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.).
An effective tool to assess embryo quality in the assisted reproduction clinical practice will enhance successful implantation rates and mitigate high risks of multiple pregnancies. Potential ...biomarkers secreted into culture medium (CM) during embryo development enable rapid and noninvasive methods of assessing embryo quality. However, small volumes, low biomolecule concentrations, and impurity interference collectively preclude the identification of quality-related biomarkers in single blastocyst CM. Here, we developed a noninvasive trace multiomics approach to screen for potential markers in individual human blastocyst CM. We collected 84 CM samples and divided them into high-quality (HQ) and low-quality (LQ) groups. We evaluated the differentially expressed proteins (DEPs) and metabolites (DEMs) in HQ and LQ CM. A total of 504 proteins and 189 metabolites were detected in individual blastocyst CM. Moreover, 9 DEPs and 32 DEMs were identified in different quality embryo CM. We also categorized HQ embryos into positive implantation (PI) and negative implantation (NI) groups based on ultrasound findings on day 28. We identified 41 DEPs and 4 DEMs associated with clinical implantation outcomes in morphologically HQ embryos using a multiomics analysis approach. This study provides a noninvasive multiomics analysis technique and identifies potential biomarkers for clinical embryo developmental quality assessment.
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.
This study proposes that hairpin‐spacer crRNAs (hs‐crRNA) improve the specificity of CRISPR/Cas13a system (hs‐CRISPR). Gibbs free energy analysis and experimental data demonstrate that hs‐crRNAs suppress Cas13a's activity to nonspecific targets. This work provides a fundamental understanding of Cas13a activation associated with binding energetics and an efficient strategy for high‐specificity crRNA design.
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.
A single‐cell transfection analysis chip (scTAC) is developed for expression screening, precise positioning of transfected cells, and immunoblotting analysis of multi‐target proteins. scTAC allows for true single‐cell up‐sampling and enables the acquisition of continuous single‐cell protein expression even in low co‐expression scenarios. scTAC provides a powerful tool for the analysis of biological pathways after transfection of rare cells.
Colon cancer (CC) is one of the leading causes of cancer related mortality. Research over past decades have profoundly enhanced our understanding of immunotherapy, a major clinical accomplishment, ...and its potential role toward treating CC. However, studies investigating the expression of these immune checkpoints, such as epithelial cell adhesion molecule (EpCAM), programmed death-1 (PD-1), and programmed death-ligand 1 (PD-L1), by peripheral blood mononuclear cells (PBMCs) is lacking. Here, high-dimensional mass cytometry (CyTOF) is used to investigate immune alterations and promising immunotherapeutic targets expression by PBMCs of CC patients. Results reveal that expression of EpCAM and PD-L1 on CD4
T cells significantly increased in patients with CC, compared with age- and sex- matching healthy controls and patients with colonic polyps. These differences are also validated in an independent patient cohort using flow cytometry. Further analysis revealed that EpCAM
CD4
T cells are PD-L1
CCR5
CCR6
. Immunofluorescence staining results demonstrate that the increase of EpCAM
CD4
T cells is also observed in tumor tissues, rather than para-cancerous tissues. To ascertain the functional disorders of the identified cell subset, phosphorylated signaling protein levels are assessed using imaging mass cytometry. Increases in pp38 MAPK and pMAPKAPK2 are observable, indicating abnormal activation of pp38 MAPK-pMAPKAPK2 signaling pathway. Results in this study indicate that EpCAM
CD4
T cells may play a role in CC development. Detailed knowledge on the functionality of EpCAM
CD4
T cells is of high translational relevance.
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
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.
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.
•Pre-coated interface proximity extension reaction assay (PIPER) is developed.•An 8-fold enhancement in sensitivity is achieved compared to solid-phase proximity ligation assay.•The PIPER assay for CRP and cTnI-TnC complex with 0.05 pg/mL (1.25 fM) detection limit is developed.•The PIPER assay showed good specificity and is capable of cTnI-TnC complex detection in undiluted serum.
Advances in single‐cell immunoblotting assays, which facilitate the exploration of cell‐to‐cell variation that affects biological systems from cancer development to stem cell biology, have attracted ...much attention. A tetrazole‐functionalized photoclick hydrogel is reported for single‐cell proteomic analysis. The gel serves as a molecular sieving matrix for sodium dodecyl sulfate–polyacrylamide gel electrophoresis and a protein immobilization scaffold for in‐gel immunoblotting. Upon a very short time (60 s) of long‐wavelength ultraviolet irradiation, it can effectively capture the electrophoretically separated proteins in the gel for the subsequent in situ antibody incubation. As a proof of concept, its performance is demonstrated in profiling cell‐to‐cell variations of P‐glycoprotein expression in GES‐1/MGC803 cell lines treated with different drugs. Combined with single‐cell immunoblotting method, employing this photoactive gel enables the monitoring simultaneously in ≈2000 individual cells of subtle protein expression level changes that may be concealed using conventional techniques. The proposed gel has the advantages of excellent electrophoretic separation ability, high protein photoimmobilization efficiency, low autofluorescence, and it can be used as a promising photoactive polyacrylamide gel for in‐gel/in situ capillary and microfluidic immunoblotting assays, especially for developing novel single cell immunoblotting methods.
A tetrazole‐functionalized photoclick hydrogel is developed for enhanced singe‐cell immunoblotting. With its excellent electrophoretic separation ability, high protein photoimmobilization efficiency, and low autofluorescence, the hydrogel serves as a molecular sieving matrix for gel electrophoresis and a protein immobilization scaffold for in‐gel immunoblotting, and is promising for in‐gel/in‐situ capillary and microfluidic immunoblotting assays.
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.
Rapid and portable PCR detection is essential for screening sexually transmitted infections regularly. We developed an infrared mediated RNA isothermal RT-PCR (IR-MERIT PCR) platform and its ...compatible multichamber microfluidic chip for simultaneous amplification and testing (SAT) detection. This microfluidic chip integrates RNA extraction, micropump, and multitarget detection function onto the same chip. By utilizing IR-light-emitting diode (LED) as heat source, this platform can fulfill isothermal amplification within 70 min.
PDF
