Type 1 diabetes is a chronic autoimmune disease in which the destruction of pancreatic β cells leads to hyperglycemia. The prevention of hyperglycemia is very important to avoid or at least postpone the development of micro- and macrovascular complications, also known as late complications. These include diabetic retinopathy, chronic renal failure, diabetic neuropathy, and cardiovascular diseases. The impact of long-term hyperglycemia has been shown to persist long after the normalization of blood glucose levels, a phenomenon known as metabolic memory. It is believed that epigenetic mechanisms such as DNA methylation, histone modifications, and microRNAs, play an important role in metabolic memory. The aim of this review is to address the impact of long-term hyperglycemia on epigenetic marks in late complications of type 1 diabetes. Type 1 diabetes (T1D) is one of the most common chronic diseases of childhood, however, it can be diagnosed at any age. T1D is an autoimmune disease characterized by the destruction of pancreatic β cells, leading to insulin deficiency . The goaSeveral clinical and animal studies demonstrated that the early establishment of intensive blood-glucose control reduces the risk for the development of late complications . For exaEpigenetic mechanisms—namely, DNA methylation, histone modifications, and microRNAs (miRNAs)—can influence transcription activity without changing the DNA sequence. They are essential for the regulation of all biological processes and are labile to environmental influences, such as nutrition, chemicals, stress, drugs, and infections ,12. EpigDNA methylation is a covalent modification of DNA involving the transfer of a methyl group to the fifth carbon of a cytosine residue to form 5-methylcytosine by DNA methyltransferases. Most DNA methylation occurs in a CpG dinucleotide context . DemethyDNA methylation analysis on blood DNA samples from DCCT participants showed that long-term hyperglycemia influenced DNA methylation. The association between the mean HbA1c value during DCCT and 186 CpG sites were identified. The persistence of DNA methylation patterns in monocytes collected 16 or 17 years later during the EDIC indicated a metabolic memory . HbA1c lUNC13B gene, previously linked to diabetic nephropathy. Methylation levels on this CpG were higher in the case group [Different methylation patterns were observed between individuals with T1D with end-stage kidney disease and those without kidney disease in blood-derived DNA ,25,26 anse group . Higher se group . Quantitse group .Genome-wide DNA methylation analysis in blood samples identified 349 CpG sites, corresponding to 233 genes, to be differentially methylated between individuals with proliferative diabetic retinopathy and individuals with long-term diabetes with no or mild retinopathy. Differences were observed in genes with known function in the retina and eye development, inflammation, diabetic complications, and oxidative stress. The majority of CpG sites showed decreased methylation in individuals with proliferative diabetic retinopathy. However, no difference in global methylation between the groups was observed . In the NINJ2 gene, coding a cell surface adhesion protein with a possible role in nerve regeneration. On the other hand, decreased methylation was reported in the first intron of the BRSK2 gene, involved in neuronal polarization, and in the 5′ untranslated region of the CLDN4 gene, coding a component of the tight junction [DNMT1, a deoxyribonucleic acid methyltransferase, was associated with the risk for cardiac autonomic neuropathy but not for peripheral neuropathy in women with T1D [In peripheral blood cells of T1D individuals with cardiac autonomic neuropathy, increased DNA methylation was observed in the first intron of the junction . A minorwith T1D . The agewith T1D .Lower global DNA methylation was observed in diabetic foot ulcers fibroblasts and diabetic foot fibroblasts, compared with non-diabetic foot fibroblasts. DNA methylation patterns persisted even after multiple cell culture passages under normoglycemic conditions . FurtherCovalent posttranslational modifications of histones change histone–DNA interaction and thus influence gene transcription and other DNA processes, such as repair, replication, and recombination . DiffereCOL11A1, CTGF, and SERPINE1, which contribute to the development of diabetic nephropathy [In diabetic rat mesangial cells, the decrease in the H3 lysine 9 trimethylation 3 (H3K9me3) mark was found at promoter sites of pro-fibrotic genes such as hropathy . The redhropathy . Similarhropathy . In kidnhropathy . High glhropathy . Howeverhropathy .IL-6, MCSF, and MCP-1. These aberrant chromatin changes persisted even after cells were cultured in vitro, indicating metabolic memory [p65 in aortic endothelial cells even when the cells were removed from the hyperglycemic environment [In vascular smooth muscle cells from diabetic db/db mice, decreased levels of H3K9me3 were identified at promoter sites of inflammatory genes—namely, c memory . The perironment ,57. Genoironment .Chronically high glucose levels caused changes in histone H3K4 and K9 demethylation in human monocytes . In DCCTIn the diabetic retina, contradictory findings were observed. Histone hyperacetylation was observed in the retinas of diabetic rats, together with increased expression of inflammatory proteins . On the EZH2, an H3K27me3 methyltransferase, and upregulation of H3K27me3 demethylases KDM6B and KDM6A were identified [SEV39H1 methyltransferase were decreased in diabetic conditions in vascular smooth muscle cells [SUV39H2 gene, a methyltransferase, was associated with diabetic retinopathy in individuals with T1D [p300, histone acetyltransferase and coactivator of NF-κB, and downregulation of histone deacetylase activity 2. These were reversed after the treatment with curcumin [High glucose levels caused aberrant expression of various histone-modifying enzymes. Increased levels of SET7/9, a methyltransferase, stimulated the expression of extracellular matrix genes in renal mesangial cells ,66,67, ientified . Levels le cells  and mesale cells . Single with T1D . In cultcurcumin .miRNAs are small ~22-nucleotide-long non-coding RNAs and negative regulators of targeted genes expression by binding to the 3′ untranslated region of mRNA . They arSMAD7 and PTEN by miR-21 increased the expression of pro-fibrotic and extracellular matrix genes [PTEN were also associated with increased expression of miR-214 as a consequence of high glucose [FOG2, an inhibitor of PI3K, thereby leading to activation of the PI3K–Akt pathway and glomerular mesangial hypertrophy [SOD1, SOD2, and PAK1 [Increased levels of miR-21 ,75,76,77 glucose ,83. TGF-ertrophy . Reductiertrophy ,80, and ertrophy . miR-377and PAK1 .On the contrary, levels of several miRNAs were decreased in the diabetic kidney, indicating their protective role in diabetic nephropathy ,88,89,90OXR1, miR-200b was demonstrated to play a protective role against oxidative stress [In the retina of Akita mice, upregulation of miR-200b was observed. By downregulating its target e stress . Howevere stress . miR-451e stress .Atg4B miR-39c negatively affected the growth of trigeminal sensory neurons and the diabetic corneal nerve regeneration [Levels of miR-39c were increased in the trigeminal ganglion tissue of STZ-induced diabetic mice. Targeting neration . Upregulneration .Single nucleotide polymorphisms in miRNAs were associated with late complications. An rs2910164 polymorphism in seed sequence within miR-146a, leading to a reduction in mature miR-146a, was associated with diabetic nephropathy, and retinopathy ,96. In tA case–control study identified different plasma levels of miRNA associated with diabetic nephropathy. Levels of miR-25, miR-27a, miR-126, miR-130b, miR-132, miR-152, miR-320, miR-326, miR-340, and miR-660 were elevated, and levels of miR-181a, miR-223, and miR-574-3p were lowered. After pancreas–kidney transplantation, levels of miR-25, miR-27a, miR-130b, miR-132, miR-152, miR-320, miR-326, miR-340, miR-574-3p, and miR-660 were normalized . In the Urinary miRNA profiling in diabetic nephropathy linked 18 miRNAs with the development of microalbuminuria . A set oEstablishing and maintaining normoglycemia after the onset of T1D is very important for preventing late T1D complications. The international study of glycemic control in people with T1D showed that glycemic control is still suboptimal . The useEpigenetic marks are good candidates as biomarkers for the early detection of late complications since they represent a link between the environment and gene expression  and are The use of the latest nucleotide sequencing detection techniques could give us new insights. Long-read sequencing enables the study of a wide range of modifications in single-molecule sequencing on native DNA. It can surpass traditional bisulfite sequencing for studying DNA methylation. Bisulfite sequencing, a golden standard for studying DNA methylation, is a DNA destructive method, and mapping generated short-read data to repetitive or low-complexity regions is very difficult . The sinA growing body of evidence shows the relationship between late complications of T1D and epigenetic mechanisms. Long-term hyperglycemia causes aberrant epigenetic marks that persisted even after the normoglycemic environment was established and maintained, indicating the involvement of epigenetics in metabolic memory. These marks can be used as biomarkers for the early detection of micro- and macrovascular complications. They may also serve as good targets for novel therapeutic approaches since epigenetic modifications can be reversed.


CDH1) and β-catenin (CTNNA1). In the present study, we provide an overview on the molecular basis of HDGC and outline the essential elements of genetic counseling and surveillance. We further provide a practical summary of current guidelines on clinical management and treatment of individuals at risk and patients with early disease.Gastric cancer is ranked fifth among the most commonly diagnosed cancers, and is the fourth leading cause of cancer-related deaths worldwide. The majority of gastric cancers are sporadic, while only a small percentage, less than 1%, are hereditary. Hereditary diffuse gastric cancer (HDGC) is a rare malignancy, characterized by early-onset, highly-penetrant autosomal dominant inheritance mainly of the germline alterations in the E-cadherin gene ( CDH1 tumor suppressor gene [CDH1 gene is estimated to be less than 1% at the age of 20, around 4% by the age of 30, increasing to about 20% for men and 45% for women by the age of 50 [Gastric cancer remains an important health issue worldwide, ranking fifth among the most commonly diagnosed malignancies 5.6%) and the fourth leading cause of cancer-related deaths 7.7%); it was responsible for over one million new incidents and an estimated 769,000 deaths in 2020 worldwide . There a.6% and t.7%; it wsor gene . The lifge of 50 ,17,18,19ge of 50 . Three dge of 50 . Advancege of 50 . In thisCDH1) [CDH1 gene is a 2.6 kb tumor suppressor gene located in chromosome 16q22.1 [CDH1 gene encodes a type I calcium transmembrane glycoprotein expressed on epithelial tissues, responsible for calcium-dependent cell adhesion basolateral membrane in adherent junctions, maintenance of cell differentiation and cell polarity, both during development and in adult life [2+ ions, a transmembrane domain and a highly conserved cytoplasmic domain containing binding sites for β-catenin (CTNNA1) [HDGC, although rare, is an aggressive cancer inherited in an autosomal dominant fashion . The genCDH1) . The hum 16q22.1 ,25. The ult life ,27,28. T(CTNNA1) ,29. E-ca(CTNNA1) . The bin(CTNNA1) ,32,33,34CDH1 gene are scattered. There have been over 155 different germline CDH1 mutations identified so far [CDH1 promoter hypermethylation was found in nine (32.1%) cases [CDH1 and gastric cancer was also confirmed in a meta-analysis by Jing et al. [CDH1-driven HDGC stages without evidence of metastatic disease [CDH1 gene have been associated with the development of signet ring cell carcinoma in situ (SRCC-pTis), characterized by tumor cells located between normal foveolar epithelial cells and the basement membrane. SRCC-pTis is considered the precursor lesion of HDGC [Mutated sites in the d so far , includid so far ,37. Mored so far . Almost d so far . Epigened so far ,49,50. Og et al. . In this disease . Moreove disease . Disrupt disease . Pathoge disease ,55. Muta of HDGC ,57. Pene of HDGC ,58. The  of HDGC , while t of HDGC ,15.CTNNA1  variants, thus justifying the low numbers [CTNNA1 gene [MAP3K6 should no longer be considered a gastric cancer predisposition gene since, according to their data [Another gene implicated in HDGC is CTNNA1 . The humthe cell . The A-cthe cell ,60,61. Athe cell ,63,64. Tfamilies ,39. In t numbers ,37. MajeNA1 gene ,37. MoreNA1 gene  of high NA1 gene , which hNA1 gene ,66; F-boNA1 gene , which mNA1 gene ; DOT1-liNA1 gene , which iNA1 gene ; mitogenNA1 gene , which aNA1 gene ; the patNA1 gene ,44; and NA1 gene ,44,45, weir data , two misThe International Gastric Linkage Consortium (IGCLC) set criteria to provide critical information for medical care providers and for those patients and families at risk of HDGC syndrome. The 2020 updated guidelines  recommenCDH1 mutations, and, if there is no variant identified, then they should subsequently be considered for CTNNA1 analysis [CDH1 mutation carriers have developed gastric cancer in their mid-teens; as a consequence, genetic testing begins at 16 years of age, and occasionally 1–2 years before, on a case-by-case basis [CDH1 pathogenic or likely pathogenic variants is detected in 48% of families with multiple cases of gastric cancer [CDH1 mutation detection rates [CDH1 and CTNNA1 germline variants must include sequencing of the entire open reading frame (ORF), including intron-exon boundaries [CDH1 point or small frameshift mutations can be identified in 30–50% of HDGC families, while CDH1 large deletions account for less than 5% of pathogenic variants [CDH1 gene [CDH1 variants have high prevalence in the New Zealand Māori population, all Māori with confirmed DGC are recommended to undergo CDH1 genetic testing [Hereditary cancer syndromes presuppose an elevated risk of cancer in family members ; therefoanalysis . Howeverse basis . Using tc cancer . Geneticon rates ,12,50. Avariants ,73. Avaivariants  in the lvariants . GeneticDH1 gene ,76. As p testing . Moreove testing ,78. Init testing ,79. In c testing .CDH1 and other genes of interest may also be considered using next generation sequencing techniques searching for mutations [Concerning molecular genetic analysis approaches, single-gene testing is recommended followed by gene-targeted deletion/duplication analysis if no pathogenic variant is found . A multiutations ,82,83,84utations . Other mutations . Using tutations ,86,87.CDH1 pathogenic variant [CDH1 mutation female carriers is 39%  while the combined risk of gastric cancer and breast cancer in women rises to 90% by age 80 years, indicating that family history of multiple LBCs at a young age should encourage genetic screening for CDH1 mutations [CDH1 mutations.HDGC is associated with LBC which may be the presenting pathology in a patient . The res variant . In cont variant . Interesutations . The Ameutations . Bilaterutations ,93, as wutations ,94,95, mManagement of patients with symptomatic invasive HDGC is not different to that of sporadic cases ,97,98,99Moreover, there have been several studies on cell-therapy applications in solid tumors. Chimeric antigen receptor (CAR)-T cell immunotherapy using genetic engineering to harness the anti-tumor activity of T-lymphocytes. T cells from the patients’ bloodstream were modified using viral vectors to introduce the CAR to recognize a particular tumor antigen and then were infused back into the patient . PromisiCDH1 mutation. Therefore, prophylactic total gastrectomy remains the cornerstone of HDGC management. However, the impact of gastrectomy should not be underestimated, since it may have severe consequences on a metabolic and nutritional level and, therefore, patients’ quality of life. Importantly, significant progress has been made in recent years in the identification of CDH1 mutations. Future efforts should be directed towards the development of improved genetic screening modalities and the identification of new genes, especially in CDH1-negative families, aiming at the early identification of asymptomatic carriers.Despite the fact that HDGC is a rare cancer syndrome, it is highly penetrant and difficult to diagnose and manage. HDGC requires the successful collaboration of a team of experts, including a geneticist, a gastroenterologist, a surgeon and an oncologist for the best decision making at each stage of disease, in order to achieve optimal long-term results. The IGCLC 2010 criteria have evolved to help clinicians make the most effective decisions. Genetic counseling should be performed before testing, while it is crucial to identify a mutation and to further determine whether unaffected relatives are at risk for cancer. There has also been progress in endoscopic techniques for the surveillance of patients who wish to postpone surgery or for those whose risk is not well defined. Nevertheless, endoscopy does not appear to offer a reliable reduction of risk in individuals with a proven pathogenic germline


Klebsiella pneumoniae is considered an opportunistic pathogen frequently involved with healthcare-associated infections. The genome of K. pneumoniae is versatile, harbors diverse virulence factors and easily acquires and exchanges resistance plasmids, facilitating the emergence of new threatening clones. In the last years, ST16 has been described as an emergent, clinically relevant strain, increasingly associated with outbreaks, and carrying virulence factors  and a diversity of resistance genes. However, a far-reaching phylogenetic study of ST16, including geographically, clinically and temporally distributed isolates is not available. In this work, we analyzed all publicly available ST16 K. pneumoniae genomes in terms of virulence factors, including capsular lipopolysaccharide and polysaccharide diversity, plasmids and antimicrobial resistance genes. A core genome SNP analysis shows that less than 1% of studied sites were variant sites, with a median pairwise single nucleotide polymorphism difference of 87 SNPs. The number and diversity of antimicrobial resistance genes, but not of virulence-related genes, increased consistently in ST16 strains during the studied period. A genomic comparison between ST16 and the high-risk clone ST11 K. pneumoniae, showed great similarities in their capacity to acquire resistance and virulence markers, differing mostly in the great diversity of capsular lipopolysaccharide and polysaccharide types in ST11, in comparison with ST16. While virulence and antimicrobial resistance scores indicated that ST11 might still constitute a more difficult-to-manage strain, results presented here demonstrate the great potential of the ST16 clone becoming critical in public health. Klebsiella pneumoniae, is considered an opportunistic pathogen associated with infections in hospitalized or otherwise immunocompromised individuals  difference = 87 SNPs), suggesting that less than one percent of the core genome nucleotide sites were variant sites. This nucleotide diversity is comparable to previously shown measures of genetic variation of core genome alignment of oup CG23 .https://microreact.org/project/drXbkjrfrVUP6TiEQAVh7r-tree-phylogenetic-kp-st16, accessed on 5 May 2022. Two major groups were identified, one consisting of 47.23% (452/957) of the isolates , and one by 35.94% (344/957) . The first group included isolates from 37 of the 40 countries that reported ST16 isolates, contrasting with the second group that depicted isolates from only 8 countries. The majority of samples from the second group originated from Thailand (89.53% [308/344]), corresponding to 92.21% (308/334) of the genomes reported by this country.In order to determine the phylogeny of the 957 ST16 isolates, a maximum-likelihood tree was inferred from the aligned core genomes by roary (Kp was detected in 30.75% (139/452) of isolates from group 1, where the most diversity of ICEKp was also found ; only ICEKp11 was not found in this group , but ICEKp11 was identified in a single isolate from Australia (2019), and this isolate did not cluster in either of the two groups.ICEDue to the over-representation of isolates from Thailand in group 2  we investigated their origin in more detail. The genomes derive from 8 distinct NCBI Bioprojects , 75% of them were reported in Bangkok between 2015 and 2018. The second largest dataset came from the province of Surat Thani . Taken together, the data suggest that isolates from Thailand do not correspond to a restricted location and represent the diversity of ST16 found in the country.Kp4, and this structure was only found in three isolates (3/452) belonging to group 1. Even though all these 116 isolates harbored ICEKp4, the isolates were significantly distinct in their core genome, distributed in 5 continents , and found in a total of 18 countries between 2012 and 2020.A total of 161 isolates did not cluster within groups 1 or 2. Interestingly, 72.04% (116/161) of these isolates carried ICEblaKPC-2 gene [blaKPC , with blaKPC-2  and blaKPC-3  being the most frequently found variants. However, blaNDM-1 was the most prevalent variant among ST16 isolates   , differeblaOXA-232, present in 38.24% of ST16 isolates , but most frequently found in isolates from Thailand (81.96%) (300 out of 366 isolates from Thailand carried this gene). Due to the high contribution of isolates from Thailand, we reevaluated the data considering only isolates from the other 12 locations, finding that 10.04% of the remaining ST16 isolates (66/657) carried blaOXA-232. Argimón et al. [blaOXA-232 in 2.16% (27/1247) of the evaluated ST11 isolates, mostly reported in Oman , Thailand  and India . The most common gene encoding a carbapenemase among ST16 isolates was isolates . The blablaCTX-M-65 is prevalent in ST11 isolates (28.9 to 59.61%) [blaOXA-1 was found in 40.33% ST16  ,14, but  59.61%)  and 8.3533% ST16 , againstK. pneumoniae [CPS is a key virulence determinant in eumoniae ,69. Tweneumoniae . This cheumoniae , while oKp3 e ICEKp4 as the most prevalent (rmpADC and rmpA2 were found in isolates reported in China: aerobactin (n = 35), salmochelin (n = 1), rmpADC (n = 10) and rmpA2 (n = 31) [n = 26) reported in Australia (n = 9), United States (n = 16) and India (n = 1); salmochelin (n = 2) reported in the United States (n = 1) and one of unknown origin (n = 1); rmpADC (n = 10) reported in Australia (n = 9) and the United States (n = 1); and rmpA2 (n = 6) reported in Australia (n = 5) and India (n = 1)  .ybt, clb and iuc, and is interpreted as follows: score 0 = none present, 1 = yersiniabactin only, 2 = colibactin without aerobactin , 3 = aerobactin only, 4 = aerobactin and yersiniabactin without colibactin, and 5 = all three present. Yersiniabactin facilitates immune escape and has been shown to increase virulence in various strains [clb together with ybt may increase virulence due to clb’s genotoxic activity in mammalian cells [iuc is related to sepsis since it promotes bacterial growth in blood via the acquisition of iron from transferrin [ybt, clb and the virulence plasmid when present in combination, but it is logical to score iuc + ybt with higher virulence than iuc without ybt [iuc (virulence plasmid) + ybt + clb as the highest score, is associated with hypervirulent strains in liver abscesses documented worldwide (CG23-I) [Kleborate virulence scores capture the hierarchy of virulence-associated loci that have emerged over the last two decades according to the literature . The sco strains ,70. The an cells ,71, but an cells . The iucnsferrin ,72,73. Thout ybt . The com(CG23-I) . KleboraThe resistome of ST16 is also alarmingly similar to ST11, as confirmed by the Kleborate score B, althouK. pneumoniae species complex (KpSC) [Kleborate resistance scores do not include the presence of intrinsic variants of oqxAB, chromosomal fosA and ampH since they are not associated with clinical resistance in x (KpSC) .K. pneumoniae belonging to ST16 is an important emergent lineage that can carry determinants to carbapenem resistance and is believed to have an enhanced virulence potential, as observed by patient survival analysis and in vitro studies [ studies ,16,74.According to the core SNP analysis of 957 ST16 genomes, two major groups comprised over 80% of all reported isolates , with the major group including globally distributed isolates reported in Africa, Asia, Europe, North America, South America and Oceania.p < 0.0001), a finding that highlights the importance of monitoring the emergence of extremely drug-resistant (XDR) ST16 isolates, already reported in several locations with distinct antimicrobial resistance profiles and clear evidence of horizontal gene transfers [This study showed a significant increase in the maximum number of AMR genes in a genome per year  and aph(4)-Ia (n = 10 genomes) were identified exclusively in the United States. For quinolones, there was no significant increase in the number of AMR in time, with two to nine genes or variants identified per genome per year during the studied period. Noteworthy was the presence of qnrE1, reported in five isolates from Brazil in 2018. This gene was originally found in a conjugative plasmid in a clinical K. pneumoniae isolate [A similar scenario was observed for aminoglycosides, with numbers between 2003 and 2011 varying from two to six genes or variants, and between 2012 and 2021 increasing to 10-18. Thirty-one genes associated with this type of resistance were found, the most common being aac(6′)-Ib-cr5 encoding an aminoglycoside acetyltransferase that also has activity against fluoroquinolones ,80. The  isolate . Also no isolate ,56.Kp and plasmids. Seven types of ICEKp  were identified, found in 63.21% (605/957) of the evaluated isolates, similar to a previous report that found ICEKp in 61.04% of the evaluated ST16 genomes [The contribution of horizontal gene transfer to the spread of AMR genes could be clearly illustrated by the widespread distribution of ICEn = 17) and pKPC-CAV1193 (n = 37). The analysis of 334 ST16 isolates from Thailand identified replicons ColKP3 and IncFIA/B, present in 90.11% (301/334) and 92.21% (308/334) of isolates, respectively, corroborating with findings by Abe et al. [blaNDM-1 and blaOXA-232 in Thailand and Canada [blaOXA-232 was also reported by Argimón et al. [K. pneumoniae ST16 co-carrying blaNDM-1 and blaOXA-232 is considered a successful clone in Thailand, a characteristic they fund to be exclusive to ST16 isolates among 43 STs classifying 577 carbapenem-resistant K. pneumoniae isolates in a nationwide surveillance study carried out in the country [A direct link between AMR genes and plasmids is not always possible with short-read assemblies, but their central role in the dissemination of AMR genes, and importance for epidemiological discussions, is well known ,82,83. Te et al. . These rd Canada , and Coln et al. ,85. K. p country . Here, w4401 being the most prominent transposon type associated with KPC-2 [4401-like transposon. KPC was mostly detected in Brazil and the United States, which together accounted for 78.37% (87/111) of cases. The first report of an ST16 isolate in Brazil harboring KPC-2 occurred in 2008 [KPC-encoding genes are often found in transposons, with Tnth KPC-2 . Accordi in 2008 . Since t in 2008 ,68,87. T in 2008 .K. pneumoniae strains to cause disease in humans [ybt-irp-fyuA, entA-F, fimA-H, mrkA-J, clb3 and KL51 type for CPS), (3) hypervirulence markers such as aerobactin, salmochelin, rmpADC and rmpA2, and (4) the locus ybt within ICEKp, a virulence factor that directly influences the pathogenicity of K. pneumoniae [Differently from AMR genes, the number of virulence genes detected per genome did not increase in the studied period  ST1pA2, and  the locueumoniae ,70.K. pneumoniae isolates may belong to high-risk clonal groups: hyper-virulent clonal complexes (CCs), mostly responsible for community-acquired invasive infections, and multidrug-resistant CCs, mostly involved in health-care associated infections [fections ,88,89. Tfections ,19,76. Wfections ,17. Our K. pneumoniae clone ST11 [K. pneumoniae. The great diversity of CPS and adjacent LPS O antigen of ST11 is associated with a recombination hotspot at the biosynthesis loci [Finally, due to the clinical importance of one ST11 ,34, we isis loci ,90,91. ImpA/rmpA2, iucABCD and iutA genes [Although ST11 and ST16 showed similar virulence scores, ST11 has been more studied and associated with hyper-virulence, such as the ST11-KL64 strain reported in China, carrying rtA genes . Given t


CALM1, CYCS, THBS1, MYC, GATA6, and SPRED3 was strongly associated with a poor prognosis. In HNSCC patients, overexpression of PIK3R3, GIGYF1, and BCL2L11 was substantially correlated with a good prognosis. Besides, “proteoglycans in cancer” was the most significant pathway enriched in the primary OSCC. The present study results revealed more possible mechanisms mediating primary OSCC and may be useful in the prognosis of the patients with early-stage OSCC.Oral squamous cell carcinoma (OSCC) is the most prevalent head and neck malignancy, with frequent cervical lymph-node metastasis, leading to a poor prognosis in OSCC patients. The present study aimed to identify potential markers, including microRNAs (miRNAs) and genes, significantly involved in the etiology of early-stage OSCC. Additionally, the main OSCC's dysregulated Gene Ontology annotations and significant signaling pathways were identified. The dataset GSE45238 underwent multivariate statistical analysis in order to distinguish primary OSCC tissues from healthy oral epithelium. Differentially expressed miRNAs (DEMs) with the criteria of p-value < 0.001 and |Log2 fold change| > 1.585 were identified in the two groups, and subsequently, validated targets of DEMs were identified. A protein interaction map was constructed, hub genes were identified, significant modules within the network were illustrated, and significant pathways and biological processes associated with the clusters were demonstrated. Using the GEPI2 database, the hub genes' predictive function was assessed. Compared to the healthy controls, main OSCC had a total of 23 DEMs. In patients with head and neck squamous cell carcinoma (HNSCC), upregulation of  Oral cancer (OC) is a global public health problem, accounting for the fourth highest frequency of malignancy . Oral sqProtein-coding genes include approximately 2% of the entire human genome, and therefore, the regulatory role of non-coding RNAs (ncRNAs) in signaling pathways, biological processes (BPs), and networks is inevitable ,14. The The present study hypothesized that the dysregulation of miRNAs in the early-stage OSCC tumor specimens (stages I and II) compared to their adjacent normal epithelium leads to the aberrant expression of their target genes. Therefore, differentially expressed miRNAs (DEMs) in early-stage OSCC tumor tissues compared with the adjacent healthy controls were identified using the multivariate statistical analysis | > 1.585), and the differential power of DEMs was evaluated using the support vector machine (SVM) algorithm. Additionally, it was hypothesized that the DEMs-targets, which are crucial in the pathogenesis of early-stage OSCC, might serve as prognostic indicators for the condition. In order to study the prognostic significance of the hub genes inside the network, we created a protein interaction map (PIM) based on the DEMs-targets. Furthermore, based on DEMs-targets and important clusters in the PIM, Gene Ontology (GO) annotation and pathway enrichment studies were carried out. The aims of the present study were followed by re-analyzing the dataset GSE45238, which was established by Shiah et al.  to monithttp://www.ncbi.nlm.nih.gov/geo [The miRNA expression dataset GSE45238  was atta.gov/geo . The GSEThe normalized dataset after preprocessing had 30 observations  and 858 characteristics (miRNA IDs). Using the "ropls," "limma," and "genefilter" packages from the R language (version 4.2.0) , the advhttps://cran.rproject.org/web/packages/e1071). The model's sensitivity, specificity, and accuracy were calculated using the leave-one-out (LOO) approach, and cross-validation was carried out by repeating LOO 100 times [To evaluate the differential power of DEMs in early-stage OSCC tissues compared to the healthy controls, the SVM algorithm with the linear kernel was constructed using the e1071 package (version 1.6-8) within the R environment (00 times . FurtherHTTP://string-db.org) [https://biit.cs.ut.ee/gprofiler/gost) [MiRWalk 2.0 database  was util-db.org)  was used-db.org)  was util-db.org) . Only moer/gost) , and thehttp://gepia2.cancer-pku.cn/#survival) [The prognostic impact of the hub genes associated with the early-stage OSCC was studied utilizing Kaplan-Meier curves. The survival analysis was conducted using GEPIA2 database  . Re-analurvival)  and Genourvival)  database2X = 0.223, R2Y = 0.959, and Q2 = 0.789. At a p-value of 0.001 and |Log2 FC >| 1.585, 23 DEMs, including nine up-regulated and 14 down-regulated miRNAs, were identified in the early-stage OSCC tissues compared with non-cancerous specimens (https://huygens.science.uva.nl/) [OPLS-DA was performed to differentiate early-stage OSCC tissue samples (n = 15) from healthy controls (n = 15). The model strongly classified two datasets with the following parameters: Rpecimens . Six hunuva.nl/) . Fig. 1BA classifier model was constructed using the SVM algorithm via linear kernel to evaluate the differential power of DEMs in early-stage OSCC samples compared to the normal oral epithelium. The dataset included 23 DEMs and 30 observations . The SVM model has the following parameters: cost = 0.01, number of support vectors = 16, and SVM-type = C-classification. The average value of sensitivity, specificity, and accuracy for the SVM was estimated as 100%, 95%, and 97%, respectively, after completing 100 rounds of LOO cross-validations. The ROC curve of model was also achieved using the “Epi” package in R language , in whicA total of 672 unique targets were uploaded into the STRING database, and a primary protein-protein interaction (PPI) network was constructed with a confidence score of ≥ 0.4. A PIM meeting the criterion of 605 proteins and 2,322 interactions was created after unconnected nodes were removed, and it was then imported into the Cytoscape program for further analysis. The MCODE program found five major modules in all, including clusters Nos. 1, 2, 3, 6, and 7 . BesidesCALM1, CYCS, THBS1, MYC, GATA6, and SPRED3, was strongly related with a poor prognosis in patients with HNSCC, according to the survival analysis performed using the GEPIA2 database. In contrast, upregulation of three of hubs, including PIK3R3, GIGYF1, and BCL2L11 was considerably related to a favorable prognosis in HNSCC patients . Thus, a prognostic panel including CALM1 and CYCS revealed the most significant result with the HR = 1.8 and p (HR) = 0.000042 . Crimi et al.  performeCALM1, CYCS, THBS1, MYC, GATA6, and SPRED3 was strongly associated with a poor prognosis in patients with HNSCC. Additionally, patients with HNSCC who had upregulation of PIK3R3, GIGYF1, and BCL2L11 had better prognoses. Calmodulin (CALM) is a major conserved calcium ion receptor protein in eukaryotic cells and is encoded by CALM1, CALM2, and CALM3 in humans [2+ binds to the phosphoinositide 3-kinase α (PI3Kα), leading to the hyperactivation of PI3Kα/Akt/mammalian target of rapamycin signaling pathway, cell proliferation, differentiation, motility, and development [Overexpression of the DEMs target genes n humans . The CALelopment -47. The elopment , bladderelopment , and naselopment .A significant part in promoting the signaling pathways connected to programmed cell death is played by cytochrome c (CYCS) . HoweverGATA6 (GATA binding protein 6) is a transcription factor involved in a GATA family regulating several genes participating in tissue morphogenesis and cell fate decision making . GATA6 hSPRED3) downregulates the Ras/Raf/mitogen-activated protein kinase (MAPK) signaling pathway during the organogenesis [SPRED3 is frequently loosed in glioblastoma [SPRED3 gene expression increased Ras/Raf/MAPK signaling, resulting in resistance to epidermal growth factor receptor–tyrosine kinase inhibitors. According to the present results, the has-miR-370-3p was significantly down-regulated in the early-stage OSCC tissues than the healthy controls , which may be hypothesized that SPRED3 is overexpressed in the early-stage OSCC tissues, which may be in line with the cancer response. However, this requires further confirmation.Sprouty-related, EVH1 domain-containing protein 3 . Proteoglycans are classified as highly glycosylated proteins involved in various parts of tissues, including the cell membrane, extracellular matrix, and pericellular space. These heavy molecules mediate many important BPs associated with angiogenesis, tissue regeneration, cell growth, and motility. Numerous forms of cancer have been linked to aberrant proteoglycan expression . In soliCALM1, CYCS, THBS1, MYC, GATA6, SPRED3, and the under-expression of PIK3R3, GIGYF1, and BCL2L11 in HNSCC patients. Furthermore, “proteoglycans in cancer” was demonstrated as the most salient signaling pathway affected in primary OSCC. These results may help the prognosis of the patients with early-stage OSCC, leading to more useful therapeutic approaches.In conclusion, the present study identified 23 DEMs in primary OSCC tissues compared to the normal oral epithelium. MiR-21-3p and miR-375-3p were the most salient up-regulated and down-regulated miRNAs in primary OSCC tissues compared to healthy controls, respectively. It has been hypothesized that DEMs contribute to OSCC's onset or undergo dysregulation in response to a tumor mass. Additionally, there was a strong correlation between a poor outcome and the upregulation of


Enhanced glycolysis is a major feature of cancer glycometabolism, and SLC2A1 is one of the pivotal genes in cancer glycometabolism. Although SLC2A1 plays an important role in the growth of many cancers, pan-cancer analysis allows us to more comprehensively and systematically understand the function and role of SLC2A1 in cancers. In this study, we found that SLC2A1 was highly expressed in most cancers, and resulted in poor prognosis. M6A methylation might be one of the important factors for the high expression level of SLC2A1. SLC2A1 not only enhanced cancer glycolysis, but also affected the tumor microenvironment. Notably, SLC2A1 was significantly and positively correlated with the T-cell-exhaustion biomarkers PD-L1 and CTLA4. Collectively, SLC2A1 may provide new strategies for pan-cancer treatment, especially cancer immunotherapy.SLC2A1 plays a pivotal role in cancer glycometabolism. SLC2A1 has been proposed as a putative driver gene in various cancers. However, a pan-cancer analysis of SLC2A1 has not yet been performed. In this study, we explored the expression and prognosis of SLC2A1 in pan-cancer across multiple databases. We conducted genetic alteration, epigenetic, and functional enrichment analyses of SLC2A. We calculated the correlation between SLC2A1 and tumor microenvironment using the TCGA pan-cancer dataset. We observed high expression levels of SLC2A1 with poor prognosis in most cancers. The overall genetic alteration frequency of SLC2A1 was 1.8% in pan-cancer, and the SLC2A1 promoter was hypomethylation in several cancers. Most m6A-methylation-related genes positively correlated with the expression of SLC2A1 in 33 TCGA cancers. Moreover, SLC2A1 was mainly related to the functions including epithelial–mesenchymal transition, glycolysis, hypoxia, cell-cycle regulation, and DNA repair. Finally, SLC2A1 positively associated with neutrophils and cancer-associated fibroblasts in the tumor microenvironment of most cancers and significantly correlated with TMB and MSI in various cancers. Notably, SLC2A1 was remarkably positively correlated with PD-L1 and CTLA4 in most cancers. SLC2A1 might serve as an attractive pan-cancer biomarker for providing new insights into cancer therapeutics. Cancer is one of the leading causes of death in humans. Although much progress has been made in the treatment of cancer, the overall therapeutic effect is unsatisfactory. Newly diagnosed cases are also increasing, placing a huge burden on society . In receGlucose is one of the basic metabolites needed by animal and plant cells. Cancer cells require a large amount of energy from the body for malignant proliferation . AberranSolute carrier family 2 member 1 (SLC2A1) is known as glucose transporter 1 (GLUT1) . SLC2A1 In this study, data from public databases and our own data convincingly showed that the expression of SLC2A1 was significantly increased in pan-cancer and conferred a poor prognosis. We explored the potential mechanism of SLC2A1 in pan-cancer through bioinformatics analysis. We further examined the association between SLC2A1 and the immune cell infiltration score, immune checkpoints, TMB, and MSI. Our results comprehensively revealed the potential mechanism of SLC2A1 in pan-cancer, and they highlight the impact of SLC2A1 on the tumor microenvironment (TME) and cancer immunotherapy.https://xena.ucsc.edu/, accessed on 14 July 2022) and the Genotype-Tissue Expression (GTEx) database , which included 15,776 samples of 33 cancer types and normal tissues. The abbreviations of all cancer types are shown in https://www.ncbi.nlm.nih.gov/geo/, accessed on 14 July 2022) for validation. These datasets were GSE2088, GSE13507, GSE10927, GSE39001, GSE26566, GSE18520, GSE53757, GSE62452, GSE87211, GSE15605, GSE33630, GSE3218, GSE17025, GSE47861, GSE68468, GSE53625, GSE13601, GSE57927, GSE75037, and GSE26899. Detailed information on the GEO datasets is shown in We downloaded transcriptome data and clinical information from the University of California Santa Cruz (UCSC) Xena browser  from the Sixth Affiliated Hospital of Sun Yat-Sen University. Each sample contained paired tumors and adjacent normal tissues. The study was approved by the Ethics Committees of the Sixth Affiliated Hospital of Sun Yat-Sen University.® Premix Ex Taq (TaKaRa), following the manufacturer’s protocol, which we calculated using the 2−ΔΔCT method. We used GAPDH as the endogenous control. The primers used in this study were as follows: SLC2A1 forward 5′-CTGCAACGGCTTAGACTTCGAC-3′ and reverse 5′-TCTCTGGGTAACAGGGATCAAACA-3′; GAPDH forward 5′- GCTCTCTGCTCCTCCTGTTC-3′ and reverse 5′- ACGACCAAATCCGTTGACTC-3′.Finally, we used RT-qPCR method to validate the differential expression of SLC2A1 in LUAD and ESCA between cancer tissues and paired normal tissues. Using TRIzol reagent , we extracted the total RNA from the frozen tissues, which we then reverse-transcribed into cDNA with a PrimeScript RT reagent Kit with gDNA Eraser (TaKaRa). Next, we confirmed the expression of SLC2A1 with TB Greenhttps://www.proteinatlas.org/) database. A previous study [http://ualcan.path.uab.edu, accessed on 14 July 2022) provides a pan-cancer protein expression analysis option based on the data from CPTAC. Therefore, we used the UALCAN database to perform pan-cancer protein expression analysis of SLC2A1.We extracted the expression data of SLC2A1 for each sample. We excluded cancer types with less than 3 samples. We used R software to calculate the expression differences between normal and tumor samples for each tumor by using Wilcoxon rank-sum and signed-rank tests. Moreover, we used the downloaded data to analyze the relationship between SLC2A1 level and clinicopathological parameters. We explored the protein level of SLC2A1 between human cancer and normal tissues by using the Human Protein Atlas  curve analysis for the TCGA-GTEx pan-cancer dataset. The pROC package was used to plot ROC curves and calculate the areas under the curves (AUCs) values in R.We used the Kaplan–Meier (log-rank) method and univariate Cox regression to evaluate the overall survival (OS) of the patients from the TCGA pan-cancer cohort. We also assessed the progression-free interval (PFI), disease-specific survival (DSS), and the disease-free interval (DFI) of the patients from the TCGA pan-cancer cohort with univariate Cox regression analysis. We determined the optimal cut-off value using the R package ‘survival’.http://cbioportal.org, accessed on 14 July 2022) is an open-access resource for exploring, visualizing, and analyzing multidimensional cancer genome data. It currently contained 225 cancer studies. We used cBioPortal to analyze the SLC2A1 gene genetic alterations in TCGA pan-cancer samples.cBioPortal . First, we used the “limma” package in R to screen differential expression genes (DEGs) between these two groups. We set FDR<0.05 and |log2FC|≥ 1 as the threshold values for DEG identification. After that, the enrichGO and enrichKEGG functions of the ClusterProfiler package in Bioconductor were used to perform GO/KEGG analysis on SLC2A1-related DEGs, choosing p.adj < 0.05, q-value < 0.05, and count ≥2 as cut-off values. Second, we performed GSEA based on the HALLMARK and REACTOME gene sets. Under the condition of FDR  < 0.25 and p < 0.05, the results were considered statistically significant. In addition, we used the single-cell database CancerSEA  to study the potential functions of SLC2A1. The aim of the CancerSEA database is to help researchers better understand various functional states of cancer cells at the single-cell level. This database contained 41,900 cancer single cells from 25 cancers, a total of 280 cell groups, and summarized 14 functional statuses of cancer cells.We selected the TCGA LUAD cohort as an example to explore the underlying mechanisms of SLC2A1. Based on the median expression of SLC2A1, we divided the patients into high and low groups. After that, we conducted Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG), and Gene Set Enrichment Analysis (GSEA) , which is a useful online platform for TCGA data analysis. Second, we compared the differences in ImmuneScore, StromalScore, and ESTIMATEScore between patients from the low-SLC2A1 and high- SLC2A1 groups with Wilcoxon signed-rank test. In addition, Spearman’s correlation analysis was used to evaluate the relationship between SLC2A1 and the tumor immune infiltration evaluated by the MCP counter score and EPIC algorithms.TME plays an important role in the occurrence and development of cancers. First, we used three algorithms, ESTIMATEScore, MCP-counter score, and EPIC ,19,20, tAccording to a previous study , we collp-value of less than 0.05 was considered statistically significant .We used R version 4.1.0 to perform the statistical analysis. Survival analysis was carried out according to Kaplan–Meier analysis, the log-rank test, and Cox regression analysis. We compared the continuous variables using Student’s t-test or the Wilcoxon rank-sum test, as appropriate. Categorical clinicopathological variables were compared using the chi-square test or Fisher’s exact test. Correlation analysis was performed by Pearson correlation analysis. A To preliminarily understand the expression of SLC2A1 in cancers, we first evaluated SLC2A1 mRNA expression in the TCGA-GTEx pan-cancer dataset. The results revealed that SLC2A1 expression was significantly upregulated in 22 cancer types: ACC, BLCA, BRCA, CESC, CHOL, COAD, ESCA, GBM, HNSC, KIRC, LGG, LIHC, LUAD, LUSC, OV, PAAD, READ, STAD, TGCT, THCA, UCEC, and UCS. In comparison, low SLC2A1 expression was observed in five kinds of tumors: DLBC, KICH, LAML, SKCM, and THYM . For paip < 0.0001), COAD (p < 0.0001), and ESCA (p < 0.0001) tissues was much higher than that in paired normal tissues , 56 in cell component (GO-CC), 10 in molecular function (GO-MF), and 14 in KEGG , of which 154 genes were downregulated and 192 genes were upregulated . The hea in KEGG . The bub in KEGG A–D. The  in KEGG A–C. The  in KEGG D–F. In a in KEGG G. CollecThe above results of function enrichment analysis and the protein expression of SLC2A1 across pan-cancer subtypes in the CPTAC samples suggested that SLC2A1 may be related to immune regulation, so we conducted an immune cell infiltration analysis, We used three algorithms  to explore the relationship between SLC2A1 expression and immune cell infiltration of TME. First, using the ESTIMATEscore, we found high expression levels of SLC2A1 are related to low ImmuneScore, low StromalScore, and low ESTIMATEScore in most cancers, such as ACC, BLCA, BRCA, ESCA, HNSC, LUAD, OSCC, PAAD, PRAD, SARC, SKCM, STAD, TGCT, UCEC, and UCS . Second,Immune surveillance affects the growth and development of cancer cells, and cancer cells evade immune responses by taking advantage of ICP . ICP genTMB and MSI are two new biomarkers that reflect the response of immunotherapy. So, we explored the correlation between SLC2A1 expression and TMB, and MSI. The expression of SLC2A11 is significantly positively correlated with TMB in most cancers, including PAAD, ACC, LUAD, THYM, GBM, SARC, STAD, CESC, BRCA, GBMLGG, STES, and HNSC, but negatively correlated with TMB in SKCM A. We alsSLC2A1 gene is one of the key genes in cancer glycometabolism, which promotes the glycolysis of cancer cells, thus affecting their growth and metastasis [The increase in glycolysis is one of the main characteristics of glycometabolism in cancer cells . The SLCtastasis ,30. StudIn our study, based on the TCGA-GTEx pan-cancer dataset, we found that SLC2A1 is highly expressed in most cancers compared with normal tissues, which we further confirmed via the GEO datasets, protein expression data from the HPA database, and our own data. Additionally, the expression of SLC2A1 is significantly upregulated in higher pathologic stages in various cancers. Previous studies have reported that SLC2A1 is highly expressed in a variety of cancers, such as BLCA, LUAD, LIHC, CESC, COAD, OV, UCEC, BRCA, STAD, ESCA, and PAAD ,35,36,37The evaluation of the prognostic value of SLC2A1 is an indispensable part of our study. We assessed the prognostic value of SLC2A1 in pan-cancer by using Kaplan–Meier and Cox regression analyses. The results showed that SLC2A1 is a hazard factor for the OS of patients in most cancers. Mortality from noncancerous causes may not reflect tumor biology, aggressiveness, or response to therapy. Additionally, a longer follow-up time is required for using OS. Therefore, to more accurately reflect the impact of SLC2A1 on the prognosis of patients, we further conducted a univariate COX analysis for PFI, DFI, and DSS of patients. The results also showed that SLC2A1 is a hazard factor for the PFI, DFI and DSS of patients in various cancers. These results indicated that high SLC2A1 expression mainly plays a hazardous role in patient prognosis for most cancer types. The high expression of SLC2A1 in cancers is often associated with poor prognosis ,39.To study why SLC2A1 is highly expressed in pan-cancer, we performed genetic alteration, DNA promoter methylation, and RNA m6A methylation analyses. We found that the overall mutation rate of SLC2A1 in pan-cancer is only 1.8%, which could not explain the high transcriptome expression of SLC2A1 in most cancers. Klepper et al. reported that Glut1 deficiency syndromes are due to SLC2A1 genetic variation . TherefoThe mechanism of SLC2A1 in pan-cancer has rarely been explored. LUAD is one of the most common cancer types. In our study, we found that SLC2A1 is highly expressed in LUAD, and was related to a poor prognosis in terms of OS, PFI, DSS, and DFI, so we took LUAD as the representative tumor in the GO, KEGG, and GSEA analyses of SLC2A1. The results showed that SLC2A1 is mainly associated with hypoxia, EMT, glycolysis, cell-cycle regulation, DNA repair, and neutrophil-mediated immunity. Single-cell sequencing can help us better understand the biological features of cancers, to achieve the goal of more precise cancer treatment ,47. In oThe TME plays a key role in the support of tumor progression, invasiveness, and metastasis . In our ICP genes affect the immune cell infiltration of the TME and cancer immunotherapy . As suchThe limitations in our study should be considered when generalizing the findings. First, our results were mainly generated from bioinformatics analysis. In vivo and in vitro experiments are needed to prove our results regarding the potential function of SLC2A1. Second, the microarray and sequencing data from different databases might have caused systematic bias. Third, the data used in this study were retrospective. Our results should thus be further confirmed by prospective studies in the future. Finally, no anti-SLC2A1 therapeutic monoclonal antibodies have yet been evaluated in clinical trials. Therefore, we have no specific and complete cases with data to identify the benefit of anti-SLC2A1-targeting drugs in the survival of cancer.SLC2A1 may play a crucial role in cancer cell proliferation and metastasis through multiple mechanisms, such as regulating the cell-cycle checkpoint, DNA repair process, EMT, CAFs and neutrophils in the TME, and T-cell exhaustion. SLC2A1 might serve as a novel pan-cancer diagnostic and prognostic biomarker and provide an opportunity to develop new immunotherapy strategies.


Objective: To uncover novel prognostic and therapeutic targets for BLCA, our study is the first to investigate the role of hsa-mir-183 and its up-regulated predicted target genes in bladder urothelial carcinoma.Methods: To address this issue, our study explored the roles of hsa-mir-183 predicted target genes in the prognosis of BLCA via UALCAN, Metascape, Kaplan-Meier plotter, Human Protein Atlas, TIMER2.0, cBioPortal and Genomics of Drug Sensitivity in Cancer databases.Results: High transcriptional expressions of PDCD6, GNG5, PHF6 and MAL2 were markedly relevant to favorable OS in BLCA patients, whereas SLC25A15 and PTDSS1 had opposite expression significance. Additionally, high transcriptional expression of PDCD6, GNG5, PHF6, MAL2, SLC25A15 and PTDSS1 were significantly correlated with BLCA individual cancer stages and molecular subtypes. Furthermore, high mutation rate of PDCD6, MAL2, SLC25A15 and PTDSS1 were observed. Finally, TP53 mutation of PDCD6, GNG5, PHF6, MAL2, SLC25A15 and PTDSS1 has guiding significance for drug selection in BLCA.Conclusions: PDCD6, GNG5, PHF6, MAL2, SLC25A15 and PTDSS1 could be the advanced independent indicators for prognosis of BLCA patients, and TP53-mutation might be a biomarker for drug option in BLCA patients. The  in 90%) , 5. New MicroRNA (miRNA), a small noncoding RNA with the function of negative regulating gene expression, plays an important role in the occurrence and development of various tumors –8. The fTo uncover novel prognostic and therapeutic targets for BLCA, our study is the first to investigate the role of hsa-mir-183 and its up-regulated predicted target genes and evaluate the significance of TP53-mutation related drug option in BLCA patients.http://ualcan.path.uab.edu). The Metascape was applied for GO and KEGG analysis of predicted target genes (https://metascape.org). The Overall Survival (OS) data of mRNA expression of predicted target genes were collected from Kaplan-Meier plotter (http://www.kmplot.com/). The protein expression data were collected from Human Protein Atlas (https://www.proteinatlas.org). The data of pan cancer analysis of predicted target genes were collected from TIMER2.0 (http://timer.cistrome.org). Gene mutations of predicted target genes in BLCA and their OS and Progression Free Survival (PFS) were analyzed by cBioPortal . Finally, the drug sensitivity and resistance of TP53 mutation in BLCA were analyzed by Genomics of Drug Sensitivity in Cancer (https://www.cancerrxgene.org).To estimate the prognostic and therapeutic significance of hsa-mir-183 and its up-regulated predicted target genes in BLCA patients, the clinical data, transcriptional expression data of miRNA and predicted target genes were collected from UALCAN . The expression of hsa-mir-183 were also significantly increased based on patient’s gender or age (P = 1.62E-12), Normal-vs-Female (P = 1.63E-8), Normal-vs-Age (41–60 Yrs) (P < 1E-12), Normal-vs-Age (61–80 Yrs) (P < 1E-12) and Normal-vs-Age (81–100 Yrs) (P = 7.55E-9).The result of top 10 over-expressed miRNA in BLCA patients were shown by TCGA miRNA analysis (Data from UALCAN) . The expr or age . There wThere were 247 predicted genes of hsa-mir-183 , including 15 up-regulated genes, 74 down-regulated genes and 158 unchanged genes in BLCA. 15 up-regulated genes were selected to study in our research . The expFunctions and pathways of up-expressed predicted target genes of hsa-mir-183 and their 10 frequently altered neighbor genes were analyzed by GO and KEGG in Metascape. As the Enrichment heatmap showed that pathways included hsa04726: Serotonergic synapse, hsa05219: Bladder cancer, GO:0007169: transmembrane receptor protein tyrosine kinase signaling pathway, GO:0048471: perinuclear region of cytoplasm, GO:0008324: cation transmembrane transporter activity, GO:1990234: transferase complex and GO:1905477: positive regulation of protein localization to membrane .P = 0.00013), GNG5 , PHF6  and MAL2  seem to be the favorable factor for BLCA patients’ prognosis. SLC25A15  and PTDSS1  more likely to be the unfavorable factor for BLCA patients’ prognosis. In addition, ENY2, PLAG1, SLC6A6, CELSR3, SLC22A23, SPATS2, CDK5R1, ATP2C1 and NRAS were not prognostic in BLCA patients.Prognostic value of mRNA expression of up-expressed predicted target genes of hsa-mir-183 were analyzed by Kaplan-Meier plotter . It was Protein expression of predicted target genes  in BLCA were explored by Human Protein Atlas . The proThe association of mRNA expression of predicted target genes  with clinicopathological parameters of BLCA patients were analyzed by UALCAN, containing BLCA individual cancer stages and molecular subtypes  and 9. FGene mutations of predicted target genes  in BLCA and their OS and PFS were analyzed by cBioPortal . Figure TP53 mutation of predicted target genes  were analyzed by UALCAN. The expression of TP53 mutation of GNG5, MAL2, SLC25A15, PTDSS1 in patients with BLCA were significantly increased compared to non-mutation group, and all predicted target genes were markedly increased compared to normal group . In addiP = 2.12e-06) of BLCA patients and the IC50 of Nutlin-3a (−) was significantly increased in TP53 mutation group (P < 0.001). TWS119 drug target was GSK3 and TWS119 drug target pathway was WNT signaling in BLCA. As was showed in P = 5.95e-06) of BLCA patients and the IC50 of TWS119 was significant decreased in TP53 mutation group (P < 0.001). Mitomycin-C drug target was DNA crosslinker and Mitomycin-C drug target pathway was DNA replication in BLCA. As was showed in P = 0.000322) of BLCA patients and the IC50 of Mitomycin-C was significant decreased in TP53 mutation group (P = 0.001).As was showed in BLCA was the most common histopathological type of bladder cancer, and its occurrence and development were affected by multiple complex factors such as gene transcription, gene regulation, gene mutation and epigenetic inheritance , 15. EacOur study found that up-expressed hsa-mir-183 and over transcriptional expressions of hsa-mir-183 predicted target genes  were observed in BLCA patients. Moreover, predicted functions and pathways of up-expressed predicted target genes of hsa-mir-183 and their 10 frequently altered neighbor genes indicated a link with bladder cancer. Besides, high transcriptional expressions of PDCD6, GNG5, PHF6 and MAL2 were markedly relevant to favorable OS in BLCA patients, whereas high transcriptional expression of SLC25A15 and PTDSS1 were markedly relevant to unfavorable OS in BLCA patients. Additionally, ENY2, PLAG1, SLC6A6, CELSR3, SLC22A23, SPATS2, CDK5R1, ATP2C1 and NRAS were not prognostic in BLCA patients. However, protein expression of predicted target genes  were inconsistent with the transcriptional expression. The mRNA levels were significantly increased than that observed in protein levels. There are two possible explanations for this phenomenon. miRNA do not limit gene translation but lead to rapid destruction of newborn peptides . AlternaProgrammed cell death 6, can encode the calcium binding protein, which is belonging to the penta-EF-hand protein family. High expression of PDCD6 was relevant to unfavorable prognosis in colorectal cancer patients via activating c-Raf, MEK and ERK pathway . InhibitG protein subunit gamma 5, can regulate the information from a variety of receptors. High expression of GNG5 was relevant to unfavorable prognosis in gliomas . At presPHD finger protein 6, has a function of transcriptional regulation by encoding domain with 2 PHD-type zinc fingers in protein. The high expression of PHF6 can play a role of tumor suppressor in patients with acute lymphoblastic leukemia and as a favorable prognostic factor –28. Howe+ T lymphocytes, thereby inhibiting the growth and metastasis of breast cancer [Mal, T cell differentiation protein 2, can encode multitransmembrane protein, which is a member of MAL proteolipid family. MAL2 was over-expressed in many human cancers, including breast, stomach, liver, pancreatic, colon, kidney, prostate and ovarian cancers –36. Zhent cancer . Zhang et cancer . In BLCAt cancer , which iSolute carrier family 25 member 15, can encode the protein transporter ornithine, which is a member of mitochondrial carrier family. Ji et al., found that SLC25A15 was up-expressed in melanoma patients and was negatively associated with OS and DFS . SLC25A1Phosphatidylserine synthase 1, has a function to promote the synthesis of phosphatidylserine. Li et al., found that over-expression of PTDSS1 in astroglioma involved in lipid metabolism . Wang etTo sum up, we concluded that PDCD6, GNG5, PHF6, MAL2, SLC25A15 and PTDSS1 could be the advanced independent indicators for prognosis of BLCA patients, and TP53-mutation might be a biomarker for drug option in BLCA patients.


KRAS-G12D mutations are the one of most frequent oncogenic drivers in human cancers. Unfortunately, no therapeutic agent directly targeting KRAS-G12D has been clinically approved yet, with such mutated species remaining undrugged. Notably, cofactor Mg Sotorasib was finally approved by the U.S. Food and Drug Administration for the clinical treatment of KRAS-G12C mutant cancers [KRAS proteins play an important role in various cellular processes, such as cell proliferation, differentiation, and survival ,2. As a  cancers ,27.In particular, several strategies have been proposed for targeting KRAS-G12D, such as indole-based inhibitors BI-2852) of the Switch-I/II pockets 52 of the, the pipClinical data have implicated not only that mutated RAS isoforms vary by tissue and cancer types, but also within the most frequently KRAS-mutations, the mutated amino acid residues of KRAS are also different for each cancer. For instance, among KRAS mutation predominant cancers, KRAS-G12C mutations mainly occur in lung cancer 45%), whereas KRAS-G12D mutations are related to more than 50% of pancreatic ductal adenocarcinoma cases ,41,42. T%, whereaAs one of the important RAS cofactors, the ion Mgproteins ,52,53. M500-fold ,55,56. H500-fold ,41,42. Tterature , we do nWith full use of the structural characteristics of these two dynamic pockets we designed in silico the specific inhibitor DBD15-21-22, a derivative of benzothiadiazine ,58 (DBD)Firstly, we investigated the conformational changes of the three different isoforms of KRAS in aqueous ionic solution. The only difference between these three KRAS is located at codons 12, with the sequences and initial structures of the three isoforms shown in Root mean square deviations and root mean square fluctuations of KRAS-WT and the two mutant isoforms were firstly analyzed A,B. Bothng sites ,48,59, a studies .The GLY12 mutation of KRAS protein also shows significant effects on the interaction of P-loop with Switch-II . GLY12 hnd GLN61 . In addind GLN61 . In the Noncovalent interactions, such as hydrogen bonds and coordination bonds . At the (O1/O2) . Further (O1/O2)  a comparSotorasib [7H8N2O2S, DBD), as a template. The most suitable species obtained is designed as DBD15-21-22 ). All bonds involving hydrogens were set to fixed lengths, allowing fluctuations of bond distances and angles for the remaining atoms. The crystal structure of GDP-bound KRAS proteins was downloaded from the RCSB PDB Protein Data Bank [Our main computational tool has been microsecond scale molecular dynamics. In MD, after the choice of reliable force fields, the corresponding Newton’s equations of motion are integrated numerically , allowinata Bank , file naemployed . Time stsampling ,84,85 orsampling ,87, weresampling ,89 to obware VMD  and UCSFware VMD  were usei, and RMSD) are defined by Equation (i (located at RMSF) are defined by Equation Rg=∑iEquation :(3)RMSFiTo summarize the main findings of the work, let us remark that KRAS mutations are widely present in many cancer cases, but there exist usually only molecular-scale differences between different KRAS mutants, such as in the one site GLY12 mutation. This means the direct information on atomic interactions and local structures at the all-atom level of KRAS can be extremely useful for oncogenic KRAS research. Based on powerful computer simulation tools, we focused our analysis on the local structure of KRAS proteins when associated with MgAfter deciphering the laws of conformational changes of different KRAS mutants, two druggable dynamic water pockets on KRAS-G12D-GDP-Mg


The non-structural proteins of ZIKV, such as non-structural protein 5, are involved in the evasion of the host’s immune defense mechanisms. Ocular manifestations of ZIKV arise from the virus’ ability to cross both the blood–brain barrier and blood-retinal barrier, as well as the blood-aqueous barrier. Most notably, this results in the development of GBS, a rare neurological complication in acute ZIKV infection. This can yield ocular symptoms and signs. Additionally, infants to whom ZIKV is transmitted congenitally develop congenital Zika syndrome (CZS). The ocular manifestations are widely variable, and include nonpurulent conjunctivitis, anterior uveitis, keratitis, trabeculitis, congenital glaucoma, microphthalmia, hypoplastic optic disc, and optic nerve pallor. There are currently no FDA approved therapeutic agents for treating ZIKV infections and, as such, a meticulous ocular examination is an important aspect of the diagnosis. This review utilized several published articles regarding the ocular findings of ZIKV, antiviral immune responses to ZIKV infection, and the pathogenesis of ocular manifestations in individuals with ZIKV infection. This review summarizes the current knowledge on the viral immunology of ZIKV, interactions between ZIKV and the host’s immune defense mechanism, pathological mechanisms, as well as anterior and posterior segment findings associated with ZIKV infection.Zika virus (ZIKV) is mosquito-borne flavivirus that caused a significant public health concern in French Polynesia and South America. The two major complications that gained the most media attention during the ZIKV outbreak were Guillain–Barré syndrome (GBS) and microcephaly in newborn infants. The two modes of ZIKV transmission are the vector-borne and non-vector borne modes of transmission. Aedes aegypti and Aedes albopictus are the most important vectors of ZIKV. ZIKV binds to surface receptors on permissive cells that support infection and replication, such as neural progenitor cells, dendritic cells, dermal fibroblasts, retinal pigment epithelial cells, endothelial cells, macrophages, epidermal keratinocytes, and trophoblasts to cause infection. The innate immune response to ZIKV infection is mediated by interferons and natural killer cells, whereas the adaptive immune response is mediated by CD8 Zika virus (ZIKV) is a mosquito-borne flavivirus of global medical importance that belongs to the Flaviviridae family. Other flaviviruses include dengue virus, hepatitis C virus, yellow fever virus, Japanese encephalitis virus, and West Nile virus. ZIKV acquired global health significance when it caused significant outbreaks in Yap Island, French Polynesia, and South America . ZIKV waZika virus is a positive-sense enveloped RNA virus with a genome of ~11 kilobases, open frame enclosed in an icosahedral nucleocapsid ,14. The Astrocytes, dendritic cells, dermal fibroblasts, endothelial cells, monocytes, macrophages, microglia, neural stem cells, mesenchymal stem cells, neural crest cells, neurons, epiderma keratinocytes, and trophoblasts are cells that support the infection and replication of ZIKV, as well as the subsequent dissemination of the virus to other permissive cells and tissues ,29,30. WZIKV engages DC-SIGN expressed on macrophages and dendritic cells to facilitate the infection of these activators and sentinel cells of the immune system ,31. The Toll-like receptors (TLR) serve as pattern recognition receptors that reside in the endosome of host cells. TLR3 recognizes dsRNA, a replication intermediate, and this interaction is associated with a downstream signaling cascade, in which the toll/interleukin-1 receptor (TIR) domain of TLR3 binds to the (TIR) domain of TIR-domain-containing adapter-inducing interferon-β (TRIF). This interaction between TLR3 and TRIF induces the recruitment and activation of TRIF. This is followed by the binding of the activated TRIF to tumor necrosis factor receptor associated factor 3 (TRAF3) and TRAF6. TRAF3 engages and recruits TBK1, resulting in the activation of IRF3. TRAF6 engages and recruits IKK and this results in the activation of NFκB. IRF3 and NFκB translocate to the nucleus where they stimulate genes required for coding interferons (type I IFN) and other cytokines respectively  20,38,3,40,41.Interferons are one of the major host restriction factors that inhibit the replication of ZIKV. Type I interferons (IFNI or IFN1 (JAK1) ,43. The 1 (JAK1) ,43. Thus1 (JAK1) . TLR3 en1 (JAK1) . This in1 (JAK1) ,25.ZIKV, like most members of the flaviviruses, evades host immune responses by interfering with molecules that mediate innate immunity. NS1, NS4, and NS4B inhibit the signaling pathway essential for the generation of IRF3 and IRF7. NS4 inhibit the activity of MAVS, whereas NS1 and NS4B inhibit the activity of TBK1 . HoweverType 1 IFN induces an antiviral microenvironment to limit the spread of the virus, as well as activate NK cells that mediate cytolytic and noncytolytic antiviral immune responses. Perforin and granzyme secreted by activated NK cells induce the cytolysis of ZIKV infected cells. NK cells secrete type II IFN (IFNγ) that inhibit viral replication and activate macrophages. Furthermore, IFNγ and TNFα secreted by activated NK cells facilitate the maturation and antigen presentation capability of dendritic cells .+T cells and effector CD8+T cells. IL-12 (secreted by dendritic cells and macrophages) and type II IFN (secreted by NK cells) induce activated CD4+T cells to differentiate into Th1 cells that secrete IL-2, IFNγ, and TNFα . I. I135]. ZIKV-induced anterior uveitis occurs through a combination of virus-induced cytopathic effects of the infected iris pigment epithelial cells and immune-mediated inflammation triggered by innate and adaptive immune responses to the ZIKV-infected iris pigment epithelial cells. Th1 cells recruited to the infected tissue secrete TNFα and IFNγ, which promote the cytokine-mediated inflammation of the infected tissue. CD8+T cells secrete perforin and granzyme that induce the direct cytotoxic destruction of ZIKV-infected cells ,138. IriOcular findings associated with congenital ZIKV syndrome include iris coloboma, microphthalmia, optic disc pallor, chorioretinal atrophy, lens subluxation, cataract, glaucoma, strabismus, retinal pigment epithelial mottling, maculopathy, abnormal retinal vessels, and pupillary abnormalities ,6. It isThere are currently no specific FDA approved medicines for treating ZIKV infection; however, specific pharmacotherapy is required to treat the ocular manifestations of ZIKV infections ,145,146.


Leukoencephalopathy with vanishing white matter (LVWM) is an autosomal recessive disease. Ovarioleukodystrophy is defined as LVWM in females showing signs or symptoms of gradual ovarian failure. We present a 38-year-old female with ovarioleukodystrophy who showed status epilepticus, gait instability, slurred speech, abdominal tendon hyperreflexia, and ovarian failure. Abnormal EEG, characteristic magnetic resonance, and unreported EIF2B5 compound heterozygous mutations [c.1016G>A (p.R339Q) and c.1157G>A (p.G386D)] were found. Furthermore, the present report summarizes 20 female patients with adult-onset ovarioleukodystrophy and EIF2B5 gene mutations. In conclusion, a new genetic locus for LVWM was discovered. Compared with previous cases, mutations at different EIF2B5 sites might have different clinical manifestations and obvious clinical heterogeneity. Leukoencephalopathy with vanishing white matter  is a progressive central nervous system condition that can be divided into the congenital, infant acute, early childhood-onset, juvenile-onset, and late-onset types , 2. The EIF2B1, EIF2B2, EIF2B3, EIF2B4, and EIF2B5 genes, which encode the subunits of the eIF2B protein, have been associated with LVWM , and high- sensitivity C-reactive protein (hs-CRP) at 69 (reference: 0–10) mg/L, erythrocyte sedimentation rate (ESR) at 106 (reference: 0–20) mm/h. Serum sex hormones showed ovarian failure, with estradiol at 55 pmol/L  IU/L. The other endocrine hormones in serum were abnormal either, such as total triiodothyronine (tT3) at 0.86 (reference: 1.01–2.48) nmol/L, prolactinemia at 835 (reference: 58–412) mIU/L. The other tests showed homocysteinemia at 22.5 (reference: 0–15.0) μmol/L and antinuclear antibody (1:100) (±). HIV antibody, HBV antibody, HCV antibody, glucose, lipids, glycosylated hemoglobin, tumor carbohydrate antigen, AMA profile, ANA profile, ANCA, anti-keratin antibody, anti-myeloperoxidase antibody, and anti-cyclic citrulline peptide antibody were all negative.She was admitted to our hospital emergency department. She was unconscious and was transferred to the encephalopathy department. A little wet rale was heard in both lower lungs on auscultation. Neurological examination showed that the bilateral radial periosteal reflex, biceps, and triceps tendons were hyper reflexive, but the bilateral knee reflex and Achilles tendon reflex were weak. Abdominal wall reflex was not elicited. Bilateral pathological signs were negative. There was no resistance in the neck, and meningeal irritation was negative. The other neuro-examinations could not be performed. The patient had no fever, but the blood tests indicated infection: white blood cells (WBC) at 9.65 (reference: 3.50–9.50) × 10Computed tomography (CT) scan confirmed the infection diagnosis of peritonitis, pneumonia and cholecystitis which were associated with her abdominal bloating and constipation. Much more abnormality was found on CT, such as multiple ground-glass nodules in the upper lobe of the right lung, dilated kidneys and entire bilateral ureters. Bilateral mild hydrops, dilated upper right kidney ureter, and minor ascites around the appendix were found by further ultrasound.2O and showed WBC at 35 × 106/L, glucose at 5.18 mmol/L, chlorine at 113.0 mmol/L, and immunoglobulin G at 39.8 mg/L suggesting subspinous intracranial infection associated with her slurred speech. Cerebrospinal fluid autoimmune encephalitis antibody spectrum, Bacteria (aerobic and anaerobic), fungal culture, and second-generation gene sequencing of pathogenic microorganisms were negative. Cytological pathology was negative. The electroencephalogram was abnormal. Multiple pathological waves, spikes, sharp, spike-slow, and mixed spike- slow waves were found in bilateral frontotemporal and midfrontal areas. Sharp waves were also seen in the bilateral temporal, central-parietal, and occipital areas . Variants that were clearly or potentially associated with the subjects' clinical phenotype were screened. Finally, Sanger sequencing was used to validate the results. Two mutations were located on chromosome 3 at chr3:183858378 and chr3:183859713. Sequencing revealed that the patient had one heterozygous mutation of the EIF2B5 gene in exon 7, changing the arginine into glutamine residue at position 339 . The frequency of this variant in the common people database was ExAC: 0.000099, gnomAD: 0.000060, and it was classified as pathogenic/possibly pathogenic by the ClinVar database. At the same amino acid position, two other mutations were reported as pathogenic variants   , 12. Ano1015C>T) . NM 0039LVWM is a disease related to white matter cystic degeneration and tissue loss . A cavitThe essential function of EIF2B is reflected by the evolutionary conservation of the complex in eukaryotes . EIF2B iEIF2B5 gene mutations. Almost all cases of ovarioleukodystrophy are due to exon mutations, except for patient #1. The intronic variant in the EIF2B5 gene activates a cryptic 5' donor splice site of intron 7, probably leading to the synthesis of a truncated protein   I98M mutant mice exhibited abnormal gait, male and female infertility, and epileptic seizures . These s/phyre2) , these tEIF2B5-related conditions are probably more frequent than initially thought, particularly in females  can be found in the article/supplementary material.The studies involving human participants were reviewed and approved by Shenzhen Traditional Chinese Medicine Hospital Ethics Committee. The patients/participants provided their written informed consent to participate in this study. Written informed consent was obtained from the individual(s) for the publication of any potentially identifiable images or data included in this article.FK was a major contributor in designing the case report and drafting the manuscript. JW and ZG revised the manuscript. FK, XL, HZ, and SL gave the clinical information containing medical history, neurological findings, hematological examination, electrophysiological analysis, neuroimages evaluation, and treatment. All authors commented on previous versions of the manuscript, read, and approved the final manuscript.This work was supported by the Shenzhen Municipal Commission of Health and Family Planning [grant number SZFZ2018013]; National Natural Science Foundation of China [grant number 82004284]; Guangdong Medical Science Foundation [grant number A2020370]; Guangdong Administration of Traditional Chinese Medicine Project ; Shenzhen Science and Technology Research Program [grant number RCBS20200714114959156].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.


Nr1d1 and Nr1d2 expression was upregulated in males during liver aging. Furthermore, the expression of other circadian genes, such as Arntl, Clock, Cry1/2, Per1/2, and Rora/c, decreased in males during liver aging; however, these genes showed various gene expression patterns in females during liver aging. Conclusions: Age-related elevation of Nr1d1/2 downregulates the expression of other circadian genes in males, but not females, during liver aging. Consequently, age-related upregulation of Nr1d1/2 may play a more crucial role in the change in circadian rhythms in males than in females during liver aging.Background: Circadian rhythm is associated with the aging process and sex differences; however, how age and sex can change circadian regulation systems remains unclear. Thus, we aimed to evaluate age- and sex-related changes in gene expression and identify sex-specific target molecules that can regulate aging. Methods: Rat livers were categorized into four groups, namely, young male, old male, young female, and old female, and the expression of several genes involved in the regulation of the circadian rhythm was confirmed by in silico and in vitro studies. Results: Gene Ontology and the Kyoto Encyclopedia of Genes and Genomes pathway enrichment analyses showed that the expression of genes related to circadian rhythms changed more in males than in females during liver aging. In addition, differentially expressed gene analysis and quantitative real-time polymerase chain reaction/western blotting analysis revealed that  Aging is associated with a decline and molecular changes in circadian rhythms via age-associated pathways, which aggravate the aging process ,2,3. In Sex has been reported to be one of the factors on the modulation of the circadian rhythm ,11, whicCry), period circadian regulators (Per), and other circadian genes [Nr1d1/2, Arntl, Clock, Cry 1/2, Per1/2, and Rora/c construct regulatory systems with several feedback loops; however, how age and sex can change these circadian modulation systems remains unclear.The regulation of circadian rhythms has been demonstrated. In mammals, aryl hydrocarbon receptor nuclear translocators, such as  and the clock circadian regulator (Clock), organize heterodimers to promote the expression of cryptochrome circadian regulators . Furthermore, DEGs were calculated after comparing the RNA-Seq data of the groups  . We idenTo identify the biological functions of genes whose expression changed according to age and sex, we used The Database for Annotation, Visualization, and Integrated Discovery (DAVID) to perform a GO enrichment analysis of the DEGs for each dataset that was identified in the results of the DEG calculation. Furthermore, we listed the top 15 GOs for each dataset in –cytokine receptor interactions, were upregulated in OM vs. YM, while metabolic pathways and circadian rhythms were downregulated in OM vs. YM. In addition, circadian rhythms were downregulated in OF vs. YF, although the metabolisms showed little age-related differences because the genes related to metabolic pathways were upregulated or downregulated, which indicated that metabolic genes were not changed in an age-specific manner. By contrast, immune pathways, including the T cell receptor signaling pathway and B cell receptor signaling pathway, were upregulated in OM vs. OF, while various metabolic pathways, drug metabolism, and circadian rhythms were downregulated in OM vs. OF. Finally, in YM vs. YF, both upregulated and downregulated pathways were involved in various metabolic processes, indicating that YM and YF showed no significant differences in immunity/inflammation and metabolic/circadian gene expression. These results showed that immune and inflammatory pathways were upregulated, while metabolic and circadian pathways were downregulated, in OM compared with OF. Furthermore, as in the GO analysis, KEGG enrichment analyses revealed that circadian rhythms were downregulated in both older males and females; however, these differences were more pronounced in older males than in older females. These results suggest that the circadian rhythm may play a crucial role in the liver aging of males but not females.To confirm the biological pathways of genes whose expressions changed according to age and sex, we performed KEGG enrichment analyses of the DEGs for each dataset that was identified in the results of the previous analysis and then listed pathways in which the DEGs were involved . InflammNr1d1 and Nr1d2 were upregulated in OM compared with YM, and their expression was downregulated in OF compared with YF. Conversely, the expression level of Arntl  and Clock (or Npas2) decreased in males during aging, and the expression level of these genes was elevated in females during aging. In addition, Cry1, Rora, and Rorc showed age-related decreases only in males. By contrast, Per2 expression was downregulated in both sexes during aging. Furthermore, Cry2 and Per1 showed no age-related differences between men and women. These results reveal that several circadian genes, such as Nr1d1, Nr1d2, Arntl, and Clock, showed sex-specific changes in gene expression during liver aging, which resulted in the differential expression of circadian genes between OM and OF.GO and KEGG analyses revealed that the expression of circadian rhythms showed sex differences during aging. Therefore, we selected genes related to the regulation of the circadian rhythm and confirmed their gene expression changes from each dataset. As shown in Nr1d1, Nr1d2, Arntl, and Clock, show sex differences in gene expression during the aging process. qRT-PCR was performed to validate the expression of the circadian genes , six old male rats , six young female rats , and six old female rats  were purchased from Samtako ,85,86,87n = 3 in each group). RNA integrity was assessed using an Agilent 2100 BioAnalyzer and an Infinite F200 . Libraries were prepared for 150-bp paired-end sequencing using the TruSeq stranded mRNA Sample Preparation Kit . mRNA molecules were purified and fragmented from 1 μg of the total RNA using oligo (dT) magnetic beads. Fragmented mRNAs were synthesized as single-stranded cDNAs through random hexamer priming. Double-stranded cDNA was prepared by applying it as a template for second strand synthesis. After the sequential process of end repair, A-tailing, and adapter ligation, cDNA libraries were amplified with a polymerase chain reaction (PCR). The quality of the cDNA libraries was evaluated using an Agilent 2100 BioAnalyzer . They were measured using the KAPA library quantification kit  according to the manufacturer’s library quantification protocol. After the cluster amplification of denatured templates was performed, paired-end sequencing (2 × 150 bp) was performed using Illumina NovaSeq6000 .The total RNA was isolated from the liver samples using TRIzol reagent , and samples from each group were pooled in equal quantities for RNA-Seq . The results of the GO and KEGG enrichment analyses are listed in Gene Ontology (GO) enrichment analysis was used to analyze the biological functions of the genes, and the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis was performed to investigate the signaling pathways related to the unique DEGs. The Database for Annotation, Visualization, and Integrated Discovery (DAVID) was used to perform GO and KEGG enrichment analyses (filtering options: n = 6 per group) and was reverse-transcribed using the cDNA synthesis kit from GenDEPOT. qRT-PCR analysis was performed to quantify mRNA levels using SYBR Green  and the CFX Connect System . Primer sequences are listed in Primers for qRT-PCR were synthesized by Bioneer, Inc. . Total RNA was isolated from rat liver tissues (20 mg) using TRIzol reagent  (g for 2 min at 4 °C. The supernatants were deemed cytosolic fractions.Frozen liver tissues (150–200 mg) were ground using liquid nitrogen in a mortar and pestle. Ground liver tissues were homogenized in 1 mL hypotonic lysis buffer. Buffer A was composed of KCl (10 mM), MgCl2 (2 mM), dithiothreitol (DTT) (1 mM), EDTA (0.1 mM), PMSF (0.1 mM), pepstatin (1 μM), leupeptin (2 μM), β-glycerophosphate (20 mM), NaF (20 mM), Na3VO4 (2 mM), and 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), pH 7.4 (10 mM). The tissue homogenizer was used for 20 s. After homogenates were incubated on ice for 15 min, 125 μL of 10% Nonidet P-40 (NP-40) was added to the homogenates and mixed for 15 s, and the mixture was centrifuged at 14,000× Total protein extraction samples of the rat liver tissues were boiled for 5 min in a loading buffer containing 0.2% bromophenol blue, 125 mM Tris-HCl, 10% 2-mercaptoethanol, pH 6.8, and 4% sodium dodecyl sulfate. Equal amounts of protein (8–10 μg) were loaded and separated via SDS-PAGE using 10% gels and then transferred to PVDF membranes at 25 V for 10 min using a semidry transfer method. Protein-transferred membranes were soaked in 5% nonfat milk buffer containing 100 mM NaCl, 10 mM Tris (pH 7.5), and 0.1% Tween-20 for 2 h. The membranes were washed four times in TBS-Tween buffer for 8 min and were immunoblotted with specific primary antibodies (1:1000) dilution at 4 °C overnight. This was followed by HRP-conjugated secondary incubation  according to the manufacturer’s instructions of each primary antibody for 1 h at RT. Antibody labeling was detected using enhanced chemiluminescence in accordance with the manufacturer’s instructions. Molecular weights were determined using wide-range protein markers. The resulting immunoblots were visualized using Chemiluminescent HRP Substrate , Davinchchemi CAS-400 , and ImageJ software , according to the manufacturers’ instructions. Antibodies against Nr1d1 (sc-100910), Nr1d2 (sc-398252), Arntl (sc-365645), and β-Actin (sc-47778) were purchased from Santa Cruz . Clock antibody (#5157) was obtained from Cell Signaling Technology , and secondary antibodies  were purchased from Genetax .t-test was used to analyze the differences between the two groups. Statistical significance was set at p < 0.05. Statistical analyses were performed using GraphPad Prism 5 .The Student’s Nr1d1/2 expressions were upregulated in males and downregulated in females, leading to sex-different disruptions of the negative feedback loop in the circadian rhythm via regulation of their target genes, such as Arntl and Clock, during liver aging. Therefore, upregulation of Nr1d1/2 expression may play an important role in disrupting the negative feedback loop in the circadian rhythm of sex-specific male rats during the liver aging process (Nr1d1 and Nr1d2 play an important role in the circadian rhythm of male rats, but not in female rats, in the liver aging process.In conclusion, via transcriptomic analysis, this study revealed that the genes involved in most circadian rhythms were downregulated in males, but not in females, during liver aging. In detail,  process . Our ana