All-in-one wearable drug efficacy assessment systems for bulbar muscle function using amyotrophic lateral sclerosis animal models.

Preclinical studies are crucial for developing amyotrophic lateral sclerosis drugs. Current FDA-approved drugs have been created by monitoring limb muscle function and histological analysis of amyotrophic lateral sclerosis model animals. Drug candidates for this disease have yet to be tested for bulbar-onset type due to the limitations of traditional preclinical tools: excessive animal use and discrete detection of disease progress. Here, our study introduces an all-in-one, wireless, integrated wearable system for facilitating continuous drug efficacy assessment of dysphagia-related muscles in animals during natural eating behaviors. By incorporating a kirigami-based strain-isolation mechanism, this device mounted on the skin of animals mitigates electromyography signal contamination caused by unpredictable animal movements. Our findings indicate this system, measuring the progression of motor neuron denervation, offers high precision in monitoring drug effects on dysphagia-responsible bulbar muscles. This study paves the way for more humane and efficient approaches to developing treatment solutions for degenerative neuromuscular diseases.

Large-scale smart bioreactor with fully integrated wireless multivariate sensors and electronics for long-term in situ monitoring of stem cell culture.

Achieving large-scale, cost-effective, and reproducible manufacturing of stem cells with the existing devices is challenging. Traditional single-use cell-bag bioreactors, limited by their rigid and single-point sensors, struggle with accuracy and scalability for high-quality cell manufacturing. Here, we introduce a smart bioreactor system that enables multi-spatial sensing for real-time, wireless culture monitoring. This scalable system includes a low-profile, label-free thin-film sensor array and electronics integrated with a flexible cell bag, allowing for simultaneous assessment of culture properties such as pH, dissolved oxygen, glucose, and temperature, to receive real-time feedback for up to 30 days. The experimental results show the accurate monitoring of time-dynamic and spatial variations of stem cells and myoblast cells with adjustable carriers from a plastic dish to a 2-liter cell bag. These advances open up the broad applicability of the smart sensing system for large-scale, lower-cost, reproducible, and high-quality engineered cell manufacturing for broad clinical use.

Synergistic Inhibition Guided Fragment-Linking Strategy and Quantitative Structure-Property Relationship Modeling To Design Inhalable Therapeutics for Asthma Targeting CSF1R.

The colony-stimulating factor-1 receptor (CSF1R) is a tyrosine-protein kinase that is a potential target for asthma therapeutics. We have applied a fragment-lead combination approach to identify small fragments that act synergistically with GW2580, a known inhibitor of CSF1R. Two fragment libraries were screened in combination with GW2580 by surface plasmon resonance (SPR). Binding affinity measurements confirmed that thirteen fragments bind specifically to the CSF1R, and a kinase activity assay further validated the inhibitory effect of these fragments. Several fragment compounds enhanced the inhibitory activity of the lead inhibitor. Computational solvent mapping, molecular docking, and modeling studies suggest that some of these fragments bind adjacent to the binding site of the lead inhibitor and further stabilize the inhibitor-bound state. Modeling results guided the computational fragment-linking approach to design potential next-generation compounds. The inhalability of these proposed compounds was predicted using quantitative structure-property relationships (QSPR) modeling based on an analysis of 71 drugs currently on the market. This work provides new insights into the development of inhalable small molecule therapeutics for asthma.

Evaluation of Four Rapid Antigen Tests for the Detection of SARS-CoV-2 Infection with Nasopharyngeal Swabs.

Owing to the high transmissibility of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants, the capacity of testing systems based on the gold standard real-time reverse transcription-polymerase chain reaction (rRT-PCR) is limited. Rapid antigen tests (RATs) can substantially contribute to the prevention of community transmission, but their further assessment is required. Here, using 1503 nasopharyngeal swabs, we compared the diagnostic performance of four RAT kits (Abbott Panbio™ COVID-19 Ag Rapid Test, SD Biosensor Standard™ Q COVID-19 Ag Test, Humasis COVID-19 Ag Test, and SG Medical Acrosis COVID-19 Ag Test) to the cycle threshold (Ct) values obtained from rRT-PCR. The precision values, area under the curve values, SARS-CoV-2 variant detection ability, and non-SARS-CoV-2 specificity of all four kits were similar. An assay using the Acrosis kit had a significantly better positive detection rate with a higher recall value and cut-off value than that using the other three RAT kits. During the current COVID-19 pandemic, the Acrosis kit is an effective tool to prevent the spread of SARS-CoV-2 in communities.

A Work-Related Musculoskeletal Disorders (WMSDs) Risk-Assessment System Using a Single-View Pose Estimation Model.

Musculoskeletal disorders are an unavoidable occupational health problem. In particular, workers who perform repetitive tasks onsite in the manufacturing industry suffer from musculoskeletal problems. In this paper, we propose a system that evaluates the posture of workers in the manufacturing industry with single-view 3D human pose-estimation that can estimate the posture in 3D using an RGB camera that can easily acquire the posture of a worker in a complex workplace. The proposed system builds a Duckyang-Auto Worker Health Safety Environment (DyWHSE), a manufacturing-industry-specific dataset, to estimate the wrist pose evaluated by the Rapid Limb Upper Assessment (RULA). Additionally, we evaluate the quality of the built DyWHSE dataset using the Human3.6M dataset, and the applicability of the proposed system is verified by comparing it with the evaluation results of the experts. The proposed system provides quantitative assessment guidance for working posture risk assessment, assisting the continuous posture assessment of workers.

Assessment of Various Density Functional Theory Methods for Finding Accurate Structures of Actinide Complexes.

Density functional theory (DFT) is a widely used computational method for predicting the physical and chemical properties of metals and organometals. As the number of electrons and orbitals in an atom increases, DFT calculations for actinide complexes become more demanding due to increased complexity. Moreover, reasonable levels of theory for calculating the structures of actinide complexes are not extensively studied. In this study, 38 calculations, based on various combinations, were performed on molecules containing two representative actinides to determine the optimal combination for predicting the geometries of actinide complexes. Among the 38 calculations, four optimal combinations were identified and compared with experimental data. The optimal combinations were applied to a more complicated and practical actinide compound, the uranyl complex (UO2(2,2'-(1E,1'E)-(2,2-dimethylpropane-1,3-dyl)bis(azanylylidene)(CH3OH)), for further confirmation. The corresponding optimal calculation combination provides a reasonable level of theory for accurately optimizing the structure of actinide complexes using DFT.

Design of SARS-CoV-2 PLpro Inhibitors for COVID-19 Antiviral Therapy Leveraging Binding Cooperativity.

Antiviral agents that complement vaccination are urgently needed to end the COVID-19 pandemic. The SARS-CoV-2 papain-like protease (PLpro), one of only two essential cysteine proteases that regulate viral replication, also dysregulates host immune sensing by binding and deubiquitination of host protein substrates. PLpro is a promising therapeutic target, albeit challenging owing to featureless P1 and P2 sites recognizing glycine. To overcome this challenge, we leveraged the cooperativity of multiple shallow binding sites on the PLpro surface, yielding novel 2-phenylthiophenes with nanomolar inhibitory potency. New cocrystal structures confirmed that ligand binding induces new interactions with PLpro: by closing of the BL2 loop of PLpro forming a novel "BL2 groove" and by mimicking the binding interaction of ubiquitin with Glu167 of PLpro. Together, this binding cooperativity translates to the most potent PLpro inhibitors reported to date, with slow off-rates, improved binding affinities, and low micromolar antiviral potency in SARS-CoV-2-infected human cells.

Development of an efficient Sanger sequencing-based assay for detecting SARS-CoV-2 spike mutations.

Novel strains of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) harboring nucleotide changes (mutations) in the spike gene have emerged and are spreading rapidly. These mutations are associated with SARS-CoV-2 transmissibility, virulence, or resistance to some neutralizing antibodies. Thus, the accurate detection of spike mutants is crucial for controlling SARS-CoV-2 transmission and identifying neutralizing antibody-resistance caused by amino acid changes in the receptor-binding domain. Here, we developed five SARS-CoV-2 spike gene primer pairs (5-SSG primer assay; 69S, 144S, 417S, 484S, and 570S) and verified their ability to detect nine key spike mutations (ΔH69/V70, T95I, G142D, ΔY144, K417T/N, L452R, E484K/Q, N501Y, and H655Y) using a Sanger sequencing-based assay. The 5-SSG primer assay showed 100% specificity and a conservative limit of detection with a median tissue culture infective dose (TCID50) values of 1.4 × 102 TCID50/mL. The accuracy of the 5-SSG primer assay was confirmed by next generation sequencing. The results of these two approaches showed 100% consistency. Taken together, the ability of the 5-SSG primer assay to accurately detect key SARS-CoV-2 spike mutants is reliable. Thus, it is a useful tool for detecting SARS-CoV-2 spike gene mutants in a clinical setting, thereby helping to improve the management of patients with COVID-19.

Identification of Small Molecule Inhibitors against Staphylococcus aureus Dihydroorotase via HTS.

Drug-resistant Staphylococcus aureus is an imminent threat to public health, increasing the importance of drug discovery utilizing unexplored bacterial pathways and enzyme targets. De novo pyrimidine biosynthesis is a specialized, highly conserved pathway implicated in both the survival and virulence of several clinically relevant pathogens. Class I dihydroorotase (DHOase) is a separate and distinct enzyme present in gram positive bacteria (i.e., S. aureus, B. anthracis) that converts carbamoyl-aspartate (Ca-asp) to dihydroorotate (DHO)-an integral step in the de novo pyrimidine biosynthesis pathway. This study sets forth a high-throughput screening (HTS) of 3000 fragment compounds by a colorimetry-based enzymatic assay as a primary screen, identifying small molecule inhibitors of S. aureus DHOase (SaDHOase), followed by hit validation with a direct binding analysis using surface plasmon resonance (SPR). Competition SPR studies of six hit compounds and eight additional analogs with the substrate Ca-asp determined the best compound to be a competitive inhibitor with a KD value of 11 µM, which is 10-fold tighter than Ca-asp. Preliminary structure-activity relationship (SAR) provides the foundation for further structure-based antimicrobial inhibitor design against S. aureus.

Potent, Novel SARS-CoV-2 PLpro Inhibitors Block Viral Replication in Monkey and Human Cell Cultures.

Antiviral agents blocking SARS-CoV-2 viral replication are desperately needed to complement vaccination to end the COVID-19 pandemic. Viral replication and assembly are entirely dependent on two viral cysteine proteases: 3C-like protease (3CLpro) and the papain-like protease (PLpro). PLpro also has deubiquitinase (DUB) activity, removing ubiquitin (Ub) and Ub-like modifications from host proteins, disrupting the host immune response. 3CLpro is inhibited by many known cysteine protease inhibitors, whereas PLpro is a relatively unusual cysteine protease, being resistant to blockade by such inhibitors. A high-throughput screen of biased and unbiased libraries gave a low hit rate, identifying only CPI-169 and the positive control, GRL0617, as inhibitors with good potency (IC50 < 10 lower case Greek µM). Analogues of both inhibitors were designed to develop structure-activity relationships; however, without a co-crystal structure of the CPI-169 series, we focused on GRL0617 as a starting point for structure-based drug design, obtaining several co-crystal structures to guide optimization. A series of novel 2-phenylthiophene-based non-covalent SARS-CoV-2 PLpro inhibitors were obtained, culminating in low nanomolar potency. The high potency and slow inhibitor off-rate were rationalized by newly identified ligand interactions with a 'BL2 groove' that is distal from the active site cysteine. Trapping of the conformationally flexible BL2 loop by these inhibitors blocks binding of viral and host protein substrates; however, until now it has not been demonstrated that this mechanism can induce potent and efficacious antiviral activity. In this study, we report that novel PLpro inhibitors have excellent antiviral efficacy and potency against infectious SARS-CoV-2 replication in cell cultures. Together, our data provide structural insights into the design of potent PLpro inhibitors and the first validation that non-covalent inhibitors of SARS-CoV-2 PLpro can block infection of human cells with low micromolar potency.

Identification and design of novel small molecule inhibitors against MERS-CoV papain-like protease via high-throughput screening and molecular modeling.

The development of new therapeutic agents against the coronavirus causing Middle East Respiratory Syndrome (MERS) is a continuing imperative. The initial MERS-CoV epidemic was contained entirely through public health measures, but episodic cases continue, as there are currently no therapeutic agents effective in the treatment of MERS-CoV, although multiple strategies have been proposed. In this study, we screened 30,000 compounds from three different compound libraries against one of the essential proteases, the papain-like protease (PLpro), using a fluorescence-based enzymatic assay followed by surface plasmon resonance (SPR) direct binding analysis for hit confirmation. Mode of inhibition assays and competition SPR studies revealed two compounds to be competitive inhibitors. To improve upon the inhibitory activity of the best hit compounds, a small fragment library consisting of 352 fragments was screened in the presence of each hit compound, resulting in one fragment that enhanced the IC50 value of the best hit compound by 3-fold. Molecular docking and MM/PBSA binding energy calculations were used to predict potential binding sites, providing insight for design and synthesis of next-generation compounds.

Discovery of a Novel Highly Selective Histamine H4 Receptor Antagonist for the Treatment of Atopic Dermatitis.

The histamine H4 receptor (H4R), a member of the G-protein coupled receptor family, has been considered as a potential therapeutic target for treating atopic dermatitis (AD). A large number of H4R antagonists have been disclosed, but no efficient agents controlling both pruritus and inflammation in AD have been developed yet. Here, we have discovered a novel class of orally available H4R antagonists showing strong anti-itching and anti-inflammation activity as well as excellent selectivity against off-targets. A pharmacophore-based virtual screening system constructed in-house successfully identified initial hit compound 9, and the subsequent homology model-guided optimization efficiently led us to discover pyrido[2,3- e]tetrazolo[1,5- a]pyrazine analogue 48 as a novel chemotype of a potent and highly selective H4R antagonist. Importantly, orally administered compound 48 exhibits remarkable efficacy on antipruritus and anti-inflammation with a favorable pharmacokinetic (PK) profile in several mouse models of AD. Thus, these data strongly suggest that our compound 48 is a promising clinical candidate for treatment of AD.

H5N8 Highly Pathogenic Avian Influenza in the Republic of Korea: Epidemiology During the First Wave, from January Through July 2014.

OBJECTIVES: This study describes the outbreaks of H5N8 highly pathogenic avian influenza (HPAI) in Korea during the first wave, from January 16, 2014 through July 25, 2014. Its purpose is to provide a better understanding of the epidemiology of H5N8 HPAI. METHODS: Information on the outbreak farms and HPAI positive wild birds was provided by the Animal and Plant Quarantine Agency. The epidemiological investigation sheets for the outbreak farms were examined. RESULTS: During the 7-month outbreak period (January-July 2014), H5N8 HPAI was confirmed in 212 poultry farms, 38 specimens from wild birds (stools, birds found dead or captured). Ducks were the most frequently infected poultry species (159 outbreak farms, 75.0%), and poultry in 67 (31.6%) outbreak farms was asymptomatic. CONCLUSION: As in the previous four H5N1 epidemics of HPAI that occurred in Korea, this epidemic of H5N8 proved to be associated with migratory birds. Poultry farms in Korea can hardly be free from the risk of HPAI introduced via migratory birds. The best way to overcome this geographical factor is to reinforce biosecurity to prevent exposure of farms, related people, and poultry to the pathogen.

A modified keyhole technique for minimizing size mismatches during a smaller than optimal lateral meniscal allograft transplantation: surgical technique.

Despite careful preoperative and intraoperative sizing of lateral menisci in recipient knees, size mismatches between allografts and recipient menisci are often encountered. To minimize size mismatches during the transplantation of smaller than expected lateral meniscal allografts, we devised a modified keyhole technique by dividing the central bone bridge into 2 bony fragments. The fragments are attached to the anterior and posterior horns. In the event of mismatch, the anterior bony fragment is moved anteriorly in the keyhole tunnel. Although the technique of meniscal transplantation is technically demanding, the central bone bridge can be divided without difficulty. The described technique is considered a useful alternative method during lateral meniscal transplantation.

Seasonal flux of nonylphenol in Han River, Korea.

In order to understand the behavior of nonylphenol (NP) in Han River, water, suspended particle and sediment samples were analyzed during summer, autumn and winter. Concentrations of nonylphenol in water ranged from 23.2 to 187.6 ng/l, in suspended particle from 6.8 to 190.8 ng/l and in sediment from 25.4 to 932.0 ng/gdrywt. An increasing trend in the concentration is noticed in all matrices along down the river. In case of water and suspended particle, concentrations were higher in warmer season than in colder season. Percentage of nonylphenol in the suspended particle phase decreased from 67% to 28% with decreasing temperature in water. A reasonable correlation (R2 = 0.63) was obtained for water and suspended particle. The partition coefficient Log Kp is 4.8. No seasonal variation of the concentration in sediment is noticed in this study.