Multilocus sequence analysis provides insights into molecular epidemiology of Chlamydia pecorum infections in Australian sheep, cattle and koalas

Chlamydia pecorum is a significant pathogen of domestic livestock and wildlife. We have developed a C. pecorum-specific multilocus sequence analysis (MLSA) scheme to examine the genetic diversity of and relationships between Australian sheep, cattle, and koala isolates. An MLSA of seven concatenated housekeeping gene fragments was performed using 35 isolates, including 18 livestock isolates (11 Australian sheep, one Australian cow, and six U.S. livestock isolates) and 17 Australian koala isolates. Phylogenetic analyses showed that the koala isolates formed a distinct clade, with limited clustering with C. pecorum isolates from Australian sheep. We identified 11 MLSA sequence types (STs) among Australian C. pecorum isolates, 10 of them novel, with koala and sheep sharing at least one identical ST (designated ST2013Aa). ST23, previously identified in global C. pecorum livestock isolates, was observed here in a subset of Australian bovine and sheep isolates. Most notably, ST23 was found in association with multiple disease states and hosts, providing insights into the transmission of this pathogen between livestock hosts. The complexity of the epidemiology of this disease was further highlighted by the observation that at least two examples of sheep were infected with different C. pecorum STs in the eyes and gastrointestinal tract. We have demonstrated the feasibility of our MLSA scheme for understanding the host relationship that exists between Australian C. pecorum strains and provide the first molecular epidemiological data on infections in Australian livestock hosts.

An efficient synthesis of (±)-frondosin B using a Stille–Heck reaction sequence

A concise, convergent synthesis of (±)-frondosin B has been developed based on the application of a Stille–Heck reaction sequence of 2-chloro-5-methoxybenzo[b]furan-3-yl triflate and 2-(3-butenyl)-3-(trimethylstannyl)cyclohex-2-enone giving the racemic natural product in a 34% overall yield.

Heterogeneity of clinical and environmental isolates of Mycobacterium fortuitum using repetitive element sequence-based PCR: municipal water an unlikely source of community-acquired infections

M. fortuitum is a rapidly growing mycobacterium associated with community-acquired and nosocomial wound, soft tissue, and pulmonary infections. It has been postulated that water has been the source of infection especially in the hospital setting. The aim of this study was to determine if municipal water may be the source of community-acquired or nosocomial infections in the Brisbane area. Between 2007 and 2009, 20 strains of M. fortuitum were recovered from municipal water and 53 patients’ isolates were submitted to the reference laboratory. A wide variation in strain types was identified using repetitive element sequence-based PCR, with 13 clusters of ≥2 indistinguishable isolates, and 28 patterns consisting of individual isolates. The clusters could be grouped into seven similar groups (>95% similarity). Municipal water and clinical isolates collected during the same time period and from the same geographical area consisted of different strain types, making municipal water an unlikely source of sporadic human infection.

Feral pig populations are structured at fine spatial scales in tropical Queensland, Australia

Feral pigs occur throughout tropical far north Queensland, Australia and are a significant threat to biodiversity and World Heritage values, agriculture and are a vector of infectious diseases. One of the constraints on long-lasting, local eradication of feral pigs is the process of reinvasion into recently controlled areas. This study examined the population genetic structure of feral pigs in far north Queensland to identify the extent of movement and the scale at which demographically independent management units exist. Genetic analysis of 328 feral pigs from the Innisfail to Tully region of tropical Queensland was undertaken. Seven microsatellite loci were screened and Bayesian clustering methods used to infer population clusters. Sequence variation at the mitochondrial DNA control region was examined to identify pig breed. Significant population structure was identified in the study area at a scale of 25 to 35 km, corresponding to three demographically independent management units (MUs). Distinct natural or anthropogenic barriers were not found, but environmental features such as topography and land use appear to influence patterns of gene flow. Despite the strong, overall pattern of structure, some feral pigs clearly exhibited ancestry from a MU outside of that from which they were sampled indicating isolated long distance dispersal or translocation events. Furthermore, our results suggest that gene flow is restricted among pigs of domestic Asian and European origin and non-random mating influences management unit boundaries. We conclude that the three MUs identified in this study should be considered as operational units for feral pig control in far north Queensland. Within a MU, coordinated and simultaneous control is required across farms, rainforest areas and National Park Estates to prevent recolonisation from adjacent localities.

Distamycin A affects the stability of NF-kappaB p50-DNA complexes in a sequence-dependent manner

The effect of two different DNA minor groove binding molecules, Hoechst 33258 and distamycin A, on the binding kinetics of NF-κB p50 to three different specific DNA sequences was studied at various salt concentrations. Distamycin A was shown to significantly increase the dissociation rate constant of p50 from the sequences PRDII (5′-GGGAAATTCC-3′) and Ig-κ B (5′-GGGACTTTCC-3′) but had a negligible effect on the dissociation from the palindromic target-κB binding site (5′-GGGAATTCCC-3′). By comparison, the effect of Hoechst 33258 on binding of p50 to each sequence was found to be minimal. The dissociation rates for the protein–DNA complexes increased at higher potassium chloride concentrations for the PRDII and Ig-κB binding motifs and this effect was magnified by distamycin A. In contrast, p50 bound to the palindromic target-κB site with a much higher intrinsic affinity and exhibited a significantly reduced salt dependence of binding over the ionic strength range studied, retaining a KD of less than 10 pM at 150 mM KCl. Our results demonstrate that the DNA binding kinetics of p50 and their salt dependence is strongly sequence-dependent and, in addition, that the binding of p50 to DNA can be influenced by the addition of minor groove-binding drugs in a sequence-dependent manner.

An objective study of acid reflux and cough in children using an ambulatory pHmetry-cough logger

Objective There are no objective ambulatory studies on the temporal relationship between reflux and cough in children. Commercial pHmetry loggers have slow capture rates (0.25 Hz) that limit objective quantification of reflux and cough. The authors aimed to evaluate if there is a temporal association between cough and acid pH in ambulatory children with chronic cough. setting and patients The authors studied children (aged <14 years) with chronic cough, suspected of acid reflux and considered for pHmetry using a specifically built ambulatory pHmetry–cough logger that enabled the simultaneous ambulatory recording of cough and pH with a fast (10 Hz) capture rate. Main outcome measures Coughs within (before and after) 10, 30, 60 and 120 s of a reflux episode (pH<4 for >0.5 s). Results Analysis of 5628 coughs in 20 children. Most coughs (83.9%) were independent of a reflux event. Cough–reflux (median 19, IQR 3–45) and reflux–cough (24.5, 13–51) sequences were equally likely to occur within 120 s. Within the 10 and 30 s time frame, reflux–cough (10 s=median 2.5, IQR 0–7.25; 30 s=6.5, 1.25–22.25) sequences were significantly less frequent than reflux–no cough (10 s=27, IQR 15–65; 30 s=24.5, 14.5–55.5) sequences, (p=0.0001 and p=0.001, respectively). No differences were found for 60 and 120 s time frame. Cough–reflux sequence (median 1.0, IQR 0–8) within 10 s was significantly less (p=0.0001) than no cough–reflux sequences (median 29.5, 15–67), within 30 s (p=0.006) and 60 s (p=0.048) but not within 120 s (p=0.47). Conclusions In children with chronic cough and suspected of having gastro-oesophageal reflux disease, the temporal relationship between acid reflux and cough is unlikely causal.

Analysis of expressed sequence tags from Actinidia : Applications of a cross species EST database for gene discovery in the areas of flavor, health, color and ripening

Background Kiwifruit (Actinidia spp.) are a relatively new, but economically important crop grown in many different parts of the world. Commercial success is driven by the development of new cultivars with novel consumer traits including flavor, appearance, healthful components and convenience. To increase our understanding of the genetic diversity and gene-based control of these key traits in Actinidia, we have produced a collection of 132,577 expressed sequence tags (ESTs). Results The ESTs were derived mainly from four Actinidia species (A. chinensis, A. deliciosa, A. arguta and A. eriantha) and fell into 41,858 non redundant clusters (18,070 tentative consensus sequences and 23,788 EST singletons). Analysis of flavor and fragrance-related gene families (acyltransferases and carboxylesterases) and pathways (terpenoid biosynthesis) is presented in comparison with a chemical analysis of the compounds present in Actinidia including esters, acids, alcohols and terpenes. ESTs are identified for most genes in color pathways controlling chlorophyll degradation and carotenoid biosynthesis. In the health area, data are presented on the ESTs involved in ascorbic acid and quinic acid biosynthesis showing not only that genes for many of the steps in these pathways are represented in the database, but that genes encoding some critical steps are absent. In the convenience area, genes related to different stages of fruit softening are identified. Conclusion This large EST resource will allow researchers to undertake the tremendous challenge of understanding the molecular basis of genetic diversity in the Actinidia genus as well as provide an EST resource for comparative fruit genomics. The various bioinformatics analyses we have undertaken demonstrates the extent of coverage of ESTs for genes encoding different biochemical pathways in Actinidia.

Identification and characterization of flowering genes in kiwifruit : sequence conservation and role in kiwifruit flower development

Background Flower development in kiwifruit (Actinidia spp.) is initiated in the first growing season, when undifferentiated primordia are established in latent shoot buds. These primordia can differentiate into flowers in the second growing season, after the winter dormancy period and upon accumulation of adequate winter chilling. Kiwifruit is an important horticultural crop, yet little is known about the molecular regulation of flower development. Results To study kiwifruit flower development, nine MADS-box genes were identified and functionally characterized. Protein sequence alignment, phenotypes obtained upon overexpression in Arabidopsis and expression patterns suggest that the identified genes are required for floral meristem and floral organ specification. Their role during budbreak and flower development was studied. A spontaneous kiwifruit mutant was utilized to correlate the extended expression domains of these flowering genes with abnormal floral development. Conclusions This study provides a description of flower development in kiwifruit at the molecular level. It has identified markers for flower development, and candidates for manipulation of kiwifruit growth, phase change and time of flowering. The expression in normal and aberrant flowers provided a model for kiwifruit flower development.

Coincident sequence-specific RNA degradation of linked transgenes in the plant genome

The expression of transgenes in plant genomes can be inhibited by either transcriptional gene silencing or posttranscriptional gene silencing (PTGS). Overexpression of the chalcone synthase-A (CHS-A) transgene triggers PTGS of CHS-A and thus results in loss of flower pigmentation in petunia. We previously demonstrated that epigenetic inactivation of CHS-A transgene transcription leads to a reversion of the PTGS phenotype. Although neomycin phosphotransferase II (nptII), a marker gene co-introduced into the genome with the CHS-A transgene, is not normally silenced in petunia, even when CHS-A is silenced, here we found that nptII was silenced in a petunia line in which CHS-A PTGS was induced, but not in the revertant plants that had no PTGS of CHS-A. Transcriptional activity, accumulation of short interfering RNAs, and restoration of mRNA level after infection with viruses that had suppressor proteins of gene silencing indicated that the mechanism for nptII silencing was posttranscriptional. Read-through transcripts of the CHS-A gene toward the nptII gene were detected. Deep-sequencing analysis revealed a striking difference between the predominant size class of small RNAs produced from the read-through transcripts (22 nt) and that from the CHS-A RNAs (21 nt). These results implicate the involvement of read-through transcription and distinct phases of RNA degradation in the coincident PTGS of linked transgenes and provide new insights into the destabilization of transgene expression.

Phospholipid composition of the rat lens is independent of diet

Dietary fatty acids are known to influence the phospholipid composition of many tissues in the body, with lipid turnover occurring rapidly. The aim of this study was to investigate whether changes in the fatty acid composition of the diet can affect the phospholipid composition of the lens. Male Sprague-Dawley rats were fed three diets with distinct profiles in both essential and non-essential fatty acids. After 8 weeks, lenses and skeletal muscle were removed, and the lenses sectioned into nuclear and cortical regions. In these experiments, the lens cortex was synthesised during the course of the variable lipid diet. Phospholipids were then identified by electrospray ionisation tandem mass spectrometry, and quantified via the use of internal standards. The phospholipid compositions of the nuclear and cortical regions of the lens differed slightly between the two regions, but comparison of the equivalent regions across the diet groups showed remarkable similarity. In contrast, the phospholipid composition of skeletal muscle (medial gastrocnemius) in these rats varied significantly. This study provides the first direct evidence to show that the phospholipid composition of the lens is tightly regulated and thus appears to be independent of diet. As phospholipids determine membrane fluidity and influence the activity and function of integral membrane proteins, regulation of their composition may be important for the function of the lens. Crown Copyright (C) 2008 Published by Elsevier Ltd. All rights reserved.

PPARγ-independent induction of growth arrest and apoptosis in prostate and bladder carcinoma

Background Although PPARγ antagonists have shown considerable pre-clinical efficacy, recent studies suggest PPARγ ligands induce PPARγ-independent effects. There is a need to better define such effects to permit rational utilization of these agents. Methods We have studied the effects of a range of endogenous and synthetic PPARγ ligands on proliferation, growth arrest (FACS analysis) and apoptosis (caspase-3/7 activation and DNA fragmentation) in multiple prostate carcinoma cell lines (DU145, PC-3 and LNCaP) and in a series of cell lines modelling metastatic transitional cell carcinoma of the bladder (TSU-Pr1, TSU-Pr1-B1 and TSU-Pr1-B2). Results 15-deoxy-prostaglandin J2 (15dPGJ2), troglitazone (TGZ) and to a lesser extent ciglitazone exhibited inhibitory effects on cell number; the selective PPARγ antagonist GW9662 did not reverse these effects. Rosiglitazone and pioglitazone had no effect on proliferation. In addition, TGZ induced G0/G1 growth arrest whilst 15dPGJ2 induced apoptosis. Conclusion Troglitazone and 15dPGJ2 inhibit growth of prostate and bladder carcinoma cell lines through different mechanisms and the effects of both agents are PPARγ-independent.

A platform - independent product library for BIM

There is considerable interest internationally in developing product libraries to support the use of BIM. Product library initiatives are driven by national bodies, manufacturers and private companies who see their potential. A major issue with the production and distribution of product information for BIM is that separate library objects need to be produced for all of the different software systems that are going to use the library. This increases the cost of populating product libraries and also increases the difficulty in maintaining consistency between the representations for the different software over time. This paper describes a project which uses “software transformation” technology from the field of software engineering to support the definition of a single generic representation of a product which can then be automatically converted to the format required by receiving software. The paper covers the current state of implementation of the product library, the technology underlying the transformations for the currently supported software and the business model for creating a national library in Australia. This is placed within the context of other current product library systems to highlight the differences. The responsibilities of the various actors involved in supporting the product library are also discussed.

The invasive and metastatic properties of hormone-independent but hormone-responsive variants of MCF-7 human breast cancer cells

We have previously isolated a series of MCF-7 human breast cancer cell variants which no longer require estrogen-supplementation for tumor growth in nude mice (Clarke et al. Proc Natl Acad Sci USA 86: 3649-3653, 1989). We now report that these hormone-independent and hormone-responsive variants (MIII, MCF7/LCC1) can invade locally from solid mammary fat pad tumors, and produce primary extensions on the surface of intraperitoneal structures including liver, pancreas, and diaphragm. Both lymphatic and hematogenous dissemination are observed, resulting in the establishing of pulmonary, bone, and renal metastases. The pattern of metastasis by MIII and MCF7/LCC1 cells closely resembles that frequently observed in breast cancer patients, and provides the first evidence of metastasis from MCF-7 cells growing in vivo without supplementary estrogen. The interexperimental incidence of metastases, and the time from cell inoculation to the appearance of metastatic disease are variable. The increased metastatic potential is not associated with an increase in either the level of laminin attachment, laminin receptor mRNA expression, or secreted type IV collagenolytic activity. We also did not detect a significant decrease in the steady-state mRNA levels of the metastasis inhibitor nm23 gene. However, when growing without estrogen in vitro, MCF7/LCC1 cells produce elevated levels of the estrogen-inducible cathepsin D enzyme.

MT1-MMP-dependent and -independent regulation of gelatinase A activation in long-term, ascorbate-treated fibroblast cultures: Regulation by fibrillar collagen

Human skin fibroblasts were cultured long-term in the presence of ascorbic acid to allow formation of a three-dimensional collagen matrix, and the effects of this on activation of secreted matrix metalloproteinase-2 (MMP-2) were examined. Accumulation of collagen over time correlated with increased levels of both mature MMP-2 and cell-associated membrane type 1-MMP (MT1-MMP), and subsequently increased mRNA levels for MT1-MMP, providing temporal resolution of the "nontranscriptional" and "transcriptional" effects of collagen on MT-1MMP functionality. MMP-2 activation by these cultures was blocked by inhibitors of prolyl-4-hydroxylase, or when fibroblasts derived from the collagen α1(I) gene-deficient Mov-13 mouse were used. MMP-2 activation by the Mov-13 fibroblasts was rescued by transfection of a full-length α1(I) collagen cDNA, and to our surprise, also by transfection with an α1(I) collagen cDNA carrying a mutation at the C-proteinase cleavage, which almost abrogated fibrillogenesis. Although studies with ascorbate-cultured MT1-MMP-/- fibroblasts showed that MT1-MMP played a significant role in the collagen-induced MMP-2 activation, a residual MT1-MMP-independent activation of MMP-2 was seen which resembled the level of MMP-2 activation persisting when wild-type fibroblasts were cultured in the presence of both ascorbic acid and MMP inhibitors. We were also unable to block this residual activation with inhibitors specific for serinyl, aspartyl, or cysteinyl enzymes.

The inter-relationships between ovarian-independent growth, tumorigenicity, invasiveness and antioestrogen resistance in the malignant progression of human breast cancer

Among the processes contributing to the progressive acquisition of the highly malignant phenotype in breast cancer are ovarian-independent growth, antioestrogen resistance and increased metastatic potential. We have previously observed that increased invasiveness and development of ovarian-independent growth occur independently. In an attempt to define the inter-relationships between these processes further, we have compared the phenotypes of ovarian-independent, invasive and antioestrogen-resistant sublines of the ovarian-dependent human breast cancer cell line MCF-7. Cells acquiring ovarian-independent growth can retain sensitivity to anti-oestrogens. One clone of MCF-7 cells selected for stable antioestrogen resistance has become non-tumorigenic but its invasive potential remains unaltered. Thus, acquisitions of some characteristics of the progressed phenotype can occur independently. This phenomenon of independent parameters in phenotypic progression could partly explain the considerable intra- and intertumour heterogeneity characteristic of breast tumours.