In vivo femtosecond laser nanosurgery of the cell wall enabling patch-clamp measurements on filamentous fungi.

Studying the membrane physiology of filamentous fungi is key to understanding their interactions with the environment and crucial for developing new therapeutic strategies for disease-causing pathogens. However, their plasma membrane has been inaccessible for a micron-sized patch-clamp pipette for pA current recordings due to the rigid chitinous cell wall. Here, we report the first femtosecond IR laser nanosurgery of the cell wall of the filamentous fungi, which enabled patch-clamp measurements on protoplasts released from hyphae. A reproducible and highly precise (diffraction-limited, submicron resolution) method for obtaining viable released protoplasts was developed. Protoplast release from the nanosurgery-generated incisions in the cell wall was achieved from different regions of the hyphae. The plasma membrane of the obtained protoplasts formed tight and high-resistance (GΩ) contacts with the recording pipette. The entire nanosurgical procedure followed by the patch-clamp technique could be completed in less than 1 hour. Compared to previous studies using heterologously expressed channels, this technique provides the opportunity to identify new ionic currents and to study the properties of the ion channels in the protoplasts of filamentous fungi in their native environment.

ATP modulation of osmotically activated anionic current in the membrane of Phycomyces blakesleeanus sporangiophore.

Ion channels are vital components of filamentous fungi signaling in communication with their environment. We exploited the ability of the apical region of growing sporangiophores of Phycomyces blakesleeanus to form membrane-enveloped cytoplasmic droplets (CDs), to examine ion currents in the filamentous fungi native plasma membrane. In hypoosmotic conditions, the dominant current in the CDs is ORIC, an osmotically activated, anionic, outwardly rectified, fast inactivating instantaneous current that we have previously characterized. Here, we examined the effect of ATP on ORIC. We show that CDs contain active mitochondria, and that respiration inhibition by azide accelerates ORIC inactivation. ATP, added intracellularly, reduced ORIC run-down and shifted the voltage dependence of inactivation toward depolarized potentials, in a manner that did not require hydrolysis. Notably, ATP led to slowing down of ORIC inactivation, as evidenced by an increased time constant of inactivation, τin, and slower decline of τin during prolonged recordings. Flavonoids (genistein and quercetin) had the effect on ORIC opposite to ATP, acting as current inhibitors, possibly by disrupting the stabilizing effect of ATP on ORIC. The integration of osmotic sensing with ATP dependence of the anionic current, typical of vertebrate cells, is described here for the first time in filamentous fungi.

Osmotically Activated Anion Current of Phycomyces Blakesleeanus-Filamentous Fungi Counterpart to Vertebrate Volume Regulated Anion Current.

Studies of ion currents in filamentous fungi are a prerequisite for forming a complete understanding of their physiology. Cytoplasmic droplets (CDs), obtained from sporangiophores of Phycomyces blakesleeanus, are a model system that enables the characterization of ion currents in the native membrane, including the currents mediated by the channels not yet molecularly identified. Osmotically activated anionic current with outward rectification (ORIC) is a dominant current in the membrane of cytoplasmic droplets under the conditions of hypoosmotic stimulation. We have previously reported remarkable functional similarities of ORIC with the vertebrate volume regulated anionic current (VRAC), such as dose-dependent activation by osmotic difference, ion selectivity sequence, and time and voltage dependent profile of the current. Using the patch clamp method on the CD membrane, we further resolve VRAC-like ORIC characteristics in this paper. We examine the inhibition by extracellular ATP and carbenoxolone, the permeation of glutamate in presence of chloride, selectivity for nitrates, and activation by GTP, and we show its single channel behavior in excised membrane. We propose that ORIC is a functional counterpart of vertebrate VRAC in filamentous fungi, possibly with a similar essential role in anion efflux during cell volume regulation.

RADON IN DRINKING WATER FROM ALTERNATIVE SOURCES OF WATER SUPPLY IN THE NORTH OF KOSOVO.

In this study, the results of research on radon activity concentrations in natural mineral waters, traditionally used for drinking but also for other needs, in rural and urban households in northern Kosovo are presented. Radon activity concentration in water was measured by the alpha spectrometric method with a RAD7 device. Radon activity concentrations in the 24 waters studied ranged from 1.6 ± 0.5 to 46.3 ± 6.3 Bq l-1, with an average activity concentration of 12.4 ± 2.0 Bq l-1, which was somewhat higher than the EPA recommended maximum activity concentration, but below the WHO recommended maximum. The contribution of radon activity concentrations in water was determined in relation to the total radon activity in air and enclosed space. The estimated annual effective doses of inhalation and ingestion radon from water were 109.4 ± 16.7 and 2.6 ± 0.4 µSv y-1, respectively.

In Search for an Ionic Liquid with the Best Performance during 210Pb/210Bi Cherenkov Counting in Waters on an LS Counter.

The research presented in this paper aims to investigate the performance of several newly synthesized ionic liquids during 210Pb/210Bi detection in water on a liquid scintillation spectrometer Quantulus 1220 via Cherenkov counting. These experiments have been triggered by the recent reports that certain ionic liquids can act as wavelength shifters, thus significantly increasing the detection efficiency of Cherenkov radiation. The benefit of ionic liquid's addition to the analysed samples is reflected in the detection limit's decrement during 210Pb quantification, which is pertinent considering naturally low levels of 210Pb in aqueous samples. Firstly, it was discovered that ionic liquid, 1-butyl-3-methylimidazolium salicylate, is more efficient than the previously explored 2-hydroxypropylammonium salicylate. Consequently, the impact of a few other ionic liquids on Cherenkov counting efficiency with the same cation group (1-butyl-3-methylimidazolium benzoate, 1-butyl-3-methylimidazolium 3-hydroxybenzoate and 1-butyl-3-methylimidazolium 4-hydroxybenzoate) was also explored to test their potential influence. Molecular simulations have been carried out to reveal which structures of ionic liquids assure wavelength-shifting behavior. The obtained results confirmed that, among the investigated ones, only ionic liquids with the salicylate anion exhibited a wavelength shifting effect.

The influence of building material structure on radon emanation.

In this work, the radon emanation coefficients for selected building materials that are most often used in Serbia for covering floor surfaces (concrete, concrete screed, granite, glazed ceramic tiles, marble, roofing tile, and terrazzo tile) were determined, and the influence of the material structure on their values. The concentration of226Ra activity in the samples was determined using the gamma spectrometry method. Radon emanation was measured with the RAD7 device. The porosity of the samples was tested using mercury intrusion porosimetry and water absorption methods, and the structural analysis was performed using x-ray diffraction analysis and x-ray fluorescence analysis. The measured values of226Ra activity concentrations were in the range (4.93-298) Bq kg-1, and the estimated values of the radon emanation coefficients were in the range (0.55-6.05) %. The obtained results indicate that the chemical and mineralogical composition, method of production, and the226Ra activity concentration have an influence on the emanation of radon from the material. No significant correlation was found between the radon emanation coefficient and the open porosity of the material, most likely due to the inhomogeneous presence of pores of different dimensions in the materials. It was established that the total value of the emanation coefficient depends on the emanation coefficient for pores ⩽100µm in size.

Label-free third harmonic generation imaging and quantification of lipid droplets in live filamentous fungi.

We report the utilization of Third-Harmonic Generation microscopy for label-free live cell imaging of lipid droplets in the hypha of filamentous fungus Phycomyces blakesleeanus. THG microscopy images showed bright spherical features dispersed throughout the hypha cytoplasm in control conditions and a transient increase in the number of bright features after complete nitrogen starvation. Colocalization analysis of THG and lipid-counterstained images disclosed that the cytoplasmic particles were lipid droplets. Particle Size Analysis and Image Correlation Spectroscopy were used to quantify the number density and size of lipid droplets. The two analysis methods both revealed an increase from 16 × 10-3 to 23 × 10-3 lipid droplets/µm2 after nitrogen starvation and a decrease in the average size of the droplets (range: 0.5-0.8 µm diameter). In conclusion, THG imaging, followed by PSA and ICS, can be reliably used for filamentous fungi for the in vivo quantification of lipid droplets without the need for labeling and/or fixation. In addition, it has been demonstrated that ICS is suitable for THG microscopy.

Experimental Studies to Test a Predictive Indoor Radon Model.

The accumulation of the radioactive gas radon in closed environments, such as dwellings, is the result of a quite complex set of processes related to the contribution of different sources. As it undergoes different physical mechanisms, all occurring at the same time, models describing the general dynamic turns out to be difficult to apply because of the dependence on many parameters not easy to measure or calculate. In this context, the authors developed, in a previous work, a simplified approach based on the combination of a physics-mathematical model and on-site experimental measurements. Three experimental studies were performed in order to preliminarily test the goodness of the model to simulate indoor radon concentrations in closed environments. In this paper, an application on a new experimental site was realized in order to evaluate the adaptability of the model to different house typologies and environmental contexts. Radon activity measurements were performed using a portable radon detector and results, showing again good performance of the model. Results are discussed and future efforts are outlined for the refining and implementation of the model into software.

Cherenkov Radiation Detection on a LS Counter for 226Ra Determination in Water and Its Comparison with Other Common Methods.

Reliable determination of 226Ra content in drinking water, surface water and groundwater is required for radiological health-risk assessment of populations and radiation-dose calculations after ingestion and inhalation. This study aimed to determine 226Ra presence in the untreated water samples on a liquid scintillation counter via Cherenkov radiation detection. Cherenkov counting is a faster, simpler, less expensive technique than other commonly used methods for 226Ra determination. Step-by-step optimization of this technique on the Quantulus detector is presented in this paper. Improvement of detection limit/efficiency in the presence of sodium salicylate was investigated in this study. The main parameters of the method obtained were detection efficiency 15.87 (24)% and detection limit 0.415 Bq/L achieved for 1000 min of counting in 20 mL of sample volume. When 1 g of sodium salicylate was added, efficiency increased to 38.1 (5)%, with a reduction in the detection limit to 0.248 Bq/L for 500 min of counting. A satisfactory precision level of Cherenkov counting was obtained, the results deviating between 5% and 20% from reference values. The precision and accuracy of the Cherenkov counting technique were compared to liquid scintillation counting (EPA Method 913.0 for radon determination) and gamma spectrometry (the direct method for the untreated water samples on HPGe spectrometer). An overview of the advantages/disadvantages of each technique is elaborated in this paper.

Nuclear medicine staff exposure to ionising radiation in 18F-FDG PET/CT practice: a preliminary retrospective study.

This retrospective study provides an insight into the levels of radiation exposure of six nuclear medicine (NM) staff (four technologists and two nurses) performing routine diagnostic 18F-fluorodeoxyglucose (18F-FDG) positron emission tomography-computed tomography (PET/CT) at the University Clinical Centre of the Republic of Srpska, Department of Nuclear Medicine and Thyroid Disorders, Banja Luka, Bosnia and Herzegovina. Data analysis included monthly staff exposure measured with personal thermoluminescent dosimeters (TLD) between June and December 2018, quantified in terms of normalised dose for the whole body [Hp(10)] and dominant hand [Hp(0.07)] and their comparison between each staff member and between the two groups (technologists and nurses). The study goal was to establish how our Department compared with reports from other PET/CT centres worldwide in terms of annual number of procedures and exposure limits and whether there could be room for further improvements in radiation protection. The number of procedures rose considerably from 208 in 2016 to 876 in 2019 and was 423 in the observed seven-month period. Mean individual whole-body exposure dose per GBq of injected 18F-FDG activity, [Hp(10)/A] was 18.55 µSv/GBq for the four technologists and 15.61 µSv/GBq for the two nurses. Mean dominant-hand exposure dose per GBq of injected 18F-FDG activity [Hp(0.07)/A] was 16.99 µSv/GBq and 25.44 µSv/GBq for the two groups, respectively. The average annual cumulative dose for all staff was (1.06±0.29) mSv for Hp(10) and (1.15±0.32) mSv for Hp(0.07). These results are comparable with those of similar studies. Staff doses were well below the annual limits. Nurses received slightly higher extremity doses than technologists. In view of the increasing trends in the number of PET/CT procedures, dose monitoring should be continued to identify exposure hotspots and maintain doses as low as possible.

Radium interference during radon measurements in water: comparison of one- and two-phase liquid scintillation counting.

Assessment of radiation exposure to drinking, surface, and groundwater and of the associated health risks calls for accurate and precise 226Ra and 222Rn measurements. One method that fits the bill is liquid scintillation counting (LSC), which allows measurements in one-phase (homogenous) or two-phase samples. The aim of our study was to compare the measurement efficiency with both variations in Niska Banja spa water, known for its elevated 222Rn content to get a better insight into the stability and behaviour of the samples and 226Ra interference in samples spiked with 226Ra with 222Rn measurement. 226Ra interference was more evident in homogenous, one-phase and much lower in two-phase samples. However, one-phase samples offer more accurate indirect 226Ra measurements. Water-immiscible cocktails (in two-phase samples) have shown a limited capacity for receiving 222Rn generated by Ra decay from the aqueous to organic phase when 222Rn/226Ra equilibrium is reached. We have also learned that samples with naturally high 222Rn content should not be spiked with 226Ra activities higher than the ones found in native samples and that calibration of two-phase samples can be rather challenging if measurements span over longer time. Further research would require much lower 226Ra activities for spiking to provide more practical answers to questions arising from the demonstrated phenomena.

From Burden to Depressive Symptoms in Informal Caregivers during the COVID-19 Pandemic: A Path Analysis.

BACKGROUND: The objective of this study was to assess the complex relationship between the multiple determinants of the caregiving process, the caregiver burden, and depression during the COVID-19 pandemic in Serbia. METHODS: A cross-sectional study was conducted on a nationally representative sample (n = 798) during the COVID-19 pandemic in Serbia from March to September 2020. A nine-section questionnaire designed for this study included the characteristics of caregivers, characteristics of care and care recipients, COVID-19 related questions, and the following standardized instruments: 12-Item Short-Form Health Survey, Fatigue Severity Scale, Activities of Daily Living Scale and Instrumental Activities of Daily Living Scale, Zarit Caregiver Burden Scale, and Beck Depression Inventory. Path analysis was used for the simultaneous assessment of the direct and indirect relationships of all determinants. RESULTS: More than two thirds (71.9%) of informal caregivers experienced a burden, and more than one quarter (27.1%) had depression symptomatology. Self-rated physical health, need for psychosocial support, and caregiver burden were the main direct predictors of depression. Multiple determinants of the caregiving process had indirect effects on depressive symptomatology via the caregiver burden as a mediating factor. CONCLUSIONS: The subjective burden presented a significant risk factor for depressive symptoms in caregivers during the COVID-19 pandemic. The provision of psychosocial support was identified as an important opportunity to reduce depressive risk in informal caregivers.

Synthesis and Biological Screening of New 4-Hydroxycoumarin Derivatives and Their Palladium(II) Complexes.

Two newly synthesized 4-hydroxycoumarin bidentate ligands (L1 and L2) and their palladium(II) complexes (C1 and C2) were screened for their biological activities, in vitro and in vivo. Structures of new compounds were established based on elemental analysis, 1H NMR, 13C NMR, and IR spectroscopic techniques. The obtained compounds were tested for their antioxidative and cytotoxic activities and results pointed to selective antiradical activity of palladium(II) complexes towards •OH and -•OOH radicals and anti-ABTS (2,2'-Azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) cation radical) activity comparable to that of ascorbate. Results indicated the effect of C1 and C2 on the enzymatic activity of the antioxidative defense system. In vitro cytotoxicity assay performed on different carcinoma cell lines (HCT166, A375, and MIA PaCa-2), and one healthy fibroblast cell line (MRC-5) showed a cytotoxic effect of both C1 and C2, expressed as a decrease in carcinoma cells' viability, mostly by induction of apoptosis. In vivo toxicity tests performed on zebrafish embryos indicated different effects of C1 and C2, ranging from adverse developmental effect to no toxicity, depending on tested concentration. According to docking studies, both complexes (C1 and C2) showed better inhibitory activity in comparison to other palladium(II) complexes.

Scintillating and wavelength shifting effect investigation of 3-methylpiridinium salicylate and its application in LSC measurements.

Possible application of a novel ionic liquid 3-methylpiridinium salicylate (3-MPS) in Liquid Scintillation Counting measurements has been explored. Its addition to several radionuclides' aqueous solutions shown a significant influence on 210Pb Cherenkov spectra, 210Pb and 226Ra gross alpha/beta spectra, even on 3H spectra appearance. 3-MPS manifested both wavelength shifting and scintillating effect, indicating that 3-MPS or other ionic liquids of similar structure soon might be implemented into the common LSC practise as an alternative to the commercial LSC cocktails.

The possibility of the phosphogypsum use in the production of brick: Radiological and structural characterization.

In this paper, phosphogypsum (PG) with the content of 226Ra of about 500 Bq kg-1 was used as a clay additive in mass ratios of (0-40) % and its influence on the radiological and mineralogical characteristics of the obtained brick samples was monitored. After sintering the samples at 1000 â„ƒ, the formation of the mineral phase gehlenite was observed, and its share increased with the share of PG in the samples. The Monte Carlo method was used to determine the gamma dose rates, and consequently annual effective dose, for a standard room, with dimensions 4 × 5 × 2.8 m, whose walls were built of brick with PG. The obtained values were in the range (0.22-0.35) mSv y-1. In addition, the active device RAD7 was used to determine the radon surface exhalation rates from the samples, which were found to be in the range (63-150) mBq m-2 h-1. The estimated indoor radon concentrations were found to be drastically lower than 100 Bq m-3, leading to low radon inhalation doses. However, estimated annual effective doses from external gamma exposure were found not to be insignificant.

Quality of Life of Informal Caregivers behind the Scene of the COVID-19 Epidemic in Serbia.

Background and objectives: The COVID-19 pandemic has had an unprecedented reliance on informal caregivers as one of the pillars of healthcare systems. The aim of this study was to assess the quality of life of informal caregivers during the COVID-19 epidemic in Serbia. Materials and Methods: A cross-sectional study was conducted among informal caregivers during the COVID-19 epidemic in Serbia. Physical and mental quality of life was measured by the 36-Item Short-Form Health Survey. Additional data included sociodemographic characteristics, caregiver and care recipient characteristics, and COVID-19 related concerns. The qualitative component was performed using focus groups and individual in-depth interviews. Results: Out of 112 informal caregivers enrolled, most were female (80%), and the average age was 51.1 ± 12.3 years. The majority was delivering care to one person, who was a family member, on a daily basis (86.4%, 92%, and 91.1%, respectively). In multiple regression models, significant predictors of caregivers' physical health were delivering care to a family member and a higher level of care complexity, while significant predictors of caregivers' mental health were a higher level of care complexity and increased concerns about self-health and the health of the person being cared for due to the COVID-19 epidemic. Conclusions: Informal caregivers are experiencing negative physical and mental health outcomes during the COVID-19 epidemic in Serbia.

The arches of chaos in the Solar System.

Space manifolds act as the boundaries of dynamical channels enabling fast transportation into the inner- and outermost reaches of the Solar System. Besides being an important element in spacecraft navigation and mission design, these manifolds can also explain the apparent erratic nature of comets and their eventual demise. Here, we reveal a notable and hitherto undetected ornamental structure of manifolds, connected in a series of arches that spread from the asteroid belt to Uranus and beyond. The strongest manifolds are found to be linked to Jupiter and have a profound control on small bodies over a wide and previously unconsidered range of three-body energies. Orbits on these manifolds encounter Jupiter on rapid time scales, where they can be transformed into collisional or escaping trajectories, reaching Neptune's distance in a mere decade. All planets generate similar manifolds that permeate the Solar System, allowing fast transport throughout, a true celestial autobahn.

Heavy metals and radon content in spring water of Kosovo.

Paper presents results of researches carried out on various locations and immediate vicinity of mining and industrial activities of the northern and south-eastern part of Kosovo. Concentrations of As, Cd, Cr, Cu, Fe, Mn, Ni, Pb, Zn, Rn-222, as well as temperature and pH values of natural spring water were measured at 15 measuring sites (that belong to Zvecan, Leposavic and Novo Brdo municipalities), in April-May and September-October 2019. The quantification of heavy metals' content was performed by applying ICP-OES method. In analysed samples a high content of As, Pb, Fe and Ni was found. Carcinogenic and non-carcinogenic risks due to the content of heavy metals in water were evaluated. Concentration of radon in water was measured by the alpha spectrometric method, and measured values range in the interval from 0.34 ± 0.12 to 341 ± 35 Bq/L. The yearly doses of inhalation and ingestion were determined for the measured concentrations of radon. Mutual correlation by the Pearson correlation coefficient, principal component analysis, cluster analysis and spatial distribution analysis of the researched parameters of sampled water were done. The most expressed mutual dependence of some heavy metals leads to the conclusion that they have the same anthropogenic origin.

Evaluation of different LSC methods for 222Rn determination in waters.

Monitoring of 222Rn in drinking or surface waters, as well as in groundwater has been performed regularly in connection with geological, hydrogeological and hydrological surveys and health hazard studies. Liquid scintillation counting (LSC) is often preferred analytical method for 222Rn measurements in waters as it allows multiple-sample automatic analysis. LSC method implies mixing of water samples with organic scintillation cocktail, which triggers radon diffusion from the aqueous into an organic phase for which it has a much greater affinity, eliminating the possibility of radon emanation in that manner. The main aim of this paper is calibration of the liquid scintillation counter Qunatulus 1220™ for measuring of radon in water and evaluation of two different methods (one-phase and two-phase) in order to obtain the most suitable LSC technique for radon in water measurement. In this study four different scintillation cocktails were tested: one miscible (Ultima Gold AB) and three immiscible (High Efficiency Mineral Oil Scintillator, Opti-Fluor O and Ultima Gold F). Evaluation of presented methods was based on obtained detection efficiency and achieved Minimal Detectable Activity (MDA) values. Comparison of presented methods, accuracy and precision, as well as different scintillation cocktail's performance, was considered from results of measurements of 226Ra spiked water samples with known activity and environmental samples. LSC results were compared with the results of radon in water measurement obtained by alpha spectrometer RAD7. Calibration was done as a dependence of calibration factor (CF) from Pulse Shape Analysis (PSA). According to the obtained results, with proper adjustment of calibration parameters, both methods could be used for radon in water measurements. The obtained MDA values for all four scintillation cocktails are very low, less than 0.1 Bq l-1 for measuring time of 300 min.

Improved non-destructive method for 90Sr activity determination in aqueous solutions using Monte Carlo simulation.

The development of new methods and improvements of existing methods for the specific activity determination of 90Sr and other distinct beta emitters has been of considerable interest. The reason for this interest is that the notably small number of methods that are able to meet all the set criteria, such as reliability of the results, measurement uncertainty and time, and minimum production of radioactive waste, as well as applicability to various samples with reference to their nature, geometry and composition. In this paper, two methods for rapid 90Sr activity determination based on Monte Carlo simulations are used, one for a Si semiconductor detector for beta spectrometric measurements and the other for the Geiger-Muller (GM) ionization probe. To improve the reliability of the measurement results, samples with high and low strontium activity solutions were prepared in the form of dry residues. The results of the proposed methodology were verified with a standard method using a liquid scintillation counter, and notably good agreements are achieved.