Light Microscopy Research Articles

METAL DEBRIS RELEASE MAY BE UNDER-RECOGNIZED IN TOTAL ANKLE ARTHROPLASTY

Despite advancements, revision rates following total ankle replacement (TAR) are high in comparison to other total joint replacements. This explant analysis study aimed to investigate whether there was appreciable metal particulate debris release from various contemporary TARs by describing patterns of material loss. Twenty-eight explanted TARs (9 designs: 3 fixed and 6 mobile bearing), revised for any reason, were studied. The articulating surfaces of the metal tibial and talar components as well as the polyethylene insert were assessed for damage features using light microscopy. Based on the results of the microscopic analysis, scanning electron microscopy with energy dispersive X-ray spectroscopy was performed to determine the composition of embedded debris identified, as well as non-contacting 3D profilometry. Pitting, indicative of material loss, was identified on the articulating surfaces of 54% of tibial components and 96% of talar components. Bearing constraint was not found to be a factor, with similar proportions of fixed and mobile bearing metal components showing pitting. More cobalt-chromium than titanium alloy tibial components exhibited pitting (63% versus 20%). Significantly higher average surface roughness (Sa) values were measured for pitted areas in comparison to unpitted areas of these metal components (p<0.05). Additionally, metallic embedded debris (cobalt-chromium likely due to pitting of the tibial and talar components or titanium likely from loss of their porous coatings) was identified in 18% of polyethylene inserts. The presence of hard 3rd body particles was also indicated by macroscopically visible sliding plane scratching, identified on 79% of talar components. This explant analysis study demonstrates that metal debris is released from the articulating surfaces and the coatings of various contemporary TARs, both fixed and mobile bearing. These findings suggest that metal debris release in TARs may be an under-recognised issue that should be considered in the study of painful or failed TAR moving forwards.

HISTOPATHOLOGICAL FEATURES OF DOGS SKIN WITH ATOPIC DERMATITIS AFTER TREATMENT USING ECO ENZYME

Atopic dermatitis is one of the most common diseases in dogs that is multifactorial and usually attacks dogs aged between 6-36 months. This study was aimed to determine the histopathological appearance of the skin of dogs suffering from atopic dermatitis after treated with eco enzyme. Eco enzyme is a liquid product resulting from the fermentation of organic kitchen waste product such as fruit and vegetable dregs, molasses, and water. This study used five dogs suffering from atopic dermatitis which were divided into two groups. Group A: three dogs samples were bathed with 10% eco enzyme liquid every three days for 28 days. In Group B: two samples of dogs were bathed with 10% eco enzyme every three days until the 9th day, followed by a 2% eco enzyme bath once a week (13th, 20th, 27th day). Both treatment groups started from day 0 until day 28. Skin samples were taken using the biopsy method and histopathological changes were observed by making histopathological skin preparations stained with Hematoxylin and Eosin. Observations of the preparations were carried out using a light microscope. Data were analyzed using the Analysis of Variance test followed by the Duncan multiple range test and the Kruskall Wallis test followed by the Mann-Whitney test, then the data was explained descriptively. The results of the Anova test showed that the thickness of the dogs epidermis treated with 2% and 10% eco enzyme was significantly different (P<0.05). The Kruskall Wallis test for scoring inflammatory cell infiltration and degeneration showed that there was a significant difference (P<0.05) in the administration of 2% and 10% eco enzyme. Eco enzyme liquid is able to reduce atopic dermatitis lesions in dogs. Giving eco enzyme at a concentration of 10% and followed by a concentration of 2% with an extended application time can return the histological structure of the dog’s skin to normal, especially the thickness of the epidermis, reduce inflammatory cell infiltration, and reduce degeneration.

Size and Surface Properties of Functionalized Organosilica Particles Impact Cell-Particle Interactions Including Mitochondrial Activity.

Understanding of the interactions between macrophages and multifunctional nanoparticles is important for development of novel macrophage-based immunotherapies. Here, we investigated the effects of fluorescent thiol-organosilica particle size and surface properties on cell-particle interactions, including mitochondrial activity, using the mouse macrophage cell line J774A.1. Three different sizes of thiol-organosilica particles (150, 400, and 680 nm in diameter) containing fluorescein (OS/F150, OS/F400, and OS/F680) and particles surface functionalized with polyethylenimine (PEI) (OS/F150PEI, OS/F400PEI, and OS/F680PEI) were prepared. Flow cytometric analysis, time-lapse imaging, and single-cell analysis of particle uptake and mitochondrial activity of J774A.1 cells demonstrated variations in uptake and kinetics depending on the particle size and surface as well as on each individual cell. Cells treated with OS/F150 and OS/F150PEI showed higher uptake and mitochondrial activity than those treated with other particles. The interaction between endosomes and mitochondria was observed using 3D fluorescent imaging and was characterized by the involvement of iron transport into mitochondria by iron-containing proteins adsorbed on the particle surface. Scanning electron microscopy of the cells treated with the particles revealed alterations in cell membrane morphology, depending on particle size and surface. We performed correlative light and electron microscopy combined with time-lapse and 3D imaging to develop an integrated correlation analysis of particle uptake, mitochondrial activity, and cell membrane morphology in single macrophages. These cell-specific characteristics of macrophages against functional particles and their evaluation methods are crucial for understanding the immunological functions of individual macrophages and developing novel immunotherapies.

Astrocyte coverage of excitatory synapses correlates to measures of synapse structure and function in ferret primary visual cortex.

Most excitatory synapses in the mammalian brain are contacted or ensheathed by astrocyte processes, forming tripartite synapses. Astrocytes are thought to be critical regulators of the structural and functional dynamics of synapses. While the degree of synaptic coverage by astrocytes is known to vary across brain regions and animal species, the reason for and implications of this variability remains unknown. Further, how astrocyte coverage of synapses relates to in vivo functional properties of individual synapses has not been investigated. Here, we characterized astrocyte coverage of synapses of pyramidal neurons in the ferret visual cortex and, using correlative light and electron microscopy, examined their relationship to synaptic strength and sensory-evoked Ca2+ activity. Nearly, all synapses were contacted by astrocytes, and most were contacted along the axon-spine interface. Structurally, we found that the degree of synaptic astrocyte coverage directly scaled with synapse size and postsynaptic density complexity. Functionally, we found that the amount of astrocyte coverage scaled with how selectively a synapse responds to a particular visual stimulus and, at least for the largest synapses, scaled with the reliability of visual stimuli to evoke postsynaptic Ca2+ events. Our study shows astrocyte coverage is highly correlated with structural metrics of synaptic strength of excitatory synapses in the visual cortex and demonstrates a previously unknown relationship between astrocyte coverage and reliable sensory activation.

Assessing the impact of green and roasted coffee extracts on colorectal cancer cells in a 3D cell culture model

BackgroundColorectal cancer (CRC) is one of the most common and deadly malignant neoplasms worldwide. It is associated with multiple lifestyle risk factors such as poor diet, physical inactivity, alcohol and red meat consumption, smoking, and obesity. Consequently, studies have been conducted on the protective activity of bioactive compounds present in some foods against CRC, making it necessary to understand how diet can affect human health. In this sense, coffee, the second most consumed beverage in the world, is a major source of phytochemicals associated with a possible antitumor effect on CRC cells. PurposeThis study aimed to analyze the chemopreventive potential of green and roasted coffee extracts on HT-29 CRC cells cultured in 2D and 3D models. MethodsFlow cytometry was employed to quantify propidium iodide and DiOC6 uptake, ROS production, phosphatidylserine exposure, and cell cycle progression. In addition, cells and spheroids were analyzed by light microscopy to identify morphological changes. ResultsThe results showed that, under the employed conditions, treatment with both extracts had a concentration-dependent cytotoxic effect. Decreased cell viability was associated with mitochondrial membrane hyperpolarization, prooxidant effect, increased phosphatidylserine exposure, and changes in the G1 phase of the cell cycle. ConclusionFor the evaluated cell markers, 3D cultures required higher doses to achieve significant cytotoxic effects when compared to monolayer cultures. Since 3D cultures favor the formation of complex cell–cell interactions, this model offers a closer look at in vivo conditions for evaluating the toxicity and preclinical efficacy of potential chemopreventive agents for CRC. The experimental evidence from this study supports the 3R principles of replacement, reduction, and refinement for toxicological research in the pharmaceutical industry.

Phyto-Photodynamic Therapy of Prostate Cancer Cells Mediated by Yemenite 'Etrog' Leave Extracts.

Cancer therapy, from malignant tumor inhibition to cellular eradication treatment, remains a challenge, especially regarding reduced side effects and low energy consumption during treatment. Hence, phytochemicals as cytotoxic sensitizers or photosensitizers deserve special attention. The dark and photo-response of Yemenite 'Etrog' leaf extracts applied to prostate PC3 cancer cells is reported here. An XTT cell viability assay along with light microscope observations revealed pronounced cytotoxic activity of the extract for long exposure times of 72 h upon concentrations of 175 μg/mL and 87.5 μg/mL, while phototoxic effect was obtained even at low concentration of 10.93 μg/mL and a short introduction period of 1.5 h. For the longest time incubation of 72 h and for the highest extract concentration of 175 μg/mL, relative cell survival decreased by up to 60% (below the IC50). In combined phyto-photodynamic therapy, a reduction of 63% compared to unirradiated controls was obtained. The concentration of extract in cells versus the accumulation time was inversely related to fluorescence emission intensity readings. Extracellular ROS production was also shown. Based on an ATR-FTIR analysis of the powdered leaves and their liquid ethanolic extract, biochemical fingerprints of both polar and non-polar phyto-constituents were identified, thereby suggesting their implementation as phyto-medicine and phyto-photomedicine.

Patient-derived parotid gland pleomorphic adenoma organoid model and its preliminary histological and functional characterization

Objective: To establish patient-derived organoid models of pleomorphic adenomas (PA) of the parotid gland and preliminarily characterize their histology, related biomarkers and functions. Methods: Fresh tumor tissue specimens were collected from surgical procedures of Oral and Maxillofacial Department. The harvested tissues were processed and cultured in a head and neck tumor organoid culture system to establish organoid models from parotid gland pleomorphic adenomas. The in vitro growth of PA organoids was recorded by light microscopy. The successfully established organoids were passaged and cryopreserved, and the cryopreserved PA organoids were revived and re-cultured to observe their viability and organoid regeneration ability. Histological characterization, as well as characterization and detection of related markers and functional proteins, were performed on the organoids, comparing them with the patient-derived tissues. Results: The constructed organoid model of pleomorphic adenoma exhibited a dense and compact three-dimensional spherical structure. Hematoxylin and eosin staining indicated morphological similarities between the organoid and its tissue of origin. Immunohistochemistry showed positive cytoplasmic staining for Calponin, cytokeratin 7, and epithelial membrane antigen in both the organoid and the source tumor tissue, suggesting consistent histopathological characteristics between the organoid and its tissue of origin. Periodic acid-Schiff staining of the organoid showed positive staining for glycogen, with positive staining located in the interior and periphery of the organoid, indicating that the organoid possessed secretory functions like the salivary gland. Conclusions: This study successfully constructed organoids of pleomorphic adenoma derived from patient samples. This model faithfully replicates the tissue morphology and biomarkers of the source tissue and exhibits biological functions associated with mucus secretion. It serves as a valuable in vitro model for studying the development and progression of salivary gland tumors.

Mineralogical investigation of asbestos contamination of soil near old vermiculite processing plant in Honolulu, Hawaiʻi

From 1954 to 1983, a vermiculite processing facility operated near the Honolulu airport and processed raw material from the Libby, Montana mine, which is now well known for the high asbestos content of its clay deposits. The factory was closed in 1983 due to health hazard concerns, and remediation was performed in 2001 as part of the Libby mine superfund project. However, because of close proximity of the closed-down facility to residential areas of metropolitan Honolulu, some concerns remain regarding the possible environmental persistence of the harmful contaminant. To assess the dispersion of asbestos-contaminated vermiculite and explore the impact of trade winds on its distribution, air samples, and soil samples were collected from multiple locations near the former vermiculite plant. Polarized light microscopy was employed to identify elongated minerals, including potential asbestos. Quantitative mineralogical analysis utilizing X-ray powder diffraction and Rietveld refinement revealed an average content of approximately 7% vermiculite and 4% tremolite at the site. The asbestiform nature of tremolite was confirmed through X-ray micro-diffraction. Detailed analysis of airborne samples using transmission electron microscopy revealed no detectable levels of asbestos fibers in the vicinity of the former processing facilities, but the possibility of asbestos fibers becoming airborne due to mechanical disturbance during dry weather cannot be ruled out.

Zinc-alkaline phosphatase at sites of aortic calcification

Zinc (Zn) is a normal trace element in mineralizing tissues, but it is unclear whether it is primarily bound to the mineral phase or to organic molecules involved in the mineralization process, or both. Tissue-nonspecific alkaline phosphatase (TNAP) is a Zn metalloenzyme with two Zn ions bound to the M1 and M2 catalytic sites that functions to control the phosphate/pyrophosphate ratio during biomineralization. Here, we studied aortas from Tagln-Cre +/−; HprtALP/Y TNAP overexpressor (TNAP-OE) mice that develop severe calcification. Zn histochemistry was performed using the sulfide-silver staining method in combination with a Zn partial extraction procedure to localize mineral-bound (mineral Zn) and TNAP-bound Zn (tenacious Zn), since soluble Zn (loose Zn) is extracted during fixation of the specimens. Two synthetic bone mineral composites with different Zn content, bone ash, and rat epiphyseal growth plate cartilage were used as controls for Zn staining. In order to correlate the distribution of mineral and tenacious Zn with the presence of mineral deposits, the aortas were examined histologically in unstained and stained thin sections using various light microscopy techniques. Our results show that 14 and 30 dpn, TNAP is concentrated in the calcifying matrix and loses Zn as Ca2+ progressively displaces Zn2+ at the M1 and M2 metal sites. Thus, in addition to its catalytic role TNAP has an additional function at calcifying sites as a Ca-binding protein.Graphical

Material and Microstructure Analysis of Wood Color Paintings from Shaanxi Cangjie Temple, China.

Cangjie Temple was built to commemorate Cangjie, the legendary inventor of Chinese characters. It stands as one of the few remaining temples in China dedicated to the invention and creation of writing. In this study, the material properties of wooden paintings from the Cangjie temple were characterized using Polarized Light Microscopy (PLM), Scanning Electron Microscopy coupled with Energy Dispersive X-ray Spectroscopy (SEM-EDS), Micro-confocal Raman Spectroscopy, X-ray Diffraction (XRD), Fourier Transform Infrared Spectroscopy (FTIR), and Pyrolysis-Gas Chromatography-Mass Spectrometry (Py-GC/MS). It was confirmed that the pigments of the paintings included cinnabar, lapis lazuli, lead white, Paris green, and carbon black. The proteinaceous glue was used as an adhesive in the pigment samples, with tung oil likely being utilized as a primer for the wooden structures before painting. This study not only provides valuable data support for the conservation and restoration of the architectural features of Cangjie Temple but also provides useful reference for the maintenance and inheritance of similar ancient buildings.

Production of porous titanium structures by combining hot isostatic pressing and solid-state foaming

The Hot Isostatic Pressing (HIP) process is based on the combined action of high levels of pressure and temperature. In general, such a process is used for reducing or eliminating microporosities in the component, especially when it is produced by additive manufacturing (AM) or casting. In the present work HIP is used for compacting TI6Al4V-ELI powders by means of a pressurized Argon gas acting at high temperature on a sealed can under vacuum in which Argon is previously inflated; thus, a subsequent Solid-State Foaming (SSF) heat treatment allows to produce a Titanium foam without any melting by exploiting gas entrapment. Samples extracted from billets produced setting different HIP parameters (size of Titanium particles and pressures) have been investigated in this work by means of heat treatments in furnace for the SSF: the temperature was kept constant (1020 °C), but the duration was varied in the range 1 - 6 h; the samples were finally analysed by Light Microscopy. Finally, the Response Surface Method (RSM) was used to determine the conditions able to increase both the size and the percentage area of the pores in order to fully control both the involved processes (HIP and SSF). Experimental results revealed that the porosity determined by the SSF is strongly affected by HIP parameters and by the SSF duration: the highest dimension of Ti particles and the highest level of Argon pressure determined the largest values of porosity, in terms of both percentage and pore dimension. The investigated process for producing porous titanium structures can be properly and efficiently combined with manufacturing processes able to create highly customised parts, not only in terms of geometry but also in terms of porosity.

Development and anatomy of petals with specialized nectar holder and pollen container in Fumarioideae (Papaveraceae).

Petal developmental characteristics in Fumarioideae were similar at early stages, and the specialized nectar holder/pollen container formed by the outer/inner petals. The micro-morphology of these two structures, however, shows diversity in seven species. Elaborate petals have been modified to form different types, including petal lobes, ridges, protuberances, and spurs, each with specialized functions. Nectar holder and pollen container presumably have a function in plant-pollinator interactions. In Fumarioideae, four elaborate petals of the disymmetric/zygomorphic flower present architecture forming the "nectar holder" and "pollen container" structure at the bottom and top separately. In the present study, the petals of seven species in Fumarioideae were investigated by scanning electron microscopy, light microscope, and transmission electron microscopes. The results show that petal development could divided into six stages: initiation, enlargement, adaxial/abaxial differentiation, elaborate specializations (sacs, spurs, and lobes formed), extension, and maturation, while the specialized "nectar holder" and "pollen container" structures mainly formed in stage 4. "Nectar holder" is developed from the shallow sac/spur differentiated at the base of the outer petal, eventually forming a multi-organized complex structure, together with staminal nectaries (1-2) with individual sizes. A semi-closed ellipsoidal "pollen container" is developed from the apical part of the 3-lobed inner petals fused by middle lobes and attain different sizes. The adaxial epidermis cells are specialized, with more distinct punctate/dense columnar protrusions or wavy cuticles presented on obviously thickening cell walls. In addition, a large and well-developed cavity appears between the inner and outer epidermis of the petals. As an exception, Hypecoum erectum middle lobes present stamen mimicry. Elaborate petal structure is crucial for comprehending the petal diversity in Fumarioideae and provides more evidence for further exploration of the reproductive study in Papaveraceae.

Establishment and Characterization of a New Cell Line from Enzootic Nasal Adenocarcinoma in Goats: ENA-1.

Enzootic nasal adenocarcinoma (ENA) is a contagious tumor disease of goats and sheep, which is caused by enzootic nasal tumor virus (ENTV). To better understand the pathogenesis of ENA, this study aimed to establish a goat ENA cell line (ENA-1). The cells have been characterized with regard to morphology, growth rate, ultrastructural features, chromosome number, expression of CK7 and CK18, tumorigenicity, species, and mycoplasma contamination. ENA-1 had an epithelioid cell morphology with an unstable chromosome number under a light microscope. Under an electron microscope, the cell nuclear heterogeneity was not obvious, and there were more intermediate filaments and a small number of immature retrovirus-like particles in the cytoplasm. ENA-1 had strong proliferative potential, and the cell multiplication time was about 36 h, which could make BALB/c nude mice develop tumors. CK7 and CK18 were expressed in the cytoplasm of primary goat tumors, in transplanted tumors from nude mice, and un ENA-1 cells with the same intensity. PCR revealed that ENA-1 continuously carried ENTV-2 up to the 17th generation with no germline contamination or mycoplasma contamination. In conclusion, using a serum-containing culture system, ENA-1 cells were successfully isolated, cultured, and purified from goat tumor tissues. The isolated ENA-1 cells retained robust proliferation potential and maintained their phenotype, indicating the potential application of the ENA-1 cell line as an in vitro model of ENA.

Eutectic mixtures containing the active pharmaceutical ingredient ezetimibe: Phase diagrams, solid state characterization and dissolution profiles

Cardiovascular disease represents the leading cause of death worldwide, according to the World Health Organization (WHO) report. One of the treatment strategies is the use of the drug ezetimibe (EZT). This drug is a cholesterol absorption inhibitor with low aqueous solubility, which affects its therapeutic efficacy. The purpose of this paper was to obtain eutectic materials of EZT with methylparaben (MPB), salicylic acid (SCA) and nicotinamide (NTM) coformers, and to study their phase diagrams, dissolution properties and hygroscopicity to assess possible enhancements. Phase and Tamman diagrams were constructed using differential scanning calorimetry (DSC) results. The eutectic materials were characterized by powder X-ray diffraction (PXRD) and polarized light optical microscopy (OM). The hygroscopicity of the materials was assessed at different relative humidities (RH) through dynamic vapor sorption (DVS). The dissolution study was conducted with the materials in powder form, with paddle dissolution, rotating at 50 rpm, in 900 mL of aqueous buffer solution at pH 6.8 and 37 °C. The phase diagrams demonstrated that EZT formed eutectic systems with eutectic molar fractions of 0.59, 0.67 and 0.69 for EZT-SCA, EZT-NTM and EZT-MPB, respectively. The three eutectic materials are characterized as microcrystalline conglomerates of their components. In terms of the hygroscopic behavior of EZT at different RHs, eutectic materials demonstrated lower susceptibility to monohydrate formation compared to the anhydrous form. In dissolution studies, EZT-SCA and EZT-MPB solubilized EZT in amounts 22 % higher than the pure drug in 15 min. On the other hand, EZT-NTM eutectic suppressed the dissolution of EZT down to 56 % at the same time. The results indicated the possibility of obtaining materials with enhanced properties using eutectic mixtures, with improved dissolution rates and hygroscopic stability.

Morphology and ultrastructure of cicatricose spores found in an isolated sporangium-bearing structure of a schizaealean fern from the Lower Cretaceous rocks at Casal do Borracho, Torres Vedras, western Portugal

Fossil spores provide important insights into the evolutionary history of ferns, including their morphological changes and adaptations over time. The morphology and ultrastructure of trilete spores with cicatricose sculpture composed of sets of parallel or near-parallel ribs (muri) have been studied. Observations on the spores were performed using light microscopy, scanning electron microscopy, and transmission electron microscopy. The cicatricose spores were extracted from a single sporangium-bearing structure collected from Casal do Borracho mesofossil flora near Torres Vedras (Lusitanian Basin, western Portugal) from sediments belonging to the Almargem Formation, considered to be of late Barremian or early Aptian age (and possible younger than that). The sporangium-bearing structure of a schizaealean fern and in situ cicatricose spores are compared with the family Anemiaceae. On the basis of spore size and shape, laesura length, with about 20 proximal narrow ribs, oriented perpendicular to the spore sides, and three sets of distal ribs forming V-shaped patterns, the cicatricose spores in the present study are clearly assigned to the fossil-taxon Cicatricosisporites venustus. The spore ultrastructure includes a homogeneous exospore of a blechnoid type with a slight difference in the thickness in the proximal, distal, and equatorial regions. The morphology and ultrastructure of the studied spores are corresponded to Anemia type.

Influence of Ausforming on the Micro‐ and Nanostructure of PH 13‐8 Mo Maraging Steels

Herein, the potential of using the thermomechanical treatment ausforming to optimize the microstructure of PH 13‐8 Mo maraging steels is investigated. Of particular interest are the effects on the formation of β‐NiAl precipitates and reverted austenite during subsequent aging. Ausforming experiments at deformation temperatures of 400 and 600 °C and strains of 40% and 70% are conducted using a deformation dilatometer. Afterward, the samples are subjected to aging at temperatures varying from 482 to 593 °C. This is followed by a thorough microstructural characterization ranging from light optical microscopy to X‐Ray diffraction and atom probe tomography. The results demonstrate that ausforming effectively refines the microstructure, resulting in a significant increase in hardness. Moreover, it is revealed that the ausforming parameters have a noticeable influence on the number density of β‐NiAl precipitates and the amount of reverted austenite. A well‐balanced microstructure containing relatively fine precipitates and a high amount of reverted austenite can be achieved by ausforming at 600 °C with 40% strain. Overall, the results prove a promising potential of ausforming for PH 13‐8 Mo maraging steels with regard to tailoring optimized microstructures, which could ultimately benefit the aerospace industry where these alloys are used.

Application of confocal laser microscopy for identification of modern and fossil pollen grains, an example in palm Mauritiinae

Confocal scanning laser microscopy (CSLM) is becoming a powerful tool for palynological studies. CSLM allows palynomorph image sectioning, internal and surface structures visualization, and 3D reconstruction at a higher resolution than standard light microscopy without extra processing. CSLM images are suitable for several image analysis techniques that could help improve the accuracy and reproducibility of taxa identification. Here, using the palm subtribe Mauritiinae (Arecaceae: Calamoideae: Lepidocaryeae) as a model group, we identify modern and fossil pollen grains using CSLM images coupled with ImageJ/Fiji 1.54f plugins and machine learning statistical analyses. Modern taxa pollen grains including Lepidocaryum tenue Mart., Mauritia flexuosa L.f., Mauritiella armata (Mart.) Burret and Mauritiella aculeata (Kunth) Burret were obtained from Smithsonian Tropical Research Institute (STRI) pollen collection or herbarium exsiccates. Fossil pollen of Grimsdalea magnaclavata Germeraad et al. 1968, and Mauritiidites franciscoi (van der Hammen) van der Hammen & Garcia de Mutis 1966, both from Miocene, and Mauritia pollen type from Holocene were obtained from STRI collection. We measured nine shape and exine quantitative parameters, and one qualitative parameter (pollen aperture). Pollen volume was the most important variable (28.270 mean decrease accuracy), followed by pollen aperture (15.003), Skewness (13.466), and spine density (10.246). The machine learning analysis, which included CART and Random Forests, correctly identified both fossil and extant grains. CSLM and the quantitative analysis of morphological traits are a new frontier in palynological studies.

Red foxes (Vulpes vulpes) and raccoon dogs (Nyctereutes procyonoides) as potential spreaders of Sarcocystis species.

Sarcocystis includes a global group of apicomplexan parasites with two-host life cycle frequently circulating in wildlife and domestic hosts, including humans. Two of the most important wild terrestrial carnivores acting as definitive hosts are the red fox and raccoon dog, due to their wide distribution in Europe and usage of wild and farmed animals as prey. This study was conducted to determine the prevalence of Sarcocystis in hunted red foxes and raccoon dogs from nine regions of the Czech Republic and to identify isolated sporocysts by molecular techniques. Approximately 5 g of the contents of large intestine from 200 animals (197 red foxes and three raccoon dogs) were examined by flotation centrifugation coprological method. Only samples of 50 red foxes and one raccoon dog positive to Sarcocystis spp. were used for the nested PCR (nPCR) method to amplify a fragment or partial sequence on the cox1 gene. Ten species-specific primer pairs for detection of Sarcocystis spp. using farm animals as intermediate hosts were utilized. In total, 38.1% of the red foxes and 66.7% of the raccoon dogs were positive to Sarcocystis by light microscopy. The molecular characterization resulted in the identification of five species in the red fox: S. arieticanis, S. capracanis, S. cruzi, S. miescheriana, and S. tenella, while the PCR was negative for the sole raccoon dog. The highest intraspecific variation was found for S. miescheriana, while S. tenella was the most prevalent. Co-infections occurred in the large intestine of the red fox. No zoonotic species were found in our samples. This is the first study where the potential role of the red fox and raccoon dogs as spreaders of Sarcocystis to farm animals in the Czech Republic is shown. The use of species-specific primers provides a fast and easy method for screening multiple samples for a particular Sarcocystis species.

Morphological state of lungs of rats under the influence of Vipera berus berus venom

About 5.4 million snake bite cases are registered worldwide every year. About half of them cause 81,000-138,000 deaths or disabled 400,000 people. Despite this, this problem is neglected in many countries in Asia, Africa and Latin America. It is believed that death from snakebites is associated with poverty since the lack of proper access to medical facilities in such sections of the population causes the development of fatal complications. The purpose of the study is to study the morphological state of rats' lungs under exposure to Vipera berus berus venom. Experimental studies were carried out on white, non-linear male rats. Animals were conditionally divided into control and experimental groups, ten individuals each. Experimental rats were injected intraperitoneally with a semi-lethal dose (LD50) (1.576 mg/g-1) of Vipera berus berus venom in a physiological solution. Animals of the control group were injected intraperitoneally with only a physiological solution. Rats were removed from the experiment 24 hours after exposure to the poison and anaesthetized by cervical dislocation. Lung tissue samples were taken for microscopic examination. Fixation of the material and preparation of paraffin blocks were carried out according to generally accepted methods. Staining of histological preparations of the lungs was carried out with hematoxylin and eosin, according to Masson, and the PAS+"Hale" reaction was carried out according to the Mowry method. Histological preparations were studied using an SEO SСAN light microscope. The toxic effect of Vipera berus berus venom caused significant changes in the vascular, stromal and parenchymal components, which was confirmed by histological, histochemical and morphometric data. In the lungs of experimental animals, the vascular bed is primarily restructured, forming coagulopathies and thrombosis. The development of DIC syndrome and haemorrhages accompanies an increase in the permeability of the vessel wall. Inflammatory phenomena are found in the bronchi and respiratory departments' walls. There is a remodelling of the respiratory department components, with the formation of significant areas of dis- and atelectasis and zones of emphysema, which is confirmed morphometrically.

Delving into the diversity of genus Ironus Bastian, 1865 with special reference to Ironus dentifurcatus Argo & Heyns, 1972 (Ironidae: Enoplida), collected from coal mines

Summary In the exploration of nematode diversity within the coal mine spoil of Sonebhadra district, an isolate of Ironus dentifurcatus Argo & Heyns, 1972 was collected from the soil surrounding the roots of Prosopis juliflora. This study aims to unravel taxonomic intricacies of I. dentifurcatus through analysis of morphometric data, morphological characteristics utilising both light and scanning electron microscopy, and genetic scrutiny employing the SSU 18s rDNA gene marker. The study adds valuable information to the evolutionary history of I. dentifurcatus by constructing a comprehensive phylogenetic tree. This analysis is further augmented by an exploration of phylogeography and genetic divergence within the genus Ironus Bastian, 1865. The results reveal the genetic variability within the species of Ironus and the possible adaptative radiations in the group.