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PloS One 2024Cluster regularly interspaced short palindromic repeats and CRISPR associated protein 9 (CRISPR-Cas9) is a promising tool for antimicrobial re-sensitization by...
Cluster regularly interspaced short palindromic repeats and CRISPR associated protein 9 (CRISPR-Cas9) is a promising tool for antimicrobial re-sensitization by inactivating antimicrobial resistance (AMR) genes of bacteria. Here, we programmed CRISPR-Cas9 with common spacers to target predominant blaCTX-M variants in group 1 and group 9 and their promoter in an Escherichia coli model. The CRISPR-Cas9 was delivered by non-replicative phagemid particles from a two-step process, including insertion of spacer in CRISPR and construction of phagemid vector. Spacers targeting blaCTX-M promoters and internal sequences of blaCTX-M group 1 (blaCTX-M-15 and -55) and group 9 (blaCTX-M-14, -27, -65, and -90) were cloned into pCRISPR and phagemid pRC319 for spacer evaluation and phagemid particle production. Re-sensitization and plasmid clearance were mediated by the spacers targeting internal sequences of each group, resulting in 3 log10 to 4 log10 reduction of the ratio of resistant cells, but not by those targeting the promoters. The CRISPR-Cas9 delivered by modified ΦRC319 particles were capable of re-sensitizing E. coli K-12 carrying either blaCTX-M group 1 or group 9 in a dose-dependent manner from 0.1 to 100 multiplicity of infection (MOI). In conclusion, CRISPR-Cas9 system programmed with well-designed spacers targeting multiple variants of AMR gene along with a phage-based delivery system could eliminate the widespread blaCTX-M genes for efficacy restoration of available third-generation cephalosporins by reversal of resistance in bacteria.
Topics: CRISPR-Cas Systems; Escherichia coli; Bacteriophages; beta-Lactamases; Escherichia coli Proteins; Plasmids; Promoter Regions, Genetic; Gene Editing; Anti-Bacterial Agents
PubMed: 38753729
DOI: 10.1371/journal.pone.0303555 -
Journal For Immunotherapy of Cancer May 2024Cancer-intrinsic type I interferon (IFN-I) production triggered by radiotherapy (RT) is mainly dependent on cytosolic double-stranded DNA (dsDNA)-mediated cGAS/STING...
BACKGROUND
Cancer-intrinsic type I interferon (IFN-I) production triggered by radiotherapy (RT) is mainly dependent on cytosolic double-stranded DNA (dsDNA)-mediated cGAS/STING signaling and increases cancer immunogenicity and enhances the antitumor immune response to increase therapeutic efficacy. However, cGAS/STING deficiency in colorectal cancer (CRC) may suppress the RT-induced antitumor immunity. Therefore, we aimed to evaluate the importance of the dsRNA-mediated antitumor immune response induced by RT in patients with CRC.
METHODS
Cytosolic dsRNA level and its sensors were evaluated via cell-based assays (co-culture assay, confocal microscopy, pharmacological inhibition and immunofluorescent staining) and in vivo experiments. Biopsies and surgical tissues from patients with CRC who received preoperative chemoradiotherapy (neoCRT) were collected for multiplex cytokine assays, immunohistochemical analysis and SNP genotyping. We also generated a cancer-specific adenovirus-associated virus (AAV)-IFNβ1 construct to evaluate its therapeutic efficacy in combination with RT, and the immune profiles were analyzed by flow cytometry and RNA-seq.
RESULTS
Our studies revealed that RT stimulates the autonomous release of dsRNA from cancer cells to activate TLR3-mediated IFN-I signatures to facilitate antitumor immune responses. Patients harboring a dysfunctional TLR3 variant had reduced serum levels of IFN-I-related cytokines and intratumoral CD8 immune cells and shorter disease-free survival following neoCRT treatment. The engineered cancer-targeted construct AAV-IFNβ1 significantly improved the response to RT, leading to systematic eradication of distant tumors and prolonged survival in defective TLR3 preclinical models.
CONCLUSION
Our results support that increasing cancer-intrinsic IFNβ1 expression is an immunotherapeutic strategy that enhances the RT-induced antitumor immune response in locally patients with advanced CRC with dysfunctional TLR3.
Topics: Humans; Colorectal Neoplasms; RNA, Double-Stranded; Interferon-beta; Mice; Animals; Interferon Type I; Signal Transduction; Female; Male
PubMed: 38749537
DOI: 10.1136/jitc-2023-008515 -
Clinical and Translational Radiation... Jul 2024Stereotactic body radiotherapy (SBRT) is an effective treatment for oligometastatic disease in multiple sites. However, the optimal radiation dose for long-term local...
PURPOSE/OBJECTIVES
Stereotactic body radiotherapy (SBRT) is an effective treatment for oligometastatic disease in multiple sites. However, the optimal radiation dose for long-term local control of adrenal metastases has yet to be determined. The aim of this study is to evaluate outcomes of adrenal SBRT and to evaluate factors that correlate with local control.
MATERIALS/METHODS
After IRB approval, a retrospective data review of patients treated with SBRT for adrenal metastases at a medical center in Israel between 2015 and 2021 was conducted. A biological effective dose was calculated using an alpha beta ratio of 10. Kaplan Meier and Cox regression were calculated using SPSS software to describe the hazard ratio for local control and survival.
RESULTS
83 cases of adrenal SBRT were identified. The average age was 67 (range 42-92 years old). Non-small cell lung cancer was the primary site in 44 % of patients. A total of 70 % of the patients had oligometastatic disease (less than five lesions), and the rest were polymetastatic, responding to systemic therapy with oligo progression in the adrenal. The average gross tumor volume (GTV) was 42 ml. Respiratory control was applied in 88 % of cases; 49.3 % used 4-D/ITV, and 38.5 % used breath-hold or continuous positive airway pressure (CPAP) with free breathing. On multivariable analysis, Dose above 75 Gy (biological effective Dose) (HR = 0.41, p = 0.031), Dose above 8 Gy per fraction (HR = 0.53p = 0.038), and breath-holds or CPAP (HR = 0.65, p = 0.047) were significant for local control. From multivariable analysis, we computed a predicted nomogram curve using seven clinical parameters to evaluate local control odds.
CONCLUSION
In this single institution series reported to date, we found unilateral adrenal SBRT safe, yet bilateral treatment harbors a risk of adrenal insufficiency. Biological effective Dose > 75 Gy (BED), motion management with breath-hold or CPAP, and Dose per fraction > 8 Gy were the enhanced local controls. We propose a nomogram to help in decision-making regarding total Dose and Dose per fraction when treating adrenal SBRT.
PubMed: 38745963
DOI: 10.1016/j.ctro.2024.100788 -
Journal of Radioanalytical and Nuclear... 2024Liquid Scintillation Counting (LSC) gross alpha/beta screening is a valuable tool for providing rapid laboratory response for the analysis of human clinical urine...
Liquid Scintillation Counting (LSC) gross alpha/beta screening is a valuable tool for providing rapid laboratory response for the analysis of human clinical urine samples during a large-scale radiation incident event. Verification of method performance, as required for clinical laboratory testing, is accomplished by the evaluation of routine, periodic measurements of radioactive spiked samples for quality control, performance testing, and accuracy checks. Radionuclide stability of alpha and beta emitters in urine for LSC analysis is an important consideration. The purpose of this work is to demonstrate optimal preparations and storage conditions of samples used for method verification.
PubMed: 38737916
DOI: 10.1007/s10967-024-09433-6 -
Frontiers in Oncology 2024Prostate cancer (PCa) is a prevalent malignancy in European men, often treated with radiotherapy (RT) for localized disease. While modern RT achieves high success rates,...
INTRODUCTION
Prostate cancer (PCa) is a prevalent malignancy in European men, often treated with radiotherapy (RT) for localized disease. While modern RT achieves high success rates, concerns about late gastrointestinal (GI) toxicities persist. This retrospective study aims to identify predictors for late GI toxicities following definitive conventionally fractionated external beam RT (EBRT) for PCa, specifically exploring the dose to the rectal wall.
MATERIALS AND METHODS
A cohort of 96 intermediate- to high-risk PCa patients underwent EBRT between 2008 and 2016. Rectum and rectum wall contours were delineated, and 3D dose matrices were extracted. Volumetric and dosimetric indices were computed, and statistical analyses were performed to identify predictors using the Mann-Whitney U-rank test, logistic regression, and recursive feature elimination.
RESULTS
In our cohort, 15 out of 96 patients experienced grade II late proctitis. Our analysis reveals distinct optimal predictors for rectum and rectum wall (RW) structures varying with values (3.0 and 2.3 Gy) across prescribed doses of 68 to 76 Gy. Despite variability, RW predictors demonstrate greater consistency, notably V68Gy[%] to V74Gy[%] for 3.0 Gy, and V68Gy[%] to V70Gy[%] for 2.3 Gy. The model with 2.3 Gy, featuring RW volume receiving 70 Gy (V70Gy[%]), stands out with a BIC value of 62.92, indicating its superior predictive effectiveness. Finally, focusing solely on the rectum structure, the V74Gy[%] emerges the best predictor for 3.0 Gy, with a BIC value of 66.73.
CONCLUSION
This investigation highlights the critical role of V70Gy[%] in the rectum wall as a robust predictor for grade II late gastrointestinal (GI) toxicity following external beam radiation therapy (EBRT) for prostate cancer (PCa). Furthermore, our findings suggest that focusing on the rectum wall specifically, rather than the entire rectum, may offer improved accuracy in assessing proctitis development. A 2.3 Gy of ≤5% and if possible ≤1% for the rectal wall should be achieved to minimize the risk of late grade II proctitis.
PubMed: 38737910
DOI: 10.3389/fonc.2024.1371384 -
Cancer Medicine May 2024Cervical cancer is one of the most common gynecological cancers. Accumulated evidence shows that long non-coding RNAs (lncRNAs) play essential roles in cervical cancer...
BACKGROUND
Cervical cancer is one of the most common gynecological cancers. Accumulated evidence shows that long non-coding RNAs (lncRNAs) play essential roles in cervical cancer occurrence and progression, but their specific functions and mechanisms remain to be further explored.
METHODS
The RT-qPCR assay was used to detect the expression of NEAT1 in cervical cancer tissues and cell lines. CCK-8, colony formation, flow cytometry, western blotting, and Transwell assays were used to evaluate the impact of NEAT1 on the malignant behavior of cervical cancer cells. Glucose consumption, lactate production, ATP levels, ROS levels, MMP levels, and the mRNA expressions of glycolysis-related genes and tricarboxylic acid cycle-related genes were detected to analyze the effect of NEAT1 on metabolism reprograming in cervical cancer cells. The expressions of PDK1, β-catenin and downstream molecules of the WNT/β-catenin signaling pathway in cervical cancer cells and tissues were detected by western blotting, RT-qPCR, immunofluorescence and immunohistochemistry assays.
RESULTS
This study investigated the role and possible molecular mechanism of lncRNA nuclear paraspeckle assembly transcript 1 (NEAT1) in cervical cancer. Our results showed that NEAT1 was highly expressed in cervical cancer tissues and cell lines. Downregulation of NEAT1 inhibited the proliferation, migration, invasion and glycolysis of cervical cancer cells, while overexpression of NEAT1 led to the opposite effects. Mechanistically, NEAT1 upregulated pyruvate dehydrogenase kinase (PDK1) through the WNT/β-catenin signaling pathway, which enhanced glycolysis and then facilitated cervical cancer metastasis. Furthermore, NEAT1 maintained the protein stability of β-catenin but did not affect its mRNA level. We also excluded the direct binding of NEAT1 to the β-catenin protein via RNA pull-down assay. The suppressive impact of NEAT1 knockdown on cell proliferation, invasion, and migration was rescued by β-catenin overexpression. The WNT inhibitor iCRT3 attenuated the carcinogenic effect induced by NEAT1 overexpression.
CONCLUSION
In summary, these findings indicated that NEAT1 may contribute to the progression of cervical cancer by activating the WNT/β-catenin/PDK1 signaling axis.
Topics: Humans; RNA, Long Noncoding; Uterine Cervical Neoplasms; Female; Pyruvate Dehydrogenase Acetyl-Transferring Kinase; Wnt Signaling Pathway; Disease Progression; Cell Proliferation; Gene Expression Regulation, Neoplastic; Cell Line, Tumor; beta Catenin; Glycolysis; Cell Movement
PubMed: 38733179
DOI: 10.1002/cam4.7221 -
Diagnostics (Basel, Switzerland) Apr 2024Oxygen-15 (β+, t = 122 s) radiolabeled diatomic oxygen, in conjunction with positron emission tomography, is the gold standard to quantitatively measure the metabolic...
Oxygen-15 (β+, t = 122 s) radiolabeled diatomic oxygen, in conjunction with positron emission tomography, is the gold standard to quantitatively measure the metabolic rate of oxygen consumption in the living human brain. We present herein a protocol for safe and effective delivery of [O]O over 200 m to a human subject for inhalation. A frugal quality control testing procedure was devised and validated. This protocol can act as a blueprint for other sites seeking to implement similar imaging programs.
PubMed: 38732316
DOI: 10.3390/diagnostics14090902 -
International Journal of Molecular... May 2024Herpes simplex virus (HSV) infections are highly widespread among humans, producing symptoms ranging from ulcerative lesions to severe diseases such as blindness and...
Herpes simplex virus (HSV) infections are highly widespread among humans, producing symptoms ranging from ulcerative lesions to severe diseases such as blindness and life-threatening encephalitis. At present, there are no vaccines available, and some existing antiviral treatments can be ineffective or lead to adverse effects. As a result, there is a need for new anti-HSV drugs. In this report, the in vitro anti-HSV effect of 9,9'-norharmane dimer (nHo-dimer), which belongs to the β-carboline (βC) alkaloid family, was evaluated. The dimer exhibited no virucidal properties and did not impede either the attachment or penetration steps of viral particles. The antiviral effect was only exerted under the constant presence of the dimer in the incubation media, and the mechanism of action was found to involve later events of virus infection. Analysis of fluorescence lifetime imaging data showed that the nHo-dimer internalized well into the cells when present in the extracellular incubation medium, with a preferential accumulation into perinuclear organelles including mitochondria. After washing the host cells with fresh medium free of nHo-dimer, the signal decreased, suggesting the partial release of the compound from the cells. This agrees with the observation that the antiviral effect is solely manifested when the alkaloid is consistently present in the incubation media.
Topics: Antiviral Agents; Chlorocebus aethiops; Humans; Vero Cells; Animals; Simplexvirus; Herpes Simplex; Carbolines; Herpesvirus 1, Human; Harmine
PubMed: 38732185
DOI: 10.3390/ijms25094966 -
International Journal of Molecular... Apr 2024Monitoring inflammatory cytokines is crucial for assessing healing process and photobiomodulation (PBM) enhances wound healing. Meanwhile, cAMP response element-binding...
Investigating the Relevance of Cyclic Adenosine Monophosphate Response Element-Binding Protein to the Wound Healing Process: An In Vivo Study Using Photobiomodulation Treatment.
Monitoring inflammatory cytokines is crucial for assessing healing process and photobiomodulation (PBM) enhances wound healing. Meanwhile, cAMP response element-binding protein (CREB) is a regulator of cellular metabolism and proliferation. This study explored potential links between inflammatory cytokines and the activity of CREB in PBM-treated wounds. A total of 48 seven-week-old male SD rats were divided into four groups (wound location, skin or oral; treatment method, natural healing or PBM treatment). Wounds with a 6 mm diameter round shape were treated five times with an 808 nm laser every other day (total 60 J). The wound area was measured with a caliper and calculated using the elliptical formula. Histological analysis assessed the epidermal regeneration and collagen expression of skin and oral tissue with H&E and Masson's trichrome staining. Pro-inflammatory (TNF-α) and anti-inflammatory (TGF-β) cytokines were quantified by RT-PCR. The ratio of phosphorylated CREB (p-CREB) to unphosphorylated CREB was identified through Western blot. PBM treatment significantly reduced the size of the wounds on day 3 and day 7, particularly in the skin wound group ( < 0.05 on day 3, < 0.001 on day 7). The density of collagen expression was significantly higher in the PBM treatment group (in skin wound, < 0.05 on day 3, < 0.001 on day 7, and < 0.05 on day 14; in oral wound, < 0.01 on day 7). The TGF-β/TNF-α ratio and the p-CREB/CREB ratio showed a parallel trend during wound healing. Our findings suggested that the CREB has potential as a meaningful marker to track the wound healing process.
Topics: Animals; Wound Healing; Low-Level Light Therapy; Male; Rats; Cyclic AMP Response Element-Binding Protein; Rats, Sprague-Dawley; Skin; Cytokines; Phosphorylation; Tumor Necrosis Factor-alpha; Collagen; Transforming Growth Factor beta
PubMed: 38732058
DOI: 10.3390/ijms25094838 -
International Journal of Molecular... Apr 2024Based on the need for radiobiological databases, in this work, we mined experimental ionizing radiation data of human cells treated with X-rays, γ-rays, carbon ions,...
Based on the need for radiobiological databases, in this work, we mined experimental ionizing radiation data of human cells treated with X-rays, γ-rays, carbon ions, protons and α-particles, by manually searching the relevant literature in PubMed from 1980 until 2024. In order to calculate normal and tumor cell survival and coefficients of the linear quadratic (LQ) established model, as well as the initial values of the double-strand breaks (DSBs) in DNA, we used WebPlotDigitizer and Python programming language. We also produced complex DNA damage results through the fast Monte Carlo code MCDS in order to complete any missing data. The calculated / values are in good agreement with those valued reported in the literature, where shows a relatively good association with linear energy transfer (LET), but not . In general, a positive correlation between DSBs and LET was observed as far as the experimental values are concerned. Furthermore, we developed a biophysical prediction model by using machine learning, which showed a good performance for , while it underscored LET as the most important feature for its prediction. In this study, we designed and developed the novel radiobiological 'RadPhysBio' database for the prediction of irradiated cell survival ( and coefficients of the LQ model). The incorporation of machine learning and repair models increases the applicability of our results and the spectrum of potential users.
Topics: Humans; Cell Survival; Radiation, Ionizing; Radiobiology; DNA Breaks, Double-Stranded; Linear Energy Transfer; Databases, Factual; Monte Carlo Method
PubMed: 38731948
DOI: 10.3390/ijms25094729