Tracking single-cell evolution using clock-like chromatin accessibility loci.

Single-cell chromatin accessibility sequencing (scATAC-seq) reconstructs developmental trajectory by phenotypic similarity. However, inferring the exact developmental trajectory is challenging. Previous studies showed age-associated DNA methylation (DNAm) changes in specific genomic regions, termed clock-like differential methylation loci (ClockDML). Age-associated DNAm could either result from or result in chromatin accessibility changes at ClockDML. As cells undergo mitosis, the heterogeneity of chromatin accessibility on clock-like loci is reduced, providing a measure of mitotic age. In this study, we developed a method, called EpiTrace, that counts the fraction of opened clock-like loci from scATAC-seq data to determine cell age and perform lineage tracing in various cell lineages and animal species. It shows concordance with known developmental hierarchies, correlates well with DNAm-based clocks and is complementary with mutation-based lineage tracing, RNA velocity and stemness predictions. Applying EpiTrace to scATAC-seq data reveals biological insights with clinically relevant implications, ranging from hematopoiesis, organ development, tumor biology and immunity to cortical gyrification.

Integrative Single Cell Atlas Revealed Intratumoral Heterogeneity Generation from an Adaptive Epigenetic Cell State in Human Bladder Urothelial Carcinoma.

Intratumor heterogeneity (ITH) of bladder cancer (BLCA) contributes to therapy resistance and immune evasion affecting clinical prognosis. The molecular and cellular mechanisms contributing to BLCA ITH generation remain elusive. It is found that a TM4SF1-positive cancer subpopulation (TPCS) can generate ITH in BLCA, evidenced by integrative single cell atlas analysis. Extensive profiling of the epigenome and transcriptome of all stages of BLCA revealed their evolutionary trajectories. Distinct ancestor cells gave rise to low-grade noninvasive and high-grade invasive BLCA. Epigenome reprograming led to transcriptional heterogeneity in BLCA. During early oncogenesis, epithelial-to-mesenchymal transition generated TPCS. TPCS has stem-cell-like properties and exhibited transcriptional plasticity, priming the development of transcriptionally heterogeneous descendent cell lineages. Moreover, TPCS prevalence in tumor is associated with advanced stage cancer and poor prognosis. The results of this study suggested that bladder cancer interacts with its environment by acquiring a stem cell-like epigenomic landscape, which might generate ITH without additional genetic diversification.

A NR2E1-interacting peptide of LSD1 inhibits the proliferation of brain tumour initiating cells.

OBJECTIVES: Elimination of brain tumour initiating cells (BTICs) is important for the good prognosis of malignant brain tumour treatment. To develop a novel strategy targeting BTICs, we studied NR2E1(TLX) involved self-renewal mechanism of BTICs and explored the intervention means. MATERIALS AND METHODS: NR2E1 and its interacting protein-LSD1 in BTICs were studied by gene interference combined with cell growth, tumour sphere formation, co-immunoprecipitation and chromatin immunoprecipitation assays. NR2E1 interacting peptide of LSD1 was identified by Amide Hydrogen/Deuterium Exchange and Mass Spectrometry (HDX-MS) and analysed by in vitro functional assays. The in vivo function of the peptide was examined with intracranial mouse model by transplanting patient-derived BTICs. RESULTS: We found NR2E1 recruits LSD1, a lysine demethylase, to demethylate mono- and di-methylated histone 3 Lys4 (H3K4me/me2) at the Pten promoter and repress its expression, thereby promoting BTIC proliferation. Using Amide Hydrogen/Deuterium Exchange and Mass Spectrometry (HDX-MS) method, we identified four LSD1 peptides that may interact with NR2E1. One of the peptides, LSD1-197-211 that locates at the LSD1 SWIRM domain, strongly inhibited BTIC proliferation by promoting Pten expression through interfering NR2E1 and LSD1 function. Furthermore, overexpression of this peptide in human BTICs can inhibit intracranial tumour formation. CONCLUSION: Peptide LSD1-197-211 can repress BTICs by interfering the synergistic function of NR2E1 and LSD1 and may be a promising lead peptide for brain tumour therapy in future.

[Laparoscopic management of persistent Müllerian duct syndrome: A case report and pedigree investigation].

OBJECTIVE: To investigate the clinical characteristics, diagnosis, treatment and etiology of persistent Müllerian duct syndrome (PMDS). METHODS: A 3-year-old boy was diagnosed with PMDS according to the clinical manifestations and the results of ultrasonography, laboratory examinations and earlier surgical examination. We performed genetic tests for the patient and his family members, removed the infantile uterus by laparoscopic wedge hysterectomy, biopsied and descended the bilateral testes, and ligated the bilateral internal rings, followed by a retrospective analysis and review of relevant literature. RESULTS: The operation was successful. Gonad biopsy revealed testis tissue, and PMDS was confirmed by intraoperative findings and related examinations. Good bilateral testicular blood supply was found during the 6-month follow-up after surgery. Medical exome sequencing showed the AMHR2 gene c.1499G > A (p.Cys500Tyr) mutant homozygote (A/A) in the patient and his sister and mutant heterozygote (G/A) in his parents. CONCLUSIONS: Laparoscopy is definitely effective for the treatment of PMDS. In surgery, the infantile uterus should be removed in case of good blood supply to the testis, and so were the bilateral testes if they cannot be descended. The homozygous mutation in the AMHR2 gene c. 1499G > A (p. Cys500Tyr) can lead to male PMDS. Pedigree investigation may provide some evidence for possible fertility in PMDS patients.

COVID-19 immune features revealed by a large-scale single-cell transcriptome atlas.

A dysfunctional immune response in coronavirus disease 2019 (COVID-19) patients is a recurrent theme impacting symptoms and mortality, yet a detailed understanding of pertinent immune cells is not complete. We applied single-cell RNA sequencing to 284 samples from 196 COVID-19 patients and controls and created a comprehensive immune landscape with 1.46 million cells. The large dataset enabled us to identify that different peripheral immune subtype changes are associated with distinct clinical features, including age, sex, severity, and disease stages of COVID-19. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA was found in diverse epithelial and immune cell types, accompanied by dramatic transcriptomic changes within virus-positive cells. Systemic upregulation of S100A8/A9, mainly by megakaryocytes and monocytes in the peripheral blood, may contribute to the cytokine storms frequently observed in severe patients. Our data provide a rich resource for understanding the pathogenesis of and developing effective therapeutic strategies for COVID-19.

A novel germline EGFR variant p.R831H causes predisposition to familial CDK12-mutant prostate cancer with tandem duplicator phenotype.

5-10% of total prostate cancer (PCa) cases are hereditary. Particularly, immune checkpoint inhibitor-sensitive tandem duplicator phenotype (TDP) accounts for 6.9% of PCa cases, whereas genetic susceptibility genes remain completely unknown. We identified a Chinese family with two PCa patients, in which the PCa phenotype co-segregated with a rare germline variant EGFRR831H. Patient-derived conditionally reprogrammed cells (CRC) exhibited increased EGFR and AKT phosphorylation, and a sensitivity to EGFR antagonist Afatinib in migration assays, suggesting the EGFR allele was constitutively active. Both EGFRR831H-mutant tumours contained biallelic CDK12 inactivation, together with prominent tandem duplication across the genome. These somatic mutations could be detected in urine before surgery. Analysis of public databases showed a significant correlation between the mutation status of EGFR and CDK12. Taken together, our genetic and functional analyses identified a previously undescribed link between EGFR and PCa.

HLA class II alleles differing by a single amino acid associate with clinical phenotype and outcome in patients with primary membranous nephropathy.

Genome-wide associations and HLA genotyping have revealed associations between HLA alleles and susceptibility to primary membranous nephropathy. However, associations with clinical phenotypes and kidney outcome are poorly defined. We previously identified DRB1*1501 and DRB1*0301 as independent risk alleles for primary membranous nephropathy. Here, we investigated HLA associations with demographic characteristics, anti-phospholipase A2 receptor (PLA2R) antibody, treatment response and kidney outcome after a median follow-up of 52 months in 258 patients. DRB1*0301, but not DRB1*1501, was associated with a significantly higher level of PLA2R antibody (odds ratio 1.58, 95% confidence interval 1.13-2.22). Although DRB1*1502, which differs from DRB1*1501 by a single amino acid, was not a risk allele for primary membranous nephropathy (odds ratio 1.01), it was associated with significantly lower estimated glomerular filtration rates both at baseline (1.79, 1.18-2.72) and at last follow-up (1.72, 1.17-2.53), a significantly worse renal outcome by Kaplan-Meier analysis and a significantly higher risk of end-stage renal disease by Cox regression analysis (hazard ratio 4.52, 1.22-16.74). Nevertheless, the absence of remission remained the only independent risk factor for end-stage renal disease by multivariate analysis. DRB1*1502 was also associated with a significantly higher median PLA2R antibody level [161.4 vs. 36.3 U/mL] and showed interaction with DRB1*0301 for this variable. Thus, HLA genes control PLA2R antibody production and primary membranous nephropathy severity and outcome. Additionally, DRB1*1502 behaves like a modifier gene with a strong predictor value when associated with HLA risk alleles. Other modifier genes need further investigations in larger cohorts.

CavityPlus: a web server for protein cavity detection with pharmacophore modelling, allosteric site identification and covalent ligand binding ability prediction.

CavityPlus is a web server that offers protein cavity detection and various functional analyses. Using protein three-dimensional structural information as the input, CavityPlus applies CAVITY to detect potential binding sites on the surface of a given protein structure and rank them based on ligandability and druggability scores. These potential binding sites can be further analysed using three submodules, CavPharmer, CorrSite, and CovCys. CavPharmer uses a receptor-based pharmacophore modelling program, Pocket, to automatically extract pharmacophore features within cavities. CorrSite identifies potential allosteric ligand-binding sites based on motion correlation analyses between cavities. CovCys automatically detects druggable cysteine residues, which is especially useful to identify novel binding sites for designing covalent allosteric ligands. Overall, CavityPlus provides an integrated platform for analysing comprehensive properties of protein binding cavities. Such analyses are useful for many aspects of drug design and discovery, including target selection and identification, virtual screening, de novo drug design, and allosteric and covalent-binding drug design. The CavityPlus web server is freely available at http://repharma.pku.edu.cn/cavityplus or http://www.pkumdl.cn/cavityplus.

The critical amino acids of a nephritogenic epitope on human Goodpasture autoantigen for binding to HLA-DRB1*1501.

BACKGROUND: Anti-GBM disease is caused by autoimmunity to Goodpasture antigen on α3(IV)NC1 and had strong associations with HLA-DRB1*1501. Previous studies identified α3127-148 (P14: TDIPPCPHGWISLWKGFSFIMF) as a T cell epitope. The present study was aimed to investigate the binding capacity of P14 to HLA-DRB1*1501 and the critical amino acids for this binding. METHODS: A line of EBV-transformed human B cells homozygous for HLA-DRB1*1501 was used to detect the binding capacity of peptides to HLA-DRB1*1501 using flow cytometry analysis. P14 was sequentially truncated into 8 peptides with 15 amino acids to identify the core binding motif. A set of alanine substituted peptides of P14-2 was then synthesized to identify its critical residues for binding to HLA-DRB1*1501. The structure of HLA-DR2b-Peptide-TCR complex was constructed by modeling to analyze the interaction of each amino acids of P14-2 with the HLA-DR2b molecule. RESULTS: P14 could bind to HLA-DRB1*1501 expressed on B cell surface. The N-terminus of P14 was the core binding motif and the truncated peptide P14-2 (DIPPCPHGWISLWKG) 128-142 had the strongest binding capacity. After sequential amino acid substitution, we found the binding capacity of P14-2 was completely lost by the substitution of cysteine (C) 132 and significantly decreased by the substitution of tryptophan (W) 136, lysine (K) 141, or glycine (G) 142, but still at a high level. The modeling showed that (C) 132 had a strong interaction with pocket 4 on the ß chain of DR2b. Thus, C132, W 136, K141, and G142 were defined as the critical amino acid residues for the binding capacity of P14 to HLA-DRB1*1501. CONCLUSION: We identified α3128-142 (DIPPCPHGWISLWKG) as the core binding motif of P14 to HLA-DRB1*1501 molecule. And the critical amino acid residues for this binding were further defined as C132, W 136, K 141, and G 142.

The susceptible HLA class II alleles and their presenting epitope(s) in Goodpasture's disease.

Goodpasture's disease is closely associated with HLA, particularly DRB1*1501. Other susceptible or protective HLA alleles are not clearly elucidated. The presentation models of epitopes by susceptible HLA alleles are also unclear. We genotyped 140 Chinese patients and 599 controls for four-digit HLA II genes, and extracted the encoding sequences from the IMGT/HLA database. T-cell epitopes of α3(IV)NC1 were predicted and the structures of DR molecule-peptide-T-cell receptor were constructed. We confirmed DRB1*1501 (OR = 4·6, P = 5·7 × 10-28 ) to be a risk allele for Goodpasture's disease. Arginine at position 13 (ARG13) (OR = 4·0, P = 1·0 × 10-17 ) and proline at position 11 (PRO11) (OR = 4·0, P = 2·0 × 10-17 ) on DRß1, encoded by DRB1*1501, were associated with disease susceptibility. α134-148 (HGWISLWKGFSFIMF) was predicted as a T-cell epitope presented by DRB1*1501. Isoleucine137 , tryptophan140 , glycine142 , phenylalanine143 and phenylalanine145 , were presented in peptide-binding pockets 1, 4, 6, 7 and 9 of DR2b, respectively. ARG13 in pocket 4 interacts with tryptophan140 and forms a hydrogen bond. In conclusion, we propose a mechanism for DRB1*1501 susceptibility for Goodpasture's disease through encoding ARG13 and PRO11 on MHC-DRß1 chain and presenting T-cell epitope, α134-148 , with five critical residues.

MHC Class II Risk Alleles and Amino Acid Residues in Idiopathic Membranous Nephropathy.

Epitopes of phospholipase A2 receptor (PLA2R), the target antigen in idiopathic membranous nephropathy (iMN), must be presented by the HLA-encoded MHC class II molecules to stimulate autoantibody production. A genome-wide association study identified risk alleles at HLA and PLA2R loci, with the top variant rs2187668 within HLA-DQA1 showing a risk effect greater than that of the top variant rs4664308 within PLA2R1. How the HLA risk alleles affect epitope presentation by MHC class II molecules in iMN is unknown. Here, we genotyped 261 patients with iMN and 599 healthy controls at the HLA-DRB1, HLA-DQA1, HLA-DQB1, and HLA-DPB1 loci with four-digit resolution and extracted the encoded amino acid sequences from the IMGT/HLA database. We predicted T cell epitopes of PLA2R and constructed MHC-DR molecule-PLA2R peptide-T cell receptor structures using Modeler. We identified DRB1*1501 (odds ratio, 4.65; 95% confidence interval [95% CI], 3.39 to 6.41; P<0.001) and DRB1*0301 (odds ratio, 3.96; 95% CI, 2.61 to 6.05; P<0.001) as independent risk alleles for iMN and associated with circulating anti-PLA2R antibodies. Strong gene-gene interaction was noted between rs4664308(AA) and HLA-DRB1*1501/DRB1*0301. Amino acid positions 13 (P<0.001) and 71 (P<0.001) in the MHC-DRß1 chain independently associated with iMN. Structural models showed that arginine13 and alanine71, encoded by DRB1*1501, and lysine71, encoded by DRB1*0301, facilitate interactions with T cell epitopes of PLA2R. In conclusion, we identified two risk alleles of HLA class II genes and three amino acid residues on positions 13 and 71 of the MHC-DRß1 chain that may confer susceptibility to iMN by presenting T cell epitopes on PLA2R.

Designed inhibitor for nuclear localization signal of polo-like kinase 1 induces mitotic arrest.

Polo-like kinase 1 (Plk1), a member of polo-like kinase family, regulates multiple essential steps of the cell cycle progression. Plk1 is overexpressed in multiple cancer cell lines and considered to be a prime anticancer target. Plk1 accumulates in the nucleus during S and G2 phases by its bipartite nuclear localization signal (NLS) sequence, which is crucial for Plk1 regulation during normal cell cycle progression. Here, through combined computational and experimental studies, we identified compound D110, which inhibits Plk1 kinase activity with an IC50 of 85 nm and blocks the nuclear localization of Plk1 during S and G2 phases. D110-treated cancer cells were arrested at mitosis with monopolar spindle, indicating the inhibition of the Plk1 kinase activity in cell. As D110 interacts with both the ATP site and the NLS in Plk1, it demonstrates good selectivity toward Plk2 and Plk3. The strategy of simultaneously inhibiting kinase activity and its subcellular translocations offers a novel approach for selective kinase inhibitor design.

Discovery of Novel Polo-Like Kinase 1 Polo-Box Domain Inhibitors to Induce Mitotic Arrest in Tumor Cells.

Polo-like kinase 1(Plk1) is vital for cell mitosis and has been identified as anticancer target. Its polo-box domain (PBD) mediates substrate binding, blocking of which may offer selective Plk1 inhibition compared to kinase domain inhibitors. Although several PBD inhibitors were reported, most of them suffer from low cell activity. Here, we report the discovery of novel inhibitors to induce mitotic arrest in HeLa cells by virtual screening with Plk1 PBD and cellular activity testing. Of the 81 compounds tested in the cell assay, 10 molecules with diverse chemical scaffolds are potent to induce mitotic arrest of HeLa at low micromolar concentrations. The best compound induces mitotic arrest of HeLa cells with an EC50 of 4.4 µM. The cellular active inhibitors showed binding to Plk1 PBD and compete with PBD substrate in microscale thermophoresis analysis.

Different Effects of p52SHC1 and p52SHC3 on the Cell Cycle of Neurons and Neural Stem Cells.

SHC3 is exclusively expressed in postmitotic neurons, while SHC1 is found in neural stem cells and neural precursor cells but absent in mature neurons. In this study, we discovered that suppression of p52SHC1 expression by RNA interference resulted in proliferation defects in neural stem cells, along with significantly reduced protein levels of cyclin E and cyclin A. At the same time, p52SHC3 RNAi caused cell cycle re-entry (9.54% in S phase and 5.70% in G2-M phase) in primary neurons with significantly up-regulated expression of cyclin D1, cyclin E, cyclin A, CDK2, and phosphorylated CDK2. When p52SHC3 was overexpressed, the cell cycle of neural stem cells was arrested with reduced protein levels of cyclin D1, cyclin E, and cyclin A, while overexpression of p52SHC1 did not result in significant changes in postmitotic neurons. Our results indicate that p52SHC3 plays an important role in maintaining the mitotic quiescence of neurons, while p52SHC1 regulates the proliferation of neural stem cells.

Deep Learning for Drug-Induced Liver Injury.

Drug-induced liver injury (DILI) has been the single most frequent cause of safety-related drug marketing withdrawals for the past 50 years. Recently, deep learning (DL) has been successfully applied in many fields due to its exceptional and automatic learning ability. In this study, DILI prediction models were developed using DL architectures, and the best model trained on 475 drugs predicted an external validation set of 198 drugs with an accuracy of 86.9%, sensitivity of 82.5%, specificity of 92.9%, and area under the curve of 0.955, which is better than the performance of previously described DILI prediction models. Furthermore, with deep analysis, we also identified important molecular features that are related to DILI. Such DL models could improve the prediction of DILI risk in humans. The DL DILI prediction models are freely available at http://www.repharma.cn/DILIserver/DILI_home.php.

Molecular dynamic analysis of mutant Y195I α-cyclodextrin glycosyltransferase with switched product specificity from α-cyclodextrin to γ-cyclodextrin.

Alpha-cyclodextrin (α-CD) glycosyltransferase (α-CGTase) can convert starch into α-CD blended with various proportions of ß-cyclodextrin (ß-CD) and/or γ-cyclodextrin (γ-CD). In this study, we verified the catalytic characteristics of purified Y195I α-CGTase and elucidated the mechanism of action with molecular dynamic (MD) simulations. We found that purified Y195I α-CGTase produced less α-CD, slightly more ß-CD, and significantly more γ-CD than wild-type α-CGTase. Correspondingly, α-CD-based K m values increased, and ß-CD- and γ-CD-based K m values decreased. MD simulation studies revealed that the dynamic trajectories of the substrate oligosaccharide chain in the mutant CGTase binding site were significantly different from those in the wild-type enzyme, with reduced hydrophobic interaction, finally resulting in different product specificity and more γ-CD formation.

Discovery of novel secretory phospholipase A2 inhibitors using virtual screen.

Human non-pancreatic secretory phospholipase A2 was reported to be associated with inflammatory diseases and considered as a potential drug target for inflammation and other related disease treatment. Although many human non-pancreatic secretory phospholipase A2 inhibitors were reported, few entered into the drug development stage due to various problems. In this study, we discovered seven novel human non-pancreatic secretory phospholipase A2 inhibitors using virtual screen. Of the 99 compounds tested by continuous fluorescence assay, seven are potent human non-pancreatic secretory phospholipase A2 inhibitors with micromolar IC50 values. Typical molecules include 9-fluorenylmethoxycarbonyl protected α-phenylalanine derivatives and azo compounds, which may serve as novel scaffold for developing potent human non-pancreatic secretory phospholipase A2 inhibitors. These compounds bind to human non-pancreatic secretory phospholipase A2 by interacting with the catalytic calcium ion and the hydrophobic regions in the substrate-binding pocket.

14-3-3ζ interacts with stat3 and regulates its constitutive activation in multiple myeloma cells.

The 14-3-3 proteins are a family of regulatory signaling molecules that interact with other proteins in a phosphorylation-dependent manner and function as adapter or scaffold proteins in signal transduction pathways. One family member, 14-3-3ζ, is believed to function in cell signaling, cycle control, and apoptotic death. A systematic proteomic analysis done in our laboratory has identified signal transducers and activators of transcription 3 (Stat3) as a novel 14-3-3ζ interacting protein. Following our initial finding, in this study, we provide evidence that 14-3-3ζ interacts physically with Stat3. We further demonstrate that phosphorylation of Stat3 at Ser727 is vital for 14-3-3ζ interaction and mutation of Ser727 to Alanine abolished 14-3-3ζ/Stat3 association. Inhibition of 14-3-3ζ protein expression in U266 cells inhibited Stat3 Ser727 phosphorylation and nuclear translocation, and decreased both Stat3 DNA binding and transcriptional activity. Moreover, 14-3-3ζ is involved in the regulation of protein kinase C (PKC) activity and 14-3-3ζ binding to Stat3 protects Ser727 dephosphorylation from protein phosphatase 2A (PP2A). Taken together, our findings support the model that multiple signaling events impinge on Stat3 and that 14-3-3ζ serves as an essential coordinator for different pathways to regulate Stat3 activation and function in MM cells.

A single-molecule study of the inhibition effect of Naringenin on transforming growth factor-ß ligand-receptor binding.

With single-molecule fluorescence imaging and single-molecule force measurement, we have found that the natural compound Naringenin exerts an inhibition effect on TGF-ß ligand-receptor interaction, the initial step of TGF-ß signaling.