Role of NT-proBNP and lung ultrasound in diagnosing and classifying heart failure in a hospitalized oldest-old population: a cross-sectional study.

AIM: Diagnosing and classifying heart failure (HF) in the oldest-old patients has technical and interpretation issues, especially in the acute setting. We assessed the usefulness of both N-terminal pro-brain natriuretic peptide (NT-proBNP) and lung ultrasound (LUS) for confirming HF diagnosis and predicting, among hospitalized HF patients, those with reduced ejection fraction (HFrEF). METHODS: We performed a cross-sectional study on 148 consecutive patients aged ≥ 80 years admitted to our Internal Medicine and Geriatrics ward with at least one symptom/sign compatible with HF and NT-proBNP ≥ 125 pg/mL. We measured serum NT-proBNP levels and performed LUS and transthoracic echocardiography (TTE) on admission before diuretic therapy. We divided our cohort into three subgroups according to the left ventricular ejection fraction (LVEF): reduced (LVEF ≤ 40%), mildly-reduced (LVEF = 41-49%) and preserved (LVEF ≥ 50%). RESULTS: The mean age was 88±5 years. Male prevalence was 42%. Patients with HFrEF were 19%. Clinical features and laboratory parameters did not differ between the three subgroups, except for higher NT-proBNP in HFrEF patients, which also had a higher number of total B-lines and intercostal spaces of pleural effusion at LUS. Overall, NT-proBNP showed an inverse correlation with LVEF (r = -0.22, p = 0.007) and a direct correlation with age, total pulmonary B-lines, and intercostal spaces of pleural effusion. According to the ROCs, NT-proBNP levels, pulmonary B-lines and pleural effusion extension were poorly predictive for HFrEF. The best-performing cut-offs were 9531 pg/mL for NT-proBNP (SP 0.70, SE 0.50), 13 for total B-lines (SP 0.69, SE 0.85) and one intercostal space for pleural effusion (SP 0.55, SE 0.89). Patients with admission NT-proBNP ≥ 9531 pg/mL had a 2-fold higher risk for HFrEF (OR 2.5, 95% CI 1.3-4.9), while we did not find any association for total B-lines ≥ 13 or pleural effusion ≥ 1 intercostal space with HFrEF. A significant association with HFrEF emerged for the combination of NT-proBNP ≥ 9531 pg/mL, total B-lines ≥ 13 and intercostal spaces of pleural effusion ≥ 1 (adjusted OR 4.3, 95% CI 1.5-12.9). CONCLUSIONS: Although NT-proBNP and LUS help diagnose HF, their accuracy in discriminating HFrEF from non-HFrEF was poor in our real-life clinical study on oldest-old hospitalized patients, making the use of TTE still necessary to distinguish HF phenotypes in this peculiar setting. These data require confirmation in more extensive and longer prospective studies.

Neutrophil-to-lymphocyte ratio (NLR) predicts mortality in hospitalized geriatric patients independent of the admission diagnosis: a multicenter prospective cohort study.

BACKGROUND: The Neutrophil-to-lymphocyte ratio (NLR) is a marker of poor prognosis in hospitalized older patients with different diseases, but there is still no consensus on the optimal cut-off value to identify older patients at high-risk of in-hospital mortality. Therefore, in this study we aimed at both validating NLR as a predictor of death in older hospitalized patients and assess whether the presence of specific acute diseases can modify its predictive value. METHODS: This prospective cohort study included 5034 hospitalizations of older patients admitted to acute care units in the context of the ReportAge study. NLR measured at admission was considered as the exposure variable, while in-hospital mortality was the outcome of the study. ROC curves with Youden's method and restricted cubic splines were used to identify the optimal NLR cut-off of increased risk. Cox proportional hazard models, stratified analyses, and Kaplan-Meier survival curves were used to analyse the association between NLR and in-hospital mortality. RESULTS: Both continuous and categorical NLR value (cut-off ≥ 7.95) predicted mortality in bivariate and multivariate prognostic models with a good predictive accuracy. The magnitude of this association was even higher in patients without sepsis, congestive heart failure, and pneumonia, and those with higher eGFR, albumin, and hemoglobin (p < 0.001). A negative multiplicative interaction was found between NLR and eGFR < 45 (p = 0.001). CONCLUSIONS: NLR at admission is a readily available and cost-effective biomarker that could improve identification of geriatric patients at high risk of death during hospital stay independent of admitting diagnosis, kidney function and hemoglobin levels.

Blood circulating bacterial DNA in hospitalized old COVID-19 patients.

BACKGROUND: Coronavirus disease COVID-19 is a heterogeneous condition caused by SARS-CoV-2 infection. Generally, it is characterized by interstitial pneumonia that can lead to impaired gas-exchange, acute respiratory failure, and death, although a complex disorder of multi-organ dysfunction has also been described. The pathogenesis is complex, and a variable combination of factors has been described in critically ill patients. COVID-19 is a particular risk for older persons, particularly those with frailty and comorbidities. Blood bacterial DNA has been reported in both physiological and pathological conditions and has been associated with some haematological and laboratory parameters but, to date, no study has characterized it in hospitalized old COVID-19 patients The present study aimed to establish an association between blood bacterial DNA (BB-DNA) and clinical severity in old COVID-19 patients. RESULTS: BB-DNA levels were determined, by quantitative real-time PCRs targeting the 16S rRNA gene, in 149 hospitalized older patients (age range 65-99 years) with COVID-19. Clinical data, including symptoms and signs of infection, frailty status, and comorbidities, were assessed. BB-DNA was increased in deceased patients compared to discharged ones, and Cox regression analysis confirmed an association between BB-DNA and in-hospital mortality. Furthermore, BB-DNA was positively associated with the neutrophil count and negatively associated with plasma IFN-alpha. Additionally, BB-DNA was associated with diabetes. CONCLUSIONS: The association of BB-DNA with mortality, immune-inflammatory parameters and diabetes in hospitalized COVID-19 patients suggests its potential role as a biomarker of unfavourable outcomes of the disease, thus it could be proposed as a novel prognostic marker in the assessment of acute COVID-19 disease.

Insights on the UV-Screening Potential of Marine-Inspired Thiol Compounds.

One of the major threats to skin aging and the risk of developing skin cancer is excessive exposure to the sun's ultraviolet radiation (UVR). The use of sunscreens containing different synthetic, organic, and inorganic UVR filters is one of the most widespread defensive measures. However, increasing evidence suggests that some of these compounds are potentially eco-toxic, causing subtle damage to the environment and to marine ecosystems. Resorting to natural products produced in a wide range of marine species to counteract UVR-mediated damage could be an alternative strategy. The present work investigates marine-inspired thiol compounds, derivatives of ovothiol A, isolated from marine invertebrates and known to exhibit unique antioxidant properties. However, their potential use as photoprotective molecules for biocompatible sunscreens and anti-photo aging formulations has not yet been investigated. Here, we report on the UVR absorption properties, photostability, and in vitro UVA shielding activities of two synthetic ovothiol derivatives, 5-thiohistidine and iso-ovothiol A, by spectrophotometric and fluorimetric analysis. We found that the UVA properties of these compounds increase upon exposure to UVA and that their absorption activity is able to screen UVA rays, thus reducing the oxidative damage induced to proteins and lipids. The results of this work demonstrate that these novel marine-inspired compounds could represent an alternative eco-friendly approach for UVR skin protection.

First In Vivo Insights on the Effects of Tempol-Methoxycinnamate, a New UV Filter, as Alternative to Octyl Methoxycinnamate, on Zebrafish Early Development.

The demand for organic UV filters as active components in sunscreen products has rapidly risen over the last century, as people have gradually realized the hazards of overexposure to UV radiation. Their extensive usage has resulted in their ubiquitous presence in different aquatic matrices, representing a potential threat to living organisms. In this context, the need to replace classic UV filters such as octyl methoxycinnamate (OMC), one of the most popular UV filters reported to be a potential pollutant of aquatic ecosystems, with more environmentally friendly ones has emerged. In this study, using zebrafish, the first in vivo results regarding the effect of exposure to tempol-methoxycinnamate (TMC), a derivative of OMC, are reported. A comparative study between TMC and OMC was performed, analyzing embryos exposed to similar TMC and OMC concentrations, focusing on morphological and molecular changes. While both compounds seemed not to affect hatching and embryogenesis, OMC exposure caused an increase in endoplasmic reticulum (ER) stress response genes, according to increased eif2ak3, ddit3, nrf2, and nkap mRNA levels and in oxidative stress genes, as observed from modulation of the sod1, sod2, gpr, and trx mRNA levels. On the contrary, exposure to TMC led to reduced toxicity, probably due to the presence of the nitroxide group in the compound's molecular structure responsible for antioxidant activity. In addition, both UV filters were docked with estrogen and androgen receptors where they acted differently, in agreement with the molecular analysis that showed a hormone-like activity for OMC but not for TMC. Overall, the results indicate the suitability of TMC as an alternative, environmentally safer UV filter.

Structural Basis for Agonistic Activity and Selectivity toward Melatonin Receptors hMT1 and hMT2.

Glaucoma, a major ocular neuropathy originating from a progressive degeneration of retinal ganglion cells, is often associated with increased intraocular pressure (IOP). Daily IOP fluctuations are physiologically influenced by the antioxidant and signaling activities of melatonin. This endogenous modulator has limited employment in treating altered IOP disorders due to its low stability and bioavailability. The search for low-toxic compounds as potential melatonin agonists with higher stability and bioavailability than melatonin itself could start only from knowing the molecular basis of melatonergic activity. Thus, using a computational approach, we studied the melatonin binding toward its natural macromolecular targets, namely melatonin receptors 1 (MT1) and 2 (MT2), both involved in IOP signaling regulation. Besides, agomelatine, a melatonin-derivative agonist and, at the same time, an atypical antidepressant, was also included in the study due to its powerful IOP-lowering effects. For both ligands, we evaluated both stability and ligand positioning inside the orthosteric site of MTs, mapping the main molecular interactions responsible for receptor activation. Affinity values in terms of free binding energy (ΔGbind) were calculated for the selected poses of the chosen compounds after stabilization through a dynamic molecular docking protocol. The results were compared with experimental in vivo effects, showing a higher potency and more durable effect for agomelatine with respect to melatonin, which could be ascribed both to its higher affinity for hMT2 and to its additional activity as an antagonist for the serotonin receptor 5-HT2c, in agreement with the in silico results.

Insights on the Hypoglycemic Potential of Crocus sativus Tepal Polyphenols: An In Vitro and In Silico Study.

Post-prandial hyperglycemia typical of diabetes mellitus could be alleviated using plant-derived compounds such as polyphenols, which could influence the activities of enzymes involved in carbohydrate digestion and of intestinal glucose transporters. Here, we report on the potential anti-hyperglycemic effect of Crocus sativus tepals compared to stigmas, within the framework of valorizing these by-products of the saffron industry, since the anti-diabetic properties of saffron are well-known, but not those of its tepals. In vitro assays showed that tepal extracts (TE) had a greater inhibitory action than stigma extracts (SE) on α-amylase activity (IC50: TE = 0.60 ± 0.09 mg/mL; SE = 1.10 ± 0.08 mg/mL; acarbose = 0.051 ± 0.07) and on glucose absorption in Caco-2 differentiated cells (TE = 1.20 ± 0.02 mg/mL; SE = 2.30 ± 0.02 mg/mL; phlorizin = 0.23 ± 0.01). Virtual screening performed with principal compounds from stigma and tepals of C. sativus and human pancreatic α-amylase, glucose transporter 2 (GLUT2) and sodium glucose co-transporter-1 (SGLT1) were validated via molecular docking, e.g., for human pancreatic α-amylase, epicatechin 3-o-gallate and catechin-3-o-gallate were the best scored ligands from tepals (-9.5 kcal/mol and -9.4 kcal/mol, respectively), while sesamin and episesamin were the best scored ones from stigmas (-10.1 kcal/mol). Overall, the results point to the potential of C. sativus tepal extracts in the prevention/management of diabetes, likely due to the rich pool of phytocompounds characterized using high-resolution mass spectrometry, some of which are capable of binding and interacting with proteins involved in starch digestion and intestinal glucose transport.

Effect of Cytomegalovirus Reactivation on Inflammatory Status and Mortality of Older COVID-19 Patients.

Herpesviridae reactivation such as cytomegalovirus (CMV) has been described in severe COVID-19 (COronaVIrusDisease-2019). This study aimed to understand if CMV reactivation in older COVID-19 patients is associated with increased inflammation and in-hospital mortality. In an observational single-center cohort study, 156 geriatric COVID-19 patients were screened for CMV reactivation by RT-PCR. Participants underwent a comprehensive clinical investigation that included medical history, functional evaluation, laboratory tests and cytokine assays (TNF-α, IFN-α, IL-6, IL-10) at hospital admission. In 19 (12.2%) of 156 COVID-19 patients, CMV reactivation was detected. Multivariate Cox regression models showed that in-hospital mortality significantly increased among CMV positive patients younger than 87 years (HR: 9.94, 95% CI: 1.66-59.50). Other factors associated with in-hospital mortality were C-reactive protein (HR: 1.17, 95% CI: 1.05-1.30), neutrophil count (HR: 1.20, 95% CI: 1.01-1.42) and clinical frailty scale (HR:1.54, 95% CI: 1.04-2.28). In patients older than 87 years, neutrophil count (HR: 1.13, 95% CI: 1.05-1.21) and age (HR: 1.15, 95% CI: 1.01-1.31) were independently associated with in-hospital mortality. CMV reactivation was also correlated with increased IFN-α and TNF-α serum levels, but not with IL-6 and IL-10 serum changes. In conclusion, CMV reactivation was an independent risk factor for in-hospital mortality in COVID-19 patients younger than 87 years old, but not in nonagenarians.

Identification of Flavone Derivative Displaying a 4'-Aminophenoxy Moiety as Potential Selective Anticancer Agent in NSCLC Tumor Cells.

Five heterocyclic derivatives were synthesized by functionalization of a flavone nucleus with an aminophenoxy moiety. Their cytotoxicity was investigated in vitro in two models of human non-small cell lung cancer (NSCLC) cells (A549 and NCI-H1975) by using MTT assay and the results compared to those obtained in healthy fibroblasts as a non-malignant cell model. One of the aminophenoxy flavone derivatives (APF-1) was found to be effective at low micromolar concentrations in both lung cancer cell lines with a higher selective index (SI). Flow cytometric analyses showed that APF-1 induced apoptosis and cell cycle arrest in the G2/M phase through the up-regulation of p21 expression. Therefore, the aminophenoxy flavone-based compounds may be promising cancer-selective agents and could serve as a base for further research into the design of flavone-based anticancer drugs.

Salt effects on mixed composition membranes containing an antioxidant lipophilic edaravone derivative: a computational-experimental study.

The protection of lipid membranes against oxidation avoids diseases associated with oxidative stress. As a strategy to contrast it, functionalized lipids with antioxidant activity are used to become part of membranes thus protecting them. For this purpose, a lipophilic edaravone derivative has been synthesized, adding a C18 saturated chain to the original structure. The antioxidant activity of C18-Edv has been demonstrated in our previous work. In this study, molecular dynamics simulations have been performed to define the effects of NaCl, MgCl2, KCl, and CaCl2 salts on a palmitoyl-oleoyl-sn-glycero-phosphocholine (POPC) lipid bilayer encapsulating C18-Edv. The results showed how different salts influence POPC lateral diffusion, and the movements of C18-Edv heads, which are antioxidant moieties, were correlated to the ability of C18-Edv molecules to protect membranes. MgCl2 showed a negative impact leading to C18-Edv clusterization and membrane stretching, while KCl and NaCl showed a moderate influence on the mixed lipid membrane structure. CaCl2 increased the exposure of the C18-Edv heads to the lipid-water interface, resulting in the salt with a higher propensity to guarantee protection against radicals in the aqueous phase. Finally, C18-Edv-POPC liposomes have been prepared following the simulation conditions, and then an experimental Oxygen Radical Absorbance Capacity (ORAC) assay has been performed to confirm the in silico predicted results.

The Association between Single Nucleotide Polymorphisms, including miR-499a Genetic Variants, and Dyslipidemia in Subjects Treated with Pharmacological or Phytochemical Lipid-Lowering Agents.

Disorders of lipoprotein metabolism are among the major risk factors for cardiovascular disease (CVD) development. Single nucleotide polymorphisms (SNPs) have been associated with the individual variability in blood lipid profile and response to lipid-lowering treatments. Here, we genotyped 34 selected SNPs located in coding genes related to lipid metabolism, inflammation, coagulation, and a polymorphism in the MIR499 gene-a microRNA previously linked to CVD-to evaluate the association with lipid trait in subjects with moderate dyslipidemia not on lipid-lowering treatment (Treatment-naïve (TN) cohort, n = 125) and in patients treated with statins (STAT cohort, n = 302). We also explored the association between SNPs and the effect of a novel phytochemical lipid-lowering treatment in the TN cohort. We found that 6 SNPs (in the MIR499, TNFA, CETP, SOD2, and VEGFA genes) were associated with lipid traits in the TN cohort, while no association was found with the response to twelve-week phytochemical treatment. In the STAT cohort, nine SNPs (in the MIR499, CETP, CYP2C9, IL6, ABCC2, PON1, IL10, and VEGFA genes) were associated with lipid traits, three of which were in common with the TN cohort. Interestingly, in both cohorts, the presence of the rs3746444 MIR499 SNP was associated with a more favorable blood lipid profile. Our findings could add information to better understand the individual genetic variability in maintaining a low atherogenic lipid profile and the response to different lipid-lowering therapies.

The Natural Alkaloid Berberine Can Reduce the Number of Pseudomonas aeruginosa Tolerant Cells.

The eradication of recurrent Pseudomonas aeruginosa (PA) lung infection in cystic fibrosis (CF) patients may be hampered by the development of persistent bacterial forms, which can tolerate antibiotics through efflux pump overexpression. After demonstrating the efflux pump inhibitory effect of the alkaloid berberine on the PA MexXY-OprM efflux pump, in this study, we tested its ability (80/320 µg/mL) to enhance tobramycin (20xMIC/1000xMIC) activity against PA planktonic/biofilm cultures. Preliminary investigations of the involvement of MexY in PA tolerance to tobramycin treatment, performed on the isogenic pair PA K767 (wild type)/K1525 (ΔmexY) growing in planktonic and biofilm cultures, demonstrated that the ΔmexY mutant K1525 produced a lower (100 and 10 000 times, respectively) amount of tolerant cells than that of the wild type. Next, we grew broth cultures of PAO1, PA14, and 20 PA clinical isolates (of which 13 were from CF patients) in the presence of 20xMIC tobramycin with and without berberine 80 µg/mL. Accordingly, most strains showed a greater (from 10- to 1000-fold) tolerance reduction in the presence of berberine. These findings highlight the involvement of the MexXY-OprM system in the tobramycin tolerance of PA and suggest that berberine may be used in new valuable therapeutic combinations to counteract persister survival.

Fractal lacunarity of trabecular bone in vertebral MRI to predict osteoporotic fracture risk in over-fifties women. The LOTO study.

BACKGROUND: Osteoporotic fractures are a major cause of morbidity in the elderly. Menopausal women represent the population with the highest risk of early osteoporosis onset, often accompanied by vertebral fractures (VF). Bone mineral density (BMD) is commonly assessed by dual-energy X-ray absorptiometry (DXA) for osteoporosis diagnosis; however, BMD alone does not represent a significant predictor of fracture risk. Bone microarchitecture, instead, arises as a determinant of bone fragility independent of BMD. High-resolution magnetic resonance imaging (MRI) is an effective noninvasive/nonionizing tool for in vivo characterisation of trabecular bone microarchitecture (TBA). We have previously set up an MRI method able to characterise TBA changes in aging and osteoporosis by one parameter, trabecular bone lacunarity parameter ß (TBLß). Fractal lacunarity was used for TBA texture analysis as it describes discontinuity of bone network and size of bone marrow spaces, changes of which increase the risk of bone fracture. This study aims to assess the potential of TBLß method as a tool for osteoporotic fracture risk. METHODS: An observational, cross-sectional, and prospective study on over-50s women at risk for VF was designed. TBLß, our index of osteoporotic fracture risk, is the main outcome measure. It was calculated on lumbar vertebra axial images, acquired by 1.5 T MRI spin-echo technique, from 279 osteopenic/osteoporotic women with/without prior VF. Diagnostic power of TBLß method, by Receiver Operating Characteristics (ROC) curve and other diagnostic accuracy measurements were compared with lumbar spine DXA-BMD. RESULTS: Baseline results show that TBLß is able to discriminate patients with/without prevalent VF (p = 0.003). AUC (area under the curve from ROC) is 0.63 for TBLß, statistically higher (p = 0.012) than BMD one (0.53). Contribution of TBLß to prevalent VF is statistically higher (p < 0.001) than BMD (sensitivity: 66% vs. 52% respectively; OR: 3.20, p < 0.0001 for TBLß vs. 1.31, p = 0.297 for BMD). Preliminary 1-year prospective results suggest that TBA contribution to incident VF is even higher (sensitivity: 73% for TBLß vs. 55% for BMD; RR: 3.00, p = 0.002 for TBLß vs. 1.31, p = 0.380 for BMD). CONCLUSION: Results from this study further highlight the usefulness of TBLß as a biomarker of TBA degeneration and an index of osteoporotic fracture risk.

Molecular dynamics simulations of quinine encapsulation into biodegradable nanoparticles: A possible new strategy against Sars-CoV-2.

A new coronavirus disease, SARS-CoV-2, has spread into a global pandemic in December 2019. Since no specific therapeutic drugs for treating COVID-19 have been approved by FDA, recent studies suggest that the known antimalarial quinine and its derivatives (chloroquine and hydroxychloroquine) inhibit receptor binding of the viral particles and inhibits the strong "cytokine storm", which is the main cause of death among infected patients. In particular, the natural alkaloid quinine has shown to possess a better safety profile and greater tolerability compared to its derivatives. Dosage optimization of quinine is still necessary as the currently available dosage forms have controversial pharmacokinetics and safety profiles. Therefore, repurposing quinine dosage forms to improve its pharmacokinetics and safety profile may be necessary to support its use against SARS-CoV-2. In this context, biodegradable/biocompatible polymeric nanoparticles may provide a safe site-specific and controlled quinine delivery, reducing the frequency of drug administration and the dose. In this study, a full atomistic molecular dynamics simulation approach has been used to investigate the use of poly-(glycolic acid) and poly-(lactic acid) and their copolymer poly-(lactic-co-glycolic acid) as potential delivery systems for lipophilic quinine to get insights into the mechanism of quinine encapsulation and release at the atomic/molecular level.

Effect of Epigallocatechin-3-Gallate on EGFR Signaling and Migration in Non-Small Cell Lung Cancer.

The epidermal growth factor receptor (EGFR) is one of the most well-studied molecular targets in non-small cell lung cancer (NSCLC) and tyrosine kinase inhibitors have been shown to be effective in the treatment of advanced NSCLC. Nevertheless, the efficacy of tyrosine kinase inhibitors could be compromised by additional mutations in EGFR and compensatory activations of other pathways. Epigallocatechin-3-gallate (EGCG), the main bioactive molecule in green tea, acts as a tyrosine kinase inhibitor toward cancer cells overexpressing EGFR (wild-type). However, little information has been reported on the effect of EGCG on EGFR with activating mutations. In this study, we evaluated the ability of EGCG to inhibit EGFR signaling activation in three different NSCLC cell lines containing wild-type EGFR or EGFR with additional mutations. The effect on proliferation, apoptosis, migration, and vinculin expression was then studied. Overall, our results demonstrate that EGCG polyphenol inhibits cell proliferation and migration in NSCLC cell lines, although with different efficacy and mechanisms. These data may be of interest for an evaluation of the use of EGCG as an adjunct to NSCLC therapies.

Berberine Derivatives as Pseudomonas aeruginosa MexXY-OprM Inhibitors: Activity and In Silico Insights.

The natural alkaloid berberine has been demonstrated to inhibit the Pseudomonas aeruginosa multidrug efflux system MexXY-OprM, which is responsible for tobramycin extrusion by binding the inner membrane transporter MexY. To find a structure with improved inhibitory activity, we compared by molecular dynamics investigations the binding affinity of berberine and three aromatic substituents towards the three polymorphic sequences of MexY found in P. aeruginosa (PAO1, PA7, and PA14). The synergy of the combinations of berberine or berberine derivatives/tobramycin against the same strains was then evaluated by checkerboard and time-kill assays. The in silico analysis evidenced different binding modes depending on both the structure of the berberine derivative and the specific MexY polymorphism. In vitro assays showed an evident MIC reduction (32-fold and 16-fold, respectively) and a 2-3 log greater killing effect after 2 h of exposure to the combinations of 13-(2-methylbenzyl)- and 13-(4-methylbenzyl)-berberine with tobramycin against the tobramycin-resistant strain PA7, a milder synergy (a 4-fold MIC reduction) against PAO1 and PA14, and no synergy against the ΔmexXY strain K1525, confirming the MexY-specific binding and the computational results. These berberine derivatives could thus be considered new hit compounds to select more effective berberine substitutions and their common path of interaction with MexY as the starting point for the rational design of novel MexXY-OprM inhibitors.

A novel 3'-tRNAGlu-derived fragment acts as a tumor suppressor in breast cancer by targeting nucleolin.

tRNA-derived fragments (tRFs) have been defined as a novel class of small noncoding RNAs. tRFs have been reported to be deregulated in cancer, but their biologic function remains to be fully understood. We have identified a new tRF (named tRF3E), derived from mature tRNAGlu, that is specifically expressed in healthy mammary glands but not in breast cancer (BC). Consistently, tRF3E levels significantly decrease in the blood of patients with epidermal growth factor receptor 2 (HER2)-positive BC reflecting tumor status (control > early cancer > metastatic cancer). tRF3E down-regulation was recapitulated in Δ16HER2 transgenic mice, representing a BC preclinical model. Pulldown assays, used to search for proteins capable to selectively bind tRF3E, have shown that this tRF specifically interacts with nucleolin (NCL), an RNA-binding protein overexpressed in BC and able to repress the translation of p53 mRNA. The binding properties of NCL-tRF3E complex, predicted in silico and analyzed by EMSA assays, are congruent with a competitive displacement of p53 mRNA by tRF3E, leading to an increased p53 expression and consequently to a modulation of cancer cell growth. Here, we provide evidence that tRF3E plays an important role in the pathogenesis of BC displaying tumor-suppressor functions through a NCL-mediated mechanism.-Falconi, M., Giangrossi, M., Elexpuru Zabaleta, M., Wang, J., Gambini, V., Tilio, M., Bencardino, D., Occhipinti, S., Belletti, B., Laudadio, E., Galeazzi, R., Marchini, C., Amici, A. A novel 3'-tRNAGlu-derived fragment acts as a tumor suppressor in breast cancer by targeting nucleolin.

Acetylshikonin isolated from Lithospermum erythrorhizon roots inhibits dihydrofolate reductase and hampers autochthonous mammary carcinogenesis in Δ16HER2 transgenic mice.

Breast cancer (BC) is the most common cancer in women and, among different BC subtypes, triple negative (TN) and human epidermal growth factor receptor 2 (HER2)-positive BCs have the worst prognosis. In this study, we investigated the anticancer activity of the root ethanolic and hexane extracts from Lithospermum erythrorhizon, a traditional Chinese herbal medicine known also as tzu ts'ao or tzu-ken, against in vitro and in vivo models of TNBC and HER2-positive BC. Treatment with L. erythrorhizon root extracts resulted in a dose-dependent inhibition of BC cell viability and in a significant reduction of the growth of TNBC cells transplanted in syngeneic mice. Acetylshikonin, a naphthoquinone, was identified as the main bioactive component in extracts and was responsible for the observed antitumor activity, being able to decrease BC cell viability and to interfere with autochthonous mammary carcinogenesis in Δ16HER2 transgenic mice. Acetylshikonin anticancer effect depends on its ability to act as a potent inhibitor of dihydrofolate reductase (DHFR), to down-regulate key mediators governing cancer growth and progression, such as HER2, Src and STAT3, and to induce apoptosis by caspase-3 activation. The accumulation of acetylshikonin in blood samples as well as in brain, kidney, liver and tumor tissues was also investigated by liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) highlighting that L. erythrorhizon treatment is effective in delivering the active compound into the target tissues. These results provide evidence that L. erythrorhizon extract and in particular its main component acetylshikonin are effective against aggressive BC subtypes and reveal new acetylshikonin mechanisms of action.

Conformational Insight on WT- and Mutated-EGFR Receptor Activation and Inhibition by Epigallocatechin-3-Gallate: Over a Rational Basis for the Design of Selective Non-Small-Cell Lung Anticancer Agents.

Non-small cell lung cancer (NSCLC) represents a difficult condition to treat, due to epidermal growth factor receptor (EGFR) kinase domain mutations, which lead to ligand-independent phosphorylation. Deletion of five amino acids (ELREA) in exon 19 and mutational change from leucine to arginine at position 858 (L858R) are responsible for tyrosine kinase domain aberrant activation. These two common types of EGFR-mutated forms are clinically associated with high response with Tyrosine Kinase Inhibitors (TKI); however, the secondary T790M mutation within the Tyrosine Kinase Domain (TKD) determines a resistance to these EGFR-TKIs. Using molecular dynamic simulation (MD), the present study investigated the architectural changes of wild-type and mutants EGFR's kinase domains in order to detect any conformational differences that could be associated with a constitutively activated state and thus to evaluate the differences between the wild-type and its mutated forms. In addition, in order to evaluate to which extent the EGFR mutations affect its inhibition, Epigallocatechin 3-Gallate (EGCG) and Erlotinib (Erl), known EGFR-TKI, were included in our study. Their binding modes with the EGFR-TK domain were elucidated and the binding differences between EGFR wild-type and the mutated forms were evidenced. The aminoacids mutations directly influence the binding affinity of these two inhibitors, resulting in a different efficacy of Erl and EGCG inhibition. In particular, for the T790M/L858R EGFR, the binding modes of studied inhibitors were compromised by aminoacidic substitution confirming the experimental findings. These results may be useful for novel drug design strategies targeting the dimerization domain of the EGFR mutated forms, thus preventing receptor activation.

Natural Alkaloid Berberine Activity against Pseudomonas aeruginosa MexXY-Mediated Aminoglycoside Resistance: In Silico and in Vitro Studies.

The multidrug efflux system MexXY-OprM, inside the resistance-nodulation-division family, is a major determinant of aminoglycoside resistance in Pseudomonas aeruginosa. In the fight aimed to identify potential efflux pump inhibitors among natural compounds, the alkaloid berberine emerged as a putative inhibitor of MexXY-OprM. In this work, we elucidated its interaction with the extrusor protein MexY and assessed its synergistic activity with aminoglycosides. In particular, we built an in silico model for the MexY protein in its trimeric association using both AcrB (E. coli) and MexB (P. aeruginosa) as 3D templates. This model has been stabilized in the bacterial cytoplasmic membrane using a molecular dynamics approach and used for ensemble docking to obtain the binding site mapping. Then, through dynamic docking, we assessed its binding affinity and its synergism with aminoglycosides focusing on tobramycin, which is widely used in the treatment of pulmonary infections. In vitro assays validated the data obtained: the results showed a 2-fold increase of the inhibitory activity and 2-4 log increase of the killing activity of the association berberine-tobramycin compared to those of tobramycin alone against 13/28 tested P. aeruginosa clinical isolates. From hemolytic assays, we preliminarily assessed berberine's low toxicity.