Structures of Receptor Complexes of a North American H7N2 Influenza Hemagglutinin with a Loop Deletion in the Receptor Binding Site

Human infections with subtype H7 avian influenza viruses have been reported as early as 1979. In 1996, a genetically stable 24-nucleotide deletion emerged in North American H7 influenza virus hemagglutinins, resulting in an eight amino acid deletion in the receptor-binding site. The continuous circulation of these viruses in live bird markets, as well as its documented ability to infect humans, raises the question of how these viruses achieve structural stability and functionality. Here we report a detailed molecular analysis of the receptor binding site of the North American lineage subtype H7N2 virus A/New York/107/2003 (NY107), including complexes with an avian receptor analog (3′-sialyl-N-acetyllactosamine, 3′SLN) and two human receptor analogs (6′-sialyl-N-acetyllactosamine, 6′SLN; sialyllacto-N-tetraose b, LSTb). Structural results suggest a novel mechanism by which residues Arg220 and Arg229 (H3 numbering) are used to compensate for the deletion of the 220-loop and form interactions with the receptor analogs. Glycan microarray results reveal that NY107 maintains an avian-type (α2-3) receptor binding profile, with only moderate binding to human-type (α2-6) receptor. Thus despite its dramatically altered receptor binding site, this HA maintains functionality and confirms a need for continued influenza virus surveillance of avian and other animal reservoirs to define their zoonotic potential.     

Suitability of flow cytometry for estimating bacterial biovolume in natural plankton samples: comparison with microscopy data

The relationship between flow cytometry data and epifluorescence microscopy measurements was assessed in bacterioplankton samples from 80 lakes to estimate bacterial biovolume and cell size distribution. The total counts of 4',6'-diamidino-2-phenylindole-stained cells estimated by both methods were significantly related, and the slope of their linear regression was not significantly different from 1, indicating that both methods produce very similar estimates of bacterial abundance. The relationships between side scatter (SSC) and 4',6'-diamidino-2-phenylindole fluorescence and cell volume (microscopy values) were improved by binning of the data in three frequency classes for each, but further increases in the number of classes did not improve these relationships. Side scatter was the best cell volume predictor, and significant relationships were observed between the SSC classes and the smallest (R2 = 0.545, P < 0.001, n = 80) and the largest (R2 = 0.544, P < 0.001, n = 80) microscopy bacterial-size classes. Based on these relationships, a reliable bacterial biomass estimation was obtained from the SSC frequency classes. Our study indicates that flow cytometry can be used to properly estimate bacterioplankton biovolume, with an accuracy similar to those of more time-consuming microscopy methods.     

Pathogenesis of avian influenza (H7) virus infection in mice and ferrets: enhanced virulence of Eurasian H7N7 viruses isolated from humans

Before 2003, only occasional case reports of human H7 influenza virus infections occurred as a result of direct animal-to-human transmission or laboratory accidents; most of these infections resulted in conjunctivitis. An increase in isolation of avian influenza A H7 viruses from poultry outbreaks and humans has raised concerns that additional zoonotic transmissions of influenza viruses from poultry to humans may occur. To better understand the pathogenesis of H7 viruses, we have investigated their ability to cause disease in mouse and ferret models. Mice were infected intranasally with H7 viruses of high and low pathogenicity isolated from The Netherlands in 2003 (Netherlands/03), the northeastern United States in 2002-2003, and Canada in 2004 and were monitored for morbidity, mortality, viral replication, and proinflammatory cytokine production in respiratory organs. All H7 viruses replicated efficiently in the respiratory tracts of mice, but only Netherlands/03 isolates replicated in systemic organs, including the brain. Only A/NL/219/03 (NL/219), an H7N7 virus isolated from a single fatal human case, was highly lethal for mice and caused severe disease in ferrets. Supporting the apparent ocular tropism observed in humans following infection with viruses of the H7 subtype, both Eurasian and North American lineage H7 viruses were detected in the mouse eye following ocular inoculation, whereas an H7N2 virus isolated from the human respiratory tract was not. Therefore, in general, the relative virulence and cell tropism of the H7 viruses in these animal models correlated with the observed virulence in humans.     

Fusion induced by a class II viral fusion protein, semliki forest virus E1, is dependent on the voltage of the target cell

Cells expressing the low pH-triggered class II viral fusion protein E1 of Semliki Forest virus (SFV) were fused to target cells. Fusion was monitored by electrical capacitance and aqueous dye measurements. Electrical voltage-clamp measurements showed that SFV E1-induced cell-cell fusion occurred quickly after acidification for a trans-negative potential across the target membrane (i.e., negative potential inside the target cell) but that a trans-positive potential eliminated all fusion. Use of an ionophore to control potentials for a large population of cells confirmed the dependence of fusion on voltage polarity. In contrast, fusion induced by the class I fusion proteins of human immunodeficiency virus, avian sarcoma leukosis virus, and influenza virus was independent of the voltage polarity across the target cell. Initial pore size and pore growth were also independent of voltage polarity for the class I proteins. An intermediate of SFV E1-induced fusion was created by transient acidification at low temperature. Membranes were hemifused at this intermediate state, and raising the temperature at neutral pH allowed full fusion to occur. Capacitance measurements showed that maintaining a trans-positive potential definitely blocked fusion at steps following the creation of the hemifusion intermediate and may have inhibited fusion at prior steps. It is proposed that the trans-negative voltage across the endosomal membrane facilitates fusion after low-pH-induced conformational changes of SFV E1 have occurred.     

Heterogeneous ribonucleoprotein m is a splicing regulatory protein that can enhance or silence splicing of alternatively spliced exons

Splicing of fibroblast growth factor receptor 2 (FGFR2) alternative exons IIIb and IIIc is regulated by the auxiliary RNA cis-element ISE/ISS-3 that promotes splicing of exon IIIb and silencing of exon IIIc. Using RNA affinity chromatography, we have identified heterogeneous nuclear ribonucleoprotein M (hnRNP M) as a splicing regulatory factor that binds to ISE/ISS-3 in a sequence-specific manner. Overexpression of hnRNP M promoted exon IIIc skipping in a cell line that normally includes it, and association of hnRNP M with ISE/ISS-3 was shown to contribute to this splicing regulatory function. Thus hnRNP M, along with other members of the hnRNP family of RNA-binding proteins, plays a combinatorial role in regulation of FGFR2 alternative splicing. We also determined that hnRNP M can affect the splicing of several other alternatively spliced exons. This activity of hnRNP M included the ability not only to induce exon skipping but also to promote exon inclusion. This is the first report demonstrating a role for this abundant hnRNP family member in alternative splicing in mammals and suggests that this protein may broadly contribute to the fidelity of splice site recognition and alternative splicing regulation.     

The role of Stn1p in Saccharomyces cerevisiae telomere capping can be separated from its interaction with Cdc13p

The function of telomeres is twofold: to facilitate complete chromosome replication and to protect chromosome ends against fusions and illegitimate recombination. In the budding yeast Saccharomyces cerevisiae, interactions among Cdc13p, Stn1p, and Ten1p are thought to be critical for promoting these processes. We have identified distinct Stn1p domains that mediate interaction with either Ten1p or Cdc13p, allowing analysis of whether the interaction between Cdc13p and Stn1p is indeed essential for telomere capping or length regulation. Consistent with the model that the Stn1p essential function is to promote telomere end protection through Cdc13p, stn1 alleles that truncate the C-terminal 123 residues fail to interact with Cdc13p and do not support viability when expressed at endogenous levels. Remarkably, more extensive deletions that remove an additional 185 C-terminal residues from Stn1p now allow cell growth at endogenous expression levels. The viability of these stn1-t alleles improves with increasing expression level, indicating that increased stn1-t dosage can compensate for the loss of Cdc13p-Stn1p interaction. However, telomere length is misregulated at all expression levels. Thus, an amino-terminal region of Stn1p is sufficient for its essential function, while a central region of Stn1p either negatively regulates the STN1 essential function or destabilizes the mutant Stn1 protein.     

Autumn migration of Montagu’s harriers Circus pygargus tracked by satellite telemetry

Although there is a general understanding of Montagu’s harriers migration routes and wintering areas, detailed information on the species’ migration is still lacking. However, improvements in satellite tracking technology in recent years, have enabled the study of medium-sized species by means of satellite telemetry. In 2006, ten adult Montagu’s harriers were fitted with satellite transmitters in northeastern Spain and tracked during their autumn migration to their wintering grounds in sub-Saharan Africa. The migration took between 10 and 30 days, and the end point was determined using breakpoint regressions. Whereas some birds had stopovers of more than a week, others stayed at the same site for only 1 or 2 days at the most. The tagged birds ultimately established at wintering grounds located along the border of Mauritania with Mali and Senegal, a distance of nearly 3000 km from the breeding sites. These sites are situated within a small range of latitudes (14° and 17°N), although distributed over a wider range of longitudes (−15°E and −4°E), with some birds occupying sites more than 1000 km apart. The distance covered in 1 day during the migration ranged between 93 and 219 km, with peaks of traveling speed of up to 65 km/h. Harriers were recorded traveling only during daytime, covering the longest distances in the late afternoon, suggesting that they are daytime migrants. Most of the distance was covered between 1500 and 2000 hours, and no traveling was recorded between 2000 and 0500 hours. During migration, harriers flew close to the ground (40–100 m on average). Improved knowledge of the harriers’ exact wintering sites may provide insights on the problems Montagu’s harriers face during the winter, highlighting the need to take into account what happens in both the breeding and wintering grounds to implement successful conservation measures.     

Understanding the structural requirements of 4-anilidopiperidine analogues for biological activities at mu and delta opioid receptors

New 4-anilidopiperidine analogues in which the phenethyl group of fentanyl was replaced by several aromatic ring-contained amino acids (or acids) were synthesized to study the biological effect of the substituents on mu and delta opioid receptor interactions. These analogues showed broad (47 nM-76 microM) but selective (up to 17-fold) binding affinities at the mu opioid receptor over the delta opioid receptor, as predicted from the message-address concept.     

ADP_EM: fast exhaustive multi-resolution docking for high-throughput coverage

MOTIVATION: Efficient fitting tools are needed to take advantage of a fast growth of atomic models of protein domains from crystallography or comparative modeling, and low-resolution density maps of larger molecular assemblies. Here, we report a novel fitting algorithm for the exhaustive and fast overlay of partial high-resolution models into a low-resolution density map. The method incorporates a fast rotational search based on spherical harmonics (SH) combined with a simple translational scanning. RESULTS: This novel combination makes it possible to accurately dock atomic structures into low-resolution electron-density maps in times ranging from seconds to a few minutes. The high-efficiency achieved with simulated and experimental test cases preserves the exhaustiveness needed in these heterogeneous-resolution merging tools. The results demonstrate its efficiency, robustness and high-throughput coverage. AVAILABILITY: http://sbg.cib.csic.es/Software/ADP_EM. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.