Molecular Biology Experiments Research Articles

TruD technology for the study of epi- and endothelial tubes in vitro.

Beyond the smallest organisms, animals rely on tubes to transport cells, oxygen, nutrients, waste products, and a great variety of secretions. The cardiovascular system, lungs, gastrointestinal and genitourinary tracts, as well as major exocrine glands, are all composed of tubes. Paradoxically, despite their ubiquitous importance, most existing devices designed to study tubes are relatively complex to manufacture and/or utilize. The present work describes a simple method for generating tubes in vitro using nothing more than a low-cost 3D printer along with general lab supplies. The technology is termed "TruD", an acronym for true dimensional. Using this technology, it is readily feasible to cast tubes embedded in ECM with easy access to the lumen. The design is modular to permit more complex tube arrangements and to sustain flow. Importantly, by virtue of its simplicity, TruD technology enables typical molecular cell biology experiments where multiple conditions are assayed in replicate.

Ginsenoside Rh2 Reduced Neurogenesis Dysfunction to Alleviate Depression-like Behaviour in CSDS-induced Mice

Background: Depression is a mental health disorder with high morbidity and disability rates that has harmful effects on public health worldwide. Despite many hypotheses about its mechanism have been put forward, its deeper mechanism is still unclear. Nowadays, there are many antidepressants developed by pharmaceutical companies, such as fluoxetine, ketamine, and paroxetine used in clinical practice, but the therapeutic effects are not satisfactory, with serious adverse reactions and drug addiction. Objective: Ginsenoside Rh2 (G-Rh2) is one of the major bioactive ginsenosides extracted from Panax ginseng that has been conducted for the treatment of many inflammatory diseases. However, the possible mechanisms of G-Rh2 involved in these neuroprotective effects on neuroinflammation are currently unclear. Methods: We established a CSDS depression model in normal mice, and then the depressive symptoms of mice were evaluated by a series of behavioral testing. Finally, molecular biology experiments were conducted to demonstrate that G-Rh2 had anti-inflammatory and antidepressant effects. Results: We showed that G-Rh2 (30 mg/kg) significantly relieved depressive behavior in mice via attenuating oxidative stress injury and release of proinflammatory cytokines. Moreover, G-Rh2 reduced impaired neurogenesis in the hippocampus of CSDS-induced mice. Conclusion: We demonstrated the anti-inflammatory and antioxidative effects of G-Rh2 to alleviate depression through improved neurogenesis in depressive mice for the first time. What's more, we aimed to verify the protective effects of G-Rh2 on neuroinflammation and provide novel insights for understanding the roles of G-Rh2 in the treatment of depression

The Long Non-Coding RNA Obesity-Related (Obr) Contributes To Lipid Metabolism Through Epigenetic Regulation.

Obesity is a multifactorial disease that is part of today's epidemic and also increases the risk of other metabolic diseases. Long noncoding RNAs (lncRNAs) provide one tier of regulatory mechanisms to maintain metabolic homeostasis. Although lncRNAs are a significant constituent of the mammalian genome, studies aimed at their metabolic significance, including obesity, are only beginning to be addressed. Here, a developmentally regulated lncRNA, termed as obesity related (Obr), whose expression in metabolically relevant tissues such as skeletal muscle, liver, and pancreas is altered in diet-induced obesity, is identified. The Clone 9 cell line and high-fat diet-induced obese Wistar rats are used as a model system to verify the function of Obr. By using stable expression and antisense oligonucleotide-mediated downregulation of the expression of Obr followed by different molecular biology experiments, its role in lipid metabolism is verified. It is shown that Obr associates with the cAMP response element-binding protein (Creb) and activates different transcription factors involved in lipid metabolism. Its association with the Creb histone acetyltransferase complex, which includes the cAMP response element-binding protein (CBP) and p300, positively regulates the transcription of genes involved in lipid metabolism. In addition, Obr is regulated by Pparγ in response to lipid accumulation.

Abstract PO5-06-11: RPL27A promotes the growth and metastasis of triple negative breast cancer by regulating Ribosome biogenesis

Abstract Background: Ribosome is a complex structure in cells for protein synthesis. It has been considered always have a constant composition. Recent studies have revealed that the Ribosome composition of tumor cells is heterogeneous. The Ribosome protein composition, post-translational modification, rRNA modification, and rRNA variants can lead to the existence of specialized "onco-ribosomes" in tumor cells, which regulates the rate of translation and protein synthesis, and can affect the occurrence and development of tumors significantly. The abnormal expression of multiple Ribosome proteins in triple-negative breast cancer may be the target of its diagnosis and treatment, also, an independent factor in prognosis evaluation. Methods: To learn the expression levels of Ribosome biogenesis related genes in normal breast cells and abnormal breast cells with different pathological subtypes of breast cancer, the following studies have been done: 1. Bioinformatics analysis on TCGA database and single-cell sequencing data; 2. IHC detection on TNBC tissue and adjacent tissues; 3. Determine the expression level of RPL27A in TNBC cells, and analyze the correlation between the expression of RPL27A and clinical information of patients. To explore the effect of RPL27A on Ribosome biogenesis, protein synthesis and other signaling pathways through Proteomics and molecular biology experiments have been designed and tested. The effects of RPL27A on malignant biological behaviors such as proliferation, migration, infiltration, and clonal formation of TNBC cells were studied through cell biology experiments and mouse experiments. Results: The analysis of TCGA database and single-cell sequencing data showed that the RPL27A was significantly increased in TNBC group. Immunohistochemical experiments showed that RPL27A was highly expressed in TNBC tissue and was impressively positively correlated with tumor size, lymph node metastasis, and bone metastasis. There was significantly negatively correlated with overall survival (OS) and disease free survival (DFS). It can be an independent factor for evaluating the prognosis of TNBC. By knocking out RPL27A in TNBC cell line, label free proteomics analysis showed that Ribosome biogenesis, protein synthesis, cell cycle regulation and other signaling pathways were obviously down regulated. Cell biology experiments have implied that the high expression of RPL27A in TNBC promotes cell proliferation, enhances cell migration, infiltration, and cloning ability, leads to more malignant biological behavior of tumor cells. Further research demonstrates that the high expression of RPL27A in TNBC significantly activated the EIF3C signal, cause changes in the composition of Ribosome in TNBC cells, promoted the formation of "onco-ribosomes", and distinctly increased the rate of protein synthesis. The study of PDX mice and Organoid showed that the knock out of RPL27A significantly reduced the growth rate and transfer ability of TNBC. Conclusion: The obvious over expressing of RPL27A in TNBC, which can significantly promote the growth and metastasis of tumor cells by influencing the Ribosome biogenesis process. Citation Format: Weipeng Zhao, Jingyi Zhang, Ye Zhu, Xin Yang, Xichuan Li. RPL27A promotes the growth and metastasis of triple negative breast cancer by regulating Ribosome biogenesis [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO5-06-11.

PMAIP1 regulates autophagy in osteoblasts via the AMPK/mTOR pathway in osteoporosis.

Osteoporosis (OP) is a highly prevalent disorder characterized by low bone mass that severely reduces patient quality of life. Although numerous treatments for OP have been introduced in clinic, many have side effects and high costs. Therefore, there is still an unmet need for optimal solutions. Here, raw signal analysis was used to identify potential high-risk factors for OP, and the biological functions and possible mechanisms of action (MOAs) of these factors were explored via gene set enrichment analysis (GSEA). Subsequently, molecular biological experiments were performed to verify and analyze the discovered risk factors in vitro and in vivo. PMAIP1 was identified as a potential risk factor for OP and significantly suppressed autophagy in osteoblasts via the AMPK/mTOR pathway, thereby inhibiting the proliferation and differentiation of osteoblasts. Furthermore, we constructed an ovariectomy (OVX) model of OP in rats and simultaneously applied si-PMAIP1 for in vivo interference. si-PMAIP1 upregulated the expression of LC3B and p-AMPK and downregulated the expression of p-mTOR, and these effects were reversed by the autophagy inhibitor. Micro-CT revealed that, si-PMAIP1 significantly inhibited the development of osteoporosis in OVX model rats, and this therapeutic effect was attenuated by treatment with an autophagy inhibitor. This study explored the role and mechanism of PMAIP1 in OP and demonstrated that PMAIP1 may serve as a novel target for OP treatment.

Precise subcellular organelle-targeted analyses of the hepatotoxicity of rhubarb

Objective: Drug-induced liver injury (DILI) is the leading cause of acute liver failure and poses a significant challenge to human health. Rhubarb (Rheum officinale Baill.) DaHuang) has been clinically used for its heat-clearing and diuresis-promoting effects. However, its toxic effects on different organelles in the liver require further validation. Methods: We analyzed the potential targets affecting hepatotoxicity in rhubarb and the potential damage relationship with five major organelles, including microsomes, mitochondria, endoplasmic reticulum (ER), Golgi apparatus (GA), and lysosomes through ITCM/HERB databases and network pharmacology. We isolated and purified different organelles, incubated them with different fractions and monomers of rhubarb in an ATP culture system and examined the structural and functional changes in the organelles using particle size analysis and molecular biological experiments to investigate whether rhubarb affects the damage and rupture of major organelles in the liver. Results: By combining virtual predictions and experimental verification, our research confirmed that emodin isolated from the anthraquinone of rhubarb, catechin in the tannins of rhubarb, and palmitic acid in the organic acids of rhubarb caused the most significant functional and structural damage to the representative organelles. Among all the monomeric compounds, emodin caused the most damage to the microsome, mitochondria, ER, and lysosome; catechin induced microsome and GA damage; and palmitic acid caused the most damage to microsomes and GA in the liver, suggesting that rhubarb components may exert hepatotoxicity through multi-organelle injury. Conclusions: Our findings revealed that rhubarb has varying degrees of damaging effects on different organelles, which in turn affects cellular life activities by impairing organelle morphology and function. This study provides a theoretical basis and technical support for a refined analysis of the toxic components and targets of rhubarb.

MiR-29b-3p targetedly regulates VEGF to inhibit tumor progression and cisplatin resistance through Nrf2/HO-1 signaling pathway in non-small cell lung cancer.

Non-small cell lung carcinoma (NSCLC) is a common type of lung cancer. Prior investigations have elucidated the pivotal role of miR-29b-3p in restraining tumor growth and metastasis. Nonetheless, it remains to be determined whether miR-29b-3p can effectively suppress NSCLC progression and enhance the sensitivity of NSCLC cells to cisplatin. This investigation sought to determine the mechanism by which miR-29b-3p inhibited the advancement of NSCLC and mitigated resistance to cisplatin. We initially assessed miR-29b-3p and VEGF levels in NSCLC tissues and cell lines. Next, miR-29b-3p expression was elevated in NSCLC cell lines H1975 and A549 by overexpression plasmid transfection. Following this, a sequence of molecular biology experiments was conducted to evaluate the impact of miR-29b-3p on the biological behaviors of NSCLC cells and their resistance to cisplatin. Additionally, we predicted VEGF was a target gene of miR-29b-3p by bioinformatics analysis. We next employed western blot to evaluate the protein expression of Nrf2 and HO-1 in NSCLC cells. Finally, we elucidated the effects of VEGF and Nrf2/HO-1pathway on NSCLC progression and cisplatin resistance by in vitro assays. In comparison to paracancerous tissues and human normal lung epithelial cells, the expression of miR-29b-3p was notably reduced and VEGF expression was clearly elevated in NSCLC tissues and cells. Moreover, miR-29b-3p upregulated obviously suppressed the biological activities of NSCLC cells and increased their sensitivity to cisplatin. Furthermore, in NSCLC cells, miR-29b-3p bound to VEGF and negatively regulate its transcription. Additionally, miR-29b-3p overexpression also inhibited the Nrf2/HO-1 signaling pathway. Finally, the overexpression of VEGF and the activation of the Nrf2/HO-1 pathway reversed miR-29b-3p-mediated inhibitory effect on biological behaviors of NSCLC cells and increased the cisplatin resistance. Our findings indicate that miR-29b-3p impedes NSCLC cells' biological behaviors and augments their sensitivity to cisplatin by targeting VEGF to modulate the Nfr2/HO-1 signaling pathway.

Molecular subtyping and the construction of a predictive model of colorectal cancer based on ion channel genes

PurposeColorectal cancer (CRC) is a highly heterogeneous malignancy with an unfavorable prognosis. The purpose of this study was to address the heterogeneity of CRC by categorizing it into ion channel subtypes, and to develop a predictive modeling based on ion channel genes to predict the survival and immunological states of patients with CRC. The model will provide guidance for personalized immunotherapy and drug treatment.MethodsA consistent clustering method was used to classify 619 CRC samples based on the expression of 279 ion channel genes. Such a method was allowed to investigate the relationship between molecular subtypes, prognosis, and immune infiltration. Furthermore, a predictive modeling was constructed for ion channels to evaluate the ion channel properties of individual tumors using the least absolute shrinkage and selection operator. The expression patterns of the characteristic genes were validated through molecular biology experiments. The effect of potassium channel tetramerization domain containing 9 (KCTD9) on CRC was verified by cellular functional experiments.ResultsFour distinct ion channel subtypes were identified in CRC, each characterized by unique prognosis and immune infiltration patterns. Notably, Ion Cluster3 exhibited high levels of immune infiltration and a favorable prognosis, while Ion Cluster4 showed relatively lower levels of immune infiltration and a poorer prognosis. The ion channel score could predict overall survival, with lower scores correlated with longer survival. This score served as an independent prognostic factor and presented an excellent predictive efficacy in the nomogram. In addition, the score was closely related to immune infiltration, immunotherapy response, and chemotherapy sensitivity. Experimental evidence further confirmed that low expression of KCTD9 in tumor tissues was associated with an unfavorable prognosis in patients with CRC. The cellular functional experiments demonstrated that KCTD9 inhibited the proliferation, migration and invasion capabilities of LOVO cells.ConclusionsIon channel subtyping and scoring can effectively predict the prognosis and evaluate the immune microenvironment, immunotherapy response, and drug sensitivity in patients with CRC.

Baohuoside I inhibits virulence of multidrug-resistant Staphylococcus aureus by targeting the transcription Staphylococcus accessory regulator factor SarZ

BackgroundStaphylococcus aureus is a versatile pathogen that can cause a wide range of infections in humans. Biofilms play a crucial role in the pathogenicity of S. aureus and contribute to its ability to cause persistent and chronic infections. Baohuoside I has garnered increasing recognition as a natural flavonol glycoside with a wide spectrum of health-related activities. PurposeThe antibacterial and anti-biofilm properties of Baohuoside I have not been extensively investigated. Our study aimed to assess its inhibitory effects and the underlying mechanisms on biofilm formation and hemolytic capacity in S. aureus. Study Design/MethodsThe impact of Baohuoside I on the biofilm and virulence of S. aureus was evaluated through in vitro experiments and Galleria mellonella as an in vivo infection model. The mechanisms were explored by Drug affinity responsive target stability (DARTS) and validated in genetic knockout strain and through molecular biological experiments using DARTS, molecular docking, electrophoretic mobility shift assay (EMSA), and bio-layer interferometry (BLI). ResultsBaohuoside I significantly inhibits the formation of S. aureus biofilms and hemolytic activity at 6.25 µM. Proteomics analysis revealed that treatment with Baohuoside I led to a reduction in the expression of quorum-sensing system agr-regulated genes. DARTS analysis identified Staphylococcus accessory regulator factor (SarZ), a key regulator involved in the expression of virulence factors in S. aureus by acting as activator of the agr quorum-sensing system, was the direct target of Baohuoside I. Molecular docking, DARTS, BLI and EMSA assays collectively confirmed the direct binding of Baohuoside I to SarZ, inhibiting its binding to downstream promoters. Furthermore, it is found through site-directed protein mutagenesis that the Tyr27 and Phe117 residues are key for Baohuoside I binding to SarZ. Additionally, the knockout of SarZ significantly diminished the hemolytic ability of S. aureus, underscoring its crucial role as a pivotal regulator of virulence. Lastly, in vivo tests utilizing the G. mellonella infection model demonstrated the efficacy of Baohuoside I. ConclusionThis study provides valuable insights into the mechanism by which Baohuoside I inhibits the virulence of S. aureus through its interaction with SarZ. These findings highlight the significance of SarZ as an effective target against the virulence of S. aureus.

Potential value of expression of receptor accessory protein 4 for evaluating the prognosis of lower-grade glioma patients.

REEP4 is involved in the regulation of the biological process of mitosis. Lower grade glioma (LGG), as a malignant tumor, is accompanied by abnormalities in mitosis, but there have been no reports of REEP4 so far. We collected transcriptome data, DNA methylation data and the clinical characteristics of thousands of patients with LGG. Various big data analysis methods and molecular biology experiments were employed to reveal the impact of REEP4 on the pathological process of LGG. It was found that the expression of REEP4 was significantly elevated and negatively regulated by its methylation site. Therefore, both the high expression of REEP4 and low methylation state of cg16311504 showed that the patients are correlated with lower patient survival rate. In addition, high REEP4 expression participates in the regulation of various cancer-related cellular signaling pathways, such as the cell cycle, MAPK signaling pathway, NOD-like receptor signaling pathway, etc. More importantly, the level of immune cell infiltration significantly increased in the high expression group of REEP4 in the LGG tumor microenvironment and REEP4 has a high positive correlation with PD-L1 and other immune checkpoints. In brief, this study is the first to introduce REEP4 in malignant tumors, which can be used as an independent risk factor that participates in the malignant process of LGG. More importantly, REEP4 has the potential to become a new star in the field of anti-tumor treatment.

CircHAS2 activates CCNE2 to promote cell proliferation and sensitizes the response of colorectal cancer to anlotinib

BackgroundTyrosine kinase inhibitors (TKIs) are crucial in the targeted treatment of advanced colorectal cancer (CRC). Anlotinib, a multi-target TKI, has previously been demonstrated to offer therapeutic benefits in previous studies. Circular RNAs (circRNAs) have been implicated in CRC progression and their unique structural stability serves as promising biomarkers. The detailed molecular mechanisms and specific biomarkers related to circRNAs in the era of targeted therapies, however, remain obscure.MethodsThe whole transcriptome RNA sequencing and function experiments were conducted to identify candidate anlotinib-regulated circRNAs, whose mechanism was confirmed by molecular biology experiments. CircHAS2 was profiled in a library of patient-derived CRC organoids (n = 22) and patient-derived CRC tumors in mice. Furthermore, a prospective phase II clinical study of 14 advanced CRC patients with anlotinib-based therapy was commenced to verify drug sensitivity (ClinicalTrials.gov identifier: NCT05262335).ResultsAnlotinib inhibits tumor growth in vitro and in vivo by downregulating circHAS2. CircHAS2 modulates CCNE2 activation by acting as a sponge for miR-1244, and binding to USP10 to facilitate p53 nuclear export as well as degradation. In parallel, circHAS2 serves as a potent biomarker predictive of anlotinib sensitivity, both in patient-derived organoids and xenograft models. Moreover, the efficacy of anlotinib inclusion into the treatment regimen yields meaningful clinical responses in patients with high levels of circHAS2. Our findings offer a promising targeted strategy for approximately 52.9% of advanced CRC patients who have high circHAS2 levels.ConclusionsCircHAS2 promotes cell proliferation via the miR-1244/CCNE2 and USP10/p53/CCNE2 bidirectional axes. Patient-derived organoids and xenograft models are employed to validate the sensitivity to anlotinib. Furthermore, our preliminary Phase II clinical study, involving advanced CRC patients treated with anlotinib, confirmed circHAS2 as a potential sensitivity marker.

Pan-cancer analysis of NUP155 and validation of its role in breast cancer cell proliferation, migration, and apoptosis

NUP155 is reported to be correlated with tumor development. However, the role of NUP155 in tumor physiology and the tumor immune microenvironment (TIME) has not been previously examined. This study comprehensively investigated the expression, immunological function, and prognostic significance of NUP155 in different cancer types. Bioinformatics analysis revealed that NUP155 was upregulated in 26 types of cancer. Additionally, NUP155 upregulation was strongly correlated with advanced pathological or clinical stages and poor prognosis in several cancers. Furthermore, NUP155 was significantly and positively correlated with DNA methylation, tumor mutational burden, microsatellite instability, and stemness score in most cancers. Additionally, NUP155 was also found to be involved in TIME and closely associated with tumor infiltrating immune cells and immunoregulation-related genes. Functional enrichment analysis revealed a strong correlation between NUP155 and immunomodulatory pathways, especially antigen processing and presentation. The role of NUP155 in breast cancer has not been examined. This study, for the first time, demonstrated that NUP155 was upregulated in breast invasive carcinoma (BRCA) cells and revealed its oncogenic role in BRCA using molecular biology experiments. Thus, our study highlights the potential value of NUP155 as a biomarker in the assessment of prognostic prediction, tumor microenvironment and immunotherapeutic response in pan-cancer.

CircALMS1 Alleviates Pulmonary Microvascular Endothelial Cell Dysfunction in Pulmonary Hypertension.

Circular RNAs can serve as regulators influencing the development of pulmonary hypertension (PH). However, their function in pulmonary vascular intimal injury remains undefined. Thus, we aimed to identify specifically expressed circular RNAs in pulmonary microvascular endothelial cells (PMECs) under hypoxia and PH. Deep RNA sequencing and quantitative real-time polymerase chain reaction revealed that circALMS1 (circular RNA Alstrom syndrome protein 1) was reduced in human PMECs under hypoxia (P<0.0001). Molecular biology and histopathology experiments were used to elucidate the roles of circALMS1 in regulating PMEC dysfunction among patients with PH. The circALMS1 expression was decreased in the plasma of patients with PH (P=0.0315). Patients with lower circALMS1 levels had higher risk of death (P=0.0006). Moreover, the circALMS1 overexpression of adeno-associated viruses improved right ventricular function and reduced pulmonary vascular remodeling in monocrotaline-PH and sugen/hypoxia-PH rats (P<0.05). Furthermore, circALMS1 overexpression promoted apoptosis and inhibited PMEC proliferation and migration under hypoxia by directly downregulating miR-17-3p (P<0.05). Dual luciferase assay confirmed the direct binding of circALMS1 to miR-17-3p and miR-17-3p binding to its target gene YT521-B homology domain-containing family protein 2 (YTHDF2) (P<0.05). The YTHDF2 levels were also downregulated in hypoxic PMECs (P<0.01). The small interfering RNA YTHDF2 reversed the effects of miR-17-3p inhibitors on PMEC proliferation, migration, and apoptosis. Finally, the results indicated that, although YTHDF2, as an N(6)-methyladenosine reader protein, contributes to the degradation of many circular RNAs, it could not regulate the circALMS1 levels in PMECs (P=0.9721). Our study sheds new light on circALMS1-regulated dysfunction of PMECs by the miR-17-3p/YTHDF2 pathway under hypoxia and provides insights into the underlying pathogenesis of PH.

GW9508 ameliorates cognitive dysfunction via autophagy pathway in streptozotocin-induced mouse model of Alzheimer's disease.

G protein-coupled receptor 40 (GPR40) is a potential drug target for Alzheimer's disease (AD), and its agonist GW9508 ameliorates cognitive impairment by intravenous administration. The present study was conducted to investigate the efficacy of GW9508 administered peripherally on cognitive dysfunction in streptozotocin (STZ)-induced AD mice. Seventy male ICR mice were randomly divided into seven groups: vehicle sham group, model, Donepezil, GW9508-L, GW9508-M, GW9508-H, and GW1100 + GW9508-H groups, and administered either vehicle (artificial cerebrospinal fluid [aCSF]) or STZ (3mg/kg in the vehicle) once a day (9:00 a.m.) by intracerebroventricular injection bilaterally on day 1 and day 3, respectively. After 2weeks of recovery, all mice were given drug treatment. Behavioral experiments were applied to test the recognition and spatial memory of mice, while molecular biology experiments such as Western blot, ELISA, and Nissl staining were used to detect the corresponding changes of signaling pathways. Intraperitoneal administration of GW9508 prevented STZ-induced cognitive impairment as well as decreased the level of p-tau and Aβ1-42 in plasma and brain. GW9508 upregulated the expression of gut-brain peptides like PYY, CCK, IGF-1, and GLP-1 both in blood circulation and brain and downregulated the expression level of autophagy-related proteins through activating Akt/mTOR signaling pathway. Meanwhile, the treatment effect of GW9508 was reversed by GPR40 antagonist GW1100 significantly. Peripheral administration of GW9508 exhibits neuroprotective effects, and it could be a promising therapy for AD. The neuroprotective mechanism of GW9508 was based on promoting gut-brain peptide secretion, activating Akt/mTOR signal pathway, and regulating neuronal autophagy.

Preliminary exploration of animal models of congenital choledochal cysts.

Various animal models have been used to explore the pathogenesis of choledochal cysts (CCs), but with little convincing results. Current surgical techniques can achieve satisfactory outcomes for treatment of CCs. Consequently, recent studies have focused more on clinical issues rather than basic research. Therefore, we need appropriate animal models to further basic research. To establish an appropriate animal model that may contribute to the investigation of the pathogenesis of CCs. Eighty-four specific pathogen-free female Sprague-Dawley rats were randomly allocated to a surgical group, sham surgical group, or control group. A rat model of CC was established by partial ligation of the bile duct. The reliability of the model was confirmed by measurements of serum biochemical indices, morphology of common bile ducts of the rats as well as molecular biology experiments in rat and human tissues. Dilation classified as mild (diameter, ≥ 1 mm to < 3 mm), moderate (≥ 3 mm to < 10 mm), and severe (≥ 10 mm) was observed in 17, 17, and 2 rats in the surgical group, respectively, while no dilation was observed in the control and sham surgical groups. Serum levels of alanine aminotransferase, aspartate aminotransferase, total bilirubin, direct bilirubin, and total bile acids were significantly elevated in the surgical group as compared to the control group 7 d after surgery, while direct bilirubin, total bilirubin, and gamma-glutamyltransferase were further increased 14 d after surgery. Most of the biochemical indices gradually decreased to normal ranges 28 d after surgery. The protein expression trend of signal transducer and activator of transcription 3 in rat model was consistent with the human CC tissues. The model of partial ligation of the bile duct of juvenile rats could morphologically simulate the cystic or fusiform CC, which may contribute to investigating the pathogenesis of CC.

Natural intestinal metabolite xylitol reduces BRD4 levels to mitigate renal fibrosis.

Renal fibrosis is a typical pathological change from chronic kidney disease (CKD) to end-stage renal failure, which presents significant challenges in prevention and treatment. The progression of renal fibrosis is closely associated with the "gut-kidney axis," therefore, although clinical intervention to modulate the "gut-kidney axis" imbalance associated with renal fibrosis brings hope for its treatment. In this study, we first identified the close relationship between renal fibrosis development and the intestinal microenvironment through fecal microtransplantation and non-absorbable antibiotics experiments. Then, we analyzed the specific connection between the intestinal microenvironment and renal fibrosis using microbiomics and metabolomics, screening for the differential intestinal metabolite. Potential metabolite action targets were initially identified through network simulation of molecular docking and further verified by molecular biology experiment. We used flow cytometry, TUNEL apoptosis staining, immunohistochemistry, and Western blotting to assess renal injury and fibrosis extent, exploring the potential role of gut microbial metabolite in renal fibrosis development. We discovered that CKD-triggered alterations in the intestinal microenvironment exacerbate renal injury and fibrosis. When metabolomic analysis was combined with experiments invivo, we found that the differential metabolite xylitol delays renal injury and fibrosis development. We further validated this hypothesis at the cellular level. Mechanically, bromodomain-containing protein 4 (BRD4) protein exhibits strong binding with xylitol, and xylitol alleviates renal fibrosis by inhibiting BRD4 and its downstream transforming growth factor-β (TGF-β) pathway. In summary, our findings suggest that the natural intestinal metabolite xylitol mitigates renal fibrosis by inhibiting the BRD4-regulated TGF-β pathway.

Comprehensive multi-omics analysis and experimental verification reveal PFDN5 is a novel prognostic and therapeutic biomarker for gastric cancer

Prefoldin Subunit 5 (PFDN5) plays a critical role as a member of the prefoldins (PFDNs) in maintaining a finely tuned equilibrium between protein production and degradation. However, there has been no comprehensive analysis specifically focused on PFDN5 thus far. Here, a comprehensive multi-omics (transcriptomics, genomics, and proteomics) analysis, systematic molecular biology experiments (in vitro and in vivo), transcriptome sequencing and PCR Array were performed for identifying the value of PFDN5 in pan-cancer, especially in Gastric Cancer (GC). We found PFDN5 had the potential to serve as a prognostic and therapeutic biomarker in GC. And PFDN5 could promote the proliferation of GC cells, primarily by affecting the cell cycle, cell death and immune process etc. These findings provide novel insights into the molecular mechanisms and precise treatments of in GC.

Changes in arterial myocyte excitability induced by subarachnoid hemorrhage in a rat model

Aneurismal subarachnoid hemorrhage (aSAH) is a neurovascular disease produced by the rupture of the cerebral arteries and the extravasation of blood to the subarachnoid space and is accompanied by severe comorbidities. Secondarily associated vasospasm is one of the main side effects after hydrocephalus and possible rebleeding. Here, we analyze the alterations in function in the arteries of a rat model of SAH.For this, autologous blood was injected into the cisterna magna. We performed electrophysiological, microfluorimetric, and molecular biology experiments at different times after SAH to determine the functional and molecular changes induced by the hemorrhage. Our results confirmed that in SAH animals, arterial myocytes were depolarized on days 5 and 7, had higher [Ca2+]i on baseline, peaks and plateaus, and were more excitable at low levels of depolarization on day 7, than in the control and sham animals. Microarray analysis showed that, on day 7, the sets of genes related to voltage-dependent Ca2+ channels and K+ dynamics in SAH animals decreased, while the voltage-independent Ca2+ dynamics genes were over-represented. In conclusion, after SAH, several mechanisms involved in arterial reactivity were altered in our animal model, suggesting that there is no unique cause of vasospasm and alterations in several signaling pathways are involved in its development.

Predicting potential target genes in molecular biology experiments using machine learning and multifaceted data sources

Experimental analysis of functionally related genes is key to understanding biological phenomena. The selection of genes to study is a crucial and challenging step, as it requires extensive knowledge of the literature and diverse biomedical data resources. Although software tools that predict relationships between genes are available to accelerate this process, they do not directly incorporate experiment information derived from the literature. Here, we develop LEXAS, a target gene suggestion system for molecular biology experiments. LEXAS is based on machine learning models trained with diverse information sources, including 24 million experiment descriptions extracted from full-text articles in PubMed Central by using a deep-learning-based natural language processing model. By integrating the extracted experiment contexts with biomedical data sources, LEXAS suggests potential target genes for upcoming experiments, complementing existing tools like STRING, FunCoup, and GOSemSim. A simple web interface enables biologists to consider newly derived gene information while planning experiments.

Sophoricoside ameliorates cerebral ischemia-reperfusion injury dependent on activating AMPK

AimsIschemic stroke accounts for 87% of all strokes, and its death and disability bring a huge burden to society. Brain injury caused by ischemia-reperfusion (I/R) is also a major difficulty in clinical treatment and prognosis. Sophoricoside (SOP) is an isoflavone glycoside isolated from the seed of medical herb Sophora japonica L. Previously, SOP was found to be effective in anti-inflammation and glucose-lipid metabolism-related diseases. In order to investigate whether SOP has a regulatory effect on cerebral I/R injury, we conducted this study. MethodsHere, by application of SOP into MCAO (transient middle cerebral artery occlusion)-induced mice and OGD/R (oxygen glucose deprivation/reperfusion)-induced primary neurons, the regulation effects of SOP was analyzed by detecting neurological score of post-stroke mice, phenotypes of brains and brain sections, cell viabilities, and apoptosis- and inflammation-regulation. RNA sequencing and molecular biology experiments were performed to explore the mechanism of SOP regulating cerebral I/R injury. ResultsSOP administration decreased the infarct size, neurological deficit score, neuronal cell injury, inflammation and apoptosis. Mechanistically, SOP exerted its protective effect by activating the AMP-activated protein kinase (AMPK) signaling pathway. ConclusionSOP inhibits cerebral I/R injury by promoting the phosphorylation of AMPK.