Multidisciplinary single-cell optical cytometry.

FAK Inhibition Reduces Tumor Infiltration of Tregs via Restricting CCL5 Release and Boosts the Combination Therapy for BRAF ^V600E Mutant Colorectal Carcinoma

Laser scanning cytometry (LSC) is a slide-based technique combining advantages of flow and image cytometry: automated, high-throughput detection of optical signals with subcellular resolution. Fluorescence resonance energy transfer (FRET) is a spectroscopic method often used for studying molecular interactions and molecular distances. FRET has been measured by various microscopic and flow cytometric techniques. We have developed a protocol for a commercial LSC instrument to measure FRET on a cell-by-cell or pixel-by-pixel basis on large cell populations, which adds a new modality to the use of LSC. As a reference sample for FRET, we used a fusion protein of a single donor and acceptor (ECFP-EYFP connected by a seven-amino acid linker) expressed in HeLa cells. The FRET efficiency of this sample was determined via acceptor photobleaching and used as a reference value for ratiometric FRET measurements. Using this standard allowed the precise determination of an important parameter (the alpha factor, characterizing the relative signal strengths from a single donor and acceptor molecule), which is indispensable for quantitative FRET calculations in real samples expressing donor and acceptor molecules at variable ratios. We worked out a protocol for the identification of adherent, healthy, double-positive cells based on light-loss and fluorescence parameters, and applied ratiometric FRET equations to calculate FRET efficiencies in a semi-automated fashion. To test our protocol, we measured the FRET efficiency between Fos-ECFP and Jun-EYFP transcription factors by LSC, as well as by confocal microscopy and flow cytometry, all yielding nearly identical results. Our procedure allows for accurate FRET measurements and can be applied to the fast screening of protein interactions. A pipeline exemplifying the gating and FRET analysis procedure using the CellProfiler software has been made accessible at our web site.

Large-scale multiparameter screening has become increasingly feasible and straightforward to perform thanks to developments in technologies such as high-content microscopy and high-throughput flow cytometry. The automated toolkits for analyzing similarities and differences between large numbers of tested conditions have not kept pace with these technological developments. Thus, effective analysis of multiparameter screening datasets becomes a bottleneck and a limiting factor in unbiased interpretation of results. Here we introduce compaRe, a toolkit for large-scale multiparameter data analysis, which integrates quality control, data bias correction, and data visualization methods with a mass-aware gridding algorithm-based similarity analysis providing a much faster and more robust analyses than existing methods. Using mass and flow cytometry data from acute myeloid leukemia and myelodysplastic syndrome patients, we show that compaRe can reveal interpatient heterogeneity and recognizable phenotypic profiles. By applying compaRe to high-throughput flow cytometry drug response data in AML models, we robustly identified multiple types of both deep and subtle phenotypic response patterns, highlighting how this analysis could be used for therapeutic discoveries. In conclusion, compaRe is a toolkit that uniquely allows for automated, rapid, and precise comparisons of large-scale multiparameter datasets, including high-throughput screens.

<p>In the published version of this article [1], the author identified the error in the reported funding grant number. This has now been corrected. </p> <p> The original article can be found online at: https://www.eurekaselect.com/article/150528ss </p> <p> Details of the error and a correction are provided here. </p> <p> ORIGINAL: </p> <p> FUNDING </p> <p> This research was financially supported by the National Natural Science Foundation of China (grant number: 321600034), the Guangxi Natural Science Foundation of China (grant number: 2021GXNSFAA075026), and the Baise Science and Tech-nology project of Guangxi, China (grant numbers: 20212346 and 20224146). </p> <p> CORRECTED: </p> <p> FUNDING </p> <p> This research was financially supported by the National Natural Science Foundation of China (grant number: 32160034), the Guangxi Natural Science Foundation of China (grant number: 2021GXNSFAA075026), and the Baise Science and Tech-nology project of Guangxi, China (grant numbers: 20212346 and 20224146). The authors apologize for any inconvenience caused.</p>

Lentiviral-mediated ho-1 gene transduced bone mesenchymal stem cells protects against acute graft-Versus-host disease in vitro and vivo

The BiFC (bimolecular fluorescence complementation) assay and BiFC combined with FRET (fluorescence resonance energy transfer) technique have become important tools for molecular interaction studies in live cells. However, the real detection and cellular imaging performances of most existing red fluorescent protein-derived BiFC assays still suffer from relatively low ensemble brightness, high cytotoxicity, the red fluorescent proteins being prone-to-aggregation or severe residual dimerization, inefficient complementation and slow maturation at 37 °C physiological temperature in live mammalian cells. We developed a BiFC assay based on a recently evolved truly monomeric red fluorescent protein (FP) mScarlet-I with excellent cellular performances such as low cytotoxicity, fast and efficient chromophore maturation and the highest in-cell brightness among all previously reported monomeric red fluorescent proteins. In this work, a classic β-Fos/β-Jun constitutive heterodimerization model and a rapamycin-inducible FRB/FKBP interaction system were used to establish and test the performance of the mScarlet-I-based BiFC assay in live mammalian cells. Furthermore, simply by adopting the large-Stokes-shift fluorescent protein mAmetrine as the donor, β-Jun-β-Fos-NFAT1 ternary protein complex formation could be readily and efficiently detected and visualized with minimal spectral cross-talk in live HeLa cells by combining live-cell sensitized-emission FRET measurement with the mScarlet-I-based BiFC assay. The currently established BiFC assay in this work was also shown to be able to detect and visualize various protein-protein interactions (PPIs) at different subcellular compartments with high specificity and sensitivity at 37 °C physiological temperature in live mammalian cells.

High-throughput flow cytometry for drug discovery: principles, applications, and case studies

Cytometry is the characterization and measurement of cells and cellular constituents for biological, diagnostic and therapeutic purposes, embracing the fields of cell and molecular biology, biochemistry, biophysics, cell physiology, pathology, immunology, genetics, biotechnology, plant biology and microbiology. Cytometry is based on quantitative measurements of the molecular and phenotypic properties of cells using flow and image cytometry, microarrays, and proteomics. Cytomics (molecular cell systems research) aims at the understanding of the molecular architecture and functionality of cell systems (cytomes) by single-cell analysis in combination with exhaustive bioinformatic knowledge extraction. The cytomics concept has been significantly advanced by a multitude of current developments like confocal and laser scanning microscopy, multiphoton fluorescence excitation, spectral imaging, fluorescence resonance energy transfer (FRET), fast imaging in flow, optical stretching in flow, and miniaturized flow and image cytometry within laboratories on a chip or laser microdissection, as well as the use of bead arrays [1, 2]. Data sieving or data mining of the vast amounts of collected multiparameter data for exhaustive multilevel bioinformatic knowledge extraction avoids the inadvertent loss of information from unknown molecular relations being inaccessible to an a priori hypothesis. These approaches will become powerful tools for such important fields as individualized medicine, drug discovery and drug development [3–6]. This special issue is focused on state-of-the-art research in advanced cytometry and application areas with particular emphasis on novel biophotonic methods, disease diagnosis, and monitoring of disease treatment at single-cell level in stationary and flow conditions. It seeks to advance scholarly research that spans from fundamental interactions between light, cells, vascular tissue, and labeling particles, to strategies and opportunities for preclinical and clinical research. Recent advances in slide-based cytometry for in vitro application are summarized by Gerstner et al. [7]. They show that single cell based quantitation using microscope based cytometry instruments is making its way from basic research into clinical use. Calibration and quality control are essential in every cytometric analysis. Basics of standardization and calibration in cytometry are comprehensively summarized by Mittag and colleague [8]. Leif [9] demonstrates how data formats produced by cytometric measurements can be integrated into clinical diagnostic system standards for improved data management in the clinical environment. Flow cytometry (FCM) is based both on cell detection as whole and the use of high-speed imaging of individual cells and nanoparticles. Tychinski and co-workers [10] analyze the metabolic component of cellular refractivity and its importance for optical cytometry. The refractivity decrease in response to energy depletion was demonstrated for mitochondria, chloroplasts, spores, and cancer cells using the coherent phase microscope “Airyscan”. Estimations indicated high values for the equivalent refractive index of structured water in cells. To reduce the interference from tissue light scattering background, gold nanoparticles with strong plasmon scattering resonance properties can be applied as in vivo FCM contrast agents. This technique requires an understanding of light interaction features of cells with and without nanoparticles. Tanev and coworkers [11] compare light scattering of these cells in controlled refractive index matching conditions. They analyze the optical schematics including phase contrast microscopy as a prospective modality for in vivo FCM. Jacobs et al. [12] consider some important features of the diffraction imaging of spheres and melanoma cells with a microscope objective. Using a confocal imaging-based method to reconstruct and analyze the 3D structure, they demonstrated that genetic modifications in these cells can induce morphological changes, and the modified cells can be used as an experimental model to study the correlation between 3D morphology features and diffraction image data. Galanzha et al. [13, 14] introduce in vivo FCM with photoacoustic and photothermal detection schematics. Conventional FCM provides fast multi-parameter quantification of the biological properties of individual cells at subcellular and molecular levels. Nevertheless, the invasive extraction of cells from a living system modifies their properties, preventing their long-term study in their native environment. In vivo FCM of blood and lymph vessels with natural bioflows overcomes these problems. Cells can be detected label-free, by endogenous chromophores and pigments or by advanced strongly absorbing nanoparticles as novel labels. Both approaches are demonstrated for noninvasive rapid detection and treatment of metastases in lymph flow and sentinel lymph nodes by an integrated system of multicolor photoacoustic lymph FCM/lymphography and photothermal therapy. Bykov et al. [15] employ Doppler OCT imaging of cytoplasm shuttle flow in a slime mold Physarum polycephalum. Radial contractions of the gel-like walls of the strands and the velocity distributions in the sol-like endoplasm streaming along the plasmodial strands are imaged. The motility inhibitor effect of carbon dioxide on the cytoplasm shuttle flow and strand wall contraction is shown.

Is aberrant expression of gene associated with retinoid-interferon-induced mortality-19 (GRIM-19) associated with unexplained recurrent spontaneous abortion (URSA)? GRIM-19 deficiency may regulate regulatory T cell/T helper 17 cell (Treg/Th17) balance partly through reactive oxygen species (ROS)-mammalian target of rapamycin (mTOR) signaling axis in URSA. Immunological disorders may cause impaired maternal immune tolerance to the fetus and result in fetal rejection. The differentiation of Treg and Th17 cells is controlled by phosphoinositide 3-kinase (PI3K)/Akt/mTOR signaling pathway. GRIM-19 participates in the immune response, but its role in URSA is largely unknown. The current study included 28 URSA patients and 30 non-pregnant healthy women. The proportion of Treg and Th17 cells in peripheral blood of URSA patients and control subjects were assessed with flow cytometry. The expression of GRIM-19 in peripheral blood lymphocytes (PBLs) was measured with quantitative real-time PCR and western blot analysis. Furthermore, the ROS level in the PBLs of URSA patients and control subjects were assessed by 2',7'-dichlorodihydrofluorescein diacetate (DCFH-DA) staining. Then, Akt/mTOR expression in the PBLs was measured. Downregulation of GRIM-19 in Jurkat cells was performed by specific siRNA. Then, intracellular ROS production and the expression of p-mTOR, which is known to enhance Th17 differentiation and decrease Treg cell differentiation, were detected. Finally, N-acetylcysteine (NAC) was used to decrease the intracellular ROS level, and the expression of p-mTOR was measured. The proportion of Treg cells was reduced in URSA patients, whereas the proportion of Th17 cells was increased. The expression of GRIM-19 was significantly lower in PBLs of URSA patients. Furthermore, there is a considerable increase in intracellular ROS production and a high level of p-Akt and p-mTOR expression in the PBLs of URSA patients compared with the control subjects. In parallel to this, downregulation of GRIM-19 in the Jurkat cells by siRNA results in an increased ROS production and an increased expression of p-mTOR. Importantly, the upregulation of p-mTOR resulting from GRIM-19 loss was significantly reversed in the cells treatment with ROS inhibitor N-acetyl-l-cysteine (NAC), indicating that ROS was indeed required for GRIM-19 depletion induced p-mTOR expression. None. A large number of researches have confirmed that the differentiation of Treg and Th17 cells is controlled by PI3K/Akt/mTOR signaling pathway. We have not shown the regulatory role of ROS and PI3K/Akt/mTOR in Treg and Th17 differentiation in this study. Our study has demonstrated that GRIM-19 deficiency may play a role in regulating Treg/Th17 balance partly through ROS-mTOR signaling axis in URSA. The present study offers a new perspective to the roles of GRIM-19 in immunoregulation. This work was supported by the National Natural Science Foundation of China (Grant numbers 81571511, 81701528, 81370711 and 30901603), the Shandong Provincial Natural Science Foundation (Grant numbers ZR2017PH052 and ZR2013HM090) and the Science Foundation of Qilu Hospital of Shandong University, Fundamental Research Funds of Shandong University (Grant numbers 2015QLQN50 and 2015QLMS24). The authors declare that there is no conflict of interest that could prejudice the impartiality of the present research.

Monocytes have emerged as critical driving force of acute inflammation. Here, we show that inhibition of Toll-like receptor 2(TLR2) dimerization by a TLR2 transmembrane peptide (TLR2-p) ameliorated DSS-induced colitis by interfering specifically with the activation of Ly6C(+) monocytes without affecting their recruitment to the colon. We report that TLR2-p directly interacts with TLR2 within the membrane, leading to inhibition of TLR2-TLR6/1 assembly induced by natural ligands. This was associated with decreased levels of extracellular signal-regulated kinases (ERK) signaling and reduced secretion of pro-inflammatory cytokines, such as interleukin (IL)-6, IL-23, IL-12, and IL-1β. Altogether, our study provides insights into the essential role of TLR2 dimerization in the activation of pathogenic pro-inflammatory Ly6C(hi) monocytes and suggests that inhibition of this aggregation by TLR2-p might have therapeutic potential in the treatment of acute gut inflammation.

‘Forbidden’ States Boost Fluorescence Resonance Energy Transfer (FRET) Studies of Membrane Receptors in Live Cells

Single-Cell Imaging

Bone is the most common site for metastasis in prostate cancer (PCa), and identifying clinically useful biomarkers to predict the risk of currently incurable bone metastases is a major priority in PCa research. Extracellular adenosine triphosphate (ATP) was shown to be a major biochemical constituent of the tumor microenvironment and regulates the tumor and host interactions by acting at P2 purinergic receptors. Among the P2 receptors, the ion channel P2X purinergic receptor 7 (P2X7R) has an unusually long C-terminus containing 200 amino acids, which forms the molecular basis for the unique bi-function of P2X7R promoting cancer cell proliferation or inducing membrane permeabilization and apoptosis, upon activation by a low or high concentration of ATP, respectively. A cysteine-rich region and a guanosine diphosphate or triphosphate (GDP/GTP)-binding site located in the C-terminus are pivotal for the structural rearrangement of the second transmembrane domain helix and the globular ballast underneath during the membrane pore permeabilization [1]. Among the 10 human P2X7R splice variants (P2X7RA-J), the P2X7RA variant is the full-length "wild-type (WT)" receptor, while the truncated P2X7RB variant lacks the intracellular C-terminal tail. This naturally occurring P2X7RB isoform maintains ATP-stimulated channel activity but not pore permeabilization [2, 3]. P2X7RB is upregulated in epithelial cancer cells [3] and associated with the trophic activity of primary bone cancer [2, 4, 5]. These findings have highlighted the importance of defining the role of P2X7R C-terminal truncated variants in cancer progression, particularly considering that data on P2X7R in PCa bone metastasis is lacking, whereas clinical evidence offers contradictory results [6, 7]. To clarify the role of P2X7R in PCa, we collected biopsies and surgical specimens from 77 patients with PCa and 9 patients with benign prostatic hyperplasia (BPH) from three local hospitals in Qingdao, Shandong, China. Among these PCa patients, 20 (26.0%) had bone metastases at the time of diagnosis, while 57 (74.0%) had no bone metastases (Supplementary Table S1). To distinguish the full-length WT P2X7RA and the C-terminus truncated P2X7RB splice variants, we used two antibodies specifically against different domains of the P2X7R, including anti-P2X7R-Cter (C-terminal) antibody against the P2X7RA-specific C-terminal tail, thus detecting the P2X7RA isoform, whilst the anti-P2X7R-ec (extracellular) antibody against the extracellular domain and detecting both P2X7RA and P2X7RB [4]. The detailed Methods can be found in the Supplementary file. Therefore, P2X7RA was defined as positive staining for both anti-P2X7R-Cter and anti-P2X7R-ec, while P2X7RB was positive for anti-P2X7R-ec but negative for anti-P2X7R-Cter (Figure 1A). Of the 66 P2X7R expressing PCa specimens (anti-P2X7R-ec-positive), 47 (71.2%) expressed P2X7RA, and 19 (28.8%) expressed P2X7RB. Comparing the Gleason scores, P2X7RB-positive cases were 8.2% higher than P2X7RA-positive cases (P = 0.047, Figure 1B). P2X7RB was not expressed in BPH specimens (0/9) but was expressed in 25.9% of PCa cases without bone metastases (14/54) and 41.7% of cases with bone metastases (5/12). The fraction of P2X7RB-positive cases were positively correlated with PCa progression (r = 0.997, P = 0.048, Figure 1C), based on the average Gleason scores of different disease stages (PCa without bone metastases = 8.0, bone metastatic PCa = 8.9, Supplementary Figure S1A). Immunoreactivity analysis showed no significant difference in P2X7RA expression level among specimens from the BPH group and groups of PCa with or without bone metastases (Supplementary Figure S1B-C), which was consistent with the retrospective analysis of clinical datasets (Supplementary Figure S1D-E). Therefore, C-terminus truncated P2X7RB variants, but not full-length P2X7RA, were positively associated with PCa progression and bone metastases. As P2RX7 exon 13 encodes 85% length of the entire C-terminus and is key to C-terminal functions, CRISPR/Cas9 technique was used to generate a frameshift after residue position 451 in exon 13 (∆C) (Supplementary Figure S2, S3), to understand how P2X7R C-terminal disruptions affect PCa cell biology. In WT PCa cells, exposure to 300 μmol/L P2X7R-specific agonist BzATP induced ethidium bromide uptake, suggesting that P2X7R mediated membrane permeabilization to large molecules, a unique property of the P2X7R C-terminus. While in ∆C cells, this was significantly reduced (P < 0.001, Figure 1D). To examine whether ∆C P2X7R is still functional in PCa cells, we tested the BzATP-induced calcium influx. BzATP was able to induce a calcium influx in PC3 ∆C cells, which was inhibited by the P2X7R antagonist A740003 (Figure 1E). There was no significant difference in BzATP-induced calcium influx peak between PC3 WT and ∆C cells (P = 0.388, Figure 1F). In addition, PC3 ∆C cells showed a significantly reduced presence of the P2X7R C-terminus at the protein level (Supplementary Figure S3F-I). These data have confirmed that our P2X7R ∆C resembled the unique characteristics of C-terminal truncated P2X7RB isoform, i.e., maintaining channel activity but not membrane pore permeabilization [2]. To test whether the disruption of P2RX7 exon 13 altered the metastatic ability of PCa cells, we tracked bone tumors formed by WT and ∆C PC3 cells following intra-cardiac injection (Figure 1G). Mice injected with ∆C cells had almost twice the skeletal tumor frequency (number of skeletal tumors/mouse) of the WT group (4.2 vs. 2.3, P = 0.016, Figure 1H), with a significant 4-day reduction in survival compared to the WT group (median survival: 26 vs. 30 days, P = 0.042, Figure 1I). However, there were no significant differences in the individual tumor size (Supplementary Figure S4A), number of non-skeletal tumors (Supplementary Figure S4B), or tumor-induced bone destruction determined ex vivo using micro-CT (Supplementary Figure S4C-F). These were consistent with data from cellular assays showing that disrupting P2X7R C-terminus did not impair proliferation (Supplementary Figure S5) and suggested that the pro-bone metastasis effects in C-terminal disrupted PCa cells were bone tropism related rather than tumor growth per se. To provide an insight into what led to the enhanced metastatic potential of ∆C cells, we performed a metastasis-initiating cell assay [8] and found that PC3 ∆C cells had significantly increased numbers of bone tropic metastasis-initiating cells by 74.1% (P = 0.012, Figure 1J). We examined these cells' response to stimulation of pore-inducing high concentration of extracellular BzATP (300 μmol/L) and showed a significant reduction of induced apoptosis in PC3 ∆C cells (P = 0.030, Figure 1K) but an increased secretion of pro-metastatic cytokine IL-6 (Figure 1L). The enhanced presence of the metastasis-initiating cell subpopulation in P2X7R ∆C cells was probably linked to their ability to escape apoptosis. The P2X7R C-terminus, with the presence of palmitoylated cysteine residues, could be inserted into the plasma membrane and facilitated pore formation by preventing cholesterol interaction with P2X7R [9]. Disruption of the C-terminus, like in the P2X7RB isoform or ∆C cells, could significantly reduce the induction of apoptosis in cells under the stimulation of pore-inducing high concentration of extracellular nucleotides (e.g., 100-500 μmol/L extracellular ATP within solid tumors) [10]. Furthermore, when stimulated by extracellular BzATP, only ∆C cells secreted IL-6, a pro-inflammatory cytokine with strong pro-metastatic effects on bone metabolism, tumor cell survival, and angiogenesis. These findings warrant further investigation to clarify the exact regulatory mechanisms determining the metastatic fate of P2X7R ∆C cells. In conclusion, using both clinical and pre-clinical data, we showed that the disruption of the C-terminus of P2X7R was positively associated with PCa progression and bone metastasis. The C-terminal truncated P2X7RB isoform, rather than the full-length P2X7RA, could potentially be used as a prognostic marker to predict PCa bone metastasis. Haiping Song: Investigation, Formal analysis, Visualization and Funding acquisition; Hector Manuel Arredondo Carrera: Investigation, Formal analysis and Visualization; Alexandria Sprules: Investigation, Validation and Methodology; Ying Ji: Investigation and Validation; Tongsong Zhang: Investigation and Validation; Jiepei He: Investigation; Eleanor Lawrence: Investigation; Alison Gartland: Writing - Review & Editing; Jian Luo: Writing - Review & Editing and Funding acquisition; Ning Wang: Conceptualization, Writing - Original Draft, Supervision, Project administration, Funding acquisition. These studies received supports from the Royal Society/the National Natural Science Foundation of China (NSFC) and Qingdao Science and Technology Bureau Research Fund. We also wish to thank the following colleagues for their excellent technical support: Dr. Bin Ding, Pathology Department, Qingdao Tumor Hospital, China; Dr. Changqing Jiang, Pathology Department, Qingdao Municipal Hospital, China; Dr. Yunqing Chen, Pathology Department, the Affiliated Hospital of Medical College, Qingdao University, China; and Dr. Karan M Shah, Department of Oncology & Metabolism, The University of Sheffield, UK. The authors declare that they have no competing interests. This research was funded by the International Exchange Scheme – Cost Share Programme, jointly sponsored by the Royal Society, UK (grant number IEC∖NSFC∖170113) and the National Natural Science Foundation of China (NSFC) (grant number 8171101485). This research was also funded by the Qingdao Science and Technology Bureau Research Fund, China, grant number 18-6-1-98-nsh. Raw data for retrospective analysis can be accessed through the Gene Expression Omnibus (GEO) database (Accession Number: GDS1439 and GDS3289). The datasets used and/or analyzed during the current study are available from the corresponding author upon reasonable request. For clinical samples, written informed consent was obtained from all patients, and the study was approved by the Ethical Review Committee of the Qingdao Tumor Hospital to ensure that rights and privacy of patients were protected (Approval Number: [Y]KY20200001). For animal studies, all procedures complied with the UK Animals (Scientific Procedures) Act 1986 and were reviewed and approved by the local Research Ethics Committees of the University of Sheffield under Home Office project license 70/8799 (Sheffield, UK). Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.

The combination of magnetic trap (MT) and fluorescence resonant energy transfer (FRET) allows for nanoscale measurements of configurational changes of biomolecules under force. However, the magnetic bead involved in MT experiments introduces a substantial amount of background fluorescence which reduces the signal-to-noise ratio (SNR) of FRET significantly. Moreover, the short lifetime of the dye used in FRET limits the total sampling time when combined with MT. Here we use a moveable tube lens to adjust the wave front in the light pathway of MT so that both images of the magnetic bead and the fluorescent signals can be detected when long DNA handles are used to reduce the auto-fluorescence of the magnetic bead. We utilize the internal trigger of an electron multiplying charge-coupled device camera to control a shutter so that the dye can be excited intermittently when long time measurement of FRET is needed. As a demonstration of the hybrid technique, we observe the unfolding/refolding dynamics of a DNA hairpin and measure the DNA unwinding activity of the saccharomyces cerevisiae Pif1 (Pif1). Our results show that the unwinding burst of Pif1 under external force is different from that without the force. In addition, the improvement provides a better SNR and a longer sampling time in experiments in the MT-FRET assay.

Circulating CXCR3 Tfh Cells Positively Correlate with Neutralizing Antibodies Responses During HCV Infection