Characterization of a pseudohemocyanin gene (PtPhc1) and its immunity function in response to Vibrio parahaemolyticus infection in the swimming crab Portunus trituberculatus.

Pseudohemocyanin is a member of the hemocyanin superfamily, but little research is available on its function in immunology. In this study, a Portunus trituberculatus pseudohemocyanin gene, named PtPhc1, was obtained by gene cloning. The PtPhc1 cDNA was 2312 bp in length, encoding 684 amino acids while exhibiting a characteristic hemocyanin structural domain. Tissue expression analysis revealed ubiquitous expression of PtPhc1 across all tissues, with the highest level of expression observed in the hepatopancreas. The expression pattern of PtPhc1 in response to Vibrio parahaemolyticus infection was clarified using RT-qPCR in swimming crabs. Notably, the expression peaked at 24 h, and increased 1435-fold compared to the control group in the hepatopancreas. While the expression level reached the maximum value at 72 h, which was 3.24 times higher than that of the control group in hemocytes. Remarkably, the reduction in PtPhc1 expression led to a noteworthy 30% increase in the mortality rate of P. trituberculatus when exposed to V. parahaemolyticus. In addition, in vitro bacterial inhibition assays exhibited a dose-dependent suppression of bacterial proliferation by recombinant PtPhc1 protein, with a notable inhibition rate of 48.33% against V. parahaemolyticus at a concentration of 0.03 mg/mL. To the best of our knowledge, the results establish the function of pseudohaemocyanin in immunity for the first time, contributing to a deeper comprehension of innate immune regulatory mechanisms in aquatic organisms and advancing strategies for disease-resistant breeding.

Identification of novel tetrahydroquinoxaline derived phenyl ureas as modulators of the hepatitis B virus nucleocapsid assembly.

A key step of hepatitis B virus (HBV) replication is the selective packaging of pregenomic RNA (pgRNA) by core protein (Cp) dimers, forming a nucleocapsid where the reverse transcriptional viral DNA replication takes place. One approach in the development of new anti-HBV drugs is to disrupt the assembly of HBV nucleocapsids by misdirecting Cp dimers to assemble morphologically normal capsids devoid of pgRNA. In this study, we built upon our previous discovery of benzamide-derived HBV capsid assembly modulators by exploring fused bicyclic scaffolds with an exocyclic amide that is ß, γ to the fused ring, and identified 1,2,3,4-tetrahydroquinoxaline derived phenyl ureas as a novel scaffold. Structure-activity relationship studies showed that a favorable hydrophobic substitution can be tolerated at the 2-position of the 1,2,3,4-tetrahydroquinoxaline core, and the resulting compound 88 demonstrated comparable or improved antiviral potencies in mouse and human hepatocyte-derived HBV-replicating cell lines compared to our previously reported benzamide compound, 38017 (8). In addition, a novel bis-urea series based on 1,2,3,4-tetrahydroquinoxaline was also found to inhibit HBV DNA replication with sub-micromolar EC50 values. The mode of action of these compounds is consistent with specific inhibition of pgRNA encapsidation into nucleocapsids in hepatocytes.

Machine Learning Prediction Models for Mechanically Ventilated Patients: Analyses of the MIMIC-III Database.

Background: Mechanically ventilated patients in the intensive care unit (ICU) have high mortality rates. There are multiple prediction scores, such as the Simplified Acute Physiology Score II (SAPS II), Oxford Acute Severity of Illness Score (OASIS), and Sequential Organ Failure Assessment (SOFA), widely used in the general ICU population. We aimed to establish prediction scores on mechanically ventilated patients with the combination of these disease severity scores and other features available on the first day of admission. Methods: A retrospective administrative database study from the Medical Information Mart for Intensive Care (MIMIC-III) database was conducted. The exposures of interest consisted of the demographics, pre-ICU comorbidity, ICU diagnosis, disease severity scores, vital signs, and laboratory test results on the first day of ICU admission. Hospital mortality was used as the outcome. We used the machine learning methods of k-nearest neighbors (KNN), logistic regression, bagging, decision tree, random forest, Extreme Gradient Boosting (XGBoost), and neural network for model establishment. A sample of 70% of the cohort was used for the training set; the remaining 30% was applied for testing. Areas under the receiver operating characteristic curves (AUCs) and calibration plots would be constructed for the evaluation and comparison of the models' performance. The significance of the risk factors was identified through models and the top factors were reported. Results: A total of 28,530 subjects were enrolled through the screening of the MIMIC-III database. After data preprocessing, 25,659 adult patients with 66 predictors were included in the model analyses. With the training set, the models of KNN, logistic regression, decision tree, random forest, neural network, bagging, and XGBoost were established and the testing set obtained AUCs of 0.806, 0.818, 0.743, 0.819, 0.780, 0.803, and 0.821, respectively. The calibration curves of all the models, except for the neural network, performed well. The XGBoost model performed best among the seven models. The top five predictors were age, respiratory dysfunction, SAPS II score, maximum hemoglobin, and minimum lactate. Conclusion: The current study indicates that models with the risk of factors on the first day could be successfully established for predicting mortality in ventilated patients. The XGBoost model performs best among the seven machine learning models.

Applicability of free drug hypothesis to drugs with good membrane permeability that are not efflux transporter substrates: A microdialysis study in rats.

In clinical pharmacology, the free drug hypothesis has been widely applied in the interpretation of the relationship between pharmacokinetics and pharmacodynamics (PK/PD). The free drug hypothesis assumes that the unbound drug concentration in blood is the same as that in the site of action at steady state. The objective of this study is to demonstrate whether the free drug hypothesis is universally applicable for all drugs. The unbound concentrations of the 18 compounds in blood and in brain interstitial fluids (ISF) at steady state following constant intravenous infusion were simultaneously monitored up to 6 hours via in vivo microdialysis technique. Based on the permeability and efflux ratio (ER), the test compounds can be divided into two classes. Class I includes the compounds with good membrane permeability that are not substrates of efflux transporters (eg, P-gp, BCRP, and MRPs), whereas Class II includes the compounds that are substrates of efflux transporters. The steady-state unbound drug concentrations in blood, brain, and CSF are quantitatively very similar for Class I compounds, whereas the steady-state unbound concentrations in the brain and CSF are significantly lower than those in blood for Class II compounds. These results strongly suggest that the free drug hypothesis is not universal for all drugs but is only applicable for drugs with good permeability that are not substrates of efflux transporters.