The concentrations of dioxins /g), dioxin-like PCBs (0.95 ± 0.48 pg TEQ/g), mercury (56.3 ± 12.9 µg/kg) and arsenic (2.56 ± 0.87 mg/kg) were three times higher in wild compared to farmed salmon, but all well below EU-uniform maximum levels for contaminants in food. The six ICES  PCBs concentrations (5.09 ± 0.83 ng/g) in wild salmon were higher than in the farmed fish (3.34 ± 0.46 ng/g). The protein content was slightly higher in wild salmon (16%) compared to the farmed fish (15%), and the amount of essential amino acids were similar. The fat content of farmed salmon (18%) was three times that of the wild fish, and the proportion of marine long-chain omega-3 fatty acids was a substantially lower (8.9 vs. 24.1%). The omega-6 to omega-3 fatty acid ratio was higher in farmed than wild salmon (0.7 vs. 0.05). Both farmed and wild Atlantic salmon are still valuable sources of eicosapentaenoic acid and docosahexaenoic acid. One 150 g portion per week will contribute to more (2.1 g and 1.8 g) than the recommended weekly intake for adults.In this paper, we present updated data on proximate composition, amino acid, and fatty acid composition, as well as concentrations of dioxins, polychlorinated biphenyls (PCBs), and selected heavy metals, in fillets from farmed ( The United Nations declaration “Transforming our World: The 2030 Agenda for Sustainable Development”, with the seventeen sustainable Development Goals (SDG), emphasizes the need to achieve food safety, food security and enhanced nutrition for everybody in a sustainable manner. As land-based resources are scarce, and food production is one of the major greenhouse gas (GHG) emitters, one strategy would be shifting human diets from high carbon and GHG land-based sources (red meat) of protein to low-carbon-based sources ,2. The cFish and seafood consumption has traditionally been recognized to lower the risk of cardiometabolic diseases ,4. This Seafood is important in a balanced diet and contributes also with a wide range of other vital nutrients, such as iodine, selenium, vitamins, and high-quality proteins . The nutIncreased attention has been drawn to the presence of persistent organic pollutants (POPs), such as dioxins and polychlorinated biphenyls (PCBs), both dioxin-like (dl) and non-dioxin-like (ndl) PCBs ,12, alonSalmo salar L.), the main fatty fish species farmed in Europe, has traditionally been fed on feeds with high inclusion of fish oil and fishmeal from small, pelagic marine fatty fish. Because the global aquaculture production has increased and wild fish stocks are under pressure, the marine resources have become too expensive. Therefore, it has become common to replace most of the fish oils and fishmeal in the feed with terrestrial feed substitutes, and today fish oil is included at minimum levels to cover the salmon’s omega-3 PUFA requirements [Atlantic salmon  were obtained from Lerøy Aurora, one of the largest farmed Atlantic salmon producers in Norway, in July 2017 . Wild salmon caught with pond nets at Lopphavet off the coast of Finnmark, Norway, in June 2017 and June 2018 were purchased from a local fishing company. Fifteen scale samples from each side of the fish, behind the dorsal fin and above the lateral line, were collected and sent to Norwegian Institute for Nature Research (NINA) for classification as originating from wild or farmed salmon using the methods described by Lund et al. [Farmed Atlantic salmon , using heptadecanoic acid  as an internal standard for fatty acids, and determined gravimetrically. Protein content was determined as the sum of individual amino acid residues (the molecular weight of each amino acid after subtraction of the molecular weight of H2O), using norleucine as internal standard, as described previously [Total lipids in the minced fillets were extracted  with diceviously . Water aeviously .v:v)) before methylation [Fatty acid composition was determined by gas chromatography (GC-FID) of fatty acid methyl esters (FAMEs) as previously described  after dihylation .2O and 0.5 mL 20 mM norleucine  and hydrolyzed as previously described [The amino acid composition was analyzed by dissolving approximately 200 mg of fish samples in 0.7 mL distilled Hescribed ,30. Follescribed .Samples were analyzed for polychlorinated dibenzo-p-dioxins (PCDD), including 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), 1,2,3,7,8-pentachlorodibenzo-p-dioxin (PeCDD), 1,2,3,4,7,8-hexachlorodibenzo-p-dioxin (HxCDD), 1,2,3,6,7,8-HxCDD, 1,2,3,7,8,9-HxCDD, 1,2,3,4,6,7,8-heptachlorodibenzo-p-dioxin (HpCDD) and octachlorodibenzo-p-dioxin (OCDD) and polychlorinated dibenzofurans (PCDF), including 2,3,7,8- tetrachlorodibenzofuran (TCDF), 1,2,3,7,8-pentachlorodibenzofuran (PeCDF), 2,3,4,7,8-PeCDF, 1,2,3,4,7,8-hexachlorodibenzofuran (HxCDF), 1,2,3,6,7,8-HxCDF, 1,2,3,7,8,9-HxCDF, 2,3,4,6,7,8-HxCDF, 1,2,3,4,6,7,8-heptachlorodibenzofuran (HpCDF), 1,2,3,4,7,8,9-HpCDF, and octachlorodibenzofuran (OCDF). The non-ortho polychlorinated biphenyls (PCB) analyzed were PCB 77, PCB 81, PCB 126, and PCB 169, and the mono-ortho PCBs analyzed were PCB 105, PCB 114, PCB 118, PCB 123, PCB 156, PCB 157, PCB 167, and PCB 189. Further, PCB 28, PCB 52, PCB 101, PCB 138, PCB 153, and PCB 180, which are commonly referred to as ICE-6 PCB, were also analyzed. These congeners have been assigned a WHO-TEF2005 and are included in the current EU maximum limit  and US Ep < 0.05 were considered significantly different.The results are presented on wet weight as arithmetic mean of 10–23 parallels ± standard deviation (SD). Statistical analyses were performed using the Statistical package for the social sciences v. 25 . Shapiro-Wilk’s test for normality and Levene’s test for homogeneity of variance were performed, and one-way analysis of variance (ANOVA) was performed on normally distributed. For non-normally distributed data, the non-parametric Mann–Whitney U test was applied. For evaluation of statistics, Tukey and Dunnett’s T3 post-hoc tests were run for equal and unequal variances, respectively. Variables with The gutted weights of wild and farmed Atlantic salmon were similar: 4.3 kg, whereas escapees were significantly smaller: 3.6 kg . Wild AtThe proximate composition of the fillet of wild, farmed, and escaped salmon, is shown in The fatty acid composition  and the total amount of fatty acids per 100 g of fillets of wild, farmed, and escaped salmon, are presented in The amino acid composition of wild, farmed and escaped Atlantic salmon are presented in The highest dioxins and furan levels and dl-PCB levels were found in wild salmon, with levels of 1.48 pg TEQ/g fillet compared to 0.57 and 0.9 pg TEQ/g fillet of farmed and escaped salmon, respectively . The levThe elements mercury, arsenic, lead, and cadmium were analyzed in this study. No specification analysis of mercury was conducted, and it was, therefore, appraised as if all mercury present is methylmercury. The mercury and arsenic concentrations were significantly different between all groups. They were highest in wild Atlantic salmon , lowest in farmed salmon , and intermediate in escaped farmed salmon  . The conS.salar) compared to wild Atlantic salmon. As a considerable proportion (43%) of the wild fish indeed was shown to be farmed escapees caught at sea, they were included as a separate group in the study. Further, we wanted to evaluate the possible health impacts of the high inclusion of vegetable oils in the feed.The study was initiated to analyze and update data on proximate composition, precisely, the amino acid composition, fatty acid composition and content of dioxins, dl-PCBs, ICES-6 PCB, and selected metals in farmed Atlantic salmon  salmon harvested at sea. This is reflected by larger variation in the latter group(s).Observed differences in lipid content, fatty acid composition, and contaminants between wild, farmed, and escaped Atlantic salmon are caused by the different diets and feeding strategies between the three salmon groups.As expected, the most striking difference between farmed and wild salmon was the fat content three times higher in farmed salmon than its wild counterpart. This reflects intensive production and typical commercial conditions, and the abundant supply of energy dense feed and salmon confinement in net pens adds to fat accumulation in salmon flesh because of the reduced activity levels. The escapees had a fat content between farmed and wild salmon, which are caused by the sudden need for the escapees to adapt to a wild diet (in addition to higher activity levels in the wild) .http://tromsoundersokelsen.no), with data from 4528 individuals and 13 years follow-up, showed that individuals consuming fatty fish at least once per week had increased waist circumference compared with those eating fatty fish less than once a week [Compared to our previously reported results on the lipid content of flesh from Atlantic salmon, it is evident that the lipid content of farmed salmon has increased from 12.3% (2010) to 17.9% (2017/2018). In contrast, the fat content in wild Atlantic salmon fillets is relatively stable (6.3% in 2010  and 6.0%The high content of LA (18:2n-6) and alpha-linolenic acid (ALA) (18:3n-3) in the farmed salmon illustrates the substantial inclusion of vegetable oils in the feed. The high amount of LA in some wild-caught salmon indicated that these individuals were escaped farmed salmon. The scale analysis confirmed this notion. The content of LA in the farmed salmon was almost four times higher compared to our results from 2010  and 20 tThe nutritional quality of proteins depends on the presence and concentration of essential amino acids . Nine amIn addition to increasing the dietary intake of long-chain omega-3 PUFAs, lowering the intake of omega-6 PUFAs has been considered beneficial to human health. The omega-6 to omega-3 ratio of the present Western diet has been calculated to be as high as 15–17/1 . Even thDioxins (PCDD/Fs) and dl-PCBs are known to cause adverse effects on the immune, endocrine and nervous systems, and impairing reproductive function and may cause cancer . In 2018This study showed that wild salmon had the highest average concentrations of dioxins, furans, and dl-PCB, whereas escaped farmed salmon had the highest content of ICES-6 PCB. The uptake of dioxins and PCBs by fish occurs both via gills and diet. Both the initial diet and farming location of the escapees are unknown. The three groups’ diets could be very different, and different prey/diet components may differ substantially for any group of compounds. All species had a content of dioxins and dl-PCB and ICES-6 PCB well below the EU maximum levels of 6.5 and 75 µg TEQ/kg fish filet, respectively. The capital contributors to the sum of dioxins and dl-PCB were the non-ortho congeners and PCB 126 that constituted 60% of dioxins and dl-PCBs in wild salmon and 45 and 27% of dioxins and dl-PCBs in farmed and escaped salmon, respectively. As for salmon in this study, PCB-126 is the dl-PCB contributing most to the current intake of PCDD/Fs and DL-PCBs. Concerning the critical effect of dioxins on semen quality, no association was observed when including dl-PCB-TEQ. This is also supported by in vitro experiments with human cells indicating that PCB-126 is less potent in humans than a TEF of 0.1 suggested by the WHO 2005 . Thus, tOur results confirm that mercury and arsenic levels are lower in farmed than in wild salmon, as previously presented by Lundeby et al. . The merAs mentioned, the UN 2030 Agenda emphasizes the need for food security and improved nutrition.It is anticipated that sustainable well-managed ocean resources will contribute through dietary shifts from land-based protein sources towards marine protein sources . While cOne portion of farmed Atlantic salmon still provides equal amounts of EPA and DHA compared to wild salmon. However, the same portion would provide a high amount of fat/energy and omega-6 fatty acids but a lower amount of contaminants. Thus, farmed Atlantic salmon is a positive contribution to our diet regarding intake of marine omega-3 and reduced intake of contaminants but may be perceived as a negative contribution when energy restriction is taken into account.


Iron (Fe) deficiency is a common problem in citrus production. As the second largest superfamily of transcription factors (TFs), the basic/helix-loop-helix (bHLH) proteins have been shown to participate in the regulation of Fe homeostasis and a series of other biological and developmental processes in plants. However, this family of members in citrus and their functions in citrus Fe deficiency are still largely unknown.Citrus grandis) genome that were classified into 18 subfamilies by phylogenetic comparison with Arabidopsis thaliana bHLH proteins. All of these CgbHLHs were randomly distributed on nine known (125 genes) and one unknown (3 genes) chromosomes, and 12 and 47 of them were identified to be tandem and segmental duplicated genes, respectively. Sequence analysis showed detailed characteristics of their intron-exon structures, bHLH domain and conserved motifs. Gene ontology (GO) analysis suggested that most of CgbHLHs were annotated to the nucleus, DNA-binding transcription factor activity, response to abiotic stimulus, reproduction, post-embryonic development, flower development and photosynthesis. In addition, 27 CgbHLH proteins were predicted to have direct or indirect protein-protein interactions. Based on GO annotation, RNA sequencing data in public database and qRT-PCR results, several of CgbHLHs were identified as the key candidates that respond to iron deficiency.In this study, we identified a total of 128 CgbHLHs from pummelo (CgbHLH genes in citrus.In total, 128 CgbHLH proteins were identified from pummelo, and their detailed sequence and structure characteristics and putative functions were analyzed. This study provides comprehensive information for further functional elucidation of  However, citrus production is also continuously influenced by many environmental factors, such as diseases, cold, drought, heat, and nutrient disorders, among which iron (Fe) deficiency is a common problem that can cause severe chlorosis of leaves, impaired tree vigor, and reduction of fruit yield and quality in citrus production , r (0.46%) , poplar  , the cl (0.46%) , and gra (0.46%) . With re (0.46%) . In citr (0.46%) , and it A. thaliana [In the CgbHLH family, 19 amino acid residues are highly conserved, with a consensus ratio higher than 50%. These residues are also highly conserved in bHLHs of thaliana , maize [thaliana  and grapthaliana , indicatthaliana . Based othaliana . A previPtrbHLH, an ortholog of AtbHLH33 (ICE2), that was isolated from trifoliate orange (Poncirus trifoliata) and proved to confer cold tolerance by regulating both POD- and CAT-mediated reactive oxygen species (ROS) scavenging [CsbHLH18 of sweet orange was also proved to modulate cold tolerance and ROS homeostasis [CubHLH1 of Satsuma mandarin (Citrus unshiu Marc.) was overexpressed in transgenic tomato fruit, resulting in modulation of carotenoid metabolism [AtbHLH29 (FIT), AtbHLH33 (ICE2), AtbHLH116 (ICE1), AtbHLH93 (NFL), AtbHLH61, and AtbHLH21 (AMS) have been shown to be involved in Fe deficiency, cold tolerance and flower development [AtbHLH genes such as AtbHLH70 (MYC70) and AtbHLH45 (MUTE) in group 2, AtbHLH10, AtbHLH89 and AtbHLH91 in group 3, AtbHLH38, AtbHLH39, AtbHLH100 and AtbHLH101 in group 4, etc. have also been functionally characterized [CgbHLH genes could be predicted. Furthermore, GO annotation and protein-protein interaction analysis of the CgbHLHs could help us to understand their possible functions.To date, only very limited citrus bHLH genes have been functionally characterized, including avenging , 45. Csbeostasis . Moreovetabolism . Generalelopment , 53–55, cterized –58. BaseCgbHLH genes were significantly differentially expressed at two or more time points under Fe deficiency. Among them, CgbHLH29.4, CgbHLH39, CgbHLH104, CgbHLH105.1 and CgbHLH105.2 are orthologs of known Fe deficiency-responsive AtbHLHs [CgbHLH6, CgbHLH13, CgbHLH14.1 and CgbHLH14.2 are orthologs of AtHLH6 (MYC2), AtHLH13 (JAM2) and AtHLH14, respectively, and these three AtHLHs of A. thaliana have been shown to participate in jasmonic acid (JA) signaling [CgbHLH genes may also be involved in the response to Fe deficiency. Interestingly, several of the significantly expressed CgbHLHs such as CgbHLH105.1, CgbHLH104, CgbHLH13 and CgbHLH130.2 also showed a high expression level in roots based on RNAseq data, which further indicates their possible functions in Fe deficiency. Moreover, we found that CgbHLH104 and CgbHLH105.1 belong to the same subfamily (Group 6) and have a similar gene structure, pI and exon number. Their orthologous bHLHs of A. thaliana, AtbHLH104 and AtbHLH105, as well as all the other members in group 6 (AtbHLH47 and AtbHLH121) have been functionally identified in Fe deficiency [CgbHLH104 and CgbHLH105.1 also showed significant up-regulation under Fe deficiency. Thus, we speculate that these two CgbHLHs most likely have similar functions with AtbHLH104 and AtbHLH105 under Fe deficiency. Apart from these known Fe deficiency-responsive bHLHs, there were also significantly differentially expressed CgbHLHs, but direct evidence could not be found regarding their orthologs that have a function in plant Fe deficiency, such as CgbHLH16, CgbHLH48, CgbHLH62, CgbHLH63, CgbHLH79, CgbHLH80, CgbHLH123, CgbHLH128 and CgbHLH130.2. New and essential bHLHs that regulate Fe homeostasis are expected to be identified from these candidates in future. In particular, CgbHLH16 was continuously up-regulated at all tested times of –Fe treatment, and it was also predicted to interact with an important Fe deficiency-responsive TF, PYE (bHLH47), based on protein-protein interaction prediction  genome and were classified into 18 subfamilies based on the phylogenetic relationship with AtbHLH proteins. The detailed analysis of Mw, pI, sequence alignment, phylogeny, gene duplication, chromosomal distribution, gene structure and protein motif suggested basic properties of each CgbHLH. GO annotation and protein-protein interaction analysis further revealed the potential functions of CgbHLHs. qRT-PCR showed that 34 of the tested CgbHLH genes were up- or down-regulated under Fe deficiency, and some of them such as CgbHLH6, CgbHLH14.2, CgbHLH16, CgbHLH48, CgbHLH63, CgbHLH79, CgbHLH80, CgbHLH104, CgbHLH105.1, CgbHLH123, CgbHLH128, CgbHLH93.1, CgbHLH62, CgbHLH130.2, CgbHLH3, CgbHLH29.4, CgbHLH14.1, CgbHLH69.1, CgbHLH153 and CgbHLH77 were considered the key candidates that respond to Fe deficiency. This study lays the foundation for further functional elucidation of CgbHLH genes in citrus.In summary, 128 CgbHLH proteins were identified from pummelo (Citrus grandis) were collected from the Citrus Research Institute, Southwest University, Chongqing, China. After surface disinfection with 2% sodium hypochlorite, seed coats were removed, and seeds were then germinated on wet filter papers that filled 96-cell plastic boxes with lids. After 2 weeks germination, uniform seedlings were transferred into modified Hoagland’s solution for hydroponics as described by Fu et al. (2017) [− 2 s− 1). Both –Fe and CK groups had three biological replicates, and each replicate contained 14 seedlings. After 0.5 d, 1.5 d, 2 d, 7 d, and 12 d of treatment, roots of –Fe and CK were sampled and then frozen in liquid nitrogen immediately.Seeds of pummelo  Genome Annotation Project (http://citrus.hzau.edu.cn/orange/download/index.php). Then, we used two methods to search for all bHLHs of pummelo. Firstly, the hidden Markov model (HMM) file of the HLH domain (PF00010) obtained from the Pfam database (http://pfam.xfam.org/) was used as the query to scan the pummelo genome with HMMER software (http://hmmer.org/). Secondly, the bHLH domain sequences of A. thaliana (AtbHLHs) were used as queries to blast against the pummelo genome. After removal of redundant sequences, all putative bHLHs obtained from the two methods were submitted to the NCBI  conserved domain database  to further verify the existence of the conserved bHLH domain. All the identified bHLH proteins of pummelo were named as CgbHLHs.The genome data of pummelo were downloaded from the Orange (http://web.expasy.org/compute_pi/). Conserved domain sequences of CgbHLHs were submitted to ClustalW to perform multiple sequence alignment, and the BioEdit was used to edit the aligned sequences and shade the conserved sites. To visualize the conserved motifs, the sequences were analyzed with WEBLOGO programs (http://weblogo.berkeley.edu). The neighbor-joining phylogenetic tree was constructed by using MEGA7 and 1000 replicates in bootstrap analysis. In addition, the full-length protein sequences of all CgbHLHs and known AtbHLHs were also aligned in Clustal Omega  to obtain their sequence identity (%) data, and the CgbHLH-AtbHLH pairs with highest percent identity were considered orthologs.Molecular weights (MWs) and isoelectric points (pIs) of CgbHLH proteins were analyzed with ExPASy (Gene structure information of each CgbHLH was obtained from the generic feature format (GFF) file downloaded from the pummelo genome database, and TBtools  was usedA. thaliana was chosen as the reference. To evaluate the expression levels of CgbHLH genes in pummelo, the published RNAseq raw data (SRR4050288/SRR4050289) of pummelo [https://www.ncbi.nlm.nih.gov/sra/). Then, the raw data were analyzed with the linux system of the fastqc and trimommatic programs to remove adaptors and low quality sequences. The kallisto program was used to map sequence data to the pummelo genome, and the featurecount program was used to calculate the values of the transcripts per million reads (TPM).GO annotation of all the CgbHLH proteins was performed with the Blast2GO program, and  pummelo  were dowhttp://string-db.org) website that contains known and predicted protein-protein interactions of different organisms [A. thaliana was chosen as the reference organism for blasting because pummulo has no protein-protein interaction database and is not included in STRING. After blasting, the matched homologs of A. thaliana with the highest scores (Bitscore) and higher than 40% identity were used to construct the network. To ensure reliability, only the predicted interactions with interaction scores > 0.7 (high confidence level was set in STRING) were shown in the network.The interaction network of CgbHLHs was predicted in STRING . All the primers of CgbHLH genes are listed in Additional file t-test was used to analyze the statistical differences between -Fe and corresponding CK samples, taking P < 0.05 as significantly different.Total RNA was extracted from the –Fe and CK roots of pummelo by using the RNAprep pure plant kit , and the RNA concentration and quality were determined with a Nanodrop 2000 spectrophotometer . Then, 1 μg of high-quality RNA was used for cDNA synthesis with HiScript II Q RT Super Mix  according to the manufacturer’s instructions. qRT-PCR was performed on the Bio-Rad CFX Connect RealTime system by using ChamQ™ Universal SYBR® qPCR Master Mix . Each PCR reaction contained 5.0 μL SYBR mix, 0.2 μM primers, and 1.0 μL diluted cDNA in a final volume of 10 μL. The 2Additional file 1: Figure S1. Sequence alignment of 128 CgbHLH proteins.Additional file 2: Table S1. Predicted MW, pI, exon number, UTR and motifs of 128 CgbHLH proteins.Additional file 3: Table S2. The primers of CgbHLH genes used for qRT-PCR.


N,N-diethyl-cis-2,6-dimethylpiperidinium (DMP) as both a structure directing agent and crystal growth inhibitor, aluminosilicate FER zeolite nanosheets, with a variety of crystal thicknesses, ranging from 6 to 200 nm, are successfully synthesized under hydrothermal conditions. Very interestingly, the amount of DMP in the starting gel is the key factor for crystal thickness control of aluminosilicate FER zeolite nanosheets. The obtained FER products, with different thicknesses, are well characterized by X-ray powder diffraction (XRD), scanning electron microscopy (SEM), N2 sorption, thermogravimetric analysis (TG), inductively coupled plasma (ICP), and magic angle spinning nuclear magnetic resonance (MAS NMR) techniques. This simple strategy might provide a novel avenue for the synthesis of other zeolite nanosheets with controllable thickness.Preparation of aluminosilicate ferrierite (FER) zeolite nanosheets with controllable thickness in the presence of a sole organic ammonium is attractive, but still challenging. In this report, with the employment of  Zeolites, especially aluminosilicate zeolites, have been widely applied in the fields of adsorption, separation, ion exchange, and catalysis due to their uniform micropore distribution, large surface areas, and highly thermal and hydrothermal stabilities ,5,6,7,8.x reduction [FER zeolite is a medium-pore zeolite containing 10-Membered Ring (10-MR) channels (0.42 × 0.54 nm) in the [001] direction and 8-MR channels (0.35 × 0.48 nm) in the [010] direction ,29,30,31eduction ,38,39,40eduction . Meanwhieduction . DespiteN,N-diethyl-cis-2,6-dimethylpiperidinium, DMP). Very interestingly, the DMP molecules not only direct the FER zeolite structure, but also inhibit the growth of FER zeolite on [100] direction, according to the theoretical calculation [More recently, we reported a simple method for synthesizing ultrathin nanosheets of aluminosilicate FER zeolite with the thickness of 6–8 nm by using a sole small organic ammonium  into the starting gel, the thickness of FER-0.015 nanosheets turned to around 50–100 nm  with a thickness of 6–8 nm would be obtained , while the mesopore volumes of these samples are increased with the decreasing of the crystal thickness, as shown in 29Si magic angle spinning nuclear magnetic resonance (MAS NMR) spectra of the aluminosilicate FER zeolite samples. These samples exhibit peaks at around −114, −111, −109, −106, and −103 ppm, which can be reasonably assigned to Si(4Si) , Si(3Si) (−106 ppm), and Si(2Si) (−103 ppm) species, respectively. According to the spectra and the structural information in 27Al MAS NMR spectra of the aluminosilicate FER zeolite samples. All of the samples give the one peak with the chemical shift at about 54 ppm associated with the tetrahedrally coordinated aluminum species in the FER zeolite framework. At the same time, there is no signal with the chemical shift at about zero ppm, suggesting the absence of extra-framework aluminum species in these samples. This result shows that the addition of different amounts of DMP molecules in the synthesis has no effect on the aluminum coordination of the samples.3-TPD technique. The NH3-TPD curves in The acidity of the aluminosilicate H-FER zeolite nanosheet samples is investigated by the NH2, AR, 99%, Sinopharm Chemical Reagent Co., Ltd., Shanghai, China), sodium hydroxide , colloidal silica , cis-2,6-dimethylpiperidine , potassium bicarbonate , iodoethane , methanol , diethyl ether , anion-exchange resin , and ammonium nitrate  were used without further purification. The deionized water was homemade.Sodium metaaluminate  was added and the mixture was stirred for 2 h. Next, 0.024 g of FER zeolite seeds was added. Finally, the mixture was transferred into a Teflon-lined autoclave oven and crystallized at 140 °C for 48 h under rotation conditions (50 rpm). After filtering, washing, and drying, the product was gained.In a typical run for synthesizing aluminosilicate FER zeolite nanosheets without the addition of DMP molecules, 0.165 g of NaAlO2 and 0.254 g of NaOH was dissolved into deionized water. Next, a certain amount of DMP solution and 2.95 g of colloidal silica (40 wt% SiO2 in water) were added. After stirring for 2 h, the mixture was transferred into a Teflon-lined autoclave oven and crystallized at 140 °C for 48 h under rotation conditions (50 rpm). After filtering, washing, and drying, the products were gained.In a typical run for synthesizing aluminosilicate FER zeolite nanosheets with the use of DMP, 0.165 g of NaAlO2 in the starting gel). The H-form of the samples (H-FER-x) were obtained by calcining and ion-exchanging the FER-x samples with the NH4NO3 solution.The as-made form of the products were denoted as FER-x (x represent the ratio of DMP/SiOKα (λ = 1.5406 Å) radiation. Scanning electron microscopy (SEM) experiments were performed on Hitachi SU-8010 electron microscopes. The N2 sorption isotherms at the temperature of liquid nitrogen were measured using Micromeritics ASAP 2020M and Tristar system. The thermogravimetric analysis (TGA) experiments were carried out on a Perkin-Elmer TGA 7 unit in air, at a heating rate of 10 °C/min, in the temperature range from room temperature to 800 °C. 29Si and 27Al MAS NMR spectra were recorded on a Varian Infinity Plus 400 spectrometer. The sample composition was determined by inductively coupled plasma (ICP) with a Perkin-Elmer 3300DV emission spectrometer. The acidity of the samples was measured by the temperature-programmed-desorption of ammonia (NH3-TPD). The 50 mg of samples were placed in a quartz tube and pretreated in He flow at 600 °C for 30 min. Then the temperature was reduced to 150 °C. NH3 passed through the samples until it reached equilibrium for 30 min. When the baseline was stable, the signal of NH3 desorption was monitored by the thermal conductivity detector (TCD) in He flow at a heating rate of 20 °C/min from 150 to 650 °C.X-ray powder diffraction (XRD) patterns were measured with a Rigaku Ultimate VI X-ray diffractometer  using CuIn summary, aluminosilicate FER zeolite nanosheets with controllable thickness are successfully prepared with the use of a sole small organic ammonium (DMP). The addition of different amounts of DMP molecules in the starting gel is the key factor for successful control of the FER nanosheet crystal thickness. All of the nanosheets of the FER zeolite samples have good crystallinity, uniform morphology, large BET surface area, four-coordinated aluminum species, and abundant acidic sites. This simple synthesis strategy might be applied when it comes to preparing, and improving the performance, of other zeolite nanosheets with controllable thicknesses.


Metazoan development from a one-cell zygote to a fully formed organism requires complex cellular differentiation and communication pathways. To coordinate these processes, embryos frequently encode signaling information with the small protein modifier ubiquitin, which is typically attached to lysine residues within substrates. During ubiquitin signaling, a three-step enzymatic cascade modifies specific substrates with topologically unique ubiquitin modifications, which mediate changes in the substrate’s stability, activity, localization, or interacting proteins. Ubiquitin signaling is critically regulated by deubiquitylases (DUBs), a class of ~100 human enzymes that oppose the conjugation of ubiquitin. DUBs control many essential cellular functions and various aspects of human physiology and development. Recent genetic studies have identified mutations in several DUBs that cause developmental disorders. Here we review principles controlling DUB activity and substrate recruitment that allow these enzymes to regulate ubiquitin signaling during development. We summarize key mechanisms of how DUBs control embryonic and postnatal differentiation processes, highlight developmental disorders that are caused by mutations in particular DUB members, and describe our current understanding of how these mutations disrupt development. Finally, we discuss how emerging tools from human disease genetics will enable the identification and study of novel congenital disease-causing DUBs. Deubiquitylases (DUBs) are a class of ~100 human enzymes that regulate ubiquitin signaling by processing ubiquitin precursors, hydrolyzing ubiquitin chains, and cleaving ubiquitin modifications from substrates.Intricate regulatory mechanisms ensure spatial and temporal regulation of DUB activity and substrate recruitment to allow DUBs to integrate signals during development and coordinate developmental cell-fate decision.DUBs regulate gene expression (through deubiquitylating histones and modulating the stability of chromatin regulators/transcription factors) and signaling pathways to control metazoan development.Mutations in particular DUBs cause developmental disorders, but the molecular mechanisms and cognate substrates or E3 ligases are often unknown.Many DUBs are intolerant to haploinsufficiency and missense mutations in the general human population, suggesting that their dysregulation likely causes developmental diseases.How are specific DUBs regulated during embryonic and postnatal development to achieve their functions in cell-fate determination?What are the mechanisms and substrates of DUBs whose mutations underlie developmental diseases?Which other DUBs cause developmental diseases? Can we utilize tools from human genetics to identify these DUBs and study their (patho-)physiological functions and mechanisms?During metazoan development, stem cells of the embryo undergo self-renewal, commit to differentiation programs, and produce and react to signaling molecules to ensure proper formation of specialized cell types, tissues, and organs. The precise execution of these processes is often controlled by ubiquitylation, an essential posttranslational modification (PTM) that regulates the stability, activity, localization, or interaction landscape of substrates –3. The dUbiquitylation regulates biological pathways in a highly specific and reversible manner, which enables ubiquitin signaling to control cellular behavior and decision-making during embryonic development , 3. SpecUSP7 [USP8 [USP9X [USP16 [USP22 [BAP1 [OTUB1 [AMSH [OTUD6B [OTUD5 [DUBs can deubiquitylate a broad range of substrates in fundamental cellular processes including transcription, translation, cell cycle progression, vesicular trafficking, autophagy, proteasomal degradation, and intracellular signaling to control various aspects of stem cell maintenance, differentiation, and development , 25–30. USP7 , USP8 [3P7 [USP8 , USP9X [8 [USP9X , 34, USPX [USP16 , USP22 [6 [USP22 , BAP1 [322 [BAP1 , OTUB1 [1 [OTUB1 , AMSH [3B1 [AMSH , OTUD6B  [OTUD6B , and OTUB [OTUD5 , 42) andB [OTUD5 , 44. It B [OTUD5 , 45–47. Here, we review structural, functional, and regulatory features of DUBs that allow this class of enzymes to fulfill their roles during development. We summarize general mechanisms of how DUBs regulate stem cell self-renewal and differentiation processes and our current understanding of how mutations in particular DUBs cause congenital diseases. Finally, we discuss how recently developed genetic resources can help identify candidate DUBs critical for development.Since the discovery of DUBs in the mid-1980s –50, exteSeveral in-depth discussions on DUB enzymology, structure, and substrate specificity have recently been published and we refer interested readers to these seminal reviews , 57–60. exo cleavage, e.g., MINDY [exo-cleavage, e.g., USP5 [To maintain free ubiquitin pools for conjugation and to regulate ubiquitin signaling, DUBs hydrolyze peptide or isopeptide bonds between ubiquitin and a substrate or within ubiquitin chains. In this process, DUBs generally utilize their catalytic domain to recognize and remove the distal  ubiquitin from the proximal (lysine- or methionine-contributing) ubiquitin or the substrate. By utilizing additional ubiquitin-binding sites and/or substrate interaction motifs, DUBs have evolved specificities for cleavage at particular positions in the ubiquitin chain or linkage types , 57–59 . For ins., MINDY , 62, andg., USP5 , respectg., USP5 , JAMMs [g., USP5 –66, MINDg., USP5 , ZUP1 [5g., USP5 –55 and tg., USP5 –69 and Cg., USP5 , 71) andg., USP5 ) are morUbiquitylation frequently orchestrates core signaling networks essential for stem cell maintenance and differentiation. During these processes, it is important that ubiquitin signals are tightly controlled. Similar to their E3-ligase counterparts , 74, DUBDuring development, similar to other signaling proteins, DUBs are commonly controlled at the level of their synthesis and degradation , 76–78. Another frequent mode of regulation in eukaryotic cells is targeted localization . ExperimIn addition to control of abundance and localization, DUBs are also subject to regulation at the level of their activity and substrate recruitment cis or trans can activate or inhibit the activity of DUBs that regulate important aspects of differentiation. However, in most cases, how these mechanisms are implemented to control ubiquitin signaling during embryonic and postnatal development remain unclear and will require further investigation.Catalytic activities of DUBs can be modified through interaction with accessory domains or proteins. For instance, ubiquitin-binding and activity of BAP1, an essential, histone-directed DUB that regulates gene expression networks during development , 111, isIn addition to interacting with allosteric regulators, DUBs also frequently associate with ubiquitin E3 ligases in cells . This coDUB catalytic activity can be further controlled by reversible PTMs such as phosphorylation, ubiquitylation, SUMOylation, or oxidation , 59. RegSeveral DUBs have been shown to control different aspects of embryonic development by diverse mechanisms. This includes AMSH and USP7, which regulate endosomal sorting and membrane trafficking required for faithful neuronal differentiation , 146 se. In the Dynamic changes in chromatin architecture are required for driving developmental gene expression programs. These changes are brought about by reversible histone PTMs, which alter the physical properties of chromatin and/or recruit effector proteins to alter transcription. Monoubiquitylation of H2A and H2B are an abundant and critical means for ensuring accurate gene expression during metazoan development , 27. CatIn addition to controlling gene expression at the level of histone deubiquitylation, DUBs also frequently target chromatin regulators and transcription factors to modulate their stability and function during stem cell maintenance and differentiation , 41, 161Multiple signaling pathways—such as FGF, Hedgehog, WNT, TGF-β/BMP, and Notch—orchestrate development, operating repeatedly at different times and regions in the embryo to regulate germ layer specification, patterning, and organogenesis . These cIn the canonical pathway, secreted TGF-β and BMP ligands elicit their functions by activating transmembrane serine/threonine kinase receptors and intracellular second messengers known as SMADs Fig. . BindingThus, as outlined in the above examples, multiple DUBs modulate TGF-β/BMP signaling at the receptor or effector level through prevention of degradation or control of protein–protein interactions. In this context, to achieve a certain biological outcome, the same DUB can regulate the pathway at different levels  or multiple DUBs can act on the same target . Future experiments are required to further examine how DUB interplay at these different levels is spatially and temporally regulated to ensure proper TGF-β/BMP signaling responses during embryonic development.Extensive studies over the last decades have established that dysregulation of DUBs leads to human diseases, in particular cancer, neurodegeneration, and inflammation , 45, 46.STAMBP, also known as AMSH, causes MIC-CAP syndrome [Amsh-deficient mice exhibit defects in cortical development similar to those in patients [Recessive loss-of-function mutations in syndrome . These psyndrome , 180–183patients . AMSH ispatients , controlpatients , 185. Thpatients  or in thpatients , 185. Dupatients . HoweverUSP7 encodes an essential DUB for which disruption of one allele, whether via heterozygous deletions or nonsense/missense mutations, results in Hao-Foutain syndrome, a developmental disorder with seizures, behavioral abnormalities, hypogonadism, and hypotonia [MAGE-L2 is located within the Prader–Willi imprinting region [USP7-deficiency-induced patient phenotypes are caused by aberrant endosomal sorting.ypotonia , 188. Suypotonia , 190, buypotonia . Elegantypotonia . USP7 acypotonia . MAGE-L2g region  and was g region , 194. ThUSP9X, encoding an X-linked DUB, cause syndromic and non-syndromic intellectual disability. Initial studies reported three male individuals with non-syndromic X-linked intellectual disability, all carrying missense variants in USP9X [Usp9x causes aberrant cortical architecture similar to that found in patients [STIL and ICQB1 encoding for NPHP5) frequently result in primary microcephaly [USP9X patients, thus suggesting that aberrant centrosome duplication and cilia assembly could contribute to MRX99. Third, USP9X has been shown to regulate dendritic spine development and maintenance [ANK, encoding for ankyrin-G are associated with neurodevelopmental disorders [USP9X patient mutations were shown to reduce interaction with ankyrin-G, strongly suggesting that abnormal ankyrin-G degradation is a pathogenic mechanism in MRX99. Consistent with this, Usp9X knockout mice exhibit synaptic abnormalities, ankyrin-G aggregates, and hyperactivity [Mutations in in USP9X . Consistpatients . Reijindpatients . Togethepatients , 199. USpatients . Dysregupatients –203 as wpatients . Mutatioocephaly , 206 andocephaly , 208, rentenance . This ocntenance –211. Varisorders  and USP9activity .It is interesting to note that TGF-β promotes cortical spine development through promoting USP9X-dependent stabilization of ankyrin-G  and thatOTUD6B causes global developmental delay, feeding difficulties, structural brain abnormalities, and congenital heart disease [OTUD6B patients.Bi-allelic loss-of-function of  disease , 214. OT disease . It has  disease  and may  disease ; howeverOTUD7A, encoding a poorly studied K11-specfic OTU DUB [OTUD7A is the most likely candidate to cause associated epilepsy. First, studies in mice have shown that OTUD7A controls dendritic branching of cortical neurons [Otud7a recapitulated neurodevelopmental deficits including abnormal EEGs [OTUD7A missense variants has been reported [ OTU DUB , is loca OTU DUB –219. 15q OTU DUB . Recent  neurons . Second,mal EEGs . Third, reported . These fOTUD5, encoding an X-linked OTU DUB that prefers cleavage of K48- and K63-linked ubiquitin chains [Otud5 is embryonic lethal in mice and OTUD5-depleted hESCs are defective in neuroectodermal differentiation, which can be rescued by re-expression of wild-type OTUD5 [Hemizygous missense and deletion variants in n chains , 222–224n chains . Affecten chains ; howeverpe OTUD5 . Interespe OTUD5 , 228, X-pe OTUD5 ) that exOTUD6B and OTUD7A). Fourth, knockout or knockdown of tens of DUBs has been shown to be lethal or to cause severe defects during embryogenesis of model organisms such as zebrafish and mice [OTUD4, USP25).Since the initial discovery of DUBs almost 40 years ago, numerous studies have provided insights into their structures, substrate/cleavage specificities, and regulatory mechanisms that allow this versatile enzyme family to contribute to diverse cellular processes. In particular, we here highlight principles of how DUBs modulate ubiquitin signaling during embryonic and postnatal development and the emerging roles of their dysregulation in congenital disorders. Despite many recent advances in our understanding of DUBs in these (patho-)physiological processes, many open questions remain. First, for more than half of the human DUBs, substrates and linkage specificities have remained unclear . Moreoveand mice , 44. In OTUD5 and LINKED syndrome [USP24, USP48, and USP32. It would be equally attractive to apply this methodology to the linkage-specific OTU DUBs OTUD4, OTUB1, VCPIP, and ZRANB1 [With the rapid increase in databases of exome and genome sequences from healthy individuals, it has now become possible to quantify the tolerance of genes to loss-of-function and missense mutations in control populations. –233 Genesyndrome , such ged ZRANB1  to uncovd ZRANB1 , 45, 234


In conclusion, FAM83A, FAM83D, FAM83E, and FAM83H have significant prognostic value in PDAC and they may play important roles in regulating tumor progression and the immune cell infiltration.Family with sequence similarity 83 (FAM83) members were shown recently to have oncogenic effect in a variety of cancer types, but the biological roles and prognostic value of FAM83 family in pancreatic ductal adenocarcinoma remain unknown. In the current study, the clinical significance and molecular function of the FAM83 family were assessed by multiple bioinformatics analysis. Besides, potential associations between differentially expressed genes (DEGs) of FAM83 family and antitumor immunity were evaluated using TIMER and TISIDB analyses. As the results show, FAM83A, FAM83D, FAM83E, and FAM83H were significantly upregulated in PDAC and were identified as DEGs. Higher expression of FAM83A, FAM83B, FAM83D, FAM83E, and FAM83H were associated with advanced tumor stage or worse patient prognosis. Importantly, the overexpression of DEGs was found to be significantly correlated with activated KRAS and loss of SMAD4, which are important drivers for PDAC. Further, FAM83A, FAM83D, and FAM83H were associated with CD8 Pancreatic cancer (PC) is considered to be one of the most aggressive cancers, leading to 4.7% of all cancer-related deaths globally . PancreaIn recent studies, some of the family with sequence similarity 83 (FAM83) family members have been demonstrated significantly upregulated in a variety of human cancer types . There aIn the current study, we first analyzed the transcriptional levels of FAM83 family members in PDAC. Then, we assessed the associations between FAM83 family expression with pathological stage or patient survival to evaluate the value of the FAM83 family in the progression and prognosis of PDAC. Differentially expressed genes (DEGs) in FAM83 family members of PDAC were integrated to DAVID 6.7 to perform functional enrichment analyses. Further, we explored the biological roles of FAM83 family members in the immune infiltration of PDAC.https://www.oncomine.org) is a publicly accessible online bioinformatics database that contains 715 datasets, as well as 86,733 normal and tumor samples, and provides powerful genome-wide expression analysis [p value of 0.05 were set as the significance thresholds. Student's t-test was used to evaluate the difference of FAM83 family members in PDAC.Oncomine  is a web-based tool for analyzing transcriptional data of 9,736 tumors and 8,587 normal samples from The Cancer Genome Atlas (TCGA) and the Genotype-Tissue Expression (GTEx) projects [t-test was used to analyze the expressions or pathological stages. The fold change cutoff was 1.5, and the p value cutoff was 0.05. Prognostic analysis was performed using a Kaplan-Meier curve.Gene Expression Profiling Interactive Analysis (GEPIA) (projects . GEPIA phttps://cancergenome.nih.gov/). Of the 177 PDAC cases obtained, the correlation of mRNA expression levels among FAM83 family was evaluated by using R software (version 3.5.3) with “corrplot” package.The transcriptional expression data based on fragments Per kilobase per million (FPKM) for PDAC were obtained from TCGA database  is a web resource for visualizing and analyzing a wide variety of cancer genomics data retrieved from TCGA database [p value < 0.05).cBioPortal Database (database . It lowehttps://david-d.ncifcrf.gov/) to perform Gene Ontology (GO) analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis [p value < 0.05 was considered statistically significant.Coexpressed genes screened from cBioPortal database were integrated to DAVID 6.7  is a user-friendly website for exploring internal relationships of gene sets [GeneMANIA database  is a webserver for systematical analysis of immune infiltration across a wild variety of cancer types [+ T cell, Gamma Delta T cell, follicular helper T cell, and CD4+ T cell. Spearman's correlation coefficients were used, and p values < 0.05 were considered statistically significant.TIMER 2.0 (er types . The webhttp://cis.hku.hk/TISIDB) is an integrated repository web for tumor immunity, through which biologists can crosscheck a gene about its role in tumor-immune interactions through literature mining and high-throughput data analysis [p values < 0.05 were considered statistically significant.TISIDB  was significantly increased with a fold change of 2.838 in PDAC [p value of 2.41e-07 [p value = 0.047) and Badea et al.  in PDAC [p value = 3.07e-06) [Eight FAM83 family members were analyzed using the Oncomine database in various cancer types, and the results showed that there were a total of 297, 230, 238, 287, 341, 254, 132, and 290 unique analyses for FAM83A, FAM83B, FAM83C, FAM83D, FAM83E, FAM83F, FAM83G, and FAM83H, respectively. Based on the data from Oncomine, the transcriptional levels of FAM83A, FAM83D, FAM83E, and FAM83H were significantly upregulated in PDAC tissues compared to normal pancreas tissues . In the  in PDAC . Pei et  in PDAC , 25. As The relative expression levels of FAM83 family members were compared in PDAC. The results showed that the expression of FAM83E was the highest while the expression of FAM83C was the lowest . We nextp = 0.00333), FAM83D (p = 0.0385), and FAM83E (p = 0.00676) was correlated with more advanced pathological stage , FAM83B , FAM83D , and FAM83H  were significantly associated with worse OS , FAM83D , and FAM83H  was dramatically correlated with worse DFS , and 40 coexpression genes for FAM83A, 57 coexpression genes for FAM83D, 301 coexpression genes for FAM83E, and 572 coexpression genes for FAM83H were enrolled into DAVID 6.7 and subjected to functional and pathway enrichment analyses. GO enrichment analysis showed that FAM83A may be involved in “ectoderm and epidermis development, constitution of plasma membrane, intermediate filament, cell junction and cytoskeleton, regulation of cell adhesion, and integrin-mediated signaling pathway”  in FAM83 family members by analyzing its potential biological pathways in PDAC. The coexpression analyses for DEGs were performed by using cBioPortal dataset . SMAD4 expression level was negatively associated with these members   . However+ T cell, Gamma Delta T cell, follicular helper T cell, and CD4+ T cell in PDAC by using TIMER 2.0 platforms , FAM83A , and FAM83E  , FAM83E , and FAM83A   . Besides 0.0124) .+ T cell , eosinophil , mast cell , and follicular helper T cell  , LGALS9 , IL10RB  , ENTPD1 , CD28 , and KLPK1  , HLA-DPB1 , HLA-DOA , and HLA-DRA   were analyzed by using the TISIDB database. As shown in 2.2e-16) . For the.29e-05) . For the2.2e-16) . For the.88e-10) . These rAlthough FAM83 family members were proven to play a significant role in several kinds of cancer, their biological roles and prognostic value in PDAC have rarely been characterized. In the current study, we first showed that FAM83A, FAM83D, FAM83E, and FAM83H were significantly overexpressed in PDAC. And higher expression of FAM83B, FAM83D, and FAM83E was associated with tumor stage. Further, upregulation of FAM83B was significantly associated with worse OS, while upregulation of FAM83A, FAM83D, and FAM83H was correlated with both worse OS and DFS. These data suggested the predictive value of FAM83A, FAM83B, FAM83D, FAM83E, and FAM83H for the prognosis of PDAC.α1, α3, α5, β4, and β5 integrins in CaSki and HeLa cells. In our study, FAM83D and its neighboring genes were mainly enriched in “cell cycle (M phase), DNA replication, nuclear division, oocyte meiosis, ATP and nucleotide binding, and p53 signaling pathway” in PDAC. In other study, exogenous FAM83D overexpression promoted, while FAM83D silencing inhibited non-small-cell lung cancer (NSCLC) cell proliferation, epithelial-mesenchymal transition, and invasion through regulating the AKT/mTOR pathway [FAM83 proteins are characterized by an N-terminal “domain of unknown function” called DUF1669. However, each member has a unique C terminus of variable length and their biological function and related mechanism may be distinct. By performing functional and pathway enrichment analyses, we showed that FAM83A was involved in regulation of cell adhesion, integrin-mediated signaling pathway, and ECM-receptor interaction in PDAC. Studies have proved that elevated FAM83A expression maintained essential MEK/ERK survival signaling and prevented cell death in PC cells . Upregul pathway . Besides pathway . These iThrough functional enrichment analyses, we found that FAM83E and FAM83H were involved in “Ras GTPase binding, regulation of Ras GTPase activity, and Ras protein signal transduction.” As an important member of the Ras family, KRAS is a critical driver gene in PDAC, which is characterized by a nearly 100% KRAS mutation frequency. In this study, KRAS expression level was found positively correlated with FAM83A, FAM83D, FAM83E, and FAM83H. Our result also demonstrated that the expression of SMAD4, another driver gene of PDAC, was negatively associated with FAM83A, FAM83D, FAM83E, and FAM83H. Taking these together, FAM83 members may regulate or be regulated by KRAS or SMAD4 in PDAC, leading to cancer progression. It will be of great interest to investigate the detailed mechanism between FAM83 members and KARS or SMAD4 in future study.+ T cell with FAM83A, D, E, and H in PDAC. Findings from the current research have indicated that subsets of TILs, especially CD8+ T cell, are strongly associated with long-term oncological outcomes in patients with PDAC and limited CD8+ T cell infiltration is found in most PDAC tumor centers [γ production, which inhibits tumor progression [αβ T cell activation and promote pancreatic oncogenesis [The immune cell infiltration and tumor microenvironment have been verified to play vital roles in PDAC progression and tumor evasion . In rece centers , 34. Gamgression . Howeverogenesis . The exp+ T cell, eosinophil, mast cell, follicular helper T cell), immunoinhibitors , immunostimulators , and MHC molecules . PVRL2 (CD112), known as the ligand of PVRIG and CD226, is overexpressed in different types of tumor cell and plays an immunosuppressive role in T cell function [+ T cells, and the triple-combination blockade of PVRIG-PVRL2, TIGIT, and PD-1 resulted in the greatest increase in IFNγ, which means mostly enhancement of CD8+ effector function [+ T cells [+ CD8+ TILs were enriched in genes related to cell proliferation and exhaustion, which are characteristics of chronically stimulated T cells. At the protein level, CD39+ CD8+ TILs also showed characteristics of exhausted cells in terms of both phenotypic and functional markers in colon and lung cancers [+ T cell and required for antiPD-1/PD-L1 activity in melanoma; agents that induce MHC-II positivity can be combined with PD-1/PD-L1-targeted therapy to improve response rates [By assessing the correlation between FAM83H expression with tumor-infiltrating lymphocytes and immunomodulators, we found a broad correlation between FAM83H with immune cell infiltration (such as effector memory CD4function . Blockadfunction . Besidesfunction . CD39 (Ese rates . Thus, FHowever, there are several limitations of our study. Although the mRNA expression of some FAM83 family members was identified as prognostic biomarkers for DFS and OS in the study, the changes of protein levels and their prognostic implication were not demonstrated. Besides, our analyses can reflect some respects of immune status in PDAC but not the global changes. Further prospective experiments and in vitro or in vivo studies are needed to validate our results and explore underlying molecular mechanisms.In conclusion, FAM83 family members display differential degrees of overexpression and play vital roles in tumor progression and the regulation of immune cell infiltration of PDAC. Further, our study explored the correlation between DEGs in FAM83 family with tumor-infiltrating lymphocytes and highlight FAM83H as a prognostic biomarker and a potential immunotherapeutic target to treat PDAC.


Palmitic acid (PA), a long-chain saturated fatty acid, might activate innate immune cells. PA plays a role in chronic liver disease, diabetes and Crohn’s disease, all of which are associated with impaired intestinal permeability. We investigated the effect of PA, at physiological postprandial intestinal concentrations, on gut epithelium as compared to lipopolysaccharide (LPS) and ethanol, using an in vitro gut model, the human intestinal epithelial cell line Caco-2 grown on transwell inserts. Cytotoxicity and oxidative stress were evaluated; epithelial barrier integrity was investigated by measuring the paracellular flux of fluorescein, and through RT-qPCR and immunofluorescence of tight junction (TJ) and adherens junction (AJ) mRNAs and proteins, respectively. In PA-exposed Caco-2 monolayers, cytotoxicity and oxidative stress were not detected. A significant increase in fluorescein flux was observed in PA-treated monolayers, after 90 min and up to 360 min, whereas with LPS and ethanol, this was only observed at later time-points. Gene expression and immunofluorescence analysis showed TJ and AJ alterations only in PA-exposed monolayers. In conclusion, PA affected intestinal permeability without inducing cytotoxicity or oxidative stress. This effect seemed to be faster and stronger than those with LPS and ethanol. Thus, we hypothesized that PA, besides having an immunomodulatory effect, might play a role in inflammatory and functional intestinal disorders in which the intestinal permeability is altered. In the last two decades, several studies have reported the association between disruption of intestinal permeability and different clinical disorders, such as inflammatory bowel disease (IBD), celiac disease, irritable bowel syndrome (IBS), diabetes, and obesity ,2. A groIt has been widely shown, both in humans and in animal models, that intestinal barrier function is highly sensitive to stress, dietary, and microbiota changes ,9,10. ChFurthermore, it was reported that the bacterial lipopolysaccharide (LPS), an endotoxin originating from the cell wall of Gram-negative bacteria ,18, whicIn recent years, a growing interest in understanding the role of lifestyle-related factors in disrupting the intestinal barrier functions has emerged, and the effect of several dietary products are now the object of several studies ,22,23.Over the past decade, we have learned about the adverse effects of ethanol intake on the functional and structural integrity of the intestinal mucosa, with loss of the intestinal barrier function ,25,26.Among the dietary products, palmitic acid (PA) has also drawn much attention in the scientific community, due to its well-known lipotoxic effects on different organs in humans ,28,29. P2, in Dulbecco’s modified eagle medium  containing 10% (vol/vol) fetal bovine serum , 2 mM L-glutamine, 100 U/mL penicillin, and 100 μg/mL streptomycin (both from Euroclone). Cells seeded at a density of 6.0 × 104 per insert were grown on transwell chambers  placed in a 12-well plate. The cells were regularly monitored using a fully motorized epifluorescence inverted microscope , by measuring the epithelial resistance. Experiments were performed 21 days after seeding when the cells reached confluence and differentiation. Fresh media was changed every other day in the apical and basolateral compartments of the well, until the day of experimentation.Human epithelial colorectal adenocarcinoma (Caco-2) cells purchased from the American Type Culture Collection , were maintained under standard cell culture conditions, in a humidified 37 °C incubator, with 5% COw/v, Sigma-Aldrich, Milan, Italy) [w/v, Sigma-Aldrich, Milan, Italy) was dissolved in methanol (vehicle) as 50 mM stock solution, and then diluted in a free fatty acid (FFA) medium that was DMEM supplemented with 2 mM L-glutamine (Euroclone), 1% bovine serum albumin (BSA) Cohn fraction V , and 10% charcoal-stripped FBS , as previously reported [v/v) for 1 h in complete DMEM [2+ and Mg2+ , and used for different assays. Undernatants were collected for further analysis, as described below.Cells were challenged apically with different chemicals for 24 h  and 1 h (ethanol) at 37 °C. LPS   was dissreported . The conreported ,40,41,43reported  used in reported ,46,47,48ete DMEM ,50, as eete DMEM . After t® Cell Viability Imaging Kit , for 30 min (min) at 37 °C in complete FluoroBrite DMEM . In brief, the NucBlue® Live reagent stained the nuclei of all cells, whereas the NucGreen® Dead reagent selectively detected only the nuclei of dead cells, with compromised plasma membranes. Micrographs were acquired and analyzed using the Eclipse Ti-E , equipped with a high-sensitivity camera  and automated acquisition/analysis software . Cell viability was determined by counting the total vs. dead cells in at least three microscopic random fields/well. Results were plotted as a percentage of live cells in the treated vs. control cultures referred to as 100% in the graphs. Cell viability/cytotoxicity was further assessed using a Vybrant Cytotoxicity Assay Kit , as described previously [After challenges with different chemicals, Caco-2 monolayers were rinsed in PBS and incubated with the ReadyProbeseviously . In brieeviously .2 DCFDA, Thermo Fisher Scientific, Waltham, MA, USA), according to the method described in [2 DCFDA for 30 min at 37 °C, in complete FluoroBrite DMEM , to exclude hydrogen peroxide generation in a medium containing phenol red, before fluorescence analysis. Incubation with 400 μM H2O2  for 3 h was used as positive control for ROS [2 DCFDA, the cells were rinsed in PBS and left in fresh FluoroBrite DMEM for a recovery time of 1 h. Images were acquired under an epifluorescence inverted microscope . The ROIs occupied by cells were identified from phase contrast micrographs and used for fluorescence analysis. Mean fluorescence intensity (MFI) of the positive cells (FITC filter set) was quantified through the NIS Elements AR software (Nikon). All treated cells were normalized to their own internal controls, after background subtraction. Representative graphs were intensity surface plots of each treatment.Oxidative stress was measured by assessing the intracellular reactive oxygen species (ROS) levels generated after exposure to LPS, ethanol, and PA, through the green-fluorescent ROS detection reagent 6-carboxy-2’,7’-dichlorodihydrofluorescein diacetate, di-(acetoxymethyl ester)  from the apical to basolateral compartments. This assay was conducted by slightly modifying the protocol from . In brieExpression of TJ and adherens junction (AJ) mRNAs was evaluated through quantitative reverse transcription polymerase chain reaction (RT-qPCR), using the comparative cycle threshold (Ct) method of relative quantification (ΔΔCt). Isolation of mRNA was performed using acid guanidinium thiocyanate–phenol–chloroform extraction . Extracted mRNA was quantified spectrophotometrically  and reverse-transcribed using the High Capacity cDNA Reverse Transcription kit , according to the manufacturer’s instructions. Amplification was performed on 20 ng of cDNA in a total reaction volume of 10 μL on an ABI 7900HT Fast Real-Time PCR System  using the TaqMan universal Master Mix II  and TaqMan Gene Expression Assay primers  for TJP1 (Hs01551861_m1); CDH1 (Hs01023894_m1); and CTNNB1 (Hs00355049_m1). After verifying their stable expression through the geNorm software , 18S rRNTo assess the epithelial barrier integrity, immunofluorescence stainings against cell-to-cell adhesion proteins  were performed using the following primary antibodies—anti-Zonula Occludens-1  diluted 1:50; anti-E-cadherin  diluted 1:50; and anti-β-catenin  diluted 1:50. All antibody dilutions were done in BlockAid blocking solution . Indirect immunostainings were done as follows—Caco-2 cell monolayers were washed in PBS and fixed in 4% paraformaldehyde in PBS, for 15 min at room temperature (RT). To block prolonged fixation, the samples were washed in 1 mM glycine, and subsequently permeabilized with ice cold methanol for 5 min. After washing in PBS, the monolayers were blocked in BlockAid , for 45 min at RT. Incubation with primary antibodies was done overnight at 4 °C. After careful washing with PBS, goat anti-rabbit IgG (H + L) Alexa Fluor-488 and goat anti-mouse IgG (H+L) Alexa Fluor-488  secondary antibodies were incubated for 1 h at RT, in the dark in BlockAid . Nuclei were counterstained with DAPI . The samples were rinsed three times in PBS before mounting on microscope glass slides , and were covered with ProLong Diamond Antifade Mountant . Images were captured using an epifluorescence inverted microscope  and analyzed through the NIS Elements AR software  for evaluating the continuity of distribution of each protein on the cell surface, and quantifying their corresponding fluorescence intensity by counting three microscopic random fields per well. Each experiment was performed in triplicate.t-test and two-way ANOVA, followed by post-hoc test (Bonferroni’s), when appropriate. Significance was at the 0.05 level.Data are presented as mean ± standard error of the mean (SEM), of at least three independent experiments. Data were analyzed using Origin v. 9  and GraphPad Prism v. 6,  software tools. Normally distributed data were analyzed for significance by unpaired ® Cell Viability Imaging Kit, under an epifluorescence inverted microscope. For all three chemicals used, there was no significant difference in cell viability (%) between the treated cell monolayers .Caco-2 cell viability and cytotoxicity of the treatments were first evaluated using the ReadyProbes2 DCFDA fluorescent probe, according to previous works [2O2 [With the aim of investigating the oxidative stress caused by exogenous exposure to the three chemicals, we evaluated intracellular ROS levels in LPS-, ethanol-, and PA-treated Caco2 monolayers, via the carboxy-Hus works ,59. Cacorks [2O2 ,54 were p < 0.05) and at 360 min (p < 0.01), and was in agreement with the literature [p < 0.01) onwards, compared to its internal control. Remarkably, upon treating cells with PA at 1 mM for 24 h, there was an increase in FD-4 permeability (p < 0.05) and up to 360 min (p < 0.001), compared to the internal control. To evaluate the persistence of the PA-induced effect on the permeability to FD-4, we performed a washout experiment and observed that a 24 h recovery period did not restore the original epithelial barrier integrity (p < 0.001), as compared to its internal control and even more significantly over time . Therefore, the effect of PA on intestinal permeability was not reversible.Previous studies ,50,60,61terature ,17,19,41eability c, which ntegrity c. In facNext, the gene expression patterns of the tight junction protein TJP1, and the adherens junction proteins E-cadherin and β-catenin were evaluated after the different treatments. In the LPS-treated cells, the expression of the mRNAs of TJP1, E-cadherin, and β-catenin, was not significantly affected as compared to the control cells . However, the mRNA expression of TJP1 was not downregulated .In ethanol-treated cells , only thegulated d. Remarkegulated . The TJPegulated g, togethegulated h and β-cegulated i mRNAs, Finally, we analyzed the protein expression of the two multiprotein junctional complexes through immunohistochemistry. Thus, the pericellular distribution and arrangement of TJP1, CDH1, and CTNNB1 were assessed at the Caco-2 cellular membranes using immunofluorescence microscopy . LPS-treInterestingly, only PA was able to induce a discontinuous pericellular distribution of TJP1, CDH1, and CTNNB1  and has been widely used for investigating the intestinal responses to drugs and toxins ,62.Our evidence showed that the concentrations of the three substances used did not induce any toxic insult to the Caco-2 cell monolayers, in agreement with previous works, at least as far as LPS and ethanol ,49,61,63The role of TJ and AJ protein complexes in the increased Caco-2 monolayer permeability observed above was also evaluated, as it is known that these proteins play a pivotal role in epithelium formation and maintenance of tissue integrity, as well as in the regulation of TJ and AJ permeability ,69. DefeFinally, immunohistochemical analysis showed that only PA was able to induce a discontinuous pericellular distribution of TJP1, CDH1, and CTNNB1, compared to the controls. Moreover, the immunostaining against CDH1 showed a diverse cell morphology and an enlarged size in the PA-treated cells, which appeared to be slightly looser with each other, as compared to the tighter and more compact monolayer of the control cells. As in the case of LPS, PA is known to activate proinflammatory pathways in various tissues, including the gut, via transmembrane receptors such as TLR4 ,31,32,35Based on the data obtained from cell viability, oxidative stress, and permeability assays, we can infer that, the herein tested concentration (1 mM) of PA affects the intestinal permeability without inducing cytotoxicity or oxidative stress. PA insult on the integrity of gut epithelium seems likely to cause a functional change in the TJ and AJ barrier, through a putative effect on the disassembly of the TJ and AJ proteins , as reported previously , rather In conclusion, based on the present results it can be hypothesized, for the first time, that palmitic acid, along with its immunomodulatory effect, might play a role in several gastrointestinal disorders in which the intestinal permeability is altered, such as IBD, IBS, and celiac disease. Thus, it would be interesting to verify if there is a correlation between people with impaired intestinal permeability and high consumption of PA-rich food in their daily diet.Our study has some limitations. First, although Caco-2 cells represent a widely accepted model of the intestinal barrier utilized in many in vitro works, they are also a heterogeneous cell line that do not always express the same characteristics and the identical behavior of the parental cell line, as extensively reviewed by Sambuy et al. . Therefo


BRAF skin melanoma, in light of recent data deriving from Whole-Exome Sequencing (WES) or Whole-Genome Sequencing (WGS) studies on melanoma cohorts for which information on the mutation rate of each gene was available, for a total of 10 NGS studies and 992 samples, focusing on available, or in experimentation, targeted therapies beyond those targeting mutated BRAF. Namely, we describe 33 established and candidate driver genes altered with frequency greater than 1.5%, and the current status of targeted therapy for each gene. Only 1.1% of the samples showed no coding mutations, whereas 30% showed at least one mutation in the RAS genes (mostly NRAS) and 70% showed mutations outside of the RAS genes, suggesting potential new roads for targeted therapy. Ongoing clinical trials are available for 33.3% of the most frequently altered genes.Melanoma is one of the most aggressive tumors of the skin, and its incidence is growing worldwide. Historically considered a drug resistant disease, since 2011 the therapeutic landscape of melanoma has radically changed. Indeed, the improved knowledge of the immune system and its interactions with the tumor, and the ever more thorough molecular characterization of the disease, has allowed the development of immunotherapy on the one hand, and molecular target therapies on the other. The increased availability of more performing technologies like Next-Generation Sequencing (NGS), and the availability of increasingly large genetic panels, allows the identification of several potential therapeutic targets. In light of this, numerous clinical and preclinical trials are ongoing, to identify new molecular targets. Here, we review the landscape of mutated non- BRAF mutation, observed in about 50% of cutaneous melanoma, can receive a targeted treatment with BRAF and MEK inhibitors   analysis, focusing on available, or in experimentation, targeted therapies. The advent of MPS, allowing the simultaneous analysis of several genes, led, in the past two decades, to Whole-Exome Sequencing (WES) and Whole-Genome Sequencing (WGS) studies that found several mutated genes in human cancers. The evolution of molecular testing in melanoma, as well as the main techniques and MPS platforms currently in use for BRAF mutation testing, have been recently reviewed  has been proposed  in our dataset, and ranked their effect on melanoma according to the DisGeNET Gene–Disease Association (GDA) Score  Figure .NRAS, neuroblastoma ras viral oncogene homolog; KRAS, Kirsten rat sarcoma viral oncogene homolog; HRAS, Harvey rat sarcoma viral oncogene homolog) are frequently mutated in cancers. In cutaneous melanoma, NRAS is mutated in about 15–30% of cases, while KRAS (∼2%) and HRAS (∼1%) play a minor role  to several downstream signaling pathways, in particularly MAPK (mitogen-activated protein kinase) and PI3K (phosphoinositide 3-kinase). The three RAS tissue specific isoforms  (NRAS mutations primarily occur at position 61 (Q61R/K/L accounts for about 80%) and, less frequently, at positions 12 and 13 (G12/13 accounts for about 6%) (The  of 30%) . NRAS mubout 6%) . These mbout 6%) .NRAS display different characteristics compared to those harboring mutated BRAF: they are older (>55 years) and have a story of UV exposure, thicker primary tumors and a higher rate of mitosis. Several studies showed that NRAS mutations may result in an inferior clinical outcome with a shorter Melanoma-Specific Survival (MSS)  , althougal (MSS) .NRAS mutations in cutaneous melanoma did not allow the development of effective drugs: targeting directly NRAS remains a great challenge, and the target therapy for NRAS mutant melanoma is focused on MEK inhibitors.The high number of KRAS encodes for two proteins resulting from alternative splicing of exon 4, KRAS4A and KRAS4B. These proteins have different structures in their C-terminal region and use different mechanisms to localize to cellular membranes  occurring almost exclusively in codon G12  and point mutations   , 2000. Tpathways . Spitz n outcome .NRAS, KRAS or HRAS and RAS-mutated patients are usually treated with immunotherapy. However, several trials are ongoing  of 10% and a high incidence of adverse events . Subsequoutcomes .Later, other MEK inhibitors were developed. Binimetinib (MEK162) is an allosteric, selective, non-ATP competitive MEK 1/2 inhibitor.NRAS and BRAF mutated melanoma tumor cells ] versus 1.5 months, respectively . Interestingly, immunotherapy pretreated patients had a longer median PFS (5.5 months). However, no differences in terms of OS were noted, and binimetinib as single agent did not receive regulatory agencies approval for the treatment of NRAS-mutated melanoma.In preclinical studies binimetinib inhibited the growth of or cells . In the or cells  MEK162 oor cells . Phase Ior cells  comparedFinally, pimasertib  has been evaluated in a phase I trial with encouraging results. Phase II study enrolled 194 patients to be treated with pimasertib or dacarbazine. Results showed a significant benefit for pimasertib, with a median PFS of 13.0 versus 6.9 weeks and a DCR of 37.7% versus 26.6%. Unfortunately, no difference in OS was showed (8.9 vs. 10.6 months) .Combination of MEK inhibition with other targets is currently being evaluated. Among all, the results of a combination trial performed on 14 patients treated with ribociclib (LEE001) and MEK162 are particularly interesting. Indeed, the combination obtained six partial response and six stable disease, with a DCR of 85% .As we mentioned before, NRAS has to undergo some post-translational modifications, like farnesylation . FarnesyPREX2 gene plays oncogenic roles in human cancers, such as melanoma, since it is involved in PIK3CA-PTEN-AKT signaling pathway   . In our PREX2 as a key player in melanoma, suggesting that PREX2 may be a potential therapeutic target, to date no clinical trials are available.Although these evidences point to TP53 tumor suppressor gene, which is universally recognized as the “guardian of the genome,” prevents the cell from dividing and promotes apoptosis. Moreover, it is the most frequently mutated gene in human cancer with a significant prevalence of missense mutations  that converts the active RAS-GTP to RAS-GDP , a calcium-dependent neutral cysteine protease, as a novel NF1 binding partner that regulates NF1 degradation in melanoma cells. ShRNA-mediated knockdown of CAPN1 or treatment with a CAPN1 inhibitor showed to stabilize NF1 protein levels, downregulate AKT signaling and melanoma cell growth. Moreover, combination treatment of Calpain inhibitor I with MEK inhibitor Trametinib in different melanoma cells seemed to be more effective in reducing melanoma cell growth compared to treatment with Trametinib alone, suggesting that this combination may have a therapeutic potential in melanoma , p. 1. TARID2 encodes a subunit of the SWI/SNF chromatin remodeling complexes (polybromo- and BGR1-associated factor [PBAF]), which facilitates ligand-dependent transcriptional activation by nuclear receptors. The SWI/SNF multiprotein chromatin remodeling complex is involved in regulating cell growth and proliferation. Mutations in nine genes encoding for subunits of the SWI/SNF chromatin remodeling complex are found in 20.0% of human cancers  gene encodes two alternatively spliced variants known to function as inhibitors of G1 progression through different mechanisms, e.g., the tumor suppressor p16INK4A and p14ARF.The INK4A blocks the G1 progression, binding CDK4 and CDK6, and preventing them from interaction with D-cyclins and phosphorylation of RB. p14ARF, whose name derives from the presence of an Alternate Reading Frame (ARF) in the transcript, binds MDM2 (Mouse double minute 2 homolog or E3 ubiquitin-protein ligase) whose role is the degradation of p53; therefore, it also plays an indirect role in G1 progression. Furthermore, p14ARF has a role in p53-independent cell functions through the interaction with other proteins  and 12%, respectively  encodes a member of the Ser/Thr protein kinase family responsible for the phosphorylation and regulation of transcription factors, including RB1, SMAD3, MYC, FOXM1, MEP50, able to mediate cell-cycle progression , a potent and ATP-competitive multitargeted kinase inhibitor that inhibits Aurora C, Aurora B, and Aurora A and that suppresses RET tyrosine kinase, PDGFRβ and Flt1; palbociclib and SHR6390, two selective inhibitor of the cyclin-dependent kinases CDK4 and CDK6.To date, several clinical trials are ongoing, attempting to find a way to modulate this pathway  Table 2Table 2. CDK4 amplification and/or CCND1 amplification and/or CDKN2A loss. Finally, SHR6390 showed a promising activity in preclinical studies performed on cell lines and human tumor xenograft models.Phase I trial with ilorasertib showed two clinical responses among 58 treated patients, and confirmed a good tolerability and safety profile of the drug . PalbociCDK4/6 alterations, several drugs are being testing.Considering P276-00 is a novel inhibitor for CDK-1, CDK4 and CDK9 that has been tested in 16 tumor cell lines from different human cancers, showing a significant antiproliferative effect compared to cisplatin. Interestingly, some cancers showed to be particularly sensitive to P279-00, like tumors of central nervous system, NSCLC, breast cancer and melanoma .BRAF or NRAS.Ribociclib also showed some activity in melanomas with activating mutations of A phase Ib/II study with ribociclib plus MEK162 has been performed in 2013. Among 14 NRAS mutant advanced or metastatic melanoma patients, the combinations of drugs allowed to obtain six partial responses and six stable disease .Abemaciclib, another CDK4/6 inhibitor, structurally different from ribociclib and palbociclib, showed to be effective among several human tumors, including melanoma, in preclinical models . Of notePTEN tumor-suppressor gene encodes for a phosphatidyl-inositol-3,4,5-triphosphate 3-phosphatase which negatively regulate the phosphatidylinositol 3-kinase (PI3K)/protein kinase B (AKT) pathway. The PTEN gene, which is also known as MMAC1 (mutated in multiple advanced cancers) and TEP1  exhibits both protein and lipid phosphatase activities. Therefore, deletion or inactivation of PTEN results in constitutive AKT activation. Loss of PTEN function through deletion, mutation, and/or decreased expression, has been found in human sporadic cancers, including melanoma  metastatic melanoma tumors  ( factor) , althoug factor) .CTNNB1 but also in APC (adenomatous polyposis coli gene) or ICAT (inhibitor of beta-catenin) genes  genes . Howeverh arrest . Furtherh arrest .CTNNB1  are relatively infrequent (around 7%)  .CTNNB1 (S33C) mutation was found to confer resistance to imatinib in a metastatic melanoma patients harboring KIT L576P mutation ; 12.2% (26/212) of melanomas were NF1-mutant/BRAF-RAS-wild-type and nine of them harbored co-mutations in RASA2 (2 nonsense and 3 R551C) (3 R551C) .RASA2 and NRAS mutations were confirmed to be mutually exclusive, with NF1 mutations significantly co-occurring with RASA2 mutations in BRAF and NRAS wild-type melanomas, since RASA2 inhibits NRAS and NF1 inhibits KRAS and HRAS  that participates in the splicing of pre-mRNAs. Mutations in SF3B1 can lead to alternative splicing events for multiple genes and are found in several cancers, including uveal (frequency of 10–20%) (SFRB1 mutations (particularly at codon 625) are rare in cutaneous melanoma despite in uveal, mucosal, leptomeningeal and blue naevi-like cutaneous melanomas are reported  , p. 1, m 10–20%) , and cut 10–20%) . SFRB1 mreported . OverallSF3B1 mutations were found to induce cancer cells to produce an abnormal form of the BRD9 RNA molecule, including noncoding DNA sequences or “junk DNA,” which garbled the genetic message  was found to be more prevalent in male patients and similar between primary (9.2%) and metastatic tumors (8.6%)  . Indeed,iagnosis . In geneectively .PREX2. Indeed, truncating mutations in PREX2 have been shown to activate the GTPase RAC1 abolishing the binding to PTEN and activating the PI3K (phosphatidyl inositol 3 kinase)/Akt signaling pathway , mitogen-activated protein kinase 2, encode for mitogen-activated protein (MAP) kinase involved in many cellular processes, such as proliferation, transcription regulation, differentiation and development, and represent the downstream targets of the RAS-RAF-MAPK cascade. Activating mutations in MAP2K1 and MAP2K2 represent one of the multiple mechanisms of resistance to BRAF and MEK inhibitors , a greater OS (6-months OS 81% vs. 67%), and a higher rate of responses (22% vs. 8%). Based on these data, trametinib was approved as single agent by the FDA in May 2013. Despite this, currently MEK inhibitors do not play a role in monotherapy in NRAS-mutated or in WT patients but could play a role in combination with immunotherapy or with other target agents.Based on the encouraging results obtained in phase I  and phasFinally, TAK-733, a selective MEK1/2 inhibitor, showed a broad antitumor activity in melanoma cell lines and in 10 out of 11 patient-derived xenografts models . In phasKIT encodes for a class III tyrosine kinase receptor that is expressed on several cell types, including melanoblasts and differentiated melanocytes, but also hematopoietic progenitors and mast cells. The binding to the stem cell grow factor causes c-kit homodimerization that leads to the phosphorylation of tyrosine residues by activating the MAPK/ERK and the PI3K/AKT/mTOR pathways , acral (36%), and melanomas arose on chronically sun-damaged skin (28%) (KIT mutations in 497 (9.5%) melanoma patients analyzing 5,224 patients from 32 studies selected  or exon 13 (K642E)  . Interesselected . Moreoveselected . Approxi (K642E) . Mutatioectively .KIT mutated melanoma patients was explored through three single arm phase II trials  .KIT mutated melanoma. With regard to clinical activity, four partial responses and seven stable diseases were obtained. The median PFS was 4.7 months and median OS 12.3 months  in acral, mucosal, and unknown origin melanomas, as well as cutaneous ones arising on chronically damaged skin, to offer an additional therapeutic option.In conclusion, to date it is recommended to test RB1 acts as a tumor suppressor gene by regulating cell cycle division: when dephosphorylated, it interacts directly with E2F1 and inhibits its transcriptional activity with cell cycle arrest. Thus, the cell cycle is finely regulated by CDKs with CDKIs varying levels of RB phosphorylation, E2F family and TP53; alterations that disrupt the p16INK4A:cyclinD-CDK4/6:RB functional pathway may the first critical step leading to melanomagenesis 1. In our dataset, the most common somatic alterations in RB1 are mutations with a frequency of 4.4% while CNVs are reported in 1.9% of skin melanoma patients  independently of BRAF or NRAS mutation . PI3K signaling has a role in many cell activities, e.g., cell proliferation, migration, survival.PIK3CA have an oncogenic effect mostly related to activating variants in two hotspots located in the regions of helical and kinase domains. The PI3K-AKT pathway plays a significant role in melanomagenesis, frequently by activating RAS-RAF-MEK-ERK pathway, e.g., for NRAS activating variants or loss of PTEN  to cooperate with BRAF V600E promoting melanomagenesis in mouse models  is an oral selective inhibitor of PI3K isoform-α that showed to be active against the somatic PI3Kα mutations and wild-type PI3Kα . Based oresponse .Parsaclisib is a novel Pi3kδ inhibitor that exhibited an excellent profile in xenograft models , 0504. TThe combination between pimasertib, a MEK1/2 inhibitor, and voxtalisib, a dual PI3K/mTOR inhibitor among patients affected by solid tumors, including melanoma, was tested in a phase Ib trial in 146 patients. In December 2018, data from this experimentation were published, showing a DCR of 52% with one complete response and five partial responses. Unfortunately, the toxicity profile of this combination was considered not acceptable .EZH2 encodes a histone methyltransferase that constitutes the catalytic component of the polycomb repressive complex-2 (PRC2) which has a role in epigenetic silencing during cell differentiation, in particular in the development of the hematopoietic and central nervous systems. EZH2 can also induce an epithelial-to-mesenchymal transition in the cancer cells, increases their metastatic potential  showed EZH2 gain. Moreover, a significant difference in overall survival and disease-free survival between no EZH2 copy number gain and gain groups was reported  and c-KIT (CD117)], and modulating vascularity, immune response and metastasis formation  in an NADP+ dependent manner, protecting cells against reactive oxygen species (ROS) and radiations (IDH1 and IDH2 (isocitrate dehydrogenase 2 (NADP+), mitochondrial) were described in several cancers, including melanoma  with a relative production of oncometabolite 2-hydroxylglutarate (D-2HG) that it is likely to play a major role in the pathophysiology of tumors blocking cellular differentiation by competitively inhibiting αKG-dependent dioxygenases involved in histone and DNA demethylation , a DH2-specific inhibitor, and Ivosidenib (AG-120), a IDH1-specific inhibitor, have been approved by FDA for patients with relapsed or refractory acute myeloid leukemia  was able to inhibit NRAS activation in NRAS-STK19 mutant mice preventing NRAS-driven melanoma development and growth -leucine zipper protein that plays a role in the development of neural crest-derived melanocytes and retinal pigment epithelial cells, and is encoded by ITF gene . MITF isITF gene , DNA repITF gene . This weITF gene . DespiteITF gene , its actITF gene . MITF lo of 1.7% . MITF amhibition . Recentlhibition . To date in vivo , this ab in vivo . Results in vivo .TERT gene needs to be addressed separately from the other genes, since the most common mutations in TERT are localized in TERT promoter and have not been systematically investigated in most of the studies included in our dataset. TERT gene codes for a telomere reverse transcriptase catalytic subunit, the principal mechanism of telomere maintenance in cancer cells.TERT promoter mutations were performed in melanoma patients. The most common TERT promoter mutations are −57A/C, −124C/T, −146C/T, upstream the TERT gene ATG  and CNVs (5.5%) are described in TERT coding variants found in our dataset were all not-pathogenic variants in melanoma, underlying TERT as a polymorphic gene.Then, a discordancy on the mutational status between the primary and metastatic lesion was found in a cohort of 194 primary nodular melanomas matched with 72 loco-regional metastases, while TERT protein expression was found to correlate with reduced patient survival . A frequmelanoma  and invemelanoma . Moreove therapy . Recentl therapy , 2020. Iutations .9% and CTERT mutated patients or with TERT inhibitors are ongoing.No clinical trials for MTOR gene encodes a protein belonging to the phosphatidylinositol kinase-related kinases family, which regulates cell growth, proliferation, motility and survival, protein synthesis and transcription , and unknown primary subtypes, with a frequency of 11.0, 14.3, 3.4–6.7, and 11.1%, respectively (∗ and L552F) in MTOR gene . Interestingly, these mutations have previously been identified as activating mutations in cancer patients using publicly available databases of cancer genome sequencing data  of mTOR. Both studies are ongoing, and thus results are not yet available.Regarding the use of everolimus in mTOR mutated patients, only two clinical trials exist to date, to our knowledge. The first one is a phase II clinical trial on everolimus in cancer patients with Temsirolimus was tested in combination with sorafenib in a phase I  and in aTACC1 gene belongs to the TACC gene family that encodes centrosomal proteins that may play roles in microtubule regulation and spindle function, and, thus, may be an important driver of genomic instability in cancer cells  in melanoma samples  co-occurring with GNAQ R183Q and GNA11 R183C  from a primary cutaneous melanoma patient carrying BRAF V600E and RAC1 P29S mutation. In vitro study confirmed that YAP-7SA encodes a hyperactive version of the YAP protein  showed no coding mutations, whereas 298 (30%) showed a mutation in one of the RAS genes (mostly NRAS) and most of the samples studied  showed mutations in non-RAS genes  are currently ongoing to prove this concept, enrolling hundreds of patients regardless of their neoplasm, and treating them with the specific drug targeting the specific identified mutation. Hopefully, the results of these experimentations will be a milestone in medical oncology because will allow us to put into practice a real personalized therapy, tailored on what is written in tumor genome.IV and ET contributed equally in conceiving the review focus, conducting the literature review and summarizing the manuscript, analyzed data, wrote the first draft, and finalized the manuscript. PG designed and coordinated the review. BD, LP, VA, WB, AB, FS, and PG reviewed the literature, revised and made corrections to the manuscript. All authors have read and agreed to the final version of the manuscript.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.