RESUMEN
In eukaryotes, gene expression is performed by three RNA polymerases that are targeted to promoters by molecular complexes. A unique common factor, the TATA-box binding protein (TBP), is thought to serve as a platform to assemble pre-initiation complexes competent for transcription. Here, we describe a novel molecular mechanism of nutrient regulation of gene transcription by dynamic O-GlcNAcylation of TBP. We show that O-GlcNAcylation at T114 of TBP blocks its interaction with BTAF1, hence the formation of the B-TFIID complex, and its dynamic cycling on and off of DNA. Transcriptomic and metabolomic analyses of TBPT114A CRISPR/Cas9-edited cells showed that loss of O-GlcNAcylation at T114 increases TBP binding to BTAF1 and directly impacts expression of 408 genes. Lack of O-GlcNAcylation at T114 is associated with a striking reprogramming of cellular metabolism induced by a profound modification of the transcriptome, leading to gross alterations in lipid storage.
Asunto(s)
Glucosa/metabolismo , Gotas Lipídicas/metabolismo , Metabolismo de los Lípidos , Factores Asociados con la Proteína de Unión a TATA/metabolismo , Proteína de Unión a TATA-Box/metabolismo , Factor de Transcripción TFIID/metabolismo , Animales , Cromatina/genética , Cromatina/metabolismo , Diabetes Mellitus Experimental/genética , Diabetes Mellitus Experimental/metabolismo , Regulación de la Expresión Génica , Glicosilación , Células HEK293 , Células HeLa , Humanos , Metabolismo de los Lípidos/genética , Masculino , Complejos Multiproteicos , Ratas Sprague-Dawley , Transducción de Señal , Factores Asociados con la Proteína de Unión a TATA/genética , Proteína de Unión a TATA-Box/genética , Factores de Tiempo , Factor de Transcripción TFIID/genética , Transcripción Genética , TranscriptomaRESUMEN
BACKGROUND: Heart failure is a leading cause of death worldwide and is associated with the rising prevalence of obesity, hypertension, and diabetes. O-GlcNAcylation (the attachment of O-linked ß-N-acetylglucosamine [O-GlcNAc] moieties to cytoplasmic, nuclear, and mitochondrial proteins) is a posttranslational modification of intracellular proteins and serves as a metabolic rheostat for cellular stress. Total levels of O-GlcNAcylation are determined by nutrient and metabolic flux, in addition to the net activity of 2 enzymes: O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA). Failing myocardium is marked by increased O-GlcNAcylation, but whether excessive O-GlcNAcylation contributes to cardiomyopathy and heart failure is unknown. METHODS: We developed 2 new transgenic mouse models with myocardial overexpression of OGT and OGA to control O-GlcNAcylation independent of pathologic stress. RESULTS: We found that OGT transgenic hearts showed increased O-GlcNAcylation and developed severe dilated cardiomyopathy, ventricular arrhythmias, and premature death. In contrast, OGA transgenic hearts had lower O-GlcNAcylation but identical cardiac function to wild-type littermate controls. OGA transgenic hearts were resistant to pathologic stress induced by pressure overload with attenuated myocardial O-GlcNAcylation levels after stress and decreased pathologic hypertrophy compared with wild-type controls. Interbreeding OGT with OGA transgenic mice rescued cardiomyopathy and premature death, despite persistent elevation of myocardial OGT. Transcriptomic and functional studies revealed disrupted mitochondrial energetics with impairment of complex I activity in hearts from OGT transgenic mice. Complex I activity was rescued by OGA transgenic interbreeding, suggesting an important role for mitochondrial complex I in O-GlcNAc-mediated cardiac pathology. CONCLUSIONS: Our data provide evidence that excessive O-GlcNAcylation causes cardiomyopathy, at least in part, attributable to defective energetics. Enhanced OGA activity is well tolerated and attenuation of O-GlcNAcylation is beneficial against pressure overload-induced pathologic remodeling and heart failure. These findings suggest that attenuation of excessive O-GlcNAcylation may represent a novel therapeutic approach for cardiomyopathy.
Asunto(s)
Muerte Súbita/patología , Insuficiencia Cardíaca/fisiopatología , N-Acetilglucosaminiltransferasas/efectos adversos , Animales , Modelos Animales de Enfermedad , Humanos , Ratones , Ratones TransgénicosRESUMEN
Elevated mitochondrial O-GlcNAcylation caused by hyperglycemia, as occurs in diabetes, significantly contributes to mitochondrial dysfunction and to diabetic cardiomyopathy. However, little is known about the enzymology of mitochondrial O-GlcNAcylation. Herein, we investigated the enzymes responsible for cycling O-GlcNAc on mitochondrial proteins and studied the mitochondrial transport of UDP-GlcNAc. Analyses of purified rat heart mitochondria from normal and streptozocin-treated diabetic rats show increased mitochondrial O-GlcNAc transferase (OGT) and a concomitant decrease in the mito-specific O-GlcNAcase (OGA). Strikingly, OGT is mislocalized in cardiac mitochondria from diabetic rats. Interaction of OGT and complex IV observed in normal rat heart mitochondria is visibly reduced in diabetic samples, where OGT is mislocalized to the matrix. Live cell OGA activity assays establish the presence of O-GlcNAcase within the mitochondria. Furthermore, we establish that the inner mitochondrial membrane transporter, pyrimidine nucleotide carrier, transports UDP-GlcNAc from the cytosol to the inside of the mitochondria. Knockdown of this transporter substantially lowers mitochondrial O-GlcNAcylation. Inhibition of OGT or OGA activity within neonatal rat cardiomyocytes significantly affects energy production, mitochondrial membrane potential, and mitochondrial oxygen consumption. These data suggest that cardiac mitochondria not only have robust O-GlcNAc cycling, but also that dysregulation of O-GlcNAcylation likely plays a key role in mitochondrial dysfunction associated with diabetes.
Asunto(s)
Diabetes Mellitus Experimental/metabolismo , Mitocondrias Cardíacas/fisiología , N-Acetilglucosaminiltransferasas/metabolismo , Adenosina Trifosfato/química , Animales , Proteínas del Complejo de Cadena de Transporte de Electrón/metabolismo , Hexosaminidasas/metabolismo , Concentración 50 Inhibidora , Lisosomas/metabolismo , Potenciales de la Membrana , Microscopía Electrónica de Transmisión , Mitocondrias/metabolismo , Proteínas de Transporte de Membrana Mitocondrial/metabolismo , Miocitos Cardíacos/citología , Nucleótidos/química , Oxígeno/química , Consumo de Oxígeno , Unión Proteica , Proteolípidos/química , Ratas , Succinato Deshidrogenasa/metabolismoRESUMEN
The phylum Apicomplexa includes serious pathogens of humans and animals. Understanding the distribution and population structure of these protozoan parasites is of fundamental importance to explain disease epidemiology and develop sustainable controls. Predicting the likely efficacy and longevity of subunit vaccines in field populations relies on knowledge of relevant preexisting antigenic diversity, population structure, the likelihood of coinfection by genetically distinct strains, and the efficiency of cross-fertilization. All four of these factors have been investigated for Plasmodium species parasites, revealing both clonal and panmictic population structures with exceptional polymorphism associated with immunoprotective antigens such as apical membrane antigen 1 (AMA1). For the coccidian Toxoplasma gondii only genomic diversity and population structure have been defined in depth so far; for the closely related Eimeria species, all four variables are currently unknown. Using Eimeria tenella, a major cause of the enteric disease coccidiosis, which exerts a profound effect on chicken productivity and welfare, we determined population structure, genotype distribution, and likelihood of cross-fertilization during coinfection and also investigated the extent of naturally occurring antigenic diversity for the E. tenella AMA1 homolog. Using genome-wide Sequenom SNP-based haplotyping, targeted sequencing, and single-cell genotyping, we show that in this coccidian the functionality of EtAMA1 appears to outweigh immune evasion. This result is in direct contrast to the situation in Plasmodium and most likely is underpinned by the biology of the direct and acute coccidian life cycle in the definitive host.
Asunto(s)
Variación Antigénica , Eimeria tenella/genética , Eimeria tenella/inmunología , Animales , Antígenos de Protozoos/inmunología , Secuencia de Bases , Pollos/parasitología , Coccidiosis/parasitología , Cruzamientos Genéticos , Heces , Variación Genética , Genética de Población , Genotipo , Geografía , Datos de Secuencia Molecular , Oocistos , Filogenia , Plasmodium/genética , Plasmodium/inmunología , Polimorfismo de Nucleótido Simple , Enfermedades de las Aves de Corral/parasitología , Vacunas AntiprotozoosRESUMEN
The enzymatic addition of a single ß-D-N-acetylglucosamine sugar molecule on serine and/or threonine residues of protein chains is referred to as O-GlcNAcylation. This novel form of post-translational modification, first reported in 1984, is extremely abundant on nuclear and cytoplasmic proteins and has site specific cycling dynamics comparable to that of protein-phosphorylation. A nutrient and stress sensor, O-GlcNAc abnormalities underlie insulin resistance and glucose toxicity in diabetes, neurodegenerative disorders and dysregulation of tumor suppressors and oncogenic proteins in cancer. Recent advances have helped understand the biochemical mechanisms of GlcNAc addition and removal and have opened the door to developing key inhibitors towards this type of protein modification. Advanced methods in detecting and measuring O-GlcNAcylation have assisted in delineating its biological roles in a variety of cellular processes and diseased states. Availability of facile glycomic techniques are allowing for the exponential growth in the study of protein O-GlcNAcylation and are helping to elucidate key biological roles of this novel PTM.
Asunto(s)
Acetilglucosamina/química , Acetilglucosamina/análisis , Acetilglucosamina/metabolismo , Hexosaminas/química , Hexosaminas/metabolismo , Humanos , Mitocondrias/metabolismo , N-Acetilglucosaminiltransferasas/metabolismo , Proteómica , Transducción de Señal , beta-N-Acetilhexosaminidasas/metabolismoRESUMEN
Background: Cocaine use disorder (CUD) is characterized by the continued use of cocaine despite serious impacts on life. This study focused on understanding the perspective of individuals with current CUD, individuals in CUD remission, and their supporters regarding current therapies, future therapies, and views on clinical trials for CUD. Methods: The online bulletin board (OBB) is a qualitative tool where participants engage in an interactive discussion on a virtual forum. Following completion of a screening questionnaire to determine eligibility, individuals in CUD remission and their supporters logged in to the OBB and responded to questions posed by the moderator. Individuals with current CUD participated in a one-time virtual focus group. Results: All individuals with current CUD and 94% of those in CUD remission reported a diagnosis consistent with CUD or substance use disorder during screening. Individuals with current CUD and their supporters were recruited from the United States (US). Individuals in CUD remission were recruited from five countries, including the US. Individuals with current CUD reported hesitation about seeking treatment due to stigma, a lack of privacy, and being labeled as a drug seeker; barriers to therapy included time, cost, and a lack of privacy. Participants wanted a safe therapy to stop cravings and withdrawal symptoms. Seven clinical trial outcomes, including long-term abstinence and craving control, were suggested based on collected insights. Conclusion: This study can help inform the design of clinical trials and emphasize the need for effective, safe, and accessible therapies. Recruiting participants will require significant trust building.
RESUMEN
Background: There is a paucity of literature describing experiences and journey of individuals with cocaine use disorder (CUD) and supporters who care for them. The aim of this study was to understand and document the journey of individuals with current CUD, those in CUD remission, and supporters. Methods: The online bulletin board (OBB) is a qualitative tool where participants engage in an interactive discussion on a virtual forum. After completing a 15-minute screening questionnaire determining eligibility, individuals in CUD remission and supporters participated in an OBB for 60 minutes, split across 8 days over 2 weeks. Individuals with current CUD participated in a one-time virtual focus group discussion for 90 minutes. Results: Individuals in CUD remission (n=35) were from Brazil, France, Spain, the UK, and the US; those with current CUD (n=5) and supporters (n=6) were from the US. Key insights were that individuals with current CUD were seeking a 'euphoric high' that cocaine provides. Those in CUD remission described a 'euphoric high' when they first tried cocaine, but over time it became harder to re-create this feeling. Individuals in CUD remission expressed a 'rollercoaster' of emotions from when they first started using cocaine to when they stopped. Supporters were sad, isolated, and worried about a potential cocaine overdose for their loved ones with CUD. Conclusion: The study provides valuable insights into the experiences and journey of individuals with CUD and their supporters. Data generated from this study gives insights into this under-served and growing population.
RESUMEN
Oncolytic adenoviruses (Ads) are an emerging alternative therapy for cancer; however, clinical trial have not yet demonstrated sufficient efficacy. When oncolytic Ads are used in combination with taxoids a synergistic increase in both cytotoxicity and viral replication is observed. In order to generate a next generation oncolytic adenovirus, virion were physically conjugated to a highly potent taxoid, SB-T-1214, and a folate targeting motif. Conjugation was enabled via the metabolic incorporation of non-canonical monosaccharides (O-GlcNAz) and amino acids (homopropargylglycine), which served as sites for chemoselective modification.
Asunto(s)
Adenoviridae/fisiología , Viroterapia Oncolítica , Adenoviridae/química , Animales , Western Blotting , Línea Celular Tumoral , RatonesRESUMEN
Diabetes mellitus (DM) and atrial fibrillation (AF) are major unsolved public health problems, and diabetes is an independent risk factor for AF. However, the mechanism(s) underlying this clinical association is unknown. ROS and protein O-GlcNAcylation (OGN) are increased in diabetic hearts, and calmodulin kinase II (CaMKII) is a proarrhythmic signal that may be activated by ROS (oxidized CaMKII, ox-CaMKII) and OGN (OGN-CaMKII). We induced type 1 (T1D) and type 2 DM (T2D) in a portfolio of genetic mouse models capable of dissecting the role of ROS and OGN at CaMKII and global OGN in diabetic AF. Here, we showed that T1D and T2D significantly increased AF, and this increase required CaMKII and OGN. T1D and T2D both required ox-CaMKII to increase AF; however, we did not detect OGN-CaMKII or a role for OGN-CaMKII in diabetic AF. Collectively, our data affirm CaMKII as a critical proarrhythmic signal in diabetic AF and suggest ROS primarily promotes AF by ox-CaMKII, while OGN promotes AF by a CaMKII-independent mechanism(s). These results provide insights into the mechanisms for increased AF in DM and suggest potential benefits for future CaMKII and OGN targeted therapies.
Asunto(s)
Fibrilación Atrial/enzimología , Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina/metabolismo , Complicaciones de la Diabetes/enzimología , Diabetes Mellitus Experimental/enzimología , Diabetes Mellitus Tipo 1/enzimología , Diabetes Mellitus Tipo 2/enzimología , Acilación , Animales , Fibrilación Atrial/genética , Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina/genética , Complicaciones de la Diabetes/genética , Diabetes Mellitus Experimental/genética , Diabetes Mellitus Tipo 1/genética , Diabetes Mellitus Tipo 2/genética , Ratones Noqueados , Oxidación-ReducciónRESUMEN
Psychostimulant drugs are widely used in children for the treatment of attention-deficit/hyperactivity disorder. Recent animal studies have suggested that exposure to these agents in early life could be detrimental to brain development. Here, for the first time, the effect of methylphenidate (MPH) and D-amphetamine (AMPH) on the expression of two key genes for neuronal development and plasticity, brain-derived neurotrophic factor (bdnf) and the effector immediate early gene activity-regulated, cytoskeletal-associated protein (Arc), was examined in both juvenile and adult rats. Both MPH [2 mg/kg, intraperitoneal (i.p.)] and AMPH (0.5 mg/kg, i.p.) induced marked decreases of bdnf mRNA in hippocampal and cortical brain regions of juveniles, whereas effects in adults were significantly less (hippocampus) or opposite (frontal cortex). In comparison, Arc mRNA was decreased (hippocampus and parietal cortex), largely unaffected (frontal cortex) or increased (striatum) in juveniles, whereas in adults, Arc mRNA increased in most brain regions. MPH-induced locomotion was also measured, and showed a much smaller increase in juveniles than in adults. In summary, our data show that the effects of MPH and AMPH on expression of the neurodevelopmentally important genes, bdnf and Arc, differ markedly in juvenile and adult rats, with juveniles showing evidence of brain region-specific decreases in both genes. These age-dependent effects on gene expression may be linked with the reported long-term harmful effects of psychostimulants in animal models.
Asunto(s)
Factor Neurotrófico Derivado del Encéfalo/genética , Encéfalo/efectos de los fármacos , Estimulantes del Sistema Nervioso Central/farmacología , Proteínas del Citoesqueleto/genética , Regulación de la Expresión Génica/efectos de los fármacos , Genes Inmediatos-Precoces/efectos de los fármacos , Proteínas del Tejido Nervioso/genética , Envejecimiento/genética , Envejecimiento/metabolismo , Anfetamina/farmacología , Animales , Encéfalo/crecimiento & desarrollo , Encéfalo/metabolismo , Regulación hacia Abajo/efectos de los fármacos , Regulación hacia Abajo/genética , Regulación de la Expresión Génica/genética , Genes Inmediatos-Precoces/genética , Masculino , Metanfetamina/farmacología , Ratas , Ratas Sprague-DawleyRESUMEN
This study describes development and evaluation of a multiplex PCR assay for simultaneous detection of Theileria annulata, Babesia bigemina and Anaplasma marginale infections in bovines. The assay was developed using parasites specific genomic DNA and three sets of PCR primers targeting the Tams1, 18S rRNA and 16S rRNA genes of T. annulata, B. bigemina and A. marginale, respectively. Blood samples collected from a total of 461 bovines, suspected for haemoparasitic infections, were examined microscopically to record the status of infection and simultaneously, genomic DNA extracted from these blood samples were utilized for the optimization and validation of multiplex PCR assay. Microscopic examination of blood samples revealed presence of single and multiple species of haemoparasites in 25.8% and 2.4% samples, respectively. Results of multiplex PCR revealed the presence of single haemoparasitic species infection in 159 cases (34.5%), whereas mixed infection was recorded in 82 (17.8%) samples. Occurrence of individual species infection detected by mPCR in the study was 26.03% (120/461) for T. annulata, 3.25% (15/461) for B. bigemina and 5.20% (24/461) for A. marginale. The detection limit of multiplex PCR assay was at the template dilutions of 10-6, 10-6 and 10-4, which corresponded to 0.1 pg, 0.1 pg and 10.0 pg of DNA for T. annulata, A. marginale, and B. bigemina, respectively. Based on the high diagnostic sensitivity and throughput, multiplex PCR assay developed in the present study could be exploited as a tool to conduct large-scale epidemiological survey for tick-borne haemoparasitic infection of bovines.
Asunto(s)
Enfermedades de los Bovinos/diagnóstico , Reacción en Cadena de la Polimerasa Multiplex/veterinaria , Enfermedades por Picaduras de Garrapatas/veterinaria , Anaplasma/genética , Anaplasma/aislamiento & purificación , Anaplasmosis/diagnóstico , Animales , Antígenos de Protozoos/genética , Babesia/genética , Babesia/aislamiento & purificación , Babesiosis/diagnóstico , Babesiosis/parasitología , Bovinos , Enfermedades de los Bovinos/parasitología , Clonación Molecular , ADN Bacteriano/sangre , ADN Bacteriano/química , ADN Bacteriano/aislamiento & purificación , ADN Protozoario/sangre , ADN Protozoario/química , ADN Protozoario/aislamiento & purificación , ARN Ribosómico 16S/genética , ARN Ribosómico 18S/genética , Reproducibilidad de los Resultados , Sensibilidad y Especificidad , Theileria annulata/genética , Theileria annulata/aislamiento & purificación , Theileriosis/diagnóstico , Theileriosis/parasitología , Enfermedades por Picaduras de Garrapatas/diagnósticoRESUMEN
O-GlcNAcylation, a dynamic nutrient and stress sensitive post-translational modification, occurs on myriad proteins in the cell nucleus, cytoplasm and mitochondria. O-GlcNAcylation serves as a nutrient sensor to regulate signaling, transcription, translation, cell division, metabolism, and stress sensitivity in all cells. Aberrant protein O-GlcNAcylation plays a critical role both in the development, as well as in the progression of a variety of age related diseases. O-GlcNAcylation underlies the etiology of diabetes, and changes in specific protein O-GlcNAc levels and sites are responsible for insulin expression and sensitivity and glucose toxicity. Abnormal O-GlcNAcylation contributes directly to diabetes related dysfunction of the heart, kidney and eyes and affects progression of cardiomyopathy, nephropathy and retinopathy. O-GlcNAcylation is a critical modification in the brain and plays a role in both plaque and tangle formation, thus making its study important in neurodegenerative disorders. O-GlcNAcylation also affects cellular growth and metabolism during the development and metastasis of cancer. Finally, alterations in O-GlcNAcylation of transcription factors in macrophages and lymphocytes affect inflammation and cytokine production. Thus, O-GlcNAcylation plays key roles in many of the major diseases associated with aging. Elucidation of its specific functions in both normal and diseased tissues is likely to uncover totally novel avenues for therapeutic intervention.
Asunto(s)
Acetilglucosamina , Envejecimiento , Enfermedad Crónica , Diabetes Mellitus , Cardiopatías , N-Acetilglucosaminiltransferasas/metabolismo , Enfermedades Neurodegenerativas , Envejecimiento/metabolismo , Envejecimiento/fisiología , Diabetes Mellitus/metabolismo , Diabetes Mellitus/fisiopatología , Cardiopatías/metabolismo , Cardiopatías/fisiopatología , Humanos , Hiperglucemia/metabolismo , Hiperglucemia/fisiopatología , Inflamación/metabolismo , Inflamación/fisiopatología , Neoplasias/metabolismo , Neoplasias/fisiopatología , Enfermedades Neurodegenerativas/metabolismo , Enfermedades Neurodegenerativas/fisiopatologíaRESUMEN
Indirect ELISA and dot-ELISA using recombinant BgP12 (rBgP12) were developed for the diagnosis of Babesia gibsoni infected dogs. The complete open reading frame of BgP12 gene (378bp) was cloned in pET-32a(+) expression vector and expressed in Escherichia coli as a soluble thioredoxin (Trx) fusion protein. The purified rBgP12 was used for production of anti-rBgP12 rabbit serum, which recognized a native 12-kDa protein in B. gibsoni infected erythrocyte by Western blot analysis. To evaluate the potential of rBgP12 for the serodiagnosis of B. gibsoni, a panel of serum/plasma samples from dogs infected with B. gibsoni (n=13), uninfected sera (n=13) and sera from dogs infected with other haemoparasites viz., Babesia canis vogeli (n=3), Ehrlichia canis (n=3), Hepatozoon canis (n=1) and Dirofilaria immitis (n=1) were used in ELISA formats. In addition, the performance of rBgP12 based indirect ELISA and dot-ELISA were evaluated using 75 serum/plasma samples collected from suspected dogs, in respect to the nested PCR as reference test. The diagnostic sensitivities of indirect ELISA and dot-ELISA were 94.59% and 89.18%, respectively, while their specificities were 84.21% and 81.57%, respectively. Moreover, both the assays using rBgP12 showed no cross reaction with sera from dogs infected with other common haemoparasites indicating their high specificity. High kappa values of indirect ELISA and dot-ELISA indicated the potentials of these assays with substantial agreement at 95% confidence level. It is concluded that indirect ELISA and dot ELISA using rBgP12 might be used in large scale epidemiological surveys and clinical diagnosis of B. gibsoni infection in dogs.
Asunto(s)
Babesiosis/diagnóstico , Enfermedades de los Perros/diagnóstico , Ensayo de Inmunoadsorción Enzimática/veterinaria , Proteínas Protozoarias/inmunología , Animales , Enfermedades de los Perros/inmunología , Enfermedades de los Perros/parasitología , Perros , Genes Protozoarios , Proteínas Protozoarias/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/inmunología , Pruebas Serológicas/veterinariaRESUMEN
The phylum Apicomplexa includes parasites of medical, zoonotic and veterinary significance. Understanding the global distribution and genetic diversity of these protozoa is of fundamental importance for efficient, robust and long-lasting methods of control. Eimeria spp. cause intestinal coccidiosis in all major livestock animals and are the most important parasites of domestic chickens in terms of both economic impact and animal welfare. Despite having significant negative impacts on the efficiency of food production, many fundamental questions relating to the global distribution and genetic variation of Eimeria spp. remain largely unanswered. Here, we provide the broadest map yet of Eimeria occurrence for domestic chickens, confirming that all the known species (Eimeria acervulina, Eimeria brunetti, Eimeria maxima, Eimeria mitis, Eimeria necatrix, Eimeria praecox, Eimeria tenella) are present in all six continents where chickens are found (including 21 countries). Analysis of 248 internal transcribed spacer sequences derived from 17 countries provided evidence of possible allopatric diversity for species such as E. tenella (FST values ⩽0.34) but not E. acervulina and E. mitis, and highlighted a trend towards widespread genetic variance. We found that three genetic variants described previously only in Australia and southern Africa (operational taxonomic units x, y and z) have a wide distribution across the southern, but not the northern hemisphere. While the drivers for such a polarised distribution of these operational taxonomic unit genotypes remains unclear, the occurrence of genetically variant Eimeria may pose a risk to food security and animal welfare in Europe and North America should these parasites spread to the northern hemisphere.
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ADN Protozoario/genética , Eimeria/genética , Enfermedades de las Aves de Corral/parasitología , Animales , Biodiversidad , Pollos/parasitología , Clasificación , Coccidiosis/epidemiología , Coccidiosis/parasitología , Coccidiosis/veterinaria , Genotipo , Filogenia , Filogeografía , Enfermedades de las Aves de Corral/epidemiología , Análisis de Secuencia de ADNRESUMEN
Diagnosis of canine babesiosis, caused by Babesia gibsoni is difficult, especially in chronically infected dogs. A loop mediated isothermal amplification (LAMP) assay was developed and standardized by using four oligonucleotide primers targeting the hypervariable region of 18S rRNA gene (GenBank Acc. no. KC461261). The primers specifically amplified B. gibsoni DNA, while no amplification was detected with DNA from non-infected dogs as well as from dogs infected with Babesia canis vogeli, Hepatozoon canis, Ehrlichia canis and Trypanosoma evansi. The assay could detect 1.35 × 10(-7) parasitaemia and 10(-4) dilution of recombinant plasmid, equivalent to 12 pg of target DNA. All the samples were tested by nested PCR as well as LAMP assay. LAMP was found to be 10 times more sensitive than nested PCR targeting the same gene. Out of 75 suspected field samples, collected from different parts of the country, LAMP could detect B. gibsoni in 43 samples, while nested PCR and microscopy could detect 37 and 23 samples, respectively. High sensitivity, specificity and rapidity of LAMP assay may be exploited for screening large number of samples in a field setting.
Asunto(s)
Babesia/aislamiento & purificación , Babesiosis/diagnóstico , Enfermedades de los Perros/parasitología , Técnicas de Amplificación de Ácido Nucleico/veterinaria , Animales , Babesia/clasificación , ADN Bacteriano/genética , Enfermedades de los Perros/diagnóstico , Perros , Técnicas de Amplificación de Ácido Nucleico/métodos , ARN Bacteriano/genética , ARN Ribosómico 18S/genética , Sensibilidad y Especificidad , Especificidad de la EspecieRESUMEN
Indirect ELISA, dot-ELISA and double antibody sandwich ELISA (DAS-ELISA) using truncated recombinant BgSA1 (rBgSA1) were developed for detecting Babesia gibsoni infection in naturally infected dogs. Truncated BgSA1 gene of 858 bp, encoding 32 kDa protein was cloned in pET-32a(+) expression vector, expressed in Escherichia coli and the recombinant protein was purified under native conditions. To evaluate the ability of the truncated rBgSA1as serodiagnostic reagent for B. gibsoni infection, a panel of sera/plasma samples from dogs infected with B. gibsoni (n = 13), uninfected sera (n = 13) and sera from dogs infected with other haemoparasites namely, Babesia canis vogeli (n = 3), Ehrlichia canis (n = 3), Hepatozoon canis (n = 1) and Dirofilaria immitis (n = 1) were used. Besides these, 75 samples collected from dogs suspected for babesiosis were used to evaluate the performance of rBgSA1 based serological assays in comparison to nested PCR. Based on the results, the diagnostic sensitivity of indirect ELISA, dot-ELISA and DAS-ELISA were 97.3%, 91.9% and 100%, respectively, when nested PCR was taken as a reference test, while their specificities were 81.6%, 84.2% and 97.4%, respectively. Further, DAS-ELISA had a quantitation limit of 0.03 µg/ml of the rBgSA1. High kappa values of indirect ELISA, dot-ELISA and DAS-ELISA were recorded, indicating that these assays had substantial to almost perfect agreement at 95% confidence level. There was no cross-reactivity with sera from dogs infected with B. canis vogeli, E. canis, H. canis and D. immitis. The results suggest that the indirect ELISA, dot-ELISA and DAS-ELISA with rBgSA1 may be used in large scale epidemiological surveys and clinical diagnosis of B. gibsoni infection in dogs. DAS-ELISA has advantages over indirect or dot-ELISA in the detection of current infection as well as monitoring the parasite burden.
Asunto(s)
Anticuerpos Antiprotozoarios/sangre , Antígenos de Protozoos/inmunología , Babesia/inmunología , Babesiosis/diagnóstico , Enfermedades de los Perros/parasitología , Ensayo de Inmunoadsorción Enzimática/veterinaria , Animales , Babesia/aislamiento & purificación , Babesiosis/parasitología , Perros , Escherichia coli/genética , Escherichia coli/metabolismo , Expresión Génica , Proteínas Recombinantes , Sensibilidad y EspecificidadRESUMEN
Molecular approaches supporting identification of Eimeria parasites infecting chickens have been available for more than 20 years, although they have largely failed to replace traditional measures such as microscopy and pathology. Limitations of microscopy-led diagnostics, including a requirement for specialist parasitological expertise and low sample throughput, are yet to be outweighed by the difficulties associated with accessing genomic DNA from environmental Eimeria samples. A key step towards the use of Eimeria species-specific PCR as a sensitive and reproducible discriminatory tool for use in the field is the production of a standardised protocol that includes sample collection and DNA template preparation, as well as primer selection from the numerous PCR assays now published. Such a protocol will facilitate development of valuable epidemiological datasets which may be easily compared between studies and laboratories. The outcome of an optimisation process undertaken in laboratories in India and the UK is described here, identifying four steps. First, samples were collected into a 2% (w/v) potassium dichromate solution. Second, oocysts were enriched by flotation in saturated saline. Third, genomic DNA was extracted using a QIAamp DNA Stool mini kit protocol including a mechanical homogenisation step. Finally, nested PCR was carried out using previously published primers targeting the internal transcribed spacer region 1 (ITS-1). Alternative methods tested included sample processing in the presence of faecal material, DNA extraction using a traditional phenol/chloroform protocol, the use of SCAR multiplex PCR (one tube and two tube versions) and speciation using the morphometric tool COCCIMORPH for the first time with field samples.
Asunto(s)
Coccidiosis/veterinaria , Eimeria/genética , Reacción en Cadena de la Polimerasa/veterinaria , Enfermedades de las Aves de Corral/parasitología , Animales , Pollos/parasitología , Coccidiosis/diagnóstico , Coccidiosis/parasitología , ADN Protozoario/química , ADN Protozoario/genética , ADN Espaciador Ribosómico/genética , Heces/parasitología , Reacción en Cadena de la Polimerasa Multiplex , Reacción en Cadena de la Polimerasa/normas , Enfermedades de las Aves de Corral/diagnóstico , ARN Ribosómico 18S/genética , ARN Ribosómico 5.8S/genética , Sensibilidad y EspecificidadRESUMEN
The inherent difficulty of performing chemical modifications of proteins in a truly site-specific fashion is often compounded by the need to work within complex biological settings. In order to alleviate this complication, targets can be "prelabeled" metabolically with unnatural residues, which allow access to highly selective bioorthogonal reactions. Due to their small size, permissibility within biosynthetic pathways and access to reactions with high specificity, azides provide excellent bioorthogonal handles. This two-step labeling process is emerging as a highly effective means to modify therapeutic proteins. In this chapter, we take this strategy a step further and apply chemoselective ligation to remodel the surfaces of adenoviruses. Despite the large number of ongoing clinical trials involving these complex mammalian viruses, new methods for their facile, flexible surface modification are necessary to drive the development of next-generation therapeutics. Here we demonstrate the modification of azides on adenoviral surfaces via a straightforward chemoselective protocol based on copper-assisted "click" chemistry. This method provides access to a wide array of effector functionalities without sacrificing infectivity.