YknWXYZ is an unusual four-component transporter with a role in protection against sporulation-delaying-protein-induced killing of Bacillus subtilis

YknXYZ is the ATP-binding cassette export complex from Bacillus subtilis, where YknX is a membrane fusion protein, YknY is an ATPase, and YknZ is a permease. The yknXYZ genes are arranged into an operon that also includes yknW, encoding a membrane protein with four putative transmembrane segments. Previous studies suggested that the yknWXYZ operon belongs to the σ(w) regulon and protects cells from the endogenous toxin SDP (sporulation-delaying protein) encoded by sdpC. In this study, we investigated the composition and function of YknW and YknXYZ. We report that the yknWXYZ operon is constitutively expressed in growing B. subtilis cells independently from sdpC. Chemical cross-linking in vivo and copurification approaches established that YknX interacts with YknYZ, whereas YknW binds YknXYZ, indicating that all four proteins form a complex in vivo. The complex assembly is modulated by YknW but proceeds in the absence of SdpC. When overproduced alone, YknW provides partial protection against SDP toxin, but all four Ykn proteins are required for full protection against both endogenous and exogenous SDP. We conclude that YknWXYZ is an unusual four-component transporter with a role in the starvation-induced killing of B. subtilis cells.     

Comparison of enhanced bioluminescence energy transfer donors for protease biosensors

Bioluminescence energy transfer (BRET) is a powerful tool for the study of protein-protein interactions and conformational changes within proteins. We directly compared two recently developed variants of Renilla luciferase (RLuc), RLuc2 and RLuc8, as BRET donors using an in vitro thrombin assay. The comparison was carried out by placing a thrombin-specific cleavage sequence between the donor luciferase and a green fluorescent protein (GFP(2)) acceptor. Substitution of native RLuc with the RLuc mutants, RLuc2 and 8, in a BRET(2) fusion protein increased the light output by a factor of ~10. Substitution of native RLuc with either of the RLuc mutants resulted in a decrease in BRET(2) ratio by a factor of ~2 when BRET(2) components were separated by the thrombin cleavage sequence. BRET(2) ratios changed by factors of 18.8±1.2 and 18.2±0.4 for GFP(2)-RG-RLuc2 and GFP(2)-RG-RLuc8 fusion proteins, respectively, on thrombin cleavage compared to 28.8±0.20 for GFP(2)-RG-RLuc. The detection limits for thrombin were 0.23 and 0.26 nM for RLuc2 and RLuc8 BRET(2) systems, respectively, and 15 pM for GFP(2)-RG-RLuc. However, overall, the mutant BRET systems remain more sensitive than FRET and brighter than standard BRET(2).     

Time‐Varying Latent Effect Model for Longitudinal Data with Informative Observation Times

Summary In analysis of longitudinal data, it is not uncommon that observation times of repeated measurements are subject‐specific and correlated with underlying longitudinal outcomes. Taking account of the dependence between observation times and longitudinal outcomes is critical under these situations to assure the validity of statistical inference. In this article, we propose a flexible joint model for longitudinal data analysis in the presence of informative observation times. In particular, the new procedure considers the shared random‐effect model and assumes a time‐varying coefficient for the latent variable, allowing a flexible way of modeling longitudinal outcomes while adjusting their association with observation times. Estimating equations are developed for parameter estimation. We show that the resulting estimators are consistent and asymptotically normal, with variance–covariance matrix that has a closed form and can be consistently estimated by the usual plug‐in method. One additional advantage of the procedure is that it provides a unified framework to test whether the effect of the latent variable is zero, constant, or time‐varying. Simulation studies show that the proposed approach is appropriate for practical use. An application to a bladder cancer data is also given to illustrate the methodology.     

Okazaki fragment processing-independent role for human Dna2 enzyme during DNA replication

Dna2 is an essential helicase/nuclease that is postulated to cleave long DNA flaps that escape FEN1 activity during Okazaki fragment (OF) maturation in yeast. We previously demonstrated that the human Dna2 orthologue (hDna2) localizes to the nucleus and contributes to genomic stability. Here we investigated the role hDna2 plays in DNA replication. We show that Dna2 associates with the replisome protein And-1 in a cell cycle-dependent manner. Depletion of hDna2 resulted in S/G(2) phase-specific DNA damage as evidenced by increased γ-H2AX, replication protein A foci, and Chk1 kinase phosphorylation, a readout for activation of the ATR-mediated S phase checkpoint. In addition, we observed reduced origin firing in hDna2-depleted cells consistent with Chk1 activation. We next examined the impact of hDna2 on OF maturation and replication fork progression in human cells. As expected, FEN1 depletion led to a significant reduction in OF maturation. Strikingly, the reduction in OF maturation had no impact on replication fork progression, indicating that fork movement is not tightly coupled to lagging strand maturation. Analysis of hDna2-depleted cells failed to reveal a defect in OF maturation or replication fork progression. Prior work in yeast demonstrated that ectopic expression of FEN1 rescues Dna2 defects. In contrast, we found that FEN1 expression in hDna2-depleted cells failed to rescue genomic instability. These findings suggest that the genomic instability observed in hDna2-depleted cells does not arise from defective OF maturation and that hDna2 plays a role in DNA replication that is distinct from FEN1 and OF maturation.     

Epoxycarotenoid cleavage by NCED5 fine-tunes ABA accumulation and affects seed dormancy and drought tolerance with other NCED family members

Carotenoid cleavage, catalyzed by the 9-cis-epoxycarotenoid dioxygenase (NCED) constitutes a key step in the regulation of ABA biosynthesis. In Arabidopsis, this enzyme is encoded by five genes. NCED3 has been shown to play a major role in the regulation of ABA synthesis in response to water deficit, whereas NCED6 and NCED9 have been shown to be essential for the ABA production in the embryo and endosperm that imposes dormancy. Reporter gene analysis was carried out to determine the spatiotemporal pattern of NCED5 and NCED9 gene expression. GUS activity from the NCED5 promoter was detected in both the embryo and endosperm of developing seeds with maximal staining after mid-development. NCED9 expression was found at early stages in the testa outer integument layer 1, and after mid-development in epidermal cells of the embryo, but not in the endosperm. In accordance with its temporal- and tissue-specific expression, the phenotypic analysis of nced5 nced6 nced9 triple mutant showed the involvement of the NCED5 gene, together with NCED6 and NCED9, in the induction of seed dormancy. In contrast to nced6 and nced9, however, nced5 mutation did not affect the gibberellin required for germination. In vegetative tissues, combining nced5 and nced3 mutations reduced vegetative growth, increased water loss upon dehydration, and decreased ABA levels under both normal and stressed conditions, as compared with nced3. NCED5 thus contributes, together with NCED3, to ABA production affecting plant growth and water stress tolerance.     

Characterisation of innate fungal recognition in the lung

The innate recognition of fungi by leukocytes is mediated by pattern recognition receptors (PRR), such as Dectin-1, and is thought to occur at the cell surface triggering intracellular signalling cascades which lead to the induction of protective host responses. In the lung, this recognition is aided by surfactant which also serves to maintain the balance between inflammation and pulmonary function, although the underlying mechanisms are unknown. Here we have explored pulmonary innate recognition of a variety of fungal particles, including zymosan, Candida albicans and Aspergillus fumigatus, and demonstrate that opsonisation with surfactant components can limit inflammation by reducing host-cell fungal interactions. However, we found that this opsonisation does not contribute directly to innate fungal recognition and that this process is mediated through non-opsonic PRRs, including Dectin-1. Moreover, we found that pulmonary inflammatory responses to resting Aspergillus conidia were initiated by these PRRs in acidified phagolysosomes, following the uptake of fungal particles by leukocytes. Our data therefore provides crucial new insights into the mechanisms by which surfactant can maintain pulmonary function in the face of microbial challenge, and defines the phagolysosome as a novel intracellular compartment involved in the innate sensing of extracellular pathogens in the lung.