-
Immunopharmacology and Immunotoxicology Jul 2024This study aimed to investigate the use of 5,7,3',4'-tetramethoxyflavone (TMF) to treat pulmonary fibrosis (PF), a chronic and fatal lung disease. and models were used...
OBJECTIVE
This study aimed to investigate the use of 5,7,3',4'-tetramethoxyflavone (TMF) to treat pulmonary fibrosis (PF), a chronic and fatal lung disease. and models were used to examine the impact of TMF on PF.
METHODS
NIH-3T3 (Mouse Embryonic Fibroblast) were exposed to transforming growth factor‑β1 (TGF-β1) and treated with or without TMF. Cell growth was assessed using the MTT method, and cell migration was evaluated with the scratch wound assay. Protein and messenger ribonucleic acid (mRNA) levels of extracellular matrix (ECM) genes were analyzed by western blotting and quantitative reverse transcription-polymerase chain reaction (RT-PCR), respectively. Downstream molecules affected by TGF-β1 were examined by western blotting. , mice with bleomycin-induced PF were treated with TMF, and lung tissues were analyzed with staining techniques.
RESULTS
The results showed that TMF had no significant impact on cell growth or migration. However, it effectively inhibited myofibroblast activation and ECM production induced by TGF-β1 in NIH-3T3 cells. This inhibition was achieved by suppressing various signaling pathways, including Smad, mitogen-activated protein kinase (MAPK), phosphoinositide 3-kinase/AKT (PI3K/AKT), and WNT/β-catenin. The experiments demonstrated the therapeutic potential of TMF in reducing PF induced by bleomycin in mice, and there was no significant liver or kidney toxicity observed.
CONCLUSION
These findings suggest that TMF has the potential to effectively inhibit myofibroblast activation and could be a promising treatment for PF. TMF achieves this inhibitory effect by targeting TGF-β1/Smad and non-Smad pathways.
PubMed: 38951964
DOI: 10.1080/08923973.2024.2371150 -
BMC Medical Genomics Jul 2024This study investigates the distribution and characteristics of linezolid and vancomycin susceptibilities among Enterococcus faecalis (E. faecalis) and Enterococcus...
BACKGROUND
This study investigates the distribution and characteristics of linezolid and vancomycin susceptibilities among Enterococcus faecalis (E. faecalis) and Enterococcus faecium (E. faecium) and explores the underlying resistance mechanisms.
METHODS
A total of 2842 Enterococcus clinical isolates from patients were retrospectively collected, and their clinical data were further analyzed. The minimum inhibitory concentrations (MICs) of vancomycin and linezolid were validated by broth dilution method. The resistance genes optrA, cfr, vanA, vanB and vanM were investigated using polymerase chain reaction (PCR). Housekeeping genes and resistance genes were obtianed through whole-genome sequencing (WGS).
RESULTS
Of the 2842 Enterococcus isolates, 88.5% (2516) originated from urine, with E. faecium accounted for 60.1% of these. The vanA gene was identified in 27/28 vancomycin resistant Enterococcus (VRE) isolates, 4 of which carried both vanA and vanM genes. The remaining strain was vanM positive. The optrA gene was identified in all E. faecalis isolates among linezolid resistant Enterococcus (LRE). E. faecium showed a higher multiple antibiotic resistance index (MAR index) compared to E. faecalis. The multi-locus sequence typing (MLST) showed the sequence type of E. faecium mainly belongs to clonal complex (CC) 17, nearly E. faecalis isolates analyzed were differentiated into 7 characteristics of sequence types (STs), among which ST16 of CC16 were the major lineage.
CONCLUSION
Urine was the primary source of VRE and LRE isolates in this study. E. faecium showed higher levels of resistance compared to E. faecalis. OptrA gene was detected in 91.6% of LRE, which could explain linezolid resistance, and van genes were detected in all vancomycin resistant Enterococcus strains, while vanA was a key resistance mechanism in VRE identified in this study.
Topics: Linezolid; Humans; China; Microbial Sensitivity Tests; Enterococcus faecium; Gram-Positive Bacterial Infections; Male; Middle Aged; Enterococcus faecalis; Female; Vancomycin; Anti-Bacterial Agents; Molecular Epidemiology; Adult; Vancomycin Resistance; Aged; Retrospective Studies; Vancomycin-Resistant Enterococci; Young Adult; Enterococcus
PubMed: 38951840
DOI: 10.1186/s12920-024-01948-x -
Communications Biology Jun 2024Light is a significant factor for living organisms with photosystems, like microbial rhodopsin-a retinal protein that functions as an ion pump, channel, and sensory...
Light is a significant factor for living organisms with photosystems, like microbial rhodopsin-a retinal protein that functions as an ion pump, channel, and sensory transduction. Gloeobacter violaceus PCC7421, has a proton-pumping rhodopsin gene, the Gloeobacter rhodopsin (GR). The helix-turn-helix family of transcriptional regulators has various motifs, and they regulate gene expression in the presence of various metal ions. Here, we report that active proton outward pumping rhodopsin interacted with the helix-turn-helix transcription regulator and regulated gene expression. This interaction is confirmed using ITC analysis (K of 8 μM) and determined the charged residues required. During in vitro experiments using fluorescent and luciferase reporter systems, ATP-binding cassette (ABC) transporters and the self-regulation of G. violaceus transcriptional regulator (GvTcR) are regulated by light, and gene regulation is observed in G. violaceus using the real-time polymerase chain reaction. These results expand our understanding of the natural potential and limitations of microbial rhodopsin function.
Topics: ATP-Binding Cassette Transporters; Light; Gene Expression Regulation, Bacterial; Transcription Factors; Cyanobacteria; Proton Pumps; Bacterial Proteins; Rhodopsins, Microbial; Rhodopsin
PubMed: 38951607
DOI: 10.1038/s42003-024-06471-4 -
Mikrochimica Acta Jul 2024A stable DNA signal amplification sensor was developed on account of rolling circle amplification (RCA). This sensor includes target DNA-controlled rolling circle...
A stable DNA signal amplification sensor was developed on account of rolling circle amplification (RCA). This sensor includes target DNA-controlled rolling circle amplification technology and locking probe DNA replacement technology, which can be used to detect DNA fragments with genetic information, thus constructing a biosensor for universal detection of DNA. This study takes the homologous DNA of human immunodeficiency virus (HIV) and let-7a as examples to describe this biosensor. The padlock probe is first cyclized by T4 DNA ligase in response to the target's reaction with it. Then, rolling cycle amplification is initiated by Phi29 DNA polymerase, resulting in the formation of a lengthy chain with several triggers. These triggers can open the locked probe LP1 with the fluorescence signal turned off, so that it can continue to react with H2 to form a stable H1-H2 double strand. This regulates the distance between B-DNA modified by the quenching group and H1 modified by fluorescent group, and the fluorescence signal is recovered.
Topics: Biosensing Techniques; Nucleic Acid Amplification Techniques; Humans; DNA Probes; Fluorescent Dyes; DNA, Viral; DNA; Spectrometry, Fluorescence; Fluorescence; DNA-Directed DNA Polymerase; Limit of Detection; HIV
PubMed: 38951284
DOI: 10.1007/s00604-024-06501-2 -
Applied Microbiology and Biotechnology Jun 2024Over the past years, several methods have been developed for gene cloning. Choosing a cloning strategy depends on various factors, among which simplicity and...
Over the past years, several methods have been developed for gene cloning. Choosing a cloning strategy depends on various factors, among which simplicity and affordability have always been considered. The aim of this study, on the one hand, is to simplify gene cloning by skipping in vitro assembly reactions and, on the other hand, to reduce costs by eliminating relatively expensive materials. We investigated a cloning system using Escherichia coli harboring two plasmids, pLP-AmpR and pScissors-CmR. The pLP-AmpR contains a landing pad (LP) consisting of two genes (λ int and λ gam) that allow the replacement of the transformed linear DNA using site-specific recombination. After the replacement process, the inducible expressing SpCas9 and specific sgRNA from the pScissors-CmR (CRISPR/Cas9) vector leads to the removal of non-recombinant pLP-AmpR plasmids. The function of LP was explored by directly transforming PCR products. The pScissors-CmR plasmid was evaluated for curing three vectors, including the origins of pBR322, p15A, and pSC101. Replacing LP with a PCR product and fast-eradicating pSC101 origin-containing vectors was successful. Recombinant colonies were confirmed following gene replacement and plasmid curing processes. The results made us optimistic that this strategy may potentially be a simple and inexpensive cloning method. KEY POINTS: •The in vivo cloning was performed by replacing the target gene with the landing pad. •Fast eradication of non-recombinant plasmids was possible by adapting key vectors. •This strategy is not dependent on in vitro assembly reactions and expensive materials.
Topics: Escherichia coli; Cloning, Molecular; Plasmids; Recombination, Genetic; Polymerase Chain Reaction; Genetic Vectors; CRISPR-Cas Systems
PubMed: 38951186
DOI: 10.1007/s00253-024-13239-7 -
ACS Applied Materials & Interfaces Jul 2024Differentiation of induced pluripotent stem cells (iPSCs) is an extremely complex process that has proven difficult to study. In this research, we utilized...
Differentiation of induced pluripotent stem cells (iPSCs) is an extremely complex process that has proven difficult to study. In this research, we utilized nanotopography to elucidate details regarding iPSC differentiation by developing a nanodot platform consisting of nanodot arrays of increasing diameter. Subjecting iPSCs cultured on the nanodot platform to a cardiomyocyte (CM) differentiation protocol revealed several significant gene expression profiles that were associated with poor differentiation. The observed expression trends were used to select existing small-molecule drugs capable of modulating differentiation efficiency. BRD K98 was repurposed to inhibit CM differentiation, while iPSCs treated with NSC-663284, carmofur, and KPT-330 all exhibited significant increases in not only CM marker expression but also spontaneous beating, suggesting improved CM differentiation. In addition, quantitative polymerase chain reaction was performed to determine the gene regulation responsible for modulating differentiation efficiency. Multiple genes involved in extracellular matrix remodeling were correlated with a CM differentiation efficiency, while genes involved in the cell cycle exhibited contrasting expression trends that warrant further studies. The results suggest that expression profiles determined via short time-series expression miner analysis of nanodot-cultured iPSC differentiation can not only reveal drugs capable of enhancing differentiation efficiency but also highlight crucial sets of genes related to processes such as extracellular matrix remodeling and the cell cycle that can be targeted for further investigation. Our findings confirm that the nanodot platform can be used to reveal complex mechanisms behind iPSC differentiation and could be an indispensable tool for optimizing iPSC technology for clinical applications.
PubMed: 38951110
DOI: 10.1021/acsami.4c04451 -
Zhonghua Yi Xue Za Zhi Jul 2024To investigate the role and underlying mechanisms of intercellular adhesion molecule-1 (ICAM-1) in the adhesion and migration of mesenchymal stem cells (MSCs) in...
To investigate the role and underlying mechanisms of intercellular adhesion molecule-1 (ICAM-1) in the adhesion and migration of mesenchymal stem cells (MSCs) in patients with ankylosing spondylitis (AS). Bone marrow and ligament tissues were collected during surgery from patients with AS and thoracolumbar fractures (as controls, HC) treated from October 2021 to October 2022 at Nanjing University Medical School Affiliated Nanjing Drum Tower Hospital. MSCs were isolated and cultured from the bone marrow using the Ficoll separation method. Cell morphology was observed under high-resolution microscopy, and differences in the cytoskeletal features between AS-and HC-MSCs were analyzed through immunofluorescence staining. The expression of ICAM-1 was quantified in both groups using real-time quantitative polymerase chain reaction (RT-qPCR) and flow cytometry. Transwell migration assays and wound healing experiments were conducted to evaluate the differences in migration rates between the two groups of MSCs. The interspinous ligament and bone marrow was acquired in AS (2 males and 1 female; 33, 37, 32 years old, respectively) and no-AS patients (2 males and 1 female; 35, 32, 38 years old, respectively). AS-MSCs exhibited broader cell morphology compared to HC-MSCs under bright field and fluorescence microscopy. Immunofluorescence staining of the interspinous ligament showed higher expression of ICAM-1 (68.38±3.42 vs 48.31±2.43) and CD105 (37.97±2.16 vs 23.36±2.06) in AS patients (both <0.001). Western blot and RT-qPCR analysis revealed significantly stronger protein expression and transcription levels of ICAM-1 in AS-MSCs when compared to those in HC-MSCs (both <0.001). Flow cytometry confirmed greater mean fluorescence intensity of ICAM-1 in AS-MSCs than in that in HC-MSCs (924.30±54.99 vs 636.47±40.03, =0.002). Regarding cell adhesion efficiency, it showed no significant difference between AS-MSCs and HC-MSCs in the early stage of adhesion (0.5 h: 1 496±213 vs 1 205±163, =0.133), but they were all significantly higher in AS-MSCs in the later stage (1 h: 2 894±172 vs 1 908±155, =0.002; 2 h: 4 540±286 vs 3 334±188, =0.004; 3 h: 5 212±281 vs 4 208±303, =0.014). Finally, cell migration experiments demonstrated a stronger migration capability of AS-MSCs compared to HC-MSCs (5 449±172 vs 4 016±155, <0.001), and the inhibition efficiency of A-205804 on the migration rate of AS-MSCs was stronger than that on HC-MSCs (2 145±239 vs 3 539±316, =0.004). The aberrant expression of ICAM-1 markedly influences the adhesion and migration dynamics of MSCs. Elevated ICAM-1 levels in MSCs derives from patients with AS significantly enhance their migratory capabilities.
Topics: Humans; Intercellular Adhesion Molecule-1; Spondylitis, Ankylosing; Mesenchymal Stem Cells; Cell Movement; Adult; Cell Adhesion; Female; Male; Bone Marrow Cells; Retrospective Studies; Cells, Cultured
PubMed: 38951108
DOI: 10.3760/cma.j.cn112137-20231115-01101 -
Journal of Ethnopharmacology Jun 2024Shegan-Mahuang Decoction (SMD) is a classical formula that has been used to effectively treat cold-induced asthma (CA) for 1800 years. Airway smooth muscle cells (ASMCs)...
Shegan-Mahuang Decoction ameliorates cold-induced asthma via regulating the proliferation and apoptosis of airway smooth muscle cells through TAS2R10: An in vivo and in vitro study.
ETHNOPHARMACOLOGICAL RELEVANCE
Shegan-Mahuang Decoction (SMD) is a classical formula that has been used to effectively treat cold-induced asthma (CA) for 1800 years. Airway smooth muscle cells (ASMCs) play a crucial role in airway remodeling of CA and can be modulated through bitter taste-sensing type 2 receptors (TAS2Rs). Given that SMD contains numerous bitter herbs and TAS2R10 expression in ASMCs remains consistently high, it is pertinent to explore whether SMD regulates ASMCs via TAS2R10 to exert its CA mechanism.
AIM OF THE STUDY
This study investigated the efficacy as well as the potential mechanism of SMD in CA.
MATERIALS AND METHODS
In this study, experiments in vivo were conducted using the CA rat model induced by ovalbumin (OVA) along with cold stimulation. The effects of SMD and TAS2R10 expression in CA rats were evaluated using the following methods: clinical symptoms, weights, pathological staining, immunofluorescence staining (IF), enzyme-linked immunosorbent assay (ELISA), real-time quantitative polymerase chain reaction (RT-qPCR) and western blot (WB). Assays in vitro including cell counting Kit-8 (CCK-8), ELISA, flow cytometry, TUNEL staining, RT-qPCR and WB were performed to investigate potential mechanism of SMD on the proliferation and apoptosis of ASMCs through upregulation of TAS2R10.
RESULTS
The administration of SMD resulted in a notable improvement in the symptoms, trends in weight, airway inflammation and airway remodeling observed in CA rats with upregulated TAS2R10. Mechanistically, we furtherly confirmed that SMD inhibits p70S6K/CyclinD1 pathway by upregulating TAS2R10. SMD furthermore blocked the G0/G1 phase, suppressed the proliferation and inducted apoptosis in ASMCs induced by platelet-derived growth factor-BB (PDGF-BB). Erythromycin (EM), a TAS2R10 agonist, can intensify these effects.
CONCLUSIONS
SMD significantly ameliorates CA by upregulating TAS2R10 and inhibiting the p70S6K/CyclinD1 pathway, thereby modulating ASMCs' proliferation and apoptosis. Inspired by the Five Flavors Theory of Traditional Chinese Medicine, this study provides an updated treatment perspective for treating CA.
PubMed: 38950796
DOI: 10.1016/j.jep.2024.118504 -
International Immunopharmacology Jun 2024Imbalanced intestinal microbiota and damage to the intestinal barrier contribute to the development of necrotizing enterocolitis (NEC). Autoinducer-2 (AI-2) plays a...
BACKGROUND
Imbalanced intestinal microbiota and damage to the intestinal barrier contribute to the development of necrotizing enterocolitis (NEC). Autoinducer-2 (AI-2) plays a crucial role in repairing intestinal damage and reducing inflammation.
OBJECTIVE
This study aimed to investigate the impact of AI-2 on the expression of intestinal zonula occludens-1 (ZO-1) and occludin proteins in NEC. We evaluated its effects in vivo using NEC mice and in vitro using lipopolysaccharide (LPS)-stimulated intestinal cells.
METHODS
Pathological changes in the intestines of neonatal mice were assessed using histological staining and scoring. Cell proliferation was measured using the cell counting kit-8 (CCK-8) assay to determine the optimal conditions for LPS and AI-2 interventions. Real-time quantitative polymerase chain reaction (RT-qPCR) was used to analyze the mRNA levels of matrix metalloproteinase-3 (MMP3), protease activated receptor-2 (PAR2), interleukin-1β (IL-1β), and IL-6. Protein levels of MMP3, PAR2, ZO-1, and occludin were evaluated using western blot, immunohistochemistry, or immunofluorescence.
RESULTS
AI-2 alleviated NEC-induced intestinal damage (P < 0.05) and enhanced the proliferation of damaged IEC-6 cells (P < 0.05). AI-2 intervention reduced the mRNA and protein expressions of MMP3 and PAR2 in intestinal tissue and cells (P < 0.05). Additionally, it increased the protein levels of ZO-1 and occludin (P < 0.05), while reducing IL-1β and IL-6 mRNA expression (P < 0.05).
CONCLUSION
AI-2 intervention enhances the expression of tight junction proteins (ZO-1 and occludin), mitigates intestinal damage in NEC neonatal mice and IEC-6 cells, potentially by modulating PAR2 and MMP3 signaling. AI-2 holds promise as a protective intervention for NEC. AI-2 plays a crucial role in repairing intestinal damage and reducing inflammation.
PubMed: 38950458
DOI: 10.1016/j.intimp.2024.112567 -
Cancer Biotherapy & Radiopharmaceuticals Jul 2024This study focuses on acute myeloid leukemia (AML), a condition with a 5-year survival rate below 30% despite various treatment options. Recent strides in targeted...
This study focuses on acute myeloid leukemia (AML), a condition with a 5-year survival rate below 30% despite various treatment options. Recent strides in targeted therapies have shown promise, leading to better outcomes with minimal toxicity. These advances underscore the importance of discovering new diagnostic and prognostic targets for AML. In this context, the authors investigated the expression of microRNA-106b-5p (miR-106b-5p), Rab10 mRNA, and Rab10 proteins in peripheral blood and bone marrow (BM) samples from both healthy individuals and AML patients at different stages of the disease (initial diagnosis, recurrence, and complete remission). This examination aimed to identify potential biomarkers for AML diagnosis, treatment, and prognosis. From June 2021 to December 2022, they collected 100 BM and peripheral blood samples. The relative expression of miR-106b-5p and Rab10 mRNA in the BM of AML patients was measured using Real-time polymerase chain reaction (qRT-PCR), while the relative expression of Rab10 protein in serum was determined using the ELISA method. The chromosomal karyotype of initially diagnosed patients was analyzed using the R tape. The qRT-PCR results revealed that the expression of miR-106b-5p and Rab10 mRNA were significantly higher in patients at initial diagnosis and recurrence compared with healthy individuals and those in complete remission ( < 0.001). They observed a significant reduction in the expression of miR-106b-5p, Rab10 mRNA, and Rab10 protein in the BM and peripheral blood of patients during complete remission ( < 0.05), as demonstrated by dynamic monitoring of five patients in the initial group. Furthermore, they found a close association between the expression of miR-106b-5p and the number of white blood cells at the initial diagnosis in AML patients ( < 0.05). Spearman correlation analysis revealed a positive correlation among miR-106b-5p, Rab10 mRNA, and Rab10 proteins ( < 0.05). The diagnostic potential of miR-106b-5p and Rab10 proteins was underscored by Receiver Operating Characteristic (ROC) curve analysis, which demonstrated their high accuracy in AML diagnosis (AUC: 0.944 and 0.853, respectively; < 0.0001). Additionally, Kaplan-Meier survival analysis suggested that lower expression of these markers was associated with better prognoses ( < 0.05). In summary, their findings propose miR-106b-5p and Rab10 proteins as promising biomarkers for AML, offering insights for diagnosis, treatment, and prognosis.
PubMed: 38949985
DOI: 10.1089/cbr.2023.0191