Magnetic resonance imaging focused on the ferritin heavy chain 1 reporter gene detects neuronal differentiation in stem cells

The neuronal differentiation of mesenchymal stem cells offers a new strategy for the treatment of neurological disorders.Thus,there is a need to identify a noninvasive and sensitive in vivo imaging approach for real-time monitoring of transplanted stem cells.Our previous study confirmed that magnetic resonance imaging,with a focus on the ferritin heavy chain 1 reporter gene,could track the proliferation and differentiation of bone marrow mesenchymal stem cells that had been transduced with lentivirus carrying the ferritin heavy chain 1 reporter gene.However,we could not determine whether or when bone marrow mesenchymal stem cells had undergone neuronal differentiation based on changes in the magnetic resonance imaging signal.To solve this problem,we identified a neuron-specific enolase that can be differentially expressed before and after neuronal differentiation in stem cells.In this study,we successfully constructed a lentivirus carrying the neuron-specific enolase promoter and expressing the ferritin heavy chain 1 reporter gene;we used this lentivirus to transduce bone marrow mesenchymal stem cells.Cellular and animal studies showed that the neuron-specific enolase promoter effectively drove the expression of ferritin heavy chain 1 after neuronal differentiation of bone marrow mesenchymal stem cells;this led to intracellular accumulation of iron and corresponding changes in the magnetic resonance imaging signal.In summary,we established an innovative magnetic resonance imaging approach focused on the induction of reporter gene expression by a neuron-specific promoter.This imaging method can be used to noninvasively and sensitively detect neuronal differentiation in stem cells,which may be useful in stem cell-based therapies.

Identifying the stability of housekeeping genes to be used for the quantitative real-time PCR normalization in retinal tissue of streptozotocin-induced diabetic rats

AIM:To investigate the stability of the seven housekeeping genes:beta-actin(ActB),glyceraldehyde-3-phosphate dehydrogenase(GAPDH),18s ribosomal unit 5(18s),cyclophilin A(CycA),hypoxanthine-guanine phosphoribosyl transferase(HPRT),ribosomal protein large P0(36B4)and terminal uridylyl transferase 1(U6)in the diabetic retinal tissue of rat model.METHODS:The expression of these seven genes in rat retinal tissues was determined using real-time quantitative reverse transcription polymerase chain reaction(RT-qPCR)in two groups;normal control rats and streptozotocininduced diabetic rats.The stability analysis of gene expression was investigated using geNorm,NormFinder,BestKeeper,and comparative delta-Ct(ΔCt)algorithms.RESULTS:The 36B4 gene was stably expressed in the retinal tissues of normal control animals;however,it was less stable in diabetic retinas.The 18s gene was expressed consistently in both normal control and diabetic rats’retinal tissue.That this gene was the best reference for data normalisation in RT-qPCR studies that used the retinal tissue of streptozotocin-induced diabetic rats.Furthermore,there was no ideal gene stably expressed for use in all experimental settings.CONCLUSION:Identifying relevant genes is a need for achieving RT-qPCR validity and reliability and must be appropriately achieved based on a specific experimental setting.

Analysis of the autophagy gene expression profile of pancreatic cancer based on autophagy-related protein microtubule-associated protein 1A/1B-light chain 3

BACKGROUND Pancreatic cancer is a highly invasive malignant tumor. Expression levels of the autophagy-related protein microtubule-associated protein 1 A/1 B-light chain 3(LC3) and perineural invasion(PNI) are closely related to its occurrence and development. Our previous results showed that the high expression of LC3 was positively correlated with PNI in the patients with pancreatic cancer. In this study, we further searched for differential genes involved in autophagy of pancreatic cancer by gene expression profiling and analyzed their biological functions in pancreatic cancer, which provides a theoretical basis for elucidating the pathophysiological mechanism of autophagy in pancreatic cancer and PNI.AIM To identify differentially expressed genes involved in pancreatic cancer autophagy and explore the pathogenesis at the molecular level.METHODS Two sets of gene expression profiles of pancreatic cancer/normal tissue(GSE16515 and GSE15471) were collected from the Gene Expression Omnibus.Significance analysis of microarrays algorithm was used to screen differentially expressed genes related to pancreatic cancer. Gene Ontology(GO) analysis and Kyoto Encyclopedia of Genes and Genomes(KEGG) pathway analysis were used to analyze the functional enrichment of the differentially expressed genes. Protein interaction data containing only differentially expressed genes was downloaded from String database and screened. Module mining was carried out by Cytoscape software and ClusterOne plug-in. The interaction relationship between the modules was analyzed and the pivot nodes between the functional modules were determined according to the information of the functional modules and the data of reliable protein interaction network.RESULTS Based on the above two data sets of pancreatic tissue total gene expression, 6098 and 12928 differentially expressed genes were obtained by analysis of genes with higher phenotypic correlation. After extracting the intersection of the two differential gene sets, 4870 genes were determined. GO analysis showed that 14 significant functional items including negative regulation of protein ubiquitination were closely related to autophagy. A total of 986 differentially expressed genes were enriched in these functional items. After eliminating the autophagy related genes of human cancer cells which had been defined, 347 differentially expressed genes were obtained. KEGG pathway analysis showed that the pathways hsa04144 and hsa04020 were related to autophagy. In addition,65 clustering modules were screened after the protein interaction network was constructed based on String database, and module 32 contains the LC3 gene,which interacts with multiple autophagy-related genes. Moreover, ubiquitin C acts as a pivot node in functional modules to connect multiple modules related to pancreatic cancer and autophagy.CONCLUSION Three hundred and forty-seven genes associated with autophagy in human pancreatic cancer were concentrated, and a key gene ubiquitin C which is closely related to the occurrence of PNI was determined, suggesting that LC3 may influence the PNI and prognosis of pancreatic cancer through ubiquitin C.

Study of differential polymerase chain reaction of C-erbB-2 oncogene amplification in gastric cancer

AIM To study the significance of C-erbB-2 oncogene amplification in gastric cancer.METHODS C-erbB-2 oncogene amplification was examined by using differential polymerase chain reaction (dPCR) in surgical and endoscopic specimens of 83 cases of gastric cancer and 101 metastatic lymph nodes.RESULTS C-erbB-2 amplification was found in 28.9% (24/ 83) surgical specimens and 20.5% (17/ 83) endoscopic ones of gastric cancer patients. The amplification was significant in both types of specimens of advanced cancer cases (P＜0.05) and surgical specimens with lymph node metastasis (P＜0.01). The incidence of C-erbB-2 amplification in lymph nodes with metastasis was higher than in primary sites (surgical specimens, P＜0.05). The patients with amplification tumors had poorer 5-year survival rates than those with unamplification ones in the early cancers and well to moderately differentiated adenocarcinomas (P＜0.05). The same surgical samples were tested again by Southern blot hybridization to ascertain C-erbB-2 amplification, and the positive rate of C-erbB-2 amplification (15.7%) was lower than that of dPCR (28.9%, P＜0.05).CONCLUSION Examining C-erbB-2 amplification by dPCR is a quick, simple, reliable and independent method, and is helpful in predicting prognosis and metastatic potential of gastric cancer.

Clonal immunoglobulin heavy chain and T-cell receptor γ gene rearrangements in primary gastric lymphoma

AIM:To study the diagnostic value of immunoglobulin heavy chain(IgH)and T-cell receptorγ (TCR-γ)gene monoclonal rearrangements in primary gastric lymphoma(PGL).METHODS:A total of 48 patients with suspected PGL at our hospital were prospectively enrolled in this study from January 2009 to December 2011.The patients were divided into three groups(a PGL group,a gastric linitis plastica group,and a benign gastric ulcer group)based on the pathological results(gastric mucosal specimens obtained by endoscopy or surgery)and follow-up.Endoscopic ultrasonography(EUS)and EUSguided biopsy were performed in all the patients.The tissue specimens were used for histopathological examination and for IgH and TCR-γ gene rearrangement polymerase chain reaction analyses.RESULTS:EUS and EUS-guided biopsy were successfully performed in all 48 patients.In the PGL group(n=21),monoclonal IgH gene rearrangements were detected in 14(66.7%)patients.A positive result for each set of primers was found in 12(57.1%),8(38.1%),and 4(19.0%)cases using FR1/JH,FR2/JH,and FR3/JH primers,respectively.Overall,12(75%)patients with mucosal-associated lymphoid tissue lymphoma(n=16)and 2(40%)patients with diffuse large B-cell lymphoma(n=5)were positive for monoclonal IgH gene rearrangements.No patients in the gastric linitis plastica group(n=17)and only one(10%)patient in the benign gastric ulcer group(n=10)were positive for a monoclonal IgH gene rearrangement.No TCRgene monoclonal rearrangements were detected.The sensitivity of monoclonal IgH gene rearrangements was 66.7%for a PGL diagnosis,and the specificity was96.4%.In the PGL group,8(100%)patients with stage IIE PGL(n=8)and 6(46.1%)patients with stage IE PGL(n=13)were positive for monoclonal IgH gene rearrangements.CONCLUSION:IgH gene rearrangements may be associated with PGL staging and may be useful for the diagnosis of PGL and for differentiating between PGL and gastric linitis plastica.

Vitreoscilla hemoglobin gene(vgb)improves lutein production in Chlorella vulgaris

Vitreoscilla hemoglobin is an oxygen-binding protein that promotes oxygen delivery and reduces oxygen consumption under low oxygen conditions to increase the effi ciency of cell respiration and metabolism.In this study,we introduced a Vitreoscilla hemoglobin gene(vgb)into Chlorella vulgaris by Agrobacterium tumefaciens-mediated transformation(ATMT).PCR analysis confi rmed that the vgb gene was successfully integrated into the Chlorella vulgaris genome.Analysis of biomass obtained in shake fl asks revealed transformant biomass concentrations as high as 3.28 g/L,which was 38.81% higher than that of the wild-type strain.Lutein content of transformants also increased slightly.Further experiments recovered a maximum lutein yield of 2.91 mg/L from the transformants,which was 36.77% higher than that of the wild-type strain.The above results suggest that integrated expression of the vgb gene may improve cell growth and lutein yield in Chlorella vulgaris,with applications to lutein production from Chlorella during fermentation.

Application of Landscape Gene Information Chain in Tourism Development of Traditional Human Settlements:A Case Study of Daqitou Village in Sanshui District,Foshan City

Based on the aspect of historical and cultural geography, the value of "landscape gene information chain" in the tourism development of ancient villages was verified by taking cultural landscape as the principal line, historical and cultural settlement as the carriers, and traditional buildings as the entry points. Taking Daqintou Village in Sanshui District, Foshan City for example, the theory of "landscape gene information chain" was applied to design a tourism planning scheme for Daqitou Village.

Antibiotic susceptibility and molecular characterization of resistance genes among Escherichia coli and among Salmonella subsp. in chicken food chains

Objective: To investigate the occurrence of resistance genes among Escherichia coli(E. coli) and Salmonella subsp. isolated in chicken food chains in Phnom Penh, 2012–2013.Methods: Six hundred eighty two E. coli and 181 Salmonella Albany, Corvallis, and Kentucky strains were examined for susceptibilities to eight antimicrobials and following resistance genes were identified by PCR: blaTem, Str A, aad A, sul1, sul2, gyr A, Tet(A), and Tet(B).Results: E. coli presented high resistances to tetracycline, amoxicillin, and sulfamethoxazole(63.1%–76.1%). Salmonella Albany and Salmonella Kentucky traduced high resistance percentages to amoxicillin, tetracycline, sulfamethoxazole, and nalidixic acid(84.6%–100%). Among amoxicillin-resistant isolates, blaTemgenes were observed for 62% of E. coli isolates and 20% of 65 Salmonella Kentucky. The Str A gene was prevalent in 36% of 331 aminoglycoside-resistant E. coli and 90% of 40 aminoglycoside-resistant Salmonella Corvallis. The sul2 gene was predominant among sulfamethoxazole-resistant isolates, for 56% of 431 E. coli and 53% of 66 Salmonella Corvallis; the sul1 gene was observed in 54% of Salmonella Albany. The Tet(A) resistance gene was prevalent in E.coli(86%), Salmonella Corvallis(82%), Salmonella Kentucky(84%). High percentages of gyr A genes observed among nalidixic-acid resistant E. coli(91%), Salmonella Albany(92%), Salmonella Corvallis(75%) and Salmonella Kentucky(85%).Conclusions: Important occurrences of resistance gene were observed among E. coli and Salmonella in chicken food chains in Cambodia.

Insertional mutagenesis and cloning of HIV env 125 peptide gene by polymerase chain reaction

One oligonueleotide probe,HIV env 101-1,and two oligonucleotide primers,HIV env125-1 and HIV env 125-2,were designed with the aid of a computer and synthesized by a DNAsynthesizer.The env 125 peptide gene was amplified by polymerase chain reaetion(PCR).Beingidentified by electrophoresis and Southern blotting,the PCR product was cloned into plasmidpUC 19.The recombinant pENV 125,identified with X-gal selection and restriction mapping, wassequenced.The results showed that the cloned env 125 peptide gene contained the inserted EcoRⅠ site and ATG at the 5’end,Hind Ⅲ site and TAG at the 3’end.The sequence and readingframe were proved to be correct.

Exogenous reference gene normalization for real-time reverse transcription-polymerase chain reaction analysis under dynamic endogenous transcription

Quantitative real-time reverse transcription-polymerase chain reaction （qPCR） is widely used to investigate transcriptional changes following experimental manipulations to the nervous system. Despite the widespread utilization of qPCR, the interpretation of results is marred by the lack of a suitable reference gene due to the dynamic nature of endogenous transcription. To address this inherent deficiency, we investigated the use of an exogenous spike-in mRNA, luciferase, as an internal reference gene for the 2ct normalization method. To induce dynamic transcription, we systemically administered capsaicin, a neurotoxJn selective for C-type sensory neurons expressing the TRPV-1 receptor, to adult male Sprague-Dawley rats. We later isolated nodose ganglia for qPCR analysis with the reference being either exogenous luciferase mRNA or the commonly used endogenous reference 13-111 tubulin. The exogenous luciferase mRNA reference clearly demonstrated the dynamic expression of the endogenous reference. Furthermore, variability of the endogenous reference would lead to misinterpretation of other genes of interest. In conclusion, traditional reference genes are often unstable under physiologically normal situations, and certainly unstable following the damage to the nervous system. The use of exogenous spike-in reference provides a consistent and easily implemented alternative for the analysis of qPCR data.

Two－step multiplex polymerase chain reaction for gene diagnosis of progressive pseudohypertrophic muscular dystrophy

in the present study,9 exon-containing DNA segments of dystrophin gene with 9 sets of oligonucleotide primers by two-step multiplex polymerase chain reaction (mPCR) were amplified. Subsequently,gene analysis was performed in 36 cases of Duchenne mascular dystroply (DMD) and 4 cases of Becker muscular dystrophy(BMD). The findings showed that 17 cases of deletion were detected by using the first 5 sets of primers with a relatively high incidence of deletion detection and 2 more cases of deletion were detected by using the remaining 4 sets of primers. The total deletion rate detected by mPCR with 9 cases of primers was 47. 5% of the patients examined,suggesting that about 79. 1% of the patients with gene deletion could be detected. Thus,as a preliminary screening, the two-step mPCR can be used in the gene diagnosis of DMD/BMD. The method is not only simple, convenient and rapid,but also free from radiosotope trouble.

GENE DIAGNOSIS OF HEMOGLOBINOPATHY IN CHINESE BY AMPLIFIED DNA

This paper describes the application of DNA amplification in vitro with the polymerase chain reaction on the prenatal diagnosis of thalassemia syndromes and on detection of HbS and HbD Punjab genes. DNA polymerase chain reaction was performed on lysed amniotic fluid cells or chorionic villus samples without prior DNA extraction and on DNA sampling from dried blood spots on filter paper blotters, α-thalassemia was prenatally diagnosed by direct analysis of amplified fetal target sequences on gel electrophoresis; and β-thalassemia was predicted by Hgi AI RFLP linkage analysis with amplified β-globin DNA. HbS and HbD Punjab genes were identified by Mst Ⅱor Eco RI mapping of the amplified β-globin DNA directly on the electrophoretic gels. The analysis of the amplified DNA does not require radioactive DNA probes and Southern hybridization. The total procedure can be completed within five hours.

CHARACTERIZATION OF THE β－MYOSIN HEAVY CHAIN GENE MISSENSE MUTATIONIN A CHINESE PATINENT WITH HYPERTROPHIC CARDIOMYOPHTHY

We have analyzed the exons 13, 16, 21 and 23 of cardiac myosin heavy chain gene in 32 Chinese patients with hypertrophic cardiomyopathy by using PCR-single strand conformation polymorphism (PCR-SSCP) procedure. The result showed an altered SSCP of the exon 13 in one patient. Sequencing analysis revealed that the patient had a G to T transversion in the codon 383, resulting in the substitution of Lys by Asn. Beacause the missense mutation was found at the residue highly conserved through species evolution, this mutation is likely, to be the cause of hypertrophic cardiomyopathy in this patient. This is the first report of a mutant cardiac β-MHC gene in the Chinese population. Also, it is a novel missense mutation of the cardiac β-MHC gene.

Cloning, sequencing and analyzing of the heavy chain V region genes of human polyreactive antibodies

The heavy chain variable region genes of 5 human polyreactive mAbs generated in our laboratory have been cloned and sequenced using polymerase chain reaction (PCR) technique. We found that 2 and 3 mAbs utilized genes of the VHIV and VHIII families, respectively. The former 2 VH segments were in germline configuration. A common VH segment, with the best similarity of 90.1 % to the published VHIII germline genes, was utilized by 2 different rearranged genes encoding the V regions of other 3 mAbs. This strongly suggests that the common VH segment is a unmutated copy of an unidentified germline VHIII gene. All these polyreactive mAbs displayed a large NDN region (VH-D-JH junction). The entire H chain V regions of these polyreactive mAbs are unusually basic. The analysis of the charge properties of these mAbs as well as those of other poly- and mono- reactive mAbs from literatures prompts us to propose that the charged amino acids with a particular distribution along the H chain V region,especially the binding sites (CDRs), may be an important structural feature involved in antibody polyreactivity.

Therapeutic gene editing strategies using CRISPR-Cas9 for theβ-hemoglobinopathies

With advancements in gene editing technologies,our ability to make precise and efficient modifications to the genome is increasing at a remarkable rate,paving the way for scientists and clinicians to uniquely treat a multitude of previously irremediable diseases.CRISPR-Cas9,short for clustered regularly interspaced short palindromic repeats and CRISPR-associated protein 9,is a gene editing platform with the ability to alter the nucleotide sequence of the genome in living cells.This technology is increasing the number and pace at which new gene editing treatments for genetic disorders are moving toward the clinic.Theβ-hemoglobinopathies are a group of monogenic diseases,which despite their high prevalence and chronic debilitating nature,continue to have few therapeutic options available.In this review,we will discuss our existing comprehension of the genetics and current state of treatment forβ-hemoglobinopathies,consider potential genome editing therapeutic strategies,and provide an overview of the current state of clinical trials using CRISPR-Cas9 gene editing.

Construction and Control of Genetic Regulatory Networks:A Multivariate Markov Chain Approach

In the post-genomic era, the construction and control of genetic regulatory networks using gene expression data is a hot research topic. Boolean networks (BNs) and its extension Probabilistic Boolean Networks (PBNs) have been served as an effective tool for this purpose. However, PBNs are difficult to be used in practice when the number of genes is large because of the huge computational cost. In this paper, we propose a simplified multivariate Markov model for approximating a PBN The new model can preserve the strength of PBNs, the ability to capture the inter-dependence of the genes in the network, qnd at the same time reduce the complexity of the network and therefore the computational cost. We then present an optimal control model with hard constraints for the purpose of control/intervention of a genetic regulatory network. Numerical experimental examples based on the yeast data are given to demonstrate the effectiveness of our proposed model and control policy.

Cloning and Sequencing of Kappa Light Chain Gene of a Mouse Monoclonal Antibody Directed Against Potato Virus Y

A cDNA library was constructed in λgt11 vectors, complementary to the mRNA isolated from a mouse hybridoma raised against potato virus Y(PVY). Thirty cDNA clones were selected from the cDNA library by in situ immunohybridization with goat anti-mouse kappa-chain-specific antibody conjugated to alkaline phosphatase, from which one clone, k6, having the largest insert was characterized by sequence analysis. The result shows that the immunoglobulin messenger RNA corresponding to k6 is 956 nucleotides in length excluding the poly(A) region, among which 31 bases code for the 5’ non-coding region, 57 for the leader sequence of the protein, 657 for the mature protein and 211 for the 3’ non-coding region. Comparison of deduced amino acid sequences of the protein and other kappa light chains shows that they share a 100% identity in their constant regions(CL) and 93.7% identity in their variable regions(VL). The kappa light chain encoded by k6 is considered to be specific to PVY since only one type of light chain is expressed in the hybridoma.

Detection of Gene Alteration for Color Vision Defects by Polymerase Chain Reaction

According to the fact that the abnormalities of visual pigment genes were always involved in the changing of the exon 5, two oligonucleotide primers were designed to amplify the exon 5 of red pigment gene and green pigment gene. After electrophoresis of the PCR products digested with Rsal or Sau3A, the DNA fragments from the exon 5 of red pigment gene (RPG) and green pigment gene (GPG) were separated since there are different restriction endonuclease sites. On the other hand, we analyzed the exon 5 rela...

Detection of <i>β</i>-Hemoglobin Gene and Sickle Cell Disorder from Umbilical Cord Blood

Sickle cell disease (SCD) is one of the most common hemoglobinopathies, which is caused by the replacement of glutamic acid with valine at the sixth position of the beta-globin amino acid chain which sickling of the entire red blood cells in the homozygous (Hb S/S) condition. There are many analyses and screening procedures were developed to detect sickle cell anemia in the early age of birth, especially from heel prick blood, but in case of developing countries, it would be more acceptable to detect sickle cell disorder using umbilical cord blood just after birth rather than using heel prick blood. In this study, umbilical cord blood (UCB) was used to detect β-hemoglobin gene and sickle cell disorder. Polymerase chain reaction (PCR) based analysis was done using two primers (wild-type and mutant type) to detect this disorder. A total number of 22 samples were enrolled in this experiment for PCR amplification among which nineteen samples were identified by amplification of both 267 bp and 517 bp fragments revealing heterozygous sickle cell trait (Hb A/S), whereas three samples were found to amplify of 517 bp only revealing healthy individuals. The result from PCR analysis was then collaborated with the information of the mothers of each sample to analyze the result more conveniently and found that the mothers of all individuals except the three samples had anemia or mild form of anemia, thus it was expected that the newborn might have anemia trait (Hb A/S) the exception was found in case of sample No. 9 and sample No. 15. Both samples showed the bands on 267 bp and 517 bp thus expressed the sickle cell disease trait although the mothers of these samples were not anemic. However, no samples were recorded having sickle cell anemia (9 Hb S/S). The inherent simplicity and low cost of this PCR based analysis with umbilical cord blood will be considered as an effective tool in future newborn screening in Bangladesh.

RT-qPCR normalization of reference genes in different lifehistory stages of Gracilaria vermiculophylla(Rhodophyta)

The quantitative realtime polymerase chain reaction(RT-qPCR)is a powerful and sensitive method to measure expression of targeted gene but it highly relies on the use of suitable reference genes for data normalization.We evaluated the expressions of 8 housekeeping genes:18S ribosomal rDNA(18S rDNA),28S ribosomal r DNA(28S rDNA),rubisco large subunit(rbc-L),β-actin(ACT),glyceraldehyde-3-phosphate dehydrogenase(GAPDH),elongation factor 1(EF1),β-tubulin(Tub B),and P-phycoerythrin B(PEB),to select the suitable reference genes for different life-history stages(tetrasporophyte,carposporophyte,and male/female gametophyte)of Gracilaria vermiculophylla by absolute quantitative method.Softwares geNorm and BestKeeper were used to verify the results acquired from copy number analysis.Results show that the expression of identified reference genes varied in comparing groups composed of different type of life stages.It is suggested that 18S rDNA and TubB could be used for highly complex samples composed of mixed ploidy and phases.18S rDNA and 28S rDNA were also preferred for using among the matured isomorphic samples.But for samples with different maturities,TubB and ACT were recommended for tetrasporophytes and gametophytes respectively.