Heterochromatin formation and remodeling by IRTKS condensates counteract cellular senescence.

Heterochromatin, a key component of the eukaryotic nucleus, is fundamental to the regulation of genome stability, gene expression and cellular functions. However, the factors and mechanisms involved in heterochromatin formation and maintenance still remain largely unknown. Here, we show that insulin receptor tyrosine kinase substrate (IRTKS), an I-BAR domain protein, is indispensable for constitutive heterochromatin formation via liquid‒liquid phase separation (LLPS). In particular, IRTKS droplets can infiltrate heterochromatin condensates composed of HP1α and diverse DNA-bound nucleosomes. IRTKS can stabilize HP1α by recruiting the E2 ligase Ubc9 to SUMOylate HP1α, which enables it to form larger phase-separated droplets than unmodified HP1α. Furthermore, IRTKS deficiency leads to loss of heterochromatin, resulting in genome-wide changes in chromatin accessibility and aberrant transcription of repetitive DNA elements. This leads to activation of cGAS-STING pathway and type-I interferon (IFN-I) signaling, as well as to the induction of cellular senescence and senescence-associated secretory phenotype (SASP) responses. Collectively, our findings establish a mechanism by which IRTKS condensates consolidate constitutive heterochromatin, revealing an unexpected role of IRTKS as an epigenetic mediator of cellular senescence.

Stevia rebaudiana Is a New Host of Stagonosporopsis pogostemonis Associated with Leaf Spot Disease in China.

Stevia rebaudiana is a promising medicinal and edible plant, widely cultivated in China. In 2022-2023, a new leaf spot disease occurred in S. rebaudiana in Hongxing country (32°34'55″N, 118°2'12″E), Dingyuan city, Anhui province. Symptoms were observed on 10 to 15% of plants in three S. rebaudiana nursery beds (0.1 ha in total). The typical symptoms included dark brown spots on the leaves and foliar wilting, the development of brown stems with dieback of top buds, and occasional plant death (Fig. 1a). To identify the pathogen, twenty diseased leaves were collected, cut into small pieces, surface sterilized with 75% ethanol for 30 s, in 0.5% sodium hypochlorite for 2 min, washed three times in sterile water, placed on PDA, and incubated at 25℃ for 5 days. Pure cultures were prepared by subculturing hyphal tips. Twenty-five Stagonosporopsis-like isolates with similar morphology were obtained. After 7 days growth on PDA, colonies had a regular margin, were cottony, and formed concentric circles on the surface that were gray-green. The reverse side of the culture was dark brown with a creme-orange and white, margin. The growth rate was 9.5 mm/day on PDA. Pycnidia were mostly solitary, globose or subglobose, pale to dark brown, thin-walled, glabrous, ostiolate, 95.735~250.851×90.93~266.32 µm (n=50). Conidia were oblong, cylindrical to ellipsoidal, smooth-walled, aseptate, with rounded ends and two polar guttules and measured 3.41 to 5.83 × 1.78 to 3.07 µm (n = 50) (Fig.1 b-d). For molecular identification, the internal transcribed spacer (ITS) rDNA, large ribosomal subunit (LSU) gene, ß-tubulin (TUB2) gene and RNA polymerase II (RPB2) gene sequences of two representative isolates (TYJ-SP1 and TYJ-SP2) were amplified by PCR (Woudenberg et al. 2009; Dong et al. 2021). The sequences were deposited in GenBank (accession nos.: OR506193 and OR506194 for ITS, OR533526 and OR533527 for LSU, OR545221and OR545222 for TUB; OR545223 and OR545224 for RPB2) and showed 99.60% to 99.2% similarity to ITS (502/504 bp and 507/511 bp; MZ156571), 100% similarity to LSU (857/857 bp and 857/857 bp; MZ191532), 98.67% to 99.3% similarity to TUB2 (296/300 bp and 298/300 bp; MZ203132) and 99.78% (888/890 bp and 868/870 bp; MZ203135) of S. pogostemonis strain ZHKUCC 21-0001. A maximum likelihood phylogenetic analysis based on the concatenated sequences of ITS, LSU, TUB2 and RPB2 using MEGA 11.0 showed the strains TYJ-SP1 and TYJ-SP2 formed a clade with S. pogostemonis (Fig. 2). Thus, the strains were identified as S. pogostemonis (Dong et al. 2021). To test pathogenicity, the strain TYJ-SP1 was inoculated onto 30-day-old S. rebaudiana seedlings which were surface sterilized with 70% alcohol and washed 3 times with water and air dried prior to inoculation. Ten seedlings were sprayed with a conidial suspension (105 conidia/mL) and ten seedlings were sprayed with sterile water to serve as the negative control. All seedlings were maintained in a growth chamber (25°C, 90% relative humidity) with a 16 h photoperiod. Brown spots were first observed on inoculated leaves 48 h after inoculation; typical symptoms appeared by 7 days post inoculation. All inoculated plants developed symptoms similar to naturally infected plants in the nursery beds, and the disease incidence reached 100% while control plants remained symptom free (Fig. 1 e-f). The same Stagonosporopsis isolates were reisolated from the inoculated plants and identified based on morphological and phylogenetic analyses. S. pogostemonis has been reported to cause leaf spot in Pogostemon cablin and Brassica oleracea var. botrytis (Dong et al. 2021; Habib et al. 2024). To our knowledge, this is the first report of S. pogostemonis causing leaf spot on S. rebaudiana in China. As a medicinal and economic plant, S. rebaudiana is widely planted in China and other Asian countries. The occurrence of this leaf spot disease seriously affects its medicinal and economic value. Therefore, it is crucial to establish and implement effective disease management practices to reduce the impact of the disease.

First report of Stagonosporopsis cucurbitacearum Causing Gummy Stem Blight on Trichosanthes kirilowii Maxim. in China.

Trichosanthes kirilowii Maxim. (Cucurbitaceae), one of the Chinese herbal medicines, is an economically important crop in Anhui Province, China. In recent years, gummy stem blight disease, a major disease of cucurbits, was widespread in many T. kirilowii plantations. The initial symptoms on the naturally infected stems appeared as dark brown water-soaked lesions, and as the disease progressed, vines of T. kirilowii gradually withered. On leaves, brown water-soaked lesions were visible initially, and then lesions enlarged and coalesced, resulting in extensive necrosis of leaves. On fruit, lesions covered with the white mycelium were nearly circular and tan to brown initially. Subsequently, the diseased fruit turned black and rotten commonly known as fruit rot or black rot. A Stagonosporopsis-like organism was consistently isolated from symptomatic stems, leaves and fruits. Fungal isolates were initially white and later turned dark grey or black with woolly to floccose aerial mycelium on PDA medium. Twenty-four isolates from different plantations were selected for further morphological studies. Pycnidia and conidia were formed after inoculating on cucumber fruit for 3 days. Pycnidia were globose to sub-globose, brown, ostiolate and 106.7 to 213.6 µm (average 160.1 µm, n = 50) in diameter. Conidia were hyaline, ellipsoidal, aseptate or one-septate, slightly constricted at the septa, 6.1 to 13.6 × 3.5 to 4.8 µm (average 9.9 × 4.1 µm, n = 50), and contained two or more oil drops. Three different loci of the genomic DNA, including the nuclear ribosome DNA internal transcribed spacer (ITS), RNA polymerase II second-largest subunit (RPB2), and ß-tubulin (TUB2) genes., were amplified using primers ITS1/ITS4 (White et al. 1990), RBP2DF/RBP2DR (Lawrence et al. 2013), and T1/ß-Sandy-R (O' Donnell and Cigelnik 1997; Stukenbrock et al. 2012), respectively and sequenced. A phylogenetic tree was built based on analysis of ITS, RPB2, and TUB2 sequences that deposited in GenBank (MW485497-MW485502 for ITS, MW531661-MW531666 for RPB2, and MW531667-MW531672 for TUB2), using the maximum likelihood method. The phylogenetic tree showed that the isolates fell into a single clade with S. cucurbitacearum. On the basis of morphological and molecular characteristics, the isolates obtained from T. kirilowii were identified as Stagonosporopsis cucurbitacearum. Pathogenicity tests were carried out on stems and leaves of 4-week-old T. kirilowii seedlings and on immature fruit collected from adult T. kirilowii plants. The epidermis, previously injured with a syringe needle, was inoculated with 5-mm-diameter mycelial plugs, and the inoculated areas were then wrapped in water-soaked cotton. Controls were similarly inoculated with agar plugs. The diameters of lesions were measured in two perpendicular directions. Re-isolations from the stem and leaf lesions were performed on the PDA medium. Stagonosporopsis cucurbitacearum, was re-identified based on its colony and conidial characteristics and, therefore, completed Koch's postulates. Gummy stem blight caused by S. cucurbitacearum has been reported in a wide range of hosts, including cucumber, luffa, pumpkin, gourd, muskmelon, cantaloupe, and watermelon (Jiang et al. 2015; Keinath 2011; Zhao et al. 2019). To our knowledge, this is the first report of gummy Stem blight disease on T. kirilowii caused by S. cucurbitacearum in China. The research provides a basis for the development and implementation of effective management strategies. Pathogenicity tests were carried out on stems and leaves of 4-week-old T. kirilowii seedlings and on immature fruits collected from adult T. kirilowii plants. The epidermis, previously injured with a syringe needle, was inoculated with 5-mm-diameter mycelial plugs, and the inoculated areas were then wrapped in water-soaked cotton. Controls were treated similarly but inoculated with agar plugs. Diameters of lesions were measured in two mutually perpendicular directions. Reisolations from the lesions were performed on PDA medium, and was re-identified based on its colony and conidial characteristics to complete Koch's postulates. Gummy stem blight caused by S. cucurbitacearum have been reported in a wide range of hosts, including cucumber, luffa, pumpkin, gourd, muskmelon, cantaloupe, and watermelon (Jiang et al. 2015; Keinath 2011; Zhao et al. 2019). To our knowledge, this is the first report of gummy Stem blight disease on T. kirilowii caused by S. cucurbitacearum in China. The research provides a basis for the development and implementation of effective management strategies.

The integration model of hepatitis B virus genome in hepatocellular carcinoma cells based on high-throughput long-read sequencing.

HBV integration and function has gradually been expanding. However, the exact mode of HBV integration remains unclear. In our research, the high-throughput long-read sequencing was combined with bioinformatics to study the complete mode of HBV integration in hepatocellular carcinoma (HCC) cells. The results demonstrated that: 1) The HBV insertion sequences of HBV integration events accounted for 49.5% of the total HBV sequences. 2) Short insertion segments with the length of 0-1 kbp accounted for 50% and the long insertion segments (>3 kbp) accounted for 25% of HBV insertion events. 3)There were different HBV insertion length in the breakpoints formed within different regions. 4) The occurrence of HBV integration events was accompanied by more frequent structural variations. 5)Furthermore, multiple HBV integration patterns were confirmed based on complete HBV insertion sequences. Our research not only clarified a variety of perfect HBV integration models but also determined multiple specific features of HBV integration.

Profile of different Hepatitis B virus integration frequency in hepatocellular carcinoma patients.

Hepatitis B virus (HBV) DNA integration is closely related to the occurrence of liver cancer. However, current studies mostly focus on the detection of the viral integration sites, ignoring the relationship between the frequency of viral integration and liver cancer. Thus, this study uses previous data to distinguish the breakpoints according to the integration frequency and analyzes the characteristics of different groups. This analysis revealed that three sets of breakpoints were characterized by its own integrated sample frequency, breakpoint distribution, and affected gene pathways. This result indicated an evolution in the virus integration sites in the process of tumor formation and development. Therefore, our research clarified the characteristics and differences in the sites of viral integration in tumors and adjacent tissues, and clarified the key signaling pathways affected by viral integration. Hence, these findings might be of great significance in the understanding of the role of viral integration frequency in hepatocellular carcinoma.

[Study on mechanism of enhancing antioxidant activity in fermented Ziziphi Spinosae Semen with Poria cocos].

In this study, the antioxidant property changes in fermented Ziziphi Spinosae Semen(FZSS) with Poria cocos were analyzed by DPPH, ABTS and FRAP methods. Then the content determination of active ingredients and ~1H nuclear magnetic resonance(~1H-NMR) spectroscopy were also used to investigate the mechanism of FZSS with P. cocos in enhancing the antioxidant activity. The results showed that the content of active ingredients such as total phenols, total saponins and total polysaccharides were significantly increased during the fermentation time. The results of ~1H-NMR metabonomics showed that the contents of amino acids such as leucine, lysine, valine and alanine, nitrogen compounds such as creatine, creatinine, and betaine, and secondary metabolites, for instance, jujuboside A and spinosin were higher after fermentation, and above components showed positive correlation with antioxidant capacity in Pearson correlation analysis. Therefore, it was inferred that the enhancement of antioxidant activity of FZSS may be the result of the joint action of various chemical components. This study preliminarily clarified the mechanism of FZSS in enhancing the antioxidant activity, and provided new research ideas for the product development and utilization of FZSS.

Mutational and transcriptomic landscapes of a rare human prostate basal cell carcinoma.

BACKGROUND: As a rare subtype of prostate carcinoma, basal cell carcinoma (BCC) has not been studied extensively and thus lacks systematic molecular characterization. METHODS: Here, we applied single-cell genomic amplification and RNA-Seq to a specimen of human prostate BCC (CK34ßE12+ /P63+ /PAP- /PSA- ). The mutational landscape was obtained via whole exome sequencing of the amplification mixture of 49 single cells, and the transcriptomes of 69 single cells were also obtained. RESULTS: The five putative driver genes mutated in BCC are CASC5, NUTM1, PTPRC, KMT2C, and TBX3, and the top three nucleotide substitutions are C>T, T>C, and C>A, similar to common prostate cancer. The distribution of the variant allele frequency values indicated that these single cells are from the same tumor clone. The 69 single cells were clustered into tumor, stromal, and immune cells based on their global transcriptomic profiles. The tumor cells specifically express basal cell markers like KRT5, KRT14, and KRT23 and epithelial markers EPCAM, CDH1, and CD24. The transcription factor covariance network analysis showed that the BCC tumor cells have distinct regulatory networks. By comparison with current prostate cancer datasets, we found that some of the bulk samples exhibit basal cell signatures. Interestingly, at single-cell resolution the gene expression patterns of prostate BCC tumor cells show uniqueness compared with that of common prostate cancer-derived circulating tumor cells. CONCLUSIONS: This study, for the first time, discloses the comprehensive mutational and transcriptomic landscapes of prostate BCC, which lays a foundation for the understanding of its tumorigenesis mechanism and provides new insights into prostate cancers in general.

Profile of HBV Integration in the Plasma DNA of Hepatocellular Carcinoma Patients.

BACKGROUND: Hepatitis B Viral (HBV) infection is one of the major causes of Hepatocellular Carcinoma (HCC). Mounting evidence had provided that the HBV integration might be a critical con-tributor of HCC carcinogenesis. OBJECTIVE AND METHODS: To explore the profile of HBV integration in the plasma DNA, the method of next-generation sequencing, HBV capture and bioinformatics had been employed to screen for HBV in-tegration sites in the plasma samples. RESULTS: In the initial experiment, a total of 87 breakpoints were detected in the 20 plasma samples. The distribution of breakpoints showed that there was significant enrichment of breakpoints in the region of intron. Furthermore, the HBV breakpoints were prone to occur in the region of X protein (1,700-2,000bp) in the plasma samples. The pathway analysis had revealed that the HBV integrations sites were specifically enriched in the cancer pathway. CONCLUSION: Altogether, our results had provided direct evidence for the HBV integration in plasma DNA, and they might be potentially useful for future HCC prognosis and diagnosis.

IRTKS Promotes Insulin Signaling Transduction through Inhibiting SHIP2 Phosphatase Activity.

Insulin signaling is mediated by a highly integrated network that controls glucose metabolism, protein synthesis, cell growth, and differentiation. Our previous work indicates that the insulin receptor tyrosine kinase substrate (IRTKS), also known as BAI1-associated protein 2-like 1 (BAIAP2L1), is a novel regulator of insulin network, but the mechanism has not been fully studied. In this work we reveal that IRTKS co-localizes with Src homology (SH2) containing inositol polyphosphate 5-phosphatase-2 (SHIP2), and the SH3 domain of IRTKS directly binds to SHIP2's catalytic domain INPP5c. IRTKS suppresses SHIP2 phosphatase to convert phosphatidylinositol 3,4,5-triphosphate (PI(3,4,5)P3, PIP3) to phosphatidylinositol (3,4) bisphosphate (PI(3,4)P2). IRTKS-knockout significantly increases PI(3,4)P2 level and decreases cellular PI(3,4,5)P3 content. Interestingly, the interaction between IRTKS and SHIP2 is dynamically regulated by insulin, which feeds back and affects the tyrosine phosphorylation of IRTKS. Furthermore, IRTKS overexpression elevates PIP3, activates the AKT-mTOR signaling pathway, and increases cell proliferation. Thereby, IRTKS not only associates with insulin receptors to activate PI3K but also interacts with SHIP2 to suppress its activity, leading to PIP3 accumulation and the activation of the AKT-mTOR signaling pathway to modulate cell proliferation.

IRTKS is correlated with progression and survival time of patients with gastric cancer.

BACKGROUND AND OBJECTIVES: IRTKS functions as a novel regulator of tumour suppressor p53; however, the role of IRTKS in pathogenesis of gastric cancer is unclear. DESIGN: We used immunohistochemistry to detect IRTKS levels in 527 human gastric cancer specimens. We generated both IRTKS-deficient and p53-deficient mice to observe survival time of these mice and to isolate mouse embryonic fibroblasts (MEFs) for evaluating in vivo tumorigenicity. Co-immunoprecipitation was used to study the interaction among p53, MDM2 and IRTKS, as well as the ubiquitination of p53. RESULTS: IRTKS was significantly overexpressed in human gastric cancer, which was conversely associated with wild-type p53 expression. Among patients with wild-type p53 (n=206), those with high IRTKS expression (n=141) had a shorter survival time than those with low IRTKS (n=65) (p=0.0153). Heterozygous p53+/- mice with IRTKS deficiency exhibited significantly delayed tumorigenesis and an extended tumour-free survival time. p53+/- MEFs without IRTKS exhibited attenuated in vivo tumorigenicity. IRTKS depletion upregulated p53 and its target genes, such as BAX and p21. Intriguingly, IRTKS overexpression promoted p53 ubiquitination and degradation in MEFs and gastric cancer cells. Under DNA damage conditions, IRTKS was phosphorylated at Ser331 by the activated Chk2 kinase and then dissociated from p53, along with the p53-specific E3 ubiquitin ligase MDM2, resulting in attenuated p53 ubiquitination and degradation. CONCLUSION: IRTKS overexpression is negatively correlated with progression and overall survival time of patients with gastric cancer with wild-type p53 through promotion of p53 degradation via the ubiquitin/proteasome pathway.

Seneca Valley Virus Suppresses Host Type I Interferon Production by Targeting Adaptor Proteins MAVS, TRIF, and TANK for Cleavage.

Seneca Valley virus (SVV) is an oncolytic RNA virus belonging to the Picornaviridae family. Its nucleotide sequence is highly similar to those of members of the Cardiovirus genus. SVV is also a neuroendocrine cancer-selective oncolytic picornavirus that can be used for anticancer therapy. However, the interaction between SVV and its host is yet to be fully characterized. In this study, SVV inhibited antiviral type I interferon (IFN) responses by targeting different host adaptors, including mitochondrial antiviral signaling (MAVS), Toll/interleukin 1 (IL-1) receptor domain-containing adaptor inducing IFN-ß (TRIF), and TRAF family member-associated NF-κB activator (TANK), via viral 3C protease (3Cpro). SVV 3Cpro mediated the cleavage of MAVS, TRIF, and TANK at specific sites, which required its protease activity. The cleaved MAVS, TRIF, and TANK lost the ability to regulate pattern recognition receptor (PRR)-mediated IFN production. The cleavage of TANK also facilitated TRAF6-induced NF-κB activation. SVV was also found to be sensitive to IFN-ß. Therefore, SVV suppressed antiviral IFN production to escape host antiviral innate immune responses by cleaving host adaptor molecules.IMPORTANCE Host cells have developed various defenses against microbial pathogen infection. The production of IFN is the first line of defense against microbial infection. However, viruses have evolved many strategies to disrupt this host defense. SVV, a member of the Picornavirus genus, is an oncolytic virus that shows potential functions in anticancer therapy. It has been demonstrated that IFN can be used in anticancer therapy for certain tumors. However, the relationship between oncolytic virus and innate immune response in anticancer therapy is still not well known. In this study, we showed that SVV has evolved as an effective mechanism to inhibit host type I IFN production by using its 3Cpro to cleave the molecules MAVS, TRIF, and TANK directly. These molecules are crucial for the Toll-like receptor 3 (TLR3)-mediated and retinoic acid-inducible gene I (RIG-I)-like receptor (RLR)-mediated signaling pathway. We also found that SVV is sensitive to IFN-ß. These findings increase our understanding of the interaction between SVV and host innate immunity.

Tafa-2 plays an essential role in neuronal survival and neurobiological function in mice.

Tafa is a family of small secreted proteins with conserved cysteine residues and restricted expression in the brain. It is composed of five highly homologous genes referred to as Tafa-1 to -5. Among them, Tafa-2 is identified as one of the potential genes responsible for intellectual deficiency in a patient with mild mental retardation. To investigate the biological function of Tafa-2 in vivo, Tafa-2 knockout mice were generated. The mutant mice grew and developed normally but exhibited impairments in spatial learning and memory in Morris water maze test and impairments in short- and long-term memory in novel object recognition test, accompanied with increased level of anxiety-like behaviors in open-field test and elevated plus maze test, and decreased level of depression-like behaviors in forced-swim test and tail-suspension test. Further examinations revealed that Tafa-2 deficiency causes severe neuronal reduction and increased apoptosis in the brain of Tafa-2-/- mice via downregulation of PI3K/Akt and MAPK/Erk pathways. Conformably, the expression levels of CREB target genes including BDNF, c-fos and NF1, and CBP were found to be reduced in the brain of Tafa-2-/- mice. Taken together, our data indicate that Tafa-2 may function as a neurotrophic factor essential for neuronal survival and neurobiological functions.

Single-cell RNA-Seq analysis reveals dynamic trajectories during mouse liver development.

BACKGROUND: The differentiation and maturation trajectories of fetal liver stem/progenitor cells (LSPCs) are not fully understood at single-cell resolution, and a priori knowledge of limited biomarkers could restrict trajectory tracking. RESULTS: We employed marker-free single-cell RNA-Seq to characterize comprehensive transcriptional profiles of 507 cells randomly selected from seven stages between embryonic day 11.5 and postnatal day 2.5 during mouse liver development, and also 52 Epcam-positive cholangiocytes from postnatal day 3.25 mouse livers. LSPCs in developing mouse livers were identified via marker-free transcriptomic profiling. Single-cell resolution dynamic developmental trajectories of LSPCs exhibited contiguous but discrete genetic control through transcription factors and signaling pathways. The gene expression profiles of cholangiocytes were more close to that of embryonic day 11.5 rather than other later staged LSPCs, cuing the fate decision stage of LSPCs. Our marker-free approach also allows systematic assessment and prediction of isolation biomarkers for LSPCs. CONCLUSIONS: Our data provide not only a valuable resource but also novel insights into the fate decision and transcriptional control of self-renewal, differentiation and maturation of LSPCs.

The role of the hepatitis B virus genome and its integration in the hepatocellular carcinoma.

The integration of Hepatitis B Virus (HBV) is now known to be closely associated with the occurrence of liver cancer and can impact the functionality of liver cells through multiple dimensions. However, despite the detailed understanding of the characteristics of HBV integration and the mechanisms involved, the subsequent effects on cellular function are still poorly understood in current research. This study first systematically discusses the relationship between HBV integration and the occurrence of liver cancer, and then analyzes the status of the viral genome produced by HBV replication, highlighting the close relationship and structure between double-stranded linear (DSL)-HBV DNA and the occurrence of viral integration. The integration of DSL-HBV DNA leads to a certain preference for HBV integration itself. Additionally, exploration of HBV integration hotspots reveals obvious hotspot areas of HBV integration on the human genome. Virus integration in these hotspot areas is often associated with the occurrence and development of liver cancer, and it has been determined that HBV integration can promote the occurrence of cancer by inducing genome instability and other aspects. Furthermore, a comprehensive study of viral integration explored the mechanisms of viral integration and the internal integration mode, discovering that HBV integration may form extrachromosomal DNA (ecDNA), which exists outside the chromosome and can integrate into the chromosome under certain conditions. The prospect of HBV integration as a biomarker was also probed, with the expectation that combining HBV integration research with CRISPR technology will vigorously promote the progress of HBV integration research in the future. In summary, exploring the characteristics and mechanisms in HBV integration holds significant importance for an in-depth comprehension of viral integration.

Characteristics of Hepatitis B virus integration and mechanism of inducing chromosome translocation.

Hepatitis B virus (HBV) integration is closely associated with the onset and progression of tumors. This study utilized the DNA of 27 liver cancer samples for high-throughput Viral Integration Detection (HIVID), with the overarching goal of detecting HBV integration. KEGG pathway analysis of breakpoints was performed using the ClusterProfiler software. The breakpoints were annotated using the latest ANNOVAR software. We identified 775 integration sites and detected two new hotspot genes for virus integration, N4BP1 and WASHP, along with 331 new genes. Furthermore, we conducted a comprehensive analysis to determine the critical impact pathways of virus integration by combining our findings with the results of three major global studies on HBV integration. Meanwhile, we found common characteristics of virus integration hotspots among different ethnic groups. To specify the direct impact of virus integration on genomic instability, we explained the causes of inversion and the frequent occurrence of translocation due to HBV integration. This study detected a series of hotspot integration genes and specified common characteristics of critical hotspot integration genes. These hotspot genes are universal across different ethnic groups, providing an effective target for better research on the pathogenic mechanism. We also demonstrated more comprehensive key pathways affected by HBV integration and elucidated the mechanism for inversion and frequent translocation events due to virus integration. Apart from the great significance of the rule of HBV integration, the current study also provides valuable insights into the mechanism of virus integration.

Multiple HPV integration mode in the cell lines based on long-reads sequencing.

Background: The integration of human papillomavirus (HPV) is closely related to the occurrence of cervical cancer. However, little is known about the complete state of HPV integration into the host genome. Methods: In this study, three HPV-positive cell lines, HeLa, SiHa, and CaSki, were subjected to NANOPORE long-read sequencing to detect HPV integration. Analysis of viral integration patterns using independently developed software (HPV-TSD) yielded multiple complete integration patterns for the three HPV cell lines. Results: We found distinct differences between the integration patterns of HPV18 and HPV16. Furthermore, the integration characteristics of the viruses were significantly different, even though they all belonged to HPV16 integration. The HPV integration in the CaSki cells was relatively complex. The HPV18 integration status in HeLa cells was the dominant, whereas the percentage of integrated HPV 16 in SiHa and CaSki cells was significantly lower. In addition, the virus sequences in the HeLa cells were incomplete and existed in an integrated state. We also identified a large number of tandem repeats in HPV16 and HPV18 integration. Our study not only clarified the feasibility of high-throughput long-read sequencing in the study of HPV integration, but also explored a variety of HPV integration models, and confirmed that viral integration is an important form of HPV in cell lines. Conclusion: Elucidating HPV integration patterns will provide critical guidance for developing a detection algorithm for HPV integration, as well as the application of virus integration in clinical practice and drug research and development.

Metformin suppresses cardiac fibroblast proliferation under high-glucose conditions via regulating the mitochondrial complex I protein Grim-19 involved in the Sirt1/Stat3 signaling pathway.

Hyperglycemia associated with myocardial oxidative stress and fibrosis is the main cause of diabetic cardiomyopathy. Currently, no approved drug is available for preventing or treating diabetes-induced cardiac fibrosis. Metformin has been reported to improve glycemic control and ameliorate diabetic cardiomyopathy. This study aimed to investigate the effects and mechanism of metformin on diabetes-induced cardiac fibrosis and high glucose-induced proliferation of cardiac fibroblasts (CFs). In this study, db/db mice were treated with metformin [250 mg/kg⋅d, gavage]. CFs were cultured in high-glucose medium to mimic an in vitro diabetes model and then subjected to treatment with or without metformin. Cardiac fibrosis was analyzed using immunohistochemistry, Masson's trichrome staining, and Western blot analysis. Cell Counting Kit-8 (CCK-8) assays and cell colony formation assays were used to examine cell proliferation capacity. Transwell and scratch-wound assays were used to detect the migration ability of CFs. Retinoid-interferon-induced mortality-19 (Grim-19), sirtuin1 (Sirt1), and signal transducer and activator of transcription 3 (Stat3) were detected using Western blot analysis. The genes downstream of the Stat3 pathway were detected using quantitative reverse transcription PCR (qRT‒PCR). Metformin treatment markedly attenuated cardiac fibrosis in db/db mice and the proliferation and migration of CFs under high-glucose conditions. Mechanistically, we found an intersection between metformin and Grim-19 using bioinformatics. Metformin was found to suppress the expression of p-Stat3 and elevate the expression of mitochondrial complex I protein Grim-19 and Sirt1, thus inhibiting the proliferation and migration of CFs under high-glucose conditions. Our data suggested that metformin inhibited the proliferation and migration of CFs by regulating the expression of mitochondrial complex I Grim-19 protein involved in the Sirt1/Stat3 signaling pathway under high-glucose conditions, thus providing new ideas for treating diabetes-induced cardiac fibrosis.

Cooperation between IRTKS and deubiquitinase OTUD4 enhances the SETDB1-mediated H3K9 trimethylation that promotes tumor metastasis via suppressing E-cadherin expression.

Elevated expression and genetic aberration of IRTKS, also named as BAIAP2L1, have been observed in many tumors, especially in tumor progression. however, the molecular and cellular mechanisms involved in the IRTKS-enhanced tumor progression are obscure. Here we show that higher IRTKS level specifically increases histone H3 lysine 9 trimethylation (H3K9me3) by promoting accumulation of the histone methyltransferase SETDB1. Furthermore, we reveal that IRTKS recruits the deubiquitinase OTUD4 to remove Lys48-linked polyubiquitination at K182/K1050 sites of SETDB1, thus blocking SETDB1 degradation via the ubiquitin-proteasome pathway. Interestingly, the enhanced IRTKS-OTUD4-SETDB1-H3K9me3 axis leads to a general decrease in chromatin accessibility, which inhibits transcription of CDH1 encoding E-cadherin, a key molecule essential for maintaining epithelial cell phenotype, and therefore results in epithelial-mesenchymal transition (EMT) and malignant cell metastasis. Clinically, the elevated IRTKS levels in tumor specimens correlate with SETDB1 levels, but negatively associate with survival time. Our data reveal a novel mechanism for the IRTKS-enhanced tumor progression, where IRTKS cooperates with OTUD4 to enhance SETDB1-mediated H3K9 trimethylation that promotes tumor metastasis via suppressing E-cadherin expression. This study also provides a potential approach to reduce the activity and stability of the known therapeutic target SETDB1 possibly through regulating IRTKS or deubiquitinase OTUD4.

Retinol dehydrogenase 13 protects the mouse retina from acute light damage.

PURPOSE: To investigate whether retinol dehydrogenase 13 (RDH13) can protect the retina from acute light-induced damage. METHODS: We generated Rdh13 knockout mice using molecular biologic methods and assessed the associated morphological and functional changes under room-light conditions by hematoxylin-eosin (H&E), transmission electron microscopy (TEM), and scotopic electroretinography. Then, the light-damage model was established by exposure to diffuse white light (3,000 lx) for 48 h. Twenty-four h after light exposure, H&E was used for the histological evaluation. The thickness of the outer-plus-inner-segment and the outer nuclear layer was measured on sections parallel to the vertical meridian of the eye. An electroretinography test was performed to assess the functional change. Furthermore, the impairment of mitochondria was detected by TEM. Finally, the expression of cytochrome c (CytC) and other apoptosis-related proteins was detected by western blot. RESULTS: We found that there was no obvious difference in phenotype or function between Rdh13 knockout and wild-type mice. In Rdh13(-/-) mice subjected to intense light exposure, the photoreceptor outer-plus-inner-segment and outer nuclear layer were dramatically shorter, and the amplitudes of a- and b-waves under scotopic conditions were significantly attenuated. Distinctly swollen mitochondria with disrupted cristae were observed in the photoreceptor inner segments of Rdh13(-/-) mice. Increased expression levels of CytC, CytC-responsive apoptosis proteinase activating factor-1 (Apaf-1) and caspases 3, and other mitochondria apoptosis-related genes (nuclear factor-kappa B P65 [P65] and B-cell lymphoma 2-associated X protein [Bax]) were observed in Rdh13(-/-) mice. CONCLUSIONS: Rdh13 can protect the retina against acute light-induced retinopathy. The mechanism may involve inhibition of the mitochondrial apoptosis pathway.

Preparation and Characterization of Calcium-Incorporated Rosa roxburghii Tratt and Its Efficacy on Bone Mineral Density in Rats.

The deficiency of traditional calcium preparation will gradually be replaced by the new type of calcium preparation. Rosa roxburghii fruit (R. roxburghii) is popular for its rich nutrients and functional ingredients. The fermentation broth of R. roxburghii, involving amino acids, flavonoids, triterpenes, polysaccharides, and other compounds, is favorable for calcium chelation. Thus, this study fabricated calcium-incorporated R. roxburghii (FECa) and further illustrated its efficacy on bone mineral density (BMD) in rats. The calcium holding capacity of FECa was identified and confirmed using AAS. Ion complexation of FECa was characterized using 1H-NMR, UV, SEM and EDS, and FTIR. The calcium contents of femurs were increased by 36%, and the bone trabeculae of femurs were significantly increased. Net calcium balance was enhanced to further improve BMD by oral administration of FECa. The above results indicate that FECa can be a potential and efficient calcium supplementation agent.