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Bio-protocol Jun 2024The Auxin-inducible degron (AID) system is a genetic tool that induces rapid target protein depletion in an auxin-dependent manner. Recently, two advanced AID...
The Auxin-inducible degron (AID) system is a genetic tool that induces rapid target protein depletion in an auxin-dependent manner. Recently, two advanced AID systems-the super-sensitive AID and AID 2-were developed using an improved pair of synthetic auxins and mutated TIR1 proteins. In these AID systems, a nanomolar concentration of synthetic auxins is sufficient as a degradation inducer for target proteins. However, despite these advancements, AID systems still require the fusion of an AID tag to the target protein for degradation, potentially affecting its function and stability. To address this limitation, we developed an affinity linker-based super-sensitive AID (AlissAID) system using a single peptide antibody known as a nanobody. In this system, the degradation of GFP- or mCherry-tagged target proteins is induced in a synthetic auxin (5-Ad-IAA)-dependent manner. Here, we introduce a simple method for generating AlissAID strains targeting GFP or mCherry fusion proteins in budding yeasts. Key features • AlissAID system enables efficient degradation of the GFP or mCherry fusion proteins in a 5-Ad-IAA-depending manner. • Transforming the pAlissAID plasmids into strains with GFP- or mCherry- tagged proteins.
PubMed: 38948262
DOI: 10.21769/BioProtoc.5019 -
Journal of Cancer 2024Tumor hypoxia has been frequently detected in nasopharyngeal carcinoma (NPC) and is intently associated with therapeutic resistance. The aim of the study is to...
Establishment and Application of Novel Hypoxia-driven Dual-reporter Model to Investigate Hypoxic Impact on Radiation Sensitivity in Human Nasopharyngeal Carcinoma Xenografts.
Tumor hypoxia has been frequently detected in nasopharyngeal carcinoma (NPC) and is intently associated with therapeutic resistance. The aim of the study is to establish a clonogenically stable hypoxia-inducible dual reporter model and apply it to investigate the effect of tumor hypoxia on DNA double strand break (DSB) and synergistic effect of irradiation in combination with chemotherapy or targeted therapy. The plasmid vector consisting of hypoxia response elements to regulate HSV1-TK and GFP genes, was constructed and stably transfected into human NPC cells. The expected clone was identified and validated by and assay. DSB repair was measured by γH2AX foci formation. Tumor growth delay assay and spatial biodistribution of various biomarkers was designed to investigate the anti-tumor effect. The system has the propensity of high expression of reporter genes under hypoxia and low to no expression under normoxia. Intratumoral biodistributions of GFP and classic hypoxic biomarkers were identical in poor-perfused region. Upon equilibration with 10% O, the xenografts showed higher expression of hypoxic biomarkers. Cisplatin radiosensitized SUNE-1/HRE cells under hypoxia by suppressing DSB repair while the addition of PI3K/mTOR inhibitor further enhanced the anti-tumoral therapeutic efficacy. Combination of IR, DDP and NVP-BEZ235 exhibited most effective anti-tumor response . These observations underline the importance of dual reporter model for imaging tumor hypoxia in therapeutic study. Our preclinical model enables the investigation of heterogeneous tumor hypoxic regions in xenograft tissues and explores the treatment efficacy of combinations of various therapeutic approaches to overcome hypoxia.
PubMed: 38947402
DOI: 10.7150/jca.96378 -
Journal of Cancer 2024Gastric cancer (GC) is one of the most common malignancies worldwide, with high incidence and mortality rate. Tripartite motif-containing 28 (TRIM28) is an important...
Gastric cancer (GC) is one of the most common malignancies worldwide, with high incidence and mortality rate. Tripartite motif-containing 28 (TRIM28) is an important molecule that affects the occurrence and development of tumors, but its function in GC has not been elucidated clearly. The purpose of this study is to explore the molecular mechanism by which TRIM28 affect the GC. TRIM28 expression was tested in RNA-seq data from TCGA database, tumor tissue samples from patients and GC cell lines. Genes were silenced or overexpressed by siRNA, lentivirus-mediated shRNA, or plasmids. Cell Counting Kit-8 (CCK-8) and colony formation assays were performed to explore the proliferation of GC cells after TRIM28 knockdown. RNA-seq and TCGA database were used to identify target genes. Luciferase report assay was employed to detect the possible mechanism between TRIM28 and Indoleamine 2,3-dioxygenase (IDO1). Tryptophan concentration in cell supernatant was measured using a fluorometric assay kit. MGC-803 and 746T cells were injected into mice to establish xenograft animal models. The expression of TRIM28 was positively correlated with tumor size and poorer prognosis. Upregulation of TRIM28 was observed in GC tissues and cells. , we proved that knockdown of TRIM28 significantly inhibited the proliferation of GC cells. Then TRIM28 was found to be positively correlated with the expression of IDO1 in GC cells. In accordance with this, tryptophan levels in cell supernatants were increased in TRIM28 knockdown GC cells and overexpression of IDO1 could reverse this phenotype. Serum response factor (SRF), a reported regulator of IDO1, was also regulated by TRIM28 in GC cells. And decreased expression of IDO1 induced by TRIM28 knockdown could be partly reversed through overexpression of serum response factor (SRF) in GC cells. Functional research demonstrated that the expression of IDO1 was increased in GC and IDO1 knockdown could also inhibited the proliferation of GC cells. Furthermore, overexpression of IDO1 could partly reverse proliferation inhibited by TRIM28 knockdown in GC cells. , knockdown of TRIM28 significantly inhibited the tumor growth and overexpression of IDO1 and SRF both could reverse proliferation inhibited by TRIM28 knockdown. TRIM28 is crucial in the development of GC, and may regulate IDO1 through SRF. TRIM28 promote GC cell proliferation through SRF/IDO1 axis.
PubMed: 38947391
DOI: 10.7150/jca.95094 -
Frontiers in Cellular and Infection... 2024Convergence of (KP) pathotypes has been increasingly reported in recent years. These pathogens combine features of both multidrug-resistant and hypervirulent KP....
BACKGROUND
Convergence of (KP) pathotypes has been increasingly reported in recent years. These pathogens combine features of both multidrug-resistant and hypervirulent KP. However, clinically used indicators for hypervirulent KP identification, such as hypermucoviscosity, appear to be differentially expressed in convergent KP, potential outbreak clones are difficult to identify. We aimed to fill such knowledge gaps by investigating the temperature dependence of hypermucoviscosity and virulence in a convergent KP strain isolated during a clonal outbreak and belonging to the high-risk sequence type (ST)307.
METHODS
Hypermucoviscosity, biofilm formation, and mortality rates in larvae were examined at different temperatures (room temperature, 28°C, 37°C, 40°C and 42°C) and with various phenotypic experiments including electron microscopy. The underlying mechanisms of the phenotypic changes were explored via qPCR analysis to evaluate plasmid copy numbers, and transcriptomics.
RESULTS
Our results show a temperature-dependent switch above 37°C towards a hypermucoviscous phenotype, consistent with increased biofilm formation and mortality, possibly reflecting a bacterial response to fever-like conditions. Furthermore, we observed an increase in plasmid copy number for a hybrid plasmid harboring carbapenemase and genes. However, transcriptomic analysis revealed no changes in expression at higher temperatures, suggesting alternative regulatory pathways.
CONCLUSION
This study not only elucidates the impact of elevated temperatures on hypermucoviscosity and virulence in convergent KP but also sheds light on previously unrecognized aspects of its adaptive behavior, underscoring its resilience to changing environments.
Topics: Klebsiella pneumoniae; Biofilms; Virulence; Animals; Klebsiella Infections; Temperature; Larva; Plasmids; Moths; Humans; Virulence Factors; Bacterial Proteins; Lepidoptera; Viscosity; Phenotype; Gene Expression Profiling
PubMed: 38947124
DOI: 10.3389/fcimb.2024.1411286 -
Frontiers in Microbiology 2024Cotrimoxazole, the combined formulation of sulfamethoxazole and trimethoprim, is one of the treatments of choice for several infectious diseases, particularly urinary...
Cotrimoxazole, the combined formulation of sulfamethoxazole and trimethoprim, is one of the treatments of choice for several infectious diseases, particularly urinary tract infections. Both components of cotrimoxazole are synthetic antimicrobial drugs, and their combination was introduced into medical therapeutics about half a century ago. In Gram-negative bacteria, resistance to cotrimoxazole is widespread, being based on the acquisition of genes from the auxiliary genome that confer resistance to each of its antibacterial components. Starting from previous knowledge on the genotype of resistance to sulfamethoxazole in a collection of cotrimoxazole resistant uropathogenic strains, this work focused on the identification of the genetic bases of the trimethoprim resistance of these same strains. Molecular techniques employed included PCR and Sanger sequencing of specific amplicons, conjugation experiments and NGS sequencing of the transferred plasmids. Mobile genetic elements conferring the trimethoprim resistance phenotype were identified and included integrons, transposons and single gene cassettes. Therefore, strains exhibited several ways to jointly resist both antibiotics, implying different levels of genetic linkage between genes conferring resistance to sulfamethoxazole () and trimethoprim (). Two structures were particularly interesting because they represented a highly cohesive arrangements ensuring cotrimoxazole resistance. They both carried a single gene cassette, or , integrated in two different points of a conserved cluster , carried on transferable plasmids. The results suggest that the pressure exerted by cotrimoxazole on bacteria of our environment is still promoting the evolution toward increasingly compact gene arrangements, carried by mobile genetic elements that move them in the genome and also transfer them horizontally among bacteria.
PubMed: 38946902
DOI: 10.3389/fmicb.2024.1395953 -
Frontiers in Microbiology 2024The dissemination of carbapenem-resistant Enterobacteriales (CRE) in nosocomial settings is primarily associated with the horizontal transfer of plasmids. However,...
INTRODUCTION
The dissemination of carbapenem-resistant Enterobacteriales (CRE) in nosocomial settings is primarily associated with the horizontal transfer of plasmids. However, limited research has focused on the in-host transferability of carbapenem resistance. In this study, ten isolates were collected from gut specimens of five individuals, each hosting two different species, including , , , , or .
METHODS
Species identification and antimicrobial susceptibility were determined by MALDI-TOF MS and broth microdilution method. Carbapenemase genes were detected and localized using PCR, S1-PFGE and southern blot. The transferability of carbapenemase genes between species was investigated through filter mating experiments, and the genetic contexts of the plasmids were analyzed using whole genome sequencing.
RESULTS AND DISCUSSION
Our results revealed that each of the ten isolates harbored a carbapenemase gene, including , , or , on a plasmid. Five different plasmids were successfully transferred to recipient cells of or by transconjugation. The genetic contexts of the carbapenemase gene were remarkably similar between the two CRE isolates from each individual. This study highlights the potential for interspecies plasmid transmission in human gut, emphasizing the colonization of CRE as a significant risk factor for the dissemination of carbapenemase genes within the host. These findings underscore the need for appropriate intestinal CRE screening and colonization prevention.
PubMed: 38946899
DOI: 10.3389/fmicb.2024.1416454 -
World Journal of Clinical Oncology Jun 2024Tankyrase 2 (TNKS2) is a potential candidate molecular target for the prognosis and treatment of non-small cell lung cancer (NSCLC), but its biological functions are...
BACKGROUND
Tankyrase 2 (TNKS2) is a potential candidate molecular target for the prognosis and treatment of non-small cell lung cancer (NSCLC), but its biological functions are unclear.
AIM
To investigate the biological functions of TNKS2 in NSCLC.
METHODS
Using a lentiviral vector, we generated H647 model cells with TNKS2 knockdown by RNA interference and A549 model cells with TNKS2 overexpression by transfection with a TNKS2 overexpressing plasmid. Increased and decreased expression levels of TNKS2 in the two cell lines were verified using real-time reverse transcriptase-polymerase chain reaction and Western blot analyses. Cell apoptosis, proliferation, and migration were determined using flow cytometry, carboxyfluorescein succinimidyl ester staining, and scratch assay, respectively. Immunofluorescence staining was conducted to examine TNKS2 and β-catenin expression levels in the two transfected cell lines and the non-transfected cells.
RESULTS
TNKS2 mRNA and protein expression was significantly higher in the highly malignant NCI-H647 cells, while it remained at a low level in the less malignant A549 cells. Lentivirus-mediated overexpression of TNKS2 in A549 cells resulted in a 3-fold increase in gene expression and a 1.7-fold increase in protein expression ( < 0.01). Conversely, shRNA interference targeting Led to an 8-fold decrease in gene expression and a 3-fold decrease in protein expression ( < 0.01) in NCI-H647 cells. Furthermore, the cell apoptosis rate was significantly reduced (50%) and cell migration rate was increased (35%) in the TNKS2 overexpression group than in the control group ( < 0.05). In contrast, sh promoted apoptosis by more than one fold and reduced migration by 60% ( < 0.05). Immunofluorescence analysis revealed enhanced nuclear localization of β-catenin fluorescence signal associated with high TNKS2 expression levels. Western blot analysis investigating TNKS2/β-catenin-related proteins indicated consistent changes between TNKS2 and β-catenin expression in lung cancer cells, whereas Axin displayed an opposite trend ( < 0.05).
CONCLUSION
The obtained results revealed that TNKS2 may serve as an adverse prognostic factor and a potential therapeutic target in NSCLC.
PubMed: 38946832
DOI: 10.5306/wjco.v15.i6.755 -
Developmental Dynamics : An Official... Jun 2024The Tol2 transposable element is the most widely used transgenesis tool in zebrafish. However, its high activity almost always leads to multiple unlinked integrations of...
BACKGROUND
The Tol2 transposable element is the most widely used transgenesis tool in zebrafish. However, its high activity almost always leads to multiple unlinked integrations of the transgenic cassette in F fish. Each of these transgenes is susceptible to positional effects from the surrounding regulatory landscape, which can lead to altered expression and, consequently, activity. Scientists therefore must strike a balance between the need to maximize reproducibility by establishing single-insertion transgenic lines and the need to complete experiments within a reasonable timeframe.
RESULTS
In this article, we introduce a simple competitive dilution strategy for rapid generation of single-insertion transgenics. By using cry:BFP reporter plasmid as a competitor, we achieved a nearly fourfold reduction in the number of the transgene of interest integrations while simultaneously increasing the proportion of single-insertion F generation transgenics to over 50%. We also observed variations in transgene of interest expression among independent single-insertion transgenics, highlighting that the commonly used ubiquitous ubb promoter is susceptible to position effects.
CONCLUSIONS
Wide application of our competitive dilution strategy will save time, reduce animal usage, and improve reproducibility of zebrafish research.
PubMed: 38946125
DOI: 10.1002/dvdy.719 -
Food Research International (Ottawa,... Aug 2024Kinema, a traditional fermented soybean food from the Himalayas, is well-liked for its sticky texture and flavourful umami taste. Among 175 bacterial strains from...
Kinema, a traditional fermented soybean food from the Himalayas, is well-liked for its sticky texture and flavourful umami taste. Among 175 bacterial strains from spontaneously fermented kinema samples, Bacillus subtilis Tamang strain stood out for its high stickiness and viscosity. The strain's Poly-γ-glutamic acid (γ-PGA) contains various groups of glutamic acid and has a molecular weight of 660 kDa. It demonstrates the ability to solubilize iron, preserve ferritin in Caco-2 cells, and exhibit antibacterial properties. The genome of B. subtilis Tamang is devoid of plasmid elements but does feature nine insert elements. Noteworthy is the presence of unique secondary metabolites with potential antimicrobial effects, such as amyloliquecidin GF610, bogorol A, and thermoactinoamide A. A total of 132 carbohydrate-active enzymes (CAZy) were identified, hinting at possible prebiotic characteristics. The genome analysis revealed genes responsible for γ-PGA production via the capBCA complex. Furthermore, genes associated with fibrinolytic activity, taste enhancement, biopeptides, immunomodulators, and vitamins like B12 and K2 were found, along with probiotics and various health benefits. The genetic material for L-asparaginase production, known for its anti-cancer properties, was also detected, as well as CRISPR-Cas systems. The absence of virulence factors and antimicrobial resistance genes confirms the safety of consuming B. subtilis Tamang as a food-grade bacterium.
Topics: Bacillus subtilis; Polyglutamic Acid; Fermentation; Whole Genome Sequencing; Genome, Bacterial; Caco-2 Cells; Humans; Food Microbiology; Fermented Foods; Soy Foods; Anti-Bacterial Agents
PubMed: 38945588
DOI: 10.1016/j.foodres.2024.114655 -
European Journal of Pharmaceutics and... Jun 2024In the current "era of lipid carriers," numerous strategies have been developed to manufacture lipid nanoparticles (LNPs). Nevertheless, the potential impact of various...
In the current "era of lipid carriers," numerous strategies have been developed to manufacture lipid nanoparticles (LNPs). Nevertheless, the potential impact of various preparation methods on the characteristics, use, and/or stability of these LNPs remains unclear. In this work, we attempted to compare the effects of three different preparation methods: microfluidics (MF), reverse phase evaporation (RV), and ouzo (OZ) on lipid-peptide NPs (LPNPs) as plasmid DNA delivery carriers. These LPNPs had the same components, namely DOTMA cationic lipid, DSPC, cholesterol, and protamine. Subsequently, we compared the LPNPs in terms of their physicochemical features, functionality as gene delivery vehicles in two distinct cell lines (NT2 and D1-MSCs), and finally, their storage stability over a six-month period. It was clear that all three LPNP formulations worked to deliver EGFP-pDNA while keeping cells alive, and their physicochemical stability was high for 6 months. However, the preparation technique had a significant impact on their physicochemical characteristics. The MF produced LPNPs with a lesser size, polydispersity index, and zeta potential than the other synthesis methods. Additionally, their DNA entrapment efficiency, cell viability, and functional stability profiles were generally superior. These findings provide new insights for comparing different manufacturing methods to create LPNPs with the desired characteristics for effective and safe gene delivery.
PubMed: 38945408
DOI: 10.1016/j.ejpb.2024.114385