Revisiting Recombination Signal in the Tick-Borne Encephalitis Virus: A Simulation Approach

PubMed Central

Johansson, Magnus; Norberg, Peter

2016-01-01

The hypothesis of wide spread reticulate evolution in Tick-Borne Encephalitis virus (TBEV) has recently gained momentum with several publications describing past recombination events involving various TBEV clades. Despite a large body of work, no consensus has yet emerged on TBEV evolutionary dynamics. Understanding the occurrence and frequency of recombination in TBEV bears significant impact on epidemiology, evolution, and vaccination with live vaccines. In this study, we investigated the possibility of detecting recombination events in TBEV by simulating recombinations at several locations on the virus’ phylogenetic tree and for different lengths of recombining fragments. We derived estimations of rates of true and false positive for the detection of past recombination events for seven recombination detection algorithms. Our analytical framework can be applied to any investigation dealing with the difficult task of distinguishing genuine recombination signal from background noise. Our results suggest that the problem of false positives associated with low detection P-values in TBEV, is more insidious than generally acknowledged. We reappraised the recombination signals present in the empirical data, and showed that reliable signals could only be obtained in a few cases when highly genetically divergent strains were involved, whereas false positives were common among genetically similar strains. We thus conclude that recombination among wild-type TBEV strains may occur, which has potential implications for vaccination with live vaccines, but that these events are surprisingly rare. PMID:27760182

How good are indirect tests at detecting recombination in human mtDNA?

PubMed

White, Daniel James; Bryant, David; Gemmell, Neil John

2013-07-08

Empirical proof of human mitochondrial DNA (mtDNA) recombination in somatic tissues was obtained in 2004; however, a lack of irrefutable evidence exists for recombination in human mtDNA at the population level. Our inability to demonstrate convincingly a signal of recombination in population data sets of human mtDNA sequence may be due, in part, to the ineffectiveness of current indirect tests. Previously, we tested some well-established indirect tests of recombination (linkage disequilibrium vs. distance using D' and r(2), Homoplasy Test, Pairwise Homoplasy Index, Neighborhood Similarity Score, and Max χ(2)) on sequence data derived from the only empirically confirmed case of human mtDNA recombination thus far and demonstrated that some methods were unable to detect recombination. Here, we assess the performance of these six well-established tests and explore what characteristics specific to human mtDNA sequence may affect their efficacy by simulating sequence under various parameters with levels of recombination (ρ) that vary around an empirically derived estimate for human mtDNA (population parameter ρ = 5.492). No test performed infallibly under any of our scenarios, and error rates varied across tests, whereas detection rates increased substantially with ρ values > 5.492. Under a model of evolution that incorporates parameters specific to human mtDNA, including rate heterogeneity, population expansion, and ρ = 5.492, successful detection rates are limited to a range of 7-70% across tests with an acceptable level of false-positive results: the neighborhood similarity score incompatibility test performed best overall under these parameters. Population growth seems to have the greatest impact on recombination detection probabilities across all models tested, likely due to its impact on sequence diversity. The implications of our findings on our current understanding of mtDNA recombination in humans are discussed.

How Good Are Indirect Tests at Detecting Recombination in Human mtDNA?

PubMed Central

White, Daniel James; Bryant, David; Gemmell, Neil John

2013-01-01

Empirical proof of human mitochondrial DNA (mtDNA) recombination in somatic tissues was obtained in 2004; however, a lack of irrefutable evidence exists for recombination in human mtDNA at the population level. Our inability to demonstrate convincingly a signal of recombination in population data sets of human mtDNA sequence may be due, in part, to the ineffectiveness of current indirect tests. Previously, we tested some well-established indirect tests of recombination (linkage disequilibrium vs. distance using D′ and r2, Homoplasy Test, Pairwise Homoplasy Index, Neighborhood Similarity Score, and Max χ2) on sequence data derived from the only empirically confirmed case of human mtDNA recombination thus far and demonstrated that some methods were unable to detect recombination. Here, we assess the performance of these six well-established tests and explore what characteristics specific to human mtDNA sequence may affect their efficacy by simulating sequence under various parameters with levels of recombination (ρ) that vary around an empirically derived estimate for human mtDNA (population parameter ρ = 5.492). No test performed infallibly under any of our scenarios, and error rates varied across tests, whereas detection rates increased substantially with ρ values > 5.492. Under a model of evolution that incorporates parameters specific to human mtDNA, including rate heterogeneity, population expansion, and ρ = 5.492, successful detection rates are limited to a range of 7−70% across tests with an acceptable level of false-positive results: the neighborhood similarity score incompatibility test performed best overall under these parameters. Population growth seems to have the greatest impact on recombination detection probabilities across all models tested, likely due to its impact on sequence diversity. The implications of our findings on our current understanding of mtDNA recombination in humans are discussed. PMID:23665874

Report on LEReC Recombination Monitor APEX Study on June 15th 2016

DOE Office of Scientific and Technical Information (OSTI.GOV)

Drees, A.; Bruno, D.; Curcio, T.

2016-12-21

During the prospective Low Energy RHIC electron Cooling (LEReC) operation, the electron beam will overlap and interact with the low energy ion beam to provide transverse cooling. Cooling is needed to facilitate reaching the BES-2 (Beam Energy Scan 2) program goals of an average store luminosity of 5 × 10 24 cm -2 s -1 at 3.85 GeV/n and 17.3 × 10 24 cm -2 s -1 at 9.1 GeV/n. The RHIC phase of BES-2 is currently planned for the RHIC runs in 2019 and 2020. Effective cooling will depend on the accuracy of velocity matching between the two beams.more » Another process, the rate of ion-electron recombination, is also maximized when the velocities are matched, but the exact matching requirement is less stringent. Therefore, as suggested by one of us, detecting and maximizing recombination signals should be helpful in finding the narrow velocity matching window conducive to cooling. When 197Au 79+ RHIC ions pick up an electron from the LEREeC electron beam they are converted into 197Au 78+ ions with nearly the same momentum while having about 1.3% higher magnetic rigidity than the original 197Au 79+ particles. The detection of the recombined ions can be done by driving the 197Au 78+ beam into the beam pipe wall, creating showers of secondary particles which then can be detected outside the cryostat by using appropriately positioned detectors. For the purpose of forcing losses of the expected off-momentum particles a dedicated lattice with large horizontal dispersion in one arc was proposed and designed.« less

Sero-detection of Toxocara canis infection in human with T.canis recombinant arginine kinase, cathepsin L-1 and TES-26 antigens.

PubMed

Varghese, Anju; Raina, Opinder K; Chandra, Dinesh; Mirdha, Bijay R; Kelawala, Naresh H; Solanki, Jayesh B; Kumar, Niranjan; Ravindran, Reghu; Arun, Anandanarayanan; Rialch, Ajayta; Lalrinkima, Hniang; Kelawala, Rohan N; Samanta, Subhamoy

2017-12-20

Three recombinant antigens viz. arginine kinase, cathepsin L-1 and TES-26 of Toxocara canis were expressed in Escherichia coli and evaluated for their potential in the detection of T. canis larval infection in human in immunoglobulin G-enzyme linked immunosorbent assay (IgG-ELISA). Results of the IgG-ELISA with the above recombinant antigens were confirmed with commercially available IgG detection kit for T. canis infection used as a standard test. All three recombinant antigens were 100% sensitive in the detection of positive cases (n = 6) of T. canis infection in human and were screened for their cross-reactivity in human patients with history of Toxoplasma gondii, Plasmodium vivax, Entamoeba histolytica, hydatid and hookworm infections. The recombinant TES-26 antigen showed higher specificity and cross-reacted with T. gondii infection sera only. However, arginine kinase and cathepsin L-1 recombinant antigens showed cross-reactions with sera of patients infected with T. gondii, P. vivax and E. histolytica but not with the patient sera infected with hydatid and hookworm. These results show that recombinant TES-26 is a potential diagnostic candidate antigen for human toxocarosis caused by migrating T. canis larvae.

Enterovirus D68 in Viet Nam (2009-2015).

PubMed

Ny, Nguyen Thi Han; Anh, Nguyen To; Hang, Vu Thi Ty; Nguyet, Lam Anh; Thanh, Tran Tan; Ha, Do Quang; Minh, Ngo Ngoc Quang; Ha, Do Lien Anh; McBride, Angela; Tuan, Ha Manh; Baker, Stephen; Tam, Pham Thi Thanh; Phuc, Tran My; Huong, Dang Thao; Loi, Tran Quoc; Vu, Nguyen Tran Anh; Hung, Nguyen Van; Minh, Tran Thi Thuy; Xang, Nguyen Van; Dong, Nguyen; Nghia, Ho Dang Trung; Chau, Nguyen Van Vinh; Thwaites, Guy; van Doorn, H Rogier; Anscombe, Catherine; Le Van, Tan

2017-01-01

Since 1962, enterovirus D68 (EV-D68) has been implicated in multiple outbreaks and sporadic cases of respiratory infection worldwide, but especially in the USA and Europe with an increasing frequency between 2010 and 2014. We describe the detection, associated clinical features and molecular characterization of EV-D68 in central and southern Viet Nam between 2009 and 2015. Enterovirus/rhinovirus PCR positive respiratory or CSF samples taken from children and adults with respiratory/central nervous system infections in Viet Nam were tested by an EV-D68 specific PCR. The included samples were derived from 3 different observational studies conducted at referral hospitals across central and southern Viet Nam between 2009 and 2015. Whole-genome sequencing was carried out using a MiSeq based approach. Phylogenetic reconstruction and estimation of evolutionary rate and recombination were carried out in BEAST and Recombination Detection Program, respectively. EV-D68 was detected in 21/625 (3.4%) enterovirus/rhinovirus PCR positive respiratory samples but in none of the 15 CSF. All the EV-D68 patients were young children (age range: 11.8 - 24.5 months) and had moderate respiratory infections. Phylogenetic analysis suggested that the Vietnamese sequences clustered with those from Asian countries, of which 9 fell in the B1 clade, and the remaining sequence was identified within the A2 clade. One intra sub-clade recombination event was detected, representing the second reported recombination within EV-D68. The evolutionary rate of EV-D68 was estimated to be 5.12E -3 substitutions/site/year. Phylogenetic analysis indicated that the virus was imported into Viet Nam in 2008. We have demonstrated for the first time EV-D68 has been circulating at low levels in Viet Nam since 2008, associated with moderate acute respiratory infection in children. EV-D68 in Viet Nam is most closely related to Asian viruses, and clusters separately from recent US and European viruses that were suggested to be associated with acute flaccid paralysis.

Theobroma cacao cystatins impair Moniliophthora perniciosa mycelial growth and are involved in postponing cell death symptoms.

PubMed

Pirovani, Carlos Priminho; da Silva Santiago, André; dos Santos, Lívia Santana; Micheli, Fabienne; Margis, Rogério; da Silva Gesteira, Abelmon; Alvim, Fátima Cerqueira; Pereira, Gonçalo Amarante Guimarães; de Mattos Cascardo, Júlio Cézar

2010-11-01

Three cystatin open reading frames named TcCys1, TcCys2 and TcCys3 were identified in cDNA libraries from compatible interactions between Theobroma cacao (cacao) and Moniliophthora perniciosa. In addition, an ORF named TcCys4 was identified in the cDNA library of the incompatible interaction. The cDNAs encoded conceptual proteins with 209, 127, 124, and 205 amino acid residues, with a deduced molecular weight of 24.3, 14.1, 14.3 and 22.8 kDa, respectively. His-tagged recombinant proteins were purified from Escherichia coli expression, and showed inhibitory activities against M. perniciosa. The four recombinant cystatins exhibited K(i) values against papain in the range of 152-221 nM. Recombinant TcCYS3 and TcCYS4 immobilized in CNBr-Sepharose were efficient to capture M. perniciosa proteases from culture media. Polyclonal antibodies raised against the recombinant TcCYS4 detected that the endogenous protein was more abundant in young cacao tissues, when compared with mature tissues. A ~85 kDa cacao multicystatin induced by M. perniciosa inoculation, MpNEP (necrosis and ethylene-inducing protein) and M. perniciosa culture supernatant infiltration were detected by anti-TcCYS4 antibodies in cacao young tissues. A direct role of the cacao cystatins in the defense against this phytopathogen was proposed, as well as its involvement in the development of symptoms of programmed cell death.

Recombination Analysis of Herpes Simplex Virus 1 Reveals a Bias toward GC Content and the Inverted Repeat Regions

PubMed Central

Lee, Kyubin; Kolb, Aaron W.; Sverchkov, Yuriy; Cuellar, Jacqueline A.; Craven, Mark

2015-01-01

ABSTRACT Herpes simplex virus 1 (HSV-1) causes recurrent mucocutaneous ulcers and is the leading cause of infectious blindness and sporadic encephalitis in the United States. HSV-1 has been shown to be highly recombinogenic; however, to date, there has been no genome-wide analysis of recombination. To address this, we generated 40 HSV-1 recombinants derived from two parental strains, OD4 and CJ994. The 40 OD4-CJ994 HSV-1 recombinants were sequenced using the Illumina sequencing system, and recombination breakpoints were determined for each of the recombinants using the Bootscan program. Breakpoints occurring in the terminal inverted repeats were excluded from analysis to prevent double counting, resulting in a total of 272 breakpoints in the data set. By placing windows around the 272 breakpoints followed by Monte Carlo analysis comparing actual data to simulated data, we identified a recombination bias toward both high GC content and intergenic regions. A Monte Carlo analysis also suggested that recombination did not appear to be responsible for the generation of the spontaneous nucleotide mutations detected following sequencing. Additionally, kernel density estimation analysis across the genome found that the large, inverted repeats comprise a recombination hot spot. IMPORTANCE Herpes simplex virus 1 (HSV-1) virus is the leading cause of sporadic encephalitis and blinding keratitis in developed countries. HSV-1 has been shown to be highly recombinogenic, and recombination itself appears to be a significant component of genome replication. To date, there has been no genome-wide analysis of recombination. Here we present the findings of the first genome-wide study of recombination performed by generating and sequencing 40 HSV-1 recombinants derived from the OD4 and CJ994 parental strains, followed by bioinformatics analysis. Recombination breakpoints were determined, yielding 272 breakpoints in the full data set. Kernel density analysis determined that the large inverted repeats constitute a recombination hot spot. Additionally, Monte Carlo analyses found biases toward high GC content and intergenic and repetitive regions. PMID:25926637

Construction of Potent Recombinant Strain Through Intergeneric Protoplast Fusion in Endophytic Fungi for Anticancerous Enzymes Production Using Rice Straw.

PubMed

El-Gendy, Mervat Morsy Abbas Ahmed; Al-Zahrani, Salha Hassan Mastour; El-Bondkly, Ahmed Mohamed Ahmed

2017-09-01

Among all fungal endophytes isolates derived from different ethno-medical plants, the hyper-yield L-asparaginase and L-glutaminase wild strains Trichoderma sp. Gen 9 and Cladosporium sp. Gen 20 using rice straw under solid-state fermentation (SSF) were selected. The selected strains were used as parents for the intergeneric protoplast fusion program to construct recombinant strain for prompt improvement production of these enzymes in one recombinant strain. Among 21 fusants obtained, the recombinant strain AYA 20-1, with 2.11-fold and 2.58-fold increase in L-asparaginase and L-glutaminase activities more than the parental isolates Trichoderma sp. Gen 9 and Cladosporium sp. Gen 20, respectively, was achieved using rice straw under SSF. Both therapeutic enzymes L-asparaginase and L-glutaminase were purified and characterized from the culture supernatant of the recombinant AYA 20-1 strain with molecular weights of 50.6 and 83.2 kDa, respectively. Both enzymes were not metalloenzymes. Whereas thiol group blocking reagents such as p-chloromercurybenzoate and iodoacetamide totally inhibited L-asparaginase activity, which refer to sulfhydryl groups and cysteine residues involved in its catalytic activity, they have no effect toward L-glutaminase activity. Interestingly, potent anticancer, antioxidant, and antimicrobial activities were detected for both enzymes.

76 FR 44309 - Notice of Intent To Grant a Partially Exclusive Patent License; TransMembrane Bioscience, Inc.

Federal Register 2010, 2011, 2012, 2013, 2014

2011-07-25

... Coxiella burnetii; U.S. Patent No. 7,824,875: Recombinant antigens for the detection of Coxiella burnetii; and U.S. Patent No. 7,824,909: Recombinant antigens for the detection of Coxiella burnetii in the... Coxiella burnetii infection by antibody-based assays using recombinant, immunodominant C. burnetii...

Construction, expression, purification and biotin labeling of a single recombinant multi-epitope antigen for double-antigen sandwich ELISA to detect hepatitis C virus antibody.

PubMed

He, Jing; Xiu, Bingshui; Wang, Guohua; Chen, Kun; Feng, Xiaoyan; Song, Xiaoguo; Zhu, Cuixia; Yang, Xiqin; Bai, Guanzhong; Ling, Shigan; Zhang, Heqiu

2011-08-01

Based on B cell epitope predictions, a recombinant antigen with multiple epitopes from four Hepatitis C Virus fragments (C, NS3, NS4 and NS5) were engineered. The recombinant gene was then highly expressed in E. coli. The non-modified and C-terminal-modified recombinant proteins were used for coating and biotin labeling, respectively, to establish the double-antigen sandwich ELISA. Ten positive reference samples confirmed by the CHIRON RIBA HCV 3.0 SIA kit were detected positive, Forty one plasma samples were positive among samples from 441 volunteers, which indicated that the recombinant antigen could readily react well with plasma HCV antibody. As critical reagents of double-antigen sandwich ELISA, the recombinant multi-epitope antigen and the C-terminal-modified and biotin-conjugated antigen show good antigenicity. In this study, we provide a simple approach to produce multiple epitopes within one recombinant protein in order to avoid the costly expression of less-effective pools of multiple proteins, which is the conventional strategy of diagnostic antigen production for HCV antibody detection.

Influence of vaccine strains on the evolution of canine distemper virus.

PubMed

da Fontoura Budaszewski, Renata; Streck, André Felipe; Nunes Weber, Matheus; Maboni Siqueira, Franciele; Muniz Guedes, Rafael Lucas; Wageck Canal, Cláudio

2016-07-01

Canine distemper virus (CDV) is a major dog pathogen belonging to the genus Morbillivirus of the family Paramyxoviridae. CDV causes disease and high mortality in dogs and wild carnivores. Although homologous recombination has been demonstrated in many members of Paramyxoviridae, these events have rarely been reported for CDV. To detect potential recombination events, the complete CDV genomes available in GenBank up to June 2015 were screened using distinct algorithms to detect genetic conversions and incongruent phylogenies. Eight putative recombinant viruses derived from different CDV genotypes and different hosts were detected. The breakpoints of the recombinant strains were primarily located on fusion and hemagglutinin glycoproteins. These results suggest that homologous recombination is a frequent phenomenon in morbillivirus populations under natural replication, and CDV vaccine strains might play an important role in shaping the evolution of this virus.

Stability of Yellow Fever Virus under Recombinatory Pressure as Compared with Chikungunya Virus

PubMed Central

McGee, Charles E.; Tsetsarkin, Konstantin A.; Guy, Bruno; Lang, Jean; Plante, Kenneth; Vanlandingham, Dana L.; Higgs, Stephen

2011-01-01

Recombination is a mechanism whereby positive sense single stranded RNA viruses exchange segments of genetic information. Recent phylogenetic analyses of naturally occurring recombinant flaviviruses have raised concerns regarding the potential for the emergence of virulent recombinants either post-vaccination or following co-infection with two distinct wild-type viruses. To characterize the conditions and sequences that favor RNA arthropod-borne virus recombination we constructed yellow fever virus (YFV) 17D recombinant crosses containing complementary deletions in the envelope protein coding sequence. These constructs were designed to strongly favor recombination, and the detection conditions were optimized to achieve high sensitivity recovery of putative recombinants. Full length recombinant YFV 17D virus was never detected under any of the experimental conditions examined, despite achieving estimated YFV replicon co-infection levels of ∼2.4×106 in BHK-21 (vertebrate) cells and ∼1.05×105 in C710 (arthropod) cells. Additionally YFV 17D superinfection resistance was observed in vertebrate and arthropod cells harboring a primary infection with wild-type YFV Asibi strain. Furthermore recombination potential was also evaluated using similarly designed chikungunya virus (CHIKV) replicons towards validation of this strategy for recombination detection. Non-homologus recombination was observed for CHIKV within the structural gene coding sequence resulting in an in-frame duplication of capsid and E3 gene. Based on these data, it is concluded that even in the unlikely event of a high level acute co-infection of two distinct YFV genomes in an arthropod or vertebrate host, the generation of viable flavivirus recombinants is extremely unlikely. PMID:21826243

Stability of yellow fever virus under recombinatory pressure as compared with chikungunya virus.

PubMed

McGee, Charles E; Tsetsarkin, Konstantin A; Guy, Bruno; Lang, Jean; Plante, Kenneth; Vanlandingham, Dana L; Higgs, Stephen

2011-01-01

Recombination is a mechanism whereby positive sense single stranded RNA viruses exchange segments of genetic information. Recent phylogenetic analyses of naturally occurring recombinant flaviviruses have raised concerns regarding the potential for the emergence of virulent recombinants either post-vaccination or following co-infection with two distinct wild-type viruses. To characterize the conditions and sequences that favor RNA arthropod-borne virus recombination we constructed yellow fever virus (YFV) 17D recombinant crosses containing complementary deletions in the envelope protein coding sequence. These constructs were designed to strongly favor recombination, and the detection conditions were optimized to achieve high sensitivity recovery of putative recombinants. Full length recombinant YFV 17D virus was never detected under any of the experimental conditions examined, despite achieving estimated YFV replicon co-infection levels of ∼2.4 x 10⁶ in BHK-21 (vertebrate) cells and ∼1.05 x 10⁵ in C₇10 (arthropod) cells. Additionally YFV 17D superinfection resistance was observed in vertebrate and arthropod cells harboring a primary infection with wild-type YFV Asibi strain. Furthermore recombination potential was also evaluated using similarly designed chikungunya virus (CHIKV) replicons towards validation of this strategy for recombination detection. Non-homologus recombination was observed for CHIKV within the structural gene coding sequence resulting in an in-frame duplication of capsid and E3 gene. Based on these data, it is concluded that even in the unlikely event of a high level acute co-infection of two distinct YFV genomes in an arthropod or vertebrate host, the generation of viable flavivirus recombinants is extremely unlikely.

On the Genealogy of Asexual Diploids

NASA Astrophysics Data System (ADS)

Lam, Fumei; Langley, Charles H.; Song, Yun S.

Given molecular genetic data from diploid individuals that, at present, reproduce mostly or exclusively asexually without recombination, an important problem in evolutionary biology is detecting evidence of past sexual reproduction (i.e., meiosis and mating) and recombination (both meiotic and mitotic). However, currently there is a lack of computational tools for carrying out such a study. In this paper, we formulate a new problem of reconstructing diploid genealogies under the assumption of no sexual reproduction or recombination, with the ultimate goal being to devise genealogy-based tools for testing deviation from these assumptions. We first consider the infinite-sites model of mutation and develop linear-time algorithms to test the existence of an asexual diploid genealogy compatible with the infinite-sites model of mutation, and to construct one if it exists. Then, we relax the infinite-sites assumption and develop an integer linear programming formulation to reconstruct asexual diploid genealogies with the minimum number of homoplasy (back or recurrent mutation) events. We apply our algorithms on simulated data sets with sizes of biological interest.

The HIV-1 epidemic in Bolivia is dominated by subtype B and CRF12_BF "family" strains.

PubMed

Guimarães, Monick L; Velarde-Dunois, Ketty G; Segurondo, David; Morgado, Mariza G

2012-01-16

Molecular epidemiological studies of HIV-1 in South America have revealed the occurrence of subtypes B, F1 and BF1 recombinants. Even so, little information concerning the HIV-1 molecular epidemiology in Bolivia is available. In this study we performed phylogenetic analyses from samples collected in Bolivia at two different points in time over a 10 year span. We analyzed these samples to estimate the trends in the HIV subtype and recombinant forms over time. Fifty one HIV-1 positive samples were collected in Bolivia over two distinct periods (1996 and 2005). These samples were genetically characterized based on partial pol protease/reverse transcriptase (pr/rt) and env regions. Alignment and neighbor-joining (NJ) phylogenetic analyses were established from partial env (n = 37) and all pol sequences using Mega 4. The remaining 14 env sequences from 1996 were previously characterized based on HMA-env (Heteroduplex mobility assay). The Simplot v.3.5.1 program was used to verify intragenic recombination, and SplitsTree 4.0 was employed to confirm the phylogenetic relationship of the BF1 recombinant samples. Phylogenetic analysis of both env and pol regions confirmed the predominance of "pure" subtype B (72.5%) samples circulating in Bolivia and revealed a high prevalence of BF1 genotypes (27.5%). Eleven out of 14 BF1 recombinants displayed a mosaic structure identical or similar to that described for the CRF12_BF variant, one sample was classified as CRF17_BF, and two others were F1pol/Benv. No "pure" HIV-1 subtype F1 or B" variant of subtype B was detected in the present study. Of note, samples characterized as CRF12_BF-related were depicted only in 2005. HIV-1 genetic diversity in Bolivia is mostly driven by subtype B followed by BF1 recombinant strains from the CRF12_BF "family". No significant temporal changes were detected between the mid-1990s and the mid-2000s for subtype B (76.2% vs 70.0%) or BF1 recombinant (23.8% vs 30.0%) samples from Bolivia.

The HIV-1 epidemic in Bolivia is dominated by subtype B and CRF12_BF "family" strains

PubMed Central

2012-01-01

Background Molecular epidemiological studies of HIV-1 in South America have revealed the occurrence of subtypes B, F1 and BF1 recombinants. Even so, little information concerning the HIV-1 molecular epidemiology in Bolivia is available. In this study we performed phylogenetic analyses from samples collected in Bolivia at two different points in time over a 10 year span. We analyzed these samples to estimate the trends in the HIV subtype and recombinant forms over time. Materials and methods Fifty one HIV-1 positive samples were collected in Bolivia over two distinct periods (1996 and 2005). These samples were genetically characterized based on partial pol protease/reverse transcriptase (pr/rt) and env regions. Alignment and neighbor-joining (NJ) phylogenetic analyses were established from partial env (n = 37) and all pol sequences using Mega 4. The remaining 14 env sequences from 1996 were previously characterized based on HMA-env (Heteroduplex mobility assay). The Simplot v.3.5.1 program was used to verify intragenic recombination, and SplitsTree 4.0 was employed to confirm the phylogenetic relationship of the BF1 recombinant samples. Results Phylogenetic analysis of both env and pol regions confirmed the predominance of "pure" subtype B (72.5%) samples circulating in Bolivia and revealed a high prevalence of BF1 genotypes (27.5%). Eleven out of 14 BF1 recombinants displayed a mosaic structure identical or similar to that described for the CRF12_BF variant, one sample was classified as CRF17_BF, and two others were F1pol/Benv. No "pure" HIV-1 subtype F1 or B" variant of subtype B was detected in the present study. Of note, samples characterized as CRF12_BF-related were depicted only in 2005. Conclusion HIV-1 genetic diversity in Bolivia is mostly driven by subtype B followed by BF1 recombinant strains from the CRF12_BF "family". No significant temporal changes were detected between the mid-1990s and the mid-2000s for subtype B (76.2% vs 70.0%) or BF1 recombinant (23.8% vs 30.0%) samples from Bolivia. PMID:22248191

Binary Programming Models of Spatial Pattern Recognition: Applications in Remote Sensing Image Analysis

DTIC Science & Technology

1991-12-01

9 2.6.1 Multi-Shape Detection. .. .. .. .. .. .. ...... 9 Page 2.6.2 Line Segment Extraction and Re-Combination.. 9 2.6.3 Planimetric Feature... Extraction ............... 10 2.6.4 Line Segment Extraction From Statistical Texture Analysis .............................. 11 2.6.5 Edge Following as Graph...image after image, could benefit clue to the fact that major spatial characteristics of subregions could be extracted , and minor spatial changes could be

Development of immunodetection system for botulinum neurotoxin type B using synthetic gene based recombinant protein

PubMed Central

Jain, Swati; Ponmariappan, S.; Kumar, Om

2011-01-01

Background & objectives: Botulinum neurotoxins (A-G) are among most poisonous substances in the world, produced by obligate anaerobic bacteria Clostridum botulinum. Among the seven serotypes A, B, E and F are of human importance. In India, the prevalence of C. botulinum as well as botulism outbreaks have been reported. Due to its extreme toxicity it has been classified in the Category A of biological warfare agent. So far, there is no commercial detection system available in India to detect botulism. The present study aims to develop an immuno detection system for botulinum neurotoxin serotype B using synthetic gene approach. Methods: The truncated fragment of the botulinum neurotoxin type B from amino acid 1-450 was synthesized using PCR overlap primers; the constructed gene was cloned in the pQE30UA vector and transformed to Escherichia coli SG 13009. The recombinant protein expression was optimized using various concentration of isopropylthiogalactoside (IPTG) induction, further the expression was confirmed by Western blot analysis using anti-His antibody. Recombinant protein was purified under denatured condition using Ni-NTA affinity chromatography. Antibody was generated against the recombinant protein using alum adjuvant in BALB/c mice and tested for cross reactivity with other serotypes of C. botulinum as well as closely related clostridia. An ELISA test was developed for the detection of botulinum neurotoxin and the minimum detection limit was also estimated. Results: The recombinant protein was expressed at maximum yield at 4.3 h of post-induction with 0.5 mM IPTG concentration. The recombinant protein was purified using Ni-NTA affinity chromatography up to the homogeneity level. The polyclonal antibodies were raised in mice with a titre of 1:2048000. The developed antibody was highly specific with a sensitivity of detecting approximately 15 ng/ml of recombinant protein and not showing any cross-reactivity with other serotypes. Interpretation & conclusions: There is no commercial immunodetection system available in India to detect botulism. The developed detection system is highly specific. It will be useful for growing food industry to detect botulinum neurotoxin in food samples as well as in clinical samples. PMID:21808132

Recombinant Temporal Aberration Detection Algorithms for Enhanced Biosurveillance

PubMed Central

Murphy, Sean Patrick; Burkom, Howard

2008-01-01

Objective Broadly, this research aims to improve the outbreak detection performance and, therefore, the cost effectiveness of automated syndromic surveillance systems by building novel, recombinant temporal aberration detection algorithms from components of previously developed detectors. Methods This study decomposes existing temporal aberration detection algorithms into two sequential stages and investigates the individual impact of each stage on outbreak detection performance. The data forecasting stage (Stage 1) generates predictions of time series values a certain number of time steps in the future based on historical data. The anomaly measure stage (Stage 2) compares features of this prediction to corresponding features of the actual time series to compute a statistical anomaly measure. A Monte Carlo simulation procedure is then used to examine the recombinant algorithms’ ability to detect synthetic aberrations injected into authentic syndromic time series. Results New methods obtained with procedural components of published, sometimes widely used, algorithms were compared to the known methods using authentic datasets with plausible stochastic injected signals. Performance improvements were found for some of the recombinant methods, and these improvements were consistent over a range of data types, outbreak types, and outbreak sizes. For gradual outbreaks, the WEWD MovAvg7+WEWD Z-Score recombinant algorithm performed best; for sudden outbreaks, the HW+WEWD Z-Score performed best. Conclusion This decomposition was found not only to yield valuable insight into the effects of the aberration detection algorithms but also to produce novel combinations of data forecasters and anomaly measures with enhanced detection performance. PMID:17947614

Direct phenotypic and genotypic detection of a recombinant pseudomonad population released into lake water.

PubMed Central

Morgan, J A; Winstanley, C; Pickup, R W; Jones, J G; Saunders, J R

1989-01-01

As a system for studying the fate of genetically engineered microorganisms in the environment, we have previously constructed recombinant plasmids encoding a xylE marker gene (C. Winstanley, J. A. W. Morgan, R. W. Pickup, J. G. Jones, and J. R. Saunders, Appl. Environ. Microbiol. 55:771-777, 1989). A series of direct membrane filter methods have been developed which facilitate the detection of bacterial cells harboring the xylE gene, its product, catechol 2,3-dioxygenase, and catechol 2,3-dioxygenase enzyme activity directly from water samples. These methods enable detection of recombinant populations at concentrations as low as 10(3) to 10(4) cells ml of lake water-1. Direct detection facilitates ecological studies of a range of bacterial strains containing the marker system in aquatic environments. The fate of a recombinant pseudomonad population in lake water was assessed by a combination of colony-forming ability, direct counts, and direct detection of the xylE gene and phenotypic expression of its product. Images PMID:2604395

Tab2, a novel recombinant polypeptide tag offering sensitive and specific protein detection and reliable affinity purification.

PubMed

Crusius, Kerstin; Finster, Silke; McClary, John; Xia, Wei; Larsen, Brent; Schneider, Douglas; Lu, Hong-Tao; Biancalana, Sara; Xuan, Jian-Ai; Newton, Alicia; Allen, Debbie; Bringmann, Peter; Cobb, Ronald R

2006-10-01

The detection and purification of proteins are often time-consuming and frequently involve complicated protocols. The addition of a peptide tag to recombinant proteins can make this process more efficient. Many of the commonly used tags, such as Flagtrade mark, Myc, HA and V5 are recognized by specific monoclonal antibodies and therefore, allow immunoaffinity-based purification. Enhancing the current scope of flexibility in using diverse peptide tags, we report here the development of a novel, short polypeptide tag (Tab2) for detection and purification of recombinant proteins. The Tab2 epitope corresponds to the NH2-terminal seven amino acid residues of human TGFalpha. A monoclonal anti-Tab2 antibody was raised and characterized. To investigate the potential of this peptide sequence as a novel tag for recombinant proteins, we expressed several different recombinant proteins containing this tag in E. coli, baculovirus, and mammalian cells. The data presented demonstrates the Tab2 tag-anti-Tab2 antibody combination is a reliable tool enabling specific Western blot detection, FACS analysis, and immunoprecipitation as well as non-denaturing protein affinity purification.

Photodetector having high speed and sensitivity

DOEpatents

Morse, Jeffrey D.; Mariella, Jr., Raymond P.

1991-01-01

The present invention provides a photodetector having an advantageous combination of sensitivity and speed; it has a high sensitivity while retaining high speed. In a preferred embodiment, visible light is detected, but in some embodiments, x-rays can be detected, and in other embodiments infrared can be detected. The present invention comprises a photodetector having an active layer, and a recombination layer. The active layer has a surface exposed to light to be detected, and comprises a semiconductor, having a bandgap graded so that carriers formed due to interaction of the active layer with the incident radiation tend to be swept away from the exposed surface. The graded semiconductor material in the active layer preferably comprises Al.sub.1-x Ga.sub.x As. An additional sub-layer of graded In.sub.1-y Ga.sub.y As may be included between the Al.sub.1-x Ga.sub.x As layer and the recombination layer. The recombination layer comprises a semiconductor material having a short recombination time such as a defective GaAs layer grown in a low temperature process. The recombination layer is positioned adjacent to the active layer so that carriers from the active layer tend to be swept into the recombination layer. In an embodiment, the photodetector may comprise one or more additional layers stacked below the active and recombination layers. These additional layers may include another active layer and another recombination layer to absorb radiation not absorbed while passing through the first layers. A photodetector having a stacked configuration may have enhanced sensitivity and responsiveness at selected wavelengths such as infrared.

Nonmutagenic carcinogens induce intrachromosomal recombination in dividing yeast cells.

PubMed

Schiestl, R H

1993-12-01

A large number of animal and human carcinogens without apparent genotoxic activity exist (nonmutagenic carcinogens) that are difficult or impossible to detect with the currently used short-term tests. Because of the association of carcinogenesis with genome rearrangement, a system selecting for intrachromosomal recombination (DEL recombination) that results in genome rearrangement has been constructed in the yeast Saccharomyces cerevisiae. Because DEL recombination is under different genetic control than interchromosomal recombination and meiotic recombination, it is probably due to a different mechanism. It has been found that DEL recombination is readily inducible by 10 mutagenic carcinogens and 17 nonmutagenic carcinogens that are not detectable (false negatives) with the Ames assay. In addition, three out of four mutagens that do not cause cancer (false positives in the Ames assay) do not induce DEL recombination. DEL recombination is inducible by UV only in dividing cells but not in cells synchronized in the G1 or G2 phase of the cell cycle. Interchromosomal recombination, on the other hand, is inducible in G1 but not in G2. The nonmutagenic carcinogens induce DEL recombination only in actively growing cells, which may give some indication as to their mechanism. Further characterization of the mechanism involved in induction of DEL recombination may contribute to the understanding of the biological activity of nonmutagenic carcinogens.

A Simple and Robust Statistical Test for Detecting the Presence of Recombination

PubMed Central

Bruen, Trevor C.; Philippe, Hervé; Bryant, David

2006-01-01

Recombination is a powerful evolutionary force that merges historically distinct genotypes. But the extent of recombination within many organisms is unknown, and even determining its presence within a set of homologous sequences is a difficult question. Here we develop a new statistic, Φw, that can be used to test for recombination. We show through simulation that our test can discriminate effectively between the presence and absence of recombination, even in diverse situations such as exponential growth (star-like topologies) and patterns of substitution rate correlation. A number of other tests, Max χ2, NSS, a coalescent-based likelihood permutation test (from LDHat), and correlation of linkage disequilibrium (both r2 and |D′|) with distance, all tend to underestimate the presence of recombination under strong population growth. Moreover, both Max χ2 and NSS falsely infer the presence of recombination under a simple model of mutation rate correlation. Results on empirical data show that our test can be used to detect recombination between closely as well as distantly related samples, regardless of the suspected rate of recombination. The results suggest that Φw is one of the best approaches to distinguish recurrent mutation from recombination in a wide variety of circumstances. PMID:16489234

Recombination elevates the effective evolutionary rate and facilitates the establishment of HIV-1 infection in infants after mother-to-child transmission

DOE Office of Scientific and Technical Information (OSTI.GOV)

Sanborn, Keri B.; Somasundaran, Mohan; Luzuriaga, Katherine

Some previous studies have demonstrated that single HIV-1 genotypes are commonly transmitted from mother to child, but such analyses primarily used single samples from mother and child. It is possible that in a single sample, obtained early after infection, only the most replication competent virus is detected even when other forms may have been transmitted. Such forms may have advantages later in infection, and may thus be detected in follow-up samples. Furthermore, because HIV-1 frequently recombines, phylogenetic analyses that ignore recombination may miss transmission of multiple forms if they recombine after transmission. Moreover, recombination may facilitate adaptation, thus providing anmore » advantage in establishing infection. The effect of recombination on viral evolution in HIV-1 infected children has not been well defined.« less

Recombination elevates the effective evolutionary rate and facilitates the establishment of HIV-1 infection in infants after mother-to-child transmission

DOE PAGES

Sanborn, Keri B.; Somasundaran, Mohan; Luzuriaga, Katherine; ...

2015-11-16

Some previous studies have demonstrated that single HIV-1 genotypes are commonly transmitted from mother to child, but such analyses primarily used single samples from mother and child. It is possible that in a single sample, obtained early after infection, only the most replication competent virus is detected even when other forms may have been transmitted. Such forms may have advantages later in infection, and may thus be detected in follow-up samples. Furthermore, because HIV-1 frequently recombines, phylogenetic analyses that ignore recombination may miss transmission of multiple forms if they recombine after transmission. Moreover, recombination may facilitate adaptation, thus providing anmore » advantage in establishing infection. The effect of recombination on viral evolution in HIV-1 infected children has not been well defined.« less

IRiS: construction of ARG networks at genomic scales.

PubMed

Javed, Asif; Pybus, Marc; Melé, Marta; Utro, Filippo; Bertranpetit, Jaume; Calafell, Francesc; Parida, Laxmi

2011-09-01

Given a set of extant haplotypes IRiS first detects high confidence recombination events in their shared genealogy. Next using the local sequence topology defined by each detected event, it integrates these recombinations into an ancestral recombination graph. While the current system has been calibrated for human population data, it is easily extendible to other species as well. IRiS (Identification of Recombinations in Sequences) binary files are available for non-commercial use in both Linux and Microsoft Windows, 32 and 64 bit environments from https://researcher.ibm.com/researcher/view_project.php?id = 2303 parida@us.ibm.com.

Within-Host Dynamics of the Emergence of Tomato Yellow Leaf Curl Virus Recombinants

PubMed Central

Urbino, Cica; Gutiérrez, Serafin; Antolik, Anna; Bouazza, Nabila; Doumayrou, Juliette; Granier, Martine; Martin, Darren P.; Peterschmitt, Michel

2013-01-01

Tomato yellow leaf curl virus (TYLCV) is a highly damaging begomovirus native to the Middle East. TYLCV has recently spread worldwide, recombining with other begomoviruses. Recent analysis of mixed infections between TYLCV and Tomato leaf curl Comoros begomovirus (ToLCKMV) has shown that, although natural selection preserves certain co-evolved intra-genomic interactions, numerous and diverse recombinants are produced at 120 days post-inoculation (dpi), and recombinant populations from different tomato plants are very divergent. Here, we investigate the population dynamics that lead to such patterns in tomato plants co-infected with TYLCV and ToLCKMV either by agro-inoculation or using the natural whitefly vector Bemisia tabaci. We monitored the frequency of parental and recombinant genotypes independently in 35 plants between 18 and 330 dpi and identified 177 recombinants isolated at different times. Recombinants were detected from 18 dpi and their frequency increased over time to reach about 50% at 150 dpi regardless of the inoculation method. The distribution of breakpoints detected on 96 fully sequenced recombinants was consistent with a continuous generation of new recombinants as well as random and deterministic effects in their maintenance. A severe population bottleneck of around 10 genomes was estimated during early systemic infection–a phenomenon that could account partially for the heterogeneity in recombinant patterns observed among plants. The detection of the same recombinant genome in six of the thirteen plants analysed beyond 30 dpi supported the influence of selection on observed recombination patterns. Moreover, a highly virulent recombinant genotype dominating virus populations within one plant has, apparently, the potential to be maintained in the natural population according to its infectivity, within-host accumulation, and transmission efficiency - all of which were similar or intermediate to those of the parent genotypes. Our results anticipate the outcomes of natural encounters between TYLCV and ToLCKMV. PMID:23472190

The CAF-1 and Hir Histone Chaperones Associate with Sites of Meiotic Double-Strand Breaks in Budding Yeast

PubMed Central

Brachet, Elsa; Béneut, Claire; Serrentino, Maria-Elisabetta; Borde, Valérie

2015-01-01

In the meiotic prophase, programmed DNA double-strand breaks (DSB) are introduced along chromosomes to promote homolog pairing and recombination. Although meiotic DSBs usually occur in nucleosome-depleted, accessible regions of chromatin, their repair by homologous recombination takes place in a nucleosomal environment. Nucleosomes may represent an obstacle for the recombination machinery and their timely eviction and reincorporation into chromatin may influence the outcome of recombination, for instance by stabilizing recombination intermediates. Here we show in budding yeast that nucleosomes flanking a meiotic DSB are transiently lost during recombination, and that specific histone H3 chaperones, CAF-1 and Hir, are mobilized at meiotic DSBs. However, the absence of these chaperones has no effect on meiotic recombination, suggesting that timely histone reincorporation following their eviction has no influence on the recombination outcome, or that redundant pathways are activated. This study is the first example of the involvement of histone H3 chaperones at naturally occurring, developmentally programmed DNA double-strand breaks. PMID:25938567

Recombination within the nonstructural genes of the parvovirus minute virus of mice (MVM) generates functional levels of wild-type NS1, which can be detected in the absence of selective pressure following transfection of nonreplicating plasmids.

PubMed

Pearson, J L; Pintel, D J

2000-03-30

Recombination within the coding region of the nonstructural genes of minute virus of mice (MVM), which generates functional levels of wild-type NS1, was observed in the absence of selective pressure following cotransfection of nonreplicating plasmids. P38 activity was used as a measure of recombinant NS1 production, which, together with direct detection of recombinant-generated products by RT-PCR, allowed an estimation of recombination efficiency. In addition, we show that very low levels of wild-type NS1 were able to significantly transactivate P38. Given that recombination following cotransfection can generate NS1 at these levels, our observations have implications for the study of parvoviral genetics, the construction of recombinant parvoviral vectors for gene therapy applications, and perhaps other systems using cotransfection of plasmids that share homologous sequences. Copyright 2000 Academic Press.

Doping in the recombinant era: strategies and counterstrategies.

PubMed

Azzazy, Hassan M E; Mansour, Mai M H; Christenson, Robert H

2005-11-01

Advances in recombinant DNA technology have created one of the most powerful weapons in the current doping arsenal: recombinant proteins [Sweeney HL. Gene doping. Sci Am 2004;291:62-9; Unal M, Ozer Unal D. Gene doping in sports. Sports Med 2004;34:357-62]. Recombinant erythropoietin (EPO) and human growth hormone (hGH) are currently being abused but are fortunately detectable either directly by employing isoelectric focusing and immunoassays or indirectly by assessing changes in selected hematopoietic parameters. The detection is technically demanding due to the extent of similarity between the recombinant proteins and their endogenous counterparts. Another issue facing detection efforts is the speed and conditions at which blood samples are collected and analyzed in a sports setting. Recently, gene doping, which stemmed out of legitimate gene therapy trials, has emerged as the next level of doping. Erythropoietin (EPO), human growth hormone (hGH), insulin-like growth factor-1 (IGF-1), peroxisome proliferator-activated receptor-delta (PPAR delta), and myostatin inhibitor genes have been identified as primary targets for doping. Sports clinical scientists today are racing against the clock because assuring the continued integrity of sports competition depends on their ability to outpace the efforts of dopers by developing new detection strategies.

Putative recombination events and evolutionary history of five economically important viruses of fruit trees based on coat protein-encoding gene sequence analysis.

PubMed

Boulila, Moncef

2010-06-01

To enhance the knowledge of recombination as an evolutionary process, 267 accessions retrieved from GenBank were investigated, all belonging to five economically important viruses infecting fruit crops (Plum pox, Apple chlorotic leaf spot, Apple mosaic, Prune dwarf, and Prunus necrotic ringspot viruses). Putative recombinational events were detected in the coat protein (CP)-encoding gene using RECCO and RDP version 3.31beta algorithms. Based on RECCO results, all five viruses were shown to contain potential recombination signals in the CP gene. Reconstructed trees with modified topologies were proposed. Furthermore, RECCO performed better than the RDP package in detecting recombination events and exhibiting their evolution rate along the sequences of the five viruses. RDP, however, provided the possible major and minor parents of the recombinants. Thus, the two methods should be considered complementary.

Development, evaluation, and laboratory validation of immunoassays for the diagnosis of equine infectious anemia (EIA) using recombinant protein produced from a synthetic p26 gene of EIA virus.

PubMed

Singha, Harisankar; Goyal, Sachin K; Malik, Praveen; Khurana, Sandip K; Singh, Raj K

2013-12-01

Equine infectious anemia (EIA)-a retroviral disease caused by equine infectious anemia virus (EIAV)-is a chronic, debilitating disease of horses, mules, and donkeys. EIAV infection has been reported worldwide and is recognized as pathogen of significant economic importance to the horse industry. This disease falls under regulatory control program in many countries including India. Control of EIA is based on identification of inapparent carriers by detection of antibodies to EIAV in serologic tests and "Stamping Out" policy. The current internationally accepted test for diagnosis of EIA is the agar gel immune-diffusion test (AGID), which detects antibodies to the major gag gene (p26) product. The objective of this study was to develop recombinant p26 based in-house immunoassays [enzyme linked immunosorbent assays (ELISA), and AGID] for EIA diagnosis. The synthetic p26 gene of EIAV was expressed in Escherichia coli and diagnostic potential of recombinant p26 protein were evaluated in ELISA and AGID on 7,150 and 1,200 equine serum samples, respectively, and compared with commercial standard AGID kit. The relative sensitivity and specificity of the newly developed ELISA were 100 and 98.6 %, respectively. Whereas, relative sensitivity and specificity of the newly developed AGID were in complete agreement in respect to commercial AGID kit. Here, we have reported the validation of an ELISA and AGID on large number of equine serum samples using recombinant p26 protein produced from synthetic gene which does not require handling of pathogenic EIAV. Since the indigenously developed reagents would be economical than commercial diagnostic kit, the rp26 based-immunoassays could be adopted for the sero-diagnosis and control of EIA in India.

Production of recombinant flagellin to develop ELISA-based detection of Salmonella Enteritidis.

PubMed

Mirhosseini, Seyed Ali; Fooladi, Abbas Ali Imani; Amani, Jafar; Sedighian, Hamid

Food-borne diseases, caused by the pathogenic bacteria, are highly prevalent in the world. Salmonella is one of the most important bacterial genera responsible for this. Salmonella Enteritidis (SE) is one of the non-typhoid Salmonellae that can be transmitted to human from poultry products, water, and contaminated food. In recent years, new and rapid detection methods such as enzyme-linked immunosorbent assay (ELISA) and polymerase chain reaction (PCR) have been developed. In this study, recombinant FliC (rFliC) was produced to be used as an antigen. The immunization was conducted in mice with the purified recombinant FliC (rFliC). The mice were subcutaneously immunized with rFliC and elicited significant rFliC specific serum IgG antibodies. An indirect ELISA system was established for the detection of Salmonella Enteritidis. Our results confirmed that the recombinant flagellin can be one of the excellent indicators for the detection of Salmonella Enteritidis. Copyright © 2017 Sociedade Brasileira de Microbiologia. Published by Elsevier Editora Ltda. All rights reserved.

[Genetic evidence for recombination and mutation in the emergence of human enterovirus 71].

PubMed

Liu, Ai-Ping; Tan, Hui; Xie, Qun; Chen, Bai-Tang; Liu, Xiao-Feng; Zhang, Yong

2014-09-01

We wished to understand the genetic recombination and phylogenetic characteristics of human en- terovirus A71 (EV-A71) and to explore its potential virulence-related sites. Full-length genomes of three EV-A71 strains isolated from patients in Chenzhou City (China) were sequenced and analyzed. Possible re- combination events and crossover sites were analyzed with Recombination Detection Program v4. 1. 6 by comparison with the complete genome sequences of 231 strains of EV-A71. Similarly, plot and bootscanning analyses were undertaken with SimPlot v3. 5. 1. Phylogenetic trees based on the sequences of VP1 regions were constructed with MEGA v5. 2 using the Kimura two-parameter model and neighbor-joining method. Results suggested that recombination events were detected among the three EV-A71 isolates from Chenzhou City. The common main parent sequence was from JF799986 isolated from samples in Guang- zhou City (China) in 2009, and the minor parent sequence was TW/70516/08. Intertypic recombination e- vents were found in the C4b strain (strain SHZH98 isolated in 1998) and C4a strain (Fuyang strain isola- ted in 2008) with the prototype strains of CVA4 and CVA14 in the 3D region. The chi-square test was used to screen-out potential virulence-related sites with nucleotide substitutions of different types of hand, foot, and mouth disease (HFMD) cases using SPSS v19.0. Results suggested that there were no significant nucleotide substitutions between death cases and severe-HFMD cases. Eighteen significant nucleotide substitutions were found between death/severe-HFMD cases and mild-HFMD cases, and all these 18 substitutions were distributed only in P2 and P3 regions. Intertypic recombination among the predominant circulating EV-A71 strains in the Chinese mainland and other EV-A strains probably dates before 1998, and intratypic recombination might have occurred frequently in the HFMD outbreak from 2008 to 2012. Substitutions in the non-capsid region may be correlated with the changes in virulence of EV-A71. These data suggest that researchers should pay more attention to the relationships between substitutions in the noncapsid region and the virulence of the virus.

Immunogenic Salivary Proteins of Triatoma infestans: Development of a Recombinant Antigen for the Detection of Low-Level Infestation of Triatomines

PubMed Central

Schwarz, Alexandra; Helling, Stefan; Collin, Nicolas; Teixeira, Clarissa R.; Medrano-Mercado, Nora; Hume, Jen C. C.; Assumpção, Teresa C.; Marcus, Katrin; Stephan, Christian; Meyer, Helmut E.; Ribeiro, José M. C.; Billingsley, Peter F.; Valenzuela, Jesus G.; Sternberg, Jeremy M.; Schaub, Günter A.

2009-01-01

Background Triatomines are vectors of Trypanosoma cruzi, the etiological agent of Chagas disease in Latin America. The most effective vector, Triatoma infestans, has been controlled successfully in much of Latin America using insecticide spraying. Though rarely undertaken, surveillance programs are necessary in order to identify new infestations and estimate the intensity of triatomine bug infestations in domestic and peridomestic habitats. Since hosts exposed to triatomines develop immune responses to salivary antigens, these responses can be evaluated for their usefulness as epidemiological markers to detect infestations of T. infestans. Methodology/Principal Findings T. infestans salivary proteins were separated by 2D-gel electrophoresis and tested for their immunogenicity by Western blotting using sera from chickens and guinea pigs experimentally exposed to T. infestans. From five highly immunogenic protein spots, eight salivary proteins were identified by nano liquid chromatography-electrospray ionization-tandem mass spectrometry (nanoLC-ESI-MS/MS) and comparison to the protein sequences of the National Center for Biotechnology Information (NCBI) database and expressed sequence tags of a unidirectionally cloned salivary gland cDNA library from T. infestans combined with the NCBI yeast protein sub-database. The 14.6 kDa salivary protein [gi|149689094] was produced as recombinant protein (rTiSP14.6) in a mammalian cell expression system and recognized by all animal sera. The specificity of rTiSP14.6 was confirmed by the lack of reactivity to anti-mosquito and anti-sand fly saliva antibodies. However, rTiSP14.6 was recognized by sera from chickens exposed to four other triatomine species, Triatoma brasiliensis, T. sordida, Rhodnius prolixus, and Panstrongylus megistus and by sera of chickens from an endemic area of T. infestans and Chagas disease in Bolivia. Conclusions/Significance The recombinant rTiSP14.6 is a suitable and promising epidemiological marker for detecting the presence of small numbers of different species of triatomines and could be developed for use as a new tool in surveillance programs, especially to corroborate vector elimination in Chagas disease vector control campaigns. PMID:19841746

Homologous Recombination—Experimental Systems, Analysis and Significance

PubMed Central

Kuzminov, Andrei

2014-01-01

Homologous recombination is the most complex of all recombination events that shape genomes and produce material for evolution. Homologous recombination events are exchanges between DNA molecules in the lengthy regions of shared identity, catalyzed by a group of dedicated enzymes. There is a variety of experimental systems in E. coli and Salmonella to detect homologous recombination events of several different kinds. Genetic analysis of homologous recombination reveals three separate phases of this process: pre-synapsis (the early phase), synapsis (homologous strand exchange) and post-synapsis (the late phase). In E. coli, there are at least two independent pathway of the early phase and at least two independent pathways of the late phase. All this complexity is incongruent with the originally ascribed role of homologous recombination as accelerator of genome evolution: there is simply not enough duplication and repetition in enterobacterial genomes for homologous recombination to have a detectable evolutionary role, and therefore not enough selection to maintain such a complexity. At the same time, the mechanisms of homologous recombination are uniquely suited for repair of complex DNA lesions called chromosomal lesions. In fact, the two major classes of chromosomal lesions are recognized and processed by the two individual pathways at the early phase of homologous recombination. It follows, therefore, that homologous recombination events are occasional reflections of the continual recombinational repair, made possible in cases of natural or artificial genome redundancy. PMID:26442506

Detection of isotype switch rearrangement in bulk culture by PCR.

PubMed

Max, E E; Mills, F C; Chu, C

2001-05-01

When a B lymphocyte changes from synthesizing IgM to synthesizing IgG, IgA, or IgE, this isotype switch is generally accompanied by a unique DNA rearrangement. The protocols in this unit describe two polymerase chain reaction (PCR)-based strategies for detecting switch rearrangements in bulk culture. The first involves direct PCR across the switch junctions, providing the opportunity for characterizing the recombination products by nucleotide sequence analysis; however, because of characteristics inherent to the PCR methodology this strategy cannot easily be used as a quantitative assay for recombination. A support protocol details the preparation of the 5' Su PCR probe for this protocol. The second basic protocol describes a method known as digestion-circularization PCR (DCPCR) that is more amenable to quantitation but yields no information on structure of the recombination products. Both techniques should be capable of detecting reciprocal deletion circles as well as functional recombination products remaining on the expressed chromosome.

Radiofrequency recombination lines from the interstellar medium

NASA Technical Reports Server (NTRS)

Dupree, A. K.

1971-01-01

Observations of recombination lines form normal H II regions, extended H II regions, nonthermal sources, and the H I medium are discussed. Detection of recombination lines from elements other than hydrogen may provide a means of identifying fossil Stromgren spheres at high temperature.

Effect of manipulating recombination rates on response to selection in livestock breeding programs.

PubMed

Battagin, Mara; Gorjanc, Gregor; Faux, Anne-Michelle; Johnston, Susan E; Hickey, John M

2016-06-22

In this work, we performed simulations to explore the potential of manipulating recombination rates to increase response to selection in livestock breeding programs. We carried out ten replicates of several scenarios that followed a common overall structure but differed in the average rate of recombination along the genome (expressed as the length of a chromosome in Morgan), the genetic architecture of the trait under selection, and the selection intensity under truncation selection (expressed as the proportion of males selected). Recombination rates were defined by simulating nine different chromosome lengths: 0.10, 0.25, 0.50, 1, 2, 5, 10, 15 and 20 Morgan, respectively. One Morgan was considered to be the typical chromosome length for current livestock species. The genetic architecture was defined by the number of quantitative trait variants (QTV) that affected the trait under selection. Either a large (10,000) or a small (1000 or 500) number of QTV was simulated. Finally, the proportions of males selected under truncation selection as sires for the next generation were equal to 1.2, 2.4, 5, or 10 %. Increasing recombination rate increased the overall response to selection and decreased the loss of genetic variance. The difference in cumulative response between low and high recombination rates increased over generations. At low recombination rates, cumulative response to selection tended to asymptote sooner and the genetic variance was completely eroded. If the trait under selection was affected by few QTV, differences between low and high recombination rates still existed, but the selection limit was reached at all rates of recombination. Higher recombination rates can enhance the efficiency of breeding programs to turn genetic variation into response to selection. However, to increase response to selection significantly, the recombination rate would need to be increased 10- or 20-fold. The biological feasibility and consequences of such large increases in recombination rates are unknown.

Recombination in the evolution of enterovirus C species sub-group that contains types CVA-21, CVA-24, EV-C95, EV-C96 and EV-C99.

PubMed

Smura, Teemu; Blomqvist, Soile; Vuorinen, Tytti; Ivanova, Olga; Samoilovich, Elena; Al-Hello, Haider; Savolainen-Kopra, Carita; Hovi, Tapani; Roivainen, Merja

2014-01-01

Genetic recombination is considered to be a very frequent phenomenon among enteroviruses (Family Picornaviridae, Genus Enterovirus). However, the recombination patterns may differ between enterovirus species and between types within species. Enterovirus C (EV-C) species contains 21 types. In the capsid coding P1 region, the types of EV-C species cluster further into three sub-groups (designated here as A-C). In this study, the recombination pattern of EV-C species sub-group B that contains types CVA-21, CVA-24, EV-C95, EV-C96 and EV-C99 was determined using partial 5'UTR and VP1 sequences of enterovirus strains isolated during poliovirus surveillance and previously published complete genome sequences. Several inter-typic recombination events were detected. Furthermore, the analyses suggested that inter-typic recombination events have occurred mainly within the distinct sub-groups of EV-C species. Only sporadic recombination events between EV-C species sub-group B and other EV-C sub-groups were detected. In addition, strict recombination barriers were inferred for CVA-21 genotype C and CVA-24 variant strains. These results suggest that the frequency of inter-typic recombinations, even within species, may depend on the phylogenetic position of the given viruses.

Wide variety of recombinant strains of norovirus GII in pediatric patients hospitalized with acute gastroenteritis in Thailand during 2005 to 2015.

PubMed

Supadej, Kanittapon; Khamrin, Pattara; Kumthip, Kattareeya; Kochjan, Pakawat; Yodmeeklin, Arpaporn; Ushijima, Hiroshi; Maneekarn, Niwat

2017-08-01

Norovirus (NoV) has been reported as being a common cause of acute gastroenteritis both in children and adults worldwide. Of the many variants, NoV GII.4 is the most predominant genotype. One of the mechanisms that drives the evolution and emergence of new variants of NoV is homologous recombination. This study describes the genetic recombination involved in cases of NoV GII detected in pediatric patients with acute gastroenteritis in Chiang Mai, Thailand during 2005 to 2015. From a total of 1938 stool samples, 3 (0.15%) were positive for NoV GI and 298 (15.38%) were identified as NoV GII. The genotypes detected in this study were GI.6, GI.14, GII.1, GII.2, GII.3, GII.4, GII.6, GII.7, GII.12, GII.13, GII.14, GII.15, GII.16, GII.17, GII.20, and GII.21. The NoV recombinant strains were verified by analysis of the partial sequence of ORF1 (RdRp)/ORF2 (capsid) junction. Phylogenetic analyses of partial ORF1 and ORF2 regions resulted in the identification of 21 (6.98%) NoV recombinant strains. Among these, 9 recombination patterns were detected in this study; GII.Pe/GII.4, GII.Pg/GII.1, GII.Pg/GII.12, GII.P7/GII.6, GII.P7/GII.14, GII.P12/GII.4, GII.P16/GII.2, GII.P16/GII.13, and GII.P21/GII.3. The findings demonstrated the wide variety of recombinant strains of NoV GII strains detected in pediatric patients admitted to the hospitals with acute gastroenteritis in Chiang Mai, Thailand during the past decade. Copyright © 2017 Elsevier B.V. All rights reserved.

Recombinant expression of the alternate reading frame protein (ARFP) of hepatitis C virus genotype 4a (HCV-4a) and detection of ARFP and anti-ARFP antibodies in HCV-infected patients.

PubMed

Shehat, Michael G; Bahey-El-Din, Mohammed; Kassem, Mervat A; Farghaly, Faten A; Abdul-Rahman, Medhat H; Fanaki, Nourhan H

2015-08-01

HCV is a single-stranded RNA virus with a single open reading frame (ORF) that is translated into a polyprotein that is then processed to form 10 viral proteins. An additional eleventh viral protein, the alternative reading frame protein (ARFP), was discovered relatively recently. This protein results from a translational frameshift in the core region during the expression of the viral proteins. Recombinant expression of different forms of ARFP was previously done for HCV genotypes 1 and 2, and more recently, genotype 3. However, none of the previous studies addressed the expression of ARFP of HCV genotype 4a, which is responsible for 80 % of HCV infections in the Middle East and Africa. Moreover, the direct detection of the ARFP antigen in HCV-infected patients was never studied before for any HCV genotype. In the present study, recombinant ARFP derived from HCV genotype 4a was successfully expressed in E. coli and purified using metal affinity chromatography. The recombinant ARFP protein and anti-ARFP antibodies were used for detection of ARFP antigen in patients' sera, employing competitive enzyme-linked immunosorbent assay (ELISA) procedures. Furthermore, the recombinant antigen was also used to detect and quantify anti-ARFP antibodies in HCV-infected Egyptian patients at different stages of pegylated interferon/ribavirin therapy, using an ELISA assay. The ARFP antigen was detectable in 69.4 % of RNA-positive sera, indicating that ARFP antigen is produced during the natural course of HCV infection. In addition, significant levels of anti-ARFP antibodies were present in 41 % of the serum samples tested. The important diagnostic value of the recombinant ARFP antigen was also demonstrated.

Most HIV Type 1 Non-B Infections in the Spanish Cohort of Antiretroviral Treatment-Naïve HIV-Infected Patients (CoRIS) Are Due to Recombinant Viruses

PubMed Central

Yebra, Gonzalo; de Mulder, Miguel; Martín, Leticia; Rodríguez, Carmen; Labarga, Pablo; Viciana, Isabel; Berenguer, Juan; Alemán, María Remedios; Pineda, Juan Antonio; García, Federico

2012-01-01

HIV-1 group M is classified into 9 subtypes, as well as recombinants favored by coinfection and superinfection events with different variants. Although HIV-1 subtype B is predominant in Europe, intersubtype recombinants are increasing in prevalence and complexity. In this study, phylogenetic analyses of pol sequences were performed to detect the HIV-1 circulating and unique recombinant forms (CRFs and URFs, respectively) in a Spanish cohort of antiretroviral treatment-naïve HIV-infected patients included in the Research Network on HIV/AIDS (CoRIS). Bootscanning and other methods were used to define complex recombinants not assigned to any subtype or CRF. A total of 670 available HIV-1 pol sequences from different patients were collected, of which 588 (87.8%) were assigned to HIV-1 subtype B and 82 (12.2%) to HIV-1 non-B variants. Recombinants caused the majority (71.9%) of HIV-1 non-B infections and were found in 8.8% of CoRIS patients. Eleven URFs (accounting for 13.4% of HIV-1 non-B infections), presenting complex mosaic patterns, were detected. Among them, 10 harbored subtype B fragments. Four of the 11 URFs were found in Spanish natives. A cluster of three B/CRF02_AG recombinants was detected. We conclude that complex variants, including unique recombinant forms, are being introduced into Spain through both immigrants and natives. An increase in the frequency of mosaic viruses, reflecting the increasing heterogeneity of the HIV epidemic in our country, is expected. PMID:22162552

Most HIV type 1 non-B infections in the Spanish cohort of antiretroviral treatment-naïve HIV-infected patients (CoRIS) are due to recombinant viruses.

PubMed

Yebra, Gonzalo; de Mulder, Miguel; Martín, Leticia; Rodríguez, Carmen; Labarga, Pablo; Viciana, Isabel; Berenguer, Juan; Alemán, María Remedios; Pineda, Juan Antonio; García, Federico; Holguín, Africa

2012-02-01

HIV-1 group M is classified into 9 subtypes, as well as recombinants favored by coinfection and superinfection events with different variants. Although HIV-1 subtype B is predominant in Europe, intersubtype recombinants are increasing in prevalence and complexity. In this study, phylogenetic analyses of pol sequences were performed to detect the HIV-1 circulating and unique recombinant forms (CRFs and URFs, respectively) in a Spanish cohort of antiretroviral treatment-naïve HIV-infected patients included in the Research Network on HIV/AIDS (CoRIS). Bootscanning and other methods were used to define complex recombinants not assigned to any subtype or CRF. A total of 670 available HIV-1 pol sequences from different patients were collected, of which 588 (87.8%) were assigned to HIV-1 subtype B and 82 (12.2%) to HIV-1 non-B variants. Recombinants caused the majority (71.9%) of HIV-1 non-B infections and were found in 8.8% of CoRIS patients. Eleven URFs (accounting for 13.4% of HIV-1 non-B infections), presenting complex mosaic patterns, were detected. Among them, 10 harbored subtype B fragments. Four of the 11 URFs were found in Spanish natives. A cluster of three B/CRF02_AG recombinants was detected. We conclude that complex variants, including unique recombinant forms, are being introduced into Spain through both immigrants and natives. An increase in the frequency of mosaic viruses, reflecting the increasing heterogeneity of the HIV epidemic in our country, is expected.

Detection of antibodies reacting with the antithetical duffy blood group antigens Fy(a) and Fy(b) using recombinant fusion proteins containing the duffy extracellular domain.

PubMed

Sheffield, William P; Bhakta, Varsha; Branch, Donald R; Denomme, Gregory A

2006-12-01

Detecting blood group-specific antibodies in patient sera is essential to the management of blood transfusions or pregnancies. We produced the antithetical forms of the 65 amino acid extracellular domain (ECD) of the Duffy (Fy) blood group protein fused to glutathione sulfotransferase (GST): GST-Fy(a); and GST-Fy(b), differing only in Gly or Asp at position 44, respectively. The purified recombinant proteins were recognized more effectively by reference polyclonal or monoclonal antibodies than the antithetical Fy specificity by either ELISA or immunoblotting. Combined immunoblot and ELISA tests performed at 1:200 dilutions of sera using the recombinant proteins gave results in agreement with undiluted sera and agglutination for 17/19 alloimmunized patients. At 1:200, agglutination detected anti-Fy(a) or anti-Fy(b) in only three of 12 samples that were positive by ELISA. Recombinant ECD-Fy proteins are suitable and sensitive reagents for the detection of anti-Fy that use technology amenable to automation and/or miniaturization and avoid the need for intact red cells.

Precise genotyping and recombination detection of Enterovirus

PubMed Central

2015-01-01

Enteroviruses (EV) with different genotypes cause diverse infectious diseases in humans and mammals. A correct EV typing result is crucial for effective medical treatment and disease control; however, the emergence of novel viral strains has impaired the performance of available diagnostic tools. Here, we present a web-based tool, named EVIDENCE (EnteroVirus In DEep conception, http://symbiont.iis.sinica.edu.tw/evidence), for EV genotyping and recombination detection. We introduce the idea of using mixed-ranking scores to evaluate the fitness of prototypes based on relatedness and on the genome regions of interest. Using phylogenetic methods, the most possible genotype is determined based on the closest neighbor among the selected references. To detect possible recombination events, EVIDENCE calculates the sequence distance and phylogenetic relationship among sequences of all sliding windows scanning over the whole genome. Detected recombination events are plotted in an interactive figure for viewing of fine details. In addition, all EV sequences available in GenBank were collected and revised using the latest classification and nomenclature of EV in EVIDENCE. These sequences are built into the database and are retrieved in an indexed catalog, or can be searched for by keywords or by sequence similarity. EVIDENCE is the first web-based tool containing pipelines for genotyping and recombination detection, with updated, built-in, and complete reference sequences to improve sensitivity and specificity. The use of EVIDENCE can accelerate genotype identification, aiding clinical diagnosis and enhancing our understanding of EV evolution. PMID:26678286

Recombinant Measles AIK-C Vaccine Strain Expressing the prM-E Antigen of Japanese Encephalitis Virus.

PubMed

Higuchi, Akira; Toriniwa, Hiroko; Komiya, Tomoyoshi; Nakayama, Tetsuo

2016-01-01

An inactivated Japanese encephalitis virus (JEV) vaccine, which induces neutralizing antibodies, has been used for many years in Japan. In the present study, the JEV prM-E protein gene was cloned, inserted at the P/M junction of measles AIK-C cDNA, and an infectious virus was recovered. The JEV E protein was expressed in B95a cells infected with the recombinant virus. Cotton rats were inoculated with recombinant virus. Measles PA antibodies were detected three weeks after immunization. Neutralizing antibodies against JEV developed one week after inoculation, and EIA antibodies were detected three weeks after immunization. The measles AIK-C-based recombinant virus simultaneously induced measles and JEV immune responses, and may be a candidate for infant vaccines. Therefore, the present strategy of recombinant viruses based on a measles vaccine vector would be applicable to the platform for vaccine development.

Recombination in the Evolution of Enterovirus C Species Sub-Group that Contains Types CVA-21, CVA-24, EV-C95, EV-C96 and EV-C99

PubMed Central

Smura, Teemu; Blomqvist, Soile; Vuorinen, Tytti; Ivanova, Olga; Samoilovich, Elena; Al-Hello, Haider; Savolainen-Kopra, Carita; Hovi, Tapani; Roivainen, Merja

2014-01-01

Genetic recombination is considered to be a very frequent phenomenon among enteroviruses (Family Picornaviridae, Genus Enterovirus). However, the recombination patterns may differ between enterovirus species and between types within species. Enterovirus C (EV-C) species contains 21 types. In the capsid coding P1 region, the types of EV-C species cluster further into three sub-groups (designated here as A–C). In this study, the recombination pattern of EV-C species sub-group B that contains types CVA-21, CVA-24, EV-C95, EV-C96 and EV-C99 was determined using partial 5′UTR and VP1 sequences of enterovirus strains isolated during poliovirus surveillance and previously published complete genome sequences. Several inter-typic recombination events were detected. Furthermore, the analyses suggested that inter-typic recombination events have occurred mainly within the distinct sub-groups of EV-C species. Only sporadic recombination events between EV-C species sub-group B and other EV-C sub-groups were detected. In addition, strict recombination barriers were inferred for CVA-21 genotype C and CVA-24 variant strains. These results suggest that the frequency of inter-typic recombinations, even within species, may depend on the phylogenetic position of the given viruses. PMID:24722726

The effects of recombination, mutation and selection on the evolution of the Rp1 resistance genes in grasses.

PubMed

Jouet, Agathe; McMullan, Mark; van Oosterhout, Cock

2015-06-01

Plant immune genes, or resistance genes, are involved in a co-evolutionary arms race with a diverse range of pathogens. In agronomically important grasses, such R genes have been extensively studied because of their role in pathogen resistance and in the breeding of resistant cultivars. In this study, we evaluate the importance of recombination, mutation and selection on the evolution of the R gene complex Rp1 of Sorghum, Triticum, Brachypodium, Oryza and Zea. Analyses show that recombination is widespread, and we detected 73 independent instances of sequence exchange, involving on average 1567 of 4692 nucleotides analysed (33.4%). We were able to date 24 interspecific recombination events and found that four occurred postspeciation, which suggests that genetic introgression took place between different grass species. Other interspecific events seemed to have been maintained over long evolutionary time, suggesting the presence of balancing selection. Significant positive selection (i.e. a relative excess of nonsynonymous substitutions (dN /dS >1)) was detected in 17-95 codons (0.42-2.02%). Recombination was significantly associated with areas with high levels of polymorphism but not with an elevated dN /dS ratio. Finally, phylogenetic analyses show that recombination results in a general overestimation of the divergence time (mean = 14.3%) and an alteration of the gene tree topology if the tree is not calibrated. Given that the statistical power to detect recombination is determined by the level of polymorphism of the amplicon as well as the number of sequences analysed, it is likely that many studies have underestimated the importance of recombination relative to the mutation rate. © 2015 John Wiley & Sons Ltd.

The potential of shifting recombination hotspots to increase genetic gain in livestock breeding.

PubMed

Gonen, Serap; Battagin, Mara; Johnston, Susan E; Gorjanc, Gregor; Hickey, John M

2017-07-04

This study uses simulation to explore and quantify the potential effect of shifting recombination hotspots on genetic gain in livestock breeding programs. We simulated three scenarios that differed in the locations of quantitative trait nucleotides (QTN) and recombination hotspots in the genome. In scenario 1, QTN were randomly distributed along the chromosomes and recombination was restricted to occur within specific genomic regions (i.e. recombination hotspots). In the other two scenarios, both QTN and recombination hotspots were located in specific regions, but differed in whether the QTN occurred outside of (scenario 2) or inside (scenario 3) recombination hotspots. We split each chromosome into 250, 500 or 1000 regions per chromosome of which 10% were recombination hotspots and/or contained QTN. The breeding program was run for 21 generations of selection, after which recombination hotspot regions were kept the same or were shifted to adjacent regions for a further 80 generations of selection. We evaluated the effect of shifting recombination hotspots on genetic gain, genetic variance and genic variance. Our results show that shifting recombination hotspots reduced the decline of genetic and genic variance by releasing standing allelic variation in the form of new allele combinations. This in turn resulted in larger increases in genetic gain. However, the benefit of shifting recombination hotspots for increased genetic gain was only observed when QTN were initially outside recombination hotspots. If QTN were initially inside recombination hotspots then shifting them decreased genetic gain. Shifting recombination hotspots to regions of the genome where recombination had not occurred for 21 generations of selection (i.e. recombination deserts) released more of the standing allelic variation available in each generation and thus increased genetic gain. However, whether and how much increase in genetic gain was achieved by shifting recombination hotspots depended on the distribution of QTN in the genome, the number of recombination hotspots and whether QTN were initially inside or outside recombination hotspots. Our findings show future scope for targeted modification of recombination hotspots e.g. through changes in zinc-finger motifs of the PRDM9 protein to increase genetic gain in production species.

Nonreplicative RNA Recombination of an Animal Plus-Strand RNA Virus in the Absence of Efficient Translation of Viral Proteins

PubMed Central

Kleine Büning, Maximiliane; Meyer, Denise; Austermann-Busch, Sophia; Roman-Sosa, Gleyder; Rümenapf, Tillmann

2017-01-01

RNA recombination is a major driving force for the evolution of RNA viruses and is significantly implicated in the adaptation of viruses to new hosts, changes of virulence, as well as in the emergence of new viruses including drug-resistant and escape mutants. However, the molecular details of recombination in animal RNA viruses are only poorly understood. In order to determine whether viral RNA recombination depends on translation of viral proteins, a nonreplicative recombination system was established which is based on cotransfection of cells with synthetic bovine viral diarrhea virus (family Flaviviridae) RNA genome fragments either lacking the internal ribosome entry site required for cap-independent translation or lacking almost the complete polyprotein coding region. The emergence of a number of recombinant viruses demonstrated that IRES-mediated translation of viral proteins is dispensable for efficient recombination and suggests that RNA recombination can occur in the absence of viral proteins. Analyses of 58 independently emerged viruses led to the detection of recombinant genomes with duplications, deletions and insertions in the 5′ terminal region of the open reading frame, leading to enlarged core fusion proteins detectable by Western blot analysis. This demonstrates a remarkable flexibility of the pestivirus core protein. Further experiments with capped and uncapped genome fragments containing a luciferase gene for monitoring the level of protein translation revealed that even a ∼1,000-fold enhancement of translation of viral proteins did not increase the frequency of RNA recombination. Taken together, this study highlights that nonreplicative RNA recombination does not require translation of viral proteins. PMID:28338950

Molecular detection and characterization of sapovirus in hospitalized children with acute gastroenteritis in the Philippines.

PubMed

Liu, Xiaofang; Yamamoto, Dai; Saito, Mariko; Imagawa, Toshifumi; Ablola, Adrianne; Tandoc, Amado O; Segubre-Mercado, Edelwisa; Lupisan, Socorro P; Okamoto, Michiko; Furuse, Yuki; Saito, Mayuko; Oshitani, Hitoshi

2015-07-01

Human sapovirus (SaV) is a causative agent of acute gastroenteritis. Recently, SaV detection has been increasing worldwide due to the emerging SaV genotype I.2. However, SaV infection has not been reported in the Philippines. To evaluate the prevalence and genetic diversity of SaV in hospitalized children aged less than 5 years with acute gastroenteritis. Stool samples were collected from children with acute gastroenteritis at three hospitals in the Philippines from June 2012 to August 2013. SaV was detected by reverse transcription real-time PCR, and the polymerase and capsid gene sequences were analyzed. Full genome sequencing and recombination analysis were performed on possible recombinant viruses. SaV was detected in 7.0% of the tested stool samples (29/417). In 10 SaV-positive cases, other viruses were also detected, including rotavirus (n=6), norovirus (n=2), and human astrovirus (n=2). Four known SaV genotypes (GI.1 [7], GI.2 [2], GII.1 [12], and GV [2]) and one novel recombinant (n=3) were identified by polymerase and capsid gene sequence analysis. Full genome sequencing revealed that the 5' nontranslated region (NTR) and nonstructural protein region of the novel recombinant were closely related to the GII.1 Bristol/98/UK variant, whereas the structural protein region and 3' NTR were closely related to the GII.4 Kumamoto6/Mar2003/JPN variant. SaV was regularly detected in hospitalized children due to acute gastroenteritis during the study period. A novel recombinant, SaV GII.1/GII.4, was identified in three cases at two different study sites. Copyright © 2015 The Authors. Published by Elsevier B.V. All rights reserved.

7 CFR 331.13 - Restricted experiments. 5

Code of Federal Regulations, 2010 CFR

2010-01-01

... Recombinant DNA Molecules.” This document is available on the Internet at http://www.aphis.usda.gov/programs...) Experiments utilizing recombinant DNA that involve the deliberate transfer of a drug resistance trait to...) Experiments involving the deliberate formation of recombinant DNA containing genes for the biosynthesis of...

7 CFR 331.13 - Restricted experiments. 5

Code of Federal Regulations, 2011 CFR

2011-01-01

... Recombinant DNA Molecules.” This document is available on the Internet at http://www.aphis.usda.gov/programs...) Experiments utilizing recombinant DNA that involve the deliberate transfer of a drug resistance trait to...) Experiments involving the deliberate formation of recombinant DNA containing genes for the biosynthesis of...

7 CFR 331.13 - Restricted experiments. 5

Code of Federal Regulations, 2012 CFR

2012-01-01

... Recombinant DNA Molecules.” This document is available on the Internet at http://www.aphis.usda.gov/programs...) Experiments utilizing recombinant DNA that involve the deliberate transfer of a drug resistance trait to...) Experiments involving the deliberate formation of recombinant DNA containing genes for the biosynthesis of...

Genome Sequence Analysis of New Isolates of the Winona Strain of Plum pox virus and the First Definitive Evidence of Intrastrain Recombination Events.

PubMed

James, Delano; Sanderson, Dan; Varga, Aniko; Sheveleva, Anna; Chirkov, Sergei

2016-04-01

Plum pox virus (PPV) is genetically diverse with nine different strains identified. Mutations, indel events, and interstrain recombination events are known to contribute to the genetic diversity of PPV. This is the first report of intrastrain recombination events that contribute to PPV's genetic diversity. Fourteen isolates of the PPV strain Winona (W) were analyzed including nine new strain W isolates sequenced completely in this study. Isolates of other strains of PPV with more than one isolate with the complete genome sequence available in GenBank were included also in this study for comparison and analysis. Five intrastrain recombination events were detected among the PPV W isolates, one among PPV C strain isolates, and one among PPV M strain isolates. Four (29%) of the PPV W isolates analyzed are recombinants; one of which (P2-1) is a mosaic, with three recombination events identified. A new interstrain recombinant event was identified between a strain M isolate and a strain Rec isolate, a known recombinant. In silico recombination studies and pairwise distance analyses of PPV strain D isolates indicate that a threshold of genetic diversity exists for the detectability of recombination events, in the range of approximately 0.78×10(-2) to 1.33×10(-2) mean pairwise distance. RDP4 analyses indicate that in the case of PPV Rec isolates there may be a recombinant breakpoint distinct from the obvious transition point of strain sequences. Evidence was obtained that indicates that the frequency of PPV recombination is underestimated, which may be true for other RNA viruses where low genetic diversity exists.

The recombinant expression and activity detection of MAF-1 fusion protein.

PubMed

Fu, Ping; Wu, Jianwei; Gao, Song; Guo, Guo; Zhang, Yong; Liu, Jian

2015-10-01

This study establishes the recombinant expression system of MAF-1 (Musca domestica antifungal peptide-1) and demonstrates the antifungal activity of the expression product and shows the relationship between biological activity and structure. The gene segments on mature peptide part of MAF-1 were cloned, based on the primers designed according to the cDNA sequence of MAF-1. We constructed the recombinant prokaryotic expression plasmid using prokaryotic expression vector (pET-28a(+)) and converted it to the competent cell of BL21(DE3) to gain recombinant MAF-1 fusion protein with His tag sequence through purifying affinity chromatographic column of Ni-NTA. To conduct the Western Blotting test, recombinant MAF-1 fusion protein was used to produce the polyclonal antibody of rat. The antifungal activity of the expression product was detected using Candida albicans (ATCC10231) as the indicator. The MAF-1 recombinant fusion protein was purified to exhibit obvious antifungal activity, which lays the foundation for the further study of MAF-1 biological activity, the relationship between structure and function, as well as control of gene expression.

An 11-Year Surveillance of HIV Type 1 Subtypes in Nagoya, Japan.

PubMed

Fujisaki, Seiichiro; Ibe, Shiro; Hattori, Junko; Shigemi, Urara; Fujisaki, Saeko; Shimizu, Kayoko; Nakamura, Kazuyo; Yokomaku, Yoshiyuki; Mamiya, Naoto; Utsumi, Makoto; Hamaguchi, Motohiro; Kaneda, Tsuguhiro

2009-01-01

Abstract To monitor active HIV-1 transmission in Nagoya, Japan, we have been determining the subtypes of HIV-1 infecting therapy-naive individuals who have newly visited the Nagoya Medical Center since 1997. The subtypes were determined by phylogenetic analyses using the base sequences in three regions of the HIV-1 genes including gag p17, pol protease (PR) and reverse transcriptase (RT), and env C2V3. Almost all HIV-1 subtypes from 1997 to 2007 and 93% of all HIV-1 isolates in 2007 were subtype B. HIV-1 subtypes A, C, D, and F have been detected sporadically since 1997, almost all in Africans and South Americans. The first detected circulating recombinant form (CRF ) was CRF01_AE (11-year average annual detection rate, 7.7%). Only two cases of CRF02_AG were detected in 2006. A unique recombinant form (URF ) was first detected in 1998 and the total number of URFs reached 25 by year 2007 (average annual detection rate, 4.7%). Eleven of these 25 were detected from 2000 to 2005 and had subtypes AE/B/AE as determined by base sequencing of the gag p17, pol PR and RT, and env C2V3 genes (average annual detection rate, 3.7%). Unique subtype B has been detected in six cases since 2006. All 17 of these patients were Japanese. Other recombinant HIV-1s have been detected intermittently in eight cases since 1998. During the 11-year surveillance, most HIV-1s in Nagoya, Japan were of subtype B. We expect that subtype B HIV-1 will continue to predominate for the next several years. Active recombination between subtype B and CRF01_AE HIV-1 and its transmission were also shown.

Absence of detectable mitochondrial recombination in Paramecium.

PubMed

Adoutte, A; Knowles, J K; Sainsard-Chanet, A

1979-12-01

An extensive search for recombination between mitochondrial markers was carried out in Paramecium tetraurelia. Thirty-two combinations, altogether involving 24 different markers, were studied. The markers belonged to the three main categories of mitochondrial mutations presently available in this organism, (a) Spontaneous or UV-induced antibiotic resistance mutations, most probably affecting mitochondrial ribosomes, (b) nitrosoguanidine-induced antibiotic resistance markers displaying thermosensitivity or slow growth, enabling easy selection of possible wild-type recombinants, and (c) mitochondrial partial suppressors of a nuclear gene, probably corresponding to molecular alterations distinct from the preceding two categories. In addition, different genetic configurations were analyzed (i.e., mutant X mutant, double-mutant X wild-type, etc.).--None of the combinations yielded any evidence for the occurrence of recombined genomes despite the fact that: (1) all of them were studied on a large scale involving the screening of at least several thousand mitochondrial genomes (often several millions), (2) in many of them the detection level was sufficiently high to enable the isolation of spontaneous mutants in control cells, and (3) in several of them, reconstitution experiments carried out in parallel show that the conditions were fully adequate to detect recombinant genotypes. The results are in marked contrast with those obtained on the few other organisms in which mitochondrial recombination has been studied, particularly Saccharomyces cerevisiae, in which mitochondrial recombination is intense.--The most likely basis for the various manifestations of mitochondrial genetic autonomy in Paramecium, described in this as well as in previous publications, is that the chondriome of this organism is made up of thousands of structurally discrete, noninteracting units.

The typical RB76 recombination breakpoint of the invasive recombinant tomato yellow leaf curl virus of Morocco can be generated experimentally but is not positively selected in tomato.

PubMed

Belabess, Z; Urbino, C; Granier, M; Tahiri, A; Blenzar, A; Peterschmitt, M

2018-01-02

TYLCV-IS76 is an unusual recombinant between the highly recombinogenic tomato yellow leaf curl virus (TYLCV) and tomato yellow leaf curl Sardinia virus (TYLCSV), two Mediterranean begomoviruses (Geminiviridae). In contrast with the previously reported TYLCV/TYLCSV recombinants, it has a TYLCSV derived fragment of only 76 nucleotides, and has replaced its parental viruses in natural conditions (Morocco, Souss region). The viral population shift coincided with the deployment of the popular Ty-1 resistant tomato cultivars, and according to experimental studies, has been driven by a strong positive selection in such resistant plants. However, although Ty-1 cultivars were extensively used in Mediterranean countries, TYLCV-IS76 was not reported outside Morocco. This, in combination with its unusual recombination pattern suggests that it was generated through a rare and possibly multistep process. The potential generation of a recombination breakpoint (RB) at locus 76 (RB76) was investigated over time in 10 Ty-1 resistant and 10 nearly isogenic susceptible tomato plants co-inoculated with TYLCV and TYLCSV clones. RB76 could not be detected in the recombinant progeny using the standard PCR/sequencing approach that was previously designed to monitor the emergence of TYLCV-IS76 in Morocco. Using a more sensitive PCR test, RB76 was detected in one resistant and five susceptible plants. The results are consistent with a very low intra-plant frequency of RB76 bearing recombinants throughout the test and support the hypothesis of a rare emergence of TYLCV-IS76. More generally, RBs were more scattered in resistant than in susceptible plants and an unusual RB at position 141 (RB141) was positively selected in the resistant cultivar; interestingly, RB141 bearing recombinants were detected in resistant tomato plants from the field. Scenarios of TYLCV-IS76 pre-emergence are proposed. Copyright © 2017 Elsevier B.V. All rights reserved.

Widespread recombination in published animal mtDNA sequences.

PubMed

Tsaousis, A D; Martin, D P; Ladoukakis, E D; Posada, D; Zouros, E

2005-04-01

Mitochondrial DNA (mtDNA) recombination has been observed in several animal species, but there are doubts as to whether it is common or only occurs under special circumstances. Animal mtDNA sequences retrieved from public databases were unambiguously aligned and rigorously tested for evidence of recombination. At least 30 recombination events were detected among 186 alignments examined. Recombinant sequences were found in invertebrates and vertebrates, including primates. It appears that mtDNA recombination may occur regularly in the animal cell but rarely produces new haplotypes because of homoplasmy. Common animal mtDNA recombination would necessitate a reexamination of phylogenetic and biohistorical inference based on the assumption of clonal mtDNA transmission. Recombination may also have an important role in producing and purging mtDNA mutations and thus in mtDNA-based diseases and senescence.

Stepwise detection of recombination breakpoints in sequence alignments.

PubMed

Graham, Jinko; McNeney, Brad; Seillier-Moiseiwitsch, Françoise

2005-03-01

We propose a stepwise approach to identify recombination breakpoints in a sequence alignment. The approach can be applied to any recombination detection method that uses a permutation test and provides estimates of breakpoints. We illustrate the approach by analyses of a simulated dataset and alignments of real data from HIV-1 and human chromosome 7. The presented simulation results compare the statistical properties of one-step and two-step procedures. More breakpoints are found with a two-step procedure than with a single application of a given method, particularly for higher recombination rates. At higher recombination rates, the additional breakpoints were located at the cost of only a slight increase in the number of falsely declared breakpoints. However, a large proportion of breakpoints still go undetected. A makefile and C source code for phylogenetic profiling and the maximum chi2 method, tested with the gcc compiler on Linux and WindowsXP, are available at http://stat-db.stat.sfu.ca/stepwise/ jgraham@stat.sfu.ca.

Nonreplicative RNA Recombination of an Animal Plus-Strand RNA Virus in the Absence of Efficient Translation of Viral Proteins.

PubMed

Kleine Büning, Maximiliane; Meyer, Denise; Austermann-Busch, Sophia; Roman-Sosa, Gleyder; Rümenapf, Tillmann; Becher, Paul

2017-04-01

RNA recombination is a major driving force for the evolution of RNA viruses and is significantly implicated in the adaptation of viruses to new hosts, changes of virulence, as well as in the emergence of new viruses including drug-resistant and escape mutants. However, the molecular details of recombination in animal RNA viruses are only poorly understood. In order to determine whether viral RNA recombination depends on translation of viral proteins, a nonreplicative recombination system was established which is based on cotransfection of cells with synthetic bovine viral diarrhea virus (family Flaviviridae) RNA genome fragments either lacking the internal ribosome entry site required for cap-independent translation or lacking almost the complete polyprotein coding region. The emergence of a number of recombinant viruses demonstrated that IRES-mediated translation of viral proteins is dispensable for efficient recombination and suggests that RNA recombination can occur in the absence of viral proteins. Analyses of 58 independently emerged viruses led to the detection of recombinant genomes with duplications, deletions and insertions in the 5' terminal region of the open reading frame, leading to enlarged core fusion proteins detectable by Western blot analysis. This demonstrates a remarkable flexibility of the pestivirus core protein. Further experiments with capped and uncapped genome fragments containing a luciferase gene for monitoring the level of protein translation revealed that even a ∼1,000-fold enhancement of translation of viral proteins did not increase the frequency of RNA recombination. Taken together, this study highlights that nonreplicative RNA recombination does not require translation of viral proteins. © The Author(s) 2017. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.

Use of bovine recombinant prion protein and real-time quaking-induced conversion to detect cattle transmissible mink encephalopathy prions and discriminate classical and atypical L- and H-Type bovine spongiform encephalopathy.

PubMed

Hwang, Soyoun; Greenlee, Justin J; Nicholson, Eric M

2017-01-01

Prions are amyloid-forming proteins that cause transmissible spongiform encephalopathies through a process involving conversion from the normal cellular prion protein to the pathogenic misfolded conformation (PrPSc). This conversion has been used for in vitro assays including serial protein misfolding amplification and real-time quaking induced conversion (RT-QuIC). RT-QuIC can be used for the detection of prions in a variety of biological tissues from humans and animals. Extensive work has been done to demonstrate that RT-QuIC is a rapid, specific, and highly sensitive prion detection assay. RT-QuIC uses recombinant prion protein to detect minute amounts of PrPSc. RT-QuIC has been successfully used to detect PrPSc from different prion diseases with a variety of substrates including hamster, human, sheep, bank vole, bovine and chimeric forms of prion protein. However, recombinant bovine prion protein has not been used to detect transmissible mink encephalopathy (TME) or to differentiate types of bovine spongiform encephalopathy (BSE) in samples from cattle. We evaluated whether PrPSc from TME and BSE infected cattle can be detected with RT-QuIC using recombinant bovine prion proteins, and optimized the reaction conditions to specifically detect cattle TME and to discriminate between classical and atypical BSE by conversion efficiency. We also found that substrate composed of the disease associated E211K mutant protein can be effective for the detection of TME in cattle and that wild type prion protein appears to be a practical substrate to discriminate between the different types of BSEs.

The high mobility group protein Abf2p influences the level of yeast mitochondrial DNA recombination intermediates in vivo.

PubMed

MacAlpine, D M; Perlman, P S; Butow, R A

1998-06-09

Abf2p is a high mobility group (HMG) protein found in yeast mitochondria that is required for the maintenance of wild-type (rho+) mtDNA in cells grown on fermentable carbon sources, and for efficient recombination of mtDNA markers in crosses. Here, we show by two-dimensional gel electrophoresis that Abf2p promotes or stabilizes Holliday recombination junction intermediates in rho+ mtDNA in vivo but does not influence the high levels of recombination intermediates readily detected in the mtDNA of petite mutants (rho-). mtDNA recombination junctions are not observed in rho+ mtDNA of wild-type cells but are elevated to detectable levels in cells with a null allele of the MGT1 gene (Deltamgt1), which codes for a mitochondrial cruciform-cutting endonuclease. The level of recombination intermediates in rho+ mtDNA of Deltamgt1 cells is decreased about 10-fold if those cells contain a null allele of the ABF2 gene. Overproduction of Abf2p by >/= 10-fold in wild-type rho+ cells, which leads to mtDNA instability, results in a dramatic increase in mtDNA recombination intermediates. Specific mutations in the two Abf2p HMG boxes required for DNA binding diminishes these responses. We conclude that Abf2p functions in the recombination of rho+ mtDNA.

Genetic structure and variability of virus populations in cross-protected grapevines superinfected by Grapevine fanleaf virus.

PubMed

Vigne, Emmanuelle; Marmonier, Aurélie; Komar, Véronique; Lemaire, Olivier; Fuchs, Marc

2009-09-01

Recombination was assessed in a vineyard site in which grapevines cross-protected with mild strains GHu of Grapevine fanleaf virus (GFLV) or Ta of Arabis mosaic virus (ArMV) were superinfected with GFLV field isolates following transmission by the nematode vector Xiphinema index. The genetic structure and variability within RNA2 of isolates from grapevines co-infected with GFLV field isolates and either GFLV-GHu or ArMV-Ta were characterized to identify intra- and interspecies recombinants. Sequence analysis and phylogenetic relationships inferred intraspecies recombination among GFLV field isolates but not between field isolates and GFLV-GHu. SISCAN analysis confirmed a mosaic structure for two GFLV field isolates for which recombination sites were located in the movement protein and coat protein genes. One of the recombinants was found in eight grapevines that were in close spatial proximity within the vineyard site, suggesting its transmission by X. index. No interspecies recombination was detected between GFLV field isolates and ArMV-Ta. Altogether, our findings suggest that mild protective strains GFLV-GHu and ArMV-Ta did not assist the emergence of viable recombinants to detectable level during a 12-year cross-protection trial. To our knowledge, this is the first extensive characterization of the genetic structure and variability of virus isolates in cross-protected plants.

Relative abundance of deformed wing virus, Varroa destructor virus 1, and their recombinants in honey bees (Apis mellifera) assessed by kmer analysis of public RNA-Seq data

USGS Publications Warehouse

Cornman, Robert S.

2017-01-01

Deformed wing virus (DWV) is a major pathogen of concern to apiculture, and recent reports have indicated the local predominance and potential virulence of recombinants between DWV and a related virus, Varroa destructor virus 1 (VDV). However, little is known about the frequency and titer of VDV and recombinants relative to DWV generally. In this study, I assessed the relative occurrence and titer of DWV and VDV in public RNA-seq accessions of honey bee using a rapid, kmer-based approach. Three recombinant types were detectable graphically and corroborated by de novo assembly. Recombination breakpoints did not disrupt the capsid-encoding region, consistent with previous reports, and both VDV- and DWV-derived capsids were observed in recombinant backgrounds. High abundance of VDV kmers was largely restricted to recombinant forms. Non-metric multidimensional scaling identified genotypic clusters among DWV isolates, which was corroborated by read mapping and consensus generation. The recently described DWV-C lineage was not detected in the searched accessions. The data further highlight the utility of high-throughput sequencing to monitor viral polymorphisms and statistically test biological predictors of titer, and point to the need for consistent methodologies and sampling schemes.

Comparative evaluation of the diagnostic potential of recombinant envelope proteins and native cell culture purified viral antigens of Chikungunya virus.

PubMed

Khan, Mohsin; Dhanwani, Rekha; Kumar, Jyoti S; Rao, P V Lakshmana; Parida, Manmohan

2014-07-01

Despite the fact that Chikungunya resurgence is associated with epidemic of unprecedented magnitude, there are challenges in the field of its clinical diagnosis. However, serological tests in an ELISA format provide a rapid tool for the diagnosis of Chikungunya infection. Indeed, ELISAs based on recombinant proteins hold a great promise as these methods are cost effective and are free from the risk of handling biohazardous material. In this study, the performance of recombinant CHIKV antigens was compared in various ELISA formats for the diagnosis of Chikungunya. Two recombinant antigens derived from the envelope proteins of Chikungunya virus were prepared and evaluated by comparing their competence for detecting circulating antibodies in serum samples of patients infected with CHIKV using MAC-ELISA and indirect IgM-ELISA. The efficacy of the recombinant antigens was also compared with the native antigen. The indirect antibody capture IgM microplate ELISA revealed ≥90% concordance with the native antigen in detecting the CHIKV specific IgM antibodies whereas the recombinant antigen based MAC-ELISA showed 100% specificity. The recombinant antigens used in this study were effective and reliable targets for the diagnosis of CHIKV infection and also provide an alternative for native antigen use which is potentially biohazardous. © 2013 Wiley Periodicals, Inc.

Generation and characterization of a Leishmania tarentolae strain for site-directed in vivo biotinylation of recombinant proteins.

PubMed

Klatt, Stephan; Hartl, Daniela; Fauler, Beatrix; Gagoski, Dejan; Castro-Obregón, Susana; Konthur, Zoltán

2013-12-06

Leishmania tarentolae is a non-human-pathogenic Leishmania species of growing interest in biotechnology, as it is well-suited for the expression of human recombinant proteins. For many applications it is desirable to express recombinant proteins with a tag allowing easy purification and detection. Hence, we adopted a scheme to express recombinant proteins with a His6-tag and, additionally, to site-specifically in vivo biotinylate them for detection. Biotinylation is a relatively rare modification of endogenous proteins that allows easy detection with negligible cross-reactivity. Here, we established a genetically engineered L. tarentolae strain constitutively expressing the codon-optimized biotin-protein ligase from Escherichia coli (BirA). We thoroughly analyzed the strain for functionality using 2-D polyacrylamide-gel electrophoresis (PAGE), mass spectrometry, and transmission electron microscopy (TEM). We could demonstrate that neither metabolic changes (growth rate) nor structural abnormalities (TEM) occurred. To our knowledge, we show the first 2-D PAGE analyses of L. tarentolae. Our results demonstrate the great benefit of the established L. tarentolae in vivo biotinylation strain for production of dual-tagged recombinant proteins. Additionally, 2-D PAGE and TEM results give insights into the biology of L. tarentolae, helping to better understand Leishmania species. Finally, we envisage that the system is transferable to human-pathogenic species.

Detection of biomarkers using recombinant antibodies coupled to nanostructured platforms

PubMed Central

Kierny, Michael R.; Cunningham, Thomas D.; Kay, Brian K.

2012-01-01

The utility of biomarker detection in tomorrow's personalized health care field will mean early and accurate diagnosis of many types of human physiological conditions and diseases. In the search for biomarkers, recombinant affinity reagents can be generated to candidate proteins or post-translational modifications that differ qualitatively or quantitatively between normal and diseased tissues. The use of display technologies, such as phage-display, allows for manageable selection and optimization of affinity reagents for use in biomarker detection. Here we review the use of recombinant antibody fragments, such as scFvs and Fabs, which can be affinity-selected from phage-display libraries, to bind with both high specificity and affinity to biomarkers of cancer, such as Human Epidermal growth factor Receptor 2 (HER2) and Carcinoembryonic antigen (CEA). We discuss how these recombinant antibodies can be fabricated into nanostructures, such as carbon nanotubes, nanowires, and quantum dots, for the purpose of enhancing detection of biomarkers at low concentrations (pg/mL) within complex mixtures such as serum or tissue extracts. Other sensing technologies, which take advantage of ‘Surface Enhanced Raman Scattering’ (gold nanoshells), frequency changes in piezoelectric crystals (quartz crystal microbalance), or electrical current generation and sensing during electrochemical reactions (electrochemical detection), can effectively provide multiplexed platforms for detection of cancer and injury biomarkers. Such devices may soon replace the traditional time consuming ELISAs and Western blots, and deliver rapid, point-of-care diagnostics to market. PMID:22833780

Genome-wide detection of conservative site-specific recombination in bacteria

PubMed Central

Mathias Garrett, Elizabeth; Camilli, Andrew

2018-01-01

The ability of clonal bacterial populations to generate genomic and phenotypic heterogeneity is thought to be of great importance for many commensal and pathogenic bacteria. One common mechanism contributing to diversity formation relies on the inversion of small genomic DNA segments in a process commonly referred to as conservative site-specific recombination. This phenomenon is known to occur in several bacterial lineages, however it remains notoriously difficult to identify due to the lack of conserved features. Here, we report an easy-to-implement method based on high-throughput paired-end sequencing for genome-wide detection of conservative site-specific recombination on a single-nucleotide level. We demonstrate the effectiveness of the method by successfully detecting several novel inversion sites in an epidemic isolate of the enteric pathogen Clostridium difficile. Using an experimental approach, we validate the inversion potential of all detected sites in C. difficile and quantify their prevalence during exponential and stationary growth in vitro. In addition, we demonstrate that the master recombinase RecV is responsible for the inversion of some but not all invertible sites. Using a fluorescent gene-reporter system, we show that at least one gene from a two-component system located next to an invertible site is expressed in an on-off mode reminiscent of phase variation. We further demonstrate the applicability of our method by mining 209 publicly available sequencing datasets and show that conservative site-specific recombination is common in the bacterial realm but appears to be absent in some lineages. Finally, we show that the gene content associated with the inversion sites is diverse and goes beyond traditionally described surface components. Overall, our method provides a robust platform for detection of conservative site-specific recombination in bacteria and opens a new avenue for global exploration of this important phenomenon. PMID:29621238

Recombinant HT{sub m4} gene, protein and assays

DOEpatents

Lim, B.; Adra, C.N.; Lelias, J.M.

1996-09-03

The invention relates to a recombinant DNA molecule which encodes a HT{sub m4} protein, a transformed host cell which has been stably transfected with a DNA molecule which encodes a HT{sub m4} protein and a recombinant HT{sub m4} protein. The invention also relates to a method for detecting the presence of a hereditary atopy. 2 figs.

Recombinant HT.sub.m4 gene, protein and assays

DOEpatents

Lim, Bing; Adra, Chaker N.; Lelias, Jean-Michel

1996-01-01

The invention relates to a recombinant DNA molecule which encodes a HT.sub.m4 protein, a transformed host cell which has been stably transfected with a DNA molecule which encodes a HT.sub.m4 protein and a recombinant HT.sub.m4 protein. The invention also relates to a method for detecting the presence of a hereditary atopy.

Termini of human chromosomes display elevated rates of mitotic recombination.

PubMed

Cornforth, M N; Eberle, R L

2001-01-01

The strand-specific in situ hybridization technique of CO-FISH was used to probe telomeres of human mitotic cells in order to determine the spontaneous frequency of crossover. This approach allowed the detection of recombinational crossovers occurring anywhere along the length of individual chromosomes, including reciprocal events taking place between sister chromatids. Although the process of sister chromatid exchange (SCE) is the most prominent type of recombination in somatic mammalian cells, our results show that SCEs accounted for less than a third of the recombinational events revealed by CO-FISH. It is concluded that chromosomal regions near the termini of chromosome arms undergo extraordinarily high rates of spontaneous recombination, producing terminal crossovers whose small size precludes detection by standard cytogenetic methods. That similar results were observed for transformed epithelial cells, as well as primary fibroblasts, suggests that the phenomenon is a common characteristic of human cells. These findings are noteworthy because, although telomeric and subtelomeric DNA is known to be preferentially involved in certain types of recombination, the tips of somatic mammalian chromosomes have not previously been identified as preferred sites for crossover. Implications of these results are discussed in terms of limitations imposed on CO-FISH for its proposed use in directional hybridization mapping.

Production of recombinant dengue non-structural 1 (NS1) proteins from clinical virus isolates.

PubMed

Yohan, Benediktus; Wardhani, Puspa; Aryati; Trimarsanto, Hidayat; Sasmono, R Tedjo

2017-01-01

Dengue is a febrile disease caused by infection of dengue virus (DENV). Early diagnosis of dengue infection is important for better management of the disease. The DENV Non-Structural Protein 1 (NS1) antigen has been routinely used for the early dengue detection. In dengue epidemic countries such as Indonesia, clinicians are increasingly relying on the NS1 detection for confirmation of dengue infection. Various NS1 diagnostic tests are commercially available, however different sensitivities and specificities were observed in various settings. This study was aimed to generate dengue NS1 recombinant protein for the development of dengue diagnostic tests. Four Indonesian DENV isolates were used as the source of the NS1 gene cloning, expression, and purification in bacterial expression system. Recombinant NS1 proteins were successfully purified and their antigenicities were assessed. Immunization of mice with recombinant proteins observed the immunogenicity of the NS1 protein. The generated recombinant proteins can be potentially used in the development of NS1 diagnostic test. With minimal modifications, this method can be used for producing NS1 recombinant proteins from isolates obtained from other geographical regions. Copyright © 2016 Elsevier Inc. All rights reserved.

Low pathogenic avian influenza (H9N2) in chicken: Evaluation of an ancestral H9-MVA vaccine.

PubMed

Ducatez, Mariette F; Becker, Jens; Freudenstein, Astrid; Delverdier, Maxence; Delpont, Mattias; Sutter, Gerd; Guérin, Jean-Luc; Volz, Asisa

2016-06-30

Modified Vaccinia Ankara (MVA) has proven its efficacy as a recombinant vector vaccine for numerous pathogens including influenza virus. The present study aimed at evaluating a recombinant MVA candidate vaccine against low pathogenic avian influenza virus subtype H9N2 in the chicken model. As the high genetic and antigenic diversity of H9N2 viruses increases vaccine design complexity, one strategy to widen the range of vaccine coverage is to use an ancestor sequence. We therefore generated a recombinant MVA encoding for the gene sequence of an ancestral hemagglutinin H9 protein (a computationally derived amino acid sequence of the node of the H9N2 G1 lineage strains was obtained using the ANCESCON program). We analyzed the genetics and the growth properties of the MVA vector virus confirming suitability for use under biosafety level 1 and tested its efficacy when applied either as an intra-muscular (IM) or an oral vaccine in specific pathogen free chickens challenged with A/chicken/Tunisia/12/2010(H9N2). Two control groups were studied in parallel (unvaccinated and inoculated birds; unvaccinated and non-inoculated birds). IM vaccinated birds seroconverted as early as four days post vaccination and neutralizing antibodies were detected against A/chicken/Tunisia/12/2010(H9N2) in all the birds before challenge. The role of local mucosal immunity is unclear here as no antibodies were detected in eye drop or aerosol vaccinated birds. Clinical signs were not detected in any of the infected birds even in absence of vaccination. Virus replication was observed in both vaccinated and unvaccinated chickens, suggesting the MVA-ancestral H9 vaccine may not stop virus spread in the field. However vaccinated birds showed less histological damage, fewer influenza-positive cells and shorter virus shedding than their unvaccinated counterparts. Copyright © 2016 Elsevier B.V. All rights reserved.

Expression of Hepatitis C Virus Core and E2 antigenic recombinant proteins and their use for development of diagnostic assays.

PubMed

Ali, Amjad; Nisar, Muhammad; Idrees, Muhammad; Rafique, Shazia; Iqbal, Muhammad

2015-05-01

Early diagnosis of HCV infection is based on detection of antibodies against HCV proteins using recombinant viral antigens. The present study was designed to select, clone and express the antigenic regions of Core and E2 genes from local HCV-3a genotype and to utilize the antigenic recombinant proteins (Core & E2) to develop highly sensitive, specific and economical diagnostic assays for detection of HCV infection. The antigenic sites were determined within Core and E2 genes and were then cloned in pET-28a expression vector. The right orientation of the desired inserted fragments of Core and E2 were confirmed via sequencing prior to expression and were then transformed in BL21 (DE3) pLysS strains of E. coli and induced with 0.5mM Isopropyl-b-D-thiogalactopyranoside (IPTG) for the production of antigenic recombinant proteins. The produced truncated antigens were then purified by Nickel affinity chromatography and were confirmed by western blotting, immunoblotting and enzyme-linked immunosorbent assay (ELISA). The expressed Core and E2 recombinant antigens were used to develop immunoblotting assay for the detection of anti-HCV antibodies in sera. With immunoblotting, a total of 93-HCV infected sera and 35-HCV negative individuals were tested for the presence of anti-HCV antibodies to the Core and E2 antigens. Recombinant antigen showed 100% reactivity against HCV infected sera, with no cross reactivity against HCV-negative sera. The immunoblot assay mixture of recombinant antigens (Core+E2) showed a strong reaction intensity in the test area (TA) as compared to the individual truncated Core and E2 recombinant antigens. In the in-house ELISA assay, mixed Core and E2 recombinant antigens showed 100% reactivity against a standardized panel of 150-HCV-positive sera and non reactivity against a standardized panel of 150 HCV-negative sera while also being non reactive to sera positive for other viral infections. The antigenic recombinant antigens also were tested for the 30 sera of known genotypes. The antigens did not detect antibodies to genotype-3a, but detected antibodies to all genotypes and did not discriminate them genotype wise. A panel of 175 of HCV-suspected serum samples was subjected to comparative analysis with our in-house ELISA assay and with commercial HCV screening assays. After subjecting the results to the formulas for determining the quality parameters, immunoblot assay had 100% sensitivity and specificity, while the ELISA assay had 100% sensitivity and 98.8% specificity as compared to commercially available assays. This study indicates that a mixture of Core and E2 antigens are potentially valuable antigens and there is the possibility of developing serological assays for monitoring HCV infection. Copyright © 2015 The Authors. Published by Elsevier Ltd.. All rights reserved.

[The preparation of recombinant adenovirus Ad-Rad50-GFP and detection of the optimal multiplicity of infection in CNE1 transfected hv Ad-Rad50-GFP].

PubMed

Yan, Ruicheng; Huang, Jiancong; Zhu, Ling; Chang, Lihong; Li, Jingjia; Wu, Xifu; Ye, Jin; Zhang, Gehua

2015-12-01

The optimal multiplicity of infection (MOI) of the recombinant adenovirus Ad-Rad50-GFP carrying a mutant Rad50 gene expression region on the cell growth of nasopharyngeal carcinoma and the viral amplification efficiency of CNE1 cell infected by this adenovirus were studied. The biological titer of Ad-Rad50-GFP was measured by end point dilution method. The impact of recombinant adenoviral vector transfection on the growth of CNE1 cells was observed by cell growth curve. Transfection efficacy of recombinant adenoviral vector was observed and calculated through fluorescence microscope. The expression f mutant Rad50 in the Ad-Rad50-GFP transfected CNE1 cells with optimal MOI was detected by Western Blot after transfection. The biological titer of Ad-Rad50-GFP was 1.26 x 10¹¹ pfu/ml. CNE1 cell growth was not influenced significantly as they were transfected by recombinant adenoviral vector with MOI less than 50. Transfection efficacy of recombinant adenoviral vector was most salient at 24 hours after transfection, with the high expression of mutant Rad50, and the efficiency still remained about 70% after 72 hours. Recombinant adenoviral vector Ad-Rad50-GFP could transfect CNE1 cells as well as result in the expression of mutant Rad50 in CNE1 cells effectively. MOI = 50 was the optimal multiplicity of infection of CNE1 cells transfected by recombinant adenoviral vector Ad-Rad50-GFP.

Characterization of novel human papillomavirus types 157, 158 and 205 from healthy skin and recombination analysis in genus γ-Papillomavirus.

PubMed

Bolatti, Elisa M; Chouhy, Diego; Casal, Pablo E; Pérez, Germán R; Stella, Emma J; Sanchez, Adriana; Gorosito, Mario; Bussy, Ramón Fernandez; Giri, Adriana A

2016-08-01

Gammapapillomavirus (γ-PV) is a diverse and rapidly expanding genus, currently consisting of 79 fully characterized human PV (HPV) types. In this study, three novel types, HPV157, HPV158 and HPV205, obtained from healthy sun-exposed skin of two immunocompetent individuals, were amplified by the "Hanging droplet" long PCR technique, cloned, sequenced and characterized. HPV157, HPV158 and HPV205 genomes comprise 7154-bp, 7192-bp and 7298-bp, respectively, and contain four early (E1, E2, E6 and E7) and two late genes (L1 and L2). Phylogenetic analysis of the L1 ORF placed all novel types within the γ-PV genus: HPV157 was classified as a new member of species γ-12 while HPV158 and HPV205 belong to species γ-1. We then explored potential recombination events in genus γ-PV with the RDP4 program in a dataset of 74 viruses (71 HPV types with available full-length genomes and the 3 novel types). Two events, both located in the E1 ORF, met the inclusion criterion (p-values <0.05 with at least four methods) and persisted in different ORF combinations: an inter-species recombination in species γ-8 (major and minor parents: species γ-24 and γ-11, respectively), and an intra-species recombination in species γ-7 (recombinant strain: HPV170; major and minor parents: HPV-109 and HPV-149, respectively). These findings were confirmed by phylogenetic tree incongruence analysis. An additional incongruence was found in members of species γ-9 but it was not detected by the RDP4. This report expands our knowledge of the family Papillomaviridae and provides for the first time in silico evidence of recombination in genus γ-PV. Copyright © 2016 Elsevier B.V. All rights reserved.

Construction of live vaccines by using genetically engineered poxviruses: biological activity of recombinant vaccinia virus expressing influenza virus hemagglutinin.

PubMed Central

Panicali, D; Davis, S W; Weinberg, R L; Paoletti, E

1983-01-01

Recombinant vaccinia viruses containing the cloned hemagglutinin (HA) gene from influenza virus were constructed. The biological activity of these poxvirus vectors was demonstrated both in vitro and in vivo. Expression of HA in cells infected with recombinant vaccinia was detected by using specific anti-HA antiserum and 125I-labeled protein A, showing that HA synthesized under the regulation of vaccinia virus was antigenic. Immunization of rabbits with these recombinant poxviruses resulted in the production of antibodies reactive with authentic influenza HA as detected by radioimmunoassay, by inhibition of HA erythrocyte agglutination, and by neutralization of influenza virus infectivity. The production of antibodies directed against influenza HA suggested that the HA gene expressed in vaccinia is immunogenic. These data indicate the potential of genetically engineered poxviruses for use as generic live vaccine vehicles that have both human and veterinary applications. Images PMID:6310573

Atomic Interferometric Gravitational-Wave Space Observatory (AIGSO)

NASA Astrophysics Data System (ADS)

Gao, Dong-Feng; Wang, Jin; Zhan, Ming-Sheng

2018-01-01

We propose a space-borne gravitational-wave detection scheme, called atom interferometric gravitational-wave space observatory (AIGSO). It is motivated by the progress in the atomic matter-wave interferometry, which solely utilizes the standing light waves to split, deflect and recombine the atomic beam. Our scheme consists of three drag-free satellites orbiting the Earth. The phase shift of AIGSO is dominated by the Sagnac effect of gravitational-waves, which is proportional to the area enclosed by the atom interferometer, the frequency and amplitude of gravitational-waves. The scheme has a strain sensitivity < {10}-20/\\sqrt{{Hz}} in the 100 mHz-10 Hz frequency range, which fills in the detection gap between space-based and ground-based laser interferometric detectors. Thus, our proposed AIGSO can be a good complementary detection scheme to the space-borne laser interferometric schemes, such as LISA. Considering the current status of relevant technology readiness, we expect our AIGSO to be a promising candidate for the future space-based gravitational-wave detection plan. Supported by the National Key Research Program of China under Grant No. 2016YFA0302002, the National Science Foundation of China under Grant Nos. 11227803 and 91536221, and the Strategic Priority Research Program of the Chinese Academy of Sciences under Grant No. XDB21010100

A programmable Cas9-serine recombinase fusion protein that operates on DNA sequences in mammalian cells

PubMed Central

Chaikind, Brian; Bessen, Jeffrey L.; Thompson, David B.; Hu, Johnny H.; Liu, David R.

2016-01-01

We describe the development of ‘recCas9’, an RNA-programmed small serine recombinase that functions in mammalian cells. We fused a catalytically inactive dCas9 to the catalytic domain of Gin recombinase using an optimized fusion architecture. The resulting recCas9 system recombines DNA sites containing a minimal recombinase core site flanked by guide RNA-specified sequences. We show that these recombinases can operate on DNA sites in mammalian cells identical to genomic loci naturally found in the human genome in a manner that is dependent on the guide RNA sequences. DNA sequencing reveals that recCas9 catalyzes guide RNA-dependent recombination in human cells with an efficiency as high as 32% on plasmid substrates. Finally, we demonstrate that recCas9 expressed in human cells can catalyze in situ deletion between two genomic sites. Because recCas9 directly catalyzes recombination, it generates virtually no detectable indels or other stochastic DNA modification products. This work represents a step toward programmable, scarless genome editing in unmodified cells that is independent of endogenous cellular machinery or cell state. Current and future generations of recCas9 may facilitate targeted agricultural breeding, or the study and treatment of human genetic diseases. PMID:27515511

A novel strain of porcine deltacoronavirus in Vietnam.

PubMed

Le, Van Phan; Song, Sok; An, Byung-Hyun; Park, Gyu-Nam; Pham, Ngoc Thach; Le, Dinh Quyen; Nguyen, Van Tam; Vu, Thi Thu Hang; Kim, Ki-Sun; Choe, SeEun; An, Dong-Jun

2018-01-01

Two porcine deltacoronavirus (PDCoV) strains (Binh21 and HaNoi6) were isolated from two pig farms in North Vietnam. Phylogenetic analysis of the complete genomes and the Spike and Membrane genes revealed that the two Vietnam PDCoVs belong to the same lineage as PDCoVs from Thailand and Laos; however, the N genes belonged to the same lineage as PDCoVs from the USA, Korea, China, and Hong Kong. The recombination detection program subsequently identified the major parent (S5011 strain) and minor parent (HKU15-44 strain) of the two Vietnam PDCoV strains (p < 0.01).

Variation block-based genomics method for crop plants.

PubMed

Kim, Yul Ho; Park, Hyang Mi; Hwang, Tae-Young; Lee, Seuk Ki; Choi, Man Soo; Jho, Sungwoong; Hwang, Seungwoo; Kim, Hak-Min; Lee, Dongwoo; Kim, Byoung-Chul; Hong, Chang Pyo; Cho, Yun Sung; Kim, Hyunmin; Jeong, Kwang Ho; Seo, Min Jung; Yun, Hong Tai; Kim, Sun Lim; Kwon, Young-Up; Kim, Wook Han; Chun, Hye Kyung; Lim, Sang Jong; Shin, Young-Ah; Choi, Ik-Young; Kim, Young Sun; Yoon, Ho-Sung; Lee, Suk-Ha; Lee, Sunghoon

2014-06-15

In contrast with wild species, cultivated crop genomes consist of reshuffled recombination blocks, which occurred by crossing and selection processes. Accordingly, recombination block-based genomics analysis can be an effective approach for the screening of target loci for agricultural traits. We propose the variation block method, which is a three-step process for recombination block detection and comparison. The first step is to detect variations by comparing the short-read DNA sequences of the cultivar to the reference genome of the target crop. Next, sequence blocks with variation patterns are examined and defined. The boundaries between the variation-containing sequence blocks are regarded as recombination sites. All the assumed recombination sites in the cultivar set are used to split the genomes, and the resulting sequence regions are termed variation blocks. Finally, the genomes are compared using the variation blocks. The variation block method identified recurring recombination blocks accurately and successfully represented block-level diversities in the publicly available genomes of 31 soybean and 23 rice accessions. The practicality of this approach was demonstrated by the identification of a putative locus determining soybean hilum color. We suggest that the variation block method is an efficient genomics method for the recombination block-level comparison of crop genomes. We expect that this method will facilitate the development of crop genomics by bringing genomics technologies to the field of crop breeding.

Recombinant antibodies and their use in biosensors.

PubMed

Zeng, Xiangqun; Shen, Zhihong; Mernaugh, Ray

2012-04-01

Inexpensive, noninvasive immunoassays can be used to quickly detect disease in humans. Immunoassay sensitivity and specificity are decidedly dependent upon high-affinity, antigen-specific antibodies. Antibodies are produced biologically. As such, antibody quality and suitability for use in immunoassays cannot be readily determined or controlled by human intervention. However, the process through which high-quality antibodies can be obtained has been shortened and streamlined by use of genetic engineering and recombinant antibody techniques. Antibodies that traditionally take several months or more to produce when animals are used can now be developed in a few weeks as recombinant antibodies produced in bacteria, yeast, or other cell types. Typically most immunoassays use two or more antibodies or antibody fragments to detect antigens that are indicators of disease. However, a label-free biosensor, for example, a quartz-crystal microbalance (QCM) needs one antibody only. As such, the cost and time needed to design and develop an immunoassay can be substantially reduced if recombinant antibodies and biosensors are used rather than traditional antibody and assay (e.g. enzyme-linked immunosorbant assay, ELISA) methods. Unlike traditional antibodies, recombinant antibodies can be genetically engineered to self-assemble on biosensor surfaces, at high density, and correctly oriented to enhance antigen-binding activity and to increase assay sensitivity, specificity, and stability. Additionally, biosensor surface chemistry and physical and electronic properties can be modified to further increase immunoassay performance above and beyond that obtained by use of traditional methods. This review describes some of the techniques investigators have used to develop highly specific and sensitive, recombinant antibody-based biosensors for detection of antigens in simple or complex biological samples.

Molecular Mechanisms of Bcl10-Mediated NF-kappaB Signal Transduction

DTIC Science & Technology

2004-04-04

recombinant expressed protein, used for detection with specific anti -FLAG antibodies GFP (green fluorescent protein): a variant of the gene first...influenza virus hemagglutin tag added to a recombinant expressed protein, used for detection with specific anti -HA antibodies HEK-293T cell: an SV40...proteins by size SH3 (Src homology 3): conserved amino acid domain, consisting to two anti -parallel β sheets, that mediates interactions between

Genetic recombination of tick-borne flaviviruses among wild-type strains.

PubMed

Norberg, Peter; Roth, Anette; Bergström, Tomas

2013-06-05

Genetic recombination has been suggested to occur in mosquito-borne flaviviruses. In contrast, tick-borne flaviviruses have been thought to evolve in a clonal manner, although recent studies suggest that recombination occurs also for these viruses. We re-analyzed the data and found that previous conclusions on wild type recombination were probably falsely drawn due to misalignments of nucleotide sequences, ambiguities in GenBank sequences, or different laboratory culture histories suggestive of recombination events in laboratory. To evaluate if reliable predictions of wild type recombination of tick-borne flaviviruses can be made, we analyzed viral strains sequenced exclusively for this study, and other flavivirus sequences retrieved from GenBank. We detected genetic signals supporting recombination between viruses within the three clades of TBEV-Eu, TBEV-Sib and TBEV-Fe, respectively. Our results suggest that the tick-borne encephalitis viruses may undergo recombination under natural conditions, but that geographic barriers restrict most recombination events to involve only closely genetically related viruses. Copyright © 2013 Elsevier Inc. All rights reserved.

Oral vaccination of wildlife using a vaccinia-rabies-glycoprotein recombinant virus vaccine (RABORAL V-RG®): a global review.

PubMed

Maki, Joanne; Guiot, Anne-Laure; Aubert, Michel; Brochier, Bernard; Cliquet, Florence; Hanlon, Cathleen A; King, Roni; Oertli, Ernest H; Rupprecht, Charles E; Schumacher, Caroline; Slate, Dennis; Yakobson, Boris; Wohlers, Anne; Lankau, Emily W

2017-09-22

RABORAL V-RG ® is an oral rabies vaccine bait that contains an attenuated ("modified-live") recombinant vaccinia virus vector vaccine expressing the rabies virus glycoprotein gene (V-RG). Approximately 250 million doses have been distributed globally since 1987 without any reports of adverse reactions in wildlife or domestic animals since the first licensed recombinant oral rabies vaccine (ORV) was released into the environment to immunize wildlife populations against rabies. V-RG is genetically stable, is not detected in the oral cavity beyond 48 h after ingestion, is not shed by vaccinates into the environment, and has been tested for thermostability under a range of laboratory and field conditions. Safety of V-RG has been evaluated in over 50 vertebrate species, including non-human primates, with no adverse effects observed regardless of route or dose. Immunogenicity and efficacy have been demonstrated under laboratory and field conditions in multiple target species (including fox, raccoon, coyote, skunk, raccoon dog, and jackal). The liquid vaccine is packaged inside edible baits (i.e., RABORAL V-RG, the vaccine-bait product) which are distributed into wildlife habitats for consumption by target species. Field application of RABORAL V-RG has contributed to the elimination of wildlife rabies from three European countries (Belgium, France and Luxembourg) and of the dog/coyote rabies virus variant from the United States of America (USA). An oral rabies vaccination program in west-central Texas has essentially eliminated the gray fox rabies virus variant from Texas with the last case reported in a cow during 2009. A long-term ORV barrier program in the USA using RABORAL V-RG is preventing substantial geographic expansion of the raccoon rabies virus variant. RABORAL V-RG has also been used to control wildlife rabies in Israel for more than a decade. This paper: (1) reviews the development and historical use of RABORAL V-RG; (2) highlights wildlife rabies control programs using the vaccine in multiple species and countries; and (3) discusses current and future challenges faced by programs seeking to control or eliminate wildlife rabies.

High Prevalence of Co-Infections by Invasive and Non-Invasive Chlamydia trachomatis Genotypes during the Lymphogranuloma Venereum Outbreak in Spain

PubMed Central

Rodriguez-Dominguez, Mario; Gonzalez-Alba, Jose Maria; Puerta, Teresa; Menendez, Blanca; Sanchez-Diaz, Ana Maria; Canton, Rafael; del Romero, Jorge; Galan, Juan Carlos

2015-01-01

The evolution of Chlamydia trachomatis is mainly driven by recombination events. This fact can be fuelled by the coincidence in several European regions of the high prevalence of non-invasive urogenital genotypes and lymphogranuloma venereum (LGV) outbreaks. This scenario could modify the local epidemiology and favor the selection of new C. trachomatis variants. Quantifying the prevalence of co-infection could help to predict the potential risk in the selection of new variants with unpredictable results in pathogenesis or transmissibility. In the 2009-2013 period, 287 clinical samples with demonstrated presence of C. trachomatis were selected. They were divided in two groups. The first group was constituted by 137 samples with C. trachomatis of the LGV genotypes, and the second by the remaining 150 samples in which the presence of LGV genotypes was previously excluded. They were analyzed to detect the simultaneous presence of non-LGV genotypes based on pmpH and ompA genes. In the first group, co-infections were detected in 10.9% of the cases whereas in the second group the prevalence was 14.6%, which is the highest percentage ever described among European countries. Moreover, bioinformatic analyses suggested the presence among men who have sex with men of a pmpH-recombinant variant, similar to strains described in Seattle in 2002. This variant was the result of genetic exchange between genotypes belonging to LGV and members of G-genotype. Sequencing of other genes, phylogenetically related to pathotype, confirmed that the putative recombinant found in Madrid could have a common origin with the strains described in Seattle. Countries with a high prevalence of co-infections and high migration flows should enhance surveillance programs in at least their vulnerable population. PMID:25965545

High Prevalence of Co-Infections by Invasive and Non-Invasive Chlamydia trachomatis Genotypes during the Lymphogranuloma Venereum Outbreak in Spain.

PubMed

Rodriguez-Dominguez, Mario; Gonzalez-Alba, Jose Maria; Puerta, Teresa; Menendez, Blanca; Sanchez-Diaz, Ana Maria; Canton, Rafael; del Romero, Jorge; Galan, Juan Carlos

2015-01-01

The evolution of Chlamydia trachomatis is mainly driven by recombination events. This fact can be fuelled by the coincidence in several European regions of the high prevalence of non-invasive urogenital genotypes and lymphogranuloma venereum (LGV) outbreaks. This scenario could modify the local epidemiology and favor the selection of new C. trachomatis variants. Quantifying the prevalence of co-infection could help to predict the potential risk in the selection of new variants with unpredictable results in pathogenesis or transmissibility. In the 2009-2013 period, 287 clinical samples with demonstrated presence of C. trachomatis were selected. They were divided in two groups. The first group was constituted by 137 samples with C. trachomatis of the LGV genotypes, and the second by the remaining 150 samples in which the presence of LGV genotypes was previously excluded. They were analyzed to detect the simultaneous presence of non-LGV genotypes based on pmpH and ompA genes. In the first group, co-infections were detected in 10.9% of the cases whereas in the second group the prevalence was 14.6%, which is the highest percentage ever described among European countries. Moreover, bioinformatic analyses suggested the presence among men who have sex with men of a pmpH-recombinant variant, similar to strains described in Seattle in 2002. This variant was the result of genetic exchange between genotypes belonging to LGV and members of G-genotype. Sequencing of other genes, phylogenetically related to pathotype, confirmed that the putative recombinant found in Madrid could have a common origin with the strains described in Seattle. Countries with a high prevalence of co-infections and high migration flows should enhance surveillance programs in at least their vulnerable population.

HIV-1 drug resistance in recently HIV-infected pregnant mother's naïve to antiretroviral therapy in Dodoma urban, Tanzania.

PubMed

Vairo, Francesco; Nicastri, Emanuele; Liuzzi, Giuseppina; Chaula, Zainab; Nguhuni, Boniface; Bevilacqua, Nazario; Forbici, Federica; Amendola, Alessandra; Fabeni, Lavinia; De Nardo, Pasquale; Perno, Carlo Federico; Cannas, Angela; Sakhoo, Calistus; Capobianchi, Maria Rosaria; Ippolito, Giuseppe

2013-09-21

HIV resistance affects virological response to therapy and efficacy of prophylaxis in mother-to-child-transmission. The study aims to assess the prevalence of HIV primary resistance in pregnant women naïve to antiretrovirals. Cross sectional baseline analysis of a cohort of HIV + pregnant women (HPW) enrolled in the study entitled Antiretroviral Management of Antenatal and Natal HIV Infection (AMANI, peace in Kiswahili language). The AMANI study began in May 2010 in Dodoma, Tanzania. In this observational cohort, antiretroviral treatment was provided to all women from the 28th week of gestation until the end of the breastfeeding period. Baseline CD4 cell count, viral load and HIV drug-resistance genotype were collected. Drug-resistance analysis was performed on 97 naïve infected-mothers. The prevalence of all primary drug resistance and primary non-nucleoside reverse-transcriptase inhibitors resistance was 11.9% and 7.5%, respectively. K103S was found in two women with no M184V detection. HIV-1 subtype A was the most commonly identified, with a high prevalence of subtype A1, followed by C, D, C/D recombinant, A/C recombinant and A/D recombinant. HIV drug- resistance mutations were detected in A1 and C subtypes. Our study reports an 11.9% prevalence rate of primary drug resistance in naïve HIV-infected pregnant women from a remote area of Tanzania. Considering that the non-nucleoside reverse-transcriptase inhibitors are part of the first-line antiretroviral regimen in Tanzania and all of Africa, resistance surveys should be prioritized in settings where antiretroviral therapy programs are scaled up.

Low-Frequency Carbon Recombination Lines in the Orion Molecular Cloud Complex

NASA Astrophysics Data System (ADS)

Tremblay, Chenoa D.; Jordan, Christopher H.; Cunningham, Maria; Jones, Paul A.; Hurley-Walker, Natasha

2018-05-01

We detail tentative detections of low-frequency carbon radio recombination lines from within the Orion molecular cloud complex observed at 99-129 MHz. These tentative detections include one alpha transition and one beta transition over three locations and are located within the diffuse regions of dust observed in the infrared at 100 μm, the Hα emission detected in the optical, and the synchrotron radiation observed in the radio. With these observations, we are able to study the radiation mechanism transition from collisionally pumped to radiatively pumped within the H ii regions within the Orion molecular cloud complex.

Mobile DNA in the pathogenic Neisseria

PubMed Central

Obergfell, Kyle P.; Seifert, H. Steven

2015-01-01

The genus Neisseria contains two pathogenic species of notable public health concern: Neisseria gonorrhoeae and Neisseria meningitidis. These pathogens display a notable ability to undergo frequent programmed recombination events. The recombination mediated pathways of transformation and pilin antigenic variation in the Neisseria are well studied systems that are critical for pathogenesis. Here we will detail the conserved and unique aspects of transformation and antigenic variation in the Neisseria. Transformation will be followed from initial DNA binding through recombination into the genome with consideration to the factors necessary at each step. Additional focus is paid to the unique type IV secretion system that mediates donation of transforming DNA in the pathogenic Neisseria. The pilin antigenic variation system uses programed recombinations to alter a major surface determinant which allows immune avoidance and promotes infection. We discuss the trans- and cis- acting factors which facilitate pilin antigenic variation and present the current understanding of the mechanisms involved in the process. PMID:25866700

Detection and analysis of recombination in GII.4 norovirus strains causing gastroenteritis outbreaks in Alberta.

PubMed

Hasing, Maria E; Hazes, Bart; Lee, Bonita E; Preiksaitis, Jutta K; Pang, Xiaoli L

2014-10-01

Recombination is an important mechanism generating genetic diversity in norovirus (NoV) that occurs commonly at the NoV polymerase-capsid (ORF1/2) junction. The genotyping method based on partial ORF2 sequences currently used to characterize circulating NoV strains in gastroenteritis outbreaks in Alberta cannot detect such recombination events and provides only limited information on NoV genetic evolution. The objective of this study was to determine whether any NoV GII.4 strains causing outbreaks in Alberta are recombinants. Twenty stool samples collected during outbreaks occurring between July 2004 and January 2012 were selected to include the GII.4 variants Farmington Hills 2002, Hunter 2004, Yerseke 2006a, Den Haag 2006b, Apeldoorn 2007, New Orleans 2009, and Sydney 2012 based on previous NoV ORF2-genotyping results. Near full-length NoV genome sequences were obtained, aligned with reference sequences from GenBank and analyzed with RDPv4.13. Two sequences corresponding to Apeldoorn 2007, and Sydney 2012 were identified as recombinants with breakpoints near the ORF1/2 junction and putative parental strains as previously reported. We also identified, for the first time, a non-recombinant sequence resembling the ORF2-3 parent of the recombinant cluster Sydney 2012 responsible for the most recent pandemic. Our results confirmed the presence of recombinant NoV GII.4 strains in Alberta, and highlight the importance of including additional genomic regions in surveillance studies to trace the evolution of pandemic NoV GII.4 strains. Copyright © 2014 Elsevier B.V. All rights reserved.

Development and immunochemical evaluation of a novel chicken IgY antibody specific for KLK6.

PubMed

Sotiropoulou, Georgia; Pampalakis, Georgios; Prosnikli, Evangelia; Evangelatos, Gregory P; Livaniou, Evangelia

2012-12-05

Human kallikrein-related peptidase 6 (KLK6) has been implicated in various types of cancer and in neurodegenerative and demyelinating diseases including multiple sclerosis. Further, anti-KLK6 antibodies attenuated disease manifestations in the mouse model of multiple sclerosis. Availability of specific antibodies against KLK6 is fundamental to the development of improved diagnostic and/or immunotherapeutic applications. Here, we exploited the enhanced immunogenicity of mammalian proteins in avian species to generate a polyclonal antibody against KLK6. Chicken were immunized with recombinant KLK6 and antibodies Y (IgYs) were purified from egg yolk with a simple procedure and evaluated for KLK6 detection by ELISA and Western blot using recombinant proteins and human cell lysates and supernatants. The anti-KLK6 Y polyclonal exhibited high affinity for KLK6 with a detection limit of 30 fmol. On the other hand, the widely used rabbit polyclonal antibody that was raised against the same recombinant KLK6 had a detection limit of 300 fmol. Moreover, the IgYs did not display any crossreactivity with recombinant KLKs or endogenous KLKs and other cellular proteins. Based on its high specificity and sensitivity the developed anti-KLK6 IgY is expected to aid the development of improved diagnostic tools for the detection of KLK6 in biological and clinical samples.

Development and immunochemical evaluation of a novel chicken IgY antibody specific for KLK6

PubMed Central

2012-01-01

Background Human kallikrein-related peptidase 6 (KLK6) has been implicated in various types of cancer and in neurodegenerative and demyelinating diseases including multiple sclerosis. Further, anti-KLK6 antibodies attenuated disease manifestations in the mouse model of multiple sclerosis. Availability of specific antibodies against KLK6 is fundamental to the development of improved diagnostic and/or immunotherapeutic applications. Here, we exploited the enhanced immunogenicity of mammalian proteins in avian species to generate a polyclonal antibody against KLK6. Results Chicken were immunized with recombinant KLK6 and antibodies Y (IgYs) were purified from egg yolk with a simple procedure and evaluated for KLK6 detection by ELISA and Western blot using recombinant proteins and human cell lysates and supernatants. The anti-KLK6 Y polyclonal exhibited high affinity for KLK6 with a detection limit of 30 fmol. On the other hand, the widely used rabbit polyclonal antibody that was raised against the same recombinant KLK6 had a detection limit of 300 fmol. Moreover, the IgYs did not display any crossreactivity with recombinant KLKs or endogenous KLKs and other cellular proteins. Conclusions Based on its high specificity and sensitivity the developed anti-KLK6 IgY is expected to aid the development of improved diagnostic tools for the detection of KLK6 in biological and clinical samples. PMID:23216878

Development of Prototype Filovirus Recombinant Antigen Immunoassays

PubMed Central

Boisen, Matt L.; Oottamasathien, Darin; Jones, Abigail B.; Millett, Molly M.; Nelson, Diana S.; Bornholdt, Zachary A.; Fusco, Marnie L.; Abelson, Dafna M.; Oda, Shun-ichiro; Hartnett, Jessica N.; Rowland, Megan M.; Heinrich, Megan L.; Akdag, Marjan; Goba, Augustine; Momoh, Mambu; Fullah, Mohammed; Baimba, Francis; Gbakie, Michael; Safa, Sadiki; Fonnie, Richard; Kanneh, Lansana; Cross, Robert W.; Geisbert, Joan B.; Geisbert, Thomas W.; Kulakosky, Peter C.; Grant, Donald S.; Shaffer, Jeffery G.; Schieffelin, John S.; Wilson, Russell B.; Saphire, Erica Ollmann; Branco, Luis M.; Garry, Robert F.; Khan, S. Humarr; Pitts, Kelly R.

2015-01-01

Background. Throughout the 2014–2015 Ebola outbreak in West Africa, major gaps were exposed in the availability of validated rapid diagnostic platforms, protective vaccines, and effective therapeutic agents. These gaps potentiated the development of prototype rapid lateral flow immunodiagnostic (LFI) assays that are true point-of-contact platforms, for the detection of active Ebola infections in small blood samples. Methods. Recombinant Ebola and Marburg virus matrix VP40 and glycoprotein (GP) antigens were used to derive a panel of monoclonal and polyclonal antibodies. Antibodies were tested using a multivariate approach to identify antibody-antigen combinations suitable for enzyme-linked immunosorbent assay (ELISA) and LFI assay development. Results. Polyclonal antibodies generated in goats were superior reagents for capture and detection of recombinant VP40 in test sample matrices. These antibodies were optimized for use in antigen-capture ELISA and LFI assay platforms. Prototype immunoglobulin M (IgM)/immunoglobulin G (IgG) ELISAs were similarly developed that specifically detect Ebola virus–specific antibodies in the serum of experimentally infected nonhuman primates and in blood samples obtained from patients with Ebola from Sierra Leone. Conclusions. The prototype recombinant Ebola LFI assays developed in these studies have sensitivities that are useful for clinical diagnosis of acute ebolavirus infections. The antigen-capture and IgM/IgG ELISAs provide additional confirmatory assay platforms for detecting VP40 and other ebolavirus-specific immunoglobulins. PMID:26232440

Auger recombination in sodium iodide

NASA Astrophysics Data System (ADS)

McAllister, Andrew; Kioupakis, Emmanouil; Åberg, Daniel; Schleife, André

2014-03-01

Scintillators are an important tool used to detect high energy radiation - both in the interest of national security and in medicine. However, scintillator detectors currently suffer from lower energy resolutions than expected from basic counting statistics. This has been attributed to non-proportional light yield compared to incoming radiation, but the specific mechanism for this non-proportionality has not been identified. Auger recombination is a non-radiative process that could be contributing to the non-proportionality of scintillating materials. Auger recombination comes in two types - direct and phonon-assisted. We have used first-principles calculations to study Auger recombination in sodium iodide, a well characterized scintillating material. Our findings indicate that phonon-assisted Auger recombination is stronger in sodium iodide than direct Auger recombination. Computational resources provided by LLNL and NERSC. Funding provided by NA-22.

Identification of Quantitative Trait Loci Controlling Root and Shoot Traits Associated with Drought Tolerance in a Lentil (Lens culinaris Medik.) Recombinant Inbred Line Population

PubMed Central

Idrissi, Omar; Udupa, Sripada M.; De Keyser, Ellen; McGee, Rebecca J.; Coyne, Clarice J.; Saha, Gopesh C.; Muehlbauer, Fred J.; Van Damme, Patrick; De Riek, Jan

2016-01-01

Drought is one of the major abiotic stresses limiting lentil productivity in rainfed production systems. Specific rooting patterns can be associated with drought avoidance mechanisms that can be used in lentil breeding programs. In all, 252 co-dominant and dominant markers were used for Quantitative Trait Loci (QTL) analysis on 132 lentil recombinant inbred lines based on greenhouse experiments for root and shoot traits during two seasons under progressive drought-stressed conditions. Eighteen QTLs controlling a total of 14 root and shoot traits were identified. A QTL-hotspot genomic region related to a number of root and shoot characteristics associated with drought tolerance such as dry root biomass, root surface area, lateral root number, dry shoot biomass and shoot length was identified. Interestingly, a QTL (QRSratioIX-2.30) related to root-shoot ratio, an important trait for drought avoidance, explaining the highest phenotypic variance of 27.6 and 28.9% for the two consecutive seasons, respectively, was detected. This QTL was closed to the co-dominant SNP marker TP6337 and also flanked by the two SNP TP518 and TP1280. An important QTL (QLRNIII-98.64) related to lateral root number was found close to TP3371 and flanked by TP5093 and TP6072 SNP markers. Also, a QTL (QSRLIV-61.63) associated with specific root length was identified close to TP1873 and flanked by F7XEM6b SRAP marker and TP1035 SNP marker. These two QTLs were detected in both seasons. Our results could be used for marker-assisted selection in lentil breeding programs targeting root and shoot characteristics conferring drought avoidance as an efficient alternative to slow and labor-intensive conventional breeding methods. PMID:27602034

Development of SYBR Green I Based Real-Time RT-PCR Assay for Specific Detection of Watermelon silver mottle Virus.

PubMed

Rao, Xueqin; Sun, Jie

2015-09-01

Watermelon silver mottle virus (WSMoV), which belongs to the genus Tospovirus , causes significant loss in Cucurbitaceae plants. Development of a highly sensitive and reliable detection method for WSMoV. Recombinant plasmids for targeting the sequence of nucleocapsid protein gene of WSMoV were constructed. SYBR Green I real-time PCR was established and evaluated with standard recombinant plasmids and 27 watermelon samples showing WSMoV infection symptoms. The recombinant plasmid was used as template for SYBR Green I real-time PCR to generate standard and melting curves. Melting curve analysis indicated no primer-dimers and non-specific products in the assay. No cross-reaction was observed with Capsicum chlorosis virus (genus Tospovirus ) and Cucumber mosaic virus (genus Cucumovirus). Repeatability tests indicated that inter-assay variability of the Ct values was 1.6%. A highly sensitive, reliable and rapid detection method of SYBR Green I real-time PCR for timely detection of WSMoV plants and vector thrips was established, which will facilitate disease forecast and control.

Development of Two FhSAP2 Recombinant-Based Assays for Immunodiagnosis of Human Chronic Fascioliasis.

PubMed

Shin, Sun Hee; Hsu, Angel; Chastain, Holly M; Cruz, Lorna A; Elder, Eric S; Sapp, Sarah G H; McAuliffe, Isabel; Espino, Ana M; Handali, Sukwan

2016-10-05

In the United States, infection with Fasciola hepatica has been identified as an emerging disease, primarily in immigrants, refugees, and travelers. The laboratory test of choice for diagnosis of fascioliasis is detection of disease specific antibodies, most commonly uses excretory-secretory antigens for detection of IgG antibodies. Recently, recombinant proteins such as F. hepatica antigen (FhSAP2) have been used to detect IgG antibodies. The glutathione S-transferase (GST)-FhSAP2 recombinant antigen was used to develop Western blot (WB) and fluorescent bead-based (Luminex) assays to detect F. hepatica total IgG and IgG 4 antibodies. The sensitivity and specificity of GST-FhSAP2 total IgG and IgG 4 WB were similar at 94% and 98%, respectively. For the IgG Luminex assay, the sensitivity and specificity were 94% and 97%, and for the IgG 4 , the values were 100% and 99%, respectively. In conclusion, the GST-FhSAP2 antigen performs well in several assay formats and can be used for clinical diagnosis. © The American Society of Tropical Medicine and Hygiene.

Issues encountered in development of enzyme-linked immunosorbent assay for use in detecting Influenza A virus subtype H5N1 exposure in swine.

PubMed

Buehler, Jason; Lager, Kelly; Vincent, Amy; Miller, Cathy; Thacker, Eileen; Janke, Bruce

2014-03-01

A potential mechanism by which highly pathogenic avian Influenza A virus subtype H5N1 could more readily infect human beings is through the infection of and adaptation in pigs. To detect the occurrence of such infection, monitoring of pig populations through serological screening would be highly desirable. In the current study, hemagglutination inhibition assays were able to detect antibodies against H5N1 developed in pigs, but because of antigenic variation between clades, the use of multiple virus strains were required. Whole recombinant virus and recombinant hemagglutinin antigen enzyme-linked immunosorbent assays (ELISAs) were generated that could detect antibody against multiple H5N1 strains, but which also detected antibody against endemic swine influenza viruses. A recombinant hemagglutinin antigen-based ELISA was as effective as the whole virus antigen ELISAs in detecting antibody against the H5N1 virus strains used and eliminated nearly all of the cross-reactivity with non-H5N1 virus antibody. The current study also highlighted the difficulty in establishing a decision (cutoff) value that would effectively counterbalance nonspecific reactivity against sensitivity. The results provide important information and considerations for the development of serological screening assays for highly pathogenic avian H5N1 viruses.

An Automated ELISA Using Recombinant Antigens for Serologic Diagnosis of B Virus Infections in Macaques

PubMed Central

Katz, David; Shi, Wei; Patrusheva, Irina; Perelygina, Ludmila; Gowda, Manjunath S; Krug, Peter W; Filfili, Chadi N; Ward, John A; Hilliard, Julia K

2012-01-01

B virus (Macacine herpesvirus 1) occurs naturally in macaques and can cause lethal zoonotic infections in humans. Detection of B virus (BV) antibodies in macaques is essential for the development of SPF breeding colonies and for diagnosing infection in macaques that are involved in human exposures. Traditionally, BV infections are monitored for presence of antibodies by ELISA (a screening assay) and western blot analysis (WBA; a confirmatory test). Both tests use lysates of infected cells as antigens. Because WBA often fails to confirm the presence of low-titer serum antibodies detected by ELISA, we examined a recombinant-based ELISA as a potential alternative confirmatory test. We compared a high-throughput ELISA using 384-well plates for simultaneous antibody screening against 4 BV-related, recombinant proteins with the standard ELISA and WBA. The recombinant ELISA results confirmed more ELISA-positive sera than did WBA. The superiority of the recombinant ELISA over WBA was particularly prominent for sera with low (<500 ELISA units) antibody titers. Among low-titer sera, the relative sensitivity of the recombinant ELISA ranged from 36.7% to 45.0% as compared with 3.3% to 10.0% for WBA. In addition, the screening and confirmatory assays can be run simultaneously, providing results more rapidly. We conclude that the recombinant ELISA is an effective replacement for WBA as a confirmatory assay for the evaluation of macaque serum antibodies to BV. PMID:23561887

Genetic variations in merozoite surface antigen genes of Babesia bovis detected in Vietnamese cattle and water buffaloes.

PubMed

Yokoyama, Naoaki; Sivakumar, Thillaiampalam; Tuvshintulga, Bumduuren; Hayashida, Kyoko; Igarashi, Ikuo; Inoue, Noboru; Long, Phung Thang; Lan, Dinh Thi Bich

2015-03-01

The genes that encode merozoite surface antigens (MSAs) in Babesia bovis are genetically diverse. In this study, we analyzed the genetic diversity of B. bovis MSA-1, MSA-2b, and MSA-2c genes in Vietnamese cattle and water buffaloes. Blood DNA samples from 258 cattle and 49 water buffaloes reared in the Thua Thien Hue province of Vietnam were screened with a B. bovis-specific diagnostic PCR assay. The B. bovis-positive DNA samples (23 cattle and 16 water buffaloes) were then subjected to PCR assays to amplify the MSA-1, MSA-2b, and MSA-2c genes. Sequencing analyses showed that the Vietnamese MSA-1 and MSA-2b sequences are genetically diverse, whereas MSA-2c is relatively conserved. The nucleotide identity values for these MSA gene sequences were similar in the cattle and water buffaloes. Consistent with the sequencing data, the Vietnamese MSA-1 and MSA-2b sequences were dispersed across several clades in the corresponding phylogenetic trees, whereas the MSA-2c sequences occurred in a single clade. Cattle- and water-buffalo-derived sequences also often clustered together on the phylogenetic trees. The Vietnamese MSA-1, MSA-2b, and MSA-2c sequences were then screened for recombination with automated methods. Of the seven recombination events detected, five and two were associated with the MSA-2b and MSA-2c recombinant sequences, respectively, whereas no MSA-1 recombinants were detected among the sequences analyzed. Recombination between the sequences derived from cattle and water buffaloes was very common, and the resultant recombinant sequences were found in both host animals. These data indicate that the genetic diversity of the MSA sequences does not differ between cattle and water buffaloes in Vietnam. They also suggest that recombination between the B. bovis MSA sequences in both cattle and water buffaloes might contribute to the genetic variation in these genes in Vietnam. Copyright © 2015 Elsevier B.V. All rights reserved.

[Study on construction and immune protective effect of recombinant nucleic acid vaccine of Toxoplasma gondii].

PubMed

Wei, Qing-Kuan

2012-04-01

To construct the polyvalent recombinant nucleic acid vaccine of Toxoplasma gondii and measure its protective immune effect. The gene of heat shock protein (HSP70) was amplified by PCR and inserted into the recombinant plasmid of pcDNA3-ROP2-p30 to construct recombinant polyvalent nucleic vaccine (pcDNA3-ROP2-p30-Hsp70). BALB/c mice were immunized with the constructed recombinant nucleic vaccine. CD4+ and CD8+ in the splenic lymphocytes and the lymphocytes in anticoagulant whole blood, the immune indices such as antibodies (IgG, IgM and IgA) and IFN-gamma, TNF, IL-2, IL-4, IL-12 in serum and splenic lymphocytes culture medium were detected, along with the challenge experiment. The protective immune responses that caused by the vaccine was measured by detecting the changes of immune indices of mice and the challenge experiment. 916 bp fragment of HSP70 gene was amplified by PCR. The recombinant polyvalent nucleic vaccine pcDNA3-ROP2-p30-HSP70 that included the whole open reading frame sequence of HSP gene was successfully constructed. The immunization results also showed this polyvalent nucleic vaccine could induce strong cellular and humoral responses by the detection of higher antibody titer in the experimental mice group, the increasing proliferation of CD4+ and CD8+ cells with significant deviations among the groups (F(CD4+) = 45.00, F(CD8+) = 15.01, all P < 0.01) and the apparent up-regulated levels of several cytokines IFN-gamma, IL-2 and IL-12 in serum and cultural supernatant of spleen cells, with more striking effect in serum. As a result of the challenge experiment, the immunized mice showed a longer survival time. The recombinant nucleic acid vaccine pcDNA3-ROP2-p30-HSP70 possesses a strong immunogenicity and is able to induce an immune protection.

Cl atom recombination on silicon oxy-chloride layers deposited on chamber walls in chlorine-oxygen plasmas

DOE Office of Scientific and Technical Information (OSTI.GOV)

Khare, Rohit; Srivastava, Ashutosh; Donnelly, Vincent M.

2012-09-15

Chlorine atom recombination coefficients were measured on silicon oxy-chloride surfaces deposited in a chlorine inductively coupled plasma (ICP) with varying oxygen concentrations, using the spinning wall technique. A small cylinder embedded in the walls of the plasma reactor chamber was rapidly rotated, repetitively exposing its surface to the plasma chamber and a differentially pumped analysis chamber housing a quadruple mass spectrometer for line-of-sight desorbing species detection, or an Auger electron spectrometer for in situ surface analysis. The spinning wall frequency was varied from 800 to 30 000 rpm resulting in a detection time, t (the time a point on themore » surface takes to rotate from plasma chamber to the position facing the mass or Auger spectrometer), of {approx}1-40 ms. Desorbing Cl{sub 2}, due to Langmuir-Hinshelwood (LH) Cl atom recombination on the reactor wall surfaces, was detected by the mass spectrometer and also by a pressure rise in one of the differentially pumped chambers. LH Cl recombination coefficients were calculated by extrapolating time-resolved desorption decay curves to t = 0. A silicon-covered electrode immersed in the plasma was either powered at 13 MHz, creating a dc bias of -119 V, or allowed to electrically float with no bias power. After long exposure to a Cl{sub 2} ICP without substrate bias, slow etching of the Si wafer coats the chamber and spinning wall surfaces with an Si-chloride layer with a relatively small amount of oxygen (due to a slow erosion of the quartz discharge tube) with a stoichiometry of Si:O:Cl = 1:0.38:0.38. On this low-oxygen-coverage surface, any Cl{sub 2} desorption after LH recombination of Cl was below the detection limit. Adding 5% O{sub 2} to the Cl{sub 2} feed gas stopped etching of the Si wafer (with no rf bias) and increased the oxygen content of the wall deposits, while decreasing the Cl content (Si:O:Cl = 1:1.09:0.08). Cl{sub 2} desorption was detectable for Cl recombination on the spinning wall surface coated with this layer, and a recombination probability of {gamma}{sub Cl} = 0.03 was obtained. After this surface was conditioned with a pure oxygen plasma for {approx}60 min, {gamma}{sub Cl} increased to 0.044 and the surface layer was slightly enriched in oxygen fraction (Si:O:Cl = 1:1.09:0.04). This behavior is attributed to a mechanism whereby Cl LH recombination occurs mainly on chlorinated oxygen sites on the silicon oxy-chloride surface, because of the weak Cl-O bond compared to the Cl-Si bond.« less

The Contribution of Genetic Recombination to CRISPR Array Evolution

PubMed Central

Kupczok, Anne; Landan, Giddy; Dagan, Tal

2015-01-01

CRISPR (clustered regularly interspaced short palindromic repeats) is a microbial immune system against foreign DNA. Recognition sequences (spacers) encoded within the CRISPR array mediate the immune reaction in a sequence-specific manner. The known mechanisms for the evolution of CRISPR arrays include spacer acquisition from foreign DNA elements at the time of invasion and array erosion through spacer deletion. Here, we consider the contribution of genetic recombination between homologous CRISPR arrays to the evolution of spacer repertoire. Acquisition of spacers from exogenic arrays via recombination may confer the recipient with immunity against unencountered antagonists. For this purpose, we develop a novel method for the detection of recombination in CRISPR arrays by modeling the spacer order in arrays from multiple strains from the same species. Because the evolutionary signal of spacer recombination may be similar to that of pervasive spacer deletions or independent spacer acquisition, our method entails a robustness analysis of the recombination inference by a statistical comparison to resampled and perturbed data sets. We analyze CRISPR data sets from four bacterial species: two Gammaproteobacteria species harboring CRISPR type I and two Streptococcus species harboring CRISPR type II loci. We find that CRISPR array evolution in Escherichia coli and Streptococcus agalactiae can be explained solely by vertical inheritance and differential spacer deletion. In Pseudomonas aeruginosa, we find an excess of single spacers potentially incorporated into the CRISPR locus during independent acquisition events. In Streptococcus thermophilus, evidence for spacer acquisition by recombination is present in 5 out of 70 strains. Genetic recombination has been proposed to accelerate adaptation by combining beneficial mutations that arose in independent lineages. However, for most species under study, we find that CRISPR evolution is shaped mainly by spacer acquisition and loss rather than recombination. Since the evolution of spacer content is characterized by a rapid turnover, it is likely that recombination is not beneficial for improving phage resistance in the strains under study, or that it cannot be detected in the resolution of intraspecies comparisons. PMID:26085541

Detection of anti-PL-12 autoantibodies by ELISA using a recombinant antigen; study of the immunoreactive region

PubMed Central

García-Lozano, J R; González-Escribano, M F; Rodríguez, R; Rodriguez-Sanchez, J L; Targoff, I N; Wichmann, I; Núñez-Roldán, A

1998-01-01

Autoantibodies to aminoacyl-tRNA synthetases are highly associated with myositis and detection is important in clinical diagnosis; however, current methods of screening limit its clinical utility. In the present study, alanyl-tRNA synthetase (PL-12) recombinant protein was obtained by immunological screening of a HeLa expression library and used in an ELISA with 22 anti-PL-12 sera, 200 autoimmune sera negative for PL-12 and 100 healthy individual sera. Sensitivity of the method was 95% (21/22) and specificity 100%. Mapping of the immunoreactive region was carried out using three anti-PL-12 sera and different recombinant protein-derived peptides. Results show that the same conformational epitope located within amino acids 730–951 of the PL-12 antigen outside the catalytic region was recognized by the three anti-PL-12 sera tested. We conclude that ELISA using recombinant protein is an effective and useful method for routine screening for anti-PL-12 autoantibodies. PMID:9822271

Molecular farming of human cytokines and blood products from plants: challenges in biosynthesis and detection of plant-produced recombinant proteins.

PubMed

da Cunha, Nicolau B; Vianna, Giovanni R; da Almeida Lima, Thaina; Rech, Elíbio

2014-01-01

Plants have emerged as an attractive alternative to the traditional mammalian cell cultures or microbial cell-based systems system for the production of valuable recombinant proteins. Through recombinant DNA technology, plants can be engineered to produce large quantities of pharmaceuticals and industrial proteins of high quality at low costs. The recombinant production, by transgenic plants, of therapeutic proteins normally present in human plasma, such as cytokines, coagulation factors, anticoagulants, and immunoglobulins, represents a response to the ongoing challenges in meeting the demand for therapeutic proteins to treat serious inherited or acquired bleeding and immunological diseases. As the clinical utilization of fractionated plasma molecules is limited by high production costs, using recombinant biopharmaceuticals derived from plants represents a feasible alternative to provide efficient treatment. Plant-derived pharmaceuticals also reduce the potential risks to patients of infection with pathogens or unwanted immune responses due to immunogenic antigens. In this review, we summarize the recent advances in molecular farming of cytokines. We also examine the technological basis, upcoming challenges, and perspectives for the biosynthesis and detection of these molecules in different plant production platforms. Copyright © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Detecting Recombination Hotspots from Patterns of Linkage Disequilibrium.

PubMed

Wall, Jeffrey D; Stevison, Laurie S

2016-08-09

With recent advances in DNA sequencing technologies, it has become increasingly easy to use whole-genome sequencing of unrelated individuals to assay patterns of linkage disequilibrium (LD) across the genome. One type of analysis that is commonly performed is to estimate local recombination rates and identify recombination hotspots from patterns of LD. One method for detecting recombination hotspots, LDhot, has been used in a handful of species to further our understanding of the basic biology of recombination. For the most part, the effectiveness of this method (e.g., power and false positive rate) is unknown. In this study, we run extensive simulations to compare the effectiveness of three different implementations of LDhot. We find large differences in the power and false positive rates of these different approaches, as well as a strong sensitivity to the window size used (with smaller window sizes leading to more accurate estimation of hotspot locations). We also compared our LDhot simulation results with comparable simulation results obtained from a Bayesian maximum-likelihood approach for identifying hotspots. Surprisingly, we found that the latter computationally intensive approach had substantially lower power over the parameter values considered in our simulations. Copyright © 2016 Wall and Stevison.

Assessment of Recombination in the S-segment Genome of Crimean-Congo Hemorrhagic Fever Virus in Iran.

PubMed

Chinikar, Sadegh; Shah-Hosseini, Nariman; Bouzari, Saeid; Shokrgozar, Mohammad Ali; Mostafavi, Ehsan; Jalali, Tahmineh; Khakifirouz, Sahar; Groschup, Martin H; Niedrig, Matthias

2016-03-01

Crimean-Congo Hemorrhagic Fever Virus (CCHFV) belongs to genus Nairovirus and family Bunyaviridae. The main aim of this study was to investigate the extent of recombination in S-segment genome of CCHFV in Iran. Samples were isolated from Iranian patients and those available in GenBank, and analyzed by phylogenetic and bootscan methods. Through comparison of the phylogenetic trees based on full length sequences and partial fragments in the S-segment genome of CCHFV, genetic switch was evident, due to recombination event. Moreover, evidence of multiple recombination events was detected in query isolates when bootscan analysis was used by SimPlot software. Switch of different genomic regions between different strains by recombination could contribute to CCHFV diversification and evolution. The occurrence of recombination in CCHFV has a critical impact on epidemiological investigations and vaccine design.

Novel canine circovirus strains from Thailand: Evidence for genetic recombination.

PubMed

Piewbang, Chutchai; Jo, Wendy K; Puff, Christina; van der Vries, Erhard; Kesdangsakonwut, Sawang; Rungsipipat, Anudep; Kruppa, Jochen; Jung, Klaus; Baumgärtner, Wolfgang; Techangamsuwan, Somporn; Ludlow, Martin; Osterhaus, Albert D M E

2018-05-14

Canine circoviruses (CanineCV's), belonging to the genus Circovirus of the Circoviridae family, were detected by next generation sequencing in samples from Thai dogs with respiratory symptoms. Genetic characterization and phylogenetic analysis of nearly complete CanineCV genomes suggested that natural recombination had occurred among different lineages of CanineCV's. Similarity plot and bootscaning analyses indicated that American and Chinese viruses had served as major and minor parental viruses, respectively. Positions of recombination breakpoints were estimated using maximum-likelihood frameworks with statistical significant testing. The putative recombination event was located in the Replicase gene, intersecting with open reading frame-3. Analysis of nucleotide changes confirmed the origin of the recombination event. This is the first description of naturally occurring recombinant CanineCV's that have resulted in the circulation of newly emerging CanineCV lineages.

Highly sensitive detection of mutations in CHO cell recombinant DNA using multi-parallel single molecule real-time DNA sequencing.

PubMed

Cartwright, Joseph F; Anderson, Karin; Longworth, Joseph; Lobb, Philip; James, David C

2018-06-01

High-fidelity replication of biologic-encoding recombinant DNA sequences by engineered mammalian cell cultures is an essential pre-requisite for the development of stable cell lines for the production of biotherapeutics. However, immortalized mammalian cells characteristically exhibit an increased point mutation frequency compared to mammalian cells in vivo, both across their genomes and at specific loci (hotspots). Thus unforeseen mutations in recombinant DNA sequences can arise and be maintained within producer cell populations. These may affect both the stability of recombinant gene expression and give rise to protein sequence variants with variable bioactivity and immunogenicity. Rigorous quantitative assessment of recombinant DNA integrity should therefore form part of the cell line development process and be an essential quality assurance metric for instances where synthetic/multi-component assemblies are utilized to engineer mammalian cells, such as the assessment of recombinant DNA fidelity or the mutability of single-site integration target loci. Based on Pacific Biosciences (Menlo Park, CA) single molecule real-time (SMRT™) circular consensus sequencing (CCS) technology we developed a rDNA sequence analysis tool to process the multi-parallel sequencing of ∼40,000 single recombinant DNA molecules. After statistical filtering of raw sequencing data, we show that this analytical method is capable of detecting single point mutations in rDNA to a minimum single mutation frequency of 0.0042% (<1/24,000 bases). Using a stable CHO transfectant pool harboring a randomly integrated 5 kB plasmid construct encoding GFP we found that 28% of recombinant plasmid copies contained at least one low frequency (<0.3%) point mutation. These mutations were predominantly found in GC base pairs (85%) and that there was no positional bias in mutation across the plasmid sequence. There was no discernable difference between the mutation frequencies of coding and non-coding DNA. The putative ratio of non-synonymous and synonymous changes within the open reading frames (ORFs) in the plasmid sequence indicates that natural selection does not impact upon the prevalence of these mutations. Here we have demonstrated the abundance of mutations that fall outside of the reported range of detection of next generation sequencing (NGS) and second generation sequencing (SGS) platforms, providing a methodology capable of being utilized in cell line development platforms to identify the fidelity of recombinant genes throughout the production process. © 2018 Wiley Periodicals, Inc.

[Recombinant OspC identification and antigenicity detection from Borrelia burgdorferi PD91 in China].

PubMed

Chen, Jian; Wan, Kang-Lin

2003-10-01

To recombine OspC gene from Borrelia burgdorferi PD91 of China and expressed it in E. coli for early diagnosis of Lyme disease. The OspC gene was amplified from the genome of Borrelia burgdorferi PD91 strain by polymerase chain reaction and recombined with plasmid PET-11D. The recombinant plasmid PET-11D-OspC was identified with PCR, restriction endonuclease analysis and sequencing. The antigenicity was verified with Western Blot. OspC gene was cloned correctly into vector PET-11D. The resultant sequence was definitely different from the published sequence. The recombinant OspC seemed to have had strong antigenicity. The findings laid basis for the studies on early diagnosis of Lyme disease.

Meiotic homoeologous recombination-based alien gene introgression in the genomics era of wheat

USDA-ARS?s Scientific Manuscript database

Wheat (Triticum spp.) has a narrow genetic basis due to its allopolyploid origin. However, wheat has numerous wild relatives usable for expanding genetic variability of its genome through meiotic homoeologous recombination. Traditionally, laborious cytological analyses have been employed to detect h...

Development of a multiplex RT-PCR assay for the identification of recombination types at different genomic regions of vaccine-derived polioviruses.

PubMed

Dimitriou, T G; Kyriakopoulou, Z; Tsakogiannis, D; Fikatas, A; Gartzonika, C; Levidiotou-Stefanou, S; Markoulatos, P

2016-08-01

Polioviruses (PVs) are the causal agents of acute paralytic poliomyelitis. Since the 1960s, poliomyelitis has been effectively controlled by the use of two vaccines containing all three serotypes of PVs, the inactivated poliovirus vaccine and the live attenuated oral poliovirus vaccine (OPV). Despite the success of OPV in polio eradication programme, a significant disadvantage was revealed: the emergence of vaccine-associated paralytic poliomyelitis (VAPP). VAPP is the result of accumulated mutations and putative recombination events located at the genome of attenuated vaccine Sabin strains. In the present study, ten Sabin isolates derived from OPV vaccinees and environmental samples were studied in order to identify recombination types located from VP1 to 3D genomic regions of virus genome. The experimental procedure that was followed was virus RNA extraction, reverse transcription to convert the virus genome into cDNA, PCR and multiplex-PCR using specific designed primers able to localize and identify each recombination following agarose gel electrophoresis. This multiplex RT-PCR assay allows for the immediate detection and identification of multiple recombination types located at the viral genome of OPV derivatives. After the eradication of wild PVs, the remaining sources of poliovirus infection worldwide would be the OPV derivatives. As a consequence, the immediate detection and molecular characterization of recombinant derivatives are important to avoid epidemics due to the circulation of neurovirulent viral strains.

Activated recombinative desorption: A potential component in mechanisms of spacecraft glow

NASA Technical Reports Server (NTRS)

Cross, J. B.

1985-01-01

The concept of activated recombination of atomic species on surfaces can explain the production of vibrationally and translationally excited desorbed molecular species. Equilibrium statistical mechanics predicts that the molecular quantum state distributions of desorbing molecules is a function of surface temperature only when the adsorption probability is unity and independent of initial collision conditions. In most cases, the adsorption probability is dependent upon initial conditions such as collision energy or internal quantum state distribution of impinging molecules. From detailed balance, such dynamical behavior is reflected in the internal quantum state distribution of the desorbing molecule. This concept, activated recombinative desorption, may offer a common thread in proposed mechanisms of spacecraft glow. Using molecular beam techniques and equipment available at Los Alamos, which includes a high translational energy 0-atom beam source, mass spectrometric detection of desorbed species, chemiluminescence/laser induced fluorescence detection of electronic and vibrationally excited reaction products, and Auger detection of surface adsorbed reaction products, a fundamental study of the gas surface chemistry underlying the glow process is proposed.

Recombinant plasmid-based quantitative Real-Time PCR analysis of Salmonella enterica serotypes and its application to milk samples.

PubMed

Gokduman, Kurtulus; Avsaroglu, M Dilek; Cakiris, Aris; Ustek, Duran; Gurakan, G Candan

2016-03-01

The aim of the current study was to develop, a new, rapid, sensitive and quantitative Salmonella detection method using a Real-Time PCR technique based on an inexpensive, easy to produce, convenient and standardized recombinant plasmid positive control. To achieve this, two recombinant plasmids were constructed as reference molecules by cloning the two most commonly used Salmonella-specific target gene regions, invA and ttrRSBC. The more rapid detection enabled by the developed method (21 h) compared to the traditional culture method (90 h) allows the quantitative evaluation of Salmonella (quantification limits of 10(1)CFU/ml and 10(0)CFU/ml for the invA target and the ttrRSBC target, respectively), as illustrated using milk samples. Three advantages illustrated by the current study demonstrate the potential of the newly developed method to be used in routine analyses in the medical, veterinary, food and water/environmental sectors: I--The method provides fast analyses including the simultaneous detection and determination of correct pathogen counts; II--The method is applicable to challenging samples, such as milk; III--The method's positive controls (recombinant plasmids) are reproducible in large quantities without the need to construct new calibration curves. Copyright © 2016 Elsevier B.V. All rights reserved.

Quantitative real-time PCR technique for the identification of E. coli residual DNA in streptokinase recombinant product.

PubMed

Fazelahi, Mansoureh; Kia, Vahid; Kaghazian, Hooman; Paryan, Mahdi

2017-11-26

Recombinant streptokinase is a biopharmaceutical which is usually produced in E. coli. Residual DNA as a contamination and risk factor may remain in the product. It is necessary to control the production procedure to exclude any possible contamination. The aim of the present study was to develop a highly specific and sensitive quantitative real-time PCR-based method to determine the amount of E. coli DNA in recombinant streptokinase. A specific primers and a probe was designed to detect all strains of E. coli. To determine the specificity, in addition to using NCBI BLASTn, 28 samples including human, bacterial, and viral genomes were used. The results confirmed that the assay detects no genomic DNA but E. coli's and the specificity was determined to be 100%. To determine the sensitivity and limit of detection of the assay, a 10-fold serial dilution (10 1 to 10 7 copies/µL) was tested in triplicate. The sensitivity of the test was determined to be 101 copies/µL or 35 fg/µL. Inter-assay and intra-assay were determined to be 0.86 and 1.69%, respectively. Based on the results, this assay can be used as an accurate method to evaluate the contamination of recombinant streptokinase in E. coli.

Absence of SUN-domain protein Slp1 blocks karyogamy and switches meiotic recombination and synapsis from homologs to sister chromatids

PubMed Central

Vasnier, Christelle; de Muyt, Arnaud; Zhang, Liangran; Tessé, Sophie; Kleckner, Nancy E.; Zickler, Denise; Espagne, Eric

2014-01-01

Karyogamy, the process of nuclear fusion is required for two haploid gamete nuclei to form a zygote. Also, in haplobiontic organisms, karyogamy is required to produce the diploid nucleus/cell that then enters meiosis. We identify sun like protein 1 (Slp1), member of the mid–Sad1p, UNC-84–domain ubiquitous family, as essential for karyogamy in the filamentous fungus Sordaria macrospora, thus uncovering a new function for this protein family. Slp1 is required at the last step, nuclear fusion, not for earlier events including nuclear movements, recognition, and juxtaposition. Correspondingly, like other family members, Slp1 localizes to the endoplasmic reticulum and also to its extensions comprising the nuclear envelope. Remarkably, despite the absence of nuclear fusion in the slp1 null mutant, meiosis proceeds efficiently in the two haploid “twin” nuclei, by the same program and timing as in diploid nuclei with a single dramatic exception: the normal prophase program of recombination and synapsis between homologous chromosomes, including loading of recombination and synaptonemal complex proteins, occurs instead between sister chromatids. Moreover, the numbers of recombination-initiating double-strand breaks (DSBs) and ensuing recombinational interactions, including foci of the essential crossover factor Homo sapiens enhancer of invasion 10 (Hei10), occur at half the diploid level in each haploid nucleus, implying per-chromosome specification of DSB formation. Further, the distribution of Hei10 foci shows interference like in diploid meiosis. Centromere and spindle dynamics, however, still occur in the diploid mode during the two meiotic divisions. These observations imply that the prophase program senses absence of karyogamy and/or absence of a homolog partner and adjusts the interchromosomal interaction program accordingly. PMID:25210014

Single-Step qPCR and dPCR Detection of Diverse CRISPR-Cas9 Gene Editing Events In Vivo.

PubMed

Falabella, Micol; Sun, Linqing; Barr, Justin; Pena, Andressa Z; Kershaw, Erin E; Gingras, Sebastien; Goncharova, Elena A; Kaufman, Brett A

2017-10-05

Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-CRISPR-associated protein 9 (Cas9)-based technology is currently the most flexible means to create targeted mutations by recombination or indel mutations by nonhomologous end joining. During mouse transgenesis, recombinant and indel alleles are often pursued simultaneously. Multiple alleles can be formed in each animal to create significant genetic complexity that complicates the CRISPR-Cas9 approach and analysis. Currently, there are no rapid methods to measure the extent of on-site editing with broad mutation sensitivity. In this study, we demonstrate the allelic diversity arising from targeted CRISPR editing in founder mice. Using this DNA sample collection, we validated specific quantitative and digital PCR methods (qPCR and dPCR, respectively) for measuring the frequency of on-target editing in founder mice. We found that locked nucleic acid (LNA) probes combined with an internal reference probe (Drop-Off Assay) provide accurate measurements of editing rates. The Drop-Off LNA Assay also detected on-target CRISPR-Cas9 gene editing in blastocysts with a sensitivity comparable to PCR-clone sequencing. Lastly, we demonstrate that the allele-specific LNA probes used in qPCR competitor assays can accurately detect recombinant mutations in founder mice. In summary, we show that LNA-based qPCR and dPCR assays provide a rapid method for quantifying the extent of on-target genome editing in vivo , testing RNA guides, and detecting recombinant mutations. Copyright © 2017 Falabella et al.

Rapid Diagnostic Assay for Intact Influenza Virus Using a High Affinity Hemagglutinin Binding Protein.

PubMed

Anderson, Caitlin E; Holstein, Carly A; Strauch, Eva-Maria; Bennett, Steven; Chevalier, Aaron; Nelson, Jorgen; Fu, Elain; Baker, David; Yager, Paul

2017-06-20

Influenza is a ubiquitous and recurring infection that results in approximately 500 000 deaths globally each year. Commercially available rapid diagnostic tests are based upon detection of the influenza nucleoprotein, which are limited in that they are unable to differentiate by species and require an additional viral lysis step. Sample preprocessing can be minimized or eliminated by targeting the intact influenza virus, thereby reducing assay complexity and leveraging the large number of hemagglutinin proteins on the surface of each virus. Here, we report the development of a paper-based influenza assay that targets the hemagglutinin protein; the assay employs a combination of antibodies and novel computationally designed, recombinant affinity proteins as the capture and detection agents. This system leverages the customizability of recombinant protein design to target the conserved receptor-binding pocket of the hemagglutinin protein and to match the trimeric nature of hemagglutinin for improved avidity. Using this assay, we demonstrate the first instance of intact influenza virus detection using a combination of antibody and affinity proteins within a porous network. The recombinant head region binder based assays yield superior analytical sensitivity as compared to the antibody based assay, with lower limits of detection of 3.54 × 10 7 and 1.34 × 10 7 CEID 50 /mL for the mixed and all binder stacks, respectively. Not only does this work describe the development of a novel influenza assay, it also demonstrates the power of recombinant affinity proteins for use in rapid diagnostic assays.

Sex in a test tube: testing the benefits of in vitro recombination.

PubMed

Pesce, Diego; Lehman, Niles; de Visser, J Arjan G M

2016-10-19

The origin and evolution of sex, and the associated role of recombination, present a major problem in biology. Sex typically involves recombination of closely related DNA or RNA sequences, which is fundamentally a random process that creates but also breaks up beneficial allele combinations. Directed evolution experiments, which combine in vitro mutation and recombination protocols with in vitro or in vivo selection, have proved to be an effective approach for improving functionality of nucleic acids and enzymes. As this approach allows extreme control over evolutionary conditions and parameters, it also facilitates the detection of small or position-specific recombination benefits and benefits associated with recombination between highly divergent genotypes. Yet, in vitro approaches have been largely exploratory and motivated by obtaining improved end products rather than testing hypotheses of recombination benefits. Here, we review the various experimental systems and approaches used by in vitro studies of recombination, discuss what they say about the evolutionary role of recombination, and sketch their potential for addressing extant questions about the evolutionary role of sex and recombination, in particular on complex fitness landscapes. We also review recent insights into the role of 'extracellular recombination' during the origin of life.This article is part of the themed issue 'Weird sex: the underappreciated diversity of sexual reproduction'. © 2016 The Author(s).

[Construction and expression of a recombinant adenovirus with LZP3].

PubMed

Chen, Bang-dang; Zhang, Fu-chun; Sun, Mei-yu; Li, Yi-jie; Ma, Zheng-hai

2007-08-01

To explore a new immunocontraceptive vaccine and construct an attenuated recombinant adenoviral vaccine against Lagurus lagurus zona pellucida 3(LZP3). LZP3 gene was subcloned into the shuttle vector pShuttle-CMV, and then a two-step transformation procedure was employed to construct a recombinant adenoviral plasmid with LZP3, which was digested with Pac I and transfected into HEK293 cells to package recombinant adenovirus particles. Finally, HeLa cells were infected by the recombinant adenovirus. LZP3 gene was detected from the recombinant virus by PCR, and its transcription and expression were analyzed by RT-PCR and Western blot. Recombinant adenovirus vector pAd-LZP3 with LZP3 gene was constructed by homologous recombination in E.coli, and a recombinant adenovirus was obtained by transfecting HEK293 cells with pAd-LZP3. PCR test indicated that LZP3 gene was successfully integrated into the adenoviral genome, and the titer of the recombinant adenovirus reached 1.2x10(10) pfu/L. The transcription and expression of LZP3 gene in the infected HeLa cells were confirmed by RT-PCR and Western blot. The recombinant adenovirus RAd-LZP3 can be successfully expressed in the infected HeLa cells, which lays the foundation for further researches into immunizing animals with RAd-LZP3.

Measurement of Yields and Fluctuations using Background and Calibration Data from the LUX Detector

NASA Astrophysics Data System (ADS)

Pease, Evan; LUX Collaboration

2016-03-01

The Large Underground Xenon (LUX) detector is a 350-kg liquid xenon (LXe) time-projection chamber designed for the direct detection of weakly-interacting massive particles (WIMPs), a leading dark matter candidate. LUX operates on the 4850-foot level of the Sanford Underground Research Facility in Lead, SD. Monoenergetic electronic recoil (ER) peaks in the WIMP search and calibration data from the first underground science run of the LUX detector have been used to measure ER light and charge yields in LXe between 5.2 keV and 662 keV. The energy resolution of the LUX detector at these energies will also be presented. Recombination fluctuations are observed to follow a linear dependence on the number of ions for the energies in this study, and this dependence is consistent with low-energy measurements made with a tritium beta source in the LUX detector. Using these results and additional measurements of the recoil bands from tritium and D-D neutron calibrations, I will compare recombination fluctuations in LXe response to electronic and nuclear recoils. The presenter is supported by the U.S. Department of Energy, Office of Science Graduate Student Research (SCGSR) program. The SCGSR program is administered by the Oak Ridge Institute for Science and Education for the DOE under contract DE-AC05-06OR23100.

MosaicSolver: a tool for determining recombinants of viral genomes from pileup data

PubMed Central

Wood, Graham R.; Ryabov, Eugene V.; Fannon, Jessica M.; Moore, Jonathan D.; Evans, David J.; Burroughs, Nigel

2014-01-01

Viral recombination is a key evolutionary mechanism, aiding escape from host immunity, contributing to changes in tropism and possibly assisting transmission across species barriers. The ability to determine whether recombination has occurred and to locate associated specific recombination junctions is thus of major importance in understanding emerging diseases and pathogenesis. This paper describes a method for determining recombinant mosaics (and their proportions) originating from two parent genomes, using high-throughput sequence data. The method involves setting the problem geometrically and the use of appropriately constrained quadratic programming. Recombinants of the honeybee deformed wing virus and the Varroa destructor virus-1 are inferred to illustrate the method from both siRNAs and reads sampling the viral genome population (cDNA library); our results are confirmed experimentally. Matlab software (MosaicSolver) is available. PMID:25120266

Use of low energy hydrogen ion implants in high efficiency crystalline silicon solar cells

NASA Technical Reports Server (NTRS)

Fonash, S. J.; Singh, R.

1985-01-01

This program is a study of the use of low energy hydrogen ion implantation for high efficiency crystalline silicon solar cells. The first quarterly report focuses on two tasks of this program: (1) an examination of the effects of low energy hydrogen implants on surface recombination speed; and (2) an examination of the effects of hydrogen on silicon regrowth and diffusion in silicon. The first part of the project focussed on the measurement of surface properties of hydrogen implanted silicon. Low energy hydrogen ions when bombarded on the silicon surface will create structural damage at the surface, deactivate dopants and introduce recombination centers. At the same time the electrically active centers such as dangling bonds will be passivated by these hydrogen ions. Thus hydrogen is expected to alter properties such as the surface recombination velocity, dopant profiles on the emitter, etc. In this report the surface recombination velocity of a hydrogen emplanted emitter was measured.

The spatial regulation of meiotic recombination hotspots: are all DSB hotspots crossover hotspots?

PubMed

Serrentino, Maria-Elisabetta; Borde, Valérie

2012-07-15

A key step for the success of meiosis is programmed homologous recombination, during which crossovers, or exchange of chromosome arms, take place. Crossovers increase genetic diversity but their main function is to ensure accurate chromosome segregation. Defects in crossover number and position produce aneuploidies that represent the main cause of miscarriages and chromosomal abnormalities such as Down's syndrome. Recombination is initiated by the formation of programmed double strand breaks (DSBs), which occur preferentially at places called DSB hotspots. Among all DSBs generated, only a small fraction is repaired by crossover, the other being repaired by other homologous recombination pathways. Crossover maps have been generated in a number of organisms, defining crossover hotspots. With the availability of genome-wide maps of DSBs as well as the ability to measure genetically the repair outcome at several hotspots, it is becoming more and more clear that not all DSB hotspots behave the same for crossover formation, suggesting that chromosomal features distinguish different types of hotspots. Copyright © 2012. Published by Elsevier Inc.

Recombination of mitochondrial DNA in skeletal muscle of individuals with multiple mitochondrial DNA heteroplasmy.

PubMed

Zsurka, Gábor; Kraytsberg, Yevgenia; Kudina, Tatiana; Kornblum, Cornelia; Elger, Christian E; Khrapko, Konstantin; Kunz, Wolfram S

2005-08-01

Experimental evidence for human mitochondrial DNA (mtDNA) recombination was recently obtained in an individual with paternal inheritance of mtDNA and in an in vitro cell culture system. Whether mtDNA recombination is a common event in humans remained to be determined. To detect mtDNA recombination in human skeletal muscle, we analyzed the distribution of alleles in individuals with multiple mtDNA heteroplasmy using single-cell PCR and allele-specific PCR. In all ten individuals who carried a heteroplasmic D-loop mutation and a distantly located tRNA point mutation or a large deletion, we observed a mixture of four allelic combinations (tetraplasmy), a hallmark of recombination. Twelve of 14 individuals with closely located heteroplasmic D-loop mutation pairs contained a mixture of only three types of mitochondrial genomes (triplasmy), consistent with the absence of recombination between adjacent markers. These findings indicate that mtDNA recombination is common in human skeletal muscle.

Ligand-receptor assay for evaluation of functional activity of human recombinant VEGF and VEGFR-1 extracellular fragment.

PubMed

Leopol'd, A V; Baklaushev, V P; Korchagina, A A; Shein, S A; Grinenko, N F; Pavlov, K A; Ryabukhin, I A; Chekhonin, V P

2012-04-01

cDNA encoding VEGF and Ig-like extracellular domains 2-4 of VEGFR-1 (sFlt-1(2-4)) were cloned into prokaryotic expression vectors pET32a and pQE60. Recombinant proteins were purified (metal affinity chromatography) and renatured. Chemiluminescent study for the interaction of recombinant VEGF and sFlt-1(2-4) showed that biotinylated VEGF specifically binds to the polystyrene-immobilized receptor extracellular fragment. Biotinylated recombinant sFlt-1 interacts with immobilized VEGF. Analysis of the interaction of immobilized recombinant VEGFR-1 and VEGF with C6 glioma cells labeled with CFDA-SE (vital fluorescent dye) showed that recombinant VEGFR-1 also binds to native membrane-associated VEGF. Recombinant VEGF was shown to bind to specific receptors expressed on the surface of C6 glioma cells. Functional activity of these proteins was confirmed by ligand-receptor assay for VEGF and VEGFR-1 (sFlt-1) and quantitative chemiluminescent detection.

Temporally-Controlled Site-Specific Recombination in Zebrafish

PubMed Central

Hans, Stefan; Kaslin, Jan; Freudenreich, Dorian; Brand, Michael

2009-01-01

Conventional use of the site-specific recombinase Cre is a powerful technology in mouse, but almost absent in other vertebrate model organisms. In zebrafish, Cre-mediated recombination efficiency was previously very low. Here we show that using transposon-mediated transgenesis, Cre is in fact highly efficient in this organism. Furthermore, temporal control of recombination can be achieved by using the ligand-inducible CreERT2. Site-specific recombination only occurs upon administration of the drug tamoxifen (TAM) or its active metabolite, 4-hydroxy-tamoxifen (4-OHT). Cre-mediated recombination is detectable already 4 or 2 hours after administration of TAM or 4-OHT, demonstrating fast recombination kinetics. In addition, low doses of TAM allow mosaic labeling of single cells. Combined, our results show that conditional Cre/lox will be a valuable tool for both, embryonic and adult zebrafish studies. Furthermore, single copy insertion transgenesis of Cre/lox constructs suggest a strategy suitable also for other organisms. PMID:19247481

Assessment of Recombination in the S-segment Genome of Crimean-Congo Hemorrhagic Fever Virus in Iran

PubMed Central

Chinikar, Sadegh; Shah-Hosseini, Nariman; Bouzari, Saeid; Shokrgozar, Mohammad Ali; Mostafavi, Ehsan; Jalali, Tahmineh; Khakifirouz, Sahar; Groschup, Martin H; Niedrig, Matthias

2016-01-01

Background: Crimean-Congo Hemorrhagic Fever Virus (CCHFV) belongs to genus Nairovirus and family Bunyaviridae. The main aim of this study was to investigate the extent of recombination in S-segment genome of CCHFV in Iran. Methods: Samples were isolated from Iranian patients and those available in GenBank, and analyzed by phylogenetic and bootscan methods. Results: Through comparison of the phylogenetic trees based on full length sequences and partial fragments in the S-segment genome of CCHFV, genetic switch was evident, due to recombination event. Moreover, evidence of multiple recombination events was detected in query isolates when bootscan analysis was used by SimPlot software. Conclusion: Switch of different genomic regions between different strains by recombination could contribute to CCHFV diversification and evolution. The occurrence of recombination in CCHFV has a critical impact on epidemiological investigations and vaccine design. PMID:27047968

ELF5 in epithelial ovarian carcinoma tissues and biological behavior in ovarian carcinoma cells.

PubMed

Yan, Hongchao; Qiu, Linglin; Xie, Xiaolei; Yang, He; Liu, Yongli; Lin, Xiaoman; Huang, Hongxiang

2017-03-01

The expression of E74-like factor 5 (ELF5) in epithelial ovarian carcinoma tissues and its effects on biological behavior in ovarian carcinoma cells were assessed in search for a new approach for gene treatment of epithelial ovarian carcinoma. RT-PCR technology was applied to detect the expression of ELF5 mRNA in epithelial ovarian carcinoma (n=49), borderline ovarian epithelial tumor (n=19), benign ovarian epithelial tumor (n=31) and normal ovarian tissues (n=40). Then, we transfected recombinant plasmid pcDNA3.1‑ELF5+EGFP into human ovarian carcinoma SKOV3 cells (recombinant plasmid group) in vitro and screened out stably transfected cells to conduct multiplication culture. Western blot analysis was performed to detect the expression of ELF5 protein in the different groups. Flow cytometry was employed to detect cell apoptosis and cycles. ELF5 mRNA in epithelial ovarian carcinoma and borderline ovarian epithelial tumor tissues were significantly lower (P<0.05) than those in benign ovarian epithelial tumor and normal ovarian tissues. ELF5 protein expression in the cells of recombinant plasmid group was significantly higher compared with empty plasmid and blank control groups. The capacity of cell reproductive recombinant plasmid group at each time point decreased (P<0.05). Flow cytometry detection showed that 67.03% of cells in recombinant plasmid group was blocked in G0/G1 phase (P<0.05), compared with empty plasmid group (37.17%) and blank control group (38.24%). Apoptotic rate of recombinant plasmid group was significantly lower (31.4±1.9%; P<0.05), compared with that of empty plasmid group (9.1±2.2%) and blank control group (8.7±1.5%), and the differences were statistically significant. In conclusion, ELF5 interfered with cell cycle of human ovarian carcinoma SKOV3 cells and promoted apoptosis of human ovarian carcinoma SKOV3 cells inhibiting their growth and invasive capacity; and thus providing a new approach to gene treatment of ovarian carcinoma.

Development and evaluation of a magnetic immunochromatographic test to detect Taenia solium, which causes taeniasis and neurocysticercosis in humans.

PubMed

Handali, Sukwan; Klarman, Molly; Gaspard, Amanda N; Dong, X Fan; Laborde, Ronald; Noh, John; Lee, Yeuk-Mui; Rodriguez, Silvia; Gonzalez, Armando E; Garcia, Hector H; Gilman, Robert H; Tsang, Victor C W; Wilkins, Patricia P

2010-04-01

Taeniasis/cysticercosis caused by Taenia solium is a frequent parasitic infection of the human brain in most of the world. Rapid and simple screening tools to identify taeniasis and cysticercosis cases are needed for control programs, mostly to identify tapeworm carriers which are the source of infection and need to be treated, or as tools for point-of-care case detection or confirmation. These screening assays should be affordable, reliable, rapid, and easy to perform. Immunochromatographic tests meet these criteria. To demonstrate proof of principle, we developed and evaluated two magnetic immunochromatographic tests (MICTs) for detection of human Taenia solium taeniasis antibodies (ES33-MICT) and neurocysticercosis antibodies (T24-MICT). These assays detected stage-specific antibodies by using two recombinant proteins, rES33 for detection of taeniasis antibodies and rT24H for detection of cysticercosis antibodies. The sensitivity and specificity of the ES33-MICT to detect taeniasis infections were 94.5% and 96%, respectively, and those of the T24-MICT to detect cases of human cysticercosis with two or more viable brain cysts were 93.9% and 98.9%, respectively. These data provide proof of principle that the ES33- and T24-MICTs provide rapid and suitable methods to identify individuals with taeniasis and cysticercosis.

Damage-induced ectopic recombination in the yeast Saccharomyces cerevisiae.

PubMed

Kupiec, M; Steinlauf, R

1997-06-09

Mitotic recombination in the yeast Saccharomyces cerevisiae is induced when cells are irradiated with UV or X-rays, reflecting the efficient repair of damage by recombinational repair mechanisms. We have used multiply marked haploid strains that allow the simultaneous detection of several types of ectopic recombination events. We show that inter-chromosomal ectopic conversion of lys2 heteroalleles and, to a lesser extent, direct repeat recombination (DRR) between non-tandem repeats, are increased by DNA-damaging agents; in contrast, ectopic recombination of the naturally occurring Ty element is not induced. We have tested several hypotheses that could explain the preferential lack of induction of Ty recombination by DNA-damaging agents. We have found that the lack of induction cannot be explained by a cell cycle control or by an effect of the mating-type genes. We also found no role for the flanking long terminal repeats (LTRs) of the Ty in preventing the induction. Ectopic conversion, DRR, and forward mutation of artificial repeats show different kinetics of induction at various positions of the cell cycle, reflecting different mechanisms of recombination. We discuss the mechanistic and evolutionary aspects of these results.

Construction of recombinant Kluyveromyces marxianus UFV-3 to express dengue virus type 1 nonstructural protein 1 (NS1).

PubMed

Bragança, Caio Roberto Soares; Colombo, Lívia Tavares; Roberti, Alvaro Soares; Alvim, Mariana Caroline Tocantins; Cardoso, Silvia Almeida; Reis, Kledna Constancio Portes; de Paula, Sérgio Oliveira; da Silveira, Wendel Batista; Passos, Flavia Maria Lopes

2015-02-01

The yeast Kluyveromyces marxianus is a convenient host for industrial synthesis of biomolecules. However, despite its potential, there are few studies reporting the expression of heterologous proteins using this yeast. Here, we report expression of a dengue virus protein in K. marxianus for the first time. The dengue virus type 1 nonstructural protein 1 (NS1) was integrated into the K. marxianus UFV-3 genome at the LAC4 locus using an adapted integrative vector designed for high-level expression of recombinant protein in Kluyveromyces lactis. The NS1 gene sequence was codon-optimized to increase the level of protein expression in yeast. The synthetic gene was cloned in frame with K. lactis α-mating factor signal peptide, and the recombinant plasmid obtained was used to transform K. marxianus UFV-3 by electroporation. The transformed cells, selected in yeast extract peptone dextrose containing 200 μg mL(-1) Geneticin, were mitotically stable. Analysis of recombinant strains by RT-PCR and protein detection using blot analysis confirmed both transcription and expression of extracellular NS1 polypeptide. After induction with galactose, the NS1 protein was analyzed by sodium dodecyl sulfate-PAGE and immunogenic detection. Protein production was investigated under two conditions: with galactose and biotin pulses at 24-h intervals during 96 h of induction and without galactose and biotin supplementation. Protease activity was not detected in post-growth medium. Our results indicate that recombinant K. marxianus is a good host for the production of dengue virus NS1 protein, which has potential for diagnostic applications.

WATER MASERS IN THE ANDROMEDA GALAXY. I. A SURVEY FOR WATER MASERS, AMMONIA, AND HYDROGEN RECOMBINATION LINES

DOE Office of Scientific and Technical Information (OSTI.GOV)

Darling, Jeremy; Gerard, Benjamin; Amiri, Nikta

We report the results of a Green Bank Telescope survey for water masers, ammonia (1, 1) and (2, 2), and the H66 α recombination line toward 506 luminous compact 24 μ m emitting regions in the Andromeda Galaxy (M31). We include the 206 sources observed in the Darling water maser survey for completeness. The survey was sensitive enough to detect any maser useful for ∼10 μ as yr{sup 1} astrometry. No new water masers, ammonia lines, or H66 α recombination lines were detected individually or in spectral stacks reaching rms noise levels of ∼3 mJy and ∼0.2 mJy, respectively, inmore » 3.1–3.3 km s{sup 1} channels. The lack of detections in individual spectra and in the spectral stacks is consistent with Galactic extrapolations. Contrary to previous assertions, there do not seem to be any additional bright water masers to be found in M31. The strong variability of water masers may enable new maser detections in the future, but variability may also limit the astrometric utility of known (or future) masers because flaring masers must also fade.« less

A cellulosic responsive "living" membrane.

PubMed

Qin, Guokui; Panilaitis, Bruce J; Kaplan, Zhongyuan Sun David L

2014-01-16

Bacterial cellulose has been demonstrated to be a remarkably versatile biomaterial and widely used in biomedical applications due to its unique physical properties. Here we reported for the first time a "living membrane" system based on recombinant Escherichia coli bacterial strains entrapped in cellulosic membranes produced by Gluconacetobacter xylinus. Biologically driven detection and identification of a range of target molecules presents unique challenges, and requires that detection methods are developed to be rapid, specific and sensitive. The compatibility of G. xylinus and recombinant E. coli strains was first investigated for co-cultivation, and the relationship between the number of entrapped E. coli and the level of inducible signal achieved was further explored by fluorescent signal observation in confocal microscopy. Finally to amplify the response to inducers for maximum fluorescent signal, a positive-feedback genetic amplifier was designed within recombinant E. coli strain entrapped in the living cellulosic membrane system, allowing for the detection mechanism to be extremely sensitive and resulting in a significant fluorescent signal from a single receptor binding event. The living membrane system proposed here will create devices of greater complexity in function for applications in biological and chemical detection. Copyright © 2013. Published by Elsevier Ltd.

Poliovirus RNA recombination: mechanistic studies in the absence of selection.

PubMed Central

Jarvis, T C; Kirkegaard, K

1992-01-01

Direct and quantitative detection of recombinant RNA molecules by polymerase chain reaction (PCR) provides a novel method for studying recombination in RNA viruses without selection for viable progeny. The parental poliovirus strains used in this study contained polymorphic marker loci approximately 600 bases apart; both exhibited wild-type growth characteristics. We established conditions under which the amount of PCR product was linearly proportional to the amount of input template, and the reproducibility was high. Recombinant progeny were predominantly homologous and arose at frequencies up to 2 x 10(-3). Recombination events increased in frequency throughout replication, indicating that there is no viral RNA sequestration or inhibition of recombination late in infection as proposed in earlier genetic studies. Previous studies have demonstrated that poliovirus recombination occurs by a copy-choice mechanism in which the viral polymerase switches templates during negative-strand synthesis. Varying the relative amount of input parental virus markedly altered reciprocal recombination frequencies. This, in conjunction with the kinetics data, indicated that acceptor template concentration is a determinant of template switching frequency. Since positive strands greatly outnumber negative strands throughout poliovirus infection, this would explain the bias toward recombination during negative-strand synthesis. Images PMID:1379178

Sabin Vaccine Reversion in the Field: a Comprehensive Analysis of Sabin-Like Poliovirus Isolates in Nigeria

PubMed Central

Chang, Stewart; Iber, Jane; Zhao, Kun; Adeniji, Johnson A.; Bukbuk, David; Baba, Marycelin; Behrend, Matthew; Burns, Cara C.; Oberste, M. Steven

2015-01-01

ABSTRACT To assess the dynamics of genetic reversion of live poliovirus vaccine in humans, we studied molecular evolution in Sabin-like poliovirus isolates from Nigerian acute flaccid paralysis cases obtained from routine surveillance. We employed a novel modeling approach to infer substitution and recombination rates from whole-genome sequences and information about poliovirus infection dynamics and the individual vaccination history. We confirmed observations from a recent vaccine trial that VP1 substitution rates are increased for Sabin-like isolates relative to the rate for the wild type due to increased nonsynonymous substitution rates. We also inferred substitution rates for attenuating nucleotides and confirmed that reversion can occur in days to weeks after vaccination. We combine our observations for Sabin-like virus evolution with the molecular clock for VP1 of circulating wild-type strains to infer that the mean time from the initiating vaccine dose to the earliest detection of circulating vaccine-derived poliovirus (cVDPV) is 300 days for Sabin-like virus type 1, 210 days for Sabin-like virus type 2, and 390 days for Sabin-like virus type 3. Phylogenetic relationships indicated transient local transmission of Sabin-like virus type 3 and, possibly, Sabin-like virus type 1 during periods of low wild polio incidence. Comparison of Sabin-like virus recombinants with known Nigerian vaccine-derived poliovirus recombinants shows that while recombination with non-Sabin enteroviruses is associated with cVDPV, the recombination rates are similar for Sabin isolate-Sabin isolate and Sabin isolate–non-Sabin enterovirus recombination after accounting for the time from dosing to the time of detection. Our study provides a comprehensive picture of the evolutionary dynamics of the oral polio vaccine in the field. IMPORTANCE The global polio eradication effort has completed its 26th year. Despite success in eliminating wild poliovirus from most of the world, polio persists in populations where logistical, social, and political factors have not allowed vaccination programs of sustained high quality. One issue of critical importance is eliminating circulating vaccine-derived polioviruses (cVDPVs) that have properties indistinguishable from those of wild poliovirus and can cause paralytic disease. cVDPV emerges due to the genetic instability of the Sabin viruses used in the oral polio vaccine (OPV) in populations that have low levels of immunity to poliovirus. However, the dynamics responsible are incompletely understood because it has historically been difficult to gather and interpret data about evolution of the Sabin viruses used in OPV in regions where cVDPV has occurred. This study is the first to combine whole-genome sequencing of poliovirus isolates collected during routine surveillance with knowledge about the intrahost dynamics of poliovirus to provide quantitative insight into polio vaccine evolution in the field. PMID:26468545

Recombinant Plants Provide a New Approach to the Production of Bacterial Polysaccharide for Vaccines

PubMed Central

Smith, Claire M.; Fry, Stephen C.; Gough, Kevin C.; Patel, Alexandra J. F.; Glenn, Sarah; Goldrick, Marie; Roberts, Ian S.; Andrew, Peter W.

2014-01-01

Bacterial polysaccharides have numerous clinical or industrial uses. Recombinant plants could offer the possibility of producing bacterial polysaccharides on a large scale and free of contaminating bacterial toxins and antigens. We investigated the feasibility of this proposal by cloning and expressing the gene for the type 3 synthase (cps3S) of Streptococcus pneumoniae in Nicotinia tabacum, using the pCambia2301 vector and Agrobacterium tumefaciens-mediated gene transfer. In planta the recombinant synthase polymerised plant-derived UDP-glucose and UDP-glucuronic acid to form type 3 polysaccharide. Expression of the cps3S gene was detected by RT-PCR and production of the pneumococcal polysaccharide was detected in tobacco leaf extracts by double immunodiffusion, Western blotting and high-voltage paper electrophoresis. Because it is used a component of anti-pneumococcal vaccines, the immunogenicity of the plant-derived type 3 polysaccharide was tested. Mice immunised with extracts from recombinant plants were protected from challenge with a lethal dose of pneumococci in a model of pneumonia and the immunised mice had significantly elevated levels of serum anti-pneumococcal polysaccharide antibodies. This study provides the proof of the principle that bacterial polysaccharide can be successfully synthesised in plants and that these recombinant polysaccharides could be used as vaccines to protect against life-threatening infections. PMID:24498433

Expression and the antigenicity of recombinant coat proteins of tungro viruses expressed in Escherichia coli.

PubMed

Yee, Siew Fung; Chu, Chia Huay; Poili, Evenni; Sum, Magdline Sia Henry

2017-02-01

Rice tungro disease (RTD) is a recurring disease affecting rice farming especially in the South and Southeast Asia. The disease is commonly diagnosed by visual observation of the symptoms on diseased plants in paddy fields and by polymerase chain reaction (PCR). However, visual observation is unreliable and PCR can be costly. High-throughput as well as relatively cheap detection methods are important for RTD management for screening large number of samples. Due to this, detection by serological assays such as immunoblotting assays and enzyme-linked immunosorbent assay are preferred. However, these serological assays are limited by lack of continuous supply of antibodies as reagents due to the difficulty in preparing sufficient purified virions as antigens. This study aimed to generate and evaluate the reactivity of the recombinant coat proteins of Rice tungro bacilliform virus (RTBV) and Rice tungro spherical virus (RTSV) as alternative antigens to generate antibodies. The genes encoding the coat proteins of both viruses, RTBV (CP), and RTSV (CP1, CP2 and CP3) were cloned and expressed as recombinant fusion proteins in Escherichia coli. All of the recombinant fusion proteins, with the exception of the recombinant fusion protein of the CP2 of RTSV, were reactive against our in-house anti-tungro rabbit serum. In conclusion, our study showed the potential use of the recombinant fusion coat proteins of the tungro viruses as alternative antigens for production of antibodies for diagnostic purposes. Copyright © 2016 Elsevier B.V. All rights reserved.

A recombinase polymerase amplification-based assay for rapid detection of African swine fever virus.

PubMed

Wang, Jianchang; Wang, Jinfeng; Geng, Yunyun; Yuan, Wanzhe

2017-10-01

A recombinase polymerase amplification (RPA)-based method was developed for rapid and specific detection of African swine fever virus (ASFV), the etiologic agent of African swine fever, a devastating disease of swine. Primers and the exo probe targeting the conserved region of the P72 gene of ASFV were designed and the reaction was run on the Genie III scanner device. Using recombinant plasmid DNA containing the P72 gene as template, we showed that the amplified product could be detected in less than 10 min and that the detection limit was 10 2 copies DNA/reaction [same detection limit as real-time polymerase chain reaction (PCR)]. The RPA assay did not cross-detect CSFV, PCV-2, PRV, PRRSV, or FMDV, common viruses seen in pigs. Tests of recombinant plasmid-spiked serum samples revealed that RPA and real-time PCR had the same diagnostic rate. The RPA assay, which is simple, cost-effective, and fast, is a promising alternative to real-time PCR for ASFV detection.

A recombinase polymerase amplification-based assay for rapid detection of African swine fever virus

PubMed Central

Wang, Jianchang; Wang, Jinfeng; Geng, Yunyun; Yuan, Wanzhe

2017-01-01

A recombinase polymerase amplification (RPA)-based method was developed for rapid and specific detection of African swine fever virus (ASFV), the etiologic agent of African swine fever, a devastating disease of swine. Primers and the exo probe targeting the conserved region of the P72 gene of ASFV were designed and the reaction was run on the Genie III scanner device. Using recombinant plasmid DNA containing the P72 gene as template, we showed that the amplified product could be detected in less than 10 min and that the detection limit was 102 copies DNA/reaction [same detection limit as real-time polymerase chain reaction (PCR)]. The RPA assay did not cross-detect CSFV, PCV-2, PRV, PRRSV, or FMDV, common viruses seen in pigs. Tests of recombinant plasmid-spiked serum samples revealed that RPA and real-time PCR had the same diagnostic rate. The RPA assay, which is simple, cost-effective, and fast, is a promising alternative to real-time PCR for ASFV detection. PMID:29081590

Molecular characterization of a novel Muscovy duck parvovirus isolate: evidence of recombination between classical MDPV and goose parvovirus strains.

PubMed

Wang, Jianye; Ling, Jueyi; Wang, Zhixian; Huang, Yu; Zhu, Jianzhong; Zhu, Guoqiang

2017-11-09

Muscovy duck parvovirus (MDPV) and Goose parvovirus (GPV) are important etiological agents for Muscovy duck parvoviral disease and Derzsy's disease, respectively; both of which can cause substantial economic losses in waterfowl industry. In contrast to GPV, the complete genomic sequence data of MDPV isolates are still limited and their phylogenetic relationships largely remain unknown. In this study, the entire genome of a pathogenic MDPV strain ZW, which was isolated from a deceased Muscovy duckling in 2006 in China, was cloned, sequenced, and compared with that of other classical MDPV and GPV strains. The genome of strain ZW comprises of 5071 nucleotides; this genome was shorter than that of the pathogenic MDPV strain YY (5075 nt). All the four deleted nucleotides produced in strain ZW are located at the base-pairing positions in the palindromic stem of inverted terminal repeats (ITR) without influencing the formation of a hairpin structure. Recombination analysis revealed that strain ZW originated from genetic recombination between the classical MDPV and GPV strain. The YY strain of MDPV acts as the major parent, whereas the virulent strains YZ99-6 and B and the vaccine strain SYG61v of GPV act as the minor parents in varying degrees. Two recombination sites were detected in strain ZW, with the small recombination site surrounding the P9 promoter, and the large recombination site situated in the middle of the VP3 gene. The SYG61V strain is a vaccine strain used for preventing goose parvoviral disease. This strain was found to be solely involved in the recombination event detected in the P9 promoter region. Phylogenetic analyses between strain ZW and other classical strains of MDPV and GPV were performed. The results supported the in silico recombination analysis conclusion. MDPV Strain ZW is a novel recombinant parvovirus, and the bulk of its genome originates from the classical MDPV strain. Two virulent strains and a vaccine strain of GPV were involved in the recombination process in varying degrees.

The Contribution of Genetic Recombination to CRISPR Array Evolution.

PubMed

Kupczok, Anne; Landan, Giddy; Dagan, Tal

2015-06-16

CRISPR (clustered regularly interspaced short palindromic repeats) is a microbial immune system against foreign DNA. Recognition sequences (spacers) encoded within the CRISPR array mediate the immune reaction in a sequence-specific manner. The known mechanisms for the evolution of CRISPR arrays include spacer acquisition from foreign DNA elements at the time of invasion and array erosion through spacer deletion. Here, we consider the contribution of genetic recombination between homologous CRISPR arrays to the evolution of spacer repertoire. Acquisition of spacers from exogenic arrays via recombination may confer the recipient with immunity against unencountered antagonists. For this purpose, we develop a novel method for the detection of recombination in CRISPR arrays by modeling the spacer order in arrays from multiple strains from the same species. Because the evolutionary signal of spacer recombination may be similar to that of pervasive spacer deletions or independent spacer acquisition, our method entails a robustness analysis of the recombination inference by a statistical comparison to resampled and perturbed data sets. We analyze CRISPR data sets from four bacterial species: two Gammaproteobacteria species harboring CRISPR type I and two Streptococcus species harboring CRISPR type II loci. We find that CRISPR array evolution in Escherichia coli and Streptococcus agalactiae can be explained solely by vertical inheritance and differential spacer deletion. In Pseudomonas aeruginosa, we find an excess of single spacers potentially incorporated into the CRISPR locus during independent acquisition events. In Streptococcus thermophilus, evidence for spacer acquisition by recombination is present in 5 out of 70 strains. Genetic recombination has been proposed to accelerate adaptation by combining beneficial mutations that arose in independent lineages. However, for most species under study, we find that CRISPR evolution is shaped mainly by spacer acquisition and loss rather than recombination. Since the evolution of spacer content is characterized by a rapid turnover, it is likely that recombination is not beneficial for improving phage resistance in the strains under study, or that it cannot be detected in the resolution of intraspecies comparisons. © The Author(s) 2015. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.

The effect of DNA priming-protein boosting on enhancing humoral immunity and protecting mice against lethal HSV infections.

PubMed

Soleimanjahi, Hoorieh; Roostaee, Mohammad Hassan; Rasaee, Mohammad Javad; Mahboudi, Fereidoon; Kazemnejad, Anooshirvan; Bamdad, Taravat; Zandi, Keivan

2006-02-01

Herpes simplex virus produces primary and latent infections with periodic recurrency. The prime-boost immunization strategies were studied using a DNA vaccine carrying the full-length glycoprotein D-1 gene and a baculovirus-derived recombinant glycoprotein D, both expressing herpes simplex virus glycoprotein D-1 protein. Immunization with recombinant DNAs encoding antigenic proteins could induce cellular and humoral responses by providing antigen expression in vivo. Higher immune response, however, occurred when the recombinant proteins followed DNA inoculation. While all groups of the immunized mice and positive control group could resist virus challenge, a higher virus neutralizing antibody level was detected in the animals receiving recombinant protein following DNA vaccination.

Phosphoethanolamine-N-methyltransferase is a potential biomarker for the diagnosis of P. knowlesi and P. falciparum malaria

PubMed Central

2018-01-01

Background Plasmodium knowlesi is recognised as the main cause of human malaria in Southeast Asia. The disease is often misdiagnosed as P. falciparum or P. malariae infections by microscopy, and the disease is difficult to eliminate due to its presence in both humans and monkeys. P. knowlesi infections can rapidly cause severe disease and require prompt diagnosis and treatment. No protein biomarker exists for the rapid diagnostic test (RDT) detection of P. knowlesi infections. Plasmodium knowlesi infections can be diagnosed by PCR. Methods and principal findings Phosphoethanolamine-N-methyltransferase (PMT) is involved in malaria lipid biosynthesis and is not found in the human host. The P. falciparum, P. vivax and P. knowlesi PMT proteins were recombinantly expressed in BL21(DE3) Escherichia coli host cells, affinity purified and used to raise antibodies in chickens. Antibodies against each recombinant PMT protein all detected all three recombinant proteins and the native 29 kDa P. falciparum PMT protein on western blots and in ELISA. Antibodies against a PMT epitope (PLENNQYTDEGVKC) common to all three PMT orthologues detected all three proteins. Antibodies against unique peptides from each orthologue of PMT, PfCEVEHKYLHENKE, PvVYSIKEYNSLKDC, PkLYPTDEYNSLKDC detected only the parent protein in western blots and P. falciparum infected red blood cell lysates or blood lysates spiked with the respective proteins. Similar concentrations of PfPMT and the control, PfLDH, were detected in the same parasite lysate. The recombinant PfPMT protein was detected by a human anti-malaria antibody pool. Conclusion PMT, like the pan-specific LDH biomarker used in RDT tests, is both soluble, present at comparable concentrations in the parasite and constitutes a promising antimalarial drug target. PMT is absent from the human proteome. PMT has the potential as a biomarker for human malaria and in particular as the first P. knowlesi specific protein with diagnostic potential for the identification of a P. knowlesi infection. PMID:29505599

Phosphoethanolamine-N-methyltransferase is a potential biomarker for the diagnosis of P. knowlesi and P. falciparum malaria.

PubMed

Krause, Robert G E; Goldring, J P Dean

2018-01-01

Plasmodium knowlesi is recognised as the main cause of human malaria in Southeast Asia. The disease is often misdiagnosed as P. falciparum or P. malariae infections by microscopy, and the disease is difficult to eliminate due to its presence in both humans and monkeys. P. knowlesi infections can rapidly cause severe disease and require prompt diagnosis and treatment. No protein biomarker exists for the rapid diagnostic test (RDT) detection of P. knowlesi infections. Plasmodium knowlesi infections can be diagnosed by PCR. Phosphoethanolamine-N-methyltransferase (PMT) is involved in malaria lipid biosynthesis and is not found in the human host. The P. falciparum, P. vivax and P. knowlesi PMT proteins were recombinantly expressed in BL21(DE3) Escherichia coli host cells, affinity purified and used to raise antibodies in chickens. Antibodies against each recombinant PMT protein all detected all three recombinant proteins and the native 29 kDa P. falciparum PMT protein on western blots and in ELISA. Antibodies against a PMT epitope (PLENNQYTDEGVKC) common to all three PMT orthologues detected all three proteins. Antibodies against unique peptides from each orthologue of PMT, PfCEVEHKYLHENKE, PvVYSIKEYNSLKDC, PkLYPTDEYNSLKDC detected only the parent protein in western blots and P. falciparum infected red blood cell lysates or blood lysates spiked with the respective proteins. Similar concentrations of PfPMT and the control, PfLDH, were detected in the same parasite lysate. The recombinant PfPMT protein was detected by a human anti-malaria antibody pool. PMT, like the pan-specific LDH biomarker used in RDT tests, is both soluble, present at comparable concentrations in the parasite and constitutes a promising antimalarial drug target. PMT is absent from the human proteome. PMT has the potential as a biomarker for human malaria and in particular as the first P. knowlesi specific protein with diagnostic potential for the identification of a P. knowlesi infection.

Evaluation of the Recombinant Protein TpF1 of Treponema pallidum for Serodiagnosis of Syphilis

PubMed Central

Jiang, Chuanhao; Zhao, Feijun; Xiao, Jinhong; Zeng, Tiebing; Yu, Jian; Ma, Xiaohua; Wu, Haiying

2013-01-01

Syphilis is a chronic infection caused by Treponema pallidum subsp. pallidum, and diagnosis with sensitive and specific methods is a challenging process that is important for its prevention and treatment. In the present study, we established a recombinant protein TpF1-based indirect immunoglobulin G (IgG) enzyme-linked immunosorbent assay (ELISA) and a Western blot assay for human and rabbit sera. The 20-kDa recombinant protein TpF1 was detected by Western blotting performed with sera from rabbits immunized with recombinant TpF1 and infected with the T. pallidum Nichols strain and T. pallidum clinical isolates but was not detected by Western blotting with sera from uninfected rabbits. The sensitivity of the recombinant protein was determined by screening sera from individuals with primary, secondary, latent, and congenital syphilis (n = 82). The specificity of the recombinant protein was determined by screening sera from uninfected controls (n = 30) and individuals with potentially cross-reactive infections, including Lyme disease (n = 30) and leptospirosis (n = 5). The sensitivities of TpF1-based ELISAs were 93.3%, 100%, 100%, and 100% for primary, secondary, latent, and congenital syphilis, respectively, and the specificities were all 100% for sera from uninfected controls and individuals with potentially cross-reactive infections. In Western blot assays, the sensitivities and specificities of TpF1 for human sera were all 100%. The reactivities of TpF1 with syphilitic sera were proportional to the titers of the T. pallidum particle agglutination (TPPA) assay. These data indicate that the recombinant protein TpF1 is a highly immunogenic protein in human and rabbit infections and a promising marker for the screening of syphilis. PMID:23945159

Development of a Rapid Immunochromatographic Lateral Flow Device Capable of Differentiating Phytase Expressed from Recombinant Aspergillus niger phyA2 and Genetically Modified Corn.

PubMed

Zhou, Xiaojin; Hui, Elizabeth; Yu, Xiao-Lin; Lin, Zhen; Pu, Ling-Kui; Tu, Zhiguan; Zhang, Jun; Liu, Qi; Zheng, Jian; Zhang, Juan

2015-05-06

Phytase is a phosphohydrolase considered highly specific for the degradation of phytate to release bound phosphorus for animal consumption and aid in the reduction of environmental nutrient loading. New sources of phytase have been sought that are economically and efficiently productive including the construction of genetically modified (GM) phytase products designed to bypass the costs associated with feed processing. Four monoclonal antibodies (EH10a, FA7, AF9a, and CC1) raised against recombinant Aspergillus niger phyA2 were used to develop a highly specific and sensitive immunochromatographic lateral flow device for rapid detection of transgenic phytase, such as in GM corn. Antibodies sequentially paired and tested along lateral flow strips showed that the EH10a-FA7 antibody pair was able to detect the recombinant yeast-phytase at 5 ng/mL, whereas the AF9a-CC1 antibody pair to GM phytase corn was able to detect at 2 ng/mL. Concurrent to this development, evidence was revealed which suggests that antibody binding sites may be glycosylated.

[Targeted detecting HER2 expression with recombinant anti HER2 ScFv-GFP fusion antibody].

PubMed

Gao, Guohui; Chen, Chong; Yang, Yanmei; Yang, Han; Wang, Jindan; Zheng, Yi; Huang, Qidi; Hu, Xiaoqu

2012-08-01

To verify the reliability of targeted detecting HER2 positive cancer cells and clinical pathological tissue specimens with a recombinant anti HER2 single chain antibody in single chain Fv fragment (scFv) format, we have constructed the fusion variable regions of the ScFv specific for HER2/neu. labeled a green-fluorescent protein(GFP). The humanized recombinant Anti HER2 ScFv-GFP gene was inserted into pFast Bac HT A, and expressed in insect cells sf9. Then the recombinant fusion protein Anti HER2 ScFv-GFP was properly purified with Ni2+-NTA affinity chromatography from the infected sf9 cells used to test the specificity of the fusion antibody for HER2 positive cancer cells. Firstly, the purified antibody incubated with HER2 positive breast cancer cells SKBR3, BT474 and HER2 negative breast cancer cells MCF7 for 12 h/24 h/48 h at 37 degrees C, in order to confirm targeted detecting HER2 positive breast cancer cells by Laser Confocal Microscopy. Furthermore, the same clinical pathological tissue samples were assessed by immunohistochemistry (IHC) and the fusion antibody Anti HER2 ScFv-GFP in the meanwhile. The data obtained indicated that the recombinant eukaryotic expression plasmid pFast Bac HT A/Anti HER2 ScFv-GFP was constructed successfully In addition, obvious green fluorescent was observed in insect cells sf9. When the purified fusion antibody was incubated with different cancer cells, much more green fluorescent was observed on the surface of the HER2 positive cancer cells SKBR3 and BT474. In contrast, no green fluorescent on the surface of the HER2 negative cancer cells MCF7 was detected. The concentration of the purified fusion antibody was 115.5 microg/mL, of which protein relative molecular weight was 60 kDa. The analysis showed the purity was about 97% and the titer was about 1:64. The detection results of IHC and fusion antibody testing indicated the conformity. In summary, the study showed that the new fusion antibody Anti HER2 ScFv-GFP can test HER2 positive cancer cells, indicating a potential candidate method for clinical HER2 positive specimens detection.

ARG-walker: inference of individual specific strengths of meiotic recombination hotspots by population genomics analysis.

PubMed

Chen, Hao; Yang, Peng; Guo, Jing; Kwoh, Chee Keong; Przytycka, Teresa M; Zheng, Jie

2015-01-01

Meiotic recombination hotspots play important roles in various aspects of genomics, but the underlying mechanisms for regulating the locations and strengths of recombination hotspots are not yet fully revealed. Most existing algorithms for estimating recombination rates from sequence polymorphism data can only output average recombination rates of a population, although there is evidence for the heterogeneity in recombination rates among individuals. For genome-wide association studies (GWAS) of recombination hotspots, an efficient algorithm that estimates the individualized strengths of recombination hotspots is highly desirable. In this work, we propose a novel graph mining algorithm named ARG-walker, based on random walks on ancestral recombination graphs (ARG), to estimate individual-specific recombination hotspot strengths. Extensive simulations demonstrate that ARG-walker is able to distinguish the hot allele of a recombination hotspot from the cold allele. Integrated with output of ARG-walker, we performed GWAS on the phased haplotype data of the 22 autosome chromosomes of the HapMap Asian population samples of Chinese and Japanese (JPT+CHB). Significant cis-regulatory signals have been detected, which is corroborated by the enrichment of the well-known 13-mer motif CCNCCNTNNCCNC of PRDM9 protein. Moreover, two new DNA motifs have been identified in the flanking regions of the significantly associated SNPs (single nucleotide polymorphisms), which are likely to be new cis-regulatory elements of meiotic recombination hotspots of the human genome. Our results on both simulated and real data suggest that ARG-walker is a promising new method for estimating the individual recombination variations. In the future, it could be used to uncover the mechanisms of recombination regulation and human diseases related with recombination hotspots.

Effect of copper on the recombination activity of extended defects in silicon

DOE Office of Scientific and Technical Information (OSTI.GOV)

Feklisova, O. V., E-mail: feklisov@iptm.ru; Yakimov, E. B.

2015-06-15

The effect of copper atoms introduced by high-temperature diffusion on the recombination properties of dislocations and dislocation trails in p-type single-crystal silicon is studied by the electron-beam-induced current technique. It is shown that, in contrast to dislocations, dislocation trails exhibit an increase in recombination activity after the introduction of copper. Bright contrast appearance in the vicinity of dislocation trails is detected after the diffusion of copper and quenching of the samples. The contrast depends on the defect density in these trails.

Detecting Genetic Introgression: High Levels of Intersubspecific Recombination Found in Xylella fastidiosa in Brazil

PubMed Central

Yuan, Xiaoli; Bromley, Robin E.; Stouthamer, Richard

2012-01-01

Documenting the role of novel mutation versus homologous recombination in bacterial evolution, and especially in the invasion of new hosts, is central to understanding the long-term dynamics of pathogenic bacteria. We used multilocus sequence typing (MLST) to study this issue in Xylella fastidiosa subsp. pauca from Brazil, a bacterium causing citrus variegated chlorosis (CVC) and coffee leaf scorch (CLS). All 55 citrus isolates typed (plus one coffee isolate) defined three similar sequence types (STs) dominated by ST11 (85%), while the remaining 22 coffee isolates defined two STs, mainly ST16 (74%). This low level of variation masked unusually large allelic differences (>1% divergence with no intermediates) at five loci (leuA, petC, malF, cysG, and holC). We developed an introgression test to detect whether these large differences were due to introgression via homologous recombination from another X. fastidiosa subspecies. Using additional sequencing around these loci, we established that the seven randomly chosen MLST targets contained seven regions of introgression totaling 2,172 bp of 4,161 bp (52%), only 409 bp (10%) of which were detected by other recombination tests. This high level of introgression suggests the hypothesis that X. fastidiosa subsp. pauca became pathogenic on citrus and coffee (crops cultivated in Brazil for several hundred years) only recently after it gained genetic variation via intersubspecific recombination, facilitating a switch from native hosts. A candidate donor is the subspecies infecting plum in the region since 1935 (possibly X. fastidiosa subsp. multiplex). This hypothesis predicts that nonrecombinant native X. fastidiosa subsp. pauca (not yet isolated) does not cause disease in citrus or coffee. PMID:22544234

ABPA diagnosis in cystic fibrosis patients: the clinical utility of IgE specific to recombinant Aspergillus fumigatus allergens.

PubMed

Almeida, Marina B; Bussamra, Maria Helena C F; Rodrigues, Joaquim C

2006-01-01

Allergic bronchopulmonary aspergillosis (ABPA) is a complicating factor of cystic fibrosis which can result in a devastating combination as lung disease progresses. The overlap between the signs and symptoms of the two conditions makes diagnosis problematic, even if standardized criteria are used. The objective of this study was to identify, in a group of cystic fibrosis patients, cases of ABPA by assaying IgE specific to recombinant Aspergillus fumigatus antigens and to compare the method with the Cystic Fibrosis Foundation diagnostic criteria. Fifty-four patients, aged 2 to 20 years, presenting characteristics that could occur with ABPA in isolation, were systematically assessed based on the following: clinical data, a chest CT scan, immediate hypersensitivity skin test for A. fumigatus, total serum IgE assay, RAST for A. fumigatus and serum IgE specific for the recombinant allergens Asp f1, f2, f3, f4 and f6. Thirty-nine patients were eligible for the study. Thirty-two of these were investigated. Sensitization to A. fumigatus was observed in 34%. Both the Cystic Fibrosis Foundation criteria and the recombinant antigen specific IgE assay defined three patients as suffering from ABPA; however, only two of these patients were diagnosed by both methods. The detection of A. fumigatus recombinant antigen specific IgE was a useful tool for the early detection of sensitization and diagnosis of ABPA. Nevertheless, diagnostic confirmation cannot be divorced from clinical findings, and before this method can be used for ABPA diagnosis, for detecting relapses and for defining cure criteria, longitudinal studies with larger numbers of patients are required.

Detecting genetic introgression: high levels of intersubspecific recombination found in Xylella fastidiosa in Brazil.

PubMed

Nunney, Leonard; Yuan, Xiaoli; Bromley, Robin E; Stouthamer, Richard

2012-07-01

Documenting the role of novel mutation versus homologous recombination in bacterial evolution, and especially in the invasion of new hosts, is central to understanding the long-term dynamics of pathogenic bacteria. We used multilocus sequence typing (MLST) to study this issue in Xylella fastidiosa subsp. pauca from Brazil, a bacterium causing citrus variegated chlorosis (CVC) and coffee leaf scorch (CLS). All 55 citrus isolates typed (plus one coffee isolate) defined three similar sequence types (STs) dominated by ST11 (85%), while the remaining 22 coffee isolates defined two STs, mainly ST16 (74%). This low level of variation masked unusually large allelic differences (>1% divergence with no intermediates) at five loci (leuA, petC, malF, cysG, and holC). We developed an introgression test to detect whether these large differences were due to introgression via homologous recombination from another X. fastidiosa subspecies. Using additional sequencing around these loci, we established that the seven randomly chosen MLST targets contained seven regions of introgression totaling 2,172 bp of 4,161 bp (52%), only 409 bp (10%) of which were detected by other recombination tests. This high level of introgression suggests the hypothesis that X. fastidiosa subsp. pauca became pathogenic on citrus and coffee (crops cultivated in Brazil for several hundred years) only recently after it gained genetic variation via intersubspecific recombination, facilitating a switch from native hosts. A candidate donor is the subspecies infecting plum in the region since 1935 (possibly X. fastidiosa subsp. multiplex). This hypothesis predicts that nonrecombinant native X. fastidiosa subsp. pauca (not yet isolated) does not cause disease in citrus or coffee.

Characterization of a naturally occurring recombinant isolate of Grapevine fanleaf virus.

PubMed

Vigne, E; Demangeat, G; Komar, V; Fuchs, M

2005-11-01

The naturally occurring Grapevine fanleaf virus (GFLV) recombinant isolate A17b was recovered from its grapevine host by sap inoculation and serial passages onto Gomphrena globosa, a pseudo local lesion herbaceous host, and Chenopodium quinoa, a systemic herbaceous host, to characterize some of its biological properties. Sequence analysis of the CP gene, in which a recombinational event was previously detected, demonstrated the genetic stability of recombinant isolate A17b over a 5-year period in its natural host as well as in C. quinoa. Also, recombinant isolate A17b was graft transmissible, as shown by an in vitro heterologous approach, and transmitted by the nematode Xiphinema index as readily as nonrecombinant GFLV isolates. Furthermore, despite a lower pathogenicity on Chenopodium amaranticolor, recombinant isolate A17b had a similar host range and induced similar symptoms in type and severity to nonrecombinant GFLV isolates. Interestingly, the use of infectious chimeric RNA2 transcripts in combination to RNA1 transcripts of GFLV strain F13 suggested no implication of the recombination event in the CP gene of isolate A17b in the reduced pathogenicity on C. amaranticolor. Altogether, recombinant isolate A17b had similar biological properties to GFLV nonrecombinant isolates.

Whole-genome analysis of genetic recombination of hepatitis delta virus: molecular domain in delta antigen determining trans-activating efficiency.

PubMed

Chao, Mei; Lin, Chia-Chi; Lin, Feng-Ming; Li, Hsin-Pai; Iang, Shan-Bei

2015-12-01

Hepatitis delta virus (HDV) is the only animal RNA virus that has an unbranched rod-like genome with ribozyme activity and is replicated by host RNA polymerase. HDV RNA recombination was previously demonstrated in patients and in cultured cells by analysis of a region corresponding to the C terminus of the delta antigen (HDAg), the only viral-encoded protein. Here, a whole-genome recombination map of HDV was constructed using an experimental system in which two HDV-1 sequences were co-transfected into cultured cells and the recombinants were analysed by sequencing of cloned reverse transcription-PCR products. Fifty homologous recombinants with 60 crossovers mapping to 22 junctions were identified from 200 analysed clones. Small HDAg chimeras harbouring a junction newly detected in the recombination map were then constructed. The results further indicated that the genome-replication level of HDV was sensitive to the sixth amino acid within the N-terminal 22 aa of HDAg. Therefore, the recombination map established in this study provided a tool for not only understanding HDV RNA recombination, but also elucidating the related mechanisms, such as molecular elements responsible for the trans-activation levels of the small HDAg.

Depleted Nanocrystal-Oxide Heterojunctions for High-Sensitivity Infrared Detection

DTIC Science & Technology

2015-08-28

from surface dangling bonds and behave as effective nonradiative recombination centers.17 Upon the growth of CdSe, the main PL peak exhibits a redshift...as nonradiative recombination sites and cause PL degradation. With a 4.5 ML CdSe shell, the QY drops to 4%. As seen in Fig. 6, the PL QY is

Antibodies specific for HT.sub.m4

DOEpatents

Lim, Bing; Adra, Chaker N.; Lelias, Jean-Michel

1998-01-01

The invention relates to a recombinant DNA molecule which encodes a HT.sub.m4 protein, a transformed host cell which has been stably transfected with a DNA molecule which encodes a HT.sub.m4 protein and a recombinant HT.sub.m4 protein. The invention also relates to a method for detecting the presence of a hereditary atopy.

An integrative 'omics' solution to the detection of recombinant human erythropoietin and blood doping.

PubMed

Pitsiladis, Yannis P; Durussel, Jérôme; Rabin, Olivier

2014-05-01

Administration of recombinant human erythropoietin (rHumanEPO) improves sporting performance and hence is frequently subject to abuse by athletes, although rHumanEPO is prohibited by the WADA. Approaches to detect rHumanEPO doping have improved significantly in recent years but remain imperfect. A new transcriptomic-based longitudinal screening approach is being developed that has the potential to improve the analytical performance of current detection methods. In particular, studies are being funded by WADA to identify a 'molecular signature' of rHumanEPO doping and preliminary results are promising. In the first systematic study to be conducted, the expression of hundreds of genes were found to be altered by rHumanEPO with numerous gene transcripts being differentially expressed after the first injection and further transcripts profoundly upregulated during and subsequently downregulated up to 4 weeks postadministration of the drug; with the same transcriptomic pattern observed in all participants. The identification of a blood 'molecular signature' of rHumanEPO administration is the strongest evidence to date that gene biomarkers have the potential to substantially improve the analytical performance of current antidoping methods such as the Athlete Biological Passport for rHumanEPO detection. Given the early promise of transcriptomics, research using an 'omics'-based approach involving genomics, transcriptomics, proteomics and metabolomics should be intensified in order to achieve improved detection of rHumanEPO and other doping substances and methods difficult to detect such a recombinant human growth hormone and blood transfusions.

Microwell hybridization assay for detection of PCR products from Mycobacterium tuberculosis complex and the recombinant Mycobacterium smegmatis strain 1008 used as an internal control.

PubMed Central

Kox, L F; Noordhoek, G T; Kunakorn, M; Mulder, S; Sterrenburg, M; Kolk, A H

1996-01-01

A microwell hybridization assay was developed for the detection of the PCR products from both Mycobacterium tuberculosis complex bacteria and the recombinant Mycobacterium smegmatis strain 1008 that is used as an internal control to monitor inhibition in the PCR based on the M. tuberculosis complex-specific insertion sequence IS6110. The test is based on specific detection with digoxigenin-labeled oligonucleotide probes of biotinylated PCR products which are captured in a microtiter plate coated with streptavidin. The captured PCR products are hybridized separately with two probes, one specific for the PCR product from IS6110 from M. tuberculosis complex and the other specific for the PCR fragment from the modified IS6110 fragment from the recombinant M. smegmatis 1008. The microwell hybridization assay discriminates perfectly between the two types of amplicon. The amount of PCR product that can be detected by this assay is 10 times less than that which can be detected by agarose gel electrophoresis. The test can be performed in 2 h. It is much faster and less laborious than Southern blot hybridization. Furthermore, the interpretation of results is objective. The assay was used with 172 clinical samples in a routine microbiology laboratory, and the results were in complete agreement with those of agarose gel electrophoresis and Southern blot hybridization. PMID:8862568

ENDOGENOUS RETROVIRUSES MOBILIZED DURING FRIEND MURINE LEUKEMIA VIRUS INFECTION

PubMed Central

Hansen, Ethan; Hendrick, Duncan; Malik, Frank; Evans, Leonard H.

2016-01-01

We have demonstrated in a mouse model that infection with a retrovirus can lead not only to the generation of recombinants between exogenous and endogenous gammaretrovirus, but also to the mobilization of endogenous proviruses by pseudotyping entire polytropic proviral transcripts and facilitating their infectious spread to new cells. However, the frequency of this occurrence, the kinetics, and the identity of mobilized endogenous proviruses was unclear. Here we find that these mobilized transcripts are detected after only one day of infection. They predominate over recombinant polytropic viruses early in infection, persist throughout the course of disease and are comprised of multiple different polytropic proviruses. Other endogenous retroviral elements such as intracisternal A particles (IAPs) were not detected. The integration of the endogenous transcripts into new cells could result in loss of transcriptional control and elevated expression which may facilitate pathogenesis, perhaps by contributing to the generation of polytropic recombinant viruses. PMID:27657834

Two-Photon Laser-Induced Fluorescence O and N Atoms for the Study of Heterogeneous Catalysis in a Diffusion Reactor

NASA Technical Reports Server (NTRS)

Pallix, Joan B.; Copeland, Richard A.; Arnold, James O. (Technical Monitor)

1995-01-01

Advanced laser-based diagnostics have been developed to examine catalytic effects and atom/surface interactions on thermal protection materials. This study establishes the feasibility of using laser-induced fluorescence for detection of O and N atom loss in a diffusion tube to measure surface catalytic activity. The experimental apparatus is versatile in that it allows fluorescence detection to be used for measuring species selective recombination coefficients as well as diffusion tube and microwave discharge diagnostics. Many of the potential sources of error in measuring atom recombination coefficients by this method have been identified and taken into account. These include scattered light, detector saturation, sample surface cleanliness, reactor design, gas pressure and composition, and selectivity of the laser probe. Recombination coefficients and their associated errors are reported for N and O atoms on a quartz surface at room temperature.

A Novel Conductometric Urea Biosensor with Improved Analytical Characteristic Based on Recombinant Urease Adsorbed on Nanoparticle of Silicalite

NASA Astrophysics Data System (ADS)

Velychko, T. P.; Soldatkin, O. O.; Melnyk, V. G.; Marchenko, S. V.; Kirdeciler, S. K.; Akata, B.; Soldatkin, A. P.; El'skaya, A. V.; Dzyadevych, S. V.

2016-02-01

Development of a conductometric biosensor for the urea detection has been reported. It was created using a non-typical method of the recombinant urease immobilization via adsorption on nanoporous particles of silicalite. It should be noted that this biosensor has a number of advantages, such as simple and fast performance, the absence of toxic compounds during biosensor preparation, and high reproducibility (RSD = 5.1 %). The linear range of urea determination by using the biosensor was 0.05-15 mM, and a lower limit of urea detection was 20 μM. The bioselective element was found to be stable for 19 days. The characteristics of recombinant urease-based biomembranes, such as dependence of responses on the protein and ion concentrations, were investigated. It is shown that the developed biosensor can be successfully used for the urea analysis during renal dialysis.

Segregation and recombination of a multipartite mitochondrial DNA in populations of the potato cyst nematode Globodera pallida.

PubMed

Armstrong, Miles R; Husmeier, Dirk; Phillips, Mark S; Blok, Vivian C

2007-06-01

The discovery that the potato cyst nematode Globodera pallida has a multipartite mitochondrial DNA (mtDNA) composed, at least in part, of six small circular mtDNAs (scmtDNAs) raised a number of questions concerning the population-level processes that might act on such a complex genome. Here we report our observations on the distribution of some scmtDNAs among a sample of European and South American G. pallida populations. The occurrence of sequence variants of scmtDNA IV in population P4A from South America, and that particular sequence variants are common to the individuals within a single cyst, is described. Evidence for recombination of sequence variants of scmtDNA IV in P4A is also reported. The mosaic structure of P4A scmtDNA IV sequences was revealed using several detection methods and recombination breakpoints were independently detected by maximum likelihood and Bayesian MCMC methods.

Immunocytological analysis of meiotic recombination in two anole lizards (Squamata, Dactyloidae).

PubMed

Lisachov, Artem P; Trifonov, Vladimir A; Giovannotti, Massimo; Ferguson-Smith, Malcolm A; Borodin, Pavel M

2017-01-01

Although the evolutionary importance of meiotic recombination is not disputed, the significance of interspecies differences in the recombination rates and recombination landscapes remains under-appreciated. Recombination rates and distribution of chiasmata have been examined cytologically in many mammalian species, whereas data on other vertebrates are scarce. Immunolocalization of the protein of the synaptonemal complex (SYCP3), centromere proteins and the mismatch-repair protein MLH1 was used, which is associated with the most common type of recombination nodules, to analyze the pattern of meiotic recombination in the male of two species of iguanian lizards, Anolis carolinensis Voigt, 1832 and Deiroptyx coelestinus (Cope, 1862). These species are separated by a relatively long evolutionary history although they retain the ancestral iguanian karyotype. In both species similar and extremely uneven distributions of MLH1 foci along the macrochromosome bivalents were detected: approximately 90% of crossovers were located at the distal 20% of the chromosome arm length. Almost total suppression of recombination in the intermediate and proximal regions of the chromosome arms contradicts the hypothesis that "homogenous recombination" is responsible for the low variation in GC content across the anole genome. It also leads to strong linkage disequilibrium between the genes located in these regions, which may benefit conservation of co-adaptive gene arrays responsible for the ecological adaptations of the anoles.

Inference of Ancestral Recombination Graphs through Topological Data Analysis

PubMed Central

Cámara, Pablo G.; Levine, Arnold J.; Rabadán, Raúl

2016-01-01

The recent explosion of genomic data has underscored the need for interpretable and comprehensive analyses that can capture complex phylogenetic relationships within and across species. Recombination, reassortment and horizontal gene transfer constitute examples of pervasive biological phenomena that cannot be captured by tree-like representations. Starting from hundreds of genomes, we are interested in the reconstruction of potential evolutionary histories leading to the observed data. Ancestral recombination graphs represent potential histories that explicitly accommodate recombination and mutation events across orthologous genomes. However, they are computationally costly to reconstruct, usually being infeasible for more than few tens of genomes. Recently, Topological Data Analysis (TDA) methods have been proposed as robust and scalable methods that can capture the genetic scale and frequency of recombination. We build upon previous TDA developments for detecting and quantifying recombination, and present a novel framework that can be applied to hundreds of genomes and can be interpreted in terms of minimal histories of mutation and recombination events, quantifying the scales and identifying the genomic locations of recombinations. We implement this framework in a software package, called TARGet, and apply it to several examples, including small migration between different populations, human recombination, and horizontal evolution in finches inhabiting the Galápagos Islands. PMID:27532298

Recombinational DSBs-intersected genes converge on specific disease- and adaptability-related pathways.

PubMed

Yang, Zhi-Kai; Luo, Hao; Zhang, Yanming; Wang, Baijing; Gao, Feng

2018-05-03

The budding yeast Saccharomyces cerevisiae is a model species powerful for studying the recombination of eukaryotes. Although many recombination studies have been performed for this species by experimental methods, the population genomic study based on bioinformatics analyses is urgently needed to greatly increase the range and accuracy of recombination detection. Here, we carry out the population genomic analysis of recombination in S. cerevisiae to reveal the potential rules between recombination and evolution in eukaryotes. By population genomic analysis, we discover significantly more and longer recombination events in clinical strains, which indicates that adverse environmental conditions create an obviously wider range of genetic combination in response to the selective pressure. Based on the analysis of recombinational DSBs-intersected genes (RDIGs), we find that RDIGs significantly converge on specific disease- and adaptability-related pathways, indicating that recombination plays a biologically key role in the repair of DSBs related to diseases and environmental adaptability, especially the human neurological disorders (NDs). By evolutionary analysis of RDIGs, we find that the RDIGs highly prevailing in populations of yeast tend to be more evolutionarily conserved, indicating the accurate repair of DSBs in these RDIGs is critical to ensure the eukaryotic survival or fitness. fgao@tju.edu.cn. Supplementary data are available at Bioinformatics online.

Development and evaluation of porcine cysticercosis QuickELISA in Triturus EIA analyzer.

PubMed

Handali, Sukwan; Pattabhi, Sowmya; Lee, Yeuk-Mui; Silva-Ibanez, Maria; Kovalenko, Victor A; Levin, Andrew E; Gonzalez, Armando E; Roberts, Jacquelin M; Garcia, Hector H; Gilman, Robert H; Hancock, Kathy; Tsang, Victor C W

2010-01-01

We evaluated three diagnostic antigens (recombinant GP50, recombinant T24H, and synthetic Ts18var1) for cysticercosis and found that all three performed well in detecting cysticercosis in humans and pigs in several assay formats. These antigens were adapted to a new antibody detection format (QuickELISA). With one single incubation step which involves all reactants except the enzyme substrate, the QuickELISA is particularly suited for automation. We formatted the QuickELISA for the Triturus EIA analyzer for testing large numbers of samples. We found that in QuickELISA formats rGP50 and rT24H have better sensitivity and specificity than sTs18var1 for detecting porcine cysticercosis.

Transport-related triplet states and hyperfine couplings in organic tandem solar cells probed by pulsed electrically detected magnetic resonance spectroscopy

NASA Astrophysics Data System (ADS)

Kraffert, Felix; Bahro, Daniel; Meier, Christoph; Denne, Maximilian; Colsmann, Alexander; Behrends, Jan

2017-09-01

Tandem solar cells constitute the most successful organic photovoltaic devices with power conversion efficiencies comparable to thin-film silicon solar cells. Especially their high open-circuit voltage - only achievable by a well-adjusted layer stacking - leads to their high efficiencies. Nevertheless, the microscopic processes causing the lossless recombination of charge carriers within the recombination zone are not well understood yet. We show that advanced pulsed electrically detected magnetic resonance techniques such as electrically detected (ED)-Rabi nutation measurements and electrically detected hyperfine sublevel correlation (ED-HYSCORE) spectroscopy help to understand the role of triplet excitons in these microscopic processes. We investigate fully working miniaturised organic tandem solar cells and detect current-influencing doublet states in different layers as well as triplet excitons located on the fullerene-based acceptor. We apply ED-HYSCORE in order to study the nuclear spin environment of the relevant electron/hole spins and detect a significant amount of the low abundant 13C nuclei coupled to the observer spins.

A Patient with Crimean-Congo Hemorrhagic Fever Serologically Diagnosed by Recombinant Nucleoprotein-Based Antibody Detection Systems

PubMed Central

Tang, Qing; Saijo, Masayuki; Zhang, Yuzhen; Asiguma, Muer; Tianshu, Dong; Han, Lei; Shimayi, Bawudong; Maeda, Akihiko; Kurane, Ichiro; Morikawa, Shigeru

2003-01-01

We treated a male patient with Crimean-Congo hemorrhagic fever (CCHF). The diagnosis of CCHF was confirmed by reverse transcription-PCR and recombinant nucleoprotein (rNP)-based immunoglobulin G (IgG) and IgM capture enzyme-linked immunosorbent assays of serially collected serum samples. The patient was treated with intravenous ribavirin and recovered with no consequences. The study indicates that rNP-based CCHF virus antibody detection systems are useful for confirming CCHF virus infections. This case also suggests that intravenous ribavirin therapy may be promising for the treatment of CCHF patients. PMID:12738657

Oral administration of myostatin-specific recombinant Saccharomyces cerevisiae vaccine in rabbit.

PubMed

Liu, Zhongtian; Zhou, Gang; Ren, Chonghua; Xu, Kun; Yan, Qiang; Li, Xinyi; Zhang, Tingting; Zhang, Zhiying

2016-04-29

Yeast is considered as a simple and cost-effective host for protein expression, and our previous studies have proved that Saccharomyces cerevisiae can deliver recombinant protein and DNA into mouse dendritic cells and can further induce immune responses as novel vaccines. In order to know whether similar immune responses can be induced in rabbit by oral administration of such recombinant S. cerevisiae vaccine, we orally fed the rabbits with heat-inactivated myostatin-recombinant S. cerevisiae for 5 weeks, and then myostatin-specific antibody in serum was detected successfully by western blotting and ELISA assay. The rabbits treated with myostatin-recombinant S. cerevisiae vaccine grew faster and their muscles were much heavier than that of the control group. As a common experimental animal and a meat livestock with great economic value, rabbit was proved to be the second animal species that have been successfully orally immunized by recombinant S. cerevisiae vaccine after mice. Copyright © 2016 Elsevier Ltd. All rights reserved.

Induction of potential protective immunity against enterotoxemia in calves by single or multiple recombinant Clostridium perfringens toxoids.

PubMed

Jiang, Zhigang; De, Yanyan; Chang, Jitao; Wang, Fang; Yu, Li

2014-11-01

Cattle enterotoxemia caused by Clostridium perfringens toxins is a noncontagious, sporadic, and fatal disease characterized by sudden death. Strategies for controlling and preventing cattle enterotoxemia are based on systematic vaccination of herds with toxoids. Because the process of producing conventional clostridial vaccines is dangerous, expensive, and time-consuming, the prospect of recombinant toxoid vaccines against diseases caused by C. perfringens toxins is promising. In this study, nontoxic recombinant toxoids derived from α-, β- and ε-toxins of C. perfringens, namely, rCPA247-370 , rCPB and rEtxHP, respectively, were expressed in Escherichia coli. High levels of specific IgG antibodies and neutralizing antibodies against the toxins were detected in sera from calves vaccinated with either a single recombinant toxoid or a mixed cocktail of all three recombinant toxoids, indicating the potential of these recombinant toxoids to provide calves with protective immunity against enterotoxemia caused by C. perfringens. © 2014 The Societies and Wiley Publishing Asia Pty Ltd.

Safety evaluation of a recombinant myxoma-RHDV virus inducing horizontal transmissible protection against myxomatosis and rabbit haemorrhagic disease.

PubMed

Torres, J M; Ramírez, M A; Morales, M; Bárcena, J; Vázquez, B; Espuña, E; Pagès-Manté, A; Sánchez-Vizcaíno, J M

2000-09-15

We have recently developed a transmissible vaccine to immunize rabbits against myxomatosis and rabbit haemorrhagic disease based on a recombinant myxoma virus (MV) expressing the rabbit haemorrhagic disease virus (RHDV) capsid protein [Bárcena et al. Horizontal transmissible protection against myxomatosis and rabbit haemorragic disease using a recombinant myxoma virus. J. Virol. 2000;74:1114-23]. Administration of the recombinant virus protects rabbits against lethal RHDV and MV challenges. Furthermore, the recombinant virus is capable of horizontal spreading promoting protection of contact animals, thus providing the opportunity to immunize wild rabbit populations. However, potential risks must be extensively evaluated before considering its field use. In this study several safety issues concerning the proposed vaccine have been evaluated under laboratory conditions. Results indicated that vaccine administration is safe even at a 100-fold overdose. No undesirable effects were detected upon administration to immunosuppressed or pregnant rabbits. The recombinant virus maintained its attenuated phenotype after 10 passages in vivo.

Fcγ1 fragment of IgG1 as a powerful affinity tag in recombinant Fc-fusion proteins: immunological, biochemical and therapeutic properties.

PubMed

Soleimanpour, Saman; Hassannia, Tahereh; Motiee, Mahdieh; Amini, Abbas Ali; Rezaee, S A R

2017-05-01

Affinity tags are vital tools for the production of high-throughput recombinant proteins. Several affinity tags, such as the hexahistidine tag, maltose-binding protein, streptavidin-binding peptide tag, calmodulin-binding peptide, c-Myc tag, glutathione S-transferase and FLAG tag, have been introduced for recombinant protein production. The fragment crystallizable (Fc) domain of the IgG1 antibody is one of the useful affinity tags that can facilitate detection, purification and localization of proteins and can improve the immunogenicity, modulatory effects, physicochemical and pharmaceutical properties of proteins. Fcγ recombinant forms a group of recombinant proteins called Fc-fusion proteins (FFPs). FFPs are widely used in drug discovery, drug delivery, vaccine design and experimental research on receptor-ligand interactions. These fusion proteins have become successful alternatives to monoclonal antibodies for drug developments. In this review, the physicochemical, biochemical, immunological, pharmaceutical and therapeutic properties of recombinant FFPs were discussed as a new generation of bioengineering strategies.

Recombinant fowlpox viruses coexpressing chicken type I IFN and Newcastle disease virus HN and F genes: influence of IFN on protective efficacy and humoral responses of chickens following in ovo or post-hatch administration of recombinant viruses.

PubMed

Karaca, K; Sharma, J M; Winslow, B J; Junker, D E; Reddy, S; Cochran, M; McMillen, J

1998-10-01

We have constructed recombinant (r) fowl pox viruses (FPVs) coexpressing chicken type I interferon (IFN) and/or hemagglutinin-neuraminidase (HN) and fusion (F) proteins of Newcastle disease virus (NDV). We administered rFPVs and FPV into embryonated chicken eggs at 17 days of embryonation or in chickens after hatch. Administration of FPV or rFPVs did not influence hatchability and survival of hatched chicks. In ovo or after hatch vaccination of chickens with the recombinant viruses resulted in protection against challenge with virulent FPV and NDV. Chickens vaccinated with FPV or FPV-NDV recombinant had significantly lower body weight 2 weeks following vaccination. This loss in body weight was not detected in chickens receiving FPV-IFN and FPV-NDV-IFN recombinants. Chickens vaccinated with FPV coexpressing IFN and NDV genes produced less antibodies against NDV in comparison with chickens vaccinated with FPV expressing NDV genes.

Recombinant blood group proteins for use in antibody screening and identification tests.

PubMed

Seltsam, Axel; Blasczyk, Rainer

2009-11-01

The present review elucidates the potentials of recombinant blood group proteins (BGPs) for red blood cell (RBC) antibody detection and identification in pretransfusion testing and the achievements in this field so far. Many BGPs have been eukaryotically and prokaryotically expressed in sufficient quantity and quality for RBC antibody testing. Recombinant BGPs can be incorporated in soluble protein reagents or solid-phase assays such as ELISA, color-coded microsphere and protein microarray chip-based techniques. Because novel recombinant protein-based assays use single antigens, a positive reaction of a serum with the recombinant protein directly indicates the presence and specificity of the target antibody. Inversely, conventional RBC-based assays use panels of human RBCs carrying a huge number of blood group antigens at the same time and require negative reactions of samples with antigen-negative cells for indirect determination of antibody specificity. Because of their capacity for single-step, direct RBC antibody determination, recombinant protein-based assays may greatly facilitate and accelerate the identification of common and rare RBC antibodies.

Recombination gives a new insight in the effective population size and the history of the old world human populations.

PubMed

Melé, Marta; Javed, Asif; Pybus, Marc; Zalloua, Pierre; Haber, Marc; Comas, David; Netea, Mihai G; Balanovsky, Oleg; Balanovska, Elena; Jin, Li; Yang, Yajun; Pitchappan, R M; Arunkumar, G; Parida, Laxmi; Calafell, Francesc; Bertranpetit, Jaume

2012-01-01

The information left by recombination in our genomes can be used to make inferences on our recent evolutionary history. Specifically, the number of past recombination events in a population sample is a function of its effective population size (Ne). We have applied a method, Identifying Recombination in Sequences (IRiS), to detect specific past recombination events in 30 Old World populations to infer their Ne. We have found that sub-Saharan African populations have an Ne that is approximately four times greater than those of non-African populations and that outside of Africa, South Asian populations had the largest Ne. We also observe that the patterns of recombinational diversity of these populations correlate with distance out of Africa if that distance is measured along a path crossing South Arabia. No such correlation is found through a Sinai route, suggesting that anatomically modern humans first left Africa through the Bab-el-Mandeb strait rather than through present Egypt.

Population-specific recombination sites within the human MHC region.

PubMed

Lam, T H; Shen, M; Chia, J-M; Chan, S H; Ren, E C

2013-08-01

Genetic rearrangement by recombination is one of the major driving forces for genome evolution, and recombination is known to occur in non-random, discreet recombination sites within the genome. Mapping of recombination sites has proved to be difficult, particularly, in the human MHC region that is complicated by both population variation and highly polymorphic HLA genes. To overcome these problems, HLA-typed individuals from three representative populations: Asian, European and African were used to generate phased HLA haplotypes. Extended haplotype homozygosity (EHH) plots constructed from the phased haplotype data revealed discreet EHH drops corresponding to recombination events and these signatures were observed to be different for each population. Surprisingly, the majority of recombination sites detected are unique to each population, rather than being common. Unique recombination sites account for 56.8% (21/37 of total sites) in the Asian cohort, 50.0% (15/30 sites) in Europeans and 63.2% (24/38 sites) in Africans. Validation carried out at a known sperm typing recombination site of 45 kb (HLA-F-telomeric) showed that EHH was an efficient method to narrow the recombination region to 826 bp, and this was further refined to 660 bp by resequencing. This approach significantly enhanced mapping of the genomic architecture within the human MHC, and will be useful in studies to identify disease risk genes.

Impact of exogenous sequences on the characteristics of an epidemic type 2 recombinant vaccine-derived poliovirus.

PubMed

Riquet, Franck B; Blanchard, Claire; Jegouic, Sophie; Balanant, Jean; Guillot, Sophie; Vibet, Marie-Anne; Rakoto-Andrianarivelo, Mala; Delpeyroux, Francis

2008-09-01

Pathogenic circulating vaccine-derived polioviruses (cVDPVs) have become a major obstacle to the successful completion of the global polio eradication program. Most cVDPVs are recombinant between the oral poliovirus vaccine (OPV) and human enterovirus species C (HEV-C). To study the role of HEV-C sequences in the phenotype of cVDPVs, we generated a series of recombinants between a Madagascar cVDPV isolate and its parental OPV type 2 strain. Results indicated that the HEV-C sequences present in this cVDPV contribute to its characteristics, including pathogenicity, suggesting that interspecific recombination contributes to the phenotypic biodiversity of polioviruses and may favor the emergence of cVDPVs.

Recombinant Protein Production from TPO Gen Cloning and Expression for Early Detection of Autoimmune Thyroid Diseases

NASA Astrophysics Data System (ADS)

Aulanni'am, Aulanni'am; Kinasih Wuragil, Dyah; Wahono Soeatmadji, Djoko; Zulkarnain; Marhendra, Agung Pramana W.

2018-01-01

Autoimmune Thyroid Disease (AITD) is an autoimmune disease that has many clinical symptoms but is difficult to detect at the onset of disease progression. Most thyroid autoimmune disease patients are positive with high titre of thyroid autoantibodies, especially thyroid peroxidase (TPO). The detection AITD are still needed because these tests are extremely high cost and have not regularly been performed in most of clinical laboratories. In the past, we have explored the autoimmune disease marker and it has been developed as source of polyclonal antibodies from patient origin. In the current study, we develop recombinant protein which resulted from cloning and expression of TPO gene from normal person and AITD patients. This work flows involves: DNA isolation and PCR to obtain TPO gene from human blood, insertion of TPO gene to plasmid and transformation to E. coli BL21, Bacterial culture to obtain protein product, protein purification and product analysis. This products can use for application to immunochromatography based test. This work could achieved with the goal of producing autoimmune markers with a guaranteed quality, sensitive, specific and economically. So with the collaboration with industries these devices could be used for early detection. Keywords: recombinant protein, TPO gene, Autoimmune thyroid diseases (AITD)ction of the diseases in the community.

Strong Artificial Selection in Domestic Mammals Did Not Result in an Increased Recombination Rate

PubMed Central

Muñoz-Fuentes, Violeta; Marcet-Ortega, Marina; Alkorta-Aranburu, Gorka; Linde Forsberg, Catharina; Morrell, Jane M.; Manzano-Piedras, Esperanza; Söderberg, Arne; Daniel, Katrin; Villalba, Adrian; Toth, Attila; Di Rienzo, Anna; Roig, Ignasi; Vilà, Carles

2015-01-01

Recombination rates vary in intensity and location at the species, individual, sex and chromosome levels. Despite the fundamental biological importance of this process, the selective forces that operate to shape recombination rate and patterns are unclear. Domestication offers a unique opportunity to study the interplay between recombination and selection. In domesticates, intense selection for particular traits is imposed on small populations over many generations, resulting in organisms that differ, sometimes dramatically, in morphology and physiology from their wild ancestor. Although earlier studies suggested increased recombination rate in domesticates, a formal comparison of recombination rates between domestic mammals and their wild congeners was missing. In order to determine broad-scale recombination rate, we used immunolabeling detection of MLH1 foci as crossover markers in spermatocytes in three pairs of closely related wild and domestic species (dog and wolf, goat and ibex, and sheep and mouflon). In the three pairs, and contrary to previous suggestions, our data show that contemporary recombination rate is higher in the wild species. Subsequently, we inferred recombination breakpoints in sequence data for 16 genomic regions in dogs and wolves, each containing a locus associated with a dog phenotype potentially under selection during domestication. No difference in the number and distribution of recombination breakpoints was found between dogs and wolves. We conclude that our data indicate that strong directional selection did not result in changes in recombination in domestic mammals, and that both upper and lower bounds for crossover rates may be tightly regulated. PMID:25414125

Antigen-capture blocking enzyme-linked immunosorbent assay based on a baculovirus recombinant antigen to differentiate Transmissible gastroenteritis virus from Porcine respiratory coronavirus antibodies.

PubMed

López, Lissett; Venteo, Angel; García, Marga; Camuñas, Ana; Ranz, Ana; García, Julia; Sarraseca, Javier; Anaya, Carmen; Rueda, Paloma

2009-09-01

A new commercially available antigen-capture, blocking enzyme-linked immunosorbent assay (antigen-capture b-ELISA), based on baculovirus truncated-S recombinant protein of Transmissible gastroenteritis virus (TGEV) and 3 specific monoclonal antibodies, was developed and evaluated by examining a panel of 453 positive Porcine respiratory coronavirus (PRCoV), 31 positive TGEV, and 126 negative field sera by using another commercially available differential coronavirus b-ELISA as the reference technique to differentiate TGEV- from PRCoV-induced antibodies. The recombinant S protein-based ELISA appeared to be 100% sensitive for TGEV and PRCoV detection and highly specific for TGEV and PRCoV detection (100% and 92.06%, respectively), when qualitative results (positive or negative) were compared with those of the reference technique. In variability experiments, the ELISA gave consistent results when the same serum was evaluated on different wells and different plates. These results indicated that truncated recombinant S protein is a suitable alternative to the complete virus as antigen in ELISA assays. The use of recombinant S protein as antigen offers great advantages because it is an easy-to-produce, easy-to-standardize, noninfectious antigen that does not require further purification or concentration. Those advantages represent an important improvement for antigen preparation, in comparison with other assays in which an inactivated virus from mammalian cell cultures is used.

[Experimental study on human periodontal ligament cells transfected with human amelogenin gene].

PubMed

Yu, Guang; Shu, Rong; Sun, Ying; Cheng, Lan; Song, Zhong-Chen; Zhang, Xiu-Li

2008-02-01

To construct the recombinant lentiviral vector of human amelogenin gene, infect human periodontal ligament cells with the recombinant lentivirus, and evaluate the feasibility of applying modified PDLCs as seeds for a further periodontal reconstruction. The mature peptide of hAm cDNA was cloned and linked into the vector plasmid, the recombinant plasmid FUAmW was confirmed by double enzyme digestion and sequence analysis. Recombinant lentivirus was prepared from 293T cells by polytheylenimine (PEI)-mediated transient cotransfection. The hPDLCs and 293T cells were infected with the generated lentivirus. The infection efficiency was analysed by detection of green fluorescence protein (GFP) with fluorescent microscope and flow cytometer 72 hours later. The expression of hAm gene was detected by reverse transcription polymerase chain reaction (RT-PCR). The sequence of inserted fragment in recombinant plasmid was identical to the hAm sequence reported in Genebank. Green fluorescence was visible under fluorescent microscope, FCM assay showed that positive percentage was 69.46% and 33.99% in 293T and hPDLCs, respectively. The targeted gene was obtained in the experimental groups by RT-PCR. The recombinan lentiviral vector of hAm gene is constructed successfully and it could be transfected into cultured hPDLCs. hAm gene and seed cells may be used for further study in the fields periodontal tissue engineering. Supported by National Natural Science Foundation of China (Grant No. 30672315).

Detection and characterization of hepatitis A virus circulating in Egypt.

PubMed

Hamza, Hazem; Abd-Elshafy, Dina Nadeem; Fayed, Sayed A; Bahgat, Mahmoud Mohamed; El-Esnawy, Nagwa Abass; Abdel-Mobdy, Emam

2017-07-01

Hepatitis A virus (HAV) still poses a considerable problem worldwide. In the current study, hepatitis A virus was recovered from wastewater samples collected from three wastewater treatment plants over one year. Using RT-PCR, HAV was detected in 43 out of 68 samples (63.2%) representing both inlet and outlet. Eleven positive samples were subjected to sequencing targeting the VP1-2A junction region. Phylogenetic analysis revealed that all samples belonged to subgenotype IB with few substitutions at the amino acid level. The complete sequence of one isolate (HAV/Egy/BI-11/2015) showed that the similarity at the amino acid level was not reflected at the nucleotide level. However, the deduced amino acid sequence derived from the complete nucleotide sequence showed distinct substitutions in the 2B, 2C, and 3A regions. Recombination analysis revealed a recombination event between X75215 (subgenotype IA) and AF268396 (subgenotype IB) involving a portion of the 2B nonstructural protein coding region (nucleotides 3757-3868) assuming the herein characterized sequence an actual recombinant. Despite the role of recombination in picornaviruses evolution, its involvement in HAV evolution has rarely been reported, and this may be due to the limited available complete HAV sequences. To our knowledge, this represents the first characterized complete sequence of an Egyptian isolate and the described recombination event provides an important update on the circulating HAV strains in Egypt.

[An indirect ELISA using Legionella pneumophila recombinant MOMP protein and its application in serological diagnosis].

PubMed

Wang, Tao; Zhang, Caixia; Cao, Xiuqin; Yang, Zhiwei

2013-12-01

To express and purify the recombinant major outer membrane protein (MOMP) of Legionella pneumophila (Lp) as diagnostic antigen, and explore its practical value in the serological diagnosis of Lp infection. The recombinant plasmid pET-momp was transformed into the E.coli BL21 competent cells. The recombinant MOMP was induced to express, and then analyzed by SDS-PAGE electrophoresis, purified by affinity chromatography. We screened and obtained 58 positive blood serum and 32 negative blood serum using the DRG (Germany, IgG/IgM/IgA) Lp kit. The blood serum samples were detected for IgG, IgM, IgA antibody levels by indirect ELISA that we had established with the purified MOMP as the coating antigen, as well as by R&D (USA, IgG/IgM/IgA) Lp kit. Then using the receiver operating characteristic (ROC) curve, we compared these two methods in the sensitivity, specificity and consistency of the test results, for evaluating the application value of the indirect ELISA of recombinant MOMP. The approximately 45 000 recombinant MOMP was successfully expressed and purified. Compared with the indirect ELISA we established with the R&D Lp kit for detecting Lp antibody IgG, IgM and IgA in blood serum, the sensitivity of the indirect ELISA of recombinant MOMP to IgG was 90.9%, the specificity was 91.7%, the Kappa value was 0.784 (P < 0.05), and the area under the ROC curve was 0.913; the sensitivity to IgM was 91.4% and the specificity was 90.6%, the Kappa value was 0.809 (P < 0.05), and the area under the ROC curve was 0.910; the sensitivity to IgA was 92.1% and the specificity was 88.9%, the Kappa value was 0.793(P < 0.05), and the area under the ROC curve was 0.905. The recombinant MOMP was successfully induced to express and purified. The indirect ELISA we established with the recombinant MOMP protein as a diagnostic antigen showed good specificity, sensitivity and consistency, which laid a foundation for the development of serological diagnosis kit of Legionnaires' disease.

Isolation of an intertypic poliovirus capsid recombinant from a child with vaccine-associated paralytic poliomyelitis.

PubMed

Martín, Javier; Samoilovich, Elena; Dunn, Glynis; Lackenby, Angie; Feldman, Esphir; Heath, Alan; Svirchevskaya, Ekaterina; Cooper, Gill; Yermalovich, Marina; Minor, Philip D

2002-11-01

The isolation of a capsid intertypic poliovirus recombinant from a child with vaccine-associated paralytic poliomyelitis is described. Virus 31043 had a Sabin-derived type 3-type 2-type 1 recombinant genome with a 5'-end crossover point within the capsid coding region. The result was a poliovirus chimera containing the entire coding sequence for antigenic site 3a derived from the Sabin type 2 strain. The recombinant virus showed altered antigenic properties but did not acquire type 2 antigenic characteristics. The significance of the presence in nature of such poliovirus chimeras and the consequences for the current efforts to detect potentially dangerous vaccine-derived poliovirus strains are discussed in the context of the global polio eradication initiative.

A Multiplex PCR Approach for Detecting Dual Infections and Recombinants Involving Major HIV Variants

PubMed Central

Cappy, Pierre; De Oliveira, Fabienne; Gueudin, Marie; Alessandri-Gradt, Elodie

2016-01-01

The cocirculation of different HIV types and groups can lead to dual infections and recombinants, which hinder diagnosis and therapeutic management. We designed two multiplex PCRs (mPCRs) coupled with capillary electrophoresis to facilitate the detection of such infections. The first, MMO2, targets three variants (HIV-1/M, HIV-1/O, and HIV-2), and the second, MMO, targets HIV-1/M and HIV-1/O. These mPCRs were validated on DNA and RNA extracts from 19 HIV-1/M, 12 HIV-1/O, and 13 HIV-2 cultures and from mixtures simulating dual infections. They were then assessed with DNA and RNA extracts from samples of 47 clinical monoinfections and HIV-1/M+O dual infections or infections with HIV-1/MO recombinants. Both mPCRs had excellent specificity. Sensitivities ranged from 80 to 100% for in vitro samples and from 58 to 100% for clinical samples, with the results obtained depending on the material used and the region of the genome concerned. Sensitivity was generally lower for DNA than for RNA and for amplifications of the integrase and matrix regions. In terms of global detection (at least one target gene for each strain), both mPCRs yielded a detection rate of 100% for in vitro samples. MMO2 detected 100% of the clinical strains from DNA and 97% from RNA, whereas MMO detected 100% of the strains from both materials. Thus, for in vitro and clinical samples, MMO2 was a useful tool for detecting dual infections with HIV-1 and HIV-2 (referred to as HIV-1+HIV-2) and HIV-1/M+O, and MMO was useful for detecting both MO dual infections and MO mosaic patterns. PMID:26912747

The role of recombination in the origin and evolution of Alu subfamilies.

PubMed

Teixeira-Silva, Ana; Silva, Raquel M; Carneiro, João; Amorim, António; Azevedo, Luísa

2013-01-01

Alus are the most abundant and successful short interspersed nuclear elements found in primate genomes. In humans, they represent about 10% of the genome, although few are retrotransposition-competent and are clustered into subfamilies according to the source gene from which they evolved. Recombination between them can lead to genomic rearrangements of clinical and evolutionary significance. In this study, we have addressed the role of recombination in the origin of chimeric Alu source genes by the analysis of all known consensus sequences of human Alus. From the allelic diversity of Alu consensus sequences, validated in extant elements resulting from whole genome searches, distinct events of recombination were detected in the origin of particular subfamilies of AluS and AluY source genes. These results demonstrate that at least two subfamilies are likely to have emerged from ectopic Alu-Alu recombination, which stimulates further research regarding the potential of chimeric active Alus to punctuate the genome.

[Study on the anti-NTHi infection of Hap recombinant protein in vivo].

PubMed

Li, Wan-yi; Wang, Bao-ning; Zuo, Feng-qiong; Zeng, Wei; Feng, Feng; Kuang, Yu; Jiang, Zhong-hua; Li, Ming-yuan

2010-07-01

To observe the immune effect of Hap recombinant protein on murine model of bronchopneumonia infected with NTHi, and explore the mechanism about the anti-NTHi infection. The C57BL/6 mice intranasally immunized with purified Hap recombinant protein and CT-B were challenged by NTHi encased in agar beads. The immunifaction of anti-infection was observed through encocyte counting of BALF, bacteria detection of lung and the pathologyical change of lung tissue. In the challenge with NTHi experiment, the inflammatory exudation of the infected murine and pathological change of lung tissue was relieved by combined immunization of Hap recombinant protein and CT-B, and quantity of NTHi in lung of the infected murine was reduced obviously. The Hap recombinant protein also had good ability of anti-NTHi infection in the murine model of NTHi bronchopneumonia. This study could offer the oretical and experimental basis for development of new vaccine against NTHi.

Spin-dependent recombination probed through the dielectric polarizability

PubMed Central

Bayliss, Sam L.; Greenham, Neil C.; Friend, Richard H.; Bouchiat, Hélène; Chepelianskii, Alexei D

2015-01-01

Despite residing in an energetically and structurally disordered landscape, the spin degree of freedom remains a robust quantity in organic semiconductor materials due to the weak coupling of spin and orbital states. This enforces spin-selectivity in recombination processes which plays a crucial role in optoelectronic devices, for example, in the spin-dependent recombination of weakly bound electron-hole pairs, or charge-transfer states, which form in a photovoltaic blend. Here, we implement a detection scheme to probe the spin-selective recombination of these states through changes in their dielectric polarizability under magnetic resonance. Using this technique, we access a regime in which the usual mixing of spin-singlet and spin-triplet states due to hyperfine fields is suppressed by microwave driving. We present a quantitative model for this behaviour which allows us to estimate the spin-dependent recombination rate, and draw parallels with the Majorana–Brossel resonances observed in atomic physics experiments. PMID:26439933

A reanalysis of the indirect evidence for recombination in human mitochondrial DNA.

PubMed

Piganeau, G; Eyre-Walker, A

2004-04-01

In an attempt to resolve the controversy about whether recombination occurs in human mtDNA, we have analysed three recently published data sets of complete mtDNA sequences along with 10 RFLP data sets. We have analysed the relationship between linkage disequilibrium (LD) and distance between sites under a variety of conditions using two measures of LD, r2 and /D'/. We find that there is a negative correlation between r2 and distance in the majority of data sets, but no overall trend for /D'/. Five out of six mtDNA sequence data sets show an excess of homoplasy, but this could be due to either recombination or hypervariable sites. Two additional recombination detection methods used, Geneconv and Maximum Chi-Square, showed nonsignificant results. The overall significance of these findings is hard to quantify because of nonindependence, but our results suggest a lack of evidence for recombination in human mtDNA.

Development and Evaluation of a Magnetic Immunochromatographic Test To Detect Taenia solium, Which Causes Taeniasis and Neurocysticercosis in Humans▿

PubMed Central

Handali, Sukwan; Klarman, Molly; Gaspard, Amanda N.; Dong, X. Fan; LaBorde, Ronald; Noh, John; Lee, Yeuk-Mui; Rodriguez, Silvia; Gonzalez, Armando E.; Garcia, Hector H.; Gilman, Robert H.; Tsang, Victor C. W.; Wilkins, Patricia P.

2010-01-01

Taeniasis/cysticercosis caused by Taenia solium is a frequent parasitic infection of the human brain in most of the world. Rapid and simple screening tools to identify taeniasis and cysticercosis cases are needed for control programs, mostly to identify tapeworm carriers which are the source of infection and need to be treated, or as tools for point-of-care case detection or confirmation. These screening assays should be affordable, reliable, rapid, and easy to perform. Immunochromatographic tests meet these criteria. To demonstrate proof of principle, we developed and evaluated two magnetic immunochromatographic tests (MICTs) for detection of human Taenia solium taeniasis antibodies (ES33-MICT) and neurocysticercosis antibodies (T24-MICT). These assays detected stage-specific antibodies by using two recombinant proteins, rES33 for detection of taeniasis antibodies and rT24H for detection of cysticercosis antibodies. The sensitivity and specificity of the ES33-MICT to detect taeniasis infections were 94.5% and 96%, respectively, and those of the T24-MICT to detect cases of human cysticercosis with two or more viable brain cysts were 93.9% and 98.9%, respectively. These data provide proof of principle that the ES33- and T24-MICTs provide rapid and suitable methods to identify individuals with taeniasis and cysticercosis. PMID:20181766

The Genetic Programming of Industrial Microorganisms.

ERIC Educational Resources Information Center

Hopwood, David A.

1981-01-01

Traces the development of the field of industrial microbial genetics, describing a range of techniques for genetic programing. Includes a discussion of site-directed mutagenesis, protoplast fusion, and recombinant DNA manipulations. (CS)

Development of the National Institutes of Health Guidelines for Recombinant DNA Research.

PubMed Central

Talbot, B

1983-01-01

Recombinant DNA is a technique of major importance in basic biomedical research and, increasingly, in industrial applications. Although the risks of this research remain hypothetical, scientists working in the field have spearheaded discussions of safety. The original National Institutes of Health (NIH) Guidelines for Recombinant DNA Research were issued in June 1976. They assigned each type of recombinant DNA experiment a specific level of "physical containment" and of "biological containment." Responsibility for overseeing the application of the guidelines belongs to the NIH Recombinant DNA Advisory Committee (RAC)--composed of scientists and laymen, including non-voting representatives from many Federal agencies--and local institutional biosafety committees at each university where recombinant DNA research is conducted. The NIH guidelines were subsequently adopted by other Federal agencies, but congressional proposals aimed at extending the guidelines to private industry did not result in national legislation. Some States and localities regulate recombinant DNA research, however, and many private companies have voluntarily submitted information on their recombinant DNA work for RAC and NIH approval. The NIH guidelines underwent a major revision in December 1978 and have been revised approximately every 3 months since then. NIH supports experiments to assess recombinant DNA risks and publishes and updates a plan for a risk assessment program. PMID:6611823

The mouse lymphoma assay detects recombination, deletion, and aneuploidy.

PubMed

Wang, Jianyong; Sawyer, Jeffrey R; Chen, Ling; Chen, Tao; Honma, Masamitsu; Mei, Nan; Moore, Martha M

2009-05-01

The mouse lymphoma assay (MLA) uses the thymidine kinase (Tk) gene of the L5178Y/Tk(+/-)-3.7.2C mouse lymphoma cell line as a reporter gene to evaluate the mutagenicity of chemical and physical agents. The MLA is recommended by both the United States Food and Drug Administration and the United States Environmental Protection Agency as the preferred in vitro mammalian cell mutation assay for genetic toxicology screening because it detects a wide range of genetic alterations, including both point mutations and chromosomal mutations. However, the specific types of chromosomal mutations that can be detected by the MLA need further clarification. For this purpose, three chemicals, including two clastogens and an aneugen (3'-azido-3'-deoxythymidine, mitomycin C, and taxol), were used to induce Tk mutants. Loss of heterozygosity (LOH) analysis was used to select mutants that could be informative as to whether they resulted from deletion, mitotic recombination, or aneuploidy. A combination of additional methods, G-banding analysis, chromosome painting, and a real-time PCR method to detect the copy number (CN) of the Tk gene was then used to provide a detailed analysis. LOH involving at least 25% of chromosome 11, a normal karyotype, and a Tk CN of 2 would indicate that the mutant resulted from recombination, whereas LOH combined with a karyotypically visible deletion of chromosome 11 and a Tk CN of 1 would indicate a deletion. Aneuploidy was confirmed using G-banding combined with chromosome painting analysis for mutants showing LOH at every microsatellite marker on chromosome 11. From this analysis, it is clear that mouse lymphoma Tk mutants can result from recombination, deletion, and aneuploidy.

Serological assays based on recombinant viral proteins for the diagnosis of arenavirus hemorrhagic fevers.

PubMed

Fukushi, Shuetsu; Tani, Hideki; Yoshikawa, Tomoki; Saijo, Masayuki; Morikawa, Shigeru

2012-10-12

The family Arenaviridae, genus Arenavirus, consists of two phylogenetically independent groups: Old World (OW) and New World (NW) complexes. The Lassa and Lujo viruses in the OW complex and the Guanarito, Junin, Machupo, Sabia, and Chapare viruses in the NW complex cause viral hemorrhagic fever (VHF) in humans, leading to serious public health concerns. These viruses are also considered potential bioterrorism agents. Therefore, it is of great importance to detect these pathogens rapidly and specifically in order to minimize the risk and scale of arenavirus outbreaks. However, these arenaviruses are classified as BSL-4 pathogens, thus making it difficult to develop diagnostic techniques for these virus infections in institutes without BSL-4 facilities. To overcome these difficulties, antibody detection systems in the form of an enzyme-linked immunosorbent assay (ELISA) and an indirect immunofluorescence assay were developed using recombinant nucleoproteins (rNPs) derived from these viruses. Furthermore, several antigen-detection assays were developed. For example, novel monoclonal antibodies (mAbs) to the rNPs of Lassa and Junin viruses were generated. Sandwich antigen-capture (Ag-capture) ELISAs using these mAbs as capture antibodies were developed and confirmed to be sensitive and specific for detecting the respective arenavirus NPs. These rNP-based assays were proposed to be useful not only for an etiological diagnosis of VHFs, but also for seroepidemiological studies on VHFs. We recently developed arenavirus neutralization assays using vesicular stomatitis virus (VSV)-based pseudotypes bearing arenavirus recombinant glycoproteins. The goal of this article is to review the recent advances in developing laboratory diagnostic assays based on recombinant viral proteins for the diagnosis of VHFs and epidemiological studies on the VHFs caused by arenaviruses.

Discrimination between spin-dependent charge transport and spin-dependent recombination in π-conjugated polymers by correlated current and electroluminescence-detected magnetic resonance

NASA Astrophysics Data System (ADS)

Kavand, Marzieh; Baird, Douglas; van Schooten, Kipp; Malissa, Hans; Lupton, John M.; Boehme, Christoph

2016-08-01

Spin-dependent processes play a crucial role in organic electronic devices. Spin coherence can give rise to spin mixing due to a number of processes such as hyperfine coupling, and leads to a range of magnetic field effects. However, it is not straightforward to differentiate between pure single-carrier spin-dependent transport processes which control the current and therefore the electroluminescence, and spin-dependent electron-hole recombination which determines the electroluminescence yield and in turn modulates the current. We therefore investigate the correlation between the dynamics of spin-dependent electric current and spin-dependent electroluminescence in two derivatives of the conjugated polymer poly(phenylene-vinylene) using simultaneously measured pulsed electrically detected (pEDMR) and optically detected (pODMR) magnetic resonance spectroscopy. This experimental approach requires careful analysis of the transient response functions under optical and electrical detection. At room temperature and under bipolar charge-carrier injection conditions, a correlation of the pEDMR and the pODMR signals is observed, consistent with the hypothesis that the recombination currents involve spin-dependent electronic transitions. This observation is inconsistent with the hypothesis that these signals are caused by spin-dependent charge-carrier transport. These results therefore provide no evidence that supports earlier claims that spin-dependent transport plays a role for room-temperature magnetoresistance effects. At low temperatures, however, the correlation between pEDMR and pODMR is weakened, demonstrating that more than one spin-dependent process influences the optoelectronic materials' properties. This conclusion is consistent with prior studies of half-field resonances that were attributed to spin-dependent triplet exciton recombination, which becomes significant at low temperatures when the triplet lifetime increases.

Development of a single-antigen magnetic bead assay (SAMBA) for the sensitive detection of HPA-1a alloantibodies using tag-engineered recombinant soluble β3 integrin.

PubMed

Skaik, Younis; Battermann, Anja; Hiller, Oliver; Meyer, Oliver; Figueiredo, Constanca; Salama, Abdulgabar; Blasczyk, Rainer

2013-05-31

Timely and accurate testing for human platelet antigen 1a (HPA-1a) alloantibodies is vital for clinical diagnosis of neonatal alloimmune thrombocytopenia (NAIT). Current antigen-specific assays used for the detection of HPA-1 alloantibodies are technically very complex and cumbersome for most diagnostic laboratories. Hence, we designed and applied recombinant soluble (rs) β3 integrins displaying HPA-1a or HPA-1b epitopes for the development of a single-antigen magnetic bead assay (SAMBA). Soluble HPA-1a and HPA-1b were produced recombinantly in human embryonic kidney 293 (HEK293) cells and differentially tagged. The recombinant soluble proteins were then immobilized onto paramagnetic beads and used for analysis of HPA-1 alloantibodies by enzyme-linked immunosorbent assay (ELISA). HPA-1a serum samples (n=7) from NAIT patients, inert sera and sera containing non-HPA-1a antibodies were used to evaluate the sensitivity and specificity of the SAMBA. Fusion of V5-His or GS-SBP-His tags to the rsβ3 integrins resulted in high-yield expression. SAMBA was able to detect all HPA-1a and -1b alloantibodies recognized by monoclonal antibody-specific immobilization of platelet antigens assay (MAIPA). No cross-reactions between the sera were observed. Two out of seven of the HPA-1a alloantibody-containing sera demonstrated weak to moderate reactivity in MAIPA but strong signals in SAMBA. SAMBA provides a very reliable method for the detection of HPA-1 antibodies with high specificity and sensitivity. This simple and rapid assay can be adapted for use in any routine laboratory and can be potentially adapted for use on automated systems. Copyright © 2013 Elsevier B.V. All rights reserved.

Recombinant nucleocapsid protein-based enzyme-linked immunosorbent assay for detection of antibody to turkey coronavirus.

PubMed

Abdelwahab, Mohamed; Loa, Chien Chang; Wu, Ching Ching; Lin, Tsang Long

2015-06-01

Nucleocapsid (N) protein gene of turkey coronavirus (TCoV) was expressed in a prokaryotic system and used to develop an enzyme-linked immunosorbent assay (ELISA) for detection of antibody to TCoV. Anti-TCoV hyperimmune turkey serum and normal turkey serum were used as positive or negative controls for optimization of the ELISA. Goat anti-turkey IgG (H+L) conjugated with horseradish peroxidase was used as detector antibody. Three hundred and twenty two turkey sera from the field were used to evaluate the performance of ELISA and determine the cut-off point of ELISA. The established ELISA was also examined with serum samples obtained from turkeys experimentally infected with TCoV. Those serum samples were collected at various time intervals from 1 to 63 days post-infection. The optimum conditions for differentiation between anti-TCoV hyperimmune serum and normal turkey serum were recombinant TCoV N protein concentration at 20 μg/ml, serum dilution at 1:800, and conjugate dilution at 1:10,000. Of the 322 sera from the field, 101 were positive for TCoV by immunofluorescent antibody assay (IFA). The sensitivity and specificity of the ELISA relative to IFA test were 86.0% and 96.8%, respectively, using the optimum cut-off point of 0.2 as determined by logistic regression method. Reactivity of anti-rotavirus, anti-reovirus, anti-adenovirus, or anti-enterovirus antibodies with the recombinant N protein coated on the ELISA plates was not detected. These results indicated that the established antibody-capture ELISA in conjunction with recombinant TCoV N protein as the coating protein can be utilized for detection of antibodies to TCoV in turkey flocks. Copyright © 2015 Elsevier B.V. All rights reserved.

Antibodies specific for HT{sub m4}

DOEpatents

Lim, B.; Adra, C.N.; Lelias, J.M.

1998-01-06

The invention relates to a recombinant DNA molecule which encodes a HT{sub m4} protein, a transformed host cell which has been stably transfected with a DNA molecule which encodes a HT{sub m4} protein and a recombinant HT{sub m4} protein. The invention also relates to a method for detecting the presence of a hereditary atopy. 2 figs.

Method and Apparatus for Measuring Near-Angle Scattering of Mirror Coatings

NASA Technical Reports Server (NTRS)

Chipman, Russell A. (Inventor); Daugherty, Brian J. (Inventor); McClain, Stephen C. (Inventor); Macenka, Steven A. (Inventor)

2013-01-01

Disclosed herein is a method of determining the near angle scattering of a sample reflective surface comprising the steps of: a) splitting a beam of light having a coherence length of greater than or equal to about 2 meters into a sample beam and a reference beam; b) frequency shifting both the sample beam and the reference beam to produce a fixed beat frequency between the sample beam and the reference beam; c) directing the sample beam through a focusing lens and onto the sample reflective surface, d) reflecting the sample beam from the sample reflective surface through a detection restriction disposed on a movable stage; e) recombining the sample beam with the reference beam to form a recombined beam, followed by f) directing the recombined beam to a detector and performing heterodyne analysis on the recombined beam to measure the near-angle scattering of the sample reflective surface, wherein the position of the detection restriction relative to the sample beam is varied to occlude at least a portion of the sample beam to measure the near-angle scattering of the sample reflective surface. An apparatus according to the above method is also disclosed.

Genomic Analysis of 15 Human Coronaviruses OC43 (HCoV-OC43s) Circulating in France from 2001 to 2013 Reveals a High Intra-Specific Diversity with New Recombinant Genotypes

PubMed Central

Kin, Nathalie; Miszczak, Fabien; Lin, Wei; Ar Gouilh, Meriadeg; Vabret, Astrid

2015-01-01

Human coronavirus OC43 (HCoV-OC43) is one of five currently circulating human coronaviruses responsible for respiratory infections. Like all coronaviruses, it is characterized by its genome’s high plasticity. The objectives of the current study were to detect genetically distinct genotypes and eventually recombinant genotypes in samples collected in Lower Normandy between 2001 and 2013. To this end, we sequenced complete nsp12, S, and N genes of 15 molecular isolates of HCoV-OC43 from clinical samples and compared them to available data from the USA, Belgium, and Hong-Kong. A new cluster E was invariably detected from nsp12, S, and N data while the analysis of nsp12 and N genes revealed the existence of new F and G clusters respectively. The association of these different clusters of genes in our specimens led to the description of thirteen genetically distinct genotypes, among which eight recombinant viruses were discovered. Identification of these recombinant viruses, together with temporal analysis and tMRCA estimation, provides important information for understanding the dynamics of the evolution of these epidemic coronaviruses. PMID:26008694

Vectorization in an oncolytic vaccinia virus of an antibody, a Fab and a scFv against programmed cell death -1 (PD-1) allows their intratumoral delivery and an improved tumor-growth inhibition.

PubMed

Kleinpeter, Patricia; Fend, Laetitia; Thioudellet, Christine; Geist, Michel; Sfrontato, Nathalie; Koerper, Véronique; Fahrner, Catherine; Schmitt, Doris; Gantzer, Murielle; Remy-Ziller, Christelle; Brandely, Renée; Villeval, Dominique; Rittner, Karola; Silvestre, Nathalie; Erbs, Philippe; Zitvogel, Laurence; Quéméneur, Eric; Préville, Xavier; Marchand, Jean-Baptiste

2016-01-01

We report here the successful vectorization of a hamster monoclonal IgG (namely J43) recognizing the murine Programmed cell death-1 (mPD-1) in Western Reserve (WR) oncolytic vaccinia virus. Three forms of mPD-1 binders have been inserted into the virus: whole antibody (mAb), Fragment antigen-binding (Fab) or single-chain variable fragment (scFv). MAb, Fab and scFv were produced and assembled with the expected patterns in supernatants of cells infected by the recombinant viruses. The three purified mPD-1 binders were able to block the binding of mPD-1 ligand to mPD-1 in vitro . Moreover, mAb was detected in tumor and in serum of C57BL/6 mice when the recombinant WR-mAb was injected intratumorally (IT) in B16F10 and MCA 205 tumors. The concentration of circulating mAb detected after IT injection was up to 1,900-fold higher than the level obtained after a subcutaneous (SC) injection (i.e., without tumor) confirming the virus tropism for tumoral cells and/or microenvironment. Moreover, the overall tumoral accumulation of the mAb was higher and lasted longer after IT injection of WR-mAb1, than after IT administration of 10 µg of J43. The IT injection of viruses induced a massive infiltration of immune cells including activated lymphocytes (CD8 + and CD4 + ). Interestingly, in the MCA 205 tumor model, WR-mAb1 and WR-scFv induced a therapeutic control of tumor growth similar to unarmed WR combined to systemically administered J43 and superior to that obtained with an unarmed WR. These results pave the way for next generation of oncolytic vaccinia armed with immunomodulatory therapeutic proteins such as mAbs.

Vectorization in an oncolytic vaccinia virus of an antibody, a Fab and a scFv against programmed cell death -1 (PD-1) allows their intratumoral delivery and an improved tumor-growth inhibition

PubMed Central

Kleinpeter, Patricia; Fend, Laetitia; Thioudellet, Christine; Geist, Michel; Sfrontato, Nathalie; Koerper, Véronique; Fahrner, Catherine; Schmitt, Doris; Gantzer, Murielle; Remy-Ziller, Christelle; Brandely, Renée; Villeval, Dominique; Rittner, Karola; Silvestre, Nathalie; Erbs, Philippe; Zitvogel, Laurence; Quéméneur, Eric; Préville, Xavier; Marchand, Jean-Baptiste

2016-01-01

ABSTRACT We report here the successful vectorization of a hamster monoclonal IgG (namely J43) recognizing the murine Programmed cell death-1 (mPD-1) in Western Reserve (WR) oncolytic vaccinia virus. Three forms of mPD-1 binders have been inserted into the virus: whole antibody (mAb), Fragment antigen-binding (Fab) or single-chain variable fragment (scFv). MAb, Fab and scFv were produced and assembled with the expected patterns in supernatants of cells infected by the recombinant viruses. The three purified mPD-1 binders were able to block the binding of mPD-1 ligand to mPD-1 in vitro. Moreover, mAb was detected in tumor and in serum of C57BL/6 mice when the recombinant WR-mAb was injected intratumorally (IT) in B16F10 and MCA 205 tumors. The concentration of circulating mAb detected after IT injection was up to 1,900-fold higher than the level obtained after a subcutaneous (SC) injection (i.e., without tumor) confirming the virus tropism for tumoral cells and/or microenvironment. Moreover, the overall tumoral accumulation of the mAb was higher and lasted longer after IT injection of WR-mAb1, than after IT administration of 10 µg of J43. The IT injection of viruses induced a massive infiltration of immune cells including activated lymphocytes (CD8+ and CD4+). Interestingly, in the MCA 205 tumor model, WR-mAb1 and WR-scFv induced a therapeutic control of tumor growth similar to unarmed WR combined to systemically administered J43 and superior to that obtained with an unarmed WR. These results pave the way for next generation of oncolytic vaccinia armed with immunomodulatory therapeutic proteins such as mAbs. PMID:27853644

A Genome-Wide Map of Mitochondrial DNA Recombination in Yeast

PubMed Central

Fritsch, Emilie S.; Chabbert, Christophe D.; Klaus, Bernd; Steinmetz, Lars M.

2014-01-01

In eukaryotic cells, the production of cellular energy requires close interplay between nuclear and mitochondrial genomes. The mitochondrial genome is essential in that it encodes several genes involved in oxidative phosphorylation. Each cell contains several mitochondrial genome copies and mitochondrial DNA recombination is a widespread process occurring in plants, fungi, protists, and invertebrates. Saccharomyces cerevisiae has proved to be an excellent model to dissect mitochondrial biology. Several studies have focused on DNA recombination in this organelle, yet mostly relied on reporter genes or artificial systems. However, no complete mitochondrial recombination map has been released for any eukaryote so far. In the present work, we sequenced pools of diploids originating from a cross between two different S. cerevisiae strains to detect recombination events. This strategy allowed us to generate the first genome-wide map of recombination for yeast mitochondrial DNA. We demonstrated that recombination events are enriched in specific hotspots preferentially localized in non-protein-coding regions. Additionally, comparison of the recombination profiles of two different crosses showed that the genetic background affects hotspot localization and recombination rates. Finally, to gain insights into the mechanisms involved in mitochondrial recombination, we assessed the impact of individual depletion of four genes previously associated with this process. Deletion of NTG1 and MGT1 did not substantially influence the recombination landscape, alluding to the potential presence of additional regulatory factors. Our findings also revealed the loss of large mitochondrial DNA regions in the absence of MHR1, suggesting a pivotal role for Mhr1 in mitochondrial genome maintenance during mating. This study provides a comprehensive overview of mitochondrial DNA recombination in yeast and thus paves the way for future mechanistic studies of mitochondrial recombination and genome maintenance. PMID:25081569

A genome-wide map of mitochondrial DNA recombination in yeast.

PubMed

Fritsch, Emilie S; Chabbert, Christophe D; Klaus, Bernd; Steinmetz, Lars M

2014-10-01

In eukaryotic cells, the production of cellular energy requires close interplay between nuclear and mitochondrial genomes. The mitochondrial genome is essential in that it encodes several genes involved in oxidative phosphorylation. Each cell contains several mitochondrial genome copies and mitochondrial DNA recombination is a widespread process occurring in plants, fungi, protists, and invertebrates. Saccharomyces cerevisiae has proved to be an excellent model to dissect mitochondrial biology. Several studies have focused on DNA recombination in this organelle, yet mostly relied on reporter genes or artificial systems. However, no complete mitochondrial recombination map has been released for any eukaryote so far. In the present work, we sequenced pools of diploids originating from a cross between two different S. cerevisiae strains to detect recombination events. This strategy allowed us to generate the first genome-wide map of recombination for yeast mitochondrial DNA. We demonstrated that recombination events are enriched in specific hotspots preferentially localized in non-protein-coding regions. Additionally, comparison of the recombination profiles of two different crosses showed that the genetic background affects hotspot localization and recombination rates. Finally, to gain insights into the mechanisms involved in mitochondrial recombination, we assessed the impact of individual depletion of four genes previously associated with this process. Deletion of NTG1 and MGT1 did not substantially influence the recombination landscape, alluding to the potential presence of additional regulatory factors. Our findings also revealed the loss of large mitochondrial DNA regions in the absence of MHR1, suggesting a pivotal role for Mhr1 in mitochondrial genome maintenance during mating. This study provides a comprehensive overview of mitochondrial DNA recombination in yeast and thus paves the way for future mechanistic studies of mitochondrial recombination and genome maintenance. Copyright © 2014 by the Genetics Society of America.

Whole-genome patterns of linkage disequilibrium across flycatcher populations clarify the causes and consequences of fine-scale recombination rate variation in birds.

PubMed

Kawakami, Takeshi; Mugal, Carina F; Suh, Alexander; Nater, Alexander; Burri, Reto; Smeds, Linnéa; Ellegren, Hans

2017-08-01

Recombination rate is heterogeneous across the genome of various species and so are genetic diversity and differentiation as a consequence of linked selection. However, we still lack a clear picture of the underlying mechanisms for regulating recombination. Here we estimated fine-scale population recombination rate based on the patterns of linkage disequilibrium across the genomes of multiple populations of two closely related flycatcher species (Ficedula albicollis and F. hypoleuca). This revealed an overall conservation of the recombination landscape between these species at the scale of 200 kb, but we also identified differences in the local rate of recombination despite their recent divergence (<1 million years). Genetic diversity and differentiation were associated with recombination rate in a lineage-specific manner, indicating differences in the extent of linked selection between species. We detected 400-3,085 recombination hotspots per population. Location of hotspots was conserved between species, but the intensity of hotspot activity varied between species. Recombination hotspots were primarily associated with CpG islands (CGIs), regardless of whether CGIs were at promoter regions or away from genes. Recombination hotspots were also associated with specific transposable elements (TEs), but this association appears indirect due to shared preferences of the transposition machinery and the recombination machinery for accessible open chromatin regions. Our results suggest that CGIs are a major determinant of the localization of recombination hotspots, and we propose that both the distribution of TEs and fine-scale variation in recombination rate may be associated with the evolution of the epigenetic landscape. © 2017 The Authors. Molecular Ecology Published by John Wiley & Sons Ltd.

New Subtypes and Genetic Recombination in HIV Type 1-Infecting Patients with Highly Active Antiretroviral Therapy in Peru (2008–2010)

PubMed Central

Acuña, Maribel; Gazzo, Cecilia; Salinas, Gabriela; Cárdenas, Fanny; Valverde, Ada; Romero, Soledad

2012-01-01

Abstract HIV-1 subtype B is the most frequent strain in Peru. However, there is no available data about the genetic diversity of HIV-infected patients receiving highly active antiretroviral therapy (HAART) here. A group of 267 patients in the Peruvian National Treatment Program with virologic failure were tested for genotypic evidence of HIV drug resistance at the Instituto Nacional de Salud (INS) of Peru between March 2008 and December 2010. Viral RNA was extracted from plasma and the segments of the protease (PR) and reverse transcriptase (RT) genes were amplified by reverse transcriptase polymerase chain reaction (RT-PCR), purified, and fully sequenced. Consensus sequences were submitted to the HIVdb Genotypic Resistance Interpretation Algorithm Database from Stanford University, and then aligned using Clustal X v.2.0 to generate a phylogenetic tree using the maximum likelihood method. Intrasubtype and intersubtype recombination analyses were performed using the SCUEAL program (Subtype Classification by Evolutionary ALgo-rithms). A total of 245 samples (91%) were successfully genotyped. The analysis obtained from the HIVdb program showed 81.5% resistance cases (n=198). The phylogenetic analysis revealed that subtype B was predominant in the population (98.8%), except for new cases of A, C, and H subtypes (n=4). Of these cases, only subtype C was imported. Likewise, recombination analysis revealed nine intersubtype and 20 intrasubtype recombinant cases. This is the first report of the presence of HIV-1 subtypes C and H in Peru. The introduction of new subtypes and circulating recombinants forms can make it difficult to distinguish resistance profiles in patients and consequently affect future treatment strategies against HIV in this country. PMID:22559065

New subtypes and genetic recombination in HIV type 1-infecting patients with highly active antiretroviral therapy in Peru (2008-2010).

PubMed

Yabar, Carlos Augusto; Acuña, Maribel; Gazzo, Cecilia; Salinas, Gabriela; Cárdenas, Fanny; Valverde, Ada; Romero, Soledad

2012-12-01

HIV-1 subtype B is the most frequent strain in Peru. However, there is no available data about the genetic diversity of HIV-infected patients receiving highly active antiretroviral therapy (HAART) here. A group of 267 patients in the Peruvian National Treatment Program with virologic failure were tested for genotypic evidence of HIV drug resistance at the Instituto Nacional de Salud (INS) of Peru between March 2008 and December 2010. Viral RNA was extracted from plasma and the segments of the protease (PR) and reverse transcriptase (RT) genes were amplified by reverse transcriptase polymerase chain reaction (RT-PCR), purified, and fully sequenced. Consensus sequences were submitted to the HIVdb Genotypic Resistance Interpretation Algorithm Database from Stanford University, and then aligned using Clustal X v.2.0 to generate a phylogenetic tree using the maximum likelihood method. Intrasubtype and intersubtype recombination analyses were performed using the SCUEAL program (Subtype Classification by Evolutionary ALgo-rithms). A total of 245 samples (91%) were successfully genotyped. The analysis obtained from the HIVdb program showed 81.5% resistance cases (n=198). The phylogenetic analysis revealed that subtype B was predominant in the population (98.8%), except for new cases of A, C, and H subtypes (n=4). Of these cases, only subtype C was imported. Likewise, recombination analysis revealed nine intersubtype and 20 intrasubtype recombinant cases. This is the first report of the presence of HIV-1 subtypes C and H in Peru. The introduction of new subtypes and circulating recombinants forms can make it difficult to distinguish resistance profiles in patients and consequently affect future treatment strategies against HIV in this country.

Assessing the complex architecture of polygenic traits in diverged yeast populations.

PubMed

Cubillos, Francisco A; Billi, Eleonora; Zörgö, Enikö; Parts, Leopold; Fargier, Patrick; Omholt, Stig; Blomberg, Anders; Warringer, Jonas; Louis, Edward J; Liti, Gianni

2011-04-01

Phenotypic variation arising from populations adapting to different niches has a complex underlying genetic architecture. A major challenge in modern biology is to identify the causative variants driving phenotypic variation. Recently, the baker's yeast, Saccharomyces cerevisiae has emerged as a powerful model for dissecting complex traits. However, past studies using a laboratory strain were unable to reveal the complete architecture of polygenic traits. Here, we present a linkage study using 576 recombinant strains obtained from crosses of isolates representative of the major lineages. The meiotic recombinational landscape appears largely conserved between populations; however, strain-specific hotspots were also detected. Quantitative measurements of growth in 23 distinct ecologically relevant environments show that our recombinant population recapitulates most of the standing phenotypic variation described in the species. Linkage analysis detected an average of 6.3 distinct QTLs for each condition tested in all crosses, explaining on average 39% of the phenotypic variation. The QTLs detected are not constrained to a small number of loci, and the majority are specific to a single cross-combination and to a specific environment. Moreover, crosses between strains of similar phenotypes generate greater variation in the offspring, suggesting the presence of many antagonistic alleles and epistatic interactions. We found that subtelomeric regions play a key role in defining individual quantitative variation, emphasizing the importance of the adaptive nature of these regions in natural populations. This set of recombinant strains is a powerful tool for investigating the complex architecture of polygenic traits. © 2011 Blackwell Publishing Ltd.

Genealogy-based methods for inference of historical recombination and gene flow and their application in Saccharomyces cerevisiae.

PubMed

Jenkins, Paul A; Song, Yun S; Brem, Rachel B

2012-01-01

Genetic exchange between isolated populations, or introgression between species, serves as a key source of novel genetic material on which natural selection can act. While detecting historical gene flow from DNA sequence data is of much interest, many existing methods can be limited by requirements for deep population genomic sampling. In this paper, we develop a scalable genealogy-based method to detect candidate signatures of gene flow into a given population when the source of the alleles is unknown. Our method does not require sequenced samples from the source population, provided that the alleles have not reached fixation in the sampled recipient population. The method utilizes recent advances in algorithms for the efficient reconstruction of ancestral recombination graphs, which encode genealogical histories of DNA sequence data at each site, and is capable of detecting the signatures of gene flow whose footprints are of length up to single genes. Further, we employ a theoretical framework based on coalescent theory to test for statistical significance of certain recombination patterns consistent with gene flow from divergent sources. Implementing these methods for application to whole-genome sequences of environmental yeast isolates, we illustrate the power of our approach to highlight loci with unusual recombination histories. By developing innovative theory and methods to analyze signatures of gene flow from population sequence data, our work establishes a foundation for the continued study of introgression and its evolutionary relevance.

Genealogy-Based Methods for Inference of Historical Recombination and Gene Flow and Their Application in Saccharomyces cerevisiae

PubMed Central

Jenkins, Paul A.; Song, Yun S.; Brem, Rachel B.

2012-01-01

Genetic exchange between isolated populations, or introgression between species, serves as a key source of novel genetic material on which natural selection can act. While detecting historical gene flow from DNA sequence data is of much interest, many existing methods can be limited by requirements for deep population genomic sampling. In this paper, we develop a scalable genealogy-based method to detect candidate signatures of gene flow into a given population when the source of the alleles is unknown. Our method does not require sequenced samples from the source population, provided that the alleles have not reached fixation in the sampled recipient population. The method utilizes recent advances in algorithms for the efficient reconstruction of ancestral recombination graphs, which encode genealogical histories of DNA sequence data at each site, and is capable of detecting the signatures of gene flow whose footprints are of length up to single genes. Further, we employ a theoretical framework based on coalescent theory to test for statistical significance of certain recombination patterns consistent with gene flow from divergent sources. Implementing these methods for application to whole-genome sequences of environmental yeast isolates, we illustrate the power of our approach to highlight loci with unusual recombination histories. By developing innovative theory and methods to analyze signatures of gene flow from population sequence data, our work establishes a foundation for the continued study of introgression and its evolutionary relevance. PMID:23226196

Oral immunization of BALB/c mice with Giardia duodenalis recombinant cyst wall protein inhibits shedding of cysts.

PubMed

Larocque, R; Nakagaki, K; Lee, P; Abdul-Wahid, A; Faubert, G M

2003-10-01

The process of encystation is a key step in the Giardia duodenalis life cycle that allows this intestinal protozoan to survive between hosts during person-to-person, animal-to-person, waterborne, or food-borne transmission. The release of cysts from infected persons and animals is the main contributing factor to contamination of the environment. Genes coding for cyst wall proteins (CWPs), which could be used for developing a transmission-blocking vaccine, have been cloned. Since the immunogenicity of recombinant Giardia CWP is unknown, we have investigated the immunogenicity of recombinant CWP2 (rCWP2) and its efficacy in interfering with the phenomenon of encystation taking place in the small bowels of BALB/c mice vaccinated with the recombinant protein. Here we report that the immunization of BALB/c mice with rCWP2 stimulated the immune system in a manner comparable to that for a live infection with Giardia muris cysts. Fecal and serum anti-rCWP2 immunoglobulin A (IgA) antibodies were detected in the immunized mice. In addition, anti-rCWP2 IgG1 and IgG2a antibodies were detected in the serum. mRNAs coding for Th1 and Th2 types of cytokines were detected in spleen and Peyer's patch cells from immunized mice. When the vaccinated mice were challenged with live cysts, the animals shed fewer cysts. We conclude that rCWP2 is a possible candidate antigen for the development of a transmission-blocking vaccine.

Detection of anti-Yta antibodies using a sensitive and specific enzyme-linked immunosorbent assay.

PubMed

Geen, J; Hullin, D A; Hogg, S I

1999-01-01

A specific, sensitive and semi-quantitative enzyme-linked immunosorbent assay (ELISA) is described to detect anti-Yta antibodies in human serum. Recombinant acetylcholinesterase (AChE E.C.3.1.1.7) was employed as the coating antigen in the microtitre plate and horseradish peroxidase (HRP)-conjugated specific antibody (IgG) was used as the secondary antibody. The method developed showed excellent sensitivity, detecting a titre > 1 in 600,000 (3.5 ng/mL mouse IgG protein) for mouse monoclonal (mMAb) anti-AChE antibody. No cross-reaction was seen with other common blood group antibodies, confirming the specificity of the method. The recombinant antigen's AChE phenotype was confirmed as Yta, as no reaction was detected with anti-Ytb-positive sera. The ELISA method correlated closely with the established serological grading system used routinely in blood transfusion laboratories.

Enhanced production and purification of recombinant surface array protein (Sap) for use in detection of Bacillus anthracis.

PubMed

Puranik, Nidhi; Tripathi, N K; Pal, V; Goel, Ajay Kumar

2018-05-01

Surface array protein (Sap) can be an important biomarker for specific detection of Bacillus anthracis , which is released by the bacterium during its growth in culture broth. In the present work, we have cloned and expressed Sap in Escherichia coli . The culture conditions and cultivation media were optimized and used in batch fermentation process for scale up of Sap in soluble form. The recombinant Sap was purified employing affinity chromatography followed by diafiltration. The final yield of purified protein was 20 and 46 mg/l of culture during shake flasks and batch fermentation, respectively. The protein purity and its reactivity were confirmed employing SDS-PAGE and Western blot, respectively. The antibodies raised against purified Sap were evaluated by Western blotting for detection of Sap released by B. anthracis . Our results showed that the Sap could be a novel marker for detection and confirmation of B. anthracis .

Bioluminescent bioreporter integrated circuit devices and methods for detecting estrogen

DOEpatents

Simpson, Michael L.; Paulus, Michael J.; Sayler, Gary S.; Applegate, Bruce M.; Ripp, Steven A.

2006-08-15

Bioelectronic devices for the detection of estrogen include a collection of eukaryotic cells which harbor a recombinant lux gene from a high temperature microorganism wherein the gene is operably linked with a heterologous promoter gene. A detectable light-emitting lux gene product is expressed in the presence of the estrogen and detected by the device.

Detection of rabies-specific antigens by egg yolk antibody (IgY) to the recombinant rabies virus proteins produced in Escherichia coli.

PubMed

Motoi, Yurie; Inoue, Satoshi; Hatta, Hajime; Sato, Kozue; Morimoto, Kinjiro; Yamada, Akio

2005-04-01

We obtained rabies-specific egg yolk antibodies (IgY) by immunizing hens with recombinant His-tagged nucleoprotein and phosphoprotein (rN, rP) of the rabies virus (CVS-11 strain) expressed in Escherichia coli. The anti-rN and rP IgY were shown to bind specifically to the respective proteins of the CVS-11 strain of rabies virus by Western blotting, immune fluorescent assay and immunohistochemistry, indicating that IgY to rabies recombinant proteins could serve as a reagent for diagnosis of rabies virus infection.

Sabin Vaccine Reversion in the Field: a Comprehensive Analysis of Sabin-Like Poliovirus Isolates in Nigeria.

PubMed

Famulare, Michael; Chang, Stewart; Iber, Jane; Zhao, Kun; Adeniji, Johnson A; Bukbuk, David; Baba, Marycelin; Behrend, Matthew; Burns, Cara C; Oberste, M Steven

2016-01-01

To assess the dynamics of genetic reversion of live poliovirus vaccine in humans, we studied molecular evolution in Sabin-like poliovirus isolates from Nigerian acute flaccid paralysis cases obtained from routine surveillance. We employed a novel modeling approach to infer substitution and recombination rates from whole-genome sequences and information about poliovirus infection dynamics and the individual vaccination history. We confirmed observations from a recent vaccine trial that VP1 substitution rates are increased for Sabin-like isolates relative to the rate for the wild type due to increased nonsynonymous substitution rates. We also inferred substitution rates for attenuating nucleotides and confirmed that reversion can occur in days to weeks after vaccination. We combine our observations for Sabin-like virus evolution with the molecular clock for VP1 of circulating wild-type strains to infer that the mean time from the initiating vaccine dose to the earliest detection of circulating vaccine-derived poliovirus (cVDPV) is 300 days for Sabin-like virus type 1, 210 days for Sabin-like virus type 2, and 390 days for Sabin-like virus type 3. Phylogenetic relationships indicated transient local transmission of Sabin-like virus type 3 and, possibly, Sabin-like virus type 1 during periods of low wild polio incidence. Comparison of Sabin-like virus recombinants with known Nigerian vaccine-derived poliovirus recombinants shows that while recombination with non-Sabin enteroviruses is associated with cVDPV, the recombination rates are similar for Sabin isolate-Sabin isolate and Sabin isolate-non-Sabin enterovirus recombination after accounting for the time from dosing to the time of detection. Our study provides a comprehensive picture of the evolutionary dynamics of the oral polio vaccine in the field. The global polio eradication effort has completed its 26th year. Despite success in eliminating wild poliovirus from most of the world, polio persists in populations where logistical, social, and political factors have not allowed vaccination programs of sustained high quality. One issue of critical importance is eliminating circulating vaccine-derived polioviruses (cVDPVs) that have properties indistinguishable from those of wild poliovirus and can cause paralytic disease. cVDPV emerges due to the genetic instability of the Sabin viruses used in the oral polio vaccine (OPV) in populations that have low levels of immunity to poliovirus. However, the dynamics responsible are incompletely understood because it has historically been difficult to gather and interpret data about evolution of the Sabin viruses used in OPV in regions where cVDPV has occurred. This study is the first to combine whole-genome sequencing of poliovirus isolates collected during routine surveillance with knowledge about the intrahost dynamics of poliovirus to provide quantitative insight into polio vaccine evolution in the field. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Targeted Multiplex Imaging Mass Spectrometry with Single Chain Fragment Variable (scfv) Recombinant Antibodies

NASA Astrophysics Data System (ADS)

Thiery, Gwendoline; Mernaugh, Ray L.; Yan, Heping; Spraggins, Jeffrey M.; Yang, Junhai; Parl, Fritz F.; Caprioli, Richard M.

2012-10-01

Recombinant scfv antibodies specific for CYP1A1 and CYP1B1 P450 enzymes were combined with targeted imaging mass spectrometry to simultaneously detect the P450 enzymes present in archived, paraffin-embedded, human breast cancer tissue sections. By using CYP1A1 and CYP1B1 specific scfv, each coupled to a unique reporter molecule (i.e., a mass tag) it was possible to simultaneously detect multiple antigens within a single tissue sample with high sensitivity and specificity using mass spectrometry. The capability of imaging multiple antigens at the same time is a significant advance that overcomes technical barriers encountered when using present day approaches to develop assays that can simultaneously detect more than a single antigen in the same tissue sample.

Genetics of Genome-Wide Recombination Rate Evolution in Mice from an Isolated Island.

PubMed

Wang, Richard J; Payseur, Bret A

2017-08-01

Recombination rate is a heritable quantitative trait that evolves despite the fundamentally conserved role that recombination plays in meiosis. Differences in recombination rate can alter the landscape of the genome and the genetic diversity of populations. Yet our understanding of the genetic basis of recombination rate evolution in nature remains limited. We used wild house mice ( Mus musculus domesticus ) from Gough Island (GI), which diverged recently from their mainland counterparts, to characterize the genetics of recombination rate evolution. We quantified genome-wide autosomal recombination rates by immunofluorescence cytology in spermatocytes from 240 F 2 males generated from intercrosses between GI-derived mice and the wild-derived inbred strain WSB/EiJ. We identified four quantitative trait loci (QTL) responsible for inter-F 2 variation in this trait, the strongest of which had effects that opposed the direction of the parental trait differences. Candidate genes and mutations for these QTL were identified by overlapping the detected intervals with whole-genome sequencing data and publicly available transcriptomic profiles from spermatocytes. Combined with existing studies, our findings suggest that genome-wide recombination rate divergence is not directional and its evolution within and between subspecies proceeds from distinct genetic loci. Copyright © 2017 by the Genetics Society of America.

Oral Administration of Recombinant Lactococcus lactis Expressing the Cellulase Gene Increases Digestibility of Fiber in Geese.

PubMed

Zhou, Haizhu; Gao, Yunhang; Gao, Guang; Lou, Yujie

2015-12-01

Enhancing cellulose digestibility in animals is important for improving the utilization of forage, which can decrease the amount of food used in animal production. The aim of the present study was to achieve recombinant expression of the cellulase gene in Lactococcus lactis and evaluate the effects of oral administration of the recombinant L. lactis on fiber digestibility in geese. Cellulase (Cell) and green fluorescent protein (GFP) genes were cloned into a L. lactis expression vector (pNZ8149) to construct the recombinant expression plasmid (pNZ8149-GFP-Cell). Then, the recombinant expression plasmid was transformed into L. lactis (NZ3900) competent cells by electroporation to obtain recombinant L. lactis (pNZ8149-GFP-Cell/NZ3900) in which protein expression was induced by Nisin. Expression of GFP and Cell by the recombinant L. lactis was confirmed using SDS-PAGE, fluorescence detection, and Congo red assays. A feeding experiment showed that oral administration of pNZ8149-GFP-Cell/NZ3900 significantly increased the digestibility of dietary fiber in geese fed either a maize stalk diet or a rice chaff diet. Therefore, oral administration of recombinant L. lactis cells expressing the cellulase gene increases fiber digestibility in geese, offering a way to increase the utilization of dietary fiber in geese.

Incorporation of a lambda phage recombination system and EGFP detection to simplify mutagenesis of Herpes simplex virus bacterial artificial chromosomes

PubMed Central

Schmeisser, Falko; Weir, Jerry P

2007-01-01

Background Targeted mutagenesis of the herpesvirus genomes has been facilitated by the use of bacterial artificial chromosome (BAC) technology. Such modified genomes have potential uses in understanding viral pathogenesis, gene identification and characterization, and the development of new viral vectors and vaccines. We have previously described the construction of a herpes simplex virus 2 (HSV-2) BAC and the use of an allele replacement strategy to construct HSV-2 recombinants. While the BAC mutagenesis procedure is a powerful method to generate HSV-2 recombinants, particularly in the absence of selective marker in eukaryotic culture, the mutagenesis procedure is still difficult and cumbersome. Results Here we describe the incorporation of a phage lambda recombination system into an allele replacement vector. This strategy enables any DNA fragment containing the phage attL recombination sites to be efficiently inserted into the attR sites of the allele replacement vector using phage lambda clonase. We also describe how the incorporation of EGFP into the allele replacement vector can facilitate the selection of the desired cross-over recombinant BACs when the allele replacement reaction is a viral gene deletion. Finally, we incorporate the lambda phage recombination sites directly into an HSV-2 BAC vector for direct recombination of gene cassettes using the phage lambda clonase-driven recombination reaction. Conclusion Together, these improvements to the techniques of HSV BAC mutagenesis will facilitate the construction of recombinant herpes simplex viruses and viral vectors. PMID:17501993

A novel recombinant pseudorabies virus expressing parvovirus VP2 gene: Immunogenicity and protective efficacy in swine.

PubMed

Chen, Yang; Guo, Wanzhu; Xu, Zhiwen; Yan, Qigui; Luo, Yan; Shi, Qian; Chen, Dishi; Zhu, Ling; Wang, Xiaoyu

2011-06-16

Porcine parvovirus (PPV) VP2 gene has been successfully expressed in many expression systems resulting in self-assembly of virus-like particles (VLPs) with similar morphology to the native capsid. Here, a pseudorabies virus (PRV) system was adopted to express the PPV VP2 gene. A recombinant PRV SA215/VP2 was obtained by homologous recombination between the vector PRV viral DNA and a transfer plasmid. Then recombinant virus was purified with plaque purification, and its identity confirmed by PCR amplification, Western blot and indirect immunofluorescence (IFA) analyses. Electronic microscopy of PRV SA215/VP2 confirmed self-assembly of both pseudorabies virus and VLPs from VP2 protein. Immunization of piglets with recombinant virus elicited PRV-specific and PPV-specific humoral immune responses and provided complete protection against a lethal dose of PRV challenges. Gilts immunized with recombinant viruses induced PPV-specific antibodies, and significantly reduced the mortality rate of (1 of 28) following virulent PPV challenge compared with the control (7 of 31). Furthermore, PPV virus DNA was not detected in the fetuses of recombinant virus immunized gilts. In this study, a recombinant PRV SA215/VP2 virus expressing PPV VP2 protein was constructed using PRV SA215 vector. The safety, immunogenicity, and protective efficacy of the recombinant virus were demonstrated in piglets and primiparous gilts. This recombinant PRV SA215/VP2 represents a suitable candidate for the development of a bivalent vaccine against both PRV and PPV infection.

Transient Expression and Cellular Localization of Recombinant Proteins in Cultured Insect Cells.

PubMed

Fabrick, Jeffrey A; Hull, J Joe

2017-04-20

Heterologous protein expression systems are used for the production of recombinant proteins, the interpretation of cellular trafficking/localization, and the determination of the biochemical function of proteins at the sub-organismal level. Although baculovirus expression systems are increasingly used for protein production in numerous biotechnological, pharmaceutical, and industrial applications, nonlytic systems that do not involve viral infection have clear benefits but are often overlooked and underutilized. Here, we describe a method for generating nonlytic expression vectors and transient recombinant protein expression. This protocol allows for the efficient cellular localization of recombinant proteins and can be used to rapidly discern protein trafficking within the cell. We show the expression of four recombinant proteins in a commercially available insect cell line, including two aquaporin proteins from the insect Bemisia tabaci, as well as subcellular marker proteins specific for the cell plasma membrane and for intracellular lysosomes. All recombinant proteins were produced as chimeras with fluorescent protein markers at their carboxyl termini, which allows for the direct detection of the recombinant proteins. The double transfection of cells with plasmids harboring constructs for the genes of interest and a known subcellular marker allows for live cell imaging and improved validation of cellular protein localization.

Qualitative and Quantitative Assays of Transposition and Homologous Recombination of the Retrotransposon Tf1 in Schizosaccharomyces pombe.

PubMed

Sangesland, Maya; Atwood-Moore, Angela; Rai, Sudhir K; Levin, Henry L

2016-01-01

Transposition and homologous recombination assays are valuable genetic tools to measure the production and integration of cDNA from the long terminal repeat (LTR) retrotransposon Tf1 in the fission yeast (Schizosaccharomyces pombe). Here we describe two genetic assays, one that measures the transposition activity of Tf1 by monitoring the mobility of a drug resistance marked Tf1 element expressed from a multi-copy plasmid and another assay that measures homologous recombination between Tf1 cDNA and the expression plasmid. While the transposition assay measures insertion of full-length Tf1 cDNA mediated by the transposon integrase, the homologous recombination assay measures levels of cDNA present in the nucleus and is independent of integrase activity. Combined, these assays can be used to systematically screen large collections of strains to identify mutations that specifically inhibit the integration step in the retroelement life cycle. Such mutations can be identified because they reduce transposition activity but nevertheless have wild-type frequencies of homologous recombination. Qualitative assays of yeast patches on agar plates detect large defects in integration and recombination, while the quantitative approach provides a precise method of determining integration and recombination frequencies.

Application of crude and recombinant ELISAs and immunochromatographic test for serodiagnosis of animal trypanosomosis in the Umkhanyakude district of KwaZulu-Natal province, South Africa

PubMed Central

NGUYEN, Thu-Thuy; MOTSIRI, Mono Sophie; TAIOE, Moeti Oriel; MTSHALI, Moses Sibusiso; GOTO, Yasuyuki; KAWAZU, Shin-Ichiro; THEKISOE, Oriel Matlhahane Molifi; INOUE, Noboru

2014-01-01

A total of 231 serum samples were collected from sheep (n=9), goats (n=99) and cattle (n=123) in northeastern KwaZulu-Natal, South Africa. Trypanosome infection was detected using Trypanosoma brucei brucei crude antigen (TbbCA) and T. congolense crude antigen (TcoCA) ELISA assays. Recombinant antigen (T. evansi GM6 which consisted of 4 repeat domains, TeGM6-4r) ELISA and immunochromatographic test (ICT) were also used. Crude antigen ELISA, TeGM6-4r-ELISA and ICT detected 27.3%, 29% and 19.9% of trypanosome seropositive samples, respectively. Trypanosome infection prevalence in cattle and goats was 35.8–46.3% and 0–9.1%, respectively. Out of 9 sheep serum samples, 2–4 sera (22.2–44.4%) were positive. The detection performance of crude and recombinant antigen ELISAs was relatively similar (K=0.6–0.7); both are recommended for reference diagnosis and large scale epidemiological surveys. There is potential application for ICT in on-site diagnosis, but its sensitivity should be improved. PMID:25342634

Homologous Recombination between Genetically Divergent Campylobacter fetus Lineages Supports Host-Associated Speciation

PubMed Central

Duim, Birgitta; van der Graaf-van Bloois, Linda; Wagenaar, Jaap A; Zomer, Aldert L

2018-01-01

Abstract Homologous recombination is a major driver of bacterial speciation. Genetic divergence and host association are important factors influencing homologous recombination. Here, we study these factors for Campylobacter fetus, which shows a distinct intraspecific host dichotomy. Campylobacter fetus subspecies fetus (Cff) and venerealis are associated with mammals, whereas C. fetus subsp. testudinum (Cft) is associated with reptiles. Recombination between these genetically divergent C. fetus lineages is extremely rare. Previously it was impossible to show whether this barrier to recombination was determined by the differential host preferences, by the genetic divergence between both lineages or by other factors influencing recombination, such as restriction-modification, CRISPR/Cas, and transformation systems. Fortuitously, a distinct C. fetus lineage (ST69) was found, which was highly related to mammal-associated C. fetus, yet isolated from a chelonian. The whole genome sequences of two C. fetus ST69 isolates were compared with those of mammal- and reptile-associated C. fetus strains for phylogenetic and recombination analysis. In total, 5.1–5.5% of the core genome of both ST69 isolates showed signs of recombination. Of the predicted recombination regions, 80.4% were most closely related to Cft, 14.3% to Cff, and 5.6% to C. iguaniorum. Recombination from C. fetus ST69 to Cft was also detected, but to a lesser extent and only in chelonian-associated Cft strains. This study shows that despite substantial genetic divergence no absolute barrier to homologous recombination exists between two distinct C. fetus lineages when occurring in the same host type, which provides valuable insights in bacterial speciation and evolution. PMID:29608720

Diagnostic value of detecting specific IgA and IgM with recombinant Trypanosoma cruzi antigens in congenital Chagas' disease.

PubMed

Lorca, M; Veloso, C; Munoz, P; Bahamonde, M I; Garcia, A

1995-06-01

The present study compares the early diagnosis of congenital Chagas' disease with a DOT assay using recombinant antigens with immunofluorescence antibody testing (IFAT) and an enzyme-linked immunosorbent assay (ELISA). The studies were performed using cord blood and sera of 12 infected newborns (group I) and 12 uninfected ones born to Trypanosoma cruzi-infected mothers (group II). Conventional IFAT and ELISA showed positive results for IgG at high titers, in infants and mothers of both groups; IgA antibodies were detected by ELISA in four of the infected infants and IgM was detected in two of them. All sera of the uninfected infants were negative for IgA and IgM in the ELISA. Application of a DOT assay using eight recombinant T. cruzi antigens allowed detection of specific IgA in the cord blood of six of the infected cases and IgM in eight of them. Repetition of these serologic tests in samples obtained during a monthly follow-up gave positive results for IgA in two of the initially negative infants of group I and for IgM in four of them. This means that diagnosis of congenital T. cruzi infection was confirmed, through demonstration of specific IgM, in all infected infants, and of IgA in eight of them. The importance of late detection of IgM in siblings born of infected mothers is discussed. The detection of IgM and IgA in sera obtained after birth is believed to be due to a congenital transmission of the parasite that occurred late in pregnancy. No IgA or IgM antibodies could be detected by the DOT assay in the sera of the negative controls.(ABSTRACT TRUNCATED AT 250 WORDS)

Fluorescence-based recombination assay for sensitive and specific detection of genotoxic carcinogens in human cells.

PubMed

Ireno, Ivanildce C; Baumann, Cindy; Stöber, Regina; Hengstler, Jan G; Wiesmüller, Lisa

2014-05-01

In vitro genotoxicity tests are known to suffer from several shortcomings, mammalian cell-based assays, in particular, from low specificities. Following a novel concept of genotoxicity detection, we developed a fluorescence-based method in living human cells. The assay quantifies DNA recombination events triggered by DNA double-strand breaks and damage-induced replication fork stalling predicted to detect a broad spectrum of genotoxic modes of action. To maximize sensitivities, we engineered a DNA substrate encompassing a chemoresponsive element from the human genome. Using this substrate, we screened various human tumor and non-transformed cell types differing in the DNA damage response, which revealed that detection of genotoxic carcinogens was independent of the p53 status but abrogated by apoptosis. Cell types enabling robust and sensitive genotoxicity detection were selected for the generation of reporter clones with chromosomally integrated DNA recombination substrate. Reporter cell lines were scrutinized with 21 compounds, stratified into five sets according to the established categories for identification of carcinogenic compounds: genotoxic carcinogens ("true positives"), non-genotoxic carcinogens, compounds without genotoxic or carcinogenic effect ("true negatives") and non-carcinogenic compounds, which have been reported to induce chromosomal aberrations or mutations in mammalian cell-based assays ("false positives"). Our results document detection of genotoxic carcinogens in independent cell clones and at levels of cellular toxicities <60 % with a sensitivity of >85 %, specificity of ≥90 % and detection of false-positive compounds <17 %. Importantly, through testing cyclophosphamide in combination with primary hepatocyte cultures, we additionally provide proof-of-concept for the identification of carcinogens requiring metabolic activation using this novel assay system.

Isolation of an Intertypic Poliovirus Capsid Recombinant from a Child with Vaccine-Associated Paralytic Poliomyelitis

PubMed Central

Martín, Javier; Samoilovich, Elena; Dunn, Glynis; Lackenby, Angie; Feldman, Esphir; Heath, Alan; Svirchevskaya, Ekaterina; Cooper, Gill; Yermalovich, Marina; Minor, Philip D.

2002-01-01

The isolation of a capsid intertypic poliovirus recombinant from a child with vaccine-associated paralytic poliomyelitis is described. Virus 31043 had a Sabin-derived type 3-type 2-type 1 recombinant genome with a 5′-end crossover point within the capsid coding region. The result was a poliovirus chimera containing the entire coding sequence for antigenic site 3a derived from the Sabin type 2 strain. The recombinant virus showed altered antigenic properties but did not acquire type 2 antigenic characteristics. The significance of the presence in nature of such poliovirus chimeras and the consequences for the current efforts to detect potentially dangerous vaccine-derived poliovirus strains are discussed in the context of the global polio eradication initiative. PMID:12368335

HT.sub.m4 methods of treatment and assays, agonists and antagonists

DOEpatents

Lim, Bing; Adra, Chaker N.; Lelias, Jean-Michel

1999-01-01

The invention relates to a recombinant DNA molecule which encodes a HT.sub.m4 protein, a transformed host cell which has been stably transfected with a DNA molecule which encodes a HT.sub.m4 protein and a recombinant HT.sub.m4 protein. The invention also relates to a method for detecting the presence of a hereditary atopy.

Characterization of a novel Aspergillus oryzae tannase expressed in Pichia pastoris.

PubMed

Koseki, Takuya; Ichikawa, Kyotaro; Sasaki, Katsuto; Shiono, Yoshihito

2018-05-31

We report the characterization of tannase-encoding gene, AotanB, from Aspergillus oryzae and its recombinant enzyme expressed in Pichia pastoris. The gene except for the signal sequence was cloned into a vector pPICZαA and the recombinant protein was secreted into the medium as an active enzyme. Recombinant AoTanB highly expressed in the incubation at 18°C compared to 30°C. Purified recombinant protein exhibited smeared band with molecular mass of approximately 90-120 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The recombinant protein yielded molecular mass of 65 kDa after N-deglycosylation. Purified recombinant enzyme had a pH and a temperature optima of 6.0 and 30-35°C, respectively, and was stable up to 40°C. Recombinant AoTanB was able to release gallic acid from natural substrates, such as (-)-catechin gallate, (-)-epicatechin gallate, (-)-gallochatechin gallate, and (-)-epigallocatechin gallate. The enzyme also hydrolyzed ethyl protocatechuate. Meanwhile, no activity was detected toward ethyl 4-hydroxybenzoate. The activity of recombinant AoTanB was lower toward natural substrates compared to that of AoTanA from A. oryzae. The lower catalytic efficiency (k cat /K m value) toward ethyl protocatechuate was due to a combination of increased K m and considerably decreased k cat . Kinetic analysis of the recombinant AoTanB showed that k cat values toward natural substrates decreased compared to those of recombinant AoTanA. Therefore, recombinant AoTanB showed a decrease in catalytic efficiency (k cat /K m value) compared to recombinant AoTanA was due to considerably lower k cat value. Copyright © 2018 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

Genomic features of intertypic recombinant sabin poliovirus strains excreted by primary vaccinees.

PubMed

Cuervo, N S; Guillot, S; Romanenkova, N; Combiescu, M; Aubert-Combiescu, A; Seghier, M; Caro, V; Crainic, R; Delpeyroux, F

2001-07-01

The trivalent oral poliomyelitis vaccine (OPV) contains three different poliovirus serotypes. It use therefore creates particularly favorable conditions for mixed infection of gut cells, and indeed intertypic vaccine-derived recombinants (VdRec) have been frequently found in patients with vaccine-associated paralytic poliomyelitis. Nevertheless, there have not been extensive searches for VdRec in healthy vaccinees following immunization with OPV. To determine the incidence of VdRec and their excretion kinetics in primary vaccinees, and to establish the general genomic features of the corresponding recombinant genomes, we characterized poliovirus isolates excreted by vaccinees following primary immunization with OPV. Isolates were collected from 67 children 2 to 60 days following vaccination. Recombinant strains were identified by multiple restriction fragment length polymorphism assays. The localization of junction sites in recombinant genomes was also determined. VdRec excreted by vaccinees were first detected 2 to 4 days after vaccination. The highest rate of recombinants was on day 14. The frequency of VdRec depends strongly on the serotype of the analyzed isolates (2, 53, and 79% of recombinant strains in the last-excreted type 1, 2, and 3 isolates, respectively). Particular associations of genomic segments were preferred in the recombinant genomes, and recombination junctions were found in the genomic region encoding the nonstructural proteins. Recombination junctions generally clustered in particular subgenomic regions that were dependent on the serotype of the isolate and/or on the associations of genomic segments in recombinants. Thus, VdRec are frequently excreted by vaccinees, and the poliovirus replication machinery requirements or selection factors appear to act in vivo to shape the features of the recombinant genomes.

Unusual Structure of the attB Site of the Site-Specific Recombination System of Lactobacillus delbrueckii Bacteriophage mv4

PubMed Central

Auvray, Frédéric; Coddeville, Michèle; Ordonez, Romy Catoira; Ritzenthaler, Paul

1999-01-01

The temperate phage mv4 integrates its genome into the chromosome of Lactobacillus delbrueckii subsp. bulgaricus by site-specific recombination within the 3′ end of a tRNASer gene. Recombination is catalyzed by the phage-encoded integrase and occurs between the phage attP site and the bacterial attB site. In this study, we show that the mv4 integrase functions in vivo in Escherichia coli and we characterize the bacterial attB site with a site-specific recombination test involving compatible plasmids carrying the recombination sites. The importance of particular nucleotides within the attB sequence was determined by site-directed mutagenesis. The structure of the attB site was found to be simple but rather unusual. A 16-bp DNA fragment was sufficient for function. Unlike most genetic elements that integrate their DNA into tRNA genes, none of the dyad symmetry elements of the tRNASer gene were present within the minimal attB site. No inverted repeats were detected within this site either, in contrast to the lambda site-specific recombination model. PMID:10572145

Exploiting persistent infection for selection of bovine herpesvirus 4 recombinants.

PubMed

Donofrio, G; Martignani, E; Cavirani, S; Flammini, C F

2005-09-01

Bovine herpesvirus 4 (BoHV-4) is a gamma-herpesvirus with no clear disease association, and due to its biological characteristics, has been suggested as a gene delivery vector. It was demonstrated previously that recombinant BoHV-4 carrying a neomycin-resistance gene was able to infect a human rhabdomyosarcoma cell line (RD-4), resulting in no detectable cytopathic effect (CPE) and allowing selection of G418-resistant persistently-infected cells containing circular episomal viral DNA [Donofrio, G., Cavirani, S., van Santen, V.L., 2000a. Establishment of a cell line persistently infected with recombinant BoHV-4. J. Gen. Virol. 81, 1807-1814.]. Those cells produce infectious virus and infection is predominantly non-permissive and non-cytopathic. Starting from these results, the ability of RD-4 cells to sustain persistent infection was combined with positive selection activity conferred by the neomycin-expression cassette insert, as an easier way to select recombinants of BoHV-4 following homologous recombination in permissive cells. A tool for selecting BoHV-4 recombinants was developed by drug positive selection.

Recombination or mutational hot spots in human mtDNA?

PubMed

Innan, Hideki; Nordborg, Magnus

2002-07-01

Awadalla, Eyre-Walker, and Maynard Smith (1999) recently argued that there might be recombination in human mitochondrial DNA (mtDNA). Their claim was based on their observation of decaying linkage disequilibrium (LD) as a function of physical distance. Their study was much criticized, and follow-up studies have failed to find any evidence for recombination. We argue that the criticisms levied, even if correct, could not possibly explain the findings of Awadalla, Eyre-Walker, and Maynard Smith (1999). Nonetheless, the test proposed by Awadalla, Eyre-Walker, and Maynard Smith (1999 ) is not robust because recombination is not the only explanation for decay of LD. We show that such a pattern can be caused by mutational hot spots as well. However, a closer look at the data suggests that the pattern observed was not caused by mutational hot spots but rather by chance. Thus, there appears to be no evidence for recombination in the mtDNA polymorphism data. In conclusion, we discuss the possibility of detecting recombination in mtDNA and the implications of its existence.

Recombinant Saccharomyces cerevisiae serves as novel carrier for oral DNA vaccines in Carassius auratus.

PubMed

Yan, Nana; Xu, Kun; Li, Xinyi; Liu, Yuwan; Bai, Yichun; Zhang, Xiaohan; Han, Baoquan; Chen, Zhilong; Zhang, Zhiying

2015-12-01

Oral delivery of DNA vaccines represents a promising vaccinating method for fish. Recombinant yeast has been proved to be a safe carrier for delivering antigen proteins and DNAs to some species in vivo. However, whether recombinant yeast can be used to deliver functional DNAs for vaccination to fish is still unknown. In this study, red crucian carp (Carassius auratus) was orally administrated with recombinant Saccharomyces cerevisiae harboring CMV-EGFP expression cassette. On day 5 post the first vaccination, EGFP expression in the hindgut was detected under fluorescence microscope. To further study whether the delivered gene could induce specific immune responses, the model antigen ovalbumin (OVA) was used as immunogen, and oral administrations were conducted with recombinant S. cerevisiae harboring pCMV-OVA mammalian gene expression cassette as gene delivery or pADH1-OVA yeast gene expression cassette as protein delivery. Each administration was performed with three different doses, and the OVA-specific serum antibody was detected in all the experimental groups by western blotting and enzyme-linked immunosorbent assay (ELISA). ELISA assay also revealed that pCMV-OVA group with lower dose (pCMV-OVA-L) and pADH1-OVA group with moderate dose (pADH1-OVA-M) triggered relatively stronger antibody response than the other two doses. Moreover, the antibody level induced by pCMV-OVA-L group was significantly higher than pADH1-OVA-M group at the same serum dilutions. All the results suggested that recombinant yeast can be used as a potential carrier for oral DNA vaccines and would help to develop more practical strategies to control infectious diseases in aquaculture. Copyright © 2015 Elsevier Ltd. All rights reserved.

Rosa26-GFP Direct Repeat (RaDR-GFP) Mice Reveal Tissue- and Age-Dependence of Homologous Recombination in Mammals In Vivo

PubMed Central

Kay, Jennifer E.; Na, Li; Rowland, Elizabeth A.; Winther, Kelly E.; Chow, Danielle N.; Kimoto, Takafumi; Matsuguchi, Tetsuya; Jonnalagadda, Vidya S.; Maklakova, Vilena I.; Singh, Vijay R.; Wadduwage, Dushan N.; Rajapakse, Jagath; So, Peter T. C.; Collier, Lara S.; Engelward, Bevin P.

2014-01-01

Homologous recombination (HR) is critical for the repair of double strand breaks and broken replication forks. Although HR is mostly error free, inherent or environmental conditions that either suppress or induce HR cause genomic instability. Despite its importance in carcinogenesis, due to limitations in our ability to detect HR in vivo, little is known about HR in mammalian tissues. Here, we describe a mouse model in which a direct repeat HR substrate is targeted to the ubiquitously expressed Rosa26 locus. In the Rosa26 Direct Repeat-GFP (RaDR-GFP) mice, HR between two truncated EGFP expression cassettes can yield a fluorescent signal. In-house image analysis software provides a rapid method for quantifying recombination events within intact tissues, and the frequency of recombinant cells can be evaluated by flow cytometry. A comparison among 11 tissues shows that the frequency of recombinant cells varies by more than two orders of magnitude among tissues, wherein HR in the brain is the lowest. Additionally, de novo recombination events accumulate with age in the colon, showing that this mouse model can be used to study the impact of chronic exposures on genomic stability. Exposure to N-methyl-N-nitrosourea, an alkylating agent similar to the cancer chemotherapeutic temozolomide, shows that the colon, liver and pancreas are susceptible to DNA damage-induced HR. Finally, histological analysis of the underlying cell types reveals that pancreatic acinar cells and liver hepatocytes undergo HR and also that HR can be specifically detected in colonic somatic stem cells. Taken together, the RaDR-GFP mouse model provides new understanding of how tissue and age impact susceptibility to HR, and enables future studies of genetic, environmental and physiological factors that modulate HR in mammals. PMID:24901438

Serologic reactivity to purified recombinant and native 29-kilodalton peripheral membrane protein of pathogenic Entamoeba histolytica.

PubMed Central

Flores, B M; Reed, S L; Ravdin, J I; Torian, B E

1993-01-01

The 29-kDa peripheral membrane protein of Entamoeba histolytica has recently been demonstrated to have epitopes on pathogenic clinical isolates which were not detected by monoclonal antibodies on nonpathogenic isolates. To analyze the serological response to this protein, we tested 93 serum specimens (from 33 patients with amebic liver abscess, 7 patients with colitis, 2 patients with ameboma, 18 individuals harboring a nonpathogenic zymodeme strain, 10 healthy Mexican migrant workers, and 23 healthy controls) by enzyme-linked immunosorbent assay (ELISA) using immunoaffinity-purified native or recombinant protein. When tested by ELISA with the native antigen, 79% (26 of 33) of the serum specimens from patients with amebic liver abscess, 4 of 9 serum specimens from symptomatic patients with colitis or ameboma, and serum from one migrant worker were positive. None of the 18 subjects harboring a nonpathogenic strain or 23 control individuals were seropositive to the native antigen (sensitivity, 71%; specificity, 98%). Of 30 serum specimens from patients with amebic liver abscess tested with recombinant antigen, 27 were seropositive (90%). In addition, six patients with colitis or ameboma and two individuals who harbored a nonpathogenic strain were seropositive to the recombinant antigen. One healthy Mexican migrant worker tested positive by both ELISAs (sensitivity, 87%; specificity, 94%). Immunoblotting of 51 serum specimens to sodium dodecyl sulfate-denatured native 29-kDa protein was less sensitive (65%) than ELISA in detecting serum antibodies to the antigen. These results suggest a similar antibody response to native and recombinant antigens (r = 0.86) and support the potential utility of a quantitative assay with defined recombinant antigen for the serodiagnosis of invasive amebiasis in nonendemic areas in conjunction with other diagnostic tools. Images PMID:8314979

Cloning, optimization of induction conditions and purification of Mycobacterium tuberculosis Rv1733c protein expressed in Escherichia coli

PubMed Central

Ashayeri-Panah, Mitra; Eftekhar, Fereshteh; Kazemi, Bahram; Joseph, Joan

2017-01-01

Background and Objectives: Rv1733c is a latency antigen from Mycobacterium tuberculosis, a probable integral-membrane protein with promiscuous T-cell and B-cell epitopes, making it a potential vaccine candidate against tuberculosis. This study aimed to clone and optimize the expression of recombinant Rv1733c in Escherichia coli for purification. Materials and Methods: Chemically synthesized rv1733c coding sequence was cloned in pET-23a(+) followed by transforming E. coli BL21 (DE3) cells. To evaluate the induction conditions for optimized expression, factorial design of experiments was employed using four different media as well as four levels of isopropyl-b-D-thiogalactopyranosid [IPTG] concentration and duration of induction. The recombinant protein was then purified using a His-tag purification kit and detected through western blotting. Results: Recombinant Rv1733c (> 24 kDa) was expressed and accumulated in the cytoplasm of the E. coli cells. Medium composition showed the most significant effect on the yield of the recombinant protein (P = 0.000). The highest yield of recombinant Rv1733c occurred in the presence of 0.4 mM of IPTG in Terrific Broth medium (containing 1.2% tryptone, 2.4% yeast extract, 72 mM K 2 HPO 4 , 17 mM KH 2 PO 4 and 0.4% glycerol) after 10 h at 37°C. Under these conditions, the expression level was around 0.5 g/L of culture medium. Purified Rv1733c was detected by an anti-polyhistidine antibody and a tuberculosis patient’s serum. Systematic optimization of induction conditions gave us high yield of recombinant polyhistidine-tagged Rv1733c in E. coli which was successfuly purified. Conclusion: We believe that the purified Rv1733c recombinant protein from M. tuberculosis might be a good candidate for vaccine production against tuberculosis. PMID:29213997

[The lysate and recombinant antigens in ELISA-test-systems for diagnostic of herpes simplex].

PubMed

Ganova, L A; Kovtoniuk, G V; Korshun, L N; Kiseleva, E K; Tereshchenko, M I; Vudmaska, M I; Moĭsa, L N; Shevchuk, V A; Spivak, N Ia

2014-08-01

The lysate and recombinant antigens of various production included informula of ELISA-test-systems were analyzed. The ELISA-test-systems are used for detection of IgG to Herpes simplex virus type I and II. For testing the panel of serums PTH 201 (BBI Inc.) were used. The samples of this panel contain antibodies to Herpes simplex virus type I and II in mixed titers. The 69 serums of donors were used too (17 samples had IgG to Herpes simplex virus type I, 23 samples to Herpes simplex virus type II and 29 samples had no antibodies to Herpes simplex virus). The diagnostic capacity of mixture of recombinant antigens gG1 Herpes simplex virus type I and gG2 Herpes simplex virus type II (The research-and-production complex "DiaprofMed") was comparable with mixture of lysate antigen Herpes simplex virus type I and II (Membrane) EIE Antigen ("Virion Ltd."). In the test-systems for differentiation of IgG to Herpes simplex virus type I the recombinant antigen gG1 Herpes simplex virus type I proved to be comparable with commercial analogue Herpes simplex virus-1 gG1M ("Viral Therapeutics Inc."'). At the same time, capacity to detect IgG to Herpes simplex virus type II in recombinant protein gG2 Herpes simplex virus type II is significantly higher than in its analogue Herpes simplex virus-2 gG2c ("Viral Therapeutics Inc.").

Defining the proximal border of the Huntington disease candidate region by multipoint recombination analyses

DOE Office of Scientific and Technical Information (OSTI.GOV)

Skraastad, M.I.; De Rooij, K.E.; De Koning Gans, P.A.M.

1993-06-01

The candidate region for the Huntington disease (HD) gene has been narrowed down to a 2.2-Mb region between D4S10 and D4S98 on the short arm of chromosome 4. To map the HD gene within this candidate region 65 Dutch HD families were studied. In total 338 informative meioses were analyzed and 11 multiple informative crossovers were detected. Assuming a minimum number of recombinations and no double recombinations, the multiple informative crossovers are consistent with one specific genetic order for 12 loci: D4S10-(D4S81,D4S126)-D4S125-(D4S127,D4S95)-D4S43-(D4S115, D4S96, D4S111, D4S90, D4S141).This is in agreement with the known data derived from similar and other methods. Themore » loci between brackets could not be mapped relative to each other. In the family material, two informative three-point marker recombination events were detected in the proximal HD candidate region, which are also informative for HD. Both recombination events map the HD gene distal to D4S81 and most likely distal to D4S125, narrowing down the HD candidate region to a 1.7-Mb region between D4S125 and D4S98. 39 refs., 3 figs., 2 tabs.« less

Multiplex detection of pathogen biomarkers in human blood, serum, and saliva using silicon photonic microring resonators

NASA Astrophysics Data System (ADS)

Estrada, I. A.; Burlingame, R. W.; Wang, A. P.; Chawla, K.; Grove, T.; Wang, J.; Southern, S. O.; Iqbal, M.; Gunn, L. C.; Gleeson, M. A.

2015-05-01

Genalyte has developed a multiplex silicon photonic chip diagnostics platform (MaverickTM) for rapid detection of up to 32 biological analytes from a drop of sample in just 10 to 20 minutes. The chips are manufactured with waveguides adjacent to ring resonators, and probed with a continuously variable wavelength laser. A shift in the resonant wavelength as mass binds above the ring resonators is measured and is directly proportional to the amount of bound macromolecules. We present here the ability to multiplex the detection of hemorrhagic fever antigens in whole blood, serum, and saliva in a 16 minute assay. Our proof of concept testing of a multiplex antigencapture chip has the ability to detect Zaire Ebola (ZEBOV) recombinant soluble glycoprotein (rsGP), Marburg virus (MARV) Angola recombinant glycoprotein (rGP) and dengue nonstructural protein I (NS1). In parallel, detection of 2 malaria antigens has proven successful, but has yet to be incorporated into multiplex with the others. Each assay performs with sensitivity ranging from 1.6 ng/ml to 39 ng/ml depending on the antigen detected, and with minimal cross-reactivity.

Recombination in diverse maize is stable, predictable, and associated with genetic load.

PubMed

Rodgers-Melnick, Eli; Bradbury, Peter J; Elshire, Robert J; Glaubitz, Jeffrey C; Acharya, Charlotte B; Mitchell, Sharon E; Li, Chunhui; Li, Yongxiang; Buckler, Edward S

2015-03-24

Among the fundamental evolutionary forces, recombination arguably has the largest impact on the practical work of plant breeders. Varying over 1,000-fold across the maize genome, the local meiotic recombination rate limits the resolving power of quantitative trait mapping and the precision of favorable allele introgression. The consequences of low recombination also theoretically extend to the species-wide scale by decreasing the power of selection relative to genetic drift, and thereby hindering the purging of deleterious mutations. In this study, we used genotyping-by-sequencing (GBS) to identify 136,000 recombination breakpoints at high resolution within US and Chinese maize nested association mapping populations. We find that the pattern of cross-overs is highly predictable on the broad scale, following the distribution of gene density and CpG methylation. Several large inversions also suppress recombination in distinct regions of several families. We also identify recombination hotspots ranging in size from 1 kb to 30 kb. We find these hotspots to be historically stable and, compared with similar regions with low recombination, to have strongly differentiated patterns of DNA methylation and GC content. We also provide evidence for the historical action of GC-biased gene conversion in recombination hotspots. Finally, using genomic evolutionary rate profiling (GERP) to identify putative deleterious polymorphisms, we find evidence for reduced genetic load in hotspot regions, a phenomenon that may have considerable practical importance for breeding programs worldwide.

Local and sex-specific biases in crossover vs. noncrossover outcomes at meiotic recombination hot spots in mice

PubMed Central

de Boer, Esther; Jasin, Maria; Keeney, Scott

2015-01-01

Meiotic recombination initiated by programmed double-strand breaks (DSBs) yields two types of interhomolog recombination products, crossovers and noncrossovers, but what determines whether a DSB will yield a crossover or noncrossover is not understood. In this study, we analyzed the influence of sex and chromosomal location on mammalian recombination outcomes by constructing fine-scale recombination maps in both males and females at two mouse hot spots located in different regions of the same chromosome. These include the most comprehensive maps of recombination hot spots in oocytes to date. One hot spot, located centrally on chromosome 1, behaved similarly in male and female meiosis: Crossovers and noncrossovers formed at comparable levels and ratios in both sexes. In contrast, at a distal hot spot, crossovers were recovered only in males even though noncrossovers were obtained at similar frequencies in both sexes. These findings reveal an example of extreme sex-specific bias in recombination outcome. We further found that estimates of relative DSB levels are surprisingly poor predictors of relative crossover frequencies between hot spots in males. Our results demonstrate that the outcome of mammalian meiotic recombination can be biased, that this bias can vary depending on location and cellular context, and that DSB frequency is not the only determinant of crossover frequency. PMID:26251527

Scedosporium boydii CatA1 and SODC recombinant proteins, new tools for serodiagnosis of Scedosporium infection of patients with cystic fibrosis.

PubMed

Mina, Sara; Staerck, Cindy; Marot, Agnès; Godon, Charlotte; Calenda, Alphonse; Bouchara, Jean-Philippe; Fleury, Maxime J J

2017-12-01

Scedosporium species rank the second among the filamentous fungi colonizing the airways of patients with cystic fibrosis (CF), after Aspergillus fumigatus. In CF, these fungi may cause various respiratory infections similar to those caused by A. fumigatus, including bronchitis and allergic broncho-pulmonary mycoses. Diagnosis of these infections relies on the detection of serum antibodies using crude antigenic extracts. However, many components of these extracts are common to Scedosporium and Aspergillus species, leading to cross-reactions. Here, 5 recombinant proteins from S. apiospermum or S. boydii were produced, and their value in serodiagnosis of Scedosporium infections was investigated by enzyme-linked immunosorbent assay. Two of them, corresponding to the Scedosporium catalase A1 or cytosolic Cu,Zn-superoxyde dismutase, allowed the detection of Scedosporium infection, and the differentiation with an Aspergillus infection. These recombinant proteins therefore may serve as a basis for the development of a standardized serological test. Copyright © 2017 Elsevier Inc. All rights reserved.

Prominent mitochondrial DNA recombination intermediates in human heart muscle.

PubMed

Kajander, O A; Karhunen, P J; Holt, I J; Jacobs, H T

2001-11-01

Recombination intermediates containing four-way (Holliday) junctions are generated during DNA repair and replication in many systems, including yeast mitochondrial DNA (mtDNA). In contrast, convincing evidence for recombination in mammalian mtDNA is lacking. We have used two-dimensional agarose-gel electrophoresis to analyse non-linear forms of mtDNA in human heart muscle. Replication intermediates from both the coupled and strand-asynchronous mtDNA replication pathways were detected. An additional class of non-linear molecules, with the electrophoretic properties of four-way junctions, was also prominent. These molecules were insensitive to topoisomerase I or RNase H, but were diminished by branch migration or RuvC treatment. Junctional molecules were detected in all regions of the mitochondrial genome, were found in myocardial DNA from young and old adults, but were present at lower levels in skeletal muscle and placenta. We suggest that they could represent intermediates of mtDNA repair, given their prevalence in the oxyradical-rich environment of heart muscle mitochondria.

Recombinant VP1 protein as a potential marker for the diagnosis of acute hepatitis A virus infection.

PubMed

da Silva Junior, Haroldo Cid; da Silva, Edimilson Domingos; Lewis-Ximenez de Souza Rodrigues, Lia Laura; Medeiros, Marco Alberto

2017-07-01

Since hepatitis A virus (HAV) production is time-consuming and expensive, the use of recombinant proteins may represent an alternative source of antigens for diagnostic purposes. The present study aimed to express, purify and evaluate the potential of recombinant VP1 protein (rVP1) as a marker for the diagnosis of acute HAV infection. The rVP1 was expressed and purified successfully from Escherichia coli. The purified rVP1 was used to establish an in-house enzyme-linked immunosorbent assay (ELISA-rVP1) for detection of IgM antibodies in sera from HAV-positive patients. For a cut-off point of 0.351, the sensitivity and specificity of ELISA-rVP1 were 100.0% and 95.0%, respectively. These results indicate that rVP1 may be a useful antigen for detection of IgM antibodies against HAV. Copyright © 2017 Elsevier B.V. All rights reserved.

Display of adenoregulin with a novel Pichia pastoris cell surface display system.

PubMed

Ren, Ren; Jiang, Zhengbing; Liu, Meiyun; Tao, Xinyi; Ma, Yushu; Wei, Dongzhi

2007-02-01

Two Pichia pastoris cell surface display vectors were constructed. The vectors consisted of the flocculation functional domain of Flo1p with its own secretion signal sequence or the alpha-factor secretion signal sequence, a polyhistidine (6xHis) tag for detection, an enterokinase recognition site, and the insertion sites for target proteins. Adenoregulin (ADR) is a 33-amino-acid antimicrobial peptide isolated from Phyllomedusa bicolor skin. The ADR was expressed and displayed on the Pichia pastoris KM71 cell surface with the system reported. The displayed recombinant ADR fusion protein was detected by fluorescence microscopy and confocal laser scanning microscopy (CLSM). The antimicrobial activity of the recombinant adenoregulin was detected after proteolytic cleavage of the fusion protein on cell surface. The validity of the Pichia pastoris cell surface display vectors was proved by the displayed ADR.

Bovine Pancreatic Trypsin Inhibitor-Trypsin Complex as a Detection System for Recombinant Proteins

NASA Astrophysics Data System (ADS)

Borjigin, Jimo; Nathans, Jeremy

1993-01-01

Bovine pancreatic trypsin inhibitor (BPTI) binds to trypsin and anhydrotrypsin (an enzymatically inactive derivative of trypsin) with affinities of 6 x 10-14 and 1.1 x 10-13 M, respectively. We have taken advantage of the high affinity and specificity of this binding reaction to develop a protein tagging system in which biotinylated trypsin or biotinylated anhydrotrypsin is used as the reagent to detect recombinant fusion proteins into which BPTI has been inserted. Two proteins, opsin and growth hormone, were used as targets for insertional mutagenesis with BPTI. In each case, both domains of the fusion protein appear to be correctly folded. The fusion proteins can be specifically and efficiently detected by biotinylated trypsin or biotinylated anhydrotrypsin, as demonstrated by staining of transfected cells, protein blotting, affinity purification, and a mobility shift assay in SDS/polyacrylamide gels.

The Role of Recombination in the Origin and Evolution of Alu Subfamilies

PubMed Central

Teixeira-Silva, Ana; Silva, Raquel M.; Carneiro, João; Amorim, António; Azevedo, Luísa

2013-01-01

Alus are the most abundant and successful short interspersed nuclear elements found in primate genomes. In humans, they represent about 10% of the genome, although few are retrotransposition-competent and are clustered into subfamilies according to the source gene from which they evolved. Recombination between them can lead to genomic rearrangements of clinical and evolutionary significance. In this study, we have addressed the role of recombination in the origin of chimeric Alu source genes by the analysis of all known consensus sequences of human Alus. From the allelic diversity of Alu consensus sequences, validated in extant elements resulting from whole genome searches, distinct events of recombination were detected in the origin of particular subfamilies of AluS and AluY source genes. These results demonstrate that at least two subfamilies are likely to have emerged from ectopic Alu-Alu recombination, which stimulates further research regarding the potential of chimeric active Alus to punctuate the genome. PMID:23750218

[Construction of the eukaryotic recombinant vector and expression of the outer membrane protein LipL32 gene from Leptospira serovar Lai].

PubMed

Huang, Bi; Bao, Lang; Zhong, Qi; Shang, Zheng-ling; Zhang, Hui-dong; Zhang, Ying

2008-02-01

To construct the eukaryotic experssion vector of LipL32 gene from Leptospira serovar Lai and express the recombinant plasmid in COS-7 cell. The LipL32 gene was amplified from Leptospira strain 017 genomic DNA by PCR and cloned into pcDNA3.1, through restriction nuclease enzyme digestion. Then the recombinant plasmid was transformed into E.coli DH5alpha. After identified by nuclease digestion, PCR and sequencing analysis, the recombinant vector was transfected into COS-7 cell with lipsome. The expression of the target gene was detected by RT-PCR and Western blot. The eukaryotic experssion vector pcDNA3.1-LipL32 was successfully constructed and stably expressed in COS-7 cell. The eukaryotic recombinant vector of outer membrane protein LipL32 gene from Leptospira serovar Lai can be expressed in mammalian cell, which provides an experimental basis for the application of the Leptospira DNA vaccine.

Cloning and expression of recombinant, functional ricin B chain

DOE Office of Scientific and Technical Information (OSTI.GOV)

Chang, M.S.; Russell, D.W.; Uhr, J.W.

1987-08-01

The cDNA encoding the B chain of the plant toxin ricin has been cloned and expressed in monkey kidney COS-M6 cells. The recombinant B chain was detected by labeling the transfected cells with (/sup 35/S)methionine and (/sup 35/S)-cysteine and demonstrating the secretion of a protein with a M/sub r/ of 30,000-32,000 that was not present in the medium of mock-transfected COS-M6 cells. This protein was specifically immunoprecipitated by an anti-ricin or anti-B-chain antibody and the amount of recombinant B chain secreted by the COS-M6 cells was determined by a radioimmunoassay. Virtually all of the recombinant B chain formed active ricinmore » when mixed with native A chain; it could also bind to the galactose-containing glycoprotein asialofetuin as effectively as native B chain.These results indicate that the vast majority of recombinant B chains secreted into the medium of the COS-M6 cells retain biological function« less

Strand invasion structures in the inverted repeat of Candida albicans mitochondrial DNA reveal a role for homologous recombination in replication.

PubMed

Gerhold, Joachim M; Aun, Anu; Sedman, Tiina; Jõers, Priit; Sedman, Juhan

2010-09-24

Molecular recombination and transcription are proposed mechanisms to initiate mitochondrial DNA (mtDNA) replication in yeast. We conducted a comprehensive analysis of mtDNA from the yeast Candida albicans. Two-dimensional agarose gel electrophoresis of mtDNA intermediates reveals no bubble structures diagnostic of specific replication origins, but rather supports recombination-driven replication initiation of mtDNA in yeast. Specific species of Y structures together with DNA copy number analyses of a C. albicans mutant strain provide evidence that a region in a mainly noncoding inverted repeat is predominantly involved in replication initiation via homologous recombination. Our further findings show that the C. albicans mtDNA forms a complex branched network that does not contain detectable amounts of circular molecules. We provide topological evidence for recombination-driven mtDNA replication initiation and introduce C. albicans as a suitable model organism to study wild-type mtDNA maintenance in yeast. Copyright © 2010 Elsevier Inc. All rights reserved.

HIV genetic diversity in Cameroon: possible public health importance.

PubMed

Ndongmo, Clement B; Pieniazek, Danuta; Holberg-Petersen, Mona; Holm-Hansen, Carol; Zekeng, Leopold; Jeansson, Stig L; Kaptue, Lazare; Kalish, Marcia L

2006-08-01

To monitor the evolving molecular epidemiology and genetic diversity of HIV in a country where many distinct strains cocirculate, we performed genetic analyses on sequences from 75 HIV-1-infected Cameroonians: 74 were group M and 1 was group O. Of the group M sequences, 74 were classified into the following env gp41 subtypes or recombinant forms: CRF02 (n = 54), CRF09 (n = 2), CRF13 (n = 2), A (n = 5), CRF11 (n = 4), CRF06 (n = 1), G (n = 2), F2 (n = 2), and E (n = 1, CRF01), and 1 was a JG recombinant. Comparison of phylogenies for 70 matched gp41 and protease sequences showed inconsistent classifications for 18 (26%) strains. Our data show that recombination is rampant in Cameroon with recombinant viruses continuing to recombine, adding to the complexity of circulating HIV strains. This expanding genetic diversity raises public health concerns for the ability of diagnostic assays to detect these unique HIV mosaic variants and for the development of broadly effective HIV vaccines.

The Analytical Chemistry of Drug Monitoring in Athletes

NASA Astrophysics Data System (ADS)

Bowers, Larry D.

2009-07-01

The detection and deterrence of the abuse of performance-enhancing drugs in sport are important to maintaining a level playing field among athletes and to decreasing the risk to athletes’ health. The World Anti-Doping Program consists of six documents, three of which play a role in analytical development: The World Anti-Doping Code, The List of Prohibited Substances and Methods, and The International Standard for Laboratories. Among the classes of prohibited substances, three have given rise to the most recent analytical developments in the field: anabolic agents; peptide and protein hormones; and methods to increase oxygen delivery to the tissues, including recombinant erythropoietin. Methods for anabolic agents, including designer steroids, have been enhanced through the use of liquid chromatography/tandem mass spectrometry and gas chromatography/combustion/isotope-ratio mass spectrometry. Protein and peptide identification and quantification have benefited from advances in liquid chromatography/tandem mass spectrometry. Incorporation of techniques such as flow cytometry and isoelectric focusing have supported the detection of blood doping.

Mechanisms and Regulation of Mitotic Recombination in Saccharomyces cerevisiae

PubMed Central

Symington, Lorraine S.; Rothstein, Rodney; Lisby, Michael

2014-01-01

Homology-dependent exchange of genetic information between DNA molecules has a profound impact on the maintenance of genome integrity by facilitating error-free DNA repair, replication, and chromosome segregation during cell division as well as programmed cell developmental events. This chapter will focus on homologous mitotic recombination in budding yeast Saccharomyces cerevisiae. However, there is an important link between mitotic and meiotic recombination (covered in the forthcoming chapter by Hunter et al. 2015) and many of the functions are evolutionarily conserved. Here we will discuss several models that have been proposed to explain the mechanism of mitotic recombination, the genes and proteins involved in various pathways, the genetic and physical assays used to discover and study these genes, and the roles of many of these proteins inside the cell. PMID:25381364

A novel variant of the infectious bronchitis virus resulting from recombination events in Italy and Spain.

PubMed

Moreno, Ana; Franzo, G; Massi, P; Tosi, G; Blanco, A; Antilles, N; Biarnes, M; Majó, N; Nofrarías, M; Dolz, R; Lelli, D; Sozzi, E; Lavazza, A; Cecchinato, M

2017-02-01

Infectious bronchitis is considered to be one of the most devastating diseases in poultry. Control of its spread is typically attempted through biosecurity measures and extensive vaccination. However, the remarkable genetic and antigenic variability of the virus, which originate from both mutations and recombination events, represents an unsolved challenge for this disease. The present study reports on the emergence and spread of recombinant clusters detected in Italy and Spain between 2012 and 2014. A total of 36 Spanish and Italian infectious bronchitis virus (IBV) field strains were investigated and genetically characterized using phylogenetic, molecular, recombination and selection pressure analyses of the complete S1 gene. Based on the partial S1 sequencing, 27 IBV strains originating from Spain and nine from Italy were initially classified as being closely related to the Guandong/Xindadi (XDN) genotype. Phylogenetic analysis of the complete S1 gene revealed that the XDN strains formed a homogeneous clade with the Spanish IBV isolates within the QX genotype, whereas there was higher variability within the Italian strains. Recombination analysis determined that these strains belonged to four groups, which originated from independent recombination events between the QX and 793B IBV genotypes. Our data support the hypothesis of two different scenarios: firstly, in Spain, the large and homogeneous clade probably originated from a single offspring of the recombinant founder, which became dominant and spread throughout the country. Secondly, the nine Italian recombinants, which are characterized by three different recombination patterns, probably represent less fitted strains, because they were less viable with respect to their recombinant parents.

A novel recombinant pseudorabies virus expressing parvovirus VP2 gene: Immunogenicity and protective efficacy in swine

PubMed Central

2011-01-01

Background Porcine parvovirus (PPV) VP2 gene has been successfully expressed in many expression systems resulting in self-assembly of virus-like particles (VLPs) with similar morphology to the native capsid. Here, a pseudorabies virus (PRV) system was adopted to express the PPV VP2 gene. Methods A recombinant PRV SA215/VP2 was obtained by homologous recombination between the vector PRV viral DNA and a transfer plasmid. Then recombinant virus was purified with plaque purification, and its identity confirmed by PCR amplification, Western blot and indirect immunofluorescence (IFA) analyses. Electronic microscopy of PRV SA215/VP2 confirmed self-assembly of both pseudorabies virus and VLPs from VP2 protein. Results Immunization of piglets with recombinant virus elicited PRV-specific and PPV-specific humoral immune responses and provided complete protection against a lethal dose of PRV challenges. Gilts immunized with recombinant viruses induced PPV-specific antibodies, and significantly reduced the mortality rate of (1 of 28) following virulent PPV challenge compared with the control (7 of 31). Furthermore, PPV virus DNA was not detected in the fetuses of recombinant virus immunized gilts. Conclusions In this study, a recombinant PRV SA215/VP2 virus expressing PPV VP2 protein was constructed using PRV SA215 vector. The safety, immunogenicity, and protective efficacy of the recombinant virus were demonstrated in piglets and primiparous gilts. This recombinant PRV SA215/VP2 represents a suitable candidate for the development of a bivalent vaccine against both PRV and PPV infection. PMID:21679423

Identification and analysis of recombineering functions from Gram-negative and Gram-positive bacteria and their phages

PubMed Central

Datta, Simanti; Costantino, Nina; Zhou, Xiaomei; Court, Donald L.

2008-01-01

We report the identification and functional analysis of nine genes from Gram-positive and Gram-negative bacteria and their phages that are similar to lambda (λ) bet or Escherichia coli recT. Beta and RecT are single-strand DNA annealing proteins, referred to here as recombinases. Each of the nine other genes when expressed in E. coli carries out oligonucleotide-mediated recombination. To our knowledge, this is the first study showing single-strand recombinase activity from diverse bacteria. Similar to bet and recT, most of these other recombinases were found to be associated with putative exonuclease genes. Beta and RecT in conjunction with their cognate exonucleases carry out recombination of linear double-strand DNA. Among four of these foreign recombinase/exonuclease pairs tested for recombination with double-strand DNA, three had activity, albeit barely detectable. Thus, although these recombinases can function in E. coli to catalyze oligonucleotide recombination, the double-strand DNA recombination activities with their exonuclease partners were inefficient. This study also demonstrated that Gam, by inhibiting host RecBCD nuclease activity, helps to improve the efficiency of λ Red-mediated recombination with linear double-strand DNA, but Gam is not absolutely essential. Thus, in other bacterial species where Gam analogs have not been identified, double-strand DNA recombination may still work in the absence of a Gam-like function. We anticipate that at least some of the recombineering systems studied here will potentiate oligonucleotide and double-strand DNA-mediated recombineering in their native or related bacteria. PMID:18230724

[Eukaryotic Expression and Immunogenic Research of Recombination Ebola Virus Membrane Protein Gp-Fc].

PubMed

Zhang, Xiaoguang; Yang, Ren; Wang, Jiao; Wang, Xuan; Hou, Mieling; An, Lina; Zhu, Ying; Cao, Yuxi; Zeng, Yi

2016-01-01

We used 293 cells to express the recombinant membrane protein of the Ebola virus. Then, the immunogenicity of the recombinant protein was studied by immunized BALB/c mice. According to the codon use frequency of humans, the gene encoding the extracellular domain of the Ebola virus membrane protein was optimized, synthesized, and inserted into the eukaryotic expression plasmid pXG-Fc to construct the human IgG Fc and Ebola GP fusion protein expression plasmid pXG-modGP-Fc. To achieve expression, the fusion protein expression vector was transfected into high-density 293 cells using transient transfection technology. The recombinant protein was purified by protein A affinity chromatography. BALB/c mice were immunized with the purified fusion protein, and serum antibody titers evaluated by an indirect enzyme-linked immunosorbent assay (ELISA). Purification and analyses of the protein revealed that the eukaryotic expression vector could express the recombinant protein GP-Fc effectively, and that the recombinant protein in the supernatant of the cell culture was present as a dimer. After immunization with the purified recombinant protein, a high titer of antigen-specific IgG could be detected in the serum of immunized mice by indirect ELISA, showing that the recombinant protein had good immunogenicity. These data suggest that we obtained a recombinant protein with good immunogenicity. Our study is the basis for development of a vaccine against the Ebola virus and for screening of monoclonal antibodies.

[Construction and identification of recombinant human platelet-derived growth factor-B adenoviral vector and transfection into periodontal ligament stem cells].

PubMed

Shang, Shu-huan; Zhang, Yu-feng; Shi, Bin; Cheng, Xiang-rong

2008-10-01

To construct a recombinant human platelet-derived growth factor-B (PDGF-B) adenoviral vector and to transfect it into human periodontal ligament stem cells (PDLSC). The recombinant plasmid pAd-PDGF-B was constructed by homologous recombination and confirmed by restriction endonucleases digestion. Recombinant adenovirus was packaged in HEK293 cells. PDLSC were transfected with recombinant adenovirus and PDGF-B expression was confirmed. Expression of collagen type I gene was determined by quantitative analysis of the products of RT-PCR. The cell proliferation was determined with MTT colorimetric assay. The recombinant plasmid pAd-PDGF-B was confirmed by restriction endonucleases digestion. EGFP expression was observed on the third day after transfecting, and the expression of PDGF-B was detected. Immunohistochemical methods revealed that PDGF-B was expressed in PDLSC. Levels of expression of collagen type I gene were increased significantly by transfer of the exogenous PDGF-B gene to PDLSC. At the same time, findings indicated that Ad-PDGF-B stimulated PDLSC proliferation. MTT assay indicated the absorbance of PDLSC by stimulating with Ad-PDGF-B was (0.68 +/- 0.02), P < 0.01. Using the AdEasy system, the human PDGF-B recombinant adenovirus can be rapidly obtained. These results indicate that recombinant adenoviruses encoding PDGF-B transgenes could modulate proliferative activity of PDLSC, enhance the high expression of collagen type I and lay the foundation for periodontal tissue regeneration and dental implant gene therapy.

Cloning and Expressing Recombinant Protective Antigen Domains of B. anthracis

DTIC Science & Technology

2011-09-01

future predictive modeling toolkits. 1 1. Introduction The use of Bacillus anthracis as a bio - weapon in the United States in 2001 affirmed the need...for improved sensing and detection of biological weapons of mass destruction (WMD). Protective Antigen (PA) protein of Bacillus anthracis is the...Cloning and Expressing Recombinant Protective Antigen Domains of B. anthracis by Deborah A. Sarkes, Joshua M. Kogot, Irene Val-Addo

When do letter features migrate? A boundary condition for feature-integration theory.

PubMed

Butler, B E; Mewhort, D J; Browse, R A

1991-01-01

Feature-integration theory postulates that a lapse of attention will allow letter features to change position and to recombine as illusory conjunctions (Treisman & Paterson, 1984). To study such errors, we used a set of uppercase letters known to yield illusory conjunctions in each of three tasks. The first, a bar-probe task, showed whole-character mislocations but not errors based on feature migration and recombination. The second, a two-alternative forced-choice detection task, allowed subjects to focus on the presence or absence of subletter features and showed illusory conjunctions based on feature migration and recombination. The third was also a two-alternative forced-choice detection task, but we manipulated the subjects' knowledge of the shape of the stimuli: In the case-certain condition, the stimuli were always in uppercase, but in the case-uncertain condition, the stimuli could appear in either upper- or lowercase. Subjects in the case-certain condition produced illusory conjunctions based on feature recombination, whereas subjects in the case-uncertain condition did not. The results suggest that when subjects can view the stimuli as feature groups, letter features regroup as illusory conjunctions; when subjects encode the stimuli as letters, whole items may be mislocated, but subletter features are not. Thus, illusory conjunctions reflect the subject's processing strategy, rather than the architecture of the visual system.

Bloom syndrome helicase in meiosis: Pro-crossover functions of an anti-crossover protein.

PubMed

Hatkevich, Talia; Sekelsky, Jeff

2017-09-01

The functions of the Bloom syndrome helicase (BLM) and its orthologs are well characterized in mitotic DNA damage repair, but their roles within the context of meiotic recombination are less clear. In meiotic recombination, multiple repair pathways are used to repair meiotic DSBs, and current studies suggest that BLM may regulate the use of these pathways. Based on literature from Saccharomyces cerevisiae, Arabidopsis thaliana, Mus musculus, Drosophila melanogaster, and Caenorhabditis elegans, we present a unified model for a critical meiotic role of BLM and its orthologs. In this model, BLM and its orthologs utilize helicase activity to regulate the use of various pathways in meiotic recombination by continuously disassembling recombination intermediates. This unwinding activity provides the meiotic program with a steady pool of early recombination substrates, increasing the probability for a DSB to be processed by the appropriate pathway. As a result of BLM activity, crossovers are properly placed throughout the genome, promoting proper chromosomal disjunction at the end of meiosis. This unified model can be used to further refine the complex role of BLM and its orthologs in meiotic recombination. © 2017 WILEY Periodicals, Inc.

Homologous genetic recombination in the yellow head complex of nidoviruses infecting Penaeus monodon shrimp.

PubMed

Wijegoonawardane, Priyanjalie K M; Sittidilokratna, Nusra; Petchampai, Natthida; Cowley, Jeff A; Gudkovs, Nicholas; Walker, Peter J

2009-07-20

Yellow head virus (YHV) is a highly virulent pathogen of Penaeus monodon shrimp. It is one of six known genotypes in the yellow head complex of nidoviruses which also includes mildly pathogenic gill-associated virus (GAV, genotype 2) and four other genotypes (genotypes 3-6) that have been detected only in healthy shrimp. In this study, comparative phylogenetic analyses conducted on replicase- (ORF1b) and glycoprotein- (ORF3) gene amplicons identified 10 putative natural recombinants amongst 28 viruses representing all six genotypes from across the Indo-Pacific region. The approximately 4.6 kb genomic region spanning the two amplicons was sequenced for three putative recombinant viruses from Vietnam (genotype 3/5), the Philippines (genotype 5/2) and Indonesia (genotype 3/2). SimPlot analysis using these and representative parental virus sequences confirmed that each was a recombinant genotype and identified a recombination hotspot in a region just upstream of the ORF1b C-terminus. Maximum-likelihood breakpoint analysis predicted identical crossover positions in the Vietnamese and Indonesian recombinants, and a crossover position 12 nt upstream in the Philippine recombinant. Homologous genetic recombination in the same genome region was also demonstrated in recombinants generated experimentally in shrimp co-infected with YHV and GAV. The high frequency with which natural recombinants were identified indicates that genetic exchange amongst genotypes is occurring commonly in Asia and playing a significant role in expanding the genetic diversity in the yellow head complex. This is the first evidence of genetic recombination in viruses infecting crustaceans and has significant implications for the pathogenesis of infection and diagnosis of these newly emerging invertebrate pathogens.

Effects of Inversions on Within- and Between-Species Recombination and Divergence

PubMed Central

Stevison, Laurie S.; Hoehn, Kenneth B.; Noor, Mohamed A. F.

2011-01-01

Chromosomal inversions disrupt recombination in heterozygotes by both reducing crossing-over within inverted regions and increasing it elsewhere in the genome. The reduction of recombination in inverted regions facilitates the maintenance of hybridizing species, as outlined by various models of chromosomal speciation. We present a comprehensive comparison of the effects of inversions on recombination rates and on nucleotide divergence. Within an inversion differentiating Drosophila pseudoobscura and Drosophila persimilis, we detected one double recombinant among 9,739 progeny from F1 hybrids screened, consistent with published double-crossover frequencies observed within species. Despite similar rates of exchange within and between species, we found no sequence-based evidence of ongoing gene exchange between species within this inversion, but significant exchange was inferred within species. We also observed greater differentiation at regions near inversion breakpoints between species versus within species. Moreover, we observed strong “interchromosomal effect” (higher recombination in inversion heterozygotes between species) with up to 9-fold higher recombination rates along collinear segments of chromosome two in hybrids. Further, we observed that regions most susceptible to changes in recombination rates corresponded to regions with lower recombination rates in homokaryotypes. Finally, we showed that interspecies nucleotide divergence is lower in regions with greater increases in recombination rate, potentially resulting from greater interspecies exchange. Overall, we have identified several similarities and differences between inversions segregating within versus between species in their effects on recombination and divergence. We conclude that these differences are most likely due to lower frequency of heterokaryotypes and to fitness consequences from the accumulation of various incompatibilities between species. Additionally, we have identified possible effects of inversions on interspecies gene exchange that had not been considered previously. PMID:21828374

[Experimental study of human umbilical cord blood derived stromal cells transfected with recombinant adenoviral vector co-expressing VCAM-1 and GFP].

PubMed

Zhang, Xi; Si, Ying-Jian; Chen, Xing-Hua; Liu, Yao; Gao, Li; Gao, Lei; Peng, Xian-Gui; Wang, Qing-Yu

2008-06-01

This study was aimed to investigate the effect of vcam-1 gene-modified human umbilical cord blood derived stromal cells (CBDSCs) on hematopoietic regulation so as to establish the experimental foundation for further study. The target gene vcam-1 was cloned into the shuttle plasmid with the report gene GFP. The recombinant shuttle plasmid was transformed into BJ5183 bacteria to recombine with backbone vector pAdeasy-l, and the recombinant adenoviral vector ad-vcam-1-gfp was confirmed after transfection with CBDSCs. The results indicated that two fragments of about 9 kb and 2 kb were obtained after digestion of recombinant plasmid pAdTrack-vcam-1 with NotIand XhoI, and single fragment of 600 bp was obtained after amplification with PCR; two fragments of about 31 kb and 4 kb were obtained after digestion of recombinant plasmid pad-vcam-1-gfp with PacI, which suggested a successful homologous recombination. The expression of vcam-1 gene in ad-vcam-1-gfp transfected CBDSCs could be detected by immunocytochemistry, RT-PCR and fluorescent microscopy. It is concluded that the recombinant adenoviral vector ad-vcam-1-gfp has been constructed successfully, and the expression of vcam-1 is up-regulated in CBDSCs transfected by gene ad-vcam-1-gfp.

Sex-chromosome differentiation parallels postglacial range expansion in European tree frogs (Hyla arborea).

PubMed

Dufresnes, Christophe; Bertholet, Youna; Wassef, Jérôme; Ghali, Karim; Savary, Romain; Pasteur, Baptiste; Brelsford, Alan; Rozenblut-Kościsty, Beata; Ogielska, Maria; Stöck, Matthias; Perrin, Nicolas

2014-12-01

Occasional XY recombination is a proposed explanation for the sex-chromosome homomorphy in European tree frogs. Numerous laboratory crosses, however, failed to detect any event of male recombination, and a detailed survey of NW-European Hyla arborea populations identified male-specific alleles at sex-linked loci, pointing to the absence of XY recombination in their recent history. Here, we address this paradox in a phylogeographic framework by genotyping sex-linked microsatellite markers in populations and sibships from the entire species range. Contrasting with postglacial populations of NW Europe, which display complete absence of XY recombination and strong sex-chromosome differentiation, refugial populations of the southern Balkans and Adriatic coast show limited XY recombination and large overlaps in allele frequencies. Geographically and historically intermediate populations of the Pannonian Basin show intermediate patterns of XY differentiation. Even in populations where X and Y occasionally recombine, the genetic diversity of Y haplotypes is reduced below the levels expected from the fourfold drop in copy numbers. This study is the first in which X and Y haplotypes could be phased over the distribution range in a species with homomorphic sex chromosomes; it shows that XY-recombination patterns may differ strikingly between conspecific populations, and that recombination arrest may evolve rapidly (<5000 generations). © 2014 The Author(s). Evolution © 2014 The Society for the Study of Evolution.

Health Reports

DTIC Science & Technology

1992-11-01

Food and Drug Recombinant Bovine Growth Hormone: FDA Approval Should Be Withheld Administration Until the Mastitis Issue Is Resolved (Report, Aug. 6...Care: Demonstration Project Concerning Future Structure of Veterans’ Health Program (Testimony, Aug. 11, 1992, GA(Yr-HRD-92-53). Recombinant Bovine ...Growth Hormone: FDA Approval Should Be Withheld Until the Mastitis Issue Is Resolved (Report, Aug. 6, 1992, GAO/PEMD-92-26). Page 10 GAD/RRD-98-38 Health

Development and Characterization of Monoclonal Antibodies to Yellow Fever Virus and Application in Antigen Detection and IgM Capture Enzyme-Linked Immunosorbent Assay

PubMed Central

Adungo, Ferdinard; Kamau, David; Inoue, Shingo; Hayasaka, Daisuke; Posadas-Herrera, Guillermo; Sang, Rosemary; Mwau, Matilu

2016-01-01

Yellow fever (YF) is an acute hemorrhagic viral infection transmitted by mosquitoes in Africa and South America. The major challenge in YF disease detection and confirmation of outbreaks in Africa is the limited availability of reference laboratories and the persistent lack of access to diagnostic tests. We used wild-type YF virus sequences to generate recombinant envelope protein in an Escherichia coli expression system. Both the recombinant protein and sucrose gradient-purified YF vaccine virus 17D (YF-17D) were used to immunize BALB/c mice to generate monoclonal antibodies (MAbs). Eight MAbs were established and systematically characterized by indirect enzyme-linked immunosorbent assay (ELISA), Western blot analysis, and immunofluorescence assay (IFA). The established MAbs showed strong reactivity with wild-type YF virus and recombinant protein with no detectable cross-reactivity to dengue virus or Japanese encephalitis virus. Epitope mapping showed strong binding of three MAbs to amino acid positions 1 to 51, while two MAbs mapped to amino acid positions 52 to 135 of the envelope protein. The remaining three MAbs did not show reactivity to envelope fragments. The established MAbs exert no neutralization against wild-type YF and 17D viruses (titer of <10 for both strains). The applicability of MAbs 8H3 and 3F4 was further evaluated using IgM capture ELISA. A total of 49 serum samples were analyzed, among which 12 positive patient and vaccinee samples were correctly identified. Using serum samples that were 2-fold serially diluted, the IgM capture ELISA was able to detect all YF-positive samples. Furthermore, MAb-based antigen detection ELISA enabled the detection of virus in culture supernatants containing titers of about 1,000 focus-forming units. PMID:27307452

Development and Use of an Internal Positive Control for Detection of Bordetella pertussis by PCR

PubMed Central

Herwegh, Stéphanie; Carnoy, Christophe; Wallet, Frédéric; Loïez, Caroline; Courcol, René J.

2005-01-01

An internal control of amplification was constructed by recombinant PCR to detect PCR inhibitors. This exogenous DNA was included in the reaction mixture and coamplified with the target gene. This detection was successfully applied to the diagnosis of whooping cough by amplification of a fragment of Bordetella pertussis IS481. PMID:15872283

Evaluation of a magnetic microsphere antibody detection assay for the investigation of exposure to Cryptosporidium parvum and Cryptosporidium hominis

EPA Science Inventory

Anti-Cryptosporidium IgA and IgG are useful markers of exposure to Cryptosporidium in human populations, but detection in saliva may be difficult. To evaluate a magnetic microsphere assay for detection of anti-Cryptosporidium IgA and IgG in saliva, recombinant sporozoite gp15 (1...

Recent Recombination Events in the Core Genome Are Associated with Adaptive Evolution in Enterococcus faecium

PubMed Central

de Been, Mark; van Schaik, Willem; Cheng, Lu; Corander, Jukka; Willems, Rob J.

2013-01-01

Reasons for the rising clinical impact of the bacterium Enterococcus faecium include the species’ rapid acquisition of adaptive genetic elements. Here, we focused on the impact of recombination on the evolution of E. faecium. We used the recently developed BratNextGen algorithm to detect recombinant regions in the core genome of 34 E. faecium strains, including three newly sequenced clinical strains. Recombination was found to have a significant impact on the E. faecium genome: of the original 1.2 million positions in the core genome, 0.5 million were predicted to have been affected by recombination in at least one strain. Importantly, strains in one of the two major E. faecium clades (clade B), which contains most of the E. faecium human gut commensals, formed the most important reservoir for donating foreign DNA to the second major E. faecium clade (clade A), which contains most of the clinical isolates. Also, several genomic regions were found to mainly recombine in specific hospital-associated E. faecium strains. One of these regions (the epa-like locus) likely encodes the biosynthesis of cell wall polysaccharides. These findings suggest a crucial role for recombination in the emergence of E. faecium as a successful hospital-associated pathogen. PMID:23882129

Coexistence of minicircular and a highly rearranged mtDNA molecule suggests that recombination shapes mitochondrial genome organization.

PubMed

Mao, Meng; Austin, Andrew D; Johnson, Norman F; Dowton, Mark

2014-03-01

Recombination has been proposed as a possible mechanism to explain mitochondrial (mt) gene rearrangements, although the issue of whether mtDNA recombination occurs in animals has been controversial. In this study, we sequenced the entire mt genome of the megaspilid wasp Conostigmus sp., which possessed a highly rearranged mt genome. The sequence of the A+T-rich region contained a number of different types of repeats, similar to those reported previously in the nematode Meloidogyne javanica, in which recombination was discovered. In Conostigmus, we detected the end products of recombination: a range of minicircles. However, using isolated (cloned) fragments of the A+T-rich region, we established that some of these minicircles were found to be polymerase chain reaction (PCR) artifacts. It appears that regions with repeats are prone to PCR template switching or PCR jumping. Nevertheless, there is strong evidence that one minicircle is real, as amplification primers that straddle the putative breakpoint junction produce a single strong amplicon from genomic DNA but not from the cloned A+T-rich region. The results provide support for the direct link between recombination and mt gene rearrangement. Furthermore, we developed a model of recombination which is important for our understanding of mtDNA evolution.

Detection of African Swine Fever Virus Antibodies in Serum and Oral Fluid Specimens Using a Recombinant Protein 30 (p30) Dual Matrix Indirect ELISA.

PubMed

Giménez-Lirola, Luis G; Mur, Lina; Rivera, Belen; Mogler, Mark; Sun, Yaxuan; Lizano, Sergio; Goodell, Christa; Harris, D L Hank; Rowland, Raymond R R; Gallardo, Carmina; Sánchez-Vizcaíno, José Manuel; Zimmerman, Jeff

2016-01-01

In the absence of effective vaccine(s), control of African swine fever caused by African swine fever virus (ASFV) must be based on early, efficient, cost-effective detection and strict control and elimination strategies. For this purpose, we developed an indirect ELISA capable of detecting ASFV antibodies in either serum or oral fluid specimens. The recombinant protein used in the ELISA was selected by comparing the early serum antibody response of ASFV-infected pigs (NHV-p68 isolate) to three major recombinant polypeptides (p30, p54, p72) using a multiplex fluorescent microbead-based immunoassay (FMIA). Non-hazardous (non-infectious) antibody-positive serum for use as plate positive controls and for the calculation of sample-to-positive (S:P) ratios was produced by inoculating pigs with a replicon particle (RP) vaccine expressing the ASFV p30 gene. The optimized ELISA detected anti-p30 antibodies in serum and/or oral fluid samples from pigs inoculated with ASFV under experimental conditions beginning 8 to 12 days post inoculation. Tests on serum (n = 200) and oral fluid (n = 200) field samples from an ASFV-free population demonstrated that the assay was highly diagnostically specific. The convenience and diagnostic utility of oral fluid sampling combined with the flexibility to test either serum or oral fluid on the same platform suggests that this assay will be highly useful under the conditions for which OIE recommends ASFV antibody surveillance, i.e., in ASFV-endemic areas and for the detection of infections with ASFV isolates of low virulence.

Intravital Fluorescence Excitation in Whole-Animal Optical Imaging.

PubMed

Nooshabadi, Fatemeh; Yang, Hee-Jeong; Bixler, Joel N; Kong, Ying; Cirillo, Jeffrey D; Maitland, Kristen C

2016-01-01

Whole-animal fluorescence imaging with recombinant or fluorescently-tagged pathogens or cells enables real-time analysis of disease progression and treatment response in live animals. Tissue absorption limits penetration of fluorescence excitation light, particularly in the visible wavelength range, resulting in reduced sensitivity to deep targets. Here, we demonstrate the use of an optical fiber bundle to deliver light into the mouse lung to excite fluorescent bacteria, circumventing tissue absorption of excitation light in whole-animal imaging. We present the use of this technology to improve detection of recombinant reporter strains of tdTomato-expressing Mycobacterium bovis BCG (Bacillus Calmette Guerin) bacteria in the mouse lung. A microendoscope was integrated into a whole-animal fluorescence imager to enable intravital excitation in the mouse lung with whole-animal detection. Using this technique, the threshold of detection was measured as 103 colony forming units (CFU) during pulmonary infection. In comparison, the threshold of detection for whole-animal fluorescence imaging using standard epi-illumination was greater than 106 CFU.

Intravital Fluorescence Excitation in Whole-Animal Optical Imaging

PubMed Central

Bixler, Joel N.; Kong, Ying; Cirillo, Jeffrey D.; Maitland, Kristen C.

2016-01-01

Whole-animal fluorescence imaging with recombinant or fluorescently-tagged pathogens or cells enables real-time analysis of disease progression and treatment response in live animals. Tissue absorption limits penetration of fluorescence excitation light, particularly in the visible wavelength range, resulting in reduced sensitivity to deep targets. Here, we demonstrate the use of an optical fiber bundle to deliver light into the mouse lung to excite fluorescent bacteria, circumventing tissue absorption of excitation light in whole-animal imaging. We present the use of this technology to improve detection of recombinant reporter strains of tdTomato-expressing Mycobacterium bovis BCG (Bacillus Calmette Guerin) bacteria in the mouse lung. A microendoscope was integrated into a whole-animal fluorescence imager to enable intravital excitation in the mouse lung with whole-animal detection. Using this technique, the threshold of detection was measured as 103 colony forming units (CFU) during pulmonary infection. In comparison, the threshold of detection for whole-animal fluorescence imaging using standard epi-illumination was greater than 106 CFU. PMID:26901051

Development and evaluation of a truncated recombinant NS3 antigen-based indirect ELISA for detection of pestivirus antibodies in sheep and goats.

PubMed

Kalaiyarasu, Semmannan; Mishra, Niranjan; Rajukumar, Katherukamem; Nema, Ram Kumar; Behera, Sthita Pragnya

2015-01-01

The aim of this study was to develop an indirect ELISA using the helicase domain of bovine viral diarrhoea virus (BVDV) NS3 protein instead of full-length NS3 protein for detection of BVDV and BDV antibodies in sheep and goats and its validation by comparing its sensitivity and specificity with virus neutralization test (VNT) as the reference test. The purified 50 kDa recombinant NS3 protein was used as the coating antigen in the ELISA. The optimal concentration of antigen was 320 ng/well at a serum dilution of 1:20 and the optimal positive cut-off optical density value was 0.40 based on test results of 418 VNT negative sheep and goat sera samples. When 569 serum samples from sheep (463) and goats (106) were tested, the ELISA showed a sensitivity of 91.71% and specificity of 94.59% with BVDV VNT. A good correlation (93.67%) was observed between the two tests. It showed a sensitivity of 85% and specificity of 86.6% with VNT in detecting BDV antibody positive or negative samples. This study demonstrates the efficacy of truncated recombinant NS3 antigen based ELISA for seroepidemiological study of pestivirus infection in sheep and goats.

Experimental reinfection of BALB/c mice with different recombinant type I/III strains of Toxoplasma gondii: involvement of IFN-gamma and IL-10.

PubMed

Brandão, Geane Peroni; Melo, Maria Norma; Gazzinelli, Ricardo Tostes; Caetano, Braulia Costa; Ferreira, Adriana Melo; Silva, Letícia Azevedo; Vitor, Ricardo Wagner Almeida

2009-03-01

To assess reinfection of BALB/c mice with different Toxoplasma gondii strains, the animals were prime infected with the non-virulent D8 strain and challenged with virulent recombinant strains. Thirty days after challenge, brain cysts were obtained from surviving BALB/c mice and inoculated in Swiss mice to obtain tachyzoites for DNA extraction and PCR-RFLP analysis to distinguish the different T. gondii strains present in possible co-infections. Anti-Toxoplasma immune responses were evaluated in D8-primed BALB/c mice by detecting IFN-gamma and IL-10 produced by T cells and measuring immunoglobulin levels in serum samples. PCR-RFLP demonstrated that BALB/c mice were reinfected with the EGS strain at 45 days post prime infection (dpi) and with the EGS and CH3 strains at 180 dpi. High levels of IFN-gamma were detected after D8 infection, with no significant difference between 45 and 180-day intervals. However, higher IL-10 levels and higher plasmatic IgG1 and IgA were detected from samples obtained 180 days after infection. BALB/c mice were susceptible to reinfection with different recombinant T. gondii strains and this susceptibility correlated with enhancement of IL-10 production.

[Identification of occult disseminated tumor cells by recombinant herpes simplex virus expressing GFP (HSV(GFP))].

PubMed

Han, Xiang-ping; Shi, Gui-lan; Wang, Cheng-feng; Li, Jie; Zhang, Jian-wei; Zhang, Yu; Zhang, Shu-ren; Liu, Bin-lei

2012-12-01

To develop a novel rapid protocol for the detection of occult disseminated tumor cells by a recombinant herpes simplex virus expressing GFP (HSV(GFP)). Tumor cells of seven cell lines were exposed to HSV(GFP) and then examined for GFP expression by fluorescence microscopy. Various numbers of tumor cells (10, 100, 1000, 10 000) were mixed into 2 ml human whole blood, separated with lymphocytes separation medium, exposed to HSV(GFP), incubated at 37°C for 6 - 24 h and then counted for the number of green cells under the fluorescence microscope. Some clinical samples including peripheral blood, pleural effusion, ascites, spinal fluid from tumor-bearing patients were screened using this protocol in parallel with routine cytological examination. HSV(GFP) was able to infect all 7 tumor cell lines indicating that the HSV(GFP) can be used to detect different types of tumor cells. The detection sensitivity was 10 cancer cells in 2 ml whole blood. In the clinical samples, there were 4/15 positive by routine cytological examination but 11/15 positive by HSV(GFP), indicating a higher sensitivity of this new protocol. Recombinant herpes simplex virus-mediated green fluorescence is a simple and sensitive technique for the identification of occult disseminated cancer cells including circulating tumor cells (CTCs).

Characterization of a New HIV-1 CRF01_AE/ CRF07_BC recombinant virus in Tianjin, China.

PubMed

Zhou, Zhehua; Ma, Ping; Feng, Yi; Ou, Weidong; Qian, Jing; Gao, Liying; Zhang, Defa; Shao, Yiming; Wei, Min

2018-05-04

Human immunodeficiency virus (HIV) is notorious for its rapid evolving since its transmissions from money to human. Currently, HIV contains multiple subtypes, circulating recombinant forms (CRFs) and unique recombinant forms (URFs). Here, from an HIV-positive mother and her child in Tianjin, China, we identified a novel HIV-1 second-generation recombinant virus (TJ20170316 and TJ20170317) between CRF01_AE and CRF07_BC. Near full-length genomes were obtained from both samples, and they shared very close sequences, except some point mutations. Phylogenetic analyses of the near full-length genomes showed that they consist of CRF01_AE backbone and part CRF07_BC sequences. Recombinant Identification Program (RIP) and Simplot software identified four breakpoints in gag, pol, vif, tat genes in TJ20170316, totally different from other reported CRFs and URFs. The emergence of such URF in Tianjin, China, highlights the complexity of HIV-1 epidemic and more measures should be taken to prevent HIV transmissions.

[Effect of endonuclease G depletion on plasmid DNA uptake and levels of homologous recombination in hela cells].

PubMed

Misic, V; El-Mogy, M; Geng, S; Haj-Ahmad, Y

2016-01-01

Endonuclease G (EndoG) is a mitochondrial apoptosis regulator that also has roles outside of programmed cell death. It has been implicated as a defence DNase involved in the degradation of exogenous DNA after transfection of mammalian cells and in homologous recombination of viral and endogenous DNA. In this study, we looked at the effect of EndoG depletion on plasmid DNA uptake and the levels of homologous recombination in HeLa cells. We show that the proposed defence role of EndoG against uptake of non-viral DNA vectors does not extend to the cervical carcinoma HeLa cells, as targeting of EndoG expression by RNA interference failed to increase intracellular plasmid DNA levels. However, reducing EndoG levels in HeLa cells resulted in a statistically significant reduction of homologous recombination between two plasmid DNA substrates. These findings suggest that non-viral DNA vectors are also substrates for EndoG in its role in homologous recombination.

Local and sex-specific biases in crossover vs. noncrossover outcomes at meiotic recombination hot spots in mice.

PubMed

de Boer, Esther; Jasin, Maria; Keeney, Scott

2015-08-15

Meiotic recombination initiated by programmed double-strand breaks (DSBs) yields two types of interhomolog recombination products, crossovers and noncrossovers, but what determines whether a DSB will yield a crossover or noncrossover is not understood. In this study, we analyzed the influence of sex and chromosomal location on mammalian recombination outcomes by constructing fine-scale recombination maps in both males and females at two mouse hot spots located in different regions of the same chromosome. These include the most comprehensive maps of recombination hot spots in oocytes to date. One hot spot, located centrally on chromosome 1, behaved similarly in male and female meiosis: Crossovers and noncrossovers formed at comparable levels and ratios in both sexes. In contrast, at a distal hot spot, crossovers were recovered only in males even though noncrossovers were obtained at similar frequencies in both sexes. These findings reveal an example of extreme sex-specific bias in recombination outcome. We further found that estimates of relative DSB levels are surprisingly poor predictors of relative crossover frequencies between hot spots in males. Our results demonstrate that the outcome of mammalian meiotic recombination can be biased, that this bias can vary depending on location and cellular context, and that DSB frequency is not the only determinant of crossover frequency. © 2015 de Boer et al.; Published by Cold Spring Harbor Laboratory Press.

Recombinant protein-based assays for detection of antibodies to severe acute respiratory syndrome coronavirus spike and nucleocapsid proteins.

PubMed

Haynes, Lia M; Miao, Congrong; Harcourt, Jennifer L; Montgomery, Joel M; Le, Mai Quynh; Dryga, Sergey A; Kamrud, Kurt I; Rivers, Bryan; Babcock, Gregory J; Oliver, Jennifer Betts; Comer, James A; Reynolds, Mary; Uyeki, Timothy M; Bausch, Daniel; Ksiazek, Thomas; Thomas, William; Alterson, Harold; Smith, Jonathan; Ambrosino, Donna M; Anderson, Larry J

2007-03-01

Recombinant severe acute respiratory syndrome (SARS) nucleocapsid and spike protein-based immunoglobulin G immunoassays were developed and evaluated. Our assays demonstrated high sensitivity and specificity to the SARS coronavirus in sera collected from patients as late as 2 years postonset of symptoms. These assays will be useful not only for routine SARS coronavirus diagnostics but also for epidemiological and antibody kinetic studies.

Recombinant Protein-Based Assays for Detection of Antibodies to Severe Acute Respiratory Syndrome Coronavirus Spike and Nucleocapsid Proteins▿

PubMed Central

Haynes, Lia M.; Miao, Congrong; Harcourt, Jennifer L.; Montgomery, Joel M.; Le, Mai Quynh; Dryga, Sergey A.; Kamrud, Kurt I.; Rivers, Bryan; Babcock, Gregory J.; Oliver, Jennifer Betts; Comer, James A.; Reynolds, Mary; Uyeki, Timothy M.; Bausch, Daniel; Ksiazek, Thomas; Thomas, William; Alterson, Harold; Smith, Jonathan; Ambrosino, Donna M.; Anderson, Larry J.

2007-01-01

Recombinant severe acute respiratory syndrome (SARS) nucleocapsid and spike protein-based immunoglobulin G immunoassays were developed and evaluated. Our assays demonstrated high sensitivity and specificity to the SARS coronavirus in sera collected from patients as late as 2 years postonset of symptoms. These assays will be useful not only for routine SARS coronavirus diagnostics but also for epidemiological and antibody kinetic studies. PMID:17229882

Rhizosphere Competitiveness of Trichloroethylene-Degrading, Poplar-Colonizing Recombinant Bacteria

PubMed Central

Shim, Hojae; Chauhan, Sadhana; Ryoo, Doohyun; Bowers, Kally; Thomas, Stuart M.; Canada, Keith A.; Burken, Joel G.; Wood, Thomas K.

2000-01-01

Indigenous bacteria from poplar tree (Populus canadensis var. eugenei ‘Imperial Carolina’) and southern California shrub rhizospheres, as well as two tree-colonizing Rhizobium strains (ATCC 10320 and ATCC 35645), were engineered to express constitutively and stably toluene o-monooxygenase (TOM) from Burkholderia cepacia G4 by integrating the tom locus into the chromosome. The poplar and Rhizobium recombinant bacteria degraded trichloroethylene at a rate of 0.8 to 2.1 nmol/min/mg of protein and were competitive against the unengineered hosts in wheat and barley rhizospheres for 1 month (colonization occurred at a level of 1.0 × 105 to 23 × 105 CFU/cm of root). In addition, six of these recombinants colonized poplar roots stably and competitively with populations as large as 79% ± 12% of all rhizosphere bacteria after 28 days (0.2 × 105 to 31 × 105 CFU/cm of root). Furthermore, five of the most competitive poplar recombinants (e.g., Pb3-1 and Pb5-1, which were identified as Pseudomonas sp. strain PsK recombinants) retained the ability to express TOM for 29 days as 100% ± 0% of the recombinants detected in the poplar rhizosphere expressed TOM constitutively. PMID:11055909

Near Full-Length Identification of a Novel HIV-1 CRF01_AE/B/C Recombinant in Northern Myanmar.

PubMed

Zhou, Yan-Heng; Chen, Xin; Liang, Yue-Bo; Pang, Wei; Qin, Wei-Hong; Zhang, Chiyu; Zheng, Yong-Tang

2015-08-01

The Myanmar-China border appears to be the "hot spot" region for the occurrence of HIV-1 recombination. The majority of the previous analyses of HIV-1 recombination were based on partial genomic sequences, which obviously cannot reflect the reality of the genetic diversity of HIV-1 in this area well. Here, we present a near full-length characterization of a novel HIV-1 CRF01_AE/B/C recombinant isolated from a long-distance truck driver in Northern Myanmar. It is the first description of a near full-length genomic sequence in Myanmar since 2003, and might be one of the most complicated HIV-1 chimeras ever detected in Myanmar, containing four CRF01_AE, six B segments, and five C segments separated by 14 breakpoints throughout its genome. The discovery and characterization of this new CRF01_AE/B/C recombinant indicate that intersubtype recombination is ongoing in Myanmar, continuously generating new forms of HIV-1. More work based on near full-length sequence analyses is urgently needed to better understand the genetic diversity of HIV-1 in these regions.

Homosubtypic and heterosubtypic antibodies against highly pathogenic avian influenza H5N1 recombinant proteins in H5N1 survivors and non-H5N1 subjects.

PubMed

Noisumdaeng, Pirom; Pooruk, Phisanu; Prasertsopon, Jarunee; Assanasen, Susan; Kitphati, Rungrueng; Auewarakul, Prasert; Puthavathana, Pilaipan

2014-04-01

Six recombinant vaccinia viruses containing HA, NA, NP, M or NS gene insert derived from a highly pathogenic avian influenza H5N1 virus, and the recombinant vaccinia virus harboring plasmid backbone as the virus control were constructed. The recombinant proteins were characterized for their expression and subcellular locations in TK(-) cells. Antibodies to the five recombinant proteins were detected in all 13 sequential serum samples collected from four H5N1 survivors during four years of follow-up; and those directed to rVac-H5 HA and rVac-NA proteins were found in higher titers than those directed to the internal proteins as revealed by indirect immunofluorescence assay. Although all 28 non-H5N1 subjects had no neutralizing antibodies against H5N1 virus, they did have cross-reactive antibodies to those five recombinant proteins. A significant increase in cross-reactive antibody titer to rVac-H5 HA and rVac-NA was found in paired blood samples from patients infected with the 2009 pandemic virus. Copyright © 2014 Elsevier Inc. All rights reserved.

Estimating diversifying selection and functional constraint in the presence of recombination.

PubMed

Wilson, Daniel J; McVean, Gilean

2006-03-01

Models of molecular evolution that incorporate the ratio of nonsynonymous to synonymous polymorphism (dN/dS ratio) as a parameter can be used to identify sites that are under diversifying selection or functional constraint in a sample of gene sequences. However, when there has been recombination in the evolutionary history of the sequences, reconstructing a single phylogenetic tree is not appropriate, and inference based on a single tree can give misleading results. In the presence of high levels of recombination, the identification of sites experiencing diversifying selection can suffer from a false-positive rate as high as 90%. We present a model that uses a population genetics approximation to the coalescent with recombination and use reversible-jump MCMC to perform Bayesian inference on both the dN/dS ratio and the recombination rate, allowing each to vary along the sequence. We demonstrate that the method has the power to detect variation in the dN/dS ratio and the recombination rate and does not suffer from a high false-positive rate. We use the method to analyze the porB gene of Neisseria meningitidis and verify the inferences using prior sensitivity analysis and model criticism techniques.

Estimating Diversifying Selection and Functional Constraint in the Presence of Recombination

PubMed Central

Wilson, Daniel J.; McVean, Gilean

2006-01-01

Models of molecular evolution that incorporate the ratio of nonsynonymous to synonymous polymorphism (dN/dS ratio) as a parameter can be used to identify sites that are under diversifying selection or functional constraint in a sample of gene sequences. However, when there has been recombination in the evolutionary history of the sequences, reconstructing a single phylogenetic tree is not appropriate, and inference based on a single tree can give misleading results. In the presence of high levels of recombination, the identification of sites experiencing diversifying selection can suffer from a false-positive rate as high as 90%. We present a model that uses a population genetics approximation to the coalescent with recombination and use reversible-jump MCMC to perform Bayesian inference on both the dN/dS ratio and the recombination rate, allowing each to vary along the sequence. We demonstrate that the method has the power to detect variation in the dN/dS ratio and the recombination rate and does not suffer from a high false-positive rate. We use the method to analyze the porB gene of Neisseria meningitidis and verify the inferences using prior sensitivity analysis and model criticism techniques. PMID:16387887

Identification of N-Terminally Truncated Derivatives of Insulin Analogs Formed in Pharmaceutical Formulations.

PubMed

Zielińska, Joanna; Stadnik, Jacek; Bierczyńska-Krzysik, Anna; Stadnik, Dorota

2018-05-16

Isolation and identification of unknown impurities of recombinant insulin lispro (produced at IBA) formed during accelerated stability testing of pharmaceutical solutions. For comparative purposes also commercially available formulations of recombinant human insulin (Humulin S®; Lilly), recombinant insulin lispro (Humalog®; Lilly), recombinant insulin aspart (NovoRapid® Penfill®; Novo Nordisk), recombinant insulin detemir (Levemir®; Novo Nordisk) and recombinant insulin glargine (Lantus®; Sanofi-Aventis) were analyzed. The impurities of insulin analogs were isolated by RP-HPLC and identified with peptide mass fingerprinting using MALDI-TOF/TOF mass spectrometry. The identified derivatives were N-terminally truncated insulin analog impurities of decreased molecular mass of 119, 147 and 377 Da related to the original protein. The modifications resulting in a mass decrease were detected at the N-terminus of B chains of insulin lispro, insulin aspart, human insulin, insulin glargine, insulin detemir in all tested formulations. To our knowledge it is the first time that these impurities are reported. The following derivatives formed by truncation of the B chain in insulin analogs were identified in pharmaceutical formulations: desPhe B1 -N-formyl-Val B2 derivative, desPhe B1 derivative, pyroGlu B4 derivative.

Characterization of an Sf-rhabdovirus-negative Spodoptera frugiperda cell line as an alternative host for recombinant protein production in the baculovirus-insect cell system.

PubMed

Maghodia, Ajay B; Geisler, Christoph; Jarvis, Donald L

2016-06-01

Cell lines derived from the fall armyworm, Spodoptera frugiperda (Sf), are widely used as hosts for recombinant protein production in the baculovirus-insect cell system (BICS). However, it was recently discovered that these cell lines are contaminated with a virus, now known as Sf-rhabdovirus [1]. The detection of this adventitious agent raised a potential safety issue that could adversely impact the BICS as a commercial recombinant protein production platform. Thus, we examined the properties of Sf-RVN, an Sf-rhabdovirus-negative Sf cell line, as a potential alternative host. Nested RT-PCR assays showed Sf-RVN cells had no detectable Sf-rhabdovirus over the course of 60 passages in continuous culture. The general properties of Sf-RVN cells, including their average growth rates, diameters, morphologies, and viabilities after baculovirus infection, were virtually identical to those of Sf9 cells. Baculovirus-infected Sf-RVN and Sf9 cells produced equivalent levels of three recombinant proteins, including an intracellular prokaryotic protein and two secreted eukaryotic glycoproteins, and provided similar N-glycosylation patterns. In fact, except for the absence of Sf-rhabdovirus, the only difference between Sf-RVN and Sf9 cells was SF-RVN produced higher levels of infectious baculovirus progeny. These results show Sf-RVN cells can be used as improved, alternative hosts to circumvent the potential safety hazard associated with the use of Sf-rhabdovirus-contaminated Sf cells for recombinant protein manufacturing with the BICS. Copyright © 2016 Elsevier Inc. All rights reserved.

Utilizing protein-lean coproducts from corn containing recombinant pharmaceutical proteins for ethanol production.

PubMed

Paraman, Ilankovan; Moeller, Lorena; Scott, M Paul; Wang, Kan; Glatz, Charles E; Johnson, Lawrence A

2010-10-13

Protein-lean fractions of corn (maize) containing recombinant (r) pharmaceutical proteins were evaluated as a potential feedstock to produce fuel ethanol. The levels of residual r-proteins in the coproduct, distillers dry grains with solubles (DDGS), were determined. Transgenic corn lines containing recombinant green fluorescence protein (r-GFP) and a recombinant subunit vaccine of Escherichia coli enterotoxin (r-LTB), primarily expressed in endosperm, and another two corn lines containing recombinant human collagen (r-CIα1) and r-GFP, primarily expressed in germ, were used as model systems. The kernels were either ground and used for fermentation or dry fractionated to recover germ-rich fractions prior to grinding for fermentation. The finished beers of whole ground kernels and r-protein-spent endosperm solids contained 127-139 and 138-155 g/L ethanol concentrations, respectively. The ethanol levels did not differ among transgenic and normal corn feedstocks, indicating the residual r-proteins did not negatively affect ethanol production. r-Protein extraction and germ removal also did not negatively affect fermentation of the remaining mass. Most r-proteins were inactivated during the mashing process used to prepare corn for fermentation. No functionally active r-GFP or r-LTB proteins were found after fermentation of the r-protein-spent solids; however, a small quantity of residual r-CIα1 was detected in DDGS, indicating that the safety of DDGS produced from transgenic grain for r-protein production needs to be evaluated for each event. Protease treatment during fermentation completely hydrolyzed the residual r-CIα1, and no residual r-proteins were detectable in DDGS.

Inclusion bodies of recombinant Epstein-Barr virus capsid antigen p18 as potential immobilized antigens in enzyme immunoassays for detection of nasopharyngeal carcinoma.

PubMed

Lim, Chun Shen; Goh, Siang Ling; Kariapper, Leena; Krishnan, Gopala; Lim, Yat-Yuen; Ng, Ching Ching

2015-08-25

Development of indirect enzyme-linked immunosorbent assays (ELISAs) often utilizes synthetic peptides or recombinant proteins from Escherichia coli as immobilized antigens. Because inclusion bodies (IBs) formed during recombinant protein expression in E. coli are commonly thought as misfolded aggregates, only refolded proteins from IBs are used to develop new or in-house diagnostic assays. However, the promising utilities of IBs as nanomaterials and immobilized enzymes as shown in recent studies have led us to explore the potential use of IBs of recombinant Epstein-Barr virus viral capsid antigen p18 (VCA p18) as immobilized antigens in ELISAs for serologic detection of nasopharyngeal carcinoma (NPC). Thioredoxin fusion VCA p18 (VCA-Trx) and IBs of VCA p18 without fusion tags (VCA-IBs) were purified from E. coli. The diagnostic performances of IgG/VCA-IBs, IgG/VCA-Denat-IBs (using VCA-IBs coated in 8mol/l urea), IgG/VCA-Trx, and IgG/VCA-Peptide assays were compared by screening 100 NPC case-control pairs. The IgG/VCA-Denat-IBs assay showed the best area under the receiver operating characteristic curve (AUC: 0.802; p<0.05), while the AUCs for the IgG/VCA-IBs, IgG/VCA-Trx, and IgG/VCA-Peptide assays were comparable (AUC: 0.740, 0.727, and 0.741, respectively). We improved the diagnostic performance of the ELISA significantly using IBs of recombinant VCA p18. Copyright © 2015 Elsevier B.V. All rights reserved.

[Cloning and expressing of cyclophilin B gene from Schistosoma japonnicum and the analysis of immunoprotective effect].

PubMed

Peng, Jinbiao; Han, Hongxiao; Hong, Yang; Wang, Yan; Guo, Fanji; Shi, Yaojun; Fu, Zhiqiang; Liu, Jinming; Cheng, Guofeng; Lin, Jiaojiao

2010-03-01

The present study was intend to clone and express the cDNA encoding Cyclophilin B (CyPB) of Schistosoma japonicum, its preliminary biological function and further immunoprotective effect against schistosome infection in mice. RT-PCR technique was applied to amplify a full-length cDNA encoding protein Cyclophilin B (Sj CyPB) from schistosomula cDNA. The expression profiles of Sj CyPB were determined by Real-time PCR using the template cDNAs isolated from 7, 13, 18, 23, 32 and 42 days parasites. The cDNA containing the Open Reading Frame of CyPB was then subcloned into a pGEX-6P-1 vector and transformed into competent Escherichia coli BL21 for expressing. The recombinant protein was renaturated, purified and its antigenicity were detected by Western blotting, and the immunoprotective effect induced by recombinant Sj CyPB was evaluated in Balb/C mice. The cDNA containing the ORF of Sj CyPB was cloned with the length of 672 base pairs, encoding 223 amino acids. Real-time PCR analysis revealed that the gene had the highest expression in 18-day schistosomula, suggesting that Sj CyPB was schistosomula differentially expressed gene. The recombinant protein showed a good antigenicity detected by Western blotting. Animal experiment indicated that the vaccination of recombinant CyPB protein in mice led to 31.5% worm and 41.01% liver egg burden reduction, respectively, compared with those of the control. A full-length cDNA differentially expressed in schistosomula was obtained. The recombinant Sj CyPB protein could induce partial protection against schistosome infection.

Progressive genomic convergence of two Helicobacter pylori strains during mixed infection of a patient with chronic gastritis

PubMed Central

Cao, Qizhi; Didelot, Xavier; Wu, Zhongbiao; Li, Zongwei; He, Lihua; Li, Yunsheng; Ni, Ming; You, Yuanhai; Lin, Xi; Li, Zhen; Gong, Yanan; Zheng, Minqiao; Zhang, Minli; Liu, Jie; Wang, Weijun; Bo, Xiaochen; Falush, Daniel; Wang, Shengqi; Zhang, Jianzhong

2015-01-01

Objective To study the detailed nature of genomic microevolution during mixed infection with multiple Helicobacter pylori strains in an individual. Design We sampled 18 isolates from a single biopsy from a patient with chronic gastritis and nephritis. Whole-genome sequencing was applied to these isolates, and statistical genetic tools were used to investigate their evolutionary history. Results The genomes fall into two clades, reflecting colonisation of the stomach by two distinct strains, and these lineages have accumulated diversity during an estimated 2.8 and 4.2 years of evolution. We detected about 150 clear recombination events between the two clades. Recombination between the lineages is a continuous ongoing process and was detected on both clades, but the effect of recombination in one clade was nearly an order of magnitude higher than in the other. Imputed ancestral sequences also showed evidence of recombination between the two strains prior to their diversification, and we estimate that they have both been infecting the same host for at least 12 years. Recombination tracts between the lineages were, on average, 895 bp in length, and showed evidence for the interspersion of recipient sequences that has been observed in in vitro experiments. The complex evolutionary history of a phage-related protein provided evidence for frequent reinfection of both clades by a single phage lineage during the past 4 years. Conclusions Whole genome sequencing can be used to make detailed conclusions about the mechanisms of genetic change of H. pylori based on sampling bacteria from a single gastric biopsy. PMID:25007814

Marek's disease virus protein kinase gene identified within the short unique region of the viral genome is not essential for viral replication in cell culture and vaccine-induced immunity in chickens.

PubMed

Sakaguchi, M; Urakawa, T; Hirayama, Y; Miki, N; Yamamoto, M; Zhu, G S; Hirai, K

1993-07-01

The open reading frame (ORF) of 1206 bp within the short unique region (Us) of Marek's disease virus type 1 (MDV1) shows significant homology with the herpes simplex virus type 1 US3 gene encoding protein kinase (PK). The lacZ gene of Escherichia coli was inserted within the ORF, designated MDV1-US3, of MDV1 K544 strain DNA by homologous recombination. The plaque-purified recombinant MDV1 stably expressed the beta-galactosidase encoded by the inserted lacZ gene in infected cells and replicated well as the parental K544 strain. Antibodies against both MDV1 antigen and beta-galactosidase were detected in the sera of chickens immunized with recombinant MDV1. Chickens vaccinated with the recombinant MDV1 were protected from challenge with virulent MDV1. The MDV1 US3 gene expressed by a baculovirus vector encoded a 44-kDa protein. Mouse antisera against the 44-kDa protein reacted with two proteins of 44 and 45 kDa in extracts of cells infected with MDV1 but not with MDV types 2 or 3. The PK activity was detected in immune complexes of the anti-44-kDa sera with extracts of cells infected with MDV1 but not with the recombinant MDV1. Thus, PK encoded from the MDV1-US3 is not essential for virus replication in cell culture and vaccine-induced immunity.

Detection and molecular characterization of the novel recombinant norovirus GII.P16-GII.4 Sydney in southeastern Brazil in 2016

PubMed Central

Tonini, Marco André Loureiro; Volpini, Lays Paula Bondi; Santos, Rodrigo Pratte; Ribeiro, Anézia Lima Chaves; Leite, José Paulo Gagliardi; Souza, Márcia Terezinha Baroni de Moraes e; Brasil, Patrícia; da Cunha, Denise Cotrim; Miagostovich, Marize Pereira; Spano, Liliana Cruz

2017-01-01

Noroviruses are the leading cause of acute gastroenteritis (AGE) in all age groups worldwide. Despite the high genetic diversity of noroviruses, most AGE outbreaks are caused by a single norovirus genotype: GII.4. Since 1995, several different variants of norovirus GII.4 have been associated with pandemics, with each variant circulating for 3 to 8 years. The Sydney_2012 variant was first reported in Australia and then in other countries. A new variant, GII.P16-GII.4, was recently described in Japan and South Korea and then in the USA, France, Germany and England. In our study, 190 faecal specimens were collected from children admitted to a paediatric hospital and a public health facility during a surveillance study of sporadic cases of AGE conducted between January 2015 and July 2016. The norovirus was detected by RT-qPCR in 51 samples (26.8%), and in 37 of them (72.5%), the ORF1-2 junction was successfully sequenced. The new recombinant GII.P16-GII.4 Sydney was revealed for the first time in Brazil in 2016 and predominated among other strains (9 GII.Pe-GII.4, 3 GII.P17-GII.17, 1 GII.Pg-GII.1, 1 GII.P16-GII.3 and 1 GII.PNA-GII.4). The epidemiological significance of this new recombinant is still unknown, but continuous surveillance studies may evaluate its impact on the population, its potential to replace the first recombinant GII.Pe-GII.4 Sydney 2012 variant, and the emergence of new recombinant forms of GII.P16. PMID:29236779

High levels of multiple infections, recombination and horizontal transmission of Wolbachia in the Andricus mukaigawae (Hymenoptera; Cynipidae) communities.

PubMed

Yang, Xiao-Hui; Zhu, Dao-Hong; Liu, Zhiwei; Zhao, Ling; Su, Cheng-Yuan

2013-01-01

Wolbachia are maternally inherited endosymbiotic bacteria of arthropods and nematodes. In arthropods, they manipulate the reproduction of their hosts to facilitate their own spread in host populations, causing cytoplasmic incompatibility, parthenogenesis induction, feminization of genetic males and male-killing. In this study, we investigated Wolbachia infection and studied wsp (Wolbachia surface protein) sequences in three wasp species associated with the unisexual galls of A. mukaigawae with the aim of determining the transmission mode and the reason for multiple infections of Wolbachia. Frequency of Wolbachia infected populations for A. mukaigawae, Synergus japonicus (inquiline), and Torymus sp. (parasitoid) was 75%, 100%, and 100%, respectively. Multiple Wolbachia infections were detected in A. mukaigawae and S. japonicus, with 5 and 8 Wolbachia strains, respectively. The two host species shared 5 Wolbachia strains and were infected by identical strains in several locations, indicating horizontal transmission of Wolbachia. The transmission potentially takes place through gall tissues, which the larvae of both wasps feed on. Furthermore, three recombination events of Wolbachia were observed: the strains W8, W2 and W6 apparently have derived from W3 and W5a, W6 and W7, W4 and W9, respectively. W8 and W2 and their respective parental strains were detected in S. japonicus. W6 was detected with only one parent (W4) in S. japonicus; W9 was detected in Torymus sp., suggesting horizontal transmission between hosts and parasitoids. In conclusion, our research supports earlier studies that horizontal transmission of Wolbachia, a symbiont of the Rickettsiales order, may be plant-mediated or take place between hosts and parasitoids. Our research provides novel molecular evidence for multiple recombination events of Wolbachia in gall wasp communities. We suggest that genomic recombination and potential plant-mediated horizontal transmission may be attributable to the high levels of multiple Wolbachia infections observed in A. mukaigawae and S. japonicus.

The Southern H ii Region Discovery Survey (SHRDS): Pilot Survey

DOE Office of Scientific and Technical Information (OSTI.GOV)

Brown, C.; Dickey, John M.; Jordan, C.

The Southern H ii Region Discovery Survey is a survey of the third and fourth quadrants of the Galactic plane that will detect radio recombination line (RRL) and continuum emission at cm-wavelengths from several hundred H ii region candidates using the Australia Telescope Compact Array. The targets for this survey come from the WISE Catalog of Galactic H ii Regions and were identified based on mid-infrared and radio continuum emission. In this pilot project, two different configurations of the Compact Array Broad Band receiver and spectrometer system were used for short test observations. The pilot surveys detected RRL emission frommore » 36 of 53 H ii region candidates, as well as seven known H ii regions that were included for calibration. These 36 recombination line detections confirm that the candidates are true H ii regions and allow us to estimate their distances.« less

[Establishment of chemiluminescent enzyme immunoassay for detecting antibodies against foot-and-mouth disease virus serotype O in swine].

PubMed

Cui, Chen; Huang, Ligang; Li, Jing; Zou, Xingqi; Zhu, Yuanyuan; Xie, Lei; Zhao, Qizu; Yang, Limin; Liu, Wenjun

2016-11-25

Recombinant structural protein VP1 of foot-and-mouth disease virus serotype O was expressed in Escherichia coli and then purified using Nickel affinity chromatography. A chemiluminescent enzyme immunoassay (CLEIA) method was established using the purified recombinant protein as coating antigen to detect antibody of foot-and-mouth disease virus serotype O in swine. The specificity of VP1-CLEIA method is 100%. The coefficients of variation in the plate and between plates are 1.10%-6.70% and 0.66%-4.80%, respectively. Comparing with the commercial indirect ELISA kit or liquid phase block ELISA kit, the calculated coincidence rate is 93.50% or 94.00%. The high specificity and stability suggested this detection method can be used to monitor the antibody level of foot-and-mouth disease virus serotype O in swine.

The evaluation of a nucleoprotein ELISA for the detection of equine influenza antibodies and the differentiation of infected from vaccinated horses (DIVA).

PubMed

Galvin, Pamela; Gildea, Sarah; Arkins, Sean; Walsh, Cathal; Cullinane, Ann

2013-12-01

Antibodies against equine influenza virus (EIV) are traditionally quantified by haemagglutination inhibition (HI) or single radial haemolysis (SRH). To evaluate an ELISA for the detection of antibodies against influenza nucleoprotein in the diagnosis and surveillance of equine influenza (EI). The ELISA was compared with the SRH and HI tests. Serial serum samples from 203 naturally and 14 experimentally infected horses, from 60 weanlings following primary vaccination with five different vaccines (two whole inactivated vaccines, two ISCOM-based subunit vaccines and a recombinant canarypox virus vaccine) and from 44 adult horses following annual booster vaccination with six different vaccines were analysed. Fewer seroconversions were detected in clinical samples by ELISA than by SRH or HI but ELISA was more sensitive than SRH in naïve foals post-experimental infection. The ELISA did not detect the antibody response to vaccination with the recombinant canarypox virus vaccine confirming the usefulness of the combination of this kit and vaccine to differentiate between naturally infected and vaccinated horses, that is, DIVA. No DIVA capacity was evident with the other vaccines. The results suggest that this ELISA is a useful supplementary test for the diagnosis of EI although less sensitive than HI or SRH. It is an appropriate test for EI surveillance in a naïve population and may be combined with the recombinant canarypox virus vaccine but not with other commercially available subunit vaccines, in a DIVA strategy. © 2013 Blackwell Publishing Ltd.

Genetically Engineered Virulent Phage Banks in the Detection and Control of Emergent Pathogenic Bacteria

PubMed Central

Blois, Hélène; Iris, François

2010-01-01

Natural outbreaks of multidrug-resistant microorganisms can cause widespread devastation, and several can be used or engineered as agents of bioterrorism. From a biosecurity standpoint, the capacity to detect and then efficiently control, within hours, the spread and the potential pathological effects of an emergent outbreak, for which there may be no effective antibiotics or vaccines, become key challenges that must be met. We turned to phage engineering as a potentially highly flexible and effective means to both detect and eradicate threats originating from emergent (uncharacterized) bacterial strains. To this end, we developed technologies allowing us to (1) concurrently modify multiple regions within the coding sequence of a gene while conserving intact the remainder of the gene, (2) reversibly interrupt the lytic cycle of an obligate virulent phage (T4) within its host, (3) carry out efficient insertion, by homologous recombination, of any number of engineered genes into the deactivated genomes of a T4 wild-type phage population, and (4) reactivate the lytic cycle, leading to the production of engineered infective virulent recombinant progeny. This allows the production of very large, genetically engineered lytic phage banks containing, in an E. coli host, a very wide spectrum of variants for any chosen phage-associated function, including phage host-range. Screening of such a bank should allow the rapid isolation of recombinant T4 particles capable of detecting (ie, diagnosing), infecting, and destroying hosts belonging to gram-negative bacterial species far removed from the original E. coli host. PMID:20569057

Recombination Does Not Hinder Formation or Detection of Ecological Species of Synechococcus Inhabiting a Hot Spring Cyanobacterial Mat

DOE Office of Scientific and Technical Information (OSTI.GOV)

Melendrez, Melanie C.; Becraft, Eric D.; Wood, Jason M.

Recent studies of bacterial speciation have claimed to support the biological species concept—that reduced recombination is required for bacterial populations to diverge into species. This conclusion has been reached from the discovery that ecologically distinct clades show lower rates of recombination than that which occurs among closest relatives. However, these previous studies did not attempt to determine whether the more-rapidly recombining close relatives within the clades studied may also have diversified ecologically, without benefit of sexual isolation. Here we have measured the impact of recombination on ecological diversification within and between two ecologically distinct clades (A and B’) of Synechococcusmore » in a hot spring microbial mat in Yellowstone National Park, using a cultivation-free, multi-locus approach. Bacterial artificial chromosome (BAC) libraries were constructed from mat samples collected at 60°C and 65°C. Analysis of multiple linked loci near Synechococcus 16S rRNA genes showed little evidence of recombination between the A and B’ lineages, but a record of recombination was apparent within each lineage. Recombination and mutation rates within each lineage were of similar magnitude, but recombination had a somewhat greater impact on sequence diversity than mutation, as also seen in many other bacteria and archaea. Despite recombination within the A and B’ lineages, there was evidence of ecological diversification within each lineage. The algorithm Ecotype Simulation identified sequence clusters consistent with ecologically distinct populations (ecotypes), and several hypothesized ecotypes were distinct in their habitat associations and in their adaptations to different microenvironments. We conclude that sexual isolation is more likely to follow ecological divergence than to precede it. Thus, an ecology-based model of speciation appears more appropriate than the biological species concept for bacterial and archaeal diversification.« less

Recombination Does Not Hinder Formation or Detection of Ecological Species of Synechococcus Inhabiting a Hot Spring Cyanobacterial Mat

DOE PAGES

Melendrez, Melanie C.; Becraft, Eric D.; Wood, Jason M.; ...

2016-01-14

Recent studies of bacterial speciation have claimed to support the biological species concept—that reduced recombination is required for bacterial populations to diverge into species. This conclusion has been reached from the discovery that ecologically distinct clades show lower rates of recombination than that which occurs among closest relatives. However, these previous studies did not attempt to determine whether the more-rapidly recombining close relatives within the clades studied may also have diversified ecologically, without benefit of sexual isolation. Here we have measured the impact of recombination on ecological diversification within and between two ecologically distinct clades (A and B’) of Synechococcusmore » in a hot spring microbial mat in Yellowstone National Park, using a cultivation-free, multi-locus approach. Bacterial artificial chromosome (BAC) libraries were constructed from mat samples collected at 60°C and 65°C. Analysis of multiple linked loci near Synechococcus 16S rRNA genes showed little evidence of recombination between the A and B’ lineages, but a record of recombination was apparent within each lineage. Recombination and mutation rates within each lineage were of similar magnitude, but recombination had a somewhat greater impact on sequence diversity than mutation, as also seen in many other bacteria and archaea. Despite recombination within the A and B’ lineages, there was evidence of ecological diversification within each lineage. The algorithm Ecotype Simulation identified sequence clusters consistent with ecologically distinct populations (ecotypes), and several hypothesized ecotypes were distinct in their habitat associations and in their adaptations to different microenvironments. We conclude that sexual isolation is more likely to follow ecological divergence than to precede it. Thus, an ecology-based model of speciation appears more appropriate than the biological species concept for bacterial and archaeal diversification.« less

A novel capture-ELISA for detection of anti-neutrophil cytoplasmic antibodies (ANCA) based on c-myc peptide recognition in carboxy-terminally tagged recombinant neutrophil serine proteases.

PubMed

Lee, Augustine S; Finkielman, Javier D; Peikert, Tobias; Hummel, Amber M; Viss, Margaret A; Specks, Ulrich

2005-12-20

Testing for antineutrophil cytoplasmic antibodies (ANCA) reacting with proteinase 3 (PR3) is part of the routine diagnostic evaluation of patients with small vessel vasculitis. For PR3-ANCA detection, capture ELISAs are reported to be superior to direct ELISAs. Standard capture ELISAs, in which PR3 is anchored by anti-PR3 monoclonal antibodies (moAB), have two potential disadvantages. First, the capturing moAB may compete for epitopes recognized by some PR3-ANCA, causing occasional false-negative results. Second, the capture of recombinant PR3 mutant molecules becomes unpredictable as modifications of specific conformational epitopes may not only affect the binding of PR3-ANCA, but also the affinity of the capturing anti-PR3 moAB. Here, we describe a new capture ELISA, and its application for PR3-ANCA detection. This new assay is based on the standardized capture of a variety of different carboxy-terminally c-myc tagged recombinant ANCA target antigens using anti-c-myc coated ELISA plates. Antigen used include c-myc tagged human rPR3 variants (mature and pro-form conformations), mouse mature rPR3 and human recombinant neutrophil elastase. This new anti-c-myc-capture ELISA for PR3-ANCA detection has an intra- and inter-assay coefficient of variation of 3.6% to 7.7%, and 15.8% to 18.4%, respectively. The analytical sensitivity and specificity for PR3-ANCA positive serum samples were 93% and 100%, respectively when rPR3 with mature conformation was used as target antigen, and 83% and 100% when the pro-enzyme conformation was employed. In conclusion, this new anti-c-myc capture ELISA compares favorably to our standard capture ELISA for PR3-ANCA detection, enables the unified capture of different ANCA target antigens through binding to a c-myc tag, and allows capture of rPR3 mutants necessary for PR3-ANCA epitope mapping studies.

Recombination of Globally Circulating Varicella-Zoster Virus

PubMed Central

Depledge, Daniel P.; Kundu, Samit; Atkinson, Claire; Brown, Julianne; Haque, Tanzina; Hussaini, Yusuf; MacMahon, Eithne; Molyneaux, Pamela; Papaevangelou, Vassiliki; Sengupta, Nitu; Koay, Evelyn S. C.; Tang, Julian W.; Underhill, Gillian S.; Grahn, Anna; Studahl, Marie; Breuer, Judith; Bergström, Tomas

2015-01-01

ABSTRACT Varicella-zoster virus (VZV) is a human herpesvirus, which during primary infection typically causes varicella (chicken pox) and establishes lifelong latency in sensory and autonomic ganglia. Later in life, the virus may reactivate to cause herpes zoster (HZ; also known as shingles). To prevent these diseases, a live-attenuated heterogeneous vaccine preparation, vOka, is used routinely in many countries worldwide. Recent studies of another alphaherpesvirus, infectious laryngotracheitis virus, demonstrate that live-attenuated vaccine strains can recombine in vivo, creating virulent progeny. These findings raised concerns about using attenuated herpesvirus vaccines under conditions that favor recombination. To investigate whether VZV may undergo recombination, which is a prerequisite for VZV vaccination to create such conditions, we here analyzed 115 complete VZV genomes. Our results demonstrate that recombination occurs frequently for VZV. It thus seems that VZV is fully capable of recombination if given the opportunity, which may have important implications for continued VZV vaccination. Although no interclade vaccine-wild-type recombinant strains were found, intraclade recombinants were frequently detected in clade 2, which harbors the vaccine strains, suggesting that the vaccine strains have already been involved in recombination events, either in vivo or in vitro during passages in cell culture. Finally, previous partial and complete genomic studies have described strains that do not cluster phylogenetically to any of the five established clades. The additional VZV strains sequenced here, in combination with those previously published, have enabled us to formally define a novel sixth VZV clade. IMPORTANCE Although genetic recombination has been demonstrated to frequently occur for other human alphaherpesviruses, herpes simplex viruses 1 and 2, only a few ancient and isolated recent recombination events have hitherto been demonstrated for VZV. In the present study, we demonstrate that VZV also frequently undergoes genetic recombination, including strains belonging to the clade containing the vOKA strain. PMID:25926648

Recombinant vaccinia/Venezuelan equine encephalitis (VEE) virus expresses VEE structural proteins.

PubMed

Kinney, R M; Esposito, J J; Johnson, B J; Roehrig, J T; Mathews, J H; Barrett, A D; Trent, D W

1988-12-01

cDNA molecules encoding the structural proteins of the virulent Trinidad donkey and the TC-83 vaccine strains of Venezuelan equine encephalitis (VEE) virus were inserted under control of the vaccinia virus 7.5K promoter into the thymidine kinase gene of vaccinia virus. Synthesis of the capsid protein and glycoproteins E2 and E1 of VEE virus was demonstrated by immunoblotting of lysates of CV-1 cells infected with recombinant vaccinia/VEE viruses. VEE glycoproteins were detected in recombinant virus-infected cells by fluorescent antibody (FA) analysis performed with a panel of VEE-specific monoclonal antibodies. Seven E2-specific epitopes and two of four E1-specific epitopes were demonstrated by FA.

Expression of the Bacillus anthracis protective antigen gene by baculovirus and vaccinia virus recombinants.

PubMed Central

Iacono-Connors, L C; Schmaljohn, C S; Dalrymple, J M

1990-01-01

The gene encoding Bacillus anthracis protective antigen (PA) was modified by site-directed mutagenesis, subcloned into baculovirus and vaccinia virus plasmid transfer vectors (pAcYM1 and pSC-11, respectively), and inserted via homologous recombinations into baculovirus Autographa californica nuclear polyhedrosis virus or vaccinia virus (strains WR and Connaught). Expression of PA was detected in both systems by immunofluorescence assays with antisera from rabbits immunized with B. anthracis PA. Western blot (immunoblot) analysis showed that the expressed product of both systems was slightly larger (86 kilodaltons) than B. anthracis-produced PA (83.5 kilodaltons). Analysis of trypsin digests of virus-expressed and authentic PA suggested that the size difference was due to the presence of a signal sequence remaining with the virus-expressed protein. Immunization of mice with either recombinant baculovirus-infected Spodoptera frugiperda cells or with vaccinia virus recombinants elicited a high-titer, anti-PA antibody response. Images PMID:2105271

Establishment of an ELISA to detect Kaposi's sarcoma-associated herpesvirus using recombinant ORF73.

PubMed

Ouyang, Xin-xing; Fu, Bi-shi; Li, Bao-lin; Zeng, Yan; Xu, Fan-hong; Wang, Lin-ding

2010-06-01

Kaposi's sarcoma-associated herpesvirus (KSHV) is causally related to Kaposi's sarcoma (KS), primary effusion lymphoma (PEL) and a proportion of cases of multicentric Castleman's disease (MCD). The ORF73 protein was cloned into pQE80L-orf73 and expressed in E.coli and purified. The expressed recombinant ORF73 was identified by sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE). A protein of about 27 kDa was expressed as expected. Western Blotting showed that the purified recombinant ORF73 reacted with KSHV positive serum. The immunogenicity of the recombinant ORF73 was further analysed by ELISA and the optimal conditions were determined. The ORF73 ELISA was used to compare the KSHV seroprevalence between Hubei and Xinjiang Han people. The Han people in Xinjiang have significantly higher KSHV seroprevalence than their counterparts in Hubei (6.7% vs 2.9%, P = 0.005).

First field trial of a transmissible recombinant vaccine against myxomatosis and rabbit hemorrhagic disease.

PubMed

Torres, J M; Sánchez, C; Ramírez, M A; Morales, M; Bárcena, J; Ferrer, J; Espuña, E; Pagès-Manté, A; Sánchez-Vizcaíno, J M

2001-08-14

As a novel approach for immunisation of wild rabbits, we have recently developed a transmissible vaccine against myxomatosis and rabbit hemorrhagic disease (RHD) based on a recombinant myxoma virus (MV) expressing the RHDV capsid protein [J. Virol. 74 (2000) 1114]. The efficacy and safety of the vaccine have been extensively evaluated under laboratory conditions. In this study, we report the first limited field trial of the candidate vaccine that was undertaken in an island of 34 Has containing a population of around 300 rabbits. Following administration by the subcutaneous route to 76 rabbits, the vaccine induced specific antibody responses against both myxomatosis and RHDV in all the inoculated rabbits. Furthermore, the recombinant virus exhibited a limited horizontal transmission capacity, promoting seroconversion of around 50% of the uninoculated rabbit population. No evidence of undesirable effects due to the recombinant virus field release was detected.

Photoluminescence of amorphous and crystalline silicon nanoclusters in silicon nitride and oxide superlattices

DOE Office of Scientific and Technical Information (OSTI.GOV)

Shuleiko, D. V., E-mail: shuleyko.dmitriy@physics.msu.ru; Zabotnov, S. V.; Zhigunov, D. M.

2017-02-15

The photoluminescence properties of silicon nitride and oxide superlattices fabricated by plasmaenhanced chemical vapor deposition are studied. In the structures annealed at a temperature of 1150°C, photoluminescence peaks at about 1.45 eV are recorded. The peaks are defined by exciton recombination in silicon nanocrystals formed upon annealing. Along with the 1.45-eV peaks, a number of peaks defined by recombination at defects at the interface between the nanocrystals and silicon-nitride matrix are detected. The structures annealed at 900°C exhibit a number of photoluminescence peaks in the range 1.3–2.0 eV. These peaks are defined by both the recombination at defects and excitonmore » recombination in amorphous silicon nanoclusters formed at an annealing temperature of 900°C. The observed features of all of the photoluminescence spectra are confirmed by the nature of the photoluminescence kinetics.« less

Pathogenic analysis of the pandemic 2009 H1N1 influenza A viruses in ferrets.

PubMed

Tsuda, Yoshimi; Weisend, Carla; Martellaro, Cynthia; Feldmann, Friederike; Haddock, Elaine

2017-08-18

The pandemic 2009 H1N1 influenza A virus emerged in humans and caused the first influenza pandemic of the 21st century. Mexican isolates, A/Mexico/4108/2009 (H1N1) (Mex4108) and A/Mexico/InDRE4478/2009 (H1N1) (Mex4487) derived from a mild case and from a cluster of severe cases, showed heterogeneity in virulence in a cynomolgus macaque model. To compare the more pathogenic differences, we generated recombinant viruses and compared their virulence in ferrets. Ferrets infected with recombinant Mex4487 displayed a slightly higher rate of viral replication and severe pneumonia in the early stage of infection. In contrast, prolonged lower virus shedding of recombinant Mex4108 than that of recombinant Mex4487 was detected in throat swabs. Thus, Mex4487 induces severe pneumonia in infected individuals, whereas Mex4108 might have wide-spreading potential with mild disease.

[Current status and prospects of gene doping detection].

PubMed

Wang, Wenjun; Zhang, Sichun; Xu, Jingjuan; Xia, Xinghua; Tian, Yaping; Zhang, Xinrong; Chen, Hong-Yuan

2008-07-01

The fast development of biotechnology promotes the development of doping. From recombinant protein to gene doping, there is a great challenge to their detection. The improvement of gene therapy and potential to enhance athletic performance open the door for gene doping. After a brief introduction of the concept of gene doping, the current status and prospects of gene doping detection are reviewed.

Sensitive and specific detection of classical scrapie prions in the brain of goats by real-time quaking-induced conversion

USDA-ARS?s Scientific Manuscript database

The real-time quaking-induced conversion (RT-QuIC) is a rapid, specific, and sensitive prion seeding activity detection assay that uses recombinant prion protein (rPrPSen) to detect sub-infectious levels of the abnormal isoforms of the prion protein (PrPSc). Although RT-QuIC has been successfully us...

phiC31 Integrase-Mediated Site-Specific Recombination in Barley

PubMed Central

Rubtsova, Myroslava; Kumlehn, Jochen; Gils, Mario

2012-01-01

The Streptomyces phage phiC31 integrase was tested for its feasibility in excising transgenes from the barley genome through site-specific recombination. We produced transgenic barley plants expressing an active phiC31 integrase and crossed them with transgenic barley plants carrying a target locus for recombination. The target sequence involves a reporter gene encoding green fluorescent protein (GFP), which is flanked by the attB and attP recognition sites for the phiC31 integrase. This sequence disruptively separates a gusA coding sequence from an upstream rice actin promoter. We succeeded in producing site-specific recombination events in the hybrid progeny of 11 independent barley plants carrying the above target sequence after crossing with plants carrying a phiC31 expression cassette. Some of the hybrids displayed fully executed recombination. Excision of the GFP gene fostered activation of the gusA gene, as visualized in tissue of hybrid plants by histochemical staining. The recombinant loci were detected in progeny of selfed F1, even in individuals lacking the phiC31 transgene, which provides evidence of stability and generative transmission of the recombination events. In several plants that displayed incomplete recombination, extrachromosomal excision circles were identified. Besides the technical advance achieved in this study, the generated phiC31 integrase-expressing barley plants provide foundational stock material for use in future approaches to barley genetic improvement, such as the production of marker-free transgenic plants or switching transgene activity. PMID:23024817

Development of ELISA using recombinant antigens for specific detection of mouse parvovirus infection.

PubMed

Kunita, Satoshi; Chaya, Miyuki; Hagiwara, Kozue; Ishida, Tomoko; Takakura, Akira; Sugimoto, Tatsuya; Iseki, Hiroyoshi; Fuke, Kumiko; Sugiyama, Fumihiro; Yagami, Ken-ichi

2006-04-01

Nucleotide sequences of mouse parvovirus (MPV) isolate, named MPV/UT, and mouse minute virus (MMV) were analyzed and used for expressing recombinant proteins in E. coli. ELISA tests using recombinant major capsid protein (rVP2) and recombinant major non-structural protein (rNS1) as antigens were developed and their performance in serologic detection of rodent parvovirus infection was assessed. MPV-rVP2 and MMV-rVP2 ELISAs reacted specifically with anti-MPV and anti-MMV mouse sera, respectively. MMV-rNS1 antigen had a wide reaction range with antisera to rodent parvoviruses including MPV, MMV, Kilham rat virus (KRV) and H-1 virus. All mice oronasally infected with MPV were seropositive at 4 weeks post-infection in screening by ELISAs using MPV-rVP2 and MMV-rNS1 antigens, but were negative by conventional ELISA using whole MMV antigen. A contact transmission experiment revealed that transmission of MPV occurred up to 4 weeks post-infection, and all cage mates were seropositive in screening with MPV-rVP2 and MMV-rNS1 ELISAs. These results indicate that MPV-rVP2 and MMV-rVP2 are specific ELISA antigens which distinguish between MPV and MVM infection, while MMV-rNS1 antigen can be used in generic ELISA for a variety of rodent parvoviruses. The higher sensitivity of MPV-rVP2 ELISA than conventional ELISA for detecting seroconversion to MPV in oronasally infected mice as well as in cage mates suggests the usefulness of MPV-rVP2 ELISA in quarantine and microbiological monitoring of MPV infection in laboratory mice.

Isolated lymphoid follicles are not IgA inductive sites for recombinant Salmonella

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hashizume, Tomomi; Momoi, Fumiki; Kurita-Ochiai, Tomoko

2007-08-24

In this study, we investigated whether isolated lymphoid follicles (ILF) play a role in the regulation of intestinal IgA antibody (Ab) responses. The transfer of wild type (WT) bone marrow (BM) to lymphotoxin-{alpha}-deficient (LT{alpha}{sup -/-}) mice resulted in the formation of mature ILF containing T cells, B cells, and FDC clusters in the absence of mesenteric lymph nodes and Peyer's patches. Although the ILF restored total IgA Abs in the intestine, antigen (Ag)-specific IgA responses were not induced after oral immunization with recombinant Salmonella expressing fragment C of tetanus toxin. Moreover, Ag-specific cell proliferation was not detected in the ILF.more » Interestingly, no IgA anti-LPS Abs were detected in the fecal extracts of LT{alpha}{sup -/-} mice reconstituted with WT BM. On the basis of these findings, ILF can be presumed to play a role in the production of IgA Abs, but lymphoid nodules are not inductive sites for the regulation of Ag-specific intestinal IgA responses to recombinant Salmonella.« less

Identification and characterization of an SPO11 homolog in the mouse.

PubMed

Metzler-Guillemain, C; de Massy, B

2000-01-01

The SPO11/TOPVIA family includes proteins from archaebacteria and eukaryotes. The protein member from the archaebacterium Sulfulobus shibatae is the catalytic subunit of TopoVI DNA topoisomerase. In Saccharomyces cerevisiae, Schizosaccharomyces pombe, Caenorhabditis elegans and Drosophila melanogaster, SPO11 is required for meiotic recombination, suggesting a conserved mechanism for the initiation step of this process. Indeed, S. cerevisiae SPO11 has been shown to be directly involved in the formation of meiotic DNA double-strand breaks that initiate meiotic recombination. Here, we report the identification of a Mus musculus Spo11 cDNA, which encodes a protein closely related to all members of the SPO11/TOPVIA family. cDNAs resulting from alternative splicing were detected, suggesting that there are potential variants of the mouse SPO11 protein. By RNA-blotting analysis, expression of the mouse Spo11 gene was detected only in the testis, in agreement with its predicted function in the initiation of meiotic recombination. We mapped the mouse Spo11 gene to chromosome 2, band H2-H4.

Oral vaccine of Lactococcus lactis harbouring pandemic H1N1 2009 haemagglutinin1 and nisP anchor fusion protein elevates anti-HA1 sIgA levels in mice.

PubMed

Joan, Stella Siaw Xiu; Pui-Fong, Jee; Song, Adelene Ai-Lian; Chang, Li-Yen; Yusoff, Khatijah; AbuBakar, Sazaly; Rahim, Raha Abdul

2016-05-01

An oral lactococcal-based vaccine which haboured the haemagglutinin1 (HA1) antigen fused to nisP anchor protein for the purpose of surface displaying the HA1 antigen was developed against H1N1 virus. Recombinant L. lactis strains expressed HA1-nisP fusion proteins when induced with nisin, as confirmed through western blotting. However, immunofluorescense did not detect any surface-displayed proteins, suggesting that the protein was either unsuccessfully translocated or improperly displayed. Despite this, oral administration of recombinant L. lactis strains to BALB/c mice revealed that significant levels of anti-HA1 sIgA antibodies were detected in mice fecal suspension samples of mice group NZ9000 (pNZ:HN) when compared to the negative control NZ9000 (pNZ8048) group. Specific anti-HA1 sIgA antibodies were locally produced and live recombinant lactococcal vaccine was able to elicit humoral response of BALB/c mice despite unsuccessful surface display of the HA1 epitope.

A New Approach for Detection Improvement of the Creutzfeldt-Jakob Disorder through a Specific Surface Chemistry Applied onto Titration Well

PubMed Central

Mille, Caroline; Debarnot, Dominique; Zorzi, Willy; Moualij, Benaissa El; Quadrio, Isabelle; Perret-Liaudet, Armand; Coudreuse, Arnaud; Legeay, Gilbert; Poncin-Epaillard, Fabienne

2012-01-01

This work illustrates the enhancement of the sensitivity of the ELISA titration for recombinant human and native prion proteins, while reducing other non-specific adsorptions that could increase the background signal and lead to a low sensitivity and false positives. It is achieved thanks to the association of plasma chemistry and coating with different amphiphilic molecules bearing either ionic charges and/or long hydrocarbon chains. The treated support by 3-butenylamine hydrochloride improves the signal detection of recombinant protein, while surface modification with the 3,7-dimethylocta-2,6-dien-1-diamine (geranylamine) enhances the sensitivity of the native protein. Beside the surface chemistry effect, these different results are associated with protein conformation. PMID:25586034

Methods for efficient high-throughput screening of protein expression in recombinant Pichia pastoris strains.

PubMed

Camattari, Andrea; Weinhandl, Katrin; Gudiminchi, Rama K

2014-01-01

The methylotrophic yeast Pichia pastoris is becoming one of the favorite industrial workhorses for protein expression. Due to the widespread use of integration vectors, which generates significant clonal variability, screening methods allowing assaying hundreds of individual clones are of particular importance. Here we describe methods to detect and analyze protein expression, developed in a 96-well format for high-throughput screening of recombinant P. pastoris strains. The chapter covers essentially three common scenarios: (1) an enzymatic assay for proteins expressed in the cell cytoplasm, requiring cell lysis; (2) a whole-cell assay for a fungal cytochrome P450; and (3) a nonenzymatic assay for detection and quantification of tagged protein secreted into the supernatant.

Simple method to detect triacylglycerol biosynthesis in a yeast-based recombinant system

USDA-ARS?s Scientific Manuscript database

Standard methods to quantify the activity of triacylglycerol (TAG) synthesizing enzymes DGAT and PDAT (TAG-SE) require a sensitive but rather arduous laboratory assay based on radio-labeled substrates. Here we describe two straightforward methods to detect TAG production in baker’s yeast Saccharomyc...

Development of an Enzyme Linked Immunosorbent Assay to Detect Chicken Parvovirus Specific Antibodies

USDA-ARS?s Scientific Manuscript database

Here we report the development and application of an enzyme linked immunosorbent assay to detect parvovirus-specific antibodies in chicken sera. We used an approach previously described for other parvoviruses to clone and express viral structural proteins in insect cells from recombinant baculovirus...

Laboratory spectroscopy of methoxymethanol in the millimeter-wave range

NASA Astrophysics Data System (ADS)

Motiyenko, Roman A.; Margulès, Laurent; Despois, Didier; Guillemin, Jean-Claude

2018-02-01

Methoxymethanol, CH3OCH2OH is a very interesting candidate for detection in the interstellar medium since it can be formed in the recombination reaction between two radicals considered as intermediates in methanol formation: CH3O (already detected in the ISM) and CH2OH.

Purification of active chloroplast sedoheptulose-1,7-bisphosphatase expressed in Escherichia coli.

PubMed

Dunford, R P; Catley, M A; Raines, C A; Lloyd, J C; Dyer, T A

1998-10-01

Sedoheptulose-1,7-bisphosphatase (SBPase) is an enzyme unique to photosynthetic organisms and has a key role in regulating the photosynthetic Calvin cycle through which nearly all carbon enters the biosphere. This makes SBPase an appropriate target for intensive study. We have expressed wheat SBPase in Escherichia coli either with or without an N-terminal polyhistidine tag. The identity of the recombinant SBPases was confirmed by SDS-PAGE analysis and immunological detection with a specific antibody. Recombinant SBPase with a polyhistidine tag (His-SBPase) was obtained in soluble, active form and purified by one-step metal-chelate chromatography. Like the native enzyme, recombinant His-SBPase was specific for the substrate sedoheptulose-1,7-bisphosphate and required the presence of a reducing agent for activity. Polyclonal antibodies were raised against recombinant SBPase and were then used to determine relative levels of the enzyme in plant extracts. The availability of large amounts of active recombinant SBPase will also allow detailed structural studies by site-directed mutagenesis and X-ray crystallography. Copyright 1998 Academic Press.

Mus81 and Yen1 promote reciprocal exchange during mitotic recombination to maintain genome integrity in budding yeast

PubMed Central

Ho, Chu Kwen; Mazón, Gerard; Lam, Alicia F.; Symington, Lorraine S.

2010-01-01

Holliday junction (HJ) resolution is required for segregation of chromosomes and for formation of crossovers during homologous recombination. The identity of the resolvase(s) that functions in vivo has yet to be established, although several proteins able to cut HJs in vitro have been identified as candidates in yeasts and mammals. Using an assay to detect unselected products of mitotic recombination we found a significant decrease in crossovers in the Saccharomyces cerevisiae mus81Δ mutant. Yen1 serves a back-up function responsible for resolving intermediates in mus81Δ mutants, or when conversion tracts are short. In the absence of both Mus81 and Yen1 intermediates are not channeled exclusively to non-crossover recombinants, but instead are processed by Pol32-dependent break-induced replication (BIR). The channeling of recombination from reciprocal exchange to BIR results in greatly increased spontaneous loss of heterozygosity (LOH) and chromosome mis-segregation in the mus81Δ yen1Δ mutant, typical of the genomic instability found in tumor cells. PMID:21172663

[Expression of goat IL-18 mature protein in insect/baculovirus and determination of bioactivity of the recombinant protein].

PubMed

Wang, Ting-Ting; Wang, Xi-Hui; Fan, Zhong-Ling; Chen, Jin-Long; Cao, Bing-Lei; Kong, Na; Hu, Jing-Dong; Zhao, Hong-Kun

2011-02-01

To express goat IL-18 in insect/baculovirus and detect the bioactivity of the recombinant protein. The mature goat interleukin-18(gIL-18) gene was cloned into the baculovirus transfer vector pFastBac Dual, and then the resulting eukaryotic expression plasmid pFastBac Dual-gIL18 was transformed into DH10Bac, followed by the identification of Bacmid-gIL18 recombinat plosmid by three antibiotics and blue-white patch. Finally, the recombinant bacmid was transfected into sf9 insect cells by Cellfectin and the transfected cells were harvested at different times. Then the expressed protein was identified by SDS-PAGE, Western blot and bioactivity assay. The recombinant protein recognized and bound to its specific antibody. Bioactivity assay showed that the recombinant protein stimulated the proliferation of lymphocytes and induced IFN-γproduction in spleen lymphocytes. The mature gIL-18 protein has been expressed successfully in insect/baculovirus expression system, and have good immunogenicity and bioactivity. The study paves a way for application of gIL-18 as an immunomodulator or immune adjuvant.

Generation of Recombinant Porcine Parvovirus Virus-Like Particles in Saccharomyces cerevisiae and Development of Virus-Specific Monoclonal Antibodies

PubMed Central

Tamošiūnas, Paulius Lukas; Petraitytė-Burneikienė, Rasa; Lasickienė, Rita; Sereika, Vilimas; Lelešius, Raimundas; Žvirblienė, Aurelija; Sasnauskas, Kęstutis

2014-01-01

Porcine parvovirus (PPV) is a widespread infectious virus that causes serious reproductive diseases of swine and death of piglets. The gene coding for the major capsid protein VP2 of PPV was amplified using viral nucleic acid extract from swine serum and inserted into yeast Saccharomyces cerevisiae expression plasmid. Recombinant PPV VP2 protein was efficiently expressed in yeast and purified using density gradient centrifugation. Electron microscopy analysis of purified PPV VP2 protein revealed the self-assembly of virus-like particles (VLPs). Nine monoclonal antibodies (MAbs) against the recombinant PPV VP2 protein were generated. The specificity of the newly generated MAbs was proven by immunofluorescence analysis of PPV-infected cells. Indirect IgG ELISA based on the recombinant VLPs for detection of PPV-specific antibodies in swine sera was developed and evaluated. The sensitivity and specificity of the new assay were found to be 93.4% and 97.4%, respectively. In conclusion, yeast S. cerevisiae represents a promising expression system for generating recombinant PPV VP2 protein VLPs of diagnostic relevance. PMID:25045718

A new RT-PCR assay for the identification of the predominant recombination types in 2C and 3D genomic regions of vaccine-derived poliovirus strains.

PubMed

Pliaka, V; Dedepsidis, E; Kyriakopoulou, Z; Mpirli, K; Tsakogiannis, D; Pratti, A; Levidiotou-Stefanou, S; Markoulatos, P

2010-06-01

In the post-eradication era of wild polioviruses, the only remaining sources of poliovirus infection worldwide would be the vaccine-derived polioviruses (VDPVs). As the preponderance of countries certified to be polio-free has switched from OPV (oral poliovirus vaccine) to IPV (inactivated poliovirus vaccine), importation of recombinant evolved derivatives of vaccinal strains would have serious implication for public health. To test the robustness of the proposed RT-PCR screening analysis, eleven recombinant vaccine-derived polioviruses that were characterized previously by sequencing by our group, in addition to three recently identified recombinant environmental isolates were assayed. Although the most definitive characterization of VDPVs is by genomic sequencing, in this study we describe a new, inexpensive and broadly applicable RT-PCR assay for the identification of the predominant recombination types S3/Sx in 2C and S2/Sx in 3D genomic regions respectively of VDPVs, that can be readily implemented in laboratories lacking sequencing facilities as a first approach for the early detection of vaccine-derived poliovirus (VDPVs).

[Recombinant hFOXA2 and hPDX1 lentivirus induced dental pulp stem cells from deciduous teeth reprogramming for insulin-producing cells].

PubMed

Shi, Jian-feng; Zhu, Chun-hui; Liu, Jin; Sun, Jun-yi; Rao, Guo-zhou; Li, Ang

2013-12-01

The purpose of this study was to culture and identify dental pulp stem cells(DPSCs) from deciduous teeth in vitro and construct the recombinant hFOXA2 and hPDX1 lentivirus vectors and transfect the DPSCs to induce insulin-producing cells (IPCs). DPSCs were separated and cultured by enzyme digest method, and purified by limited dilution method. Flow cytometry was used to determine the surface marker expression of the DPSCs, and the ability of multiple differentiations was determined by specific staining. hFOXA2 and hPDX1 genes were amplified by PCR, and the recombinant hFOXA2 and hPDX1 lentivirus vectors were reconstructed and transfected into 293T cells by lipofectamine2000 for virus packaging. The viral infection efficiency and titer were determined through fluorescence cell count. The recombinant virus was used to infect the DPSCs cells via multiplicity of infection (MOI) and induce the DPSCs reprogramming for IPCs. Immunofluorescence staining was used to measure the expression of proinsulin, FOXA2 and PDX1. ELISA method was used to detect the insulin secretion. The data was analyzed Using SPSS13.0 software package. DPSCs were isolated and cultured successfully. Cell surface highly expressed STRO-1 (98.01%), CDl46 (98.51%), CD34 (99.54%) and CD45 (24.08%). The multi-lineage differentiation capacity into osteoblasts, chondrocytes, and adipose was achieved. The recombinant hFOXA2 and hPDX1 lentivirus vectors were successfully constructed. Double enzyme digestion and sequencing appraisal showed that the sequence was fully consistent with GenBank retrieval. Virus packing efficiency was (96.15±0.17) % and (95.49±0.21) % respectively, and the infection titer was about 1.80±108 GTU/mL. The best MOI of the virus was 20. After inducing the cells to express proinsulin, FOXA2 and PDX1, insulin secretion volume was about 1.92 μmol/L. Compared with the uninduced group and control group, insulin secretion increased significantly (P<0.01). The recombinant transcription factor virus can activate cell reprogramming mechanism, form insulin-producing cells, and can be used for gene therapy of diabetes seed cells. Supported by Science and Technology Research Program of Shaanxi Province (2009K17-06) and Science and Technology Innovation as a Whole Plan Resources Leading Industry Key Technology (Chain) Project of Shaanxi Province (2011KTCL03-24).

Genetic diversity and recombination analysis of sweepoviruses from Brazil

PubMed Central

2012-01-01

Background Monopartite begomoviruses (genus Begomovirus, family Geminiviridae) that infect sweet potato (Ipomoea batatas) around the world are known as sweepoviruses. Because sweet potato plants are vegetatively propagated, the accumulation of viruses can become a major constraint for root production. Mixed infections of sweepovirus species and strains can lead to recombination, which may contribute to the generation of new recombinant sweepoviruses. Results This study reports the full genome sequence of 34 sweepoviruses sampled from a sweet potato germplasm bank and commercial fields in Brazil. These sequences were compared with others from public nucleotide sequence databases to provide a comprehensive overview of the genetic diversity and patterns of genetic exchange in sweepoviruses isolated from Brazil, as well as to review the classification and nomenclature of sweepoviruses in accordance with the current guidelines proposed by the Geminiviridae Study Group of the International Committee on Taxonomy of Viruses (ICTV). Co-infections and extensive recombination events were identified in Brazilian sweepoviruses. Analysis of the recombination breakpoints detected within the sweepovirus dataset revealed that most recombination events occurred in the intergenic region (IR) and in the middle of the C1 open reading frame (ORF). Conclusions The genetic diversity of sweepoviruses was considerably greater than previously described in Brazil. Moreover, recombination analysis revealed that a genomic exchange is responsible for the emergence of sweepovirus species and strains and provided valuable new information for understanding the diversity and evolution of sweepoviruses. PMID:23082767

Polarity of recombination in transformation of Streptococcus pneumoniae

PubMed Central

Pasta, Franck; Sicard, Michel A.

1999-01-01

In transformation of Streptococcus pneumoniae DNA enters the cell as single-strand fragments and integrates into the chromosome by homologous recombination. Deletions and insertions of a few hundred base pairs frequently stop the recombination process of a donor strand. In this work we took advantage of such interruptions of recombination to compare the transformation efficiencies of the segments 5′- and 3′-ward from a deletion. The deletion was created in the center of a fragment of the ami locus, and sites around the deletion were labeled by a frameshift generating a restriction site. Heteroduplexes were constructed containing two restriction sites on one strand and two different ones on the complementary strand. ami+ bacteria were transformed with such heteroduplexes. ami− transformants were isolated and individually underwent amplification of the transformed ami region. We have obtained two kinds of amplification products: short when the deletion was integrated, long when recombination stops at the deletion. Each long fragment was tested by the four restriction enzymes to detect which strand and which side of the deletion had recombined. We found that 80% of the cuts were located 5′ to the deletion, showing that, in vivo, the 5′ side is strongly favored by recombination. Further results suggest that exchanges occurring from 5′ to 3′ relative to the donor strand are more efficient than in the opposite direction, thus accounting for the 5′ preference. PMID:10077616

Polarity of recombination in transformation of Streptococcus pneumoniae.

PubMed

Pasta, F; Sicard, M A

1999-03-16

In transformation of Streptococcus pneumoniae DNA enters the cell as single-strand fragments and integrates into the chromosome by homologous recombination. Deletions and insertions of a few hundred base pairs frequently stop the recombination process of a donor strand. In this work we took advantage of such interruptions of recombination to compare the transformation efficiencies of the segments 5'- and 3'-ward from a deletion. The deletion was created in the center of a fragment of the ami locus, and sites around the deletion were labeled by a frameshift generating a restriction site. Heteroduplexes were constructed containing two restriction sites on one strand and two different ones on the complementary strand. ami+ bacteria were transformed with such heteroduplexes. ami- transformants were isolated and individually underwent amplification of the transformed ami region. We have obtained two kinds of amplification products: short when the deletion was integrated, long when recombination stops at the deletion. Each long fragment was tested by the four restriction enzymes to detect which strand and which side of the deletion had recombined. We found that 80% of the cuts were located 5' to the deletion, showing that, in vivo, the 5' side is strongly favored by recombination. Further results suggest that exchanges occurring from 5' to 3' relative to the donor strand are more efficient than in the opposite direction, thus accounting for the 5' preference.

Development and Evaluation of a Sensitive and Specific Assay for Diagnosis of Human Toxocariasis by Use of Three Recombinant Antigens (TES-26, TES-30USM, and TES-120)▿

PubMed Central

Mohamad, Suharni; Azmi, Norhaida Che; Noordin, Rahmah

2009-01-01

Diagnosis of human toxocariasis currently relies on serologic tests that use Toxocara excretory-secretory (TES) antigen to detect immunoglobulin G (IgG) antibodies to the larvae. In general, however, these assays do not have adequate specificity for use in countries in which other soil-transmitted helminths are endemic. The use of recombinant antigens in these assays, however, is promising for improving the specificity of the diagnosis of toxocariasis. Toward this goal, we developed an IgG4 enzyme-linked immunosorbent assay (ELISA) involving three recombinant antigens: rTES-30USM (previously produced), rTES-26, and rTES-120. The latter two antigens were produced by reverse transcription-PCR cloning; subcloned into glutathione S-transferase (GST)-tagged and His-tagged prokaryotic expression vectors, respectively; and expressed in Escherichia coli. The recombinant proteins were subsequently purified by affinity chromatography using GST and His-Trap resins. The diagnostic potential of each purified recombinant antigen was tested with various immunoglobulin classes (IgG, IgM, and IgE) and IgG subclasses. The IgG4 ELISA was determined to have the highest specificity and was further evaluated using a panel of serum samples. The rTES-26 IgG4 ELISA showed 80.0% (24/30 samples positive) sensitivity, and both the rTES-30USM IgG4 ELISA and rTES-120 IgG4 ELISA had 93.0% (28/30) sensitivity. Combined use of rTES-120 and rTES-30 IgG4 ELISA for the diagnosis of toxocariasis provided 100% sensitivity. The specificities of rTES-26, rTES-30USM, and rTES-120 antigens were 96.2%, 93.9%, and 92.0%, respectively. These results indicate that the development of a diagnostic test using the three recombinant antigens will allow for more-accurate detection of toxocariasis. PMID:19369434

Development and evaluation of a sensitive and specific assay for diagnosis of human toxocariasis by use of three recombinant antigens (TES-26, TES-30USM, and TES-120).

PubMed

Mohamad, Suharni; Azmi, Norhaida Che; Noordin, Rahmah

2009-06-01

Diagnosis of human toxocariasis currently relies on serologic tests that use Toxocara excretory-secretory (TES) antigen to detect immunoglobulin G (IgG) antibodies to the larvae. In general, however, these assays do not have adequate specificity for use in countries in which other soil-transmitted helminths are endemic. The use of recombinant antigens in these assays, however, is promising for improving the specificity of the diagnosis of toxocariasis. Toward this goal, we developed an IgG4 enzyme-linked immunosorbent assay (ELISA) involving three recombinant antigens: rTES-30USM (previously produced), rTES-26, and rTES-120. The latter two antigens were produced by reverse transcription-PCR cloning; subcloned into glutathione S-transferase (GST)-tagged and His-tagged prokaryotic expression vectors, respectively; and expressed in Escherichia coli. The recombinant proteins were subsequently purified by affinity chromatography using GST and His-Trap resins. The diagnostic potential of each purified recombinant antigen was tested with various immunoglobulin classes (IgG, IgM, and IgE) and IgG subclasses. The IgG4 ELISA was determined to have the highest specificity and was further evaluated using a panel of serum samples. The rTES-26 IgG4 ELISA showed 80.0% (24/30 samples positive) sensitivity, and both the rTES-30USM IgG4 ELISA and rTES-120 IgG4 ELISA had 93.0% (28/30) sensitivity. Combined use of rTES-120 and rTES-30 IgG4 ELISA for the diagnosis of toxocariasis provided 100% sensitivity. The specificities of rTES-26, rTES-30USM, and rTES-120 antigens were 96.2%, 93.9%, and 92.0%, respectively. These results indicate that the development of a diagnostic test using the three recombinant antigens will allow for more-accurate detection of toxocariasis.

The recombination landscape around forensic STRs: Accurate measurement of genetic distances between syntenic STR pairs using HapMap high density SNP data.

PubMed

Phillips, C; Ballard, D; Gill, P; Court, D Syndercombe; Carracedo, A; Lareu, M V

2012-05-01

Family studies can be used to measure the genetic distance between same-chromosome (syntenic) STRs in order to detect physical linkage or linkage disequilibrium. However, family studies are expensive and time consuming, in many cases uninformative, and lack a reliable means to infer the phase of the diplotypes obtained. HapMap provides a more comprehensive and fine-scale estimation of recombination rates using high density multi-point SNP data (average inter-SNP distance: 900 nucleotides). Data at this fine scale detects sub-kilobase genetic distances across the whole recombining human genome. We have used the most recent HapMap SNP data release 22 to measure and compare genetic distances, and by inference fine-scale recombination rates, between 29 syntenic STR pairs identified from 39 validated STRs currently available for forensic use. The 39 STRs comprise 23 core loci: SE33, Penta D & E, 13 CODIS and 7 non-CODIS European Standard Set STRs, plus supplementary STRs in the recently released Promega CS-7™ and Qiagen Investigator HDplex™ kits. Also included were D9S1120, a marker we developed for forensic use unique to chromosome 9, and the novel D6S1043 component STR of SinoFiler™ (Applied Biosystems). The data collated provides reliable estimates of recombination rates between each STR pair, that can then be placed into haplotype frequency calculators for short pedigrees with multiple meiotic inputs and which just requires the addition of allele frequencies. This allows all current STR sets or their combinations to be used in supplemented paternity analyses without the need for further adjustment for physical linkage. The detailed analysis of recombination rates made for autosomal forensic STRs was extended to the more than 50 X chromosome STRs established or in development for complex kinship analyses. Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.

A baculovirus-mediated strategy for full-length plant virus coat protein expression and purification.

PubMed

Ardisson-Araújo, Daniel Mendes Pereira; Rocha, Juliana Ribeiro; da Costa, Márcio Hedil Oliveira; Bocca, Anamélia Lorenzetti; Dusi, André Nepomuceno; de Oliveira Resende, Renato; Ribeiro, Bergmann Morais

2013-08-15

Garlic production is severely affected by virus infection, causing a decrease in productivity and quality. There are no virus-free cultivars and garlic-infecting viruses are difficult to purify, which make specific antibody production very laborious. Since high quality antisera against plant viruses are important tools for serological detection, we have developed a method to express and purify full-length plant virus coat proteins using baculovirus expression system and insects as bioreactors. In this work, we have fused the full-length coat protein (cp) gene from the Garlic Mite-borne Filamentous Virus (GarMbFV) to the 3'-end of the Polyhedrin (polh) gene of the baculovirus Autographa californica multiple nucleopolyhedrovirus (AcMNPV). The recombinant baculovirus was amplified in insect cell culture and the virus was used to infect Spodoptera frugiperda larvae. Thus, the recombinant fused protein was easily purified from insect cadavers using sucrose gradient centrifugation and analyzed by Western Blotting. Interestingly, amorphous crystals were produced in the cytoplasm of cells infected with the recombinant virus containing the chimeric-protein gene but not in cells infected with the wild type and recombinant virus containing the hexa histidine tagged Polh. Moreover, the chimeric protein was used to immunize rats and generate antibodies against the target protein. The antiserum produced was able to detect plants infected with GarMbFV, which had been initially confirmed by RT-PCR. The expression of a plant virus full-length coat protein fused to the baculovirus Polyhedrin in recombinant baculovirus-infected insects was shown to produce high amounts of the recombinant protein which was easily purified and efficiently used to generate specific antibodies. Therefore, this strategy can potentially be used for the development of plant virus diagnostic kits for those viruses that are difficult to purify, are present in low titers or are present in mix infection in their plant hosts.

Identification of new antigen candidates for the early diagnosis of Mycobacterium avium subsp. paratuberculosis infection in goats.

PubMed

Souriau, Armel; Freret, Sandrine; Foret, Benjamin; Willemsen, Peter T J; Bakker, Douwe; Guilloteau, Laurence A

2017-12-01

Currently Mycobacterium avium subsp. paratuberculosis (MAP) infection is diagnosed through indirect tests based on the immune response induced by the infection. The antigens commonly used in IFN-γ release assays (IGRA) are purified protein derivative tuberculins (PPD). However, PPDs, lack both specificity (Sp) and sensitivity (Se) in the early phase of infection. This study investigated the potential of 16 MAP recombinant proteins and five lipids to elicit the release of IFN-γ in goats from herds with or without a history of paratuberculosis. Ten recombinant proteins were selected as potential candidates for the detection of MAP infection in young goats. They were found to detect 25 to 75% of infected shedder (IS) and infected non-shedder (INS) kids younger than 10months of age. In comparison, PPD was shown to detect only 10% of INS and no IS kids. For seven antigens, Se (21-33%) and Sp (≥90%) of IGRA were shown to be comparable with PPD at 20months old. Only three antigens were suitable candidates to detect IS adult goats, although Se was lower than that obtained with PPD. In paratuberculosis-free herds, IGRA results were negative in 97% of indoor goats and 86% of outdoor goats using the 10 antigens. However, 22 to 44% of one-year-old outdoor goats were positive suggesting that they may be infected. In conclusion, this study showed that ten MAP recombinant proteins are potential candidates for early detection of MAP infected goats. Combining these antigens could form a possible set of MAP antigens to optimize the Se of caprine IGRA. Copyright © 2017 Elsevier Ltd. All rights reserved.

Development of a recombinant human ovarian (BG1) cell line containing estrogen receptor α and β for improved detection of estrogenic/antiestrogenic chemicals.

PubMed

Brennan, Jennifer C; Bassal, Arzoo; He, Guochun; Denison, Michael S

2016-01-01

Estrogenic endocrine-disrupting chemicals are found in environmental and biological samples, commercial and consumer products, food, and numerous other sources. Given their ubiquitous nature and potential for adverse effects, a critical need exists for rapidly detecting these chemicals. The authors developed an estrogen-responsive recombinant human ovarian (BG1Luc4E2) cell line recently accepted by the US Environmental Protection Agency (USEPA) and Organisation for Economic Co-operation and Development (OECD) as a bioanalytical method to detect estrogen receptor (ER) agonists/antagonists. Unfortunately, these cells appear to contain only 1 of the 2 known ER isoforms, ERα but not ERβ, and the differential ligand selectivity of these ERs indicates that the currently accepted screening method only detects a subset of total estrogenic chemicals. To improve the estrogen screening bioassay, BG1Luc4E2 cells were stably transfected with an ERβ expression plasmid and positive clones identified using ERβ-selective ligands (genistein and Br-ERβ-041). A highly responsive clone (BG1LucERβc9) was identified that exhibited greater sensitivity and responsiveness to ERβ-selective ligands than BG1Luc4E2 cells, and quantitative reverse-transcription polymerase chain reaction confirmed the presence of ERβ expression in these cells. Screening of pesticides and industrial chemicals identified chemicals that preferentially stimulated ERβ-dependent reporter gene expression. Together, these results not only demonstrate the utility of this dual-ER recombinant cell line for detecting a broader range of estrogenic chemicals than the current BG1Luc4E2 cell line, but screening with both cell lines allows identification of ERα- and ERβ-selective chemicals. © 2015 SETAC.

Development of a recombinant human ovarian (BG1) cell line containing estrogen receptor α and β for improved detection of estrogenic/antiestrogenic chemicals

PubMed Central

Brennan, Jennifer C.; Bassal, Arzoo; He, Guochun; Denison, Michael S.

2016-01-01

Estrogenic endocrine disrupting chemicals are found in environmental and biological samples, commercial and consumer products, food, and numerous other sources. Given their ubiquitous nature and potential for adverse effects, there is a critical need for rapidly detecting these chemicals. We developed an estrogen-responsive recombinant human ovarian (BG1Luc4E2) cell line recently accepted by the USEPA and OECD as a bioanalytical method to detect estrogen receptor (ER) agonists/antagonists. Unfortunately, these cells appear to contain only one of the two known ER isoforms, ERα but not ERβ, and the differential ligand selectivity of these ERs indicates that the currently accepted screening method only detects a subset of total estrogenic chemicals. To improve the estrogen screening bioassay, BG1Luc4E2 cells were stably transfected with an ERβ expression plasmid and positive clones identified using ERβ-selective ligands (genistein and Br-ERβ-041). A highly responsive clone (BG1LucERβc9) was identified that exhibited greater sensitivity and responsiveness to ERβ-selective ligands than BG1Luc4E2 cells and qRT-PCR confirmed the presence of ERβ expression in these cells. Screening of pesticides and industrial chemicals identified chemicals that preferentially stimulated ERβ-dependent reporter gene expression. Together, these results not only demonstrate the utility of this dual ER recombinant cell line for detecting a broader range of estrogenic chemicals than the current BG1Luc4E2 cell line, but screening with both cell lines allows identification of ERα and ERβ-selective chemicals. PMID:26139245

Identification of Novel Recombinant Forms of Hepatitis B Virus Generated from Genotypes Ae and G in HIV-1-Positive Japanese Men Who Have Sex with Men.

PubMed

Kojima, Yoko; Kawahata, Takuya; Mori, Haruyo; Furubayashi, Keiichi; Taniguchi, Yasushi; Itoda, Ichiro; Komano, Jun

2015-07-01

The rare hepatitis B virus (HBV) genotype G (HBV/G) coinfects HIV-1-positive individuals along with HBV/A and generates recombinants. However, the circulation of HBV A/G recombinants remains poorly understood. This molecular epidemiologic study examined HBV A/G recombinants in Japanese HIV-1-positive men who have sex with men (MSM). Initially, blood specimens submitted for confirmatory tests of HIV infection in Osaka and Tokyo, Japan, from 2006 to 2013 were examined for HIV-1, and HIV-1-positive specimens were screened for HBV. Among 817 specimens from HIV-1-positive individuals, HBsAg was detected in 59 specimens; of these, HBV/Ae (alternatively A2), a subgenotype of HBV/A prevalent in Europe and North America, was identified in 70.2%, HBV/C in 17.5%, and HBV/G in 10.5%, and HBV/E in 1.8% according to the core gene sequence. The full-length genome analysis of HBV was performed on HBV/G-positive specimens because some HBV A/G recombinants were historically overlooked by genotyping based on a partial genome analysis. It revealed that five of the specimens contained novel Ae/G recombinants, the core gene of which had a high sequence similarity to HBV/G. Detailed analyses showed that novel recombinants were coinfected with HBV/Ae in a recombinant-dominant fashion. No major drug-resistant mutations were found in the newly identified HBV Ae/G recombinants. Some of the individuals asymptomatically coinfected with HIV/HBV suffered mild liver injury. This study demonstrated that novel Ae/G HBV recombinants were identified in Japanese HIV-1-positive MSM. The pathogenicity of novel HBV Ae/G recombinants should be examined in a future longitudinal study. Surveillance of such viruses in HIV-1-positive individuals should be emphasized.

LDsplit: screening for cis-regulatory motifs stimulating meiotic recombination hotspots by analysis of DNA sequence polymorphisms.

PubMed

Yang, Peng; Wu, Min; Guo, Jing; Kwoh, Chee Keong; Przytycka, Teresa M; Zheng, Jie

2014-02-17

As a fundamental genomic element, meiotic recombination hotspot plays important roles in life sciences. Thus uncovering its regulatory mechanisms has broad impact on biomedical research. Despite the recent identification of the zinc finger protein PRDM9 and its 13-mer binding motif as major regulators for meiotic recombination hotspots, other regulators remain to be discovered. Existing methods for finding DNA sequence motifs of recombination hotspots often rely on the enrichment of co-localizations between hotspots and short DNA patterns, which ignore the cross-individual variation of recombination rates and sequence polymorphisms in the population. Our objective in this paper is to capture signals encoded in genetic variations for the discovery of recombination-associated DNA motifs. Recently, an algorithm called "LDsplit" has been designed to detect the association between single nucleotide polymorphisms (SNPs) and proximal meiotic recombination hotspots. The association is measured by the difference of population recombination rates at a hotspot between two alleles of a candidate SNP. Here we present an open source software tool of LDsplit, with integrative data visualization for recombination hotspots and their proximal SNPs. Applying LDsplit on SNPs inside an established 7-mer motif bound by PRDM9 we observed that SNP alleles preserving the original motif tend to have higher recombination rates than the opposite alleles that disrupt the motif. Running on SNP windows around hotspots each containing an occurrence of the 7-mer motif, LDsplit is able to guide the established motif finding algorithm of MEME to recover the 7-mer motif. In contrast, without LDsplit the 7-mer motif could not be identified. LDsplit is a software tool for the discovery of cis-regulatory DNA sequence motifs stimulating meiotic recombination hotspots by screening and narrowing down to hotspot associated SNPs. It is the first computational method that utilizes the genetic variation of recombination hotspots among individuals, opening a new avenue for motif finding. Tested on an established motif and simulated datasets, LDsplit shows promise to discover novel DNA motifs for meiotic recombination hotspots.

LDsplit: screening for cis-regulatory motifs stimulating meiotic recombination hotspots by analysis of DNA sequence polymorphisms

PubMed Central

2014-01-01

Background As a fundamental genomic element, meiotic recombination hotspot plays important roles in life sciences. Thus uncovering its regulatory mechanisms has broad impact on biomedical research. Despite the recent identification of the zinc finger protein PRDM9 and its 13-mer binding motif as major regulators for meiotic recombination hotspots, other regulators remain to be discovered. Existing methods for finding DNA sequence motifs of recombination hotspots often rely on the enrichment of co-localizations between hotspots and short DNA patterns, which ignore the cross-individual variation of recombination rates and sequence polymorphisms in the population. Our objective in this paper is to capture signals encoded in genetic variations for the discovery of recombination-associated DNA motifs. Results Recently, an algorithm called “LDsplit” has been designed to detect the association between single nucleotide polymorphisms (SNPs) and proximal meiotic recombination hotspots. The association is measured by the difference of population recombination rates at a hotspot between two alleles of a candidate SNP. Here we present an open source software tool of LDsplit, with integrative data visualization for recombination hotspots and their proximal SNPs. Applying LDsplit on SNPs inside an established 7-mer motif bound by PRDM9 we observed that SNP alleles preserving the original motif tend to have higher recombination rates than the opposite alleles that disrupt the motif. Running on SNP windows around hotspots each containing an occurrence of the 7-mer motif, LDsplit is able to guide the established motif finding algorithm of MEME to recover the 7-mer motif. In contrast, without LDsplit the 7-mer motif could not be identified. Conclusions LDsplit is a software tool for the discovery of cis-regulatory DNA sequence motifs stimulating meiotic recombination hotspots by screening and narrowing down to hotspot associated SNPs. It is the first computational method that utilizes the genetic variation of recombination hotspots among individuals, opening a new avenue for motif finding. Tested on an established motif and simulated datasets, LDsplit shows promise to discover novel DNA motifs for meiotic recombination hotspots. PMID:24533858

Biochemical and biophysical investigations of the interaction between human glucokinase and pro-apoptotic BAD.

PubMed

Rexford, Alix; Zorio, Diego A R; Miller, Brian G

2017-01-01

The glycolytic enzyme glucokinase (GCK) and the pro-apoptotic protein BAD reportedly reside within a five-membered complex that localizes to the mitochondria of mammalian hepatocytes and pancreatic β-cells. Photochemical crosslinking studies using a synthetic analog of BAD's BH3 domain and in vitro transcription/translation experiments support a direct interaction between BAD and GCK. To investigate the biochemical and biophysical consequences of the BAD:GCK interaction, we developed a method for the production of recombinant human BAD. Consistent with published reports, recombinant BAD displays high affinity for Bcl-xL (KD = 7 nM), and phosphorylation of BAD at S118, within the BH3 domain, abolishes this interaction. Unexpectedly, we do not detect association of recombinant, full-length BAD with recombinant human pancreatic GCK over a range of protein concentrations using various biochemical methods including size-exclusion chromatography, chemical cross-linking, analytical ultracentrifugation, and isothermal titration calorimetry. Furthermore, fluorescence polarization assays and isothermal titration calorimetry detect no direct interaction between GCK and BAD BH3 peptides. Kinetic characterization of GCK in the presence of high concentrations of recombinant BAD show modest (<15%) increases in GCK activity, observable only at glucose concentrations well below the K0.5 value. GCK activity is unaffected by BAD BH3 peptides. These results raise questions as to the mechanism of action of stapled peptide analogs modeled after the BAD BH3 domain, which reportedly enhance the Vmax value of GCK and stimulate insulin release in BAD-deficient islets. Based on our results, we postulate that the BAD:GCK interaction, and any resultant regulatory effect(s) upon GCK activity, requires the participation of additional members of the mitochondrial complex.

Progressive genomic convergence of two Helicobacter pylori strains during mixed infection of a patient with chronic gastritis.

PubMed

Cao, Qizhi; Didelot, Xavier; Wu, Zhongbiao; Li, Zongwei; He, Lihua; Li, Yunsheng; Ni, Ming; You, Yuanhai; Lin, Xi; Li, Zhen; Gong, Yanan; Zheng, Minqiao; Zhang, Minli; Liu, Jie; Wang, Weijun; Bo, Xiaochen; Falush, Daniel; Wang, Shengqi; Zhang, Jianzhong

2015-04-01

To study the detailed nature of genomic microevolution during mixed infection with multiple Helicobacter pylori strains in an individual. We sampled 18 isolates from a single biopsy from a patient with chronic gastritis and nephritis. Whole-genome sequencing was applied to these isolates, and statistical genetic tools were used to investigate their evolutionary history. The genomes fall into two clades, reflecting colonisation of the stomach by two distinct strains, and these lineages have accumulated diversity during an estimated 2.8 and 4.2 years of evolution. We detected about 150 clear recombination events between the two clades. Recombination between the lineages is a continuous ongoing process and was detected on both clades, but the effect of recombination in one clade was nearly an order of magnitude higher than in the other. Imputed ancestral sequences also showed evidence of recombination between the two strains prior to their diversification, and we estimate that they have both been infecting the same host for at least 12 years. Recombination tracts between the lineages were, on average, 895 bp in length, and showed evidence for the interspersion of recipient sequences that has been observed in in vitro experiments. The complex evolutionary history of a phage-related protein provided evidence for frequent reinfection of both clades by a single phage lineage during the past 4 years. Whole genome sequencing can be used to make detailed conclusions about the mechanisms of genetic change of H. pylori based on sampling bacteria from a single gastric biopsy. Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://group.bmj.com/group/rights-licensing/permissions.

Evaluation of a novel Dot-ELISA assay utilizing a recombinant protein for the effective diagnosis of Taenia pisiformis larval infections.

PubMed

Chen, Lin; Yang, Deying; Gu, Xiaobin; Peng, Xuerong; Yang, Guangyou

2014-08-29

Cysticercosis, caused by the larvae of Taenia pisiformis, is a common disease in domestic breeds of the rabbit Oryctolagus cuniculus that results in economic losses. At present, there is no convenient and effective method for the rapid detection of T. pisiformis larvae. Here, we developed and tested the efficacy of a Dot-ELISA assay for the diagnosis of T. pisiformis larval infections in rabbits, based on the expression of the recombinant fusion protein (rTp1) from the Tp1 gene. Rapid amplification of cDNA ends (RACE) was used to amplify the 3' ends of the Tp1 gene, based on the unigene similar to Ts1 gene (EU009656.1) which comes from transcriptome sequencing of T. pisiformis. The Tp1 gene was successfully amplified, cloned and expressed in BL21 (DE3). Western blot analysis revealed that the recombinant Tp1 protein is specifically recognized by rabbit T. pisiformis cysticercosis antisera. This purified recombinant fusion protein, rTp1, was probed by Dot-ELISA with sera from rabbits infected with T. pisiformis larvae and with other parasitic infections. Results showed that this Dot-ELISA assay had both high sensitivity (92.9-97.6%) and specificity (95.2-98.4%) to detect T. pisiformis larval infections. We also found very low levels of cross-reaction with other parasitic infections. This study has revealed that our novel Dot-ELISA assay utilizing the recombinant fusion protein, rTp1, has a strong potential for the effective diagnosis of T. pisiformis infections in rabbits. Copyright © 2014 Elsevier B.V. All rights reserved.

Detection of Inter-Lineage Natural Recombination in Avian Paramyxovirus Serotype 1 Using Simplified Deep Sequencing Platform

PubMed Central

Satharasinghe, Dilan A.; Murulitharan, Kavitha; Tan, Sheau W.; Yeap, Swee K.; Munir, Muhammad; Ideris, Aini; Omar, Abdul R.

2016-01-01

Newcastle disease virus (NDV) is a prototype member of avian paramyxovirus serotype 1 (APMV-1), which causes severe and contagious disease in the commercial poultry and wild birds. Despite extensive vaccination programs and other control measures, the disease remains endemic around the globe especially in Asia, Africa, and the Middle East. Being a single serotype, genotype II based vaccines remained most acceptable means of immunization. However, the evidence is emerging on failures of vaccines mainly due to evolving nature of the virus and higher genetic gaps between vaccine and field strains of APMV-1. Most of the epidemiological and genetic characterizations of APMVs are based on conventional methods, which are prone to mask the diverse population of viruses in complex samples. In this study, we report the application of a simple, robust, and less resource-demanding methodology for the whole genome sequencing of NDV, using next-generation sequencing (NGS) on the Illumina MiSeq platform. Using this platform, we sequenced full genomes of five virulent Malaysian NDV strains collected during 2004–2013. All isolates clustered within highly prevalent lineage 5 (specifically in lineage 5a); however, a significantly greater genetic divergence was observed in isolates collected from 2004 to 2011. Interestingly, genetic characterization of one isolate collected in 2013 (IBS025/13) shown natural recombination between lineage 2 and lineage 5. In the event of recombination, the isolate (IBS025/13) carried nucleocapsid protein consist of 55–1801 nucleotides (nts) and near-complete phosphoprotein (1804–3254 nts) genes of lineage 2 whereas surface glycoproteins (fusion, hemagglutinin-neuraminidase) and large polymerase of lineage 5. Additionally, the recombinant virus has a genome size of 15,186 nts which is characteristics for the old genotypes I–IV isolated from 1930 to 1960. Taken together, we report the occurrence of a natural recombination in circulating strains of NDV in commercial poultry using NGS methodology. These findings will not only highlight the potential of RNA viruses to evolve but also to consider the application of NGS in revealing the genetic diversity of these viruses in clinical materials. Factors that drive these evolutionary events and subsequent impact of these divergences on clinical outcome of the disease warrant future investigations. PMID:27965642

Detection of Inter-Lineage Natural Recombination in Avian Paramyxovirus Serotype 1 Using Simplified Deep Sequencing Platform.

PubMed

Satharasinghe, Dilan A; Murulitharan, Kavitha; Tan, Sheau W; Yeap, Swee K; Munir, Muhammad; Ideris, Aini; Omar, Abdul R

2016-01-01

Newcastle disease virus (NDV) is a prototype member of avian paramyxovirus serotype 1 (APMV-1), which causes severe and contagious disease in the commercial poultry and wild birds. Despite extensive vaccination programs and other control measures, the disease remains endemic around the globe especially in Asia, Africa, and the Middle East. Being a single serotype, genotype II based vaccines remained most acceptable means of immunization. However, the evidence is emerging on failures of vaccines mainly due to evolving nature of the virus and higher genetic gaps between vaccine and field strains of APMV-1. Most of the epidemiological and genetic characterizations of APMVs are based on conventional methods, which are prone to mask the diverse population of viruses in complex samples. In this study, we report the application of a simple, robust, and less resource-demanding methodology for the whole genome sequencing of NDV, using next-generation sequencing (NGS) on the Illumina MiSeq platform. Using this platform, we sequenced full genomes of five virulent Malaysian NDV strains collected during 2004-2013. All isolates clustered within highly prevalent lineage 5 (specifically in lineage 5a); however, a significantly greater genetic divergence was observed in isolates collected from 2004 to 2011. Interestingly, genetic characterization of one isolate collected in 2013 (IBS025/13) shown natural recombination between lineage 2 and lineage 5. In the event of recombination, the isolate (IBS025/13) carried nucleocapsid protein consist of 55-1801 nucleotides (nts) and near-complete phosphoprotein (1804-3254 nts) genes of lineage 2 whereas surface glycoproteins (fusion, hemagglutinin-neuraminidase) and large polymerase of lineage 5. Additionally, the recombinant virus has a genome size of 15,186 nts which is characteristics for the old genotypes I-IV isolated from 1930 to 1960. Taken together, we report the occurrence of a natural recombination in circulating strains of NDV in commercial poultry using NGS methodology. These findings will not only highlight the potential of RNA viruses to evolve but also to consider the application of NGS in revealing the genetic diversity of these viruses in clinical materials. Factors that drive these evolutionary events and subsequent impact of these divergences on clinical outcome of the disease warrant future investigations.

The Time Scale of Recombination Rate Evolution in Great Apes

PubMed Central

Stevison, Laurie S.; Woerner, August E.; Kidd, Jeffrey M.; Kelley, Joanna L.; Veeramah, Krishna R.; McManus, Kimberly F.; Bustamante, Carlos D.; Hammer, Michael F.; Wall, Jeffrey D.

2016-01-01

Abstract We present three linkage-disequilibrium (LD)-based recombination maps generated using whole-genome sequence data from 10 Nigerian chimpanzees, 13 bonobos, and 15 western gorillas, collected as part of the Great Ape Genome Project (Prado-Martinez J, et al. 2013. Great ape genetic diversity and population history. Nature 499:471–475). We also identified species-specific recombination hotspots in each group using a modified LDhot framework, which greatly improves statistical power to detect hotspots at varying strengths. We show that fewer hotspots are shared among chimpanzee subspecies than within human populations, further narrowing the time scale of complete hotspot turnover. Further, using species-specific PRDM9 sequences to predict potential binding sites (PBS), we show higher predicted PRDM9 binding in recombination hotspots as compared to matched cold spot regions in multiple great ape species, including at least one chimpanzee subspecies. We found that correlations between broad-scale recombination rates decline more rapidly than nucleotide divergence between species. We also compared the skew of recombination rates at centromeres and telomeres between species and show a skew from chromosome means extending as far as 10–15 Mb from chromosome ends. Further, we examined broad-scale recombination rate changes near a translocation in gorillas and found minimal differences as compared to other great ape species perhaps because the coordinates relative to the chromosome ends were unaffected. Finally, on the basis of multiple linear regression analysis, we found that various correlates of recombination rate persist throughout the African great apes including repeats, diversity, and divergence. Our study is the first to analyze within- and between-species genome-wide recombination rate variation in several close relatives. PMID:26671457

Chaperokine function of recombinant Hsp72 produced in insect cells using a baculovirus expression system is retained.

PubMed

Zheng, Hongying; Nagaraja, Ganachari M; Kaur, Punit; Asea, Edwina E; Asea, Alexzander

2010-01-01

Extracellular heat shock protein 72 (Hsp72; inducible form of the 70-kDa heat shock protein) plays a critical role in innate and adaptive immune responses and has shown promise as an ideal adjuvant for the optimization of antigen-specific anti-tumor vaccines. Recent studies suggest that to correctly elucidate the mechanisms by which Hsp72 exerts its beneficial effects in vitro, great care must be taken to ensure that endotoxin by-products do not invalidate the findings. In this study, we have taken advantage of the baculovirus expression vector system for production of endotoxin-free recombinant Hsp72. The coding sequence of human hsp72 was recombined into the baculovirus immediately downstream of the strong polyhedron gene promoter. Ninety-six h post-infection of Sf9 insect cells with recombinant baculovirus, maximal levels of Hsp72 protein were detected. The recombinant human Hsp72 was purified by affinity chromatography from insect cells, and purity was confirmed by SDS-PAGE and mass spectrometry. The purified human recombinant Hsp72(bv) (Hsp72 produced using the BEVS) was demonstrated to have no endotoxin contamination and was shown to have stimulated potent calcium flux in the human monocytic cell line. Furthermore, recombinant Hsp72(bv) enhanced the tolerance of neuroblastoma cells to heat stress-induced cell death and displayed classical chaperokine functions including augmentation of inflammatory cytokine productions in mouse splenocytes. The production of functional, endotoxin-free recombinant human Hsp72(bv) in insect cells is inexpensive and convenient and eliminates the need of special procedures for endotoxin depletion. Endotoxin-free recombinant human Hsp72(bv) can now be used to unlock the important role Hsp72 plays in modulating immune function.

Chaperokine Function of Recombinant Hsp72 Produced in Insect Cells Using a Baculovirus Expression System Is Retained*

PubMed Central

Zheng, Hongying; Nagaraja, Ganachari M.; Kaur, Punit; Asea, Edwina E.; Asea, Alexzander

2010-01-01

Extracellular heat shock protein 72 (Hsp72; inducible form of the 70-kDa heat shock protein) plays a critical role in innate and adaptive immune responses and has shown promise as an ideal adjuvant for the optimization of antigen-specific anti-tumor vaccines. Recent studies suggest that to correctly elucidate the mechanisms by which Hsp72 exerts its beneficial effects in vitro, great care must be taken to ensure that endotoxin by-products do not invalidate the findings. In this study, we have taken advantage of the baculovirus expression vector system for production of endotoxin-free recombinant Hsp72. The coding sequence of human hsp72 was recombined into the baculovirus immediately downstream of the strong polyhedron gene promoter. Ninety-six h post-infection of Sf9 insect cells with recombinant baculovirus, maximal levels of Hsp72 protein were detected. The recombinant human Hsp72 was purified by affinity chromatography from insect cells, and purity was confirmed by SDS-PAGE and mass spectrometry. The purified human recombinant Hsp72bv (Hsp72 produced using the BEVS) was demonstrated to have no endotoxin contamination and was shown to have stimulated potent calcium flux in the human monocytic cell line. Furthermore, recombinant Hsp72bv enhanced the tolerance of neuroblastoma cells to heat stress-induced cell death and displayed classical chaperokine functions including augmentation of inflammatory cytokine productions in mouse splenocytes. The production of functional, endotoxin-free recombinant human Hsp72bv in insect cells is inexpensive and convenient and eliminates the need of special procedures for endotoxin depletion. Endotoxin-free recombinant human Hsp72bv can now be used to unlock the important role Hsp72 plays in modulating immune function. PMID:19861412

Fed batch fermentation and purification strategy for high yield production of Brucella melitensis recombinant Omp 28 kDa protein and its application in disease diagnosis.

PubMed

Karothia, B S; Athmaram, T N; D, Thavaselvam; Ashu, Kumar; Tiwari, Sapna; Singh, Anil K; Sathyaseelan, K; Gopalan, N

2013-07-01

Brucellosis is a disease caused by bacteria belonging to the genus Brucella. It affects cattle, goat, sheep, dog and humans. The serodiagnosis of brucellosis involves detection of antibodies generated against the LPS or whole cell bacterial extracts, however these tests lack sensitivity and specificity. The present study was performed to optimize the culture condition for the production of recombinant Brucella melitensis outer membrane protein 28 kDa protein in E.coli via fed batch fermentation. Expression was induced with 1.5mM isopropyl β thiogalactoside and the expressed recombinant protein was purified using Ni-NTA affinity chromatography. After fed-batch fermentation the dry cell weight of 17.81 g/L and a purified protein yield of 210.10 mg/L was obtained. The purified Brucella melitensis recombinant Omp 28 kDa protein was analyzed through SDS- poly acrylamide gel electrophoresis and western blotting. The obtained recombinant protein was evaluated for its diagnostic application through Indirect ELISA using brucellosis suspected human sera samples. Our results clearly indicate that recombinant Omp28 produced via fed batch fermentation has immense potential as a diagnostic reagent that could be employed in sero monitoring of brucellosis.

Genetic recombination variation in wild Robertsonian mice: on the role of chromosomal fusions and Prdm9 allelic background.

PubMed

Capilla, Laia; Medarde, Nuria; Alemany-Schmidt, Alexandra; Oliver-Bonet, Maria; Ventura, Jacint; Ruiz-Herrera, Aurora

2014-07-07

Despite the existence of formal models to explain how chromosomal rearrangements can be fixed in a population in the presence of gene flow, few empirical data are available regarding the mechanisms by which genome shuffling contributes to speciation, especially in mammals. In order to shed light on this intriguing evolutionary process, here we present a detailed empirical study that shows how Robertsonian (Rb) fusions alter the chromosomal distribution of recombination events during the formation of the germline in a Rb system of the western house mouse (Mus musculus domesticus). Our results indicate that both the total number of meiotic crossovers and the chromosomal distribution of recombination events are reduced in mice with Rb fusions and that this can be related to alterations in epigenetic signatures for heterochromatinization. Furthermore, we detected novel house mouse Prdm9 allelic variants in the Rb system. Remarkably, mean recombination rates were positively correlated with a decrease in the number of ZnF domains in the Prdm9 gene. The suggestion that recombination can be modulated by both chromosomal reorganizations and genetic determinants that control the formation of double-stranded breaks during meiosis opens new avenues for understanding the role of recombination in chromosomal speciation. © 2014 The Author(s) Published by the Royal Society. All rights reserved.

Evaluation of whole blood IFNgamma test using PPD and recombinant antigen challenge for diagnosis of pulmonary and extra-pulmonary tuberculosis.

PubMed

Kalantri, Yatiraj; Hemvani, Nanda; Chitnis, D S

2009-06-01

Quantiferon TB gold (QFT-G) with recombinant antigen cocktail is well evaluated for diagnosis of pulmonary tuberculosis (PTB). However, diagnosis of extra-pulmonary tuberculosis (EPTB) is more difficult due to limitations of conventional techniques. This study compares recombinant antigens based QFT-G and low cost PPD based interferon test for the diagnosis of PTB and EPTB. IFNgamma release, with recombinant antigens and PPD, was assayed by ELISA from 140 cases of EPTB, 100 cases of PTB along with acid fast bacillus (AFB) detection, AFB culture on LJ and MGIT BACTEC. Sensitivity and specificity for QFT-G recombinant antigens was 84.29% and 96%, while for PPD based interferon was 70% and 84% for EPTB group. The sensitivity was far superior to AFB smear and culture for both the antigens. Nine samples were identified as non-tubercular mycobacteria (NTM) in the EPTB group and all were negative for QFT-G, but six of them were positive for PPD based test. Results of the study show that QFT-G using recombinant antigen is sensitive and specific for both PTB and EPTB diagnosis. The PPD based test is economic and offers comparable performance for PTB and EPTB diagnosis and also useful for diagnosis of NTM.

A nuclear mutation defective in mitochondrial recombination in yeast.

PubMed

Ling, F; Makishima, F; Morishima, N; Shibata, T

1995-08-15

Homologous recombination (crossing over and gene conversion) is generally essential for heritage and DNA repair, and occasionally causes DNA aberrations, in nuclei of eukaryotes. However, little is known about the roles of homologous recombination in the inheritance and stability of mitochondrial DNA which is continuously damaged by reactive oxygen species, by-products of respiration. Here, we report the first example of a nuclear recessive mutation which suggests an essential role for homologous recombination in the stable inheritance of mitochondrial DNA. For the detection of this class of mutants, we devised a novel procedure, 'mitochondrial crossing in haploid', which has enabled us to examine many mutant clones. Using this procedure, we examined mutants of Saccharomyces cerevisiae that showed an elevated UV induction of respiration-deficient mutations. We obtained a mutant that was defective in both the omega-intron homing and Endo.SceI-induced homologous gene conversion. We found that the mutant cells are temperature sensitive in the maintenance of mitochondrial DNA. A tetrad analysis indicated that elevated UV induction of respiration-deficient mutations, recombination deficiency and temperature sensitivity are all caused by a single nuclear mutation (mhr1) on chromosome XII. The pleiotropic characteristics of the mutant suggest an essential role for the MHR1 gene in DNA repair, recombination and the maintenance of DNA in mitochondria.

Antigenic evaluation of a recombinant baculovirus-expressed Sarcocystis neurona SAG1 antigen.

PubMed

Gupta, G D; Lakritz, J; Saville, W J; Livingston, R S; Dubey, J P; Middleton, J R; Marsh, A E

2004-10-01

Sarcocystis neurona is the primary parasite associated with equine protozoal myeloencephalitis (EPM). This is a commonly diagnosed neurological disorder in the Americas that infects the central nervous system of horses. Current serologic assays utilize culture-derived parasites as antigen. This method requires large numbers of parasites to be grown in culture, which is labor intensive and time consuming. Also, a culture-derived whole-parasite preparation contains conserved antigens that could cross-react with antibodies against other Sarcocystis species and members of Sarcocystidae such as Neospora spp., Hammondia spp., and Toxoplasma gondii. Therefore, there is a need to develop an improved method for the detection of S. neurona-specific antibodies. The sera of infected horses react strongly to surface antigen 1 (SnSAG1), an approximately 29-kDa protein, in immunoblot analysis, suggesting that it is an immunodominant antigen. The SnSAG1 gene of S. neurona was cloned, and recombinant S. neurona SAG1 protein (rSnSAG1-Bac) was expressed with the use of a baculovirus system. By immunoblot analysis, the rSnSAG1-Bac antigen detected antibodies to S. neurona from naturally infected and experimentally inoculated equids, cats, rabbit, mice, and skunk. This is the first report of a baculovirus-expressed recombinant S. neurona antigen being used to detect anti-S. neurona antibodies in a variety of host species.

Recombinations in Staphylococcal Cassette Chromosome mec Elements Compromise the Molecular Detection of Methicillin Resistance in Staphylococcus aureus

PubMed Central

Hill-Cawthorne, Grant A.; Hudson, Lyndsey O.; El Ghany, Moataz Fouad Abd; Piepenburg, Olaf; Nair, Mridul; Dodgson, Andrew; Forrest, Matthew S.

2014-01-01

Clinical laboratories are increasingly using molecular tests for methicillin-resistant Staphylococcus aureus (MRSA) screening. However, primers have to be targeted to a variable chromosomal region, the staphylococcal cassette chromosome mec (SCCmec). We initially screened 726 MRSA isolates from a single UK hospital trust by recombinase polymerase amplification (RPA), a novel, isothermal alternative to PCR. Undetected isolates were further characterised using multilocus sequence, spa typing and whole genome sequencing. 96% of our tested phenotypically MRSA isolates contained one of the six orfX-SCCmec junctions our RPA test and commercially available molecular tests target. However 30 isolates could not be detected. Sequencing of 24 of these isolates demonstrated recombinations within the SCCmec element with novel insertions that interfered with the RPA, preventing identification as MRSA. This result suggests that clinical laboratories cannot rely solely upon molecular assays to reliably detect all methicillin-resistance. The presence of significant recombinations in the SCCmec element, where the majority of assays target their primers, suggests that there will continue to be isolates that escape identification. We caution that dependence on amplification-based molecular assays will continue to result in failure to diagnose a small proportion (∼4%) of MRSA isolates, unless the true level of SCCmec natural diversity is determined by whole genome sequencing of a large collection of MRSA isolates. PMID:24972080

Using Recombinant Proteins from Lutzomyia longipalpis Saliva to Estimate Human Vector Exposure in Visceral Leishmaniasis Endemic Areas

PubMed Central

Souza, Ana Paula; Andrade, Bruno Bezerril; Aquino, Dorlene; Entringer, Petter; Miranda, José Carlos; Alcantara, Ruan; Ruiz, Daniel; Soto, Manuel; Teixeira, Clarissa R.; Valenzuela, Jesus G.; de Oliveira, Camila Indiani; Brodskyn, Cláudia Ida; Barral-Netto, Manoel; Barral, Aldina

2010-01-01

Background Leishmania is transmitted by female sand flies and deposited together with saliva, which contains a vast repertoire of pharmacologically active molecules that contribute to the establishment of the infection. The exposure to vector saliva induces an immune response against its components that can be used as a marker of exposure to the vector. Performing large-scale serological studies to detect vector exposure has been limited by the difficulty in obtaining sand fly saliva. Here, we validate the use of two sand fly salivary recombinant proteins as markers for vector exposure. Methodology/principal findings ELISA was used to screen human sera, collected in an area endemic for visceral leishmaniasis, against the salivary gland sonicate (SGS) or two recombinant proteins (rLJM11 and rLJM17) from Lutzomyia longipalpis saliva. Antibody levels before and after SGS seroconversion (n = 26) were compared using the Wilcoxon signed rank paired test. Human sera from an area endemic for VL which recognize Lu. longipalpis saliva in ELISA also recognize a combination of rLJM17 and rLJM11. We then extended the analysis to include 40 sera from individuals who were seropositive and 40 seronegative to Lu. longipalpis SGS. Each recombinant protein was able to detect anti-saliva seroconversion, whereas the two proteins combined increased the detection significantly. Additionally, we evaluated the specificity of the anti-Lu. longipalpis response by testing 40 sera positive to Lutzomyia intermedia SGS, and very limited (2/40) cross-reactivity was observed. Receiver-operator characteristics (ROC) curve analysis was used to identify the effectiveness of these proteins for the prediction of anti-SGS positivity. These ROC curves evidenced the superior performance of rLJM17+rLJM11. Predicted threshold levels were confirmed for rLJM17+rLJM11 using a large panel of 1,077 serum samples. Conclusion Our results show the possibility of substituting Lu. longipalpis SGS for two recombinant proteins, LJM17 and LJM11, in order to probe for vector exposure in individuals residing in endemic areas. PMID:20351785

E3 ligase Hei10: a multifaceted structure-based signaling molecule with roles within and beyond meiosis

PubMed Central

De Muyt, Arnaud; Zhang, Liangran; Piolot, Tristan; Kleckner, Nancy; Espagne, Eric; Zickler, Denise

2014-01-01

Human enhancer of invasion-10 (Hei10) mediates meiotic recombination and also plays roles in cell proliferation. Here we explore Hei10’s roles throughout the sexual cycle of the fungus Sordaria with respect to localization and effects of null, RING-binding, and putative cyclin-binding (RXL) domain mutations. Hei10 makes three successive types of foci. Early foci form along synaptonemal complex (SC) central regions. At some of these positions, depending on its RING and RXL domains, Hei10 mediates development and turnover of two sequential types of recombination complexes, each demarked by characteristic amplified Hei10 foci. Integration with ultrastructural data for recombination nodules further reveals that recombination complexes differentiate into three types, one of which corresponds to crossover recombination events during or prior to SC formation. Finally, Hei10 positively and negatively modulates SUMO localization along SCs by its RING and RXL domains, respectively. The presented findings suggest that Hei10 integrates signals from the SC, associated recombination complexes, and the cell cycle to mediate both the development and programmed turnover/evolution of recombination complexes via SUMOylation/ubiquitination. Analogous cell cycle-linked assembly/disassembly switching could underlie localization and roles for Hei10 in centrosome/spindle pole body dynamics and associated nuclear trafficking. We suggest that Hei10 is a unique type of structure-based signal transduction protein. PMID:24831702

Mapping of new quantitative trait loci for sudden death syndrome and soybean cyst nematode resistance in two soybean populations.

PubMed

Swaminathan, Sivakumar; Abeysekara, Nilwala S; Knight, Joshua M; Liu, Min; Dong, Jia; Hudson, Matthew E; Bhattacharyya, Madan K; Cianzio, Silvia R

2018-05-01

Novel QTL conferring resistance to both the SDS and SCN was detected in two RIL populations. Dual resistant RILs could be used in breeding programs for developing resistant soybean cultivars. Soybean cultivars, susceptible to the fungus Fusarium virguliforme, which causes sudden death syndrome (SDS), and to the soybean cyst nematode (SCN) (Heterodera glycines), suffer yield losses valued over a billion dollars annually. Both pathogens may occur in the same production fields. Planting of cultivars genetically resistant to both pathogens is considered one of the most effective means to control the two pathogens. The objective of the study was to map quantitative trait loci (QTL) underlying SDS and SCN resistances. Two recombinant inbred line (RIL) populations were developed by crossing 'A95-684043', a high-yielding maturity group (MG) II line resistant to SCN, with 'LS94-3207' and 'LS98-0582' of MG IV, resistant to both F. virguliforme and SCN. Two hundred F 7 derived recombinant inbred lines from each population AX19286 (A95-684043 × LS94-3207) and AX19287 (A95-684043 × LS98-0582) were screened for resistance to each pathogen under greenhouse conditions. Five hundred and eighty and 371 SNP markers were used for mapping resistance QTL in each population. In AX19286, one novel SCN resistance QTL was mapped to chromosome 8. In AX19287, one novel SDS resistance QTL was mapped to chromosome 17 and one novel SCN resistance QTL was mapped to chromosome 11. Previously identified additional SDS and SCN resistance QTL were also detected in the study. Lines possessing superior resistance to both pathogens were also identified and could be used as germplasm sources for breeding SDS- and SCN-resistant soybean cultivars.

Multicentre evaluations of two new rapid IgG4 tests (WB rapid and panLF rapid) for detection of lymphatic filariasis

PubMed Central

Noordin, Rahmah; Itoh, Makoto; Kimura, Eisaku; Abdul Rahman, Rohana; Ravindran, Balachandran; Mahmud, Rohela; Supali, Taniawati; Weerasooriya, Mirani

2007-01-01

In the global effort to eliminate lymphatic filariasis (LF), rapid field-applicable tests are useful tools that will allow on-site testing to be performed in remote places and the results to be obtained rapidly. Exclusive reliance on the few existing tests may jeopardize the progress of the LF elimination program, thus the introduction of other rapid tests would be useful to address this issue. Two new rapid immunochromatographic IgG4 cassette tests have been produced, namely WB rapid and panLF rapid, for detection of bancroftian filariasis and all three species of lymphatic filaria respectively. WB rapid was developed using BmSXP recombinant antigen, while PanLF rapid was developed using BmR1 and BmSXP recombinant antigens. A total of 165 WB rapid and 276 panLF rapid tests respectively were evaluated at USM and the rest were couriered to another university in Malaysia (98 WB rapid, 129 panLF rapid) and to universities in Indonesia (56 WB rapid, 62 panLF rapid), Japan (152 of each test) and India (18 of each test) where each of the tests underwent independent evaluations in a blinded manner. The average sensitivities of WB rapid and panLF rapid were found to be 97.6% (94%–100%) and 96.5% (94%–100%) respectively; while their average specificities were both 99.6% (99%–100%). Thus this study demonstrated that both the IgG4 rapid tests were highly sensitive and specific, and would be useful additional tests to facilitate the global drive to eliminate this disease. PMID:17961262

Insecticidal activity of two proteases against Spodoptera frugiperda larvae infected with recombinant baculoviruses

PubMed Central

2010-01-01

Background Baculovirus comprise the largest group of insect viruses most studied worldwide, mainly because they efficiently kill agricutural insect pests. In this study, two recombinant baculoviruses containing the ScathL gene from Sarcophaga peregrina (vSynScathL), and the Keratinase gene from the fungus Aspergillus fumigatus (vSynKerat), were constructed. and their insecticidal properties analysed against Spodoptera frugiperda larvae. Results Bioassays of third-instar and neonate S. frugiperda larvae with vSynScathL and vSynKerat showed a decrease in the time needed to kill the infected insects when compared to the wild type virus. We have also shown that both recombinants were able to increase phenoloxidase activity in the hemolymph of S. frugiperda larvae. The expression of proteases in infected larvae resulted in destruction of internal tissues late in infection, which could be the reason for the increased viral speed of kill. Conclusions Baculoviruses and their recombinant forms constitute viable alternatives to chemical insecticides. Recombinant baculoviruses containing protease genes can be added to the list of engineered baculoviruses with great potential to be used in integrated pest management programs. PMID:20587066

Meiotic recombination hotspots - a comparative view.

PubMed

Choi, Kyuha; Henderson, Ian R

2015-07-01

During meiosis homologous chromosomes pair and undergo reciprocal genetic exchange, termed crossover. Meiotic recombination has a profound effect on patterns of genetic variation and is an important tool during crop breeding. Crossovers initiate from programmed DNA double-stranded breaks that are processed to form single-stranded DNA, which can invade a homologous chromosome. Strand invasion events mature into double Holliday junctions that can be resolved as crossovers. Extensive variation in the frequency of meiotic recombination occurs along chromosomes and is typically focused in narrow hotspots, observed both at the level of DNA breaks and final crossovers. We review methodologies to profile hotspots at different steps of the meiotic recombination pathway that have been used in different eukaryote species. We then discuss what these studies have revealed concerning specification of hotspot locations and activity and the contributions of both genetic and epigenetic factors. Understanding hotspots is important for interpreting patterns of genetic variation in populations and how eukaryotic genomes evolve. In addition, manipulation of hotspots will allow us to accelerate crop breeding, where meiotic recombination distributions can be limiting. © 2015 The Authors The Plant Journal © 2015 John Wiley & Sons Ltd.

Development of a Luminex Bead Based Assay for Diagnosis of Toxocariasis Using Recombinant Antigens Tc-CTL-1 and Tc-TES-26.

PubMed

Anderson, John P; Rascoe, Lisa N; Levert, Keith; Chastain, Holly M; Reed, Matthew S; Rivera, Hilda N; McAuliffe, Isabel; Zhan, Bin; Wiegand, Ryan E; Hotez, Peter J; Wilkins, Patricia P; Pohl, Jan; Handali, Sukwan

2015-01-01

The clinical spectrum of human disease caused by the roundworms Toxocara canis and Toxocara cati ranges from visceral and ocular larva migrans to covert toxocariasis. The parasite is not typically recovered in affected tissues, so detection of parasite-specific antibodies is usually necessary for establishing a diagnosis. The most reliable immunodiagnostic methods use the Toxocara excretory-secretory antigens (TES-Ag) in ELISA formats to detect Toxocara-specific antibodies. To eliminate the need for native parasite materials, we identified and purified immunodiagnostic antigens using 2D gel electrophoresis followed by electrospray ionization mass spectrometry. Three predominant immunoreactive proteins were found in the TES; all three had been previously described in the literature: Tc-CTL-1, Tc-TES-26, and Tc-MUC-3. We generated Escherichia coli expressed recombinant proteins for evaluation in Luminex based immunoassays. We were unable to produce a functional assay with the Tc-MUC-3 recombinant protein. Tc-CTL-1 and Tc-TES-26 were successfully coupled and tested using defined serum batteries. The use of both proteins together generated better results than if the proteins were used individually. The sensitivity and specificity of the assay for detecting visceral larval migrans using Tc-CTL-1 plus Tc-TES-26 was 99% and 94%, respectively; the sensitivity for detecting ocular larval migrans was 64%. The combined performance of the new assay was superior to the currently available EIA and could potentially be employed to replace current assays that rely on native TES-Ag.

Identification and characterisation of the proteins bound by specific phage-displayed recombinant antibodies (scFv) obtained against Brazil nut and almond extracts.

PubMed

de la Cruz, Silvia; Madrid, Raquel; García-García, Aina; Alcocer, Marcos; Martín, Rosario; González, Isabel; García, Teresa

2018-03-01

Almonds and Brazil nuts are widely consumed allergenic nuts whose presence must be declared according to food labelling regulations. Their detection in food products has been recently achieved by ELISA methods with recombinant antibodies (scFv) isolated against complete Brazil nut and almond protein extracts. The screening of phage-scFv libraries against complete protein extracts confers a series of advantages over the use of purified proteins, as recombinant proteins might alter their native folding. However, using this strategy, the nature of the target detected by phage-displayed antibodies remains unknown, and requires further research to identify whether they are nut allergens or other molecules present in the extract, but not related to their allergenic potential. Electrophoretic, chromatographic, immunological and spectrometric techniques revealed that the Brazil nut (BE95) and almond (PD1F6 and PD2C9) specific phage-scFvs detected conformational epitopes of the Brazil nut and almond 11S globulins, recognised by WHO/IUIS as Ber e 2 and Pru du 6 major allergens. Circular dichroism data indicated that severe heat treatment would entail loss of epitope structure, disabling scFv for target detection. The presence of important Brazil nut and almond allergens (Ber e 2 and Pru du 6) in foodstuffs can be determined by using phage-display antibodies BE95, PD1F6 and PD2C9 as affinity probes in ELISA. © 2017 Society of Chemical Industry. © 2017 Society of Chemical Industry.

Development of a Luminex Bead Based Assay for Diagnosis of Toxocariasis Using Recombinant Antigens Tc-CTL-1 and Tc-TES-26

PubMed Central

Anderson, John P.; Rascoe, Lisa N.; Levert, Keith; Chastain, Holly M.; Reed, Matthew S.; Rivera, Hilda N.; McAuliffe, Isabel; Zhan, Bin; Wiegand, Ryan E.; Hotez, Peter J.; Wilkins, Patricia P.; Pohl, Jan; Handali, Sukwan

2015-01-01

The clinical spectrum of human disease caused by the roundworms Toxocara canis and Toxocara cati ranges from visceral and ocular larva migrans to covert toxocariasis. The parasite is not typically recovered in affected tissues, so detection of parasite-specific antibodies is usually necessary for establishing a diagnosis. The most reliable immunodiagnostic methods use the Toxocara excretory-secretory antigens (TES-Ag) in ELISA formats to detect Toxocara-specific antibodies. To eliminate the need for native parasite materials, we identified and purified immunodiagnostic antigens using 2D gel electrophoresis followed by electrospray ionization mass spectrometry. Three predominant immunoreactive proteins were found in the TES; all three had been previously described in the literature: Tc-CTL-1, Tc-TES-26, and Tc-MUC-3. We generated Escherichia coli expressed recombinant proteins for evaluation in Luminex based immunoassays. We were unable to produce a functional assay with the Tc-MUC-3 recombinant protein. Tc-CTL-1 and Tc-TES-26 were successfully coupled and tested using defined serum batteries. The use of both proteins together generated better results than if the proteins were used individually. The sensitivity and specificity of the assay for detecting visceral larval migrans using Tc-CTL-1 plus Tc-TES-26 was 99% and 94%, respectively; the sensitivity for detecting ocular larval migrans was 64%. The combined performance of the new assay was superior to the currently available EIA and could potentially be employed to replace current assays that rely on native TES-Ag. PMID:26485145

Progressive Recombination Suppression and Differentiation in Recently Evolved Neo-sex Chromosomes

PubMed Central

Natri, Heini M.; Shikano, Takahito; Merilä, Juha

2013-01-01

Recombination suppression leads to the structural and functional differentiation of sex chromosomes and is thus a crucial step in the process of sex chromosome evolution. Despite extensive theoretical work, the exact processes and mechanisms of recombination suppression and differentiation are not well understood. In threespine sticklebacks (Gasterosteus aculeatus), a different sex chromosome system has recently evolved by a fusion between the Y chromosome and an autosome in the Japan Sea lineage, which diverged from the ancestor of other lineages approximately 2 Ma. We investigated the evolutionary dynamics and differentiation processes of sex chromosomes based on comparative analyses of these divergent lineages using 63 microsatellite loci. Both chromosome-wide differentiation patterns and phylogenetic inferences with X and Y alleles indicated that the ancestral sex chromosomes were extensively differentiated before the divergence of these lineages. In contrast, genetic differentiation appeared to have proceeded only in a small region of the neo-sex chromosomes. The recombination maps constructed for the Japan Sea lineage indicated that recombination has been suppressed or reduced over a large region spanning the ancestral and neo-sex chromosomes. Chromosomal regions exhibiting genetic differentiation and suppressed or reduced recombination were detected continuously and sequentially in the neo-sex chromosomes, suggesting that differentiation has gradually spread from the fusion point following the extension of recombination suppression. Our study illustrates an ongoing process of sex chromosome differentiation, providing empirical support for the theoretical model postulating that recombination suppression and differentiation proceed in a gradual manner in the very early stage of sex chromosome evolution. PMID:23436913

Protective efficacy of six immunogenic recombinant proteins of Vibrio anguillarum and evaluation them as vaccine candidate for flounder (Paralichthys olivaceus).

PubMed

Xing, Jing; Xu, Hongsen; Wang, Yang; Tang, Xiaoqian; Sheng, Xiuzhen; Zhan, Wenbin

2017-06-01

Vibrio anguillarum is a severe bacterium that causes terminal haemorrhagic septicaemia in freshwater and marine fish. Virulence-associated proteins play an important role in bacterial pathogenicity and could be applied for immunoprophylaxis. In this study, six antigenic proteins from V. anguillarum were selected and the immune protective efficacy of their recombinant proteins was investigated. VirA, CheR, FlaC, OmpK, OmpR and Hsp33 were recombinantly produced and the reactions of recombinant proteins to flounder-anti-V. anguillarum antibodies (fV-ab) were detected, respectively. Then the recombinant proteins were injected to fish, after immunization, the percentages of surface membrane immunoglobulin-positive (sIg+) cell in lymphocytes, total antibodies, antibodies against V. anguillarum, antibodies against recombinant proteins and relative percent survival (RPS) were analyzed, respectively. The results showed that all the recombinant proteins could react to fV-ab, proliferate sIg + cells in lymphocytes and induce production of total antibodies, specific antibodies against V. anguillarum or the recombinant proteins; the RPS of rVirA, rCheR, rFlaC, rOmpK, rOmpR and rHsp33 against V. anguillarum was 70.27%, 27.03%, 16.22%, 62.16%, 45.95% and 81.08%, respectively. The results revealed that rHsp33, rVirA and rOmpK have good protections against V. anguillarum and could be vaccine candidates against V. anguillarum. Copyright © 2017 Elsevier Ltd. All rights reserved.

The highly obscured nucleus of 3C 219

NASA Technical Reports Server (NTRS)

Fabbiano, G.; Willner, S. P.; Carleton, N. P; Elvis, M.

1986-01-01

The detection of a strong, and possibly broad, Paschen-alpha line from the narrow-line radio galaxy 3C 219 is reported. The detected flux is larger than predicted from the H-alpha line and the case B recombination. This implies the presence of a highly reddened line-emitting region in the nucleus.

Carbon lines at limitedly low frequencies

NASA Technical Reports Server (NTRS)

Valtts, I. Y.

1983-01-01

Detection of several absorption recombination radio lines of carbon at 26 MHz in Cas A direction resulted in an attempt to select similar situations (a gas cloud projection on the intense source of the nonthermal radio emission) that are promising for detecting lines of such a kind. Recommendations are given for observations to be made.

Affinity Pulldown of Biotinylated RNA for Detection of Protein-RNA Complexes.

PubMed

Panda, Amaresh C; Martindale, Jennifer L; Gorospe, Myriam

2016-12-20

RNA-binding proteins (RBPs) have recently emerged as crucial players in the regulation of gene expression. The interactions of RBPs with target mRNAs control the levels of gene products by altering different regulatory steps, including pre-mRNA splicing and maturation, nuclear mRNA export, and mRNA stability and translation (Glisovic et al. , 2008). There are several methodologies available today to identify RNAs bound to specific RBPs; some detect only recombinant molecules in vitro , others detect recombinant and endogenous molecules, while others detect only endogenous molecules. Examples include systematic evolution of ligands by exponential enrichment (SELEX), biotinylated RNA pulldown assay, RNA immunoprecipitation (RIP) assay, electrophoretic mobility shift assay (EMSA), RNA footprinting analysis, and various UV crosslinking and immunoprecipitation (CLIP) methods such as CLIP, PAR-CLIP, and iCLIP (Popova et al. , 2015). Here, we describe a simple and informative method to study and identify the RNA region of interaction between an RBP and its target transcript (Panda et al. , 2014 and 2016). Its reproducibility and ease of use make this protocol a fast and useful method to identify interactions between RBPs and specific RNAs.

Nucleoprotein-based indirect enzyme-linked immunosorbent assay (indirect ELISA) for detecting antibodies specific to Ebola virus and Marbug virus.

PubMed

Huang, Yi; Zhu, Youjie; Yang, Mengshi; Zhang, Zhenqing; Song, Donglin; Yuan, Zhiming

2014-12-01

Full-length nucleoproteins from Ebola and Marburg viruses were expressed as His-tagged recombinant proteins in Escherichia coli and nucleoprotein-based enzyme-linked immunosorbent assays (ELISAs) were established for the detection of antibodies specific to Ebola and Marburg viruses. The ELISAs were evaluated by testing antisera collected from rabbit immunized with Ebola and Marburg virus nucleoproteins. Although little cross-reactivity of antibodies was observed in anti-Ebola virus nucleoprotein rabbit antisera, the highest reactions to immunoglobulin G (IgG) were uniformly detected against the nucleoprotein antigens of homologous viruses. We further evaluated the ELISA's ability to detect antibodies to Ebola and Marburg viruses using human sera samples collected from individuals passing through the Guangdong port of entry. With a threshold set at the mean plus three standard deviations of average optical densities of sera tested, the ELISA systems using these two recombinant nucleoproteins have good sensitivity and specificity. These results demonstrate the usefulness of ELISA for diagnostics as well as ecological and serosurvey studies of Ebola and Marburg virus infection.

Development of Two FhSAP2 Recombinant–Based Assays for Immunodiagnosis of Human Chronic Fascioliasis

PubMed Central

Shin, Sun Hee; Hsu, Angel; Chastain, Holly M.; Cruz, Lorna A.; Elder, Eric S.; Sapp, Sarah G. H.; McAuliffe, Isabel; Espino, Ana M.; Handali, Sukwan

2016-01-01

In the United States, infection with Fasciola hepatica has been identified as an emerging disease, primarily in immigrants, refugees, and travelers. The laboratory test of choice for diagnosis of fascioliasis is detection of disease specific antibodies, most commonly uses excretory-secretory antigens for detection of IgG antibodies. Recently, recombinant proteins such as F. hepatica antigen (FhSAP2) have been used to detect IgG antibodies. The glutathione S-transferase (GST)–FhSAP2 recombinant antigen was used to develop Western blot (WB) and fluorescent bead-based (Luminex) assays to detect F. hepatica total IgG and IgG4 antibodies. The sensitivity and specificity of GST-FhSAP2 total IgG and IgG4 WB were similar at 94% and 98%, respectively. For the IgG Luminex assay, the sensitivity and specificity were 94% and 97%, and for the IgG4, the values were 100% and 99%, respectively. In conclusion, the GST-FhSAP2 antigen performs well in several assay formats and can be used for clinical diagnosis. PMID:27549636

Modeling ionization and recombination from low energy nuclear recoils in liquid argon

DOE PAGES

Foxe, M.; Hagmann, C.; Jovanovic, I.; ...

2015-03-27

Coherent elastic neutrino-nucleus scattering (CENNS) is an as-yet undetected, flavor-independent neutrino interaction predicted by the Standard Model. Detection of CENNS could offer benefits for detection of supernova and solar neutrinos in astrophysics, or for detection of antineutrinos for nuclear reactor monitoring and nuclear nonproliferation. One challenge with detecting CENNS is the low energy deposition associated with a typical CENNS nuclear recoil. In addition, nuclear recoils result in lower ionization yields than those produced by electron recoils of the same energy. While a measurement of the nuclear recoil ionization yield in liquid argon in the keV energy range has been recentlymore » reported, a corresponding model for low-energy ionization yield in liquid argon does not exist. For this reason, a Monte Carlo simulation has been developed to predict the ionization yield at sub-10 keV energies. The model consists of two distinct components: (1) simulation of the atomic collision cascade with production of ionization, and (2) the thermalization and drift of ionization electrons in an applied electric field including local recombination. As an application of our results we report updated estimates of detectable ionization in liquid argon from CENNS at a nuclear reactor.« less

Developing a HER3 Vaccine to Prevent Resistance to Endocrine Therapy

DTIC Science & Technology

2013-10-01

linearized using digestion with Pme I, recombined into linearized (E1-,E2b-,E3-) serotype 5 pAd construct in BJ 5183 bacterial recombination-based system...activity Clinical activity was assessed according to Response Evalu- ation Criteria in Solid Tumors ( RECIST 1.0 criteria [22]) using computed tomography...tumors: revised RECIST guideline (version 1.1). Eur J Cancer 45:228–247 23. CTEP Cancer Therapy Evaluation Program. CTCAE and CTC Website (2010) http

Finding trans-regulatory genes and protein complexes modulating meiotic recombination hotspots of human, mouse and yeast.

PubMed

Wu, Min; Kwoh, Chee-Keong; Li, Xiaoli; Zheng, Jie

2014-09-11

The regulatory mechanism of recombination is one of the most fundamental problems in genomics, with wide applications in genome wide association studies (GWAS), birth-defect diseases, molecular evolution, cancer research, etc. Recombination events cluster into short genomic regions called "recombination hotspots". Recently, a zinc finger protein PRDM9 was reported to regulate recombination hotspots in human and mouse genomes. In addition, a 13-mer motif contained in the binding sites of PRDM9 is found to be enriched in human hotspots. However, this 13-mer motif only covers a fraction of hotspots, indicating that PRDM9 is not the only regulator of recombination hotspots. Therefore, the challenge of discovering other regulators of recombination hotspots becomes significant. Furthermore, recombination is a complex process. Hence, multiple proteins acting as machinery, rather than individual proteins, are more likely to carry out this process in a precise and stable manner. Therefore, the extension of the prediction of individual trans-regulators to protein complexes is also highly desired. In this paper, we introduce a pipeline to identify genes and protein complexes associated with recombination hotspots. First, we prioritize proteins associated with hotspots based on their preference of binding to hotspots and coldspots. Second, using the above identified genes as seeds, we apply the Random Walk with Restart algorithm (RWR) to propagate their influences to other proteins in protein-protein interaction (PPI) networks. Hence, many proteins without DNA-binding information will also be assigned a score to implicate their roles in recombination hotspots. Third, we construct sub-PPI networks induced by top genes ranked by RWR for various species (e.g., yeast, human and mouse) and detect protein complexes in those sub-PPI networks. The GO term analysis show that our prioritizing methods and the RWR algorithm are capable of identifying novel genes associated with recombination hotspots. The trans-regulators predicted by our pipeline are enriched with epigenetic functions (e.g., histone modifications), demonstrating the epigenetic regulatory mechanisms of recombination hotspots. The identified protein complexes also provide us with candidates to further investigate the molecular machineries for recombination hotspots. Moreover, the experimental data and results are available on our web site http://www.ntu.edu.sg/home/zhengjie/data/RecombinationHotspot/NetPipe/.

Analysis of human reticulocyte genes reveals altered erythropoiesis: potential use to detect recombinant human erythropoietin doping.

PubMed

Varlet-Marie, Emmanuelle; Audran, Michel; Lejeune, Mireille; Bonafoux, Béatrice; Sicart, Marie-Therese; Marti, Jacques; Piquemal, David; Commes, Thérèse

2004-08-01

Enhancement of oxygen delivery to tissues is associated with improved sporting performance. One way of enhancing oxygen delivery is to take recombinant human erythropoietin (rHuEpo), which is an unethical and potentially dangerous practice. However, detection of the use of rHuEpo remains difficult in situations such as: i) several days after the end of treatment ii) when a treatment with low doses is conducted iii) if the rHuEpo effect is increased by other substances. In an attempt to detect rHuEpo abuse, we selected erythroid gene markers from a SAGE library and analyzed the effects of rHuEpo administration on expression of the HBB, FTL and OAZ genes. Ten athletes were assigned to the rHuEpo or placebo group. The rHuEpo group received subcutaneous injections of rHuEpo (50 UI/kg three times a week, 4 weeks; 20 UI/kg three times a week, 2 weeks). HBB, FTL and OAZ gene profiles were monitored by real time-polymerase chain reaction (PCR) quantification during and for 3 weeks after drug administration. The global analysis of these targeted genes detected in whole blood samples showed a characteristic profile of subjects misusing rHuEpo with a increase above the threshold levels. The individual analysis of OAZ mRNA seemed indicative of rHuEpo treatment. The performance-enhancing effect of rHuEpo treatment is greater than the duration of hematologic changes associated with rHuEpo misuse. Although direct electrophoretic methods to detect rHuEpo have been developed, recombinant isoforms of rHuEpo are not detectable some days after the last subcutaneous injection. To overcome these limitations indirect OFF models have been developed. Our data suggest that, in the near future, it will be possible to consolidate results achievable with the OFF models by analyzing selected erythroid gene markers as a supplement to indirect methods.

Transformative Colloidal Nanomaterials for Mid- Infrared Devices

DTIC Science & Technology

2015-06-11

like. Luminescence has been detected up to the detection limit of our MCT detector (11 microns). Inorganic ligand exchange using ammonium sulfide...biexciton lifetime is not changing significantly, being about 30-50 ps. One issue with CQDs films for mid-IR detection will be to achieve noise ...limited by generation/recombination. Our first studies showed that at 1kHz, 1/f noise dominated starting at temperatures of 100K and increased

Population Genetic Analysis of Streptomyces albidoflavus Reveals Habitat Barriers to Homologous Recombination in the Diversification of Streptomycetes

PubMed Central

Cheng, Kun; Rong, Xiaoying; Pinto-Tomás, Adrián A.; Fernández-Villalobos, Marcela; Murillo-Cruz, Catalina

2014-01-01

Examining the population structure and the influence of recombination and ecology on microbial populations makes great sense for understanding microbial evolution and speciation. Streptomycetes are a diverse group of bacteria that are widely distributed in nature and a rich source of useful bioactive compounds; however, they are rarely subjected to population genetic investigations. In this study, we applied a five-gene-based multilocus sequence analysis (MLSA) scheme to 41 strains of Streptomyces albidoflavus derived from diverse sources, mainly insects, sea, and soil. Frequent recombination was detected in S. albidoflavus, supported by multiple lines of evidence from the pairwise homoplasy index (Φw) test, phylogenetic discordance, the Shimodaira-Hasegawa (SH) test, and network analysis, underpinning the predominance of homologous recombination within Streptomyces species. A strong habitat signal was also observed in both phylogenetic and Structure 2.3.3 analyses, indicating the importance of ecological difference in shaping the population structure. Moreover, all three habitat-associated groups, particularly the entomic group, demonstrated significantly reduced levels of gene flow with one another, generally revealing habitat barriers to recombination. Therefore, a combined effect of homologous recombination and ecology is inferred for S. albidoflavus, where dynamic evolution is at least partly balanced by the extent that differential distributions of strains among habitats limit genetic exchange. Our study stresses the significance of ecology in microbial speciation and reveals the coexistence of homologous recombination and ecological divergence in the evolution of streptomycetes. PMID:25416769

HIV classification using the coalescent theory

PubMed Central

Bulla, Ingo; Schultz, Anne-Kathrin; Schreiber, Fabian; Zhang, Ming; Leitner, Thomas; Korber, Bette; Morgenstern, Burkhard; Stanke, Mario

2010-01-01

Motivation: Existing coalescent models and phylogenetic tools based on them are not designed for studying the genealogy of sequences like those of HIV, since in HIV recombinants with multiple cross-over points between the parental strains frequently arise. Hence, ambiguous cases in the classification of HIV sequences into subtypes and circulating recombinant forms (CRFs) have been treated with ad hoc methods in lack of tools based on a comprehensive coalescent model accounting for complex recombination patterns. Results: We developed the program ARGUS that scores classifications of sequences into subtypes and recombinant forms. It reconstructs ancestral recombination graphs (ARGs) that reflect the genealogy of the input sequences given a classification hypothesis. An ARG with maximal probability is approximated using a Markov chain Monte Carlo approach. ARGUS was able to distinguish the correct classification with a low error rate from plausible alternative classifications in simulation studies with realistic parameters. We applied our algorithm to decide between two recently debated alternatives in the classification of CRF02 of HIV-1 and find that CRF02 is indeed a recombinant of Subtypes A and G. Availability: ARGUS is implemented in C++ and the source code is available at http://gobics.de/software Contact: ibulla@uni-goettingen.de Supplementary Information: Supplementary data are available at Bioinformatics online. PMID:20400454

The contribution of alu elements to mutagenic DNA double-strand break repair.

PubMed

Morales, Maria E; White, Travis B; Streva, Vincent A; DeFreece, Cecily B; Hedges, Dale J; Deininger, Prescott L

2015-03-01

Alu elements make up the largest family of human mobile elements, numbering 1.1 million copies and comprising 11% of the human genome. As a consequence of evolution and genetic drift, Alu elements of various sequence divergence exist throughout the human genome. Alu/Alu recombination has been shown to cause approximately 0.5% of new human genetic diseases and contribute to extensive genomic structural variation. To begin understanding the molecular mechanisms leading to these rearrangements in mammalian cells, we constructed Alu/Alu recombination reporter cell lines containing Alu elements ranging in sequence divergence from 0%-30% that allow detection of both Alu/Alu recombination and large non-homologous end joining (NHEJ) deletions that range from 1.0 to 1.9 kb in size. Introduction of as little as 0.7% sequence divergence between Alu elements resulted in a significant reduction in recombination, which indicates even small degrees of sequence divergence reduce the efficiency of homology-directed DNA double-strand break (DSB) repair. Further reduction in recombination was observed in a sequence divergence-dependent manner for diverged Alu/Alu recombination constructs with up to 10% sequence divergence. With greater levels of sequence divergence (15%-30%), we observed a significant increase in DSB repair due to a shift from Alu/Alu recombination to variable-length NHEJ which removes sequence between the two Alu elements. This increase in NHEJ deletions depends on the presence of Alu sequence homeology (similar but not identical sequences). Analysis of recombination products revealed that Alu/Alu recombination junctions occur more frequently in the first 100 bp of the Alu element within our reporter assay, just as they do in genomic Alu/Alu recombination events. This is the first extensive study characterizing the influence of Alu element sequence divergence on DNA repair, which will inform predictions regarding the effect of Alu element sequence divergence on both the rate and nature of DNA repair events.

Oral or parenteral administration of replication-deficient adenoviruses expressing the measles virus haemagglutinin and fusion proteins: protective immune responses in rodents.

PubMed

Fooks, A R; Jeevarajah, D; Lee, J; Warnes, A; Niewiesk, S; ter Meulen, V; Stephenson, J R; Clegg, J C

1998-05-01

The genes encoding the measles virus (MV) haemagglutinin (H) and fusion (F) proteins were placed under the control of the human cytomegalovirus immediate early promoter in a replication-deficient adenovirus vector. Immunofluorescence and radioimmune precipitation demonstrated the synthesis of each protein and biological activity was confirmed by the detection of haemadsorption and fusion activities in infected cells. Oral as well as parenteral administration of the H-expressing recombinant adenovirus elicited a significant protective response in mice challenged with MV. While the F-expressing adenovirus failed to protect mice, cotton rats immunized with either the H- or F-expressing recombinant showed reduced MV replication in the lungs. Antibodies elicited in mice following immunization with either recombinant had no in vitro neutralizing activity, suggesting a protective mechanism involving a cell-mediated immune response. This study demonstrates the feasibility of using oral administration of adenovirus recombinants to induce protective responses to heterologous proteins.

Clonal reproduction with androgenesis and somatic recombination: the case of the ant Cardiocondyla kagutsuchi

NASA Astrophysics Data System (ADS)

Okita, Ichiro; Tsuchida, Koji

2016-04-01

In haplodiploid insects such as ants, male sexuals develop from unfertilised haploid eggs, while female sexuals and workers develop from fertilized diploid eggs. However, some ant species do not exchange their gene pool between sexes; both male and female sexuals are clonally produced, while workers are sexually produced. To date, three ant species, Wasmannia auropunctata, Vollenhovia emeryi, and Paratrechina longicornis, have been reported to reproduce using such reproductive systems. In this study, we reveal that in one lineage of the ant Cardiocondyla kagutsuchi, male and female sexuals are also clonally produced. In contrast to the abovementioned three species, the workers were not only sexually produced but had recombinant sequences in their nuclear internal transcribed spacer regions, although the recombinant sequences were not detected in male or female sexuals. These results suggest that the lineage likely possesses a mechanism to compensate for the reduction in genetic variation due to clonal reproduction with somatic recombination that occurs within the workers.

Clonal reproduction with androgenesis and somatic recombination: the case of the ant Cardiocondyla kagutsuchi.

PubMed

Okita, Ichiro; Tsuchida, Koji

2016-04-01

In haplodiploid insects such as ants, male sexuals develop from unfertilised haploid eggs, while female sexuals and workers develop from fertilized diploid eggs. However, some ant species do not exchange their gene pool between sexes; both male and female sexuals are clonally produced, while workers are sexually produced. To date, three ant species, Wasmannia auropunctata, Vollenhovia emeryi, and Paratrechina longicornis, have been reported to reproduce using such reproductive systems. In this study, we reveal that in one lineage of the ant Cardiocondyla kagutsuchi, male and female sexuals are also clonally produced. In contrast to the abovementioned three species, the workers were not only sexually produced but had recombinant sequences in their nuclear internal transcribed spacer regions, although the recombinant sequences were not detected in male or female sexuals. These results suggest that the lineage likely possesses a mechanism to compensate for the reduction in genetic variation due to clonal reproduction with somatic recombination that occurs within the workers.

Molecular cloning and expression in mammalian cells of ricin B chain

DOE Office of Scientific and Technical Information (OSTI.GOV)

Chang, M.

1987-01-01

In these studies, the cDNA encoding the B chain of ricin has been cloned and expressed in monkey kidney COS-M6 cells. The recombinant B chain was detected by labeling the transfected cells with {sup 35}S-methionine and {sup 35}S-cysteine and demonstrating secretion of a protein with a Mr of 30-32,000 which was not present in the medium of mock-transfected COS-M6 cells. This protein was specifically immunoprecipitated by an anti-ricin or anti-B chain antibody. The amount of recombinant B chain secreted by the COS-M6 cells was determined by radioimmunoassay to be 1-10 ng/ml of media. Virtually all the recombinant B chain formedmore » active ricin when mixed with native A chain; it could also bind as effectively as native B chain to the galactose-containing glycoprotein, asialofetuin. These results indicate that the vast majority of recombinant B chains secreted into the medium of the COS-M6 cells retain biological function.« less

Construction of Poxviruses as Cloning Vectors: Insertion of the Thymidine Kinase Gene from Herpes Simplex Virus into the DNA of Infectious Vaccinia Virus

NASA Astrophysics Data System (ADS)

Panicali, Dennis; Paoletti, Enzo

1982-08-01

We have constructed recombinant vaccinia viruses containing the thymidine kinase gene from herpes simplex virus. The gene was inserted into the genome of a variant of vaccinia virus that had undergone spontaneous deletion as well as into the 120-megadalton genome of the large prototypic vaccinia variant. This was accomplished via in vivo recombination by contransfection of eukaryotic tissue culture cells with cloned BamHI-digested thymidine kinase gene from herpes simplex virus containing flanking vaccinia virus DNA sequences and infectious rescuing vaccinia virus. Pure populations of the recombinant viruses were obtained by replica filter techniques or by growth of the recombinant virus in biochemically selective medium. The herpes simplex virus thymidine kinase gene, as an insert in vaccinia virus, is transcribed in vivo and in vitro, and the fidelity of in vivo transcription into a functional gene product was detected by the phosphorylation of 5-[125I]iodo-2'-deoxycytidine.

Enzyme-linked immunosorbent assay using a recombinant baculovirus-expressed Bacillus anthracis protective antigen (PA): measurement of human anti-PA antibodies.

PubMed Central

Iacono-Connors, L C; Novak, J; Rossi, C; Mangiafico, J; Ksiazek, T

1994-01-01

We developed an antigen capture enzyme-linked immunosorbent assay (ELISA) which does not require purified protective antigen (PA) for detection of human antibodies to Bacillus anthracis PA. Lysates of Spodoptera frugiperda (Sf-9) cells infected with recombinant baculovirus containing the PA gene were used as the source of PA to develop the ELISA. Recombinant PA from crude Sf-9 cell lysates or PA purified from B. anthracis Sterne strain was captured by an anti-PA monoclonal antibody coated onto microtiter plates. We demonstrated that human serum antibody titers to PA were identical in the ELISA whether we used crude Sf-9 cell lysates containing recombinant baculovirus-expressed PA or purified Sterne PA. Finally, false-positive results observed in a direct ELISA were eliminated with this antigen capture ELISA. Thus, the antigen capture ELISA with crude preparations of baculovirus-expressed PA is reliable, safe, and inexpensive for determining anti-PA antibody levels in human sera. PMID:7496927

Roles of silkworm endoplasmic reticulum chaperones in the secretion of recombinant proteins expressed by baculovirus system.

PubMed

Imai, Saki; Kusakabe, Takahiro; Xu, Jian; Li, Zhiqing; Shirai, Shintaro; Mon, Hiroaki; Morokuma, Daisuke; Lee, Jae Man

2015-11-01

Baculovirus expression vector system (BEVS) is widely used for production of recombinant eukaryotic proteins in insect larvae or cultured cells. BEVS has advantages over bacterial expression system in producing post-translationally modified secreted proteins. However, for some unknown reason, it is very difficult for insects to secrete sufficiently for certain proteins of interest. To understand the reasons why insect cells fail to secrete some kinds of recombinant proteins, we here employed three mammalian proteins as targets, EPO, HGF, and Wnt3A, with different secretion levels in BEVS and investigated their mRNA transcriptions from the viral genome, subcellular localizations, and interactions with silkworm ER chaperones. Moreover, we observed that no significantly influence on the secretion amounts of all three proteins when depleting or overexpressing most endogenous ER chaperone genes in cultured silkworm cells. However, among all detected ER chaperones, the depletion of BiP severely decreased the recombinant protein secretion in BEVS, indicating the possible central role of Bip in silkworm secretion pathway.

Transgenic bovine as bioreactors: Challenges and perspectives

PubMed Central

Monzani, Paulo S.; Adona, Paulo R.; Ohashi, Otávio M.; Meirelles, Flávio V.; Wheeler, Matthew B.

2016-01-01

ABSTRACT The use of recombinant proteins has increased in diverse commercial sectors. Various systems for protein production have been used for the optimization of production and functional protein expression. The mammary gland is considered to be a very interesting system for the production of recombinant proteins due to its high level of expression and its ability to perform post-translational modifications. Cows produce large quantities of milk over a long period of lactation, and therefore this species is an important candidate for recombinant protein expression in milk. However, transgenic cows are more difficult to generate due to the inefficiency of transgenic methodologies, the long periods for transgene detection, recombinant protein expression and the fact that only a single calf is obtained at the end of each pregnancy. An increase in efficiency for transgenic methodologies for cattle is a big challenge to overcome. Promising methodologies have been proposed that can help to overcome this obstacle, enabling the use of transgenic cattle as bioreactors for protein production in milk for industry. PMID:27166649

Ornithine aminotransferase (OAT): recombination between an X-linked OAT sequence (7.5 kb) and the Norrie disease locus.

PubMed

Ngo, J T; Bateman, J B; Spence, M A; Cortessis, V; Sparkes, R S; Kivlin, J D; Mohandas, T; Inana, G

1990-01-01

A human ornithine aminotransferase (OAT) locus has been mapped to the Xp11.2, as has the Norrie disease locus. We used a cDNA probe to investigate a 3-generation UCLA family with Norrie disease; a 4.2-kb RFLP was detected and a maximum lod score of 0.602 at zero recombination fraction was calculated. We used the same probe to study a second multigeneration family with Norrie disease from Utah. A different RFLP of 7.5 kb in size was identified and a recombinational event between the OAT locus represented by this RFLP and the disease loci was observed. Linkage analysis of these two loci in this family revealed a maximum load score of 1.88 at a recombination fraction of 0.10. Although both families have affected members with the same disease, the lod scores are reported separately because the 4.2- and 7.5-kb RFLPs may represent two different loci for the X-linked OAT.

Lymphoproliferative and Gamma Interferon Responses to Stress-Regulated Mycobacterium avium subsp. paratuberculosis Recombinant Proteins

PubMed Central

Gurung, Ratna B.; Begg, Douglas J.; Purdie, Auriol C.; de Silva, Kumudika; Bannantine, John P.

2014-01-01

Johne's disease in ruminants is a chronic infection of the intestines caused by Mycobacterium avium subsp. paratuberculosis. An important strategy to control disease is early detection, and a potentially efficient method for early detection is measurement of cell-mediated immune responses developed by the host in response to exposure or infection. One method is to measure lymphoproliferation and cytokine release from the host cells when exposed to the organism or parts of the organism. In this study, 10 recombinant M. avium subsp. paratuberculosis proteins known to be upregulated under in vitro stress conditions were evaluated by examining their ability to evoke memory as a result of exposure by vaccination or oral challenge with live Mycobacterium avium subsp. paratuberculosis. Out of 10 proteins, MAP2698c was found to induce higher cell-mediated immune responses in vaccinated and challenged sheep in comparison to healthy controls. The findings suggest that not all stress-regulated proteins have the diagnostic potential to detect cell-mediated immune responses in ovine paratuberculosis. PMID:24695774

Red fluorescent genetically encoded indicator for intracellular hydrogen peroxide

NASA Astrophysics Data System (ADS)

Ermakova, Yulia G.; Bilan, Dmitry S.; Matlashov, Mikhail E.; Mishina, Natalia M.; Markvicheva, Ksenia N.; Subach, Oksana M.; Subach, Fedor V.; Bogeski, Ivan; Hoth, Markus; Enikolopov, Grigori; Belousov, Vsevolod V.

2014-10-01

Reactive oxygen species (ROS) are conserved regulators of numerous cellular functions, and overproduction of ROS is a hallmark of various pathological processes. Genetically encoded fluorescent probes are unique tools to study ROS production in living systems of different scale and complexity. However, the currently available recombinant redox sensors have green emission, which overlaps with the spectra of many other probes. Expanding the spectral range of recombinant in vivo ROS probes would enable multiparametric in vivo ROS detection. Here we present the first genetically encoded red fluorescent sensor for hydrogen peroxide detection, HyPerRed. The performance of this sensor is similar to its green analogues. We demonstrate the utility of the sensor by tracing low concentrations of H2O2 produced in the cytoplasm of cultured cells upon growth factor stimulation. Moreover, using HyPerRed we detect local and transient H2O2 production in the mitochondrial matrix upon inhibition of the endoplasmic reticulum Ca2+ uptake.

Immunofluorescence Technique Using HeLa Cells Expressing Recombinant Nucleoprotein for Detection of Immunoglobulin G Antibodies to Crimean-Congo Hemorrhagic Fever Virus

PubMed Central

Saijo, Masayuki; Qing, Tang; Niikura, Masahiro; Maeda, Akihiko; Ikegami, Tetsuro; Sakai, Koji; Prehaud, Christophe; Kurane, Ichiro; Morikawa, Shigeru

2002-01-01

A HeLa cell line continuously expressing recombinant nucleoprotein (rNP) of the Crimean-Congo hemorrhagic fever virus (CCHFV) was established by transfection with an expression vector containing the cDNA of CCHFV NP (pKS336-CCHFV-NP). These cells were used as antigens for indirect immunofluorescence (IF) to detect immunoglobulin G antibodies to CCHFV. The sensitivity and specificity of this IF technique were examined by using serum samples and were compared to those of the IF technique using CCHFV-infected Vero E6 cells (authentic antigen). Staining of the CCHFV rNP expressed in HeLa cells showed a unique granular pattern similar to that of CCHFV-infected Vero E6 cells. Positive staining could easily be distinguished from a negative result. All 13 serum samples determined to be positive by using the authentic antigen were also determined to be positive by using CCHFV rNP-expressing HeLa cells (recombinant antigen). The 108 serum samples determined to be negative by using the authentic antigen were also determined to be negative by using the recombinant antigen. Thus, both the sensitivity and the specificity of this IF technique were 100% compared to the IF with authentic antigen. The novel IF technique using CCHFV rNP-expressing HeLa cells can be used not only for diagnosis of CCHF but also for epidemiological studies on CCHFV infections. PMID:11825944

Analysis of the N-glycans of recombinant human Factor IX purified from transgenic pig milk

PubMed Central

Gil, Geun-Cheol; Velander, William H; Van Cott, Kevin E

2008-01-01

Glycosylation of recombinant proteins is of particular importance because it can play significant roles in the clinical properties of the glycoprotein. In this work, the N-glycan structures of recombinant human Factor IX (tg-FIX) produced in the transgenic pig mammary gland were determined. The majority of the N-glycans of transgenic pig-derived Factor IX (tg-FIX) are complex, bi-antennary with one or two terminal N-acetylneuraminic acid (Neu5Ac) moieties. We also found that the N-glycan structures of tg-FIX produced in the porcine mammary epithelial cells differed with respect to N-glycans from glycoproteins produced in other porcine tissues. tg-FIX contains no detectable Neu5Gc, the sialic acid commonly found in porcine glycoproteins produced in other tissues. Additionally, we were unable to detect glycans in tg-FIX that have a terminal Galα(1,3)Gal disaccharide sequence, which is strongly antigenic in humans. The N-glycan structures of tg-FIX are also compared to the published N-glycan structures of recombinant human glycoproteins produced in other transgenic animal species. While tg-FIX contains only complex structures, antithrombin III (goat), C1 inhibitor (rabbit), and lactoferrin (cow) have both high mannose and complex structures. Collectively, these data represent a beginning point for the future investigation of species-specific and tissue/cell-specific differences in N-glycan structures among animals used for transgenic animal bioreactors. PMID:18456721

[Cloning of human CD45 gene and its expression in Hela cells].

PubMed

Li, Jie; Xu, Tianyu; Wu, Lulin; Zhang, Liyun; Lu, Xiao; Zuo, Daming; Chen, Zhengliang

2015-11-01

To clone human CD45 gene PTPRC and establish Hela cells overexpressing recombinant human CD45 protein. The intact cDNA encoding human CD45 amplified using RT-PCR from the total RNA extracted from peripheral blood mononuclear cells (PBMCs) of a healthy donor was cloned into pMD-18T vector. The CD45 cDNA fragment amplified from the pMD-18T-CD45 by PCR was inserted to the coding region of the PcDNA3.1-3xflag vector, and the resultant recombinant expression vector PcDNA3.1-3xflag-CD45 was transfected into Hela cells. The expression of CD45 in Hela cells was detected by flow cytometry and Western blotting, and the phosphastase activity of CD45 was quantified using an alkaline phosphatase assay kit. The cDNA fragment of about 3 900 bp was amplified from human PBMCs and cloned into pMD-18T vector. The recombinant expression vector PcDNA3.1-3xflag-CD45 was constructed, whose restriction maps and sequence were consistent with those expected. The expression of CD45 in transfected Hela cells was detected by flow cytometry and Western blotting, and the expressed recombinant CD45 protein in Hela cells showed a phosphastase activity. The cDNA of human CD45 was successfully cloned and effectively expressed in Hela cells, which provides a basis for further exploration of the functions of CD45.

PRDM9 and Its Role in Genetic Recombination.

PubMed

Paigen, Kenneth; Petkov, Petko M

2018-04-01

PRDM9 is a zinc finger protein that binds DNA at specific locations in the genome where it trimethylates histone H3 at lysines 4 and 36 at surrounding nucleosomes. During meiosis in many species, including humans and mice where PRDM9 has been most intensely studied, these actions determine the location of recombination hotspots, where genetic recombination occurs. In addition, PRDM9 facilitates the association of hotspots with the chromosome axis, the site of the programmed DNA double-strand breaks (DSBs) that give rise to genetic exchange between chromosomes. In the absence of PRDM9 DSBs are not properly repaired. Collectively, these actions determine patterns of genetic linkage and the possibilities for chromosome reorganization over successive generations. Copyright © 2017 Elsevier Ltd. All rights reserved.

Synthesis of recombinant human parainfluenza virus 1 and 3 nucleocapsid proteins in yeast Saccharomyces cerevisiae.

PubMed

Juozapaitis, Mindaugas; Zvirbliene, Aurelija; Kucinskaite, Indre; Sezaite, Indre; Slibinskas, Rimantas; Coiras, Mayte; de Ory Manchon, Fernando; López-Huertas, María Rosa; Pérez-Breña, Pilar; Staniulis, Juozas; Narkeviciute, Irena; Sasnauskas, Kestutis

2008-05-01

Human parainfluenza virus types 1 and 3 (HPIV1 and HPIV3, respectively), members of the virus family Paramyxoviridae, are common causes of lower respiratory tract infections in infants, young children, the immunocompromised, the chronically ill, and the elderly. In order to synthesize recombinant HPIV1 and HPIV3 nucleocapsid proteins, the coding sequences were cloned into the yeast Saccharomyces cerevisiae expression vector pFGG3 under control of GAL7 promoter. A high level of recombinant virus nucleocapsid proteins expression (20-24 mg l(-1) of yeast culture) was obtained. Electron microscopy demonstrated the assembly of typical herring-bone structures of purified recombinant nucleocapsid proteins, characteristic for other paramyxoviruses. These structures contained host RNA, which was resistant to RNase treatment. The nucleocapsid proteins were stable in yeast and were easily purified by caesium chloride gradient ultracentrifugation. Therefore, this system proved to be simple, efficient and cost-effective, suitable for high-level production of parainfluenza virus nucleocapsids as nucleocapsid-like particles. When used as coating antigens in an indirect ELISA, the recombinant N proteins reacted with sera of patients infected with HPIV1 or 3. Serological assays to detect HPIV-specific antibodies could be designed on this basis.

Interspecific Plastome Recombination Reflects Ancient Reticulate Evolution in Picea (Pinaceae).

PubMed

Sullivan, Alexis R; Schiffthaler, Bastian; Thompson, Stacey Lee; Street, Nathaniel R; Wang, Xiao-Ru

2017-07-01

Plastid sequences are a cornerstone in plant systematic studies and key aspects of their evolution, such as uniparental inheritance and absent recombination, are often treated as axioms. While exceptions to these assumptions can profoundly influence evolutionary inference, detecting them can require extensive sampling, abundant sequence data, and detailed testing. Using advancements in high-throughput sequencing, we analyzed the whole plastomes of 65 accessions of Picea, a genus of ∼35 coniferous forest tree species, to test for deviations from canonical plastome evolution. Using complementary hypothesis and data-driven tests, we found evidence for chimeric plastomes generated by interspecific hybridization and recombination in the clade comprising Norway spruce (P. abies) and 10 other species. Support for interspecific recombination remained after controlling for sequence saturation, positive selection, and potential alignment artifacts. These results reconcile previous conflicting plastid-based phylogenies and strengthen the mounting evidence of reticulate evolution in Picea. Given the relatively high frequency of hybridization and biparental plastid inheritance in plants, we suggest interspecific plastome recombination may be more widespread than currently appreciated and could underlie reported cases of discordant plastid phylogenies. © The Author 2017. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.

Molecular adaptation within the coat protein-encoding gene of Tunisian almond isolates of Prunus necrotic ringspot virus.

PubMed

Boulila, Moncef; Ben Tiba, Sawssen; Jilani, Saoussen

2013-04-01

The sequence alignments of five Tunisian isolates of Prunus necrotic ringspot virus (PNRSV) were searched for evidence of recombination and diversifying selection. Since failing to account for recombination can elevate the false positive error rate in positive selection inference, a genetic algorithm (GARD) was used first and led to the detection of potential recombination events in the coat protein-encoding gene of that virus. The Recco algorithm confirmed these results by identifying, additionally, the potential recombinants. For neutrality testing and evaluation of nucleotide polymorphism in PNRSV CP gene, Tajima's D, and Fu and Li's D and F statistical tests were used. About selection inference, eight algorithms (SLAC, FEL, IFEL, REL, FUBAR, MEME, PARRIS, and GA branch) incorporated in HyPhy package were utilized to assess the selection pressure exerted on the expression of PNRSV capsid. Inferred phylogenies pointed out, in addition to the three classical groups (PE-5, PV-32, and PV-96), the delineation of a fourth cluster having the new proposed designation SW6, and a fifth clade comprising four Tunisian PNRSV isolates which underwent recombination and selective pressure and to which the name Tunisian outgroup was allocated.

Rapid detection of food-borne Salmonella contamination using IMBs-qPCR method based on pagC gene.

PubMed

Wang, Jiashun; Li, Yi; Chen, Jia; Hua, Deping; Li, Yi; Deng, Hui; Li, Ying; Liang, Zhixuan; Huang, Jinhai

Detection of Salmonella is very important to minimize the food safety risk. In this study, the recombinant PagC protein and PagC antibody were prepared and coupled with immunomagnetic beads (IMBs) to capture Salmonella cells from pork and milk samples. And then the SYBR Green qualitative PCR was developed to detect the pathogenic Salmonella. The results showed that the PagC polyclonal antiserum is of good specificity and the capture rate of 0.1mg IMBs for Salmonella tended to be stable at the range of 70-74% corresponding to the concentrations between 10 1 and 10 4 CFU/mL. The method developed demonstrated high specificity for the positive Salmonella samples when compared to non-specific DNA samples, such as Escherichia coli, Staphylococcus aureus, Yersinia enterocolitica, and Yersinia pseudotuberculosis. The limit of detection of this assay was 18CFU/mL. Detection and quantitative enumeration of Salmonella in samples of pork or milk shows good recoveries of 54.34% and 52.07%. In conclusion, the polyclonal antibody of recombinant PagC protein is effective to capture Salmonella from detected samples. The developed pagC antibody IMBs-qPCR method showed efficiency, sensitivity and specificity for 30 Salmonella detection, enabling detection within 10h, which is a promising rapid method to detect Salmonella in emergency. Copyright © 2017 Sociedade Brasileira de Microbiologia. Published by Elsevier Editora Ltda. All rights reserved.

Advances in Biological Science.

ERIC Educational Resources Information Center

Oppenheimer, Steven B.; And Others

1988-01-01

Reviews major developments in areas that are at the cutting edge of biological research. Areas include: human anti-cancer gene, recombinant DNA techniques for the detection of Huntington disease carriers, and marine biology. (CW)

Development of a competitive ELISA using a truncated E2 recombinant protein as antigen for detection of antibodies to classical swine fever virus.

PubMed

Clavijo, A; Lin, M; Riva, J; Mallory, M; Lin, F; Zhou, E M

2001-02-01

The sequence encoding a truncated E2 glycoprotein of the Alfort/187 strain of classical swine fever virus (CSFV) was expressed in Escherichia coli using the pET expression system and the recombinant product purified by Ni-NTA agarose affinity chromatography. The antigenicity of this recombinant protein was demonstrated by immunoblot using anti- CSFV-specific antibodies. A monoclonal antibody was produced against the truncated E2 protein and used as competitor in an ELISA for the detection of antibodies to CSFV. Specific antibodies were demonstrated by competitive ELISA (C-ELISA) as early as 21 days post-infection (dpi) in experimentally infected pigs. Seroconversion was demonstrated by C-ELISA and neutralising peroxidase-linked assay (NPLA) in all infected animals by 4 weeks. No cross-reaction with antibodies to bovine viral diarrhoea virus (BVDV) was seen in the C-ELISA using sera from experimentally infected pigs. The C-ELISA is not intended as a substitute for the NPLA. However, it is expected it will be useful for monitoring and prevalence studies. It will also assist in testing a large number of samples in the event of an outbreak. Copyright 2001 Harcourt Publishers Ltd.

[Study on serological cross-reactivity of six pathogenic phleboviruses].

PubMed

Wu, Wei; Zhang, Shuo; Zhang, Quan-Fu; Li, Chuan; Liang, Mi-Fang; Li, De-Xin

2014-07-01

This article aimed to study the antigenicity of nucleocapsid proteins (NPs) in six pathogenic phleboviruses and to provide theoretical evidence for the development of serological diagnostic reagents. NPs of six pathogenic phleboviruses were expressed and purified using a prokaryotic expression system and rabbits were immunized with individual recombinant NPs. Cross-reactions among NPs and rabbit sera were determined by both indirect ELISA and Western blotting analyses, and the sera titer was determined by indirect ELISA. Furthermore, sera from SFTS patients were also detected by each recombinant NP as a coating antigen using indirect ELISA. The cross-reactions and the sera titer were subsequently determined. Both the concentration and purity of recombinant NPs of six pathogenic phleboviruses met the standards for immunization and detection. The results of indirect ELISA and Western blotting showed that each anti-phlebovirus NP rabbit immune serum had potential serological cross-reactivity with the other five virus NP antigens. Furthermore, the sera from SFTS patients also had cross-reactivity with the other five NP antigens to a certain extent. Our preliminary study evaluated the antigenicity and immune reactivity of six pathogenic phleboviruses NPs and laid the foundation for the development of diagnostic reagents.

In-Field Implementation of a Recombinant Factor C Assay for the Detection of Lipopolysaccharide as a Biomarker of Extant Life within Glacial Environments

PubMed Central

Barnett, Megan J.; Wadham, Jemma L.; Jackson, Miriam; Cullen, David C.

2012-01-01

The discovery over the past two decades of viable microbial communities within glaciers has promoted interest in the role of glaciers and ice sheets (the cryosphere) as contributors to subglacial erosion, global biodiversity, and in regulating global biogeochemical cycles. In situ or in-field detection and characterisation of microbial communities is becoming recognised as an important approach to improve our understanding of such communities. Within this context we demonstrate, for the first time, the ability to detect Gram-negative bacteria in glacial field-environments (including subglacial environments) via the detection of lipopolysaccharide (LPS); an important component of Gram-negative bacterial cell walls. In-field measurements were performed using the recently commercialised PyroGene® recombinant Factor C (rFC) endotoxin detection system and used in conjunction with a handheld fluorometer to measure the fluorescent endpoint of the assay. Twenty-seven glacial samples were collected from the surface, bed and terminus of a low-biomass Arctic valley glacier (Engabreen, Northern Norway), and were analysed in a field laboratory using the rFC assay. Sixteen of these samples returned positive LPS detection. This work demonstrates that LPS detection via rFC assay is a viable in-field method and is expected to be a useful proxy for microbial cell concentrations in low biomass environments. PMID:25585634

Reciprocal Translocation Observed in End-of-Production Cells of a Commercial CHO-Based Process.

PubMed

Rouiller, Yolande; Kleuser, Beate; Toso, Emiliano; Palinksy, Wolf; Rossi, Mara; Rossatto, Paola; Barberio, Davide; Broly, Hervé

2015-01-01

During the validation of an additional working cell bank derived from a validated master cell bank to support the commercial production continuum of a recombinant protein, we observed an unexpected chromosomal location of the gene of interest in some end-of-production cells. This event-identified by fluorescence in situ hybridization and multicolour chromosome painting as a reciprocal translocation involving a chromosome region containing the gene of interest with its integral coding and flanking sequences-was unique, occurred probably during or prior to multicolour chromosome painting establishment, and was transmitted to the descending generations. Cells bearing the translocation had a transient and process-independent selective advantage, which did not affect process performance and product quality. However, this first report of a translocation affecting the gene of interest location in Chinese Hamster Ovary cells used for producing a biotherapeutic indicates the importance of the demonstration of the integrity of the gene of interest in end-of-production cells. The expression of recombinant therapeutic proteins in mammalian cells depends on the establishment of a cell line with the gene of interest integrated in the host genome and stably expressed over time. Before being used for commercial production, cell lines are submitted to a qualification program in order to ensure their phenotypic and genotypic characteristics and the efficacy and safety of the product. During the production life cycle of a therapeutic protein, additional cells banks have to be validated after exhaustion of the current qualified cell bank in order to support the commercial production continuum of the recombinant protein. It is during the validation of an additional working cell bank derived from a validated master cell bank that we detected a different chromosome bearing the gene of interest in a portion of cells at the end of the upstream production phase. In our case, this event did not affect the process performance, the product quality, or its safety profile, but it highlights the need to characterize the integrity of the gene of interest in end-of-production cells when producing recombinant proteins for human use. © PDA, Inc. 2015.

An efficient method for native protein purification in the selected range from prostate cancer tissue digests

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ahmad, Rumana; Nicora, Carrie D.; Shukla, Anil K.

Prostate cancer (CP) cells differ from their normal counterpart in gene expression. Genes encoding secreted or extracellular proteins with increased expression in CP may serve as potential biomarkers. For their detection and quantification, assays based on monoclonal antibodies are best suited for development in a clinical setting. One approach to obtain antibodies is to use recombinant proteins as immunogen. However, the synthesis of recombinant protein for each identified candidate is time-consuming and expensive. It is also not practical to generate high quality antibodies to all identified candidates individually. Furthermore, non-native forms (e.g., recombinant) of proteins may not always lead tomore » useful antibodies. Our approach was to purify a subset of proteins from CP tissue specimens for use as immunogen.« less

Recombinant protein expression and purification: a comprehensive review of affinity tags and microbial applications.

PubMed

Young, Carissa L; Britton, Zachary T; Robinson, Anne S

2012-05-01

Protein fusion tags are indispensible tools used to improve recombinant protein expression yields, enable protein purification, and accelerate the characterization of protein structure and function. Solubility-enhancing tags, genetically engineered epitopes, and recombinant endoproteases have resulted in a versatile array of combinatorial elements that facilitate protein detection and purification in microbial hosts. In this comprehensive review, we evaluate the most frequently used solubility-enhancing and affinity tags. Furthermore, we provide summaries of well-characterized purification strategies that have been used to increase product yields and have widespread application in many areas of biotechnology including drug discovery, therapeutics, and pharmacology. This review serves as an excellent literature reference for those working on protein fusion tags. Copyright © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Subgenomic Reporter RNA System for Detection of Alphavirus Infection in Mosquitoes

PubMed Central

Steel, J. Jordan; Franz, Alexander W. E.; Sanchez-Vargas, Irma; Olson, Ken E.; Geiss, Brian J.

2013-01-01

Current methods for detecting real-time alphavirus (Family Togaviridae) infection in mosquitoes require the use of recombinant viruses engineered to express a visibly detectable reporter protein. These altered viruses expressing fluorescent proteins, usually from a duplicated viral subgenomic reporter, are effective at marking infection but tend to be attenuated due to the modification of the genome. Additionally, field strains of viruses cannot be visualized using this approach unless infectious clones can be developed to insert a reporter protein. To circumvent these issues, we have developed an insect cell-based system for detecting wild-type sindbis virus infection that uses a virus inducible promoter to express a fluorescent reporter gene only upon active virus infection. We have developed an insect expression system that produces sindbis virus minigenomes containing a subgenomic promoter sequence, which produces a translatable RNA species only when infectious virus is present and providing viral replication proteins. This subgenomic reporter RNA system is able to detect wild-type Sindbis infection in cultured mosquito cells. The detection system is relatively species specific and only detects closely related viruses, but can detect low levels of alphavirus specific replication early during infection. A chikungunya virus detection system was also developed that specifically detects chikungunya virus infection. Transgenic Aedes aegypti mosquito families were established that constitutively express the sindbis virus reporter RNA and were found to only express fluorescent proteins during virus infection. This virus inducible reporter system demonstrates a novel approach for detecting non-recombinant virus infection in mosquito cell culture and in live transgenic mosquitoes. PMID:24367703

Immune and histopathological responses in animals vaccinated with recombinant vaccinia viruses that express individual genes of human respiratory syncytial virus.

PubMed

Stott, E J; Taylor, G; Ball, L A; Anderson, K; Young, K K; King, A M; Wertz, G W

1987-12-01

Previous reports have established that vaccinia virus (VV) recombinants expressing G, F, or N protein of respiratory syncytial (RS) virus protect small animals against intranasal challenge with live RS virus. This work demonstrates that a variety of parameters affect the protection induced by recombinant viruses. The route of vaccination, the subtype of challenge virus, and the species used influenced the antibody titers and extent of protection. During these studies, observations were also made on the subclass of antibody generated, and pulmonary histopathological changes induced by challenge after vaccination were noted. The effect of route of inoculation on host response was examined by vaccinating mice intranasally, intraperitoneally, or by scarification with a recombinant VV expressing the RS virus G glycoprotein. Intranasal vaccination induced 25-fold-higher titers of antibody to RS virus in the lung than the intraperitoneal route did, but both routes resulted in complete suppression of virus replication after intranasal challenge 21 days after vaccination. Scarification was a less effective method of vaccination. The antibody induced by recombinant VV in mice was mostly immunoglobulin G2a (IgG2a) with some IgG2b. No antibody to RS virus was detected in the IgA, IgM, IgG1, or IgG3 subclass irrespective of the vaccination route. The G and F glycoproteins were shown to elicit similar subclasses of antibody. However, animals vaccinated with the G and F vectors differed strikingly in their response to challenge by heterologous virus. Mice or cotton rats vaccinated with recombinant VV carrying the G gene of RS virus were protected against challenge only with homologous subtype A virus. Vaccination with a recombinant VV expressing the F glycoprotein induced protection against both homologous and heterologous subtype B virus challenge. The protection induced in mice was greater than that detected in cotton rats, indicating that the host may also affect immunity. Finally, this report describes histological examination of mouse lungs after vaccination and challenge. Vaccinated mice that were subsequently challenged had significantly greater lung lesion scores than unvaccinated challenged mice. The lesions were primarily peribronchiolar and perivascular infiltrations of polymorphonuclear cells and lymphocytes. Further work will establish whether these pulmonary changes are a desirable immune response to virus invasion or a potential immunopathogenic hazard. The results have important implications for planning a strategy of vaccination against RS virus and emphasize potential dangers that may attend the use of recombinant VV as vaccines.

The physics of background discrimination in liquid xenon, and first results from Xenon10 in the hunt for WIMP dark matter

NASA Astrophysics Data System (ADS)

Dahl, Carl Eric

2009-06-01

The WIMP limit set by the Xenon10 experiment in 2007 signals a new era in direct detection of dark matter, with several large-scale liquid target detectors now under construction. A major challenge in these detectors will be to understand backgrounds at the level necessary to claim a positive WIMP signal. In liquid xenon, these backgrounds are dominated by electron recoils, which may be distinguished from the WIMP signal (nuclear recoils) by their higher charge-to-light ratio. During the construction and operation of Xenon10, the prototype detector Xed probed the physics of this discrimination. Particle interactions in liquid xenon both ionize and excite xenon atoms, giving charge and scintillation signals, respectively. Some fraction of ions recombine, reducing the charge signal and creating additional scintillation. The charge-to-light ratio, determined by the initial exciton-ion ratio and the ion recombination fraction, provides the basis for discrimination between electron and nuclear recoils. Intrinsic fluctuations in the recombination fraction limit discrimination. Changes in recombination induce an exact anti-correlation between charge and light, and when calibrated this anti-correlation distinguishes recombination fluctuations from uncorrelated fluctuations in the measured signals. We determine the mean recombination and recombination fluctuations as a function of energy and applied field for electron and nuclear recoils, finding that recombination fluctuations are already the limiting factor for discrimination above ~12 keVr (nuclear recoil energy). Below 12 keVr statistical fluctuations in the number of scintillation photons counted dominate, and we project a x6 improvement in background rejection with a x2 increase in light collection efficiency. We also build a simple recombination model that successfully reproduces the mean recombination in electron and nuclear recoils, including the surprising reversal of the expected trend for recombination with ionization density in low energy electron recoils. The model also reproduces the measured recombination fluctuations to within a factor of two at high energies. Surprisingly, the model suggests that recombination at low energies is independent of ionization density, and our observed discrimination is due not to the different stopping powers of electrons and nuclei as was thought, but rather to a different initial exciton-ion ratio. We suggest two possible physical models for this new result.

Evidence of natural interspecific recombinant viruses between bovine alphaherpesviruses 1 and 5.

PubMed

Maidana, Silvina Soledad; Craig, Patricio Oliver; Craig, María Isabel; Ludwig, Louisa; Mauroy, Axel; Thiry, Etienne; Romera, Sonia Alejandra

2017-10-15

Closely related bovine alphaherpesviruses 1 (BoHV-1) and 5 (BoHV-5) co-circulate in certain countries, rendering cattle co-infection possible. This is a prerequisite for BoHV recombination. Here, we report the first identification of homologous recombination between field isolates of BoHV-1 and BoHV-5, two alphaherpesviruses belonging to two distinct species with an average genomic similarity of 82.3%. Three isolates of BoHV-5, previously classified as subtype "BoHV-5b", were phylogenetically studied and analyzed via eight PCR sequencing assays dispersed at regular intervals throughout the genome to discriminate between BoHV-1 and BoHV-5. In the phylogenetic analysis, differences of clustering were found in the UL27 gene which encodes the glycoprotein B (gB). We detected two recombination breakpoints in the open reading frame of the UL27 gene. We compared the amino acid sequences of the gB of BoHV-1.1 and 1.2, BoHV-5a and recombinant formerly named BoHV-5b (chimeric gB) and subsequently performed molecular modeling. All structures were alike and, simultaneously, similar to the chimeric gB. Neutralizing antibodies against BoHV-1, BoHV-5 and recombinant viruses were analyzed via serum virus neutralization test using polyclonal sera and a monoclonal antibody against gB to demonstrate an absence of viral escape for both assays. Our results show that homologous recombination between two related species of ruminant alphaherpesviruses can occur in natural field conditions. We found three recombinant field isolates, previously classified as BoHV-5b subtypes, between BoHV-1 and BoHV-5. Copyright © 2017 Elsevier B.V. All rights reserved.

Genetic relatedness and recombination analysis of Allorhizobium vitis strains associated with grapevine crown gall outbreaks in Europe.

PubMed

Kuzmanović, N; Biondi, E; Bertaccini, A; Obradović, A

2015-09-01

To analyse genetic diversity and epidemiological relationships among 54 strains of Allorhizobium vitis isolated in Europe during an 8-year period and to assess the relative contribution of mutation and recombination in shaping their diversity. By using random amplified polymorphic DNA (RAPD) PCR, strains studied were distributed into 12 genetic groups. Sequence analysis of dnaK, gyrB and recA housekeeping genes was employed to characterize a representative subcollection of 28 strains. A total of 15 different haplotypes were found. Nucleotide sequence analysis suggested the presence of recombination events in A. vitis, particularly affecting dnaK locus. Although prevalence of mutation over recombination was found, impact of recombination was about two times greater than mutation in the evolution of the housekeeping genes analysed. The RAPD analysis indicated high degree of genetic diversity among the strains. However, the most abundant RAPD group was composed of 35 strains, which could lead to the conclusion that they share a common origin and were distributed by the movement of infected grapevine planting material as a most common way of crossing long distances. Furthermore, it seems that recombination is acting as an important driving force in the evolution of A. vitis. As no substantial evidence of recombination was detected within recA gene fragment, this phylogenetic marker could be reliable to characterize phylogenetic relationships among A. vitis strains. We demonstrated clear epidemiological relationship between majority of strains studied, suggesting a need for more stringent phytosanitary measures in international trade. Moreover, this is the first study to report recombination in A. vitis. © 2015 The Society for Applied Microbiology.

A Protein Microarray ELISA for the Detection of Botulinum neurotoxin A

DOE Office of Scientific and Technical Information (OSTI.GOV)

Varnum, Susan M.

An enzyme-linked immunosorbent assay (ELISA) microarray was developed for the specific and sensitive detection of botulinum neurotoxin A (BoNT/A), using high-affinity recombinant monoclonal antibodies against the receptor binding domain of the heavy chain of BoNT/A. The ELISA microarray assay, because of its sensitivity, offers a screening test with detection limits comparable to the mouse bioassay, with results available in hours instead of days.

An amorphous silicon photodiode microfluidic chip to detect nanomolar quantities of HIV-1 virion infectivity factor.

PubMed

Vistas, Cláudia R; Soares, Sandra S; Rodrigues, Rogério M M; Chu, Virginia; Conde, João P; Ferreira, Guilherme N M

2014-08-07

A hydrogenated amorphous silicon (a-Si:H) photosensor was explored for the quantitative detection of a HIV-1 virion infectivity factor (Vif) at a detection limit in the single nanomolar range. The a-Si:H photosensor was coupled with a microfluidic channel that was functionalized with a recombinant single chain variable fragment antibody. The biosensor selectively recognizes HIV-1 Vif from human cell extracts.

HYBRIDCHECK: software for the rapid detection, visualization and dating of recombinant regions in genome sequence data.

PubMed

Ward, Ben J; van Oosterhout, Cock

2016-03-01

HYBRIDCHECK is a software package to visualize the recombination signal in large DNA sequence data set, and it can be used to analyse recombination, genetic introgression, hybridization and horizontal gene transfer. It can scan large (multiple kb) contigs and whole-genome sequences of three or more individuals. HYBRIDCHECK is written in the r software for OS X, Linux and Windows operating systems, and it has a simple graphical user interface. In addition, the r code can be readily incorporated in scripts and analysis pipelines. HYBRIDCHECK implements several ABBA-BABA tests and visualizes the effects of hybridization and the resulting mosaic-like genome structure in high-density graphics. The package also reports the following: (i) the breakpoint positions, (ii) the number of mutations in each introgressed block, (iii) the probability that the identified region is not caused by recombination and (iv) the estimated age of each recombination event. The divergence times between the donor and recombinant sequence are calculated using a JC, K80, F81, HKY or GTR correction, and the dating algorithm is exceedingly fast. By estimating the coalescence time of introgressed blocks, it is possible to distinguish between hybridization and incomplete lineage sorting. HYBRIDCHECK is libré software and it and its manual are free to download from http://ward9250.github.io/HybridCheck/. © 2015 John Wiley & Sons Ltd.

Production of specific IgY antibody to the recombinant FanC protein produced in Escherichia coli.

PubMed

Nasiri, Khadijeh; Zibaee, Saeed; Nassiri, Mohammadreza; Tahmoorespur, Mojtaba; Haghparast, Alireza

2016-08-01

Enterotoxigenic Escherichia coli (ETEC) strains are one of the primary causes of diarrhea in newborn calves and in humans, pigs, and sheep. IgY technology has been identified as a promising alternative to generating a mass amount of specific antibody for use in immunotherapy and immunodiagnostics. The purpose of this study was to produce specific antibody by egg yolk antibody (IgY) to recombinant FanC protein from ETEC. FanC (K99) gene was amplified from ETEC by specific primers and polymerase chain reaction. The gene was cloned and subcloned into pTZ57R/T and pET32a (+) vectors, respectively. Recombinant vector was transferred into E. coli BL21 CodonPlus (DE3). Protein expression was investigated by 1 mM IPTG induction. Hens were immunized by the purified recombinant FanC protein. The activity and specificity of the IgY antibody were detected by dot-blotting, Western blotting, and indirect ELISA. We obtained FanC specific IgYs by immunizing the hens with the recombinant FanC protein. The anti-FanC IgY showed binding specifically to the FanC protein of ETEC. The results emphasize that specific IgY against the recombinant FanC protein could be recommended as a candidate for passive immunization against ETEC infection in animals and humans.

Production of specific IgY antibody to the recombinant FanC protein produced in Escherichia coli

PubMed Central

Nasiri, Khadijeh; Zibaee, Saeed; Nassiri, Mohammadreza; Tahmoorespur, Mojtaba; Haghparast, Alireza

2016-01-01

Objective(s): Enterotoxigenic Escherichia coli (ETEC) strains are one of the primary causes of diarrhea in newborn calves and in humans, pigs, and sheep. IgY technology has been identified as a promising alternative to generating a mass amount of specific antibody for use in immunotherapy and immunodiagnostics. The purpose of this study was to produce specific antibody by egg yolk antibody (IgY) to recombinant FanC protein from ETEC. Materials and Methods: FanC (K99) gene was amplified from ETEC by specific primers and polymerase chain reaction. The gene was cloned and subcloned into pTZ57R/T and pET32a (+) vectors, respectively. Recombinant vector was transferred into E. coli BL21 CodonPlus (DE3). Protein expression was investigated by 1 mM IPTG induction. Hens were immunized by the purified recombinant FanC protein. The activity and specificity of the IgY antibody were detected by dot-blotting, Western blotting, and indirect ELISA. Results: We obtained FanC specific IgYs by immunizing the hens with the recombinant FanC protein. The anti-FanC IgY showed binding specifically to the FanC protein of ETEC. Conclusion: The results emphasize that specific IgY against the recombinant FanC protein could be recommended as a candidate for passive immunization against ETEC infection in animals and humans. PMID:27746871

Recombination in Eukaryotic Single Stranded DNA Viruses

PubMed Central

Martin, Darren P.; Biagini, Philippe; Lefeuvre, Pierre; Golden, Michael; Roumagnac, Philippe; Varsani, Arvind

2011-01-01

Although single stranded (ss) DNA viruses that infect humans and their domesticated animals do not generally cause major diseases, the arthropod borne ssDNA viruses of plants do, and as a result seriously constrain food production in most temperate regions of the world. Besides the well known plant and animal-infecting ssDNA viruses, it has recently become apparent through metagenomic surveys of ssDNA molecules that there also exist large numbers of other diverse ssDNA viruses within almost all terrestrial and aquatic environments. The host ranges of these viruses probably span the tree of life and they are likely to be important components of global ecosystems. Various lines of evidence suggest that a pivotal evolutionary process during the generation of this global ssDNA virus diversity has probably been genetic recombination. High rates of homologous recombination, non-homologous recombination and genome component reassortment are known to occur within and between various different ssDNA virus species and we look here at the various roles that these different types of recombination may play, both in the day-to-day biology, and in the longer term evolution, of these viruses. We specifically focus on the ecological, biochemical and selective factors underlying patterns of genetic exchange detectable amongst the ssDNA viruses and discuss how these should all be considered when assessing the adaptive value of recombination during ssDNA virus evolution. PMID:21994803

The evolution of temperature and bolometric luminosity in Type II supernovae

NASA Astrophysics Data System (ADS)

Faran, T.; Nakar, E.; Poznanski, D.

2018-01-01

In this work, we present a uniform analysis of the temperature evolution and bolometric luminosity of a sample of 29 Type II supernovae (SNe), by fitting a blackbody model to their multiband photometry. Our sample includes only SNe with high quality multiband data and relatively well-sampled time coverage. Most of the SNe in our sample were detected less than a week after explosion so their light curves cover the evolution both before and after recombination starts playing a role. We use this sample to study the signature of hydrogen recombination, which is expected to appear once the observed temperature drops to ≈7000 K. Theory predicts that before recombination starts affecting the light curve, both the luminosity and the temperature should drop relatively fast, following a power law in time. Once the recombination front reaches inner parts of the outflow, it sets the observed temperature to be nearly constant, and slows the decline of the luminosity (or even leads to a re-brightening). We compare our data to analytic studies and find strong evidence for the signature of recombination. We also find that the onset of the optical plateau in a given filter, is effectively the time at which the blackbody peak reaches the central wavelength of the filter, as it cools, and it does not correspond to the time at which recombination starts affecting the emission.

Isolation of recombinant antibodies directed against surface proteins of Clostridium difficile.

PubMed

Shirvan, Ali Nazari; Aitken, Robert

2016-01-01

Clostridium difficile has emerged as an increasingly important nosocomial pathogen and the prime causative agent of antibiotic-associated diarrhoea and pseudomembranous colitis in humans. In addition to toxins A and B, immunological studies using antisera from patients infected with C. difficile have shown that a number of other bacterial factors contribute to the pathogenesis, including surface proteins, which are responsible for adhesion, motility and other interactions with the human host. In this study, various clostridial targets, including FliC, FliD and cell wall protein 66, were expressed and purified. Phage antibody display yielded a large panel of specific recombinant antibodies, which were expressed, purified and characterised. Reactions of the recombinant antibodies with their targets were detected by enzyme-linked immunosorbent assay; and Western blotting suggested that linear rather than conformational epitopes were recognised. Binding of the recombinant antibodies to surface-layer proteins and their components showed strain specificity, with good recognition of proteins from C. difficile 630. However, no reaction was observed for strain R20291-a representative of the 027 ribotype. Binding of the recombinant antibodies to C. difficile M120 extracts indicated that a component of a surface-layer protein of this strain might possess immunoglobulin-binding activities. The recombinant antibodies against FliC and FliD proteins were able to inhibit bacterial motility. Copyright © 2016. Published by Elsevier Editora Ltda.

Cloning structural genes for Treponema pallidum immunogens and characterisation of recombinant treponemal surface protein, P2 (P2 star).

PubMed Central

Peterson, K M; Baseman, J B; Alderete, J F

1987-01-01

A genomic library consisting of partially digested 10 to 20 kilobase pair fragments of Treponema pallidum deoxyribonucleic acid (DNA) was constructed using bacteriophage lambda EMBL-3 as the vector. Positive clones expressing T pallidum antigens were detected with sera from experimentally infected rabbits. Treponemal proteins ranging in molecular weight from 37,000 daltons to 120,000 daltons were identified by sodium dodecyl sulphate-polyacrylamide gel electrophoresis and immunoblotting of phage lysate proteins. One recombinant phage was examined further and contained an insert encoding a prominent treponemal 37,000 dalton protein. The recombinant protein was not recognised by antiserum directed against a fibronectin binding treponemal adhesion that contained the same electrophoretic mobility. Neither did antibody to the recombinant 37,000 dalton protein react with any treponemal proteins purified by fibronectin affinity chromatography. The recombinant protein in Escherichia coli lysates was labelled P2 (P2 star) to differentiate it from the comigrating adhesin protein called P2. Native P2 protein was present on T pallidum surfaces as shown by radioimmunoprecipitation assays with extrinsically labelled organisms. A cross reactive molecule like P2 was not synthesised by the avirulent spirochaete, T phagedenis biotype Reiter, which indicated that P2 is a protein specific to virulent T pallidum organisms. Finally, only sera of patients with primary syphilis possessed appreciable concentrations of antibody to recombinant P2 protein. Images PMID:3315959

Characterization of Helper Virus-Independent Cytopathogenic Classical Swine Fever Virus Generated by an In Vivo RNA Recombination System

PubMed Central

Gallei, Andreas; Rümenapf, Till; Thiel, Heinz-Jürgen; Becher, Paul

2005-01-01

Molecular analyses revealed that most cytopathogenic (cp) pestivirus strains evolve from noncytopathogenic (noncp) viruses by nonhomologous RNA recombination. In contrast to bovine viral diarrhea virus (BVDV), cp classical swine fever virus (CSFV) field isolates were rarely detected and always represented helper virus-dependent subgenomes. To investigate RNA recombination in more detail, we recently established an in vivo system allowing the efficient generation of recombinant cp BVDV strains in cell culture after transfecting a synthetic subgenomic and nonreplicatable transcript into cells being infected with noncp BVDV (A. Gallei, A. Pankraz, H.-J. Thiel, and P. Becher, J. Virol. 78:6271-6281, 2004). Using an analogous approach, the first helper virus-independent cp CSFV strain (CP G1) has now been generated by RNA recombination. Accordingly, this study demonstrates the applicability of RNA recombination for designing new viral RNA genomes. The genomic RNA of CP G1 has a calculated size of 18.139 kb, almost 6 kb larger than all previously described CSFV genomes. It contains cellular sequences encoding a polyubiquitin fragment directly upstream of the nonstructural protein NS3 coding gene together with a duplication of viral sequences. CP G1 induces a cytopathic effect on different tissue culture cell lines from pigs and cattle. Subsequent analyses addressed growth kinetics, expression of NS3, and genetic stability of CP G1. PMID:15681445

Development of vaccines for poultry against H5 avian influenza based on turkey herpesvirus vector

USDA-ARS?s Scientific Manuscript database

Avian influenza (AI) remains a major threat to public health as well as to the poultry industry. AI vaccines are considered a suitable tool to support AI control programs in combination with other control measures such as good biosecurity and monitoring programs. We constructed recombinant turkey he...

Atomic Structure of Interface States in Silicon Heterojunction Solar Cells

NASA Astrophysics Data System (ADS)

George, B. M.; Behrends, J.; Schnegg, A.; Schulze, T. F.; Fehr, M.; Korte, L.; Rech, B.; Lips, K.; Rohrmüller, M.; Rauls, E.; Schmidt, W. G.; Gerstmann, U.

2013-03-01

Combining orientation dependent electrically detected magnetic resonance and g tensor calculations based on density functional theory we assign microscopic structures to paramagnetic states involved in spin-dependent recombination at the interface of hydrogenated amorphous silicon crystalline silicon (a-Si:H/c-Si) heterojunction solar cells. We find that (i) the interface exhibits microscopic roughness, (ii) the electronic structure of the interface defects is mainly determined by c-Si, (iii) we identify the microscopic origin of the conduction band tail state in the a-Si:H layer, and (iv) present a detailed recombination mechanism.

Method for detection of dental caries and periodontal disease using optical imaging

DOEpatents

Nathel, Howard; Kinney, John H.; Otis, Linda L.

1996-01-01

A method for detecting the presence of active and inactive caries in teeth and diagnosing periodontal disease uses non-ionizing radiation with techniques for reducing interference from scattered light. A beam of non-ionizing radiation is divided into sample and reference beams. The region to be examined is illuminated by the sample beam, and reflected or transmitted radiation from the sample is recombined with the reference beam to form an interference pattern on a detector. The length of the reference beam path is adjustable, allowing the operator to select the reflected or transmitted sample photons that recombine with the reference photons. Thus radiation scattered by the dental or periodontal tissue can be prevented from obscuring the interference pattern. A series of interference patterns may be generated and interpreted to locate dental caries and periodontal tissue interfaces.

Generation of a rabbit single-chain fragment variable (scFv) antibody for specific detection of Bradyrhizobium sp. DOA9 in both free-living and bacteroid forms

PubMed Central

Srila, Witsanu; Yuttavanichakul, Watcharin; Teamtisong, Kamonluck; Teaumroong, Neung; Boonkerd, Nantakorn; Tittabutr, Panlada

2017-01-01

A simple and reliable method for the detection of specific nitrogen-fixing bacteria in both free-living and bacteroid forms is essential for the development and application of biofertilizer. Traditionally, a polyclonal antibody generated from an immunized rabbit was used for detection. However, the disadvantages of using a polyclonal antibody include limited supply and cross-reactivity to related bacterial strains. This is the first report on the application of phage display technology for the generation of a rabbit recombinant monoclonal antibody for specific detection and monitoring of nitrogen-fixing bacteria in both free-living form and in plant nodules. Bradyrhizobium sp. DOA9, a broad host range soil bacteria, originally isolated from the root nodules of Aeschynomene americana in Thailand was used as a model in this study. A recombinant single-chain fragment variable (scFv) antibody library was constructed from the spleen of a rabbit immunized with DOA9. After three rounds of biopanning, one specific phage-displayed scFv antibody, designated bDOA9rb8, was identified. Specific binding of this antibody was confirmed by phage enzyme-linked immunosorbent assay (phage ELISA). The phage antibody could bind specifically to DOA9 in both free-living cells (pure culture) and bacteroids inside plant nodules. In addition to phage ELISA, specific and robust immunofluorescence staining of both free-living and bacteroid forms could also be observed by confocal-immunofluorescence imaging, without cross-reactivity with other tested bradyrhizobial strains. Moreover, specific binding of free scFv to DOA9 was also demonstrated by ELISA. This recombinant antibody can also be used for the study of the molecular mechanism of plant–microbe interactions in the future. PMID:28654662

Generation of a rabbit single-chain fragment variable (scFv) antibody for specific detection of Bradyrhizobium sp. DOA9 in both free-living and bacteroid forms.

PubMed

Vu, Nguyen Xuan; Pruksametanan, Natcha; Srila, Witsanu; Yuttavanichakul, Watcharin; Teamtisong, Kamonluck; Teaumroong, Neung; Boonkerd, Nantakorn; Tittabutr, Panlada; Yamabhai, Montarop

2017-01-01

A simple and reliable method for the detection of specific nitrogen-fixing bacteria in both free-living and bacteroid forms is essential for the development and application of biofertilizer. Traditionally, a polyclonal antibody generated from an immunized rabbit was used for detection. However, the disadvantages of using a polyclonal antibody include limited supply and cross-reactivity to related bacterial strains. This is the first report on the application of phage display technology for the generation of a rabbit recombinant monoclonal antibody for specific detection and monitoring of nitrogen-fixing bacteria in both free-living form and in plant nodules. Bradyrhizobium sp. DOA9, a broad host range soil bacteria, originally isolated from the root nodules of Aeschynomene americana in Thailand was used as a model in this study. A recombinant single-chain fragment variable (scFv) antibody library was constructed from the spleen of a rabbit immunized with DOA9. After three rounds of biopanning, one specific phage-displayed scFv antibody, designated bDOA9rb8, was identified. Specific binding of this antibody was confirmed by phage enzyme-linked immunosorbent assay (phage ELISA). The phage antibody could bind specifically to DOA9 in both free-living cells (pure culture) and bacteroids inside plant nodules. In addition to phage ELISA, specific and robust immunofluorescence staining of both free-living and bacteroid forms could also be observed by confocal-immunofluorescence imaging, without cross-reactivity with other tested bradyrhizobial strains. Moreover, specific binding of free scFv to DOA9 was also demonstrated by ELISA. This recombinant antibody can also be used for the study of the molecular mechanism of plant-microbe interactions in the future.

Amplification and pyrosequencing of near-full-length hepatitis C virus for typing and monitoring antiviral resistant strains.

PubMed

Trémeaux, P; Caporossi, A; Ramière, C; Santoni, E; Tarbouriech, N; Thélu, M-A; Fusillier, K; Geneletti, L; François, O; Leroy, V; Burmeister, W P; André, P; Morand, P; Larrat, S

2016-05-01

Directly acting antiviral drugs have contributed considerable progress to hepatitis C virus (HCV) treatment, but they show variable activity depending on virus genotypes and subtypes. Therefore, accurate genotyping including recombinant form detection is still of major importance, as is the detection of resistance-associated mutations in case of therapeutic failure. To meet these goals, an approach to amplify the HCV near-complete genome with a single long-range PCR and sequence it with Roche GS Junior was developed. After optimization, the overall amplification success rate was 73% for usual genotypes (i.e. HCV 1a, 1b, 3a and 4a, 16/22) and 45% for recombinant forms RF_2k/1b (5/11). After pyrosequencing and subsequent de novo assembly, a near-full-length genomic consensus sequence was obtained for 19 of 21 samples. The genotype and subtype were confirmed by phylogenetic analysis for every sample, including the suspected recombinant forms. Resistance-associated mutations were detected in seven of 13 samples at baseline, in the NS3 (n = 3) or NS5A (n = 4) region. Of these samples, the treatment of one patient included daclatasvir, and that patient experienced a relapse. Virus sequences from pre- and posttreatment samples of four patients who experienced relapse after sofosbuvir-based therapy were compared: the selected variants seem too far from the NS5B catalytic site to be held responsible. Although tested on a limited set of samples and with technical improvements still necessary, this assay has proven to be successful for both genotyping and resistance-associated variant detection on several HCV types. Copyright © 2016 European Society of Clinical Microbiology and Infectious Diseases. Published by Elsevier Ltd. All rights reserved.

Production of monoclonal antibodies and development of a quantitative immuno-polymerase chain reaction assay to detect and quantify recombinant Glutathione S-transferase.

PubMed

Abud, J E; Luque, E H; Ramos, J G; Rodriguez, H A

2017-07-01

GST-tagged proteins are important tools for the production of recombinant proteins. Removal of GST tag from its fusion protein, frequently by harsh chemical treatments or proteolytic methods, is often required. Thus, the monitoring of the proteins in tag-free form requires a significant effort to determine the remnants of GST during purification process. In the present study, we developed both a conventional enzyme-linked immunosorbent assay (ELISA) and an immuno-polymerase chain reaction (IPCR) assay, both specific for detection of recombinant GST (rGST). rGST was expressed in Escherichia coli JM109, using a pGEX4T-3 vector, and several anti-rGST monoclonal antibodies were generated using hybridoma technology. Two of these were rationally selected as capture and detection antibodies, allowing the development of a sandwich ELISA with a limit of detection (LOD) of 0.01 μg/ml. To develop the rGST-IPCR assay, we selected "Universal-IPCR" format, comprising the biotin-avidin binding as the coupling system. In addition, the rGST-IPCR was developed in standard PCR tubes, and the surface adsorption of antibodies on PCR tubes, the optimal neutravidin concentrations, the generation of a reporter DNA and the concentration effect were studied and determined. Under optimized assay conditions, the rGST-IPCR assay provided a 100-fold increase in the LOD as well as an expanded working range, in comparison with rGST-ELISA. The proposed method exhibited great potentiality for application in several fields in which measurement of very low levels of GST is necessary, and might provide a model for other IPCR assays. Copyright © 2017 Elsevier Inc. All rights reserved.

Detection and genetic characterization of norovirus strains circulating among infants and children with acute gastroenteritis in Japan during 2004-2005.

PubMed

Phan, Tung Gia; Takanashi, Sayaka; Kaneshi, Kunio; Ueda, Yuichi; Nakaya, Shigekazu; Nishimura, Shuichi; Sugita, Kumiko; Nishimura, Tadashi; Yamamoto, Atsuko; Yagyu, Fumihiro; Okitsu, Shoko; Maneekarn, Niwat; Ushijima, Hiroshi

2006-01-01

A total of 752 fecal specimens collected during the period of July 2004 to June 2005 from infants and children with acute gastroenteritis from four different regions (Maizuru, Tokyo, Sapporo, and Osaka) of Japan were tested for the presence of norovirus by RT-PCR. It was found that 139 (18.5%) fecal specimens were positive for norovirus. Norovirus infection was detected almost all year round with the highest prevalence in January. Norovirus GII was the most predominant genogroup (98.6%; 137 of 139). The genotypes detected in this study were GI/1, GII/1, GII/3, GII/4, and GII/6. Of these, NoV GII/4 (known as the Lordsdale virus cluster) was re-emerging and became the leading genotype (77.7%). Meanwhile, the incidence of NoV GII/3 (known as the Arg320 virus cluster) has dropped rapidly, accounting for only 15.8%. Another interesting feature of the study was the identification of Picton03/AU-like recombinant NoV for the first time in Japan. Based on the genetic analysis, it was interesting to note that NoV GII/4 in 2004-2005 made a distinct cluster in comparison to other NoV GII/4 circulating in 2002-2003 and 2003-2004. Of note, "new recombinant variant designated GIIb" within NoV GII/3, which was first detected in Saga City, Japan in 2003-2004 in only one case, had increased, spreading widely in Japan and representing 45.5% (10 of 22). Further epidemiological studies should be conducted to determine whether this new recombinant variant strain will be dominant in Japan in the coming year.

Recombinant Nucleoprotein-Based Enzyme-Linked Immunosorbent Assay for Detection of Immunoglobulin G Antibodies to Crimean-Congo Hemorrhagic Fever Virus

PubMed Central

Saijo, Masayuki; Qing, Tang; Niikura, Masahiro; Maeda, Akihiko; Ikegami, Tetsuro; Prehaud, Christophe; Kurane, Ichiro; Morikawa, Shigeru

2002-01-01

The full-length nucleoprotein of Crimean-Congo hemorrhagic fever virus (CCHFV; 482 amino acid residues) was expressed as a His-tagged recombinant protein (His-CCHFV rNP) in the baculovirus system. The His-CCHFV rNP was efficiently expressed in insect cells and purified by Ni2+ column chromatography. Using this substrate, an immunoglobulin G (IgG) enzyme-linked immunosorbent assay (ELISA) was developed. We evaluated the sensitivity and specificity of the IgG ELISA, using serum samples previously determined to be antibody positive or negative by immunofluorescence tests on CCHFV-infected Vero E6 cells. We found very good correlation between the two tests: 87% for the positive sera (13 of 15) and 99% for the negative sera (107 of 108). These results indicate that the new IgG ELISA using His-CCHFV rNP has high sensitivity and specificity for detecting CCHFV antibodies. The CCHF patients' sera with high titers reacted only with the NP fragment containing amino acid residues between 201 and 306 in Western blotting. It is known that amino acid homologies are high in this region among various isolates. Thus, it is expected that this ELISA can detect antibodies not only for Chinese strains of CCHFV but also for other strains circulating in the world. These results suggest that the IgG ELISA system developed with the recombinant CCHFV NP is a valuable tool for diagnosis and epidemiological investigations of CCHFV infections. PMID:11980926

Generation and Characterization of Six Recombinant Botulinum Neurotoxins as Reference Material to Serve in an International Proficiency Test.

PubMed

Weisemann, Jasmin; Krez, Nadja; Fiebig, Uwe; Worbs, Sylvia; Skiba, Martin; Endermann, Tanja; Dorner, Martin B; Bergström, Tomas; Muñoz, Amalia; Zegers, Ingrid; Müller, Christian; Jenkinson, Stephen P; Avondet, Marc-Andre; Delbrassinne, Laurence; Denayer, Sarah; Zeleny, Reinhard; Schimmel, Heinz; Åstot, Crister; Dorner, Brigitte G; Rummel, Andreas

2015-11-26

The detection and identification of botulinum neurotoxins (BoNT) is complex due to the existence of seven serotypes, derived mosaic toxins and more than 40 subtypes. Expert laboratories currently use different technical approaches to detect, identify and quantify BoNT, but due to the lack of (certified) reference materials, analytical results can hardly be compared. In this study, the six BoNT/A1-F1 prototypes were successfully produced by recombinant techniques, facilitating handling, as well as improving purity, yield, reproducibility and biosafety. All six BoNTs were quantitatively nicked into active di-chain toxins linked by a disulfide bridge. The materials were thoroughly characterized with respect to purity, identity, protein concentration, catalytic and biological activities. For BoNT/A₁, B₁ and E₁, serotypes pathogenic to humans, the catalytic activity and the precise protein concentration were determined by Endopep-mass spectrometry and validated amino acid analysis, respectively. In addition, BoNT/A₁, B₁, E₁ and F₁ were successfully detected by immunological assays, unambiguously identified by mass spectrometric-based methods, and their specific activities were assigned by the mouse LD50 bioassay. The potencies of all six BoNT/A1-F1 were quantified by the ex vivo mouse phrenic nerve hemidiaphragm assay, allowing a direct comparison. In conclusion, highly pure recombinant BoNT reference materials were produced, thoroughly characterized and employed as spiking material in a worldwide BoNT proficiency test organized by the EQuATox consortium.

The fate of deleted DNA produced during programmed genomic deletion events in Tetrahymena thermophila.

PubMed Central

Saveliev, S V; Cox, M M

1994-01-01

Thousands of DNA deletion events occur during macronuclear development in the ciliate Tetrahymena thermophila. In two deleted genomic regions, designated M and R, the eliminated sequences form circles that can be detected by PCR. However, the circles are not normal products of the reaction pathway. The circular forms occur at very low levels in conjugating cells, but are stable. Sequencing analysis showed that many of the circles (as many as 50% of those examined) reflected a precise deletion in the M and R regions. The remaining circles were either smaller or larger and contained varying lengths of sequences derived from the chromosomal DNA surrounding the eliminated region. The chromosomal junctions left behind after deletion were more precise, although deletions in either the M or R regions can generate any of several alternative junctions (1). Some new chromosomal junctions were detected in the present study. The results suggest that the deleted segment is released as a linear DNA species that is degraded rapidly. The species is only rarely converted to the stable circles we detect. The deletion mechanism is different from those proposed for deletion events in hypotrichous ciliates (2-4), and does not reflect a conservative site-specific recombination process such as that promoted by the bacteriophage lambda integrase (5). Images PMID:7838724

Whole genome analysis of porcine astroviruses detected in Japanese pigs reveals genetic diversity and possible intra-genotypic recombination.

PubMed

Ito, Mika; Kuroda, Moegi; Masuda, Tsuneyuki; Akagami, Masataka; Haga, Kei; Tsuchiaka, Shinobu; Kishimoto, Mai; Naoi, Yuki; Sano, Kaori; Omatsu, Tsutomu; Katayama, Yukie; Oba, Mami; Aoki, Hiroshi; Ichimaru, Toru; Mukono, Itsuro; Ouchi, Yoshinao; Yamasato, Hiroshi; Shirai, Junsuke; Katayama, Kazuhiko; Mizutani, Tetsuya; Nagai, Makoto

2017-06-01

Porcine astroviruses (PoAstVs) are ubiquitous enteric virus of pigs that are distributed in several countries throughout the world. Since PoAstVs are detected in apparent healthy pigs, the clinical significance of infection is unknown. However, AstVs have recently been associated with a severe neurological disorder in animals, including humans, and zoonotic potential has been suggested. To date, little is known about the epidemiology of PoAstVs among the pig population in Japan. In this report, we present an analysis of nearly complete genomes of 36 PoAstVs detected by a metagenomics approach in the feces of Japanese pigs. Based on a phylogenetic analysis and pairwise sequence comparison, 10, 5, 15, and 6 sequences were classified as PoAstV2, PoAstV3, PoAstV4, and PoAstV5, respectively. Co-infection with two or three strains was found in individual fecal samples from eight pigs. The phylogenetic trees of ORF1a, ORF1b, and ORF2 of PoAstV2 and PoAstV4 showed differences in their topologies. The PoAstV3 and PoAstV5 strains shared high sequence identities within each genotype in all ORFs; however, one PoAstV3 strain and one PoAstV5 strain showed considerable sequence divergence from the other PoAstV3 and PoAstV5 strains, respectively, in ORF2. Recombination analysis using whole genomes revealed evidence of multiple possible intra-genotype recombination events in PoAstV2 and PoAstV4, suggesting that recombination might have contributed to the genetic diversity and played an important role in the evolution of Japanese PoAstVs. Copyright © 2017 Elsevier B.V. All rights reserved.

Very deep spectroscopy of the bright Saturn nebula NGC 7009 - II. Analysis of the rich optical recombination spectrum

NASA Astrophysics Data System (ADS)

Fang, X.; Liu, X.-W.

2013-03-01

In Paper I, we presented a deep, long-slit spectrum of the bright Saturn nebula NGC 7009. Numerous permitted lines emitted by the C+, N+, O+ and Ne+ ions were detected. Gaussian profile fitting to the spectrum yielded more than 1000 lines, the majority of which are optical recombination lines (ORLs) of heavy-element ions. In the current paper, we present a critical analysis of the rich optical recombination spectrum of NGC 7009, in the context of the bi-abundance nebular model proposed by Liu et al. Transitions from individual multiplets are checked carefully for potential blended lines. The observed relative intensities are compared with the theoretical predictions based on high-quality effective recombination coefficients, now available for the recombination line spectrum of a number of heavy-element ions. The possibility of plasma diagnostics using the ORLs of various heavy-element ions is discussed in detail. The line ratios that can be used to determine electron temperature are presented for each ion, although there is still a lack of adequate atomic data and some of the lines are still not detected in the spectrum of NGC 7009 due to weakness and/or line blending. Plasma diagnostics based on the N II and O II recombination spectra both yield electron temperatures close to 1000 K, which is lower than those derived from the collisionally excited line (CEL) ratios (e.g. the [O III] and [N II] nebular-to-auroral line ratios; see Paper I for details) by nearly one order of magnitude. The very low temperatures yielded by the O II and N II ORLs indicate that they originate from very cold regions. The C2+/H+, N2+/H+, O2+/H+ and Ne2+/H+ ionic abundance ratios derived from ORLs are consistently higher, by about a factor of 5, than the corresponding values derived from CELs. In calculating the ORL ionic abundance ratios, we have used the newly available high-quality effective recombination coefficients, and adopted an electron temperature of ˜1000 K, as given by the ORL diagnostics and as a consequence presumably representing the physical conditions prevailing in the regions where the heavy-element ORLs arise. Measurements of the ultraviolet (UV) and infrared (IR) CELs from the literature are used to calculate CEL ionic abundance ratios when optical data are not available for the ionic species. A comparison of results of plasma diagnostics and abundance determinations for NGC 7009 points to the existence of `cold', metal-rich (i.e. H-deficient) inclusions embedded in the hot, diffuse ionized gas, first postulated by Liu et al. At electron temperatures yielded by the N II and O II ORLs, the predicted relative intensities of ORLs agree well with the observed values, indicating that the current quantum calculations of the recombination spectra of those two ionic species well represent the recombination processes under nebular conditions. Deviations from the LS coupling, noticed in an earlier quantitative spectroscopy by Liu et al. for the same object, are again confirmed, especially for recombination lines of the 4f-3d transition array. For N II, as well as for O II, the ionic abundances derived from different J-resolved transitions within a multiplet, or from the transitions belonging to different multiplets, agree with each other. This is another evidence that the new effective recombination coefficients are reliable. New calculations of the effective recombination coefficients for the Ne II lines at nebular temperatures and densities are needed.

In utero and acute exposure to benzene: investigation of DNA double-strand breaks and DNA recombination in mice.

PubMed

Lau, Annette; Belanger, Christine Lea; Winn, Louise M

2009-05-31

Benzene, a ubiquitous pollutant, has been identified as a human leukemogen and early exposure to environmental carcinogens such as benzene has been linked to childhood leukemia. It is known that genotoxic agents can increase the frequency of DNA double-strand breaks (DSBs), which can initiate DNA recombinational repair mechanisms. In this study we investigated the induction of micronuclei, the formation of gamma-H2A.X as a marker of DNA DSBs, and the induction of somatic DNA recombination events in hematopoietic tissue from pKZ1 transgenic mice exposed acutely or in utero to benzene. Adult male C57Bl/6N mice were treated with a single i.p. injection of benzene, and timed-pregnant females pKZ1 were treated with daily i.p. injections of 200 mg/kg or 400 mg/kg benzene through gestational days 7-15. Acute exposure to 400 mg/kg benzene resulted in a statistically significant increase in the percentage of micronucleated cells in adult male bone marrow cells and in fetal liver and post-natal day 9 bone marrow cells of mice exposed in utero. Immunoblotting techniques did not detect benzene-induced increases in the formation of gamma-H2A.X in bone marrow cells of adult male mice and in maternal bone marrow, fetal liver, and post-natal bone marrow cells after specific time-point exposures. Finally, no recombination events were detected in adult pKZ1 mouse tissue; however, in post-natal day 9 pups in utero exposure to 400 mg/kg of benzene caused a trend towards increasing recombination frequency although this did not reach statistical significance. These results demonstrate that in utero exposure increases the frequency of micronuclei and DNA recombination events in hematopoietic tissue of fetal and post-natal mice and may be an initiating event in the etiology of childhood leukemias. Further investigations into different types of DNA damage and repair pathways are warranted to fully elucidate the role of genotoxic mechanisms in the etiology of benzene-induced childhood leukemias.

Use of a Recombinant Gamma-2 Herpesvirus Vaccine Vector against Dengue Virus in Rhesus Monkeys.

PubMed

Bischof, Georg F; Magnani, Diogo M; Ricciardi, Michael; Shin, Young C; Domingues, Aline; Bailey, Varian K; Gonzalez-Nieto, Lucas; Rakasz, Eva G; Watkins, David I; Desrosiers, Ronald C

2017-08-15

Research on vaccine approaches that can provide long-term protection against dengue virus infection is needed. Here we describe the construction, immunogenicity, and preliminary information on the protective capacity of recombinant, replication-competent rhesus monkey rhadinovirus (RRV), a persisting herpesvirus. One RRV construct expressed nonstructural protein 5 (NS5), while a second recombinant expressed a soluble variant of the E protein (E85) of dengue virus 2 (DENV2). Four rhesus macaques received a single vaccination with a mixture of both recombinant RRVs and were subsequently challenged 19 weeks later with 1 × 10 5 PFU of DENV2. During the vaccine phase, plasma of all vaccinated monkeys showed neutralizing activity against DENV2. Cellular immune responses against NS5 were also elicited, as evidenced by major histocompatibility complex class I (MHC-I) tetramer staining in the one vaccinated monkey that was Mamu-A*01 positive. Unlike two of two unvaccinated controls, two of the four vaccinated monkeys showed no detectable viral RNA sequences in plasma after challenge. One of these two monkeys also showed no anamnestic increases in antibody levels following challenge and thus appeared to be protected against the acquisition of DENV2 following high-dose challenge. Continued study will be needed to evaluate the performance of herpesviral and other persisting vectors for achieving long-term protection against dengue virus infection. IMPORTANCE Continuing studies of vaccine approaches against dengue virus (DENV) infection are warranted, particularly ones that may provide long-term immunity against all four serotypes. Here we investigated whether recombinant rhesus monkey rhadinovirus (RRV) could be used as a vaccine against DENV2 infection in rhesus monkeys. Upon vaccination, all animals generated antibodies capable of neutralizing DENV2. Two of four vaccinated monkeys showed no detectable viral RNA after subsequent high-dose DENV2 challenge at 19 weeks postvaccination. Furthermore, one of these vaccinated monkeys appeared to be protected against the acquisition of DENV2 infection on the basis of undetectable viral loads and the lack of an anamnestic antibody response. These findings underscore the potential utility of recombinant herpesviruses as vaccine vectors. Copyright © 2017 American Society for Microbiology.

[A comparative study on characterizations of genetic recombination hotspots in PPARG gene between Kirgiz and Uyghur ethnic groups in Xinjiang].

PubMed

Zohra, Rozi; Song, M S; Iliham, Nizam; Dolikun, Mamatyusupu

2016-08-16

To investigate the characterizations of genetic recombination hotspots and linkage disequilibrium (LD) patterns in peroxisome proliferative activated receptor gamma (PPARG) gene in Kirgiz and Uyghur ethnic groups. Blood samples were collected from 100 Kirgiz (50 healthy controls and 50 patients with type 2 diabetes mellitus) residents in Halajun County, Artux City, Kizilsu Kirgiz Autonomous Prefecture, Xinjiang in August 2013, and 50 healthy Uyghur residents in Hotan Prefecture of Xinjiang Uygur Autonomous Region in May 2012.Thirty-one tagSNPs in PPARG gene were genotyped using Matrix-Assisted Laser Desorption/Ionization Time of Flight Mass Spectrometry (MALDI-TOF-MS) method.The recombination hotspots and LD patterns within the PPARG gene were estimated by analyzing the SNP genotying data using the Hotspot Fisher program and Haploview software, respectively. Eighteen tagSNPs (rs1151999, rs1175540, rs1875796, rs1899951, rs2292101, rs2921190, rs2938397, rs2959272, rs2959273, rs2972162, rs3856806, rs4135247, rs4135275, rs709151, rs4135354, rs6805419, rs17036700 and rs4135304) were same with relatively higher recombination rates between the patients with type 2 diabetes mellitus (T2DM) and healthy controls of Kirgiz ethnic group, and healthy controls of Uyghur ethnic group.Five haplotype blocks with LD coefficient D' value of 1, indicating no genetic recombination occurred within the region, were observed in the healthy controls of Kirgiz ethnic groups, whereas five haplotype blocks with LD coefficient D' value less than 1 were observed in the Kirgiz patients with T2DM, indicating historical recombination events occurred within the region.Four haplotype blocks with LD coefficient D' value of 1 were observed in the Uyghur healthy controls, indicating no genetic recombination occurred within the region.There were significantly different recombination hotspot profiles between the Kirgiz, Uyghur, Utah residents with Northern and Western European ancestry (CEU), Yoruban in Ibadan, Nigeria (YRI) and Han Chinese in Beijing (CHB) and Japanese in Tokyo (JPT) samples.There are six recombination hotspots in the HapMap profile of genetic recombination.The last 5 SNPs within the PPARG gene were shown with lower recombination rates in the Kirgiz, whereas no recombination hotspot was found in the Uyghur. Variable recombination rates may be present in certain chromosome region between patients and healthy controls within the same or between the different ethnic groups.There may be presence of recombination hotspots of ethnic specificity and with variable recombination rates.

Field safety assessment of recombination in transgenic grapevines expressing the coat protein gene of Grapevine fanleaf virus.

PubMed

Vigne, Emmanuelle; Komar, Véronique; Fuchs, Marc

2004-04-01

One of the major environmental safety issues over transgenic crops containing virus-derived genes relates to the outcome of recombination events between viral transgene transcripts and RNAs from indigenous virus populations. We addressed this issue by assessing the emergence of viable Grapevine fanleaf virus (GFLV) recombinants in transgenic grapevines expressing the GFLV coat protein (CP) gene. Test plants consisted of nontransgenic scions grafted onto transgenic and nontransgenic rootstocks that were exposed over 3 years to nematode-mediated GFLV infection in two distinct vineyard sites. The CP gene of challenging GFLV isolates was amplified from scions by IC-RT-PCR, and characterized by RFLP and nucleotide sequencing using strain F13 as reference since it provided the CP transgene. Analysis of EcoRI and StyI RFLP banding patterns from 347 challenging GFLV isolates and sequence data from 85 variants revealed no characteristics similar to strain F13 and no difference in the molecular variability among isolates from 190 transgenic and 157 nontransgenic plants, or from plants within (253 individuals) or outside (94 individuals) of the two sites. Interestingly, five GFLV recombinants were identified in three nontransgenic plants located outside of the two field settings. This survey indicates that transgenic grapevines did not assist the emergence of viable GFLV recombinants to detectable levels nor did they affect the molecular diversity of indigenous GFLV populations during the trial period. This is the first report on safety assessment of recombination with a transgenic crop expressing a CP gene under field conditions of heavy disease pressure but low, if any, selection pressure against recombinant viruses.

Efficient secretory expression of recombinant proteins in Escherichia coli with a novel actinomycete signal peptide.

PubMed

Cui, Yanbing; Meng, Yiwei; Zhang, Juan; Cheng, Bin; Yin, Huijia; Gao, Chao; Xu, Ping; Yang, Chunyu

2017-01-01

In well-established heterologous hosts, such as Escherichia coli, recombinant proteins are usually intracellular and frequently found as inclusion bodies-especially proteins possessing high rare codon content. In this study, successful secretory expression of three hydrolases, in a constructed inducible or constitutive system, was achieved by fusion with a novel signal peptide (Kp-SP) from an actinomycete. The signal peptide efficiently enabled extracellular protein secretion and also contributed to the active expression of the intracellular recombinant proteins. The thermophilic α-amylase gene of Bacillus licheniformis was fused with Kp-SP. Both recombinants, carrying inducible and constitutive plasmids, showed remarkable increases in extracellular and intracellular amylolytic activity. Amylase activity was observed to be > 10-fold in recombinant cultures with the constitutive plasmid, pBSPPc, compared to that in recombinants lacking Kp-SP. Further, the signal peptide enabled efficient secretion of a thermophilic cellulase into the culture medium, as demonstrated by larger halo zones and increased enzymatic activities detected in both constructs from different plasmids. For heterologous proteins with a high proportion of rare codons, it is difficult to obtain high expression in E. coli owing to the codon bias. Here, the fusion of an archaeal homologue of the amylase encoding gene, FSA, with Kp-SP resulted in > 5-fold higher extracellular activity. The successful extracellular expression of the amylase indicated that the signal peptide also contributed significantly to its active expression and signified the potential value of this novel and versatile signal peptide in recombinant protein production. Copyright © 2016 Elsevier Inc. All rights reserved.

A biosensor based on electroactive dipyrromethene-Cu(II) layer deposited onto gold electrodes for the detection of antibodies against avian influenza virus type H5N1 in hen sera.

PubMed

Jarocka, Urszula; Sawicka, Róża; Stachyra, Anna; Góra-Sochacka, Anna; Sirko, Agnieszka; Zagórski-Ostoja, Włodzimierz; Sączyńska, Violetta; Porębska, Anna; Dehaen, Wim; Radecki, Jerzy; Radecka, Hanna

2015-10-01

This paper describes the development of a biosensor for the detection of anti-hemagglutinin antibodies against the influenza virus hemagglutinin. The steps of biosensor fabrications are as follows: (i) creation of a mixed layer containing the thiol derivative of dipyrromethene and 4-mercapto-1-butanol, (ii) complexation of Cu(II) ions, (iii) oriented immobilization of the recombinant histidine-tagged hemagglutinin, and (iv) filling free spaces with bovine serum albumin. The interactions between recombinants hemagglutinin from the highly pathogenic avian influenza virus type H5N1 and anti-hemagglutinin H5 monoclonal antibodies were explored with Osteryoung square-wave voltammetry. The biosensor displayed a good detection limit of 2.4 pg/mL, quantification limit of 7.2 pg/mL, and dynamic range from 4.0 to 100.0 pg/mL in buffer. In addition, this analytical device was applied for the detection of antibodies in hen sera from individuals vaccinated and non-vaccinated against the avian influenza virus type H5N1. The limit of detection for the assay was the dilution of sera 1: 7 × 10(6), which is about 200 times better than the enzyme-linked immunosorbent assay.

Development and modification of a recombinant cell bioassay to directly detect halogenated and polycyclic aromatic hydrocarbons in serum.

PubMed

Ziccardi, M H; Gardner, I A; Denison, M S

2000-03-01

Polycyclic and halogenated aromatic hydrocarbons (PAHs/HAHs) are a diverse group of widespread and persistent environmental contaminants that can cause a variety of detrimental effects in vertebrates. As most available methods to detect these contaminants are expensive, labor and time intensive, and require large amounts of tissue for extraction and analysis, several rapid mechanistically based bioassay systems have been developed to detect these chemicals. Here we describe application and optimization of a recently developed recombinant mouse cell bioassay system that responds to both PAHs and HAHs with the rapid induction of firefly luciferase for the detection of these chemicals in whole serum samples. This chemically activated luciferase expression (CALUX) bioassay has been modified to allow rapid (4-h) and direct analysis of small volumes (25-50 microl) of whole serum in a 96-well microtiter plate format without the need for solvent extraction. This bioassay can detect as little as 10 parts per trillion of the most potent HAH, 2,3,7,8-TCDD, and is also sensitive to other HAHs and PAHs. The use of simple procedures corrects for interplate and intraplate variability and the Ah receptor dependence of the induction response is accounted for by use of the antagonist 4-amino-3-methoxyflavone.

Immunodiagnostic monoclonal antibody-based sandwich ELISA of fasciolosis by detection of Fasciola gigantica circulating fatty acid binding protein.

PubMed

Anuracpreeda, Panat; Chawengkirttikul, Runglawan; Sobhon, Prasert

2016-09-01

Up to now, parasitological diagnosis of fasciolosis is often unreliable and possesses low sensitivity. Hence, the detection of circulating parasite antigens is thought to be a better alternative for diagnosis of fasciolosis, as it reflects the real parasite burden. In the present study, a monoclonal antibody (MoAb) against recombinant Fasciola gigantica fatty acid binding protein (rFgFABP) has been produced. As well, a reliable sandwich enzyme-linked immunosorbent assay (sandwich ELISA) has been developed for the detection of circulating FABP in the sera of mice experimentally and cattle naturally infected with F. gigantica. MoAb 3A3 and biotinylated rabbit anti-recombinant FABP antibody were selected due to their high reactivities and specificities. The lower detection limit of sandwich ELISA was 5 pg mL-1, and no cross-reaction with other parasite antigens was observed. This assay could detect F. gigantica infection from day 1 post infection. In experimental mice, the sensitivity, specificity and accuracy of this assay were 93·3, 100 and 98·2%, while in natural cattle they were 96·7, 100 and 99·1%. Hence, this sandwich ELISA method showed high efficiencies and precisions for diagnosis of fasciolosis by F. gigantica.

Detection of exogenous gene doping of IGF-I by a real-time quantitative PCR assay.

PubMed

Zhang, Jin-Ju; Xu, Jing-Feng; Shen, Yong-Wei; Ma, Shi-Jiao; Zhang, Ting-Ting; Meng, Qing-Lin; Lan, Wen-Jun; Zhang, Chun; Liu, Xiao-Mei

2017-07-01

Gene doping can be easily concealed since its product is similar to endogenous protein, making its effective detection very challenging. In this study, we selected insulin-like growth factor I (IGF-I) exogenous gene for gene doping detection. First, the synthetic IGF-I gene was subcloned to recombinant adeno-associated virus (rAAV) plasmid to produce recombinant rAAV2/IGF-I-GFP vectors. Second, in an animal model, rAAV2/IGF-I-GFP vectors were injected into the thigh muscle tissue of mice, and then muscle and blood specimens were sampled at different time points for total DNA isolation. Finally, real-time quantitative PCR was employed to detect the exogenous gene doping of IGF-I. In view of the characteristics of endogenous IGF-I gene sequences, a TaqMan probe was designed at the junction of exons 2 and 3 of IGF-I gene to distinguish it from the exogenous IGF-I gene. In addition, an internal reference control plasmid and its probe were used in PCR to rule out false-positive results through comparison of their threshold cycle (Ct) values. Thus, an accurate exogenous IGF-I gene detection approach was developed in this study. © 2016 International Union of Biochemistry and Molecular Biology, Inc.

Meiotic recombination modulates the structure and dynamics of the synaptonemal complex during C. elegans meiosis

PubMed Central

2017-01-01

During meiotic prophase, a structure called the synaptonemal complex (SC) assembles at the interface between aligned pairs of homologous chromosomes, and crossover recombination events occur between their DNA molecules. Here we investigate the inter-relationships between these two hallmark features of the meiotic program in the nematode C. elegans, revealing dynamic properties of the SC that are modulated by recombination. We demonstrate that the SC incorporates new subunits and switches from a more highly dynamic/labile state to a more stable state as germ cells progress through the pachytene stage of meiotic prophase. We further show that the more dynamic state of the SC is prolonged in mutants where meiotic recombination is impaired. Moreover, in meiotic mutants where recombination intermediates are present in limiting numbers, SC central region subunits become preferentially stabilized on the subset of chromosome pairs that harbor a site where pro-crossover factors COSA-1 and MutSγ are concentrated. Polo-like kinase PLK-2 becomes preferentially localized to the SCs of chromosome pairs harboring recombination sites prior to the enrichment of SC central region proteins on such chromosomes, and PLK-2 is required for this enrichment to occur. Further, late pachytene nuclei in a plk-2 mutant exhibit the more highly dynamic SC state. Together our data demonstrate that crossover recombination events elicit chromosome-autonomous stabilizing effects on the SC and implicate PLK-2 in this process. We discuss how this recombination-triggered modulation of SC state might contribute to regulatory mechanisms that operate during meiosis to ensure the formation of crossovers while at the same time limiting their numbers. PMID:28339470

The SMC-5/6 Complex and the HIM-6 (BLM) Helicase Synergistically Promote Meiotic Recombination Intermediate Processing and Chromosome Maturation during Caenorhabditis elegans Meiosis

PubMed Central

Hong, Ye; Sonneville, Remi; Agostinho, Ana; Meier, Bettina; Wang, Bin; Blow, J. Julian; Gartner, Anton

2016-01-01

Meiotic recombination is essential for the repair of programmed double strand breaks (DSBs) to generate crossovers (COs) during meiosis. The efficient processing of meiotic recombination intermediates not only needs various resolvases but also requires proper meiotic chromosome structure. The Smc5/6 complex belongs to the structural maintenance of chromosome (SMC) family and is closely related to cohesin and condensin. Although the Smc5/6 complex has been implicated in the processing of recombination intermediates during meiosis, it is not known how Smc5/6 controls meiotic DSB repair. Here, using Caenorhabditis elegans we show that the SMC-5/6 complex acts synergistically with HIM-6, an ortholog of the human Bloom syndrome helicase (BLM) during meiotic recombination. The concerted action of the SMC-5/6 complex and HIM-6 is important for processing recombination intermediates, CO regulation and bivalent maturation. Careful examination of meiotic chromosomal morphology reveals an accumulation of inter-chromosomal bridges in smc-5; him-6 double mutants, leading to compromised chromosome segregation during meiotic cell divisions. Interestingly, we found that the lethality of smc-5; him-6 can be rescued by loss of the conserved BRCA1 ortholog BRC-1. Furthermore, the combined deletion of smc-5 and him-6 leads to an irregular distribution of condensin and to chromosome decondensation defects reminiscent of condensin depletion. Lethality conferred by condensin depletion can also be rescued by BRC-1 depletion. Our results suggest that SMC-5/6 and HIM-6 can synergistically regulate recombination intermediate metabolism and suppress ectopic recombination by controlling chromosome architecture during meiosis. PMID:27010650

The SMC-5/6 Complex and the HIM-6 (BLM) Helicase Synergistically Promote Meiotic Recombination Intermediate Processing and Chromosome Maturation during Caenorhabditis elegans Meiosis.

PubMed

Hong, Ye; Sonneville, Remi; Agostinho, Ana; Meier, Bettina; Wang, Bin; Blow, J Julian; Gartner, Anton

2016-03-01

Meiotic recombination is essential for the repair of programmed double strand breaks (DSBs) to generate crossovers (COs) during meiosis. The efficient processing of meiotic recombination intermediates not only needs various resolvases but also requires proper meiotic chromosome structure. The Smc5/6 complex belongs to the structural maintenance of chromosome (SMC) family and is closely related to cohesin and condensin. Although the Smc5/6 complex has been implicated in the processing of recombination intermediates during meiosis, it is not known how Smc5/6 controls meiotic DSB repair. Here, using Caenorhabditis elegans we show that the SMC-5/6 complex acts synergistically with HIM-6, an ortholog of the human Bloom syndrome helicase (BLM) during meiotic recombination. The concerted action of the SMC-5/6 complex and HIM-6 is important for processing recombination intermediates, CO regulation and bivalent maturation. Careful examination of meiotic chromosomal morphology reveals an accumulation of inter-chromosomal bridges in smc-5; him-6 double mutants, leading to compromised chromosome segregation during meiotic cell divisions. Interestingly, we found that the lethality of smc-5; him-6 can be rescued by loss of the conserved BRCA1 ortholog BRC-1. Furthermore, the combined deletion of smc-5 and him-6 leads to an irregular distribution of condensin and to chromosome decondensation defects reminiscent of condensin depletion. Lethality conferred by condensin depletion can also be rescued by BRC-1 depletion. Our results suggest that SMC-5/6 and HIM-6 can synergistically regulate recombination intermediate metabolism and suppress ectopic recombination by controlling chromosome architecture during meiosis.

MOLED: Simulation of multilayer organic light emitting diodes

NASA Astrophysics Data System (ADS)

Houili, H.; Tutiš, E.; Lütjens, H.; Bussac, M. N.; Zuppiroli, L.

2003-12-01

MOLED solves the dynamics of electrons and holes in multilayer Organic Light Emitting Diodes (OLED). The carriers are injected on the positive and negative electrodes of the device by tunneling through a potential barrier. Thermal excitation processes across the barrier are also included. In the interior of the device the electron-hole recombination occurs when the two carriers are close enough, according to a model inspired from the one of Langevin. A fraction of these recombined pairs gives photons. The charge transport inside the organic material occurs through hopping. Several choices of mobility formulae are available in the code. MOLED can be used for OLEDs with an arbitrary number of layers. The output consists of numerous fields that describe the device performance. For example, there are the current, the recombination and the charge density distributions, the electric field distribution, the current-voltage characteristics and the device internal quantum efficiency. Program summaryTitle of program: MOLED Catalogue identifier: ADSG Program summary URL:http://cpc.cs.qub.ac.uk/summaries/ADSG Program obtainable from: CPC Program Library, Queen's University of Belfast, N. Ireland Operating systems under which the program has been tested: Unix, Linux Programming language used: FORTRAN 90 Memory required to execute with typical data: 2 MB No. of bytes in distributed program: 26 942 No. of bits in a word: 64 Peripherals used: permanent disk storage No. of lines in distributed program, including test data, etc.: 3695 Distribution format: tar gzip file Nature of the physical problem: Injection of electrons and holes into an organic electroluminescent material occurs through tunneling from metal electrodes. The transport of carriers inside the molecular medium proceeds by hopping from one molecule to another. The emission of light is a result of their radiative Langevin recombination (for a review see [Scott et al., Synthetic Metals 111-112 (2000) 289; Friend et al., Nature 397 (1999) 121]). Method of solution: The equations governing the time evolution of current, charge density and electric field in the device are solved after discretization in time. An implicit procedure is used to perform the time step. The nonlinear effects, originating from strong dependence of the hopping frequency on charge distribution, are treated within the first order in the implicit method [Press et al., Numerical Recipes, Cambridge Univ. Press, 1986, Ch. 17]. The molecular energy levels are updated at each time step to take into account the Coulomb interactions [Tutiš et al., J. Appl. Phys. 89 (2001) 430]. Typical running time: A typical calculation takes a few minutes on DEC Alpha 500 MHz or Pentium 4 1.8 GHz machines. The execution time may vary considerably, depending on the complexity of the problem. Unusual features of the program: This code has a GUI interface for input/output that can be obtained from the URL: http://lomm.epfl.ch/.

Novel HBV recombinants between genotypes B and C in 3'-terminal reverse transcriptase (RT) sequences are associated with enhanced viral DNA load, higher RT point mutation rates and place of birth among Chinese patients.

PubMed

Liu, Baoming; Yang, Jing-Xian; Yan, Ling; Zhuang, Hui; Li, Tong

2018-01-01

As one of the major global public health concerns, hepatitis B virus (HBV) can be divided into at least eight genotypes, which may be related to disease severity and treatment response. We previously demonstrated that genotypes B and C HBV, with distinct geographical distribution in China, had divergent genotype-dependent amino acid polymorphisms and variations in reverse transcriptase (RT) gene region, a target of antiviral therapy using nucleos(t)ide analogues. Recently recombination between HBV genotypes B and C was reported to occur in the RT region. However, their frequency and clinical significance is poorly understood. Here full-length HBV RT sequences from 201 Chinese chronic hepatitis B (CHB) patients were amplified and sequenced, among which 31.34% (63/201) were genotype B whereas 68.66% (138/201) genotype C. Although no intergenotypic recombination was detected among C-genotype HBV, 38.10% (24/63) of B-genotype HBV had recombination with genotype C in the 3'-terminal RT sequences. The patients with B/C intergenotypic recombinants had significantly (P<0.05) higher serum HBV DNA level than the "pure" B-genotype cohort did. Moreover, the B/C intergenotypic recombinants were prone to more substitutions at several specific residues in the RT region than genotype B or C. Besides, unlike their parental genotypes, the recombinant HBV appeared to display an altered geographic distribution feature in China. Our findings provide novel insight into the virological, clinical and epidemiological features of new HBV B/C intergenotypic recombinants at the 3' end of RT sequences among Chinese CHB patients. The highly complex genetic background of the novel recombinant HBV carrying new mutations affecting RT protein may contribute to an enhanced heterogeneity in treatment response or prognosis among CHB patients. Published by Elsevier B.V.

Joint Molecule Resolution Requires the Redundant Activities of MUS-81 and XPF-1 during Caenorhabditis elegans Meiosis

PubMed Central

O'Neil, Nigel J.; Martin, Julie S.; Youds, Jillian L.; Ward, Jordan D.; Petalcorin, Mark I. R.; Rose, Anne M.; Boulton, Simon J.

2013-01-01

The generation and resolution of joint molecule recombination intermediates is required to ensure bipolar chromosome segregation during meiosis. During wild type meiosis in Caenorhabditis elegans, SPO-11-generated double stranded breaks are resolved to generate a single crossover per bivalent and the remaining recombination intermediates are resolved as noncrossovers. We discovered that early recombination intermediates are limited by the C. elegans BLM ortholog, HIM-6, and in the absence of HIM-6 by the structure specific endonuclease MUS-81. In the absence of both MUS-81 and HIM-6, recombination intermediates persist, leading to chromosome breakage at diakinesis and inviable embryos. MUS-81 has an additional role in resolving late recombination intermediates in C. elegans. mus-81 mutants exhibited reduced crossover recombination frequencies suggesting that MUS-81 is required to generate a subset of meiotic crossovers. Similarly, the Mus81-related endonuclease XPF-1 is also required for a subset of meiotic crossovers. Although C. elegans gen-1 mutants have no detectable meiotic defect either alone or in combination with him-6, mus-81 or xpf-1 mutations, mus-81;xpf-1 double mutants are synthetic lethal. While mus-81;xpf-1 double mutants are proficient for the processing of early recombination intermediates, they exhibit defects in the post-pachytene chromosome reorganization and the asymmetric disassembly of the synaptonemal complex, presumably triggered by crossovers or crossover precursors. Consistent with a defect in resolving late recombination intermediates, mus-81; xpf-1 diakinetic bivalents are aberrant with fine DNA bridges visible between two distinct DAPI staining bodies. We were able to suppress the aberrant bivalent phenotype by microinjection of activated human GEN1 protein, which can cleave Holliday junctions, suggesting that the DNA bridges in mus-81; xpf-1 diakinetic oocytes are unresolved Holliday junctions. We propose that the MUS-81 and XPF-1 endonucleases act redundantly to process late recombination intermediates to form crossovers during C. elegans meiosis. PMID:23874209

The Recent Recombinant Evolution of a Major Crop Pathogen, Potato virus Y

PubMed Central

Visser, Johan Christiaan; Bellstedt, Dirk Uwe; Pirie, Michael David

2012-01-01

Potato virus Y (PVY) is a major agricultural disease that reduces crop yields worldwide. Different strains of PVY are associated with differing degrees of pathogenicity, of which the most common and economically important are known to be recombinant. We need to know the evolutionary origins of pathogens to prevent further escalations of diseases, but putatively reticulate genealogies are challenging to reconstruct with standard phylogenetic approaches. Currently available phylogenetic hypotheses for PVY are either limited to non-recombinant strains, represent only parts of the genome, and/or incorrectly assume a strictly bifurcating phylogenetic tree. Despite attempts to date potyviruses in general, no attempt has been made to date the origins of pathogenic PVY. We test whether diversification of the major strains of PVY and recombination between them occurred within the time frame of the domestication and modern cultivation of potatoes. In so doing, we demonstrate a novel extension of a phylogenetic approach for reconstructing reticulate evolutionary scenarios. We infer a well resolved phylogeny of 44 whole genome sequences of PVY viruses, representative of all known strains, using recombination detection and phylogenetic inference techniques. Using Bayesian molecular dating we show that the parental strains of PVY diverged around the time potatoes were first introduced to Europe, that recombination between them only occurred in the last century, and that the multiple recombination events that led to highly pathogenic PVYNTN occurred within the last 50 years. Disease causing agents are often transported across the globe by humans, with disastrous effects for us, our livestock and crops. Our analytical approach is particularly pertinent for the often small recombinant genomes involved (e.g. HIV/influenza A). In the case of PVY, increased transport of diseased material is likely to blame for uniting the parents of recombinant pathogenic strains: this process needs to be minimised to prevent further such occurrences. PMID:23226339

Analysis of four microsatellite markers on the long arm of chromosome 9 by meiotic recombination in flow-sorted single sperm

DOE Office of Scientific and Technical Information (OSTI.GOV)

Furlong, R.A.; Goudie, D.R.; Carter, N.P.

1993-06-01

Meiotic recombination in flow-sorted single sperm was used to analyze four highly polymorphic microsatellite markers on the long arm of chromosome 9. The microsatellites comprised three tightly linked markers: 9CMP1 (D9S109), 9CMP2 (D9S127), and D9S53, which map to 9q31, and a reference marker, ASS, which is located in 9q34.1. Haplotypes of single sperm were assessed by using PCR in a single-step multiplex reaction to amplify each locus. Recombinant haplotypes were identified by their relative infrequency and were analyzed using THREELOC, a maximum-likelihood-analysis program, and an adaptation of CRI-MAP. The most likely order of these markers was cen-D9S109-D9S127-D9S53-ASS-tel with D9S109, D9S127,more » and D9S53 being separated by a genetic distance of approximately 3%. The order of the latter three markers did not however achieve statistical significance using the THREELOC program. 21 refs., 2 figs., 4 tabs.« less

Novel recombinant insulin analogue with flexible C-terminus in B chain. NMR structure of biosynthetic engineered A22G-B31K-B32R human insulin monomer in water/acetonitrile solution.

PubMed

Borowicz, Piotr; Bocian, Wojciech; Sitkowski, Jerzy; Bednarek, Elżbieta; Mikiewicz-Syguła, Diana; Błażej-Sosnowska, Sylwia; Bogiel, Monika; Rusek, Dorota; Kurzynoga, Dariusz; Kozerski, Lech

2011-11-01

A tertiary structure of recombinant A22(G)-B31(K)-B32(R)-human insulin monomer (insulin GKR) has been characterized by (1)H, (13)C NMR at natural isotopic abundance using NOESY, TOCSY, (1)H/(13)C-GHSQC, and (1)H/(13)C-GHSQC-TOCSY spectra. Translational diffusion studies indicate the monomer structure in water/acetonitrile (65/35vol.%). CSI analysis confirms existence of secondary structure motifs present in human insulin standard (HIS). Both techniques allow to establish that in this solvent recombinant insulin GKR exists as a monomer. Starting from structures calculated by the program CYANA, two different refinement protocols used molecular dynamics simulated annealing with the program AMBER; in vacuum (AMBER_VC), and including a generalized Born solvent model (AMBER_GB). From these calculations an ensemble of 20 structures of lowest energy was chosen which represents the tertiary structure of studied insulin. Here we present novel insulin with added A22(G) amino acid which interacts with β-turn environment resulting in high flexibility of B chain C-terminus. Copyright © 2011 Elsevier B.V. All rights reserved.

Major quality trait analysis and QTL detection in hexaploid wheat in humid rain-fed agriculture.

PubMed

Li, H M; Tang, Z X; Zhang, H Q; Yan, B J; Ren, Z L

2013-05-21

Humid rain-fed agriculture is a special environment for wheat (Triticum aestivum) culture that tends to negatively affect wheat yield and quality. To identify quality characters of wheat in a humid environment, we conducted quality analysis and quantitative trait loci (QTL) detection in a recombinant inbred line whose parent had a high level of quality for several years. We found that high-quality wheat had less gluten content and lower protein content. Apparently, wheat quality and associated quantity traits were in a dynamic state of equilibrium. We detected 83 QTL for 10 wheat quality traits in this recombinant inbred line population. Nine QTL were detected in both evaluation years; Q.DT.scau-2A, linked to Xwmc522-2A, was detected at the same genetic location in both years. Other QTL for different traits were detected simultaneously in more than one location. Consequently, there appeared to be pleiotropic genes that control wheat quality. Based on previous studies and our research on QTL analysis of grain protein content, we conclude that there must be one or more genes for grain protein content on chromosome 6B, whose expression was little affected by environment. We constructed a consensus map and projected the QTL on it. It was useful for choosing optimal markers for marker-assisted breeding and map-based cloning.

Potential technology transfers of research on low-temperature carbon monoxide-oxygen recombination catalysts

NASA Technical Reports Server (NTRS)

Poziomek, Edward J.

1990-01-01

Results from research on catalytic recombination of CO-O2 for stable closed-cycle operation of CO2 lasers hold much promise for a variety of technology transfer. Expansion of CO2 laser remote sensing applications toward chemical detection and pollution monitoring would certainly be expected. However, the catalysts themselves may be especially effective in low-temperature oxidation of a number of chemicals in addition to CO. It is therefore of interest to compare the CO-O2 catalysts with chemical systems designed for chemical sensing, air purification and process catalysis. Success in understanding the catalytic mechanisms of the recombination of CO-O2 could help to shed light on how catalyst systems operate. New directions in low-temperature oxidation catalysts, coatings for chemical sensors and sorbents for air purification could well emerge.

Current Protocols in Protein Science

PubMed Central

Huynh, Kathy

2015-01-01

The purification of recombinant proteins for biochemical assays and structural studies is time-consuming and presents inherent difficulties that depend on the optimization of protein stability. The use of dyes to monitor thermal denaturation of proteins with sensitive fluorescence detection enables the rapid and inexpensive determination of protein stability using real-time PCR instruments. By screening a wide range of solution conditions and additives in 96-well format, the thermal shift assay easily identifies conditions that significantly enhance the stability of recombinant proteins. The same approach can be used as a low cost, initial screen to discover new protein:ligand interactions by capitalizing on increases in protein stability that typically occur upon ligand binding. This unit presents a methodological workflow for the small-scale, high-throughout thermal denaturation of recombinant proteins in the presence of SYPRO Orange dye. PMID:25640896

Processing of human cytomegalovirus glycoprotein B in recombinant adenovirus-infected cells.

PubMed

Marshall, G S; Fenger, D P; Stout, G G; Knights, M E; Hunt, L A

1996-07-01

Intracellular processing of human cytomegalovirus (HCMV) glycoprotein B (gB; gpUL55) expressed by a recombinant adenovirus (Ad-gB) was studied in human A549 cells as processing events could affect immunogenicity when such viruses are used as live-recombinant vaccines. Cleavage of [35S]methionine-labelled gp13O into gp93 and gp55 reached a maximum after a 3 h chase. Cleavage was completely inhibited by brefeldin A, suggesting that processing normally occurs as a late Golgi or post-Golgi event. Uncleaved gp 130 remained completely sensitive to endo-beta-N-acetylglucosaminidase H (Endo-H) in untreated cells following long chase periods, indicating high-mannose oligosaccharides at all of the 18 N-linked glycosylation sites (Asn-X-Ser/Thr) and retention in the endoplasmic reticulum. Endo-H analysis of gp55 from swainsonine-treated and untreated cells was consistent with glycosylation at all three potential sites, with two oligosaccharides remaining sensitive to Endo-H and one being processed to Endo-H resistance. The heavily glycosylated N-terminal gp93 subunit was not detected by [35S]methionine-labelling but was easily detected along with gp55 after labelling with [3H]mannose. No cleavage of gp 130 was observed in analogous pulse-chase radiolabelling of Ad-gB-infected human fibroblasts, even though these cells are permissive for HCMV replication and can process the native gB molecule. Processing of gB in recombinant adenovirus-infected A549 cells was generally similar to that previously reported for native gB in HCMV-infected fibroblasts.

Targeted delivery of CYP2E1 recombinant adenovirus to malignant melanoma by bone marrow-derived mesenchymal stem cells as vehicles.

PubMed

Wang, Jishi; Ma, Dan; Li, Yan; Yang, Yuan; Hu, Xiaoyan; Zhang, Wei; Fang, Qin

2014-03-01

The aim of this study was to explore the effects of bone marrow-derived mesenchymal stem cells (BMSCs) as intermediate carriers on targeting of P450 gene recombinant adenovirus to malignant melanoma in vitro and in vivo. BMSCs were transduced with pAd5-CMV-CYP2E1 recombinant adenovirus. BMSC migration was detected by Transwell plates in vitro and by superparamagnetic iron oxide particles in vivo. Growth-inhibitory effect and apoptosis were determined by MTT and immunity fluorescence staining. Anticancer effects were examined by a human melanoma nude mouse model in vivo. BMSCs moved toward A375 cells in Transwell plates. Numerous superparamagnetic MSCs labeled with iron oxide were identified in the peripheral areas of the tumor, but were detected in primary organs by Prussian blue staining. BMSC-CYP2E1 cells mediated a bystander killing effect on CYP2E1-negative A375 cells during coculture (IC50 values for A375 cells cocultured with BMSC-EGFP and BMSC-CYP2E1 were 4.08 and 2.68 mmol/l, respectively). Intravenously injecting CYP2E1 recombinant adenovirus-loaded BMSCs in mice with established human melanoma managed to target the tumor site, and BMSCs with forced expression of CYP2E1 inhibited the growth of malignant cells in vivo by activating 5-(3,3-dimethyl-1-triazeno)imidazole-4-carboxamide. BMSCs may serve as a platform of P450 gene-directed enzyme prodrug therapy for the delivery of chemotherapeutic prodrugs to tumors.

Construction and characterization of recombinant adenovirus carrying a mouse TIGIT-GFP gene.

PubMed

Zheng, J M; Cui, J L; He, W T; Yu, D W; Gao, Y; Wang, L; Chen, Z K; Zhou, H M

2015-12-29

Recombinant adenovirus vector systems have been used extensively in protein research and gene therapy. However, the construction and characterization of recombinant adenovirus is a tedious and time-consuming process. TIGIT is a recently discovered immunosuppressive molecule that plays an important role in maintaining immunological balance. The construction of recombinant adenovirus mediating TIGIT expression must be simplified to facilitate its use in the study of TIGIT. In this study, the TIGIT gene was combined with green fluorescent protein (GFP); the TIGIT-GFP gene was inserted into a gateway plasmid to construct a TIGIT-GFP adenovirus. HEK 293A cells were infected with the adenovirus, which was then purified and subjected to virus titering. TIGIT-GFP adenovirus was characterized by flow cytometry and immunofluorescence, and its expression in mouse liver was detected by infection through caudal vein injection. The results showed the successful construction of the TIGIT-GFP adenovirus (5 x 10(10) PFU/mL). Co-expression of TIGIT and GFP was identified in 293A and liver cells; synthesis and positioning of TIGIT-GFP was viewed under a fluorescence microscope. TIGIT-GFP was highly expressed on liver cells 1 day (25.53%) after infection and faded 3 days (11.36%) after injection. In conclusion, the fusion of TIGIT with GFP allows easy, rapid, and uncomplicated detection of TIGIT translation. The construction of a TIGIT-GFP adenovirus, mediating TIGIT expression in vitro and in vivo, lays the foundation for further research into TIGIT function and gene therapy. Moreover, the TIGIT-GFP adenovirus is a helpful tool for studying other proteins (which could replace the TIGIT gene).

Positive selection and functional divergence of farnesyl pyrophosphate synthase genes in plants.

PubMed

Qian, Jieying; Liu, Yong; Chao, Naixia; Ma, Chengtong; Chen, Qicong; Sun, Jian; Wu, Yaosheng

2017-02-04

Farnesyl pyrophosphate synthase (FPS) belongs to the short-chain prenyltransferase family, and it performs a conserved and essential role in the terpenoid biosynthesis pathway. However, its classification, evolutionary history, and the forces driving the evolution of FPS genes in plants remain poorly understood. Phylogeny and positive selection analysis was used to identify the evolutionary forces that led to the functional divergence of FPS in plants, and recombinant detection was undertaken using the Genetic Algorithm for Recombination Detection (GARD) method. The dataset included 68 FPS variation pattern sequences (2 gymnosperms, 10 monocotyledons, 54 dicotyledons, and 2 outgroups). This study revealed that the FPS gene was under positive selection in plants. No recombinant within the FPS gene was found. Therefore, it was inferred that the positive selection of FPS had not been influenced by a recombinant episode. The positively selected sites were mainly located in the catalytic center and functional areas, which indicated that the 98S and 234D were important positively selected sites for plant FPS in the terpenoid biosynthesis pathway. They were located in the FPS conserved domain of the catalytic site. We inferred that the diversification of FPS genes was associated with functional divergence and could be driven by positive selection. It was clear that protein sequence evolution via positive selection was able to drive adaptive diversification in plant FPS proteins. This study provides information on the classification and positive selection of plant FPS genes, and the results could be useful for further research on the regulation of triterpenoid biosynthesis.

Recombination and evolution of duplicate control regions in the mitochondrial genome of the Asian big-headed turtle, Platysternon megacephalum.

PubMed

Zheng, Chenfei; Nie, Liuwang; Wang, Jue; Zhou, Huaxing; Hou, Huazhen; Wang, Hao; Liu, Juanjuan

2013-01-01

Complete mitochondrial (mt) genome sequences with duplicate control regions (CRs) have been detected in various animal species. In Testudines, duplicate mtCRs have been reported in the mtDNA of the Asian big-headed turtle, Platysternon megacephalum, which has three living subspecies. However, the evolutionary pattern of these CRs remains unclear. In this study, we report the completed sequences of duplicate CRs from 20 individuals belonging to three subspecies of this turtle and discuss the micro-evolutionary analysis of the evolution of duplicate CRs. Genetic distances calculated with MEGA 4.1 using the complete duplicate CR sequences revealed that within turtle subspecies, genetic distances between orthologous copies from different individuals were 0.63% for CR1 and 1.2% for CR2app:addword:respectively, and the average distance between paralogous copies of CR1 and CR2 was 4.8%. Phylogenetic relationships were reconstructed from the CR sequences, excluding the variable number of tandem repeats (VNTRs) at the 3' end using three methods: neighbor-joining, maximum likelihood algorithm, and Bayesian inference. These data show that any two CRs within individuals were more genetically distant from orthologous genes in different individuals within the same subspecies. This suggests independent evolution of the two mtCRs within each P. megacephalum subspecies. Reconstruction of separate phylogenetic trees using different CR components (TAS, CD, CSB, and VNTRs) suggested the role of recombination in the evolution of duplicate CRs. Consequently, recombination events were detected using RDP software with break points at ≈290 bp and ≈1,080 bp. Based on these results, we hypothesize that duplicate CRs in P. megacephalum originated from heterological ancestral recombination of mtDNA. Subsequent recombination could have resulted in homogenization during independent evolutionary events, thus maintaining the functions of duplicate CRs in the mtDNA of P. megacephalum.

Recombination and Evolution of Duplicate Control Regions in the Mitochondrial Genome of the Asian Big-Headed Turtle, Platysternon megacephalum

PubMed Central

Zheng, Chenfei; Nie, Liuwang; Wang, Jue; Zhou, Huaxing; Hou, Huazhen; Wang, Hao; Liu, Juanjuan

2013-01-01

Complete mitochondrial (mt) genome sequences with duplicate control regions (CRs) have been detected in various animal species. In Testudines, duplicate mtCRs have been reported in the mtDNA of the Asian big-headed turtle, Platysternon megacephalum, which has three living subspecies. However, the evolutionary pattern of these CRs remains unclear. In this study, we report the completed sequences of duplicate CRs from 20 individuals belonging to three subspecies of this turtle and discuss the micro-evolutionary analysis of the evolution of duplicate CRs. Genetic distances calculated with MEGA 4.1 using the complete duplicate CR sequences revealed that within turtle subspecies, genetic distances between orthologous copies from different individuals were 0.63% for CR1 and 1.2% for CR2app:addword:respectively, and the average distance between paralogous copies of CR1 and CR2 was 4.8%. Phylogenetic relationships were reconstructed from the CR sequences, excluding the variable number of tandem repeats (VNTRs) at the 3′ end using three methods: neighbor-joining, maximum likelihood algorithm, and Bayesian inference. These data show that any two CRs within individuals were more genetically distant from orthologous genes in different individuals within the same subspecies. This suggests independent evolution of the two mtCRs within each P. megacephalum subspecies. Reconstruction of separate phylogenetic trees using different CR components (TAS, CD, CSB, and VNTRs) suggested the role of recombination in the evolution of duplicate CRs. Consequently, recombination events were detected using RDP software with break points at ≈290 bp and ≈1,080 bp. Based on these results, we hypothesize that duplicate CRs in P. megacephalum originated from heterological ancestral recombination of mtDNA. Subsequent recombination could have resulted in homogenization during independent evolutionary events, thus maintaining the functions of duplicate CRs in the mtDNA of P. megacephalum. PMID:24367563

RNA Interference towards the Potato Psyllid, Bactericera cockerelli, Is Induced in Plants Infected with Recombinant Tobacco mosaic virus (TMV)

PubMed Central

Wuriyanghan, Hada; Falk, Bryce W.

2013-01-01

The potato/tomato psyllid, Bactericera cockerelli (B. cockerelli), is an important plant pest and the vector of the phloem-limited bacterium Candidatus Liberibacter psyllaurous (solanacearum), which is associated with the zebra chip disease of potatoes. Previously, we reported induction of RNA interference effects in B. cockerelli via in vitro-prepared dsRNA/siRNAs after intrathoracic injection, and after feeding of artificial diets containing these effector RNAs. In order to deliver RNAi effectors via plant hosts and to rapidly identify effective target sequences in plant-feeding B. cockerelli, here we developed a plant virus vector-based in planta system for evaluating candidate sequences. We show that recombinant Tobacco mosaic virus (TMV) containing B. cockerelli sequences can efficiently infect and generate small interfering RNAs in tomato (Solanum lycopersicum), tomatillo (Physalis philadelphica) and tobacco (Nicotiana tabacum) plants, and more importantly delivery of interfering sequences via TMV induces RNAi effects, as measured by actin and V-ATPase mRNA reductions, in B. cockerelli feeding on these plants. RNAi effects were primarily detected in the B. cockerelli guts. In contrast to our results with TMV, recombinant Potato virus X (PVX) and Tobacco rattle virus (TRV) did not give robust infections in all plants and did not induce detectable RNAi effects in B. cockerelli. The greatest RNA interference effects were observed when B. cockerelli nymphs were allowed to feed on leaf discs collected from inoculated or lower expanded leaves from corresponding TMV-infected plants. Tomatillo plants infected with recombinant TMV containing B. cockerelli actin or V-ATPase sequences also showed phenotypic effects resulting in decreased B. cockerelli progeny production as compared to plants infected by recombinant TMV containing GFP. These results showed that RNAi effects can be achieved in plants against the phloem feeder, B. cockerelli, and the TMV-plant system will provide a faster and more convenient method for screening of suitable RNAi target sequences in planta. PMID:23824081

Selective constraints, molecular recombination structure and phylogenetic reconstruction of isometric plant RNA viruses of the families Luteoviridae and Tymoviridae.

PubMed

Boulila, Moncef

2011-02-01

In an effort to enhance the knowledge on molecular evolution of currently the known members of the families Luteoviridae and Tymoviridae, in-depth molecular investigations in the entire genome of 147 accessions retrieved from the international databases, were carried out. Two algorithms (RECCO and RDP version 3.31β) adapted to the mosaic structure of viruses were utilized. The recombination frequency along the sequences was dissected and demonstrated that the three virus genera of the family Luteoviridae comprise numerous members subjected to recombination. It has pointed out that the major viruses swapped a few but long genomic segments. In addition, in Barley yellow dwarf virus, heredity material might be exchanged between two different serotypes. Even more, putative recombination events occurred between two different genera. Based on Fisher's Exact Test of Neutrality, positive selection acting on protein expression was detected only in the poleroviruses Cereal yellow dwarf virus, Potato leafroll virus and Wheat yellow dwarf virus. In contrast, several components of the family Tymoviridae were highly recombinant. Genomic portion exchange arose in many positions consisting of short fragments. Furthermore, no positive selection was detected. The evolutionary history showed, in the Luteoviridae, that all screened isolates split into three clusters corresponding to the three virus genera forming this family. Moreover, in the serotype PAV of Barley yellow dwarf virus, two major clades corresponding to PAV-USA and PAV-China, were delineated. Similarly, in the Tymoviridae, all analyzed isolates fell into four groups corresponding to the three virus genera composing this family along with the unclassified Tymoviridae. Inferred phylogenies reshuffled the existing classification and showed that Wheat yellow dwarf virus-RPV was genetically closely related to Cereal yellow dwarf virus and the unclassified Tymoviridae Grapevine syrah virus-1 constituted an integral part of the genus Marafivirus. Copyright © 2010 Elsevier Masson SAS. All rights reserved.

Level of tissue differentiation influences the activation of a heat-inducible flower-specific system for genetic containment in poplar (Populus tremula L.).

PubMed

Hoenicka, Hans; Lehnhardt, Denise; Nunna, Suneetha; Reinhardt, Richard; Jeltsch, Albert; Briones, Valentina; Fladung, Matthias

2016-02-01

Differentiation level but not transgene copy number influenced activation of a gene containment system in poplar. Heat treatments promoted CRE gene body methylation. The flower-specific transgene deletion was confirmed. Gene flow between genetic modified trees and their wild relatives is still motive of concern. Therefore, approaches for gene containment are required. In this study, we designed a novel strategy for achieving an inducible and flower-specific transgene removal from poplar trees but still expressing the transgene in the plant body. Hence, pollen carrying transgenes could be used for breeding purposes under controlled conditions in a first phase, and in the second phase genetic modified poplars developing transgene-free pollen grains could be released. This approach is based on the recombination systems CRE/loxP and FLP/frt. Both gene constructs contained a heat-inducible CRE/loxP-based spacer sequence for in vivo assembling of the flower-specific FLP/frt system. This allowed inducible activation of gene containment. The FLP/frt system was under the regulation of a flower-specific promoter, either CGPDHC or PTD. Our results confirmed complete CRE/loxP-based in vivo assembling of the flower-specific transgene excision system after heat treatment in all cells for up to 30 % of regenerants derived from undifferentiated tissue cultures. Degradation of HSP::CRE/loxP spacer after recombination but also persistence as extrachromosomal DNA circles were detected in sub-lines obtained after heat treatments. Furthermore, heat treatment promoted methylation of the CRE gene body. A lower methylation level was detected at CpG sites in transgenic sub-lines showing complete CRE/loxP recombination and persistence of CRE/loxP spacer, compared to sub-lines with incomplete recombination. However, our results suggest that low methylation might be necessary but not sufficient for recombination. The flower-specific FLP/frt-based transgene deletion was confirmed in 6.3 % of flowers.

Evidence for positive selection and recombination hotspots in Deformed wing virus (DWV).

PubMed

Dalmon, A; Desbiez, C; Coulon, M; Thomasson, M; Le Conte, Y; Alaux, C; Vallon, J; Moury, B

2017-01-25

Deformed wing virus (DWV) is considered one of the most damaging pests in honey bees since the spread of its vector, Varroa destructor. In this study, we sequenced the whole genomes of two virus isolates and studied the evolutionary forces that act on DWV genomes. The isolate from a Varroa-tolerant bee colony was characterized by three recombination breakpoints between DWV and the closely related Varroa destructor virus-1 (VDV-1), whereas the variant from the colony using conventional Varroa management was similar to the originally described DWV. From the complete sequence dataset, nine independent DWV-VDV-1 recombination breakpoints were detected, and recombination hotspots were found in the 5' untranslated region (5' UTR) and the conserved region encoding the helicase. Partial sequencing of the 5' UTR and helicase-encoding region in 41 virus isolates suggested that most of the French isolates were recombinants. By applying different methods based on the ratio between non-synonymous (dN) and synonymous (dS) substitution rates, we identified four positions that showed evidence of positive selection. Three of these positions were in the putative leader protein (Lp), and one was in the polymerase. These findings raise the question of the putative role of the Lp in viral evolution.

Serum from Nipah Virus Patients Recognises Recombinant Viral Proteins Produced in Escherichia coli.

PubMed

Tiong, Vunjia; Lam, Chui-Wan; Phoon, Wai-Hong; AbuBakar, Sazaly; Chang, Li-Yen

2017-01-24

The genes for Nipah virus (NiV) proteins were amplified from viral RNA, cloned into the plasmid pTriEx-3 Hygro, expressed, and purified using immobilized metal affinity chromatography. The recombinant N, F, and G NiV proteins (rNiV-N, rNiV-F, and rNiV-G), were successfully expressed in Escherichia coli and purified with a yield of 4, 16, and 4 mg/L, respectively. All 3 recombinant viral proteins reacted with all 19 samples of NiV-positive human sera. The rNiV-N and rNiV-G proteins were the most immunogenic. The recombinant viral proteins did not react with any of the 12 NiV-negative sera. However, serum from a patient with a late-onset relapsing NiV infection complication was found to be primarily reactive to rNiV-G only. Additionally, there is a distinctive variation in the profile of antigen-reactive bands between the sample from a case of relapsing NiV encephalitis and that of acute NiV infection. The overall findings of this study suggest that the recombinant viral proteins have the potential to be developed further for use in the detection of NiV infection, and continuous biosurveillance of NiV infection in resource-limited settings.

Oral Immunization with Recombinant Lactobacillus acidophilus Expressing the Adhesin Hp0410 of Helicobacter pylori Induces Mucosal and Systemic Immune Responses

PubMed Central

Hongying, Fan; Xianbo, Wu; Fang, Yu; Yang, Bai

2014-01-01

Helicobacter pylori infection is relatively common worldwide and is closely related to gastric mucosa-associated lymphoid tissue (MALT) lymphoma, chronic gastritis, and stomach ulcers. Therefore, a safe and effective method for preventing H. pylori infection is urgently needed. Given that developing an effective vaccine against H. pylori is one of the best alternatives, H. pylori adhesin Hp0410 was expressed in the food-grade bacterium Lactobacillus acidophilus. The recombinant live bacterial vaccine was then used to orally vaccinate mice, and the immunoprotective effects of Hp0410-producing strains were investigated. H. pylori colonization in the stomach of mice immunized with the recombinant L. acidophilus was significantly reduced, in comparison with that in control groups. Furthermore, mucosal secretory IgA antibodies were elicited in the mucosal tissue of mice immunized with the recombinant bacteria, and specific anti-Hp0410 IgG responses were also detected in mouse serum. There was a significant increase in the level of protection against gastric Helicobacter infection following a challenge with H. pylori Sydney strain 1 (SS1). Our results collectively indicate that adhesin Hp0410 is a promising candidate vaccine antigen, and recombinant L. acidophilus expressing Hp0410 is likely to constitute an effective, low-cost, live bacterial vaccine against H. pylori. PMID:24285819

Highly diverse recombining populations of Vibrio cholerae and Vibrio parahaemolyticus in French Mediterranean coastal lagoons

PubMed Central

Esteves, Kévin; Mosser, Thomas; Aujoulat, Fabien; Hervio-Heath, Dominique; Monfort, Patrick; Jumas-Bilak, Estelle

2015-01-01

Vibrio parahaemolyticus and Vibrio cholerae are ubiquitous to estuarine and marine environments. These two species found in Mediterranean coastal systems can induce infections in humans. Environmental isolates of V. cholerae (n = 109) and V. parahaemolyticus (n = 89) sampled at different dates, stations and water salinities were investigated for virulence genes and by a multilocus sequence-based analysis (MLSA). V. cholerae isolates were all ctxA negative and only one isolate of V. parahaemolyticus displayed trh2 gene. Most Sequence Types (ST) corresponded to unique ST isolated at one date or one station. Frequent recombination events were detected among different pathogenic species, V. parahaemolyticus, V. cholerae, Vibrio mimicus, and Vibrio metoecus. Recombination had a major impact on the diversification of lineages. The genetic diversity assessed by the number of ST/strain was higher in low salinity condition for V. parahaemolyticus and V. cholerae whereas the frequency of recombination events in V. cholerae was lower in low salinity condition. Mediterranean coastal lagoon systems housed V. cholerae and V. parahaemolyticus with genetic diversities equivalent to the worldwide diversity described so far. The presence of STs found in human infections as well as the frequency of recombination events in environmental vibrios populations could predict a potential epidemiological risk. PMID:26236294

Dissociative recombination of HCl+, H2Cl+, DCl+, and D2Cl+ in a flowing afterglow

NASA Astrophysics Data System (ADS)

Wiens, Justin P.; Miller, Thomas M.; Shuman, Nicholas S.; Viggiano, Albert A.

2016-12-01

Dissociative recombination of electrons with HCl+, H2Cl+, DCl+, and D2Cl+ has been measured under thermal conditions at 300, 400, and 500 K using a flowing afterglow-Langmuir probe apparatus. Measurements for HCl+ and DCl+ employed the variable electron and neutral density attachment mass spectrometry (VENDAMS) method, while those for H2Cl+ and D2Cl+ employed both VENDAMS and the more traditional technique of monitoring electron density as a function of reaction time. At 300 K, HCl+ and H2Cl+ recombine with kDR = 7.7±2.14.5 × 10-8 cm3 s-1 and 2.6 ± 0.8 × 10-7 cm3 s-1, respectively, whereas D2Cl+ is roughly half as fast as H2Cl+ with kDR = 1.1 ± 0.3 × 10-7 cm3 s-1 (2 σ confidence intervals). DCl+ recombines with a rate coefficient below the approximate detection limit of the method (≲5 × 10-8 cm3 s-1) at all temperatures. Relatively slow dissociative recombination rates have been speculated to be responsible for the large HCl+ and H2Cl+ abundances in interstellar clouds compared to current astrochemical models, but our results imply that the discrepancy must originate elsewhere.

A VNTR element associated with steroid sulfatase gene deletions stimulates recombination in cultured cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Gong, Y.; Li, X.M.; Shapiro, L.J.

1994-09-01

Steroid sulfatase deficiency is a common genetic disorder, with a prevalence of approximately one in every 3500 males world wide. About 90% of these patients have complete gene deletions, which appear to result from recombination between members of a low-copy repeat family (CRI-232 is the prototype) that flank the gene. RU1 and RU2 are two VNTR elements found within each of these family members. RU1 consists of 30 bp repeating units and its length shows minimal variation among individuals. The RU2 element consists of repeating sequences which are highly asymmetric, with about 90% purines and no C`s on one strand,more » and range from 0.6 kb to over 23 kb among different individuals. We conducted a study to determine if the RU1 or RU2 elements can promote recombination in an in vivo test system. We inserted these elements adjacent to the neo gene in each of two pSV2neo derivatives, one of which has a deletion in the 5{prime} portion of the neo gene and the other having a deletion in the 3{prime} portion. These plasmids were combined and used to transfect EJ cells. Survival of cells in G418 indicates restoration of a functional neo gene by recombination between two deletion constructs. Thus counting G418 resistant colonies gives a quantitative measure of the enhancement of recombination by the inserted VNTR elements. The results showed no effect on recombination by the inserted RU1 element (compared to the insertion of a nonspecific sequence), while the RU2 element stimulated recombination by 3.5-fold (P<0.01). A separate set of constructs placed RU1 or RU2 within the intron of an exon trapping vector. Following tranfection of cells, recombination events were monitored by a PCR assay that detected the approximation of primer binding sites (as a result of recombination). These studies showed that, as in the first set of experiments, the highly variable RU2 element is capable of stimulating somatic recombination in mammalian cells.« less

The frequency of occurrence and nature of recombinant feline leukemia viruses in the induction of multicentric lymphoma by infection of the domestic cat with FeLV-945.

PubMed

Ahmad, Shamim; Levy, Laura S

2010-08-01

During feline leukemia virus (FeLV) infection in the domestic cat, viruses with a novel envelope gene arise by recombination between endogenous FeLV-related elements and the exogenous infecting species. These recombinant viruses (FeLV-B) are of uncertain disease association, but have been linked to the induction of thymic lymphoma. To assess the role of FeLV-B in the induction of multicentric lymphoma and other non-T-cell disease, the frequency of occurrence and nature of FeLV-B were examined in diseased tissues from a large collection of FeLV-infected animals. Diseased tissues were examined by Southern blot and PCR amplification to detect the presence of FeLV-B. Further analysis was performed to establish the recombination junctions and infectivity of FeLV-B in diseased tissues. The results confirmed the frequent association of FeLV-B with thymic lymphoma but showed infrequent generation, low levels and lack of infectivity of FeLV-B in non-T-cell diseases including multicentric lymphoma.

Development of a sensitive and specific indirect enzyme-linked immunosorbent assay based on a baculovirus recombinant antigen for detection of specific antibodies against Ehrlichia canis.

PubMed

López, Lissett; Venteo, Angel; Aguirre, Enara; García, Marga; Rodríguez, Majosé; Amusátegui, Inmaculada; Tesouro, Miguel A; Vela, Carmen; Sainz, Angel; Rueda, Paloma

2007-11-01

An indirect enzyme-linked immunosorbent assay (ELISA) based on baculovirus recombinant P30 protein of Ehrlichia canis and the 1BH4 anticanine IgG monoclonal antibody was developed and evaluated by examining a panel of 98 positive and 157 negative sera using the indirect fluorescent antibody (IFA) test as the reference technique. The P30-based ELISA appeared to be sensitive and specific (77.55% and 95.54%, respectively) when qualitative results (positive/negative) were compared with those of the IFA test; the coefficient of correlation (R) between the 2 tests was 0.833. Furthermore, it was possible to establish a mathematical formula for use in comparing the results of both techniques. These results indicate that recombinant P30 antigen-based ELISA is a suitable alternative of the IFA test for simple, consistent, and rapid serodiagnosis of canine ehrlichiosis. Moreover, the use of this recombinant protein as antigen offers a great advantage for antigen preparation in comparison with other techniques in which the whole E. canis organism is used as antigen.