Suchergebnisse

An undecamer oligonucleotide probe based on a pair of deoxythymidines flanked by several modified nucleotides is a specific and highly efficient biosensor for micrococcal nuclease (MNase), an endonuclease produced by Staphylococcus aureus. Herein, the interaction mode and cleavage process on such oligonucleotide probes are identified and described for the first time. Also, we designed truncated pentamer probes as the minimum-length substrates required for specific and efficient biosensing. By means of computational (virtual docking) and experimental (ultra-performance liquid chromatography-mass spectrometry and matrix-assisted laser desorption ionization time-of-flight) techniques, we perform a sequence/structure-activity relationship analysis, propose a catalytically active substrate-enzyme complex, and elucidate a novel two-step phosphodiester bond hydrolysis mechanism, identifying the cleavage sites and detecting and quantifying the resulting probe fragments. Our results unravel a picture of both the enzyme-biosensor complex and a two-step cleavage/biosensing mechanism, key to the rational oligonucleotide design process. ; Funding Agencies|Wallenberg Centre for Molecular Medicine (WCMM) Linkoping, Sweden; Swedish Government Strategic Research Area in Materials Science on Advanced Functional Materials at Linkoping University (Faculty grant SFO-Mat-LiU) [2009-00971]; Maria de Maeztu Units of Excellence Program from the Spanish State Research Agency [MDM-2017-0720]; industrial doctorate program (MINECO) [DI-16-08891]; program Torres Quevedo (MINECO) [PTQ-17-09382]

Lysosomes are known as key players in cellular signalling and act as terminal degradation stations involved in a multitude of cellular processes. Being a highly influential physiological factor, pH is essential in the regulation of lysosome-mediated physiological and pathological processes. Aberrant pH fluctuations are highly related to lysosomal dysfunction that correlates to lysosomal storage diseases and neurodegenerative disorders. As such, real-time quantitative monitoring of lysosomal pH (pHL) is crucial for gaining insight into lysosomal dysfunction but challenging by the lack of effective lysosome-specific probes with high signal fidelity. Toward this end, we have proposed a lysosomal fluorogenic nanoprobe (TR-MP) for reliable ratiometric measuring of pHL. It is fabricated by rational manipulation of fluorescence resonance energy transfer (FRET) in a tailorable nanoplatform. The nanoprobe consists of biocompatible silica nanoparticles assembled with a pH-sensitive rhodamine derivative (RDM-TEOS) as an acceptor and aggregation-induced emission (AIE) fluorophore (TPE-OMe) as a donor to ensure high energy transfer efficiency. Further equipped with cell-penetrating facilitator and morpholine to enable effective cell-internalization and high lysosome affinity of TR-MP. Results show that TR-MP can quantitatively measure pH in a range of 3.0 - 7.0 and detect pHL fluctuations in live cells under various stimuli, as well as real-time monitor pHL during apoptosis. ; Funding Agencies|STINT Joint China-Sweden Mobility Project [CH2017-7243]; Swedish Research Council (VR)Swedish Research Council [VR 2019-02409, 2020-05437]; China Scholarship Council (CSC)China Scholarship Council; Carl Tryggers Stiftelse [CTS 19:379]; Swedish Government strategic faculty grant in material science (SFO, MATLIU) in Advanced Functional Materials (AFM) (VR) [5.1-2015-5959]; Centre in Nano Science and technology at LiTH (CeNano); LiU Cancer network at Linkoping University

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the cause of the COVID-19 pandemic with severe consequences for afflicted individuals and the society as a whole. The biology and infectivity of the virus has been intensively studied in order to gain a better understanding of the molecular basis of virus-host cell interactions during infection. It is known that SARS-CoV-2 binds to angiotensin-converting enzyme 2 (ACE2) via its spike protein. Priming of the virus by specific proteases leads to viral entry via endocytosis and to the subsequent steps in the life cycle of SARS-CoV-2. Sphingosine and ceramide belong to the sphingolipid family and are abundantly present in cell membranes. These lipids were recently shown to interfere with the uptake of virus particles of SARS-CoV-2 into epithelial cell lines and primary human nasal cells in culture. The mechanisms of action were partly different, as sphingosine blocked, whilst ceramide facilitated viral entry. Acid sphingomyelinase (ASM) is vital for the generation of ceramide and functional inhibition of ASM by drugs like amitriptyline reduced SARS-CoV-2 entry into the epithelial cells. Recent data indicates that serum level of sphingosine-1-phosphate (S1P) is a prognostic factor for COVID-2 severity. Further, stimulation of sphingosine-1-phosphate receptor 1 (S1PR1) might also constrain the hyper-inflammatory conditions linked to SARS-CoV-2. Here, we review recent exciting findings regarding sphingolipids in the uptake of SARS-CoV-2 and in the course of COVID-19 disease. More studies are required on the mechanisms of action and the potential use of antidepressant drugs and sphingolipid modifiers in SARS-CoV-2 infections and in the treatment of the more serious and fatal consequences of the disease. ; Funding: Magnus Ehrnrooth Foundation; Liv and Halsa Foundation; Finska Lakaresallskapet; Finnish Society of Sciences and Letters; Minerva Foundation; European Union, H2020-MSCA-RISE-2016 [73479]; Swedish Research CouncilSwedish Research CouncilEuropean ...

Electronic control of biological processes with bioelectronic devices holds promise for sophisticated regulation of physiology, for gaining fundamental understanding of biological systems, providing new therapeutic solutions, and digitally mediating adaptations of organisms to external factors. The organic electronic ion pump (OEIP) provides a unique means for electronically-controlled, flow-free delivery of ions, and biomolecules at cellular scale. Here, a miniaturized OEIP device based on glass capillary fibers (c-OEIP) is implanted in a biological organism. The capillary form factor at the sub-100 mu m scale of the device enables it to be implanted in soft tissue, while its hyperbranched polyelectrolyte channel and addressing protocol allows efficient delivery of a large aromatic molecule. In the first example of an implantable bioelectronic device in plants, the c-OEIP readily penetrates the leaf of an intact tobacco plant with no significant wound response (evaluated up to 24 h) and effectively delivers the hormone abscisic acid (ABA) into the leaf apoplast. OEIP-mediated delivery of ABA, the phytohormone that regulates plants tolerance to stress, induces closure of stomata, the microscopic pores in leafs epidermis that play a vital role in photosynthesis and transpiration. Efficient and localized ABA delivery reveals previously unreported kinetics of ABA-induced signal propagation. ; Funding Agencies|Knut and Alice Wallenberg FoundationKnut & Alice Wallenberg Foundation; Swedish Research Council (VR)Swedish Research Council; Swedish Foundation for Strategic Research (SSF)Swedish Foundation for Strategic Research; Onnesjo Foundation; VINNOVAVinnova; Swedish Government Strategic Research Area in Materials Science on Functional Materials at Linkoping University [2009-00971]; European UnionEuropean Union (EU) [800926]; Marie Sklodowska Curie Individual Fellowship (MSCA-IFEF-ST, Trans-Plant) [702641]

The scavenger receptor class B type 1 (SR-B1) is an important HDL receptor involved in cholesterol uptake and efflux, but its physiological role in human lipoprotein metabolism is not fully understood. Heterozygous carriers of the SR-B1P297S mutation are characterized by increased HDL cholesterol levels, impaired cholesterol efflux from macrophages and attenuated adrenal function. Here, the composition and function of lipoproteins were studied in SR-B1P297S heterozygotes. Lipoproteins from six SR-B1P297S carriers and six family controls were investigated. HDL and LDL/VLDL were isolated by ultracentrifugation and proteins were separated by two-dimensional gel electrophoresis and identified by mass spectrometry. HDL antioxidant properties, paraoxonase 1 activities, apoA-I methionine oxidations and HDL cholesterol efflux capacity were assessed. Multivariate modeling separated carriers from controls based on lipoprotein composition. Protein analyses showed a significant enrichment of apoE in LDL/VLDL and of apoL-1 in HDL from heterozygotes compared to controls. The relative distribution of plasma apoE was increased in LDL and in lipid-free form. There were no significant differences in paraoxonase 1 activities, HDL antioxidant properties or HDL cholesterol efflux capacity but heterozygotes showed a significant increase of oxidized methionines in apoA-I. The SR-B1P297S mutation affects both HDL and LDL/VLDL protein compositions. The increase of apoE in carriers suggests a compensatory mechanism for attenuated SR-B1 mediated cholesterol uptake by HDL. Increased methionine oxidation may affect HDL function by reducing apoA-I binding to its targets. The results illustrate the complexity of lipoprotein metabolism that has to be taken into account in future therapeutic strategies aiming at targeting SR-B1. ; Funding agencies: EUs Sixth Framework Program [037631]; European Union [FP7-603091-2]; CardioVascular Research Initiative [CVON2011-16]; Research Council of South East Sweden [FORSS-3755]; County Council of Ostergotland (C-ALF); Faculty of Health Sciences in Linkoping; Ven

Background: Cardiovascular diseases (CVDs) and cancers are the major causes of chronic arsenic exposure-related morbidity and mortality. Matrix metalloproteinase-2 (MMP-2) and −9 (MMP-9) are deeply involved in the pathogenesis of CVDs and cancers. This study has been designed to evaluate the interactions of arsenic exposure with serum MMP-2 and MMP-9 concentrations especially in relation to the circulating biomarkers of CVDs. Methods: A total of 373 human subjects, 265 from arsenic-endemic and 108 from non-endemic areas in Bangladesh were recruited for this study. Arsenic concentrations in the specimens were measured by inductively coupled plasma mass spectroscopy (ICP-MS) and serum MMPs were quantified by immunoassay kits. Results: Serum MMP-2 and MMP-9 concentrations in arsenic-endemic population were significantly (p 50 μg/L) groups based on the regulatory upper limit of water arsenic concentration set by WHO and Bangladesh Government. MMPs were also found to be significantly (p < 0.05) associated with each other. Finally, the concentrations of both MMPs were correlated with several circulating markers related to CVDs. Conclusions: This study showed the significant positive associations and dose–response relationships of arsenic exposure with serum MMP-2 and MMP-9 concentrations. This study also showed the interactions of MMP-2 and MMP-9 concentrations with the circulating markers of CVDs suggesting the MMP-2 and MMP-9 -mediated mechanism of arsenic-induced CVDs.

Background: Cardiovascular diseases (CVDs) and cancers are the major causes of chronic arsenic exposure-related morbidity and mortality. Matrix metalloproteinase-2 (MMP-2) and −9 (MMP-9) are deeply involved in the pathogenesis of CVDs and cancers. This study has been designed to evaluate the interactions of arsenic exposure with serum MMP-2 and MMP-9 concentrations especially in relation to the circulating biomarkers of CVDs. Methods: A total of 373 human subjects, 265 from arsenic-endemic and 108 from non-endemic areas in Bangladesh were recruited for this study. Arsenic concentrations in the specimens were measured by inductively coupled plasma mass spectroscopy (ICP-MS) and serum MMPs were quantified by immunoassay kits. Results: Serum MMP-2 and MMP-9 concentrations in arsenic-endemic population were significantly (p 50 μg/L) groups based on the regulatory upper limit of water arsenic concentration set by WHO and Bangladesh Government. MMPs were also found to be significantly (p < 0.05) associated with each other. Finally, the concentrations of both MMPs were correlated with several circulating markers related to CVDs. Conclusions: This study showed the significant positive associations and dose–response relationships of arsenic exposure with serum MMP-2 and MMP-9 concentrations. This study also showed the interactions of MMP-2 and MMP-9 concentrations with the circulating markers of CVDs suggesting the MMP-2 and MMP-9 -mediated mechanism of arsenic-induced CVDs.

The acceleration of the process of understanding the pharmacological application of new marine bioactive compounds requires identifying the compound protein targets leading the molecular mechanisms in a living cell. The thermal proteome profiling (TPP) methodology does not fulfill the requirements for its application to any bioactive compound lacking chemical and functional characterization. Here, we present a modified method that we called bTPP for bioactive thermal proteome profiling that guarantees target specificity from a soluble subproteome. We showed that the precipitation of the microsomal fraction before the thermal shift assay is crucial to accurately calculate the melting points of the protein targets. As a probe of concept, the protein targets of 13(2)-hydroxy-pheophytin, a compound previously isolated from a marine cyanobacteria for its lipid reducing activity, were analyzed on the hepatic cell line HepG2. Our improved method identified 9 protein targets out of 2500 proteins, including 3 targets (isocitrate dehydrogenase, aldehyde dehydrogenase, phosphoserine aminotransferase) that could be related to obesity and diabetes, as they are involved in the regulation of insulin sensitivity and energy metabolism. This study demonstrated that the bTPP method can accelerate the field of biodiscovery, revealing protein targets involved in mechanisms of action (MOA) connected with future applications of bioactive compounds. ; Funding Agencies|ERA-NET Marine Biotechnology project CYANOBESITY; FORMAS, Sweden [2016-02004]; FCT Foundation of Science and Technology, Portugal [ERA-MBT/0001/2015]; IKERBASQUE; Basque Government [IT-971-16]; FCT [SFRH/BPD/112287/2015, SFRH/BD/116009/2016, UID/Multi/04423/2019]

Reactive oxygen species (ROS) regulating the release of free zinc ions (Zn2+) in cellular lysosome is closely related to various pathways of cellular signal transduction, such as inflammation and oxidative stress. Directly visualizing Zn2+ release in lysosome is essential for in-depth understanding these physiological processes, and is still an atelic challenge. In this work, we successfully fabricate a lysosome-specific Zn2+ fluorescent probe and achieve the visualization of ROS-induced Zn2+ release in lysosome of inflammatory cells. The as-prepared probe combines a green fluorophore, an ionophore with five-dentate sites, and a morpholine as the lysosome-specific localization moiety. The fluorescence of the fluorophore in the free probe is suppressed by a photoinduced electron transfer (PET) process from nitrogen atoms in the ionophore. Upon the addition of Zn2+, the fluorescence can be promoted immediately, achieving the real-time detection. Meanwhile, the probe is sensitive and selective to Zn2+, which provides the capability to detect low-concentration of free Zn2+ in lysosomes. Accordingly, the Zn2+ release was clearly observed in lysosome with the increase of ROS levels when the inflammation occurred in living cells. (c) 2018 Published by Elsevier B.V. ; Funding Agencies|Swedish Research Council (VR) [621-2013-5357]; National Natural Science Foundation of China [21705001, 21577098]; Swedish Government strategic faculty grant in material science (SFO, MATLIU) in Advanced Functional Materials (AFM) (VR) [5.1-2015-5959]; Knut and Alice Wallenberg Foundation (KAW) [2012.0083]; Centre in Nanoscience and Nanotechnology (CeNano)

Seminal plasma (SP), the non-cellular component of semen, is a heterogeneous composite fluid built by secretions of the testis, the epididymis and the accessory sexual glands. Its composition, despite species-specific anatomical peculiarities, consistently contains inorganic ions, specific hormones, proteins and peptides, including cytokines and enzymes, cholesterol, DNA and RNA-the latter often protected within epididymis- or prostate-derived extracellular vesicles. It is beyond question that the SP participates in diverse aspects of sperm function pre-fertilization events. The SP also interacts with the various compartments of the tubular genital tract, triggering changes in gene function that prepares for an eventual successful pregnancy; thus, it ultimately modulates fertility. Despite these concepts, it is imperative to remember that SP-free spermatozoa (epididymal or washed ejaculated) are still fertile, so this review shall focus on the differences between the in vivo roles of the SP following semen deposition in the female and those regarding additions of SP on spermatozoa handled for artificial reproduction, including cryopreservation, from artificial insemination to in vitro fertilization. This review attempts, including our own results on model animal species, to critically summarize the current knowledge of the reproductive roles played by SP components, particularly in our own species, which is increasingly affected by infertility. The ultimate goal is to reconcile the delicate balance between the SP molecular concentration and their concerted effects after temporal exposure in vivo. We aim to appraise the functions of the SP components, their relevance as diagnostic biomarkers and their value as eventual additives to refine reproductive strategies, including biotechnologies, in livestock models and humans. ; Funding Agencies|Research Council FORMAS, Stockholm [2017-00946, 2019-00288]; Swedish Research Council (Vetenskapsradet, VR) Stockholm, Sweden [2015-05919]; MICINN (Spain)Spanish Government; FEDER EU-funds, Madrid, Spain [AGL201569735-R, AGL2015-69738-R, PID2020-113493RB-100, RTI2018-093525-B-I00]

Following the worsening energy crisis of unreliable electricity and unaffordable petroleum products coupled with the increase number of poverty-stricken people in Nigeria, the populace is desperately in need of cheap alternative energy supplies that will replace or complement the existing energy sources. Previous efforts by the government in tackling the challenge by citizenship sensitization of the need for introduction of biofuel into the country's energy mix have not yielded the expected results because of a lack of sustained government effort. In light of the shortcomings, this study assesses the current potential of available biomass feedstock for biogas production in Nigeria, and further proposes appropriate biogas plants, depending on feedstock type and quantity, for the six geopolitical zones in Nigeria. Besides, the study proposes government-driven biogas development systems that could be effectively used to harness, using biogas technology, the estimated 270 TWh of potential electrical energy from 181 million tonnes of available biomass, in the advancement of electricity generation and consequent improvement of welfare in Nigeria. ; Sponsorship : Swedish Energy Agency, Sweden and Lagos State University, Nigeria

Human brain tissue models such as cerebral organoids are essential tools for developmental and biomedical research. Current methods to generate cerebral organoids often utilize Matrigel as an external scaffold to provide structure and biologically relevant signals. Matrigel however is a nonspecific hydrogel of mouse tumor origin and does not represent the complexity of the brain protein environment. In this study, we investigated the application of a decellularized adult porcine brain extracellular matrix (B-ECM) which could be processed into a hydrogel (B-ECM hydrogel) to be used as a scaffold for human embryonic stem cell (hESC)-derived brain organoids. We decellularized pig brains with a novel detergent- and enzyme-based method and analyzed the biomaterial properties, including protein composition and content, DNA content, mechanical characteristics, surface structure, and antigen presence. Then, we compared the growth of human brain organoid models with the B-ECM hydrogel or Matrigel controls in vitro. We found that the native brain source material was successfully decellularized with little remaining DNA content, while Mass Spectrometry (MS) showed the loss of several brain-specific proteins, while mainly different collagen types remained in the B-ECM. Rheological results revealed stable hydrogel formation, starting from B-ECM hydrogel concentrations of 5 mg/mL. hESCs cultured in B-ECM hydrogels showed gene expression and differentiation outcomes similar to those grown in Matrigel. These results indicate that B-ECM hydrogels can be used as an alternative scaffold for human cerebral organoid formation, and may be further optimized for improved organoid growth by further improving protein retention other than collagen after decellularization. ; CC BY 4.0 © 2021 Simsa et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. ; European Union's Horizon 2020 Research and Innovation Program under the Marie SklodowskaCurie Grant

Industrial biotechnology has been announced by several organizations and governments as a key enabling technology for the enhanced economic growth in a low-carbon and knowledge-based bioeconomy. An important goal to promote an environment friendly and sustainable industrial biotechnology is the discovery of new enzymes. To date, almost all enzymes used in industry have been discovered by pure culturing of microorganisms, however, it is known that less than 1% of all microorganisms can be obtained in pure cultures. The remaining majority of microorganisms is only viable by close biological interactions provided in microbial communities and is not available for enzyme discovery using the classical pure culture approaches. The investigation of microbial communities, which can be viewed as metaorganisms, has been enabled during the last two decades by refining established methods for the analysis of genes, mRNA or proteins and are called metagenomics, metatranscriptomics and metaproteomics, respectively. To date, these techniques have mostly been used in the field of microbial ecology for the understanding of the composition, function and metabolism of microbial communities but not for the purpose of bioprospecting for novel enzymes. Identification of genes that code for possible enzyme candidates is hindered, due to the fact that 30-40% of the sequenced metagenomes contain genes coding for unidentified proteins. Additionally, the -omics techniques generate large amounts of data that need to be analyzed and the outcome of the analysis does not necessarily lead to the discovery of novel applicable enzymes. The work presented in this thesis describes the establishment of the necessary conditions for a metaproteomics-based method that allows for a straightforward and targeted identification of novel enzymes with desired activity from microbial communities. The approach provides a valuable alternative to the incomplete and inefficient analysis of non-targeting data and laborious workflow, which is typically generated by the established meta-omics techniques. In developing the methods presented in this thesis, microbial communities in constructed environments were established, which allowed for the controlled expression of extracellular hydrolytic enzymes under defined conditions. By combination and modulation of advanced metaproteomics and metagenomics techniques, we were able to directly identify the enzymes and the corresponding gene sequences of several cellulolytic enzymes as a first example for the feasibility of this approach.

Modern healthcare is the result of scientific advancement across disciplines and has enabled us to understand the rationale behind many diseases and how to treat or cure them; but still a myriad of unanswered questions remains. Especially infectious diseases play an important role in healthcare as they pose a constant threat for global health and well-being. This was painfully highlighted in this year's ongoing COVID-19 pandemic with more than 40 million people infected and over 1 million deaths. Pandemics like this have not only devastating effects on global health but also economy. Therefore, scientific research in the field of infectious diseases is paramount to ensure outbreak control and surveillance of emerging threats. Current healthcare relies heavily on the diagnosis of infectious diseases in centralized healthcare centers thereby overlooking the access of molecular diagnostics for other areas such as airports, home-testing and especially the developing world with its limited resources. Towards this, various isothermal nucleic acid amplification technologies have been developed that hold the promise to bring state-of-the-art molecular diagnostics into these areas as they are versatile, sensitive and specific, and cost-effective. One such technique is rolling circle amplification which was used in this thesis. This research work provides an overview of the developments in biochemistry, related disciplines and their combination to design methods for diagnostic platforms tackling infectious diseases. The studies conducted in this work can be considered as individual modules for addressing challenges, like typing of pathogens and disease-related antibodies, and inexpensive bulk as well as digital quantification and simplified assay schemes. These approaches and their combinations aim to bring rolling circle amplification-based assay schemes into the molecular diagnostic field and towards decentralized healthcare.