Nonparametric estimation for the three-stage irreversible illness-death model

In this paper, we present new nonparametric estimators of the stage-occupation probabilities in the three-stage irreversible illness-death model. These estimators use a fractional risk set and a reweighting approach and are valid under stage-dependent censoring. Using a simulated data set, we compare the behavior of our estimators with previously proposed estimators. We also apply our estimators to data on time to Pneumocystis pneumonia and death obtained from an AIDS cohort study.     

Structural perturbation of α-crystallin and its chaperone-like activity

α-Crystallin is a multimeric lenticular protein that has recently been shown to be expressed in several non-lenticular tissues as well. It is shown to prevent aggregation of non-native proteins as a molecular chaperone. By using a non-thermal aggregation model, we could show that this process is temperature-dependent. We investigated the chaperone-like activity of α-crystallin towards photo-induced aggregation of γ-crystallin, aggregation of insulin and on the refolding induced aggregation of β- and γ-crystallins. We observed that α-crystallin could prevent photo-aggregation of γ-crystallin and this chaperone-like activity of α-crystallin is enhanced several fold at temperatures above 30°C. This enhancement parallels the exposure of its hydrophobic surfaces as a function of temperature, probed using hydrophobic fluorescent probes such as pyrene and 8-anilinonaphthalene-1-sulfonate. We, therefore, concluded that α-crystallin prevents the aggregation of other proteins by providing appropriately placed hydrophobic surfaces; a structural transition above 30°C involving enhanced or re-organized hydrophobic surfaces of α-crystallin is important for its chaperone-like activity. We also addressed the issue of conformational aspects of target proteins and found that their aggregation prone molten globule states bind to α-crystallin. We trace these developments and discuss some new lines that suggest the role of tertiary structural aspects in the chaperone process.     

Co-ordinated expression of multiple enzymes in different subcellular compartments in plants

A gene expression system designed for coordinated expression of multiple genes in plants and their targeting to specified subcellular locations was tested. A series of genes encoding polyproteins containing the tobacco vein mottling virus (TVMV) NIa proteinase along with two other reporter genes (those encoding the Escherichia coli acetate kinase (ACK) and Tn9 chloramphenicol acetyl transferase (CAT) enzymes) were assembled. The respective coding sequences of these genes were separated by a TVMV NIa proteinase recognition sequence. In addition, in some instances, chloroplast targeting information (a transit peptide (TP) from a pea rbcS gene) was incorporated into the polyprotein. We found that the NIa proteinase can be used to express, as individual polypeptides, the ACK and CAT proteins, and that these proteins retain enzymatic activity. Polyproteins with the structure TP-NIa-ACK-CAT or TP-ACK-CAT-NIa failed to yield chloroplast-localized ACK and CAT proteins, although the latter did give rise to a chloroplast-localized ACK-CAT polyprotein. These results indicate that the NIa proteinase acts in cis more rapidly than transport of proteins into the chloroplast, but that chloroplast localization can take place before complete processing of the polyprotein. Polyproteins with the structures ACK-NIa-TP-CAT and TP-ACK-NIa-TP-CAT yielded appropriately processed and targeted ACK and CAT. Our results show that subcellular localization signals can be effectively recognized in the context of a polyprotein, and they suggest an appropriate strategy for simultaneous engineering of multiple subcellular compartments in plants.     

以基因枪介导获转ps1—barnase基因的工程雄性不育水稻植株

以ps1-barnase(brn)为目的基因,pHcintG(PG)为选择/标记基因进行共转化,以PDS-1000-氦气基因枪介导,将brn及PG基因转化到水稻台北309及秋光的核DNA中,得到了转ps1-barnase基因的工程雄性不育植株。以悬浮细胞作为基因枪轰击的靶材料,转化植株再生频率较初级愈伤组织的为高。转brn基因植株的其他主要性状与供体亲本无显著差异,但却表现不育。其不育的程度在不同的植株之间表现不同。在转brn基因植株中观察到全不育(占全部brn阳性植株的40.6％)、高不育(占15.6％)及半不育的个体(占43.7％)。全不育的转基因植株自交完全不能结实(结实率为零),除个别植株外,花粉完全不被I-KI染色;而人工授以正常的花粉则可以获得杂交种子。而brn基因的阴性植株及未进行转化的对照植株则完全可育,表明转基因植株之雄性不育乃brn基因所致。结果表明,brn基因在水稻中是完全可以正常表达的,其表达的时期推测在花粉母细胞减数分裂前至花粉形成之间的整个时期。     

Thermal stresses from large volumetric expansion during freezing of biomaterials.

Thermal stresses were studied in freezing of biomaterials containing significant amounts of water. An apparent specific heat formulation of the energy equation and a viscoelastic model for the mechanics problem were used to analyze the transient axi-symmetric freezing of a long cylinder. Viscoelastic properties were measured in an Instron machine. Results show that, before phase change occurs at any location, both radial and circumferential stresses are tensile and keep increasing until phase change begins. The maximum principal tensile stress during phase change increases with a decrease in boundary temperature (faster cooling). This is consistent with experimentally observed fractures at a lower boundary temperature. Large volumetric expansion during water to ice transformation was shown to be the primary contributor to large stress development. For very rapid freezing, relaxation may not be significant, and an elastic model may be sufficient.     

Mechanistic studies of initiator-initiator interaction and replication initiation.

Unlike the chromosome of Escherichia coli that needs only one replication initiator protein (origin recognition protein) called DnaA, many plasmid replicons require dual initiators: host-encoded DnaA and a plasmid-encoded origin recognition protein, which is believed to be the major determinant of replication control. Hitherto, the relative mechanistic roles of dual initiators in DNA replication were unclear. Here, we present the first evidence that DnaA communicates with the plasmid-encoded pi initiator of R6K and contacts the latter at a specific N-terminal region. Without this specific contact, productive unwinding of plasmid ori gamma and replication is abrogated. The results also show that DnaA performs different roles in host and plasmid replication as revealed by the finding that the ATP-activated form of DnaA, while indispensable for oriC replication, was not required for R6K replication. We have analyzed the accessory role of the DNA bending protein, integration host factor (IHF), in promoting initiator-origin interaction and have found that IHF significantly enhances the binding of DnaA to its cognate site. Collectively, the results further advance our understanding of replication initiation.     

Developing the wool proteome

The wool proteome has been largely uncharted due to a lack of database coverage, poor protein extractability and dynamic range issues. Yet, investigating correlations between wool physical properties and protein content, or characterising UV-, heat- or processing-induced protein damage requires the availability of an identifiable and identified proteome.In this study we have achieved unprecedented wool proteome identification through a strategy of comprehensive data acquisition, iterative protein identification/validation and concurrent augmentation of the sequence database. Data acquisition comprised a range of different hyphenated MS techniques including LC–MS/MS, LC–MALDI, 2D-LC–MS/MS and SDS-PAGE LC–MS. Using iterative searching of databases and search result combination using ProteinScape, a systematic expansion of identifiable proteins in the sequence database was achieved. This was followed by extensive validation and rationalisation of the protein identifications. In total, 72 complete and 30 partial ovine-specific protein sequences were added to the database, and 113 wool proteins were identified.Enhanced access to ovine-specific protein identification and characterisation will facilitate all wool fibre protein chemistry and proteomics research.     

Reduced Hornbill Abundance Associated with Low Seed Arrival and Altered Recruitment in a Hunted and Logged Tropical Forest

Logging and hunting are two key direct threats to the survival of wildlife in the tropics, and also disrupt important ecosystem processes. We investigated the impacts of these two factors on the different stages of the seed dispersal cycle, including abundance of plants and their dispersers and dispersal of seeds and recruitment, in a tropical forest in north-east India. We focused on hornbills, which are important seed dispersers in these forests, and their food tree species. We compared abundances of hornbill food tree species in a site with high logging and hunting pressures (heavily disturbed) with a site that had no logging and relatively low levels of hunting (less disturbed) to understand logging impacts on hornbill food tree abundance. We compared hornbill abundances across these two sites. We, then, compared the scatter-dispersed seed arrival of five large-seeded tree species and the recruitment of four of those species. Abundances of hornbill food trees that are preferentially targeted by logging were two times higher in the less disturbed site as compared to the heavily disturbed site while that of hornbills was 22 times higher. The arrival of scatter-dispersed seeds was seven times higher in the less disturbed site. Abundances of recruits of two tree species were significantly higher in the less disturbed site. For another species, abundances of younger recruits were significantly lower while that of older recruits were higher in the heavily disturbed site. Our findings suggest that logging reduces food plant abundance for an important frugivore-seed disperser group, while hunting diminishes disperser abundances, with an associated reduction in seed arrival and altered recruitment of animal-dispersed tree species in the disturbed site. Based on our results, we present a conceptual model depicting the relationships and pathways between vertebrate-dispersed trees, their dispersers, and the impacts of hunting and logging on these pathways.     

CARD9-Dependent Neutrophil Recruitment Protects against Fungal Invasion of the Central Nervous System

Candida is the most common human fungal pathogen and causes systemic infections that require neutrophils for effective host defense. Humans deficient in the C-type lectin pathway adaptor protein CARD9 develop spontaneous fungal disease that targets the central nervous system (CNS). However, how CARD9 promotes protective antifungal immunity in the CNS remains unclear. Here, we show that a patient with CARD9 deficiency had impaired neutrophil accumulation and induction of neutrophil-recruiting CXC chemokines in the cerebrospinal fluid despite uncontrolled CNS Candida infection. We phenocopied the human susceptibility in Card9 ^-/- mice, which develop uncontrolled brain candidiasis with diminished neutrophil accumulation. The induction of neutrophil-recruiting CXC chemokines is significantly impaired in infected Card9 ^-/- brains, from both myeloid and resident glial cellular sources, whereas cell-intrinsic neutrophil chemotaxis is Card9-independent. Taken together, our data highlight the critical role of CARD9-dependent neutrophil trafficking into the CNS and provide novel insight into the CNS fungal susceptibility of CARD9-deficient humans.