RESUMEN
Collagen scaffolds have been widely employed as a dermal equivalent to induce fibroblast infiltrations and dermal regeneration in the treatment of chronic wounds and diabetic foot ulcers. Cross-linking methods have been developed to address the disadvantages of the rapid degradation associated with collagen-based scaffolds. To eliminate the potential drawbacks associated with glutaraldehyde cross-linking, methods using a water soluble carbodiimide have been developed. In the present study, the glycosaminoglycan (GAG) hyaluronic acid (HA), was covalently attached to an equine tendon derived collagen scaffold using 1-ethyl-3-(3-dimethyl aminopropyl) carbodiimide (EDC) to create ntSPONGE The HA was shown to be homogeneously distributed throughout the collagen matrix. In vitro analyses of the scaffold indicated that the cross-linking enhanced the biological stability by decreasing the enzymatic degradation and increasing the thermal denaturation temperature. The material was shown to support the attachment and proliferation of mouse L929 fibroblast cells. In addition, the cross-linking decreased the resorption rate of the collagen as measured in an intramuscular implant model in rabbits. The material was also shown to be biocompatible in a variety of in vitro and in vivo assays. These results indicate that this cross-linked collagen-HA scaffold, ntSPONGE has the potential for use in chronic wound healing.
Asunto(s)
Vendas Hidrocoloidales , Colágeno/química , Colágeno/toxicidad , Fibroblastos/efectos de los fármacos , Fibroblastos/fisiología , Ácido Hialurónico/efectos adversos , Ácido Hialurónico/química , Animales , Materiales Biocompatibles/efectos adversos , Materiales Biocompatibles/síntesis química , Línea Celular , Supervivencia Celular/efectos de los fármacos , Reactivos de Enlaces Cruzados/efectos adversos , Reactivos de Enlaces Cruzados/química , Fibroblastos/citología , Ensayo de Materiales , Ratones , Estrés Mecánico , Resistencia a la TracciónRESUMEN
Smallpox is considered a biological threat based upon the possibility of deliberate reintroduction into the population, creating an urgent need for effective antivirals. The antiviral drug cidofovir (Cr) has shown to be effective against poxviruses, although route-specific nephrotoxicity has hampered its development for emergency post-exposure prophylaxis (PEP). In this study, we use a micronized dry powder formulation of pharmaceutical-grade Cr (NanoFOVIRTM; Nf) to treat rabbits exposed to aerosolized rabbitpox virus (RPXV) to further evaluate the effectiveness of direct drug delivery to the lung. Naïve rabbits were infected with RPXV by aerosol; three subsets received aerosolized Nf at 0.5, 1.0 or 1.75mg/kg daily for 3days post-exposure, positive and negative control groups received intravenous (IV) Cr treatments and no treatment, respectively. Nf groups showed an antiviral-dose associated survival of 50% (0.5mg/kg), 80% (1.0mg/kg) and 100% (1.75mg/kg). All animals (100%) from the IV-Cr treatment group and none (0%) from the untreated controls survived. Nf (1.75) protected rabbits from RPX at approximately 10% of the equivalent IV-Cr dose. A dose-related effect was observed in clinical development of RPX disease in Nf groups. Significant reduction of RPX-induced pathological changes was observed in Nf (1.75) and IV-Cr groups. Results suggest that Nf may be a viable antiviral for emergency post-exposure prophylaxis and should be evaluated in other models of poxviral disease.
Asunto(s)
Antivirales/administración & dosificación , Citosina/análogos & derivados , Organofosfonatos/administración & dosificación , Profilaxis Posexposición , Virus Vaccinia , Vaccinia/prevención & control , Administración por Inhalación , Animales , Línea Celular , Cidofovir , Citosina/administración & dosificación , Modelos Animales de Enfermedad , Femenino , Pulmón/patología , Pulmón/virología , Masculino , Conejos , Vaccinia/mortalidad , Vaccinia/virologíaRESUMEN
Citrus canker disease is caused by five groups of Xanthomonas citri strains that are distinguished primarily by host range: three from Asia (A, A*, and A(w)) and two that form a phylogenetically distinct clade and originated in South America (B and C). Every X. citri strain carries multiple DNA fragments that hybridize with pthA, which is essential for the pathogenicity of wide-host-range X. citri group A strain 3213. DNA fragments that hybridized with pthA were cloned from a representative strain from all five groups. Each strain carried one and only one pthA homolog that functionally complemented a knockout mutation of pthA in 3213. Every complementing homolog was of identical size to pthA and carried 17.5 nearly identical, direct tandem repeats, including three new genes from narrow-host-range groups C (pthC), A(w) (pthAW), and A* (pthA*). Every noncomplementing paralog was of a different size; one of these was sequenced from group A* (pthA*-2) and was found to have an intact promoter and full-length reading frame but with 15.5 repeats. None of the complementing homologs nor any of the noncomplementing paralogs conferred avirulence to 3213 on grapefruit or suppressed avirulence of a group A* strain on grapefruit. A knockout mutation of pthC in a group C strain resulted in loss of pathogenicity on lime, but the strain was unaffected in ability to elicit an HR on grapefruit. This pthC- mutant was fully complemented by pthA, pthB, or pthC. Analysis of the predicted amino-acid sequences of all functional pthA homologs and nonfunctional paralogs indicated that the specific sequence of the 17th repeat may be essential for pathogenicity of X. citri on citrus.
Asunto(s)
Proteínas Bacterianas/fisiología , Citrus/microbiología , Xanthomonas/patogenicidad , Secuencia de Aminoácidos , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Datos de Secuencia Molecular , Hojas de la Planta/microbiología , Alineación de Secuencia , Análisis de Secuencia de Proteína , Homología de Secuencia de Aminoácido , Especificidad de la Especie , Secuencias Repetidas en Tándem , Virulencia , Xanthomonas/genética , Xanthomonas/fisiologíaRESUMEN
Xylella fastidiosa infects a wide range of hosts and causes serious diseases on some of them. The complete genomic sequences of both a citrus variegated chlorosis (CVC) and a Pierce's disease (PD) strain revealed two type I protein secretion plus two multidrug resistance efflux systems, and all evidently were dependent on a single tolC homolog. Marker exchange mutagenesis of the single tolC gene in PD strain Temecula resulted in a total loss of pathogenicity on grape. Importantly, the tolC- mutant strains were not recovered after inoculation into grape xylem, strongly indicating that multidrug efflux is critical to survival of this fastidious pathogen. Both survival and pathogenicity were restored by complementation using tolC cloned in shuttle vector pBBR1MCS-5, which was shown to replicate autonomously, without selection, for 60 days in Temecula growing in planta. These results also demonstrate the ability to complement mutations in X. fastidiosa.
Asunto(s)
Proteínas Bacterianas/fisiología , Enfermedades de las Plantas/microbiología , Vitis/microbiología , Xylella/patogenicidad , Proteínas Bacterianas/genética , Prueba de Complementación Genética , Proteínas de Transporte de Membrana/genética , Proteínas de Transporte de Membrana/fisiología , Viabilidad Microbiana/efectos de los fármacos , Compuestos de Organosilicio/farmacología , Virulencia , Vitis/química , Xylella/genética , Xylella/crecimiento & desarrollo , Xilema/microbiologíaRESUMEN
Suppression subtractive hybridization (SSH) was used to identify genes present in the systemic crucifer black rot pathogen Xanthomonas campestris pv. campestris 528T but missing from the nonsystemic crucifer leaf spot pathogen, X. campestris pv. armoraciae 417. Among the DNA fragments unique to 528T was Xcc2109, one of eight putative avr genes identified in the published 528T genome (NC_003902). Individual and sequential deletion, insertion mutations, or both of all eight 528T avr gene loci were made, but no change in pathogenicity was observed with any combination of avr mutations, including a strain with all eight avr genes deleted. However, insertion or deletion mutants affecting the Xcc2109 locus lost avirulence (i.e., became virulent) on Florida Mustard, an X. campestris pv. campestris race-determining, differential host. The Xcc2109 open reading frame as annotated was cloned and found to be nonfunctional. A longer gene, encompassing Xcc2109 and here designated avrXccFM, was cloned and found to complement the Xcc2109 mutants and to confer avirulence to two additional wild-type X. campestris pv. campestris strains, thereby changing their races. Resistance in Florida Mustard to 528T strains carrying avrXccFM occurred without a typical hypersensitive response (HR) on leaves, although a vascular HR was observed in seedlings.