Homogeneous antibody and CAR-T cells with improved effector functions targeting SSEA-4 glycan on pancreatic cancer.

Pancreatic cancer is usually asymptomatic in the early stages; the 5-y survival rate is around 9%; and there is a lack of effective treatment. Here we show that SSEA-4 is more expressed in all pancreatic cancer cell lines examined but not detectable in normal pancreatic cells; and high expression of SSEA-4 or the key enzymes B3GALT5 + ST3GAL2 associated with SSEA-4 biosynthesis significantly lowers the overall survival rate. To evaluate potential new treatments for pancreatic cancer, homogeneous antibodies with a well-defined Fc glycan for optimal effector functions and CAR-T cells with scFv construct designed to target SSEA-4 were shown highly effective against pancreatic cancer in vitro and in vivo. This was further supported by the finding that a subpopulation of natural killer (NK) cells isolated by the homogeneous antibody exhibited enhancement in cancer-cell killing activity compared to the unseparated NK cells. These results indicate that targeting SSEA-4 by homologous antibodies or CAR-T strategies can effectively inhibit cancer growth, suggesting SSEA-4 as a potential immunotherapy target for treating pancreatic disease.

miR-4433a-3p promotes granulosa cell apoptosis by targeting peroxisome proliferator-activated receptor alpha and inducing immune cell infiltration in polycystic ovarian syndrome.

BACKGROUND: Granulosa cell (GC) proliferation and apoptosis are critical events of the ovum energy supply, which lead to follicular growth retardation or atresia, and various ovulatory obstacles, eventually resulting in the development of ovarian disorders such as polycystic ovarian syndrome (PCOS). Apoptosis and dysregulated miRNA expression in GCs are manifestations of PCOS. miR-4433a-3p has been reported to be involved in apoptosis. However, there is no study reporting the roles of miR-4433a-3p in GC apoptosis and PCOS progression. METHODS: miR-4433a-3p and peroxisome proliferator-activated receptor alpha (PPAR-α) levels in GCs of PCOS patients or in tissues of a PCOS rat model were examined by quantitative polymerase chain reaction and immunohistochemistry. Bioinformatics analyses and luciferase assays were used to examine the association between miR-4433a-3p and PPAR-α, as well as PPAR-α and immune cell infiltration, in PCOS patients. RESULTS: miR-4433a-3p expression in GCs of PCOS patients was increased. miR-4433a-3p overexpression inhibited the growth of the human granulosa-like tumor cell line (KGN) and promoted apoptosis, while co-treatment with PPAR-α and miR-4433a-3p mimic rescued miR-4433a-3p-induced apoptosis. PPAR-α was a direct target of miR-4433a-3p and its expression was decreased in PCOS patients. PPAR-α expression was also positively correlated with the infiltration of activated CD4+ T cells, eosinophils, B cells, gamma delta T cells, macrophages, and mast cells, but negatively correlated with the infiltration of activated CD8+ T cells, CD56+ bright natural killer cells, immature dendritic cells, monocytes, plasmacytoid dendritic cells, neutrophils, and type 1 T helper cells in PCOS patients. CONCLUSION: The miR-4433a-3p/PPAR-α/immune cell infiltration axis may function as a novel cascade to alter GC apoptosis in PCOS.

Interdomain Flexibility of Chikungunya Virus nsP2 Helicase-Protease Differentially Influences Viral RNA Replication and Infectivity.

Chikungunya virus (CHIKV) is a mosquito-borne alphavirus responsible for chikungunya fever. Nonstructural protein 2 (nsP2), a multifunctional protein essential for viral replication, has an N-terminal helicase region (nsP2h), which has both nucleotide triphosphatase and RNA triphosphatase activities, as well as a C-terminal cysteine protease region (nsP2p), which is responsible for nonstructural polyprotein processing. The two functional units are connected through a linker of 14 residues. Although crystal structures of the helicase and protease regions of CHIKV nsP2 have been solved separately, the conformational arrangement of the full-length nsP2 and the biological role of the linker remain elusive. Using the small-angle X-ray scattering (SAXS) method, we demonstrated that the full-length nsP2 is elongated and partially folded in solution. The reconstructed model of the structure of nsP2 contains a flexible interdomain linker, and there is no direct interaction between the two structured regions. To examine the function of the interdomain linker, we constructed and characterized a set of CHIKV mutants. The deletion of three or five amino acid residues in the linker region resulted in a modest defect in viral RNA replication and transcription but completely abolished viral infectivity. In contrast, increasing the flexibility of nsP2 by lengthening the interdomain linker increased both genomic RNA replication and viral infectivity. The enzymatic activities of the corresponding mutant proteins were largely unaffected. This work suggests that increasing the interdomain flexibility of nsP2 could facilitate the assembly of the replication complex (RC) with increased efficiency and promote virus production.IMPORTANCE CHIKV nsP2 plays multiple roles in viral RNA replication and virus-host interactions. The helicase and protease regions of nsP2 are connected through a short linker. Here, we determined that the conformation of full-length CHIKV nsP2 is elongated and that the protein is flexible in solution. We also highlight the importance of the flexibility of the interdomain of nsP2 on viral RNA synthesis and infectivity. CHIKV mutants harboring shortened linkers fail to produce infectious virus particles despite showing only relatively mild defects in genomic and subgenomic RNA synthesis. Mutations increasing the length of the interdomain linker have only mild and generally beneficial impacts on virus replication. Thus, our findings link interdomain flexibility with the regulation of viral RNA replication and infectivity of the viral genome.

Structural insights into RNA recognition by the Chikungunya virus nsP2 helicase.

Chikungunya virus (CHIKV) is transmitted to humans through mosquitoes and causes Chikungunya fever. Nonstructural protein 2 (nsP2) exhibits the protease and RNA helicase activities that are required for viral RNA replication and transcription. Unlike for the C-terminal protease, the structure of the N-terminal RNA helicase (nsP2h) has not been determined. Here, we report the crystal structure of the nsP2h bound to the conserved 3'-end 14 nucleotides of the CHIKV genome and the nonhydrolyzable transition-state nucleotide analog ADP-AlF4 Overall, the structural analysis revealed that nsP2h adopts a uniquely folded N-terminal domain followed by a superfamily 1 RNA helicase fold. The conserved helicase motifs establish polar contacts with the RNA backbone. There are three hydrophobic residues (Y161, F164, and F287) which form stacking interactions with RNA bases and thereby bend the RNA backbone. An F287A substitution that disrupted these stacking interactions increased the basal ATPase activity but decreased the RNA binding affinity. Furthermore, the F287A substitution reduced viral infectivity by attenuating subgenomic RNA synthesis. Replication of the mutant virus was restored by pseudoreversion (A287V) or adaptive mutations in the RecA2 helicase domain (T358S or V410I). Y161A and/or F164A substitutions, which were designed to disrupt the interactions with the RNA molecule, did not affect the ATPase activity but completely abolished the replication and transcription of viral RNA and the infectivity of CHIKV. Our study sheds light on the roles of the RNA helicase region in viral replication and provides insights that might be applicable to alphaviruses and other RNA viruses in general.

Structural determinants for peptide-bond formation by asparaginyl ligases.

Asparaginyl endopeptidases (AEPs) are cysteine proteases which break Asx (Asn/Asp)-Xaa bonds in acidic conditions. Despite sharing a conserved overall structure with AEPs, certain plant enzymes such as butelase 1 act as a peptide asparaginyl ligase (PAL) and catalyze Asx-Xaa bond formation in near-neutral conditions. PALs also serve as macrocyclases in the biosynthesis of cyclic peptides. Here, we address the question of how a PAL can function as a ligase rather than a protease. Based on sequence homology of butelase 1, we identified AEPs and PALs from the cyclic peptide-producing plants Viola yedoensis (Vy) and Viola canadensis (Vc) of the Violaceae family. Using a crystal structure of a PAL obtained at 2.4-Å resolution coupled to mutagenesis studies, we discovered ligase-activity determinants flanking the S1 site, namely LAD1 and LAD2 located around the S2 and S1' sites, respectively, which modulate ligase activity by controlling the accessibility of water or amine nucleophile to the S-ester intermediate. Recombinantly expressed VyPAL1-3, predicted to be PALs, were confirmed to be ligases by functional studies. In addition, mutagenesis studies on VyPAL1-3, VyAEP1, and VcAEP supported our prediction that LAD1 and LAD2 are important for ligase activity. In particular, mutagenesis targeting LAD2 selectively enhanced the ligase activity of VyPAL3 and converted the protease VcAEP into a ligase. The definition of structural determinants required for ligation activity of the asparaginyl ligases presented here will facilitate genomic identification of PALs and engineering of AEPs into PALs.

From Chinese tourists to Taiwanese campers: Impacts of tourism policies on campsite land use/cover change.

The rapid increase in camping activities and campsites has had negative environmental impacts in mountainous areas. Tourism policies may be an important factor in changing recreational behavior and increasing campsites. The purpose of this study was to explore the effects of tourism policies on campsite-related landscape changes in Taiwan. The study area consisted of 276 campsites in the Jianshi and Wufeng Townships in Hsinchu County. The tourism policy periods were divided into 2001-2007 (Taiwan's agri-tourism policy), 2008-2015 (China and Taiwan's travel permit policy), and 2016-2019 (China's travel restriction policy), based on a reference review and relative theories. The 2000, 2008, 2016, and 2019 campsite landscapes were classified into forestland and non-forestland through object-based classification. This study established a general linear model to analyze the effect of tourism policy period on campsite forestland and non-forestland landscape change, according to the 50, 100, 250, 500, and 1000 m radii of 276 campsites. The results showed that tourism policies had a significant effect on campsite forestland and non-forestland landscape changes. The effect sizes ranged from medium to large. The Chinese tourist travel permit policy was significantly associated with increased non-forestland in campsites from 2008 to 2016. This policy likely affected recreational behavior indirectly, promoting camping and increasing non-forestland through the crowding-out effects of the many Chinese tourists, which was not the original purpose of the policy. Tourism policy decision-makers should consider the potential negative landscape change effects of changes in recreational behavior, and provide supporting measures to maintain recreational quality and avoid crowding-out effects. Campsite development should also be regulated to prevent forestland changes and achieve sustainable management.

MicroRNA-665 facilitates cell proliferation and represses apoptosis through modulating Wnt5a/ß-Catenin and Caspase-3 signaling pathways by targeting TRIM8 in LUSC.

BACKGROUND: MicroRNAs (miRNAs) are involved in the oncogenesis, development and transformation of lung squamous cell carcinoma (LUSC). miR-665 is clinically significant and acts as a pivotal function in some cancers. Nevertheless, the effects and the potential mechanisms of miR-665 in human LUSC are still unknown. METHODS: To analyse the clinical significant of miR-665 in human LUSC, quantitative real-time PCR (qRT-PCR) was use to measure miR-665 expression in LUSC specimen tissues and cell lines. Tripartite motif 8 (TRIM8) was verified a target of miR-665 by performing bioinformatic prediction and luciferase reporter assay. The expression levels of TRIM8 were examined through qRT-PCR and Western blotting in LUSC specimen tissues. CCK8 assay was fulfilled for analyzing the function in LUSC cell proliferation. Flow cytometry was used to detect cell and apoptosis. TRIM8 silencing and overexpression further verified the biological effects as those caused by miR-665. RESULTS: Here we reported that miR-665 expression was upregulated in LUSC specimen tissues and cell lines. High miR-665 levels were related to differentiation, tumor size and TNM stage. miR-665 mimics facilitated LUSC cell growth and cell cycle G1-S transition and repressed apoptosis. miR-665 inhibitor suppressed cell proliferation and G1-S transition and promoted apoptosis. miR-665 expression was negatively correlated with TRIM8 mRNA expression in LUSC. Luciferase reporter assay confirmed that TRIM8 was a direct target gene of miR-665. miR-665 mimics downregulated the TRIM8 levels, and miR-665 inhibitor upregulated the TRIM8 levels in LUSC cells. Particularly, silencing TRIM8 led to the similar effects of miR-665 mimics in LUSC cells. Overexpression of TRIM8 inhibited LUSC cell proliferation in vitro and in vivo. Furthermore, miR-665 promoted LUSC cell proliferation through facilitating the Wnt5a/ß-catenin signaling pathway and restrained apoptosis via inhibiting Caspase-3 signaling pathway, whereas TRIM8 suppressed cell growth by repressing the Wnt5a/ß-catenin signaling pathway and induced apoptosis through activating Caspase-3 signaling pathway. CONCLUSIONS: The current study demonstrates that miR-665 facilitates LUSC cell proliferation and cell cycle transition by regulation of the Wnt5a/ß-Catenin signaling pathway and represses cell apoptosis via modulation of Caspase-3 signaling pathway by directly targeting TRIM8. These findings suggest that miR-665 might be a potential new target for LUSC therapy.

System Biology-Guided Chemical Proteomics to Discover Protein Targets of Monoethylhexyl Phthalate in Regulating Cell Cycle.

Chemical proteomics methods have been used as effective tools to identify novel protein targets for small molecules. These methods have great potential to be applied as environmental toxicants to figure out their mode of action. However, these assays usually generate dozens of possible targets, making it challenging to validate the most important one. In this study, we have integrated the cellular thermal shift assay (CETSA), quantitative proteomics, metabolomics, computer-assisted docking, and target validation methods to uncover the protein targets of monoethylhexyl phthalate (MEHP). Using the mass spectrometry implementation of CETSA (MS-CETSA), we have identified 74 possible protein targets of MEHP. The Gene Ontology (GO) enrichment integration was further conducted for the target proteins, the cellular dysregulated proteins, and the metabolites, showing that cell cycle dysregulation could be one primary change due to the MEHP-induced toxicity. Flow cytometry analysis confirmed that hepatocytes were arrested at the G1 stage due to the treatment with MEHP. Subsequently, the potential protein targets were ranked by their binding energy calculated from the computer-assisted docking with MEHP. In summary, we have demonstrated the development of interactomics workflow to simplify the redundant information from multiomics data and identified novel cell cycle regulatory protein targets (CPEB4, ANAPC5, and SPOUT1) for MEHP.

Laser-Induced Fluorescence Spectroscopy of Large Secondary Alkoxy Radicals: Part II. Rotational and Fine Structure.

Selected vibronic bands of the B̃ ← X̃ laser-induced fluorescence (LIF) spectra of jet-cooled 2-pentoxy and 2-hexoxy, including the origin and CO-stretch bands, have been measured with rotational resolution and analyzed using (1) an effective Hamiltonian that comprises a rotational part and a spin-rotation (SR) part (the "isolated-states model") and (2) a recently developed Hamiltonian in which the nearly degenerate à and X̃ states are treated together (the "coupled-states model") (see Liu, J., J. Chem. Phys. 2018, 148, 124112). The observed rotational and fine structures of the strongest vibronic bands have first been simulated using a genetic algorithm with the isolated-states model. The parameters for the simulation include rotational constants for both the X̃ and B̃ states, which can be calculated from the electronic structure theory, as well as the electronic SR constants of the X̃ state and the transition dipole moments (TDMs), both of which are predicted based on their transferability in an "orbital-fixed coordinate system" using iso-propoxy as the reference molecule. Quantum chemistry calculations suggest that the lowest two electronic (X̃ and Ã) states of secondary alkoxy radicals have small energy separations on the order of 100 cm-1 (see Part I of this series: J. Phys. Chem. A 2021, DOI: 10.1021/acs.jpca.0c10662). The electron configurations of these two nearly degenerate states have been determined by comparing the experimentally determined rotational constants and the TDMs to the ones predicted for the X̃ and à states. The experimental LIF spectra were also simulated with the coupled-states model, in which the effective spin-orbit (SO) constants (aζed) and the SO-free separation between the à and the X̃ states (ΔE0) have been determined. Molecular constants derived from fitting the rotational and fine structures of the experimental LIF spectra enabled unambiguous assignment of the observed vibronic bands to specific conformers of 2-pentoxy and 2-hexoxy as reported in Part I.

Molecular Imaging and Preclinical Studies of Radiolabeled Long-Term RGD Peptides in U-87 MG Tumor-Bearing Mice.

The Arg-Gly-Asp (RGD) peptide shows a high affinity for αvß3 integrin, which is overexpressed in new tumor blood vessels and many types of tumor cells. The radiolabeled RGD peptide has been studied for cancer imaging and radionuclide therapy. We have developed a long-term tumor-targeting peptide DOTA-EB-cRGDfK, which combines a DOTA chelator, a truncated Evans blue dye (EB), a modified linker, and cRGDfK peptide. The aim of this study was to evaluate the potential of indium-111(111In) radiolabeled DOTA-EB-cRGDfK in αvß3 integrin-expressing tumors. The human glioblastoma cell line U-87 MG was used to determine the in vitro binding affinity of the radiolabeled peptide. The in vivo distribution of radiolabeled peptides in U-87 MG xenografts was investigated by biodistribution, nanoSPECT/CT, pharmacokinetic and excretion studies. The in vitro competition assay showed that 111In-DOTA-EB-cRGDfK had a significant binding affinity to U-87 MG cancer cells (IC50 = 71.7 nM). NanoSPECT/CT imaging showed 111In-DOTA-EB-cRGDfK has higher tumor uptake than control peptides (111In-DOTA-cRGDfK and 111In-DOTA-EB), and there is still a clear signal until 72 h after injection. The biodistribution results showed significant tumor accumulation (27.1 ± 2.7% ID/g) and the tumor to non-tumor ratio was 22.85 at 24 h after injection. In addition, the pharmacokinetics results indicated that the 111In-DOTA-EB-cRGDfK peptide has a long-term half-life (T1/2λz = 77.3 h) and that the calculated absorbed dose was safe for humans. We demonstrated that radiolabeled DOTA-EB-cRGDfK may be a promising agent for glioblastoma tumor imaging and has the potential as a theranostic radiopharmaceutical.

Lipopolysaccharide inhibits GPR120 expression in macrophages via Toll-like receptor 4 and p38 MAPK activation.

Free fatty acid receptor G protein-coupled receptor 120 (GPR120) is highly expressed in macrophages and was reported to inhibit lipopolysaccharide (LPS)-stimulated cytokine expression. Under inflammation, macrophages exhibit striking functional changes, but changes in GPR120 expression and signaling are not known. In this study, the effects of LPS treatment on macrophage GPR120 expression and activation were investigated. The results showed that LPS inhibited GPR120 expression in mouse macrophage cell line Ana-1 cells. Moreover, LPS treatment inhibited GPR120 expression in mouse alveolar macrophages both in vitro and in vivo. The inhibitory effect of LPS on GPR120 expression was blocked by Toll-like receptor 4 (TLR4) inhibitor TAK242 and p38 mitogen-activated protein kinase inhibitor LY222820, but not by ERK1/2 inhibitor U0126 and c-Jun N-terminal kinase inhibitor SP600125. LPS-induced inhibition of GPR120 expression was not attenuated by GPR120 agonists TUG891 and GW9508. TUG891 inhibited the phagocytosis of alveolar macrophages, and LPS treatment counteracted the effects of TUG891 on phagocytosis. These results indicate that pretreatment with LPS inhibits GPR120 expression and activation in macrophages. It is suggested that LPS-induced inhibition of GPR120 expression is a reaction enhancing the LPS-induced pro-inflammatory response of macrophages.

Cytotoxic monoterpenoid indole alkaloids from the aerial parts of Kopsia officinalis.

Three new monoterpenoid indole alkaloids, kopsiaofficines A-D (1-3), were isolated from the 95% EtOH extract of the aerial parts of Kopsia officinalis. Their structures were established on the basis of spectroscopic data. The isolated alkaloids were tested in vitro for cytotoxic activity against seven lung cancer cell lines. Consequently, alkaloids 1 and 3 exhibited some cytotoxic activities against all the tested tumor cell lines with IC50 values less than 20 µM.[Formula: see text].

To Boldly Look Where No One Has Looked Before: Identifying the Primary Photoproducts of Acetylacetone.

We investigate the gas-phase photochemistry of the enolone tautomer of acetylacetone (pentane-2,4-dione) following S2(ππ*) ← S0 excitation at λ = 266 and 248 nm, using three complementary time-resolved spectroscopic methods. Contrary to earlier reports, which claimed to study one-photon excitation of acetylacetone and found OH and CH3 as the only important gas-phase products, we detect 15 unique primary photoproducts and demonstrate that five of them, including OH and CH3, arise solely by multiphoton excitation. We assign the one-photon products to six photochemical channels and show that the most significant pathway is phototautomerization to the diketone form, which is likely an intermediate in several of the other product channels. Furthermore, we measure the equilibrium constant of the tautomerization of the enolone to diketone on S0 from 320 to 600 K and extract Δ H = 4.1 ± 0.3 kcal·mol-1 and Δ S = 6.8 ± 0.5 cal·mol-1·K-1 using a van't Hoff analysis. We correct the C-OH bond dissociation energy in acetylacetone, previously determined as 90 kcal·mol-1 by theory and experiment, to a new value of 121.7 kcal·mol-1. Our experiments and electronic structure calculations provide evidence that some of the product channels, including phototautomerization, occur on S0, while others likely occur on excited triplet surfaces. Although the large oscillator strength of the S2 ← S0 transition results from the (ππ*) excitation of the C═C-C═O backbone, similar to conjugated polyenes, the participation of triplets in the dissociation pathways of acetylacetone appears to have more in common with ketone photochemistry.

Direct measurement of ˙OH and HO2˙ formation in ˙R + O2 reactions of cyclohexane and tetrahydropyran.

Formation of the key general radical chain carriers, ˙OH and HO2˙, during pulsed-photolytic ˙Cl-initiated oxidation of tetrahydropyran and cyclohexane are measured with time-resolved infrared absorption in a temperature-controlled Herriott multipass cell in the temperature range of 500-750 K at 20 Torr. The experiments show two distinct timescales for HO2˙ and ˙OH formation in the oxidation of both fuels. Analysis of the timescales reveals striking differences in behavior between the two fuels. In both cyclohexane and tetrahydropyran oxidation, a faster timescale is strongly related to the "well-skipping" (˙R + O2 → alkene + HO2˙ or cyclic ether + ˙OH) mechanism and is expected to have, at most, a weak temperature dependence. Indeed, the fast HO2˙ formation timescale is nearly temperature independent both for cyclohexyl + O2 and for tetrahydropyranyl + O2 below 700 K. A slower HO2˙ formation timescale in cyclohexane oxidation is shown to be linked to the sequential ˙R + O2 → ROO˙ → alkene + HO2˙ pathway, and displays a strong temperature dependence mainly from the final step (with energy barrier ∼32.5 kcal mol-1). In contrast, the slower HO2˙ formation timescale in tetrahydropyran oxidation is surprisingly temperature insensitive across all measured temperatures. Although the ˙OH formation timescales in tetrahydropyran oxidation show a temperature dependence similar to the cyclohexane oxidation, the temperature dependence of ˙OH yield is opposite in both cases. This significant difference of HO2˙ formation kinetics and ˙OH formation yield for the tetrahydropyran oxidation can arise from contributions related to ring-opening pathways in the tetrahydropyranyl + O2 system that compete with the typical ˙R + O2 reaction scheme. This comparison of two similar fuels demonstrates the consequences of differing chemical mechanisms on ˙OH and HO2˙ formation and shows that they can be highlighted by analysis of the eigenvalues of a system of simplified kinetic equations for the alkylperoxy-centered ˙R + O2 reaction pathways. We suggest that such analysis can be more generally applied to complex or poorly known oxidation systems.

Products of Criegee intermediate reactions with NO2: experimental measurements and tropospheric implications.

The reactions of Criegee intermediates with NO2 have been proposed as a potentially significant source of the important nighttime oxidant NO3, particularly in urban environments where concentrations of ozone, alkenes and NOx are high. However, previous efforts to characterize the yield of NO3 from these reactions have been inconclusive, with many studies failing to detect NO3. In the present work, the reactions of formaldehyde oxide (CH2OO) and acetaldehyde oxide (CH3CHOO) with NO2 are revisited to further explore the product formation over a pressure range of 4-40 Torr. NO3 is not observed; however, temporally resolved and [NO2]-dependent signal is observed at the mass of the Criegee-NO2 adduct for both formaldehyde- and acetaldehyde-oxide systems, and the structure of this adduct is explored through ab initio calculations. The atmospheric implications of the title reaction are investigated through global modelling.

Nucleation of shear bands in amorphous alloys.

The initiation and propagation of shear bands is an important mode of localized inhomogeneous deformation that occurs in a wide range of materials. In metallic glasses, shear band development is considered to center on a structural heterogeneity, a shear transformation zone that evolves into a rapidly propagating shear band under a shear stress above a threshold. Deformation by shear bands is a nucleation-controlled process, but the initiation process is unclear. Here we use nanoindentation to probe shear band nucleation during loading by measuring the first pop-in event in the load-depth curve which is demonstrated to be associated with shear band formation. We analyze a large number of independent measurements on four different bulk metallic glasses (BMGs) alloys and reveal the operation of a bimodal distribution of the first pop-in loads that are associated with different shear band nucleation sites that operate at different stress levels below the glass transition temperature, Tg. The nucleation kinetics, the nucleation barriers, and the density for each site type have been determined. The discovery of multiple shear band nucleation sites challenges the current view of nucleation at a single type of site and offers opportunities for controlling the ductility of BMG alloys.

Nanotwinned Boron Suboxide (B6O): New Ground State of B6O.

Nanotwinned structures in superhard ceramics rhombohedral boron suboxide (R-B6O) have been examined using a combination of transmission electron microscopy (TEM) and quantum mechanics (QM). QM predicts negative relative energies to R-B6O for various twinned R-B6O (denoted as τ-B6O, 2τ-B6O, and 4τ-B6O), consistent with the recently predicted B6O structure with Cmcm space group (τ-B6O) which has an energy 1.1 meV/B6O lower than R-B6O. We report here TEM observations of this τ-B6O structure, confirming the QM predictions. QM studies under pure shear deformation and indentation conditions are used to determine the deformation mechanisms of the new τ-B6O phase which are compared to R-B6O and 2τ-B6O. The lowest stress slip system of τ-B6O is (010)/⟨001⟩ which transforms τ-B6O to R-B6O under pure shear deformation. However, under indentation conditions, the lowest stress slip system changes to (001)/⟨110⟩, leading to icosahedra disintegration and hence amorphous band formation.

NBM-T-BMX-OS01, an Osthole Derivative, Sensitizes Human Lung Cancer A549 Cells to Cisplatin through AMPK-Dependent Inhibition of ERK and Akt Pathway.

BACKGROUND: Drug combination therapies using cisplatin and natural products are common practice in the treatment of human lung cancer. Osthole is a natural compound extracted from a number of medicinal plants and has been shown to exert strong anticancer activities with low toxicity. METHODS: In the present study, NBM-T-BMX-OS01 (BMX), derived from the semi-synthesis of osthole, was evaluated in cisplatin treated A549 cells to investigate its effect on cisplatin resistance in human lung cancer. The anticancer effect of BMX were measured by cell viablity' colony formation' TUNEL staining' flow cytometry and cell cycle assay. The fluorescence staining was performed to detect intracellular and mitochondrial reactive oxygen species (ROS) generation. Western blot analysis, antagonists pretreatment and small interfering RNA (siRNA) transfection were used to determine the potential mechanism. RESULTS: It was found that, in comparison with single cisplatin treatment, the combination of BMX and cisplatin resulted in greater efficacy in inhibition of proliferation and colony formation, apoptosis induction and cell cycle arrest. The results of fluorescence staining showed that the combination effect of BMX and cisplatin was due to oxidative stress induced by mitochondrial ROS generation. In addition, BMX significantly attenuated the phosphorylation of ERK and Akt, two important pro-survival kinases. In contrast, BMX inhibited the activation of AMPK, and knockdown of AMPK using specific siRNA partially reversed BMX-induced inhibition of ERK and Akt, as well as its synthetic effects on cisplatin induced anticancer activity in A549 cells. CONCLUSION: Taken together, this study provides that BMX might modulate cisplatin resistance through AMPK-ERK and AMPK-Akt pathways. These results also support the role of BMX as a potential drug candidate for use in combination with cisplatin in the treatment of human lung cancer.

Jet cooled cavity ringdown spectroscopy of the Ã(2)E(″)←X˜(2)A2 (') transition of the NO3 radical.

The A˜(2)E(″)←X˜(2)A2 (') spectrum of NO3 radical from 7550 cm(-1) to 9750 cm(-1) has been recorded and analyzed. Our spectrum differs from previously recorded spectra of this transition due to jet-cooling, which narrows the rotational contours and eliminates spectral interference from hot bands. Assignments of numerous vibronic features can be made based on both band contour and position including the previously unassigned 30 (1) band and several associated combination bands. We have analyzed our spectrum first with an independent anharmonic oscillator model and then by a quadratic Jahn-Teller vibronic coupling model. The fit achieved with the quadratic Jahn-Teller model is excellent, but the potential energy surface obtained with the fitted parameters is in only qualitative agreement with one obtained from ab initio calculations.

Broadly neutralizing DNA vaccine with specific mutation alters the antigenicity and sugar-binding activities of influenza hemagglutinin.

The rapid genetic drift of influenza virus hemagglutinin is an obstacle to vaccine efficacy. Previously, we found that the consensus hemagglutinin DNA vaccine (pCHA5) can only elicit moderate neutralization activities toward the H5N1 clade 2.1 and clade 2.3 viruses. Two approaches were thus taken to improve the protection broadness of CHA5. The first one was to include certain surface amino acids that are characteristic of clade 2.3 viruses to improve the protection profiles. When we immunized mice with CHA5 harboring individual mutations, the antibodies elicited by CHA5 containing P157S elicited higher neutralizing activity against the clade 2.3 viruses. Likewise, the viruses pseudotyped with hemagglutinin containing 157S became more susceptible to neutralization. The second approach was to update the consensus sequence with more recent H5N1 strains, generating a second-generation DNA vaccine pCHA5II. We showed that pCHA5II was able to elicit higher cross-neutralization activities against all H5N1 viruses. Comparison of the neutralization profiles of CHA5 and CHA5II, and the animal challenge studies, revealed that CHA5II induced the broadest protection profile. We concluded that CHA5II combined with electroporation delivery is a promising strategy to induce antibodies with broad cross-reactivities against divergent H5N1 influenza viruses.