Stereo-selectivity of human serum albumin to enantiomeric and isoelectronic pollutants dissected by spectroscopy, calorimetry and bioinformatics

1-naphthol (1N), 2-naphthol (2N) and 8-quinolinol (8H) are general water pollutants. 1N and 2N are the configurational enantiomers and 8H is isoelectronic to 1N and 2N. These pollutants when ingested are transported in the blood by proteins like human serum albumin (HSA). Binding of these pollutants to HSA has been explored to elucidate the specific selectivity of molecular recognition by this multiligand binding protein. The association constants (K(b)) of these pollutants to HSA were moderate (10(4)-10(5) M(-1)). The proximity of the ligands to HSA is also revealed by their average binding distance, r, which is estimated to be in the range of 4.39-5.37 nm. The binding free energy (ΔG) in each case remains effectively the same for each site because of enthalpy-entropy compensation (EEC). The difference observed between ΔC(p) (exp) and ΔC(p) (calc) are suggested to be caused by binding-induced flexibility changes in the HSA. Efforts are also made to elaborate the differences observed in binding isotherms obtained through multiple approaches of calorimetry, spectroscopy and bioinformatics. We suggest that difference in dissociation constants of pollutants by calorimetry, spectroscopic and computational approaches could correspond to occurrence of different set of populations of pollutants having different molecular characteristics in ground state and excited state. Furthermore, our observation of enhanced binding of pollutants (2N and 8H) in the presence of hemin signifies that ligands like hemin may enhance the storage period of these pollutants in blood that may even facilitate the ill effects of these pollutants.     

The ascomycete Verticillium longisporum is a hybrid and a plant pathogen with an expanded host range

Hybridization plays a central role in plant evolution, but its overall importance in fungi is unknown. New plant pathogens are thought to arise by hybridization between formerly separated fungal species. Evolution of hybrid plant pathogens from non-pathogenic ancestors in the fungal-like protist Phytophthora has been demonstrated, but in fungi, the most important group of plant pathogens, there are few well-characterized examples of hybrids. We focused our attention on the hybrid and plant pathogen Verticillium longisporum, the causal agent of the Verticillium wilt disease in crucifer crops. In order to address questions related to the evolutionary origin of V. longisporum, we used phylogenetic analyses of seven nuclear loci and a dataset of 203 isolates of V. longisporum, V. dahliae and related species. We confirmed that V. longisporum was diploid, and originated three different times, involving four different lineages and three different parental species. All hybrids shared a common parent, species A1, that hybridized respectively with species D1, V. dahliae lineage D2 and V. dahliae lineage D3, to give rise to three different lineages of V. longisporum. Species A1 and species D1 constituted as yet unknown taxa. Verticillium longisporum likely originated recently, as each V. longisporum lineage was genetically homogenous, and comprised species A1 alleles that were identical across lineages.     

The inheritance of resistance to Verticillium wilt caused by race 1 isolates of <Emphasis Type="Italic">Verticillium dahliae</Emphasis> in the lettuce cultivar La Brillante

Verticillium wilt of lettuce caused by Verticillium dahliae can cause severe economic damage to lettuce producers. Complete resistance to race 1 isolates is available in Lactuca sativa cultivar (cv.) La Brillante and understanding the genetic basis of this resistance will aid development of new resistant cultivars. F₁ and F₂ families from crosses between La Brillante and three iceberg cultivars as well as a recombinant inbred line population derived from L. sativa cv. Salinas 88 × La Brillante were evaluated for disease incidence and disease severity in replicated greenhouse and field experiments. One hundred and six molecular markers were used to generate a genetic map from Salinas 88 × La Brillante and for detection of quantitative trait loci. Segregation was consistent with a single dominant gene of major effect which we are naming Verticillium resistance 1 (Vr1). The gene described large portions of the phenotypic variance (R ² = 0.49–0.68) and was mapped to linkage group 9 coincident with an expressed sequence tag marker (QGD8I16.yg.ab1) that has sequence similarity with the Ve gene that confers resistance to V. dahliae race 1 in tomato. The simple inheritance of resistance indicates that breeding procedures designed for single genes will be applicable for developing resistant cultivars. QGD8I16.yg.ab1 is a good candidate for functional analysis and development of markers suitable for marker-assisted selection.     

Structural and biochemical studies of serine acetyltransferase reveal why the parasite Entamoeba histolytica cannot form a cysteine synthase complex

Cysteine (Cys) plays a major role in growth and survival of the human parasite Entamoeba histolytica. We report here the crystal structure of serine acetyltransferase (SAT) isoform 1, a cysteine biosynthetic pathway enzyme from E. histolytica (EhSAT1) at 1.77 Å, in complex with its substrate serine (Ser) at 1.59 Å and inhibitor Cys at 1.78 Å resolution. EhSAT1 exists as a trimer both in solution as well as in crystal structure, unlike hexamers formed by other known SATs. The difference in oligomeric state is due to the N-terminal region of the EhSAT1, which has very low sequence similarity to known structures, also differs in orientation and charge distribution. The Ser and Cys bind to the same site, confirming that Cys is a competitive inhibitor of Ser. The disordered C-terminal region and the loop near the active site are responsible for solvent-accessible acetyl-CoA binding site and, thus, lose inhibition to acetyl-CoA by the feedback inhibitor Cys. Docking and fluorescence studies show that EhSAT1 C-terminal-mimicking peptides can bind to O-acetyl serine sulfhydrylase (EhOASS), whereas native C-terminal peptide does not show any binding. To test further, C-terminal end of EhSAT1 was mutated and found that it inhibits EhOASS, confirming modified EhSAT1 can bind to EhOASS. The apparent inability of EhSAT1 to form a hexamer and differences in the C-terminal region are likely to be the major reasons for the lack of formation of the large cysteine synthase complex and loss of a complex regulatory mechanism in E. histolytica.     

Evaluation of Replication and Cross-Reactive Antibody Responses of H2 Subtype Influenza Viruses in Mice and Ferrets

H2 influenza viruses have not circulated in humans since 1968, and therefore a large segment of the population would likely be susceptible to infection should H2 influenza viruses reemerge. The development of an H2 pandemic influenza virus vaccine candidate should therefore be considered a priority in pandemic influenza preparedness planning. We selected a group of geographically and temporally diverse wild-type H2 influenza viruses and evaluated the kinetics of replication and compared the ability of these viruses to induce a broadly cross-reactive antibody response in mice and ferrets. In both mice and ferrets, A/Japan/305/1957 (H2N2), A/mallard/NY/1978 (H2N2), and A/swine/MO/2006 (H2N3) elicited the broadest cross-reactive antibody responses against heterologous H2 influenza viruses as measured by hemagglutination inhibition and microneutralization assays. These data suggested that these three viruses may be suitable candidates for development as live attenuated H2 pandemic influenza virus vaccines.Influenza pandemics occur when a novel influenza virus enters a population with little preexisting immunity (36). During the pandemics of the last century, novel influenza viruses were introduced either directly from an avian reservoir (34) or were the result of reassortment between contemporaneously circulating human, avian, and swine influenza viruses (5, 29, 36). Due to the lack of preexisting immunity to the novel virus, morbidity and mortality rates are typically higher than in epidemics caused by seasonal influenza viruses (4).Although pandemic preparedness planning has largely focused on the highly pathogenic H5 and H7 avian influenza virus subtypes, the recent emergence of the 2009 pandemic H1N1 viruses underscores the need to consider other influenza virus subtypes as well. Of the 16 hemagglutinin (HA) influenza A virus subtypes that have been identified to date, H1, H2, and H3 have been known to cause influenza pandemics (7, 27), suggesting that these viruses are capable of sustained transmission and can cause disease in humans. While the H1 and H3 subtypes have cocirculated in humans since 1977, H2 influenza viruses have not circulated in humans since 1968 (36) and therefore a large segment of the population would likely be susceptible to infection should H2 influenza viruses reemerge. The 1957 H2 pandemic virus was a reassortant that derived the HA, neuraminidase (NA), and PB1 genes from an avian virus and the remaining gene segments from the circulating H1N1 virus (15, 30). As H2 subtype viruses continue to circulate in avian reservoirs worldwide (12, 17, 18, 22, 33), they remain a potential pandemic threat. The development of an H2 influenza virus vaccine candidate should therefore be considered a priority in future pandemic influenza preparedness planning.Given the low likelihood that a previously selected vaccine virus will exactly match the pandemic virus, the ability to elicit a broadly cross-reactive antibody response to antigenically distinct viruses within a subtype is an important consideration in the selection of a pandemic influenza vaccine candidate. Previous studies have examined the ability of inactivated H2 influenza viruses to provide cross-protection against mouse-adapted variants of reassortant human viruses and an avian H2 influenza virus from 1978 (9, 14). Given the potential for live attenuated influenza virus vaccines to confer a great breadth of heterologous cross-protection (1, 2, 6, 35), we recently conducted a study evaluating cold-adapted A/Ann Arbor/6/1960 (AA CA), an H2 influenza virus used as the backbone of the seasonal live attenuated influenza A virus vaccine currently licensed in the United States (3). However, as H2 influenza virus continues to circulate widely and appear in migratory birds (10, 24, 26), in poultry markets (20), and in swine (21), with evidence of interregional gene transmission (19, 22), a more extensive evaluation of recent isolates may be warranted in the selection of a potential H2 pandemic vaccine candidate.H2 influenza viruses fall into three main lineages: a human lineage, a North American avian lineage, and a Eurasian avian lineage (29). In addition to viruses whose replicative ability in mammals has previously been established (11, 21, 23, 25), we selected a group of geographically and temporally diverse H2 influenza viruses from each lineage. We evaluated the kinetics of replication of each of these viruses in mice and ferrets and compared the abilities of these viruses to induce a broadly cross-reactive antibody response to determine which of these viruses would be suitable for further development as an H2 pandemic influenza vaccine candidate.     

An Adjuvant for the Induction of Potent,Protective Humoral Responses to an H5N1 Influenza Virus Vaccine with Antigen-Sparing Effect in Mice

Intramuscular administration of inactivated influenza virus vaccine is the main vaccine platform used for the prevention of seasonal influenza virus infection. In clinical trials, inactivated H5N1 vaccines have been shown to be safe and capable of eliciting immune correlates of protection. However, the H5N1 vaccines are poorly immunogenic compared to seasonal influenza virus vaccines. Needle-free vaccination would be more efficient and economical in a pandemic, and the development of an effective and safe mucosal adjuvant will be an important milestone. A stabilized chemical analog of double-stranded RNA, PIKA, was previously reported to be a potent mucosal adjuvant in a murine model. While PIKA stimulates dendritic cells in vitro, little was known about its receptor and adjuvanting mechanism in vivo. In this study, we demonstrated that the immunostimulatory effect of PIKA resulted in an increased number of mature antigen-presenting cells, with the induction of proinflammatory cytokines at the inoculation site. In addition, coadministration of PIKA with a poorly immunogenic H5N1 subunit vaccine led to antigen sparing and quantitative and qualitative improvements of the immune responses over those achieved with an unadjuvanted vaccine in mice. The adjuvanted vaccine provided protection against lethal challenge with homologous and heterologous H5N1 wild-type viruses. Mice lacking functional TLR3 showed diminished cytokine production with PIKA stimulation, diminished antibody responses, and reduced protective efficacy against wild-type virus challenge following vaccination. These data suggest that TLR3 is important for the optimal performance of PIKA as an adjuvant. With its good safety profile and antigen-sparing effect, PIKA could be an attractive adjuvant for use in future pandemics.Influenza is an acute respiratory disease associated with significant morbidity and mortality worldwide. The newly emerged swine-origin H1N1 virus has caused the first influenza pandemic of this century (4). Since its appearance in April 2009, the virus has spread to every continent and caused significant morbidity and mortality (WHO website, http://gamapserver.who.int/h1n1/cases-deaths/h1n1_casesdeaths.html). The sporadic transmission of highly pathogenic avian influenza (HPAI) viruses (H5N1 influenza A viruses) from poultry to humans in Asia also raises concerns about a possible pandemic (2, 28).Although vaccination is the most effective tool for the control of influenza (7, 33), the combined production capacity of global vaccine suppliers is not sufficient to meet the demand during a pandemic, so a vaccine shortage is expected. Any strategy that can maximize vaccine coverage will be valuable in a pandemic.Inactivated seasonal influenza virus vaccines are administered mainly by the intramuscular (i.m.) route; however, it has been demonstrated that intranasal (i.n.) administration of inactivated influenza virus vaccines is more effective at inducing nasal IgA responses and protecting the respiratory epithelium (1, 47). Induction of immunity by the intranasal route often requires a high dose of vaccine or the inclusion of an adjuvant. Although a number of compounds have been identified as promising mucosal adjuvants, there is a need to continue to develop safe mucosal adjuvants, because some compounds, such as Escherichia coli heat-labile toxin and poly(I:C), are associated with significant side effects (27, 37).We previously demonstrated the potency of a stabilized chemical analog of double-stranded RNA (dsRNA), PIKA, as an adjuvant for a seasonal influenza virus vaccine with a substantial antigen-sparing effect in mice (25). While we and others have shown that PIKA activates dendritic cells (DC) in culture (25, 38), there are no reports on this effect in vivo, and the protective efficacy of PIKA-adjuvanted vaccine against wild-type (wt) virus challenge has not been demonstrated. The current study was designed to evaluate changes in the number and phenotypic expression of local antigen-presenting cells (APC) and in cytokine expression at the inoculation site and to evaluate the adjuvanting potency of PIKA in a lethal-challenge model using a wt influenza virus with pandemic potential. The A/Vietnam/1203/2004 (H5N1) virus was chosen over the A/California/04/2009 (H1N1) virus as the challenge virus for two reasons. First, the H5N1 virus is more virulent than the 2009 H1N1 pandemic virus in mice (the 50% mouse lethal doses [MLD₅₀] of the H5N1 and the H1N1 viruses are 10^0.4 and 10^5.8 50% tissue culture infective doses [TCID₅₀], respectively [20, 41]), which allows a higher lethal-challenge dose to be used in the experiments. Second, the unadjuvanted split-virion H5N1 vaccine was poorly immunogenic in humans, requiring 12 times more antigen (two doses of 90 μg) than the typical seasonal influenza virus vaccine (15 μg) in order to generate immunity associated with protection against influenza in humans (42), while data from the H1N1 human vaccine trial show that the unadjuvanted H1N1 vaccine is able to elicit robust immune responses after a single dose (14, 51). Our results show that administration of PIKA with inactivated H5N1 vaccine elicited a rapid production of proinflammatory cytokines with infiltration of mature DC at the site of administration. This vaccine formulation allowed significant antigen sparing and provided protection against lethal challenge with the wt HPAI viruses A/Vietnam/1203/2004 and A/Indonesia/05/2005 (H5N1).     

The Open Reading Frame 3a Protein of Severe Acute Respiratory Syndrome-Associated Coronavirus Promotes Membrane Rearrangement and Cell Death

The genome of the severe acute respiratory syndrome-associated coronavirus (SARS-CoV) contains eight open reading frames (ORFs) that encode novel proteins. These accessory proteins are dispensable for in vitro and in vivo replication and thus may be important for other aspects of virus-host interactions. We investigated the functions of the largest of the accessory proteins, the ORF 3a protein, using a 3a-deficient strain of SARS-CoV. Cell death of Vero cells after infection with SARS-CoV was reduced upon deletion of ORF 3a. Electron microscopy of infected cells revealed a role for ORF 3a in SARS-CoV induced vesicle formation, a prominent feature of cells from SARS patients. In addition, we report that ORF 3a is both necessary and sufficient for SARS-CoV-induced Golgi fragmentation and that the 3a protein accumulates and localizes to vesicles containing markers for late endosomes. Finally, overexpression of ADP-ribosylation factor 1 (Arf1), a small GTPase essential for the maintenance of the Golgi apparatus, restored Golgi morphology during infection. These results establish an important role for ORF 3a in SARS-CoV-induced cell death, Golgi fragmentation, and the accumulation of intracellular vesicles.The severe acute respiratory syndrome-associated coronavirus (SARS-CoV) genome encodes several smaller open reading frames (ORFs) located in the 3′ region of the genome that are predicted to express eight novel proteins termed accessory proteins. The accessory proteins are designated ORFs 3a, 3b, 6, 7a, 7b, 8a, 8b, and 9b and range in size from 39 to 274 amino acids (35, 50). These SARS-CoV-specific ORFs are not present in other coronaviruses and do not display significant homology with any known proteins in the NCBI database. Five of these are predicted to code for polypeptides of greater than 50 amino acids (35, 50). Antibodies reactive against all of the SARS-CoV proteins have been detected in sera isolated from SARS patients, indicating that these proteins are expressed by the virus in vivo (7, 9, 17-19, 45, 59). Expression of three of the ORF proteins has been demonstrated during infection using protein-specific antibodies and include the ORFs 3a, 6, and 7a (12, 37, 41, 60). Six of the eight group-specific ORFs, including ORFs 3a, 3b, 6, 7a, 7b, and 9b, were deleted from recombinant SARS-CoV and shown to be dispensable for in vitro and in vivo replication (66).Related coronaviruses also encode unique accessory proteins in the 3′ region of the genome, often referred to as group-specific ORFs. Similar to SARS-CoV, several of these proteins are dispensable for viral replication. Murine hepatitis virus (MHV) expresses accessory proteins ORFs 2a, 4, and 5a. A recombinant virus in which ORF 2a was deleted replicated normally in vitro but caused attenuated disease in vivo (55). Deletion of the group-specific ORF 7 in porcine coronavirus TGEV also results in reduced replication and virulence in vivo despite normal replication in vitro (38). Similarly, in feline infectious peritonitis virus (FIPV), group-specific proteins are dispensable for replication in cell culture but contribute to pathogenesis in vivo (20). Thus, while the SARS-CoV group specific proteins are unnecessary for in vitro and in vivo replication, their expression may underlie the devastating pathology associated with SARS disease. Detailed characterization of these novel proteins may contribute to a better understanding of SARS pathogenesis and host-virus interactions.The ORF 3a protein is expressed from subgenomic RNA3, which contains the 3a and 3b ORFs (35, 50). The 3a protein, which is the largest group-specific SARS-CoV accessory protein at 274 amino acids, has been reported to localize to the Golgi apparatus, the plasma membrane, and intracellular vesicles of unknown origin (67, 68). The protein is efficiently transported to the cell surface and is also internalized during the process of endocytosis (60).The mechanism of SARS-CoV-induced cell death has been investigated by several groups. Studies to date have used overexpression of individual SARS-CoV ORFs to evaluate their intrinsic cytotoxicity. Using this approach, the following proteins have been reported to cause apoptosis: the 3CL-like protease; spike; ORFs 3a, 3b, and 7a; and the envelope (E), membrane (M), and nucleocapsid (N) proteins (23, 31, 32, 36, 46, 58, 61, 65, 69). However, since all of these reports utilize overexpression of individual proteins, it is unclear whether these effects may be attributable to high, nonphysiological levels of protein and whether they occur during infection. Analysis of recombinant viruses with specific mutations or deletions is necessary to determine the relative contribution of these proteins to the cytotoxicity of SARS-CoV during infection (63). Therefore, the cytotoxic component(s) of SARS-CoV have not been fully defined.Here, we have investigated the function of the ORF 3a protein in the context of SARS-CoV infection and by overexpression. We confirm that ORF 3a contributes to SARS-CoV cytotoxicity using a recombinant strain deficient for expression of ORF 3a. While characterizing this deficient strain, we observed that SARS-CoV-induced vesicle formation, a feature that has been documented in cells from infected SARS patients, is dependent on ORF 3a. Furthermore, we observed that SARS-CoV infection causes Golgi fragmentation by ORF 3a. Additional characterization of 3a in transfected cells revealed that the protein colocalizes with markers of the trans-Golgi network (TGN) and late endosomal pathways and causes an accumulation of these vesicles. Finally, we report that Arf1 overexpression rescued SARS-CoV or 3a-induced Golgi fragmentation, suggesting that the ORF 3a protein may perturb Arf1-mediated vesicle trafficking.     

Scientific barriers to developing vaccines against avian influenza viruses

The increasing number of reports of direct transmission of avian influenza viruses to humans underscores the need for control strategies to prevent an influenza pandemic. Vaccination is the key strategy to prevent severe illness and death from pandemic influenza. Despite long-term experience with vaccines against human influenza viruses, researchers face several additional challenges in developing human vaccines against avian influenza viruses. In this Review, we discuss the features of avian influenza viruses, the gaps in our understanding of infections caused by these viruses in humans and of the immune response to them that distinguishes them from human influenza viruses, and the current status of vaccine development.     

Quality assessment of buccal versus blood genomic DNA using the Affymetrix 500 K GeneChip

Background

Cystic fibrosis (CF) mice, created with a genetically engineered mutation in the Cystic fibrosis transmembrane conductance regulator (Cftr) gene, may develop intestinal plugs which limit their survival past weaning. In a studied population of genetically mixed CF mice differences in allelic ratios at particular loci, between surviving CF mice and mice with the lethal intestinal defect, were used to map cystic fibrosis modifier gene one, Cfm1. Using this approach, we previously identified an X chromosome locus which may influence the survival to weaning of C57BL/6J × BALB/cJ F2 CF mice. We also detected two regions of transmission ratio distortion, independent of Cftr genotype, in a limited dataset. To investigate these findings, in this study we have genotyped 1208 three-week old F2 mice, and 186 day E15.5 embryos, derived from a congenic (C57BL/6J × BALB/cJ) F1 Cftr +/- intercross, for the putative distortion regions.

Results

An excess of homozygous BALB genotypes, compared to Mendelian expectations, was detected on chromosomes 5 (p = 5.7 × 10^-15) and X (p = 3.0 × 10^-35) in three-week old female mice but transmission ratio distortion was not evident in the tested region of chromosome 3 (p = 0.39). Significant pre-weaning lethality of CF mice occurred as 11.3% (137/1208) of the three-week old offspring were identified as CF mice. X chromosome genotypes were not, however, distorted in the female CF mice (p = 0.62), thus the significant non-Mendelian inheritance of this locus was dependent on CF status. The survival of CF embryos to day E15.5 was consistent with Mendelian expectations (42/186 = 23%), demonstrating the loss of CF mice to have occurred between E15.5 and three weeks of age. The excess of X chromosome homozygous BALB genotypes was recorded in female embryos (p = 0.0048), including CF embryos, indicating the distortion to be evident at this age.

Conclusion

Two of three previously suggested loci of transmission ratio distortion were replicated as distorted in this mouse cross. The non-Mendelian inheritance of X chromosome genotypes implicates this region in the survival to weaning of non-CF mice.