Carboxylesterase (CXE) is a type of hydrolase with α/β sheet hydrolase activity widely found in animals, plants and microorganisms, which plays an important role in plant growth, development and resistance to stress.CXE genes were identified in Gossypium barbadense, Gossypium hirsutum, Gossypium raimondii and Gossypium arboreum, respectively. The gene structure and expression pattern were analyzed. The GBCXE genes were divided into 6 subgroups, and the chromosome distribution of members of the family were mapped. Analysis of promoter cis-acting elements showed that most GBCXE genes contain cis-elements related to plant hormones  or abiotic stress. These 6 genes we screened out were expressed in the root, stem and leaf tissues. Combined with the heat map, GBCXE49 gene was selected for subcellular locate and confirmed that the protein was expressed in the cytoplasm.A total of 72, 74, 39, 38 CXE genes of the four cotton species in this family indicated that tandem replication played an indispensable role in the evolution of the CXE gene family. The expression patterns of GBCXE gene under different stress treatments indicated that GBCXE gene may significantly participate in the response to salt and alkaline stress through different mechanisms. Through the virus-induced gene silencing technology (VIGS), it was speculated that GBCXE49 gene was involved in the response to alkaline stress in G. barbadense.The collinearity analysis of the The online version contains supplementary material available at 10.1186/s12870-022-03579-9. CXE genes have been found in Arabidopsis thaliana [Malus domestica [Vitis flexuosa Thunb [Prunus persica [CXE genes were actively involved in different developmental processes. CXE had a variety of physiological functions, such as participating in the degradation of nerve signal transmission pheromones and the degradation of other harmful foreign bodies in animals [Thousands of metabolites were synthesized in higher plants, these metabolites played an important role in plant growth and development, hormone signal transmission, and stress defense response . Plant hthaliana , Malus domestica , Vitis fsa Thunb , Prunus  persica  and othe animals . In plan animals , activat animals , and resAtCXE) genes, AtCXE8 and AtCXE9 from Arabidopsis thaliana, the AtCXE8 and AtCXE9 proteins possess carboxylesterase motifs (-GXSXG-) and catalytic triads . Overexpression of the AtCXE8 gene in transgenic Arabidopsis plants led to enhanced disease resistance against B. cinerea [PepEST genes containing -HGGGF-and-GXSXG-motifs and a catalytic triad, when the fruit is infected with C. gloeosporioides, PepEST accumulation was localized in epidermal and cortical cell layers in infected ripe fruit, but rarely even in epidermal cells in infected unripe one, so it proved that PepEST was involved in the resistance of ripe fruit against C. gloeosporioides infection [If a type of protein or gene is a domain with a common origin, it belongs to a gene family, superfamily refers to the same gene can belong to two or more gene families . Plant C cinerea . Similarnfection .Arabidopsis, which are widely present in the Arabidopsis genome (4 of 5 chromosomes), 18 of which were expressed in most tissues of Arabidopsis plants, AtCXE13 was only expressed in flowers and fruits, AtCXE1 was not expressed in leaves at all stages [CXE genes identified from Malus domestica, MdCXE1 and MdCXE12 had very high homology with Arabidopsis AtCXE18 genes, while MdCXE12 was more closely related to rice gibberellin receptor GID1. MdCXE1 was in the early stage of Malus domestica fruit development, it was expressed at a low level of harvested and mature, while a highest level in the fruit, it may affect the flavor of Malus domestica through the ability to hydrolyze the relevant flavor esters in the mature fruit. During the entire fruit development process, MdCXE16 was in the wild constitutive expression [Actinidia eriantha (AeCXE1) can hydrolyze a range of carboxylester substrates with acyl groups ranging from C2 to C16, with a preference for butyryl moieties [Early studies found that plant CXE isoenzymes were widely present in different tissues, organs, developmental stages and different parts of cells . Carboxyl stages . The carl stages . Among tpression . In addimoieties . This diGossypium barbadense, as a cultivated species of Gossypium, is well-known not only for high-quality fibers, but also for pioneer species in the saline and alkaline land. However, the growth and yield of cotton will be severely restricted by external and internal environmental factors. More and more studies have shown that CXE family proteins play an important role in hormone signal transduction, defense response and participation in biological stress. There are few studies on the CXE protein in the process of cotton growth and development. This study conducted a comprehensive review of the cotton CXE family, and initially identified the CXE genes of the four major cotton species. In G. barbadense, one of the CXE genes was identified. Further research was carried out, and a preliminary identification of the alkali resistance of the gene was made. This study provided a new perspective for the evolution and in-depth study of this gene family by analyzing the possible involvement of some CXE genes in environmental stress tolerance. Our results provide an identifiable basis for further study of the mechanism of action of this family of genes and the biological function of CXE gene in the growth and development of G. barbadense.CXEs family in cotton, we used the hidden Markov model of PF07859 as the query condition, and compared the proteome and genome of G. barbadense, G. hirsutum, G. arboreum, and G. raimondii by using local blast software. Screening was performed according to the structural domain Abhydrolase_3, and the genes that are incomplete and do not contain this structural domain are eliminated to determine the family members. We finally identified 72, 74, 38, 39 genes in G. barbadense (GBCXEs), G. hirsutum (GHCXEs), G. arboretum (GaCXEs), and G. raimondii (GrCXEs), respectively , isoelectric point (pIs), and protein hydrophilicity and hydrophobicity) were analyzed  to 462 aa (GBCXE71), MWs ranged from 28.44 kDa (GBCXE25) to 50.86 kDa (GBCXE34), and pIs ranged from 4.85 (GBCXE7) to 9.15 (GBCXE68), the range of the charge was -10.5–15, and the hydrophilic and hydrophobic coefficients were all negative, which proved that the proteins in this family were all hydrophilic proteins. Among the three family members of GHCXEs, GaCXEs, and GrCXEs of other cotton species, the physicochemical properties of the encoded proteins ranged from 227 aa (GHCXE72) to 462 aa (GHCXE73), 260 aa (GaCXE6) to 462 aa (GaCXE35), 244 aa (GrCXE9) -462 aa (GrCXE38), MWs from 25.77 kDa (GHCXE72) -50.86 kDa (GHCXE34), 29.33 kDa (GaCXE6) -50.77 kDa (GaCXE35), 27.10 kDa (GrCXE9)-50.75 kDa (GrCXE38). PIs ranged from 4.85 (GHCXE7)-9.48 (GHCXE70), 4.91 (GaCXE8)-9.10 (GaCXE31), 4.99 (GrCXE32)-9.36 (GrCXE27), the charge number ranged from -10.5 to 16.5, -9.5–15, 8.5–16, all family members were hydrophilic proteins. The prediction of subcellular localization shown that there were 46 genes in the GBCXEs family were located at the cytoplasm, 2 at the extracellular matrix, 4 at the inner membrane, 2 at the outer membrane, and 18 at the periplasmic.The physical and chemical properties of the d Table S. The resArabidopsis were used for sequence alignment. By using MEGA7 software, the evolutionary relationship between each sequence was constructed and the rootless phylogenetic tree was generated  provide evidence. Interestingly, the homologous genes of G. barbadense, whether it was G. arboreum or G. raimondii, a large proportion of the CXE protein was carried out under the influence of stable selection, so these genes evolve slowly after repeated events, and conservation at the protein level. In the evolutionary tree, all species have gene pairs from the same node, which indicates that the CXE genes of all the species we studied have experienced gene duplication events, which eventually led to the expansion of CXE genes. However, in different species, the gene duplication between different groups was different. The genes number of G. hirsutum and G. barbadense was much higher than that of other species, indicating that the CXE gene family in these two cotton species has shown a large-scale expansion during the evolution process. These results indicated that gene duplication was the main reason for the expansion of the CXE gene family in plants.In addition, to study the relationship between the common ancestors of diploid and allotetraploid cotton, we have constructed four phylogenetic trees of CXE proteins of cotton species Fig. A. ReferrCXE genes of G. barbadense, G. hirsutum, G. arboreum, and G. raimondii were located on the chromosomes of the corresponding cotton species, 221 family members were located on different specific chromosomes, of which only GBCXE10, GaCXE38 gene were mapped to scaffold. Through the distribution of genes and subgenome of allotetraploid species [G. barbadense, this discovery was the same as G. hirsutum, and in terms of evolutionary relationship, GBCXE1 and GBCXE37 gene were a homologous gene, and the two genes had similar promoter elements . Among the 74 CXE genes of G. hirsutum, there are 90 gene pairs , GHCXE41/42, GHCXE47/48, GHCXE55/56). The tandem duplications of the CXE genes of the two tetraploid cotton species all occured on chromosomes 3, 5, 13 of subgroup A, and chromosomes 2, 5, and 8 of subgroup D, respectively. Moreover, there were two pairs of tandem repeats on chromosome 2 in subgroup D.To describe the positional relationship of genes on the same chromosome, the homology of genes, the amplification mechanism and the sequence of arrangement, we performed a collinearity analysis on the  MCScanX  was used MCScanX , the resnse Fig. -g, of whnse Fig.   occured on chromosomes 4, 5, and 9 of subgroup D, and a total of 3 pairs of tandem duplications occur on chromosome 5. Meanwhile, 39 genes of G. arboreum formed 25 CXE homologous gene pairs , tandem duplication occured on chromosomes 3, 8, and 13 of subgroup A. These results indicated that fragment duplication is the main cause of gene amplification, and tandem duplication played an indispensable role in the evolution of the CXE gene family. Analysis between diploid and diploid , sub-functionalization , and new functionalization (acquisition of new functions) were the three evolutionary directions of proteins in which replication genes experience functional differences . CalculaG. barbadense CXE gene sequence were analyzed through the online website MEME. A total of 10 motifs were identified . The function of GBCXE protein was speculated to be based on the study of AtCXE protein. In our results, the promoters of most GBCXE members had GA response elements, and these proteins interact with other pathways. The GBCXE49 homologous AtCXE15 protein (AT5G06570) interacted with CXE13, CXE12, CXE18, and CXE16, also interacted with the proteins GA2ox1, GA2ox2, GA2ox3, GA2ox4, GA2ox6, and GA2ox8. In addition, in the interaction network, the carboxylesterase protein AtCXE15 was also closely related to the ABC transporter, the nucleic acid-binding protein, and the EXPA13 protein that regulated the relaxation and extension of plant cell walls. Combined with our analysis of the CXE protein promoter's action elements and protein network interactions, it would help us understand the protein's mechanism of action.To study the function of GBCXE protein, sis Fig.  by usingGBCXE31 and GBCXE69 were expressed at a higher level in roots, and GBCXE11, GBCXE24, GBCXE26, and GBCXE49 were expressed at a higher level in leaves. It was worth noting that we found that GBCXE49 had a higher expression level in stems and leaves. This may mean that different GBCXE genes played different roles in plant growth and development.We randomly selected 6 genes based on different mechanisms in response to salt and alkaline abiotic stress responses and used qRT-PCR to detect their expression patterns in different tissues . The results showed Fig. A that GBGBCXE49 gene with high expression levels in roots and leaves for in-depth study. To determine the expression characteristics of GBCXE49 protein in cells, subcellular localization analysis of the gene was performed, a GFP vector for subcellular localization analysis of GBCXE49 protein was constructed. The experiment was carried out by injecting GBCXE49-GFP fusion protein into tobacco leaves. at the same time, GFP empty fusion protein was used as a control, after the leaves were injected for 3 days, the position of the fusion protein was observed by a fluorescent confocal microscope. The result , EM and VIGS plants, the results showed  as a control. After treatment for 2 days, we found that all three plants had wilted and the infested VIGS and EM plants were significantly more stressed than WT plants, and the stress was the heaviest in VIGS, followed by EM, and then WT plants. This showed to a certain extent that the silencing of the GBCXE49 gene affects the tolerance of G. barbadense plants to alkaline stress.When the cotton cotyledons fully unfolded, the cotton cotyledons were infected with the resuspension of no-load (EM) and VIGE respectively. About 10–15 days after infestation by injection, when true leaves appeared albino Fig. D, the qRwed Fig. C that thCXE genes we identified in four cotton species  were 72, 74, 38, 39, respectively. Here, the number of genes in tetraploid cotton was about the sum of the numbers of two diploids  played an important role in gene amplification , 34. TheCXEs are involved in the activation of herbicide active substances, the metabolism of plant hormone signal substances and biological stress and other processes to play important biological functions. Early research found that plant CXE can demethylate inactive methyl salicylate and methyl jasmonate to form active salicylic acid and jasmonic acid [VvMJE1 with methyl jasmonate (MeJA) esterase activity was cloned in Vitis vinifera, which was significantly up-regulated after cold treatment and UV-B treatment [GBCXE gene promoter . We speculated that CXE family genes may be related to abiotic stress and hormone regulation pathways. In Zea mays, CXE protein can also regulate the metabolism of gibberellin (GA20) glycosyl [CXE regulates IAA metabolism in the immature endosperm tissue of Zea mays [CXE can act on sugar ester methyl esters to generate active auxins to maintain the balance of plant growth and development [GA2ox was an oxidase with regulatory function in the third stage of gibberellin synthesis [GBCXE family genes in response to the two stresses are basically the same, but the degree of response was different, and the expression levels were different. We speculated that this result was caused by the difference in pH between the two sodium salts. This also confirmed the difference in the response of G. barbadense to the two stresses. The analysis of our promoter action elements combined with the research obtained further shows that carboxyl esterase may also play a role in plant signal transduction pathways.Plant nic acid , 41. In nic acid , other snic acid , 44. Latreatment . In our glycosyl . KowalczZea mays . Previouelopment . GA2ox wynthesis . In our CXE had a wide range of substrate catalytic activity, a wide range of functions, and participates in a variety of different biological processes [CXE with different functions would have different positioning in plants, moreover, the expression and role of different CXE genes in different tissues of plants were not the same. The GBCXEs we selected, GBCXE31, GBCXE69 were highly expressed in roots, GBCXE11, GBCXE24, GBCXE26 were highly expressed in leaves, and GBCXE49 was highly expressed in stems and leaves, combined with the evolutionary relationship between species, it was not difficult to find that the GBCXE49 gene and the CXE6, CXE17 genes of Arabidopsis were located in the same group. In addition, the subcellular localization results showed that GBCXE49 was localized in the cytoplasm. We learned that the PpCXE1 gene in peaches, and its subcellular location indicates that it was also present in the cytoplasm [Arabidopsis contained a variety of carboxylesterases with biological activity of foreign organisms (herbicide esters), and it has been verified by tDNA knock-out that the role of AtCXE12 on the bioactivation of herbicides in Arabidopsis was confirmed [Cucurbita moschata was involved in the metabolism of di-n-butyl phthalate (DnBP) [GBCXE49 gene that actively responds to alkaline stress and is highly expressed in roots and leaves, through the VIGS technology to silence it in cotton, it was finally discovered that the gene may be involved in the regulation mechanism of cotton alkaline stress.rocesses , 50, andytoplasm . Arabidoonfirmed . Anothere (DnBP) . We combCXE family genes in four cotton species and analyzed their evolutionary relationship. The gene structure, chromosome distribution, phylogenetic relationship, cis-acting elements, and collinearity relationship of G. barbadense GBCXEs were compared, which greatly enriched our understanding of the cotton CXE gene family. Through the analysis of the cis-acting elements in the promoter of GBCXE genes, it was speculated that GBCXE genes may be involved in stress defense response, abiotic stress and plant hormone signal transduction mechanism. Combined with transcriptome analysis, we selected 6 GBCXE genes for simple tissue-specific analysis, and VIGS silenced the GBCXE49 gene that significantly responded to alkaline stress, and finally determined that GBCXE49 gene may be involved in the alkaline stress response of G. barbadense, which provided a basis for the study of the molecular mechanism of CXE genes in abiotic stress in plants.In summary, we have identified the G. barbadense from Cotton FGD  (http://www.cottonfgd.org/). The Hidden Markov Model (HMM) (version 3.0) configuration file of PF07859 was downloaded from Pfam (https://pfam.xfam.org/). Then, we used the HMMER 3.0 software with default parameters and settings to obtain the CXE gene and protein sequence of Pfam PF07859. This type of gene was likely to belong to the CXE gene family. We used the online websites Pfam (https://pfam.xfam.org/) and SMART (http://smart.emblheidelberg.de/) to analyze the gene structure, then conduct a comprehensive analysis, and further screen to determine the gene family member. Online tools, such as CELLO ver.2.5 (http://cello.life.nctu.edu.tw/) [http://www.cbs.dtu.dk/services/TargetP/), WOLF-PSORT (https://wolfpsort.hgc.jp/)were used to predict the subcellular location information of GBCXEs. We also searched for other features of the G. barbadense CXE gene by using the Cotton Functional Genome Database (CottonFGD). The online website ExPASy-ProtParam (https://web.expasy.org/protparam/) was used to analyze Isoelectric point (pIs), Molecular weight (MWs), exon/intron length, mean hydrophilicity and charge.We download the genome annotation file of edu.tw/) , TargetPCXE genes of different cotton species, similarly, we used the bio-analysis software HMMER 3.0 to obtain the homologous genes of Gossypium hirsutum, Gossypium arboreum, and Gossypium raimondii. The Arabidopsis genome data was obtained from the database TAIR (https://www.arabidopsis.org/), and the AtCXE gene protein sequence was obtained using the Hidden Markov Model Abhydrolase_3 of PF07859 as the screening condition. We used the online tool CD-Search (https://www.ncbi.nlm.nih.gov/Structure/bwrpsb/bwrpsb.cgi) to screen and obtain AtCXE gene members, all the protein sequences were further analyzed in the SMART (http://smart.embl-heidelberg.de/). The protein sequence was named according to its position on the chromosome [GBCXEs intraspecific and interspecific phylogenetic relationship have been constructed in the same way.To study the evolutionary relationship between romosome , alignedCXE family genes of G. barbadense, we used the MEME (http://meme-suite.org/) website to identify the conserved sequence of the protein. The maximum motif parameter of the gene was 10, the rest of the parameters remain unchanged, and the domain files of the GBCXE family genes were obtained accordingly. The domain MAST file, the gene evolution relationship file, and the G. barbadense genome gff3 file downloaded from the MEME website were used to draw the protein domain picture through the TBtools tool.For the G. barbadense reference genome gff3 file and gene ID files were used to map the chromosome positions of family members through TBtools software, the position of CXEs in the chromosomes of G. hirsutum, G. arboreum and G. raimondii was successfully mapped by the same method. The genome sequences of G. hirsutum, G. arboreum, and G. raimondii are all downloaded from Cotton FGD (http://www.cottonfgd.org/).GBCXEs using the software Tbtools. The Plant CARE website (http://bioinformatics.psb.ugent.be/webtools/plantcare/html/) was used to predict the cis-regulatory elements in the promoter region of the gene GBCXEs. Metabolism, plant growth and development related cis-acting elements were screened and analyzed. Similarly, the software TBtools was used for mapping [We used the reference genome and the genome sequence file, and obtained the 2000 bp DNA sequence upstream of the  mapping .G. barbadense under salt  and alkaline  stress were analyzed. The genes that GBCXE family members respond to stress were screened out, and heat maps, phylogenetic trees and cis-acting elements were generated using TBtool software by using the number of fragments per kilobase exon (FPKM). The seeds of G. barbadense alkali-resistant material (Jiza 67) were provided by the Crop Genetics and Breeding Laboratory of Xinjiang Agricultural University, according to the RN38 EASYspin Plus plant RNA rapid extraction kit instructions of Beijing Adelaide Biotechnology, the total RNA of the root, stem, and leaf tissues were extracted separately, according to HiScript® III RT SuperMix for qPCR (+ gDNA wiper) instructions for preparing cDNA. The equipment used for qRT-PCR was Applied Biosystems@ 7500 Fast, and the fluorescence quantification kit was ChamQ Universal SYBR qPCR Master Mix. Primers were listed in supplementary Table SAccording to RNA-seq data, the differentially expressed genes of G. barbadense, G. hirsutum, G. arboreum, G. raimondii). The chromosome length file and the genome alignment filewere used for finally the collinearity result visualization.We the gff file of the target genome was prepared and merged with the TBtool software, then the protein sequence alignment of the genome and then the gene pair files of each two species were obtained . The TBtool software was used to analyze the collinearity of the repetitive gene pairs of 4 cotton species , and then incubated in the dark at 28 °C for 24 h. Ensure the normal growth of seedlings at a temperature (28 14 h/25 10 h).Using the cloning vector Additional file 1:Figure S1. Distribution of CXE genes in four cotton species. Figure S2. A breakdown of the number of CXE genes among species. Table S1. Attached table of physical and chemical properties. Table S2. Primer sequence. Table S3. Tandem repeats and fragment repeats in four cotton species. Table S4. Gene pairs of ten combinatorial. Table S5. Prediction of duplicated gene pairs involved in different combinations from four Gossypium species. Table S6. Statistics of promoter cis-element. Table S7. AlkVSCK_Gene_differential_expression. Table S8. SalVSCK_Gene_differential_expression.


Background: Abdominal obesity, together with atherogenic dyslipidemia, increased blood pressure and impaired glucose tolerance, was included in the components of metabolic syndrome identifying patients at high risk of cardiovascular diseases. Subcutaneous adipose tissue is a buffer for dietary fatty acids (FAs). It was reported that the fatty acid composition of adipose tissue reflects the dietary intake of FAs in the previous 6 to 9 months. Therefore, we decided to evaluate the early and long-term metabolic effects of lipocavitation and/or thermolipolysis on abdominal fat reduction. Material and Methods: The study population comprised 60 women. Subjects were randomly allocated into one of three subgroups, 20 women per subgroup, and each subgroup received 10 treatments with ultrasound (U group), radiofrequency (RF group), or combined radiofrequency and ultrasound (RF/U group) for the abdominal region. Treatments were provided three times a week using the multifunctional device . Each treatment to reduce adipose tissue, regardless of the method used, involved 20 min of massage with the dedicated applicator head on a rectangular 20 cm × 10 cm area of the abdominal region. Fatty acid composition and biochemical and anthropometric parameters were measured before the first, after 10 treatments and 6 months after the last treatment. Results and discussion: The series of 10 treatments to reduce abdominal adipose tissue using ultrasound, radiofrequency or both methods resulted in a cosmetic effect which was reflected in weight loss and BMI reduction. Reduced waist circumference was also found in patients who received radiofrequency or two types of intervention (RF + U) but not ultrasound treatments. The long-term cosmetic effect (lasting for at least 6 months) was achieved only with RF treatment and was reflected in reduced body weight, BMI and waist circumference. None of the treatments had a direct, short or long-term effect on the lipid profile, insulin resistance markers, inflammation markers, or blood pressure. Consequently, did not modify the risk of cardiovascular diseases. Abdominal obesity is a recognised, significant factor increasing the risk of cardiovascular diseases, both through the development of major classical risk factors and through other mechanisms, including dyslipidaemia or insulin resistance, eventually leading to atherosclerosis . TherefoSubcutaneous adipose tissue acts as a buffer for dietary fatty acids (FAs), which are stored and released under the influence of many different factors. It was reported that the fatty acid composition of adipose tissue reflects the dietary intake of FAs in the previous 6 to 9 months . RecentlRecent years have increased the popularity of aesthetic medicine treatments ,7,8,9,10Ultrasound used for body sculpting can be divided into two categories: relatively low-intensity/low-frequency nonthermal ultrasound and high-intensity focused ultrasound (HIFU). Ultrasound can lyse adipocytes through mechanical and thermal mechanisms. With nonthermal ultrasound, a low frequency is chosen to increase the likelihood of cavitation while generating little heat through absorption mechanisms. At lower frequencies, ultrasound can easily cause cavitation, creating holes (cavities) when the ultrasound wave has sufficient negative pressure to overcome the adhesion of the medium molecules to each other, causing cell death ,11. Low-Used for body sculpting, HIFU delivers focused, high-intensity ultrasonic energy to deep subcutaneous tissue, producing heat capable of ablating adipose tissue and thermally modifying collagen . ExposurAnother non-invasive method, radiofrequency, generates heat in different tissues by transforming energy through three basic mechanisms from an electromagnetic field. These mechanisms include (i) orientation of electric dipoles that already exist in the atoms and molecules in the tissue; (ii) polarisation of atoms and molecules to produce dipole moments; and (iii) displacement of conduction electrons and ions in the tissue . RadiofrThere are no numerous reports in the literature presenting the effects of non-invasive techniques for adipose tissue reduction on metabolic parameters significantly contributing to the pathogenesis of atherosclerosis. Therefore, we decided to evaluate the early and long-term metabolic effects of lipocavitation and/or thermolipolysis on abdominal fat reduction. 2 spot area, generating ultrasound with 1 MHz frequency and 1.4 W/cm2 intensity  as not normally distributed. The Shapiro–Wilk test was used to check if a continuous variable The baseline clinical, anthropometric, and laboratory characteristics of the study population, along with the short and long-term effects of all interventions applied for adipose tissue reduction, are presented in There were no significant differences between baseline anthropometric and biochemical measurements in the U, RF, and RF/U subgroups. When considering the effect of individual interventions to reduce adipose tissue on the analysed parameters, significant differences were found between treatments. However, they were limited to body weight, waist circumference, and BMI. All treatments resulted in a reduction in body weight and BMI after 10 treatments, but the long-term effect evaluated 6 months after the last treatment was only found in the RF subgroup. None of the methods used alone resulted in short- and/or long-term hip circumference reduction. Short-term reduction in waist circumference was found for the RF and RF/U interventions, but not for U. Long-term reduction in waist circumference assessed 6 months after treatment was only achieved for the RF intervention . n-6, and decreased levels of C10:0 were found. In the U subgroup, reduced levels of C:16, C18:1w9ct, C18:1trans1, C18:2n6c, and C20:4n6 were noted , there was no change in the concentration of free FAs one and 4 h after the procedure, nor after 10 treatments. RF and U treatments, but not RF/U, had a significant long-term effect on the levels of some FAs measured 6 months after the completion of treatments. In the RF subgroup, increased levels of C:15, C:18, C18:3re noted . A series of 10 treatments to reduce abdominal adipose tissue using ultrasound, radiofrequency, or both methods resulted in a cosmetic effect reflected in weight loss and BMI reduction. Reduction in waist circumference was also found in patients who received radiofrequency or two types of intervention (RF + U), but not ultrasound treatments.A long-term cosmetic effect (lasting for at least 6 months) was achieved only with RF treatment and was reflected in reduced body weight, BMI, and waist circumference.None of the treatments had a direct, short-, or long-term effect on the lipid profile, insulin resistance markers, inflammation markers, or blood pressure.Adipose tissue is a unique tissue adapted to lipid storage and involved in immune, nervous and endocrine functions . AdiposeDetermination of fatty acid composition in adipose tissue and blood has been recognised as the gold standard for evaluating the quality of FAs consumed by individuals who do not experience sudden changes in their body weight. It was reported that the fatty acid composition of adipose tissue reflects the dietary intake of FAs in the previous 6 to 9 months . AdditioExcessive body weight is becoming one of the major global health care problems. It contributes to increased morbidity and mortality and generates high costs of health care . Therefo2 indicates overweight, and values higher than 30 kg/m2 indicate obesity. However, BMI does not provide information on the location of adipose tissue in the body [Adipose tissue in a healthy woman should account for 20–25% of body mass. Obesity in adults is diagnosed when the content of fat in the body, regardless of age, exceeds 25% of normal body weight in men and 30% in women. The content of adipose tissue is 14–28% in women with normal body weight, 29–32% in overweight women, and more than 32% in those with obesity. The state of overweight and obesity is determined by the Body Mass Index (BMI), calculated as the quotient of body weight (in kilograms) and the square of height expressed in meters. For the adult population, normal BMI is in the range of 18.5–24.9, 25–29.9 kg/m2), the waist circumference (81.9 cm) showed a tendency to accumulate subcutaneous abdominal fat. In addition, the mean HOMA-IR index was 2.2  and sugsistance .It seems that the women who received treatments in our study began to pay attention to their diet, which was confirmed by the changes in the content of individual FAs in the blood. Our findings suggest that especially women who received ultrasound treatments reduced their daily fat intake, which was reflected in a significantly reduced concentration of the following FAs in the blood 6 months after the first treatment: C10:0 (14.2 vs. 5.4), C14:0 (27.3 vs. 14.5), C18:1w9ct (393 vs. 155.8), C18:1trans11 (55.3 vs. 34.9), C18:2n6c (523 vs. 195.4), C20:4n6 (147.8 vs. 53). Similar changes in the RF group were found only for C12: 0 (124.1 vs. 64.9). In the RF group, there was a significant decrease in the content of stearic acid in the blood after the last treatment (143.7 vs. 126.4), as well as an increase in its content after 6 months to the baseline value (143.7 vs. 204.3). This suggests that once patients achieved a satisfactory outcome after 10 treatments (reduction of the waist circumference from 82.4 to 80.9 cm and body weight from 73.2 to 72.4 kg), they liberalised their approach to a healthy diet. Consequently, six months after the completion of the study, the waist circumference of women from the RF group increased to 81.1 cm. That is why it is so important for specialists who offer cosmetology treatments to make their patients aware that maintaining reduced body weight and reducing the risk of developing the components of metabolic syndrome is possible only through a permanent change of diet. Cosmetological treatments can only serve as an addition to a healthy lifestyle, improve skin firmness, and sculpt the problematic parts of the body .The content of adipose tissue can be precisely measured, for example, by densitometry or magnetic resonance ,30,31. HIn our study, we focused on two treatments, i.e., lipocavitation and thermolipolysis, which are used for body contouring, so they can mainly affect subcutaneous fat content in a selected body area.In cosmetology, ultrasound is used, for example, for body sculpting and fat reduction treatments known as non-invasive liposuction ,7,8,9,10Ultrasounds can ablate the adipose tissue by its nonthermal and thermal effects. Nonthermal-focused ultrasound applied for body sculpting uses mechanical stress generated from inertial cavitation to disrupt adipose tissue ,11. The 2. Lipocavitation can also be stimulated by intensities only slightly higher than 1 W/cm2 but with an adequately longer exposure time. This procedure damages adipocytes and causes the release of fat into the extracellular compartment. It is presumed that triglycerides released in this way are transported via vessels of the lymphatic system to the liver and are metabolised further [According to some researchers, ultrasound induces lipocavitation with a frequency of 1 MHz and an intensity of 2 W/cm further ,9,11,32.2. It can be assumed that the unfocused ultrasound used in the present study had a weaker effect on the lipocavitation process, which could have resulted in a slight reduction in the values of the analysed parameters. This is in agreement with the results of other authors [Treatments with ultrasound alone had a less pronounced effect on reducing the values of the analysed parameters compared to the other two modalities. In our study, it might be related to unfocused ultrasound with a frequency of 1 MHz and intensity of 1.4 W/cm authors .On the other hand, electromagnetic (radiofrequency) waves with a frequency of 1 MHz and a power of 100 W (bipolar method) used in this study showed a stronger effect on adipose tissue compared to that produced by ultrasound. Endogenous heat is produced in tissues exposed to an electromagnetic field. The local increase in skin temperature results in the destruction of some cross-links between collagen fibres. Collagen is partially denatured, but more thermostable chemical bonds are unaffected. Collagen fibres shorten, which is clinically manifested by improved skin firmness ,9,11. PaOne study subgroup received combined radiofrequency and ultrasound treatments. First, massage was performed with a bipolar applicator head that generates electromagnetic (radiofrequency) waves. The heat generation in the tissue exposed to radiofrequency waves probably created optimal conditions for ultrasound waves by enhancing their effect. It could also have had a more favourable effect on lowering the values of the analysed parameters compared to one type of treatment alone. Radiofrequency waves cause thermolipolysis and boost metabolism in adipocytes ,9. On thOur study evaluating the effects of electromagnetic (radiofrequency) waves showed their significant impact on the body weight and BMI reduction of the studied women, contrary to some previously reported findings .In some clinical trials, 6–10 radiofrequency treatments have produced 2- to 3.5 cm reductions in waist and thigh circumference. However, newer devices are more effective; in one study, a reduction in thigh circumference of 2.43 cm after only four weekly treatments was observed . HIFU anThe non-invasive physical methods used in our study can be applied primarily to prevent the excessive deposition of adipose tissue, as well as to support the treatment of overweight and obesity. However, they cannot be the basic method for reducing the volume of deposited adipose tissue, regardless of its location. Importantly, the combination of radiofrequency and ultrasound affected adipose tissue, resulting in a partial reduction of fat content, as well as improved skin elasticity. Other authors also revealed that some of the non-invasive body contouring devices in animal and human studies, such as cryolipolysis, RF, and HIFU, showed statistically significant effects on body contouring, removing unwanted fat in some body areas. However, the clinical effects are mild to moderate, for example, 2–4 cm circumference reduction as a sign of subcutaneous fat reduction during total treatment sessions . Importantly, all the treatments mentioned in this study, because of the use of lower energy levels, can be performed by cosmetologists. The legal aspects of procedures performed by cosmetologists, ultrasound and radiofrequency treatments included, are at present not regulated in local legislation. Theoretical and practical training on these treatments and an evaluation of risk/benefits for each patient are necessary to perform these procedures effectively and safely.An important informational and educational issue, which should be considered in beauty salons offering non-invasive physical treatments, is making their patients aware that long-term reduction of adipose tissue in individuals with overweight or obesity requires a change in lifestyle, including an appropriate diet and modification of energy expenditure by physical activity . EvidencThe study design did not include a control group. It is likely that the inclusion of individuals who did not undergo any abdominal adipose tissue intervention might have elucidated whether observed blood FAs changes were due to the treatment or caused by other interventions such as changes in dietary habits.Treatments to reduce abdominal adipose tissue with ultrasound, radiofrequency, or both techniques provided a desired cosmetic effect. RF treatment was the most efficient modality since it produced a long-term cosmetic effect (lasting for at least 6 months), reflected in reduced body weight, BMI, and waist circumference. However, many biochemical cardiovascular risk factors remained intact. In fact, it is too early to determine whether a total cardiovascular risk has been modified due to a relatively short follow-up.The techniques used in our study did not increase the content of pro-inflammatory free fatty acids in the blood (such as arachidonic acid), which suggests the safety of these treatments.


We report the first case of a patient with large pericardial cyst occurring concurrently with a very large thoracic aortic aneurysm. There have been no patients reported in the literature with this constellation of syndromes. Additionally, this case was followed through a period of 4 years, enabling the natural evolution of both pathologies to be visualised. A 76-year-old female presented to the Emergency Department in December 2019 with a 1-day history of chest and epigastric pain which became more severe during a routine dental check-up. She was an ex-smoker and had known hypertension, peripheral artery disease, and previous non-ST elevation MI.Previous chest X-ray in March 2015 had shown a widened mediastinum with localised well-demarcated soft tissue opacity over the right heart border, suggestive of aortic dilatation and pericardial cyst, respectively . PreviouThis admission, chest X-ray showed increased soft tissue opacity and increased mediastinal widening d. CardiaShe underwent urgent repair of ascending aorta and hemiarch, aortic valve replacement using a size 21 mm perimount valve, along with vein graft to left circumflex artery. In cardiac intensive care unit, she made a slow recovery, was successfully weaned off the ventilator, and remains well at follow up.1 The diameter of most pericardial cysts is usually between 1 and 5 cm. Large pericardial cysts are rare, with only data from 12 cases available from 2000 to 2014 in PubMed. Most are asymptomatic and are found incidentally on radiological imaging. However, they can be associated with compression of adjacent structures, leading to chest pain and breathlessness, and more serious complications such as cardiac tamponade and sudden death.2 The pericardial cyst in our patient appears to be congenital and grew in size over years.Pericardial cysts are rare benign intrathoracic lesions, with an incidence of 1 in 100,000.3 In total, 60% of thoracic aortic aneurysms involve annuloaortic ectasia of the aortic root or the ascending aorta, such as in this case. The giant aneurysm in our patient was due to hypertension along with atherosclerosis; there was no evidence of syphilis, connective tissue disorder or other predisposing factors.Giant thoracic aortic aneurysms are less benign, often from cystic degeneration of the media, leading to weakening of the vessel wall, aortic dilation and aneurysm formation.4 The risk of rupture is exponentially linked to diameter. Generally, the literature supports considering intervention in aneurysms with a diameter of 4-5.5 cm.5 Due to this, our patient underwent urgent elective cardiothoracic repair of the aortic root, and she was fortunate not to have rupture or dissection at this massive size.Thoracic aneurysms are frequently asymptomatic and discovered incidentally on imaging. However, they can present with mass effects, aortic regurgitation if at the aortic root, and lead to life-threatening complications such as dissection or rupture. Aneurysms greater than 6 cm in diameter have a rupture or dissection rate of 6.9% per year and a death rate of 11.8%.


Building ribosomes for cellular protein translation is a massive, energy-consuming undertaking. The process is executed by all cells to replenish the relevant pools of proteins required for cellular functions. Cancer cells are strikingly dependent on this activity, as they need continuous protein synthesis for sustained proliferation and growth. Ribosome biogenesis requires the activities of three RNA polymerases, which transcribe essential RNAs that make up the backbone of the ribosome, and hundreds of proteins, which provide structural and functional support. Of the three RNA polymerases, RNA polymerase I executes a critical and rate-limiting step by transcribing three key ribosomal RNAs. Pol I transcription is pervasively deregulated in cancers, enabling unlimited protein synthesis. Here, we review this enzyme and provide examples of current efforts to target Pol I transcription therapeutically.RNA polymerase I is a highly processive enzyme with fast initiation and elongation rates. The structure of Pol I, with its in-built RNA cleavage ability and incorporation of subunits homologous to transcription factors, enables it to quickly and efficiently synthesize the enormous amount of rRNA required for ribosome biogenesis. Each step of Pol I transcription is carefully controlled. However, cancers have highjacked these control points to switch the enzyme, and its transcription, on permanently. While this provides an exceptional benefit to cancer cells, it also creates a potential cancer therapeutic vulnerability. We review the current research on the regulation of Pol I transcription, and we discuss chemical biology efforts to develop new targeted agents against this process. Lastly, we highlight challenges that have arisen from the introduction of agents with promiscuous mechanisms of action and provide examples of agents with specificity and selectivity against Pol I. Ribosome biogenesis is a complex and metabolically costly process involving the activities of three cellular RNA polymerases, their RNA products, and hundreds of proteins ,2,3,4,5.The ribosome is a ribonucleoprotein complex responsible for translating mRNA into proteins ,5. The eIn 1969, Roeder and Rutter made the seminal discovery that eukaryotes possess three different DNA-dependent RNA polymerases (Pols), in contrast to the single RNA polymerase found in prokaryotes . Over thThe nucleolus forms around the active sites of rRNA gene transcription, which are defined by the multicopy rRNA gene clusters termed as nucleolar organizing regions (NORs) . Human NXenopus, where they were first discovered and are best studied, the enhancers influence the number of activated promoters, but are not absolutely required for basal transcription or polymerase loading [cis and inhibit unlinked promoters in trans, but related regulatory factors remain to be identified. We do not know the relevance of these elements and whether they regulate transcription in humans. The spacer promoter is separated from the functional promoter by transcription termination recognition sequences essentially blocking read-through transcription [The ribosomal DNA (rDNA) gene repeats are arranged head-to-tail ,18,23 FA. Each r loading ,26,27,28cription . In micecription ,30.The first, rate-limiting step of ribosome biogenesis is the transcription of rDNA by Pol I into a 47S rRNA precursor ,31,32. TWhile Pol I machinery is predominantly localized in the fibrillar center of the nucleolus, Pol I transcription occurs on the border of the fibrillar center and dense fibrillar component . The PolSaccharomyces cerevisiae. The nomenclature for yeast and human subunits is different and has evolved over time. We refer the reader to a recent review for the naming convention [Structural studies of Pol I have detailed the enzyme and the preinitiation complex in the various steps in the transcription cycle, namely, preinitiation, initiation, and elongation  and with Pol III AC40, AC19), which provide mostly structural support for the core assembly [reviewed in ,39. The , AC19, wPol I contains four peripheral subunits unique to this enzyme: A43 (human RPA43), A14, A49, and A34.5. The A43-A14 heterodimer forms the stalk, a structure that protrudes from the core. Preinitiation factor Rrn3 (human RRN3) binds to the A43-A14 stalk and recruits the Rrn3-Pol I complex to the rDNA promoter ,45,46,47Within the past year, two groups have elucidated the structure of human Pol I for the first time ,51 Figu. OverallPol I is a highly efficient RNA polymerase that has evolved for the transcription of its long 13 kb transcript. The structure of Pol I, with its in-built RNA cleavage ability and incorporation of subunits homologous to transcription factors, enables it to quickly and efficiently synthesize the enormous amount of rRNA required for ribosome biogenesis ,53. ThisInitiation. The 140-160-base pair rRNA gene promoter contains two key elements, a core element and the UCE [I110, TAFI48, TAFI63, TAFI12, and TAFI41 [I63 and TAFI110 interact with RRN3 on the RRN3-Pol I complex and recruit Pol I to the promoter. Together, these proteins form the PIC. This process is relatively conserved in yeast, as yeast has Rrn3, TBP, and a core complex homologous to SL-1 [ the UCE ,31. Bind the UCE ,56. Whild TAFI41 ,31. RRN3d TAFI41 . Similard TAFI41 ,46,47,51 to SL-1 ,45,46,47 to SL-1 .The successful assembly of the PIC bends the promoter to an angle, remarkably different from that of the Pol II promoter, favorable for transcription initiation. Pol I stutters on the first few nucleotides, during which the DNA duplex melts to favor the formation of the transcription bubble and promoter escape. This process is further assisted by A49 and A12.2, which support the binding of Pol I to the promoter and the formation of the open elongation complex, respectively ,43. Pol Elongation. In the active site, two magnesium cations in the catalytic aspartate triad coordinate an NTP condensation reaction. During this reaction, one nucleoside monophosphate (NMP) is added to the nascent RNA strand as pyrophosphate is released. Pol I then translocates down the DNA template by one nucleotide [cleotide . This trcleotide . As eachcleotide . Pol I ncleotide . The meccleotide ,60,61,62Pausing and Backtracking. Given the ratchet motion during elongation, the enzyme is prone to backtracking. Furthermore, if the polymerase encounters an obstacle, such as DNA damage or an incorrect nucleotide, it will pause. The polymerase will then backtrack. Since the “gating tyrosine” in the active site remains “open”, the polymerase can backtrack as much as it needs to. The C-terminus of A12.2 inserts into the active site and cleaves the RNA [ the RNA ,64. This the RNA . Ineffic the RNA , and the the RNA .Termination. Transcription termination elements are positioned on two separate sites on the rDNA gene repeat: at the 3′ end of the transcribed region and upstream of the transcription start site. Transcription termination factor I (TTF-I) binds to the termination element at the 3′ end of the transcribed region, bends the DNA, and triggers Pol I to pause [to pause . Howeverto pause . Given tto pause ,68.Pol I Complex Stability. Pol I processivity is facilitated by enzyme stability. However, the mechanistic understanding of which factors govern mammalian Pol I assembly, stability, and localization is incomplete. In yeast, the interaction between A190 and A135 is stable [s stable , and in s stable .In yeast, A49 stabilizes the expression of A34.5 and is mediated by a protease-sensitive linker domain. Conversely, depletion of A34.5 also destabilizes A49, suggesting that this complex is inherently unstable if either subunit is missing . In contExternal factors, such as zinc availability and temperature, also mediate Pol I stability. Zinc depletion has been shown to induce vacuolar proteolysis of Pol I in yeast . Cold te1 phase, and resumes full capacity during S phase and G2 phase [Pol I transcription is regulated by metabolic states and environmental factors, such as cell growth, nutrient availability, stress, and the cell cycle [reviewed in ,7,8,9. CG2 phase . There aG2 phase ,83.PIC factors are targets of post-translational modifications. These regulatory events affect UBF, SL-1, and RRN3 ,84, and I110 during mitosis prevents SL-1 from interacting with UBF and prevents transcription initiation [1 phase. Both SL-1 and UBF are fully active during S phase and G2 phase [Cell cycle-dependent transcription of Pol I is also achieved by post-translational modifications of the transcription factors. The phosphorylation of SL-1 subunit TAFitiation . SL-1 isG2 phase ,93.Strikingly, Pol I transcription activity and ribosome biogenesis are also under the control of the circadian clock ,95. TranChanging the number of active (versus silent) rRNA genes at an rDNA repeat is regulated by epigenetic mechanisms. The rDNA of actively transcribed genes exists in a nucleosome-deplete, “open” euchromatin configuration that is characterized by DNA hypomethylation, H4ac, and H3K4me2 . In Approximately half of the rRNA genes are maintained in an active state. However, the number of active rRNA genes differs based on cell type, suggesting that the number of active genes is passed down through cell lineages during development and differentiation . This inOf all genes, the rRNA genes are the most heavily transcribed. Given the high transcriptional activity and the speed of the polymerase on the long 13 kb transcript, this polymerase is particularly sensitive to transcriptional stresses . A surroRNA polymerases encounter obstacles generated by altered chromatin conformation or DNA adducts, such as those caused by DNA damage and UV irradiation, that stall or arrest transcription. Each polymerase has its own error rate, with Pol II having the highest fidelity, closely matched by Pol I, while Pol III error rates are the highest . To resoThe largest subunit of yeast Pol III, C160, is also ubiquitinated and degraded by the proteasome upon transcription stalling. To induce cell stress, Lesniewska et al.  treated Pol I transcription is a critical step in ribosome biogenesis and protein translation. As such, it is essential, and proportional, to cancer cell growth and a rate-limiting factor for cancer cell proliferation ,12. OverStaining of the NOR using silver, which recognizes both nucleolar proteins and RNA, is termed as argyrophilic nucleolar organizing region (AgNOR) staining, and it has been used extensively to assess tumor specimens ,131. TheMore recently, we introduced specific detection of the 47S rRNA precursor using chromogenic in situ hybridization for visualization and quantification of Pol I transcription activity in human tissue samples . Using tSeveral studies have used surrogate markers for Pol I activity, such as the expression of rRNA methyltransferase fibrillarin (FBL) ,137 and Cancer cells employ several different mechanisms to increase Pol I transcription and ribosome biogenesis ,142,143.mTOR drives multiple steps in ribosome biogenesis and regulates both transcription of rRNA and mRNA synthesis of ribosomal proteins  and cyclins are also deregulated in many cancers due to their mutations, amplifications, and uncontrolled activation. Their deregulation leads also to interference of Pol I transcription by phosphorylation of SL-1 and UBF to maintain constitutively high Pol I transcription rates ,143,165 Additionally, epigenetic modification of the rDNA promoter may contribute to the high Pol I transcription activity in cancer cells. For example, hepatocellular carcinomas are characterized by hypomethylation of the rDNA promoter, leading to a persistently “open” euchromatin state and upregulation of Pol I transcription . rDNA hyI110 to prevent their interaction and productive initiation [Schizosaccharomyces pombe, Dicer and other RNA interference mutants are inviable in a manner dependent on Pol I transcription [Several tumor suppressors, which are mutated and inactivated in cancers, control and attenuate Pol I transcription activity ,9. The Ritiation . PTEN diitiation . BRCA1, itiation . It has itiation . Nucleolitiation . Furtheritiation . These aitiation . Cellulaitiation ,92. MYBBitiation . RUNX2 titiation ,176. DICcription . Deletiocription . The ovecription .Given the essential role of ribosome biogenesis in cancer cell growth and the activated state of Pol I in many cancers, Pol I is a rational target for cancer therapeutics. The high DNA metabolic activity due to the activated transcriptional rates creates unique vulnerabilities at this genetic locus, especially to events that cause transcriptional impediments. Enzyme stability is required for effective transcription elongation. This is especially critical for Pol I, given the high initiation rates and fast processivity over the long precursor transcript. Furthermore, the polymerase density, analyzed in detail in yeast, is very high and renders this enzyme highly vulnerable to perturbations caused by lesions or conformational impediments. In fact, several classes of chemotherapeutic drugs—including alkylating agents, anti-metabolites, antibiotics, and topoisomerase inhibitors—disrupt Pol I transcription . Hence, Alkylating agents  attach alkyl groups to DNA to form DNA adducts. These adducts trigger a DNA damage response and induce cell cycle arrest and cell death . CisplatAnti-metabolites  mimic cellular metabolites, interfering with enzymatic processes and incorporating into DNA and RNA and interfere with rRNA processing . MethotrChemotherapeutic agents in the class of antibiotics, such as Actinomycin D, intercalate with DNA and induce DNA damage ,185,186.Topoisomerases (TOP) are enzymes that are required to resolve DNA supercoiling and facilitate processes such as transcription, replication, and recombination ,188. TOPG quadruplexes are unusual four-stranded secondary DNA structures that are formed by G-G base pairs via Hoogsteen hydrogen bonding . These sWe have summarized these drugs in It is evident that many challenges remain in the quest to develop effective Pol I inhibitors. Most of the current drugs induce nonspecific, widespread DNA damage. It would be optimal to test drugs that target Pol I transcription directly. Yet, the mechanisms that govern the stability and regulation of the Pol I enzyme itself remain unclear, making it an elusive enzyme to target. While rRNA synthesis is upregulated in many types of cancer, it is also essential for normal cells. An effective cancer therapeutic must therefore selectively target Pol I transcription to exploit cancer cell vulnerabilities without affecting normal cells. Currently there are only few pharmacological tools that qualify as specific and selective Pol I inhibitors for this purpose.We recently discovered a first-in-class small molecule, BMH-21, that specifically and selectively blocks Pol I transcription ,210. BMHBMH-21 blocks three critical steps in the transcription cycle, namely initiation, promoter escape, and elongation ,218 Fig. BMH-21 RPA1 degradation is prominently observed in cancer cells and is associated with BMH-21-induced cancer cell death . The degFBXL14 as an E3 ligase involved in the BMH-21-induced degradation of RPA1 [By conducting an unbiased RNAi screen against ubiquitome proteins, we recently identified SCF of RPA1 . We show of RPA1 . However of RPA1 . We infeThe discovery of BMH-21 is exciting for both fundamental and translational reasons. The compound induces the proteasome-mediated degradation of the Pol I catalytic subunit, analogous to prior studies of the proteasome-mediated degradation of the large subunits of Pols II and III. Prior studies focused on the regulation of Pol I activity through the modification of its transcription factors and the rDNA, but these results show that the Pol I enzyme itself can also be regulated through the degradation of its catalytic subunit. This finding thus provides essential knowledge about the stability and regulation of the Pol I complex relevant for the mechanistic understanding of the enzyme function. BMH-21 is the first compound to specifically and selectively inhibit Pol I without having off-target effects, such as those by other chemotherapeutic drugs. This class of small molecules thus has very promising potential in the clinic, and preclinical studies are currently underway on BMH-21 analogs.Pol I is responsible for the first and rate-limiting step of ribosome biogenesis—the transcription of rDNA into a 47S rRNA precursor. To match their proliferative and biosynthetic activities, cancer cells possess abnormally high rates of ribosome biogenesis. Since major cancer drivers constitutively activate Pol I transcription, Pol I is a rational target for cancer therapeutics. Current studies focus on the regulation of Pol I activity through the modification of its transcription factors and the rDNA, but very little is known about the stability and regulation of the enzyme itself. The finding that the Pol I complex can be regulated through the degradation of its catalytic subunit provides fundamental knowledge about the stability and regulation of the enzyme relevant for the mechanistic understanding of the enzyme function and the development of Pol I—targeting agents. Preclinical studies have detailed the precise mechanisms of how the BMH-21 class of small molecules inhibits Pol I transcription. Development of these molecules for clinical testing is ongoing. These findings open up a new path for the discovery of other molecules that take advantage of this new regulatory mechanisms.


Primary intrahepatic rhabdomyosarcoma is an extremely rare malignant tumor. Here, we describe a case of embryonal rhabdomyosarcoma of the liver in a 7-year-old boy without any symptoms. Serologically, the patient showed abnormal levels of serum tumor markers and liver function. Imaging revealed a large mass in the left lobe of the liver and no lesions elsewhere. At first, the patient was misdiagnosed by percutaneous liver biopsy as having clear cell sarcoma. However, the final diagnosis was established to be hepatic embryonal rhabdomyosarcoma based on postoperative histopathology and typical immunohistochemical staining, which was positive for desmin and myogenin. For treatment, the patient received two cycles of preoperative chemotherapy, prophylactic radiotherapy, and 13 cycles of combined postoperative chemotherapy. Routine follow-ups after all treatment conducted by imaging examinations showed no sign of recurrence or metastasis over 13 months, and the patient survives more than 38 months since initial diagnosis. To our knowledge, this patient is the first with hepatic rhabdomyosarcoma to receive neoadjuvant chemotherapy (preoperative chemotherapy) combined with relative comprehensive treatment and achieve a favorable result. Rhabdomyosarcoma is the most common soft-tissue sarcoma in children and adolescents, accounting for 3% of all pediatric tumors . HoweverA 7-year-old boy was admitted to our hospital to examine an abdominal mass. He had no obvious clinical symptoms such as abdominal pain or weight loss. In addition, he had no significant personal or family medical history except that he was born premature. Physical examination revealed a mass (~9 × 8 cm) in the upper abdomen that was firm with poor mobility and no obvious tenderness. Laboratory test results showed normal levels of white blood cells, hemoglobin, platelets, and transaminase. However, the following levels were elevated: alkaline phosphatase, 244.1 U/L (range: 45–125 U/L); lactase dehydrogenase, 482 U/L (range: 120–250 U/L); C-reactive protein, 34.4 mg/L (range: 0–3.5 mg/L). In addition, the virus markers of hepatitis B and C were negative, and serum tumor markers showed normal levels of alpha-fetoprotein, carcinoembryonic antigen, and CA19-9. However, neuron-specific enolase was 30.45 ng/mL (range: 0–16.3 ng/mL), and CA72-4 was 7.94 U/mL (range: 0–6.9 U/mL). The rest of the laboratory tests were normal.An ultrasound revealed a solid mass (83.3 × 79.4 × 106 mm) in the front of the upper abdomen spine, which is located behind the lesser curvature of the stomach, and the boundary with the left lobe of the liver was still clear. Abdomen contrast-enhanced computed tomography scans showed a mass-like high-density shadow (7.5 × 7.3 × 7.7 cm) in the left lobe of the liver, next to the lesser curvature of the stomach . SubsequHe underwent percutaneous liver biopsy, and histopathological examination of the tumor cells displayed large and small round cells, haphazardly oriented, containing variable amount of cytoplasm and prominent nucleolus . ImmunohThe patient received two cycles neoadjuvant chemotherapy: 1.55 mg vincristine , 1,500 mg ifosfamide (given on days 1–5 in a 21-day cycle), 100 mg etoposide (given on days 1–3 in a 21-day cycle), and responded well, including decreased tumor volume, so he was able to undergo surgery. Intraoperatively, a tumor was confirmed occupying the caudate lobe of the liver, rather than the left lobe of the liver as the abdominal computed tomography scan showed, and no infiltration of the surrounding organs and the cutting edge was identified. The gross specimen revealed a well-defined tenacious texture mass , the following postsurgical chemotherapy regimen was initiated: 1.55 mg vincristine , 1,800 mg ifosfamide (given on days 1–5 in a 21-day cycle), and 100 mg etoposide (given on days 1–5 in a 21-day cycle). Chemotherapy was repeated every 3 weeks, and this therapy was well-tolerated without severe side effects. After three cycles, prophylactic radiotherapy in the liver tumor bed was started to obtain long-term tumor-free survival, and the total radiation therapy dose was 5040 Gy. After this therapy, he received another 10 cycles of chemotherapy: the first three cycles matched the previous postsurgical regimen; from the fourth cycle and on, the regimen changed to 1.55 mg vincristine , cyclophosphamide 1,300 mg (given only on day 1), and 475 μg actinomycin D (given on days 1–5 in a 21-day cycle).To conclude, the patient was totally recievied 15 cycles of chemotherapy, including two cycles neoadjuvant chemotherapy and 13 cycles postsurgical chemotherapy. The overall treatment time which include surgical treatment, radiotherapy and chemotherapy was nearly 15 months.Although it was difficult to avoid infection or varying degrees of bone marrow suppression during chemotherapy, no serious life-threatening side effects occurred, and the life quality was guaranteed. Regarding the prognosis, the patient survives for 38 months since first being accepted to the hospital, and to date, no recurrence or metastasis has been found by abdominal computed tomography or abdominal ultrasound after treatment .Primary rhabdomyosarcoma of the liver is extremely rare. According to these reports, 11 cases presented clinical onsets with varying degrees of epigastric pain; three, with fever; two, with abdominal distension, two, with fatigue; and two, with shortness of breath. Imaging usually showed lesions in the right liver lobe. In addition, serological test results often indicated levels only slightly outside the normal range, with an occasional abnormal tumor marker. However, our patient had no clinical symptoms, and imaging revealed a mass on the caudate lobe of the liver, with an unclear boundary between the liver and pancreas. In addition, serum tumor markers showed abnormal levels of neuron-specific enolase and CA72-4. No reports to date have described the typical clinical symptoms and imaging findings of rhabdomyosarcoma, so the pathologic findings are regarded as the gold standard for diagnosis. For prognosis, the mean survival time of patients with hepatic rhabdomyosarcoma was 9.25 months (time span of 2–31 months) since initial diagnosis or symptoms. Notably, our patient was the only one alive for sure and had the longest disease-free survival by comprehensive treatment.In a comparison analysis among reported previously cases, we calculated a misdiagnosis rate of 40% for patients with hepatic rhabdomyosarcoma. There are three main reasons for misdiagnosis of these patients. First, clinical manifestation can be highly variable which lack of typical clinical representations. Most symptoms depend on the location of the primary tumor, the presence or absence of metastases, and whether any complications developed. Second, the liver as a rare location of rhabdomyosarcoma has an insidious onset, make it easy to misdiagnose. Third, imaging findings of rhabdomyosarcoma have little diagnostic significance. However, they can provide useful assessment of the primary tumor, its size, and local and distant spread  and can Quite specially, our case is the only one misdiagnosed by a pathologic diagnosis rather than imaging findings or clinical symptoms despite the liver histopathological examination was seen as a gold standard in diagnosis of RMS. The reason might be pathological tissue collected by needle biopsy was too limited or contained too much necrosis material and it is hard for pathologists to effectively view the lesions and select the targeted protein for immunohistochemical staining. This episode highlights an important lesson: for disease diagnosis, we strongly suggest incisional biopsy rather than needle biopsy to ensure the retrieval of complete tissue for pathological diagnosis . If needRegarding treatment and prognosis, our patient was the only one who received the most comprehensive treatment, had the longest survival since diagnosis, and achieved a favorable curative effect. As Malempati and Hawkin , 18 suggAmong the cases reported, we grouped the cases by the diameter of tumor, which set 10 cm as a critical value. A total of eight cases were reported with explicit tumor size and survival state, including five cases with the largest tumor diameter of 10 cm or larger and three cases with a tumor diameter of <10 cm. We calculated and compared the overall survival time  for eachIn addition, we analyzed the cases according to different treatment regimens. The results indicated that despite the value of surgical care and the sensitivity of rhabdomyosarcoma to chemotherapy and radiotherapy, a single therapeutic strategy often fails to achieve the expected result regardless of the size of the tumor. Multidisciplinary comprehensive treatment can significantly prolong survival time . Among tRhabdomyosarcoma, a malignant tumor with striated muscle differentiation, rarely occurs in liver. Here, we summarized all published hepatic rhabdomyosarcoma cases and reported a patient with the longest survival time for this cancer type with no recurrence or metastasis. This case report suggests incisional biopsy as the top preference in diagnosis, larger diameter puncture needle biopsy or repeat puncture biopsy is second preference. In terms of remedy, a comprehensive treatment based on surgery and chemotherapy have better effectiveness, and we strongly suggest that surgery should be carried out whenever resection is possible.The original contributions presented in the study are included in the article/Ethical review and approval was not required for the study of human participants in accordance with the local legislation and institutional requirements. Written informed consent was obtained from the patients' parents before clinical samples were collected. The patient and their parents were informed of the test results. Written informed consent was obtained from the patients' parents for the publication of any potentially identifiable images or data included in this article.XuL and XiL developed the original idea, reviewed the literature, and wrote the manuscript. D-FY was the patient's doctor in charge and was responsible for collecting medical history. ML analyzed the histological characteristics of all pathologic sections. H-QX was responsible for collecting medical history and analyzing the statistical issues involved in this case. SZ was responsible for analyzing the imaging data. P-JG reviewed the literature and contributed to manuscript revision. All authors have read and approved the manuscript.This study was supported by the Science and Technology Development Program of Jilin Province; Grant Number: 20190103079JH and Youth Development Foundation of the First Hospital of Jilin University; Grant Number: JDYY102019004.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.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Background: It is important to be able to predict, for each individual patient, the likelihood of later metastatic occurrence, because the prediction can guide treatment plans tailored to a specific patient to prevent metastasis and to help avoid under-treatment or over-treatment. Deep neural network (DNN) learning, commonly referred to as deep learning, has become popular due to its success in image detection and prediction, but questions such as whether deep learning outperforms other machine learning methods when using non-image clinical data remain unanswered. Grid search has been introduced to deep learning hyperparameter tuning for the purpose of improving its prediction performance, but the effect of grid search on other machine learning methods are under-studied. In this research, we take the empirical approach to study the performance of deep learning and other machine learning methods when using non-image clinical data to predict the occurrence of breast cancer metastasis (BCM) 5, 10, or 15 years after the initial treatment. We developed prediction models using the deep feedforward neural network (DFNN) methods, as well as models using nine other machine learning methods, including naïve Bayes (NB), logistic regression (LR), support vector machine (SVM), LASSO, decision tree (DT), k-nearest neighbor (KNN), random forest (RF), AdaBoost (ADB), and XGBoost (XGB). We used grid search to tune hyperparameters for all methods. We then compared our feedforward deep learning models to the models trained using the nine other machine learning methods. Results: Based on the mean test AUC (Area under the ROC Curve) results, DFNN ranks 6th, 4th, and 3rd when predicting 5-year, 10-year, and 15-year BCM, respectively, out of 10 methods. The top performing methods in predicting 5-year BCM are XGB (1st), RF (2nd), and KNN (3rd). For predicting 10-year BCM, the top performers are XGB (1st), RF (2nd), and NB (3rd). Finally, for 15-year BCM, the top performers are SVM (1st), LR and LASSO (tied for 2nd), and DFNN (3rd). The ensemble methods RF and XGB outperform other methods when data are less balanced, while SVM, LR, LASSO, and DFNN outperform other methods when data are more balanced. Our statistical testing results show that at a significance level of 0.05, DFNN overall performs comparably to other machine learning methods when predicting 5-year, 10-year, and 15-year BCM. Conclusions: Our results show that deep learning with grid search overall performs at least as well as other machine learning methods when using non-image clinical data. It is interesting to note that some of the other machine learning methods, such as XGB, RF, and SVM, are very strong competitors of DFNN when incorporating grid search. It is also worth noting that the computation time required to do grid search with DFNN is much more than that required to do grid search with the other nine machine learning methods. In 2020, female breast cancer surpassed lung cancer as the most commonly diagnosed cancer worldwide, with an estimated 2.3 million new cases in 2020 . Breast Women rarely die of breast cancer confined to the breast or draining lymph nodes; rather, they die mainly due to metastasis, a condition in which cancer spreads to other vital organs, such as the lung and brain. Metastatic breast cancer (MBC) is the cause of over 90% of breast cancer related deaths  and remaClinicians face uncertainty in determining the ideal treatment course for individual patients with breast cancer. For example, image-guided core needle biopsy of the breast is a common procedure that can return non-definitive results in 5% to 15% of women. In these cases, it is difficult to determine the subtype of the breast cancer. Variation in breast cancer subtypes has been known to be associated with a patient’s drug response, progression of the tumor, and survival of the patient ,9. ThereVarious learning methods have been developed and applied in biomedical prediction ,16,17,18A neural network (NN) is one of the machine learning methods that can be used to conduct prediction and classification. A NN consists of layers of artificial neurons, also called nodes, structurally mimicking, in a sense, the impulse propagation mechanism in the human nervous system ,20, so iArtificial neural networks (ANNs), including DNNs, are widely used in science and information technology due to their notable properties, including parallelism, distributed storage, and adaptive self-learning capability ,27,28,29Precision medicine promises to help us improve patient outcomes by tailoring healthcare to the individual patient . The eleThe LSDS (Lynn Sage Dataset) was a de-identified and publicly available clinical dataset about breast cancer that was created and published via previous studies ,39. It wIn this research, we took the empirical approach to study the performance of DFNN models when predicting BCM using clinical data. Note that we describe the structure of our feedforward deep learning models in detail later in the Methods Section, and we refer to these models as the DFNN  models throughout the text. We applied DFNN method to learn prediction models from LSM datasets. These models can be used to predict 5-, 10-, and 15-year BCM. The performance of a DFNN model is affected by the number of hidden layers and number of nodes per hidden layer, which are called hyperparameters. In addition, there are other hyperparameters that can be used to adjust the prediction performance of deep learning. For example, “epochs” is a hyperparameter we consider. One epoch means a deep learning model is trained by each of the training set samples exactly once. The learning might not converge when epochs is too low, and model overfitting tends to get severe when it is too high. Tuning hyperparameters is the process of identifying the set of hyperparameter values that are expected to produce the best prediction model out of all sets of hyperparameter values examined. Grid search is designed to conduct hyperparameter tuning in a systematic way by going through a possible set of hyperparameter values automatically during learning. In this study, we optimized DFNN model performance by conducting hyperparameter tuning via grid search. To evaluate the performance of the DFNN, we compared our DFNN models with the ones that we trained using nine other well-known machine learning methods. We applied hyperparameter tuning and grid search to optimize model performance for each of the nine comparison methods. We conjectured that the performance of our DFNN models with grid search would be comparable to that of other machine learning methods when predicting the binary status of BCM. We posit this conjecture, because deep learning is a very powerful tool for prediction and has been successful in other applications, such as image recognition ,46,47,48In this study, we used three LSM datasets about breast cancer metastasis: LSM-5Year, LSM-10Year, and LSM-15Year. Missing data were filled in using the nearest neighbor (NN) imputation algorithm . Metasta0 represents the node for the bias passing from the input layer to the first hidden layer. The activation function x. We used a rectifier linear unit (ReLU), in which Our DFNN models are fully connected feedforward neural networks composed of more than one hidden layer. ut layer ,47,48,49 problem ,47,48,49ut layer ,47,48,49ut layer . He_norm a layer . TensorF a layer  is an op a layer ,53. Our Deep learning is a powerful machine learning method due to its large number of hyperparameters that can be optimized . See TabHyperparameter tuning is the process of identifying the set of hyperparameter values that is expected to produce the best prediction model from all sets of hyperparameter values being examined. Hyperparameter tuning gives us the power to optimize model performance but tuning a large number of hyperparameters presents a major challenge in terms of computation time ,53,54. GWe tried to improve model performance by conducting grid search implemented in Python using the scikit-learn package ,52. In aOverfitting is a phenomenon in which the model performs well on training data but generalizes poorly to unseen data ,59,60. OIt is not possible to completely eliminate overfitting, but we took multiple approaches to minimize the effect of overfitting. First, we tuned “dropout rate” and “epochs” to reduce the effect of overfitting . The “drWe designed an output format for grid search and recorded 64 different output values for each of the models trained in a grid search. Among the output values are information about the computer system used, computation time, and measures for model performance. For a given binary diagnostic test, a receiver operator characteristic (ROC) curve plots the true positive rate against the false positive rate for all possible cutoff values . The area under an ROC curve (AUC) measures the discrimination performance of a model. We conducted a 5-fold cross-validation to train and evaluate each model in a grid search. The entire dataset was split evenly into 5 portions. The division was mostly executed randomly, except that each portion had approximately 20% of the positive cases and 20% of the negative cases to ensure that it was a representative fraction of the dataset. Training and testing were repeated five times. Each time, a unique portion was used as the validation set to test the model learned from the training set, which combined the remaining four portions. Training and testing AUCs were reported. The average training and testing AUC across all five times was also derived and reported. The best-performing set of hyperparameter values was chosen based on the highest mean test AUC. The best model would be the one refitted from the entire dataset using the best-performing set of hyperparameters values. We used this procedure for all methods involved in this study.We compared the performance of the best-performing DFNN model to that of a representative set of machine-learning methods, each obtained via grid search. The representative set of methods include naïve Bayes (NB), logistic regression (LR), decision tree (DT), support vector machine (SVM), the least absolute shrinkage and selection operator (LASSO), k-nearest neighbor (KNN), eXtreme gradient boosting (XGB), adaptive boosting (AdaBoost), and random forest (RF). We used the scikit-learn ,52 packaNB (naïve bayes) [A BN (bayesian network) consists of a directed acyclic graph (DAG) V contains random variables and whose edges E represent relationships among the random variables [X∈ V given each combination of values of its parent nodes. Each node V in a BN is conditionally independent of all its nondescendents given its parents in the BN. NB is a simplified BN which normally only contains one parent node and a set of children nodes. In a basic NB model, there is an edge from the parent to each of the children. When a NB model is used to conduct classification, it is called a NB classifier. We used the BernoulliNB classifier in this study because we have binary classes. Alpha is the Laplace smoothing parameter that deals with the problems of zero probability and regularize complexity, the larger the alpha, the stronger the smoothing and the lower the complexity of the model. We tested 500 alpha values, which are all positive integers from 1 to 500.e bayes) ,62,63,64LR (logistic regression) [C is the inverse of regularization strength (ression) ,66 is a ression) . C is th dataset ,66. We uDT (decision tree) [on tree) ,68 is onon tree) . It can SVM (support vector machine) [−5, 2−3… 215. The parameter γ defines how far the influence of a single training example reaches (inverse of the radius of influence of samples selected by the model as support vectors). Low values mean “far” and high values mean “close”. We tested values in the range 2−15, 2−13… 23.machine) ,73,74,75machine) ,70,71. Tmachine) . Smallermachine) ,73,74,75LASSO (the least absolute shrinkage and selection operator) is a regression-based method classifier that is capable of conducting variable selection and regularization in order to enhance prediction performance and control overfitting [rfitting . The parKNN (k-nearest neighbor) [eighbor) ,78,79 iseighbor) . KNN assRF (random forest) [ forest) ,81,82,83 forest) ,80. An aADB (adaptive boosting) [oosting) ,83,84,85XGB (eXtreme gradient boosting) [oosting) ,90,91,92oosting) ,87,88,89We conducted the Wilcoxon rank sum tests to determine the statistical significance of the AUC results. We conjectured that deep learning with grid search would perform comparably to other methods when predicting the binary status of 5-, 10-, and 15-year BCM. We paired the DFNN with each of the 9 other machine learning methods, and conducted both the right-tailed (greater) and left-tailed (less) Wilcoxon tests for each pair of the methods and repeated these tests for each of the three datasets separately. The null hypothesis for all the Wilcoxon tests is that the two methods perform indifferently. The alternative hypothesis of the right-tailed Wilcoxon tests is DFNN does better (greater) than the comparison method, and this is to test whether DFNN performs better than other methods. The alternative hypothesis of the left-tailed Wilcoxon tests is DFNN does worse (less) than other methods, and this is to test whether DFNN performs worse than the comparison method. We conducted the Wilcoxon rank sum test in R using the wilcox.test function included in the R package.p-value, and the 95% confidence interval (CI) for each of the Wilcoxon tests we conducted. W is the test statistic used in the Wilcoxon rank sum test.We compared side by side the ROC curves of the best-performing models of DFNN and the nine comparison methods. Based on the mean test AUC values shown in p-values we obtained for each pair methods range from 0.111 (DFNN vs. NB) to 0.925 (DFNN vs. KNN) in predicting 5-year BCM, which indicates that at a significance level of 0.05, we are not confident in rejecting the null hypothesis which states that DFNN performs no difference from the comparison methods. p-value 0.028) and SVM , but no difference from other methods at a significance level of 0.05 when predicting 10-year BCM. Again, according to p-value of 0.011 and 0.030 each, respectively, but no difference from other methods at a significance level of 0.05 when predicting 15-year BCM. Based on We notice that in each of the three metastasis categories, the mean test AUC values of the top performers are quite close to each other. For instance, when predicting 15-year BCM, the mean test AUC values of the top four performers are 0.842 (DFNN), 0.844 (LR), 0.844 (LASSO), and 0.845 (SVM). We further look at the statistical testing results shown in As demonstrated in Based on the statistical testing results, we conclude that at a significance level of 0.05, DFNN performs comparably to any of the nine other methods when predicting 5-, 10-, and 15-year BCM. This is consistent with our conjecture that deep learning with grid search performs comparably to the other methods when predicting the binary status of BCM. On the other hand, it is interesting to learn that some of the other machine learning methods, such as XGB, RF, and SVM, are very strong competitors of DFNN. Additionally, obtaining the best-performing DFNN models required much more computation time than doing so for the nine comparison methods.


Fish is among the foods exerting favourable effects on colorectal cancer (CRC), but the possible role of canned fish has been insufficiently investigated. We aimed to investigate the relationship between canned fish consumption and CRC risk. We analysed data from two case–control studies conducted between 1992 and 2010 in several Italian areas, comprising a total of 2419 incident cases and 4723 hospital controls. Canned fish consumption was analysed according to the weekly frequency of consumption as <1 serving per week (s/w) (reference category), 1 < 2 s/w, and ≥2 s/w. We calculated odds ratios (ORs) and 95% confidence intervals (CIs) using unconditional logistic regression models, adjusting for several recognised confounding factors. Overall, canned fish consumption was lower among cases than among controls (23.8% vs. 28.6%). An inverse association was found between canned fish consumption and CRC risk with a significant trend in risk , which was consistent across strata of several covariates. This study is the first to offer a basis of support for canned fish consumption as a component of a healthy diet, and it has relevant public health implications given the high ranking of CRC in incidence and mortality worldwide. In 2020, colorectal cancer (CRC) accounted for 1.9 million new cases of cancer worldwide, ranking second for women and third for men in terms of incidence, and it was the second leading cause of cancer mortality, with 935,000 deaths ,5. Fish In some studies, fish consumption comprises canned fish consumption, i.e., fish that has been steamed, cooled, cleaned, sealed in a can with covering of olive oil or brine, and then heated for a determined time for sterilisation in order to last up to several years . Canned Household consumption of processed fish and seafood, such as canned fish, has been increasing in recent decades, totalling 727,000 tons in 2018 . In partThis study aims to investigate the relationship between canned fish consumption and CRC risk, using data from two large case–control studies conducted in Italy.Data for this study were obtained from two Italian case–control studies on CRC: the first study  was carrAmong controls, 1260 (26.7%) were admitted for non-traumatic orthopaedic disorders, 1134 (24.0%) for surgical conditions, 953 (20.2%) for trauma, 842 (17.8%) for eye diseases, and 534 (11.3%) for a miscellanea of other diseases. The study protocols were approved by the local ethical committees of the respective centres, and all participants signed an informed consent according to the rules at the time when the study was conducted.Trained staff collected data through face-to-face interviews. The data include sociodemographic and anthropometric factors, physical activity, smoking habits, selected medical conditions, family history of CRC, and dietary habits in the 2 years before the interview. Subjects were also asked to report height and average weight at ages 30 and 50 years, as well as before diagnosis/interview. Occupational physical activity at ages 15–19, 30–39, and 50–59 years was self-reported as very heavy, heavy, intermediate, standing, or sedentary.Usual diet was assessed through a structured, validated , and rep2), years of education , and family history of cancer (yes/no) among first-degree relatives  were added. In the third model, tobacco smoking , alcohol consumption , level of occupational physical activity at age 30–39 years , fruit  and vegetable  consumption, and total energy intake  were also considered. The cut-off values for categories were identified based on the distribution of the variables among the controls. In the fourth model, fresh fish was added as a confounding factor. ORs and 95% CIs were also computed for type of fish consumption  in a separate model.Canned fish was analysed according to the weekly frequency of consumption as <1 serving per week (s/w) (used as reference category), 1 < 2 s/w, ≥2 s/w, and as a continuous variable where the average serving of canned fish in the Italian diet was 80 gr/day. Odds ratios (OR) and corresponding 95% confidence intervals (CI) were estimated using an unconditional multivariable logistic regression model . Three hMissing values for confounding variables were imputed with the most frequent categories, according to case/control and gender. A complete case analysis was also conducted. Tests for linear trend were performed by including exposure covariate as continuous in the model.2 test with 1 degree of freedom.Stratified analyses were carried out according to sex; age; education; smoking; alcohol consumption; physical activity at the workplace; energy intake; and fruit, vegetable, and fresh fish consumption. Heterogeneity across strata was assessed for canned fish intake as a continuous variable (10 gr increase intake) using the Wald χAll the analyses were carried out using SAS software 9.4 .Selected characteristics of 2419 CRC cases and 4723 controls are reported in Canned fish consumption in the cases and controls in the two studies are reported in p-value for trend <0.0001). When considering canned fish intake as a continuous variable, we found an OR of 0.86 (95% CI: 0.79–0.93) for a 10 gr increase in daily intake. These estimates were substantially similar to those derived from models 1, 2, and 3.p-value for trend = 0.0004); for rectal cancer, the OR for 1 < 2 s/w was 0.80 (95% CI: 0.67–0.97), and for ≥2 s/w, the OR was 0.65 (95% CI: 0.44–0.95)  (data not shown). Similar results were also found for specific subsites, although larger confidence intervals were found, and the results were no longer statistically significant.When we estimated the risks for colon and rectal cancers separately, an inverse association with canned fish consumption was confirmed in both sites: for colon cancer, the OR for 1 < 2 s/w was 0.82 (95% CI: 0.70–0.95), and for ≥2 s/w, the OR was 0.66 (95% CI: 0.49–0.90) . No other significant interactions between canned fish intake or other factors emerged. The inverse relationship with canned fish was consistently significant in strata of age, smoking habits, alcohol drinking, education, energy intake, vegetable intake, fruit intake, and fresh fish consumption  in males and 0.75 (95% CI: 0.65–0.85) in females, with a significant interaction between canned fish and sex (sumption .We found an inverse association between canned fish consumption and the risk of CRC and colon and rectum cancers separately, which persisted after adjustment for several recognised confounding factors and for the strata of covariates. The risk decreased with an increase in the consumption of canned fish.Previous studies reported a protective effect of fresh fish on CRC. A case–control study conducted in China found an almost halved risk of CRC for the highest level of fish consumption . In a EuN-3 fatty acids, in particular, the long-chain polyunsaturated fatty acids eicosapentaenoic and docosahexaenoic acids, which are present in cold-water fish and fish oil, may inhibit carcinogenesis . PreclinGenerally, canned and fresh fish present similar nutritional characteristics. The industrial process carried out to produce canned fish is likely to preserve the most important nutrients and nutritional properties of fresh fish. For example, the tuna parts selected for canning are rich in fat, and, consequently, they present a high concentration of n-3 PUFAs . MoreoveOur study shows a stronger inverse relationship for women than for men. A cohort study of fresh fish consumption and CRC found similar inverse associations both in men and women , and a mper se, and its effect was not driven by other selected dietary habits. In particular, canned fish consumption was also inversely related to CRC in individuals who did not consume fresh fish. Interestingly, canned fish had a borderline significant association in subjects with a low intake of fruit and vegetables, suggesting that the lack of an adequate consumption of fruit and vegetables and the consequent effect on CRC risk were not completely blunted by other healthy dietary habits.Canned fish showed a protective role independently of other dietary habits, suggesting that canned fish can be protective In this study, the protective effect of canned fish on CRC substantially equalled that of fresh fish, and the consumption of both types of fish provided an even greater effect, suggesting that fish consumption offers protection from CRC, however processed.The satisfactory reproducibility  and valiThe questionnaire asked about the consumption of tuna, mackerel, and sardines in oil, and, therefore, our results cannot be generalised to canned fish without oil or to other fishes. Indeed, particularly in relation to the time of the first study, canned fish was usually limited to those same fishes in olive oil, and our data should well represent the canned fish consumption of the Italian population in the two periods when the studies took place.Overall, few studies considered canned fish, and only a few of them considered fresh fish in addition to canned fish as a unique item , therefo