ESTIMATION OF SINGLE GENERATION MIGRATION DISTANCES FROM GEOGRAPHIC VARIATION IN ANIMAL MITOCHONDRIAL DNA

A new approach is introduced for the analysis of dispersal from the geographic distributions of mtDNA lineages. The method is based on the expected spatial distributions of lineages arising under a multigeneration random walk process. Unlike previous methods based on the predicted equilibria between genetic drift and gene flow, this approach is appropriate for non-equilibrium conditions, and yields an estimate of dispersal distance rather than dispersal rate. The theoretical basis for this method is examined, and an analysis of mtDNA restriction site data for Peromyscus maniculatus is presented as an example of how this approach can be applied to empirical data.     

Compatibility of Stabilized Whole Blood Products with CD4 Technologies and Their Suitability for Quality Assessment Programs

Background

CD4 T cell enumeration is the most widely used prognostic marker for management of HIV disease. Internal quality control and external quality assessment (EQA) programs are critical to ensure reliability of clinical measurements. The utility of stabilized whole blood products (SWBP) as a test reagent for EQA programs such as Quality Assessment and Standardization for Immunological measures relevant to HIV/AIDS (QASI) program have been demonstrated previously. Since then, several new commercial SWBPs and alternative CD4 enumeration technologies have become available. Seven SWBPs were evaluated on seven different enumeration platforms to determine which product(s) are most suitable for EQA programs that support multiple analytical technologies.

Method

Assessment of SWBPs was based on two criteria: (1) accuracy of CD4 T cell measurements and; (2) stability under sub optimal storage conditions.

Results

Three SWBPs (Multi-Check, StatusFlow and CD4 Count) showed accurate CD4 T-cell absolute count and percentage values across six of the enumeration platforms. All products retain stability up to 18 days at 21–23°C with the exception of Multi-Check-high on FacsCount and Multi-Check-Low and StatusFlow-Low on Pima. One of the products (CD4 Count) retained stability for three days on all platforms tested when stored at 37°C.

Conclusion

This study demonstrated that the characteristics of commercially available SWBPs vary across multiple CD4 platforms. The compatibility of testing panels for EQA programs with multiple analytical platforms needs to be carefully considered, especially in large multiplatform CD4 EQA programs. The selection of a suitable cross-platform SWBP is an increasing challenge as more reagents and platforms are introduced for CD4 T-cell enumeration.     

Ethenoguanines Undergo Glycosylation by Nucleoside 2′-Deoxyribosyltransferases at Non-Natural Sites

Deoxyribosyl transferases and functionally related purine nucleoside phosphorylases are used extensively for synthesis of non-natural deoxynucleosides as pharmaceuticals or standards for characterizing and quantitating DNA adducts. Hence exploring the conformational tolerance of the active sites of these enzymes is of considerable practical interest. We have determined the crystal structure at 2.1 Å resolution of Lactobacillus helveticus purine deoxyribosyl transferase (PDT) with the tricyclic purine 8,9-dihydro-9-oxoimidazo[2,1-b]purine (N ²,3-ethenoguanine) at the active site. The active site electron density map was compatible with four orientations, two consistent with sites for deoxyribosylation and two appearing to be unproductive. In accord with the crystal structure, Lactobacillus helveticus PDT glycosylates the 8,9-dihydro-9-oxoimidazo[2,1-b]purine at N7 and N1, with a marked preference for N7. The activity of Lactobacillus helveticus PDT was compared with that of the nucleoside 2′-deoxyribosyltransferase enzymes (DRT Type II) from Lactobacillus leichmannii and Lactobacillus fermentum, which were somewhat more effective in the deoxyribosylation than Lactobacillus helveticus PDT, glycosylating the substrate with product profiles dependent on the pH of the incubation. The purine nucleoside phosphorylase of Escherichia coli, also commonly used in ribosylation of non-natural bases, was an order of magnitude less efficient than the transferase enzymes. Modeling based on published active-site structures as templates suggests that in all cases, an active site Phe is critical in orienting the molecular plane of the purine derivative. Adventitious hydrogen bonding with additional active site residues appears to result in presentation of multiple nucleophilic sites on the periphery of the acceptor base for ribosylation to give a distribution of nucleosides. Chemical glycosylation of O ⁹-benzylated 8,9-dihydro-9-oxoimidazo[2,1-b]purine also resulted in N7 and N1 ribosylation. Absent from the enzymatic and chemical glycosylations is the natural pattern of N3 ribosylation, verified by comparison of spectroscopic and chromatographic properties with an authentic standard synthesized by an unambiguous route.     

Centromere silencing and function in fission yeast is governed by the amino terminus of histone H3

BACKGROUND: Centromeric domains often consist of repetitive elements that are assembled in specialized chromatin, characterized by hypoacetylation of histones H3 and H4 and methylation of lysine 9 of histone H3 (K9-MeH3). Perturbation of this underacetylated state by transient treatment with histone deacetylase inhibitors leads to defective centromere function, correlating with delocalization of the heterochromatin protein Swi6/HP1. Likewise, deletion of the K9-MeH3 methyltransferase Clr4/Suvar39 causes defective chromosome segregation. Here, we create fission yeast strains retaining one histone H3 and H4 gene; the creation of these strains allows mutation of specific N-terminal tail residues and their role in centromeric silencing and chromosome stability to be investigated. RESULTS: Reduction of H3/H4 gene dosage to one-third does not affect cell viability or heterochromatin formation. Mutation of lysines 9 or 14 or serine 10 within the amino terminus of histone H3 impairs centromere function, leading to defective chromosome segregation and Swi6 delocalization. Surprisingly, silent centromeric chromatin does not require the conserved lysine 8 and 16 residues of histone H4. CONCLUSIONS: To date, mutation of conserved N-terminal residues in endogenous histone genes has only been performed in budding yeast, which lacks the Clr4/Suvar39 histone methyltransferase and Swi6/HP1. We demonstrate the importance of conserved residues within the histone H3 N terminus for the maintenance of centromeric heterochromatin in fission yeast. In sharp contrast, mutation of two conserved lysines within the histone H4 tail has no impact on the integrity of centromeric heterochromatin. Our data highlight the striking divergence between the histone tail requirements for the fission yeast and budding yeast silencing pathways.     

Direct cell contact influences bone marrow mesenchymal stem cell fate

Adult bone marrow-derived mesenchymal stem cells (MSC) can differentiate into various cell types of mesenchymal origin, but mechanisms regulating such cellular changes are unclear. We have conducted co-culture experiments to examine whether mesenchymal stem cell differentiation is influenced by indirect or direct contact with differentiated cells. Cultured adult mesenchymal stem cells showed some characteristics of synthetic state vascular smooth muscle cells (SMC). When co-cultured with vascular endothelial cells (EC) without cell contact, they exhibited abundant well-organised smooth muscle alpha-actin (alpha-actin) filaments. Direct co-culture with endothelial cells resulted in increased smooth muscle alpha-actin mRNA and protein, yet also comprehensive disruption of smooth muscle alpha-actin filament organisation. In order to assess whether these cell contact effects on mesenchymal stem cells were cell type specific, we also analysed direct co-cultures of mesenchymal stem cells with dermal fibroblasts. However, these experiments were characterised by the appearance of abundant spindle-shaped myofibroblast-like cells containing organised smooth muscle alpha-actin filaments. Thus, direct contact with distinct differentiated cells may be a critical determinant of mesenchymal stem cell fate in blood vessels and other connective tissues.     

The mechanism of angiotensin II-induced extracellular signal-regulated kinase-1/2 activation is independent of angiotensin AT(1A) receptor internalisation

The aim of this study was to determine whether internalisation of the angiotensin II (Ang II) AT(1A) receptor (AT(1A)R) was a prerequisite for Ang II-induced activation of the extracellular signal-regulated kinases, ERK-1/2. The human embryonic kidney (HEK293) cell line stably transfected with either the wild-type rat AT(1A)R or an internalisation-deficient C-terminal truncated mutant of the AT(1A)R (AT(1A)T318R) was used as a model for these studies. Inhibition of AT(1A)R internalisation by treatment with an inhibitor of clathrin-mediated endocytosis, Concanavalin A (Con A), did not inhibit Ang II-induced ERK-1/2 activation. Furthermore, cells transfected with the internalisation-deficient AT(1A)T318R mutant readily activated ERK-1/2 in response to Ang II. Ang II activated ERK-1/2 via two distinct signalling pathways in HEK-AT(1A)R cells. Approximately half of Ang II-induced ERK-1/2 activation was protein kinase C (PKC)-dependent, and the remainder was calcium- and c-Src-dependent and involved transactivation of the epidermal growth factor receptor (EGFR). In summary, Ang II-induced activation of ERK-1/2 occurs via two distinct pathways in HEK293 cells, neither of which requires AT(1A)R internalisation.     

'Bellography': Life and Contributions of Ross and Joyce Bell, two New England Naturalists

The lives and contributions of Ross and Joyce Bell are described with particular attention to studies of invertebrate natural history in the state of Vermont and carabid beetles of several groups, including the world rhysodine fauna. Their work, all done at the University of Vermont, was mainly taxonomic in nature and included aspects of the biology of the species considered. During their careers they described more than 75% of the c. 340 rhysodine species known to science. Ross Bell also wrote a number of seminal papers about the basal relationships of the Adephaga and the comparative anatomy of carabid coxal cavities. Ross and Joyce inspired several generations of students at UVM to take up advanced work in entomology and natural history.     

Mandibles and labrum-epipharynx of tiger beetles: basic structure and evolution (Coleoptera, Carabidae, Cicindelitae)

Using for comparison with, and as outgroups for, supertribe Cicindelitae, we describe and illustrate the mandibles and labrum-epipharynx of the basal geadephagans Trachypachus gibbsii LeConte, 1861 (family Trachypachidae), and family Carabidae: Pelophila rudis (LeConte, 1863) (supertribe Nebriitae, tribe Pelophilini) and Ceroglossus chilensis (Eschscholtz, 1829) (supertribe Carabitae, tribe Ceroglossini). The range and pattern of variation in structure of mandibles and labrum-epipharynx within the supertribe Cicindelitae was assessed using scanning-electron (SEM) images of these structures in nine exemplar taxa: Amblycheila baroni (Rivers, 1890), Omus californicus (Eschscholtz, 1829) and Picnochile fallaciosa (Chevrolat, 1854) (representing the Amblycheilini); Manticora tuberculata (DeGeer, 1778) (representing the Manticorini): Tetracha carolina (Linnaeus, 1767) (representing the Megacephalini); Pogonostoma chalybeum (Klug, 1835) (representing the Collyridini); and Therates basalis Dejean, 1826, Oxycheila species, and Cicindela longilabris Say, 1824 (representing the Cicindelini). An evolutionary transformation series was postulated for the mandibles and labrum-epipharynx, based on a reconstructed phylogenetic sequence, which, in turn, was based on morphological and DNAevidence.Principal features of the transformation series for the mandibles included development of a densely setose basal face; wide quadridentate retinaculum; a lengthened incisor tooth; a multidentate terebra (one to five teeth; two-three most frequent), followed by subsequent loss of one or more such teeth; development of a diastema in the occlusal surface; development and subsequent loss of scrobal setae, and reduction and loss of the scrobe. Principal features of the transformation series for the labrum included evolution of form from transverse, sub-rectangular to elongate almost square, to triangular; position and number of setae evolved from dorsal to insertion on the apical margin, the number increased from 8-10 to as many as 36, and decreased to as few as four. The epipharynx broadened evolutionarily, the pedium evolving in form from narrow, triangular and nearly flat, to broad, palatiform, and markedly convex; anterior parapedial setae both increased and decreased in number, and in orientation, from a row parallel to the parapedial ridge to a setal row extended forward at about a right angle to the latter.     

Strengthening medical appraisal: reactions to proposals for a new appraisal system to support revalidation

This paper introduces the first stage of the NHS Revalidation Support Team's (RST) proposals to strengthen medical appraisal. It reports on four focus groups held at London Deanery in 2010, with the aim of gauging initial reactions from general practitioners (GPs). The four groups consisted of two groups of appraisers and two groups of appraisees. After presentation of the proposals to strengthen appraisal, participants were invited to make comparisons between existing appraisal, and the new proposals, Interestingly, the matter which attracted most discussion was a proposal to include an element of self-assessment by the appraisee prior to appraisal, and not, as might have been expected, the proposals for assessment of the appraisee's progress towards revalidation by the appraiser. Since these focus groups, the model of strengthened medical appraisal referred to in this paper has been the subject of testing in the pathfinder pilot, a large scale pilot involving 3000 doctors in various settings in England. The evaluation of the pathfinder pilot was published (July 2011). After further refinement of the appraisal process, including taking into account new GMC and Royal College publications and more testing and piloting, the final version of medical appraisal to support revalidation, known as the Medical Appraisal Guide (MAG) is due to be published in March 2012, in time to permit the expected commencement of revalidation in late 2012.     

98例慢性肝衰竭患者的护理体会

目的：探讨慢性肝衰竭的护理方法。方法：通过对98例慢性肝衰竭患者在抗乙型肝炎病毒、护肝和输血和白蛋白等治疗的基础上,根据疾病的不同临床表现及并发症、心理变化采取不同的个体化护理措施,同时给予患者健康教育指导。结果：治愈33例,好转28例,病情恶化自动出院17（17．34％）例,死亡20（20．40％）例。结论：根据疾病的不同临床表现、并发症采和心理改变取不同的个体护理措施,同时加强对患者的健康教育,可有助于疾病恢复及减少并发症的发生,降低患者的死亡率。