Role of Novel Immunoregulatory Long Noncoding RNAs in Airway Epithelial Pathophysiology and Chronic Pulmonary Disease

COPD is currently the third leading cause of death globally, accounting for approximately 6% of all deaths in 2019, and cigarette smoke (CS) is the primary risk factor for disease development. Transcriptomic analysis of a 3D in vitro model using differentiated human airway epithelial cells (AECs) identified a novel lncRNA on the antisense strand of ICAM-1 or LASI that showed increased expression upon CS exposure. The lncRNA was significantly upregulated in CS-induced Rhesus macaque airways and in human COPD airways that exhibited higher mucus expression and goblet cell hyperplasia, which was recapitulated in vitro. Blocking lncRNA expression in cell culture setting suppressed the smoke-induced and COPD-associated dysregulated mucoinflammatory response suggesting that this airway specific immunomodulatory lncRNA may represent a novel target to mitigate the smoke-mediated inflammation and mucus hyperexpression. Additionally, not much is known about contribution of airway lncRNAs in COVID-19. RNA-sequencing analysis of nasal samples from COVID-19 patients showed significantly higher expression of secretory mucin and inflammatory gene signatures compared to the uninfected controls. COVID-19 patients showed elevated expression of inflammatory factors, airway mucins and associated transcription factors. LASI was induced in COVID-19 patients with high viral-load. A SARS-CoV-2 infected 3D-airway model largely recapitulated these clinical findings. Molecular dynamic modeling further suggested a stable interaction between viral RNA and LASI lncRNA. Notably, blocking LASI lncRNA reduced SARS-CoV-2 viral load and suppressed MUC5AC mucin levels. LASI lncRNA represents an essential facilitator of SARS-CoV-2 infection and associated airway mucoinflammatory response. Altogether, LASI lncRNA may represent a novel target to control the smoke-mediated dysregulation in airway responses and COPD exacerbations, as well as in viral infection-related inflammatory responses

Defining the level of subsurface water with electromagnetic (VLF) and electrical resistance (VES) methods

The purpose of the investigations presented in the paper is the determination of the level of underground water for a geologically researched area. On the basis of the geological data are determined the vertical faults and contacts that can be assessed as accumulating areas with possible presence of exploitable amount of underground water.The geophysical approach is based on the preliminary geological investigations as well as the measurable physical parameters, and ultimately is reduced to the application of methods that records electromagnetic waves with very low frequency (VLF) and geoelectrical investigations performed with the method of vertical electrical sounding. The geophysical surveys are applied in stages where the VLF method initially is used as a prospective investigation obtained through three profile lines (positioned transverse of the assumed fault zones), whereas the electrical resistance research is used as an investigative method to define the depth to the assumed water bearing structure. The VLF surveys are obtained through evaluation of the electromagnetic field and filtering the obtained data through different determined depths and are performed with the instrument WADI. The electrical investigations are performed with the instrument TERRASAS 1000

Effect of poplar trees on nitrogen and water balance in outdoor pig production – A case study in Denmark

Nitrate leaching from outdoor pig production is a long-standing environmental problem for surface and groundwater pollution. In this study, the effects of inclusion of poplar trees in paddocks for lactating sows on nitrogen (N) balances were studied for an organic pig farm in Denmark. Vegetation conditions, soil water and nitrate dynamics were measured in poplar and grass zones of paddocks belonging to main treatments: access to trees (AT), no access to trees (NAT) and a control without trees (NT), during the hydrological year April 2015 to April 2016. Soil water drainage for each zone, simulated by two simulation models (CoupModel and Daisy), was used to estimate nitrate leaching from the zones in each paddock. N balances (input minus output) for the treatments were computed and compared

Geo-electrical measurements in environment with presence of underground water

The coal mine “Suvodol” is chosen for geo-electrical examination because it has been most thoroughlyresearched and has the densest network of mapped boreholes. Through examination of the parameters fromthe boreholes and the geological map of the coal mine we determinate the layered structure of the ore body that providesfavorable conditions for applying the method of geo-electrical sounding. In the process of production of themodels particular attention is given to the level of underground water that has dominant influence in the conductivityproperties of the geological structures.The curves of apparent electrical resistance are interpreted while solving the directtask. For acquiring precise and concrete data while interpreting the synthetic curves it is important to obtain accuratedata for the mapped geological material

Ihmisen UDP-glukuronosyylitransferaasien aktiivisuus ja entsyymikinetiikka : Psilosiinin glukuronidaatio sekä albumiinin vaikutus entsyymikinetiikan mekanismiin

Human UDP-glucuronosyltransferases (UGTs) are important in the metabolic elimination of xenobiotics and endogenous compounds from the body. These enzymes transfer glucuronic acid moiety from the cosubstrate, UDP-glucuronic acid (UDPGA), to nucleophilic groups of small organic molecules, such as hydroxyl, carboxylic, or amino group. The conjugation of these molecules with polar glucuronic acid usually diminishes their pharmacodynamic activity, promotes aqueous solubility and enhances recognition by efflux transporters in the cells, all of which contributes to the efficient metabolic elimination and excretion of the conjugate from the body. Due to these unique properties, UGT enzymes play major roles in drug metabolism and pharmacokinetics. The main goal of this thesis was to investigate the activity and enzyme kinetics of UGTs, as well as the in vitro assay conditions needed to accurately determine the enzyme kinetic parameters. Particular attention focused on the glucuronidation of psilocin, the enhancement of UGT activity by the inclusion of purified bovine serum albumin (BSA), and the enzyme kinetic mechanism of UGT1A9. These goals are especially important in the early phases of preclinical drug development, where in vitro assays serve to explain and predict the glucuronidation of the drug in vivo, both qualitatively and quantitatively. As a starting point, we studied the glucuronidation of psilocin, the hallucinogenic indole alkaloid from mushrooms of the genus Psilocybe, by all the human UGTs of subfamilies 1A, 2A, and 2B. To understand the substrate selectivity of human UGTs, we also studied the glucuronidation of 4-hydroxyindole, a chemically simpler analog of psilocin which lacks the N,N-dimethylaminoethyl side chain. We successfully prevented the oxidative degradation of psilocin, a problem we encountered early in the study, by including an antioxidant (1 mM dithiothreitol) in the assays. Our results showed that psilocin is glucuronidated mainly by UGTs 1A10 and 1A9, whereas the activities of UGTs 1A8, 1A7, and 1A6 were lower. On the other hand, 4-hydroxyindole was glucuronidated mainly by UGT1A6, whereas the activities of UGTs 1A7 1A10 closely correlated with their respective rates of psilocin glucuronidation. To understand in which human tissues psilocin glucuronidation takes place, we studied the expression levels of mRNA for UGTs 1A7 1A10; this work was performed in collaboration with Dr. Michael H. Court of the Tufts University School of Medicine, Boston, Massachusetts. The combined results of the activity and expression studies indicate that although the intestinal enzyme UGT1A10 shows the highest glucuronidation clearance, UGT1A9, an enzyme abundantly expressed in both the liver and kidneys, may be the main contributor to psilocin glucuronidation in vivo. The inclusion of purified albumin is known to significantly enhance glucuronidation rates in vitro. In subsequent studies, we focused our attention on the scope and mechanism of this activity enhancement and investigated albumin effects in a total of 11 human UGTs. Before proceeding with enzyme kinetic assays, we carefully measured the binding of substrates to BSA by either ultrafiltration or rapid equilibrium dialysis. Our results showed that the inclusion of BSA significantly enhances the in vitro glucuronidation activity of almost all the UGTs we tested, either by increasing the apparent substrate affinity (lower Km) or the reaction-limiting velocity (higher Vmax), or both. The nature of albumin effects, however, varied greatly and depended both on the UGT enzyme and the substrate employed. The highest activity increases in the presence of BSA were observed in UGTs 1A7, 1A9, 1A10, 2A1, and 2B7, whereas BSA stimulation was comparatively less pronounced in UGTs 1A1, 1A6, 1A8, 2B4, and 2B15. On the other hand, depending on the substrate used, the addition of BSA to UGTs 1A1, 1A6, and 2B17 sometimes resulted in a lack of any stimulatory effects. Moreover, the activity enhancement by BSA appears independently of the enzyme source used, since both native enzymes in human liver microsomes and recombinant enzymes expressed in Sf9 insect cells yielded similar results. To investigate the mechanism of albumin effects, as well as to elucidate the enzyme kinetic mechanism of human UGTs, we studied bisubstrate enzyme kinetics, the product inhibition, and dead-end inhibition kinetics of UGT1A9. For this purpose, we employed 4-methylumbelliferone as the aglycone substrate and investigated both forward- and reverse-direction UGT-catalyzed reactions. The combined results of our experiments strongly suggest that UGT1A9 follows the compulsory-order ternary-complex mechanism with UDPGA binding first. The addition of BSA quantitatively changes the enzyme kinetic parameters, presumably by removing internal inhibitors that bind to binary (enzyme-UDPGA) or ternary (enzyme-UDPGA-aglycone) complexes, but the underlying compulsory-order ternary-complex mechanism remains unaffected. In addition, based on enzyme kinetic parameters measured in the forward and reverse reaction, we elucidated the thermodynamic equilibrium constant of the overall reaction (Keq = 574), as well as the relative magnitude of the individual rate constants. In summary, the results obtained deepen our current knowledge of UGT enzyme kinetics and set new guidelines for performing in vitro UGT assays. The study of psilocin and 4-hydroxyindole glucuronidation revealed that relatively small structural modifications, such as the loss of the side chain, lead to major changes in UGT substrate selectivity. And provided the substrate binding to BSA is accounted for, the addition of BSA significantly enhances the activities of almost all the UGTs tested and improves the accuracy of the measured enzyme kinetic parameters. These features are especially important for the prediction of UGT activity in vivo. Finally, our results deepen our current understanding of the UGT enzyme kinetic mechanism and conclusively show that UDPGA is the first, and the aglycone substrate is the second binding substrate to form a ternary complex in UGT1A9-catalyzed reactions.Useilla lääkkeillä ja elimistön molekyyleillä on huono liukoisuus veteen sekä korkea affiniteetti kehon rasvaan. Tällaisten aineiden poisto virtsan kautta on hankalaa, mikä saattaa johtaa niiden haitalliseen kertymiseen. Tämän ongelman ratkaisemiseksi ihmisen elimistöstä löytyy joukko erilaisia metaboliaentsyymejä. Näiden päätehtävänä on kemiallisesti muokata rasvaliukoisten aineiden rakennetta enemmän vesiliukoisiksi ja biologisesti inaktiivisiksi. Metaboliaentsyymien toiminta on siten hyvin tärkeää paitsi elimistön normaalille toiminnalle myös farmakoterapialle ja lääkekehitykselle. Ihmisen UDP-glukuronosyylitransferaasit (UGT:t) ovat yksi keskeisistä metaboliaentsyymeistä, ja ne vastaavat terapeuttisten lääkkeiden, ravintoaineiden, steroidihormonien, sappihappojen sekä punasolupigmenttien eliminaatiosta. UGT:t lisäävät sokeriryhmän (glukuronihapon) rasvaliukoisiin molekyyleihin, jolloin näiden vesiliukoisuus paranee huomattavasti. Tämän väitöskirjan päätavoitteena oli tutkia kuinka ihmisen UGT:t toimivat. Keskityimme erityisesti (1) psilosiinin, sienistä löytyvän hallusinogeenisen alkaloidin, glukuronidaatioon, (2) UGT-entsyymien laboratoriokoeolosuhteisiin, sekä (3) mekanismiin, millä sokeriryhmä siirretään rasvaliukoisiin molekyyleihin. Tuloksemme osoittavat, että psilosiinin glukuronisaatiosta vastaa pääosin UGT1A9 maksassa ja munuaisissa, sekä pienemmissä määrin suoliston UGT1A10. Havaitsimme myös, että albumiinin lisääminen nostaa merkittävästi monien yksittäisten ihmisen UGT-entsyymien aktiivisuuksia, millä on vaikutusta UGT tutkimuksille laboratorioissa. Lisäksi edistyimme huomattavasti UGT-entsyymin kinetiikkamekanismin ymmärtämisessä. Kaiken kaikkiaan, tulokset syventävät nykyistä tietämystämme ihmisen UGT-entsyymeistä ja asettavat uusia ohjeistuksia UGT laboratoriotutkimuksille. Uskomme tämän väitöskirjan parantavan käsitystämme ihmisen lääkeainemetaboliasta ja turvallisesta farmakoterapiasta

Prothrombin expression in cancer-derived cell lines

The link between thrombotic disorders and cancer has been known for over 150 years, although the precise mechanism of this relationship has not yet been resolved. Current data show that thrombin has a significant role in cancer metabolism, invasiveness, adhesion and survival. However, data regarding the expression of the thrombin precursor prothrombin in various cancer cell lines are scarce. Therefore, it was our objective to determine whether common cancer-derived cell lines (Caco-2, MCF-7, SK-BR-3, U-87 and U-251) express prothrombin. The prothrombin RNA expression level was assessed by qPCR, and the presence of prothrombin was analyzed by Western blot analysis. Our results show that Caco-2 cells originating from colorectal adenocarcinoma express prothrombin, whereas other analyzed cell lines do not. Our results provide a background for further research into the role of (pro) thrombin in cancer etiopathology

Lactobacillus brevis BGZLS10-17 and Lb. plantarum BGPKM22 Exhibit Anti-Inflammatory Effect by Attenuation of NF-kappa B and MAPK Signaling in Human Bronchial Epithelial Cells

Bronchial epithelial cells are exposed to environmental influences, microbiota, and pathogens and also serve as a powerful effector that initiate and propagate inflammation by the release of proinflammatory mediators. Recent studies suggested that lung microbiota differ between inflammatory lung diseases and healthy lungs implicating their contribution in the modulation of lung immunity. Lactic acid bacteria (LAB) are natural inhabitants of healthy human lungs and also possess immunomodulatory effects, but so far, there are no studies investigating their anti-inflammatory potential in respiratory cells. In this study, we investigated immunomodulatory features of 21 natural LAB strains in lipopolysaccharide (LPS)-stimulated human bronchial epithelial cells (BEAS-2B). Our results show that several LAB strains reduced the expression of pro-inflammatory cytokine and chemokine genes. We also demonstrated that two LAB strains, Lactobacillus brevis BGZLS10-17 and Lb. plantarum BGPKM22, effectively attenuated LPS-induced nuclear factor-kappa B (NF-kappa B) nuclear translocation. Moreover, BGZLS10-17 and BGPKM22 reduced the activation of p38, extracellular signal-related kinase (ERK), and c-Jun amino-terminal kinase (JNK) signaling cascade resulting in a reduction of pro-inflammatory mediator expressions in BEAS-2B cells. Collectively, the LAB strains BGZLS10-17 and BGPKM22 exhibited anti-inflammatory effects in BEAS-2B cells and could be employed to balance immune response in lungs and replenish diminished lung microbiota in chronic lung diseases

Integrated modelling of crop production and nitrate leaching with the Daisy model

An integrated modelling strategy was designed and applied to the Soil-Vegetation-Atmosphere Transfer model Daisy for simulation of crop production and nitrate leaching under pedo-climatic and agronomic environment different than that of model original parameterisation. The points of significance and caution in the strategy are: • Model preparation should include field data in detail due to the high complexity of the soil and the crop processes simulated with process-based model, and should reflect the study objectives. Inclusion of interactions between parameters in a sensitivity analysis results in better account for impacts on outputs of measured variables. • Model evaluation on several independent data sets increases robustness, at least on coarser time scales such as month or year. It produces a valuable platform for adaptation of the model to new crops or for the improvement of the existing parameters set. On daily time scale, validation for highly dynamic variables such as soil water transport remains challenging. • Model application is demonstrated with relevance for scientists and regional managers. The integrated modelling strategy is applicable for other process-based models similar to Daisy. It is envisaged that the strategy establishes model capability as a useful research/decision-making, and it increases knowledge transferability, reproducibility and traceability

Accurate estimates of land surface energy fluxes and irrigation requirements from UAV-based thermal and multispectral sensors

The two-source energy balance model estimates canopy transpiration (Tr) and soil evaporation (E) traditionally from satellite partitions of remotely sensed land surface temperature (LST) and the Priestley-Taylor equation (TSEB-PT) at seasonal time with limited accuracy. The high spatial–temporal resolution spectral data collected by unmanned aerial vehicles (UAVs) provide valuable opportunity to estimate Tr and E precisely, improve the understanding of the seasonal and the diurnal cycle of evapotranspiration (ET), and timely detect agricultural drought. The UAV data vary in spatial resolution and the uncertainty imposed on the TSEB-PT outcome has thus far not being considered. To address these challenges and prospects, a new energy flux modelling framework based on TSEB-PT for high spatial resolution thermal and multispectral UAV data is proposed in this paper. Diurnal variations of LST in agricultural fields were recorded with a thermal infrared camera installed on an UAV during drought in 2018 and 2019. Observing potato as a test crop, LST, plant biophysical parameters derived from corresponding UAV multispectral data, and meteorological forcing variables were employed as input variables to TSEB-PT. All analyses were conducted at different pixelation of the UAV data to quantify the effect of spatial resolution on the performance. The 1 m spatial resolution produced the highest correlation between Tr modelled by TSEB-PT and measured by sap flow sensors (R2 = 0.80), which was comparable to the 0.06, 0.1, 0.5 and 2 m pixel sizes (R2 = 0.76–0.78) and markedly higher than the lower resolutions of 2 to 24 m (R2 = 0.30–0.72). Modelled Tr was highly and significantly correlated with measured leaf water potential (R2 = 0.81) and stomatal conductance (R2 = 0.74). The computed irrigation requirements (IRs) reflected the field irrigation treatments, ET and conventional irrigation practices in the area with high accuracy. It was also found that using a net primary production model with explicit representation of temperature influences made it possible to distinguish effects of drought vis-a-vis heat stress on crop productivity and water use efficiency. The results showed excellent model performance for retrieving Tr and ET dynamics under drought stress and proved that the proposed remote sensing based TSEB-PT framework at UAV scale is a promising tool for the investigation of plant drought stress and water demand; this is particularly relevant for local and regional irrigations scheduling.info:eu-repo/semantics/publishedVersio

Accurate estimates of land surface energy fluxes and irrigation requirements from UAV-based thermal and multispectral sensors

The two-source energy balance model estimates canopy transpiration (Tr) and soil evaporation (E) traditionally from satellite partitions of remotely sensed land surface temperature (LST) and the Priestley-Taylor equation (TSEB-PT) at seasonal time with limited accuracy. The high spatial-temporal resolution spectral data collected by unmanned aerial vehicles (UAVs) provide valuable opportunity to estimate Tr and E precisely, improve the understanding of the seasonal and the diurnal cycle of evapotranspiration (ET), and timely detect agricultural drought. The UAV data vary in spatial resolution and the uncertainty imposed on the TSEB-PT outcome has thus far not being considered. To address these challenges and prospects, a new energy flux modelling framework based on TSEB-PT for high spatial resolution thermal and multispectral UAV data is proposed in this paper. Diurnal variations of LST in agricultural fields were recorded with a thermal infrared camera installed on an UAV during drought in 2018 and 2019. Observing potato as a test crop, LST, plant biophysical parameters derived from corresponding UAV multispectral data, and meteorological forcing variables were employed as input variables to TSEB-PT. All analyses were conducted at different pixelation of the UAV data to quantify the effect of spatial resolution on the performance. The 1 m spatial resolution produced the highest correlation between Tr modelled by TSEB-PT and measured by sap flow sensors (R2 = 0.80), which was comparable to the 0.06, 0.1, 0.5 and 2 m pixel sizes (R2 = 0.76-0.78) and markedly higher than the lower resolutions of 2 to 24 m (R2 = 0.30-0.72). Modelled Tr was highly and significantly correlated with measured leaf water potential (R2 = 0.81) and stomatal conductance (R2 = 0.74). The computed irrigation requirements (IRs) reflected the field irrigation treatments, ET and conventional irrigation practices in the area with high accuracy. It was also found that using a net primary production model with explicit representation of temperature influences made it possible to distinguish effects of drought vis-a-vis heat stress on crop productivity and water use efficiency. The results showed excellent model performance for retrieving Tr and ET dynamics under drought stress and proved that the proposed remote sensing based TSEB-PT framework at UAV scale is a promising tool for the investigation of plant drought stress and water demand; this is particularly relevant for local and regional irrigations scheduling