Female-age-dependent changes in the lipid fingerprint of the mammalian oocytes.

STUDY QUESTION: Can oocyte functionality be assessed by observing changes in their intracytoplasmic lipid droplets (LDs) profiles? SUMMARY ANSWER: Lipid profile changes can reliably be detected in human oocytes; lipid changes are linked with maternal age and impaired developmental competence in a mouse model. WHAT IS KNOWN ALREADY: In all cellular components, lipid damage is the earliest manifestation of oxidative stress (OS), which leads to a cascade of negative consequences for organelles and DNA. Lipid damage is marked by the accumulation of LDs. We hypothesized that impaired oocyte functionality resulting from aging and associated OS could be assessed by changes in LDs profile, hereafter called lipid fingerprint (LF). STUDY DESIGN, SIZE, DURATION: To investigate if it is possible to detect differences in oocyte LF, we subjected human GV-stage oocytes to spectroscopic examinations. For this, a total of 48 oocytes derived from 26 young healthy women (under 33 years of age) with no history of infertility, enrolled in an oocyte donation program, were analyzed. Furthermore, 30 GV human oocytes from 12 women were analyzed by transmission electron microscopy (TEM). To evaluate the effect of oocytes' lipid profile changes on embryo development, a total of 52 C57BL/6 wild-type mice and 125 Gnpat+/- mice were also used. PARTICIPANTS/MATERIALS, SETTING, METHODS: Human oocytes were assessed by label-free cell imaging via coherent anti-Stokes Raman spectroscopy (CARS). Further confirmation of LF changes was conducted using spontaneous Raman followed by Fourier transform infrared (FTIR) spectroscopies and TEM. Additionally, to evaluate whether LF changes are associated with developmental competence, mouse oocytes and blastocysts were evaluated using TEM and the lipid dyes BODIPY and Nile Red. Mouse embryonic exosomes were evaluated using flow cytometry, FTIR and FT-Raman spectroscopies. MAIN RESULTS AND THE ROLE OF CHANCE: Here we demonstrated progressive changes in the LF of oocytes associated with the woman's age consisting of increased LDs size, area, and number. LF variations in oocytes were detectable also within individual donors. This finding makes LF assessment a promising tool to grade oocytes of the same patient, based on their quality. We next demonstrated age-associated changes in oocytes reflected by lipid peroxidation and composition changes; the accumulation of carotenoids; and alterations of structural properties of lipid bilayers. Finally, using a mouse model, we showed that LF changes in oocytes are negatively associated with the secretion of embryonic exosomes prior to implantation. Deficient exosome secretion disrupts communication between the embryo and the uterus and thus may explain recurrent implantation failures in advanced-age patients. LIMITATIONS, REASONS FOR CAUTION: Due to differences in lipid content between different species' oocytes, the developmental impact of lipid oxidation and consequent LF changes may differ across mammalian oocytes. WIDER IMPLICATIONS OF THE FINDINGS: Our findings open the possibility to develop an innovative tool for oocyte assessment and highlight likely functional connections between oocyte LDs and embryonic exosome secretion. By recognizing the role of oocyte LF in shaping the embryo's ability to implant, our original work points to future directions of research relevant to developmental biology and reproductive medicine. STUDY FUNDING/COMPETING INTEREST(S): This research was funded by National Science Centre of Poland, Grants: 2021/41/B/NZ3/03507 and 2019/35/B/NZ4/03547 (to G.E.P.); 2022/44/C/NZ4/00076 (to M.F.H.) and 2019/35/N/NZ3/03213 (to L.G.). M.F.H. is a National Agency for Academic Exchange (NAWA) fellow (GA ULM/2019/1/00097/U/00001). K.F. is a Diamond Grant fellow (Ministry of Education and Science GA 0175/DIA/2019/28). The open-access publication of this article was funded by the Priority Research Area BioS under the program "Excellence Initiative - Research University" at the Jagiellonian University in Krakow. The authors declare no competing interest. TRIAL REGISTRATION NUMBER: N/A.

New Highly Sensitive and Specific Raman Probe for Live Cell Imaging of Mitochondrial Function.

For Raman hyperspectral detection and imaging in live cells, it is very desirable to create novel probes with strong and unique Raman vibrations in the biological silent region (1800-2800 cm-1). The use of molecular probes in Raman imaging is a relatively new technique in subcellular research; however, it is developing very rapidly. Compared with the label-free method, it allows for a more sensitive and selective visualization of organelles within a single cell. Biological systems are incredibly complex and heterogeneous. Directly visualizing biological structures and activities at the cellular and subcellular levels remains by far one of the most intuitive and powerful ways to study biological problems. Each organelle plays a specific and essential role in cellular processes, but importantly for cells to survive, mitochondrial function must be reliable. Motivated by earlier attempts and successes of biorthogonal chemical imaging, we develop a tool supporting Raman imaging of cells to track biochemical changes associated with mitochondrial function at the cellular level in an in vitro model. In this work, we present a newly synthesized highly sensitive RAR-BR Raman probe for the selective imaging of mitochondria in live endothelial cells.

Raman microscopy reveals how cell inflammation activates glucose and lipid metabolism.

Metabolism of endothelial cells (ECs) depends on the availability of the energy substrates. Since the endothelium is the first line of defence against inflammation in the cardiovascular system and its dysfunction can lead to the development of cardiovascular diseases, it is important to understand how glucose metabolism changes during inflammation. In this work, glucose uptake was studied in human microvascular endothelial cells (HMEC-1) in high glucose (HG), and additionally in an inflammatory state, using Raman imaging. HG state was induced by incubation of ECs with a deuterated glucose analogue, while the EC inflammation was caused by TNF-α pre-treatment. Spontaneous and stimulated Raman scattering spectroscopy provided comprehensive information on biochemical changes, including lipids and the extent of unsaturation induced by excess glucose in ECs., induced by excess glucose in ECs. In this work, we indicated spectroscopic markers of metabolic changes in ECs as a strong increase in the ratio of the intensity of lipids / (proteins + lipids) bands and an increase in the level of lipid unsaturation and mitochondrial changes. Inflamed ECs treated with HG, revealed enhanced glucose uptake, and intensified lipid production i.a. of unsaturated lipids. Additionally, increased cytochrome c signal in the mitochondrial region indicated higher mitochondrial activity and biogenesis. Raman spectroscopy is a powerful method for determining the metabolic markers of ED which will better inform understanding of disease onset, development, and treatment.

Modified glucose as a sensor to track the metabolism of individual living endothelial cells - Observation of the 1602 cm-1 band called "Raman spectroscopic signature of life".

A relatively new approach to subcellular research is Raman microscopy with the application of sensors called Raman probes. This paper describes the use of the sensitive and specific Raman probe, 3-O-propargyl-d-glucose (3-OPG), to track metabolic changes in endothelial cells (ECs). ECs play a significant role in a healthy and dysfunctional state, the latter is correlated with a range of lifestyle diseases, particularly with cardiovascular disorders. The metabolism and glucose uptake may reflect the physiopathological conditions and cell activity correlated with energy utilization. To study metabolic changes at the subcellular level the glucose analogue, 3-OPG was used, which shows a characteristic and intense Raman band at 2124 cm-1.3-OPG was applied as a sensor to track both, its accumulation in live and fixed ECs and then metabolism in normal and inflamed ECs, by employing two spectroscopic techniques, i.e. spontaneous and stimulated Raman scattering microscopies. The results indicate that 3-OPG is a sensitive sensor to follow glucose metabolism, manifested by the Raman band of 1602 cm-1. The 1602 cm-1 band has been called the "Raman spectroscopic signature of life" in the cell literature, and here we demonstrate that it is attributed to glucose metabolites. Additionally, we have shown that glucose metabolism and its uptake are slowed down in the cellular inflammation. We showed that Raman spectroscopy can be classified as metabolomics, and its uniqueness lies in the fact that it allows the analysis of the processes of a single living cell. Gaining further knowledge on metabolic changes in the endothelium, especially in pathological conditions, may help in identifying markers of cellular dysfunction, and more broadly in cell phenotyping, better understanding of the mechanism of disease development and searching for new treatments.

Reliable cell preparation protocol for Raman imaging to effectively differentiate normal leukocytes and leukemic blasts.

Leukemias are a remarkably diverse group of malignancies originating from abnormal progenitor cells in the bone marrow. Leukemia subtypes are classified according to the cell type that has undergone neoplastic transformation using demanding and time-consuming methods. Alternative is Raman imaging that can be used both for living and fixed cells. However, considering the diversity of leukemic cell types and normal leukocytes, and the availability of different sample preparation protocols, the main objective of this work was to verify them for leukemia and normal blood cell samples for Raman imaging. The effect of glutaraldehyde (GA) fixation in a concentration gradient (0.1 %, 0.5 %, and 2.5 % GA) on the molecular structure of T-cell acute lymphoblastic leukemia (T-ALL) and peripheral blood mononuclear cells (PBMCs) was verified. Changes in the secondary structure of proteins within cells were indicated as the main effect of fixation, as shown by an increase in band intensity at 1041 cm-1, characteristic for in-plane δ(CH) deformation in phenylalanine (Phe). Different sensitivity of mononuclear and leukemic cells to fixation was observed. While the 0.1 % concentration of GA was too low to preserve the cell structure for an extended period of time, a GA concentration of 0.5 % seemed optimal for both normal and malignant cells. Chemical changes in PBMCs samples stored for 11 days were also investigated, which manifested in numerous modifications in the secondary structure of proteins and the content of nucleic acids. The impact of cell preculturing for 72 h after unbanking was verified, and there was no significant effect on the molecular structure of cells fixed with 0.5 % GA. In summary, the developed protocol for the preparation of samples for Raman imaging allows for the effective differentiation of fixed normal leukocytes from malignant T lymphoblasts.

Changes in the mitochondrial membrane potential in endothelial cells can be detected by Raman microscopy.

The role of mitochondria goes beyond their capacity to create molecular fuel and includes e.g. the production of reactive oxygen species and the regulation of cell death. In endothelial cells, mitochondria have a significant impact on cellular function under both healthy and pathological conditions. Endothelial dysfunction contributes to the development of various lifestyle diseases and the key players in their pathogenesis are among others vascular inflammation and oxidative stress. The latter is very closely related to mitochondrial dysfunction; however, it is not straightforward. First, because mitochondria are small cellular structures, and second, it requires a sensitive method to follow the subtle biochemical changes. For this purpose, Raman microscopy (RM) was used here, which is considered a high-resolution method and can be applied in situ, usually as a non-labeled technique. In this work, we show that RM can not only locate mitochondria in the cell but also track their functional changes. Moreover, we test if labeling cells with Raman probes (Rp) can improve the specificity and sensitivity of RM (compared to conventional labeled techniques such as fluorescence, and the non-labeled Raman technique). MitoBADY Rp was used to detect changes in mitochondrial membrane potential as an indicator of mitochondrial activity, e.g. hyperpolarization or distortion of the proton gradient in the intermembrane space (depolarization). Thus, we show and compare RM, in the form of a label and non-labeled, to such a subtle cellular analysis.

Raman-based spectrophenotyping of the most important cells of the immune system.

INTRODUCTION: Human peripheral blood mononuclear cells (PBMCs) are a heterogeneous population of cells that includes T and B lymphocytes. The total number of lymphocytes and their percentage in the blood can be a marker for the diagnosis of several human diseases. Currently, cytometric methods are widely used to distinguish subtypes of leukocytes and quantify their number. These techniques use cell immunophenotyping, which is limited by the number of fluorochrome-labeled antibodies that can be applied simultaneously. OBJECTIVE: B and T lymphocytes were isolated from peripheral blood obtained from healthy human donors. METHODS: The immunomagnetic negative selection was used for the enrichment of B and T cells fractions, and their purity was assessed by flow cytometry. Isolated cells were fixed with 0.5% glutaraldehyde and measured using confocal Raman imaging. K-means cluster analysis, principal component analysis and partial least squares discriminant methods were applied for the identification of spectroscopic markers to distinguish B and T cells. HPLC was the reference method for identifying carotene in T cells. RESULTS: Reliable discrimination between T and B lymphocytes based on their spectral profile has been demonstrated using label-free Raman imaging and chemometric analysis. The presence of carotene in T lymphocytes (in addition to the previously reported in plasma) was confirmed and for the first time unequivocally identified as ß-carotene. In addition, the molecular features of the lymphocytes nuclei were found to support the discriminant analysis. It has been shown that although the presence of carotenoids in T cells depends on individual donor variability, the reliable differentiation between lymphocytes is possible based on Raman spectra collected from individual cells. CONCLUSIONS: This proves the potential of Raman spectroscopy in clinical diagnostics to automatically differentiate between cells that are an important component of our immune system.

What is the ability of inflamed endothelium to uptake exogenous saturated fatty acids? A proof-of-concept study using spontaneous Raman, SRS and CARS microscopy.

Endothelial cells (EC) in vivo buffer and regulate the transfer of plasma fatty acid (FA) to the underlying tissues. We hypothesize that inflammation could alter the functionality of the EC, i.e., their capacity and uptake of different FA. The aim of this work is to verify the functionality of inflamed cells by analyzing their ability to uptake and accumulate exogenous saturated FA. Control and inflammatory human microvascular endothelial cells stimulated in vitro with two deuterium-labeled saturated FA (D-FA), i.e., palmitic (D31-PA) and myristic (D27-MA) acids. Cells were measured both by spontaneous and stimulated Raman imaging to extract detailed information about uptaken FA, whereas coherent anti-Stokes Raman scattering and fluorescence imaging showed the global content of FA in cells. Additionally, we employed atomic force microscopy to obtain a morphological image of the cells. The results indicate that the uptake of D-FA in inflamed cells is dependent on their concentration and type. Cells accumulated D-FA when treated with a low concentration, and the effect was more pronounced for D27-MA, in normal cells, but even more so, in inflamed cells. In the case of D31-PA, a slightly increased uptake was observed for inflamed cells when administered at higher concentration. The results provide a better understanding of the EC inflammation and indicate the impact of the pathological state of the EC on their capacity to buffer fat. All the microscopic methods used showed complementarity in the analysis of FA uptake by EC, but each method recognized this process from a different perspective.

Multiplex Raman imaging of organelles in endothelial cells.

Raman imaging using molecular reporters is a relatively new approach in subcellular investigations. It enables the visualization of organelles in cells with better selectivity and sensitivity compared to the label-free approach. Essentially Raman reporters possess in their structure an alkyne molecular group that can be selectively identified in the spectral region silent for biomolecules, hence facilitate the localization of individual organelles. The aim of this work is to visualize the main cell organelles in endothelial cells (HMEC-1) using established reporters (EdU and MitoBADY), but also to test a new one, namely falcarinol, which exhibits lipophilic properties. Moreover, we tested the possibility to use Raman reporters as a probe to detect changes in distribution of certain organelles after induced endothelial dysfunction (ED) in in vitro models. In both cases, induced ED is characterized by the formation of lipid droplets in the cells, which is why a good tool for the detection of lipid-rich organelles is so important in these studies. Two-dimensional Raman images were obtained, visualizing the distribution of selected organic compounds in the cell, such as proteins, lipids, and nucleic acids. Additionally, the distribution of EdU, MitoBADY and falcarinol in endothelial cells (ECs) was determined. Moreover, we highlight some drawback of established Raman reporter and the need for testing them in various physiological state of the cell.

[Prevalence IgG antibodies against Bartonella henselae in children with lymphadenopathy]. / Przeciwciala klasy IgG swoiste dla Bartonella henselae u dzieci z limfadenopatia.

Bartonella henselae is a cat scratch disease's etiological agent which is usually manifestated as regional lymphadenopathy. In differential diagnosis of lymphadenopathy infections about etiology B. henselae are rarely taken into consideration. Enlargement of lymph nodes observed in children more often than in adults are caused by bacterial, virus or parasitic factors. In this study immunoglobulines G class antibodies to B. henselae were determined among children with limphadenopathy. At 53 children with recognized lymphadenopathy IgG antibodies were determined by indirect immunofluoroscence method specific for B. henselae. Of the 53 subjects examined, positive results were got at 29 (55%) children. Of the 23 children with negative results of IgG antibodies in 9 children study was repeated. In 5 (56%) cases the increase of IgG antibodies were shown with relation to the first research. The cat scratch disease should be considered as a cause of lymphadenopathy at children because the frequency of occurance of antibodies IgG specific for B. henselae is high. In case of getting negative results, participation of B. henselae should not be out of question in limphadenopathy etiology at children and second determination should be repeted after 10-21 days since the first one.