Identification of novel subgroup a variants with enhanced receptor binding and replicative capacity in primary isolates of anaemogenic strains of feline leukaemia virus

<b>BACKGROUND:</b> The development of anaemia in feline leukaemia virus (FeLV)-infected cats is associated with the emergence of a novel viral subgroup, FeLV-C. FeLV-C arises from the subgroup that is transmitted, FeLV-A, through alterations in the amino acid sequence of the receptor binding domain (RBD) of the envelope glycoprotein that result in a shift in the receptor usage and the cell tropism of the virus. The factors that influence the transition from subgroup A to subgroup C remain unclear, one possibility is that a selective pressure in the host drives the acquisition of mutations in the RBD, creating A/C intermediates with enhanced abilities to interact with the FeLV-C receptor, FLVCR. In order to understand further the emergence of FeLV-C in the infected cat, we examined primary isolates of FeLV-C for evidence of FeLV-A variants that bore mutations consistent with a gradual evolution from FeLV-A to FeLV-C.<p></p> <b>RESULTS:</b> Within each isolate of FeLV-C, we identified variants that were ostensibly subgroup A by nucleic acid sequence comparisons, but which bore mutations in the RBD. One such mutation, N91D, was present in multiple isolates and when engineered into a molecular clone of the prototypic FeLV-A (Glasgow-1), enhanced replication was noted in feline cells. Expression of the N91D Env on murine leukaemia virus (MLV) pseudotypes enhanced viral entry mediated by the FeLV-A receptor THTR1 while soluble FeLV-A Env bearing the N91D mutation bound more efficiently to mouse or guinea pig cells bearing the FeLV-A and -C receptors. Long-term in vitro culture of variants bearing the N91D substitution in the presence of anti-FeLV gp70 antibodies did not result in the emergence of FeLV-C variants, suggesting that additional selective pressures in the infected cat may drive the subsequent evolution from subgroup A to subgroup C.<p></p> <b>CONCLUSIONS:</b> Our data support a model in which variants of FeLV-A, bearing subtle differences in the RBD of Env, may be predisposed towards enhanced replication in vivo and subsequent conversion to FeLV-C. The selection pressures in vivo that drive the emergence of FeLV-C in a proportion of infected cats remain to be established

Kinetic Pathway of Pyrophosphorolysis by a Retrotransposon Reverse Transcriptase

DNA and RNA polymerases use a common phosphoryl transfer mechanism for base addition that requires two or three acidic amino acid residues at their active sites. We previously showed, for the reverse transcriptase (RT) encoded by the yeast retrotransposon Ty1, that one of the three conserved active site aspartates (D211) can be substituted by asparagine and still retain in vitro polymerase activity, although in vivo transposition is lost. Transposition is partially restored by second site suppressor mutations in the RNAse H domain. The novel properties of this amino acid substitution led us to express the WT and D211N mutant enzymes, and study their pre-steady state kinetic parameters. We found that the kpol was reduced by a factor of 223 in the mutant, although the Kd for nucleotide binding was unaltered. Further, the mutant enzyme had a marked preference for Mn2+ over Mg2+. To better understand the functions of this residue within the Ty1 RT active site, we have now examined the in vitro properties of WT and D211N mutant Ty1 RTs in carrying out pyrophosphorolysis, the reverse reaction to polymerization, where pyrophosphate is the substrate and dNTPs are the product. We find that pyrophosphorolysis is efficient only when the base-paired primer template region is >14 bases, and that activity increases when the primer end is blunt-ended or recessed by only a few bases. Using pre-steady state kinetic analysis, we find that the rate of pyrophosphorolysis (kpyro) in the D211N mutant is nearly 320 fold lower than the WT enzyme, and that the mutant enzyme has an ∼170 fold lower apparent Kd for pyrophosphate. These findings indicate that subtle substrate differences can strongly affect the enzyme's ability to properly position the primer-end to carry out pyrophosphorolysis. Further the kinetic data suggests that the D211 residue has a role in pyrophosphate binding and release, which could affect polymerase translocation, and help explain the D211N mutant's transposition defect

Association and Linkage Analysis of Aluminum Tolerance Genes in Maize

Aluminum (Al) toxicity is a major worldwide constraint to crop productivity on acidic soils. Al becomes soluble at low pH, inhibiting root growth and severely reducing yields. Maize is an important staple food and commodity crop in acidic soil regions, especially in South America and Africa where these soils are very common. Al exclusion and intracellular tolerance have been suggested as two important mechanisms for Al tolerance in maize, but little is known about the underlying genetics. linkage populations with approximately 200 individuals each were used to study genetic variation in this complex trait. Al tolerance was measured as net root growth in nutrient solution under Al stress, which exhibited a wide range of variation between lines. Comparative and physiological genomics-based approaches were used to select 21 candidate genes for evaluation by association analysis.). These four candidate genes are high priority subjects for follow-up biochemical and physiological studies on the mechanisms of Al tolerance in maize. Immediately, elite haplotype-specific molecular markers can be developed for these four genes and used for efficient marker-assisted selection of superior alleles in Al tolerance maize breeding programs

Derivation of Myoepithelial Progenitor Cells from Bipotent Mammary Stem/Progenitor Cells

There is increasing evidence that breast and other cancers originate from and are maintained by a small fraction of stem/progenitor cells with self-renewal properties. Recent molecular profiling has identified six major subtypes of breast cancer: basal-like, ErbB2-overexpressing, normal breast epithelial-like, luminal A and B, and claudin-low subtypes. To help understand the relationship among mammary stem/progenitor cells and breast cancer subtypes, we have recently derived distinct hTERT-immortalized human mammary stem/progenitor cell lines: a K5+/K19− type, and a K5+/K19+ type. Under specific culture conditions, bipotent K5+/K19− stem/progenitor cells differentiated into stable clonal populations that were K5−/K19− and exhibit self-renewal and unipotent myoepithelial differentiation potential in contrast to the parental K5+/K19− cells which are bipotent. These K5−/K19− cells function as myoepithelial progenitor cells and constitutively express markers of an epithelial to mesenchymal transition (EMT) and show high invasive and migratory abilities. In addition, these cells express a microarray signature of claudin-low breast cancers. The EMT characteristics of an un-transformed unipotent mammary myoepithelial progenitor cells together with claudin-low signature suggests that the claudin-low breast cancer subtype may arise from myoepithelial lineage committed progenitors. Availability of immortal MPCs should allow a more definitive analysis of their potential to give rise to claudin-low breast cancer subtype and facilitate biological and molecular/biochemical studies of this disease

Muramyl Dipeptide Induces NOD2-Dependent Ly6Chigh Monocyte Recruitment to the Lungs and Protects Against Influenza Virus Infection

Bacterial peptidoglycan-derived muramyl dipeptide (MDP) and derivatives have long-recognized antiviral properties but their mechanism of action remains unclear. In recent years, the pattern-recognition receptor NOD2 has been shown to mediate innate responses to MDP. Here, we show that MDP treatment of mice infected with Influenza A virus (IAV) significantly reduces mortality, viral load and pulmonary inflammation in a NOD2-dependent manner. Importantly, the induction of type I interferon (IFN) and CCL2 chemokine was markedly increased in the lungs following MDP treatment and correlated with a NOD2-dependent enhancement in circulating monocytes. Mechanistically, the protective effect of MDP could be explained by the NOD2-dependent transient increase in recruitment of Ly6Chigh “inflammatory” monocytes and, to a lesser extent, neutrophils to the lungs. Indeed, impairment in both Ly6Chigh monocyte recruitment and survival observed in infected Nod2-/- mice treated with MDP was recapitulated in mice deficient for the chemokine receptor CCR2 required for CCL2-mediated Ly6Chigh monocyte migration from the bone marrow into the lungs. MDP-induced pulmonary monocyte recruitment occurred normally in IAV-infected and MDP-treated Ips-1-/- mice. However, IPS-1 was required for improved survival upon MDP treatment. Finally, mycobacterial N-glycolyl MDP was more potent than N-acetyl MDP expressed by most bacteria at reducing viral burden while both forms of MDP restored pulmonary function following IAV challenge. Overall, our work sheds light on the antiviral mechanism of a clinically relevant bacterial-derived compound and identifies the NOD2 pathway as a potential therapeutic target against IAV

Tuberculosis chemotherapy: current drug delivery approaches

Tuberculosis is a leading killer of young adults worldwide and the global scourge of multi-drug resistant tuberculosis is reaching epidemic proportions. It is endemic in most developing countries and resurgent in developed and developing countries with high rates of human immunodeficiency virus infection. This article reviews the current situation in terms of drug delivery approaches for tuberculosis chemotherapy. A number of novel implant-, microparticulate-, and various other carrier-based drug delivery systems incorporating the principal anti-tuberculosis agents have been fabricated that either target the site of tuberculosis infection or reduce the dosing frequency with the aim of improving patient outcomes. These developments in drug delivery represent attractive options with significant merit, however, there is a requisite to manufacture an oral system, which directly addresses issues of unacceptable rifampicin bioavailability in fixed-dose combinations. This is fostered by the need to deliver medications to patients more efficiently and with fewer side effects, especially in developing countries. The fabrication of a polymeric once-daily oral multiparticulate fixed-dose combination of the principal anti-tuberculosis drugs, which attains segregated delivery of rifampicin and isoniazid for improved rifampicin bioavailability, could be a step in the right direction in addressing issues of treatment failure due to patient non-compliance

Distinct External Signals Trigger Sequential Release of Apical Organelles during Erythrocyte Invasion by Malaria Parasites

The invasion of erythrocytes by Plasmodium merozoites requires specific interactions between host receptors and parasite ligands. Parasite proteins that bind erythrocyte receptors during invasion are localized in apical organelles called micronemes and rhoptries. The regulated secretion of microneme and rhoptry proteins to the merozoite surface to enable receptor binding is a critical step in the invasion process. The sequence of these secretion events and the external signals that trigger release are not known. We have used time-lapse video microscopy to study changes in intracellular calcium levels in Plasmodium falciparum merozoites during erythrocyte invasion. In addition, we have developed flow cytometry based methods to measure relative levels of cytosolic calcium and study surface expression of apical organelle proteins in P. falciparum merozoites in response to different external signals. We demonstrate that exposure of P. falciparum merozoites to low potassium ion concentrations as found in blood plasma leads to a rise in cytosolic calcium levels through a phospholipase C mediated pathway. Rise in cytosolic calcium triggers secretion of microneme proteins such as the 175 kD erythrocyte binding antigen (EBA175) and apical membrane antigen-1 (AMA-1) to the merozoite surface. Subsequently, interaction of EBA175 with glycophorin A (glyA), its receptor on erythrocytes, restores basal cytosolic calcium levels and triggers release of rhoptry proteins. Our results identify for the first time the external signals responsible for the sequential release of microneme and rhoptry proteins during erythrocyte invasion and provide a starting point for the dissection of signal transduction pathways involved in regulated exocytosis of these key apical organelles. Signaling pathway components involved in apical organelle discharge may serve as novel targets for drug development since inhibition of microneme and rhoptry secretion can block invasion and limit blood-stage parasite growth

Growth Performance and Root Transcriptome Remodeling of Arabidopsis in Response to Mars-Like Levels of Magnesium Sulfate

Martian regolith (unconsolidated surface material) is a potential medium for plant growth in bioregenerative life support systems during manned missions on Mars. However, hydrated magnesium sulfate mineral levels in the regolith of Mars can reach as high as 10 wt%, and would be expected to be highly inhibitory to plant growth. at 180 min. after initiation of treatment. mutants exhibit partial tolerance to magnesium sulfate, and by elucidating a small subset (500 vs. >10,000) of candidate genes for mutation or metabolic engineering that will enhance tolerance to magnesium sulfate soils