Mass spectrometry-based proteomic strategy for ecchymotic skin examination in forensic pathology.

Mass spectrometry (MS)-based proteomics has recently attracted the attention from forensic pathologists. This work is the first report of the development of a shotgun bottom-up proteomic approach based on rapid protein extraction and nano-liquid chromatography/high-resolution mass spectrometry applied to full-thickness human skin for the differential analysis of normal and ecchymotic tissues to identify new biomarkers for bruise characterization and dating. We identified around 2000 proteins from each pooled extract. The method showed excellent precision on independent replicates, with Pearson correlation coefficients always higher than 95%. Glycophorin A, a known biomarker of vital wounds from immunochemical studies, was identified only in ecchymotic tissues, as confirmed by Western blotting analysis. This finding suggests that this protein can be used as a MS-detectable biomarker of wound vitality. By focusing on skin samples from individuals with known wound dating, besides Glycophorin A, other proteins differentially expressed in ecchymotic samples and dependant on wound age were identified, although further analysis on larger datasets are needed to validate these findings. This study paves the way for an in-depth investigation of the potential of MS-based techniques for wound examination in forensic pathology, overcoming the limitations of immunochemical assays.

A Hybrid In Silico/In Vitro Target Fishing Study to Mine Novel Targets of Urolithin A and B: A Step Towards a Better Comprehension of Their Estrogenicity.

SCOPE: Urolithin A and B are gut metabolites of ellagic acid and ellagitannins associated with many beneficial effects. Evidence in vitro pointed to their potential as estrogenic modulators. However, both molecular mechanisms and biological targets involved in such activity are still poorly characterized, preventing a comprehensive understanding of their bioactivity in living organisms. This study aimed at rationally identifying novel biological targets underlying the estrogenic-modulatory activity of urolithins. METHODS AND RESULTS: The work relies on an in silico/in vitro target fishing study coupling molecular modeling with biochemical and cell-based assays. Estrogen sulfotransferase and 17ß-hydroxysteroid dehydrogenase are identified as potentially subject to inhibition by the investigated urolithins. The inhibition of the latter undergoes experimental confirmation either in a cell-free or cell-based assay, validating computational outcomes. CONCLUSIONS: The work describes target fishing as an effective tool to identify unexpected targets of food bioactives detailing the interaction at a molecular level. Specifically, it described, for the first time, 17ß-hydroxysteroid dehydrogenase as a target of urolithins and highlighted the need of further investigations to widen the understanding of urolithins as estrogen modulators in living organisms.

Primary pulmonary arterial hypertension: Protocol to assess comprehensively in the rat the response to pharmacologic treatments.

The identification of new treatments for primary pulmonary arterial hypertension (PAH) is a critical unmet need since there is no a definitive cure for this disease yet. Due to the complexity of PAH, a wide set of methods are necessary to assess the response to a pharmacological intervention. Thus, a rigorous protocol is crucial when experimental studies are designed. In the present experimental protocol, a stepwise approach was followed in a monocrotaline-induced PAH model in the rat, moving from the dose finding study of treatment compounds to the recognition of the onset of disease manifestation, in order to identify when to start a curative treatment. A complete multidimensional evaluation of treatment effects represented the last step. The primary study endpoint was the change in right ventricular systolic pressure after 14 days of treatment; echocardiographic and biohumoral markers together with heart and pulmonary arterial morphometric parameters were considered as secondary efficacy and/or safety endpoints and for the evaluation of the biologic coherence in the different results.

Monocrotaline-induced pulmonary arterial hypertension: Time-course of injury and comparative evaluation of macitentan and Y-27632, a Rho kinase inhibitor.

Novel pharmacological approaches are needed to improve outcomes of patients with idiopathic pulmonary hypertension. Rho-associated protein kinase (ROCK) inhibitors have shown beneficial effects in preclinical models of pulmonary arterial hypertension (PAH), because of their role in the regulation of pulmonary artery vasoconstrictor tone and remodeling. We compared a ROCK inhibitor, Y-27632, for the first time with the dual endothelin receptor antagonist, macitentan, in a monocrotaline-induced rat pulmonary hypertension model. Different methods (echocardiography, hemodynamics, histology of right ventricle and pulmonary vessels, and circulating biomarkers) showed consistently that 100 mg/kg daily of Y-27632 and 10 mg/kg daily of macitentan slowed the progression of PAH both at the functional and structural levels. Treatments started on day 14 after monocrotaline injection and lasted 14 days. The findings of all experimental methods show that the selective ROCK inhibitor Y-27632 has more pronounced effects than macitentan, but a major limitation to its use is its marked peripheral vasodilating action.

Downregulation of SLC7A7 Triggers an Inflammatory Phenotype in Human Macrophages and Airway Epithelial Cells.

Lysinuric protein intolerance (LPI) is a recessively inherited aminoaciduria caused by mutations of SLC7A7, the gene encoding y+LAT1 light chain of system y+L for cationic amino acid transport. The pathogenesis of LPI is still unknown. In this study, we have utilized a gene silencing approach in macrophages and airway epithelial cells to investigate whether complications affecting lung and immune system are directly ascribable to the lack of SLC7A7 or, rather, mediated by an abnormal accumulation of arginine in mutated cells. When SLC7A7/y+LAT1 was silenced in human THP-1 macrophages and A549 airway epithelial cells by means of short interference RNA (siRNA), a significant induction of the expression and release of the inflammatory mediators IL1ß and TNFα was observed, no matter the intracellular arginine availability. This effect was mainly regulated at transcriptional level through the activation of NFκB signaling pathway. Moreover, since respiratory epithelial cells are the important sources of chemokines in response to pro-inflammatory stimuli, the effect of IL1ß has been addressed on SLC7A7 silenced A549 cells. Results obtained indicated that the downregulation of SLC7A7/y+LAT1 markedly strengthened the stimulatory effect of the cytokine on CCL5/RANTES expression and release without affecting the levels of CXCL8/IL8. Consistently, also the conditioned medium of silenced THP-1 macrophages activated airway epithelial cells in terms of CCL5/RANTES expression due to the presence of elevated amount of proinflammatory cytokines. In conclusion, our results point to a novel thus far unknown function of SLC7A7/y+LAT1, that, under physiological conditions, besides transporting arginine, may act as a brake to restrain inflammation.

Human macrophage differentiation induces OCTN2-mediated L-carnitine transport through stimulation of mTOR-STAT3 axis.

l-Carnitine, in addition to playing a fundamental role in the ß-oxidation of fatty acids, has been recently identified as a modulator of immune function, although the mechanisms that underlie this role remain to be clarified. In this study, we addressed the modulation of l-carnitine transport and expression of related transporters during differentiation of human monocytes to macrophages. Whereas monocytes display a modest uptake of l-carnitine, GM-CSF-induced differentiation massively increased the saturable Na+-dependent uptake of l-carnitine. Kinetic and inhibition analyses demonstrate that in macrophage l-carnitine transport is mediated by a high-affinity component (Km ∼4 µM) that is identifiable with the operation of OCTN2 transporter and a low-affinity component (Km > 10 mM) that is identifiable with system A for neutral amino acids. Consistently, both SLC22A5/OCTN2 and SLC38A2/SNAT2 are induced during the differentiation of monocytes to macrophages at gene and protein levels. Elucidation of GM-CSF signaling demonstrates that the cytokine causes the activation of mTOR kinase, leading to the phosphorylation and activation of STAT3, which, in turn, is responsible for OCTN2 transcription. SLC22A5/OCTN2 therefore emerges as a novel member of the set of genes markers of macrophage differentiation.

An innovative method based on quick, easy, cheap, effective, rugged, and safe extraction coupled to desorption electrospray ionization-high resolution mass spectrometry for screening the presence of paralytic shellfish toxins in clams.

The capabilities of desorption electrospray ionization-high resolution mass spectrometry (DESI-HRMS) were tested for screening the presence of some paralytic shellfish toxins in clams. A quick, easy, cheap, effective, rugged, and safe (QuEChERS) approach is proposed for sample clean-up. QuEChERS extraction was optimized by using a full factorial design followed by the multicriteria method of the desirability functions. Quantitation limits in the microgram per kilogram range proved reliability of the method for the detection of the investigated toxins in accordance to the rules laid down by European legislation. The optimized QuEChERS-DESI-HRMS based-method allowed for a rapid reliable screening of the investigated compounds at levels of interest, thus being useful for high-throughput analyses.

Quantitative proteomics analysis of platelet-derived microparticles reveals distinct protein signatures when stimulated by different physiological agonists.

Platelet-derived MPs (PMPs) are a heterogeneous population of microvesicles released from platelets upon activation and apoptosis. Different platelet activations may affect PMP protein profiles and roles in intercellular communication. Here, we performed a quantitative proteomics study to characterize the protein content of PMPs generated by four differentially activated platelet samples. We selected known physiological agonists for platelet activation such as ADP, thrombin and collagen. Thrombin, which is mostly used to generate PMPs in vitro, was set as control. Platelets were activated by following a known agonist strength scale in which ADP was the weakest activation and thrombin and collagen stimulations were the strongest ones. Our proteomic analysis allowed the quantification of 3383 proteins, of which 428 membrane and 131 soluble proteins were found as significantly different in at least one of the analyzed conditions. Activation with stronger agonists led to the enrichment of proteins related to platelet activation in PMPs. In addition, proteins involved in platelet degranulation and proteins from the electron transport chain were less abundant in PMPs when stronger activation was used. Collectively, our data describe the most detailed characterization of PMPs after platelet physiological activation. Furthermore, we show that PMP protein content is highly dependent on the type of physiological agonist involved in platelet stimulation. BIOLOGICAL SIGNIFICANCE: Platelet-derived MPs (PMPs) are a population of vesicles generated upon platelet activation by various stimuli known to be involved in several physiological and pathological processes. This manuscript investigates the protein profile of PMPs obtained by performing four different activation protocols using mass spectrometry-based quantitative proteomics. By following a known physiological agonist strength scale our findings suggest a biological link between agonist strength and proteins associated to platelet mediated processes such as activation and degranulation. These data may provide new insights for understanding PMP biological role and formation.