BS148 Reduces the Aggressiveness of Metastatic Melanoma via Sigma-2 Receptor Targeting.

The management of advanced-stage melanoma is clinically challenging, mainly because of its resistance to the currently available therapies. Therefore, it is important to develop alternative therapeutic strategies. The sigma-2 receptor (S2R) is overexpressed in proliferating tumor cells and represents a promising vulnerability to target. Indeed, we have recently identified a potent S2R modulator (BS148) that is effective in melanoma. To elucidate its mechanism of action, we designed and synthesized a BS148 fluorescent probe that enters SK-MEL-2 melanoma cells as assessed using confocal microscopy analysis. We show that S2R knockdown significantly reduces the anti-proliferative effect induced by BS148 administration, indicating the engagement of S2R in BS148-mediated cytotoxicity. Interestingly, BS148 treatment showed similar molecular effects to S2R RNA interference-mediated knockdown. We demonstrate that BS148 administration activates the endoplasmic reticulum stress response through the upregulation of protein kinase R-like ER kinase (PERK), activating transcription factor 4 (ATF4) genes, and C/EBP homologous protein (CHOP). Furthermore, we show that BS148 treatment downregulates genes related to the cholesterol pathway and activates the MAPK signaling pathway. Finally, we translate our results into patient-derived xenograft (PDX) cells, proving that BS148 treatment reduces melanoma cell viability and migration. These results demonstrate that BS148 is able to inhibit metastatic melanoma cell proliferation and migration through its interaction with the S2R and confirm its role as a promising target to treat cancer.

Thermodynamics and Kinetics of Electron Transfer of Electrode-Immobilized Small Laccase from Streptomyces coelicolor.

The thermodynamic and kinetic properties for heterogeneous electron transfer (ET) were measured for the electrode-immobilized small laccase (SLAC) from Streptomyces coelicolor subjected to different electrostatic and covalent protein-electrode linkages, using cyclic voltammetry. Once immobilized electrostatically onto a gold electrode using mixed carboxyl- and hydroxy-terminated alkane-thiolate SAMs or covalently exploiting the same SAM subjected to N-hydroxysuccinimide+1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide (NHS-EDC) chemistry, the SLAC-electrode electron flow occurs through the T1 center. The E°' values (from +0.2 to +0.1 V vs. SHE at pH 7.0) are lower by more than 0.2 V compared to the protein either in solution or immobilized with different anchoring strategies using uncharged SAMs. For the present electrostatic and covalent binding, this effect can, respectively, be ascribed to the negative charge of the SAM surfaces and to deletion of the positive charge of Lys/Arg residues due to amide bond formation which both selectively stabilize the more positively charged oxidized SLAC. Observation of enthalpy/entropy compensation within the series indicates that the immobilized proteins experience different reduction-induced solvent reorganization effects. The E°' values for the covalently attached SLAC are sensitive to three acid base equilibria, with apparent pKa values of pKa1ox = 5.1, pKa1red = 7.5, pKa2ox = 8.4, pKa2red = 10.9, pKa2ox = 8.9, pKa2red = 11.3 possibly involving one residue close to the T1 center and two residues (Lys and/or Arg) along with moderate protein unfolding, respectively. Therefore, the E°' value of immobilized SLAC turns out to be particularly sensitive to the anchoring mode and medium conditions.

Assessing the Functional and Structural Stability of the Met80Ala Mutant of Cytochrome c in Dimethylsulfoxide.

The Met80Ala variant of yeast cytochrome c is known to possess electrocatalytic properties that are absent in the wild type form and that make it a promising candidate for biocatalysis and biosensing. The versatility of an enzyme is enhanced by the stability in mixed aqueous/organic solvents that would allow poorly water-soluble substrates to be targeted. In this work, we have evaluated the effect of dimethylsulfoxide (DMSO) on the functionality of the Met80Ala cytochrome c mutant, by investigating the thermodynamics and kinetics of electron transfer in mixed water/DMSO solutions up to 50% DMSO v/v. In parallel, we have monitored spectroscopically the retention of the main structural features in the same medium, focusing on both the overall protein structure and the heme center. We found that the organic solvent exerts only minor effects on the redox and structural properties of the mutant mostly as a result of the modification of the dielectric constant of the solvent. This would warrant proper functionality of this variant also under these potentially hostile experimental conditions, that differ from the physiological milieu of cytochrome c.

How to Turn an Electron Transfer Protein into a Redox Enzyme for Biosensing.

Cytochrome c is a small globular protein whose main physiological role is to shuttle electrons within the mitochondrial electron transport chain. This protein has been widely investigated, especially as a paradigmatic system for understanding the fundamental aspects of biological electron transfer and protein folding. Nevertheless, cytochrome c can also be endowed with a non-native catalytic activity and be immobilized on an electrode surface for the development of third generation biosensors. Here, an overview is offered of the most significant examples of such a functional transformation, carried out by either point mutation(s) or controlled unfolding. The latter can be induced chemically or upon protein immobilization on hydrophobic self-assembled monolayers. We critically discuss the potential held by these systems as core constituents of amperometric biosensors, along with the issues that need to be addressed to optimize their applicability and response.

Binding of S. cerevisiae iso-1 cytochrome c and its surface lysine-to-alanine variants to cardiolipin: charge effects and the role of the lipid to protein ratio.

The interaction of cytochrome c with cardiolipin (CL) is a critical step in the initial stages of apoptosis and is mediated by a positively charged region on the protein surface comprising several lysine residues (site A). Here, the interaction of wt S. cerevisiae cytochrome c (ycc) and its K72A/K73A, K72A/K79A, K73A/K79A and K72A/K73A/K79A variants with CL was studied through UV-Vis and MCD spectroscopies at pH 7 and molecular dynamics (MD) simulations, to clarify the role of the mutated lysines. Moreover, the influence of the lipid to protein ratio on the interaction mechanism was investigated using low (0.5-10) and high (5-60) CL/ycc molar ratios, obtained with small and gradual or large and abrupt CL additions, respectively. Although all proteins bind to CL, switching from the native low-spin His/Met-ligated form to a low-spin bis-His conformer and to a high-spin species at larger CL concentrations, the two schemes of CL addition show relevant differences in the CL/ycc molar ratios at which the various conformers appear, due to differences in the interaction mechanism. Extended lipid anchorage and peripheral binding appear to prevail at low and high CL/ycc molar ratios, respectively. Simultaneous deletion of two or three surface positive charges from Site A does not abolish CL binding, but instead increases protein affinity for CL. MD calculations suggest this unexpected behavior results from the mutation-induced severe weakening of the H-bond connecting the Nε of His26 with the backbone oxygen of Glu44, which lowers the conformational stability compared to the wt species, overcoming the decreased surface electrostatic interaction.

Probing the Effect of Sildenafil on Progesterone and Testosterone Production by an Intracellular FRET/BRET Combined Approach.

Forster resonance energy transfer (FRET)-based biosensors have been recently applied to the study of biological pathways. In this study, a new biosensor was validated for the first time in live HEK293 and steroidogenic MLTC-1 cell lines for studying the effect of the PDE5 inhibitor on the hCG/LH-induced steroidogenic pathway. The sensor improves FRET between a donor (D), the fluorescein-like diarsenical probe that can covalently bind a tetracysteine motif fused to the PDE5 catalytic domain, and an acceptor (A), the rhodamine probe conjugated to the pseudosubstrate cGMPS. Affinity constant ( Kd) values of 5.6 ± 3.2 and 13.7 ± 0.8 µM were obtained with HEK293 and MLTC-1 cells, respectively. The detection was based on the competitive displacement of the cGMPS-rhodamine conjugate by sildenafil; the Ki values were 3.6 ± 0.3 nM (IC50 = 2.3 nM) in HEK293 cells and 10 ± 1.0 nM (IC50 = 3.9 nM) in MLTC-1 cells. The monitoring of both cAMP and cGMP by bioluminescence resonance energy transfer allowed the exploitation of the effects of PDE5i on steroidogenesis, indicating that sildenafil enhanced the gonadotropin-induced progesterone-to-testosterone conversion in a cAMP-independent manner.

Helicobacter pylori Infection and Gastric Dysbiosis: Can Probiotics Administration Be Useful to Treat This Condition?

Helicobacter pylori (Hp) is responsible for one of the most common infections in the world. The prevalence exceeds 50% of the population in developing countries, and approximately one-third of the adults are colonized in North Europe and North America. It is considered a major pathogenic agent of chronic gastritis, peptic ulcer, atrophic gastritis, gastric cancer, and mucosa-associated lymphoid tissue lymphoma (MALT). Hp colonization modifies the composition of gastric microbiota that could drive the development of gastric disorders. Currently, an emerging problem in Hp treatment is represented by the increasing rate of antimicrobial therapy resistance. In this context, the search for adjuvant agents can be very useful to overcome this issue and probiotics administration can represent a valid option. The aim of this review is to describe the gastric microbiota changes during Hp colonization, the mechanisms of action, and a possible role of probiotics in the treatment of this infection.

Computational evidence support the hypothesis of neuroglobin also acting as an electron transfer species.

Neuroglobin (Ngb) is a recently identified hexa-coordinated globin, expressed in the nervous system of humans. Its physiological role is still debated: one hypothesis is that Ngb serves as an electron transfer (ET) species, possibly by reducing cytochrome c and preventing it to initiate the apoptotic cascade. Here, we use the perturbed matrix method (PMM), a mixed quantum mechanics/molecular dynamics approach, to investigate the redox thermodynamics of two neuroglobins, namely the human Ngb and GLB-6 from invertebrate Caenorhabditis elegans. In particular, we calculate the reduction potential of the two globins, resulting in an excellent agreement with the experimental values, and we predict the reorganization energies, λ, which have not been determined experimentally yet. The calculated λ values match well those reported for known ET proteins and thereby support a potential involvement in vivo of the two globins in ET processes.

Immobilized cytochrome c bound to cardiolipin exhibits peculiar oxidation state-dependent axial heme ligation and catalytically reduces dioxygen.

Mitochondrial cytochrome c (cytc) plays an important role in programmed cell death upon binding to cardiolipin (CL), a negatively charged phospholipid of the inner mitochondrial membrane (IMM). Although this binding has been thoroughly investigated in solution, little is known on the nature and reactivity of the adduct (cytc-CL) immobilized at IMM. In this work, we have studied electrochemically cytc-CL immobilized on a hydrophobic self-assembled monolayer (SAM) of decane-1-thiol. This construct would reproduce the motional restriction and the nonpolar environment experienced by cytc-CL at IMM. Surface-enhanced resonance Raman (SERR) studies allowed the axial heme iron ligands to be identified, which were found to be oxidation state dependent and differ from those of cytc-CL in solution. In particular, immobilized cytc-CL experiences an equilibrium between a low-spin (LS) 6c His/His and a high-spin (HS) 5c His/- coordination states. The former prevails in the oxidized and the latter in the reduced form. Axial coordination of the ferric heme thus differs from the (LS) 6c His/Lys and (LS) 6c His/OH(-) states observed in solution. Moreover, a relevant finding is that the immobilized ferrous cytc-CL is able to catalytically reduce dioxygen, likely to superoxide ion. These findings indicate that restriction of motional freedom due to interaction with the membrane is an additional factor playing in the mechanism of cytc unfolding and cytc-mediated peroxidation functional to the apoptosis cascade.

Effects of removal of the axial methionine heme ligand on the binding of S. cerevisiae iso-1 cytochrome c to cardiolipin.

The cleavage of the axial S(Met) - Fe bond in cytochrome c (cytc) upon binding to cardiolipin (CL), a glycerophospholipid of the inner mitochondrial membrane, is one of the key molecular changes that impart cytc with (lipo)peroxidase activity essential to its pro-apoptotic function. In this work, UV - VIS, CD, MCD and fluorescence spectroscopies were used to address the role of the Fe - M80 bond in controlling the cytc-CL interaction, by studying the binding of the Met80Ala (M80A) variant of S. cerevisiae iso-1 cytc (ycc) to CL liposomes in comparison with the wt protein [Paradisi et al. J. Biol. Inorg. Chem. 25 (2020) 467-487]. The results show that the integrity of the six-coordinate heme center along with the distal heme site containing the Met80 ligand is a not requisite for cytc binding to CL. Indeed, deletion of the Fe - S(Met80) bond has a little impact on the mechanism of ycc-CL interaction, although it results in an increased heme accessibility to solvent and a reduced structural stability of the protein. In particular, M80A features a slightly tighter binding to CL at low CL/cytc ratios compared to wt ycc, possibly due to the lift of some constraints to the insertion of the CL acyl chains into the protein hydrophobic core. M80A binding to CL maintains the dependence on the CL-to-cytc mixing scheme displayed by the wt species.

Ustekinumab safety and effectiveness in patients with ulcerative colitis: results from a large real-life study.

BACKGROUND: Ustekinumab (UST) is an interleukin-12/interleukin-23 receptor antagonist recently approved for treating ulcerative colitis (UC) but with limited real-world data. Therefore, we evaluated the effectiveness and safety of UST in patients with UC in a real-world setting. RESEARCH DESIGN AND METHODS: This is a multicenter, retrospective, observational cohort study. The primary endpoints were the clinical remission rate (partial Mayo score, PMS, ≤1) and the safety of UST. Other endpoints were corticosteroid-free remission (CSFR) rate, clinical response rate (PMS reduction of at least 2 points), and fecal calprotectin (FC) reduction at week 24. RESULTS: We included 256 consecutive patients with UC (M/F 139/117, median age 52). The clinical remission and clinical response rates at eight weeks were 18.7% (44/235) and 53.2% (125/235), respectively, and 27.6% (42/152) and 61.8% (94/152) at 24 weeks, respectively. At 24 weeks, CSFR was 20.3% (31/152), and FC significantly dropped at week 12 (p = 0.0004) and 24 (p = 0.038). At eight weeks, patients naïve or with one previous biologic treatment showed higher remission (p = 0.002) and clinical >response rates (p = 0.018) than patients previously treated with ≥ 2. Adverse events occurred in six patients (2.3%), whereas four patients (1.6%) underwent colectomy. CONCLUSION: This real-world study shows that UST effectively and safely treats patients with UC.

Axial iron coordination and spin state change in a heme c upon electrostatic protein-SAM interaction.

A bacterial di-heme cytochrome c binds electrostatically to a gold electrode surface coated with a negatively charged COOH-terminated SAM adopting a sort of 'perpendicular' orientation. Cyclic voltammetry, Resonance Raman and SERRS spectroscopies indicate that the high-potential C-terminal heme center proximal to the SAM's surface undergoes an adsorption-induced swapping of one axial His ligand with a water molecule, which is probably lost in the reduced form, and a low- to high-spin transition. This coordination change for a bis-His ligated heme center upon an electrostatically-driven molecular recognition is as yet unprecedented, as well as the resulting increase in reduction potential. We discuss it in comparison with the known methionine ligand lability in monoheme cytochromes c occurring upon interaction with charged molecular patches. One possible implication of this finding in biological ET is that mobile redox partners do not behave as rigid and invariant bodies, but in the ET complex are subjected to molecular changes and structural fluctuations that affect in a complex way the thermodynamics and the kinetics of the process.

Hydrogen peroxide induces heme degradation and protein aggregation in human neuroglobin: roles of the disulfide bridge and hydrogen-bonding in the distal heme cavity.

In the present study, human neuroglobin (hNgb) was found to undergo H2 O2 -induced breakdown of the heme center at a much slower rate than other globins, namely in the timescale of hours against minutes. We investigated how the rate of the process is affected by the Cys46/Cys55 disulfide bond and the network of non-covalent interactions in the distal heme side involving Tyr44, Lys67, the His64 heme iron axial ligand and the heme propionate-7. The rate is increased by the Tyr44 to Ala and Phe mutations; however the rate is lowered by Lys67 to Ala swapping. The absence of the disulfide bridge slows down the reaction further. Therefore, the disulfide bond-controlled accessibility of the heme site and the residues at position 44 and 67 affect the activation barrier of the reaction. Wild-type and mutated species form ß-amyloid aggregates in the presence of H2 O2 producing globular structures. Furthermore, the C46A/C55A, Y44A, Y44F and Y44F/C46A/C55A variants yield potentially harmful fibrils. Finally, the nucleation and growth kinetics for the aggregation of the amyloid structures can be successfully described by the Finke-Watzky model.

A PETase enzyme synthesised in the chloroplast of the microalga Chlamydomonas reinhardtii is active against post-consumer plastics.

Polyethylene terephthalate hydrolases (PETases) are a newly discovered and industrially important class of enzymes that catalyze the enzymatic degradation of polyethylene terephatalate (PET), one of the most abundant plastics in the world. The greater enzymatic efficiencies of PETases compared to close relatives from the cutinase and lipase families have resulted in increasing research interest. Despite this, further characterization of PETases is essential, particularly regarding their possible activity against other kinds of plastic. In this study, we exploited for the first time the use of the microalgal chloroplast for more sustainable synthesis of a PETase enzyme. A photosynthetic-restoration strategy was used to generate a marker-free transformant line of the green microalga Chlamydomonas reinhardtii in which the PETase from Ideonella sakaiensis was constitutively expressed in the chloroplast. Subsequently, the activity of the PETase against both PET and post-consumer plastics was investigated via atomic force microscopy, revealing evidence of degradation of the plastics.

Use of tofacitinib as first or second-line therapy is associated with better outcomes in patients with ulcerative colitis: data from a real-world study.

BACKGROUND: Data regarding the real-world (RW) use of tofacitinib (TOF) in patients with ulcerative colitis (UC) are limited. We aimed to investigate TOF's RW efficacy and safety in Italian UC patients. RESEARCH DESIGN AND METHODS: A retrospective assessment of clinical and endoscopic activity was performed according to the Mayo score. The primary endpoints were to evaluate the effectiveness and safety of TOF. RESULTS: We enrolled 166 patients with a median follow-up of 24 (IQR 8-36) weeks. Clinical remission was achieved in 61/166 (36.7%) and 75/166 (45.2%) patients at 8-week and 24-week follow-ups, respectively. The optimization was requested in 27 (16.3%) patients. Clinical remission was achieved more frequently when TOF was used as a first/second line rather than a third/fourth line treatment (p = 0.007). Mucosal healing was reported in 46% of patients at the median follow-up time. Colectomy occurred in 8 (4.8%) patients. Adverse events occurred in 12 (5.4%) patients and severe in 3 (1.8%). One case of simple Herpes Zoster and one of renal vein thrombosis were recorded. CONCLUSIONS: Our RW data confirm that TOF is effective and safe in UC patients. It performs remarkably better when used as the first/second line of treatment.

Real-world efficacy and safety of vedolizumab in managing ulcerative colitis versus Crohn's disease: results from an Italian multicenter study.

BACKGROUND: Vedolizumab (VDZ) can be used to treat refractory ulcerative colitis (UC) and Crohn's disease (CD). We assessed whether there are differences in treating UC vs CD with VDZ. RESEARCH DESIGN AND METHODS: Mayo score in UC and the Harvey-Bradshaw Index (HBI) in CD scored the clinical activity. Achievement and maintenance of clinical remission during the follow-up, and safety were the primary endpoints. RESULTS: 729 patients (475 with UC and 254 with CD), median follow-up of 18 (IQR 6-36) months, were enrolled. Clinical remission at the 6th month of treatment was achieved in 488 (66.9%) patients (74.4% in CD vs 62.9% in UC, p<0.002) while, during the follow-up, no difference was found (81.5% in the UC group and 81.5% pts in the CD group; p=0.537). The clinical remission at the 6th month of treatment (p=0.001) and being naïve to biologics (p<0.0001) were significantly associated with prolonged clinical remission. The clinical response was significantly higher in UC (90.1%) vs CD (84.3%) (p=0.023), and surgery occurred more frequently in CD (1.9% in UC vs 5.1% in CD, p=0.016). CONCLUSION: We found differences when using VDZ in UC vs CD in real life. These parameters can help the physician predict this drug's longterm efficacy.

Role of Met80 and Tyr67 in the low-pH conformational equilibria of cytochrome c.

The low-pH conformational equilibria of ferric yeast iso-1 cytochrome c (ycc) and its M80A, M80A/Y67H, and M80A/Y67A variants were studied from pH 7 to 2 at low ionic strength through electronic absorption, magnetic circular dichroism, and resonance Raman spectroscopies. For wild-type ycc, the protein structure, axial heme ligands, and spin state of the iron atom convert from the native folded His/Met low-spin (LS) form to a molten globule His/H(2)O high-spin (HS) form and a totally unfolded bis-aquo HS state, in a single cooperative transition with an apparent pK(a) of ~3.0. An analogous cooperative transition occurs for the M80A and M80A/Y67H variants. This is preceded by protonation of heme propionate-7, with a pK(a) of ~4.2, and by an equilibrium between a His/OH(-)-ligated LS and a His/H(2)O-ligated HS conformer, with a pK(a) of ~5.9. In the M80A/Y67A variant, the cooperative low-pH transition is split into two distinct processes because of an increased stability of the molten globule state that is formed at higher pH values than the other species. These data show that removal of the axial methionine ligand does not significantly alter the mechanism of acidic unfolding and the ranges of stability of low-pH conformers. Instead, removal of a hydrogen bonding partner at position 67 increases the stability of the molten globule and renders cytochrome c more susceptible to acid unfolding. This underlines the key role played by Tyr67 in stabilizing the three-dimensional structure of cytochrome c by means of the hydrogen bonding network connecting the Ω loops formed by residues 71-85 and 40-57.

pH and solvent H/D isotope effects on the thermodynamics and kinetics of electron transfer for electrode-immobilized native and urea-unfolded stellacyanin.

The thermodynamics of Cu(II) to Cu(I) reduction and the kinetics of the electron transfer (ET) process for Rhus vernicifera stellacyanin (STC) immobilized on a decane-1-thiol coated gold electrode have been measured through cyclic voltammetry at varying pH and temperature, in the presence of urea and in D(2)O. Immobilized STC undergoes a limited conformational change that mainly results in an enhanced exposure of one or both copper binding histidines to solvent which slightly stabilizes the cupric state and increases histidine basicity. The large immobilization-induced increase in the pK(a) for the acid transition (from 4.5 to 6.3) makes this electrode-SAM-protein construct an attractive candidate as a biomolecular ET switch operating near neutral pH in molecular electronics. Such a potential interest is increased by the robustness of this interface against chemical unfolding as it undergoes only moderate changes in the reduction thermodynamics and in the ET rate in the presence of up to 8 M urea. The sensitivity of these parameters to solvent H/D isotope effects testifies to the role of protein solvation as effector of the thermodynamics and kinetics of ET.

Anti-Spoilage Activity and Exopolysaccharides Production by Selected Lactic Acid Bacteria.

In this study, eight lactic acid bacteria (LAB) strains, previously isolated from traditional and gluten-free sourdoughs, and selected for their potential in improving the sensory and rheological quality of bakery products, were screened against some common spoilage agents. The anti-mould activity was tested using strains of the species Fusarium graminearum, Aspergillus flavus, Penicillium paneum and Aspergillus niger. Regarding the antibacterial activity, it was assessed against four strains of the species Escherichia coli, Campylobacter jejuni, Salmonella typhimurium and Listeria monocytogenes. Furthermore, LAB strains were evaluated for their ability to produce exopolysaccharides, which are gaining considerable attention for their functional properties and applicability in different food industrial applications. A strain-specific behaviour against the moulds was observed. In particular, F. graminearum ITEM 5356 was completely inhibited by all the LAB strains. Regarding the antibacterial activity, the strains Leuconostoc citreum UMCC 3011, Lactiplantibacillus plantarum UMCC 2996, and Pediococcus pentosaceus UMCC 3010 showed wide activity against the tested pathogens. Moreover, all the LAB strains were able to produce exopolysaccharides, which were preliminarily characterized. The assessed features of the LAB strains allow us to consider them as promising candidates for single or multiple starter cultures for food fermentation processes.

Activity and substrate specificity of lytic polysaccharide monooxygenases: An ATR FTIR-based sensitive assay tested on a novel species from Pseudomonas putida.

Pseudomonas putida W619 is a soil Gram-negative bacterium commonly used in environmental studies thanks to its ability in degrading many aromatic compounds. Its genome contains several putative carbohydrate-active enzymes such as glycoside hydrolases and lytic polysaccharide monooxygenases (PMOs). In this study, we have heterologously produced in Escherichia coli and characterized a new enzyme belonging to the AA10 family, named PpAA10 (Uniprot: B1J2U9), which contains a chitin-binding type-4 module and showed activity toward ß-chitin. The active form of the enzyme was produced in E. coli exploiting the addition of a cleavable N-terminal His tag which ensured the presence of the copper-coordinating His as the first residue. Electron paramagnetic resonance spectroscopy showed signal signatures similar to those observed for the copper-binding site of chitin-cleaving PMOs. The protein was used to develop a versatile, highly sensitive, cost-effective and easy-to-apply method to detect PMO's activity exploiting attenuated total reflection-Fourier transform infrared spectroscopy and able to easily discriminate between different substrates.