Kidney renal clear cell carcinoma (KIRC) is one of the most prevalent and deadly types of renal cancer in adults. Recent research has identified circadian genes as being involved in the development and progression of KIRC by altering their expression. This study aimed to identify circadian genes that are differentially expressed in KIRC and assess their role in KIRC progression. In KIRC, there were 553 differentially expressed rhythm genes (DERGs), with 300 up-regulated and 253 down-regulated DERGs. Functional enrichment analyses showed that DERGs were greatly enriched in the circadian rhythm and immune response pathways. Survival analyses indicated that higher expression levels of CSNK1E were related to shorter overall survival of KIRC patients, whereas lower expression levels of GNA11, KLF9, and THRAP3 were associated with shorter overall survival of KIRC patients. Through cell assay verification, the mRNA level of CSNK1E was significantly up-regulated, whereas the mRNA levels of GNA11, KLF9, and THRAP3 were dramatically down-regulated in KIRC cells, which were consistent with the bioinformatics analysis of KIRC patient samples. Age, grade, stage, TM classification, and CSNK1E expression were all shown to be high-risk variables, whereas GNA11, KLF9, and THRAP3 expression were found to be low-risk factors in univariate Cox analyses. Multivariate Cox analyses showed that CSNK1E and KLF9 were also independently related to overall survival. Immune infiltration analysis indicated that the proportion of immune cells varied greatly between KIRC tissues and normal tissue, whereas CSNK1E, GNA11, KLF9, and THRAP3 expression levels were substantially linked with the infiltration abundance of immune cells and immunological biomarkers. Moreover, interaction networks between CSNK1E/GNA11/KLF9/THRAP3 and immune genes were constructed to explore the stream connections. The findings could help us better understand the molecular mechanisms of KIRC progression, and CSNK1E/GNA11/KLF9/THRAP3 might be used as molecular targets for chronotherapy in KIRC patients in the near future. Kidney renal clear cell carcinoma (KIRC) is the most common histological subtype of renal cell carcinoma, accounting for 80–90% of all cases, with a high degree of immune infiltration ,2,3. A bThe circadian rhythm is critical in maintaining human homeostasis balance. Recent research has discovered that circadian rhythm disorders can lead to a variety of diseases, including endocrine disorders, sleep disorders, metabolic disorders, decreased immune function, neurasthenia, premature aging, and tumors. Circadian rhythm disorders have been linked to an increased risk of cancer in epidemiological studies, and abnormal expression levels of abundant clock genes and circadian reset have also been found in tumor patients ,7. In reHere, we used bioinformatics methods to identify the differentially expressed rhythm genes in kidney renal clear cell carcinoma and perform functional enrichment analysis, survival analysis, expression analysis, clinical correlation analysis, immune infiltration analysis, and PPI network analysis . Our stuhttps://portal.gdc.cancer.gov/, access date: 2 October 2021) was used to obtain gene expression data and clinical information for 539 KIRC tumor samples and 72 normal samples. IMMPORT  provided the list of immunological genes. The data were analyzed using R software (version 4.0.3) with a threshold of p < 0.05.The Cancer Genome Atlas  constructed by our team [p. adjust value.We used R software to screen gene expression data and obtained 6233 differentially expressed genes (DEGs) representing differences with |log2-Fold Change (FC)| > 2 and p. adjust < 0.05 was considered statistically significant.For GO and KEGG pathway analysis, we used the Cluster Profiler program. First, we utilized the limma package to determine the |Log2(Fold Change)| > 2 threshold value of the mRNA expression difference. First, we utilized the limma package to determine the |Log2(Fold Change)| > 2 threshold value of the mRNA expression difference. Subsequently, we used the ggplot2 package in R software for functional enrichment analyses. p < 0.05 was considered statistically significant). Then, using the Kaplan–Meier plotter online tool , the survival curve analyses of the CSNK1E, GNA11, KLF9, and THRAP3 genes were validated . The median gene expression was used as the cut-off value for the 539 KIRC samples, which were separated into high and low groups. Using the survminer and survival R packages, we performed the overall survival (OS) analyses of 12 DERGs enriched in the circadian pathway . Moreover, we extracted the expression levels of CSNK1E, GNA11, KLF9 and THRAP3 genes and drew matching graphs in R software (version 4.0.3).https://www.proteinatlas.org/, access date: 8 June 2022). We entered the gene name in the search field and clicked ‘SEARCH’. The search results are as follows: first a description of the gene, and then the gene was expressed in tissue, cell, and pathology tumor tissue.The Human Protein Atlas (HPA) provided information on the tissue and cellular distribution of 26,000 human proteins , 489 samples were retained for univariate and multivariate COX analyses to evaluate the combined effect of clinical characteristics and genes. The prognostic model was evaluated using area under the curve (AUC) values and the receiver operating characteristic (ROC) curve.https://cistrome.shinyapps.io/timer/, access date: 14 October 2021). Moreover, we used TIMER2.0 to analyze the correlation between CSNK1E/GNA11/KLF9/THRAP3 and immune cell gene biomarkers.We applied CIBERSORT to calculate the abundance and correlations of 22 distinct functional immune cell types in KIRC. The relationship between DERGs and immune-infiltrating cells was detected with TIMER2.0, an online analysis website . CytoScape software was used to map the network between DERGs and differential immune genes.2.HEK-293T (Procell CL-0005), A-498 (Procell CL-0254) and 786-O (Procell CL-0010) cell lines were kindly provided by Procell Life Science and Technology Co., Ltd. , and were correctly identified by STR. RPTEC (FY-22FN1580) cells were kindly provided by Fuyu Biotechnology Co., Ltd. , and were correctly identified by STR. HEK-293T and RPTEC cells were cultured in Dulbecco’s modified Eagle’s medium ; A-498 and 786-O cells were cultured in Minimum Essential Medium  and Roswell Park Memorial Institute  separately with 10% fetal bovine serum  and 1% penicillin and streptomycin  in a 37 °C incubator containing 5% COTrizol was used to lyse cells and extract total RNA. Quantitative real-time PCR (qRT-PCR) was performed with the TransScript Green One-Step qRT-PCR SuperMix (Trans Gen Biotechnoloy) using the Step One Plus Real-Time PCR System (Life Technologies). The primers of four DERGs were as follows: CSNK1E-fw: ggagatatctacctgggtgcc. CSNK1E-rv: tctcgatgtgcagctggg. GNA11-fw: gaagagcacgttcatcaagc. GNA11-rv: agaggatcttgagcgtctcc. KLF9-fw: cagagtgcatacaggtgaagc. KLF9-rv: tctcacacagcggacagc. THRAP3-fw: gttcttttggggtagtgtctgg. THRAP3-rv: agagcgagatccagacttgg.t-test to compare the two groups of data. p < 0.05 or p. adjust < 0.05 was considered statistically significant. All the data were processed and analyzed using the R software (version 4.0.3). We used the Mann–Whitney test and A total of 6233 differentially expressed genes and 1368 circadian genes were noted after analyzing the KIRC-FRPK and CGDB datasets, separately. In total, 553 DERGs were detected in both datasets . In KIRCp < 0.05 , were high-risk factors, whereas GNA11 expression , KLF9 expression , and THRAP3 expression  were low-risk factors (p < 0.001) and KLF9  were also independently related to overall survival in KIRC patients  curves for DERGs were derived from clinical data from 489 KIRC patients from the TCGA. Univariate Cox analyses revealed that age, grade, stage, TM classification, CSNK1E expression , Macrophages M0, Macrophages M1, and Macrophages M2 than paired normal tissues. However, KIRC tissues had reduced numbers of B cells naive, T cells CD4 memory resting, and dendritic cells activated B. The la+ T cells  and neutrophils  but was negatively associated with the infiltration abundance of B cells  (p = 8.60 × 10−3), CD8+ T cells , CD4+ T cells , macrophages , neutrophils  and dendritic cells  , CD4+ T cells , macrophages , neutrophils  and dendritic cells  (p = 1.62 × 10−8), CD8+ T cells , CD4+ T cells , macrophages , neutrophils  and dendritic cells  were correlated with THRAP3 expression  A. GNA11  × 10−8) A. Moreov × 10−2) A. B cellpression A. Furthe B cells B. The fiTo elucidate the regulatory relationships between CSNK1E/GNA11/KLF9/THRAP3 and important immune gene modules, we constructed the PPI network of CSNK1E/GNA11/KLF9/THRAP3 and 101 differentially expressed genes associated with survival . The intIn epidemiological research, it has been discovered that a disruption in circadian rhythm may increase the risk of cancer, and tumor patients have been found to exhibit an aberrant expression of abundant circadian genes, as well as a reset of their circadian rhythm. In studies examining the role of molecular clocks in human cancers, dysfunctions or polymorphisms in multiple or all of the core circadian genes have been identified. For example, dysregulations or polymorphisms of PER1, PER2, PER3, CLOCK, BMAL1, CRY1, CRY2, and NPAS2 were common in ovarian cancer, prostate cancer, leukemia, gliomas, hepatocellular carcinoma, and colorectal cancer ,22,23,24+ T cells and Neutrophils and negatively associated with the infiltration abundance of B cells. CSNK1E positively regulated the expression of differentially expressed immune genes related to survival such as AGER, GNRH1, SHC1, IRF9, TNFRSF25, LMBR1L, LTB4R, and LTB4R2. A recent study reported that AGER, GNRH1, SHC1, IRF9, and LTB4R could be used as prognostic factors of KIRC [The protein encoded by CSNK1E was a serine/threonine protein kinase that belonged to the casein kinase I protein family. Members of this family have been linked to cytoplasmic and nuclear activities such as DNA replication and repair. CK1 kinases participated in multiple cellular processes such as apoptosis, differentiation, and cell division . In glio of KIRC ,30,31. + T cells, CD4+ T cells, macrophages, neutrophils, and dendritic cells. Moreover, we analyzed the PD-L1(CD274) expression in KIRC tissues and normal tissues from the TCGA and analyzed the correlation between PD-L1(CD274) expression and circadian gene expression using the TIMER website. Renal tumors are highly immunogenic (they attract immune cells); however, they are rendered dysfunctional through the expression of PD-L1 (and other immune inhibitory molecules) on tumor cells. Therefore, we analyzed the PD-L1(CD274) expression in KIRC tissues and normal tissue from the TCGA and analyzed the correlation between PD-L1(CD274) expression and circadian gene expression using the TIMER website. The results showed that the mRNA levels of PD-L1(CD274) were significantly up-regulated compared with normal tissues. The PD-L1(CD274) expression was positively correlated with the GNA11/KLF9/THRAP3 expression but was not significantly correlated with the CSNK1E expression (The protein encoded by GNA11 was a member of the guanine nucleotide-binding protein (G protein) family, which served as modulators or transducers in a variety of transmembrane signaling systems. The protein encoded by KLF9 was a transcription factor that bound to a GC box element located in the promoter. The encoded protein binding to a single GC box inhibited transcription, while binding to tandem repeating GC Box elements activated transcription. Moreover, KLF9 was a circadian transcription factor in the human epidermis that controlled keratinocyte growth . The propression .Many anticancer drugs have significant time-of-administration effects, and their biological characteristics, such as absorption, distribution, metabolism, and elimination, are synchronized with people’s circadian rhythms. Once these drugs entered tumor cells, their functions would be regulated by the tumor cells’ circadian cycle, resulting in anticancer drugs with variable anticancer effects and adverse consequences depending on the time of administration. A new mode of chronotherapy has been proposed and combined with clinical treatment of tumor, forming chronotherapy of tumor; that is, radiotherapy and chemotherapy could be carried out at a specific time according to the circadian rhythm, so as to maximize the killing of tumor cells and reduce the toxic and side effects related to radiotherapy and chemotherapy ,36,37. B+ T cell responses were mediated by an internal cellular circadian oscillator, which could drive rhythmic CD4+ T cell immunological responses [There is increasing evidence that the circadian clock is involved in regulating the tumor microenvironment and immune system. Deng’s research found that the immune checkpoint pathway in macrophages was regulated by circadian genes, which could be potential therapeutic targets for lethal infection . Chen’s esponses . Our invAlthough we have found four circadian genes with prognostic value in KIRC using the TCGA database and validated them using the KM online website, we will conduct more cell experiments to investigate in depth the expression of these four genes and the mechanism of action on KIRC in the future.In this study, we identified 553 rhythm genes that were differentially expressed in kidney renal clear cell carcinoma. Among them, the expression levels of CSNK1E, GNA11, KLF9, and THRAP3 were not only significantly correlated with overall survival and other clinical parameters of KIRC patients but also strongly associated with the infiltration abundance of immune cells and immunological biomarkers. In summary, circadian genes, cancer hallmark pathways and immune infiltration are closely related . Our fin


USP7 gene (*602519) mutations on chromosome 16p13.2. So far, only 15 cases with 14 deleterious variants in the USP7 gene have been reported.Hao-fountain syndrome (HAFOUS) is a neurodevelopmental syndrome characterized by global developmental and severe language delays, behavioral abnormalities (including autism), and mild dysmorphic impairment of intellectual development. It is a dominant genetic disease caused by USP7 variants. Besides, we identified novel de novo heterozygous USP7 variants using trio-whole exome sequencing and verified by Sanger sequencing. Furthermore, clinical characteristics were evaluated by reviewing the medical records.This study describes three unrelated patients with The three identified variants, i.e., one frameshift variant  and two missense variants  are unreported. The predominant clinical manifestations of the three patients included: DD/ID; language impairment; abnormal behavior; abnormal brain magnetic resonance ; some also had facial abnormalities.USP7 gene using whole-genome sequence data. This work expands the USP7 gene mutation spectrum and provides additional clinical data on the clinical phenotype of HAFOUS.In summary, DD/ID is the most prevalent clinical phenotype of HAFOUS, although some patients also exhibit language and behavioral abnormalities. For the first time in China, we identified three variants of the  Hao-fountain syndrome  is a neurodevelopmental syndrome characterized by impaired intellectual development, including global developmental and language delays, behavioral abnormalities, and mild deformities. Other variable features include: hypotonia, feeding problems, delayed walking with an unstable gait, hypogonadism in males, and eye abnormalities, including strabismus. Patients develop epilepsy; others present mild white matter abnormalities on brain imaging .USP7 gene cause HAFOUS. Notably, the USP7 gene is located in chromosome 16p13.2, has 31 exons, and encodes Ubiquitin carboxyl-terminal hydrolase 7 protein comprising 1,102 amino acids. The USP7 gene encodes a deubiquitinase, a component of the MAGEL2 (605283)/TRIM27 (602165) ubiquitin ligase complex. Moreover, the USP7 gene is essential for WASH (613632)-mediated endosomal actin assembly and protein recycling. Only 15 HAFOUS cases with 14 deleterious point variants in the USP7 gene have been reported in two articles. Tyr143Ter) in the USP7 gene of a 13-year-old girl diagnosed with HAFOUS (in vitro knockdown of the USP7 gene in HeLa cells reduced protein levels of TRIM27 (602165), endosomal protein recycling, and F-actin accumulation  (P1), xGen Exome Research Panel v2.0  (P2 and P3), and target enrichment kits. Paired-end reads were utilized for sequencing on the Illumina NovaSeq6000 and BGI DNBSEQ-T7 platforms.Trio-WES and Variant Assessment were analyzed as follows .After the removal of adapters and low-quality reads, the resultant clean data were aligned to the NCBI human reference genome (hgl9) using the Burrows-Wheeler alignment (BWA). The variants (SNPs and indels) were called using GATK. Libraries were sequenced to an average depth of × 100, and each sample had at least 95% of target bases sequenced by 20 reads. de novo, recessive, compound heterozygous, or inherited X-linked variants. Sanger sequencing confirmed the de novo occurrence of all USP7 gene variants from each patient. Sanger sequencing was carried out using the primers: 247-F1: GCTGATCAAATTTGGCTTCCAGTA, 247-R1: AGCACTCTGAGTTTTGGACTTCAT; 992-F2: ATGCCA TTAGATACACACAACACT, 992-R2: TGGTGGTATGTGGCT ACCAG; 835-F3: AGCCTCCCTAAATTAAGTGCCTAC, 835-R3: TCGTTAAAAGCCCTAACAACAACC. Variant annotation was based on the USP7 gene transcript NM_003470.The databases for MAFs annotation included: gnomAD, dbSNP, 1000 Genomes Project, ExAC, ESP, and an in-house database . The variants were predicted to the effects on protein product structure by SIFT, Polyphen-2, Provean, REVEL, CADD, and MutationTaster. spliceAI, MaxEntScan, dbscSNV, and GTAG software packages were used to predict a functional change of variants on the splicing sites. As a prioritized pathogenicity annotation to the American College of Medical Genetics and Genomics (ACMG) criteria and guidelines , OMIM, HThe variant sites were mapped onto the protein structure (PBD: 2F1Z) to evaluate the effect of the identified missense variants on Ubiquitin carboxyl-terminal hydrolase 7.Patient 1 (P1) was a 5 years and 1 month old boy born to healthy, non-consanguineous parents. The patient could sit at 7 months and walk at 16, respectively. Facial features include a depressed nasal bridge, abnormal dental pulp morphology, narrow palpebral fissure, and an abnormal pinna. At 2 years, the child uttered the first words; by 4 years, he could speak only single words. The patient presented with impaired social interactions and had suboptimal self-care ability. The patient underwent surgery for hydrocele testis at the age of 2. Brain MRI at 4 years 6 months and 5 years 1 month revealed lateral ventricles dilation, dilation of Virchow-Robin spaces, and the third ventricle .Patient 2 (P2) was a 22-month-old boy admitted to the hospital with painful urination with no obvious cause. Laboratory test results showed increased alkaline phosphatase, lactate dehydrogenase, alpha-hydroxybutyrate dehydrogenase, lactate dehydrogenase isoenzyme 1, and decreased creatinine. Blood ammonia level was 45 μmol/L . Chordee and hypospadias were discovered at 18 months of age. The speech was limited to a few words at 1 year and 10 months. A brain MRI revealed abnormal cerebral white matter morphology in bilateral occipital lobes, hypodysplasia of the corpus callosum, and an arachnoid cyst.via the cesarean section at the 35th week of gestation due to membrane rupture. His development delay was apparent by 4 months. At 7 months, the child could not hold his head upright or turn over. An MRI of the brain revealed delayed myelination and a widened subarachnoid space (Patient 3 (P3) was 7 months old and was born to healthy non-consanguineous parents. The patient was delivered with a weight of 2,000 g and a length of 45 cm id space . Echocarde novo variants in USP7, i.e., one frameshift variant and two missense variants. P1 had a frameshift variant c.247_250del (p.Glu83Argfs × 18). The missense variants c.992A > G (p.Tyr331Cys) and c.835T > G  were identified in P2 and P3, respectively. Sanger sequencing confirmed the result of trio-WES . Some patients had language and behavioral abnormalities , and some had genitourinary abnormalities.The present study describes three novel patients with pathogenic/likely pathogenic missense and truncating variants in the patients . All theUSP7 protein is a component of the MAGEL2/TRIM27 ubiquitin ligase complex essential for WASH-mediated endosomal actin assembly and protein recycling (magel2-trim27 ubiquitin ligase complex is responsible for polyubiquitination. Patients with Prader-Willi (PWS) and Schaaf-Yang syndrome, neurodevelopmental disorders, intellectual disability with attributes, autism, and low muscle tone have magel2 functional deficits  is a deubiquitinating enzyme that causes neurodevelopmental disorders (USP7 protein (Pfam: Q93009) contains the following important functional domains: MATH, UCH, USP7_ICP0_bdg, and USP_C2. MATH domain is a binding domain, originally defined by functionally unrelated domains, i.e., the region of homology between the intracellular TRAF-C domain of the TRAF protein and the C-terminal regions of the extracellular meprins A and B. Although intracellular TRAFs and extracellular meprins are functionally unrelated, they share a conserved region of approximately 180 residues known as the meprin and TRAF homology (MATH) domain , also known as Deubiquitinating enzymes (DUBs), is a thiol protease that recognizes and hydrolyzes the peptide bond at the C-terminal glycine of ubiquitin . However, its function remains unclear  domain . Meprins) domain . Cytokin) domain . TRAF pr) domain . They prterminus . Ubiquitbiquitin . UCH thixtension . Ubiquitractions . The USP unclear . PathogeUSP7 gene in three Chinese males using whole-exome sequencing. Our findings enrich the mutation spectrum of the USP7 gene and improve the understanding of HAFOUS disease. Also, this work demonstrates the importance of whole-exome sequencing in the precise diagnosis of genetic diseases.In conclusion, we identified three novel variants in the https://www.ncbi.nlm.nih.gov/, SCV002588724, SCV002588725, and SCV002588726.The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found below: The studies involving human participants were reviewed and approved by the Ethics Committee of Henan Province People’s Hospital. Written informed consent to participate in this study was provided by the participants’ legal guardian/next of kin. Written informed consent was obtained from the individual(s), and minor(s)’ legal guardian/next of kin, for the publication of any potentially identifiable images or data included in this article.HZ and SM wrote the manuscript. FL, LW, YW, and WG were responsible for genetic analysis. JRH, FZ, JH, YL, and HL provided clinical information. HL and WG critically reviewed the manuscript. All authors read and approved the final manuscript.


TIN is typically characterized by a (sub)acute rise in creatininemia, which is seldom specific.Yet, quantitative and qualitative data about the association of TIN with various drugs are scarce. Therefore, we aimed to rank the drugs having the strongest association with TIN, based on the international pharmacovigilance database and a disproportionality analysis.Between November 14, 1967, and August 31, 2021, a total of 15,696 cases of TIN  were reported in VigiBase .S23-S29 Among 15,696 reports, 12,481 (79.5%) were deemed serious, including 977 fatal cases (6.2%). The median time to onset was 20 (interquartile range: 5–103) days. The MedDRA terms most frequently co-reported were acute kidney injury, in 6350 reports (40.5%), and chronic kidney disease in 4292 cases (27.3%). Pyrexia , nausea , rash , and eosinophilia  were the most frequently coreported extrarenal terms. Among 8221 cases with an available outcome, TIN completely recovered in 3019 reports (36.7%), did not recover in 2676 reports (32.6%), and was recovering or recovered with sequelae (without returning to baseline kidney function) in 2526 reports (30.7%).Most of the reported drugs belong to the “alimentary tract and metabolism” and the “antiinfectives for systemic use” categories , with 8204 (52.3%) and 4525 (28.8%) notifications, respectively. Specifically, proton pump inhibitors (PPIs) were the most frequently reported drugs, with 5769 reports (36.8%). Omeprazole was the most reported , followed by lansoprazole and esomeprazole. Aside from PPIs, the most reported active ingredient was ciprofloxacin with 529 reports (3.4%), followed by ibuprofen , vancomycin , diclofenac , and sulfamethoxazole/trimethoprim . As a whole, β-lactams accounted for 1461 reports.inter alia. Penicillin antibiotics, such as nafcillin  and flucloxacillin , were also characterized by strong ROR. As a whole, β-lactam was characterized by a ROR of 4.8 (95% CI 4.6–5.1). Other drugs significantly associated with TIN included sodium phosphate  and mesalazine . The ranking of active ingredients with IC025 > 0 and ≥30 reports is provided in In the disproportionality analysis, 223 active ingredients were characterized by a positive IC025. PPI  reached the 9 strongest ROR, including dexlansoprazole , lansoprazole , and rabeprazole , Although virtually any drug can be associated with a risk of TIN, this analysis of the World Health Organization safety database highlights the most frequently involved. These drugs should alert the clinician facing suspicion of TIN or acute renal failure of undetermined cause. PPIs, antibiotics, and nonsteroidal anti-inflammatory drugs are the most reported drugs for TIN. This ranking is confirmed by the disproportionality analysis, which is a more reliable indicator of the comparative risk for a drug to be associated with TIN.,,S5,Previous studiesS3,As expected,S6,S9–S12In line with previous studies,S2,S6,S16–S20The association of nonsteroidal anti-inflammatory drugs  with TIN was foreseeable.Sodium phosphate can elicit acute or chronic phosphate nephropathy,To our knowledge, this study about TIN is the first to rely on a comprehensive analysis of >15,000 reports from the World Health Organization safety database, with drugs used in a real-life setting. Though, pharmacovigilance studies are hindered by under-reporting.In this comprehensive analysis of the World Health Organization safety database, a ranking of drugs associated with TIN has been established. The strongest associations with TIN involve PPIs, antibiotics , and nonsteroidal anti-inflammatory drugs. Early identification of a potential causative drug is pivotal to increase chances of recovery. Hence, pharmacovigilance data can foster the efficient management of (sub)acute renal failure of undetermined origin.All the authors declared no competing interests.


Sickle cell disease (SCD) is a group of inherited blood disorders affecting the β-globin gene, resulting in the polymerization of hemoglobin and subsequent sickling of the red blood cell. Renal disease, the most common complication in SCD, begins in childhood with glomerular hyperfiltration and then progresses into albuminuria, a fast decline of glomerular filtration, and renal failure in adults. This mini-review focuses on glomerular filtration abnormalities and the mechanisms of hyperfiltration, explores genetic modifiers and methods of estimating glomerular filtration rates, and examines novel biomarkers of glomerular filtration in SCD. Sickle cell disease (SCD) is caused by a single nucleotide mutation in the β-globin gene resulting in the substitution of valine for glutamic acid leading to the polymerization of hemoglobin and sickling of red blood cells (RBCs) under low oxygen conditions. The pathophysiology of SCD is characterized by chronic hemolysis, vaso-occlusion, and organ damage. SCD may occur as homozygous hemoglobin S (HbSS), compound heterozygous inheritance of HbS with mutant hemoglobin C (HbSC), or mutations that result in decreased or absent β-globin . HbSS and HbSβ0 genotypes are clinically similar disorders associated with severe anemia and disease complications, whereas HbSC and HbSβ+ thalassemia are relatively less severe. SCD is widespread in the malaria-endemic belt: sub-Saharan Africa; South America, the Caribbean, and Central America; Saudi Arabia; India; and the Mediterranean countries . It is aRenal disease is one of the most common complications of SCD , 7. Appr2 for women and 140 ml/min/1.73 m2 for men. There is no definition for hyperfiltration based on measured GFR (mGFR). The assessment of mGFR in 176 SCD infants demonstrated significantly higher values than in non-SCD infants (125.2 ± 34.4 in SCD vs. 91.5 ± 17.8 ml/min/1.73 m2 in controls). Schwartz formula for eGFR produced even higher values (184.4 ± 55.5 ml/min/1.73 m2)  –27, with1.73 m2) , 29.In young SCD adult patients, hyperfiltration is common, particularly in early adulthood but declines into a normal range. The decline into a normal range is faster in men than women and represents a drop in renal function rather than an improvement. Hyperfiltration assessed by mGFR occurred in 51% of adult SCD patients, and the prevalence of hyperfiltration was higher in men (60%) than in women (42%) . We receGlomerular enlargement and congestion, mainly in the juxtamedullary glomeruli, are more severe in children  than in 2 for severe genotypes (HbSS and HbSβ0) and 1.16 ml/min/1.73 m2 for mild genotypes (HbSC and HbSβ+) (2) was observed by Xu et al. in 193 out of 288 SCD patients over the course of 5-years follow-up; a rate two-fold higher than in African American adults without SCD  . A highehout SCD . The sexhout SCD , 29. Evehout SCD .2 that defines stage 3 CKD, and severe kidney injury may occur at higher GFR levels  levels are associated with hyperfiltration in diabetic and obese patients , 59. In APOL1, HMOX1, MYH9, HbA1, and HbA2 variants, are implicated in the development and progression of SCN  were significantly associated with hyperfiltration, but HMOX1 GT-dinucleotide long repeats were associated with lower eGFR  is a best-characterized modifier of SCD that negatively correlates with the severity of complications. Low HbF levels are associated with more hemolysis . Single n of SCN , 68–70. wer eGFR . In contwer eGFR .Currently, no consensus exists regarding the optimal eGFR equation for SCD patients , 74. TheIn children with SCD, Schwartz equation based on height and serum creatinine is commonly used for eGFR calculation. A study in 176 HbSS infants found no correlation between mGFR and eGFR estimated by Swartz equation . Additio2 , the Modification of Diet in Renal Disease (MDRD), and Chronic Kidney Disease Epidemiology-Collaboration (CKD-EPI) equations with and without adjustment for race , 78–80. 2 . The use2 , 79. New2 , 80. Alsfunction , 81, 82.Glomerular hyperfiltration in SCD plays an essential role in the progression of renal disease; however, to date, no reliable biomarkers of hyperfiltration exist . Common Chronic sickling and hemolysis of RBCs induce multiple mechanisms that cause kidney injury. Thus, hemolysis markers such as hemoglobin (Hb), bilirubin, lactate dehydrogenase (LDH) and reticulocyte, and RBCs Hb levels were explored. Studies provided conflicting evidence of the correlation between GFR and hemolysis: Some studies did not find a significant relationship , 66, 83,Metabolomics analysis identified two metabolites (asymmetric dimethylarginine – ADMA and quinolinic acid) associated with a rapid decline of GFR . ADMA isIn our study, mass-spectrometry analysis of urine samples collected from HbSS patients identified orosomucoid (ORM)  and ceruIn summary, SCN is a common cause of morbidity and mortality in SCD patients , 94, 95.NA and MJ wrote the manuscript. Both authors contributed to the article and approved the submitted version.


The ubiquitin-proteasome system (UPS) is involved in various aspects of cell processes, including cell proliferation, differentiation, and cell cycle progression. F-box and WD repeat domain-containing protein 7 (FBW7), as a key component of UPS proteins and a critical tumor suppressor in human cancers, controls proteasome-mediated degradation by ubiquitinating oncoproteins such as c-Myc, Mcl-1, cyclin E, and Notch. It also plays a role in the development of various cancers, including solid and hematological malignancies, such as T-cell acute lymphoblastic leukemia, diffuse large B-cell lymphoma, and multiple myeloma. This comprehensive review emphasizes the functions, substrates, and expression of FBW7 in malignant lymphoproliferative disorders. Malignant lymphoproliferative disorders are a heterogeneous group of disorders characterized by the abnormal proliferation of lymphocytes, ranging from indolent to highly aggressive neoplasms, including chronic lymphocytic leukemia (CLL), diffuse large B-cell lymphoma (DLBCL), multiple myeloma (MM), and acute lymphoblastic leukemia (ALL). Although targeted drugs have significantly improved the prognosis of patients with malignant lymphoproliferative disorders, a subset of patients have a remarkably poor outcome. Therefore, exploring the underlying mechanisms of malignant lymphoproliferative disorders is critical .F-box and WD repeat domain-containing protein 7 (FBW7), also known as FBXW7, hCDC4, and AGO, is located at chromosome 4q31, which is a genomic region deleted in more than 30% of cancers . Oberg aFBW7 mutations have been detected in a wide range of human malignancies, including lung cancer, ovarian cancer, colorectal cancer, T acute lymphoblastic leukemia (T-ALL), and MM. The percentage of FBW7 mutations varies across cancers, being up to 20% in T-ALL , and CCAAT/enhancer binding protein-δ (C/EBP δ) (Mutations or deletions of C/EBP δ) . P53 is C/EBP δ) . AnotherC/EBP δ) . FurtherC/EBP δ) . In addiT lymphoblastic lymphoma/acute lymphoblastic leukemia (T-LBL/ALL) is a highly aggressive hematologic malignancy caused by the malignant transformation of T−cell progenitors. T−ALL accounts for 15% and 25% of the total number of childhood and adult cases of ALL, respectively. Adults with T−ALL have poor long-term survival. Approximately 50% of them develop recurrence within 1 year, and the remission rate of second-line chemotherapy is only 30%–45%, which ultimately develops refractory leukemia , 39. TheNotch1 is the most common oncogene for T-ALL, and mutations in Notch1 have been reported in more than 60% of T-ALL. Besides, the mutation rate of Notch1 in adults is higher than children. Mutations of Notch1 occur mainly in the heterodimeric domain (HD) region and the proline, glutamine, serine, and threonine domain (PEST). Mutations in the HD domain can result in Notch1 activation by reducing the interaction of the Notch1 subunit. The PEST region regulates protein turnover by targeting proteins to ubiquitin–proteasome complex for subsequent degradation  of patients with ATL had mutations in the FBW7 WD40 domain, region of D510E and D527G, which had ability to target and degrade cyclin E, Mcl-1, and c-Myc. Mutation in FBW7 failed to degrade NICD, activating the Notch signaling pathway and ultimately promoting ATL cell proliferation. However, FBW7 expression in wild-type, proliferation of cells was inhibited (Adult T-cell leukemia/lymphoma (ATL) is a rare malignant T-cell monoclonal proliferative disease caused by human T-cell leukemia virus type 1 (HTLV1). Southwestern Japan is one of the most endemic areas for malignancy, along with the Caribbean basin, Central and South America. The Japan Clinical Oncology Group-Lymphoma Study Group proposed four clinical subtypes of ATL: acute, lymphoma, chronic, and smoldering. Nonetheless, the course of ATL is highly variable. Patients with acute, lymphoma, and chronic type with unfavorable prognostic factors, defined by levels of blood urea nitrogen or lactate dehydrogenase at higher than normal or having albumin levels lower than normal, are categorized as having aggressive clinical course; however, chronic type without unfavorable prognostic factors and smoldering type are indolent ATL , 57. FBWnhibited . Patientnhibited . Moreovenhibited . Therefonhibited Figure 1DLBCL is the most common type of non-Hodgkin lymphoma in adults, accounting for 30%–40% of new diagnoses. DLBCL is classified as germinal center B-cell (GCB) subtype and activated B-cell (ABC) subtype by genetic profiling, and patients with the ABC subtype have significantly poorer outcomes . ApproxiTyr705 which are associated with poor survival in ABC-DLBCL, were reduced following FBW7 overexpression in ABC-DLBCL cell lines. The downstream antiapoptotic target genes of activated Stat3, including c-Myc, Mcl-1, Bcl-2, and Bcl-xl, showed decreased mRNA expression following exogenous FBW7 overexpression. Similarly, the negative relationship between FBW7 and Stat3 levels was also confirmed in DLBCL patient samples  . FBW7 ov samples   were most frequently observed, followed by mutations in ARID1A (8.7%), CREBBP (8.4%), KMT2C (8.2%), TET2 (7.8%), EP300 (6.8%), and EZH2 (2.9%)  of 619 patients with DLBCL revealed that somatic mutations in 2 (2.9%) . Somaticmutation , 60. The pathway   , and Del(11q) chromosomal aberrations and emission , 63. Apprognosis . Patienton (57%) . In addind G423V . FBW7 neradation . Moreovetination . These f pathway Figure 2Primary effusion lymphoma (PEL) is a highly aggressive B-cell lymphoma with a poor prognosis and median survival of 6.2 months . The etiMM is a hematologic malignancy characterized by the presence of abnormal clonal plasma cells in the bone marrow, accounting for 15% of adult hematologic malignancies. It is characterized by anemia, hypercalcemia, bone lesions, and kidney dysfunction. It remains an incurable disease, with a 5-year OS of approximately 45% –73. Therin vivo and in vitro. Thus, FBW7α and GSK3 function as pro-survival factors by controlling p100 degradation in MM .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.


Juvenile idiopathic arthritis (JIA) is the most common chronic rheumatic disease in the paediatric population. JIA comprises a heterogeneous group of disorders with different onset patterns and clinical presentations with the only element in common being chronic joint inflammation. This review sought to evaluate the most relevant and up-to-date evidence on current knowledge regarding the pathogenesis of JIA subtypes to provide a better understanding of these disorders. Despite significant improvements over the past decade, the aetiology and molecular mechanisms of JIA remain unclear. It has been suggested that the immunopathogenesis is characterised by complex interactions between genetic background and environmental factors that may differ between JIA subtypes. Human leukocyte antigen (HLA) haplotypes and non-HLA genes play a crucial role in the abnormal activation of both innate and adaptive immune cells that cooperate in causing the inflammatory process. This results in the involvement of proinflammatory cytokines, including tumour necrosis factor (TNF)α, interleukin (IL)-1, IL-6, IL-10, IL-17, IL-21, IL-23, and others. These mediators, interacting with the surrounding tissue, cause cartilage stress and bone damage, including irreversible erosions. The purpose of this review is to provide a comprehensive overview of the genetic background and molecular mechanisms of JIA. Juvenile idiopathic arthritis (JIA) is the most common chronic rheumatic disease in childhood . Under tGrowing evidence suggests that while some of these JIA subtypes are homogeneous and have a counterpart in the adult population, others are heterogeneous and occur exclusively in children, and thus they do not represent the childhood onset of an adult illness ,3,8. AltIn JIA, several types of cells, such as macrophages, neutrophils, natural killer (NK) cells, and lymphocytes, secrete proinflammatory mediators, including interleukins and chemotactic cytokines, that attract and promote the differentiation of immune cells in the inflammation site, that in JIA is usually, but not only, represented by joints ,23. In cThe last group consists of Th17 cells, which have a leading role in the development of various autoimmune and inflammatory disorders, mostly producing IL-17 and IL-22 ,30. HoweTNFα is a major regulator of inflammation and lead actor in the pathogenesis of both inflammatory and autoimmune disorders . TNFα siTissue macrophages, blood monocytes, and dendritic cells represent the major sources of IL-1β . IL-1β pIL-6 is a pleiotropic cytokine that takes an active role in the acute phase of the pathology and has been detected at high concentrations both in blood and synovial fluid of JIA patients ,52. Its IL-10 is a pleomorphic cytokine produced by a wide variety of both innate and adaptive immune cells, including granulocytes, dendritic cells, macrophages, and T and B cells ,69. IL-1T follicular helper (Tfh) cells are a novel subgroup of CD4+ Th cells, secreting IL-21 as a signature cytokine ,75. The The pathophysiology of JIA is characterised by a strong genetic background . EpidemiFor several years, the MHC region has been considered as the most significant genetic risk factor for JIA. The development of innovative approaches for the analysis of conventional HLA alleles from genotyping array data is providing more detailed knowledge of the JIA molecular background . The impPTPN22 and PTPN2 [PTPN22) is a gene located on chromosome 1p13.2, encoding the homonym protein PTPN22, also called “lymphoid phosphatase” (LYP) in humans, which is expressed nearly exclusively by haematopoietic cells, especially T lymphocytes, in the thymus and spleen [PTPN22-knockout mice exhibit increased T cell dependent immunoglobulin response to antigens, as well as enhanced effector and memory T cell proliferation [PTPN22 missense mutation R620W  has been associated with an increased risk of several autoimmune disorders, such as SLE, type 1 diabetes mellitus (T1DM), vitiligo, RA, and finally JIA, probably by not promoting type 1 IFN-driven inhibition of inflammation [PTPN2 is a gene located on chromosome 18p11.21, encoding the homonym tyrosine phosphatase PTPN2, which also has its own role in the regulation of both T and B lymphocytes like PTPN22, modulating immunity through the Janus kinases/signal transducer and activator of transcription proteins (JAK/STAT) signalling pathways [PTPN22, PTPN2, STAT4, C12orf30, COG6, ANGPT1, ADAD1-IL2-IL21 [TNFAIP3, PTPN22, and STAT4 [PTPN22 and PTPN2, whose molecular aspects are better understood, and on genes related not to simple susceptibility but to monogenic forms of the disease.Two associated non-HLA loci were found in JIA genetic investigations prior to 2013: nd PTPN2 . Proteind spleen . LYP is d spleen . PTPN22-feration ,92,93. Mammation ,97,98,99ammation . Howeverammation ,100. PTPpathways . Haematopathways . In recepathways ,90,102. pathways . The maipathways ,105. ManIL2-IL21 ,101. Accnd STAT4 ,106. In nd STAT4 . HoweverLACC1, LRBA, NFIL3, and UNC13D has significantly increased the understanding of crucial molecular pathways of JIA [LACC1) gene encodes C13orf31/FAMIN, a key protein in the modulation of mitochondrial functionality [LACC1 [LACC1 mutation I254V  causes a serious impairment of FAMIN and is considered associated with the development of non-systemic JIA, inflammatory bowel diseases, and Behçet disease [LACC1 pathogenic mutations have been associated with JIA, such as C284R , which has been found in several consanguineous sJIA patients, and the frameshift p.Cys43Tyrfs*6 and p.T276fs*2 mutations, which have been confirmed respectively in severe oJIA and RF − pJIA patients [LRBA) gene. It encodes the protein LRBA, a central molecule in the negative modulation of the degradation of cytotoxic T-lymphocyte-associated antigen 4 (CTLA-4) proteins [LRBA could be a lead actor in the autoantigen tolerance and immune system modulation. Its loss of function is responsible for a subset of common variable immunodeficiency (CVID), an immune dysregulation disorder in which up to 10% of patients develop an autoimmune disorder, and among these JIA is frequently observed, both in oligoarticular and polyarticular forms, probably due to a reduced CTLA-4 activity [NFIL3, which plays a role in the development and modulation of NK cells and cytokine production by Th2 cells [NFIL3-knockout mice showed increased IL-1β expression and more severe joint symptoms and disease course [UNC13D) gene is essential in the immunology synapse for the correct cytolytic granule secretion, and its reduced expression has been found to be associated with a decreased NK cell degranulation [UNC13D intronic region has been regarded as responsible for development of sJIA and recurrent macrophage activation syndrome (MAS), probably due to an impaired lymphocyte-specific downregulation of the NF-kB [In view of the uncertainty surrounding JIA’s pathophysiology and categorisation, the discovery of four monogenic variants of JIA attributed to the genes s of JIA . The lacionality . Severaly [LACC1 ,109,110.y [LACC1 ,107. The disease ,111. In proteins ,113. Groactivity ,114. Rech2 cells . Studiese course . The unche NF-kB ,117,118.Together with the genetic susceptibility, several environmental factors may trigger and influence JIA with different mechanisms, including epigenetic changes, imbalance of microbiota, and immunity modulation . BreastmJIA is a complex paediatric disease with multiple subtypes. Understanding the differences in the genetic background of the various subtypes, evaluating both HLA haplotypes and non-HLA genes, is critical to providing a better understanding of these disorders. It is known that oJIA and RF − pJIA share several genetic similarities in susceptible HLA haplotypes (mainly HLA-DRB1*08) and are characterised by a pathogenetic centrality of classic and non-classic Th1 cells and TNFα, while ErA appears to have peculiar molecular mechanisms, mainly involving HLA-B27 and IL-17. Although RF + pJIA may be considered the paediatric counterpart of adult RA, with a quite similar genetic background , several differences have been highlighted with the other JIA subtypes, including the poor involvement of RF and ACPA in JIA pathogenesis, the centrality of CD4+ Th17/Th1 cells in oJIA, the importance of HLA-B27 in ErA and the autoinflammatory characteristics of sJIA. Furthermore, growing evidence suggests an inflammatory phenotype for sJIA, considering both genetic and molecular implications, such as the driving role of IL-1 and the different therapeutic strategies used to treat this form. Further genetic investigations and more detailed studies focusing on the molecular pathways of JIA will improve our understanding of the disease and help achieve more accurate classification criteria. A more comprehensive understanding of the molecular pathogenesis of JIA is a key goal to deliver more accurate targeted therapies and improve disease course and outcome.


Intensive research has focused on minimizing the infarct area and stimulating endogenous regeneration after myocardial infarction. Our group previously elucidated that apicidin, a histone deacetylase (HDAC) inhibitor, robustly accelerates the cardiac commitment of naïve mesenchymal stem cells (MSCs) through acute loss of YAP1. Here, we propose the novel regulation of YAP1 in MSCs. We found that acute loss of YAP1 after apicidin treatment resulted in the mixed effects of transcriptional arrest and proteasomal degradation. Subcellular fractionation revealed that YAP1 was primarily localized in the cytoplasm. YAP1 was acutely relocalized into the nucleus and underwent proteasomal degradation. Interestingly, phosphor-S127 YAP1 was shuttled into the nucleus, suggesting that a mechanism other than phosphorylation governed the subcellular localization of YAP1. Apicidin successfully induced acetylation and subsequent dissociation of YAP1 from 14-3-3, an essential molecule for cytoplasmic restriction. HDAC6 regulated both acetylation and subcellular localization of YAP1. An acetylation-dead mutant of YAP1 retarded nuclear redistribution upon apicidin treatment. We failed to acquire convincing evidence for polyubiquitination-dependent degradation of YAP1, suggesting that a polyubiquitination-independent regulator determined YAP1 fate. Nuclear PSME4, a subunit of the 26 S proteasome, recognized and degraded acetyl YAP1 in the nucleus. MSCs from PSME4-null mice were injected into infarcted heart, and aberrant sudden death was observed. Injection of immortalized human MSCs after knocking down PSME4 failed to improve either cardiac function or the fibrotic scar area. Our data suggest that acetylation-dependent proteasome subunit PSME4 clears acetyl-YAP1 in response to apicidin treatment in the nucleus of MSCs. Cardiovascular disease (CVD) such as myocardial infarction (MI) is a major cause of global death worldwide . CurrentMSC-based regeneration therapies are currently used to treat both acute MI and chronic ischemic cardiomyopathy . The sugIn 2010, Terzic et al. showed that treatment of MSCs with a cocktail of TGF-β, BMP-4, Activin-A, IGF-1, IL-6, FGF2, thrombin, and retinoic acid could successfully upregulate cardiac transcription factors to facilitate cardiopoiesis . CardiopThe follow-up phase 3 CHART-1 trial (congestive heart failure cardiopoietic regenerative therapy) was a double-blind study designed to assess cardiac outcomes in ischemic heart failure patients treated with endomyocardial injection of lineage-specified MSCs. Two years of clinical data demonstrated safety, but the treatment did achieve the primary endpoint compared with sham control. Several questions—including cell dosage, delivery route, and cell state—remain unsolved to meet therapeutic efficacy. Therapeutic benefit was observed in the heart failure with cardiac dilation group .We previously showed that apicidin treatment induces early cardiac gene expression by reducing YAP1 (Yes-associated protein 1) in MSCs . YAP1 isAntibodies used were as follows: anti-YAP1, anti-phosphor-YAP1 (S127), and anti-acetyl-lysine were from Cell Signaling Technology . Anti-PSME4, anti-histone H3, and anti-HDAC2 were from Abcam . Anti-14-3-3 epsilon was from Thermo Fisher Scientific . Anti-HSP90, anti-HDAC6, anti-acetylated-tubulin, and anti-GAPDH were from Santa Cruz Biotechnology . Anti-actin, anti-tubulin, anti-HA, and anti-flag were from Sigma . Alexa Fluor 568-conjugated goat anti-mouse IgG was from Molecular Probes .Reagents used were as follows: apicidin, cycloheximide, chloroquine, actinomycin D, leptomycin B, nicotinamide, tubastatin A, and doxycycline were from Sigma . MG132 was purchased from Cayman . Puromycin and blasticidin were from InvivoGen .siRNAs targeting HDAC6, FBXW7, SOCS6, and PSME4 were purchased from Dharmacon . Scramble siRNA was purchased from Bioneer .2. hTERT-MSCs express CD90 but are negative for CD45, CD34, CD14, and CD11b. Differentiation properties into adipogenic, osteogenic, or chondrogenic lineages were previously confirmed  × LVIDs3, where LVIDd is LV interventricular dimension at end diastole and LVIDs is that at end systole. Fractional shortening was measured by the formula FS = (LVIDd − LVIDs)/LVIDd.Cardiac function was measured by ultrasonography . Mice were anesthetized with intramuscular injection of ketamine (50 mg/kg)/xylazine (5 mg/kg), and ultrasound gel was applied to the chest after it was shaved. At the papillary muscle level, two-dimensional M-mode was acquired from the parasternal long-axis view or parasternal short-axis view. Ejection fraction was determined using the Teichholz formula, EF (%) = (Vd − Vs)/Vd, where Vd indicates LV volume at end diastole and Vs is at end systole, and Vd = [7/(2.4 + LVIDd)] × LVIDdStatistical significance was analyzed with PASW Statistics 27 . One-way analysis of variance (ANOVA) with post hoc multiple comparison was used. When there were two or more main effects, two-way ANOVA was applied. If an interaction of the main effects was confirmed as being significant, further stratification was carried out to perform pairwise comparison. For post hoc tests, Tukey’s HSD (honestly significant difference) was applied for multiple comparisons in equal variance.We reported previously that overnight treatment of primary cultures of MSCs or immortalized human MSCs (hTERT-MSCs) with apicidin induces acute ablation of YAP1 and subsequent early cardiac gene expression . Here, wWe next questioned whether apicidin solely regulated the transcription of YAP1 or whether another mechanism was also involved. De novo protein synthesis was halted by overnight treatment with either cycloheximide  or actinomycin D , and YAP1 protein was measured. The natural clearance of YAP1 in hTERT-MSCs after CHX or ActD treatment was relatively slower than after apicidin treatment b. We furWe next examined the two main pathways for protein degradation: the lysosome (or autophagy) pathway and the proteasome pathway. Chloroquine, which functions in the lysosome pathway, failed to alleviate YAP1 degradation c. Next, Next, we questioned in which subcellular fraction YAP1 was degraded. We briefly fractionated the subcellular component into cytoplasm and nucleus after MG132 treatment, and the fractions were tested with Western blot. Restored YAP1 accumulated predominantly in the nucleus, whereas MG132 alone tended to halt degradation of YAP1 in the cytoplasm e. BecausAmong the numerous posttranslational modifications of YAP1, the mechanism of S127 phosphorylation is well established; Lats1/2 phosphorylates S127 of YAP1, which leads to 14-3-3 recognition and, thus, cytoplasmic restriction . Hence, Apicidin is a potent HDAC inhibitor (HDACi) that regulates acetylation of histone or nonhistone proteins . Hence, To further determine the subtype of HDAC(s) involved in YAP1 deacetylation, we tested YAP1 degradation by treatment with various HDACi including apicidin. Among those tested, tubastatin A (TST), a selective HDAC6 inhibitor , successAs noted, TST is a specific HDAC6i. To specify the role of HDAC6 in YAP1 regulation, endogenous HDAC6 was specifically targeted by use of siRNA transfection. As with overnight treatment with apicidin or TST, total YAP1 was significantly reduced by 3 days after siRNA transfection d. IntereTo clarify the role of acetyl-YAP1, we mutated candidate residues, lysine 494 and lysine 497, according to a previous reference . As repoSo far, we delineated that apicidin induced YAP1 acetylation by inhibiting cytoplasmic HDAC6  and thenNext, we generated rtTA-shPSME4 that bound to endogenous PSME4 and confirmed efficiency in infected cells . TargeteNext, we introduced PSME4-null mice to clarify its role in YAP1 regulation. As reported, PSME4 homozygous males were infertile as reported , and oveOn the basis of the results shown in Our findings are summarized below . In MSCsYAP1 was originally highlighted as a functional activator of the Hippo signal, which determines organ growth and size in mammals . ConstitAlthough overexpression of YAP1 improves cardiac dysfunction after MI, YAP1 can exacerbate fibrotic changes. Selective overexpression of YAP1 in cardiac fibroblasts promotes proliferation of fibroblasts and differentiation into a mature form called myofibroblasts. Furthermore, specific deletion of YAP1 in the fibroblast results in less fibrosis after MI . OverallMSCs are multipotent cells that can differentiate into adipocytes, chondrocytes, and osteocytes . ESCs anOne of the most well-established modifications of YAP1 is phosphorylation. Phosphorylation of YAP1 results in cytoplasmic restriction. LAT1/2 initially phosphorylates S127 of YAP1, which triggers sequential phosphorylation at S61, S109, S164, and S397 ,38. PhosLysine methylation also regulates YAP1 activity. SET1A monomethylates nuclear-imported YAP1 at K342, which interferes with recognition of CRM1, a nuclear export enzyme. K342-methylated YAP1 activates its target gene expression, and prolonged activation of YAP1 in the nucleus accelerates tumorigenesis . In addiWe delineated the role of acetylation in the nuclear targeting and activity of YAP1. Target amino acids for acetylation were confirmed by use of YAP1 containing point mutations. It is interesting that dual modification was possible at a single lysine: methylation and acetylation. We utilized point mutant protein (2KA or 2KR) for the study and the variants were also methylation-resistant . Indeed,As expected, we observed accumulation of YAP1 in the nucleus when PSME4 was decreased . We alsoWe are interested in cardiac commitment of MSCs to differentiate mature cardiomyocytes in the heart, as it is still controversial whether the preconditioned MSCs successfully ameliorate contractile impairment of failing heart as functional cardiomyocytes . In factIn conclusion, our study showed that, in MSCs, YAP1 is restricted in the cytoplasm in the quiescent status. Apicidin, a potent HDACi, inactivates cytosolic HDAC6, which in turn allows acetylation of YAP1 in the cytosol and stimulates transcription of p21. Acetylation of YAP1 leads to dissociation from the 14-3-3 molecule that governs cytosolic restriction. Acetyl-YAP1 is shuttled into the nucleus and transiently activates its target genes. Robustly activated p21 arrests the transcription of YAP1, and PSME4 degrades acetyl-YAP1 simultaneously.


Inhibitors of phosphodiesterase 4 (PDE4), a group of isoenzymes that hydrolyze and inactivate the second messenger cAMP, produce promising therapeutic benefits, including anti-inflammatory and memory-enhancing effects. Here, we report that, unexpectedly, PDE4 inhibitors also reduce serum potassium levels in mice. As both the total potassium content of the body, as well as the distribution of potassium between intra- and extracellular compartments, are critical for normal cellular functions, we further explored this observation. Several structurally distinct PDE4 inhibitors reduce serum potassium levels in mice, suggesting it is a class effect of these drugs. Serum potassium levels decrease within 15 min of drug injection, suggesting that PDE4 inhibition lowers serum potassium levels by promoting a transcellular shift of potassium from the blood into cells. This shift is a characteristically fast process, compared to a loss of total-body potassium via the kidneys or digestive tract . Indeed, stimulating cAMP synthesis with β-adrenoceptor agonists is known to rapidly shift potassium into cells, and PDE4 inhibitors appear to mimic this process by preventing PDE4-mediated cAMP degradation. Our findings reveal that the various acute physiologic effects of PDE4 inhibitors are paralleled and/or may be affected by reduced serum potassium levels.2-blocker Yohimbine, and is partially blocked by pre-treatment with the β-blocker Propranolol. Together, these data suggest that PDE4 inhibitors reduce serum potassium levels by modulating the adrenergic regulation of cellular potassium uptake.The analysis of blood samples from mice treated with the PDE4 inhibitor Roflumilast revealed an unexpected reduction in serum potassium levels, while sodium and chloride levels were unaffected. Treatment with several structurally distinct PAN-PDE4 inhibitors, including Roflumilast, Rolipram, RS25344, and YM976 dose-dependently reduced serum potassium levels, indicating the effect is a class-characteristic property. PDE4 inhibition also induces hypothermia and hypokinesia in mice. However, while general anesthesia abrogates these effects of PDE4 inhibitors, potassium levels decrease to similar extents in both awake as well as in fully anesthetized mice. This suggests that the hypokalemic effects of PDE4 inhibitors occur independently of hypothermia and hypokinesia. PDE4 inhibition reduces serum potassium within 15 min of treatment, consistent with a rapid transcellular shift of potassium. Catecholamines promote the uptake of potassium into the cell via increased cAMP signaling. PDE4 appears to modulate these adrenoceptor-mediated effects, as PDE4 inhibition has no additional effects on serum potassium in the presence of saturating doses of the β-adrenoceptor agonist Isoprenaline or the α The proper regulation of total-body potassium, and its relative distribution between intra- and extracellular compartments, is critical for normal cellular functions and hence maintained within a narrow homeostatic range ,2,3,4. F+/K+-ATPase —the latter being the principal mechanism of transporting potassium into cells [+/K+-ATPase [Total-body potassium levels are principally maintained by adapting to potassium intake and/or unusual potassium loss  with changes in the rate of potassium reabsorption/excretion by the kidneys—a process that occurs over an hours-long time scale ,2,3,4. Ito cells ,10. Give+-ATPase . Increas+-ATPase . Indeed,+-ATPase ,12,13,14β-adrenoceptors mediate their effects through increasing production of the ubiquitous second messenger cAMP. This increase in the rate of cellular cAMP synthesis is balanced by the rate of cAMP degradation by phosphodiesterases (PDEs), which cleave its cyclic phosphodiester bond to produce 5′-AMP, thus terminating cAMP signaling ,16,17. PTreatment of mice with PAN-PDE4 inhibitors produces several acute effects, including hypokinesia, hypothermia, and gastroparesis ,32,33,34Rolipram  . All mice were maintained in a temperature-controlled (22–23 °C) vivarium with a 12 h light/dark cycle. Animals were group-housed up to four mice per cage and maintained with ad libitum access to food and water. Adult mice ≥ 18 g of body weight, ≥10 weeks of age, and of either sex were used for experimentation by evenly and randomly dividing cage littermates into experimental groups. On the day of an experiment, mice were routinely fasted for 5 h with ad libitum access to water before drug injection and/or experimentation. Experimenters were blinded to the identity of injected drugs until data acquisition and analyses were completed. All experiments and procedures were approved by the University of South Alabama Institutional Animal Care and Use Committee and were conducted in accordance with the guidelines described in the Guide for the Care and Use of Laboratory Animals .2 for <5 min). The blood was collected into lithium-heparin blood collection tubes  and subsequently subjected to blood chemistry analysis . Mice were then euthanized using EUTHASOL® euthanasia solution  followed by cervical dislocation. For experiments that were intended to abrogate the effects of PDE4 inhibition on body temperature regulation or locomotion, mice were anesthetized using Isoflurane (3% Isoflurane in 1.5 L/min O2) for 10 min before drug injection, and anesthesia was maintained throughout the experiments until after blood samples had been collected.At the end of the specified treatment duration, blood was collected by submandibular bleed under light Isoflurane anesthesia (3% Isoflurane in 1.5 L/min OCore body temperature was measured using a thermocouple thermometer (MicroTherma 2T) with a mouse rectal probe (RET-3), both from Braintree Scientific , following the manufacturer’s instructions and as reported previously ,34.p > 0.05), * (p < 0.05), ** (p < 0.01), and *** (p < 0.001).All data are expressed as the mean ± SEM. In scatter dot plots, each dot represents a different animal. For time courses, the n numbers indicate the number of individual animals assessed. GraphPad Prism software  was used to perform statistical analyses. The Mann–Whitney test with a 95% confidence interval was used to compare two treatment groups and the Kruskal–Wallis test followed by Dunn’s post hoc test were used to determine differences between more than two treatment groups. Experiments comparing the effects of multiple drugs or effects across multiple time points were analyzed using a two-way ANOVA with Sidak’s post hoc test. Statistical differences are indicated as ns  using two-way ANOVA. Together, these data support the hypothesis that PDE4 inhibitors lower serum potassium levels, at least in part, by modulating an adrenoceptor-dependent, cAMP-mediated transcellular shift of potassium into cells.It is well established that increased cAMP signaling, such as upon β-adrenergic stimulation, can rapidly reduce serum potassium levels by shifting potassium into cells (transcellular shift). Indeed, β-agonists, such as Albuterol, are used clinically for the treatment of hyperkalemia ,14,52,53m levels   but this would not be expected to significantly increase serum sodium levels, given its negligible addition to the already very high concentration of extracellular sodium under baseline conditions (~145 mmol/L).Serum potassium levels decrease rapidly but transiently upon i.p. injection of a PDE4 inhibitor, producing a maximal reduction within 15 min, and then slowly recover to normal levels within a few hours . A reducm uptake ,10,61, am uptake , which w+-ATPase , such asincrease ,65,66. Hincrease ,70,71,722-adrenoceptors) promotes cellular potassium uptake, whereas decreasing cAMP levels, such as via activation of Gi-coupled α2-adrenoceptors, promotes potassium release by tissues [2-blocker Yohimbine (+/K+-ATPase or inhibition of potassium leak currents). Moreover, when pre-treated with the β-blocker Propranolol, the hypokalemic effect of PDE4 inhibitors is lessened, suggesting that PDE4 acts, at least in part, by controlling subcellular pools of cAMP controlled by β-adrenoceptor signaling.The rapid rate with which PDE4 inhibitor treatment lowers serum potassium itself suggests a mechanism that produces a rapid shift of potassium into cells, rather than a loss of total-body potassium. Additionally, it is established that increasing cellular cAMP levels, such as via activation of β-adrenoceptors , the somatic nervous system (hypokinesia), and the autonomic nervous system , to effects on reproduction and cardiac output ,76,77,78Conversely, as a tight regulation of serum potassium levels is particularly critical for the normal function of excitable cells, such as muscle and nerve cells ,58,79,80+/K+-ATPase and/or the inhibition of potassium leak channels [2-adrenoceptor antagonists; Taken together, PAN-selective inhibition of PDE4 enzymes affects a wide array of physiologic paradigms, and exerts many potentially therapeutic benefits, in experimental animals and humans alike ,75,76,81channels ,9,10,62.


The study aims to identify potential diagnostic markers of idiopathic pulmonary fibrosis (IPF) and analyze the significance of immune cell infiltration in this pathology.Download two publicly available gene expression profiles (GSE10667 and GSE24206 datasets) from the GEO database including 48 Idiopathic pulmonary fibrosis (IPF) samples and 21 human control samples and select for distinctly expressed genes (DEG) from them. Lasso regression model and support vector machine recursive feature elimination S,V,R,F analysis were used to check candidate biomarkers. The area under the subject’s work characteristic curve (AUC) value is used to evaluate its recognition ability. The GSE53845 dataset (40 IPF patients and 8 controls) continue to validate the expression level and diagnostic value of biomarkers in IPF. Comprehensive analysis of immune infiltrated cells of IPF was performed using R software and immune cell infiltration estimation analysis tool- deconvolution algorithm (CIBERSORT).43 DEGs were identified in total. The identified DEGs mostly involve pneumonia, lung disease, collagen disease, obstructive pulmonary disease and other diseases. The activation of IL-17 signaling pathways, amoebic disease, interaction of viral proteins with cytokines and cytokine receptors, protein digestion and absorption, and flaccid hormone signaling pathways in IPF were different from the control group. The expression degree of CRTAC1, COL10A1, COMP, RPS4Y1, IGFL2, NECAB1, SCG5, SLC6A4, and SPP1 in IPF tissue were prominently higher than the normal group. Immune cell infiltration analysis showed that CRTAC1, COL10A1, COMP, IGFL2, NECAB1, SCG5, SLC6A4, and SPP1 were associated with monocytes, plasma cells, neutrophils, and regulatory (treg) T cells.CRTAC1, COL10A1, COMP, IGFL2, NECAB1, SCG5, SLC6A4, and SPP1 can be used as diagnostic markers for IPF, providing new ideas for the future study of IPF occurrence and molecular mechanisms. IPF c2 and next employ “limma” and “SVA” Two software packages combine two microarray datasets, GSE10667 and GSE24206, into a single dataset as a training dataset. Since the two datasets of GSE10667 and GSE24206 contained samples of IPF tissue and normal lung tissue, they were used for subsequent difference analysis, and the GSE53845 microarray dataset was selected as the model validation dataset for follow-up research.This study used gene chip data from GEO (see text footnote 1) data from the National Biotechnology Information Centre in the United States, serial numbers GSE10667, GSE24206, and GSE53845. Among them, the GSE10667 dataset collected 31 pulmonary fibrosis tissue samples and 15 normal tissue samples, the GSE24206 dataset collected 17 pulmonary fibrotic tissue samples and 6 normal lung tissue samples, and the GSE53845 dataset collected 40 pulmonary fibrosis tissue specimens and 8 healthy lung tissue samples. The Original Data is operated using the Robost Multiarray Averaging function of the oligoR packageP value and fold change (FC). P < 0.05 and | log2FC| > 2 were used as thresholds for screening DEGs, and IPF-related differential genes were screened out by volcano maps and heat maps.Differentially expressed genes (DEGs) were selected for the combined GSE10667 dataset and GSE24206 dataset using the R language “Limma” and “pheatmap” software packages, and the differences in gene expression were represented by the log2FC of 3 conducted gene ontology (GO) function and Kyoto encyclopedia of genes and genomes (KEGG) pathway enrichment analysis to comprehensively annotate the biological function information of genes  enrichment analysis of DEGs using “clusterprofiler” and the DOSE software package in R of genes . GO funcof genes , 15. Theage in R , 17. If To identify major prognostic variables, we use two machine learning algorithms to calculate disease status. Minimum Absolute contraction selection operator (LASSO) is a regression statistics method, which uses regularization way to ameliorate degree of accuracy. Make use of the LASSO in the “glmnet” software package in R to distinguish genes that were prominently relevanted with UC and normal sample discrimination . SupportTo examine the predictive value of the identified biomarkers, We used mRNA expression data from 48 IPF and 21 check samples to create ROC curves. The area under ROC curve (AUC) value was used to determine the diagnostic validity of IPF relative to the control sample, and was further validated in the GSE53845 dataset.4 was made use of calculating immunocyte infiltration. A reference set containing 22 immunocyte subtypes (LM22) and 1000 permutations was used to estimate the hypothetical immunocyte abundance . The LIMMA package is used to perform DEG analysis on the metadata after the batch effect is removed. A total of 43 DEGs are obtained, as shown in P < 0.05 as the filter, involving 192 biological processes. The first 20 biological processes include 10 BP, 5 CC, 5 MF, as shown in Gene ontology biological process analysis of DEGs, with via the LASSO returning method to obtain 10 variables as diagnostic biomarkers for IPF, as shown in P < 0.05). Whereas, there was no obviously discrepancies in IL-6 expression between the two groups. Thus, we applied the logistic regression algorithm to build the diagnostic model of nine identified genes in the metadata queue.We use two distinctive algorithms to sifting potential biomarkers. Narrow the DEGs As presentation in P = 0.009), regulatory (Tregs) T cells (P = 0017), resting NK cells (P = 0019), monocytes (P < 0.001), M0 macrophages (P = 0.012), M2 macrophages (P = 0.024), Eosinophils (P = 0.033) in IPF tissues were apparently lower than in normal tissues. Nevertheless, the percentage of memory B cells (P = 0.010), CD4 memory resting T cells (P = 0.001), Neutrophils (P < 0.001) in IPF tissues was obviously higher than that in normal tissues, as shown in First of all, we investigated the constitution of immunocyte in IPF tissues vs. normal control tissues. The ratio of Plasma cells (Correlation of ratio of each immunocyte in IPF tissue samples as presentation in p = 0.0018), T cells CD8  and negatively correlated with Plasma cells , Macrophages M0 . COL10A1 was positively correlated with T cells CD4 memory activated , and negatively correlated with Neutrophils . COMP was positively correlated with Plasma cells , B cells memory  and negatively correlated with Monocytes , Neutrophils , NK cells resting , Eosinophils , T cells CD4 memory resting .IGFL2 was positively correlated with B cells memory , T cells regulatory (Tregs) , Plasma cells , Dendritic cells resting , Macrophages M0  and negatively correlated with NK cells resting , Monocytes , T cells CD4 memory resting , Neutrophils . NECAB1 was positively correlated with Eosinophils , Monocytes , Neutrophils , T cells CD4 memory resting , Dendritic cells activated  and negatively correlated with Plasma cells , B cells memory , T cells regulatory (Tregs) , T cells gamma delta . SCG5 was positively related to Plasma cells , B cells memory , Dendritic cells resting , Macrophages M0 , T cells regulatory (Tregs)  and negatively correlated with NK cells resting , Monocytes , Neutrophils , T cells CD4 memory resting . SLC6A4 was positively correlated with Monocytes , NK cells resting , NK cells activated , Eosinophils , Dendritic cells activated  and negatively correlated with Plasma cells , B cells memory , T cells follicular helper , T cells regulatory (Tregs) , T cells gamma delta. SPP1 was positively correlated with B cells memory , Plasma cells , Macrophages M0 , T cells regulatory (Tregs) , T cells follicular helper , T cells gamma delta  and negatively related to Monocytes , Eosinophils , NK cells activated , T cells CD8 , T cells CD4 memory resting .As shown in Idiopathic pulmonary fibrosis is a rare, progressive, and often lethal type of chronic fibrotic interstitial lung illness . IPF is In this study, the raw data of the two datasets of GSE10667 and GSE24206 were collected from the GEO database and the datasets were comprehensively analyzed. Altogether 43 DEGs were selected, of which 35 were highly expressed in IPF and 8 DEGs were lowly expressed in IPF. Subsequently, DEGs were analyzed for GO function, KEGG enrichment and disease enrichment, which were consistent with the results of previous mechanism studies, and the biological functions of DEGs were mainly concentrated in cell adhesion, biological adhesion, collagen metabolism, cytoskeletal development, extracellular matrix changes, cytokine interactions, etc. The results of KEGG pathway enrichment analysis show that DEGs are mainly related to ECM receptor interactions, protein digestion and absorption, PI3K-Akt signaling pathway, and cytokine–cytokine receptor mutual influence and other signaling pathways. The results of disease enrichment analysis showed that DEGs were mainly involved in diseases such as pneumonia, lung disease, collagen disease, sarcoidosis, osteoarthritis, integumentary system disease, systemic scleroderma, and so on.Based on the LASSO regression model and SVM-RFE analysis, COMP, SPP1, SLC6A4, COL10A1, CRTAC1, IGFL2, NECAB1, and SCG5 were identified as diagnostic markers of IPF. TGF-β1 is generally believed to be a key cytokine driving fibrosis , and theThe categories of immunocyte penetration in IPF and normal samples were evaluate using CIBERSOTR. The results showed that various immunocyte subtypes were closely related to the vital biological processes of IPF. An increased permeability of Plasma cells, regulatory (Tregs) T cells, resting NK cells, Monocytes, M0 macrophages, M2 macrophages, and Eosinophils, and a decreased penetration of memory B cells, CD4 memory resting T cells, and neutrophils has been found to be associated with the onset and development of IPF. Moreover, by utilizing correlation analysis between COMP, SPP1, SLC6A4, COL10A1, CRTAC1, IGFL2, NECAB1, SCG5 and immune cells, COMP, SPP1, SLC6A4, COL10A1, CRTAC1, IGFL2, NECAB1, and SCG5 were found to be correlated with Plasma cells, monocytes, Neutrophils, regulatory (Tregs) T cells. Chronic inflammation and the immune response is significantly important in the progression of the IPF , Immune in vivo and in vitro to obtain experimental confirmation. This study illustrates a trustworthy and comprehensive perspective on the pathogenesis and progress of IPF, and a large number of potential molecules relevant to the etiopathogenesis of IPF can be aquired through bioinformatics analysis, and can be verified by later experiments to provide a more detailed basis for the diagnosis and treatment of IPF.Large-scale studies of high-throughput sequencing techniques and molecular mechanisms have provided clues to the origin and development of IPF, but further research is needed to elucidate the pathogenesis of IPF. Considering the potential for false-positive results, limited sample sizes, and potential heterogeneity in the analysis of a single microarray dataset, we have integrated data from two datasets (GSE10667 and GSE24206), and the molecular mechanisms of IPF pathogenesis involved in the obtained DEGs still need to be further explored. The limitation of this study is that although the DEGs associated with IPF were extracted through data mining and detailed bioinformatics analysis of multiple datasets, and the diagnostic characteristic genes of IPF were obtained according to THE LASO regression model and SVM-RFE analysis, it was still necessary to perform related experiments for example western blotting and immunohistochemical analysis to verify the level of these genes in IPF. In addition to identifying IPF-associated DEGs and characteristic genes by analyzing two datasets, more reliable and accurate DGEs need to be explored through new techniques and bioinformatics analysis methods. In addition, further loss of function and function must be carried out In a word, CRTAC1, COL10A1, COMP, IGFL2, NECAB1, SCG5, SLC6A4, and SPP1 were identified as diagnostic biomarkers of IPF. Monocytes, Plasma cells, Neutrophils, Regulatory (Tregs) T cells, M2 macrophages, Eosinophils, and CD4 memory resting T cells are tightly relevant to the appearance and progress of IPF. These immune cells may be developed as targets for immunotherapy in patients with IPF. In summary, these research have shown that neutrophils are related to the formation of IPF pulmonary fibrosis, and through in-depth study of the immune regulation mechanism of sextrophils in IPF, IPF target therapeutic drugs can be developed.The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found in the article/supplementary material.RS and YW conceived the study. XD and ZY wrote the manuscript. WZ, SL, QZ, and TL edited the pictures. LC and LL revised the manuscript. All authors read and approved the final manuscript.