Drug utilization and medication adherence for the treatment of psoriatic arthritis: an Italian study.

Introduction: This study aims to evaluate the persistence, treatment adherence and drug cost associated with biologic and targeted synthetic disease-modifying antirheumatic drugs (b/tsDMARDs) in the management of psoriatic arthritis (PsA) in Italy, with a focus on biosimilar drugs. Methods: This was a retrospective observational study involving eight hospital pharmacies, between January 2017 and December 2020, on naïve patients with at least one b/tsDMARD dispensation indicated for PsA. Patients were followed up for 12 months and persistence and adherence were evaluated by proportion of days covered (PDC). The originator and biosimilar for adalimumab and etanercept were compared. Furthermore, the real annual cost per patient based on adherence to therapy was calculated. Results: Patients initiating b/tsDMARDs for PsA had a mean persistence of 263 days and 48.6% remained persistent for 1 year. Adherent patients (PDC ≥ 0.8) were 47.6% for the overall population. Similar persistence and adherence were observed between patients treated with the adalimumab originator and its biosimilar, while patients treated with the etanercept originator showed lower persistence and adherence compared to those treated with its biosimilar (mean persistence: 222 vs. 267 days, patient persistent at 1 year: 29.4% vs. 51.5%, mean PDC: 0.53 vs. 0.70, adherent patients: 23.5% vs. 51.5%). The average annual drug cost ranged from €8,724 (etanercept) to €14,783 (ustekinumab), with an annual saving of more than €2,500 by using biosimilars. Conclusion: Poor adherence to medications contributes to suboptimal clinical outcomes. The comparison between biosimilar and originator offers further evidence in support of the biosimilar to optimizing resources in healthcare.

Intramolecular Disulfide Bridges in Voltage-Dependent Anion Channel 2 (VDAC2) Protein from Rattus norvegicus Revealed by High-Resolution Mass Spectrometry.

Voltage-Dependent Anion Channel isoforms (VDAC1, VDAC2, and VDAC3) are relevant components of the outer mitochondrial membrane (OMM) and play a crucial role in regulation of metabolism and in survival pathways. As major players in the regulation of cellular metabolism and apoptosis, VDACs can be considered at the crossroads between two broad families of pathologies, namely, cancer and neurodegeneration, the former being associated with elevated glycolytic rate and suppression of apoptosis in cancer cells, the latter characterized by mitochondrial dysfunction and increased cell death. Recently, we reported the characterization of the oxidation pattern of methionine and cysteines in rat and human VDACs showing that each cysteine in these proteins is present with a preferred oxidation state, ranging from the reduced to the trioxidized form, and such an oxidation state is remarkably conserved between rat and human VDACs. However, the presence and localization of disulfide bonds in VDACs, a key point for their structural characterization, have so far remained undetermined. Herein we have investigated by nanoUHPLC/High-Resolution nanoESI-MS/MS the position of intramolecular disulfide bonds in rat VDAC2 (rVDAC2), a protein that contains 11 cysteines. To this purpose, extraction, purification, and enzymatic digestions were carried out at slightly acidic or neutral pH in order to minimize disulfide bond interchange. The presence of six disulfide bridges was unequivocally determined, including a disulfide bridge linking the two adjacent cysteines 4 and 5, a disulfide bridge linking cysteines 9 and 14, and the alternative disulfide bridges between cysteines 48, 77, and 104. A disulfide bond, which is very resistant to reduction, between cysteines 134 and 139 was also detected. In addition to the previous findings, these results significantly extend the characterization of the oxidation state of cysteines in rVDAC2 and show that it is highly complex and presents unusual features. Data are available via ProteomeXchange with the identifier PXD044041.

Human VDAC pseudogenes: an emerging role for VDAC1P8 pseudogene in acute myeloid leukemia.

BACKGROUND: Voltage-dependent anion selective channels (VDACs) are the most abundant mitochondrial outer membrane proteins, encoded in mammals by three genes, VDAC1, 2 and 3, mostly ubiquitously expressed. As 'mitochondrial gatekeepers', VDACs control organelle and cell metabolism and are involved in many diseases. Despite the presence of numerous VDAC pseudogenes in the human genome, their significance and possible role in VDAC protein expression has not yet been considered. RESULTS: We investigated the relevance of processed pseudogenes of human VDAC genes, both in physiological and in pathological contexts. Using high-throughput tools and querying many genomic and transcriptomic databases, we show that some VDAC pseudogenes are transcribed in specific tissues and pathological contexts. The obtained experimental data confirm an association of the VDAC1P8 pseudogene with acute myeloid leukemia (AML). CONCLUSIONS: Our in-silico comparative analysis between the VDAC1 gene and its VDAC1P8 pseudogene, together with experimental data produced in AML cellular models, indicate a specific over-expression of the VDAC1P8 pseudogene in AML, correlated with a downregulation of the parental VDAC1 gene.

Is the secret of VDAC Isoforms in their gene regulation? Characterization of human VDAC genes expression profile, promoter activity, and transcriptional regulators.

VDACs (voltage-dependent anion-selective channels) are pore-forming proteins of the outer mitochondrial membrane, whose permeability is primarily due to VDACs' presence. In higher eukaryotes, three isoforms are raised during the evolution: they have the same exon-intron organization, and the proteins show the same channel-forming activity. We provide a comprehensive analysis of the three human VDAC genes (VDAC1-3), their expression profiles, promoter activity, and potential transcriptional regulators. VDAC isoforms are broadly but also specifically expressed in various human tissues at different levels, with a predominance of VDAC1 and VDAC2 over VDAC3. However, an RNA-seq cap analysis gene expression (CAGE) approach revealed a higher level of transcription activation of VDAC3 gene. We experimentally confirmed this information by reporter assay of VDACs promoter activity. Transcription factor binding sites (TFBSs) distribution in the promoters were investigated. The main regulators common to the three VDAC genes were identified as E2F-myc activator/cell cycle (E2FF), Nuclear respiratory factor 1 (NRF1), Krueppel-like transcription factors (KLFS), E-box binding factors (EBOX) transcription factor family members. All of them are involved in cell cycle and growth, proliferation, differentiation, apoptosis, and metabolism. More transcription factors specific for each VDAC gene isoform were identified, supporting the results in the literature, indicating a general role of VDAC1, as an actor of apoptosis for VDAC2, and the involvement in sex determination and development of VDAC3. For the first time, we propose a comparative analysis of human VDAC promoters to investigate their specific biological functions. Bioinformatics and experimental results confirm the essential role of the VDAC protein family in mitochondrial functionality. Moreover, insights about a specialized function and different regulation mechanisms arise for the three isoform gene.

NRF-1 and HIF-1α contribute to modulation of human VDAC1 gene promoter during starvation and hypoxia in HeLa cells.

VDAC (Voltage Dependent Anion Channel) is a family of pore forming protein located in the outer mitochondrial membrane. Its channel property ensures metabolites exchange between mitochondria and the rest of the cell resulting in metabolism and bioenergetics regulation, and in cell death and life switch. VDAC1 is the best characterized and most abundant isoform, and is involved in many pathologies, as cancer or neurodegenerative diseases. However, little information is available about its gene expression regulation in normal and/or pathological conditions. In this work, we explored VDAC1 gene expression regulation in normal conditions and in the contest of some metabolic and energetic mitochondrial dysfunction and cell stress as example. The core of the putative promoter region was characterized in terms of transcription factors responsive elements both by bioinformatic studies and promoter activity experiments. In particular, we found an abundant presence of NRF-1 sites, together with other transcription factors binding sites involved in cell growth, proliferation, development, and we studied their prevalence in gene activity. Furthermore, upon depletion of nutrients or controlled hypoxia, as detected in various pathologies, we found that VDAC1 transcripts levels were significantly increased in a time related manner. VDAC1 promoter activity was also validated by gene reporter assays. According to PCR real-time experiments, it was confirmed that VDAC1 promoter activity is further stimulated when cells are exposed to stress. A bioinformatic survey suggested HIF-1α, besides NRF-1, as a most active TFBS. Their validation was obtained by TFBS mutagenesis and TF overexpression experiments. In conclusion, we experimentally demonstrated the involvement of both NRF-1 and HIF-1α in the regulation of VDAC1 promoter activation at basal level and in some peculiar cell stress conditions.

Cysteine Oxidations in Mitochondrial Membrane Proteins: The Case of VDAC Isoforms in Mammals.

Cysteine residues are reactive amino acids that can undergo several modifications driven by redox reagents. Mitochondria are the source of an abundant production of radical species, and it is surprising that such a large availability of highly reactive chemicals is compatible with viable and active organelles, needed for the cell functions. In this work, we review the results highlighting the modifications of cysteines in the most abundant proteins of the outer mitochondrial membrane (OMM), that is, the voltage-dependent anion selective channel (VDAC) isoforms. This interesting protein family carries several cysteines exposed to the oxidative intermembrane space (IMS). Through mass spectrometry (MS) analysis, cysteine posttranslational modifications (PTMs) were precisely determined, and it was discovered that such cysteines can be subject to several oxidization degrees, ranging from the disulfide bridge to the most oxidized, the sulfonic acid, one. The large spectra of VDAC cysteine oxidations, which is unique for OMM proteins, indicate that they have both a regulative function and a buffering capacity able to counteract excess of mitochondrial reactive oxygen species (ROS) load. The consequence of these peculiar cysteine PTMs is discussed.

Comments on Scheffler et al. Cytotoxic Evaluation of E-Liquid Aerosol using Different Lung Derived Cell Models. Int. J. Environ. Res. Public Health, 2015, 12, 12466-12474.

There is merit in considering a simple toxicological screening method that evaluates the total cytotoxic potential of e-liquids or electronic cigarettes (ECs) aerosol emissions in one single testing. However, there is growing confusion, with several researchers endorsing their personal solution to the problem. Here, we discuss as an example the recent paper by Scheffler and colleagues, in which the authors suggest that more relevant and well differentiated cell lines from human airways could be the most suitable candidates for toxicological evaluation of ECs aerosol emissions. We advance recommendations for validated protocols and advocate for an international coordinated effort aimed at establishing consensus on methodology.

VDAC3 as a sensor of oxidative state of the intermembrane space of mitochondria: the putative role of cysteine residue modifications.

Voltage-Dependent Anion selective Channels (VDAC) are pore-forming mitochondrial outer membrane proteins. In mammals VDAC3, the least characterized isoform, presents a set of cysteines predicted to be exposed toward the intermembrane space. We find that cysteines in VDAC3 can stay in different oxidation states. This was preliminary observed when, in our experimental conditions, completely lacking any reducing agent, VDAC3 presented a pattern of slightly different electrophoretic mobilities. This observation holds true both for rat liver mitochondrial VDAC3 and for recombinant and refolded human VDAC3. Mass spectroscopy revealed that cysteines 2 and 8 can form a disulfide bridge in native VDAC3. Single or combined site-directed mutagenesis of cysteines 2, 8 and 122 showed that the protein mobility in SDS-PAGE is influenced by the presence of cysteine and by the redox status. In addition, cysteines 2, 8 and 122 are involved in the stability control of the pore as shown by electrophysiology, complementation assays and chemico-physical characterization. Furthermore, a positive correlation between the pore conductance of the mutants and their ability to complement the growth of porin-less yeast mutant cells was found. Our work provides evidence for a complex oxidation pattern of a mitochondrial protein not directly involved in electron transport. The most likely biological meaning of this behavior is to buffer the ROS load and keep track of the redox level in the inter-membrane space, eventually signaling it through conformational changes.

VDAC3 As a Potential Marker of Mitochondrial Status Is Involved in Cancer and Pathology.

VDAC3 is the least known isoform of the mammalian voltage-dependent anion selective channels of the outer mitochondrial membrane. It has been recently shown that cysteine residues of VDAC3 are found over-oxidized. The VDAC3 cysteine over-oxidation was associated with the oxidizing environment and the abundance of reactive oxygen species (ROS) in the intermembrane space. In this work, we have examined the role of VDAC3 in general pathogenic mechanisms at the basis of mitochondrial dysfunction and involving the mitochondrial quality control. Many of the diseases reported here, including cancer and viral infections, are often associated with significant changes in the intracellular redox state. In this sense, VDAC3 bearing oxidative modifications could become marker of the oxidative load in the mitochondria and part of the ROS signaling pathway.

Oncoprotein E7 from beta human papillomavirus 38 induces formation of an inhibitory complex for a subset of p53-regulated promoters.

Our previous studies on cutaneous beta human papillomavirus 38 (HPV38) E6 and E7 oncoproteins highlighted a novel activity of IκB kinase beta (IKKß) in the nucleus of human keratinocytes, where it phosphorylates and stabilizes ΔNp73α, an antagonist of p53/p73 functions. Here, we further characterize the role of the IKKß nuclear form. We show that IKKß nuclear translocation and ΔNp73α accumulation are mediated mainly by HPV38 E7 oncoprotein. Chromatin immunoprecipitation (ChIP)/Re-ChIP experiments showed that ΔNp73α and IKKß are part, together with two epigenetic enzymes DNA methyltransferase 1 (DNMT1) and the enhancer of zeste homolog 2 (EZH2), of a transcriptional regulatory complex that inhibits the expression of some p53-regulated genes, such as PIG3. Recruitment to the PIG3 promoter of EZH2 and DNMT1 resulted in trimethylation of histone 3 on lysine 27 and in DNA methylation, respectively, both events associated with gene expression silencing. Decreases in the intracellular levels of HPV38 E7 or ΔNp73α strongly affected the recruitment of the inhibitory transcriptional complex to the PIG3 promoter, with consequent restoration of p53-regulated gene expression. Finally, the ΔNp73α/IKKß/DNMT1/EZH2 complex appears to bind a subset of p53-regulated promoters. In fact, the complex is efficiently recruited to several promoters of genes encoding proteins involved in DNA repair and apoptosis, whereas it does not influence the expression of the prosurvival factor Survivin. In summary, our data show that HPV38 via E7 protein promotes the formation of a multiprotein complex that negatively regulates the expression of several p53-regulated genes.

Circulating endothelial-coagulative activation markers after smoking cessation: a 12-month observational study.

BACKGROUND: Cigarette smoking is associated with cardiovascular morbidity and mortality. Exposure to cigarette smoke can cause endothelial dysfunction with impaired endothelium-dependent vasodilation and 'endothelial activation', which predispose to atherothrombosis. The effects of continued smoking and smoking cessation on the level of endothelial, platelet and clotting activation have not been described previously. Here, we prospectively monitored changes in circulating endothelial-coagulative activation markers in smokers undertaking smoking cessation. METHOD: This 12-month prospective study of 174 smokers with no commonly acquired atherothrombotic risk factors underwent an intensive smoking-cessation programme investigating the effect of quitting on circulating levels of von Willebrand's Factor Antigen (vWF:Ag), soluble Thrombomodulin (sTM), d-Dimer (d-D), prothrombin fragment F1+2 (F1+2), platelet factor-4 (PF4) and ß-Thromboglobulin (ß-TG). Blood samples and study measures were collected and compared at baseline and at 2, 6 and 12months after smoking cessation from quitters and relapsers'. RESULTS: No significant differences in demographic or laboratory parameters at baseline were observed between the study groups. Significant changes in von Willebrand's Factor activity were observed at 2months after smoking cessation, with levels decreasing from 141·8% to 113·6%. Substantial modifications in d-Dimer, prothrombin fragment F1 +2, platelet factor-4 and ß-thromboglobulin concentrations were observed only at 6 and 12months after smoking cessation. Positive associations between baseline levels of these biomarkers and number of pack per years have been demonstrated. CONCLUSIONS: Chronic exposure to cigarette smoke sustains the activation of the endothelial-coagulative system and abstinence may result in the improvement of several endothelial-coagulative abnormalities in regular smokers. This may translate into an overall decline in cardiovascular risk.

Cigarette smoke extract immobilizes human spermatozoa and induces sperm apoptosis.

Cigarette smoking by the male partner adversely affects assisted reproductive techniques, suggesting that it may damage sperm chromatin/DNA and consequently embryo development. The effects of graded concentrations of research cigarettes smoke extract (CSE) on motility, mitochondrial membrane potential (MMP), chromatin integrity and apoptosis were evaluated in spermatozoa obtained from 13 healthy, non-smoking men with normal sperm parameters, by flow cytometry. CSE suppressed sperm motility in a concentration- and time-dependent manner and increased the number of spermatozoa with low MMP, the main source of energy for sperm motility. In addition, CSE had a detrimental effect on sperm chromatin condensation and apoptosis. Indeed, it increased the number of spermatozoa with phosphatidylserine externalization, an early apoptotic sign, and fragmented DNA, a late apoptotic sign, in a concentration- and time-dependent manner. These effects of CSE were of similar or even greater magnitude to those obtained following incubation with tumour necrosis factor-alpha, a cytokine known for its negative impact on sperm function, used as positive control. Since transmission of smoking-induced sperm DNA alterations has been found in pre-implantation embryos, and this may predispose offspring to a greater risk of malformations, cancer and genetic diseases, men seeking to father a child are recommended to give up smoking.

Carnosinase levels in aging brain: redox state induction and cellular stress response.

Carnosinase is a dipeptidase found almost exclusively in brain and serum. Its natural substrate carnosine, present at high concentration in the brain, has been proposed as an antioxidant in vivo. We investigated the role of carnosinase in brain aging to establish a possible correlation with age-related changes in cellular stress response and redox status. In addition, a stable HeLa cell line expressing recombinant human serum carnosinase CN1 was established. The enzyme was purified from transfected cells, and specific antibodies were produced against it. Brain expression of CN1, Hsp72, heme oxygenase-1, and thioredoxin reductase increased with age, with a maximal induction in hippocampus and substantia nigra, followed by cerebellum, cortex, septum, and striatum. Hsps induction was associated with significant changes in total SH groups, GSH redox state, carbonyls, and HNE levels. A positive correlation between decrease in GSH and increase in Hsp72 expression was observed in all brain regions examined during aging. Increased carnosinase activity in the brain can lead to decreased carnosine levels and GSH/GSSG ratio. These results, consistent with the current notion that oxidative stress and cellular damage are characteristic hallmarks of the aging process, sustain the critical role of cellular stress-response mechanisms as possible targets for novel antiaging strategies.

Structure of the voltage dependent anion channel: state of the art.

The eukaryotic porin or Voltage Dependent Anion-selective Channels (VDAC) is the protein forming the aqueous pore channel in the mitochondrial outer membrane. It can modulate the energy-dependent metabolism of the cell forming a diffusion barrier to ions, adenine-nucleotides and other metabolites and it is probably involved in the regulation of apoptotic-relevant events. For these reasons, VDAC co-responsibility in unphysiological events leading to important pathologies such as onset or sustainment of cancer has been envisaged very early. The knowledge of the VDAC atomic structure is thus a relevant step in the design of modern drugs acting upon the mitochondrial function and its related apoptotic balance. This goal, despite many efforts, has not been gained until now. Several predictive or descriptive techniques have been employed to obtain models or representations of the pore-structure. The results obtained are reported in this review. The emerging picture arising from these many results is coherent and sufficiently informative. From these efforts it appears that VDAC is functionally monomeric but can cluster in tight but regular groups; it is asymmetric with larger exposed domains on the cytosolic side of the outer mitochondrial membrane; the diameter of the pore is between 2.5-3.0 nm and it is apparently free from obstructions (in the open state); the channel wall is mainly formed by typical amphipathic beta-strands; mobile components (the N-terminal ?) can have functional relevance to the pore regulation.

Relevance of endothelial-haemostatic dysfunction in cigarette smoking.

Cigarette smoking plays a major role in the development of atherosclerosis and is associated with increased morbidity and mortality for coronary heart disease, stroke and peripheral vascular disease. In spite of the abundance of epidemiological evidence that links cigarette smoking to vascular disease, the pathologic mechanisms for such interaction are not clear. The endothelium is a major target organ that undergoes activation when exposed to common vascular triggers, including hypertension, hypercholesterolemia, hyperglycaemia and smoking. Changes in endothelial function may lead to a dysfunctional vascular phenotype characterized by anomalous responses of the vascular tone, abnormal endothelial proliferation and prothrombotic activation. Several studies have demonstrated that smoking may alter endothelial function by a direct toxic effect and consequently trigger haemostatic activation and thrombosis. In this article we will review the evidence that loss of normal endothelial function may result in a loss of the balance of the haemostatic system and in changes of the platelet physiology that may be relevant for the pathogenic effect of smoking on the development of atherothrombosis.