Fibulin-4A in zebrafish development

Fibulin-4 is an extracellular matrix protein required for the formation of the elastic fibers. Human mutations in the fibulin-4 gene cause autosomal recessive cutis laxa with widespread systemic involvement and congenital presentation. To study the developmental functions of fibulin-4, I studied one of the two fibulin-4 genes, fbln4a. The mRNA for fbln4a RNA was expressed in the adaxial cells around the notochord during somite formation, and in the myosepta, head, and heart at later stages. Fbln4a protein was localized to the notochord sheath starting from somite formation suggesting differential expression of fbln4a RNA and protein in the midline structures during development. To inactivate fbln4a in vivo, we used a retroviral mutant and an antisense morpholino oligonucleotide (MO) for transient knockdown. Embryos homozygous for the fbln4a mutation showed a complete loss of the Fbln4a protein, but presented no obvious gross abnormalities. However, fbln4a knockdown yielded cardiovascular and musculoskeletal defects at 2 days post fertilization including pooling of blood at the caudal vein plexus, vascular hemorrhage in the head, reduced circulation and heart rate, bent notochord, rounded somites and reduced embryo length. All of these features were rescued by co-injection of fbln4a mRNA with the MO. Early cardiac and vascular progenitor markers showed an expansion of the heart field and reduction of the vascular fields in knockdown embryos. Homozygous mutants were resistant, whereas heterozygotes were sensitized to the effect of fbln4a MO, protective compensatory mechanisms as a possible reason for phenotypic discrepancy between fbln4a mutant and knockdown animals. Inhibition of transforming growth factor beta (Tgfb) signaling with a small molecule rescued both the cardiovascular and connective tissue anomalies in knockdown embryos. By varying the period of treatment, I identified late gastrulation and early segmentation as the critical periods. I conclude that Fbln4a inhibits Tgfb signals emanating from the notochord and regulates cardiac and vascular progenitor pools. The public health significance of this work is the identification of Tgfb inhibition as a candidate approach for treating of fibulin-4-related cutis laxa, an orphan disease for which no treatment has been available to date

Elastic Stable Intramedullary Nailing for Treatment of Pediatric Tibial Fractures

Introduction: Tibia fractures in the skeletally immature patient can usually be treated with above knee cast or patellar tendon bearing cast. The purpose of our study was to evaluate epidemiology and outcome of Elastic stable intramedullary nailing fixation of pediatric tibial shaft fractures treated at our institution. Methods: Over a period of one year, fifty pediatric patients of tibial shaft fractures, with average age of 9.68 yr (SD=2.37), were treated with elastic stable intramedullary nail. Demographic data, union and complication rate were evaluated. Results: There were 36 closed and 14 open fractures. The average time to union was 11.6 weeks  (SD=2.65) for close and  14.3 weeks (SD=2.62) for open fracture. There were no instances of growth arrest, remanipulations, or refracture. Conclusion: We conclude that flexible intramedullary fixation is an easy and effective method of management of both open and closed unstable fractures of the tibia in children

Pharmacokinetics and safety of co-administered paritaprevir plus ritonavir, ombitasvir, and dasabuvir in hepatic impairment

Background & Aims: Paritaprevir, ombitasvir, and dasabuvir are direct-acting antivirals for treatment of chronic hepatitis C virus (HCV) infection. The aim of this study was to characterize the effects of mild, moderate, and severe hepatic impairment on the pharmacokinetics of these drugs. Methods: HCV-negative subjects with normal hepatic function (n = 7) or mild (Child-Pugh A, n = 6), moderate (Child-Pugh B, n = 6), or severe (Child-Pugh C, n = 5) hepatic impairment received a single-dose of the combination of paritaprevir plus ritonavir (paritaprevir/r, 200/100 mg), ombitasvir (25 mg), and dasabuvir (400 mg). Plasma samples were collected through 144 hours after administration for pharmacokinetic assessments. Results: Paritaprevir, ombitasvir, dasabuvir, and ritonavir exposures (maximal plasma concentration, C max , and area under the concentration-time curve, AUC) were minimally affected in subjects with mild or moderate hepatic impairment. Differences in exposures between healthy controls and subjects with mild or moderate hepatic impairment were less than 35%, except for 62% higher paritaprevir AUC in subjects with moderate hepatic impairment. Paritaprevir and dasabuvir AUC were significantly higher in subjects with severe hepatic impairment (950% and 325%, respectively). However, ombitasvir AUC was 54% lower and ritonavir AUC was comparable. Adverse events included eye stye, insomnia, and pain from an infiltrated intravenous line. Conclusions: The changes observed in paritaprevir, ritonavir, ombitasvir, and dasabuvir exposures in subjects with mild or moderate hepatic impairment do not necessitate dose adjustment. Subjects with severe hepatic impairment had substantially higher paritaprevir and dasabuvir exposures.

Characterization of New Species of Begomoviruses and Their Satellites, Infecting Eclipta prostrata and Rosa chinensis

Satellites associated with begomoviruses are causing huge crop losses throughout the World, particularly in South Asia. Although much is known about the diversity of begomoviruses and their satellites infecting major crops such as cotton, tomato and chilies, other hosts such as weeds and ornamental plants are not well studied. During 2006-2007, ornamental rose plants (Rosa chinensis) were found with highly stunted growth and upward leaf curling in Faisalabad, Pakistan. Another commonly growing weed (Eclipta prostrata) showing vein yellowing was also analyzed for begomovirus infection. By using the rolling circle amplification method (which does not require prior sequence information for amplifying circular single stranded DNA molecules) followed by complete nucleotide sequencing, we discovered two new begomoviruses and their satellites infecting these hosts. In line with taxonomic conventions for begomovirus species, new names Rose leaf curl virus (RoLCuV) and Eclipta yellow vein virus (EcYVV) are proposed. Phylogenetic analysis based on their complete nucleotide sequences suggested that both viruses could be clustered with already identified monopartite begomoviruses from the region. EcYVV is closely related to Cotton leaf curl Bangalore virus (84%) and RoLCuV shows close identity (83%) with Tomato leaf curl Pakistan virus. One betasatellite isolated from these hosts showed less than 78% identity with other known betasatellites and can therefore be considered as a member of a new species. The second betasatellite isolated with EcYVV was found to be an isolate of the species Kenaf leaf curl betasatellite. The infectious molecules for each of the virus/satellite were prepared and inoculated through Agrobacterium to model host Nicotiana benthamiana plants, which induced severe vein yellowing and leaf curling. We conducted further experiments to understand the flexibility of betasatellite trans-replication in the presence of non-cognate begomoviruses. The betasatellites identified in this study are promiscuous in their ability to be trans-replicated by a non-cognate, already known bipartite virus; Tomato leaf curl New Delhi virus. The potential threat and replication flexibility of replication of the different molecules in the begomovirus disease complexes studied here is discussed