Microsatellite Enrichment from AFLP Fragments by Magnetic Beads

A simple and efficient method was presented for isolating microsatellite DNA markers from peanut (Arachis hypogaea L.) genome. The genomic DNA was converted into pre-amplified AFLP fragments and hybridized with biotin-labeled SSR probes. Then the hybrid mixture was used to incubate with magnetic beads coated with streptavidin. After washing to remove the non-SSR fragments, the eluted single-strand DNA, which was cloned and sequenced, was largely enriched for microsatellites. Primers can then be designed according to the sequence flanking the repeat motifs and used for polymorphism analysis. The whole experiment can be completed within one week and can be employed as a reliable option for any molecular laboratory to develop SSR markers.

Highly efficient generation of GGTA1 knockout pigs using a combination of TALEN m RNA and magnetic beads with somatic cell nuclear transfer

The transcription activator-like effector nuclease （TALEN） technique combined with the somatic cel nuclear transfer （SCNT） method has been successfuly applied for creating geneticaly modiifed pigs. However, methods for isolating cels with bialelic indels requires further improvement because of the relatively low enrichment efifciency of mutated somatic cels. Moreover, little is known regarding the off-target effects of the TALEN system and the heredity of TALEN-modiifed pigs. In this study, an efifcient method to increase the enrichment efifciency of TALEN-mediated bialelic knockout （KO） cels was established, and corresponding geneticaly modiifed pigs with the expected genotype were generated whose off-target effect, fertility and heredity characteristics were aslo evaluated. Two TALEN pairs were constructed to target the porcine α-1,3-galactosyltransferase （GGTA1） gene locus. TALEN mRNA was transfected into the ear ifbroblasts folowed by the enrichment of α-Gal nul cels of minipigs using isolectin B4 （IB4） lectin and magnetic beads. A total of 115 cel colonies were formed and validated to beGGTA1 KO cels by sequencing and 10 bialelic KO cel colonies were used as nuclear donors for SCNT. ThirtyGGTA1 bialelic KO piglets were successfuly delivered and grew normaly. Seventeen potential off-target sites were investigated, and no off-target events were detected in the live piglets. To determine the fertility and heredity characteristics of TALEN-modiifed pigs, 10 mature founders were mated with each other and the mutations were determined to be transmitted to the F1 piglets. We established a robust and safe technology for developing geneticaly modiifed pig lines with expected genotypes for agricultural breeding and biomedical application.

Preparation of Immuno-magnetic Beads and Their Separation & Detection to Ovary Cancer Cells

The organic monomer-molecule with nanometer magnetic powder by means of reforming the surface of nanometer magnetic powder have been synthesized. Magnetic beads in diameter of 2 mum or so am obtained by controlling conditions. Ovary cancer cells of ascites are separated and ovary cancer cells of blood arc detected by using immuno-magnetic beads linked with ovary cancer cell mono-antibodies. Results show that the specificity is 85% sensitivity is 87%, accuracy is 84%, cells acquiring purity is 90%, cells activity is 92% and detection sensitivity is 25 x 10(-7).

A preparation strategy for protein-oriented immobilized silica magnetic beads with Spy chemistry for ligand fishing

Due to the complexity of bioactive ingredients in biological samples,the screening of target proteins is a complex process.Herein,a feasible strategy for directing protein immobilization on silica magnetic beads for ligand fishing based on SpyTag/SpyCatcher(ST/SC)-mediated anchoring is presented.Carboxyl functional groups on the surface of silica-coated magnetic beads(SMBs)were coupled with SC using the 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride/N-hydroxysulfosuccinimide method,named SC-SMBs.The green fluorescent protein(GFP),as the capturing protein model,was ST-labeled and anchored at a specific orientation onto the surface of SC-SMBs directly from relevant cell lysates via ST/SC self-ligation.The characteristics of the SC-SMBs were studied via electron microscopy,energy dispersive spectroscopy,and Fourier transform infrared spectroscopy.The spontaneity and site-specificity of this unique reaction were confirmed via electrophoresis and fluorescence analyses.Although the alkaline stability of ST-GFP-ligated SC-SMBs was not ideal,the formed isopeptide bond was unbreakable under acidic conditions(0.05 M glycine-HCl buffer,pH 1e6)for 2 h,under 20%ethanol solution within 7 days,and at most temperatures.We,therefore,present a simple and universal strategy for the preparation of diverse protein-functionalized SMBs for ligand fishing,prompting its usage on drug screening and target finding.

Applied Study on Magnetic Nanometer Beads in Preparation of Genechip Samples

A protocol for enrichment and adsorption of karyocyte from whole blood by using magnetic nanometer beads as solid phase absorbents was presented. The PCR amplification could be accomplished by using the nanobeads with karyocyte as template directly and the PCR products were applied on an oligonucleotide array to do gene typing. The HLA A PCR amplification system and a small HLA A oligonucleotide microarray were applied as the platform and an experiment protocol of separating karyocyte from whole blood using the magnetic nanometer beads (Fe 2O 3) were set up. The experimental conditions were also discussed. It showed that pH level of PBS eluent, Taq enzyme quantity and fragment length of products could influent the amplification results, and the magnetic nano beads could succeed in sample preparation in microarray to provide a promising way in automatic detection and lab on a chip.

Effect of superstrong magnetic field on electron screening at the crusts of neutron stars

The influences of electron screening （ES） and electron energy correction （EEC） are investigated by superstrong magnetic field （SMF）. We also discuss in detail the discrepant factor between our results and those of Fushiki, Gudmundsson and Pethick （FGP） in SMF. The results show that SMF has only a slight effect on ES when B 〈 10^9 T on the surfaces of most neutron stars. Whereas for some magnetars, SMF influence ES greatly when B 〉 10^9 T . For instance, due to SMF the ES potential may be increased about 23.6% and the EEC may be increased about 4 orders of magnitude at ρ/μe = 1.0 × 10^6 mol/cm^3 and T9 = 1. On the other hand, the discrepant factor shows that our results are in good agreement with FGP＇s when B 〈 10^9 T . But the difference will be increased with increasing SMF.

Usefulness of Immuno-Magnetic Beads Conjugated with Anti-EpCAM Antibody for Detecting Endometrial Cancer Cells

A simple and non-invasive method for detecting endometrial cancer in women with abnormal uterine bleeding is required. For this purpose, we prepared immuno-magnetic beads conjugated with anti-human EpCAM rat monoclonal antibody (mAb) for isolating exfoliated endometrial cells including endometrial cancer cells in vaginal discharge. The affinities of the anti-human EpCAM rat mAbs were analyzed by flow cytometry and immunocytochemistry and then magnetic beads were conjugated with the mAbs. The rate of retrieval of endometrial cells using the immuno-magnetic beads was calculated. Endometrial cells were isolated using the immuno-magnetic beads from the vaginal discharges of 22 patients with endometrial cancer and 16 non-malignant controls. The isolated cells were stained using endometrial cancer specific-mAbs and analyzed by flow cytometry and imaging cytometry. The immuno-magnetic beads conjugated with high-affinity mAb (clone 1456) appeared to have very low auto-fluorescence. Sufficient enrichment of Ep-CAMpositive cells using immuno-magnetic beads was observed in both simulation and clinical samples. The overall sensitivities of flow cytometry and imaging cytometry to detect endometrial cancer cells were 72.7% and 45.5%, respectively. Meanwhile, the overall specificities of flow cytometry and imaging cytometry for healthy controls were 75.0% and 81.3%, respectively. Our immuno-magnetic beads have very low auto-fluorescence, so they could be useful for fluorescent analysis, such as fluorescent immunochemical staining. In the future, these novel immuno-magnetic beads could be used for cytological study.

Selective adsorption behavior of ion-imprinted magnetic chitosan beads for removal of Cu(Ⅱ) ions from aqueous solution

Heavy metal ion is one of the major environmental pollutants.In this study,a Cu(Ⅱ)ions imprinted magnetic chitosan beads are prepared to use chitosan as functional monomer,Cu(Ⅱ)ions as template,Fe_(3)O_(4) as magnetic core and epichlorohydrin and glutaraldehyde as crosslinker,which can be used for removal Cu(Ⅱ)ions from wastewater.The kinetic study shows that the adsorption process follows the pseudosecond-order kinetic equations.The adsorption isotherm study shows that the Langmuir isotherm equation best fits for the monolayer adsorption processes.The selective adsorption properties are performed in Cu(Ⅱ)/Zn(Ⅱ),Cu(Ⅱ)/Ni(Ⅱ),and Cu(Ⅱ)/Co(Ⅱ)binary systems.The results shows that the ⅡMCD has a high selectivity for Cu(Ⅱ)ions in binary systems.The mechanism of ⅡMCD recognition Cu(Ⅱ)ions is also discussed.The results show that the ⅡMCD adsorption Cu(Ⅱ)ions is an enthalpy controlled process.The absolute value of DH(Cu(Ⅱ))and DS(Cu(Ⅱ))is greater than DH(Zn(Ⅱ),Ni(Ⅱ),Co(Ⅱ))and DS(Zn(Ⅱ),Ni(Ⅱ),Co(Ⅱ)),respectively,this indicates that the Cu(Ⅱ)ions have a good spatial matching with imprinted holes on ⅡMCD.The FTIR and XPS also demonstrates the strongly combination of function groups on imprinted holes in the suitable space position.Finally,the ⅡMCD can be regenerated and reused for 10 times without a significantly decreasing in adsorption capacity.This information can be used for further application in the selective removal of Cu(Ⅱ)ions from industrial wastewater.

Effects of Magnetic Particles Entrance Arrangements on Mixing Efficiency of a Magnetic Bead Micromixer

In this study, a computer code is developed to numerically investigate a magnetic bead micromixer under different conditions. The micromixer consists of a microchannel and numerous micro magnetic particles which enter the micromixer by fluid flows and are actuated by an alternating magnetic field normal to the main flow. An important feature of micromixer which is not considered before by researchers is the particle entrance arrangement into the micromixer. This parameter could effectively affect the micromixer efficiency. There are two general micro magnetic particle entrance arrangements in magnetic bead micromixers: determined position entrance and random position entrance. In the case of determined position entrances, micro magnetic particles enter the micromixer at specific positions of entrance cross section. However, in a random position entrance,particles enter the microchannel with no order. In this study mixing efficiencies of identical magnetic bead micromixers which only differ in particle entrance arrangement are numerically investigated and compared.The results reported in this paper illustrate that the prepared computer code can be one of the most powerful and beneficial tools for the magnetic bead micromixer performance analysis. In addition, the results show that some features of the magnetic bead micromixer are strongly affected by the entrance arrangement of the particles.

Suppressing of humping bead using an external magnetic field in high-speed gas metal arc welding

During high-speed gas metal arc welding （GMAW）, the backward flowing molten jet with high momentum in the weld pool is considered to be responsible for the occurrence of humping bead. To suppress humping bead, an electromagnetic device is developed and coupled with the welding system. By adjusting the conditions of external magnetic field, forward electromagnetic force is obtained to reduce the momentum of the backward flow of molten metal in weld pool. Consequently, the humping bead can be suppressed by adjusting the external magnetic field. Bead-on-plate welding experiment was conducted on mild steel plates, and the influence of magnetic flux density on the arc deflection angle and weld bead quality is investigated. It is found that external magnetic field can remarkably adjust the momentum of backward flow jet and significantly improve the quality of weld bead.

Preliminary Application of WCX Magnetic Bead-Based Matrix-Assisted Laser Desorption Ionization Time-of-Flight Mass Spectrometry in Analyzing the Urine of Renal Clear Cell Carcinoma

Objective To evaluate the application of weak cation exchange (WCX) magnetic bead-based Matrix-Assisted Laser Desorption Ionization Time-of-Flight Mass Spectrometry (MALDI-TOF MS) in detecting differentially expressed proteins in the urine of renal clear cell carcinoma (RCCC) and its value in the early diagnosis of RCCC.Methods Eleven newly diagnosed patients (10 males and 1 female, aged 46-78, mean 63 years) of renal clear cell carcinoma by biopsy and 10 healthy volunteers (all males, aged 25-32, mean 29.7 years) were enrolled in this study. Urine samples of the RCCC patients and healthy controls were collected in the morning.Weak cation exchange (WCX) bead-based MALDI-TOF MS technique was applied in detecting differential protein peaks in the urine of RCCC. ClinProTools2.2 software was utilized to determine the characteristic proteins in the urine of RCCC patients for the predictive model of RCCC.Results The technique identified 160 protein peaks in the urine that were different between RCCC patients and health controls; and among them, there was one peak (molecular weight of 2221.71 Da) with statistical significance (P=0.0304). With genetic algorithms and the support vector machine, we screened out 13 characteristic protein peaks for the predictive model.Conclusions The application of WCX magnetic bead-based MALDI-TOF MS in detecting differentiallyexpressed proteins in urine may have potential value for the early diagnosis of RCCC.

Peptidome profiling of human serum of uveal melanoma patients based on magnetic bead fractionation and mass spectrometry

AIM： To find new biomarkers for uveal melanoma （UM） by analyzing the serum peptidome profile. METHODS： Proteomic spectra in patients with UM before and after operation were analyzed and compared with those of healthy controls. Magnetic affinity beads were used to capture serum peptides and matrix-assisted laser desorption/ionization time-of-flight （MALDI-TOF） mass spectrometer were used to compile serum peptide profiles. RESULTS： A panel of 49 peptides were differentially expressed between UM patients and controls, of which 33 peptides were of higher intensities in patient group and 16 peptides were of higher intensities in control group. Based on combined use of these potential markers, peptides with mean molecular masses of 1467 and 9289.0 Da provide high sensitivity （83.3%）, specificity （100%） and accuracy rate （93.0%） together to differentiate melanoma patients from healthy controls. At the time point of 6mo postoperatively, the levels of many peptides differentially expressed before surgery showed no more statistical difference between the patients and the control group. Fibrinogen o-chain precursors were identified as potential UM markers. CONCLUSION： We have shown that a convenient and fast proteomic technique, affinity bead separation and MALDI- TOF analysis combined with bioinformatic software, facilitates the identification of novel biomarkers for UM.

Screening-current-induced magnetic fields and strains in a compact REBCO coil in self field and background field

REBa_(2)Cu_(3)O_(7−x)(REBCO)coated conductors,owing to its high tensile strength and current‐carrying ability in a background field,are widely regarded a promising candidate in high‐field applications.Despite the great potentials,recent studies have highlighted the challenges posed by screening currents,which are featured by a highly nonuniform current distribution in the superconducting layer.In this paper,we report a comprehensive study on the behaviors of screening currents in a compact REBCO coil,specifically the screeningcurrent‐induced magnetic fields and strains.Experiments were carried out in the self‐generated magnetic field and a background field,respectively.In the self‐field condition,the full hysteresis of the magnetic field was obtained by applying current sweeps with repeatedly reversed polarity,as the nominal center field reached 9.17 T with a maximum peak current of 350 A.In a background field of 23.15 T,the insert coil generated a center field of 4.17 T with an applied current of 170 A.Ultimately,a total center field of 32.58 T was achieved before quench.Both the sequential model and the coupled model considering the perpendicular field modification due to conductor deformation are applied.The comparative study shows that,for this coil,the electromagnetic–mechanical coupling plays a trivial role in self‐field conditions up to 9 T.In contrast,with a high axial field dominated by the background field,the coupling effect has a stronger influence on the predicted current and strain distributions.Further discussions regarding the role of background field on the strains in the insert suggest potential design strategies to maximize the total center field.

STUDY ON THE SEPARATION AND UTILIZATION TECHNOLOGY OF MAGNETIC BEAD IN FLY ASH

On the basis of study on physical and chemical properties of magnetic bead (MB) in fly ash (FA), the paper gives out the separation methods of MB and results of three separating process. The result of comparative test in size, density, stability, magnetic material content, specific magnetic susceptibility (SMS), medium recovery oxidation resistance and wear resistance between MB and magnetic fines currently used in dense medium separation leads to that using MB recovered from fly ash is used as medium solids in coal cleaning in stead of magnetic fines not only have no influence upon taryests of separation, but can bring good economic and social benefits.

Construction Fractional Genomic Libraries and Screening Microsatellites DNA of Esox reieherti Dybowski

Esox reieherti Dybowsk genomic microsateUites were developed by using enrichment protocols combined with radioactive hybridization protocol. Four hundred to nine hundred base pair fragments were selected for the whole genome. DNA PCR amplification after digestion with restriction endonuclease Sau 3A Ⅰ, and （CA）12, （GA）12 probes marked with biotin were used for microsateUite DNA enrichment. The product fragments were connected with carder pGEM-T and transferred into DH5α Escherichia coli competent cells, and radioactive isotope probes marked with γ^-32 p were used for the second hybridization. As a result, a total of 1600 bacteria were obtained in the microsatellite genomic libraries, positive clones accounted for 90.91% before hybridization and 81.25% after hybridization, amounting to 1300. One hundred and ninety-six positive clones were selected for sequencing, and 192 clones included microsateUite sequences. The microsateUite sequences obtained, mono-nucleotide, quad-nucleotide and quint-nucleotide repeat motifs were observed beside double-base-pairs CA/GT, GA/CT. Seventy primers were designed according to the flanking sequences by using software Primer Premier 5.0, and 32 primers were selected to be synthesized. After optimizing PCR reaction conditions, 28 primers were amplified and produced clear purpose bands. The aim of our research was to promote the development and utilization of E. reieherti genomic resource, and lay the foundation for optimizing E. reieherti breeding strain in order to detect the genetic diversity and construct a genetic map.

Screening current in ultra-high-field non-insulated superconducting magnets

Screening current is recognized as one of the critical elements limiting the progression of superconducting magnets toward achieving higher magnetic fields. Currently, most non-insulated(NI) superconducting magnets consider the magnet as insulated when addressing the issue of screening current. However, the bypass current in the NI magnet can modify the actual history of magnetization, so the screening current in NI magnet will be different from that in the insulated magnet. This paper presents a novel method based on the homogenized T-A formulation(T is the current vector potential, and A is the magnetic vector potential), which enables real-time simulation of both the bypass current behavior and the implications of screening current in NI superconducting magnets, even when these magnets contain tens of thousands of turns. We have developed a 32 T NI hybrid superconducting magnet and validated the effectiveness of this method through experiments. Employing this efficacious method,we conducted a comprehensive calculation of screening current in NI magnets, comparing them with insulated magnets in terms of screening current-induced stress(SCIS), screening current-induced field(SCIF), and losses. The results indicate that in the NI insert coils, the sequential excitation of background coils and insert coils induces a reverse screening current, resulting in slightly lower SCIF and SCIS compared to those in the insulated magnets. The method and results can contribute to the enhancement of magnet design and provide valuable insights for the development of ultra-high fields(UHF) NI magnets.

Methods and outcomes of screening for pancreatic adenocarcinoma in high-risk individuals

Pancreatic ductal adenocarcinoma(PDAC) is a lethal neoplasia, for which secondary prevention(i.e., screening) is advisable for high-risk individuals with "familiar pancreatic cancer" and with other specific genetic syndromes(Peutz-Jeghers, p16, BRCA2, PALB and mismatch repair gene mutation carriers). There is limited evidence regarding the accuracy of screening tests, their acceptability, costs and availability, and agreement on whom to treat. Successful target of screening are small resectable PDAC, intraductal papillary mucinous neoplasms with high-grade dysplasia and advanced pancreatic intraepithelial neoplasia. Both magnetic resonance imaging(MRI) and endoscopic ultrasound(EUS) are employed for screening, and the overall yield for pre-malignant or malignant pancreatic lesions is of about 20% with EUS and 14% with MRI/magnetic resonance colangiopancreatography. EUS performs better for solid and MRI for cystic lesions. However, only 2% of these detected lesions can be considered a successful target, and there are insufficient data demonstrating that resection of benign or low grade lesions improves survival. Many patients in the published studies therefore seemed to have received an overtreatment by undergoing surgery. It is crucial to better stratify the risk of malignancy individually, and to better define optimal screening intervals and methods either with computerized tools or molecular biomarkers, possibly in large multicentre studies. At the moment, screening should be carefully performed within research protocols at experienced centres, offering involved individuals medical and psychological advice.

The detection of tetrahydrofuran hydrate formation and saturation using magnetic resonance imaging technique

Tetrahydrofuran（THF） hydrate was formed in bulk as well as in glass beads pack with a mean diameter of 3.0 mm by controlling the temperature under ambient pressure.Images of THF hydrate formation procedure were obtained using the magnetic resonance imaging（MRI） technique.The experiment results showed that MRI is an effective method for the detection of hydrate formation.Saturation of hydrate formed both in bulk and glass beads can be confirmed by intensity integration of MRI images.

A magnetic nanoparticles-based method for DNA extraction from the saliva of stroke patients

C677T polymorphism in the methylenetetrahydrofolate reductase （MTHFR） gene is a risk factor for stroke, suggesting that widespread detection could help to prevent stroke. DNA from 70 stroke pa- tients and 70 healthy controls was extracted from saliva using a magnetic nanoparticles-based method and from blood using conventional methods. Real-time PCR results revealed that the C677T polymorphism was genotyped by PCR using DNA extracted from both saliva and blood samples. The genotype results were confirmed by gene sequencing, and results for saliva and blood samples were consistent. The mutation TT genotype frequency was significantly higher in the stroke group than in controls. Homocysteine levels were significantly higher than controls in both TT genotype groups. Therefore, this noninvasive magnetic nanoparticles-based method using saliva samples could be used to screen for the MTHFR C677T polymorphism in target populations.