Identification of a set of genes potentially responsible for resistance to ferroptosis in lung adenocarcinoma cancer stem cells.

Scientific literature supports the evidence that cancer stem cells (CSCs) retain inside low reactive oxygen species (ROS) levels and are, therefore, less susceptible to cell death, including ferroptosis, a type of cell death dependent on iron-driven lipid peroxidation. A collection of lung adenocarcinoma (LUAD) primary cell lines derived from malignant pleural effusions (MPEs) of patients was used to obtain 3D spheroids enriched for stem-like properties. We observed that the ferroptosis inducer RSL3 triggered lipid peroxidation and cell death in LUAD cells when grown in 2D conditions; however, when grown in 3D conditions, all cell lines underwent a phenotypic switch, exhibiting substantial resistance to RSL3 and, therefore, protection against ferroptotic cell death. Interestingly, this phenomenon was reversed by disrupting 3D cells and growing them back in adherence, supporting the idea of CSCs plasticity, which holds that cancer cells have the dynamic ability to transition between a CSC state and a non-CSC state. Molecular analyses showed that ferroptosis resistance in 3D spheroids correlated with an increased expression of antioxidant genes and high levels of proteins involved in iron storage and export, indicating protection against oxidative stress and low availability of iron for the initiation of ferroptosis. Moreover, transcriptomic analyses highlighted a novel subset of genes commonly modulated in 3D spheroids and potentially capable of driving ferroptosis protection in LUAD-CSCs, thus allowing to better understand the mechanisms of CSC-mediated drug resistance in tumors.

Influence of Chemical Composition and Structure on the Cooperative Fluctuation in Supercooled Glass-Forming Liquids.

The kinetics of the glass transition and the characteristic size of the fluctuating spatio-temporal domains in supercooled glass-forming liquids, i.e., the Cooperatively Rearranging Regions (CRR), were measured upon cooling over a broad range of cooling rates using Differential Scanning Calorimetry (DSC) and chip-based Fast Scanning Calorimetry (FSC). The investigations were conducted on a selection of fragile glass formers (fragility indices between 80 and 140), with a large variance in the atomic or molecular structure but comparable thermal glass transition temperatures Tg, with the aim of evaluating the influence of chemical composition and structure on the CRR size and the associated temperature fluctuation. The selected materials are two polymers (poly(vinyl acetate) (PVAc), poly(lactic acid) (PLA)) as well as the simplest chalcogenide glass-former (selenium). It turned out that the CRR size plotted against the reduced temperature T/Tg follows the same trend, irrespective of the considered glass-former.

ALDOC- and ENO2- driven glucose metabolism sustains 3D tumor spheroids growth regardless of nutrient environmental conditions: a multi-omics analysis.

BACKGROUND: Metastases are the major cause of cancer-related morbidity and mortality. By the time cancer cells detach from their primary site to eventually spread to distant sites, they need to acquire the ability to survive in non-adherent conditions and to proliferate within a new microenvironment in spite of stressing conditions that may severely constrain the metastatic process. In this study, we gained insight into the molecular mechanisms allowing cancer cells to survive and proliferate in an anchorage-independent manner, regardless of both tumor-intrinsic variables and nutrient culture conditions. METHODS: 3D spheroids derived from lung adenocarcinoma (LUAD) and breast cancer cells were cultured in either nutrient-rich or -restricted culture conditions. A multi-omics approach, including transcriptomics, proteomics, and metabolomics, was used to explore the molecular changes underlying the transition from 2 to 3D cultures. Small interfering RNA-mediated loss of function assays were used to validate the role of the identified differentially expressed genes and proteins in H460 and HCC827 LUAD as well as in MCF7 and T47D breast cancer cell lines. RESULTS: We found that the transition from 2 to 3D cultures of H460 and MCF7 cells is associated with significant changes in the expression of genes and proteins involved in metabolic reprogramming. In particular, we observed that 3D tumor spheroid growth implies the overexpression of ALDOC and ENO2 glycolytic enzymes concomitant with the enhanced consumption of glucose and fructose and the enhanced production of lactate. Transfection with siRNA against both ALDOC and ENO2 determined a significant reduction in lactate production, viability and size of 3D tumor spheroids produced by H460, HCC827, MCF7, and T47D cell lines. CONCLUSIONS: Our results show that anchorage-independent survival and growth of cancer cells are supported by changes in genes and proteins that drive glucose metabolism towards an enhanced lactate production. Notably, this finding is valid for all lung and breast cancer cell lines we have analyzed in different nutrient environmental conditions. broader Validation of this mechanism in other cancer cells of different origin will be necessary to broaden the role of ALDOC and ENO2 to other tumor types. Future in vivo studies will be necessary to assess the role of ALDOC and ENO2 in cancer metastasis.

A successful defence strategy in grapevine cultivar 'Tocai friulano' provides compartmentation of grapevine Flavescence dorée phytoplasma.

BACKGROUND: Flavescence dorée (FD) is a grapevine disease caused by phytoplasma and it is one of the most destructive pathologies in Europe. Nowadays, the only strategies used to control the epidemics are insecticides against vector, but more sustainable techniques are required. Completely resistant Vitis vinifera varieties have not been uncovered yet, but differences in susceptibility among cultivars and spontaneous recovery from FD symptoms have been observed. The grapevine cultivar 'Tocai friulano' shows very low susceptibility to FD but its defence strategy to counteract the phytoplasma spread has not been deciphered yet. In this work, the mechanisms occurring within 'Tocai friulano' FD-infected plants were examined in depth to identify the phytoplasma distribution and the defence pathways involved. RESULTS: In 'Tocai friulano' symptoms of FD-infection remained confined near the area where they appeared during all the vegetative season. Analyses of secondary phloem showed a total absence of FD phytoplasma (FDp) in the trunk and its disappearance in 2-year-old arms from July to November, which was different from 'Pinot gris', a highly susceptible variety. Diverse modulations of defence genes and accumulation of metabolites were revealed in 1-year-old canes of 'Tocai friulano' FD-infected plants, depending on the sanitary status. Symptomatic portions showed high activation of both jasmonate- and salicylate-mediated responses, together with a great accumulation of resveratrol. Whereas activation of jasmonate-mediated response and high content of ε-viniferin were identified in asymptomatic 1-year-old cane portions close to the symptomatic ones. CONCLUSION: Successful defence mechanisms activated near the symptomatic areas allowed the compartmentation of FD symptoms and phytoplasmas within the infected 'Tocai friulano' plants. These results could suggest specific agronomical practices to be adopted during FD management of this variety, and drive research of resistance genes against FD.

Effects of antiepileptic therapy on bone mineral status evaluated by phalangeal quantitative ultrasound in pediatric patients with epilepsy and motor impairment.

BACKGROUND: In epileptic patients with motor disability, it's difficult to disentangle the effects of antiepileptic drugs (AEDs) on bone health from those provoked by impaired mobility. The aim of this study was to evaluate the effects of AEDs on bone mineral status by phalangeal quantitative ultrasound (QUS), a no-radiation and non-invasive method, in pediatric patients with motor impairment and epilepsy. METHODS: We enrolled 56 patients (31 females, 25 males) with epilepsy and motor impairment and 24 children with only motor disability (13 females, 11 males). Patients were stratified by Gross Motor Function Classification System Scale (GMFCS) in 4 groups: group A1 with epilepsy and mild motor impairment (GMFCS levels I-II), group A2 with only mild motor impairment, group B1 with epilepsy and severe motor impairment (GMFCS levels III-V), group B2 with only severe motor impairment. The bone mineral status was evaluated by phalangeal QUS and amplitude-dependent speed of sound (AD-SoS) Z-score was calculated for each patient. RESULTS: The four groups showed no significant differences in age, gender and 25-hydroxyvitamin D levels. The group B1 had a statistically lower amplitude-dependent speed of sound Z-score as compared to group A2 (P<0.05). The multivariate analysis of independent factors revealed a significant correlation between amplitude-dependent speed of sound Z-score and Gross Motor Function Classification System levels (P=0.004). The mean Z-score value decreased by 0.53, increasing the motor impairment. CONCLUSIONS: The bone mineral status measured as AD-SoS strongly correlates with severity of motor disability evaluated by GMFCS as compared to antiepileptic therapy and 25-hydroxyvitamin D levels.

Evaluation of the CNC® prosthetic system in recurrent breast cancer patients with chemotherapy-induced alopecia: a pilot study.

BACKGROUND: Chemotherapy-induced alopecia (CIA), although generally reversible, is felt as extremely distressing by patients with breast cancer. A certified medical device (Capelli Naturali a Contatto®-CNC®) was produced to provide patients with a personalized scalp prosthesis, reproducing the patient's original hair, resistant to any type of everyday or sporting activity, and hairdressing. AIMS: The present study aimed to evaluate the impact of the CNC® device on the patient's perception of their body image, psychological wellbeing, satisfaction, strengths and weakness of the CNC® device. METHOD: A pilot study was carried out on 21 patients affected by CIA due to recurrent breast cancer. A mixed quantitative/qualitative method was used, including administering a questionnaire and a focus group. RESULTS: Based on the Body Image Scale, body image perception improved after 3 and 6 months using the device in the 20 patients who answered the questionnaire. No significant change over time emerged for the six dimensions investigated by the Italian version of the Psychological Well-Being Scale. The thematic analysis of the focus groups showed six themes: definition of the prosthetic device, acceptance of the proposal, experience with the conventional wig, strengths, weaknesses, economic issues. CONCLUSION: Compared to the previous experience of CIA and the standard wig, the use of the CNC® device improved everyday life and may be proposed to women undergoing chemotherapy and expecting alopecia to prevent discomfort, social embarrassment, and compromised body image.

B4GALT1 as a New Biomarker of Idiopathic Pulmonary Fibrosis.

Idiopathic pulmonary fibrosis (IPF) is a disease characterized by progressive scarring of the lung that involves the pulmonary interstitium. The disease may rapidly progress, leading to respiratory failure, and the long-term survival is poor. There are no accurate biomarkers available so far. Our aim was to evaluate the expression of the B4GALT1 in patients with IPF. Analysis of B4GALT1 gene expression was performed in silico on two gene sets, retrieved from the Gene Expression Omnibus database. Expression of B4GALT1 was then evaluated, both at the mRNA and protein levels, on lung specimens obtained from lung biopsies of 4 IPF patients, on one IPF-derived human primary cell and on 11 cases of IPF associated with cancer. In silico re-analysis demonstrated that the B4GALT1 gene was overexpressed in patients and human cell cultures with IPF (p = 0.03). Network analysis demonstrated that B4GALT1 upregulation was correlated with genes belonging to the EMT pathway (p = 0.01). The overexpression of B4GALT1 was observed, both at mRNA and protein levels, in lung biopsies of our four IPF patients and in the IPF-derived human primary cell, in other fibrotic non-lung tissues, and in IPF associated with cancer. In conclusion, our results indicate that B4GALT1 is overexpressed in IPF and could represent a novel marker of this disease.

Deconvolution of malignant pleural effusions immune landscape unravels a novel macrophage signature associated with worse clinical outcome in lung adenocarcinoma patients.

BACKGROUND: Immune checkpoint inhibitors are still unable to provide clinical benefit to the large majority of non-small cell lung cancer (NSCLC) patients. A deeper characterization of the tumor immune microenvironment (TIME) is expected to shed light on the mechanisms of cancer immune evasion and resistance to immunotherapy. Here, we exploited malignant pleural effusions (MPEs) from lung adenocarcinoma (LUAD) patients as a model system to decipher TIME in metastatic NSCLC. METHODS: Mononuclear cells from MPEs (PEMC) and peripheral blood (PBMC), cell free pleural fluid and/or plasma were collected from a total of 24 LUAD patients and 12 healthy donors. Bulk-RNA sequencing was performed on total RNA extracted from PEMC and matched PBMC. The DEseq2 Bioconductor package was used to perform differential expression analysis and CIBERSORTx for the regression-based immune deconvolution of bulk gene expression data. Cytokinome analysis of cell-free pleural fluid and plasma samples was performed using a 48-Plex Assay panel. THP-1 monocytic cells were used to assess macrophage polarization. Survival analyses on NSCLC patients were performed using KM Plotter (LUAD, N=672; lung squamous cell carcinoma, N=271). RESULTS: Transcriptomic analysis of immune cells and cytokinome analysis of soluble factors in the pleural fluid depicted MPEs as a metastatic niche in which all the components required for an effective antitumor response are present, but conscripted in a wound-healing, proinflammatory and tumor-supportive mode. The bioinformatic deconvolution analysis revealed an immune landscape dominated by myeloid subsets with the prevalence of monocytes, protumoral macrophages and activated mast cells. Focusing on macrophages we identified an MPEs-distinctive signature associated with worse clinical outcome in LUAD patients. CONCLUSIONS: Our study reports for the first time a wide characterization of MPEs LUAD microenvironment, highlighting the importance of specific components of the myeloid compartment and opens new perspectives for the rational design of new therapies for metastatic NSCLC.

Italian telephone-based Mini-Mental State Examination (Itel-MMSE): item-level psychometric properties.

BACKGROUND: The Italian telephone-based Mini-Mental State Examination (Itel-MMSE), despite being psychometrically sound, has shown relevant ceiling effects, which may negatively impact the interpretation of its scores. In address to overcome such an issue, this study aimed at providing item-level insights on the Itel-MMSE through Item Response Theory (IRT) analyses. METHODS: Five-hundred and sixty-seven healthy Italian adults (227 males, 340 females; mean age: 51 ± 17 years, range 18-96; mean education: 13.31 ± 4.3 years). A two-parameter logistic IRT model was implemented to assess item discrimination and difficulty of the Itel-MMSE. Construct unidimensionality, statistical independence of items, and model and item fit were tested. Informativity levels were also assessed graphically. RESULTS: With respect to the Itel-MMSE total score, ceiling effects were found in 92.7% of participants. Unidimensionality was violated; both model and item fit were poor; a few items showed statistical dependence. Both the whole test and its items proved to be scarcely informative, especially for medium-to-high levels of ability, except for attention and spatial orientation subtests, which consistently yielded the highest discriminative capability. DISCUSSION: The Itel-MMSE appears to be most informative in low-performing healthy individuals. However, the present findings should not lead practitioners to aprioristically equate ceiling effects/low informativity to clinical uselessness. Items assessing attention and, to a lesser extent, spatial orientation appear to be the most informative.

Diagnostic properties of the Frontal Assessment Battery (FAB) in Italian healthy adults.

BACKGROUND: Sub-clinical cognitive efficiency deficits of a dysexecutive nature are moderately prevalent in healthy older adults and negatively affect their functional outcomes. To screen for such dysfunctions, the Frontal Assessment Battery (FAB) provided promising evidence, although its diagnostic properties have not been tested to date. This study thus aimed at exploring the performance on the FAB of a large sample of Italian healthy adults and comparing it in individuals aged < 75 years vs. ≥ 75 years. METHODS: Four hundred and seventy-five healthy adults (169 males, 306 females, age: 61.1 ± 15.1; education 11.7 ± 4.6) were administered the FAB and the Montreal Cognitive Assessment (MoCA). Sensitivity, specificity, positive and negative predictive values and likelihood ratios were computed through receiver-operating characteristics analyses by addressing an above- vs. below-cutoff performance on the MoCA as the state variable (as including measures of executive functioning). RESULTS: The FAB overall showed good accuracy (AUC = 0.71-0.76), although higher for healthy older adults. A trend towards higher specificity (64.4-80.3%) than sensitivity (61.1-77.8%) was found, despite these metrics being comparable in healthy older adults. Negative predictive values (0.98-0.99) were systematically higher than positive predictive values (0.05-0.24), whereas consistent post-test probabilities were detected (positive likelihood ratios: 2.19-3.35; negative likelihood ratios: 0.28-0.48). DISCUSSION: The FAB is an accurate test for the first-level assessment of dysexecutive-related global cognitive inefficiency in the general population, despite being moderately conservative as far as both its pre- and post-test features are concerned. Its diagnostic value is more informative for individuals aged ≥ 75 years.

ALS Cognitive Behavioral Screen-Phone Version (ALS-CBS™-PhV): norms, psychometrics, and diagnostics in an Italian population sample.

BACKGROUND: Up to 50% of motor neuron disease (MND) patients show neuropsychological deficits which negatively affect prognosis and care. However, disability-related logistical issues and uneven geographical coverage of healthcare services may prevent MND patients from accessing neuropsychological evaluations. This study thus aimed to standardize for the Italian population the ALS Cognitive Behavioral Screen-Phone Version (ALS-CBS™-PhV), an MND-specific, telephone-based screening for frontotemporal dysfunction. METHODS: The cognitive section of the ALS-CBS™-PhV, the Italian telephone-based Mini-Mental State Examination (Itel-MMSE), and the Telephone Interview for Cognitive Status (TICS) was administered to 359 healthy individuals (143 males, 216 females; age, 52.7 ± 15.8; education, 13.1 ± 4.4). Norms were derived through equivalent scores. Validity, factorial structure, reliability, diagnostic accuracy, and item difficulty and discrimination were examined. Statistical equivalence between the telephone-based and in-person versions was tested. RESULTS: ALS-CBS™-PhV measures were predicted by age and education. The ALS-CBS™-PhV reflected a mono-component structure, converged with Itel-MMSE and TICS scores (rs = .23-.51) and was equivalent to its in-person format (t = .37; p = .72). Good internal (Cronbach's α = .61), test-retest (ICC = .69), and inter-rater (ICC = .96) reliability was detected. High accuracy was found when tested against both the Itel-MMSE and the TICS (AUC = .82-89). Backward digit span items were the most discriminative. DISCUSSION: The ALS-CBS™-PhV is a statistically solid screening test for frontotemporal disorders featuring MND. Its standardization allows for (1) improvements in tele-healthcare for MND patients, (2) epidemiological applications, and (3) effective assessments in decentralized clinical trials. The ALS-CBS™-PhV can be also suitable for assessing bedridden and visually impaired patients with motor disorders.

The Montreal Cognitive Assessment (MoCA): updated norms and psychometric insights into adaptive testing from healthy individuals in Northern Italy.

BACKGROUND: The availability of fine-grained, culture-specific psychometric outcomes can favor the interpretation of scores of the Montreal Cognitive Assessment (MoCA), the most frequently used instrument to screen for mild cognitive dysfunctions in both instrumental and non-instrumental domains. This study thus aimed at providing: (i) updated, region-specific norms for the Italian MoCA, by also (ii) comparing them to pre-existing ones with higher geographical coverage; (iii) information on sensitivity and discriminative capability at the item level. METHODS: Five hundred and seventy nine healthy individuals from Northern Italy (208 males, 371 females; age: 63.4 ± 15, 21-96; education: 11.3 ± 4.6, 1-25) were administered the MoCA. Item Response Theory (IRT) was adopted to assess item difficulty and discrimination. Normative values were derived by means of the Equivalent Scores (ESs) method, applied to the MoCA and its sub-scales. Average ESs were also computed. Agreement with previous ESs classification was assessed via Cohen's k. RESULTS: Age and education significantly predicted all MoCA measures except for Orientation, which was related to age only. No sex differences were detected when tested along with age and education. Substantial disagreements with previous ESs classifications were detected. Several items proved to be scarcely sensitive, especially the place item from Orientation and the letter detection task. Memory items showed high discriminative capability, along with certain items assessing executive functions and orientation. DISCUSSION: Item-level information herewith provided for the Italian MoCA can help interpret its scores by Italian practitioners. Italian practitioners should consider an adaptive use of region-specific norms for the MoCA.

The Frontal Assessment Battery (FAB) and its sub-scales: validation and updated normative data in an Italian population sample.

BACKGROUND: Deficits of executive functioning (EF) are frequently found in neurological disorders. The Frontal Assessment Battery (FAB) is one of the most widespread and psychometrically robust EF screeners in clinical settings. However, in Italy, FAB norms date back to 15 years ago; moreover, its validity against "EF-loaded" global cognitive screeners (e.g., the Montreal Cognitive Assessment, MoCA) has yet to be tested. This study thus aimed at (a) providing updated normative data for the Italian FAB and (b) assessing its convergent validity with the MoCA. METHODS: Four-hundred and seventy-five healthy Italian native speakers (306 females, 169 males; mean age: 61.08 ± 15.1; mean education: 11.67 ± 4.57) were administered by the MoCA and the FAB. FAB items were divided into three subscales: FAB-1 (linguistically mediated EF), FAB-2 (planning), and FAB-3 (inhibition). Regression-based norms were derived (equivalent scores) for all FAB measures. RESULTS: Age and education were predictive of all FAB measures, whereas no gender differences were detected. The FAB and its sub-scales were related to MoCA measures-the strongest associations being found with MoCA total and MoCA-EF scores. FAB sub-scales were both internally related and associated with FAB total scores. DISCUSSION: The FAB proved to have convergent validity with both global cognitive and EF measures in healthy individuals. The present study provides updated normative data for the FAB and its sub-scales in an Italian population sample, and thus supports an adaptive usage of this EF screener.

Telephone Interview for Cognitive Status (TICS): Italian adaptation, psychometrics and diagnostics.

BACKGROUND: Telephone-based cognitive screening (TBCS) is crucial to telehealth care of neurological patients, prevention campaigns, and epidemiological studies on cognitive impairment. The Telephone Interview for Cognitive Status (TICS) is one of the most widespread and psychometrically/diagnostically sound TBCS test, with several versions developed worldwide (e.g., with and without a delayed recall item). In Italy, only attempts of adaptation and preliminary evidence of its statistical features have been provided so far. This study thus aimed at (1) developing an Italian version of the TICS and assessing its (2) psychometric and (3) diagnostic properties. METHODS: A back-translated and culturally adapted version of the TICS was developed. Three-hundred and sixty-five healthy individuals from different regions of Italy (147 males, 216 females; age: 53.2 ± 16 years; education: 13 ± 4.5 years) were administered the TICS and the Italian telephone-based Mini-Mental State Examination (Itel-MMSE). Validity was tested by convergence and at the structure level, whereas reliability as internal consistency, test-retest, and inter-rater. Diagnostic accuracy, item difficulty, and discrimination were also examined. RESULTS: The TICS featured a single component and its score converged with that of the Itel-MMSE (rs = .37). Reliability was excellent as inter-rater (ICC = .94), good as test-retest (ICC = .78), and acceptable as internal consistency (Cronbach's α = .63). Accuracy was high as tested against the Itel-MMSE (AUC = .83) and did not improve when adding the delayed recall. Backward subtraction was the most difficult and discriminative task. DISCUSSION: The Italian TICS is a valid, reliable, and diagnostically accurate TBCS test. The original format of the TICS can be thus adopted in both clinical and research settings.