Synthesis and Biological Characterization of a New Norbormide Derived Bodipy FL-Conjugated Fluorescent Probe for Cell Imaging

Background: Norbormide (NRB) is a selective rat toxicant endowed with vasoconstrictor activity confined to the rat peripheral arteries. In a recent work we used a fluorescent derivative of NRB (NRB-AF12), obtained by coupling the NBD fluorophore to the parent molecule via a linker, in order to gain information about the possible site of action of the unlabeled compound. We found that NRB-AF12 labeled intracellular organelles in both NRB-sensitive and -insensitive cells and we accordingly proposed its use as a scaffold for the development of a new class of fluorescent probes. In this study, we examined the fluorescent properties of a BODIPY FL-conjugated NRB probe (MC009) developed: (A) to verify if NRB distribution could be influenced by the attached fluorophore; (B) to improve the fluorescent performance of NRB-AF12. Methods: MC009 characteristics were investigated by confocal fluorescence microscopy, in freshly isolated rat caudal artery myocytes (FIRCAM) and in LX2 cells, representative of NRB-sensitive and insensitive cells, respectively. Main results: In both FIRCAM and LX2 cells MC009 stained endoplasmic reticulum, mitochondria, Golgi apparatus and lipid droplets, revealing the same intracellular distribution as NRB-AF12, and, at the same time, had both improved photostability and gave a more intense fluorescent signal at lower concentrations than was possible with NRB-AF12, which resulted in a better and finer visualization of intracellular structures. Furthermore, MC009 was effective in cellular labeling in both living and fixed cells. At the concentration used to stain the cells, MC009 did not show any cytotoxic effect and did not affect the regular progression of cell cycle and division. Conclusions: This study demonstrates that the distribution of fluorescently labeled NRB is not affected by the type of fluorophore attached to the parent compound, supporting the idea that the localization of the fluorescent derivatives may reasonably reflect that of the parent compound. In addition, we observed a marked improvement in the fluorescent properties of BODIPY FL-conjugated NRB (MC009) over its NBD-derived counterpart (NRB-AF12), confirming NRB as a scaffold for the development of new, high performance, non-toxic fluorescent probes for the labeling of intracellular structures in both living and fixed cells

Surveillance of Influenza and Other Airborne Transmission Viruses during the 2021/2022 Season in Hospitalized Subjects in Tuscany, Italy

Winter in the northern hemisphere is characterized by the circulation of influenza viruses, which cause seasonal epidemics, generally from October to April. Each influenza season has its own pattern, which differs from one year to the next in terms of the first influenza case notification, the period of highest incidence, and the predominant influenza virus subtypes. After the total absence of influenza viruses in the 2020/2021 season, cases of influenza were again recorded in the 2021/2022 season, although they remained below the seasonal average. Moreover, the co-circulation of the influenza virus and the SARS-CoV-2 pandemic virus was also reported. In the context of the DRIVE study, oropharyngeal swabs were collected from 129 Tuscan adults hospitalized for severe acute respiratory infection (SARI) and analyzed by means of real-time polymerase chain reaction (RT-PCR) for SARS-CoV-2 and 21 different airborne pathogens, including influenza viruses. In total, 55 subjects tested positive for COVID-19, 9 tested positive for influenza, and 3 tested positive for both SARS-CoV-2 and the A/H3N2 influenza virus. The co-circulation of different viruses in the population requires strengthened surveillance that is no longer restricted to the winter months. Indeed, constant, year-long monitoring of the trends of these viruses is needed, especially in at-risk groups and elderly people

An NBD derivative of the selective rat toxicant norbormide as a new probe for living cell imaging

Norbormide (NRB) is a unique compound that acts directly on rat vascular myocytes to trigger a contractile process, through an as yet unknown mechanism, which results in the selective contraction of rat peripheral arteries. To gain insight into the mechanisms involved in NRB rat-selective activity, we investigated the subcellular distribution of NRB-AF12, a nitrobenzoxadiazole (NBD)-derivative of NRB, in living NRB-sensitive and NRB-insensitive cells. In both cell types, NRB-AF12 localized to the endoplasmic reticulum (ER), Golgi apparatus, mitochondria, lysosomes, and endosomes; however, in NRB-sensitive cells, the fluorescence also extended to the plasma membrane. NRB-AF12 was rapidly internalized into the cells, could easily be washed out and then reloaded back into the same cells, all with a high degree of reproducibility. Cells exposed for 24 h to NRB-AF12 did not show apparent signs of toxicity, even at concentrations of the dye (10 μM) much higher than those required for fluorescence labeling (500 ηM). The distribution pattern of NRB-AF12 fluorescence was near identical to that of ER-Tracker® (Er-Tr), a fluorescent derivative of glibenclamide, a known KATP channel blocker. Displacement tests did not demonstrate, but at the same time did not rule out the possibility of a common target for ER-Tr, NRB-AF12, NRB, and glibenclamide. On the basis of these results we hypothesize a common target site for NRB-AF12 and ER-Tr, and a similar target profile for NRB and glibenclamide, and propose NRB-AF12 as an alternative fluorescence probe to ER-Tracker. Furthermore, NRB-based fluorescence derivatives could be designed to selectively label single cellular structures

The PANGEA project: a qualitative analysis for Intimate Partner Violence through anti violence workers during COVID-19 pandemic : Il progetto PANGEA: un’analisi qualitativa dell’Intimate Partner Violence attraverso i lavoratori della rete antiviolenza durante la pandemia da COVID-19

Intimate Partner Violence (IPV) is the most difficult gender-based violence’s form to identify and to stop. The pandemic conditions may have caused an increase of this form but it is difficult to measure. The anti-violence workers could be a valuable and reliable source to obtain informations about women and working conditions during the pandemic. Tuscany region is suitable to conduct this study because has an high rate of IPV and had a long period of lockdown. Thanks to the regional interest it was possible to make a project able to study many aspects of this phenomenon. In the first phase, described in this article, it was possible to understand the major sentiments, emotions, feelings and moods of the anti-violence workers respect of the issues in question.La violenza di genere è un fenomeno molto frequente e l’Intimate Partner Violence (IPV) è la forma più difficile da identificare e contrastare. Le condizioni pandemiche potrebbero aver causato un incremento di questa forma, ma misurarla risulta estremamente complesso. Gli operatori anti violenza sono delle valide ed affidabili risorse per ottenere informazioni riguardo alle condizioni delle donne, ma anche riguardo alle condizioni lavorative durantela pandemia. Considerando che la regione toscana ha un alto tasso di IPV e che in questa regione il lockdown è durato a lungo, è una delle migliori regioni in Italia dove condurre uno studio di questo tipo. Grazie all’interesse mostrato dalla regione è stato possibile iniziare un progetto in grado di valutare molti aspetti di questo fenomeno. Nella prima fase, che sarà descritta in questo articolo, è possibile comprendere i principali sentimenti, emozioni,stati d’animo e atteggiamenti degli operatori anti violenza rispetto alle tematiche in questione

Acute effects of caffeine and cigarette smoking on ventricular long-axis function in healthy subjects

<p>Abstract</p> <p>Background</p> <p>Few data exist regarding the direct effects of caffeine and smoking on cardiac function. We sought to explore the acute effects of caffeine assumption, cigarette smoking, or both on left ventricular (LV) and right ventricular (RV) function in a population of young normal subjects.</p> <p>Methods</p> <p>Forty-five healthy subjects aged 25 ± 2 years underwent echocardiography. Fifteen of them were non-smokers and habitual coffee consumers (group 1), 15 were smokers and not habitual coffee consumers (group 2), and 15 were smokers and habitual coffee consumers (group 3). Peak systolic (S_a), early diastolic E_a, and late diastolic (A_a) velocity of mitral annulus were measured by pulsed Tissue Doppler, and left atrioventricular plane displacement was determined by M-mode. Tricuspid annular velocities and systolic excursion (TAPSE) were also determined. Measurements were performed at baseline and after oral assumption of caffeine 100 mg in group 1, one cigarette smoking in group 2, and both in group 3.</p> <p>Results</p> <p>No changes in ventricular function were observed in group 1 after caffeine administration. In group 2, cigarette smoking yielded an acute increase in mitral A_a(+12.1%, p = 0.0026), tricuspid S_a(+9.8%, p = 0.012) and TAPSE (+7.9%, p = 0.017), and a decrease in the mitral E_a/A_aratio (-8.5%, p = 0.0084). Sequential caffeine assumption and cigarette smoking in group 3 was associated with an acute increase in mitral A_a(+13.0%, p = 0.015) and tricuspid A_a(+11.6%, p < 0.0001) and a reduction in mitral E_a/A_aratio (-8.5%, p = 0.0084) tricuspid E_a(-6.6%, p = 0.048) and tricuspid E_a/A_aratio (-9.6%, p = 0.0003). In a two-way ANOVA model controlling for hemodynamic confounding factors, changes in the overall population remained significant for mitral A_aand E_a/A_aratio, and for tricuspid A_aand E_a/A_aratio.</p> <p>Conclusion</p> <p>In young healthy subjects, one cigarette smoking is associated to an acute impairment in LV diastolic function and a hyperdynamic RV systolic response. Caffeine assumption alone does not exert any acute effect on ventricular long-axis function, but potentiates the negative effect of cigarette smoking by abolishing RV supernormal response and leading to a simultaneous impairment in both LV and RV diastolic function.</p

Guidelines for the use and interpretation of diagnostic methods in adult food allergy

Food allergy has an increasing prevalence in the general population and in Italy concerns 8 % of people with allergies. The spectrum of its clinical manifestations ranges from mild symptoms up to potentially fatal anaphylactic shock. A number of patients can be diagnosed easily by the use of first- and second-level procedures (history, skin tests and allergen specific IgE). Patients with complex presentation, such as multiple sensitizations and pollen-food syndromes, frequently require a third-level approach including molecular diagnostics, which enables the design of a component-resolved sensitization profile for each patient. The use of such techniques involves specialists' and experts' skills on the issue to appropriately meet the diagnostic and therapeutic needs of patients. Particularly, educational programs for allergists on the use and interpretation of molecular diagnostics are needed

Follow-up strategies for patients with splenic trauma managed non-operatively : the 2022 World Society of Emergency Surgery consensus document

Background In 2017, the World Society of Emergency Surgery published its guidelines for the management of adult and pediatric patients with splenic trauma. Several issues regarding the follow-up of patients with splenic injuries treated with NOM remained unsolved. Methods Using a modified Delphi method, we sought to explore ongoing areas of controversy in the NOM of splenic trauma and reach a consensus among a group of 48 international experts from five continents (Africa, Europe, Asia, Oceania, America) concerning optimal follow-up strategies in patients with splenic injuries treated with NOM. Results Consensus was reached on eleven clinical research questions and 28 recommendations with an agreement rate >= 80%. Mobilization after 24 h in low-grade splenic trauma patients (WSES Class I, AAST Grades I-II) was suggested, while in patients with high-grade splenic injuries (WSES Classes II-III, AAST Grades III-V), if no other contraindications to early mobilization exist, safe mobilization of the patient when three successive hemoglobins 8 h apart after the first are within 10% of each other was considered safe according to the panel. The panel suggests adult patients to be admitted to hospital for 1 day (for low-grade splenic injuries-WSES Class I, AAST Grades I-II) to 3 days (for high-grade splenic injuries-WSES Classes II-III, AAST Grades III-V), with those with high-grade injuries requiring admission to a monitored setting. In the absence of specific complications, the panel suggests DVT and VTE prophylaxis with LMWH to be started within 48-72 h from hospital admission. The panel suggests splenic artery embolization (SAE) as the first-line intervention in patients with hemodynamic stability and arterial blush on CT scan, irrespective of injury grade. Regarding patients with WSES Class II blunt splenic injuries (AAST Grade III) without contrast extravasation, a low threshold for SAE has been suggested in the presence of risk factors for NOM failure. The panel also suggested angiography and eventual SAE in all hemodynamically stable adult patients with WSES Class III injuries (AAST Grades IV-V), even in the absence of CT blush, especially when concomitant surgery that requires change of position is needed. Follow-up imaging with contrast-enhanced ultrasound/CT scan in 48-72 h post-admission of trauma in splenic injuries WSES Class II (AAST Grade III) or higher treated with NOM was considered the best strategy for timely detection of vascular complications. Conclusion This consensus document could help guide future prospective studies aiming at validating the suggested strategies through the implementation of prospective trauma databases and the subsequent production of internationally endorsed guidelines on the issue.Peer reviewe

Retrospective evaluation of whole exome and genome mutation calls in 746 cancer samples

Funder: NCI U24CA211006Abstract: The Cancer Genome Atlas (TCGA) and International Cancer Genome Consortium (ICGC) curated consensus somatic mutation calls using whole exome sequencing (WES) and whole genome sequencing (WGS), respectively. Here, as part of the ICGC/TCGA Pan-Cancer Analysis of Whole Genomes (PCAWG) Consortium, which aggregated whole genome sequencing data from 2,658 cancers across 38 tumour types, we compare WES and WGS side-by-side from 746 TCGA samples, finding that ~80% of mutations overlap in covered exonic regions. We estimate that low variant allele fraction (VAF < 15%) and clonal heterogeneity contribute up to 68% of private WGS mutations and 71% of private WES mutations. We observe that ~30% of private WGS mutations trace to mutations identified by a single variant caller in WES consensus efforts. WGS captures both ~50% more variation in exonic regions and un-observed mutations in loci with variable GC-content. Together, our analysis highlights technological divergences between two reproducible somatic variant detection efforts