Immunolocalization and histochemical evidence for the association of two different<Emphasis Type="Italic"> Arabidopsis</Emphasis> annexins with secretion during early seedling growth and development

Annexins are a multigene, multifunctional family of calcium-dependent, membrane-binding proteins found in animal and plant cells. In plants, annexins have been localized in the cytoplasm and at the cell periphery of highly secretory cell types, and in the tip region of polarly growing cells. Consequently, one proposed function for annexins in plant cells is participation in the Golgi-mediated secretion of new wall materials. In Arabidopsis, there are eight different annexin cDNAs, which share between 30% and 81% deduced amino acid sequence identity. We have used two monospecific Arabidopsis anti-annexin antibodies, raised against divergent 31-mer peptides from AnnAt1 and AnnAt2 and a previously characterized pea anti-annexin p35 antibody, for Western blot and immunolocalization studies in Arabidopsis. Western blot analyses of various Arabidopsis protein fractions showed that the two Arabidopsis antibodies are able to specifically recognize annexins in both soluble and membrane fractions. Immunofluorescence results with the three annexin antibodies show staining of secretory cells, especially at the cell periphery in developing sieve tubes, outer root cap cells, and in root hairs, consistent with previous results. In developmentally different stages some staining was also seen near the apical meristem, in some leaf cells, and in phloem-associated cells. Autoradiography following ³H-galactose incorporation was used to more clearly correlate active secretion of wall materials with the localization patterns of a specific individual annexin protein in the same cells at the same developmental stage. The results obtained in this study provide further support for the hypothesis that these two Arabidopsis annexins function in Golgi-mediated secretion during early seedling growth and development.     

The 2.0 A crystal structure of cephalosporin acylase

BACKGROUND: Semisynthetic cephalosporins are primarily synthesized from 7-aminocephalosporanic acid (7-ACA), which is usually obtained by chemical deacylation of cephalosporin C (CPC). The chemical production of 7-ACA includes, however, several expensive steps and requires thorough treatment of chemical wastes. Therefore, an enzymatic conversion of CPC to 7-ACA by cephalosporin acylase is of great interest. The biggest obstacle preventing this in industrial production is that cephalosporin acylase uses glutaryl-7ACA as a primary substrate and has low substrate specificity for CPC. RESULTS: We have solved the first crystal structure of a cephalosporin acylase from Pseudomonas diminuta at 2.0 A resolution. The overall structure looks like a bowl with two "knobs" consisting of helix- and strand-rich regions, respectively. The active site is mostly formed by the distinctive structural motif of the N-terminal (Ntn) hydrolase superfamily. Superposition of the 61 residue active-site pocket onto that of penicillin G acylase shows an rmsd in Calpha positions of 1.38 A. This indicates structural similarity in the active site between these two enzymes, but their overall structures are elsewhere quite different. CONCLUSION: The substrate binding pocket of the P. diminuta cephalosporin acylase provides detailed insight into the ten key residues responsible for the specificity of the cephalosporin C side chain in four classes of cephalosporin acylases, and it thereby forms a basis for the design of an enzyme with an improved conversion rate of CPC to 7-ACA. The structure also provides structural evidence that four of the five different classes of cephalosporin acylases can be grouped into one family of the Ntn hydrolase superfamily.     

Analyse des pedigrees et calcul des coefficients d'identité par les arbres géniques

Two genes in a pedigree are identical by descent if they are two copies of a common ancestor gene. To obtain an unambiguous definition of the set of genes, at some autosomal locus, any gene is defined as an ordered pair of zygotes: the zygote who carries the gene, and the parent who transmitted it. The natural ordered structure on the set of zygotes yields an ordered structure upon the set of genes. Any event of the mendelian segregation splits down the set of genes into non-overlaping classes of identical genes: when considered as an ordered sub-set of genes, each class is shown to have the algebraic properties of a tree. Given a sub-set ?? of genes, a family of exclusive events ensuring identity between all genes of ?? is identified as a family of genic trees with some property. This relationship between segregational events and genic trees is extended to the case where two sub-sets ?? and ??′ of genes are considered together. As a consequence, a general method is obtained to compute either identity coefficients involving any number of genes splitted into one or two identity classes, or the fifteen coefficients defined among four genes, whichever the relationships between zygotes and genes might be. Using this approach to deal with the allelic structure in a set of genes carried by related zygotes is suggested.     

A triple-drug nanotherapy to target breast cancer cells,cancer stem cells,and tumor vasculature

Triple-negative breast cancer (TNBC) is the most aggressive subtype of breast cancer, accounting for the majority of breast cancer-related death. Due to the lack of specific therapeutic targets, chemotherapeutic agents (e.g., paclitaxel) remain the mainstay of systemic treatment, but enrich a subpopulation of cells with tumor-initiating capacity and stem-like characteristics called cancer stem cells (CSCs); thus development of a new and effective strategy for TNBC treatment is an unmet medical need. Cancer nanomedicine has transformed the landscape of cancer drug development, allowing for a high therapeutic index. In this study, we developed a new therapy by co-encapsulating clinically approved drugs, such as paclitaxel, verteporfin, and combretastatin (CA4) in polymer-lipid hybrid nanoparticles (NPs) made of FDA-approved biomaterials. Verteporfin is a drug used in the treatment of macular degeneration and has recently been found to inhibit the Hippo/YAP (Yes-associated protein) pathway, which is known to promote the progression of breast cancer and the development of CSCs. CA4 is a vascular disrupting agent and has been tested in phase II/III of clinical trials. We found that our new three drug-NP not only effectively inhibited TNBC cell viability and cell migration, but also significantly diminished paclitaxel-induced and/or CA4-induced CSC enrichment in TNBC cells, partially through inhibiting the upregulated Hippo/YAP signaling. Combination of verteporfin and CA4 was also more effective in suppressing angiogenesis in an in vivo zebrafish model than single drug alone. The efficacy and application potential of our triple drug-NPs were further assessed by using clinically relevant patient-derived xenograft (PDX) models. Triple drug-NP effectively inhibited the viability of PDX organotypic slide cultures ex vivo and stopped the growth of PDX tumors in vivo. This study developed an approach capable of simultaneously inhibiting bulk cancer cells, CSCs, and angiogenesis.Subject terms: Breast cancer, Cancer stem cells     

Characterizing RNA–protein interaction using cross-linking and metabolite supplemented nuclear RNA-immunoprecipitation

RNA-immunoprecipitation (RNA-IP) is a method used to isolate and identify RNA molecules specifically associated with an RNA-binding protein. Non-coding RNAs are emerging as key regulators of many biological and developmental pathways and RNA-IP has become an important tool in studying their function(s). While RNA-IP is successfully used to determine protein-RNA interaction, specific details regarding the level of this association and the metabolic requirement of this interaction which can influence the success of RNA-IP remain unclear. Here, we investigate the conditions required for efficient nuclear RNA-IP using Arabidopsis AGO4 (Argonaute 4) and siRNA binding as the study model. We showed that formaldehyde cross-linking, but not UV cross-linking, allowed for efficient pull-down of 24-nt siRNAs, suggesting that AGO4–siRNA interaction involves other protein(s). We also showed that, while formaldehyde cross-linking could also be performed on purified nuclei, ATP supplementation to the nuclei isolation buffer was needed to efficiently pull down 24-nt siRNAs. This result indicates that ATP is required for efficient siRNA loading onto AGO4. As most of the known RNA-mediated regulatory processes occur in the nucleus, our findings on cross-linking conditions and metabolite requirement for successful AGO4 nuclear RNA-IP provide a valuable insight and future consideration when studying the function of protein–RNA interactions in plants.     

Parahellenia,a new genus segregated from Hellenia (Costaceae) based on phylogenetic and morphological evidence

Previous studies recognized three major lineages of the family Costaceae: a South American clade, an Asian clade and a Costus clade. However, the genus Hellenia within the Asian clade has been shown to be non-monophyletic and its morphology has not been studied carefully. Therefore, the complete plastid genomes of Hellenia species were obtained and the monophyly of Hellenia was tested through four different datasets in this study. Plastid phylogenomic analyses of Costaceae revealed that Hellenia is strongly supported as paraphyletic. Two major clades are recovered, namely the Hellenia s.s. subclade and the Parahellenia subclade. Phylogenetic analyses based on an enlarged taxon sampling of the Asian clade using a two chloroplast markers dataset (trnK intron and trnL-F spacer) confirmed the paraphyly of Hellenia. Meanwhile, morphological analyses suggested that members of the Parahellenia subclade differ from the remaining Hellenia species in many characters including inflorescences, bracts, stigma, axillary buds, floral tubes and labellum. According to the present molecular and morphological evidence, the latter subclade is recognized as a new genus, Parahellenia. Two new species are described, four new combinations are made, and identification keys are also provided.     

How often do physicians review medication charts on ward rounds?

Background  

Prescribing errors are common in hospital settings. Regular review of medication charts is recommended as a way to reduce errors but it is not clear how often this happens. The aim of this study was to determine the frequency with which specialist physicians reviewed medication charts during ward rounds.     

Height and death in the Antebellum United States: A view through the lens of geographically weighted regression

Factors related to geography such as climate, natural resources or waterways often affect human activities. However, traditional approaches such as ordinary least squares (OLS) have limitations in investigating such patterns. Unlike OLS regression, geographically weighted regression (GWR) allows the coefficients of explanatory variables to differ by locality by giving relatively more weight to geographically close observations. GWR depicts spatial patterns. This paper examines the pattern of height and crude death rate in the United States prior to the Civil War by this method. The GWR results show that access to water transportation increased mortality and decreased stature in the food exporting areas of the Midwest, and the opposite pattern appeared in the food importing areas of the Northeast.     

Synthesis and SAR study of novel sarsasapogenin derivatives as potent neuroprotective agents and NO production inhibitors

Sarsasapogenin, isolated from rhizomes of Anemarrhena asphodeloides, was found to be able to enhance memory. On the basis of the structure of Sarsasapogenin, a series of derivatives were synthesized and evaluated for their neuroprotective activity in PC12 cells and NO production inhibitory activity in RAW264.7 cell lines. The preliminary structure-activity relationship of them indicated that introduction of carbamate groups at the 3-hydroxyl position of sarsasapogenin might improve neuroprotective activity. Some synthesized derivatives such as AA3, AA4, AA9 and AA13 exhibited both notably neuroprotective activity and NO production inhibitory activity.