((2-(piperidin-1-yl)ethyl)aminomethyl)benzyl-l-glutamate) for the delivery of Cas9 expression plasmid and sgRNA to various cell types and gene-editing scenarios. The cell-penetrating α-helical polypeptide enhanced cellular uptake and promoted escape of pCas9 and/or sgRNA from the endosome and transport into the nucleus. The colloidally stable P-HNPs achieved a Cas9 transfection efficiency up to 60% and sgRNA uptake efficiency of 67.4%, representing an improvement over existing polycation-based gene delivery systems. After performing single or multiplex gene editing with an efficiency up to 47.3% in vitro, we demonstrated that P-HNPs delivering Cas9 plasmid/sgRNA targeting the polo-like kinase 1 (Plk1) gene achieved 35% gene deletion in HeLa tumor tissue to reduce the Plk1 protein level by 66.7%, thereby suppressing the tumor growth by >71% and prolonging the animal survival rate to 60% within 60 days. Capable of delivering Cas9 plasmids to various cell types to achieve multiplex gene knock-out, gene knock-in, and gene activation in vitro and in vivo, the P-HNP system offers a versatile gene-editing platform for biological research and therapeutic applications.

Testicular damage during cryopreservation and transplantation.

The aim of this study is to do a study of cryoinjury and ischaemic injury on testicular graft during cryopreservation and transplantation. According to time at 1, 3, 7 and 14 days after transplantation, the grafts were collected for immunohistochemistry assay for CD34 (blood vessel marker), VEGF (neoangiogenesis marker), caspase-3 (apoptosis marker) MAGE-A4 (germ cell marker). A significant increase was observed in the density of VEGF-positive blood vessels on day 3, reached a peak on day 7. On post-transplant day 3, a sharp increase occurred in the rate of spermatogonia-expressing caspase-3 until the day 7. At 14th day after transplantation, the spermatogonia number per round tubule of nonfrozen grafts was 41 ± 5.9% from that of fresh control tissues, while, in frozen-thawed grafts, the spermatogonia number per round tubule was 36.8 ± 4.6% from that of fresh control tissues. In testicular grafts, angiogenesis initiated reperfusion from day 3, and the formation of new blood vessel generally is completed about 7 days after transplantation. Angiogenesis in grafts after transplantation plays a crucial role in the restoration of function. Therefore, minimising ischaemic injury as well as improvement of cryopreservation protocols are needed to improve testicular graft after freezing, thawing and grafting.

Targeting against the activity of the NLRP3 inflammasome is a potential therapy for rat testicular tissue cryopreservation and transplantation.

The objective of the present experiment was to explore the role of NLRP3 inflammasome in the testicular tissue freezing, thawing and grafting; furthermore, the potential effect of a NLRP3 inhibitor on the function of testis transplant was explored. Tissues from male Wistar rats in pre-pubertal age were cryopreserved, thawed and auto-transplanted into the scrotum treated or not treated with the MCC950 (a NLRP3 inhibitor). After grafting, cryopreserved tissue was removed and analysed. Quantitative morphometric, immunohistochemical techniques and Western blotting were used to evaluate the survival of spermatogonia and the activation of the NLRP3 inflammasome after freezing/thawing/grafting. Moreover, serum IL-1ß level was assessed with ELISA kits. The testicular transplants exhibited upregulated expression of the NLRP3 pathway meditors (NLRP3, IL-1ß). In NLRP3 inhibition group, the rate of recovered grafts, the percentage of intact tubules and spermatogonial number were significantly higher than that in cryopreserved graft group. Moreover, serum concentration of IL-1ß in NLRP3 inhibition group was significantly lower than that in cryopreserved graft group. Testicular tissue cryopreservation and transplantation exhibited upregulated expression of NLRP3 pathway and NLRP3 inflammasome blockade improves testicular graft function. These finding suggest that NLRP3 inflammasome is a therapeutic target for testicular tissue cryopreservation and transplantation.

Tripartite motif 10 regulates cardiac hypertrophy by targeting the PTEN/AKT pathway.

The pathogenesis of cardiac hypertrophy is tightly associated with activation of intracellular hypertrophic signalling pathways, which leads to the synthesis of various proteins. Tripartite motif 10 (TRIM10) is an E3 ligase with important functions in protein quality control. However, its role in cardiac hypertrophy was unclear. In this study, neonatal rat cardiomyocytes (NRCMs) and TRIM10-knockout mice were subjected to phenylephrine (PE) stimulation or transverse aortic constriction (TAC) to induce cardiac hypertrophy in vitro and in vivo, respectively. Trim10 expression was significantly increased in hypertrophied murine hearts and PE-stimulated NRCMs. Knockdown of TRIM10 in NRCMs alleviated PE-induced changes in the size of cardiomyocytes and hypertrophy gene expression, whereas TRIM10 overexpression aggravated these changes. These results were further verified in TRIM10-knockout mice. Mechanistically, we found that TRIM10 knockout or knockdown decreased AKT phosphorylation. Furthermore, we found that TRIM10 knockout or knockdown increased ubiquitination of phosphatase and tensin homolog (PTEN), which negatively regulated AKT activation. The results of this study reveal the involvement of TRIM10 in pathological cardiac hypertrophy, which may occur by prompting of PTEN ubiquitination and subsequent activation of AKT signalling. Therefore, TRIM10 may be a promising target for treatment of cardiac hypertrophy.

Genetic ablation and pharmacological inhibition of immunosubunit ß5i attenuates cardiac remodeling in deoxycorticosterone-acetate (DOCA)-salt hypertensive mice.

Hypertensive cardiac remodeling is a major cause of heart failure. The immunoproteasome is an inducible form of the proteasome and its catalytic subunit ß5i (also named LMP7) is involved in angiotensin II-induced atrial fibrillation; however, its role in deoxycorticosterone-acetate (DOCA)-salt-induced cardiac remodeling remains unclear. C57BL/6 J wild-type (WT) and ß5i knockout (ß5i KO) mice were subjected to uninephrectomy (sham) and DOCA-salt treatment for three weeks. Cardiac function, fibrosis, and inflammation were evaluated by echocardiography and histological analysis. Protein and gene expression levels were analyzed by quantitative real-time PCR and immunoblotting. Our results showed that after 21 days of DOCA-salt treatment, ß5i expression and chymotrypsin-like activity were the most significantly increased factors in the heart compared with the sham control. Moreover, DOCA-salt-induced elevation of blood pressure, adverse cardiac function, chamber and myocyte hypertrophy, interstitial fibrosis, oxidative stress, and inflammation were markedly attenuated in ß5i KO mice. These findings were verified in ß5i inhibitor PR-957-treated mice. Moreover, blocking of PTEN (the gene of phosphate and tensin homolog deleted on chromosome ten) markedly attenuated the inhibitory effect of ß5i knockout on DOCA-salt-induced cardiac remodeling. Mechanistically, DOCA-salt stress upregulated the expression of ß5i, which promoted the degradation of PTEN and the activation of downstream signals (AKT/mTOR, TGF-ß1/Smad2/3, NOX, and NF-κB), which ultimately led to cardiac hypertrophic remodeling. This study provides new evidence of the critical role of ß5i in DOCA-salt-induced cardiac remodeling through the regulation of PTEN stability, and indicates that the inhibition of ß5i may be a promising therapeutic target for the treatment of hypertensive heart diseases.

mTORC1 in Thymic Epithelial Cells Is Critical for Thymopoiesis, T-Cell Generation, and Temporal Control of γδT17 Development and TCRγ/δ Recombination.

Thymus is crucial for generation of a diverse repertoire of T cells essential for adaptive immunity. Although thymic epithelial cells (TECs) are crucial for thymopoiesis and T cell generation, how TEC development and function are controlled is poorly understood. We report here that mTOR complex 1 (mTORC1) in TECs plays critical roles in thymopoiesis and thymus function. Acute deletion of mTORC1 in adult mice caused severe thymic involution. TEC-specific deficiency of mTORC1 (mTORC1KO) impaired TEC maturation and function such as decreased expression of thymotropic chemokines, decreased medullary TEC to cortical TEC ratios, and altered thymic architecture, leading to severe thymic atrophy, reduced recruitment of early thymic progenitors, and impaired development of virtually all T-cell lineages. Strikingly, temporal control of IL-17-producing γδT (γδT17) cell differentiation and TCRVγ/δ recombination in fetal thymus is lost in mTORC1KO thymus, leading to elevated γδT17 differentiation and rearranging of fetal specific TCRVγ/δ in adulthood. Thus, mTORC1 is central for TEC development/function and establishment of thymic environment for proper T cell development, and modulating mTORC1 activity can be a strategy for preventing thymic involution/atrophy.

CRISPR/Cas9-Based Genome Editing for Disease Modeling and Therapy: Challenges and Opportunities for Nonviral Delivery.

Genome editing offers promising solutions to genetic disorders by editing DNA sequences or modulating gene expression. The clustered regularly interspaced short palindromic repeats (CRISPR)/associated protein 9 (CRISPR/Cas9) technology can be used to edit single or multiple genes in a wide variety of cell types and organisms in vitro and in vivo. Herein, we review the rapidly developing CRISPR/Cas9-based technologies for disease modeling and gene correction and recent progress toward Cas9/guide RNA (gRNA) delivery based on viral and nonviral vectors. We discuss the relative merits of delivering the genome editing elements in the form of DNA, mRNA, or protein, and the opportunities of combining viral delivery of a transgene encoding Cas9 with nonviral delivery of gRNA. We highlight the lessons learned from nonviral gene delivery in the past three decades and consider their applicability for CRISPR/Cas9 delivery. We also include a discussion of bioinformatics tools for gRNA design and chemical modifications of gRNA. Finally, we consider the extracellular and intracellular barriers to nonviral CRISPR/Cas9 delivery and propose strategies that may overcome these barriers to realize the clinical potential of CRISPR/Cas9-based genome editing.

Chemokine (C-X-C motif) receptor 2 blockade by SB265610 inhibited angiotensin II-induced abdominal aortic aneurysm in Apo E-/- mice.

Inflammation plays a critical role in the development of abdominal aortic aneurysm (AAA). Chemokine receptor CXCR2 mediates inflammatory cell chemotaxis in several diseases. However, the role of CXCR2 in AAA and the underlying mechanisms remain unknown. In this study, we found that the CXCR2 expressions in AAA tissues from human and angiotensin II (Ang II)-infused apolipoprotein E knockout (Apo E-/-) mice were significantly increased. The pharmacological inhibition of CXCR2 (SB265610) markedly reduced Ang II-induced AAA formation. Furthermore, SB265610 treatment significantly reduced collagen deposition, elastin degradation, the metal matrix metalloprotease expression and accumulation of macrophage cells. In conclusion, these results showed CXCR2 plays a pathogenic role in AAA formation. Inhibition of CXCR2 pathway may represent a novel therapeutic approach to treat AAA.

Stimuli-responsive clustered nanoparticles for improved tumor penetration and therapeutic efficacy.

A principal goal of cancer nanomedicine is to deliver therapeutics effectively to cancer cells within solid tumors. However, there are a series of biological barriers that impede nanomedicine from reaching target cells. Here, we report a stimuli-responsive clustered nanoparticle to systematically overcome these multiple barriers by sequentially responding to the endogenous attributes of the tumor microenvironment. The smart polymeric clustered nanoparticle (iCluster) has an initial size of ∼100 nm, which is favorable for long blood circulation and high propensity of extravasation through tumor vascular fenestrations. Once iCluster accumulates at tumor sites, the intrinsic tumor extracellular acidity would trigger the discharge of platinum prodrug-conjugated poly(amidoamine) dendrimers (diameter ∼5 nm). Such a structural alteration greatly facilitates tumor penetration and cell internalization of the therapeutics. The internalized dendrimer prodrugs are further reduced intracellularly to release cisplatin to kill cancer cells. The superior in vivo antitumor activities of iCluster are validated in varying intractable tumor models including poorly permeable pancreatic cancer, drug-resistant cancer, and metastatic cancer, demonstrating its versatility and broad applicability.

[Self-made metal freezing plate for cryopreservation of human epididymal sperm: An experimental study].

OBJECTIVE: To study the effect of a new human sperm freezing method on the sperm recovery rate and search for an optimal method for cryopreservation of human epididymal sperm. METHODS: We collected semen samples from 76 men with obstructive azoospermia by percutaneous epididymal sperm aspiration and divided each sample into two parts to be cryopreserved with a self-made metal freezing plate (the experimental group) or by slow freezing (the control group), respectively. We measured the percentage of progressively motile sperm (PMS) with the computer-assisted semen analysis system and compared the membrane function, DNA fragmentation index (DFI), acrosin activity and morphological abnormality of the sperm between the two groups before and after cryopreservation. RESULTS: After thawing, both the percentages of PMS and hypotonically swollen sperm were significantly higher in the experimental than in the control group (ï¼»12.0 ± 7.5ï¼½% vs ï¼»8.0 ± 5.1ï¼½%, P < 0.05; ï¼»22.0 ± 17.5ï¼½% vs ï¼»18.0 ± 20.5ï¼½%, P < 0.05), though both decreased in comparison with the pre-freezing parameters (ï¼»20.7 ± 8.8ï¼½% and ï¼»30.0 ± 13.5ï¼½%) (P < 0.05). The sperm acrosin activity was remarkably higher in the experimental than in the control group after thawing (ï¼»75.2 ± 9.5ï¼½ vs ï¼»55.7 ± 8.3ï¼½ µIU/106sperm, P < 0.05), though decreased as compared with the baseline (ï¼»120.0 ± 10.5ï¼½ µIU/106 sperm, P < 0.05). No statistically significant differences were observed between the experimental and the control groups after thawing in the percentage of morphologically abnormal sperm (ï¼»98.7 ± 8.8ï¼½% vs ï¼»98.5±9.2ï¼½%, P > 0.05) or sperm DFI ï¼»38.2 ± 8.5ï¼½% vs ï¼»39.5 ± 10.2ï¼½%, P > 0.05), though both markedly elevated in comparison with the pre-freezing parameters (ï¼»97.2 ± 9.5ï¼½% and ï¼»30.8 ± 9.7ï¼½%) (P < 0.05). The post-thaw recovery rate of sperm was significantly higher in the experimental than in the control group (ï¼»65.2 ± 12.0ï¼½% vs ï¼»52.3 ± 18.0ï¼½%, P < 0.05). CONCLUSIONS: The self-made metal freezing plate, with its advantages of low cost, high efficiency, and easy operation, can be used as an effective method for cryopreservation of human sperm to achieve a high post-thaw sperm recovery rate, progressive sperm motility, and sperm acrosin activity.

Cardiac Ablation of SOCS3 Aggravates DOCA-Salt-Induced Hypertrophic Remodeling by Activation of Gp130-Dependent Signaling in Mice.

BACKGROUND/AIMS: Cardiac remodeling is a critical pathogenetic process leading to heart failure. Suppressor of cytokine signaling-3 (SOCS3) is demonstrated as a key negative regulator of the gp130 receptor to inhibit cardiac hypertrophy. However, the role of SOCS3 in deoxycorticosterone-acetate (DOCA)-salt-induced cardiac remodeling remains unclear. METHODS: Cardiac-specific SOCS3 knockout (SOCS3cKO) and wild-type (WT) C57BL/6J mice were subjected to uninephrectomy and DOCA-salt for 3 weeks. The effect of SOCS3 on cardiac remodeling and inflammation was evaluated by histological analysis. Gene and protein levels were measured by real-time PCR and immunoblotting analysis. RESULTS: After DOCA-salt treatment, the expression of SOCS3, activation of gp130/JAK/STAT3, cardiac dysfunction and fibrosis in DOCA-salt mice were significantly elevated, which were markedly attenuated by eplerenone, a specific mineralocorticoid receptor (MR) blocker. Moreover, DOCA-salt-induced cardiac dysfunction, hypertrophy, fibrosis and inflammation were aggravated in SOCS3cKO mice, but were significantly reduced in AAV9-SOCS3-injected mice. These effects were mostly associated with activation of gp130/STAT3/AKT/ERK1/2, TGF-ß/Smad2/3 and NF-κB signaling pathways. CONCLUSIONS: Our data demonstrate that loss of SOCS3 in cardiomyocytes promotes DOCA-salt-induced cardiac remodeling and inflammation, and it may be a novel potential therapeutic target for hypertensive heart disease.

BET-inhibition by JQ1 alleviates streptozotocin-induced diabetic cardiomyopathy.

Diabetic cardiomyopathy is a cascade of complex events leading to eventual heart failure in diabetes. JQ1, one of Bromodomain and extra-terminal domain (BET) protein inhibitors, has exerted therapeutic effects on cancer proliferation, inflammation and cardiovascular disease. Recently, JQ1 was reported to protect mice from bleomycin-induced lung fibrosis and reverse the fibrotic response in carbon tetrachloride-induced liver fibrosis. However, its role in diabetic cardiomyopathy remains to be clarified. Our results indicated that JQ1 treatment suppressed cardiac fibrosis and improved cardiac function in a STZ-induced diabetic mouse model. We further used both cardiofibroblasts and cardiomyocytes in vitro to investigate the protective mechanism of JQ1. JQ1 significantly suppressed hyperglycemia-induced cardiofibroblasts proliferation and migration, myofibroblast differentiation, and collagen production. Moreover, JQ1 reduced hyperglycemia-induced apoptosis of cardiomyocytes in vitro and in vivo. Mechanistically, JQ1 treatment could reverse the expression of Caveolin-1, which modulates transforming growth factor-ß1 (TGF-ß1) signaling in cardiofibroblasts and inhibits cardiomyocytes apoptosis. Our findings identify BET inhibitor JQ1 as promising agent for diabetic cardiomyopathy.

I2/TBHP-Mediated Oxidative Coupling of Amino-Based Bisnucleophiles and Isocyanides: Access to 2-Aminobenzoxazinones, 2-Aminobenzoxazines, and 2-Aminoquinazolines under Metal-Free Conditions.

An I2/ tert-butyl hydroperoxide (TBHP)-mediated oxidative coupling reaction of isocyanides with amino-based bisnucleophiles is described for the synthesis of 2-aminobenzoxazinones, 2-aminobenzoxazines, and 2-aminoquinozolines in moderate to excellent yields. Furthermore, this method provides a simple and practical method to construct potential functionalized biologically active molecules.

mTORC2 in Thymic Epithelial Cells Controls Thymopoiesis and T Cell Development.

Thymic epithelial cells (TECs) play important roles in T cell generation. Mechanisms that control TEC development and function are still not well defined. The mammalian or mechanistic target of rapamycin complex (mTORC)2 signals to regulate cell survival, nutrient uptake, and metabolism. We report in the present study that mice with TEC-specific ablation of Rictor, a critical and unique adaptor molecule in mTORC2, display thymic atrophy, which accompanies decreased TEC numbers in the medulla. Moreover, generation of multiple T cell lineages, including conventional TCRαß T cells, regulatory T cells, invariant NKT cells, and TCRγδ T cells, was reduced in TEC-specific Rictor-deficient mice. Our data demonstrate that mTORC2 in TECs is important for normal thymopoiesis and efficient T cell generation.

Two-stage orthogonality based estimation for semiparametric varying-coefficient models and its applications in analyzing AIDS data.

Semiparametric smoothing methods are usually used to model longitudinal data, and the interest is to improve efficiency for regression coefficients. This paper is concerned with the estimation in semiparametric varying-coefficient models (SVCMs) for longitudinal data. By the orthogonal projection method, local linear technique, quasi-score estimation, and quasi-maximum likelihood estimation, we propose a two-stage orthogonality-based method to estimate parameter vector, coefficient function vector, and covariance function. The developed procedures can be implemented separately and the resulting estimators do not affect each other. Under some mild conditions, asymptotic properties of the resulting estimators are established explicitly. In particular, the asymptotic behavior of the estimator of coefficient function vector at the boundaries is examined. Further, the finite sample performance of the proposed procedures is assessed by Monte Carlo simulation experiments. Finally, the proposed methodology is illustrated with an analysis of an acquired immune deficiency syndrome (AIDS) dataset.