Dual nanoenzymes loaded hollow mesoporous organotantalum nanospheres for chemo-radio sensitization.

Chemo-radiotherapy has been extensively used in clinics, displaying substantial advantages in treatment and prognosis. Stimuli-responsive biodegradable nanoagents that can achieve not only delivery and controlled release of chemotherapeutics, but also hypoxia alleviation to enhance chemoradiotherapy therefore has tremendous potential. Herein, glutathione (GSH)-responsive, biodegradable, doxorubicin-carrying hollow mesoporous organotantalum nanospheres modified with Au and Pt dual nanoenzymes (HMOTP@Pt@Au@Dox) were constructed for chemo-radio sensitization. Degradation of HMOTP@Pt@Au@Dox can be self-activated through GSH stimulation and on-demand release packaged Dox owing to the disulfide bond in the hybrid framework of organotantalum nanospheres. Au and Pt nanoenzymes triggered cascade catalytic reactions that could alleviate hypoxia by utilizing ß-d-glucose and H2O2, thereby sensitizing ROS-based chemoradiotherapy with synergistic starving therapy. Given the radiosensitization of high-Z elements (Ta, Pt, Au), nanoenzymes induced cascade catalytic reaction for hypoxia relief, and the depletion of the predominant antioxidant GSH, desirable tumor suppression could be achieved both in vitro and in vivo, indicating that HMOTP@Pt@Au@Dox is a promising nanoagent to boost chemo-radiotherapy.

The ubiquitin ligase Cul5 regulates CD4+ T cell fate choice and allergic inflammation.

Antigen encounter directs CD4+ T cells to differentiate into T helper or regulatory cells. This process focuses the immune response on the invading pathogen and limits tissue damage. Mechanisms that govern T helper cell versus T regulatory cell fate remain poorly understood. Here, we show that the E3 ubiquitin ligase Cul5 determines fate selection in CD4+ T cells by regulating IL-4 receptor signaling. Mice lacking Cul5 in T cells develop Th2 and Th9 inflammation and show pathophysiological features of atopic asthma. Following T cell activation, Cul5 forms a complex with CIS and pJak1. Cul5 deletion reduces ubiquitination and subsequent degradation of pJak1, leading to an increase in pJak1 and pSTAT6 levels and reducing the threshold of IL-4 receptor signaling. As a consequence, Cul5 deficient CD4+ T cells deviate from Treg to Th9 differentiation in low IL-4 conditions. These data support the notion that Cul5 promotes a tolerogenic T cell fate choice and reduces susceptibility to allergic asthma.

Tailoring the Photoelectrochemical Activity of Hexametaphosphate-Capped CdS Quantum Dots by Ca2+-Triggered Surface Charge Regulation: A New Signaling Strategy for Sensitive Immunoassay.

The development of efficient signaling strategies is highly important for photoelectrochemical (PEC) immunoassay. We report here a new and efficient strategy for sensitive PEC immunoassay by tailoring the electrostatic interaction between the photoactive material and the electron donor. The photoelectric conversion of hexametaphosphate (HMP)-capped CdS quantum dots (QDs) in Na2SO3 solution is significantly boosted after Ca2+ incubation. The negative surface charges on CdS@HMP QDs decrease because of the complexation reaction between HMP and Ca2+, and the electrostatic repulsion between CdS@HMP QDs and electron donor (SO32-) becomes weak accordingly, leading to an improved electron-hole separation efficiency. Inspired by the PEC response of CdS@HMP QDs to Ca2+, a novel "signal-on" PEC immunoassay platform is established by employing CaCO3 nanoparticles as labels. By regulating the surface charge of CdS@HMP QDs with in situ-generated Ca2+ from CaCO3 labels, sensitive detection of the carcinoembryonic antigen (CEA) is achieved. The linear detection range is 0.005-50 ng mL-1 and the detection limit is 1 pg mL-1 for CEA detection. Our work not only provides a facile route to tailor the photoelectric conversion but also lays the foundation for sensitive PEC immunoassay by simply regulating the surface charge of photoactive materials.

Comparison of Three Immunoassay Systems for Determining Serum Anti-Mullerian Hormone.

BACKGROUND: Serum anti-Mullerian hormone (AMH) is a dimeric glycoprotein of the transforming growth factor-ß (TGF-ß) family of growth and serves as a key biomarker of folliculogenesis as well as steroidogenesis within ovaries. Cobas E601, UniCel DxI 800 and iFlash3000 are automated immunoassay systems which are widely used to measure serum AMH levels in the blood. However, the correlations of serum AMH measured by the three automated immunoassay systems are not well recognized, the aim of this study is to compare the performance of the three automated immunoassay systems and record the correlation of serum AMH concentrations. METHODS: Serum AMH concentrations were measured in 100 serum samples using the UniCel DxI 800, Cobas E601, and iFlash 3000 automated immunoassay systems on the same day. Passing-Bablok regression analysis and Bland-Altman plot analysis were used to compare the system methods. The concordance correlation coefficient was analyzed to explain interrater agreement between the three immunoassay systems. RESULTS: Bland-Altman plot showed that the concentrations of AMH measured by UniCel DxI 800 were about 0.15 times higher than that measured using Cobas E601, the concentrations of AMH measured by UniCel DxI 800 were about 0.05 times higher than that measured using iFlash 3000, serum AMH concentrations measured by the iFlash3000 were about 0.19 times higher than that measured using Cobas E601. The concordance correlation coefficient ρc was 0.921, 0.909 and 0.978 for the UniCel DxI 800 versus the Cobas E601, the iFlash 3000 versus the UniCel DxI 800, and the iFlash3000 versus the Cobas E601, respectively. CONCLUSIONS: The three measurement systems have good correlation with each other for determining serum AMH.

Hypoxia-induced CNPY2 upregulation promotes glycolysis in cervical cancer through activation of AKT pathway.

This study aimed to investigate the function and mechanism of the protein-coding gene CNPY2 in the glycolysis of cervical cancer cells. Cells were exposed to normoxia and hypoxia conditions. Knockdown and ectopic overexpression of CNPY2 were achieved by transfection of small interfering RNA (siRNA) specific to CNPY2 or CNPY2 overexpression vectors, respectively. Quantitative real-time PCR and Western blot were used to evaluate CNPY2 expression in patient specimens and different cervical cancer cell lines under normoxia or hypoxia conditions. Cell viability was assessed by MTT and colony formation assays. Glucose consumption, lactate production, oxygen consumption and ATP production were analyzed by enzyme-linked immunosorbent assays. Dual-luciferase reporter assay and chromatin immunoprecipitation assay were performed to detect interaction between hypoxia-induced factor 1α (HIF-1α) on CNPY2 promoter. CNPY2 upregulation was a characteristic of cervical cancer and correlated with poor prognosis. Knockdown and overexpression of CNPY2 inhibited and promoted proliferation glucose consumption, lactate production, oxygen consumption and ATP production in cervical cancer cells, respectively. CNPY2 was transcriptionally regulated by HIF-1α. The hypoxia-induced "Warburg effect" in cervical cancer cells was at least partially dependent on the CNPY2/AKT signaling pathway. Hypoxia-induced CNPY2 promoted glycolysis in cervical cancer cells by activating the AKT pathway. CNPY2 may serve as a potential diagnostic marker and therapeutic target for cervical cancer patients.

CdS Quantum-Dots-Decorated V2O5 Nanosheets as Chemically Etchable Active Materials for Sensitive Photoelectrochemical Immunoassay of Carcinoembryonic Antigen.

We report here CdS quantum-dots (QDs)-decorated V2O5 nanosheets as high-performance and chemically etchable photoelectric active materials for constructing a photoelectrochemical (PEC) immunoassay platform. CdS QDs-decorated V2O5 nanosheets as new photoelectric materials can show superior photocurrent to V2O5 nanosheets and CdS QDs under visible-light irradiation because of the promoted photogenerated electron-hole separation and the increased visible-light absorption. V2O5 nanosheets can be etched by ascorbic acid (AA) because of the reduction of V2O5 to V4+, and the photocurrent of CdS/V2O5-nanocomposite-modified indium tin oxide electrode decreases significantly after being etched by AA. Inspired by this phenomenon, a PEC immunoassay platform is constructed for carcinoembryonic antigen (CEA) detection by using CdS/V2O5 nanocomposite as the photoelectric material and AA-encapsulated liposome immunonanocapsules as labels. The linear detection range for detecting CEA is from 0.5 pg mL-1 to 1 ng mL-1, with a limit of detection of 0.1 pg mL-1. The proposed method also shows good selectivity, excellent reproducibility, and satisfactory recovery in detection of CEA in human serum samples. We believe that this work will lay the foundation for the future development of V2O5-based materials for PEC analysis, and also provide a reasonable design and implementation for the development of PEC immunoassay.

Core-shell nanostructures introduce multiple potential barriers to enhance energy filtering for the improvement of the thermoelectric properties of SnTe.

Using dispersed nanostructures to induce an energy filtering effect is an easy and effective mechanism to optimize the performance of bulk thermoelectric materials. Compared with other nanostructures, core-shell nanostructures possess more interfaces and multiple potential barriers, which would lead to a significant impact on the thermal and electrical properties of materials. In this paper, after BiCuSeO alloy doping into SnTe, SnO2 layers were formed at the interfaces and the BiCuSeO nanoparticles were wrapped in the SnO2 shell during the following high temperature solid state reaction. The formation of SnO2 layers could be observed and confirmed by X-ray diffraction (XRD) and scanning electron microscopy (SEM). BiCuSeO@SnO2 core-shell nanostructures can introduce multiple potential barriers to enhance the energy filtering effect. Once the BiCuSeO doping concentration was over 3%, the carrier concentration could decrease to about 10% while the mobility increases to 350% compared to the values of the undoped sample at room temperature. Meanwhile, the Seebeck coefficients were improved to 176.05 µV K-1 at 835 K. Additionally, due to the scattering of core-shell nanostructures for the phonons, a lower thermal conductivity is achieved with a value of 1.04 W m-1 K-1 at 835 K in Sn1.03Te-5% BiCuSeO. Combined with the improvement of thermal and electrical properties by the BiCuSeO@SnO2 core-shell, a high ZT value of ∼1.21 was achieved for Sn1.03Te-5% BiCuSeO at 835 K, which was enhanced by 190% compared to pristine SnTe.

Hollow Mesoporous Tantalum Oxide Based Nanospheres for Triple Sensitization of Radiotherapy.

Radiotherapy (RT) is one of the most widely used cancer treatments in the clinical setting, while hypoxia-associated resistance often occurs. Herein, a PEGylated TaOx-based oxygen-carrying nanoplatform was constructed for triple sensitizing tumor radiotherapy. The high-Z element based hollow mesoporous TaOx nanospheres were prepared following the in situ growth of ultrasmall CuS nanocrystals and then packaged with O2-saturated perfluoropentane (PFP). NIR laser-triggered mild hyperthermia would lead to the increase of intratumoral blood flow, together with the release of O2, the radiotherapeutic efficiency would be enhanced. Alternatively, radiant energy would be deposited inside the tumor by the Ta element, therefore triple sensitization of radiotherapy could be achieved. The in vivo studies showed that the as-prepared nanospheres could achieve almost total inhibition of tumor growth without obvious side effects, which provides new possibilities for multisensitizing tumor radiotherapy.

Expression and clinical significance of PD-L1 and BRAF expression in nasopharyngeal carcinoma.

BACKGROUND: The prognostic value of programmed death-ligand 1 (PD-L1) and BRAF expression in nasopharyngeal carcinoma (NPC) is not well-defined. In this study we investigated alterations in PD-L1, BRAF and EGFR by using immunohistochemistry analysis in a cohort of consecutively enrolled NPC patients. METHODS: A retrospective review of 154 NPC patients form our previous study (BMC Cancer. 2013; 13:226) were conducted. Survival and prognostic impacts were analyzed based on PD-L1, BRAF and EGFR expression levels. RESULTS: One hundred fifty four patients were included in this study. PD-L1 expression was detected in 87.7% of patients; 14.3% had 1-5% PD-L1 expression, 47.4% had 5-49% expression while 26% had ≥50% expression Higher PD-L1 expression was significantly associated with shorter PFS and OS. The median PFS was 25 months (95% CI 15.7-34.3 months) and OS was 35 months (95% CI 22.60-47.4 months) for patients with PD-L1 expression ≥50%; both median PFS and OS were not yet reached for patients with PD-L1 expression < 50%. PFS was significantly higher in BRAF mutation positive patients (5-year PFS: 55.1% vs. 30.8%, P = 0.044). CONCLUSION: Tumor PD-L1 expression and BRAF mutation are associated with poor outcomes in patients with NPC. This study was retrospectively registered in ClinicalTrials.gov (NCT03989297) on 2019-6-18.

Development of a quantitative biochemical and cellular sphingomyelin synthase assay using mass spectrometry.

The last step in sphingolipid biosynthesis is the conversion of ceramide (Cer) to sphingomyelin (SM), which is catalyzed by sphingomyelin synthase (SMS). Two isoforms of SMS have been identified with differential subcellular localizations. It is not clear whether the two isoforms have any differences in biochemical or cellular SMS activities. This report describes a mass spectrometry (MS)-based method that was used to characterize biochemical and cellular SMS activities of the two isoforms of SMS, namely SMS1 and SMS2. Cellular extracts of SMS1 or SMS2 expressed in SF9 cells displayed significant SMS activity. When these activities were measured by MS, both SMS1 and SMS2 demonstrated similar time- and substrate-dependent SMS activity. A previously reported SMS inhibitor, D609, inhibited both SMS1 and SMS2 activity. In HEK293 cells, overexpression of either SMS1 or SMS2 significantly increased SMS activity. These studies using MS methods to measure SMS activity of SMS1 and SMS2 represent the first quantitative measurement of SMS activities. The establishment of quantitative biochemical and cellular SMS assays may help to facilitate the discovery of novel SMS1- or SMS2-specific inhibitors.

A SNX10/V-ATPase pathway regulates ciliogenesis in vitro and in vivo.

Sorting nexins (SNXs) are phosphoinositide-binding proteins implicated in the sorting of various membrane proteins in vitro, but the in vivo functions of them remain largely unknown. We reported previously that SNX10 is a unique member of the SNX family genes in that it has vacuolation activity in cells. We investigate the biological function of SNX10 by loss-of-function assay in this study and demonstrate that SNX10 is required for the formation of primary cilia in cultured cells. In zebrafish, SNX10 is involved in ciliogenesis in the Kupffer's vesicle and essential for left-right patterning of visceral organs. Mechanistically, SNX10 interacts with V-ATPase complex and targets it to the centrosome where ciliogenesis is initiated. Like SNX10, V-ATPase regulates ciliogenesis in vitro and in vivo and does so synergistically with SNX10. We further discover that SNX10 and V-ATPase regulate the ciliary trafficking of Rab8a, which is a critical regulator of ciliary membrane extension. These results identify an SNX10/V-ATPase-regulated vesicular trafficking pathway that is crucial for ciliogenesis, and reveal that SNX10/V-ATPase, through the regulation of cilia formation in various organs, play an essential role during early embryonic development.

Role of ubiquitination in PCSK9-mediated low-density lipoprotein receptor degradation.

The proprotein convertases subtilisin kexin 9 (PCSK9) binds to the epidermal growth factor domain A (EGF-A) of low-density lipoprotein receptor (LDLR) and leads to its destruction. However, the intracellular processes leading to LDLR degradation have not been fully delineated. In this report, we show that PCSK9 treatment can lead to ubiquitination of LDLR, which was enhanced in the presence of proteasome inhibitor MG132. Furthermore, LDLR protein carrying mutations in the C-terminal ubiquitination sites was resistant to PCSK9-mediated degradation. Our data suggest that the ubiquitination system is involved in PCSK9-induced LDLR degradation.

The microsomal cardiolipin remodeling enzyme acyl-CoA lysocardiolipin acyltransferase is an acyltransferase of multiple anionic lysophospholipids.

Phospholipids are subjected to remodeling through the Lands cycle to attain appropriate FA compositions. In recent years, at least two families of lysophospholipid acyltransferases have been identified. Acyl-CoA lysocardiolipin acyltransferase 1 (ALCAT1) was initially identified as a microsomal lysocardiolipin acyltransferase. However, the physiological relevance of how this enzyme is involved in cardiolipin remodeling has not been elucidated. We report in this study that ALCAT1 possesses acyltransferase activities toward lysophosphatidylinositol (LPI) and lysophosphatidylglycerol (LPG). Membrane preparations from human embryonic kidney 293 (HEK293) cells overexpressing human ALCAT1 demonstrated significant increases in LPI acyltransferase (LPIAT) and LPG acyltransferase (LPGAT) activities using a variety of fatty acyl-CoAs. The enzyme affinities toward LPI and LPG were determined through kinetic studies suggesting that the LPI binding affinity to ALCAT1 depends on fatty acyl-CoA. Reduced expression of ALCAT1 in Hela cells resulted in significant reductions of LPIAT and LPGAT activities, but not ALCAT activity. Through structural and functional studies, we have identified critical amino acids D168 and L169 within ALCAT1 that are potentially involved in lysophospholipid substrate binding. Our studies provide the molecular basis for future investigations of the physiological function of ALCAT1 and offer evidence of critical amino acids involved in substrate binding for the family of glycerolipid acyltransferases.

Peroxisome proliferator-activated receptor alpha agonists regulate cholesterol ester transfer protein.

Peroxisome proliferator-activated receptor alpha (PPARalpha) belongs to the nuclear receptor superfamily that regulates multiple target genes involved in lipid metabolism. Cholesterol ester transfer protein (CETP) is a secreted glycoprotein that modifies high-density lipoprotein (HDL) particles. In humans, plasma CETP activity is inversely correlated with HDL cholesterol levels. We report here that PPARalpha agonists increase CETP mRNA, protein and accordingly its activity. In a human CETP transgenic animal model harboring the natural flanking regions (Jiang et al. in J Clin Investigat 90:1290-1295, 1992), both fenofibrate and a specific synthetic PPARalpha agonist LY970 elevated human CETP mRNA in liver, serum protein and CETP activity. In hamsters, the endogenous liver CETP mRNA level and the serum CETP activity were dose-dependently upregulated by fenofibrate. In addition Wy14643, a PPARalpha agonist, also significantly elevated CETP mRNA and activity. In a carcinoma cell line of hepatic origin, HepG2 cells, overexpression of PPARalpha resulted in increased CETP mRNA and agonist treatment further elevated CETP mRNA levels. We conclude that PPARalpha agonists upregulate CETP expression and activity and may play an important role in PPARalpha (agonist mediated HDL cholesterol homeostasis in humans.

Identification and characterization of a major liver lysophosphatidylcholine acyltransferase.

Phosphatidylcholine (PC) is synthesized through the Kennedy pathway, but more than 50% of PC is remodeled through the Lands cycle, i.e. the deacylation and reacylation of PC to attain the final and proper fatty acids within PC. The reacylation step is catalyzed by lysophosphatidylcholine acyltransferase (LPCAT), and we report here the identification of a novel LPCAT, which we named LPCAT3. LPCAT3 belongs to the membrane-bound O-acyltransferase (MBOAT) family and encodes a protein of 487 amino acids with a calculated molecular mass of 56 kDa. Membranes from HEK293 cells overexpressing LPCAT3 showed significantly increased LPCAT activity as assessed by thin layer chromatography analysis with substrate preference toward unsaturated fatty acids. LPCAT3 is localized within the endoplasmic reticulum and is primarily expressed in metabolic tissues including liver, adipose, and pancreas. In a human hepatoma Huh7 cells, RNA interference-mediated knockdown of LPCAT3 resulted in virtually complete loss of membrane LPCAT activity, suggesting that LPCAT3 is primarily responsible for hepatic LPCAT activity. Furthermore, peroxisome proliferator-activated receptor alpha agonists dose-dependently regulated LPCAT3 in liver in a peroxisome proliferator-activated receptor alpha-dependent fashion, implicating a role of LPCAT3 in lipid homeostasis. Our studies identify a long-sought enzyme that plays a critical role in PC remodeling in metabolic tissues and provide an invaluable tool for future investigations on how PC remodeling may potentially impact glucose and lipid homeostasis.