Live-cell System Research Articles

Single-Nucleobase-Resolved Nanoruler Determines the Surface Energy Transfer Radius on the Living Cell Membrane.

Investigations about surface energy transfer radius (r0) are limited to the aqueous solution system, and it is quite limited on experimental values of r0 between dyes and the corresponding gold particle (AuNP) sizes, especially for living cell systems. Hence, the selection of suitable AuNP-dye pairs is restricted when designing nanometal surface energy transfer (NSET) strategies in analytical sciences. Here, we developed a single-nucleobase-resolved NSET strategy to in situ measure the r0 value between a specific dye and different-sized AuNPs on the living cell membrane. Using the aptamer-dye complex (XQ-2d-nTA-FAM) and antiCD71 antibody-coupled AuNP conjugate (Au@antiCD71) as two working elements to bind two different sites on CD71 receptors on living cell membranes, we modified the nTA spacer between FAM and the terminal of aptamer to change the distance (r) from FAM to AuNP center and further adjusted the quenching efficiency (Φ) between them. Different r0 values of various AuNP-FAM pairs in living cells are determined by this in situ detection strategy. Based on this single-nucleobase-resolved NSET strategy, we established a simple and efficient universal method for measuring r0 in the living cell system, which greatly expanded the selection range of AuNP-dye pairs during the construction of the NSET model at the nanoscale.

Novel Antibacterial and Biocompatible Nanostructured Gels Based on One-step Synthesis as a Potential Disinfectant for Endodontic Infection Control

AimThe objective of this study was to develop nanostructured gels as biocompatible intracanal disinfectants by one-step microwave radiation-assisted synthesis. MethodsPolyvinyl alcohol (PVA) and polyvinyl pyrrolidone (PVP) were used as a support network, and polyethylene glycol (PEG) was used as a reducing agent. The gels were characterized by measuring the swelling ratio (SR) and rheological properties and by scanning electron microscopy (SEM), transmission electron microscopy (TEM) and atomic force microscopy (AFM). The antibacterial effects of each gel were evaluated against the endodontic clinical strain Enterococcus faecalis. Then, the viability of the 21-day mature multispecies bacterial biofilm was assessed using confocal microscopy in an ex vivo model, where the biofilm was exposed to the mix of nanogels. The cell proliferation, viability, and morphology of human periodontal ligament (HPDL) cells were quantified using a real-time IncuCyte® S3 Live-Cell System. Viability was measured by confocal microscopy using an ex vivo model exposing a 21-day mature multispecies bacterial biofilm to the mix of nanogels. ResultsThe antibacterial activity of the gels coincided with the superficial characterization and the solubility of the gel in the growth medium. Gels with higher viscosity (327.85-980.58 Pa s), higher dissolution (42-70%SR), and lower porosity (no porosity and 611.63 nm) showed excellent antibacterial activity against E. faecalis. Despite their physicochemical characteristics, CuNPs gels showed greater effectiveness against E. faecalis.These nanostructured gels with high PVA concentrations promote HPDL cells proliferation while still exerting antibacterial properties. Mix of nanogels showed an increase non-viable cells biomass from at of application. ConclusionsThe use of biocompatible polymers influences the physicochemical, bactericidal, and cytotoxic response, making these materials potential disinfectant agents against resistant bacteria with good biocompatibility and improved HPDL cells proliferation.

Real-time evaluation of the biocompatibility of calcium silicate-based endodontic cements: An in vitro study.

An ideal filling material should hermetically seal the communication pathways between the canal system and surrounding tissues. Therefore, during the last few years, the development of obturation materials and techniques to create optimal conditions for the proper healing of apical tissues has been a focus of interest. The effects of calcium silicate-based cements (CSCs) on periodontal ligament cells have been investigated, and promising results have been obtained. To date, there are no reports in the literature that have evaluated the biocompatibility of CSCs using a real-time live cell system. Therefore, this study aimed to evaluate the real-time biocompatibility of CSCs with human periodontal ligament cells (hPDLCs). hPDLC were cultured with testing media of endodontic cements for 5 days: TotalFill-BC Sealer, BioRoot RCS, Tubli-Seal, AH Plus, MTA ProRoot, Biodentine, and TotalFill-BC RRM Fast Set Putty. Cell proliferation, viability, and morphology were quantified using real-time live cell microscopy with the IncuCyte S3 system. Data were analyzed using the one-way repeated measures (RM) analysis of variancemultiple comparison test (p < .05). Compared to the control group, cell proliferation in the presence of all cements was significantly affected at 24 h (p < .05). ProRoot MTA and Biodentine lead to an increase in cell proliferation; there were no significant differences with the control group at 120 h. In contrast, Tubli-Seal and TotalFill-BC Sealer inhibited cell growth in real-time and significantly increased cell death compared to all groups. hPDLC co-cultured with sealer and repair cements showed a spindle-shaped morphology except with cements Tubli-Seal and TotalFill-BC Sealer where smaller and rounder cells were obtained. The biocompatibility of the endodontic repair cements performed better than the sealer cements, highlighting the cell proliferation of the ProRoot MTA and Biodentine in real-time. However, the calcium silicate-based TotalFill-BC Sealer presented a high percentage of cell death throughout the experiment similar to that obtained.

Binding affinity-based intracellular drug detection enabled by a unimolecular cucurbit[7]uril-dye conjugate.

Label-free fluorescence-based chemosensing has been increasingly brought into focus due to its simplicity and high sensitivity for intracellular monitoring of molecules. Currently used methods, such as conventional indicator displacement assays (IDAs), pose limitations related to dissociation upon dilution, random diffusion of the released indicators, and high sensitivity to interference by agents from the ambient cellular environment (e.g., salts, enzymes, and proteins). Herein we report a potentially widely applicable strategy to overcome the limitations of conventional IDAs by employing a macrocyclic cucurbit[7]uril (CB7) host covalently coupled to a nitrobenzoxadiazole (NBD) fluorescent dye (CB7-NBD conjugate). As a proof of concept, we demonstrated that the CB7-NBD unimolecular conjugate responded to various target analytes even in the complex live cell system. Moreover, the sensing system was compatible with fluorescence imaging, fluorescence-assisted cell sorting (FACS), and fluorescence spectrometry with a microplate reader. These experiments demonstrated an application of covalently bound unimolecular CB7-NBD conjugate as a sensor for detecting diverse analytes in the intracellular compartment of live cells.

ALFA-PRF: a novel approach to detect murine perforin release from CTLs into the immune synapse.

When killing through the granule exocytosis pathway, cytotoxic lymphocytes release key effector molecules into the immune synapse, perforin and granzymes, to initiate target cell killing. The pore-forming perforin is essential for the function of cytotoxic lymphocytes, as its pores disrupt the target cell membrane and allow diffusion of pro-apoptotic serine proteases, granzyme, into the target cell, where they initiate various cell death cascades. Unlike human perforin, the detection of its murine counterpart in a live cell system has been problematic due its relatively low expression level and the lack of sensitive antibodies. The lack of a suitable methodology to visualise murine perforin secretion into the synapse hinders the study of the cytotoxic lymphocyte secretory machinery in murine models of human disease. Here, we describe a novel recombinant technology, whereby a short ALFA-tag sequence has been fused with the amino-terminus of a mature murine perforin, and this allowed its detection by the highly specific FluoTag®-X2 anti-ALFA nanobodies using both Total Internal Reflection Fluorescence (TIRF) microscopy of an artificial synapse, and confocal microscopy of the physiological immune synapse with a target cell. This methodology can have broad application in the field of cytotoxic lymphocyte biology and for the many models of human disease.

OR22-4 Homing of Antigen-specific Engineered Regulatory T Cells To Human Pancreatic Islets

Abstract The therapeutic application of regulatory T cells (Tregs) for the treatment of autoimmune disorders, although efficacious, has been limited by the scarcity of antigen-specific Tregs. If antigen-specific Tregs, capable of reaching the desired autoimmune target, could be produced on demand, antigen-specific immune suppression of autoimmune diseases would be achievable. One approach to endow T cells with a desired antigen-specificity uses chimeric T cell antigen-receptors (CAR) with antibody-type specificity. Adoptive cell transfer therapies with CAR-re-directed cytotoxic T cells, have shown impressive efficacy in the treatment of hematologic malignancies. Accordingly, employing such technology to re-direct Tregs to sites of autoimmune attack may be a useful therapeutic approach to alleviate a broad spectrum of diseases in which uncontrolled auto and alloimmune responses play a major role. We recently developed pancreatic beta-cell, antigen-specific, CAR Tregs (1) and explored their therapeutic potential against T1D in our humanized mouse model (2). Ours is the first successful, antigen-specific CAR-Treg treatment of T1D in a humanized mouse model that closely resembles the human disease. Based on our mice data, we believe treatment with pancreatic beta-cell, antigen-specific CAR-Tregs will allow for recovery and reconstitution of beta cells in human T1D patients as well. The purpose of this study was to determine if antigen-specific human CAR Tregs could also identify target and home to human pancreatic islets in culture as they do in mice. The study involved drawing 10 cc of blood 1-2 weeks prior to pancreas surgery; followed by collection of a small piece of pancreas (5 cc wedge) once the pancreas was removed for a clinical indication (cancer, pancreatitis). We first isolated Tregs from peripheral blood of these human donors and expanded them in vitro. Treg cells were genetically modified to express either a beta-cell antigen-specific (GAD65) CAR or an irrelevant (EPCAM) CAR construct together with GFP marker. CAR Tregs were selectively expanded in the presence of rhGAD65 antigen. Once pancreas tissue became available, it was processed for islet separation using the collagenase method. Pancreatic islets were then co-cultured with syngeneic CAR Tregs for 7 days. IncuCyte S3 Live-cell System (Sartorius) is a real-time system that automatically acquires and analyzes HD, phase and fluorescent images of cell cultures, around the clock, for 7 days, while cells remain undisturbed. Live immunofluorescence microscopy demonstrated the distinct homing of GAD65 CAR Tregs to islets as compared to control EPCAM CAR Tregs as early as 24 hours of co-culture. Importantly, proliferation of GAD65 CAR Tregs was clearly demonstrated within 72 hours. GAD65 CAR Treg cytokine profile from co-culture supernatant was characteristic of activated Tregs. 1 PATENT #WO2020097546 2 PATENT #US20200037586 Presentation: Monday, June 13, 2022 11:45 a.m. - 12:00 p.m.

Abstract 3084: Label-free, real-time live cell assays for 3D organoids embedded in Matrigel®

Abstract Organoid technologies are increasingly being used as in-vitro models of human development and disease as they exhibit structural, morphogenetic, and functional properties that recapitulate in vivo pathophysiology. To successfully use these models across a variety of research disciplines and applications, approaches that reduce variability and technology pipelines to image &amp; quantify these complex cell models are required. Here, we demonstrate simple, robust workflows for monitoring and automatically quantifying features, such as morphology, growth and death of organoids using real time live cell analysis. To quantitatively optimize and characterize organoid cultures in-vitro, mouse hepatic, intestinal and pancreatic organoids were embedded in Matrigel® domes (50% or 100%) in 24-well plates and imaged over time in an Incucyte® Live Cell System. Organoid growth, differentiation, and maturation was measured using Incucyte’ s automated Organoid Software Analysis Module, which tracks changes in size and morphology. Integrated metrics enabled objective determination of cell-type specific growth conditions and passaging regimes. To illustrate the utility of the Incucyte® Organoid Analysis Software Module to track organoid growth and death in 96-well plates, intestinal and hepatic organoid fragments were embedded in Matrigel® (50%) for 3 days prior to treatment with protein kinase inhibitor staurosporine (1 µM, STP). Vehicle treated organoids increased in size (10-fold; intestinal or 3-fold; hepatic) over time while marked reduction was observed in the presence of STP. Using label-free size and morphology metrics we could distinguish between cytotoxic and cytostatic mechanisms of action (MoA) of known chemotherapeutic compounds. STP, cisplatin (CIS, DNA synthesis inhibitor) or fluorouracil (5-FU, thymidylate synthetase inhibitor) exhibited concentration dependent inhibition of hepatic organoid growth, yielding IC50 values of 3 nM for STP, 9.7 µM for CIS and 0.78 µM for 5-FU. Whilst attenuation of size was observed across all compounds, increases in eccentricity and darkness indictive of 3D structure disruption and cell death respectively were only observed in CIS and STP-treated organoids (cytotoxic MoA). Differences between the size and morphology readouts illustrated the cytostatic mechanism of 5-FU. Use of this approach was extended to visualize and quantify CFTR function. Following forskolin stimulation, a concentration-dependent increase in intestinal organoid size was observed. In the presence of CFTR inhibitor CFTRinh-172 the maximal response was reduced by &amp;gt;50% (~150% at 10 µM) illustrating that swelling was CFTR-dependent. These data demonstrate the capability to kinetically visualize and quantify distinct organoid morphologies, assess drug-induced cellular changes label-free and illustrates the amenability of this approach across a range of disease areas. Citation Format: Tim R. Jackson, Miniver Oliver, Daniel Appledorn, Tim Dale, Kalpana Barnes. Label-free, real-time live cell assays for 3D organoids embedded in Matrigel® [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3084.

Dual-response fluorescence sensor for detecting Cu2+ and Pd2+ based on bis-tetraphenylimidazole Schiff-base

Although the fluorescence sensors for Cu2+ or Pd2+ had been prepared, the sensor of in-situ detecting both Cu2+ ion and Pd2+ ion in aqueous media was rarely concerned. Herein, a bis-tetraphenylimidazole Schiff-base (Bis-TPI) with aggregation-induced emission was designed and prepared in yield of 88%. Bis-TPI displayed strong red fluorescence at 550–650 nm in aqueous media and selectively response on detecting Cu2+ and Pd2+ with quenched fluorescence. The detection limits attained 8.3 × 10-7 M for Cu2+ and 9.2 × 10-7 M for Pd2+, respectively. The mutual interference on detecting Cu2+ and Pd2+ in their coexistence system was eliminated by the masking reagent of adenosine triphosphate for Cu2+, realizing the detection of Cu2+ and Pd2+ sequentially. The sensing mechanism of 1:1 binding system was supported by FT-IR spectra, binding MS spectra, 1H NMR spectra and fluorescence Job’s plot. Moreover, Bis-TPI showed good fluorescence bioimaging property and achieved in-situ detection on Cu2+ and Pd2+ in living-cell system, implying the application prospect on in-situ sensing Cu2+ and Pd2+ in both vitro environment and vivo assay.

A Water-Soluble Organic Photocatalyst Discovered for Highly Efficient Additive-Free Visible-Light-Driven Grafting of Polymers from Proteins at Ambient and Aqueous Environments.

Since the pioneering discovery of a protein bound to poly(ethylene glycol), the utility of protein-polymer conjugates (PPCs) is rapidly expanding to currently emerging applications. Photoinduced energy/electron-transfer reversible addition-fragmentation chain-transfer (PET-RAFT) polymerization is a very promising method to prepare structurally well-defined PPCs, as it eliminates high-cost and time-consuming deoxygenation processes due to its oxygen tolerance. However, the oxygen-tolerance behavior of PET-RAFT polymerization is not well-investigated in aqueous environments, and thereby the preparation of PPCs using PET-RAFT polymerization needs a substantial amount of sacrificial reducing agents or inert-gas purging processes. Herein a novel water-soluble and biocompatible organic photocatalyst (PC) is reported, which enables visible-light-driven additive-free "grafting-from" polymerizations of a protein in ambient and aqueous environments. Interestingly, the developed PC shows unconventional "oxygen-acceleration" behavior for a variety of acrylic and acrylamide monomers in aqueous conditions without any additives, which are apparently distinct from previously reported systems. With such a PC, "grafting-from" polymerizations are successfully performed from protein in ambient buffer conditions under green light-emitting diode (LED) irradiation, which result in various PPCs that have neutral, anionic, cationic, and zwitterionic polyacrylates, and polyacrylamides. It is believed that this PC will be widely employed for a variety of photocatalysis processes in aqueous environments, including the living cell system.

A fluorescence-based, gain-of-signal, live cell system to evaluate SARS-CoV-2 main protease inhibition

The likelihood of continued circulation of COVID-19 and its variants, and novel coronaviruses due to future zoonotic transmissions, combined with the current paucity of coronavirus antivirals, emphasize the need for improved screening in developing effective antivirals for the treatment of infection by SARS-CoV-2 (CoV2) and other coronaviruses. Here we report the development of a live-cell based assay for evaluating the intracellular function of the critical, highly-conserved CoV2 target, the Main 3C-like protease (Mpro). This assay is based on expression of native wild-type mature CoV2 Mpro, the function of which is quantitatively evaluated in living cells through cleavage of a biosensor leading to loss of fluorescence. Evaluation does not require cell harvesting, allowing for multiple measurements from the same cells facilitating quantification of Mpro inhibition, as well as recovery of function upon removal of inhibitory drugs. The pan-coronavirus Mpro inhibitor, GC376, was utilized in this assay and effective inhibition of intracellular CoV2 Mpro was found to be consistent with levels required to inhibit CoV2 infection of human lung cells. We demonstrate that GC376 is an effective inhibitor of intracellular CoV2 Mpro at low micromolar levels, while other predicted Mpro inhibitors, bepridil and alverine, are not. Results indicate this system can provide a highly effective high-throughput coronavirus Mpro screening system.

A novel multifunctional NCQDs-based injectable self-crosslinking and in situ forming hydrogel as an innovative stimuli responsive smart drug delivery system for cancer therapy

In this work, we offer an easy approach to develop a novel injectable, pH sensitive and in situ smart drug delivery system for use in cancer treatments. The developed hydrogels containing nitrogen doped carbon quantum dots (NCQD), doxorubicin (Dox) and hydroxyapatite (HA) were obtained by in situ self-crosslinking. Characterization of the synthesized nanomaterials, interactions between NCQD/Dox/HA hydrogel structure were carried out by TEM, FESEM, EDS, FTIR, XPS, XRD, Zeta potential, DLS, UV–Vis, SEM, gelation time, injectability and DIST measurements. In addition, antibacterial evaluation which was performed against Staphylococcus aureus realized that HA compound significantly increased the antibacterial activity of the hybrid hydrogel. The anticancer drug release to the tumor cell microenvironment with a pH of 5.5 was found to be higher compared to the release in the normal physiological range of pH 6.5 and 7.4. MTT and live/dead assays were also performed using L929 fibroblastic cell lines to investigate the cytotoxic behavior of NCQDs, and NCQDs/Dox/HA hydrogels. Furthermore, the NCQDs/Dox/HA hydrogel could transport Dox within a MCF-7 cancerous cell at specifically acidic pH. Additionally, imaging of cell line was observed using NCQDs and their use in imaging applications and multicolor features in the living cell system were evaluated. The overall study showed that in situ formed NCQDs/Dox/HA hydrogel represented a novel and multifunctional smart injectable controlled-release drug delivery system with great potential, which may be considered as an attractive minimal invasive smart material for future intelligent delivery of chemotherapeutic drug and disease therapy applications.

Optically activated, customizable, excitable cells.

Genetically encoded fluorescent biosensors are powerful tools for studying complex signaling in the nervous system, and now both Ca2+ and voltage sensors are available to study the signaling behavior of entire neural circuits. There is a pressing need for improved sensors, but improving them is challenging because testing them involves a low throughput, labor-intensive processes. Our goal was to create synthetic, excitable cells that can be activated with brief pulses of blue light and serve as a medium throughput platform for screening the next generation of sensors. In this live cell system, blue light activates an adenylyl cyclase enzyme (bPAC) that increases intracellular cAMP (Stierl M et al. 2011). In turn, the cAMP opens a cAMP-gated ion channel. This produces slow, whole-cell Ca2+ transients and voltage changes. To increase the speed of these transients, we add the inwardly rectifying potassium channel Kir2.1, the bacterial voltage-gated sodium channel NAVROSD, and Connexin-43. The result is a highly reproducible, medium-throughput, live cell system that can be used to screen voltage and Ca2+ sensors.

Abstract P067: Piezo1 Activation Inhibits Adipogenesis And Lipogenesis In Perivascular Adipose Tissue Preadipocytes

In adipogenesis, perivascular adipose tissue (PVAT) preadipocytes turn into adipocytes. In non-PVAT preadipocytes, mechanical forces affect the commitment and lipogenic stages of adipogenesis. The mechanism may involve PIEZO1, a mechanosensor, that boosts differentiation of progenitor cells towards osteogenic and fibroblastic lineages. Since hypertension causes changes in the vascular forces that could affect adipogenesis in PVAT, our objective was to evaluate PIEZO1 expression patterns in PVAT and the effects of PIEZO1 activation on the adipogenic potential of preadipocytes. We hypothesize that PIEZO1 activation limits the adipogenic potential of PVAT preadipocytes. PVAT from the thoracic aorta (APVAT) was collected from male Sprague Dawley rats at 10 weeks of age (n=5). Preadipocytes were obtained by Liberase digestion followed by serial passage of adherent cells. Preadipocyte progenitors, CD34+PDGFRα+, were harvested by magnetic-activated sorting. PIEZO1 expression was assessed by RT-qPCR and immunofluorescence (IF). Preadipocytes were induced to differentiate for 14 d in standard media (CON) or in the presence of PIEZO1 agonist Yoda (10μM) and inhibitor Dooku (5μM) during days 0-2 (commitment), 3-14 (lipogenesis), and 0-14 (overall adipogenesis). Adipogenesis was evaluated with IncuCyte Live-Cell system using the triglyceride stain Bodipy 493503. Triglyceride content is reported as Bodipy Intensity/Adipocyte Count. Piezo1 RNA was expressed in adipocytes and the stromal vascular fraction of APVAT. PIEZO1 IF signal was detected in SVF and preadipocyte. Triglyceride was reduced by Yoda (62 ± 14.3) and Dooku (49.3 ± 14) during 0-2 d compared to CON (312.5 ± 165.6). Neither Yoda nor Dooku for 12 d affected triglyceride accumulation compared to CON. In contrast, the lipid content of Yoda (77.7 ± 21.3) and Dooku (48.9 ± 15.2) treated cells during 14 d was reduced vs CON (312.5 ± 165.6). The expression of PIEZO1 in all PVAT fractions suggests mechanosensitivity. PIEZO1 activation during adipogenesis commitment impaired adipocyte maturation. These data provide evidence for the capacity of mechanosensory expressed in PVAT preadipocytes to modulate adipogenesis, underpinning the deleterious impact of hypertension on PVAT function.

Construction and application of a high-content analysis for identifying human carboxylesterase 2 inhibitors in living cell system.

Human carboxylesterase 2 (hCE2), one of the most principal drug-metabolizing enzymes, catalyzes the hydrolysis of a variety of endogenous esters, anticancer agents, and environmental toxicants. The significant roles of hCE2 in both endobiotic and xenobiotic metabolism sparked great interest in the discovery and development of efficacious and selective inhibitors. However, the safe and effective inhibitors of hCE2 are scarce, due to the lack of efficient screening and evaluation systems for complex biological systems. To offer a solution to this problem, a high-content analysis (HCA)-based cell imaging and multiparametric assay method was constructed for evaluating the inhibitory effect and safety of hCE2 inhibitors in living cell system. In this study, we first established a cell imaging-based method for identifying hCE2 inhibitors at the living cell level with hCE2 fluorescent probe NCEN. Meanwhile, two nuclear probes, Hoechst 33342 and PI, were integrated to evaluate the potential cytotoxicity of compounds simultaneously. Then, the accuracy of the HCA-based method was verified by the LC-FD-based method with a positive inhibitor BNPP, and the results showed that the HCA-based method exhibited excellent precision, robustness, and reliability. Finally, the newly established HCA-based multiparametric assay panel was successfully applied to re-evaluate a series of reported hCE2 inhibitors in living cells. In summary, the HCA-based multiparametric method could serve as an efficient tool for the accuracy measurement inhibitory effect and cytotoxicity of compounds against hCE2 in living cell system. Graphical abstract.

Dual nitrogen-oxygen isotopic analysis and kinetic model for enzymatic nitrate reduction coupled with Fe(II) oxidation by Pseudogulbenkiania sp. strain 2002

While microbially mediated coupling process of nitrate reduction and Fe(II) oxidation (NRFO) plays important role in widespread nitrogen and iron biogeochemical cycles in the natural environments, the underlying mechanism of NRFO is still not clearly understood. Here, N and O isotope fractionation during microbial nitrate reduction by Pseudogulbenkiania sp. strain 2002 with or without Fe(II) were investigated. The nitrate reduction rate decreased substantially in the presence of Fe(II), and cells became encrusted with Fe(III) minerals. However, the N isotope enrichment factors (ε) with or without Fe(II) were identical within uncertainty (24.1 ± 2.4‰ and 25.1 ± 2.4‰), implying that Fe(II) oxidation had little influence on the extent of N isotope fractionation and that cell encrustation was unlikely to cause additional mass transfer limitation. Isotopic composition ratios of 18O to 15N (18ε: 15ε) were 0.73 ± 0.13 with and 0.50 ± 0.08 without Fe(II), close to reported values for nitrate reduction by periplasmic nitrate reductases, implying that enzymatic nitrate reduction mainly occurs in strain 2002's periplasmic space. A kinetic model based on elementary reactions was established, and rate constants of enzymatic nitrate reducing reactions were obtained in a living cell system. Combining dual N-O isotope fractionation analysis with kinetic modeling is a promising approach for interpreting diverse enzymatic pathways during NRFO processes.

Probing Deviation of Adhered Membrane Dynamics between Reconstituted Liposome and Cellular System.

The dynamics of cell-cell adhesion are complicated due to complexities in cellular interactions and intra-membrane interactions. In the present work, we have reconstituted a liposome-based model system to mimic the cell-cell adhesion process. Our model liposome system consists of one fluorescein-tagged and one TRITC (tetramethyl-rhodamine isothiocyanate)-tagged liposome, adhered through biotin-neutravidin interaction. We monitored the adhesion process in liposomes using Förster Resonance Energy Transfer (FRET) between fluorescein (donor) and TRITC (acceptor). Occurrence of FRET is confirmed by the decrease in donor lifetime as well as distinct rise time of the acceptor fluorescence. Interestingly, the acceptor's emission exhibits fluctuations in the range of ≈3±1 s. This may be attributed to structural oscillations associated in two adhered liposomes arising from the flexible nature of biotin-neutravidin interaction. We have compared the dynamics in a cell-mimicking liposome system with that in an in vitro live cell system. In the adhered live cell system, we used CPM (7-diethylamino-3-(4-maleimido-phenyl)-4-methylcoumarin, donor) and nile red (acceptor), which are known to stain the membrane of CHO (Chinese Hamster Ovary) cells. The dynamics of the adhered membranes of two live CHO cells were observed through FRET between CPM and nile red. The acceptor fluorescence intensity exhibits an oscillation in the time-scale of ≈1±0.75 s, which is faster compared to the reconstituted liposome system, indicating the contributions and involvement of multiple dynamic protein complexes around the cell membrane. This study offers simple reconstituted model systems to understand the complex membrane dynamics using a FRET-based physical chemistry approach.

α-l-Threose Nucleic Acids as Biocompatible Antisense Oligonucleotides for Suppressing Gene Expression in Living Cells.

Because of the chemical simplicity of α-l-threose nucleic acid (TNA) and its ability to exchange genetic information between itself and RNA, it has attracted significant interest as the RNA ancestor. We herein explore the biological properties and evaluate the potency of sequence-designed TNA polymers to suppress the gene expression in living environments. We found that sequence-specific TNA macromolecules exhibit strong affinity and specificity toward the complementary RNA targets, are highly biocompatible and nontoxic in a living cell system, and readily enter a number of cell lines without using transfecting agents. Particularly, TNA exhibited much stronger enzymatic resistance toward fetal bovine serum or human serum as compared to traditional antisense oligonucleotides, which means that the intrinsic structure of TNA is thoroughly resistant to biological degradation. Importantly, the efficacy of the TNA molecule with green fluorescent protein (GFP) target sequence (anti-GFP TNAs) as antisense agents was first demonstrated in living cells in which these polymers revealed high antisense activity in terms of the degree of inhibition of GFP gene expression. The GFP gene inhibition studies in HeLa and HEK293 cells characterize sequence-controlled TNA as a functional biomaterial and a valuable alternative to traditional antisense oligonucleotides such as peptide nucleic acids, phosphorodiamidate morpholino oligomers, and locked nucleic acids for a wide range of applications in drug discovery and life science research. Additionally, we also first reported the cost-efficient approach to synthesize the four TNA phosphoramidite monomers using 2-cyanoethyl N, N, N', N'-tetraisopropylphosphoramidite as a key reagent. Furthermore, by increasing the frequency of the deblocking and coupling reactions together with extending their reaction time in each synthesis cycle, sequence-controlled TNAs can be easily synthesized in a quantitative yield and high purity.

Interaction between a Unique Minor Protein and a Major Capsid Protein of Bluetongue Virus Controls Virus Infectivity.

ABSTRACTAmong the Reoviridae family of double-stranded RNA viruses, only members of the Orbivirus genus possess a unique structural protein, termed VP6, within their particles. Bluetongue virus (BTV), an important livestock pathogen, is the prototype Orbivirus. BTV VP6 is an ATP-dependent RNA helicase, and it is indispensable for virus replication. In the study described in this report, we investigated how VP6 might be recruited to the virus capsid and whether the BTV structural protein VP3, which forms the internal layer of the virus capsid core, is involved in VP6 recruitment. We first demonstrated that VP6 interacts with VP3 and colocalizes with VP3 during capsid assembly. A series of VP6 mutants was then generated, and in combination with immunoprecipitation and size exclusion chromatographic analyses, we demonstrated that VP6 directly interacts with VP3 via a specific region of the C-terminal portion of VP6. Finally, using our reverse genetics system, mutant VP6 proteins were introduced into the BTV genome and interactions between VP6 and VP3 were shown in a live cell system. We demonstrate that BTV strains possessing a mutant VP6 are replication deficient in wild-type BSR cells and fail to recruit the viral replicase complex into the virus particle core. Taken together, these data suggest that the interaction between VP3 and VP6 could be important in the packaging of the viral genome and early stages of particle formation.IMPORTANCE The orbivirus bluetongue virus (BTV) is the causative agent of bluetongue disease of livestock, often causing significant economic and agricultural impacts in the livestock industry. In the study described in this report, we identified the essential region and residues of the unique orbivirus capsid protein VP6 which are responsible for its interaction with other BTV proteins and its subsequent recruitment into the virus particle. The nature and mechanism of these interactions suggest that VP6 has a key role in packaging of the BTV genome into the virus particle. As such, this is a highly significant finding, as this new understanding of BTV assembly could be exploited to design novel vaccines and antivirals against bluetongue disease.

Redox Transformation of Arsenic and Antimony in Soils Mediated by Pantoea sp. IMH

Soil contamination with co-existing arsenic (As) and antimony (Sb) has become a serious environmental problem worldwide. Microorganisms play a dominant role in the redox transformation and mobilization of As and Sb. As and Sb belong to the same family; they are alike in nature and have related microbial oxidation-reduction mechanisms. However, limited knowledge is available about the impact of As-reducing bacteria on the fate of As and Sb in their co-existing soils. The purpose of this study was to explore the redox transformation and release of As and Sb in the presence of an arsC carrier, Pantoea sp. IMH, which has high As(Ⅴ)-reducing capability. In addition to the IMH incubation system, the dead cell system and abiotic control experiments were conducted. The results showed that the IMH incubation reduced all soluble As(Ⅴ) (72.7 μg·L-1) to As(Ⅲ) form, whereas soluble Sb(Ⅴ) (364.8 μg·L-1) was not reduced, indicating that the As (V)-reducing pathway mediated by arsC genesis was not able to reduce Sb(Ⅴ). The amounts of total As (506.8 μg·L-1) and total Sb (821.1 μg·L-1) in the dead cell system were approximately four times higher than in the living cell system (As=155.2 μg·L-1; Sb=364.8 μg·L-1) and the abiotic control (As=57.6 μg·L-1; Sb=271.1 μg·L-1) because of the biomolecules released from the dead cells which enhanced the release of As and Sb. The correlation analysis of dissolved As and Sb showed that the release of total As and Sb was correlated (P<0.05) in three systems. Our study shed new light on the impact of bacteria on the fate of As and Sb in soils.

Biocompatible yogurt carbon dots: evaluation of utilization for medical applications

In this study, carbon dots (CDs) were produced from yogurt, a fermented milk product, via microwave-assisted process (800 W) in 30 min without using any additional chemical agents. Yogurt CDs had outstanding nitrogen and oxygen ratios. These dots were monodisperse and about 2 nm sized. The toxicological assessments of yogurt carbon dots in human cancer cells and normal epithelial cells and their fluorescence imaging in living cell system were carried out. Yogurt carbon dots had intense fluorescent signal under confocal microscopy and good fluorescence stability in living cell system. The resulting yogurt carbon dots exhibited high biocompatibility up to 7.1 mg/mL CD concentration which may find utilization in medical applications such as cellular tracking, imaging and drug delivery. Yogurt carbon dots have potential to be good diagnostic agents to visualize cancer cells which may be developed as a therapeutic carrier.