Ultrastructural Organization Research Articles

Nothobranchius as a model for anorexia of aging research: an evolutionary, anatomical, histological, immunohistochemical, and molecular study

BackgroundAnorexia of aging, defined as a decrease in appetite and a preponderant loss of body weight occurring in late life, is one of the most common diseases affecting older people.The peptide hormone cholecystokinin (Cck) is known to play a key role in regulating food intake and satiety in higher vertebrates. In humans as well as in rats, an increased concentration of Cck was described as the basis of appetite loss in elderly. However, the role of increased plasma Cck concentrations in mediating the age-related decrease in appetite remains to be established.Although in vitro studies are an excellent resource for investigating aging, the use of a model organism that shares and imitates the human physiological processes guarantees a better understanding of the in vivo mechanisms.African annual fishes from the genus Nothobranchius are emerging as a prominent model organism in biogerontology and developmental biology due to their short captive lifespan.Therefore, in the current study, we aimed to investigate the possibility of using the genus Nothobranchius to model the anorexia of aging and their potential contribution to better understanding the pathway by which Cck induce appetite loss in older people providing a comparative/evolutionary localization of the current study model among the aging canonicals models, the morphology of its gastrointestinal tract and its Cck expression pattern. MethodsThe comparative/evolutionary investigation was conducted using the NCBI blastp (protein-protein BLAST) and NCBI Tree Viewer. The macroscopic morphology, histological features, ultrastructural organization of Nothobranchius rachovii gastrointestinal tract were investigated using stereomicroscope, Masson's trichrome and alcian blue–PAS staining, and transmission electron microscopy, respectively.The cck expression pattern was studied through immunofluorescence labeling, western blotting, and quantitative RT-PCR. ResultsThe intestine was folded into different segments divided into an anterior intestine made of a rostral intestinal bulb and an intestinal annex of lower diameter, mid and posterior intestine. The gradual transition from the rostral intestinal bulb to the posterior intestine sections's epithelium is characterized by a gradual reduction in the striated muscular bundles, villi height, and goblet mucous cells count. The lining epithelium of the intestinal villi was characterized by a typical brush border enterocytes full of mitochondria. Moreover, Cck expression was detected in scattered intraepithelial cells concentrated in the anterior tract of the intestine. ConclusionsOur study introduces Nothobranchius rachovii as a model for anorexia of aging, giving the first bases on the gastrointestinal tract morphology and cck expression pattern. Future studies on young and elderly Notobranchius can divulge the contribution of cck in the mechanisms of anorexia associated with aging.

A human septin octamer complex sensitive to membrane curvature drives membrane deformation with a specific mesh-like organization.

Septins are cytoskeletal proteins interacting with the inner plasma membrane and other cytoskeletal partners. Being key in membrane remodeling processes, they often localize at specific micrometric curvatures. To analyze the behavior of human septins at the membrane and decouple their role from other partners, we used a combination of bottom-up in vitro methods. We assayed their ultrastructural organization, their curvature sensitivity, as well as their role in membrane reshaping. On membranes, human septins organize into a two-layered mesh of orthogonal filaments, instead of generating parallel sheets of filaments observed for budding yeast septins. This peculiar mesh organization is sensitive to micrometric curvature and drives membrane reshaping as well. The observed membrane deformations together with the filamentous organization are recapitulated in a coarse-grained computed simulation to understand their mechanisms. Our results highlight the specific organization and behavior of animal septins at the membrane as opposed to those of fungal proteins.

Influence of protein-vitamin mineral supplements on the splenic morphometric parameters of quails

Currently, a growing industrial poultry sphere in many countries is quail farming. Quails have a complex of unique economic-productive advantages, as compared with other poultry, – high body temperature, intensive metabolism, small size, fast maturation, and high egg productivity. In the study, we examined the specifics of microscopic structure of morphometry of the spleen of the control and experimental birds as characteristics sensitive to the action of supplements. The proposed methods are intended for studying effects of protein-vitamin mineral supplements on poultry farming. Therefore, for 21 days, with the main diet, the quails were given mineral supplements produced by the Multilife trade mark (crude protein, amino acids (lysine, threonine, methionine, cysteine), calcium, available phosphorus, sodium, chlorine, vitamins (A, D3, E)), by the Missi trademark (protein, vitamins, minerals), and by the Standard Agro trademark (soybean press cake, vitamins А, D3, Е, В1, В2, В6, В12), multi-enzyme complex, salt, ground limestone, phosphate monocalcium, amino acids (lysine, threonine, methionine with cysteine). This is the first study of the ultrastructural organization of the spleen of Japanese quail (Coturnix japonica) fed with protein-vitamin mineral supplements. The tested supplements were beneficial for the productivity of the quails, as evidenced by increase in the body and spleen weight. Body weight of the quails increased by 5.8% in the group that was fed additionally with the Multilife supplement, and by 12.5% in the group that consumed the Missi supplement added to the fodder, and by 8.1% in the group that consumed the Standard Agro supplement with the diet. At the same time, the spleen weight increased by 7.2%, 16.0%, and 10.5%, respectively. However, the relative weight (0.1%) underwent no changes, which characterizes this parameter as permanent for age and species of animals. Analysis of the data of our morphological studies revealed that the splenic microscopic structure of the quails was characterized by presence of support-contractile apparatus (capsule and trabeculae) with the relative area of 4.6 ± 0.8% and single radial trabecula. The parenchyma was formed by white and red pulps without distinct boundaries. In the white pulp, there were designated lymphoid nodes with no light centers, and also periarterial lymphoid sheaths and ellipsoids. In the reticular stroma of the white pulp of the spleen, we found blood cells of various maturity, in particular, lymphocytes, which were clearly divided into large, average, and small; mononuclear and multinuclear macrophages, which contained hemosiderin and melanin, leukocytes, mostly eosinophilous. The ultrastructural organization of the spleen of the quails had the following peculiarities: nuclei of leukocytes contained large nuclei, endotheliocytes of the central artery of lymphoid follicle performed phagocyte function by accumulating autophagosomes in its cytoplasm. In the quails that had consumed the diet containing supplements, the macroscopic, microscopic, and ultramicroscopic structures underwent no changes. At the same time, there was seen a tendency towards increase in the relative area of white pulp and the support-contractile apparatus. The relative area of white pulp of the spleen increased by 0.5% in the group of quails that had received the diet with the Multilife supplement, by 1% in the group that had consumed the diet with the Missi supplement, and by 0.6% in the group that had consumed the diet with the Standard Agro supplement. The relative area of the red pulp in the spleen of the quails was observed to have the tendency towards decrease: by 0.4% in the group that received the Multilife supplement, by 1% in the group that consumed the Missi supplement, and by 0.5% in the group that consumed the Standard Agro supplement. The relative area of the support-contractile apparatus of the spleen increased by 0.4% in the group of quails that consumed the Multilife supplement, by 1% in the group that consumed the Missi supplement, and 0.5% in the group that consumed the Standard Agro supplement. Taking into account the data we obtained, we may conclude that our studies confirm the benefits of using modern protein-vitamin supplements in poultry farming for stimulation of growth and development. Especially valuable is the absence of negative effect on the morphofunctional parameters of the spleen, which we used as biomarkers.

Effect of immobilized hyaluronidase and subtilisin enzymes on the ultrastructure of human conjunctival epithelial cells

The use of enzyme preparations is a traditional trend in various fields of medicine. The usage of immobilized enzymes hyaluronidase (IG) and subtilisin (IS) seems to be very promising for the treatment of damage to the ocular surface. The aim of the study was to establish the nature of changes in the ultrastructural organization of human conjunctival epithelium under the influence of immobilized hyaluronidase and subtilisin in vitro. Material and methods. In the experiment, a culture of normal cells of the human conjunctiva Chanqconjunctiva, clone 1–5 C-4, was seeded in 96well plates in the amount of 2 × 104 cells/well, after 24 h the medium was removed, Eagle’s medium MEM and fetal calf serum were added and cells were cultured for another 48 h. 5 experimental groups were formed: group 1 – without drugs (except for the composition of the incubation scheme), groups 2 and 3 – with IS at a concentration of 37 and 150 U/ml, respectively, groups 4 and 5 – with IG at a concentration of 37 and 150 U/ml, respectively. After preparation of cell preparations under a JEM 1400 electron microscope (Japan), ultrathin sections of 70–100 nm were studied at ×1000 magnification. Results. The article presents the results of electron microscopy of a culture of normal cells of the human conjunctiva in 5 experimental groups of cells with a description of changes in cytoarchitectonics under the influence of IG and IS. Conclusions. The introduction of immobilized enzymes into human conjunctival cell culture at a low dose (37 U/ml) affects the organization of cells without cytotoxic effects, while increasing the dose directly correlates with the occurrence of a cytotoxic effect.

Effect of Viral Infection on the Ultrastructural Organization of Black Currant Leaf Tissue Cells

Effect of Viral Infection on the Ultrastructural Organization of Black Currant Leaf Tissue Cells

Molecular resolution imaging reveals step-wise reduction of axonemal diameter and ultrastructural architecture of intraflagellar transport proteins.

Primary cilia are microtubule-based organelles essential for mediating mechano-/photo-/chemo-sensations in mammalian cells. Intraflagellar transport (IFT) proteins are crucial components to support ciliary assembly, maintenance, and cellular signaling. The ciliary microtubule doublets of the ‘9 + 0’ arrangement serve as tracks for molecular motor-based transport along cilia. Although the tapering structure of primary cilia has been examined by electron microscopy, the spatial correlation between the IFT pattern and axonemal structure remains elusive due to insufficient light resolution. Here, we integrated single-molecule localization microscopy and sample expansion techniques achieving two-color imaging with effective localization precision of sub-5 nm to explore the axonemal ultrastructure and molecular organization of IFT and IFT-associated proteins in retinal pigment epithelial cilia. Our results revealed the step-wise reduction of ciliary diameter, different from the well-known paradigm showing a gradual change in the width of axonemal bundles. Surprisingly, our data showed that the IFT proteins congested near the end of the microtubule doubles within the ciliary compartment. In addition, we resolved the molecular architecture of IFT complexes, cargo, and motor proteins to characterize their relative localizations. We found that IFT proteins presented distinct distributions, including isolated units, paired configurations, or alternating arrangements in IFT trains. Together, our ultrastructural light nanoscopic imaging enables us to perform morphological analysis of primary cilia, which guides the localization of IFT complexes at segmented sites of cilia, suggesting the asynchronous axonemal growth of microtubule doubles.

A systematic investigation of the cardiac intercalated disk in health and disease.

During each heartbeat, action potential (AP) propagation at intercalated discs (ID), specialized structures that electrically (gap junctions [GJ]) and mechanically (adherens junctions [AJ], desmosomes [Des]) couple cardiomyocytes, coordinates the contraction of billions of cardiomyocytes. Electrogenic proteins underlying excitability (cardiac sodium channels [NaV1.5], inward-rectifier potassium channels [Kir2.1] and sodium-potassium ATPase [NKA]) enriched within ID nanodomains are emerging as vital machinery for AP propagation. ID disruption is a hallmark of arrhythmic disease, yet, IDs are omitted from physiological models due to lack of structural data. We used indirect correlative light and electron microscopy [iCLEM] to quantify ID ultrastructure and molecular organization in health and disease and link them with functional properties (conduction velocity, arrhythmia vulnerability). Additional studies in hearts treated with adhesion inhibitor peptides targeting different ID nanodomains helped bridge health with complex disease. In healthy mouse hearts, transmission electron microscopy uncovered smaller, more frequent junctions and closer intermembrane distance at peri-junctional nanodomains in atria vs. ventricles. Confocal microscopy revealed greater NaV1.5, Kir2.1, NKA expression with more preferential ID enrichment in atrial myocytes than ventricular. STORM single molecule localization microscopy, a super-resolution technique, identified chamber-specific differences in NaV1.5, Kir2.1 and NKA clustering and distribution relative to AJ, Des, GJ as well as each other. Adhesion inhibitor peptides Nadp1 (target: AJ), dadp1 (target: Des) and βadp1 (target: GJ-adjacent perinexi), but not corresponding inactive control peptides, acutely (60 minutes) disrupted their respective target nanodomains and dispersed NaV1.5 clusters from those sites. These structural impacts led to conduction slowing and reentrant arrhythmias with severity proportional to the amount of NaV1.5 present at the target site. These data parallel our previous observations in atrial fibrillation (mouse models and human patients) and highlight NaV1.5-rich ID nanodomains as potential targets for antiarrhythmic therapy.

Biochar and metal-tolerant bacteria in alleviating ZnO nanoparticles toxicity in barley

The constant use of zinc oxide nanoparticles (ZnO NPs) in agriculture could increase their concentration in soil, and cause a threat to sustainable crop production. The present study was designed to determine the role of spore-forming and metal-tolerant bacteria, and biochar in alleviating the toxic effects of a high dose of ZnO NPs (2000 mg kg−1) spiked to the soil (Haplic Chernozem) on barley (Hordeum sativum L). The mobile compounds of Zn in soil and their accumulation in H. sativum tissues were increased significantly. The addition of biochar (2.5% of total soil) and bacteria (1010 CFU kg−1) separately and in combination showed a favorable impact on H. sativum growth in ZnO NPs polluted soil. The application of bacteria (separately) to the contaminated soil reduced the mobility of Zn compounds by 7%, due to loosely bound Zn compounds, whereas only biochar inputs lowered Zn mobile compounds mobility by 33%, even the combined application of biochar and bacteria also suppressed the soil Zn mobile compounds. Individual application of biochar and bacteria reduced the Zn plant uptake, i.e., underground parts (roots) by 44% and 20%, and in the above-ground parts of H. sativum plants by 39% and 13%, respectively, compared to ZnO NPs polluted soil treatments. Biochar, both separately and in combination with bacteria improved the root length by 48 and 85%, and plant height by 53 and 40%, respectively, compared to the polluted control. The root length and plant height decreased by 52 and 40% in ZnO NPs spiked soil compared clean soil treatments. Anatomical results showed an improvement in the structural organization of cellular–sub–cellular tissues of root and leaf. The changes in ultrastructural organization of assimilation tissue cells were noted all treatments due to the toxic effects of ZnO NPs compared with control treatment. The results indicate that metal-tolerant bacteria and biochar could be effective as a soil amendment to reduce metal toxicity, enhance crop growth, and improve soil health.

Morphometrie assessment of ultrastructural organization Escherichia coli bacteria after exposure to gamma radiation

Morphometrie assessment of ultrastructural organization Escherichia coli bacteria after exposure to gamma radiation

Ultrastructural organization of liver hepatocytes of the Anglo-Nubian goat

The aim of the study was to establish the ultrastructural organization of hepatocytes of the Anglo-Nubian goat. Livers of adult goats of the Anglo-Nubian breed served as the material for the study. The study was carried out using the electron microscopic method. For this purpose, fragments of liver parenchyma tissues, no larger than 2.0 mm3, were selected. The tissue samples were fixed in a 2.0% glutaraldehyde solution on a cacodylate buffer (pH 7.2-7.4) for 2 hours. Then, they were washed in three portions of the same buffer and post-fixed in a 1.0% solution of osmium tetrachloride (prepared in cacodylate buffer, pH 7.2-7.4) for 1 hour. Samples were dehydrated in increasing concentrations of alcohol and anhydrous acetone. The tissue fragments were then embedded in Epon-812. Ultrathin sections were obtained using an ultramicrotome (LKB-III, Sweden) and processed with 2.0% aqueous uranyl acetate and lead citrate solutions. The resulting ultrathin sections were photographed using a Jem-1011 electron microscope (JEOL, Japan) at 2500-3000? magnification. It was established that two morphological types of hepatocytes can be distinguished in the liver parenchyma of the Anglo-Nubian goat at the ultrastructural level ? dark and light, both with characteristic morphological features. Dark hepatocytes have a high electron-optical density due to the presence of a larger number of organelles. Light hepatocytes are characterized by a smaller number of organelles and a lower electron-optical density than dark hepatocytes.

Ультраструктура эпителия проксимальных канальцев почки при пероральном введении карбоната лития в условиях опухолевого роста

Introduction. Although new biological effects of lithium salts are being constantly discovered, they are still used as nephrotoxins. Recently, experimental data have appeared on regenerative and protective properties of short-term lithium administration in various kidney pathologies. The paper aimed to evaluate ultrastructural organization of the kidney proximal tubule cells in peripheral tumor growth and lithium carbonate peroral administration. Materials and methods. The study was carried out on CBA male mice (n=20). We formed four groups, 5 animals each: “control”, “control + lithium”, “tumor” and “tumor + lithium.” To stimulate tumor growth in the thigh muscle tissue, we used a cell line of hepatocellular carcinoma-29. Lithium carbonate was administered per os at a dose of 125 mg/kg of body weight every other day, starting from day 3 after inoculation of tumor cells. Animals were sacrificed on day 30 of tumor development, and kidney fragments were collected for electron microscopy. Results. We compared and analyzed ultrastructural organization of the apical and perinuclear zones as well as autophagic structures of the kidney proximal tubule cells. The animals with peripheral tumor growth showed decreased numerical density of endosomes and dense apical tubules as well as decreased volume density of the granular endoplasmic reticulum and mitochondria. In these animals, we also revealed increased volume density of autolysosomes in the cytoplasm of the epitheliocytes. The intact animals that received lithium carbonate demonstrated an increase in the volume density of free polysomal ribosome complexes. The animals that developed tumors and were administered lithium carbonate showed a growth in both numerical density of dense apical tubules and the volume density of autophagic structures. Conclusion. Peroral administration of lithium carbonate to intact animals every other day for 30 days did not cause any destructive changes to the kidney proximal tubule cells. The administration of lithium carbonate to animals with peripheral tumor growth had a protective effect on the ultrastructural organization of the kidney proximal tubules cells (probably due to autophagy activation). Keywords: peripheral tumor, lithium carbonate, renal proximal tubules, ultrastructure, endocytosis, autophagy

Features of morphological and ultrastructural organization of the cornea (literature review)

The human cornea – the anterior fibrous membrane of the eye, is a unique ordered optical-biological system that is avascular, saturated with nerve endings, includes tissue-specific cells, consists mainly of various types of collagen. An exceptional feature of the collagen layers of the cornea, including the collagen plates of the stroma, is transparency, which provides physiological refraction and light transmission due to the stable supporting properties of the cornea. The data on the morphological structure of the cornea, which is an important element of the optical system of the eye, are of considerable interest not only from theoretical, but also from practical positions. This is due to the fact that the identification of the first signs of deviation from normal physiological morphological and ultrastructural criteria in the cornea allows us to establish the nature of its pathological changes, which can be caused by both hereditary predisposition and local and general disorders. It has been shown that the thinning of the layers of the cornea, a decrease in the density of endotheliocytes or keratocytes signal the development of dystrophic processes in it. In addition to evaluating quantitative morphometric data, changes in qualitative ultrastructural indicators play an important role. In particular it was found that a decrease in the density of endothelial cells is accompanied by an increase in their size and a decrease in the cell nucleus. In addition, a number of degenerative pathological conditions are characterized by a decrease in the diameter of collagen fibrils and a change in the density of fibrillary packaging.This literature review presents basic information, features of morphology, ultrastructural organization and functional purpose of layers and cells of the human cornea.

Ultrastructural reorganization of the constituent components of the rat periodontium under conditions of chronic opioid exposure

Opioid addiction has acquired the characteristics of an epidemic in recent years and is a serious problem in modern medicine. In users of narcotic substances, a significant prevalence of diseases of the oral cavity and periodontal tissues is noted in comparison with the general population. The aim of the work was to investigate the ultrastructural organization of the periodontal tissues of rats at the end of the fourteenth week of the experimental opioid effect. The animals were administered intramuscularly, daily, once, the opioid analgesic nalbuphine (pharmacotherapeutic group: analgesics, opioids, morphinan derivatives) in increasing doses for 14 weeks: 1-2 weeks – 0.212 mg/kg, 3-4 weeks – 0.225 mg/kg, 5-6 weeks – 0.252 mg/kg, 7-8 weeks – 0.260 mg/kg, 9-10 weeks – 0.283 mg/kg; 11-12 – 0.300 mg/kg, 13-14 weeks – 0.450 mg/kg. At the end of the fourteenth week of opioid exposure, ultrastructural changes in the epithelium of the mucous membrane of the gums, periodontium, and hemomicrovessels with the development of decompensation and pronounced dystrophic-destructive changes were established. With the long-term effect of an opioid analgesic, signs of karyorrhexis, segregation of nucleoli, spongiosis, apoptosis, violation of the integrity of intercellular contacts, as well as vacuole-like formations, which were a manifestation of focal necrosis of epitheliocytes, were revealed. The development of necrotic changes was caused by significant damage and destruction of organelles, as well as fragmentation or lysis of bundles of tonofilaments. The intercellular spaces were mostly expanded and uneven, which was caused by edematous phenomena. Fragmentation and destruction of periodontal collagen fibers, destructive changes in fibrocytes and fibroblasts were found in the periodontium. The translucency of the amorphous component of the intercellular substance of the connective tissue was determined. The ultrastructural reorganization of the cellular components of the periodontium was caused by vascular disorders of a systemic nature, which were manifested by the phenomena of dilatation, hemoptysis, sludge phenomenon, dystrophic changes in the endotheliocytes of hemocapillaries and venules, and the progression of sclerotic changes, which significantly disturbed the trophism of the periodontal tissues of rats.

Ultrastructural organization of the respiratory part of the lungs in streptozotocin-induced diabetes

Background. Diabetes mellitus is a metabolic disease characterized by persistent hyperglycemia, the prevalence and incidence of which have dramatically increased worldwide. Our research aimed to study the dynamics of changes in the components of the respiratory part of the lungs in streptozotocin-induced diabetes. Materials and methods. The experiments were performed on 88 white male Wistar rats weighing 170-210 g. The animals were divided into three groups: 1 - intact (n=10); 2 - control (n=40); 3 - experimental (n=38) with a model of diabetes mellitus, which was reproduced by intraperitoneal injection of streptozotocin company "Sigma" (USA), diluted in 0.1 M citrate buffer with pH 4.5, at a rate of 60 mg/kg body weight. The control group of animals received an intraperitoneal injection with an equivalent dose of 0.1 M citrate buffer solution with a pH of 4.5. Pulmonary tissue collection for electron microscopic examination was performed under thiopental anesthesia 14, 28, 42, and 70 days after streptozotocin injection. Pieces of lung tissue were fixed in 2.5% glutaraldehyde solution, followed by fixation in 1% osmium tetroxide solution. After dehydration, the material was poured into epon-araldite. Sections obtained on an ultramicrotome "Tesla BS-490" were studied in an electron microscope «PEM-125K». All studies were performed under sodium thiopental anesthesia at the rate of 60 mg/kg of body weight. Results. Our research showed that 14 days after the modeling of streptozotocin-induced diabetes, mainly reactive changes were observed in the components of the respiratory part of the lungs. With an increase in the duration of the study (28-70 days), changes of a dystrophic-destructive nature were noted in alveolocytes of types I and II, and endotheliocytes of hemocapillaries. At the same time, cells with increased functional activity were determined in the components of the respiratory part of the lungs. Conclusion. Streptozotocin-induced diabetes leads to violations of the ultrastructural organization of components of the respiratory part of the lungs. The nature and severity of structural changes in type I, II alveolocytes, and endotheliocytes of hemocapillaries depend on the duration of diabetes.

Antennal sensory structures of Phenacoccus solenopsis (Hemiptera: Pseudococcidae)

BackgroundThe cotton mealybug Phenacoccus solenopsis Tinsley (Hemiptera: Pseudococcidae) is one of the most devastating sap-sucking pests of cultivated plants. The success of P. solenopsis is attributable to its ecological resilience and insecticide resistance, making its control extremely difficult and expensive. Thus, alternative safe approaches are needed to prevent the pest population from reaching the economic threshold. One of these novel approaches is based on the fact that chemical communication via the olfactory system drives critical behaviors required for the survival and development of the species. This knowledge can be useful for controlling insect pests using traps based on semiochemicals. The antennae of insects are an invaluable model for studying the fundamentals of odor perception. Several efforts have been made to investigate the histological and ultrastructural organization of the olfactory organs, such as the antennae and maxillary palps, in many insect species. However, studies on the antennal sensory structures of Phenacoccus species are lacking. Furthermore, although enormous progress has been made in understanding the antennal structures of many mealybug species, the olfactory sensilla in the antennae of P. solenopsis have not yet been described. In this study, we describe, for the first time, the morphology and distribution of the antennal sensilla in male and female P. solenopsis using scanning electron microscopy.ResultsOur results revealed that the entire antennae length and the number of flagellar segments were different between the sexes. Eight morphological types of sensilla were identified on male antennae: trichoid sensilla, chaetic sensilla (three subtypes), basiconic sensilla (two subtypes), and campaniform sensilla (two subtypes). Six morphological types of sensilla were found on female antennae. Sensilla chaetica of subtype 2 and campaniform sensilla of subtype 1 were distributed only on male antennae, suggesting that these sensilla are involved in the recognition of female sex pheromones. The subtype 1 of sensilla chaetica was significantly more abundant on female antennae than on male ones, while subtype 3 was only located on the terminal flagellar segment of the antenna in both sexes.ConclusionsThis study provides insightful information for future electrophysiological and behavioral studies on chemical communication in insects, particularly the cotton mealybug, P. solenopsis that could help in developing new strategies for controlling this economically important insect species.

Forms of Bacterial Survival in Model Biofilms

Bacterial survival upon sharp fluctuations of environmental parameters and exposure to lethal doses of stressors (antibiotics, disinfectants, heat shock, and others) is ensured by the use of different strategies of resistance, an important place among which is occupied by the forms with reduced or stopped metabolism, antibiotic tolerant (AT) persister (P) cells, anabiotic dormant forms (DFs), and viable but non-culturable (VBNC) cells. Elucidating the role of these forms of bacterial resistance to an impact of chemical and biological toxicants and physical stressors is of great fundamental and practical interest. The aim of this research was to study the dynamics of the resistance forms in bacteria developing in biofilms and, for comparison in liquid media, upon exposure to lethal doses of antibiotics and heat shock (80 °C, 15 min). In the trials, the experimental model of the development of monospecies and binary forms of bacterial biofilms including contaminants of meat products (eight strains of genera Pseudomonas, Escherichia, Salmonella, Staphylococcus, and Kocuria) on the fiberglass filters was used. It was established that survival of populations in the presence of lethal doses of antibiotics and upon heating was ensured by persister cells forming in bacterial populations and, at the late stages of the development of biofilm or planktonic cultures (28 days of incubation), by anabiotic DFs. With that, the number of thermoresistant (TR) DFs (103–104 CFU/mL) in dying biofilms (28 days) developing in the standard conditions (composition and volume of a medium, pH, growth temperature) weakly depended on the bacterial taxonomic status. This study demonstrates the heterogeneity of DF populations of biofilm bacterial cultures in terms of the depth of dormancy, as a result of which the number of thermoresistant DFs after heating can exceed their total number before heating (due to the effect of DF revival, resuscitation). When studying the dynamics of TR cells and P cells in bacterial biofilm and planktonic cultures, it was found that their number (CFU/mL) in populations decreased up to the absence of TR cells and P cells on the 21st day of growth and was restored on the 28th day of growth. The revealed phenomenon can be explained only by cardinal changes in the ultrastructural organization of cells, namely, cytoplasm vitrification due to a sharp decrease in an amount of free water in a cell, which, according to the results obtained, occurs in the period between the 21st and 28th days of incubation. A high degree of correlation between the number of AT cells and TR cells (0.5–0.92) confirms the hypothesis that regards P cells as precursors of DFs.

Visualizing extracellular vesicle biogenesis in gram-positive bacteria using super-resolution microscopy

BackgroundRecently, bacterial extracellular vesicles (EVs) have been considered to play crucial roles in various biological processes and have great potential for developing cancer therapeutics and biomedicine. However, studies on bacterial EVs have mainly focused on outer membrane vesicles released from gram-negative bacteria since the outermost peptidoglycan layer in gram-positive bacteria is thought to preclude the release of EVs as a physical barrier.ResultsHere, we examined the ultrastructural organization of the EV produced by gram-positive bacteria using super-resolution stochastic optical reconstruction microscopy (STORM) at the nanoscale, which has not been resolved using conventional microscopy. Based on the super-resolution images of EVs, we propose three major mechanisms of EV biogenesis, i.e., membrane blebbing (mechanisms 1 and 2) or explosive cell lysis (mechanism 3), which are different from the mechanisms in gram-negative bacteria, despite some similarities.ConclusionsThese findings highlight the significant role of cell wall degradation in regulating various mechanisms of EV biogenesis and call for a reassessment of previously unresolved EV biogenesis in gram-positive bacteria.

Morpho-functional Analysis of the Head Glands in Three Auchenorrhyncha Species and Their Possible Biological Significance.

The Cicadomorpha Philaenus spumarius, Neophilaenus campestris, and Cicadella viridis are known transmitters of the bacterium Xylella fastidiosa. Here, we studied the ultrastructural organization of their cephalic glands. Our investigations with scanning, transmission, focused ion beam-scanning electron microscopes and light microscope revealed for the first time in Auchenorrhyncha the presence of two types of cephalic glands. Both belonged to the Class III epidermal glands, according to the Noirot and Quennedey classification. Type A glands were the most common, being mainly located around antennae, lorum, and gena. Moreover, these glands were observed also on the abdomen and thorax, always in association with sensilla trichoidea. The second type of glands (type B) were located exclusively at the apical part of the postclypeus in P. spumarius and N. campestris. The ultrastructural organization was similar in both types, being composed of a secretory cell and a conducting canal. Differences were observed in the width of the cuticular opening, being smaller in the type II glands. In addition, we have recorded the presence of a maxillary sensory pit in all species and described sensilla trichoidea ultrastructural organization. Finally, we discussed the ultrastructural organization of the glands and their potential biological role.

Super-resolution microscopy reveals nanoscale architecture and regulation of podosome clusters in primary macrophages

Podosomes, an important actin-based adhesive architecture, play critical roles in cell migration and matrix invasiveness. Here, we elucidate the ultrastructural organization and regulation of podosome clusters in primary macrophages. With three-dimensional stochastic optical reconstruction microscopy (3D-STORM), we achieve ∼20/50nm (lateral/axial) spatial resolution to resolve the mutual localization of podosome core and ring components, and further show that microtubules pass through podosomes at the layer of myosin IIA. The microtubule disruption-caused podosome dissolution is previously ascribed to Rho/ROCK-myosin signaling, yet inhibiting this pathway with Y27632 or blebbistatin only partially recovers podosome assembly, thus suggesting the contribution of the physical supporting of microtubules in stabilizing podosome structures. Through improved substrate-coating technique, we further corroborate that the matrix-degrading capability of macrophages depends on the formation of podosome clusters. Together, 3D-STORM super-resolution microscopy reveals the nanoscale spatial arrangement and the microtubule-dependent regulation of the matrix-degrading podosome clusters in macrophages.

Sarcomere maturation: function acquisition, molecular mechanism, and interplay with other organelles.

During postnatal cardiac development, cardiomyocytes mature and turn into adult ones. Hence, all cellular properties, including morphology, structure, physiology and metabolism, are changed. One of the most important aspects is the contractile apparatus, of which the minimum unit is known as a sarcomere. Sarcomere maturation is evident by enhanced sarcomere alignment, ultrastructural organization and myofibrillar isoform switching. Any maturation process failure may result in cardiomyopathy. Sarcomere function is intricately related to other organelles, and the growing evidence suggests reciprocal regulation of sarcomere and mitochondria on their maturation. Herein, we summarize the molecular mechanism that regulates sarcomere maturation and the interplay between sarcomere and other organelles in cardiomyocyte maturation.This article is part of the theme issue ‘The cardiomyocyte: new revelations on the interplay between architecture and function in growth, health, and disease’.