Genetic transformation of apple (Malus pumila Mill.) using a disarmed Ti-binary vector

The disamed Ti-binary vector pBIN 6 in Agrobacterium tumefaciens has been used in leaf disc transfomations to produce transgenic apple (Malus pumila Mill.) plants with a nomal phenotype except for a somewhat reduced capacity to root. The presence of the genes for nopaline synthase and neomycin phosphotrans ferase (conferring kanamycin resistance), inserted into the host genome by the vector, was confirmed by Southern blot analysis, the detection of nopaline synthase activity and rooting in the presence of the antibiotic.The nopaline synthase gene continued to be expressed in glasshouse-grown plants several months after removal from in vitro growth conditions.     

Human Mre11/human Rad50/Nbs1 and DNA ligase IIIalpha/XRCC1 protein complexes act together in an alternative nonhomologous end joining pathway

Recent studies have implicated a poorly defined alternative pathway of nonhomologous end joining (alt-NHEJ) in the generation of large deletions and chromosomal translocations that are frequently observed in cancer cells. Here, we describe an interaction between two factors, hMre11/hRad50/Nbs1 (MRN) and DNA ligase IIIα/XRCC1, that have been linked with alt-NHEJ. Expression of DNA ligase IIIα and the association between MRN and DNA ligase IIIα/XRCC1 are altered in cell lines defective in the major NHEJ pathway. Most notably, DNA damage induced the association of these factors in DNA ligase IV-deficient cells. MRN interacts with DNA ligase IIIα/XRCC1, stimulating intermolecular ligation, and together these proteins join incompatible DNA ends in a reaction that mimics alt-NHEJ. Thus, our results provide novel mechanistic insights into the alt-NHEJ pathway that not only contributes to genome instability in cancer cells but may also be a therapeutic target.     

Lymphatic ontogeny and effect of hypoplasia in developing lung

The pulmonary lymphatic vasculature plays a vital role in maintaining fluid homeostasis required for efficient gas exchange at capillary alveolar barriers and contributes to lung fluid clearance at birth. To further understanding of pulmonary lymphatic function at birth, lineage-tracing analysis of mouse lung was used. Lineage analysis confirmed that lymphatic endothelial cells (LEC) bud from extrapulmonary lymphatics and demonstrated that LEC migrate into developing lung along precise pathways. LEC cluster first in the primary bronchovascular region then along the secondary broncho-arterial regions and along veins. Small lymphatic vessels in distal lung develop from LEC that have migrated into lung mesenchyme from the extrapulmonary lymphatics. Finally, proximal and distal lymphatics remodel to form vessels with lumens in stereotypical locations. Loss of function analysis with lung-specific expression of a secreted form of the extracellular domain of vascular endothelial growth factor receptor-3 (dnR3) caused significant embryonic pulmonary lymphatic hypoplasia with fourfold reduction in distal LEC. Lung-specific expression of dnR3 did not affect blood vascular development, overall lung organogenesis or lymphatic development in other organs. Neonatal mice with pulmonary lymphatic hypoplasia developed respiratory distress with significantly increased mortality. During the transition to air breathing, lymphatic hypoplasia adversely affected fetal lung fluid clearance as determined by wet/dry weight analysis and morphometric analysis of bronchovascular cuffing and mesenchymal thickening. Surfactant synthesis was unaffected. Together, these data demonstrate that lung lymphatics develop autonomously and that pulmonary lymphatic hypoplasia is detrimental to survival of the neonate due to impaired lung fluid clearance.     

Tobacco smoke mediated induction of sinonasal microbial biofilms

Cigarette smokers and those exposed to second hand smoke are more susceptible to life threatening infection than non-smokers. While much is known about the devastating effect tobacco exposure has on the human body, less is known about the effect of tobacco smoke on the commensal and commonly found pathogenic bacteria of the human respiratory tract, or human respiratory tract microbiome. Chronic rhinosinusitis (CRS) is a common medical complaint, affecting 16% of the US population with an estimated aggregated cost of $6 billion annually. Epidemiologic studies demonstrate a correlation between tobacco smoke exposure and rhinosinusitis. Although a common cause of CRS has not been defined, bacterial presence within the nasal and paranasal sinuses is assumed to be contributory. Here we demonstrate that repetitive tobacco smoke exposure induces biofilm formation in a diverse set of bacteria isolated from the sinonasal cavities of patients with CRS. Additionally, bacteria isolated from patients with tobacco smoke exposure demonstrate robust in vitro biofilm formation when challenged with tobacco smoke compared to those isolated from smoke naïve patients. Lastly, bacteria from smoke exposed patients can revert to a non-biofilm phenotype when grown in the absence of tobacco smoke. These observations support the hypothesis that tobacco exposure induces sinonasal biofilm formation, thereby contributing to the conversion of a transient and medically treatable infection to a persistent and therapeutically recalcitrant condition.     

Protection of HIV neutralizing aptamers against rectal and vaginal nucleases: implications for RNA-based therapeutics

RNA-based drugs are an emerging class of therapeutics. They have the potential to regulate proteins, chromatin, as well as bind to specific proteins of interest in the form of aptamers. These aptamers are protected from nuclease attack by chemical modifications that enhance their stability for in vivo usage. However, nucleases are ubiquitous, and as we have yet to characterize the entire human microbiome it is likely that many nucleases are yet to be identified. Any novel, unusual enzymes present in vivo might reduce the efficacy of RNA-based therapeutics, even when they are chemically modified. We have previously identified an RNA-based aptamer capable of neutralizing a broad spectrum of clinical HIV-1 isolates and are developing it as a vaginal and rectal microbicide candidate. As a first step we addressed aptamer stability in the milieu of proteins present in these environments. Here we uncover a number of different nucleases that are able to rapidly degrade 2'-F-modified RNA. We demonstrate that the aptamer can be protected from the nuclease(s) present in the vaginal setting, without affecting its antiviral activity, by replacement of key positions with 2'-O-Me-modified nucleotides. Finally, we show that the aptamer can be protected from all nucleases present in both vaginal and rectal compartments using Zn(2+) cations. In conclusion we have derived a stable, antiviral RNA-based aptamer that could form the basis of a pre-exposure microbicide or be a valuable addition to the current tenofovir-based microbicide candidate undergoing clinical trials.     

Crucial role for prion protein membrane anchoring in the neuroinvasion and neural spread of prion infection

In nature prion diseases are usually transmitted by extracerebral prion infection, but clinical disease results only after invasion of the central nervous system (CNS). Prion protein (PrP), a host-encoded glycosylphosphatidylinositol (GPI)-anchored membrane glycoprotein, is necessary for prion infection and disease. Here, we investigated the role of the anchoring of PrP on prion neuroinvasion by studying various inoculation routes in mice expressing either anchored or anchorless PrP. In control mice with anchored PrP, intracerebral or sciatic nerve inoculation resulted in rapid CNS neuroinvasion and clinical disease (154 to 156 days), and after tongue, ocular, intravenous, or intraperitoneal inoculation, CNS neuroinvasion was only slightly slower (193 to 231 days). In contrast, in anchorless PrP mice, these routes resulted in slow and infrequent CNS neuroinvasion. Only intracerebral inoculation caused brain PrPres, a protease-resistant isoform of PrP, and disease in both types of mice. Thus, anchored PrP was an essential component for the rapid neural spread and CNS neuroinvasion of prion infection.     

Availability of a pediatric trauma center in a disaster surge decreases triage time of the pediatric surge population: a population kinetics model

Background

The concept of disaster surge has arisen in recent years to describe the phenomenon of severely increased demands on healthcare systems resulting from catastrophic mass casualty events (MCEs) such as natural disasters and terrorist attacks. The major challenge in dealing with a disaster surge is the efficient triage and utilization of the healthcare resources appropriate to the magnitude and character of the affected population in terms of its demographics and the types of injuries that have been sustained.

Results

In this paper a deterministic population kinetics model is used to predict the effect of the availability of a pediatric trauma center (PTC) upon the response to an arbitrary disaster surge as a function of the rates of pediatric patients' admission to adult and pediatric centers and the corresponding discharge rates of these centers. We find that adding a hypothetical pediatric trauma center to the response documented in an historical example (the Israeli Defense Forces field hospital that responded to the Haiti earthquake of 2010) would have allowed for a significant increase in the overall rate of admission of the pediatric surge cohort. This would have reduced the time to treatment in this example by approximately half. The time needed to completely treat all children affected by the disaster would have decreased by slightly more than a third, with the caveat that the PTC would have to have been approximately as fast as the adult center in discharging its patients. Lastly, if disaster death rates from other events reported in the literature are included in the model, availability of a PTC would result in a relative mortality risk reduction of 37%.

Conclusions

Our model provides a mathematical justification for aggressive inclusion of PTCs in planning for disasters by public health agencies.     

Gene Descent, Duplication, and Horizontal Transfer in the Evolution of Glutamyl- and Glutaminyl-tRNA Synthetases

In translation, separate aminoacyl-tRNA synthetases attach the 20 different amino acids to their cognate tRNAs, with the exception of glutamine. Eukaryotes and some bacteria employ a specific glutaminyl-tRNA synthetase (GlnRS) which other Bacteria, the Archaea (archaebacteria), and organelles apparently lack. Instead, tRNA^Gln is initially acylated with glutamate by glutamyl-tRNA synthetase (GluRS), then the glutamate moiety is transamidated to glutamine. Lamour et al. [(1994) Proc Natl Acad Sci USA 91:8670–8674] suggested that an early duplication of the GluRS gene in eukaryotes gave rise to the gene for GlnRS—a copy of which was subsequently transferred to proteobacteria. However, questions remain about the occurrence of GlnRS genes among the Eucarya (eukaryotes) outside of the ``crown' taxa (animals, fungi, and plants), the distribution of GlnRS genes in the Bacteria, and their evolutionary relationships to genes from the Archaea. Here, we show that GlnRS occurs in the most deeply branching eukaryotes and that putative GluRS genes from the Archaea are more closely related to GlnRS and GluRS genes of the Eucarya than to those of Bacteria. There is still no evidence for the existence of GlnRS in the Archaea. We propose that the last common ancestor to contemporary cells, or cenancestor, used transamidation to synthesize Gln-tRNA^Gln and that both the Bacteria and the Archaea retained this pathway, while eukaryotes developed a specific GlnRS gene through the duplication of an existing GluRS gene. In the Bacteria, GlnRS genes have been identified in a total of 10 species from three highly diverse taxonomic groups: Thermus/Deinococcus, Proteobacteria γ/β subdivision, and Bacteroides/Cytophaga/Flexibacter. Although all bacterial GlnRS form a monophyletic group, the broad phyletic distribution of this tRNA synthetase suggests that multiple gene transfers from eukaryotes to bacteria occurred shortly after the Archaea–eukaryote divergence.     

Cementum annulation and age determination in Homo sapiens. II. Estimates and accuracy

The cementum annulation aging technique was evaluated in a sample of 80 clinically extracted premolars (age range 11–70 years). Demineralized thin sections (7μm) stained with hematoxylin were used. The correlation (r) between age and adjusted count (number of annulations added to age of tooth eruption) was 0.78 for the entire sample (N = 73) and 0.86 for a subsample in which teeth with periodontal disease were excluded (N = 55). Standard error of the estimates ranged from 4.7 to 9.7 years depending on sex and health status of the tooth. The technique provided significantly better estimates for females than for males. The overall inaccuracy (mean absolute error) of the technique was 6.0 years, with a bias (mean error) of 0.26 years. Reduced major axis regression of adjusted count on age produced a slope of 0.797 for the entire sample and 0.889 for the nonperiodontal disease subsample. These slopes are consistent with a hypothesis of annual deposition of cementum rings given a decrease in cementogenesis with increasing age.