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Genetically encoded fluorescent proteins have been used for metal ion detections by combining fluorescent proteins with metal-binding proteins or peptides. However, their applications are largely restricted to a limited number of metal ions, such as Ca(2+) and Zn(2+), due to the lack of available metal-binding proteins or peptides that can be fused to fluorescent proteins and the difficulty in transforming the binding of metal ions into a change of fluorescent signal. To overcome these limitations, we report herein the use of Mg(2+)-specific 10-23 or Zn(2+)-specific 8-17 RNA-cleaving DNAzymes to regulate the expression of fluorescent proteins as a new class of ratiometric fluorescent sensors for metal ions. Specifically, we demonstrate the use of DNAzymes to suppress the expression of Clover2, a variant of the green fluorescent protein, by cleaving the mRNA of Clover2, while the expression of Ruby2, a mutant of the red fluorescent protein, is not affected. The Mg(2+) or Zn(2+) in Hela cells can be detected using both fluorescent confocal imaging and flow cytometry. Since a wide variety of metal-specific DNAzymes, such as for Mg(2+), Na(+), Cu(2+), Zn(2+), Pb(2+), Hg(2+), Ag(+), and UO(2)(2+), can be obtained through in vitro selection, and the resulting DNAzymes often share a similar secondary structure and reaction mechanism, the method described in this work can likely be applied to imaging many other metal ions and thus significantly expand beyond the range of the current genetically-encoded fluorescent proteins, allowing this class of sensors to be even more powerful in providing deeper understanding of the roles of metal ions in biology.

A tumor-related hypoxic microenvironment can promote the proliferation of gastric cancer cells, and hypoxic-induced autophagy is the main mechanism of protection against hypoxia in gastric cancer cells. Isorhamnetin (ISO) is a chemical substance derived from plants, mainly from the sea buckthorn. Previous studies have shown that ISO has antitumor effects, but the effects of ISO against gastric cancer in a hypoxic environment are still unknown. In this study, we investigated the effects of ISO against gastric cancer in a hypoxic environment and the mechanisms underlying ISO-induced gastric cancer cell death. The results show that ISO targeted PI3K and blocked the PI3K-AKT-mTOR signaling pathway, significantly inhibiting gastric cancer cell autophagy in a hypoxic environment, inhibiting cell proliferation, decreasing mitochondrial membrane potential, and promoting mitochondria-mediated apoptosis. ISO, a functional food component, is a promising candidate for the treatment of gastric cancer.

Oriented “shish-kebab” structures could be obtained by shearing to enhance the mechanical properties of polymer samples markedly. However, the effect of shear mode on mechanical properties is still uncertain. The study of stepped hoop shear field on the isotactic polypropylene (iPP) pipe was developed through applying a self-designed rotational shear system (RSS). The effect of stepped shear field on the microstructure and comprehensive properties of iPP pipe was investigated by the comparison with continuous shear. It could be found that the loosely-assembled shish-kebabs with the larger size were formed in the continuous shear pipes, but the smaller and tightly-stacked ones existed in the pipes with stepped shear. Surprisingly, due to differential morphologies under different shear modes, better comprehensive mechanical properties were obtained in the pipes with stepped shear.

Owing to splendid properties, trinitrophenol-based ionic liquid consisting of ammonium–imidazolium cation, 3,5-diamino-1,2,4-triazole picrate (DATPA), has been investigated concerning the thermal stability in this study. Differential scanning calorimeter (DSC) and thermogravimetry (TG) are used to explore the thermal behavior such as melting point, decomposition temperature, and the temperature of 10% mass loss which are the target to evaluate the thermal stability of other two picrates (picrate and 4-amino-1,2,4-triazole picrate). The results show that DATPA has better thermal stability at 251–252 °C than the other two picrates, and the melt volatility has decreased as well. Moreover, thermal kinetics parameters are calculated by changing the heating rates of DSC and TG. ASTM standard E698 and Kissinger methods are utilized to calculate, and double extrapolation is accepted to explore the mechanism function in the decomposition course, which has been defined as $$f(\alpha ) = 2.105(1{-}\alpha )[\ln (1{-}\alpha )]^{0.525}$$ in this study. Finally, an accelerating rate calorimeter is employed to study the thermal hazard of DATPA under the adiabatic condition and evaluate the level of runaway reaction through the risk matrix. According to Townsend method, we achieve the thermal kinetics parameters of DATPA under adiabatic condition. The results show that the apparent activation energy (Ea) of DATPA is 273.2 kJ mol−1 higher than many other explosives. Besides, according to the theoretical calculation, we find that DATPA has good detonation properties with high stability.

Type 1 copper (T1Cu) proteins play important roles in electron transfer in biology, largely due to the unique structure of the T1Cu center, which is reflected by its spectroscopic properties. Previous reports have suggested a correlation between a high ratio of electronic absorbance at ∼450 nm to that at ∼600 nm (

Sleep deprivation (SD) has detrimental effects on the physiological function of the brain. However, the underlying mechanism remains elusive. In the present study, we investigated the expression of candidate plasticity-related gene 15 (cpg15), a neurotrophic gene, and its potential role in SD using a REM-SD mouse model. Immunofluorescent and Western blot analysis revealed that the expression of cpg15 protein decreased in the hippocampus, ventral group of the dorsal thalamus (VENT), and somatosensory area of cerebral cortex (SSP) after 24–72 h of REM-SD, and the oxidative stress in these brain regions was increased in parallel, as indicated by the ratio of glutathione (GSH) to its oxidative product (GSSG). Over-expression of cpg15 in thalamus, hippocampus, and cerebral cortex mediated by AAV reduced the oxidative stress in these regions, indicating that the decrease of cpg15 might be a cause that augments oxidative stress in the sleep deprived mouse brain. Collectively, the results imply that cpg15 may play a protective function in the SD-subjected mouse brain via an anti-oxidative function. To our knowledge, this is the first time to provide evidences in the role of cpg15 against SD-induced oxidative stress in the brain.

Somatic cell nuclear transfer (SCNT) technology has been applied in the construction of disease model, production of transgenic animals, therapeutic cloning, and other fields. However, the cloning ...

Pancreatic cancer is a highly malignant disease that is associated with poor prognosis. One hallmark of pancreatic cancer is excessive desmoplasia, characterized by fibrous or connective tissue growth and altered tumor stroma. Pancreatic stellate cells (PSCs) comprise a mesenchymal cell type that contributes to pancreas fibrosis and cancer progression. PSME3 is a regulatory subunit of the proteasome that is expressed in various cancers such as breast, ovarian, and pancreatic. Notably, PSME3 modulates lactate secretion in pancreatic cancer, suggesting a potential function in regulating pancreas fibrosis. However, the role of PSME3 in pancreatic cancer cell (PCC)-PSC interactions remains unclear. The current study, for the first time, explored the mechanism involved in PSME3-mediated PCC-PSC interactions. IHC showed that PSME3 is highly expressed in PCCs, and this was found to correlate with tumor differentiation. RNA interference (RNAi) indicated that PSME3 is involved in PCC apoptosis. PCR array and cell co-culture experiments suggested that conditioned culture medium (CM) from PSME3-knockdown PCCs could suppress PSC proliferation by down-regulating TGFB1 secretion. Transcription factor (TF) activation assays showed that PSME3 regulates TGFB1 production by inhibiting activation protein-1 (AP-1). Together, these data demonstrate that PSME3 interacts with AP-1 to regulate TGFB1 secretion in PCCs and promote PSC proliferation. Our results indicate a novel PSME3-regulated association between PSCs and PCCs and provide a promising therapeutic strategy for this malignancy.

Gastric cancer (GC) is a globally important disease. Targeted therapies licensed to treat gastric cancer include trastuzumab, which targets HER2, and nivolumab or pembrolizumab, which block PD-1/PD-L1. The main challenge of tumor molecule-targeted drugs is resistance. Based on the multicomponent and multitarget characteristics of Traditional Chinese Medicine (TCM) and our previous research on Guiqi Baizhu Prescription (GQBZP) in targeting HER2/PD-L1, we used different cell models to verify the effect of quercetin targeting of HER2 and formononetin targeting of PD-L1. The results show that quercetin can inhibit MKN-45 cell proliferation by targeting HER2 to inhibit the PI3K-AKT pathway, and formononetin can regulate T-cell function by inhibiting the PD-1/PD-L1 pathway. We also used gastric cancer MKN-45 cells in a T lymphocyte co-culture model to show that quercetin and formononetin co-treatment can regulate T cell function and inhibit MKN-45 cell proliferation. More importantly, quercetin with formononetin treatment had a greater effect than treatment with either quercetin or formononetin alone. We believe that quercetin and formononetin may be useful to target HER2 and inhibit PD-1/PD-L1 in gastric cancer.

In vitro developmental competence of sheep oocytes is still relatively low. In this study, we investigated cathepsin B activity in sheep cumulus-oocyte complexes, the expression of cathepsin B and apoptosis rates in sheep cumulus cells, and the correlations between these indicies and in vitro developmental competence of sheep oocytes. The present result suggested that the active form of cathepsin B in cumulus-oocyte complexes was predominantly present in cumulus cells rather than oocytes. Cathepsin B activity in cumulus-oocyte complexes was strongly inhibited by cathepsin inhibitor E-64. RT-PCR analysis showed that cathepsin B expression in cumulus cells decreased during in vitro maturation. TUNEL staining and flow cytometry of cumulus cells showed that the apoptosis rate increased after in vitro maturation, and treatment with E-64 reduced both cathepsin B expression and apoptosis levels. Furthermore, E-64 treatment increased blastocyst rates of sheep.

In this paper, the effect of anhydrite on the cement-based self-leveling mortar performance was studied. The construction operation of mortar and its mechanical property testing were based on the standard JC/T 981-2005. The results showed that the anhydrite played a significant role on the performance of compressive strength and flexural strength. Furthermore, the best performance of dimensional change rate, abrasion resistance and adhesive strength was obtained with 8% anhydrite content. This study has guiding significance for the construction and application of cement-based self-leveling mortar.

The current paper experimentally investigated the effect of different kinds re-dispersible polymer powers (RPP) on cement-based self-leveling mortars. The construction operation of mortar and its mechanical property was tested in accordance with the standard JC/T 981-2005. Besides, the micro-structure surface of self-leveling mortar was characterized by means of SEM to reflect the microscopic mechanism of the performance. The results showed the dispersible polymer powders could significantly improve performance of fluidity, adhesion property and abrasion resistance on cement-based self-leveling mortar; Meanwhile, there is no bad impact on its compressive strength and flexural strength. This study has guiding significance for the construction and application of cement based self-leveling mortar.

A novel DNAzymatic amplifier nanomachine that is reconstructed in response to an mRNA input has been developed to enable the functions of concurrent sensitive mRNA imaging and activatable gene therapy in living cells.

Physical damage and oxidative stress may occur in prepubertal cumulus cells, due to insufficient glutathione synthesis. To determine potential epigenetic mechanisms related to antioxidant effects of melatonin on ovine prepubertal cumulus cells, 30 lambs, 4-wk-old were randomly allocated into two groups: a control (C, n = 20) group and a melatonin (M, n = 10) group given a subcutaneous implant containing 18 mg melatonin. All lambs were superovulated (250 IU FSH and 250 IU eCG). Cumulus cells from germinal vesicle stage cumulus oocyte complexes (COCs) were collected by ovarian follicular aspiration and dissociated with hyaluronidase. Compared to the C group, the M group had greater superovulation, better antioxidant capacity, a higher proportion of fully expanded COCs and a lower proportion of apoptotic cumulus cells (P 0.05). Melatonin up-regulated mRNA expression of genes for melatonin receptors MT1 and nuclear binding site RORα, antioxidants (SOD1, GPx4 and CAT) and cumulus cell expansion (PTX3, HAS2 and PTGS2), as well as Bcl2, but down-regulated expression of Bax (P 0.05). Regarding epigenetics, there were less methylation at five CpG sites of SOD1, three CpG sites of GPx4 and two CpG sites of CAT in M versus C groups (P 0.05), leading to lower total methylation of SOD1, GPx4 and CAT promoters region on M group (P 0.05). In a mechanistic study, addition of MT1 or RORα antagonist increased ROS and MDA concentrations, but decreased T-AOC, GPx, CAT and T-SOD concentrations (P 0.05), whereas there were no significant difference between the melatonin and MT2 antagonist treatment groups for T-AOC, GPx, CAT and T-SOD concentrations. Furthermore, addition of RORα agonist decreased total DNA methylation of SOD1, GPx4 and CAT, with no significant difference after MT1 agonist treatment. These studies provided new information regarding epigenetic mechanisms by which melatonin promoted ovine prepubertal cumulus cells antioxidant through RORα, both in vitro and in vivo.

Loop-mediated isothermal amplification (LAMP) is a useful platform for nucleic acids detection in point-of-care (POC) situations, and development of single-step, close-tube LAMP reactions for specific detection of single nucleotide mutations (SNMs) remains a challenge. We develop a novel primer-activatable LAMP (PA-LAMP) strategy that enables highly specific and sensitive SNM detection using single-step, close-tube reactions. This strategy designs a terminal-blocked inner primer with a ribonucleotide insertion, which is cleaved and activated specifically to perfectly matched targets by ribonuclease (RNase) H2, to realize efficient amplification of mutant genes. It has shown dynamic responses of mutant target in a linear range from 220 aM to 22 pM with a lowest detectable concentration of 22 aM. It also demonstrates very high specificity in identifying the mutant in a large excess of the wild-type with a discrimination ratio as high as ∼10,000. It has been successfully applied to mutation detection of genomic DNA in tumor cells. The PA-LAMP strategy provides a useful, portable and affordable POC platform for highly sensitive and specific detection of genetic mutations in clinical applications.

This study presents a simple and label-free biosensor for sensitive and selective detection of microRNA based on the formation of the adenosine2–coralyne–adenosine2 complex in the presence of poly(A) polymerase and coralyne.

A nickel-based compound layer was prepared on a nickel plate by anodization in a 75 wt% H3PO4 solution containing NH4F. This layer was then treated by galvanostatic charge/discharge (GCD) until a black outer layer was detached, leaving behind a film on the nickel plate as a binder-free electrode material for supercapacitors. The microstructural characterization shows that the film consists of Ni(OH)2 and NiO, and no fluoride is found in the as-obtained film. Electrochemical tests demonstrate that this fluoride-free film electrode exhibits a high capacitance of 954 F·g−1 at 7.5 A·g−1, excellent rate capability (a 19.5 % capacitance reduction with the current density increasing to 120 A·g−1) and cycling stability. Within 3500 cycles, the specific capacitance does not decrease, but rather increases from 840 F·g−1 to approximately 1092 F·g−1 in the first 100 cycles at 60 A·g−1, and remains stable until the aforementioned layer is detached.

Community-associated infections due to Escherichia coli producing CTX-M-type extended-spectrum β-lactamases are increasingly recognized in the United States. The bla CTX-M genes are frequently carried on IncF group plasmids. In this study, bla CTX-M-15 -harboring plasmids pCA14 (sequence type 131 [ST131]) and pCA28 (ST44) and bla CTX-M-14 -harboring plasmid pCA08 (ST131) were sequenced and characterized. The three plasmids were closely related to other IncFII plasmids from continents outside the United States in the conserved backbone region and multiresistance regions (MRRs). Each of the bla CTX-M-15 -carrying plasmids pCA14 and pCA28 belonged to F31:A4:B1 (FAB [FII, FIA, FIB] formula) and showed a high level of similarity (92% coverage of pCA14 and 99% to 100% nucleotide identity), suggesting a possible common origin. The bla CTX-M-14 -carrying plasmid pCA08 belonged to F2:A2:B20 and was highly similar to pKF3-140 from China (88% coverage of pCA08 and 99% to 100% nucleotide identity). All three plasmids carried multiple antimicrobial resistance genes and modules associated with virulence and biochemical pathways, which likely confer selective advantages for their host strains. The bla CTX-M -carrying IncFII-IA-IB plasmids implicated in community-associated infections in the United States shared key structural features with those identified from other continents, underscoring the global nature of this plasmid epidemic.

In this work the effect of argon filling ratio and air space thickness on the heat transfer coefficient of double-glazing was studied. The heat transfer coefficient of double-glazing was measured by heat flow meter method (based on standards ISO10291, ISO 10292, ISO8301 and DIN EN 12939), during this process the testings of argon filling ratio and emissivity of coated glass surface were involved. For the thermal performance of double-glazing, the influence of various factors on heat transfer coefficient was analyzed. The comparison was conducted between coated double-glazing (one glass pane coated with low-emissivity) and non-coated double-glazing. Within the thickness of air space in the range of 9–18 mm, the higher the air space thickness, the higher the filling ratio of argon gas, the better the thermal insulation performance achieved. Under the same gas filling ratio, the heat transfer coefficient of coated double-glazing (emissivity is 0.13) which is much lower than that of non-coated double-glazing, can effectively meet the requirement of building energy efficiency in China.

Aberrant DNA methylation reduces the developmental competence of mammalian somatic cell nuclear transfer (SCNT) embryos. Thus, hypomethylation-associated drugs are beneficial for improving reprogramming efficiency. Therefore, in the present study we investigated the effect of zebularine, a relatively novel DNA methyltransferase inhibitor, on the developmental potential of ovine SCNT embryos. First, reduced overall DNA methylation patterns and gene-specific DNA methylation levels at the promoter regions of pluripotency genes (octamer-binding transcription factor 4 (Oct4), SRY (sex determining region Y)-box 2 (Sox2) and Nanog) were found in zebularine-treated cumulus cells. In addition, the DNA methylation levels in SCNT embryos derived from zebularine-treated cumulus cells were significantly reduced at the 2-, 4-, 8-cell, and blastocyst stages compared with their corresponding controls (P

Uracil-DNA glycosylase (UDG) plays essential roles in base excision repair (BER) pathway by eliminating uracil from DNA to sustain the genome integrity. Sensitive detection of UDG activity is of great significance in the study of many fundamental biochemical processes and clinical applications. We develop a label-free method for UDG activity detection using stem-loop primer-mediated exponential amplification (SPEA). In the presence of active UDG, the uracil base in helper hairpin probe (HP) can be excised to generate an abasic site (AP site), which can be cleaved by endonuclease IV (Endo IV) with a blocked primer released. This primer then triggers the strand displacement reaction to produce a dumb-bell structure DNA, which can initiate a loop-mediated isothermal amplification (LAMP) reaction. This reaction generates a large number of long double-strand DNA replicates, which can be stained by SYBR Green (SG) I to deliver enhanced fluorescence for quantitative detection of UDG activity. A linear range from 0.001 U/mL to 1 U/mL and a detection limit down to 0.00068 U/mL are achieved. This strategy has also been demonstrated for UDG assay in complex cell lysates, implying its great potential for UDG based clinical diagnostics and therapeutics.

This study was conducted to determine the effect of meiotic status, cumulus cells and cytoskeleton stabilizer on ovine oocyte vitrification. Oocytes at various developmental stages including GV (germinal vesicle), GVBD (GV breakdown), MI (metaphase I) and MII (metaphase II) were vitrified using open pulled straw (OPS) method. After warming, the survival rates were determined based on the morphological appearance and 3′,6′-diacetyl fluorescein staining. The developmental potential of treated oocytes was evaluated by their ability to undergo successful in vitro fertilization (IVF) and support embryo development in culture after in vitro maturation. In the first experiments, we evaluated the effect of meiosis status on oocytes vitrification. Survival rates of oocytes after warming were not different among all groups. However, significantly higher proportion of cleavages and blastocysts were obtained from vitrified MII oocytes than those from vitrified immature oocytes. Next, we selected MII oocytes to determine the influence of cumulus cells on vitrification and the results showed that survival rates were not affected by the absence of cumulus cells. Furthermore, the cleavage rates and blastocyst rates were not different with or without cumulus cells. Lastly, we examined the effect of cytoskeleton stabilizer on MII oocyte vitrification. Compared with the vehicle treated controls, pretreatment with Taxol significantly improved the survival rates (81.91% vs. 66.00%), cleavage rates ((52.29% vs . 34.25%) and blastocyst rates (9.72% vs. 4.86%). Pretreatment of MII oocytes with another cytoskeleton stabilizer Cytochalansin B had no effect on oocyte survival and in vitro embryo development. Collectively, the meiotic status affected the developmental potential of oocytes after vitrification. MII stage oocytes showed better resistance to cryopreservation compared with the oocytes at immatured stages. Taxol treatment prior to vitrification was beneficial to vitrified/warmed ovine matured oocytes.

Three new molecular magnets [DiFBzPid][Ni(mnt)2]·0.5CH3COCH3(1), [DiBrBzPid][Ni(mnt)2](2) and [DiNO2BzPid][Ni(mnt)2](3) have been obtained by self-assembly of the [Ni(mnt)2]− (mnt2− = maleonitriledithiolate) anion and [DiRBzPid]+ ([DiRBzPid]+ = N,N′-diRbenzylpiperidinium; R = F, Br, NO2) cation. The Ni(III) ions of the anions in 1 and 3 form a 1D alternating magnetic chain within a [Ni(mnt)2]− column through Ni⋯S, S⋯S, S⋯N, S⋯C or C⋯N short interactions, while the anions in 2 form a dimer. The cations in three compounds stack into a 1D column in different shapes via p⋯π, F⋯F, Br⋯Br, and O⋯O weak interactions or C H⋯π hydrogen bonds. In addition, the [DiNO2BzPid]+ cations (C) and [Ni(mnt)2]− anions (A) of 3 stack into a 1D alternative column with a A–CC–A–CC–A sequence. The change of the substituent group in the benzyl ring from F(1) to Br(2) and NO2(3) results in the significant differences in the anion–anion, anion–cation and cation–cation stacking modes of 1, 2 and 3. Magnetic susceptibility measurements in the temperature range of 2–300 K show that 1 and 3 show characteristic of spin-gap (Δ/kb = 524.6 K or 1706.6 K), while 2 exhibits a strong antiferromagnetic coupling feature with J/kb = −663.2 K.

Compelling evidence suggests that acetylation at histone H4 lysine 12 and 16 (acH4K12 and acH4K16) influence oocyte competence. The current study was conducted to determine the effect of vitrification on acH4K12 and acH4K16 patterns in sheep oocytes. Vitrified/warmed oocytes or fresh oocytes were collected at GV, GVBD, MI and MII, and acH4K12 and acH4k16 were assessed using fluorescent staining. Confocal microscopy analyses revealed that acH4K12 and acH4K16 at GV, MI and MII were significantly higher in vitrified/warmed oocytes than in fresh oocytes ( P P > 0.05). In summary, vitrification alters the acH4K12 and acH4K16 levels in mature and immature sheep oocytes, which likely contributes to the impaired development of vitrified oocytes after fertilization.

A novel ligation-based loop-mediated isothermal amplification (ligation-LAMP) method has been developed for miRNA detection, which enables highly selective and sensitive quantitative detection of miR-21 in a dynamic range from 1 fM to 1 nM with the ability to discriminate single-base mismatches.

In this paper, we report the positive bias temperature instability (PBTI) effects of the back-gated MoS2 n-MOSFET with Al2O3 gate dielectric. Multilayer MoS2 was used and all measurements are carried in vacuum to avoid the electric signal contamination by the top MoS2 surface water or oxygen molecules absorption. In the stress phase, the Id-Vg curve shifts to the positive gate bias direction. In the recovery phase, the Id-Vg shifts back completely to the fresh device curve. This indicates that voltage shift is purely due to trapping or detrapping of the oxide border traps at the MoS2/Al2O3 interface and no new traps are generated during the stress time. When applying different stress voltage and measuring the Id-Vg shift, we can calculate the oxide border trap energy density Ξ(E), which has a peak value of 6×1013cm−2eV−1 at 0.035eV above EC of MoS2 conduction band.

Vitrification by using two-step exposures to combined cryoprotective agents (CPAs) has become one of the most common methods for oocyte cryopreservation. By quantitatively examining the status of oocytes during CPA additions and dilutions, we can analyze the degree of the associated osmotic damages. The osmotic responses of mouse MII oocyte in the presence of the combined CPAs (ethylene glycol, EG, and dimethyl sulfoxide, DMSO) were recorded and analyzed. A two-parameter model was used in the curve-fitting calculation to determine the values of hydraulic conductivity (L(p)) and permeability (P(s)) to the combined CPAs at 25°C and 37°C. The effects of exposure durations and the exposure temperatures on the cryopreservation in terms of frozen-thawed cell survival rates and subsequent development were examined in a series of cryopreservation experiments. Mouse MII oocytes were exposed to pretreatment solution (PTS) and vitrification solution (VS) at specific temperatures. The PTS used in our experiment was 10% EG and 10% DMSO dissolved in modified PBS (mPBS), and the VS was EDFS30 (15% EG, 15% DMSO, 3 × 10(-3) M Ficoll, and 0.35 M sucrose in mPBS).The accumulative osmotic damage (AOD) and intracellular CPA concentrations were calculated under the different cryopreservation conditions, and for the first time, the quantitative interactions between survival rates, subsequent development rates, and values of AOD were investigated.

The anti-allergic drug, N-(3,4-dimethoxycinnamonyl) anthranilic acid (3,4-DAA), is a synthetic anthranilic acid derivative that has been used therapeutically in Japan for many years. In this study, to investigate the effects of 3,4-DAA in allograft immunorejection model, liver orthotopic transplants were performed using inbred male Dark Agouti donors and Lewis rat recipients (allografts). The levels of indoleamine 2,3-dioxygenases (IDO) enzymic activities in five groups, allografts (control), dimethyl sulphoxide-treated group (vehicle control), 200 mg·kg–1·day–1 of 3,4-DAA-treated group and 200 mg·kg–1·day–1 of 3,4-DAA + 5 mg·ml–1 of 1-methyl-D-tryptophan (1-MT)-treated group were confirmed by determination of L-kynurenine (L-Kyn) concentrations. The serum alanine aminotransferase levels in 3,4-DAA-treated rats significantly decreased compared with those in mock and control group, whereas treatment of 1-MT in allografts led to the opposite effect. Administration of 3,4-DAA reduced histological severity of allograft immunorejection, decreased serum levels of cytokines tumour necrosis factor-alpha (TNF-α) and interferon-gamma (IFN-γ), and raised serum levels of interleukin-10 (IL-10), suggesting that 3,4-DAA has both anti-inflammatory and anti-immunorejection properties through IDO in immune regulation and may therefore be useful in filling an unmet need, in the treatment of allograft immunorejection. Copyright © 2011 John Wiley & Sons, Ltd.

The objective was to determine whether adding L-carnitine in IVM/IVC medium enhanced maturation and developmental competence of porcine oocytes in vitro. Oocyte maturation rates did not differ significantly among groups supplemented with 0, 0.25, 0.5, or 1 mg/mL of L-carnitine added during IVM (although 2 mg/mL of L-carnitine reduced maturation rate). Compared with control oocytes, those treated with 0.5 mg/mL of L-carnitine during IVM had greater (P < 0.05) rates of blastocyst formation after parthenogenetic activation, and these blastocysts had less (P < 0.05) apoptosis. Adding 0.5 mg/mL of L-carnitine during IVM also significantly reduced intracellular reactive oxygen species (ROS), and increased glutathione (GSH) concentrations. With or without glucose supplementation, 0.5 mg/mL of L-carnitine in the IVM medium significantly hastened nuclear maturation of oocytes. Moreover, supplementing the IVM medium with either glucose or L-carnitine increased (P < 0.05) percentages of oocytes that reached the metaphase II (MII) stage, relative to a control group. Final maturation rates in IVM medium containing either glucose or L-carnitine were not significantly different. Adding L-carnitine (0 to 2 mg/mL) to IVC medium for activated porcine oocytes did not significantly affect development. However, 0.5 mg/mL of L-carnitine in IVC medium significantly reduced reactive oxygen species levels and apoptosis in activated blastocysts, although glutathione concentrations were not significantly altered. In conclusion, adding L-carnitine during IVM/IVC improved developmental potential of porcine oocytes, and also the quality of parthenogenetic embryos, probably by accelerating nuclear maturation, and preventing oxidative damage and apoptosis.

In order to examine its effect on oocyte lipid content and cryosurvival, Forskolin was added to the medium for in vitro maturation of porcine oocytes. Treatments were control (IVM without Forskolin during the 42 h incubation period), addition of 10 μM Forskolin for the entire 42 h (0-42) and addition of 10 μM Forskolin between 24 and 42 h only (24-42). In Experiment 1, treatments did not differ significantly in cleavage rate, but the blastocyst formation rate was lower in the 0-42 group than for control and 24-42 group oocytes (17, 32 and 40%, respectively; P0.05). It was shown in Experiment 2 that Forskolin treatment from 0-42 h and from 24-42 h significantly reduced lipid content of oocytes compared to that of control cells (65 and 99 vs. 140 μm(2) intensity of fluorescence, respectively; P0.05). In Experiment 3, the percentage of oocyte survival after cryopreservation and thawing was significantly higher in both Forskolin treatment groups than in control oocytes (72% for 0-42, 65% for 24-42 and 52% for control; P0.05). However, Forskolin treatment did not increase cleavage rates of vitrified in vitro matured porcine oocytes (Control group 28%, 0-42 h group 0%, 24-42 h group 26.67%). Addition of Forskolin affected the nuclear maturation of porcine oocytes. The percentage of PBE (polar body extrusion) were significantly reduced in the 0-42 h group (0-42 h group 42.00 ± 2.08 vs. Control group 79.70 ± 2.82 and 24-42 h group 70.60 ± 2.83; P0.05). The 24-42 h group showed similar nuclear status to that of the Control group. We propose that delipation engendered by incubation with 10 μM Forskolin during 24-42 hours of maturation increased cryosurvival of in vitro-maturated porcine oocytes and that attendant chemical lipolysis did not impair their further development as it may have done in oocytes incubated with Forskolin for the full 42 h.

Recent advances in optical detection methods and techniques applied in cell-based microfluidic systems are reviewed, and these methods mainly focused on microsystems that integrate on-chip cell culture, real-time detection, and recent advances in correlated detection methods and techniques. The comparison of six optical detection methods and techniques are summarized, and their research applicability for cell study is proposed. The trend and the development perspective of each technique applied for label-free, real-time detection and sensing of living cell are discussed. Finally, an assessment of future directions for lab chip-based cell research and sensing microsystem is concluded.

In this work, we demonstrate a simple and reliable method for selectively separating single wall carbon nanotubes (SWNTs). This method is based on a preferable adsorption of linear polynuclear aromatic dyes on metallic SWNTs. With different adsorption amount of soluble dyes, the metallic and semconducting SWNTs show different solubility in solvents, hence can be separated. The types of the obtained SWNTs and the separative efficiency using different dyes have been studied using Raman spectroscopy. It was found that the polynuclear molecules with more benzene rings show higher selectivity. Meanwhile, the separative efficiency is also sensitive to the choice of solvents.

Systematic studies on noncovalent functionalization of multiwalled carbon nanotubes (MWNTs) were assisted by a series of aromatic dye molecules with different structural characteristics. The effects of molecular structure on the affinities of different dyes to the MWNTs were evaluated. Two main factors have been found to play the key roles for the dye–MWNTs interactions, which are molecular geometry and charge. It was found that molecules with planar structures and high charge load are favored for the adsorption. This work not only demonstrates a simple and effective method to functionalize the sidewalls of carbon nanotubes but also give insight to the adsorption mechanism between the carbon nanotubes and aromatic molecules.

Conducting polyaniline (PANI) was in situ polymerized at the surface of electrospun poly(methyl methacrylate) (PMMA) fibers to obtain flexible composite fibers. The electrical conductivity of an individual PANI/PMMA composite fiber was estimated to be 2.0 × 10−1 S cm−1at room temperature. The ammonia sensing properties of the samples were tested by impedance analysis. The PANI/PMMA fibers could obviously respond to low concentration of ammonia at ppb level and could respond to relatively high concentration of ammonia at 10 ppm level quickly. In addition, the sensitivity exhibited a good linear relationship to the ammonia concentration. Particularly, the flexible PANI/PMMA fibers showed a reversible change in electrical resistance with repeated cycles of bending and relaxing, and the electrical resistance decreased with the increase of curvature. These results indicate that the flexible PANI/PMMA composite fibers may be used in toxic ammonia gas detection, strain sensing, and flexible electronic devices.

A novel fluorescent nanosensor has been developed by combining super fluorescence quenching ability of graphene oxide and hybridization chain reaction amplification, which enables highly sensitive detection of base excision repair enzyme activity with a wide dynamic range from 0.0001 to 100 U mL(-1) and a detection limit of 0.00006 U mL(-1).

RmtE is a rare 16S-RMTase which was first reported in an aminoglycoside-resistant Escherichia coli strain of calf origin ([1][1]). Subsequently, we reported the first human case of infection caused by RmtE-producing E. coli ([2][2]). The rmtE gene is carried on a self-conjugative plasmid (pYDC637)

Nanofibrous sugar sticks with linear PANCGAMA and cyclic PANCAG glucose pendants were fabricated by electrospinning from acrylonitrile-based glycopolymers. Field emission scanning electron microscope was used to characterize the morphologies of the nanofibers. Utilizing the specific carbohydrate-protein interaction, these two kinds of nanofiber mats were applied to separate the protein mixtures. Con A and BSA were used as model proteins. The Con A/BSA mixture solution was isolated successfully by selective adsorption of Con A to the cyclic glucose pendants on the PANCAG nanofibrous sugar sticks. However, PANCGAMA nanofibers showed almost no selectivity for these two proteins due to the poor specificity between linear glucose pendants and Con A.

Titanium dioxide (TiO 2 ) films have been successfully deposited on metal alloy substrates by radio-frequency magnetron reactive sputtering in an Ar+O 2 gas mixture. The effects of gas total pressure on the structure and phase transition of TiO 2 films were studied by X-ray diffraction and Raman spectra. It is suggested that the film structure changes from rutile to anatase while work gas total pressure changes from 0.2 to 2 Pa. The structure of TiO 2 films is not affected by the film thickness.

Background/Aims : Chronic ethanol consumption results in the induction of hepatic cytochrome P4502E1 (CYP2E1) in man, which is believed to play an important role in the pathogenesis of alcoholic liver disease. However, the amount and duration of alcohol intake associated with CYP2E1 induction is not known but limited information is available on the disappearance of CYP2E1 following alcohol withdrawal. Methods : To study these questions, five healthy male volunteers received ethanol daily (40g/day) over 4 weeks. CYP2E1 induction was monitored by using the chlorzoxazone test before and every week following the start of alcohol ingestion. In addition, CYP2E1 was also determined in five alcoholics 1, 3, 8 and 15 days following ethanol withdrawal and in five patients with non-alcoholic liver disease. Results : A significant CYP2E1 induction occurred 1 week following the ingestion of 40g ethanol per day and increased further after 4 weeks. The disappearance of CYP2E1 was found to be significant 3 days following ethanol withdrawal and further decreased up to day 8. Thereafter, no significant change occurred and CYP2E1 activities were comparable with those in patients with non-alcoholic liver disease. Conclusions : These data show a significant and quick induction of CYP2E1 activity, already at moderate alcohol consumption, which may be of importance in the pathogenesis of alcoholic liver disease, of ethanol, drug and vitamin A interactions and in alcohol associated carcinogenesis.

Paclitaxel-containing treatment regimens are standard chemotherapy schemes for breast cancer patients. The use of oncolytic herpes simplex virus (oHSV) vectors has been shown to be a safe and effective therapeutic approach for different types of cancer. We hypothesized that paclitaxel in combination with an oHSV vector would present an enhanced killing effect when used against breast cancer cells. In the present study, we demonstrated that the combined use of the oHSV vector G47Δ and paclitaxel produced a synergistic effect against breast cancer cells both in vitro and in vivo. In vitro studies demonstrated that paclitaxel and G47Δ both caused dose-dependent cytotoxicity against the human breast cancer cell lines MCF-7 and MDA-MB-468. G47Δ and paclitaxel also demonstrated synergistic cytotoxicity when applied together, with Chou-Talalay combination indices ranging from 0.44 to 0.77 for MCF-7 cells and 0.68 to 0.83 for MDA-MB‑468 cells. Paclitaxel did not enhance viral replication or viral spread among tumor cells. However, G47Δ increased the antitumor ability of paclitaxel by inducing mitotic arrest and apoptosis. In vivo studies indicated that when combined with G47Δ, the dose of paclitaxel could be reduced at least 5-fold while maintaining levels of tumor reduction similar to those achieved with the administration of paclitaxel alone. Combination therapy resulted in no morbidity in vivo. Our data demonstrated that G47Δ and paclitaxel combination therapy had synergistic effects in the treatment of breast cancer. This combination therapy may be promising for breast cancer patients.

SummaryThis study was conducted to investigate the pattern of DNA methylation in vitrified–thawed mouse oocytes and theirin vitrofertilized early embryos. Firstly, mouse oocytes at metaphase II (MII) stage of meiosis were allocated randomly into three groups: (1) untreated (control); (2) exposed to vitrification solution without being plunged into liquid nitrogen (toxicity); or (3) vitrified by open-pulled straw (OPS) method (vitrification). Oocytes from all three groups were fertilized subsequentlyin vitro. The level of DNA methylation in the MII oocytes and their early embryos was then examined by immunofluorescence using an anti-5-methylcytosine (anti-5-MeC) monoclonal antibody and fluorescein isothiocyanate (FITC)-conjugated goat anti-mouse IgG. Developmental rates to 2-cell embryos (62.28%) and blastocysts (43.68%) of the vitrified–thawed oocytes were lower (P< 0.01) than those of fresh oocytes (81.47%, 61.99%) and vitrification solution treated (79.20%, 60.04%) oocytes. DNA methylation (as reflected by 5-MeC fluorescence intensity) in the vitrification group was less (P< 0.01) for MII oocyte and 2- to 8-cell stages compared with that in the control and toxicity groups. Accordingly, a reduction in global genomic methylation due to vitrification of MII oocytes may result in compromisedin vitrodevelopmental potential in early mouse embryos.

Accumulating evidence suggests that breast cancer originates from cancer stem cells (CSCs), which comprise a small percentage of the overall tumor but are highly tumorigenic and pluripotent with unlimited proliferation potential. Furthermore, CSCs are highly resistant to conventional treatment, which may explain certain difficulties in treating cancer with current therapy options. In this study, the third generation oncolytic herpes simplex virus (oHSV) vector G47∆ effectively killed different subtypes of breast cancer cells, with more than 98% of the tumor cells killed by Day 5. Moreover, G47∆ targeted equally non-cancer stem cells (NCSCs) and CSCs which showed resistance to paclitaxel. We demonstrated that G47∆ effectively replicated and spread among CSCs. G47∆ also impaired the self-renewal ability of CSCs, as the viable cells were unable to form secondary tumor spheres. We also showed that G47∆ was able to induce the regression of tumor xenografts in BALB/c nude mice and demonstrated the ability of G47∆ to synergize with paclitaxel by killing both NCSCs and CSCs, suggesting that oHSV may be an effective treatment modality for patients with breast cancer.

To investigate the effect of exogenous normal lymph on kidney injury in disseminated intravascular coagulation (DIC) rats and to probe its mechanism.The DIC model was established by intravenous injection of Dextran 500. After 6 min, normal lymph without cell components was infused in the lymph group. After 40 min, the renal and coagulation function indices and renal histomorphology were observed.Serum urea and creatinine in the model group were significantly higher than in the control and lymph groups. Renal morphological study showed red blood cell silting and casts forming in the model group. The prothrombin time (PT), prothrombin time ratio (PTR), activated partial thromboplastin time (APTT), and thrombin time of lymph and model groups were higher than those in the control group, whereas fibrinogen was lower. The PT, PTR, and APTT were prolonged in the lymph group than in the model group. The platelet functions of the lymph and model groups were higher than in the control group, but platelet aggregation rate and thrombosis-forming indices were lower than in the control group; the platelet adhesive and aggregation rates and thrombosis dry weight of the lymph group were lower than those of the model group.Exogenous normal lymph could alleviate kidney injury in DIC rats, which may be related to the improving coagulation function.

Cell surface carbohydrates, usually binding with other biomacromolecules (such as lipids and proteins), are involved in numerous biological functions, including cellular recognition, adhesion, cell growth regulation, and inflammation. Synthetic carbohydrate-based polymers, so-called glycopolymers, are emerging as important well-defined tools for investigating carbohydrate-based biological processes and for simulating various functions of carbohydrates. In this study, a novel two-step sequence for the generation of a glycopolymer layer tethered on a polypropylene microporous membrane is described. First, a UV-induced graft polymerization of 2-aminoethyl methacrylate hydrochloride (AEMA) was carried out on the membrane to generate an amino-functionalized surface, and the effects of polymerization factors (monomer/initiator concentration and UV irradiation time) on the grafting density were studied. Second, sugar moieties were bound with the grafted functional layer to form glycopolymer by the reaction between the amino groups on the membrane surface and carbohydrate lactones. Chemical analysis by Fourier transform infrared spectroscopy and X-ray photoelectron spectroscopy combined with surface morphology observation by scanning electron microscopy confirmed the graft polymerization of AEMA and the formation of glycopolymer. The decreases of water contact angle and protein adsorption on the membrane revealed the enhancement of hydrophilicity and protein resistance due to the typical characteristics of the glycopolymer tethered on the surface. These results indicated that the novel sequence reported in this work is a facile process to form glycopolymer-modified surfaces.