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Akt is a serine-threonine kinase that has an important role in transducing survival signals. Akt also regulates a number of proteins involved in the apoptotic process. To find new Akt interactors, we performed a two-hybrid screening in yeast using full-length Akt cDNA as bait and a human cDNA heart library as prey. Among 200 clones obtained, two of them were identified as coding for the c-FLIPL protein. c-FLIPL is an endogenous inhibitor of death receptor-induced apoptosis through the caspase-8 pathway. Using co-immunoprecipitation experiments of either transfected or endogenous proteins, we confirmed the interaction between Akt and c-FLIPL. Furthermore, we observed that c-FLIPL overexpression interferes with Gsk3-β phosphorylation levels. Moreover, through its effects on Gsk3β, c-FLIPL overexpression in cancer cells induced resistance to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL). This effect was mediated by the regulation of p27Kip1 and caspase-3 expression. These results indicate the existence of a new mechanism of resistance to TRAIL in cancer cells, and unexpected functions of c-FLIPL. [ABSTRACT FROM AUTHOR]

In recent years, significant progress has been made in the development of fluorescent contrast agents for clinical applications. For the development of a fluorescent probe, it is crucial to evaluate its safety profile, including biodistribution. Specific methods need to be developed for the absolute quantification of fluorescent probes in tissue specimens from animals administered with test compounds in the framework of biodistribution/efficacy/toxicity studies. Here, we describe a new method for the absolute quantification of fluorescent probes in tissue specimens from animals administered with compounds that have absorption and emission wavelength in the Near-Infrared region (600-800 nm). The protocol is based on the standard addition approach in order to minimize the interference of the matrix on the analyte signal causing inaccuracy in the absolute determination of the concentration. The measurement of the fluorescence intensity is done via a microplate reader. The method has been fully validated and applied for the quantification of a fluorescence-guided surgery targeted contrast agent in a Good Laboratory Practice (GLP) biodistribution study. Results clearly demonstrate that this procedure is fully applicable in a preclinical setting and that it overcomes common issues associated with fluorescence signal quantification in tissue extracts., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests:All the authors are employees of Bracco Imaging SpA, which is the applicant of a pending patent claiming, among else, the Test Article described in this manuscript., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Hypoxia plays a crucial role in tumorigenesis and drug resistance, and it is recognised as a major factor affecting patient clinical outcome. Therefore, the detection of hypoxic areas within the tumour micro-environment represents a useful way to monitor tumour growth and patients' responses to treatments, properly guiding the choice of the most suitable therapy. To date, non-invasive hypoxia imaging probes have been identified, but their applicability in vivo is strongly limited due to an inadequate resistance to the low oxygen concentration and the acidic pH of the tumour micro-environment. In this regard, nucleic acid aptamers represent very powerful tools thanks to their peculiar features, including high stability to harsh conditions and a small size, resulting in easy and efficient tumour penetration. Here, we describe a modified cell-SELEX (Systematic Evolution of Ligands by EXponential enrichment) approach that allows the isolation of specific RNA aptamers for the detection of the hypoxic phenotype in breast cancer (BC) cells. We demonstrated the effectiveness of the proposed method in isolating highly stable aptamers with an improved and specific binding to hypoxic cells. To our knowledge, this is the first example of a cell-SELEX approach properly designed and modified to select RNA aptamers against hypoxia-related epitopes expressed on tumour cell surfaces. The selected aptamers may provide new effective tools for targeting hypoxic areas within the tumour with great clinical potential., Competing Interests: Author MI was employed by Bracco Imaging S.p.A. Author AA was employed by Percuros B.V. The remaining 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 Nuzzo, Iaboni, Ibba, Rienzo, Musumeci, Franzese, Roscigno, Affinito, Petrillo, Quintavalle, Ciccone, Esposito and Catuogno.)

Our research explores serum extracellular circulating miRNAs (ecmiRNAs) involved in dog stress response immediately after the search and rescue (SAR) of missing people. The experimental plan considers four arduous SAR simulations. The SAR dogs are trained by the Alpine School of the Military Force of Guardia di Finanza (Passo Rolle, Italy). The First SAR Trial analyzed dog serum samples at rest time (T0), and immediately after SAR performance (T1) using the miRNome-wide screening next-generation sequencing (NGS). T1 versus T0 NGS results revealed a different expression level of let-7a and let-7f. Subsequently, in a large sample size including: 1st (n = 6), 2nd (n = 6), 3rd (n = 6), and 4th (n = 4) trials, let-7a and let-7f were validated by qPCR. Bioinformatics analysis with TarBase (v.8) and the Diana-mirPath (v.3) revealed a functional role of let-7a and let-7f in the p53 pathway to restore cellular homeostasis. Let-7a and let-7f, highly expressed at T1, could stop MDMs-p53 inhibition inducing the p53 increase in level. In addition, let-7a and let-7f, via p53 post-transcriptional regulation, buffers p53 transcription spikes. During SAR stress, the possibility of p53 preconditioning could explain the phenomenon of "stress hardening" where the tolerance of particular stress increases after preconditioning., (© 2022. The Author(s).)

This study aimed to assess the heart rate (HR) responses of avalanche SAR dogs and handlers under working field conditions. Thirteen SAR units (dogs and handlers) performed an exercise ( Endurance ) consisting of approximately 5.5 km of rough tracks through deep snow, at an altitude of 1991-2250 m.a.s.l. The exercise was repeated twice for each of the two different tracks. Both handlers and dogs were equipped with a global positioning satellite/heart rate (GPS/HR) system (Polar ® ). Multivariable models were used to evaluate the effects of environmental (i.e., gradient, altitude, track, and time) and intrinsic (i.e., speed, repetition, and breed) factors on changes from baseline HR (Δ%HR). The dog's Δ%HR was greater in the flat and uphill compared with downhill, and increased progressively as the speed increased ( p < 0.001). Moreover, it rose at altitudes above 2100 m.a.s.l. and peaked after 30 min of the Endurance activity ( p < 0.01). These findings indicated that HR monitors could be a valuable tool to contribute to the evaluation of avalanche dogs' fitness in their real working environment. In contrast, the lack of correlation between the dogs' and handlers' HR changes suggests that handlers might not perceive the physical conditions of their dog in real-time. Thus, implementing protocols to monitor avalanche SAR dogs' fitness using a GPS/HR monitoring system could help handlers to tailor the training and workload and to detect the risk factors for physical distress of working dogs.

The strong molecular interaction between biotin and streptavidin is widely used in the growing field of nucleic acid nanotechnology. Several biotin labeled oligonucleotide tools have been developed for the detection of biological molecules as well as for protein purification. For these reasons, biotinylation can be considered one of the main chemical reactions for nucleic acid labeling. However, despite its widespread application and the presence on the market of many reagents for the conjugation of biotin to oligonucleotides, it is not yet available a cheap, easy and sensitive system able to assess the effectiveness and reproducibility of this reaction. Here, we present an accurate and reliable method to achieve a qualitative and quantitative analysis of oligonucleotide biotinylation. The protocol employs basic laboratory instruments and standard software for molecular biology applications and does not require advanced expertise for its execution. Most importantly, our method is independent from complex kinetic equilibrium parameters and shows a limit of detection more than one order of magnitude lower than the current fluorometric gold standard assay. Therefore, this method could become a standard, inexpensive and routinely used quality test for post-synthesis evaluation of biotin conjugation reactions., Competing Interests: Declaration of competing interest None., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Small therapeutic peptides represent a promising field for the treatment of pathologies such as cardiac diseases. However, the lack of proper target-selective carriers hampers their translation towards a potential clinical application. Aptamers are cell-specific carriers that bind with high affinity to their specific target. However, some limitations on their conjugation to small peptides and the functionality of the resulting aptamer-peptide chimera exist. Here, we generated a novel aptamer-peptide chimera through conjugation of the PDGFRβ-targeting Gint4.T aptamer to MP, a small mimetic peptide that via targeting of the Cavβ2 subunit of the L-type calcium channel (LTCC) can recover myocardial function in pathological heart conditions associated with defective LTCC function. The conjugation reaction was performed by click chemistry in the presence of N,N,N',N',N"-pentamethyldiethylenetriamine as a Cu (I) stabilizing agent in a DMSO-free aqueous buffer. When administered to cardiac cells, the Gint4.T-MP aptamer-peptide chimera was successfully internalized in cells, allowing the functional targeting of MP to LTCC. This approach represents the first example of the use of an internalizing aptamer for selective delivery of a small therapeutic peptide to cardiac cells.

Cancer-associated fibroblasts (CAFs) are the major components of the tumor microenvironment. They may drive tumor progression, although the mechanisms involved are still poorly understood. Exosomes have emerged as important mediators of intercellular communication in cancer. They mediate horizontal transfer of microRNAs (miRs), mRNAs and proteins, thus affecting breast cancer progression. Differential expression profile analysis identified three miRs (miRs -21, -378e, and -143) increased in exosomes from CAFs as compared from normal fibroblasts. Immunofluorescence indicated that exosomes may be transferred from CAFs to breast cancer cells, releasing their cargo miRs. Breast cancer cells (BT549, MDA-MB-231, and T47D lines) exposed to CAF exosomes or transfected with those miRs exhibited a significant increased capacity to form mammospheres, increased stem cell and epithelial-mesenchymal transition (EMT) markers, and anchorage-independent cell growth. These effects were reverted by transfection with anti-miRs. Similarly to CAF exosomes, normal fibroblast exosomes transfected with miRs -21, -378e, and -143 promoted the stemness and EMT phenotype of breast cancer cells. Thus, we provided evidence for the first time of the role of CAF exosomes and their miRs in the induction of the stemness and EMT phenotype in different breast cancer cell lines. Indeed, CAFs strongly promote the development of an aggressive breast cancer cell phenotype.

Nucleic acid-based aptamers are emerging as therapeutic antagonists of disease-associated proteins such as receptor tyrosine kinases. They are selected by an in vitro combinatorial chemistry approach, named Systematic Evolution of Ligands by Exponential enrichment (SELEX), and thanks to their small size and unique chemical characteristics, they possess several advantages over antibodies as diagnostics and therapeutics. In addition, aptamers that rapidly internalize into target cells hold as well great potential for their in vivo use as delivery tools of secondary therapeutic agents. Here, we describe a nuclease resistant RNA aptamer, named GL56, which specifically recognizes the insulin receptor (IR). Isolated by a cell-based SELEX method that allows enrichment for internalizing aptamers, GL56 rapidly internalizes into target cells and is able to discriminate IR from the highly homologous insulin-like growth factor receptor 1. Notably, when applied to IR expressing cancer cells, the aptamer inhibits IR dependent signaling. Given the growing interest in the insulin receptor as target for cancer treatment, GL56 reveals a novel molecule with great translational potential as inhibitor and delivery tool for IR-dependent cancers.

Saving human lives is of paramount importance in avalanche rescue missions. Avalanche military dogs represent an invaluable resource in these operations. However, their performance can be influenced by several environmental, social and transport challenges. If too severe, these are likely to activate a range of responses to stress, which might put at risk the dogs' welfare. The aim of this study was to assess the physiological and behavioural responses of a group of military dogs to a Simulated Avalanche Search and Rescue mission (SASR). Seventeen avalanche dogs from the Italian Military Force Guardia di Finanza (SAGF dogs) were monitored during a simulated search for a buried operator in an artificial avalanche area (SASR). Heart rate (HR), body temperature (RBT) and blood samples were collected at rest the day before the trial (T0), immediately after helicopter transport at the onset of the SASR (T1), after the discovery of the buried operator (T2) and 2h later (T3). Heart rate (HR), rectal body temperature (RBT), cortisol, aspartate aminotransferase (AST), creatine kinase (CK), non-esterified fatty acids (NEFA) and lactate dehydrogenase (LDH) were measured. During the search mission the behaviour of each SAGF dog was measured by focal animal sampling and qualitatively assessed by its handler and two observers. Inter-rater agreement was evaluated. Snow and environmental variables were also measured. All dogs successfully completed their search for the buried, simulated victim within 10min. The SASR was shown to exert significant increases on RBT, NEFA and cortisol (P<0.001), CK and HR (P<0.01), AST and LDH (P<0.05). These indicate the activation of a response to stress probably induced by the addition of factors such as helicopter transport, disembarking, and the search and rescue exercise. However, changes were moderate and limited over time, progressively decreasing with complete recovery at T3 except for sera cortisol that showed a slightly slower decline. More time walking within the search was related to lower RBT, conversely to walking. Standing still with head up and exploring with head-up were inversely related with HR. Agreement between handler and observers' opinions on a dog's search mission ability was found only for motivation, signalling behaviour, signs of stress and possessive reward playing. More time signalling was related to shorter search time. In conclusion, despite extreme environmental and training conditions only temporary physiological and behavioural changes were recorded in the avalanche dogs. Their excellent performance in successful simulated SASR may be attributable to extensive training and good dog-handler relationships. Simulated SASR did not seem to impair SAGF dogs' performance or welfare., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Glioblastoma is the most common primary brain tumor in adults; with a survival rate of 12 months from diagnosis. However, a small subgroup of patients, termed long-term survivors (LTS), has a survival rate longer then 12-14 months. There is thus increasing interest in the identification of molecular signatures predicting glioblastoma prognosis and in how to improve the therapeutic approach. Here, we report miR-340 as prognostic tumor-suppressor microRNA for glioblastoma. We analyzed microRNA expression in > 500 glioblastoma patients and found that although miR-340 is strongly down-regulated in glioblastoma overall, it is up-regulated in LTS patients compared to short-term survivors (STS). Indeed, miR-340 expression predicted better prognosis in glioblastoma patients. Coherently, overexpression of miR-340 in glioblastoma cells was found to produce a tumor-suppressive activity. We identified NRAS mRNA as a critical, direct target of miR-340: in fact, miR-340 negatively influenced multiple aspects of glioblastoma tumorigenesis by down-regulating NRAS and downstream AKT and ERK pathways. Thus, we demonstrate that expression of miR-340 in glioblastoma is responsible for a strong tumor-suppressive effect in LTS patients by down-regulating NRAS. miR-340 may thus represent a novel marker for glioblastoma diagnosis and prognosis, and may be developed into a tool to improve treatment of glioblastoma.

TNF-related apoptosis-inducing ligand (TRAIL) is a promising antitumor agent for its remarkable ability to selectively induce apoptosis in cancer cells, without affecting the viability of healthy bystander cells. The TRAIL tumor suppressor pathway is deregulated in many human malignancies including lung cancer. In human non-small cell lung cancer (NSCLC) cells, sensitization to TRAIL therapy can be restored by increasing the expression levels of the tumor suppressor microRNA-212 (miR-212) leading to inhibition of the anti-apoptotic protein PED/PEA-15 implicated in treatment resistance. In this study, we exploited a previously described RNA aptamer inhibitor of the tyrosine kinase receptor Axl (GL21.T) expressed on lung cancer cells, as a means to deliver miR-212 into human NSCLC cells expressing Axl. We demonstrate efficient delivery of miR-212 following conjugation of the miR to GL21.T (GL21.T-miR212 chimera). We show that the chimera downregulates PED and restores TRAIL-mediate cytotoxicity in cancer cells. Importantly, treatment of Axl+ lung cancer cells with the chimera resulted in (i) an increase in caspase activation and (ii) a reduction of cell viability in combination with TRAIL therapy. In conclusion, we demonstrate that the GL21.T-miR212 chimera can be employed as an adjuvant to TRAIL therapy for the treatment of lung cancer.

Cancer stem cells (CSCs) are a small part of the heterogeneous tumor cell population possessing self-renewal and multilineage differentiation potential as well as a great ability to sustain tumorigenesis. The molecular pathways underlying CSC phenotype are not yet well characterized. MicroRNAs (miRs) are small noncoding RNAs that play a powerful role in biological processes. Early studies have linked miRs to the control of self-renewal and differentiation in normal and cancer stem cells. We aimed to study the functional role of miRs in human breast cancer stem cells (BCSCs), also named mammospheres. We found that miR-221 was upregulated in BCSCs compared to their differentiated counterpart. Similarly, mammospheres from T47D cells had an increased level of miR-221 compared to differentiated cells. Transfection of miR-221 in T47D cells increased the number of mammospheres and the expression of stem cell markers. Among miR-221's targets, we identified DNMT3b. Furthermore, in BCSCs we found that DNMT3b repressed the expression of various stemness genes, such as Nanog and Oct 3/4, acting on the methylation of their promoters, partially reverting the effect of miR-221 on stemness. We hypothesize that miR-221 contributes to breast cancer tumorigenicity by regulating stemness, at least in part through the control of DNMT3b expression.

Glioblastoma multiforme (GBM) is one of the most deadly types of cancer. To date, the best clinical approach for treatment is based on administration of temozolomide (TMZ) in combination with radiotherapy. Much evidence suggests that the intracellular level of the alkylating enzyme O(6)-methylguanine-DNA methyltransferase (MGMT) impacts response to TMZ in GBM patients. MGMT expression is regulated by the methylation of its promoter. However, evidence indicates that this is not the only regulatory mechanism present. Here, we describe a hitherto unknown microRNA-mediated mechanism of MGMT expression regulation. We show that miR-221 and miR-222 are upregulated in GMB patients and that these paralogues target MGMT mRNA, inducing greater TMZ-mediated cell death. However, miR-221/miR-222 also increase DNA damage and, thus, chromosomal rearrangements. Indeed, miR-221 overexpression in glioma cells led to an increase in markers of DNA damage, an effect rescued by re-expression of MGMT. Thus, chronic miR-221/222-mediated MGMT downregulation may render cells unable to repair genetic damage. This, associated also to miR-221/222 oncogenic potential, may poor GBM prognosis.

PED/PEA-15 (PED) is a death effector domain family member of 15 kDa with a broad antiapoptotic function found overexpressed in a number of different human tumors, including lung cancer. To date, the mechanisms that regulate PED expression are unknown. Therefore, we address this point by the identification of microRNAs that in non-small cell lung cancer (NSCLC) modulate PED levels. In this work, we identify miR-212 as a negative regulator of PED expression. We also show that ectopic expression of this miR increases tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-induced cell death in NSCLC cells. In contrast, inhibition of endogenous miR-212 by use of antago-miR results in increase of PED protein expression and resistance to TRAIL treatment. Besides, in NSCLC, we show both in vitro and in vivo that PED and miR-212 expressions are inversely correlated, that is, PED is upregulated and miR-212 is rarely expressed. In conclusion, these findings suggest that miR-212 should be considered as a tumor suppressor because it negatively regulates the antiapoptotic protein PED and regulates TRAIL sensitivity., ((c)2010 AACR.)