JADE1 is dispensable for the brain development in mice.

In mammalian brain development, WNT signaling balances proliferation and differentiation of neural progenitor cells, and is essential for the maintenance of regular brain development. JADE1 is a candidate transcription co-factor essential for DNA replication, cell division, and cell cycle regulation. In 293T cells, JADE1 is stabilized by von Hippel-Lindau protein pVHL, promotes the ß-catenin ubiquitination and thus blunts canonical WNT signaling. Furthermore, JADE1 inhibits ß-catenin-induced ectopic axis formation in Xenopus embryos. However, JADE1's role in mammalian brain development remains unknown. Here, we generated a new Jade1 knockout mouse line using CRISPR-Cas9 technology. We found that JADE1 null resulted in decreased survival rate, reduced body weight and brain weight in mice. However, histological analysis revealed a normal brain development. Furthermore, Jade1 null neural progenitor cells proliferated normally in vivo and in vitro. RNA-seq analysis further showed that JADE1 loss did not affect the cerebral cortex gene expression. Our findings indicate that JADE1 is dispensable for developing the cerebral cortex in mice.

Strain-Regulated Pt-NiO@Ni Sub-Micron Particles Achieving Bifunctional Electrocatalysis for Zinc-Air Battery.

Highly active bifunctional electrocatalysts for oxygen evolution reaction (OER) and oxygen reduction reaction (ORR) have always been the key factors to affect the performance of zinc-air batteries. However, integrating the independent reaction sites of ORR and OER in a catalyst remains a major challenge. Herein, a collaborative strategy based on defect induction and doping is proposed to prepare the strain-regulated Pt-NiO@Ni sub-micron particles (Pt-NiO@Ni SP). Benefiting from the synergistic effect of tensile strain and Pt-doped, the metallic Ni-based sub-micron particles with tensile strain as the catalyst carriers can effectively optimize the electronic distribution of atomic structures in Pt and NiO on the surface of particles, leading to reduce the energy barrier of intermediates for ORR and OER. Consequently, the Pt-NiO@Ni SP exhibits outstanding bifunctional catalytic activity with the ΔE index of 0.65 V under a low Pt loading, outperforming that of the benchmark Pt/C+IrO2 catalysts (0.76 V). Impressively, the Pt-NiO@Ni SP-based liquid zinc-air battery develops a high open-circuit potential (1.47 V), excellent energy density (188.2 mW cm-2 ), and favorable cyclic charge-discharge cycling durability (200 h at 20 mA cm-2 ). This work provides an innovative avenue for the rational construction of highly active bifunctional electrocatalysts for practical applications.

Research on Key Technologies to Improve Cementing Displacement Efficiency.

The annulus has a wide and narrow clearance due to casing eccentricity in the cementing process and due to the eccentricity of casing in the process of cementing. Because the flow resistance of the drilling fluid in the wide gap is less than that in the narrow gap, the phenomena of a delayed flow or even an overall nonflow occurs in the narrow gap. Based on the existing displacement efficiency calculation model, this paper establishes the cementing displacement efficiency model under the condition of oil-based drilling fluid, explores the residual layer thickness of drilling fluid on the casing side and the sidewall side, and then links the annular displacement efficiency to the injection displacement in combination with the circulating mode resistance pressure drop formula so as to explore the change in the cementing displacement efficiency under different displacements. Considering the change in the physical parameters of annulus fluid, the change in annulus displacement efficiency is obtained. On this basis, the relationship between the wellhead cement injection flow and the annulus retention layer is studied; then, the displacement is calculated. The reasonable cementing displacement is calculated by combining the displacement with annulus displacement efficiency. The results show that the thickness of the annular detention layer increases with the increase in the casing eccentricity in the same well depth, and the growth rate of the detention layer on the wellbore side is greater than that on the casing side at the same circumferential angle. The greater the displacement, the greater the annular circulation pressure drop and circulation equivalent density, thus increasing the cementing risk. The displacement is reasonably designed. The research results in this paper have a certain guiding significance for improving the displacement rate of isolation fluid under oil-based drilling fluid conditions.

Complement component 3 haplotypes influence serum complement activity and milk production traits in Chinese Holstein cattle.

Complement component 3 (C3) is the key molecule of the three pathways of complement activation (alternative, classical, and lectin pathways), which are involved in phagocytosis, inflammation, and immunoregulation processes to destroy infectious microorganisms. In this study, three novel single-nucleotide polymorphisms (SNPs) (g.-1293C>G located in the 5'-flanking region, g.56T>C in exon I, and g.7017C>T in exon XII) of the C3 gene were detected using created restriction site polymerase chain reaction, restriction fragment length polymorphism, and DNA sequencing in 952 cattle from three Chinese breeds. The genotypes and haplotypes were analyzed to investigate the polymorphisms and their possible implications, with particular investigative focus on their associations with serum C3 level, complement hemolytic activity (CH50 and ACH50), and milk production traits. The g.56T>C SNP in exon I affected the serum ACH50 (P<0.01) and the milk somatic cell score (SCS) (P<0.05), and the g.7017C>T SNP in exon XII significantly affected the serum ACH50 values (P<0.01). Moreover, statistical analyses revealed that individuals with genotypic combination CCC/GCC showed significantly lower SCS and the lowest C3 concentration in serum compared with cows with CCC/GTT (P = 0.0007) and CTT/CTT (P = 0.0021); the individuals with CCC/CCT had significantly higher ACH50 values than those with CCC/CTC (P = 0.0008) and CTC/GTC (P = 0.001); cows with CCT/CTT had higher values of CH50 and 305-day milk yield (P>0.05). The C3 expression levels were significantly increased in lung and mammary tissues (P<0.05), while significantly decreased in heart, spleen, liver, and kidney tissues in mastitis cows compared with those in healthy animals (P<0.01), respectively. Bacterial counts of serum antibacterial activities were also completed to verify the effect of SNPs on resistance to mastitis pathogens. Genetically resistant cows (CCC/GCC) had serum with noticeably higher antibacterial activity against S. aureus and E. coli in vitro than the genetically susceptible CCC/GTT cows (P<0.05). Results from this study imply that the C3 gene plays a role in resistance to bacterial infection and that it can be used as a molecular marker for complement activity and traits related to milk production.

The payment scheme for ecosystem services in the coastal city based on the ecosystem services value and current payment efficiency: a case study in Jimo of Qingdao city, China.

The reasonable strategies of payment for ecosystem services (PES) play a key role in solving the contradiction between ecological protection and economic development in coastal cities. However, at present, the payment efficiency is relatively low and the determination method of PES is lack. Therefore, in this study, the Jimo district of Qingdao city in China, a typical coastal city, was selected as study area, and the equivalent factor method and marine ecological capital assessment method were used to obtain terrestrial and marine ecosystem services value (ESV) and its tempo-spatial variations from 2010 to 2018. Moreover, the payment efficiency of the sea area and 15 towns over 8 years was measured using the Super-efficient SBM-DEA model based on undesired output. ESV presented a clear reduction over 8 years. The marine ecosystem provided the main service value, and waste treatment, water regulation, and soil formation were the top 3 main functions in the land ecosystem. Regulating services provided the highest component among all functions. The high-value areas were mainly distributed in the northeast and northwest regions and the low-value areas were in the south-central regions. The average payment efficiency of the sea area and 15 towns over 8 years shown a stable trend at a low level. On the basis of the evaluation of ESV and efficiency, a new possible payment scheme including payment order and quota was proposed. The total quota of marine ecosystem was 5.226 billion RMB (88% of the total) and of terrestrial ecosystem was 0.713 billion RMB. Tian Heng, Jin Kou, Yi Fengdian, and Ling Shan are the first 4 priority towns, with compensation amounts of 0.205, 0.083, and 0.063 billion RMB respectively, while the last 3 towns are Tong Ji, Huan Xiu, and Chao Hai accounting for 6.02% of the total. The theoretical payment total quota was 5.939 billion RMB, and which accounted for a low proportion of local GDP. The study can provide some recommendations for making the reasonable and feasible payment schemes for ecosystem services in coastal city, and it is feasible in the practice of ecological environment protection and sustainable development.

Evaluation of Flushing Ability of Cementing Flushing Fluids.

During cementing operation with oil-based drilling fluids, the casing and well wall are in the "oil wet" environment, and the cement slurry cannot achieve good cementation with the second interface, which seriously affects the cementation quality of the cementing interface. Therefore, it is very important to select a flushing fluid with good performance and high flushing efficiency for cementing operation with an oil-based drilling fluid. An integrated measurement system of interface parameters and a rotating liquid interface tension tester is selected in the room. A set of evaluation methods of wetting reversal is developed to evaluate the wetting reversal ability of type A and type-B flushing fluids on site to optimize the reasonable type and concentration of the flushing fluid. The experimental results show that under the same concentration, the surface tension and interfacial tension of type A flushing solution are lower than those of type-B; that is, the hydrophilic effect of type A flushing solution is better than that of type-B. The wettability reduction of type A is better than that of type-B. The wetting reversal ability of type A flushing solution with a surfactant concentration of 30% is better than that of type-B. The evaluation methods include surface tension, interface tension, dewetting ability, and oil washing ability of the flushing fluid, and the evaluation of the wetting reversal ability is carried out on the basis of the sandstone, sheet metal (steel for three open casings on site), and slide glass (as a reference). Then, according to the optimal concentration of the flushing fluid, a mixing experiment of the two kinds of flushing fluids is carried out to evaluate the ability of wetting reversal, and compared with the experimental results of A and B, the reasonable flushing fluid formula is finally selected. A proper analysis of the adaptability of the evaluation method of the flushing fluid is conducive to the reliable evaluation of the performance of the flushing fluid and the enhancement of the operator's ability to grasp the cementing quality. To guide the field practice has a very important practical significance, but it also has a guiding role for the development of new pre-liquid products.

Three-Dimensional Morphology and Size Measurement of High-Temperature Metal Components Based on Machine Vision Technology: A Review.

The three-dimensional (3D) size and morphology of high-temperature metal components need to be measured in real time during manufacturing processes, such as forging and rolling. Since the surface temperature of a metal component is very high during the forming and manufacturing process, manually measuring the size of a metal component at a close distance is difficult; hence, a non-contact measurement technology is required to complete the measurement. Recently, machine vision technology has been developed, which is a non-contact measurement technology that only needs to capture multiple images of a measured object to obtain the 3D size and morphology information, and this technology can be used in some extreme conditions. Machine vision technology has been widely used in industrial, agricultural, military and other fields, especially fields involving various high-temperature metal components. This paper provides a comprehensive review of the application of machine vision technology in measuring the 3D size and morphology of high-temperature metal components. Furthermore, according to the principle and method of measuring equipment structures, this review highlights two aspects in detail: laser scanning measurement and multi-view stereo vision technology. Special attention is paid to each method through comparisons and analyses to provide essential technical references for subsequent researchers.

Sevoflurane inhibits malignant progression of colorectal cancer via hsa_circ_0000231-mediated miR-622.

BACKGROUND: Sevoflurane (Sev), a commonly used volatile anesthetic, has been reported to inhibit the process of colorectal cancer (CRC). Circular RNAs (circRNAs) are revealed to participate in the pathogenesis of CRC. This study aims to reveal the mechanism of hsa_circ_0000231 in Sev-mediated CRC progression. METHODS: The expression of hsa_circ_0000231 and microRNA-622 (miR-622) was detected by quantitative real-time polymerase chain reaction (qRT-PCR). Protein level was determined by western blot analysis. Cell proliferation was investigated by 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), cell colony formation and DNA content quantitation assays. Cell apoptosis was detected by Annexin V-fluorescein isothiocyanate and propidium iodide double staining and caspase 3 activity assays. Cell migration and invasion were investigated by wound-healing and transwell invasion assays, respectively. The putative relationship between hsa_circ_0000231 and miR-622 was predicted by circular RNA Interactome online database, and identified by dual-luciferase reporter and RNA immunoprecipitation assays. The impacts of hsa_circ_0000231 on Sev-mediated tumor formation in vivo were presented by in vivo assay. RESULTS: Hsa_circ_0000231 expression was upregulated, while miR-622 was downregulated in CRC tissues and cells compared with control groups. Sev treatment decreased hsa_circ_0000231 expression, but increased miR-622 expression in CRC cells. Sev treatment suppressed cell proliferation, migration and invasion, and induced cell apoptosis. Hsa_circ_0000231 overexpression restored Sev-mediated CRC progression in vitro. Additionally, hsa_circ_0000231 acted as a sponge of miR-622, and miR-622 inhibitors reversed the impacts of hsa_circ_0000231 silencing on CRC process. Furthermore, Sev treatment inhibited tumor growth by regulating hsa_circ_0000231 in vivo. CONCLUSION: Hsa_circ_0000231 attenuated Sev-aroused repression impacts on CRC development by sponging miR-622. This findings may provide an appropriate anesthetic protocol for CRC sufferers undergoing surgery.

Temporal variation of oxidative potential of water soluble components of ambient PM2.5 measured by dithiothreitol (DTT) assay.

The exposure to ambient fine particulate matter (PM2.5) can induce oxidative stress, contributing to global burden of diseases. The evaluation of the oxidative potential (OP) of PM2.5 is thus critical for the health risk assessment. We collected ambient PM2.5 samples in Hangzhou city, China for four consecutive quarters in the year 2017 and investigated the oxidation property of PM2.5 components by the dithiothreitol (DTT) assay. The annual mean of ambient PM2.5 mass concentrations in 2017 was 63.05 µg m-3 (median: 57.34, range: 6.67-214.33 µg m-3) with the significant seasonal variations ranking as winter > spring > summer > autumn. Secondary inorganic aerosol (SIA) species including SO42-, NO3- and NH4+ totally account for >50% of PM2.5 mass. The annual mean volume-normalized DTT activity (DTTv) showed a relatively high value of 0.62 nmol/min/m3 (median: 0.62, range: 0.11-1.66 nmol/min/m3) and DTTv of four seasons was roughly at the same level, indicating a high annual exposure level of ambient PM2.5. SIA species were correlated well with the corresponding DTTv and showed significant diurnal variations with strong or moderate correlations at day and weak correlations at night, suggesting strong secondary formation in daytime with contribution to the particulate OP. The annual mean mass-normalized DTT activity (DTTm) had a relatively low value of 6.39 pmol/min/µg (median: 5.63, range: 1.99-22.70 pmol/min/µg), indicating low intrinsic oxidative toxicity. The DTTm of four seasons ranked as autumn > winter > spring > summer, indicating seasonal variations of the DTT-active components. The PM2.5 mass concentration is more related to exposure levels than intrinsic properties of components, while OP is determined by the components rather than PM2.5 mass concentration. Our results provide an insight into reactive oxygen species-induced health risk of PM2.5 exposure and decision for subsequent emission control.

Thyroid Disruption by Bisphenol S Analogues via Thyroid Hormone Receptor ß: in Vitro, in Vivo, and Molecular Dynamics Simulation Study.

Bisphenol S (4-hydroxyphenyl sulfone, BPS) is increasingly used as a bisphenol A (BPA) alternative. The global usage of BPS and its analogues (BPSs) resulted in the frequent detection of their residues in multiple environmental media. We investigated their potential endocrine-disrupting effects toward thyroid hormone receptor (TR) ß. The molecular interaction of BPSs toward TRß ligand binding domain (LBD) was probed by fluorescence spectroscopy and molecular dynamics (MD) simulations. BPSs caused the static fluorescence quenching of TRß LBD. The 100 ns MD simulations revealed that the binding of BPSs caused significant changes in the distance between residue His435 at helix 11(H11) and residue Phe459 at H12 in comparison to no ligand-bound TRß LBD, indicating relative repositioning of H12. The recombinant two-hybrid yeast assay showed that tetrabromobisphenol S (TBBPS) and tetrabromobisphenol A (TBBPA) have potent antagonistic activity toward TRß, with an IC10 of 10.1 and 21.1 nM, respectively. BPS and BPA have the antagonistic activity with IC10 of 312 and 884 nM, respectively. BPSs significantly altered the expression level of mRNA of TRß gene in zebrafish embryos. BPS and TBBPS at environmentally relevant concentrations have antagonistic activity toward TRß, implying that BPSs are not safe BPA alternatives in many BPA-free products. Future health risk assessments for TR disruption and other adverse effects should focus more on the structure-activity relationship in the design of environmentally benign BPA alternatives.

The evaluation of endocrine disrupting effects of tert-butylphenols towards estrogenic receptor α, androgen receptor and thyroid hormone receptor ß and aquatic toxicities towards freshwater organisms.

The phenolic compounds have posed public concern for potential threats to human health and ecosystem. Tert-butylphenols (TBPs), as one group of emerging contaminants, showed potential endocrine disrupting effects and aquatic toxicities. In the present study, we detected concentrations of 2,4-DTBP ranging from <0.001 to 0.057 µg/L (detection limit: 0.001 µg/L) in drinking water source from the Qiantang River in East China in April 2016. The endocrine disrupting effects of 2-TBP, 2,4-DTBP and 2,6-DTBP toward human estrogen receptor α (ERα), androgen receptor (AR) and thyroid hormone receptor ß (TRß) were evaluated using human recombinant two-hybrid yeast bioassay. Their aquatic toxicities were investigated with indicator organisms including Photobacterium phosphoreum, Vibrio fischeri and freshwater green alga Chlamydomonas reinhardtii. 2-TBP and 2,4-DTBP exhibited moderate antagonistic effects toward human ERα and AR in a concentration-dependent manner. 2-TBP significantly inhibited the light emission of P. phosphoreum. 2-TBP, 2,4-DTBP and 2,6-DTBP significantly inhibited the growth of C. reinhardtii and reduced the chlorophyll content. Our results suggest the potential adverse effects of TBPs on human health and aquatic organisms. The data will facilitate further risk assessment of TBPs and related contaminants.

Side Chains of Parabens Modulate Antiandrogenic Activity: In Vitro and Molecular Docking Studies.

Parabens have been widely used in packaged foods, pharmaceuticals, and personal-care products. Considering their potential hydrolysis, we herein investigated structural features leading to the disruption of human androgen receptor (AR) and whether hydrolysis could alleviate such effects using the recombinant yeast two-hybrid assay. Parabens with an aryloxy side chain such as benzyl paraben and phenyl paraben have the strongest antiandrogenic activity. The antiandrogenic activity of parabens with alkyloxyl side chains decreases as the side chain length increases from 1 to 4, and no antiandrogenic effect occurred for heptyl, octyl, and dodecyl parabens with the number of alkoxyl carbon atoms longer than 7. The antiandrogenic activity of parabens correlates significantly with their binding energies (R2 = 0.84, p = 0.01) and were completely diminished after the hydrolysis, particularly for parabens with aryloxy side chains. The Km for the hydrolysis of parabens with aromatic moiety side chain is 1 order of magnitude higher than that of the parabens with alkyl side chains. Both in vitro and in silico data, for the first time, suggest parabens with aromatic side chains are less prone to hydrolysis. Our results provide an insight into risk of various paraben and considerations for design of new paraben-related substitutes.

In vitro and in silico investigations of the binary-mixture toxicity of phthalate esters and cadmium (II) to Vibrio qinghaiensis sp.-Q67.

Phthalate esters (PAEs) are widely used as plasticizers and have become one of the emerging contaminants with an increasing public concern. The residues of PAEs frequently co-exist with heavy metals such as cadmium (Cd) in waters; however, their joint ecotoxicity remains largely unknown. We herein investigated the single and joint toxicity of commonly used PAEs and Cd using freshwater luminescent bacteria Vibrio qinghaiensis sp.-Q67. The median effective concentration (EC50) of benzyl butyl phthalate (BBP), dibutyl phthalate (DBP), diethyl phthalate (DEP), dimethyl phthalate (DMP), diisooctyl phthalate (DIOP) and di-n-octyl phthalate (DOP) were determined to be in the range from 134.4mg/L to as high as 1000mg/L, indicating very weak toxicity to Vibrio qinghaiensis sp.-Q67. The toxicity of single PAEs showed a significant linear relationship with Log Kow, indicating the dependence of the elevated toxicity on the increasing hydrophilicity. The toxicity of binary mixture of PAEs was further evaluated in silico using the independent action (IA) model and concentration addition (CA) model. DMP-DEP, DEP-DBP or DMP-DBP exhibited antagonistic effects with the toxic unit value higher than 1.2. The CA and IA models poorly predicted the joint toxicity of DMP-DEP, DEP-DBP or DMP-DBP. The joint toxicity of the binary mixtures of DMP, DEP or DBP with Cd was simple additive as predicted by the CA and IA models. Our results indicated the potentially higher risk of PAEs in the presence of Cd, emphasizing the importance of determining the impact of their joint effects on aquatic organisms. The integrated in vitro and in silico methods employed in this study will be beneficial to study the joint toxicity and better assess the aquatic ecological risk of PAEs.

Benzotriazole UV 328 and UV-P showed distinct antiandrogenic activity upon human CYP3A4-mediated biotransformation.

Benzotriazole ultraviolet stabilizers (BUVSs) are prominent chemicals widely used in industrial and consumer products to protect against ultraviolet radiation. They are becoming contaminants of emerging concern since their residues are frequently detected in multiple environmental matrices and their toxicological implications are increasingly reported. We herein investigated the antiandrogenic activities of eight BUVSs prior to and after human CYP3A4-mediated metabolic activation/deactivation by the two-hybrid recombinant human androgen receptor yeast bioassay and the in vitro metabolism assay. More potent antiandrogenic activity was observed for the metabolized UV-328 in comparison with UV-328 at 0.25 µM ((40.73 ± 4.90)% vs. (17.12 ± 3.00)%), showing a significant metabolic activation. In contrast, the metabolized UV-P at 0.25 µM resulted in a decreased antiandrogenic activity rate from (16.08 ± 0.95)% to (6.91 ± 2.64)%, indicating a metabolic deactivation. Three mono-hydroxylated (OH) and three di-OH metabolites of UV-328 were identified by ultra-performance liquid chromatography quadrupole time of flight mass spectrometry (UPLC-Q-TOF-MS/MS), which were not reported previously. We further surmised that the hydroxylation of UV-328 occurs mainly at the alicyclic hydrocarbon atoms based on the in silico prediction of the lowest activation energies of hydrogen abstraction from C-H bond. Our results for the first time relate antiandrogenic activity to human CYP3A4 enzyme-mediated hydroxylated metabolites of BUVSs. The biotransformation through hydroxylation should be fully considered during the health risk assessment of structurally similar analogs of BUVSs and other emerging contaminants.

Genome Alignment Spanning Major Poaceae Lineages Reveals Heterogeneous Evolutionary Rates and Alters Inferred Dates for Key Evolutionary Events.

Multiple comparisons among genomes can clarify their evolution, speciation, and functional innovations. To date, the genome sequences of eight grasses representing the most economically important Poaceae (grass) clades have been published, and their genomic-level comparison is an essential foundation for evolutionary, functional, and translational research. Using a formal and conservative approach, we aligned these genomes. Direct comparison of paralogous gene pairs all duplicated simultaneously reveal striking variation in evolutionary rates among whole genomes, with nucleotide substitution slowest in rice and up to 48% faster in other grasses, adding a new dimension to the value of rice as a grass model. We reconstructed ancestral genome contents for major evolutionary nodes, potentially contributing to understanding the divergence and speciation of grasses. Recent fossil evidence suggests revisions of the estimated dates of key evolutionary events, implying that the pan-grass polyploidization occurred ∼96 million years ago and could not be related to the Cretaceous-Tertiary mass extinction as previously inferred. Adjusted dating to reflect both updated fossil evidence and lineage-specific evolutionary rates suggested that maize subgenome divergence and maize-sorghum divergence were virtually simultaneous, a coincidence that would be explained if polyploidization directly contributed to speciation. This work lays a solid foundation for Poaceae translational genomics.

Direct in situ observation of synergism between cellulolytic enzymes during the biodegradation of crystalline cellulose fibers.

High-resolution atomic force microscopy (AFM) was used to image the real-time in situ degradation of crystalline by three types of T. reesei cellulolytic enzymes-TrCel6A, TrCel7A, and TrCel7B-and their mixtures. TrCel6A and TrCel7A are exo-acting cellobiohydrolases processing cellulose fibers from the nonreducing and reducing ends, respectively. TrCel7B is an endoglucanase that hydrolyzes amorphous cellulose within fibers. When acting alone on native cellulose fibers, each of the three enzymes is incapable of significant degradation. However, mixtures of two enzymes exhibited synergistic effects. The degradation effects of this synergism depended on the order in which the enzymes were added. Faster hydrolysis rates were observed when TrCel7A (exo) was added to fibers pretreated first with TrCel7B (endo) than when adding the enzymes in the opposite order. Endo-acting TrCel7B removed amorphous cellulose, softened and swelled the fibers, and exposed single microfibrils, facilitating the attack by the exo-acting enzymes. AFM images revealed that exo-acting enzymes processed the TrCel7B-pretreated fibers preferentially from one specific end (reducing or nonreducing). The most efficient (almost 100%) hydrolysis was observed with the mixture of the three enzymes. In this mixture, TrCel7B softened the fiber and TrCel6A and TrCel7A were directly observed to process it from the two opposing ends. This study provides high-resolution direct visualization of the nature of the synergistic relation between T. reesei exo- and endo-acting enzymes digesting native crystalline cellulose.

The synergistic anticancer effect of artesunate combined with allicin in osteosarcoma cell line in vitro and in vivo.

BACKGROUND: Artesunate, extracted from Artemisia annua, has been proven to have anti-cancer potential. Allicin, diallyl thiosulfinate, the main biologically active compound derived from garlic, is also of interest in cancer treatment research. This object of this report was to document synergistic effects of artesunate combined with allicin on osteosarcoma cell lines in vitro and in vivo. METHODS: After treatment with artesunate and allicin at various concentrations, the viability of osteosarcoma cells was analyzed by MTT method, with assessment of invasion and motility, colony formation and apoptosis. Western Blotting was performed to determine the expression of caspase-3/9, and activity was also detected after drug treatment. Moreover, in a nude mouse model established with orthotopic xenograft tumors, tumor weight and volume were monitored after drug administration via the intraperitoneal (i.p.) route. RESULTS: The viability of osteosarcoma cells in the combination group was significantly decreased in a concentration and time dependent manner; moreover, invasion, motility and colony formation ability were significantly suppressed and the apoptotic rate was significantly increased through caspase-3/9 expression and activity enhancement in the combination group. Furthermore, suppression of tumor growth was evident in vivo. CONCLUSION: Our results indicated that artesunate and allicin in combination exert synergistic effects on osteosarcoma cell proliferation and apoptosis.

Real-time observation of the swelling and hydrolysis of a single crystalline cellulose fiber catalyzed by cellulase 7B from Trichoderma reesei.

The biodegradation of cellulose involves the enzymatic action of cellulases (endoglucanases), cellobiohydrolases (exoglucanases), and ß-glucosidases that act synergistically. The rate and efficiency of enzymatic hydrolysis of crystalline cellulose in vitro decline markedly with time, limiting the large-scale, cost-effective production of cellulosic biofuels. Several factors have been suggested to contribute to this phenomenon, but there is considerable disagreement regarding the relative importance of each. These earlier investigations were hampered by the inability to observe the disruption of crystalline cellulose and its subsequent hydrolysis directly. Here, we show the application of high-resolution atomic force microscopy to observe the swelling of a single crystalline cellulose fiber and its-hydrolysis in real time directly as catalyzed by a single cellulase, the industrially important cellulase 7B from Trichoderma reesei. Volume changes, the root-mean-square roughness, and rates of hydrolysis of the surfaces of single fibers were determined directly from the images acquired over time. Hydrolysis dominated the early stage of the experiment, and swelling dominated the later stage. The high-resolution images revealed that the combined action of initial hydrolysis followed by swelling exposed individual microfibrils and bundles of microfibrils, resulting in the loosening of the fiber structure and the exposure of microfibrils at the fiber surface. Both the hydrolysis and swelling were catalyzed by the native cellulase; under the same conditions, its isolated carbohydrate-binding module did not cause changes to crystalline cellulose. We anticipate that the application of our AFM-based analysis on other cellulolytic enzymes, alone and in combination, will provide significant insight into the process of cellulose biodegradation and greatly facilitate its application for the efficient and economical production of cellulosic ethanol.

Atomic force microscopy studies of a floating-bilayer lipid membrane on a Au(111) surface modified with a hydrophilic monolayer.

The surface of a gold electrode was functionalized with a hydrophilic monolayer of 1-thio-ß-D-glucose formed by spontaneous self-assembly. The Langmuir-Blodgett/Langmuir-Schaefer (LB/LS) method was then used to assemble a bilayer onto the modified Au(111) surface. The bilayer lipid membrane (BLM) was separated from the Au(111) electrode surface by incorporating the monosialoganglioside GM1 into the inner leaflet of a bilayer composed of 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) and cholesterol. To make the inner leaflet, monolayers of GM1/DMPC/cholesterol with mole ratios of 1:6:3, 2:5:3, and 3:4:3 were used. The outer leaflet was composed of a 7:3 mole ratio of DMPC/cholesterol. Because of the amphiphilic properties of GM1, the hydrophobic acyl chains were incorporated into the BLM, whereas the large hydrophilic carbohydrate headgroups were physically adsorbed to the Au(111) electrode surface, creating a "floating" BLM (fBLM). This model contained a water-rich reservoir between the BLM and the gold surface. In addition, because of the bilayer being physically adsorbed onto the support, the fluidity of the BLM was maintained. The compression isotherms were measured at the air/water interface to determine the phase behavior and optimal transfer conditions. The images acquired using atomic force microscopy (AFM) and the force-distance measurements showed that the structure of the fBLM evolved with increasing GM1 content from 10 to 30 mol %, undergoing a transition from a corrugated to a homogeneous phase. This change was associated with a significant increase in bilayer thickness (from ∼5.3 to 7.3 nm). The highest-quality fBLM was produced with 30 mol % GM1.