Microscopic Evidence of Malaria Infection in Visceral Tissue from Medici Family, Italy.

Microscopy of mummified visceral tissue from a Medici family member in Italy identified a potential blood vessel containing erythrocytes. Giemsa staining, atomic force microscopy, and immunohistochemistry confirmed Plasmodium falciparum inside those erythrocytes. Our results indicate an ancient Mediterranean presence of P. falciparum, which remains responsible for most malaria deaths in Africa.

An RB-1 loss of function gene signature as a tool to predict response to neoadjuvant chemotherapy plus anti-HER2 agents: a substudy of the NeoALTTO trial (BIG 1-06).

BACKGROUND: Chemotherapy added to anti-HER2 agents (H) is the treatment of choice in patients with HER2+ early breast cancer. However, HER2+ tumours are clinically and biologically heterogeneous, and treatment response varies significantly by hormone receptor (HR) status and molecular subtype. Predictive biomarkers are needed in this context. This study assessed whether an RB-1 loss of function gene signature (RBsig) is predictive of response to neoadjuvant chemotherapy in combination with trastuzumab, lapatinib or both, within the NeoALTTO trial. METHODS: We collected RNA-sequencing data from pretreatment biopsies derived from the NeoALTTO trial. RBsig expression was computed retrospectively and correlated with pathological complete response (pCR) using receiver-operating characteristic (ROC) curves. The RBsig was dichotomised as High/Low in correspondence to the 25th percentile. Reported p values resulted from Fisher's exact test. RESULTS: Of 455 NeoALTTO patients, 244 were eligible for this substudy (HR+ n = 129; HR- n = 115). Overall, pCR rate was significantly higher in patients with RBsig High tumours than those with RBsig Low (35% versus 18% respectively; p = 0.01). The area under the ROC curve (AUC) was 0.60 (95% CI 0.52-0.67). A remarkably low pCR rate of 11% was seen in HR+/RBsig Low patients versus 28% in HR+/RBsig High. CONCLUSIONS: These results indicate RBsig may add valuable information to HER2 and HR expression, which may in turn inform treatment choices. HR+/HER2+/RBsig Low breast cancers exhibited the poorest pathological response following chemotherapy plus H. Accordingly, in such patients, endocrine therapy in combination with H and, possibly, a CDK4/6 inhibitor, may potentially prove to be a more effective treatment.

Prognostic role of serum thymidine kinase 1 activity in patients with hormone receptor-positive metastatic breast cancer: Analysis of the randomised phase III Evaluation of Faslodex versus Exemestane Clinical Trial (EFECT).

BACKGROUND: Thymidine kinase 1 (TK1) plays a critical role in DNA synthesis and cell proliferation. Recent studies have shown potential for serum TK1 activity (sTKa) as a prognostic marker and indicator of early response to endocrine therapy in advanced breast cancer. The aim of this study is to assess the correlation between sTKa and patient outcome. PATIENTS AND METHODS: The Evaluation of Faslodex versus Exemestane Clinical Trial (EFECT) was a double-blind, double-dummy, randomised trial of fulvestrant versus exemestane after progression on non-steroidal aromatase inhibitor therapy, in postmenopausal women with advanced breast cancer. Retrospective analyses of serum archived from EFECT were conducted. sTKa was assessed using the DiviTum® assay on samples collected at baseline, after three and six months of endocrine therapy, and at disease progression. RESULTS: The median time to progression (mTTP) for patients with low baseline sTKa levels was 5.03 months (95% confidence interval [CI]: 3.91-5.89) versus 2.57 months (95% CI: 2.04-3.52) in patients with high sTKa baseline levels (P < 0.0001). On treatment, patients whose sTKa increased from baseline had a significantly shorter mTTP (3.39 months, 95% CI: 2.14-4.11) than those without an sTKa increase (5.39 months, 95% CI: 4.01-6.68) (P = 0.0045). Similar results were observed in the separate EFECT treatment arms. After adjusting for major prognostic factors, sTKa remained an independent marker. CONCLUSION: sTKa is a potential circulating prognostic marker in patients with advanced breast cancer treated with endocrine therapy. It may also represent a tool for upfront identification of endocrine therapy resistance and early positive response to therapy. Independent validation of these results is warranted.

Cyclin E1 and Rb modulation as common events at time of resistance to palbociclib in hormone receptor-positive breast cancer.

CDK4/6 inhibitors represent a new treatment standard for hormone receptor-positive (HR+), HER2-negative advanced breast cancer (BC) patients. Although efficacious, resistance to these agents is universal. Here, we profiled a large panel of HR+ BC cell lines with conditioned resistance to the CDK4/6 inhibitor palbociclib, and analyzed cell cycle-related markers by gene expression profiles (GEP) and western blot (WB). GEP showed high molecular heterogeneity among the models, with E2F targets being significantly enriched both during treatment and at the time of resistance. By both WB and GEP, a common molecular feature at the time of palbociclib resistance was the concomitant overexpression of cyclin E1 and down-regulation of Rb. CCNE1 was the only significantly up-regulated gene among E2F targets at resistance with CCNE1 genomic amplification being observed in two resistant models. Rb was downregulated in all resistant models; a reduction of RB1 copy number was observed in three resistant cell lines. In silico analyses showed that CCNE1/RB1 ratio correlated with palbociclib IC50 in different datasets of both breast and non-breast cancer cell lines, performing better than CCNE1 or RB1 taken separately. Finally, the CCNE1/RB1 ratio was shown to be an adverse prognostic factor in patients with ER+ BC and to be able to discriminate palbociclib-sensitive versus resistant among patients enrolled in the NeoPalAna trial, a neoadjuvant trial testing palbociclib, performing better than CCNE1 or RB1 alone. Our data suggest that the CCNE1/RB1 ratio may be a viable biomarker of palbociclib resistance, warranting further clinical validation.

ddSeeker: a tool for processing Bio-Rad ddSEQ single cell RNA-seq data.

BACKGROUND: New single-cell isolation technologies are facilitating studies on the transcriptomics of individual cells. Bio-Rad ddSEQ is a droplet-based microfluidic system that, when coupled with downstream Illumina library preparation and sequencing, enables the monitoring of thousands of genes per cell. Sequenced reads show unique features that do not permit the use of freely available tools to perform single cell demultiplexing. RESULTS: We present ddSeeker, a tool to perform initial processing and quality metrics of reads generated through Bio-Rad ddSEQ/Illumina experiments. Its application to the Illumina test dataset demonstrates that ddSeeker performs better than Illumina BaseSpace software, enabling a higher recovery of valid reads. We also show its utility in the analysis of an in-house dataset including two read sets characterized by low and high sequencing quality. ddSeeker and its source code are available at https://github.com/cgplab/ddSeeker . CONCLUSIONS: ddSeeker is a freely available tool to perform initial processing and quality metrics of reads generated through Bio-Rad ddSEQ/Illumina single cell transcriptomic experiments.

Plasma thymidine kinase-1 activity predicts outcome in patients with hormone receptor positive and HER2 negative metastatic breast cancer treated with endocrine therapy.

The aim of this study was to investigate if thymidine kinase-1 (TK1), a well-known proliferation marker, could represent a valid circulating biomarker to identify hormone receptor positive (HR+)/HER2 negative (HER2neg) metastatic breast cancer (MBC) patients most likely to benefit from endocrine therapy (ET). We used the DiviTum™ assay to analyze TK1 activity in cell lysates of three HR+/HER2neg BC cell lines and in plasma of 31 HR+/HER2neg MBC patients receiving ET. Blood samples were collected at treatment initiation, after one month and at disease progression. CTCs count and ESR1/PIK3CA mutations in circulating tumor DNA were performed and correlated with TK1 activity. TK1 activity was reduced in the two endocrine-sensitive cell lines after 2 days of treatment. In patients, high baseline TK1 activity correlated with CTCs positivity (p-value=0.014). Patients with low baseline levels of TK1 activity had a significantly better PFS compared to those with high baseline TK1 activity (p-value=0.012). Patients with an early drop of TK1 activity after one month of treatment had a significantly better PFS compared to those who experienced an increase (p-value=0.0026). Our study suggests that TK1 could be a potential prognostic, predictive and monitoring marker of early ET response in HR+/HER2neg MBC patients.

Model lipid bilayers mimic non-specific interactions of gold nanoparticles with macrophage plasma membranes.

Understanding the interaction between nanomaterials and biological interfaces is a key unmet goal that still hampers clinical translation of nanomedicine. Here we investigate and compare non-specific interaction of gold nanoparticles (AuNPs) with synthetic lipid and wild type macrophage membranes. A comprehensive data set was generated by systematically varying the structural and physicochemical properties of the AuNPs (size, shape, charge, surface functionalization) and of the synthetic membranes (composition, fluidity, bending properties and surface charge), which allowed to unveil the matching conditions for the interaction of the AuNPs with macrophage plasma membranes in vitro. This effort directly proved for the first time that synthetic bilayers can be set to mimic and predict with high fidelity key aspects of nanoparticle interaction with macrophage eukaryotic plasma membranes. It then allowed to model the experimental observations according to classical interface thermodynamics and in turn determine the paramount role played by non-specific contributions, primarily electrostatic, Van der Waals and bending energy, in driving nanoparticle-plasma membrane interactions.

Relaxin induces up-regulation of ADAM10 metalloprotease in RXFP1-expressing cells by PI3K/AKT signaling.

ADAM10 metalloprotease is required for activation of Notch-1, a transmembrane receptor regulating cell differentiation, proliferation and apoptosis, whose intracellular proteolytic fragment NICD mediates some key cardiovascular effects of the hormone relaxin (RLX). This study demonstrates the involvement of ADAM10 and PI3K/Akt signaling in mediating RLX-induced Notch-1 activation. H9c2 cardiomyocytes and NIH3T3 fibroblasts were incubated with human RLX-2 (17 nmol/l, 24 h) in presence or absence of the PI3K or Akt inhibitors wortmannin (WT, 100 nmol/l) and triciribine (TCN, 1 µmol/l). Cyclohexanedione-inactivated RLX (iRLX) served as negative control. RLX significantly increased Akt phosphorylation, ADAM10 and NICD expression, which were abolished by WT or TCN and did not occur with iRLX. These findings highlight a new receptor-specific signal transduction pathway of RLX.

Mechanisms of Resistance to CDK4/6 Inhibitors in Breast Cancer and Potential Biomarkers of Response.

Randomized clinical trials demonstrated that CDK4/6 inhibitors are highly effective in patients with hormone receptor-positive (HR+), HER2-negative (HER2-) metastatic breast cancer in combination with endocrine therapy. The use of CDK4/6 inhibitors in clinics is becoming common for patients with HR+/HER2- metastatic breast cancer and will certainly increase in the near future. However, patients might show de novo or acquired resistance to these drugs. Molecular alterations have been suggested as determinants for de novo resistance to CDK4/6 inhibitors, but have never been validated in a clinical setting. In addition, molecular mechanisms of acquired resistance to palbociclib have been analyzed only in preclinical studies. Here we review the current knowledge on the available preclinical data about the mechanisms of de novo and acquired resistance to CDK4/6 inhibitors in breast cancer, and clinical data about potential biomarkers of response.

Methylene blue-containing liposomes as new photodynamic anti-bacterial agents.

Methylene blue (MB) can be employed as a photo-activatable antimicrobial drug in photodynamic therapy (PDT) due to its ability to release oxygen free radicals upon photo-activation. However, its poor ability to penetrate bacterial cell walls and bacterial biofilms limits its antimicrobial activity. To overcome these limitations, we propose some formulations of MB based on different cationic liposomes. The liposome-MB systems were characterized using dynamic light scattering (DLS), zeta potential analysis and UV-visible spectroscopy. Their ability to penetrate inside the cytoplasm of E. coli, taken as a bacterial model for Gram-negative strains, was investigated through laser scanning confocal microscopy (CLSM) and compared to the penetration of naked MB. Then, MB-loaded liposomes were photo-activated and their antimicrobial activity was tested against E. coli, showing a strong improvement with respect to MB solutions. The liposomal formulations dramatically enhance MB penetration in bacterial biofilms and reduce the inflammatory response due to lipopolysaccharide exposure in mammalian cells. The observed antimicrobial and anti-inflammatory efficacies show a clear correlation with some structural features of the carriers, namely the size and the surface charge density. Overall, these results provide fundamental knowledge that enables the design of novel efficient PDT treatments, which potentially overcome the rising incidence of antibiotic resistance of bacterial strains.

HYDAMTIQ, a selective PARP-1 inhibitor, improves bleomycin-induced lung fibrosis by dampening the TGF-ß/SMAD signalling pathway.

Idiopathic pulmonary fibrosis is a severe disease characterized by excessive myofibroblast proliferation, extracellular matrix and fibrils deposition, remodelling of lung parenchyma and pulmonary insufficiency. Drugs able to reduce disease progression are available, but therapeutic results are unsatisfactory; new and safe treatments are urgently needed. Poly(ADP-ribose) polymerases-1 (PARP-1) is an abundant nuclear enzyme involved in key biological processes: DNA repair, gene expression control, and cell survival or death. In liver and heart, PARP-1 activity facilitates oxidative damage, collagen deposition and fibrosis development. In this study, we investigated the effects of HYDAMTIQ, a potent PARP-1 inhibitor, in a murine model of lung fibrosis. We evaluated the role of PARP on transforming growth factor-ß (TGF-ß) expression and TGF-ß/SMAD signalling pathway in lungs. Mice were intratracheally injected with bleomycin and then treated with either vehicle or different doses of HYDAMTIQ for 21 days. Airway resistance to inflation and lung static compliance, markers of lung stiffness, were assayed. Histochemical and biochemical parameters to evaluate TGF-ß/SMAD signalling pathway with alpha-smooth muscle actin (αSMA) deposition and the levels of a number of inflammatory markers (tumour necrosis factor-α, interleukin-1ß, iNOS and COX-2) were performed. Bleomycin administration increased lung stiffness. It also increased lung PARP activity, TGF-ß levels, pSMAD3 expression, αSMA deposition and content of inflammatory markers. HYDAMTIQ attenuated all the above-mentioned physiological, biochemical and histopathological markers. Our findings support the proposal that PARP inhibitors could have a therapeutic potential in reducing the progression of signs and symptoms of the disease by decreasing TGF-ß expression and the TGF-ß/SMAD transduction pathway.

Analysis of Lipoasiprated Following Centrifugation: Wet Versus Dry Harvesting Technique.

BACKGROUND: The success of lipotransfer strongly depends on the harvesting, processing, and placement of the lipoaspirated samples. This study was designed to assess the histomorphometric characteristics and viability of fat harvested using different techniques (wet and dry) following centrifugation, as described by Coleman. METHODS: The study enrolled 85 consecutive, nonrandomized, healthy patients from March 2010 to December 2014 (45 males and 40 females). The mean age was 40 years (range, 18-59 years), and the mean body mass index was 25.8 (range, 24-32). The authors performed a histological analysis (hematoxylin/eosin), morphometry (ImageJ 1.33 free-share image analysis software), and a viability assessment (Trypan Blue exclusion test; Sigma-Aldrich, Milan, Italy) of the lipoaspirated samples. RESULTS: The hematoxylin and eosin-stained sections exhibited similar features; in particular, clear-cut morphological signs of adipocyte disruption, apoptosis, or necrosis were not detected in the examined samples. Morphometry confirmed the visual findings, and the values of the mean surface area of the adipocyte vacuoles were not significantly different. Additionally, the adipocyte viability was not significantly different in the analyzed fat tissue samples. CONCLUSIONS: The results from this study showed, for the first time, that there is not a reduction in the viability of fat grafts harvested with the dry or wet technique following centrifugation according to Coleman technique. Both methods of fat harvesting collect viable cells, which are not influenced by standard centrifugation. The fat grafts harvested and processed by this technique could be used in clinical settings without increasing the reabsorption rate. LEVEL OF EVIDENCE: V.

Protection from Cigarette Smoke-Induced Lung Dysfunction and Damage by H2 Relaxin (Serelaxin).

Cigarette smoke (CS) is the major etiologic factor of chronic obstructive pulmonary disease (COPD), which is characterized by airway remodeling, lung inflammation and fibrosis, emphysema, and respiratory failure. The current therapies can improve COPD management but cannot arrest its progression and reduce mortality. Hence, there is a major interest in identifying molecules susceptible of development into new drugs to prevent or reduce CS-induced lung injury. Serelaxin (RLX), or recombinant human relaxin-2, is a promising candidate because of its anti-inflammatory and antifibrotic properties highlighted in lung disease models. Here, we used a guinea pig model of CS-induced lung inflammation, and remodeling reproducing some of the hallmarks of COPD. Animals exposed chronically to CS (8 weeks) were treated with vehicle or RLX, delivered by osmotic pumps (1 or 10 µg/day) or aerosol (10 µg/ml/day) during CS treatment. Controls were nonsmoking animals. RLX maintained airway compliance to a control-like pattern, likely because of its capability to counteract lung inflammation and bronchial remodeling. In fact, treatment of CS-exposed animals with RLX reduced the inflammatory recruitment of leukocytes, accompanied by a significant reduction of the release of proinflammatory cytokines (tumor necrosis factor α and interleukin-1ß). Moreover, RLX was able to counteract the adverse bronchial remodeling and emphysema induced by CS exposure by reducing goblet cell hyperplasia, smooth muscle thickening, and fibrosis. Of note, RLX delivered by aerosol has shown a comparable efficacy to systemic administration in reducing CS-induced lung dysfunction and damage. In conclusion, RLX emerges as a new molecule to counteract CS-induced inflammatory lung diseases.

Protection from cigarette smoke-induced vascular injury by recombinant human relaxin-2 (serelaxin).

Smoking is regarded as a major risk factor for the development of cardiovascular diseases (CVD). This study investigates whether serelaxin (RLX, recombinant human relaxin-2) endowed with promising therapeutic properties in CVD, can be credited of a protective effect against cigarette smoke (CS)-induced vascular damage and dysfunction. Guinea pigs exposed daily to CS for 8 weeks were treated with vehicle or RLX, delivered by osmotic pumps at daily doses of 1 or 10 µg. Controls were non-smoking animals. Other studies were performed on primary guinea pig aortic endothelial (GPAE) cells, challenged with CS extracts (CSE) in the absence and presence of 100 ng/ml (17 nmol/l) RLX. In aortic specimens from CS-exposed guinea pigs, both the contractile and the relaxant responses to phenylephrine and acetylcholine, respectively, were significantly reduced in amplitude and delayed, in keeping with the observed adverse remodelling of the aortic wall, endothelial injury and endothelial nitric oxide synthase (eNOS) down-regulation. RLX at both doses maintained the aortic contractile and relaxant responses to a control-like pattern and counteracted aortic wall remodelling and endothelial derangement. The experiments with GPAE cells showed that CSE significantly decreased cell viability and eNOS expression and promoted apoptosis by sparkling oxygen free radical-related cytotoxicity, while RLX counterbalanced the adverse effects of CSE. These findings demonstrate that RLX is capable of counteracting CS-mediated vascular damage and dysfunction by reducing oxidative stress, thus adding a tile to the growing mosaic of the beneficial effects of RLX in CVD.

Protection of coronary endothelial cells from cigarette smoke-induced oxidative stress by a new Mn(II)-containing polyamine-polycarboxilate scavenger of superoxide anion.

Oxidative stress plays a major role in cardiovascular injury and dysfunction induced by cigarette smoke. Smoke-borne pro-oxidants impair endothelial function and predispose to thrombosis, inflammation and atherosclerosis. This in vitro study evaluates whether Mn(II)(4,10-dimethyl-1,4,7,10-tetraazacyclododecane-1,7-diacetate).2H2O (Mn(II)(Me2DO2A)), a polyamine-polycarboxilate, Mn(II)-containing O2(-) scavenger, has a direct protective action on guinea pig coronary endothelial (GPCE) cells exposed to cigarette smoke extracts (CSE). Mn(II)(Me2DO2A) (1-10µmol/l) was added to the culture medium together with CSE and maintained for 4h. In parallel experiments, the inactive congener Zn(II)(Me2DO2A), in which Zn(II) replaced the functional Mn(II) center in the same organic scaffold, was used as negative control. Mn(II)(Me2DO2A), mostly at the higher doses (5 and 10µmol/l), significantly increased GPCE cell viability (trypan blue assay), improved mitochondrial activity (MTT test, mitochondrial membrane potential Δψ), reduced cellular apoptosis (mPTP, caspase-3 activity, TUNEL assay), decreased intracellular ROS levels (H2DCFDA), lipoperoxidation (BODIPY 581/591) and decreased protein nitrosylation. Of note, Zn(II)(Me2DO2A) did not preserve cell viability. These findings suggest that Mn(II)(Me2DO2A) is a promising O2(-) scavenging compound able to protect from cigarette smoke-induced oxidative cell injury. In perspective, should its efficacy be confirmed in future in vivo studies, this molecule might represent a therapeutic or preventive drug to counteract cigarette smoke toxicity.

Relaxin protects cardiac muscle cells from hypoxia/reoxygenation injury: involvement of the Notch-1 pathway.

In animal models, the cardiotropic hormone relaxin has been shown to protect the heart against ischemia and reperfusion-induced damage, acting by multiple mechanisms that primarily involve the coronary vessels. This in vitro study evaluates whether relaxin also has a direct protective action on cardiac muscle cells. H9c2 rat cardiomyoblasts and primary mouse cardiomyocytes were subjected to hypoxia and reoxygenation. In some experiments, relaxin was added preventatively before hypoxia; in others, at reoxygenation. To elucidate its mechanisms of action, we focused on Notch-1, which is involved in heart pre- and postconditioning to ischemia. Inactivated RLX was used as negative control. Relaxin (17 nmol/L, EC50 4.7 nmol/L), added 24 h before hypoxia or at reoxygenation, protected against cardiomyocyte injury. In fact, relaxin significantly increased cell viability (assayed by trypan blue and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide), decreased apoptosis (assayed by TUNEL and bax/bcl-2 ratio), and reduced nitroxidative damage (assayed by nitrotyrosine expression and 8-hydroxy-deoxyguanosine levels). These effects were partly attributable to the ability of relaxin to upregulate Notch-1 signaling; indeed, blockade of Notch-1 activation with the specific inhibitor DAPT reduced relaxin-induced cardioprotection during hypoxia and reoxygenation. This study adds new mechanistic insights on the cardioprotective role of relaxin on ischemic and oxidative damage.