Characterization of the polycaprolactone melt crystallization: complementary optical microscopy, DSC, and AFM studies.

The first stages of the crystallization of polycaprolactone (PCL) were studied using several techniques. The crystallization exotherms measured by differential scanning calorimetry (DSC) were analyzed and compared with results obtained by polarized optical microscopy (POM), rheology, and atomic force microscope (AFM). The experimental results suggest a strong influence of the observation scale. In particular, the AFM, even if limited on time scale, appears to be the most sensitive technique to detect the first stages of crystallization. On the contrary, at least in the case analysed in this work, rheology appears to be the least sensitive technique. DSC and POM provide closer results. This suggests that the definition of induction time in the polymer crystallization is a vague concept that, in any case, requires the definition of the technique used for its characterization.

New insights into Chlamydiae persistence: an energy metabolism strategy?

Chlamydiaceae is a family of obligate intracellular bacteria generally considered energy parasites. Several studies have suggested that Chlamydiae are capable of independently producing energy and, more importantly, several genes involved in the energy metabolism are up-regulated during the persistent state. Thus, it has been suggested that chlamydial persistence could be a complex and flexible metabolic strategy designed to favor a lengthy survival in the host cell by evading the immune response. In conclusion, more detailed studies on the shift in the chlamydial energy metabolism, from the active to the persistent form, may be helpful in future to determine whether chlamydial persistence observed in vitro does occur in vivo and whether chronic sequelae of chlamydial diseases may be related to the persistence.

Resveratrol in Chlamydia pneumoniae-induced foam cell formation and interleukin-17A synthesis.

The involvement of Chlamydia pneumoniae in the pathogenesis of atherosclerosis has been suggested by numerous seroepidemiological, in vivo and in vitro studies. In particular, it has been shown that C. pneumoniae is able to promote the accumulation of low-density lipoproteins into macrophages, thus facilitating foam cell formation. The aim of our study was to investigate the effects of resveratrol on macrophage derived foam cell formation induced by C. pneumoniae, examining its underlying biochemical mechanisms. Our results showed a relevant decrease in the number of foam cells, in the production of thiobarbituric acid reactive substances, superoxide anion and IL 17A while treating C. pneumoniae infected macrophages with resveratrol. Furthermore, the inhibition of Peroxisome Proliferator-Activated Receptors gamma by a specific antagonist (GW 9662), in presence of resveratrol and C. pneumoniae, enhanced intracellular lipid and cholesterol accumulation and the subsequent foam cell formation. In conclusion, the main result of our study is the evidence of an antiatherogenic effect of resveratrol on macrophage-derived foam cell formation and IL-17A production induced by C. pneumoniae.

Analysis of gene expression in penicillin G induced persistence of Chlamydia pneumoniae.

Chlamydia pneumoniae is responsible for respiratory tract infections and has been associated to chronic diseases such as atherosclerosis. The involvement of C. pneumoniae in chronic diseases may be correlated to its ability to induce persistent forms in which Chlamydiae remain viable but are not cultivable. The aim of our study is to investigate C. pneumoniae specific gene activities associated with the development of Chlamydial persistence in a cell culture system in the presence of penicillin G. Chlamydia-infected HEp 2 cells were incubated with or without penicillin G for up to 72 hours. The relative mRNA expression levels of early and late genes in treated and untreated cell cultures were determined by Real-time RT-PCR. Our results revealed a consistent down-regulation of Chlamydial hctA and hctB genes (p=0.012 and p=0.003 respectively) in association with up-regulation of htrA gene (p=0.002) during penicillin G-induced persistence suggesting these gene sets as leading candidate for in vivo investigation of the development of persistent Chlamydial infection. In conclusion, the Chlamydial expression pattern of hctA, hctB, and htrA genes may be helpful to identify target molecules to diagnose and treat Chlamydia-associated chronic diseases.

Arterial entrapment syndrome in the cubital fossa: a rare cause of acute stress-related arterial thrombosis in a patient with brachial artery duplication.

Arterial entrapment syndrome (AES) at elbow level is very rare and to our knowledge no case of AES by lacertus fibrosus in the cubital fossa in presence of brachial artery duplication has been described to date. We describe a rare case of acute arterial thrombosis of one of two brachial arteries highlighted in the cubital fossa which developed after strenuous right elbow flexor muscle activity and hyper-extensions presumably related to AES by lacertus fibrosus at elbow level. A 43-year-old right-handed woman, experienced paleness, coldness and numbness of the right hand, after 8 consecutive hours of gardening. As she worked, her ipsilateral flexor elbow muscles remained in prolonged and inappropriate tension. Clinical examination evidenced the absence of radial artery pulse in the wrist and mild hypothermia in the second and third finger. During surgical exploration two anastomosed brachial arteries were detected in the cubital fossa under the lacertus fibrosus. The lateral superficial brachial artery was occluded. Intraoperative arteriography evidenced brachial artery duplication at the third superior of the arm and normal vascular pattern at the forearm level. In cases of unexplained atypical intermittent upper extremity claudication or acute ischemic symptoms an AES should always be ruled out, particularly when symptoms are exacerbated by strenuous upper extremity activity or when upper limb muscular hypertrophy is evident. In these cases a thorough dynamic clinical and instrumental examination is mandatory to confirm a diagnosis of AES and to avoid possible future ischemic complications.

The "blind age assessment": applicability of Greulich and Pyle, Demirjian and Mincer aging methods to a population of unknown ethnic origin.

PURPOSE: Age estimation is one of the most crucial issues in case of unknown deceased as well as in the living and is very frequently of radiological interest. Three methods for age estimation have been designated as the most reliable among the others: Greulich and Pyle, Demirjian and Mincer. The literature provides several studies concerning their applicability in different geographic contexts. However, not always can ancestry be ascertained, for example, in the case of badly preserved corpses. In these cases, age assessment must be performed without the corrections suggested by the literature for different ethnic groups. One may therefore wonder how reliable the result of age assessment performed without knowing the racial group to which the subject belongs may be. This study aimed at testing the applicability of the Greulich and Pyle Atlas, the Demirjian and the Mincer methods on a mixed population to compare skeletal and dental methods of age estimation. MATERIALS AND METHODS: X-ray films of 167 subjects aged between 4 and 31 years from more than 18 countries were recruited. One hundred and nine orthopantomographs (OPG) of children aged between 4 and 15.5 years were evaluated by Demirjian's method; whenever the highest Demirjian score was reached (31 cases), the Mincer method was applied. The skeletal maturation of 54 subjects aged between 7 and 19 years was determined by the Greulich and Pyle method. RESULTS: The lowest average variance from chronological age was shown by the Greulich and Pyle method, followed by Demirjian. The Mincer method showed very high mean variances. CONCLUSIONS: Mean variances from the different methods do not significantly differ from data reported in the literature and demonstrate that the reliability of Demirjian, and Greulich and Pyle as they stand may be applied satisfactorily to remains or individuals of unknown ethnic origin.

Anticancer innovative therapy: Highlights from the ninth annual meeting.

The Ninth Annual Conference of "Anticancer Innovative Therapy", organized by Fondazione IRCCS Istituto Nazionale dei Tumori di Milano (Fondazione IRCCS INT) and hosted by Hotel Michelangelo, was held in Milan on 25 January 2019. Cutting-edge science was presented in two main scientific sessions: i) pre-clinical evidences and new targets, and ii) clinical translation. The Keynote lecture entitled "Cancer stem cells (CSCs): metabolic strategies for their identification and eradication" presented by M. Lisanti, was one of the highlights of the conference. One key concept of the meeting was how the continuous advances in our knowledge about molecular mechanisms in various fields of research (cancer metabolism reprogramming, epigenetic regulation, transformation/invasiveness, and immunology, among others) are driving cancer research towards more effective personalized antineoplastic strategies. Specifically, recent preclinical data on the following topics were discussed: 1. Polycomb group proteins in cancer; 2. A d16HER2 splice variant is a flag of HER2 addiction across HER2-positive cancers; 3. Studying chromatin as a nexus between translational and basic research; 4. Metabolomic analysis in cancer patients; 5. CDK4-6 cyclin inhibitors: clinical activity and future perspectives as immunotherapy adjuvant; and 6. Cancer stem cells (CSCs): metabolic strategies for their identification and eradication. In terms of clinical translation, several novel approaches were presented: 1. Developing CAR-T cell therapies: an update of preclinical and clinical development at University of North Carolina; 2. Vγ9Vδ2 T-cell activation and immune suppression in multiple myeloma; 3. Predictive biomarkers for real-world immunotherapy: the cancer immunogram model in the clinical arena; and 4. Mechanisms of resistance to immune checkpoint blockade in solid tumors. Overall, the pre-clinical and clinical findings presented could pave the way to identify novel actionable therapeutic targets to significantly enhance the care of persons with cancer.

Innovative therapy, monoclonal antibodies, and beyond: Highlights from the eighth annual meeting.

The eighth annual conference of "Innovative therapy, monoclonal antibodies, and beyond" was held in Milan on Jan. 26, 2018, and hosted by Fondazione IRCCS-Istituto Nazionale dei Tumori (Fondazione IRCCS INT). The conference was divided into two main scientific sessions, of i) pre-clinical assays and novel biotargets, and ii) clinical translation, as well as a third session of presentations from young investigators, which focused on recent achievements within Fondazione IRCCS INT on immunotherapy and targeted therapies. Presentations in the first session addressed the issue of cancer immunotherapy activity with respect to tumor heterogeneity, with key topics addressing: 1) tumor heterogeneity and targeted therapy, with the definition of the evolutionary Index as an indicator of tumor heterogeneity in both space and time; 2) the analysis of cancer evolution, with the introduction of the TRACERx Consortium-a multi-million pound UK research project focused on non-small cell lung cancer (NSCLC); 3) the use of anti-estrogen agents to boost immune recognition of breast cancer cells; and 4) the high degree of functional plasticity within the NK cell repertoire, including the expansion of adaptive NK cells following viral challenges. The second session addressed: 1) the effectiveness of radiotherapy to enhance the proportion of patients responsive to immune-checkpoint blockers (ICBs); 2) the use of MDSC scores in selecting melanoma patients with high probability to be responsive to ICBs; and 3) the relevance of the gut microbiome as a predictive factor, and the potential of its perturbation in increasing the immune response rate to ICBs. Overall, a picture emerged of tumor heterogeneity as the main limitation that impairs the effectiveness of anti-cancer therapies. Thus, the choice of a specific therapy based on reproducible and selective predictive biomarkers is an urgent unmet clinical need that should be addressed in order to increase the proportion of long-term responding patients and to improve the sustainability of novel drugs.

In vitro selection and characterization of mutants in TEM-1-producing Escherichia coli by ceftazidime and ceftibuten.

The present work was undertaken to study the ability of ceftazidime and ceftibuten to selectin vitro Escherichia coli HB101 harboring bla(TEM-1) beta-lactamase gene. Minimum inhibitory concentrations (MICs) of ceftazidime and ceftibuten were increased by a factor of 32, overcoming in the case of ceftazidime the breakpoint for clinical resistance. Outer membrane protein analysis and PCR for bla(TEM )alleles revealed that ceftazidime and ceftibuten select for different resistance mechanisms. Ceftazidime created mutants that encode an extended-spectrum beta-lactamase (TEM-12) and exhibit decreased expression of OmpF. Ceftibuten was unable to select for extended-spectrum beta-lactamase expressing mutants but reduced the expression of two porins, OmpC and OmpF. The stability of ceftibuten to hydrolysis and the difference in the structure of these beta-lactam antibiotics could be responsible for the selection of different mechanisms of resistance.

[Role of endovascular surgery in presence of rupture of the thoracic aorta in patients with severe multisystemic post-traumatic surgical lesions]. / Ruolo della chirurgia endovascolare nelle rotture aortiche del politraumatizzato con lesioni polidistrettuali di pertinenza chirurgica.

The endovascular treatment (ET) of traumatic rupture of the thoracic aorta (TRTA) may represent, particularly in patients with severe multisystemic post-traumatic surgical lesions, an alternative approach to traditional surgery. We observed (October 2001- November 2004) 5 male patients (age: range 23-42 years - average 32,4) affected by TRTA (3 isthmic aortic ruptures - 2 distal descending thoracic aorta ruptures), all successfully treated with an endovascular approach. The Glasgow Coma Score (GCS) ranged between 5 and 13. After performing resuscitation manoeuvres, all patients were investigated with total body CT scans in order to evaluate the thoracic aorta and to identify associated visceral lesions. In 4 cases were evident associated visceral lesions (3 cases: bone, abdominal and neurosurgical trauma - 1 case: bone, abdominal, neurosurgical and thoracic trauma). All the procedures were performed in the operative room using DSA (Digital Subtraction Angiography). The mean operating time was 105 minutes (range 80 - 125). We didn't observed early and late complications (follow-up: average 24 months, range 12-36). In conclusion the ET of TRTA represents in 'critical' patients with severe polytrauma an alternative approach to traditional surgery in order to 'stabilizing' the cardiovascular clinical parameters and to treating 'safety' the other associated surgical lesions.

Clonal analysis of gene loss of function and tissue-specific gene deletion in zebrafish via CRISPR/Cas9 technology.

In the last few years the development of CRISPR/Cas 9-mediated genome editing techniques has allowed the efficient generation of loss-of-function alleles in several model organisms including zebrafish. However, these methods are mainly devoted to target-specific genomic loci leading to the creation of constitutive knock-out models. On the contrary, the analysis of gene function via tissue- or cell-specific mutagenesis remains challenging in zebrafish. To circumvent this limitation, we present here a simple and versatile protocol to achieve tissue-specific gene disruption based on the Cas9 expression under the control of the Gal4/upstream activating sequence binary system. In our method, we couple Cas9 with green fluorescent protein or Cre reporter gene expression. This strategy allows us to induce somatic mutations in genetically labeled cell clones or single cells, and to follow them in vivo via reporter gene expression. Importantly, because none of the tools that we present here are restricted to zebrafish, similar approaches are readily applicable in virtually any organism where transgenesis and DNA injection are feasible.

Stroke in young adults in the community-based L'Aquila registry: incidence and prognosis.

BACKGROUND AND PURPOSE: Stroke type in the young may influence the outcome and may have a dramatic impact on the quality of life in survivors. This study aimed to evaluate the incidence and prognosis of first-ever stroke in the young and to make comparisons with older patients within a well-defined population. METHODS: All first-ever strokes occurring in the L'Aquila district, central Italy, were traced by active monitoring of inpatient and outpatient health services. Incidence rates were standardized to the 1996 European population according to the direct method. Long-term survival was estimated by the Kaplan-Meier method; outcome in survivors was evaluated by the modified Rankin scale. RESULTS: Of 4353 patients who had a first-ever stroke, 89 patients <45 years of age (55 men and 34 women) (2%) were identified in a 5-year period. Mean age+/-SD was 36.1+/-8.1 years. Twenty patients (22.5%) had a subarachnoid hemorrhage, 18 (20.2%) an intracerebral hemorrhage, and 51 (57.3%) a cerebral infarction. The corresponding proportions in patients >45 years of age were 2.4%, 13.3%, and 83.1%. Neuroimaging studies of the brain detected 14 intracranial aneurysms and 6 arteriovenous malformations in 20 of 38 patients (52.6%) with either subarachnoid (n=17) or intracerebral (n=3) hemorrhage. The crude annual incidence rate was 10.18/100,000 (95% CI, 8.14 to 12.57) and 10.23/100,000 when standardized to the 1996 European population. The 30-day case-fatality rate was 11.2% (95% CI, 6.2 to 19.4). Patients with subarachnoid hemorrhage had the highest proportion of good recovery (60%), patients with intracerebral hemorrhage had the highest mortality (44%), and patients with cerebral infarction had the highest proportion of severe disability (47%). CONCLUSIONS: Stroke patients <45 years of age showed a disproportionate cumulative high prevalence (42.7%) of subarachnoid and intracerebral hemorrhage with respect to older patients (15.7%), mainly (52.6%) due to aneurysms and arteriovenous malformations. Therefore, screening procedures and preventive strategies in the young should also be addressed to subjects at risk of subarachnoid and intracerebral hemorrhage.

Diagnostic and prognostic value of dipyridamole and dobutamine stress echocardiography in patients with Q-wave acute myocardial infarction.

The aim of this study was to compare dipyridamole and dobutamine stress echocardiography, performed early in patients with acute myocardial infarction (AMI) to evaluate residual ischemia, viability, and prognosis. Fifty patients (mean age 55 +/- 9 years, 47 men, 3 women) with AMI, all treated with thrombolytic therapy, underwent standard dipyridamole and dobutamine tests, within the fifth day of the event. Wall motion score index and the 16 segments model were used to evaluate contractility. Forty-seven patients underwent coronary angiography within the tenth day of the event. The mean follow-up was 24 +/- 12 months. No side effects occurred during both tests. Both dipyridamole and dobutamine tests were positive for ischemia, in 32 and 33 of 47 patients, respectively (sensitivity 73% and 75%; specificity 67% and 67%); these tests induced an improvement of contractility in 23 and 38 of 139 abnormal segments at baseline, respectively (sensitivity 52% and 86%; specificity 100% and 100%). Cardiac events occurred in 26 of 50 patients, 22 with a positive dipyridamole test and 21 with positive dobutamine test. Thus, both tests were feasible, safe, and useful to evaluate residual ischemia, viability, and prognosis. No significant differences were found in sensitivity and specificity between tests.

Epigenetic gene silencing in acute promyelocytic leukemia.

The recent explosion in our knowledge of how chromatin organization modulates gene transcription has highlighted the importance of epigenetic mechanisms in the initiation and progression of human cancer. These epigenetic changes--in particular, aberrant promoter hypermethylation that is associated with inappropriate gene silencing--affect virtually every step in tumor progression. Intriguingly, methylation patterns are severely altered in tumors, with an overall hypomethylation of the genome and hypermethylation of islands of CpGs clusters within specific DNA regions. Though overexpression of DNA methyltransferases (DNMTs) has been proposed to be a mechanism for aberrant genome methylation, it does not explain the specific regional hypermethylation in cancer cells. We have analyzed the role of chromatin modifying activities in cell transformation using acute promyelocytic leukemia as a model system. This disease is caused by expression of the PML-RARalpha fusion protein, thus offering the opportunity of studying the mechanisms of leukemogenesis through molecular investigation of the activity of the directly transforming protein. Recent evidence suggests that PML-RARalpha as well as other leukemia-associated fusion proteins induce changes in the chromatin structure. Specifically, aberrant recruitment of different chromatin modifying enzymes to specific promoters induces DNA hypermethylation and heterochromatin formation, which consequentially leads to the transcriptional silencing of that genes. Importantly, these epigenetic modifications were found to contribute to the leukemogenic potential of PML-RARalpha. These observations suggest that epigenetic alterations could actively contribute to the development of APL and other hyperproliferative diseases.

Tricuspid regurgitation due to sinus of Valsalva-right heart fistula diagnosed by two-dimensional echocardiography.

The case of a 31-year-old woman with severe right heart failure in the course of bacterial endocarditis and systolic and diastolic murmur at the third left intercostal space is described. Two-dimensional echocardiography showed a vegetation moving from the noncoronary aortic sinus of Valsalva to the right atrium, encroaching upon the septal leaflet of the tricuspid valve. An acquired fistula was confirmed by aortography and surgery. This is an unusual case of tricuspid regurgitation due to acquired aortic sinus of Valsalva-right heart fistula diagnosed by two-dimensional echocardiography.

The mucosa of the rabbit vagina: a proposed experimental model for correlated morphofunctional studies in humans.

In order to suggest a valid experimental model for correlated studies regarding the complex phenomena of reproductive biology in humans, light (LM), transmission (TEM) and scanning electron microscopic (SEM) observations were carried out on vaginae of sexually mature New-Zealand albino rabbits, both in estrus and at 5 h after mating. To avoid artifacts depending upon the operative times for specimen removal and, in particular, to well visualize the inner surface of this organ, vaginal irrigation with fixative was performed prior to the surgical removal. Our data demonstrate that the upper 2/3 of the vaginal duct form a kind of 'cervico-vaginal canal' which shares many histological features with the human endocervix. In fact, this portion is lined within by a monostratified epithelium provided with tall microvillous and ciliated cells and often invaginated in crypts. After mating, the microvillous cells showed evidence of secretory activity, exhibiting dome-like luminal surfaces. Secretory droplets often contacted cilia and spermatozoa present in the mucosal furrows and crypts. Because of these singular features and the morphofunctional interactions between vaginal epithelium and male gametes, we believe the rabbit to be a helpful model for comparative biology in humans, concerning sperm capacitation and the general reactivity of the female genital tract during the reproductive cycle.

Negative interference of Teflon sampling devices in the determination of nitric acid and particulate nitrate.

A laboratory study was performed to evaluate the transmission of nitric acid through different materials. Copper, stainless steel, sodium glass and chromium-plated stainless steel proved to be poor choices for the construction of size classifiers, transfer lines and filter holder plates of air sampling devices, as they adsorb significant amounts of nitric acid. Tubes made of Teflon or pyrex exhibited good HNO3 transmission but, unexpectedly, devices machined from Teflon rods (cyclones, manifolds) showed a noticeable sorption capacity. Moreover, the Teflon of different manufacturers behaved differently. The features of a sampling unit capable of accurate measurements of nitric acid and ammonium nitrate in the atmosphere are discussed.

Preliminary results of a complete study protocol on synthetic vascular graft healing and its complications.

The microscopic and anatomic features and bacteriologic culture results of different portions of single, explanted dacron synthetic vascular grafts (SVG) were studied together with patient clinical data. With this complete study protocol a better understanding of the healing process and its associated pathology can be achieved. We studied three, amply distanced graft portions from each of five patients (15 total graft portions) undergoing revision for infectious and non-infectious reasons. We divided the SVG portions studied into a Group 1, with high degrees of graft healing and into a Group 2, with both infection-dependent, early healing complications and perigraft chronic inflammatory reaction-dependent, late healing complications. These late healing complications were found dependent upon a host vs graft reaction. This study confirmed in humans the important role of an internal and external fibrotic graft incorporation in the definitive healing of a SVG. A host vs graft reaction was suggested to be an alternative to the frequently cited low virulent infection pathogenesis of late SVG healing complications. A sure definition and treatment of late SVG healing complications will only be established by means of a complete study protocol performed on a large number of explanted SVGs.

Corrosive effects from the deposition of gaseous pollutants on surfaces of cultural and artistic value inside museums.

The objectives of the project were to assess the critical relationships between environmental factors and damage of the artifacts and other cultural property exposed inside museums, by studying: (a) the outdoor/indoor pollutant concentration and their transfer inside the museum; (b) the distribution and circulation of pollutants inside the museum influenced by various factors; (c) chemical interactions between pollutants in the gas phase leading to removal and/or formation of secondary pollutants; (d) the final deposition of the indoor pollutants on surfaces of artistic interest and the damage on them, governed by strictly defined physicochemical parameters. All the above information, together with the main factors influencing each stage, were obtained by applying the methodology developed and described in detail here. Measurements of rate constants of reactions in the gas phase, of physicochemical deposition parameters on artefacts, and the synergistic effects of pollutants on the deposition parameters, were conducted. Seven PC programmes for analysing the experimental data were written and used. The pollutants, the solid materials and the museums chosen in this programme are only examples needed to develop the necessary methodology. The numerical results obtained serve the purpose of exemplifying the procedures and not enriching the world's bibliography with useless empirical information. Two commercially available protectives for marble were investigated from the point of view of their reactivity towards SO2 by using a diffusional technique. From measurements of SO2 concentration carried out on three types of marble, the deposition velocities have been calculated. Indoor monitoring of the church of San Luigi dei Francesi and of the Museo della Civiltá Romana in Rome has shown that indoor production of nitrous acid most likely results from heterogeneous reactions indoors, on the walls and the exposed surfaces.

The use of a new passive sampler for ozone and nitrogen oxides monitoring in ecological effects research.

A simple, cost-effective diffusive sampler is described that is suitable for measuring parts per billion (ppb) levels of ozone and nitrogen oxides. The diffusive sampler makes use of nitrite for ozone determination whereas for nitrogen oxides and nitrogen dioxide an active carbon tissue impregnated with sodium carbonate is used. Nitrate and nitrite, the formation of which is proportional to the pollutant concentration and sampling duration, are the two species analysed, respectively. Diffusion tubes have the advantage of being a low- cost, convenient way of mapping spatial distributions and investigating long-term trends of ozone and nitrogen oxides. The method is extremely useful for assessing long-term concentrations such as the annual mean for nitrogen oxides, as required by the Daughter Directive 1999/30/EC. Field tests to validate the method have been carried out at an urban background location with co-located passive samplers and continuous measurements of O3 and NOx. An application in ecological effects monitoring for ozone is also presented.