羌活与宽叶羌活的群体遗传结构和种间分化研究

为了合理利用羌活和宽叶羌活的药用植物资源,同时保护其物种多样性,该研究利用SSR分子标记技术对羌活与宽叶羌活邻域及异域分布的23个自然种群,共计227个个体进行多样性和种间分化研究.结果显示:(1)两个物种具有中等水平的遗传多样性;羌活的平均等位基因数(Na)、有效等位基因数(Ne)和期望杂合度(He)分别为2.603...     

Informing evidence-based decision-making for patients with comorbidity: availability of necessary information in clinical trials for chronic diseases

Background

The population with multiple chronic conditions is growing. Prior studies indicate that patients with comorbidities are frequently excluded from trials but do not address whether information is available in trials to draw conclusions about treatment effects for these patients.

Methods and Findings

We conducted a literature survey of trials from 11 Cochrane Reviews for four chronic diseases (diabetes, heart failure, chronic obstructive pulmonary disease, and stroke). The Cochrane Reviews systematically identified and summarized trials on the effectiveness of diuretics, metformin, anticoagulants, longacting beta-agonists alone or in combination with inhaled corticosteroids, lipid lowering agents, exercise and diet. Eligible studies were reports of trials included in the Cochrane reviews and additional papers that described the methods of these trials. We assessed the exclusion and inclusion of people with comorbidities, the reporting of comorbidities, and whether comorbidities were considered as potential modifiers of treatment effects. Overall, the replicability of both the inclusion criteria (mean [standard deviation (SD)]: 6.0 (2.1), range (min-max): 1–9.5) and exclusion criteria(mean(SD): 5.3 (2.1), range: 1–9.5) was only moderate. Trials excluded patients with many common comorbidities. The proportion of exclusions for comorbidities ranged from 0–42 percent for heart failure, 0–55 percent for COPD, 0–44 percent for diabetes, and 0–39 percent for stroke. Seventy of the 161 trials (43.5%) described the prevalence of any comorbidity among participants with the index disease. The reporting of comorbidities in trials was very limited, in terms of reporting an operational definition and method of ascertainment for the presence of comorbidity and treatments for the comorbidity. It was even less common that the trials assessed whether comorbidities were potential modifiers of treatment effects.

Conclusions

Comorbidities receive little attention in chronic disease trials. Given the public health importance of people with multiple chronic conditions, trials should better report on comorbidities and assess the effect comorbidities have on treatment outcomes.     

Archaeal ApbC/Nbp35 Homologs Function as Iron-Sulfur Cluster Carrier Proteins

Iron-sulfur clusters may have been the earliest catalytic cofactors on earth, and most modern organisms use them extensively. Although members of the Archaea produce numerous iron-sulfur proteins, the major cluster assembly proteins found in the Bacteria and Eukarya are not universally conserved in archaea. Free-living archaea do have homologs of the bacterial apbC and eukaryotic NBP35 genes that encode iron-sulfur cluster carrier proteins. This study exploits the genetic system of Salmonella enterica to examine the in vivo functionality of apbC/NBP35 homologs from three archaea: Methanococcus maripaludis, Methanocaldococcus jannaschii, and Sulfolobus solfataricus. All three archaeal homologs could correct the tricarballylate growth defect of an S. enterica apbC mutant. Additional genetic studies showed that the conserved Walker box serine and the Cys-X-X-Cys motif of the M. maripaludis MMP0704 protein were both required for function in vivo but that the amino-terminal ferredoxin domain was not. MMP0704 protein and an MMP0704 variant protein missing the N-terminal ferredoxin domain were purified, and the Fe-S clusters were chemically reconstituted. Both proteins bound equimolar concentrations of Fe and S and had UV-visible spectra similar to those of known [4Fe-4S] cluster-containing proteins. This family of dimeric iron-sulfur carrier proteins evolved before the archaeal and eukaryal lineages diverged, representing an ancient mode of cluster assembly.Members of the Archaea produce many proteins that require iron-sulfur cluster cofactors, including redox proteins, aconitase-like dehydratases, radical S-adenosylmethionine enzymes, and RNA polymerase (9, 13, 18, 32). Methanogenic archaea are obligate anaerobes, and many heterotrophic archaea grow anaerobically, indicating that oxidative stress has not limited the proliferation of iron-sulfur proteins in these lineages. Archaea must have a mechanism to assemble Fe-S clusters, but many members lack homologs of the known bacterial and eukaryotic Nif or Isc systems, suggesting that an alternative system is present (see Table S1 in the supplemental material) (14, 15).Some euryarchaea have homologs of the bacterial genes iscS (encoding cysteine desulfurase) and iscU (encoding a scaffold protein). Yet many other archaea, including the euryarchaea Pyrococcus furiosus, Methanocaldococcus jannaschii, and Methanococcus maripaludis, plus most crenarchaea, either lack a homologous cysteine desulfurase gene or have no homologs of A-type or U-type scaffold genes. Due to their sulfide-rich environments, it is reasonable that the anaerobic archaea may use an inorganic sulfur source to assemble Fe-S clusters. Most archaeal genome sequences do carry homologs of the sufBC genes that are part of the alternative Suf system for Fe-S cluster biosynthesis (36). Biochemical studies have shown that SufC is an ABC-type ATPase and that SufB is a persulfide acceptor that may act as a site for Fe-S cluster assembly (20). The SufB and SufC proteins interact, and SufB stimulates the ATPase activity of SufC. We hypothesize that the Archaea share a common mechanism for Fe-S cluster biosynthesis, supplemented with genes acquired by horizontal gene transfer in some lineages.A screen for Salmonella enterica bacteria defective in thiamine biosynthesis identified lesions in the apbC locus (28) that compromised Fe-S metabolism (33). An abpC mutant cannot grow with tricarballylate as a carbon and energy source, which may be due to a defect in assembling or repairing [4Fe-4S] clusters in the membrane-bound TcuB protein (21, 22). ApbC is a 40-kDa cytoplasmic protein with Walker A and B nucleotide-binding domains and two conserved carboxy-terminal cysteine residues separated by two amino acids (Cys-X-X-Cys). Mutational analyses have shown that ApbC proteins with directed changes in the Cys-X-X-Cys or Walker A motifs are not active in vivo (6). Suppressor analysis allowed the conclusion that a degree of functional redundancy between ApbC and the Fe-S scaffold protein IscU exists (4, 38). Although purified ApbC does not contain iron or sulfur, biochemical studies showed that ApbC can bind an Fe-S cluster and rapidly transfer it to an apoprotein (5).It is thought that in eukaryotes, Fe-S clusters are assembled by the mitochondrial iron-sulfur cluster (ISC) system (23). The clusters are transported into the cytosol and delivered by the cytosolic iron-sulfur protein assembly system. Two components of this system, Nbp35 and Cfd1, are homologs of bacterial ApbC (Fig. ​(Fig.1)1) and act as intermediate Fe-S cluster-trafficking proteins in the cytosol (16, 27, 30). Electron paramagnetic resonance, Mössbauer, and absorbance spectra of the Saccharomyces cerevisiae, human, and Arabidopsis Nbp35 holoproteins suggest that these holoproteins form dimers with stable amino-terminal [4Fe-4S] clusters and a shared carboxy-terminal [4Fe-4S] cluster (10, 34).Open in a separate windowFIG. 1.A protein sequence alignment of bacterial, archaeal, and eukaryotic ApbC/Nbp35 homologs was constructed using the ClustalW program (version 1.83) (37). The sequence of the S. enterica serovar Typhimurium protein (ApbC; RefSeq accession no. NP_461098.1) is shown without the amino-terminal domain that is not homologous to the amino-terminal domains of the archaeal and eukaryotic proteins. The archaeal homologs are from S. solfataricus (SSO0460; accession no. NP_341994.1), P. furiosus (PF1145; accession no. NP_578874.1), Methanosarcina acetivorans (MA4246; accession no. NP_619111.1), M. jannaschii (MJ0283; accession no. NP_247256.1), and M. maripaludis (MMP0704; accession no. NP_987824.1). The two paralogs from S. cerevisiae are Nbp35 (accession no. NP_011424.1) and Cfd1 (accession no. NP_012263.1). Conserved amino acid residues are shown in white on a black background. Similar residues are shown in black on a gray background. The four conserved amino-terminal cysteine residues shared by the MMP0704 and Nbp35p proteins are boxed. Asterisks above the sequences indicate MMP0704 residues replaced by mutagenesis in this study. A vertical bar indicates the N termini of the truncated proteins MJ0283(19-290) and MMP0704(20-289).Archaeal homologs of bacterial ApbC and eukaryotic Nbp35 are underannotated as nucleotide-binding proteins or misannotated as cobyrinic acid a,c-diamide synthases in sequence databases. The hallmarks of the Nbp35 sequences are an amino-terminal ferredoxin-like domain, an ATP-binding motif, and two conserved carboxy-terminal cysteine residues that are believed to bind an Fe-S cluster. The amino-terminal ferredoxin-like domain is absent in the ApbC family of proteins. The ApbC and Nbp35 proteins belong to a large superfamily of P-loop-containing nucleoside triphosphate hydrolases that also includes the bacterial MinD and CooC proteins. The M. maripaludis MMP0704 protein shows approximately 40% amino acid identity to both the S. enterica ApbC and S. cerevisiae Nbp35 proteins (Fig. ​(Fig.1).1). However, the MMP0704 protein also shows 30% sequence identity to two paralogous proteins from M. maripaludis. The genome sequence of M. maripaludis encodes at least nine paralogs, although only the MMP0704 protein contains the conserved cysteine residues found in most ApbC/Nbp35 proteins.The experiments described herein identified the first archaeal proteins that form functional Fe-S carrier proteins. The apbC/NBP35 homologs from M. maripaludis (MMP0704), M. jannaschii (MJ0283), and Sulfolobus solfataricus (SSO0460) allowed an S. enterica strain with an apbC null mutation to grow on tricarballylate. Genetic studies showed that the Walker A box and at least one cysteine residue from the Cys-X-X-Cys motif were required for in vivo functionality. The unique amino-terminal ferredoxin-like domains of the MMP0704 and MJ0283 proteins were not required. Purified MMP0704 proteins bound Fe-S clusters. Orthologs of ApbC/Npb35 proteins were found in all of the available genomes of free-living archaea, identifying this protein family as an ancient part of the Fe-S assembly system that evolved before the divergence of Archaea and Eukarya.     

Expression and self-assembly of norwalk virus capsid protein from venezuelan equine encephalitis virus replicons

The Norwalk virus (NV) capsid protein was expressed using Venezuelan equine encephalitis virus replicon particles (VRP-NV1). VRP-NV1 infection resulted in large numbers of recombinant NV-like particles that were primarily cell associated and were indistinguishable from NV particles produced from baculoviruses. Mutations located in the N-terminal and P1 domains of the NV capsid protein ablated capsid self-assembly in mammalian cells.     

Diminished Reovirus Capsid Stability Alters Disease Pathogenesis and Littermate Transmission

Reovirus is a nonenveloped mammalian virus that provides a useful model system for studies of viral infections in the young. Following internalization into host cells, the outermost capsid of reovirus virions is removed by endosomal cathepsin proteases. Determinants of capsid disassembly kinetics reside in the viral σ3 protein. However, the contribution of capsid stability to reovirus-induced disease is unknown. In this study, we found that mice inoculated intramuscularly with a serotype 3 reovirus containing σ3-Y354H, a mutation that reduces viral capsid stability, succumbed at a higher rate than those infected with wild-type virus. At early times after inoculation, σ3-Y354H virus reached higher titers than wild-type virus at several sites within the host. Animals inoculated perorally with a serotype 1 reassortant reovirus containing σ3-Y354H developed exaggerated myocarditis accompanied by elaboration of pro-inflammatory cytokines. Surprisingly, unchallenged littermates of mice infected with σ3-Y354H virus displayed higher titers in the intestine, heart, and brain than littermates of mice inoculated with wild-type virus. Together, these findings suggest that diminished capsid stability enhances reovirus replication, dissemination, lethality, and host-to-host spread, establishing a new virulence determinant for nonenveloped viruses.     

Protein expression in the obligate hydrocarbon-degrading psychrophile Oleispira antarctica RB-8 during alkane degradation and cold tolerance

In cold marine environments, the obligate hydrocarbon-degrading psychrophile Oleispira antarctica RB-8, which utilizes aliphatic alkanes almost exclusively as substrates, dominates microbial communities following oil spills. In this study, LC–MS/MS shotgun proteomics was used to identify changes in the proteome induced during growth on n-alkanes and in cold temperatures. Specifically, proteins with significantly higher relative abundance during growth on tetradecane (n-C₁₄) at 16°C and 4°C have been quantified. During growth on n-C₁₄, O. antarctica expressed a complete pathway for the terminal oxidation of n-alkanes including two alkane monooxygenases, two alcohol dehydrogenases, two aldehyde dehydrogenases, a fatty-acid-CoA ligase, a fatty acid desaturase and associated oxidoreductases. Increased biosynthesis of these proteins ranged from 3- to 21-fold compared with growth on a non-hydrocarbon control. This study also highlights mechanisms O. antarctica may utilize to provide it with ecological competitiveness at low temperatures. This was evidenced by an increase in spectral counts for proteins involved in flagella structure/output to overcome higher viscosity, flagella rotation to accumulate cells and proline metabolism to counteract oxidative stress, during growth at 4°C compared with 16°C. Such species-specific understanding of the physiology during hydrocarbon degradation can be important for parameterizing models that predict the fate of marine oil spills.     

Stereoselective formation of benzo(c)phenanthrene (+)-(3S,4R) and (+)-(5S,6R)-oxides by cytochrome P450c in a highly purified and reconstituted system

The principal oxidative metabolites formed from benzo(c)phenanthrene (B(c)Ph) by the cytochromes P450 in liver microsomes from control and treated rats are the 3,4- and 5,6-arene oxides. A procedure is described which allows determination of the enantiomer composition and absolute configuration of these arene oxides based on HPLC separation of isomeric thiolate adducts formed with N-acetyl-L-cysteine in base. Incubation of [3H]-B(c)Ph with highly purified cytochrome P450c in a reconstituted monooxygenase system followed by trapping of the metabolically formed arene oxides as above indicated that the 3,4-oxide was predominantly the (+)-(3S,4R)-enantiomer (90%) and that the 5,6-oxide consisted mainly of the (+)-(5S,6R)-enantiomer (76%). The results are discussed in terms of their implications about the catalytic binding site of cytochrome P450c.     

Movements of wintering surf scoters: predator responses to different prey landscapes

The distribution of predators is widely recognized to be intimately linked to the distribution of their prey. Foraging theory suggests that predators will modify their behaviors, including movements, to optimize net energy intake when faced with variation in prey attributes or abundance. While many studies have documented changes in movement patterns of animals in response to temporal changes in food, very few have contrasted movements of a single predator species naturally occurring in dramatically different prey landscapes. We documented variation in the winter movements, foraging range size, site fidelity, and distribution patterns of a molluscivorous sea duck, the surf scoter (Melanitta perspicillata), in two areas of coastal British Columbia with very different shellfish prey features. Baynes Sound has extensive tidal flats with abundant clams, which are high-quality and temporally stable prey for scoters. Malaspina Inlet is a rocky fjord-like inlet where scoters consume mussels that are superabundant and easily accessible in some patches but are heavily depleted over the course of winter. We used radio telemetry to track surf scoter movements in both areas and found that in the clam habitats of Baynes Sound, surf scoters exhibited limited movement, small winter ranges, strong foraging site fidelity, and very consistent distribution patterns. By contrast, in mussel habitats in the Malaspina Inlet, surf scoters displayed more movement, larger ranges, little fidelity to specific foraging sites, and more variable distribution patterns. We conclude that features associated with the different prey types, particularly the higher depletion rates of mussels, strongly influenced seasonal space use patterns. These findings are consistent with foraging theory and confirm that predator behavior, specifically movements, is environmentally mediated.     

Interactions and quantitative analysis of immunoregulatory cells in the chicken thymus

Step-wise dilution of chicken thymus cell suspensions has been used to sequentially reveal suppressor, effector, and helper cells in these suspensions. The cells were tested either alone or in autologous mixture combinations with peripheral blood lymphocytes (PBL) as a source of effector cells. The assays studied were graft-vs-host reaction (GvHR) and mixed lymphocyte (MLR) reaction, spontaneous cellular cytotoxicity and antibody-dependent cell-mediated cytotoxicity, and mitogen responsiveness to Con A, PHA, and PWM. When tested alone, high numbers of thymus cells (1 X 10(7) gave weak or low responses, with the exception of GvHR, which was high. When this number of thymocytes was mixed with a strongly responding PBL effector population, there was marked suppression of the latter. Nonspecific crowding was excluded as a cause for the decreased responsiveness, and the data therefore demonstrated the presence of suppressor cells in the thymus. With gradual reduction of the thymus cell number in the mixtures, the suppressor activity was lost, but concomitant with this was the appearance of, or a gradual increase in, thymus effector cells giving good responses. Further dilutions of the thymus (to, e.g., 1 X 10(5) cells) depleted the suspension of effector cells, but helper cells capable of markedly amplifying the effector potential of PBL were revealed. The suppressor/helper function of the thymus was not only dependent on the absolute numbers of thymus cells present, but also on the degree of inherent responsiveness of the effector PBL. If the response of PBL alone was strong, a thymus suspension containing both helper and suppressor cells (e.g., 1 X 10(6) cells) caused suppression of the PBL; if the PBL alone were weak, this same thymus cell suspension caused enhancement. The outcome of an immune response is therefore dependent not only on the presence or absence of particular cell types, but also on the ratios between these cells. An imbalance in these ratios in vivo may underlie diseases of immunologic origin, e.g., autoimmunity.     

A DNA vaccine co-expressing antigen and an anti-apoptotic molecule further enhances the antigen-specific CD8+ T-cell immune response

We have shown that DNA encoding the anti-apoptotic protein Bcl-xL enhances E7-specific CD8+ T-cell responses and DNA encoding pro-apoptotic protein caspase-3 suppresses E7-specific CD8+ T-cell responses when co-administered intradermally via gene gun with DNA encoding human papillomavirus type 16 (HPV-16) E7 linked to the sorting signal of the lysosome-associated membrane protein type 1 (LAMP-1). E7 and LAMP-1 are linked to form the chimeric Sig/E7/LAMP-1 (SEL). Because co-administration does not ensure delivery of both constructs to a single cell, we used pVITRO, a mammalian expression vector with double promoters, to ensure expression of both molecules in the same cell. We vaccinated C57BL/6 mice with pVITRO-SEL-Bcl-xL, pVITRO-SEL-mtBcl-xL, pVITRO-SEL, or pVITRO-SEL-caspase-3 intradermally via gene gun and intramuscularly via injection. We demonstrated that vaccination with pVITRO achieved similar results to a co-administration strategy: that Bcl-xL enhanced the E7-specific CTL response and caspase-3 suppressed the E7-specific CTL response. In addition, we found intradermal vaccination elicited significantly higher numbers of E7-specific CD8+ T cells compared to intramuscular vaccination. Thus, intradermal vaccination with a pVITRO vector combining an anti-apoptotic strategy (Bcl-xL) and an intracellular targeting strategy (SEL) further enhances the E7-specific CD8+ T-cell response and guarantees co-expression of both encoded molecules in transfected cells.T.W.K. and C.-F.H. contributed equally to this work.