Bombyx mori nucleopolyhedrovirus LEF-2 disrupts the cell cycle in the G2/M phase by triggering a host cell DNA damage response.

It is a common strategy for viruses to block the host cell cycle to favour their DNA replication. Baculovirus, being a double-stranded DNA virus, can arrest the cell cycle in the G2/M phase to facilitate its replication. However, the key viral genes and mechanisms crucial for inducing cell cycle arrest remain poorly understood. Here, we initially examined the impacts of several Bombyx mori nucleopolyhedrovirus (BmNPV) DNA replication-associated genes: ie1, lef-1, lef-2, lef-3, lef-4, odv-ec27 and dbp. We assessed their effects on both the host cells' DNA replication and cell cycle. Our findings reveal that when the lef-2 gene was overexpressed, it led to a significant increase in the number of cells in the G2/M phase and a reduction in the number of cells in the S phase. Furthermore, we discovered that the LEF-2 protein is located in the virogenic stroma and confirmed its involvement in viral DNA replication. Additionally, by employing interference and overexpression experiments, we found that LEF-2 influences host cell DNA replication and blocks the cell cycle in the G2/M phase by regulating the expression of CyclinB and CDK1. Finally, we found that BmNPV lef-2 triggered a DNA damage response in the host cell, and inhibiting this response removed the cell cycle block caused by BmNPV LEF-2. Thus, our findings indicate that the BmNPV lef-2 gene plays a crucial role in viral DNA replication and can regulate host cell cycle processes. This study furthers our understanding of baculovirus-host cell interactions and provides new insight into the molecular mechanisms of antiviral research.

Engineering Visible to Near-Infrared Luminescence through a Selective Doping Strategy for High-Performance Temperature Sensing.

Luminescence nanothermometers have garnered considerable attention due to their noncontact measurement, high spatial resolution, and rapid response. However, many nanothermometers employing single-mode measurement encounter challenges regarding their relative sensitivity. Herein, a unique class of tunable upconversion (UC) and downshifting (DS) luminescence covering the visible to near-infrared range (400-1700 nm) is reported, characterized by the superior Tm3+, Ho3+, and Er3+ emissions induced by efficient energy transfer. The outstanding negative thermal expansion characteristic of ScF3 nanocrystals has been found to guide excitation energy toward the relevant emitting states in the Yb3+-Ho3+-Tm3+-codoped system, consequently resulting in remarkable near-infrared III (NIR-III) luminescence at ∼1625 nm (Tm3+:3F4 → 3H6 transition), which in turn presents numerous opportunities for designing multimode ratiometric luminescence thermometry. Furthermore, by facilitating phonon-assisted energy transfer in Er3+-Ho3+-codoped systems, the luminescence intensity ratio (LIR) of 4I13/2 of Er3+ and 5I6 of Ho3+ in ScF3:Yb3+/Ho3+/Er3+ exhibits a strong temperature dependence, enabling NIR-II/III luminescence thermometry with superior thermal sensitivity and resolution (Sr = 0.78% K-1, δT = 0.64 K). These findings not only underscore the distinctive and ubiquitous attributes of lanthanide ion-doped nanomaterials but also hold significant implications for crafting luminescence thermometers with unparalleled sensitivity.

Toward Ultra-High Sensitivity Optical Thermometers and Bright Yellow LEDs Based on Phonon-Assisted Energy Transfer in Rare Earth-Doped La2ZnTiO6 Double Perovskite.

Double perovskites, a class of ceramic oxides with unique crystal structures and diverse physical properties, show promise for various technological applications including solar cells, photodetectors, and light-emitting diodes (LEDs). Despite limited research on rare earth-doped double perovskites, leveraging their ultrahigh luminous efficiency to achieve bright yellow LED emission and addressing energy transfer challenges between Yb3+ and Nd3+ ions in double perovskite La2ZnTiO6 with moderate phonon energy are explored in this work. Through phonon-assisted energy transfer, an ultrasensitive optical thermometer covering a wide temperature range is developed by utilizing the different temperature responses of Er3+ emission in the visible light region and Nd3+ emission in the near-infrared region based on the luminescence intensity ratio (LIR). All the results demonstrate that the rare earth (Yb-Er, Yb-Nd, and Yb-Nd-Er)-doped La2ZnTiO6 phosphors can be effectively utilized for ultrabright LED illumination and ultrahigh sensitivity self-calibrated temperature sensing. This research underscores the significance of phonon-assisted energy transfer in improving material properties and provides valuable insights for the advancement of multifunctional materials.

Achieving Near-Infrared Highly Sensitive Temperature Sensing in Lanthanide-Doped Lead-Free Double Perovskite NaLaMgWO6 with Significant Thermal Enhancement.

The significant temperature response of lanthanide-doped up-conversion luminescent materials is typically characterized by a severe thermal quenching of the luminescence intensity at elevated ambient temperatures, which severely restricts materials' capability in temperature sensing. Herein, the influence of matrix phonon properties on the remarkable thermal enhancement effect in the thermosensitive material NaLaMgWO6:Yb3+/Nd3+ is reported. It is elucidated that achieving a significant thermal enhancement of Nd3+ with a higher phonon energy oxide matrix is easier than a halide matrix, which has lower phonon energy by comparison with previous findings. Interestingly, the emission of thermally related levels gets enhanced to various extents through phonon-assisted thermal population. In light of this, a three-model thermometer is constructed based on luminescence intensity ratio (LIR) technology. Given that Sr and ΔE possess a positive correlation, it is feasible to acquire greater temperature monitoring sensitivity Sr in Nd3+, which has a larger ΔE. At 313 K, this thermometry model may achieve a maximum sensitivity of 2.84%·K-1. This work not only provides guidance for the selection of efficient near-infrared up-conversion material but also opens up a prospect for the realization of ultrasensitive thermally coupled luminescent thermometers.

Design of a High-Power Nanosecond Electromagnetic Pulse Radiation System for Verifying Spaceborne Detectors.

The Spaceborne Global Lightning Location Network (SGLLN) serves the purpose of identifying transient lightning events occurring beneath the ionosphere, playing a significant role in detecting and warning of disaster weather events. To ensure the effective functioning of the wideband electromagnetic pulse detector, which is a crucial component of the SGLLN, it must be tested and verified with specific signals. However, the inherent randomness and unpredictability of lightning occurrences pose challenges to this requirement. Consequently, a high-power electromagnetic pulse radiation system with a 20 m aperture reflector is designed. This system is capable of emitting nanosecond electromagnetic pulse signals under pre-set spatial and temporal conditions, providing a controlled environment for assessing the detection capabilities of SGLLN. In the design phase, an exponentially TEM feed antenna has been designed firstly based on the principle of high-gain radiation. The feed antenna adopts a pulser-integrated design to mitigate insulation risks, and it is equipped with an asymmetric protective loading to reduce reflected energy by 85.7%. Moreover, an innovative assessment method for gain loss, based on the principle of Love's equivalence, is proposed to quantify the impact of feed antenna on the radiation field. During the experimental phase, a specialized E-field sensor is used in the far-field experiment at a distance of 400 m. The measurements indicate that at this distance, the signal has a peak field strength of 2.2 kV/m, a rise time of 1.9 ns, and a pulse half-width of 2.5 ns. Additionally, the beamwidth in the time domain is less than 10°. At an altitude of 500 km, the spaceborne detector records a signal with a peak field strength of approximately 10 mV/m. Particularly, this signal transformed into a nonlinear frequency-modulated signal in the microsecond range across its frequency spectrum, which is consistent with the law of radio wave propagation in the ionosphere. This study offers a stable and robust radiation source for verifying spaceborne detectors and establishes an empirical foundation for investigating the impact of the ionosphere on signal propagation characteristics.

scRNASequest: an ecosystem of scRNA-seq analysis, visualization, and publishing.

BACKGROUND: Single-cell RNA sequencing is a state-of-the-art technology to understand gene expression in complex tissues. With the growing amount of data being generated, the standardization and automation of data analysis are critical to generating hypotheses and discovering biological insights. RESULTS: Here, we present scRNASequest, a semi-automated single-cell RNA-seq (scRNA-seq) data analysis workflow which allows (1) preprocessing from raw UMI count data, (2) harmonization by one or multiple methods, (3) reference-dataset-based cell type label transfer and embedding projection, (4) multi-sample, multi-condition single-cell level differential gene expression analysis, and (5) seamless integration with cellxgene VIP for visualization and with CellDepot for data hosting and sharing by generating compatible h5ad files. CONCLUSIONS: We developed scRNASequest, an end-to-end pipeline for single-cell RNA-seq data analysis, visualization, and publishing. The source code under MIT open-source license is provided at https://github.com/interactivereport/scRNASequest . We also prepared a bookdown tutorial for the installation and detailed usage of the pipeline: https://interactivereport.github.io/scRNAsequest/tutorial/docs/ . Users have the option to run it on a local computer with a Linux/Unix system including MacOS, or interact with SGE/Slurm schedulers on high-performance computing (HPC) clusters.

Architecture of the human interactome defines protein communities and disease networks.

The physiology of a cell can be viewed as the product of thousands of proteins acting in concert to shape the cellular response. Coordination is achieved in part through networks of protein-protein interactions that assemble functionally related proteins into complexes, organelles, and signal transduction pathways. Understanding the architecture of the human proteome has the potential to inform cellular, structural, and evolutionary mechanisms and is critical to elucidating how genome variation contributes to disease. Here we present BioPlex 2.0 (Biophysical Interactions of ORFeome-derived complexes), which uses robust affinity purification-mass spectrometry methodology to elucidate protein interaction networks and co-complexes nucleated by more than 25% of protein-coding genes from the human genome, and constitutes, to our knowledge, the largest such network so far. With more than 56,000 candidate interactions, BioPlex 2.0 contains more than 29,000 previously unknown co-associations and provides functional insights into hundreds of poorly characterized proteins while enhancing network-based analyses of domain associations, subcellular localization, and co-complex formation. Unsupervised Markov clustering of interacting proteins identified more than 1,300 protein communities representing diverse cellular activities. Genes essential for cell fitness are enriched within 53 communities representing central cellular functions. Moreover, we identified 442 communities associated with more than 2,000 disease annotations, placing numerous candidate disease genes into a cellular framework. BioPlex 2.0 exceeds previous experimentally derived interaction networks in depth and breadth, and will be a valuable resource for exploring the biology of incompletely characterized proteins and for elucidating larger-scale patterns of proteome organization.

Uncovering the prevalence and drivers of antibiotic resistance genes in soils across different land-use types.

The emergence and spread of antibiotic resistance genes (ARGs) in soil due to animal excreta and organic waste is a major threat to human health and ecosystems, and global efforts are required to tackle the issue. However, there is limited knowledge of the variation in ARG prevalence and diversity resulting from different land-use patterns and underlying driving factors in soils. This study aimed to comprehensively characterize the profile of ARGs and mobile genetic elements and their drivers in soil samples collected from 11 provinces across China, representing three different land-use types, using high-throughput quantitative polymerase chain reaction and 16S rRNA amplicon sequencing. Our results showed that agricultural soil had the highest abundance and diversity of ARGs, followed by tea plantation and forest land. A total of 124 unique ARGs were detected in all samples, with shared subtypes among different land-use patterns indicating a common origin or high transmission frequency. Moreover, significant differences in ARG distribution were observed among different geographical regions, with the greatest enrichment of ARGs found in southern China. Biotic and abiotic factors, including soil properties, climatic factors, and bacterial diversity, were identified as the primary drivers associated with ARG abundance, explaining 71.8% of total ARG variation. The findings of our study demonstrate that different land-use patterns are associated with variations in ARG abundance in soil, with agricultural practices posing the greatest risk to human health and ecosystems regarding ARGs. Our identification of biotic and abiotic drivers of ARG abundance provides valuable insights into strategies for mitigating the spread of these genes. This study emphasizes the need for coordinated and integrated approaches to address the global antimicrobial resistance crisis.

Integrated single-cell transcriptomics of cerebrospinal fluid cells in treatment-naïve multiple sclerosis.

Multiple sclerosis (MS) is a chronic and often disabling autoimmune disease of the central nervous system (CNS). Cerebrospinal fluid (CSF) surrounds and protects the CNS. Analysis of CSF can aid the diagnosis of CNS diseases, help to identify the prognosis, and underlying mechanisms of diseases. Several recent studies have leveraged single-cell RNA-sequencing (scRNA-seq) to identify MS-associated changes in CSF cells that are considerably more altered than blood cells in MS. However, not all alterations were replicated across all studies. We therefore integrated multiple available scRNA-seq datasets of CSF cells from MS patients with early relapsing-remitting (RRMS) disease. We provide a searchable and interactive resource of this integrated analysis ( https://CSFinMS.bxgenomics.com ) facilitating diverse visualization and analysis methods without requiring computational skills. In the present joint analysis, we replicated the known expansion of B lineage and the recently described expansion of natural killer (NK) cells and some cytotoxic T cells and decrease of monocytes in the CSF in MS. The previous observation of the abundance of Th1-like Th17 effector memory cells in the CSF was not replicated. Expanded CSF B lineage cells resembled class-switched plasmablasts/-cells (e.g., SDC1/CD138, MZB1) as expected. Our integrative analysis thus validates increased cell type diversity and B cell maturation in the CSF in MS and improves accessibility of available data.

Changes in microbial community composition drive the response of ecosystem multifunctionality to elevated ozone.

Increasing tropospheric ozone poses a potential threat to both above- and belowground components of the terrestrial biosphere. Microorganisms are the main drivers of soil ecological processes, however, the link between soil microbial communities and ecological functions under elevated ozone remains poorly understood. In this study, we assessed the responses of three crop seedlings (i.e., soybean, maize, and wheat) growth and soil microbial communities to elevated ozone (40 ppb O3 above ambient air) in a pot experiment in the solardomes. Results showed that elevated ozone adversely affected ecosystem multifunctionality by reducing crop biomass, inhibiting soil extracellular enzyme activities, and altering nutrient availability. Elevated ozone increased bacterial and fungal co-occurrence network complexity, negatively correlated with ecosystem multifunctionality. Changes in the relative abundance of some specific bacteria and fungi were associated with multiple ecosystem functioning. In addition, elevated ozone significantly affected fungal community composition but not bacterial community composition and microbial alpha-diversity. Crop type played a key role in determining bacterial alpha-diversity and microbial community composition. In conclusion, our findings suggest that short-term elevated ozone could lead to a decrease in ecosystem multifunctionality associated with changes in the complexity of microbial networks in soils.

Baculovirus LEF-11 interacts with BmIMPI to induce cell cycle arrest in the G2/M phase for viral replication.

Viruses arrest the host cell cycle and using multiple functions of host cells is an important approach for their replication. Baculovirus arrests infected insect cells at both the late S and G2/M phase, but the strategy employed by baculovirus is not clearly understood. Our research suggests that the Bombyx mori nucleopolyhedrovirus (BmNPV) could arrest the cell cycle in the G2/M phase to promote virus replication, and also that the viral protein LEF-11 could inhibit host cell proliferation and arrest the cell cycle by inhibiting the cell cycle checkpoint proteins BmCyclinB and BmCDK1. Furthermore, we found that LEF-11 interacts with BmIMPI to regulate cell proliferation, but not by direct interaction with BmCyclinB or BmCDK1. In addition, our findings showed that BmIMPI was important and necessary for LEF-11 induced cell cycle arrest in the G2/M phase. Moreover, BmIMPI was found to interact with BmCyclinB and BmCDK1, and down-regulate the expression of BmCyclinB and BmCDK1 to compromise the cell cycle and cell proliferation. Taken together, the data presented demonstrated that baculovirus LEF-11 regulates BmIMPI to inhibit host cell proliferation and provide a new insight into the molecular mechanisms employed by viruses to induce cell cycle arrest.

Identification of the Key Functional Domains of Bombyx mori Nucleopolyhedrovirus IE1 Protein.

The immediate early protein 1 (IE1) acts as a transcriptional activator and is essential for viral gene transcription and viral DNA replication. However, the key regulatory domains of IE1 remain poorly understood. Here, we analyzed the sequence characteristics of Bombyx mori nucleopolyhedrovirus (BmNPV) IE1 and identified the key functional domains of BmNPV IE1 by stepwise truncation. Our results showed that BmNPV IE1 was highly similar to Autographa californica nucleopolyhedrovirus (AcMNPV) IE1, but was less conserved with IE1 of other baculoviruses, the C-terminus of IE1 was more conserved than the N-terminus, and BmNPV IE1 was also necessary for BmNPV proliferation. Moreover, we found that IE1158-208 was a major nuclear localization element, and IE11-157 and IE1539-559 were minor nuclear localization elements, but the combination of these two minor elements was equally sufficient to fully mediate the nuclear entry of IE1. Meanwhile, IE11-258, IE1560-584, and the association of amino acids 258 and 259 were indispensable for the transactivation activity of BmNPV IE1. These results systematically resolve the functional domains of BmNPV IE1, which contribute to the understanding of the mechanism of baculovirus infection and provide a possibility to synthesize a small molecule IE1-truncated mutant as an agonist or antagonist.

Radix Asteris: Traditional Usage, Phytochemistry and Pharmacology of An Important Traditional Chinese Medicine.

Radix Asteris (RA), also known as 'Zi Wan', is the dried root and rhizome of Aster tataricus L. f., which has been used to treat cough and asthma in many countries such as China, Japan, Korea and Vietnam. This article summarizes the available information on RA in ancient Chinese medicine books and modern research literature: its botanical properties, traditional uses, chemical composition, pharmacological activity, toxicity and quality control. Studies have shown that RA extracts contain terpenes, triterpenoid saponins, organic acids, peptides and flavonoids, and have various pharmacological activities such as anti-inflammatory, anti-tumor, anti-oxidation, and anti-depression. RA is considered to be a promising medicinal plant based on its traditional use, chemical constituents and pharmacological activities. However, there are few studies on its toxicity and the consistency of its components, which indicates the need for further in-depth studies on the toxicity and quality control of RA and its extracts.

Development of a TEM-cell-integrated CO2 incubator for cell-based transient electromagnetic field bioeffect study.

To facilitate the cell-based experiment for pulsed electromagnetic field biological effect study, a novel TEM-cell-integrated CO2 incubator was developed. The integrated experimental system could simultaneously meet the requirement of standard cell culture condition and the various Transient Electromagnetic Field (TEF) exposure, which made it possible to study the relationship between different electromagnetic pulse exposure and the cellular responses in a reliable way. During the research, a comparison experiment was carried out to evaluate the necessity of the integrated incubator system: firstly, two different types of cell lines, which are the human prostate cancer cell line (PC3) and the pancreatic ß cell line (MIN6) were chosen and exposed in the TEM-cell which located in the open area and the integrated system, respectively, with the same EFT radiation conditions; then, the cells' viability, the cellular ROS level and the mitochondrial membrane potential (MMP) were detected, respectively. The results showed that in the same parameter of the EFT radiation, the processes of the cells had a significant difference and even opposite in the incubator and open area, and all the results could be reproducible. The phenomenon indicated the stability of the TEM-cell-integrated CO2 incubator, and also demonstrated the necessity to strictly control the cell culture condition when carrying out the precise mechanism study of the TEF bioresponse at the cellular levels.

An integrated transcriptomics and proteomics analysis reveals functional endocytic dysregulation caused by mutations in LRRK2.

BACKGROUND: Mutations in LRRK2 are the most common cause of autosomal dominant Parkinson's disease, and the relevance of LRRK2 to the sporadic form of the disease is becoming ever more apparent. It is therefore essential that studies are conducted to improve our understanding of the cellular role of this protein. Here we use multiple models and techniques to identify the pathways through which LRRK2 mutations may lead to the development of Parkinson's disease. METHODS: A novel integrated transcriptomics and proteomics approach was used to identify pathways that were significantly altered in iPSC-derived dopaminergic neurons carrying the LRRK2-G2019S mutation. Western blotting, immunostaining and functional assays including FM1-43 analysis of synaptic vesicle endocytosis were performed to confirm these findings in iPSC-derived dopaminergic neuronal cultures carrying either the LRRK2-G2019S or the LRRK2-R1441C mutation, and LRRK2 BAC transgenic rats, and post-mortem human brain tissue from LRRK2-G2019S patients. RESULTS: Our integrated -omics analysis revealed highly significant dysregulation of the endocytic pathway in iPSC-derived dopaminergic neurons carrying the LRRK2-G2019S mutation. Western blot analysis confirmed that key endocytic proteins including endophilin I-III, dynamin-1, and various RAB proteins were downregulated in these cultures and in cultures carrying the LRRK2-R1441C mutation, compared with controls. We also found changes in expression of 25 RAB proteins. Changes in endocytic protein expression led to a functional impairment in clathrin-mediated synaptic vesicle endocytosis. Further to this, we found that the endocytic pathway was also perturbed in striatal tissue of aged LRRK2 BAC transgenic rats overexpressing either the LRRK2 wildtype, LRRK2-R1441C or LRRK2-G2019S transgenes. Finally, we found that clathrin heavy chain and endophilin I-III levels are increased in human post-mortem tissue from LRRK2-G2019S patients compared with controls. CONCLUSIONS: Our study demonstrates extensive alterations across the endocytic pathway associated with LRRK2 mutations in iPSC-derived dopaminergic neurons and BAC transgenic rats, as well as in post-mortem brain tissue from PD patients carrying a LRRK2 mutation. In particular, we find evidence of disrupted clathrin-mediated endocytosis and suggest that LRRK2-mediated PD pathogenesis may arise through dysregulation of this process.

Predictive value of lymphocyte-to-monocyte ratio (LMR) and neutrophil-to-lymphocyte ratio (NLR) in patients with oesophageal cancer undergoing concurrent chemoradiotherapy.

BACKGROUND AND OBJECTIVES: The survival rate of patients with advanced oesophageal cancer is very low and can vary significantly, even among patients with the same TNM stage. It is important to look for indicators that are economical and readily available to predict overall survival. The aim of this study was to determine whether lymphocyte-to-monocyte ratio (LMR) and neutrophil-to-lymphocyte ratio (NLR) could be potential predictors of survival in patients with advanced oesophageal squamous cell carcinoma (ESCC) undergoing concurrent chemoradiotherapy. METHODS: Differences in survival among 204 patients with advanced oesophageal cancer who underwent concurrent chemoradiotherapy were collected and analysed. Univariate and multivariate COX regression analyses were used to investigate the association between blood inflammatory markers and patient survival before treatment. RESULTS: Univariate COX regression analyses showed that a history of alcohol use, neutrophil count, LMR, NLR, tumour length, and N stage were significantly associated with the survival of tumour patients receiving concurrent chemoradiotherapy. Multivariate COX regression analysis showed that NLR and LMR were predictors of outcome in tumour patients receiving chemoradiotherapy. According to receiver operating characteristic (ROC) curve analysis, the AUC of LMR and NLR was 0.734 and 0.749, and the best cutoff point for LMR and NLR was 3.03 and 2.64, respectively. CONCLUSIONS: LMR and NLR can be used to predict the survival of patients with advanced oesophageal cancer receiving concurrent chemoradiotherapy, thereby providing clinicians with suggestions for further treatment options.

Reduced nitrogen supply enhances the cellular antioxidant potential of phenolic extracts through alteration of the phenolic composition in lettuce (Lactuca sativa L.).

BACKGROUND: Nitrogen availability is an important environmental factor that determines the production of phenolic compounds in vegetables, but the relationship between low nitrogen-induced alterations of phenolic compounds in vegetable crops and the cellular antioxidant activities of these compounds remains unclear. This study investigated the effect of reduced nitrogen supply (0.05 mmol L-1 nitrate) on phenolic metabolism in lettuce and the protective role of phenolic extracts against H2 O2 -induced oxidative stress in Caco-2 cells by determining cell damage, reactive oxygen species (ROS) content and antioxidant enzyme activities. RESULTS: Reduced nitrogen supply significantly improved the accumulation of phenolic compounds in lettuce, which was partially correlated with the upregulation of genes related to the phenolic synthesis pathway. Phenolic extracts from lettuce cultivated in low-nitrogen medium exhibited a better protective effect against H2 O2 -induced oxidative damage in Caco-2 cells than those from lettuce cultivated with adequate nitrogen. These extracts act by increasing the activities of antioxidant enzymes and, subsequently, by inhibiting ROS overproduction, which leads to a decrease in mitochondrial membrane and DNA damage. The results of HPLC and correlation analyses implied that the improvement in the protective capacity of lettuce extracts after low-nitrogen treatment may be related, not only to the increased content of phenolic compounds, but also to the increased percentage contribution of chlorogenic acid and quercetin derivatives to the total phenolic content. CONCLUSION: Reduction in nitrogen supply can be a powerful strategy to modify phenolic metabolism and composition in lettuce and, consequently, to improve their antioxidant capacity. © 2019 Society of Chemical Industry.

Toward performance-diverse small-molecule libraries for cell-based phenotypic screening using multiplexed high-dimensional profiling.

High-throughput screening has become a mainstay of small-molecule probe and early drug discovery. The question of how to build and evolve efficient screening collections systematically for cell-based and biochemical screening is still unresolved. It is often assumed that chemical structure diversity leads to diverse biological performance of a library. Here, we confirm earlier results showing that this inference is not always valid and suggest instead using biological measurement diversity derived from multiplexed profiling in the construction of libraries with diverse assay performance patterns for cell-based screens. Rather than using results from tens or hundreds of completed assays, which is resource intensive and not easily extensible, we use high-dimensional image-based cell morphology and gene expression profiles. We piloted this approach using over 30,000 compounds. We show that small-molecule profiling can be used to select compound sets with high rates of activity and diverse biological performance.

Human genetics in rheumatoid arthritis guides a high-throughput drug screen of the CD40 signaling pathway.

Although genetic and non-genetic studies in mouse and human implicate the CD40 pathway in rheumatoid arthritis (RA), there are no approved drugs that inhibit CD40 signaling for clinical care in RA or any other disease. Here, we sought to understand the biological consequences of a CD40 risk variant in RA discovered by a previous genome-wide association study (GWAS) and to perform a high-throughput drug screen for modulators of CD40 signaling based on human genetic findings. First, we fine-map the CD40 risk locus in 7,222 seropositive RA patients and 15,870 controls, together with deep sequencing of CD40 coding exons in 500 RA cases and 650 controls, to identify a single SNP that explains the entire signal of association (rs4810485, P = 1.4×10(-9)). Second, we demonstrate that subjects homozygous for the RA risk allele have ∼33% more CD40 on the surface of primary human CD19+ B lymphocytes than subjects homozygous for the non-risk allele (P = 10(-9)), a finding corroborated by expression quantitative trait loci (eQTL) analysis in peripheral blood mononuclear cells from 1,469 healthy control individuals. Third, we use retroviral shRNA infection to perturb the amount of CD40 on the surface of a human B lymphocyte cell line (BL2) and observe a direct correlation between amount of CD40 protein and phosphorylation of RelA (p65), a subunit of the NF-κB transcription factor. Finally, we develop a high-throughput NF-κB luciferase reporter assay in BL2 cells activated with trimerized CD40 ligand (tCD40L) and conduct an HTS of 1,982 chemical compounds and FDA-approved drugs. After a series of counter-screens and testing in primary human CD19+ B cells, we identify 2 novel chemical inhibitors not previously implicated in inflammation or CD40-mediated NF-κB signaling. Our study demonstrates proof-of-concept that human genetics can be used to guide the development of phenotype-based, high-throughput small-molecule screens to identify potential novel therapies in complex traits such as RA.

Development of a Biomedical Micro/Nano Robot for Drug Delivery.

Flagellated bacteria have been utilized as potential swimming micro-robotic bodies for propulsion of spherical liposome by attaching several bacteria on their surface. Liposome as a drug delivery vehicle can contain biologically active compounds. In this work, the antibody binding technique is developed to attach bacteria to liposome's surface. Consequently, the stochastic effect of bacterial propulsion of liposome is investigated analytically and experimentally. It is shown that the mobility of liposome with bacteria was higher than that of liposome without bacteria. Experimental data matches well with statistical calculation.