Bioluminescence is a natural light source based on luciferase catalysis of its substrate luciferin. We performed directed evolution on firefly luciferase using a red-shifted and highly deliverable ...luciferin analog to establish AkaBLI, an all-engineered bioluminescence in vivo imaging system. AkaBLI produced emissions in vivo that were brighter by a factor of 100 to 1000 than conventional systems, allowing noninvasive visualization of single cells deep inside freely moving animals. Single tumorigenic cells trapped in the mouse lung vasculature could be visualized. In the mouse brain, genetic labeling with neural activity sensors allowed tracking of small clusters of hippocampal neurons activated by novel environments. In a marmoset, we recorded video-rate bioluminescence from neurons in the striatum, a deep brain area, for more than 1 year. AkaBLI is therefore a bioengineered light source to spur unprecedented scientific, medical, and industrial applications.
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BFBNIB, NMLJ, NUK, ODKLJ, PNG, SAZU, UL, UM, UPUK
In preclinical cancer research, bioluminescence imaging with firefly luciferase and D-luciferin has become a standard to monitor biological processes both in vitro and in vivo. However, the emission ...maximum (λmax) of bioluminescence produced by D-luciferin is 562 nm where light is not highly penetrable in biological tissues. This emphasizes a need for developing a red-shifted bioluminescence imaging system to improve detection sensitivity of targets in deep tissue. Here we characterize the bioluminescent properties of the newly synthesized luciferin analogue, AkaLumine-HCl. The bioluminescence produced by AkaLumine-HCl in reactions with native firefly luciferase is in the near-infrared wavelength ranges (λmax=677 nm), and yields significantly increased target-detection sensitivity from deep tissues with maximal signals attained at very low concentrations, as compared with D-luciferin and emerging synthetic luciferin CycLuc1. These characteristics offer a more sensitive and accurate method for non-invasive bioluminescence imaging with native firefly luciferase in various animal models.
Although the current murine model of bone metastasis using intracardiac (IC) injection successfully recapitulates the process of bone metastasis, further progress in the study of bone metastasis ...requires a new model to circumvent some limitations of this model. Here, we present a new murine model of bone metastasis achieved by injecting cancer cells through the intra-caudal arterial (CA). This model does not require high technical proficiency, predominantly delivers cancer cells to bone marrow of hind limbs with much higher efficiency than IC injection, and greatly shortens the period of overt bone metastasis development. Moreover, CA injection barely causes acute death of mice, enabling us to inject a larger number of cancer cells to further accelerate the development of bone metastasis with a wide variety of cell lines. Our model may open a new avenue for understanding the bone metastatic processes and development of drugs preventing bone metastasis and recurrence.
Quantitative depth estimation of tumor invasion in early gastric cancer by scattering of circularly polarized light is computationally investigated using the Monte Carlo method. Using the optical ...parameters of the human stomach wall and its carcinoma, the intensity and circular polarization of light scattered from pseudo‐healthy and cancerous tissues were calculated over a wide spectral range. Large differences in the circular polarization with opposite signs, together with the large intensity, are obtained at wavelengths 600 nm and 950 nm. At these two wavelengths, the sampling depth of the biological tissues can be modulated by tuning the detection angle. In bi‐layered pseudo‐tissues with a cancerous layer on a healthy layer and vice versa, the degree of circular polarization of scattered light shows systematic changes depending on the thickness and depth of the cancerous layer, which indicates the feasibility of in vivo quantitative estimation of cancer progression in early gastric cancer.
Quantitative depth estimation of tumor invasion in early gastric cancer by scattering of circularly polarized light is computationally investigated using the Monte Carlo method. At the optimized two wavelengths, the degrees of circular polarization of scattered light show systematic changes depending on the cancer thickness in bi‐layered pseudo‐tissues with a cancerous layer on a healthy layer, which indicates the feasibility of in vivo quantitative estimation of cancer progression.
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FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SBCE, SBMB, UL, UM, UPUK
The Western pattern diet is rich not only in fat and calories but also in phosphate. The negative effects of excessive fat and calorie intake on health are widely known, but the potential harms of ...excessive phosphate intake are poorly recognized. Here, we show the mechanism by which dietary phosphate damages the kidney. When phosphate intake was excessive relative to the number of functioning nephrons, circulating levels of FGF23, a hormone that increases the excretion of phosphate per nephron, were increased to maintain phosphate homeostasis. FGF23 suppressed phosphate reabsorption in renal tubules and thus raised the phosphate concentration in the tubule fluid. Once it exceeded a threshold, microscopic particles containing calcium phosphate crystals appeared in the tubule lumen, which damaged tubule cells through binding to the TLR4 expressed on them. Persistent tubule damage induced interstitial fibrosis, reduced the number of nephrons, and further boosted FGF23 to trigger a deterioration spiral leading to progressive nephron loss. In humans, the progression of chronic kidney disease (CKD) ensued when serum FGF23 levels exceeded 53 pg/mL. The present study identified calcium phosphate particles in the renal tubular fluid as an effective therapeutic target to decelerate nephron loss during the course of aging and CKD progression.
Bioluminescence imaging (BLI) is an indispensable technique for visualizing the dynamics of diverse biological processes in mammalian animal models, including cancer, viral infections, and immune ...responses. However, a critical scientific challenge remains: non-invasively visualizing homeostatic and disease mechanisms in freely moving animals to understand the molecular basis of exercises, social behavior, and other phenomena. Classical BLI relies on prolonged camera exposure to accumulate the limited number of photons that traveled from deep tissues in anesthetized or constrained animals. Recent advancements in synthetic bioluminescence reactions, utilizing artificial luciferin–luciferase pairs, have considerably increased the number of detectable photons from deep tissues, facilitating high-speed BLI to capture moving objects. In this review, I provide an overview of emerging synthetic bioluminescence reactions that enable the non-invasive imaging of freely moving animals. This approach holds the potential to uncover unique physiological processes that are inaccessible with current methodologies.
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IZUM, KILJ, NUK, PILJ, PNG, SAZU, UL, UM, UPUK
Abstract
During the development of analgesic tolerance to morphine, the V1b vasopressin receptor has been proposed to bind to β-arrestin 2 and the µ-opioid receptor to enable their interaction. ...However, direct evidence of such a high-order complex is lacking. Using bioluminescent resonance energy transfer between a split Nanoluciferase and the Venus fluorescent protein, the NanoBit-NanoBRET system, we found that β-arrestin 2 closely located near the heteromer µ-V1b receptor in the absence of an agonist and moved closer to the receptor carboxyl-termini upon agonist stimulation. An additive effect of the two agonists for opioid and vasopressin receptors was detected on the NanoBRET between the µ-V1b heteromer and β-arrestin 2. To increase the agonist response of NanoBRET, the ratio of the donor luminophore to the acceptor fluorophore was decreased to the detection limit of luminescence. In the first phase of access, β-arrestin 2 was likely to bind to the unstimulated V1b receptor in both its phosphorylated and unphosphorylated forms. In contrast, the second-phase access of β-arrestin 2 was agonist dependent, indicating a possible pharmacological intervention strategy. Therefore, our efficient method should be useful for evaluating chemicals that directly target the vasopressin binding site in the µ-V1b heteromer to reduce the second-phase access of β-arrestin 2 and thereby to alleviate tolerance to morphine analgesia.
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IZUM, KILJ, NUK, PILJ, PNG, SAZU, UL, UM, UPUK
Small antibody mimetics that contain high-affinity target-binding peptides can be lower cost alternatives to monoclonal antibodies (mAbs). We have recently developed a method to create small antibody ...mimetics called FLuctuation-regulated Affinity Proteins (FLAPs), which consist of a small protein scaffold with a structurally immobilized target-binding peptide. In this study, to further develop this method, we established a novel screening system for FLAPs called monoclonal antibody-guided peptide identification and engineering (MAGPIE), in which a mAb guides selection in two manners. First, antibody-guided design allows construction of a peptide library that is relatively small in size, but sufficient to identify high-affinity binders in a single selection round. Second, in antibody-guided screening, the fluorescently labeled mAb is used to select mammalian cells that display FLAP candidates with high affinity for the target using fluorescence-activated cell sorting. We demonstrate the reliability and efficacy of MAGPIE using daclizumab, a mAb against human interleukin-2 receptor alpha chain (CD25). Three FLAPs identified by MAGPIE bound CD25 with dissociation constants of approximately 30 nM as measured by biolayer interferometry without undergoing affinity maturation. MAGPIE can be broadly adapted to any mAb to develop small antibody mimetics.
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The circular polarization of light scattered by biological tissues provides valuable information and has been considered as a powerful tool for the diagnosis of tumor tissue. We propose a ...non-staining, non-invasive and in vivo cancer diagnosis technique using an endoscope equipped with circularly polarized LEDs (spin-LEDs). We studied the scattering process of the circularly polarized light against cell nuclei in pseudo-healthy and cancerous tissues using the existing Monte Carlo method. The calculation results indicate that the resultant circular polarizations of light scattered in pseudo tissues shows a clear difference in a wide range of detection angles, and the sampling depth depends on those detection angles. The structure of the endoscope probe comprising spin-LEDs is designed based on the calculation results, providing structural and depth information regarding biological tissues simultaneously.
Small extracellular vesicles (sEV) contain various microRNAs (miRNAs) and play crucial roles in the tumor metastatic process. Although miR‐29b levels in peritoneal exosomes were markedly reduced in ...patients with peritoneal metastases (PM), their role has not been fully clarified. In this study, we asked whether the replacement of miR‐29b can affect the development of PM in a murine model. UE6E7T‐12, human bone marrow‐derived mesenchymal stem cells (BMSCs), were transfected with miR‐29b‐integrating recombinant lentiviral vector and sEV were isolated from culture supernatants using ultracentrifugation. The sEV contained markedly increased amounts of miR‐29b compared with negative controls. Treatment with transforming growth factor‐β1 decreased the expression of E‐cadherin and calretinin with increased expression of vimentin and fibronectin on human omental tissue‐derived mesothelial cells (HPMCs). However, the effects were totally abrogated by adding miR‐29b‐rich sEV. The sEV inhibited proliferation and migration of HPMCs by 15% (p < 0.005, n = 6) and 70% (p < 0.005, n = 6), respectively, and inhibited adhesion of NUGC‐4 and MKN45 to HPMCs by 90% (p < 0.0001, n = 5) and 77% (p < 0.0001, n = 5), respectively. MicroRNA‐29b‐rich murine sEV were similarly obtained using mouse BMSCs and examined for in vivo effects with a syngeneic murine model using YTN16P, a highly metastatic clone of gastric cancer cell. Intraperitoneal (IP) transfer of the sEV every 3 days markedly reduced the number of PM from YTN16P in the mesentery (p < 0.05, n = 6) and the omentum (p < 0.05, n = 6). Bone marrow mesenchymal stem cell‐derived sEV are a useful carrier for IP administration of miR‐29b, which can suppress the development of PM of gastric cancer.
miR‐29b incorporated in mesenchymal stem cells (MSCs) derived exsosomes strongly inhibits mesothelial mesenchymal transition in vitro. Intraperitoneal transfer of the miR‐29b‐rich exosomes suppresses the development of peritoneal metastasis from gastric cancer in murine model.
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