RESUMO
BACKGROUND: Intestinal schistosomiasis is of public health importance in Uganda but communities living above 1400 m are not targeted for control as natural transmission is thought unlikely. To assess altitudinal boundaries and at-risk populations, conjoint malacological and epidemiological surveys were undertaken on Mount Elgon (1139 m-3937 m), in Fort Portal crater lakes and in the Rwenzori Mountains (1123 m-4050 m). METHODS: Seventy freshwater habitats [Mount Elgon (37), Fort Portal crater lakes (23), Rwenzori Mountains (8) and Lake Albert (2)] were inspected for Biomphalaria species. Water temperature, pH and conductivity were recorded. A parasitological examination of 756 schoolchildren [Mount Elgon (300), Fort Portal crater lakes (456)] by faecal microscopy of duplicate Kato-Katz smears from two consecutive stool samples was bolstered by antigen (urine-CCA dipstick) and antibody (SEA-ELISA) diagnostic assays. RESULTS: Biomphalaria spp. was found up to 1951 m on Mount Elgon and 1567 m in the Fort Portal crater lakes. Although no snail from Mount Elgon shed cercariae, molecular analysis judged 7.1% of snails sampled at altitudes above 1400 m as having DNA of Schistosoma mansoni; in Fort Portal crater lakes three snails shed schistosome cercariae. Prevalence of intestinal schistosomiasis as measured in schoolchildren by Kato-Katz (Mount Elgon = 5.3% v. Fort Portal crater lakes = 10.7%), CCA urine-dipsticks (18.3% v. 34.4%) and SEA-ELISA (42.3% v. 63.7%) showed negative associations with increasing altitude with some evidence of infection up to 2000 m. CONCLUSIONS: Contrary to expectations, these surveys clearly show that natural transmission of intestinal schistosomiasis occurs above 1400 m, possibly extending up to 2000 m. Using spatial epidemiological predictions, this now places some extra six million people at-risk, denoting an expansion of preventive chemotherapy needs in Uganda.
Assuntos
Esquistossomose mansoni/epidemiologia , Esquistossomose mansoni/parasitologia , Esquistossomose mansoni/transmissão , Adolescente , Altitude , Animais , Anti-Helmínticos , Criança , Sistemas de Informação Geográfica , Humanos , Lagos/parasitologia , Praziquantel , Prevalência , Medicina Preventiva , Fatores de Risco , Schistosoma mansoni/genética , Schistosoma mansoni/isolamento & purificação , Caramujos/parasitologia , Uganda/epidemiologiaRESUMO
Metallothioneins are proteins that are involved in intracellular zinc storage and transport. Their expression levels have been reported to be elevated in several settings of skeletal muscle atrophy. We therefore investigated the effect of metallothionein blockade on skeletal muscle anabolism in vitro and in vivo We found that concomitant abrogation of metallothioneins 1 and 2 results in activation of the Akt pathway and increases in myotube size, in type IIb fiber hypertrophy, and ultimately in muscle strength. Importantly, the beneficial effects of metallothionein blockade on muscle mass and function was also observed in the setting of glucocorticoid addition, which is a strong atrophy-inducing stimulus. Given the blockade of atrophy and the preservation of strength in atrophy-inducing settings, these results suggest that blockade of metallothioneins 1 and 2 constitutes a promising approach for the treatment of conditions which result in muscle atrophy.
Assuntos
Metalotioneína/metabolismo , Força Muscular/fisiologia , Músculo Esquelético/metabolismo , Animais , Biomarcadores/metabolismo , Peso Corporal , Tamanho Celular , Inativação Gênica , Glucocorticoides/efeitos adversos , Humanos , Hipertrofia , Camundongos , Desenvolvimento Muscular , Fibras Musculares Esqueléticas/metabolismo , Fibras Musculares Esqueléticas/patologia , Músculo Esquelético/patologia , Músculo Esquelético/fisiopatologia , Atrofia Muscular , Tamanho do Órgão , Proteínas Proto-Oncogênicas c-akt/metabolismo , Ratos , Sarcopenia/metabolismo , Sarcopenia/patologia , Sarcopenia/fisiopatologia , Transdução de Sinais , Serina-Treonina Quinases TOR/metabolismo , Regulação para Cima , Zinco/metabolismoRESUMO
BACKGROUND: Hearing loss is the most common sensory defect afflicting several hundred million people worldwide. In most cases, regardless of the original cause, hearing loss is related to the degeneration and death of hair cells and their associated spiral ganglion neurons. Despite this knowledge, relatively few studies have reported regeneration of the auditory system. Significant gaps remain in our understanding of the molecular mechanisms underpinning auditory function, including the factors required for sensory cell regeneration. Recently, the identification of transcriptional activators and repressors of hair cell fate has been augmented by the discovery of microRNAs (miRNAs) associated with hearing loss. As miRNAs are central players of differentiation and cell fate, identification of miRNAs and their gene targets may reveal new pathways for hair cell regeneration, thereby providing new avenues for the treatment of hearing loss. RESULTS: In order to identify new genetic elements enabling regeneration of inner ear sensory hair cells, next-generation miRNA sequencing (miRSeq) was used to identify the most prominent miRNAs expressed in the mouse embryonic inner ear cell line UB/OC-1 during differentiation towards a hair cell like phenotype. Based on these miRSeq results eight most differentially expressed miRNAs were selected for further characterization. In UB/OC-1, miR-210 silencing in vitro resulted in hair cell marker expression, whereas ectopic expression of miR-210 resulted in new hair cell formation in cochlear explants. Using a lineage tracing mouse model, transdifferentiation of supporting epithelial cells was identified as the likely mechanism for this new hair cell formation. Potential miR-210 targets were predicted in silico and validated experimentally using a miR-trap approach. CONCLUSION: MiRSeq followed by ex vivo validation revealed miR-210 as a novel factor driving transdifferentiation of supporting epithelial cells to sensory hair cells suggesting that miR-210 might be a potential new factor for hearing loss therapy. In addition, identification of inner ear pathways regulated by miR-210 identified potential new drug targets for the treatment of hearing loss.
Assuntos
Transdiferenciação Celular , Células Ciliadas Auditivas Internas/citologia , MicroRNAs/metabolismo , Órgão Espiral/citologia , Regeneração , Animais , Linhagem Celular , Técnicas de Introdução de Genes , Sequenciamento de Nucleotídeos em Larga Escala , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , MicroRNAs/genética , Técnicas de Cultura de Órgãos , Fatores de Transcrição SOXB1/genética , Análise de Sequência de RNARESUMO
TLQP-21, a peptide derived from VGF (non-acronymic) by proteolytic processing, has been shown to modulate energy metabolism, differentiation, and cellular response to stress. Although extensively investigated, the receptor for this endogenous peptide has not previously been described. This study describes the use of a series of studies that show G protein-coupled receptor-mediated biological activity of TLQP-21 signaling in CHO-K1 cells. Unbiased genome-wide sequencing of the transcriptome from responsive CHO-K1 cells identified a prioritized list of possible G protein-coupled receptors bringing about this activity. Further experiments using a series of defined receptor antagonists and siRNAs led to the identification of complement C3a receptor-1 (C3AR1) as a target for TLQP-21 in rodents. We have not been able to demonstrate so far that this finding is translatable to the human receptor. Our results are in line with a large number of physiological observations in rodent models of food intake and metabolic control, where TLQP-21 shows activity. In addition, the sensitivity of TLQP-21 signaling to pertussis toxin is consistent with the known signaling pathway of C3AR1. The binding of TLQP-21 to C3AR1 not only has effects on signaling but also modulates cellular functions, as TLQP-21 was shown to have a role in directing migration of mouse RAW264.7 cells.
Assuntos
Movimento Celular/efeitos dos fármacos , Fragmentos de Peptídeos/farmacologia , Receptores de Complemento/metabolismo , Transdução de Sinais/efeitos dos fármacos , Animais , Sequência de Bases , Células CHO , Cricetinae , Cricetulus , Estudo de Associação Genômica Ampla , Células HEK293 , Humanos , Camundongos , Dados de Sequência Molecular , Fatores de Crescimento Neural/farmacologia , Neuropeptídeos/farmacologia , Toxina Pertussis/farmacologia , Ratos , Receptores de Complemento/agonistas , Receptores de Complemento/genética , Especificidade da Espécie , Transcriptoma/efeitos dos fármacosRESUMO
BACKGROUND: To clarify the extent and putative transmission zone of bovine fasciolosis on the slopes of Mount Elgon, Uganda, conjoint parasitological and malacological surveys, inclusive of inspection of animals at slaughter, were undertaken at increasing altitudes. RESULTS: A total of 239 cattle were sampled across eight locations ranging in elevation from 1112-2072 m. Faecal material was examined for presence of Fasciola eggs and sera were tested by ELISA for antibodies against Fasciola antigens. Bolstering this, 38 cattle at slaughter from 2 abattoir sites at 1150 m and 1947 m were inspected; in addition, wild buffalo stool (n=10) opportunistically picked within Mount Elgon National Park (MENP) at 3640 m was examined. By faecal egg detection, prevalence of Fasciola gigantica at low (<1500 m) and high (>1500 m) altitude sites was 43.7% (95% CI 35.4-52.2) and 1.1% (95% CI 0.0-6.0), respectively, while by ELISA was much higher, low altitude--77.9% (95% CI 69.7-85.4) and high altitude--64.5% (95% CI 51.3-76.3). The decline in prevalence with increasing altitude was corroborated by abattoir sampling. Thirty seven aquatic habitats, ranging from 1139-3937 m in altitude were inspected for freshwater snails, 12 of which were within MENP. At lower altitudes, Lymnaea (Radix) natalensis was common, and often abundant, but at higher altitudes became much rarer ceasing to be found above 1800 m. On the other hand, Lymnaea (Galba) truncatula was found only at altitudes above 3000 m and within MENP alone. The snail identifications were confirmed by DNA analysis of the ribosomal 18S gene. CONCLUSIONS: Active infections of F. gigantica in cattle are common in lower altitude settings but appear to diminish with increasing elevation. This is likely due to a growing paucity of intermediate hosts, specifically populations of L. natalensis for which a natural boundary of 1800 m appeared. Although F. hepatica was not encountered, the presence of several populations of L. truncatula at elevations over 3000 m point towards a potential transmission zone within MENP should this parasite be introduced.
Assuntos
Altitude , Doenças dos Bovinos/epidemiologia , Doenças dos Bovinos/parasitologia , Fasciolíase/veterinária , Caramujos/fisiologia , Matadouros , Animais , Anticorpos Anti-Helmínticos/sangue , Bovinos , Primers do DNA/genética , Vetores de Doenças , Ensaio de Imunoadsorção Enzimática/veterinária , Fasciolíase/epidemiologia , Fezes/parasitologia , Fígado/parasitologia , Reação em Cadeia da Polimerase , RNA Ribossômico 18S/genética , Análise de Regressão , Caramujos/genética , Uganda/epidemiologiaRESUMO
Studies under nonphysiological conditions suggest that long receptor residency time is responsible for the 24-h duration of action of the long-acting muscarinic antagonist (LAMA) tiotropium. Our aim was to determine how clinically relevant dissociation rates under more physiological conditions influence the differences in onset of action between tiotropium and 3-[(cyclopentylhydroxyphenylacetyl oxy]-1,1-dimethyl-pyrrolidinium bromide (NVA237), a once-daily dry-powder formulation of the LAMA glycopyrronium bromide in development for chronic obstructive pulmonary disease. In addition, we have investigated kinetic selectivity at each of the muscarinic receptor subtypes to determine whether the improved cardiovascular therapeutic index obtained with NVA237 in animal models is attributable to differences in kinetic rate constants. The binding of radioligand [3H]N-methyl-scopolamine was measured in the presence/absence of several concentrations of unlabeled competitors, and data were analyzed using a competition kinetic model to provide on/off rates for the competitor. We found shorter dissociation half-lives for NVA237 and tiotropium under physiological (11.4 and 46.2 min, respectively) versus nonphysiological conditions (173 and 462 min, respectively). NVA237 had a more rapid onset of action (3-4.8 times) versus tiotropium, determined in an vitro calcium and rat tracheal strip assay. Simulations suggested that the more rapid onset of NVA237 action could be explained by differences in kinetic parameters. NVA237 had greater equilibrium binding and kinetic selectivity for muscarinic type 3 (M3) versus muscarinic type 2 (M2) receptors, with a faster off rate from M2 versus M3 receptors than tiotropium, potentially affording it a more favorable therapeutic index. This study suggests that the 24-h duration of action of NVA237 and tiotropium is not solely the result of their slow dissociation from the M3 receptor and highlights the importance of conducting in vitro experiments in conditions reflecting those in vivo.
Assuntos
Broncodilatadores/farmacologia , Broncodilatadores/uso terapêutico , Glicopirrolato/farmacologia , Antagonistas Muscarínicos/farmacologia , Antagonistas Muscarínicos/uso terapêutico , Receptor Muscarínico M3/antagonistas & inibidores , Derivados da Escopolamina/farmacologia , Derivados da Escopolamina/uso terapêutico , Animais , Broncodilatadores/farmacocinética , Células CHO , Cálcio/metabolismo , Simulação por Computador , Cricetinae , Cricetulus , Interpretação Estatística de Dados , Glicopirrolato/farmacocinética , Glicopirrolato/uso terapêutico , Técnicas In Vitro , Cinética , Ensaio Radioligante , Ratos , Receptor Muscarínico M2/antagonistas & inibidores , Derivados da Escopolamina/farmacocinética , Sódio/farmacologia , Temperatura , Brometo de Tiotrópio , Traqueia/efeitos dos fármacosRESUMO
The Wnt/ß-catenin signaling pathbway controls many important biological processes. R-Spondin (RSPO) proteins are a family of secreted molecules that strongly potentiate Wnt/ß-catenin signaling, however, the molecular mechanism of RSPO action is not yet fully understood. We performed an unbiased siRNA screen to identify molecules specifically required for RSPO, but not Wnt, induced ß-catenin signaling. From this screen, we identified LGR4, then an orphan G protein-coupled receptor (GPCR), as the cognate receptor of RSPO. Depletion of LGR4 completely abolished RSPO-induced ß-catenin signaling. The loss of LGR4 could be compensated by overexpression of LGR5, suggesting that LGR4 and LGR5 are functional homologs. We further demonstrated that RSPO binds to the extracellular domain of LGR4 and LGR5, and that overexpression of LGR4 strongly sensitizes cells to RSPO-activated ß-catenin signaling. Supporting the physiological significance of RSPO-LGR4 interaction, Lgr4-/- crypt cultures failed to grow in RSPO-containing intestinal crypt culture medium. No coupling between LGR4 and heterotrimeric G proteins could be detected in RSPO-treated cells, suggesting that LGR4 mediates RSPO signaling through a novel mechanism. Identification of LGR4 and its relative LGR5, an adult stem cell marker, as the receptors of RSPO will facilitate the further characterization of these receptor/ligand pairs in regenerative medicine applications.
Assuntos
Receptores Acoplados a Proteínas G/metabolismo , Trombospondinas/fisiologia , Proteínas Wnt/metabolismo , beta Catenina/metabolismo , DNA Complementar/metabolismo , Proteínas de Ligação ao GTP/metabolismo , Células HEK293 , Humanos , Mucosa Intestinal/metabolismo , Ligantes , Modelos Biológicos , Fases de Leitura Aberta , Estrutura Terciária de Proteína , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Transdução de Sinais , Células-Tronco/citologiaRESUMO
A library of chemokine antagonists has been synthesized using a combination of solid and solution-phase chemistry. Structures of known chemokine antagonists were used to produce a pharmacophore which served to guide monomer selection. Several combinations of monomers have resulted in providing novel chemokine antagonists which in some cases display dual chemokine receptor antagonism.
Assuntos
Quimiocinas/antagonistas & inibidores , Desenho de Fármacos , Bibliotecas de Moléculas Pequenas , Animais , Linhagem Celular , Cricetinae , CricetulusRESUMO
Host factor pathways are known to be essential for hepatitis C virus (HCV) infection and replication in human liver cells. To search for novel host factor proteins required for HCV replication, we screened a subgenomic genotype 1b replicon cell line (Luc-1b) with a kinome and druggable collection of 20,779 siRNAs. We identified and validated several enzymes required for HCV replication, including class III phosphatidylinositol 4-kinases (PI4KA and PI4KB), carbamoyl-phosphate synthetase 2, aspartate transcarbamylase, and dihydroorotase (CAD), and mevalonate (diphospho) decarboxylase. Knockdown of PI4KA could inhibit the replication and/or HCV RNA levels of the two subgenomic genotype 1b clones (SG-1b and Luc-1b), two subgenomic genotype 1a clones (SG-1a and Luc-1a), JFH-1 genotype 2a infectious virus (JFH1-2a), and the genomic genotype 1a (FL-1a) replicon. In contrast, PI4KB knockdown inhibited replication and/or HCV RNA levels of Luc-1b, SG-1b, and Luc-1a replicons. The small molecule inhibitor, PIK93, was found to block subgenomic genotype 1b (Luc-1b), subgenomic genotype 1a (Luc-1a), and genomic genotype 2a (JFH1-2a) infectious virus replication in the nanomolar range. PIK93 was characterized by using quantitative chemical proteomics and in vitro biochemical assays to demonstrate PIK93 is a bone fide PI4KA and PI4KB inhibitor. Our data demonstrate that genetic or pharmacological modulation of PI4KA and PI4KB inhibits multiple genotypes of HCV and represents a novel druggable class of therapeutic targets for HCV infection.
Assuntos
1-Fosfatidilinositol 4-Quinase/metabolismo , Hepacivirus/genética , Hepacivirus/metabolismo , Fígado/virologia , Replicação Viral , 1-Fosfatidilinositol 4-Quinase/química , Antivirais/farmacologia , Ligação Competitiva , Linhagem Celular , Inativação Gênica , Genótipo , Humanos , Concentração Inibidora 50 , Espectrometria de Massas/métodos , Proteômica/métodos , RNA Interferente Pequeno/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Tiazóis/farmacologiaRESUMO
The advent of miniaturized assay formats has made possible the screening of large numbers of compounds against a single target, known as high-throughput screening. Despite this clear advantage, assay miniaturization also increases the risk of ligand depletion, where the actual concentration of free ligand is significantly lower than that added. This, in turn, complicates the interpretation of data from such assays, potentially introducing significant error if not recognized. In this study, the effects of reducing assay volume on radioligand Kd and competitor Ki values have been investigated, using the muscarinic M(3) receptor as a model system. It was found that assay miniaturization caused dramatic effects, with up to a 30-fold underestimation of ligand affinity. A theoretical model was developed and shown to accurately predict both the degree of ligand depletion in any given assay volume and the effect of this depletion on affinity estimates for competing ligands. Importantly, it was found that in most cases, errors introduced by ligand depletion could be largely corrected for by the use of appropriate analysis methods. In addition to those previously described by others, the authors propose a simple method capable of correcting errors in competition binding experiments performed in conditions of ligand depletion.
Assuntos
Avaliação Pré-Clínica de Medicamentos/métodos , Receptor Muscarínico M3/análise , Animais , Ligação Competitiva , Células CHO , Simulação por Computador , Cricetinae , Cricetulus , Cinética , Ligantes , Miniaturização , Ensaio Radioligante , TransfecçãoRESUMO
Here, we describe the preclinical pharmacological profile of 5-[(R)-2-(5,6-diethyl-indan-2-ylamino)-1-hydroxy-ethyl]-8-hydroxy-1H-quinolin-2-one (indacaterol), a novel, chirally pure inhaled beta(2) adrenoceptor agonist, in comparison with marketed drugs. Indacaterol is close to a full agonist at the human beta(2) adrenoceptor (E(max) = 73 +/- 1% of the maximal effect of isoprenaline; pEC(50) = 8.06 +/- 0.02), whereas salmeterol displays only partial efficacy (38 +/- 1%). The functional selectivity profile of indacaterol over beta(1) human adrenoceptors is similar to that of formoterol, whereas its beta(3) adrenoceptor selectivity profile is similar to that of formoterol and salbutamol. In isolated superfused guinea pig trachea, indacaterol has a fast onset of action (30 +/- 4 min) similar to formoterol and salbutamol, and a long duration of action (529 +/- 99 min) comparable with salmeterol. In the conscious guinea pig, when given intratracheally as a dry powder, indacaterol inhibits 5-hydroxytryptamine-induced bronchoconstriction for at least 24 h, whereas salmeterol, formoterol, and salbutamol have durations of action of 12, 4, and 2 h, respectively. When given via nebulization to anesthetized rhesus monkeys, all of the compounds dose-dependently inhibit methacholine-induced bronchoconstriction, although indacaterol produces the most prolonged bronchoprotective effect and induces the lowest increase in heart rate for a similar degree of antibronchoconstrictor activity. In conclusion, the preclinical profile of indacaterol suggests that this compound has a superior duration of action compatible with once-daily dosing in human, together with a fast onset of action and an improved cardiovascular safety profile over marketed inhaled beta(2) adrenoceptor agonists.
Assuntos
Agonistas de Receptores Adrenérgicos beta 2 , Agonistas Adrenérgicos beta/farmacologia , Broncoconstrição/efeitos dos fármacos , Broncodilatadores/farmacologia , Indanos/farmacologia , Quinolonas/farmacologia , Administração por Inalação , Agonistas Adrenérgicos beta/administração & dosagem , Agonistas Adrenérgicos beta/efeitos adversos , Animais , Broncodilatadores/administração & dosagem , Broncodilatadores/efeitos adversos , Células Cultivadas , Clonagem Molecular , Cricetinae , Cricetulus , Relação Dose-Resposta a Droga , Avaliação Pré-Clínica de Medicamentos , Feminino , Cobaias , Frequência Cardíaca/efeitos dos fármacos , Humanos , Indanos/administração & dosagem , Indanos/efeitos adversos , Macaca mulatta , Masculino , Ovário/citologia , Quinolonas/administração & dosagem , Quinolonas/efeitos adversos , Taquifilaxia , Fatores de TempoRESUMO
The mechanism by which ATP primes for subsequent macrophage-derived chemokine (MDC) mediated intracellular calcium (Ca2+(i)) responses at the human CCR4 receptor stably expressed in Chinese hamster ovary (CHO) cells was investigated. MDC alone was unable to elicit a Ca2+(i) response, but pre-stimulation of cells with ATP enabled a subsequent MDC-mediated Ca2+(i) response with a pEC50 of 8.66+/-0.16. The maximal response elicited by MDC was dependent upon the concentration of ATP used to prime, but the pEC50 was stable at all ATP concentrations tested. Pertussis toxin pre-treatment did not effect the ATP response, but abolished that to MDC, demonstrating that priming with ATP did not alter G protein-coupling specificity of the CCR4 receptor. Ionomycin and thapsigargin both increased Ca2+(i) concentrations (pEC50s of 7.59+/-0.57 and 6.81+/-0.31 respectively), but were unable to prime for MDC responses, suggesting the priming mechanism was not dependent upon increases in Ca2+(i) concentrations. Priming of the MDC response was still observed when experiments were performed with low Ca2+(e) (70 microM), indicating that Ca2+ influx was not required for ATP to prime the CCR4 receptor. Neither Ro31-8220 nor wortmannin affected priming, suggesting that protein kinase C and phosphoinositol 3-kinase were not involved. In conclusion, pre-stimulation of endogenous P2Y receptors with ATP facilitates Ca2+ signalling at the recombinant CCR4 receptor in CHO cells, although the mechanism by which this occurs remains to be defined.