The rich stereochemistry of the self-assembled monolayers (SAMs) of the four butanethiols on Au(111) is described, SAMs containing up to 12 individual C, S, or Au chiral centers per surface unit cell. This is facilitated by synthesis of enantiomerically pure 2-butanethiol (the smallest unsubstituted chiral alkanethiol), followed by in situ scanning tunneling microscopy (STM) imaging combined with density-functional theory (DFT) molecular dynamics STM-image simulations. Even though butanethiol SAMs manifest strong head-group interactions, steric interactions are shown to dominate SAM structure and chirality. Indeed, steric interactions are shown to dictate the nature of the head-group itself: whether it takes on the adatom-bound motif RS•Au(0)S•R or else involves direct binding of RS• to face-centered cubic (FCC) or hexagonal close-packed (HCP) sites. Binding as RS• produces large organizationally chiral domains even when R is achiral, while adatom binding leads to rectangular plane groups that suppress long-range expression of chirality. Binding as RS• also inhibits the pitting intrinsically associated with adatom binding, desirably producing more regularly structured SAMs.
A temperature compensated magnetic field strength optical fiber sensor has been proposed and experimentally demonstrated. A fiber Bragg grating (FBG) is cascaded to modal interferometer (MI), which is fabricated by dual S-bend splicing between thin fiber (TF) and single mode fiber (SMF) with intentionally controlled misalignment between cores. We established a modified numerical model to describe the multi-mode interference of this exceptional S-bend and misalignment structure, together with the simulation based on beam propagation method to gain insight into its operation mechanism. The FBG is used to interrogate the temperature change, and then compensate the perturbation of temperature on transmission of the MI. Thanks to the proposed dual S-bend structure and the diameter-thinned TF used here; we have obtained high magnetic sensitivity of -0.0678 dB/Oe using only 4 mm TF after the elimination of ambient temperature change.
Study Design. Biomechanical in vitro studyObjective. To determine whether the peak pullout force (PPF) of Anterior Cervical Transpedicular Screw (ATPS) fixed in osteoporotic vertebrae positively influence screw stability or not before and after fatigue.Summary of Background Data. Multilevel cervical spine procedures with osteoporosis can challenge the stability of current screw-and-plate systems. A second surgical posterior approach is coupled with potential risks of increased morbidity and complications. Hence, anterior cervical instrumentation that increases primary construct stability, while avoiding the need for posterior augmentation, would be valuable.Methods. Sixty formalin-fixed vertebrae at different levels were randomly selected. The vertebrae were divided into healthy controls (group A1, A2), osteoporotic controls (B1, B2), healthy ATPS groups (C1, C2), osteoporotic ATPS groups (D1, D2) and osteoporotic restoration controls (E1, E2). The procedure of ATPS insertion was simulated with 2 pilot holes being drilled on each side of 20 vertebral bodies that were implanted with either vertebral screw or PMMA. Each side randomly received either instant PPF or PPF beyond fatigue (2.5 Hz; 20,000 times).Results. The pre-fatigue PPFs were significantly higher than the post-fatigue PPFs in all groups (group A: 366.06±58.78 vs. 248.93±57.21N; group B: 275.58±23.18 vs. 142.79±44.78 N; group C: 635.99±185.28 vs. 542.57±136.58N; group D: 519.22±122.12 vs. 393.16±192.07N, and group E: 431.78±75.77 vs. 325.74±95.10 N). The post-fatigue PPFs were reduced by 32.00% (group A), 48.19% (group B), 14.69% (group C), 24.28% (group D) and 24.72% (group E). The acute and post-fatigue PPFs of both control groups were significantly lower than that of ATPS groups (P<0.05). The cyclic osteoporosis ATPS group achieved the same PPF compared to the vertebral restoration screw (VBRS) group.Conclusions. The findings of this study suggest that instant PPF and fatigue resistance capability of an ATPS fixation were significantly better than other control groups, especially in the osteoporotic vertebrae.
We report a highly sensitive fiber-optic sensor based on two cascaded intrinsic fiber Fabry-Perot interferometers (IFFPIs). The cascaded IFFPIs have different free spectral ranges (FSRs) and are formed by a short section of hollow core photonic crystal fiber sandwiched by two single mode fibers. With the superposition of reflective spectrum with different FSRs, the Vernier effect will be generated in the proposed sensor and we found that the strain sensitivity of the proposed sensor can be improved from 1.6 pm/?? for a single IFFPI sensor to 47.14 pm/?? by employing the Vernier effect. The sensor embed with a metglas ribbon can be also used to measure the magnetic field according to the similar principle. The sensitivity of the magnetic field measurement is achieved to be 71.57 pm/Oe that is significantly larger than the 2.5 pm/Oe for a single IFFPI sensor.
A method of comprehensive chemical pattern recognition of Atractylodis Rhizoma was established by GC-MS fingerprint, principal component analysis, cluster analysis and discriminant analysis. A DB-wax column (0.25 mm x 60 m, 0.25 microm) with El ion source and 70 V electron multiplier were used for GC-MS analysis. Using principal component analysis, cluster analysis, and discriminant analysis, 15 common peaks of sample fingerprints for chemical pattern recognition research were analysed. The same results were obtained from the fingerprint, principal component analysis and cluster analysis, which could use to distinguish genuine Atractylodes lancea, ungenuine A. lancea and A. chinensis. Thus, this method could be used for the quality control and comprehensive evaluation of Atractylodis Rhizoma.
Objectives: Previous research has rarely examined the intervening and buffering effects of leisure on the relationship between age-related stress and health among institutionalized elders, especially in the Chinese context. This study thus examines the extent to which participation in leisure activities mediates and moderates the impact of functional disability on depression among older adults living in residential care homes in China. Method: A total of 1429 participants (858 men) aged over 60 living in residential care homes, of which 46.1% experienced depression using a cut-off score ? 5 on the 15-item Geriatric Depression Scale, were selected from a national survey across China by using the probability proportional to size sampling method. Results: The findings showed that depression was positively predicted by functional disability and negatively predicted by participation in leisure activities. The results of the mediation analysis showed that participation in leisure activities partially mediated the relationship between functional disability and depression. Functional disability predicted depression both directly and indirectly through its negative influence on participation in leisure activities. Participation in leisure activities also significantly buffered the relationship between functional disability and depression such that the impact of functional disability was weaker for those who participated in leisure activities more frequently. Conclusion: These results provide support for the mediating and moderating roles of leisure in the stress-health relationship among institutionalized elders. To enhance residents' psychological health, residential care homes are recommended to organize more leisure activities.
We sought to determine the comparative diagnostic performance of standard b-value (800-1000 s/mm2) versus low b-value (400-500 s/mm2) diffusion-weighted magnetic resonance imaging (DW-MRI) in the detection of renal cell carcinoma (RCC).
Miniature mass spectrometry analytical systems of backpack configuration fitted with sampling probes could potentially be of significant interest for in-field, real-time chemical analysis. In this study, various configurations were explored in which a long narrow tube was used to connect the turbo and backing pumps used to create and maintain vacuum. Also, for the first time we introduced two new types of pumps for miniature mass spectrometers, the Creare 130 g drag pump and Creare 350 g scroll backing pump. These pumps, along with another Creare 550 turbo pump and the commercially available Pfeiffer HiPace 10 turbo and KnF diaphragm backing pumps, were tested with the backpack configurations. The system performance, especially the scan time, was characterized when used with a discontinuous atmospheric pressure interface (DAPI) for ion introduction. The pumping performance in the pressure region above 1 mtorr is critical for DAPI operation. The 550 g turbo pump was shown to have a relatively higher pumping speed above 1 mtorr and gave a scan time of 300 ms, almost half the value obtained with the larger, heavier HiPace 10 often used with miniature mass spectrometers. The 350 g scroll pump was also found to be an improvement over the diaphragm pumps generally used as backing pumps. With a coaxial low temperature plasma ion source, direct analysis of low volatility compounds glass slides was demonstrated, including 1 ng DNP (2,4-Dinitrophenol) and 10 ng TNT (2,4,6-trinitrotoluene) with Creare 550 g turbo pump as well as 10 ng cocaine and 20 ng DNP with Creare 130 g drag pump.
Direct mass spectrometry (MS) analysis of biofluids with simple procedures represents a key step in the translation of MS techniques to clinical and point-of-care applications. The current study reports the development of a single-step method using slug-flow microextraction and nano-electrospray ionization for MS analysis of organic compounds in blood and urine. High sensitivity and quantitation precision have been achieved in the analysis of therapeutic and illicit drugs in 5 ?L samples. Real-time chemical derivatization has been incorporated for analyzing anabolic steroids. The monitoring of enzymatic functions has also been demonstrated with cholinesterase in wet blood. The reported study encourages the future development of disposable cartridges, which function with simple operation to replace the traditional complex laboratory procedures for MS analysis of biological samples.
Mass spectrometers are complex instrumentation systems where ions are transferred though different pressure regions and mass-analyzed under high vacuum. In this work, we have investigated the impact of the gas flows that exit almost universally in all pressure regions. We developed a method that incorporates the dynamic gas field with the electric field in the simulation of ion trajectories. The scope of the electro-hydrodynamic simulation (EHS) method was demonstrated for characterizing the ion optical systems at atmospheric pressure interfaces. With experimental validation, the trapping of the externally injected ions in a linear ion trap at low pressure was also studied. Further development of the EHS method and the knowledge acquired in this research are expected to be useful in the design of hybrid instruments and the study of ion energetics.
The HMG-CoA reductase (HMGCR) pathway is an important metabolic route that is present in almost every organism. However, whether HMGCR affects the expression of IFN-responsive genes is unclear. In this study, expression levels of IFN-responsive genes were monitored by real time PCR and ELISA. The results showed that expression levels of IFN-responsive genes were significantly increased in HMGCR-downregulated cells and HMGCR inhibitor-treated cells, indicating that inhibition of HMGCR activates the expression of IFN-responsive genes. The result in this study will give new insight into the role of HMG-CoA reductase in antiviral research. This article is protected by copyright. All rights reserved.
Tandem mass spectrometry (MS/MS) plays an essential role in modern chemical analysis. It is used for differentiating isomers and isobars and suppressing chemical noise, which allows high precision quantitation. The MS/MS analysis has been typically applied by isolating the target precursor ions, while disregarding other ions, followed by a fragmentation process that produces the product ions. In this study, configurations of dual linear ion traps were explored to develop high efficiency MS/MS analysis. The ions trapped in the first linear ion trap were axially, mass-selectively transferred to the second linear ion trap for MS/MS analysis. Ions from multiple compounds simultaneously introduced into the mass spectrometer could be sequentially analyzed. This development enables highly efficient use of the sample. For miniature ion trap mass spectrometers with discontinuous atmospheric pressure interfaces, the analysis speed and the quantitation precision can be significantly improved.
Mass spectrometry (MS) is known for highly specific and sensitive analysis. The general applicability of this technique makes it a good candidate for biological applications over a much broader range than is now the case. The limiting factors preventing MS from being applied at the biologist's bench or in a physician's office are identified as the large size of the systems, as well as the complicated analytical procedures required. An approach for developing miniature MS analysis systems with simplified operational procedures is described and the associated technical developments are discussed.
The domestic pig has been widely used as an important large animal model. Precise and efficient genetic modification in pig provides a great promise in biomedical research. Recently, clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated (Cas) system has been successfully used to produce many gene-targeted animals. However, these animals have been generated by co-injection of Cas9 mRNA and single-guide RNA (sgRNA) into one-cell stage embryos, which mostly resulted in mosaicism of the modification. One or two rounds of further breeding should be performed to obtain homozygotes with identical genotype and phenotype. To address this issue, gene-targeted somatic cells can be used as donor for somatic cell nuclear transfer (SCNT) to produce gene-targeted animals with single and identical mutations. In this study, we applied Cas9/sgRNAs to effectively direct gene editing in porcine fetal fibroblasts and then mutant cell colonies were used as donor to generate homozygous gene-targeted pigs through single round of SCNT. As a result, we successfully obtained 15 tyrosinase (TYR) biallelic mutant pigs and 20 PARK2 and PINK1 double-gene knockout (KO) pigs. They were all homozygous and no off-target mutagenesis was detected by comprehensive analysis. TYR (-/-) pigs showed typical albinism and the expression of parkin and PINK1 were depleted in PARK2 (-/-)/PINK1 (-/-) pigs. The results demonstrated that single- or double-gene targeted pigs can be effectively achieved by using the CRISPR/Cas9 system combined with SCNT without mosaic mutation and detectable off-target effects. This gene-editing system provides an efficient, rapid, and less costly manner to generate genetically modified pigs or other large animals.
It is generally believed that aberrant expression of imprinted genes participates in growth retardation of mammalian parthenogenesis. Neuronatin (NNAT), a paternally expressed gene, plays important roles in neuronal growth and metabolic regulation. Here we have compared the gene expression and promoter methylation pattern of NNAT between pig normally fertilized (Con) and parthenogenetic (PA) embryos. The results showed loss of NNAT expression (p<0.001) and hypermethylation of NNAT promoter in PA samples. Additionally, partial methylation was observed in Con fetuses, while almost full methylation and unmethylation of NNAT promoter were apparent in Metaphase II (MII) oocytes and mature sperms, respectively, which identified the CpG promoter region as a putative differentially methylated region (DMR) of NNAT. The data demonstrate that promoter hypermethylation is associated with the silencing of NNAT in pig PA fetuses, which may be related to developmental failure of pig parthenogenesis at early stages.
Previous studies have shown that preoperative anemia is correlated with the prognoses of various solid tumors. This study was performed to determine the effect of preoperative anemia on relapse and survival in patients with breast cancer.
Determination of eight drugs of abuse in blood has been performed using paper spray or extraction spray mass spectrometry in under 2 min with minimal sample preparation. A method has been optimized for quantification of amphetamine, methamphetamine, 3,4-methylenedioxyamphetamine (MDA), 3,4-methylenedioxy-N-methylamphetamine (MDMA), 3,4-methylenedioxy-N-ethylamphetamine (MDEA), morphine, cocaine, and ?9-tetrahydrocannabinol (THC) from a single blood spot. Sample to sample variations of 1-5% relative standard deviation were achieved using stable isotope-labeled internal standards and tandem mass spectrometry. Limits of detection for all drugs were below typical physiological and toxicological levels. Paper spray and extraction spray each used less than 10 ?L of whole blood. These methods exhibit the potential for performing rapid and high-throughput assays for selective on-site multicompound quantitative screening of illicit drugs.
Pigs share many physiological, biochemical, and anatomical similarities with humans and have emerged as valuable large animal models for biomedical research. Considering the advantages in immune system resemblance, suitable size, and longevity for clinical practical and monitoring purpose, SCID pigs bearing dysfunctional RAG could serve as important experimental tools for regenerative medicine, allograft and xenograft transplantation, and reconstitution experiments related to the immune system. In this study, we report the generation and phenotypic characterization of RAG1 and RAG2 knockout pigs using transcription activator-like effector nucleases. Porcine fetal fibroblasts were genetically engineered using transcription activator-like effector nucleases and then used to provide donor nuclei for somatic cell nuclear transfer. We obtained 27 live cloned piglets; among these piglets, 9 were targeted with biallelic mutations in RAG1, 3 were targeted with biallelic mutations in RAG2, and 10 were targeted with a monoallelic mutation in RAG2. Piglets with biallelic mutations in either RAG1 or RAG2 exhibited hypoplasia of immune organs, failed to perform V(D)J rearrangement, and lost mature B and T cells. These immunodeficient RAG1/2 knockout pigs are promising tools for biomedical and translational research.
A rapid, selective, and sensitive ultra-fast liquid chromatography with tandem mass spectrometry method was developed for the determination of ochratoxin A in traditional Chinese medicines based on vortex-assisted solid-liquid microextraction and aptamer-affinity column clean-up. Through optimizing the sample pretreatment procedures and chromatographic conditions, good linearity (r(2) ? 0.9993), low limit of detection (0.5-0.8 ?g/kg), and satisfactory recovery (83.54-94.44%) expressed the good reliability and applicability of the established method in various traditional Chinese medicines. Moreover, the aptamer-affinity column, prepared in-house, showed an excellent feasibility owing to its specific identification of ochratoxin A in various kinds of selected traditional Chinese medicines. The maximum adsorption amount and applicability value were 188.96 ± 10.56 ng and 72.3%, respectively. The matrix effects were effectively eliminated, especially for m/z 404.2?358.0 of ochratoxin A. The application of the developed method for screening the natural contamination levels of ochratoxin A in 25 random traditional Chinese medicines on the market in China indicated that only eight samples were contaminated with low levels below the legal limit (5.0 ?g/kg) set by the European Union. This study provided a preferred choice for the rapid and accurate monitoring of ochratoxin A in complex matrices.
In this study, the concept of ion sponge has been explored for developing 3D arrays of large numbers of ion traps but with simple configurations. An ion sponge device with 484 trapping units in a volume of 10 × 10 × 3.2 cm has been constructed by simply stacking 9 meshes together. A single rf was used for trapping ions and mass-selective ion processing. The ion sponge provides a large trapping capacity and is highly transparent for transfer of ions, neutrals, and photons for gas phase ion processing. Multiple layers of quadrupole ion traps, with 121 trapping units in each layer, can operate as a single device for MS or MS/MS analysis, or as a series of mass-selective trapping devices with interlayer ion transfers facilitated by AC and DC voltages. Automatic sorting of ions to different trapping layers based on their mass-to-charge (m/z) ratios was achieved with traps of different sizes. Tandem-in-space MS/MS has also been demonstrated with precursor ions and fragment ions trapped in separate locations.
In the present study, 89 porcine reproductive and respiratory syndrome virus (PRRSV) isolates in China during 2007 to 2012 were randomly selected from the GenBank genetic sequence database. Evolutionary characteristics of these isolates were analyzed based on the sequences of non-structural protein 2 (Nsp2) and glycoprotein 5 (GP5). The genetic variations of the isolates were also compared with six representative strains. The results showed that a high degree of genetic diversity exists among the PRRSV population in China. Highly pathogenic PRRSV isolates, with a discontinuous deletion of a 30 amino acid residue in the Nsp2 region, remained the most dominant virus throughout 2007-2012 in China. Owing to the extensive use of representative vaccine strains, natural recombination events occurred between strains. Three isolates - HH08, DY, and YN-2011 - were more closely related to vaccine strains than the other isolates. Both YN-2011 and DY were the evolutionary products of recombination events between strains SP and CH-1R. The results of the present study provide useful information for the epidemiology of PRRSV as well as for vaccine development.
Nitro-oleic acid (OA-NO2), acting as anti-inflammatory signaling mediators, are involved in multiple signaling pathways. Lipoprotein-associated phospholipase A2 (Lp-PLA2) is well known as a cardiovascular risk biomarker. Our results showed that OA-NO2 downregulated the expression of Lp-PLA2 in a time- and dose-dependent manner, whereas native OA had no such effect. Furthermore, OA-NO2 could repress Lp-PLA2 expression in the peripheral blood mononuclear cells of apo CIII-transgenic (apo CIII TG) pigs, which exhibited higher Lp-PLA2 expression and activity than did wild-type (WT) pigs. OA-NO2 inhibited Lp-PLA2 expression in macrophages, independent of nitric oxide formation and PPAR?-activation. However, OA-NO2 downregulates Lp-PLA2 by inhibiting the p42/p44 mitogen-activated protein kinase (MAPK) and the nuclear factor ?B (NF?B) pathways. When used to mediate anti-inflammatory signaling, the regulation of inflammatory cytokines and SOD by OA-NO2 might be associated with the reduction of Lp-PLA2. These results suggested that OA-NO2 might exert a vascular-protective effect partially via Lp-PLA2 inhibition.
Acute lymphoblastic leukemia (ALL) is the most common type of leukemia of childhood. Over the last 50 years there have been tremendous scientific advances in understanding the pathogenesis and the mechanisms that control cellular proliferation in ALL. These discoveries led to the development of efficient therapeutic regimens that greatly improved survival of children with ALL. Recently, several genes have been demonstrated to play a key role in tumor suppression and that their deregulation leads to malignant transformation and can affect overall survival. This review summarizes the role of Ikaros (IKZF1) in tumor suppression and regulation of gene expression in leukemia. Deletions and/or mutations of Ikaros have been detected in a large percentage of pediatric and adult ALL and reduced Ikaros function has been associated with poor outcome in ALL. Ikaros function in chromatin remodeling and epigenetic regulation of gene transcription emphasizes the important role of this protein in controlling cellular proliferation. In this review, we particularly focus on the role of signaling pathways in the regulation of Ikaros activity and its transcriptional control in leukemia.
We examined the role of HMG-CoA reductase (HMGCR) during porcine circovirus 2 (PCV2) infection. The results demonstrated that levels of endogenous HMGCR were not significantly different in PCV2-infected cells and mock-infected cells. However, the level of phosphorylated HMGCR, an inactivated form of HMGCR, was increased in PCV2-infected cells. Furthermore, HMGCR was upregulated by overexpression, silenced by siRNA or inactivated using its dominant-negative form in PK-15 cells. The results showed that PCV2 infection was inhibited by HMGCR overexpression, whereas it was significantly increased in HMGCR-silenced cells and HMGCR inhibitor-treated cells. Moreover, there was a robust apoptotic response at 48 h post-infection (p.i.) in HMGCR-inactivated cells, and this response was significantly greater than that observed in PK-15 cells. A modest apoptotic response was also observed in HMGCR-silenced cells. Caspase-3 activity was also analysed in PCV2-infected cells at 48 h p.i. As expected, caspase-3 activity was significantly increased in HMGCR-inactivated and -silenced cells compared with PK-15 cells. PCV2 replication was dose-dependently increased in HMGCR-inactivated cells when treated with increasing amounts of caspase-3 inhibitor. Altogether, HMGCR was negatively associated with PCV2 infection and PCV2-induced apoptotic cell death. These data demonstrated that HMGCR can be used as a candidate target for PCV2 disease control and antivirus research. Furthermore, the cells generated in this study can be used to evaluate the potential effects of HMGCR on PCV2 replication.
To gain insight into parthenogenesis in pigs, we report for the first time that using parthenogenetic somatic cells as nuclear donors (PSCNT), the porcine parthenogenetic fetus can develop to gestational day 39. Weight and morphological analysis revealed that PSCNT fetuses were smaller and developmentally retarded when compared to normally fertilized controls. Quantitative gene expression analysis indicated that in PSCNT fetuses, H19 was over-expressed, whereas Igf2 was significantly reduced (p < 0.05) compared with their controls. In addition, bisulfite-sequencing PCR results demonstrated that H19 differentially DNA methylated regions (DMRs) were hypomethylated in PSCNT fetuses, while Igf2 DMRs were hypermethylated in both PSCNT and control fetuses. Our results suggest that extended development of the porcine parthenogenetic fetus can be accomplished using PSCNT and that abnormal DNA methylation of H19 DMRs might contribute to the critical barrier of parthenogenesis in pigs.
Ikaros is a DNA-binding protein that acts as master-regulator of hematopoiesis and a tumor suppressor. In thymocytes and T-cell leukemia, Ikaros negatively regulates transcription of terminal deoxynucleotide transferase (TdT), a key protein in lymphocyte differentiation. The signaling pathways that regulate Ikaros-mediated repression of TdT are unknown. Our previous work identified Casein Kinase II (CK2) and Protein Phosphatase 1 (PP1) as regulators of Ikaros DNA binding activity. Here, we investigated the role of PP1 and CK2 in regulating Ikaros-mediated control of TdT expression.
A major design objective of portable mass spectrometers is the ability to perform in situ chemical analysis on target samples in their native states in the undisturbed environment. The miniature instrument described here is fully contained in a wearable backpack (10 kg) with a geometry-independent low-temperature plasma (LTP) ion source integrated into a hand-held head unit (2 kg) to allow direct surface sampling and analysis. Detection of chemical warfare agent (CWA) simulants, illicit drugs, and explosives is demonstrated at nanogram levels directly from surfaces in near real time including those that have complex geometries, those that are heat-sensitive, and those bearing complex sample matrices. The instrument consumes an average of 65 W of power and can be operated autonomously under battery power for ca. 1.5 h, including the initial pump-down of the manifold. The maximum mass-to-charge ratio is 925 Th with mass resolution of 1-2 amu full width at half-maximun (fwhm) across the mass range. Multiple stages of tandem analysis can be performed to identify individual compounds in complex mixtures. Both positive and negative ion modes are available. A graphical user interface (GUI) is available for novice users to facilitate data acquisition and real-time spectral matching.
A benchtop miniature mass spectrometer system, Mini 12, with ambient ionization source and tandem mass spectrometry capabilities has been developed and characterized. This instrument was developed as a self-contained system to produce quantitative results for unprocessed samples of small volumes including nonvolatile analytes. The ion processing system, vacuum system, and control system are detailed. An integrated sample loading system facilitates automated operation. A user interface has been developed to acquire and to interpret analytical results for personnel who have limited mass spectrometry knowledge. Peak widths of ?m/z 0.6 Th (full width at half-maximum) and a mass range of up to m/z 900 are demonstrated with the rectilinear ion trap mass analyzer. Multistage experiments up to MS(5) are accomplished. Consumable cartridges have been designed for use in ambient paper spray ionization, and the recently developed extraction spray ionization method has been employed to improve the quantitative performance. Monitoring of trace-levels of chemicals in therapeutic drugs, as well as in food safety and environmental protection operations is demonstrated. Dual MS/MS scans are implemented to obtain the intensities of the fragment ions from the analyte and its internal standard, and the ratio is used in quantitative analysis of complex samples. Limits of quantitation (LOQ) of 7.5 ng/mL, with relative standard deviations below 10%, have been obtained for selected therapeutic drugs in whole blood throughout their individual therapeutic ranges.
In the human immunodeficiency virus (HIV)-associated sensory neuropathy, neuropathic pain associated with the use of nucleoside reverse transcriptase inhibitors (NRTIs) in patients with HIV/acquired immunodeficiency syndrome is clinically common. While evidence demonstrates that neuropathic pain is influenced by neuroinflammatory events that include the proinflammatory molecules, tumor necrosis factor-? (TNF-?), stromal cell-derived factor 1-? (SDF1-?), and C-X-C chemokine receptor type 4 (CXCR4), the detailed mechanisms by which NRTIs contribute to the development of neuropathic pain are not known. In this study, we investigated the role of these proinflammatory molecules in the dorsal root ganglion (DRG) and the spinal dorsal horn in NRTIs-mediated neuropathic pain state.
Most mammalian parthenogenetic embryos are unable to develop to term due to placental defects, potentially caused by decreased vasculogenesis and angiogenesis of the parthenogenetic placenta. Here we have compared the expression status of vascular endothelial growth factor (VEGF) and angiopoietin family members between normally developing and parthenogenetic porcine placentas. The result showed significantly reduced expression of these genes but elevated expression of VEGF 120 in the parthenogenetic porcine placenta (p < 0.05). We postulate that the abnormal expression levels of VEGF and angiopoietin family members and, especially, the elevated expression of VEGF 120 observed in parthenogenetic porcine placentas are related to the early miscarriage of parthenogenetic embryos in pigs.
3-Deoxy-d-arabino-heptulonate-7-phosphate (DAHP) synthase is one of the key enzymes, which catalyzes the first step in the aromatic amino acid biosynthetic pathway and yields the three amino acids tyrosine (Tyr), tryptophan (Typ), and phenylalanine (Phe). In Escherichia coli (E. coli), three differently regulated DAHP synthases carry out the first regulated step in the aromatic amino acid biosynthetic pathway. The three DAHP synthases encoded by the genes aroG, aroF, and aroH are inhibited by phenylalanine, tyrosine and tryptophan, respectively. In this work, the aroG gene was cloned and mutated by site-directed mutagenesis using overlap extension PCR (SOE-PCR) technique. The feedback-resistant DAHP synthase encoded by aroG was achieved by replacing the residue Pro150 of aroG with Leu as to increase net carbon flow down the common pathway. SDS-PAGE and Western blots were used to assess the protein expression level of aroGM which showed the strain harboring the mutated aroGM150 gene achieving over-expression compared to the strain containing an empty plasmid pET-28b((+)).
Capsid protein (Cap) of porcine circovirus 2 (PCV2) contained critical epitopes for inducing a protective immune response. Here, different fragments of PCV2 Cap protein were cloned, expressed, purified and used to raise polyclonal antibodies. The result showed the recombinant plasmids expressed efficiently in the prokaryotic system. Western blot and ELISA showed the recombinant protein had antigenicity and immunogenicity. Furthermore, efficiency of different constructs to produce antibody against PCV2 was compared. Reactivity and specificity of the polyclonal antibody were characterized by Western blot and indirect immunofluorescent assays. The results indicated that polyclonal antiserum prepared from protein ?Cap17-233 had better reactivity and specificity against PCV2 in comparison to that of protein ?Cap51-233 and the inactivated vaccine. These results will contribute to further studies focusing on the gene and vaccine development against PCV2.
Aptamers are single-stranded oligonucleotides with high affinity and specificity and are widely used in targets separation and enrichment. Here, an aptamer-affinity column (AAC) was firstly prepared in-house through a covalent immobilization strategy. Then, ochratoxin A (OTA) in ginger powder was absorbed and enriched using the new aptamer-based clean-up technology for the first time, and was further analyzed by ultra high performance liquid chromatography with fluorescence detection. After optimization, the average recoveries for blank samples spiked with OTA at 5, 15, and 45 ?g/kg ranged from 85.36 to 96.83%. Furthermore, the AAC exhibited a similar accuracy as an immunoaffinity column to clean up OTA in ginger powder. Above all, it exhibited better reusability, twice that of the immunoaffinity column, had lower toxicity and cost, and took less time. Of 25 contaminated ginger powder samples, OTA contamination levels ranged from 1.51 to 4.31 ?g/kg, which were lower than the European Union (EU) regulatory limits. All the positive samples were further confirmed by ultra-fast LC with MS/MS. In conclusion, the method of clean-up based on the AAC coupled to ultra-HPLC with fluorescence detection was rapid, specific, and sensitive for the quantitative analysis of OTA in a complex matrix.
Mutations in the human copper/zinc superoxide dismutase 1 (hSOD1) gene cause familial amyotrophic lateral sclerosis (ALS). It remains unknown whether large animal models of ALS mimic more pathological events seen in ALS patients via novel mechanisms. Here, we report the generation of transgenic pigs expressing mutant G93A hSOD1 and showing hind limb motor defects, which are germline transmissible, and motor neuron degeneration in dose- and age-dependent manners. Importantly, in the early disease stage, mutant hSOD1 did not form cytoplasmic inclusions, but showed nuclear accumulation and ubiquitinated nuclear aggregates, as seen in some ALS patient brains, but not in transgenic ALS mouse models. Our findings revealed that SOD1 binds PCBP1, a nuclear poly(rC) binding protein, in pig brain, but not in mouse brain, suggesting that the SOD1-PCBP1 interaction accounts for nuclear SOD1 accumulation and that species-specific targets are key to ALS pathology in large mammals and in humans.
Here, we report a convenient and efficient miRNA inhibition strategy employing the CRISPR system. Using specifically designed gRNAs, miRNA gene has been cut at a single site by Cas9, resulting in knockdown of the miRNA in murine cells. Using a modified CRISPR interference system (CRISPRi), inactive Cas9 can reversibly prevent the expression of both monocistronic miRNAs and polycistronic miRNA clusters. Furthermore, CRISPR/CRISPRi is also capable of suppressing genes in porcine cells.
NF?B transcription activation leads to malfunction of endothelial cells, which is the main reason for pig xenograft rejection. Overexpression of a dominant negative mutant of porcine NF?B p65 (pp65RHD) could inhibit NF?B activation in endothelial cells. This study presents an advanced tetracycline-regulated system for pp65RHD spatiotemporal expression in porcine iliac endothelial cell line. In this system, an endothelial specific promoter ICAM-2 is used to improve pTet-On and internal ribosome entry site as well as enhanced green fluorescent protein (EGFP) elements are used to facilitate the result observation in pTRE-Tight. Through transfection and drug selection, we obtained 7 single cell clones containing the advanced Tet-On system, in which pp65RHD expression is under tight regulated by doxycycline and can be visualized easily through EGFP. The distribution of induced pp65RHD was verified by immunocytochemical assays test. Then, NF?B activity was tested. Luciferase reporter assays showed that NF?B activity in two clones was influenced by the Dox-induced pp65RHD expression, but other clones weren't influenced. Therefore, we picked up 2 cell clones from the uninfluenced clones for further investigation by immunocytochemical assays and RT-PCR detection. The final results supported the overexpression of pp65RHD in one clone could successfully inhibit NF?B activity. The success of pp65RHD spatiotemporal expression system is helpful to regulate NF?B activity and conquer cell-mediated immunity and could be used for preparation of transgenic pig, contributing to xenotransplantation.
The myxovirus resistance gene (Mx1) has a broad spectrum of antiviral activities. It is therefore an interesting candidate gene to improve disease resistance in farm animals. In this study, we report the use of somatic cell nuclear transfer (SCNT) to produce transgenic pigs over-expressing the Mx1 gene. These transgenic pigs express approximately 15-25 times more Mx1 mRNA than non-transgenic pigs, and the protein level of Mx1 was also markedly enhanced. We challenged fibroblast cells isolated from the ear skin of transgenic and control pigs with influenza A virus and classical swine fever virus (CFSV). Indirect immunofluorescence assay (IFA) revealed a profound decrease of influenza A proliferation in Mx1 transgenic cells. Growth kinetics showed an approximately 10-fold reduction of viral copies in the transgenic cells compared to non-transgenic controls. Additionally, we found that the Mx1 transgenic cells were more resistant to CSFV infection in comparison to non-transgenic cells. These results demonstrate that the Mx1 transgene can protect against viral infection in cells of transgenic pigs and indicate that the Mx1 transgene can be harnessed to develop disease-resistant pigs.
Nanosilver particles (NSPs), are among the most attractive nanomaterials, and have been widely used in a range of biomedical applications, including diagnosis, treatment, drug delivery, medical device coating, and for personal health care. With the increasing application of NSPs in medical contexts, it is becoming necessary for a better understanding of the mechanisms of NSPs' biological interactions and their potential toxicity. In this review, we first introduce the synthesis routes of NSPs, including physical, chemical, and biological or green synthesis. Then the unique physiochemical properties of NSPs, such as antibacterial, antifungal, antiviral, and anti-inflammatory activity, are discussed in detail. Further, some recent applications of NSPs in prevention, diagnosis, and treatment in medical fields are described. Finally, potential toxicology considerations of NSPs, both in vitro and in vivo, are also addressed.
Direct mass spectrometry analysis of untreated samples of volumes as low as 0.2 µL were achieved using fast extraction and nanoESI (electrospray ionization) in a combined fashion. The analytes in dried samples on paper substrates were extracted by organic solvent in a nanoESI tube and ionized with a high voltage applied for generating a spray. The ionization source produced stable signals for different atmospheric pressure interfaces of triple quadrupole instruments. Analysis time more than 20 minutes were available with 10 µL solvent consumed for the entire analysis process. The performance in qualitative and quantitative analysis was characterized with a wide variety of samples. Limits of detection as low as 0.1 ng/mL (corresponding to an absolute amount of 0.05 pg) were obtained for analysis of atrazine in river water, thiabendazole in orange homogenate, and methamphetamine in blood.
A sampling probe based on ambient desorption ionization was designed for in vivo chemical analysis by mass spectrometry in surgical and endoscopic procedures. Sampling ionization of analytes directly from tissue was achieved by sealing the sampling tip against the tissue surface without allowing leakage of the auxiliary gas used for desorption ionization. The desorbed charged species were transferred over a long distance (up to 4 m) through a flexible tube of internal diameter as small as 1/16 in. to the inlet of the mass spectrometer used for analysis. The conditions used for desorption electrospray ionization (DESI) were optimized to achieve biocompatibility for clinical applications while obtaining adequate efficiency for the analysis. This optimization involved the removal of high voltage and use of pure water as a spray solvent instead of the organic solvents or aqueous mixtures normally used. Improved sensitivity was achieved under these conditions by increasing the gas flow rate in the transfer tube. The destructive effect on tissue surfaces associated with typical desorption ionization was avoided by altering the local gas dynamics in the sample area without compromising the overall analysis efficiency.
The determination of tobacco derived nicotine alkaloids in biofluid samples is of great importance to testing for tobacco use, tobacco cessation treatment, and studies on exposure to secondhand smoke. Paper spray mass spectrometry (MS) has been adapted for direct, quantitative analysis of tobacco alkaloids from biofluid samples, such as blood, urine, and saliva in liquid and dried form. Limits of quantitation as low as several nanograms per milliliter were obtained for nicotine, cotinine, trans-3-hydroxycotinine, and anabasine. Direct analysis of fresh blood samples has also been achieved with improved sensitivity using print paper substrates of high density. Quantitation of the cotinine in the blood of a rat was performed with both direct analysis using paper spray and a traditional analysis protocol using liquid chromatography MS. Comparable results were obtained and the precision of the two methods was similar. The paper spray MS method is rapid and shows potential for significantly improved analytical efficiency in clinical laboratories as well as for point-of-care tobacco use assessment.
The successful generation of pigs via somatic cell nuclear transfer depends on reducing risk factors in several aspects. To provide an overview of some influencing factors related to embryo transfer, the follow-up data related to cloned pig production collected in our laboratory was examined. (i) Spring showed a higher full-term pregnancy rate compared with winter (33.6% vs 18.6%, P?=?0.006). Furthermore, a regression equation can be drawn between full-term pregnancy numbers and pregnancy numbers in different months (y?=?0.692x-3.326). (ii) There were no significant differences detected in the number of transferred embryos between surrogate sows exhibiting full-term development compared to those that did not. (iii) Non-ovulating surrogate sows presented a higher percentage of full-term pregnancies compared with ovulating sows (32.0% vs 17.5%, P?=?0.004; respectively). (iv) Abortion was most likely to take place between Day 27 to Day 34. (v) Based on Life Table Survival Analysis, delivery in normally fertilized and surrogate sows is expected to be completed before Day 117 or Day 125, respectively. Additionally, the length of pregnancy in surrogate sows was negatively correlated with the average litter size, which was not found for normally fertilized sows. In conclusion, performing embryo transfer in appropriate seasons, improving the quality of embryos transferred, optimizing the timing of embryo transfer, limiting the occurrence of abortion, combined with ameliorating the management of delivery, is expected to result in the harvest of a great number of surviving cloned piglets.
An urgent unmet need exists for early-stage treatment of spinal cord injury (SCI). Currently methylprednisolone is the only therapeutic agent used in clinics, for which the efficacy is controversial and the side effect is well-known. We demonstrated functional restoration of injured spinal cord by self-assembled nanoparticles composed of ferulic acid modified glycol chitosan (FA-GC). Chitosan and ferulic acid are strong neuroprotective agents but their systemic delivery is difficult. Our data has shown a prolonged circulation time of the FA-GC nanoparticles allowing for effective delivery of both chitosan and ferulic acid to the injured site. Furthermore, the nanoparticles were found both in the gray matter and white matter. The in vitro tests demonstrated that nanoparticles protected primary neurons from glutamate-induced excitotoxicity. Using a spinal cord contusion injury model, significant recovery in locomotor function was observed in rats that were intravenously administered nanoparticles at 2 h post injury, as compared to non-improvement by methylprednisolone administration. Histological analysis revealed that FA-GC treatment significantly preserved axons and myelin and also reduced cavity volume, astrogliosis, and inflammatory response at the lesion site. No obvious adverse effects of nanoparticles to other organs were found. The restorative effect of FA-GC presents a promising potential for treating human SCIs.
Paper spray has been developed as a fast sampling ionization method for direct analysis of raw biological and chemical samples using mass spectrometry (MS). Quantitation of therapeutic drugs in blood samples at high accuracy has also been achieved using paper spray MS without traditional sample preparation or chromatographic separation. The paper spray ionization is a process integrated with a fast extraction of the analyte from the raw sample by a solvent, the transport of the extracted analytes on the paper, and a spray ionization at the tip of the paper substrate with a high voltage applied. In this study, the influence on the analytical performance by the solvent-substrate systems and the selection of the elution methods was investigated. The protein hemoglobin could be observed from fresh blood samples on silanized paper or from dried blood spots on silica-coated paper. The on-paper separation of the chemicals during the paper spray was characterized through the analysis of a mixture of the methyl violet 2B and methylene blue. The mode of applying the spray solvent was found to have a significant impact on the separation. The results in this study led to a better understanding of the analyte elution, on-paper separation, as well as the ionization processes of the paper spray. This study also help to establish a guideline for optimizing the analytical performance of paper spray for direct analysis of target analytes using mass spectrometry.
Selective activation of benzene has been mainly limited to the C-H activation. Simple replacement of one carbon in benzene with another atom remains unresolved due to the high dissociation energy. Herein, we demonstrate a direct breakage of the particularly strong C = C bond in benzene through ion-molecule reaction in a low-temperature plasma, in which one carbon atom was replaced by one atomic nitrogen with the formation of pyridine. The mechanism for the formation of pyridine from benzene has been proposed based on the extensive investigation with tandem mass spectrometry. The reaction pathway also works to other aromatics such as toluene and o-xylene. This finding provides a new avenue for selective conversion of aromatics into nitrogen-containing compounds.
Rotator cuff injuries are a common clinical problem either as a result of overuse or aging. Biological approaches to tendon repair that involve use of scaffolding materials or cell-based approaches are currently being investigated. The cell-based approaches are focused on applying multipotent mesenchymal stem cells (MSCs) mostly harvested from bone marrow. In the present study, we focused on characterizing cells harvested from tissues associated with rotator cuff tendons based on an assumption that these cells would be more appropriate for tendon repair. We isolated MSCs from bursa tissue associated with rotator cuff tendons and characterized them for multilineage differentiation in vitro and in vivo. Human bursa was obtained from patients undergoing rotator cuff surgery and cells within were isolated using collagenase and dispase digestion. The cells isolated from the tissues were characterized for osteoblastic, adipogenic, chondrogenic, and tenogenic differentiation in vitro and in vivo. The results showed that the cells isolated from bursa tissue exhibited MSCs characteristics as evidenced by the expression of putative cell surface markers attributed to MSCs. The cells exhibited high proliferative capacity and differentiated toward cells of mesenchymal lineages with high efficiency. Bursa-derived cells expressed markers of tenocytes when treated with bone morphogenetic protein-12 (BMP-12) and assumed aligned morphology in culture. Bursa cells pretreated with BMP-12 and seeded in ceramic scaffolds formed extensive bone, as well as tendon-like tissue in vivo. Bone formation was demonstrated by histological analysis and immunofluorescence for DMP-1 in tissue sections made from the scaffolds seeded with the cells. Tendon-like tissue formed in vivo consisted of parallel collagen fibres typical of tendon tissues. Bursa-derived cells also formed a fibrocartilagenous tissue in the ceramic scaffolds. Taken together, the results demonstrate a new source of MSCs with a high potential for application in tendon repair.
Microorganisms and higher plants possess their own omega-3 and omega-6 polyunsaturated fatty acid (PUFAs) biosynthetic pathways. The n-6 fatty acid desaturase gene fad-2 codes for the n-6 desaturase enzyme that coverts oleic acid (OA 18:1 n-9) into linoleic acid (LA 18:2 n-6). The n-3 fatty acid desaturase gene fat-1 codes for the n-3 desaturase enzyme that converts n-6 PUFAs into n-3 PUFAs. Mammals lack n-3 and n-6 desaturase enzymes; therefore, they must obtain their omega-3 and omega-6 fatty acids by consuming plants or seafood. The beneficial effects of n-3 and n-6 PUFAs on human development and cardiovascular health have been well documented.
The discontinuous atmospheric pressure interface (DAPI) has been developed as a facile means for efficiently introducing ions generated at atmospheric pressure to an ion trap in vacuum [e.g., a rectilinear ion trap (RIT)] for mass analysis. Introduction of multiple beams of ions or neutral species through two DAPIs into a single RIT has been previously demonstrated. In this study, a home-built instrument with a DAPI-RIT-DAPI configuration has been characterized for the study of gas-phase ion/molecule and ion/ion reactions. The reaction species, including ions or neutrals, can be introduced from both ends of the RIT through the two DAPIs without complicated ion optics or differential pumping stages. The primary reactant ions were isolated prior to reaction and the product ions were mass analyzed after controlled reaction time period. Ion/molecule reactions involving peptide radical ions and proton-transfer ion/ion reactions have been carried out using this instrument. The gas dynamic effect due to the DAPI operation on internal energy deposition and the reactivity of peptide radical ions has been characterized. The DAPI-RIT-DAPI system also has a unique feature for allowing the ion reactions to be carried out at significantly elevated pressures (in 10(-1) Torr range), which has been found to be helpful to speed up the reactions. The viability and flexibility of the DAPI-RIT-DAPI system for the study of gas-phase ion reactions have been demonstrated.
The important differences in physiological parameters and anatomical characteristics of the kidney between humans and mice make it difficult to replicate the precise progression of human renal cystic diseases in gene modification mouse models. In contrast to mice, pigs are a better animal model of human diseases, as they are more similar in terms of organ size, structure, and physiological parameters. Here, we report the generation and initial examination of an AQP2-Cre transgenic (Tg) Chinese miniature (mini)-pig line that expresses Cre recombinase exclusively in kidney collecting duct cells. An 8-kb fragment of the mini-pig aquaporin 2 (AQP2) 5-flanking region was utilized to direct Cre expression in Tg mini-pigs. Two Tg mini-pigs were generated by pig somatic cell nuclear transfer and both carried the entire coding sequence of Cre recombinase. RT-PCR and western blotting analysis revealed that Cre recombinase was uniquely expressed in the kidney, while immunohistochemical studies located its expression in kidney collecting duct cells. Furthermore, six integration sites and 12-14 copies of the Cre gene were detected in various tissues by high-efficiency thermal asymmetric interlaced PCR and absolute quantitative real-time PCR, respectively. Combined with previous studies of Cre recombinase activity, we believe that this AQP2-Cre Tg mini-pig line will be a useful tool to generate kidney collecting duct cell-specific gene knockout mini-pig models, thereby allowing the investigation of gene functions in kidney development and the mechanisms of human renal cystic disease.
We describe a sampling method using glass capillaries for quantitative analysis of trace analytes in small volumes of complex mixtures (~1 ?L) using ambient ionization mass spectrometry. The internal surface of a sampling glass capillary was coated with internal standard then used to draw liquid sample and so transfer both the analyte and internal standard in a single fixed volume onto a substrate for analysis. The internal standard was automatically mixed into the sample during this process and the volumes of the internal standard solution and sample are both fixed by the capillary volume. Precision in quantitation is insensitive to variations in length of the capillary, making the preparation of the sampling capillary simple and providing a robust sampling protocol. Significant improvements in quantitation accuracy were obtained for analysis of 1 ?L samples using various ambient ionization methods.
Paper spray (PS) ambient ionization is implemented using a portable mass spectrometer and applied to the detection of alkyl quaternary ammonium salts in a complex oil matrix. These salts are commonly used as active components in the formulation of corrosion inhibitors. They were identified in oil and confirmed by their fragmentation patterns recorded using tandem mass spectrometry (MS/MS). The cations of alkyl and benzyl-substituted quaternary ammonium salts showed characteristic neutral losses of CnH2n (n carbon number of the longest chain) and C7H8, respectively. Individual quaternary ammonium compounds were detected at low concentrations (<1 ng ?L(-1)) and over a dynamic range of ?5 pg ?L(-1) to 500 pg ?L(-1) (ppb). Direct detection of these compounds in complex oil samples without prior sample preparation or pre-concentration was also demonstrated using a home-built miniature mass spectrometer at levels below 1 ng ?L(-1).
This paper reports the development of arrays of capillary-based low-temperature plasma (LTP) probes for direct sample analysis. These probe arrays allow a higher surface area to be analyzed, increasing the throughput in large sample analysis. Validation of these arrays was performed on illicit, cathinone-based drugs marketed as bath salts.
Acrolein, an ?,?-unsaturated aldehyde and a reactive product of lipid peroxidation, has been suggested as a key factor in neural post-traumatic secondary injury in spinal cord injury (SCI), mainly based on in vitro and ex vivo evidence. Here, we demonstrate an increase of acrolein up to 300%; the elevation lasted at least 2 weeks in a rat SCI model. More importantly, hydralazine, a known acrolein scavenger can provide neuroprotection when applied systemically. Besides effectively reducing acrolein, hydralazine treatment also resulted in significant amelioration of tissue damage, motor deficits, and neuropathic pain. This effect was further supported by demonstrating the ability of hydralazine to reach spinal cord tissue at a therapeutic level following intraperitoneal application. This suggests that hydralazine is an effective neuroprotective agent not only in vitro, but in a live animal model of SCI as well. Finally, the role of acrolein in SCI was further validated by the fact that acrolein injection into the spinal cord caused significant SCI-like tissue damage and motor deficits. Taken together, available evidence strongly suggests a critical causal role of acrolein in the pathogenesis of spinal cord trauma. Since acrolein has been linked to a variety of illness and conditions, we believe that acrolein-scavenging measures have the potential to be expanded significantly ensuring a broad impact on human health. The pathological role of acrolein in spinal cord injury (SCI) is demonstrated through its prolonged elevation and subsequent reduction by hydralazine, an acrolein scavenger, which leads to amelioration of tissue damage, motor deficits, and neuropathic pain. Acrolein injection into the spinal cord caused similar SCI pathologies that further support its role as an effective therapeutic target in SCI.
The aberrant expression of imprinted genes induces parthenogenetic fetal and placental dysplasia, thus leading to failures in embryonic development. Igf2 and H19 are co-expressed in endoderm and mesoderm-derived tissues and play an important role in normal embryo and extraembryonic development. In this study, the expression and methylation of Igf2/H19 in porcine parthenogenetic fetuses and placentas which had grown 28 days was examined first time to further characterize mammalian parthenogenesis. Weight and morphological comparisons were conducted between parthenogenetic embryos on Day 28 and normal fertilized embryos (control). The results indicated that parthenogenetic fetuses and placentas had smaller weights and volumes than those of the control. In addition, quantitative RT-PCR (qRT-PCR) analysis was performed to determine Igf2/H19 expression levels, showing that the expression of H19 was up-regulated, while Igf2 expression was almost undetectable in both parthenogenetic fetuses and placentas. As a potential mechanism underlying this disrupted expression, the methylation of Igf2/H19 DMR3 was detected using bisulfite sequencing PCR analysis, which revealed the significant hypomethylation of DMR3 in parthenogenetic fetuses and placentas. These results suggest that disruption of Igf2/H19 expression in parthenogenetic fetuses and placentas contributes to implantation failure and/or abortion in swine parthenogenesis, which might be associated with differential methylation patterns in the imprinting control region of imprinted genes.
The purpose of this study was to investigate the pharmacological effect of fraction of Atractylodes lancea (Thunb.) DC. (A. lancea) extract. In this study, we isolated different polarity fractions, including petroleum ether (PE), ethyl acetate, n-butanol, and the remaining H2O fractions from the water extract of A. lancea. The antigastric cancer properties of the different fractions in BGC-823 and SGC-7901 cells were evaluated. Apoptotic cells were treated with PE fraction and stained with Hoechst 33342 and 5,5,6,6-tetrachloro-1,1,3,3-tetraethylbenzimidazolylcarbocyanine iodide. The cell cycle was analyzed via flow cytometry. The main compounds of PE fraction were determined by HPLC-ESI-MS. Results of this study showed that the PE fraction of A. lancea inhibited the growth of BGC-823 and SGC-7901 cells in a dose- and time-dependent manner. The morphological and mitochondrial transmembrane potential changes suggested that the cells showed preliminary apoptosis characteristics after treatment with the three different polarities. The main compounds of PE fraction include two sesquiterpene compounds: eudesm-4(15),7-diene-9?,11-diol and eudesm-4(15)-ene-7a,11-diol; three sesquiterpene lactone compounds: atractylenolid I, atractylenolid III and 3-?-acetyl-atractylenolid III and one polyacetylenic compound: 4,6,12-tetradecatriene-8,10-diyne-1,3,14-triol.
Previous studies have demonstrated that recipient-derived immature dendritic cells transfected by recombinant adenovirus-mediated IKK2dn (Adv?IKK2dn) and loaded with donor splenocyte lysate generate CD4+CD25- T cells (Adv-IKK2dn-CD4+CD25- T cells). These cells may inhibit T cell responses in vitro. In the present study, Lewis (LW) rats were administered with an intravenous injection of naive CD4+ T cells, empty adenovirus (Adv-0)-dendritic cell-generated CD4+CD25- T cells (Adv-0-CD4+CD25- T cells), Adv-IKK2dn-CD4+CD25- T cells or an equal volume of normal saline, seven days prior to transplantation. The potency and the mechanism of action of Adv-IKK2dn-CD4+CD25- T cells was analyzed, as well as an investigation of their tolerogenic properties in vivo. Administration of Adv-IKK2dn-CD4+CD25- T cells in vivo to LW rats was observed to markedly prolong the survival of a kidney allograft from Brown Norway rats. Furthermore, the Adv-IKK2dn-CD4+CD25- T cell-treated group exhibited significantly reduced levels of interleukin (Il)-2 and interferon-? production and increased Il-10 and transforming growth factor-? (TGF-?) secretion. The serum creatinine levels remained at low levels in the Adv-IKK2dn-CD4+CD25- T cell-treated group. Their ability to induce allogeneic T cell proliferation was markedly reduced compared with the other groups. These observations indicated that Adv-IKK2dn-CD4+CD25- T cells induce prolongation of kidney allograft survival in vivo, which is hypothesized to be due to the high expression levels of Il-10 and TGF-?.
Porcine CD4 proximal promoter and enhancer sequences were cloned and aligned with the corresponding human and murine sequences. The alignment showed nucleotide homology between porcine and human sequences was 62.4 % for the CD4 promoter and 56.6 % for the CD4 enhancer. The nucleotide homology between porcine and murine sequences was 42.5 % for the CD4 promoter and 25.4 % for the CD4 enhancer. The proximal enhancer and promoter regions of the CD4 gene from porcine, murine and human cells were compared for their ability to direct foreign gene expression in transiently transfected human cell lines. The results indicated the porcine CD4 promoters and enhancers could effectively direct expression of a foreign gene in human cells. The porcine promoter was equally efficient as CMV and EF-1? in directing gene expression.
The understanding of the gas dynamics of the atmospheric pressure interface is very important for the development of mass spectrometry systems with high sensitivity. While the gas flows at high pressure (>1 Torr) and low pressure (<10(-3) Torr) stages are relatively well understood and could be modeled using continuum and molecular flows, respectively, the theoretical modeling or numeric simulation of gas flow through the transition pressure stage (1 to 10(-3) Torr) remains challenging. In this study, we used the direct simulation Monte Carlo (DMSC) method to develop the gas dynamic simulations for the continuous and discontinuous atmospheric pressure interfaces (API), with different focuses on the ion transfer by gas flows through a skimmer or directly from the atmospheric pressure to a vacuum stage, respectively. The impacts by the skimmer location in the continuous API and the temporal evolvement of the gas flow with a discontinuous API were characterized, which provide a solid base for the instrument design and performance improvement.
Understanding Ostwald ripening and disintegration of supported metal particles under operating conditions has been of central importance in the study of sintering and dispersion of heterogeneous catalysts for long-term industrial implementation. To achieve a quantitative description of these complicated processes, an atomistic and generic theory taking into account the reaction environment, particle size and morphology, and metal-support interaction is developed. It includes (1) energetics of supported metal particles, (2) formation of monomers (both the metal adatoms and metal-reactant complexes) on supports, and (3) corresponding sintering rate equations and total activation energies, in the presence of reactants at arbitrary temperature and pressure. The thermodynamic criteria for the reactant assisted Ostwald ripening and induced disintegration are formulated, and the influence of reactants on sintering kinetics and redispersion are mapped out. Most energetics and kinetics barriers in the theory can be obtained conveniently by first-principles theory calculations. This allows for the rapid exploration of sintering and disintegration of supported metal particles in huge phase space of structures and compositions under various reaction environments. General strategies of suppressing the sintering of the supported metal particles and facilitating the redispersions of the low surface area catalysts are proposed. The theory is applied to TiO(2)(110) supported Rh particles in the presence of carbon monoxide, and reproduces well the broad temperature, pressure, and particle size range over which the sintering and redispersion occurred in such experiments. The result also highlights the importance of the metal-carbonyl complexes as monomers for Ostwald ripening and disintegration of supported metal catalysts in the presence of CO.
The development of technologies for mass spectrometry imaging is of substantial research interest. Mass spectrometry is potentially capable of providing highly specific information about the distribution of compounds in tissues, with high sensitivity. The in-situ analysis needed for tissue imaging requires MS to be performed under conditions different from the traditional ones, typically with intensive sample preparation and optimized for pharmaceutical applications. In this paper we critically review the current status of MS imaging with different methods of sample ionization and discuss the 3D and quantitative imaging capabilities which need further development, the importance of the multi-modal imaging, and the balance between the pursuit of high-resolution imaging and the practical application of MS imaging in biomedicine.
Automated sample extraction for regulated bioanalysis by liquid chromatography/tandem mass spectrometry (LC-MS/MS) still presents significant challenges. A new sample preparation methodology with a simplified and completely automated workflow was developed to overcome these challenges using cap piercing for direct biofluid transfer and evaporation-free solid phase extraction (SPE). Using pierceable cap sample tubes, a robotic liquid handler was able to sample without uncapping or recapping during sample preparation. Evaporation for SPE was eliminated by using a mobile phase-compatible elution solvent followed by sample dilution prior to LC-MS/MS analysis. Presented here are three LC-MS/MS assays validated using this methodology to support three CNS drug development programs: (1) BMS-763534 and its metabolite, BMS-790318, in dog plasma; (2) BMS-694153 in monkey plasma; and (3) Pexacerfont (BMS-562086) and two metabolites, BMS-749241 and DPH-123554, in human plasma. These assays were linear from 1.00 to 1000 or 2.00 to 2000ng/mL for each analyte with excellent assay accuracy, precision and reproducibility. These assays met acceptance criteria for regulated bioanalysis and have been successfully applied to drug development study samples. The methodology described here successfully eliminated all manual intervention steps achieving fully automated sample preparation without compromising assay performance. Importantly, this methodology eliminates the potential exposure of the bioanalyst to any infectious biofluids during sample preparation.
Ambient ionization techniques allow complex chemical samples to be analyzed in their native state with minimal sample preparation. This brings the obvious advantages of simplicity, speed, and versatility to mass spectrometry: Desorption electrospray ionization (DESI), for example, is used in chemical imaging for tumor margin diagnosis. This review on the extractive methods of ambient ionization focuses on chemical aspects, mechanistic considerations, and the accelerated chemical reactions occurring in charged liquid droplets generated in the spray process. DESI uses high-velocity solvent droplets to extract analytes from surfaces. Nano-DESI employs liquid microjunctions for analyte dissolution, whereas paper-spray ionization uses DC potentials applied to wet porous material such as paper or biological tissue to field emit charged analyte-containing solvent droplets. These methods also operate in a reactive mode in which added reagents allow derivatization during ionization. The accelerated reaction rates seen in charged microdroplets are useful in small-scale rapid chemical synthesis.
Asunaprevir (BMS-650032) is a potent hepatitis C virus (HCV) non-structural protein protease inhibitor currently in Phase III clinical trials for the treatment of HCV infection. A rugged and accurate LC-MS/MS method was developed and validated for the quantitation of asunaprevir in rat, dog, monkey, rabbit and mouse plasma. A systematic method screening and optimization strategy was applied to achieve optimized mass spectrometry, chromatography, and sample extraction conditions. The validated method utilized stable-isotope labeled D9-asunaprevir as the internal standard. The samples were extracted by liquid-liquid extraction using 10% ethyl acetate in hexane. Chromatographic separation was achieved with gradient elution on a Waters Atlantis dC18 analytical column. Analyte and its internal standard were detected by positive ion electrospray tandem mass spectrometry. The standard curve, which ranged from 5.00 to 2000ng/mL for asunaprevir, was fitted to a 1/x(2) weighted linear regression model. The intra-assay precision was within ±3.6% CV, inter-assay precision was within ±4.0% CV, and the assay accuracy was within ±8.1% of the nominal values in all the species. The method was successfully applied to support multiple pre-clinical toxicokinetic studies in different species.
The sensitivity of mass analysis is highly dependent on the efficiencies of the sample introduction, analyte ionization, and subsequent ion transfer to the mass analyzer. For miniature mass spectrometry (MS) systems, it is highly desirable to maximize the amount of the samples that can be introduced at the pumping capability highly compromised and to have the MS analysis performed with simple electronics and robust in-vacuum components. In this study, a discharge ionization source synchronized with the sample introduction has been developed for hand-held mass spectrometers with discontinuous atmospheric pressure interfaces (DAPI). The flow rate of the sample introduced with a DAPI can be much higher than that allowed with a conventional continuous atmospheric pressure interface. The pressure variation associated with the DAPI operation was used to turn on and off the synchronized discharge ionization (SDI). For analysis of volatile organic compounds (VOCs) in air samples, the SDI has been shown to be soft and molecular ions of the VOCs have been observed as the major species. Since the ions could be transferred directly to the ion trap mass analyzer without a barrier for maintaining pressure differences, high sensitivity in chemical analysis was also achieved, with limits of detection (LODs) better than 1 ppbv obtained for PAHs (polycyclic aromatic hydrocarbons) in air.
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