The rapid proximity labeling system PhastID identifies ATP6AP1 as an unconventional GEF for Rheb.

Rheb is a small G protein that functions as the direct activator of the mechanistic target of rapamycin complex 1 (mTORC1) to coordinate signaling cascades in response to nutrients and growth factors. Despite extensive studies, the guanine nucleotide exchange factor (GEF) that directly activates Rheb remains unclear, at least in part due to the dynamic and transient nature of protein-protein interactions (PPIs) that are the hallmarks of signal transduction. Here, we report the development of a rapid and robust proximity labeling system named Pyrococcus horikoshii biotin protein ligase (PhBPL)-assisted biotin identification (PhastID) and detail the insulin-stimulated changes in Rheb-proximity protein networks that were identified using PhastID. In particular, we found that the lysosomal V-ATPase subunit ATP6AP1 could dynamically interact with Rheb. ATP6AP1 could directly bind to Rheb through its last 12 amino acids and utilizes a tri-aspartate motif in its highly conserved C-tail to enhance Rheb GTP loading. In fact, targeting the ATP6AP1 C-tail could block Rheb activation and inhibit cancer cell proliferation and migration. Our findings highlight the versatility of PhastID in mapping transient PPIs in live cells, reveal ATP6AP1's role as an unconventional GEF for Rheb, and underscore the importance of ATP6AP1 in integrating mTORC1 activation signals through Rheb, filling in the missing link in Rheb/mTORC1 activation.

A Comprehensive Pan-Cancer Analysis of the Potential Biological Functions and Prognosis Values of RICTOR.

The importance of the network defined by phosphatidylinositol-3-kinase (PI3K), AKT and mammalian target of rapamycin (mTOR) downstream of Receptor Tyrosine Kinase (RTK) has been recognized for many years. However, the central role of RICTOR (rapamycin-insensitive companion of mTOR) in this pathway has only recently come to light. The function of RICTOR in pan-cancer still needs to be systematically elucidated. In this study, we examined RICTOR's molecular characteristics and clinical prognostic value by pan-cancer analysis. Our findings indicate that RICTOR was overexpressed in twelve cancer types, and a high RICTOR expression was linked to poor overall survival. Moreover, the CRISPR Achilles' knockout analysis revealed that RICTOR was a critical gene for the survival of many tumor cells. Function analysis revealed that RICTOR-related genes were mainly involved in TOR signaling and cell growth. We further demonstrated that the RICTOR expression was significantly influenced by genetic alteration and DNA-methylation in multiple cancer types. Additionally, we found a positive relationship between RICTOR expression and the immune infiltration of macrophages and cancer-associated fibroblasts in Colon adenocarcinoma and Head and Neck squamous cell carcinoma. Finally, we validated the ability of RICTOR in sustaining tumor growth and invasion in the Hela cell line using cell-cycle analysis, the cell proliferation assay, and wound-healing assay. Our pan-cancer analysis highlights the critical role of RICTOR in tumor progression and its potential as a prognostic marker for various cancer types.

Comprehensive RNA-Seq Analysis Pipeline for Non-Model Organisms and Its Application in Schmidtea mediterranea.

RNA sequencing (RNA-seq) is a high-throughput technology that provides in-depth information on transcriptome. The advancement and dropping costs of RNA sequencing, accompanied by more available reference genomes for different species, make transcriptome analysis in non-model organisms possible. Current obstacles in analyzing RNA-seq data include a lack of functional annotation, which may complicate the process of linking genes to corresponding functions. Here, we provide a one-stop RNA-seq analysis pipeline, PipeOne-NM, for transcriptome functional annotation, non-coding RNA identification, and transcripts alternative splicing analysis of non-model organisms, intended for use with Illumina platform-based RNA-seq data. We performed PipeOne-NM on 237 Schmidtea mediterranea RNA-seq runs and assembled a transcriptome with 84,827 sequences from 49,320 genes, identifying 64,582 mRNA from 35,485 genes, 20,217 lncRNA from 17,084 genes, and 3481 circRNAs from 1103 genes. In addition, we performed a co-expression analysis of lncRNA and mRNA and identified that 1319 lncRNA co-express with at least one mRNA. Further analysis of samples from S. mediterranea sexual and asexual strains revealed the role of sexual reproduction in gene expression profiles. Samples from different parts of asexual S. mediterranea revealed that differential expression profiles of different body parts correlated with the function of conduction of nerve impulses. In conclusion, PipeOne-NM has the potential to provide comprehensive transcriptome information for non-model organisms on a single platform.

CRISPR-assisted transcription activation by phase-separation proteins.

The clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 system has been widely used for genome engineering and transcriptional regulation in many different organisms. Current CRISPR-activation (CRISPRa) platforms often require multiple components because of inefficient transcriptional activation. Here, we fused different phase-separation proteins to dCas9-VPR (dCas9-VP64-P65-RTA) and observed robust increases in transcriptional activation efficiency. Notably, human NUP98 (nucleoporin 98) and FUS (fused in sarcoma) IDR domains were best at enhancing dCas9-VPR activity, with dCas9-VPR-FUS IDR (VPRF) outperforming the other CRISPRa systems tested in this study in both activation efficiency and system simplicity. dCas9-VPRF overcomes the target strand bias and widens gRNA designing windows without affecting the off-target effect of dCas9-VPR. These findings demonstrate the feasibility of using phase-separation proteins to assist in the regulation of gene expression and support the broad appeal of the dCas9-VPRF system in basic and clinical applications.

Transplantation of Wharton's jelly mesenchymal stem cells encapsulated with Hydroactive® Gel promotes diabetic wound antifibrotic healing in type 2 diabetic rats.

Diabetic cutaneous ulcers (DCU) are a complication for diabetes patients, mostly occurring in the foot and causing non-healing diabetic foot ulcers. Mesenchymal stem cell (MSC)-based therapy is currently being investigated as a therapeutic avenue for chronic diabetic ulcers. However, poor engraftment, short retention, and low survival still limit the treatment effectiveness. Hydroactive® Gel is a sterile transparent gel made of natural hydrocolloid, which has been widely used for wound management. Whether transplantation of Wharton's jelly mesenchymal stem cells (WJMSCs) encapsulated with Hydroactive® Gel is helpful to diabetic ulcers wound healing remains to be explored. The biocompatibility experiments showed that WJMSCs embedded in Hydroactive® Gel did not influence the cell viability, survival, proliferation, and apoptosis of WJMSCs in vitro. RNA-seq results also implied that Hydroactive® Gel + WJMSCs transplantation activated the "cytokine-cytokine receptor interaction", "mononuclear cell differentiation", "regulation of cell-cell adhesion", and "chemokine receptor activity" to accelerate the inflammatory reaction and epidermis regeneration in diabetic wounds. Histological analysis results demonstrated that Hydroactive® Gel encapsulated WJMSCs transplantation promoted diabetic wound healing and regeneration, indicating improved dermis regeneration, sebaceous gland formation, and type III collagen fiber deposition. Besides, immunohistochemical analysis results showed that Hydroactive® Gel + WJMSCs transplantation also facilitated the transformation of pro-inflammatory M1 macrophages to anti-inflammatory M2 macrophages, cell proliferation, and neovascularization at the wound site. Hydroactive® Gel encapsulation further prolonged the retention time of WJMSCs at the diabetic wound site. Above all, Hydroactive® Gel accelerates WJMSCs-mediated diabetic wound healing by promoting macrophage transformation, facilitating cell proliferation and angiogenesis, and prolonging cell retention time. Our findings may potentially provide a useful therapeutic strategy based on the combination of WJMSCs and biomedical materials for patients with diabetic cutaneous ulcers.

APOBEC Alteration Contributes to Tumor Growth and Immune Escape in Pan-Cancer.

The accumulating evidence demonstrates that the apolipoprotein B mRNA editing enzyme catalytic polypeptide-like (APOBEC), DNA-editing protein plays an important role in the molecular pathogenesis of cancer. In particular, the APOBEC3 family was shown to induce tumor mutations by an aberrant DNA editing mechanism. However, knowledge regarding the reconstitution of the APOBEC family genes across cancer types is still lacking. Here, we systematically analyzed the molecular alterations, immuno-oncological features, and clinical relevance of the APOBEC family in pan-cancer. We found that APOBEC genes were widely and significantly differentially expressed between normal and cancer samples in 16 cancer types, and that their expression levels are significantly correlated with the prognostic value in 17 cancer types. Moreover, two patterns of APOBEC-mediated stratification with distinct immune characteristics were identified in different cancer types, respectively. In ACC, for example, the first pattern of APOBEC-mediated stratification was closely correlated with the phenotype of immune activation, which was characterized by a high immune score, increased infiltration of CD8 T cells, and higher survival. The other pattern of APOBEC-mediated stratification was closely correlated with the low-infiltration immune phenotype, which was characterized by a low immune score, lack of effective immune infiltration, and poorer survival. Further, we found the APOBEC-mediated pattern with low-infiltration immune was also highly associated with the advanced tumor subtype and the CIMP-high tumor subtype (CpG island hypermethylation). Patients with the APOBEC-mediated pattern with immune activation were more likely to have therapeutic advantages in ICB (immunological checkpoint blockade) treatment. Overall, our results provide a valuable resource that will be useful in guiding oncologic and therapeutic analyses of the role of APOBEC family in cancer.

FPIA: A database for gene fusion profiling and interactive analyses.

As one of the hallmarks of cancer, gene fusions play an important role in tumorigenesis, and have been established as biomarkers and therapeutic targets. Although recent years have witnessed the development of gene fusion databases, a tool with interactive analytic functions remains lacking. Here, we introduce fusion profiling interactive analysis (FPIA), a web server to perform interactive and customizable analysis on fusion genes. With this platform, researchers can easily explore fusion-associated biological and molecular differences including gene expression, tumor purity and ploidy, mutation, copy number variations, protein expression, immune cell infiltration, stemness, telomere length, microsatellite instability, survival and novel peptides based on 33 cancer types from The Cancer Genome Atlas (TCGA) data. Currently, it contains 31 633 fusion events from 6910 patients. FPIA complements the existing gene fusion annotation databases with its multiomics analytic capacity, integrated analysis features, customized analysis selection and user-friendly design. The comprehensive data analyses by FPIA will greatly facilitate data mining, hypothesis generation and therapeutic target discovery. FPIA is available at http://bioinfo-sysu.com/fpia.

High prevalence of vitamin D deficiency in Shenzhen pregnant women.

BACKGROUND: Vitamin D deficiency is a public health problem worldwide. Vitamin D deficiency in pregnant women often leads to negative clinical consequence and has been distributed differently in certain latitudes. Here, we aimed to determine the prevalence of vitamin D deficiency in pregnant women in Shenzhen City and investigate the influencing factors. METHODS: A total of 27,166 healthy pregnant women, undergoing prenatal examinations in our hospital between July 2014 and December 2018, were enrolled in our study. Maternal characteristics, including the duration of pregnancy, age and enrollment time, were recorded. The concentrations of serum 25(OH)D in the blood samples were detected by immunochemistry assays. RESULTS: For the total study population, the median serum 25(OH)D concentration was 23.36 [17.98-29.51] ng/mL, and 34.3% and 42.4% of the participants exhibited vitamin D deficiency (serum 25(OH) D < 20 ng/mL) and insufficiency (serum 25(OH)D 21-29 ng/mL), respectively. Vitamin D deficiency decreased with gestation (37.83%, 33.8%, and 29.3% for the first trimester, second trimester and third trimester, respectively, p < .001) and decreased by age (36.03%, 35.20%, 31.86% and 29.83%, for the age groups 18-24, 25-29, 30-34 and 35-46 years, respectively, p < .001). This prevalence had conspicuous seasonality (winter vs. autumn, OR 3.69, 95% CI: 3.42-3.99, p < .001). Temperature was positively associated with women's serum 25(OH)D level (r = 0.48, p < .001). CONCLUSIONS: Overall, we demonstrated that vitamin D deficiency in pregnant women in Shenzhen was common and was affected by gestation, age and season/temperature.

Comprehensive Analysis of Large-Scale Transcriptomes from Multiple Cancer Types.

Various abnormalities of transcriptional regulation revealed by RNA sequencing (RNA-seq) have been reported in cancers. However, strategies to integrate multi-modal information from RNA-seq, which would help uncover more disease mechanisms, are still limited. Here, we present PipeOne, a cross-platform one-stop analysis workflow for large-scale transcriptome data. It was developed based on Nextflow, a reproducible workflow management system. PipeOne is composed of three modules, data processing and feature matrices construction, disease feature prioritization, and disease subtyping. It first integrates eight different tools to extract different information from RNA-seq data, and then used random forest algorithm to study and stratify patients according to evidences from multiple-modal information. Its application in five cancers (colon, liver, kidney, stomach, or thyroid; total samples n = 2024) identified various dysregulated key features (such as PVT1 expression and ABI3BP alternative splicing) and pathways (especially liver and kidney dysfunction) shared by multiple cancers. Furthermore, we demonstrated clinically-relevant patient subtypes in four of five cancers, with most subtypes characterized by distinct driver somatic mutations, such as TP53, TTN, BRAF, HRAS, MET, KMT2D, and KMT2C mutations. Importantly, these subtyping results were frequently contributed by dysregulated biological processes, such as ribosome biogenesis, RNA binding, and mitochondria functions. PipeOne is efficient and accurate in studying different cancer types to reveal the specificity and cross-cancer contributing factors of each cancer.It could be easily applied to other diseases and is available at GitHub.

Identification of differentially expressed circulating exosomal lncRNAs in IgA nephropathy patients.

BACKGROUND: Although immunoglobulin A nephropathy (IgAN) is one of the foremost primary glomerular disease, treatment of IgAN is still in infancy. Non-invasive biomarkers are urgently needed for IgAN diagnosis. We investigate the difference in expression profiles of exosomal long non-coding-RNAs (lncRNAs) in plasma from IgAN patients compared with their healthy first-degree relatives, which may reveal novel non-invasive IgAN biomarkers. METHODS: We isolated exosomes from the plasma of both IgAN patients and their healthy first-degree relatives. High-throughput RNA sequencing and real-time quantitative polymerase chain reaction (qRT-PCR) was used to validate lncRNA expression profiles. Pathway enrichment analysis was used to predict their nearest protein-coding genes. RESULTS: lncRNA-G21551 was significantly down-regulated in IgAN patients. Interestingly, the nearest protein-coding gene of lncRNA-G21551 was found to be encoding the low affinity receptor of the Fc segment of immunoglobulin G (FCGR3B). CONCLUSIONS: Exosomal lncRNA-G21551, with FCGR3B as the nearest protein-coding gene, was down-regulated in IgAN patients, indicating its potential to serve as a non-invasive biomarker for IgAN.