Exploring the impact of nonylphenol exposure on Litopenaeus vannamei at the histological and molecular levels.

Nonylphenol (NP) is one of the common pollutants in the environment that have toxic effects on aquatic animals. Nevertheless, little is known about the possible toxicity mechanism of NP on the hepatopancreas of Litopenaeus vannamei. In the present study, the detrimental effects of NP on the hepatopancreas of the L. vannamei were explored at the histological and transcriptomic levels. The findings indicated that after NP exposed for 3, 12, and 48 h, the hepatopancreas histology was changed significantly. Transcriptomic analysis showed that a total of 4302, 3651, and 4830 differentially expressed genes (DEGs) were identified at 3, 12, and 48 h following NP exposure. All these DEGs were classified into 12 clusters according to the expression patterns at different time points. GO and KEGG enrichment analyses of DEGs were also performed, immunological, metabolic, and inflammatory related pathways, including arachidonic acid metabolism (ko00590), the PPAR signaling pathway (ko03320), and the regulation of TRP channels by inflammatory mediators (ko04750) were significantly enriched. Six DEGs were selected for validation by quantitative real-time PCR (qRT-PCR) and the results confirmed the reliability of transcriptome data. All results indicated that NP is toxic to L. vannamei by damaging the histopathological structure and disrupting the biological function. The findings would provide a theoretical framework for lowering or limiting the detrimental impacts of NP on aquaculture and help us to further study the molecular toxicity of NP in crustaceans.

Comparative Transcriptome Analysis of the Hepatopancreas from Macrobrachium rosenbergii Exposed to the Heavy Metal Copper.

The contamination of aquatic ecosystems by the heavy metal copper (Cu) is an important environmental issue and poses significant risks to the physiological functions of aquatic organisms. Macrobrachium rosenbergii is one of the most important freshwater-cultured prawns in the world. The hepatopancreas of crustaceans is a key organ for immune defense, heavy metal accumulation, and detoxification, playing a pivotal role in toxicological research. However, research on the molecular response of the hepatopancreas in M. rosenbergii to Cu exposure is still lacking. In this study, the transcriptomic response in the hepatopancreas of M. rosenbergii was studied after Cu exposure for 3 and 48 h. Compared with the control group, 11,164 (7288 up-regulated and 3876 down-regulated genes) and 10,937 (6630 up-regulated and 4307 down-regulated genes) differentially expressed genes (DEGs) were identified after 3 and 48 h exposure, respectively. Most of these DEGs were up-regulated, implying that gene expressions were largely induced by Cu. Functional enrichment analysis of these DEGs revealed that immunity, copper homeostasis, detoxification, DNA damage repair, and apoptosis were differentially regulated by Cu. Seven genes involved in immunity, detoxification, and metabolism were selected for validation by qRT-PCR, and the results confirmed the reliability of RNA-Seq. All these findings suggest that M. rosenbergii attempts to resist the toxicity of Cu by up-regulating the expression of genes related to immunity, metabolism, and detoxification. However, with the excessive accumulation of reactive oxygen species (ROS), the antioxidant enzyme system was destroyed. As a result, DNA damage repair and the cellular stress response were inhibited, thereby exacerbating cell damage. In order to maintain the normal function of the hepatopancreas, M. rosenbergii removes damaged cells by activating the apoptosis mechanism. Our study not only facilitates an understanding of the molecular response mechanisms of M. rosenbergii underlying Cu toxicity effects but also helps us to identify potential biomarkers associated with the stress response in other crustaceans.

Biochemical, histological and transcriptional response of intestines in Litopenaeus vannamei under chronic zinc exposure.

Zinc (Zn) is an essential trace element for the normal physiological function of aquatic organisms, but it could become toxic to organisms when the concentration increased in water. As the first line of defense, the shrimp intestines are the most susceptible organ to environmental stress. In this study, the chronic toxicity of 0 (control, IC), 0.01(IL), 0.1(IM) and 1 mg/L (IH) Zn in intestines of Litopenaeus vannamei was investigated from the perspectives of biochemical, histological and transcriptional changes after exposure for 30 days. The results showed that the intestinal tissue basement membrane is swollen in the IM and IH groups and detached in the IH group. The total antioxidant capacities (T-AOC) were reduced while the content of malondialdehyde (MDA) were increased significantly in IM and IH groups. The production of reactive oxygen species (ROS) was increased significantly in IH group. Many differentially expressed genes (DEGs) were identified in IL, IM and IH groups, respectively. GO and KEGG enrichment analyses were conducted on the DEGs to obtain the underlying biological processes and pathways. The gene modules related to the sample were identified by weighted gene co-expression network analysis (WGCNA), and genes in modules highly corelated with IH group were mainly enriched in immune related pathways. Nine DEGs were selected for validation by quantitative real time PCR (qRT-PCR) and the expression profiles of these DEGs kept a well consistent with the high-throughput data, which confirmed reliability of transcriptome results. Additionally, 10 DEGs were screened to detect the changes of expression level in different groups. All these results indicated that Zn exposure could damage the intestinal barrier, provoke oxidative stress, reduce the immune function, increase the susceptibility to bacterial infections of L. vannamei and cause inflammation, ultimately result in cell apoptosis. Our study provides more perspective on the stress response of crustacean under Zn exposure.

Gene doping detection in the era of genomics.

Recent progress in gene editing has enabled development of gene therapies for many genetic diseases, but also made gene doping an emerging risk in sports and competitions. By delivery of exogenous transgenes into human body, gene doping not only challenges competition fairness but also places health risk on athletes. World Anti-Doping Agency (WADA) has clearly inhibited the use of gene and cell doping in sports, and many techniques have been developed for gene doping detection. In this review, we will summarize the main tools for gene doping detection at present, highlight the main challenges for current tools, and elaborate future utilizations of high-throughput sequencing for unbiased, sensitive, economic and large-scale gene doping detections. Quantitative real-time PCR assays are the widely used detection methods at present, which are useful for detection of known targets but are vulnerable to codon optimization at exon-exon junction sites of the transgenes. High-throughput sequencing has become a powerful tool for various applications in life and health research, and the era of genomics has made it possible for sensitive and large-scale gene doping detections. Non-biased genomic profiling could efficiently detect new doping targets, and low-input genomics amplification and long-read third-generation sequencing also have application potentials for more efficient and straightforward gene doping detection. By closely monitoring scientific advancements in gene editing and sport genetics, high-throughput sequencing could play a more and more important role in gene detection and hopefully contribute to doping-free sports in the future.

Mutational signature and prognosis in adenocarcinoma of the bladder.

Adenocarcinoma of the bladder is a rare urinary bladder carcinoma with limited therapy options due to lack of molecular characterization. Here, we aimed to reveal the mutational and transcriptomic landscapes of adenocarcinoma of the bladder and assess any relationship with prognosis. Between February 2015 and June 2021, a total of 23 patients with adenocarcinoma of the bladder were enrolled. These included 16 patients with primary bladder adenocarcinomas and seven patients with urachal adenocarcinoma. Whole exome sequencing (16 patients), whole genome sequencing (16 patients), bulk RNA sequencing (RNA-seq) (19 patients), and single-cell RNA-seq (5 patients) were conducted for the specimens. Correlation analysis, survival analysis, and t-tests were also performed. Prevalent T>A substitutions were observed among somatic mutations, and major trinucleotide contexts included 5'-CTC-3' and 5'-CTG-3'. This pattern was mainly contributed by COSMIC signature 22 related to chemical carcinogen exposure (probably aristolochic acid), which has not been reported in bladder adenocarcinoma. Moreover, genes with copy number changes were also enriched in the KEGG term 'chemical carcinogenesis'. Transcriptomic analysis suggested high immune cell infiltration and luminal-like features in the majority of samples. Interestingly, a small fraction of samples with an APOBEC-derived mutational signature exhibited a higher risk of disease progression compared with samples with only a chemical carcinogen-related signature, confirming the molecular and prognostic heterogeneity of bladder adenocarcinoma. This study presents mutational and transcriptomic landscapes of bladder adenocarcinoma, and indicates that a chemical carcinogen-related mutational signature may be related to a better prognosis compared with an APOBEC signature in adenocarcinoma of the bladder. © 2024 The Pathological Society of Great Britain and Ireland.

Heavy metal contamination in Shanghai agricultural soil.

As heavy metals in soil could enrich in biomass and pose health risk to human, it is vital to monitor their contaminations to ensure qualified agricultural production. In this study, we collected >4000 soil samples from agricultural fields in Shanghai during 2010∼2020, and unveiled heavy metal contamination status in this metropolitan. We found that although Shanghai has a long industrialization history, the heavy metal levels in agricultural soil are within safe ranges according to national standard. Specifically, the median levels of Cd, Hg, As, Pb, Cr and Cu are 0.11, 0.13, 7.47, 23.80, 41.00 and 28.30 mg/kg, respectively, which are as good as, or even better than national averages. However, there are spatial and temporal heterogeneities for heavy metal contaminations in Shanghai. For example, the levels of Cd, Hg and Cr are relatively higher in some districts with high industry density, which should be further monitored in the future. Moreover, while the levels for Cd, Cr and Pb have decreased, the level for Hg has mildly increased during this period which needs counteractive measures. Correlation analysis of heavy metal levels and soil fertility parameters suggested overuse of fertilizers may be related to heavy metal contamination in some regions. In summary, our study present by far the largest and most comprehensive landscape of heavy metal contamination in Shanghai agricultural soil, which will be useful for future policy-design and land use planning to ensure safe agricultural production.

scCURE identifies cell types responding to immunotherapy and enables outcome prediction.

A deep understanding of immunotherapy response/resistance mechanisms and a highly reliable therapy response prediction are vital for cancer treatment. Here, we developed scCURE (single-cell RNA sequencing [scRNA-seq] data-based Changed and Unchanged cell Recognition during immunotherapy). Based on Gaussian mixture modeling, Kullback-Leibler (KL) divergence, and mutual nearest-neighbors criteria, scCURE can faithfully discriminate between cells affected or unaffected by immunotherapy intervention. By conducting scCURE analyses in melanoma and breast cancer immunotherapy scRNA-seq data, we found that the baseline profiles of specific CD8+ T and macrophage cells (identified by scCURE) can determine the way in which tumor microenvironment immune cells respond to immunotherapy, e.g., antitumor immunity activation or de-activation; therefore, these cells could be predictive factors for treatment response. In this work, we demonstrated that the immunotherapy-associated cell-cell heterogeneities revealed by scCURE can be utilized to integrate the therapy response mechanism study and prediction model construction.

Single-cell RNA sequencing reveals the heterogeneity and intercellular communication of hepatic stellate cells and macrophages during liver fibrosis.

Uncontrolled and excessive progression of liver fibrosis is thought to be the prevalent pathophysiological cause of liver cirrhosis and hepatocellular cancer, and there are currently no effective antifibrotic therapeutic options available. Intercellular communication and cellular heterogeneity in the liver are involved in the progression of liver fibrosis, but the exact nature of the cellular phenotypic changes and patterns of interregulatory remain unclear. Here, we performed single-cell RNA sequencing on nonparenchymal cells (NPCs) isolated from normal and fibrotic mouse livers. We identified eight main types of cells, including endothelial cells, hepatocytes, dendritic cells, B cells, natural killer/T (NK/T) cells, hepatic stellate cells (HSCs), cholangiocytes and macrophages, and revealed that macrophages and HSCs exhibit the most variance in transcriptional profile. Further analyses of HSCs and macrophage subpopulations and ligand-receptor interaction revealed a high heterogeneity characterization and tightly interregulated network of these two groups of cells in liver fibrosis. Finally, we uncovered a profibrotic Thbs1+ macrophage subcluster, which expands in mouse and human fibrotic livers, activating HSCs via PI3K/AKT/mTOR signaling pathway. Our findings decode unanticipated insights into the heterogeneity of HSCs and macrophages and their intercellular crosstalk at a single-cell level, and may provide potential therapeutic strategies in liver fibrosis.

Heterocyclic Boron Acid Catalyzed Dehydrative Amidation of Aliphatic/Aromatic Carboxylic Acids with Amines.

A commercially available and versatile dehydrative amidation catalyst, featuring a thianthrene boron acid structure, has been developed. The catalyst shows high catalytic activity to both aliphatic and less reactive aromatic carboxylic acid substrates, including several bioactive or clinical molecules with a carboxylic acid group.

Discovery of Novel PTP1B Inhibitors with Once-Weekly Therapeutic Potential for Type 2 Diabetes: Design, Synthesis, and In Vitro and In Vivo Investigations of BimBH3 Peptide Analogues.

Poor medication adherence in patients with type 2 diabetes mellitus has become one of the main causes of suboptimal glycemic control. Once-weekly drugs can markedly improve the convenience, adherence, and quality of life of T2DM patients; thus, they are clinically needed and preferred. PTP1B plays a negative role in both insulin and leptin signaling pathways, which makes it an important target for diabetes. Herein, we design and synthesize 35 analogues of core BimBH3 peptide via lipidation/acylation strategy based on our previous work and evaluate their PTP1B inhibitory activity, obtaining the primary structure-activity relationship. Five compounds with good PPT1B inhibitory activity, target selectivity, and significantly improved stability were selected for molecular docking study and searching candidate molecules with long-acting antidiabetic potential. The in vivo anti-T2DM evaluation validated the once-weekly therapeutic potential of analogues 19, 26, 27, 31, and 33, which were comparable with semaglutide and therefore presented as promising drug candidates.

Solvent-Controlled Regioselective Reaction of 2-Methyleneaziridines with Acrylic/Propargylic Acids: Synthesis of Carboxylate Aziridine/Acetone Esters.

Herein, we report a convenient solvent-controlled regioselective esterification to access two types of carboxylate esters without any additive or non-green activation strategy. In this transformation, 2-methyleneaziridines served as an ester reagent, providing two alternative electrophilic carbon centers. Notably, this protocol is suitable for some structure-complicated clinical molecules with a carboxylic acid group, presenting remarkable application potential.

Accurate tumor clonal structures require single-cell analysis.

Tumor clonal structure is closely related to future progression, which has been mainly investigated as mutation abundance clustering in bulk samples. With relatively limited studies at single-cell resolution, a systematic comparison of the two approaches is still lacking. Here, using bulk and single-cell mutational data from the liver and colorectal cancers, we checked whether co-mutations determined by single-cell analysis had corresponding bulk variant allele frequency (VAF) peaks. While bulk analysis suggested the absence of subclonal peaks and, possibly, neutral evolution in some cases, the single-cell analysis identified coexisting subclones. The overlaps of bulk VAF ranges for co-mutations from different subclones made it difficult to separate them. Complex subclonal structures and dynamic evolution could be hidden under the seemingly clonal neutral pattern at the bulk level, suggesting single-cell analysis is necessary to avoid underestimation of tumor heterogeneity.

Inhibition of Arid1a increases stem/progenitor cell-like properties of liver cancer.

ARID1A, a key subunit of the SWI/SNF chromatin remodeling complex, exhibits recurrent mutations in various types of human cancers, including liver cancer. However, the function of ARID1A in the pathogenesis of liver cancer remains controversial. Here, we demonstrate that Arid1a knockout may result in states of different cell differentiation, as indicated by single-cell RNA sequencing (scRNA-seq) analysis. Bulk RNA-seq also revealed that Arid1a deficiency upregulated these genes related to cell stemness and differentiation, but downregulated genes related to the hepatic functions. Furthermore, we confirmed that deficiency of Arid1a increased the expression of hepatic stem/progenitor cell markers, such as Cd133 and Epcam, and enhanced the self-renewal ability of cells. Mechanistic studies revealed that Arid1a loss remodeled the chromatin accessibility of some genes related to liver functions. Thus, Arid1a deficiency might contribute to cancer development by increasing the number of stem/progenitor-like cells through dysregulating the expression of these genes related to cell stemness, differentiation and liver functions.

Cinobufagin Is a Selective Anti-Cancer Agent against Tumors with EGFR Amplification and PTEN Deletion.

Glioblastoma multiforme (GBM) is the most common and malignant brain tumor, and almost half of the patients carrying EGFR-driven tumor with PTEN deficiency are resistant to EGFR-targeted therapy. EGFR amplification and/or mutation is reported in various epithelial tumors. This series of studies aimed to identify a potent compound against EGFR-driven tumor. We screened a chemical library containing over 600 individual compounds purified from Traditional Chinese Medicine against GBM cells with EGFR amplification and found that cinobufagin, the major active ingredient of Chansu, inhibited the proliferation of EGFR amplified GBM cells and PTEN deficiency enhanced its anti-proliferation effects. Cinobufagin also strongly inhibited the proliferation of carcinoma cell lines with wild-type or mutant EGFR expression. In contrast, the compound only weakly inhibited the proliferation of cancer cells with low or without EGFR expression. Cinobufagin blocked EGFR phosphorylation and its downstream signaling, which additionally induced apoptosis and cytotoxicity in EGFR amplified cancer cells. In vivo, cinobufagin blocked EGFR signaling, inhibited cell proliferation, and elicited apoptosis, thereby suppressing tumor growth in both subcutaneous and intracranial U87MG-EGFR xenograft mouse models and increasing the median survival of nude mice bearing intracranial U87MG-EGFR tumors. Cinobufagin is a potential therapeutic agent for treating malignant glioma and other human cancers expressing EGFR.

Discovery of Novel PTP1B Inhibitors Derived from the BH3 Domain of Proapoptotic Bcl-2 Proteins with Antidiabetic Potency.

BH3 peptide analogues are generally believed to exhibit great potency as cancer therapeutics via targeting antiapoptotic Bcl-2 proteins. Here, we describe the synthesis and identification of a new class of palmitoylated peptide BH3 analogues derived from the core region (h1-h4) of BH3 domains of proapoptotic Bcl-2 proteins and as alternative PTP1B inhibitors with antidiabetic potency in vitro and in vivo. PTP1B inhibitors are attractive for treatment of type 2 diabetes. We design the analogues using a simple lipidation approach and discovered novel lead analogues with promising antidiabetic potency in vitro and in vivo. The results presented here expanded the alternative target and function for the BH3 peptide analogues from one member Bim to other members of the proapoptotic Bcl-2 proteins and emphasize their therapeutic potential in T2DM. Furthermore, our findings may provide new proof of the regulatory function of Bcl-2 family proteins in mitochondrial nutrient and energy metabolism.

Clonal evolution in liver cancer at single-cell and single-variant resolution.

Genetic heterogeneity of tumor is closely related to its clonal evolution, phenotypic diversity and treatment resistance, and such heterogeneity has only been characterized at single-cell sub-chromosomal scale in liver cancer. Here we reconstructed the single-variant resolution clonal evolution in human liver cancer based on single-cell mutational profiles. The results indicated that key genetic events occurred early during tumorigenesis, and an early metastasis followed by independent evolution was observed in primary liver tumor and intrahepatic metastatic portal vein tumor thrombus. By parallel single-cell RNA-Seq, the transcriptomic phenotype of HCC was found to be related with genetic heterogeneity. For the first time we reconstructed the single-cell and single-variant clonal evolution in human liver cancer, and dissection of both genetic and phenotypic heterogeneity will facilitate better understanding of their relationship.

LZP is required for hepatic triacylglycerol transportation through maintaining apolipoprotein B stability.

The conserved zona pellucida (ZP) domain is found in hundreds of extracellular proteins that are expressed in various organs and play a variety of roles as structural components, receptors and tumor suppressors. A liver-specific zona pellucida domain-containing protein (LZP), also named OIT3, has been shown to be mainly expressed in human and mouse hepatocytes; however, the physiological function of LZP in the liver remains unclear. Here, we show that Lzp deletion inhibited very low-density lipoprotein (VLDL) secretion, leading to hepatic TG accumulation and lower serum TG levels in mice. The apolipoprotein B (apoB) levels were significantly decreased in the liver, serum, and VLDL particles of LZP-deficient mice. In the presence of LZP, which is localized to the endoplasmic reticulum (ER) and Golgi apparatus, the ER-associated degradation (ERAD) of apoB was attenuated; in contrast, in the absence of LZP, apoB was ubiquitinated by AMFR, a known E3 ubiquitin ligase specific for apoB, and was subsequently degraded, leading to lower hepatic apoB levels and inhibited VLDL secretion. Interestingly, hepatic LZP levels were elevated in mice challenged with a high-fat diet and humans with simple hepatic steatosis, suggesting that LZP contributes to the physiological regulation of hepatic TG homeostasis. In general, our data establish an essential role for LZP in hepatic TG transportation and VLDL secretion by preventing the AMFR-mediated ubiquitination and degradation of apoB and therefore provide insight into the molecular function of LZP in hepatic lipid metabolism.

A novel neoantigen discovery approach based on chromatin high order conformation.

BACKGROUND: High-throughput sequencing technology has yielded reliable and ultra-fast sequencing for DNA and RNA. For tumor cells of cancer patients, when combining the results of DNA and RNA sequencing, one can identify potential neoantigens that stimulate the immune response of the T cell. However, when the somatic mutations are abundant, it is computationally challenging to efficiently prioritize the identified neoantigen candidates according to their ability of activating the T cell immuno-response. METHODS: Numerous prioritization or prediction approaches have been proposed to address this issue but none of them considers the original DNA loci of the neoantigens from the perspective of 3D genome. Based on our previous discoveries, we propose to investigate the distribution of neoantigens with different immunogenicity abilities in 3D genome and propose to adopt this important information into neoantigen prediction. RESULTS: We retrospect the DNA origins of the immuno-positive and immuno-negative neoantigens in the context of 3D genome and discovered that DNA loci of the immuno-positive neoantigens and immuno-negative neoantigens have very different distribution pattern. Specifically, comparing to the background 3D genome, DNA loci of the immuno-positive neoantigens tend to locate at specific regions in the 3D genome. We thus used this information into neoantigen prediction and demonstrated the effectiveness of this approach. CONCLUSION: We believe that the 3D genome information will help to increase the precision of neoantigen prioritization and discovery and eventually benefit precision and personalized medicine in cancer immunotherapy.

Investigating higher-order interactions in single-cell data with scHOT.

Single-cell genomics has transformed our ability to examine cell fate choice. Examining cells along a computationally ordered 'pseudotime' offers the potential to unpick subtle changes in variability and covariation among key genes. We describe an approach, scHOT-single-cell higher-order testing-which provides a flexible and statistically robust framework for identifying changes in higher-order interactions among genes. scHOT can be applied for cells along a continuous trajectory or across space and accommodates various higher-order measurements including variability or correlation. We demonstrate the use of scHOT by studying coordinated changes in higher-order interactions during embryonic development of the mouse liver. Additionally, scHOT identifies subtle changes in gene-gene correlations across space using spatially resolved transcriptomics data from the mouse olfactory bulb. scHOT meaningfully adds to first-order differential expression testing and provides a framework for interrogating higher-order interactions using single-cell data.

DeepAntigen: a novel method for neoantigen prioritization via 3D genome and deep sparse learning.

MOTIVATION: The mutations of cancers can encode the seeds of their own destruction, in the form of T-cell recognizable immunogenic peptides, also known as neoantigens. It is computationally challenging, however, to accurately prioritize the potential neoantigen candidates according to their ability of activating the T-cell immunoresponse, especially when the somatic mutations are abundant. Although a few neoantigen prioritization methods have been proposed to address this issue, advanced machine learning model that is specifically designed to tackle this problem is still lacking. Moreover, none of the existing methods considers the original DNA loci of the neoantigens in the perspective of 3D genome which may provide key information for inferring neoantigens' immunogenicity. RESULTS: In this study, we discovered that DNA loci of the immunopositive and immunonegative MHC-I neoantigens have distinct spatial distribution patterns across the genome. We therefore used the 3D genome information along with an ensemble pMHC-I coding strategy, and developed a group feature selection-based deep sparse neural network model (DNN-GFS) that is optimized for neoantigen prioritization. DNN-GFS demonstrated increased neoantigen prioritization power comparing to existing sequence-based approaches. We also developed a webserver named deepAntigen (http://yishi.sjtu.edu.cn/deepAntigen) that implements the DNN-GFS as well as other machine learning methods. We believe that this work provides a new perspective toward more accurate neoantigen prediction which eventually contribute to personalized cancer immunotherapy. AVAILABILITY AND IMPLEMENTATION: Data and implementation are available on webserver: http://yishi.sjtu.edu.cn/deepAntigen. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.