A MiR181/Sirtuin1 regulatory circuit modulates drug response in biliary cancers.

Despite recent advances, biliary tract cancer (BTC) remains one of the most lethal tumor worldwide due to late diagnosis, limited therapeutic strategies and resistance to conventional therapies. In recent years, high-throughput technologies have enabled extensive genome, and transcriptome sequencing unveiling, among others, the regulatory potential of microRNAs (miRNAs). Compelling evidence shown that miRNA are attractive therapeutic targets and promising candidates as biomarkers for various therapy-resistant tumors. The analysis of miRNA profile successfully identified miR-181c and -181d as significantly downregulated in BTC patients. Low miR-181c and -181d expression levels were correlated with worse prognosis and poor treatment efficacy. In fact, progression-free survival analysis indicated poor survival rates in miR-181c and -181d low expressing patients. The expression profile of miR-181c and -181d in BTC cell lines revealed that both miRNAs were dysregulated. Functional in vitro experiments in BTC cell lines showed that overexpression of miR-181c and -181d affected cell viability and increased sensitivity to chemotherapy compared to controls. In addition, by using bioinformatic tools we showed that the miR-181c/d functional role is determined by binding to their target SIRT1 (Sirtuin 1). Moreover, BTC patients expressing high levels of miR-181 and low SIRT1 shown an improved survival and treatment response. An integrative network analysis demonstrated that, miR-181/SIRT1 circuit had a regulatory effect on several important metabolic tumor-related processes. Our study demonstrated that miR-181c and -181d act as tumor suppressor miRNA in BTC, suggesting the potential use as therapeutic strategy in resistant cancers and as predictive biomarker in the precision medicine of BTC.

Role of viable but non culturable cells in patients with cystic fibrosis in the era of highly effective modulator therapy.

BACKGROUND: Lung infections antibiotic treatment in Cystic Fibrosis patients (pwCF) is often complicated by bacterial persisters, including the so-called Viable but Non Culturable (VBNC) forms, live cells undetected by the routine cultural microbiological methods. This study investigated the occurrence of VBNC cells of five CF bacterial pathogens in 94 pwCF over one year and the possible associations with the patients' clinical features. METHODS: Sputum samples, recovered at routine visits and during exacerbation episodes, were analyzed for the presence of the five pathogens by both routine culture-based assays and species-specific qPCR. VBNC cells were estimated as the difference between molecular and cultural counts and their presence was matched with the clinical data in particular the therapeutic regimens. RESULTS: All but ten pwCF showed the presence of VBNC cells at least once during the study. Pseudomonas aeruginosa and methicillin-susceptible Staphylococcus aureus were the species most frequently found in the VBNC state. Only the former showed a significant association between chronic infection and VBNC cells presence; VBNC-MSSA positive patients significantly increased overtime. The presence of non culturable bacteria was generally concurrent with poor lung functionality and more frequent pulmonary exacerbations. No significant association with modulator treatment was evidenced. CONCLUSIONS: The obtained data demonstrated the overwhelming occurrence of bacterial VBNC cells in CF lung infections, warranting a constant monitoring of pwCF and underlining the need of implementing the routine culture-based assays with culture-independent techniques. This is pivotal to understand the CF bacterial population dynamics and to efficiently contrast the lung infection progression and worsening.

De novo missense variants in phosphatidylinositol kinase PIP5KIγ underlie a neurodevelopmental syndrome associated with altered phosphoinositide signaling.

Phosphoinositides (PIs) are membrane phospholipids produced through the local activity of PI kinases and phosphatases that selectively add or remove phosphate groups from the inositol head group. PIs control membrane composition and play key roles in many cellular processes including actin dynamics, endosomal trafficking, autophagy, and nuclear functions. Mutations in phosphatidylinositol 4,5 bisphosphate [PI(4,5)P2] phosphatases cause a broad spectrum of neurodevelopmental disorders such as Lowe and Joubert syndromes and congenital muscular dystrophy with cataracts and intellectual disability, which are thus associated with increased levels of PI(4,5)P2. Here, we describe a neurodevelopmental disorder associated with an increase in the production of PI(4,5)P2 and with PI-signaling dysfunction. We identified three de novo heterozygous missense variants in PIP5K1C, which encodes an isoform of the phosphatidylinositol 4-phosphate 5-kinase (PIP5KIγ), in nine unrelated children exhibiting intellectual disability, developmental delay, acquired microcephaly, seizures, visual abnormalities, and dysmorphic features. We provide evidence that the PIP5K1C variants result in an increase of the endosomal PI(4,5)P2 pool, giving rise to ectopic recruitment of filamentous actin at early endosomes (EEs) that in turn causes dysfunction in EE trafficking. In addition, we generated an in vivo zebrafish model that recapitulates the disorder we describe with developmental defects affecting the forebrain, including the eyes, as well as craniofacial abnormalities, further demonstrating the pathogenic effect of the PIP5K1C variants.

An Effective Model for Estimating Age in Unaccompanied Minors under the Italian Legal System.

This article presents an effective model for estimating the age of subjects without identification documents, in accordance with Italian legislation covering unaccompanied minors, using instrumental methods recognized by the scientific community for age estimation. A decision-making tree has been developed, in which the first step is a physical examination. If secondary sexual characteristics are fully developed and there are no obvious signs of abnormal growth, dental X-rays are the next step. If the roots of the seven left mandibular teeth between the central incisor and the second molar are completely developed, the focus then moves to the third molar. If the index of the third molar (I3M) value is less than 0.08, or if third molars are not assessable, the following step is to study the clavicle which, if fully formed, indicates that the subject is an adult with 99.9% probability; otherwise, the probability is 96%. In all other cases (where the I3M is over 0.08), the probability that the subject has reached 18 years is less than 60%. The research, carried out initially on x-rays of the wrist, teeth and clavicle, highlighted the uselessness of the x-ray of the wrist for determining the age of majority, because in our sample, all subjects with incomplete maturity of hand/wrist bones were under 16 years of age; thus, OPT was necessary anyway. What we propose is a practical, easily feasible, fast, economical, and extremely reliable method, which can be used on Caucasian populations and beyond for multiple forensic purposes.

Combining Bayesian Calibration and Copula Models for Age Estimation.

Accurately estimating and predicting chronological age from some anthropometric characteristics of an individual without an identity document can be crucial in the context of a growing number of forced migrants. In the related literature, the prediction of chronological age mostly relies upon the use of a single predictor, which is usually represented by a dental/skeletal maturity index, or multiple independent ordinal predictor (stage of maturation). This paper is the first attempt to combine a robust method to predict chronological age, such as Bayesian calibration, and the use of multiple continuous indices as predictors. The combination of these two aspects becomes possible due to the implementation of a complex statistical tool as the copula. Comparing the forecasts from our copula-based method with predictions from an independent model and two single predictor models, we showed that the accuracy increased.

One-lung ventilation in obese patients undergoing thoracoscopic lobectomy for lung cancer.

BACKGROUND: We evaluated the safety and feasibility of one-lung ventilation in obese patients undergoing thoracoscopic lobectomy and whether obesity affected peri- and postoperative outcomes. METHODS: This was a retrospective single center study including consecutive patients undergoing thoracoscopic lobectomy between October 2019 and February 2022. Obese patients were statistically compared to a control group to evaluate any differences in relation to one-lung ventilation and peri- and postoperative outcomes. RESULTS: Our study population included 111 patients; of these, 26 (23%) were included in the obese group, while 85 (77%) were included within the nonobese group. To obtain one-lung ventilation in nonobese patients, a double-lumen tube was more frequently used than a single-lumen tube with bronchial blocker (61% vs. 39%; p = 0.02), while in obese patients a single-lumen tube with bronchial blocker was used more than a double-lumen tube (81% vs. 19%, p = 0.001). Intergroup comparison showed that a double-lumen tube was the preferred method in nonobese patients, while a single-lumen tube with bronchial blockers was the strategy of choice in obese patients (p = 0.0002). Intubation time was longer in the obese group than in the nonobese group (94.0 ± 6.1 vs. 85.0 ± 7.0 s; p = 0.0004) and failure rate of first attempt at intubation was higher in the obese group (23% vs. 5%; p = 0.01). Obesity was not associated with increased intra-, peri- and postoperative complications and/or mortality. CONCLUSIONS: One-lung ventilation is a feasible and safe procedure also in obese patients and obesity did not negatively affect peri- and postoperative outcomes after lung resection.

Targeting the MITF/APAF-1 axis as salvage therapy for MAPK inhibitors in resistant melanoma.

Melanoma is a deadly form of cancer characterized by remarkable therapy resistance. Analyzing the transcriptome of MAPK inhibitor sensitive- and resistant-melanoma, we discovered that APAF-1 is negatively regulated by MITF in resistant tumors. This study identifies the MITF/APAF-1 axis as a molecular driver of MAPK inhibitor resistance. A drug-repositioning screen identified quinacrine and methylbenzethonium as potent activators of apoptosis in a context that mimics drug resistance mediated by APAF-1 inactivation. The compounds showed anti-tumor activity in in vitro and in vivo models, linked to suppression of MITF function. Both drugs profoundly sensitize melanoma cells to MAPK inhibitors, regulating key signaling networks in melanoma, including the MITF/APAF-1 axis. Significant activity of the two compounds in inhibiting specific epigenetic modulators of MITF/APAF-1 expression, such as histone deacetylases, was observed. In summary, we demonstrate that targeting the MITF/APAF-1 axis may overcome resistance and could be exploited as a potential therapeutic approach to treat resistant melanoma.

Occurrence of Idiopathic Pulmonary Fibrosis in Italy: Latest Evidence from Real-World Data.

The aim of the study was to evaluate the trend in the incidence of idiopathic pulmonary fibrosis (IPF) in a real-world setting of the Marche region, a region of Central Italy, between 2014 and 2019. This observational prospective study was based on administrative databases of hospital discharges and drug prescriptions. All adult residents in the Marche Region with a first prescription of antifibrotic drugs, or a first hospitalization with a diagnosis of IPF during the study period, were identified as incident cases of IPF. A multiple Poisson regression analysis was used to estimate the IPF incidence trend, adjusted for age, sex, and health conditions. The mean incidence rate was 9.8 cases per 100,000 person-years. A significant increasing trend of 6% per year was observed. The incidence rates were significantly higher in males than females, older subjects, and those with poorer health conditions. To our knowledge, this is the first study evaluating incidences of IPF over a 6-year period in Italy, combining hospital discharge and drug prescription databases. The study highlights that the combined use of two secondary sources is a reliable strategy to accurately identify new cases of IPF when the appropriate disease registry is lacking.

Correlation study between anatomopathological data and levels of blood morphine concentrations in heroin-related deaths.

Heroin-related mortality is a complex phenomenon involving several factors that make an individual more susceptible to opioid toxicity. Among these, pre-existing pathological conditions play an important role. The current paper reviewed 51 autopsied cases performed over the last 30 years, each subject ("frequent heroin user") having taken heroin intravenously alone before death. For each case, total blood morphine concentration determined by gas chromatography-mass spectrometry (GC-MS) was obtained from the archives. For histopathological evaluation we examined microscopic sections of the heart, lung, brain, liver and kidney, and for each organ a score table with a numerical value from 1 to 5, as an indicator of increasing severity, was drawn up. Finally, we created a scoring system based on the Injury Severity Score (ISS), generated by the sum of the squares of the three highest organ scores. We investigated the relationship between total blood morphine value (mg/l) and pathological score by inserting the two variables in a linear regression model: Score = a + b·Morphinemia (p-value <0.001). The results demonstrated a statistically significant correlation between the two variables: individuals with low total blood morphine values tended to have a higher pathological score. Our study provides useful assessment tools for forensic practice, confirming the need to combine two important disciplines in drug death investigation: forensic pathology and toxicology.

Dental age estimation in children affected by juvenile rheumatoid arthritis.

Dental root calcification has proven to be a reliable biological evidence to estimate chronological age of children. The development of structures usually examined in the age estimation forensic practice (e.g. skeleton, teeth) is supposed to be influenced by diseases and nutritional, environmental, ethnic, and ultimately even socioeconomic factors. This research aims to study the age estimation in children affected by juvenile rheumatoid arthritis (JRA) with and without steroids treatment and compared with healthy subjects. MATERIAL AND METHODS: Dental age estimations based on 752 OPGs, 420 girls and 332 boys, aged from 3.3 to 15.99 years, were provided by applying Demirjian and Willems' original methods. Of the whole sample, 103 individuals were affected by JRA and 40 received a continuous corticosteroid therapy, over 1 year long. CONCLUSIONS: Willems' and Demirjian's original methods, as methods commonly applied to estimate age for sub-adults with unremarkable medical history, can be used for medico-legal purposes to children affected by JRA. Willems' method tended to underestimate age while Demirjian's method resulted to be prone to overestimation for both healthy and JRA-affected children. JRA showed to have no influence on root calcification process even in children that received steroid treatment for 1 year or longer.

Comparison of Frontal Sinuses for Personal Identification in 3 Populations Using Cameriere's Code Number.

ABSTRACT: The identification of unknown individuals is of crucial importance to society, especially in the context of mass disasters involving individuals of different nationalities. The comparison of frontal sinus patterns is an important tool for personal identification. In Cameriere's method, a new personal code number (codC) containing 8 digits was elaborated, based on the measurements of frontal sinus patterns. The present study is an update of this previous work. Digital radiographic images of the skulls of 299 individuals of different nationalities (100 Italians, 99 Kosovars, and 100 Turks) were analyzed, adopting the same maximum-weight dependence tree used in the original work. For comparisons between groups, Fisher exact test and the χ2 test were carried out. The method, applied to different population groups, significantly increases the likelihood of identifying a person by comparison of antemortem and postmortem frontal sinus radiographs. Results demonstrated that the model is more discriminative in identifying individuals of different nationalities.

Regulation of autophagosome biogenesis by OFD1-mediated selective autophagy.

Autophagy is a lysosome-dependent degradation pathway essential to maintain cellular homeostasis. Therefore, either defective or excessive autophagy may be detrimental for cells and tissues. The past decade was characterized by significant advances in molecular dissection of stimulatory autophagy inputs; however, our understanding of the mechanisms that restrain autophagy is far from complete. Here, we describe a negative feedback mechanism that limits autophagosome biogenesis based on the selective autophagy-mediated degradation of ATG13, a component of the ULK1 autophagy initiation complex. We demonstrate that the centrosomal protein OFD1 acts as bona fide autophagy receptor for ATG13 via direct interaction with the Atg8/LC3/GABARAP family of proteins. We also show that patients with Oral-Facial-Digital type I syndrome, caused by mutations in the OFD1 gene, display excessive autophagy and that genetic inhibition of autophagy in a mouse model of the disease, significantly ameliorates polycystic kidney, a clinical manifestation of the disorder. Collectively, our data report the discovery of an autophagy self-regulated mechanism and implicate dysregulated autophagy in the pathogenesis of renal cystic disease in mammals.

Italian Response to Coronavirus Pandemic in Dental Care Access: The DeCADE Study.

The aim of the study was to evaluate the impact of the coronavirus pandemic on the willingness, anxiety and concerns of Italian people on undergoing dental appointments. An anonymous survey was posted online on social media on 11 May 2020 and was completed by 1003 respondents in one week. Multiple correspondence analysis and multiple logistic regression were used to evaluate the association between socio-demographic characteristics, dental care access, contagion fear of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), trust in dentists regarding sanitization procedures and perception of the impact of the risk of contagion on dental care. Subjects with a high level of education, attending public dental offices and that are used to go to dental offices for urgent care would not feel comfortable in undergoing a dental appointment and would prefer to postpone or cancel dental visits, waiting for a decrease in the number of the contagions. Moreover, the risk of canceling or postponing the appointment at the dentist was 1.59 times greater in those who claimed to be strongly influenced by SARS-CoV-2. Fear of coronavirus disease (COVID-19), new cases decrease and the not urgent nature of dental visits influenced more than the lowered income household on upcoming or resuming dental appointments. In the next months, despite the forecasted economic crisis caused by coronavirus pandemic, fear and anxiety generated by the spread of the virus will impact more than the lowered familiar income with regards to access to dental care.

Analysis of carpal bones on MR images for age estimation: First results of a new forensic approach.

Current multifactorial age estimation methods are based on radiography, however, in the forensic field there is growing interest in using magnetic resonance imaging (MRI). With regard to the carpal region, MRI provides more information for defining the individual ossification nuclei and the cartilage surrounding single bones. During the phase of bone growth, the progressive reduction of the cartilage layer is accompanied by the development of a cartilage-bone interface. The aim of our study was to create a new model for age estimation, based on the ratio between the area occupied by the nucleus of ossification (NO) and the surface of growth (SG) of each carpal bone, the latter derived by adding NO to the area of cartilage-bone interface. We analyzed 57 MRI scans of Italian subjects aged between 12 and 20 years, without growth diseases, endocrine disorders or osteodystrophy. Measurements of NO and SG areas were extracted using ImageJ software, and the ratio between the NO and SG of each bone (NOSG) was calculated. A multiple linear regression model was used to estimate the individual's age as a function of the variables: gender and wrist bone measurements. The results showed that the best model was obtained with 6 predictors (nvmax=6): Gender, and the NOSG of the Trapezoid, Trapezium, Scaphoid, Pisiform, and Capitate. The median of the residuals (observed age minus predicted age) was -0.025 years, with an IQR of 0.19 years. Thus a new forensic approach to age assessment using MRI is introduced in this paper, which gives the preliminary results.

TGF-ß1 secreted by pancreatic stellate cells promotes stemness and tumourigenicity in pancreatic cancer cells through L1CAM downregulation.

Pancreatic stellate cells (PSCs) secrete high levels of transforming growth factor-ß1 (TGF-ß1) that contributes to the development of pancreatic ductal adenocarcinoma (PDAC). TGF-ß1 modulates the expression of L1 cell adhesion molecule (L1CAM), but its role in tumour progression still remains controversial. To clarify L1 function in PDAC and cellular phenotypes, we performed L1CAM cell sorting, silencing and overexpression in several primary pancreatic cancer cells. PSCs silenced for TGF-ß1 were used for crosstalk experiments. We found that TGF-ß1 secreted by PSCs negatively regulates L1CAM expression, through canonical TGF-ß-Smad2/3 signalling, leading to a more aggressive PDAC phenotype. Cells with reduced expression of L1CAM harboured enhanced stemness potential and tumourigenicity. Inactivation of TGF-ß1 signalling in PSCs strongly reduced the aggressiveness of PDAC cells. Our data provide functional proof and mechanistic insights for the tumour-suppressive function of L1CAM via reducing stemness. Rescuing L1CAM expression in cancer cells through targeting of TGF-ß1 reverses stemness and bears the potential to improve the still miserable prognosis of PDAC patients.

Validation of the third molar maturity index cut-off value of <0.08 for indicating legal age of 18 years in Eastern Chinese region.

Having a reliable, inexpensive and simple method to estimate 18 years of age would be of help in the forensic field. This study aimed to test the accuracy of the third molar maturity index (I3M) in indicating the legal adult age of 18 years. This retrospective cross-sectional study analysed 450 digital panoramic images of Eastern Chinese children and young adults (226 females and 224 males) aged between 14 and 22 years. A cut-off value of I3M < 0.08 was tested in discriminating adults from minors for both sex. For females, the sensitivity of the test (Se) was 75.0%, with a 95% confidence interval (95%CI) of 67.5% to 82.5%. The specificity of the test (Sp) was 100%. The proportion accurately classified (Ac) individuals was 85.8% (95%CI, 81.3% to 90.4%). The Bayes post-test probability was 100% (93.6% to 100%). For males, Se, Sp and Ac were 91.9% (95%CI, 87.1% to 96.7%), 92.0%(95%CI, 86.7% to 97.3%) and 92% (95%CI, 88.4% to 95.5%), respectively. The Bayes post-test probabilities was 92% (95%CI, 88.4% to 95.5%). Males were ahead in the development of third molars comparing to females according to I3M. A stepwise logistic regression analysis showed that both I3M and sex contribute to the regression model to discriminate adults (≥18 years) from minors (<18 years), while a receiver operating curve (ROC) analysis indicated some better accuracy of I3M < 0.12 in females, without statistically significant difference when compared to I3M < 0.08. The results of this study show that the cut-off value of I3M < 0.08 may help to discriminate Eastern Chinese adults from minors. However, further study should evaluate the usefulness and possible variability of I3M cut-off value in a specific population before used for legal and forensic procedures.

Age estimation in Turkish children and young adolescents using fourth cervical vertebra.

This study aimed to test radiographic projections of the posterior and anterior sides of the C4 vertebral body for age estimation in a cross-sectional sample of Turkish children and adolescents from the Baskent University Faculty of Dentistry. A sample of 232 x-rays from individuals aged between 5 and 15 years (101 boys and 131 girls) was analysed to investigate the correlation of age and changes in the projection ratio of the anterior and posterior sides of the body of the fourth cervical vertebra (Vba) in lateral cephalograms. The Normal Bayesian Calibration (NBC) model for age estimation was used to assess the relationship between Vba and age. The intra-observer repeatability and inter-observer agreement were 0.95 and 0.94, respectively. Age distribution gradually increased as Vba increased up to 14 years in both sexes. The difference between chronological and estimated ages did not increase with the increase in age. Calibration distributions of the dataset by the evaluation of Vba suggested mean absolute errors (MAEs) of 0.879 years and 0.906 years (mean interquartile ranges (MIQRs) 1.290 and 1.435 years)) in boys and girls, respectively. The biases of the estimates (ßERR) were 0.013 and 0.027 for boys and girls, respectively. In conclusion, the NBC method on Vba variable appears to be suitable for age estimation in Turkish children and adolescents.

Analysis of Frontal Sinuses for Personal Identification in a Chinese Sample Using a New Code Number.

The study of frontal sinuses for personal identification is a considered approach in the forensic field. In Yoshino's system, the frontal sinus patterns of a given person were formulated as a code number (codY) obtained by arranging the class numbers in each classification item. The aim of this work was to use a new code number (codC) with eight digits that includes other two continuous variables obtained as ratios SOR1 (left frontal sinus area/left orbit area) and SOR2 (right frontal sinus area/right orbit area), comparing the results with that of Yoshino. Digital radiographic images of the skulls of 150 Chinese people were analyzed. Spearman's partial correlation coefficients were evaluated between variables characterizing frontal sinus patterns. We used a maximum-weight dependence tree for statistical analysis. Results demonstrated that codC significantly reduced the probability of having the same personal code number compared with codY. Our scientific approach results valid for personal identification purposes.

The prognostic value of anterior commissure involvement in T1 glottic cancer: A systematic review and meta-analysis.

OBJECTIVE: The anterior commissure is considered a laryngeal subsite, but it is not taken into account in the current T-staging system. The impact of anterior commissure involvement (ACI) on the oncologic outcomes of T1 glottic tumors has not been well defined and remains controversial. The aim of this study was to assess the influence of ACI in the prognosis of T1 glottic tumors. METHODS: A systematic review and meta-analysis of observational studies was performed by including studies published up to July 2019. Studies were selected if they had investigated the local control rate (LCR) at 5 years of clinical stage I (T1 and N0) glottic squamous cell carcinoma with and without ACI. The difference in LCR at 5 years between T1 glottic tumors without and with ACI was evaluated. RESULTS: The meta-analysis showed that the difference in LCR at 5 years between T1 glottic tumors without and with ACI is 12% (95% confidence interval: 8%-16%, p < 0,0001, I2 = 34.81%). CONCLUSIONS: Our study pointed out that the anterior commissure involvement is a negative prognostic factor in LCR at 5 years for T1 glottic tumors. Our results suggest the need to consider ACI in the T staging of glottic tumors. Laryngoscope, 130:1932-1940, 2020.

Segmented Bayesian calibration approach for estimating age in forensic science.

Forensic age estimation is receiving growing attention from researchers in the last few years. Accurate estimates of age are needed both for identifying real age in individuals without any identity document and assessing it for human remains. The methods applied in such context are mostly based on radiological analysis of some anatomical districts and entail the use of a regression model. However, estimating chronological age by regression models leads to overestimated ages in younger subjects and underestimated ages in older ones. We introduced a full Bayesian calibration method combined with a segmented function for age estimation that relied on a Normal distribution as a density model to mitigate this bias. In this way, we were also able to model the decreasing growth rate in juveniles. We compared our new Bayesian-segmented model with other existing approaches. The proposed method helped producing more robust and precise forecasts of age than compared models while exhibited comparable accuracy in terms of forecasting measures. Our method seemed to overcome the estimation bias also when applied to a real data set of South-African juvenile subjects.