Bioluminescent Pseudomonas aeruginosa and Escherichia coli for whole-cell screening of antibacterial and adjuvant compounds.

Continued efforts to discover new antibacterial molecules are critical to achieve a robust pre-clinical pipeline for new antibiotics. Screening of compound or natural product extract libraries remains a widespread approach and can benefit from the development of whole cell assays that are robust, simple and versatile, and allow for high throughput testing of antibacterial activity. In this study, we created and validated two bioluminescent reporter strains for high-throughput screening, one in Pseudomonas aeruginosa, and another in a hyperporinated and efflux-deficient Escherichia coli.

Recent Advances in Models of Immune-Mediated Drug-Induced Liver Injury.

Hepatic inflammation is a key feature of a variety of liver diseases including drug-induced liver injury (DILI), orchestrated by the innate immune response (Kupffer cells, monocytes, neutrophils, dendritic cells) and the adaptive immune system (T cells and natural killer T cells). In contrast to acute DILI, prediction of immune-mediated DILI (im-DILI) has been more challenging due to complex disease pathogenesis, lack of reliable models and limited knowledge of underlying mechanisms. This review summarizes and systems that have been used to model im-DILI.

Strain maps characterize the symmetry of convergence and extension patterns during zebrafish gastrulation.

During gastrulation of the zebrafish embryo, the cap of blastoderm cells organizes into the axial body plan of the embryo with left-right symmetry and head-tail, dorsal-ventral polarities. Our labs have been interested in the mechanics of early development and have investigated whether these large-scale cell movements can be described as tissue-level mechanical strain by a tectonics-based approach. The first step is to image the positions of all nuclei from mid-epiboly to early segmentation by digital sheet light microscopy, organize the surface of the embryo into multi-cell spherical domains, construct velocity fields from the movements of these domains and extract strain rate maps from the change in density of the domains.

Strategies to Avoid Artifacts in Mass Spectrometry-Based Epitranscriptome Analyses.

In this report, we perform structure validation of recently reported RNA phosphorothioate (PT) modifications, a new set of epitranscriptome marks found in bacteria and eukaryotes including humans. By comparing synthetic PT-containing diribonucleotides with native species in RNA hydrolysates by high-resolution mass spectrometry (MS), metabolic stable isotope labeling, and PT-specific iodine-desulfurization, we disprove the existence of PTs in RNA from E. coli, S.

All-Atom MD Simulations of the HBV Capsid Complexed with AT130 Reveal Secondary and Tertiary Structural Changes and Mechanisms of Allostery.

The hepatitis B virus (HBV) capsid is an attractive drug target, relevant to combating viral hepatitis as a major public health concern. Among small molecules known to interfere with capsid assembly, the phenylpropenamides, including AT130, represent an important antiviral paradigm based on disrupting the timing of genome packaging. Here, all-atom molecular dynamics simulations of an intact AT130-bound HBV capsid reveal that the compound increases spike flexibility and improves recovery of helical secondary structure in the spike tips.

Direct and Label-Free Cell Status Monitoring of Spheroids and Microcarriers Using Microfluidic Impedance Cytometry.

3D cellular spheroids/microcarriers (100 µm-1 mm) are widely used in biomanufacturing, and non-invasive biosensors are useful to monitor cell quality in bioprocesses. In this work, a novel microfluidic approach for label-free and continuous-flow monitoring of single spheroid/microcarrier (hydrogel and Cytodex) based on electrical impedance spectroscopy using co-planar Field's metal electrodes is reported. Through numerical simulation and experimental validation, two unique impedance signatures (|Z | (60 kHz), |Z | (1 MHz)) which are optimal for spheroid growth and viability monitoring are identified.

Humanized Mouse as a Tool to Predict Immunotoxicity of Human Biologics.

Advancements in science enable researchers to constantly innovate and create novel biologics. However, the use of non-human animal models during the development of biologics impedes identification of precise interactions between the human immune system and treatments. Due to lack of this understanding, adverse effects are frequently observed in healthy volunteers and patients exposed to potential biologics during clinical trials.

Microfluidic label-free bioprocessing of human reticulocytes from erythroid culture.

In vitro erythroid cultures from human hematopoietic stem cells produce immature red blood cells (RBCs) called reticulocytes, which are important for RBCs production, and are widely used in scientific studies of malaria pathology, hematological diseases and protein translation. However, in vitro reticulocyte cultures contain expelled cell nuclei and erythroblasts as undesirable by-products and current purification methods such as density gradient centrifugation and fluorescence-activated cell sorting (FACS) are not optimal for integrated bioprocessing and downstream therapeutic applications. Developments in Dean flow fractionation (DFF) and deterministic lateral displacement (DLD) microfluidic sorting methods are ideal alternatives due to label-free size sorting, throughput scalability and low manufacturing cost.

Epigenetic competition reveals density-dependent regulation and target site plasticity of phosphorothioate epigenetics in bacteria.

Phosphorothioate (PT) DNA modifications-in which a nonbonding phosphate oxygen is replaced with sulfur-represent a widespread, horizontally transferred epigenetic system in prokaryotes and have a highly unusual property of occupying only a small fraction of available consensus sequences in a genome. Using as a model, we asked a question of fundamental importance: How do the PT-modifying DndA-E proteins select their GAAC/GTTC targets? Here, we applied innovative analytical, sequencing, and computational tools to discover a novel behavior for DNA-binding proteins: The Dnd proteins are "parked" at the GATC Dam methyltransferase consensus sequence instead of the expected GAAC/GTTC motif, with removal of the A permitting extensive PT modification of GATC sites. This shift in modification sites further revealed a surprising constancy in the density of PT modifications across the genome.

Upcycling chitin-containing waste into organonitrogen chemicals via an integrated process.

Chitin is the most abundant renewable nitrogenous material on earth and is accessible to humans in the form of crustacean shell waste. Such waste has been severely underutilized, resulting in both resource wastage and disposal issues. Upcycling chitin-containing waste into value-added products is an attractive solution.

Temperature-Induced Catch-Slip to Slip Bond Transit in Plasmodium falciparum-Infected Erythrocytes.

Plasmodium falciparum malaria-infected red blood cells (IRBCs), or erythrocytes, avoid splenic clearance by adhering to host endothelium. Upregulation of endothelial receptors intercellular adhesion molecule-1 (ICAM-1) and cluster of differentiation 36 (CD36) are associated with severe disease pathology. Most in vitro studies of IRBCs interacting with these molecules were conducted at room temperature.

Culture and Differentiation of Lung Bronchiolar Epithelial Cells In Vitro.

Club cells are a major bronchiolar epithelial cell type in the lung. Using genetic lineage tracing in mice and in vitro culture of purified cells, we have shown that club cells can differentiate into alveolar type I and II cells. Here we describe the detailed protocol for culturing and differentiating club cells in 3-dimensional culture.

Establishing multiple omics baselines for three Southeast Asian populations in the Singapore Integrative Omics Study.

The Singapore Integrative Omics Study provides valuable insights on establishing population reference measurement in 364 Chinese, Malay, and Indian individuals. These measurements include > 2.5 millions genetic variants, 21,649 transcripts expression, 282 lipid species quantification, and 284 clinical, lifestyle, and dietary variables.

Soft tubular microfluidics for 2D and 3D applications.

Microfluidics has been the key component for many applications, including biomedical devices, chemical processors, microactuators, and even wearable devices. This technology relies on soft lithography fabrication which requires cleanroom facilities. Although popular, this method is expensive and labor-intensive.

Differentiation of Club Cells to Alveolar Epithelial Cells In Vitro.

Club cells are known to function as regional progenitor cells to repair the bronchiolar epithelium in response to lung damage. By lineage tracing in mice, we have shown recently that club cells also give rise to alveolar type 2 cells (AT2s) and alveolar type 1 cells (AT1s) during the repair of the damaged alveolar epithelium. Here, we show that when highly purified, anatomically and phenotypically confirmed club cells are seeded in 3-dimensional culture either in bulk or individually, they proliferate and differentiate into both AT2- and AT1-like cells and form alveolar-like structures.

Human CD34(lo)CD133(lo) fetal liver cells support the expansion of human CD34(hi)CD133(hi) hematopoietic stem cells.

We have recently discovered a unique CD34(lo)CD133(lo) cell population in the human fetal liver (FL) that gives rise to cells in the hepatic lineage. In this study, we further characterized the biological functions of FL CD34(lo)CD133(lo) cells. Our findings show that these CD34(lo)CD133(lo) cells express markers of both endodermal and mesodermal lineages and have the capability to differentiate into hepatocyte and mesenchymal lineage cells by ex vivo differentiation assays.

ADAR-Mediated RNA Editing Predicts Progression and Prognosis of Gastric Cancer.

Backgroud & Aims: Gastric cancer (GC) is the third leading cause of global cancer mortality. Adenosine-to-inosine RNA editing is a recently described novel epigenetic mechanism involving sequence alterations at the RNA but not DNA level, primarily mediated by ADAR (adenosine deaminase that act on RNA) enzymes. Emerging evidence suggests a role for RNA editing and ADARs in cancer, however, the relationship between RNA editing and GC development and progression remains unknown.

Comparative Plasmodium gene overexpression reveals distinct perturbation of sporozoite transmission by profilin.

Plasmodium relies on actin-based motility to migrate from the site of infection and invade target cells. Using a substrate-dependent gliding locomotion, sporozoites are able to move at fast speed (1-3 μm/s). This motility relies on a minimal set of actin regulatory proteins and occurs in the absence of detectable filamentous actin (F-actin).

Capsules of virulent pneumococcal serotypes enhance formation of neutrophil extracellular traps during in vivo pathogenesis of pneumonia.

Neutrophil extracellular traps (NETs) are released by activated neutrophils to ensnare and kill microorganisms. NETs have been implicated in tissue injury since they carry cytotoxic components of the activated neutrophils. We have previously demonstrated the generation of NETs in infected murine lungs during both primary pneumococcal pneumonia and secondary pneumococcal pneumonia after primary influenza.

Stiffening of Red Blood Cells Induced by Cytoskeleton Disorders: A Joint Theory-Experiment Study.

The functions and elasticities of the cell are largely related to the structures of the cytoskeletons underlying the lipid bilayer. Among various cell types, the red blood cell (RBC) possesses a relatively simple cytoskeletal structure. Underneath the membrane, the RBC cytoskeleton takes the form of a two-dimensional triangular network, consisting of nodes of actins (and other proteins) and edges of spectrins.

Delineation of Natural Killer Cell Differentiation from Myeloid Progenitors in Human.

Understanding of natural killer (NK) cell development in human is incomplete partly because of limited access to appropriate human tissues. We have developed a cytokine-enhanced humanized mouse model with greatly improved reconstitution and function of human NK cells. Here we report the presence of a cell population in the bone marrow of the cytokine-treated humanized mice that express both NK cell marker CD56 and myeloid markers such as CD36 and CD33.

Characterisation of liver pathogenesis, human immune responses and drug testing in a humanised mouse model of HCV infection.

Objective: HCV infection affects millions of people worldwide, and many patients develop chronic infection leading to liver cancers. For decades, the lack of a small animal model that can recapitulate HCV infection, its immunopathogenesis and disease progression has impeded the development of an effective vaccine and therapeutics. We aim to provide a humanised mouse model for the understanding of HCV-specific human immune responses and HCV-associated disease pathologies.

Dengue subgenomic RNA binds TRIM25 to inhibit interferon expression for epidemiological fitness.

The global spread of dengue virus (DENV) infections has increased viral genetic diversity, some of which appears associated with greater epidemic potential. The mechanisms governing viral fitness in epidemiological settings, however, remain poorly defined. We identified a determinant of fitness in a foreign dominant (PR-2B) DENV serotype 2 (DENV-2) clade, which emerged during the 1994 epidemic in Puerto Rico and replaced an endemic (PR-1) DENV-2 clade.

Microstructured dextran hydrogels for burst-free sustained release of PEGylated protein drugs.

Hydrogels have gained significant attention as ideal delivery vehicles for protein drugs. However, the use of hydrogels for protein delivery has been restricted because their porous structures inevitably cause a premature leakage of encapsulated proteins. Here, we report a simple yet effective approach to regulate the protein release kinetics of hydrogels through the creation of microstructures, which serve as a reservoir, releasing their payloads in a controlled manner.