Median Tests for Censored Survival Data; a Contingency Table Approach

Summary The median failure time is often utilized to summarize survival data because it has a more straightforward interpretation for investigators in practice than the popular hazard function. However, existing methods for comparing median failure times for censored survival data either require estimation of the probability density function or involve complicated formulas to calculate the variance of the estimates. In this article, we modify a K ‐sample median test for censored survival data ( Brookmeyer and Crowley, 1982 , Journal of the American Statistical Association 77, 433–440) through a simple contingency table approach where each cell counts the number of observations in each sample that are greater than the pooled median or vice versa. Under censoring, this approach would generate noninteger entries for the cells in the contingency table. We propose to construct a weighted asymptotic test statistic that aggregates dependent χ² ‐statistics formed at the nearest integer points to the original noninteger entries. We show that this statistic follows approximately a χ² ‐distribution with k? 1 degrees of freedom. For a small sample case, we propose a test statistic based on combined p ‐values from Fisher’s exact tests, which follows a χ² ‐distribution with 2 degrees of freedom. Simulation studies are performed to show that the proposed method provides reasonable type I error probabilities and powers. The proposed method is illustrated with two real datasets from phase III breast cancer clinical trials.     

A Dominant Negative Heterozygous G87R Mutation in the Zinc Transporter, ZnT-2 (SLC30A2), Results in Transient Neonatal Zinc Deficiency

Zinc is an essential mineral, and infants are particularly vulnerable to zinc deficiency as they require large amounts of zinc for their normal growth and development. We have recently described the first loss-of-function mutation (H54R) in the zinc transporter ZnT-2 (SLC30A2) in mothers with infants harboring transient neonatal zinc deficiency (TNZD). Here we identified and characterized a novel heterozygous G87R ZnT-2 mutation in two unrelated Ashkenazi Jewish mothers with infants displaying TNZD. Transient transfection of G87R ZnT-2 resulted in endoplasmic reticulum-Golgi retention, whereas the WT transporter properly localized to intracellular secretory vesicles in HC11 and MCF-7 cells. Consequently, G87R ZnT-2 showed decreased stability compared with WT ZnT-2 as revealed by Western blot analysis. Three-dimensional homology modeling based on the crystal structure of YiiP, a close zinc transporter homologue from Escherichia coli, revealed that the basic arginine residue of the mutant G87R points toward the membrane lipid core, suggesting misfolding and possible loss-of-function. Indeed, functional assays including vesicular zinc accumulation, zinc secretion, and cytoplasmic zinc pool assessment revealed markedly impaired zinc transport in G87R ZnT-2 transfectants. Moreover, co-transfection experiments with both mutant and WT transporters revealed a dominant negative effect of G87R ZnT-2 over the WT ZnT-2; this was associated with mislocalization, decreased stability, and loss of zinc transport activity of the WT ZnT-2 due to homodimerization observed upon immunoprecipitation experiments. These findings establish that inactivating ZnT-2 mutations are an underlying basis of TNZD and provide the first evidence for the dominant inheritance of heterozygous ZnT-2 mutations via negative dominance due to homodimer formation.     

Computational selection, identification and structural analysis of ω-aminotransferases with various substrate specificities from the genome sequence of Mesorhizobium loti MAFF303099

ω-Aminotransferase (ω-AT) is an important class of enzymes for the synthesis of chiral amines or β-amino acids. Family profile analysis was applied to screen putative ω-ATs from Mesorhizobium loti MAFF303099, a nitrogen fixation bacterium that has a larger number of ATs than other microorganisms. By family profile analysis, we selected 10 putative ω-ATs according to E-value. The functions of the putative ω-ATs were investigated by examining activities towards amines and/or β-amino acids. 10 putative proteins were found to have ω-AT activity with narrow or broad substrate specificity. Structure analysis using crystal structure of mll7127 and homology models of mll1632 and mll3663 indicated that the structures of active sites of the enzymes were very similar and highly conserved, but their substrate specificities appeared to be determined by residues positioned at the entrance region of the active site binding pockets.     

Bioethanol production from the acid hydrolysate of the carrageenophyte Kappaphycus alvarezii (cottonii)

Seaweed resources can be used as raw materials to produce bioethanol, a renewable biofuel, to overcome fossil fuel depletion and environmental problems. Red seaweeds possess high amount of bioethanol-producible carbohydrates. Among 55 species tested, the carrageenophyte Kappaphycus alvarezii (also known as cottonii) was selected as the best resource for bioethanol production. This species is one of the most abundant and easily cultured red seaweeds. The main components of carrageenan are d-galactose-4-sulfate and 3,6-anhydro-d-galactose-2-sulfate, which are potentially fermentable d-typed carbohydrates. The seaweed powder was hydrolyzed with 0.2?M sulfuric acid and fermented with brewer’s yeast. The ethanol yield from the K. alvarezii hydrolysate was 0.21?g?g^?1-galactose, which corresponded to a 41% theoretical yield. It revealed a relative ethanol production of 66% comparing to that of pure galactose.     

A coordination polymer nanobelt (CPNB)-based aptasensor for sulfadimethoxine

A polymer-based aptasensor, which consisted of fluorescein amidite (FAM)-modified aptamers and coordination polymer nanobelts (CPNBs), was developed utilizing the fluorescence quenching effect to detect sulfadimethoxine residue in food products. A single-stranded DNA (ssDNA) aptamer, which was a specific bio-probe for sulfadimethoxine (Su13; 5'-GAGGGCAACGAGTGTTTATAGA-3'), was discovered by a magnetic bead-based systematic evolution of ligands by exponential enrichment (SELEX) technique, and the fluorescent quenchers CPNBs were produced by mixing AgNO(3) and 4,4'-bipyridine. This aptasensor easily and sensitively detected sulfadimethoxine in solution with a limit of detection (LOD) of 10ng/mL. Furthermore, the antibiotic dissolved in milk was also effectively detected with the same LOD value. In addition, this aptamer probe offered high specificity for sulfadimethoxine compared to other antibiotics. These valuable results provide ample evidence that the CPNB-based aptasensor can be used to quantify sulfadimethoxine residue in food products.     

Roles of DCL4 and DCL3b in rice phased small RNA biogenesis

Higher plants have evolved multiple proteins in the RNase III family to produce and regulate different classes of small RNAs with specialized molecular functions. In rice (Oryza sativa), numerous genomic clusters are targeted by one of two microRNAs (miRNAs), miR2118 and miR2275, to produce secondary small interfering RNAs (siRNAs) of either 21 or 24 nucleotides in a phased manner. The biogenesis requirements or the functions of the phased small RNAs are completely unknown. Here we examine the rice Dicer-Like (DCL) family, including OsDCL1, -3a, -3b and -4. By deep sequencing of small RNAs from different tissues of the wild type and osdcl4-1, we revealed that the processing of 21-nucleotide siRNAs, including trans-acting siRNAs (tasiRNA) and over 1000 phased small RNA loci, was largely dependent on OsDCL4. Surprisingly, the processing of 24-nucleotide phased small RNA requires the DCL3 homolog OsDCL3b rather than OsDCL3a, suggesting functional divergence within DCL3 family. RNA ligase-mediated 5' rapid amplification of cDNA ends and parallel analysis of RNA ends (PARE)/degradome analysis confirmed that most of the 21- and 24-nucleotide phased small RNA clusters were initiated from the target sites of miR2118 and miR2275, respectively. Furthermore, the accumulation of the two triggering miRNAs requires OsDCL1 activity. Finally, we show that phased small RNAs are preferentially produced in the male reproductive organs and are likely to be conserved in monocots. Our results revealed significant roles of OsDCL4, OsDCL3b and OsDCL1 in the 21- and 24-nucleotide phased small RNA biogenesis pathway in rice.     

XIAO is involved in the control of organ size by contributing to the regulation of signaling and homeostasis of brassinosteroids and cell cycling in rice

Organ size is determined by cell number and size, and involves two fundamental processes: cell proliferation and cell expansion. Although several plant hormones are known to play critical roles in shaping organ size by regulating the cell cycle, it is not known whether brassinosteroids (BRs) are also involved in regulating cell division. Here we identified a rice T-DNA insertion mutant for organ size, referred to as xiao, that displays dwarfism and erect leaves, typical BR-related phenotypes, together with reduced seed setting. XIAO is predicted to encode an LRR kinase. The small stature of the xiao mutant resulted from reduced organ sizes due to decreased cell numbers resulting from reduced cell division rate, as supported by the observed co-expression of XIAO with a number of genes involved in cell cycling. The xiao mutant displayed a tissue-specific enhanced BR response and greatly reduced BR contents at the whole-plant level. These results indicated that XIAO is a regulator of BR signaling and cell division. Thus, XIAO may provide a possible connection between BRs and cell-cycle regulation in controlling organ growth.     

Functional characterization of ObgC in ribosome biogenesis during chloroplast development

The Spo0B-associated GTP-binding protein (Obg) GTPase, essential for bacterial viability, is also conserved in eukaryotes, but its primary role in eukaryotes remains unknown. Here, our functional characterization of Arabidopsis and rice obgc mutants strongly underlines the evolutionarily conserved role of eukaryotic Obgs in organellar ribosome biogenesis. The mutants exhibited a chlorotic phenotype, caused by retarded chloroplast development. A plastid DNA macroarray revealed a plastid-encoded RNA polymerase (PEP) deficiency in an obgc mutant, caused by incompleteness of the PEP complex, as its western blot exhibited reduced levels of RpoA protein, a component of PEP. Plastid rRNA profiling indicated that plastid rRNA processing is defective in obgc mutants, probably resulting in impaired ribosome biogenesis and, in turn, in reduced levels of RpoA protein. RNA co-immunoprecipitation revealed that ObgC specifically co-precipitates with 23S rRNA in vivo. These findings indicate that ObgC functions primarily in plastid ribosome biogenesis during chloroplast development. Furthermore, complementation analysis can provide new insights into the functional modes of three ObgC domains, including the Obg fold, G domain and OCT.     

Identification of marneral synthase,which is critical for growth and development in Arabidopsis

Plants produce structurally diverse triterpenoids, which are important for their life and survival. Most triterpenoids and sterols share a common biosynthetic intermediate, 2,3‐oxidosqualene (OS), which is cyclized by 2,3‐oxidosqualene cyclase (OSC). To investigate the role of an OSC, marneral synthase 1 (MRN1), in planta, we characterized a Arabidopsis mrn1 knock‐out mutant displaying round‐shaped leaves, late flowering, and delayed embryogenesis. Reduced growth of mrn1 was caused by inhibition of cell expansion and elongation. Marnerol, a reduced form of marneral, was detected in Arabidopsis overexpressing MRN1, but not in the wild type or mrn1. Alterations in the levels of sterols and triterpenols and defects in membrane integrity and permeability were observed in the mrn1. In addition, GUS expression, under the control of the MRN1 gene promoter, was specifically detected in shoot and root apical meristems, which are responsible for primary growth, and the mRNA expression of Arabidopsis clade II OSCs was preferentially observed in roots and siliques containing developing seeds. The eGFP:MRN1 was localized to the endoplasmic reticulum in tobacco protoplasts. Taken together, this report provides evidence that the unusual triterpenoid pathway via marneral synthase is important for the growth and development of Arabidopsis.     

Structure-based de novo design and biochemical evaluation of novel BRAF kinase inhibitors

VRAF murine sarcoma viral oncogene homologue B1 (BRAF) kinase has been considered to be a promising therapeutic target for various human cancers. We have been able to identify 24 novel BRAF kinase inhibitors with K(d) values ranging from 0.4 to 10μM utilizing a structure-based de novo design method with the two known inhibitor scaffolds. Because these discovered inhibitors were also screened for having desirable physicochemical properties as a drug candidate, they deserve consideration for further investigation as anticancer agents. Structural features relevant to the stabilization of the newly identified inhibitors in the ATP-binding site of BRAF are discussed in detail.