Pub Date : 2020-12-18DOI: 10.4236/ajmb.2021.111001
I. Rosales, Laura Salazar, Daniel Luna, A. Negrón, I. Bdikin, B. Rodriguez, A. Heredia
Investigating amyloid nanofibril self-assembly, with an emphasis on the electromechanical property of amyloid peptides, namely, piezoelectricity, may have several important implications: 1) the self-assembly process can hinder the biological stability and give rise to the formation of amyloid structures associated with neurodegenerative diseases; 2) investigations in this field may lead to an improved understanding of high-performance, functional biological nanomaterials, 3) new technologies could be established based on peptide self-assembly and the resultant functional properties, e.g., in the creation of a piezoelectric device formed with vertical diphenylalanine peptide tubes as a piezoelectric biosensor, and 4) new knowledge can be generated about neurodegenerative disorders, potentially yielding new therapies. Therefore, in this review, we will present the current investigations associated with self-assembly of amyloid-beta, the mechanisms that generate new structures, as well as theoretical calculations exploring the functionality of the structures under physiological pressure and electric field.
{"title":"Self-Assembly of Amyloid-Beta and Its Piezoelectric Properties","authors":"I. Rosales, Laura Salazar, Daniel Luna, A. Negrón, I. Bdikin, B. Rodriguez, A. Heredia","doi":"10.4236/ajmb.2021.111001","DOIUrl":"https://doi.org/10.4236/ajmb.2021.111001","url":null,"abstract":"Investigating amyloid nanofibril self-assembly, with an emphasis on the electromechanical property of amyloid peptides, namely, piezoelectricity, may have several important implications: 1) the self-assembly process can hinder the biological stability and give rise to the formation of amyloid structures associated with neurodegenerative diseases; 2) investigations in this field may lead to an improved understanding of high-performance, functional biological nanomaterials, 3) new technologies could be established based on peptide self-assembly and the resultant functional properties, e.g., in the creation of a piezoelectric device formed with vertical diphenylalanine peptide tubes as a piezoelectric biosensor, and 4) new knowledge can be generated about neurodegenerative disorders, potentially yielding new therapies. Therefore, in this review, we will present the current investigations associated with self-assembly of amyloid-beta, the mechanisms that generate new structures, as well as theoretical calculations exploring the functionality of the structures under physiological pressure and electric field.","PeriodicalId":65391,"journal":{"name":"美国分子生物学期刊(英文)","volume":"1 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2020-12-18","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"42052727","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2020-09-09DOI: 10.4236/ajmb.2020.104017
H. Ahmed, Nassreldeen Khalid Abdelrahman, M. A. Adam
Objective: To determine the prevalence of human metapneumovirus (hMPV) among children with acute respiratory tract infection and the correlation between (hMPV) and age group. Methods: This prospective, descriptive cross-sectional hospital-based study is carried out from January to May 2019 among children from under five years old who were admitted to El-Obeid pediatric teaching Hospital, North kordofan, Sudan with acute respiratory tract infection. Results: Fifty hospitalized children with acute respiratory tract infection were enrolled in the study. Two of these (4%) were tested positive for (hMPV) in the age group (7 months - 2 years) old. Moreover, the study showed no significant correlation between infection with (hMPV) and age.
{"title":"The Prevalence of Human Metapneumovirus among Children with Acute Respiratory Tract Infection in North Kordofan State, Sudan","authors":"H. Ahmed, Nassreldeen Khalid Abdelrahman, M. A. Adam","doi":"10.4236/ajmb.2020.104017","DOIUrl":"https://doi.org/10.4236/ajmb.2020.104017","url":null,"abstract":"Objective: To determine the prevalence of human metapneumovirus (hMPV) among children with acute respiratory tract infection and the correlation between (hMPV) and age group. Methods: This prospective, descriptive cross-sectional hospital-based study is carried out from January to May 2019 among children from under five years old who were admitted to El-Obeid pediatric teaching Hospital, North kordofan, Sudan with acute respiratory tract infection. Results: Fifty hospitalized children with acute respiratory tract infection were enrolled in the study. Two of these (4%) were tested positive for (hMPV) in the age group (7 months - 2 years) old. Moreover, the study showed no significant correlation between infection with (hMPV) and age.","PeriodicalId":65391,"journal":{"name":"美国分子生物学期刊(英文)","volume":"10 1","pages":"259-264"},"PeriodicalIF":0.0,"publicationDate":"2020-09-09","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"49149239","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2020-09-09DOI: 10.4236/ajmb.2020.104019
K. Tun, A. Z. Latt, Win Naing, S. Htwe, Y. K. Ko, W. Mar, S. Hlaing, Wai Wai Han, S. Win
Myeloproliferative neoplasms (MPNs) are a group of clonal haematopoietic stem cell disorders characterized by the proliferation of one or more myeloid cell lineages. According to WHO classification, the Janus associated kinase 2 (JAK2) V617F mutation is one of the major diagnostic criteria in BCR-ABL1 negative myeloproliferative neoplasms. The aim of this study is to detect the JAK2 (V617F) mutation in patients with myeloproliferative neoplasms to get accurate diagnosis and proper management. A total of 90 clinically diagnosed MPN patients attending to Department of Clinical Haematology, Yangon General Hospital were enrolled in this study. The mean age was 53.4 ± 14 years which ranged from 16 to 81 years old and male and female ratio was 2.4:1. The identification of JAK2 (V617F) point mutation was found to be positive in 44/90 MPN patients (48.9%). According to MPN subtypes, the JAK2 mutation positivity was found in 19 out of 46 polycythemia vera patients (41.3%), 17 out of 25 essential thrombocythemia patients (68%), 8 out of 15 primary myelofibrosis patients (53.3%), 0 of 4 others myeloproliferative neoplasms (0%). Confirmation of each of nine JAK2 mutation positive and negative samples was done by Sanger sequencing. The arterial or venous thrombotic attack was found in 32/44 JAK2 mutation positive cases (72.7%) and 12/44 JAK2 mutation negative cases (27.3%). The association between thrombotic attack and presence of JAK2 mutation was statistically significance with p = 0.000. The diagnosis of myeloproliferative neoplasms mainly relies on the molecular genetics according to WHO classification. The Allele specific PCR reaction is sensitive, simple test and relatively cost-effective. Therefore, the identification of JAK2 (V617F) somatic point mutation by AS-PCR should be implemented as a routine diagnosis procedure for patients with chronic and suspected myeloproliferative neoplasms.
{"title":"Identification of JAK2 (V617F) Mutation in Myeloproliferative Neoplasms by Using Allele Specific Polymerase Chain Reaction (AS-PCR)","authors":"K. Tun, A. Z. Latt, Win Naing, S. Htwe, Y. K. Ko, W. Mar, S. Hlaing, Wai Wai Han, S. Win","doi":"10.4236/ajmb.2020.104019","DOIUrl":"https://doi.org/10.4236/ajmb.2020.104019","url":null,"abstract":"Myeloproliferative neoplasms (MPNs) are a group of clonal haematopoietic stem cell disorders characterized by the proliferation of one or more myeloid cell lineages. According to WHO classification, the Janus associated kinase 2 (JAK2) V617F mutation is one of the major diagnostic criteria in BCR-ABL1 negative myeloproliferative neoplasms. The aim of this study is to detect the JAK2 (V617F) mutation in patients with myeloproliferative neoplasms to get accurate diagnosis and proper management. A total of 90 clinically diagnosed MPN patients attending to Department of Clinical Haematology, Yangon General Hospital were enrolled in this study. The mean age was 53.4 ± 14 years which ranged from 16 to 81 years old and male and female ratio was 2.4:1. The identification of JAK2 (V617F) point mutation was found to be positive in 44/90 MPN patients (48.9%). According to MPN subtypes, the JAK2 mutation positivity was found in 19 out of 46 polycythemia vera patients (41.3%), 17 out of 25 essential thrombocythemia patients (68%), 8 out of 15 primary myelofibrosis patients (53.3%), 0 of 4 others myeloproliferative neoplasms (0%). Confirmation of each of nine JAK2 mutation positive and negative samples was done by Sanger sequencing. The arterial or venous thrombotic attack was found in 32/44 JAK2 mutation positive cases (72.7%) and 12/44 JAK2 mutation negative cases (27.3%). The association between thrombotic attack and presence of JAK2 mutation was statistically significance with p = 0.000. The diagnosis of myeloproliferative neoplasms mainly relies on the molecular genetics according to WHO classification. The Allele specific PCR reaction is sensitive, simple test and relatively cost-effective. Therefore, the identification of JAK2 (V617F) somatic point mutation by AS-PCR should be implemented as a routine diagnosis procedure for patients with chronic and suspected myeloproliferative neoplasms.","PeriodicalId":65391,"journal":{"name":"美国分子生物学期刊(英文)","volume":"10 1","pages":"273-282"},"PeriodicalIF":0.0,"publicationDate":"2020-09-09","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"43417587","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2020-09-09DOI: 10.4236/ajmb.2020.104018
Leticia Surian Batalini, S. Castro, Carla Geórgia Rodrigues Guimarães Souto, H. Neitzke-Abreu, M. Lima-Júnior
There are several methods used to obtain DNA from cells; however, the quantity, integrity, and purity of DNA vary among the methods, which may interfere with the polymerase chain reaction (PCR) results. The objective was to determine the most efficient and cost-effective method that provides the best DNA yield and PCR results. Three methods of DNA isolation were compared: 20% sodium dodecyl sulfate (SDS), guanidine isothiocyanate-phenol-chloroform (GTPC), and DNA extraction using a commercial kit (GE Healthcare GenomicPrep Blood DNA Isolation KitTM). Human peripheral blood samples were inoculated with 104 promastigotes of Leishmania infantum. DNA was quantified and PCR was performed with 13A/13B primers. The results showed that a higher DNA yield was obtained using the GTPC technique (214.51 ng/μL), followed by SDS (26.16 ng/μL) and the commercial kit (10.99 ng/μL). We concluded that while all of the techniques were effective for obtaining DNA, the GTPC method provided the best yield and the brightest bands.
有几种方法可以用来从细胞中获得DNA；然而，DNA的数量、完整性和纯度因方法而异，这可能会干扰聚合酶链式反应（PCR）的结果。目的是确定提供最佳DNA产量和PCR结果的最有效和最具成本效益的方法。比较了三种DNA分离方法：20%十二烷基硫酸钠（SDS）、异硫氰酸胍苯酚氯仿（GTPC）和使用商业试剂盒（GE Healthcare GenomicPrep Blood DNA isolation KitTM）提取DNA。用104个婴儿利什曼原虫前体接种人外周血样本。对DNA进行定量，并用13A/13B引物进行PCR。结果表明，GTPC技术的DNA产量较高（214.51ng/ml），SDS（26.16ng/ml）和商业试剂盒（10.99ng/ml）次之。我们得出的结论是，虽然所有的技术都能有效地获得DNA，但GTPC方法提供了最好的产量和最亮的条带。
{"title":"Evaluation of DNA Extraction Methods for Detection of Leishmania by Polymerase Chain Reaction","authors":"Leticia Surian Batalini, S. Castro, Carla Geórgia Rodrigues Guimarães Souto, H. Neitzke-Abreu, M. Lima-Júnior","doi":"10.4236/ajmb.2020.104018","DOIUrl":"https://doi.org/10.4236/ajmb.2020.104018","url":null,"abstract":"There are several methods used to obtain DNA from cells; however, the quantity, integrity, and purity of DNA vary among the methods, which may interfere with the polymerase chain reaction (PCR) results. The objective was to determine the most efficient and cost-effective method that provides the best DNA yield and PCR results. Three methods of DNA isolation were compared: 20% sodium dodecyl sulfate (SDS), guanidine isothiocyanate-phenol-chloroform (GTPC), and DNA extraction using a commercial kit (GE Healthcare GenomicPrep Blood DNA Isolation KitTM). Human peripheral blood samples were inoculated with 104 promastigotes of Leishmania infantum. DNA was quantified and PCR was performed with 13A/13B primers. The results showed that a higher DNA yield was obtained using the GTPC technique (214.51 ng/μL), followed by SDS (26.16 ng/μL) and the commercial kit (10.99 ng/μL). We concluded that while all of the techniques were effective for obtaining DNA, the GTPC method provided the best yield and the brightest bands.","PeriodicalId":65391,"journal":{"name":"美国分子生物学期刊(英文)","volume":" ","pages":""},"PeriodicalIF":0.0,"publicationDate":"2020-09-09","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"47967756","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2020-06-01DOI: 10.4236/ajmb.2020.103010
J. Kusnadi, N. Ashari, E. L. Arumingtyas
Polymerase Chain Reaction (PCR) is an accurate, simple and fast analytical method. This technique is widely used in the identification of meat adulteration and meat-based processed food products. Three Mitochondrial DNA (mt-DNA) primers NADH Dehydrogenase sub unit 5 (ND5), D-Loop, and Cytochrome b (Cyt-b) were tested for their specificity in detecting of pig (Sus scrofa) DNA fragments. DNA genome from 6 meat samples (pork, beef, goat, lamb, and chicken) was amplified by PCR technique using three pairs of primers (ND5, D-Loop, and Cyt-b) and sequenced. The results of amplification using the three primers produced specific DNA bands with the lengths of 232 bp, 951 bp, and 404 bp, respectively. Comparison results with ND5, D-Loop, and Cyt-b gene sequences resulted in similarity values of 100%, 97%, and 99%, respectively. These showed that the mt-DNA primers of ND5, D-Loop, and Cyt-b genes can be recommended as specific primers in detecting pig (Sus scrofa) DNA fragments.
{"title":"Specificity of Various Mitochondrial DNA (mtDNA), ND5, D-Loop, and Cty-b DNA Primers in Detecting Pig (Sus scrofa) DNA Fragments","authors":"J. Kusnadi, N. Ashari, E. L. Arumingtyas","doi":"10.4236/ajmb.2020.103010","DOIUrl":"https://doi.org/10.4236/ajmb.2020.103010","url":null,"abstract":"Polymerase Chain Reaction (PCR) is an accurate, simple and fast analytical method. This technique is widely used in the identification of meat adulteration and meat-based processed food products. Three Mitochondrial DNA (mt-DNA) primers NADH Dehydrogenase sub unit 5 (ND5), D-Loop, and Cytochrome b (Cyt-b) were tested for their specificity in detecting of pig (Sus scrofa) DNA fragments. DNA genome from 6 meat samples (pork, beef, goat, lamb, and chicken) was amplified by PCR technique using three pairs of primers (ND5, D-Loop, and Cyt-b) and sequenced. The results of amplification using the three primers produced specific DNA bands with the lengths of 232 bp, 951 bp, and 404 bp, respectively. Comparison results with ND5, D-Loop, and Cyt-b gene sequences resulted in similarity values of 100%, 97%, and 99%, respectively. These showed that the mt-DNA primers of ND5, D-Loop, and Cyt-b genes can be recommended as specific primers in detecting pig (Sus scrofa) DNA fragments.","PeriodicalId":65391,"journal":{"name":"美国分子生物学期刊(英文)","volume":" ","pages":""},"PeriodicalIF":0.0,"publicationDate":"2020-06-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"46475314","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2020-06-01DOI: 10.4236/ajmb.2020.103011
I. Eto
Introduction. The molecular biological mechanism of the increased incidence of the various types of cancer in obesity or type 2 diabetes in rodents or humans has largely been resolved in recent years. By contrast, the molecular biological mechanism of the decreased, not increased, incidence of the various types of cancer in the homozygous long-lived Ames dwarf mice still remains unresolved. Objective. The first objective of the present study was to investigate whether the decrease in the incidence of cancer in the homozygous long-lived Ames dwarf mice is due to the increase, not decrease, in the expression of p27Kip1, a cell cycle repressor protein. The second objective was to investigate whether the decrease in the incidence of cancer in the homozygous long-lived Ames dwarf mice is due to the decrease, not increase, in the levels of glucose or insulin. Methods. To achieve these objectives, we first performed western immunoblot analysis of the hepatic expression of p27Kip1 protein. We then performed, using a human breast cancer cell line in vitro, the luciferase reporter plasmid assay to determine whether the translation initiation activity of the p27Kip1 mRNA is increased when the concentrations of either glucose or insulin are decreased. Results and Conclusion. The results of the first objective indicated that the hepatic expression of p27Kip1 protein was up-regulated in the homozygous long-lived Ames dwarf mice as expected. We also found that the lower concentrations of glucose or insulin increased the translation initiation activity of the p27Kip1 mRNA.
{"title":"Lower Concentrations of Glucose or Insulin Decrease the Risk of Various Types of Cancer in the Long-Lived Ames Dwarf Mouse by Increasing the Expression of p27Kip1, a Cell-Cycle Repressor Protein","authors":"I. Eto","doi":"10.4236/ajmb.2020.103011","DOIUrl":"https://doi.org/10.4236/ajmb.2020.103011","url":null,"abstract":"Introduction. The molecular biological mechanism of the increased \u0000incidence of the various types of cancer in obesity or type 2 diabetes in \u0000rodents or humans has largely been resolved in recent years. By contrast, the \u0000molecular biological mechanism of the decreased, not increased, incidence of \u0000the various types of cancer in the homozygous long-lived Ames dwarf mice still \u0000remains unresolved. Objective. The first objective of the present study \u0000was to investigate whether the decrease in the incidence of cancer in the homozygous \u0000long-lived Ames dwarf mice is due to the increase, not decrease, in the \u0000expression of p27Kip1, a cell cycle repressor protein. The second objective was \u0000to investigate whether the decrease in the incidence of cancer in the \u0000homozygous long-lived Ames dwarf mice is due to the decrease, not increase, in \u0000the levels of glucose or insulin. Methods. To achieve these objectives, \u0000we first performed western immunoblot analysis of the hepatic expression of \u0000p27Kip1 protein. We then performed, using a human breast cancer cell line in vitro, \u0000the luciferase reporter plasmid assay to determine whether the translation \u0000initiation activity of the p27Kip1 mRNA is increased when the concentrations of \u0000either glucose or insulin are decreased. Results and Conclusion. The \u0000results of the first objective indicated that the hepatic expression of p27Kip1 \u0000protein was up-regulated in the homozygous long-lived Ames dwarf mice as \u0000expected. We also found that the lower concentrations of glucose or insulin \u0000increased the translation initiation activity of the p27Kip1 mRNA.","PeriodicalId":65391,"journal":{"name":"美国分子生物学期刊(英文)","volume":"30 1","pages":"148-164"},"PeriodicalIF":0.0,"publicationDate":"2020-06-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"70513533","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2020-06-01DOI: 10.4236/ajmb.2020.103016
N. Sharif, M. Mehmood, S. Naqvi, Huma Anwar ul-Haq, S. S. Ahmed, M. Ghani, M. Shoaib, M. Hussain
Hydro-Pericardium Hepatitis (HPH) is an emerging infectious disease of commercial poultry, caused by different serotypes of Fowl Adeno Virus. The vertical transmission of the virus into the progeny may results in devastating damage, causing huge economic losses to its farmers. In present study, molecular typing is performed on basis of partially conserved hexon gene sequences, using a unique set of primers having common reverse oligo for simultaneous detection of FAdV1, FAdV-4 and FAdV-11. A total of 14 fowl adeno virus strains were isolated from 100 suspected adeno virus liver samples, collected from different districts in Pakistan, between 2018 and 2019. FASTA’s sequence alignment and phylogenetic analysis revealed that out of the 14, one isolate which belonged to group A showed 27% similarity with FAdV-1, while three isolates showed 99%, 95% & 45% similarity to FAdV-4 (Group C). Whereas, ten isolates showed more than 99% similarity to FAdV-11 (Group D). The serotypes FAdV1, FAdV-4 and FAdV-11 are prevailing in the breeder and broilers. These results hold great importance in rapid, reliable and simultaneous detection of the three FAdV serotypes. Therefore, fowl adeno virus vaccine production for commercial poultry shall be according to the prevalent field serotypes.
{"title":"PCR Based Detection and Phylogenetic Analysis of Fowl Adenovirus Strains Isolated from 2019 Epidemic from Punjab and Sindh, Pakistan","authors":"N. Sharif, M. Mehmood, S. Naqvi, Huma Anwar ul-Haq, S. S. Ahmed, M. Ghani, M. Shoaib, M. Hussain","doi":"10.4236/ajmb.2020.103016","DOIUrl":"https://doi.org/10.4236/ajmb.2020.103016","url":null,"abstract":"Hydro-Pericardium Hepatitis (HPH) is an emerging infectious disease of commercial poultry, caused by different serotypes of Fowl Adeno Virus. The vertical transmission of the virus into the progeny may results in devastating damage, causing huge economic losses to its farmers. In present study, molecular typing is performed on basis of partially conserved hexon gene sequences, using a unique set of primers having common reverse oligo for simultaneous detection of FAdV1, FAdV-4 and FAdV-11. A total of 14 fowl adeno virus strains were isolated from 100 suspected adeno virus liver samples, collected from different districts in Pakistan, between 2018 and 2019. FASTA’s sequence alignment and phylogenetic analysis revealed that out of the 14, one isolate which belonged to group A showed 27% similarity with FAdV-1, while three isolates showed 99%, 95% & 45% similarity to FAdV-4 (Group C). Whereas, ten isolates showed more than 99% similarity to FAdV-11 (Group D). The serotypes FAdV1, FAdV-4 and FAdV-11 are prevailing in the breeder and broilers. These results hold great importance in rapid, reliable and simultaneous detection of the three FAdV serotypes. Therefore, fowl adeno virus vaccine production for commercial poultry shall be according to the prevalent field serotypes.","PeriodicalId":65391,"journal":{"name":"美国分子生物学期刊(英文)","volume":" ","pages":""},"PeriodicalIF":0.0,"publicationDate":"2020-06-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"49564010","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2020-06-01DOI: 10.4236/ajmb.2020.103013
Tombari Pius Monsi, A. Ben-Chioma, Donaltus Onukwufor Onwuli
Background: Overexpression of efflux pumps is the drug resistance and adaptation mechanism employed by some eukaryotes and bacteria to transport endogenous and chemotherapeutic compounds from the intracellular to the extracellular environment. Aim: The study aimed at establishing a fluorescent cell-based assay to monitor the efflux activities of an ABC-transporter, multi-drug resistance protein 4 (MRP4). Methods: DH5α competent E. coli cells were transformed with pcDNA-MRP4 by the heat-shock process. The presence of the MRP4 gene was analyzed by the digestion of plasmid using EcoRI and analyzed on a 1% agarose gel. HEK 293 cells were transfected with purified pcDNA-MRP4 under optimized conditions using a Polyethylenimine (PEI) protocol. The level of MRP4 in the HEK 293 cells was characterized by western blotting analysis using M4I-10 anti-MRP4 and anti-Rat IgG (whole molecule)-Alkaline phosphatase antibodies. The fluorescent uptake study was performed by the incubation of 0.02 mM 8-[fluo-cAMP] with the MRP4-transfected and control HEK 293 cells for 1 h. The level of fluorescence was analyzed using fluorescence microscopy and spectrometer. Results: The agarose gel analysis showed a plasmid of 9.4 kb and restriction product of 5 kb, which correspond with the pcDNA and MRP4 sizes respectively. The western blot results of the transfection showed 4 μg pcDNA-MRP4 and the N/P ratio of 9 was the optimized condition to transfect our HEK 293 cells as it showed the broadest band. In the efflux studies, the fluorescence images of the MRP4-transfected HEK 293 cells were very low compared to the untransfected control. The level of fluorescence accumulation was significantly (P ≤ 0.0001) higher 228.6 ± 13.1 RFU in the untransfected cells than the MRP4-transfected cells 8.6 ± 1.8 RFU. Conclusion: The higher levels of fluorescence detected in the control in both the fluorescent microscopy and spectrophotometer showed that MRP4-transfected cells had effluxed the 8-[fluo-cAMP] substrate out of the cell. This method could be employed in the detection of MRP4 functions in bacteria and cancer cells.
{"title":"A Fluorescent Cell-Based Technique for Monitoring Efflux of MRP4","authors":"Tombari Pius Monsi, A. Ben-Chioma, Donaltus Onukwufor Onwuli","doi":"10.4236/ajmb.2020.103013","DOIUrl":"https://doi.org/10.4236/ajmb.2020.103013","url":null,"abstract":"Background: Overexpression of efflux pumps is the drug resistance and adaptation mechanism employed by some eukaryotes and bacteria to transport endogenous and chemotherapeutic compounds from the intracellular to the extracellular environment. Aim: The study aimed at establishing a fluorescent cell-based assay to monitor the efflux activities of an ABC-transporter, multi-drug resistance protein 4 (MRP4). Methods: DH5α competent E. coli cells were transformed with pcDNA-MRP4 by the heat-shock process. The presence of the MRP4 gene was analyzed by the digestion of plasmid using EcoRI and analyzed on a 1% agarose gel. HEK 293 cells were transfected with purified pcDNA-MRP4 under optimized conditions using a Polyethylenimine (PEI) protocol. The level of MRP4 in the HEK 293 cells was characterized by western blotting analysis using M4I-10 anti-MRP4 and anti-Rat IgG (whole molecule)-Alkaline phosphatase antibodies. The fluorescent uptake study was performed by the incubation of 0.02 mM 8-[fluo-cAMP] with the MRP4-transfected and control HEK 293 cells for 1 h. The level of fluorescence was analyzed using fluorescence microscopy and spectrometer. Results: The agarose gel analysis showed a plasmid of 9.4 kb and restriction product of 5 kb, which correspond with the pcDNA and MRP4 sizes respectively. The western blot results of the transfection showed 4 μg pcDNA-MRP4 and the N/P ratio of 9 was the optimized condition to transfect our HEK 293 cells as it showed the broadest band. In the efflux studies, the fluorescence images of the MRP4-transfected HEK 293 cells were very low compared to the untransfected control. The level of fluorescence accumulation was significantly (P ≤ 0.0001) higher 228.6 ± 13.1 RFU in the untransfected cells than the MRP4-transfected cells 8.6 ± 1.8 RFU. Conclusion: The higher levels of fluorescence detected in the control in both the fluorescent microscopy and spectrophotometer showed that MRP4-transfected cells had effluxed the 8-[fluo-cAMP] substrate out of the cell. This method could be employed in the detection of MRP4 functions in bacteria and cancer cells.","PeriodicalId":65391,"journal":{"name":"美国分子生物学期刊(英文)","volume":" ","pages":""},"PeriodicalIF":0.0,"publicationDate":"2020-06-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"43000818","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2020-06-01DOI: 10.4236/ajmb.2020.103009
Chinwe Obiomah, G. Amilo, Israel Ndulue
Hepatitis B is an infectious disease of great public health importance. Nigeria is one of the countries with the highest incidence of Hepatitis B Virus (HBV) infection worldwide. However, the accessibility and affordability of HBV DNA quantification (viral load) assay is the key laboratory test for therapy initiation, and monitoring is a challenge to HBV management. This study aimed at determining the relationship between HBV DNA quantification and routine haemato-serological parameters in order to develop a more cost-effective diagnostic algorithm for Hepatitis B management. Cross sectional study design was used with a total of 264 subjects comprising of 88 HBsAg seropositive treatment naïve subjects, 88 HBsAg seropositive subjects on antiviral therapy as case subjects and 88 age-matched apparently healthy HBsAg seronegative individuals were recruited as control subjects. Hepatitis B Virus DNA assay was performed using real time PCR technique while ELISA technique was used for Hepatitis B surface antigen quantification. HBsAg quantification showed strong positive correlation with HBV DNA viral load both in treatment and non-treatment groups (r = 0.673; p
{"title":"Evaluation of HBsAg Quantification as Surrogate to HBV DNA Viral Load in Hepatitis B Infected Patients in Anambra State, Nigeria","authors":"Chinwe Obiomah, G. Amilo, Israel Ndulue","doi":"10.4236/ajmb.2020.103009","DOIUrl":"https://doi.org/10.4236/ajmb.2020.103009","url":null,"abstract":"Hepatitis B is an infectious disease of great public health importance. Nigeria \u0000is one of the countries with the highest incidence of Hepatitis B Virus (HBV) \u0000infection worldwide. However, the accessibility and affordability of HBV \u0000DNA quantification (viral load) assay is the key laboratory test for therapy initiation, \u0000and monitoring is a challenge to HBV management. This study \u0000aimed at determining the relationship between HBV DNA quantification \u0000and routine haemato-serological parameters in order to develop a more \u0000cost-effective diagnostic algorithm for Hepatitis B management. Cross \u0000sectional study design was used with a total of 264 subjects comprising of 88 \u0000HBsAg seropositive treatment naïve subjects, 88 HBsAg seropositive subjects \u0000on antiviral therapy as case subjects and 88 age-matched apparently healthy \u0000HBsAg seronegative individuals were recruited as control subjects. Hepatitis \u0000B Virus DNA assay was performed using real time PCR technique while \u0000ELISA technique was used for Hepatitis B surface antigen quantification. \u0000HBsAg quantification showed strong positive correlation with HBV DNA \u0000viral load both in treatment and non-treatment groups (r = 0.673; p","PeriodicalId":65391,"journal":{"name":"美国分子生物学期刊(英文)","volume":" ","pages":""},"PeriodicalIF":0.0,"publicationDate":"2020-06-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"42814861","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
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