Fission yeast: in shape to divide.

How are cell morphogenesis and cell cycle coordinated? The fission yeast is a rod-shaped unicellular organism widely used to study how a cell self-organizes in space and time. Here, we discuss recent advances in understanding how the cell acquires and maintains its regular rod shape and uses it to control cell division. The cellular body plan is established by microtubules, which mark antipodal growth zones and medial division. In turn, cellular dimensions are defined by the small GTPase Cdc42 and downstream regulators of vesicle trafficking. Yeast cells then repetitively use their simple rod shape to orchestrate the position and timing of cell division.

Cell-free reconstitution of microautophagy in yeast.

Microautophagy is the direct uptake of soluble or particulate cellular constituents into lysosomes. Here, I describe methods to reconstitute and study this process in vitro, using vacuoles (lysosomes) from the yeast Saccharomyces cerevisiae as model organelles. Protocols to grow the cells, isolate vacuoles from them, and to induce microautophagy of soluble tracers are presented.

IPH response to DSD consultation on Marine Gardens, Bangor

A development has been proposed in Bangor, Co Down.  The Department of Social Development (DSD) consulted on the issue and IPH has responded as below.  IPH has also carried out a Health Impact Assessment (HIA) screening on the proposal which can be accessed below.  The proposed development is bounded by Main Street, King Street, Southwell Road, Queens Street and the Marine Gardens Car Park. The scheme includes a mix of retail units within a new covered street; office space and over 200 residential homes. The development proposal also contains multiple leisure aspects with plans to incorporate a 120-150 bed hotel; a 400 seat multi-use arts, performance and convention space; a family entertainment centre and restaurants, in addition to enhancing the public realm and civic uses of the waterfront area with the creation of a promenade, an outdoor performance space, public parks and tourist facilities.

Guidance on the transfer of mentally disordered patients detained under the Mental Health (NI) Order 1986 to and from Hospitals in Great Britain - August 2011 (PDF 101 KB)

Guidance on the transfer of mentally disordered patients detained under the Mental Health (NI) Order 1986 to and from Hospitals in Great Britain - August 2011.

Analysis of parity between protein-based electrophoretic methods for the characterization of oral Candida species

Electrophoretic studies of multilocus-enzymes (MLEE) and whole-cell protein (SDS-PAGE) were carried out in order to evaluate the parity between different methods for the characterization of five Candida species commonly isolated from oral cavity of humans by numerical taxonomy methods. The obtained data revealed that sodium dodecyl sulfate polyacrylamide gel electrophoresis is more efficient in grouping strains in their respective species while MLEE has much limited resolution in organizing all strains in their respective species-specific clusters. MLEE technique must be regarded for surveys in which just one species of Candida is involved.

Genomics of wine yeast: functional analysis of new and variable genes

Projecte de recerca elaborat a partir d’una estada a l’Institut National de la Recherche Agronomique, França, entre 2007 i 2009. Saccharomyces cerevisiae ha estat el llevat utilitzat durant mil.lenis en l'elaboració de vins. Tot i així, es té poc coneixement sobre les pressions de selecció que han actuat en la modelització del genoma dels llevats vínics. S’ha seqüenciat el genoma d'una soca vínica comercial, EC1118, obtenint 31 supercontigs que cobreixen el 97% del genoma de la soca de referència, S288c. S’ha trobat que el genoma de la soca vínica es diferencia bàsicament en la possessió de 3 regions úniques que contenen 34 gens implicats en funcions claus per al procés fermentatiu. A banda, s’han dut a terme estudis de filogènia i synteny (ordre dels gens) que mostren que una d'aquestes tres regions és pròxima a una espècie relacionada amb el gènere Saccharomyces, mentre que les altres dos regions tenen un origen no-Saccharomyces. S’ha identificat mitjançant PCR i seqüenciació a Zygosaccharomyces bailii, una espècie contaminant de les fermentacions víniques, com a espècie donadora d'una de les dues regions. Les hibridacions naturals entre soques de diferents espècies dins del grup Saccharomyces sensu stricto ja han estat descrites. El treball és el primer que presenta hibridacions entre espècies Saccharomyces i no-Saccharomyces (Z. bailii, en aquest cas). També s’assenyala que les noves regions es troben freqüent i diferencialment presents entre els clades de S. cerevisiae, trobant-se de manera gairebé exclusiva en el grup de les soques víniques, suggerint que es tracta d'una adquisició recent de transferència gènica. En general, les dades demostren que el genoma de les soques víniques pateix una constant remodelació mitjançant l'adquisició de gens exògens. Els resultats suggereixen que aquests processos estan afavorits per la proximitat ecològica i estan implicats en l'adaptació molecular de les soques víniques a les condicions d'elevada concentració en sucres, poc nitrogen i elevades concentracions en etanol.

Tackling Equality and Targeting Social Need - Improving the Infrastucture (PDF 101 KB)

This paper considers two recent initiatives in Northern Ireland relating to targeting social need and inequalities. While the paper deals with each separately they should be seen as complementary. Therefore, many of the points made in relation to one are also pertinent to the other. åÊ

PAP1 [poly(A) polymerase 1] homozygosity and hyperadenylation are major determinants of increased mRNA stability of CDR1 in azole-resistant clinical isolates of Candida albicans.

Using genetically matched azole-susceptible (AS) and azole-resistant (AR) clinical isolates of Candida albicans, we recently demonstrated that CDR1 overexpression in AR isolates is due to its enhanced transcriptional activation and mRNA stability. This study examines the molecular mechanisms underlying enhanced CDR1 mRNA stability in AR isolates. Mapping of the 3' untranslated region (3' UTR) of CDR1 revealed that it was rich in adenylate/uridylate (AU) elements, possessed heterogeneous polyadenylation sites, and had putative consensus sequences for RNA-binding proteins. Swapping of heterologous and chimeric lacZ-CDR1 3' UTR transcriptional reporter fusion constructs did not alter the reporter activity in AS and AR isolates, indicating that cis-acting sequences within the CDR1 3' UTR itself are not sufficient to confer the observed differential mRNA decay. Interestingly, the poly(A) tail of the CDR1 mRNA of AR isolates was approximately 35-50 % hyperadenylated as compared with AS isolates. C. albicans poly(A) polymerase (PAP1), responsible for mRNA adenylation, resides on chromosome 5 in close proximity to the mating type-like (MTL) locus. Two different PAP1 alleles, PAP1-a/PAP1-alpha, were recovered from AS (MTL-a/MTL-alpha), while a single type of PAP1 allele (PAP1-alpha) was recovered from AR isolates (MTL-alpha/MTL-alpha). Among the heterozygous deletions of PAP1-a (Deltapap1-a/PAP1-alpha) and PAP1-alpha (PAP1-a/Deltapap1-alpha), only the former led to relatively enhanced drug resistance, to polyadenylation and to transcript stability of CDR1 in the AS isolate. This suggests a dominant negative role of PAP1-a in CDR1 transcript polyadenylation and stability. Taken together, our study provides the first evidence, to our knowledge, that loss of heterozygosity at the PAP1 locus is linked to hyperadenylation and subsequent increased stability of CDR1 transcripts, thus contributing to enhanced drug resistance.

Cryo-negative staining reveals conformational flexibility within yeast RNA polymerase I.

The structure of the yeast DNA-dependent RNA polymerase I (RNA Pol I), prepared by cryo-negative staining, was studied by electron microscopy. A structural model of the enzyme at a resolution of 1.8 nm was determined from the analysis of isolated molecules and showed an excellent fit with the atomic structure of the RNA Pol II Delta4/7. The high signal-to-noise ratio (SNR) of the stained molecular images revealed a conformational flexibility within the image data set that could be recovered in three-dimensions after implementation of a novel strategy to sort the "open" and "closed" conformations in our heterogeneous data set. This conformational change mapped in the "wall/flap" domain of the second largest subunit (beta-like) and allows a better accessibility of the DNA-binding groove. This displacement of the wall/flap domain could play an important role in the transition between initiation and elongation state of the enzyme. Moreover, a protrusion was apparent in the cryo-negatively stained model, which was absent in the atomic structure and was not detected in previous 3D models of RNA Pol I. This structure could, however, be detected in unstained views of the enzyme obtained from frozen hydrated 2D crystals, indicating that this novel feature is not induced by the staining process. Unexpectedly, negatively charged molybdenum compounds were found to accumulate within the DNA-binding groove, which is best explained by the highly positive electrostatic potential of this region of the molecule, thus, suggesting that the stain distribution reflects the overall surface charge of the molecule.

Myosin Vs organize actin cables in fission yeast.

Myosin V motors are believed to contribute to cell polarization by carrying cargoes along actin tracks. In Schizosaccharomyces pombe, Myosin Vs transport secretory vesicles along actin cables, which are dynamic actin bundles assembled by the formin For3 at cell poles. How these flexible structures are able to extend longitudinally in the cell through the dense cytoplasm is unknown. Here we show that in myosin V (myo52 myo51) null cells, actin cables are curled, bundled, and fail to extend into the cell interior. They also exhibit reduced retrograde flow, suggesting that formin-mediated actin assembly is impaired. Myo52 may contribute to actin cable organization by delivering actin regulators to cell poles, as myoV defects are partially suppressed by diverting cargoes toward cell tips onto microtubules with a kinesin 7-Myo52 tail chimera. In addition, Myo52 motor activity may pull on cables to provide the tension necessary for their extension and efficient assembly, as artificially tethering actin cables to the nuclear envelope via a Myo52 motor domain restores actin cable extension and retrograde flow in myoV mutants. Together these in vivo data reveal elements of a self-organizing system in which the motors shape their own tracks by transporting cargoes and exerting physical pulling forces.

Antimicrobial activity in vitro of plumbagin isolated from Plumbago species

Plumbagin is a naturally occurring naphthoquinone isolated from roots of Plumbago scandens. The plant was collected at the Campus of Fundação Oswaldo Cruz, Rio de Janeiro, Brazil. P. scandens is used as a traditional medicine for the treatment of several diseases. The antimicrobial activity of plumbagin was evaluated using the macrodilution method. The compound exhibited relatively specific activity against bacteria and yeast. The minimum inhibitory concentration test showed the growth inhibiton of Staphylococcus aureus at a concentration of 1.56 µg/ml and of Candida albicans at a concentration of 0.78 µg/ml. These results suggest the naphthoquinone plumbagin as a promising antimicrobial agent.

Use of molecular methods in identification of Candida Species and evaluation of fluconazole resistance

The aim of this study was to evaluate the use of one of the molecular typing methods such as PCR (polymerase chain reaction) following by RFLP (restriction fragment length polymorphism) analysis in the identification of Candida species and then to differentiate the identified azole susceptible and resistant Candida albicans strains by using AP-PCR (arbitrarily primed-polymerase chain reaction). The identification of Candida species by PCR and RFLP analysis was based on the size and primary structural variation of rDNA intergenic spacer regions (ITS). Forty-four clinical Candida isolates comprising 5 species were included to the study. The amplification products were digested individually with 3 different restriction enzymes: HaeIII, DdeI, and BfaI. All the isolates tested yielded the expected band patterns by PCR and RFLP analysis. The results obtained from this study demonstrate that Candida species can be differentiated as C. albicans and non-C. albicans strains only by using HaeIII restriction enzyme and BfaI maintains the differentiation of these non-C. albicans species. After identification Candida species with RFLP analysis, C. albicans strains were included to the AP-PCR test. By using AP-PCR, fluconazole susceptible and resistant strains were differentiated. Nine fluconazole susceptible and 24 fluconazole resistant C. albicans were included to the study. Fluconazole resistant strains had more bands when evaluating with the agarose gel electrophoresis but there were no specific discriminatory band patterns to warrant the differentiation of the resistance. The identification of Candida species with the amplification of intergenic spacer region and RFLP analysis is a practical, short, and a reliable method when comparing to the conventional time-consuming Candida species identification methods. The fluconazole susceptibility testing with AP-PCR seems to be a promising method but further studies must be performed for more specific results.

Ku-deficient yeast strains exhibit alternative states of silencing competence.

In Saccharomyces cerevisiae, efficient silencer function requires telomere proximity, i.e. compartments of the nucleoplasm enriched in silencing factors. Accordingly, silencers located far from telomeres function inefficiently. We show here that cells lacking yKu balance between two mitotically stable states of silencing competence. In one, a partial delocalization of telomeres and silencing factors throughout the nucleoplasm correlates with enhanced silencing at a non-telomeric locus, while in the other, telomeres retain their focal pattern of distribution and there is no repression at the non-telomeric locus, as observed in wild-type cells. The two states also differ in their level of residual telomeric silencing. These findings indicate the existence of a yKu-independent pathway of telomere clustering and Sir localization. Interestingly, this pathway appears to be under epigenetic control.

Oral Candida flora from Brazilian human immunodeficiency virus-infected patients in the highly active antiretroviral therapy era

One of the main opportunistic fungal infections amongst immunocompromised individuals is oral candidosis, which has been found in up to 90% of human immunodeficiency virus (HIV)-infected patients. This study employed yeasts isolated from the saliva and oral cavities of 114 HIV-infected patients living in Campinas, São Paulo. Of the isolates, 57.8% were identified as Candida albicans and 42.1% as non-C. albicans. The latter isolates were subsequently identified as C. krusei (7.5%), C. lusitaniae (5.2%), C. tropicalis (4.6%), C. parapsilosis (4.6%), C. glabrata (2.8%), C. kefyr (1.7%), C. guilliermondii (1.7%), C. intermedia (1.1%), C. norvegensis (0.5%), and Rhodotorula rubra (1.7%). Susceptibility of the isolates to amphotericin B, fluconazole, miconazole, and itraconazole was also determined by a microdilution method adopted by the National Committee for Clinical Laboratory Standards. The isolates demonstrated various susceptibilities to the antifungal agents. In particular 29 C. albicans and 13 non-C. albicans isolates showed low susceptibility to FLCZ (> 64 µg/ml). This study revealed huge diversity of Candida species, in particular the increasing emergence of non-C. albicans associated with the oral flora of HIV-infected patients.