Alina E Grigor’eVa

Mitochondrial proteins (mtPs) packaged into extracellular vesicles (EVs), particularly exosomes, have emerged as promising biomarkers and functional mediators in cancer biology. In this study, we investigated whether breast cancer (BC)-derived exosomes selectively incorporate mtPs, thereby providing insights into their diagnostic potential and role in tumor progression. Exosomes were isolated from conditioned media of multiple BC cell lines and non-tumorigenic breast epithelial cells using ultracentrifugation and were characterized by transmission electron microscopy and flow cytometry for canonical markers CD9 and CD81. Proteomic profiling by MALDI-TOF mass spectrometry revealed a distinct enrichment of mtPs in exosomes derived exclusively from tumor cells, while non-tumorigenic cells lacked such cargo. Identified proteins were predominantly associated with the oxidative phosphorylation system, with Complex I subunits most frequently detected, whereas Complex II components were entirely absent. These findings highlight a cancer-specific mechanism of exosomal mtP incorporation, potentially reflecting altered energy metabolism and stress responses in malignant cells. We conclude that mtPs in exosomes represent both functional contributors to tumor progression and promising candidates for liquid biopsy-based BC diagnostics.

The structure of the nucleoid observed after standard aldehyde fixation did not correspond to modern ideas about its spatial organization: DNA strands are

Background: Breast cancer (BC) is the most common cancer among women worldwide; therefore, the efforts of many scientists are aimed at finding effective biomarkers for this disease. It is known that exosomes are nanosized extracellular vesicles (EVs) that are released from various cell types, including cancer cells. Exosomes are directly involved in governing the physiological and pathological processes of an organism through the horizontal transfer of functional molecules (proteins, microRNA, etc.) from producing to receiving cells. Since the diagnosis and treatment of BC have been improved substantially with exosomes, in this study, we isolated breast carcinoma cell-derived exosomes, primary endotheliocyte-derived exosomes, and blood exosomes from BC patients (BCPs) in the first stage of disease and investigated their proteomic profiles. Methods: Exosomes were isolated from the samples by ultrafiltration and ultracentrifugation, followed by mass spectrometric and bioinformatics analyses of the data. The exosomal nature of vesicles was verified using transmission electron microscopy and flow cytometry. Results: Exosome proteins secreted by MCF-7 and BT-474 cells were found to form two clusters, one of which enhanced the malignant potential of cancer cells, while the other coincided with a cluster of HUVEC-derived exosome proteins. Despite the different ensembles of proteins in exosomes from the MCF-7 and BT-474 lines, the relevant portions of these proteins are involved in similar biological pathways. Comparison analysis revealed that more BC-associated proteins were found in the exosomal fraction of blood from BCPs than in the exosomal fraction of conditioned medium from cells mimicking the corresponding cancer subtype (89% and 81% for luminal A BC and MCF-7 cells and 86% and 80% for triple-positive BC and BT-474 cells, respectively). Conclusions: Tumor-associated proteins should be sought not in exosomes secreted by cell lines but in the composition of blood exosomes from cancer patients, while the contribution of endotheliocyte exosomes to the total pool of blood exosomes can be neglected.

A protein corona is present on any nanoparticle (NP) entering biological fluids; however, the existence of a natural protein corona on natural NPs has not been experimentally confirmed. We used our previously developed photomodification method to fix the natural corona on “biological nanoparticles” (bio-NPs) in fetal bovine serum and newborn bovine serum; native sera served as a control. To isolate photomodified bio-NPs, we used ultracentrifugation (UC), sucrose gradient (12%, 30%, and 50%), and sucrose cushion (30%) methods. Isolated bio-NPs were visualized using transmission electron microscopy. The results showed that a protein corona is present on extracellular vesicles and lipoproteins isolated by UC. The isolation of bio-NPs through sucrose gradient or cushion did not preserve the protein corona. At the same time, we observed signs of a negative effect of the sucrose gradient on bio-NPs of intact and photomodified serum; when isolating these particles on a sucrose cushion, no negative effects were observed. We believe that the data we present will be useful to researchers using sucrose solutions to isolate bio-NPs and working on the properties of the protein corona. In this work, we have obtained direct images of a “natural” protein corona on natural bio-NPs of blood serum for the first time

Breast cancer (BC) is the most common cancer among women worldwide, therefore, the efforts of many scientists are aimed at finding effective biomarkers of this disease. It is known that exosomes are nanosized extracellular vesicles (EVs), which are released from various cell types, including cancer cells. Exosomes are directly involved in governing physiological and pathological processes of an organism by horizontal transfer of functional molecules (proteins, microRNA, etc.) from producing to receiving cells. Since the diagnosis and treatment of BC were improved substantially with exosomes, in this study, we isolated breast carcinoma cells‐derived exosomes, primary endoteliocytes-derived exosomes and blood exosomes from BC patients at 1st stage of disease and investigated their proteomic profile. Exosomes were isolated from the samples by ultrafiltration and ultracentrifugation, followed by mass spectrometric and bioinformatics analysis of the data. Exosomal nature of vesicles has been verified using transmission electron microscopy and flow cytometry. Exosome proteins secreted by MCF-7 and BT-474 cells were found to form 4 clusters, three of which enhanced the malignant potential of cancer cells, and one cluster coincided with a cluster of HUVEC-derived exosome proteins. Despite the different ensembles of proteins in exosomes from the MCF-7 and BT-474 lines, these portions of these proteins are involved in similar biological pathways. Comparison analysis reveals more BC-associated proteins were found in the exosomal content from the blood of BCPs than in the content of conditioned medium of cells mimicking the corresponding cancer subtype (89% and 82% for luminal A BC and MCF-7 cells and 86% and 75% for triple-positive BC and BT-474 cells, respectively). Thus, tumor-associated proteins should be sought not in exosomes secreted by cell lines, but in the composition of blood exosomes of cancer patients, while the contribution of endotheliocyte exosomes to the total pool of blood exosomes can be neglected.

Background: Overweight and especially obesity are associated with the risk of the development and progression of colorectal cancer. It can be assumed that there are multifaceted interactions between the tumor and adipose tissue during anti-tumor treatment. Cancer cells secrete exosomes, extracellular vesicles affecting the microenvironment of the tumor and promoting its progression or regression. The presence of transcription/translation/folding factors (heat shock proteins (HSPs), matrix metalloproteinases (MMPs) and their tissue inhibitors (TIMPs) in exosomes secreted by irradiated cells and cells exposed to hyperthermia, indicates the cell adaptation to the thermal and radiation stress. Aim: To analyze the MMPs, TIMP1, and HSPs on CD9-positive (CD9+) exosomes, as well as on exosomes of adipocytic origin (FABP4+) in rectal cancer patients with overweight/obesity under thermoradiochemotherapy (TRCT) and their association with the immediate treatment efficacy. Methods: Since 2021, 20 patients (of those 8 men; median age 59.0 [52.0; 63.0] years, median body mass index 29.6 [28.5; 33.1] kg/m2) with morphologically verified rectal cancer (T3-4N0M0 and T3-4N1M0, differentiation grade G1–G3) have been participating in the study. They were treated with TRCT: external gamma therapy (2 Gy, 1 fraction/day, 5 days/week, total focal dose 54 Gy), chemotherapy with capecitabine (825 mg/m2 twice daily) combined with local hyperthermia (42–44 °C, 60 min, 3 times/week, 10 sessions). The TRCT efficacy was assessed by RECIST 1.1 and ESGAR criteria. Blood samples for exosomes were taken from the patients at baseline (point 1), in the middle of the treatment course (point 2), at 6 to 10 weeks after the end of TRCT (point 3), and at 6 months after point 1 (point 4). Small extracellular vesicles were isolated from plasma by ultrafiltration with double ultracentrifugation. The isolated exosomes were characterized by transmission electronic microscopy, flow cytometry and nanoparticle trajectory analysis (NTA). Results: TRCT resulted in complete tumor regression in 13/20 of the rectal cancer patients and partial regression or stabilization in 7/20. Four subpopulations of CD9+ and FABP4+ exosomes associated with the TRCT efficacy were identified (CD9+MMP2+, СD9+MMP2+9+TIMP1+, СD9+MMP2+9+TIMP1-, and FABP4+MMP2+9-TIMP1+). Compared to the CD9+ exosomes, the adipocytic vesicles had higher MMP2 expression (p = 0.026); however, the adipocyte vesicles subpopulation were virtually free of vesicles with combined MMP2 and MMP9 gelatinase expression. The HSPs expression by circulating exosomes at various TRCT steps was associated neither with direct treatment efficacy nor with the vesicle type. Conclusion: The expression of MMPs and TIMP1 on CD9+ and FABP4+ exosomes is associated with TRCT efficacy. In the future, vesicular markers could be used to build prognostic models, to identify patient groups with an unfavorable prognosis, and to personalize treatment and follow-up.

Different methods for fixing biofilms of Staphylococcus aureus and Salmonella enterica for light and electron microscopy were compared. Paraformaldehyde fi

Spontaneous sorption of proteins on the nanoparticles’ surface leads to the fact that nanoparticles in biological media are always enveloped by a layer of proteins—the protein corona. Corona proteins affect the properties of nanoparticles and their behavior in a biological environment. In this regard, knowledge about the composition of the corona is a necessary element for the development of nanomedicine. Because proteins have different sorption efficacy, isolating particles with a full corona and characterizing the full corona is challenging. In this study, we propose a photo-activated cross-linker for full protein corona fixation. We believe that the application of our proposed approach will make it possible to capture and visualize the full corona on nanoparticles coated with a lipid shell.

The purposeful development of synthetic antibacterial compounds requires an understanding of the relationship between effects of compounds and their chemical structure. This knowledge can be obtained by studying changes in bacteria ultrastructure under the action of antibacterial compounds of a certain chemical structure. Our study was aimed at examination of ultrastructural changes in S. aureus cells caused by polycationic amphiphile based on 1,4‒diazabicyclo[2.2.2]octane (DL₄12), ciprofloxacin and their hybrid (DL₅Cip6); the samples were incubated for 15 and 45 min. DL₄12 first directly interacted with bacterial cell wall, damaging it, then penetrated into the cell and disrupted cytoplasm. Ciprofloxacin penetrated into cell without visually damaging the cell wall, but altered the cell membrane and cytoplasm, and inhibited the division of bacteria. The ultrastructural characteristics of S. aureus cells damaged by the hybrid clearly differed from those under ciprofloxacin or DL₄12 action. Signs associated with ciprofloxacin predominated in cell damage patterns from the hybrid. We studied the effect of ciprofloxacin, DL₄12 and their hybrid on S. aureus biofilm morphology using paraffin sections. Clear differences in compound effects on S. aureus biofilm (45 min incubation) were observed. The results obtained allow us to recommend this simple and cheap approach for the initial assessment of antibiofilm properties of synthesized compounds.

Extracellular vesicles (EVs), carriers of molecular signals, are considered a critical link in maintaining homeostasis in mammals. Currently, there is growing interest in studying the role of EVs, including exosomes (subpopulation of EVs), in animals of other evolutionary levels, including marine invertebrates. We have studied the possibility of obtaining appropriate preparations of EVs from whole-body extract of holothuria Eupentacta fraudatrix using a standard combination of centrifugation and ultracentrifugation. However, the preparations were heavily polluted, which did not allow us to conclude that they contained vesicles. Subsequent purification by FLX gel filtration significantly reduced the pollution but did not increase vesicle concentration to a necessary level. To detect EVs presence in the body of holothurians, we used transmission electron microscopy of ultrathin sections. Late endosomes, producing the exosomes, were found in the cells of the coelom epithelium covering the gonad, digestive tube and respiratory tree, as well as in the parenchyma cells of these organs. The study of purified homogenates of these organs revealed vesicles (30–100 nm) morphologically corresponding to exosomes. Thus, we can say for sure that holothurian cells produce EVs including exosomes, which can be isolated from homogenates of visceral organs.