A Reference Profile of N-Glycosylation for Human Kidney and the Identification of Cell-Cell Interactions between Parietal Epithelial Cells and Capillary Endothelial Cells by Single-Cell Glycosylation-Sequencing.

BACKGROUND: N-glycosylation is one of the most common posttranslational modifications in humans, and these alterations are associated with kidney diseases. METHODS: A novel technological approach, single-cell N-acetyllactosamine sequencing (scLacNAc-seq), was applied to simultaneously detect N-glycosylation expression and the transcriptome at single-cell resolution in three human kidney tissues from zero-time biopsy. Cell clusters, glycation abundance in each cell cluster, functional enrichment analysis, cell-cell crosstalk, and pseudotime analysis were applied. RESULTS: Using scLacNAc-seq, 24,247 cells and 22 cell clusters were identified, and N-glycan abundance in each cell was obtained. Transcriptome analysis revealed a close connection between capillary endothelial cells (CapECs) and parietal epithelial cells (PECs). PECs and CapECs communicate with each other through several pairs of ligand receptors (e.g., TGFB1-EGFR, GRN-EGFR, TIMP1-FGFR2, VEGFB-FLT1, ANGPT2-TEK, and GRN-TNFRSF1A). Finally, a regulatory network of cell-cell crosstalk between PECs and CapECs was constructed, which is involved in cell development. CONCLUSIONS: We here, for the first time, constructed the glycosylation profile of 22 cell clusters in the human kidney from zero-time biopsy. Moreover, cell-cell communication between PECs and CapECs through the ligand-receptor system may play a crucial regulatory role in cell proliferation.

Functional CYP3A variants affecting tacrolimus trough blood concentrations in Chinese renal transplant recipients.

The aim of this study was to identify novel genetic variants affecting tacrolimus trough blood concentrations. We analyzed the association between 58 single nucleotide polymorphisms (SNPs) across the CYP3A gene cluster and the log-transformed tacrolimus concentration/dose ratio (log (C0/D)) in 819 renal transplant recipients (Discovery cohort). Multivariate linear regression was used to test for associations between tacrolimus log (C0/D) and clinical factors. Luciferase reporter gene assays were used to evaluate the functions of select SNPs. Associations of putative functional SNPs with log (C0/D) were further tested in 631 renal transplant recipients (Replication cohort). Nine SNPs were significantly associated with tacrolimus log (C0/D) after adjustment for CYP3A5*3 and clinical factors. Dual luciferase reporter assays indicated that the rs4646450 G allele and rs3823812 T allele were significantly associated with increased normalized luciferase activity ratios (p < 0.01). Moreover, CYP3A7*2 was associated with higher TAC log(C0/D) in the group of CYP3A5 expressers. Age, serum creatinine and hematocrit were significantly associated with tacrolimus log (C0/D). CYP3A7*2, rs4646450, and rs3823812 are proposed as functional SNPs affecting tacrolimus trough blood concentrations in Chinese renal transplant recipients. Clinical factors also significantly affect tacrolimus metabolism.

Assessment of variation in B-cell receptor heavy chain repertoire in patients with end-stage renal disease by high-throughput sequencing.

Background/Aims: End-stage renal disease (ESRD), characterized by progressive loss of rental function during the disease course, has been reported to be correlated with immune dysregulation. To date, a majority of previous studies on immune response to ESRD have been focused on the T-cell response. This prospective study was to assess the B-cell receptor (BCR) heavy chain repertoire in ESRD patients.Materials and methods: A total of 10 ESRD patients and six healthy controls were prospectively enrolled in this study. BCR immunoglobulin heavy chain (IGH) repertoire in the peripheral blood from ESRD patients and healthy individuals were analyzed by means of next generation sequencing (NGS) in combination with multiplex PCR, Illumina sequencing, and the international ImMunoGeneTics database (IMGT).Results: Abnormal BCR complementary-determining region 3 (CDR3) sequences were identified in relation to ESRD. We also found that the degree of the B-cell clonal expansion in the ESRD group was significantly greater than that in the control group (p < .05), whereas the distributions of BCR CDR3, V, D, J, and V-J gene segments were comparable between the ESRD and control groups. T-test for analysis of the distribution ratio of the V, D, J, and V-J genes revealed five up-regulated genes and nine down-regulated genes associated with ESRD, and there were significant differences between the ESRD and control groups (p < .05).Conclusions: We have provided a successful approach to analyzing peripheral B-cell repertoire in ESRD patients, and the results suggest a direct correlation between the BCR repertoire and ESRD. The ESRD-specific BCR CDR3 sequences may hold promise for potentially therapeutic benefit.

Identification of lysine acetylome of oral squamous cell carcinoma by label-free quantitative proteomics.

Lysine acetylation (Kac) on histone promotes relaxation of the chromatin conformation and favors gene transcription to regulate oncogenesis, whereas the total acetylation profiling of oral squamous cell carcinoma (OSCC) is unknown. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) was utilised to investigate lysine acetylation features of tumor tissues and adjacent normal tissues from 9 patients with OCSS. 282 upregulated Kac sites in 234 proteins and 235 downregulated Kac sites in 162 proteins between OSCC tissues and paired adjacent normal tissues were identified. Different acetylation proteins (DAPs) were analyzed through KEGG-based and MCODE. These DAPs are enriched in the ribosome biogenesis pathway. Survival Analysis of hub genes with TCGA database was performed. In addition, IPA software was used to explore the connection between 9 core DAPs (RPS3, RPL24, RPL19, EIF4A2, RPL12, MYBPC1, RPS6, ARCN1, and TMEM9) and the different expression of KATs and KDACs identified in our proteomic. The study is the first comparative study of Kac modification on oral squamous cell carcinoma. We propose to put forward the hypothesis that the dysfunction of ribosome biogenesis caused by the change of Lysine acetylation, especially downregulated acetylation on RPS6 and RPS3 may associated with the pathogenesis of OSCC. SIGNIFICANCE: The study is the first comparative study of Kac modification on oral squamous cell carcinoma through LC-MS/MS-based modified proteomic. These DAPs are high enriched in the ribosome biogenesis pathway. Used MCODE and survival analysis, 9 core DAPs (RPS3, RPL24, RPL19, EIF4A2, RPL12, MYBPC1, RPS6, ARCN1, and TMEM9) were screened. IPA software was used to explore the connection between 9 core DAPs and the different expression of KATs and KDACs identified in our proteomic. In addition, we propose to put forward the hypothesis that the dysfunction of ribosome biogenesis caused by the change of Lysine acetylation, especially downregulated acetylation on RPS6 and RPS3 may associated with the pathogenesis of OSCC.

Intestinal microbiome and metabolome analyses reveal metabolic disorders in the early stage of renal transplantation.

Renal transplantation is the most effective treatment for end-stage renal disease, but the long-term prognosis of organs after transplantation is not ideal. In recent years, the importance of gut microbes and metabolites in the study of disease mechanisms has gradually received attention. However, the coordination between gut microbes and the metabolism of renal transplant patients needs further study. We integrated 16s sequencing and metabolomics data to describe the changes in the serum and fecal metabolites of renal transplant patients. Our data revealed that the gut microbial diversity decreased and the relative abundance of many bacteria, such as Enterococcus and Streptococcus, significantly changed after transplantation. In addition, a large number of amino acids and peptides in serum and feces significantly changed, suggesting an abnormal amino acid metabolism after transplantation. Spearman's correlation analysis revealed the changes in the co-metabolism pattern between gut microbes and the host metabolism after transplantation. Furthermore, Enterococcus was found to be correlated with renal functions and metabolites reflecting renal damage. This study provides potential gut microbes and metabolites impacting renal health, which helps in understanding the renal damage in patients with kidney transplantation.

Lysine 2-hydroxyisobutyrylation proteomics reveals protein modification alteration in the actin cytoskeleton pathway of oral squamous cell carcinoma.

As the most commonplace malignant carcinoma in the oral cavity, oral squamous cell carcinoma (OSCC) is highly invasive and prone to recurrence. The nosogenesis of OSCC are affected by epigenetics. Recently, a newly-found post-translational modification of lysine, 2-hydroxyisobutylation (Khib), has been proved to play a critical role in biological regulation. However, no research has evaluated the mechanism of Khib in oral cancer. Here, we performed liquid chromatography-mass spectrometry-based quantitative proteomics combined with bioinformatics analysis to reveal and evaluate Khib protein alterations in OSCC. Numerous proteins in OSCC undergo up-regulated modification of Khib. We quantified and identified 967 proteins with differential expression levels, and 617 2-hydroxyisobutylated proteins with 938 Khib sites. Among them, 125 proteins both differentially expressed and accompanied by obvious Khib modification were further identified and analyzed through KEGG-based and ingenuity pathway analysis (IPA). These proteins are enriched in the actin cytoskeleton regulatory pathway, and IPA predicted that they alter the state of actin aggregation and stability, hence impacting and regulating the actin cytoskeleton in OSCC. This is the first 2-hydroxyisobutylated modification proteomics performed for OSCC. Khib protein is significantly concentrated in the actin cytoskeleton regulatory pathway, indicating that this pathway may mediate the tumorigenesis or exacerbation of OSCC. SIGNIFICANCE: This is the first study that revealed the alterations of Khib protein in oral squamous cell carcinoma through LC-MS/MS-based modified proteomic. Our data showed that the protein in the actin cytoskeleton regulatory pathway was underwent significant Khib modification and abundance changes. We applied predictive function in IPA software to analyze and clarify that the aggregation of actin and the regulation of actin stability that mediated by the actin cytoskeleton regulatory pathway may be the potential mechanism of the occurrence and development of oral squamous cell carcinoma. Our research broadens the understanding of the pathogenesis of oral squamous cell carcinoma and provides new insights for future research.

Dynamic Metabolomics Study of the Bile Acid Pathway During Perioperative Primary Hepatic Carcinoma Following Liver Transplantation.

BACKGROUND There are many situations of abnormal metabolism influencing liver graft function. This study aims to provide data for the development of liver function recovery after liver transplantation by dynamically analyzing metabolites of bile acids pathway in serum. MATERIAL AND METHODS A comprehensive metabolomics profiling of serum of 9 liver transplantation patients before transplantation, on the 1st, 3rd, and 7th days after liver transplantation, and healthy individuals were performed by ultra-performance liquid chromatography-mass spectrometry (UPLC-MS). Multivariate data and dynamic analysis were used to search for biomarkers between the metabolomics profiles present in perioperative liver transplantation and normal controls. RESULTS Thirty-three differential endogenous metabolites were screened by the threshold of variable importance in the projection (VIP) from an orthogonal partial least square discriminant analysis (OPLS-DA) greater than 1.0, q-value <0.05, and fold change (FC) ≤0.8 or ≥1.2 between the preoperative group and the normal controls in negative mode. The metabolite intensities of taurocholic acid, taurochenodeoxycholic acid, chenodeoxycholic acid glycine conjugate, and glycocholic acid pre-transplantation were significantly higher than those of normal controls. The average metabolite intensities of taurocholic acid and taurochenodesoxycholic acid on the first day after liver transplantation were lower than those observed pre-transplantation. The average metabolite intensities on day 3 after liver transplantation showed a sudden increase and then decreased after 7 postoperative days. The average metabolite intensities of glycocholic acid and chenodeoxycholic acid glycine conjugate showed an increasing trend on the 1st, 3rd, and 7th days after liver transplantation. CONCLUSIONS Use of taurocholic acid and taurochenodeoxycholic acid-related bile secretion, liver regeneration, and de novo bile acid synthesis may help clinical evaluation and provide data for the development of liver function recovery after liver transplantation.

Differential expression of transfer RNA-derived small RNAs in IgA nephropathy.

BACKGROUND: IgA nephropathy (IgAN) is one of the most common forms of primary glomerulonephritis. Recent studies have indicated that small noncoding RNAs, such as tRNA-derived small RNAs (tsRNAs), might be novel biomarkers for glomerulonephritis. We therefore investigated the potential roles and possible functions of the tsRNAs in IgAN. METHOD: Peripheral blood mononuclear cells (PBMCs) were extracted from blood samples of the patients with IgAN and healthy control groups. The expression profiles of tsRNAs were assessed by small RNA sequencing (RNA-Seq) in PBMCs of the IgAN and control groups. Dysregulated tsRNAs were selected for validation by quantitative real-time polymerase chain reaction (qRT-PCR). Target gene prediction and enrichment were performed by bioinformatics analysis. RESULTS: The results revealed that 143 significantly upregulated and 202 significantly downregulated tsRNAs were differentially altered in the IgAN group compared with the control group. Five upregulated tsRNAs (tRF-Val-AAC-007, tRF-Ala-AGC-063, tRF-Gln-CTG-010, tRF-Tyr-GTA-011 and tRF-Thr-AGT-007) and 3 downregulated tsRNAs (tiRNA-Val-TAC-004, tRF-Gly-CCC-005 and tRF-His-GTG-006) were selected for validation by qRT-PCR; the results were consistent with the sequencing data. Gene Ontology (GO) analysis revealed that the target genes predicted by upregulated tsRNAs were mostly enriched in "nucleic acid metabolic process,' "intracellular part,' and "ion binding,' whereas the target genes predicted by downregulated tsRNAs were mostly enriched in "regulation of cellular component organization,' "membrane-bound organelle,' and "ion binding.' Kyoto Encyclopedia of Genes and Genomes pathway analysis revealed that the target genes predicted by upregulated tsRNAs were mostly enriched in "herpes simplex virus 1 infection,' whereas the target genes predicted by downregulated tsRNAs were mostly enriched in "circadian rhythm CONCLUSIONS:: The present study confirmed the differential expression of tsRNAs in patients with IgAN, and these dysregulated tsRNAs might be novel potential targets for the diagnosis and treatment of IgAN.

Proteomic analysis of differentially expressed proteins in the serum of patients with acute renal allograft rejection using iTRAQ labelling technology.

Transplantation is currently the best treatment for patients with end­stage renal disease. However, acute rejection (AR) is the major source of failure in renal transplantation. The current best practice for the diagnosis of AR involves renal biopsy, but it is invasive, time­consuming, costly and inconvenient. Sensitive and less invasive detection of AR episodes in renal transplant patients is essential to preserve allograft function. The present study applied isobaric tags for relative and absolute quantitation (iTRAQ) mass spectrometry to analyze serum protein expression in patients with AR and healthy controls. Overall, 1,399 proteins were identified. Using a cut­off of Q<0.05 and a fold change of >1.2 for the variation in expression, 109 proteins were identified to be differentially expressed between the AR and control groups, 72 of which were upregulated and 37 were downregulated. Several proteins, including properdin, keratin 1, lipoprotein(a) and vitamin D­binding protein, may have roles in the pathogenesis of AR. The present study focused on iTRAQ­based proteomic profiling of serum samples in AR. Insight from the present study may help advance the understanding of the molecular mechanisms of AR and identify potential novel biomarkers of AR for further characterization.

Integrated analysis of quantitative proteome and transcriptional profiles reveals abnormal gene expression and signal pathway in bladder cancer.

BACKGROUND: Bladder cancer (BCa) is a tumor associated with high morbidity and mortality and its incidence is increasing worldwide. However, the pathogenesis of bladder cancer is not well understood. OBJECTIVE: To further illustrate the molecular mechanisms involved in the pathogenesis of BCa and identify potential therapeutic targets, we combined the transcriptomic analysis with RNA sequencing and tandem mass tags (TMT)-based proteomic methods to quantitatively screen the differentially expressed genes and proteins between bladder cancer tissues (BC) and adjacent normal tissues (AN). RESULTS: Transcriptome and proteome studies indicated 7094 differentially expressed genes (DEGs) and 596 differentially expressed proteins (DEPs) between BC and AN, respectively. GO enrichment analyses revealed that cell adhesion, calcium ion transport, and regulation of ATPase activity were highly enriched in BCa. Moreover, several key signaling pathway were identified as of relevance to BCa, in particular the ECM-receptor interaction, cell adhesion molecules (CAMs), and PPAR signaling pathway. Interestingly, 367 genes were shared by DEGs and DEPs, and a significant positive correlation between mRNA and translation profiles was found. CONCLUSION: In summary, this joint analysis of transcript and protein profiles provides a comprehensive reference map of gene activity regarding the disease status of BCa.

Next generation sequencing reveals novel alterations in B-cell heavy chain receptor repertoires associated with acute-on-chronic liver failure.

Acute­on­chronic liver failure (ACLF) is a newly­defined serious syndrome with major features of acute decompensation (AD) of hepatic cirrhosis, liver failure and failure of multiple other organs. To date, the mechanism underlying the development and progression of ACLF remains to be fully elucidated. It has been noted that ACLF is associated with immune dysregulation. However, studies have mainly focused on T­cell responses. The present study aimed to determine the composition and alterations of B­cell receptor (BCR) heavy chain repertoires associated with ACLF using next generation sequencing (NGS). A total of six patients with hepatitis B virus (HBV)­related ACLF and six healthy control subjects were prospectively enrolled in the present study. The B­cell immunoglobulin heavy chain (IGH) repertoires in peripheral blood mononuclear cells (PBMCs) obtained from the patients with HBV­related ACLF and the control subjects were analyzed using NGS, coupled with multiplex polymerase chain reaction, were Illumina sequenced, and were further characterized using the international ImMunoGeneTics database. The distribution of the BCR complementarity­determining region 3 (CDR3) variable (V), diversity (D) and joining (J) and V­J gene segments were found to be comparable between the ACLF and control groups. Of note, the degree of clonal expansion in the ACLF group was significantly higher than that in the control group (P<0.05). Furthermore, a t­test of the distribution ratio of the V, D, J and V­J combinations in patients with ACLF and control subjects revealed differentially expressed genes. In total, six genes were upregulated and 19 genes were downregulated in response to ACLF. The difference between these two groups was statistically significant (P<0.05). The approach used in the present study was feasible and effective for analyzing peripheral B­cell repertoires in HBV­related ACLF. These results provide direct evidence that the BCR repertoire is important in immune responses, autoimmunity and alloreactivity, and that there is a link between the BCR repertoire and HBV­ACLF. Therefore, ACLF­specific BCR CDR3 sequences hold promise for therapeutic benefit to HBV­ACLF in the future.

Composition and diversity analysis of the B-cell receptor immunoglobulin heavy chain complementarity-determining region 3 repertoire in patients with acute rejection after kidney transplantation using high-throughput sequencing.

The aim of the present study was to assess the genetic diversity of the B-cell receptor (BCR) in kidney transplant recipients with acute rejection. A total of three patients with acute rejection after kidney transplantation were examined by performing a composition and diversity analysis of the BCR immunoglobulin heavy chain (IGH) complementarity-determining region 3 (H-CDR3) repertoire. The peripheral blood mononuclear cells of patients were collected at 1 day prior to (Pre1), as well as 1 day (Post1) and 7 days (Post7) after the transplantation, and DNA was extracted. High-throughput sequencing technology was applied to determine the BCR repertoire. Raw sequences in FASTQ format were analyzed with the Basic Local Alignment Search Tool. The diversity of the BCR repertoire was assessed by calculating Shannon entropy, Simpson's diversity index, the Gini coefficient and highly expanded clone distributions. The diversity of the BCR repertoire at Pre1 was greater than that at Post1 or Post7. The diversity of the BCR repertoire was the lowest at Post1 and increased at Post7 but failed to reach the pre-transplantation levels. Patients exhibited the loss of seven IGH variable (IGHV)3 family genes, while five new genes were expressed at a low frequency. Furthermore, five IGHV-IGH joining (IGHJ) gene pairings, including IGHJ6-IGHV3-11, were detected in the patients. Up- and downregulated genes were assessed by calculating the expression frequencies of the IGH diversity and IGHV gene families at Post1 and Post7. The results of the H-CDR3 length distribution and H-CDR3 amino acid (AA) usage analyses indicated that in Case 1 and 2, the AA length was similar at mostly 14-18 AA, while that in Case 3 was relatively stable at 12-16 AA. In conclusion, the present results illustrate the diversity of H-CDR3 in patients with acute rejection after kidney transplantation may provide novel ideas, methods and means of monitoring and analyzing the immune status of patients under physiological and pathological conditions.

Comparative proteomic analysis of human serum before and after liver transplantation using quantitative proteomics.

Liver cancer is the second leading cause of cancer mortality worldwide. Safer and more effective diagnostic methods for liver cancer are desirable, and biomarkers represent a potentially alternative method for diagnosis. The present study was designed to identify liver cancer biomarkers. We quantified the changes in serum protein levels between liver transplantation and healthy (control) females using isobaric tags for relative and absolute quantitation (iTRAQ) as well as proteomic analysis. A total of 1399 proteins were identified; of these, three proteins showed significantly different concentrations between the before transplantation group and the control group. These proteins may thus be relevant to liver cancer and constitute potential liver cancer biomarkers.

T cell repertoire following kidney transplantation revealed by high-throughput sequencing.

Delayed T cell recovery and restricted T cell receptor (TCR) diversity after kidney transplantation are associated with increased risks of infection and malignancy. Technical challenges limit the faithful measurement of TCR diversity after kidney transplantation. In this study, we used a combination of multiplex-PCR, Illumina sequencing and IMGT/HighV-QUEST to directly assess millions of TCRs per individual before and at two time points after kidney transplantation (1days and 7days after transplantation) in a cohort of 10 patients compared to a normal control (NC) group (n=10). We identified the most commonly observed CDR3 length, VD indel length, and DJ indel length in transplantation group and normal group. In addition, we found that the TCR repertoire diversity of transplantation groups was relatively lower compared to NC group. T cell depletion in Post-1 group can be observed, which resulted in the altered distribution characteristics of clonotype abundance. A modest proportion of high abundance clones were shared among the pre-1 group, post-1 group and post-7 group, and it did not exist in the NC group, which exhibited a signature of antigen selection. Moreover, our results also demonstrated that various TRBV expression increased and some public sequences at different time points after kidney transplantation, which may provide biomarkers to monitor the immune status of transplant patients.

Composition and variation analysis of the TCR ß-chain CDR3 repertoire in systemic lupus erythematosus using high-throughput sequencing.

The ability of T lymphocytes to mount an immune response against a diverse array of pathogens is primarily conveyed by the amino acid (aa) sequence of the hypervariable complementarity-determining region 3 (CDR3) segments of the T cell receptor (TCR). In this study, we used a combination of multiplex-PCR, Illumina sequencing and IMGT/HighV-QUEST for a standardized analysis of the characteristics and polymorphisms of the T-cell receptor BV complementarity-determining region 3 (TCR BV CDR3) gene in peripheral blood mononuclear cells (PBMCs) from SLE patients and healthy donors (NC). We found the distributions of CDR3, VD indel, and DJ indel lengths to be comparable between the SLE and NC groups. The degree of clonal expansion in the SLE group was significantly greater than in the NC group, and the expression levels of 10 TRßV segments and 6 TRßJ segments were also significantly different in the SLE group. Regarding public T cell responses, 3CDR3 DNA sequences and 4 aa sequences were shared by all SLE patients and may serve as biomarkers for SLE disease risk, diagnosis and/or prognosis.