Silencing of Transcription Factor Sp1 Promotes SN1 Transporter Regulation by Ammonia in Mouse Cortical Astrocytes

The involvement of the astrocytic SN1 (SNAT3) transporter in ammonia-induced l-glutamine retention was recently documented in mouse-cultured astrocytes. Here we investigated the involvement of specificity protein 1 (Sp1) transcription factor in SN1 regulation in ammonium chloride (“ammonia”)-treated astrocytes. Sp1 expression and its cellular localization were determined using real-time qPCR, Western blot, and confocal microscopy. Sp1 binding to Snat3 promoter was analyzed by chromatin immunoprecipitation. The role of Sp1 in SN1 expression and SN1-mediated [3H]glutamine uptake in ammonia-treated astrocytes was verified using siRNA and mithramycin A. The involvement of protein kinase C (PKC) isoforms in Sp1 level/phosphorylation status was verified using siRNA technology. Sp1 translocation to the nuclei and its enhanced binding to the Snat3 promoter, along with Sp1 dependence of system N-mediated [3H]glutamine uptake, were observed in astrocytes upon ammonia exposure. Ammonia decreased the level of phosphorylated Sp1, and the effect was reinforced by long-term incubation with PKC modulator, phorbol 12-myristate 13-acetate, which is a treatment likely to dephosphorylate Sp1. Furthermore, silencing of the PKCδ isoform appears to enhance the ammonia effect on the Sp1 level. Collectively, the results demonstrate the regulatory role of Sp1 in regulation of SN1 expression and activity in ammonia-treated astrocytes and implicate altered Sp1 phosphorylation status in this capacity

Road users’ behaviour study in areas of zebra crosswalks

Pedestrian safety on crosswalks is extremely vital in Poland since 2015, when the first study on road users’ behaviour on driver–pedestrian encounters in areas of zebra crosswalks were conducted. The second study was carried out in 2018 and its results are published in this article for the first time. The results of the project helped lead to changes in pedestrians’ safety regulations in Poland, increasing the safety of pedestrians in areas of crossing. Since June, the 1st, 2021, drivers of oncoming cars approaching a zebra crossing are obliged to stop to allow pedestrians approaching from the sidewalk to cross the zebra crossing. Data to assess pedestrian safety presented in this article combine a new approach that is a combination of different diagnostic techniques: a surrogate safety measure (nonaccident-based indicators) and the traditional approach: statistical analysis. As a result of the study, the most frequent categories of interactions between drivers and pedestrians and pedestrians itself were characterized on crossing facilities. Videos recording pedestrians’ and drivers’ behaviours, and vehicle speed measurements at pedestrian crossings from 2018 allow to assess the safety of 7 000 pedestrians during behaviour observation in onsite fields: on all crossing facilities, except with light signalization, 55% of pedestrians had to stop and wait at the sidewalks to cross, before an oncoming car. Only 45% of drivers approaching not signalised zebra crossings gave way to pedestrians. Pedestrians aged 60+ entering the road on marked crossings without traffic light waited longer to cross than younger. In residential areas with high traffic volume more drivers stopped at non signalised crossings and gave way to pedestrians. At low speed of vehicle in urban areas pedestrians felt safer and were ready to enter the crossing; their behaviour was more predictable. Results showed dangerous pedestrian behaviours on signal-regulated crossing facilities who failed to obey the traffic lights (7% of red-light crossings). 8% of observed pedestrians crossed the street outside designated pedestrian crossings. Video-recorded speed measurements of over 32 000 vehicles on-site study fields of pedestrian crossings showed that the speed of vehicles was higher than permitted. This article presents the newest characteristics of traffic and pedestrian’ behavior at crossings together with measurements of pedestrians’ speed and loss of pedestrians’ time on different road cross sections in Poland in 2018

The Role of Nrf2 Transcription Factor and Sp1-Nrf2 Protein Complex in Glutamine Transporter SN1 Regulation in Mouse Cortical Astrocytes Exposed to Ammonia

Ammonia toxicity in the brain primarily affects astrocytes via a mechanism in which oxidative stress (OS), is coupled to the imbalance between glutamatergic and GABAergic transmission. Ammonia also downregulates the astrocytic N system transporter SN1 that controls glutamine supply from astrocytes to neurons for the replenishment of both neurotransmitters. Here, we tested the hypothesis that activation of Nrf2 is the process that links ammonia-induced OS formation in astrocytes to downregulation and inactivation of SN1 and that it may involve the formation of a complex between Nrf2 and Sp1. Treatment of cultured cortical mouse astrocytes with ammonia (5 mM NH4Cl for 24 h) evoked Nrf2 nuclear translocation, increased its activity in a p38 MAPK pathway-dependent manner, and enhanced Nrf2 binding to Slc38a3 promoter. Nrf2 silencing increased SN1 mRNA and protein level without influencing astrocytic [3H]glutamine transport. Ammonia decreased SN1 expression in Nrf2 siRNA treated astrocytes and reduced [3H]glutamine uptake. In addition, while Nrf2 formed a complex with Sp1 in ammonia-treated astrocytes less efficiently than in control cells, treatment of astrocytes with hybrid-mode inactivated Sp1-Nrf2 complex (Nrf2 silencing + pharmacological inhibition of Sp1) did not affect SN1 protein level in ammonia-treated astrocytes. In summary, the results document that SN1 transporter dysregulation by ammonia in astrocytes involves activation of Nrf2 but does not require the formation of the Sp1-Nrf2 complex

Modelling Net Ecosystem Exchange in the Biebrza Wetlands using satellite and meteorological data

The CO2 flux on Biebrza Wetlands in Poland is analysed by ground measurements and satellite data. The main goal of this study was to determine the Net Ecosystem Exchange (NEE) for sedges, reeds and grasses using meteorological, soil and vegetation parameters and satellite indicators. The calculations are based on chamber measurements scattered over the main habitats and meteorological registrations, and compared to the 2015–2022 eddy covariance (EC) measurements. The NEE model, to get its distribution throughout the day, was elaborated using modelled gross primary production GPP and modelled respiration RESP. The model of ecosystem respiration was developed applying air temperature, soil temperature, soil moisture and NDVI. The satellite vegetation index NDVI, surface and air difference temperature (TS-TA), and NDII proved to be good predictors for the daily mean GPP. The data from the EC tower was used to compare the results from modelled NEE and CO2 flux

Różnorodność gatunkowa użytków zielonych w Dolinie Środkowego Wieprza (PLH060005) w zależności od typu łąki i częstotliwości koszenia

The studies were conducted on grasslands in the middle Wieprz valley in 2017. The aim of study was to assess the diversity of the species composition of selected grassland, depending on the frequency of mowing. The investigations focused on the meadow vegetation of Molinietalia order, Arrhenatheretalia order, the Molinio-Arrhenatheretea class and Phragmitetea class. The floristic diversity of the grassland communities was determined based on two indices, namely the number of species (SN) and the Shannon-Wiener index H'. In order to detect significant differences between the obtained values of these indexes for the particular grasslands (taking into account the research factors), a variance analysis was carried out, taking into account the Linear Model (LM) for the Shannon-Wiener index and a Generalised Linear Model (GLM) for the number of species on the assumption of the Poisson distribution of this variable. In addition, a Multidimensional principal component analysis (PCA) (Jolliffe 2002) was carried out to detect the variation of the analysed dataset with regard to the values of the ecological indices (Ellenberg et al. 1992). In the grassland under study, the number of species varied depending on the type of meadow and mowing frequency. Meadows with vegetation of the Arrhenatheretalia order had the greatest number of species. Furthermore, the number of species was found to increase with the growing frequency of use. The least numerous communities occurred in rush meadows (Phragmitetea class). A more frequent use of the sward of these communities led to a decreased number of species. Similar trends were observed in the case of the Shannon-Wiener index values. A variance analysis was carried out and its results indicate a significant influence of both research factors (meadow type and cutting frequency) on the floristic diversity of the communities under study. The biggest significant differences were confirmed between meadow vegetation of the Arrhenatheretalia order and Phragmitetea class. The biggest influence of cutting frequency on the increased species diversity occurred in meadows with communities of the Arrhenatheretalia and Molinietalia order. According to a multidimensional principal component analysis (PCA) indications, habitat humidity is the main factor determining the type of meadow community and its species diversity.Badania fitosocjologiczne przeprowadzono w 2017 roku na użytkach zielonych w dolinie środkowego Wieprza. Dolina Środkowego Wieprza (PHL060005) stanowi mozaikę siedlisk o zróżnicowanym uwilgotnieniu, co sprzyja występowaniu różnorodnych zespołów roślinnych. Celem badań była ocena zróżnicowania składu gatunkowego wybranych użytków zielonych, w zależności od częstotliwości koszenia. Przedmiotem badań była szata roślinna łąk rzędu Molinietalia, Arrhenatheretalia, klasy Molinio-Arrhenatheretea i Phragmitetea. Różnorodność florystyczną badanych zbiorowisk roślinnych oceniono za pomocą dwóch wskaźników, jakimi są liczba gatunków (SN) oraz indeks Shannona-Wienera H'. W celu wykrycia istotnych różnic między uzyskanymi wartościami tych wskaźników dla poszczególnych zbiorowisk (z uwzględnieniem czynników badań) wykonano analizę wariancji z uwzględnieniem modelu liniowego (LM) dla indeksu Shannona-Wienera oraz uogólnionego modelu liniowego (GLM) dla liczebności gatunków przy założeniu rozkładu Poissona tej cechy. Dodatkowo wykonano wielowymiarową analizę głównych składowych PCA (Jolliffe 2002) w celu wykrycia zmienności analizowanego zbioru danych, ze względu na wartości wskaźników ekologicznych (Ellenberg et al. 1992). Na badanych użytkach zielonych, wartość tego parametru wykazywała zróżnicowanie w zależności od typu łąki oraz częstotliwości koszenia. Łąki porośnięte roślinnością rzędu Arrhenatheretalia były najbardziej liczne gatunkowo. Stwierdzono ponadto zwiększanie się liczby gatunków w zbiorowisku wraz z rosnącą częstotliwością użytkowania. Najmniej liczne zbiorowiska występowały na łąkach z roślinnością klasy Phragmitetea. Częstsze użytkowanie runi tych zbiorowisk przyczyniało się do zmniejszenia liczby gatunków. Podobne trendy stwierdzono w przypadku wartości indeksu Shannona-Wienera. Wykonano analizę wariancji, której wyniki świadczą o istotnym wpływie obu uwzględnionych czynników (typ łąki oraz częstotliwość koszenia) na zróżnicowanie florystyczne badanych zbiorowisk. Największe istotne różnice potwierdzono między szatą roślinną rzędu Arrhenatheretalia i klasy Phragmitetea. Największy wpływ częstotliwości koszenia na wzrost różnorodności gatunkowej zaznaczył się na łąkach z roślinnością rzędu Arrhenatheretalia i Molinietalia. Wskazaniem wielowymiarowej analizy głównych składowych PCA jest, że wilgotność siedliska jest tu głównym czynnikiem decydującym o typie szaty roślinnej i jego zróżnicowaniu gatunkowym