N6-Methyladenine DNA Modification in the Human Genome.

DNA N6-methyladenine (6mA) modification is the most prevalent DNA modification in prokaryotes, but whether it exists in human cells and whether it plays a role in human diseases remain enigmatic. Here, we showed that 6mA is extensively present in the human genome, and we cataloged 881,240 6mA sites accounting for ∼0.051% of the total adenines. [G/C]AGG[C/T] was the most significantly associated motif with 6mA modification. 6mA sites were enriched in the coding regions and mark actively transcribed genes in human cells. DNA 6mA and N6-demethyladenine modification in the human genome were mediated by methyltransferase N6AMT1 and demethylase ALKBH1, respectively. The abundance of 6mA was significantly lower in cancers, accompanied by decreased N6AMT1 and increased ALKBH1 levels, and downregulation of 6mA modification levels promoted tumorigenesis. Collectively, our results demonstrate that DNA 6mA modification is extensively present in human cells and the decrease of genomic DNA 6mA promotes human tumorigenesis.

High Contrast Bioimaging of Tumor and Inflammation with a Bicyclic Dioxetane Chemiluminescent Probe.

The link between inflammation and the evolution of cancer is well established. Visualizing and tracking both tumor proliferation and the associated inflammatory response within a living organism are vital for dissecting the nexus between these two processes and for crafting precise treatment modalities. We report the creation and synthesis of an advanced NIR chemiluminescence probe that stands out for its exceptional selectivity, extraordinary sensitivity at nanomolar concentrations, swift detection capabilities, and broad application prospects. Crucially, this probe has been successfully utilized to image endogenous ONOO- across different inflammation models, including abdominal inflammation triggered by LPS, subcutaneous inflammatory conditions, and tumors grafted onto mice. These findings highlight the significant promise of chemiluminescence imaging in enhancing our grasp of the intricate interplay between cancer and inflammation and in steering the development of potent, targeted therapeutic strategies.

CD56bright NK cell expansion correlated with EBV reactivation control post allogeneic hematopoietic stem cell transplantation.

Natural killer (NK) cells are equipped with anti-Epstein-Barr virus (EBV) function, however, whether EBV infection will affect NK cells reconstitution after allogeneic hematopoietic stem cell transplantation (allo-HSCT) remains unclear. To identify the characteristics of NK cells, we prospectively enrolled 11 patients who occurred EBV reactivation post allo-HSCT and 11 patients without EBV infection as control. We found that that EBV infection induced the expansion of CD56bright and NKG2A+KIR- NK subsets,and decreased the cytotoxicity function of NK cells. The frequency of NKG2A+KIR- NK cells were higher in patients who progressed into post-transplant lymphoproliferative disorder (PTLD) than EBV viremia patients, which also correlated with decreased proliferation and cytotoxic function. By screening the activation receptors of NK cells, we found the DNAM-1+CD56bright NK cells is significantly increased after EBV stimulation, further we demonstrated that DNAM-1 is essential for EBV induced NK cells activation as the cytokine release against EBV-transformed lymphoblastoid cell lines(EBV-LCLs) of CD56bright NK cells were significantly decreased after DNAM-1 blockade. NK cells infusion suppressed the progression of EBV-related tumor mice model. A prospective cohort indicated that old donor age was an independent risk factor for EBV infection. Rapid CD56bri expansion and high expression of DNAM-1 on CD56bri NK cells in response to EBV reactivation correlated with rapid EBV clearance post allo-HSCT in patients with younger donors. In summary, our data showed that high expression of DNAM-1 receptors on NK cell may participate protective CD56bri NK cells response to EBV infection after allo-HSCT.

Functional Competence of NK Cells via the KIR/MHC Class I Interaction Correlates with DNAM-1 Expression.

The interaction of inhibitory receptors with self-MHC class I (MHC-I) molecules is responsible for NK cell education. The intensity of DNAM-1 expression correlates with NK cell education. However, whether DNAM-1 expression directly influences the functional competence of NK cells via the KIR/MHC-I interaction remains unclear. Based on allogeneic haploidentical hematopoietic stem cell transplantation, we investigated the intensity of DNAM-1 expression on reconstituted NK cells via the interaction of KIR with both donor HLA and recipient HLA at days 30, 90, and 180 after hematopoietic stem cell transplantation. The reconstituted NK cells educated by donor and recipient HLA molecules showed the highest DNAM-1 expression, whereas DNAM-1 expression on educated NK cells with only recipient HLA molecules was higher than that on educated NK cells with only donor HLA molecules, indicating that NK cells with donor or recipient HLA molecules regulate DNAM-1 expression and thereby affect NK cell education. Additionally, the effects of recipient cells on NK cell education were greater than those of donor cells. However, only when the DNAM-1, NKP30, and NKG2D receptors were blocked simultaneously was the function of educated and uneducated NK cells similar. Therefore, activating receptors may collaborate with DNAM-1 to induce educated NK cell hyperresponsiveness. Our data, based on in vitro and in vivo studies, demonstrate that the functional competence of NK cells via the KIR/MHC-I interaction correlates with DNAM-1 expression in human NK cells.

CFVisual: an interactive desktop platform for drawing gene structure and protein architecture.

BACKGROUND: When researchers perform gene family analysis, they often analyze the structural characteristics of the gene, such as the distribution of introns and exons. At the same time, characteristic structural analysis of amino acid sequence is also essential, for example, motif and domain features. Researchers often integrate these analyses into one image to dig out more information, but the tools responsible for this integration are lacking. RESULTS: Here, we developed a tool (CFVisual) for drawing gene structure and protein architecture. CFVisual can draw the phylogenetic tree, gene structure, and protein architecture in one picture, and has rich interactive capabilities, which can meet the work needs of researchers. Furthermore, it also supports arbitrary stitching of the above analysis images. It has become a useful helper in gene family analysis. The CFVisual package was implemented in Python and is freely available from https://github.com/ChenHuilong1223/CFVisual/ . CONCLUSION: CFVisual has been used by some researchers and cited by some articles. In the future, CFVisual will continue to serve as a good helper for researchers in the study of gene structure and protein architecture.

Donor activating killer cell immunoglobulin-like receptors genes correlated with Epstein-Barr virus reactivation after haploidentical haematopoietic stem cell transplantation.

Natural killer (NK) cells exert anti-viral effects after haematopoietic stem cell transplantation (HSCT). The balance between inhibition and activation of NK cells determined by the inherited repertoire of killer cell immunoglobulin-like receptors (KIR) genes may influence Epstein-Barr virus (EBV) reactivation after transplantation. To evaluate the relative contributions of KIR genotypes to EBV reactivation, we prospectively enrolled 300 patients with malignant haematological disease who were suitable for haploidentical HSCT. Univariate analysis showed that donors with KIR2DS1, KIR2DS3 or KIR3DS1 genes were associated with an increased risk of EBV reactivation [hazard ratio (HR) 1·86, 95% confidence interval (CI) 1·19-2·9, P = 0·0067; HR 1·78, 95% CI 1·07-2·97, P = 0·027; HR 1·86, 95% CI 1·19-2·91, P = 0·0065 respectively]. Multivariate analysis revealed that the presence of KIR2DS1, KIR2DS3 or KIR3DS1 genes was associated with increased EBV reactivation after HSCT. This effect was more evident in the absence of the cognate ligands for the corresponding activating receptors. Our present data firstly showed that donors with activating KIR genes, specifically activating KIR2DS1, KIR2DS3 and KIR3DS1, had an increased risk of EBV reactivation. Precaution for patients whose donors carry activating genes will help prevent EBV reactivation and improve patient prognosis after HSCT.

Dioscin exhibits protective effects on in vivo and in vitro asthma models via suppressing TGF-ß1/Smad2/3 and AKT pathways.

Dioscin is a natural product that possesses protective effects on multiple chronic injuries, but its effects on asthma are not fully understood. Herein, we evaluated its effects on asthmatic mice established by ovalbumin (OVA) sensitization and challenges and further explored the mechanism. Inflammatory cells in bronchoalveolar lavage fluids (BALFs) were analyzed using Diff-Quik staining. OVA-specific immunoglobulin E (IgE)/IgG1 in serum and inflammatory cytokines (interleukin 4[IL-4], IL-5, IL-13, and tumor necrosis factor-α) in BALFs and lung tissues were measured using Enzyme-Linked Immunosorbent Assay Kits. Hematoxylin and eosin, periodic acid-Schiff, and immunohistochemistry staining showed histopathological changes in lung tissues. Epithelial-mesenchymal transition (EMT) in human bronchial epithelial (16HBE) cells was assessed by immunofluorescence staining. Hydroxyproline content was used to evaluate collagen deposition. Polymerase chain reaction and Western blot were performed to measure messenger RNA and protein expression. We found that dioscin treatment (particularly at the dose of 80 mg/kg) significantly inhibited pulmonary inflammation in asthmatic mice, as evidenced by the decreased serum OVA-specific IgE/IgG1 and the reduced inflammatory cells and cytokines in BALFs and lung tissues. Moreover, dioscin effectively ameliorated the goblet cell hyperplasia, mucus hypersecretion, collagen deposition, and smooth muscle hyperplasia in the airways of asthmatic mice. Mechanistically, dioscin restrained the activated TGF-ß1/Smad2/3 and protein kinase B (AKT) signal pathways in lung tissues and potently reversed the TGF-ß1-induced EMT and phosphorylation of Smad2/3 and AKT in 16HBE cells. Collectively, dioscin displayed protective effects on OVA-induced asthmatic mice via adjusting TGF-ß1/Smad2/3 and AKT signal pathways, supporting the fact that dioscin could be a candidate for chronic asthma prevention in the future.

Genome-wide codon usage pattern analysis reveals the correlation between codon usage bias and gene expression in Cuscuta australis.

The protein-coding genes and pseudogenes of Cuscuta australis had the diverse contribution to the formation and evolution of parasitism. The codon usage pattern analysis of these two type genes could be used to understand the gene transcription and translation. In this study, we systematically analyzed the codon usage patterns of protein-coding sequences and pseudogenes sequences in C. australis. The results showed that the high frequency codons of protein coding sequences and pseudogenes had the same A/U bias in the third position. However, these two sequences had converse bias at the third base in optimal codons: the protein coding sequences preferred G/C-ending codons while pseudogene sequences preferred A/U-ending codons. Neutrality plot and effective number of codons plot revealed that natural selection played a more important role than mutation pressure in two sequences codon usage bias. Furthermore, the gene expression level had a significant positive correlation with codon usage bias in C. australis. Highly-expressed protein coding genes exhibited a higher codon bias than lowly-expressed genes. Meanwhile, the high-expression genes tended to use G/C-ending synonymous codons. This result further verified the optimal codons usage bias and its correlation with the gene expression in C. australis.

MECAT: fast mapping, error correction, and de novo assembly for single-molecule sequencing reads.

We present a tool that combines fast mapping, error correction, and de novo assembly (MECAT; accessible at https://github.com/xiaochuanle/MECAT) for processing single-molecule sequencing (SMS) reads. MECAT's computing efficiency is superior to that of current tools, while the results MECAT produces are comparable or improved. MECAT enables reference mapping or de novo assembly of large genomes using SMS reads on a single computer.

[Updated reproductive toxicology of polybrominated diphenyl ether].

Since 1970, polybrominated diphenyl ether (PBDE) has been widely used as additive flame retardants in everyday consumer products, including polyurethane foam and electronic products like mattresses and upholstered furniture. Thermoplastics, mixed in polymers, do not chemically bond with plastics, textiles, etc., and therefore can be separated from the product into the environment. Because of its high lipophilicity, accumulation, degradation-resistance and biochemical toxicity, PBDE can invade the human body in a variety of ways and is toxic to multiple systems in the human body. PBDE affects the male reproductive system in many aspects, as by causing sperm quality decline, epididymis, seminal vesicle and prostate dysplasia, sperm head deformity, and decreased levels of testosterone and other reproductive hormones. PBDE also affects male reproductive function from the genetic aspect, as by altering the sperm DNA methylation level, inducing sperm chromatin damage, etc. Some environmental factors, such as high-fat diet and indoor dust increase, can indirectly promote the reproductive toxicity of PBDE. This article reviews the impacts of PBDE on exposed populations and the animal reproductive system and the latest research progress at home and abroad.

[Clinical characteristics and treatment strategies of prostatic mucinous adenocarcinoma: A report of 10 cases and literature review].

OBJECTIVE: To investigate the clinical characteristics and treatment strategies of prostatic mucinous adenocarcinoma (PMAC). METHODS: We retrospectively analyzed the clinical data on 10 cases of PMAC treated in the First Affiliated Hospital of Nanjing Medical University from January 2014 to June 2018. The patients were aged 51－79 (65 ± 14) years, with a medium PSA level of 89 (14.63－128.05) µg/L and Gleason scores of 3 + 3 in 1 case, 3 + 4 in 2, 4 + 3 in 1 and 8 in 6 cases preoperatively, 1 treated by robot-assisted radical prostatectomy and the other 9 by laparoscopic radical prostatectomy. We conducted pelvic cavity lymph node dissection for all the patients and analyzed their prognosis and survival. RESULTS: Operations were successfully completed in all the cases. Pathological examination revealed 2 cases of mucinous adenocarcinoma with signet ring cell carcinoma in the 10 PMAC patients, 2 at stage ≤T2b, 5 at stage ≥T2c, 3 positive at pelvic lymph node dissection and 5 positive at the incision margin. The patients were followed up for 6－48 (median 26) months. Four of the patients were found with biochemical recurrence within 2 years after operation and treated by androgen-deprivation therapy, radiotherapy and chemotherapy, which reduced the PSA level to <1.0 µg/ml in all the 4 cases. CONCLUSIONS: PMAC has a good prognosis. Radical surgery is recommended for moderate and low-risk PMAC and the patients with postoperative biochemical recurrence can benefit from comprehensive treatment of total androgen blockade.

DNA N6-Methyladenosine modification role in transmitted variations from genomic DNA to RNA in Herrania umbratica.

BACKGROUND: DNA methylation is an important epigenetic modification. Recently the developed single-molecule real-time (SMRT) sequencing technology provided an efficient way to detect DNA N6-methyladenine (6mA) modification that played an important role in epigenetic and positively regulated gene expression. In addition, the gene expression was also regulated by genetic variation. However, the relationship between DNA 6mA modification and variation is still unknown. RESULTS: We collected the SMRT long-reads DNA, Illumina short reads DNA and RNA datasets from the young leaves of Herrania umbratica, and used them to detect 35,654 6mA modification sites, 829,894 DNA variations and 60,672 RNA variations respectively, among which, there are 303 DNA variations and 19 RNA variations with 6mA modification, and 57,468 transmitted genetic variations from DNA to RNA. The results illustrated that the genes with 6mA modification were significant disadvantage to mutate than those genes without modification (p-value< 4.9e-08). And result from the linear regression model showed the 6mA densities of genes were associated with the transmitted variations type 0/1 to 1/1 (p-value < 0.001). CONCLUSIONS: The variations of DNA and RNA in genes with 6mA modification were significant less than those in unmodified genes. Furthermore, the variations in 6mA modified genes were easily transmitted from DNA to RNA, especially the transmitted variation from DNA heterozygote to RNA homozygote.

[Establishment of Ace2 knockout mouse model with CRISPR/Cas9 gene targeting technology].

The aim of the study was to establish Ace2 (angiotensin-converting enzyme 2) knockout mouse model with CRISPR/Cas9 gene targeting technology. A vector targeting Ace2 gene knockout was constructed with the primers of single-guide RNA (gRNA), and then transcribed gRNA/Cas9 mRNA was micro-injected into the mouse zygote. The deletion of exons 3 to 18 of Ace2 gene in mice was detected and identified by PCR and gene sequencing. The Ace2 gene knock-out mice were bred and copulated. Ace2 protein and mRNA expression were detected by Western blot and qRT-PCR in F3 progeny knock-out male mice. The gRNA expression vector was successfully constructed and transcribed in vitro, and active gRNA and Cas9 mRNA were injected directly into zygote. The deletion of exons 3 to 18 of Ace2 gene in six positive founder mice as the F0 generation were confirmed by PCR and gene sequencing. Six founder mice were mated with wild-type mice, then achieved F1 generation were mated and produced F2 generation. The female positive mouse of F2 was selected to mate with wild-type mice and produce Ace2-/Y mice of F3 generation. Ace2 mRNA and protein were not detected in tissues of these Ace2-/Y mice. In conclusion, a mouse model with Ace2 deficiency has been successfully established with CRISPR/Cas9 technique, which shall lay a foundation for future investigation of Ace2.

[Glans-preserving surgery for superficial penile squamous cell carcinoma ≥2 cm in diameter].

OBJECTIVE: To explore the feasibility of glans-preserving surgery (GPS) in the treatment of superficial penile squamous cell carcinoma (PSCC) with the lesion diameter of ≥2 cm. METHODS: We retrospectively analyzed the clinical data on 69 cases of superficial PSCC (≤T1aN0) treated by GPS (n = 36) or radical surgery (total or partial penectomy, n = 33) from July 2007 to July 2017. RESULTS: The mean tumor diameter and depth of invasion were 3.16 (2.0－6.0) cm and 0.89 (0.5－2.0) cm in the GPS group and 3.56 (2.0－6.0) cm and 1.89 (0.6－4.0) cm respectively in the radical surgery group. The patients were followed up for 10－102 (mean 42) months, during which, 5 patients in the GPS group developed local recurrence at 40 days and 2, 4, 7 and 9 months postoperatively, again underwent gansectomy, partial penectomy or GPS, and experienced no more recurrence during the follow-up of 54, 34, 39, 66 and 70 months. No local recurrence was observed in the radical surgery group, and none of the 69 patients experienced lymph node metastasis or died during the follow-up. CONCLUSIONS: GPS is safe and efficient for the treatment of superficial PSCC with the lesion diameter of ≥2 cm.

Gene-to-Gene Network Analysis of the Mediation of Plant Innate Immunity by the Eliciting Plant Response-Like 1 (Epl1) Elicitor of Trichoderma formosa.

A new clade, Trichoderma formosa, secretes eliciting plant response-like 1 (Epl1), a small peptide elicitor that stimulates plant immunity. Nicotiana benthamiana pretreated with Epl1 for 3 days developed immunity against Tomato mosaic virus (ToMV) infection. The transcriptome profiles of T. formosa and N. benthamiana were obtained by deep sequencing; the transcript of Epl1 is 736 nt in length and encodes a 12-kDa peptide. Identifying critical genes in Epl1-mediated immunity was challenging due to high similarity between the transcriptome expression profiles of Epl1-treated and ToMV-infected N. benthamiana samples. Therefore, an efficient bioinformatics data mining approach was used for high-throughput transcriptomic assays in this study. We integrated gene-to-gene network analysis into the ContigViews transcriptome database, and genes related to jasmonic acid and ethylene signaling, salicylic acid signaling, leucine-rich repeats, transcription factors, and histone variants were hubs in the gene-to-gene networks. In this study, the Epl1 of T. formosa triggers plant immunity against various pathogen infections. Moreover, we demonstrated that high-throughput data mining and gene-to-gene network analysis can be used to identify critical candidate genes for further studies on the mechanisms of plant immunity.

RPFdb: a database for genome wide information of translated mRNA generated from ribosome profiling.

Translational control is crucial in the regulation of gene expression and deregulation of translation is associated with a wide range of cancers and human diseases. Ribosome profiling is a technique that provides genome wide information of mRNA in translation based on deep sequencing of ribosome protected mRNA fragments (RPF). RPFdb is a comprehensive resource for hosting, analyzing and visualizing RPF data, available at www.rpfdb.org or http://sysbio.sysu.edu.cn/rpfdb/index.html. The current version of database contains 777 samples from 82 studies in 8 species, processed and reanalyzed by a unified pipeline. There are two ways to query the database: by keywords of studies or by genes. The outputs are presented in three levels. (i) Study level: including meta information of studies and reprocessed data for gene expression of translated mRNAs; (ii) Sample level: including global perspective of translated mRNA and a list of the most translated mRNA of each sample from a study; (iii) Gene level: including normalized sequence counts of translated mRNA on different genomic location of a gene from multiple samples and studies. To explore rich information provided by RPF, RPFdb also provides a genome browser to query and visualize context-specific translated mRNA. Overall our database provides a simple way to search, analyze, compare, visualize and download RPF data sets.

Sulforaphane attenuates di-N-butylphthalate-induced reproductive damage in pubertal mice: Involvement of the Nrf2-antioxidant system.

di-N-butylphthalate (DBP) is a ubiquitous environmental pollutant used for plastic coating and in the cosmetics industry. It has toxic effects on body health, especially the male reproductive system. Here, we investigated the effects of DBP on the male reproductive system of pubertal mice and explored the protective role of sulforaphane (SFN). The results showed that DBP significantly reduced the anogenital distance, testicular weight, sperm count and motility, and plasma and testicular testosterone levels and significantly increased the oxidative stress, sperm abnormalities, and testicular cell apoptosis. SFN supplementation ameliorated these effects. After DBP stimulation, the transcription factor nuclear factor erythroid-related factor 2 (Nrf2) was adaptively increased together with its target genes, such as HO-1 and NQO1. Upregulation of Nrf2 by SFN reduced the DBP-mediated intracellular oxidative toxicity and also increased testosterone secretion and spermatogenesis, which were decreased by DBP. These findings indicate that SFN can attenuate DBP-induced reproductive damage in pubertal mice via Nrf2-associated pathways.

Event-related lateralized readiness potential correlates of the emotion-priming Simon effect.

The Simon effect indicates that the reaction time (RT) is shorter when the stimulus and response locations are congruent than when they are not. This study used a priming-target paradigm to explore the emotion-priming Simon effect with event-related potential techniques. The technique of residue iteration decomposition was employed to analyze the lateralized readiness potential (LRP) component, which contributed to disentangling the overlap between LRP and N2 central contralateral in the Simon task with horizontal stimulus-response arrangements. The behavioral result revealed significant Simon effect in RT. In the neural process, the Simon effect was reflected by both the stimulus-locked LRP (S-LRP) and the response-locked LRP (R-LRP), with the incongruent condition showing longer onset latency, larger Gratton-dip, and smaller negative-going deflection of S-LRP and smaller negative-going deflection of R-LRP. These findings suggest that the interference of irrelevant location information is located at the perceptual-encoding (indicated by S-LRP) and response-execution stages (indicated by R-LRP), providing evidence for both the perceptual-interference and response-interference accounts. However, the further linear regression result signaled that the Simon effect might be more closely related to the response-execution stage than the perceptual-encoding stage. In addition, the influence of emotion on the Simon effect was salient only in the incongruent condition, showing longer onset latency of S-LRP and larger Gratton-dip of R-LRP in the negative emotion-priming condition than in the neutral emotion-priming condition, which revealed that the emotional interference effect arose from the stages of perceptual encoding and early response execution only when the locations of a stimulus and the corresponding response were incongruent.

Discovery of a Flexible Triazolylbutanoic Acid as a Highly Potent Uric Acid Transporter 1 (URAT1) Inhibitor.

In order to systematically explore and understand the structure-activity relationship (SAR) of a lesinurad-based hit (1c) derived from the replacement of the S atom in lesinurad with CH2, 18 compounds (1a-1r) were designed, synthesized and subjected to in vitro URAT1 inhibitory assay. The SAR exploration led to the discovery of a highly potent flexible URAT1 inhibitor, 1q, which was 31-fold more potent than parent lesinurad (IC50 = 0.23 µM against human URAT1 for 1q vs 7.18 µM for lesinurad). The present study discovered a flexible molecular scaffold, as represented by 1q, which might serve as a promising prototype scaffold for further development of potent URAT1 inhibitors, and also demonstrated that the S atom in lesinurad was not indispensable for its URAT1 inhibitory activity.