Scophthalmus maximus), commercially important flatfish species, is widely cultivated in Europe and China. With the continuous expansion of the intensive breeding scale, turbot is exposed to various stresses, which greatly impedes the healthy development of turbot industry. Here, we present an improved high-quality chromosome-scale genome assembly of turbot using a combination of PacBio long-read and Illumina short-read sequencing technologies. The genome assembly spans 538.22 Mb comprising 27 contigs with a contig N50 size of 25.76 Mb. Annotation of the genome assembly identified 104.45 Mb repetitive sequences, 22,442 protein-coding genes and 3,345 ncRNAs. Moreover, a total of 345 stress responsive candidate genes were identified by gene co-expression network analysis based on 14 published stress-related RNA-seq datasets consisting of 165 samples. Significantly improved genome assembly and stress-related candidate gene pool will provide valuable resources for further research on turbot functional genome and stress response mechanism, as well as theoretical support for the development of molecular breeding technology for resistant turbot varieties.Turbot (  Scophthalmus maximus (FishBase ID: 1348), as known as turbot, an economically important flatfish (Pleuronectiformes), is native to Northeast Atlantic throughout the Mediterranean and along the European coasts to Arctic Circle1, and now is the most widely cultivated commercial flatfish around the world with the highest annual aquaculture production2. Since its firstly introduction into China in 1992, turbot aquaculture industry has made great progress, leading to the rise of the fourth wave of mariculture industry in China2. However, turbot was affected by various biotic and abiotic stresses during the breeding process, which seriously threatened the healthy development of turbot aquaculture industry and caused huge economic losses. Therefore, carrying out research on the resistance of turbot and obtaining genetic resources related to stress resistance will contribute to the research on the resistance molecular mechanism of turbot and provide theoretical support for the subsequent genetic improvement of turbot germplasm.Enteromyxum scophthalmi4, Vibrio anguillarum5), heat stress6, oxygen stress7, crowding stress8, salinity stress9, and feeding stress10. All these researches were solely focused on the identification of differentially expressed genes (DEGs), whereas connectivity analysis has not yet been taken into account. Instead of focusing only on DEGs, gene co-expression network (GCN) analysis provides new insight into the identification of co-expressed gene modules, their correlation with specific traits, and the pinpointing of key hub genes12, which cannot be detected by standard transcriptome analysis. This powerful approach has been widely applied to detect diverse stresses response in Nibea albiflora13, Oysters14, Scophthalmus maximus6, etc.In recent years, numerous RNA-seq studies have been conducted to explore the stress responsive genes and molecular mechanisms under various stresses, such as pathogens stress  and Illumina short-read sequencing technologies. Based on this improved genome assembly, we re-annotated the protein-coding genes, repetitive sequences and ncRNAs. In addition, we re-analyzed multiple stress-related RNA-seq datasets from National Center for Biotechnology Information (NCBI) Sequence Read Archive (SRA) database by gene co-expression network analysis, and identified multiple gene modules and candidate genes response to various stresses in turbot. Taken together, these resources will not only serve as key resources for studying genomics and further research into the stress response mechanisms, but will also promote the progress of genetic improvement and comprehensive stress-resistant molecular breeding of turbot.Genomic DNA was extracted from the muscle samples of a super-female (WW) turbot using Puregene Tissue Core Kit A  according to the manufacturer’s instruction. The quality of the extracted genomic DNA was checked using electrophoresis on 1% agarose gel and the concentration was quantified using a NanoDrop 2000 to ensure the DNA samples met libraries sequencing requirements.The extracted DNA molecules were firstly used to construct an Illumina pair-end (PE) library with 350 bp insert size using standard protocols provided by Illumina . The PE library was then sequenced using the Illumina HiSeq 4000 platform with 150 bp PE mode according to the manufacturer’s instructions. Finally, a total of 51.80 Gb raw reads, accounting ~90X coverage of whole genome, were generated  and sequenced it using the PacBio Sequel II platform with the continuous long-read (CLR) mode following the manufacturer’s instruction. In total, we obtained 150.30 Gb (~265X) PacBio long reads (Table 15 with default parameters. And then corrected reads were subsequently assembled by Flye (v2.7)16 (--pacbio-corr -- threads 80 --genome-size 568 m). To obtain the final assembly, the draft assembly was removed haplotypic duplication by purge_dups17 and polished by gcpp (https://github.com/PacificBiosciences/gcpp) with default parameters using PacBio data, then Pilon18(--fix bases) was used to further polish the genome using Illumina data 22 based on the RepBase TE library (version 10/26/2018)23. In addition, de novo prediction was conducted using RepeatMasker to further detect novel repeats, which based on the de novo repeats library of the turbot genome constructed with RepeatModeler (http://www.repeatmasker.org/RepeatModeler/) and LTR-FINDER24. Finally, a total of 104.45 Mb of non-redundant repetitive sequences (Combined TEs) were obtained, accounting for 19.41% of the assembled genome . Exonerate (v2.4.0)27 was used to align homologous protein sequences to turbot genome. Homologous genes were predicted ranging from 35,093 to 48,770 in above species reference sequences erate v2..027 was erate v2..027 was erate v2..027 was 30, Pfam31, Swissprot32 and TrEMBL32 databases were respectively used to predict protein function based on the conserved protein domains by InterProScan (v5.46)33. BLASTP  was used for the homolog search in multiple databases, such as Gene Ontology (GO)34, Kyoto Encyclopedia of Genes and Genomes (KEGG)35, and NCBI non-redundant protein (NR)36 databases. Ultimately, a total of 21,360 genes  could be functionally annotated by at least one of the abovementioned databases 41 was used to calculate transcripts per million (TPM) values for all genes using sorted bam files obtained from reads alignment. Subsequently, we used BioNERO42 to preprocess the gene expression data according to the following steps: I) Replacing missing values (NAs) with 0 using replace_na function; II) Removing the genes whose average gene expression was less than 1 with remove_nonexp function; III) Removing outlying samples with ZKfiltering function; IV) Adjusting for confounding artifacts with PC_correction function to make every gene follow an approximate normal distribution. After filtering and processing   reaches 0.8 and mean connectivity tends to 0. Next, we used the exp2gcn function to infer the GCN with power 11. As a result, a total of 24 co-expression modules were eventually identified  Fig. . First oied Fig. , with thp-value < 0.01) positively or negatively correlated with particular traits (stresses) by calculating module-trait spearman correlation coefficients using the module_trait_cor function from BioNERO. As shown in Fig. 43  < 0.5).We then identified modules that were extremely significant  < 0.05 and |log2FC| > 1. DEGs, related to the same stress, were merged as DEGs set for this stress. Finally, genes, included in both hub genes set and DEGs set corresponding to the same stress, were defined as the candidate key hub genes for this stress. Candidate key hub genes related to crowding, feeding, heat, oxygen, pathogens and salinity stress were 0, 128, 40, 7, 90 and 80, respectively.To identify candidate key hub genes related to every stress, we firstly constructed hub genes set. Hub genes, defined as the top 10% genes with highest degree  that have module membership (MM)  > 0.8, were identified using the function get_hubs_gcn. Then, hub genes belonging to modules that were extremely significant associated with same stress were merged as hub genes set for this stress. Following, we set up the differentially expressed genes (DEGs) set. Firstly, we used featureCounts47) in modules that were extremely significant correlated with heat stress. Meanwhile, KEGG enrichment analyses were employed on the same modules, and the results manifested that metabolism, lipid metabolism, carbohydrate metabolism, glycolysis/gluconeogenesis, PPAR signaling pathway, proteasome, digestive system, fat digestion and absorption, peroxisome, cell growth and death, transport and catabolism, cellular processes, and protein kinases were the significantly enriched pathways (KEGG enrichment.xlsx47). Finally, we identified 40 candidate heat-related key hub genes, of which 7 genes including ABI148, CD4449, CCDC15350, G2e351, PATJ52, HYKK53 and occludin54 has been verified to contribute to heat stress. For instance, G2e3 was one of the candidate genes in the liver of heat-treated large yellow croaker51. Exposure to heat stress (39 °C or 41 °C) resulted in increased expression of occludin protein in Caco-2 cells54.To heat stress, GO enrichment analyses illustrated that metabolic process, cellular process, catabolic process, catalytic activity, hydrolase activity, oxidoreductase activity, cellular response to stress, biosynthetic process, and binding were the significantly enriched terms (GO enrichment.xlsx47) in modules that were extremely significant correlated with oxygen stress. Furthermore, we employed KEGG enrichment analyses on the same modules, and the results demonstrated that metabolism, oxidative phosphorylation, environmental adaptation, energy metabolism, and peroxisome were significantly enriched pathways (KEGG enrichment.xlsx47). In addition, we obtained 7 candidate oxygen-related hub genes, among which AMBP55 and CNN156 had been confirmed to conduce to heat stress. Such as, the gene for A1M is denoted AMBP, which has a physiological role as a protective antioxidant55. Five percent oxygen concentration significantly increased the expression levels of CNN1 in adipose-derived stem cell cultures after 2 weeks of induction56.To oxygen stress, GO enrichment results showed that aerobic respiration, aerobic electron transport chain, metabolic process, oxidoreductase activity, mitochondrial inner membrane, mitochondrial respirasome, respiratory chain complex, oxidative phosphorylation, oxidoreductase complex, catabolic process, catalytic activity, response to stress, response to external stimulus were the significantly enriched terms (GO enrichment.xlsx47) in modules that were extremely significant related with pathogens stress. Meanwhile, KEGG enrichment analyses were employed on the same modules, and the results manifested that immune system, human diseases, complement and coagulation cascades, CD molecules, lysosome, phagosome, B cell receptor signaling pathway, hematopoietic cell lineage metabolism, glycosaminoglycan binding proteins, exosome, neutrophil extracellular trap formation, were the significantly enriched pathways (KEGG enrichment.xlsx47). Ultimately, we determined 90 candidate pathogens-relevant hub genes, thereinto, 18 genes, such as CMKLR157, CSF3R58, SIGLEC1059, RAP1GAP260, Cd300lf61, NPTN62, MRC163, LILRA664, BLNK65, CXCL1266, PIGR67, SIGLEC1568, GULP169, MARCO70, NLRP1271, CRP72, FGG73, and lysozyme74, had been proved to be conducive to pathogens stress. For example, LILRA6 is essential for macrophage-mediated immune responses and it has the potential to complement the innate and adaptive immune system against pathogens64. Siglec-15 probably plays a conserved, regulatory role in the immune system of vertebrates68. In addition to its direct antimicrobial role, more recent evidence has shown that lysozyme modulates the host immune response to infection74.To pathogens stress, GO enrichment results showed that immune response, immune system process, response to wound healing, blood coagulation, hemostasis, response to external stimulus, response to stress, biosynthetic process, catalytic activity, metabolic process, and cellular process were the significantly enriched terms (GO enrichment.xlsx47), such as, ion binding, small molecule binding, anion binding, proteasome complex, proteasome-activating activity, catabolic process, metabolic process, cellular process, cellular response to stress, binding, and ATP binding were significantly enriched in modules that were extremely significant related with salinity stress. Simultaneously, we employed KEGG enrichment analyses on the same modules, and the results indicated that proteasome pathway, protein kinases were the significantly enriched pathways (KEGG enrichment.xlsx47). After taking the intersection of hub genes set and DEGs set, 80 genes were defined as candidate salinity-associated hub genes, and six genes  had been certified to be related to salinity stress. For example, RBBP6 was one of the identified candidate genes for freshwater vs. marine adaptation in threespine stickleback77. MAP3K7, also known as TAK1, could be highly activated by osmotic stress79.To salinity stress, according to GO enrichment analyses, terms 85 with 4,584 single-copy orthologs from actinopterygii_odb9 database. BUSCO analysis revealed that 97.4%  complete BUSCOs (94.9% single-copy and 2.5% duplicated BUSCOs) and 1.1% (49) fragmented BUSCOs were identified in the assembled genome of turbot. Thirdly, we further evaluated the assembly quality using Inspector86 by aligning PacBio long reads to the assembled contigs for generating read-to-contig alignment and performing downstream assembly evaluation. As a result, read-to-contig mapping rate and quality value (QV) were 91.93% and 45.41, respectively. All these indicators suggested a high-quality and complete genome assembly for the further research in genetics and genomics of turbot.The quality and completeness of the new assembly were evaluated through three independent approaches. Firstly, the base-level accuracy and completeness were estimated using Merqury


The multi-ligand receptor for advanced glycation end-products (RAGE) and its ligands are contributing factors in autoimmunity, cancers, and infectious disease. RAGE activation is increased in chronic kidney disease (CKD) and coronavirus disease 2019 (COVID-19). CKD may increase the risk of COVID-19 severity and may also develop in the form of long COVID. RAGE is expressed in essentially all kidney cell types. Increased production of RAGE isoforms and RAGE ligands during CKD and COVID-19 promotes RAGE activity. The downstream effects include cellular dysfunction, tissue injury, fibrosis, and inflammation, which in turn contribute to a decline in kidney function, hypertension, thrombotic disorders, and cognitive impairment. In this review, we discuss the forms and mechanisms of RAGE and RAGE ligands in the kidney and COVID-19. Because various small molecules antagonize RAGE activity in animal models, targeting RAGE, its co-receptors, or its ligands may offer novel therapeutic approaches to slowing or halting progressive kidney disease, for which current therapies are often inadequate. Chronic kidney disease (CKD) is caused by both systemic diseases  and primary kidney diseases  . CKD alsAGER, located on human chromosome 6. The gene is within the major histocompatibility class III locus and near the genes for the transcriptional activator PBX2 and cell surface receptor neurogenic locus notch homolog protein-3 (NOTCH3)  .More than 20 mRNA splice variants of RAGE have been detected in human tissues . HoweverThere are four known naturally-occurring RAGE protein isoforms:- full length (fl)-RAGE- dominant negative (dn)-RAGE, which lacks a functional intracellular domain- endogenous secretory (es)-RAGE, which lacks a transmembrane and intracellular domain- cRAGE, generated by proteolytic cleavage from the cell-membrane, carried out by a disintegrin and metalloproteinase domain-containing protein 10 (ADAM10)RAGE isoforms in serum and plasma are termed soluble RAGE (sRAGE) and include cleaved RAGE (cRAGE) and endogenous secretory RAGE (esRAGE) , 12 Fig. RAGE plMβ2-integrin, and as complement receptor 3 (RAGE isoforms form homo- and hetero-dimers that bind a diverse array of ligands . These lceptor 3 .RAGE and RAGE ligands are strongly implicated in severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infections, which may initiate or exacerbate kidney injury. Acute and chronic inflammation in COVID-19 and CKD, respectively, drive pathogenesis, suggesting that RAGE activity, identified in both diseases, may illustrate the molecular mechanisms in which these syndromes interact. In order to explore the role of RAGE and its ligands in COVID-19-induced CKD, we review here the ligands and their interaction with RAGE in the kidney and with respect to SARS-CoV-2.RAGE is expressed in various cells in kidney, including podocytes, mesangial cells, tubular epithelial cells, neurons, endothelial cells, and infiltrating immune cells . In normRAGE expression increases with age. In renal tissue from patients undergoing radical nephrectomy for urological tumors or trauma, younger patients  expressed less RAGE and pSTAT5  compared to older patients , as assessed by immunoblots . In C57B2), plasma sRAGE is elevated compared to healthy controls and is inversely associated with estimated glomerular filtration rate, echocardiographic intima–media thickness, and the total number of atherosclerotic plaques  serum sRAGE levels are also elevated in comparison to 21 healthy subjects and 25 CKD patients . Becausedb/db mouse model of type 2 diabetes, RAGE expression is elevated in podocytes and there is increased recruitment of S100A8/S100A9 -expressing leukocytes into glomeruli. Therapeutic administration of sRAGE to these mice reduces inflammation and improves renal function   . This in (NICD1) , suggestFurther, mouse metanephroi cultured in the presence of a γ-secretase inhibitor, N-S-phenyl-glycine-t-butyl ester (DAPT), manifest impaired NOTCH1 signaling and podocyte development . IncreasDiaph1 in streptozotocin-induced diabetic C57BL/6 mice suppresses glomerular inflammation and reduces podocyte foot-process effacement (The angiotensin II type 1 receptor (AT1R) forms a heteromeric complex with RAGE, and Ang II-induced activation of the AT1R transactivates RAGE . Both ATfacement . Expressfacement  is requifacement . A downsfacement . In hypefacement . PossiblRAGE also co-immunoprecipitates with the αVβ3-integrin , which iStudies of suPAR in murine MPC-5 podocytes indicate that the ligand increases the production of reactive oxygen species (ROS) through the NADPH oxidase 2 (NOX2) in a Rac-1-dependent manner. This oxidative stress then promotes the activation of Src kinases and the increased production of TRPC6 calcium channels , which aIn summary, RAGE is expressed in podocytes, which may encourage the recruitment of S100A8/S100A9-positive leukocytes. Activation of RAGE in podocytes induces γ-secretase and NOTCH activity associated with injury. Ang II promotes RAGE expression through AT2R and induces RAGE transactivation via AT1R. A downstream signal of RAGE and AT1R is DIAPH1, which can induce the activation of Rac1 in macrophages and possibly other cell types, such as podocytes. In podocytes, activation of Rac1 promotes injury. RAGE and αVβ3 complex and regulate the activity of TRPC6, which is associated with calcium transport, adhesion, motility, and podocyte homeostasis. Changes in TRPC6 function are associated with podocyte injury .In normal rat glomerular mesangial cells, AGE induce Smad2 and Smad3 phosphorylation, which in turn promote profibrotic type I collagen production . In primIn the murine mesangial cell line SV40 MES 13, both glyoxal-lysine dimers and methyl glyoxal-lysine dimers (AGE) increase production of RAGE and ROS , 51. TheIn summary, mesangial RAGE promotes the activity of SMAD2/3 and production of type I collagen, similar to TGF-β1. RAGE-induced ROS contribute to ACE/Ang II/AT1R pathway activation, which also activates SMAD2/3 indirectly through TGF-β1 production. Mesangial AT1R and RAGE activation cooperate in promoting inflammation and fibrosis in glomerulosclerosis, indicating that inhibitors to AT1R and/or RAGE may impede disease progression .in vitro -induced/TGF-β1-independent and TGF-β1-dependent Smad cell signals . AGE-indMoreover, both AGE-induced MAPK activity and epithelial mesenchymal transition (EMT) in rat NRK-52E cells are blocked by treatment with AT1R blockers  . In NRK-In summary, RAGE activation in epithelial cells induces cell signals  and promotes the production of molecules  commonly associated with fibrogenesis, EMT, and inflammation. Because RAGE complexes with AT1R in these cells, AT1R blockers antagonize RAGE cell signals. Metformin inhibits the formation of α-dicarbonyl AGE and may antagonize RAGE-induced ROS production and NF-κB activity through activation of AMPK-α. Molecules that block AT1R and RAGE and/or promote AMPK-α activation may attenuate the fibrotic and inflammatory responses of RAGE in tubular epithelial cells .2+ and Zn2+ binding dimer , estimated at more than 40% and 5% of cytosolic and total proteins of neutrophils, respectively. These proteins bind tubulin to regulate cytoskeleton organization, phagocytosis, and migration and are abundantly released in various non-infectious and infectious diseases to promote chemotaxis, cell signaling, and apoptosis , interleukin (IL)-6, and C-C motif chemokine ligand 2 (CCL2), the latter accumulating to the highest levels  derived from either wild-type or S100A9t levels . Pre-inct levels , indicatLastly, Ang II-induced NF-κB in HUVECs increases production of AT1R and promotes autocrine RAGE activation by upregulating HMGB1 and RAGE. Ang II-induces DIAPH1 expression and downstream pSRC-induced endothelial layer permeability in cultured HUVECs through RAGE activation. Blocking RAGE with sRAGE or blocking the AT1R with a specific antagonist  reduces endothelial permeability  Figure .In summary, S100A8/S100A9 molecules and S100A8/S100A9-positive myeloid cells increase in circulation during CKD. RAGE ligands induce endothelial chemokine production associated with the recruitment of immune cells, upregulate myeloid Mac-1 cell surface expression, and increase endothelial cell surface ICAM-1 expression. Ang II supports leukocyte adhesion to the endothelium through NF-κB activation and enhances RAGE signaling. Activation of Src kinases, downstream of RAGE-associated DIAPH1, promotes endothelial permeability. Inhibitors to RAGE, AT1R, and DIAPH1 may therefore protect the endothelium during CKD.As described above, RAGE is implicated in CKD pathogenesis through the expression and function of the protein in various cells of the kidney and immune system. Small molecules and biologics that target RAGE, RAGE ligands, and RAGE downstream cell signals in models of CKD repeatedly have demonstrated reduced kidney pathology and improved function . Crosstain vitro induce NOTCH1 cleavage and formation of NICD1 through the production of γ-secretase (AGE-exposed human podocytes ecretase . RAGE acecretase . In a precretase . The rolThe angiotensin-converting enzyme (ACE)-2 is also regulated by γ-secretase. After ACE2 cell surface cleavage by ADAM10 or TMPRSS2, the γ-secretase enzyme forms an ACE2 intracellular domain, which like NICD1, may serve as a cell signaling molecule . ACE2 isIn humans and experimental animals, ACE2 is expressed in renal tubular cells, podocytes, mesangial cells, and endothelial cells. In renal biopsies from patients with type 2 diabetes, immunohistochemical expression of ACE is higher but ACE2 is lower in the glomeruli and tubulointerstitium compared to non-diabetic control biopsies . In diabBecause SARS-CoV-2 binding to ACE2 induces ACE2 degradation , the renACE2 transgenic mice infected with SARS-CoV-2, treatment with the RAGE inhibitor, FPS-ZM1, reduced overall inflammation and significantly increased survival , asthma, kidney disease, cerebrovascular disease, cirrhosis, dementia, severe obesity, and higher age were identified as factors that increased the risk of hospitalization . In a meThe N(ε)-Carboxymethyllysine (CML) advanced glycation end-product is an indicator of microvascular inflammation and dysfunction. In comparing autopsied hearts from non-COVID-19 patients and those from the COVID-19 first wave (February–June 2020) and second wave (September–December 2020), CML was found in the endothelium and smooth muscle cells of intramyocardial blood vessels and was elevated in the first wave in association with increased deposits of FVII and FXII clotting factors .Because serum AGE levels are increased in CKD patients and are associated with decreased glomerular filtration rates , we hypo1−6), RAGE, and peroxisome proliferator-activated receptor gamma (PPAR-γ)  . LPA cel(PPAR-γ) . This biAR-γ) (5 . In CKD AR-γ) (5  and in CAR-γ) , binds soluble ligands  and cell surface receptors  to regulate cell signaling, adhesion/extravasation, phagocytosis, thrombosis, and inflammation -1  and is eAmyloid precursor protein (APP), produced by neurons and platelets, is cleaved by secretases  to form Aβ peptides, which bind various lipid, proteoglycan, and neurological receptors, in addition to RAGE. The accumulation of these peptides forms aggregates (fibrils) that may further assemble into plaques , 135. ThHMGB1, also known as amphoterin, is a nuclear protein that binds chromosomal DNA, various transcription factors, tissue-type plasminogen activator (tPA), urokinase-type plasminogen (uPA) activator and plasminogen . HMGB1 iIn CKD, HMGB1 expression in renal tissue and HMBG1 levels in blood and urine are elevated . In COVIHMGB1 activation of RAGE in various human epithelial cell lines promotes mRNA expression of the SARS-CoV-2 receptor, ACE2, and this effect can be blocked by the RAGE inhibitor, FPS-ZM1 . The binThe RAGE ligand and complement factor, C1q, recognizes pathogens and apoptotic cells, either directly by binding to antigen-antibody complexes, or indirectly, through associations with pentraxins, such as C reactive protein (CRP). The C1q molecule consists of a collagen-like (cC1q) region that binds the receptor cC1qR and a globular head (gC1q) region that binds gC1qR . Anotherin vitro studies indicate that gC1qR binds to SARS-CoV-2 viral proteins   , which mocapsid) . Howeverocapsid) . It rematrans  and sRAGE are elevated in CKD, COVID-19 and many of the co-morbidities that increase the risk of disease severity. AKI and CKD increase the mortality risk in COVID-19 patients. Post-COVID-19 patients with long COVID are at heightened risk for AKI or CKD, and these conditions further increase the mortality risk. Diverse RAGE isoforms, present in plasma and in tissues, and a variety of RAGE ligands contribute to the pathogenesis of these syndromes. RAGE pathway activation is associated with cell injury and dysfunction, in diverse renal cell types, including glomerular cells , tubular epithelial cells, and endothelial cells. RAGE is expressed by infiltrating immune cells, where it promotes immune cell differentiation, recruitment, and activation. RAGE also forms complexes with AT1R, αVβ3, and LAIR-1, which may promote RAAS activity, oxidative stress, and anti-inflammatory responses, respectively. Additional downstream consequences of RAGE activity can include increased TGF-β1 production, epithelial mesenchymal transition, endothelial permeability, and significant systematic inflammation. These effects culminate in glomerulosclerosis, tubulointerstitial fibrosis, cell injury and ultimately organ failure. The accumulation of circulating RAGE ligands coupled with reduced kidney function promotes cognitive impairments and thrombotic complications, increasing disease severity and mortality. Interventions to reduce RAGE and RAGE ligand plasma and tissue levels, as tested in animal models, may offer novel approaches to slow or halt progression of CKD that develops in the absence, presence, or as a consequence of COVID-19.CC: methodology, conceptualization, investigation, table formatting, study curation, illustrations, and writing an original draft. JK: methodology, conceptualization, investigation, writing, and editing. All authors contributed to the manuscript and approved the submitted version.This work was supported by the National Institute of Diabetes and Digestive and Kidney Diseases, Intramural Research Program, and the National Institutes of Health Clinical Center.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.


S) which will certainly influence and expand therapeutic approaches in the future treatment of patients. Alpha or beta particles and Auger electrons are used for therapy purposes, and each has advantages and disadvantages. The radionuclides labeled drug delivery system will deliver the particles to the specific targeting cell. Different radioligands can be chosen to uniquely target molecular receptors or intracellular components, making them suitable for personal patient-tailored therapy in modern cancer therapy management. Advances in nanotechnology have enabled nanoparticle drug delivery systems that can allow for specific multivalent attachment of targeted molecules of antibodies, peptides, or ligands to the surface of nanoparticles for therapy and imaging purposes. This review presents fundamental radionuclide properties with particular reference to tumor biology and receptor characteristic of radiopharmaceutical targeted therapy development.Advances in the field of molecular biology have had an impact on biomedical applications, which provide greater hope for both imaging and therapeutics. Work has been intensified on the development of radionuclides and their application in radiopharmaceuticals (RP Therapeutic applications immediately followed this discovery . For manmia vera ,5,6. Radmia vera ,8. The dIn recent times, RPs use in nuclear medicine has become popular in theranostics. These are used in therapeutic interventions after imaging verifies the presence of a biological target ,9,10. UnRadiopharmaceutical therapy (RPT) is a novel modality that can be effective with minimal toxicity ,7. The aRPT can be used as adjuvant therapy with or after other treatment options such as chemotherapy and surgery . It is bThe physical characteristics of a radionuclide should be considered when selecting it for therapy purposes. These include physical half-life, radiation energy, type of emissions, daughter product(s), production method, and radionuclide purity ,9. IdealFurthermore, in vivo stability, toxicity, and the biological half-life within the patient’s body must be considered ,16, alonRadionuclides used in RPT are primarily beta (β)-particle 0.2 keV/μm) or alpha (α)-particle 50–230 keV/μm) emitters  keV/μm o,11,15,230 keV/μm 32P, 89Sr, 90Y, and 169Er [Beta particles have been used in cancer therapy over the last 40 years . They ar131I). Hertz and Roberts used radioiodine I-130 (130I) for hyperthyroid therapy in 1941, which rose at the birth of nuclear medicine [130I was replaced by 131I because it was much cheaper [131I is a β and γ emitter with a half-life of 8.05 days. The β particle has a peak energy of 0.606 MeV, with a maximum range of ~3 mm in the tissue, and it is used for therapy. The peak energy of the γ ray is 0.364 MeV and is used for imaging [131I has been used countless times for therapy for hyperthyroid and thyroid cancer [131I-iobenguane  was introduced as a diagnostic agent, and in 1984, it was used for treating malignant phaeochromocytoma [131I, and, in 2003, FDA approved 131I-tositumomab (Bexxar) for the treatment of refractory non-Hodgkin’s lymphoma (NHL) [90Y) and Lutetium-177 (177Lu), for more effective therapy purposes [The most familiar and frequently used β particle is iodine-131  . Severalpurposes ,33.90Y) or Rhenium-188 (188Re) is preferable for treating higher volume solid and poorly perfused tumors and is less suited for targeting micro-metastases to avoid crossfire doses to neighbor cells [90Y, widely available like 131I, is a popular radionuclide for liver cancer and metastases [S) have been treated with radionuclide therapy (PPRT) targeting peptide receptors with radiopharmaceuticals labeled with 90Y. Antibodies also labeled with 90Y, have been introduced for ovarian and hematological cancers [177Lu), is more efficient for small tumors [177Lu is becoming a popular ẞ-particle source for treating small tumors [177Lu has a half-life of 6.73 days and is compatible with antibodies and peptides [153Sm) is used to treat palliative bone metastases and other primary cancers [153Sm through six ligands (four phosphate groups and two amines). It has been widely used since FDA approval in various osteoblastic metastatic lesions, especially in prostate and breast cancer [The high-energy β from Yttrium-90  gained approval in 2013 . Alpha p4He nucleus without its surrounding electrons (sometimes denoted as (He2+)) [An alpha particle is a  (He2+)) ,45. Alph (He2+)) ,32,45,46 (He2+)) ,26,32. MFurthermore, intracellular accumulation of the α particles effectively creates double-strand breaks (DSBs) in DNA, and numerous clusters of DSBs in target cells, making cellular repair systems ineffectual ,32,47. T213Bi) and astatine-211 (211At) and are used to treat leukemia and brain tumors [213Bi successfully treated ovarian cancer in animal models with no signs of toxicity [213Bi has a short half-life and is produced using a generator and labeling to produce TAT compounds is therefore completed on-site [213Bi needs to be delivered directly into tumor tissue, and it can be given at a high dose over a short period, which is more effective than low dose rates given over a more extended period [213Bi has been used to label DOTA peptides in preclinical and clinical trials with >99% purity [213Bi and 225Ac have been used to label somatostatin receptors [Improvements in understanding molecular tumor biology, labeling techniques, technology development, and other related disciplines have paved the way for significant new clinical applications of α radiation as a novel therapeutic agent ,15,51. An tumors . The montoxicity . 213Bi h on-site ,54. Becad period ,32,55. 2% purity . In prececeptors ,32.223Ra can generate irreparable DNA double-strand breaks in the adjacent osteoblasts and osteoclasts, which has a detrimental effect on the adjacent cells and inhibits abnormal bone formation [223Ra is being studied as a radioactive label for other cytotoxic agents such as poly (ADP-ribose) polymerase inhibitors (olaparib), docetaxel , and new androgen axis inhibitors as enzalutamide and abiraterone citrate. The recently high number of 223Ra and in combination with other therapeutics, showed promising results [Radium-223 dichloride (Xofigo), a α particle emitter used for bone pain palliation in prostate and breast cancer patients, was approved by FDA in 2013 ,26,32,45ormation . 223Ra i results .225Ac, the parent of 213Bi, which is relatively long-lived, with a half-life of 9.9 days [255Ac is produced via the neutron transmutation of 225Ra or decay of 233U [225Ac can be used to treat neuroendocrine tumors. It has been used to label PSMA with a radiochemical purity of >98% for prostate cancer therapy [225Ac-labeled PSMA-ligands have gained popularity as an alternative to 177Lu-PSMA [225Ac may damage the healthy cells due to daughter radionuclides such as 221Fr, 217At, and 213Bi [Another alpha particle attracting increasing interest is 9.9 days . 255Ac i of 233U ,54,55. 2 therapy ,54,56. I therapy ,32,55. R therapy . Lately,7Lu-PSMA ,54,56. Hnd 213Bi . Danger 77Br), indium-111 (111In), iodine-123 (123I), and iodine-125 (125I) are the most commonly used radionuclides sources [Auger electrons (AE) have an even shorter range than alpha particles delivering radiation of only 1–1000 nm through the tissue causing potent tumor cell death if they can be conjugated with suitable ligands that effectively target micro-metastasis, particularly of DNA and cell membranes ,32,34,52 sources ,59,60. H125I-anti-CEA mAbs and in non-exposed cells through a bystander effect. 125I-labeled anti-CEA 35A7 was also found to be effective in vivo for treating small peritoneal tumors in mice [Despite the short range of AE, local cross-dose effects occur in cells adjacent to the radionuclide decay mediated by the several micrometer ranges of higher energy AEs and internal conversion (IC) that causes the death of distant non-irradiated cells through the bystander effect ,25,27,32 in mice ,63. Toxi in mice ,25,57.So, AE nuclear targeting is essential but not always required for RPT . The absRadiopharmaceutical or radioligand therapy includes systemic radiation therapy, molecular radiotherapy, targeted radiation therapy, or peptide receptor radionuclide therapy (PRRT) and there are examples of where RPT is applied in optimizing and balancing the therapeutic index (TI). Various radioligands are being developed and investigated to target molecular receptors or intracellular components in personal therapy .90Y, and tositumomab (Bexxar) labeled with 131I) is used for lymphoma therapy [The monoclonal antibodies (mAbs) are labeled with radionuclides. The smaller fragments and new fusion proteins are directed against tumor antigens to deliver radionuclides to the targeted tumor ,9. The F therapy ,55,64,65The effect of the RPT depends on the radiation’s energy and the antibody’s affinity, antigen target concentration on the cells, tissue vascularity, and antibody/antigen rate constants . Novel a227Th-anti CD22 and 225Ac-PSMA-617 [223Ra and 227Th do [Patient selection for RPT should be based on the predetermined expression of specific tumor antigens or diagnostic results . SeveralPSMA-617 . However227Th do . Unfortu227Th do ,64.Radionuclide-labeled mAbs demonstrate more efficacy in inducing cancer remissions than unlabeled molecules and are also more effective than chemotherapy . They ha131I-labeled prostate-specific membrane antigen (PSMA) ligands showed promise for prostate cancer therapy and were further developed to ”the 177Lu-PSMA” introduced in 2015 [177Lu-PSMA is safe and effectively reduces the tumor burden. It has low toxicity [177Lu-PSMA, including the salivary glands and the kidneys. However, the radiation dose to bone marrow, spleen, and liver is below critical limits [ in 2015 . PSMA is in 2015 ,66,67,68 in 2015 ,66. Radi in 2015 ,56,66,69177Lu [225Ac maybe have an advantage compared to PSMA-targeting ligands using β particles. Clinical studies using 225Ac-labeled PSMA-ligands (PSMA-617 or PSMA-I&T) have demonstrated remarkable therapeutic results recently. Data on treatment with 225Ac-PSMA-617 indicate an excellent effect on tumor control in both early and late-stage mCRPC [227Th-PSMA has shown potency in in vitro studies and efficacy in xenograft models of prostate cancer [Currently, the two most frequently used PSMA ligands are PSMA-617 and PSMA-I&T (imaging and therapy), labeled with 177Lu . PSMA- tge mCRPC . A novele cancer ,67. Howee cancer . Concerne cancer ,68. Combe cancer ,71,72.177Lu and 90Y. 177Lu–SSTR ligand is more effective in small-sized tumors, whereas, for larger tumor volumes, 90Y might be a better choice [90Y-labeled DOTATOC and DOTATATE. However, significant permanent kidney damage has been reported [177Lu-labeled DOTATATE or DOTATOC was the next PRRT radiopharmaceutical, causing less nephrotoxicity compared to 90Y [177Lu-DOTATATE (Lutathera®) has also become the most widely used PRRT radiopharmaceutical at present [Receptor-based radionuclide therapies (PRRT) targeting the somatostatin receptor (SSTR), have since early 1990 been an important treatment modality for neuroendocrine tumors ,26. The r choice ,73. The reported ,74. 177L present .213Bi and 225Ac have been clinically tested for brain tumors, neuroendocrine tumors, and prostate cancer therapy [213Bi and 225Ac-DOTA chelated peptides have been developed for peptide receptor radiotherapies, such as DOTA-Substance P targeting the neurokinin-1 receptor and somatostatin-analogs  [Overall, α-emitters PRRT has shown good results. However, crossfire effects on small-size tumors have a significant impact. Additionally, hypoxia tumor tissue could be resistant to β-emitters treatment. α particles with high LET over a short range can minimize damage to surrounding healthy tissue.  therapy . 213Bi aOTATATE) . However131I has been used for adjuvant therapy to manage well-differentiated thyroid cancer (DTC) for more than 60 years. It is used to destroy remaining thyroid cells post-thyroidectomy, including in metastases, and is relatively inexpensive and widely available [vailable ,75. It ivailable , indicatvailable ,77.+) and one anion of iodide (I-) into the cells. This process is facilitated by an enzyme Na+/K+ ATPase [A sodium iodide symporter (NIS) transports iodine through the cell membrane. Iodine is transported into follicular thyroid cells against the electrochemical gradient ,75,76. I+ ATPase ,29,75,76131I in managing radioiodine refractory disease in DTC [Genetic alteration causes the mitogen-activated protein kinase (MAPK) and phosphoinositide 3-kinase (PI3K) pathways associated with the silencing of solute carrier family five-member 5 (SLC5A5), which encodes NIS. The condition causes the cancer cell failure to take radioiodine . A clinie in DTC .131I therapeutic effect [Furthermore, NIS transgene has been successfully transferred selectively into extra-thyroidal tumor cells or cells in the tumor environment using various gene delivery systems . An advac effect .In the last three decades, there has been a rapid increase in the use of new nanomaterials and radionuclides to enhance cancer diagnosis and therapies ,79. ManyNanotargeted radionuclides have three main components, the nanoparticle core, the targeting biomolecule , and the radionuclide . NanoparThe targeting biomolecule must have a high affinity for the targeted epitopes. For ligands to bind effectively, each radionuclide can be conjugated directly on the nanoparticle surface, with or without a spacer, or can be attached to the nanoparticle during chemical synthesis. The spacer groups between the nanoparticle surface, the radionuclide, or the biomolecule can be a simple hydrocarbon chain, a peptide sequence, or a PEG linker ,81,82. I131I labeled nanoparticles for targeted therapy of different tumor types, according to the targeting strategy of the prepared NPs in which 131I is incorporated. The targeting strategy of these NPs depends on either passive or active targeting. 131I labeled NPs (silver or polymeric) shows 131I accumulation in different tumor types [131I labeled NPs targeting integrin have been studied. This protein is essential in regulating angiogenesis processes and tumor progression. Radionuclide labeled arginine–glycine–aspartate (RGD) can specifically target tumor integrin receptors [188Re, Holmium-166 (166Ho), 90Y, and gold-198 (198Au)-NPs have also been investigated for tumor-targeted radiotherapy [88Re-liposome has been shown to have a therapeutic effect in various animal models and translational clinical research [166Ho nanoparticles have also been prepared and studied in radionuclide tumor therapy for skin cancer and ovarian cancer metastases [90Y has been investigated for colon and melanoma tumors in animal models [198Au NP have been shown to be potential prostate cancer therapeutic agents in an animal model [There are many passive and active targeted nanoparticle therapies being developed. Most development is still at the in vitro or animal study stage. The most significant development is of or types . 131I laeceptors . Other βotherapy ,82. 88Reresearch . 166Ho ntastases ,82. Lipol models . Gum araal model .211At has been studied as a prospective NP’s alpha particle emitter, but the main disadvantage of 211At for NTR is low in vivo stability [223Ra, showing a cytotoxic effect on glioma cells [225Ac-Au@TADOTAGA administrated intratumoral delayed tumor growth in glioma xenografts, and it is the first reported study using 225Ac-labeled gold nanoparticles [225Ac use remains challenging for insufficient retention of its daughter’s products due to the α recoil effect observed upon release of an α particle [Nanoparticles labeled with α-particle emitters have been synthesized to enhance therapeutic efficiency with minimum danger to healthy tissues. tability . The sodma cells . A preliarticles . Howeverparticle . So, theparticle .In the last three decades, there has been a growing interest in radioiodine, a beta and gamma emitter, as new RPs are introduced for therapy and imaging (theranostics) for specific target tumor cells. However, one must be aware of issues related to the crossfire effect and toxicity of ß particles. The high LET and short range of α particles enable effective and rapid cancer therapies but are hindered by short half-lives and rarely emit gamma radiation for imaging ,15,55. CThe development of intelligent drug delivery agents such as peptides, small molecules, mAb, and mAb fragments, and especially nanoparticle cores offer the promise of better diagnostic and therapeutic options ,81. HoweRadiopharmaceuticals provide effective cancer treatment, particularly when other standard therapeutic approaches have failed. However, even after more than four decades of clinical investigation, RPs have still not become a standard part of cancer management therapy, which is peculiar, especially in light that other “targeted therapy” have clinical trial failure rates of 97% and is more popular than RPs that ,85. FurtRadiopharmaceutical therapy can be a safe and effective targeted approach to treating many types of cancer. RPT has shown high efficacy with minimal toxicity compared to other systemic cancer treatment options. Different radioligands can be chosen to uniquely target molecular receptors or intracellular components, making them suitable for personal patient-tailored therapy in modern cancer therapy management. Further research is still needed regarding specific targets, radioligand stability in vivo, toxic effects, crossfire, dosimetry, and bond stability with daughter nuclides, particularly for alpha emitters.However, new particle drug delivery systems continue to enhance targeted therapy efficacy and safety, including the use of nanoparticles. The number of successful studies exploring new drug delivery agents’ different delivery systems of radionuclide particles will probably increase the effectiveness and range of applicants. With a growing positive track record, public understanding and perception of the safety and success of RPT may improve.If this occurs, then RPT will be adopted as an increasingly mainstream cancer therapy approach and the investment needed to resolve issues of radionuclides supply. In the coming decades, RPT may provide an increasing variety of rapid, personalized, practical, effective, and affordable treatments that offer new hope to cancer patients.


Atherosclerosis is a progressive inflammatory disease leading to mortality and morbidity in the civilized world. Atherosclerosis manifests as an accumulation of plaques in the intimal layer of the arterial wall that, by its subsequent erosion or rupture, triggers cardiovascular diseases. Diabetes mellitus is a well-known risk factor for atherosclerosis. Indeed, Type 2 diabetes mellitus patients have an increased risk of atherosclerosis and its associated-cardiovascular complications than non-diabetic patients. Sodium-glucose co-transport 2 inhibitors (SGLT2i), a novel anti-diabetic drugs, have a surprising advantage in cardiovascular effects, such as reducing cardiovascular death in a patient with or without diabetes. Numerous studies have shown that atherosclerosis is due to a significant inflammatory burden and that SGLT2i may play a role in inflammation. In fact, several experiment results have demonstrated that SGLT2i, with suppression of inflammatory mechanism, slows the progression of atherosclerosis. Therefore, SGLT2i may have a double benefit in terms of glycemic control and control of the atherosclerotic process at a myocardial and vascular level. This review elaborates on the anti-inflammatory effects of sodium-glucose co-transporter 2 inhibitors on atherosclerosis. Atherosclerosis is a widespread chronic inflammatory disorder of large- and medium-caliber artery walls with a complex biochemical and cellular etiology . It is cTherefore, to date, it still represents the major potential risk factor of most CVDs, including myocardial infarction (MI), heart failure (HF), stroke, and peripheral arterial disease, causing disability and mortality worldwide .Cardiovascular diseases are the leading cause of death globally. Over 17.9 million people died yearly from CVDs in 2019, representing 32% of all deaths worldwide. 85% of these deaths were due to heart attack and stroke . CVDs acAtherosclerosis results from predisposing genetic factors and exposure to oxidative and inflammatory damage mediators. In addition, modifiable and non-modifiable risk factors, which include age, sex, cigarette smoking, unhealthy diet, physical inactivity, dyslipidemia, diabetes mellitus, hypertension, and obesity, contribute to its beginning and development . DiabeteThis review reports experimental and clinical evidence to clarify the anti-inflammatory mechanisms underlying the anti-atherosclerotic effect of SGLT2i.In vitro and animal studies, clinical trials, reviews, meta-analyses, and guidelines were reviewed. We evaluated the anti-inflammatory effects of SGLT2i from experimental evidence  and clinical trials. For the clinical trials, we considered the cardiovascular outcomes, including myocardial infarction, stroke, peripheral artery disease, cardiovascular death, or hospitalization for heart failure. Articles not in the English language or meeting abstracts were excluded.A literature search was conducted by searching updated and relevant publications on SGLT2i and atherosclerosis in databases, including PubMed and Google Scholar. During this research, keywords such as “SGLT2 inhibitor, empagliflozin, dapagliflozin, canagliflozin, ertugliflozin, ipragliflozin, luseogliflozin, sotagliflozin, atherosclerosis, atherosclerosis cardiovascular diseases, inflammation, coronary heart disease, stroke, angina pectoris, myocardial infarction and peripheral artery disease, major adverse cardiac events (MACE), cardiovascular (CV) mortality” were used. Atherosclerosis disease can be schematized in three stages, starting from the endothelium activation/dysfunction and resulting in plaque formation .In the initial stages of the lesion, endothelial dysfunction occurs under harmful stimuli such as hypertension, dyslipidemia, and disturbed shear stress resulting in a chronic inflammatory state. Parallel changes in endothelial permeability promote oxidation of low-density lipoproteins (LDL), followed by infiltration of monocytes in the intimal layer . OxidizeInversely, M2 macrophages are associated with an anti-inflammatory phenotype being polarized in response to IL-4 and IL-13 and producing anti-inflammatory factors such as the IL-1 receptor agonist, transforming growth factor beta (TGF-β), and IL-10 . ConsequIn addition, the lipids accumulation in macrophages, with associated foam cell formation, results in the activation of the nucleotide-binding domain-like receptor protein 3 (NLRP3) inflammasome complex, which promotes the release of pro-inflammatory cytokines contributing to the development and progression of atherosclerosis . Indeed,Moreover, in the atherogenesis process, oxidative stress and cellular senescence contribute to the maintenance of inflammation and endothelial dysfunction. Indeed, reactive oxygen species (ROS) induce both the synthesis of pro-inflammatory cytokines and stimulate the expression of adhesion molecules, thus allowing monocytes to transmigrate into the vessel wall . In addiSenescence-inducing stress can result from various cardiovascular risk factors . Indeed,In the second stage, characterized by plaque progression, smooth muscle cells produce extracellular matrix components contributing to plaque thickening and progressive growth in the vessel's lumen. In addition, macrophages and smooth muscle cells undergo apoptosis by going on to form a central lipid-rich core. Therefore, in the late stage, the plaque consists of a central lipid core and a fibrous cap . Pro-infTherefore, it is possible to distinguish two types of plaques:- Stable plaques, which possess a thick fibrous cap. It can regress with the proper lifestyle and/or drug therapy or may progress by occluding the vessel lumen.- Vulnerable or unstable plaques are rich in macrophages and possess a thick lipid core and a thin fibrous cap. It is more likely to undergo rupture and complications .As a result, the cardiac complications of atherosclerotic disease are related to (I) reduced blood flow due to an insufficient oxygen supply, such as angina pectoris; (II) plaque can occlude the vessel leading to ischemia of downstream tissues, for example, in myocardial infarction or stroke; (III) detachment of part of the thrombus, termed an embolus can result in occlusions of distal arteries as pulmonary embolism .The SGLT2i are a new category of anti-diabetic drugs, approved by the Food and Drug Administration (FDA), used to treat patients with type 2 diabetes . SGLT2i Sodium-glucose co-transport 2 inhibitors, targeting the major glucose transporter SGLT2 in the kidney, block glucose reabsorption in the proximal renal tubule, increasing glycosuria independently of insulin sensitivity and secretion . The SGL+, inhibits proteins in a reversible, competitive way  and the FDA: canagliflozin, dapagliflozin, and empagliflozin .In addition to structural differences, these compounds present variable selectivity to SGLT1 and SGLT2. Empagliflozin has the greatest selectivity for SGLT2  compared to SGLT1. The binding affinity of dapagliflozin for SGLT2 is 100-fold higher than that for SGLT1, and Canagliflozin shows an SGLT2 selectivity of 250-fold .Despite different selectivity, SGLT2i share similar pharmaco-kinetic characteristics, such as rapid oral absorption, a long half-life, extensive hepatic metabolism, low renal elimination, and the absence of drug-drug interactions .Besides their effects on blood glucose, preclinical and clinical studies have demonstrated that SGLT2i therapy provides extra-glycemic effects and clinical advantages in patients with diabetes , such asIn addition, SGLT2 inhibitors have been convincingly demonstrated to reduce main adverse cardiovascular events and the occurrence of heart failure with reduced ejection fraction (HFrEF), independently of glycemic status . InteresSeveral studies have been conducted to establish underlying mechanisms of SGLT2i cardio-protective effects. In particular, studies conducted in animal models and patients with diabetes demonstrated the anti-atherosclerotic action of SGLT2i, revealing some potential mechanisms.Nevertheless, the molecular mechanisms are still unclear . This gaThe mechanisms underlying the SGLT2 benefit on atherosclerosis could be multifactorial and due to attenuation of secretion of inflammatory molecules, reduction of macrophage infiltration, improvement of autophagy impairment, and inhibition of endothelial dysfunction  out of tTherefore, considering the clinically beneficial effects of SGLT2 on atherosclerosis, we provide an insightful overview focusing on mechanisms by which SGLT2i may act on inflammation in atherosclerosis and atherosclerosis cardiovascular diseases.As described before, inflammation is a significant factor in vascular cell dysfunction, causing the development and progression of atherosclerosis in diabetes .The atherosclerosis treatment from an inflammatory perspective could be a valid therapeutic strategy. Clinical trials have demonstrated that modulation of inflammation can prevent atherosclerosis and its complications.Recent studies have shown an improvement in inflammatory and oxidative status in subjects with T2DM treated with SGLT2i regardless of glycemic control . These dTherefore, inflammation may be considered a mechanism by which SGLT2i can exert their protective effects against atherosclerosis. This role can be discussed regarding the impact of SGLT-2i on systemic inflammation and immune signaling pathways, including changes in local atherosclerotic tissues.Experimental evidence revealed that SGLT2i reduced the expression of circulating inflammatory molecules, such as TNF-α, monocyte chemoattractant protein 1 (MCP-1), platelet endothelial cell adhesion molecule-1 (PECAM-1), VCAM-1, intercellular adhesion molecule 1 (ICAM-1), IL-1β, and IL-6 –50. SeveHuman umbilical vein endothelial cells (HUVECs) and macrophages exposed to dapagliflozin and LPS showed attenuate levels of LPS-induced TLR-4 expression, NF-κB p65 phosphorylation, and miR-155 and elevated levels of miR-146a. Moreover, dapagliflozin shifted from inflammatory M1 macrophages toward M2-dominant macrophages .In LPS-stimulated RAW 264.7 macrophages, empagliflozin in association with gemigliptin and Dipeptidyl peptidase-4 (DPP-IV) inhibitor reduced gene expression and pro-inflammatory cytokine and chemokine release through the IKK/NF-κB/JAK2 - STAT1/3, and MKK4/7- JNK pathways .Similar results were also obtained in mouse models. In nicotinamide and streptozotocin (NA/STZ)-treated ApoE KO mice, the treatment with luseogliflozin reduces the expression of inflammation-related genes, including F4/80, TNFα, IL-1β, IL-6, ICAM-1, PECAM-1, MMP2 and MMP9 .In STZ-treated ApoE–/– mice, dapagliflozin inhibited IL-1β and IL-18 secretion, blocking the ROS-NLRP3-caspase-1 pathway showing that NLRP3 inflammasome complex could be considered an SGLT2i target , 54.ex vivo experiments with macrophages demonstrated that empagliflozin inhibited NLRP3, increasing BHB levels and reducing glucose, uric acid, and insulin , and the other is Ca+ dependent. Specifically,  insulin . Empaglirecovery .Moreover, it was demonstrated that also dapagliflozin reduced the production of NLRP3 protein by activating mTOR Complex 2 (mTORC2), leading to the activation of AMPK and Forkhead box 3 (FOXO3) , 58.Considerable evidence showed that SGLT2i ameliorated endothelial dysfunction and improved endothelium-dependent vasodilation. In particular, in diabetic ApoE –/– mice, empagliflozin improved endothelial function by reducing CD68, MCP-1, ICAM-1, and TNF-α, suppressing the development and progression of atherosclerotic lesions . In a HiEvidence revealed that SGLT2i attenuates macrophage infiltration and inflammation, foam cell formation, and M1 polarization, crucial steps in the development of atherosclerosis. It was demonstrated that empagliflozin reduced macrophage infiltration and CD36 gene expression resulting in the loss of macrophage capability to scavenger ox-LDL and foam cell formation in db/db mice . A similMoreover, dapagliflozin reduced macrophage infiltration and induced M2 polarization with a concomitant decrease in M1 through a mechanism depending on a RONS-dependent STAT3 pathway. These data demonstrated that dapagliflozin induced the production of anti-inflammatory factors participating in inflammation prevention and tissue repair .Similar results were demonstrated in white rabbits, where dapagliflozin increased M2 macrophages and inhibited the expression of TLR4 and NF-κB .However, the exact mechanism by which SGLT2i can induce M2 polarization and reduce macrophage infiltration is not fully elucidated, and many possible hypotheses are formulated. Indeed, reducing glucose levels, the principal energy source of macrophages, might have a role  Figure ; Table 1Several clinical studies have been conducted to evaluate the changes in the main inflammatory and oxidative stress biomarkers to evaluate the potential role of SGLT2i in protecting against atherosclerosis. The inflammatory markers implicated and therefore assessed in the atherosclerosis process are CRP, IL-6, and TNF-α.In a single-center, open-label, randomized, prospective study, the administration of empagliflozin (10 mg/day for 12 months) to 51 diabetic patients induced a significant reduction of blood hs-CRP levels associated compared with baseline and placebo (−74.4% vs. placebo and −55.6% vs. baseline) .p = 0.002, 6 months: p = 0.001, 12 months: p = 0.007)  induced a significant decrease in hs-CRP after 3, 6, and 12 months compared with baseline (3 months: = 0.007) .p = 0.002 vs. p = 0.27) and TNF-alpha (p = 0.002 vs. p = 0.29), while canagliflozin was more effective in reducing HbA1c   .Moreover, atherosclerotic plaques of diabetic patients treated with canagliflozin displayed increased SIRT 6 expression and lower oxidative stress and inflammation markers  Table 2Table 2.Atherosclerotic cardiovascular diseases (ASCVD) represent a very important cause of death and disability, especially in subjects affected by T2DM. The main manifestations of ASCVD are coronary heart disease, ischemic stroke, peripheral artery disease, and heart failure .Coronary artery disease (CAD) is the most common atherosclerotic vascular disease, and it includes two main clinical phenotypes: stable/unstable angina and acute MI (AMI). Data regarding MI in patients treated with SGLT2i are conflicting. In the EMPA-REG OUTCOME study and CANVAS study, no significant difference in MI incidence was observed between the treated and placebo group  , 75. HowThe effects of early SGLT2i treatment in patients with recent AMI were not well-studied.Early SGLT2i treatment might improve cardiovascular outcomes through its beneficial effects on endothelial function, neurohormonal activation, cardiomyocyte necrosis, and reperfusion injury . Three s- EMMY  trial (NCT03087773)- DAPA-MI (Dapagliflozin Effects on Cardiovascular Events in Patients With an Acute Heart Attack) (NCT04564742)- EMPACT-MI  trial (NCT04509674).The recent European clinical guidelines on HF have approved SGLT2 inhibitors for treating HFrEF and preventing HF hospitalizations independently of diabetes , 81–83. However, in the VERTIS-CV study, the reduction of HF hospitalization was not statistically significant because the endpoint was a composite of cardiovascular death and hospitalization for HF .Other studies, such as DAPA-HF and EMPEROR-Reduced, also provide convincing evidence of the beneficial effects of SGLT2i on HF , 87. A mData regarding the risk of stroke in patients treated with SGLT2i are conflicting. The EMPAREG OUTCOME study did not show a significant reduction in the incidence of stroke in patients treated with empagliflozin compared to the placebo group  , 85.However, the CVD-REAL and the CVD-REAL2 studies showed a lower risk of stroke associated with SGLT2i therapy   , 77.However, the analysis conducted by these studies did not consider the SGLT2i effect on different types of stroke , making the data conflicting.Therefore, a systematic review and meta-analysis were performed to evaluate the stroke subtypes. Considering five studies, CANAVAS, CREDENCE, DECLARE-TIMI, EMPAREG AND VERTIS- CV, they found that SGLT2i reduced the incidence of hemorrhagic stroke and Atrial Fibrillation (AF) by 0.5%. This could be related to the natriuretic effect and subsequent reduction in blood pressure. However, Further studies aimed at evaluating the impact of SGLT2 on this pathology will be needed .Peripheral artery disease (PAD) is a manifestation of systemic atherosclerosis, characterized by progressive narrowing to occlusion of arterial vessels in the limbs, resulting in ulceration and subsequent gangrene and amputation.Peripheral artery disease increases the risk of coronary artery and cerebrovascular disease and is an independent predictor of CVD death . The EMPHowever, the CANAVAS and CANAVAS-R studies reported that amputation risk was twice in the treated group compared to the placebo group  . AlthougThe most important cardiovascular trials for SGLT2 evaluated the incidence of major adverse cardiac events (MACEs), such as cardiovascular death, non-fatal myocardial infarction, and non-fatal stroke. A meta-analysis of 6 placebo-controlled clinical outcomes trials  , 92, 93,Four large cardiovascular outcome studies have recently been completed: EMPAREG OUTCOME, CANVAS, DECLARE-TIMI, and VERTIS CV , 92, 93.Sodium-glucose co-transport 2 inhibitors exert anti-atherosclerotic properties attenuating inflammatory factors, alleviating inflammation, mitigating insulin resistance, and reducing stress on vessels, inhibiting atherosclerosis development and progression . Recent Moreover, cardiovascular safety studies conducted with SGLT2i suggested that subjects affected by T2DM could benefit from SGLT2i treatments with positive effects on cardiovascular events . UnfortuAtherosclerosis is a chronic inflammatory disorder representing the major potential risk factor of CVDs. The progress in managing the atherosclerosis complications has extended life, but many individuals still present impaired cardiac function, straining healthcare systems and resources . In thisTherefore, in conclusion, SGLT2 inhibitors can represent up-and-coming therapeutic drugs with pleiotropic effects in terms of metabolic control and reduction of associated cardiovascular complications.LSc, VC, and MB: concept and design. LSc, VC, FT, RF, APe, PG, MF, APu, AD, LSp, RM, and MB: drafting of the manuscript. All authors have read and agreed to the published version of the manuscript.This study was funded by PON Ricerca e Innovazione 2014–2020 ARS01_01270.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.


In-situ hybridization provides a convenient and reliable method to detect human papillomavirus (HPV) infection in formalin-fixed paraffin-embedded tissue. Cases of conjunctival papillomas, conjunctival intraepithelial neoplasia (CIN), conjunctival carcinoma in situ (cCIS), and invasive squamous cell carcinoma (SCC), in which low-risk (LR) and/or high-risk (HR) HPV types were evaluated by RNA or DNA in-situ hybridization, were retrospectively identified. LR HPV types were frequently detected in conjunctival papillomas , including 17/18 (94%) with RNA probes, compared to 8/12 (75%) with DNA probes. None of the CIN/cCIS or SCC cases were positive for LR HPV by either method. HR HPV was detected by RNA in-situ hybridization in 1/16 (6%) of CIN/cCIS cases and 2/4 (50%) of SCC cases, while DNA in-situ hybridization failed to detect HPV infection in any of the CIN/cCIS lesions. Reactive atypia and dysplasia observed in papillomas was generally associated with the detection of LR HPV types. Collectively, our findings indicate RNA in-situ hybridization may provide a high-sensitivity approach for identifying HPV infection in squamous lesions of the conjunctiva and facilitate the distinction between reactive atypia and true dysplasia. There was no clear association between HPV infection and atopy in papillomas or dysplastic lesions. The ocular surface barrier, predominately comprising the conjunctival epithelium, is uniquely vulnerable to chronic environmental exposure and microbial infection ,2. As a Atopy has also been reported as a risk factor in OSSN development. Several proposed mechanisms for atopy-associated tumorigenesis have been hypothesized, including pruritus-induced epithelial microtrauma with secondary HPV infection, type I hypersensitivity-mediated inflammation and tissue remodeling, and competitive binding of IgE receptors by allergen-specific IgE, limiting anti-tumor immune responses ,24,25,26It has been estimated that as many as 400 types of HPV, a small, spherical, non-enveloped, double-stranded DNA virus, exist, and approximately 200 have been fully characterized, with mucosal types grouped into low- (LR HPV) and high-risk (HR HPV) subtypes based on potential for malignant transformation ,31,32,33A number of studies suggest HPV, alone, is sufficient as a causative agent for conjunctival lesions; however, others propose a potential two-hit etiology, requiring prior UVR damage, chemical mutagenesis, or immunosuppression in addition to HPV infection ,43,44,45RNA in-situ hybridization permits direct visualization of intralesional transcriptionally active virus, with probes capable of detecting E6 and E7 mRNA, and provides morphologic evaluation of nuclear HPV localization within virally infected neoplastic cells ,65,66,67HPV in-situ hybridization was performed on 53 lesions, including 33 papillomas, 16 CIN/cCIS, and 4 invasive SCCs. Demographic and clinicopathologic information for these patients is provided in Representative clinical photographs and photomicrographs of each type of lesion are presented in In-situ hybridization using DNA probes was performed in 14/33 (42%) of papillomatous lesions and 4/16 (25%) CIN/cCIS lesions. In-situ hybridization using RNA probes was performed in the remaining lesions: (19/33 (58%) papillomas, 12/16 (75%) CIN/cCIS, and 3/3 invasive SCCs). Low-risk types were detected in 25/30 (83%) papillomas, 0/11 CIN/cCIS, and 0/1 invasive SCCs. High-risk types were detected in 2/28 (7%) papillomas, 1/16 (6%) CIN/cCIS, and 2/4 (50%) invasive SCCs. Detection of LR and HR HPV types was mutually exclusive in papillomatous lesions, and four lesions (12%) were negative for both LR and HR HPV.HPV signal presented in epithelial cells as dot-like foci visible at 100× magnification in the nucleus. Multiple dots in at least a subset of cells were required to call a case positive. In some cases, the small intracellular signals were quite numerous, and could become almost confluent within a single nucleus. Staining was restricted to epithelial cells, and stromal elements, including blood vessels and mesenchymal cells, were negative in all cases examined. In cases containing normal conjunctiva adjacent to a lesion, the non-neoplastic epithelium was also always negative.With respect to the extent of staining within individual lesions, HPV was present diffusely in most papillomas. Focal HPV positivity was observed in 8/25 (32%) LR HPV-positive papillomas and 1/2 (50%) HR HPV-positive papillomas, while diffuse staining was noted in 17/25 (68%) and 1/2 (50%) LR and HR HPV-positive papillomas, respectively. In contrast, HPV expression was more localized in the OSSN cases that contained virus. Focal HPV positivity was observed in the single HR HPV-positive CIN (Case 39) and both HR HPV-positive invasive SCC lesions. Further details of HPV in-situ hybridization are summarized in Conjunctival papillomas can become quite inflamed, leading to reactive epithelial atypia, in which epithelial cells show generally mild nuclear enlargement and pleomorphism, along with some chromatin changes and more prominent nucleoli. Reactive atypia can sometimes be difficult to distinguish from dysplasia, although the latter process is often less uniform and more severe. Detection of LR HPV could potentially be helpful in such cases and support a less aggressive biological potential if positive. In 18 of 33 (55%) papilloma cases, intraepithelial inflammation and epithelial atypia diagnosed as reactive were detected. Dysplasia was rarer, and noted in 4/25 (16%) LR HPV-positive papillomas, 1/2 (50%) HR-positive papillomas (Case 29), and none of the HPV-negative papillomas. In three LR HPV-positive papillomas diagnosed with dysplasia, the focal in-situ hybridization signal corresponded to the foci of dysplastic epithelium, while in Case 33 and Case 39, discrete foci of epithelial dysplasia were observed, but more diffuse LR and HR HPV signal was noted. An inflamed papilloma with reactive atypia and LR HPV-positive epithelium is demonstrated in Koilocytic cells were identified in all lesion types but were relatively rare, with focal change observed in 2/6 (33%) of HPV-negative papillomas, 10/25 (40%) LR HPV-positive papillomas, 1/15 (7%) HPV-negative CIN/cCIS, and 1/2 (50%) HR HPV-positive invasive SCCs. More diffuse koilocytic-appearing cells were observed in 1/25 (4%) LR HPV-positive papillomas, as shown in Patients returned for follow-up care in 17/33 (52%) papilloma cases, 8/16 (50%) CIN/cCIS cases, and none of the invasive SCCs. The length of follow-up (mean ± SD) was 605 ± 1219 days and 887 ± 1534 days for papilloma and CIN/cCIS cases, respectively. Recurrence was noted in only one case (Case 14) following an initial diagnosis of an HPV-negative papilloma. The recurrent lesion was originally treated with excision and subsequently observed.History of atopic disease, including asthma, eczema, or allergic rhinitis or conjunctivitis, was also recorded for patients when possible. Electronic medical records were available for review for 18/33 (56%) of all papillomas, including 3 HPV-negative lesions, 14 LR HPV-positive lesions, and 1 HR HPV-positive lesion. Atopic disease, including asthma (1/3), rhinitis/conjunctivitis (1/3), and a combination of the two (1/3), was reported in patients with HPV-negative papillomas. A history of asthma was present in 9/14 (64%) patients with LR HPV-positive papillomas, 2 of whom also had additional atopic disease, including eczema and rhinitis/conjunctivitis, respectively. Two additional patients with LR HPV-positive papillomas had histories of eczema and rhinitis/conjunctivitis, alone. There was no reported history of atopic disease in the single HR HPV-positive papilloma. For cases of CIN/cCIS, there was no history of atopic disease in the HR HPV-positive lesion that had medical records available for review.The prevalence of HPV infection in conjunctival epithelial proliferations has been difficult to establish, due, at least in part, to the range of detection methods used ,72. To sConjunctival papillomas can occasionally become inflamed and show prominent reactive atypia, which can be difficult to distinguish from clinically worrisome dysplasia. Among the 33 papillomas we examined, generally mild dysplasia was identified in 5 cases (15%). This number may be elevated due to selection bias, as papillomas with concerning microscopic features were more likely to have had HPV status analyzed during the study period. LR HPV was detected in four of five potentially dysplastic papillomas, while HR HPV was only detected in one. This suggests that the majority of conjunctival papillomas with atypia or dysplasia are associated with LR HPV, and that in-situ analysis could be useful in supporting limited malignant potential in many of these cases. Similar to these findings at the ocular surface, LR HPV was detected in the majority of papillomas, including those with atypical features of the nasopharynx, oral cavity, and larynx, while HR types are more commonly detected in invasive SCCs of these tissues ,82,83,84The reported prevalence of HPV in CIN and other more aggressive OSSN is more variable, with positive cases almost always containing high-risk types ,34,35,85Immunohistochemical approaches, including direct detection of HPV-encoded proteins or alterations in p16 expression, have been investigated, but more recent studies suggest that these methods are prone to false-positive results ,58,59,82Many demographic and clinical associations in our study were similar to prior reports. Papillomas of the conjunctiva reportedly occur more commonly in younger patients, while more dysplastic OSSN lesions tend to present in older ones, and our cases had a mean age of 43, 68, and 74 years at the time of presentation for papillomas, CIN/cCIS, and invasive SCCs, respectively ,77,87. PLimitations of the current study include the relatively small number of invasive SCC cases, precluding the possibility of statistical comparisons between groups. In addition, detailed review of electronic medical records was not possible for all cases in this series, as many were older pathology consultations submitted by outside physicians. While RNA in archived formalin-fixed paraffin-embedded tissue is labile and sensitive to degradation, potentially affecting the sensitivity of in-situ hybridization, we do not believe this is of concern in our study, as all in-situ hybridization assays were performed around the time of initial histologic review ,65.A retrospective review of the Ophthalmic Pathology Archives of The Johns Hopkins Hospital for cases matching keyword searches including “conjunctiva,” “in-situ hybridization,” and “human papillomavirus” between 2007–2021 was conducted. Electronic medical records were reviewed for the following clinical and pathologic information: age at presentation, gender, lesion type and location, clinical treatment modalities, length of clinical follow-up, history of recurrence, history of atopic disease, HPV in-situ hybridization assay details, and details regarding any additional immunohistochemical stains. Any case for which in-situ hybridization was performed to detect LR and/or HR HPV in a conjunctival papilloma, CIN/cCIS, or invasive squamous cell carcinoma was included. Cases were reviewed to confirm the initial diagnosis, and intralesional koilocytic or dysplastic features were noted.HPV in-situ hybridization was performed by the Johns Hopkins Hospital clinical laboratories at the time of initial review of histology using standard protocols. Lesions assayed prior to 2015 were subject to DNA ISH , and those evaluated from 2015 and onward were analyzed using RNA probes . Both DNA and RNA probes for LR HPV detect the same two types , while DNA probes for HR HPV detect 12 types  and RNA probes detect 18 HR HPV types . While 37/53 lesions (70%) were evaluated for both LR and HR types, evaluation by either DNA or RNA approaches was mutually exclusive. The commercially available probes utilized in this study were designed to hybridize with E6/E7 in the various HPV types. All determinations of in-situ hybridization positivity were performed by an ocular pathologist (CGE).We confirmed that LR HPV is frequently detected in conjunctival papillomas while HR HPV is rarely detected in these lesions. The fact that most papillomas with histopathological dysplasia were positive for LR HPV suggests that their malignant potential is limited, and that in-situ hybridization may help with prognostic assessment, although analysis of more cases with long-term follow-up will be needed to confirm this. HR HPV was detected by RNA in-situ hybridization in a relatively small number of CIN and SCC, but was not clearly linked to atopy or other clinical and histopathological features.


The trans-perineal and suprapubic approaches were adopted in 12 and 6 cases, respectively. Surgical PCa treatment in these cases involved endoscopic extraperitoneal RP, robot-assisted RP, and not otherwise specified RP in 30, 15, and 2 cases, respectively. Biochemical recurrence rates ranged from 20% to 28%. Non-surgical PCa treatment options included brachytherapy, RT plus androgen deprivation therapy, and RT alone in 23, 2 and 4 patients, respectively. PCa specific survival was reported by one study and was 100%.The management of patients with prostate cancer (PCa) and previous or synchronous colorectal cancer (CRC) represents a challenging issue. A systematic review was performed in May 2022 to summarize available evidence about the diagnosis, management, and outcomes of these patients. Twenty-seven studies involving 252 patients were identified. Overall, 163 (64.7%) and 89 (35.3%) patients had synchronous and metachronous PCa and CRC, respectively. In patients with synchronous diseases, PCa treatment involved active surveillance in 1 patient, radical prostatectomy (RP) in 36 patients, radiotherapy (RT) in 60 patients, RP plus RT in 1 patient, proton beam therapy in 1 patient, and cryoablation in 1 patient. In patients with previous CRC treatment, prostate biopsy was mostly performed by transrectal approach ( Prostate cancer (PCa) and colorectal cancer (CRC) represent two of the most common cancers in males contributing to 15% and 9% of new cancers, respectively ,3,4,5,6.The present analysis was conducted and reported according to the general guidelines recommended by the Primary Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) statement . This prThe search was performed in the Medline , Scopus , and Web of Science Core Collection  databases up to May 2022. The following terms were combined to capture relevant publications: “prostate cancer”, “colorectal cancer”, “rectal cancer”, “colon cancer” “synchronous”, “simultaneous”, and “metachronous”. Reference lists in relevant articles and reviews were also screened for additional studies. Conference abstracts were also considered.Two authors (L.N. and G.C.) reviewed the records separately and individually to select relevant publications, with any discrepancies resolved by a third author (M.C.). To assess eligibility for the systematic review, PICOS  criteria were used . PICOS cThe search strategy revealed a total of 124 results. The screening of the titles and abstracts determined 112 papers eligible for inclusion. Further assessment of eligibility, based on the study of the full-text papers, led to the exclusion of 83 papers. Finally, 27 studies involving 252 patients were included in the final analysis  1,2,7,1,38,39,40Study characteristics, patients’ demographics and tumors’ features are summarized in Diagnostic features and treatment outcomes in patients diagnosed with synchronous PCa and CRC are reported in The details of diagnosis, treatment modalities and outcomes of surgical and non-surgical treatments for PCa in patients with a history of CRC are reported in n = 24). The transperineal approach was adopted in 12 cases. In six cases, computed tomography was used to guide suprapubic biopsy. The time between CRC surgery and PCa diagnosis was described in eight studies and ranged from 1 month to 7 years. Overall, GS was available in 71 patients . Surgical PCa treatment involved endoscopic extraperitoneal radical prostatectomy (EERPE) in 30 patients, RALP in 15 patients, and not otherwise specified RP in 2 cases. Mean OT ranged from 168 to 235 min. Conversion to open surgery was described in three patients undergoing RALP. LOS ranged from 6.0 to 10.8 days and catheterization time ranged from 6.0 to 7.8 days. None of the patients undergoing surgery required adjuvant therapy. None of the studies described the incidence of positive surgical margins. Median follow-up of patients undergoing surgery for PCa ranged from 6.0 to 53.1 months and biochemical recurrence (BCR) rates ranged from 20% to 28%. Non-surgical PCa treatment options included brachytherapy (BT) in 23 patients, radiotherapy (RT) plus ADT in 2, and RT alone in 4 patients. ADT alone was prescribed in five cases. Median follow-up of patients undergoing non-surgical PCa treatment ranged from 18.6 to 85.7 months. PCa specific survival was reported by one study and was 100% at a median follow-up of 85.7 months [Mean PSA ranged from 4.8 to 29.5 ng/dL. The technique of prostate biopsy was described in 42 cases, and it was mostly performed by transrectal approach , resection of the right colon (n = 4), resection of the rectum (n = 4)) after an interval of 5 years. Interestingly, most prostatectomies were completed robotically (n = 11), median blood loss was low, no major postoperative complication was reported, and hospital stay was in line with standards . Eleven (78%) patients were continent at 1 year. Four patients with non-organ-confined disease (pT3a-b N0-1 Gleason score 8 or higher) experienced biochemical persistence or recurrence and underwent adjuvant RT during a median follow-up of 3.5 years [Current options with curative intent to manage PCa patients include active surveillance, RP and RT. The choice strongly depends on life expectancy, PCa risk group and patients’ preference. To our knowledge, we performed the first systematic review of studies evaluating the management and outcomes of PCa diagnosis and treatment in patients with synchronous or metachronous CRC. Interestingly, most available data is about patients with synchronous tumours. Of note, this condition is expected to increase in the next years due to improved life expectancy, increased screening programs for both malignancies, and increased use of pelvic magnetic resonance imaging . Therefo.5 years . Alterna.5 years . The preDespite the growing epidemiological relevance of double/multiple primary malignant tumors, current evidence about the diagnosis, management, and outcomes of synchronous or metachronous PCa in CRC patients remains suboptimal, deriving only from retrospective small series and case reports. In patients with synchronous diseases, single-session RP, and CRC surgery, as well as RT with or without ADT, represent treatment options with promising short-term oncologic PCa outcomes. In patients with a history of CRC who receive a diagnosis of PCa, minimally invasive RP techniques have been investigated with promising perioperative outcomes. RT with or without ADT represents a potential alternative. However, the level of evidence is still suboptimal, and results from well conducted, multi-institutional series with adequate follow-up are required.