283 research outputs found

    Effects of municipal smoke-free ordinances on secondhand smoke exposure in the Republic of Korea

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    ObjectiveTo reduce premature deaths due to secondhand smoke (SHS) exposure among non-smokers, the Republic of Korea (ROK) adopted changes to the National Health Promotion Act, which allowed local governments to enact municipal ordinances to strengthen their authority to designate smoke-free areas and levy penalty fines. In this study, we examined national trends in SHS exposure after the introduction of these municipal ordinances at the city level in 2010.MethodsWe used interrupted time series analysis to assess whether the trends of SHS exposure in the workplace and at home, and the primary cigarette smoking rate changed following the policy adjustment in the national legislation in ROK. Population-standardized data for selected variables were retrieved from a nationally representative survey dataset and used to study the policy action’s effectiveness.ResultsFollowing the change in the legislation, SHS exposure in the workplace reversed course from an increasing (18% per year) trend prior to the introduction of these smoke-free ordinances to a decreasing (−10% per year) trend after adoption and enforcement of these laws (β2 = 0.18, p-value = 0.07; β3 = −0.10, p-value = 0.02). SHS exposure at home (β2 = 0.10, p-value = 0.09; β3 = −0.03, p-value = 0.14) and the primary cigarette smoking rate (β2 = 0.03, p-value = 0.10; β3 = 0.008, p-value = 0.15) showed no significant changes in the sampled period. Although analyses stratified by sex showed that the allowance of municipal ordinances resulted in reduced SHS exposure in the workplace for both males and females, they did not affect the primary cigarette smoking rate as much, especially among females.ConclusionStrengthening the role of local governments by giving them the authority to enact and enforce penalties on SHS exposure violation helped ROK to reduce SHS exposure in the workplace. However, smoking behaviors and related activities seemed to shift to less restrictive areas such as on the streets and in apartment hallways, negating some of the effects due to these ordinances. Future studies should investigate how smoke-free policies beyond public places can further reduce the SHS exposure in ROK

    The development of efficient hemi-autotrophic carbon fixation in Escherichia Coli

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    Carbon fixation is a process vital to any life and as by far its most prevalent variant, the Calvin Benson Bassham (CBB) cycle is vital to virtually all known terrestrial life. Mostly occurring in plants, it uses light energy to sequester atmospheric carbon dioxide (CO2) and convert it into biomass. As the most inefficient natural carboxylation process and source of most biomass documented, even a small increase of its performance could have vast downstream effects. Such a development could assimilate the abundantly available atmospheric CO2 while generating minimal amounts of waste for any biosynthesized product. The Escherichia coli bacterium was previously shown to functionally express the CBB cycle upon the addition of phosphoribulokinase (PRK) and ribulose 1,5-bisphosphate carboxylase/oxygenase (RuBisCO). Further knock-outs severed its energetic metabolism from the carbon metabolism resulted CO2-dependent biomass accumulation. This carbon fixation is driven by the energy independently generated in the TCA cycle from a supply of pyruvate. This unique, split metabolism was dubbed hemi-autotrophy. The hemi-autotrophic strain of E. coli serves as a model organism for the CBB cycle, but lacking any of the difficulties of light-dependent or multi-cellular organisms. A pyrophosphate-dependent 6-phosphofructokinase (PFP) originating from Methylococcus capsulatus Bath was characterised as catalyzing three reactions of the typical CBB cycle. Where PRK completes its catalysis with a dependency on energy-carrier adenosine triphosphate (ATP), PFP was shown to complete this reaction with the less energetic pyrophosphate (PPi) that is partially generated in its FBPase and SBPase-equivalent reactions. Successful integration of this synthetic CBB cycle would conserve 33% of all ATP expended in the native CBB cycle. The hemi-autrophic E. coli strain’s unique culturing requirements proved challenging but methods with increased dependability were established. Transformations without the relief of these conditions remain elusive, requiring pre-cultures in rich media and heterotrophic metabolism. The consecutive sub-culturing of the strain to increase its hampered growth characteristics resulted in mild improvements. Despite observing modest culturing characteristic and a relatively high chromosomal mutation rate, the strain did not demonstrated an increase in transformation efficiency. The attempted replacements of the plasmid-encoded prkA by pfp did not result in hemi-autotrophic growth in any of its constructs, despite modulation of their expression. Troubled by high mutation rates, it remains unknown whether the expression range of the significantly less efficient PFP was sufficient or if the cytoplasmic availability of PPi remained below its functionally required concentration. The putative H+-pyrophosphatase pump (HPP), natively expressed as the second gene in the pfp-hpp operon, remains uncharacterised but its co-expression did not manage to compensate for this deficiency either. Though native fbp was successfully knocked-out, the essential inorganic pyrophosphatase gene of E. coli remains. Thorough analysis of the components in the CBB system led to several design improvements and pathway modelling indicates the proposed synthetic CBB cycle is a viable alternative to its natural variant. Thermodynamic feasibility of the synthetic pathway was confirmed and kinetic analysis also predicted it to perform at reduced efficiencies while still indicating culture viability. Growth rates approximating those of the hemi-autotrophic strain were produced in a kinetic model of the central carbon metabolism while incorporating minimal assumptions. Modifying it to support the synthetic CBB cycle suggested its viability at a nominal reduction of growth, while suggesting further directions of research for the system

    Precision mapping of gene expression and proteins in the brain using gene editing and barcoded viral vectors

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    The human brain is a masterpiece of intricate design and impeccable functionality. It serves as the ultimate command center for our thoughts, sensations, and actions, which define our very existence. This organ operates flawlessly, with billions of neurons working in perfect harmony to process information, create memories, and regulate our emotions. The brain's neural network is composed of trillions of connections, consisting of interconnected cells that communicate through electrical impulses and chemical signals at remarkable speeds. These connections, also known as synapses, serve as the means of communication that allow for information to travel uninterrupted throughout the brain. This intricate network enables us to think, learn, reason, and react to our surroundings. However, neurological disorders have the potential to disrupt this delicate balance, leading to a range of manifestations. These can include gradual memory erosion in Alzheimer's disease to the slow progression of motor and cognitive impairment in Parkinson's disease. Each condition presents a unique puzzle for scientists and researchers to decipher. The intricate interactions of genes, proteins, and neural circuits create a complex landscape that holds the key to understanding these disorders' origins and potential treatments.In this thesis, we worked on understanding a new type of neuronal communication based on the retrotransposon protein of Arc. The investigation was conducted using a gene editing technique based on the CRISPR/Cas9 system, next-generation sequencing technologies, and refined immunohistochemistry protocol. Using a mouse animal model, our findings reinforced the hypothesis that Arc has the capacity for inter-neuronal transport, as previously proposed in vitro studies. An additional objective of the thesis has been the investigation of molecular changes occurring within the Substantia Nigra throughout the progression of Parkinson's disease. At the core of this disorder's pathophysiology lies the alpha-synuclein protein. With this objective in mind, we developed a single- cell methodology to effectively investigate modifications in gene expression provoked by an overload of alpha- synuclein in animal models of rodents. From this data set, the overarching goal is to train a machine learning able to predict the disease course and to establish possible therapeutic interventions

    High-throughput Single-Entity Analysis Methods: From Single-Cell Segmentation to Single-Molecule Force Measurements

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    This work is focused on the development of new microscopy-based analysis methods with single-entity resolution and high-throughput capabilities from the cellular to the molecular level to study biomembrane-associated interactions. Currently, there is a variety of methods available for obtaining quantitative information on cellular and molecular responses to external stimuli, but many of them lack either high sensitivity or high throughput. Yet, the combination of both aspects is critical for studying the weak but often complex and multivalent interactions at the interface of biological mem-branes. These interactions include binding of pathogens such as some viruses (e.g., influenza A virus, herpes simplex virus, and SARS-CoV-2), transmembrane signaling such as ligand-based oli-gomerization processes, and transduction of mechanical forces acting on cells. The goal of this work was to overcome the shortcomings of current methods by developing and es-tablishing new methods with unprecedented levels of automation, sensitivity, and parallelization. All methods are based on the combination of optical (video) microscopy followed by highly refined data analysis to study single cellular and molecular events, allowing the detection of rare events and the identification and quantification of cellular and molecular populations that would remain hidden in ensemble-averaging approaches. This work comprises four different projects. At the cellular level, two methods have been developed for single-cell segmentation and cell-by-cell readout of fluorescence reporter systems, mainly to study binding and inhibition of binding of viruses to host cells. The method developed in the first pro-ject features a high degree of automation and automatic estimation of sufficient analysis parameters (background threshold, segmentation sensitivity, and fluorescence cutoff) to reduce the manual ef-fort required for the analysis of cell-based infection assays. This method has been used for inhibition potency screening based on the IC50 value of various virus binding inhibitors. With the method used in the second project, the sensitivity of the first method is extended by providing an estimate of the number of fluorescent nanoparticles bound to the cells. The image resolution was chosen to allow many cells to be imaged in parallel. This allowed for the quantification of cell-to-cell heterogeneity of particle binding, at the expense of resolution of the individual fluorescent nanoparticles. To account for this, a new approach was developed and validated by simulations to estimate the number of fluo-rescent nanoparticles below the diffraction limit with an accuracy of about 80 to 100 %. In the third project, an approach for the analysis and refinement of two-dimensional single-particle tracking ex-periments was presented. It focused on the quality assessment of the derived tracks by providing a guide for the selection of an appropriate maximal linking distance. This tracking approach was used in the fourth project to quantify small molecule responses to hydrodynamic shear forces with sub-nm resolution. Here, the combination of TIRF microscopy, microfluidics, and single particle tracking enabled the development of a new single molecule force spectroscopy method with high resolution and parallelization capabilities. This method was validated by quantifying the mechanical response of well-defined PEG linkers and subsequently used to study the energy barriers of dissociation of mul-tivalent biotin-NeutrAvidin complexes under low (~ 1.5 to 12 pN) static forces. In summary, with this work, the repertoire of appropriate methods for high-throughput investigation of the properties and interactions of cells, nanoparticles, and molecules at single resolution is expand-ed. In the future, the methods developed here will be used to screen for additional virus binding inhib-itors, to study the oligomerization of membrane receptors on cells and model membranes, and to quantify the mechanical response of force-bearing proteins and ligand-receptor complexes under low force conditions

    Rationales Design von polyfluorierten und enzymatisch abbaubaren Biomaterialien auf Peptidbasis

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    Amphiphilic peptide-based biomaterials are of great interest for pharmaceutical and biomedical applications and mainly associated with pronounced biocompatibility and biodegradability. In fact, introducing fluorine-containing amino acids into peptides & proteins offers an unique opportunity to enhance their biophysical properties such as membrane permeability. Through its influence on hydrophobicity and polarity, the degree of fluorination dictates the extent of fluorine-specific interactions on peptide folding and stability, intermolecular interactions, and biological activity. The first study of this doctoral thesis describes the folding, self-assembly, and hydrogelation of single-strand amphipathic peptides with different degrees of fluorination on the amino acid side chains by the iterative incorporation of monofluoroethylglycine (MfeGly), difluoroethylglycine (DfeGly), and trifluoroethylglycine (TfeGly). A combination of experimental and theoretical approaches proved a higher degree of side chain fluorination to promote β-sheet formation and the rheological stability of peptide-based hydrogels in physiological conditions, whereas secondary structure formation was inhibited at a low fluorine content due to fluorine-induced polarity. In a follow-up study, the selective modification of antimicrobial peptides (AMPs) by fluorinated amino acids was investigated. A β-hairpin-forming peptide motif, whose amphipathic structure enables the targeted disruption of bacterial cell membranes, was therefore examined. Extensive MIC screening with Gram-negative and Gram-positive bacteria confirmed highly fluorinated amino acids such as trifluoroethylglycine (TfeGly) or pentafluoropropylglycine (PfpGly) to strengthen the bioactivity of the AMPs through enhanced intrinsic hydrophobicity without causing a simultaneous increase in toxic & hemolytic properties. Numerous studies on the singular incorporation of fluorinated amino acids have been published to date, whereas synthetic peptides with larger or exclusive amounts of these building blocks remained unexplored. That drove the motivation for the herein-described development and characterization of so-called "fluoropeptides". In brief, β-sheet to α-helix or fluorine-induced PPII-helix transitions were observed in SDS-supplemented buffer (pH 7.4). In situ SEIRAS experiments with POPC:POPG-based membrane models functioned to investigate the fluoropeptide’s lipid insertion and (re)folding. Thus, the highest α-helical secondary structure content was found for the nonfluorinated homooligopeptide and decreased in the order of tri-, di-, and mono-fluorination of the side chains. An important focus of this doctoral thesis was the evaluation of biodegradability for especially higher polyfluorinated sequences. In fact, all peptides prepared in this work could be hydrolyzed by various proteases regardless of the fluorine content. In cooperation with the University College Dublin, first data on the microbial digestion of fluorinated peptides and individual amino acids could be generated. The enzyme-catalyzed cleavage of the C-F bond on the side chain for both kind of substrates was, for instance, proven by detection of released fluoride ions in solution. The results of this work will contribute to the rational design and potential application of polyfluorinated peptides, whose enzymatic degradability is going to be of great interest for the future development of fluorinated biomaterials.Amphiphile peptidbasierte Biomaterialien sind vom großen Interesse für pharmazeutische und biomedizinische Anwendungen und überzeugen zumeist durch ihre Biokompatibilität und Bioabbaubarkeit. Die Einführung von fluorhaltigen Aminosäuren in Peptide & Proteine bietet hierbei die einzigartige Möglichkeit, ihre biophysikalischen Eigenschaften wie etwa die Membranpermeabilität zu verstärken. Insbesondere der Fluorierungsgrad spielt eine entscheidende Rolle, da er durch seinen Einfluss auf die Hydrophobie und Polarität die Gesamtheit fluor-spezifischer Wechselwirkungen auf die Peptidfaltung und -stabilität, intermolekularen Wechselwirkungen und biologische Aktivität steuern kann. Die erste Studie dieser Doktorarbeit beschreibt die Faltung, Selbstassemblierung und Hydrogelierung von einzelsträngigen amphipathischen Peptiden mit unterschiedlichen Fluorierungsgraden der Aminosäureseitenketten durch den iterativen Einbau von Monofluorethylglycin (MfeGly), Difluorethylglycin (DfeGly) und Trifluorethylglycin (TfeGly). Mittels einer Kombination aus experimentellen und theoretischen Ansätzen konnte gezeigt werden, dass bei physiologischen Bedingungen ein höherer Fluorierungsgrad die Bildung von β-Faltblattstrukturen und die rheologische Stabilität der peptid-basierten Hydrogele fördert, jedoch diese Sekundärstruktur von Peptiden mit niedrigem Fluorgehalt durch die fluor-induzierte Polarität inhibiert wird. In einer weiteren Studie wurde die gezielte Modifizierung der biologischen Eigenschaften antimikrobieller Peptide (AMP) durch den Einbau fluorierter Aminosäuren untersucht. Hierzu wurde ein β-Hairpin bildendes Peptidmotiv ausgewählt, dessen amphipathische Struktur die zielgerichtete Disruption bakterieller Zellmembrane ermöglicht. Die ermittelten minimalen Hemmkonzentrationen (MHK) gegen verschiedene Gram-negative und Gram-positive Bakterien zeigen, dass hochfluorierte Aminosäuren wie Trifluorethylglycin (TfeGly) und Pentafluorpropylglycin (PfpGly) die Bioaktivität antimikrobieller Peptide durch Erhöhung der intrinsischen Hydrophobie selektiv verstärken können, ohne eine gleichzeitige Zunahme toxischer & hämolytischer Eigenschaften zu verursachen. Zahlreiche Studien zum singulären Einbau fluorierter Aminosäuren wurden bis dato veröffentlicht, während synthetische Peptide mit größeren bzw. ausschließlichen Mengen dieser Bausteine unerforscht blieben. Dies war die Motivation zur Entwicklung und Charakterisierung sogenannter "Fluoropeptide". In SDS-beinhaltenden Puffer (pH 7.4) wurden, unter anderem, Übergänge von β-Faltblatt Strukturen zu α-Helices oder Fluor-induzierte PPII-Helices beobachtet. In-situ SEIRAS-Studien mit POPC:POPG-basierten Membranmodellen dienten zum Studium der Lipidinsertion und (Rück-)-Faltung der Fluoropeptide in Abhängigkeit zum gesamten Fluoranteil. Hierbei wurde der höchste Gehalt an α-helikaler Sekundärstruktur für das nichtfluorierte Homooligopeptid bestimmt, welcher in der Reihenfolge der Tri-, Di- und Monofluorierung der Seitenkette abnahm. Ein wichtiger Schwerpunkt dieser Doktorarbeit war die Bewertung der biologischen Abbaubarkeit für insbesondere höher polyfluorierte Sequenzen. Tatsächlich konnten alle in dieser Arbeit hergestellten Peptide unabhängig vom Fluorgehalt durch verschiedene Proteasen hydrolysiert werden. In Zusammenarbeit mit dem University College Dublin konnten zudem erste Daten zum mikrobiellen Verdau fluorierter Peptide und Aminosäuren generiert werden. Die enzymkatalysierte Spaltung der C-F-Bindung an der Seitenkette für beide Substratarten wurde beispielsweise durch den Nachweis von freigesetzten Fluorid-Ionen in Lösung nachgewiesen. Die Ergebnisse dieser Arbeit werden zum rationalen Design und potenzieller Anwendbarkeit neuartiger polyfluorierter Peptide beitragen, deren enzymatische Abbaubarkeit von großem Interesse für die künftige Entwicklung fluorhaltiger Biomaterialien sein wird

    Molecular Mechanisms and Therapies of Colorectal Cancer

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    Colorectal cancer (CRC) is currently the third leading cause of cancer-related mortality, with 1.9 million incidence cases and 0.9 million deaths worldwide. The global number of new CRC cases is predicted to reach 3.2 million in 2040, based on the projection of aging, population growth, and human development.In clinics, despite advances of diagnosis and surgical procedures, 20% of the patients with CRC present with metastasis at the time of diagnosis, caused by residual tumor cells that have spread to distant organs prior to surgery, affecting the patient survival rate. Standard systemic chemotherapy, alternative therapies that target mechanisms involved in cancer progression and metastasis, immunotherapy, and combination therapies are the major CRC-treatment strategies. In the advanced stage of CRC the transforming growth factor-beta (TGF-β) plays an oncogenic role by promoting cancer cell proliferation, cancer cell self-renewal, epithelial-to-mesenchymal transition, invasion, tumor progression, metastatic spread, and immune escape. Furthermore, high levels of TGF-β1 confers poor prognosis and is associated with early recurrence after surgery, resistance to chemo- or immunotherapy, and shorter survival. Based on the body of experimental evidence indicating that TGF-β signaling has the potential to be a good therapeutic target in CRC, several anti-TGF-β drugs have been investigated in cancer clinical trials. Here, we presented a comprehensive collection of manuscripts regarding studies on targeting the TGF-β signaling in CRC to improve patient’s prognosis and personalized treatments

    Body Condition and Productivity, Health and Welfare

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    This reprint focuses on applied research on growth traits, fat reserves, fatty acid profiles and enriched diets and their effects on reproduction, health status and welfare. The studies contained in the volume have been carried out in both monogastric organisms and ruminants, fish, broilers or pigeons. Fundamental studies with rats or mice are also presented. The reader will find novel research about the effects of the degree of fatness and muscularity determined by ultrasound method on sows´ reproductive performance, genetic studies on fatty acid profiles and growth traits in rabbits and Gilthead Seabream; nutritional studies addressing diets enriched with bee pollen in rats, postbiotics in male rabbits, concentrate and bypass supplementations in buffaloes; and interesting studies on the welfare assessment of meat sheep, rabbits, racing pigeons, broilers, alpacas and llamas using body condition measurements

    Structure-based design of a phosphotyrosine-masked covalent ligand targeting the E3 ligase SOCS2

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    The Src homology 2 (SH2) domain recognizes phosphotyrosine (pY) post translational modifications in partner proteins to trigger downstream signaling. Drug discovery efforts targeting the SH2 domains have long been stymied by the poor drug-like properties of phosphate and its mimetics. Here, we use structure-based design to target the SH2 domain of the E3 ligase suppressor of cytokine signaling 2 (SOCS2). Starting from the highly ligand-efficient pY amino acid, a fragment growing approach reveals covalent modification of Cys111 in a co-crystal structure, which we leverage to rationally design a cysteine-directed electrophilic covalent inhibitor MN551. We report the prodrug MN714 containing a pivaloyloxymethyl (POM) protecting group and evidence its cell permeability and capping group unmasking using cellular target engagement and in-cell 19F NMR spectroscopy. Covalent engagement at Cys111 competitively blocks recruitment of cellular SOCS2 protein to its native substrate. The qualified inhibitors of SOCS2 could find attractive applications as chemical probes to understand the biology of SOCS2 and its CRL5 complex, and as E3 ligase handles in proteolysis targeting chimera (PROTACs) to induce targeted protein degradation.</p

    Terapias basadas en surfactante pulmonar : viajando por la interfase respiratoria para administrar fármacos

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    Tesis inédita de la Universidad Complutense de Madrid, Facultad de Ciencias Biológicas, leída el 28-10-2022Lungs are highly specialised organs in charge of gas exchange between the environment and the bloodstream. To obtain the necessary oxygen, lungs take more than 10000 L of air every day that come into close contact with around 6000L of blood through a very extensive and thin air-blood barrier. These features convert the respiratory system in a promising route for administering therapeutic molecules to the body. Delivering drugs to the distal airways could serve to treat both local and systemic diseases. However, evolution has developed various strategies to protect this enormous surface area from the entrance of external particles and their possible injuries. These barriers are multiple and should be considered when intending to administer inhalable therapies. A proper design of the drug and the delivery system could make the difference between reaching or not the alveoli. In this line, pulmonary surfactant has been proposed as a potent drug carrier to overcome lung barriers and enhance the biomedical application of different therapies...Los pulmones son órganos especializados en el intercambio gaseoso entre el exterior y la sangre. Para obtener el oxígeno necesario, los pulmones toman más de 10000 L de aire al día, que han de ponerse en contacto con unos 6000 L desangre a través de una extensísima y fina barrera aire-sangre. Esto hace que el sistema respiratorio se haya convertido en una prometedora ruta de administración de moléculas terapéuticas. Transportar fármacos a las vías aéreas distales puede servir tanto para tratamientos locales como sistémicos. Sin embargo, la evolución ha desarrollado varias estrategias para proteger la enorme área superficial del sistema respiratorio de la entrada de patógenos externos y sus posibles daños. Estas barreras son variadas y deben ser consideradas cuando se pretende administrar terapias inhaladas. Un diseño adecuado de los fármacos y sistemas de transporte puede marcar la diferencia entre alcanzar o no los alveolos. En esta línea, el surfactante pulmonar se ha propuesto como un potente vehículo de fármacos capaz de superar las barreras pulmonares y potenciar la aplicación biomédica de distintas terapias...Fac. de Ciencias BiológicasTRUEunpu
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