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Background : We compared the performance of 21 different assays performed by the Wantai Wan200+ (Wantai BioPharm, Beijing, China) with respect to other methods in use at the University Hospital of Padova (AOPD), Italy. Methods : The plasma (P) or serum (S) of 5027 leftover samples, collected from May to Sept 2023, was either analyzed or frozen at -20 °C. Beckman DXI800 (DXI), Roche Cobas 8000 e801 (RC), Snibe Maglumi 4000 plus (SM), DiaSorin Liaison XL (DL) and Binding Site Optilite (BS) equipment were used at the AOPD. P-procalcitonin (PCT), DXI; P-Troponin I (TnI), DXI; S-CA125, DXI; S-free PSA (f-PSA), DXI; S-total PSA (t-PSA), DXI; S-IL6, SM; P-Troponin T (TnT), RC; P-NT-proBNP, RC; P-Neuron-Specific Enolase (NSE), RC; S-CA15-3, DL; S-CA19-9, DL; S-AFP, DL; and S-CEA, DL were tested in fresh samples. P-Myoglobin (Myo), DXI; P-Cyfra21-1, RC; S-β2 microglobulin (B2MIC), BS; S-HE4, SM; S-PGI, SM; S-PGII, SM; S-CA72-4, SM; and S-CA50, SM were analyzed in frozen and thawed samples. Bland-Altman (BA), Passing-Bablok (PB) and Cohen's Kappa (CKa) metrics were used as statistics. Results : An excellent comparability profile was found for 11 analytes. For example, the t-PSA CKa was 0.94 (95%CI: 0.90 to 0.98), and the PB slope and intercept were 1.02 (95%CI: 0.99 to 1.03) and 0.02 (95%CI: 0.01 to 0.03), respectively; the BA bias was 2.25 (95%CI: -0.43 to 4.93). Ten tested measurands demonstrated a suboptimal comparability profile. Biological variation in EFLM (EuBIVAS) performance specifications was evaluated to assess the clinical relevance of measured biases. Conclusions : Evaluation of the Wantai Wan200+'s performance suggests that between-method differences did not exceed the calculated bias. Metrological traceability may influence the comparisons obtained for some measurands.

Histone chaperones control nucleosome density and chromatin structure. In yeast, the H3-H4 chaperone Spt2 controls histone deposition at active genes but its roles in metazoan chromatin structure and organismal physiology are not known. Here we identify the Caenorhabditis elegans ortholog of SPT2 (CeSPT-2) and show that its ability to bind histones H3-H4 is important for germline development and transgenerational epigenetic gene silencing, and that spt-2 null mutants display signatures of a global stress response. Genome-wide profiling showed that CeSPT-2 binds to a range of highly expressed genes, and we find that spt-2 mutants have increased chromatin accessibility at a subset of these loci. We also show that SPT2 influences chromatin structure and controls the levels of soluble and chromatin-bound H3.3 in human cells. Our work reveals roles for SPT2 in controlling chromatin structure and function in Metazoa., (© 2024. The Author(s).)

Saliva is a promising matrix with several purposes. Our aim is to verify if salivary anti-SARS-CoV-2 antibody determination is suitable for monitoring immune responses. One hundred eighty-seven subjects were enrolled at University-Hospital Padova: 105 females (56.1%) and 82 males (43.9%), 95 (50.8%) children and 92 (49.2%) adults. Subjects self-collected saliva using Salivette; nineteen subjects collected three different samples within the day. A serum sample was obtained for all individuals. The N/S anti-SARS-CoV-2 salivary IgG (sal-IgG) and serum anti-SARS-CoV-2 S-RBD IgG (ser-IgG) were used for determining anti-SARS-CoV-2 antibodies. The mean (min-max) age was 9.0 (1-18) for children and 42.5 (20-61) for adults. Of 187 samples, 63 were negative for sal-IgG (33.7%), while 7 were negative for ser-IgG (3.7%). Spearman's correlation was 0.56 ( p < 0.001). Sal-IgG and ser-IgG levels were correlated with age but not with gender, comorbidities, prolonged therapy, previous SARS-CoV-2 infection, or time from last COVID-19 infection/vaccination. The repeatability ranged from 23.8% (7.4 kAU/L) to 4.0% (3.77 kAU/L). The linearity of the assay was missed in 4/6 samples. No significant intrasubject differences were observed in sal-IgG across samples collected at different time points. Sal-IgG has good agreement with ser-IgG. Noninvasive saliva collection represents an alternative method for antibody measurement, especially in children.

Objectives: In this study, we describe the analytical and clinical performances of the SNIBE Maglumi SARS-CoV-2 antigen fully-automated chemiluminescent immunoassay (MAG-CLIA) on salivary samples., Methods: Limit of detection (LOD), linearity and precision were tested for values close to or below the declared LOD. Clinical performance of MAG-CLIA was evaluated on leftover salivary samples from the healthcare workers (HCW) surveillance program, at the University-Hospital of Padova. Salivary samples were analyzed by Lumipulse G SARS-CoV-2 Ag, and in case where the values exceeded 0.41 ng/L, further testing was conducted using TaqPathTM COVID-19 RT-PCR (Applied Biosystems, Thermo Fisher Scientific)., Results: The estimated MAG-CLIA LOD was 3 ng/L, with repeatability of 7.5 %. Good linearity was demonstrated by diluting two samples at 52.7 ng/L and 211.4 ng/L. Of the 228 HCW samples, 59/228 (25.9 %) were positive, 169/228 (74.1 %) were negative. MAG-CLIA SARS-CoV-2 sAg median level (and interquartile range [IQR]) was 5.03 ng/L (<0.001-35.8 ng/L) for positive and <0.001 ng/L (<0.001 ng/L) for negative samples. MAG-CLIA AUC was 0.795 (95 % CI: 0.720-0.871). Using the best cut-off, 3.5 ng/L, sensitivity and specificity were 57.1 % (95 % CI: 42.2-71.2 %) and 97.0 % (95 % CI: 93.2-99.0 %), respectively. The agreement with the molecular assay was 88.1 % (Cohen's kappa 0.606 [SE=0.066, p<0.001])., Conclusions: The analytical performances of MAG-CLIA are satisfactory, also when values below LOD were tested. In saliva samples, although specificity was elevated, clinical performance was not comparable with that on nasopharyngeal swabs (NPS)., (© 2023 the author(s), published by De Gruyter, Berlin/Boston.)

Embryos across metazoan lineages can enter reversible states of developmental pausing, or diapause, in response to adverse environmental conditions. The molecular mechanisms that underlie this remarkable dormant state remain largely unknown. Here we show that N 6 -methyladenosine (m 6 A) RNA methylation by Mettl3 is required for developmental pausing in mouse blastocysts and embryonic stem (ES) cells. Mettl3 enforces transcriptional dormancy through two interconnected mechanisms: (1) it promotes global mRNA destabilization and (2) it suppresses global nascent transcription by destabilizing the mRNA of the transcriptional amplifier and oncogene N-Myc, which we identify as a crucial anti-pausing factor. Knockdown of N-Myc rescues pausing in Mettl3 -/- ES cells, and forced demethylation and stabilization of Mycn mRNA in paused wild-type ES cells largely recapitulates the transcriptional defects of Mettl3 -/- ES cells. These findings uncover Mettl3 as a key orchestrator of the crosstalk between transcriptomic and epitranscriptomic regulation during developmental pausing, with implications for dormancy in adult stem cells and cancer., (© 2023. The Author(s), under exclusive licence to Springer Nature Limited.)

Objectives: The SARS-CoV-2 immune response is mediated by both humoral and cellular immunity. In this study, SARS-CoV-2 specific cellular immunity was tested by a novel direct real-time PCR (dRT-PCR) assay, targeting mRNA of CXCL10 , and compared with respect to an ELISA measuring interferon gamma (IFN-γ) release., Methods: Whole blood (Li-He) and serum samples were collected from 92 healthcare workers (HCW), with three doses of homologous (Pfizer/BioNTech, n=74) or heterologous (Pfizer/BioNTech and Vaxzevria or Moderna, n=18) vaccinations. Li-He samples were incubated with SCV2 PANEL-1-T-ACTIVATION (Hyris srl, Lodi, Italy), or CoV-2 IGRA TUBE ELISA (Euroimmune, Lubeck, Germany). CXCL10 mRNA expression was analyzed by bCube/bApp (Hyris), while IFN-γ was evaluated by quant-T-Cell SARS-CoV-2 ELISA (Euroimmune). Anti-SARS-CoV-2 S-RBD IgG levels were measured in sera using a CLIA assay (Snibe, Shenzen, China)., Results: Imprecision of dRT-PCR assay was found to be satisfactory, and the two methods for measuring T cell immunity to SARS-CoV-2 peptides agreed in 82/87 (94.2%) of results. At qualitative dRT-PCR analyses, 81 subjects (93.2%) resulted as reactive to SARS-CoV-2 peptides, 3 (3.4%) were borderline and 3 were negative (3.4%). At univariate and multivariate analyses of quantitative dRT-PCR mRNA of CXCL10 and IFN-γ release results showed no difference between HCW with previous infection, homologous/heterologous vaccination, or demographical features. Anti-SARS-CoV-2 S-RBD IgG was associated with the previous infection and the time between the last vaccination or positivity., Conclusions: Direct RT-PCR appeared accurate for determining the presence or absence of immunoreactivity of SARS-CoV-2 specific T cells, especially when rapid analyses are required, such as for organ transplantation., (© 2023 Walter de Gruyter GmbH, Berlin/Boston.)

The paper reports the results of the analyses on mortar-based materials from the Roman theatre of Aquileia (Friuli Venezia Giulia, Northern Italy), recently dated between the mid-1st Century BCE and the mid-1st Century CE. Samples were characterized by Polarized Light Microscopy on thin sections (PLM), Scanning Electron Microscopy with Energy Dispersive Spectroscopy (SEM-EDS) and Quantitative Phase Analysis by X-Ray Powder Diffraction (QPA-XRPD). Pyroclastic aggregates (mainly pumices and scattered tuffs), incompatible with the regional geology, were found in two samples from the preparation layers of the ground floor of the building. Their provenance was determined by means of QPA-XRPD, SEM-EDS, X-Ray Fluorescence (XRF) and Laser-Ablation Inductively-Coupled-Plasma Mass-Spectrometry (LA-ICP-MS). Mineralogical and geochemical analyses demonstrated their provenance from the Bay of Naples, thus recognizing them as pulvis puteolana, a type of pozzolanic aggregate outcropping around the modern town of Pozzuoli and prescribed by Vitruvius (De Architectura, 2.6.1) in mortar-based materials to strengthen masonries and produce hydraulic concrete for harbor piers. This evidence represents the oldest analytically-established case of pulvis puteolana exploitation in Northern Italy up to now, and an early use of the material out of Campania adapted for civil constructions in a non-strictly maritime-related environment. Indeed, the theatre was built in the low-lying Aquileia's deltaic plain, prone to water infiltrations that are typical in lagoon-like environments. The data highlight the craftsmen's resilience in adapting and reinterpreting the traditional use of the Neapolitan volcanic materials to deal with the geomorphological challenges of Aquileia's lowland., (© 2023. The Author(s).)

A challenge during development is to ensure lineage segregation while preserving plasticity. Using pluripotency progression as a paradigm, we review how developmental transitions are coordinated by redeployments, rather than global resettings, of cellular components. We highlight how changes in response to extrinsic cues (FGF, WNT, Activin/Nodal, Netrin-1), context- and stoichiometry-dependent action of transcription factors (Oct4, Nanog) and reconfigurations of epigenetic regulators (enhancers, promoters, TrxG, PRC) may confer robustness to naïve to primed pluripotency transition. We propose the notion of Molecular Versatility to regroup mechanisms by which molecules are repurposed to exert different, sometimes opposite, functions in close stem cell configurations., (© 2023. The Author(s).)

Background: Lung cancer represents a heterogeneous group of neoplasms, with the highest frequency and mortality in both sexes combined. In a clinical scenario characterized by the widespread of multidetector-row spiral CT, core-needle biopsy under tomographic guidance is one of the main and safest methods to obtain tissue specimens, even though there are relatively high rates of pneumothorax (0-60% incidence) and pulmonary hemorrhage (4-27% occurrence rates). The aim of this retrospective study is to assess the diagnostic accuracy of CT-guided core-needle biopsy in the diagnosis of primary lung malignancies and to compare our results with evidence from the literature., Materials and Methods: Our analysis included 350 thoracic biopsies, performed from 2017 to 2022 with a 64-row CT guidance and 16/18 G needles mounted on a biopsy gun. We included in the final cohort all samples with evidence of primary lung malignancies, precursor lesions, and atypia, as well as inconclusive and negative diagnoses., Results: There was sensitivity of 90.07% (95% CI 86.05-93.25%), accuracy of 98.87% (95% CI 98.12-99.69%), positive predictive value of 100%, and negative value of 98.74% (95% CI 98.23-99.10%). Specificity settled at 100% (93.84-100%). The AUC was 0.952 (95% CI 0.924-0.972). Only three patients experienced major complications after the procedure. Among minor complications, longer distances from the pleura, the presence of emphysema, and the lower dimensions of the lesions were correlated with the development of pneumothorax after the procedure, while longer distances from the pleura and the lower dimensions of the lesions were correlated with intra-alveolar hemorrhage. Immunohistochemistry analysis was performed in 51% of true positive cases, showing TTF-1, CK7, and p40 expression, respectively, in 26%, 24%, and 10% of analyzed samples., Conclusions: The CT-guided thoracic core-needle biopsy is an extremely accurate and safe diagnostic procedure for the histological diagnosis of lung cancer, a first-level interventional radiology exam for peripheral and subpleural lesions of the lung, which is also able to provide adequate samples for advanced pathologic assays (e.g., FISH, PCR) to assess molecular activity and genetic sequencing.

Coordinated changes of cellular plasticity and identity are critical for pluripotent reprogramming and oncogenic transformation. However, the sequences of events that orchestrate these intermingled modifications have never been comparatively dissected. Here, we deconvolute the cellular trajectories of reprogramming (via Oct4/Sox2/Klf4/c-Myc) and transformation (via Ras/c-Myc) at the single-cell resolution and reveal how the two processes intersect before they bifurcate. This approach led us to identify the transcription factor Bcl11b as a broad-range regulator of cell fate changes, as well as a pertinent marker to capture early cellular intermediates that emerge simultaneously during reprogramming and transformation. Multiomics characterization of these intermediates unveiled a c-Myc/Atoh8/Sfrp1 regulatory axis that constrains reprogramming, transformation and transdifferentiation. Mechanistically, we found that Atoh8 restrains cellular plasticity, independent of cellular identity, by binding a specific enhancer network. This study provides insights into the partitioned control of cellular plasticity and identity for both regenerative and cancer biology., (© 2022. The Author(s).)

Alternative splicing is central to metazoan gene regulation, but the regulatory mechanisms are incompletely understood. Here, we show that G-quadruplex (G4) motifs are enriched ~3-fold near splice junctions. The importance of G4s in RNA is emphasised by a higher enrichment for the non-template strand. RNA-seq data from mouse and human neurons reveals an enrichment of G4s at exons that were skipped following depolarisation induced by potassium chloride. We validate the formation of stable RNA G4s for three candidate splice sites by circular dichroism spectroscopy, UV-melting and fluorescence measurements. Moreover, we find that sQTLs are enriched at G4s, and a minigene experiment provides further support for their role in promoting exon inclusion. Analysis of >1,800 high-throughput experiments reveals multiple RNA binding proteins associated with G4s. Finally, exploration of G4 motifs across eleven species shows strong enrichment at splice sites in mammals and birds, suggesting an evolutionary conserved splice regulatory mechanism., (© 2022. The Author(s).)

The human terminal uridyl transferases TUT4 and TUT7 (TUT4/7) catalyze the additions of uridines at the 3' end of RNAs, including the precursors of the tumor suppressor miRNA let-7 upon recruitment by the oncoprotein LIN28A. As a consequence, let-7 family miRNAs are down-regulated. Disruption of this TUT4/7 activity inhibits tumorigenesis. Hence, targeting TUT4/7 could be a potential anticancer therapy. In this study, we investigate TUT4/7-mediated RNA regulation in two cancer cell lines by establishing catalytic knockout models. Upon TUT4/7 mutation, we observe a significant reduction in miRNA uridylation, which results in defects in cancer cell properties such as cell proliferation and migration. With the loss of TUT4/7-mediated miRNA uridylation, the uridylated miRNA variants are replaced by adenylated isomiRs. Changes in miRNA modification profiles are accompanied by deregulation of expression levels in specific cases. Unlike let-7s, most miRNAs do not depend on LIN28A for TUT4/7-mediated regulation. Additionally, we identify TUT4/7-regulated cell-type-specific miRNA clusters and deregulation in their corresponding mRNA targets. Expression levels of miR-200c-3p and miR-141-3p are regulated by TUT4/7 in a cancer cell-type-specific manner. Subsequently, BCL2, which is a well-established target of miR-200c is up-regulated. Therefore, TUT4/7 loss causes deregulation of miRNA-mRNA networks in a cell-type-specific manner. Understanding of the underlying biology of such cell-type-specific deregulation will be an important aspect of targeting TUT4/7 for potential cancer therapies., (© 2022 Medhi et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.)

Early in development, placental and marsupial mammals harbouring at least two X chromosomes per nucleus are faced with a choice that affects the rest of their lives: which of those X chromosomes to transcriptionally inactivate. This choice underlies phenotypical diversity in the composition of tissues and organs and in their response to the environment, and can determine whether an individual will be healthy or affected by an X-linked disease. Here, we review our current understanding of the process of choice during X-chromosome inactivation and its implications, focusing on the strategies evolved by different mammalian lineages and on the known and unknown molecular mechanisms and players involved.

The collection and assessment of individual case safety reports (ICSRs) is important to detect unknown adverse drug reactions particularly in the first decade after approval of new chemical entities. However, regulations require that these activities are routinely undertaken for all medicinal products, including older medicines such as generic medicinal products with a well-established safety profile. For the latter, the risk management plans no longer contain important risks, considered important safety concerns, on the basis that routine pharmacovigilance activity would not allow their further characterisation. Society assumes that unexpected adverse reactions causally related to pharmacological activity are very unlikely to be detected for such well-established medicines, but important risks can still occur. For these products, a change in the safety profile which is brand or source specific and usually local in nature, associated with failures with the adequate control of quality of manufacturing or distribution are important safety issues. These may be the consequence of manufacturing and pharmacovigilance quality systems that are not fully integrated over the product life cycle (e.g. inadequate control of quality defects affecting one or multiple batches; inadequate impact assessment of change/variation of manufacturing, quality control testing, storage and distribution processes; inadequate control over the distribution channels including the introduction of counterfeit or falsified products into the supply chain). Drug safety hazards caused by the above-mentioned issues have been identified with different products and formulations, from small molecules to complex molecules such as biological products extracted from animal sources, biosimilars and advanced therapy medicinal products. The various phases of the drug manufacturing and distribution of pharmaceutical products require inputs from pharmacovigilance to assess any effects of quality-related issues and to identify proportionate risk minimisation measures that often have design implications for a medicine which requires a close link between proactive vigilance and good manufacturing practice. To illustrate our argument for closer organisational integration, some examples of drug safety hazards originating from quality, manufacturing and distribution issues are discussed., Plain Language Summary: Monitoring the manufacturing and quality of medicines: the fundamental task of pharmacovigilance Pharmacovigilance is the science relating to the collection, detection, assessment, monitoring, and prevention of adverse reactions with pharmaceutical products. The collection and assessment of adverse reactions are particularly important in the first decade after marketing authorisation of a drug as the information gathered in this period could help, for example, to identify complications from its use which were unknown before its commercialization. However, when it comes to medicines that have been on the market for a long time there is general acceptance that their safety profile is already well-established and unknown adverse reactions unlikely to occur. Nevertheless, even older medicines, such as generic drugs, can generate new risks. For these drugs a change in the safety profile could be the result of inadequate control of their quality, manufacturing and distribution systems. To overcome such an obstacle, it is necessary to fully integrate manufacturing and pharmacovigilance quality systems in the medicine life-cycle. This could help detect safety hazards and prevent the development of new complications which may arise due to the poor quality of a drug. Pharmacovigilance activities should indeed be included in all phases of the drugs' manufacturing and distribution process, regardless of their chemical complexity to detect quality-related matters in good time and reduce the risk of safety concerns to a minimum., Competing Interests: Conflict of interest statement: The authors declared the following potential conflicts of interest with respect to the research, authorship and/or publication of this article: The Editor-in-Chief of Therapeutic Advances in Drug Safety is an author of this paper; therefore, the peer-review process was managed by alternative members of the Board and the submitting Editor had no involvement in the decision-making process., (© The Author(s), 2021.)

Hemp cultivation is living a period of renewed interest worldwide after long years of opposition and abandonment. The European Union (EU) allows and subsidizes the growing of fiber and oilseed cultivars of Cannabis sativa L. with respect to the THC content limit of 0.2%. The EU method for the quantitative determination of Δ9-tetrahydrocannabinol (THC) content in hemp varieties provides to apply a tolerance of 0.03 g of THC per 100 g of sample concerning compliance assessment to that limit. However, the method does not report any precision data, especially useful as a function of THC content to evaluate measurement uncertainty and therefore to establish the conformity of hemp at different THC legal limits. Measurement uncertainty of the method by both bottom-up and top-down approach, besides repeatability and reproducibility, was investigated and estimated in the THC concentration range 0.2-1.0%, which includes the different legal limits set out for hemp around the world. We proposed Decision Rules for conformity of hemp showing that a non-compliant declaration beyond reasonable doubt should be stated when the THC content, as a mean result on a duplicate analysis, exceeds the limit by at least 11-15%, depending on THC limit. We highlighted other issues concerning practical aspects of hemp analysis, from sampling to evaluation of results, as well as the need to carry out collaborative studies on the EU method.

During the immune response, activation of the secretory pathway is key to mounting an effective response, while gauging its output is important to maintain cellular homeostasis. The Exo70 subunit of the exocyst functions as a spatiotemporal regulator by mediating numerous interactions with proteins and lipids. However, a molecular understanding of the exocyst regulation remains challenging. We show that, in Arabidopsis thaliana, Exo70B2 behaves as a bona fide exocyst subunit. Conversely, treatment with the salicylic acid (SA) defence hormone analog benzothiadiazole (BTH), or the immunogenic peptide flg22, induced Exo70B2 transport into the vacuole. We reveal that Exo70B2 interacts with AUTOPHAGY-RELATED PROTEIN 8 (ATG8) via two ATG8-interacting motives (AIMs) and its transport into the vacuole is dependent on autophagy. In line with its role in immunity, we discovered that Exo70B2 interacted with and was phosphorylated by the kinase MPK3. Mimicking phosphorylation had a dual impact on Exo70B2: first, by inhibiting localization at sites of active secretion, and second, it increased the interaction with ATG8. Phosphonull variants displayed higher effector-triggered immunity (ETI) and were hypersensitive to BTH, which induce secretion and autophagy. Our results suggest a molecular mechanism by which phosphorylation diverts Exo70B2 from the secretory into the autophagy pathway for its degradation, to dampen secretory activity., (© American Society of Plant Biologists 2020. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

PIWI-interacting RNAs (piRNAs) are genome-encoded small RNAs that regulate germ cell development and maintain germline integrity in many animals. Mature piRNAs engage Piwi Argonaute proteins to silence complementary transcripts, including transposable elements and endogenous genes. piRNA biogenesis mechanisms are diverse and remain poorly understood. Here, we identify the RNA polymerase II (RNA Pol II) core subunit RPB-9 as required for piRNA-mediated silencing in the nematode Caenorhabditis elegans. We show that rpb-9 initiates heritable piRNA-mediated gene silencing at two DNA transposon families and at a subset of somatic genes in the germline. We provide genetic and biochemical evidence that RPB-9 is required for piRNA biogenesis by recruiting the Integrator complex at piRNA genes, hence promoting transcriptional termination. We conclude that, as a part of its rapid evolution, the piRNA pathway has co-opted an ancient machinery for high-fidelity transcription., (© 2021 The Authors. Published under the terms of the CC BY 4.0 license.)

Patients and prescribers need to be aware of adverse drug events to minimize the risk of their occurrence and the severity with which they appear. However, numerous studies show that being informed about adverse events can increase the possibility of suffering from them. Patients tend to overestimate the likelihood of experiencing the adverse events included in the label, and this can contribute to worsening the negative expectations which are at the root of the nocebo effect. In fact, patients can become anxious after reading the undesirable effects section of the leaflet and, in addition to suffering from the nocebo effect, might not take a drug they could benefit from due to the fear of experiencing adverse events. In addition, patients' attention can focus towards non-specific symptoms of daily living that can be misattributed to the drug and included in the labelling. This article proposes a number of suggestions to reduce the abovementioned unintended effects associated with labelling, namely, an increased focus on the excess risk of experiencing adverse events rather than crude incidence, using attribute framing to help patients to better understand the risk of experiencing adverse events, dividing the undesirable effect section of the leaflet into subsections according to the level of evidence supporting causal relationships and, finally, restricting the addition of non-specific adverse events that are also symptoms of daily living to only those where there is enough evidence to show they have been caused by the drug. More studies on how to minimize the nocebo effect induced by adverse event information should be performed, and these should be done in collaboration with health authorities, to reach a shared consensus on how to better present adverse event information in the label.

Elderly patients are the main users of drugs and they differ from younger patients. They are a heterogeneous population that cannot be defined only by age but should rather be stratified based on their frailty. The elderly have distinctive pharmacokinetic and pharmacodynamic characteristics, are frequently polymorbid, and are therefore treated with multiple drugs. They may experience adverse reactions that are difficult to recognize, since some of them present non-specific symptoms easily mistaken for geriatric conditions. Paradoxically, the elderly are underrepresented in clinical trials, especially the frail individuals whose pharmacological response and expected treatment outcome can be different from those of non-frail patients. This means that the benefit-risk balance of drugs used in frail elderly patients is frequently unknown. We present some proposals to overcome the barriers preventing the enrollment of frail elderly patients in clinical trials, and strategies for monitoring their therapy to minimize the risk of adverse reactions. Automated alerts for drug and drug-disease interactions could help appropriate prescribing but should flag only clinically relevant interactions. Pharmaceutical forms should be designed to allow easy dose adjustment and, together with packaging and labeling, should account for the physical and cognitive limitations of frail elderly patients. Aggregate pharmacovigilance reports should summarize the safety profile in the elderly, but rather than presenting the results by age they should focus on patients' frailty, perhaps using the number of comorbidities as a proxy when information on frailty is not available.

In mouse embryonic stem cells (mESCs), chemical blockade of Gsk3α/β and Mek1/2 (2i) instructs a self-renewing ground state whose endogenous inducers are unknown. Here we show that the axon guidance cue Netrin-1 promotes naive pluripotency by triggering profound signalling, transcriptomic and epigenetic changes in mESCs. Furthermore, we demonstrate that Netrin-1 can substitute for blockade of Gsk3α/β and Mek1/2 to sustain self-renewal of mESCs in combination with leukaemia inhibitory factor and regulates the formation of the mouse pluripotent blastocyst. Mechanistically, we reveal how Netrin-1 and the balance of its receptors Neo1 and Unc5B co-regulate Wnt and MAPK pathways in both mouse and human ESCs. Netrin-1 induces Fak kinase to inactivate Gsk3α/β and stabilize β-catenin while increasing the phosphatase activity of a Ppp2r2c-containing Pp2a complex to reduce Erk1/2 activity. Collectively, this work identifies Netrin-1 as a regulator of pluripotency and reveals that it mediates different effects in mESCs depending on its receptor dosage, opening perspectives for balancing self-renewal and lineage commitment.

Background: Encased cantilevers are novel force sensors that overcome major limitations of liquid scanning probe microscopy. By trapping air inside an encasement around the cantilever, they provide low damping and maintain high resonance frequencies for exquisitely low tip-sample interaction forces even when immersed in a viscous fluid. Quantitative measurements of stiffness, energy dissipation and tip-sample interactions using dynamic force sensors remain challenging due to spurious resonances of the system. Results: We demonstrate for the first time electrostatic actuation with a built-in electrode. Solely actuating the cantilever results in a frequency response free of spurious peaks. We analyze static, harmonic, and sub-harmonic actuation modes. Sub-harmonic mode results in stable amplitudes unaffected by potential offsets or fluctuations of the electrical surface potential. We present a simple plate capacitor model to describe the electrostatic actuation. The predicted deflection and amplitudes match experimental results within a few percent. Consequently, target amplitudes can be set by the drive voltage without requiring calibration of optical lever sensitivity. Furthermore, the excitation bandwidth outperforms most other excitation methods. Conclusion: Compatible with any instrument using optical beam deflection detection electrostatic actuation in encased cantilevers combines ultra-low force noise with clean and stable excitation well-suited for quantitative measurements in liquid, compatible with air, or vacuum environments.

Accumulation of the Xist long noncoding RNA (lncRNA) on one X chromosome is the trigger for X chromosome inactivation (XCI) in female mammals. Xist expression, which needs to be tightly controlled, involves a cis-acting region, the X-inactivation center (Xic), containing many lncRNA genes that evolved concomitantly to Xist from protein-coding ancestors through pseudogeneization and loss of coding potential. Here, we uncover an essential role for the Xic-linked noncoding gene Ftx in the regulation of Xist expression. We show that Ftx is required in cis to promote Xist transcriptional activation and establishment of XCI. Importantly, we demonstrate that this function depends on Ftx transcription and not on the RNA products. Our findings illustrate the multiplicity of layers operating in the establishment of XCI and highlight the diversity in the modus operandi of the noncoding players., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Neodymium-iron-boron (NdFeB) magnets offer the strongest magnetic field per unit volume, and thus, are widely used in clean energy applications such as electric vehicle motors. However, rare earth elements (REEs), which are the key materials for creating NdFeB magnets, have been subject to significant supply uncertainty in the past decade. NdFeB magnet-to-magnet recycling has recently emerged as a promising strategy to mitigate this supply risk. This paper assesses the environmental footprint of NdFeB magnet-to-magnet recycling by directly measuring the environmental inputs and outputs from relevant industries and compares the results with production from "virgin" materials, using life cycle assessments. It was found that magnet-to-magnet recycling lowers environmental impacts by 64-96%, depending on the specific impact categories under investigation. With magnet-to-magnet recycling, key processes that contribute 77-95% of the total impacts were identified to be (1) hydrogen mixing and milling (13-52%), (2) sintering and annealing (6-24%), and (3) electroplating (6-75%). The inputs from industrial sphere that play key roles in creating these impacts were electricity (24-93% of the total impact) and nickel (5-75%) for coating. Therefore, alternative energy sources such as wind and hydroelectric power are suggested to further reduce the overall environmental footprint of NdFeB magnet-to-magnet recycling.

Spatiotemporal variations of neurogenesis are thought to account for the evolution of brain shape. In the dorsal telencephalon (pallium) of vertebrates, it remains unresolved which ancestral neurogenesis mode prefigures the highly divergent cytoarchitectures that are seen in extant species. To gain insight into this question, we developed genetic tools to generate here the first 4-dimensional (3D + birthdating time) map of pallium construction in the adult teleost zebrafish. Using a Tet-On-based genetic birthdating strategy, we identify a "sequential stacking" construction mode where neurons derived from the zebrafish pallial germinal zone arrange in outside-in, age-related layers from a central core generated during embryogenesis. We obtained no evidence for overt radial or tangential neuronal migrations. Cre-lox-mediated tracing, which included following Brainbow clones, further demonstrates that this process is sustained by the persistent neurogenic activity of individual pallial neural stem cells (NSCs) from embryo to adult. Together, these data demonstrate that the spatiotemporal control of NSC activity is an important driver of the macroarchitecture of the zebrafish adult pallium. This simple mode of pallium construction shares distinct traits with pallial genesis in mammals and non-mammalian amniotes such as birds or reptiles, suggesting that it may exemplify the basal layout from which vertebrate pallial architectures were elaborated., (Copyright © 2017 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Crosstalk between posttranslational modifications, such as ubiquitination and phosphorylation, play key roles in controlling the duration and intensity of signaling events to ensure cellular homeostasis. However, the molecular mechanisms underlying the regulation of negative feedback loops remain poorly understood. Here, we uncover a pathway in Arabidopsis thaliana by which a negative feedback loop involving the E3 ubiquitin ligase PUB22 that dampens the immune response is triggered by MITOGEN-ACTIVATED PROTEIN KINASE3 (MPK3), best known for its function in the activation of signaling. PUB22's stability is controlled by MPK3-mediated phosphorylation of residues localized in and adjacent to the E2 docking domain. We show that phosphorylation is critical for stabilization by inhibiting PUB22 oligomerization and, thus, autoubiquitination. The activity switch allows PUB22 to dampen the immune response. This regulatory mechanism also suggests that autoubiquitination, which is inherent to most single unit E3s in vitro, can function as a self-regulatory mechanism in vivo., (© 2017 The author(s).)

Ubiquitination is a central posttranslational modification that impinges on the fate of proteins. While attachment of K48-linked chains onto soluble proteins marks them for proteolysis via the 26S proteasome, mono-ubiquitination or K63-linked chains result in the endocytosis and sorting through the endomembrane system of integral membrane proteins, such as pattern recognition receptors. In vitro ubiquitination assays allow the biochemical analysis of all individual components of the ubiquitination machinery and its potential substrates. Here, we describe how to reconstitute the ubiquitination cascade in vitro and detail different variations of the assay, the required controls and how to interpret the obtained results.

Neurogenesis in the post-embryonic vertebrate brain varies in extent and efficiency between species and brain territories. Distinct neurogenesis modes may account for this diversity, and several neural progenitor subtypes, radial glial cells (RG) and neuroepithelial progenitors (NE), have been identified in the adult zebrafish brain. The neurogenic sequences issued from these progenitors, and their contribution to brain construction, remain incompletely understood. Here we use genetic tracing techniques based on conditional Cre recombination and Tet-On neuronal birthdating to unravel the neurogenic sequence operating from NE progenitors in the zebrafish post-embryonic optic tectum. We reveal that a subpopulation of her5-positive NE cells of the posterior midbrain layer stands at the top of a neurogenic hierarchy involving, in order, the amplification pool of the tectal proliferation zone (TPZ), followed by her4-positive RG cells with transient neurogenic activity. We further demonstrate that the adult her5-positive NE pool is issued in lineage from an identically located NE pool expressing the same gene in the embryonic neural tube. Finally, we show that these features are reminiscent of the neurogenic sequence and embryonic origin of the her9-positive progenitor NE pool involved in the construction of the lateral pallium at post-embryonic stages. Together, our results highlight the shared recruitment of an identical neurogenic strategy by two remote brain territories, where long-lasting NE pools serve both as a growth zone and as the life-long source of young neurogenic RG cells., (Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.)

X-chromosome inactivation (XCI) is a chromosome-wide regulatory process that ensures dosage compensation for X-linked genes in Theria. XCI is established during early embryogenesis and is developmentally regulated. Different XCI strategies exist in mammalian infraclasses and the regulation of this process varies also among closely related species. In Eutheria, initiation of XCI is orchestrated by a cis-acting locus, the X-inactivation center (Xic), which is particularly enriched in genes producing long noncoding RNAs (lncRNAs). Among these, Xist generates a master transcript that coats and propagates along the future inactive X-chromosome in cis, establishing X-chromosome wide transcriptional repression through interaction with several protein partners. Other lncRNAs also participate to the regulation of X-inactivation but the extent to which their function has been maintained in evolution is still poorly understood. In Metatheria, Xist is not conserved, but another, evolutionary independent lncRNA with similar properties, Rsx, has been identified, suggesting that lncRNA-mediated XCI represents an evolutionary advantage. Here, we review current knowledge on the interplay of X chromosome-encoded lncRNAs in ensuring proper establishment and maintenance of chromosome-wide silencing, and discuss the evolutionary implications of the emergence of species-specific lncRNAs in the control of XCI within Theria. WIREs RNA 2016, 7:702-722. doi: 10.1002/wrna.1359 For further resources related to this article, please visit the WIREs website., (© 2016 Wiley Periodicals, Inc.)

Raman spectroscopy followed by multivariate data analysis was used to analyze cotton fibers dyed using similar formulations and submitted to different aging conditions. Spectra were collected on a commercial instrument using a near-infrared laser with a 780nm light source. Discriminant analysis allowed to correctly classify the aged fibers 100% of the time. The prediction ability of the calculated model was estimated to be 100% by the "leave-one-out" cross-validation for 3 out of the 4 series under investigation. Finally, reliability of the developed approach for the discrimination of aged vs new fibers was confirmed by the analysis of commercial polyamide and polyester textiles submitted to the same aging process., (Copyright © 2016 Elsevier B.V. All rights reserved.)

T cell receptor (TCR) phosphorylation requires the kinase Lck and phosphatase CD45. CD45 activates Lck by dephosphorylating an inhibitory tyrosine of Lck to relieve autoinhibition. However, CD45 also dephosphorylates the TCR, and the spatial exclusion of CD45 from TCR clustering in the plasma membrane appears to attenuate this negative effect of CD45. To further investigate the role of CD45 in signal initiation, we reconstituted membrane TCR clusters in vitro on supported lipid bilayers. Fluorescence microscopy of single clusters showed that incorporation of CD45 enhanced phosphorylation of TCR clusters, but only when Lck co-clustered with TCR. We found that clustered Lck autophosphorylated the inhibitory tyrosine and thus could be activated by CD45, whereas diffusive Lck molecules did not. In the TCR-Lck clusters and at low CD45 density, we speculate that the effect of Lck activation may overcome dephosphorylation of TCR, resulting in a net positive regulation. The CD45 density in physiological TCR clusters is also low because of the exclusion of CD45. Thus, we propose that the spatial organization of TCR/Lck/CD45 in T cell membranes is important not only for modulating the negative role of CD45 but also for creating conditions in which CD45 has a positive role in signal initiation., (© 2014 by The American Society for Biochemistry and Molecular Biology, Inc.)

Severe hyperbilirubinemia causes neurological damage both in humans and rodents. The hyperbilirubinemic Gunn rat shows a marked cerebellar hypoplasia. More recently bilirubin ability to arrest the cell cycle progression in vascular smooth muscle, tumour cells, and, more importantly, cultured neurons has been demonstrated. However, the involvement of cell cycle perturbation in the development of cerebellar hypoplasia was never investigated before. We explored the effect of sustained spontaneous hyperbilirubinemia on cell cycle progression and apoptosis in whole cerebella dissected from 9 day old Gunn rat by Real Time PCR, Western blot and FACS analysis. The cerebellum of the hyperbilirubinemic Gunn rats exhibits an increased cell cycle arrest in the late G0/G1 phase (p < 0.001), characterized by a decrease in the protein expression of cyclin D1 (15%, p < 0.05), cyclin A/A1 (20 and 30%, p < 0.05 and 0.01, respectively) and cyclin dependent kinases2 (25%, p < 0.001). This was associated with a marked increase in the 18 kDa fragment of cyclin E (67%, p < 0.001) which amplifies the apoptotic pathway. In line with this was the increase of the cleaved form of Poly (ADP-ribose) polymerase (54%, p < 0.01) and active Caspase3 (two fold, p < 0.01). These data indicate that the characteristic cerebellar alteration in this developing brain structure of the hyperbilirubinemic Gunn rat may be partly due to cell cycle perturbation and apoptosis related to the high bilirubin concentration in cerebellar tissue mainly affecting granular cells. These two phenomena might be intimately connected.

Clinical and drugs safety organisations run their operation independently and use separate databases designed to comply with different data standards. This separation is neither efficient nor effective since investigators need to report serious adverse events both to the clinical and drug safety departments, causing the respective databases to contain partially overlapping data sets containing common elements that need to be reconciled. Electronic data capture provides the opportunity to avoid duplicate storage and obviate reconciliation. It also introduces the risk of non-compliance due to late submission of unexpected serious adverse reactions to competent authorities. This raises the potential for a clinical department to receive a case that the drug safety department is unaware of. However, the most significant inefficiency probably lies in the preparation of aggregate reports and regulatory documents that need to be prepared using data originating from both databases. In a resource-constrained world, unnecessary activities and associated costs are unwelcome, particularly when they are avoidable. The Clinical Data Interchange Consortium (CDISC) has set the standards for clinical trial data, while the International Conference of Harmonization (ICH) dictates drug safety ones. CDISC is expanding its Clinical Data Acquisition Standards Harmonisation (CDASH) model to capture adverse event data associated with ICH E2B. All common data items have two labels that have been mapped. This exercise is showing that there is no scientific justification for data segregation. The differences between these two standards can be attributed to conventions or arise from new technology that renders unnecessary the keying in of certain context information (dates, times and recorder ID). Once this mapping is completed then a common data acquisition process will become feasible. This is the prerequisite to ultimately unifying the two databases and to implementing more efficient processes. The Authors also propose a new workflow to provide safety with the array of benefits that technology and process harmonisation offers and ultimately unifying the clinical drug safety processes.

From pathogen perception and the activation of signal transduction cascades to the deployment of defense responses, protein ubiquitination plays a key role in the modulation of plant immunity. Ubiquitination is mediated by three enzymes, of which the E3 ubiquitin ligases, the substrate determinants, have been the major focus of attention. Accumulating evidence suggests that ubiquitination modulates signaling mediated by pattern recognition receptors and is important for the accumulation of nucleotide-binding leucine-rich repeat type intracellular immune sensors. Recent studies also indicate that ubiquitination directs vesicle trafficking, a function that has been clearly established for immune signaling in animals. In this mini review, we discuss these and other recent advances and highlight important open questions.

Current regulations require a description of the overall safety profile or the specific risks of a drug in multiple documents such as the Periodic and Development Safety Update Reports, Risk Management Plans (RMPs) and Signal Detection Reports. In a resource-constrained world, the need for preparing multiple documents reporting the same information results in shifting the focus from a thorough scientific and medical evaluation of the available data to maintaining compliance with regulatory timelines. Since the aim of drug safety is to understand and characterize product issues to take adequate risk minimization measures rather than to comply with bureaucratic requirements, there is the need to avoid redundancy. In order to identify core drug safety activities that need to be undertaken to protect patient safety and reduce the number of documents reporting the results of these activities, the author has reviewed the main topics included in the drug safety guidelines and templates. The topics and sources that need to be taken into account in the main regulatory documents have been found to greatly overlap and, in the future, as a result of the new Periodic Safety Update Report structure and requirements, in the author's opinion this overlap is likely to further increase. Many of the identified inter-document differences seemed to be substantially formal. The Development Safety Update Report, for example, requires separate presentation of the safety issues emerging from different sources followed by an overall evaluation of each safety issue. The RMP, instead, requires a detailed description of the safety issues without separate presentation of the evidence derived from each source. To some extent, however, the individual documents require an in-depth analysis of different aspects; the RMP, for example, requires an epidemiological description of the indication for which the drug is used and its risks. At the time of writing this article, this is not specifically required by other documents. The author has identified signal detection (intended not only as adverse event disproportionate reporting, but including non-clinical, laboratory, clinical analysis data and literature screening) and characterization as the basis for the preparation of all drug safety documents, which can be viewed as different ways of presenting the results of this activity. Therefore, the author proposes to merge all the aggregate reports required by current regulations into a single document - the Drug Safety Master File. This report should contain all the available information, from any source, regarding the potential and identified risks of a drug. It should be a living document updated and submitted to regulatory authorities on an ongoing basis.

Society has been increasingly intolerant of excuses for systems breakdown in many areas of public life. This is hardly surprising given that there is overwhelming evidence behind why processes fail and mistakes are made, and so, based on this evidence, processes should be designed to mitigate risk. The main root cause of many process failures can be attributed to the human factor, which encompasses all those factors that can influence people and their behaviour. Based on experience from other safety-conscious industries, there is a major move to manage the human factor as part of delivery of safety culture in healthcare systems. Since pharmaceutical companies are healthcare companies, it makes sense that the principles underlying a pharmaceutical safety culture are aligned with those of the healthcare sector. A good place to start applying human factor management to a pharmaceutical safety process would be the complex process required to produce a good quality Periodic Safety Update Report (PSUR) on time and to an acceptable format. This can be achieved by a process aimed at building on an ongoing learning cycle through planning, observing if execution matches expectations and learning from mistakes and through the interdependent teamwork of PSUR contributors providing mutual support. Such a framework of teamwork and communication principles can be applied to the entire process for the preparation and submission of PSURs.

Dermatofibrosarcoma protuberans (DFSP) is an uncommon skin tumour characterized by a slow, infiltrative growth and marked tendency towards local recurrences. Wide surgical excision of 3-5 cm from the margins including the fascia is the recommended treatment. Mohs Micrographic Surgery has been shown to afford lower rates of recurrence. The Mohs-Tübingen technique is a variant indicated for very large excisions that allows a complete eradication of the tumour, preserving healthy tissues. We report the case of a 45 year-old man affected by DFSP of the right shoulder deeply infiltrating the muscles, referred to us for a recurrence after a large excision. We submitted the patient to Mohs-Tübingen surgery in collaboration with an orthopaedic surgeon due to the presence of muscular involvement of DFSP. Two surgical operations were necessary to obtain negative histology as the tumor was deeply infiltrating the prescapular muscles. After three years of follow-up, the patient did not have any recurrence and the normal mobility of the shoulder was preserved. Precocious diagnosis and adequate therapy are necessary for DFSP as not only the margins, but also the deep invasion of the tumor have to be carefully controlled.

Several pathological studies have revealed a prominent involvement of the cerebral cortex in patients with multiple sclerosis (MS). In order to better understand the events that lead to the progressive neuronal dysfunction in MS, herein we explore the contribution of the glutamatergic release in cerebral cortex synaptosomes isolated from rats with experimental autoimmune encephalomyelitis, an animal model reproducing many features of MS. We found that the Ca(2+)-dependent but not the Ca(2+)-independent glutamate release induced by KCl and 4-aminopyridine was significantly decreased during the acute stage of the disease. This inhibited release coincides with the onset of the clinical signs and after 24 h tends to recover the level of the control animals. The results also showed an inhibition of the glutamate release stimulated by ionomycin. When the animals were totally recovered from clinical signs, the neurotransmitter release stimulated by the different inductors was similar to the controls. Examination of the cytosolic Ca(2+) using fura-2-acetoxymethyl ester revealed that the inhibition of glutamate release could not be attributed to a reduction in voltage-dependent Ca(2+) influx. However, this inhibition was concomitant with a lower phosphorylation of synapsin I at P-site1. Our results show that the inhibition observed on the Ca(2+)-dependent neurotransmitter release from cerebral cortex synaptosomes in experimental autoimmune encephalomyelitis is specific and correlates with the beginning of the clinical disease. Moreover, they suggest an alteration in the metabolism of proteins involved in the vesicular glutamate release more than a deregulation in the influx of cytosolic Ca(2+).

Is sampling really effective in staging non-small cell lung cancer? The aim of the study was to assess if systematic nodal dissection is necessary in order to stage non-small cell lung cancer correctly or whether mediastinal lymph node sampling can be used and whether in selected cases it could replace systematic nodal dissection for the treatment of lung cancer. A prospective study was conducted in 94 patients affected by clinically resectable non-small cell lung cancer (stages I-IIIB) who were surgically treated by the same team of surgeons. During surgery mediastinal lymph node sampling was done first and then another surgeon completed the systematic nodal dissection and performed the lung resection. One hundred and ninety-three mediastinal nodal stations were investigated using the American Thoracic Society lymph node map to identify them. On analysing the 193 mediastinal nodal stations investigated, it emerged that in 181 cases (94%) mediastinal lymph node sampling and systematic nodal dissection yielded the same histopathological findings, whereas in 12 cases (6%) there was no agreement between the two techniques. The negative predictive value of mediastinal lymph node sampling was 92.8% (103/111). The results of the study show no statistical difference between mediastinal lymph node sampling and systematic nodal dissection in staging non-small cell lung cancer. However, it is possible that in a limited percentage of cases a nodal station could be understaged and thus the surgical resection could prove incomplete if mediastinal lymph node sampling alone is performed. Moreover, in those cases where mediastinal lymph node sampling detects N2 disease and systematic nodal dissection has not been completed, the intervention cannot be considered radical.

The main indications for cervical mediastinoscopy are preoperative staging of lung cancer and diagnostic biopsy of mediastinal mass (lymphoma, sarcoidosis, tuberculosis etc.). We undertook a retrospective review of our experience of mediastinal exploration by cervical media-stinoscopy: 253 mediastinoscopies were performed on 252 patients (195 male and 57 female; mean age 53 years, range 14-88 years) between 1995 and June 2003. Four extended mediastinoscopies were performed and 1 patient had a re-mediastinoscopy following a non-diagnostic procedure. 319 lymph nodal stations were investigated in 253 procedures. We observed no mortality, while 2 patients had major bleeding (0.7%), with the need for open surgical treatment in order to achieve haemostasis. The median length of hospital-stay was one day, with discharge in the first postoperative day. 69 out of 170 patients, who eventually resulted to be affected by a histologically proven lung cancer, had a negative mediastinoscopy. Fifteen of them resulted N2 at the time of surgery: 8 patients with a false negativity in a biopsied station (4 in station 4R and 4 in station 7), while 7 cases showed infiltration in stations which were not sampled (5 in station 5, 1 in station 8 and 1 in station 7, the latter being the only one in which a standard cervical mediastincoscopy could have been able to stage it correctly). So, having observed 61 true negatives and 8 false-negatives in the sampled stations, in our experience the negative predictive value of cervical mediastinoscopy was 88.4%, with 78.2% of patients correctly staged without using other diagnostic tools. In conclusion, mediastinoscopy is an important procedure for the diagnostic biopsy of mediastinal mass and a useful tool in preoperative staging of lung cancer, especially if associated with chest CT-scan and Positron Emission Tomography (PET). In our experience, the spreading of PET does not lead to a reduction of cervical mediastinoscopies, both for the contemporary introduction of new chemotherapeutic preoperative protocols and, above all, for the not negligible incidence of false-positive results using PET, suggesting that media-stinoscopy should always be performed in patients affected by a PET-positive mediastinal growth.

Pituitary stalk thickening has a wide differential diagnosis, but almost all infundibular diseases present with diabetes insipidus (DI). We present a child with metastatic involvement of the pituitary stalk from a primary pontine tumour and a child with tuberculous infiltration of the pituitary stalk and associated meningeal inflammation. Neither child presented with DI. Even though both metastatic disease and tuberculous infiltration of the stalk have been reported in adults, these are the first reports with accompanying cross-sectional images of pituitary stalk involvement by these diseases in children.

Objective: The objective of this study was to assess the results of completion pneumonectomy performed for non-small cell lung cancer, classified as second primary or recurrence/metastasis., Methods: From 1982 to 2000, 59 patients underwent completion pneumonectomy for lung cancer, classified second primary or recurrence/metastasis according to a modified form of Martini's criteria, after a mean interval from first resection of 60 months for second primary lung cancers and 19 months for recurrences/metastases., Results: Operative mortality was 3.4% and complications occurred in 30% of patients. Five-year survival rate for completely resected patients was 25% (median 20 months). No significant difference in long-term survival was detected between second primary and recurrent tumors; survival was not adversely affected by a resection interval of less than 2 years or less than 12 months., Conclusions: Completion pneumonectomy for non-small cell lung cancer is a safe surgical procedure in experienced hands; long-term survival is acceptable and the best results are obtained for stage I lung cancer. Distinction between second primary lung cancer and recurrence failed to demonstrate a prognostic value.

Objective: To assess operative mortality (OM), morbidity and long-term results of sleeve lobectomies performed for non-small cell lung cancer (NSCLC) and carcinoids during a 35-year period., Methods: A retrospective review of patients who underwent a sleeve lobectomy for NSCLC and carcinoids was undertaken, univariate and multivariate analyses of factors influencing early mortality in NSCLC were performed and for this purpose the series was split into an early and a contemporary phase, the Kaplan-Meier method was used to calculate the cumulative survival rate, and statistical significance was calculated with the log-rank test. Causes of death were evaluated in relation to the stage of the disease., Results: OM for NSCLC was 14.6% in the early phase and 6% in the contemporary one; late stenosis occurred in 7.7% of NSCLC patients in the early phase and in 2% in the contemporary one. No OM or late stenosis occurred in carcinoid patients. Three, 5 and 10-year survival rates excluding carcinoids were 77, 62 and 31% for stage I(A-B), 45, 34 and 27% for stage II(A-B), 33, 22 and 0% for stage III(A-B). The 10-year survival rate for carcinoids was 100%. There was no significant difference in long-term survival between stages II and III, while the difference between stage I and stages II and III was significant (P<0.001). When survival was analyzed in relation to nodal status, 3, 5 and 10-year survival rates were 71, 57 and 33% for N0 disease, 42, 33 and 22% for N1 disease, and 34 and 19% with the last observation at 82 months of 19% for N2 disease; there was no significant difference in survival between N1 and N2 disease. A second primary lung cancer occurred in six patients (3.7%) who underwent resection. Late mortality was not related to cancer in most stage I patients while in stages II and III patients it was related to local and distant recurrences., Conclusions: Sleeve lobectomy is a valid alternative to pneumonectomy: careful patient selection and surgical technique make it possible to achieve a mortality rate comparable to or lower than that for pneumonectomy along with a better quality of life. In addition, it allows further lung resection, if necessary.

During mast cell degranulation the soluble component of the granule is released into extracellular fluid, whereas two neutral proteases and heparin proteoglycans form the extracellular granule remnants. These structures are negatively charged and bind with high affinity LDL and other basic molecules. In this study we show that granule remnants expelled into extracellular fluid are able to bind the aminoglycoside antibiotic gentamicin and the anticancer agent doxorubicin in a dose-dependent manner. In addition, granule remnants loaded with the two basic substances are subsequently phagocytosed by macrophages. Indeed, when cells are incubated for 24 h with 1 mg/ml gentamicin, the intracellular concentration of the drug, which in basal conditions is extremely low, increases significantly in the presence of degranulating mast cells (from 5.1 +/- 1.0 to 25.4 +/- 2.5 microg/mg protein) and a good correlation between histamine release and gentamicin uptake is evident. The antineoplastic agent doxorubicin can penetrate cells by passive diffusion; however, when mast cells are added to macrophage monolayer, incubated for 30 min with 50 microM of the antineoplastic agent, a significant increase in intracellular doxorubicin concentration is observed (from 3.5 +/- 0.2 to 4.7 +/- 0.2 microg/mg protein). Internalization of granule remnants carrying gentamicin or doxorubicin is also evident in smooth muscle cells of the synthetic phenotype. In particular, when smooth muscle cells are incubated for 24 h with 1 mg/ml gentamicin, addition of isolated granules increases the uptake from 2.4 +/- 0.2 to 4.8 +/- 0.4 microg/mg protein. Similar results are obtained in smooth muscle cells incubated for 4 h with doxorubicin 50 microM (from 3.3 +/- 0.2 to 4.8 +/- 0.5 microg/mg protein). Data are confirmed by microscopic experiments by means of fluorescence microscopy and electron microscopic studies. The study demonstrates that basic substances can enter phagocytic cells when loaded to granule remnants. The phenomenon can be of particular interest for substances like the aminoglycosides that do not cross biological membranes; indeed, the storage of these antibiotics in phagocytic cells could have important consequences on their antibacterial activity in vivo. Macrophages and smooth muscle cells can also act as a reservoir for doxorubicin. High concentrations of the antineoplastic agent in these cells could be responsible for toxicity, as well as play an important role in the transport of the drug to tumor cells., (Copyright 2000 Academic Press.)

This work aimed to evaluate the Deladenus siricidicola, Bedding (Neotylenchidae) parasitism in adults Sirex noctilio, Fabricius, 1793 (Hymenoptera: Siricidae). Timber was sampled by cutting out pieces of 0.80 m in length in Sirex noctilio attacked and Deladenus siricidicola inoculated Pinus taeda. Longs were 15-20 cm in diameter, according to the tree age. Samples were packed in gauze-cages, for daily observations, till S. noctilio adults emergence. The emerged insects were transported, in plastic containers to the laboratory, where they were sectioned and dissected under stereoscopic microscopy to observ the nematode occurrence. From the initially proposed ten units, nine of them were evaluated in a total of 1,810 emerged adult insects, being 1,441 males and 369 females. Nematode parasitism was shown in 267 males and 74 females, in a total of 341 infected insects (18.84%).

The mechanisms by which aminoglycosides are accumulated in renal proximal tubular cells remain unclear. Adsorptive mediated endocytosis, via a common pathway for cationic proteins, or receptor endocytosis, mediated by the glycoprotein 330/megalin, have been proposed to be involved in gentamicin transport in renal cells. We used the LLC-PK1 cell line, derived from the pig proximal tubule, to explore further the regulation of gentamicin endocytosis in these cells and to determine the role of clathrin mediated endocytosis and G proteins in this function. Gentamicin endocytosis was strictly temperature dependent, whereas total uptake (endocytosis plus binding) did not significantly differ at 4 or 37 degrees C. Substances that suppress receptor mediated, clathrin dependent endocytosis, such as monensin, phenylarsine oxide and dansylcadaverine, or inhibit caveolae mediated endocytosis, such as nystatin, did not affect gentamicin entrance in LLC-PK1 cells. Among substances that disrupt the actin cytoskeleton, only cytochalasin D, that is active also on fluid phase endocytosis, significantly reduced the intracellular concentrations of the aminoglycoside. Other maneuvers that perturb clathrin dependent endocytosis without affecting clathrin independent pathway, such as acidification of cytosol or incubation in hypertonic medium, were also without effect. Mastoparan, a well known stimulator of heterotrimeric G proteins, strongly increased endocytosis of gentamicin, and the same effect was evident with two other G protein stimulators, aluminum fluoride and fluoride alone; however the effect seems not to be mediated by an activation of adenylyl cyclase. In conclusion, gentamicin endocytosis in LLC-PK1 cells is probably clathrin independent, limited by cytochalasin D, which interacts with cytoskeleton, and increased by substances like mastoparan and aluminum fluoride, which activate heterotrimeric G proteins.