Rotavirus (RV) is an important pathogen causing acute gastroenteritis in young humans and animals. Attachment to the host receptor is a crucial step for the virus infection. The recent advances in illustrating the interactions between RV and glycans promoted our understanding of the host range and epidemiology of RVs. VP8*, the distal region of the RV outer capsid spike protein VP4, played a critical role in the glycan recognition. Group A RVs were classified into different P genotypes based on the VP4 sequences and recognized glycans in a P genotype-dependent manner. Glycans including sialic acid, gangliosides, histo-blood group antigens (HBGAs), and mucin cores have been reported to interact with RV VP8*s. The glycan binding specificities of VP8*s of different RV genotypes have been studied. Here, we mainly discussed the structural basis for the interactions between RV VP8*s and glycans, which provided molecular insights into the receptor recognition and host tropism, offering new clues to the design of RV vaccine and anti-viral agents. Reoviridae, is an important pathogen leading to acute gastroenteritis (AGE) in children under 5 years old and caused ∼200,000 deaths worldwide each year (Rotavirus (RV), belonging to the ach year . RV genoach year . The RV ach year . Based oach year . Groups ach year . Among thttps://rega.kuleuven.be/cev/viralmetagenomics/virus-classification). Different combinations of G and P genotypes have been reported in human infections whereas G9P[8], G1P[8], G3P[8], G2P[4], G8P[8], are the widely prevalent RVAs  and N-glycolylneuraminic acid (Neu5Gc) are the most common neuraminic acids in nature and widely expressed on the surface of most mammalian cells. Several RV strains of the P[I] genogroup, including Nebraska calf diarrhea virus , rhesus rotavirus , SA11 , porcine rotavirus strain  were sialidase sensitive and showed distinct preference for Neu5Ac or Neu5Gc  . N-acetyr Neu5Gc . The infr Neu5Gc . Canine r Neu5Gc . In addir Neu5Gc . VP8*s or Neu5Gc .Later studies revealed that most animal RVs and human RVs are SA independent . RecentlRV VP8*s recognized HBGAs in a genotype dependent manner. The P[8] and P[4] RVs that are widely prevalent in humans interacted with mucin cores, lewis b, and type I HBGA, including H type-1 antigen (H1), H1 precursor, lacto-N-tetraose (LNT), Lacto-N-fucopentaose I (LNFP1) . Human PMucins are large glycoproteins containing a protein core and a high number of O-linked oligosaccharides . Mucin cThe glycan binding specificity influenced the host tropism and prevalence of RVs. Animal RVs could recognize sialic acid and mucin core 2, whereas human RVs bound to HBGAs and mucin cores. The RVs that can infect both human and porcine such as P[6] and P[19] showed distinct glycan binding preference. Porcine P[6] and P[19] VP8*s recognized mucin core 2, while human P[6] and P[19] VP8*s interacted with the H1 HBGA, indicating an evolutionary path from animal to human. P[11] RV VP8* recognized type I and type II precursors that are developmentally regulated in neonates, consistent with the fact that P[11] RVs are mainly identified in neonates. P[8] and P[4] infections were mainly identified in secretors and lewis positive children , consistThe crystal structures of VP8*and VP8*/glycans are listed in The structural basis of P[14] interacting with A type HBGA has been illustrated  Figure . The widP[11] VP8*, clustered in P[IV] genogroup, possessed a quite different conformation comparing to other VP8*. Human P[11] VP8* interacted with type I and type II precursors using a distinct glycan binding site consisting of N153, R154, N155, Y156, I158, W178, G179, S180, Y183, D185, and R187  Figure . The typHuman P[4]/P[6]/P[8]/P[19] VP8*s belonging to P[II] genogroup all interacted with type I HBGA . Human Pc VP8* bound more intensively to H1 (affinity constants KD = 27.9 ± 0.7 μM) compared to LNB (KD = 52.1 ± 4.3 μM)  were determined . LNB and 4.3 μM) , implyinThe interactions between P[8]/P[19] VP8* and mucin core 2 have been illustrated . Mucin cb) (b based on nuclear magnetic resonance (NMR) spectroscopy-based titration experiments and NMR-derived high ambiguity driven docking (HADDOCK) method (b HBGA in another pocket consisting of the edge of two β-sheets (named ββ binding site)  . A recen) method . Unlike ng site) . The potng site) . FurtherAccording to the crystal structures of VP8* and glycans, three glycan binding sites are identified in different RV VP8*s so far . The firGroup/species C rotaviruses (RVCs) have been identified as important pathogens of acute gastroenteritis in children, family-based outbreaks, as well as animal infections . Human RHuman group B (RVB) and group H rotavirus (RVH) caused outbreaks in China in the 1980s and mainly infected adults. Infections of human RVB and RVH have constantly reported in some areas such as Southeast Asia . The recSome animal RVs recognized sialic acid, such as P[3], P[7]. Some animal RVs were reported to bind sialic acid and αGal, such as bovine P[5] RVs . The ide


This study aims to investigate the underlying mechanisms of KAT2A/MCT1 axis in renal cell carcinoma (RCC), providing potential therapeutic targets.in vitro and in vivo assays were further conducted to assess the oncogenic roles of KAT2A. The transcriptome sequencing and chromatin immunoprecipitation (ChIP) sequencing were conducted to screen KAT2A downstream targets. The dose-effect curves were used to detect the 50% inhibiting concentration (IC50) of AZD3965. Data analysis was performed in the Graphpad Prism (Version 8.3.0) and R software (Version 3.6.1).We obtained the expression data of KAT2A and MCT1 from The Cancer Genome Atlas Kidney Clear Cell Carcinoma (TCGA-KIRC) and International Cancer Genome Consortium (ICGC) databases. Differential analysis was conducted via the limma package. The CCK8 assay, soft agar assay, clone formation assay, and patients-derived organoid models were used to detect cell growth. The transwell and wound-healing assays were utilized to detect cell migration. The in vitro and in vivo. Transcriptome analysis and ChIP-seq were combined to find that KAT2A mainly regulated the glycolytic process. Validation and rescue assays revealed that MCT1 was the downstream target of KAT2A, and KAT2A depended on MCT1 to promote RCC malignant phenotypes. Lastly, MCT1 inhibitor (AZD3965) was effective to treat KAT2A-induced RCC progression.Our study found that KAT2A was highly expressed in RCC versus normal samples. Prognostic analysis indicated that a high KAT2A was an independent biomarker and associated with poor survival outcomes. KAT2A could promote RCC proliferation and distal metastasis Our study indicated that KAT2A was an oncogenic chromatin modifier that promotes RCC progression by inducing MCT1 expression. We proposed that MCT1 inhibitor (AZD3965) was useful for suppressing RCC. Renal cell carcinoma (RCC) belongs to one of the most common urological malignancies, accounting for ∼2–3% of all malignant diseases worldwide . LaparosAberrant epigenetic remodeling represents a molecular hallmark that contributes to tumorigenesis and progression . High-thMetabolic reprogramming of RCC has a significant impact on the tumor progression and distal metastasis, which shed light on the alternative strategies for the treatment of RCC . WarburgIn the current study, we discovered the oncogenic role of KAT2A in RCC and described the underlying mechanisms between KAT2A and MCT1, proposing that KAT2A/MCT1 axis may be a novel predictive biomarker and therapeutic target for RCC.2°According to the expression data in the CCLE dataset, Caki-2, SLR-21, and A498 are divided into the KAT2Ahigh cells, whereas 786-O belongs to the KAT2Alow cells.The human RCC cell lines  and 293 T were derived from the American Type Culture Collection (ATCC). The cells were maintained in RPMI-1640  containing 10% FBS  and 100 μg/ml of streptomycin under 37°C with 5% COThe 15 pairs of RCC tumor samples and matched normal kidney tissues were obtained from the department of urology in the first affiliated hospital of Bengbu medical college from July 2017 to September 2020. The tissues were frozen and maintained in liquid nitrogen for following analysis. Besides, FFPE samples of RCC and the corresponding normal samples were collected upon resection and stored in formalin. The protein levels of genes in fresh RCC samples were determined via Western blotting. Meanwhile, the clinical parameters of RCC were further collected correspondingly including TNM stages, tumor grades, pathological subtypes, age, and gender. The study was reviewed and approved by the Ethics Committee of the first affiliated hospital of Bengbu Medical College, and informed consents were all written by the RCC patients in this study.The guide oligos (sgRNAs) targeting KAT2A gene were designed and cloned into the pX459 plasmid. RCC cell lines (A498 and Caki-2) were plated and transfected pX459 constructs for 24 h. Then, 1 μg/ml of puromycin was added to screen cells for 3 days. The monoclonal cell line was derived when seeding the living cells into 96-well plates with limited dilution. Western blot assays and corresponding sanger sequencing were utilized to confirm the knockout efficiency of genes. The specific sgRNAs targeting KAT2A were summarized as follows: sgRNA (KAT2A-KO#1) F: 5-CACCGCCTGAGACCTCTGCCGAAAC-3; R: 5-AAACGTTTCGGCAGAGGTCTCAGGC-3; sgRNA (KAT2A-KO#2) F: 5-CACCGCCTACAAGGTCAATTACACC-3; R: 5-AAACGGTGTAATTGACCTTGTAGGC-3.5 cells per well. The number of soft agar colonies was scored and compared by the ImageJ software.Cells (400 cells per well) were seeded into a 6-well plate and incubated for 2 weeks until cell clones appeared. Then, the colonies of cells were fixed with methanol and stained with Giemsa. Cell counting kit-8 (CCK-8) assay  was used to determine the short-term cell proliferation. Briefly, the cells were seeded onto the 96-well plates . A total of 10 μl of the CCK-8 solution was added into the culture and incubated for 2 h. The optical density was assessed using a microplate reader at 450 nm. Each assay was carried out in triplicate. To conduct the soft agar colony formation experiment, 10% FBS and 0.7% agar were contained with 2 ml of gel. The cells were then seeded into the medium including 10% FBS with 0.35% agar and incubated at 37°C for 21 days with the density of 1 × 104 cells were seeded in a serum-free medium in the upper chamber . Besides, the lower chamber was filled with RPMI1640 containing 5% FBS. The cells were then fixed in 4% paraformaldehyde for 15 min and stained with crystal violet after 48 h. For the invasion assay, the membranes were covered with Matrigel (BD Biosciences). The migrated cells were stained and counted in nine different fields.Caki-2 and A498 cells were maintained in a serum-free medium for 2 days. For the migration assay, a total of 3 × 10We purchased the pLKO.3G GFP-shRNA plasmids from Addgene. After 48 h of transfection, the viruses were collected from the medium. Cells were infected with the collected viruses over 2 days with the company of polybrene, which were then sorted via GFP signals for 4 days. The specific shRNA sequences were listed as follows: KAT2A-shRNA#1 Sense: 5-GATCCGTACCGGAGACACCAAGCAGGTCTATTTCTCGAG AAATAGACCTGCTTGGTGTCTTTTTTTGG-3; Anti-sense: 5-AATTCCAAAAAAAGACACCAAGCAGGTCTATTTCTCGAG AAATAGACCTGCTTGGTGTCTCCGGTACG-3. KAT2A-shR NA#2 Sense: 5-GATCCCCGGGCTGAACTTTGTGCAGTACAA CTCGAGTTGTACTGCACAAAGTTCAGCTTTTTGG-3; Anti -sense: 5-AATTCCAAAAAGCTGAACTTTGTGCAGTACAAC TCGAGTTGTACTGCACAAAGTTCAGCCCGGG-3. MCT1- shRNA#1 Sense: 5-GATCCCCGGGCTCCGTATTGTTTGA AACATCTCGAGATGTTTCAAACAATACGGAGCTTTTTGG-3; Anti-sense: 5-AATTCCAAAAAGCTCCGTATTGTTTGA  A A C A T CTCGAGATGTTTCAAACAATACGGAGCCCGGG-3; MCT1-shRNA#2 Sense: 5-GATCCCCGGTATGTTTCTGCTAG CTATATACTC G A G T A TATAGCTAGCAGAAACATATTTTTG G-3; Anti-sense: 5-AATTCCAAAAATATGTTTCTGCTAGCTA TATACTCGAGTATATAGCTAGCAGAAACATACCGGG-3.TM Western Blotting system kit . The detailed information of the primary antibodies was summarized as follows: KAT2A ; MCT1 ; β-Actin (CST#4970).The cells were collected and lysed in the lysis buffer which was mixed with 0.1% sodium dodecyl sulfate (SDS), 50 mM TrisHCl, sodium deoxycholate, 150 mM NaCl, 1% NP-40, and a protease inhibitor (PH = 7.5). Then, the protein samples were loaded to 8% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a polyvinylidene difluoride membrane (PVDF). After blocking with 5% non-fat dry milk for 1 h, the membranes were incubated with the specific primary antibody overnight. After washing in TBST for three times in 15 min, the membranes were then incubated with the secondary antibodies (peroxidase-labeled anti-mouse and anti-rabbit antibodies) for 1 h. Briefly, the primary antibodies were utilized at a dilution of 1:2,000, while secondary antibodies were diluted with 1:5,000. After washing in TBST for three times in 15 min, the membranes were finally visualized in an ECL + plusTotal RNA from Caki-2 cells were extracted from with or without KAT2A deficiency, which were then subjected to HiSeq RNA-Seq based on the Jiayin Tech Solutions Co platform. Each RNA sample was designed to three biological replicas and was finally mixed to analyze for further minimizing the variations among samples. The genome (hg19) based on the Bowtie tool was utilized to map the final transcriptome reads. The gene expression data was further normalized by the RSEM software. For the ChIP-seq experiment, the Caki-2 cells were cross-lined and lysed, and sonication was used to shear the DNA fragments into 200–800 bp. The ChIP-seq assays were conducted via Active Motif using the KAT2A antibody . The nucleotide reads derived from the Illumina sequencing were mapped on the genome through the BWA algorithm, and the average profile for peak distributions was generated via deeptools. For the ChIP-qPCR assays, Quantitative PCR analysis was conducted following the ChIP experiment. Relative occupancy of the promoter region of MCT1 and CCND1 was normalized to the normal rabbit IgG.2O2 (15%) to suppress the endogenous peroxidase activity for 15 min under room temperature. The slides were blocked using the normal goat serum for 1 h and incubated with primary antibody  overnight. After washing three times in 1 × PBS, the sections were incubated with biotinylated goat-anti–rabbit IgG secondary antibodies for 30 min. After washing three times in 1 × PBS, the samples were further incubated with HRP. After washing three times in 1 × PBS for 15 min, specific samples were finally developed with diaminobenzidine. Lastly, the IHC staining of KAT2A was scored and analyzed by two independent pathologists.In total, 15 paired RCC samples and paired normal tissues were collected from the first affiliated hospital of Bengbu Medical College via laparoscopic surgery. The formalin-fixed paraffin-embedded (FFPE) tissues and hematoxylin and eosin (H&E) slides were confirmed by a pathologist. Then, the samples were incubated with H6 indicated A498 and Caki-2 RCC cells were suspended in 150 μl of PBS buffer and injected into the flanks of male nude mice. Tumor growth of mice was monitored every 2 days, for a total period of 30 days. At the end of 4 weeks, all mice were sacrificed, and in vivo solid tumors were dissected and analyzed. All experimental protocols were reviewed and approved by the Ethics Review Committee for Animal Experimentation of Bengbu medical college.The 4–6-week-old BALB/c nu/nu mice were purchased from Mu Tu Biological Technology , which were maintained in our institutional pathogen-free mouse facilities. Briefly, 6 × 101, ICGC-RCC2, and GSE404353. The differential analysis was performed via the limma package. The univariate Cox regression analysis and Kaplan-Meier analysis were mainly conducted by the survival package. The enriched biological items were obtained via the clusterprofiler package. The ATAC-seq data in RCC was obtained and analyzed from the TCGA dataset4.The expression data of RCC patients were obtained and collected from the public dataset, including TCGA-KIRCt-test was used to compare the difference between two groups. The log-rank test was used to analyze the Kaplan-Meier curves. Spearman’s rank correlation analysis was conducted to analyze the associations between KAT2A and clinical parameters. All data analysis was performed in the R studio (Version 3.6.0) and Graphpad prism (Version 8.0). The P-values < 0.05 were considered to be statistically significant.All data derived in this study were shown as Mean ± SD for assays which were conducted with at least three replicates. The Student’s P < 0.001  for the following studies, in which KAT2A was preferentially expressed  dataset and selected two KAT2Axpressed . We thenblotting . The CCKblotting . Besidesblotting . Additioblotting . Moreoveg scores . Lastly,g scores . Taken tin vivo assays were further conducted to assess the effects of KAT2A on RCC metastasis. The luciferase-labeled RCC cells with a modified KAT2A were established and injected into the tail vein of BALB/c nude mice. Then, we detected the bioluminescence signals with a regular interval to capture the metastatic RCC cells into the lung. After 8 weeks, we observed that KAT2 overexpression significantly promoted the ability of RCC cells to metastasize into the lung compared with the control group in the Caki-2 metastatic model as quantified by BLI signals and numbers of lung metastases, whereas KAT2 ablation significantly inhibited the distal metastasis in the A498 metastatic model  were found with the cutoff of | Log(fold change)| > 1 and FDR < 0.01 . The topr = 0.29 and P < 0.0001  is an active marker through releasing paused RNA polymerase II to facilitate gene expressions. H3K9 acetylation is previously reported to be mainly mediated by KAT2A. We thus performed the ChIP-seq analysis using the H3K9ac antibody in Caki-2, parallelly. We integrated the ATAC-seq data of the RCC samples and the corresponding Chip-seq data to detect the significantly enriched peaks of KAT2A across MCT1. We observed the co-occupancy of KAT2A and H3K9ac at the promoter regions of MCT1 in the IGV plot, in accordance with our speculations . Besides< 0.0001 . We also< 0.0001 . As expe< 0.0001 . In addi< 0.0001 . The 3D < 0.0001 . Lastly,< 0.0001 . Collectin vitro growth of A498 and Caki-2 cells in a dose-dependent manner  could effectively inhibit the oncogenic phenotypes induced by KAT2A in RCC.The morbidity and mortality of RCC has gradually increased yearly and the overall prognosis of RCC remains unsatisfactory . How to bona fide target of KAT2A. As well known, the solute carrier 16 (SLC16) family of genes mainly encode these MCTs, including MCT1/SLC16A1, MCT2/SLC16A7, MCT3/SLC16A8, and MCT4/SLC16A3  were warranted to assess the potential differences of inhibitory efficacy of AZD3965 in the KAT2Ahigh and KAT2Alow RCC samples.However, there still existed several limitations in the current study. First of all, owing to the limited patients in our hospital, we were still uncertain to identify the exact cutoff to discriminate the high-KAT2A RCC samples. Secondly, apart from the proliferation and distal metastasis, whether KAT2A mediated other oncogenic processes, like stemness, angiogenesis, or drug resistance, was interesting to systematically elucidated. Furthermore, we mainly conducted the experimental assays in KAT2A-KO and KAT2A-OE cell models, without considering the genetic heterogeneity across cells. We should obtain more cell lines to assess the expression levels of KAT2A in RCC. The associations between KAT2A expression levels and AZD3965 drug sensitivity should be quantitatively assessed for pharmacological researches. How to define the cutoff to clarify the KAT2Ahigh and KAT2Alow RCC and assess the pharmacological security of AZD3965 will be of great importance to deal with in following researches.Taken together, these findings derived from the TCGA analysis, functional validations, and high-throughput sequencing data highlighted that KAT2A was an independent biomarker and a potentially therapeutic target in RCC. KAT2A depended on MCT1 to drive malignant phenotypes. and AZD3965 was significantly effective to suppress the tumor progression induced by KAT2A. How to reasonably define the KAT2APRJNA732761).The data presented in the study are deposited in the Sequence Read Archive (SRA) data repository, accession number (The studies involving human participants were reviewed and approved by the Ethics Committee of the first affiliated hospital of Bengbu Medical College. The patients/participants provided their written informed consent to participate in this study. The animal study was reviewed and approved by the Ethics Committee of the first affiliated hospital of Bengbu Medical College.LC conceived the concept of this research. YG, BL, and YL performed the experimental research and data analysis. WS, WG, and SM collected the samples and data analysis. LC and YG wrote and reviewed the draft of the manuscript. All authors approved the final manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The present study is aimed at evaluating the functional and clinical values of P3H4 in lung adenocarcinoma. Moreover, we also investigated the downstream pathways that P3H4 might participate in.  The differential expression analysis was used to identify genes differentially expressed in lung adenocarcinoma tissues as compared with normal tissues. Survival analysis was used to test the association between P3H4 and survival time. Gene set enrichment analysis was conducted to explore the downstream pathways. CCK8 and transwell were employed to examine the impact of P3H4 on cell phenotypes.  P3H4 was highly upregulated in LUAD tissues at both RNA and protein levels. Moreover, the LUAD patients, who had high expression of P3H4, were also observed to have shorter disease-free survival and overall survival. These results demonstrated that P3H4 could be used as a prognostic biomarker for LUAD. Moreover, we also found that it was the copy number alterations (CNAs), not DNA methylation, that regulated the RNA expression of P3H4, indicating that its upregulation might be partially resulted from the CNAs. Furthermore, functional experiments revealed that the A549 and H1299 cells with siRNA treatment (siP3H4) exhibited significantly decreased cell proliferation after 24 hours, migratory ability, and invasiveness. Functionally, the upregulated proteins in the P3H4 high expression group were mainly enriched in tumor microenvironment-related pathways such as phagosome, focal adhesion, and ECM-receptor interaction and cancer-related pathways such as bladder cancer pathway, proteoglycans in cancer, and hippo signaling pathway.  The present study systematically evaluated the functional and clinical values of P3H4 in LUAD, and explored the related biological pathways. P3H4 might promote LUAD progression through regulating tumor microenvironment-related pathways. Lung adenocarcinoma (LUAD) is a major type of non-small-cell lung cancers (NSCLC) . DevelopTumor microenvironment (TME) and the organization of local extracellular matrix (ECM) are found to be essential players in tumor progression and metastasis in various cancers –8. The cProlyl 3-hydroxylation (P3H) is a rare but conserved posttranslational modification in many collagen types  and may https://xena.ucsc.edu/) with accession numbers: TCGA-LUAD [http://linkedomics.org).The gene expression dataset was obtained from public databases including UCSC Xena  was used to identify the Reactome pathways enriched by the upregulated genes . The enr2. The two small-interface RNAs specifically binding P3H4 mRNA and the negative control were denoted as si-P3H4-1, si-P3H4-2, and si-NC. The transfection was conducted on the cells in logarithmic phase using Lipofectamine 2000 (Thermo Fisher Scientific). The following are the sequences of siRNAs: si-P3H4-1—GGGCUGUGAAGCUCUACAACA; si-P3H4-2—GGCACGCUCUGGAGCAGUACG.The two human lung cancer cell lines (A549 and H1299) were purchased from Shanghai Institute of Materia Medica, Chinese Academy of Sciences (CAS). These cells were cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum (Gibco) and 1% penicillin-streptomycin and incubated at 37°C with 5% COTotal RNAs from A549 and H1299 cells were separated using a TRizol reagent. Following the manufacturer's protocol, we performed the reverse transcription using PrimeScript RT reagent kit . P3H4 mRNA expression was quantitatively analyzed using an ABI Prism 7900HT , with GAPDH as an internal reference. The following are the primers for P3H4: forward—5′-CATGAGCAGGTGGACTTCAAGG-3′, reverse—5′-ACTTGTCCACGAAGTAGCCACC-3′; and the primers for GAPDH: forward—5′-AGGCTGTTGGGAAAGTTCTTC-3′, reverse—5′-ACTGTTGGAACTCGGAATGC-3′. All these experiments were conducted in triplicate.3 cells/well. Using a microplate reader , we detected the absorbance at 450 nm following the manual. All these experiments were conducted in triplicate.The CCK8 assays were used to detect the cell proliferation. The experiments were conducted in 96-well plates with 2 × 10μm pore size; Millipore) and chambers coated with Matrigel were used to conduct cell migration and invasion assays, respectively. The cells (5 × 104 cells) with 48 h of transfection were planted into the upper chamber, and 500 μL of medium containing 10% FBS was filled into the lower chambers. The migrated or invaded cells with 4% paraformaldehyde, which were incubated at 37°C for 24 h, were fixed for 30 min and strained with 0.1% crystal violet for another 20 min. All these experiments were conducted in triplicate.The transwell chambers  and Student's P value < 0.001), with about 2-fold than the normal tissues. The survival analysis revealed that shorter overall survival was observed in samples with high mRNA expression of P3H4 as compared with those with low mRNA expression .To investigate the RNA expression levels of P3H4 in lung adenocarcinoma (LUAD) and normal tissues, we compared its expression of LUAD with that of normal tissues using The Cancer Genome Atlas (TCGA) LUAD cohort. Specifically, P3H4 RNA expression was highly upregulated in LUAD . Moreover, the comparison of P3H4 RNA expression between the tumor samples with P3H4 gain and those without P3H4 gain revealed that P3H4 was expressed higher in those with P3H4 gain . These results indicated that P3H4 was highly upregulated in LUAD tissues and its upregulation might be partially resulted from the CNAs.To further explore the transcription of P3H4, we investigated whether the copy number alteration (CNAs) and DNA methylation levels were associated with its RNA expression. The Pearson correlation between the DNA methylation and RNA expression of P3H4 was about -0.081, suggesting that P3H4 mRNA expression was poorly associated with DNA methylation . The further survival analysis of the P3H4 protein expression confirmed that P3H4 protein expression was also highly associated with both disease-free survival (DFS) and overall survival (OS) , suggesting that P3H4 protein expression was an indicator of poor prognosis in LUAD. To test the independence of P3H4 protein expression about the prediction of survival in LUAD, we conducted multivariable Cox regression analysis using TNM stage, age, and differentiation levels as cofactors. Remarkably, the P3H4 protein expression was also statistically significant in the multivariable Cox model , indicating that this siRNA could efficiently decrease the P3H4 RNA expression. Compared with the negative control (siNC), the two cell lines with siRNAs treatment (si-P3H4-1 and si-P3H4-2) exhibited significantly decreased cell proliferation , suggesting that P3H4 gene silence could suppress the uncontrolled proliferation of A549 and H1299 cells.To explore whether P3H4 regulated the functionalities of lung cancer cells, we silenced the P3H4 gene by two small interface RNAs (siRNAs) using A549 and H1299 cell lines. Specifically, the RNA expression levels of P3H4 were significantly suppressed by the siRNAs . Notably, only the upregulated proteins were successfully enriched in the KEGG pathways by gene set enrichment analysis, as the number of downregulated proteins was small. Specifically, the upregulated proteins in the high P3H4 protein expression group were mainly enriched in the pathways regulating the tumor microenvironment such as phagosome, focal adhesion, and ECM-receptor interaction and cancer-related pathways such as bladder cancer pathway, proteoglycans in cancer, and hippo signaling pathway , the cells with siRNA treatment (siP3H4) exhibited significantly decreased cell proliferation, migratory ability, and invasiveness using A549 and H1299 cell lines. Consistently, the inhibition of proliferation and invasion by the knockdown of P3H4 were also observed in bladder cancer .Functionally, we also explored the biological function of P3H4 protein in LUAD. The upregulated proteins in the P3H4 high expression group were mainly enriched in tumor microenvironment-related pathways such as phagosome, focal adhesion, and ECM-receptor interaction and cancer-related pathways such as bladder cancer pathway, proteoglycans in cancer, and hippo signaling pathway. Notably, the molecules localizing to the extracellular matrix such as ITGA11, PDGFRA, THBS1/2/3, and COMP were coexpressed with P3H4 and might be the direct target of P3H4. ITGA11 was found to enhance tumorigenicity of human non-small-cell lung cancer cells by regulating IGF2 expression in fibroblasts . PDGFRA In conclusion, the present study systematically evaluated the expression levels and clinical values of P3H4 in LUAD and explored the related biological pathways. P3H4 might promote LUAD progression through regulating tumor microenvironment-related pathways.


Based on the antigenic distances, subgroup II and III strains resulted in two clear antigenic clusters 2 and 3, separated from the vaccine strain F98, cluster 1. New antigenic properties of subgroup III viruses were associated with 11 amino acid changes in the HA protein, suggesting antigenic drift in H9N2 viruses. Our phylogenetic and antigenic analyses of the H9N2 strains circulating in local markets in Southern China provide new insights on the antigenic diversification of H9N2 viruses.The H9N2 subtype avian influenza virus (AIV) has become endemic in poultry globally; however due to its low pathogenicity, it is not under primary surveillance and control in many countries. Recent reports of human infection caused by H9N2 AIV has increased public concern. This study investigated the genetic and antigenic characteristics of H9N2 AIV isolated from local markets in nine provinces in Southern China from 2013 to 2018. We detected an increasing annual isolation rate of H9N2 AIV. Phylogenetic analyses of hemagglutinin (HA) genes suggests that isolated strains were rooted in BJ94 lineage but have evolved into new subgroups (II and III), which derived from subgroup I. The estimated substitution rate of the subgroup III strains was 6.23 × 10IMPORTANCE The H9N2 low pathogenicity avian influenza (LPAI) virus has become endemic in poultry globally. In several Asian countries, vaccination against H9N2 avian influenza virus (AIV) was approved to reduce economic losses in the poultry industry. However, surveillance programs initiated after the introduction of vaccination identified the persistence of H9N2 AIV in poultry . Recent reports of human infection caused by H9N2 AIV has increased public concern. Surveillance of H9N2 circulating in poultry in the fields or markets was essential to update the vaccination strategies. This study investigated the genetic and antigenic characteristics of H9N2 AIVs isolated from local markets in nine provinces in Southern China from 2013 to 2018. The discovery of mutations in the hemagglutinin (HA) gene that result in antigenic changes provides a baseline reference for evolutionary studies of H9N2 viruses and vaccination strategies in poultry. Currently, H9N2 avian influenza virus (AIV) is one of the predominant subtypes (together with H5 and H7 subtype AIVs) circulating in both wild birds and poultry , 2. Ever5–Surveillance of H9N2 AIV is carried out in many countries, especially China and other Asian countries in which live poultry markets (LPMs) are potential areas for human-poultry transmission or reassortment . GeneticIn addition to surveillance programs, some countries have also implemented poultry vaccination to prevent H9N2 AIV from becoming endemic –18. Afte–Since 2003, monitoring programs of LPAI H9N2 in Southern China were conducted by the Research Team of the Etiologic Ecology of Animal Influenza and Avian Emerging Viral Disease in Shanghai Veterinary Research Institute (SHVRI). Swab samples from domestic poultry  were collected annually in the local markets in Southern China. The most recent samples collected from 2013 to 2018 showed an increasing annual isolation rate of H9N2 AIV in Southern China. The phylogenetic and antigenic analyses of the H9N2 isolates were combined to identify a common clustering change. The most recent isolates clustered together but were far from the vaccine strains. Isolated strains forming a novel antigenic group in antigenic assays also clustered in a new subgroup in the phylogenetic tree. The evolution rate of strains in the new antigenic group was estimated to be higher than the average rate of H9N2 in China. Moreover, amino acid changes on HA protein indicating potential antigenic sites provide new evidence for the molecular evolution of H9N2.To monitor the prevalence of H9N2 AIV in Southern China, we collected oropharynx and cloaca samples of poultry from LPMs in nine cities of different provinces in Southern China. From 2013 to 2018, a total of 13,981 samples were collected from chickens , ducks , geese (81 samples), and pigeons (32 samples) in different LPMs . H4, H6,To analyze the genetic evolution of the HA gene of the H9N2 isolates, 113 representative isolates were selected to perform HA gene sequencing and further analysis based on geographical and host differences. These included 21 viruses isolated in 2013, 42 in 2014, 23 in 2015, 7 in 2016, 11 in 2017, and 9 in 2018. Online HA sequences were used for the phylogenetic tree, including 4 in 2013, 3 in 2014, 7 in 2015, 11 in 2016, 8 in 2017, and 9 in 2018. Phylogenetic analysis showed that the HA genes of the isolates from 2013 to 2018 were all distributed in subgroup II or subgroup III, except for A/chicken/Guangdong/D2703/2013 and A/chicken/Fujian/F1132/2015 and the commercial vaccine strain A/chicken/Shanghai/F/1998 (F/98), which were colocated in subgroup I . Among tIn addition, a new branch of evolution, subgroup III, began to appear after 2013, including 14 viruses  isolated in 2013, 28  in 2014, 19  in 2015, 6  in 2016, 7  in 2017, and 9  in 2018.To estimate the time when subgroup I, subgroup II, and subgroup III viruses started to appear, the most recent common ancestor (tMRCA) for these time points was estimated based on the HA genes of all H9N2 isolates in the time-scaled phylogenic tree . Subgroup I lineage viruses first emerged in August 1992, with a 95% highest posterior density (HPD) interval from September 1991 to May 1993. The tMRCA of subgroup II viruses was September 1996 (July 1995 to November 1996). The most recently isolated H9N2 strains from 2018 were in subgroup III, of which the tMRCA was estimated as September 2010 .To monitor the antigenic map of the H9N2 isolated viruses, 27 representative isolates were selected for crossing hemagglutination inhibition (HI) tests, including three viruses isolated in 2013, two in 2014, two in 2015, seven in 2016, seven in 2017, and six in 2018 . A vacci−3  substitutions/site/year over a 25-year period , with only 18.75% of the average evolution rate of the HA gene of H9N2 AIVs. In contrast, the HA genes of antigenic cluster 3 viruses (subgroup III) evolved most rapidly with 6.23 × 10−3 substitutions/site/year, 1.5-fold faster than that of the average substitution rate of HA genes for H9N2 viruses  of the viral effective population size . The popThe homology of HA protein from different antigenic clusters was analyzed to assess the potential amino acid residues resulting in antigen drift. Eleven amino acid residues were conserved in vaccine cluster viruses  but mutated into the same amino acid residues both in cluster 2 and 3 viruses, including M96L, T205A, D208E, Q226L, N273K, S274R, N275S, V276T, R285K, P306S, and A325S (H3 numbering), indicating that these residues in HA may contribute to the antigen change between cluster 1 and clusters 2/3 ; Fig. 4aInvestigation of the key amino acids characterizing antigenic properties of strains in cluster 2 showed 10 mutated amino acid residues in the HA protein between cluster 2 viruses and cluster 3 viruses, including N/H57Q, G81E, S/N133D, K137T, D145G, Q156G, N159G, A160D/N/E, V/A190T, and T192R ; Fig. 4bTo gain information on the evolution of potential antigenic amino acid residues on HA from H9N2 over time in nature, we analyzed HA proteins of H9N2 isolates and other viruses submitted to NCBI. Of the 11 potential antigenic amino acids characteristic of cluster 1, 10 were dominant patterns in China, including M96L, T205A, D208E, Q226L, N273K, N275R, V276T, R285K, P306S, and A325S in HA protein (H3 numbering). Currently, S274R is the predominant amino acid for H9N2 HA, mixed with some S274 . MoreoveThe H9N2 virus is a predominant subtype of AIV circulating in Asia and other countries all over the world –33. H9N2–35–Our surveys of LPMs conducted in China from 2013 to 2018 demonstrate that H9N2 AIVs are still prevalent, as described previously . An incrTogether with the high isolation rate and long-term endemic circulation, H9N2 AIVs in mainland China are undergoing a rapid antigenic drift. The hypothesis that vaccination triggered rapid evolution leading to antigenic drift was reported in Korea only 4 years after the national vaccination program was applied in poultry in 2007 . Similar−3 substitutions/site/year. This substitution rate increased almost 1.5-fold (6.23 × 10−3 substitutions/site/year) when antigenic differences were involved.Phylogenic analysis of 113 H9N2 strains isolated from LPMs in Southern China from 2013 to 2018 provided sufficient circulating and evolutionary information of H9N2 in local poultry. Consistent with previous studies indicating G9 (A/chicken/Hong Kong/G9/1997) lineage (h9.4.2)  as the d−3 substitutions/site/year)  using the sequences from the influenza database has been estimated. These rates are similar to the estimations using the same method for H3 (HA1 domain) in humans (5.7 × 10te/year) . For betWe found 21 amino acid mutations on H9 antigenic sites (in region 66 to 334 on the H9 protein sequence) specific to strains in antigenic cluster 2, different from those in cluster 1 as well as three vaccine strains. Referring to the global online database of AIV H9N2 in Nextstrain, all these mutations evolved after 2013 and became dominant in Southern China. This explains why the isolation ratios in LPMs from 2013 to 2018 did not decrease even though vaccines had been used long term in poultry, with reports that the H9N2 virus continues to circulate in vaccinated chickens , 46.Structural analyses of the HA1 protein to predict key potential antigenic sites dominating the subgroup of current H9N2 isolates from field markets in Southern China were performed. The four surface locations  at the top of the HA head also show high entropy in real-time tracing . This inIn summary, our study shows that the currently circulating H9N2 viruses form a new antigenic group to those from vaccine strains and are also clearly divided into new subgroups in phylogenic trees. The declining diversity together with the high substitution rate of H9N2 strains in the new antigenic group suggest that vaccination triggers the evolution of H9N2 into new antigenic groups. Our study provides a baseline reference for evolutionary studies of H9N2 viruses and vaccination strategies in poultry.All animal experiments were conducted in accordance with the recommendations of the Guide for the Care and Use of Laboratory Animals of the Ministry of Science and Technology of the People’s Republic of China and the Netherlands. The protocols (SHVRI-SZ-20190730-01 and SHVRI-SZ-20190906-02) used in the study were approved by the Animal Care and Use Committee of Shanghai Veterinary Research Institute (SHVRI).TTCTAACCGAGGTCGAAAC-3′ (M-229U) and 5′-AAGCGTCTACGCTGCAGTCC-3′ (M-229L). Hemagglutination inhibition (HI) assays were then used as antiserums specific for H9 subtype influenza viruses.Between 2013 and 2018, a total of 13,981 oropharyngeal swabs and cloaca swabs were collected from chickens, ducks, geese, and pigeons in local markets from nine provinces in China . Swab samples were inoculated in the allantoic cavity of 10-day-old specific-pathogen-free (SPF) chicken egg embryos. The harvested allantoic fluids were tested by hemagglutination assay. Total viral RNAs of HA-positive allantoic fluids were extracted to detect influenza viruses by amplifying the M gene by PCR. The primers used for this detection were 5′-The viral RNA was extracted, and reverse transcription was performed with the Uni12 primer as previously described , 50. ComFJ190132, EPI_ISL_260531, AF156378, CY005632, EPI_ISL_146723, and KF188265) were identified and removed from the data set. We then constructed time-scaled phylogenies using a Bayesian Markov chain Monte Carlo (MCMC) framework implemented in BEAST (v1.10.2) .To generate a time scale tree of the whole lineage containing the H9N2 strains isolated from this lab , we inclAccording to the phylogenetic tree of the HA gene, 27 H9N2 viruses were selected from the isolated strains from 2013 to 2018; six stored virus strains from previous research  were selected for the antigenic analysis. Briefly, specific antisera with inactivated virus isolates were produced in SPF Leghorn chickens with adjuvant Montanide ISA 71VG (SEPPIC). Four-week-old SPF Leghorn chickens were vaccinated with a total of 1.5 mL by intramuscular injection. After 3 weeks, sera were collected from vaccinated chickens. The hemagglutination inhibition (HI) assay was conducted to test cross-reactivity between virus and antisera as described previously by the World Organization for Animal Health (OIE) . First, 1JSD)  position of the identified molecular changes on the sites of the HA protein, the HA1 structure of H9 was simulated. A/swine/Hong Kong/9/98 (H9N2) (1JSD)  was used1JSD) .https://nextstrain.org)  . To zoomhttps://platform.gisaid.org/epi3/cfrontend#80368) with isolate IDs from EPI_ISL_462482 to EPI_ISL_462520. These HA sequences were submitted to the GenBank database (OK563122 to OK563235).All the nucleotide sequences of HA and NA genes from H9N2 isolated in Southern China from 2013 to 2018 were submitted in the GISAID database (http://www.ncbi.nlm.nih.gov/genomes/FLU/FLU.html) hosted by NCBI. Details are at https://doi.org/10.1371/journal.pcbi.1007189.s012 (The G9 lineage strains recovered from the data set in reference 189.s012 .https://nextstrain.org/flu/avian/h9n2/ha) consisted of 1,092 of 1,332 genomes sampled between January 1976 and June 2019. The data used in this research were filtered to avian  from Africa (115), China (358), Europe (55), South Asia (126), Southeast Asia (93), and West Asia (395).Data and analysis from Nextstrain (


Despite its impact on patient survival, the mechanism underlying chemoresistance against 5-FU remains poorly understood. Here, we identified serine hydroxymethyltransferase-2 (SHMT2) as a critical regulator of 5-FU chemoresistance in CRC. SHMT2 inhibits autophagy by binding cytosolic p53 instead of metabolism. SHMT2 prevents cytosolic p53 degradation by inhibiting the binding of p53 and HDM2. Under 5-FU treatment, SHMT2 depletion promotes autophagy and inhibits apoptosis. Autophagy inhibitors decrease low SHMT2-induced 5-FU resistance in vitro and in vivo. Finally, the lethality of 5-FU treatment to CRC cells was enhanced by treatment with the autophagy inhibitor chloroquine in patient-derived and CRC cell xenograft models. Taken together, our findings indicate that autophagy induced by low SHMT2 levels mediates 5-FU resistance in CRC. These results reveal the SHMT2–p53 interaction as a novel therapeutic target and provide a potential opportunity to reduce chemoresistance. Colorectal cancer (CRC) is the third leading cause of cancer mortality worldwide due to its metastatic properties and resistance to current treatments . 5-FluorAutophagy, a catabolic process, is thought to buffer metabolic stress, thereby promoting cell survival , 5. In tBecause of the rapid proliferation of CRC cells, moderate serine amounts must be converted to glycine to support nucleotide biosynthesis and proliferation –19. SeriIn this study, we found via Gene Expression Omnibus (GEO) and TCGA database analysis that SHMT2 is tightly related to CRC progression , 31. StrFifty paired fresh-frozen samples of primary colorectal carcinoma (CRC) and adjacent normal colon tissues were collected from the Department of Surgery at Shanghai General Hospital, School of Medicine, Shanghai Jiaotong University. A total of 378 paraffin-embedded samples of stage II–III primary colorectal carcinoma were collected between 2003 and 2011. Tissue microarrays were constructed from these samples. All the samples were obtained during surgery. This research was approved by the Ethics Committee of Shanghai General Hospital (2016KY069), and written informed consent was obtained from all patients before enrollment in the study.6) were injected subcutaneously into the flanks of nude mice. 5-FU (20 mg/kg/day) was injected intraperitoneally weekly for 3 weeks. CQ (10 mg/kg) was administered as a daily oral gavage. All animal procedures were conducted in accordance with the Hospital Animal Care guidelines of Shanghai General Hospital. All efforts were made to minimize animal suffering.CRC xenografts were established in 5-week-old female BALB/c nude mice purchased from the Institute of Zoology, Chinese Academy of Sciences, Shanghai. Tumor-bearing and the calculation of tumor volumes were performed as previously described . Brieflyscid Il2rgtm1/Bcgen (B-NSG) mice (Biocytogen) with a 10-gauge trocar needle. Once established, solid tumor xenografts were serially passaged using the same technique. A primary tissue sample was anonymized and obtained by the Shanghai General Hospital  Institutional Review Board.Fresh tissues from 4 CRC patients (two with high SHMT2 expression and two with low SHMT2 expression) undergoing surgical treatment were obtained and implanted subcutaneously into the flanks of female NOD-Prkdc2. The SHMT2, HDM2, p53 and p53 mutant (NLS- and NES-) sequences [ACAAGTACTCGGAGGGTTATCCTCGAGGATAACCCTCCGAGTACTTGTTTTTTG (SHMT2-sh-1), 5ʹCCGGTAGGGCAAGAGCCAGGTATAGCTCGAGTAGGGCAA-GAGCCAGGTATAGTTTTTG (SHMT2-sh-2), and 5ʹCCGGGTCTGACGTCAAGCGGAT-ATCCTCGAGGATATCCGCTTGACGTCAGACTTTTTTG (SHMT2-sh-3).The human cell lines HCT116, SW480 and 293 T were purchased from the American Type Culture Collection . All the cell lines were maintained in DMEM supplemented with 10% FBS  at 37°C, 95% humidity and 5% COequences  were cloCGGCGCACAGAGGAAGAGAATCTCGAGATTCTCTTCCTCTGTGC-GCCGTTTTTG (p53-Sh) . 3-MA, CQ and sodium formate  were obtained from Sigma. Antibodies specific for LC3 , SHMT2 , p62 , p53  and actin  were used.The sequence of the shRNA targeting p53 was as follow: 5ʹCCGGhttp://crispr.mit.edu/). The guide sequences targeting human SHMT2 were 5ʹ- CACCGCGAGTACTTGTTGTTCAGAC-3ʹ and 5ʹ-CACCGGTTGCTGTGCTGAGC-CCGAA-3ʹ.The sgRNA sequences targeting SHMT2 were designed with CRISPR Designer  can be identified through their contents and double bilayers with narrow electron-lucent clefts. Autolysosomes (ALs) can be identified by their partially degraded, electron-dense contents.® In Situ PLA probes and Duolink® In Situ detection reagents (Cat. No: DUO92101) were obtained from Sigma-Aldrich . The PLA assay was performed according to the manufacturer’s instructions. Cells were incubated with PLA probes for 1 h. Cells were then incubated with the ligation mix for 30 min, and the Cy3 amplification mix was applied to the slides for 100 min at 37°C. The samples were mounted using Duolink® In Situ Mounting Medium with DAPI.Cells were permeabilized and treated with primary antibodies. Duolink0.5 μg of the mCherry-GFP-LC3B plasmid and 1.5 μg of pLKO.1-shRNA plasmids expressing Scramble-sh or SHMT2-sh were cotransfected into HCT116 cells. The cells were incubated in mock medium or medium containing 100 μM CQ for 8 h and were then fixed with 4% paraformaldehyde (Sigma-Aldrich). The cells were subsequently stained with DAPI, and the formation of intracellular puncta was monitored with a Leica TCS SP8 microscope.Cells were treated with sodium formate (Sigma–Aldrich) at the indicated concentrations and collected as previously described after 12 h for western blotting. WT SHMT2 plasmids and plasmids carrying the sequences for the SHMT2 K95Q , K280Q avac). Puncta were counted in a minimum of 100 cells per sample in three replicates. Cells exhibiting a mostly diffusive distribution of GFP-LC3 in the cytoplasm and nucleus were considered nonautophagic, whereas cells showing several intense punctate GFP-LC3 aggregates but no nuclear GFP-LC3 were classified as autophagic. Each GFP-LC3 stained sample was evaluated by two independent investigators.Cellular autophagic activities were assessed by determining the formation of GFP-LC3 aggregates in HCT116 cells and were quantified by counting the percentage of cells exhibiting the accumulation of GFP-LC3 in dots or vacuoles  database harbors the most comprehensive datasets for the gene expression profiles of all cancer types. To identify key genes that might significantly contribute to the prognosis of CRC, we first analyzed differentially expressed genes (DEGs) between CRC and adjacent normal tissues (control) using a machine learning approach based on five algorithms . A total of 66 genes were identified using the GSE9348 datasets  as the tAlthough SHMT2 is an essential enzyme in one-carbon metabolism, it is found in complex with many other important proteins, including BRISC , 29. HerCytosolic p53 mediates the inhibition of autophagy, as deletion, depletion, or pharmacological inhibition of p53 induces autophagy in mouse, human and nematode cells . Having Typically, SHMT2 functions to regulate one-carbon metabolism , 48. We Next, we evaluated whether SHMT2 regulates autophagy via cytosolic p53. SHMT2 overexpression affected neither the LC3-II/I ratio nor the p62 level in the absence of p53 Fig. . SimilarInduction of autophagy has been reported to stimulate proteasome-mediated degradation of p53 through a pathway dependent on the E3 ubiquitin ligase HDM2 , 43. TheGiven that cytosolic p53 triggers apoptosis and inhibits autophagy , we furtNext, we investigated whether CQ increases sensitivity to 5-FU treatment in SHMT2-sh xenograft tumors. Control and SHMT2-sh HCT116 cells were injected subcutaneously into nude mice above the left and right hind legs, respectively. Tumor growth was markedly inhibited in SHMT2-sh–injected mice after CQ and 5-FU treatment compared with CQ treatment alone Fig. . 5-FU trSHMT2 is a potential cancer driver gene and promotes colorectal carcinogenesis , 26; morn = 378) to explore the function of SHMT2 in response to 5-FU–based adjuvant chemotherapy (Table n = 164) of the CRC tissues exhibited low SHMT2 expression  treated with 5-FU–based adjuvant chemotherapy had worse DFS and OS than those with SHMT2-high CRC  were implanted subcutaneously Fig. . A similWe screened 66 differentially expressed genes associated with CRC progression in 224 colon cancer tissues and 165 adjacent normal tissues from three GEO data sets and found that SHMT2 is important in CRC metabolism. SHMT2, responsible for the conversion of serine to glycine, supports cancer cell proliferation in various cancers , 21, 53.Autophagy plays opposing and context-dependent roles in cancer, and the therapeutic targeting of autophagy in cancer is sometimes viewed as controversial . In our –/– cells. Some inducers of autophagy stimulate proteasome-mediated degradation of p53 via the E3 ubiquitin ligase HDM2. However, the factors regulating the binding of HDM2 to p53 require exploration. Here, we found that SHMT2 competitively bound to cytosolic p53 to exclude HDM2 and thus inhibited autophagy. Treatment with 5-FU increased the binding of p53 to HDM2 to induce autophagy but decreased the binding of cytosolic p53 to SHMT2. In summary, we verified a new autophagy regulation mechanism involving the SHMT2–p53–HDM2 competitive binding system and confirmed the importance of this mechanism in mediating the response to CRC 5-FU–based chemotherapy. Indeed, it was unlikely that the p53 dependent role of SHMT2 in autophagy regulation and its sensitizing effect in 5-FU treatment was only limited to CRC. Further study is warranted to determine whether this mechanism holds true in other tumor types.The factors inducing autophagy are complex  , 55. InhSHMT2 is located not only in mitochondria but also in the cytoplasm, as shown by our data and data from other studies . SHMT2 iGiven the complexity of tumorigenesis, mechanisms affecting chemotherapy require further exploration. Our study showed that low expression of SHMT2 is related to 5-FU resistance in CRC, implying that SHMT2 expression could be used as a therapeutic marker for clinical 5-FU resistance. Investigation of the molecular mechanism showed that SHMT2 competitively binds to cytosolic p53 to exclude the E3 ubiquitin ligase HDM2 and SHMT2 depletion decreases the stability of cytosolic p53 to induce autophagy, which maintains the survival of cancer cells treated with 5-FU. These findings reveal the SHMT2–p53 interaction as a novel oncotherapeutic target and provide a potential opportunity to reduce 5-FU resistance using autophagy inhibitors in chemotherapy.Supplemental FiguresSupplemental Table


Finding new technologies and materials that provide real alternatives to the environmental and energy-related issues represents a key point on the future sustainability of the industrial activities and society development. The water contamination represents an important problem considering that the quantity and complexity of organic pollutant  molecules can not be efficiently addressed by the traditional wastewater treatments. The use of fossil fuels presents two major disadvantages: (1) environmental pollution and (2) limited stock, which inevitably causes the energy shortage in various countries. A possible answer to the above issues is represented by the photocatalytic technology based on S-scheme heterostructures characterized by the use of light energy in order to degrade organic pollutants or to split the water molecule into its components. The present mini-review aims to outline the most recent achievements in the production and optimization of S-scheme heterostructures for photocatalytic applications. The paper focuses on the influence of heterostructure components and photocatalytic parameters  on the pollutant removal efficiency and hydrogen evolution rate. Additionally, based on the systematic evaluation of the reported results, several perspectives regarding the future of S-scheme heterostructures were included. In recent years, due to the expansion of industrial activities, the problem of energy demands and water contamination have become major issues to be solved by the scientific community in order to increase the quality of life and human health ,2. The wA possible answer to the above issues is represented by the photocatalytic technology, which is characterized by the use of light energy in order to degrade organic pollutants or to split the water molecule into its components. Fast charge carriers recombination and limited light absorption spectra represent key issues of the mono-component photocatalysts ,8. The dThe literature mentions four typical mechanisms to describe heterostructures: type I, type II, Schottky junctions, and Z-scheme heterostructures see . HoweverThe present mini-review aims to outline the most recent achievements (2019–2021) in the production and optimization of S-scheme heterostructures for photocatalytic applications. There may be many others scientific papers reporting similar or different information that are not included here due to the space limitation. The paper focuses on the influence of heterostructure components and photocatalytic parameters  on the pollutant removal efficiency and hydrogen evolution rate. Additionally, based on the systematic evaluation of the reported results, several perspectives regarding the future of S-scheme heterojunctions were included.The need to adopt sustainable energy sources and remedy wastewater environmental issues has encouraged the researchers to find new materials and technologies that are able to give alternatives to the traditional ones. Semiconductors are considered as versatile materials that have or can be optimized to exhibit photocatalytic properties, alone or in combination with other materials ,21,22. TRecently, the S-scheme heterojunctions mechanism  containiThe S-scheme heterostructure has emerged as a suitable solution for the limitations induced by the previous photoactive mechanisms that use two or more semiconductors, coupled together using hydrothermal , solvoth2− from reduction reactions and •OH from oxidation reactions) for photochemical reactions [If compared with the Z-scheme heterostructure, the S-scheme mechanism is driven by the built-in electric field that is able to reduce the transmission distance of photogenerated carriers based on the synergetic interface between the two semiconductors ,42. The eactions ,44.The type-II heterostructure weakens the redox capabilities of the photogenerated electrons and holes with detrimental effect on the hydrogen production applications. Contrary, the S-scheme mechanism can maintain the photogenerated carriers with firm redox capability distinctly on two semiconductors, which is beneficial for hydrogen evolution during the water splitting process ,46,47. This mini-review has selected two major photocatalytic applications of the S-scheme heterostructures: organic pollutant removal from wastewater and hydrogen production. It should be mentioned that there may be other papers that are not included here due to the space limitation or insufficient experimental data required to describe or compare the results. There are also papers describing the use of S-scheme heterostructures in other applications that were not considered in this work.Photocatalytic technology is based on a chemical reaction mediated by mono-component or composite semiconductors that efficiently use the light radiation to promote the reduction and oxidation (redox) reaction rates due to the photogenerated carriers ,49,50. I2−) radicals are developed. These highly reactive oxygen species will decompose the organic pollutants to complete mineralization . Most of the organic pollutants contain also other atoms, and the formation of additional by-products is possible [As presented in possible ,59. Tabl2O4/ZnFe2O4 [18O49 [2O4 nanoparticles (EG = 1.88 eV)/ZnFe2O4 nanoparticles (EG = 1.78 eV) heterostructure with a specific active surface of 68.79 m2/g. The SnFe2O4/ZnFe2O4 shows enhanced Vis light absorbance and a direct S-scheme path of charge separation and transfer. However, the TC concentration was 5× lower (10 mg/L) compared with BP nanosheets (EG = 1.68 eV)/BiOBr nanosheets (EG = 2.73 eV) photocatalytic activity, where the efficiency was 85%. The two-dimensional BP/BiOBr nano-heterojunction has a good performance on boosting the spatial charge separation in order to use holes–electrons with higher redox ability. The TiO2 nanosheets (EG = 3.00 eV)/W18O49 spindle-like (EG = 2.78 eV) heterostructure was able to remove 80.3% from the initial TC concentration (30 mg/L). In order to have an objective evaluation of the photocatalytic activity, the pollutant concentration, photocatalyst dosage, and irradiation time must be correlated. Based on these characteristics, the BP/BiOBr using a 100 mg/100 mL photocatalytst dosage was able to remove the highest tetracycline (TC) quantity (42.5 mg/L) during 90 min of irradiation with 300 W Vis light compared with SnFe2O4/ZnFe2O4 (9.3 mg/L in 120 min) and TiO2/W18O49 (22.5 mg/L in 75 min). The TiO2/W18O49 heterostructure was comparatively evaluated for Rhodamine B (RhB) photocatalytic removal, and the efficiency was higher (82.1%) than that of TC removal.Pharmaceutical active compounds are helpful tools to eradicate human diseases but the pollution induced by the metabolized or partially metabolized pharmaceutical wastes represents an important bio-hazard ,61. The /ZnFe2O4 , and TiO4 [18O49 . The hig4@MoS2 [G = 3.23 eV)/BiOI flower-like microspheres (EG = 1.74 eV) exhibited 90% RhB photocatalytic removal . The photocatalytic efficiency remains unchanged after 5 cycles of 1 h each. The macroporous/microspheric hierarchical system allows light multi-scattering, which increases the photogenerated carrier’s concentration. The same photocatalytic efficiency (90%) was recorded for the BiVO4 nanorods @MoS2 sheets heterostructure but after 20 min of irradiation with 500 W Vis light intensity and using 20 mg/L RhB concentration. The BiVO4@MoS2 presents an improved photocurrent density that promotes the spatial distribution of BiVO4 oxidation sites and MoS2 reduction sites. The solvothermal method was also used to obtain Bi2O3 microspheres (EG = 2.40 eV)/Bi2SiO5 flower-like (EG = 3.64 eV) S-scheme heterostructure [2/g. The photocatalytic activity was evaluated against phenol and methyl orange (MO) organic pollutants at the same concentration (10 mg/L) and irradiation scenario (500 W Vis light). The results indicate an accelerated photocatalytic activity toward MO molecule  and lower oxidation rate on phenol (0.0001 min−1 and 30% efficiency). The heterostructure exhibit a small decrease of the phohotocatalytic efficiency after 5 cycles of 6 h each. The synergic effect induced by the heterostructure components enhances the production of predominant active radicals responsible for pollutant mineralization.Similar studies for RhB photocatalytic removal were done with NiO/BiOI  and BiVO4@MoS2  S-schemetructure  with a s3 nanorods (EG = 2.76 eV)/CdIn2S4 nanosheets (EG = 1.94 eV) were able to remove 95% of the initial TC concentration (50 mg/L). Compared with WO3, the CdIn2S4 shows strong Vis light absorption, suggesting that it is a good candidate to develop large-scale photocatalytic technologies. After 20 min of irradiation with a 500 W Vis light source, the CdS (EG = 2.42 eV)/UiO-66 (EG = 2.75 eV) removal efficiency reaches 80% toward the 4-nitroaniline molecule (20 mg/L). Using the pore-size performance of metal organic frameworks, the CdS/UiO-66 photocatalyst has a fast light excitation response and short carrier transport distances. The SnNb2O6 flaky structure (EG = 2.10 eV)/Ag3VO4 nanoparticles (EG = 2.16 eV) heterostructure behaves as an energy-efficient material that is able to remove 99% of MB (20 mg/L) in 10 min using a 50 W Vis light source (light emitting diode (LED)). The development of Ag3VO4 nanoparticles on 2D SnNb2O6 nanosheets favors the migration and separation of photogenerated carriers and increases the exposure of more active sites.The hydrothemal method  was empl2MoO6 (EG = 2.64 eV)/g-C3N4 (EG = 2.76 eV) S-scheme heterostructure [3N4. As mentioned before, the use of noble metals raises economic issues when up-scaling will be considered. The BiOI flakes (EG = 1.68 eV)/Bi2WO6 nanosheets (EG = 2.60 eV) S-scheme heterostructure [−1) compared with MO . The mechanism of pollutant degradation preference was not completely elucidated.Photoreduction and hydrothermal coupled methods were involved in the development of a Bitructure  for RhB tructure  possesesG = 2.75 eV)/I-BiOAc nanosheets (EG = 2.35 eV) S-scheme heterostructure [G = 2.62 eV)/BiO(HCOO)Br-x tube (EG = 2.96 eV) S-scheme heterostructure [2/g for AgI/I-BiOAc and 9.65 m2/g for BiOBr/BiO(HCOO)Br-x). In terms of photocatalytic activity, the BiOBr/BiO(HCOO)Br-x reaches 100% MG degradation efficiency in 60 min. However, if MO is involved as a pollutant molecule, the photocatalytic efficiency decreases at 30% after 120 min of irradiation. These results demonstrate that the photocatalytic efficiency of the S-scheme heterostructure is dependent of the surface/interface chemistry with the pollutant molecule. The construction of interfacial-close-contact BiOBr/BiORBr allows the photo-generated electrons on the CB of BiOBr to migrate on the VB of BiORBr. Using charge carriers with higher redox capacity, the heterostructure produce oxidative species is able to induce 98% RhB and 80% TC photodegradation efficiencies.An interesting study was done on an AgI nanoparticles /MoO3 (EG = 3.01 eV)/g-C3N4 (EG = 2.63 eV) lump-like heterostructure [2/g. The cumulative photogenerated electrons present in the conduction band of C3N4 and Bi2S3, and the photogenerated holes will enrich in the valence band of MoO3. Additionally, the cumulative photoinduced holes with positive potentials (3.4 eV) in the valence band of MoO3 can be directly involved in the oxidation reaction of MO. Consequently, after 120 min of light irradiation , the photocatalytic removal efficiency of MO (10 mg/L) in the presence of Bi2S3/MoO3/g-C3N4  was 78% (0.0091 min−1). The Bi2S3 nanorods (EG = 2.50 eV)/porous g-C3N4 nanosheets (EG = 2.70 eV) heterostructure [−1) MB removal with lower energy consumption and a faster degradation rate, which significantly increases the technological sustainability. The photogenerated holes on the Bi2S3 valence band can directly mineralize the MB molecule. The potential of photoinduced electrons on the g-C3N4 conduction band is −1.21 eV, which is lower than the O2/•O2− reduction potential (−0.33 eV). Consequently, the S-scheme has an important contribution in promoting the MB photocatalytic degradation.The one-pot method was used to develop two g-Ctructure  has the tructure  was test3 (EG = 2.34 eV)/g-C3N4 (EG = 2.65 eV) nanosheets [G = 2.83 eV) nanosheets/BiOAc1−xBrx (EG = 3.28 eV) flower-like [3/g-C3N4 sample obtained by the template-assisted polymer method exhibited a 43.03 m2/g active surface area using the combined advantage of a g-C3N4 thin planar structure and WO3 oxygen deficit, which enhanced the charge transfer and increased the quantum efficiency. Based on these characteristics, the WO3/g-C3N4 photocatalyst in 50 mg/50 mL dosage is able to remove 90.54% of TC (20 mg/L) after 60 min of irradiation with a 300 W Vis light source. The stability tests indicate that the WO3/g-C3N4 photocatalytic efficiency remains unchanged after 6 cycles. The second heterostructure with a 33.9 m2/g active surface area obtained by co-precipitation benefits from the formation of BiOAc1−xBrx solid solution, which enlarges the visible light response of BiOAc and prolongs the lifetime of photogenerated carriers with stronger redox ability. Using a 20 mg/50 mL photocatalyst dosage, the TC (20 mg/L) removal efficiency was 99.2% after 120 min of irradiation with a 500W Vis source. Comparing the rate constant of the two experiments, it became obvious that WO3/g-C3N4 possesses a faster removal rate (0.0378 min−1) than that of BiOBr/BiOAc1−xBrx (0.0230 min−1). However, the photocatalytic activity of BiOBr/BiOAc1−xBrx increases when TC is replaced with RhB. In the same experimental conditions, the photocatalytic efficiency for RhB removal was 99.4%, and the rate constant was 0.0330 min−1.The photocatalytic removal of ciprofloxacin and TC was evaluated using WOnosheets  and BiOBwer-like  S-scheme3 (EG = 2.41 eV) nanoparticles/TiO2 (EG = 3.20 eV) nanoparticles S-scheme heterostructure [2, the irradiation was done using both UV (300W Hg) and Vis (300 W Xe) sources during 210 min. The development of a binary structure can hinder the crystal grain growth and particles agglomeration, inducing the formation of a smaller particle size; it is also able to reduce the photogenerated carriers transfer route from the semiconductor inner to the surface and to produce enough oxidative species in order to reach 54% CIP photodegradation. The experiment was repeated using MO instead of CIP, and the results were even better. At 20 mg/L MO concentration, the photocatalytic efficiency after 150 min of irradiation was 92%. Using half of the MO concentration, the LaNiO3/TiO2 is able to completely remove the organic pollutant when a 100 mg/50 mL photocatalyst dosage is used. Similar results were obtained for RhB removal with g-C3N4 (EG = 2.83 eV) nanosheets/Bi nanoparticles/BiVO4 (EG = 2.40 eV) nanoparticles S-scheme heterostructure [2/g specific active surface obtained by an in situ reduction process. After 70 min of irradiation with a 350W Vis light source, the g-C3N4/Bi/BiVO4 photocatalyst (50 mg/50 mL) can completely eliminate the RhB (10 mg/L) from the aqueous solution. It is worth noting that this heterostructure uses three active Vis light absorption centers  inducing the direction of the built-in electric field from the g-C3N4 surface to the BiVO4 surface, which favors the vectorial interfacial transition of the photogenerated carriers from the BiVO4 conduction band to the g-C3N4 valence band during the photocatalytic activity.Ciprofloxacin (CIP) is an extensively used antibiotic for a large number of bacterial infections. The LaNiOtructure  obtainedtructure  with 50 3N4 (EG = 2.70 eV) nanosheets/ZrO2 (EG = 2.60 eV) nanoparticles S-scheme heterostructure [2/g) due to the dispersed nanoparticles being able to provide more active sites and higher Vis light absorption capacity. The g-C3N4 structure has the ability to enlarge the distribution area. All these factors will be beneficial on the production of reactive free radicals during the 150 min of irradiation with a 300 W Vis light source. Using the same experimental conditions in terms of pollutant concentration (10 mg/L), photocatalyst dosage (30 mg/50 mL), and irradiation scenario, the photocatalytic removal efficiencies were 98% for AO II, 82% for RhB, and 50% for MO. The efficiency differences are mainly attributed to the dye molecule and surface chemical compatibility. The S-scheme charge transfer mechanism is based on the smaller work function (4.18 eV) and higher Fermi level of g-C3N4, contrary to the ZrO2, which will enhance the photocurrent response and decrease the emission PL spectrum intensity.A comparative study of the photocatalytic removal efficiency of three dye molecules ) was done on a g-Ctructure  obtained2O3 based S-scheme heterostructures were developed to remove ethinylestradiol and phenol. The photodeposition method was used to obtain Bi2O3 (EG = 2.80 eV) nanoplates/CuBi2O4 (EG = 1.87 eV) nanoparticles/Ag nanoparticles heterostructure [2O3 conduction band to the CuBi2O4 valence band, while silver metal is an electron reservoir as well as surface plasmon, ensuring the achievement of higher charge carrier separation. Based on this mechanism, the photocatalyst (40 mg/100 mL) reaches 94.6% ethinylestradiol removal efficiency after 120 min of irradiation with a 250W Vis light source. A lower photocatalytic efficiency (50%) was obtained for TC removal using a Bi2O3 (EG = 2.77 eV) rod-like/TiO2 (EG = 3.0 eV) nanofiber S-scheme heterostructure [2/g. The photocatalytic experimental conditions use extreme values in terms of pollutant concentration (100 mg/L) and photocatalyst dosage (50 mg/15 mL). As mentioned before describing other similar reports [2 as recombination charges for the electrons from Bi2O3, resulting in more available photogenerated electrons in the TiO2 conduction band for •O2− production. Another outcome is represented by the increased availability of holes on the Bi2O3 valence band, which favors the phenol oxidation by reactive oxidative species.Two Bitructure , and thetructure . The pho reports ,73, the Photochemical (PC) and photoelectrochemical (PEC) water splitting are considered as potential candidates to generate clean and cost-effective hydrogen using a green pathway of solar energy conversion. However, there are still important difficulties to overcome: (i) limited light spectral absorption, (ii) efficient conversion of the photogenerated carriers during the water-splitting process, (iii) suitable conduction and valence band-edge potentials for redox reactions. 2/CoAl [3/TiO2/reduced graphene oxide (rGO) [GMoS2 = 1.8 eV and EGCoAl = 2.1 eV), and the heterostructure is able to exhibit 17.1 µmol/h hydrogen evolution under 300W light irradiation. Benefiting from the advantages of 2D/2D architecture and high photocurrent intensity, the MoS2/CoAl represents a promising candidate for hydrogen technology. The WO3/TiO2/rGO heterostructure have the disadvantage of using two semiconductors active in the UV range (EGWO3 = 3.2 eV and EGTiO2 = 2.6 eV). However, by inserting rGO in the heterostructure composition, it was possible to increase the specific surface area up to 165 m2/g and the absorption range. The hydrogen evolution rate (HyER) was optimized at 12.29 µmol/h using 50 mg/80 mL photocatayst dosage. The combined effect of the S-scheme heterostructure, formed between semiconductors metal oxides, and the Schottky heterojunction, formed between TiO2 and graphene sheets, is able to suppress the recombination of useful photogenerated carriers and increase the number of active surface sites for the reduction reaction.The hydrothermal method was employed to develop MoS2/CoAl  and WO3/de (rGO)  heterost3N4 heterostructure consisting of hydrothermally treated melamine and urea was developed [3N4 (MCN) and urea g-C3N4 (UCN) with different morphologies (comb-like for MCN and laminar for UCN) but a compatible band structure (EGMCN = 2.69 eV and EGUCN = 2.81 eV) were irradiated with 300 W Vis light for 180 min. The photogenerated electrons can migrate from MCN to UCN due to the MCN large work function, resulting in a positive interface on MCN and negative interface on UCN. The stability test indicated that the HyER remains unchanged after 4 cycles. The HyER using a triethanolamine (TEOA) sacrificial agent was significant (29.9 µmol/h) considering the heterostructure specific surface area of 46 m2/g. Using the same sacrificial agent and synthesis method, the CoAl layered double hydroxides (LDH)@Ni-metal–organic frameworks (MOF)-74 [An isotype g-Ceveloped  to work (MOF)-74  heterost2O6 nanosheets and CdS diethylenetriamine nanosheets with band gap values between 2.25 eV (SnNb2O6) and 2.51 eV (CdS). Using a photocatalyst dosage of 30 mg in 50 mL and Na2S + Na2SO3 as sacrificial agents, the heterostructure is able to produce 234.24 µmol/h HyER under 300 W Vis light irradiation. In similar experimental conditions in terms of light radiation and sacrificial agents, the Mn0.5Cd0.5S nanoparticles (EG = 2.48 eV)/WO3 nanorods (EG = 2.7 eV) heterostructure obtained by the chemical deposition method exhibits a significantly higher HyER (517.13 µmol/h) even if the photocatalyst dosage is lower (50 mg/100 mL). The reason consists of the Mn0.5Cd0.5S/WO3 multichannel-enhanced charge transfer combined with the Au nanoparticles insertion, which acts as an electron storage and provider for driving the hydrogen evolution reaction from water reduction. On the contrary, the SnNb2O6/CdS is a noble metal-free photocatalyst with interfaces toward a 2D S-scheme and with the potential to be cost effective for large-scale implementation.Two S-scheme heterostructures with similar band gap values were obtained by hydrothermal  and chem3/g-C3N4 heterostructure obtained by the one-pot method is characterized by uniformly distributed MoO3 nanoparticles on the g-C3N4 nanosheets. The HyER reaches 25.62 µmol/h due to the g-C3N4 structure change taking place during the one-pot process, which will increase the Vis radiation absorption capacity and the charge carrier’s photogeneration. A higher HyER (32.92 µmol/h) corresponds to the S-doped g-C3N4 nanosheets/N-doped MoS2 nanobelts heterostructure obtained by thermal condensation and having a 4.9 m2/g specific surface area. It must be underlined that in this case, the photocatalyst dosage was double compared with the MoO3/g-C3N4 heterostructure, which may explain the HyER differences. Additionally, the N-doped MoS2 was involved not only as a co-catalyst during the H2 production but also to extend the light absorption spectra, facilitating the charge carrier transfer and separation through N-Mo and C-S-Mo chemical bonds.One-pot  and ther3N4/CdS-diethylenetriamine [2S3/g-C3N4 [2S + Na2SO3 sacrificial agents and 300 W Vis light radiation). The highest HyER value (486.9 µmol/h) corresponds to the g-C3N4 nanosheets (EG = 2.61 eV)/CdS-diethylenetriamine nanosheets (EG = 2.68 eV) heterostructure with a specific active surface of 36.6 m2/g. The synergic activity of the nanocomposite induces fast interfacial charge separation and diffusion of the photogenerated charge. The Bi2S3 nanorods (EG = 1.60 eV)/g-C3N4 nanosheets (EG = 2.78 eV) heterostructure attempts 101.8 µmol/h HyER, even if the specific active surface is higher (58.1 m2/g) due to the reduced photocatalyts dosage (30 mg/100 mL). However, the advantage of the second heterostructure is given by its composition characterized by earth-abundant elements with low environmental impact. Using the same sacrificial agents and irradiation conditions, the g-C3N4 nanosheets (EG = 2.85 eV)/CdSe-amine flower-like (EG = 1.86 eV) S-scheme heterostructure [3N4/CdSe-amine sample is characterized by a 73.1 m2/g specific active surface, and the photocatalyst dosage was 20 mg/50 mL. The incorporation of Cl atoms will promote interlayer charge transfer and a conduction band up-shifted value. Additionally, the S atom can be used to replace the N atom into the CN framework, which allows tailoring the band gap.The influence of photocatalyst dosage can be observed by comparing the HyER values of g-Ctriamine  and Bi2S3/g-C3N4  obtainedtructure  obtained2 nanoparticles (EG = 3.7 eV)/SnS2 nanosheets (EG = 2.2 eV) S-scheme heterostructure [2 nanoparticles developed on the surfaces and edges of 2D SnS2 nanosheets will increase the carrier’s recombination rate corresponding to relatively weaker redox capacity and favor the separation of photoinduced carriers with stronger redox capacity.Finally, the SnOtructure  was obtaThese results indicate that the increase of HyER values can be obtained by correlating several simultaneous conditions: suitable position of the energy bands, specific active surface, catalyst dosage, and irradiation scenarios.Finding new technologies and materials that provide real alternatives to the environmental and energy-related issues represents a key point in the future sustainability of the industrial activities and social development. Photocatalytic technologies possess several advantages: (i) environmentally “friendly” materials, (ii) renewable energy source, (iii) long life-time cycles, and (iv) high versatility for both wastewater treatment and hydrogen production. The use of environmentally friendly materials will be a significant contribution in implementing a sustainable new large-scale technology with good social acceptance and relatively easy integration in the wastewater plants.2O6/Ag3VO4 or Bi2S3/porous g-C3N4 S-scheme heterostructures. Similarly, a 5W light energy source can be used to irradiate the CoAl LDH@Ni-MOF-74 S-scheme heterostructure in order to reach a 213 µmol/h hydrogen evolution rate. Reducing the irradiation time and photocatalysis dosage is essential for increasing the overall efficiency of the photocatalytic process. The BiVO4@MoS2 heterostructure was able to remove 90% of RhB (20 mg/L) in 20 min. By increasing the irradiation time to 40 or 100 min in order to gain a few extra percentages may not be cost effective due to the higher energy consumption. The photocatalyst recovery as well as standardization procedures remain an important issue to be solved in the near future. The S-scheme heterostructure mechanism describes a new pathway to induce the partition and migration of the photoinduced charge carriers preserving their redox ability under the influence of an internal electric field due to the differences of the Fermi level corresponding to each semiconductor partner. Based on this pathway, several works were presented in order to evaluate the photocatalytic efficiency of the S-scheme heterostructure on organic pollutant removal and hydrogen production. The systemic evaluation indicates that the photocatalytic efficiency should be correlated with other aspects  in order to have a comprehensive view on the future feasibility of the process. The high photocatalytic efficiency with the expense of large energy or material consumption is not sustainable when future large-scale implementation is considered. Using low energy light sources (LED) allows eliminating 90% of MB (20 mg/L) using SnNbThe main advantages of using S-scheme heterostructures are related to the efficient transfer and conversion of photogenerated charge carriers in order to be involved in the mineralization reaction of organic pollutant. Additionally, the S-scheme is a useful tool to design future photocatalyt materials for the extensive use of a solar spectrum in various energy applications. The disadvantage is given by the limitations induced by the materials type that can be used and tailored to increase the conversion efficiency and light absorbance. As perspectives, the use of coupled techniques and procedures may increase the overall performance in both wastewater treatment and hydrogen production. There are already many papers showing the advantages of simultaneous adsorption–photocatalysis processes. Biodegradation may be considered as well based on the use on non-polluting materials and low energy costs.3N4) is a metal-free semiconductor and nontoxic photocatalyst exhibiting good chemical stability and suitable band gap, allowing the use Vis light energy. Other materials can be optimized by doping, surface sensitization, exfoliation, or defect engineering in order to improve their quantum efficiency and photocatalytic activity. Doping can target specific materials properties without affecting the entire structure and composition. It can be used to modify the conduction type, the absorbance spectra, or even the surface energy. The size of the photocatalytic materials plays an important role considering two aspects: area/volume ratio and photogenerated charge carriers migration. The photocatalytic process requires large surfaces on small volumes that favor the interface development of oxidative species.The integration of new or optimized materials can contribute to the increase of the photocatalytic efficiency. Graphitic carbon nitride (g-C


Multiple observational studies have reported aninverse relationship between 25-hydroxyvitaminD concentrations (25(OH)D) and type 2 diabetes (T2D). However, the results ofshort- and long-term interventional trials concerning the relationship between 25(OH)D and T2D risk have beeninconsistent.To evaluate the causal role of reduced blood25(OH)D in T2D, here we have performed a bidirectional Mendelian randomizationstudy using 59,890 individuals  fromEuropean and Asian Indian ancestries. We used six known SNPs, including threeT2D SNPs and three vitamin D pathway SNPs, as a genetic instrument to evaluatethe causality and direction of the association between T2D and circulating25(OH)D concentration.–32; Z score 11.86, which, however, hadno significant association with 25(OH)D status . Likewise, the geneticallyinstrumented composite score of 25(OH)D lowering alleles significantlydecreased 25(OH)D concentrations  but was notassociated with increased risk for T2D . However, using 25(OH)D synthesis SNP  as anindividual genetic instrument, a per allele reduction of 25(OH)D concentration(-4.2nmol/L ± SE 0.3nmol/L)was predicted to increase T2D risk by 5%, p = 0.004;Z score 2.84. This effect, however, was not seen in other 25(OH)D SNPs  when used as an individual instrument.Results of the combined meta-analysis of eightparticipating studies showed that a composite score of three T2D SNPs wouldsignificantly increase T2D risk by an odds ratio (OR) of 1.24, p = 1.82 × 10Our new data on this bidirectional Mendelianrandomization study suggests that genetically instrumented T2D risk does notcause changes in 25(OH)D levels. However, genetically regulated 25(OH)Ddeficiency due to vitamin D synthesis gene (DHCR7) may influence the risk ofT2D.The online version contains supplementary material available at 10.1186/s12937-021-00725-1. Type 2 diabetes mellitus (T2D) has become a global health epidemic of twenty-first century. The International Diabetes Federation (IDF) data showed that the number of people affected with T2D would rise from 382 million in 2011 to 592 million by 2035 . In addiVitamin D deficiency increases in conjunction with T2D, Type 1 diabetes mellitus (T1D), obesity, and cardiovascular disease . The invThe blood level of 25(OH)D is the best and commonly used method to determine vitamin D status. According to the US Endocrinology Society, vitamin D deficiency is defined as a 25(OH)D level of ≤ 20 ng/mL; insufficiency as 21 to 29 ng/mL; and sufficiency as > 30 ng/mL or more . Low 25(Common genetic variants in ~ six loci have been identified to affect blood 25(OH)D concentrations in genome-wide association studies (GWAS) performed in European whites , HispaniHere, we have used genetic instrumental variable methods to obtain estimates of the causal association between circulating 25(OH)D concentrations and T2D, including the direction of causality, by performing two-directional reciprocal Mendelian randomization study , 26–28. All participating studies were reviewed and approved by the respective Universities or Institutional Review Boards, including the primary institute of the University of Oklahoma Health Sciences Center’s Institutional Review Board, as well as the Human Subject Protection Committees at the participating hospitals and institutes in India. All participants provided written informed consent for investigations.http://www.cpc.ncep.noaa.gov) and by averaging of the previous three months UV index as described previously [Our study with data from up to 59,890 participants from eight cohorts included: the Asian Indian Diabetic Heart Study/Sikh Diabetes Study (AIDHS/SDS); the Indian Migration Study (IMS); Twins UK; The British 1958 birth cohort (1958 BC); the Copenhagen City Heart Study (CCHS), the Copenhagen City General Population Study (CGPS), and the Copenhagen Ischemic Heart Disease Study (CIHDS); The UK Household Longitudinal Study (UKHLS); and The Prevention of Renal and Vascular End-Stage Disease (PREVEND) study. Details of study design and experimental work flow is presented in the Flow Chart . Phenotyeviously . The clir2 = 0.001). None of the instrumental variables were associated with the outcome via exposure to other factors .The selection of SNPs as genetic instruments was based on their strong global association with respective exposure phenotypes of T2D and 25(OH)D reported in multiethnic GWAS and meta-analysis studies , 26–28. For 25(OH)D analysis, linear regression and additive genetic model were used with the natural log-transformed 25(OH)D level adjusted with age, gender, BMI, and study-specific covariates . Similarly, the three GWAS SNPs known to affect T2D risk were analyzed for their association with T2D using logistic regression and an additive genetic model. The T2D binary trait was the dependent variable with covariates age, gender, BMI. Each cohort analysis was limited to a single ancestral group . If selected SNPs were unavailable, the best proxy SNP  with the reference SNP) was used using SNAP .Upon completing the association analysis, the final list of SNPs was determined for gene score construction for each trait. The SNP, risk allele, number of risk alleles, and effect size D levels) were required to construct a weighted gene score using the PLINK program . Sampleshttp://ntp.niehs.nih.gov/ntp/ohat/forestplot/), and PRISM (https://www.graphpad.com/scientific-software/prism/) was used to generate the forest plots. We did not observe heterogeneity of association of the genetic instruments of T2D and 25(OH)D with their respective phenotypes between studies; thus, fixed-effect models were used in metanalysis to drive odds ratios and confidence intervals. Finally, to assess the robustness of our conclusions, we applied conservative Bonferroni’s correction and used a corrected significant threshold of 0.0063 D concentration)An association analysis was performed on the constructed gene scores for each cohort. A linear regression and an additive genetic model were used with blood 25(OH)D levels as the dependent variable adjusted by gender, age, BMI, and T2D. Similarly, a logistic regression and an additive genetic model were used with T2D as the dependent variable adjusted by the gender, age, BMI. To combine the association analysis results of each trait by SNP for each cohort, a fixed effect inverse variance meta-analysis implemented in METAL  was perfDemographics and clinical characteristics of all study cohorts are summarized in Table P= 7.2x10-7, and Z score = 4.96  with 25(OH)D concentration Beta ± SE , P= 0.26, Z score = -1.18 after adjusting for age, gender, and T2D  with T2D  showed an OR (95% CI) of 1.12 , 470579 wiP= 2.87x10-61; and Z score = -16.52 nmol/L after adjusting for age, gender and BMI. For CYP2R1, age, gender, and BMI adjusted association was (Beta -0.039 ± SE 0.0032); P=7.56x10-34; and Z score -11.66 nmol/L, while the association of the DHCR7 variant (rs12785878) resulted in Beta -0.042 ± SE 0.0032, P= 9.0x10-32; and Z score -11.73 nmol/L for a total of 49,433 samples  allele of GC (rs2282679) and CYP2R1 (rs12794714) and DHCR7 (rs12785878) with blood 25(OH)D were largely consistent across chorts , and 1.01 , P=0.453, Z score = 1.09 for the CYP2R1 (rs12794714). However, the per-allele effect of the DHCR7 (rs12785878) variant showed a significantly increased risk for T2D OR 1.05 95% CI , P=0.004, and Z score =2.84 in the meta-analysis . These results appear to suggest that diabetes genetic loci increase T2D risk independently without modulating 25(OH)D concentrations , TCF7L2 rs7903146), and KCNQ1 (rs2237896) showed a significant association with increased risk for T2D in all studies OR (95% CI) 1.24 , P= 1.82x10470579, T146, and -78, and Z score = -18.68 D concentrations and gene variants associated with T2D risk by constructing a gene score. Table p = 0.004, with a Z score of 2.84 (Supplementary Table p trend = 0.04), whereas no association was observed for CYP2R1  in that study [Here we performed a Mendelian randomization study to investigate whether genetically reduced vitamin D levels could be causally related to increasing T2D risk using data from up to 59,890 participants . Essentially, our findings suggest that even though the candidate genetic variants  or via composite gene score are associated with increased susceptibility to T2D, these are not involved in affecting blood 25(OH)D concentration. This means that the genetically instrumented T2D risk could be independent of the pathway (s) linked with vitamin D synthesis, transport, or early stages of metabolism. Additionally, our data showed that the genetically instrumented reduction of 25(OH)D levels does not increase T2D susceptibility in a composite gene score. More specifically, gene variants in the vitamin D transporter (GC) and metabolism (hydroxylation) (CYP2R1) regulate 25(OH)D concentrations independent of influencing T2D risk. On the other hand, in the D synthesis), the genetically instrumented decrease of -4.2 nmol/L ± 0.3 nmol/L SE of 25(OH)D concentration , is converted to cholesterol by the action of ultraviolet light (UV) on the skin through a thermal isomerization. Hydroxylation of pre-vitamin D3 occurs in the liver from the actions of CYPs (CYP2R1). The CYP27A1 drives the conversion of 25(OH)D to 1,25(OH)D in the kidney  in meta-analysis despite using a sample size of up to 476,099 of Chinese and European adults.Presumably, the differences in linkage disequilibrium patterns among different populations, population-specific risk factors, and pleiotropy may have masked the potential cumulative effects of gene scores. It appears that perhaps due to similar limitations, the study of Lu et al.  also cou. [p = 1.26 × 10–98, Z score -21.08 D assays between cohorts as described earlier . Limitat0 to 550 N)  for effecting 25(OH)D concentration in most studies were not available except for our Punjabi Sikh cohort (AIDHS/SDS). Nonetheless, the geographic location of most participating European cohorts was nearby (Northern Europe), which might not have confounded the results due to latitude (to derive distance from the equator) variation D metabolism and transport genes, but the association with synthesis gene (DHCR7) could suggest that any association with T2D may be through a UV-dependent, vitamin D-independent effect as described . The powFor the first time, we have performed a bidirectional multiethnic Mendelian randomization study to determine the causal relationship between T2D and vitamin D concentrations, including data from the population of South Asians with Europeans. Even though our research has not found strong evidence of causation, it also does not rule out the possible contribution of genetic influence of vitamin D synthesis SNPs in increasing T2D risk. As the long-standing question of the role of vitamin D in influencing the risk of T2D remains unaddressed, our data stresses the need for the population-specific design for future observational and randomized clinical trials on this important and controversial topic of immense clinical importance.In summary, our bidirectional Mendelian randomization study having data from South Asian Indians with Europeans suggests that genetically instrumented T2D risk may not be causing changes in 25(OH)D levels. However, we cannot entirely exclude the likelihood of DHCR7 genetic variants to influence the risk of T2D.Additional file 1: Supplementary Table 1. Association results of individual T2D SNPs used as genetic instruments in Mendelian randomization for their association with T2D. Supplementary Table 2. Association of individual T2D SNPs used as genetic instruments in Mendelian randomization analyses for their association with 25(OH)D concentrations. Supplementary Table 3. Association results of individual vitamin D SNPs used as genetic instruments in Mendelian randomization for their association with circulating 25(OH)D concentration. Supplementary Table 4. Association results of individual vitamin D SNPs used as genetic instruments in Mendelian randomization for their association with T2D. Supplementary Table 5. Association of vitamin D SNPs used as genetic instruments for affecting 25(OH)D concentrations using UV index as a covariate. Supplementary Table 6. Association of vitamin D SNPs used as genetic instruments showing their effect on Type 2 diabetes risk using UV index as a covariate. Supplementary Table 7 A-B. Results of joint metanalysis of current study and a published study  showing significant associations of variants in vitamin D candidate genes (GC and DHCR7) for effecting 25(OH)D concentrations. Supplementary Table 8 A-B. Results of joint metanalysis of current study and a published study  showing effects of genetically instrumented vitamin D candidate genes (GC and DHCR7) SNPs for their effects on T2D risk. Supplementary Table 9. Information on samples with missing 25(OH)D levels from 8 different cohorts. Supplementary Table 10. Differences in latitude, 25(OH)D concentration and distribution of allele frequencies of vitamin D and T2D SNPs among South Asian and European cohorts.Additional file 2: Supplementary (Flow Chart). Experimental plan including the details of participating cohorts and candidate gene SNPs used as genetic instrument for performing a bidirectional Mendelian randomization study. Supplementary Figure 1 (A-B). Association analysis of composite gene score of T2D SNPs  used as a genetic instrument for their joint effect on T2D and 25(OH)D concentrations (https://www.graphpad.com/scientific-software/prism/). Supplementary Figure 2 (A-B). Association analysis of composite gene score of vitamin D SNPs  used as a genetic instrument for their effect on 25(OH)D concentrations and T2D (https://www.graphpad.com/scientific-software/prism/). Supplementary Figure 3. This figure illustrates pathways of synthesis, absorption, metabolism, and transportation of vitamin D. Genetic factors known to influence circulating levels of vitamin D are shown in the figure. DHCR7 expresses a reductase which uses nicotinamide adenine dinucleotide phosphate-oxidase to catalyzes the production of cholesterol to 7-dehydrocholesterol (7-DHC). The GC gene catalyzes the vitamin D binding protein formation. The enzyme (25-hydroxylase) responsible for the first hydroxylation step is encoded by CYP2R1. The enzyme in the kidneys responsible for the second hydroxylation is catalyzed by the CYP27B1 gene product (not used in this study). 1,25(OH)2D3 is the most effective and commonly measured vitamin D deficiency marker. Genetic instruments used in this study include gene pathways involved in vitamin D synthesis (DHCR7), metabolism (CYP2R1) and transport (GC) and genetic loci linked with increased T2D risk . Note that in our results causality appears to be inferred by direct association of 25(OH)D variants (i.e. DHCR7) with 25(OH)D levels and the T2D phenotype. In contrast the T2D genes are associated with phenotype but not with the intermediate phenotype measured as 25(OH)D.Additional file 3. Online Supplemental Information: Phenotypic measurements including T2D, 25(OH)D, and other recruitment details are presented separately for each cohort.