101 research outputs found
Recommended from our members
Novel molecular engineering approaches for genotyping and DNA sequencing
The completion of the Human Genome Project has increased the need for investigation of genetic sequences and their biological functions, which will significantly contribute to the advances in biomedical sciences, human genetics and personalized medicine. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) offers an attractive option for DNA analysis due to its high accuracy, sensitivity and speed. In the first part of the thesis, we report the design, synthesis and evaluation of a novel set of mass tagged, cleavable biotinylated dideoxynucleotides (ddNTP-N3-biotins) for DNA polymerase extension reaction and its application in DNA sequencing and single nucleotide polymorphism (SNP) genotyping by mass spectrometry. These nucleotide analogs have a biotin moiety attached to the 5 position of the pyrimidines (C and U) or the 7 position of the purines (A and G) via a chemically cleavable azido-based linker, with different length linker arms serving as mass tags that contribute large mass differences among the nucleotides to increase resolution in MS analysis. It has been demonstrated that these modified nucleotides can be efficiently incorporated by DNA polymerase, and the DNA strand bearing biotinylated nucleotides can be captured by streptavidin coated beads and efficiently released using tris(2-carboxyethyl) phosphine in aqueous solution which is compatible with DNA and downstream procedures. Reversible solid phase capture (SPC) mass spectrometry sequencing using ddNTP-N3-biotins was performed, and various DNA templates, including biological samples, were accurately sequenced achieving a read-length of 37 bases. In mass spectrometric SNP genotyping, we have successfully exploited our reversible solid phase capture (SPC)-single base extension (SBE) assay and been able to detect as low as 2.5% heteroplasmy in mitochondrial DNA samples, with interrogation of human mitochondrial genome position 8344 which is associated with an important mitochondrial disease (myoclonic epilepsy with ragged red fibers, MERRF); we have also quantified the heteroplasmy level of a real MERRF patient and determined several mitochondrial MERRF mutations in a multiplex approach. These results demonstrated that our improved mass spectrometry genotyping technologies have great potential in DNA analysis, with particular applications in sequencing short-length targets or detecting SNPs with high accuracy and sensitivity requirements, such as DNA fragments with small indels, or SNPs in pooled samples. To truly implement this mass spectrometry-based genotyping method, we further explored the use of a lab-on-a-chip microfluidic device with the potential for high throughput, miniaturization, and automation. The microdevice primarily consists of a micro-reaction chamber for single base extension and cleavage reactions with an integrated micro heater and temperature sensor for on-chip temperature control, a microchannel loaded with streptavidin magnetic beads for solid phase capture, and a microchannel packed with C18-modified reversed-phase silica particles as a stationary phase for desalting before MALDI-TOF analysis. By performing each functional step, we have demonstrated 100% on-chip single base incorporation, sufficient capture and release of the biotin-ddNTP terminated single base extension products, and high sample recovery from the C18 reverse-phase microchannel with as little as 0.5 pmol DNA molecules. The feasibility of the microdevice has shown its promise to improve mass spectrometric DNA sequencing and SNP genotyping to a new paradigm. DNA sequencing by synthesis (SBS) appears to be a very promising molecular tool for genome analysis with the potential to achieve the $1000 Genome goal. However, the current short read-length is still a challenge. Therefore, the second part of the thesis focuses on strategies to overcome the short read-length of SBS. We developed a novel primer walking strategy to increase the read-length of SBS with cleavable fluorescent nucleotide reversible terminators (CF-NRTs) and nucleotide reversible terminators (NRTs) or hybrid-SBS with cleavable fluorescent nucleotide permanent terminators and NRTs. The idea of the walking strategy is to recover the initial template after one round of sequencing and re-initiate a second round of sequencing at a downstream base to cover more bases overall. The combination of three natural nucleotides and one NRT effectively regulated the primer walking: the primer extension temporarily paused when the NRT was incorporated, and resumed after removing the 3' capping group to restore the 3'-OH group. We have successfully demonstrated the integration of this primer walking strategy into the sequencing by synthesis approach, and were able to obtain a total read-length of 53 bases, nearly doubling the read-length of the previous sequencing. On the other hand, we explored the sequencing bead-on-chip approach to increase the throughput of SBS and hence the total genome coverage per run. The various prerequisite conditions have been optimized, allowing the accurate sequencing of several bases on the bead surface, which demonstrated the feasibility of this approach. Both of these approaches could be integrated into current SBS platforms, allowing increased overall coverage and lowering overall costs. As a step beyond genotyping, the in vivo visualization of biomolecules, like DNA and its encoded RNA and proteins, provides further information about their biological functions and mechanisms. The third part of the thesis focuses on the development of a novel quantum dot (QD)-based binary molecular probe, which takes advantage of fluorescent resonance energy transfer (FRET), for detection of nucleic acids, aiming at their eventual use for detection of mRNAs involved in long term memory studies in the model organism Aplysia californica. We reported the design, synthesis, and characterization of a binary probe (BP) that consists of carboxylic quantum dot (CdSe/ZnS core shell)-DNA (QD-DNA) conjugated donor and a cyanine-5 (Cy5)-DNA acceptor for the detection of a sensorin mRNA-based synthetic DNA molecule. We have demonstrated that in the absence of target DNA, the QD fluorescence is the main signal observed (605 nm); in the presence of the complementary target DNA sequence, a decrease of QD emission and an increase of Cy5 emission at 667 nm was observed. We have demonstrated the distance dependence of FRET, with the finding that the target with 16 base separation between the QD and Cy5 after probe hybridization gave the most efficient FRET. Further studies are in progress to evaluate the effectiveness of this QD-based probe inside a cell extract and in living cells
Orexin-A protects against oxygen-glucose deprivation/reoxygenation-induced cell damage by inhibiting endoplasmic reticulum stress-mediated apoptosis via the Gi and PI3K signaling pathways
The neuropeptide orexin-A (OXA) has a neuroprotective effect, acting as an anti-apoptotic factor in response to multiple stimuli. Apoptosis induced by endoplasmic reticulum stress (ERS) underlies oxygen-glucose deprivation and reoxygenation (OGD/R)-induced cell damage, an in vitro model of ischemia/reperfusion injury. However, that OXA inhibits ERS-induced apoptosis in the OGD/R model has not been reported. In the present study, we investigated the neuroprotective effect of OXA (0.1 μM) on OGD/R-induced damage in the human neuroblastoma cell line SH-SY5Y. After OXA treatment following 4 h oxygen-glucose deprivation (OGD) and then 4 h reoxygenation (R), cell morphology, viability, and apoptosis were analyzed by histology, Cell Counting Kit-8 assay, and flow cytometry, respectively. Western blotting was used to measure expression levels of ERS- and apoptosis-related proteins. To determine signaling pathways involved in OXA-mediated neuroprotection, the Gi pathway inhibitor pertussis toxin (PTX; 100 ng/mL) and PI3K inhibitor LY294002 (LY; 10 μM) were added. In addition, in order to prove the specificity of these characteristics, the OXA antagonist Suvorexant (DORA; Ki of 0.55 nM and 0.35 nM for OX1R and OX2R) was used for intervention. Our results showed that OGD/R induced cell damage, manifested as morphological changes and a significant decrease in viability. Furthermore, Western blotting detected an increase in ERS-related proteins GRP78, p-IRE1α, p-JNK, and Cleaved caspase-12, as well as apoptosis-related proteins Cleaved caspase-3 and Bax, and a decrease in the anti-apoptosis factor Bcl-2. OXA intervention alleviated the degree of cellular damage, and protein expression was also reversed. In addition, the protective effect of OXA was reduced by adding PTX and LY. Meanwhile, after the use of DORA, changes in the expression of related proteins were detected, and it was found that the protective effect of OXA was weakened. Collectively, our results indicate that OXA has a neuroprotective effect on OGD/R-induced cell damage by inhibiting ERS-induced apoptosis through the combined action of Gi and PI3K signaling pathways. These findings help to clarify the mechanism underlying the neuroprotective action of OXA, which should aid the development of further candidate drugs, and provide a new therapeutic direction for the treatment of ischemic stroke
Service Life Prediction of Electrolytic Capacitors in Urban Rail Transit Based on Analytical Iteration and GM (
In the power supply of urban rail transit system, electrolytic capacitors are used in large numbers. They suffer from inevitable ripple currents; such ripple currents generate heat dissipation, which shortens their service life seriously. To increase the reliability of the whole power supply system, the aging process of electrolytic capacitors must be evaluated, so that their service life could be predicted and measures could be taken in advance before their failures. When they are applied into the power supply system, the accuracy of conventional approaches is somewhat lowered, owing to the existence of unpredictable ripple current and ambient temperature variations. In this paper, we build an analytical ripple model to offer online aging monitoring of electrolytic capacitors. After that, a GM(2,1) model is adopted to predict service life with higher accuracy than conventional approaches
Imaging and Pathological Features of Percutaneous Cryosurgery on Normal Lung Evaluated in a Porcine Model
Background and objective Lung cancer is one of the most commonly occurring malignancies and frequent causes of death in the world. Cryoablation is a safe and alternative treatment for unresectable lung cancer. Due to the lung being gas-containing organ and different from solid organs such as liver and pancreas, it is difficult to achieve the freezing range of beyond the tumor edge 1 cm safety border. The aim of this study is to examine the effect of different numbers of freeze cycles on the effectiveness of cryoablation on normal lung tissue and to create an operation guideline that gives the best effect. Methods Six healthy Tibetan miniature pigs were given a CT scan and histological investigation after percutaneous cryosurgery. Cryoablation was performed as 2 cycles of 10 min of active freezing in the left lung; each freeze followed by a 5 min thaw. In the right lung, we performed the same 2 cycles of 5 min of freezing followed by 5 min of thawing. However, for the right lung, we included a third cycle of consisting of 10 min of freezing followed by 5 min of thawing. Three cryoprobes were inserted into the left lung and three cryoprobes in the right lung per animal, one in the upper and two in the lower lobe, so as to be well away from each other. Comparison under the same experimental condition was necessary. During the experiment, observations were made regarding the imaging change of ice-ball. The lungs were removed postoperatively at 3 intervals: 4 h, 3 d of postoperation and 7 d of postoperation, respectively, to view microscopic and pathological change. Results The ice-ball grew gradually in relation to the increase in time, and the increase in number of cycles. The size of the cryolesion (hypothesis necrotic area) in specimens, over time, became larger in size than the size of the ice-ball during operation, regardless of whether 2 or 3 freeze-thaw cycles were performed. The area of necrosis was gradually increased over the course of time. The hypothesis necrotic area was equal to necrosis area 3 d after cryosurgery. Conclusion Percutaneous cryoablation of the lung can achieve complete ablation of target tissue. The freezing technique may be different depending on the individual circumstances of each tumor. In technology, 3 freeze-thaw cycles are recommended, and the range of cryoablation’s effective diameter may be not necessarily beyond the tumor edge at least 1 cm safe border during cryosurgery
The Effect of Raw Soybean on Oxidative Status of Digestive Organs in Mice
The present study was undertaken to specify the effect of raw soybean on oxidative status of digestive organs in mice. For this purpose, thirty male (C57BL/6J) mice were randomly divided into three groups and fed on different diets as follows: Group 1 was fed on control diet, Group 2 was fed on raw soybean diet and Group 3 was fed on raw soybean diet supplemented with 30 mg/kg cysteamine. After two weeks of feeding, duodenum, liver and pancreas samples were collected to measure oxidative and antioxidative parameters. The results show that ingestion of raw soybean markedly increased contents of superoxide anion and malondialdehyde (MDA) and activity of inducible nitric oxide synthase (iNOS), decreased activity of superoxide dismutase (SOD), T-AOC and content of reduced glutathione (GSH) in digestive organs of mice (P < 0.05). In the group fed with raw soybean diet supplemented with cysteamine, oxidative stress was mitigated. However, oxidative parameter levels were still higher than those of control diet-fed group. The present study indicates that ingestion of raw soybean could result in an imbalance between oxidant and antioxidant, and thus induce oxidative stress in digestive organs of mice
Distribution of HLA-A, -B and -DRB1 Genes and Haplotypes in the Tujia Population Living in the Wufeng Region of Hubei Province, China
BACKGROUND: The distribution of HLA alleles and haplotypes varies widely between different ethnic populations and geographic areas. Before any genetic marker can be used in a disease-associated study it is therefore essential to investigate allelic frequencies and establish a genetic database. METHODOLOGY/PRINCIPAL FINDINGS: This is the first report of HLA typing in the Tujia group using the Luminex HLA-SSO method HLA-A, -B and -DRB1 allelic distributions were determined in 124 unrelated healthy Tujia individuals, and haplotypic frequencies and linkage disequilibrium parameters were estimated using the maximum-likelihood method. In total 10 alleles were detected at the HLA-A locus, 21 alleles at the HLA-B locus and 14 alleles at the HLA-DRB1 locus. The most frequently observed alleles in the HLA-I group were HLA-A*02 (35.48%), A*11 (28.23%), A*24 (15.73%); HLA-B*40 (25.00%), B*46 (16.13%), and B*15 (15.73%). Among HLA-DRB1 alleles, high frequencies of HLA-DRB1*09 (25.81%) were observed, followed by HLA-DRB1*15 (12.9%), and DRB1*12 (10.89%). The two-locus haplotypes at the highest frequency were A*02-B*46A (8.47%), followed by A*11-B*40 (7.66%), A*02-B*40 (8.87%), A*11-B*15 (6.45%), A*02-B*15 (6.05%), B*40-DRB1*09 (9.27%) and B*46-DRB1*09 (6.45%). The most common three-locus haplotypes found in the Tujia population were A*02-B*46-DRB1*09 (4.84%) and A*02-B*40-DRB1*09 (4.03%). Fourteen two-loci haplotypes had significant linkage disequilibrium. Construction of a neighbor-joining phylogenetic tree and principal component analysis using the allelic frequencies at HLA-A was performed to compare the Tujia group and twelve other previously reported populations. The Tujia population in the Wufeng of Hubei Province had the closest genetic relationship with the central Han population, and then to the Shui, the Miao, the southern Han and the northern Han ethnic groups. CONCLUSIONS/SIGNIFICANCE: These results will become a valuable source of data for tracing population migration, planning clinical organ transplantation, carrying out HLA-linked disease-associated studies and forensic identification
- …