Abstract Boron neutron capture therapy (BNCT) is one of the promising treatment methods for cancer. BNCT is based on the unique high ability of the non-radioactive boron-10 ( 10 B) nucleus to absorb thermal neutrons. The absorption of a neutron by boron results in a nuclear reaction 10 B(n,α) 7 Li with 84% of the energy being released within the cell, thereby inducing cancer cell death. We propose a novel approach based on using lithium instead of boron in neutron capture therapy: the neutron capture reaction cross-section for lithium is 4 times smaller than that of boron, while the energy release is 2 times higher, and the primary advantage of lithium is that 100% of the energy is released inside the cell. A series of in vivo neutron irradiation experiments using the murine B16 skin melanoma model demonstrated the efficacy of lithium neutron capture therapy (LiNCT). Higher lithium concentrations in the tumor achieved through intraperitoneal administration, compared to the oral route, contributed to a significant increase in animal survival and a significant decrease in tumor growth. The results of our study provide entirely new opportunities for the development of neutron capture therapy, both with lithium and with a possible lithium-boron combination.

Chloroquine (CQ), an autophagy inhibitor, is increasingly studied for its antitumor effects, but its impact on neuronal ultrastructure in tumor conditions remains unclear. We investigated prefrontal cortex pyramidal neurons in the melanoma mice model after CQ treatment. Transmission electron microscopy revealed reduced endoplasmic reticulum (ER) stress, manifested in decreased ER cisterns volume density, and a significant reduction in clathrin-coated and transport vesicles, possibly due to PICALM inhibition and disrupted clathrin-mediated endocytosis. CQ impaired autophagic flux in tumor-bearing mice, as indicated by autophagosome and lysosome accumulation alongside decreased autolysosomes. LC3β expression was increased in CQ-treated control mice but remained unchanged in tumor-bearing mice as compared to control. Lipofuscin accumulation in the Tumor + CQ group suggested possible neurodegenerative changes associated with autophagy. Restoration of synapse number and increased synaptic vesicle density in the Tumor + CQ group may reflect compensatory synaptogenesis. These findings suggest that, in addition to its antitumor effect, CQ significantly alters neuronal homeostasis, warranting further investigation of its neurotoxic and neuroprotective potential.

Neoadjuvant chemoradiotherapy (nCRT) followed by surgery is the standard treatment for locally advanced rectal cancer. However, the response to nCRT is variable and not always associated with improved survival. Colorectal cancer (CRC) is characterized by a complex tumor microenvironment (TME), a key component of which is cancer-associated fibroblasts (CAF). While their functions and interactions with tumor cells are under active investigation, the role of distinct CAF subpopulations and their plasticity remains largely undefined. A comprehensive characterization of CAFs during cancer progression could therefore contribute to the development of novel anticancer diagnostic and therapeutic strategies. In this study, we used immunohistochemistry to demonstrate that nCRT induces a significant reorganization of the TME in rectal adenocarcinoma. This reorganization was characterized by a redistribution of myofibroblasts (alpha-smooth muscle actin, αSMA+) and tumor-associated fibroblasts (fibroblast activation protein, FAP+; and fibroblast-specific protein 1, FSP1+), leading to pronounced fibrosis in both central and peripheral tumor regions. A decrease in E-cadherin expression, coupled with increased vimentin and transforming growth factor beta (TGFβ) levels in a subset of patients, indicated activation of epithelial-mesenchymal transition and the emergence of vasculogenic mimicry as an alternative mechanism of tumor vascularization. Ultrastructural analysis revealed structural changes in the cytoplasm of fibroblasts suggestive of active membrane remodeling and fibroblast–myofibroblast transition, particularly at the tumor periphery. Collectively, these findings suggest that nCRT may be accompanied by a reorganization of the tumor stroma, leading to fibroblast activation, epithelial-mesenchymal transition, and vasculogenic mimicry, all of which could potentially contribute to tumor progression.

Protein deficiency in the diet during pregnancy and lactation has a serious impact on the offspring by programming a predisposition to such serious diseases as hypertension and type 2 diabetes mellitus. In our study, we examined liver ultrastructure of rat pups at ages 2, 21, and 40 days with maternal protein deficiency. Body weight of the pups progressively lagged behind the control throughout the experiment, and the timing of eye opening indicated a slowdown of development. In the liver of 2-day-old animals, the proportion of hematopoietic cells at early stages of differentiation was higher as compared to the control. At the ultrastructural level, no obvious pathological changes were revealed, but a decrease in the amount of organelles was observed simultaneously with accumulation of lipids and glycogen. In the course of the experiment, a progressive decrease in the amount of the rough endoplasmic reticulum and ribosomes and increasing accumulation of glycogen in the cytoplasm of hepatocytes were noted. The most pronounced difference in ultrastructure between periportal and pericentral hepatocytes of control rat pups was detected on the 40th day of development, whereas in the low-protein diet group, the difference was weakly pronounced throughout the experiment. Thus, we showed that with prenatal and early postnatal protein deficiency, the growth and development of rat pups slows down, and glycogen accumulates excessively in the liver concurrently with a decrease in the amount of organelles.

BACKGROUND:Skin melanoma is a highly metastatic cancer with an increasing global incidence. Despite advancements in immunotherapy, new treatment strategies based on tumor biology are essential for improving outcomes and developing novel therapies. Autophagy plays a critical role in melanoma cell metabolism and affects the tumor microenvironment (TME). This study aims to evaluate the impact of autophagy-modifying drugs on extracellular matrix (ECM) remodeling and changes in the TME cytokine profile. METHODS:Immunohistochemical analysis was performed using paraffin-embedded tumor samples of B16-bearing C57BL/6 mice to assess the effects of autophagy-modifying drugs, lithium or chloroquine, on the matrix degradation proteins, their main substrates, lysyl oxidase and collagen fibril formation-associated proteins. The cytokine profile of the tumor was defined to estimate the effect of autophagy-modifying drugs on the TME. RESULTS:Chloroquine and lithium administration caused a decrease in the expression of matrix metalloproteinases, and chloroquine contributed to the accumulation of collagen type I. Moreover, chloroquine dramatically decreased LOX levels. Decorin expression levels were reduced in tumors of mice treated with chloroquine or lithium. Significant changes in the cytokine profile were detected after chloroquine treatment, with increased expression of IL1, IL4, IL6, M-CSF, TGFβ2 and TNF-α genes observed in the tumors. CONCLUSION:Autophagy-modifying drugs affect the TME, in particular, chloroquine promotes ECM remodeling, accumulation of collagen type I deposits and probably the formation of abnormal collagen fibril structures. In addition, chloroquine-treated mice showed high expression of pro-tumorigenic cytokines and growth factors, such as IL1, IL4, IL6, M-CSF, TGFβ2 and TNF-α in the TME.

The expression of cancer-associated fibroblast (CAF) and epithelial–mesenchymal transition marker proteins was assessed in tumor samples of B16 skin melanoma-bearing mice after chloroquine or lithium administration. Chloroquine increased the expression of CAF markers (αSMA and FAP). Hence, chloroquine can contribute to the activation of individual CAF populations in melanoma. In addition, a decrease in the expression of the epithelial marker E-cadherin and an increase in the expression of the mesenchymal marker vimentin were observed after chloroquine administration, which may indicate activation of epithelial–mesenchymal transition processes in the tumor.

High levels of autophagy can increase the viability of tumor cells as well as their resistance to chemotherapy. Evaluation of the dynamics of autophagy processes at different stages of carcinogenesis can extend our understanding of melanoma pathogenesis to develop new therapeutic approaches. We performed a comparative study of tumor cell autophagy in stages II and III human skin melanoma. Tumor cells were characterized by high content of structures associated with autophagy (autophagosomes and autolysosomes). In stage III melanoma characterized by the presence of regional metastases in the lymph nodes, tumor cells showed higher expression of the autophagy marker protein LC3beta in comparison with stage II melanoma cells, which can indicate the involvement of autophagy processes in tumor progression and the formation of metastases in the lymph nodes.

Uveal melanoma is an ocular tumor with a high risk of developing metastases. The endo-lysosomal system can affect the melanoma progression by accelerating and facilitating invasion or metastasis. This study aims to conduct comparative analysis of normal choroidal melanocytes and uveal melanoma cells ultrastructure with a focus on intracellular transport system, and to examine the patterns of autophagy- and vesicular trafficking-related proteins expression in a case series of uveal melanomas. Transmission electron microscopy was used to assess the ultrastructure of normal choroidal melanocytes and uveal melanoma cells. The expression levels of autophagy- and vesicular trafficking-related proteins in three histological types of uveal melanoma were analyzed by immunofluorescence staining. Electron microscopy results showed that the autophagic vacuoles were more abundant in normal choroidal melanocytes, than in uveal melanoma cells. The normal choroidal melanocytes were characterized by active intracellular vesicular trafficking; however, the proportion of caveolae was higher in uveal melanoma cells. The spindle type of tumor was characterized by a high expression levels of LC3 beta, while Rab7 and Rab11 proteins expression was significantly up-regulated in the mixed-type tumor cells. The results indicate that uveal melanoma cells probably have lower basal levels of autophagy and higher receptor-mediated endocytic trafficking-associated with caveolae than normal choroidal melanocytes. Research Highlights _ The autophagic vacuoles are abundant in normal choroidal melanocytes. _ Uveal melanoma cells are characterized by a high proportion of caveolae. _ The high expression levels of LC3 beta were revealed in a spindle type of tumor, while Rab7 and Rab11 proteins expression was up-regulated in the mixed-type tumor cells.

The expression of marker proteins of acute kidney injury after administration of high doses of lithium carbonate was assessed to evaluate the possibility of lithium use in neutron capture therapy. In mice with implanted skin melanoma B16, the expression of Kim1 (kidney injury molecule 1) and NGAL (neutrophil gelatinase-associated lipocalin) proteins in the kidneys was evaluated immunohistochemically 15, 30, 90, 180 min, and 7 days after peroral administration of lithium carbonate at single doses of 300 and 400 mg/kg. An increase in the expression of the studied proteins was found in 30 and 90 min after administration of 400 mg/kg lithium carbonate, however, 7 days after the drug administration, the expression returned to the level observed in the control group. It can be suggested that single administration of lithium carbonate in the studied doses effective for lithium neutron capture therapy will not significantly affect the renal function.

The incidence of the most malignant form of skin cancer, melanoma, is increasing. Tumor cells and tumor microenvironment cells produce proinflammatory cytokines, which can contribute to damage to the blood-brain barrier and, as a result, impairment of neuronal function. Lithium and chloroquine drugs are recognized as antitumor therapy agents. Lithium carbonate is used in the treatment of psychoemotional disorders and exhibits neuroprotective properties. The antitumor effect of chloroquine is manifested in the inhibition of autophagy in tumor cells. The purpose of the study was to evaluate the effects of lithium carbonate and chloroquine on the structural organization of prefrontal cortex pyramidal neurons in the melanoma mice model. Experiments were performed on male C57BL/6 mice, injected with Melanoma B16 tumor cells, and treated with lithium carbonate and chloroquine for 7 days. Using morphometric analysis, we calculated the quantitative and volume density of organelles of prefrontal cortex pyramidal neurons. Lithium carbonate has been shown to exhibit neuroprotective properties in melanoma: reduced signs of ER stress and oxidative stress, reduced autophagy. Chloroquine has been noted to block autophagic activity also leads to the accumulation of lipofuscin bodies, which may be one of the factors leading to neurodegeneration.

Cell-based therapies using corneal stromal stem cells (CSSC), corneal keratocytes, or a combination of both suppress corneal scarring. The number of quiescent keratocytes in the cornea is small; it is difficult to expand them in vitro in quantities suitable for transplantation. This study examined the therapeutic effect of corneal fibroblasts reversed into keratocytes (rCF) in a mouse model of mechanical corneal injury. The therapeutic effect of rCF was studied in vivo (slit lamp, optical coherence tomography) and ex vivo (transmission electron microscopy and immunofluorescence staining). Injection of rCF into the injured cornea was accompanied by recovery of corneal thickness, improvement of corneal transparency, reduction of type III collagen in the stroma, absence of myofibroblasts, and the improvement in the structural organization of collagen fibers. TEM results showed that 2 months after intrastromal injection of cells, there was a decrease in the fibril density and an increase in the fibril diameter and the average distance between collagen fibrils. The fibrils were well ordered and maintained the short-range order and the number of nearest-neighbor fibrils, although the averaged distance between them increased. Our results demonstrated that the cell therapy of rCF from ReLEx SMILe lenticules promotes the recovery of transparent corneal stroma after injury.

Tumor-produced pro-inflammatory cytokines and toxic substances passing through the blood-brain barrier can cause disturbances of brain homeostasis and neuronal damage. Correction of brain homeostasis under peripheral tumor growth conditions is an important task. In the present study, a cytokine expression in the brain and ultrastructural features of the prefrontal cortex pyramidal neurons in animals with skin melanoma and after lithium carbonate treatment were detected by PCR analysis, transmission electron microscopy, and immunohistochemical staining. The low expression levels of the neurotrophic factor BDNF and an increase expression of colony-stimulating factors G-CSF and GM-CSF were noted in animals with skin melanoma. At the same time, as welling of mitochondria, inner mitochondrial membrane damage and dilated endoplasmic reticulum were revealed in the prefrontal cortex neurons. Furthermore, the predominance of autophagosomes was revealed under peripheral tumor growth conditions. Lithium carbonate administration had a corrective effect on the cytokine expression in the brain and the ultrastructure of the prefrontal cortex neurons in animals with skin melanoma.

Background. Benign prostatic hyperplasia (BPH) is a common disease in older men. Transurethral surgery in BPH is the gold standard for treatment, but the techniques differ in the energy used. Considering the different mechanisms of action of bipolar plasma and thulium fiber laser energy on prostate tissue, the study of structural changes under their influence is relevant.The aim. To study the features of structural changes in the prostate during bipolar plasma and thulium fiber laser enucleation of benign prostatic hyperplasia.Methods. Thirty one patients with BPH were divided into two groups. Group 1 (n = 17) underwent transurethral plasma enucleation of the prostate; Group 2 (n = 14) underwent transurethral thulium fiber laser enucleation. Fragments of the surgical prostate capsule were collected intraoperatively and were processed according to standard examination protocols using light and electron microscopy. The width of coagulation necrosis, the relief of the dissection line, the severity of cell and the prostate intercellular matrix destruction were taken into account.Results. Studies have shown the clinical and functional perioperative homology of the compared methods of surgical treatment of BPH. Data from light and electron prostate microscopy showed a greater damaging effect of bipolar plasma energy, which is manifested by a larger width of the zone of coagulation necrosis, a torn and raised appearance of the dissection line, and pronounced integrity violations of cellular elements and intercellular matrix components. Laser exposure causes less pronounced changes, which indicates a more gentle effect of the thulium fiber laser on the prostate components.Conclusion. The obtained microscopy results indicate that the intraoperative action of a thulium fiber laser is less traumatic for the cells and intercellular matrix of the surgical prostate capsule compared to bipolar plasma exposure.

One of the hallmarks of cancer is tumor angiogenesis, so the development and testing of angiogenesis inhibitors is the focus of fundamental and clinical research. However, some mechanisms of tumor evasion from anti-angiogenic therapy have been identified. They include activation of compensatory/alternative angiogenesis pathways, vasculogenic mimicry, and vessel co-option.

Introduction. The development of a novel coronavirus infection (COVID-19) is not limited to respiratory damage. Adverse outcomes in patients with COVID-19 may be associated with cardiac damage. Aim. Study of the structural changes in the myocardium and the molecular biological characteristics of the endothelium of blood capillaries in severe forms of COVID-19. Materials and methods. An analysis of the medical documentation – inpatient records of 73 deceased with bilateral multisegmental viral pneumonia caused by the SARS-CoV-2 virus, as well as data from 43 autopsies of patients who died (sudden coronary death) between September 2020 to July 2021, was performed. An assessment of some clinical and laboratory data (degree of lung damage; levels of C-reactive protein, troponin I, D-dimer; left ventricular ejection fraction, etc.), structural changes in the myocardium and molecular biological characteristics (CD31, CD34) of the endothelium of blood capillaries in severe forms of COVID-19 was carried out. Results. The analysis of clinical and laboratory data has shown that a decrease in myocardial contractility in severe forms of COVID-19 due to an extensive lung damage was accompanied by an increase in the level of C-reactive protein, D-dimer, troponin I and indicated acute myocardial damage. According to electrocardiography (ECG) monitoring and echocardiography, repolarization disorders and a decrease in myocardial contractility were noted, which was accompanied by a decrease in ejection fraction by 32%. In more than 70% of cases, various arrhythmic disorders (atrial fibrillation, etc.) were observed during ECG. Light microscopy revealed the presence of sludge phenomenon, as well as swelling, desquamation and proliferation of the endothelium of blood capillaries, formation of diapedetic and focal hemorrhages, in some places leukocyte margination and their migration beyond vascular bed. Fibrin masses were observed in the lumen of blood vessels. Polarization microscopy, along with contractures in cardiomyocytes, revealed groups of cells with myocytolysis and primary granularity. In all observations, lipofuscinosis of cardiomyocytes was detected. Immunohistochemical examination showed a marked decrease in the expression level of CD31 (PECAM-1) and CD34 proteins – markers of blood vessels. Conclusion. The performed clinical and morphological analysis in severe forms of COVID-19 allowed us to obtain new data on degenerative changes in the myocardium and the structure of endothelial cells of blood capillaries, which can be used as a basis for methodological approaches to studying the mechanisms of development of heart failure in a novel coronavirus infection.

The structural organization of the extracellular matrix of rectal adenocarcinoma of different differentiation degrees without and after neoadjuvant radiation therapy was studied on postoperative material using immunohistochemistry and electron microscopy. The differences in the expression of types I and III collagens, as well as in the ultrastructural organization of the extracellular matrix of rectal adenocarcinoma of different differentiation degrees without and after neoadjuvant radiation therapy were revealed. We observed high expression of collagen I and wide channels in the collagen matrix in the central areas of the well differentiated adenocarcinomas without neoadjuvant radiation therapy and in poorly differentiated adenocarcinomas after neoadjuvant radiation therapy, which can be associated with metastasis and poor prognosis for the patients.

The expression of markers associated with epithelial-mesenchymal transition (EMT) and extracellular matrix degradation in human uveal melanoma tissue samples and postequatorial zone of the choroid was assessed by immunohistochemical staining. Increased expression of EMT markers E-cadherin and vimentin was observed in the tumor. The ratio of MMP-9 to TIMP-1 proteins related to the extracellular matrix degradation was higher in the tumor. These results may indicate activation of EMT-like process in the uveal melanoma cells and degradation of the extracellular matrix, which can contribute to the development of collective invasion in uveal melanoma.

It is known that the unfavorable outcome in patients infected with SARS-CoV-2 may be associated with the development of complications caused by heart damage due to the direct virus action. The mechanism of these cardiovascular injuries caused by SARS-CoV-2 infection has not been fully understood; however, the study of COVID-19-associated myocardial microcirculatory dysfunction can represent the useful strategy to solving this challenge. Thus, here we aimed to study the ultrastructural organization of endothelial cells of myocardial capillaries in patients with COVID-19. The morphology of endotheliocytes of the myocardial blood capillaries in patients with COVID-19 was studied on cardiac autopsy material using transmission electron microscopy. The endotheliocytes of myocardial capillaries in patients with COVID-19 were characterized by the abundant rough endoplasmic reticulum (ER) membranes, the Golgi complex, and free polysomal complexes of ribosomes and lipids. The presence of double membrane vesicles with virions and zippered ER was detected in the cytoplasm of endotheliocytes. The revealed endothelial ultrastructural changes indicate the remodeling of intracellular membranes during SARS-CoV-2 infection. Our findings confirm the formation of virus-induced structures in myocardial endothelial cells considered critical for viral replication and assembly. The data may elucidate the mechanisms of endothelial dysfunction development in patients with COVID-19 to provide potential targets for drug therapy.

In the treatment of coronary heart disease, the most promising approach for replacing lost contractile elements involves obtaining cardiomyocytes through cardiac differentiation of pluripotent cells. The objective of this study is to develop a technology for creating a functional layer of cardiomyocytes derived from iPSCs, capable of generating rhythmic activity and synchronous contractions. To expedite the maturation of cardiomyocytes, a renal subcapsular transplantation model was employed in SCID mice. Following explantation, the formation of the cardiomyocyte contractile apparatus was assessed using fluorescence and electron microscopy, while the cytoplasmic oscillation of calcium ions was evaluated through visualization using the fluorescent calcium binding dye Fluo-8. The results demonstrate that transplanted human iPSC-derived cardiomyocyte cell layers, placed under the fibrous capsules of SCID mouse kidneys (for up to 6 weeks), initiate the development of an organized contractile apparatus and retain functional activity along with the ability to generate calcium ion oscillations even after removal from the body.