Bioactive Thymosin Alpha-1 Does Not Influence F508del-CFTR Maturation and Activity.

Deletion of phenylalanine 508 (F508del) in the cystic fibrosis transmembrane conductance regulator (CFTR) anion channel is the most frequent mutation causing cystic fibrosis (CF). F508del-CFTR is misfolded and prematurely degraded. Recently thymosin a-1 (Tα-1) was proposed as a single molecule-based therapy for CF, improving both F508del-CFTR maturation and function by restoring defective autophagy. However, three independent laboratories failed to reproduce these results. Lack of reproducibility has been ascribed by the authors of the original paper to the use of DMSO and to improper handling. Here, we address these potential issues by demonstrating that Tα-1 changes induced by DMSO are fully reversible and that Tα-1 peptides prepared from different stock solutions have equivalent biological activity. Considering the negative results here reported, six independent laboratories failed to demonstrate F508del-CFTR correction by Tα-1. This study also calls into question the autophagy modulator cysteamine, since no rescue of mutant CFTR function was detected following treatment with cysteamine, while deleterious effects were observed when bronchial epithelia were exposed to cysteamine plus the antioxidant food supplement EGCG. Although these studies do not exclude the possibility of beneficial immunomodulatory effects of thymosin α-1, they do not support its utility as a corrector of F508del-CFTR

The integrity of the HMR complex is necessary for centromeric binding and reproductive isolation in Drosophila

Postzygotic isolation by genomic conflict is a major cause for the formation of species. Despite its importance, the molecular mechanisms that result in the lethality of interspecies hybrids are still largely unclear. The genus Drosophila, which contains over 1600 different species, is one of the best characterized model systems to study these questions. We showed in the past that the expression levels of the two hybrid incompatibility factors Hmr and Lhr diverged in the two closely related Drosophila species, D. melanogaster and D. simulans, resulting in an increased level of both proteins in interspecies hybrids. The overexpression of the two proteins also leads to mitotic defects, a misregulation in the expression of transposable elements and decreased fertility in pure species. In this work, we describe a distinct six subunit protein complex containing HMR and LHR and analyse the effect of Hmr mutations on complex integrity and function. Our experiments suggest that HMR needs to bring together components of centromeric and pericentromeric chromatin to fulfil its physiological function and to cause hybrid male lethality

Psychometric Evaluation of the Short Self-Regulation Questionnaire across Three European Countries

The aim of this study was to extend the psychometric evaluation of the Short Self-Regulation Questionnaire (SSRQ) by assessing the factor structure across three countries from Central and Eastern Europe. The sample included 1809 students from Slovakia, Lithuania and Hungary. Based on an initial confirmative factor analysis, a 2-factor structure by Neal and Carey (2005) was confirmed in the Lithuanian sample. Next, exploratory factor analyses were used on the Slovak and Hungarian subsamples separately. For both national subsamples, a very similar four factor solution was found, which was confirmed by confirmatory factor analyses on the rest of the data. Despite the reduced number of items, the abridged scale did not suffer in terms of its internal reliability and thus provides an adequate approximation of self-regulation levels as the entire scale or as the scale with the proposed 4-factor solution

Restoration of CFTR function in patients with cystic fibrosis carrying the F508del-CFTR mutation

<div><p>Restoration of BECN1/Beclin 1-dependent autophagy and depletion of SQSTM1/p62 by genetic manipulation or autophagy-stimulatory proteostasis regulators, such as cystamine, have positive effects on mouse models of human cystic fibrosis (CF). These measures rescue the functional expression of the most frequent pathogenic CFTR mutant, F508del, at the respiratory epithelial surface and reduce lung inflammation in <i>Cftr^F508del</i> homozygous mice. Cysteamine, the reduced form of cystamine, is an FDA-approved drug. Here, we report that oral treatment with cysteamine greatly reduces the mortality rate and improves the phenotype of newborn mice bearing the <i>F508del-CFTR</i> mutation. Cysteamine was also able to increase the plasma membrane expression of the F508del-CFTR protein in nasal epithelial cells from <i>F508del</i> homozygous CF patients, and these effects persisted for 24 h after cysteamine withdrawal. Importantly, this cysteamine effect after washout was further sustained by the sequential administration of epigallocatechin gallate (EGCG), a green tea flavonoid, both <i>in vivo</i>, in mice, and <i>in vitro</i>, in primary epithelial cells from CF patients. In a pilot clinical trial involving 10 <i>F508del-CFTR</i> homozygous CF patients, the combination of cysteamine and EGCG restored BECN1, reduced SQSTM1 levels and improved CFTR function from nasal epithelial cells <i>in vivo</i>, correlating with a decrease of chloride concentrations in sweat, as well as with a reduction of the abundance of <i>TNF/TNF-alpha (tumor necrosis factor)</i> and <i>CXCL8</i> (<i>chemokine [C-X-C motif] ligand 8</i>) transcripts in nasal brushing and TNF and CXCL8 protein levels in the sputum. Altogether, these results suggest that optimal schedules of cysteamine plus EGCG might be used for the treatment of CF caused by the <i>F508del-CFTR</i> mutation.</p></div

Diagnosing mucopolysaccharidosis IVA

Mucopolysaccharidosis IVA (MPS IVA; Morquio A syndrome) is an autosomal recessive lysosomal storage disorder resulting from a deficiency of N-acetylgalactosamine-6-sulfate sulfatase (GALNS) activity. Diagnosis can be challenging and requires agreement of clinical, radiographic, and laboratory findings. A group of biochemical genetics laboratory directors and clinicians involved in the diagnosis of MPS IVA, convened by BioMarin Pharmaceutical Inc., met to develop recommendations for diagnosis. The following conclusions were reached. Due to the wide variation and subtleties of radiographic findings, imaging of multiple body regions is recommended. Urinary glycosaminoglycan analysis is particularly problematic for MPS IVA and it is strongly recommended to proceed to enzyme activity testing even if urine appears normal when there is clinical suspicion of MPS IVA. Enzyme activity testing of GALNS is essential in diagnosing MPS IVA. Additional analyses to confirm sample integrity and rule out MPS IVB, multiple sulfatase deficiency, and mucolipidoses types II/III are critical as part of enzyme activity testing. Leukocytes or cultured dermal fibroblasts are strongly recommended for enzyme activity testing to confirm screening results. Molecular testing may also be used to confirm the diagnosis in many patients. However, two known or probable causative mutations may not be identified in all cases of MPS IVA. A diagnostic testing algorithm is presented which attempts to streamline this complex testing process

AI is a viable alternative to high throughput screening: a 318-target study

: High throughput screening (HTS) is routinely used to identify bioactive small molecules. This requires physical compounds, which limits coverage of accessible chemical space. Computational approaches combined with vast on-demand chemical libraries can access far greater chemical space, provided that the predictive accuracy is sufficient to identify useful molecules. Through the largest and most diverse virtual HTS campaign reported to date, comprising 318 individual projects, we demonstrate that our AtomNet® convolutional neural network successfully finds novel hits across every major therapeutic area and protein class. We address historical limitations of computational screening by demonstrating success for target proteins without known binders, high-quality X-ray crystal structures, or manual cherry-picking of compounds. We show that the molecules selected by the AtomNet® model are novel drug-like scaffolds rather than minor modifications to known bioactive compounds. Our empirical results suggest that computational methods can substantially replace HTS as the first step of small-molecule drug discovery

The Drosophila speciation factor HMR localizes to genomic insulator sites

Hybrid incompatibility between Drosophila melanogaster and D. simulans is caused by a lethal interaction of the proteins encoded by the Hmr and Lhr genes. In D. melanogaster the loss of HMR results in mitotic defects, an increase in transcription of transposable elements and a deregulation of heterochromatic genes. To better understand the molecular mechanisms that mediate HMR's function, we measured genome-wide localization of HMR in D. melanogaster tissue culture cells by chromatin immunoprecipitation. Interestingly, we find HMR localizing to genomic insulator sites that can be classified into two groups. One group belongs to gypsy insulators and another one borders HP1a bound regions at active genes. The transcription of the latter group genes is strongly affected in larvae and ovaries of Hmr mutant flies. Our data suggest a novel link between HMR and insulator proteins, a finding that implicates a potential role for genome organization in the formation of species

Identification and characterization of a protein complex involved in chromatin architecture and the evolution of Drosophila species

Centromeres and pericentromeric heterochromatin are involved in a number of essential functions including cell division, silencing of repetitive DNA elements and spatial organization of the genome. The clustering of these regions around chromocenters during interphase is required for their proper function and involves a complex and finely tuned network of protein-protein interactions. Despite their critical importance, centromeric and pericentromeric chromatin are poorly conserved even among closely related species and are, in fact, often involved in the formation of species. This apparent paradox is often the result of intrinsically fast-evolving repetitive DNA elements embedded into heterochromatin. The rapid evolution of such repetitive elements poses a threat to cellular fitness thereby exerting a selective pressure on the silencing machinery responsible for their management. Such process typically leads to the rapid coevolution of heterochromatin proteins acting like suppressors of fast-evolving DNA elements. This coevolution can, in turn, lead two populations once belonging to the same species to diverge by developing species-specific heterochromatin, that can eventually function as a postzygotic barrier between the two newly generated species. The fruit fly sibling species Drosophila melanogaster (D.mel) and Drosophila simulans (D.sim) have been a privileged model for studying the formation of species since over a century and provide an excellent example of how hybrid incompatibilities can arise within chromatin. Hybrids from D. melanogaster mothers and D. simulans fathers fail to develop because of the detrimental genetic interaction of the three hybrid incompatibility (HI) genes Hmr, Lhr and gfzf. Genetic studies of the three HI genes have revealed pleiotropic phenotypes, with Hmr and Lhr mutations disrupting oogenesis, female fertility and repetitive elements silencing and gfzf mutants having a broad spectrum of phenotypes including defects in cell cycle regulation. In addition to their genetic interaction, HMR and LHR proteins interact within a complex network of protein-protein interactions that is important for the architecture and function of centromeric and pericentromeric chromatin. For GFZF, on the other hand, few molecular data are available and there is no evidence of molecular interactions with the other two HI genes. Although the last decade has brought enormous progress in the field, the molecular details underlying both the divergent evolution of these hybrid incompatibility factors in the respective pure species and their genetic interactions in interspecific hybrids are still poorly understood. A major twist in the field has been the finding that the expression of HMR and LHR proteins has diverged during the evolution in D.mel and D.sim species and that the two proteins are both overrepresented in hybrids. However, while these findings revealed the importance of a proper quantitative balance of HMR and LHR, the HMR/LHR protein-protein interaction network could only be studied in overexpressing conditions so far. Characterizing the HMR/LHR protein complex in native conditions could therefore pave the way, on the one hand for understanding how these two proteins interact to mediate normal pericentromeric and centromeric functions in pure species, and, on the other hand, how their interaction network is altered in hybrids where they are overexpressed. To address these questions, in the first publication, we used affinity purification coupled with Mass Spectrometry (AP-MS) and revealed for the first time the existence of a stable six-subunit HMR/LHR protein complex in native conditions. In addition to HMR and LHR, the complex contains the two nucleolar proteins nucleoplasmin (NLP) and nucleophosmin (NPH), as well as the two non-characterized proteins, CG33213 and CG4788. For these last two proteins our publication provided the first molecular characterization and we named them Buddy Of HMR 1 (BOH1) and Buddy Of HMR 2 (BOH2), respectively. In addition, as a resource for the field, we published a detailed description of the intricate network of interactions (interactome) involving each complex component. After identifying the complex we went further and generated two different mutants targeting two different Hmr domains, to dissect how HMR interacts with other complex components and how disrupting such interactions affects HMR localization and function. Our results suggest that the integrity of the HMR/LHR complex is necessary for both HMR physiological function in pure species and its toxic function in hybrids. Next, we started from the HMR/LHR native complex and induced HMR/LHR overexpression to mimic a hybrid background in a cell culture system and asked how the HMR protein-protein interaction network is altered upon their overexpression. A range of new chromatin interactors, from architectural proteins like insulators to zinc-finger DNA binding proteins, appear to specifically interact when HMR is in excess, suggesting that these may be binding with low affinity and therefore only observed when HMR amount is not limiting, such as in hybrids. Finally, we set out to study HMR subnuclear localization with respect to CENP-A and HP1a, a centromeric and a pericentromeric marker, respectively. Our findings allow us to build a model that reconciles previous controversies and suggests that HMR is neither centromeric nor pericentromeric but it is rather sitting in the middle, bridging these two types of chromatin by forming a complex that interacts with both. In the second publication, we focused on the third and less known HI factor and asked how GFZF localizes in D.mel, D.sim and hybrids and how it molecularly interacts with HMR to cause hybrid incompatibility. We used in situ hybridization in polytene chromosomes to describe for the first time GFZF localization in D.mel, D.sim and hybrids. In addition, here we show the first evidence of a molecular interaction between GFZF and HMR by looking at their respective localization in both polytene chromosomes and cell lines where HMR was present in physiological amounts or overexpressed with LHR (hybrid-mimicking condition). Strikingly, while the two HI proteins HMR and GFZF occupy distinct and non-overlapping territories in pure species, their localization merges in the hybrid background

Investigation of IoT camera solutions in Smart Cities from a Technology, Information Security and Trust point of view

Smart cities utilize IoT cameras in a rushing pace. However, these high technological connected devices are back doors to safe systems. Implementation of high technological solutions without concerning other important aspects as information security and the factor of trust violates not only the quality aspect of the solutions, but also breaches the privacy of individuals. Secure solutions that reserve the trust of individuals with the help of high technological solutions is therefore significant. This study is aimed to investigate and find answer to how IoT camera solutions for smart cities can include all three of the important factors, which contributes for a deeper understanding of the whole product development process` possible improvements. Through a qualitative approach the research questions are investigated which resulted in a contribution that support the academic world of information security in presenting the importance of correct communication, collaboration and the including of the three factors during the development process of IoT camera solutions for smart cities. The results are open for further investigation as observed during research, but also key findings and recommendations are offered to the stakeholders of the IoT camera solution development industry

En undersökning av förändringar som behöver införas för att överensstämma med GDPR - I utveckling och drift av smarta kameror

Konceptet av sakernas internet handlar om att datorer ska kunna agera utan mänsklig interaktion. Detta gör att smarta larm med kameror själva kan avgöra när ett larm ska utlösas och kan då ta åtgärder som att ta bilder på hemmet som kameror är installerade i samt på individer. Detta skapar vissa etiska frågor kopplade till dataintegriteten. För att kunna kontrollera företagens användning av mängder data som kan genereras idag och för att ge individer rättigheter över hur deras data behandlas har EU tagit fram en förordning kallad General Data Protection Regulation (GDPR). Denna kommer att förändra utveckling och drift av smarta kameror, eftersom bilder är personuppgifter som omfattas av förordningen. Därför är tydliga strategier kring vilka förändringar företag som utvecklar eller driftar kameror behöver införa för att uppfylla GDPR kraven nödvändiga för att upprätthålla användarnas dataintegritet. Projektets syfte är därför att redogöra för huvudsakliga förändringar som behöver införas hos företag identiska till studieobjekten samtidigt som inställningen till förändringen beaktas, vilket är en nyckelfaktor till en lyckad implementering. Detta uppnås genom datainsamling via intervjuer som utfördes hos företag ledande inom utveckling och drift av smarta kameror. Resultatet bekräftar hypotesen om att kamera och säkerhetsföretag är sakkunniga inom säkerhet och integritet, samt visar att företag handskas med fem, följande, huvudsakliga förändringar: finna lösningar på insamling av informerade samtycken, dokumentation som måste bli mer utförlig, kontraktskrivning med leverantörer måste ges mer uppmärksamhet, att möjliggöra framförallt i backenden när en kund begär ut all data, samt privacy by design och ett helhetsperspektiv som behöver implementeras mer under utvecklingsprocessen. Vidare kan arbetet användas som beslutsunderlag i liknande organisationer som studieobjekten, eftersom det ger även förslag på förbättringsområden vilka har för avsikt att förstärka möjligheten till en lyckad tillfredsställelse av GDPR kraven.The concept of the Internet of Things is about computers being able to act without human interaction. This allows smart alarms with cameras to determine themselves when an alarm is triggered and can take action like taking pictures at home that cameras are installed in as well as on individuals. This creates some ethical issues related to data privacy. In order to control companies' use of amounts of data that can be generated today and to give individuals rights over how their data is processed, the EU has developed a regulation called the General Data Protection Regulation (GDPR). This will change the development and operation of smart cameras, as images are personal data that are covered by the regulation. Therefore, clear strategies about the changes that needs to be implemented to meet the GDPR requirements is necessary to maintain users' data privacy. The purpose of the project is therefore to account for major changes that need to be introduced to companies identical to the study objects while taking into account the attitude towards the change, which is a key factor for successful implementation. This is achieved through data acquisition through interviews conducted by companies leading in the development and operation of smart cameras. The result confirms the hypothesis that camera and security companies are experts in security and integrity, as well as demonstrating that companies are dealing with five, the following major changes: finding solutions for collecting informed consent, documentation that needs to be more detailed, contracting with suppliers must be paid more attention to, make it technically possible for a customer to request and get all data, as well as Privacy by Design and an overall perspective that needs to be implemented during the development process. In addition, the work can be used as a basis for decision making in similar organizations as the study objects who develop IoT products, as it also provides suggestions for improvement areas that intend to enhance the possibility of a successful satisfaction of the GDPR requirements