Reappraisal of the type species of Polysiphonia (Rhodomelaceae, Rhodophyta)

The genus Polysiphonia Greville, nom. cons., has had a long and confused nomenclatural history. At present, Polysiphonia has a wide circumscription, including at least 200 species, but it is heterogeneous in many vegetative and reproductive developmental features. Central to any re-evaluation of the genus is a detailed examination of the type species of Polysiphonia, P. urceolata (Lightfoot ex Dillwyn) Greville, which is conspecific with P. stricta (Dillwyn) Greville. We here report on the vegetative and reproductive morphology of P. stricta, including P, urceolata, based on type and other material from the British Isles. Thalli consist of prostrate and erect ecorticate axes with four pericentral cells, attached by unicellular rhizoids remaining in open connection with pericentral cells. Prostrate axes lack vegetative trichoblasts; trichoblasts occur seasonally on erect axes. Branch initials are cut off from the subapical cell at intervals of four or five segments in dichotomous and alternating pairs rather than being formed horn each axial cell in the spiral pattern typical of most species of Polysiphonia. Spermatangial branch initials, which are trichoblast homologues, are produced directly from each axial cell at the tips of erect branches, not subtended by trichoblasts, and have two- to five-celled sterile tips when mature. The mature carpogonial branch is four-celled with a two-celled first sterile group and a one-celled second sterile group. Following presumed fertilization, direct fusion apparently takes place between carpogonium and auxiliary tell; mature cystocarps are usually urceolate. Tetrasporangia are formed from the third pericentral cell, in straight series, and have two pre-sporangial cover cells. Previous accounts of a third, post-sporangial cover cell could not be substantiated. P. stricta and a small group of other Polysiphonia species differ in several important respects from most members of the genus, which have rhizoids cut off from pericentral cells by a cell division, abundant trichoblasts, spirally arranged tetrasporangia and a post-sporangial cover cell. The branching pattern of P. stricta highlights the difficulties of distinguishing between the tribes Polysiphonieae and Pterosiphonieae.

Ownership type and team climate in elderly care facilities: the moderating effect of stress factors

Aims. This paper is a report of a study examining the association between ownership type and perceived team climate among older people care staff. In addition, we examined whether work stress factors (time pressure, resident-related stress, role conflicts and role ambiguity) mediated or moderated the above mentioned association. Background. There has been a trend towards contracting out in older people care facilities in Finland and the number of private for-profit firms has increased. Studies suggest that there may be differences in employee well-being and quality of care according to the ownership type of older people care. Methods. Cross-sectional survey data was collected during the autumn of 2007 from 1084 Finnish female older people care staff aged 1869 years were used. Team Climate Inventory was used to measure team climate. Ownership type was divided into four categories: for-profit sheltered homes, not-for-profit sheltered homes, public sheltered homes and not-for-profit nursing homes. Analyses of covariance were used to examine the associations. Results. Team climate dimensions participative safety, vision and support for innovation were higher in not-for-profit organizations (both sheltered homes and nursing homes) compared to for-profit sheltered homes and public sheltered homes. Stress factors did not account for these associations but acted as moderators in a way that in terms of task orientation and participative safety employees working in for-profit organizations seemed to be slightly more sensitive to work-related stress than others. Conclusion. Our results suggest that for-profit organizations and public organizations may have difficulties in maintaining their team climate. In consequence, these organizations should focus more effort on improving their team climate.

Immune responses and vaccine-induced immunity against Porcine circovirus type 2

Porcine circovirus type 2 (PCV2) is essential but not sufficient for postweaning multi-systemic wasting syndrome (PMWS) occurrence in pigs. The outcome of PCV2 infection depends on the specific immune responses that are developing during the infection. Diseased pigs are immunosupressed and unable to mount effective immune responses to clear the virus from circulation. In the final stage, PMWS-affected pigs suffer from extensive lymphoid lesions and altered cytokine expression patterns in peripheral blood mononuclear cells (PBMCs) and lymphoid organs. PCV2 infection can also be asymptomatic, demonstrating that not every infection will guarantee the occurrence of severe immunopathological disturbances. Asymptomatic animals have higher virus specific and neutralising antibody titres than PMWS-affected animals. Recent results have pointed out that the mechanisms by which PCV2 can affect the immune responses involve the induction of IL-10, virus accumulation into and modulation of plasmacytoid dendritic cells and the role of viral DNA in regulation of immune cell functions. Fourteen years after the first description of PMWS in Canada, efficient commercial vaccines against PCV2 are available. The vaccine success is based on activated humoral and cellular immune responses against PCV2. This review focuses on the recent research on immunological aspects during PCV2 infections and summarizes what is currently known about the vaccine-induced immunity. (C) 2010 Elsevier B.V. All rights reserved.

Detection of a novel porcine boca-like virus in the background of porcine circovirus type 2 induced postweaning multisystemic wasting syndrome

Porcine circovirus type 2 (PCV-2) has been found to be the causative agent of postweaning multisystemic wasting syndrome (PMWS). However, PCV-2 is a ubiquitous virus in the swine population and a majority of pigs infected with PCV-2 do not develop the disease. Different factors such as age, maintenance, the genetics of PCV-2, other pathogens, etc. have been suggested to contribute to the development of PMWS. However, so far no proven connection between any of these factors and the disease development has been found. In this study we explored the possible presence of other so far unknown DNA containing infectious agents in lymph nodes collected from Swedish pigs with confirmed PMWS through random amplification and high-throughput sequencing. Although the vast majority of the amplified genetic sequences belonged to PCV-2, we also found genome sequences of Torque Teno virus (TTV) and of a novel parvovirus. The detection of TTV was expected since like PCV-2, TTV has been found to have high prevalence in pigs around the world. We were able to amplify a longer region of the parvovirus genome, consisting of the entire NP1 and partial VP1/2. By comparative analysis of the nucleotide sequences and phylogenetic studies we propose that this is a novel porcine parvovirus, with genetic relationship to bocaviruses.

Evaluation of induction of porcine dermatitis and nephropathy syndrome in gnotobiotic pigs with negative results for porcine circovirus type 2

Objective-To determine whether porcine dermatitis and nephropathy syndrome (PDNS) could be experimentally induced in gnotobiotic swine.

Lack of in vitro and in vivo effects of lipopolysaccharide on porcine circovirus type 2 infection

Porcine circovirus type 2 (PCV2) is the causative agent of postweaning multisystemic wasting syndrome (PMWS). The presence of immunostimulating factors or concurrent infections seems to be crucial for PMWS development. Lipopolysaccharide (LPS) is a potent immunological activator and has recently been suggested to enhance PCV2 replication in vitro. This study was designed to evaluate the effects of different LPS products on PCV2 in vitro replication of pulmonary macrophages (PMs), and on the potential ability to trigger PMWS in cesarean-derived, colostrum-deprived (CDCD) PCV2-inoculated piglets. In vitro studies using two different PCV2 isolates (Stoon-1010 and 1452/3) showed the presence of PCV2 antigen within the cytoplasm to a variable degree; PCV2 Stoon-1010 was barely detectable (

T lymphocyte epitope mapping of porcine circovirus type 2

Immunoreactive T lymphocyte epitopes within the ORF1, ORF2, and ORF 3 products of porcine circovirus type 2 (PCV2) were mapped. For this, overlapping linear 20-mer peptides were synthesized and tested for their ability to induce T lymphocyte proliferation in porcine peripheral blood mononuclear cells (PBMCs) isolated from experimentally PCV2-infected pigs. After a preliminary screening of 31 (ORF1), 23 (ORF2), and 10 (ORF3) peptides using PBMCs from 4 PCV2-infected pigs, none of the peptides appeared to be immunoreactive (stimulation index [SI] : 2) in all four pigs. Only 14 peptides appeared to be immunoreactive in 3 of the 4 pigs. These peptides were designated as immunodominant in the preliminary screening and selected for further analysis. The immunodominant peptides were resynthesized and purified by high-performance liquid chromatography and tested for their ability to induce T lymphocyte proliferation in PBMCs from another three PCV2-infected pigs. None of the immunodominant peptides appeared to be immunoreactive in all three pigs of the second screening. Only three peptides appeared to be immunoreactive in two of three pigs, two encoded by PCV2 ORF1 (amino acid residues 81-100 and 201-220) and one encoded by PCV2 ORF3 (amino acid residues 31-50), and were therefore considered to be immunodominant in both screenings. Although peptides encoded by ORF2 appeared to show the highest immunoreactivity in some pigs, none of these peptides displayed immunodominance in both screenings. In summary, the present study indicates that the T lymphocyte responses to PCV2 are primarily directed toward epitopes of the nonstructural proteins of ORF1 and ORF3.

A novel Kazal-type trypsin inhibitor from the skin secretion of the Central American red-eyed leaf frog, Agalychnis callidryas.

The chemical complexity of the defensive skin secretion of the red-eyed leaf frog, (Agalychnis callidryas), has not been elucidated in detail. During a systematic study of the skin secretion peptidomes of phyllomedusine frogs, we discovered a novel Kazal-type protein with potent trypsin inhibitory activity (Ki = 1.9 nM) that displays the highest degree of structural similarity with Kazal proteins from bony fishes. The protein was located in reverse-phase HPLC fractions following a screen of such for trypsin inhibition and subsequent partial Edman degradation of the peak active fraction derived the sequence: ATKPR-QYIVL-PRILRPV-GT. The molecular mass of the major component in this fraction was established by MALDI-TOF MS as 5893.09 Da. This partial sequence (assuming blank cycles to be Cys residues) was used to design a degenerate primer pool that was employed successfully in RACE-PCR to clone homologous precursor-encoding cDNA that encoded a mature Kazal protein of 52 amino acid residues with a computed molecular mass of 5892.82 Da. The protein was named A. callidryas Kazal trypsin inhibitor (ACKTI). BLAST analysis revealed that ACKTI contained a canonical Kazal motif (C-x(7)-C-x(6)-Y-x(3)-C-x(2,3)-C). This novel amphibian skin Kazal trypsin inhibitor adds to the spectrum of trypsin inhibitors of Kunitz- and Bowman Birk-type reported from this amphibian source.

BcsK(C) Is an Essential Protein for the Type VI Secretion System Activity in Burkholderia cenocepacia That Forms an Outer Membrane Complex with BcsL(B)

The type VI secretion system (T6SS) contributes to the virulence of Burkholderia cenocepacia, an opportunistic pathogen causing serious chronic infections in patients with cystic fibrosis. BcsK(C) is a highly conserved protein among the T6SSs in Gram-negative bacteria. Here, we show that BcsK(C) is required for Hcp secretion and cytoskeletal redistribution in macrophages upon bacterial infection. These two phenotypes are associated with a functional T6SS in B. cenocepacia. Experiments employing a bacterial two-hybrid system and pulldown assays demonstrated that BcsK(C) interacts with BcsL(B), another conserved T6SS component. Internal deletions within BcsK(C) revealed that its N-terminal domain is necessary and sufficient for interaction with BcsL(B). Fractionation experiments showed that BcsK(C) can be in the cytosol or tightly associated with the outer membrane and that BcsK(C) and BcsL(B) form a high molecular weight complex anchored to the outer membrane that requires BcsF(H) (a ClpV homolog) to be assembled. Together, our data show that BcsK(C)/BcsL(B) interaction is essential for the T6SS activity in B. cenocepacia.

A novel sensor kinase-response regulator hybrid controls biofilm formation and type VI secretion system activity in Burkholderia cenocepacia

Burkholderia cenocepacia is an important opportunistic pathogen causing serious chronic infections in patients with cystic fibrosis (CF). Adaptation of B. cenocepacia to the CF airways may play an important role in the persistence of the infection. We have identified a sensor kinase-response regulator (BCAM0379) named AtsR in B. cenocepacia K56-2 that shares 19% amino acid identity with RetS from Pseudomonas aeruginosa. atsR inactivation led to increased biofilm production and a hyperadherent phenotype in both abiotic surfaces and lung epithelial cells. Also, the atsR mutant overexpressed and hypersecreted an Hcp-like protein known to be specifically secreted by the type VI secretion system (T6SS) in other gram-negative bacteria. Amoeba plaque assays demonstrated that the atsR mutant was more resistant to Dictyostelium predation than the wild-type strain and that this phenomenon was T6SS dependent. Macrophage infection assays also demonstrated that the atsR mutant induces the formation of actin-mediated protrusions from macrophages that require a functional Hcp-like protein, suggesting that the T6SS is involved in actin rearrangements. Three B. cenocepacia transposon mutants that were found in a previous study to be impaired for survival in chronic lung infection model were mapped to the T6SS gene cluster, indicating that the T6SS is required for infection in vivo. Together, our data show that AtsR is involved in the regulation of genes required for virulence in B. cenocepacia K56-2, including genes encoding a T6SS.

Investigating the efficiency of fitting Coxian phase-type distributions to health care data

Coxian phase-type distributions are becoming a popular means of representing survival times within a health care environment. They are favoured as they show a distribution as a system of phases and can allow for an easy visual representation of the rate of flow of patients through a system. Difficulties arise, however, in determining the parameter estimates of the Coxian phase-type distribution. This paper examines ways of making the fitting of the Coxian phase-type distribution less cumbersome by outlining different software packages and algorithms available to perform the fit and assessing their capabilities through a number of performance measures. The performance measures rate each of the methods and help in identifying the more efficient. Conclusions drawn from these performance measures suggest SAS to be the most robust package. It has a high rate of convergence in each of the four example model fits considered, short computational times, detailed output, convergence criteria options, along with a succinct ability to switch between different algorithms.

The Conditional Phase-type distribution: Graphical Models for Continuous Non-Normal Data:The Conditional Phase-type (C-Ph) distribution:

Conditional Gaussian (CG) distributions allow the inclusion of both discrete and continuous variables in a model assuming that the continuous variable is normally distributed. However, the CG distributions have proved to be unsuitable for survival data which tends to be highly skewed. A new method of analysis is required to take into account continuous variables which are not normally distributed. The aim of this paper is to introduce the more appropriate conditional phase-type (C-Ph) distribution for representing a continuous non-normal variable while also incorporating the causal information in the form of a Bayesian network.

Association analysis of proopiomelanocortin (POMC) haplotypes in type 1 diabetes in a UK population

Aim

To assess the association of POMC haplotype-tagged single nucleotide polymorphisms (htSNPs) with the development of type 1 diabetes (T1D) in a Caucasian population.

Methods

All exons, intron 1, and approximately 6-kb upstream and 3-kb downstream of the POMC gene were bidirectionally resequenced to identify DNA polymorphisms in 30 individuals. Allele frequencies were determined (60 chromosomes) and efficient htSNPs were selected using the htSNP2 programme. Genotyping was performed in 390 cases, 339 controls and 245 T1D parent-offspring trios, using Taqman, Sequenom and direct-sequencing technologies.

Results

Thirteen polymorphisms (two novel) with a minor allele frequency greater than 1% were identified. Six POMC htSNPs (rs3754863 G>A, ss161151662 A>G, rs3754860 C>T, rs1009388 G>C, rs3769671 A>C, rs1042571 G>A) were identified. Allele and haplotype frequencies were similar between case and control groups (P>0.60 by permutation test), and assessment of allele transmission distortion from informative parents to affected offspring also failed to find any association. Stratification of these analyses for age-at-onset and HLA-DR risk group (DR3/DR4) revealed no significant associations. A haplotype block of 9.86-kb from rs3754863 to rs1042571 was identified, encompassing the POMC gene. Comparison of haplotype frequencies identified the GGCGAG haplotype as protective against T1D in 12.9% of cases vs. 18.3% of controls: ?2=8.18, Pc=0.03 by permutation test.

Conclusion

The POMC SNP haplotype GGCGAG may have a protective effect against T1D in the UK population. However, this finding needs to be replicated, and the cellular and molecular processes influenced by this POMC haplotype determined to fully appreciate its impact.

Altered cofactor binding affects stability and activity of human UDP-galactose 4′-epimerase: Implications for type III galactosemia

Deficiency of UDP-galactose 4'-epimerase is implicated in type III galactosemia. Two variants, p.K161N-hGALE and p.D175N-hGALE, have been previously found in combination with other alleles in patients with a mild form of the disease. Both variants were studied in vivo and in vitro and showed different levels of impairment. p.K161N-hGALE was severely impaired with substantially reduced enzymatic activity, increased thermal stability, reduced cofactor binding and no ability to rescue the galactose-sensitivity of gal10-null yeast. Interestingly p.K161N-hGALE showed less impairment of activity with UDP-N-acetylgalactosamine in comparison to UDP-galactose. Differential scanning fluorimetry revealed that p.K161N-hGALE was more stable than the wild-type protein and only changed stability in the presence of UDP-N-acetylglucosamine and NAD(+). p.D175N-hGALE essentially rescued the galactose-sensitivity of gal10-null yeast, was less stable than the wild-type protein but showed increased stability in the presence of substrates and cofactor. We postulate that p.K161N-hGALE causes its effects by abolishing an important interaction between the protein and the cofactor, whereas p.D175N-hGALE is predicted to remove a stabilizing salt bridge between the ends of two a-helices that contain residues that interact with NAD(+). These results suggest that the cofactor binding is dynamic and that its loss results in significant structural changes that may be important in disease causation.

The Adult Cystic Fibrosis Airway Microbiota Is Stable over Time and Infection Type, and Highly Resilient to Antibiotic Treatment of Exacerbations

Cystic fibrosis (CF) is characterized by defective mucociliary clearance and chronic airway infection by a complex microbiota. Infection, persistent inflammation and periodic episodes of acute pulmonary exacerbation contribute to an irreversible decline in CF lung function. While the factors leading to acute exacerbations are poorly understood, antibiotic treatment can temporarily resolve pulmonary symptoms and partially restore lung function. Previous studies indicated that exacerbations may be associated with changes in microbial densities and the acquisition of new microbial species. Given the complexity of the CF microbiota, we applied massively parallel pyrosequencing to identify changes in airway microbial community structure in 23 adult CF patients during acute pulmonary exacerbation, after antibiotic treatment and during periods of stable disease. Over 350,000 sequences were generated, representing nearly 170 distinct microbial taxa. Approximately 60% of sequences obtained were from the recognized CF pathogens Pseudomonas and Burkholderia, which were detected in largely non-overlapping patient subsets. In contrast, other taxa including Prevotella, Streptococcus, Rothia and Veillonella were abundant in nearly all patient samples. Although antibiotic treatment was associated with a small decrease in species richness, there was minimal change in overall microbial community structure. Furthermore, microbial community composition was highly similar in patients during an exacerbation and when clinically stable, suggesting that exacerbations may represent intrapulmonary spread of infection rather than a change in microbial community composition. Mouthwash samples, obtained from a subset of patients, showed a nearly identical distribution of taxa as expectorated sputum, indicating that aspiration may contribute to colonization of the lower airways. Finally, we observed a strong correlation between low species richness and poor lung function. Taken together, these results indicate that the adult CF lung microbiome is largely stable through periods of exacerbation and antibiotic treatment and that short-term compositional changes in the airway microbiota do not account for CF pulmonary exacerbations.