Search Results (16)

Background: Hyperbaric oxygen therapy (HBOT) is the standard treatment for moderate to severe carbon monoxide (CO) poisoning, but middle ear barotrauma (MEB) remains a common complication. This study identified risk factors associated with MEB in patients undergoing monoplace HBOT. Methods: This retrospective cohort study included patients treated for CO poisoning with monoplace HBOT at a tertiary academic hospital between May 2021 and December 2023. MEB severity was assessed before and after treatment using video otoscopy and graded according to the modified O’Neill Grading System. Results: MEB occurred predominantly at lower severity grades according to the O’Neill scale. In univariate analysis, significant risk factors for MEB included altered mental status at presentation (OR: 3.16, 95% CI: 1.35–7.40, p = 0.008), serum albumin > 4.3 g/dL (OR: 0.22, 95% CI: 0.10–0.65, p = 0.004), and magnesium levels (OR: 0.21, 95% CI: 0.05–0.98, p = 0.046). Multivariate analysis confirmed altered mental status (OR: 3.16, 95% CI: 1.05–9.52, p = 0.041), albumin > 4.3 g/dL (OR: 0.26, 95% CI: 0.10–0.65, p = 0.004), and magnesium level (OR: 0.21, 95% CI: 0.05–0.88, p = 0.033) as independent predictors of MEB. Patients with higher albumin and magnesium levels showed lower risk. Conclusions: Altered mental status, lower albumin, and lower magnesium levels predicted middle ear barotrauma in patients undergoing monoplace HBOT for CO poisoning. These findings highlight the importance of careful pre-treatment evaluation and close monitoring during therapy to reduce the incidence of MEB. Full article

The question of at which energy the transition from galactic to extra-galactic cosmic rays takes place has been a long-standing conundrum in cosmic ray physics. The sun stands out as the closest and clearest astrophysical accelerator of cosmic rays, while other objects within and beyond the galaxy remain enigmatic. It is probable that the cosmic ray spectrum and mass components from these celestial sources share similarities, offering a novel approach to study their origin. In this study, we perform joint analysis of spectra and mass in the energy range from MeV to 10 EeV, and find the following: (1) $⟨\mathrm{lnA}⟩$ demonstrates three clear peaks, tagging component transition; (2) a critical variable $\Delta$ is adopted to define the location of the transition; (3) for protons, the knee is located at ∼1.8 PeV, and the boundary between the galaxy and extra-galaxy occurs at ∼60 PeV, marked by a spectral dip; and (4) the all-particle spectrum exhibits hardening at ∼60 PeV due to the contribution of nearby galaxies, and the extra-galaxy dominates ∼0.8 EeV. We hope the LHAASO experiment can perform spectral measurements of individual species to validate these specific observations. Full article

Cutaneous melanoma is rapidly on the rise globally, surpassing the growth rate of other cancers, with metastasis being the primary cause of death in melanoma patients. Consequently, understanding the mechanisms behind this metastatic process and exploring innovative treatments is of paramount importance. Recent research has shown promise in unravelling the role of epigenetic factors in melanoma progression to metastasis. While DNA hypermethylation at gene promoters typically suppresses gene expression, we have contributed to establishing the newly understood mechanism of paradoxical activation of genes via DNA methylation, where high methylation coincides with increased gene activity. This mechanism challenges the conventional paradigm that promoter methylation solely silences genes, suggesting that, for specific genes, it might actually activate them. Traditionally, altering DNA methylation in vitro has involved using global demethylating agents, which is insufficient for studying the mechanism and testing the direct consequence of gene methylation changes. To investigate promoter hypermethylation and its association with gene activation, we employed a novel approach utilising a CRISPR-SunTag All-in-one system. Here, we focused on editing the DNA methylation of a specific gene promoter segment (EBF3) in melanoma cells using the All-in-one system. Using bisulfite sequencing and qPCR with RNA-Seq, we successfully demonstrated highly effective methylation and demethylation of the EBF3 promoter, with subsequent gene expression changes, to establish and validate the paradoxical role of DNA methylation. Further, our study provides novel insights into the function of the EBF3 gene, which remains largely unknown. Overall, this study challenges the conventional view of methylation as solely a gene-silencing mechanism and demonstrates a potential function of EBF3 in IFN pathway signalling, potentially uncovering new insights into epigenetic drivers of malignancy and metastasis. Full article

Background: This study investigated how the expression of heat shock protein 27 (HSP27), cellular FLICE-like inhibitory protein (cFLIP), and clusterin (CLU) affects the progression of cancer cells and their susceptibility to doxazosin-induced apoptosis. By silencing each of these genes individually, their effect on prostate cancer cell viability after doxazosin treatment was investigated. Methods: PC-3 prostate cancer cells were cultured and then subjected to gene silencing using siRNA targeting HSP27, cFLIP, and CLU, either individually, in pairs, or all together. Cells were then treated with doxazosin at various concentrations and their viability was assessed by MTT assay. Results: The study found that silencing the CLU gene in PC-3 cells significantly reduced cell viability after treatment with 25 µM doxazosin. In addition, the dual silencing of cFLIP and CLU decreased cell viability at 10 µM doxazosin. Notably, silencing all three genes of HSP27, cFLIP, CLU was most effective and reduced cell viability even at a lower doxazosin concentration of 1 µM. Conclusions: Taken together, these findings suggest that the simultaneous silencing of HSP27, cFLIP, and CLU genes may be a potential strategy to promote apoptosis in prostate cancer cells, which could inform future research on treatments for malignant prostate cancer. Full article

DNA methylation is critically involved in the regulation of chromatin states and cell-type-specific gene expression. The exclusive expression of imprinted genes from either the maternal or the paternal allele is regulated by allele-specific DNA methylation at imprinting control regions (ICRs). Aberrant DNA hyper- or hypomethylation at the ICR1 of the H19/IGF2 imprinting locus is characteristic for the imprinting disorders Beckwith–Wiedemann syndrome (BWS) and Silver–Russell syndrome (SRS), respectively. In this paper, we performed epigenome editing to induce targeted DNA demethylation at ICR1 in HEK293 cells using dCas9-SunTag and the catalytic domain of TET1. 5-methylcytosine (5mC) levels at the target locus were reduced up to 90% and, 27 days after transient transfection, >60% demethylation was still observed. Consistent with the stable demethylation of CTCF-binding sites within the ICR1, the occupancy of the DNA methylation-sensitive insulator CTCF protein increased by >2-fold throughout the 27 days. Additionally, the H19 expression was increased by 2-fold stably, while IGF2 was repressed though only transiently. Our data illustrate the ability of epigenome editing to implement long-term changes in DNA methylation at imprinting control regions after a single transient treatment, potentially paving the way for therapeutic epigenome editing approaches in the treatment of imprinting disorders. Full article

The SARS-CoV-2 coronavirus has caused worldwide disruption through the COVID-19 pandemic, providing a sobering reminder of the profound impact viruses can have on human well-being. Understanding virus life cycles and interactions with host cells lays the groundwork for exploring therapeutic strategies against virus-related diseases. Fluorescence microscopy plays a vital role in virus imaging, offering high spatiotemporal resolution, sensitivity, and spectroscopic versatility. In this opinion piece, we first highlight two recent techniques, SunTag and StayGold, for the in situ imaging of viral RNA translation and viral assembly. Next, we discuss a new class of genetically encoded fluorogenic protease reporters, such as FlipGFP, which can be customized to monitor SARS-CoV-2’s main (M^pro) or papain-like (PL^pro) protease activity. These assays have proven effective in identifying potential antivirals through high-throughput screening, making fluorogenic viral protease reporters a promising platform for viral disease diagnostics and therapeutics. Full article

This paper presents the deployment of a hybrid energy harvesting system that combines a wireless energy harvesting (EH) system and a 6 V, 170 mA monocrystalline solar energy derived from the Sun’s rays. The hybrid energy harvesting (HEH) system comprises the rectifier, the solar cell panel, the charging circuit, and the EM4325 embedded RFID tag. This study aims to design an efficient EH system capable of increasing the read range of an active RFID tag. The proposed approach integrates a meandered line radio frequency identification (RFID) tag with an EM4325 IC chip as the receiver antenna. A halfwave doubler RF rectifier circuit is connected to the antenna using a 50 Ω SMA connector to convert the captured RF waves into usable electrical power. A solar energy charging module equipped with a Maximum Power Point Tracking (MPPT) system, a rechargeable lithium-ion battery, and a DC-DC converter is configured to manage and store the harvested energy efficiently. The UHF tag antenna operates at 919 MHz, achieving a peak gain of 3.54 dB. The proposed rectenna achieves a maximum measured harvested power conversion efficiency (PCE) of 55.14% for an input power (Pin) of 15 dBm at a distance of 5.10 cm, while the solar cell panel realizes 3.92 W of power. Experimental results demonstrate the hybrid harvester system’s effectiveness, achieving a PCE of 86.49% at an output voltage (VDC) of 5.35 V. The main advantage of this approach is the creation of a compact hybrid RF and solar EH system by combining the solar cell panel with the antenna, thus enabling multi-functionality. Full article

A CRISPRa transcription activation system was used to upregulate insulin expression in HEK293T cells. To increase the delivery of the targeted CRISPR/dCas9a, magnetic chitosan nanoparticles, imprinted with a peptide from the Cas9 protein, were developed, characterized, and then bound to dCas9a that was complexed with a guide RNA (gRNA). The adsorption of dCas9 proteins conjugated with activators (SunTag, VPR, and p300) to the nanoparticles was monitored using both ELISA kits and Cas9 staining. Finally, the nanoparticles were used to deliver dCas9a that was complexed with a synthetic gRNA into HEK293T cells to activate their insulin gene expression. Delivery and gene expression were examined using quantitative real-time polymerase chain reaction (qRT-PCR) and staining of insulin. Finally, the long-term release of insulin and the cellular pathway related to stimulation by glucose were also investigated. Full article

Genome editing technology has become one of the hottest research areas in recent years. Among diverse genome editing tools, the Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR-associated proteins system (CRISPR/Cas system) has exhibited the obvious advantages of specificity, simplicity, and flexibility over any previous genome editing system. In addition, the emergence of Cas9 mutants, such as dCas9 (dead Cas9), which lost its endonuclease activity but maintains DNA recognition activity with the guide RNA, provides powerful genetic manipulation tools. In particular, combining the dCas9 protein and transcriptional activator to achieve specific regulation of gene expression has made important contributions to biotechnology in medical research as well as agriculture. CRISPR/dCas9 activation (CRISPRa) can increase the transcription of endogenous genes. Overexpression of foreign genes by traditional transgenic technology in plant cells is the routine method to verify gene function by elevating genes transcription. One of the main limitations of the overexpression is the vector capacity constraint that makes it difficult to express multiple genes using the typical Ti plasmid vectors from Agrobacterium. The CRISPRa system can overcome these limitations of the traditional gene overexpression method and achieve multiple gene activation by simply designating several guide RNAs in one vector. This review summarizes the latest progress based on the development of CRISPRa systems, including SunTag, dCas9-VPR, dCas9-TV, scRNA, SAM, and CRISPR-Act and their applications in plants. Furthermore, limitations, challenges of current CRISPRa systems and future prospective applications are also discussed. Full article

Hepatitis B virus infections are the main reason for hepatocellular carcinoma development. Current treatment reduces the viral load but rarely leads to virus elimination. Despite its medical importance, little is known about infection dynamics on the cellular level not at least due to technical obstacles. Regardless of infections leading to extreme viral loads, which may reach 10¹⁰ virions per mL serum, hepatitis B viruses are of low abundance and productivity in individual cells. Imaging of the infections in cells is thus a particular challenge especially for cccDNA that exists only in a few copies. The review describes the significance of microscopical approaches on genome and transcript detection for understanding hepatitis B virus infections, implications for understanding treatment outcomes, and recent microscopical approaches, which have not been applied in HBV research. Full article

DNA methylation is a key epigenetic modification implicated in the pathogenesis of numerous human diseases, including cancer development and metastasis. Gene promoter methylation changes are widely associated with transcriptional deregulation and disease progression. The advent of CRISPR-based technologies has provided a powerful toolkit for locus-specific manipulation of the epigenome. Here, we describe a comprehensive global workflow for the design and application of a dCas9-SunTag-based tool for editing the DNA methylation locus in human melanoma cells alongside protocols for downstream techniques used to evaluate subsequent methylation and gene expression changes in methylation-edited cells. Using transient system delivery, we demonstrate both highly efficacious methylation and demethylation of the EBF3 promoter, which is a putative epigenetic driver of melanoma metastasis, achieving up to a 304.00% gain of methylation and 99.99% relative demethylation, respectively. Furthermore, we employ a novel, targeted screening approach to confirm the minimal off-target activity and high on-target specificity of our designed guide RNA within our target locus. Full article

Aerial imaging technologies have been widely applied in agricultural plant remote sensing. However, an as yet unexplored challenge with field imaging is that the environmental conditions, such as sun angle, cloud coverage, temperature, and so on, can significantly alter plant appearance and thus affect the imaging sensor’s accuracy toward extracting plant feature measurements. These image alterations result from the complicated interaction between the real-time environments and plants. Analysis of these impacts requires continuous monitoring of the changes through various environmental conditions, which has been difficult with current aerial remote sensing systems. This paper aimed to propose a modeling method to comprehensively understand and model the environmental influences on hyperspectral imaging data. In 2019, a fixed hyperspectral imaging gantry was constructed in Purdue University’s research farm, and over 8000 repetitive images of the same corn field were taken with a 2.5 min interval for 31 days. Time-tagged local environment data, including solar zenith angle, solar irradiation, temperature, wind speed, and so on, were also recorded during the imaging time. The images were processed for phenotyping data, and the time series decomposition method was applied to extract the phenotyping data variation caused by the changing environments. An artificial neural network (ANN) was then built to model the relationship between the phenotyping data variation and environmental changes. The ANN model was able to accurately predict the environmental effects in remote sensing results, and thus could be used to effectively eliminate the environment-induced variation in the phenotyping features. The test of the normalized difference vegetation index (NDVI) calculated from the hyperspectral images showed that variance in NDVI was reduced by 79%. A similar performance was confirmed with the relative water content (RWC) predictions. Therefore, this modeling method shows great potential for application in aerial remote sensing applications in agriculture, to significantly improve the imaging quality by effectively eliminating the effects from the changing environmental conditions. Full article

Increasing numbers of studies seek to characterize the different cellular sub-populations present in mammalian tissues. The techniques “Isolation of Nuclei Tagged in Specific Cell Types” (INTACT) or “Fluorescence-Activated Nuclei Sorting” (FANS) are frequently used for isolating nuclei of specific cellular subtypes. These nuclei are then used for molecular characterization of the cellular sub-populations. Despite the increasing popularity of both techniques, little is known about their isolation efficiency, advantages, and disadvantages or downstream molecular effects. In our study, we compared the physical and molecular attributes of sfGFP+ nuclei isolated by the two methods—INTACT and FANS—from the neocortices of Arc-CreERT2 × CAG-Sun1/sfGFP animals. We identified differences in efficiency of sfGFP+ nuclei isolation, nuclear size as well as transcriptional (RNA-seq) and chromatin accessibility (ATAC-seq) states. Therefore, our study presents a comprehensive comparison between the two widely used nuclei sorting techniques, identifying the advantages and disadvantages for both INTACT and FANS. Our conclusions are summarized in a table to guide researchers in selecting the most suitable methodology for their individual experimental design. Full article

Overexpression of a gene of interest is a general approach used in both basic research and therapeutic applications. However, the conventional approach involving overexpression of exogenous genes has difficulty achieving complete genome coverage, and is also limited by the cloning capacity of viral vectors. Therefore, an alternative approach would be to drive the expression of an endogenous gene using an artificial transcriptional activator. Fusion proteins of dCas9 and a transcription activation domain, such as dCas9–VP64, are widely used for activation of endogenous genes. However, when using a single sgRNA, the activation range is low. Consequently, tiling of several sgRNAs is required for robust transcriptional activation. Here we describe the screening of factors that exhibit the best synergistic activation of gene expression with TET1 in the dCas9–SunTag format. All seven factors examined showed some synergy with TET1. Among them, VP64 gave the best results. Thus, simultaneous tethering of VP64 and TET1 to a target gene using an optimized dCas9–SunTag format synergistically activates gene expression using a single sgRNA. Full article

Epigenome editing is a promising technology, potentially allowing the stable reprogramming of gene expression profiles without alteration of the DNA sequence. Targeted DNA methylation has been successfully documented by many groups for silencing selected genes, but recent publications have raised concerns regarding its specificity. In the current work, we developed new EpiEditors for programmable DNA methylation in cells with a high efficiency and improved specificity. First, we demonstrated that the catalytically deactivated Cas9 protein (dCas9)-SunTag scaffold, which has been used earlier for signal amplification, can be combined with the DNMT3A-DNMT3L single-chain effector domain, allowing for a strong methylation at the target genomic locus. We demonstrated that off-target activity of this system is mainly due to untargeted freely diffusing DNMT3A-DNMT3L subunits. Therefore, we generated several DNMT3A-DNMT3L variants containing mutations in the DNMT3A part, which reduced their endogenous DNA binding. We analyzed the genome-wide DNA methylation of selected variants and confirmed a striking reduction of untargeted methylation, most pronounced for the R887E mutant. For all potential applications of targeted DNA methylation, the efficiency and specificity of the treatment are the key factors. By developing highly active targeted methylation systems with strongly improved specificity, our work contributes to future applications of this approach. Full article