Lack of Association Between Haptoglobin Phenotype and Cystic Fibrosis Outcomes

Haptoglobin (Hp), a heme-Iron chelator, has different isoforms which are associated with variable tendency toward infections: Hp 1-1, Hp 2-1, and Hp 2-2. Cystic fibrosis (CF) outcomes are variable and influenced by genetic and environmental factors. The aim of this study was to determine whether Hp phenotype influenced disease severity in CF. One hundred forty-two CF patients from two centers were analyzed for Haptoglobin phenotype using gel electrophoresis of hemoglobin enriched serum. Clinical and microbiological data including bacterial colonization status, lung function, presence of CF-related diabetes and liver disease, rate of exacerbation, and mortality were compared between Hp phenotype groups. We found a trend toward less mucoid PA among Hp 2-2 (20.4 %) compared with Hp 1-1 and Hp 2-1 individuals (33.3 %), p = 0.317. Hp 2-2 individuals also had less antibiotic courses, and lower inflammatory markers without statistical significance. Haptoglobin phenotype is unlikely to be an important modifier of CF phenotype.

You ain´t seen nothing yet. : Transparency’s (lack of) Effect on Source and Message Credibility

Transparency has been proposed to both change the way journalism is being produced and increase its credibility. However, little research has been conducted to assess the connection between transparency and credibility. This study utilizes an experimental setting (N=1320) to measure what impact transparency have on source and message credibility from the user perspective. The results reveals an almost absence of any transparency effect on both source and message credibility although some small significant effects could be observed primarily regarding internal hyperlinks, comments and contextual information. Although further research is desperately needed in this area the study suggest that transparency does not affect the credibility of journalism in the eyes of the contemporary audience and thus have limited appeal as a new norm in journalism.

Close-in planets around giant stars: lack of hot-Jupiters and prevalence of multiplanetary systems

Extrasolar planets abound in almost any possible configuration. However, until five years ago, there was a lack of planets orbiting closer than 0.5 au to giant or subgiant stars. Since then, recent detections have started to populated this regime by confirming 13 planetary systems. We discuss the properties of these systems in terms of their formation and evolution off the main sequence. Interestingly, we find that 70.0 ± 6.6% of the planets in this regime are inner components of multiplanetary systems. This value is 4.2σ higher than for main-sequence hosts, which we find to be 42.4 ± 0.1%. The properties of the known planets seem to indicate that the closest-in planets (a< 0.06 au) to main-sequence stars are massive (i.e., hot Jupiters) and isolated and that they are subsequently engulfed by their host as it evolves to the red giant branch, leaving only the predominant population of multiplanetary systems in orbits 0.06 of this emerging observational trend in the context of formation and evolution of hot Jupiters.

Influence of delayed cholecystectomy after acute gallstone pancreatitis on recurrence: consequences of lack of resources

Introduction: Acute pancreatitis is often a relapsing condition, particularly when its triggering factor persists. Our goal is to determine the recurrence rate of acute biliary pancreatitis after an initial episode, and the time to relapse, as well as to identify the risk factors for recurrence. Material and method: We included all patients admitted for a first acute gallstone pancreatitis event during four years. Primary endpoints included readmission for recurrence and time to relapse. Results: We included 296 patients admitted on a total of 386 occasions. The incidence of acute biliary pancreatitis in our setting is 17.5/100,000 population/year. In all, 19.6% of pancreatitis were severe (22.6% of severe acute pancreatitis for first episodes versus 3.6% for recurring pancreatitis), with an overall mortality of 4.4%. Overall recurrence rate was 15.5%, with a median time to relapse of 82 days. In total, 14.2% of patients relapsed after an acute pancreatitis event without cholecystectomy or endoscopic retrograde cholangio-pancreatography. Severe acute pancreatitis recur in 7.2% of patients, whereas mild cases do so in 16.3%, this being the only risk factor for recurrence thus far identified. Conclusions: Patients admitted for pancreatitis should undergo cholecystectomy as soon as possible or be guaranteed priority on the waiting list. Otherwise, endoscopic retrograde cholangio-pancreatography with sphincterotomy may be an alternative to surgery for selected patients.

RGS14 (414)-mediated prevention of an episodic memory loss: a study of molecular mechanism

A large proportion of human populations suffer memory impairments either caused by normal aging or afflicted by diverse neurological and neurodegenerative diseases. Memory enhancers and other drugs tested so far against memory loss have failed to produce therapeutic efficacy in clinical trials and thus, there is a need to find remedy for this mental disorder. In search for cure of memory loss, our laboratory discovered a robust memory enhancer called RGS14(414). A treatment in brain with its gene produces an enduring effect on memory that lasts for lifetime of rats. Therefore, current thesis work was designed to investigate whether RGS14(414) treatment can prevent memory loss and furthermore, explore through biological processes responsible for RGS-mediated memory enhancement. We found that RGS14(414) gene treatment prevented episodic memory loss in rodent models of normal aging and Alzheimer´s disease. A memory loss was observed in normal rats at 18 months of age; however, when they were treated with RGS14(414) gene at 3 months of age, they abrogated this deficit and their memory remained intact till the age of 22 months. In addition to normal aging rats, effect of memory enhancer treatment in mice model of Alzheimer´s disease (AD-mice) produced a similar effect. AD-mice subjected to treatment with RGS14(414) gene at the age of 2 months, a period when memory was intact, showed not only a prevention in memory loss observed at 4 months of age but also they were able to maintain normal memory after 6 months of the treatment. We posit that long-lasting effect on memory enhancement and prevention of memory loss mediated through RGS14(414) might be due to a permanent structural change caused by a surge in neuronal connections and enhanced neuronal remodeling, key processes for long-term memory formation. A neuronal arborization analysis of both pyramidal and non-pyramidal neurons in brain of RGS14(414)-treated rats exhibited robust rise in neurites outgrowth of both kind of cells, and an increment in number of branching from the apical dendrite of pyramidal neurons, reaching to almost three times of the control animals. To further understand of underlying mechanism by which RGS14(414) induces neuronal arborization, we investigated into neurotrophic factors. We observed that RGS14 treatment induces a selective increase in BDNF. Role of BDNF in neuronal arborization, as well as its implication in learning and memory processes is well described. In addition, our results showing a dynamic expression pattern of BDNF during ORM processing that overlapped with memory consolidation further support the idea of the implication of this neurotrophin in formation of long-term memory in RGS-animals. On the other hand, in studies of expression profiling of RGS-treated animals, we have demonstrated that 14-3-3ζ protein displays a coherent relationship to RGS-mediated ORM enhancement. Recent studies have demonstrated that the interaction of receptor for activated protein kinase 1 (RACK1) with 14-3-3ζ is essential for its nuclear translocation, where RACK1-14-3-3ζ complex binds at promotor IV region of BDNF and promotes an increase in BDNF gene transcription. These observations suggest that 14-3-3ζ might regulate the elevated level of BDNF seen in RGS14(414) gene treated animals. Therefore, it seems that RGS-mediated surge in 14-3-3ζ causes elevated BDNF synthesis needed for neuronal arborization and enhanced ORM. The prevention of memory loss might be mediated through a restoration in BDNF and 14-3-3ζ protein levels, which are significantly decreased in aging and Alzheimer’s disease. Additionally, our results demonstrate that RGS14(414) treatment could be a viable strategy against episodic memory loss.

Corporate social responsibility reporting in the mining sector of Tanzania:(Lack of) government regulatory controls and NGO activism

Purpose – The purpose of this paper is to contribute to the ongoing debate on governance, accountability, transparency and corporate social responsibility (CSR) in the mining sector of a developing country context. It examines the reporting practices of the two largest transnational gold-mining companies in Tanzania in order to draw attention to the role played by local government regulations and advocacy and campaigning by nationally organised non-governmental organisations (NGOs) with respect to promoting corporate social reporting practices. Design/methodology/approach – The paper takes a political economy perspective to consider the serious implications of the neo-liberal ideologies of the global capitalist economy, as manifested in Tanzania’s regulatory framework and in NGO activism, for the corporate disclosure, accountability and responsibility of transnational companies (TNCs). A qualitative field case study methodology is adopted to locate the largely unfamiliar issues of CSR in the Tanzanian mining sector within a more familiar literature on social accounting. Data for the case study were obtained from interviews and from analysis of documents such as annual reports, social responsibility reports, newspapers, NGO reports and other publicly available documents. Findings – Analysis of interviews, press clips and NGO reports draws attention to social and environmental problems in the Tanzanian mining sector, which are arguably linked to the manifestation of the broader crisis of neo-liberal agendas. While these issues have serious impacts on local populations in the mining areas, they often remain invisible in mining companies’ social disclosures. Increasing evidence of social and environmental ills raises serious questions about the effectiveness of the regulatory frameworks, as well as the roles played by NGOs and other pressure groups in Tanzania. Practical implications – By empowering local NGOs through educational, capacity building, technological and other support, NGOs’ advocacy, campaigning and networking with other civil society groups can play a pivotal role in encouraging corporations, especially TNCs, to adopt more socially and environmentally responsible business practices and to adhere to international and local standards, which in turn may help to improve the lives of many poor people living in developing countries in general, and Tanzania in particular. Originality/value – This paper contributes insights from gold-mining activities in Tanzania to the existing literature on CSR in the mining sector. It also contributes to political economy theory by locating CSR reporting within the socio-political and regulatory context in which mining operations take place in Tanzania. It is argued that, for CSR reporting to be effective, robust regulations and enforcement and stronger political pressure must be put in place.

Lack of geographic variation in Y-chromosomal introns of red deer (Cervus elaphus)

Intra-specific Y-chromosomal sequence variation is useful for analysing the male contribution to a species’ spatial genetic structure. In red deer (Cervus elaphus) this is especially relevant, because geographic dispersal and game translocations occur mainly through the males. However, Y-chromosomal markers for wild organisms are scarce and frequently non-polymorphic within species. We assessed the intra-specific variation of two Y-chromosomal introns in red deer, one in the DBY (or DDX3Y) gene and the other in the UBE1Y gene. The introns were amplified using previously published exonic primers and directly sequenced in individuals of five red deer subspecies from across Eurasia. However, no nucleotide polymorphism was observed, which rebuts the usefulness of these introns for studies of red deer phylogeography and on illegal transport of red deer within this region. Male-based phylogeographic studies should thus be focused on other Y-chromosomal markers for this species.

Investigation of the mechanism of resistance to glyphosate herbicide in hairy fleaban.

The resistance of weeds to herbicides is a consequence of one or more mechanisms in the plant, responsible for not allowing the herbicide to act properly at the active site.

Verification of the mechanism of glyphosate resistance in Italian ryegrass biotypes.

The intense use of glyphosate for weed control led to the emergence of several cases of resistance to this herbicide. Weeds can survive the application of herbicides due to several factors, which may or may not be related to the herbicide site of action. The objectives of this study were to quantify the accumulation of shikimate in ryegrass biotypes in response to glyphosate application; investigate possible mutations on the EPSPs gene in susceptible and resistant biotypes; and evaluate the response of ryegrass biotypes to the application of glyphosate after treatment with a metabolism inhibitor of cyt P450 monooxygenase.

Phospholipase A(2) myotoxins from Bothrops snake venoms: Structure-function relationship

Phospholipases A(2) constitute the major components from Bothrops snake venoms and have been extensively investigated not only because they are relatively very abundant in these venoms but mainly because they display a range of many relevant biological effects, including: myotoxic, cytotoxic, edema-inducing, artificial membrane disrupting, anticoagulant, neuromuscular, platelet aggregation inhibiting, hypotensive, bactericidal, anti-HIV, anti-tumoural, anti-malarial and anti-parasitic. The primary structures of several PLA(2)s have been elucidated through direct amino acid sequencing or, inderectly, through the corresponding nucleotide sequencing. Two main subgroups were thus described: (i) Asp49 PLA(2)s, showing low (basic, highly myotoxic) to relatively high (acidic, less or non myotoxic) Ca++-dependent hydrolytic activity upon artificial substrates; (ii) Lys49 PLA(2)s (basic, highly myotoxic) , showing no detectable hydrolytic activity on artificial substrates. Several crystal structures of Lys49 PLAs from genus Bothrops have already been solved, revealing very similar fold patterns. Lack of catalytic activity of myotoxic Lys49-PLA(2)s, first related solely with the fact that Lys49 occupies the position of the calcium ion in the catalyticly active site of Asp49 PLA(2)s, is now also attributed to Lys122 which interacts with the carbonyl of Cys29 hyperpolarising the peptide bond between Cys29 and Gly30 and trapping the fatty acid product in the active site, thus interrupting the catalytic cycle. This hypothesis, supported for three recent structures, is also discussed here. All Asp49 myotoxins showed to be pharmacologically more potent when compared with the Lys49 variants, but phospholipid hydrolysis is not an indispensable condition for the myotoxic, cytotoxic, bactericidal, anti-HIV, anti-parasitic, liposome disrupting or edema-inducing activities. Recent studies on site directed mutagenesis of the recombinant Lys49 myotoxin from Bothrops jararacussu revealed the participation of important amino acid residues in the membrane damaging and myotoxic activities.

Synthese und Charakterisierung biologischer Hybridpolymere zur gentechnologischen Anwendung

KurzfassungrnrnZiel der vorliegenden Arbeit war es eine gezielte, hochspezifische Inhibierung der Proteinbiosynthese zu erreichen. Dies kann durch eine Blockierung des mRNA-Strangs durch komplementäre DNA/RNA-Stränge (ähnlich zur Antisense-Methode) oder durch die Hydrolyse des mRNA-Strangs mit Hilfe spezieller Enzyme (RNasen) realisiert werden. Da jedoch beide Methoden nicht zu zufriedenstellenden Ergebnissen führen, wäre deshalb eine Kombination aus beiden Methoden ideal, welche in einer spezifischen, gezielten und permanenten Ausschaltung der Proteinbiosynthese resultieren würde. Um dieses Ziel zu verwirklichen, ist es nötig, ein Molekül zu synthetisieren, welches in der Lage ist selektiv an einer spezifischen Position an den RNA-Strang zu hybridisieren und anschließend den RNA-Strang an dieser zu hydrolysieren. Der große Vorteil dieses Konzepts liegt darin, dass die DNA-Sequenz für die Hybridisierung an die entsprechende RNA maßgeschneidert hergestellt werden kann und somit jede RNA gezielt angesteuert werden kann, was letztendlich zu einer spezifischen Inhibierung der korrespondierenden Proteinbiosynthese führen soll.rnDurch die Verwendung und Optimierung der Nativen Chemischen Ligation (NCL) als Konjugationsmethode konnten zwei Biomakromoleküle in Form einer 46-basenlangen DNA (komplementär zum RNA-Strang) und einer 31-aminosäurelangen RNase kovalent verknüpft werden. Durch unterschiedliche chemische und molekularbiologische Analysemethoden, wie PAGE, GPC, CE, MALDI-ToF-MS etc., war es zudem möglich, die erfolgreiche Synthese dieses biologischen Hybridpolymers als monodisperses, reines Produkt zu bestätigen. rnDie Synthese des ca. 800-basenlangen RNA-Strangs, der als Modell-Matrize für die selektive und spezifische Degradierung durch das DNA-RNase-Konjugat dienen sollte, konnte unter Zuhilfenahme gentechnologischer Standard-Methoden erfolgreich bewerkstelligt werden. Weiterhin konnte durch die Verwendung der radioaktiven cDNA-Synthese gezeigt werden, dass das DNA-RNase-Konjugat an die gewünschte Stelle des RNA-Strangs hybridisiert. Die Identifizierung einer anschließenden spezifischen Hydrolyse des RNA-Strangs durch die an den DNA-Strang angeknüpfte RNase war aufgrund der geringen katalytischen Aktivität des Enzyms bisher allerdings nicht möglich.rn

Molecular genetic approaches to defining lipid function.

Lipids fulfill multiple and diverse functions in cells. Establishing the molecular basis for these functions has been challenging due to the lack of catalytic activity of lipids and the pleiotropic effects of mutations that affect lipid composition. By combining molecular genetic manipulation of membrane lipid composition with biochemical characterization of the resulting phenotypes, the molecular details of novel lipid functions have been established. This review summarizes the results of such a combined approach to defining lipid function in bacteria.

Structural insights into the allosteric activation mechanism of cGMP-dependent protein kinase I

Cyclic GMP-dependent protein kinase (PKG) is a key transducer in the NO-cGMP signaling pathway. In this line, PKG has been considered an important drug target for treating hypertensive cardiovascular and pulmonary diseases. However, the investigation of PKG’s allosteric activation mechanism has been hampered by a lack of structural information. One of the fundamental questions on the cGMP-dependent activation of PKG is how the enzyme can distinguish cGMP over cAMP and selectively respond to cGMP. To ensure proper signaling, PKG must have developed unique features to ensure its activation upon the right activation signal. In this thesis, the cGMP-selective activation mechanism of PKG was studied through determining crystal structures of three truncated constructs of the regulatory domain [CNB-A (92-227), CNB-B (271-369), and CNB-A/B (92-351)] of PKG Iβ in the absence or presence of cyclic nucleotides. Herein, two individual CNB domain structures with biochemical data revealed that the C-terminal CNB domain (CNB-B) is responsible for cGMP selectivity, while the N-terminal CNB-domain (CNB-A) has a higher binding affinity for both cGMP and cAMP without showing any selectivity. Based on these crystal structures, mutagenesis studies were performed in which the critical residues for cyclic nucleotide selectivity and activation were identified. Furthermore, we discovered that the conformational changes of the C-terminal helix of the CNB-B that bridges between the regulatory and catalytic domains including the hydrophobic capping interaction are crucial for PKG activation. In addition, to observe the global conformation of the activated R-domain, I solved a co-crystal structure of the CNB-A/B with cGMP. Although a monomeric construct was crystallized, the structure displays a dimer. Strikingly, the CNB-A domain and its bound cGMP provide a key interface for this dimeric interaction. Using small angle X-ray scattering (SAXS), the existence of the cGMP-mediated dimeric interface within the CNB domains was confirmed. Furthermore, measuring cGMP-binding affinities (EC50) of the dimeric interface mutants as well as determining activation constants (Ka) revealed that the interface formation is important for PKG activation. To conclude, this thesis study provides a new mechanistic insight in PKG activation along with a newly found interface that can be targeted for designing PKG-specific activity modulators.

Gamma-secretase modulators and inhibitors in Alzheimer's disease: influence of genetic background on efficacy and mechanism of action

According to the amyloid hypothesis, Alzheimer’s disease (AD) is caused by aberrant production or clearance of the amyloid-β (Aβ) peptides, and in particular of the longer more aggregation-prone Aβ42. The Aβ peptides are generated through successive proteolytic cleavage of the amyloid precursor protein (APP) by the β-site APP cleaving enzyme (BACE) and γ-secretase. γ-secretase produces Aβ peptides with variable C-termini ranging from Aβ34 to Aβ48, presumably by sequential trimming of longer into shorter peptides. γ-secretase is a multiprotein complex consisting of at least four different proteins and the presenilin proteins (PS1 or PS2) contain the catalytic center of the complex. In 2001 several non-steroidal anti-inflammatory drugs were identified as the founding members of a new class of γ-secretase modulators (GSMs) that can selectively reduce production of Aβ42. Concomitantly, these GSMs increase Aβ38 production indicating closely coordinated generation of Aβ42 and Aβ38 and a potential precursor-product relationship between these peptides. GSMs seem to exert their activity by direct modulation of γ-secretase. Support for this hypothesis is drawn from the finding that some PS mutations associated with early-onset familial AD (FAD) can modulate the cellular response to GSMs and to γ-secretase inhibitors (GSIs), which inhibit production of all Aβ peptides and are known to directly interact with PS. A particularly interesting FAD PS mutation is PS1-ΔExon9, a complex deletion mutant that blocks endoproteolysis of PS1 and renders cells completely non-responsive to GSMs. Studies presented in this thesis show that the diminished response of PS1-ΔExon9 to GSMs is mainly caused by its lack of endoproteolytic cleavage. Furthermore, we were able to demonstrate that a reduced response to GSMs and GSIs is not limited to PS1-ΔExon9 but is a common effect of aggressive FAD-associated PS1 mutations. Surprisingly, we also found that while the Aβ42 response to GSMs is almost completely abolished by these PS1 mutations, the accompanying Aβ38 increase was indistinguishable to wild-type PS1. Finally, the reduced response to GSIs was confirmed in a mouse model with transgenic expression of an aggressive FAD-associated PS1 mutation as a highly potent GSI failed to reduce Aβ42 levels in brain of these mice. Taken together, our findings provide clear evidence for independent generation of Aβ42 and Aβ38 peptides, and argue that the sequential cleavage model might be an oversimplification of the molecular mechanism of γ-secretase. Most importantly, our results highlight the significance of genetic background in drug discovery efforts aimed at γ-secretase, and indicate that the use of cellular models with transgenic expression of FAD-associated PS mutations might confound studies of the potency and efficacy of GSMs and GSIs. Therefore, such models should be strictly avoided in the ongoing preclinical development of these promising and potentially disease-modifying therapeutics for AD.