Naturally Occurring Chalcones with Aggregation-Induced Emission Enhancement Characteristics.

In this paper, the natural chalcones: 2'-hydroxy-4,4',6'-trimethoxychalcone (HCH), cardamonin (CA), xanthohumol (XN), isobavachalcone (IBC) and licochalcone A (LIC) are studied using spectroscopic techniques such as UV-vis, fluorescence spectroscopy, scanning electron microscopy (SEM) and single-crystal X-ray diffraction (XRD). For the first time, the spectroscopic and structural features of naturally occurring chalcones with varying numbers and positions of hydroxyl groups in rings A and B were investigated to prove the presence of the aggregation-induced emission enhancement (AIEE) effect. The fluorescence studies were carried out in the aggregate form in a solution and in a solid state. As to the results of spectroscopic analyses conducted in the solvent media, the selected mixtures (CH3OH:H2O and CH3OH:ethylene glycol), as well as the fluorescence quantum yield (ϕF) and SEM, confirmed that two of the tested chalcones (CA and HCH) exhibited effective AIEE behaviour. On the other hand, LIC showed a large fluorescence quantum yield and Stokes shift in the polar solvents and in the solid state. Moreover, all studied compounds were tested for their promising antioxidant activities via the utilisation of 1,1- diphenyl-2-picrylhydrazyl as a free-radical scavenging reagent as well as potential anti-neurodegenerative agents via their ability to act as acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE) inhibitors. Finally, the results demonstrated that licochalcone A, with the most desirable emission properties, showed the most effective antioxidant (DPPH IC50 29%) and neuroprotective properties (AChE IC50 23.41 ± 0.02 µM, BuChE IC50 42.28 ± 0.06 µM). The substitution pattern and the biological assay findings establish some relation between photophysical properties and biological activity that might apply in designing AIEE molecules with the specified characteristics for biological application.

Steinernema poinari (Nematoda: Steinernematidae): a new symbiotic host of entomopathogenic bacteria Xenorhabdus bovienii.

Three strains of symbiotic bacteria were isolated from an entomopathogenic nematode Steinernema poinari retrieved from soil in eastern Poland. Using 16S rDNA, recA, gltX, gyrB, and dnaN gene sequences for phylogenetic analysis, these strains were shown to belong to the species Xenorhabdus bovienii. The nucleotide identity between the studied S. poinari microsymbionts and other X. bovienii strains calculated for 16S rDNA and concatenated sequences of four protein-coding genes was 98.7-100% and 97.9-99.5%, respectively. The phenotypic properties of the isolates also supported their close phylogenetic relationship with X. bovienii. All three tested X. bovienii strains of different Steinernema clade origin supported the recovery of infective juveniles and subsequent development of the nematode population. However, the colonization degree of new infective juvenile generations was significantly affected by the bacterial host donor/recipient. The colonization degree of infective juveniles reared on bacterial symbionts deriving from a non-cognate clade of nematodes was extremely low, but proved the possible host-switching between non-related Steinernema species.

Studies on the removal of Cd ions by gastrointestinal lactobacilli.

Accumulation of toxic metal ions in food and water is nowadays a growing health-related problem. One detoxification method involves the use of microorganisms naturally inhabiting the gastrointestinal tract (GIT). The purpose of this study was to prove that lactic acid bacteria derived from the GIT are able to effectively remove Cd2+ from water solution. Seven strains of lactobacilli, out of 11 examined, showed tolerance to high concentrations of cadmium ions. The metal-removal efficiencies of these seven lactobacilli ranged from 6 to 138.4 µg/h mg. Among these bacteria, Lactobacillus gallinarum and Lactobacillus crispatus belonged to the highest (85%) Cd-removal efficiency class. An analysis of the zeta potential (ζ) indicated that the bacterial cell surface had a negative charge at the pH ranging from 3 to 10. The presence of carboxyl, amide, and phosphate groups was favorable for Cd2+ binding to the cell surface, which found confirmation in FTIR-ATR spectra. Elemental SEM/EDS analysis and TEM imaging not only confirmed the adsorption of Cd2+ on the cell envelope but also gave us a reason to suppose that Lb. crispatus accumulates metal ions inside the cell. Our findings open perspectives for further research on the new biological function of GIT lactobacilli as natural biosorbents.

The effect of cell surface components on adhesion ability of Lactobacillus rhamnosus.

The aim of this study was to analyze the cell envelope components and surface properties of two phenotypes of Lactobacillus rhamnosus isolated from the human gastrointestinal tract. The ability of the bacteria to adhere to human intestinal cells and to aggregate with other bacteria was determined. L. rhamnosus strains E/N and PEN differed with regard to the presence of exopolysaccharides (EPS) and specific surface proteins. Transmission electron microscopy showed differences in the structure of the outer cell surface of the strains tested. Bacterial surface properties were analyzed by Fourier transform infrared spectroscopy, fatty acid methyl esters and hydrophobicity assays. Aggregation capacity and adhesion of the tested strains to the human colon adenocarcinoma cell line HT29 was determined. The results indicated a high adhesion and aggregation ability of L. rhamnosus PEN, which possessed specific surface proteins, had a unique fatty acid content, and did not synthesize EPS. Adherence of L. rhamnosus was dependent on specific interactions and was promoted by surface proteins (42-114 kDa) and specific fatty acids. Polysaccharides likely hindered bacterial adhesion and aggregation by masking protein receptors. This study provides information on the cell envelope constituents of lactobacilli that influence bacterial aggregation and adhesion to intestinal cells. This knowledge will help to understand better their specific contribution in commensal-host interactions and adaptation to this ecological niche.

Variability of S-layer proteins in Lactobacillus helveticus strains.

The presence of S-layer proteins in the cell envelope of Lactobacillus helveticus may be technologically important. S-layer proteins are the adhesion site for cell envelope proteinase, which forms the proteolytic pathway in bacteria. Eleven strains of L. helveticus were examined for the presence of S-layer proteins and slpH genes. S-layer proteins from six strains were identified and sequenced. Multiple alignments of the deduced amino acid sequences demonstrated a strong sequence conservation of all Slp studied. Transmission Electron Microscopy analysis of the cells revealed the typical cell wall architecture of the S-layer. This is the first report on characterisation of glycosylated S-layer proteins from different strains of L. helveticus. The amino acid composition, the secondary structure, and the physical properties of these proteins were found to be quite similar to those of S-layer proteins from other lactobacilli. However, PCR analysis revealed that five of the examined strains of L. helveticus did not have slpH genes. This finding suggests that S-layer protein genes cannot be considered as housekeeping genes and cannot be used as molecular markers for L. helveticus.

Quinalizarin as a potential antifungal drug for the treatment of Candida albicans fungal infection in cancer patients.

This study aims to analyze the antifungal properties of quinalizarin, a plant-derived compound with proven anticancer effects. Quinalizarin exhibited antifungal activity against opportunistic pathogenic Candida species and Geotrichum capitatum. The treatment with this anthraquinone reduced hyphal growth, inhibited biofilm formation, and damaged mature Candida albicans biofilms. Real-time RT-PCR revealed that quinalizarin downregulated the expression of hyphae-related and biofilm-specific genes. The flow cytometry method used in the study showed that both apoptosis and necrosis were the physiological mechanisms of quinalizarin-induced C. albicans cell death, depending on the dose of the antifungal agent. A further study revealed an increase in the levels of intracellular reactive oxygen species and alterations in mitochondrial membrane potential after treatment with quinalizarin. Finally, quinalizarin was found to have low toxicity in a hemolytic test using human erythrocytes. In conclusion, we have identified quinalizarin as a potential antifungal compound.IMPORTANCEThis article is a study to determine the antifungal activity of quinalizarin (1,2,5,8-tetrahydroxyanthraquinone). Quinalizarin has potential antitumor properties and is effective in different types of tumor cells. The aim of the present study was to prove that quinalizarin can be used simultaneously in the treatment of cancer and in the treatment of intercurrent fungal infections. Quinalizarin was identified as a novel antifungal compound with low toxicity. These results may contribute to the development of a new drug with dual activity in the treatment of cancer-associated candidiasis.

Green Synthesis of Iron Nanoparticles Using an Aqueous Extract of Strawberry (Fragaria × ananassa Duchesne) Leaf Waste.

In this study, we analysed the potential use of dried strawberry leaves and calyces for the production of nanoparticles using inorganic iron compounds. We used the following iron precursors FeCl3 × 6H2O, FeCl2 × 4H2O, Fe(NO3)3 × 9H2O, Fe2(SO4)3 × H2O, FeSO4 × 7H2O, FeCl3 anhydrous. It was discovered that the content of polyphenols and flavonoids in dried strawberries and their antioxidant activity in DPPH and FRAP were 346.81 µM TE/1 g and 331.71 µM TE/1 g, respectively, and were similar to these of green tea extracts. Microimages made using TEM techniques allowed for the isolation of a few nanoparticles with dimensions ranging from tens of nanometres to several micrometres. The value of the electrokinetic potential in all samples was negative and ranged from -21,300 mV to -11,183 mV. XRF analyses confirmed the presence of iron ranging from 0.13% to 0.92% in the samples with a concentration of 0.01 mol/dm3. FT-IR spectra analyses showed bands characteristic of nanoparticles. In calorimetric measurements, no increase in temperature was observed in any of the tests during exposure to the electromagnetic field. In summary, using the extract from dried strawberry leaves and calyxes as a reagent, we can obtain iron nanoparticles with sizes dependent on the concentration of the precursor.

The modified activity of prolyl 4 hydroxylases reveals the effect of arabinogalactan proteins on changes in the cell wall during the tomato ripening process.

Arabinogalactan proteins (AGPs) are proteoglycans with an unusual molecular structure characterised by the presence of a protein part and carbohydrate chains. Their specific properties at different stages of the fruit ripening programme make AGPs unique markers of this process. An important function of AGPs is to co-form an amorphous extracellular matrix in the cell wall-plasma membrane continuum; thus, changes in the structure of these molecules can determine the presence and distribution of other components. The aim of the current work was to characterise the molecular structure and localisation of AGPs during the fruit ripening process in transgenic lines with silencing and overexpression of SlP4H3 genes (prolyl 4 hydroxylase 3). The objective was accomplished through comprehensive and comparative in situ and ex situ analyses of AGPs from the fruit of transgenic lines and wild-type plants at specific stages of ripening. The experiment showed that changes in prolyl 4 hydroxylases (P4H3) activity affected the content of AGPs and the progress in their modifications in the ongoing ripening process. The analysis of the transgenic lines confirmed the presence of AGPs with high molecular weights (120-60 kDa) at all the examined stages, but a changed pattern of the molecular features of AGPs was found in the last ripening stages, compared to WT. In addition to the AGP molecular changes, morphological modifications of fruit tissue and alterations in the spatio-temporal pattern of AGP distribution at the subcellular level were detected in the transgenic lines with the progression of the ripening process. The work highlights the impact of AGPs and their alterations on the fruit cell wall and changes in AGPs associated with the progression of the ripening process.

Inherently acidic films based on chitosan lactate-doped starches and pullulan as carries of nisin: A comparative study of controlled-release and antimicrobial properties.

Nisin (NIS) Z was incorporated (0.05 %, 0.1 %, 0.2 %) into edible films based on chitosan lactate (CHL) and 75/25 blends of polysaccharides (corn starch (CS), wheat starch (WS), oxidized potato starch (OPS), pullulan (PUL)) with CHL. The increase in the NIS/polymer ratio promoted the diffusion-driven release. Compared with the fully dissolvable CHL and PUL/CHL carriers, the starch/CHL films had limited solubility (≈27-37 %) and, consequently, ensured slower/incomplete release of NIS. The assayable NIS half-release times, determined in water, ranged from <1 min to ∼13 h. Probably due to the similar pH (≈4.5), there were generally no large differences between the antibacterial activities of the formulations. The NIS-supplemented systems limited the growth of some pathogens (B. cereus, L. monocytogenes, S. aureus), phytopathogens (P. carotovorum), and bacterial starter cultures. The NIS improved the UV-blocking ability of the films, but the 0.2 % NIS addition weakened (by ≈17-32 %) the tensile strength of most of the films.

Impact of the Body Composition on Knee Osteoarthritis Assessed Using Bioimpedance Analysis.

Osteoarthritis (OA) ranks among the most prevalent inflammatory diseases affecting the musculoskeletal system and is a leading cause of disability globally, impacting approximately 250 million individuals. This study aimed to assess the relationship between the severity of knee osteoarthritis (KOA) and body composition in postmenopausal women using bioimpedance analysis (BIA). The study included 58 postmenopausal females who were candidates for total knee arthroplasty. The control group consisted of 25 postmenopausal individuals with no degenerative knee joint changes. The anthropometric analysis encompassed the body mass index (BMI), mid-arm and mid-thigh circumferences (MAC and MTC), and triceps skinfold thickness (TSF). Functional performance was evaluated using the 30 s sit-to-stand test. During the BIA test, electrical parameters such as membrane potential, electrical resistance, capacitive reactance, impedance, and phase angle were measured. Additionally, body composition parameters, including Total Body Water (TBW), Extracellular Water (ECW), Intracellular Water (ICW), Body Cellular Mass (BCM), Extracellular Mass (ECM), Fat-Free Mass (FFM), and Fat Mass (FM), were examined. The study did not find any statistically significant differences in the electrical parameters between the control (0-1 grade on the K-L scale) and study groups (3-4 grade on the K-L scale). However, statistically significant differences were observed in BMI, fat mass (FM), arm circumference, triceps skinfold thickness, and sit-to-stand test results between the analyzed groups. In conclusion, the association between overweight and obesity with KOA in postmenopausal women appears to be primarily related to the level of adipose tissue and its metabolic activity.

Inhibitory effect of a combination of baicalein and quercetin flavonoids against Candida albicans strains isolated from the female reproductive system.

Flavonoids are a diverse group of compounds originating from several natural plant sources. Various biological effects of flavonoids have been reported, including antimicrobial and antifungal activities. In this study, we showed the possibility of using commercial flavonoids, i.e. baicalein and quercetin, based on natural equivalents as compounds with antifungal properties against Candida albicans species. The effects of baicalein and/or quercetin were investigated using reference C. albicans strain and 50 clinical strains isolated from vulvovaginal candidiasis (VVC) patients. Baicalein and quercetin MIC values against C. albicans strains ranged between 0.5 and 256 µg/ml. We observed predominantly indifferent, synergistic, or partially synergistic interactions between both flavonoids and between the flavonoids and fluconazole in the treatment of planktonic cells of the C. albicans strains. Treatment with the flavonoid complex inhibited adhesion and aggregation of cells, cell surface hydrophobicity (CSH), flocculation, biofilm formation and reduced hyphal growth. Real-time RT-PCR revealed that baicalein and quercetin in combination down-regulated the expression of biofilm-specific genes. Finally, we observed increase in the cell membrane permeability of C. albicans and its physical destruction as a result of the synergistic activity of baicalein and quercetin. Our research evidences the effectiveness of baicalein and quercetin applied in combination as potential anti-Candida agents.

Surface and Structural Studies of Age-Related Changes in Dental Enamel: An Animal Model.

In the animal kingdom, continuously erupting incisors provided an attractive model for studying the enamel matrix and mineral composition of teeth during development. Enamel, the hardest mineral tissue in the vertebrates, is a tissue sensitive to external conditions, reflecting various disturbances in its structure. The developing dental enamel was monitored in a series of incisor samples extending the first four weeks of postnatal life in the spiny mouse. The age-dependent changes in enamel surface morphology in the micrometre and nanometre-scale and a qualitative assessment of its mechanical features were examined by applying scanning electron microscopy (SEM) and atomic force microscopy (AFM). At the same time, structural studies using XRD and vibrational spectroscopy made it possible to assess crystallinity and carbonate content in enamel mineral composition. Finally, a model for predicting the maturation based on chemical composition and structural factors was constructed using artificial neural networks (ANNs). The research presented here can extend the existing knowledge by proposing a pattern of enamel development that could be used as a comparative material in environmental, nutritional, and pharmaceutical research.

Corn starch and methylcellulose edible films incorporated with fireweed (Chamaenerion angustifolium L.) extract: Comparison of physicochemical and antioxidant properties.

The properties of edible films derived from corn starch (CS) and methylcellulose (MC) supplemented with fireweed extract (FE; 0.0125-0.05% w/w) were analyzed. Due to their more crystalline structure, the MC films were significantly stronger (~26 MPa) than the CS films (~4 MPa). In turn, CS produced films with lower water vapor permeability (WVP, 50.12-51.74 vs. 56.52-59.10 g mm m-2 d-1 kPa-1). The hydrothermally-disrupted starch granules contributed to high roughness and opacity of the CS films. The FE-supplemented films exhibited an intensive yellow color and improved the UV-absorbing effect. FE delayed starch retrogradation, as indicated by the reduced crystallinity and slightly improved transparency of the CS films. Incorporation of FE significantly enhanced the released radical scavenging activity (RSA) of the films, while did not affect the WVP and mechanical properties. Due to better FE-trapping capacity, the CS-based films exhibited lower antioxidant activity (RSA60min = 2.21-19.75%) as compared to the MC counterparts (RSA60min = 4.87-38.31%).

Differences in Intestinal Barrier Development between Intrauterine Growth Restricted and Normal Birth Weight Piglets.

Intrauterine growth restricted (IUGR) piglets are born at term but have low birth mass and a characteristic shape of the head. Impaired general condition, especially in intestinal function, leads to an increase in the occurrence of diarrhoea and high mortality in the first days of life. So far, the mechanical and immunological gut barrier functions in IUGR are poorly understood. The aim of this study was to microscopically evaluate the early postnatal changes in the gut mucosa occurring in IUGR piglets. Whole-tissue small intestine samples were collected from littermate pairs (IUGR and normal) on postnatal day (PD) 7, 14 and 180 and analysed by light microscopy. We found that in the IUGR piglets, the percentage of intraepithelial leukocytes was reduced in the duodenum on PD 7, but it increased in the proximal and middle jejunum both on PD 7 and PD 14, which suggested the development of an inflammatory process. The number of goblet cells was also reduced on PD 14. The average size of the Peyer's patches in the distal jejunum and ileum showed significant reduction on PD 7 as compared to normal pigs; however, on PD 14, it returned to normal. On PD 180, we did not find any differences in the measured parameters between the IUGR and the normal pigs. In conclusion, we found that in one-week-old IUGR pig neonates, the gut barrier and the immune system structures display signs of retarded development but recover within the second postnatal week of life.

Properties of Arabinogalactan Proteins (AGPs) in Apple (Malus × Domestica) Fruit at Different Stages of Ripening.

Arabinogalactan proteins (AGPs) are constituents of the cell wall-plasma membrane continuum in fruit tissue. The aim of the study was to characterise AGPs contained in fruit by determination of their chemical structure and morphological properties. The results were obtained from in and ex situ investigations and a comparative analysis of AGPs present in Malus × domestica fruit at different stages of ripening from green fruit through the mature stage to over-ripening during fruit storage. The HPLC and colorimetric methods were used for analyses of the composition of monosaccharides and proteins in AGPs extracted from fruit. We have found that AGPs from fruit mainly consists of carbohydrate chains composed predominantly of arabinose, galactose, glucose, galacturonic acid, and xylose. The protein moiety accounts for 3.15-4.58%, which depends on the various phases of ripening. Taken together, our results show that the structural and morphological properties of AGPs and calcium concentration in AGPs are related to the progress of ripening, which is correlated with proper fruit cell wall assembly. In line with the existing knowledge, our data confirmed the typical carbohydrate composition of AGPs and may be the basis for studies regarding their presumed properties of binding calcium ions.

Oxalate oxidase from Abortiporus biennis - protein localisation and gene sequence analysis.

We have described for the first time the localisation of oxalate oxidase (OXO, EC 1.2.3.4) in Abortiporus biennis cells, using histochemical and immunochemical methods coupled with transmission electron microscopy. Rabbit anti-oxalate oxidase immunoglobulins with anti-rabbit secondary antibody conjugated with 10-nm gold particles were used. Moreover, the formation of electron dense precipitation of reaction of diaminobenzidine (DAB) with horseradish peroxidase (HRP) for histochemical localisation of the enzyme was found. OXO was localised close to the membranous structures of the cell membranes, in membranous vesicles located close to the outer cell membrane, and vacuolar membranes surrounding vacuoles. The positive immunoreaction to OXO was also intense in cell wall areas. Moreover, we proved that gene coding for OXO was expressed in the same cultures. Corresponding mRNA was isolated, full length cDNA was synthesized, cloned and sequenced. Two copies of cupin domains were found in the sequence of amino-acids conserved domain coding for the cupin enzyme. Comparison of the genomic DNA and cDNA sequences has revealed the presence of seventeen introns in the gene. The isoelectric point of the protein was estimated at pH 4.5 and several possible N-glycosylation sites were predicted.

Morphological and physiological changes in Lentilactobacillus hilgardii cells after cold plasma treatment.

Atmospheric cold plasma (ACP) inactivation of Lentilactobacillus hilgardii was investigated. Bacteria were exposed to ACP dielectric barrier discharge with helium and oxygen as working gases for 5, 10, and 15 min. The innovative approach in our work for evaluation of bacterial survival was the use in addition to the classical plate culture method also flow cytometry which allowed the cells to be sorted and revealed different physiological states after the plasma treatment. Results showed total inhibition of bacterial growth after 10-min of ACP exposure. However, the analysis of flow cytometry demonstrated the presence of 14.4% of active cells 77.5% of cells in the mid-active state and 8.1% of dead cells after 10 min. In addition, some of the cells in the mid-active state showed the ability to grow again on culture medium, thus confirming the hypothesis of induction of VBNC state in L .hilgardii cells by cold plasma. In turn, atomic force microscopy (AFM) which was used to study morphological changes in L. hilgardii after plasma treatment at particular physiological states (active, mid-active, dead), showed that the surface roughness of the mid-active cell (2.70 ± 0.75 nm) was similar to that of the control sample (2.04 ± 0.55 nm). The lack of considerable changes on the cell surface additionally explains the effective cell resuscitation. To the best of our knowledge, AFM was used for the first time in this work to analyze cells which have been sorted into subpopulations after cold plasma treatment and this is the first work indicating the induction of VBNC state in L. hilgardii cells after exposure to cold plasma.

Cremophor EL Nano-Emulsion Monomerizes Chlorophyll a in Water Medium.

In this paper, the application of a non-ionic detergent Cremophor EL for monomerization of chlorophyll a in an aqueous medium is studied. The spectrophotometric properties of chlorophyll a encapsulated into the Cremophor EL nano-emulsion system were characterized by electronic absorption, steady-state and time-resolved fluorescence as well as circular dichroism spectroscopy. The results have shown that chlorophyll a dissolves more efficiently in the aqueous medium containing low-level Cremophor (5 wt%) than at an ethanolic solution even in the concentration of 10-4 M. The molecular organization of the chlorophyll a in the Cremophor EL nano-micelles was also investigated by means of Raman spectroscopy. The spectral changes in the frequency of the C=O stretching group were used to distinguish the aggregation state of chlorophyll. It was revealed that chlorophyll a exists dominantly in the monomeric form in the Cremophor EL aqueous solution. The promising aspect of the use of Cremophor EL nano-emulsion as a delivery system is to maintain stable chlorophyll monomer in an aqueous medium. It would open the potential for a new, practical application of chlorophyll a in medicine, as a dietary supplement or studies on molecular organization of chlorophyll a in the well-defined artificial system.

Microsporidia Nosema spp. - obligate bee parasites are transmitted by air.

Microsporidia Nosema are transferred among bees via the faecal-oral route. Nosema spp. spores have been detected on flowers and transferred to hives along with the bee pollen. The aim of the present study was to determine whether Nosema microsporidia are transferred by air in an apiary, in a control area (without the presence of bee colonies), and/or in a laboratory during cage experiments with artificially infected bees. The novel way of transmission by air was investigated by the volumetric method using a Hirst-type aerobiological sampler located on the ground in the apiary, in the Botanical Garden and on the laboratory floor. Concurrently, the mean rate of Nosema infections in the foragers in the apiary was estimated with the Bürker haemocytometer method. Spore-trapping tapes were imaged by means of light microscopy, Nomarski interference contrast microscopy and scanning electron microscopy. The highest concentration of Nosema spores per 1m3 of air (4.65) was recorded in August, while the lowest concentration (2.89) was noted in July. This was confirmed by a Real-Time PCR analysis. The presence of N. apis as well as N. ceranae was detected in each of the tested tapes from the apiary. The average copy number of N. apis was estimated at 14.4 × 104 copies per 1 cm2 of the tape; whereas the number of N. ceranae was 2.24 × 104 copies per tape per 1 cm2. The results indicate that Nosema microsporidia were transferred by the wind in the apiary, but not in the Botanical Garden and laboratory by air. This was confirmed by genetic analyses. DNA from immobilised biological material was isolated and subjected to a PCR to detect the Nosema species. A fragment of the 16S rRNA gene, characteristic of Nosema apis and N. ceranae, was detected. Our research adds knowledge about the transfer of Nosema spp. microsporidia in the natural environment and indicates the season associated with the greatest risk of a bee colony infection with Nosema spp.

A simplified method of preparation of mammalian intestine samples for scanning electron microscopy.

Due to strong tissue hydration and complex architecture of the mucous membrane, appropriate preparation of inhomogeneous gastrointestinal tissues, especially from the intestine, for scanning electron microscopy is still a challenge and requires constant improvement of preparation techniques. In this article, we describe a simplified method of preparation of small intestinal mucosa tissues for observations in a scanning electron microscope. We emphasized the most important points in the preparation process that, when ignored, may result in formation of numerous artifacts and the inability to analyze the samples reliably. The developed technique facilitates proper animal tissue sampling in the field conditions, reducing the time of tissue collection and sample preparation as well as the total process costs. The fixative of choice, that is, buffered formalin, fixes, and stiffens the processed tissues properly, which is especially important in preservation of long, highly hydrated intestinal villi without shrinkage artifacts. The method described has been successfully used in comparative studies of the development of small intestines in mammals (pigs, mice, rats), reptiles, and birds (hens).