Exploring the Influence of Zinc Ions on the Conformational Stability and Activity of Protein Disulfide Isomerase.

The interplay between metal ion binding and the activity of thiol proteins, particularly within the protein disulfide isomerase family, remains an area of active investigation due to the critical role that these proteins play in many vital processes. This research investigates the interaction between recombinant human PDIA1 and zinc ions, focusing on the subsequent implications for PDIA1's conformational stability and enzymatic activity. Employing isothermal titration calorimetry and differential scanning calorimetry, we systematically compared the zinc binding capabilities of both oxidized and reduced forms of PDIA1 and assessed the structural consequences of this interaction. Our results demonstrate that PDIA1 can bind zinc both in reduced and oxidized states, but with significantly different stoichiometry and more pronounced conformational effects in the reduced form of PDIA1. Furthermore, zinc binding was observed to inhibit the catalytic activity of reduced-PDIA1, likely due to induced alterations in its conformation. These findings unveil a potential regulatory mechanism in PDIA1, wherein metal ion binding under reductive conditions modulates its activity. Our study highlights the potential role of zinc in regulating the catalytic function of PDIA1 through conformational modulation, suggesting a nuanced interplay between metal binding and protein stability in the broader context of cellular redox regulation.

Tear nanoDSF Denaturation Profile Is Predictive of Glaucoma.

Primary open-angle glaucoma (POAG) is a frequent blindness-causing neurodegenerative disorder characterized by optic nerve and retinal ganglion cell damage most commonly due to a chronic increase in intraocular pressure. The preservation of visual function in patients critically depends on the timeliness of detection and treatment of the disease, which is challenging due to its asymptomatic course at early stages and lack of objective diagnostic approaches. Recent studies revealed that the pathophysiology of glaucoma includes complex metabolomic and proteomic alterations in the eye liquids, including tear fluid (TF). Although TF can be collected by a non-invasive procedure and may serve as a source of the appropriate biomarkers, its multi-omics analysis is technically sophisticated and unsuitable for clinical practice. In this study, we tested a novel concept of glaucoma diagnostics based on the rapid high-performance analysis of the TF proteome by differential scanning fluorimetry (nanoDSF). An examination of the thermal denaturation of TF proteins in a cohort of 311 ophthalmic patients revealed typical profiles, with two peaks exhibiting characteristic shifts in POAG. Clustering of the profiles according to peaks maxima allowed us to identify glaucoma in 70% of cases, while the employment of artificial intelligence (machine learning) algorithms reduced the amount of false-positive diagnoses to 13.5%. The POAG-associated alterations in the core TF proteins included an increase in the concentration of serum albumin, accompanied by a decrease in lysozyme C, lipocalin-1, and lactotransferrin contents. Unexpectedly, these changes were not the only factor affecting the observed denaturation profile shifts, which considerably depended on the presence of low-molecular-weight ligands of tear proteins, such as fatty acids and iron. Overall, we recognized the TF denaturation profile as a novel biomarker of glaucoma, which integrates proteomic, lipidomic, and metallomic alterations in tears, and monitoring of which could be adapted for rapid non-invasive screening of the disease in a clinical setting.

Toward a Hydrogen-Free Reductive Catalytic Fractionation of Wheat Straw Biomass.

The reductive catalytic fractionation (RCF) of lignocellulosic biomass is an attractive method for the conversion of lignin toward valuable low-molecular weight aromatics. A limitation to the upscaling of such technology is represented by the use ofpressurized hydrogen gas. Here, the role of hydrogen gas within the RCF of wheat straw biomass is investigated. The use of H2 is shown to enhance lignin depolymerization, by virtue of an improved hydrogenolysis and hydrogenation of lignin fragments, with a yield of phenolic monomers that increased from ca. 12 wt % of acid-insoluble lignin in the initial biomass under inert atmosphere to up to ca. 25 wt % under H2 (in methanol, at 250 °C, with Ru/C). The adoption of methanol, ethanol or isopropanol as hydrogen-donor solvents was also investigated in the absence of H2 . Ethanol was found to give the highest yield of monophenolic compounds (up to ≈20 wt %) owing to a better balance between solvolysis, hydrogenolysis, and hydrogenation of lignin. Nevertheless, a substantial loss of the carbohydrate fraction was observed. The use of a lower temperature (200 °C) in combination with H3 PO4 resulted in an improved recovery of cellulose in the pulp and in the solubilization of hemicellulose and lignin, with the formation of monosaccharides (≈14 wt % of polysaccharides in the initial biomass) and phenolic monomers (up to 18 wt %, in the absence of H2 ). Overall, a tradeoff exists between the removal of H2 from the process and the production of low-molecular weight phenolics during RCF.

Plasma nanoDSF Denaturation Profile at Baseline Is Predictive of Glioblastoma EGFR Status.

Glioblastoma (GBM) is the most frequent and aggressive primary brain tumor in adults. Recently, we demonstrated that plasma denaturation profiles of glioblastoma patients obtained using Differential Scanning Fluorimetry can be automatically distinguished from healthy controls with the help of Artificial Intelligence (AI). Here, we used a set of machine-learning algorithms to automatically classify plasma denaturation profiles of glioblastoma patients according to their EGFR status. We found that Adaboost AI is able to discriminate EGFR alterations in GBM with an 81.5% accuracy. Our study shows that the use of these plasma denaturation profiles could answer the unmet neuro-oncology need for diagnostic predictive biomarker in combination with brain MRI and clinical data, in order to allow for a rapid orientation of patients for a definitive pathological diagnosis and then treatment. We complete this study by showing that discriminating another mutation, MGMT, seems harder, and that post-surgery monitoring using our approach is not conclusive in the 48 h that follow the surgery.

Myotoxin-3 from the Pacific Rattlesnake Crotalus oreganus oreganus Venom Is a New Microtubule-Targeting Agent.

Microtubule targeting agents (MTA) are anti-cancer molecules that bind tubulin and interfere with the microtubule functions, eventually leading to cell death. In the present study, we used an in vitro microtubule polymerization assay to screen several venom families for the presence of anti-microtubule activity. We isolated myotoxin-3, a peptide of the crotamine family, and three isoforms from the venom of the Northern Pacific rattlesnake Crotalus oreganus oreganus, which was able to increase tubulin polymerization. Myotoxin-3 turned out to be a cell-penetrating peptide that slightly diminished the viability of U87 glioblastoma and MCF7 breast carcinoma cells. Myotoxin 3 also induced remodeling of the U87 microtubule network and decreased MCF-7 microtubule dynamic instability. These effects are likely due to direct interaction with tubulin. Indeed, we showed that myotoxin-3 binds to tubulin heterodimer with a Kd of 5.3 µM and stoichiometry of two molecules of peptide per tubulin dimer. Our results demonstrate that exogenous peptides are good candidates for developing new MTA and highlight the richness of venoms as a source of pharmacologically active molecules.

Binding of two zinc ions promotes liquid-liquid phase separation of Tau.

Tau is a naturally disordered microtubule associated protein which forms intraneuronal aggregates in several neurodegenerative diseases including Alzheimer's disease (AD). It was reported that zinc interaction with tau protein can trigger its aggregation. Recently we identified three zinc binding sites located in the N-terminal part, repeat region and the C-terminal part of tau. Here we characterized zinc binding to each of the three sites using isothermal titration calorimetry (ITC) and determined the impact of each site on aggregation using dynamic light scattering (DLS) assays. First, we confirmed the presence of three zinc binding sites on tau and determined the thermodynamic parameters of binding of zinc to these sites. We found a high-affinity zinc binding site located in the repeat region of tau and two N- and C-terminus binding sites with a lower binding constant for zinc. Second, we showed that tau aggregation necessitates zinc binding to the high affinity site in the R2R3 region, while LLPS necessitates zinc binding to any two binding sites. With regard to the role of zinc ions in the aggregation of proteins in neurodegenerative diseases, these findings bring new insights to the understanding of the aggregation mechanism of tau protein induced by zinc.

Tau Protein as Therapeutic Target for Cancer? Focus on Glioblastoma.

Despite being extensively studied for several decades, the microtubule-associated protein Tau has not finished revealing its secrets. For long, Tau has been known for its ability to promote microtubule assembly. A less known feature of Tau is its capability to bind to cancer-related protein kinases, suggesting a possible role of Tau in modulating microtubule-independent cellular pathways that are associated with oncogenesis. With the intention of finding new therapeutic targets for cancer, it appears essential to examine the interaction of Tau with these kinases and their consequences. This review aims at collecting the literature data supporting the relationship between Tau and cancer with a particular focus on glioblastoma tumors in which the pathological significance of Tau remains largely unexplored. We will first treat this subject from a mechanistic point of view showing the pivotal role of Tau in oncogenic processes. Then, we will discuss the involvement of Tau in dysregulating critical pathways in glioblastoma. Finally, we will outline promising strategies to target Tau protein for the therapy of glioblastoma.

High-Entropy-Alloy Nanocrystal Based Macro- and Mesoporous Materials.

High-entropy-alloy (HEA) nanoparticles are attractive for several applications in catalysis and energy. Great efforts are currently devoted to establish composition-property relationships to improve catalytic activity or selectivity. Equally importantly, developing practical fabrication methods for shaping HEA-based materials into complex architectures is a key requirement for their utilization in catalysis. However, shaping nano-HEAs into hierarchical structures avoiding demixing or collapse remains a great challenge. Herein, we overcome this issue by introducing a simple soft-chemistry route to fabricate ordered macro- and mesoporous materials based on HEA nanoparticles, with high surface area, thermal stability, and catalytic activity toward CO oxidation. The process is based on spray-drying from an aqueous solution containing five different noble metal precursors and polymer latex beads. Upon annealing, the polymer plays a double role: templating and reducing agent enabling formation of HEA nanoparticle-based porous networks at only 350 °C. The formation mechanism and the stability of the macro- and mesoporous materials were investigated by a set of in situ characterization techniques; notably, in situ transmission electron microscopy unveiled that the porous structure is stable up to 800 °C. Importantly, this process is green, scalable, and versatile and could be potentially extended to other classes of HEA materials.

APOE4 drives inflammation in human astrocytes via TAGLN3 repression and NF-κB activation.

Apolipoprotein E4 (APOEε4) is the major allelic risk factor for late-onset sporadic Alzheimer's disease (sAD). Inflammation is increasingly considered as critical in sAD initiation and progression. Identifying brain molecular mechanisms that could bridge these two risk factors remain unelucidated. Leveraging induced pluripotent stem cell (iPSC)-based strategies, we demonstrate that APOE controls inflammation in human astrocytes by regulating Transgelin 3 (TAGLN3) expression and, ultimately, nuclear factor κB (NF-κB) activation. We uncover that APOE4 specifically downregulates TAGLN3, involving histone deacetylases activity, which results in low-grade chronic inflammation and hyperactivated inflammatory responses. We show that APOE4 exerts a dominant negative effect to prime astrocytes toward a pro-inflammatory state that is pharmacologically reversible by TAGLN3 supplementation. We further confirm that TAGLN3 is downregulated in the brain of patients with sAD. Our findings highlight the APOE-TAGLN3-NF-κB axis regulating neuroinflammation in human astrocytes and reveal TAGLN3 as a molecular target to modulate neuroinflammation, as well as a potential biomarker for AD.

Zinc Modulation of Neuronal Calcium Sensor Proteins: Three Modes of Interaction with Different Structural Outcomes.

Neuronal calcium sensors (NCSs) are the family of EF-hand proteins mediating Ca2+-dependent signaling pathways in healthy neurons and neurodegenerative diseases. It was hypothesized that the calcium sensor activity of NCSs can be complemented by sensing fluctuation of intracellular zinc, which could further diversify their function. Here, using a set of biophysical techniques, we analyzed the Zn2+-binding properties of five proteins belonging to three different subgroups of the NCS family, namely, VILIP1 and neurocalcin-δ/NCLD (subgroup B), recoverin (subgroup C), as well as GCAP1 and GCAP2 (subgroup D). We demonstrate that each of these proteins is capable of coordinating Zn2+ with a different affinity, stoichiometry, and structural outcome. In the absence of calcium, recoverin and VILIP1 bind two zinc ions with submicromolar affinity, and the binding induces pronounced conformational changes and regulates the dimeric state of these proteins without significant destabilization of their structure. In the presence of calcium, recoverin binds zinc with slightly decreased affinity and moderate conformational outcome, whereas VILIP1 becomes insensitive to Zn2+. NCALD binds Zn2+ with micromolar affinity, but the binding induces dramatic destabilization and aggregation of the protein. In contrast, both GCAPs demonstrate low-affinity binding of zinc independent of calcium, remaining relatively stable even at submillimolar Zn2+ concentrations. Based on these data, and the results of structural bioinformatics analysis, NCSs can be divided into three categories: (1) physiological Ca2+/Zn2+ sensor proteins capable of binding exchangeable (signaling) zinc (recoverin and VILIP1), (2) pathological Ca2+/Zn2+ sensors responding only to aberrantly high free zinc concentrations by denaturation and aggregation (NCALD), and (3) Zn2+-resistant, Ca2+ sensor proteins (GCAP1, GCAP2). We suggest that NCS proteins may therefore govern the interconnection between Ca2+-dependent and Zn2+-dependent signaling pathways in healthy neurons and zinc cytotoxicity-related neurodegenerative diseases, such as Alzheimer's disease and glaucoma.

Identification of the three zinc-binding sites on tau protein.

Tau protein has been extensively studied due to its key roles in microtubular cytoskeleton regulation and in the formation of aggregates found in some neurodegenerative diseases. Recently it has been shown that zinc is able to induce tau aggregation by interacting with several binding sites. However, the precise location of these sites and the molecular mechanism of zinc-induced aggregation remain unknown. Here we used Nuclear Magnetic Resonance (NMR) to identify zinc binding sites on tau. These experiments revealed three distinct zinc binding sites on tau, located in the N-terminal part, the repeat region and the C-terminal part. Further analysis enabled us to show that the N-terminal and the C-terminal sites are independent of each other. Using molecular simulations, we proposed a model of each site in a complex with zinc. Given the clinical importance of zinc in tau aggregation, our findings pave the way for designing potential therapies for tauopathies.

Mechanism of Zn2+ and Ca2+ Binding to Human S100A1.

S100A1 is a member of the S100 family of small ubiquitous Ca2+-binding proteins, which participates in the regulation of cell differentiation, motility, and survival. It exists as homo- or heterodimers. S100A1 has also been shown to bind Zn2+, but the molecular mechanisms of this binding are not yet known. In this work, using ESI-MS and ITC, we demonstrate that S100A1 can coordinate 4 zinc ions per monomer, with two high affinity (KD~4 and 770 nm) and two low affinity sites. Using competitive binding experiments between Ca2+ and Zn2+ and QM/MM molecular modeling we conclude that Zn2+ high affinity sites are located in the EF-hand motifs of S100A1. In addition, two lower affinity sites can bind Zn2+ even when the EF-hands are saturated by Ca2+, resulting in a 2Ca2+:S100A1:2Zn2+ conformer. Finally, we show that, in contrast to calcium, an excess of Zn2+ produces a destabilizing effect on S100A1 structure and leads to its aggregation. We also determined a higher affinity to Ca2+ (KD~0.16 and 24 µm) than was previously reported for S100A1, which would allow this protein to function as a Ca2+/Zn2+-sensor both inside and outside cells, participating in diverse signaling pathways under normal and pathological conditions.

Green Synthesis of Iron-Doped Cobalt Oxide Nanoparticles from Palm Kernel Oil via Co-Precipitation and Structural Characterization.

In this study, a bio-derived precipitating agent/ligand, palm kernel oil, has been used as an alternative route for the green synthesis of nanoparticles of Fe-doped Co3O4 via the co-precipitation reaction. The palm oil was extracted from dried palm kernel seeds by crushing, squeezing and filtration. The reaction of the palm kernel oil with potassium hydroxide, under reflux, yielded a solution containing a mixture of potassium carboxylate and excess hydroxide ions, irrespective of the length of saponification. The as-obtained solution reacts with an aqueous solution containing iron and cobalt ions to yield the desired metallo-organic precursor, iron cobalt carboxylate. Characterization of the precursors by IR and gas chromatography (GC) attests to the presence of carboxylate fatty acids in good agreement with the proportion contained in the oil, and ICP confirms that the metallic ratios are in the proportion used during the synthesis. Analysis of the products thermally decomposed between 400 °C and 600 °C by XRD, EDX, TEM and ToF-SIMS, established that cobalt iron oxide nanoparticles (Co(1-x)Fex)3O4 were obtained for x ≤ 0.2 and a nanocomposite material (Co(1-x)Fex)3O4/Fe3O4 for x ≥ 0.2, with sizes between 22 and 9 nm. ToF-SIMS and XRD provided direct evidence of the progressive substitution of cobalt by iron in the Co3O4 crystal structure for x ≤ 0.2.

Disulfide Dimerization of Neuronal Calcium Sensor-1: Implications for Zinc and Redox Signaling.

Neuronal calcium sensor-1 (NCS-1) is a four-EF-hand ubiquitous signaling protein modulating neuronal function and survival, which participates in neurodegeneration and carcinogenesis. NCS-1 recognizes specific sites on cellular membranes and regulates numerous targets, including G-protein coupled receptors and their kinases (GRKs). Here, with the use of cellular models and various biophysical and computational techniques, we demonstrate that NCS-1 is a redox-sensitive protein, which responds to oxidizing conditions by the formation of disulfide dimer (dNCS-1), involving its single, highly conservative cysteine C38. The dimer content is unaffected by the elevation of intracellular calcium levels but increases to 10-30% at high free zinc concentrations (characteristic of oxidative stress), which is accompanied by accumulation of the protein in punctual clusters in the perinuclear area. The formation of dNCS-1 represents a specific Zn2+-promoted process, requiring proper folding of the protein and occurring at redox potential values approaching apoptotic levels. The dimer binds Ca2+ only in one EF-hand per monomer, thereby representing a unique state, with decreased α-helicity and thermal stability, increased surface hydrophobicity, and markedly improved inhibitory activity against GRK1 due to 20-fold higher affinity towards the enzyme. Furthermore, dNCS-1 can coordinate zinc and, according to molecular modeling, has an asymmetrical structure and increased conformational flexibility of the subunits, which may underlie their enhanced target-binding properties. In HEK293 cells, dNCS-1 can be reduced by the thioredoxin system, otherwise accumulating as protein aggregates, which are degraded by the proteasome. Interestingly, NCS-1 silencing diminishes the susceptibility of Y79 cancer cells to oxidative stress-induced apoptosis, suggesting that NCS-1 may mediate redox-regulated pathways governing cell death/survival in response to oxidative conditions.

An AI-Powered Blood Test to Detect Cancer Using NanoDSF.

Glioblastoma is the most frequent and aggressive primary brain tumor. Its diagnosis is based on resection or biopsy that could be especially difficult and dangerous in the case of deep location or patient comorbidities. Monitoring disease evolution and progression also requires repeated biopsies that are often not feasible. Therefore, there is an urgent need to develop biomarkers to diagnose and follow glioblastoma evolution in a minimally invasive way. In the present study, we described a novel cancer detection method based on plasma denaturation profiles obtained by a non-conventional use of differential scanning fluorimetry. Using blood samples from 84 glioma patients and 63 healthy controls, we showed that their denaturation profiles can be automatically distinguished with the help of machine learning algorithms with 92% accuracy. Proposed high throughput workflow can be applied to any type of cancer and could become a powerful pan-cancer diagnostic and monitoring tool requiring only a simple blood test.

Zinc Binds to RRM2 Peptide of TDP-43.

Transactive response DNA and RNA binding protein 43 kDa (TDP-43) is a highly conserved heterogeneous nuclear ribonucleoprotein (hnRNP), which is involved in several steps of protein production including transcription and splicing. Its aggregates are frequently observed in motor neurons from amyotrophic lateral sclerosis patients and in the most common variant of frontotemporal lobar degeneration. Recently it was shown that TDP-43 is able to bind Zn2+ by its RRM domain. In this work, we have investigated Zn2+ binding to a short peptide 256-264 from C-terminus of RRM2 domain using isothermal titration calorimetry, electrospray ionization mass spectrometry, QM/MM simulations, and NMR spectroscopy. We have found that this peptide is able to bind zinc ions with a Ka equal to 1.6 × 105 M-1. Our findings suggest the existence of a zinc binding site in the C-terminal region of RRM2 domain. Together with the existing structure of the RRM2 domain of TDP-43 we propose a model of its complex with Zn2+ which illustrates how zinc might regulate DNA/RNA binding.

In silico molecular target prediction unveils mebendazole as a potent MAPK14 inhibitor.

The concept of polypharmacology involves the interaction of drug molecules with multiple molecular targets. It provides a unique opportunity for the repurposing of already-approved drugs to target key factors involved in human diseases. Herein, we used an in silico target prediction algorithm to investigate the mechanism of action of mebendazole, an antihelminthic drug, currently repurposed in the treatment of brain tumors. First, we confirmed that mebendazole decreased the viability of glioblastoma cells in vitro (IC50 values ranging from 288 nm to 2.1 µm). Our in silico approach unveiled 21 putative molecular targets for mebendazole, including 12 proteins significantly upregulated at the gene level in glioblastoma as compared to normal brain tissue (fold change > 1.5; P < 0.0001). Validation experiments were performed on three major kinases involved in cancer biology: ABL1, MAPK1/ERK2, and MAPK14/p38α. Mebendazole could inhibit the activity of these kinases in vitro in a dose-dependent manner, with a high potency against MAPK14 (IC50  = 104 ± 46 nm). Its direct binding to MAPK14 was further validated in vitro, and inhibition of MAPK14 kinase activity was confirmed in live glioblastoma cells. Consistent with biophysical data, molecular modeling suggested that mebendazole was able to bind to the catalytic site of MAPK14. Finally, gene silencing demonstrated that MAPK14 is involved in glioblastoma tumor spheroid growth and response to mebendazole treatment. This study thus highlighted the role of MAPK14 in the anticancer mechanism of action of mebendazole and provides further rationale for the pharmacological targeting of MAPK14 in brain tumors. It also opens new avenues for the development of novel MAPK14/p38α inhibitors to treat human diseases.

CO2 Hydrogenation to Methanol with Ga- and Zn-Doped Mesoporous Cu/SiO2 Catalysts Prepared by the Aerosol-Assisted Sol-Gel Process*.

The preparation of copper-based heterogeneous catalysts dedicated to the hydrogenation of CO2 to methanol typically relies on multi-step procedures carried out in batch. These steps are precisely tailored to introduce the active phase (Cu) and the promoters (e. g., zinc, gallium) onto a preformed support to maximize catalyst performance. However, each process step can be associated with the formation of waste and with the consumption of energy, thereby negatively impacting the environmental performance of the overall catalyst preparation procedure. Here, a direct and continuous production process is proposed for the synthesis of efficient catalysts for the CO2 -to-methanol reaction. Gallium- and zinc-promoted mesoporous Cu-SiO2 catalysts were prepared in one step by the aerosol-assisted sol-gel process. The catalysts consisted of spherical microparticles and featured high specific surface area and pore volume, with interconnected pores of about 6 nm. A strong promoting effect of Ga and Zn was highlighted, boosting the selectivity for methanol at the expense of CO. Upon calcination, it was shown that Cu species (initially trapped in the silica matrix) underwent a migration towards the catalyst surface and a progressive sintering. After optimization, the catalysts obtained via such direct, continuous, simple, and scalable route could compete with the best catalysts reported in the literature and obtained via multi-step approaches.

Role of Tau as a Microtubule-Associated Protein: Structural and Functional Aspects.

Microtubules (MTs) play a fundamental role in many vital processes such as cell division and neuronal activity. They are key structural and functional elements in axons, supporting neurite differentiation and growth, as well as transporting motor proteins along the axons, which use MTs as support tracks. Tau is a stabilizing MT associated protein, whose functions are mainly regulated by phosphorylation. A disruption of the MT network, which might be caused by Tau loss of function, is observed in a group of related diseases called tauopathies, which includes Alzheimer's disease (AD). Tau is found hyperphosphorylated in AD, which might account for its loss of MT stabilizing capacity. Since destabilization of MTs after dissociation of Tau could contribute to toxicity in neurodegenerative diseases, a molecular understanding of this interaction and its regulation is essential.

Plasmatic Signature of Disease by Differential Scanning Calorimetry (DSC).

Differential scanning calorimetry (DSC) has been used for several decades to characterize thermal stability of macromolecules such as proteins and DNA. It allows to determine the denaturation temperature and enthalpy of individual domains of proteins, thus giving new insights into their domain organization and ligand interaction. Over the past decade, it has been shown that this technique can also be used to study biofluids such as plasma or cerebrospinal fluid to obtain denaturation profiles. An increasing number of studies demonstrated that such profiles obtained from patients were significantly different from profiles obtained using biofluids of healthy individuals. This opens interesting perspectives for new diagnostics and monitoring tools for a large number of diseases. Nevertheless, the extensive studies of plasma samples from patients with different pathologies as well as the development of standardized methods of data analysis are necessary to reach the promising diagnostic potential of this methodology. Using plasma samples from healthy individuals and glioblastoma patients, we outline the steps necessary to obtain a plasmatic calorimetric profile with VP-DSC instrument and describe a cluster analysis of obtained data.