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In dogs, canine parvovirus (CPV) enteritis is associated with high morbidity and fatality rates requiring early diagnosis to facilitate treatment and reduce its spread. In recent times, various commercial immunochromatographic (IC) test kits are available for its rapid diagnosis, which require an assessment of their accuracy. Therefore, precision of a point-of-care IC combination test kit for canine coronavirus (CCoV)/CPV faecal antigen detection was evaluated in this study. Multicentred random faecal samples from 115 dogs with gastroenteritis were checked for the presence of CPV antigens using the SensPERT IC combination test kit and the result was compared with polymerase chain reaction (PCR) as a reference test. Parvovirus was detected in 105 (91.3%) and 108 (93.9%) faecal samples by the point-of-care test kit and PCR, respectively. The point-of-care IC test kit showed 95.4% relative sensitivity, 71.4% specificity, 98.1% positive predictive value, 50.0% negative predictive value, and 93.9% accuracy comparable to conventional PCR in the samples tested. This point-of-care test kit also demonstrated a fair positive likelihood ratio (3.34), a very low negative likelihood ratio (0.07) and a moderate agreement (Kappaâ¯=â¯0.6) compared with conventional PCR. This test kit has shown to be very useful in the screening of dogs for CPV infection, and is a reliable alternative for diagnosing CPV both in conventional laboratories and remote areas without laboratories. Negative results in the IC testing with high suspicion of CPV infection should be further confirmed using superior test such as PCR.
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Enfermedades de los Perros , Infecciones por Parvoviridae , Animales , Enfermedades de los Perros/diagnóstico , Perros , Infecciones por Parvoviridae/diagnóstico , Infecciones por Parvoviridae/veterinaria , Pruebas en el Punto de Atención , Reacción en Cadena de la Polimerasa/veterinariaRESUMEN
This report describes the clinical presentation, pathology and molecular diagnosis of canine parvovirus infection in male Boerboel and female Alsatian puppies. The history of the dogs was considered, examined clinically for vital parameters, haemogram changes and faeces screened for parasites and canine parvovirus faecal antigen. Tissue samples were taken at necropsy for confirmatory diagnosis using histopathology, immunohistochemistry, polymerase chain reaction (PCR) and sequence analysis. There was a severe regenerative anaemia, leucopenia and lymphopaenia. The positive antigen faecal test and pathological findings of haemorrhagic enteritis suggested canine parvoviral enteritis disease. Polymerase chain reaction and sequence analysis confirmed canine parvovirus-2a as the aetiology of the disease. Informed management is important to avoid complications resulting from secondary to severe dehydration, hypovolemia from marked gastrointestinal fluid and protein loss and sepsis from bacterial translocation and leukopenia.
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Background Pueraria tuberosa (Willd) D.C. (Fabaceae) tubers are already used in traditional medicine by Ayurvedic physicians for the management of fertility disorders, general weakness, and also as anti-ageing therapies. Other known pharmacological properties include: anti-hyperglycemics, hepatoprotective, anti-hyperlipidemic, diuretic, nutritive, and anti-fertility agents in male rats. Methods The anti-proliferative effect of the aqueous tuberous root extract of Pueraria tuberosa on vascular smooth muscle cells (VSMCs) and Human Colorectal Adenocarcinoma Cell lines (HT-29) was investigated using the Cell Titer 96 MTT Proliferation Assay where the viable cells were seeded at a density of 5 × 104 (100 µL/well). For VSMC, log concentrations of the extract at 200 and 800 µg/mL were added and incubated for 24 and 48 h time points. Incubation of the extract in the presence of vascular endothelial growth factor (VEGF) and ET-1 was also conducted at different times. Concentrations of the extract (200, 400 and 700 µg/mL) were also added and incubated with the HT 29 cell lines for 24, 48 and 72 h time points. The effect of the tuber aqueous extract of the plant on nuclear factor-κB (NF-κB) expression after 2 h was also carried out using immunoblotting technique. Results The result showed that after 24 h, the effect of the extract in the presence of the mitogens and on the VSMC was more of proliferation. However, at 48 h, the 200 µg/mL dose, both alone and in the presence of VEGF caused 11.1% and 25.9% decreases respectively, in cell proliferation. In the HT 29 cytotoxic study the 200 µg/mL concentration caused the greatest cytotoxic effect at 77.1% cell inhibition followed by 400 µg/mL concentration at 71.4% after 72 h. The immunoblotting assay showed a down regulation of NF-κB expressions with 0.7 µg/mL concentration showing the greatest effect. NF-κB, a pro-inflammatory agent is increasingly recognized as a crucial player in many steps of cancer initiation and progression. Conclusions It could therefore be concluded that the aqueous root extract of Pueraria tuberosa possesses cytotoxic effect and could serve as a lead compound for anticancer and anti-inflammatory agents.
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In the present study, we investigated the occurrence of intersex condition, histopathological changes in the gonad and endocrine disruptor biomarker responses in Tilapia species (Tilaipia guineensis, Sarotherodon galileaus and Oreochromis niloticus) along the Ogun River, Nigeria. The study sites covered a length of 320 km and a total of 1074 tilapias were collected from three sampling sites (Abeokuta, Isheri and Ikorodu) with different degrees of anthropogenic contamination. Samples were also collected from an upstream putative control site (Igboho) along the Ogun River. Hepatic transcript levels for vitellogenin (Vtg), zona radiata (Zrp) and aromatase (cyp19a1) were analyzed using real-time PCR. Gross gonadal morphology revealed a 24% prevalence of intersex showing visible testis and ovary in phenotypic females (25.4%) or males (74.6%). The intersex condition paralleled histopathological changes (ovotestis or testis-ova) in the gonads of female and male fish, respectively. Plasma concentrations of luteinizing hormone (LH), follicle stimulating hormone (FSH), 11-ketotestosterone (11-KT) and estradiol-17ß (E2) were measured using enzyme immunoassay, showing that male fish from downstream of the control site had significantly higher plasma E2, LH, and FSH concentrations compared to females. Similarly, Vtg, Zrp and cyp19a1 mRNA was significantly higher in males, compared to females. Analysis of contaminants showed the presence of 15 PCB congeners, lindane and dieldrin, and 4-iso-nonylphenol (4-iso-NP) and 4-tert-octylphenol (4-tert-OP) in fish muscle and sediment samples from Ogun River. Principal component analysis (PCA) revealed site and sex relationships between measured biological responses to groups of environmental contaminants, showing that the endocrine disruptive responses in fish were associated with biota and sediment contaminant burden. In addition, strong positive correlations were observed between male fish and Zrp, cyp19a1, E2, LH, FSH, PCBs, 4-iso-NP and 4-tert-OP, suggesting possible feminization effects of these contaminants on the male. In female fish, PCBs, 4-iso-NP and 4-tert-OP showed positive relationships with 11-KT and gonadosomatic index (GSI), suggesting masculinization effects by these contaminants. Overall, our findings demonstrate a causal relationship between endocrine disruption and contaminants burden in Tilapias species from Ogun River.
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Disruptores Endocrinos/análisis , Tilapia/fisiología , Contaminantes Químicos del Agua/análisis , Animales , Cíclidos , Países en Desarrollo , Ecosistema , Estradiol/análisis , Femenino , Geografía , Gónadas/efectos de los fármacos , Hormonas/sangre , Hormona Luteinizante/sangre , Masculino , Nigeria , Ovario/efectos de los fármacos , Fenoles/análisis , Bifenilos Policlorados/análisis , Prevalencia , Análisis de Componente Principal , Ríos , Testículo/efectos de los fármacos , Testosterona/análogos & derivados , Vitelogeninas/sangreRESUMEN
BACKGROUND: One of the cardinal requirements for effective therapeutic management of tumors is the selective delivery of cancer drugs to the right site by ligand-decorated nanomedicines. Screening of 2 × 109 clone landscape phage library provides a reliable avenue for generating protein ligands specific for tumor cells. It was shown that selective phage proteins derived from landscape phage libraries against breast and prostate cancer cells are able to navigate drug or siRNA loaded liposomes to corresponding cancer cells with minimal toxicity to non-neoplastic cells. In an alternative platform, glioma cell-specific phage proteins were used for assembling in vivo cancer-specific phage-like particles, named 'phagemid infective particles' as targeted gene-delivery vehicles. METHODS: To extend the panel of anticancer cell phages, we have screened a 2 × 109 clone landscape phage library f8/8 to select phage clones specific for metastatic prostate cancer cell PC-3M. The phage clones were characterized for their selective interaction with PC-3M cells using phage capture assay, immunofluorescence microscopy and electron microscopy. A prostate cancer selective phage was converted to phage-like particles harboring emerald green fluorescent protein. RESULTS: Phage clone EPTHSWAT (designated by the sequence of inserted peptide) was found to be most selective for PC-3M cells and was observed to internalize PC-3M cells as revealed by immunofluorescence microscopy and electron microscopy. Conversion of this phage to phage-like particles harboring emerald green fluorescent protein and the expression of emerald green fluorescent protein in the phage-like particles treated PC-3M cells showed potential of adoption of this phage-like particle in prostate cancer therapeutic gene delivery. CONCLUSION: Successful employment of phage-like particles expressing emerald green fluorescent protein genes targeted to prostate cancer cells PC-3M confirms a prospect of their use for targeted delivery of therapeutic genes to cancer cells.
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Bacteriófagos/genética , Técnicas de Transferencia de Gen , Biblioteca de Péptidos , Virión/genética , Secuencia de Aminoácidos , Línea Celular Tumoral , Endocitosis , Expresión Génica , Genes Reporteros , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Humanos , Masculino , Microscopía Electrónica , Microscopía Fluorescente , Datos de Secuencia Molecular , Terapia Molecular Dirigida/métodos , Metástasis de la Neoplasia , Neoplasias de la Próstata/genética , Neoplasias de la Próstata/patología , Neoplasias de la Próstata/terapiaRESUMEN
Breast cancer is a leading cause of death among women in the USA. The efficacy of existing anticancer therapeutics can be improved by targeting them through conjugation with ligands binding to cellular receptors. Recently, we developed a novel drug targeting strategy based on the use of pre-selected cancer-specific 'fusion pVIII proteins' (fpVIII), as targeting ligands. To study the efficiency of this approach in animal models, we developed a panel of breast cancer cell-binding phages as a source of targeted fpVIIIs. Two landscape phage peptide libraries (8-mer f8/8 and 9-mer f8/9) were screened to isolate 132 phage variants that recognize breast carcinoma cells MCF-7 and ZR-75-1 and internalize into the cells. When tested for their interaction with the breast cancer cells in comparison with liver cancer cells HepG2, human mammary cells MCF-10A cells and serum, 16 of the phage probes selectively interacted with the breast cancer cells whereas 32 bound both breast and liver cancer cells. The most prominent cancer-specific phage DMPGTVLP, demonstrating sub-nanomolar Kd in interaction with target cells, was used for affinity chromatography of cellular membrane molecules to reveal its potential binding receptor. The isolated protein was identified by direct sequencing as cellular surface nucleolin. This conclusion was confirmed by inhibition of the phage-cell interaction with nucleolin antibodies. Other prominent phage binders VPTDTDYS, VEEGGYIAA, and DWRGDSMDS demonstrate consensus motifs common to previously identified cancer-specific peptides. Isolated phage proteins exhibit inherent binding specificity towards cancer cells, demonstrating the functional activity of the selected fused peptides. The selected phages, their peptide inserts and intact fusion proteins can serve as promising ligands for the development of targeted nanomedicines and their study in model mice with xenograft of human cells MCF-7 and ZR-75-1.
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Neoplasias de la Mama/metabolismo , Sistemas de Liberación de Medicamentos/métodos , Biblioteca de Péptidos , Péptidos/metabolismo , Proteínas Recombinantes de Fusión/metabolismo , Secuencia de Aminoácidos , Animales , Anticuerpos/metabolismo , Bacteriófagos/genética , Neoplasias de la Mama/tratamiento farmacológico , Proteínas de la Cápside/química , Proteínas de la Cápside/genética , Proteínas de la Cápside/metabolismo , Línea Celular Tumoral , Femenino , Células Hep G2 , Humanos , Ratones , Datos de Secuencia Molecular , Péptidos/química , Péptidos/genética , Fosfoproteínas/metabolismo , Ingeniería de Proteínas/métodos , Proteínas de Unión al ARN/metabolismo , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/genética , Ensayos Antitumor por Modelo de Xenoinjerto , NucleolinaRESUMEN
Efficacy of siRNAs as potential anticancer therapeutics can be increased by their targeted delivery into cancer cells via tumor-specific ligands. Phage display offers a unique approach to identify highly specific and selective ligands that can deliver nanocarriers to the site of disease. In this study, we proved a novel approach for intracellular delivery of siRNAs into breast cancer cells through their encapsulation into liposomes targeted to the tumor cells with preselected intact phage proteins. The targeted siRNA liposomes were obtained by a fusion of two parental liposomes containing spontaneously inserted siRNA and fusion phage proteins. The presence of pVIII coat protein fused to a MCF-7 cell-targeting peptide DMPGTVLP in the liposomes was confirmed by Western blotting. The novel phage-targeted siRNA-nanopharmaceuticals demonstrate significant down-regulation of PRDM14 gene expression and PRDM14 protein synthesis in the target MCF-7 cells. This approach offers the potential for development of new anticancer siRNA-based targeted nanomedicines. FROM THE CLINICAL EDITOR: In this study, the authors report a novel approach for targeted intracellular delivery of siRNAs into breast cancer cells through encapsulation into liposomes targeted to the tumor cells with preselected intact phage proteins.
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Bacteriófagos/metabolismo , Neoplasias de la Mama/metabolismo , Técnicas de Transferencia de Gen , Liposomas/química , Oligopéptidos/metabolismo , ARN Interferente Pequeño/metabolismo , Proteínas Virales de Fusión/metabolismo , Neoplasias de la Mama/virología , Línea Celular Tumoral , Proteínas de Unión al ADN , Femenino , Silenciador del Gen , Humanos , Especificidad de Órganos , Tamaño de la Partícula , Transporte de Proteínas , Proteínas de Unión al ARN , Proteínas Represoras/metabolismo , Electricidad Estática , Factores de Transcripción , Transcripción GenéticaRESUMEN
AIM: To explore cancer cell-specific phage fusion pVIII coat protein, identified using phage display, for targeted delivery of drug-loaded liposomes to MCF-7 breast cancer cells. MATERIAL & METHODS: An 8-mer landscape library f8/8 and a biopanning protocol against MCF-7 cells were used to select a landscape phage protein bearing MCF-7-specific peptide. Size and morphology of doxorubicin-loaded liposomes modified with the tumor-specific phage fusion coat protein (phage-Doxil) were determined by dynamic light scattering and freeze-fraction electron microscopy. Topology of the phage protein in liposomes was examined by western blot. Association of phage-Doxil with MCF-7 cells was evaluated by fluorescence microscopy and fluorescence spectrometry. Selective targeting to MCF-7 was shown by FACS using a coculture model with target and nontarget cells. Phage-Doxil-induced tumor cell killing and apoptosis were confirmed by CellTiter-Blue Assay and caspase-3/CPP32 fluorometric assay. RESULTS: A chimeric phage fusion coat protein specific towards MCF-7 cells, identified from a phage landscape library, was directly incorporated into the liposomal bilayer of doxorubicin-loaded PEGylated liposomes (Doxil) without additional conjugation with lipophilic moieties. Western blotting confirmed the presence of both targeting peptide and pVIII coat protein in the phage-Doxil, which maintained the liposomal morphology and retained a substantial part of the incorporated drug after phage protein incorporation. The binding activity of the phage fusion pVIII coat protein was retained after incorporation into liposomes, and phage-Doxil strongly and specifically targeted MCF-7 cells, demonstrating significantly increased cytotoxicity towards target cells in vitro. CONCLUSION: We present a novel and straightforward method for making tumor-targeted nanomedicines by anchoring specific phage proteins (substitute antibodies) on their surface.