Mutations in H5N1 Influenza Virus Hemagglutinin that Confer Binding to Human Tracheal Airway Epithelium

The emergence in 2009 of a swine-origin H1N1 influenza virus as the first pandemic of the 21st Century is a timely reminder of the international public health impact of influenza viruses, even those associated with mild disease. The widespread distribution of highly pathogenic H5N1 influenza virus in the avian population has spawned concern that it may give rise to a human influenza pandemic. The mortality rate associated with occasional human infection by H5N1 virus approximates 60%, suggesting that an H5N1 pandemic would be devastating to global health and economy. To date, the H5N1 virus has not acquired the propensity to transmit efficiently between humans. The reasons behind this are unclear, especially given the high mutation rate associated with influenza virus replication. Here we used a panel of recombinant H5 hemagglutinin (HA) variants to demonstrate the potential for H5 HA to bind human airway epithelium, the predominant target tissue for influenza virus infection and spread. While parental H5 HA exhibited limited binding to human tracheal epithelium, introduction of selected mutations converted the binding profile to that of a current human influenza strain HA. Strikingly, these amino-acid changes required multiple simultaneous mutations in the genomes of naturally occurring H5 isolates. Moreover, H5 HAs bearing intermediate sequences failed to bind airway tissues and likely represent mutations that are an evolutionary “dead end.” We conclude that, although genetic changes that adapt H5 to human airways can be demonstrated, they may not readily arise during natural virus replication. This genetic barrier limits the likelihood that current H5 viruses will originate a human pandemic.     

HIV-1 integrase preassembled on donor DNA is refractory to activity stimulation by LEDGF/p75

LEDGF/p75 is known to enhance the integrase strand transfer activity in vitro, but the underlying mechanism is unclear. Using an integrase assay with a chemiluminescent readout adapted to a 96-well plate format, the effect of LEDGF/p75 on both the 3'-processing and strand transfer steps was analyzed. Integrase inhibitors of the strand transfer reaction remained active in the presence of LEDGF/p75, but displayed 3- to 7-fold higher IC50 values. Our analyses indicate that, in the presence of 150 nM LEDGF/p75, active integrase/donor DNA complexes were increased by 5.3-fold during the 3'-processing step. In addition, these integrase/donor DNA complexes showed a 4.5-fold greater affinity for the target DNA during the subsequent strand transfer step. We also observed a 3.7-fold increase in the rate constant of catalysis of the strand transfer step when 150 nM LEDGF/p75 was present during the 3'-processing step. In contrast, when LEDGF/p75 was added at the beginning of the strand transfer step, no increase in either the concentration of active integrase/donor DNA complex or its rate constant of strand transfer catalysis was observed. This observation suggested that the integrase/donor DNA formed in the absence of LEDGF/p75 became refractory to the stimulatory effect of LEDGF/p75. Instead, this LEDGF/p75 added at the start of the strand transfer step was able to promote the formation of a new cohort of active integrase/donor DNA complexes which became functional with a delay of 45 min after LEDGF/p75 addition. We propose a model whereby LEDGF/p75 can only bind integrase before the latter binds donor DNA whereas donor DNA can engage either free or LEDGF/p75-bound integrase.     

Applying nonsense-mediated mRNA decay research to the clinic: progress and challenges

Premature termination codons (PTCs) are equivalent to nonsense sequences. They encode no amino acid, and their presence precludes the synthesis of full-length proteins. Furthermore, the resulting truncated proteins, if synthesized and stable, are likely to be non-functional or might even be deleterious to cellular metabolism. Approximately one third of genetic and acquired diseases are due to PTCs. In fact, PTCs are apt to cause at least some cases of all diseases that involve protein insufficiency. Cells have evolved a way to eliminate mRNAs that contain PTCs using a mechanism called nonsense-mediated mRNA decay (NMD). Here, we will review how to determine which PTCs elicit NMD, what is currently known about the mechanism of NMD, and additional information that is pertinent to establishing therapies for PTC-associated diseases.     

Breeding soybeans for the tropics capable of nodulating effectively with indigenousRhizobium spp.

Summary Most soybean varieties fail to nodulate effectively in tropical soils unless inoculated with a competitive strain ofRhizobium japonicum. Developing countries in the tropics, with few exceptions, lack inoculant industries to produce and distribute viable inoculants to small farmers and extension programs to teach them to use inoculant. Several soybean genotypes have been identified that nodulate effectively with many strains of the cowpea inoculation group which is ubiquitous in tropical soils of Africa. Soybean genotypes that nodulate and grow well without inoculant application are called promiscuous. Methodologies for incorporation of the promiscuity character into high-yielding backgrounds are discussed.Supported in part by grant 05-0560 from United Nations Development Program to the International Institute of Tropical Agriculture.     

Three-year weight change in successful weight losers who lost weight on a low-carbohydrate diet

Objective: The purpose of this study was to evaluate long‐term weight loss and eating and exercise behaviors of successful weight losers who lost weight using a low‐carbohydrate diet. Research Methods and Procedures: This study examined 3‐year changes in weight, diet, and physical activity in 891 subjects (96 low‐carbohydrate dieters and 795 others) who enrolled in the National Weight Control Registry between 1998 and 2001 and reported ≥30‐lb weight loss and ≥1 year weight loss maintenance. Results: Only 10.8% of participants reported losing weight after a low‐carbohydrate diet. At entry into the study, low‐carbohydrate diet users reported consuming more kcal/d (mean ± SD, 1895 ± 452 vs. 1398 ± 574); fewer calories in weekly physical activity (1595 ± 2499 vs. 2542 ± 2301); more calories from fat (64.0 ± 7.9% vs. 30.9 ± 13.1%), saturated fat (23.8 ± 4.1 vs. 10.5 ± 5.2), monounsaturated fat (24.4 ± 3.7 vs. 11.0 ± 5.1), and polyunsaturated fat (8.6 ± 2.7 vs. 5.5 ± 2.9); and less dietary restraint (10.8 ± 2.9 vs. 14.9 ± 3.9) compared with other Registry members. These differences persisted over time. No differences in 3‐year weight regain were observed between low‐carbohydrate dieters and other Registry members in intent‐to‐treat analyses (7.0 ± 7.1 vs. 5.7 ± 8.7 kg). Discussion: It is possible to achieve and maintain long‐term weight loss using a low‐carbohydrate diet. The long‐term health effects of weight loss associated with a high‐fat diet and low activity level merits further investigation.     

Genetic impacts of Anacapa deer mice reintroductions following rat eradication

The Anacapa deer mouse is an endemic subspecies that inhabits Anacapa Island, part of Channel Islands National Park, California. We used mitochondrial DNA cytochrome c oxidase subunit II gene (COII) and 10 microsatellite loci to evaluate the levels of genetic differentiation and variation in ~1400 Anacapa deer mice sampled before and for 4 years after a black rat (Rattus rattus) eradication campaign that included trapping, captive holding and reintroduction of deer mice. Both mitochondrial and microsatellite analyses indicated significant differentiation between Anacapa deer mice and mainland mice, and genetic variability of mainland mice was significantly higher than Anacapa mice even prior to reintroduction. Bayesian cluster analysis and Principal Coordinates Analysis indicated that East, Middle and West Anacapa mice were genetically differentiated from each other, but translocation of mice among islands resulted in the East population becoming less distinct as a result of management. Levels of heterozygosity were similar before and after management. However, numerous private alleles in the founder populations were not observed after reintroduction and shifts in allele frequencies occurred, indicating that the reintroduced populations experienced substantial genetic drift. Surprisingly, two mitochondrial haplotypes observed in an earlier study of Anacapa deer mice were lost in the 20 years prior to the rat eradication program, leaving only a single haplotype in Anacapa deer mice. This study demonstrates how genetic monitoring can help to understand the re-establishment of endemic species after the eradication of invasive species and to evaluate the effectiveness of the management strategies employed.     

Fungal biodiversity in aquatic habitats

Fungal biodiversity in freshwater, brackish and marine habitats was estimated based on reports in the literature. The taxonomic groups treated were those with species commonly found on submerged substrates in aquatic habitats: Ascomycetes (exclusive of yeasts), Basidiomycetes, Chytridiomycetes, and the non-fungal Saprolegniales in the Class Oomycetes. Based on presence/absence data for a large number and variety of aquatic habitats, about 3,000 fungal species and 138 saprolegnialean species have been reported from aquatic habitats. The greatest number of taxa comprise the Ascomycetes, including mitosporic taxa, and Chytridiomycetes. Taxa of Basidiomycetes are, for the most part, excluded from aquatic habitats. The greatest biodiversity for all groups occurs in temperate areas, followed by Asian tropical areas. This pattern may be an artifact of the location of most of the sampling effort. The least sampled geographic areas include Africa, Australia, China, South America and boreal and tropical regions worldwide. Some species overlap occurs among terrestrial and freshwater taxa but little species overlap occurs among freshwater and marine taxa. We predict that many species remain to be discovered in aquatic habitats given the few taxonomic specialists studying these fungi, the few substrate types studied intensively, and the vast geographical area not yet sampled.     

Activation-induced cytidine deaminase targets DNA at sites of RNA polymerase II stalling by interaction with Spt5

Activation-induced cytidine deaminase (AID) initiates antibody gene diversification by creating U:G mismatches. However, AID is not specific for antibody genes; Off-target lesions can activate oncogenes or cause chromosome translocations. Despite its importance in these transactions little is known about how AID finds its targets. We performed an shRNA screen to identify factors required for class switch recombination (CSR) of antibody loci. We found that Spt5, a factor associated with stalled RNA polymerase II (Pol II) and single stranded DNA (ssDNA), is required for CSR. Spt5 interacts with AID, it facilitates association between AID and Pol II, and AID recruitment to its Ig and non-Ig targets. ChIP-seq experiments reveal that Spt5 colocalizes with AID and stalled Pol II. Further, Spt5 accumulation at sites of Pol II stalling is predictive of AID-induced mutation. We propose that AID is targeted to sites of Pol II stalling in part via its association with Spt5.