Regulation of apoptotic protease activating factor-1 oligomerization and apoptosis by the WD-40 repeat region

Apoptotic protease activating factor-1 (Apaf-1) has been identified as a proximal activator of caspase-9 in cell death pathways that trigger mitochondrial damage and cytochrome c release. The mechanism of Apaf-1 action is unclear but has been proposed to involve the clustering of caspase-9 molecules, thereby facilitating autoprocessing of adjacent zymogens. Here we show that Apaf-1 can dimerize via the CED-4 homologous and linker domains of the molecule providing a means by which Apaf-1 can promote the clustering of caspase-9 and facilitate its activation. Apaf-1 dimerization was repressed by the C-terminal half of the molecule, which contains multiple WD-40 repeats, but this repression was overcome in the presence of cytochrome c and dATP. Removal of the WD-40 repeat region resulted in a constitutively active Apaf-1 that exhibited greater cytotoxicity in transient transfection assays when compared with full-length Apaf-1. These data suggest a mechanism for Apaf-1 function and reveal an important regulatory role for the WD-40 repeat region.

StaRProtein: a web server for prediction of stability of repeat proteins

Repeat proteins have become increasingly important due to their capability to bind to almost any proteins and the potential as alternative therapy to monoclonal antibodies. In the past decade repeat proteins have been designed to mediate specific protein-protein interactions. The tetratricopeptide and ankyrin repeat proteins are two classes of helical repeat proteins that form different binding pockets to accommodate various partners. It is important to understand the factors that define folding and stability of repeat proteins in order to prioritize the most stable designed repeat proteins to further explore their potential binding affinities. Here we developed distance-dependant statistical potentials using two classes of alpha-helical repeat proteins, tetratricopeptide and ankyrin repeat proteins respectively, and evaluated their efficiency in predicting the stability of repeat proteins. We demonstrated that the repeat-specific statistical potentials based on these two classes of repeat proteins showed paramount accuracy compared with non-specific statistical potentials in: 1) discriminate correct vs. incorrect models 2) rank the stability of designed repeat proteins. In particular, the statistical scores correlate closely with the equilibrium unfolding free energies of repeat proteins and therefore would serve as a novel tool in quickly prioritizing the designed repeat proteins with high stability. StaRProtein web server was developed for predicting the stability of repeat proteins.

A coiled-coil-repeat protein 'Ccrp' in Bdellovibrio bacteriovorus prevents cellular indentation, but is not essential for vibroid cell morphology

Bdellovibrio bacteriovorus are small, vibroid, predatory bacteria that grow within the periplasmic space of a host Gram-negative bacterium. The intermediate-filament (IF)-like protein crescentin is a member of a broad class of IF-like, coiled-coil-repeat-proteins (CCRPs), discovered in Caulobacter crescentus, where it contributes to the vibroid cell shape. The B. bacteriovorus genome has a single ccrp gene encoding a protein with an unusually long, stutter-free, coiled-coil prediction; the inactivation of this did not alter the vibriod cell shape, but caused cell deformations, visualized as chiselled insets or dents, near the cell poles and a general 'creased' appearance, under the negative staining preparation used for electron microscopy, but not in unstained, frozen, hydrated cells. Bdellovibrio bacteriovorus expressing 'teal' fluorescent protein (mTFP), as a C-terminal tag on the wild-type Ccrp protein, did not deform under negative staining, suggesting that the function was not impaired. Localization of fluorescent Ccrp-mTFP showed some bias to the cell poles, independent of the cytoskeleton, as demonstrated by the addition of the MreB-specific inhibitor A22. We suggest that the Ccrp protein in B. bacteriovorus contributes as an underlying scaffold, similar to that described for the CCRP protein FilP in Streptomyces coelicolor, preventing cellular indentation, but not contributing to the vibroid shape of the B. bacteriovorus cells.

PCR amplification of short tandem repeat sequences allows serial studies of chimaerism/engraftment following BMT in rodents

Animal models of bone marrow transplantation (BMT) allow evaluation of new experimental treatment strategies. One potential strategy involves the treatment of donor marrow with ultra-violet B light to allow transplantation across histocompatibility boundaries without an increase in graft rejection or graft-versus-host disease. A major requirement for a new experimental protocol, particularly if it involves manipulation of the donor marrow, is that the manipulated marrow gives rise to long-term multilineage engraftment. DNA based methodologies are now routinely used by many centres to evaluate engraftment and degree of chimaerism post-BMT in humans. We report the adaptation of this methodology to the serial study of engraftment in rodents. Conditions have been defined which allow analysis of serial tail vein samples using PCR of short tandem repeat sequences (STR-PCR). These markers have been used to evaluate the contribution of ultraviolet B treated marrow to engraftment following BMT in rodents without compromising the health of the animals under study. Chimaerism data from sequential tail vein samples and bone marrow from selected sacrificed animals showed excellent correlation, thus confirming the validity of this approach in analysing haemopoietic tissue. Thus the use of this assay may facilitate experimental studies in animal BMT.

Evaluation of mixed chimerism by in vitro amplification of dinucleotide repeat sequences using the polymerase chain reaction

The influence of mixed hematopoietic chimerism (MC) after allogeneic bone marrow transplantation remains unknown. Increasingly sensitive detection methods have shown that MC occurs frequently. We report a highly sensitive novel method to assess MC based on the polymerase chain reaction (PCR). Simple dinucleotide repeat sequences called microsatellites have been found to vary in their repeat number between individuals. We use this variation to type donor-recipient pairs following allogeneic BMT. A panel of seven microsatellites was used to distinguish between donor and recipient cells of 32 transplants. Informative microsatellites were subsequently used to assess MC after BMT in this group of patients. Seventeen of the 32 transplants involved a donor of opposite sex; hence, cytogenetics and Y chromosome-specific PCR were also used as an index of chimerism in these patients. MC was detected in bone marrow aspirates and peripheral blood in 18 of 32 patients (56%) by PCR. In several cases, only stored slide material was available for analysis but PCR of microsatellites or Y chromosomal material could be used successfully to assess the origin of cells in this archival material. Cytogenetic analysis was possible in 17 patients and MC was detected in three patients. Twelve patients received T-cell-depleted marrow and showed a high incidence of MC as revealed by PCR (greater than 80%). Twenty patients received unmanipulated marrow, and while the incidence of MC was lower (44%), this was a high percentage when compared with other studies. Once MC was detected, the percentages of recipient cells tended to increase. However, in patients exhibiting MC who subsequently relapsed, this increase was relatively sudden. The overall level of recipient cells in the group of MC patients who subsequently relapsed was higher than in those who exhibited stable MC. Thus, while the occurrence of MC was not indicative of a poor prognosis per se, sudden increases in the proportions of recipient cells may be a prelude to graft rejection or relapse.

Repeat-measures longitudinal study evaluating behavioural and gastrointestinal symptoms in children with autism before, during and after visceral osteopathic technique (VOT)

Summary: This study investigated the influence of visceral osteopathic technique (VOT) on the behaviour and gastrointestinal (GI) symptoms of children with autism using a validated questionnaire to measure outcome. Methods: The 49 recruited autistic children suffered GI symptoms and impaired social interaction and communication, but were otherwise healthy. Thirty minute VOT sessions were applied to the abdomens of the children over a 6 week period whilst their GI and behavioural parameters were recorded. Outcomes were measured using a modified Autism Research Institute Secretin Outcomes Survey Form, the ‘S.O.S Form’. Four questionnaires were completed by parents before treatment (control period), four completed during treatment (treatment period) and one completed six weeks after the last treatment (post treatment period). Subjects acted as their own controls. Results: Results from repeat ANOVA demonstrated a positive, overall significant, symptomatic improvement (p < 0.05) in ‘social behaviour and communication’ and ‘digestive signs’ subscales of the questionnaire comparing before and after VOT. Significant improvement in vomiting (p = 0.00029), poor appetite (p = 0.039) and eye contact (p = 0.035) was also demonstrated after VOT application. Discussion and conclusion: The experimental hypothesis has been supported indicating a positive effect of VOT on some of the measured GI symptoms and behavioural patterns in this group of children with autism. This data indicates that the application of VOT may be of benefit to children with autism and GI disturbance.

Development of triplet repeat primed PCR (TP-PCR) technique as a support of molecular test in Machado-Joseph disease

Dissertação de Mestrado, Ciências Biomédicas, 3 de Fevereiro de 2016, Universidade dos Açores.

Analyse de la fertilité des vaches laitières Holstein «Repeat Breeder»

L’importance de la problématique des vaches laitières non gestantes avec multiples inséminations (› 3) communément appelées « Repeat Breeder » (RB), au Québec, repose sur deux facteurs: son incidence et son impact économique. Actuellement, l’incidence du syndrome RB au Québec est de ± 25% (Rapport annuel, juin 2008, www.dsahr.ca). Les pertes monétaires associées à ce problème proviennent des frais vétérinaires et d’insémination, de la diminution de productivité ainsi que des pertes en lien avec la réforme involontaire. Afin d’avoir de meilleures connaissances sur ce syndrome, nous devons connaître les facteurs de risques généraux impliqués et ensuite explorer la condition individuelle de ces vaches problèmes. Dans la première partie de ce mémoire, une banque de données informatisées répertoriant 418 383 lactations fut analysée. L’objectif de ce projet de recherche était d’évaluer l’impact des problèmes reproducteurs post-partum et l’effet du nombre de lactations comme facteurs de risques de la vache Repeat Breeder. L’analyse a permis d’établir la dystocie comme étant la condition ayant le plus de conséquences sur la fertilité future. D’autres facteurs de risques à savoir le nombre de lactations influencent le pronostic reproducteur. La deuxième partie de ce mémoire consistait à explorer, à l’aide d’outils cliniques, la condition individuelle des vaches RB. Une étude cohorte fut menée sur des vaches Holstein en fin de période d’attente volontaire au jour 7 du cycle oestral. Les tests cliniques étudiés furent la vaginoscopie, l’examen transrectal, l’ultrasonographie du système reproducteur, la présence d’estérases leucocytaires, la bactériologie et la biochimie du liquide utérin, la cytologie endométriale et le profil de progestérone sérique. Les résultats de ces tests cliniques dévoilent que l’examen bactériologique du liquide utérin est révélateur du statut reproducteur futur.

Exploring TERRA (TElomeric Repeat-containing RNA) Expression and Regulation During Cell Growth in Saccharomyces cerevisiae

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On Queues with Interruptions and Repeat or Resumption of Service

This thesis entitled' On Queues with Interruptions and Repeat or Resumption of Service' introduces several new concepts into queues with service interruption. It is divided into Seven chapters including an introductory chapter. The following are keywords that we use in this thesis: Phase type (PH) distribution, Markovian Arrival Process (MAP), Geometric Distribution, Service Interruption, First in First out (FIFO), threshold random variable and Super threshold random variable. In the second chapter we introduce a new concept called the 'threshold random variable' which competes with interruption time to decide whether to repeat or resume the interrupted service after removal of interruptions. This notion generalizes the work reported so far in queues with service interruptions. In chapter 3 we introduce the concept of what is called 'Super threshold clock' (a random variable) which keeps track of the total interruption time of a customer during his service except when it is realized before completion of interruption in some cases to be discussed in this thesis and in other cases it exactly measures the duration of all interruptions put together. The Super threshold clock is OIl whenever the service is interrupted and is deactivated when service is rendered. Throughout this thesis the first in first out service discipline is followed except for priority queues.

Protection of Farmers’ Rights in India-Challenges for Law in the Context of Plant Breeders’ Rights

School of Legal Studies, Cochin University of Science and Technology

How do I get Word 2010 table headers to repeat on subsequent pages

When a table crosses on to 2 or more pages it can be very useful to have the headers appear on all subsequent pages. This video shows how.

Plasmodium vivax lineages: geographical distribution, tandem repeat polymorphism, and phylogenetic relationship

Background: Multi-drug resistance and severe/ complicated cases are the emerging phenotypes of vivax malaria, which may deteriorate current anti-malarial control measures. The emergence of these phenotypes could be associated with either of the two Plasmodium vivax lineages. The two lineages had been categorized as Old World and New World, based on geographical sub-division and genetic and phenotypical markers. This study revisited the lineage hypothesis of P. vivax by typing the distribution of lineages among global isolates and evaluated their genetic relatedness using a panel of new mini-satellite markers. Methods: 18S SSU rRNA S-type gene was amplified from 420 Plasmodium vivax field isolates collected from different geographical regions of India, Thailand and Colombia as well as four strains each of P. vivax originating from Nicaragua, Panama, Thailand (Pak Chang), and Vietnam (ONG). A mini-satellite marker panel was then developed to understand the population genetic parameters and tested on a sample subset of both lineages. Results: 18S SSU rRNA S-type gene typing revealed the distribution of both lineages (Old World and New World) in all geographical regions. However, distribution of Plasmodium vivax lineages was highly variable in every geographical region. The lack of geographical sub-division between lineages suggests that both lineages are globally distributed. Ten mini-satellites were scanned from the P. vivax genome sequence; these tandem repeats were located in eight of the chromosomes. Mini-satellites revealed substantial allelic diversity (7-21, AE = 14.6 +/- 2.0) and heterozygosity (He = 0.697-0.924, AE = 0.857 +/- 0.033) per locus. Mini-satellite comparison between the two lineages revealed high but similar pattern of genetic diversity, allele frequency, and high degree of allele sharing. A Neighbour-Joining phylogenetic tree derived from genetic distance data obtained from ten mini-satellites also placed both lineages together in every cluster. Conclusions: The global lineage distribution, lack of genetic distance, similar pattern of genetic diversity, and allele sharing strongly suggested that both lineages are a single species and thus new emerging phenotypes associated with vivax malaria could not be clearly classified as belonging to a particular lineage on basis of their geographical origin.