Human erythrocytes under stress. Spectroscopic fingerprints of known oxidative mechanisms and beyond.

In this work, we investigated the oxidative stress-related biochemical alterations in red blood cells (RBCs) and their membranes with the use of spectroscopic techniques. We aimed to show their great advantage for the in situ detection of lipid classes and secondary structures of proteins without the need for their extraction in the cellular environment. The exposition of the cells to peroxides, t-butyl hydroperoxide (tBOOH) or hydrogen peroxide (H2O2) led to different degradation processes encompassing the changes in the composition of membranes and structural modifications of hemoglobin (Hb). Our results indicated that tBOOH is generally a stronger oxidizing agent than H2O2 and this observation was congruent with the activity of superoxide and glutathione peroxidase. ATR-FTIR and Raman spectroscopies of membranes revealed that tBOOH caused primarily the partial loss and peroxidation of the lipids resulting in loss of the integrity of membranes. In turn, both peroxides induced several kinds of damage in the protein layer, including the partial decrease of their content and irreversible aggregation of spectrin, ankyrin, and membrane-bound globin. These changes were especially pronounced on the membrane surface where stress conditions induced the formation of ß-sheets and intramolecular aggregates, particularly for tBOOH. Interestingly, nano-FTIR spectroscopy revealed the lipid peroxidative damage on the membrane surface in both cases. As far as hemoglobin was concerned, tBOOH and H2O2 caused the increase of the oxyhemoglobin species and conformational alterations of its polypeptide chain into ß-sheets. Our findings confirm that applied spectroscopies effectively track the oxidative changes occurring in the structural components of red blood cells and the simplicity of conducting measurements and sample preparation can be readily applied to pharmacological and clinical studies.

Molecular tracking of interactions between progenitor and endothelial cells via Raman and FTIR spectroscopy imaging: a proof of concept of a new analytical strategy for in vitro research.

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.

Biomolecular composition of porcine ovarian follicles following in vitro treatment of vitamin D3 and insulin alone or in combination.

The study aimed to analyze changes in biomolecular composition of granulosa and theca interna cells of porcine ovarian follicles following in vitro treatment of vitamin D3 and insulin alone or in combination. Medium antral follicles (n = 4/each group) were cultured alone (C; control) or in the presence of 1α,25(OH)2D3 (VD; 100 ng/mL) and insulin (I; 10 ng/mL) separately or in combination, VD and I (VD+I). Then paraplast-embedded ovarian follicles were used for Fourier Transform Infrared (FTIR) spectroscopy and respective histological stainings. FTIR analysis revealed changes in the content of fibrous proteins (mainly collagens) within theca interna following vitamin D3 and insulin co-administration that was verified by Masson's trichrome staining. Treatment-dependent differences were also observed in the secondary structure of proteins, indicating enhanced conversion to α-helices in response to vitamin D3 and insulin action/interaction in both follicular compartments. In the granulosa and theca interna layers, tendency to lower DNA content in the VD+I group was noted and confirmed by Fulgen's staining. Finally, altered monosaccharides production in both follicular layers was found. Based on FTIR results, it is possible to attribute the observed alterations to biological processes that could be regulated by vitamin D3 and insulin in the porcine ovarian follicles.

Fourier transform IR imaging of primary tumors predicts lymph node metastasis of bladder carcinoma.

The process of metastasis is complex and often impossible to be recognized in conventional clinical diagnosis. Lymph node metastasis (LNM) of bladder carcinoma (BC) is often associated with muscle-invasive tumors. To prevent and recognize LNM, the standard treatment includes radical cystectomy with lymph node dissection and histological examination. Here, we propose infrared (IR) microscopy as a tool for the prediction of LNM. For this purpose, IR images of bladder biopsies from patients with diagnosed non-metastatic early (E BC) and advanced (A BC), as well as metastatic advanced (M BC) bladder cancer were first collected. Furthermore, this dataset was complemented with images of the secondary tumors from the lymph nodes (M LN) of the M BC patients. Unsupervised clustering was used to extract tissue structures from IR images to create a data set comprising 382 IR spectra of non-metastatic bladder tumors and 241 metastatic ones. Based on that, we next established discrimination models using PLS-DA with repeated random sampling double cross-validation, and permutation test to perform the classification. The accuracy of BC metastasis prediction from IR bladder biopsies was 83 % and 78 % for early and advanced BC, respectively, herein demonstrating a proof-of-concept IR detection of BC metastasis. The analysis of spectral profiles additionally showed molecular composition similarity between metastatic bladder and lymph node tumors. We also determined spectral biomarkers of LNM that are associated with sugar metabolism, remodeling of extracellular matrix, and morphological features of cancer cells. Our approach can improve clinical decision-making in urological oncology.

Evaluation of grade and invasiveness of bladder urothelial carcinoma using infrared imaging and machine learning.

Urothelial bladder carcinoma (BC) is primarily diagnosed with a subjective examination of biopsies by histopathologists, but accurate diagnosis remains time-consuming and of low diagnostic accuracy, especially for low grade non-invasive BC. We propose a novel approach for high-throughput BC evaluation by combining infrared (IR) microscopy of bladder sections with machine learning (partial least squares-discriminant analysis) to provide an automated prediction of the presence of cancer, invasiveness and grade. Cystoscopic biopsies from 50 patients with clinical suspicion of BC were histologically examined to assign grades and stages. Adjacent tissue cross-sections were IR imaged to provide hyperspectral datasets and cluster analysis segregated IR images to extract the average spectra of epithelial and subepithelial tissues. Discriminant models, which were validated using repeated random sampling double cross-validation, showed sensitivities (AUROC) ca. 85% (0.85) for the identification of cancer in epithelium and subepithelium. The diagnosis of non-invasive and invasive cases showed sensitivity values around 80% (0.84-0.85) and 76% (0.73-0.80), respectively, while the identification of low and high grade BC showed higher sensitivity values 87-88% (0.91-0.92). Finally, models for the discrimination between cancers with different invasiveness and grades showed more modest AUROC values (0.67-0.72). This proves the high potential of IR imaging in the development of ancillary platforms to screen bladder biopsies.

Endothelial-mesenchymal transition induced by metastatic 4T1 breast cancer cells in pulmonary endothelium in aged mice.

Ageing is a major risk factor for cancer metastasis but the underlying mechanisms remain unclear. Here, we characterised ageing effects on cancer-induced endothelial-mesenchymal transition (EndMT) in the pulmonary circulation of female BALB/c mice in a metastatic 4T1 breast cancer model. The effect of intravenously injected 4T1 cells on pulmonary endothelium, pulmonary metastasis, lung tissue architecture, and systemic endothelium was compared between 40-week-old and 20-week-old mice. The 40-week-old mice showed features of ongoing EndMT in their lungs before 4T1 breast cancer cell injection. Moreover, they had preexisting endothelial dysfunction in the aorta detected by in vivo magnetic resonance imaging (MRI) compared to 20-week-old mice. The injection of 4T1 breast cancer cells into 40-week-old mice resulted in rapid EndMT progression in their lungs. In contrast, injection of 4T1 breast cancer cells into 20-week-old mice resulted in initiation and less pronounced EndMT progression. Although the number of metastases did not differ significantly between 20-week-old and 40-week-old mice, the lungs of older mice displayed altered lung tissue architecture and biochemical content, reflected in higher Amide II/Amide I ratio, higher fibronectin levels, and hypoxia-inducible factor 1 subunit alpha (HIF1α) levels as well as lower nitric oxide (NO) production. Our results indicate that age-dependent pre-existing endothelial dysfunction in the pulmonary endothelium of 40-week-old mice predisposed them to rapid EndMT progression in the presence of circulating 4T1 breast cancer cells what might contribute to a more severe metastatic breast cancer phenotype in these ageing mice compared to younger mice.

Photoelectrochemical Performance of Nanotubular Fe2O3-TiO2 Electrodes under Solar Radiation.

Fe2O3-TiO2 materials were obtained by the cathodic electrochemical deposition of Fe on anodic TiO2 at different deposition times (5-180 s), followed by annealing at 450 °C. The effect of the hematite content on the photoelectrochemical (PEC) activity of the received materials was studied. The synthesized electrodes were characterized by field emission scanning electron microscopy (FE-SEM), energy dispersive spectroscopy (EDS), X-ray diffraction (XRD), Raman spectroscopy, diffuse reflectance spectroscopy (DRS), Mott-Schottky analysis, and PEC measurements. It was shown that the amount of deposited iron (ca. 0.5 at.%-30 at.%) and, consequently, hematite after a final annealing increased with the extension of deposition time and directly affected the semiconducting properties of the hybrid material. It was observed that the flat band potential shifted towards more positive values, facilitating photoelectrochemical water oxidation. In addition, the optical band gap decreased from 3.18 eV to 2.77 eV, which resulted in enhanced PEC visible-light response. Moreover, the Fe2O3-TiO2 electrodes were sensitive to the addition of glucose, which indicates that such materials may be considered as potential PEC sensors for the detection of glucose.

High-Resolution Fourier Transform Infrared (FT-IR) Spectroscopic Imaging for Detection of Lung Structures and Cancer-Related Abnormalities in a Murine Model.

Label-free molecular imaging is a promising utility to study tissues in terms of the identification of their compartments as well as chemical features and alterations induced by disease. The aim of this work was to assess if higher magnification of optics in the Fourier transform infrared (FT-IR) microscope coupled with the focal plane detector resulted in better resolution of lung structures and if the histopathological features correlated with clustering of spectral images. FT-IR spectroscopic imaging was performed on paraffinized lung tissue sections from mice with optics providing a total magnification of 61× and 36×. Then, IR images were subjected to unsupervised cluster analysis and, subsequently, cluster maps were compared with hematoxylin and eosin staining of the same tissue section. Based on these results, we observed minute features such as cellular compartments in single alveoli and bronchiole, blood cells and megakaryocytes in a vessel as well as atelectasis of the lung. In the case of the latter, differences in composition were also noted between the tissue from the non-cancerous and cancerous specimen. This study demonstrated the ability of high-definition FT-IR imaging to evaluate the chemical features of well-resolved lung structures that could complement the histological examination widely used in animal models of disease.

Dual-enhancement and dual-tag design for SERS-based sandwich immunoassays: evaluation of a metal-metal effect in 3D architecture.

The design of a sandwich-type SERS immunoassay (surface-enhanced Raman spectroscopy) is demonstrated operating in dual surface enhancement and dual-tag paradigm. The capture and detection antibodies are linked to two SERS-active substrates and form together the three-dimensional (3D) structure after specific binding to interleukin 6. A variety of metal combinations is tested (Au-Ag, Au-Au, and Ag-Ag), but an enhanced electromagnetic field is generated only due to coupling of Ag and Au nanoparticles with an Au hexagonal nanoarray. The amplified in that way Raman signals improve the limit of detection over 3 times in comparison to the assay with only one SERS-active substrate. It is also shown that the proper readout of the true-positive signal can be achieved in assays with two Raman tags, and this approach also improves LOD. For the optimal combination of the metal-metal junction and Raman tags, a linear relationship between the Raman signal and the concentration of IL-6 is obtained in the range 0-1000 pg⋅mL-1with LOD of 25.2 pg mL-1and RSD < 10%. The presented proof-of-concept of the SERS immunoassay with the dual-enhancement and dual-tag opens additional opportunities for engineering reliable SERS biosensing.

Identification of inflammatory markers in eosinophilic cells of the immune system: fluorescence, Raman and CARS imaging can recognize markers but differently.

Eosinophils (Eos) play an important role in the immune system's response releasing several inflammatory factors and contributing to allergic rhinitis, asthma, or atopic dermatitis. Since Eos have a relatively short lifetime after isolation from blood, usually eosinophilic cell line (EoL-1) is used to study mechanisms of their activation and to test therapies. In particular, EoL-1 cells are examined in terms of signalling pathways of the inflammatory response manifested by the presence of lipid bodies (LBs). Here we examined the differences in response to inflammation modelled by various factors, between isolated human eosinophils and EoL-1 cells, as manifested in the number and chemical composition of LBs. The analysis was performed using fluorescence, Raman, and coherent anti-Stokes Raman scattering (CARS) microscopy, which recognised the inflammatory process in the cells, but it is manifested slightly differently depending on the method used. We showed that unstimulated EoL-1 cells, compared to isolated eosinophils, contained more LBs, displayed different nucleus morphology and did not have eosinophilic peroxidase (EPO). In EoL-1 cells stimulated with various proinflammatory agents, including butyric acid (BA), liposaccharide (LPS), or cytokines (IL-1ß, TNF-α), an increased production of LBs with a various degree of lipid unsaturation was observed in spontaneous Raman spectra. Furthermore, stimulation of EoL-1 cells resulted in alterations of the LBs morphology. In conclusion, a level of lipid unsaturation and eosinophilic peroxidase as well as LBs distribution among cell population mainly accounted for the biochemistry of eosinophils upon inflammation.

Response of physiological parameters in Dionaea muscipula J. Ellis teratomas transformed with rolB oncogene.

BACKGROUND: Plant transformation with rol oncogenes derived from wild strains of Rhizobium rhizogenes is a popular biotechnology tool. Transformation effects depend on the type of rol gene, expression level, and the number of gene copies incorporated into the plant's genomic DNA. Although rol oncogenes are known as inducers of plant secondary metabolism, little is known about the physiological response of plants subjected to transformation. RESULTS: In this study, the physiological consequences of rolB oncogene incorporation into the DNA of Dionaea muscipula J. Ellis was evaluated at the level of primary and secondary metabolism. Examination of the teratoma (transformed shoots) cultures of two different clones (K and L) showed two different strategies for dealing with the presence of the rolB gene. Clone K showed an increased ratio of free fatty acids to lipids, superoxide dismutase activity, synthesis of the oxidised form of glutathione, and total pool of glutathione and carotenoids, in comparison to non-transformed plants (control). Clone L was characterised by increased accumulation of malondialdehyde, proline, activity of superoxide dismutase and catalase, total pool of glutathione, ratio of reduced form of glutathione to oxidised form, and accumulation of selected phenolic acids. Moreover, clone L had an enhanced ratio of total triglycerides to lipids and accumulated saccharose, fructose, glucose, and tyrosine. CONCLUSIONS: This study showed that plant transformation with the rolB oncogene derived from R. rhizogenes induces a pleiotropic effect in plant tissue after transformation. Examination of D. muscipula plant in the context of transformation with wild strains of R. rhizogenes can be a new source of knowledge about primary and secondary metabolites in transgenic organisms.

FTIR Spectroscopic Imaging Supports Urine Cytology for Classification of Low- and High-Grade Bladder Carcinoma.

Bladder urothelial carcinoma (BC) is a common, recurrent, life-threatening, and unpredictable disease which is difficult to diagnose. These features make it one of the costliest malignancies. Although many possible diagnostic methods are available, molecular heterogeneity and difficulties in cytological or histological examination induce an urgent need to improve diagnostic techniques. Herein, we applied Fourier transform infrared spectroscopy in imaging mode (FTIR) to investigate patients' cytology samples assigned to normal (N), low-grade (LG) and high-grade (HG) BC. With unsupervised hierarchical cluster analysis (UHCA) and hematoxylin-eosin (HE) staining, we observed a correlation between N cell types and morphology. High-glycogen superficial (umbrella) and low-glycogen piriform urothelial cells, both with normal morphology, were observed. Based on the spectra derived from UHCA, principal component analysis (PCA) and partial least squares discriminant analysis (PLS-DA) were performed, indicating a variation of protein content between the patient groups. Moreover, BC spectral cytology identified a low number of high-glycogen cells for which a shift of the carbohydrate/phosphate bands was also observed. Despite high cellular heterogeneity, PLS-DA was able to classify the spectra obtained. The voided urine FTIR cytology is one of the options that might be helpful in BC diagnosis, as high sensitivity and specificity up to 97% were determined.

Molecular profiling of the intestinal mucosa and immune cells of the colon by multi-parametric histological techniques.

The impact of the post-mortem interval (PMI) on the optical molecular characteristics of the colonic mucosa and the gut-associated lymphoid tissue (GALT) were examined by multi-parametric measurements techniques. Inflammatory cells were identified by immunohistochemical staining. Molecular parameters were estimated using the Raman spectroscopy (RS) and Fourier Transform Infrared (FTIR) spectroscopic imaging. The 3D refractive index (3D-RI) distributions of samples were determined using the digital holographic tomography. The distribution of immune cells between post-mortem (PM) and normal controls did show significant differences for CD4 (P = 0.0016) or CD8 (P < 0.0001), whose expression level was decreased in PM cases. No association was found between individual PMI values and inflammatory cell distribution. However, there was a tendency for a negative correlation between CD4+ cells and PMI (r = - 0.542, P = 0.032). The alterations ongoing in post-mortem tissue may suggest that PMI has a suppressive effect on the effector properties of the cell-mediated immunity. Moreover, it was confirmed that spectroscopic and digital holotomographic histology are also a useful technique for characterization of the differences in inflammation of varying intensity and in GALT imaging in a solid tissue. Anatomical location of immune cells and methods of tissue fixation determine the molecular and optical parameters of the examined cases.

Spectroscopic Signature of Red Blood Cells in a D-Galactose-Induced Accelerated Aging Model.

This work presents a semi-quantitative spectroscopic approach, including FTIR-ATR and Raman spectroscopies, for the biochemical analysis of red blood cells (RBCs) supported by the biochemical, morphological and rheological reference techniques. This multi-modal approach provided the description of the RBC alterations at the molecular level in a model of accelerated aging induced by administration of D-galactose (D-gal), in comparison to natural aging. Such an approach allowed to conclude that most age-related biochemical RBC membrane changes (a decrease in lipid unsaturation and the level of phospholipids, or an increase in acyl chain shortening) as well as alterations in the morphological parameters and RBC deformability are well reflected in the D-gal model of accelerated aging. Similarly, as in natural aging, a decrease in LDL level in blood plasma and no changes in the fraction of glucose, creatinine, total cholesterol, HDL, iron, or triglycerides were observed during the course of accelerated aging. Contrary to natural aging, the D-gal model led to an increase in cholesterol esters and the fraction of total esterified lipids in RBC membranes, and evoked significant changes in the secondary structure of the membrane proteins. Moreover, a significant decrease in the phosphorous level of blood plasma was specific for the D-gal model. On the other hand, natural aging induced stronger changes in the secondary structures of the proteins of the RBCs' interior. This work proves that research on the aging mechanism, especially in circulation-related diseases, should employ the D-gal model with caution. Nonetheless, the D-gal model enables to imitate age-related rheological alterations in RBCs, although they are partially derived from different changes observed in the RBC membrane at the molecular level.

Intracellular pH - Advantages and pitfalls of surface-enhanced Raman scattering and fluorescence microscopy - A review.

The value of pH in various parts of protoplasm can affect nearly all aspects of cell functions. Therefore, the determination of intracellular acid-base features is required in many areas of biological and biochemical studies. Because of a significant scientific importance of in vivo intracellular pH measurements, various groups carried out such experiments. In this review article we describe intracellular pH measurements using two the most sensitive optical spectroscopies: surface-enhanced Raman scattering (SERS) and fluorescence. It is reasonable to present these two techniques in one review article because the experimental approach in Raman and fluorescence experiments is relatively similar. The basic theoretical background explaining the mechanism of operation of fluorescence and SERS sensors are discussed and the motivations to carry out intracellular pH measurements are briefly described. Future perspectives in this field are also discussed.

Fourier Transform Infrared Polarization Contrast Imaging Recognizes Proteins Degradation in Lungs upon Metastasis from Breast Cancer.

The current understanding of mechanisms underlying the formation of metastatic tumors has required multi-parametric methods. The tissue micro-environment in secondary organs is not easily evaluated due to complex interpretation with existing tools. Here, we demonstrate the detection of structural modifications in proteins using emerging Fourier Transform Infrared (FTIR) imaging combined with light polarization. We investigated lungs affected by breast cancer metastasis in the orthotopic murine model from the pre-metastatic phase, through early micro-metastasis, up to an advanced phase, in which solid tumors are developed in lung parenchyma. The two IR-light polarization techniques revealed, for the first time, the orientational ordering of proteins upon the progression of pulmonary metastasis of breast cancer. Their distribution was complemented by detailed histological examination. Polarized contrast imaging recognised tissue structures of lungs and showed deformations in protein scaffolds induced by inflammatory infiltration, fibrosis, and tumor growth. This effect was recognised by not only changes in absorbance of the spectral bands but also by the band shifts and the appearance of new signals. Therefore, we proposed this approach as a useful tool for evaluation of progressive and irreversible molecular changes that occur sequentially in the metastatic process.

In Vitro Spectroscopy-Based Profiling of Urothelial Carcinoma: A Fourier Transform Infrared and Raman Imaging Study.

Markers of bladder cancer cells remain elusive, which is a major cause of the low recognition of this malignant neoplasm and its recurrence. This implies an urgent need for additional diagnostic tools which are based on the identification of the chemism of bladder cancer. In this study, we employed label-free techniques of molecular imaging-Fourier Transform Infrared and Raman spectroscopic imaging-to investigate bladder cancer cell lines of various invasiveness (T24a, T24p, HT-1376, and J82). The urothelial HCV-29 cell line was the healthy control. Specific biomolecules discriminated spatial distribution of the nucleus and cytoplasm and indicated the presence of lipid bodies and graininess in some cell lines. The most prominent discriminators are the total content of lipids and sugar moieties as well as the presence of glycogen and other carbohydrates, un/saturated lipids, cytochromes, and a level of S-S bridges in proteins. The combination of the obtained hyperspectral database and chemometric methods showed a clear differentiation of each cell line at the level of the nuclei and cytoplasm and pointed out spectral signals which differentiated bladder cancer cells. Registered spectral markers correlated with biochemical composition changes can be associated with pathogenesis and potentially used for the diagnosis of bladder cancer and response to experimental therapies.

Stem Photosynthesis-A Key Element of Grass Pea (Lathyrus sativus L.) Acclimatisation to Salinity.

Grass pea (Lathyrus sativus) is a leguminous plant of outstanding tolerance to abiotic stress. The aim of the presented study was to describe the mechanism of grass pea (Lathyrus sativus L.) photosynthetic apparatus acclimatisation strategies to salinity stress. The seedlings were cultivated in a hydroponic system in media containing various concentrations of NaCl (0, 50, and 100 mM), imitating none, moderate, and severe salinity, respectively, for three weeks. In order to characterise the function and structure of the photosynthetic apparatus, Chl a fluorescence, gas exchange measurements, proteome analysis, and Fourier-transform infrared spectroscopy (FT-IR) analysis were done inter alia. Significant differences in the response of the leaf and stem photosynthetic apparatus to severe salt stress were observed. Leaves became the place of harmful ion (Na+) accumulation, and the efficiency of their carboxylation decreased sharply. In turn, in stems, the reconstruction of the photosynthetic apparatus (antenna and photosystem complexes) activated alternative electron transport pathways, leading to effective ATP synthesis, which is required for the efficient translocation of Na+ to leaves. These changes enabled efficient stem carboxylation and made them the main source of assimilates. The observed changes indicate the high plasticity of grass pea photosynthetic apparatus, providing an effective mechanism of tolerance to salinity stress.

2-(4-Iodo-2,5-dimethoxyphenyl)-N-[(2-methoxyphenyl)methyl]ethanamine (25I-NBOME): A Harmful Hallucinogen Review.

NBOMes are N-benzylmethoxy derivatives of the 2C family compounds with N-2-methoxybenzyl moiety substituted by the methoxy group at the 2- and 5-position and the halogen group at the 4-position of the phenyl ring. These substances are a new class of potent serotonin 5-HT2A receptor agonist hallucinogens with potential harmful effects. The substitution with halogen of the already psychoactive phenethylamine produces a derivative (2C-I) with increased hallucinogenic effects. This class of hallucinogens has chemical structures very similar to natural hallucinogenic alkaloid mescaline and these are sold mainly via internet as a 'legal' alternative to other hallucinogenic drug-lysergic acid diethylamide (LSD). 25I-NBOMe is the first synthesized and one of the most common compound from NBOMes. Knowledge of pharmacological properties of 25I-NBOMe is very limited so far. There are only a few in vivo and in vitro so far published studies. The behavioral experiments are mainly related with the hallucinogenic effect of 25I-NBOMe while the in vitro studies concerning mainly the affinity for 5-HT2A receptors. The 25I-NBOMe Critical Review 2016 reported 51 non-fatal intoxications and 21 deaths associated with 25I-NBOMe across Europe. Case reports describe various toxic effects of 25I-NBOMe usage including tachycardia, hypertension, hallucinations, rhabdomyolysis, acute kidney injury and death. The growing number of fatal and non-fatal intoxication cases indicates that 25I-NBOMe should be considered as a serious danger to public health. This review aims to present the current state of knowledge on pharmacological effects and chemical properties of 25I-NBOMe and to describe reported clinical cases and analytical methods available for identification of this agent in biological material.