Supercritical Fluid Extraction of Bioactive Components from Apple Peels and Their Modulation of Complex I Activity in Isolated Mitochondria.

Supercritical fluid extraction (SFE) was used to extract bioactive compounds from apple (Malus domestica) peel waste from three different Italian cultivars. The bioactive fractions were extracted applying a temperature of 60 °C and a pressure of 250 bar for 15 min with 20% ethanol as co-solvent, at a flow rate of 2 mL/min. The total polyphenol (TP), anthocyanin (TA), ascorbic acid (AA), and antioxidant activity contents (TACs) were measured, while chromatographic analyses were performed to highlight the differences between the extracts. The Stark cultivar had the highest levels of polyphenols, anthocyanins, and ascorbic acid, while the Royal Gala cultivar showed the highest total antioxidant activity. SFE extracts were then tested for their effect on the mitochondrial NADH-ubiquinone oxidoreductase (Complex I) activity on mitochondria isolated from human embryonic kidney cells (HEK239). The Stark extract showed the most positive response in terms of NADH oxidation. The results obtained in this work highlight the potential of apple peel waste as a source of functional phytocompounds and suggest that Stark cultivar extracts may be exploited for pharmacological applications. This study supports the circular bioeconomy by promoting the use of waste products as a valuable resource.

Nucleotide substitutions at the p.Gly117 and p.Thr180 mutational hot-spots of SKI alter molecular dynamics and may affect cell cycle.

Heterozygous deleterious variants in SKI cause Shprintzen-Goldberg Syndrome, which is mainly characterized by craniofacial features, neurodevelopmental disorder and thoracic aorta dilatations/aneurysms. The encoded protein is a member of the transforming growth factor beta signaling. Paucity of reported studies exploring the SGS molecular pathogenesis hampers disease recognition and clinical interpretation of private variants. Here, the unpublished c.349G>A, p.[Gly117Ser] and the recurrent c.539C>T, p.[Thr180Met] SKI variants were studied combining in silico and in vitro approach. 3D comparative modeling and calculation of the interaction energy predicted that both variants alter the SKI tertiary protein structure and its interactions. Computational data were functionally corroborated by the demonstration of an increase of MAPK phosphorylation levels and alteration of cell cycle in cells expressing the mutant SKI. Our findings confirmed the effects of SKI variants on MAPK and opened the path to study the role of perturbations of the cell cycle in SGS.

TNFRSF13B gene mutation in familial acute myeloid leukemia: A new piece in the complex scenario of hereditary predisposition?

TNFRSF13B mutations are widely associated with common variable immunodeficiency. TNFRSF13B was recently counted among relevant genes associated with childhood-onset of hematological malignancies; nonetheless, its role in acute myeloid leukemia (AML) remains unexplored. We report the study of a family with two cases of AML, sharing a germline TNFRSF13B mutation favoring the formation of a more stable complex with its ligand TNFSF13: a positive regulator of AML-initiating cells. Our data turn the spotlight onto the TNFRSF13B role in AML onset, inserting a new fragment into the complex scenario of a hereditary predisposition to myeloid neoplasms.

Case report: biallelic DNMT3A mutations in acute myeloid leukemia.

DNMT3A gene mutations, detected in 20-25% of de novo acute myeloid leukemia (AML) patients, are typically heterozygous. Biallelic variants are uncommon, affecting ~3% of cases and identifying a worse prognosis. Indeed, two concomitant DNMT3A mutations were recently associated with shorter event-free survival and overall survival in AML. We present an AML case bearing an unusual DNMT3A molecular status, strongly affecting its function and strangely impacting the global genomic methylation profile. A 56-year-old Caucasian male with a diagnosis of AML not otherwise specified (NOS) presented a complex DNMT3A molecular profile consisting of four different somatic variants mapping on different alleles (in trans). 3D modelling analysis predicted the effect of the DNMT3A mutational status, showing that all the investigated mutations decreased or abolished DNMT3A activity. Although unexpected, DNMT3A's severe loss of function resulted in a global genomic hypermethylation in genes generally involved in cell differentiation. The mechanisms through which DNMT3A contributes to AML remain elusive. We present a unique AML case bearing multiple biallelic DNMT3A variants abolishing its activity and resulting in an unexpected global hypermethylation. The unusual DNMT3A behavior described requires a reflection on its role in AML development and persistence, highlighting the heterogeneity of its deregulation.

Deconstructing SARS-CoV-2 neutralization: A modular molecular framework for computational design and comparison of antibodies and nanobodies targeting the spike RBD.

Since 2020 the COVID-19 pandemic has led scientists to search for strategies to predict the transmissibility and virulence of new severe acute respiratory syndrome coronavirus 2 variants based on the estimation of the affinity of the spike receptor binding domain (RBD) for the human angiotensin-converting enzyme 2 (ACE2) receptor and/or neutralizing antibodies. In this context, our lab developed a computational pipeline to quickly quantify the free energy of interaction at the spike RBD/ACE2 protein-protein interface, reflecting the incidence trend observed in the transmissibility/virulence of the investigated variants. In this new study, we used our pipeline to estimate the free energy of interaction between the RBD from 10 variants, and 14 antibodies (ab), or 5 nanobodies (nb), highlighting the RBD regions preferentially targeted by the investigated ab/nb. Our structural comparative analysis and interaction energy calculations allowed us to propose the most promising RBD regions to be targeted by future ab/nb to be designed by site-directed mutagenesis of existing high-affinity ab/nb, to increase their affinity for the target RBD region, for preventing spike-RBD/ACE2 interactions and virus entry in host cells. Furthermore, we evaluated the ability of the investigated ab/nb to simultaneously interact with the three RBD located on the surface of the trimeric spike protein, which can alternatively be in up- or down- (all-3-up-, all-3-down-, 1-up-/2-down-, 2-up-/1-down-) conformations.

The Mycotoxin Patulin Inhibits the Mitochondrial Carnitine/Acylcarnitine Carrier (SLC25A20) by Interaction with Cys136 Implications for Human Health.

The effect of mycotoxin patulin (4-hydroxy-4H-furo [3,2c] pyran-2 [6H] -one) on the mitochondrial carnitine/acylcarnitine carrier (CAC, SLC25A20) was investigated. Transport function was measured as [3H]-carnitineex/carnitinein antiport in proteoliposomes reconstituted with the native protein extracted from rat liver mitochondria or with the recombinant CAC over-expressed in E. coli. Patulin (PAT) inhibited both the mitochondrial native and recombinant transporters. The inhibition was not reversed by physiological and sulfhydryl-reducing reagents, such as glutathione (GSH) or dithioerythritol (DTE). The IC50 derived from the dose-response analysis indicated that PAT inhibition was in the range of 50 µM both on the native and on rat and human recombinant protein. The kinetics process revealed a competitive type of inhibition. A substrate protection experiment confirmed that the interaction of PAT with the protein occurred within a protein region, including the substrate-binding area. The mechanism of inhibition was identified using the site-directed mutagenesis of CAC. No inhibition was observed on Cys mutants in which only the C136 residue was mutated. Mass spectrometry studies and in silico molecular modeling analysis corroborated the outcomes derived from the biochemical assays.

Targeting mitochondrial impairment for the treatment of cardiovascular diseases: From hypertension to ischemia-reperfusion injury, searching for new pharmacological targets.

Mitochondria and mitochondrial proteins represent a group of promising pharmacological target candidates in the search of new molecular targets and drugs to counteract the onset of hypertension and more in general cardiovascular diseases (CVDs). Indeed, several mitochondrial pathways result impaired in CVDs, showing ATP depletion and ROS production as common traits of cardiac tissue degeneration. Thus, targeting mitochondrial dysfunction in cardiomyocytes can represent a successful strategy to prevent heart failure. In this context, the identification of new pharmacological targets among mitochondrial proteins paves the way for the design of new selective drugs. Thanks to the advances in omics approaches, to a greater availability of mitochondrial crystallized protein structures and to the development of new computational approaches for protein 3D-modelling and drug design, it is now possible to investigate in detail impaired mitochondrial pathways in CVDs. Furthermore, it is possible to design new powerful drugs able to hit the selected pharmacological targets in a highly selective way to rescue mitochondrial dysfunction and prevent cardiac tissue degeneration. The role of mitochondrial dysfunction in the onset of CVDs appears increasingly evident, as reflected by the impairment of proteins involved in lipid peroxidation, mitochondrial dynamics, respiratory chain complexes, and membrane polarization maintenance in CVD patients. Conversely, little is known about proteins responsible for the cross-talk between mitochondria and cytoplasm in cardiomyocytes. Mitochondrial transporters of the SLC25A family, in particular, are responsible for the translocation of nucleotides (e.g., ATP), amino acids (e.g., aspartate, glutamate, ornithine), organic acids (e.g. malate and 2-oxoglutarate), and other cofactors (e.g., inorganic phosphate, NAD+, FAD, carnitine, CoA derivatives) between the mitochondrial and cytosolic compartments. Thus, mitochondrial transporters play a key role in the mitochondria-cytosol cross-talk by leading metabolic pathways such as the malate/aspartate shuttle, the carnitine shuttle, the ATP export from mitochondria, and the regulation of permeability transition pore opening. Since all these pathways are crucial for maintaining healthy cardiomyocytes, mitochondrial carriers emerge as an interesting class of new possible pharmacological targets for CVD treatments.

New Insights Regarding Hemin Inhibition of the Purified Rat Brain 2-Oxoglutarate Carrier and Relationships with Mitochondrial Dysfunction.

A kinetic analysis of the transport assays on the purified rat brain 2-oxoglutarate/malate carrier (OGC) was performed starting from our recent results reporting about a competitive inhibitory behavior of hemin, a physiological porphyrin derivative, on the OGC reconstituted in an active form into proteoliposomes. The newly provided transport data and the elaboration of the kinetic equations show evidence that hemin exerts a mechanism of partially competitive inhibition, coupled with the formation of a ternary complex hemin-carrier substrate, when hemin targets the OGC from the matrix face. A possible interpretation of the provided kinetic analysis, which is supported by computational studies, could indicate the existence of a binding region responsible for the inhibition of the OGC and supposedly involved in the regulation of OGC activity. The proposed regulatory binding site is located on OGC mitochondrial matrix loops, where hemin could establish specific interactions with residues involved in the substrate recognition and/or conformational changes responsible for the translocation of mitochondrial carrier substrates. The regulatory binding site would be placed about 6 Å below the substrate binding site of the OGC, facing the mitochondrial matrix, and would allow the simultaneous binding of hemin and 2-oxoglutarate or malate to different regions of the carrier. Overall, the presented experimental and computational analyses help to shed light on the possible existence of the hemin-carrier substrate ternary complex, confirming the ability of the OGC to bind porphyrin derivatives, and in particular hemin, with possible consequences for the mitochondrial redox state mediated by the malate/aspartate shuttle led by the mitochondrial carriers OGC and AGC.

Proline/Glycine residues of the PG-levels guide conformational changes along the transport cycle in the mitochondrial carnitine/acylcarnitine carrier (SLC25A20).

Mitochondrial carnitine/acylcarnitine carrier (CAC) is a member of the mitochondrial carrier (MC) family and imports acylcarnitine into the mitochondrial matrix in exchange for carnitine, playing a pivotal role in carnitine shuttle, crucial for fatty acid oxidation. The crystallized structure of CAC has not been solved yet, however, the availability of several in vitro/in silico studies, also based on the crystallized structures of the ADP/ATP carrier in the cytosolic-conformation and in the matrix-conformation, has made possible to confirm the hypothesis of the single-binding centered-gated pore mechanism for all the members of the MC family. In addition, our recent bioinformatics analyses allowed quantifying in silico the importance of protein residues of MC substrate binding region, of those involved in the formation of the matrix and cytosolic gates, and of those belonging to the Pro/Gly (PG) levels, proposed to be crucial for the tilting/kinking/bending of the six MC transmembrane helices, funneling the substrate translocation pathway. Here we present a combined in silico/in vitro analysis employed for investigating the role played by a group of 6 proline residues and 6 glycine residues, highly conserved in CAC, belonging to MC PG-levels. Residues of the PG-levels surround the similarly located MC common substrate binding region, and were proposed to lead conformational changes and substrate translocation, following substrate binding. For our analysis, we employed 3D molecular modeling approaches, alanine scanning site-directed mutagenesis and in vitro transport assays. Our analysis reveals that P130 (H3), G268 (H6) and G220 (H5), mutated in alanine, affect severely CAC transport activity (mutant catalytic efficiency lower than 5 % compared to the wild type CAC), most likely due to their major role in triggering CAC conformational changes, following carnitine binding. Notably, P30A (H1) and G121A (H3) CAC mutants, increase the carnitine uptake up to 217 % and 112 %, respectively, compared to the wild type CAC.

Genome-Wide Identification and Validation of Gene Expression Biomarkers in the Diagnosis of Ovarian Serous Cystadenocarcinoma.

Ovarian cancer is the second most prevalent gynecologic malignancy, and ovarian serous cystadenocarcinoma (OSCA) is the most common and lethal subtype of ovarian cancer. Current screening methods have strong limits on early detection, and the majority of OSCA patients relapse. In this work, we developed and cross-validated a method for detecting gene expression biomarkers able to discriminate OSCA tissues from healthy ovarian tissues and other cancer types with high accuracy. A preliminary ranking-based approach was applied, resulting in a panel of 41 over-expressed genes in OSCA. The RNA quantity gene expression of the 41 selected genes was then cross-validated by using NanoString nCounter technology. Moreover, we showed that the RNA quantity of eight genes (ADGRG1, EPCAM, ESRP1, MAL2, MYH14, PRSS8, ST14 and WFDC2) discriminates each OSCA sample from each healthy sample in our data set with sensitivity of 100% and specificity of 100%. For the other three genes (MUC16, PAX8 and SOX17) in combination, their RNA quantity may distinguish OSCA from other 29 tumor types.

3D structures inferred from cDNA clones identify the CD1D-Restricted γδ T cell receptor in dromedaries.

The Camelidae species occupy an important immunological niche within the humoral as well as cell mediated immune response. Although recent studies have highlighted that the somatic hypermutation (SHM) shapes the T cell receptor gamma (TRG) and delta (TRD) repertoire in Camelus dromedarius, it is still unclear how γδ T cells use the TRG/TRD receptors and their respective variable V-GAMMA and V-DELTA domains to recognize antigen in an antibody-like fashion. Here we report about 3D structural analyses of the human and dromedary γδ T cell receptor. First, we have estimated the interaction energies at the interface within the human crystallized paired TRG/TRD chains and quantified interaction energies within the same human TRG/TRD chains in complex with the CD1D, an RPI-MH1-LIKE antigen presenting glycoprotein. Then, we used the human TRG/TRD-CD1D complex as template for the 3D structure of the dromedary TRG/TRD-CD1D complex and for guiding the 3D human/dromedary comparative analysis. The choice of mutated TRG alternatively combined with mutated TRD cDNA clones originating from the spleen of one single dromedary was crucial to quantify the strength of the interactions at the protein-protein interface between the paired C. dromedarius TRG and TRD V-domains and between the C. dromedarius TRG/TRD V-domains and CD1D G-domains. Interacting amino acids located in the V-domain Complementarity Determining Regions (CDR) and Framework Regions (FR) according to the IMGT unique numbering for V-domains were identified. The resulting 3D dromedary TRG V-GAMMA combined with TRD V-DELTA protein complexes allowed to deduce the most stable gamma/delta chains pairings and to propose a candidate CD1D-restricted γδ T cell receptor complex.

Personalized Medicine in Mitochondrial Health and Disease: Molecular Basis of Therapeutic Approaches Based on Nutritional Supplements and Their Analogs.

Mitochondrial diseases (MDs) may result from mutations affecting nuclear or mitochondrial genes, encoding mitochondrial proteins, or non-protein-coding mitochondrial RNA. Despite the great variability of affected genes, in the most severe cases, a neuromuscular and neurodegenerative phenotype is observed, and no specific therapy exists for a complete recovery from the disease. The most used treatments are symptomatic and based on the administration of antioxidant cocktails combined with antiepileptic/antipsychotic drugs and supportive therapy for multiorgan involvement. Nevertheless, the real utility of antioxidant cocktail treatments for patients affected by MDs still needs to be scientifically demonstrated. Unfortunately, clinical trials for antioxidant therapies using α-tocopherol, ascorbate, glutathione, riboflavin, niacin, acetyl-carnitine and coenzyme Q have met a limited success. Indeed, it would be expected that the employed antioxidants can only be effective if they are able to target the specific mechanism, i.e., involving the central and peripheral nervous system, responsible for the clinical manifestations of the disease. Noteworthily, very often the phenotypes characterizing MD patients are associated with mutations in proteins whose function does not depend on specific cofactors. Conversely, the administration of the antioxidant cocktails might determine the suppression of endogenous oxidants resulting in deleterious effects on cell viability and/or toxicity for patients. In order to avoid toxicity effects and before administering the antioxidant therapy, it might be useful to ascertain the blood serum levels of antioxidants and cofactors to be administered in MD patients. It would be also worthwhile to check the localization of mutations affecting proteins whose function should depend (less or more directly) on the cofactors to be administered, for estimating the real need and predicting the success of the proposed cofactor/antioxidant-based therapy.

The Non-Gastric H+/K+ ATPase (ATP12A) Is Expressed in Mammalian Spermatozoa.

H+/K+ ATPase Type 2 is an heteromeric membrane protein involved in cation transmembrane transport and consists of two subunits: a specific α subunit (ATP12A) and a non-specific ß subunit. The aim of this study was to demonstrate the presence and establish the localization of ATP12A in spermatozoa from Bubalus bubalis, Bos taurus and Ovis aries. Immunoblotting revealed, in all three species, a major band (100 kDa) corresponding to the expected molecular mass. The ATP12A immunolocalization pattern showed, consistently in the three species, a strong signal at the acrosome. These results, described here for the first time in spermatozoa, are consistent with those observed for the ß1 subunit of Na+/K+ ATPase, suggesting that the latter may assemble with the α subunit to produce a functional ATP12A dimer in sperm cells. The above scenario appeared to be nicely supported by 3D comparative modeling and interaction energy calculations. The expression of ATP12A during different stages of bovine sperm maturation progressively increased, moving from epididymis to deferent ducts. Based on overall results, we hypothesize that ATP12A may play a role in acrosome reactions. Further studies will be required in order to address the functional role of this target protein in sperm physiology.

Modeling SARS-CoV-2 spike/ACE2 protein-protein interactions for predicting the binding affinity of new spike variants for ACE2, and novel ACE2 structurally related human protein targets, for COVID-19 handling in the 3PM context.

Aims: The rapid spread of new SARS-CoV-2 variants has highlighted the crucial role played in the infection by mutations occurring at the SARS-CoV-2 spike receptor binding domain (RBD) in the interactions with the human ACE2 receptor. In this context, it urgently needs to develop new rapid tools for quickly predicting the affinity of ACE2 for the SARS-CoV-2 spike RBD protein variants to be used with the ongoing SARS-CoV-2 genomic sequencing activities in the clinics, aiming to gain clues about the transmissibility and virulence of new variants, to prevent new outbreaks and to quickly estimate the severity of the disease in the context of the 3PM. Methods: In our study, we used a computational pipeline for calculating the interaction energies at the SARS-CoV-2 spike RBD/ACE2 protein-protein interface for a selected group of characterized infectious variants of concern/interest (VoC/VoI). By using our pipeline, we built 3D comparative models of the SARS-CoV-2 spike RBD/ACE2 protein complexes for the VoC B.1.1.7-United Kingdom (carrying the mutations of concern/interest N501Y, S494P, E484K at the RBD), P.1-Japan/Brazil (RBD mutations: K417T, E484K, N501Y), B.1.351-South Africa (RBD mutations: K417N, E484K, N501Y), B.1.427/B.1.429-California (RBD mutations: L452R), the B.1.141 (RBD mutations: N439K), and the recent B.1.617.1-India (RBD mutations: L452R; E484Q) and the B.1.620 (RBD mutations: S477N; E484K). Then, we used the obtained 3D comparative models of the SARS-CoV-2 spike RBD/ACE2 protein complexes for predicting the interaction energies at the protein-protein interface. Results: Along SARS-CoV-2 mutation database screening and mutation localization analysis, it was ascertained that the most dangerous mutations at VoC/VoI spike proteins are located mainly at three regions of the SARS-CoV-2 spike "boat-shaped" receptor binding motif, on the RBD domain. Notably, the P.1 Japan/Brazil variant present three mutations, K417T, E484K, N501Y, located along the entire receptor binding motif, which apparently determines the highest interaction energy at the SARS-CoV-2 spike RBD/ACE2 protein-protein interface, among those calculated. Conversely, it was also observed that the replacement of a single acidic/hydrophilic residue with a basic residue (E484K or N439K) at the "stern" or "bow" regions, of the boat-shaped receptor binding motif on the RBD, appears to determine an interaction energy with ACE2 receptor higher than that observed with single mutations occurring at the "hull" region or with other multiple mutants. In addition, our pipeline allowed searching for ACE2 structurally related proteins, i.e., THOP1 and NLN, which deserve to be investigated for their possible involvement in interactions with the SARS-CoV-2 spike protein, in those tissues showing a low expression of ACE2, or as a novel receptor for future spike variants. A freely available web-tool for the in silico calculation of the interaction energy at the SARS-CoV-2 spike RBD/ACE2 protein-protein interface, starting from the sequences of the investigated spike and/or ACE2 variants, was made available for the scientific community at: https://www.mitoairm.it/covid19affinities. Conclusion: In the context of the PPPM/3PM, the employment of the described pipeline through the provided webservice, together with the ongoing SARS-CoV-2 genomic sequencing, would help to predict the transmissibility of new variants sequenced from future patients, depending on SARS-CoV-2 genomic sequencing activities and on the specific amino acid replacement and/or on its location on the SARS-CoV-2 spike RBD, to put in play all the possible counteractions for preventing the most deleterious scenarios of new outbreaks, taking into consideration that a greater transmissibility has not to be necessarily related to a more severe manifestation of the disease. Supplementary Information: The online version contains supplementary material available at 10.1007/s13167-021-00267-w.

Zoledronic Acid as a Novel Dual Blocker of KIR6.1/2-SUR2 Subunits of ATP-Sensitive K+ Channels: Role in the Adverse Drug Reactions.

Zoledronic acid (ZOL) is used as a bone-specific antiresorptive drug with antimyeloma effects. Adverse drug reactions (A.D.R.) are associated with ZOL-therapy, whose mechanics are unknown. ZOL is a nitrogen-containing molecule whose structure shows similarities with nucleotides, ligands of ATP-sensitive K+ (KATP) channels. We investigated the action of ZOL by performing in vitro patch-clamp experiments on native KATP channels in murine skeletal muscle fibers, bone cells, and recombinant subunits in cell lines, and by in silico docking the nucleotide site on KIR and SUR, as well as the glibenclamide site. ZOL fully inhibited the KATP currents recorded in excised macro-patches from Extensor digitorum longus (EDL) and Soleus (SOL) muscle fibers with an IC50 of 1.2 ± 1.4 × 10-6 and 2.1 ± 3.7 × 10-10 M, respectively, and the KATP currents recorded in cell-attached patches from primary long bone cells with an IC50 of 1.6 ± 2.8 × 10-10 M. ZOL fully inhibited a whole-cell KATP channel current of recombinant KIR6.1-SUR2B and KIR6.2-SUR2A subunits expressed in HEK293 cells with an IC50 of 3.9 ± 2.7 × 10-10 M and 7.1 ± 3.1 × 10-6 M, respectively. The rank order of potency in inhibiting the KATP currents was: KIR6.1-SUR2B/SOL-KATP/osteoblast-KATP > KIR6.2-SUR2A/EDL-KATP >>> KIR6.2-SUR1 and KIR6.1-SUR1. Docking investigation revealed that the drug binds to the ADP/ATP sites on KIR6.1/2 and SUR2A/B and on the sulfonylureas site showing low binding energy <6 Kcal/mol for the KIR6.1/2-SUR2 subunits vs. the <4 Kcal/mol for the KIR6.2-SUR1. The IC50 of ZOL to inhibit the KIR6.1/2-SUR2A/B channels were correlated with its musculoskeletal and cardiovascular risks. We first showed that ZOL blocks at subnanomolar concentration musculoskeletal KATP channels and cardiac and vascular KIR6.2/1-SUR2 channels.

The Interaction of Hemin, a Porphyrin Derivative, with the Purified Rat Brain 2-Oxoglutarate Carrier.

The mitochondrial 2-oxoglutarate carrier (OGC), isolated and purified from rat brain mitochondria, was reconstituted into proteoliposomes to study the interaction with hemin, a porphyrin derivative, which may result from the breakdown of heme-containing proteins and plays a key role in several metabolic pathways. By kinetic approaches, on the basis of the single binding centre gated pore mechanism, we analyzed the effect of hemin on the transport rate of OGC in uptake and efflux experiments in proteoliposomes reconstituted in the presence of the substrate 2-oxoglutarate. Overall, our experimental data fit the hypothesis that hemin operates a competitive inhibition in the 0.5-10 µM concentration range. As a consequence of the OGC inhibition, the malate/aspartate shuttle might be impaired, causing an alteration of mitochondrial function. Hence, considering that the metabolism of porphyrins implies both cytoplasmic and mitochondrial processes, OGC may participate in the regulation of porphyrin derivatives availability and the related metabolic pathways that depend on them (such as oxidative phosphorylation and apoptosis). For the sake of clarity, a simplified model based on induced-fit molecular docking supported the in vitro transport assays findings that hemin was as good as 2-oxoglutarate to bind the carrier by engaging specific ionic hydrogen bond interactions with a number of key residues known for participating in the similarly located mitochondrial carrier substrate binding site.

The T Cell Receptor (TRB) Locus in Tursiops truncatus: From Sequence to Structure of the Alpha/Beta Heterodimer in the Human/Dolphin Comparison.

The bottlenose dolphin (Tursiops truncatus) belongs to the Cetartiodactyla and, similarly to other cetaceans, represents the most successful mammalian colonization of the aquatic environment. Here we report a genomic, evolutionary, and expression study of T. truncatus T cell receptor beta (TRB) genes. Although the organization of the dolphin TRB locus is similar to that of the other artiodactyl species, with three in tandem D-J-C clusters located at its 3' end, its uniqueness is given by the reduction of the total length due essentially to the absence of duplications and to the deletions that have drastically reduced the number of the germline TRBV genes. We have analyzed the relevant mature transcripts from two subjects. The simultaneous availability of rearranged T cell receptor α (TRA) and TRB cDNA from the peripheral blood of one of the two specimens, and the human/dolphin amino acids multi-sequence alignments, allowed us to calculate the most likely interactions at the protein interface between the alpha/beta heterodimer in complex with major histocompatibility class I (MH1) protein. Interacting amino acids located in the complementarity-determining region according to IMGT numbering (CDR-IMGT) of the dolphin variable V-alpha and beta domains were identified. According to comparative modelization, the atom pair contact sites analysis between the human MH1 grove (G) domains and the T cell receptor (TR) V domains confirms conservation of the structure of the dolphin TR/pMH.

Inverse Virtual Screening for the rapid re-evaluation of the presumed biological safe profile of natural products. The case of steviol from Stevia rebaudiana glycosides on farnesoid X receptor (FXR).

Nonnutritive sweeteners (NNSs) are widely employed as dietary substitutes for classical sugars thanks to their safety profile and low toxicity. In this study, a re-evaluation of the biological effects of steviol (1), the main metabolite from Stevia rebaudiana glycosides, was performed using the Inverse Virtual Screening (IVS) target fishing computational approach. Starting from well-known pharmacological properties of Stevia rebaudiana glycosides, this computational tool was employed for predicting the putative interacting targets of 1 and, afterwards, of its five synthetic ester derivatives 2-6, accounting a large panel of proteins involved in cancer and inflammation events. Applying this methodology, the farnesoid X receptor (FXR) was identified as the putative target partner of 1-6. The predicted ligand-protein interactions were corroborated by transactivation assays, specifically disclosing the agonistic activity of 1 and the antagonistic activities of 2-6 on FXR. The reported results highlight the feasibility of IVS as a fast and potent tool for predicting the interacting targets of query compounds, addressing the re-evaluation of their bioactivity. In light of the obtained results, the presumably safe profile of known compounds, such as the case of steviol (1), is critically discussed.

Major antigen and paramyosin proteins as candidate biomarkers for serodiagnosis of canine infection by zoonotic Onchocerca lupi.

Onchocerca lupi (Spirurida: Onchocercidae) is a filarial worm parasitizing domestic carnivores and humans. Adult nematodes usually localize beneath in the sclera or in the ocular retrobulbar of infected animals, whilst microfilariae are found in the skin. Therefore, diagnosis of O. lupi is achieved by microscopic and/or molecular detection of microfilariae from skin biopsy and/or surgical removal of adults from ocular tissues of infected hosts. An urgent non-invasive diagnostic tool for the diagnosis of O. lupi in dog is mandatory. In this study, an immunoproteomic analyses was performed using a combination of immunoblotting and mass spectrometry techniques. Onchocerca lupi major antigen (Ol-MJA) and paramyosin (Ol-PARA) proteins were identified as potential biomarkers for serodiagnosis. Linear epitopes were herein scanned for both proteins using high-density peptide microarray. Sera collected from dog infected with O. lupi and healthy animal controls led to the identification of 11 immunodominant antigenic peptides (n = 7 for Ol-MJA; n = 4 for Ol-PARA). These peptides were validated using sera of dogs uniquely infected with the most important filarioids infesting dogs either zoonotic (Dirofilaria repens, Dirofilaria immitis) or not (Acanthocheilonema reconditum and Cercopithifilaria bainae). Overall, six antigenic peptides, three for Ol-MJA and for Ol-PARA, respectively, were selected as potential antigens for the serological detection of canine O. lupi infection. The molecular and proteomic dataset herein reported should provide a useful resource for studies on O. lupi toward supporting the development of new interventions (drugs, vaccines and diagnostics) against canine onchocercosis.