Time-lapse proteomics unveil constant high exposure of non-antibiotic drug induces synthetic susceptibility towards regular antibiotics.

Antibiotic resistance is a significant threat to the human race, as regular consumption of antibiotics may lead to antibiotic-resistant bacterial strains. Non-antibiotic drugs also have an extensive impact on bacterial strains, where persistent uptake alters the survival mechanisms of bacteria that could lead to cross-resistance towards other antibiotics. Here, we use time-lapse proteomics shift assays to examine Gram-negative (E. coli. O157:H7 and P. aeruginosa) and Gram-positive (E. faecalis and S. aureus) strains of bacteria for short and continuous exposure to the non-antibiotic drug Hydroxychloroquine (HCQ). Proteomic transitions from wild type to HCQ-exposed strains revealed bacterial transitions and their survival adaptabilities, which were different across all strains. In addition to their structural differences, some shared pathways were enriched among Gram-negative and positive strains. We also validated the cross-resistance and sensitivity towards 24 regularly prescribed antibiotics, indicating that long-term exposure to non-antibiotic drugs may induce general proteomics alterations in the bacterial strains, promoting antibiotic resistance. We validated that HCQ exposure renders Gram-negative strains resistant to Β-lactam and susceptible to macrolides and folic acid. In contrast, Gram-positive strains become susceptible to Β-lactam and resistant to aminoglycosides. Exposure to non-antibiotic drugs causes resistance or susceptibility toward other antibiotics, providing clinicians a reason to overcome antibiotic resistance.

Expansion microscopy with ninefold swelling (NIFS) hydrogel permits cellular ultrastructure imaging on conventional microscope.

Superresolution microscopy enables probing of cellular ultrastructures. However, its widespread applications are limited by the need for expensive machinery, specific hardware, and sophisticated data processing. Expansion microscopy (ExM) improves the resolution of conventional microscopy by physically expanding biological specimens before imaging and currently provides ~70-nm resolution, which still lags behind that of modern superresolution microscopy (~30 nm). Here, we demonstrate a ninefold swelling (NIFS) hydrogel, that can reduce ExM resolution to 31 nm when using regular traditional microscopy. We also design a detachable chip that integrates all the experimental operations to facilitate the maximal reproducibility of this high-resolution imaging technology. We demonstrate this technique on the superimaging of nuclear pore complex and clathrin-coated pits, whose structures can hardly be resolved by conventional microscopy. The method presented here offers a universal platform with superresolution imaging to unveil cellular ultrastructural details using standard conventional laboratory microscopes.

Sickle-like Inertial Microfluidic System for Online Rare Cell Separation and Tandem Label-Free Quantitative Proteomics (Orcs-Proteomics).

Label-free proteomics with trace clinical samples provides a wealth of actionable insights for personalized medicine. Clinically acquired primary cells, such as circulating tumor cells (CTCs), are usually with low abundance that is prohibitive for conventional label-free proteomics analysis. Here, we present a sickle-like inertial microfluidic system for online rare cell separation and tandem label-free proteomics (namely, Orcs-proteomics). Orcs-proteomics adopts a buffer system with 0.1% N-dodecyl ß-d-maltoside (DDM), 1 mM Tris (2-carboxyethyl) phosphine (TCEP), and 2 mM 2-chloroacetamide (CAA) for cell lysis and reductive alkylation. We demonstrate the application of Orcs-proteomics with 293T cells and manage to identify 913, 1563, 2271, and 2770 protein groups with 4, 13, 68, and 119 cells, respectively. We then spike MCF7 cells with white blood cells (WBCs) to simulate the patient's blood sample. Orcs-proteomics identifies more than 2000 protein groups with an average of 61 MCF7 cells. We further recruit two advanced breast cancer patients and collect 5 and 7 CTCs from each patient through minimally invasive blood drawing. Orcs-proteomics manages to identify 973 and 1135 protein groups for each patient. Therefore, Orcs-proteomics empowers rare cells simultaneously to be separated and counted for proteomics and provides technical support for personalized treatment decision making with rare primary patient samples.

Integrated microfluidic single-cell immunoblotting chip enables high-throughput isolation, enrichment and direct protein analysis of circulating tumor cells.

Effective capture and analysis of a single circulating tumor cell (CTC) is instrumental for early diagnosis and personalized therapy of tumors. However, due to their extremely low abundance and susceptibility to interference from other cells, high-throughput isolation, enrichment, and single-cell-level functional protein analysis of CTCs within one integrated system remains a major challenge. Herein, we present an integrated multifunctional microfluidic system for highly efficient and label-free CTC isolation, CTC enrichment, and single-cell immunoblotting (ieSCI). The ieSCI-chip is a multilayer microfluidic system that combines an inertia force-based cell sorter with a membrane filter for label-free CTC separation and enrichment and a thin layer of a photoactive polyacrylamide gel with microwell arrays at the bottom of the chamber for single-cell immunoblotting. The ieSCI-chip successfully identified a subgroup of apoptosis-negative (Bax-negative) cells, which traditional bulk analysis did not detect, from cisplatin-treated cells. Furthermore, we demonstrated the clinical application of the ieSCI-chip with blood samples from breast cancer patients for personalized CTC epithelial-to-mesenchymal transition (EMT) analysis. The expression level of a tumor cell marker (EpCAM) can be directly determined in isolated CTCs at the single-cell level, and the therapeutic response to anticancer drugs can be simultaneously monitored. Therefore, the ieSCI-chip provides a promising clinical translational tool for clinical drug response monitoring and personalized regimen development.

Rapid and efficient capturing of circulating tumor cells from breast cancer Patient's whole blood via the antibody functionalized microfluidic (AFM) chip.

Accurate enumeration of circulating tumor cells (CTCs) in cancer patient's blood functions as a form of "liquid biopsy", which is pivotal for cancer screening, prognosis, and diagnosis. Herein, we demonstrate a novel antibody functionalized microfluidic (AFM) chip that rapidly and accurately qualifies CTCs from breast cancer patient's whole blood. The AFM chip consists of three buffering zones, and four main capturing zones filled with equilateral triangular pillars and periodically distributed obstacles. We validate the AFM chip with three Epithelial cell adhesion molecule (EpCAM) positive cancer cell lines, including breast (MCF-7), prostate (PC3), and lung cancer cell lines (A549), achieving capture efficiencies of 99.5%, 98.5%, and 96.72%, respectively, at a flow rate of 0.6 mL/hour. We further confirm the efficacy of the AFM chip with five advanced breast cancer patients' whole blood to capture EpCAM+/CK19+/CD45-/DAPI + CTCs. Interestingly, high number of CTCs were identified from each patient's 1 mL whole blood (595-2270), The AFM chip is highly efficient at rapidly capturing CTCs from cancer patients' whole blood without requiring extra equipment, which is critically beneficial for clinical application.

A Hashing-Based Framework for Enhancing Cluster Delineation of High-Dimensional Single-Cell Profiles.

Although many methods have been developed to explore the function of cells by clustering high-dimensional (HD) single-cell omics data, the inconspicuously differential expressions of biomarkers of proteins or genes across all cells disturb the cell cluster delineation and downstream analysis. Here, we introduce a hashing-based framework to improve the delineation of cell clusters, which is based on the hypothesis that one variable with no significant differences can be decomposed into more diversely latent variables to distinguish cells. By projecting the original data into a sparse HD space, fly and densefly hashing preprocessing retain the local structure of data, and improve the cluster delineation of existing clustering methods, such as PhenoGraph. Moreover, the analyses on mass cytometry dataset show that our hashing-based framework manages to unveil new hidden heterogeneities in cell clusters. The proposed framework promotes the utilization of cell biomarkers and enriches the biological findings by introducing more latent variables. Supplementary Information: The online version contains supplementary material available at 10.1007/s43657-022-00056-z.

SCANCell reveals diverse inter-cluster interaction patterns in systemic lupus erythematosus across the disease spectrum.

MOTIVATION: High-dimensional mass cytometry (CyTOF), which provides both cellular signatures and inter-cluster interactions like the antagonism between immune activation and suppression, and the pro-inflammatory synergy, sheds light on the cellular and molecular basis of disease pathogenesis. However, revealing the aberrance of inter-cluster communication networks in CyTOF datasets remains a significant challenge. RESULTS: Here, we developed Sample Classification and direct Association Network among Cell clusters (SCANCell) that quantifies the direct association (DA) network of cell clusters. SCANCell was applied to profile inter-cluster interaction patterns of a well-recruited systemic lupus erythematosus (SLE) cohort, including 8 healthy controls, 10 active SLE patients (APs) and 8 remission SLE patients (RPs). SCANCell identified decreased inter-cluster interactions of CD8+ T cells in APs compared with RPs, and enhanced DA of CD8+ T cells after stimulation with immunostimulatory cytokine interleukin-2 in vitro. These discoveries prove that SCANCell can uncover pathology- and drug stimulation-associated inter-cluster interactions, which potentially benefits understanding of pathogenesis and novel therapeutic strategies. AVAILABILITY AND IMPLEMENTATION: The main processing scripts of SCNACell are available at https://github.com/Lxc417/SCANCell. Other codes for the following data statistics are available from the corresponding author upon request. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Polydopamine nanospheres coated with bovine serum albumin permit enhanced cell differentiation: fundamental mechanism and practical application for protein coating formation.

Protein coating is a strategy for modifying and improving the surface functional properties of nanomaterials. However, the underlying mechanism behind protein coating formation, which is essential for its practical applications, remains largely unknown. Herein, we investigate the fundamental molecular mechanism of protein coating formation. Polydopamine nanospheres (PDANS) coated with bovine serum albumin (BSA) are examined in this study due to their wide biomedical potential. Our results demonstrate that BSAs can flexibly bind to PDANS and maintain their structural dynamicity. Our findings unveil that regular structure formation arises from BSAs lateral interactions via electrostatic forces. Notably, the protein coating modified PDANS surface enhances cell adhesion and proliferation as well as osteogenic differentiation. Such an enhancement is attributed to complementary surface properties provided by the dynamic PDANS-BSA complex and regular structure caused by BSA-BSA interactions in protein coating formation. This study provides a fundamental understanding of the molecular mechanism of protein coating formation, which facilitates the further development of functional protein-coated nanomaterials and guides the bioengineering decision making for biomedical applications, especially in bone tissue engineering.

Highly sensitive and portable mRNA detection platform for early cancer detection.

Pancreatic cancer, at unresectable advanced stages, presents poor prognoses, which could be prevented by early pancreatic cancer diagnosis methods. Recently, a promising early-stage pancreatic cancer biomarker, extracellular vesicles (EVs) related glypican-1 (GPC1) mRNA, is found to overexpress in pancreatic cancer cells. Current mRNA detection methods usually require expensive machinery, strict preservation environments, and time-consuming processes to guarantee detection sensitivity, specificity, and stability. Herein, we propose a novel two-step amplification method (CHAGE) via the target triggered Catalytic Hairpin Assembly strategy combined with Gold-Enhanced point-of-care-testing (POCT) technology for sensitive visual detection of pancreatic cancer biomarker. First, utilizing the catalyzed hairpin DNA circuit, low expression of the GPC1 mRNA was changed into amplification product 1 (AP1, a DNA duplex) as the next detection targets of the paper strips. Second, the AP1 was loaded onto a lateral flow assay and captured with the gold signal nanoparticles to visualize results. Finally, the detected results can be further enhanced by depositing gold to re-enlarge the sizes of gold nanoparticles in detection zones. As a result, the CHAGE methodology lowers the detection limit of mRNA to 100 fM and provides results within 2 h at 37 °C. Furthermore, we demonstrate the successful application in discriminating pancreatic cancer cells by analyzing EVs' GPC1 mRNA expression levels. Hence, the CHAGE methodology proposed here provides a rapid and convenient POCT platform for sensitive detection of mRNAs through unique probes designs (COVID, HPV, etc.).

One-pot pre-coated interface proximity extension assay for ultrasensitive co-detection of anti-SARS-CoV-2 antibodies and viral RNA.

In the field of in vitro diagnostics, detection of nucleic acids and proteins from biological samples is typically performed with independent platforms; however, co-detection remains a major technical challenge. Specifically, during the coronavirus disease 2019 (COVID-19) pandemic, the ability to simultaneously detect viral RNA and human antibodies would prove highly useful for efficient diagnosis and disease course management. Herein, we present a multiplex one-pot pre-coated interface proximity extension (OPIPE) assay that facilitates the simultaneous recognition of antibodies using a pre-coated antigen interface and a pair of anti-antibodies labeled with oligonucleotides. Following anti-antibody-bound nucleic acid chain extension to form templates in proximity, antibody signals can be amplified, together with that of targeted RNA, via a reverse transcription-polymerase chain reaction. Using four-color fluorescent TaqMan probes, we demonstrate the co-detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-specific antibodies and viral nucleic acids in a single bio-complex sample, including nucleocapsid protein-specific IgG and IgM, and the RNA fragments of RdRp and E genes. The serum detection limit for this platform is 100 fg/mL (0.67 fM) for the anti-SARS-CoV-2 antibody and 10 copies/µL for viral RNA. The OPIPE assay offers a practical and affordable solution for ultrasensitive co-detection of nucleic acids and antibodies from the same trace biological sample without the additional requirement of complicated equipment.

Single-Cell Microwell Platform Reveals Circulating Neural Cells as a Clinical Indicator for Patients with Blood-Brain Barrier Breakdown.

Central nervous system diseases commonly occur with the destruction of the blood-brain barrier. As a primary cause of morbidity and mortality, stroke remains unpredictable and lacks cellular biomarkers that accurately quantify its occurrence and development. Here, we identify NeuN+/CD45-/DAPI+ phenotype nonblood cells in the peripheral blood of mice subjected to middle cerebral artery occlusion (MCAO) and stroke patients. Since NeuN is a specific marker of neural cells, we term these newly identified cells as circulating neural cells (CNCs). We find that the enumeration of CNCs in the blood is significantly associated with the severity of brain damage in MCAO mice (p < 0.05). Meanwhile, the number of CNCs is significantly higher in stroke patients than in negative subjects (p < 0.0001). These findings suggest that the amount of CNCs in circulation may serve as a clinical indicator for the real-time prognosis and progression monitor of the occurrence and development of ischemic stroke and other nervous system disease.

The optimization of combinatorial drug therapies: Strategies and laboratorial platforms.

Designing optimal combinatorial drug therapies is challenging, because the drug interactions depend not only on the drugs involved, but also on their doses. With recent advances, combinatorial drug therapy is closer than ever to clinical application. Herein, we summarize approaches and advances over the past decade for identifying and optimizing drug combination therapies, with innovations across research fields, covering physical laboratory platforms for combination screening to computational models and algorithms designed for synergism prediction and optimization. By comparing different types of approach, we detail a three-step workflow that could maximize the overall optimization efficiency, thus enabling the application of personalized optimization of combinatorial drug therapy.

Single-Cell Immunoblotting based on a Photoclick Hydrogel Enables High-Throughput Screening and Accurate Profiling of Exogenous Gene Expression.

Fast and accurate profiling of exogenous gene expression in host cells is crucial for studying gene function in cellular and molecular biology, but still faces the challenge of incomplete co-expression of reporter genes and target genes. Here, a single-cell transfection analysis chip (scTAC) is presented, which is based on the in situ microchip immunoblotting method, for rapid and accurate analysis of exogenous gene expression in thousands of individual host cells. scTAC not only can assign information of exogenous gene activity to specific transfected cells, but enables the acquisition of continuous protein expression even in low co-expression scenarios. It is demonstrated that scTAC can reveal the relationship of expression level between reporter genes and target genes, which is helpful for evaluating transient transfection strategy efficiency. The advantages of this method for the study of fusion protein expression and downstream protein expression in signaling pathway in rare cells are shown. Empirically, an EGFP-TSPAN8 fusion plasmid is transfected into MCF-7 breast cancer cells and the expressions of two cancer stemness biomarkers (ALDHA1 and SOX2) are analyzed. The scTAC method clearly reveals an interesting phenomenon that transfected adherent MCF-7 cells exhibit some stem cell characteristics, but they do not have stem cell appearance.

Pre-coated interface proximity extension reaction assay enables trace protein detection with single-digit accuracy.

Advances in trace protein detection contribute to the early diagnosis of diseases and exploration of stem cell development. The pre-coated interface proximity extension reaction (PIPER) assay enables target protein detection at trace levels and was developed based on protein biomarker recognition using sets of three specific antibodies and the extension of antibody-bound nucleic acid chains in proximity, accompanied by amplification and reading of protein signals via real-time quantitative polymerase chain reaction (qPCR). Noise generated in binding reactions and enzymatic steps was decreased by transferring the liquid-liquid reactions onto a liquid-solid interface in glutaraldehyde-treated tubes pre-coated with antibodies. Nucleic acid sequences of oligo-antibody-based probes were designed for extension and qPCR without pre-amplification when binding to a target molecule. As a proof of concept, the PIPER assay was used to profile slight variations in crucial biomarkers, high-sensitivity C-reactive protein, and cardiac troponin I. The detection sensitivity of the assay for the biomarkers was 0.05 pg/mL (1.25 fM) in 10% human serum. In phosphate-buffered saline, the PIPER assay detected fewer than 10 protein molecules per µL. The simple, widely applicable PIPER assay can detect trace protein biomarkers with single-digit accuracy, making it appropriate for the development of clinical hypersensitive protein detection and single-cell protein detection technology.

Hairpin-Spacer crRNA-Enhanced CRISPR/Cas13a System Promotes the Specificity of Single Nucleotide Polymorphism (SNP) Identification.

The Cas13a system has great potential in RNA interference and molecular diagnostic fields. However, lacking guidelines for crRNA design hinders practical applications of the Cas13a system in RNA editing and single nucleotide polymorphism identification. This study posits that crRNAs with hairpin spacers improve the specificity of CRISPR/Cas13a system (termed hs-CRISPR). Gibbs free energy analysis suggests that the hairpin-spacer crRNAs (hs-crRNAs) suppress Cas13a's affinity to off-target RNA. A hepatitis B virus DNA genotyping platform is established to further validate the high-specificity of hs-CRISPR/Cas13a system. Compared to ordinary crRNA, hs-crRNAs increase the specificity by threefold without sacrificing the sensitivity of the CRISPR/Cas13a system. Furthermore, the mechanism of the Cas13a/hs-crRNA/target RNA composition is elucidated with theoretical simulations. This work builds on the fundamental understanding of Cas13a activation and offers significant improvements for the rational design of crRNA for the CRISPR/Cas13a system.

Job Tension and Emotional Sensitivity to COVID-19 Public Messaging and Risk Perception.

During the COVID-19 pandemic, government social marketing messages support strategies of suppression (often stay-at-home orders or lockdowns) and/or mitigation (through testing, isolation, and tracing). Success at lowering the virus reproduction rate (R0) depends on social marketing messaging that rapidly changes behaviors. This study explores a potential side effect of a successful antivirus public health messaging campaign, when employees are back at work but the virus threat has not disappeared, that leads to on-the-job stress. The authors surveyed office employees in Shanghai, the People's Republic of China, where a nearly 2-month COVID-19 quarantine ended in late March 2020 and work locations reopened with strong public health messaging to encourage cooperation with continued virus spread suppression strategies-an approach likely to be followed in numerous countries. This study examines the relationship of pandemic public messaging sensitivity with tension and negative emotions on the job. Canonical correlation analysis is used with a sample of 1154 respondents, 4 predictor variables (reference group, self-regulation, media, and risk), and 2 criterion variables (negative emotions and job tension). Results show employees are differentially affected by the pandemic background noise. Those more sensitive to social-level virus risks and more open to reference group influence report increased levels of negative emotions and work tension.

Progress and applications of mass cytometry in sketching immune landscapes.

Recently emerged mass cytometry (cytometry by time-of-flight [CyTOF]) technology permits the identification and quantification of inherently diverse cellular systems, and the simultaneous measurement of functional attributes at the single-cell resolution. By virtue of its multiplex ability with limited need for compensation, CyTOF has led a critical role in immunological research fields. Here, we present an overview of CyTOF, including the introduction of CyTOF principle and advantages that make it a standalone tool in deciphering immune mysteries. We then discuss the functional assays, introduce the bioinformatics to interpret the data yield via CyTOF, and depict the emerging clinical and research applications of CyTOF technology in sketching immune landscape in a wide variety of diseases.

Real-time detection of foodborne bacterial viability using a colorimetric bienzyme system in food and drinking water.

Foodborne bacterial infection poses a serious threat to human health. As most diseases are caused by living bacteria, real-time assessment of bacterial viability is vitally important to the public health sector. Herein, we developed a simple and novel colorimetric assay based on the Glucose oxidase (GOD)/Horseradish peroxidase (HRP) bienzyme system for real-time monitoring of bacterial viability in food and drinking water. This bienzyme system is free of any chemical synthesis and only requires 3 sample handling steps. The color response is easily observable with the naked eye or recordable with a smartphone for precise determination of bacterial viability. The proposed strategy was validated with various bacteria both Gram-positive and Gram-negative, indicating its capability for broad-spectrum bacteria viability detection. Therefore, the proposed strategy shows promise for rapid and reliable quality control in food and drinking water.

Recent advances in microfluidics for drug screening.

With ever increasing drug resistance and emergence of new diseases, demand for new drug development is at an unprecedented urgency. This fact has led to extensive recent efforts to develop new drugs and novel techniques for efficient drug screening. However, new drug development is commonly hindered by cost and time span. Thus, developing more accessible, cost-effective methods for drug screening is necessary. Compared with conventional drug screening methods, a microfluidic-based system has superior advantages in sample consumption, reaction time, and cost of the operation. In this paper, the advantages of microfluidic technology in drug screening as well as the critical factors for device design are described. The strategies and applications of microfluidics for drug screening are reviewed. Moreover, current limitations and future prospects for a drug screening microdevice are also discussed.

Overcoming Multidrug-Resistance in Bacteria with a Two-Step Process to Repurpose and Recombine Established Drugs.

The emergence and ongoing spread of multidrug-resistant (MDR) bacteria is a major global public health threat. MDR has extensively combated the potency of antibiotics. Development of new antibiotics requires several years with prohibitive cost that will not last. An alternative solution is to recombine failed antibiotics, which has been proven to be not only cost-effective, but also potent. However, selection of the optimal combinations of these chemicals through conventional trial-and-error methods is challenging and slow, since M candidates with N doses lead to NM possible combinations. Herein, we present a artificial intelligence (AI) guided chemical combination optimization technique, namely Streamlined Rapid Identification of Combinatorial Therapies (STRICT), which is phenotype based and can efficiently learn and identify the optimal drug-combinations with minimal experimental efforts. With the guidance of STRICT, we successfully identified potent combinations of five antibiotics from 26 antibiotics that are individually ineffective at inhibiting an artificially induced strain of MDR bacteria. Rather than examine millions of tests, STRICT accomplished this task with only 120 carefully selected tests. Our results indicate that STRICT is a powerful platform to identify efficacious multiantibiotic combinations for the treatment of MDR bacteria. The AI-guided platform introduced here is an effective tool for drug repurposing, beneficial toward large-scale drug screening for other disease models, and also has a broad application in chemical combination optimization to deliver a desired end point for a complex system.