Light Microscopic Methods Research Articles

Interconnected idioblasts in Peltaea polymorpha: a novel component of the mucilage secretory apparatus in Malvaceae

Abstract The anatomical and cytological characteristics of the mucilage secretory system have been widely studied in Malvaceae. However, conflicting information regarding the morphological nature of secretory structures exists, and some remain poorly understood. In this sense, some secretory structures in Malvaceae are not characterized as typical isolated idioblasts, canals, or cavities. Here, we describe a novel component of the mucilage-secretory apparatus in the Malvaceae family. Samples of the shoot apex, mature stem, and fully expanded leaves were obtained from adult Peltaea polymorpha, which grow in the Cerrado (Brazilian savanna). The samples were processed using standard light and transmission electron microscopy methods. Mucilage cells occurred in the cortex and pith of petioles and stems, and in the midrib of leaves. These cells originate early in the stem apex from successive divisions of cells of the fundamental meristem, resulting in a row of interconnected secretory cells enveloped by a sheath of parenchyma cells devoid of secretory activity. Mucilage is stored in both protoplast and apoplast. In a same row, some cells filled with mucilage become very swollen and compress the neighboring idioblasts that become flattened. This phenomenon results in a sandwich panel structure consisting of the swollen transversal walls of adjacent cells. As the differentiation progresses, the transversal walls of the rowed mucilage cells became very swollen, multilayered, and porous. Cytoplasmic strands cross such transversal walls connecting rowed cells. Mucilage-secreting cells in P. polymorpha are interconnected idioblasts and represent a novel component of the mucilage-secretory apparatus in Malvaceae. These findings open new avenues for understanding the structure and dynamics of mucilage-secreting cells from a functional perspective.

Functional Morphology of the Bronchial Epithelium of Rats after Exposure to Chronic Low-Intensity γ Radiation

Revealing the reactions of cells and tissues to chronic low-intensity radiation exposure plays an important role in assessing the possible biological effects of man-made radiation effects on the body. The purpose of the study was to quantitatively study the microscopic structure and motor activity of the bronchial epithelium in rats under experimental exposure to chronic low-intensity ү-radiation. Materials and methods. Using the digital technology for intravital registration of motor activity indicators of the ciliary apparatus in combination with the methods of light and transmission electron microscopy, the main, lobular and segmental bronchi of 18 male Wistar rats subjected to experimental chronic gamma irradiation for 21 days (total absorbed doses – 5 cGy and 50 cGy) and 9 control animals were studied. In conditions of natural radiation background. The frequency was measured in the layer of the main types of epithelial cells and cells with Ki-67 expression, and the size characteristics of ciliated cells were determined as well. Research results. Chronic ү-irradiation in small doses causes a decrease in the frequency of cilia beating by 17–26% due to elongation of the recovery shock phase, an increase by 23–29% in the content of differentiated cell forms in the epithelium and a decrease in the frequency of basal (cambial) cells by 2–2.5 times. Electron microscopy revealed the emergence of cells with impaired ultrastructure, areas of intercellular edema, extrusion from the epithelial cell layer or their fragments. Irradiation at a dose of 50 cGy causes a decrease in the frequency of Ki-67 expressing epithelial cells by 3–3.8 times in all types of bronchi. In the subpopulation of basal cells, a pronounced dose-dependent inhibition of their proliferation in the main and lobar bronchi and complete blockade of cellular reproduction in segmental bronchi were found. Conclusions. Chronic low-intensity ү-radiation is a potential risk factor that can cause disturbance in the structure and function of the epithelial lining in the airways. Intravital assessing the frequency of cilia beating in combination with determining the content of Ki-67-positive epithelial cells can serve as an informative method for bioindicating the effects of low-dosed ionizing radiation on the body.

In vitro screening of potential echinochrome delivery systems for the treatment of eye diseases

An important task of topical application of medicines in the treatment of eyes is to achieve a compromise between their effectiveness and safety. The development of new multifunctional local ophthalmic drug delivery systems and in vitro screening of potential medicinal eye products are key areas in solving this problem. In this study, primary in vitro screening of the effect of echinochrome (Ech), the carrageenan complex of echinochrome (CRG/Ech) and its liposomal form (CRG/Ech-Lip) was performed on cultured epithelial cells of the outer shell of the eyeball: conjunctival epithelial cells (Chang Conjunctiva, Clone 1-5c-4) and corneal epithelium human (HCE). The cell viability was assessed by their morphology and metabolic activity using light microscopy and MTT test methods. The direct dependence of the intensity of the cytotoxic effect of Ech on its concentration in the nutrient medium, the form of use, the cellular test system and the incubation time of cells was revealed. Ech in the form of an alcoholic solution in its final concentration of 0.1 mg/ml of the nutrient medium exhibits pronounced cytoxicity against both cellular test systems. The same final concentration of Ech in the nutrient medium, but already as part of the carrageenan complex of echinochrome (CRG/Ech), turned out to be critical only for the viability of corneal epithelial cells, the survival rate of conjunctival cells under these conditions was about 50 %. A high biocompatibility of the liposomal form of the carrageenan complex of echinochrome (CRG/Ech-Lip) with cells of both test systems and a stimulating cytoprotective effect against the cells of the conjunctiva epithelium was revealed.

Morphology of lecithotrophic postlarvae of genus Austropallene (Arthropoda: Chelicerata) with some notes on reproductive strategy

The family Callipallenidae Hilton, 1942 belongs to the superfamily Nymphonoidea Pocock, 1904 together with other family, Nymphonidae. The lecithotrophic postlarvae hatch from the eggs of the callipallenid sea spiders, but the data on this life stage are very scarce and fragmentary. This gives a very limited understanding of larval anatomy, morphology, and diversity. The larvae of Austropallene bucera Pushkin, 1993, Austropallene calmani Gordon, 1944, and Austropallene cristata Bouvier, 1911 have been studied and described for the first time by the methods of light (LM) and scanning electron microscopy (SEM). The main morphometry parameters have been determined in larvae and adult egg-bearing males. The general plan of the postlarvae is presented together with its specific features. The postlarvae of the studied Austropallene species combine the features of lecithotrophic and free-living pycnogonid larvae. The diversity of larvae in the Nymphonoidea superfamily has been analysed considering original and published data, and a morphological series has been developed. The complex of lecithotrophic larvae, like postlarvae of Callipallenidae, should be considered as primary for the entire superfamily. It is also suggested that sea spiders with lecithotrophic larvae tend to follow the K-strategy, but they care for their offspring to varying degrees.

Hidden diversity in the Mallomonas pseudomatvienkoae species complex (Synurales, Chrysophyceae) and description of two new species

ABSTRACT The Mallomonas pseudomatvienkoae species complex was investigated in a tropical region using light and electron microscopy and molecular methods. Based on a study of 37 algal cultures belonging to the M. pseudomatvienkoae morphotype, three clades were identified based on three datasets (SSU rDNA, rbcL and ITS rDNA). For the first time in a tropical region (Vietnam), the presence of M. pseudomatvienkoae, originally described in South Korea, was confirmed by molecular methods. The original description contains data based only on scanning electron microscopic observations. Here, we provide a description of our strains based both on scanning and transmission electron microscopy. We propose two new species, M. pustula sp. nov. and M. kapustinii sp. nov. from Vietnam based on molecular characteristics and morphological analysis of the scales and bristles ultrastructure. The distribution of these three species was analysed using 37 strains and a metabarcoding approach. The most common species found in Vietnam was M. pustula found in 28 localities in nine provinces. Mallomonas pseudomatvienkoae was confirmed by molecular methods in 15 localities in 7 provinces. Mallomonas kapustinii was only found in six localities in three provinces. Our data suggest that M. pustula and M. pseudomatvienkoae are likely to be widespread in tropical regions.

Intestinal Parasites and Hematological Parameters in Children Living in Ambatoboeny District, Madagascar.

Madagascar is one of the poorest countries in the world. The country's extreme weather conditions, poor sanitation, and weak economy facilitate the spread of parasitic diseases. Infections with intestinal parasites are particularly dangerous for children because they can cause malnutrition and anemia, which, in turn, have a negative effect on children's cognitive functions and physical development. The aim of the present study was to analyze the prevalence of intestinal parasites and to assess hematological parameters in a group of children living in northern Madagascar. The screening was conducted in May 2024 in the Clinique Medicale Beyzym in Manerinerina, the Ambatoboeny district. It involved a sample of 208 children aged 0-17 years. Single stool samples were collected from all study participants. The samples were fixed in SAF fixative and then transported from Africa to Europe for further diagnostics at the Department of Epidemiology and Tropical Medicine, the Military Institute of Medicine-the National Research Institute in Poland. First, the samples were analyzed by light microscopy methods using three different diagnostic techniques (direct smear, decantation with distilled water, and the Fülleborn method). Next, they were tested by molecular biology methods (real-time PCR). Blood samples for the assessment of hematological parameters were collected at the healthcare center in Madagascar. The prevalence of intestinal parasites in the study sample was 61.5%. Helminths were found in 15.2% of the investigated children, and Giardia intestinalis (20.5%) was found to be the most prevalent parasite in the study population. Most infections were caused by potentially pathogenic stramenopila Blastocystis spp. (32.0%). Mean Hb, HCT, MCV, MCH, and MCHC levels in the study participants were below normal values. However, no correlation was found between the presence of a parasitic infection and low hematological parameters, which are a clinical sign of anemia. High rates of infections with intestinal parasites in children living in northern Madagascar support the necessity to introduce long-term preventive measures, which would limit the spread of parasitic diseases in the Malagasy population. Low hematological parameters in non-infected children may be indicative of persistent malnutrition or infection with other parasites, e.g., malaria or schistosomiasis.

A Novel Polarized Light Microscope for the Examination of Birefringent Crystals in Synovial Fluid

Background: The gold standard for crystal arthritis diagnosis relies on the identification of either monosodium urate (MSU) or calcium pyrophosphate (CPP) crystals in synovial fluid. With the goal of enhanced crystal detection, we adapted a standard compensated polarized light microscope (CPLM) with a polarized digital camera and multi-focal depth imaging capabilities to create digital images from synovial fluid mounted on microscope slides. Using this single-shot computational polarized light microscopy (SCPLM) method, we compared rates of crystal detection and raters’ preference for image. Methods: Microscope slides from patients with either CPP, MSU, or no crystals in synovial fluid were acquired using CPLM and SCPLM methodologies. Detection rate, sensitivity, and specificity were evaluated by presenting expert crystal raters with (randomly sorted) CPLM and SCPLM digital images, from FOV above clinical samples. For each FOV and each method, each rater was asked to identify crystal suspects and their level of certainty for each crystal suspect and crystal type (MSU vs. CPP). Results: For the 283 crystal suspects evaluated, SCPLM resulted in higher crystal detection rates than did CPLM, for both CPP (51%. vs. 28%) and MSU (78% vs. 46%) crystals. Similarly, sensitivity was greater for SCPLM for CPP (0.63 vs. 0.35) and MSU (0.88 vs. 0.52) without giving up much specificity resulting in higher AUC. Conclusions: Subjective and objective measures of greater detection and higher certainty were observed for SCPLM over CPLM, particularly for CPP crystals. The digital data associated with these images can ultimately be incorporated into an automated crystal detection system that provides a quantitative report on crystal count, size, and morphology.

THIN AND ULTRATHIN STRUCTURE OF ENVELOPES OF STEPHANOSTOMUM BACCATUM (TREMATODA: ACANTHOCOLPIDAE) METACERCARIAE IN YELLOWFIN SOLE, LIMANDA ASPERA.

The structure of the envelopes (capsule and cyst) surrounding metacercariae of Stephanostomum baccatum (Nicoll, 1907) in the second intermediate host, the yellowfin sole Limanda aspera (Pallas 1814), is examined with the methods of light and transmission electron microscopy. The cyst, presumably formed by secretions of the metacercarial tegument, consists of 2 layers: the outer, very thin layer of an electron-dense, finely granular substance and the inner layer composed of loose material of a moderate electron density that includes dense bodies varying in size, shape, and localization. The capsule, formed by the host's cells, is also organized into 2 distinct layers. The inner layer of the capsule is loose, consisting of evenly spaced debris of degenerated cells and lipid droplets with inclusions of intact macrophages between them. The outer layer of the capsule consists of parallel rows of cells arranged around the parasite, with fibroblasts and macrophages being dominant types and granulocytes and lymphocytes found in smaller numbers. Aggregations of collagen fibers are located in narrow spaces between the cells. The number of lipid droplets in the outer layer is significantly smaller than in the inner layer. The capsules formed around the examined trematodes have several structural features that distinguish them from those of S. baccatum and Stephanostomum sp. metacercariae recovered from other fishes of the family Pleuronectidae. The major morphological features of such capsules are the lack of epithelioid or giant multinucleated cells and the presence of numerous lipid droplets. Investigating the structural details of the envelopes surrounding metacercariae in trematodes, as well as other helminths, contributes to our scientific understanding of parasite biology, which can, in turn, have broader implications for understanding host-parasite interactions and evolutionary biology.

Mitochondrial Structure, Dynamics, and Physiology: Light Microscopy to Disentangle the Network.

Mitochondria serve as energetic and signaling hubs of the cell: This function results from the complex interplay between their structure, function, dynamics, interactions, and molecular organization. The ability to observe and quantify these properties often represents the puzzle piece critical for deciphering the mechanisms behind mitochondrial function and dysfunction. Fluorescence microscopy addresses this critical need and has become increasingly powerful with the advent of superresolution methods and context-sensitive fluorescent probes. In this review, we delve into advanced light microscopy methods and analyses for studying mitochondrial ultrastructure, dynamics, and physiology, and highlight notable discoveries they enabled.

Morphological aspects of Sticholecitha serpentis (Plagiorchiida: Plagiorchiidae) in a new Viperidae host in the Brazilian Amazon

Introduction: Sticholecitha serpentis is a poorly studied parasite of the snakes’ esophagus, with an unclear taxonomy. Currently, there are few records of S. serpentis in snakes in Brazil. Although it has been recorded in snakes of the family Viperidae, it has not yet been reported in Bothrops atrox. Objectives: To present new morphological characters, as well as ecological and parasitic relationships of S. serpentis with B. atrox. Methods: The samples were obtained in a dryland area in the Tapajós National Forest, Pará State, Brazil. Ten specimens of B. atrox were examined manually with the aid of tweezers, and three were positive for trematodes identified as S. serpentis in the oral cavity. Results: We provide scanning electron microscopy images, with detailed images of the S. serpentis tegument and its spines, as well as details of the dorsal keel using light microscopy methods. Moreover, we report the first record of S. serpentis parasitizing an Amazonian viperid, with infection frequency data of 30 %. Conclusion: We emphasize that the new morphological data presented in this manuscript provide additional information to facilitate the identification of this trematode in future studies.

Exploring the antibacterial efficacy of Satureja hortensis and Bacillus subtilis extracts in combating Xanthomonas citri

Biofilm plays a critical role in protecting the associated bacterial colonies. Citrus canker caused by Xanthomonas citri, resulted in remarkable yield reduction in citrus orchards. This study aims to evaluate the anti-biofilm properties of S. hortensis and B. subtilis extracts against X. citri. The antibacterial effects of both extracts were evaluated using the disk diffusion method and 96-well microdilution plates. Crystal violet and XTT procedures were employed to assess the inhibition of adhesion and antibiofilm effects of both extracts. The checkboard titration method was applied to determine the synergistic effects of the plant-bacterial extracts. The antibiofilm effects were confirmed by the light microscopy method. The results showed that the antibacterial and antibiofilm properties of both extracts varied. The MIC values of S. hortensis and B. subtilis extracts were 6.25 and 50 mg/ml, respectively. In these concentrations the inhibition of adhesion effects of S. hortensis and B. subtilis were 70% and 80%, whereas their antibiofilm effects were 60% and 72%. S. hortensis and B. subtilis extracts showed 63% and 76% antibiofilm activity, respectively, using the XTT test. The extracts showed synergistic effects, resulting in higher inhibition rates in combinatorial applications. Molecular docking results also confirmed that the secondary metabolites of S. hortensis could interact with different X. citri proteins differentially. Further large-scale studies on combinatorial antibiofilm effects of plant and bacterial extracts against citrus pathogens will provide new opportunities to develop safe and applicable reagents to control these destructive diseases.

Elucidating nanostructural organization and photonic properties of butterfly wing scales using hyperspectral microscopy.

Biophotonic nanostructures in butterfly wing scales remain fascinating examples of biological functional materials, with intriguing open questions with regard to formation and evolutionary function. One particularly interesting butterfly species, Erora opisena (Lycaenidae: Theclinae), develops wing scales that contain three-dimensional photonic crystals that closely resemble a single gyroid geometry. Unlike most other gyroid-forming butterflies, E. opisena develops discrete gyroid crystallites with a pronounced size gradient hinting at a developmental sequence frozen in time. Here, we present a novel application of a hyperspectral (wavelength-resolved) microscopy technique to investigate the ultrastructural organization of these gyroid crystallites in dry, adult wing scales. We show that reflectance corresponds to crystallite size, where larger crystallites reflect green wavelengths more intensely; this relationship could be used to infer size from the optical signal. We further successfully resolve the red-shifted reflectance signal from wing scales immersed in refractive index liquids with varying refractive index, including values similar to water or cytosol. Such photonic crystals with lower refractive index contrast may be similar to the hypothesized nanostructural forms in the developing butterfly scales. The ability to resolve these fainter signals hints at the potential of this facile light microscopy method for in vivo analysis of nanostructure formation in developing butterflies.

Microalgal Diversity and Molecular Ecology: A Comparative Study of Classical and Metagenomic Approaches in Ponds of the Eifel National Park, Germany

While molecular methods have begun to transform ecology, most algal biodiversity is still studied using the classical approach of identifying microalgae by light microscopy directly in sample material or using cultures. In this study, we compare both approaches (light microscopy and metagenomics as a molecular approach) using the freshwater ponds of the Eifel National Park in Germany as a case study. The ponds were found to be rich in desmids by light microscopy. A total of 299 species representing 81 genera were identified by light microscopy. While the molecular method does not currently allow species identification in most cases, we were able to identify 207 different algal genera. In total, 157 genera were detected only by metagenomics, 50 genera were found with both methods, and 31 genera were found by light microscopy, highlighting the need to continue using light microscopy in addition to a molecular approach. The metagenomics method has several advantages over the light microscopy method: (1) deeper assessment of alpha biodiversity, (2) better abundance numbers, and (3) complete coverage of all living matter. The latter is also a significant improvement over metabarcoding, as universal PCR primers are not available.

Root Anatomy: Preparing, Imaging, and Analyzing Maize Root Cross-Sections.

Root anatomy plays a critical structural and functional role in the maize root system, and regulates edaphic stress tolerance. The function and genetic basis of several maize root anatomical traits for stress tolerance have been demonstrated. Leveraging root anatomical traits in maize thus holds great potential for developing cultivars with greater nutrient and water efficiency. Key for such approaches is the ability to characterize the root anatomy of plants of interest. Here, we outline a systematic method for preparing, imaging, and analyzing maize root cross-sections. The protocol describes root sectioning (by hand or using a vibratome), preparation of microscope slides and toluidine blue staining, imaging under a light microscope, and both manual and semiautomated methods for anatomical feature extraction from images. The protocol enables the visualization and quantification of various anatomical tissues and traits, and its simplicity, adaptability, and accessibility make it an ideal choice for both small- and large-scale phenotyping studies in maize and other plant species. This standardized protocol provides researchers with a comprehensive methodology to accurately dissect root structures, enabling in-depth analyses that are essential for understanding plant growth, development, and adaptive value for stress tolerance.

Exogenous Sodium Nitroprusside Affects the Redox System of Wheat Roots Differentially Regulating the Activity of Antioxidant Enzymes under Short-Time Osmotic Stress.

Nitric oxide (NO) is a multifunctional signalling molecule involved in the regulation of plant ontogenesis and adaptation to different adverse environmental factors, in particular to osmotic stress. Understanding NO-induced plant protection is important for the improvement of plant stress tolerance and crop productivity under global climate changes. The root system is crucial for plant survival in a changeable environment. Damages that it experiences under water deficit conditions during the initial developmental periods seriously affect the viability of the plants. This work was devoted to the comparative analysis of the pretreatment of wheat seedlings through the root system with NO donor sodium nitroprusside (SNP) for 24 h on various parameters of redox homeostasis under exposure to osmotic stress (PEG 6000, 12%) over 0.5-24 h. The active and exhausted solutions of SNP, termed as (SNP/+NO) and (SNP/-NO), respectively, were used in this work at a concentration of 2 × 10-4 M. Using biochemistry and light microscopy methods, it has been revealed that osmotic stress caused oxidative damages and the disruption of membrane cell structures in wheat roots. PEG exposure increased the production of superoxide (O2•-), hydrogen peroxide (H2O2), malondialdehyde (MDA), and the levels of electrolyte leakage (EL) and lipid peroxidation (LPO). Stress treatment enhanced the activities of superoxide dismutase (SOD), ascorbate peroxidase (APX), catalase (CAT), the excretion of proline, and the rate of cell death and inhibited their division. Pretreatment with (SNP/+NO) decreased PEG-induced root damages by differently regulating the antioxidant enzymes under stress conditions. Thus, (SNP/+NO) pretreatment led to SOD, APX, and CAT inhibition during the first 4 h of stress and stimulated their activity after 24 h of PEG exposure when compared to SNP-untreated or (SNP/-NO)-pretreated and stress-subjected plants. Osmotic stress triggered the intense excretion of proline by roots into the external medium. Pretreatment with (SNP/+NO) in contrast with (SNP/-NO) additionally increased stress-induced proline excretion. Our results indicate that NO is able to mitigate the destructive effects of osmotic stress on the roots of wheat seedlings. However, the mechanisms of NO protective action may be different at certain periods of stress exposure.

Risk of thromboembolism after intraosseous implantation of metallic devices with extracellular vesicles derived from multipotent stromal cells: preliminary results

Background. New implantation methods are of great importance due to the development of endoprostheses in traumatology and orthopedics, restorative medicine and dentistry. Equally important is the early detection and description of the implant-associated complications. The aim of the study is to find and describe thrombi and emboli in the heart and lungs formed after experimental implantation of metallic devices in the peripheral part of limb using extracellular vesicles of mesenchymal stromal cells. Methods. Outbred rabbits of both genders at the age from 4 to 6 months and of weight from 3 to 4 kg underwent experimental implantation. The study enrolled 57 species in total. They were divided into two groups: 30 animals underwent implantation of metallic devices using extracellular vesicles of mesenchymal stromal cells (EV MSCs), 27 — without their use. The rabbits’ hearts and lungs were studied by light microscopy methods at different stages after integration of screw titanium implants into the proximal condyle of the tibia using EV MSCs. Results. After implantation of metallic devices into the proximal condyle of the tibia, we detected fibrin, detritus and even the red bone marrow structures (various blast forms of hematopoietic cells: megakaryocytes, cells of the erythroid and myeloid lineages) in the right cavities of the heart. In the pulmonary arteries, we also found thrombi and emboli, which either led to the obliteration of the involved vessel or to gradual lysis, not disappearing completely within 10 days of follow-up. Conclusions. After intraosseous implantation of the metallic devices, there is an embolism risk in the right atria and ventricle of the heart and the pulmonary arteries and veins due to the debris migration with the bloodstream from the surgery site. At the same time, one cannot exclude a thrombotic risk in the heart and pulmonary arteries as a reaction to the presence of detritus. It is advisable to take measures aimed at preventing both debris releasing into the bloodstream and pulmonary embolism during any implantations into the bone tissues, even of relatively small devices. Using EV MSCs to affect the implant engraftment processes has no significant effect on the severity and frequency of thromboembolic complications.

Endohelminth diversity in the invasive Clarias gariepinus (Burchell, 1822) from two freshwater lakes: Naivasha and Ol'Bolossat, Kenya.

Fish parasitology contributes to our understanding of the potential risks posed by diverse groups of parasitic organisms on fish stocks in either wild and culture systems. This study was conducted in May 2023 and aimed at assessing the diversity of endohelminths in the invasive North African catfish Clarias gariepinus (Burchell, 1822) obtained from two freshwater lakes, Naivasha and Ol'Bolossat, in Kenya. Parasitological examination of 66 and 35 fish samples collected from the two lakes respectively was achieved using light and scanning electron microscopy methods. Results revealed endohelminth diversity broadly classified as four digeneans, two nematodes, and one cestode. Seven taxa of endohelminths were found in C. gariepinus samples, but only four of these taxa could be identified up to the species level. Six of the taxa (Diplostomum sp., Tylodelphys mashonense, Plagiorchioidea sp., Paracamallanus cyathopharynx, Contracaecum sp., and Tetracampos ciliotheca) were common in samples from the two lakes. Glossidium pedatum only occurred in samples from Lake Ol'Bolossat. Parasite prevalence ranged from 8.6 (T. mashonense) to 100% (Diplostomum sp., T. ciliotheca, and Contracaecum sp.) and mean intensity from 1.4 (T. mashonense) to 16.9 (Diplostomum sp.). The diversity and richness indices were comparatively higher in fish samples from Lake Ol'Bolossat and attributed to the occurrence of G. pedatum in the Ol'Bolossat. However, parasitic infestation of fish samples from the two lakes depicted close similarity, both in diversity and prevalence. These findings form an important baseline data for further follow-up studies, and they suggest the need for further molecular analyses to fully describe three of the taxa only identified up to the genus level.

Rapid detection of salmon louse larvae in seawater based on machine learning

The proliferation of salmon lice is one of the most challenging problems in salmon farming, seriously affecting the welfare of both farmed and wild salmonids. The detection of salmon lice in seawater, especially in the (pre-) infectious stage, supports the accurate management of salmon lice proliferation. Methods for detecting salmon lice in seawater to date involve traditional light and fluorescence microscopy and Polymerase Chain Reaction (PCR) methods. However, these methods require sample pre-processing and are laborious and time-consuming, rendering them difficult to apply for the task of continuous, rapid salmon lice detection in large water volumes. This study aims to develop a rapid and easy-to-use method for the detection of salmon lice in seawater. A novel approach was introduced for the detection of salmon lice at different larval stages in seawater by leveraging machine learning. A setup for the rapid acquisition of images of salmon lice in seawater was developed, and a dataset comprising 8316 images encompassing salmon lice “Nauplius Live,” “Nauplius Shell” and “Copepodite” was created. Then 16 object detection models including one- and two-stage Convolutional Neural Network (CNN-) based models and Transformer-based models were trained, tested, and compared on this comprehensive dataset. RT-DETR showed the best performance among all models tested with a precision of 0.972 and a recall of 0.978. The YOLO series models, except for YOLOv3, showcased remarkable inference speeds (ranging from 152 to 416 frames per second) while maintaining high precision (ranging from 0.968 to 0.977) and recall (ranging from 0.964 to 0.970). To our knowledge, this is the first time machine learning has been applied to the task of rapidly detecting salmon louse larvae at different stages in seawater samples. The results demonstrate that the machine learning approaches can achieve rapid easy-to-use and cost-effective detection of salmon lice in seawater. In the future, this study has the potential for application in measuring salmon louse larvae numbers in aquatic environments, supporting the enhancement of the welfare of both farmed and wild salmon.

Iron deposition in gastric black spots: Clinicopathological insights and NanoSuit-correlative light and electron microscopy analysis.

Black spots (BSs) are lentiginous findings observed in the gastric body and fundus during upper gastrointestinal endoscopy and are predominantly seen in patients undergoing Helicobacter pylori eradication treatment. However, the detailed patient background and exact composition are poorly understood. This study aims to clarify the clinicopathological features of BSs, examine patient demographics, and use the NanoSuit-correlative light and electron microscopy (CLEM) method combined with scanning electron microscopy-energy dispersive X-ray spectroscopy for elemental analysis. Patients who underwent upper gastrointestinal endoscopy between 2017 and 2022 were included. Data on age, medications, blood tests, and H. pylori infection status were retrospectively gathered from medical records. Univariate analysis was conducted to examine BS presence, with results then used in a multivariate model to identify associated risk factors. Additionally, pathological specimens from patients with BSs were analyzed for elemental composition using the NanoSuit-CLEM method combined with scanning electronmicroscopy-energy dispersive X-ray spectroscopy. An analysis of 6778 cases identified risk factors for BSs, including older age and using proton pump inhibitors, statins, corticosteroids, and antithrombotic drugs. Endoscopically, BSs correlated with higher gastric atrophy and lower active H. pylori infection. Iron deposition at BS sites was specifically identified using NanoSuit-CLEM. BSs on gastrointestinal endoscopy may indicate an absence of active H. pylori inflammation. The discovery of iron deposition within BSs using the NanoSuit-CLEM method has offered new insights into the possible causative factors and advances our understanding of the etiology of BSs, bringing us closer to unraveling the underlying mechanisms of their formation.

Effect of composite collagen containing hydrogel on the functional activity of fibroblasts after acid-induced <i>in vitro</i> injury

It is well known that the acidity of the microenvironment has a significant impact on the functional activity of cells involved in tissue repair. To date, the effect of biomaterials containing components of the human dermis intercellular matrix on fibroblasts under acidosis has not been fully studied.The aim of the study. To evaluate the effect of a composite hydrogel containing dermal components on the functional activity of intact fibroblasts and fibroblasts with acid-induced dysfunction.Materials and methods. To simulate the physicochemical conditions of acute inflammation, human fibroblasts were incubated for an hour at 39 °C in a nutrient medium overacidified to pH = 6. The morphometric characteristics of human skin fibroblasts were assessed using light and fluorescence microscopy methods. The number of apoptotic cells and proliferation index were assessed using flow cytometry.Results. It has been established that exposure of fibroblasts to an acidic medium inhibits their adhesive properties and reduces the rate of cell growth. The introduction of hydrogel into a cell suspension with damaged cells restores adhesive properties and cell growth. In the fibroblast population, after acid exposure, the number of living cells decreases, and the number of apoptotic cells increases. Cultivation of damaged fibroblasts in the presence of a composite hydrogel increases the number of living cells in the population and reduces the number of apoptotic cells. Acid-induced fibroblast damage reduces the proliferative activity index. It was revealed that the introduction of hydrogel into the culture medium stimulates the proliferative activity of both intact and damaged fibroblasts. The results obtained indicate that a composite hydrogel consisting of extracellular matrix components is capable of restoring the functional activity of fibroblasts damaged as a result of exposure to an acidic medium. The results obtained can be used to create biomaterials that increase the efficiency of skin regeneration in cases of excessively intense acute inflammation.