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We describe the different categories of systems and systems thinking, and illustrate why almost all clinical interactions constitute simultaneously complex and complicated systems, so-called "wicked systems". We also discuss why they are not amenable to quantitative analysis. With the use of comparisons and illustrations we show some of the problems that reductionist metrics create, and support concerns regarding quantitative fallacy. The systematic correlation of data in medicine was one of the earliest achievements of the discipline. Recently, however, the overwhelming bias towards an evidence base, which relies almost entirely on randomised controlled trials, has created a reductionist view that often excludes important aspects of medicine and, in particular, surgery. We must now move away from thinking that is controlled by the "tyranny of metrics" to embrace complex-systems thinking, and work across disciplines. We outline the arguments for this and give clinical examples from oral and maxillofacial surgery. [ABSTRACT FROM AUTHOR]

Accurate pre-Liver Transplant(LT) cardiac risk estimation is paramount to guide graft allocation and to improve post-LT outcome. CAR-OLT-score predicts major 1-year post-LT cardiovascular complications(CVD-Com), but external validation is lacking. We aimed to test the performance of the score in identifying cardiac contraindication(Ca-Con) to LT-listing and to validate CAR-OLT-score to predict major 1-year CVD-Com post-LT. We consecutive enrolled all adult cirrhotics who underwent first pre-LT evaluation in Rome-Gemelli-Center(RO-cohort, N=342, from 2015 to 2019) and Turin-Center(TU-cohort, N=302, from 2015 to 2017). The main outcome measures were Ca-Con to listing(after center-specific cardiology work-up) and major 1-year CVD-Com post-transplant(death/hospitalization for CVD event). Discriminative performance of CAR-OLT-score was evaluated by area-under-the-ROC-curve(AUROC) method. Among 644 pre-LT cirrhotics, 23(3.6%) received a Ca-Con to listing (52.2% because of coronary artery disease, CAD). CAD, heart failure and atrial fibrillation were independent predictors of Ca-Con to listing. Among 431 first-LT recipients, 38(8.8%) patients experienced 1-year major CVD-Com (39.5% atrial-fibrillation, 23.7% heart-failure, 21% myocardial infarction). Diabetes, pulmonary hypertension, non-active workers were independent predictors of 1-year major CVD-Com. RO-cohort had higher prevalence of hypertensive, obese, diabetic, alcoholic patients and higher burden of CAD, atrial-fibrillation, heart-failure, peripheral vasculopathy compared with TU-cohort. CAR-OLT-score predicts Ca-Con to listing in RO-cohort (AUROC 0.806) and TU-cohort (AUROC 0.978) and 1-year major CVD-Com in RO-cohort (AUROC 0.746) and TU-cohort (AUROC 0.640). 193(29.9%) candidates had CAR-OLT-score ≤23. This cut-off had 99% NPV for Ca-Con to listing and 95% NPV for 1-year major CVD-Com post-LT. Candidates with CAR-OLT-score ≤23 underwent 18 cardiac-exercise-stress-test, 89 myocardial-perfusion-imaging, 24 dobutamine-stress-echocardiography, 10 coronary-computed-tomography, 17 coronary-angiography pre-listing, with estimated costs of 68.090€. CAR-OLT-score ≤23 identified candidates who can be safely listed without provocative cardiac tests, allowing time and cost savings and we validated CAR-OLT-score as predictor of major 1-year CVD-Com in an Italian cohort of LT recipients. [ABSTRACT FROM AUTHOR]

We proved the ability of Fourier Transform Infrared microspectroscopy (microFTIR) complemented by Principal Component Analysis (PCA) to detect protein phosphorylation/de-phosphorylation in mammalian cells. We analyzed by microFTIR human polymorphonuclear neutrophil (PMNs) leukocytes, mouse-derived parental Ba/F3 cells (Ba/F3#PAR), Ba/F3 cells transfected with p210BCR/ABL (Ba/F3#WT) and expressing high levels of protein tyrosine kinase (PTK), and human-derived BCR/ABL positive K562 leukemic cell sub-clones engineered to differently express receptor-type tyrosine-protein phosphatase gamma (PTPRG). Synchrotron radiation (SR) and conventional (globar) IR sources were used to perform microFTIR respectively, on single cells and over several cells within the same sample. Ex vivo time-course experiments were run, inducing maximal protein phosphorylation in PMNs by 100 nM N-formylated tripeptide fMLP. Within the specific IR fingerprint 1800–850 cm−1 frequency domain, PCA identified two regions with maximal signal variance. These were used to model and test the robustness of PCA in representing the dynamics of protein phosphorylation/de-phosphorylation processes. An IR signal ratio marker reflecting the homeostatic control by protein kinases and phosphatases was identified in normal leukocytes. The models identified by microFTIR and PCA in normal leukocytes also distinguished BCR/ABL positive Ba/F3#WT from BCR/ABL negative Ba/F3#PAR cells as well as K562 cells exposed to functionally active protein tyrosine phosphatase recombinant protein ICD-Tat transduced in cells by HIV-1 Tat technology or cells treated with the PTK inhibitor imatinib mesylate (IMA) from cells exposed to phosphatase inactive (D1028A)ICD-Tat recombinant protein and untreated control cells, respectively. The IR signal marker correctly reflected the degrees of protein phosphorylation associated with abnormal PTK activity in BCR/ABL positive leukemic cells and in general was inversely related to the expression/activity of PTPRG in leukemic sub-clones. In conclusion, we have described a new, reliable and simple spectroscopic method to study the ex vivo protein phosphorylation/de-phosphorylation balance in cell models: it is suitable for biomedical and pharmacological research labs but it also needs further optimization and its evaluation on large cohorts of patients to be proposed in the clinical setting of leukemia. [ABSTRACT FROM AUTHOR]

Abstract Background Apoptosis is a critical biological phenomenon, executed under the guidance of the Apoptotic Machinery (AM), which allows the physiologic elimination of terminally differentiated, senescent or diseased cells. Because of its relevance to BioMedicine, we have sought to obtain a detailed characterization of AM Omics in Homo sapiens, namely its Genomics and Evolution, Transcriptomics, Proteomics, Interactomics, Oncogenomics, and Pharmacogenomics. Methods This project exploited the methodology commonly used in Computational Biology (i.e., mining of many omics databases of the web) as well as the High Throughput biomolecular analytical techniques. Results In Homo sapiens AM is comprised of 342 protein-encoding genes (possessing either anti- or pro-apoptotic activity, or a regulatory function) and 110 MIR-encoding genes targeting them: some have a critical role within the system (core AM nodes), others perform tissue-, pathway-, or disease-specific functions (peripheral AM nodes). By overlapping the cancer type-specific AM mutation map in the fourteen most frequent cancers in western societies (breast, colon, kidney, leukaemia, liver, lung, neuroblastoma, ovary, pancreas, prostate, skin, stomach, thyroid, and uterus) to their transcriptome, proteome and interactome in the same tumour type, we have identified the most prominent AM molecular alterations within each class. The comparison of the fourteen mutated AM networks (both protein- as MIR-based) has allowed us to pinpoint the hubs with a general and critical role in tumour development and, conversely, in cell physiology: in particular, we found that some of these had already been used as targets for pharmacological anticancer therapy. For a better understanding of the relationship between AM molecular alterations and pharmacological induction of apoptosis in cancer, we examined the expression of AM genes in K562 and SH-SY5Y after anticancer treatment. Conclusion We believe that our data on the Apoptotic Machinery will lead to the identification of new cancer genes and to the discovery of new biomarkers, which could then be used to profile cancers for diagnostic purposes and to pinpoint new targets for pharmacological therapy. This approach could pave the way for future studies and applications in molecular and clinical Medicine with important perspectives both for Oncology as for Regenerative Medicine.

Molecular imaging of muscle-invasive bladder cancer (MBC) is restricted to its locoregional and distant metastases, since most radiopharmaceuticals have a urinary excretion that limits the visualization of the primary tumor. 64 CuCl 2 , a positron-emitting radiotracer with nearly exclusive biliary elimination, could be well suited to exploring urinary tract neoplasms. In this study, we evaluated the feasibility of 64 CuCl 2 -based staging of patients with MBC; furthermore, we compared the diagnostic capability of this method with those of the current gold standards, that is, contrast-enhanced CT (ceCT) and 18 F-FDG PET/CT. Methods: We prospectively enrolled patients referred to our institution for pathology-confirmed MBC staging/restaging between September 2021 and January 2023. All patients underwent ceCT, 18 F-FDG, and 64 CuCl 2 PET/CT within 2 wk. Patient-based analysis and lesion-based analysis were performed for all of the potentially affected districts (overall, bladder wall, lymph nodes, skeleton, liver, lung, and pelvic soft tissue). Results: Forty-two patients (9 women) were enrolled. Thirty-six (86%) had evidence of disease, with a total of 353 disease sites. On patient-based analysis, ceCT and 64 CuCl 2 PET/CT showed higher sensitivity than 18 F-FDG PET/CT in detecting the primary tumor ( P < 0.001); moreover, 64 CuCl 2 PET/CT was slightly more sensitive than 18 F-FDG PET/CT in disclosing soft-tissue lesions ( P < 0.05). Both PET methods were more specific and accurate than ceCT in classifying nodal lesions ( P < 0.05). On lesion-based analysis, 64 CuCl 2 PET/CT outperformed 18 F-FDG PET/CT and ceCT in detecting disease localizations overall ( P < 0.001), in the lymph nodes ( P < 0.01), in the skeleton ( P < 0.001), and in the soft tissue ( P < 0.05). Conclusion: 64 CuCl 2 PET/CT appears to be a sensitive modality for staging/restaging of MBC and might represent a "one-stop shop" diagnostic method in these scenarios., (© 2024 by the Society of Nuclear Medicine and Molecular Imaging.)

Objective: Adrenal insufficiency (AI) is characterized by increased mortality compared to general population, mainly due to cardiovascular disease. Conventional glucocorticoid (GC) replacement therapy has a role in determining the increased mortality risk. Primary outcome of the current study was to evaluate the impact of 10 years of conventional GCs and DR-HC on body weight changes in treatment-naive patients with AI. Secondary outcomes were changes from baseline to 5 and 10 years in anthropometric and metabolic profile, insulin sensitivity, cardiovascular, and bone parameters., Design and Methods: We prospectively randomized 42 patients to conventional GCs (cortisone acetate or hydrocortisone) and 44 to DR-HC (1:1). Anthropometric, metabolic, cardiovascular, and bone parameters were evaluated at baseline and after 5 and 10 years of follow-up. This trial is registered at ClinicalTrials.gov NCT06260462., Results: At 10 years of follow-up, patients with conventional GCs had significantly higher values of BMI (P = .031), waist circumference (P = .047), systolic blood pressure (P = .039), total and LDL cholesterol (P = .041 and P = .042), HbA1c (P = .040), HOMA-IR (P = .006), AUC2h of glucose (P < .001), thickness of the interventricular septum in diastole and of the posterior wall (both P < .001) and significantly lower values of oral disposition index (P = .001) and ISI-Matsuda (P < .001), lumbar spine T score (P = .036), and femoral neck Z score (P = .026), compared to patients treated with DR-HC., Conclusions: In patients with treatment-naive AI, 10 years of conventional GC treatment is associated with a worsening of metabolic, insulin-sensitivity, cardiac, and bone outcomes, while DR-HC had no impact on them achieving a lower risk of developing comorbidities., Competing Interests: Conflict of interest: None declared., (© The Author(s) 2024. Published by Oxford University Press on behalf of European Society of Endocrinology. All rights reserved. For commercial re-use, please contact reprints@oup.com for reprints and translation rights for reprints. All other permissions can be obtained through our RightsLink service via the Permissions link on the article page on our site—for further information please contact journals.permissions@oup.com.)

Diabetic foot ulcer (DFU), one of the most significant complications of diabetes, is a condition that causes anatomical and functional alterations of the foot resulting in an important social and economic impact, related to disability and health care costs. Recently, three-dimensional bioprinting - which allows the fabrication of complex and biocompatible structures - has been identified as a promising approach in the field of regenerative medicine to promote the healing of chronic wounds, such as DFU. In this concise review we highlight the most relevant and recent attempts of using 3D bioprinted constructs in vivo - both on animals and people - in order to treat non-healing diabetic ulcers and prevent their worsening. Finally, we briefly focus on the future implications of bioprinting, suggesting its forthcoming importance not only for DFU treatment but also for other areas of clinical care., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2024 The Authors.)

Inhibitors of Apoptosis proteins (IAPs) were discovered through experiments aimed at rescuing apoptosis in insects. Classically associated with the inhibition of apoptosis, the IAP member Survivin also regulates cell cycle progression and is an essential component of the Chromosomal Passenger Complex (CPC), responsible for chromosomal segregation. Although undetectable in most adult tissues, Survivin is expressed in Adult Stem Cells (ASCs) and plays a crucial role in their maintenance. Survivin is overexpressed in most cancers, contributing to their clonal expansion. As a result, it has been proposed as a possible anticancer target for nearly two decades. In this discussion, we will explore the rationale behind Survivin as a therapeutic target, focusing on common cancer types such as carcinomas, sarcomas, and leukemias. We will delve into the modulation of Survivin by cancer pro-survival cell signaling, the association between SNPs and tumorigenesis, and its regulation by miRNAs. Finally, we will compare cell growth, clonogenic capacity, and apoptosis, along with different strategies for Survivin inhibition, including gene expression and protein activity modulation., Competing Interests: Declaration of competing interest The authors have no conflicts of interest to declare. All co-authors have seen and agree with the contents of the manuscript and there is no financial interest to report., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Background: Dedifferentiated liposarcoma is a formidable sarcoma subtype due to its high local recurrence rate and resistance to medical treatment. While 2D cell cultures are still commonly used, 3D cell culture systems have emerged as a promising alternative, particularly scaffold-based techniques that enable the creation of 3D models with more accurate cell-stroma interactions. Objective: To investigate how 3D structures with or without the scaffold existence would affect liposarcoma cell lines growth morphologically and biologically. Methods: Lipo246 and Lipo863 cell lines were cultured in 3D using four different methods; Matrigel ® ECM scaffold method, Collagen ECM scaffold method, ULA plate method and Hanging drop method, in addition to conventional 2D cell culture methods. All samples were processed for histopathological analysis (HE, IHC and DNAscope™), Western blot, and qPCR; moreover, 3D collagen-based models were treated with different doses of SAR405838, a well-known inhibitor of MDM2, and cell viability was assessed in comparison to 2D model drug response. Results: Regarding morphology, cell lines behaved differently comparing the scaffold-based and scaffold-free methods. Lipo863 formed spheroids in Matrigel ® but not in collagen, while Lipo246 did not form spheroids in either collagen or Matrigel ® . On the other hand, both cell lines formed spheroids using scaffold-free methods. All samples retained liposarcoma characteristic, such as high level of MDM2 protein expression and MDM2 DNA amplification after being cultivated in 3D. 3D collagen samples showed higher cell viability after SAR40538 treatment than 2D models, while cells sensitive to the drug died by apoptosis or necrosis. Conclusion: Our results prompt us to extend our investigation by applying our 3D models to further oncological relevant applications, which may help address unresolved questions about dedifferentiated liposarcoma biology., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The author(s) declared that they were an editorial board member of Frontiers, at the time of submission. This had no impact on the peer review process and the final decision., (Copyright © 2024 Tahara, Sharma, de Faria, Sarchet, Tomasello, Rentsch, Karna, Calore and Pollock.)

Introduction: The onset of precision medicine has led to the integration of traditional morphologic tissues evaluation with biochemical and molecular data for a more appropriate pathological diagnosis. The preanalytic phase and, particularly, timing of cold ischemia are crucial to guarantee high-quality biorepositories of formalin-fixed paraffin-embedded (FFPE) tissues for patients' needs and scientific research. However, delayed fixation using the gold-standard and carcinogenic fixative neutral-buffered formalin (NBF) can be a significant limitation to diagnosis and biopathological characterization. HistoCold (patented; Bio-Optica Milano S.p.A., Milano, Italy) is a nontoxic, stable, and refrigerated preservative solution for tissue handling. This study examined HistoCold's potential role in improving the preanalytic phase of the pathological diagnostic process. Materials and Methods: Breast, lung, or colorectal cancers (20, 25, and 10 cases, respectively) that were to be surgically resected were recruited between 2019 and 2021. Once specimens were surgically removed, three residual samples for each patient were first promptly immersed into HistoCold for 24, 48, and 72 hours and then FFPE. These were compared with routine specimens regarding morphologic features (hematoxylin and eosin) and tissue antigenicity (immunohistochemical stains). Results: Good concordance regarding both the morphologic characteristics of the neoplasms and their proteins expression between the routine and HistoCold handled tissues were found. The tissue handling with the solution never affected the histopathological diagnosis. Conclusions: The use of HistoCold for samples transporting is easy, allows for improving the management of cold ischemia time, and monitoring the fixation times in NBF, resulting in good quality tissue blocks for biobanking. Moreover, it could be a candidate to eliminate formalin from operating theaters. HistoCold looks very promising for the preanalytic phase of human tissues handling in the era of precision medicine, to provide the best service to patients, and to scientific research.

Prostate cancer still represents one of the most frequent cancers and causes of death worldwide, despite the huge therapeutic advances in the last decades. The introduction into clinical practice of prostate-specific membrane antigen positron emission tomography/computed tomography (PSMA PET/CT) has significantly improved diagnostic capacity, allowing for the identification of lesions previously undetectable. The case we are presenting is about a 90-year-old man affected by metastatic prostate cancer and treated with hormonal therapies. At the second progression, the restaging with PSMA PET/CT pointed out a millimetric cardiac intra-atrial metastasis, on which little/scarce literature data are still available. On one hand, this finding confirms the high sensitivity of this technique, which should be preferred over traditional imaging. On the other hand, it suggests that introducing next-generation imaging into clinical practice may provide novel insights about prostate cancer metastatic spread.

Adipose tissue (AT) secretes pro- and anti-inflammatory cytokines involved in AT homeostasis, including tumor necrosis factor-α (TNFα) and irisin. The functionality of AT is based on a regulated equilibrium between adipogenesis and extracellular matrix (ECM) remodeling. We investigated the contributions of adipose progenitors (ASCs) and adipocytes (AMCs) to TNFα-induced ECM remodeling and a possible implication of irisin in AT impairment in obesity. ASCs and AMCs were exposed to TNFα treatment and nuclear factor-kappa (NF-kB) pathway was investigated: Tissue Inhibitor of Metalloproteinase (TIMP-1), Twist Family Transcription Factor 1 (TWIST-1), and peroxisome proliferator-activated receptor-γ (PPARγ) expression levels were analyzed. The proteolytic activity of matrix metalloproteinases (MMPs) -2 and -9 was analyzed by zymography, and the irisin protein content was measured by ELISA. In inflamed AMCs, a TIMP-1/TWIST-1 imbalance leads to a drop in PPARγ. Adipogenesis and lipid storage ability impairment come with local tissue remodeling due to MMP-9 overactivation. In vitro and ex vivo measurements confirm positive correlations among inflammation, adipose secreting irisin levels, and circulating irisin levels in patients with visceral obesity. Our findings identify the NF-kB downstream effectors as molecular initiators of AT dysfunction and suggest irisin as a possible AT damage and obesity predictive factor.

This study analyzed the efficacy of EEG resting state and neuropsychological performances in discriminating patients with different forms of dementia, or mild cognitive impairment (MCI), compared with control subjects. Forty-four patients with dementia (nineteen patients with AD, and seven with FTD), eighteen with MCI, and nineteen healthy subjects, matched for age and gender, underwent an extensive neuropsychological test battery and an EEG resting state recording. Results showed greater theta activation in posterior areas in the Alzheimer's disease (AD) and Fronto-Temporal Dementia (FTD) groups compared with the MCI and control groups. AD patients also showed more delta band activity in the temporal-occipital areas than controls and MCI patients. By contrast, the alpha and beta bands did not discriminate among groups. A hierarchical clustering analysis based on neuropsychological and EEG data yielded a three-factor solution. The clusters differed for several neuropsychological measures, as well as for beta and theta bands. Neuropsychological tests were most sensitive in capturing an initial cognitive decline, while increased theta activity was uniquely associated with a substantial worsening of the clinical picture, representing a negative prognostic factor. In line with the Research Domains Framework (RDoC) perspective, the joint use of cognitive and neurophysiological data may provide converging evidence to document the evolution of cognitive skills in at-risk individuals.

The superior diagnostic accuracy of [68Ga]Ga-prostate-specific membrane antigen-11 (PSMA) ([68Ga]Ga-PSMA-11) compared to [18F]F-Fluorocholine Positron Emission Tomography/Computed Tomography (PET/CT) in Prostate Cancer (PCa) is established. However, it is currently unclear if the added diagnostic accuracy actually translates into improved clinical outcomes in oligometastatic PCa patients treated with [68Ga]Ga-PSMA-11 PET-guided metastasis-directed therapy (MDT). The present study aimed to assess the impact of these two imaging techniques on Progression-Free Survival (PFS) in a real-world sample of oligometastatic PCa patients submitted to PET-guided MDT. Thirty-seven oligometastatic PCa patients treated with PET-guided MDT were retrospectively enrolled. MDT was guided by [18F]F-Fluorocholine PET/CT in eleven patients and by [68Ga]Ga-PSMA-11 PET/CT in twenty-six. Progression was defined as biochemical recurrence (BR), radiological progression at subsequent PET/CT imaging, clinical progression, androgen deprivation therapy initiation, or death. Clinical and imaging parameters were assessed as predictors of PFS. [18F]F-Fluorocholine PET-guided MDT was associated with significantly lower PFS compared to the [68Ga]Ga-PSMA-11 group (median PFS, mPFS 15.47 months, 95% CI: 4.13−38.00 vs. 40.93 months, 95% CI: 40.93−40.93, respectively; p < 0.05). Coherently, the radiotracer used for PET-guided MDT resulted in predictive PFS at the univariate analysis, as well as the castration-resistant status at the time of MDT and the PSA nadir after MDT. However, in the multivariate analysis, castration resistance and PSA nadir after MDT remained the sole independent predictors of PFS. In conclusion, in the present proof-of-concept study, [68Ga]Ga-PSMA-11 provided higher PFS rates than [18F]F-Fluorocholine imaging in oligometastatic PCa patients receiving PET-guided MDT. Although preliminary, this finding suggests that enlarging the “tip of the iceberg”, by detecting a major proportion of the submerged disease thanks to next-generation imaging may favourably impact the oncological outcome of oligometastatic PCa treated with MDT.

Epitranscriptomic studies of miRNAs have added a new layer of complexity to the cancer field. Although there is fast-growing interest in adenosine-to-inosine (A-to-I) miRNA editing and alternative cleavage that shifts miRNA isoforms, simultaneous evaluation of both modifications in cancer is still missing. Here, we concurrently profiled multiple miRNA modification types, including A-to-I miRNA editing and shifted miRNA isoforms, in &gt;13,000 adult and pediatric tumor samples across 38 distinct cancer cohorts from The Cancer Genome Atlas and The Therapeutically Applicable Research to Generate Effective Treatments data sets. The differences between canonical miRNAs and the wider miRNAome in terms of expression, clustering, dysregulation, and prognostic standpoint were investigated. The combination of canonical miRNAs and modified miRNAs boosted the quality of clustering results, outlining unique clinicopathologic features among cohorts. Certain modified miRNAs showed opposite expression from their canonical counterparts in cancer, potentially impacting their targets and function. Finally, a shifted and edited miRNA isoform was experimentally validated to directly bind and suppress a unique target. These findings outline the importance of going beyond the well-established paradigm of one mature miRNA per miRNA arm to elucidate novel mechanisms related to cancer progression., Significance: Modified miRNAs may act as cancer biomarkers and function as allies or antagonists of their canonical counterparts in gene regulation, suggesting the concurrent consideration of canonical and modified miRNAs can boost patient stratification., (©2022 The Authors; Published by the American Association for Cancer Research.)

This study concerns the analysis of the modulation of Chronic Myeloid Leukemia (CML) cell model K562 transcriptome following transfection with the tumor suppressor gene encoding for Protein Tyrosine Phosphatase Receptor Type G (PTPRG) and treatment with the tyrosine kinase inhibitor (TKI) Imatinib. Specifically, we aimed at identifying genes whose level of expression is altered by PTPRG modulation and Imatinib concentration. Statistical tests as differential expression analysis (DEA) supported by gene set enrichment analysis (GSEA) and modern methods of ontological term analysis are presented along with some results of current interest for forthcoming experimental research in the field of the transcriptomic landscape of CML. In particular, we present two methods that differ in the order of the analysis steps. After a gene selection based on fold-change value thresholding, we applied statistical tests to select differentially expressed genes. Therefore, we applied two different methods on the set of differentially expressed genes. With the first method (Method 1), we implemented GSEA, followed by the identification of transcription factors. With the second method (Method 2), we first selected the transcription factors from the set of differentially expressed genes and implemented GSEA on this set. Method 1 is a standard method commonly used in this type of analysis, while Method 2 is unconventional and is motivated by the intention to identify transcription factors more specifically involved in biological processes relevant to the CML condition. Both methods have been equipped in ontological knowledge mining and word cloud analysis, as elements of novelty in our analytical procedure. Data analysis identified RARG and CD36 as a potential PTPRG up-regulated genes, suggesting a possible induction of cell differentiation toward an erithromyeloid phenotype. The prediction was confirmed at the mRNA and protein level, further validating the approach and identifying a new molecular mechanism of tumor suppression governed by PTPRG in a CML context.

Chronic myeloid leukemia (CML) is a myeloproliferative disease caused by the acquisition of t(9;22) generating the fusion tyrosine kinase BCR::ABL1. However, despite the crucial role of this protein in the dysregulation of numerous signal transduction pathways, a direct measure of BCR::ABL1 kinase activity in chronic phase (CP) CML was never accomplished due to intense degradative activity present in mature leukocytes. Therefore, we developed a procedure suitable to preserve BCR::ABL1 protein under non-denaturing, neutral pH conditions in primary, chronic phase (CP)-CML samples. As a result, specific kinase activity was detected utilizing a biotinylated peptide substrate highly selective for c-ABL1. Furthermore, through this approach, BCR::ABL1 kinase activity was barely detectable in CP-CML compared to Ph + acute lymphoblastic leukemia primary samples, where kinase activity is comparable to those measured in Ph + cell lines. These in vitro findings provide the first direct measure of BCR::ABL1 kinase activity in primary CP-CML and reveal the presence of a still uncharacterized inhibitory mechanism that maintains BCR::ABL1 in a low activity state in CP-CML despite its overexpression., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Boni, Bonifacio, Vezzalini, Scaffidi, Tomasello, Parker, Boscarino, Paladin, Krampera and Sorio.)

To assess the differences between patients with normal glucose tolerance (NGT) and prediabetes/diabetes mellitus (DM) in secondary adrenal insufficiency (SAI). We cross-sectionally evaluated 102, out of a total of 140, patients with SAI, who were on hydrocortisone (HC) (n = 50) and cortisone acetate (n = 52) replacement therapy. Clinical, anthropometric, and metabolic parameters were compared in patients with NGT (n = 60) and DM (n = 42). Patients with prediabetes/DM have a more marked family history of DM (p = 0.002), BMI (p < 0.001), higher waist circumference (p < 0.001), total cholesterol (p = 0.012), LDL-cholesterol (p = 0.004), triglycerides (p = 0.031), fasting glucose (p = 0.002), fasting insulin (p = 0.035), glutamate pyruvate transaminase (p = 0.018), HOMA-IR (p = 0.039), area under curves of glucose (p = 0.001) and insulin (p = 0.002), HbA1c (p < 0.001), Visceral adiposity index (VAI) (p = 0.038) and lower ISI-Matsuda (p = 0.008) and oral disposition index (p < 0.001) than patients with NGT. Multivariate analysis showed that family history of DM and VAI are independent predictive factors for DM in patients with SAI. Family history of DM and VAI can be predictors of the development of DM in patients with SAI and need to be investigated during steroid replacement therapy. Interestingly, the type and the dose of replacement steroid do not impact on diabetes mellitus., (© 2022. The Author(s).)

Debate is around the optimal immunization regimen for cancer vaccines since too intense vaccination schedules may exhaust reactive lymphocytes. GX301 is a telomerase-based cancer vaccine whose safety and immunological effects were tested in a phase I trial applying an eight administrations schedule. Main objective of this study was to comparatively analyse safety and immunological response to three GX301 regimens in metastatic castration-resistant prostate cancer patients with response/disease stability after docetaxel chemotherapy. This was a multicentre, randomized, parallel-group, open-label trial registered with EudraCT (2014-000095-26) and ClinicalTrials.gov (NCT02293707, 2014). Ninety-eight patients were randomized to receive either eight (regimen 1), four (regimen 2) or two (regimen 3) vaccine administrations. Sixty-three patients were assessable for the primary immunological end-point. Vaccine-specific immune responses were evaluated by intracellular staining for IFN, elispot and cytotoxic assay at 90 and 180 days from baseline. No major side effects were recorded. A 54% overall immune responder rate was observed with 95% of patients showing at least one vaccine-specific immune response. Rate of immunological responders and number of immunizations were proportionally related, suggesting superiority of regimens 1 and 2 over regimen 3. Overall survival did not differ among regimens in both immunological responders and non-responders and was inversely associated (P = 0.002) with increase in the number of circulating CD8 + T regulatory cells at 180 days. These data indicate that GX301 cancer vaccine is safe and immunogenic in metastatic castration-resistant prostate cancer patients. Schedules with high number of administrations should be preferred in future studies due to their better immunological outcome., (© 2021. The Author(s).)

MicroRNAs (miRNAs) are regulatory small non-coding RNAs that function as translational repressors. MiRNAs are involved in most cellular processes, and their expression and function are presided by several factors. Amongst, miRNA editing is an epitranscriptional modification that alters the original nucleotide sequence of selected miRNAs, possibly influencing their biogenesis and target-binding ability. A-to-I and C-to-U RNA editing are recognized as the canonical types, with the A-to-I type being the predominant one. Albeit some bioinformatics resources have been implemented to collect RNA editing data, it still lacks a comprehensive resource explicitly dedicated to miRNA editing. Here, we present MiREDiBase, a manually curated catalog of editing events in miRNAs. The current version includes 3,059 unique validated and putative editing sites from 626 pre-miRNAs in humans and three primates. Editing events in mature human miRNAs are supplied with miRNA-target predictions and enrichment analysis, while minimum free energy structures are inferred for edited pre-miRNAs. MiREDiBase represents a valuable tool for cell biology and biomedical research and will be continuously updated and expanded at https://ncrnaome.osumc.edu/miredibase ., (© 2021. The Author(s).)

MicroRNAs (miRNAs or miRs) are the most characterized class of non-coding RNAs and are engaged in many cellular processes, including cell differentiation, development, and homeostasis. MicroRNA dysregulation was observed in several diseases, cancer included. Epitranscriptomics is a branch of epigenomics that embraces all RNA modifications occurring after DNA transcription and RNA synthesis and involving coding and non-coding RNAs. The development of new high-throughput technologies, especially deep RNA sequencing, has facilitated the discovery of miRNA isoforms (named isomiRs) resulting from RNA modifications mediated by enzymes, such as deaminases and exonucleases, and differing from the canonical ones in length, sequence, or both. In this review, we summarize the distinct classes of isomiRs, their regulation and biogenesis, and the active role of these newly discovered molecules in cancer and other diseases., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Tomasello, Distefano, Nigita and Croce.)

Background: Despite the survival advantage, not all metastatic renal cell carcinoma (mRCC) patients achieve a long-term benefit from immunotherapy. Moreover, the identification of prognostic biomarkers is still an unmet clinical need., Methods: This multicenter retrospective study investigated the prognostic role of peripheral-blood inflammatory indices and clinical factors to develop a novel prognostic score in mRCC patients receiving at least second-line nivolumab. The complete blood count before the first cycle of therapy was assessed by calculating neutrophil-to-lymphocyte ratio (NLR), derived NLR (dNLR), lymphocyte-to-monocyte ratio (LMR), platelet-to-lymphocyte ratio (PLR), systemic inflammation index (SII), and systemic inflammation response index (SIRI). Clinical factors included pre-treatment International Metastatic RCC Database Consortium (IMDC) score, line of therapy, and metastatic sites., Results: From October 2015 to November 2019, 571 mRCC patients received nivolumab as second- and further-line treatment in 69% and 31% of cases. In univariable and multivariable analyses all inflammatory indices, IMDC score, and bone metastases significantly correlated with overall survival (OS). The multivariable model with NLR, IMDC score, and bone metastases had the highest c-index (0.697) and was chosen for the developing of the score (Schneeweiss scoring system). After internal validation (bootstrap re-sampling), the final index (Meet-URO score) composed by NLR, IMDC score, and bone metastases had a c-index of 0.691. It identified five categories with distinctive OSs: group 1 (median OS - mOS = not reached), group 2 (mOS = 43.9 months), group 3 (mOS = 22.4 months), group 4 (mOS = 10.3 months), and group 5 (mOS = 3.2 months). Moreover, the Meet-URO score allowed for a fine risk-stratification across all three IMDC groups., Conclusion: The Meet-URO score allowed for the accurate stratification of pretreated mRCC patients receiving nivolumab and is easily applicable for clinical practice at no additional cost. Future steps include its external validation, the assessment of its predictivity, and its application to first-line combinations., Competing Interests: Conflict of interest statement: Dr Fornarini services advisory boards for Astellas, Janssen, Pfizer, Bayer, MSD, and Merck, and received travel accommodation from Astellas, Janssen, and Bayer. Dr Buti received honoraria as a speaker at scientific events and in an advisory role by BMS, Pfizer; MSD, Ipsen, Roche, Eli Lilly, AstraZeneca, Pierre-Fabre, Novartis. Dr Banna reports personal fees from AstraZeneca, Janssen-Cilag, Boehringer Ingelheim, Roche, and non-financial support from Bristol-Myers Squibb, AstraZeneca, MedImmune, Pierre Fabre, IPSEN, outside the submitted work. Dr De Giorgi services as an advisory/board member of Astellas, Bayer, Bristol-Myers Squibb, IPSEN, Janssen, Merck, Pfizer, and Sanofi, received research grant/funding to the institution from AstraZeneca, Roche, Sanofi, and travel/accommodations/expenses from Bristol-Myers Squibb, IPSEN, Janssen, and Pfizer. Dr Zucali services advisory boards/consulting for Pfizer, Bristol-Myers Squibb, MSD, IPSEN, Novartis, Roche, Amgen, AstraZeneca, Sanofi, Janssen, and Astellas. Dr Masini received personal fees as a speaker from Astellas, as a consultant from IPSEN, MSD, and Janssen, and for travel accommodation from BMS, Pfizer, Astellas, Janssen, and IPSEN. Dr Procopio services advisory boards/consulting for Astellas, AstraZeneca, Bayer, Bristol-Myers Squibb, Janssen, IPSEN, Merk, MSD, Novartis, and Pfizer. Dr Cortellini receives speaker fees/grant consultancies from Astrazeneca, BMS, MSD, Roche, Novartis, and Astellas. Dr Morelli received grants from MSD and Pfizer. Dr Bersanelli received research funding to the institution from Roche, Pfizer, Seqirus UK, AstraZeneca, Bristol-Myers Squibb, Novartis, and Sanofi, and received personal fees for advisory role, copyright transfer, consultancies, and as speaker at scientific events from Sciclone Pharmaceuticals, Bristol-Myers Squibb, AstraZeneca, Pierre-Fabre, Novartis, and Pfizer. The other authors have no conflicts of interest to disclose., (© The Author(s), 2021.)

Adenosine to inosine (A-to-I) editing consists of an RNA modification where single adenosines along the RNA sequence are converted into inosines. Such a biochemical transformation is catalyzed by enzymes belonging to the family of adenosine deaminases acting on RNA (ADARs) and occurs either co- or post-transcriptionally. The employment of powerful, high-throughput detection methods has recently revealed that A-to-I editing widely occurs in non-coding RNAs, including microRNAs (miRNAs). MiRNAs are a class of small regulatory non-coding RNAs (ncRNAs) acting as translation inhibitors, known to exert relevant roles in controlling cell cycle, proliferation, and cancer development. Indeed, a growing number of recent researches have evidenced the importance of miRNA editing in cancer biology by exploiting various detection and validation methods. Herein, we briefly overview early and currently available A-to-I miRNA editing detection and validation methods and discuss the significance of A-to-I miRNA editing in human cancer.

Objective: It is debatable whether BCR-ABL1 transcript type has an impact on outcome of treatment of patients with CML, and it is not widely studied whether body weight influences response to treatment. In this study, we tried to find out if any of these factors has an impact on response to treatment and outcome., Methodology: We conducted a retrospective analysis of the files of 79 patients being treated in our center for CML with known BCR-ABL1 breakpoints, and patients' management and response assessment was done based on ELN 2013 guidelines. The analysis was performed based on two main groups, obese vs. normal BMI, and then based on BCR-ABL1 transcripts: e13a2 vs. e14a2. Cumulative incidence of MMR, CCyR, and DMR were estimated using the Kaplan-Meier survival curve method, and comparisons between groups were performed by the Log-rank/Gray test methods., Results/conclusion: In the patient-cohort studied, there was no statistically significant difference in molecular response between patients with CML based on body weight or transcript type although patients in the obesity group achieved higher and faster MMR with no statistical significance.

Penile cancer is a very rare malignancy and it is usually identified as a nodule or an ulceration. We report a case of locally advanced penile cancer presented as a urethral stricture and recurrent scrotal abscess. The patient had been treating for obstructive voiding symptoms and septic condition for 5 months. Because of persistent symptoms, a drainage of the abscess was performed and the histopathological examination showed infiltrating moderately differentiated squamous cell carcinoma of penis. He underwent a surgical Emasculation followed by the administration of a combination of chemotherapy (paclitaxel, ifosfamide, and cisplatin). We highlight the importance of including penile cancer in the differential diagnosis of scrotal abscess and urethral stricture. A multimodal approach is an effective strategy to manage the disease.

Parathyroid carcinoma is one of the least frequent malignant neoplasms, diagnosis and treatment are often complex; however, the prognosis is not bad and, although recurrences are frequent, the mortality rate is low. We describe the clinical case of a 42-years-old patient, the evolution of the natural history and treatment.

Background: Several studies showed that aberrant DNA methylation is involved in leukemia and cancer pathogenesis. Protein tyrosine phosphatase receptor gamma (PTPRG) expression is a natural inhibitory mechanism that is downregulated in chronic myeloid leukemia (CML) disease. The mechanism behind its downregulation has not been fully elucidated yet., Aim: This study aimed to investigate the CpG methylation status at the PTPRG locus in CML patients., Methods: Peripheral blood samples from CML patients at time of diagnosis [no tyrosine kinase inhibitors (TKIs)] (n = 13), failure to (TKIs) treatment (n = 13) and healthy controls (n = 6) were collected. DNA was extracted and treated with bisulfite treatment, followed by PCR, sequencing of 25 CpG sites in the promoter region and 26 CpG sites in intron-1 region of PTPRG. The bisulfite sequencing technique was employed as a high-resolution method., Results: CML groups (new diagnosed and failed treatment) showed significantly higher methylation levels in the promoter and intron-1 regions of PTPRG compared to the healthy group. There were also significant differences in methylation levels of CpG sites in the promoter and intron-1 regions amongst the groups., Conclusion: Aberrant methylation of PTPRG is potentially one of the possible mechanisms of PTPRG downregulation detected in CML., (© 2020 The Authors. Molecular Genetics & Genomic Medicine published by Wiley Periodicals LLC.)

Double knockout of the two miR-15/16 loci in mouse resulted in the development of acute myeloid leukemia (AML). This result suggested that, at least, a fraction of human AMLs could be due to a similar mechanism. We analyzed the role of the two miR-15/16 clusters in 93 myelodysplastic syndrome (MDS) patients divided in three subgroups: patients with MDS, patients with MDS before transforming into AML (MDS-T), and patients with AML evolving from MDS (MDS-AML). Then, we tested 139 AML cases and 14 different AML cell lines by assessing microRNA (miRNA) expression, target protein expression, genetic loss, and silencing. MDS-T and MDS-AML patients show a reduction of the expression of miR-15a/-15b/-16 compared to MDS patients. Each miRNA can significantly predict MDS and MDS-T groups. Then, 79% of primary AMLs show a reduced expression of miR-15a and/or miR-15b. The expression of miR-15a/-15b/-16 significantly stratified AML patients in two prognostic classes. Furthermore, 40% of AML cell lines showed a combined loss of the expression of miR-15a/-15b and overexpression of their direct/indirect targets. As potential mechanisms involved in the silencing of the two miR-15/16 loci, we identified a genetic loss of miR-15a and miR-15b and silencing of these two loci by methylation. We identified a potential driver oncogenic role in the loss of expression of both miR-15/16 clusters in the progression of MDS into AML and in AML pathogenesis. The stratification of AML patients, based on miR-15/16 expression, can lead to targeted and combination therapies for the treatment of this incurable disease., Competing Interests: Competing interest statement: K.R.R. and C.M.C. are co-authors on a 2017 review paper.

Background: In periodontal patients with jawbone resorption, the autologous bone graft is considered a "gold standard" procedure for the placing of dental prosthesis; however, this procedure is a costly intervention and poses the risk of clinical complications. Thanks to the use of adult mesenchymal stem cells, smart biomaterials, and active biomolecules, regenerative medicine and bone tissue engineering represent a valid alternative to the traditional procedures., Aims: In the past, mesenchymal stem cells isolated from periodontally compromised gingiva were considered a biological waste and discarded during surgical procedures. This study aims to test the osteoconductive activity of FISIOGRAFT Bone Granular ® and Matriderm ® collagen scaffolds on mesenchymal stem cells isolated from periodontally compromised gingiva as a low-cost and painless strategy of autologous bone tissue regeneration., Materials and Methods: We isolated human mesenchymal stem cells from 22 healthy and 26 periodontally compromised gingival biopsy tissues and confirmed the stem cell phenotype by doubling time assay, colony-forming unit assay, and expression of surface and nuclear mesenchymal stem cell markers, respectively by cytofluorimetry and real-time quantitative PCR. Healthy and periodontally compromised gingival mesenchymal stem cells were seeded on FISIOGRAFT Bone Granular ® and Matriderm ® scaffolds, and in vitro cell viability and bone differentiation were then evaluated., Results: Even though preliminary, the results demonstrate that FISIOGRAFT Bone Granular ® is not suitable for in vitro growth and osteogenic differentiation of healthy and periodontally compromised mesenchymal stem cells, which, instead, are able to grow, homogeneously distribute, and bone differentiate in the Matriderm ® collagen scaffold., Conclusion: Matriderm ® represents a biocompatible scaffold able to support the in vitro cell growth and osteodifferentiation ability of gingival mesenchymal stem cells isolated from waste gingiva, and could be employed to develop low-cost and painless strategy of autologous bone tissue regeneration., (Copyright © 2020 Cristaldi, Mauceri, Campisi, Pizzo, Alessandro, Tomasello, Pitrone, Pizzolanti and Giordano.)

Protein tyrosine phosphatase receptor type γ (PTPRG) is a tumor suppressor gene, down-regulated in Chronic Myeloid Leukemia (CML) cells by the hypermethylation of its promoter region. β-catenin (CTNNB1) is a critical regulator of Leukemic Stem Cells (LSC) maintenance and CML proliferation. This study aims to demonstrate the antagonistic regulation between β-catenin and PTPRG in CML cells. The specific inhibition of PTPRG increases the activation state of BCR-ABL1 and modulates the expression of the BCR-ABL1- downstream gene β-Catenin. PTPRG was found to be capable of dephosphorylating β-catenin, eventually causing its cytosolic destabilization and degradation in cells expressing PTPRG. Furthermore, we demonstrated that the increased expression of β-catenin in PTPRG-negative CML cell lines correlates with DNA (cytosine-5)-methyl transferase 1 (DNMT1) over-expression, which is responsible for PTPRG promoter hypermethylation, while its inhibition or down-regulation correlates with PTPRG re-expression. We finally confirmed the role of PTPRG in regulating BCR-ABL1 and β-catenin phosphorylation in primary human CML samples. We describe here, for the first time, the existence of a regulative loop occurring between PTPRG and β-catenin, whose reciprocal imbalance affects the proliferation kinetics of CML cells.

: Radium-223 dichloride (Ra223) represents the unique bone-directed treatment option that shows an improvement in overall survival (OS) in metastatic castrate resistant prostate cancer (mCRPC). However, there is an urgent need for the identification of reliable biomarkers to non-invasively determine its efficacy (possibly improving patients' selection or identifying responders' after therapy completion). 18F-Fluorodeoxyglucose (FDG)-avidity is low in naïve prostate cancer, but it is enhanced in advanced and chemotherapy-refractory mCRPC, providing prognostic insights. Moreover, this tool showed high potential for the evaluation of response in cancer patients with bone involvement. For these reasons, FDG Positron Emission Tomography (FDG-PET) might represent an effective tool that is able to provide prognostic stratification (improving patients selection) at baseline and assessing the treatment response to Ra223. We conducted a retrospective analysis of 28 mCRPC patients that were treated with Ra223 and submitted to bone scan and FDG-PET/CT for prognostic purposes at baseline and within two months after therapy completion. The following parameters were measured: number of bone lesions at bone scan, SUVmax of the hottest bone lesion, metabolic tumor volume (MTV), and total lesion glycolysis (TLG). In patients who underwent post-therapy 18F-Fluorodeoxyglucose Positron Emission Tomography/Computed Tomography (FDG-PET/CT), (20/28), PET Response Criteria in Solid Tumors (PERCIST), and European Organization for Research and Treatment of Cancer (EORTC) criteria were applied to evaluate the metabolic treatment response. The difference between end of therapy and baseline values was also calculated for Metabolic Tumor Volume (MTV), TLG, prostate-specific antigen (PSA), alkaline phosphatase (AP), and lactate dehydrogenase (LDH) (termed deltaMTV, deltaTLG, deltaPSA, deltaAP and deltaLDH, respectively). Predictive power of baseline and post-therapy PET- and biochemical-derived parameters on OS were assessed by Kaplan-Meier, univariate and multivariate analyses. At baseline, PSA, LDH, and MTV significantly predicted OS. However, MTV (but not PSA nor LDH) was able to identify a subgroup of patients with worse prognosis, even after adjusting for the number of lesions at bone scan (which, in turn, was not an independent predictor of OS). After therapy, PERCIST criteria were able to capture the response to Ra223 by demonstrating longer OS in patients with partial metabolic response. Moreover, the biochemical parameters were outperformed by PERCIST in the post-treatment setting, as their variation after therapy was not informative on long term OS. The present study supports the role of FDG-PET as a tool for patient's selection and response assessment in mCRPC patients undergoing Ra223 administration., Competing Interests: SM received speaker honoraria from General Electric and Eli-Lilly. The other authors declare they have no conflict of interest.

Our previous studies documented that human fibroblast-limbal stem cells (f-LSCs) possess immunosuppressive capabilities, playing a role in regulating T-cell activity. This study highlights the molecular activities by which human f-LSCs can attenuate the inflammatory responses of self-reactive peripheral blood mononuclear cells (PBMCs) collected from patients with autoimmune endocrine diseases (AEDs). Anti-CD3 activated PBMCs from twenty healthy donors and fifty-two patients with AEDs were cocultured on f-LSC monolayer. 2D-DIGE proteomic experiments, mass spectrometry sequencing and functional in vitro assays were assessed in cocultured PBMCs. We identified the downmodulation of several human heterogeneous nuclear ribonucleoprotein A2/B1 (hnRNP A2/B1) isoforms in healthy and AED activated PBMCs upon f-LSC interaction. The reduction of hnRNPA2/B1 protein expression largely affected the cycling ki67 + , CD25 + , PD-1 + reactive cells and the double marked CD8 + /hnRNPA2B1 + T cell subset. Anti-PD1 blocking experiments evoked hnRNPA2/B1 overexpression, attributing putative activation function to the protein. hnRNPA2/B2 transient silencing inverted immunopolarization of the self-reactive PBMCs from AEDs toward a M2/Th2-type background. Pharmacological inhibition and co-immunoprecipitation experiments demonstrated the involvement of NF-ĸB in hnRNPA2/B activity and turnover. Our data indicate cardinal involvement of hnRNP A2/B1 protein in peripheral mechanisms of tolerance restoration and attenuation of inflammation, identifying a novel immunoplayer potentially targetable in all AEDs., Competing Interests: The authors declare that they have no conflict of interest.

Chronic lymphocytic leukemia (CLL) is the most common human leukemia, and dysregulation of tRNA-derived short noncoding RNA (tsRNA) (tRF-1) expression is an accompanying event in the development of this disease. tsRNAs are fragments originating from the 3' end of tRNA precursors and do not contain mature tRNA sequences. In contrast to tsRNAs, mature tRFs (tRF-3s, tRF-5s, and internal tRFs) are produced from mature tRNA sequences and are redundant fragments. We investigated tsRNA expression in CLL and determined tsRNA signatures in indolent CLL and aggressive CLL vs. normal B cells. We noticed that both ts-43 and ts-44 are derived from distinct genes of pre-tRNA His , and are down-regulated in CLL 3- to 5-fold vs. normal B cells. Thus, we investigated expression levels of tRF-5 fragments from tRNA His in CLL samples and healthy controls, and determined that such fragments are down-regulated by 5-fold in CLLs vs. normal controls. Given these results, we investigated the expression of all mature tRFs in CLLs vs. normal controls. We found a drastic dysregulation of the expression of mature tRFs in CLL. In aggressive CLL, for the top 15 up-regulated fragments, linear fold change varied from 2,053- to 622-fold. For the top 15 down-regulated fragments in CLL, linear fold change varied from 314- to 52-fold. In addition, 964 mature tRFs were up-regulated at least 2-fold in CLL, while 701 fragments were down-regulated at least 2-fold. Similar results were obtained for indolent CLL. Our results suggest that mature tRFs may have oncogenic and/or tumor suppressor function in CLL., Competing Interests: Competing interest statement: N.C. and T.J.K. are coauthors on a 2018 guidelines paper. N.C. and C.M.C. are coinvestigators on a grant application but have not begun a collaboration yet.

Ex vivo limbal stem cell transplantation is the main therapeutic approach to address a complete and functional re-epithelialization in corneal blindness, the second most common eye disorder. Although important key points were defined, the molecular mechanisms involved in the epithelial phenotype determination are unclear. Our previous studies have demonstrated the pluripotency and immune-modulatory of fibroblast limbal stem cells (f-LSCs), isolated from the corneal limbus. We defined a proteomic profile especially enriched in wound healing and cytoskeleton-remodelling proteins, including Profilin-1 (PFN1). In this study we postulate that pfn-1 knock down promotes epithelial lineage by inhibiting the integrin-β1(CD29)/mTOR pathway and subsequent NANOG down-expression. We showed that it is possible modulate pfn1 expression levels by treating f-LSCs with Resveratrol (RSV), a natural compound: pfn1 decline is accompanied with up-regulation of the specific differentiation epithelial genes pax6 (paired-box 6), sox17 (sex determining region Y-box 17) and ΔNp63-α (p63 splice variant), consistent with drop-down of the principle stem gene levels. These results contribute to understand the molecular biology of corneal epithelium development and suggest that pfn1 is a potential molecular target for the treatment of corneal blindness based on epithelial cell dysfunction., (© 2019 The Authors. Journal of Cellular and Molecular Medicine published by John Wiley & Sons Ltd and Foundation for Cellular and Molecular Medicine.)

The production and characterization of a double-layer scaffold, to be used as a system for the "on-demand" release of corneal limbal stem cells, are reported here. The devices used in the clinics and proposed so far in the scientific literature, for the release of corneal stem cells in the treatment of limbal stem cell deficiency, cannot control the in vivo space-time release of cells as the biomaterial of which they are composed is devoid of the stimuli-responsive feature. Our approach was to produce a scaffold composed of two different polymeric layers that give the device the appropriate mechanical properties to be placed on the ocular surface and the possibility of releasing the stem cells following a noninvasive and cell-friendly treatment. This device consists of an electrospun microfibrillar scaffold of poly-l-lactic acid coated by a polymeric film based on an amphiphilic derivative of hyaluronic acid sensitive to the ionic strength of the external medium and to the presence of a complexing agent. The latter represents the "sacrificial" cell containing layer of the scaffold that can be dissolved "on demand" by the treatment with a solution of cyclodextrins. The rapid removal of the external polymeric film from the device is exploited to control the space-time release of the cells. In vitro and ex vivo experiments showed that fibroblast-like limbal stem cells cultured on the scaffold without the use of the feeder layer maintained their characteristics of stem cells and can be released "on demand" on the culture well coated with Matrigel or on the decellularized bovine cornea, respectively.

The core components of regenerative medicine are stem cells with high self-renewal and tissue regeneration potentials. Adult stem cells can be obtained from many organs and tissues. NANOG , SOX2 and OCT4 represent the core regulatory network that suppresses differentiation-associated genes, maintaining the pluripotency of mesenchymal stem cells. The roles of NANOG in maintaining self-renewal and undifferentiated status of adult stem cells are still not perfectly established. In this study we define the effects of downregulation of NANOG in maintaining self-renewal and undifferentiated state in mesenchymal stem cells (MSCs) derived from subcutaneous adipose tissue (hASCs). hASCs were expanded and transfected in vitro with short hairpin Lentivirus targeting NANOG . Gene suppressions were achieved at both transcript and proteome levels. The effect of NANOG knockdown on proliferation after 10 passages and on the cell cycle was evaluated by proliferation assay, colony forming unit (CFU), qRT-PCR and cell cycle analysis by flow-cytometry. Moreover, NANOG involvement in differentiation ability was evaluated. We report that downregulation of NANOG revealed a decrease in the proliferation and differentiation rate, inducing cell cycle arrest by increasing p27 / CDKN1B (Cyclin-dependent kinase inhibitor 1B) and p21 / CDKN1A (Cyclin-dependent kinase inhibitor 1A) through p53 and regulate DLK1 / PREF1 . Furthermore, NANOG induced downregulation of DNMT1 , a major DNA methyltransferase responsible for maintaining methylation status during DNA replication probably involved in cell cycle regulation. Our study confirms that NANOG regulates the complex transcription network of plasticity of the cells, inducing cell cycle arrest and reducing differentiation potential.

Developmental coordination disorder (DCD) and attention-deficit hyperactivity disorder (ADHD) are neuro-developmental disorders, starting in childhood, which can affect the planning of movements and the coordination. We investigated how and in which measure a system based on wearable inertial measurement units (IMUs) can provide an objective support to the diagnosis of motor impairments in school-aged children. The IMUs measured linear and rotational movements of 37 schoolchildren, 7-10yo, 17 patients and 20 control subjects, during the execution of motor exercises, performed under medical and psychiatric supervision, to assess different aspects of the motor coordination. The measured motor parameters showed a high degree of significance in discriminating the ADHD/DCD patients from the healthy subjects, pointing out which motor tasks are worth focusing on. So, medical doctors have a novel key lecture to state a diagnosis, gaining in objectivity with respect to the standard procedures which mainly involve subjective human judgment. Differently to other works, we propose a novel approach in terms of number of used IMUs and of performed motor tasks. Moreover, we demonstrate the meaningful parameters to be considered as more discriminant in supporting the medical diagnosis., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Noncoding RNAs (ncRNAs) have received much attention due to their central role in gene expression and translational regulation as well as due to their involvement in several biological processes and disease development. Small noncoding RNAs (sncRNAs), such as microRNAs and piwiRNAs, have been thoroughly investigated and functionally characterized. Long noncoding RNAs (lncRNAs), known to play an important role in chromatin-interacting transcription regulation, posttranscriptional regulation, cell-to-cell signaling, and protein regulation, are also being investigated to further elucidate their functional roles.Next-generation sequencing (NGS) technologies have greatly aided in characterizing the ncRNAome. Moreover, the coupling of NGS technology together with bioinformatics tools has been essential to the genome-wide detection of RNA modifications in ncRNAs. RNA editing, a common human co-transcriptional and posttranscriptional modification, is a dynamic biological phenomenon able to alter the sequence and the structure of primary transcripts (both coding and noncoding RNAs) during the maturation process, consequently influencing the biogenesis, as well as the function, of ncRNAs. In particular, the dysregulation of the RNA editing machineries have been associated with the onset of human diseases.In this chapter we discuss the potential functions of ncRNA editing and describe the knowledge base and bioinformatics resources available to investigate such phenomenon.