Vitaly Erukhimovitch

Novel spectral method for the study of viral carcinogenesis in vitro.

Fourier transform infrared (FTIR) spectroscopy is a unique technique for the laboratory diagnosis of cellular variations based on the characteristic molecular vibrational spectra of the cells. Microscopic FTIR was used to investigate spectral differences between normal and malignant fibroblasts transformed by retrovirus infection. A detailed analysis showed significant differences between cancerous and normal cells. The contents of vital cellular metabolites were significantly lower in the transformed cells than in the normal cells. In an attempt to identify the cellular components responsible for the observed spectral differences between normal and cancerous cells, we found significant differences between DNA of normal and cancerous cells.

FTIR microspectroscopy of malignant fibroblasts transformed by mouse sarcoma virus

Fourier transform infrared microspectroscopy (FTIR-MSP), which is based on the characteristic molecular vibrational spectra of cells, was used to investigate spectral differences between normal primary rabbit bone marrow (BM) cells and bone marrow cells transformed (BMT) by murine sarcoma virus (MuSV). Primary cells, rather than cell lines, were used for this research because primary cells are similar to normal tissue cells in most of their characteristics. Our results showed dramatic changes in absorbance between the control cells and MuSV124-transformed cells. Various biological markers, such as the phosphate level and the RNA/DNA obtained, based on the analysis of the FTIR-MSP spectra, also displayed significant differences between the control and transformed cells. Preliminary results suggested that the cluster analysis performed on the FTIR-MSP spectra yielded 100% accuracy in classifying both types of cells.

Spectroscopic Characterization of Human and Mouse Primary Cells, Cell Lines and Malignant Cells {

Fourier transform infrared (FTIR) spectroscopy is currently being developed as a new optical approach to the diagnosis and characterization of cell or tissue pathology. The advantage of FTIR microspectroscopy over conventional FTIR spectroscopy in the diagnosis of malignancies is that it facilitates inspection of restricted regions of the cell culture or tissue. In this study, we set out to evaluate FTIR microspectroscopy as a diagnostic tool for identifying retrovirus‐induced malignancies. Our study showed significant and consistent differences between cultures of different types of cells of both mouse and human origin, i.e. primary fibroblast cells (one to two passages in cell culture), fibroblast cell lines and malignant cells transformed by murine sarcoma virus. An impressive decrease in the levels of phosphate and other metabolites was seen in malignant cells compared with primary cells. The levels of these metabolites in the cell lines were significantly lower than in the primary cells but higher than in the malignant cells. In addition, the peak attributed to the PO2− symmetric stretching mode at 1082 cm−1 in primary cells shifted significantly to 1085 cm−1 for the cell line and to 1087 cm−1 for the malignant cells. These differences taken together with differences in the shapes of various bands throughout the spectrum strongly support the possibility of developing FTIR microspectroscopy for the detection and study of malignant—and possibly premalignant—cells.

Novel methodology for the follow-up of acute lymphoblastic leukemia using FTIR microspectroscopy

In this report, we present a novel spectroscopic method of follow-up during chemotherapy treatment for B- and T-cell childhood leukemia patients. We isolated peripheral lymphocytes from blood drawn from patients before and after the chemotherapy and collected Microscopic FTIR (FTIR-MC) spectra of the isolated lymphocytes. Our results showed that nucleic acids content decreased in both types of patients. Changes in phospholipids and proteins level could be observed. The overall effects of drugs administered to the patients can be understood at the molecular level using FTIR-MC and these results are expected to stimulate wider applications of spectroscopy in leukemia research.

The use of FTIR microscopy for the evaluation of anti-bacterial agents activity.

FTIR spectroscopy has been used by chemists as a powerful tool to characterize inorganic and organic compounds. In this study we examined the potential of FTIR microspectroscopy for early evaluation of the efficiency of anti-bacterial therapy. For this purpose, the effect of caffeic acid phenethyl ester (CAPE) and ampicillin on the development of bacterial infection in cell culture was examined. CAPE is one of the most active components of propolis which is a natural honeybee product with a potent anti-bacterial activity. Our results show early (2h post-treatment), unique and significant spectral indicators for successful treatment with CAPE although some of these biomarkers showed different trends in Gram (-) compared with Gram (+) bacteria. For instance, the intensity of bands at 682 and 1316 cm(-1) decreases in all examined Gram (-) bacterial strains while significantly increases in all examined Gram (+) bacterial strains. On the other hand, both Gram (+) and Gram (-) bacteria treated with ampicillin did not show any spectral differences compared with the control untreated bacteria. It seems that FTIR spectroscopy can be used as an effective tool for an early evaluation of the efficiency of the anti-bacterial effect of CAPE and probably other used drugs.

Spectroscopic detection and identification of infected cells with herpes viruses

Matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) and Fourier transform infrared (FTIR) microspectroscopy were previously applied for the identification of various biological samples. In the present study, normal cells in culture and cells infected with herpes simplex virus type 2 (HSV-2) or varicella-zoster virus (VZV) were analyzed by MALDI-TOF and FTIR microscopy. Specific spectral biomarkers for rapid and reliable monitoring and identification of infected cells and probably for the discrimination between these viruses were searched. The results show consistent spectral peaks in all examined normal uninfected human fibroblast cells both in MALDI-T0F and FTIR microscopy. In HSV-2- or VZV-infected cells, two unique peaks for each appeared at m/z 5397 and 5813 or at m/z 3501 and 4951, respectively, in MALDI-TOF spectra. In addition, several peaks that appeared in control uninfected cells at the region m/z 13,000-20,000 disappeared completely in all examined infected samples. When these infected cells were examined by FTIR microscopy, a band at 859 cm(-1) in control uninfected cells was significantly shifted to 854 cm(-1) in both HSV2- and VZV-infected cells. In addition, phosphate levels were considerably increased in all infected cells compared to normal uninfected cells. These parameters could be used as a basis for developing a spectral method for the detection and identification of cells infected with herpes viruses.

Spectroscopic Characterization of Normal Primary and Malignant Cells Transformed by Retroviruses

Microscopic FT-IR spectroscopy was used to investigate spectral differences between various primary cells obtained from different sources (mouse, rat, rabbit, and human) and malignant cells transformed by murine sarcoma virus (MuSV). The advantage of this method over conventional FT-IR spectroscopy is that it facilitates inspection of restricted regions of tissue. Our results showed significant and consistent differences between all the tested normal and malignant cells. An impressive decrease in the levels of vital cellular metabolites was seen in malignant cells compared to normal cells. The peak attributed to the PO2− symmetric stretching mode at 1082 cm−1 was shifted significantly to a higher frequency (1086–1087 cm−1) in all the tested malignant cells. The impressive and consistent differences between normal primary cells and malignant cells (obtained from various organs and species) in the shapes and position of various bands throughout the spectrum strongly support the possibility of developing FT-IR microscopy as a diagnostic method for the detection and study of cancer cells.

FTIR Microscopy Detection of Cells Infected With Viruses

Fourier-transform infrared (FTIR) microscopy is considered a comprehensive and sensitive method for detection of molecular changes in cells. The advantage of FTIR microspectroscopy over conventional FTIR spectroscopy is that it facilitates inspection of restricted regions of a cell culture or a tissue. We have shown that it is possible to apply FTIR microscopy as a sensitive and effective assay for the detection of cells infected with various members of the herpes family of viruses and retroviruses. Detectable and significant spectral differences between normal and infected cells were evident at early stages of the infection. Impressive changes in several spectroscopic parameters were seen in infected compared with uninfected cells. It seems that the change in spectral behavior is specific to the infecting virus, because cells infected with herpesviruses showed different spectral changes compared with cells infected with retroviruses.

Early and Rapid Detection of Potato's Fungal Infection by Fourier Transform Infrared Microscopy:

Fungi are considered serious pathogens to many plants and can cause severe economic damage. Early detection of these pathogens is very important and might be critical for their control. The available methods for detection of fungi are time consuming and not always very specific. Fourier transform infrared (FT-IR) microscopy has proved to be a reliable and sensitive method for detection of molecular changes in cells. Fungi pathogens display typical infrared spectra that differ from the spectra of substrate material such as potato. In the present study we used FT-IR microscopy for early and rapid detection of the potato fungal pathogen Colletotrichum coccodes on the surface of potato tubers. Infected potatoes with this fungal pathogen and uninfected potatoes were examined and correctly classified as infected or not infected by FT-IR microscopy at very early stages of infection when no morphological signs of infection could be seen. Unique spectral biomarkers were found in naturally infected potatoes compared to disease-free control potatoes.

Spectroscopic evaluation of the effect of a red microalgal polysaccharide on herpes-infected Vero cells.

The sulfated polysaccharide obtained from a species of red microalga has proved to be a potent antiviral agent against various members of the herpes family. In the present study, we used microscopic Fourier transform infrared spectroscopy (FT-IR) to investigate differences between normal cells, those infected with herpes viruses, and infected cells treated with red microalgal polysaccharide. FT-IR enables the characterization of cell or tissue pathology based on characteristic molecular vibrational spectra of the cells. The advantage of microscopic FT-IR spectroscopy over conventional FT-IR spectroscopy is that it facilitates inspection of restricted regions of cell cultures or tissue. Our results showed significant spectral differences at early stages of infection between infected and noninfected cells, and between infected cells treated with the polysaccharide and those not treated. In infected cells, there was an impressive decrease in sugar content and a considerable increase in phosphate levels in conjunction with the infection progress. Our results also proved that sugars penetrated and accumulated inside cells treated with the red microalgal polysaccharide. These could have been sugar fragments of low molecular weight present in the polysaccharide solution, despite purification by dialysis. Such sugar accumulation might be responsible for a breakdown in the internal steps of the viral replication cycle.