ABSTRACT
Recent efforts in the study of vector-borne parasitic diseases (VBPDs) have emphasized an increased consideration for preventing drug resistance and promoting the environmental safety of drugs, from the beginning of the drug discovery pipeline. The intensive use of the few available antileishmanial drugs has led to the spreading of hyper-resistant Leishmania infantum strains, resulting in a chronic burden of the disease. In the present work, we have investigated the biochemical mechanisms of resistance to antimonials, paromomycin, and miltefosine in three drug-resistant parasitic strains from human clinical isolates, using a whole-cell mass spectrometry proteomics approach. We identified 14 differentially expressed proteins that were validated with their transcripts. Next, we employed functional association networks to identify parasite-specific proteins as potential targets for novel drug discovery studies. We used SeqAPASS analysis to predict susceptibility based on the evolutionary conservation of protein drug targets across species. MATH-domain-containing protein, adenosine triphosphate (ATP)-binding cassette B2, histone H4, calpain-like cysteine peptidase, and trypanothione reductase emerged as top candidates. Overall, this work identifies new biological targets for designing drugs to prevent the development of Leishmania drug resistance, while aligning with One Health principles that emphasize the interconnected health of people, animals, and ecosystems.
ABSTRACT
Charcot-Marie-Tooth (CMT) disease is one of the most common inherited neuropathies and can lead to progressive muscular weakness, pes cavus, loss of deep tendon reflexes, distal sensory loss, and gait impairment. There are still no effective drugs or surgical therapies for CMT, and supportive treatment is limited to rehabilitative therapy and surgical treatment of skeletal deformities. Many rehabilitative therapeutic approaches have been proposed, but timing and cadence of rehabilitative intervention are not clearly defined, and long-term follow-up is lacking in literature. The aim of this real-practice retrospective study was to assess the effectiveness of an intensive neurorehabilitation protocol on muscle strength and functioning in CMT patients. We analyzed data of patients with diagnosis of mild to moderate CMT. The rehabilitation program lasted 2-4 h a day, 5 days a week, for 3 weeks and consisted of manual treatments, strengthening exercises, stretching, core stability, balance and resistance training, aerobic exercises, and tailored self-care training. Data were collected at baseline (T0), after treatment (T1), and at the 12-month mark (T2) in terms of the following outcome measures: muscle strength, pain, fatigue, cramps, balance, walking speed, and ability. We included 37 CMT patients with a median age of 50.72 ± 13.31 years, with different forms: demyelinating (n = 28), axonal (n = 8), and mixed (n = 1). After intensive rehabilitation treatment, all outcomes significantly improved. This improvement was lost at the 1-year mark. Taken together, these findings suggest that an intensive rehabilitation program improves short-term symptoms and functional outcomes in a cohort of inpatients affected by mild to moderate CMT.
Subject(s)
Charcot-Marie-Tooth Disease , Humans , Adult , Middle Aged , Retrospective Studies , Walking/physiology , Exercise Therapy/methods , Physical Therapy ModalitiesABSTRACT
[This corrects the article DOI: 10.1016/j.ijpx.2022.100136.].
ABSTRACT
The lactate dehydrogenase from rabbit skeletal muscle (rbLDH) is a tetrameric enzyme, known to undergo dissociation when exposed to acidic pH conditions. Moreover, it should be mentioned that this dissociation translates into a pronounced loss of enzyme activity. Notably, among the compounds able to stabilize proteins and enzymes, the disaccharide trehalose represents an outperformer. In particular, trehalose was shown to efficiently counteract quite a number of physical and chemical agents inducing protein denaturation. However, no information is available on the effect, if any, exerted by trehalose against the dissociation of protein oligomers. Accordingly, we thought it of interest to investigate whether this disaccharide is competent in preventing the dissociation of rbLDH induced by acidic pH conditions. Further, we compared the action of trehalose with the effects triggered by maltose and cellobiose. Surprisingly, both these disaccharides enhanced the dissociation of rbLDH, with maltose being responsible for a major effect when compared to cellobiose. On the contrary, trehalose was effective in preventing enzyme dissociation, as revealed by activity assays and by Dynamic Light Scattering (DLS) experiments. Moreover, we detected a significant decrease of both K0.5 and Vmax when the rbLDH activity was tested (at pH 7.5 and 6.5) as a function of pyruvate concentration in the presence of trehalose. Further, we found that trehalose induces a remarkable increase of Vmax when the enzyme is exposed to pH 5. Overall, our observations suggest that trehalose triggers conformational rearrangements of tetrameric rbLDH mirrored by resistance to dissociation and peculiar catalytic features.
Subject(s)
Maltose , Trehalose , Animals , Rabbits , Trehalose/chemistry , Maltose/chemistry , Maltose/metabolism , Cellobiose , L-Lactate Dehydrogenase/metabolism , Disaccharides/pharmacology , Disaccharides/metabolism , Hydrogen-Ion ConcentrationABSTRACT
As the world is facing increasing difficulties to treat leishmaniasis with current therapies, deeper investigation into the molecular mechanisms responsible for both drug resistance and treatment failure (TF) is essential in drug discovery and development. So far, few available drugs cause severe side effects and have developed several resistance mechanisms. Drug resistance and TF parasite strains from clinical isolates may have acquired altered expression of proteins that characterize specific mechanisms leading to therapy inefficacy. This work aims to identify the biochemical pathways of THP-1 human monocytes infected by different Leishmania infantum clinical isolates from patients with either resistance or with TF outcome, using whole cell differential Mass Spectrometry proteomics. We have adopted network enrichment analysis to integrate the transcriptomics and the proteomic results of infected cells studies. Transferrin receptor C (TFRC) and nucleoside diphosphate kinase 3 (NDK3) were discovered as overexpressed proteins in THP-1 cells infected with paromomycin, antimony, and miltefosine resistant L. infantum lines. The overall achievements represent founding concepts to confirm new targets involved in the parasitic drug resistance and TF mechanisms, and to consider in perspective the importance of a dual host-guest pharmacological approach to treat the acute stage of the disease.
Subject(s)
Antiprotozoal Agents , Leishmania infantum , Humans , Antiprotozoal Agents/pharmacology , Proteomics/methods , THP-1 Cells , Drug Resistance , Mass SpectrometryABSTRACT
Introduction: Idiopathic pulmonary fibrosis (IPF) severely affects the lung leading to aberrant deposition of extracellular matrix and parenchymal stiffness with progressive functional derangement. The limited availability of fresh tissues represents one of the major limitations to study the molecular profiling of IPF lung tissue. The primary aim of this study was to explore the proteomic profiling yield of archived formalin-fixed paraffin-embedded (FFPE) specimens of IPF lung tissues. Methods: We further determined the protein expression according to respiratory functional decline at the time of biopsy. The total proteins isolated from 11 FFPE samples of IPF patients compared to 3 FFPE samples from a non-fibrotic lung defined as controls, were subjected to label-free quantitative proteomic analysis by liquid chromatography-mass spectrometry (LC-MS/MS) and resulted in the detection of about 400 proteins. Results: After the pairwise comparison between controls and IPF, functional enrichment analysis identified differentially expressed proteins that were involved in extracellular matrix signaling pathways, focal adhesion and transforming growth factor ß (TGF-ß) signaling pathways strongly associated with IPF onset and progression. Five proteins were significantly over- expressed in the lung of IPF patients with either advanced disease stage (Stage II) or impaired pulmonary function (FVC<75, DLCO<55) compared to controls; these were lymphocyte cytosolic protein 1 (LCP1), peroxiredoxin-2 (PRDX2), transgelin 2 (TAGLN2), lumican (LUM) and mimecan (OGN) that might play a key role in the fibrogenic processes. Discussion: Our work showed that the analysis of FFPE samples was able to identify key proteins that might be crucial for the IPF pathogenesis. These proteins are correlated with lung carcinogenesis or involved in the immune landscape of lung cancer, thus making possible common mechanisms between lung carcinogenesis and fibrosis progression, two pathological conditions at risk for each other in the real life.
ABSTRACT
Drugs that target human thymidylate synthase (hTS), a dimeric enzyme, are widely used in anticancer therapy. However, treatment with classical substrate-site-directed TS inhibitors induces over-expression of this protein and development of drug resistance. We thus pursued an alternative strategy that led us to the discovery of TS-dimer destabilizers. These compounds bind at the monomer-monomer interface and shift the dimerization equilibrium of both the recombinant and the intracellular protein toward the inactive monomers. A structural, spectroscopic, and kinetic investigation has provided evidence and quantitative information on the effects of the interaction of these small molecules with hTS. Focusing on the best among them, E7, we have shown that it inhibits hTS in cancer cells and accelerates its proteasomal degradation, thus causing a decrease in the enzyme intracellular level. E7 also showed a superior anticancer profile to fluorouracil in a mouse model of human pancreatic and ovarian cancer. Thus, over sixty years after the discovery of the first TS prodrug inhibitor, fluorouracil, E7 breaks the link between TS inhibition and enhanced expression in response, providing a strategy to fight drug-resistant cancers.
Subject(s)
Ovarian Neoplasms , Thymidylate Synthase , Female , Animals , Mice , Humans , Binding Sites , Thymidylate Synthase/chemistry , Thymidylate Synthase/metabolism , Fluorouracil/pharmacology , Ovarian Neoplasms/drug therapy , Enzyme Inhibitors/pharmacologyABSTRACT
Nanoparticles (NPs) are paving the way for improved treatments for difficult to treat diseases diseases; however, much is unknown about their fate in the body. One important factor is the interaction between NPs and blood proteins leading to the formation known as the "protein corona" (PC). The PC, consisting of the Hard (HC) and Soft Corona (SC), varies greatly based on the NP composition, size, and surface properties. This highlights the need for specific studies to differentiate the PC formation for each individual NP system. This work focused on comparing the HC and SC of three NPs with different matrix compositions: a) polymeric NPs based on poly(lactic-co-glycolic) acid (PLGA), b) hybrid NPs consisting of PLGA and Cholesterol, and c) lipidic NPs made only of Cholesterol. NPs were formulated and characterized for their physico-chemical characteristics and composition, and then were incubated in human plasma. In-depth purification, identification, and statistical analysis were then performed to identify the HC and SC components. Finally, similar investigations demonstrated whether the presence of a targeting ligand on the NP surface would affect the PC makeup. These results highlighted the different PC fingerprints of these NPs, which will be critical to better understand the biological influences of the PC and improve future NP designs.
ABSTRACT
OBJECTIVES: This study aims to explore the impact of Charcot-Marie-Tooth disease type 1A (CMT1A) and its treatment on patients in European (France, Germany, Italy, Spain, and the United Kingdom) and US real-world practice. METHODS: Adults with CMT1A (n = 937) were recruited to an ongoing observational study exploring the impact of CMT. Data were collected via CMT&Me, an app through which participants completed patient-reported outcome measures. RESULTS: Symptoms ranked with highest importance were weakness in the extremities, difficulty in walking, and fatigue. Almost half of participants experienced a worsening of symptom severity since diagnosis. Anxiety and depression were each reported by over one-third of participants. Use of rehabilitative interventions, medications, and orthotics/walking aids was high. CONCLUSIONS: Patient-reported burden of CMT1A is high, influenced by difficulties in using limbs, fatigue, pain, and impaired quality of life. Burden severity appears to differ across the population, possibly driven by differences in rehabilitative and prescription-based interventions, and country-specific health care variability.
Subject(s)
Charcot-Marie-Tooth Disease , Adult , Charcot-Marie-Tooth Disease/diagnosis , Charcot-Marie-Tooth Disease/epidemiology , Fatigue/etiology , Humans , Life Style , Patient Reported Outcome Measures , Quality of LifeABSTRACT
Stability studies performed by the pharmaceutical industry are principally designed to fulfill licensing requirements. Thus, post-dilution or post-reconstitution stability data are frequently limited to 24 h only for bacteriological reasons, regardless of the true physicochemical stability which could, in many cases, be longer. In practice, the pharmacy-based centralized preparation may require preparation in advance for administration, for example, on weekends, holidays, or in general when pharmacies may be closed. We report an innovative strategy for storing resuspended solutions of azacitidine, a well-known chemotherapic agent, for which the manufacturer lists maximum stability of 22 h. By placing the syringe with the azacitidine reconstituted suspension between two refrigerant gel packs and storing it at 4 °C, we found that the concentration of azacitidine remained above 98% of the initial concentration for 48 h, and no change in color nor the physicochemical properties of the suspension were observed throughout the study period. The physicochemical and microbiological properties were evaluated by HPLC-UV and UHPLC-HRMS analysis, FTIR spectroscopy, pH determination, visual and subvisual examination, and sterility assay. The HPLC-UV method used for evaluating the chemical stability of azacitidine was validated according to ICH. Precise control of storage temperature was obtained by a digital data logger. Our study indicates that by changing the storage procedure of azacitidine reconstituted suspension, the usage window of the drug can be significantly extended to a time frame that better copes with its use in the clinical environment.
ABSTRACT
BACKGROUND: The stability of precursors and reagents is of utmost importance for developing a robust radiolabelling method that provides high and constant radiochemical yield and radiochemical purity. While performing the QC of the [68Ga]Ga-PSMA-11 injectable solutions according to Ph. Eur. Monograph that has recently been published, a trend to the instability of the standard PSMA-11, the same used as a precursor for [68Ga]Ga-PSMA-11 radiosynthesis, has been observed. This instability led to the formation of a side product in a time-dependent manner. The formation of this compound, besides making the implementation of the Ph. Eur. analytical method more difficult, negatively influenced the radiochemical yield and the radiochemical purity by increasing gallium-68 in colloidal and ionic forms. RESULTS: The nature of the side product was investigated by adding chelators, such as EDTA, to PSMA-11 solutions and using the combination of UHPLC-HRMS. The results led to the definition of the side product structure, as natFe-PSMA-11, from the combination of the high-affinity chelator HBED-CC, present in the molecule of PSMA-11, and environmental Fe (III). CONCLUSIONS: Strategies to reduce the risk of low radiolabeling yields and to increase the stability of the PSMA-11 in an aqueous solution were also discussed.
ABSTRACT
Charcot-Marie-Tooth disease (CMT) is a rare, chronic, progressive motor and sensory neuropathy affecting the peripheral nervous system. This study will explore the real-world impact of CMT. The trial is a digital study of approximately 2000 people in 6 countries with CMT ≥18 years. Participants will use a smartphone application to check eligibility, provide consent and contribute data. The dataset will include a personal profile, covering demographics, lifestyle, diagnosis and treatment and a selection of validated generic and disease-specific instruments. Participants will provide data for up to 2 years. Data analysis will be conducted upon registration of the 1000th participant and at 12-month intervals from launch. This study is designed to help researchers and clinicians understand the real-world impact of CMT and the unmet needs of patients. ClinicalTrials.gov identifier: NCT03782883.
Subject(s)
Charcot-Marie-Tooth Disease/psychology , Life Style , Patient Reported Outcome Measures , Female , Humans , Male , Prospective StudiesABSTRACT
The Charcot-Marie-Tooth Health Index (CMT-HI) is a disease-specific patient-reported outcome measure measuring overall disease burden in Charcot-Marie-Tooth (CMT) patients, designed for natural history studies and clinical trials in English-speaking affected individuals. We developed and validated its Italian Charcot-Marie-Tooth Health Index (I-CMT-HI) version. The questionnaire was translated and culturally adapted from source into Italian by two neurologists experienced in CMT and neuromuscular disorders (NMDs). The two translations were reviewed by a panel of seven experts in CMT and NMD. The provisional version was back-translated into English by a professional translator. The definitive Italian version was developed during a consensus teleconference by the panel and a patient representative from ACMT-Rete. A series of clinically and genetically characterized CMT patients completed the final questionnaire; 11 participated in a test-retest reliability assessment of the instrument. The I-CMT-HI was administered to 30 CMT patients (13 CMT1A, eight CMTX1, two CMT1B, two CMT1E, two CMT2I, one CMT2A, one CMT2N, one distal Hereditary Motor Neuropathy), with test-rest in 11:14 females and 16 males, aged (mean ± SD) 48.0 ± 16.4 years (range 18-81), with CMT Examination Score (CMTES) = 10.0 ± 4.4 (range 2-18). The I-CMT-HI mean total score was 29.4 ± 21.2 (range 0.1-60.3). The I-CMT-HI showed a high test-retest reliability: intraclass correlation coefficient = 0.95 (95% confidence interval, 0.84-0.99). No patient had difficulty in completing the questionnaire and none reported any problem with the questions' formulation. The total CMT-HI score was positively correlated with age and CMTES, with higher disease burden with increasing age and disease severity according to the CMTES. The I-CMT-HI is now ready for use in clinical studies in the Italian population.
Subject(s)
Charcot-Marie-Tooth Disease/diagnosis , Patient Reported Outcome Measures , Psychometrics/standards , Severity of Illness Index , Adolescent , Adult , Aged , Aged, 80 and over , Female , Humans , Italy , Male , Psychometrics/methods , Reproducibility of Results , Young AdultABSTRACT
Recent work has disclosed the critical role played by enamel peptides in sex classification of old skeletal remains. In particular, protein AMELY (amelogenin isoform Y) is present in the enamel dental tissue of male individuals only, while AMELX (isoform X) can be found in both sexes. AMELY can be easily detected by LC-MS/MS in the ion extracted chromatograms of the SM(ox)IRPPY peptide (monoisotopic [M + 2 H]+2 mass = 440.2233 m/z). In this paper, we exploited the dimorphic features of the amelogenin protein to determine the sex of the so-called 'Lovers of Modena', two Late Antique individuals whose skeletons were intentionally buried hand-in-hand. Upon discovery, mass media had immediately assumed they were a male-female couple, even if bad preservation of the bones did not allow an effective sex classification. We were able to extract proteins from the dental enamel of both individuals (~1600 years old) and to confidently classify them as males. Results were compared to 14 modern and archaeological control samples, confirming the reliability of the ion chromatogram method for sex determination. Although we currently have no information on the actual relationship between the 'Lovers of Modena' (affective? Kin-based?), the discovery of two adult males intentionally buried hand-in-hand may have profound implications for our understanding of funerary practices in Late Antique Italy.
Subject(s)
Amelogenin/genetics , Dental Enamel Proteins/genetics , Dental Enamel/metabolism , Paleontology/methods , Peptide Fragments/metabolism , Sex Determination Analysis/methods , Amelogenin/metabolism , Dental Enamel Proteins/metabolism , Female , Humans , Italy , Male , Peptide Fragments/analysis , Polymerase Chain ReactionABSTRACT
Proteomics and bioinformatics are a useful combined technology for the characterization of protein expression level and modulation associated with the response to a drug and with its mechanism of action. The folate pathway represents an important target in the anticancer drugs therapy. In the present study, a discovery proteomics approach was applied to tissue samples collected from ovarian cancer patients who relapsed after the first-line carboplatin-based chemotherapy and were treated with pemetrexed (PMX), a known folate pathway targeting drug. The aim of the work is to identify the proteomic profile that can be associated to the response to the PMX treatment in pre-treatement tissue. Statistical metrics of the experimental Mass Spectrometry (MS) data were combined with a knowledge-based approach that included bioinformatics and a literature review through ProteinQuest™ tool, to design a protein set of reference (PSR). The PSR provides feedback for the consistency of MS proteomic data because it includes known validated proteins. A panel of 24 proteins with levels that were significantly different in pre-treatment samples of patients who responded to the therapy vs. the non-responder ones, was identified. The differences of the identified proteins were explained for the patients with different outcomes and the known PMX targets were further validated. The protein panel herein identified is ready for further validation in retrospective clinical trials using a targeted proteomic approach. This study may have a general relevant impact on biomarker application for cancer patients therapy selection.
ABSTRACT
The heterotrimeric NF-Y complex is a pioneer factor that binds to CCAAT-genes and regulates their transcription. NF-Y cooperates with multiple transcription factors and co-regulators in order to positively or negatively influence gene transcription. The recruitment of NF-Y to CCAAT box is significantly enriched in cancer-associated gene promoters loci and positively correlates with malignancy. NF-Y subunits, in particular the DNA-binding subunit NF-YA and the histone-fold subunit NF-YC, appear overexpressed in specific types of cancer. Here we demonstrate that NF-Y subunits expression is finely regulated through transcriptional and post-translational mechanisms thus allowing control over basal expression levels. NF-Y negatively regulates the transcription of the genes encoding for its subunits. DNA pull-down/affinity purification assay coupled with Mass Spectrometry identified putative co-regulators, such as Lamin A, involved in NF-YA gene transcription level. We also evidentiate how the stability of the complex is severely affected by the absence of one subunit. Our results identified for the first time one of the mechanisms responsible for NF-Y expression, which may be involved in the aberrant expression and activity observed in tumor cells and other pathological conditions.
Subject(s)
CCAAT-Binding Factor/genetics , Lamin Type A/genetics , Neoplasms/genetics , Transcription, Genetic , Gene Expression Regulation , HCT116 Cells , Humans , Mass Spectrometry , Neoplasms/pathology , Promoter Regions, Genetic , Protein BindingABSTRACT
AIM: The investigation on protein coronas (PCs) adsorbed onto nanoparticle (NP) surface is representing an open issue due to difficulties in detection and clear isolation of the adsorbed proteins. In this study, we investigated protocols able to isolate the compositions of PCs of three polymeric NPs. MATERIALS & METHODS: Unfunctionalized NPs and two functionalized NPs were considered as proof-of-concept for the qualitative and semiquantitative analysis of both the corona levels (stably or weakly adsorbed coronas [SC/WC]) of these different nanocarriers. RESULTS: The protocols applied were able to discriminate between the SC and WC. In particular, experimental results indicated that stably adsorbed coronas are prevalently composed by ApoE, while WC by albumin in all the NPs. Otherwise, some differences in WC could be correlated with surface functionalization. CONCLUSION: This experimental approach allows characterizing the whole PCs, proposing a protocol for isolation of different types of proteins composing PCs.
Subject(s)
Antibodies, Monoclonal/chemistry , Nanoparticles/chemistry , Oligopeptides/chemistry , Protein Corona/analysis , Adsorption , Albumins/chemistry , Apolipoproteins E/chemistry , Particle Size , Polylactic Acid-Polyglycolic Acid Copolymer/chemistry , Proof of Concept Study , Surface PropertiesABSTRACT
CONTEXT: LR-peptide, a novel hydrophilic peptide synthetized and characterized in previous work, is able to reduce the multi-drug resistance response in cisplatin (cDPP) resistant cancer cells by inhibiting human thymidylate synthase (hTS) overexpressed in several tumors, including ovarian and colon-rectal cancers, but it is unable to enter the cells spontaneously. OBJECTIVE: The aim of this work was to design and characterize liposomal vesicles as drug delivery systems for the LR peptide, evaluating the possible benefits of the pH-responsive feature in improving intracellular delivery. MATERIALS AND METHODS: For this purpose, conventional and pH-sensitive liposomes were formulated, compared regarding their physical-chemical properties (size, PDI, morphology, in vitro stability and drug release) and studied for in vitro cytotoxicity against a cDDP-resistant cancer cells. RESULTS AND DISCUSSION: Results indicated that LR peptide was successfully encapsulated in both liposomal formulations but at short incubation time only LR loaded pH-sensitive liposomes showed cell inhibition activity while for long incubation time the two kinds of liposomes demonstrated the same efficacy. CONCLUSIONS: Data provide evidence that acidic pH-triggered liposomal delivery is able to significantly reduce the time required by the systems to deliver the drug to the cells without inducing an enhancement of the efficacy of the drug.