A highly conserved nuclear gene for low-level phylogenetics: elongation factor-1 alpha recovers morphology-based tree for heliothine moths

Molecular systematists need increased access to nuclear genes. Highly conserved, low copy number protein-encoding nuclear genes have attractive features for phylogenetic inference but have heretofore been applied mostly to very ancient divergences. By virtue of their synonymous substitutions, such genes should contain a wealth of information about lower-level taxonomic relationships as well, with the advantage that amino acid conservatism makes both alignment and primer definition straightforward. We tested this postulate for the elongation factor-1 alpha (EF-1 alpha) gene in the noctuid moth subfamily Heliothinae, which has probably diversified since the middle Tertiary. We sequenced 1,240 bp in 18 taxa representing heliothine groupings strongly supported by previous morphological and allozyme studies. The single most parsimonious gene tree and the neighbor-joining tree for all nucleotides show almost complete concordance with the morphological tree. Homoplasy and pairwise divergence levels are low, transition/transversion ratios are high, and phylogenetic information is spread evenly across gene regions. The EF-1 alpha gene and presumably other highly conserved genes hold much promise for phylogenetics of Tertiary age eukaryote groups.      

Plant virus infection development as affected by heavy metal stress

The work was focused on the investigation of possible dependencies between the development of viral infection in plants and the presence of high heavy metal concentrations in soil. Field experiments have been conducted in order to study the development of systemic tobacco mosaic virus (TMV) infection in Lycopersicon esculentum L. cv. Miliana plants under effect of separate salts of heavy metals Cu, Zn and Pb deposited in soil. As it is shown, simultaneous effect of viral infection and heavy metals in tenfold maximum permissible concentration leads to decrease of total chlorophyll content in experiment plants mainly due to the degradation of chlorophyll a. The reduction of chlorophyll concentration under the combined influence of both stress factors was more serious comparing to the separate effect of every single factor. Plants' treatment with toxic concentrations of lead and zinc leaded to slight delay in the development of systemic TMV infection together with more than twofold increase of virus content in plants that may be an evidence of synergism between these heavy metal's and virus' effects. Contrary, copper although decreased total chlorophyll content but showed protective properties and significantly reduced amount of virus in plants.     

Non-impactor-Based Methods for Sizing of Aerosols Emitted from Orally Inhaled and Nasal Drug Products (OINDPs)

The purpose of this article is to review non-impactor-based methods for measuring particle size distributions of orally inhaled and nasal pharmaceutical aerosols. The assessment of the size distributions of sprays and aerosols from orally inhaled and nasal drug products by methods not involving multi-stage cascade impaction may offer significant potential advantages in terms of labor savings and reducing the risk for operator-related errors associated with complex-to-undertake impactor-based methods. Indeed, in the case of nasal spray products, cascade impaction is inappropriate and alternative, and preferably non-invasive methods must be sought that minimize size-related bias associated with the measurement process for these relatively large droplets. This review highlights the options that are available to those involved with product quality assessments, providing guidance on relative strengths and weaknesses, as well as highlighting precautions that should be observed to minimize bias. The advent of Raman chemical imaging, which enables an estimate to be made of the proportion of each particle comprising active pharmaceutical ingredient(s) (APIs), necessitates a re-think about the value of classical microscopy image analysis as now being capable of providing API-relevant information from collected aerosols and sprays.     

Product quality research institute evaluation of cascade impactor profiles of pharmaceutical aerosols, part 3: Final report on a statistical procedure for determining equivalence

The purpose of this article is to report final results of the evaluation of a chi-square ratio test proposed by the US Food and Drug Administration (FDA) for demonstrating equivalence of aerodynamic particle size distribution (APSD) profiles of nasal and orally inhaled drug products. A working group of the Product Quality Research Institute previously published results demonstrating some limitations of the proposed test. In an effort to overcome the test's limited discrimination, the group proposed a supplemental test, a population bioequivalence (PBE) test for impactor-sized mass (ISM). In this final report the group compares the chi-square ratio test to the ISM-PBE test and to the combination of both tests. The basis for comparison is a set of 55 realistic scenarios of cascade impactor data, which were evaluated for equivalence by the statistical tests and independently by the group members. In many instances, the combined application of these 2 tests appeared to increase the discriminating ability of the statistical procedure compared with the chi-square ratio test alone. In certain situations the chi-square ratio test alone was sufficient to determine equivalence of APSD profiles, while in other situations neither of the tests alone nor their combination was adequate. This report describes all of these scenarios and results. In the end, the group did not recommend a statistical test for APSD profile equivalence. The group did not investigate other in vitro tests, in vivo issues, or other statistical tests for APSD profile comparisons. The studied tests are not intended for routine quality control of APSD.     

Evidence for contractile protein translocation in macrophage spreading, phagocytosis, and phagolysosome formation

Macrophage pseudopodia that surround objects during phagocytosis contain a meshwork of actin filaments and exclude organelles. Between these pseudopodia at the base of developing phagosomes, the organelle exclusion ceases, and lysosomes enter the cell periphery to fuse with the phagosomes. Macrophages also extend hyaline pseudopodia on the surface of nylon wool fibers and secrete lysosomal enzymes into the extracellular medium instead of into phagosomes. To analyze biochemically these concurrent alterations in cytoplasmic architecture, we allowed rabbit lung macrophages to spread on nylon wool fibers and then subjected the adherent cells to shear. This procedure caused the selective release of β-glucoronidase into the extracellular medium and yielded two fractions, cell bodies and isolated pseudopod blebs resembling podosomes, which are plasma-lemma-bounded sacs of cortical cytoplasm. Cytoplasmic extracts of the cell bodies eluted from nylon fibers contained two-thirds less actin-binding protein and myosin, and approximately 20 percent less actin and two-thirds of the other two proteins were accounted for in podosomes. The alterations in protein composition correlated with assays of myosin-associated EDTA-activated adenosine triphosphatase activity, and with a diminution in the capacity of extracts of nylon wool fiber-treated cell bodies to gel, a property dependent on the interaction between actin-binding protein and F-actin. However, the capacity of the remaining actin in cell bodies to polymerize did not change. We propose that actin-binding protein and myosin are concentrated in the cell cortex and particularly in pseudopodia where prominent gelation and syneresis of actin occur. Actin in the regions from which actin-binding protein and myosin are displaced disaggregates without depolymerizing, permitting lysosomes to gain access to the plasmalemma. Translocation of contractile proteins could therefore account for the concomitant differences in organelle exclusion that characterize phagocytosis.     

Improved Quality Control Metrics for Cascade Impaction Measurements of Orally Inhaled Drug Products (OIPs)

This study of aerodynamic mass-weighted particle size distribution (APSD) data from orally inhaled products (OIPs) investigated whether a set of simpler (than currently used) metrics may be adequate to detect changes in APSD for quality control (QC) purposes. A range of OIPs was examined, and correlations between mass median aerodynamic diameter and the ratio of large particle mass (LPM) to small particle mass (SPM) were calculated. For an Andersen cascade impactor, the LPM combines the mass associated with particle sizes from impactor stage 1 to a product-specific boundary size; SPM combines the mass of particles from that boundary through to terminal filter. The LPM–SPM boundary should be chosen during development based on the full-resolution impactor results so as to maximize the sensitivity of the LPM/SPM ratio to meaningful changes in quality. The LPM/SPM ratio along with the impactor-sized mass (ISM) are by themselves sufficient to detect changes in central tendency and area under the APSD curve, which are key in vitro quality attributes for OIPs. Compared to stage groupings, this two-metric approach provides better intrinsic precision, in part due to having adequate mass and consequently better ability to detect changes in APSD and ISM, suggesting that this approach should be a preferred QC tool. Another advantage is the possibility to obtain these metrics from the abbreviated impactor measurements (AIM) rather than from full-resolution multistage impactors. Although the boundary is product specific, the testing could be accomplished with a basic AIM system which can meet the needs of most or all OIPs.     

Interaction of 70 S ribosomes from Escherichia coli with spin-labeled N-Cbz-Phe-tRNAPhe. An electron paramagnetic resonance study

Two selectively spin-labeled Cbz-Phe-tRNAsPhe, one at position s4U8 and the other at position U33, have been used to study the dynamics of tRNA-ribosome interaction in the presence of poly(U) and factors washable from ribosomes. Upon binding to the ribosome, the correlation time of the spin label at position s4U8 decreases markedly while the same parameter for the label in the anticodon increases. The presence of poly(U) is not a prerequisite condition for the EPR spectral changes observed but larger variation occurs in the presence of factors washable from ribosomes. No variation in the correlation time is observed if uncharged spin-labeled tRNAPhe (on the s4U8 residue) is used in these experiments. Most of the ribosome-bound spin-labeled Cbz-Phe-tRNAPhe are puromycin-reactive, and consequently, the observed effect is manifested mainly at the ribosomal P site. These observations seem to suggest that the interaction between the N-blocked aminoacyl residue on the tRNA and the ribosome results in a conformational change on the tRNA, possibly involving tertiary interactions in a region close to s4U8. The role that the amino acid at the 3'-end can possibly play on this structural change is discussed.     

The eukaryotic replisome tolerates leading‐strand base damage by replicase switching

The high‐fidelity replicative DNA polymerases, Pol ε and Pol δ, are generally thought to be poorly equipped to replicate damaged DNA. Direct and complete replication of a damaged template therefore typically requires the activity of low‐fidelity translesion synthesis (TLS) polymerases. Here we show that a yeast replisome, reconstituted with purified proteins, is inherently tolerant of the common oxidative lesion thymine glycol (Tg). Surprisingly, leading‐strand Tg was bypassed efficiently in the presence and absence of the TLS machinery. Our data reveal that following helicase–polymerase uncoupling a switch from Pol ε, the canonical leading‐strand replicase, to the lagging‐strand replicase Pol δ, facilitates rapid, efficient and error‐free lesion bypass at physiological nucleotide levels. This replicase switch mechanism also promotes bypass of the unrelated oxidative lesion, 8‐oxoguanine. We propose that replicase switching may promote continued leading‐strand synthesis whenever the replisome encounters leading‐strand damage that is bypassed more efficiently by Pol δ than by Pol ε.     

Novel and functional regulatory SNPs in the promoter region of <Emphasis Type="Italic">FOXP3</Emphasis> gene in a Gabonese population

Parasites exert a selection pressure on their hosts and are accountable for driving diversity within gene families and immune gene polymorphisms in a host population. The overwhelming response of regulatory T cells during infectious challenges directs the host immune system to lose the ability to mount parasite specific T cell responses. The underlying idea of this study is that regulatory single nucleotide polymorphism (SNPs) can cause significant changes in gene expression in functional immune genes. We identified and investigated regulatory SNPs in the promoter region of the FOXP3 gene in a group of Gabonese individuals exposed to a variety of parasitic infections. We identified two novel and one promoter variants in 40 individual subjects. We further validated these promoter variants for their allelic gene expression using transient transfection assays. Two promoter variants, −794 (C/G) and the other variant −734/−540 (C/T) revealed a significant higher expression of the reporter gene at basal level in comparison to the major allele. The identification of SNPs that modify function in the promoter regions could provide a basis for studying parasite susceptibility in association studies.     

Association of CISH -292A/T genetic variant with hepatitis B virus infection

Cytokine-inducible SRC homology 2 domain protein (CISH) is a suppressor of cytokine signaling that controls interleukin-2 signaling pathway. We investigated the single nucleotide polymorphism (SNP) -292A>T in 473 Vietnamese hepatitis B virus (HBV) carriers and 416 healthy controls. CISH variants at -292A>T were associated to HBV infection (Allelic: OR, 1.22 95% CI, 1–1.49; P = 0.04; Recessive: OR, 1.69 95% CI 1.23–2.54; P = 0.007). A gene dose effect for the risk allele -292T was observed (P = 0.04). The level of interleukin 2 and liver enzymes such as alanine transaminase, aspartate transaminase, total bilirubin, and direct bilirubin were not associated to CISH polymorphism at position -292A>T This study associated the vital role of CISH SNP -292A>T variant to hepatitis B virus infection in a Vietnamese population.