Transfer learning for the efficient detection of COVID-19 from smartphone audio data.

Disease detection from smartphone data represents an open research challenge in mobile health (m-health) systems. COVID-19 and its respiratory symptoms are an important case study in this area and their early detection is a potential real instrument to counteract the pandemic situation. The efficacy of this solution mainly depends on the performances of AI algorithms applied to the collected data and their possible implementation directly on the users' mobile devices. Considering these issues, and the limited amount of available data, in this paper we present the experimental evaluation of 3 different deep learning models, compared also with hand-crafted features, and of two main approaches of transfer learning in the considered scenario: both feature extraction and fine-tuning. Specifically, we considered VGGish, YAMNET, and L3-Net (including 12 different configurations) evaluated through user-independent experiments on 4 different datasets (13,447 samples in total). Results clearly show the advantages of L3-Net in all the experimental settings as it overcomes the other solutions by 12.3% in terms of Precision-Recall AUC as features extractor, and by 10% when the model is fine-tuned. Moreover, we note that to fine-tune only the fully-connected layers of the pre-trained models generally leads to worse performances, with an average drop of 6.6% with respect to feature extraction. Finally, we evaluate the memory footprints of the different models for their possible applications on commercial mobile devices.

Toward a Detailed Evaluation of Wireless Industrial Data Distribution Approaches.

Data distribution is a cornerstone of efficient automation for intelligent machines in Industry 4.0. Although in the recent literature there have been several comparisons of relevant methods, we identify that most of those comparisons are either theoretical or based on abstract simulation tools, unable to uncover the specific, detailed impacts of the methods to the underlying networking infrastructure. In this respect, as a first contribution of this paper, we develop more detailed and fine-tuned solutions for robust data distribution in smart factories on stationary and mobile scenarios of wireless industrial networking. Using the technological enablers of WirelessHART, RPL and the methodological enabler of proxy selection as building blocks, we compose the protocol stacks of four different methods (both centralized and decentralized) for data distribution in wireless industrial networks over the IEEE 802.15.4 physical layer. We implement the presented methods in the highly detailed OMNeT++ simulation environment and we evaluate their performance via an extensive simulation analysis. Interestingly enough, we demonstrate that the careful selection of a limited set of proxies for data caching in the network can lead to an increased data delivery success rate and low data access latency. Next, we describe two test cases demonstrated in an industrial smart factory environment. First, we show the collaboration between robotic elements and wireless data services. Second, we show the integration with an industrial fog node which controls the shop-floor devices. We report selected results in much larger scales, obtained via simulations.

Soluble CTLA-4 as a favorable predictive biomarker in metastatic melanoma patients treated with ipilimumab: an Italian melanoma intergroup study.

CTLA-4 blockade by means of ipilimumab (IPI) potentiates the immune response and improves overall survival (OS) in a minority of metastatic melanoma (MM) patients. We investigated the role of soluble CTLA-4 (sCTLA-4) as a possible biomarker for identifying this subset of patients. sCTLA-4 levels were analyzed at baseline in sera from 113 IPI-treated MM patients by ELISA, and the median value (200 pg/ml) was used to create two equally sized subgroups. Associations of sCTLA-4 with best overall response (BOR) to IPI and immune-related adverse events (irAEs) were evaluated through logistic regression. Kaplan-Meier and Cox regression methods were used to analyze OS. A remarkable association between sCTLA-4 levels and BOR was found. Specifically, the proportion of patients with sCTLA-4 > 200 pg/ml in irSD or irPD (immune-related stable or progressive disease) was, respectively, 80% (OR = 0.23; 95%CL = 0.03-1.88) and 89% (OR = 0.11; 95%CL = 0.02-0.71) and was lower than that observed among patients in irCR/irPR (immune-related complete/partial response). sCTLA-4 levels increased during IPI treatment, since the proportion of patients showing sCTLA > 200 pg/ml after 3 cycles was 4 times higher (OR = 4.41, 95%CL = 1.02-19.1) than that after 1 cycle. Moreover, a significantly lower death rate was estimated for patients with sCTLA-4 > 200 pg/ml (HR = 0.61, 95%CL = 0.39-0.98). Higher baseline sCTLA-4 levels were also associated with the onset of any irAE (p value = 0.029), in particular irAEs of the digestive tract (p value = 0.041). In conclusion, our results suggest that high sCTLA-4 serum levels might predict favorable clinical outcome and higher risk of irAEs in IPI-treated MM patients.

Cross-contamination in canine and feline dietetic limited-antigen wet diets.

BACKGROUND: Adverse food reactions (AFRs) are defined as abnormal responses to an ingested food or food additive. Diagnosis and treatment of AFRs consist of the complete elimination of these ingredients in the dietary trial. Previous studies have demonstrated the presence of undeclared ingredients in commercial limited-antigen dry food diets that can compromise the results and efficacy of dietary elimination trails. The aim of this study was to assess a selection of commercial canine and feline dietetic limited-antigen wet foods for the potential cross-contamination of animal proteins from origins not mentioned on the label. RESULTS: Eleven canine and feline dietetic limited-antigen wet foods (9 novel animal protein foods, 1 vegetarian and 1 hydrolyzed) were analyzed by polymerase chain reaction (PCR) to detect the presence DNA of animal and vegetal origins. PCR analysis confirmed the contamination of 6 of the 11 (54.5%) limited-antigen wet diets with undeclared animal protein. One of these 6 diets was solely composed of animal protein sources completely unrelated to those declared on the label. None of the foods containing horse meat or fish were contaminated, and neither were the vegetarian or the hydrolyzed food products. Moreover, the results show that had zoological class primers only been used to check for cross-class contaminations, as are generally used in the pet food industry for in-house checks, the apparent contamination rate would have been significantly underestimated: less than 20% (3/11), instead of the actual rate of 54.7% using species-specific primers. CONCLUSION: This study reveals a high rate of cross-contamination in dietetic limited-antigen wet canine and feline foods, as previously described for dietetic dry limited-antigen foods (reported to be more than 80%). These results add new fuel to the discussion about the potential causes underlying the failure of elimination diets, since animal protein contaminants may actually be present in the commercial dietetic limited-antigen diets. AFRs may therefore occur as a result of inadequate practices in the pet food industry.

Exosome-mediated transfer of miR-222 is sufficient to increase tumor malignancy in melanoma.

BACKGROUND: Growing evidence is showing that metastatic cell populations are able to transfer their characteristics to less malignant cells. Exosomes (EXOs) are membrane vesicles of endocytic origin able to convey their cargo of mRNAs, microRNAs (miRs), proteins and lipids from donors to proximal as well as distant acceptor cells. Our previous results indicated that miR-221&222 are key factors for melanoma development and dissemination. The aim of this study was to verify whether the tumorigenic properties associated with miR-222 overexpression can be also propagated by miR-222-containing EXOs. METHODS: EXOs were isolated by UltraCentrifugation or Exoquick-TC(®) methods. Preparations of melanoma-derived vesicles were characterized by using the Nanosight™ technology and the expression of exosome markers analyzed by western blot. The expression levels of endogenous and exosomal miRNAs were examined by real time PCR. Confocal microscopy was used to evaluate transfer and uptake of microvesicles from donor to recipient cells. The functional significance of exosomal miR-222 was estimated by analyzing the vessel-like process formation, as well as cell cycle rates, invasive and chemotactic capabilities. RESULTS: Besides microvesicle marker characterization, we evidenced that miR-222 exosomal expression mostly reflected its abundance in the cells of origin, correctly paralleled by repression of its target genes, such as p27Kip1, and induction of the PI3K/AKT pathway, thus confirming its functional implication in cancer. The possible differential significance of PI3K/AKT blockade was assessed by using the BKM120 inhibitor in miR-222-transduced cell lines. In addition, in vitro cultures showed that vesicles released by miR-222-overexpressing cells were able to transfer miR-222-dependent malignancy when taken-up by recipient primary melanomas. Results were confirmed by antagomiR-221&222 treatments and by functional observations after internalization of EXOs devoid of these miRs. CONCLUSION: All together these data, besides generally confirming the role of miR-222 in melanoma tumorigenesis, supported its responsibility in the exosome-associated melanoma properties, thus further indicating this miR as potential diagnostic and prognostic biomarker and its abrogation as a future therapeutic option.

Ultrasonographic features of adrenal gland lesions in dogs can aid in diagnosis.

BACKGROUND: Ultrasonography to visualize adrenal gland lesions and evaluate incidentally discovered adrenal masses in dogs has become more reliable with advances in imaging techniques. However, correlations between sonographic and histopathological changes have been elusive. The goal of our study was to investigate which ultrasound features of adrenal gland abnormalities could aid in discriminating between benign and malignant lesions. To this end, we compared diagnosis based on ultrasound appearance and histological findings and evaluated ultrasound criteria for predicting malignancy. RESULTS: Clinical records of 119 dogs that had undergone ultrasound adrenal gland and histological examination were reviewed. Of these, 50 dogs had normal adrenal glands whereas 69 showed pathological ones. Lesions based on histology were classified as cortical adrenal hyperplasia (n = 67), adenocarcinoma (n = 17), pheochromocytoma (n = 10), metastases (n = 7), adrenal adenoma (n = 4), and adrenalitis (n = 4). Ultrasonographic examination showed high specificity (100%) but low sensitivity (63.7%) for identifying the adrenal lesions, which improved with increasing lesion size. Analysis of ultrasonographic predictive parameters showed a significant association between lesion size and malignant tumors. All adrenal gland lesions >20 mm in diameter were histologically confirmed as malignant neoplasms (pheochromocytoma and adenocarcinoma). Vascular invasion was a specific but not sensitive predictor of malignancy. As nodular shape was associated with benign lesions and irregular enlargement with malignant ones, this parameter could be used as diagnostic tool. Bilaterality of adrenal lesions was a useful ultrasonographic criterion for predicting benign lesions, as cortical hyperplasia. CONCLUSIONS: Abnormal appearance of structural features on ultrasound images (e.g., adrenal gland lesion size, shape, laterality, and echotexture) may aid in diagnosis, but these features alone were not pathognomic. Lesion size was the most direct ultrasound predictive criterion. Large and irregular masses seemed to be better predictors of malignant neoplasia and lesions <20 mm in diameter and nodular in shape were often identified as cortical hyperplastic nodules or adenomas.

Comprehensive analysis of PTEN status in Sezary syndrome.

Sézary syndrome (SS) is an incurable leukemic variant of cutaneous T-cell lymphoma characterized by recurrent chromosomal alterations, among which, chromosome 10q deletion is very frequent. In this study, we investigated the PTEN status, on locus 10q23, in 44 SS patients; our findings show that PTEN is deleted in 36% of SS cases, whereas PTEN downregulation is observed in almost all of the samples evaluated by quantitative reverse-transcriptase polymerase chain reaction and Western blotting analysis. Neither DNA sequence mutation nor promoter hypermethylation were found at the PTEN locus, but we demonstrate that PTEN level can be also reduced by a group of miRs previously found upregulated and of prognostic relevance in SS; particularly, miR-21, miR-106b, and miR-486 were able to control PTEN abundance either in vitro or in vivo. Finally, because reduced PTEN activates the PI3/AKT-mediated pathway of cell growth and survival, we demonstrate that PTEN deficiency is associated with activated AKT in skin resident but not circulating SS cells, suggesting that the cutaneous milieu may strongly contribute to the SS cell growth. To our knowledge, this is the first study fully exploring the PTEN status in a large cohort of SS patients, unveiling potential elements of clinical utility in this malignancy.

The Alpine ibex (Capra ibex) gut microbiome, seasonal dynamics, and potential application in lignocellulose bioconversion.

Aiming to shed light on the biology of wild ruminants, we investigated the gut microbiome seasonal dynamics of the Alpine ibex (Capra ibex) from the Central Italian Alps. Feces were collected in spring, summer, and autumn during non-invasive sampling campaigns. Samples were analyzed by 16S rRNA amplicon sequencing, shotgun metagenomics, as well as targeted and untargeted metabolomics. Our findings revealed season-specific compositional and functional profiles of the ibex gut microbiome that may allow the host to adapt to seasonal changes in available forage, by fine-tuning the holobiont catabolic layout to fully exploit the available food. Besides confirming the importance of the host-associated microbiome in providing the phenotypic plasticity needed to buffer dietary changes, we obtained species-level genome bins and identified minimal gut microbiome community modules of 11-14 interacting strains as a possible microbiome-based solution for the bioconversion of lignocellulose to high-value compounds, such as volatile fatty acids.

Birth weight, puppies' growth and health with limited-ingredient novel protein diet vs standard diet in late pregnancy.

Great attention has been given in the last years to the protein source of dog food, and commercial limited-ingredient diets with a single protein are available also for late pregnancy. This work compared the effect of a fish-based limited ingredient diet (LID), and of a standard mixed-protein diet (Mixed), fed to the bitches from the last three weeks of pregnancy and to the puppies at weaning, on birth weight, growth and health of the puppies. From a breeder's records, the weight of 22 Lagotto Romagnolo (LR) and 10 Appenzeller Cattle Dog (ACD) bitches on the day of mating, and of their 199 puppies, were extracted. The effect of diet on puppies' weight on day 0, 6, 30 and 60 was analyzed, considering litter size and sex. The analyses were repeated on puppies' weights normalized on the relative dam's non-pregnant bodyweight. Birth weight was available for 146 puppies, 82 LR and 64 ACD. Median birth weight of LR puppies was 287.5 g (170-400 g); sex ratio was 1.11 (males/females, N = 80). Median birth weight of ACD puppies was 390 g (240-525 g); sex ratio 1.15 (males/females, N = 58). Diet did not significantly affect birth weight in both breeds; however, it showed a significant effect on normalized birth weights (LR, P = 0.016; ACD, P = 0.034), with higher values for LID. At day 30, ACD puppies showed significantly higher weights with the Mixed diet (P = 0.002), and, at day 60, diet significantly affected the normalized weight in both breeds (LR, P = 0.019; ACD, P = 0.001), with higher values for the Mixed type. LID may help the dam to invest the energy in the growth of her litter, however, the same diet resulted in lower puppies' weights around weaning, compared to the Mixed diet. Although our results should be confirmed with larger numbers of animals and more breeds, they set some points worth to be further investigated. The choice of a limited-ingredient single-protein diet can affect litter weight and weight at weaning. Whether, administered to dams and puppies, it can prevent later pathologies, like chronic gastrointestinal diseases or food allergies, is a field of research deserving full attention.

Transcriptomic and physiological approaches to decipher cold stress mitigation exerted by brown-seaweed extract application in tomato.

Chilling temperatures represent a challenge for crop species originating from warm geographical areas. In this situation, biostimulants serve as an eco-friendly resource to mitigate cold stress in crops. Tomato (Solanum lycopersicum L.) is an economically important vegetable crop, but quite sensitive to cold stress, which it encounters in both open field and greenhouse settings. In this study, the biostimulant effect of a brown-seaweed extract (BSE) has been evaluated in tomato exposed to low temperature. To assess the product effects, physiological and molecular characterizations were conducted. Under cold stress conditions, stomatal conductance, net photosynthesis, and yield were significantly (p ≤ 0.05) higher in BSE-treated plants compared to the untreated ones. A global transcriptomic survey after BSE application revealed the impact of the BSE treatment on genes leading to key responses to cold stress. This was highlighted by the significantly enriched GO categories relative to proline (GO:0006560), flavonoids (GO:0009812, GO:0009813), and chlorophyll (GO:0015994). Molecular data were integrated by biochemical analysis showing that the BSE treatment causes greater proline, polyphenols, flavonoids, tannins, and carotenoids contents.The study highlighted the role of antioxidant molecules to enhance tomato tolerance to low temperature mediated by BSE-based biostimulant.

A dual-omics approach for profiling plant responses to biostimulant applications under controlled and field conditions.

A comprehensive approach using phenomics and global transcriptomics for dissecting plant response to biostimulants is illustrated with tomato (Solanum lycopersicum cv. Micro-Tom and Rio Grande) plants cultivated in the laboratory, greenhouse, and open field conditions. Biostimulant treatment based on an Ascophyllum nodosum extract (ANE) was applied as a foliar spray with two doses (1 or 2 l ha-1) at three different phenological stages (BBCH51, BBCH61, and BBCH65) during the flowering phase. Both ANE doses resulted in greater net photosynthesis rate, stomatal conductance, and fruit yield across all culture conditions. A global transcriptomic analysis of leaves from plants grown in the climate chamber, revealed a greater number of differentially expressed genes (DEGs) with the low ANE dose compared to the greater one. The second and third applications induced broader transcriptome changes compared to the first one, indicating a cumulative treatment effect. The functional enrichment analysis of DEGs highlighted pathways related to stimulus-response and photosynthesis, consistent with the morpho-physiological observations. This study is the first comprehensive dual-omics approach for profiling plant responses to biostimulants across three different culture conditions.

Fecal microbiota and inflammatory and antioxidant status of obese and lean dogs, and the effect of caloric restriction.

Introduction: Obesity is the most common nutritional disease in dogs, and is generally managed by caloric restriction. Gut microbiota alteration could represent a predisposing factor for obesity development, which has been associated with a low-grade inflammatory condition and an impaired antioxidant status. Besides, weight loss has been shown to influence the gut microbiota composition and reduce the inflammatory response and oxidative stress. Method: However, these insights in canine obesity have not been fully elucidated. The aim of this study was to assess the differences in serum and inflammatory parameters, antioxidant status, fecal microbiota and bacterial metabolites in 16 obese and 15 lean client-owned dogs and how these parameters in obese may be influenced by caloric restriction. First, for 30 days, all dogs received a high-protein, high-fiber diet in amounts to maintain their body weight; later, obese dogs were fed for 180 days the same diet in restricted amounts to promote weight loss. Results: Before the introduction of the experimental diet (T0), small differences in fecal microbial populations were detected between obese and lean dogs, but bacterial diversity and main bacterial metabolites did not differ. The fecal Dysbiosis Index (DI) was within the reference range (< 0) in most of dogs of both groups. Compared to lean dogs, obese dogs showed higher serum concentrations of acute-phase proteins, total thyroxine (TT4), and antioxidant capacity. Compared to T0, dietary treatment affected the fecal microbiota of obese dogs, decreasing the abundance of Firmicutes and increasing Bacteroides spp. However, these changes did not significantly affect the DI. The caloric restriction failed to exert significative changes on a large scale on bacterial populations. Consequently, the DI, bacterial diversity indices and metabolites were unaffected in obese dogs. Caloric restriction was not associated with a reduction of inflammatory markers or an improvement of the antioxidant status, while an increase of TT4 has been observed. Discussion: In summary, the present results underline that canine obesity is associated with chronic inflammation. This study highlights that changes on fecal microbiota of obese dogs induced by the characteristics of the diet should be differentiated from those that are the consequence of the reduced energy intake.

In vivo and in vitro Digestibility of an Extruded Complete Dog Food Containing Black Soldier Fly (Hermetia illucens) Larvae Meal as Protein Source.

Growing attention is being directed toward insects as a novel and sustainable source of protein for pet food. The aim of the study was to evaluate nutrient digestibility of a diet containing black soldier fly larvae as its main protein source. Moreover, the purpose of the study was to compare the traditional in vivo total collection method with the in vivo marker method and in vitro digestibility method. Two isonitrogenous and isoenergetic dry diets containing either venison meal (CTRL diet) or black soldier fly larvae meal (BSF diet) as their primary sources of proteins were fed to six adult dogs, according to a Latin square design. The digestibility of nutrients was determined using both in vivo ("total collection" and "internal marker" approaches) and in vitro methods. The two diets showed similar nutrient digestibility values for dry matter, organic matter, ether extract, ash, and phosphorus. However, a statistical trend (p = 0.066) was observed indicating greater protein digestibility in the BSF diet compared with the CTRL diet. Calcium digestibility was higher in the BSF diet compared with the CTRL diet (p = 0.018). On the contrary, fiber digestibility was lower in the insect-based diet compared with the venison diet (p < 0.001). There was no difference between total collection and internal marker methods in the assessment of in vivo digestibility for any of the nutrients considered. The in vitro digestibility values for dry matter, organic matter, and crude protein, as well as the estimated in vivo digestibility of organic matter and crude protein by the means of the predictive equation, were aligned with the in vivo results, although in vitro estimations were consistently higher compared with those obtained by in vivo analysis. Digestibility analysis of a dog food containing insect meal as the sole source of protein (36.5% inclusion) showed promising results in terms of it presenting similar values as a meat-based diet, indicating its suitability as a sustainable protein source for pet food. Moreover, the study showed that both the in vivo marker method and the in vitro method could be possible alternatives to the traditional total collection method in digestibility trials.

Effects of "fresh mechanically deboned meat" inclusion on nutritional value, palatability, shelf-life microbiological risk and digestibility in dry dog food.

Fresh mechanically deboned meat (MDM) is usually claimed as high-quality ingredient on dry pet food recipes and this aspect may positively influence consumer choice. It is important to determine the scientifically sustainability of this claim and to assess the microbiological safety of MDM inclusion in dry pet food. Objectives were: 1) to evaluate the effect of inclusion of MDM in dry dog food on fatty acid profile and in vivo and in vitro digestibility, proposing a new system (DaisyII Incubator) to measure the in vitro digestibility for dogs; 2) to compare palatability of dry dog food containing MDM with dry dog food in which meat by-products (MBP) are the only animal protein sources; 3) to determine, whether or not, the inclusion of that ingredient changes the microbiology and the storage quality. Results indicated that MDM product was characterized by significant higher nutritional value in terms of fatty acids profile, in vitro digestibility (HV-IVD method) and lower palatability than the MBP product. Microbiological risk assessment showed no microbiological hazards for either product. After 6-months storage, the total mesophilic bacterial count ranged between 1.77 and 2.09 log CFU/g feed, while polyamine values were higher in the MDM (0.37 g/kg) than in the MBP (0.27 g/kg). The DaisyII Incubator was found to be a valid instrument for studying in vitro digestibility also for dogs, providing data simply, quickly, with less variability and costs than in vivo trials. In conclusion, MDM inclusion in dry dog food is microbiologically safe and it can improve its nutritional quality, at the expense of a reduced palatability. The higher polyamine levels fount in MDM-enriched petfood after 6-months storage, however, may represent a possible hazard, and further studies are still warranted.

Reduced Interleukin-17-Expressing Cells in Cutaneous Melanoma.

Characterization of tumor associated lymphocytes (TILs) in tumor lesions is important to obtain a clear definition of their prognostic value and address novel therapeutic opportunities. In this work, we examined the presence of T helper (Th)17 lymphocytes in cutaneous melanoma. We performed an immunohistochemical analysis of a small cohort of primary melanomas, retrospectively selected. Thereafter, we isolated TILs from seven freshly surgically removed melanomas and from three basal cell carcinomas (BCC), as a comparison with a non-melanoma skin cancer known to retain a high amount of Th17 cells. In both studies, we found that, differently from BCC, melanoma samples showed a lower percentage of Th17 lymphocytes. Additionally, TIL clones could not be induced to differentiate towards the Th17 phenotype in vitro. The presence or absence of Th17 cells did not correlate with any patient characteristics. We only observed a lower amount of Th17 cells in samples from woman donors. We found a tendency towards an association between expression by melanoma cells of placenta growth factor, angiogenic factors able to induce Th17 differentiation, and presence of Th17 lymphocytes. Taken together, our data indicate the necessity of a deeper analysis of Th17 lymphocytes in cutaneous melanoma before correlating them with prognosis or proposing Th17-cell based therapeutic approaches.

Altered expression of selected microRNAs in melanoma: antiproliferative and proapoptotic activity of miRNA-155.

Altered expression of microRNAs (miRNAs) has been detected in cancer, suggesting that these small non-coding RNAs can act as oncogenes or tumor suppressor genes. In the present study, we investigated the expression of miRNA-17-5p, miRNA-18a, miRNA-20a, miRNA-92a, miRNA-146a, miRNA-146b and miRNA-155 by real-time quantitative RT-PCR in a panel of melanocyte cultures and melanoma cell lines and explored the possible role of miRNA-155 in melanoma cell proliferation and survival. The analyzed miRNAs were selected on the basis of previous studies strongly supporting their involvement in cancer development and/or progression. We found that miRNA-17-5p, miRNA-18a, miRNA-20a, and miRNA-92a were overexpressed, whereas miRNA-146a, miRNA-146b and miRNA-155 were down-regulated in the majority of melanoma cell lines with respect to melanocytes. Ectopic expression of miRNA-155 significantly inhibited proliferation in 12 of 13 melanoma cell lines with reduced levels of this miRNA and induced apoptosis in 4 out of 4 cell lines analyzed. In conclusion, our data further support the finding of altered miRNA expression in melanoma cells and establish for the first time that miRNA-155 is a negative regulator of melanoma cell proliferation and survival.

Role of key-regulator genes in melanoma susceptibility and pathogenesis among patients from South Italy.

BACKGROUND: Several genetic alterations have been demonstrated to contribute to the development and progression of melanoma. In this study, we further investigated the impact of key-regulator genes in susceptibility and pathogenesis of such a disease. METHODS: A large series (N = 846) of sporadic and familial cases originating from South Italy was screened for germline mutations in p16(CDKN2A), BRCA2, and MC1R genes by DHPLC analysis and automated DNA sequencing. Paired primary melanomas and lymph node metastases from same patients (N = 35) as well as melanoma cell lines (N = 18) were analyzed for somatic mutations in NRAS, BRAF, and p16(CDKN2A) genes. RESULTS: For melanoma susceptibility, investigations at germline level indicated that p16(CDKN2A) was exclusively mutated in 16/545 (2.9%) non-Sardinian patients, whereas BRCA2 germline mutations were observed in 4/91 (4.4%) patients from North Sardinia only. Two MC1R germline variants, Arg151Cys and Asp294His, were significantly associated with melanoma in Sardinia. Regarding genetic events involved in melanoma pathogenesis at somatic level, mutually-exclusive mutations of NRAS and BRAF genes were observed at quite same rate (about two thirds) in cultured and in vivo melanomas (either primary or metastatic lesions). Conversely, p16(CDKN2A) gene alterations were observed at increased rates moving from primary to metastatic melanomas and melanoma cell lines. Activation of the ERK gene product was demonstrated to be consistently induced by a combination of molecular alterations (NRAS/BRAF mutations and p16(CDKN2A) silencing). CONCLUSION: Our findings further clarified that: a) mutation prevalence in melanoma susceptibility genes may vary within each specific geographical area; b) multiple molecular events are accumulating during melanomagenesis.

Concomitant activation of Wnt pathway and loss of mismatch repair function in human melanoma.

Constitutive activation of the Wnt pathway plays a key role in the development of colorectal cancer and has also been implicated in the pathogenesis of other malignancies. Deregulation of Wnt signaling mainly occurs through genetic alterations of APC, the beta-catenin gene (CTNNB1), AXIN1 and AXIN2, leading to stabilization of beta-catenin. Physiologically, AXIN2 is transcriptionally induced on Wnt signaling activation and acts as a negative feedback regulator of the pathway. In colorectal cancer, mutations in CTNNB1 and AXIN2 occur preferentially in tumors with inactivation of the mismatch repair (MMR) genes MSH2, MLH1, or PMS2. In this study, the expression of beta-catenin and AXIN2, and the mutational status of CTNNB1, APC, and AXIN2 were evaluated in two MMR-deficient (PR-Mel and MR-Mel) and seven MMR-proficient human melanoma cell lines. Only PR-Mel and MR-Mel cells showed nuclear accumulation of beta-catenin and expression of the AXIN2 gene, and hence, constitutive activation of Wnt signaling. Mutational analysis identified a somatic heterozygous missense mutation in CTNNB1 exon three and a germline heterozygous deletion within AXIN2 exon seven in PR-Mel cells, and a somatic biallelic deletion within APC in MR-Mel cells. Deregulation of Wnt signaling and a defective MMR system were also present in the original tumor of PR and MR patients. Thus, we describe additional melanomas with mutations in CTNNB1 and APC, identify for the first time a germline AXIN2 mutation in a melanoma patient and suggest that inactivation of the MMR system and deregulation of the Wnt/beta-catenin signaling pathway cooperate to promote melanoma development and/or progression.

Corrigendum: Association of CTLA-4 Gene Variants with Response to Therapy and Long-term Survival in Metastatic Melanoma Patients Treated with Ipilimumab: An Italian Melanoma Intergroup Study.

[This corrects the article on p. 386 in vol. 8, PMID: 28446908.].

Combined effect of temozolomide and hyperthermia on human melanoma cell growth and O6-methylguanine-DNA methyltransferase activity.

Hyperthermic isolated limb perfusion (HILP) with L-phenylalanine mustard (L-PAM) represents an effective treatment for locally advanced melanoma of the limbs. However, regional chemotherapy of melanoma still needs to be improved. Temozolomide (TMZ) is a methylating agent that spontaneously decomposes into the active metabolite of dacarbazine, the most effective agent for the systemic treatment of melanoma. Tumor cells with high levels of O6-methylguanine-DNA methyltransferase (MGMT) and/or with a defective DNA mismatch repair (MMR) are resistant to TMZ. Inhibition of MGMT activity increases TMZ sensitivity of MMR-proficient, but not of MMR-deficient cells, while inhibition of base excision repair (BER) potentiates TMZ cytotoxicity in both cell types. Recent studies, performed in an animal model, have shown that TMZ is more effective than L-PAM when applied regionally and that hyperthermia can increase the antitumor activity of TMZ. In this study, three thermoresistant human melanoma cell lines, endowed with different MGMT activity and functional status of the MMR system, were treated with TMZ at 37 degrees C or 41.5 degrees C for 90 min, and then analyzed for cell growth and MGMT activity. Hyperthermia significantly enhanced TMZ cytotoxicity in MMR-proficient cells, either endowed or not with MGMT activity, and in MMR-deficient cells. Endogenous MGMT activity was not affected by hyperthermia that, however, enhanced the enzyme depletion induced by TMZ treatment. Moreover, MGMT recovery after drug removal was delayed in cells that had been treated at 41.5 degrees C. Taken together, these findings confirm the therapeutic potential of a combined treatment of hyperthermia and TMZ. They also suggest that inhibition of BER and/or increased DNA methylation may be involved in the thermal enhancement of TMZ cytotoxicity. Additional studies are necessary to better clarify the mechanisms underlying hyperthermia-induced potentiation of TMZ activity. However, the present investigation provides further support to the development of clinical trials of HILP with TMZ.