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Patients with COVID-19 may experience multiple conditions (e.g., fever, hyperventilation, anorexia, gastroenteritis, acid-base disorder) that may cause electrolyte imbalances. Hypokalemia is a concerning electrolyte disorder that may increase the susceptibility to various kinds of arrhythmia. This study aimed to estimate prevalence, risk factors and outcome of hypokalemia in a cohort of non-critically ill patients. A retrospective analysis was conducted on 290 hospitalized patients with confirmed COVID-19 infection at the tertiary teaching hospital of Modena, Italy.Hypokalemia (Hypokalemia is a frequent disorder in COVID-19 patients and urinary potassium loss may be the main cause of hypokalemia. The disorder was mild in the majority of the patients and was unrelated to poor outcomes. Nevertheless, hypokalemic patients required potassium supplements to dampen the risk of arrhythmias.

The catastrophic impact of the coronavirus disease 2019 (COVID-19) epidemic in Italy has been described previously [1, 2]. In a recent editorial, Nava and co-authors pay a well-deserved tribute to the almost 200 health care workers who succumbed to COVID-19 in the country in less than 3 months since the first case was reported [3]. We would like to contribute with some considerations on the shortcomings of Italy's response to the pandemic., The COVID-19 epidemic in Italy has shown many shortcomings of the national health care system but it also represents a historic opportunity to reinforce the central health care governance and reduce inequalities across the country

BackgroundThe aim of this secondary analysis of the TESEO cohort is to identify, early in the course of treatment with tocilizumab, factors associated with the risk of progressing to mechanical ventilation and death and develop a risk score to estimate the risk of this outcome according to patients' profile.MethodsPatients with COVID-19 severe pneumonia receiving standard of care + tocilizumab who were alive and free from mechanical ventilation at day 6 after treatment initiation were included in this retrospective, multicenter cohort study. Multivariable logistic regression models were built to identify predictors of mechanical ventilation or death by day-28 from treatment initiation and β-coefficients were used to develop a risk score. Secondary outcome was mortality. Patients with the same inclusion criteria as the derivation cohort from 3 independent hospitals were used as validation cohort.Results266 patients treated with tocilizumab were included. By day 28 of hospital follow-up post treatment initiation, 40 (15%) underwent mechanical ventilation or died [26 (10%)]. At multivariable analysis, sex, day-4 PaO2/FiO2 ratio, platelets and CRP were independently associated with the risk of developing the study outcomes and were used to generate the proposed risk score. The accuracy of the score in AUC was 0.80 and 0.70 in internal validation and test for the composite endpoint and 0.92 and 0.69 for death, respectively.ConclusionsOur score could assist clinicians in identifying, early after tocilizumab administration, patients who are likely to progress to mechanical ventilation or death, so that they could be selected for eventual rescue therapies.

Cancer incidence and mortality continue to increase, while the conventional chemotherapeutic drugs confer limited efficacy and relevant toxic side effects. Novel strategies are urgently needed for more effective and safe therapeutics in oncology. However, a large number of proteins are considered undruggable by conventional drugs, such as the small molecules. Moreover, the mRNA itself retains oncological functions, and its targeting offers the double advantage of blocking the tumorigenic activities of the mRNA and the translation into protein. Finally, a large family of non-coding RNAs (ncRNAs) has recently emerged that are also dysregulated in cancer, but they could not be targeted by drugs directed against the proteins. In this context, this review describes how the oligonucleotide therapeutics targeting RNA or DNA sequences, are emerging as a new class of drugs, able to tackle the limitations described above. Numerous clinical trials are evaluating oligonucleotides for tumor treatment, and in the next few years some of them are expected to reach the market. We describe the oligonucleotide therapeutics targeting undruggable proteins (focusing on the most relevant, such as those originating from the MYC and RAS gene families), and for ncRNAs, in particular on those that are under clinical trial evaluation in oncology. We highlight the challenges and solutions for the clinical success of oligonucleotide therapeutics, with particular emphasis on the peculiar challenges that render it arduous to treat tumors, such as heterogeneity and the high mutation rate. In the review are presented these and other advantages offered by the oligonucleotide as an emerging class of biotherapeutics for a new era of precision anti-cancer medicine.

Background Neuroblastoma is a deadly childhood cancer, and MYCN-amplified neuroblastoma (MNA-NB) patients have the worst prognoses and are therapy-resistant. While retinoic acid (RA) is beneficial for some neuroblastoma patients, the cause of RA resistance is unknown. Thus, there remains a need for new therapies to treat neuroblastoma. Here we explored the possibility of combining a MYCN-specific antigene oligonucleotide BGA002 and RA as therapeutic approach to restore sensitivity to RA in NB. Methods By molecular and cellular biology techniques, we assessed the combined effect of the two compounds in NB cell lines and in a xenograft mouse model MNA-NB. Results We found that MYCN-specific inhibition by BGA002 in combination with RA (BGA002-RA) act synergistically and overcame resistance in NB cell lines. BGA002-RA also reactivated neuron differentiation (or led to apoptosis) and inhibited invasiveness capacity in MNA-NB. Moreover, we found that neuroblastoma had the highest level of mRNA expression of mTOR pathway genes, and that BGA002 led to mTOR pathway inhibition followed by autophagy reactivation in MNA-NB cells, which was strengthened by BGA002-RA. BGA002-RA in vivo treatment also eliminated tumor vascularization in a MNA-NB mouse model and significantly increased survival. Conclusion Taken together, MYCN modulation mediates the therapeutic efficacy of RA and the development of RA resistance in MNA-NB. Furthermore, by targeting MYCN, a cancer-specific mTOR pathway inhibition occurs only in MNA-NB, thus avoiding the side effects of targeting mTOR in normal cells. These findings warrant clinical testing of BGA002-RA as a strategy for overcoming RA resistance in MNA-NB.

Background: Patients with acute exacerbation of idiopathic pulmonary fibrosis (AE-IPF) may experience severe acute respiratory failure, even requiring ventilatory assistance. Physiological data on lung mechanics during these events are lacking. Methods: Patients with AE-IPF admitted to Respiratory Intensive Care Unit to receive non-invasive ventilation (NIV) were retrospectively analyzed. Esophageal pressure swing (ΔPes) and respiratory mechanics before and after 2 hours of NIV were collected as primary outcome. The correlation between positive end-expiratory pressure (PEEP) levels and changes of in dynamic compliance (dynCRS) and PaO2/FiO2 ratio was assessed. Further, an exploratory comparison with a historical cohort of ARDS patients matched 1:1 by age, sequential organ failure assessment score, body mass index and PaO2/FiO2 level was performed. Results: At baseline, AE-IPF patients presented a high respiratory drive activation with ΔPes = 27 (21-34) cmH2O, respiratory rate (RR) = 34 (30-39) bpm and minute ventilation (VE) = 21 (20-26) L/min. Two hours after NIV application, ΔPes, RR and VE values showed a significant reduction (16 [14-24] cmH2O, p

Acute exacerbation (AE) is a possible manifestation of interstitial lung diseases (ILD) associated to very high mortality. It’s defined as clinically significant respiratory deterioration with evidence of new widespread alveolar abnormalities on computed tomography scan. AE is better described in idiopathic pulmonary fibrosis (IPF) but also reported in ILD secondary to connective tissue diseases (CTD) and vasculitis. The main features and the real clinical impact of this severe complication in these patients are not well defined. Aim of our study was to prospectively investigate the incidence, clinical features and outcome of AE in a population of patients with ILD related to CTD and vasculitis. We consecutively enrolled all patients, with ILD secondary to rheumatic systemic diseases, referring to our multidisciplinary outpatient clinic for rare lung diseases. All patients were followed for at least 12 months (range, 12–36 months). At baseline, all patients underwent to a core set of laboratory investigations and periodically followed; data about demographic, disease onset, clinical, serological and therapeutic features were also recorded. AE occurred in 9/78 patients, with an incidence of 5.77/100 patients/year, and 5/9 patients died because of AE. The baseline value of DLCO was significantly associated to the risk of AE at Cox regression. In patients with ILD related to rheumatic systemic diseases AE can occur with an incidence similar to IPF. Rheumatologists should carefully consider this life-threatening complication as a possible natural course of all patients with ILD secondary to systemic rheumatic disease.

Background & aims: Hydroxychloroquine (HCQ) and chloroquine (CQ) are anti-malarial drugs frequently used in the rheumatologic field. They were recently identified as potential therapeutic options for Coronavirus Disease (COVID-19). The present study aims to map and grade the diverse health outcomes associated with HCQ/CQ using an umbrella review approach. Methods: Umbrella review of systematic reviews of observational and intervention studies. For observational studies, random-effects summary effect size, 95% confidence interval, and 95% prediction interval were estimated. We also assessed heterogeneity, evidence for small-study effect, and evidence for excess significance bias. The quality of evidence was then graded using validated criteria from highly convincing to weak. The evidence from randomized controlled trials (RCTs) was graded using the Grading of Recommendations, Assessment, Development and Evaluation (GRADE) tool. Results: From 313 articles returned in the literature search, six meta-analyses were included (n = 25 outcomes). Among meta-analyses (MAs) of observational studies, HCQ/CQ are weakly associated with a reduced risk for cardiovascular events and diabetes when used for autoimmune diseases and with spontaneous abortion; they are also associated with a higher risk of death in COVID-19 patients. Among MAs of RCTs, HCQ/CQ are associated with an improvement of articular manifestations of rheumatic diseases. Conclusions: There is high evidence of the efficacy of HCQ/CQ in the rheumatologic field. The lack of evidence for efficacy and the risk of death associated with the use of HCQ/CQ for COVID-19 indicate the inappropriateness of their inclusion in recent COVID-19 therapy guidelines and the urgent need for RCTs to determine eventual appropriateness as a COVID-19 therapy. Keywords: COVID-19; Chloroquine; Hydroxychloroquine; umbrella review. https://www.sciencedirect.com/science/article/pii/S1201971220325418?via%3Dihub

Protective ventilation is the cornerstone of treatment of patients with the acute respiratory distress syndrome (ARDS); however, no studies have yet established the best ventilatory strategy to adopt when patients with acute exacerbation of interstitial lung disease (AE-ILD) are admitted to the intensive care unit. Due to the severe impairment of the respiratory mechanics, the fibrotic lung is at high risk of developing ventilator-induced lung injury, regardless of the lung fibrosis etiology. The purpose of this review is to analyze the effects of mechanical ventilation in AE-ILD and to increase the knowledge on the characteristics of fibrotic lung during artificial ventilation, introducing the concept of “squishy ball lung”. The role of positive end-expiratory pressure is discussed, proposing a “lung resting strategy” as opposed to the “open lung approach”. The review also discusses the practical management of AE-ILD patients discussing illustrative clinical cases.

Laryngotracheal stenosis (LTS) is a complex and heterogeneous disease whose pathogenesis remains unclear. LTS is considered to be the result of aberrant wound-healing process that leads to fibrotic scarring, originating from different aetiology. Although iatrogenic aetiology is the main cause of subglottic or tracheal stenosis, also autoimmune and infectious diseases may be involved in causing LTS. Furthermore, fibrotic obstruction in the anatomic region under the glottis can also be diagnosed without apparent aetiology after a comprehensive workup; in this case, the pathological process is called idiopathic subglottic stenosis (iSGS). So far, the laryngotracheal scar resulting from airway injury due to different diseases was considered as inert tissue requiring surgical removal to restore airway patency. However, this assumption has recently been revised by regarding the tracheal scarring process as a fibroinflammatory event due to immunological alteration, similar to other fibrotic diseases. Recent acquisitions suggest that different factors, such as growth factors, cytokines, altered fibroblast function and genetic susceptibility, can all interact in a complex way leading to aberrant and fibrotic wound healing after an insult that acts as a trigger. However, also physiological derangement due to LTS could play a role in promoting dysregulated response to laryngo-tracheal mucosal injury, through biomechanical stress and mechanotransduction activation. The aim of this narrative review is to present the state-of-the-art knowledge regarding molecular mechanisms, as well as mechanical and physio-pathological features behind LTS.

Only one class of targeted agents (anti-GD2 antibodies) has been incorporated into front-line therapy for neuroblastoma since the 1980s. The Neuroblastoma New Drug Development Strategy (NDDS) initiative commenced in 2012 to accelerate the development of new drugs for neuroblastoma. Advances have occurred, with eight of nine high-priority targets being evaluated in paediatric trials including anaplastic lymphoma kinase inhibitors being investigated in front-line, but significant challenges remain. This article reports the conclusions of the second NDDS forum, which expanded across the Atlantic to further develop the initiative. Pre-clinical and clinical data for 40 genetic targets and mechanisms of action were prioritised and drugs were identified for early-phase trials. Strategies to develop drugs targeting TERT, telomere maintenance, ATRX, alternative lengthening of telomeres (ALT), BRIP1 and RRM2 as well as direct targeting of MYCN are high priority and should be championed for drug discovery. Promising pre-clinical data suggest that targeting of ALT by ATM or PARP inhibition may be potential strategies. Drugs targeting CDK2/9, CDK7, ATR and telomere maintenance should enter paediatric clinical development rapidly. Optimising the response to anti-GD2 by combinations with chemotherapy, targeted agents and other immunological targets are crucial. Delivering this strategy in the face of small patient cohorts, genomically defined subpopulations and a large number of permutations of combination trials, demands even greater international collaboration. In conclusion, the NDDS provides an internationally agreed, biologically driven selection of prioritised genetic targets and drugs. Improvements in the strategy for conducting trials in neuroblastoma will accelerate bringing these new drugs more rapidly to front-line therapy. (C) 2020 Elsevier Ltd. All rights reserved.

Alessandro Marchioni,1 Roberto Tonelli,1,2 Riccardo Fantini,1 Luca Tabbì,1 Ivana Castaniere,1,2 Francesco Livrieri,1,3 Sabrina Bedogni,4 Valentina Ruggieri,1 Lara Pisani,5 Stefano Nava,5 Enrico Clini1 1University Hospital of Modena, Pneumology Unit and Center for Rare Lung Diseases, Department of Medical and Surgical Sciences, University of Modena and Reggio Emilia, Modena, Italy; 2PhD Course in Clinical and Experimental Medicine, University of Modena and Reggio Emilia, Modena, Italy; 3Respiratory Disease Unit, Hospital Carlo Poma, Mantova, Italy; 4School of Medicine, University of Modena and Reggio Emilia, Modena, Italy; 5Department of Specialistic, Diagnostic and Experimental Medicine (DIMES), University of Bologna, Bologna, ItalyCorrespondence: Roberto TonelliUniversity Hospital of Modena, Pneumology Unit and Center for Rare Lung Diseases, Department of Medical and Surgical Sciences, University of Modena and Reggio Emilia, Via del Pozzo, 71 – 41125, Modena, ItalyTel +39-342-7241672Email roberto.tonelli@me.com Skype roberto.tonelli150288Background: Although non-invasive mechanical ventilation (NIV) is the gold standard treatment for patients with acute exacerbation of COPD (AECOPD) developing respiratory acidosis, failure rates still range from 5% to 40%. Recent studies have shown that the onset of severe diaphragmatic dysfunction (DD) during AECOPD increases risk of NIV failure and mortality in this subset of patients. Although the imbalance between the load and the contractile capacity of inspiratory muscles seems the main cause of AECOPD-induced hypercapnic respiratory failure, data regarding the influence of mechanical derangement on DD in this acute phase are lacking. With this study, we investigate the impact of respiratory mechanics on diaphragm function in AECOPD patients experiencing NIV failure.Methods: Twelve AECOPD patients with respiratory acidosis admitted to the Respiratory ICU of the University Hospital of Modena from 2017 to 2018 undergoing mechanical ventilation (MV) due to NIV failure were enrolled. Static respiratory mechanics and end-expiratory lung volume (EELV) were measured after 30 mins of volume control mode MV. Subsequently, transdiaphragmatic pressure (Pdi) was calculated by means of a sniff maneuver (Pdisniff) after 30 mins of spontaneous breathing trial. Linear regression analysis and Pearson’s correlation coefficient served to assess associations.Results: Average Pdisniff was 23.3 cmH2O (standard deviation 29 cmH2O) with 3 patients presenting bilateral diaphragm palsy. Pdisniff was directly correlated with static lung elastance (r=0.69, p=0.001) while inverse correlation was found with dynamic intrinsic PEEP (r=−0.73, p=0.007). No significant correlation was found with static intrinsic PEEP (r=−0.55, p=0.06), EELV (r=−0.4, p=0.3), airway resistance (r=−0.2, p=0.54), chest wall, and total elastance (r=−0-01, p=0.96 and r=0.3, p=0.36, respectively). Significant linear inverse correlation was found between Pdisniff and the ratio betweenPdi assessed at tidal volume and Pdi sniff (r=−0.82, p=0.02).Conclusion: The causes of extreme DD in AECOPD patients who experienced NIV failure might be predominantly mechanical, driven by a severe dynamic hyperinflation that overlaps on an elastic lung substrate favoring volume overload.Keywords: acute exacerbation of COPD, non-invasive mechanical ventilation, respiratory failure, transdiaphragmatic pressure

Idiopathic pulmonary fibrosis (IPF) is a fibrotic lung disease characterized by progressive loss of lung function and poor prognosis. The so-called acute exacerbation of IPF (AE-IPF) may lead to severe hypoxemia requiring mechanical ventilation in the intensive care unit (ICU). AE-IPF shares several pathophysiological features with acute respiratory distress syndrome (ARDS), a very severe condition commonly treated in this setting. A review of the literature has been conducted to underline similarities and differences in the management of patients with AE-IPF and ARDS. During AE-IPF, diffuse alveolar damage and massive loss of aeration occurs, similar to what is observed in patients with ARDS. Differently from ARDS, no studies have yet concluded on the optimal ventilatory strategy and management in AE-IPF patients admitted to the ICU. Notwithstanding, a protective ventilation strategy with low tidal volume and low driving pressure could be recommended similarly to ARDS. The beneficial effect of high levels of positive end-expiratory pressure and prone positioning has still to be elucidated in AE-IPF patients, as well as the precise role of other types of respiratory assistance (e.g., extracorporeal membrane oxygenation) or innovative therapies (e.g., polymyxin-B direct hemoperfusion). The use of systemic drugs such as steroids or immunosuppressive agents in AE-IPF is controversial and potentially associated with an increased risk of serious adverse reactions. Common pathophysiological abnormalities and similar clinical needs suggest translating to AE-IPF the lessons learned from the management of ARDS patients. Studies focused on specific therapeutic strategies during AE-IPF are warranted.

Rhadbomyosarcoma (RMS) is the most common soft-tissue sarcoma in children and is subdivided in the embryonal (ERMS) and alveolar (ARMS) subtypes, the latter being associated with the worst prognosis. We report that sulforaphane (SFN), a broccoli-derived anticancer isothiocyanate, causes dose- and time-dependent growth inhibition and apoptosis in both ERMS and ARMS cells. In ARMS, SFN induced the modulation of expression of crucial genes and proteins: mRNA and protein levels of PAX3-FKHR, MYCN, and MET decreased, while those of p21 and TRAIL-receptor DR5 (but not DR4) increased. Since DR5 expression increased specifically in ARMS, we treated ARMS cells with TRAIL, SFN, or their combination. While ARMS cells (RH30 and RH4) proved to be TRAIL-resistant, SFN restored their sensitivity to TRAIL-induced cell-growth inhibition, leading to a stronger effect in combination with TRAIL. ARMS cells transfected with siDR5 showed that SFN-induced DR5 acts as a key regulator, being directly related to the TRAIL-induced cell-growth inhibition. The in vivo anti-tumor activity of SFN and TRAIL was evaluated in a xenograft murine model of ARMS through microPET. The results showed that the systemic treatment (3 wk) of mice with SFN or TRAIL as single agents only delayed tumor evolution, while the combined treatment of SFN and TRAIL led to tumor elimination. These findings indicate that SFN triggers the apoptotic pathway in both alveolar and embryonal rhabdomyosarcomas and that combined treatment with SFN and TRAIL might be a promising therapy for the aggressive alveolar subtype.

Chromosomal translocations involving the MLL gene on 11q23 present frequent abnormalities in pediatric, adult and therapy-related acute leukemias, and are generally associated with aggressive disease and poor prognosis. Here, we report bioluminescent acute leukemia xenograft mouse models of the most frequent and aggressive MLL-related acute leukemias (infant and adult MLL-AF9, MLL-ENL, MLL-AF4). Four acute leukemia cell lines carrying MLL-related translocations were stably transduced with a firefly luciferase transgene and injected intravenously into NOD/SCID mice. Leukemia progression was monitored by in vivo bioluminescence imaging (BLI). All mice developed MLL-related acute leukemia. The four MLL-related acute leukemia models showed a different course of infant and adult MLL-AF9 acute myeloid leukemia, and a rapid aggressiveness of MLL-ENL acute lymphoblastic leukemia and MLL-AF4 acute biphenotypic leukemia. Tissue analysis and RT-PCR of bone marrow, spleen and liver from the mice confirmed the BL results. To validate BLI for the detection of a therapeutic response, systemic treatment with an anti-luciferase-targeting siRNA (siLuc) complexed with cationic nanoparticles was administered to mice with MLL-AF4 acute lymphoblastic leukemia. The BLI signal showed a reduction following treatment with siLuc compared to the control mice. These mouse models present MLL-related acute leukemia evolution similar to the human counterparts. Moreover, they are non-invasive, rapid and sensitive models, suitable for the in vivo study of MLL-related acute leukemias. Finally, BLI showed in vivo luminescence down modulation obtained by systemic treatment with luciferase-targeting siRNA nanoparticle complexes, confirming that these MLL-related leukemia mouse models are optimal for the evaluation and selection of delivery systems for siRNA and other new biotechnological pharmaceuticals.

In this work we present an empirical study on the use of inheritance in a curated corpus of Python systems. Replicating a study preformed on Java, we analyzed a collection of 51 software systems written in Python, and investigated how inheritance is effectively used by Python developers in practice through a convenient set of inheritance metrics. Our results suggest that on average fewer classes inherit from other classes than in Java, but more classes are inherited from. We also see a sort of symmetry relating the number of ancestors and the number of descendants in each system.

ArgBP2, encoding a negative regulator of ABL, is fused to MLL in a case of infant M5 acute myeloid leukemia involving 4q35 and 11q23

TheMYCNoncogene is the major negative prognostic marker in neuroblastoma with important roles in both the pathogenesis and clinical behavior of this aggressive malignancy.MYConcogenes activate both proliferative and apoptotic cellular pathways and, accordingly, inhibition of p53-mediated apoptosis is a prerequisite for MYC-driven tumorigenesis. To identify novel transcriptional targets mediating the MYCN-dependent phenotype, we screened a MYCN-amplified neuroblastoma cell line by using chromatin immunoprecipitation (ChIP) cloning. We identified the essential p53 inhibitor and protooncogeneMDM2as a putative target. MDM2 has multiple p53-independent functions modulating cell cycle and transcriptional events. Standard ChIP with MYCN antibodies established the binding of MYCN to a consensus E-box within the humanMDM2promoter. Oligonucleotide pull-down assays further established the capacity of MYCN to bind to this promoter region, confirming the ChIP results. Luciferase reporter assays confirmed the E-box-specific, MYCN-dependent regulation of theMDM2promoter in MYCN-inducible neuroblastoma cell lines. Real-time quantitative PCR and Western blot analysis demonstrated a rapid increase in endogenousMDM2mRNA and MDM2 protein upon induction of MYCN. Targeted inhibition of MYCN in aMYCN-amplified neuroblastoma cell line resulted in decreased MDM2 expression levels with concomitant stabilization of p53 and induction of apoptosis. Our finding that MYCN directly modulates baseline MDM2 levels suggests a mechanism contributing to the pathogenesis of neuroblastoma and other MYC-driven malignancies through inhibition of MYC-stimulated apoptosis.

RHEUMATOID ARTHRITIS RELATED INTERSTITIAL LUNG DISEASE. RADIOLOGICAL PATTERNS AND CORRELATIONS WITH CLINICAL, SEROLOGICAL AND DEMOGRAPHIC FEATURES OF DISEASE

Conventional in vivo bioluminescence imaging using wild-type green-emitting luciferase is limited by absorption and scattering of the bioluminescent signal through tissues. Imaging methods using a red-shifted thermostable luciferase from Photinus pyralis were optimized to improve the sensitivity and image resolution. In vivo bioluminescence imaging performance of red- and green-emitting luciferases were compared in two different xenograft mouse models for cancer.Human hepatoblastoma cell line (HepG2) and human acute monocytic leukemia cell line (Thp1) cells were genetically engineered using retroviral vector technology to stably express the red-shifted or the wild-type green luciferase. A xenograft model of liver cancer was established by subcutaneous injection of the HepG2-engineered cells in the flank regions of mice, and a leukemia model was generated by intravenous injection of the engineered Thp1 cells. The cancer progression was monitored with an ultrasensitive charge-coupled device camera. The relative intensities of the green- and red-emitting luciferases were measured, and the resulting spatial resolutions of the images were compared. Imaging was performed with both intact and scarified live animals to quantify the absorption effects of the skin and deep tissue.The red-emitting luciferase was found to emit a bioluminescence signal with improved transmission properties compared to the green-emitting luciferase. By imaging the HepG2 models, which contained tumors just beneath the skin, before and after scarification, the percentage of light absorbed by the skin was calculated. The green bioluminescent signal was 75 +/- 8% absorbed by the skin, whereas the red signal was only 20 +/- 6% absorbed. The Thp1 model, which contains cancer cells within the bones, was likewise imaged before and after scarification to calculate the percentage of light absorbed by all tissue under the skin. This tissue was responsible for 90 +/- 5% absorption of the green signal, but only 65 +/- 6% absorption of the red signal.Two different bioluminescent mouse cancer models demonstrate the utility of a new red-shifted thermostable luciferase, Ppy RE-TS, that improved the in vivo imaging performance when compared with wild-type P. Pyralis luciferase. While wild-type luciferase is currently a popular reporter for in vivo imaging methods, this study demonstrates the potential of red-emitting firefly luciferase mutants to enhance the performance of bioluminescence imaging experiments.

The interaction with DNA of a peptide nucleic acid (PNA) oligomer (16nt) conjugated with a nuclear localization signal (NLS) peptide, which was previously found to be able to inhibit tumor cell proliferation through block of transcription of the MYCN oncogene, was studied by UV and CD spectroscopy. While data obtained by UV were not conclusive, the use of circular dichroism gave clear-cut evidence of the formation of a PNA:DNA duplex of exceptionally high stability (Tm ≥ 90°C). Using the same approach, the effect of mutations on DNA:PNA stability was evaluated, and was found in accordance with that expected for a Watson–Crick interaction. The role of the NLS peptide was evaluated by using a PNA lacking of this part, which gave rise to less stable PNA:DNA duplexes. Finally, a competition experiment carried out with a 26mer dsDNA, containing the target 16mer sequence in its middle region, in the presence of PNA-NLS gave evidence for the formation of a ternary complex at 25°, while at higher temperature, the PNA:DNA duplex and the displaced homologous DNA strand were detected. The present results support the possibility of an analogous mechanism of action of this antitumor PNA in vivo. Chirality, 2008. © 2007 Wiley-Liss, Inc.

We developed an anti-gene peptide nucleic acid (PNA) for selective inhibition of MYCN transcription in neuroblastoma cells, targeted against a unique sequence in the antisense DNA strand of exon 2 of MYCN and linked at its NH2 terminus to a nuclear localization signal peptide. Fluorescence microscopy showed specific nuclear delivery of the PNA in six human neuroblastoma cell lines: GI-LI-N and IMR-32 (MYCN-amplified/overexpressed); SJ-N-KP and NB-100 (MYCN-unamplified/low-expressed); and GI-CA-N and GI-ME-N (MYCN-unamplified/unexpressed). Antiproliferative effects were observable at 24 hours (GI-LI-N, 60%; IMR-32, 70%) and peaked at 72 hours (GI-LI-N, 80%; IMR-32, 90%; SK-N-KP, 60%; NB-100, 50%); no reduction was recorded for GI-CA-N and GI-ME-N (controls). In MYCN-amplified/overexpressed IMR-32 cells and MYCN-unamplified/low-expressed SJ-N-KP cells, inhibition was recorded of MYCN mRNA (by real-time PCR) and N-Myc (Western blotting); these inhibitory effects increased over 3 days after single treatment in IMR-32. Anti-gene PNA induced G1-phase accumulation (39–53%) in IMR-32 and apoptosis (56% annexin V–positive cells at 24 hours in IMR-32 and 22% annexin V–positive cells at 48 hours in SJ-N-KP). Selective activity of the PNA was shown by altering three point mutations, and by the observation that an anti-gene PNA targeted against the noncoding DNA strand did not exert any effect. These findings could encourage research into development of an anti-gene PNA–based tumor-specific agent for neuroblastoma (and other neoplasms) with MYCN expression.

We developed an antisense peptide nucleic acid (PNA) targeted against a unique sequence in the terminus of the 5'-UTR of N-myc, designed for selective inhibition of NMYC in neuroblastoma cells. Fluorescent microscopy showed carrier-free delivery of the PNA to two human neuro-blastoma cell lines: GI-LI-N (N-myc-amplified) and GI-CA-N (N-myc-unamplified). Only in the former, PNA treatment determined 70% cell-viability reduction (at 48 h). In N-myc-amplified GI-LI-N cells, the PNA determined NMYC-translation inhibition (Western blotting), accumulation of cells in G1, induction of differentiation and apoptosis. Selectivity of the PNA was demonstrated by altering three point mutations. These findings should encourage development of a PNA-based tumor-specific agent for neuroblastoma (or other neoplasms) with N-myc overexpression.

Thrombocytopenia with absent radii (TAR) is a rare autosomal recessive disease characterized by hypomegakaryocytic thrombocytopenia and bilateral radial aplasia. We performed mutational screening of coding and promoter regions of the c-mpl gene, encoding thrombopoietin (TPO) receptor, by sequence analysis in four unrelated patients affected by TAR syndrome. Our results indicate that c-mpl gene mutations are not a common cause of thrombocytopenia in TAR syndrome.

In the last years we have seen the rise and the fall of many version control systems. These systems collect a large amount of data spanning from the path of the files involved in changes to the exact text changed in every file. This data can be exploited to produce an overview about how the system changed over time and evolved. We have developed a tool, called VCS-Analyzer, to use this information, both for data retrieval and analysis tasks. Currently, VCS-Analyzer implements six different analyses: two based on source code for the computation of metrics and the detection of code smells, and four original analysis based on repositories metadata, which are based on the concepts of Repository Metrics and Code Repository Smells. In this paper, we describe one smell and two metrics we have defined for source code repositories analysis.