Gene transduction and cell entry pathway of fiber-modified adenovirus type 5 vectors carrying novel endocytic peptide ligands selected on human tracheal glandular cells

Monolayers of cystic fibrosis transmembrane conductance regulator (CFTR)-deficient human tracheal glandular cells (CF-KM4) were subjected to phage biopanning, and cell-internalized phages were isolated and sequenced, in order to identify CF-KM4-specific peptide ligands that would confer upon adenovirus type 5 (Ad5) vector a novel cell target specificity and/or higher efficiency of gene delivery into airway cells of patients with cystic fibrosis (CF). Three different ligands, corresponding to prototypes of the most represented families of phagotopes recovered from intracellular phages, were designed and individually inserted into Ad5-green fluorescent protein (GFP) (AdGFP) vectors at the extremities of short fiber shafts (seven repeats [R7]) terminated by scissile knobs. Only one vector, carrying the decapeptide GHPRQMSHVY (abbreviated as QM10), showed an enhanced gene transduction of CF-KM4 cells compared to control nonliganded vector with fibers of the same length (AdGFP-R7-knob). The enhancement in gene transfer efficiency was not specific to CF-KM4 cells but was observed in other mammalian cell lines tested. The QM10-liganded vector was referred to as AdGFP-QM10-knob in its knobbed version and as AdGFP-QM10 in its proteolytically deknobbed version. AdGFP-QM10 was found to transduce cells with a higher efficiency than its knob-bearing version, AdGFP-QM10-knob. Consistent with this, competition experiments indicated that the presence of knob domains was not an absolute requirement for cell attachment of the QM10-liganded vector and that the knobless AdGFP-QM10 used alternative cell-binding domains on its capsid, including penton base capsomer, via a site(s) different from its RGD motifs. The QM10-mediated effect on gene transduction seemed to take place at the step of endocytosis in both quantitative and qualitative manners. Virions of AdGFP-QM10 were endocytosed in higher numbers than virions of the control vector and were directed to a compartment different from the early endosomes targeted by members of species C Ad. AdGFP-QM10 was found to accumulate in late endosomal and low-pH compartments, suggesting that QM10 acted as an endocytic ligand of the lysosomal pathway. These results validated the concept of detargeting and retargeting Ad vectors via our deknobbing system and redirecting Ad vectors to an alternative endocytic pathway via a peptide ligand inserted in the fiber shaft domain.     

Inhibition of neutrophil apoptosis by type 1 IFN depends on cross-talk between phosphoinositol 3-kinase,protein kinase C-delta,and NF-kappa B signaling pathways

Neutrophils are abundant, short-lived leukocytes with a key role in the defense against rapidly dividing bacteria. They enter apoptosis spontaneously within 24-48 h of leaving the bone marrow. However, their life span can be extended during inflammatory responses by several proinflammatory cytokines. Inappropriate survival of neutrophils contributes to chronic inflammation and tissue damage associated with diseases such as rheumatoid arthritis. We have previously reported that type I IFNs can inhibit both cytokine deprivation and Fas-induced apoptosis in activated T cells. IFN-beta locally produced by hyperplastic fibroblasts within the pannus tissue of patients with rheumatoid arthritis contributes to the inappropriately extended life span of infiltrating T cells. Type I IFNs are equally effective at delaying spontaneous apoptosis in human neutrophils. In the work presented here we investigated the signaling pathways involved in mediating this effect. The antiapoptotic actions of IFN-beta were targeted at an early stage of neutrophil apoptosis, occurring upstream of mitochondrial permeability transition, and were phosphatidylinositol 3-kinase (PI3K) dependent, as they were blocked by the PI3K inhibitor LY294002. Analysis of signaling pathways downstream of PI3K revealed that the antiapoptotic effect of type 1 IFN was inhibited by rottlerin, SN50, and cycloheximide, indicating requirements for activation of protein kinase C-delta, NF-kappaB, and de novo protein synthesis, respectively. Moreover, EMSA was used to show that the activation of NF-kappaB occurred downstream of PI3K and protein kinase C-delta activation. We conclude that type I IFNs inhibit neutrophil apoptosis in a PI3K-dependent manner, which requires activation of protein kinase C-delta and induction of NF-kappaB-regulated genes.     

Entropy learning and relevance criteria for neural network pruning

In this paper, entropy is a term used in the learning phase of a neural network. As learning progresses, more hidden nodes get into saturation. The early creation of such hidden nodes may impair generalisation. Hence an entropy approach is proposed to dampen the early creation of such nodes by using a new computation called entropy cycle. Entropy learning also helps to increase the importance of relevant nodes while dampening the less important nodes. At the end of learning, the less important nodes can then be pruned to reduce the memory requirements of the neural network.     

The 100K-chaperone protein from adenovirus serotype 2 (Subgroup C) assists in trimerization and nuclear localization of hexons from subgroups C and B adenoviruses

Recombinant hexons from subgroup C adenoviruses (Ad2 and Ad5) and from a member of subgroup B (Ad3) adenoviruses have been expressed in insect cells. When expressed alone, all three hexons were found to be insoluble and accumulated as inclusion bodies in the cytoplasm. However, co-expression of recombinant Ad2, Ad5 or Ad3 hexon with Ad2 L4-100K protein resulted in the formation of soluble trimeric hexons. EM analysis of hexons revealed that they were indistinguishable from native hexon capsomers isolated from Ad2-infected human cells, or released from partially disrupted adenovirions. This suggests that 100K acts as a chaperone for hexon folding and self-assembly into capsomer in insect cells. Since 100K protein assists in the trimerization of subgroup C hexon, and of subgroup B hexon protein, it implies that it functions in a manner that is both homo- and heterotypic. During the course of recombinant protein expression, the 100K protein was found in association with hexon monomers and trimers within the cytoplasm. In the nucleus, however, 100K was found in complexes with hexon trimers exclusively. EM observation of purified 100K protein samples showed a dumb-bell-shaped molecule compatible with a monomeric protein. EM analysis of hexon-100K protein complexes showed that interaction of hexon with the 100K protein occurred via one of the globular domains of the 100K protein molecule. Our data confirm the role of the 100K protein as a scaffold protein for hexon, and provide evidence suggesting its function in hexon nuclear import in insect cells.     

Estimates of Lifetime Risk of Occupational Fatal Injury from Age-Specific Rates

The lifetime risk of fatal workplace injury is a critical issue in the evaluation of occupational hazards. Recently, Fosbroke, Kisner, and Myers (1997) described a metric for working lifetime risk (WLTR) to determine the probability that a worker will die due to a work-related fatal injury in a year over a certain number of years of employment. This quantity was defined assuming that the annual rate of fatal injuries will be the same each year during employment. Recognizing the fact that annual fatal injury rates differ with the age of the worker along with other factors, modification of the definition of working lifetime risk is derived. We obtain the estimates of the lifetime risk using age-categorized annual fatality rates and derive an estimate of the standard error of the WLTR estimator and a confidence interval for the WLTR. We illustrate these calculations by estimating the lifetime risk for work-related fatal injuries for workers in four high risk industries: agriculture-forestry-fishing, mining, construction, and transportation public utilities. The estimates are based on employment data from the Bureau of Labor Statistics and an updated version of fatality data from the National Traumatic Occupational Fatalities surveillance system.     

A fast assay for developing serum free media for cord blood haematopoietic cells

One of the bottlenecks in haematopoietic cell expansion is the lack of a defined serum free media. We describe a fast method of monitoring live haematopoietic cell numbers, using fluorescein diacetate, to facilitate the development of such a media. Several carbohydrates (fructose at 500g/ml and pyruvate at 300g/ml) and lipids (cholesterol and lecithin, both at 10g/ml) were tested and could replace serum in cord blood haematopoietic cell cultures.     

A fast graphics printing program for neurophysiological data

A program was written in assembly language for fast graphicsscreen dump of neurophysiological data during and after experiments.This program takes 3 s to plot a 1024 x 768 graphics image.A resident program has also been produced, which allows a screengraphics image to be printed by using a keyboard command. APascal-compatible object file is available to interface theprogram with Turbo-Pascal programs.     

Agricultural diversification as an important strategy for achieving food security in Africa

Farmers in Africa have long adapted to climatic and other risks by diversifying their farming activities. Using a multi‐scale approach, we explore the relationship between farming diversity and food security and the diversification potential of African agriculture and its limits on the household and continental scale. On the household scale, we use agricultural surveys from more than 28,000 households located in 18 African countries. In a next step, we use the relationship between rainfall, rainfall variability, and farming diversity to determine the available diversification options for farmers on the continental scale. On the household scale, we show that households with greater farming diversity are more successful in meeting their consumption needs, but only up to a certain level of diversity per ha cropland and more often if food can be purchased from off‐farm income or income from farm sales. More diverse farming systems can contribute to household food security; however, the relationship is influenced by other factors, for example, the market orientation of a household, livestock ownership, nonagricultural employment opportunities, and available land resources. On the continental scale, the greatest opportunities for diversification of food crops, cash crops, and livestock are located in areas with 500–1,000 mm annual rainfall and 17%–22% rainfall variability. Forty‐three percent of the African cropland lacks these opportunities at present which may hamper the ability of agricultural systems to respond to climate change. While sustainable intensification practices that increase yields have received most attention to date, our study suggests that a shift in the research and policy paradigm toward agricultural diversification options may be necessary.