RESUMO
Huntington's disease (HD) is a neurodegenerative disorder caused by an inherited unstable HTT CAG repeat that expands further, thereby eliciting a disease process that may be initiated by polyglutamine-expanded huntingtin or a short polyglutamine-product. Phosphorylation of selected candidate residues is reported to mediate polyglutamine-fragment degradation and toxicity. Here to support the discovery of phosphosites involved in the life-cycle of (full-length) huntingtin, we employed mass spectrometry-based phosphoproteomics to systematically identify sites in purified huntingtin and in the endogenous protein by proteomic and phosphoproteomic analyses of members of an HD neuronal progenitor cell panel. Our results bring total huntingtin phosphosites to 95, with more located in the N-HEAT domain relative to numbers in the Bridge and C-HEAT domains. Moreover, phosphorylation of C-HEAT Ser2550 by cAMP-dependent protein kinase (PKA), the top hit in kinase activity screens, was found to hasten huntingtin degradation, such that levels of the catalytic subunit (PRKACA) were inversely related to huntingtin levels. Taken together, these findings highlight categories of phosphosites that merit further study and provide a phosphosite kinase pair (pSer2550-PKA) with which to investigate the biological processes that regulate huntingtin degradation and thereby influence the steady state levels of huntingtin in HD cells.
Assuntos
Proteínas Quinases Dependentes de AMP Cíclico , Doença de Huntington , Humanos , Proteínas Quinases Dependentes de AMP Cíclico/genética , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Temperatura Alta , Proteína Huntingtina/metabolismo , Doença de Huntington/genética , Doença de Huntington/metabolismo , Fosforilação , Domínios Proteicos , ProteômicaRESUMO
Sudan ebolavirus (SUDV) is a member of the genus Ebolavirus (Family Filoviridae) and has caused sporadic outbreaks of Ebola disease (EBOD), or more specifically Sudan virus disease (SVD), with high mortality rates in Africa. Current vaccines and therapies that have been developed for filoviruses are almost all specific for Ebola virus (EBOV; of the species Zaire ebolavirus), and there is a current lack of therapeutics specific for SUDV. The recent SUDV outbreak in Uganda, which was distributed across multiple districts, including Kampala, a densely populated urban center, highlights the critical need for the development of novel SUDV-specific or pan-Ebola virus therapeutics. Previous work has characterized two monoclonal antibodies, FVM04 and CA45, which have neutralization capabilities against both EBOV and SUDV and have shown protective efficacy in animal challenge studies. Here, we expand upon this work, showing that treatment with a monoclonal antibody cocktail consisting of FVM04 and CA45 provides full protection against lethal SUDV infection in cynomolgus macaques. Studies that evaluate outcomes at late time points after infection, once clinical signs of illness are apparent, are vital for assessing the therapeutic efficacy of antibody therapeutics. We have shown that when treatment is initiated as late as 5 days after infection, with a second dose given on day 8, that treated groups showed few clinical signs or morbidity, with complete survival. This work provides further evidence that FVM04 and CA45 have strong therapeutic potential against SUDV and their development as a pan-Ebola virus therapeutic should be pursued. IMPORTANCE: There are currently no approved vaccines or therapeutics for Sudan virus, a filovirus which is highly related to Ebola virus and causes similar disease and outbreaks. In this study, a cocktail of two potent monoclonal antibodies that effectively neutralize Sudan virus was tested in a nonhuman primate model of Sudan virus disease. Treatment was highly effective, even when initiated as late as 5 days after infection, when clinical signs of infection were already evident. All treated animals showed complete recovery from infection, with little evidence of disease, while all animals that received a control treatment succumbed to infection within 8 days. The study further demonstrated the strong therapeutic potential of the antibody treatment and supported further development for use in Sudan virus outbreaks.
Assuntos
Anticorpos Monoclonais , Anticorpos Antivirais , Ebolavirus , Doença pelo Vírus Ebola , Atraso no Tratamento , Animais , Feminino , Anticorpos Monoclonais/uso terapêutico , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/administração & dosagem , Anticorpos Neutralizantes/imunologia , Anticorpos Neutralizantes/uso terapêutico , Anticorpos Antivirais/imunologia , Anticorpos Antivirais/uso terapêutico , Modelos Animais de Doenças , Ebolavirus/imunologia , Doença pelo Vírus Ebola/prevenção & controle , Doença pelo Vírus Ebola/imunologia , Doença pelo Vírus Ebola/tratamento farmacológico , Macaca fascicularis , MasculinoRESUMO
Huntington's disease pathogenesis involves a genetic gain-of-function toxicity mechanism triggered by the expanded HTT CAG repeat. Current therapeutic efforts aim to suppress expression of total or mutant huntingtin, though the relationship of huntingtin's normal activities to the gain-of-function mechanism and what the effects of huntingtin-lowering might be are unclear. Here, we have re-investigated a rare family segregating two presumed HTT loss-of-function (LoF) variants associated with the developmental disorder, Lopes-Maciel-Rodan syndrome (LOMARS), using whole-genome sequencing of DNA from cell lines, in conjunction with analysis of mRNA and protein expression. Our findings correct the muddled annotation of these HTT variants, reaffirm they are the genetic cause of the LOMARS phenotype and demonstrate that each variant is a huntingtin hypomorphic mutation. The NM_002111.8: c.4469+1G>A splice donor variant results in aberrant (exon 34) splicing and severely reduced mRNA, whereas, surprisingly, the NM_002111.8: c.8157T>A NP_002102.4: Phe2719Leu missense variant results in abnormally rapid turnover of the Leu2719 huntingtin protein. Thus, although rare and subject to an as yet unknown LoF intolerance at the population level, bona fide HTT LoF variants can be transmitted by normal individuals leading to severe consequences in compound heterozygotes due to huntingtin deficiency.
Assuntos
Regulação da Expressão Gênica , Proteína Huntingtina/genética , Mutação , Transtornos do Neurodesenvolvimento/genética , Sequência de Aminoácidos , Linhagem Celular , Criança , Pré-Escolar , Feminino , Humanos , Proteína Huntingtina/química , Proteína Huntingtina/metabolismo , Mutação com Perda de Função , Masculino , Mutação de Sentido Incorreto , Transtornos do Neurodesenvolvimento/metabolismo , Linhagem , Fenótipo , Splicing de RNA , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Alinhamento de Sequência , Análise de Sequência de DNARESUMO
Inherited alleles account for most of the genetic risk for schizophrenia. However, new (de novo) mutations, in the form of large chromosomal copy number changes, occur in a small fraction of cases and disproportionally disrupt genes encoding postsynaptic proteins. Here we show that small de novo mutations, affecting one or a few nucleotides, are overrepresented among glutamatergic postsynaptic proteins comprising activity-regulated cytoskeleton-associated protein (ARC) and N-methyl-d-aspartate receptor (NMDAR) complexes. Mutations are additionally enriched in proteins that interact with these complexes to modulate synaptic strength, namely proteins regulating actin filament dynamics and those whose messenger RNAs are targets of fragile X mental retardation protein (FMRP). Genes affected by mutations in schizophrenia overlap those mutated in autism and intellectual disability, as do mutation-enriched synaptic pathways. Aligning our findings with a parallel case-control study, we demonstrate reproducible insights into aetiological mechanisms for schizophrenia and reveal pathophysiology shared with other neurodevelopmental disorders.
Assuntos
Modelos Neurológicos , Mutação/genética , Rede Nervosa/metabolismo , Vias Neurais/metabolismo , Esquizofrenia/genética , Esquizofrenia/fisiopatologia , Sinapses/metabolismo , Transtornos Globais do Desenvolvimento Infantil/genética , Proteínas do Citoesqueleto/metabolismo , Exoma/genética , Proteína do X Frágil da Deficiência Intelectual/metabolismo , Humanos , Deficiência Intelectual/genética , Taxa de Mutação , Rede Nervosa/fisiopatologia , Proteínas do Tecido Nervoso/metabolismo , Vias Neurais/fisiopatologia , Fenótipo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Receptores de N-Metil-D-Aspartato/metabolismo , Esquizofrenia/metabolismo , Especificidade por SubstratoRESUMO
Chikungunya virus (CHIKV) is a mosquito-transmitted pathogen that causes chikungunya disease (CHIK); the disease is characterized by fever, muscle ache, rash, and arthralgia. This arthralgia can be debilitating and long-lasting, seriously impacting quality of life for years. Currently, there is no specific therapy available for CHIKV infection. We have developed a despeciated equine polyclonal antibody (CHIKV-EIG) treatment against CHIKV and evaluated its protective efficacy in mouse models of CHIKV infection. In immunocompromised (IFNAR-/-) mice infected with CHIKV, daily treatment for five consecutive days with CHIKV-EIG administered at 100 mg/kg starting on the day of infection prevented mortality, reduced viremia, and improved clinical condition as measured by body weight loss. These beneficial effects were seen even when treatment was delayed to 1 day after infection. In immunocompetent mice, CHIKV-EIG treatment reduced virus induced arthritis (including footpad swelling), arthralgia-associated cytokines, viremia, and tissue virus loads in a dose-dependent fashion. Collectively, these results suggest that CHIKV-EIG is effective at preventing CHIK and could be a viable candidate for further development as a treatment for human disease.
Assuntos
Febre de Chikungunya , Vírus Chikungunya , Animais , Cavalos , Humanos , Camundongos , Viremia/tratamento farmacológico , Viremia/prevenção & controle , Qualidade de Vida , Vírus Chikungunya/fisiologia , Anticorpos Antivirais/uso terapêutico , Artralgia/tratamento farmacológico , Artralgia/prevenção & controleRESUMO
Lognormal distribution is often used as a default model for regression analysis of particle size distribution (PSD) data; however, its goodness-of-fit to particle matter (PM) sampled from animal buildings and its comparison to other PSD models have not been well examined. This study aimed to evaluate and to compare the goodness-of-fit of six PSD models to total suspended particulate matter (TSP) samples collected from 15 animal buildings. Four particle size analyzers were used for PSD measurement. The models' goodness-of-fit was evaluated based on adjusted R2, Akaike's information criterion (AIC), and mean squared error (MSE) values. Results showed that the models' approximation of measured PSDs differed with particle size analyzer. The lognormal distribution model offered overall good approximations to measured PSD data, but was inferior to the gamma and Weibull distribution models when applied to PSD data derived from the Horiba and Malvern analyzers. Single-variable models including the exponential, Khrgian-Mazin, and Chen's empirical models provided relatively poor approximations and, thus, were not recommended for future investigations. A further examination on model-predicted PSD parameters revealed that even the best-fit model of the six could significantly misestimate mean diameter median diameter; and variance. However, compared with other models, the best-fit model still offered the relatively best estimates of mean and median diameters, whereas the best predicted variances were given by the gamma distribution model.
Assuntos
Poluentes Atmosféricos/química , Poluição do Ar em Ambientes Fechados/estatística & dados numéricos , Abrigo para Animais , Material Particulado/química , Animais , Galinhas , Modelos Químicos , Tamanho da Partícula , Suínos , PerusRESUMO
Despite ongoing vaccination efforts to prevent SARS-CoV-2 infections, treatment tools are still necessary to address the ongoing COVID-19 pandemic. We report here that COVID-HIGIV, a human immunoglobulin product for treatment of COVID-19, provided a significant survival benefit in SARS-CoV-2 infected transgenic mice compared to controls. COVID-HIGIV also has similar pharmacokinetic profiles in healthy and SARS-CoV-2 infected mice over time after intravenous administration, with identical or comparable Tmax, Cmax, AUC0-∞ and Cl. AUC0-last increased and mean residence time, T1/2, and Vd reduced in infected animals compared to healthy animals. These data suggest that COVID-HIGIV may be an effective treatment for SARS-CoV-2 infection when given early after exposure.
Assuntos
COVID-19 , Humanos , Camundongos , Animais , SARS-CoV-2 , RNA Viral , Pandemias/prevenção & controle , AnticorposRESUMO
In late 2019 the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus emerged in China and quickly spread into a worldwide pandemic. It has caused millions of hospitalizations and deaths, despite the use of COVID-19 vaccines. Convalescent plasma and monoclonal antibodies emerged as major therapeutic options for treatment of COVID-19. We have developed an anti-SARS-CoV-2 immunoglobulin intravenous (Human) (COVID-HIGIV), a potential improvement from using convalescent plasma. In this report the efficacy of COVID-HIGIV was evaluated in hamster and mouse models of SARS-CoV-2 infection. COVID-HIGIV treatment in both mice and hamsters significantly reduced the viral load in the lungs. Among COVID-HIGIV treated animals, infection-related body weight loss was reduced and the animals regained their baseline body weight faster than the PBS controls. In hamsters, COVID-HIGIV treatment reduced infection-associated lung pathology including lung inflammation, and pneumocyte hypertrophy in the lungs. These results support ongoing trials for outpatient treatment with COVID-HIGIV for safety and efficacy evaluation (NCT04910269, NCT04546581).
Assuntos
COVID-19 , Animais , Anticorpos Monoclonais , COVID-19/terapia , Vacinas contra COVID-19 , Ensaios Clínicos como Assunto , Cricetinae , Modelos Animais de Doenças , Humanos , Imunização Passiva , Pulmão/patologia , Camundongos , SARS-CoV-2 , Soroterapia para COVID-19RESUMO
The closely related flaviviruses, dengue and Zika, cause significant human disease throughout the world. While cross-reactive antibodies have been demonstrated to have the capacity to potentiate disease or mediate protection during flavivirus infection, the mechanisms responsible for this dichotomy are still poorly understood. To understand how the human polyclonal antibody response can protect against, and potentiate the disease in the context of dengue and Zika virus infection we used intravenous hyperimmunoglobulin (IVIG) preparations in a mouse model of the disease. Three IVIGs (ZIKV-IG, Control-Ig and Gamunex®) were evaluated for their ability to neutralize and/or enhance Zika, dengue 2 and 3 viruses in vitro. The balance between virus neutralization and enhancement provided by the in vitro neutralization data was used to predict the IVIG concentrations which could protect or enhance Zika, and dengue 2 disease in vivo. Using this approach, we were able to define the unique in vivo dynamics of complex polyclonal antibodies, allowing for both enhancement and protection from flavivirus infection. Our results provide a novel understanding of how polyclonal antibodies interact with viruses with implications for the use of polyclonal antibody therapeutics and the development and evaluation of the next generation flavivirus vaccines.
Assuntos
Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , Interações Hospedeiro-Patógeno/imunologia , Imunoglobulinas Intravenosas , Infecção por Zika virus/imunologia , Infecção por Zika virus/virologia , Zika virus/imunologia , Animais , Linhagem Celular , Reações Cruzadas/imunologia , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Humanos , Imunoglobulinas Intravenosas/uso terapêutico , Macrófagos/imunologia , Macrófagos/metabolismo , Camundongos , Camundongos Knockout , Testes de Neutralização , Infecção por Zika virus/sangue , Infecção por Zika virus/tratamento farmacológicoRESUMO
We report a case of a 54-year-old Caucasian woman with an earlier diagnosis of Peutz-Jeghers Syndrome (PJS) and sex cord tumor with annular tubules (SCTAT). The sex cord stromal tumors showed aggressive malignant behavior with repeated recurrence and metastasis. This is an unusual behavior of SCTAT in patients with PJS, with only 2 such cases reported earlier. Genetic analysis revealed that the patient has a new (unreported earlier) missense mutation of the LKB1 gene. A review of the literature reporting the clinicopathologic features and biologic behavior of SCTAT in patients with and without PJS is presented. We discuss the presentation and management of this case and highlight the importance of considering the possibility of aggressive behavior of these tumors in the management of patients with PJS.
Assuntos
Neoplasias Ovarianas/patologia , Síndrome de Peutz-Jeghers/complicações , Tumores do Estroma Gonadal e dos Cordões Sexuais/patologia , Quinases Proteína-Quinases Ativadas por AMP , Feminino , Humanos , Pessoa de Meia-Idade , Mutação de Sentido Incorreto , Neoplasias Ovarianas/complicações , Neoplasias Ovarianas/genética , Síndrome de Peutz-Jeghers/genética , Proteínas Serina-Treonina Quinases/genética , Tumores do Estroma Gonadal e dos Cordões Sexuais/complicações , Tumores do Estroma Gonadal e dos Cordões Sexuais/genéticaRESUMO
Seasonal influenza causes significant morbidity and mortality around the world each year, even with the use of vaccines and antivirals. There is a need for more effective treatments for severe and hospitalized cases of influenza. In this study, we have tested the efficacy of a human plasma-derived IgG product (FLU-IGIV) against seasonal influenza in mouse and ferret models of influenza infection. FLU-IGIV successfully protected mice (100% survival) against lethal influenza infection. Also, the survival rate observed with FLU-IGIV treatment was better than the survival rate observed with oseltamivir (60% survival). FLU-IGIV significantly reduced the viral load in the lungs compared to placebo (PBS) in ferrets infected with influenza A/California/07/2009 (H1N1pdm09) virus. Overall, these studies demonstrate the efficacy of human plasma-derived FLU-IGIV in relevant animal models of influenza virus infection.
Assuntos
Anticorpos Antivirais/uso terapêutico , Antivirais/uso terapêutico , Imunoglobulinas Intravenosas/uso terapêutico , Infecções por Orthomyxoviridae/terapia , Animais , Antivirais/farmacocinética , Relação Dose-Resposta Imunológica , Feminino , Furões/virologia , Humanos , Imunoglobulinas Intravenosas/farmacocinética , Vírus da Influenza A Subtipo H1N1 , Camundongos , Camundongos Endogâmicos BALB C , Pandemias , Carga Viral/efeitos dos fármacosRESUMO
BACKGROUND: Huntington's disease (HD) is an inherited neurodegenerative disorder caused by an expanded CAG repeat in the HTT gene. It is diagnosed following a standardized examination of motor control and often presents with cognitive decline and psychiatric symptoms. Recent studies have detected genetic loci modifying the age at onset of motor symptoms in HD, but genetic factors influencing cognitive and psychiatric presentations are unknown. METHODS: We tested the hypothesis that psychiatric and cognitive symptoms in HD are influenced by the same common genetic variation as in the general population by 1) constructing polygenic risk scores from large genome-wide association studies of psychiatric and neurodegenerative disorders and of intelligence and 2) testing for correlation with the presence of psychiatric and cognitive symptoms in a large sample (n = 5160) of patients with HD. RESULTS: Polygenic risk score for major depression was associated specifically with increased risk of depression in HD, as was schizophrenia risk score with psychosis and irritability. Cognitive impairment and apathy were associated with reduced polygenic risk score for intelligence. CONCLUSIONS: Polygenic risk scores for psychiatric disorders, particularly depression and schizophrenia, are associated with increased risk of the corresponding psychiatric symptoms in HD, suggesting a common genetic liability. However, the genetic liability to cognitive impairment and apathy appears to be distinct from other psychiatric symptoms in HD. No associations were observed between HD symptoms and risk scores for other neurodegenerative disorders. These data provide a rationale for treatments effective in depression and schizophrenia to be used to treat depression and psychotic symptoms in HD.
Assuntos
Doença de Huntington , Transtornos Psicóticos , Cognição , Estudo de Associação Genômica Ampla , Humanos , Doença de Huntington/complicações , Doença de Huntington/genética , Transtornos Psicóticos/complicações , Transtornos Psicóticos/genética , Fatores de RiscoRESUMO
Botulism neurotoxins are highly toxic and are potential agents for bioterrorism. The development of effective therapy is essential to counter the possible use of these toxins in military and bioterrorism scenarios, and to provide treatment in cases of natural intoxication. Guinea pigs were intoxicated with a lethal dose of botulinum neurotoxin serotypes A, B, C, D, E, F or G, and at onset of the clinical disease intoxicated animals were treated with either BAT® [Botulism Antitoxin Heptavalent (A, B, C, D, E, F, G)-(Equine)] or placebo. BAT product treatment significantly (p<0.0001) enhanced survival compared to placebo for all botulinum neurotoxin serotypes and arrested or mitigated the progression of clinical signs of botulism intoxication. These results demonstrated the therapeutic efficacy of BAT product in guinea pigs and provided supporting evidence of effectiveness for licensure of BAT product under FDA 21 CFR Part 601 (Subpart H Animal Rule) as a therapeutic for botulism intoxication to serotypes A, B, C, D, E, F or G in adults and pediatric patients.
Assuntos
Antitoxinas/farmacologia , Antitoxina Botulínica/farmacologia , Toxinas Botulínicas/antagonistas & inibidores , Botulismo/metabolismo , Neurotoxinas/antagonistas & inibidores , Animais , Bioterrorismo/prevenção & controle , Progressão da Doença , Feminino , Cobaias , Cavalos , Masculino , Camundongos , SorogrupoRESUMO
BACKGROUND: Botulism is a disease caused by neurogenic toxins that block acetylcholine release, resulting in potentially life threatening neuroparalysis. Seven distinct serotypes of botulinum neurotoxins (BoNTs) have been described and are found in nature world-wide. This, combined with ease of production, make BoNTs a significant bioweapon threat. An essential countermeasure to this threat is an antitoxin to remove circulating toxin. An antitoxin, tradename BAT (Botulism Antitoxin Heptavalent (A, B, C, D, E, F, G)-(Equine)), has been developed and its efficacy evaluated against all seven serotypes in guinea pigs. METHODS AND FINDINGS: Studies were conducted to establish the lethal dose and clinical course of intoxication for all seven toxins, and post-exposure prophylactic efficacy of BAT product. Animals were monitored for signs of intoxication and mortality for 14 days. Guinea pig intramuscular LD50s (GPIMLD50) for all BoNTs ranged from 2.0 (serotype C) to 73.2 (serotype E) of mouse intraperitoneal LD50 units. A dose of 4x GPIMLD50 was identified as the appropriate toxin dose for use in subsequent efficacy and post-exposure prophylaxis studies. The main clinical signs observed included hind limb paralysis, weak limb, change in breathing rate/pattern, and forced abdominal respiration. Mean time to onset of clinical signs ranged from 12 hours (serotype E) to 39 hours (serotype G). Twelve hours post-intoxication was selected as the appropriate time point for intervention for all serotypes apart from E where 6 hours was selected because of the rapid onset and progression of clinical signs. Post-exposure treatment with BAT product resulted in a significantly (p<0.0001) higher survival at >0.008 scaled human dose for serotypes A, B, C, F and G, at >0.2x for serotype D and >0.04x for serotype E. CONCLUSIONS: These studies confirm the efficacy of BAT as a post-exposure prophylactic therapy against all seven known BoNT serotypes.
Assuntos
Antitoxina Botulínica/uso terapêutico , Botulismo/tratamento farmacológico , Animais , Antitoxina Botulínica/administração & dosagem , Cobaias , Cavalos , SorogrupoRESUMO
Zika virus (ZIKV) is an emerging mosquito-borne flavivirus that represents a major threat to global health. ZIKV infections in adults are generally asymptomatic or present with mild symptoms. However, recent outbreaks of ZIKV have revealed that it can cause Congenital Zika Syndrome in neonates and Guillain-Barré syndrome in adults. Currently, no ZIKV-specific vaccines or antiviral treatments are available. In this study, we tested the efficacy of convalescent plasma IgG hyperimmune product (ZIKV-IG) isolated from individuals with high neutralizing anti-ZIKV titers as a therapeutic candidate against ZIKV infection using a model of ZIKV infection in Ifnar1-/- mice. ZIKV-IG successfully protected mice from lethal ZIKV challenge. In particular, ZIKV-IG treatment at 24 hours after lethal ZIKV infection improved survival by reducing weight loss and tissue viral burden and improving clinical score. Additionally, ZIKV-IG eliminated ZIKV-induced tissue damage and inflammation in the brain and liver. These results indicate that ZIKV-IG is efficacious against ZIKV, suggesting this human polyclonal antibody is a viable candidate for further development as a treatment against human ZIKV infection.
Assuntos
Anticorpos Neutralizantes/imunologia , Infecção por Zika virus/imunologia , Zika virus/imunologia , Animais , Anticorpos Antivirais/imunologia , Encéfalo/imunologia , Chlorocebus aethiops , Cricetinae , Culicidae , Humanos , Imunoglobulina G/imunologia , Inflamação/imunologia , Fígado/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Células VeroRESUMO
Integration of exogenous DNA into a host genome represents an important route to generate animal and cellular models for exploration into human disease and therapeutic development. In most models, little is known concerning structural integrity of the transgene, precise site of integration, or its impact on the host genome. We previously used whole-genome and targeted sequencing approaches to reconstruct transgene structure and integration sites in models of Huntington's disease, revealing complex structural rearrangements that can result from transgenesis. Here, we demonstrate in the R6/2 mouse, a widely used Huntington's disease model, that integration of a rearranged transgene with coincident deletion of 5,444 bp of host genome within the gene Gm12695 has striking molecular consequences. Gm12695, the function of which is unknown, is normally expressed at negligible levels in mouse brain, but transgene integration has resulted in cortical expression of a partial fragment (exons 8-11) 3' to the transgene integration site in R6/2. This transcript shows significant expression among the extensive network of differentially expressed genes associated with this model, including synaptic transmission, cell signalling and transcription. These data illustrate the value of sequence-level resolution of transgene insertions and transcription analysis to inform phenotypic characterization of transgenic models utilized in therapeutic research.
Assuntos
Modelos Animais de Doenças , Doença de Huntington/patologia , Camundongos Transgênicos , Animais , Perfilação da Expressão Gênica , Rearranjo Gênico , Humanos , Camundongos , Análise de Sequência de DNA , Deleção de SequênciaRESUMO
Leishmania minicircular deoxyribonucleic acid (DNA) is arranged into different classes according to sequence. These classes differ substantially in sequence, despite species- and genus-specific regions, and are present in widely different copy numbers within and between Leishmania strains. Homologous minicircles have been identified in different species of Leishmania by comparing sequences of known minicircles. However, it is possible to select for minicircles of the same class by amplifying Leishmania DNA with polymerase chain reaction primers from the conserved and variable regions. This approach was used with 2 different minicircle classes in the L. donovani complex. In all isolates tested it was possible to amplify minicircles of the selected class.
Assuntos
DNA de Cinetoplasto/genética , Leishmania donovani/genética , Animais , Sequência de Bases , DNA de Protozoário/genética , Dados de Sequência Molecular , Reação em Cadeia da Polimerase/métodos , Alinhamento de SequênciaRESUMO
This paper discusses the utility of a set of primers (3J1, 3J2) designed from a repetitive nuclear deoxyribonucleic acid sequence for the diagnosis of Leishmania braziliensis infection in samples obtained from humans, insect vectors and mammalian reservoir hosts from different endemic areas in Venezuela. A high incidence of Leishmania (Viannia) braziliensis infection was found in the endemic areas studied. The sensitivity and specificity of the primers used were adequate for the identification of the natural vectors and reservoir hosts of L. (V.) braziliensis. The polymerase chain reaction was more sensitive than culture and stained smear examination in the diagnosis of cutaneous leishmaniasis, detecting 80% of cases compared to 42% and 72%, respectively.
Assuntos
Doenças Endêmicas , Leishmania braziliensis/genética , Leishmaniose Cutânea/epidemiologia , Animais , Primers do DNA , DNA de Protozoário/análise , Humanos , Insetos Vetores/parasitologia , Leishmania braziliensis/classificação , Leishmaniose Cutânea/diagnóstico , Leishmaniose Cutânea/veterinária , Mamíferos/parasitologia , Reação em Cadeia da Polimerase/métodos , Psychodidae/parasitologia , Sensibilidade e Especificidade , Venezuela/epidemiologiaRESUMO
Leishmania species of the subgenus Viannia account for 88% of all cases of leishmaniasis recorded in Colombia. Correct diagnosis is essential as infection with members of this subgenus can produce disfiguring destruction of the mucosa. Several methods are available to diagnose leishmaniasis in clinical samples. More recently, the polymerase chain reaction (PCR) has been used, with varying sensitivities and specificities depending on the primers used. In this paper we report on the sensitivity and specificity of PCR primers B1/B2 used on clinical samples and compare their use to the conventional parasitological methods. PCR alone is more sensitive than any single conventional method used, but a combination of conventional methods produced comparable sensitivity. PCR is well suited for use in selected cases and as a test for mucosal leishmaniasis.
Assuntos
Leishmaniose Cutânea/diagnóstico , Reação em Cadeia da Polimerase/métodos , Adolescente , Adulto , Idoso , Animais , Criança , Colômbia , DNA de Cinetoplasto/genética , Humanos , Leishmania/genética , Pessoa de Meia-Idade , Parasitologia/métodos , Sensibilidade e EspecificidadeRESUMO
This paper reports the isolation and characterization of a complementary deoxyribonucleic acid clone showing sequence homology with genes coding for the eukaryotic elongation factor 1 gamma (EF-1 gamma). The clone encodes an open reading frame of 404 amino acids corresponding to a deduced molecular mass of 46.2 kDa. Database searches revealed 30-64% sequence identity between the Leishmania infantum EF-1 gamma and several eukaryotic homologues. Southern blot analysis indicated that 2 genes tandemly organized were present in the L. infantum genome. The 3' untranslated regions of these 2 genes differed in size. Southern hybridization and pulsed field gel electrophoresis showed that EF-1 gamma genes are highly conserved among members of the Leishmania genus and must be clustered in a single chromosomal locus.