Mitochondrial DNA Together with miR-142-3p in Plasma Can Predict Unfavorable Outcomes in Patients after Acute Myocardial Infarction

Myocardial infarction is one of the leading causes of death worldwide, despite numerous efforts to find efficient prognostic biomarkers and treatment targets. In the present study, we aimed to assess the potential of six microRNAs known to be involved in cardiovascular diseases, cell-free DNA (cfDNA), and mitochondrial DNA (mtDNA) circulating in plasma to be used as prognostic tools for the occurrence of unfavorable outcomes such as major adverse cardiovascular events (MACE) after acute ST-segment elevation myocardial infarction (STEMI). Fifty STEMI patients were enrolled and monitored for 6 months for the occurrence of MACE. Plasma was collected at three time points: upon admission to hospital (T₀), at discharge from hospital (T₁), and 6 months post-STEMI (T₆). Plasma levels of miR-223-3p, miR-142-3p, miR-155-5p, miR-486-5p, miR-125a-5p, and miR-146a-5p, as well as of cfDNA and mtDNA, were measured by RT-qPCR. Results showed that the levels of all measured miRNAs, as well as of cfDNA and mtDNA, were the most increased at T₁, compared to the other two time points. In the plasma of STEMI patients with MACE compared to those without MACE, we determined increased levels of miRNAs, cfDNA, and mtDNA at T_1. Hence, we used the levels of all measured parameters at T₁ for further statistical analysis. Statistical analysis demonstrated that all six miRNAs and cfDNA plus mtDNA levels, respectively, were associated with MACE. The minimal statistical model that could predict MACE in STEMI patients was the combination of mtDNA and miR-142-3p levels, as evidenced by ROC analysis (AUC = 0.97, p < 0.001). In conclusion, the increased plasma levels of mtDNA, along with miR-142-3p, could be used to predict unfavorable outcomes in STEMI patients.     

Implication of Irisin in Different Types of Cancer: A Systematic Review and Meta-Analysis

Cancer is a set of diseases characterized by several hallmark properties, such as increased angiogenesis, proliferation, invasion, and metastasis. The increased angiogenic activity constantly supplies the tumors with nutrients and a plethora of cytokines to ensure cell survival. Along these cytokines is a newly discovered protein, called irisin, which is released into the circulation after physical exercise. Irisin is the product of fibronectin type III domain-containing protein 5 (FNDC5) proteolytic cleavage. Recently it has been the topic of investigation in several types of cancer. In this study, we conducted a systematic review and meta-analysis to investigate its implication in different types of cancer. Our results suggest that irisin expression is decreased in cancer patients, thus it can be used as a valid biomarker for the diagnosis of several types of cancer. In addition, our results indicate that irisin may have an important role in tumor progression and metastasis since it is involved in multiple signaling pathways that promote cell proliferation and migration.     

Identification of Potential New Aedes aegypti Juvenile Hormone Inhibitors from N-Acyl Piperidine Derivatives: A Bioinformatics Approach

Aedes aegypti mosquitoes transmit several human pathogens that cause millions of deaths worldwide, mainly in Latin America. The indiscriminate use of insecticides has resulted in the development of species resistance to some such compounds. Piperidine, a natural alkaloid isolated from Piper nigrum, has been used as a hit compound due to its larvicidal activity against Aedes aegypti. In the present study, piperidine derivatives were studied through in silico methods: pharmacophoric evaluation (PharmaGist), pharmacophoric virtual screening (Pharmit), ADME/Tox prediction (Preadmet/Derek 10.0^®), docking calculations (AutoDock 4.2) and molecular dynamics (MD) simulation on GROMACS-5.1.4. MP-416 and MP-073 molecules exhibiting ΔG binding (MMPBSA −265.95 ± 1.32 kJ/mol and −124.412 ± 1.08 kJ/mol, respectively) and comparable to holo (ΔG binding = −216.21 ± 0.97) and pyriproxyfen (a well-known larvicidal, ΔG binding= −435.95 ± 2.06 kJ/mol). Considering future in vivo assays, we elaborated the theoretical synthetic route and made predictions of the synthetic accessibility (SA) (SwissADME), lipophilicity and water solubility (SwissADME) of the promising compounds identified in the present study. Our in silico results show that MP-416 and MP-073 molecules could be potent insecticides against the Aedes aegypti mosquitoes.     

Overexpression of Brain- and Glial Cell Line-Derived Neurotrophic Factors Is Neuroprotective in an Animal Model of Acute Hypobaric Hypoxia

Currently, the role of the neurotrophic factors BDNF and GDNF in maintaining the brain’s resistance to the damaging effects of hypoxia and functional recovery of neural networks after exposure to damaging factors are actively studied. The assessment of the effect of an increase in the level of these neurotrophic factors in brain tissues using genetic engineering methods on the resistance of laboratory animals to hypoxia may pave the way for the future clinical use of neurotrophic factors BDNF and GDNF in the treatment of hypoxic damage. This study aimed to evaluate the antihypoxic and neuroprotective properties of BDNF and GDNF expression level increase using adeno-associated viral vectors in modeling hypoxia in vivo. To achieve overexpression of neurotrophic factors in the central nervous system’s cells, viral constructs were injected into the brain ventricles of newborn male C57Bl6 (P0) mice. Acute hypobaric hypoxia was modeled on the 30th day after the injection of viral vectors. Survival, cognitive, and mnestic functions in the late post-hypoxic period were tested. Evaluation of growth and weight characteristics and the neurological status of animals showed that the overexpression of neurotrophic factors does not affect the development of mice. It was found that the use of adeno-associated viral vectors increased the survival rate of male mice under hypoxic conditions. The present study indicates that the neurotrophic factors’ overexpression, induced by the specially developed viral constructs carrying the BDNF and GDNF genes, is a prospective neuroprotection method, increasing the survival rate of animals after hypoxic injury.     

Development of the Method for Nusinersen and Its Metabolites Identification in the Serum Samples of Children Treated with Spinraza for Spinal Muscular Atrophy

The application of oligonucleotides as drugs for different genetic diseases is increasing rapidly. Since 2016 they are used during spinal muscular atrophy treatment with the use of nusinersen oligonucleotide. The purpose of this study was to improve methods for the analysis of serum samples of patients treated with nusinersen. The results showed that liquid-liquid extraction (with phenol/chloroform) is insufficient and an additional purification step using solid-phase extraction is necessary. The best results were obtained for microextraction by packed sorbents. Important parameters in the optimization of the method were mainly the type of amine in the mobile phase and the stationary phase. Both influenced the selectivity of metabolite separation and thus their correct identification; while amine type impacted also the intensity of signals. Finally, the highest resolution of separation and the highest peak areas were obtained for N,N-dimethylbutylamine or N,N-diisopropylthylamine with an octadecyl column with a terminal aryl group. Over a dozen of metabolites were successfully identified with the use of methods developed during the study. The 3′ exonucleases and 5′ exonucleases were mainly responsible for nusinersen metabolism, consequently, 3′end shortmers, and 5′end shortmers were observed, as well as metabolites with simultaneous loss of bases at both ends of the sequence. However, some depurination and depyrimidination products were also identified. To the best of our knowledge, this is the first report on nusinersen and its metabolite identification in serum samples by liquid chromatography and mass spectrometry.     

Quantitative Plasma Proteomics to Identify Candidate Biomarkers of Relapse in Pediatric/Adolescent Hodgkin Lymphoma

Classical pediatric Hodgkin Lymphoma (HL) is a rare malignancy. Therapeutic regimens for its management may be optimized by establishing treatment response early on. The aim of this study was to identify plasma protein biomarkers enabling the prediction of relapse in pediatric/adolescent HL patients treated under the pediatric EuroNet-PHL-C2 trial. We used untargeted liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based proteomics at the time of diagnosis—before any therapy—as semiquantitative method to profile plasma proteins specifically associated with relapse in 42 children with nodular sclerosing HL. In both the exploratory and the validation cohorts, six proteins (apolipoprotein E, C4b-binding protein α chain, clusterin, fibrinogen γ chain, prothrombin, and vitronectin) were more abundant in the plasma of patients whose HL relapsed (|fold change| ≥ 1.2, p < 0.05, Student’s t-test). Predicting protein function with the Gene Ontology classification model, the proteins were included in four biological processes (p < 0.01). Using immunoblotting and Luminex assays, we validated two of these candidate biomarkers—C4b-binding protein α chain and clusterin—linked to innate immune response function (GO:0045087). This study identified C4b-binding protein α chain and clusterin as candidate early plasma biomarkers of HL relapse, and important for the purpose of shedding light on the molecular scenario associated with immune response in patients treated under the EuroNet-PHL-C2 trial.     

11C-Methionine PET/CT in Assessment of Multiple Myeloma Patients: Comparison to 18F-FDG PET/CT and Prognostic Value

Multiple myeloma (MM) is the second most common haematological malignancy and remains incurable despite therapeutic advances. ¹⁸F-FDG (FDG) PET/CT is a relevant tool MM for staging and it is the reference imaging technique for treatment evaluation. However, it has limitations, and investigation of other PET tracers is required. Preliminary results with L-methyl-[¹¹C]- methionine (MET), suggest higher sensitivity than ¹⁸F-FDG. This study aimed to compare the diagnostic accuracy and prognostic value of ¹FDG and MET in MM patients. We prospectively compared FDG and MET PET/CT for assessment of bone disease and extramedullary disease (EMD) in a series of 52 consecutive patients (8 smoldering MM, 18 newly diagnosed MM and 26 relapsed MM patients). Bone marrow (BM) uptake patterns and the detection of focal lesions (FLs) and EMD were compared. Furthermore, FDG PET parameters with known MM prognostic value were explored for both tracers, as well as total lesion MET uptake (TLMU). Median patient age was 61 years (range, 37–83 years), 54% were male, 13% of them were in stage ISS (International Staging System) III, and 31% had high-risk cytogenetics. FDG PET/CT did not detect active disease in 6 patients, while they were shown to be positive by MET PET/CT. Additionally, MET PET/CT identified a higher number of FLs than FDG in more than half of the patients (63%). For prognostication we focussed on the relapsed cohort, due to the low number of progressions in the two other cohorts. Upon using FDG PET/CT in relapsed patients, the presence of more than 3 FLs (HR 4.61, p = 0.056), more than 10 FLs (HR 5.65, p = 0.013), total metabolic tumor volume (TMTV) p50 (HR 4.91, p = 0.049) or TMTV p75 (HR 5.32, p = 0.016) were associated with adverse prognosis. In MET PET/CT analysis, TMTV p50 (HR 4.71, p = 0.056), TMTV p75 (HR 6.27, p = 0.007), TLMU p50 (HR 8.8, p = 0.04) and TLMU p75 (HR 6.3, p = 0.007) adversely affected PFS. This study confirmed the diagnostic and prognostic value of FDG in MM. In addition, it highlights that MET has higher sensitivity than FDG PET/CT for detection of myeloma lesions, including FLs. Moreover, we show, for the first time, the prognostic value of TMTV and TLMU MET PET/CT in the imaging evaluation of MM patients.     

Open Questions in Cold Atmospheric Plasma Treatment in Head and Neck Cancer: A Systematic Review

Over the past decade, we witnessed a promising application of cold atmospheric plasma (CAP) in cancer therapy. The aim of this systematic review was to provide an exhaustive state of the art of CAP employed for the treatment of head and neck cancer (HNC), a tumor whose late diagnosis, local recurrence, distant metastases, and treatment failure are the main causes of patients’ death. Specifically, the characteristics and settings of the CAP devices and the in vitro and in vivo treatment protocols were summarized to meet the urgent need for standardization. Its molecular mechanisms of action, as well as the successes and pitfalls of current CAP applications in HNC, were discussed. Finally, the interesting emerging preclinical hypotheses that warrant further clinical investigation have risen. A total of 24 studies were included. Most studies used a plasma jet device (54.2%). Argon resulted as the mostly employed working gas (33.32%). Direct and indirect plasma application was reported in 87.5% and 20.8% of studies, respectively. In vitro investigations were 79.17%, most of them concerned with direct treatment (78.94%). Only eight (33.32%) in vivo studies were found; three were conducted in mice, and five on human beings. CAP showed pro-apoptotic effects more efficiently in tumor cells than in normal cells by altering redox balance in a way that oxidative distress leads to cell death. In preclinical studies, it exhibited efficacy and tolerability. Results from this systematic review pointed out the current limitations of translational application of CAP in the urge of standardization of the current protocols while highlighting promising effects as supporting treatment in HNC.     

Whole Body Vibration Improves Brain and Musculoskeletal Health by Modulating the Expression of Tissue-Specific Markers: FNDC5 as a Key Regulator of Vibration Adaptations

Whole body vibration (WBV) is well known to exert beneficial effects on multiple tissues, improving synaptic transmission, muscle mass, bone quality, and reducing anxiety and depressive behavior. However, the underlying molecular mechanisms are not yet fully understood, and organs and tissues may respond differently to the vibratory stimulus depending on multiple factors. Therefore, we investigated the WBV effects on the brain and musculoskeletal tissue of 4-month-old young mice, evaluating synaptic plasticity by electrophysiological recordings and tissue organization by histology and histomorphometric analysis. Specifically, WBV protocols were characterized by the same vibration frequency (45 Hz), but different in vibration exposure time (five series of 3 min for the B protocol and three series of 2 min and 30 s for the C protocol) and recovery time between two vibration sessions (1 min for the B protocol and 2 min and 30 s for the C protocol). In addition, immunohistochemistry was conducted to evaluate the expression of fibronectin type III domain-containing protein 5 (FNDC5), as well as that of tissue-specific markers, such as brain-derived neurotrophic factor (BDNF) in brain, myostatin in muscle and collagen I (COL-1) in bone. Our results suggest that the WBV effects depend closely on the type of protocol used and support the hypothesis that different organs or tissues have different susceptibility to vibration. Further studies will be needed to deepen our knowledge of physiological adaptations to vibration and develop customized WBV protocols to improve and preserve cognitive and motor functions.     

The New Microtubule-Targeting Agent SIX2G Induces Immunogenic Cell Death in Multiple Myeloma

Microtubule-targeting agents (MTAs) are effective drugs for cancer treatment. A novel diaryl [1,2]oxazole class of compounds binding the colchicine site was synthesized as cis-restricted-combretastatin-A-4-analogue and then chemically modified to have improved solubility and a wider therapeutic index as compared to vinca alkaloids and taxanes. On these bases, a new class of tricyclic compounds, containing the [1,2]oxazole ring and an isoindole moiety, has been synthetized, among which SIX2G emerged as improved MTA. Several findings highlighted the ability of some chemotherapeutics to induce immunogenic cell death (ICD), which is defined by the cell surface translocation of Calreticulin (CALR) via dissociation of the PP1/GADD34 complex. In this regard, we computationally predicted the ability of SIX2G to induce CALR exposure by interacting with the PP1 RVxF domain. We then assessed both the potential cytotoxic and immunogenic activity of SIX2G on in vitro models of multiple myeloma (MM), which is an incurable hematological malignancy characterized by an immunosuppressive milieu. We found that the treatment with SIX2G inhibited cell viability by inducing G2/M phase cell cycle arrest and apoptosis. Moreover, we observed the increase of hallmarks of ICD such as CALR exposure, ATP release and phospho-eIF2α protein level. Through co-culture experiments with immune cells, we demonstrated the increase of (i) CD86 maturation marker on dendritic cells, (ii) CD69 activation marker on cytotoxic T cells, and (iii) phagocytosis of tumor cells following treatment with SIX2G, confirming the onset of an immunogenic cascade. In conclusion, our findings provide a framework for further development of SIX2G as a new potential anti-MM agent.