Combination of chemotherapy and focused ultrasound for the treatment of unresectable pancreatic cancer: a proof-of-concept study.

OBJECTIVES: To investigate the safety and preliminary efficacy of the combined treatment of focused ultrasound (FUS) and chemotherapy (nab-paclitaxel plus gemcitabine, nPac/Gem) for patients with unresectable pancreatic cancer. METHODS: Patients pathologically diagnosed with unresectable pancreatic cancer were included. Low (Isppa = 1.5 kW/cm2), intermediate (2.0 kW/cm2), and high (2.5 kW/cm2) FUS intensity treatment groups were predefined. A 1% duty cycle and the 3+3 scheme were used. Six combined treatments were performed, and adverse events were assessed. Changes in tumor size and tumor response, CA 19-9 level, and patient-reported outcomes at the immediate follow-up (F/U) and/or at the 3-month F/U and survival were evaluated. RESULTS: Three participants were enrolled in each intensity group. No adverse device effect or dose-limiting toxicity occurred in any of the participants. Seven of the nine participants experienced a >15% tumor size decrease at the immediate F/U CT and at the 3-month F/U CT. The CA 19-9 level decreased in all of the participants at the immediate F/U. All participants in the intermediate-intensity treatment group showed a > 30% tumor size decrease, partial response, and a significant decrease in the CA 19-9 level at 3-month F/U and longer survival (p < 0.05). CONCLUSION: FUS with an intensity of 1.5 to 2.5 kW/cm2 was safe in the combined treatment of FUS and nPac/Gem. Considering the results of the change in tumor size, the change in CA 19-9 level, tumor response, and survival, these FUS parameters can be used for subsequent clinical trials. KEY POINTS: • No adverse device effect or dose-limiting toxicity occurred in any of the participants when focused ultrasound with an intensity of 1.5-2.5 kW/cm2 and a low duty cycle of 1% was combined with chemotherapy. • The intermediate-intensity group showed a >30% tumor size decrease, partial response, and a significant decrease in CA 19-9 in all of the participants at the 3-month follow-up and the longest survival. • Any focused ultrasound setting used in this study could be safe and optimal for subsequent clinical trials.

Impact of Insurance Benefits and Education on Point-of-Care Ultrasound Use in a Single Emergency Department: An Interrupted Time Series Analysis.

Background and Objectives: Point-of-care ultrasound (POCUS) is a useful tool that helps clinicians properly treat patients in emergency department (ED). This study aimed to evaluate the impact of specific interventions on the use of POCUS in the ED. Materials and Methods: This retrospective study used an interrupted time series analysis to assess how interventions changed the use of POCUS in the emergency department of a tertiary medical institute in South Korea from October 2016 to February 2021. We chose two main interventions-expansion of benefit coverage of the National Health Insurance (NHI) for emergency ultrasound (EUS) and annual ultrasound educational workshops. The primary variable was the EUS rate, defined as the number of EUS scans per 1000 eligible patients per month. We compared the level and slope of EUS rates before and after interventions. Results: A total of 5188 scanned records were included. Before interventions, the EUS rate had increased gradually. After interventions, except for the first workshop, the EUS rate immediately increased significantly (p < 0.05). The difference in the EUS rate according to the expansion of the NHI was estimated to be the largest (p < 0.001). However, the change in slope significantly decreased after the third workshop during the coronavirus disease 2019 pandemic (p = 0.004). The EUS rate increased significantly in the presence of physicians participating in intensive POCUS training (p < 0.001). Conclusion: This study found that expansion of insurance coverage for EUS and ultrasound education led to a significant and immediate increase in the use of POCUS, suggesting that POCUS use can be increased by improving education and insurance benefits.

Online Learning versus Hands-On Learning of Basic Ocular Ultrasound Skills: A Randomized Controlled Non-Inferiority Trial.

Background and objectives: Ocular ultrasound is a core application of point-of-care ultrasound (POCUS) to assist physicians in promptly identifying various ocular diseases at the bedside; however, hands-on POCUS training is challenging during a pandemic. Materials and Methods: A randomized controlled non-inferiority trial was conducted in an academic emergency department from October 2020 to April 2021. Thirty-two participants were randomly assigned to one of two groups. Group H (hands-on learning group) participated individually in a hands-on session with a standardized patient for 30 min, whereas Group O (online learning group) learned training materials and video clips for 20 min. They scanned four eyeballs of two standardized patients sequentially following the ocular POCUS scan protocol. Repeated POCUS scans were performed 2 weeks later to assess skill maintenance. Both groups completed the pre- and post-surveys and knowledge tests. Two emergency medicine faculty members blindly evaluated the data and assigned a score of 0−25. The primary endpoint was the initial total score of scan quality evaluated using non-inferiority analysis (generalized estimating equation). The secondary endpoints were total scores for scan quality after 2 weeks, scan time, and knowledge test scores. Results: The least squares means of the total scores were 21.7 (0.35) for Group O and 21.3 (0.25) for Group H, and the lower bound of the 95% confidence interval (CI) was greater than the non-inferiority margin of minus 2 (95% CI: −0.48−1.17). The second scan scores were not significantly different from those of the first scan. The groups did not differ in scanning time or knowledge test results; however, Group H showed higher subjective satisfaction with the training method (p < 0.001). Conclusion: This study showed that basic online ocular ultrasound education was not inferior to hands-on education, suggesting that it could be a useful educational approach in the pandemic era.

The Use of Point-of-care Ultrasound in Emergency Medical Centers in Korea: a National Cross-sectional Survey.

BACKGROUND: Point-of-care ultrasound (POCUS) is an essential tool in emergency medicine (EM). We aimed to investigate the current status and perception of POCUS use in emergency medical centers in Korea. METHODS: A cross-sectional, nationwide survey was conducted using a mobile survey of physicians at emergency medical centers in Korea. The first message was sent on November 27, 2020, and the second message was sent on December 3, 2020 to the non-responders. The questionnaire comprised 6 categories and 24 questionnaires on demographics, current practice, education, perception, and barriers to the use of POCUS. RESULTS: A total of 467 physicians participated in the survey (a response rate of 32% among 1,458 target physicians), of which 43% were residents and 57% were EM specialists. Most of the respondents (96%) answered that they use POCUS, of which 89% reported using it at least once a week. The most frequently used types of POCUS were focused assessment with sonography for trauma (68%) and echocardiography (66%). Musculoskeletal, male genital, and pediatric scans were rarely performed tests but ranked as of the scans physicians most wanted to learn. About 73% of the respondents received ultrasound education, and 41% received ultrasound education at their own institutions. Nevertheless, education-related barriers are still the biggest deterrent to POCUS use (60%). In addition, multivariate multinomial logistic regression analysis revealed that the greater the number of ultrasound devices and the total number of physicians in the emergency center, the more likely they were to use POCUS every day. CONCLUSION: This study found that most physicians currently working in emergency medical centers in Korea more frequently perform various types of ultrasound scans compared to those 10 years prior. To further promote the use of POCUS, it is important to have an appropriate number of ultrasound devices and physicians in the emergency center along with systematic POCUS education.

Respiratory Protection Effect of Ear-loop-type KF94 Masks according to the Wearing Method in COVID-19 Pandemic: a Randomized, Open-label Study.

BACKGROUND: Ear-loop-type Korean Filter 94 masks (KF94 masks, equivalent to the N95 and FFP2) are broadly used in health care settings in Korea for the coronavirus disease 2019 pandemic. METHODS: A prospective randomized open-label study was designed to identify differences in the fitting performance between mask wearing methods in three different types of KF94 mask with ear loops between January to March 2021. General-fitting involved wearing an ear-loop-type KF94 mask, and tight-fitting involved wearing a mask aided by a clip connecting the ear loops. Each of the 30 participants wore three types of masks according to a randomly assigned order in both methods and performed a total of six quantitative fit tests (QNFTs) according to the occupational safety and health administration protocol. RESULTS: All fit factors (FFs) measured by the QNFT were significantly higher for tight-fitting method with the clip in all KF94 masks (P < 0.001). However, the total FFs were very low, with a median (interquartile range) of 6 (3-23) and 29 (9-116) for general-fitting and tight-fitting, respectively. When wearing tightly, the horizontal 3-fold type mask with adjustable ear-loop length had the highest FF, with a median of 125, and the QNFT pass rate (FF ≥ 100) increased significantly from 4 (13%) to 18 (60%). CONCLUSION: Even with sufficient filter efficiency, ear-loop-type-KF94 masks do not provide adequate protection. However, in relatively low-risk environments, wearing a face-seal adjustable KF94 mask and tight wearing with a clip can improve respiratory protection for healthcare workers. TRIAL REGISTRATION: ClinicalTrials.gov Identifier: NCT04794556.

Portable ultrasound-guided high-intensity focused ultrasound with functions for safe and rapid ablation: prospective clinical trial for uterine fibroids-short-term and long-term results.

OBJECTIVES: To investigate the efficacy and safety of a new portable ultrasound-guided high-intensity focused ultrasound system (USgHIFU) with advanced targeting and beam steering technology for the treatment of uterine fibroids. METHODS: Fifty-nine uterine fibroids from 36 participants (mean age, 44.9 ± 4.1 years) were included from November 2013 to November 2015. All participants were treated with HIFU, with 3D electronic steering. MR imaging studies were performed before HIFU, immediately after HIFU, and 1 month and 3 (or 5) months after the HIFU treatment. The non-perfused volume ratio (NPVR), fibroid volume shrinkage (FVS), symptom improvement, quantified life quality assessment, and safety were analyzed. A long-term follow-up was conducted in July to December 2017 (mean, 32.2 months). RESULTS: The volume of the treated uterine fibroids ranged from 7.5 to 274.4 cm3 (mean, 69.8 cm3; SD, 64.3 cm3). The mean NPVR on the immediate post-HIFU MR imaging was 74.8 ± 25.2%. The mean FVS was 17.3% at 1 month, 33.3% at 3 months, and 45.1% at 5 months after HIFU treatment. The mean treatment time was 44.6 ± 28.2 min per fibroid and 72.9 ± 31.4 min per participant. Uterine fibroid-related symptoms and quality of life showed statistically significant improvement after the HIFU treatment. No significant symptoms related to safety or complications occurred. In the long-term follow-up, 78.8% of those surveyed were satisfied with their HIFU treatment. CONCLUSION: This clinical trial showed that a portable USgHIFU with advanced functions may safely and effectively treat uterine fibroids. KEY POINTS: • A portable compact ultrasound-guided high-intensity focused ultrasound (HIFU) can effectively and safely treat uterine fibroids. • Advanced functions, such as portability, targeted forecasting, electronic beam steering, and interleaved scanning, might be helpful in enhancing the clinical applicability of ultrasound-guided high-intensity focused ultrasound. • In the long-term follow-up of more than 2 years, approximately 80% of those surveyed were satisfied with their HIFU treatment.

3D-cultured neural stem cell microarrays on a micropillar chip for high-throughput developmental neurotoxicology.

Numerous chemicals including environmental toxicants and drugs have not been fully evaluated for developmental neurotoxicity. A key gap exists in the ability to predict accurately and robustly in vivo outcomes based on in vitro assays. This is particularly the case for predicting the toxicity of chemicals on the developing human brain. A critical need for such in vitro assays is choice of a suitable model cell type. To that end, we have performed high-throughput in vitro assessment of proliferation and differentiation of human neural stem cells (hNSCs). Conventional in vitro assays typically use immunofluorescence staining to quantify changes in cell morphology and expression of neural cell-specific biomarkers, which is often time-consuming and subject to variable specificities of available antibodies. To alleviate these limitations, we developed a miniaturized, three-dimensional (3D) hNSC culture with ReNcell VM on microarray chip platforms and established a high-throughput promoter-reporter assay system using recombinant lentiviruses on hNSC spheroids to assess cell viability, self-renewal, and differentiation. Optimum cell viability and spheroid formation of 3D ReNcell VM culture were observed on a micropillar chip over a period of 9 days in a mixture of 0.75% (w/v) alginate and 1 mg/mL growth factor reduced (GFR) Matrigel with 25 mM CaCl2 as a crosslinker for alginate. In addition, 3D ReNcell VM culture exhibited self-renewal and differentiation on the microarray chip platform, which was efficiently monitored by enhanced green fluorescent protein (EGFP) expression of four NSC-specific biomarkers including sex determining region Y-box 2 (SOX2), glial fibrillary acidic protein (GFAP), synapsin1, and myelin basic protein (MBP) with the promoter-reporter assay system.

High-throughput metabolism-induced toxicity assays demonstrated on a 384-pillar plate.

The US Environmental Protection Agency (EPA) launched the Transform Tox Testing Challenge in 2016 with the goal of developing practical methods that can be integrated into conventional high-throughput screening (HTS) assays to better predict the toxicity of parent compounds and their metabolites in vivo. In response to this need and to retrofit existing HTS assays for assessing metabolism-induced toxicity of compounds, we have developed a 384-pillar plate that is complementary to traditional 384-well plates and ideally suited for culturing human cells in three dimensions at a microscale. Briefly, human embryonic kidney (HEK) 293 cells in a mixture of alginate and Matrigel were printed on the 384-pillar plates using a microarray spotter, which were coupled with 384-well plates containing nine model compounds provided by the EPA, five representative Phase I and II drug metabolizing enzymes (DMEs), and one no enzyme control. Viability and membrane integrity of HEK 293 cells were measured with the calcein AM and CellTiter-Glo® kit to determine the IC50 values of the nine parent compounds and DME-generated metabolites. The Z' factors and the coefficient of variation measured were above 0.6 and below 14%, respectively, indicating that the assays established on the 384-pillar plate are robust and reproducible. Out of nine compounds tested, six compounds showed augmented toxicity with DMEs and one compound showed detoxification with a Phase II DME. This result indicates that the 384-pillar plate platform can be used to measure metabolism-induced toxicity of compounds in high-throughput with individual DMEs. As xenobiotics metabolism is a complex process with a variety of DMEs involved, the predictivity of our approach could be further improved with mixtures of DMEs.

SH003 induces apoptosis of DU145 prostate cancer cells by inhibiting ERK-involved pathway.

BACKGROUND: Herbal medicines have been used in cancer treatment, with many exhibiting favorable side effect and toxicity profiles compared with conventional chemotherapeutic agents. SH003 is a novel extract from Astragalus membranaceus, Angelica gigas, and Trichosanthes Kirilowii Maximowicz combined at a 1:1:1 ratio that impairs the growth of breast cancer cells. This study investigates anti-cancer effects of SH003 in prostate cancer cells. METHODS: SH003 extract in 30% ethanol was used to treat the prostate cancer cell lines DU145, LNCaP, and PC-3. Cell viability was determined by MTT and BrdU incorporation assays. Next, apoptotic cell death was determined by Annexin V and 7-AAD double staining methods. Western blotting was conducted to measure protein expression levels of components of cell death and signaling pathways. Intracellular reactive oxygen species (ROS) levels were measured using H2DCF-DA. Plasmid-mediated ERK2 overexpression in DU145 cells was used to examine the effect of rescuing ERK2 function. Results were analyzed using the Student's t-test and P-values < 0.05 were considered to indicate statistically-significant differences. RESULTS: Our data demonstrate that SH003 induced apoptosis in DU145 prostate cancer cells by inhibiting ERK signaling. SH003 induced apoptosis of prostate cancer cells in dose-dependent manner, which was independent of androgen dependency. SH003 also increased intracellular ROS levels but this is not associated with its pro-apoptotic effects. SH003 inhibited phosphorylation of Ras/Raf1/MEK/ERK/p90RSK in androgen-independent DU145 cells, but not androgen-dependent LNCaP and PC-3 cells. Moreover, ERK2 overexpression rescued SH003-induced apoptosis in DU145 cells. CONCLUSIONS: SH003 induces apoptotic cell death of DU145 prostate cancer cells by inhibiting ERK2-mediated signaling.

Self-assembling peptides induced by eyes absent enzyme to boost the efficacy of doxorubicin therapy in drug-resistant breast cancer cells.

Enzyme-induced self-assembly (EISA) is a recently developed nanotechnology technique in which small molecules are induced by cellular enzymes self-assembling into nanostructures inside cancer cells. This technique can boost the efficacy of chemotherapy drugs by avoiding drug efflux, inhibiting the cells' DNA repair mechanisms, and targeting the mitochondria. In this work, we study the self-assembly of a short peptide and its fluorescence analogue induced by Eyes absent (EYA) tyrosine phosphatases to boost the efficacy of doxorubicin (DOX) therapy in drug-resistant types of breast cancer cells, MDA-MB-231 and MCF-7. The peptides Fmoc-FF-YP and NBD-FF-YP were synthesized with the solid-phase peptide synthesis (SPPS) method and analyzed with HPLC and MALDI-TOF. Dynamic light scattering was used to determine the size distribution of peptides exposed to the EYA enzyme in vitro. The presence of EYA enzymes in breast cancer cells was confirmed using the western blotting assay. The intracellular location of the peptide self-assembly was studied by imaging fluorescence NBD-tagged peptides. The efficacy of the peptide alone and with DOX was determined against MCF-7 and MDA-MB-231 using MTT and LIVE-DEAD assays. Nucleus and cytoplasm F-actin (Phalloidin) staining was used to determine cell morphology changes in response to the combination therapy of peptides/DOX. At an optimal concentration, the peptides are not toxic to the cells; however, they boost the efficacy of DOX against drug-resistant breast cancer cells. We used state-of-the-art computer-aided techniques to predict the molecular structure of peptides and their interactions with EYA. This study demonstrates an approach for incorporating non-cytotoxic components into DOX combination therapy, thereby avoiding increased systemic burden or adverse effects.

Reproducible generation of human liver organoids (HLOs) on a pillar plate platform via microarray 3D bioprinting.

Human liver organoids (HLOs) hold significant potential for recapitulating the architecture and function of liver tissues in vivo. However, conventional culture methods of HLOs, forming Matrigel domes in 6-/24-well plates, have technical limitations such as high cost and low throughput in organoid-based assays for predictive assessment of compounds in clinical and pharmacological lab settings. To address these issues, we have developed a unique microarray 3D bioprinting protocol of progenitor cells in biomimetic hydrogels on a pillar plate with sidewalls and slits, coupled with a clear bottom, 384-deep well plate for scale-up production of HLOs. Microarray 3D bioprinting, a droplet-based printing technology, was used to generate a large number of small organoids on the pillar plate for predictive hepatotoxicity assays. Foregut cells, differentiated from human iPSCs, were mixed with Matrigel and then printed on the pillar plate rapidly and uniformly, resulting in coefficient of variation (CV) values in the range of 15-18%, without any detrimental effect on cell viability. Despite utilizing 10-50-fold smaller cell culture volume compared to their counterparts in Matrigel domes in 6-/24-well plates, HLOs differentiated on the pillar plate exhibited similar morphology and superior function, potentially due to rapid diffusion of nutrients and oxygen at the small scale. Day 25 HLOs were robust and functional on the pillar plate in terms of their viability, albumin secretion, CYP3A4 activity, and drug toxicity testing, all with low CV values. From three independent trials of in situ assessment, the IC50 values calculated for sorafenib and tamoxifen were 6.2 ± 1.6 µM and 25.4 ± 8.3 µM, respectively. Therefore, our unique 3D bioprinting and miniature organoid culture on the pillar plate could be used for scale-up, reproducible generation of HLOs with minimal manual intervention for high-throughput assessment of compound hepatotoxicity.

Reproducible generation of human liver organoids (HLOs) on a pillar plate platform via microarray 3D bioprinting.

Human liver organoids (HLOs) hold significant potential for recapitulating the architecture and function of liver tissues in vivo. However, conventional culture methods of HLOs, forming Matrigel domes in 6-/24-well plates, have technical limitations such as high cost and low throughput in organoid-based assays for predictive assessment of compounds in clinical and pharmacological lab settings. To address these issues, we have developed a unique microarray 3D bioprinting protocol of progenitor cells in biomimetic hydrogels on a pillar plate with sidewalls and slits, coupled with a clear bottom, 384-deep well plate for scale-up production of HLOs. Microarray 3D bioprinting, a droplet-based printing technology, was used to generate a large number of small organoids on the pillar plate for predictive hepatotoxicity assays. Foregut cells, differentiated from human iPSCs, were mixed with Matrigel and then printed on the pillar plate rapidly and uniformly, resulting in coefficient of variation (CV) values in the range of 15 - 18%, without any detrimental effect on cell viability. Despite utilizing 10 - 50-fold smaller cell culture volume compared to their counterparts in Matrigel domes in 6-/24-well plates, HLOs differentiated on the pillar plate exhibited similar morphology and superior function, potentially due to rapid diffusion of nutrients and oxygen at the small scale. Day 25 HLOs were robust and functional on the pillar plate in terms of their viability, albumin secretion, CYP3A4 activity, and drug toxicity testing, all with low CV values. From three independent trials of in situ assessment, the IC50 values calculated for sorafenib and tamoxifen were 6.2 ± 1.6 µM and 25.4 ± 8.3 µM, respectively. Therefore, our unique 3D bioprinting and miniature organoid culture on the pillar plate could be used for scale-up, reproducible generation of HLOs with minimal manual intervention for high-throughput assessment of compound hepatotoxicity.

Emergency medicine residents' learning curve in diagnosing deep vein thrombosis with 3-point venous point-of-care ultrasound.

BACKGROUND: Many cases of deep vein thrombosis (DVT) are diagnosed in the emergency department, and abbreviated lower extremity venous point-of-care ultrasound (POCUS) has already shown an accuracy comparable to that of specialists. This study aimed to identify the learning curve necessary for emergency medicine (EM) residents to achieve expertise-level accuracy in diagnosing DVT through a 3-point lower extremity venous POCUS. METHODS: This prospective study was conducted at an emergency department between May 2021 and October 2022. Four EM residents underwent a one-hour POCUS training session and performed DVT assessments in participants with DVT symptoms or confirmed pulmonary embolism. POCUS was performed at three proximal lower extremity sites to evaluate the thrombi presence and vein compressibility, with results validated by specialized radiology ultrasound. Cumulative sum (CUSUM) and the Bush and Mosteller models were used to analyze the learning curve, while generalized estimating equations were used to identify factors affecting diagnostic accuracy. RESULTS: 91 POCUS scans were conducted in 49 patients, resulting in 22% DVT confirmed by specialized venous ultrasound. In the CUSUM analysis, all four EM residents attained a 90% success rate at the common femoral vein, whereas only half achieved this rate when all three sites were considered. According to Bush and Mosteller models, 13-18 cases are required to attain 90-95% diagnostic accuracy. After 10-16 cases, the examination time for each resident decreased, and a 20% increase in examiner confidence was linked to a 2.506-fold increase in the DVT diagnosis accuracy. CONCLUSION: EM residents generally required 13-18 cases for 90-95% DVT diagnostic accuracy, but proficiency varied among individuals, particularly requiring more cases for regions outside the common femoral vein.

Regenerative human liver organoids (HLOs) in a pillar/perfusion plate for hepatotoxicity assays.

Human liver organoids (HLOs) differentiated from embryonic stem cells (ESCs), induced pluripotent stem cells (iPSCs), and adult stem cells (ASCs) can recapitulate structure and function of human fetal liver tissues, thus, considered as a promising tissue model for liver diseases and predictive compound screening. Nonetheless, there are still several technical challenges to adopt HLOs in the drug discovery process, which include relatively long-term cell differentiation with multiple culture media (3 - 4 weeks) leading to batch-to-batch variation, short-term hepatic function after maturation (3 - 5 days), low assay throughput due to Matrigel dissociation and HLO transfer to a microtiter well plate, and insufficient maturity as compared to primary hepatocytes. To address these issues, expandable HLOs (Exp-HLOs) derived from human iPSCs were generated by optimizing differentiation protocols, which were rapidly printed on a 144-pillar plate with sidewalls and slits (144PillarPlate) and dynamically cultured for up to 20 days into differentiated HLOs (Diff-HLOs) in a 144-perfusion plate with perfusion wells and reservoirs (144PerfusionPlate) for in situ organoid culture and analysis. Dynamically cultured Diff-HLOs were generated robustly and reproducibly in the pillar/perfusion plate with higher maturity as compared to those in statically cultured HLOs by differentiating Exp-HLOs for 10 days. In addition, Diff-HLOs in the pillar/perfusion plate were tested with acetaminophen and troglitazone for 3 days to assess drug-induced liver injury (DILI) and then incubated in an expansion medium for 10 days to evaluate the recovery of the liver from DILI. The assessment of liver regeneration post injury is critical to understand the mechanism of recovery and determine the threshold drug concentration beyond which there will be a sharp decrease in the liver's regenerative capacity. We envision that bioprinted Diff-HLOs in the pillar/perfusion plate could be used for high-throughput screening (HTS) of hepatotoxic compounds due to short-term differentiation of passage-able Exp-HLOs necessary, stable hepatic function after maturation, high reproducibility, and high throughput with capability of in situ organoid culture, testing, staining, imaging, and analysis.

Dynamic culture of cerebral organoids using a pillar/perfusion plate for the assessment of developmental neurotoxicity.

Despite the potential toxicity of commercial chemicals to the development of the nervous system (known as developmental neurotoxicity or DNT), conventional in vitro cell models have primarily been employed for the assessment of acute neuronal toxicity. On the other hand, animal models used for the assessment of DNT are not physiologically relevant due to the heterogenic difference between humans and animals. In addition, animal models are low-throughput, time-consuming, expensive, and ethically questionable. Recently, human brain organoids have emerged as a promising alternative to assess the detrimental effects of chemicals on the developing brain. However, conventional organoid culture systems have several technical limitations including low throughput, lack of reproducibility, insufficient maturity of organoids, and the formation of the necrotic core due to limited diffusion of nutrients and oxygen. To address these issues and establish predictive DNT models, cerebral organoids were differentiated in a dynamic condition in a unique pillar/perfusion plate, which were exposed to test compounds to evaluate DNT potential. The pillar/perfusion plate facilitated uniform, dynamic culture of cerebral organoids with improved proliferation and maturity by rapid, bidirectional flow generated on a digital rocker. Day 9 cerebral organoids in the pillar/perfusion plate were exposed to ascorbic acid (DNT negative) and methylmercury (DNT positive) in a dynamic condition for 1 and 3 weeks, and changes in organoid morphology and neural gene expression were measured to determine DNT potential. As expected, ascorbic acid didn't induce any changes in organoid morphology and neural gene expression. However, exposure of day 9 cerebral organoids to methylmercury resulted in significant changes in organoid morphology and neural gene expression. Interestingly, methylmercury did not induce adverse changes in cerebral organoids in a static condition, thus highlighting the importance of dynamic organoid culture in DNT assessment.

Real-Time Tracheal Ultrasound vs. Capnography for Intubation Confirmation during CPR Wearing a Powered Air-Purifying Respirator in COVID-19 Era.

This study aimed to compare the accuracy of real-time trans-tracheal ultrasound (TTUS) with capnography to confirm intubation in cardiopulmonary resuscitation (CPR) while wearing a powered air-purifying respirator (PAPR). This setting reflects increased caution due to contagious diseases. This single-center, prospective, comparative study enrolled patients requiring CPR while wearing a PAPR who visited the emergency department of a tertiary medical center from December 2020 to August 2022. A physician performed the TTUS in real time and recorded the tube placement assessment. Another healthcare provider attached waveform capnography to the tube and recorded end-tidal carbon dioxide (EtCO2) after five ventilations. The accuracy and agreement of both methods compared with direct laryngoscopic visualization of tube placement, and the time taken by both methods was evaluated. Thirty-three patients with cardiac arrest were analyzed. TTUS confirmed tube placement with 100% accuracy, sensitivity, and specificity, whereas capnography demonstrated 97% accuracy, 96.8% sensitivity, and 100% specificity. The Kappa values for TTUS and capnography compared to direct visualization were 1.0 and 0.7843, respectively. EtCO2 was measured in 45 (37-59) seconds (median (interquartile range)), whereas TTUS required only 12 (8-23) seconds, indicating that TTUS was significantly faster (p < 0.001). No significant correlation was found between the physician's TTUS proficiency and image acquisition time. This study demonstrated that TTUS is more accurate and faster than EtCO2 measurement for confirming endotracheal tube placement during CPR, particularly in the context of PAPR usage in pandemic conditions.

A Pillar/Perfusion Plate Enhances Cell Growth, Reproducibility, Throughput, and User Friendliness in Dynamic 3D Cell Culture.

Static three-dimensional (3D) cell culture has been demonstrated in ultralow attachment well plates, hanging droplet plates, and microtiter well plates with hydrogels or magnetic nanoparticles. Although it is simple, reproducible, and relatively inexpensive, thus potentially used for high-throughput screening, statically cultured 3D cells often suffer from a necrotic core due to limited nutrient and oxygen diffusion and waste removal and have a limited in vivo-like tissue structure. Here, we overcome these challenges by developing a pillar/perfusion plate platform and demonstrating high-throughput, dynamic 3D cell culture. Cell spheroids were loaded on the pillar plate with hydrogel by simple sandwiching and encapsulation and cultured dynamically in the perfusion plate on a digital rocker. Unlike traditional microfluidic devices, fast flow velocity was maintained within perfusion wells and the pillar plate was separated from the perfusion plate for cell-based assays. It was compatible with common lab equipment and allowed cell culture, testing, staining, and imaging in situ. The pillar/perfusion plate enhanced cell growth by rapid diffusion, reproducibility, assay throughput, and user friendliness in a dynamic 3D cell culture.

Status and perception of point-of-care ultrasound education in Korean medical schools: A national cross-sectional study.

As point-of-care ultrasound (POCUS) is increasingly being used in clinical settings, ultrasound education is expanding into student curricula. We aimed to determine the status and awareness of POCUS education in Korean medical schools using a nationwide cross-sectional survey. In October 2021, a survey questionnaire consisting of 20 questions was distributed via e-mail to professors in the emergency medicine (EM) departments of Korean medical schools. The questionnaire encompassed 19 multiple-choice questions covering demographics, current education, perceptions, and barriers, and the final question was an open-ended inquiry seeking suggestions for POCUS education. All EM departments of the 40 medical schools responded, of which only 13 (33%) reported providing POCUS education. The implementation of POCUS education primarily occurred in the third and fourth years, with less than 4 hours of dedicated training time. Five schools offered a hands-on education. Among schools offering ultrasound education, POCUS training for trauma cases is the most common. Eight schools had designated professors responsible for POCUS education and only 2 possessed educational ultrasound devices. Of the respondents, 64% expressed the belief that POCUS education for medical students is necessary, whereas 36%, including those with neutral opinions, did not anticipate its importance. The identified barriers to POCUS education included faculty shortages (83%), infrastructure limitations (76%), training time constraints (74%), and a limited awareness of POCUS (29%). POCUS education in Korean medical schools was limited to a minority of EM departments (33%). To successfully implement POCUS education in medical curricula, it is crucial to clarify learning objectives, enhance faculty recognition, and improve the infrastructure. These findings provide valuable insights for advancing ultrasound training in medical schools to ensure the provision of high-quality POCUS education for future healthcare professionals.

High-throughput assessment of metabolism-mediated neurotoxicity by combining 3D-cultured neural stem cells and liver cell spheroids.

Despite the fact that biotransformation in the liver plays an important role in the augmented toxicity and detoxification of chemicals, relatively little efforts have been made to incorporate biotransformation into in vitro neurotoxicity testing. Conventional in vitro systems for neurotoxicity tests lack the capability of investigating the qualitative and quantitative differences between parent chemicals and their metabolites in the human body. Therefore, there is a need for an in vitro toxicity screening system that can incorporate hepatic biotransformation of chemicals and predict the susceptibility of their metabolites to induce neurotoxicity. To address this need, we adopted 3D cultures of metabolically competent HepaRG cell line with ReNcell VM and established a high-throughput, metabolism-mediated neurotoxicity testing system. Briefly, spheroids of HepaRG cells were generated in an ultralow attachment (ULA) 384-well plate while 3D-cultured ReNcell VM was established on a 384-pillar plate with sidewalls and slits (384PillarPlate). Metabolically sensitive test compounds were added in the ULA 384-well plate with HepaRG spheroids and coupled with 3D-cultured ReNcell VM on the 384PillarPlate, which allowed us to generate metabolites in situ by HepaRG cells and test them against neural stem cells. We envision that this approach could be potentially adopted in pharmaceutical and chemical industries when high-throughput screening (HTS) is necessary to assess neurotoxicity of compounds and their metabolites.

A pillar/perfusion plate enhances cell growth, reproducibility, throughput, and user friendliness in dynamic 3D cell culture.

Static three-dimensional (3D) cell culture has been demonstrated in ultralow attachment well plates, hanging droplet plates, and microtiter well plates with hydrogels or magnetic nanoparticles. Although it is simple, reproducible, and relatively inexpensive, thus potentially used for high-throughput screening, statically cultured 3D cells often suffer from the necrotic core due to limited nutrient and oxygen diffusion and waste removal and have limited in vivo-like tissue structure. Here, we overcome these challenges by developing a pillar/perfusion plate platform and demonstrating high-throughput, dynamic 3D cell culture. Cell spheroids have been loaded on the pillar plate with hydrogel by simple sandwiching and encapsulation and cultured dynamically in the perfusion plate on a digital rocker. Unlike traditional microfluidic devices, fast flow rates were maintained within perfusion wells, and the pillar plate could be separated from the perfusion plate for cell-based assays. It was compatible with common lab equipment and allowed cell culture, testing, staining, and imaging in situ. The pillar/perfusion plate enhanced cell growth by rapid diffusion, reproducibility, assay throughput, and user friendliness in dynamic 3D cell culture.