Protein undernutrition affects inter alia blood parameters and immune system. These negative effects may be improved by the addition of vitamin B6 to the diet. Here, we evaluated the effect of vitamin B6 supplementation and physical exercise in rats fed a protein-deficient diet. Rats were divided into six groups: exercised or unexercised – control, protein deficient, protein deficient with vitamin B6 supplementation. Sixty days of protein malnutrition caused significant changes in the rat body weight, haematological parameters (mainly red blood cells parameters), immunological parameters (NK and NKT cells) and biochemical parameters (total protein and albumin concentration and activity of AST). The rat exercise did not intensify the negative effects of protein malnutrition. The vitamin B6 supplementation in protein-deficient groups significantly improved body weight and red blood cell parameters. These results indicate that vitamin B6 should be considered as a valuable supplement for individuals suffering from protein malnutrition.

Adipocytes derived from 3T3-L1 cells are a gold standard for analyses of adipogenesis processes and the metabolism of fat cells. A widely used histological and immunohistochemical staining and mass spectrometry lipidomics are mainly aimed for examining lipid droplets (LDs). Visualizing other cellular compartments contributing to the cellular machinery requires additional cell culturing for multiple labeling. Here, we present the localization of the intracellular structure of the 3T3-L1-derived adipocytes utilizing vibrational spectromicroscopy, which simultaneously illustrates the cellular compartments and provides chemical composition without extensive sample preparation and in the naïve state. Both vibrational spectra (FTIR—Fourier transform infrared and RS—Raman scattering spectroscopy) extended the gathered chemical information. We proved that both IR and RS spectra provide distinct chemical information about lipid content and their structure. Despite the expected presence of triacylglycerols and cholesteryl esters in lipid droplets, we also estimated the length and unsaturation degree of the fatty acid acyl chains that were congruent with known MS lipidomics of these cells. In addition, the clustering of spectral images revealed that the direct surroundings around LDs attributed to lipid-associated proteins and a high abundance of mitochondria. Finally, by using quantified markers of biomolecules, we showed that the fixative agents, paraformaldehyde and glutaraldehyde, affected the cellular compartment differently. We concluded that PFA preserves LDs better, while GA fixation is better for cytochromes and unsaturated lipid analysis. The proposed analysis of the spectral images constitutes a complementary tool for investigations into the structural and molecular features of fat cells.

The full understanding of molecular mechanisms of cell differentiation requires a holistic view. Here we combine label-free FTIR and Raman hyperspectral imaging with data mining to detect the molecular cell composition enabling noninvasive monitoring of cell differentiation and identifying biochemical heterogeneity. Mouse adipose-derived mesenchymal stem cells (AD-MSCs) undergoing adipogenesis were followed by Raman and FT-IR imaging, Oil Red, and immunofluorescence. A workflow of the data analysis (IRRSmetrics4stem) was designed to identify spectral predictors of adipogenesis and test machine-learning (ML) methods (hierarchical clustering, PCA, PLSR) for the control of the AD-MSCs differentiation degree. IRRSmetrics4stem provided insights into the chemism of adipogenesis. With single-cell tracking, we established IRRS metrics for lipids, proteins, and DNA variations during AD-MSCs differentiation. The over 90% predictive efficiency of the selected ML methods proved the high sensitivity of the IRRS metrics. Importantly, the IRRS metrics unequivocally recognize a switch from proliferation to differentiation. This study introduced a new bioassay identifying molecular markers indicating molecular transformations and delivering rapid and machine learning-based monitoring of adipogenesis that can be relevant to other differentiation processes. Thus, we introduce a novel, rapid, machine learning-based bioassay to identify molecular markers of adipogenesis. It can be relevant to identification of differentiation-related molecular processes in other cell types, and beyond the cell differentiation including progression of different cellular pathophysiologies reconstituted in vitro.

Circulating endothelial cell progenitors originating from the bone marrow are considered to be a powerful tool in the repair of endothelium damage. Due to their unique properties, endothelial progenitors are now broadly investigated to assess their clinical significance in diseases e.g., associated with brain endothelial dysfunction. However, their distinction in terms of the expression of specific markers remains ambiguous. Additionally, endothelial progenitor cells may change their repertoire of markers depending on the microenvironment of the tissue in which they are currently located. Here, we applied the label-free Raman and FTIR imaging to discriminate mice brain endothelium and endothelial progenitors. Cells cultured separately showed distinctly different spectral signatures extracted from the whole cellular interior as well as the detected intracellular compartments (nucleus, cytoplasm, perinuclear area, and lipid droplets). Then, we used these spectroscopic signals to examine the cells co-cultured for 24 h. Principal cluster analysis showed their grouping with the progenitor cells and segregation from brain endothelium at a level of the entire cell machinery (in FTIR images) which resulted from biochemical alternations in the cytoplasm and lipid droplets (in Raman images). The models included in partial least square regression indicated that lipid droplets are the key element for the classification of endothelial progenitor-brain endothelial cells interactions.

Brain microvascular endothelial cells experience hypoxic conditions in several neurodegenerative disease processes and the underlying mechanisms still need to be explored. Current imaging modalities and biochemical assays require many specific markers that should be detected to identify the hypoxic response, especially at a level of single cells. This study presents a single-cell molecular imaging approach utilizing Fourier-Transform Infrared and Raman spectroscopy. Those methods enable the simultaneous detection of proteins, lipids, and nucleic acids encoded in their unique vibrational fingerprints. By establishing ratiometric estimators, we measured upregulated lipid metabolism, structural changes of proteins and asses DNA:RNA ratio at the single-cell level induced by oxygen depletion. Moreover, this approach allows for analyzing changes within specific cellular compartments, including nuclei, providing a comprehensive understanding of how hypoxia affects cellular functions and metabolism. Our findings pave the way for future investigations into the cellular adaptations to hypoxia in brain endothelial cells, potentially revealing novel therapeutic targets for neurodegenerative diseases.

Acetylcholinesterase (AChE) and cholinergic receptors play an important role in the immune system, including lymphocyte-induced angiogenesis. However, their exact role is not fully understood. The presented work tests the influence of isopropyl methylphosphonofluoridate (IMPF), an irreversible inhibitor of AChE, on selected immune parameters associated with angiogenesis in mice. The levels of VEGF, bFGF, TNF-α, and IFN-γ production were measured, together with the ability of lymphoid spleen cells to induce local GvH reaction after a single dose of IMPF.Experiments were performed in male BALB/c mice. Acetylcholinesterase activity in erythrocytes was determined by the Ellman`s procedure. Levels of cytokines were measured in serum using standard commercial ELISA kits. Influence of IMPF intoxication upon angiogenesis was examined by the LIA test, according to the Sidky and Auerbach procedure.The results showed a 6- and 8-fold increase in VEGF at days 1 and 7 of the experiment, respectively, as well as a decrease (at days 14 and 21 after administration), followed by a significant increase (day 1) in bFGF levels. A statistically significant decrease in the concentration of IFN-γ was observed throughout all experiments. The maximum decrease in the level of TNF-α was found at days 1 and 7 after administration of IMPF. Additionally, a significant decrease was found in the ability to form new blood vessels following IMPF administration.This study revealed that IMPF has a significant effect on the regulation of lymphocyte-induced angiogenesis, which is related with the modulation of angiogenic and pro-inflammatory cytokines secretion. The observed differences suggest a possible derangement of certain elements of the neuronal and/or non-neuronal cholinergic system.