Specific and Sensitive Visual Proviral DNA Detection of Major Pathogenic Avian Leukosis Virus Subgroups Using CRISPR-Associated Nuclease Cas13a.

Avian leukosis viruses (ALVs) include a group of avian retroviruses primarily associated with neoplastic diseases in poultry, commonly referred to as avian leukosis. Belonging to different subgroups based on their envelope properties, ALV subgroups A, B, and J (ALV-A, ALV-B, and ALV-J) are the most widespread in poultry populations. Early identification and removal of virus-shedding birds from infected flocks are essential for the ALVs' eradication. Therefore, the development of rapid, accurate, simple-to-use, and cost effective on-site diagnostic methods for the detection of ALV subgroups is very important. Cas13a, an RNA-guided RNA endonuclease that cleaves target single-stranded RNA, also exhibits non-specific endonuclease activity on any bystander RNA in close proximity. The distinct trans-cleavage activity of Cas13 has been exploited in the molecular diagnosis of multiple pathogens including several viruses. Here, we describe the development and application of a highly sensitive Cas13a-based molecular test for the specific detection of proviral DNA of ALV-A, B, and J subgroups. Prokaryotically expressed LwaCas13a, purified through ion exchange and size-exclusion chromatography, was combined with recombinase polymerase amplification (RPA) and T7 transcription to establish the SHERLOCK (specific high-sensitivity enzymatic reporter unlocking) molecular detection system for the detection of proviral DNA of ALV-A/B/J subgroups. This novel method that needs less sample input with a short turnaround time is based on isothermal detection at 37 °C with a color-based lateral flow readout. The detection limit of the assay for ALV-A/B/J subgroups was 50 copies with no cross reactivity with ALV-C/D/E subgroups and other avian oncogenic viruses such as reticuloendotheliosis virus (REV) and Marek's disease virus (MDV). The development and evaluation of a highly sensitive and specific visual method of detection of ALV-A/B/J nucleic acids using CRISPR-Cas13a described here will help in ALV detection in eradication programs.

Hypomethylation-associated Sox11 upregulation promotes oncogenesis via the PI3K/AKT pathway in OLP-associated OSCC.

Oral lichen planus (OLP) is a particularly prevalent oral disorder with the potential to progress to oral squamous cell carcinoma (OSCC). SRY-box transcription factor 11 (Sox11) has been reported to serve as a prognostic marker for various cancers. However, the role and mechanism of Sox11 in OLP-related OSCC are unknown. Our results indicated that Sox11 was highly expressed, and that Sox11 promoter methylation was significantly reduced in OLP-associated OSCC tissues. High Sox11 expression and Sox11 promoter hypomethylation indicate a poor patient prognosis. According to in vivo and in vitro experiments, the knockdown of Sox11 inhibited proliferation, invasion, and migration while driving its apoptotic death in OSSC cells; Sox11 overexpression exerted the opposite effect as Sox11 knockdown. Mechanistically, knockdown of Sox11 inhibited PI3K/AKT and glycolysis pathway, and overexpression of Sox11 enhanced the PI3K/AKT and glycolysis pathways in OSCC cells. In addition, we demonstrated that Sox11 overexpression accelerated the progression of OSCC, at least in part by promoting PI3K/AKT pathway activation. In conclusion, our data indicated that the DNA hypomethylation-associated upregulation of Sox11 could promote oncogenic transformation via the PI3K/AKT pathway in OLP-associated OSCC. Therefore, Sox11 might be a reliable biomarker for predicting the progression of precancerous oral tissues.

Dihydroartemisinin induces ferroptosis in T cell acute lymphoblastic leukemia cells by downregulating SLC7A11 and activating the ATF4­CHOP signaling pathway.

The present study aimed to investigate the anti-leukemic effects of dihydroartemisinin (DHA) on T-cell acute lymphoblastic leukemia (T-ALL) cell lines, Jurkat and Molt-4, and the underlying mechanisms. Cell Counting Kit-8 was performed to measure cell viability. Cell apoptosis and cell cycle distribution were assessed by flow cytometry. The expression levels of ATF4 and CHOP mRNA were assessed by reverse transcription-quantitative PCR, while the protein abundance of SLC7A11, GPX4, ATF4 and CHOP was determined by western blotting. Moreover, malondialdehyde, glutathione (GSH) and reactive oxygen species (ROS) assays were used to detect the levels of ferroptosis. The results showed that DHA suppressed T-ALL cell viability in vitro, and induced cell cycle arrest at S or G2/M phase. DHA also induced ROS burst, activated endoplasmic reticulum (ER) stress, disrupted the system Xc--GSH-GSH peroxidase 4 antioxidant system, and increased lipid peroxide accumulation, resulting in cell death. By contrast, the pharmacological inhibition of ferroptosis alleviated DHA-induced cell death, confirming that DHA induces T-ALL cell death via ferroptosis. Mechanistically, the effect of DHA on ferroptosis was partly mediated by downregulating SLC7A11 and upregulating the ATF4-CHOP signaling pathway, which is associated with ER stress. These results indicated that DHA may induce ferroptosis in T-ALL cell lines and could represent a promising therapeutic agent for treating T-ALL.

Heterologous survey of 130 DNA transposons in human cells highlights their functional divergence and expands the genome engineering toolbox.

Experimental studies on DNA transposable elements (TEs) have been limited in scale, leading to a lack of understanding of the factors influencing transposition activity, evolutionary dynamics, and application potential as genome engineering tools. We predicted 130 active DNA TEs from 102 metazoan genomes and evaluated their activity in human cells. We identified 40 active (integration-competent) TEs, surpassing the cumulative number (20) of TEs found previously. With this unified comparative data, we found that the Tc1/mariner superfamily exhibits elevated activity, potentially explaining their pervasive horizontal transfers. Further functional characterization of TEs revealed additional divergence in features such as insertion bias. Remarkably, in CAR-T therapy for hematological and solid tumors, Mariner2_AG (MAG), the most active DNA TE identified, largely outperformed two widely used vectors, the lentiviral vector and the TE-based vector SB100X. Overall, this study highlights the varied transposition features and evolutionary dynamics of DNA TEs and increases the TE toolbox diversity.

AHNAK2 Regulates NF-κB/MMP-9 Signaling to Promote Pancreatic Cancer Progression.

Early metastasis of pancreatic cancer (PaC) is a major cause of its high mortality rate. Previous studies have shown that AHNAK2 is involved in the progression of some tumors and is predicted to be an independent prognostic factor for PaC; however, the specific mechanisms through which AHNAK2 regulates PaC remain unclear. In this study, we examined the role of AHNAK2 in PaC and its potential molecular mechanisms. AHNAK2 mRNA and protein expression in PaC tissues and cells were measured using qRT-PCR and western blot analysis. After AHNAK2 knockdown using small interfering RNA, PaC cells were subjected to CCK-8 scratch, and Transwell assays to assess cell proliferation, migration, and invasion, respectively. Furthermore, the validation of the mechanistic pathway was achieved by western blot analysis. AHNAK2 mRNA and protein levels were up-regulated in PaC and silencing AHNAK2 significantly inhibited the proliferation, migration, and invasion of PaC cells. Mechanistically, AHNAK2 knockdown decreased the expression of phosphorylated p65, phosphorylated IκBα, and matrix metalloproteinase-9 (MMP-9), suggesting that activation of the NF-κB/MMP-9 signaling pathway was inhibited. Importantly, activation of NF-κB reversed the effects of AHNAK2 knockdown. Our findings indicate that AHNAK2 promotes PaC progression through the NF-kB/MMP-9 pathway and provides a theoretical basis for targeting AHNAK2 for the treatment of PaC.

Synthesis of Boronate Affinity-Based Oriented Dummy Template-Imprinted Magnetic Nanomaterials for Rapid and Efficient Solid-Phase Extraction of Ellagic Acid from Food.

Ellagic acid (EA) is a natural polyphenol and possesses excellent in vivo bioactivity and antioxidant behaviors, which play an important role in the treatment of oxidative stress-related diseases, such as cancer. Additionally, EA is also known as a skin-whitening ingredient. The content of EA would determine its efficacy. Therefore, the accurate analysis of EA content can provide more information for the scientific consumption of EA-rich foods and cosmetics. Nevertheless, the analysis of EA in these samples is challenging due to the low concentration level and the presence of interfering components with high abundance. Molecularly imprinted polymers are highly efficient pretreatment materials in achieving specific recognition of target molecules. However, the traditional template molecule (EA) could not be absolutely removed. Hence, template leakage continues to occur during the sample preparation process, leading to a lack of accuracy in the quantification of EA in actual samples, particularly for trace analytes. In addition, another drawback of EA as an imprinting template is that EA possesses poor solubility and a high price. Gallic acid (GA), called dummy templates, was employed for the synthesis of MIPs as a solution to these challenges. The approach used in this study was boronate affinity-based oriented surface imprinting. The prepared dummy-imprinted nanoparticles exhibited several significant advantages, such as good specificity, high binding affinity ((4.89 ± 0.46) × 10-5 M), high binding capacity (6.56 ± 0.35 mg/g), fast kinetics (6 min), and low binding pH (pH 5.0) toward EA. The reproducibility of the dummy-imprinted nanoparticles was satisfactory. The dummy-imprinted nanoparticles could still be reused even after six adsorption-desorption cycles. In addition, the recoveries of the proposed method for EA at three spiked levels of analysis in strawberry and pineapple were 91.0-106.8% and 93.8-104.0%, respectively, which indicated the successful application to real samples.

Calcium-sensing receptor-mediated macrophage polarization improves myocardial remodeling in spontaneously hypertensive rats.

Chronic inflammation is a key element in the progression of essential hypertension (EH). Calcium plays a key role in inflammation, so its receptor, the calcium-sensing receptor (CaSR), is an essential mediator of the inflammatory process. Compelling evidence suggests that CaSR mediates inflammation in tissues and immune cells, where it mediates their activity and chemotaxis. Macrophages (Mφs) play a major role in the inflammatory response process. This study provided convincing evidence that R568, a positive regulator of CaSR, was effective in lowering blood pressure in spontaneously hypertensive rats (SHRs), improving cardiac function by alleviating cardiac hypertrophy and fibrosis. R568 can increase the content of CaSR and M2 macrophages (M2Mφs, exert an anti-inflammatory effect) in myocardial tissue, reduce M1 macrophages (M1Mφs), which have a pro-inflammatory effect in this process. In contrast, NPS2143, a negative state regulator of CaSR, exerted the opposite effect in all of the above experiments. Following this study, R568 increased CaSR content in SHR myocardial tissue, lowered blood pressure, promoted macrophages to M2Mφs and improved myocardial fibrosis, but interestingly, both M1Mφs and M2Mφs were increased in the peritoneal cavity of SHRs, the number of M2Mφs remained lower than M1Mφs. In vitro, R568 increased CaSR content in RAW264.7 cells (a macrophage cell line), regulating intracellular Ca2+ ([Ca2+]i) inhibited NOD-like receptor family protein 3 (NLRP3) inflammasome activation and ultimately prevented its conversion to M1Mφs. The results showed that a decrease in CaSR in hypertensive rats causes further development of hypertension and cardiac damage. EH myocardial remodeling can be improved by CaSR overexpression by suppressing NLRP3 inflammasome activation and macrophage polarization toward M1Mφs and increasing M2Mφs.

Endoscopic submucosal dissection for the treatment of a large inflammatory fibroid polyp in the gastric antrum prolapsing into the duodenum: A case report.

RATIONALE: Inflammatory fibroid polyp (IFP), also known as Vanek tumor, is a rare, benign gastrointestinal lesion characterized by its inflammatory and fibroid histological features. IFP is often discovered incidentally during endoscopic examinations. It is exceedingly rare for an IFP to prolapse into the duodenum and results in incomplete obstruction of the pylorus. PATIENT CONCERNS: A 64-year-old male patient was admitted to the hospital with recurrent episodes of melena over a 6-month period, along with complaints of dizziness and fatigue in the past 10 days. DIAGNOSES: Gastroscopy showed a giant polypoid mass on the posterior wall of the gastric antrum, prolapsing into the duodenum. Abdominal computer tomography (CT) confirmed the tumor protruding into the duodenum. Pathologic examination of the resected specimen confirmed the IFP diagnosis. INTERVENTIONS: The giant tumor was completely and successfully excised using endoscopic submucosal dissection (ESD). After the surgery, the patient underwent acid suppression and fluid replenishment therapy. OUTCOMES: The patient responded well to ESD and was discharged in stable condition. As of the submission of the case report, there has been no recurrence of the tumor after a 5-month follow-up, and the patient is still under follow-up. LESSONS: While IFPs have traditionally been managed surgically, ESD demonstrates promising treatment outcomes, avoiding the need for surgical distal gastrectomy, and emerges as a safe and effective treatment option.

Role of differentiated embryo-chondrocyte expressed gene 2 in immunity.

Differentiated embryo-chondrocyte expressed gene 2 (DEC2) is a member of the basic helix-loop-helix (bHLH) subfamily of transcription factors. DEC2 is implicated in tumor immunotherapy, immune system function regulation, and autoimmune diseases. DEC2 enhances Th2 cell differentiation by regulating the IL-2 and IL-4 signaling pathways and mediates the growth of B-1a cells, thereby promoting the occurrence and development of inflammatory responses. In this study, we review the reported roles of DEC2, including the regulation of immune cell differentiation and cytokine production in various cells in humans, and discuss its potential in treating autoimmune diseases and tumors.

Deubiquitinase USP4 suppresses antitumor immunity by inhibiting IRF3 activation and tumor cell-intrinsic interferon response in colorectal cancer.

Despite the approval of immune checkpoint blockade (ICB) therapy for various tumor types, its effectiveness is limited to only approximately 15% of patients with microsatellite instability-high (MSI-H) or mismatch repair deficiency (dMMR) colorectal cancer (CRC). Approximately 80%-85% of CRC patients have a microsatellite stability (MSS) phenotype, which features a rare T-cell infiltration. Thus, elucidating the mechanisms underlying resistance to ICB in patients with MSS CRC is imperative. In this study, we demonstrate that ubiquitin-specific peptidase 4 (USP4) is upregulated in MSS CRC tumors and negatively regulates the immune response against tumors in CRC. Additionally, USP4 represses the cellular interferon (IFN) response and antigen presentation and impairs PRR signaling-mediated cell death. Mechanistically, USP4 impedes the nuclear localization of interferon regulator Factor 3 (IRF3) by deubiquitinating the K63-polyubiquitin chain of TRAF6 and IRF3. Knockdown of USP4 enhances the infiltration of T cells in CRC tumors and overcomes ICB resistance in an MC38 syngeneic mouse model. Moreover, published datasets revealed that patients showing higher USP4 expression exhibited decreased responsiveness to anti-PD-L1 therapy. These findings highlight an essential role of USP4 in the suppression of antitumor immunity in CRC.

Interferon-γ in the tumor microenvironment promotes the expression of B7H4 in colorectal cancer cells, thereby inhibiting cytotoxic T cells.

The bioactivity of interferon-γ (IFN-γ) in cancer cells in the tumor microenvironment (TME) is not well understood in the current immunotherapy era. We found that IFN-γ has an immunosuppressive effect on colorectal cancer (CRC) cells. The tumor volume in immunocompetent mice was significantly increased after subcutaneous implantation of murine CRC cells followed by IFN-γ stimulation, and RNA sequencing showed high expression of B7 homologous protein 4 (B7H4) in these tumors. B7H4 promotes CRC cell growth by inhibiting the release of granzyme B (GzmB) from CD8+ T cells and accelerating apoptosis in CD8+ T cells. Furthermore, interferon regulatory factor 1 (IRF1), which binds to the B7H4 promoter, is positively associated with IFN-γ stimulation-induced expression of B7H4. The clinical outcome of patients with CRC was negatively related to the high expression of B7H4 in cancer cells or low expression of CD8 in the microenvironment. Therefore, B7H4 is a biomarker of poor prognosis in CRC patients, and interference with the IFN-γ/IRF1/B7H4 axis might be a novel immunotherapeutic method to restore the cytotoxic killing of CRC cells.

Liraglutide Improves PCOS Symptoms in Rats by Targeting FDX1.

BACKGROUND: Polycystic ovary syndrome (PCOS) is a gynecological endocrine disorder characterized by ovulatory disorders, hyperandrogenemia, and polycystic changes in the ovaries. FDX1 is a ferredoxin-reducing protein on human mitochondria that plays an important role in steroid anabolism. Liraglutide, a glucagon-like peptide-1 receptor agonist (GLP-1RA), has recently emerged as a potential therapeutic agent for PCOS. Recent studies have suggested that FDX1 may be associated with the development of PCOS. This study aims to explore the pivotal role of FDX1 in the amelioration of PCOS through liraglutide intervention. MATERIALS AND METHODS: A PCOS rat model was induced via subcutaneous DHEA injections. Following successful model establishment, the rats were treated with liraglutide combined with metformin, or with each drug individually, over a six-week period. After 6 weeks of treatment, we assessed changes in body weight, fasting blood glucose, sex hormone levels, estrous cycle regularity, ovarian morphology, FDX1 expression in ovarian tissue, and ovarian ROS levels. RESULTS: PCOS rats exhibited significant increases in body weight and fasting blood glucose levels, disrupted estrous cycles, and polycystic ovarian morphology. FDX1 expression was notably reduced in the ovarian tissues of PCOS rats. Treatment with liraglutide, both alone and in combination with metformin, led to improvements in body weight, fasting blood glucose, sex hormone balance, estrous cycle regularity, ovarian morphology, and ovarian ROS levels. Notably, FDX1 expression was significantly restored in all treatment groups, with the most substantial increase observed in the liraglutide-treated group. CONCLUSION: This study suggests that FDX1 could serve as a potential biomarker for elucidating the underlying mechanisms of liraglutide's therapeutic effects in PCOS management.

Non-invasive prediction of overall survival time for glioblastoma multiforme patients based on multimodal MRI radiomics.

Glioblastoma multiforme (GBM) is the most common and deadly primary malignant brain tumor. As GBM tumor is aggressive and shows high biological heterogeneity, the overall survival (OS) time is extremely low even with the most aggressive treatment. If the OS time can be predicted before surgery, developing personalized treatment plans for GBM patients will be beneficial. Magnetic resonance imaging (MRI) is a commonly used diagnostic tool for brain tumors with high-resolution and sound imaging effects. However, in clinical practice, doctors mainly rely on manually segmenting the tumor regions in MRI and predicting the OS time of GBM patients, which is time-consuming, subjective and repetitive, limiting the effectiveness of clinical diagnosis and treatment. Therefore, it is crucial to segment the brain tumor regions in MRI, and an accurate pre-operative prediction of OS time for personalized treatment is highly desired. In this study, we present a multimodal MRI radiomics-based automatic framework for non-invasive prediction of the OS time for GBM patients. A modified 3D-UNet model is built to segment tumor subregions in MRI of GBM patients; then, the radiomic features in the tumor subregions are extracted and combined with the clinical features input into the Support Vector Regression (SVR) model to predict the OS time. In the experiments, the BraTS2020, BraTS2019 and BraTS2018 datasets are used to evaluate our framework. Our model achieves competitive OS time prediction accuracy compared to most typical approaches.

Expression of Sphingosine-1-phosphate (S1P) on the cerebral vasospasm after subarachnoid hemorrhage in rabbits

PURPOSE:To demonstrate the relationship between of sphingosine-1-phosphate (S1P) expression and subarachnoid hemorrhage (SAH).METHODS:The basilar arteries from a "double-hemorrhage" rabbit model of SAH were used to investigate the relation between S1P expression and SAH. Various symptoms, including blood clots, basilar artery cross-sectional area, and S1P phosphatase expression were measured at day 3, 5, 7, 9.RESULTS: The expression of S1P was enhanced in the cerebral vasospasm after subarachnoid hemorrhage in the rabbits. And S1P expression was consistent with the basilar artery cross-sectional area changes at day 3, 5, 7, 9.CONCLUSION: Sphingosine-1-phosphate expression in the cerebral arterial may be a new indicator in the development of cerebral vasospasm after subarachnoid hemorrhage and provide a new therapeutic method for SAH.