RESUMO
To achieve a virological cure for hepatitis B virus (HBV), innovative strategies are required to target the covalently closed circular DNA (cccDNA) genome. Guanine-quadruplexes (G4s) are a secondary structure that can be adopted by DNA and play a significant role in regulating viral replication, transcription, and translation. Antibody-based probes and small molecules have been developed to study the role of G4s in the context of the human genome, but none have been specifically made to target G4s in viral infection. Herein, we describe the development of a humanized single-domain antibody (S10) that can target a G4 located in the PreCore (PreC) promoter of the HBV cccDNA genome. MicroScale Thermophoresis demonstrated that S10 has a strong nanomolar affinity to the PreC G4 in its quadruplex form and a structural electron density envelope of the complex was determined using Small-Angle X-ray Scattering. Lentiviral transduction of S10 into HepG2-NTCP cells shows nuclear localization, and chromatin immunoprecipitation coupled with next-generation sequencing demonstrated that S10 can bind to the HBV PreC G4 present on the cccDNA. This research validates the existence of a G4 in HBV cccDNA and demonstrates that this DNA secondary structure can be targeted with high structural and sequence specificity using S10.
Assuntos
DNA Circular , DNA Viral , Quadruplex G , Vírus da Hepatite B , Anticorpos de Domínio Único , Humanos , Vírus da Hepatite B/genética , Vírus da Hepatite B/imunologia , DNA Circular/genética , DNA Viral/genética , Células Hep G2 , Anticorpos de Domínio Único/genética , Anticorpos de Domínio Único/imunologia , Anticorpos de Domínio Único/química , Genoma Viral , Regiões Promotoras Genéticas , Replicação Viral , Hepatite B/virologiaRESUMO
Background:There are numerous animal models available to study bone healing as well as test strategies to accelerate bone formation. Sheep are commonly used for evaluation of long bone fractures due to similar dimensions and weight bearing environments compared to patients. Large critical-size defects can be created in sheep to facilitate the study of implantable materials, osteogenic proteins, and stem cell treatments. Studies have been published using plates to stabilize large critical size defects in femoral, tibial, and metatarsal defects. External fixators have also been used to stabilize tibial defects in sheep.Methods: The purpose of the current paper is to detail the surgical technique for creation of a 42 mm mid-diaphyseal femoral defect stabilization with an intramedullary device in sheep. Additional surgical details are provided for dynamization, reverse dynamization, and device removal.Conclusion: The article provides multiple technical tips applicable to this and other ovine osteotomy models and concludes with a discussion comparing the use of each stabilization technique in clinically significant large critical-size bone defects.
Assuntos
Consolidação da Fratura , Fraturas da Tíbia , Animais , Placas Ósseas , Fixadores Externos , Fêmur/cirurgia , Humanos , Ovinos , Tíbia/cirurgia , Fraturas da Tíbia/cirurgiaRESUMO
Se analizó la morbimortalidad materna y neonatal temprana de 343 embarazos con presentación pélvica y se establecieron las variables asociadas con el valor de Apgar a los cinco minutos del nacimiento. La morbilidad materno-neonatal en los casos atendidos por cesárea es baja, independientemente de la modalidad de presentación pélvica. La mortalidad fue ausente. Las variables asociadas significativamente con el valor de Apgar a los cinco minutos de nacimiento fueron la edad gestacional y el peso al nacimiento. Esta asociación se demostró en la totalidad de pacientes y cuando fueron analizadas por separado de acuerdo a la vía de interrupción del embarazo. El resultado perinatal de los atendidos por vía vaginal con presentación pélvica completa e incompleta franca de nalgas no mostraron diferencias con los atendidos por cesárea.