Modifications to a molt‐based ageing system proposed by Wolfe et al. (2010)

ABSTRACT Wolfe et al. (2010 . Journal of Field Ornithology 81: 186–194) proposed a coding system for ageing birds based on the sequence of molts and plumages, which is more practical than a calendar‐based system, especially in tropical and southern latitudes where species often breed across 1 January. The Wolfe–Ryder–Pyle (hereafter, W–R–P) three‐letter system is based on recognition of molt cycle (first, second, third, definitive, and so on) and plumage phase (juvenile, supplemental, formative, alternate, and basic). For example, a bird in First Cycle Formative plumage is coded as FCF. We propose the use of two additional code options that further refine age brackets. First, we suggest the use of an “after” or “A” code in place of the “C,” or cycle code, where an earlier molt cycle or plumage can be ruled out. For example, a bird that exhibits Staffelmauser might be aged as after‐third cycle basic, or TAB. Second, we suggest using “pre” or “P” in place of the “C,” or cycle code, when birds are actively molting, such as for birds undergoing the second prebasic molt or SPB. For both codes, we discuss their applicability using examples based on actual banding data. Our proposed codes will improve the utility of the W–R–P system by better refining age brackets and by expanding its applicability to a diverse array of taxa.     

Determinants of the Hepatitis C Virus Nonstructural Protein 2 Protease Domain Required for Production of Infectious Virus

The hepatitis C virus (HCV) nonstructural protein 2 (NS2) is a dimeric multifunctional hydrophobic protein with an essential but poorly understood role in infectious virus production. We investigated the determinants of NS2 function in the HCV life cycle. On the basis of the crystal structure of the postcleavage form of the NS2 protease domain, we mutated conserved features and analyzed the effects of these changes on polyprotein processing, replication, and infectious virus production. We found that mutations around the protease active site inhibit viral RNA replication, likely by preventing NS2-3 cleavage. In contrast, alterations at the dimer interface or in the C-terminal region did not affect replication, NS2 stability, or NS2 protease activity but decreased infectious virus production. A comprehensive deletion and mutagenesis analysis of the C-terminal end of NS2 revealed the importance of its C-terminal leucine residue in infectious particle production. The crystal structure of the NS2 protease domain shows that this C-terminal leucine is locked in the active site, and mutation or deletion of this residue could therefore alter the conformation of NS2 and disrupt potential protein-protein interactions important for infectious particle production. These studies begin to dissect the residues of NS2 involved in its multiple essential roles in the HCV life cycle and suggest NS2 as a viable target for HCV-specific inhibitors.An estimated 130 million people are infected with hepatitis C virus (HCV), the etiologic agent of non-A, non-B viral hepatitis. Transmission of the virus occurs primarily through blood or blood products. Acute infections are frequently asymptomatic, and 70 to 80% of the infected individuals are unable to eliminate the virus. Of the patients with HCV-induced chronic hepatitis, 15 to 30% progress to cirrhosis within years to decades after infection, and 3 to 4% of patients develop hepatocellular carcinoma (17). HCV infection is a leading cause of cirrhosis, end-stage liver disease, and liver transplantation in Europe and the United States (7), and reinfection after liver transplantation occurs almost universally. There is no vaccine available, and current HCV therapy of pegylated alpha interferon in combination with ribavirin leads to a sustained response in only about 50% of genotype 1-infected patients.The positive-stranded RNA genome of HCV is about 9.6 kb in length and encodes a single open reading frame flanked by 5′ and 3′ nontranslated regions (5′ and 3′ NTRs). The translation product of the viral genome is a large polyprotein containing the structural proteins (core, envelope proteins E1 and E2) in the N-terminal region and the nonstructural proteins (p7, nonstructural protein 2 [NS2], NS3, NS4A, NS4B, NS5A, and NS5B) in the C-terminal region. The individual proteins are processed from the polyprotein by various proteases. The host cellular signal peptidase cleaves between core/E1, E1/E2, E2/p7, and p7/NS2, and signal peptide peptidase releases core from the E1 signal peptide. Two viral proteases, the NS2-3 protease and the NS3-4A protease, cleave the remainder of the viral polyprotein in the nonstructural region (22, 27). The structural proteins package the genome into infectious particles and mediate virus entry into a naïve host cell; the nonstructural proteins NS3 through NS5B form the RNA replication complex. p7 and NS2 are not thought to be incorporated into the virion but are essential for the assembly of infectious particles (14, 36); however, their mechanisms of action are not understood.NS2 (molecular mass of 23 kDa) is a hydrophobic protein containing several transmembrane segments in the N-terminal region (5, 9, 32, 39). The C-terminal half of NS2 and the N-terminal third of NS3 form the NS2-3 protease (10, 11, 26, 37). NS2 is not required for the replication of subgenomic replicons, which span NS3 to NS5B (20). However, cleavage at the NS2/3 junction is necessary for replication in chimpanzees (16), the full-length replicon (38), and in the infectious tissue culture system (HCVcc) (14). Although cleavage can occur in vitro in the absence of microsomal membranes, synthesis of the polyprotein precursor in the presence of membranes greatly increases processing at the NS2/3 site (32). In vitro studies indicate that purified NS2-3 protease is active in the absence of cellular cofactors (11, 37). In addition to its role as a protease, NS2 has been shown to be required for assembly of infectious intracellular virus (14). The N-terminal helix of NS2 was first implicated in infectivity by the observation that an intergenotypic breakpoint following this transmembrane segment resulted in higher titers of infectious virus (28). Structural and functional characterization of the NS2 transmembrane region has shown that this domain is essential for infectious virus production (13). In particular, a central glycine residue in the first NS2 helix plays a critical role in HCV infectious virus assembly (13). The NS2 protease domain, but not its catalytic activity, is also essential for infectious virus assembly, whereas the unprocessed NS2-3 precursor is not required (13, 14).The crystal structure of the postcleavage NS2 protease domain (NS2^pro, residues 94 to 217), revealed a dimeric cysteine protease containing two composite active sites (Fig. 2C; [21]). Two antiparallel α-helices make up the N-terminal subdomain, followed by an extended crossover region, which positions the β-sheet-rich C-terminal subdomain near the N-terminal region of the partner monomer. Two of the conserved residues of the catalytic triad (His 143, Glu 163) are located in the loop region after the second N-terminal helix of one monomer, while the third catalytic residue, Cys 184, is located in the C-terminal subdomain of the other monomer. Creation of this unusual pair of composite active sites through NS2 dimerization has been shown to be essential for autoproteolytic cleavage (21). The structure of NS2^pro further demonstrated that the C-terminal residue of NS2 remains bound in the active site after cleavage, suggesting a possible mechanism for restriction of this enzyme to a single proteolytic event (21). Here we have used the crystal structure of NS2^pro, along with sequence alignments, to target conserved residues in each of the NS2^pro structural regions. Our mutational analysis revealed that the residues in the dimer crossover region and the C-terminal subdomain are important for infectious virus production. In contrast, the majority of amino acids in the active site pocket were not required for infectivity. Interestingly, we observed that the extreme C-terminal leucine of NS2 is absolutely essential for generation of infectious virus, as mutations, deletions, and extensions into NS3 are very poorly tolerated. This analysis begins to dissect the determinants of the multiple functions of this important protease in the HCV life cycle.     

Experimentally based orientational refinement of membrane protein models: A structure for the Influenza A M2 H+ channel

The 97-residue M2 protein from Influenza A virus forms H+-selective ion channels which can be attributed solely to the homo-tetrameric alpha-helical transmembrane domain. Site-directed infrared dichroism spectra were obtained for the transmembrane domain of M2, reconstituted in lipid vesicles. Data analysis yielded the helix tilt angle beta=31.6(+/-6.2) degrees and the rotational pitch angle about the helix axis for residue Ala29 omegaAla29=-59.8(+/-9.9) degrees, whereby omega is defined as zero for a residue located in the direction of the helix tilt. A structure was obtained from an exhaustive molecular dynamics global search protocol in which the orientational data are utilised directly as an unbiased refinement energy term. Orientational refinement not only allowed selection of a unique structure but could also be shown to increase the convergence towards that structure during the molecular dynamics procedure. Encouragingly, the structure obtained is highly consistent with all available mutagenesis and conductivity data and offers a direct chemical insight that relates the altered functionality of the channel to its structure.     

No-choice preference of cerotoma trifurcata (coleoptera: chrysomelidae) to potential host plants of bean pod mottle virus (Comoviridae) in Iowa

To better understand the naturally occurring host range of Bean pod mottle virus (family Comoviridae, genus Comovirus, BPMV) and its principal vector Cerotoma trifurcata (F?rster) (Coleoptera: Chrysomelidae), 18 field-collected perennial plant species were tested for the presence of BPMV. By using no-choice assays, we determined the preference of these plants by bean leaf beetle, by measuring their level of herbivory relative to soybean, Glycine max (L.). New food hosts for adult bean leaf beetles include Lespedeza capitata (Michaux), Lotus corniculatus L., Trifolium alexandrinum L., Trifolium ambiguum Bieberstein, and Trifolium incarnatum L. Desmodium illinoense Gray is discovered as a new naturally occurring host for BPMV.     

Effects of the emerald ash borer invasion on the community composition of arthropods associated with ash tree boles in Maryland,U.S.A.

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The application of GIS and spatiotemporal analyses to investigations of unusual marine mammal strandings and mortality events

In 2006–2007, an unusually high number of harbor porpoises (Phocoena phocoena) stranded along the Washington and Oregon coastlines. Spatiotemporal analyses were used to examine their ability to detect clusters of porpoise strandings during an unusual mortality event (UME) in the Pacific Northwest using stranding location data. Strandings were evaluated as two separate populations, outer coast and inland waters. The presence of global clustering was evaluated using the Knox spatiotemporal test, and the presence of local clusters was investigated using a spatiotemporal scan statistic (space–time permutation). There was evidence of global clustering, but no local clustering, supporting the hypothesis that strandings were due to more varied etiologies instead of localized causes. Further analyses at subregional levels, and concurrently assessing environmental factors, might reveal additional geographic distribution patterns. This article describes the spatial analytical tools applied in this study and how they can help elucidate the spatiotemporal epidemiology of other UMEs and assist in determining their causes. More than one spatial analytical technique should be used if the study objective is to detect and describe clustering in time and space and to generate hypotheses regarding causation of marine mammal disease and stranding events.