Elisa Mitri

Time-Resolved FT-IR Microspectroscopy of Protein Aggregation Induced by Heat-Shock in Live Cells

Maintaining the correct folding of cellular proteins is essential for preserving cellular homeostasis. Protein dishomeostasis, aberrant protein folding, and protein aggregation are indeed involved in several diseases including cancer, aging-associated, and neurodegenerative disorders. Accumulation of protein aggregates can also be induced from a variety of stressful conditions, such as temperature increase or oxidative stress. In this work, we monitored by Fourier transform-infrared (FT-IR) microspectroscopy the response of live breast cancer MCF-7 and mammary breast adenocarcinoma MDA-MB 231 cell lines to severe heat-shock (HS), caused by the rise of the cellular medium temperature from 37 ± 0.5 °C to 42 ± 0.5 °C. Through the study of the time-evolution of the second derivatives of the spectra and by the 2D correlation analysis of FT-IR absorbance data, we were able to identify a common sudden heat-shock response (HSR) among the two cell lines. The hyperfluidization of mammalian cell membranes, the transient increment of the signal lipids, as well as the alteration of proteome profile were all monitored within the first 40 min of stress application, while the persistent intracellular accumulation of extended β-folded protein aggregates was detected after 40 min up to 2 h. As a whole, this paper offers a further prove of the diagnostic capabilities of FT-IR microspectroscopy for monitoring in real-time the biochemical rearrangements undergone by live cells upon external stimulation.

Apoptotic pathways of U937 leukemic monocytes investigated by infrared microspectroscopy and flow cytometry

Apoptosis is a strictly regulated cell death mechanism that plays a pivotal role in the normal evolution of multicellular organisms. Its misregulation has been associated with many diseases, making its early and reliable detection a key point for modern cellular biology. In this paper, we propose the use of infrared microspectroscopy (IRMS) as a label-free methodology for the detection of apoptotic-related biochemical processes induced on U937 leukemic monocytes by serum starvation and CCCP-exposure. The spectroscopic results are in agreement with parallel Flow Cytometry (FC) experiments, where plasma membrane integrity and mitochondrial activity were assessed. Spectroscopic outcomes complement FC data and allow drawing a more complete picture of the apoptotic pathways. In particular, we established that the two apoptosis-inducing treatments, cell starvation and CCCP exposure, affect the cell cycle in a different way. With the former, cell death is preceded by a cell cycle arrest, whereas the latter causes an increased cell cycle progression. Spectral data demonstrate that for both conditions apoptosis proceeds through the accumulation of lipid droplets within cells. Moreover, we were able to establish a spectral marker for DNA condensation/fragmentation: the enhancement of the PhI band component centred at ~1206 cm(-1), which is more sensitive than the relative intensity of the PhII band to which phospholipids and carbohydrates also contribute significantly. In conclusion, we demonstrate that the intrinsic multi-parametric nature of IRMS and its application on cells under physiological conditions can be well exploited for the investigation of apoptotic pathways.

Vibrational mapping of sinonasal lesions by Fourier transform infrared imaging spectroscopy

Abstract. Fourier transform infrared imaging (FTIRI) is a powerful tool for analyzing biochemical changes in tumoral tissues. The head and neck region is characterized by a great variety of lesions, with different degrees of malignancy, which are often difficult to diagnose. Schneiderian papillomas are sinonasal benign neoplasms arising from the Schneiderian mucosa; they can evolve into malignant tumoral lesions (squamous cell carcinoma). In addition, they can sometimes be confused with the more common inflammatory polyps. Therefore, an early and definitive diagnosis of this pathology is mandatory. Progressing in our research on the study of oral cavity lesions, 15 sections consisting of inflammatory sinonasal polyps, benign Schneiderian papillomas, and sinonasal undifferentiated carcinomas were analyzed using FTIRI. To allow a rigorous description of these pathologies and to gain objective diagnosis, the epithelial layer and the adjacent connective tissue of each section were separately investigated by following a multivariate analysis approach. According to the nature of the lesion, interesting modifications were detected in the average spectra of the different tissue components, above all in the lipid and protein patterns. Specific band-area ratios acting as spectral markers of the different pathologies were also highlighted.

A new light on Alkaptonuria: A Fourier-transform infrared microscopy (FTIRM) and low energy X-ray fluorescence (LEXRF) microscopy correlative study on a rare disease

BACKGROUND Alkaptonuria (AKU) is an ultra-rare disease associated to the lack of an enzyme involved in tyrosine catabolism. This deficiency results in the accumulation of homogentisic acid (HGA) in the form of ochronotic pigment in joint cartilage, leading to a severe arthropathy. Secondary amyloidosis has been also unequivocally assessed as a comorbidity of AKU arthropathy. Composition of ochronotic pigment and how it is structurally related to amyloid is still unknown. METHODS We exploited Synchrotron Radiation Infrared and X-Ray Fluorescence microscopies in combination with conventional bio-assays and analytical tools to characterize chemical composition and morphology of AKU cartilage. RESULTS We evinced that AKU cartilage is characterized by proteoglycans depletion, increased Sodium levels, accumulation of lipids in the peri-lacunar regions and amyloid formation. We also highlighted an increase of aromatic compounds and oxygen-containing species, depletion in overall Magnesium content (although localized in the peri-lacunar region) and the presence of calcium carbonate fragments in proximity of cartilage lacunae. CONCLUSIONS We highlighted common features between AKU and arthropathy, but also specific signatures of the disease, like presence of amyloids and peculiar calcifications. Our analyses provide a unified picture of AKU cartilage, shedding a new light on the disease and opening new perspectives. GENERAL SIGNIFICANCE Ochronotic pigment is a hallmark of AKU and responsible of tissue degeneration. Conventional bio-assays have not yet clarified its composition and its structural relationship with amyloids. The present work proposes new strategies for filling the aforementioned gap that encompass the integration of new analytical approaches with standardized analyses.

Pitfalls and promises in FTIR spectromicroscopy analyses to monitor iron-mediated DNA damage in sperm

Many drugs, chemicals, and environmental factors can impair sperm functionality by inducing DNA damage, one of the important causes of reduced fertility potential. The use of vibrational spectromicroscopy represents a promising approach for monitoring DNA integrity in sperm, although some limitations exist, depending from the experimental conditions. Here, we report that when using FTIR spectromicroscopy to reveal oxidative stress mediated by Fentons reaction on hydrated sperm samples, DNA damage interpretation is partially compromised by unexpected cell surface precipitates. The precipitates give a broad band in the 1150-1000cm(-1) infrared region, which partially covers one of the signatures of DNA (phosphate stretching bands), and are detected as iron and oxygen containing material when using XRF spectroscopy. On the other hand, the analyses further support the potential of FTIR spectromicroscopy to reveal cellular oxidative damage events such as lipid peroxidation, protein misfolding and aggregations, as well as DNA strain breaks.

Contribution of Ribonucleic Acid (RNA) to the Fourier Transform Infrared (FTIR) Spectrum of Eukaryotic Cells

We report on an optimized protocol for the digestion of cellular RNA, which minimally affects the cell membrane integrity, maintaining substantially unaltered the vibrational contributions of the other cellular macromolecules. The design of this protocol allowed us to collect the first Fourier transform infrared (FTIR) spectra of intact hydrated B16 mouse melanoma cells deprived of RNA and to highlight the in-cell diagnostic spectral features of it. Complementing the cellular results with the FTIR analysis of extracted RNA, ds-DNA, ss-cDNA and isolated nuclei, we verified that the spectral component centered at ∼1220 cm-1 is a good qualitative and semiquantitative marker of cellular DNA, since it is minimally affected by cellular RNA removal. Conversely, the band centered at ∼1240 cm-1, conventionally attributed to RNA, is only a qualitative marker of it, since its intensity is majorly influenced by other macromolecules containing diverse phosphate groups, such as phospholipids and phosphorylated proteins. On the other hand, we proved that the spectral contribution centered at ∼1120 cm-1 is the most reliable indicator of variations in cellular RNA levels, that better correlates with cellular metabolic activity. The achievement of these results have been made possible also by the implementation of new methods for baseline correction and automated peak fitting, presented in this paper.

Fourier transform infrared microspectroscopy reveals biochemical changes associated with glioma stem cell differentiation.

According to the cancer stem cell theory malignant glioma is incurable because of the presence of the cancer stem cells - a subpopulation of cells that are resistant to therapy and cause the recurrence of a tumor after surgical resection. Several protein markers of cancer stem cell were reported but none of those is fully reliable to grade the content of stem cells in a tumor. Hereby we propose Fourier transform infrared (FTIR) microspectroscopy as an alternative, labelfree, non-damaging and fast method to identify glioma stem cells based on their own spectral characteristics. The analysis of FTIR data revealed that in NCH421k cells, a model of glioma stem cells, the relative content of lipids is higher than in their all-trans retinoic acid-differentiated counterparts. Moreover, it has been assessed that stem cells have more rigid cellular membranes and more phosphorylated proteins, whereas after differentiation glycogen level increases. The ability of FTIR to estimate the content of stem cells in a heterogeneous sample, on the base of the identified spectral markers, and to classify stem and non-stem cells into two separate populations was probed. Although it was not possible to calculate the exact percentage of each subpopulation, we could clearly see that with the increasing amount of differentiated cells in a sample, more hits occupy the PC space previously identified as a space of differentiated cells. The present study is therefore an initial step towards the development of a FTIR based protocol in clinical practice to estimate the content of stem cells in a tumor sample.