Nucleotide‐binding resistance gene signatures in sugar beet,insights from a new reference genome

Nucleotide‐binding (NB‐ARC), leucine‐rich‐repeat genes (NLRs) account for 60.8% of resistance (R) genes molecularly characterized from plants. NLRs exist as large gene families prone to tandem duplication and transposition, with high sequence diversity among crops and their wild relatives. This diversity can be a source of new disease resistance, but difficulty in distinguishing specific sequences from homologous gene family members hinders characterization of resistance for improving crop varieties. Current genome sequencing and assembly technologies, especially those using long‐read sequencing, are improving resolution of repeat‐rich genomic regions and clarifying locations of duplicated genes, such as NLRs. Using the conserved NB‐ARC domain as a model, 231 tentative NB‐ARC loci were identified in a highly contiguous genome assembly of sugar beet, revealing diverged and truncated NB‐ARC signatures as well as full‐length sequences. The NB‐ARC‐associated proteins contained NLR resistance gene domains, including TIR, CC and LRR, as well as other integrated domains. Phylogenetic relationships of partial and complete domains were determined, and patterns of physical clustering in the genome were evaluated. Comparison of sugar beet NB‐ARC domains to validated R‐genes from monocots and eudicots suggested extensive Beta vulgaris‐specific subfamily expansions. The NLR landscape in the rhizomania resistance conferring Rz region of Chromosome 3 was characterized, identifying 26 NLR‐like sequences spanning 20 MB. This work presents the first detailed view of NLR family composition in a member of the Caryophyllales, builds a foundation for additional disease resistance work in B. vulgaris, and demonstrates an additional nucleic‐acid‐based method for NLR prediction in non‐model plant species.     

Evaluating the interaction of a tracheobronchial stent in an ovine in-vivo model

Tracheobronchial stents are used to restore patency to stenosed airways. However, these devices are associated with many complications such as stent migration, granulation tissue formation, mucous plugging and stent strut fracture. Of these, granulation tissue formation is the complication that most frequently requires costly secondary interventions. In this study a biomechanical lung modelling framework recently developed by the authors to capture the lung in-vivo stress state under physiological loading is employed in conjunction with ovine pre-clinical stenting results and device experimental data to evaluate the effect of stent interaction on granulation tissue formation. Stenting is simulated using a validated model of a prototype covered laser-cut tracheobronchial stent in a semi-specific biomechanical lung model, and physiological loading is performed. Two computational methods are then used to predict possible granulation tissue formation: the standard method which utilises the increase in maximum principal stress change, and a newly proposed method which compares the change in contact pressure over a respiratory cycle. These computational predictions of granulation tissue formation are then compared to pre-clinical stenting observations after a 6-week implantation period. Experimental results of the pre-clinical stent implantation showed signs of granulation tissue formation both proximally and distally, with a greater proximal reaction. The standard method failed to show a correlation with the experimental results. However, the contact change method showed an apparent correlation with granulation tissue formation. These results suggest that this new method could be used as a tool to improve future device designs.     

SAHA overcomes FLIP-mediated inhibition of SMAC mimetic-induced apoptosis in mesothelioma

Malignant pleural mesothelioma (MPM) is a highly pro-inflammatory malignancy that is rapidly fatal and increasing in incidence. Cytokine signaling within the pro-inflammatory tumor microenvironment makes a critical contribution to the development of MPM and its resistance to conventional chemotherapy approaches. SMAC mimetic compounds (SMCs) are a promising class of anticancer drug that are dependent on tumor necrosis factor alpha (TNFα) signaling for their activity. As circulating TNFα expression is significantly elevated in MPM patients, we examined the sensitivity of MPM cell line models to SMCs. Surprisingly, all MPM cell lines assessed were highly resistant to SMCs either alone or when incubated in the presence of clinically relevant levels of TNFα. Further analyses revealed that MPM cells were sensitized to SMC-induced apoptosis by siRNA-mediated downregulation of the caspase 8 inhibitor FLIP, an antiapoptotic protein overexpressed in several cancer types including MPM. We have previously reported that FLIP expression is potently downregulated in MPM cells in response to the histone deacetylase inhibitor (HDACi) Vorinostat (SAHA). In this study, we demonstrate that SAHA sensitizes MPM cells to SMCs in a manner dependent on its ability to downregulate FLIP. Although treatment with SMC in the presence of TNFα promoted interaction between caspase 8 and the necrosis-promoting RIPK1, the cell death induced by combined treatment with SAHA and SMC was apoptotic and mediated by caspase 8. These results indicate that FLIP is a major inhibitor of SMC-mediated apoptosis in MPM, but that this inhibition can be overcome by the HDACi SAHA.     

Polyphosphate accumulation by Pseudomonas putida CA-3 and other medium-chain-length polyhydroxyalkanoate-accumulating bacteria under aerobic growth conditions

Pseudomonas putida CA-3 accumulates polyphosphate (polyP) and medium-chain-length polyhydroxyalkanoate (mclPHA) concurrently under nitrogen limitation. Five other mclPHA-accumulating Pseudomonas strains are capable of simultaneous polyP and mclPHA biosynthesis. It appears that polyP is not the rate-limiting step for mclPHA accumulation in these Pseudomonas strains.     

Role of microbial immigration in the colonization of apple leaves by Aureobasidium pullulans

The role of microbial immigration in the veinal colonization pattern of Aureobasidium pullulans on the adaxial surface of apple leaves was investigated in two experiments at two periods (early and late seasons) in 2004 by applying green fluorescent protein (GFP)-tagged blastospores to the foliage of orchard trees. Individual leaves were resampled by a semidestructive method immediately after inoculation (t(0)) and about 1 (t(1)), 2 (t(2)), and 3 (t(3)) weeks later. At t(0), there were no significant (P < or = 0.05) differences in densities (cells/mm(2)) on veinal (excluding midvein) sites and those on interveinal sites, but at all points thereafter, densities were significantly higher on veins. GFP-tagged A. pullulans cells remained primarily as singletons on interveinal regions (> or =90% at all points), while > or =20% of cells over veins at t(3) were in colonies of > or =4 cells. The colonies that developed from single cells placed on midveins and other veins were significantly larger than those that developed on interveinal regions of detached field and seedling leaves incubated under controlled conditions. Colonies primarily developed linearly along veins, reaching average colony sizes (72 h) of 24.4 +/- 12.7 (mean +/- standard deviation) cells. In contrast, colonies on interveinal regions tended to average only 2.9 +/- 1.3 cells, with less linearity. To examine the potential role of A. pullulans growth-inhibiting factors associated with interveinal features, single GFP-tagged A. pullulans cells in droplets previously incubated on interveinal sites were placed on midveins and compared to midvein colonies derived from cells in a water-only suspension. No differences in colony size resulted. Our results indicate that immigration limitation and growth-inhibiting factors are not the primary factors responsible for A. pullulans veinal colonization patterns in the field. Rather, indirect evidence suggests that growth-promoting substances occur locally in the veinal areas.     

Molecular mechanism of ligand recognition by membrane transport protein,Mhp1

The hydantoin transporter Mhp1 is a sodium‐coupled secondary active transport protein of the nucleobase‐cation‐symport family and a member of the widespread 5‐helix inverted repeat superfamily of transporters. The structure of Mhp1 was previously solved in three different conformations providing insight into the molecular basis of the alternating access mechanism. Here, we elucidate detailed events of substrate binding, through a combination of crystallography, molecular dynamics, site‐directed mutagenesis, biochemical/biophysical assays, and the design and synthesis of novel ligands. We show precisely where 5‐substituted hydantoin substrates bind in an extended configuration at the interface of the bundle and hash domains. They are recognised through hydrogen bonds to the hydantoin moiety and the complementarity of the 5‐substituent for a hydrophobic pocket in the protein. Furthermore, we describe a novel structure of an intermediate state of the protein with the external thin gate locked open by an inhibitor, 5‐(2‐naphthylmethyl)‐L‐hydantoin, which becomes a substrate when leucine 363 is changed to an alanine. We deduce the molecular events that underlie acquisition and transport of a ligand by Mhp1.     

DSM-IV “Criterion A” Schizophrenia Symptoms Across Ethnically Different Populations: Evidence for Differing Psychotic Symptom Content or Structural Organization?

There is significant variation in the expression of schizophrenia across ethnically different populations, and the optimal structural and diagnostic representation of schizophrenia are contested. We contrasted both lifetime frequencies of DSM-IV criterion A (the core symptom criterion of the internationally recognized DSM classification system) symptoms and types/content of delusions and hallucinations in transethnic schizophrenia populations from Australia (n = 776), India (n = 504) and Sarawak, Malaysia (n = 259), to elucidate clinical heterogeneity. Differences in both criterion A symptom composition and symptom content were apparent. Indian individuals with schizophrenia reported negative symptoms more frequently than other sites, whereas individuals from Sarawak reported disorganized symptoms more frequently. Delusions of control and thought broadcast, insertion, or withdrawal were less frequent in Sarawak than Australia. Curiously, a subgroup of 20 Indian individuals with schizophrenia reported no lifetime delusions or hallucinations. These findings potentially challenge the long-held view in psychiatry that schizophrenia is fundamentally similar across cultural groups, with differences in only the content of psychotic symptoms, but equivalence in structural form.