Novel pyrazolo[1,5-a]pyridines with improved aqueous solubility as p110α-selective PI3 kinase inhibitors

As part of our investigation into pyrazolo[1,5-a]pyridines as novel p110α selective PI3 kinase inhibitors, we report a range of analogues with improved aqueous solubility by the addition of a basic amine. The compounds demonstrated comparable p110α potency and selectivity to earlier compounds but with up to 1000× greater aqueous solubility, as the hydrochloride salts. The compounds also displayed good activity in a cellular assay of PI3 kinase activity.     

Marker-assisted identification of restorer gene(s) in iso-cytoplasmic restorer lines of WA cytoplasm in rice and assessment of their fertility restoration potential across environments

Iso-cytoplasmic restorers possess the same male sterile cytoplasm as the cytoplasmic male sterile (CMS) lines, thereby minimizing the potential cyto-nuclear conflict in the hybrids. Restoration of fertility of the wild abortive CMS is governed by two major genes namely, Rf3 and Rf4. Therefore, assessing the allelic status of these restorer genes in the iso-cytoplasmic restorers using molecular markers will not only help in estimating the efficiency of these genes either alone or in combination, in fertility restoration in the hybrids in different environments, but will also be useful in determining the efficacy of these markers. In the present study, the efficiency of molecular markers in identifying genotypes carrying restorer allele of the gene(s) Rf3 and Rf4, restoring male fertility of WA cytoplasm in rice was assessed in a set of 100 iso-cytoplasmic rice restorers using gene linked as well as candidate gene based markers. In order to validate the efficacy of markers in identifying the restorers, a sub-set of selected 25 iso-cytoplasmic rice restorers were crossed with four different cytoplasmic male sterile lines namely, IR 79156A, IR 58025A, Pusa 6A and RTN 12A, and the pollen and spikelet fertility of the F₁s were evaluated at three different locations. Marker analysis showed that Rf4 was the predominant fertility restorer gene in the iso-cytoplasmic restorers and Rf3 had a synergistic effect on fertility restoration. The efficiency of gene based markers, DRCG-RF4-14 and DRRM-RF3-10 for Rf4 (87%) and Rf3 (84%) genes was higher than respective gene-linked SSR markers RM6100 (80%) and RM3873 (82%). It is concluded that the gene based markers can be effectively used in identifying fertility restorer lines obviating the need for making crosses and evaluating the F₁s. Though gene based markers are more efficient, there is a need to identify functional polymorphisms which can provide 100% efficiency. Three iso-cytoplasmic restorers namely, PRR 300, PRR 363 and PRR 396 possessing both Rf4 and Rf3 genes and good fertility restoration have been identified which could be used further in hybrid rice breeding.     

Rapid biosynthesis of PVP coated silver nanoparticles by <Emphasis Type="Italic">Kocuria rosea</Emphasis> and their antimicrobial activity

The need for more effective antimicrobial agent and propitious application of nanotechnology in therapeutics and diagnostics has prompted the research on ecofriendly synthesis of silver nanoparticles. The objective of present study was to investigate the antibacterial and antifungal activity of biologically synthesized silver nanoparticles. The silver nanoparticles were synthesized by extracellular method, using soil bacteria Kocuria rosea. The synthesized silver nanoparticles were characterized by UV-Visible spectroscopy, X-ray diffractometry (XRD), transmission electron microscopy (TEM) and fourier transformation infrared spectroscopy (FTIR). On the basis of TEM analysis, the synthesized nanoparticles were found to be spherical with an average size of 30–50 nm. The biologically synthesized silver nanoparticles showed significant antimicrobial activity against pathogens.     

MCM9 binds Cdt1 and is required for the assembly of prereplication complexes

Prereplication complexes (pre-RCs) define potential origins of DNA replication and allow the recruitment of the replicative DNA helicase MCM2-7. Here, we characterize MCM9, a member of the MCM2-8 family. We demonstrate that MCM9 binds to chromatin in an ORC-dependent manner and is required for the recruitment of the MCM2-7 helicase onto chromatin. Its depletion leads to a block in pre-RC assembly, as well as DNA replication inhibition. We show that MCM9 forms a stable complex with the licensing factor Cdt1, preventing an excess of geminin on chromatin during the licensing reaction. Our data suggest that MCM9 is an essential activating linker between Cdt1 and the MCM2-7 complex, required for loading the MCM2-7 helicase onto DNA replication origins. Thus, Cdt1, with its two opposing regulatory binding factors MCM9 and geminin, appears to be a major platform on the pre-RC to integrate cell-cycle signals.     

A vector system for genomic FLAG epitope-tagging in Schizosaccharomyces pombe

The fission yeast Schizosaccharomyces pombe is a popular model organism to study various cellular processes, although research tools available for S. pombe are relatively inadequate. To facilitate genetic and biochemical investigation in S. pombe, we report here a system of vectors for genomic FLAG epitope-tagging. These vectors enable us to amplify gene-targeting fragments for integration into specific loci of the S. pombe genome. All vectors in this report were designed to express FLAG epitope-tagged proteins from their endogenous genomic loci. Vectors for N-terminal FLAG epitope-tagging allow us to control protein expression levels using the wild-type nmt1 promoter, its weaker derivatives, and the urg1 promoter. These vectors are available with various antibiotic markers including kanMX6, hphMX6, natMX6 and bleMX6, and the his3(+) marker. Vectors for C-terminal FLAG epitope-tagging were designed to express FLAG-fusion proteins under the control of their native promoters at their own genomic loci, allowing us to characterize protein functions under physiological conditions. These vectors are available with kanMX6, hphMX6, nat-MX6 and bleMX6 markers. The series of vectors described in this report should prove useful for protein studies in fission yeast.     

Is Exercise Protective Against Influenza-Associated Mortality?

Background

Little is known about the effect of physical exercise on influenza-associated mortality.

Methods and Findings

We collected information about exercise habits and other lifestyles, and socioeconomic and demographic status, the underlying cause of death of 24,656 adults (21% aged 30–64, 79% aged 65 or above) who died in 1998 in Hong Kong, and the weekly proportion of specimens positive for influenza A (H3N1 and H1N1) and B isolations during the same period. We assessed the excess risks (ER) of influenza-associated mortality due to all-natural causes, cardiovascular diseases, or respiratory disease among different levels of exercise: never/seldom (less than once per month), low/moderate (once per month to three times per week), and frequent (four times or more per week) by Poisson regression. We also assessed the differences in ER between exercise groups by case-only logistic regression. For all the mortality outcomes under study in relation to each 10% increase in weekly proportion of specimens positive for influenza A+B, never/seldom exercise (as reference) was associated with 5.8% to 8.5% excess risks (ER) of mortality (P<0.0001), while low/moderate exercise was associated with ER which were 4.2% to 6.4% lower than those of the reference (P<0.001 for all-natural causes; P = 0.001 for cardiovascular; and P = 0.07 for respiratory mortality). Frequent exercise was not different from the reference (change in ER −0.8% to 1.7%, P = 0.30 to 0.73).

Conclusion

When compared with never or seldom exercise, exercising at low to moderate frequency is beneficial with lower influenza-associated mortality.     

Heterosubtypic Neutralizing Monoclonal Antibodies Cross-Protective against H5N1 and H1N1 Recovered from Human IgM+ Memory B Cells

Background

The hemagglutinin (HA) glycoprotein is the principal target of protective humoral immune responses to influenza virus infections but such antibody responses only provide efficient protection against a narrow spectrum of HA antigenic variants within a given virus subtype. Avian influenza viruses such as H5N1 are currently panzootic and pose a pandemic threat. These viruses are antigenically diverse and protective strategies need to cross protect against diverse viral clades. Furthermore, there are 16 different HA subtypes and no certainty the next pandemic will be caused by an H5 subtype, thus it is important to develop prophylactic and therapeutic interventions that provide heterosubtypic protection.

Methods and Findings

Here we describe a panel of 13 monoclonal antibodies (mAbs) recovered from combinatorial display libraries that were constructed from human IgM⁺ memory B cells of recent (seasonal) influenza vaccinees. The mAbs have broad heterosubtypic neutralizing activity against antigenically diverse H1, H2, H5, H6, H8 and H9 influenza subtypes. Restriction to variable heavy chain gene IGHV1-69 in the high affinity mAb panel was associated with binding to a conserved hydrophobic pocket in the stem domain of HA. The most potent antibody (CR6261) was protective in mice when given before and after lethal H5N1 or H1N1 challenge.

Conclusions

The human monoclonal CR6261 described in this study could be developed for use as a broad spectrum agent for prophylaxis or treatment of human or avian influenza infections without prior strain characterization. Moreover, the CR6261 epitope could be applied in targeted vaccine strategies or in the design of novel antivirals. Finally our approach of screening the IgM⁺ memory repertoire could be applied to identify conserved and functionally relevant targets on other rapidly evolving pathogens.     

Supervised Lowess normalization of comparative genome hybridization data – application to lactococcal strain comparisons

Background

Visualising the evolutionary history of a set of sequences is a challenge for molecular phylogenetics. One approach is to use undirected graphs, such as median networks, to visualise phylogenies where reticulate relationships such as recombination or homoplasy are displayed as cycles. Median networks contain binary representations of sequences as nodes, with edges connecting those sequences differing at one character; hypothetical ancestral nodes are invoked to generate a connected network which contains all most parsimonious trees. Quasi-median networks are a generalisation of median networks which are not restricted to binary data, although phylogenetic information contained within the multistate positions can be lost during the preprocessing of data. Where the history of a set of samples contain frequent homoplasies or recombination events quasi-median networks will have a complex topology. Graph reduction or pruning methods have been used to reduce network complexity but some of these methods are inapplicable to datasets in which recombination has occurred and others are procedurally complex and/or result in disconnected networks.

Results

We address the problems inherent in construction and reduction of quasi-median networks. We describe a novel method of generating quasi-median networks that uses all characters, both binary and multistate, without imposing an arbitrary ordering of the multistate partitions. We also describe a pruning mechanism which maintains at least one shortest path between observed sequences, displaying the underlying relations between all pairs of sequences while maintaining a connected graph.

Conclusion

Application of this approach to 5S rDNA sequence data from sea beet produced a pruned network within which genetic isolation between populations by distance was evident, demonstrating the value of this approach for exploration of evolutionary relationships.     

Effect of cultivation time and growth medium on the coaggregation capability of <Emphasis Type="Italic">Acinetobacter johnsonii</Emphasis> S35

The effects of different cultivation periods and growth media on the cell surface hydrophobicity and coaggregation ability of Acinetobacter johnsonii S35 with Oligotropha carboxidovorans S23 (both sewage sludge isolates) were examined. It was observed that when cultivated in polypeptone medium, A. johnsonii S35 shows high hydrophobicity and strong coaggregation (98% aggregation index) with O. carboxidovorans S23, which remain unaltered after a 1-day, 3-day or 7-day cultivation period. When A. johnsonii S35 was cultivated with different carbon substrates, large coaggregates and a high aggregation index were observed using acetate-grown cells (96%), followed by ethanol-(83%) and vegetable oil-grown cells (72%). Coaggregates were small and narrow in ethanol-grown cells and still smaller in case of the vegetable oil-grown cells. Although no direct correlation could be observed, more hydrophobic cells showed a higher aggregation index. None of the culture supernatants of A. johnsonii S35 showed emulsification of hexadecane or a significant ability to aggregate O. carboxidovorans S23 cells, indicating that the coaggregation factor of A. johnsonii S35 is cell-bound. These results demonstrate that the coaggregation ability of A. johnsonii S35 is constitutive and is not lost under diverse cultivation conditions.     

Evaluation of the Inheritance of the Complex Vertebral Malformation Syndrome by Breeding Studies

To investigate the congenital complex vertebral malformation syndrome (CVM) in Holstein calves, two breeding studies were performed including 262 and 363 cows, respectively. Cows were selected from the Danish Cattle Database based on pedigree and insemination records. Selected cows were progeny of sires with an established heterozygous CVM genotype and pregnant after insemination with semen from another sire with heterozygous CVM genotype. Following calving the breeders should state, if the calf was normal and was requested to submit dead calves for necropsy. In both studies, significantly fewer CVM affected calves than expected were obtained; a finding probably reflecting extensive intrauterine mortality in CVM affected foetuses. The findings illustrate increased intrauterine mortality as a major potential bias in observational studies of inherited disorders.