Assessment of the Novel Anti-Seizure Potential of Validamycin A Using Zebrafish Epilepsy Model.

Epilepsy is a prevalent neurological disorder characterized by recurrent seizures. Validamycin A (VA) is an antibiotic fungicide that inhibits trehalase activity and is widely used for crop protection in agriculture. In this study, we identified a novel function of VA as a potential anti-seizure medication in a zebrafish epilepsy model. Electroencephalogram (EEG) analysis demonstrated that VA reduced pentylenetetrazol (PTZ)-induced seizures in the brains of larval and adult zebrafish. Moreover, VA reduced PTZ-induced irregular movement in a behavioral assessment of adult zebrafish. The developmental toxicity test showed no observable anatomical alteration when the zebrafish larvae were treated with VA up to 10 µM within the effective range. The median lethal dose of VA in adult zebrafish was > 14,000 mg/kg. These results imply that VA does not demonstrate observable toxicity in zebrafish at concentrations effective for generating anti-seizure activity in the EEG and alleviating abnormal behavior in the PTZ-induced epileptic model. Furthermore, the effectiveness of VA was comparable to that of valproic acid. These results indicate that VA may have a potentially safer anti-seizure profile than valproic acid, thus offering promising prospects for its application in agriculture and medicine.

Balloon neck-plasty to create a wide-necked aneurysm in the elastase-induced rabbit model.

PURPOSE: The elastase-induced aneurysm (EIA) model in rabbits has been proposed for translational research; however, the adjustment of aneurysm neck size remains challenging. In this study, the technical feasibility and safety of balloon neck-plasty to create a wide-necked aneurysm in rabbit EIA model were investigated. METHODS: Male New Zealand White rabbits (N = 15) were randomly assigned to three groups: group A, EIA creation without neck-plasty; group B, neck-plasty immediately after EIA creation; group C, neck-plasty 4 weeks after EIA creation. The diameter of balloon used for neck-plasty was determined 1 mm larger than origin carotid artery diameter. All rabbits were euthanized 4 weeks after their final surgery. Aneurysm neck, height, dome-to-neck (D/N) ratio, and histologic parameters were compared among the groups. RESULTS: Aneurysm creation was technically successful in 14 out of 15 rabbits (93.3%), with one rabbit experiencing mortality due to an adverse anesthetic event during the surgery. Saccular and wide-necked aneurysms were successfully created in all rabbits. Aneurysm neck was significantly greater in groups B and C compared to group A (all P < .05). D/N ratio was significantly lower in groups B and C compared to group A (all P < .05). Additionally, tunica media thickness, vessel area, and luminal area were significantly greater in groups B and C compared to group A (all P < .05). These variables were found to be significantly greater in group B compared to group C (all P < .05). CONCLUSION: The creation of a wide-necked aneurysm using balloon neck-plasty after elastase induction in rabbits has been determined to be technically feasible and safe.

Rapid deep learning-assisted predictive diagnostics for point-of-care testing.

Prominent techniques such as real-time polymerase chain reaction (RT-PCR), enzyme-linked immunosorbent assay (ELISA), and rapid kits are currently being explored to both enhance sensitivity and reduce assay time for diagnostic tests. Existing commercial molecular methods typically take several hours, while immunoassays can range from several hours to tens of minutes. Rapid diagnostics are crucial in Point-of-Care Testing (POCT). We propose an approach that integrates a time-series deep learning architecture and AI-based verification, for the enhanced result analysis of lateral flow assays. This approach is applicable to both infectious diseases and non-infectious biomarkers. In blind tests using clinical samples, our method achieved diagnostic times as short as 2 minutes, exceeding the accuracy of human analysis at 15 minutes. Furthermore, our technique significantly reduces assay time to just 1-2 minutes in the POCT setting. This advancement has the potential to greatly enhance POCT diagnostics, enabling both healthcare professionals and non-experts to make rapid, accurate decisions.

Generation of Protease Inhibitory Antibodies by Functional In Vivo Selection.

Targeting dysregulated protease expression and/or abnormal substrate proteolysis, highly selective inhibition of pathogenic proteases by monoclonal antibodies (mAbs) presents an attractive therapeutic approach for the treatment of diseases including cancer. Herein, we report a functional selection method for protease inhibitory mAbs by periplasmic co-expression of three recombinant proteins-a protease of interest, an antibody Fab library, and a modified ß-lactamase TEM-1. We validate this approach by isolation of highly selective and potent mAbs inhibiting human matrix metalloproteinase 9 (MMP9).

High-fidelity large-diversity monoclonal mammalian cell libraries by cell cycle arrested recombinase-mediated cassette exchange.

Mammalian cells carrying defined genetic variations have shown great potentials in both fundamental research and therapeutic development. However, their full use was limited by lack of a robust method to construct large monoclonal high-quality combinatorial libraries. This study developed cell cycle arrested recombinase-mediated cassette exchange (aRMCE), able to provide monoclonality, precise genomic integration and uniform transgene expression. Via optimized nocodazole-mediated mitotic arrest, 20% target gene replacement efficiency was achieved without antibiotic selection, and the improved aRMCE efficiency was applicable to a variety of tested cell clones, transgene targets and transfection methods. As a demonstration of this versatile method, we performed directed evolution of fragment crystallizable (Fc), for which error-prone libraries of over 107 variants were constructed and displayed as IgG on surface of CHO cells. Diversities of constructed libraries were validated by deep sequencing, and panels of novel Fc mutants were identified showing improved binding towards specific Fc gamma receptors and enhanced effector functions. Due to its large cargo capacity and compatibility with different mutagenesis approaches, we expect this mammalian cell platform technology has broad applications for directed evolution, multiplex genetic assays, cell line development and stem cell engineering.

Assessment of the viability of integrating virtual reality programs in practical tests for the Korean Radiological Technologists Licensing Examination: a survey study

PURPOSE: The objective of this study was to assess the feasibility of incorporating virtual reality/augmented reality (VR/AR) programs into practical tests administered as part of the Korean Radiological Technologists Licensing Examination (KRTLE). This evaluation is grounded in a comprehensive survey that targeted enrolled students in departments of radiology across the nation. METHODS: In total, 682 students from radiology departments across the nation were participants in the survey. An online survey platform was used, and the questionnaire was structured into 5 distinct sections and 27 questions. A frequency analysis for each section of the survey was conducted using IBM SPSS ver. 27.0. RESULTS: Direct or indirect exposure to VR/AR content was reported by 67.7% of all respondents. Furthermore, 55.4% of the respondents expressed that VR/AR could be integrated into their classes, which signified a widespread acknowledgment of VR among the students. With regards to the integration of a VR/AR or mixed reality program into the practical tests for purposes of the KRTLE, a substantial amount of the respondents (57.3%) exhibited a positive inclination and recommended its introduction. CONCLUSION: The application of VR/AR programs within practical tests of the KRTLE will be used as an alternative for evaluating clinical examination procedures and validating job skills.

Transglutaminase 2 Prevents Premature Senescence and Promotes Osteoblastic Differentiation of Mesenchymal Stem Cells through NRF2 Activation.

Transglutaminase 2 (TG2) is a multifunctional enzyme that exhibits transamidase, GTPase, kinase, and protein disulfide isomerase (PDI) activities. Of these, transamidase-mediated modification of proteins regulates apoptosis, differentiation, inflammation, and fibrosis. TG2 is highly expressed in mesenchymal stem cells (MSCs) compared with differentiated cells, suggesting a role of TG2 specific for MSC characteristics. In this study, we report a new function of TG2 in the regulation of MSC redox homeostasis. During in vitro MSC expansion, TG2 is required for cell proliferation and self-renewal by preventing premature senescence but has no effect on the expression of surface antigens and oxidative stress-induced cell death. Moreover, induction of differentiation upregulates TG2 that promotes osteoblastic differentiation. Molecular analyses revealed that TG2 mediates tert-butylhydroquinone, but not sulforaphane, -induced nuclear factor erythroid 2-related factor 2 (NRF2) activation in a transamidase activity-independent manner. Differences in the mechanism of action between two NRF2 activators suggest that PDI activity of TG2 may be implicated in the stabilization of NRF2. The role of TG2 in the regulation of antioxidant response was further supported by transcriptomic analysis of MSC. These results indicate that TG2 is a critical enzyme in eliciting antioxidant response in MSC through NRF2 activation, providing a target for optimizing MSC manufacturing processes to prevent premature senescence.

Sample-to-answer platform for the clinical evaluation of COVID-19 using a deep learning-assisted smartphone-based assay.

Since many lateral flow assays (LFA) are tested daily, the improvement in accuracy can greatly impact individual patient care and public health. However, current self-testing for COVID-19 detection suffers from low accuracy, mainly due to the LFA sensitivity and reading ambiguities. Here, we present deep learning-assisted smartphone-based LFA (SMARTAI-LFA) diagnostics to provide accurate decisions with higher sensitivity. Combining clinical data learning and two-step algorithms enables a cradle-free on-site assay with higher accuracy than the untrained individuals and human experts via blind tests of clinical data (n = 1500). We acquired 98% accuracy across 135 smartphone application-based clinical tests with different users/smartphones. Furthermore, with more low-titer tests, we observed that the accuracy of SMARTAI-LFA was maintained at over 99% while there was a significant decrease in human accuracy, indicating the reliable performance of SMARTAI-LFA. We envision a smartphone-based SMARTAI-LFA that allows continuously enhanced performance by adding clinical tests and satisfies the new criterion for digitalized real-time diagnostics.

Feasibility of a silicone vascular phantom replicating real arterial contrast filling dynamics on cerebral angiography: An in-vitro pilot study.

Some cerebral arterial silicone phantoms have been used in preclinical evaluations. However, typical silicone-based phantoms are limited in their capacity to reproduce real contrast filling dynamics of the human cerebral artery. This study aimed to develop a cerebral arterial silicone phantom to analyze the feasibility of real contrast filling dynamics. The fluid circulation phantom system consisted of a cerebral arterial silicone phantom without or with additional devices, a pump, an injection system, a pressure-monitoring system, a constant-temperature bath, and a venous drainage container. Vascular resistance was reproduced with a plastic cistern only or a plastic cistern filled with a sponge pad. Three phantom groups were constructed as follows: a) the cerebral arterial silicone phantom used as the control group (type A), b) phantom with the incorporated plastic cistern (type B), and c) phantom with the incorporated plastic cistern filled with a sponge pad (type C). The contrast concentration-time curve patterns of the three groups obtained from digital subtraction angiography (DSA) were compared. Consequently, the DSA pattern of the type C phantom was the most similar to that obtained from the control group as the reference data, which showed the broadest full-width-at-half-maximum and the area under the curve values and the highest maximum contrast concentration. In conclusion, we could emulate the arterial contrast filling dynamics of clinical cerebral angiography by applying a small cistern filled with a sponge pad at the drainage side of the phantom.

A modified method of elastase-induced saccular aneurysm creation in rabbits: Schematic representation of the method.

BACKGROUND: The purpose of our study was to investigate the safety and effectiveness of a modified technique using a short guiding catheter for instillation of elastase in comparison with the previously described method of creating elastase-induced aneurysm in rabbits. METHODS: Following right common carotid artery (RCCA) access using an arterial sheath and inflating the Fogarty balloon in the subclavian artery, a short guiding catheter was used for the instillation of the elastase in the experimental group (n = 5) while it was performed with a microcatheter in the control group (n = 5). The procedure duration was recorded from the RCCA puncture to the sheath removal. The histological changes were characterized using H&E and Masson's trichrome (MT) staining. RESULTS: The procedure time was 23 ± 2 min in the experimental group and 29 ± 2 min in the control group. All the rabbits (100%) in the experimental group survived without neurologic deficits, but two rabbits (40%) survived in the control group. All aneurysms were created in the saccular shape (100%) with a neck size of 2.3 ± 0.29 mm, a width of 2.75 ± 0.36 mm, and height of 6.37 ± 0.46 mm, and a dome to neck ratio of 1.21 ± 0.23. The aneurysm walls were partly thickened due to the degradation of the media tunica and adventitia proliferation with loss of the internal elastic lamina. CONCLUSION: By using a short guiding catheter, we could instill the elastase in a more effective and safe manner in the creation of the elastase-induced aneurysm model in rabbits.

Trends of Expanding Indications of Woven EndoBridge Devices for the Treatment of Intracranial Aneurysms: A Systematic Review and Meta-analysis.

PURPOSE: Woven EndoBridge (WEB) was introduced for the endovascular therapy of wide-neck intracranial aneurysms. The safety and efficacy have been evaluated through several meta-analyses. However, these reviews did not cover the expanding indications in detail. Therefore, we aimed to show the changing trends for intracranial aneurysm treatment using the WEB device. METHODS: A systematic review and meta-analysis was conducted with PubMed, Embase, and Cochrane databases. We searched for studies that reported baseline characteristics of aneurysms and the WEB devices, which had treated more than 20 aneurysms consecutively. The pooled proportions of aneurysm indications and used WEB device types were obtained. To evaluate the changing indications for the treated aneurysm size, including the neck diameter, a trend line and linear regression model was measured. RESULTS: A total of 27 cohorts were included encompassing 1831 aneurysms treated with the WEB. A total of 86% were used in the four major locations as on-label indications (middle cerebral artery bifurcation; 34%, anterior communicating artery; 26%, basilar tip; 18%, internal carotid artery terminus; 7%). Among off-label indications, the most common location was the posterior communicating artery (8%), followed by the anterior cerebral artery including the pericallosal artery (6%). The median aneurysm size and neck diameter was 7 mm and 4.6 mm, respectively. The WEB device has been used for the treatment of smaller aneurysms than the initial indication. Also, the proportion for ruptured aneurysm treatment was increased up to 15%. CONCLUSION: The mechanical and technical development of the WEB resulted in expanding the indications for the treatment of intracranial aneurysms. The off-label indications accounted for 14% in total and an increasing number of small aneurysms are treated with WEB devices. Moreover, the proportion for ruptured aneurysm treatment was currently increased up to 14% more than in the beginning.

Zebrafish EEG predicts the efficacy of antiepileptic drugs.

Background: Pharmacological evaluation of antiepileptic drugs (AEDs) using mammalian animals takes long time and is expensive. The zebrafish is a species commonly used to study brain functions, neurological diseases, and drug toxicity, and attracts more attention as an alternative animal model to substitute or supplement mammalian animals in drug development. Electroencephalogram (EEG) is a key indicator for diagnosing brain diseases such as epilepsy, by directly measuring the brain activity. We propose a novel method for pharmacological evaluation of AEDs based on EEG from adult zebrafish, which allows researchers to select more clinically valuable drugs at the early stage of AED screening. Methods: To evaluate the efficacy of AEDs, zebrafish EEG signals were measured after administering six AEDs (valproate acid, gabapentin, ethosuximide, oxcarbazepine, tiagabine, and topiramate) at various doses to pentylenetetrazol (PTZ)-induced seizure models. The change in seizure activity was investigated according to doses. The antiepileptic effect was determined by observing a significant decrease in at least one out of three indicators of the number, total duration, and mean duration of ictal events. Results: Using EEG signals from adult zebrafish, antiepileptic effects were observed with all six AEDs. Among them, antiepileptic effects depending on dose were confirmed with valproate acid, gabapentin, ethosuximide, and tiagabine. Moreover, the 50% effective doses (ED50) of valproate acid and tiagabine were determined based on zebrafish EEG for the first time, indicating that the quantitative inter-species comparison of the AED efficacy is possible between zebrafish and mammals such as rodents. Significance: The results show that zebrafish can be used to effectively and quantitatively evaluate the efficacy of AEDs based on EEG, the same method to evaluate antiepileptic effects in mammals, suggesting that the proposed method can contribute in reducing the cost and duration of search for AEDs and thus accelerate the drug development cycles.

Size-dependent seizurogenic effect of polystyrene microplastics in zebrafish embryos.

The effects of polystyrene microplastic (PS-MP) size on neurotoxicity remain to be evaluated at various microsizes, and the seizurogenic effects of PS-MPs are unknown. This study aimed to evaluate the swimming behavior of zebrafish larvae under light-dark transitions after exposure to four PS-MP sizes (i.e., 1, 6, 10, and 25 µm) at concentrations of 500, 5,000, and 50,000 particles/mL. Changes in electroencephalographic signals, seizure-related gene expression, and neurochemical concentrations were measured. Locomotor activity was inhibited only by 10-µm PS-MPs. According to electroencephalographic signals, the number and total duration of seizure-like events significantly increased by 10-µm PS-MPs, which was confirmed by the altered expression of seizure-related genes c-fos and pvalb5. Additionally, an increase in the levels of neurochemicals choline, betaine, dopamine, 3-methoxytyramine, and gamma-aminobutyric acid indicated that the observed hypoactivity and seizure-like behavior were associated with the dysregulation of the cholinergic, dopaminergic, and GABAergic systems. Overall, these findings demonstrate that exposure to PS-MPs can potentially cause seizurogenic effects in developing zebrafish embryos, and we highlight that PS-MPs 10 µm in size dominantly affect neurotoxicity.

Safety and Effectiveness of the Novel Catheter 3.0 System for Diagnostic Cerebral Angiography: A Pilot Study.

PURPOSE: The purpose of this study was to evaluate the safety and effectiveness of a new angiographic system (Catheter 3.0 system) using a 5 French (Fr), large-bore angiography catheter, a 0.032-inch stiff guidewire, and a continuous flushing system in diagnostic cerebral angiography. MATERIALS AND METHODS: This retrospective study included 30 consecutive patients who underwent transfemoral cerebral angiography using the Catheter 3.0 system from October 2019 to March 2020. As the control group, we included 30 consecutive patients examined before the Catheter 3.0 system was introduced. Procedural outcomes, including technical success, procedure time, dose metrics, procedure-related complications, and image quality were reviewed and analyzed. RESULTS: All transfemoral cerebral angiographies were performed for a diagnosis of unruptured intracranial aneurysms. The Catheter 3.0 system showed a significantly shorter fluoroscopy time (6.2 vs. 9.7 minutes, P=0.008) and lower fluoroscopy dose (387.2 vs. 614.4, P=0.002) compared with the conventional 4-Fr catheter system. The Catheter 3.0 system also showed better results in terms of procedural time (21.0 vs. 22.5 minutes, P=0.072) and technical success rate (98.1% vs. 94.0%, P=0.078), although a statistical significance was not reached. The complication rate and qualitative assessment of the digital subtraction angiography (DSA) image quality were similar between the two groups. CONCLUSION: The Catheter 3.0 system using a 5 Fr catheter with a large inner diameter was convenient, effective, and safe compared with the conventional system in diagnostic cerebrovascular angiography.

Monitoring In Vivo Performances of Protein-Drug Conjugates Using Site-Selective Dual Radiolabeling and Ex Vivo Digital Imaging.

In preclinical models, the development and optimization of protein-drug conjugates require accurate determination of the plasma and tissue profiles of both the protein and its conjugated drug. To this aim, we developed a bioanalytical strategy based on dual radiolabeling and ex vivo digital imaging. By combining enzymatic and chemical reactions, we obtained homogeneous dual-labeled anti-MMP-14 Fabs (antigen-binding fragments) conjugated to monomethyl auristatin E where the protein scaffold was labeled with carbon-14 (14C) and the conjugated drug with tritium (3H). These antibody-drug conjugates with either a noncleavable or a cleavable linker were then evaluated in vivo. By combining liquid scintillation counting and ex vivo dual-isotope radio-imaging, it was possible not only to monitor both components simultaneously during their circulation phase but also to quantify accurately their amount accumulated within the different organs.

Double-Stranded RNA Enhances Matrix Metalloproteinase-1 and -13 Expressions through TLR3-Dependent Activation of Transglutaminase 2 in Dermal Fibroblasts.

UV-irradiation induces the secretion of double-stranded RNA (dsRNA) derived from damaged noncoding RNAs in keratinocytes, which enhance the expression of matrix metalloproteinases (MMP) in non-irradiated dermal fibroblasts, leading to dysregulation of extracellular matrix homeostasis. However, the signaling pathway responsible for dsRNA-induced MMP expression has not been fully understood. Transglutaminase 2 (TG2) is an enzyme that modifies substrate proteins by incorporating polyamine or crosslinking of proteins, thereby regulating their functions. In this study, we showed that TG2 mediates dsRNA-induced MMP-1 expression through NF-κB activation. Treatment of poly(I:C), a synthetic dsRNA analogue binding to toll-like receptor 3 (TLR3), generates ROS, which in turn activates TG2 in dermal fibroblast. Subsequently, TG2 activity enhances translocation of p65 into the nucleus, where it augments transcription of MMP. We confirmed these results by assessing the level of MMP expression in Tlr3-/-, TG2-knockdowned and Tgm2-/- dermal fibroblasts after poly(I:C)-treatment. Moreover, treatment with quercetin showed dose-dependent suppression of poly(I:C)-induced MMP expression. Furthermore, ex vivo cultured skin from Tgm2-/- mice exhibited a significantly reduced level of MMP mRNA compared with those from wild-type mice. Our results indicate that TG2 is a critical regulator in dsRNA-induced MMP expression, providing a new target and molecular basis for antioxidant therapy in preventing collagen degradation.

Body weight regulation via MT1-MMP-mediated cleavage of GFRAL.

GDNF-family receptor a-like (GFRAL) has been identified as the cognate receptor of growth/differentiation factor 15 (GDF15/MIC-1), considered a key signaling axis in energy homeostasis and body weight regulation. Currently, little is known about the physiological regulation of the GDF15-GFRAL signaling pathway. Here we show that membrane-bound matrix metalloproteinase 14 (MT1-MMP/MMP14) is an endogenous negative regulator of GFRAL in the context of obesity. Overnutrition-induced obesity increased MT1-MMP activation, which proteolytically inactivated GFRAL to suppress GDF15-GFRAL signaling, thus modulating the anorectic effects of the GDF15-GFRAL axis in vivo. Genetic ablation of MT1-MMP specifically in GFRAL+ neurons restored GFRAL expression, resulting in reduced weight gain, along with decreased food intake in obese mice. Conversely, depletion of GFRAL abolished the anti-obesity effects of MT1-MMP inhibition. MT1-MMP inhibition also potentiated GDF15 activity specifically in obese phenotypes. Our findings identify a negative regulator of GFRAL for the control of non-homeostatic body weight regulation, provide mechanistic insights into the regulation of GDF15 sensitivity, highlight negative regulators of the GDF15-GFRAL pathway as a therapeutic avenue against obesity and identify MT1-MMP as a promising target.

Transglutaminase 2 mediates UVB-induced matrix metalloproteinase-1 expression by inhibiting nuclear p65 degradation in dermal fibroblasts.

Matrix metalloproteinases (MMPs) play a key role in tissue remodelling by cleaving extracellular matrix (ECM) components. In the skin, UV irradiation increases expression of MMPs that causes dysregulation of ECM homeostasis in dermis, leading to acceleration of skin aging. However, the mediator(s) that links UV irradiation to the upregulation of MMPs have not been fully defined. Previously, we showed that UVB irradiation activated transglutaminase 2 (TG2) in keratinocytes, eliciting an inflammatory response by activating NF-κB signalling. In this study, we reported the role of TG2 in mediating the UVB-induced expression of MMP-1. In human dermal fibroblasts, UVB irradiation enhanced the expression and activity of TG2, which in turn promotes the expression of MMP-1. Analyses of MMP-1 promoter showed that activation of the NF-κB signalling pathway, rather than AP-1, was responsible for the TG2-mediated upregulation of MMP-1. Moreover, Western blot analysis revealed that TG2 increased the activity of NF-κB by inhibiting degradation of p65 in the nucleus. Furthermore, ex vivo skin from TG2-knockout mice exhibited significantly reduced levels of MMP-1 compared to that from wild-type mice. These results indicate that TG2 functions as a mediator for the UVB-induced expression of MMP-1 in dermal fibroblasts, providing a new target for preventing skin photodamage.

Image quality assessments according to the angle of tilt of a flex tilt coil supporting device: An ACR phantom study.

In this study, we assessed how image quality depends on the angle of tilt of a flex tilt coil supporting device during an MRI examination. All measurements were performed with an American College of Radiology (ACR) MRI phantom using a flex tilt coil supporting device. All images were analyzed using an automatic assessment method following the ACR MRI accreditation guidance. Image quality was compared between acquisitions grouped according to the angle of tilt of the coil supporting device: group A (Flat mode), group B (10˚), and group C (18˚). All measured image qualities were within the ACR recommended criteria, regardless of the angle of tilt of the flex tilt coil supporting device. However, statistically significant differences between the three groups were found for slice thickness, position accuracy, image intensity uniformity, and SNR (P < 0.05, ANOVA). The flex tilt coil supporting device can provide sufficient image quality, passing the criteria of the ACR MRI guideline, despite differences in slice thickness, slice position accuracy, image intensity uniformity, and SNR according to the angle of tilt.