Evaluation of unmodified human cell-derived extracellular vesicle mitochondrial deoxyribonucleic acid-based biodistribution in rodents.

Recently, extracellular vesicles (EVs) have been developed as therapeutic targets for various diseases. Biodistribution is crucial for EVs intended for therapeutic purposes because it can determine the degree of on- and off-target effects. This study aimed to explore techniques to evaluate the biodistribution of unmodified EVs. We devised a novel quantitative polymerase chain reaction (qPCR)-based assay to detect unmodified EVs by targeting mitochondrial deoxyribonucleic acid (mtDNA), a constituent of EVs. We focused on specific mtDNA regions that exhibited homologous variations distinct from their rodent mtDNA counterparts to establish this analytical approach. Herein, we successfully designed primers and probes targeting human and rodent mtDNA sequences and developed a highly specific and sensitive qPCR method. Furthermore, the quantification range of EVs isolated from various cells differed based on the manufacturer and cell source. IRDye 800CW-labelled Expi293F EV mimetics were administered to the animals via the tail vein to compare the imaging test and mtDNA-qPCR results. The results obtained from imaging tests and mtDNA-qPCR to investigate EV biodistribution patterns revealed differences. The results revealed that our newly developed method effectively determined the biodistribution of unmodified EVs with high sensitivity and reproducibility.

The effect of early versus late remdesivir treatment in hospitalized mild to moderate COVID-19 patients in the Omicron era: A retrospective study.

Although real-world studies have found that remdesivir is effective in preventing poor prognosis, more information is needed on the optimal timing of remdesivir administration in high-risk coronavirus disease 2019 (COVID-19) patients in the Omicron era. From February 2022 to January 2023, a single-center retrospective study was performed in Korea. We compared the clinical characteristics and treatment outcomes between early (remdesivir treatment within 0-3 days from symptom onset) and late (≥ 4 days from symptom onset) treatment groups of patients who received remdesivir monotherapy. Of 284 patients, 225 were classified into the early treatment group and 59 were classified into the late treatment group. The early treatment group had a lower rate of 28-day progression to severe disease than the late treatment group (1.4% vs 7.4%, P = .03). Delaying remdesivir treatment ≥ 4 days from symptom onset (adjusted odds ratio [aOR], 6.17; 95% CI, 1.18-32.44; P = .03) and Charlson comorbidity index ≥ 3 (aOR, 9.62; 95% CI, 1.65-56.10; P = .01) were independent risk factors for 28-day progression to severe disease. Our results suggest that early administration of remdesivir could be associated with better prognosis in COVID-19 patients with the Omicron variant, and within 3 days from symptom onset seems to be the appropriate timing.

Investigation of the efficacy and safety of wild- type and triple-gene knockout pig RBC transfusions in nonhuman primates.

Introduction: Decreasing rates of blood donation and close margins between blood supply and demand pose challenges in healthcare. Genetically engineered pig red blood cells (pRBCs) have been explored as alternatives to human RBCs for transfusion, and triple-gene knockout (TKO) modification improves the compatibility of pRBCs with human blood in vitro. In this study, we assessed the efficacy and risks of transfusing wild-type (WT)- and TKO-pRBCs into nonhuman primates (NHPs). Methods: Blood from O-type WT and TKO pigs was processed to produce pRBCs for transfusion, which were transfused or not into NHPs (n=4 per group: WT, TKO, and control) after 25% total blood volume withdrawal: their biological responses were compared. Hematological, biochemical, and immunological parameters were measured before, immediately after, and at intervals following transfusion. Two months later, a second transfusion was performed in three NHPs of the transfusion group. Results: Transfusion of both WT- and TKO-pRBCs significantly improved RBC counts, hematocrit, and hemoglobin levels up to the first day post-transfusion, compared to the controls. The transfusion groups showed instant complement activation and rapid elicitation of anti-pig antibodies, as well as elevated liver enzyme and bilirubin levels post-transfusion. Despite the higher agglutination titers with WT-pRBCs in the pre-transfusion crossmatch, the differences between the WT and TKO groups were not remarkable except for less impairment of liver function in the TKO group. After the second transfusion, more pronounced adverse responses without any hematological gain were observed. Conclusions: WT- and TKO-pRBC transfusions effectively increased hematologic parameters on the first day, with rapid clearance from circulation thereafter. However, pRBC transfusion triggers strong antibody responses, limiting the benefits of the pRBC transfusion and increasing the risk of adverse reactions.

Stochastic characterization of navigation strategies in an automated variant of the Barnes maze.

Animals can use a repertoire of strategies to navigate in an environment, and it remains an intriguing question how these strategies are selected based on the nature and familiarity of environments. To investigate this question, we developed a fully automated variant of the Barnes maze, characterized by 24 vestibules distributed along the periphery of a circular arena, and monitored the trajectories of mice over 15 days as they learned to navigate towards a goal vestibule from a random start vestibule. We show that the patterns of vestibule visits can be reproduced by the combination of three stochastic processes reminiscent of random, serial, and spatial strategies. The processes randomly selected vestibules based on either uniform (random) or biased (serial and spatial) probability distributions. They closely matched experimental data across a range of statistical distributions characterizing the length, distribution, step size, direction, and stereotypy of vestibule sequences, revealing a shift from random to spatial and serial strategies over time, with a strategy switch occurring approximately every six vestibule visits. Our study provides a novel apparatus and analysis toolset for tracking the repertoire of navigation strategies and demonstrates that a set of stochastic processes can largely account for exploration patterns in the Barnes maze.

Risk Factors for Suboptimal Colon Cancer Diagnosis and Management at a Safety-Net Hospital System.

INTRODUCTION: Colon cancer (CC) is the second leading cause of cancer-related deaths in the United States. Quality measures have been introduced by the American Gastroenterological Association and Commission on Cancer for optimal management of CC. In this study, we sought to identify factors that may hinder the timely diagnosis and treatment of CC at a safety-net hospital system. METHODS: Retrospective chart review was performed for patients aged ≥18 y diagnosed with CC from 2018 to 2021. Primary outcomes were time from positive fecal immunochemical test to colonoscopy, time from diagnosis to surgery, and time from diagnosis to adjuvant chemotherapy. Secondary end points were demographic characteristics associated with suboptimal outcomes in any of the above measures. RESULTS: One hundred ninety patients were diagnosed with nonmetastatic CC. The majority were Hispanic and non-English-speaking. 74.1% of patients with a positive fecal immunochemical test received a colonoscopy within 180 d. 59.6% of nonemergent cases received surgery within 60 d of diagnosis. 77% of those eligible received adjuvant chemotherapy within 120 d of diagnosis. No clinically significant demographic factor was associated with delay in colonoscopy, surgery, or adjuvant chemotherapy. Most frequent cause of delay in surgery (38.0%) was optimization of comorbidities. Most frequent cause of delay in adjuvant chemotherapy (71.4%) was delay in surgery itself. CONCLUSIONS: No clinically significant demographic factor was associated with experiencing delays in diagnostic colonoscopy, surgery, or adjuvant chemotherapy.

Clinical profile and treatment outcomes of idiopathic intracranial hypertension: a multicenter study from Korea.

BACKGROUND: Currently, there is a relative lack of detailed reports regarding clinical presentation and outcome of idiopathic intracranial hypertension in Asians. This study aims to describe the clinical features and treatment outcomes of Korean patients with idiopathic intracranial hypertension. METHODS: We prospectively recruited patients with idiopathic intracranial hypertension from one hospital and retrospectively analyzed the medical records of 11 hospitals in Korea. We collected data regarding preceding medical conditions or suspected medication exposure, headache phenotypes, other associated symptoms, detailed neuroimaging findings, treatments, and outcomes after 1-2 and 3-6 months of treatment. RESULTS: Fifty-nine (83.1% women) patients were included. The mean body mass index was 29.11 (standard deviation, 5.87) kg/m2; only 27 patients (45.8%) had a body mass index of ≥ 30 kg/m2. Fifty-one (86.4%) patients experienced headaches, patterns of which included chronic migraine (15/51 [29.4%]), episodic migraine (8/51 [15.7%]), probable migraine (4/51 [7.8%]), chronic tension-type headache (3/51 [5.9%]), episodic tension-type headache (2/51 [3.9%]), probable tension-type headache (2/51 [3.9%]), and unclassified (17/51 [33.3%]). Medication overuse headache was diagnosed in 4/51 (7.8%) patients. After 3-6 months of treatment, the intracranial pressure normalized in 8/32 (25.0%), improved in 17/32 (53.1%), no changed in 7/32 (21.9%), and worsened in none. Over the same period, headaches remitted or significantly improved by more than 50% in 24/39 patients (61.5%), improved less than 50% in 9/39 (23.1%), and persisted or worsened in 6/39 (15.4%) patients. CONCLUSION: Our findings suggest that the features of Asian patients with idiopathic intracranial hypertension may be atypical (i.e., less likely obese, less female predominance). A wide spectrum of headache phenotypes was observed. Medical treatment resulted in overall favorable short-term outcomes; however, the headaches did not improve in a small proportion of patients.

Selection of Convolutional Neural Network Model for Bladder Tumor Classification of Cystoscopy Images and Comparison with Humans.

Purpose: An investigation of various convolutional neural network (CNN)-based deep learning algorithms was conducted to select the appropriate artificial intelligence (AI) model for calculating the diagnostic performance of bladder tumor classification on cystoscopy images, with the performance of the selected model to be compared against that of medical students and urologists. Methods: A total of 3,731 cystoscopic images that contained 2,191 tumor images were obtained from 543 bladder tumor cases and 219 normal cases were evaluated. A total of 17 CNN models were trained for tumor classification with various hyperparameters. The diagnostic performance of the selected AI model was compared with the results obtained from urologists and medical students by using the receiver operating characteristic (ROC) curve graph and metrics. Results: EfficientNetB0 was selected as the appropriate AI model. In the test results, EfficientNetB0 achieved a balanced accuracy of 81%, sensitivity of 88%, specificity of 74%, and an area under the curve (AUC) of 92%. In contrast, human-derived diagnostic statistics for the test data showed an average balanced accuracy of 75%, sensitivity of 94%, and specificity of 55%. Specifically, urologists had an average balanced accuracy of 91%, sensitivity of 95%, and specificity of 88%, while medical students had an average balanced accuracy of 69%, sensitivity of 94%, and specificity of 44%. Conclusions: Among the various AI models, we suggest that EfficientNetB0 is an appropriate AI classification model for determining the presence of bladder tumors in cystoscopic images. EfficientNetB0 showed the highest performance among several models and showed high accuracy and specificity compared to medical students. This AI technology will be helpful for less experienced urologists or nonurologists in making diagnoses. Image-based deep learning classifies bladder cancer using cystoscopy images and shows promise for generalized applications in biomedical image analysis and clinical decision making.

Disrupted Rotational Perception During Simultaneous Stimulation of Rotation and Inertia.

Two vestibular signals, rotational and inertial cues, converge for the perception of complex motion. However, how vestibular perception is built on neuronal behaviors and decision-making processes, especially during the simultaneous presentation of rotational and inertial cues, has yet to be elucidated in humans. In this study, we analyzed the perceptual responses of 20 participants after pairwise rotational experiments, comprised of four control and four test sessions. In both control and test sessions, participants underwent clockwise and counterclockwise rotations in head-down and head-up positions. The difference between the control and test sessions was the head re-orientation relative to gravity after rotations, thereby providing only rotational cues in the control sessions and both rotational and inertial cues in the test sessions. The accuracy of perceptual responses was calculated by comparing the direction of rotational and inertial cues acquired from participants with that predicted by the velocity-storage model. The results showed that the accuracy of rotational perception ranged from 80 to 95% in the four control sessions but significantly decreased to 35 to 75% in the four test sessions. The accuracy of inertial perception in the test sessions ranged from 50 to 70%. The accuracy of rotational perception improved with repetitive exposure to the simultaneous presentation of both rotational and inertial cues, while the accuracy of inertial perception remained steady. The results suggested a significant interaction between rotational and inertial perception and implied that vestibular perception acquired in patients with vestibular disorders are potentially inaccurate.

Reprogramming anchorage dependency to develop cell lines for recombinant protein expression.

As the biopharmaceutical industry continues to mature in its cost-effectiveness and productivity, many companies have begun employing larger-scale biomanufacturing and bioprocessing protocols. While many of these protocols require cells with anchorage-independent growth, it remains challenging to induce the necessary suspension adaptations in many different cell types. In addition, although transfection efficiency is an important consideration for all cells, especially for therapeutic protein production, cells in suspension are generally more difficult to transfect than adherent cells. Thus, much of the biomanufacturing industry is focused on the development of new human cell lines with properties that can support more efficient biopharmaceutical production. With this in mind, we identified a set of "Adherent-to-Suspension Transition" (AST) factors, IKZF1, BTG2 and KLF1, the expression of which induces adherent cells to acquire anchorage-independent growth. Working from the HEK293A cell line, we established 293-AST cells and 293-AST-TetR cells for inducible and reversible reprogramming of anchorage dependency. Surprisingly, we found that the AST-TetR system induces the necessary suspension adaptations with an accompanying increase in transfection efficiency and protein expression rate. Our AST-TetR system therefore represents a novel technological platform for the development of cell lines used for generating therapeutic proteins.

HSPA5 and FGFR1 genes in the mesenchymal subtype of glioblastoma can improve a treatment efficacy.

Tyrosine kinase inhibitors (TKIs) have emerged as a potential treatment strategy for glioblastoma multiforme (GBM). However, their efficacy is limited by various drug resistance mechanisms. To devise more effective treatments for GBM, genetic characteristics must be considered in addition to pre-existing treatments. We performed an integrative analysis with heterogeneous GBM datasets of genomic, transcriptomic, and proteomic data from DepMap, TCGA and CPTAC. We found that poor prognosis was induced by co-upregulation of heat shock protein family A member 5 (HSPA5) and fibroblast growth factor receptor 1 (FGFR1). Co-up regulation of these two genes could regulate the PI3K/AKT pathway. GBM cell lines with co-upregulation of these two genes showed higher drug sensitivity to PI3K inhibitors. In the mesenchymal subtype, the co-upregulation of FGFR1 and HSPA5 resulted in the most malignant subtype of GBM. Furthermore, we found this newly discovered subtype was correlated with homologous recombination deficiency (HRD) In conclusion, we discovered novel druggable candidates within the group exhibiting co-upregulation of these two genes in GBM, suggest potential strategies for combination therapy.

Monolayer, open-mesh, pristine PEDOT:PSS-based conformal brain implants for fully MRI-compatible neural interfaces.

Understanding brain function is essential for advancing our comprehension of human cognition, behavior, and neurological disorders. Magnetic resonance imaging (MRI) stands out as a powerful tool for exploring brain function, providing detailed insights into its structure and physiology. Combining MRI technology with electrophysiological recording system can enhance the comprehension of brain functionality through synergistic effects. However, the integration of neural implants with MRI technology presents challenges because of its strong electromagnetic (EM) energy during MRI scans. Therefore, MRI-compatible neural implants should facilitate detailed investigation of neural activities and brain functions in real-time in high resolution, without compromising patient safety and imaging quality. Here, we introduce the fully MRI-compatible monolayer open-mesh pristine PEDOT:PSS neural interface. This approach addresses the challenges encountered while using traditional metal-based electrodes in the MRI environment such as induced heat or imaging artifacts. PEDOT:PSS has a diamagnetic property with low electrical conductivity and negative magnetic susceptibility similar to human tissues. Furthermore, by adopting the optimized open-mesh structure, the induced currents generated by EM energy are significantly diminished, leading to optimized MRI compatibility. Through simulations and experiments, our PEDOT:PSS-based open-mesh electrodes showed improved performance in reducing heat generation and eliminating imaging artifacts in an MRI environment. The electrophysiological recording capability was also validated by measuring the local field potential (LFP) from the somatosensory cortex with an in vivo experiment. The development of neural implants with maximized MRI compatibility indicates the possibility of potential tools for future neural diagnostics.

Efficacy of vestibular rehabilitation and its facilitating and hindering factors from real-world clinical data.

Background and purpose: Customized vestibular rehabilitation improved dizziness and imbalance in several randomized controlled trials. In the present study, we determined the efficacy of customized vestibular rehabilitation using real-world observational data. Methods: In this retrospective observational study, we recruited 64 patients (median age = 60, interquartile range = 48-66.3) who completed the customized vestibular rehabilitation from January to December 2022. The outcomes of rehabilitation were evaluated using the dizziness handicap inventory (DHI) or vestibular disorders activities of daily living scale (VADL). The factors associated with outcomes were assessed with a generalized linear model, of which covariates included patients' age, sex, duration of illness, type of vestibular disorders, initial DHI and VADL scores, exercise compliance, and initial hospital anxiety and depression scale (HADS) scores. Results: After the median of 6 (4-6) weeks of rehabilitation, DHI and VADL scores significantly improved in patients with either peripheral or central vestibular disorders (Wilcoxon signed-rank test, p < 0.05). The initial DHI and VADL scores showed a positive while the sum of HADS scores showed a negative correlation with the outcome. In contrast, the age, sex, duration of illness, types of vestibular disorders, and exercise compliance did not affect the outcome. Discussion and conclusion: Customized vestibular rehabilitation is effective for central as well as peripheral disorders, especially when the symptoms are severe and the psychological distress is mild.

Sustainable production of natural products using synthetic biology: Ginsenosides.

Synthetic biology approaches offer potential for large-scale and sustainable production of natural products with bioactive potency, including ginsenosides, providing a means to produce novel compounds with enhanced therapeutic properties. Ginseng, known for its non-toxic and potent qualities in traditional medicine, has been used for various medical needs. Ginseng has shown promise for its antioxidant and neuroprotective properties, and it has been used as a potential agent to boost immunity against various infections when used together with other drugs and vaccines. Given the increasing demand for ginsenosides and the challenges associated with traditional extraction methods, synthetic biology holds promise in the development of therapeutics. In this review, we discuss recent developments in microorganism producer engineering and ginsenoside production in microorganisms using synthetic biology approaches.

Integration of in vivo electrophysiology and optogenetics in rodents with PEDOT:PSS neural electrode array.

Here, we present a protocol for the fabrication of transparent implantable electrode arrays for integrating optogenetics and electrophysiology. We describe steps for fabricating microelectrodes using the conductive polymer poly(3,4-ethylenedioxythiophene):poly(styrenesulfonate). We then detail procedures for analyzing performance of the electrodes and recording light-evoked neural activities from the transgenic mouse. This protocol utilizes photolithography rather than conventional electrodeposition. For complete details on the use and execution of this protocol, please refer to Cho et al. (2022).1.

A Comparison Between GalT-/-;hCD39;hCD55 and GalT-/-;hCD39;hCD46;hCD55;TBM Pig Kidneys Transplanted in Nonhuman Primates.

Because there is a shortage of donor kidneys, researchers are exploring the possibility of using genetically modified pig kidneys for transplantation. Approaches involving knockout of carbohydrate genes or knockin of protective proteins have been attempted to determine the best gene modifications. In this study, we utilized GalT-/-;hCD39;hCD55 and GalT-/-;hCD39;hCD46;hCD55;thrombomodulin (TBM) pigs for transplantation in nonhuman primates (NHPs). The NHPs survived for 4 weeks after kidney transplantation (4 WAT) from the GalT-/-;hCD39;hCD55 pig and for 6 WAT from the GalT-/-;hCD39;hCD46;hCD55;TBM pig. However, messenger RNA (mRNA) sequencing and immunohistochemistry analysis revealed that the 6 WAT kidney exhibited more severe apoptosis, inflammation, loss of renal function, and renal fibrosis than the 4 WAT kidney. These results indicate that additional knockin of complement regulator (hCD46) and coagulation regulator (TBM) is not enough to prevent renal damage, suggesting that improved immune suppression is needed for more prolonged survival.