Canine Epithelial Skin Tumours: Expression of the Stem Cell Markers Lgr5, Lgr6 and Sox9 in Light of New Cancer Stem Cell Theories.

Evidence is accumulating that tumour development is driven by cancer stem cells (CSCs). In order to understand the presence and potential contribution of stem cells (SCs) as tumour-initiating cells in canine cutaneous tumours, we selected three putative SC markers (Lgr5, Lgr6 and Sox9) and investigated their expression pattern, level of protein and mRNA expression, in 43 canine hair follicle (HF) and 18 canine cutaneous epidermal tumours by immunohistochemistry and qRT-PCR, using normal skin samples as controls. Lgr5 protein expression was not detected in epidermal and HF tumours; however, Lgr5 mRNA overexpression was evident in some HF tumours. Sox9 was expressed in several tumour cases, both at the protein and mRNA level. The Lgr6 antibody tested was not suitable for formalin-fixed paraffin-embedded tissue samples, but Lgr6 gene showed higher expression in several samples of both HF and epidermal tumours compared with normal skin. Significantly higher mRNA expression levels of the three SC markers were found in trichoblastomas (TB) compared with basal cell carcinomas (BCC). The present results indicated that canine HF and epidermal tumours might have common tumour-initiating cells. The mRNA expression of the three selected SC markers, especially Lgr5, could be potentially useful in the distinction between canine TB and BCC

Serum Cystatin C for the diagnosis of acute Kidney Injury in Patients Admitted in the Emergency Department

BACKGROUND: Early diagnosis of acute kidney injury (AKI) at emergency department (ED) is a challenging issue. Current diagnostic criteria for AKI poorly recognize early renal dysfunction and may cause delayed diagnosis. We evaluated the use of serum cystatin C (CysC) for the early and accurate diagnosis of AKI in patients hospitalized from the ED. METHODS: In a total of 198 patients (105 males and 93 females), serum CysC, serum creatinine (sCr), and estimated glomerular filtration rate (eGFR) were calculated at 0, 6, 12, 24, 48, and 72 hours after presentation to the ED. We compared two groups according to the presence or absence of AKI. RESULTS: Serial assessment of CysC, sCr, and eGFR was not a strong, reliable tool to distinguish AKI from non-AKI. CysC > 1.44 mg/L at admission, both alone (Odds Ratio = 5.04; 95%CI 2.20-11.52; P < 0.0002) and in combination with sCr and eGFR (Odds Ratio = 5.71; 95%CI 1.86-17.55; P < 0.002), was a strong predictor for the risk of AKI. CONCLUSIONS: Serial assessment of CysC is not superior to sCr and eGFR in distinguishing AKI from non-AKI. Admission CysC, both alone and in combination with sCr and eGFR, could be considered a powerful tool for the prediction of AKI in ED patients

H2AFZ:A Novel Prognostic Marker in Canine Melanoma and a Predictive Marker for Resistance to CDK4/6 Inhibitor Treatment

Uncontrolled proliferation is a key feature of tumor progression and malignancy. This suggests that cell-cycle related factors could be exploited as cancer biomarkers and that pathways specifically involved in the cell cycle, such as the Rb-E2F pathway, could be targeted as an effective anti-tumor therapy. We investigated 34 formalin-fixed paraffin-embedded (FFPE) tissue samples of canine cutaneous melanocytoma, cutaneous melanoma, and oral melanoma. Corresponding clinical follow-up data were used to determine the prognostic value of the mRNA expression levels of several cell cycle regulated E2F target genes (E2F1, DHFR, CDC6, ATAD2, MCM2, H2AFZ, GINS2, and survivin/BIRC5). Moreover, using four canine melanoma cell lines, we explored the possibility of blocking the Rb-E2F pathway by using a CDK4/6 inhibitor (Palbociclib) as a potential anti-cancer therapy. We investigated the expression levels of the same E2F target gene transcripts before and after treatment to determine the potential utility of these molecules as predictive markers. The E2F target gene H2AFZ was expressed in 91.43% of the primary tumors and H2AFZ expression was significantly higher in cases with unfavorable clinical outcome. Among the other tested genes, survivin/BIRC5 showed as well-promising results as a prognostic marker in canine melanoma. Three of the four tested melanoma cell lines were sensitive to the CDK4/6 inhibitor. The resistant cell line displayed higher expression levels of H2AFZ before treatment compared to the CDK4/6 inhibitor-sensitive cell lines. The present results suggest that CDK4/6 inhibitors could potentially be used as a new anti-cancer treatment for canine melanoma and that H2AFZ could serve as a prognostic and predictive marker for patient selection.</p

Reawakening the Intrinsic Cardiac Regenerative Potential: Molecular Strategies to Boost Dedifferentiation and Proliferation of Endogenous Cardiomyocytes

Despite considerable efforts carried out to develop stem/progenitor cell-based technologies aiming at replacing and restoring the cardiac tissue following severe damages, thus far no strategies based on adult stem cell transplantation have been demonstrated to efficiently generate new cardiac muscle cells. Intriguingly, dedifferentiation, and proliferation of pre-existing cardiomyocytes and not stem cell differentiation represent the preponderant cellular mechanism by which lower vertebrates spontaneously regenerate the injured heart. Mammals can also regenerate their heart up to the early neonatal period, even in this case by activating the proliferation of endogenous cardiomyocytes. However, the mammalian cardiac regenerative potential is dramatically reduced soon after birth, when most cardiomyocytes exit from the cell cycle, undergo further maturation, and continue to grow in size. Although a slow rate of cardiomyocyte turnover has also been documented in adult mammals, both in mice and humans, this is not enough to sustain a robust regenerative process. Nevertheless, these remarkable findings opened the door to a branch of novel regenerative approaches aiming at reactivating the endogenous cardiac regenerative potential by triggering a partial dedifferentiation process and cell cycle re-entry in endogenous cardiomyocytes. Several adaptations from intrauterine to extrauterine life starting at birth and continuing in the immediate neonatal period concur to the loss of the mammalian cardiac regenerative ability. A wide range of systemic and microenvironmental factors or cell-intrinsic molecular players proved to regulate cardiomyocyte proliferation and their manipulation has been explored as a therapeutic strategy to boost cardiac function after injuries. We here review the scientific knowledge gained thus far in this novel and flourishing field of research, elucidating the key biological and molecular mechanisms whose modulation may represent a viable approach for regenerating the human damaged myocardium

Cardiotoxicity of Anticancer Drugs: Molecular Mechanisms and Strategies for Cardioprotection

Chemotherapy and targeted therapies have significantly improved the prognosis of oncology patients. However, these antineoplastic treatments may also induce adverse cardiovascular effects, which may lead to acute or delayed onset of cardiac dysfunction. These common cardiovascular complications, commonly referred to as cardiotoxicity, not only may require the modification, suspension, or withdrawal of life-saving antineoplastic therapies, with the risk of reducing their efficacy, but can also strongly impact the quality of life and overall survival, regardless of the oncological prognosis. The onset of cardiotoxicity may depend on the class, dose, route, and duration of administration of anticancer drugs, as well as on individual risk factors. Importantly, the cardiotoxic side effects may be reversible, if cardiac function is restored upon discontinuation of the therapy, or irreversible, characterized by injury and loss of cardiac muscle cells. Subclinical myocardial dysfunction induced by anticancer therapies may also subsequently evolve in symptomatic congestive heart failure. Hence, there is an urgent need for cardioprotective therapies to reduce the clinical and subclinical cardiotoxicity onset and progression and to limit the acute or chronic manifestation of cardiac damages. In this review, we summarize the knowledge regarding the cellular and molecular mechanisms contributing to the onset of cardiotoxicity associated with common classes of chemotherapy and targeted therapy drugs. Furthermore, we describe and discuss current and potential strategies to cope with the cardiotoxic side effects as well as cardioprotective preventive approaches that may be useful to flank anticancer therapies

Insulin requirements and carbohydrate to insulin ratio in normal weight, overweight, and obese women with type 1 diabetes under pump treatment during pregnancy: a lesson from old technologies

Aim:The primary aim of this study was to assess insulin requirements and carbohydrateto insulin ratio (CHO/IR) in normal weight, overweight, and obese pregnant women withtype 1 diabetes across early, middle, and late pregnancy.Methods:In this multicenter, retrospective, observational study we evaluated 86 of 101pregnant Caucasian women with type 1 diabetes under pump treatment. The womenwere trained to calculate CHO/IR daily by dividing CHO grams of every single meal byinsulin units injected. Since the purpose of the study was to identify the CHO/IR able toreach the glycemic target, we only selected the CHO/IR obtained when glycemic valueswere at target. Statistics: SPSS 20.Results:We studied 45 normal weight, 31 overweight, and 10 obese women. Insulinrequirements increased throughout pregnancy (p &lt; 0.0001 and &lt;0.001 respectively) inthe normal and overweight women, while it remained unchanged in the obese women.Insulin requirements were different between groups when expressed as an absolute value,but not when adjusted for body weight. Breakfast CHO/IR decreased progressivelythroughout pregnancy in the normal weight women, from 13.3 (9.8–6.7) at thefirst stageof pregnancy to 6.2 (3.8–8.6) (p = 0.01) at the end stage, and in the overweight womenFrontiers in Endocrinology | www.frontiersin.orgFebruary 2021 | Volume 12 | Article 6108771Edited by:Elena Succurro,University of Magna Graecia, ItalyReviewed by:Cristina Bianchi,Azienda Ospedaliero-UniversitariaPisana, ItalyMaria Grazia Dalfra’,University of Padua, Italy*Correspondence:Camilla [email protected] section:This article was submitted toObesity,a section of the journalFrontiers in EndocrinologyReceived:27 September 2020Accepted:14 January 2021Published:25 February 2021Citation:Festa C,Fresa R,Visalli N,Bitterman O,Giuliani C,Suraci C,Bongiovanni M andNapoli A (2021)Insulin Requirements andCarbohydrate to Insulin Ratio inNormal Weight, Overweight, andObese Women With Type 1Diabetes Under Pump TreatmentDuring Pregnancy: A LessonFrom Old Technologies.Front. Endocrinol. 12:610877.doi: 10.3389/fendo.2021.610877ORIGINAL RESEARCHpublished: 25 February 2021doi: 10.3389/fendo.2021.610877 from 8.5 (7.1–12.6) to 5.2 (4.0–8.1) (p = 0.001), while in the obese women it remainedstable, moving from 6.0 (5.0–7.9) to 5.1 (4.1–7.4) (p = 0.7). Likewise, lunch and dinnerCHO/IR decreased in the normal weight and overweight women (p &lt; 0.03) and not in theobese women. The obese women gained less weight than the others, especially in earlypregnancy when they even lost a median of 1.25 (−1−1.1) kg (p = 0.005). In earlypregnancy, we found a correlation between pregestational BMI and insulin requirements(IU/day) or CHO/IR at each meal (p &lt; 0.001 and p = 0.001, respectively). In latepregnancy, a relationship between pre-gestational BMI and CHO/IR change was found(P = 0.004), as well as between weight gain and CHO/IR change (p=0.02). Thesignificance was lost when both variables were included in the multiple regressionanalysis. There was no difference in pregnancy outcomes except for a higher pre-termdelivery rate in the obese women.Conclusion:Pre-gestational BMI and weight gain may play a role in determining CHO/IRduring pregnancy in women with type 1 diabetes under pump treatment

Transcriptome Analysis of Canine Cutaneous Melanoma and Melanocytoma Reveals a Modulation of Genes Regulating Extracellular Matrix Metabolism and Cell Cycle

Interactions between tumor cells and tumor microenvironment are considered critical in carcinogenesis, tumor invasion and metastasis. To examine transcriptome changes and to explore the relationship with tumor microenvironment in canine cutaneous melanocytoma and melanoma, we extracted RNA from formalin-fixed, paraffin-embedded (FFPE) specimens and analyzed them by means of RNA-seq for transcriptional analysis. Melanocytoma and melanoma samples were compared to detect differential gene expressions and significant enriched pathways were explored to reveal functional relations between differentially expressed genes. The study demonstrated a differential expression of 60 genes in melanomas compared to melanocytomas. The differentially expressed genes cluster in the extracellular matrix-receptor interaction, protein digestion and absorption, focal adhesion and PI3K-Akt (phosphoinositide 3-kinase/protein kinase B) signaling pathways. Genes encoding for several collagen proteins were more commonly differentially expressed. Results of the RNA-seq were validated by qRT-PCR and protein expression of some target molecules was investigated by means of immunohistochemistry. We hypothesize that the developing melanoma actively promotes collagen metabolism and extracellular matrix remodeling as well as enhancing cell proliferation and survival contributing to disease progression and metastasis. In this study, we also detected unidentified genes in human melanoma expression studies and uncover new candidate drug targets for further testing in canine melanoma

Immunohistochemical investigation of cell cycle and apoptosis regulators (Survivin, beta-Catenin, P53, Caspase 3) in canine appendicular osteosarcoma

Background: Osteosarcoma (OSA) represents the most common canine primary bone tumour. Despite several pathways have been investigated so far, few molecules have been identified as prognostic tools or potential therapeutic targets, and there is still the need to find out molecular pathways with specific influence over OSA progression to facilitate earlier prognosis and treatment.Aims of the present study were to evaluate the immunohistochemical pattern and levels of expression of a panel of molecules (survivin, β-catenin, caspase 3 -inactive and active forms- and p53) involved in cell cycle and apoptosis regulation in canine OSA samples, known to be of interest in the study also of human OSA, and to detect specific relations among them and with histological tumour grade, disease free interval (DFI) and overall survival (OS).Results: Nuclear β-catenin immunostaining was detected in normal osteoblasts adjacent to the tumour, and in 47% of the cases. Cytoplasmic and/or membranous immunostaining were also observed. Nuclear survivin and p53 positive cells were found in all cases. Moderate/high cytoplasmic β-catenin expression (≥10% positive cells) was significantly associated with the development of metastasis (P = 0.014); moderate/high nuclear p53 expression (≥10% positive cells) was significantly associated with moderate/high histological grade (P = 0.017) and shorter OS (P = 0.049). Moderate/high nuclear survivin expression (≥15% positive cells) showed a tendency toward a longer OS (P = 0,088).Conclusions: The present results confirmed p53 as negative prognostic marker, while suggested survivin as a potential positive prognostic indicator, rather than indicative of a poor prognosis. The detection of nuclear β-catenin immunostaining in normal osteoblasts and the absent/low expression in most of the OSAs, suggested that this pathway could not play a major role in oncogenic transformation of canine osteoblasts. Further studies are needed to confirm these hypotheses

Domino reaction for the sustainable functionalization of few-layer graphene

The mechanism for the functionalization of graphene layers with pyrrole compounds was investigated. Liquid 1,2,5-trimethylpyrrole (TMP) was heated in air in the presence of a high surface area nanosized graphite (HSAG), at temperatures between 80°C and 180°C. After the thermal treatments solid and liquid samples, separated by centrifugation, were analysed by means of Raman, Fourier Transform Infrared (FT-IR) spectroscopy, X-Rays Photoelectron Spectroscopy (XPS) and1H-Nuclear Magnetic Resonance (1H NMR) spectroscopy and High Resolution Transmission Electron Microscopy (HRTEM). FT-IR spectra were interpreted with the support of Density Functional Theory (DFT) quantum chemical modelling. Raman findings suggested that the bulk structure of HSAG remained substantially unaltered, without intercalation products. FT-IR and XPS spectra showed the presence of oxidized TMP derivatives on the solid adducts, in a much larger amount than in the liquid. For thermal treatments at T ≥ 150°C, IR spectral features revealed not only the presence of oxidized products but also the reaction of intra-annular double bond of TMP with HSAG. XPS spectroscopy showed the increase of the ratio between C(sp2)N bonds involved in the aromatic system and C(sp3)N bonds, resulting from reaction of the pyrrole moiety, observed while increasing the temperature from 130°C to 180°C. All these findings, supported by modeling, led to hypothesize a cascade reaction involving a carbocatalyzed oxidation of the pyrrole compound followed by Diels-Alder cycloaddition. Graphene layers play a twofold role: at the early stages of the reaction, they behave as a catalyst for the oxidation of TMP and then they become the substrate for the cycloaddition reaction. Such sustainable functionalization, which does not produce by-products, allows us to use the pyrrole compounds for decorating sp2 carbon allotropes without altering their bulk structure and smooths the path for their wider application

Prognostic and Predictive Role of Body Composition in Metastatic Neuroendocrine Tumor Patients Treated with Everolimus: A Real-World Data Analysis

Neuroendocrine tumors (NETs) are rare neoplasms frequently characterized by an up- regulation of the mammalian rapamycin targeting (mTOR) pathway resulting in uncontrolled cell proliferation. The mTOR pathway is also involved in skeletal muscle protein synthesis and in adipose tissue metabolism. Everolimus inhibits the mTOR pathway, resulting in blockade of cell growth and tumor progression. The aim of this study is to investigate the role of body composition in- dexes in patients with metastatic NETs treated with everolimus. The study population included 30 patients with well-differentiated (G1-G2), metastatic NETs treated with everolimus at the IRCCS Romagnolo Institute for the Study of Tumors (IRST) “Dino Amadori”, Meldola (FC), Italy. The body composition indexes (skeletal muscle index [SMI] and adipose tissue indexes) were assessed by measuring on a computed tomography (CT) scan the cross-sectional area at L3 at baseline and at the first radiological assessment after the start of treatment. The body mass index (BMI) was assessed at baseline. The median progression-free survival (PFS) was 8.9 months (95% confidence interval [CI]: 3.4–13.7 months). The PFS stratified by tertiles was 3.2 months (95% CI: 0.9–10.1 months) in patients with low SMI (tertile 1), 14.2 months (95% CI: 2.3 months-not estimable [NE]) in patients with intermediate SMI (tertile 2), and 9.1 months (95% CI: 2.7 months-NE) in patients with high SMI (tertile 3) (p = 0.039). Similarly, the other body composition indexes also showed a statistically significant difference in the three groups on the basis of tertiles. The median PFS was 3.2 months (95% CI: 0.9–6.7 months) in underweight patients (BMI 18.49 kg/m2) and 10.1 months (95% CI: 3.7–28.4 months) in normal-weight patients (p = 0.011). There were no significant differences in terms of overall survival. The study showed a correlation between PFS and the body composition indexes in patients with NETs treated with everolimus, underlining the role of adipose and muscle tissue in these patients